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https://f1000research.com/articles/8-647/v1
|
10 May 19
|
{
"type": "Research Article",
"title": "The perceived benefit of a short-term service trip of students and participants while working in an HIV community center in Puerto Rico",
"authors": [
"Martin G. Rosario",
"Samantha Ewing",
"Kaitlyn Foster",
"Samantha Ewing",
"Kaitlyn Foster"
],
"abstract": "Introduction: Puerto Rico is among the top five territories in the USA to be affected by human immunodeficiency virus (HIV), which is why our goal is to help the island with service trips. Studies have reported the professional and personal benefits students can gain by participating in service activities. However, the benefits obtained by the Hispanic-Latino participants living with HIV in Puerto Rico, have not been outlined. The purpose of this study was to discuss the perceived benefits of a short-term week-long service trip for the students, participants, and personnel. Methods: A total of 11 physical therapy students and one professor travelled to Puerto Rico for a one-week service trip. The group partnered with an established organization named ‘La Perla de Gran Precio,’ which works with low-income Hispanic-Latino USA citizens participants that have been diagnosed with HIV. Students were involved in both academic and cultural components by providing physical therapy services to the participants. At the end of the week, surveys were given to all parties involved. Results: Students, personnel and participants reported the service trip as extremely positive. Students suggested that its integration should be considered in any physical therapy curriculum to improve the future of this profession further. Participants reported learning from this experience and have been able to implement the methods into their routine. Conclusions: The Puerto Rico service trip enhanced the education of physical therapy students and their ability to increase cultural awareness, boost communication skills, provide opportunities to overcome challenges, and foster a sense of purpose. Also, the Puerto Rico service trip was a beneficial and positive experience for all people involved. Consideration should be made to incorporate this initiative a much larger scale in a population that is vastly underserved.",
"keywords": [
"Service trip",
"Puerto Rico",
"HIV",
"educational service trips"
],
"content": "Introduction\n\nThe human immunodeficiency virus (HIV) is a worldwide pandemic affecting the lives of over 36.9 million individuals (see data from UNAIDS). According to the Center for Disease Control (CDC), there are more than 1.1 million people with HIV of ages of 13 years and older in the United States alone. In the United States, Texas and Puerto Rico are among the five locations with the highest rate of HIV diagnoses, with 15.4 and 13.3 per 100,000 people, respectively. According to the CDC, the number of HIV infections is four times higher in Hispanic/Latino males than in white males within these groups (USA territory). In their latest surveillance report (in 2015, revised in November 2018), the CDC stated that the number of HIV infections has increased among individuals between 25–34 years old, with the rates of infection in males being 4.8 times more than in females. The prevalence and incidence rates of HIV in Puerto Rico are deemed prominent and call for the necessity of multimodal and multifocal assistants, intervention, integration of innovative research and service projects.\n\nService projects are a component of many educational programs and have been shown to be beneficial at a personal and professional level for everyone involved (Withers et al., 2013). Service trips are also intended to aid in different health care needs in low-income regions (Sykes, 2014). Prior research demonstrated that for medical students and residents, service excursions have been favorable in creating insight into valuable experiences for personal development and diplomatic connections (Chiu et al., 2012). Additionally, service trips had been found to improve clinical skills and training competency (Campbell et al., 2011), enhance cultural awareness (Brown et al., 2012) and provide a meaningful connection with others in diverse cultural contexts (Chiu et al., 2012).\n\nMuch attention has been drawn by multiple organizations to helping in larger regions through service trips, such as Africa and Asia, where the number of people infected with HIV is higher in comparison to the Latin/Hispanic population within the US territory (see data from Go Overseas). However, prior studies have failed to discuss the advantages of these trips in an HIV low-income community center (located in Puerto Rico) which mainly treats Latino/Hispanic people. Additionally, the value or perception of the students and personnel have not been reported.\n\nTherefore, the purpose of this article is to explore and report whether short-term service trips can be beneficial in a professional and personal level to those providing the services, as well as to those being served in a Latino-Hispanic, low-income, HIV community setting.\n\n\nMethods\n\nA total of 40 people were involved in the Puerto Rico Service Trip. There were 11 physical therapy students (9 females and 2 males ranging from 23 to 26 years of age), and a Professor from Texas Woman’s University (Dallas Campus). The group partnered with an established organization in Puerto Rico, La Perla de Gran Precio (LPGP), which mainly works with low income, HIV diagnosed participants. There was a total of 19 patients (age average 59.2±1.7 years) and 9 employees who participated in the service activities. All students, employes and participants were included in this study.\n\nThe group visited an established organization in Puerto Rico, LPGP, which works mainly with participants who are low income and have been diagnosed with HIV. LPGP is a community center comprised of an inpatient facility to assist women who are unsafe to live on their own, are undergoing drug rehabilitation, a shelter for children who have parents receiving treatment from the institution, a transitional home (from supervised living to independent), independent living (own/rented apartment), and an outpatient facility. LPGP also participates in regular homeless outreach events throughout the city.\n\nThe group of physical therapy students and their professor traveled to San Juan, Puerto Rico from May 13th to May 20th, 2018. The service trip had academic and cultural elements, which are depicted in the itinerary (see Extended data, Appendix 1 (Rosario, 2019b)).\n\nThe academic components were related to the physical therapy skills students acquired during their first two years of the physical therapy program. These skills were mainly physical therapy evaluation and intervention under the supervision of the professor, a licensed physical therapist. The students provided physical therapy services to the participants in both the outpatient and inpatient facilities, updated exercise plans, and performed exercise testing within a variety of diagnoses.\n\nThe cultural component was related to group discussions on the cultural differences (Hispanic-Latino population in Texas versus Puerto Rico), the impact of HIV in Puerto Rican Hispanic-Latino people, and the participation in a daily reflection (see Extended data, Appendix 2 (Rosario, 2019b)) to evaluate the events of the day and troubleshoot potential barriers. Additionally, the group engaged in homeless outreach events; these outreach events included providing clean attire, showers, haircuts, HIV testing, and educational information to the people around the San Juan Area who were without a permanent residence. Finally, on the last day, the students developed a social lunch activity where they aided the community center’s chef in preparing and serving food for the activity. This venture provided an opportunity for the students, personnel, and participants to share a meal and life experiences.\n\nThree separate surveys were designed for the students, employees, and participants of LPGP. The student survey (see Extended data, Appendix 3 (Rosario, 2019b)) was 30 questions, 23 Likert scale style and 7 free response, intended to gauge the perceived benefits of participating in the service trip, and the employee survey was composed of 12 questions, 8 Likert scale style, and 4 free response. Both the employee (see Extended data, Appendix 4 (Rosario, 2019b)) and participant surveys (see Extended data, Appendix 5 (Rosario, 2019b)) were translated from English to Spanish by a student volunteer, and were checked for accuracy by the Spanish-fluent professor who had previously resided in Puerto Rico. The Community Center’s participants surveys included 8 questions, 5 Likert scale style and 3 free response. All Likert scale questions ranged from strongly disagree (1) to strongly agree (5). Surveys were voluntarily completed on the last day by employees and members, whereas student surveys were conducted during the students’ trip back and were completed prior to returning home the next day. All surveys were completed anonymously, allowing one to report their true perception without concern of repercussion.\n\nThe authors used the ARECCI tool to determine and justify that the service trip is classified under Program Quality Improvement, for which the ARECCI tool is recommended instead of Institutional Review Board approval. Participants provided LDGP Center written informed consent and verbally agreed to be part of the activities of the service trip and the initiative. To protect all parties involved, no photos or identifiers were collected from any participant in this project.\n\nData were gathered in a spreadsheet and Likert scale for averages and the standard deviation was analyzed by Statistical Package for Social Sciences (SPSS) version 24. Overall free response themes were analyzed.±\n\n\nResults\n\nOn a Likert scale ranging from strongly disagree (1 points) to strongly agree (5 points), students responded with strong agreement that their participation in the service activity made them more aware of their impact on others, encouraged them to overcome challenges, assisted in becoming aware of one’s own biases and prejudices, as well as foster a belief that because of this trip, they will be a better new-grad hire. Additionally, students stated that this mission trip availed them on a personal and professional level, and felt as though they made an impact on the community they worked with. Students agreed and gave the lowest score to the pre-trip reflections helped prepare them for the trip question (Likert scale 3.78±0.67). However, results showed that the overall service trip activity was a success, according to the Likert scale data, 4.71± 0.4. Raw responses are available Underlying data (Rosario, 2019a).\n\nAll of the employees strongly agreed to the questions “our group coming to serve your organization was beneficial” and “our group provided appropriate interventions and services for your organization.”\n\nEvery member strongly agreed to the questions “do you feel like you will be able to implement what you learned into your routine?” and “would you like to have a group come back again?”.\n\nThe experience had a positive effect, as demonstrated by the above results, as every person involved found value from participating in this service activity, whether it be as a volunteer or member of the organization. All students rated strongly agree on “made me more aware of my possible impact on others,” “service trips like this one can provide real aid for people in the community,” “service trips like this one can benefit other students,” and, “I felt like I had an impact with the community I worked with on this service trip.” All employees strongly agreed with all statements indicating that they felt the services provided were of great value to their organization. Similar to the employees, the members strongly agreed to nearly every question, with only one participant reporting a 4 out of 5 regarding communication between students and members. Otherwise, the members strongly agreed with all statements indicating significance from interacting with students during their service trip to Puerto Rico.\n\nThrough free response questions, students reported that the most favorable aspect of the trip was being able to connect with patients, experience all aspects of a community-based clinic, LPGP, and spending time in daily reflection. When asked what aspect of the service trip could be improved, students reported a need for an orientation to the population being served and the environment of the community service clinic, LPGP, along with strategies to reduce language barriers. When queried about the learning experience from the trip, students reported “to treat all with kindness and respect, despite their past,” and that “the smallest, most simple things can make a difference,” while remembering “how to incorporate alone time or small breaks in order to not get frustrated and to continue to work to the best of my abilities.”\n\nAll employees reported interest in having another group come back, with one employee stating “the group was very willing and had a great attitude working in the clinic, showed great professionalism and was great working with the patients and the organization.” Members of the clinic reported that they felt the group did an excellent job and that the only change would be to have the group stay for a longer period of time. Not only did both the members and employees feel that the trip was beneficial, the number of members at the outpatient clinic gym each day were found to be higher than a typical week, per report of the employees. This allowed the group to be able to perform several re-evaluations that were in need of an update, which helped bring the participants paperwork up-to-date.\n\n\nDiscussion\n\nPrior research reported the personal and academic benefits (Fisher et al., 2017) of short-term service assignment in students within different professions. For instance, in medical students and residents, service trips have been shown to be constructive in creating insight into ethical and societal issues regarding global health (Abedini et al., 2012) while creating valuable experiences for professional development (Chetta et al., 2018). Reduction of healthcare worker burnout (Campbell et al., 2009), as well as improvement in clinical skills and enhance cultural awareness in medical and pharmacy students (Chuang et al., 2015) were also found. Additionally, trips such as these have proven to be meaningful to occupational therapy students by allowing connections to be made with others in diverse cultural contexts (Humbert et al., 2012). This service trip served as motivation to aid and provide service to a Latino-Hispanic, low-income community center that specializes in helping people living with HIV in Puerto Rico. Since it is often unclear whether the community partners served during these trips experience the same sort of gain, the purpose of this article is to delineate the benefits of this short-term mission trip to Puerto Rico for both those providing the services, as well as those being served.\n\nDuring the planning stages of this service-learning experience, many negative opinions about potential harm short-term service trips can cause were discovered within our group. Concerns included western savior complex, creating a cycle of dependence, and the trip being more focused on the benefits for volunteers as opposed to the people they are serving. Moreover, concerns about language and culture barriers affecting the success of the services provided and the ability to make meaningful connections with those being served were brought up. In doing a literature review, little to no evidence was found to neither support nor negate these claims.\n\nDeliberations commenced to create an abroad mission trip course were the destination could vary depending on the areas requiring assistance. Future clinical skills will be assessed to identify quantifiable methods of measuring professional improvements and learning. Further, this service trip was composed of only physical therapy students and faculty, considerations will be made for interdisciplinary collaborations focusing on the necessity of the participants needs.\n\n\nConclusion\n\nSince a small student sample expressed interests in this initiative and were able to fund their travel to Puerto Rico, our findings and data cannot be generalized. However, as per the results of this study, along with the reports from the participants, employees, and participants, it can be supported that short-term service learning experiences should be included in graduate academic programs in order to produce well rounded, culturally aware students. As indicated above, every person involved in this event found great value in at least one aspect, indicating that similar events can be used to foster growth in students and advance global health initiatives. Further research should be conducted to assess the value of similar trips in different regions to explore the benefits of short-term physical therapy services provided to different populations.\n\n\nData availability\n\nFigshare: Puerto Rico Service Trip Database.xlsx. https://doi.org/10.6084/m9.figshare.7999370.v1 (Rosario, 2019a).\n\nThis file contains the answers from each respondent, in addition to pooled responses. Please note that some of the free response answers from the participants and employees are in Spanish.\n\nUnderlying data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: Appendices 1–5. https://doi.org/10.6084/m9.figshare.8021240.v1 (Rosario, 2019b).\n\nThis file contains the following extended data:\n\n\n\n• Appendix I: Itinerary of cultural and academic activities per day.\n\n• Appendix II: Daily Reflexion Example, day 1 and 2.\n\n• Appendix III: Student Survey and Student Survey Results.\n\n• Appendix IV: Employee Survey and Employee Survey Results.\n\n• Appendix V: Participant Survey and participant Survey results.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThank you to La Perla de Gran Precio, San Juan Puerto Rico for providing access to the facilities.\n\n\nReferences\n\nAbedini NC, Gruppen LD, Kolars JC, et al.: Understanding the effects of short-term international service-learning trips on medical students. Acad Med. 2012; 87(6): 820–828. PubMed Abstract | Publisher Full Text\n\nBrown DA, Fairclough JL, Ferrill MJ: Planning a pharmacy-led medical mission trip, part 4: an exploratory study of student experiences. Ann Pharmacother. 2012; 46(9): 1250–1255. PubMed Abstract | Publisher Full Text\n\nCampbell C, Campbell D, Krier D, et al.: Reduction in burnout may be a benefit for short-term medical mission volunteers. Mental Health, Religion & Culture. 2009; 12(7): 627–637. Publisher Full Text\n\nCampbell A, Sullivan M, Sherman R, et al.: The medical mission and modern cultural competency training. J Am Coll Surg. 2011; 212(1): 124–129. PubMed Abstract | Publisher Full Text\n\nChetta MD, Shakir A, Paek LS, et al.: Evaluating Resident Perspectives on International Humanitarian Missions. J Craniofac Surg. 2018; 29(2): 279–285. PubMed Abstract\n\nChiu YW, Weng YH, Chen CF, et al.: A comparative study of Taiwan's short-term medical missions to the South Pacific and Central America. BMC Int Health Hum Rights. 2012; 12: 37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChuang C, Khatri SH, Gill MS, et al.: Medical and pharmacy student concerns about participating on international service-learning trips. BMC Med Educ. 2015; 15(1): 232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFisher EE, Sharp RL, Bradley MJ: Perceived Benefits of Service Learning: A Comparison of Collegiate Recreation Concentrations. Journal of Experiential Education. 2017; 40(2): 187–201. Publisher Full Text\n\nHumbert TK, Burket A, Deveney R, et al.: Occupational therapy students' perspectives regarding international cross-cultural experiences. Aust Occup Ther J. 2012; 59(3): 225–234. PubMed Abstract | Publisher Full Text\n\nRosario M: Puerto Rico Service Trip Database.xlsx. figshare. Dataset. 2019a. http://www.doi.org/10.6084/m9.figshare.7999370.v1\n\nRosario M: Appendices 1-5. figshare. Journal contribution. 2019b. http://www.doi.org/10.6084/m9.figshare.8021240.v1\n\nSykes KJ: Short-term medical service trips: a systematic review of the evidence. Am J Public Health. 2014; 104(7): e38–48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWithers M, Browner CH, Aghaloo T: Promoting volunteerism in global health: lessons from a medical mission in northern Mexico. J Community Health. 2013; 38(2): 374–384. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "86336",
"date": "21 Jun 2021",
"name": "Romy Parker",
"expertise": [
"Reviewer Expertise Chronic pain management",
"physiotherapy",
"pain in HIV/AIDS"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 undertaking this ambitious project and reporting on it to share the lessons learnt. This paper clearly summarizes the work undertaken, a service project trip by a group of physical therapy students and faculty to Puerto Rico where the group partnered with a local organization who provide care for people living with HIV. This paper is useful as it informs the reader about the justification for the project, the steps followed to facilitate engagement by both students and partners, and the positive results. I believe some engagement with several issues would strengthen the work and are of benefit to raise for others contemplating similar work. My first comment relates to some of the language and terminology used in the piece. It is particularly important to ensure that the terms we use are universally understood. I was not clear about the definition or the meaning of several terms and wonder whether other terms could have been used (e.g. “the necessity of multimodal and multifocal assistants”, I was not sure if this referred to treatment techniques or to individual people or to systems), and the term “service trip/project” is not one I am familiar with. Providing a definition would be helpful, particularly for someone who works in a different academic environment. Finally, in the methods there is reference to “women who are unsafe” being provided with accommodation, it is not clear whether this infers that women may be a risk to themselves and others, or refers to women who are at risk in their communities.\nI feel that your introduction would have been strengthened with an additional paragraph focusing on the role of physical therapy in optimizing quality of life in people living with HIV. Your first paragraph clearly states the health burden of HIV in Puerto Rico, but the reader unfamiliar with HIV management may not have an understanding of the role of physical therapy in contributing to improved quality of life in this population. This would also strengthen your argument as to why the service project was particularly relevant for this group of students and the potential benefits for the community.\nIn the methods you state that you “partnered” with a local organization. Creating true partnerships is challenging and it would be useful to expand on what you mean by “partnered”. How was this relationship initiated? To ensure true partnership, power imbalances need to be considered and actively prevented or managed, what steps were taken to ensure this occurred? In addition to expanding on partnerships in the methods, engaging with this further in the discussion based on the survey responses would also be valuable. My questions about how this partnership was formed and managed also arose when reading the results, where employees felt that the project was of value to their organization. Did you come in and offer services or did the employees/organization identify areas they would like input and support in. Were the services and activities negotiated and mutually agreed?\nIn the methods there is also a description of the partner organization – LPGP. As I read this description, I realized that I am not familiar with Puerto Rico, its social and demographic characteristics. Here it would also be helpful to describe the community broadly and the community of people living with HIV in Puerto Rico. Different communities have different modes of HIV transmission (e.g. in South Africa the primary mode of transmission is through heterosexual relationships with risk factors including having low levels of income and being a woman); reading the description of the services the LPGP provides gives me some insight into who the people they serve are, and what the biopsychosocial factors which increase their vulnerability might be. But it would be helpful to have a rich description of the PLWHA in this community. After all, physical therapy treatment follows the ICF model and considers the person in their environment and this information provides valuable context. Interestingly, this sense of the reader needing more contextual information, is reflected in the results with the students requesting more orientation to the population, the environment, and the clinic.\nThe surveys used with all participants are useful and provide some concise information while also allowing for greater engagement with the experiences. It is particularly rewarding to read the students’ positive responses to reflections. It is not clear to me why you chose to report the Likert scale responses as means with standard deviation. Likert scale responses should be treated as non-parametric data and it would be more appropriate to report them as median and IQR or even in frequency tables to give a full sense of the responses, particularly in such a small group. As no comparisons between groups were conducted, a fuller descriptive reporting of the results is valuable.\nIn the discussion the term “short-term mission trip” is introduced. This variance is terminology is confusing. A “mission trip” and a ‘service trip” have fundamental differences of motivation, purpose and outcome measures. As mentioned earlier, clearly defining the terms would help the reader evaluate the entire project.\nIn the discussion there is an excellent reflection on potential negative effects of a service trip, identifying concerns I had also felt in reading this work. I hope that some of my comments relating to developing true partnerships provide some tools on how to prevent these potential negative consequences in future work. I would like to congratulate the authors for undertaking this ambitious project and wish them all of the best in training the next generation of physical therapists with a broader understanding of their role in community-based 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "97350",
"date": "17 Nov 2021",
"name": "Paulo Moreira Silva Dantas",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe purpose of this study was to discuss the perceived benefits of a short week-long service trip for students, participants and staff.\nMethods: A total of 11 physical therapy students and a teacher traveled to Puerto Rico for a week-long service trip. The group has partnered with an established organization called \"La Perla de Gran Precio,\" which works with low-income US Hispanic-Latino citizens who have been diagnosed with HIV. Students were involved in academic and cultural components, providing physiotherapy services to participants. At the end of the week, surveys were carried out with all parties involved.\nResults: Students, staff and participants reported the service trip as extremely positive. Students suggested that their integration should be considered in any physical therapy curriculum to further improve the future of this profession. Participants reported that they learned from this experience and were able to implement the methods in their routine.\nConclusions: The Puerto Rico service trip improved physiotherapy students' education and their ability to increase cultural awareness, boost communication skills, provide opportunities to overcome challenges, and foster a sense of purpose.\nThe work even initially presents clearly and with current literature information about HIV, Puerto Rico, service projects, etc, but the study design was not possible to be identified, making it impossible to verify technical appropriation. The manuscript in its current form resembles an experience report rather than a Research Article. The authors would need to offer greater detail on the methods and analyzes to allow replication by other scholars to meet these standards.\nThe sample size presented in the abstract does not correspond to that presented in the methodology text; it is not possible to identify the characteristics of the participants, it was not possible to understand the academic and cultural elements that were made possible from this short-term service trip to a community center for HIV, etc., implying in other parts (results, discussion and conclusion).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-647
|
https://f1000research.com/articles/7-1890/v1
|
04 Dec 18
|
{
"type": "Research Article",
"title": "Developing an Indonesian fertility preservation questionnaire for health care providers treating patients with cancer: A preliminary pilot study",
"authors": [
"Achmad Kemal Harzif",
"Raymond Surya",
"Mila Maidarti",
"Ana Mariana",
"Bara Tracy Lovita",
"Budi Wiweko",
"Raymond Surya",
"Mila Maidarti",
"Ana Mariana",
"Bara Tracy Lovita",
"Budi Wiweko"
],
"abstract": "Background: Early detection and advanced treatment increases the five-year survival rate of patients with cancer. However, long-term cancer therapy, such as chemotherapy and radiotherapy, can have negative effects, such as infertility. This study aimed to develop a standardized Indonesian questionnaire, which would be used to assess the quality of health care providers’ knowledge, attitude, and practice regarding fertility preservation in patients with cancer. Methods: A pilot study was performed in January and February 2018 at Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia. An existing questionnaire was translated from English to Indonesian using forward translation, back translation, expert panel, pretesting, and cognitive interviewing. Ten subspecialists in the following departments made up an expert panel, who were involved in pretesting and cognitive interviewing: pediatric hematology-oncology, hematology-oncology/internal medicine, gynecologic oncology, gynecologic immune-endocrinology, radiology-oncology, and surgical oncology. Results: The questionnaire was successfully translated. The ten respondents stated that the maximum age for women’s fertility preservation is 40 years of age (60%), 45 years of age (30%), or had no maximum age (10%). Additionally, the respondents stated that the maximum age for men’s fertility preservation is 40 years of age (30%), 50 years of age (20%), or had no maximum age (50%). The respondents’ knowledge stated that > 50% of them were aware but do not know enough about fertility preservation. The respondents stated that more than 50% of them give feedback agreeing to fertility preservation, and they always give advice about fertility preservation to their patients. Conclusion: The translation of the questionnaire followed translation steps from the World Health Organization and was adjusted based on the expert panel’s comments concerning fertility preservation. This validated questionnaire tool in Indonesian can be used for research purposes and clinical evaluation of fertility preservation among health care providers in Indonesia.",
"keywords": [
"Knowledge",
"Attitude",
"Practice",
"Indonesian Questionnaire"
],
"content": "Introduction\n\nBased on data from the US Cancer Statistics Working Group, the five most common cancers in 2014 were breast, prostate, lung and respiratory tract, colon and rectum, and uterine and ovary1. The Riset Kesehatan Dasar 2013 showed that the prevalence of cancer in Indonesia was 0.14% (347,782 people), with cervical (0.08%; 98,692 people) and breast (0.05%; 61,682 people) cancers ranked highest2. In Indonesia, more than 135,000 people below 45 years of age are diagnosed with cancer annually2.\n\nEarly detection and advanced treatments increase the five-year survival rate of patients with cancer. However, long-term cancer therapy, such as chemotherapy and radiotherapy, can have negative psychologic, economic, social, sexual, and biologic effects3,4. In 2014, the Guidelines from National Comprehensive Cancer Network stated that fertility preservation is an essential component when treating young and adolescent patients with cancer5,6. In fact, few patients are offered treatment choices based on fertility preservation due to lack of knowledge on optimal time, methods, and counselling approaches7. The American Academy of Pediatrics, American Society of Clinical Oncology, and American Society of Reproductive Medicine suggest discussing potential complications and choice of fertility preservation as early as possible8,9.\n\nBecause there are no fertility preservation questionnaires available in Indonesia, this study aimed to develop a standardized Indonesian questionnaire that can be used to assess quality of health care providers’ knowledge, attitude, and practice regarding fertility preservation in patients with cancer.\n\n\nMethods\n\nThis study was approved by The Ethics Committee of Faculty of Medicine, University of Indonesia under number 926/UN2.F1/ETIK/2017. Written informed consent was obtained from all participants prior to participation.\n\nThis study was performed in January and February 2018 at Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia. An existing questionnaire, “Fertility preservation in cancer survivors: A national survey of oncologists’ current knowledge, practice, and attitudes,” published in English in 201310 was translated to Indonesian by two independently certified medical translators whose first language is Indonesian, with permission from the publisher.\n\nTo check the accuracy of the translation, the Indonesian questionnaire was back translated to English by another medical translator (RS). Misunderstandings or unclear word choices in the initial translations were resolved by an author (RS) as appropriate to the aim of this study. After the translation had been completed, an expert (AKH) familiar with the construct of interest and methodology reviewed all versions of translations and determined whether the translation had achieved semantic, idiomatic, experiential, and conceptual equivalence.\n\nThe final translation of the Indonesian pilot questionnaire (Supplementary File 1) was given to specialists and subspecialists who completed the questionnaire and were interviewed verbally to ensure clarity of answers11. These interviews took place in Dr. Cipto Mangunkusumo Hospital. Specialist and subspecialists were chosen randomly from all staff at Dr. Cipto Mangunkusumo Hospital who fit the inclusion criteria (see below). Researchers contacted these experts directly, providing an explanation of the study and gaining informed consent from the expert to get involved in this study. Interviews were not recorded.\n\nTen subspecialists or specialists in the following departments who directly treat patients with cancer were recruited to take part in the study: pediatric hematology-oncology, hematology-oncology/internal medicine, gynecologic oncology, gynecologic immune-endocrinology, radiology-oncology, and surgical oncology. The subspecialist or specialist in Dr. Cipto Mangunkusumo Hospital were randomly chosen and recruited to this study based on their experience for at least 5 years. They were considered as an expert panel. Inclusion criteria for this study were (a) subspecialists or specialists aged 30–45 years; and (b) subspecialists or specialists who have studied in their field for at least 5 years; and exclusion criteria for this study were (a) respondents who not willing to be a participant in this study; and (b) incomplete filling of the informed consent.\n\nDue to the qualitative nature of this study, only face validity and construct validity were assessed. Face validity was used to determine whether the instrument was understandable and relevant to the targeted population. Construct validity was used to determine the reason and consequence describing the real condition. Content validity was not assessed because the purpose of this questionnaire was not to determine good/bad knowledge and positive/negative attitude about practice.\n\nThe data gathered from completed questionnaires were distributed by frequency and percentage. The analysis used SPSS Statistics for Windows, version 23.0.\n\n\nResults\n\nIn this preliminary pilot study, ten respondents from Dr Cipto Mangunkusumo Hospital (8 males) participated in this study from the following specialties: pediatric hematology-oncology (n=2), hematology-oncology/internal medicine (n=1), gynecologic oncology (n=2), gynecologic immune-endocrinology (n=2), radiology-oncology (n=1), and surgical oncology (n=2).\n\nIn total, 60% (6/10), 30% (3/10), and 10% (1/10) of respondents stated that the maximum age for women’s fertility preservation is 40 years of age, 45 years of age, or had no maximum age, respectively. A total of 30% (3/10), 20% (2/10), and 50% (5/10) of respondents stated the maximum age for men’s fertility preservation is 40 years of age, 50 years of age, or had no maximum age, respectively.\n\nTable 1 describes the frequency that health care providers encounter patients who have used/are using fertility preservation options. Table 2 describes the familiarity of health care providers about methods of fertility preservation.\n\nTable 3 shows the health care providers’ practice of giving advice about fertility preservation, and Table 4 shows their attitudes towards fertility preservation. Table 5 shows the factors influencing health care providers when initiating a discussion about fertility preservation.\n\nAll comments have been translated from Indonesian.\n\nThe following are comments made during the verbal interview about the format and definitions used in the translated questionnaire:\n\n“The questionnaire should contain [an] explanation of each fertility preservation.” (hematology-oncologic internal medicine.)\n\n“The identity [of a patient requiring fertility preservation] is only initial. [The] number of cases on Q2 should be made [into categories] and focused to the last year. [The] format [of the] questionnaire should be adjusted to [be easier for] the readers.” (hematology-oncologic pediatrician.)\n\n“The questionnaire is too difficult. It should contain the explanation of what the definition [is for] each fertility preservation.” (hematology-oncologic pediatrician.)\n\n“The length of clinical practice in the oncology field should be included to the questionnaire.” (oncology gynecologic.)\n\n“On identity column, it should add the last major educational background and radio-oncology should be included. Number of cases taking care by health care providers should be shown [by] percentage.” (radio-oncology.)\n\nThe following are comments made during the verbal interview about the use of cultural background used in the translated questionnaire:\n\n“Cultural background around social, racial, and religion should be omitted.” (hematology-oncologic internal medicine.)\n\n“On cultural background [questions], [due to the] culture [in] Indonesia [concerning] gay or lesbian, or towards social [status], race, and religion should be omitted to minimalize the possibility of conflict.” (immune endocrinology gynecologic.)\n\n\nDiscussion\n\nThis study includes the pilot results of the measures of success of a translation of an existing questionnaire to Indonesian to determine knowledge, attitude, and practice of providers regarding fertility preservation in patients with cancer. The results of this study are not applicable to all health care providers because the questionnaire is very specific for clinicians treating patients with cancer.\n\nThe World Health Organization proposes various steps to achieve different language versions of English instruments that are equivalent in each target country/culture: forward translation, expert panel, back translation, pretesting and cognitive interviewing, leading to a final version12. In this study, the expert panel, made up of ten subspecialists and specialists, provided feedback after the back translation via pretesting and cognitive interviewing. After this pilot study, we would like to distribute this questionnaire to health care providers treating patients with cancer.\n\nBased on respondents’ feedback, we conclude that, in Indonesia, fertility preservation still is not common and familiar among practitioners taking care of patients with cancer. In total, 50% of respondents were aware of but not experts in fertility preservation. Additionally, 50% of respondents never had patients who had used or were using fertility preservation; however, respondents were subspecialist oncologists or clinicians directly taking care of patients with cancer. This may be because there is still no availability of fertility preservation in Indonesia. In Hong Kong, 45.6% of clinicians were familiar with fertility preservation13.\n\nOur study also shows that most respondents had discussed the impact of treatment to future fertility with patients. In total, 30% of respondents had referred patients to a fertility specialist. A similar study in Lebanon found that 90% of clinical practitioners and 94% of oncologists agreed to discuss fertility preservation with patients before cancer treatment14. Clinicians in Hong Kong did not refer patients to fertility specialists due to lack of available time before treatment, considerable risk of recurrence, poor prognosis, financial constraints, cancer treatment as top priority at the time, and lack of awareness of such service13.\n\n\nLimitations\n\nAs shown by the quotes in the Results section, limitations of the questionnaire were that cultural background factors influenced health care providers’ decisions to initiate discussions about fertility preservation. Some respondents commented that this questionnaire included questions that are contrary to Indonesian culture; thus, they recommended that we omit these questions from the Indonesian questionnaire to limit the conflict of interest. However, according to the original English questionnaire, respondents strongly endorsed questions about culture. For example, British respondents stated that their decision to discuss fertility preservation was influenced by poor prognosis (88%) and whether the patient already had children (45%)10.\n\n\nConclusion\n\nBased on data obtained in this preliminary pilot study, we translated the English questionnaire to Indonesian and revised it following processes adopted from World Health Organization and adjusted through expert respondents’ comment. Supplementary File 1 contains the Indonesian version of the questionnaire about the quality of health care providers’ knowledge, attitude, and practice regarding fertility preservation in patients with cancer.\n\nBy having this validated tool questionnaire in Indonesian, it can be used for both research purposes and clinical evaluation of fertility preservation among health care providers in Indonesia.\n\n\nData availability\n\nF1000Research: Dataset 1. All raw data included the medical background of respondents, the health care providers’ knowledge, attitude, and practice regarding fertility preservation and also factors that health care providers consider when deciding to initiate a discussion about fertility preservation, https://doi.org/10.5256/f1000research.15948.d22787815",
"appendix": "Grant information\n\nThis work is supported by Hibah PITTA 2018 funded by DRPM Universitas Indonesia (No.5000/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\nAcknowledgments\n\nThe authors wish to thank everyone who supported this study and also to DRPM Universitas Indonesia by Hibah Pitta 2018. We also would like to thank to Monica Helton through AuthorAid for helping us improve our manuscript by copyediting and giving us thoughtful comments.\n\n\nSupplementary material\n\nSupplementary File 1: Questionnaire and informed consent form in Bahasa Indonesia.\n\nClick here to access the data\n\n\nReferences\n\nUnited States Cancer Statistics [Internet]. [cited 2017 Jul 25]. Reference Source\n\nData P Informasi: Situasi penyakit kanker. Jakarta: Departemen Kesehatan. [Internet]. [cited 2017 Aug 18]. 2014. Reference Source\n\nGoldhirsch A, Ingle JN, Gelber RD, et al.: Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009. Ann Oncol. 2009; 20(8): 1319–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCancer_fertility_effects_Jan08.pdf [Internet]. [cited 2017 Jul 25]. Reference Source\n\nCoccia PF, Altman J, Bhatia S, et al.: Adolescent and young adult oncology. Clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2012; 10(9): 1112–50. PubMed Abstract | Publisher Full Text\n\nLevine J, Canada A, Stern CJ: Fertility preservation in adolescents and young adults with cancer. J Clin Oncol. 2010; 28(32): 4831–41. PubMed Abstract | Publisher Full Text\n\nTaylor JF, Ott MA: Fertility Preservation after a Cancer Diagnosis: A Systematic Review of Adolescents’, Parents’, and Providers’ Perspectives, Experiences, and Preferences. J Pediatr Adolesc Gynecol. 2016; 29(6): 585–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoren AW, Mangu PB, Beck LN, et al.: Fertility preservation for patients with cancer: American Society of Clinical Oncology clinical practice guideline update. J Clin Oncol. 2013; 31(19): 2500–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFallat ME, Hutter J, , American Academy of Pediatrics Committee on Bioethics, et al.: Preservation of fertility in pediatric and adolescent patients with cancer. Pediatrics. 2008; 121(5): e1461–9. PubMed Abstract | Publisher Full Text\n\nAdams E, Hill E, Watson E: Fertility preservation in cancer survivors: a national survey of oncologists’ current knowledge, practice and attitudes. Br J Cancer. 2013; 108(8): 1602–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsang S, Royse CF, Terkawi AS: Guidelines for developing, translating, and validating a questionnaire in perioperative and pain medicine. Saudi J Anaesth. 2017; 11(Suppl 1): S80–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrganization WH: Process of translation and adaptation of instruments. 2009. Reference Source\n\nChung JP, Lao TT, Li TC: Evaluation of the awareness of, attitude to, and knowledge about fertility preservation in cancer patients among clinical practitioners in Hong Kong. Hong Kong Med J. 2017; 23(6): 556–61. PubMed Abstract | Publisher Full Text\n\nGhazeeri G, Zebian D, Nassar AH, et al.: Knowledge, attitudes and awareness regarding fertility preservation among oncologists and clinical practitioners in Lebanon. Hum Fertil (Camb). 2016; 19(2): 127–33. PubMed Abstract | Publisher Full Text\n\nHarzif AK, Surya R, Maidarti M, et al.: Dataset 1 in: Developing an Indonesian fertility preservation questionnaire for health care providers treating patients with cancer: A preliminary pilot study. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.15948.d227878"
}
|
[
{
"id": "42438",
"date": "22 Jan 2019",
"name": "Yutaka Osuga",
"expertise": [
"Reviewer Expertise Reproduction"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors aimed to develop a standardized Indonesian questionnaire from already published English questionnaire that was successfully used to survey oncologists’ current knowledge, practice and attitudes in a Western country. The authors translated the questionnaire, and the content was validated through the process of back translation. In addition, the authors used the translated questionnaire to interview ten sub-specialists in Indonesia. The questionnaire worked well for that purpose.\nIn summary, the translated questionnaire will be useful for future studies in the field of oncofertility in Indonesia and may be used for comparing the quality of health care providers’ knowledge, attitude, and practice regarding fertility preservation in patients with cancer in different countries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "45599",
"date": "11 Apr 2019",
"name": "Jane A. Stewart",
"expertise": [
"Reviewer Expertise Relevant area of research - Fertility preservation in children and young adults - attitudes and practice. I am a consultant in reproductive medicine in UK National Health Service."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes a pilot study of a questionnaire to assess the practice of health care providers' (namely oncologists') understanding and attitudes to fertility preservation in a local setting to consider its utility nationally. The authors describe the process of translation and verification of sense of the questionnaire and then its use in the clinical setting with a small group of oncologists. The process proved effective.\nThe initial findings are of interest and the authors have raised a number of relevant points which confirm that rolling out nationally may indeed be informative.\nThe authors see the trial as successful. They comment on the discussion around cultural questions. They have not discussed the comments around the \"difficulty\" of the questionnaire which more than one respondent made. Since the paper was designed to explore the utility of the questionnaire rather than the specific answers given by the oncologists it would be useful to see more discussion around those points and in particular whether or not they felt that any modification was needed before wider use in Indonesia. If so what and if not, given those comments, why not?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4623",
"date": "10 May 2019",
"name": "Achmad Kemal",
"role": "Author Response",
"response": "Thank you for the review.We uploaded revision on our manuscript. We hope to hear your review."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1890
|
https://f1000research.com/articles/7-76/v1
|
17 Jan 18
|
{
"type": "Research Note",
"title": "Triplex target sites of MEG3 RNA-chromatin interactions",
"authors": [
"Ivan Antonov",
"Yulia A. Medvedeva",
"Ivan Antonov"
],
"abstract": "Many long noncoding RNAs are bound to chromatin. MEG3 binds to multiple different genomic locations, containing GA-rich motifs, and form RNA-DNA triplex structures. In this work, we test whether the MEG3 binding sites are specific enough to be regulated by a particular lncRNA. We show that at least in the case of MEG3, a subset of the triplex target sites (TTS) is able to hybridize with various different RNAs almost irrespectively of their sequences. Nowadays, the role of chromatin bound RNAs in the formation of 3D chromatin structure is actively discussed. We speculate that such universal TTSs may contribute to establishing long-distance chromosomal contacts.",
"keywords": [
"MEG3 lncRNA",
"triple helix",
"triplex target sites (TTS)"
],
"content": "Introduction\n\nMany human long noncoding RNAs are localized in the nucleus and potentially can participate in chromatin formation and remodeling1. Recently, technologies such as ChIRP2, ChRIP3, ChOP4, CHART5, RAP6, MARGI7 and GRID8 have been developed to map the genomic interacting sites of various lncRNAs. Although these techniques determine location of RNA binding sites, they are unable to clarify the interaction mechanisms. LncRNAs are capable of binding chromatin proteins, nascent RNA, single-stranded or double-stranded DNA, forming R-loops or triple helixes, respectively.\n\nMaternally expressed gene 3 (MEG3) is one of the lncRNAs known to target chromatin. Genome-wide mapping of MEG3 with the chromatin oligo affinity precipitation followed by deep sequencing (ChOP-seq) method reveals that MEG3 binding sites are widespread, contain GA-rich motifs, and form RNA-DNA triplex structure4. Growing body of evidence shows that RNA-DNA triplex formation plays important role in RNA-chromatin interactions. Moreover, it has been shown earlier that triplex target sites (TTS) are frequently located near regulatory regions (including gene promoters) in the human genome9. In this work we investigate whether the DNA sites capable of triplex formation are specific enough to be regulated by a particular lncRNA.\n\n\nMethods\n\nAfter mapping 6837 ChOP-seq MEG3 peaks from hg19 to the hg38 using liftOver (the tool was downloaded from the UCSC Genome Browser on Nov 7, 2017 and ran as follows: liftOver MEG3.hg19_peaks.bed, hg19ToHg38.over.chain.gz MEG3.hg38_peaks.bed, unMapped.txt), 6694 peaks shorter than 1000 bp were used. Next, we selected 3kb regions (bins) centered at the peak middle positions using bedtools (version 2.25.0). The 3620 bins with overlapping genes (according to the GENCODE ver. 27 annotation10) were selected as true positives. Additionally, 3620 genomic regions of the same length and GC-content overlapping the GENCODE genes were randomly selected from the human genome as true negatives (the control bins). The validation set consisted of two subsets of bins without MEG3 peaks. Namely, another group of 3620 control bins were sampled and combined with the 3620 true negative regions from the test set.\n\nWe predicted triple helixes using Triplexator11 (version 1.3.2) with the settings recommended at the official website: -l 15 -e 20 -c 2 -fr off) with the following RNA queries:\n\n- MEG3 (NR_002766: length = 1595 nt, GC-content = 57.55%),\n\n- BE2L6 (NM_198183: 1620 nt, 57.59%),\n\n- LILRA3 (NM_006865: 1608 nt, 57.71%),\n\n- HMOX1 (NM_002133: 1590 nt, 57.80%).\n\nThe transcript sequences similar to the MEG3 in length and GC content were found using the RANN (version 2.5.1) R package12. UBE2L6, LILRA3 and HMOX1 were used to verify the sequence specificity of the MEG3-DNA hybridization. Additionally, the three random query sequences were obtained by mono-nucleotide shuffling the original MEG3 transcript using a custom Perl script. The Triplexator score for each genomic region was calculated as the sum of the scores of all the predictions between RNA and the genomic region.\n\n\nResults\n\nThe Triplexator tool11 was used to predict the RNA-DNA interactions between the MEG3 transcript and the 7240 genomic regions (bins) from the test set according to the Hoogsteen and reverse Hoogsteen base pairing rules. As anticipated, the triplex scores predicted for the MEG3 peak-containing bins were stronger than for the control bins – the median Triplexator SumScores were 48 and 25, respectively (p-value = 5.2e-100, see Figure 1a).\n\nStrikingly, in all cases the SumScores produced by Triplexator for 3 other human RNAs (UBE2L6, LILRA3 and HMOX1) and the MEG3 peak-containing bins were also stronger than the scores for the control bins. Moreover, the statistical significances of the observed SumScore differences for two out of the three mRNAs were higher than for the MEG3 predictions (p-values = 0, 3.0e-24 and 1.4e-174, respectively – see Figure 1b). To find out whether it is a general property of the human transcripts or the identified MEG3-TTS have a tendency to form triplexes with any RNA in a nonspecific manner, the three random sequences were generated by the mono-nucleotide shuffling of the MEG3 transcript. Once again, the statistical significant differences between the two sets of bins were observed in all three cases (p-values = 1.0e-143, 5.8e-41 and 1.3e-33 – see Figure 1c).\n\n(a–c) The distributions of the Triplexator SumScores for the 3620 control regions without peaks and 3620 genomic regions with MEG3 peaks identified in the ChOP-seq experiment. (d–f) The distributions of the Triplexator SumScores for two sets of genomic regions without MEG3 peaks. The query transcript used in Triplexator run is indicated below each image.\n\nTo rule out a possibility of overprediction, we applied our computational approach to a ’validation set’ consisting of the MEG3 peak-free bins only (see Methods). On the contrary to the test set, no significant difference between the two groups of control bins was found for any of the seven RNA sequences (all p-values > 0.05, Figures 1d–f).\n\n\nDiscussion\n\nOur results suggest that at least in some cases, the formation of the RNA-DNA triplexes may be governed by the DNA sequence alone. If it is so, such ’universal’ TTSs are able to hybridize with various different RNAs almost irrespectively of their sequences (however the length and nucleotide content are probably important). Indeed, 18 peak-containing bins were present in the top 5% of the predictions for all seven tested RNAs. In contrast, there was only one such bin among the control regions. Notably, some of the 18 identified universal bins were extremely GA-rich (for example, hg38:chr5:93580373-93583373). The presence of the universal TTSs among the MEG3 peaks may explain the phenomena observed in our computational analysis.\n\nTherefore, some parts of the human genome can hybridize with a number of different RNAs (or different regions of the same long RNA). It should be noted that one of the possible and actively discussed roles of the chromatin bound RNAs (including lncRNAs) is to bring different chromosomal parts together to enable the remote DNA-DNA interactions8. In the light of this biological role, the universal TTS can be viewed as the anchor point which can be bound by various nuclear RNAs to provide longdistance chromosomal contacts. Analysis of additional datasets is needed to further support this hypothesis.\n\n\nData availability\n\nDataset 1: Coordinates of the original hg19-based and the converted hg38-based MEG3 peaks. DOI, 10.5256/f1000research.13522.d18975013\n\nDataset 2: Sequences of the seven queries as well as all the bins from the test and the validation sets. DOI, 10.5256/f1000research.13522.d18975114\n\nDataset 3: SumScores computed by running the Triplexator for each of the queries against the test or the validation set. DOI, 10.5256/f1000research.13522.d18975215\n\nDataset 4: [universal_TTS_bins.fna.gz]. Sequences of the bins containing putative ’universal’ TTSs. DOI, 10.5256/f1000research.13522.d18975316",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Russian Science Foundation [grant 14-15-30002].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are thankful to Dr. Chandrasekhar Kanduri (University of Gothenburg, Sweden) for providing the original coordinates of the ChOP-seq peaks for the lncRNA MEG3.\n\n\nReferences\n\nKhalil AM, Guttman M, Huarte M, et al.: Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A. 2009; 106(28): 11667–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu C, Qu K, Zhong FL, et al.: Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. Mol Cell. 2011; 44(4): 667–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPandey RR, Mondal T, Mohammad F, et al.: Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell. 2008; 32(2): 232–246. PubMed Abstract | Publisher Full Text\n\nMondal T, Subhash S, Vaid R, et al.: MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures. Nat Commun. 2015; 6: 7743. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimon MD, Wang CI, Kharchenko PV, et al.: The genomic binding sites of a noncoding RNA. Proc Natl Acad Sci U S A. 2011; 108(51): 20497–502. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngreitz JM, Pandya-Jones A, McDonel P, et al.: The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science. 2013; 341(6147): 1237973. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSridhar B, Rivas-Astroza M, Nguyen TC, et al.: Systematic Mapping of RNA-Chromatin Interactions In Vivo. Curr Biol. 2017; 27(4): 602–609. PubMed Abstract | Publisher Full Text\n\nLi X, Zhou B, Chen L, et al.: GRID-seq reveals the global RNA-chromatin interactome. Nat Biotechnol. 2017; 35(10): 940–950. PubMed Abstract | Publisher Full Text\n\nGoñi JR, De La Cruz X, Orozco M: Triplex-forming oligonucleotide target sequences in the human genome. Nucleic Acids Res. 2004; 32(1): 354–360. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarrow J, Frankish A, Gonzalez JM, et al.: GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012; 22(9): 1760–1774. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuske FA, Bauer DC, Mattick JS, et al.: Triplexator: detecting nucleic acid triple helices in genomic and transcriptomic data. Genome Res. 2012; 22(7): 1372–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArya S, Mount D, Kemp SE, et al.: Package ‘RANN’. 2017. Reference Source\n\nAntonov I, Medvedeva Y: Dataset 1 in: Triplex target sites of MEG3 RNA-chromatin interactions. F1000Research. 2018. Data Source\n\nAntonov I, Medvedeva Y: Dataset 2 in: Triplex target sites of MEG3 RNA-chromatin interactions. F1000Research. 2018. Data Source\n\nAntonov I, Medvedeva Y: Dataset 3 in: Triplex target sites of MEG3 RNA-chromatin interactions. F1000Research. 2018. Data Source\n\nAntonov I, Medvedeva Y: Dataset 4 in: Triplex target sites of MEG3 RNA-chromatin interactions. F1000Research. 2018. Data Source"
}
|
[
{
"id": "29954",
"date": "23 Jan 2018",
"name": "Ingrid Grummt",
"expertise": [
"Reviewer Expertise Regulation of gene expression by noncoding RNA"
],
"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\nLong noncoding RNAs (lncRNA) can regulate gene expression by targeting specific DNA sequences via Hoogsteen base paring, forming RNA-DNA triple helical structures. Computational analyses have revealed that a large population of triplex-forming motifs is present across the genome, the majority of annotated human genes containing at least one unique and high-affinity triplex target site, preferentially in regulatory gene regions (Goni et al. 2004, Buske et al. 2012). Moreover, several studies have provided in vitro and in vivo evidence for the existence and biological relevance of RNA-DNA triplexes, including pRNA (Schmitz et al. 2010), Fendrr (Grote et al. 2013), Khps1 (Postepska-Igielska et al. 2015), PARTICLE (O’Leary et al. 2015), and MEG3 (Mondal et al. 2015). MEG3 has been shown to associate with AG-rich DNA motifs and facilitate recruitment of PRC2 to target sites. Considering the large number of purine-rich sequences in the genome, triplex-mediated targeting of lncRNAs and associated proteins to distinct genomic loci is very likely a commonly used mechanism of gene regulation.\nGiven the importance and emerging acceptance of the concept of triplex-dependent gene regulation, it is more than surprising, if not irritating, that the authors challenge this concept feeding the ‘Triplexator’ only with a few RNAs and a subset of MEG3-interacting regions rather than providing any experimental data and/or more global bioinformatic analysis.\nJust some specific comments:\nIn the abstract they claim ‘these triplex interactions might contribute to establishing long-distance chromosomal contact’ without providing any information or bioinformatic analyses. They use the term ‘hybridization’ for the interaction between RNA and dsDNA. This is wrong as hybridization refers to Watson-Crick base-pairing between RNA and ssDNA and not to Hoogsteen bonding. They took MEG3-interacting DNA peaks shorter than 1000 bp, then selected 3000 bp bins centering these regions and used these bins for analysis. There is no rationale for this selection which of course determines the final outcome of the analysis. Accordingly, the majority of these bin regions did not coincide with regions determined by ChOP-seq. Probably, a shorter binning would be more reliable to analyze the available data.\n\nThey focused on bins that overlap genes. Even if partial overlapping was accepted, they might have missed some promoters. Intergenic regions containing regulatory sequences (e.g. enhancers) were excluded. Why were only peaks overlapping with annotated genes considered to be significant (or ”real“)? Genomic regions that do not harbor annotated genes, such as enhancers, are important regulatory elements that are targeted by lncRNAs and as such are functional RNA-binding sites, highly relevant for this study. In addition, since it is known that the base composition of genic and intergenic regions is different, exclusion of intergenic regions introduces a considerable bias to the analysis. Selection of just three additional RNAs is certainly not adequate for the far-reaching conclusion: ‘TTSs are able to hybridize with various different RNAs almost irrespectively of their sequence’. It would be more convincing to show the results from scanning more RNAs, irrespective of their length and GC-content. Also, there is no attention given to the expression profiles of selected MEG3-mimicking RNAs. This is important because transcription of MEG3 is highly tissue-specific. The sum scores from the Triplexator analysis are shown which does not mean that the same regions are involved in triplex formation. It would be much more convincing to show similarities (or differences) of triplex-forming RNAs for a given TTS in a given bin.\n\nThe terms ‘universal TTS’ and ‘universal bins’ are not synonymous and interchangeable!! One bin (3000 bp) can contain many putative TTSs. If there are only 18 ‘universal bins’ out of 3620 bins among seven RNA analyses, this small number is not sufficient for claiming that there is no specificity in RNA targeting. They hypothesize that ‘the universal TTS can be viewed as the anchor point which can be bound by various nuclear RNAs to provide long-distance chromosomal contacts’. Even if this might be true, without any supportive data this is pure speculation.\n\nAltogether, the authors´ claim that triplex formation occurs almost sequence-independent is not justified but is based solely on in silico analyses. At least another available bioinformatics tool should have been used and standard in vitro assays (e.g. EMSA experiments) should have been performed to validate that the candidate RNAs are indeed capable to form triplexes. The authors do not even mention that the in vivo situation might be completely different than algorithm-based predictions and that there might be additional factors/constraints involved in triplex formation and stability.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\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": "4641",
"date": "10 May 2019",
"name": "Ivan Antonov",
"role": "Author Response",
"response": "We would like to thank Dr. Grummt for the extended comments to our work. They have helped us improve the design of our study and obtain additional results. We hope that they made our conclusions more reliable and reproducible. Given the importance and emerging acceptance of the concept of triplex-dependent gene regulation, it is more than surprising, if not irritating, that the authors challenge this concept feeding the ‘Triplexator’ only with a few RNAs and a subset of MEG3-interacting regions rather than providing any experimental data and/or more global bioinformatic analysis. We do not challenge the possibility of triplex-dependant regulation. We simply claim that RNA interactions with some genomic regions have low sequence specificity because many other RNAs may be able to bind the same loci. We investigated features of such regions and found them to be enriched in purine rich low complexity repeats. In the current version, we completely redesigned the study and incorporated the analysis of 306 RNA sequences to confirm our findings. We also modified the text so the main conclusions are clear and non-misleading. Just some specific comments: 1) In the abstract they claim ‘these triplex interactions might contribute to establishing long-distance chromosomal contact’ without providing any information or bioinformatic analyses. We believe it is reasonable to speculate about this possibility in the discussion for the following reasons. First, it has recently been shown that \"RNAs originating from super-enhancers form triplexes at distant regions\" [PMID: 30605520]. Second, we showed that the predicted Capture-seq universal TTSs were highly enriched in gene promoters (Supplementary Figure 10). Together these observations indicate that the same eRNA may be able to interact with several different universal TTSs and therefore contribute to the long-distance chromosomal (i.e. enhancer-promoter) contacts. However, additional experimental verification of this hypothesis is required. We modified the text in the abstract as follows: \"We speculated that such universal TTSs may contribute to establishing long-distance chromosomal contacts and may facilitate distal enhancer-promoter interactions.\" 2) They use the term ‘hybridization’ for the interaction between RNA and dsDNA. This is wrong as hybridization refers to Watson-Crick base-pairing between RNA and ssDNA and not to Hoogsteen bonding. We have corrected the terminology used in the manuscript. 3) They took MEG3-interacting DNA peaks shorter than 1000 bp, then selected 3000 bp bins centering these regions and used these bins for analysis. There is no rationale for this selection which of course determines the final outcome of the analysis. Accordingly, the majority of these bin regions did not coincide with regions determined by ChOP-seq. Probably, a shorter binning would be more reliable to analyze the available data. We now use the exact locations for all the ChOP-seq peaks. To compensate for the peak length variability we developed a method that estimates the statistical significance of the number of triplexes predicted for a RNA-DNA pair taking into account lengths of both sequences. 4) They focused on bins that overlap genes. Even if partial overlapping was accepted, they might have missed some promoters. Intergenic regions containing regulatory sequences (e.g. enhancers) were excluded. We now analyze all the ChOP-seq peaks without considering their overlaps with the annotated genes. 5) Why were only peaks overlapping with annotated genes considered to be significant (or ”real“)? Genomic regions that do not harbor annotated genes, such as enhancers, are important regulatory elements that are targeted by lncRNAs and as such are functional RNA-binding sites, highly relevant for this study. In addition, since it is known that the base composition of genic and intergenic regions is different, exclusion of intergenic regions introduces a considerable bias to the analysis. We now analyze all the ChOP-seq peaks. 6) Selection of just three additional RNAs is certainly not adequate for the far-reaching conclusion: ‘TTSs are able to hybridize with various different RNAs almost irrespectively of their sequence’. It would be more convincing to show the results from scanning more RNAs, irrespective of their length and GC-content. Also, there is no attention given to the expression profiles of selected MEG3-mimicking RNAs. This is important because transcription of MEG3 is highly tissue-specific. We have increased the number of the considered query RNAs to 306 and used the expressed transcripts only. 7) The sum scores from the Triplexator analysis are shown which does not mean that the same regions are involved in triplex formation. It would be much more convincing to show similarities (or differences) of triplex-forming RNAs for a given TTS in a given bin. We no longer use sum scores as a measure of triplex-based interaction. Instead, we estimate the statistical significance (p-value) of each RNA-DNA interaction based on the number of predicted triplexes. Our work was focused on the properties of the DNA sequences that may allow them to interact with various different RNAs. We therefore analyzed the parts of the ChOP-seq/Capture-seq peaks universal TTSs that allowed them to interact with various different RNAs. Our analysis indicates that such triplex-forming hot-spots frequently coincide with the purine-rich low complexity genomic regions. 8) The terms ‘universal TTS’ and ‘universal bins’ are not synonymous and interchangeable!! One bin (3000 bp) can contain many putative TTSs. We do not use the concept of bins and ‘universal bins’ in the current version of the manuscript. However, we kept the term ‘universal TTS’. 9) If there are only 18 ‘universal bins’ out of 3620 bins among seven RNA analyses, this small number is not sufficient for claiming that there is no specificity in RNA targeting. We would like to emphasize that our paper don't question the concept of the sequence specific triplex-dependent gene regulation (moreover, we support this idea and conduct research in this direction). We claim that some genomic regions may have a potential to form triple helices with a variety of different long RNAs forming \"universal\" triplex target sites (TTSs). At the same time, we do not challenge the sequence specificificity of the other triplex-based RNA-DNA interactions. 10) They hypothesize that ‘the universal TTS can be viewed as the anchor point which can be bound by various nuclear RNAs to provide long-distance chromosomal contacts’. Even if this might be true, without any supportive data this is pure speculation. We agree that at the moment our claim is a hypothesis/speculation. Nevertheless, the recent published results [PMID: 30605520] as well our own indicate the possibility of such mechanism. By discussing it in the current manuscript we hope to attract attention of experimental biologists to further study this topic. We modified the text in the paper to clarify the issue: \"One of the possible and actively discussed roles of the chromatin bound RNAs (including lncRNAs) is to bring different chromosomal parts together to enable the remote DNA-DNA contacts. Moreover, it has recently been shown that RNAs originating from super-enhancers form triplexes at distant regions. Therefore, it is possible that universal TTSs may facilitate distal enhancer-promoter interactions via engagement with the same enhancer RNA. In line with this hypothesis, we observed the statistical significant enrichment of the universal Capture- seq peaks near (< 1 kb) the transcription start sites (TSSs) of the annotated genes (Supplementary Figure 10C).\" 11) Altogether, the authors´ claim that triplex formation occurs almost sequence-independent is not justified but is based solely on in silico analyses. At least another available bioinformatics tool should have been used and standard in vitro assays (e.g. EMSA experiments) should have been performed to validate that the candidate RNAs are indeed capable to form triplexes. The authors do not even mention that the in vivo situation might be completely different than algorithm-based predictions and that there might be additional factors/constraints involved in triplex formation and stability. We added analysis of the recent in vitro data obtained by the Capture-seq method. According to this experimental data some genomic fragments can interact with all three RNA oligos used in the original study. This supports the observations obtained in the analysis of the ChOP-seq peaks. Our computational analysis classified almost 40% of these shared Capture-seq peaks as universal TTSs. Moreover, the ChOP-seq and the Capture-seq universal TTSs were similar in that they were enriched with the purine rich low complexity regions. We believe that these results indirectly support the existence of universal TTS . Yet, experimental validation of these results is beyond the scope of the current paper. Still, at the end of the manuscript, we discuss the limitations of our computational approach and mention that the obtained results resemble mostly the situation with the naked DNA in vitro, than the interactions with the chromatin in vivo. We added the following text to the discussion: \"Importantly, the current computational analysis has a number of limitations. Namely, the triplex-based interactions of the full length transcripts were predicted without taking their secondary structure into account. We are not aware of any bioinformatics tools that would be able to produce such predictions. Moreover, cellular localization of the 153 selected expressed transcripts as well as DNA binding proteins and chromatin compaction were not considered. Therefore, our simulations are more similar to the in vitro Capture-seq experiments with short oligos than to the interactions of long transcripts with the chromatin inside the nucleus.\""
}
]
},
{
"id": "29955",
"date": "26 Jan 2018",
"name": "Andrey A. Mironov",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes an application of the Triplexator software for search possible binding sites of the imprinting related MEG3 linc RNA on the human genome. The authors give a good example of the statistical analysis of the results. The main paper about this software has 62 references (google scholar data). Most of them have only reference to the software and only a few of them used the Triplexator. Only a few reports show a success story about the application of the Triplexator software and comparison the results with an experiment. In some papers, a significant enrichment of triplex targets on regions of interest was found. But they did not analyze the specificity of the predicted triplex formation. The current paper focused on a specificity of the Triplexator predictions. The authors got unexpected results that the Triplexator gives many non-specific hits for the case.\nComments:\nDescription of similar transcripts and the parameters of the Triplexator software should be rearranged because the appearance of some RNA names before definition sounds strange. The parameters of the Triplexator software contains a reference to the BE2L6 RNA while the description of the control RNA set has a reference on UBE2L6.\n\nThe di-nucleotide shuffling seems more adequate for RNA analysis.\n\nIn one paper1 the Triplexator software also was used for analysis of MEG3 RNA-DNA contacts. The comparison of the obtained results with the results of given manuscript should be provided. Seems in current manuscript a more accurate analysis with good controls was provided.\n\nIt would be good to look at the practice of using the program on literature and make sure that the program has a sufficiently low specificity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4640",
"date": "10 May 2019",
"name": "Ivan Antonov",
"role": "Author Response",
"response": "Dear reviewer, We would like to apologise for a significant delay with the reply to all the comments. To implement the changes suggested by the reviewers we had to completely redesign our study and significantly increase the number of analyzed RNAs. Particularly, we developed a new method to estimate the statistical significance of the number of triplexes predicted for each RNA-DNA pair. Moreover, we analyzed the results of the recently published Capture-seq experiment that identified interactions of three different RNA oligos with \"naked\" DNA. We hope that all these analyses improved our study. 1) Description of similar transcripts and the parameters of the Triplexator software should be rearranged because the appearance of some RNA names before definition sounds strange. The parameters of the Triplexator software contains a reference to the BE2L6 RNA while the description of the control RNA set has a reference on UBE2L6. We have made the appropriate corrections in the text 2) The di-nucleotide shuffling seems more adequate for RNA analysis. We now use the di-nucleotide shuffling to generate random RNA sequences. 3) In one paper1 the Triplexator software also was used for analysis of MEG3 RNA-DNA contacts. The comparison of the obtained results with the results of given manuscript should be provided. Seems in current manuscript a more accurate analysis with good controls was provided. In this original paper the authors focused on the triplex-based interactions of a single RNA (MEG3) with the chromatin. In the present study we are interested whether other transcripts may have a potential to interact with the same genomic regions. Taking into account the different aims (and approaches) of the studies we are not sure if it is reasonable to compare their results. 4) It would be good to look at the practice of using the program on literature and make sure that the program has a sufficiently low specificity. In our recent benchmarking study [PMID:29697742] we showed that Triplexator was the most accurate tool as of 2018. This is why we used it in the present study."
}
]
},
{
"id": "29953",
"date": "05 Feb 2018",
"name": "Hao Zhu",
"expertise": [
"Reviewer Expertise Genome analysis",
"lncRNA analysis",
"molecular evolution"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany lncRNAs can bind to DNA sequences by forming triplexes (the binding sites are often called TTS, triplex-targeting sites). Whether there are “universal TTS” as described here is interesting and unreported, and deserves a careful investigation. But I have a major concern about the work: the authors reach the conclusion upon too few examples. Also, why these lncRNAs (BE2L6, LILRA3, HMOX1) were chosen (randomly or selected for some reasons)?\nA few others issues should also be addressed. First, what is the relationship between the “universal TTSs” and base-pairing rules is untouched. For example, do the universal TTSs allow many lncRNAs to bind to them using the same rules? If very different rules are involved, what does this mean? To some extent, binding upon different rules indicates lncRNA specific TTSs, instead of universal TTSs. Second, I feel that the genomic regions used to sum scores are unreasonably long (3000 bp). Finally, it is said that “the median Triplexator SumScores were 48 and 25, respectively (p-value=5.2e-100)”. Statistically, the difference is significance, but biologically might not. I think 48 is not that large and 25 is not that small.\n\nIs the work 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? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "4642",
"date": "10 May 2019",
"name": "Ivan Antonov",
"role": "Author Response",
"response": "Many lncRNAs can bind to DNA sequences by forming triplexes (the binding sites are often called TTS, triplex-targeting sites). Whether there are “universal TTS” as described here is interesting and unreported, and deserves a careful investigation. But I have a major concern about the work: the authors reach the conclusion upon too few examples. Also, why these lncRNAs (BE2L6, LILRA3, HMOX1) were chosen (randomly or selected for some reasons)? In the current version we have significantly increased the number of query RNAs (i.e. 153 expressed transcripts and 153 random sequences obtained by di-nucleotide shuffling of MEG3 lncRNA). The 153 transcripts that we use now were chosen so that their lengths and GC contents were similar to the MEG3 lncRNA. 1) A few others issues should also be addressed. First, what is the relationship between the “universal TTSs” and base-pairing rules is untouched. For example, do the universal TTSs allow many lncRNAs to bind to them using the same rules? If very different rules are involved, what does this mean? To some extent, binding upon different rules indicates lncRNA specific TTSs, instead of universal TTSs. Our preliminary analysis indicated that different RNAs interacted with universal TTSs via mixed (G or U) motif a little bit more frequently than via the purine or pyrimidine motifs. Importantly, all the analyzed transcripts were predicted to form a lot of triple helices (using different RNA motifs) with the universal TTSs. This is what makes uTTS special rather than specific motifs . 2) Second, I feel that the genomic regions used to sum scores are unreasonably long (3000 bp). In the current version we decreased the genomic region lengths by considering the exact ChOP-seq and Capture-seq peaks. 3) Finally, it is said that “the median Triplexator SumScores were 48 and 25, respectively (p-value=5.2e-100)”. Statistically, the difference is significance, but biologically might not. I think 48 is not that large and 25 is not that small. We now use p-values instead of SumScore to estimate the statistical significance of each RNA-DNA interaction."
}
]
}
] | 1
|
https://f1000research.com/articles/7-76
|
https://f1000research.com/articles/8-161/v1
|
06 Feb 19
|
{
"type": "Study Protocol",
"title": "Protocol for a comparison study of 1-day versus 2-day prophylactic antibiotic administration in Holmium Laser enucleation of the prostate (HoLEP): a randomized controlled trial",
"authors": [
"Katsumi Shigemura",
"Fukashi Yamamichi",
"Kento Nishimoto",
"Koichi Kitagawa",
"Masato Fujisawa",
"Fukashi Yamamichi",
"Kento Nishimoto",
"Koichi Kitagawa",
"Masato Fujisawa"
],
"abstract": "Background: The best method of antimicrobial prophylaxis administration for surgical site infection (SSI) in transurethral holmium laser resection and enucleation of the prostate (HoLEP)/bipolar transurethral enucleation (TUEB) remains controversial. The purpose of this study is to compare one-day and two-day cefazolin in a randomized 2nd-phase study to help establish a protocol with a 95% confidence interval (CI) for SSI prevention. Methods: Patients undergoing HoLEP/TUEB for benign prostate hyperplasia without preoperative pyuria will be enrolled and randomized to receive prophylactic antibiotic administration for HoLEP/TUEB in two groups, 1-day cefazolin and 2-day cefazolin. The primary endpoint is the occurrence rate of postoperative urinary tract infection or urogenital infection within 30 days after HoLEP/TUEB with a statistical 95% CI in comparison between those groups. Secondary outcomes include the kind of infectious disease and evidence of diagnosis, day of diagnosis of infectious disease, performance of urine or blood culture, detection of bacteria, treatments, duration of treatments, AEs other than surgical site infection, and drug-induced AEs. Discussion: The results of this study will provide evidence for defining the optimal duration of cefazolin prophylactic antibiotic administration for SSI. Trial registration: This study was registered in the University Hospital Medical Information Network-Clinical Trial Registry (UMIN000027955) based on recommendations from the International Committee of Medical Journal Editors (ICMJE) on July 1st 2017.",
"keywords": [
"HoLEP",
"Transurethral resection of the prostate",
"prophylactic antibiotic administration"
],
"content": "Abbreviation\n\nSSI: surgical site infection; HoLEP: Holmium laser resection and enucleation of the prostate; TUEB: bipolar transurethral enucleation; CI: confidence interval; TURP: Transurethral resection of the BPH: prostate; benign prostate hyperplasia; UTI: urinary tract infection; PAA: prophylactic antibiotic administration; CEZ: cefazolin\n\n\nTrial registration\n\nThis study is registered in the University Hospital Medical Information Network-Clinical Trial Registry (UMIN000027955) based on recommendations from the International Committee of Medical Journal Editors (ICMJE).\n\n\nIntroduction\n\nTransurethral resection of the prostate (TURP) has been the gold standard for surgical treatment of benign prostate hyperplasia (BPH). However, currently enucleative surgery, such as transurethral holmium laser resection and enucleation of the prostate (HoLEP) or bipolar transurethral enucleation (TUEB), is increasingly performed as a substitution for TURP.\n\nGuidelines for prophylactic antimicrobial administration (PAA) for TURP have been published by the European Association of Urology (EAU), American Urological Association (AUA) and The Japanese Urological Association (JUA)1–3. Prophylaxis guidelines for HoLEP or TUEB are not fully established4. A meta-analysis by Ahyia et al. showed postoperative urinary tract infection (UTI) occurred in 4.1% (0–22%) of TURP cases and 0.9% (0–4.9%) of HoLEP cases, and concluded that HoLEP had a lower rate of SSI5. Similarly, our group presented data from a prospective multi-center study showing that SSI occurred in 8% of TURP cases and 5% of HoLEP cases1. Importantly, no definitive description of the duration of antibiotic dosing was shown and the optimal duration of prophylactic antibiotic administration (PAA) for SSI need to be established.\n\n\nMethods\n\nStudy sites\n\nPatients recruitment began on May 1st 2018 and will continue up to April 30th 2021 from the institutions of Kobe University Hospital, Kobe University International Clinical Cancer Research Center, Hara Genitourinary Hospital, Shinko Memorial Hospital, Hyogo prefectural Amagasaki General Medical Center, Kobe City Medical Center West Hospital, Kakogawa Central City Hospital and Hyogo Prefectural Kakogawa Medical Center. Those patients undergoing HoLEP/TUEB for BPH without preoperative pyuria will be enrolled. Preoperative pyuria will be defined as 5 or more white blood cells (WBC)/higher power field (HPF). Since PAA duration is limited to 72 h or less in TURP and 48h or less in HoLEP or TUEB6, we will carry out a randomized study of 1-day and 2-day PAA for HoLEP/TUEB using cefazolin (CEZ). This is a feasible randomized 2nd phase study to help design further confirmatory studies evaluating the differences of SSI occurrence rate with a 95% confidential interval.\n\nSelection criteria\n\nIn the period from May 1st 2018 to April 30th 2021, patients 20 years old or older undergoing HoLEP/TUEB without preoperative pyuria and bacteriuria will be enrolled. Pyuria will be defined as 5 ≥WBC/HPF or ≥10/ μl (flowcytometer) preoperatively.\n\nExclusion criteria\n\ni) Patients who have undergone another procedure such as prostate biopsy or bladder urolithiasis at the time of HoLEP/TUEB. ii) Those with an indwelling urethral catheter. iii) Those with an allergy to CEZ. iv) Hemodialysis patients.\n\nEligible patients will be randomized in equal proportions between 1-day and 2-day PAA for HoLEP/TUEB using cefazolin (CEZ).\n\nPrimary endpoint\n\nPrimary endpoint is to compare SSI occurrence rate in both 2 groups.\n\nItems of postoperative infection complications:\n\n30-days postoperative infectious complications after HoLEP\n\nIn this study, UTI and urogenital infection means prostatitis, epididymitis, pyelonephritis and urosepsis. The occurrence date of these infections and the duration (days) until disappearance of pyuria, in those cases with pyuria, will be recorded.\n\nSecondary endpoints\n\nIn cases where perioperative infection requires antibiotic therapies, the following information will be recorded. 1) The kind of infection and reasons for the diagnosis; 2) Occurrence data of such infection; 3) Blood culture and urine culture; 4) Identified bacteria; 5) Methods of therapies; 6) Duration of therapies; 7) Other postoperative complications than infectious ones; 8) Drug-induced adverse events.\n\nFeasible purpose\n\nThis is a feasible randomized study to investigate the occurrence rate of postoperative UTI or urogenital infection within 30 days after HoLEP/TUEB with an estimated 95% CI, and will be followed by confirmatory studies.\n\n\nParticipant timeline (See Table 1)\n\n○; Items to be done before antibiotic administration, ●: Items to be done after antibiotic administration\n\na: Adverse events are not necessarily associated with antibiotics.\n\nb: Hematological examination consists of hematological laboratory tests, biochemical laboratory tests and urinalysis to check the safty of the tested antibiotics and includes white blood cell (WBC) and differential white blood count as inflammatory markers. The volume for hematological laboratory tests is 8ml.\n\nc: Biochemical laboratory tests use CRP as inflammatory markers, which is performed within the range of daily clinical examination.\n\nd: Urinalysis includes white blood cell (WBC) and bacteriuria as inflammatory markers. These tests are done as a confirmation safety check for this study.\n\nFor a feasible randomized comparing study, the target sample size is n=180 (1 day: n=90 and 2 days: n=90). The sample calculation was performed as follows: this study is a feasibility study. We referred to the following study which examined their 164 TURP and HoLEP cases, and found the postoperative infectious complications in 7/72 cases (9.7%) and 2/72 cases (2.8%), respectively. Accordingly, if we estimate 3 or 4 cases of UTI or urogenital infectious complication within 30 days after HoLEP, the occurrence frequencies for a 95% confidence interval (CI) are 0.007-0.094 and 0.012-0.111, respectively. The upper limit of a 95% CI is 10% or so, and it may be useful for planning the next study to set them as in Jhanwar et al.7.\n\nStatistical analysis\n\nAnalysis of study participants’ background:\n\nThe difference between groups will be analyzed by the following methods:\n\nPearson’s chi-square: nominal variables; Fisher’s direct probability calculation method is performed where the expected frequency of less than 5 is 20% or higher; T-tests will be done for continuous variables. The Significance standard is set as 5% in two-sided tests.\n\nRecruitment will be performed from the patients with indication of HoLEP in those institutions participated in this study. Randomization will be performed by a table of random numbers as a simple randomized study under the control of the responsible party (Dr. Yuzo Nakano, Department of Urology, Kobe University Hospital).\n\nParticipants will be randomly assigned to either 1-day or 2-days antibiotic group with a 1:1 allocation as per a computer-generated randomization schedule.\n\nAssessments regarding clinical recovery will be conducted by Dr. Yuzo Nakano blind to treatment allocation. Due to the nature of the intervention neither participants nor staff can be blinded to allocation, but are strongly inculcated not to disclose the allocation status of the participant at the follow up assessments. An employee outside the research team will feed data into the computer in separate datasheets so that the researchers can analyse data without having access to information about the allocation\n\nTested antibiotics\n\n1st generation cephalosporine : Cefazolin Sodium Hydrate (J01DB04)\n\nMethod of administration\n\nPatients will be randomly divided into a 1-day group (CEZ 1g, once per i.v. just before HoLEP/TUEB) and a 2-day group (CEZ 1g, i.v. just before HoLEP/TUEB with a repeat dose the next morning). Comparison of SSI occurrence in these 2 groups is a feasible randomization study.\n\nOutline of study\n\ni) One-day dosing: i.v. initiation of CEZ (1g) 30 min prior to surgery, completed in 30–60 min (with no any other antibiotic administration)\n\nii) Two-day dosing: i.v. initiation of CEZ (1g) 30 min prior to surgery, completed in 30-60 min, repeated every 12 hours for 2 days including the surgery day. Additional dosing is necessary in cases with 3 hours or longer of surgery time. Test items and schedule of this study is shown in Table 1.\n\nStudy therapy\n\nTo investigate the inhibiting effect of cefazolin (CEZ) 1-day and 2-day administration on perioperative infectious complications in HoLEP with a calculated 95% CI.\n\nCombination medicine\n\nExclusion criteria include use of other antibiotics or cases requiring an additional antibiotic.\n\nTermination of antibiotic administration\n\nCases exhibiting or suspicious for drug-induced allergy.\n\nManagement and delivery of study drug\n\nApplicants will be contacted by fax or e-mail and then given either 1-day or 2-day CEZ under randomization as described above.\n\nIn cases where the attending physician diagnoses a perioperative infectious complication, the doctor can treat at discretion, including i.v. for severe cases and oral antibiotics for mild cases.\n\nWe will gather the following data from the medical records.\n\ni) Patients’ background factors\n\nAge, Body Mass Index (BMI), Preoperative IPSS/QOL・Qmax・residual urine\n\nvolume (ml), Estimated prostate volume(ml), Preoperative PSA, ASA-PS, Diabetes mellitus (HbA1c, blood sugar control), Chemotherapy and immune-suppressants\n\nii) Surgery-related categories\n\nSurgical time (min, including morcellation), Resected prostate weight (g), Catheterized period (days), Post-operative residual urine volume (ml): until 30 days after surgery, Duration of antibiotic administration (1 day or 2 days)\n\niii) Postoperative infectious complications\n\nInvestigation for 30 days after surgery (need to record), UTI or urogenital infection (prostatitis, epididymitis or pyelonephritis), Postoperative complication other than infectious ones, Occurrence date, Cases with infectious complications requiring additional antibiotic therapy, Detail of infection and the diagnosing evidence, Infection occurrence date, Urine culture and blood culture, Detected bacteria, Therapy, Therapeutic duration, and Days needed to reduce pyuria\n\nScreening tests will be done to check the following criteria i) and ii).\n\ni) Enrollment criteria\n\nThose patients who are older than 20 years old undergoing HoLEP for benign prostate hyperplasia (BPH) without pyuria* or bacteriuria preoperatively and with informed consent. *Definition of pyuria: WBC≧5/hpf in urine (under microscopy) or WBC≧10/ul in urine (flowcytometry)\n\nii) Exclusion criteria: see above\n\nThe following information will be recorded at the time of informed consent acquisition and screening tests.\n\ni) Date of informed consent acquisition; ii) Class card number of study participants; iii) Backgrounds of study participants, birth date, age, height, body weight, past history, complication (underlying disease), iv) Present illness: date of definitive diagnosis, risk scoring, family history\n\n\nItems for observation/ tests/ evaluation\n\nObservation of adverse events including infectious complication, blood pressure, heart rate, body weight, hematology examination, blood biochemical examination, urinalysis, chest X-ray, ECG, urine culture\n\nAge, body mass index (BMI), preoperative PSS/QOL・Qmax・residual urine volume (ml) estimated prostate weight (ml), preoperative PSA, ASA-PS, diabetes mellitus (HbA1c, blood sugar control), chemotherapy or immune-suppressants,\n\nSurgical time (min; including morcellation time), resected prostate weight (g), duration (days) to removal of urethral catheter postoperatively, Postoperative residual urine volume (ml) till 30-days after surgery, duration of prophylactic antibiotic administration (CEZ) : 1-day or 2-days group\n\nUTI and urogenital infection (prostatitis, epididymitis and pyelonephritis), postoperative adverse events including infectious ones, except postoperative infection, occurrence date.\n\nIn cases with perioperative infection who need antibiotic therapies, the details include bacterial information (see above).\n\ni) Those cases who are not willing to continue this study and/or wish to withdraw informed consent. ii) Those cases who are found not to be satisfied about their applicability to this study or who cannot continue the study owing to the occurrence of complications and/or exacerbation. iii) Those cases who cannot continue the study owing to study-related adverse events. iv) The study itself is canceled. v) Cases with uncontrollable infectious complications postoperatively. vi) PIs or the members of this research judge that cancelation is needed owing to other reasons.\n\nMay 1st 2018 to April 30th 2021; the follow-up period is 1 year afterwards.\n\n\nAnalysis of efficacy\n\nWe will estimate the difference in postoperative infection occurrence rate at the 95% CI and do the 5% two-sided test for Significant for such differences.\n\nThis study needs interim analysis because it is planned as a feasible randomized study.\n\nThat is, once 45 cases are completed, the occurrence rate of postoperative infection can be compared in the 2 groups and to determine if there is less than a 5% of difference and then to continue if the difference is 5% or less.\n\nEthical consideration and consent\n\nThis protocol was approved by the Institutional review board of the Kobe University Graduate School of Medicine (C180043). All procedures were in compliance with the relevant laws and guidelines in accordance with the ethical standards of the Declaration of Helsinki.\n\nThis study was registered in the University Hospital Medical Information Network-Clinical Trial Registry (UMIN000027955) based on recommendations from the International Committee of Medical Journal Editors (ICMJE) on July 1st 2017.\n\nProtocol amendments\n\nAny modifications to the protocol which may impact on the conduct of the study, potential benefit of the patient or may affect patient safety, including changes of study objectives, study design, patient population, sample sizes, study procedures, or significant administrative aspects will require a formal amendment to the protocol. Such amendment will be approved by the Ethics Committee/IRB (institutional review board) prior to implementation and notified to the health authorities in accordance with local regulations. Administrative changes of the protocol are minor corrections and/or clarifications that have no effect on the way the study is to be conducted. These administrative changes will be agreed upon by Ethics Committee/IRB, and will be documented in a memorandum. The Ethics Committee/IRB may be notified of administrative changes at the discretion of Helsinki Declaration.\n\nA trained research doctor will introduce the trial to patients who will be shown an informed consent form regarding the main aspects of the trial. Research doctors will discuss the trial with patients in light of the information provided in information sheets. Patients will then be able to have an informed discussion with the participating consultant. Research doctors will obtain written consent from patients willing to participate in the trial. Information sheets and consent forms are provided for all patients (Extended data8). All information sheets and consent forms transcripts have been translated into Japanese.\n\nAll study-related information will be stored securely at the study site. All participant information will be stored in locked file cabinets in areas with limited access. All laboratory specimens, reports, data collection, process, and administrative forms will be identified by a coded ID number only to maintain participant confidentiality. All records that contain names or other personal identifiers, such as locator forms and informed consent forms, will be stored separately from study records identified by code number. All local databases will be secured with password-protected access systems. Forms, lists, logbooks, appointment books, and any other listings that link participant ID numbers to other identifying information will be stored in a separate, locked file in an area with limited access.\n\nThe Data Management Coordinating Center will oversee the intra-study data sharing process, with input from the Data Management Subcommittee.\n\nAll Principal Investigators will be given access to the cleaned data sets. Project data sets will be housed on the Project Accept Web site and/or the file transfer protocol site created for the study, and all data sets will be password protected. Project Principal Investigators will have direct access to their own site’s data sets, and will have access to other sites data by request. To ensure confidentiality, data dispersed to project team members will be blinded of any identifying participant information.\n\nPatients that are enrolled into the study are covered by national health insurance in all the tests and treatments including the ones performed additionally owing to the adverse events of this study.\n\nWe plan to disseminate the information of this study through the UMIN and our department homepage.\n\nThe study is now undergoing pre-recruitment for participants (recruitment started May 1st, 2018).\n\n\nDiscussion\n\nThis study is designed to address the following issues:\n\n1) AMR: antimicrobial resistance action plan\n\nWHO suggested the AMR action plan in 2015 based on recent trends of emergence of antibiotic resistant bacteria in infectious diseases. Many researchers suggest that inappropriate antibiotics use may cause this problem. Inappropriate antibiotic prescriptions are often seen, for instance in upper respiratory infections which is caused by not only bacteria but also virus9. Accordingly, the Japanese government suggested an AMR action plan aiming at a 50% decrease in the use of oral antibiotic such as cephalosporines or macrolides10. The chief justification for the preventive use of antibiotics is for the purpose of inhibiting postoperative infectious complications. Not only surgery, but interventional examinations such as transrectal prostate biopsy require antibiotics. There are many reports on the efficacy of PAA to inhibit SSI when compared with no use of antibiotics11,12.\n\n2) Duration of PAA\n\nConsidering the two contradictory concepts mentioned above, what we should do next is to determine how to safely decrease antibiotic use without affecting or increasing the SSI occurrence rate. Another thing we need to consider is that long term antibiotic use can cause untreatable infections by antibiotic-resistant strains13. Therefore, this kind of prospective study is a valuable way to learn how to decrease antibiotic use safely.\n\n3) Semi-contaminated\n\nMany guidelines say that surgeries need to be classified according to the extent of pre-surgical pollution, infection, or bacterial colonization. Especially in urological cases, the concept of clean-contaminated operation exists when opening the urinary tract with the possibility of urine dissemination. Preoperative pyuria or bacteriuria should be checked in advance pre-operatively and treated before surgery, including confirmation of absence of such infection in order to decrease the risk of SSI.\n\n4) HoLEP in Japan\n\nMost of the prospective studies on how to decrease antibiotic use, for instance comparisons between 1-day and 3-day prophylactic antibiotic administration (PAA) for urological surgeries such as TUR-P in prospective 2-group or double-blind studies are from western countries14. We should not forget the differences in medical systems and standards between countries15. Arguably, individual studies need to be designed or undertaken in each country or region with the same or similar medical systems. For instance, one major difference between most western countries versus Korea and Japan is that high volume surgical centers are more common in the west, and thus the number of surgical cases per surgeons is higher in the west than Japan. High volume centers expect shorter surgical times, which can affect SSI occurrence16. These issues support the necessity of our study.\n\n5) Prior studies as evidence for drawing up guidelines\n\nThere are several studies regarding the optimal PAA duration period17,18. However, we need to decrease the variation in patients’ backgrounds in such studies; How many patients with preoperative UTI cases that need to be controlled before surgery. There is no definitive study for comparison between controlled and uncontrolled preoperative UTI caused by retention19 and SSI occurrence after HoLEP. Also, case numbers are limited; so, variations in patient criteria and the surgeons’ experience cannot be ignored.\n\n6) Sample size\n\nThis kind of prospective feasibility study needs a setting with the necessary case numbers for analysis. A similar study referred to above had 164 cases for comparison in 2 groups with TUR-P and HoLEP for SSI occurrence, with SSI occurrence after HoLEP being 2/72 cases (2.8%)9. We therefore anticipate 180 cases (90 cases per arm) for a feasibility study with 95% CI for the future design of a prospective double blind non-inferiority study. To our knowledge, no definitive study with one-day PAA for HoLEP has been performed so, we have designed this study as a comparison with 2-days dosing.\n\n7) Study setting\n\nIn most retrospective studies, the study period for patients’ enrollment may be different. For instance, comparing TUR-P and HoLEP, HoLEP is a comparatively newer technique leading to the possibility of selection bias for HoLEP surgeons with TUR-P experience and this may influence the results of SSI occurrence. To reduce selection bias, prospective studies where not only patients but also surgeons have similar backgrounds, should be undertaken for definitive conclusions with high evidence level.\n\n8) Significance and problems with prospective studies\n\nMany guidelines refer to prospective studies as providing a high evidence level20. However, prospective randomized studies with intervention may be more difficult to perform21. In Japan, apart from research by medical doctors, publication of scientific papers in the medical field is decreasing. IRB referees need to be strict about study approval and this can make things more complicated for prospective clinical interventional studies. Protocol papers will help researchers plan, design and perform clinical studies. This study describes a comparison study of PAA for HoLEP with 1-day and 2-day dosing and help to establish a protocol for PAA taking into account decreasing unnecessary antimicrobial use to prevent the development of AMR.\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\nThe consent form and information sheet that will be used in this study is available from Harvard Dataverse\n\nHarvard Dataverse: Extended data. Replication data for Protocol for a comparison study of 1-day versus 2-day prophylactic antibiotic administration in Holmium Laser enucleation of the prostate (HoLEP): a randomized controlled trial https://doi.org/10.7910/DVN/SGTWJN8\n\nLicense: CC0 1.0 Universal",
"appendix": "Grant information\n\nThis study is funded by research contribution in Department of Urology, Kobe University Hospital.\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\nÇek M, Tandoğdu Z, Naber K, et al.: Antibiotic prophylaxis in urology departments, 2005-2010. Eur Urol. 2013; 63(2): 386–94. PubMed Abstract | Publisher Full Text\n\nWolf JS Jr, Bennett CJ, Dmochowski RR, et al.: Best practice policy statement on urologic surgery antimicrobial prophylaxis. J Urol. 2008; 179(4): 1379–90. PubMed Abstract | Publisher Full Text\n\nMatsumoto T, Kiyota H, Matsukawa M, et al.: Japanese guidelines for prevention of perioperative infections in urological field. Int J Urol. 2007; 14(10): 890–909. PubMed Abstract | Publisher Full Text\n\nTogo Y, Shimatani K, Hanasaki T, et al.: Incidence of genitourinary tract infection following transurethral enucleation with bipolar. Japanese Journal of Endourology. 2015; 28(2): 317–21. Publisher Full Text\n\nAhyai SA, Gilling P, Kaplan SA, et al.: Meta-analysis of functional outcomes and complications following transurethral procedures for lower urinary tract symptoms resulting from benign prostatic enlargement. Eur Urol. 2010; 58(3): 384–97. PubMed Abstract | Publisher Full Text\n\nTogo Y, Tanaka S, Kanematsu A, et al.: Antimicrobial prophylaxis to prevent perioperative infection in urological surgery: a multicenter study. J Infect Chemother. 2013; 19(6): 1093–101. PubMed Abstract | Publisher Full Text\n\nIshikawa K, Maruyama T, Kusaka M, et al.: [The state of antimicrobial prophylaxis for holmium laser enucleation of the prostate : HoLEP and the results of a questionnaire survey]. Hinyokika Kiyo. 2011; 57(10): 539–43. PubMed Abstract\n\nShigemura K: Replication data for Protocol for a comparison study of 1-day versus 2-day prophylactic antibiotic administration in Holmium Laser enucleation of the prostate (HoLEP): a randomized controlled trial. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/SGTWJN\n\nJhanwar A, Sinha RJ, Bansal A, et al.: Outcomes of transurethral resection and holmium laser enucleation in more than 60 g of prostate: A prospective randomized study. Urol Ann. 2017; 9(1): 45–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Sullivan JW, Harvey RT, Glasziou PP, et al.: Written information for patients (or parents of child patients) to reduce the use of antibiotics for acute upper respiratory tract infections in primary care. Cochrane Datebase Syst Rev. 2016; 11: CD011360. PubMed Abstract | Publisher Full Text\n\nMorioka H, Nagao M, Yoshihara S, et al.: The first multi-centre point-prevalence survey in four Japanese university hospitals. J Hosp Infect. 2018; 99(3): 325–31. PubMed Abstract | Publisher Full Text\n\nKim DH, Bae SR, Choi WS, et al.: The real practice of antibiotic prophylaxis for prostate biopsy in Korea where the prevalence of quinolone-resistant Escherichia coli is high. Korean J Urol. 2014; 55(9): 593–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevy SB, Marshall B: Antibacterial resistance worldwide: causes, challenges and responses. Nat Med. 2004; 10(12 Suppl): S122–9. PubMed Abstract | Publisher Full Text\n\nValdevenito Sepúlveda JP: [Antibiotics in transurethral resection of the prostate in patients with low risk of infectious complications: randomized prospective comparative study]. Arch Esp Urol. 2004; 57(1): 48–57. PubMed Abstract\n\nBlendon RJ, Leitman R, Morrison I, et al.: Satisfaction with health systems in ten nations. Health Aff (Millwood). 1990; 9(2): 185–92. PubMed Abstract | Publisher Full Text\n\nMonn MF, El Tayeb M, Bhojani N, et al.: Predictors of Enucleation and Morcellation Time During Holmium Laser Enucleation of the Prostate. Urology. 2015; 86(2): 338–42. PubMed Abstract | Publisher Full Text\n\nTsugawa M, Hashimoto H, Monden K, et al.: [Antimicrobial prophylaxis in transurethral resection of the prostate]. Nihon Hinyokika Gakkai Zasshi. 1998; 89(4): 453–9. PubMed Abstract | Publisher Full Text\n\nTsuji Y, Okamura K, Miyake K, et al.: [Antibiotic prophylaxis for transurethral resection of the prostate--comparison of oral administration therapy with intravenous administration therapy]. Hinyokika Kiyo. 1993; 39(12): 1145–52. PubMed Abstract\n\nChen JS, Chang CH, Yang WH, et al.: Acute urinary retention increases the risk of complications after transurethral resection of the prostate: a population-based study. BJU Int. 2012; 110(11 Pt C): E896–901. PubMed Abstract | Publisher Full Text\n\nRoussey-Kesler G, Gadjos V, Idres N, et al.: Antibiotic prophylaxis for the prevention of recurrent urinary tract infection in children with low grade vesicoureteral reflux: results from a prospective randomized study. J Urol. 2008; 179(2): 674–9; discussion 679. PubMed Abstract | Publisher Full Text\n\nCardozo L, Lisec M, Millard R, et al.: Randomized, double-blind placebo controlled trial of the once daily antimuscarinic agent solifenacin succinate in patients with overactive bladder. J Urol. 2004; 172(5 Pt 1): 1919–24. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "44091",
"date": "29 Apr 2019",
"name": "Seung-Ju Lee",
"expertise": [
"Reviewer Expertise Antimicrobial prophylaxis for urological surgeries"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 aims to establish optimal duration of cefazolin antibiotic prophylaxis prior to HoLEP procedure. The rationale and objective of the study protocol is clearly defined and the overall study design looks appropriate. Regarding details of the method, I have a couple of queries.\n\nFirst, one-day dosing and single-dose dosing should be distinguished. If you look at your protocol, it's single-dose (CEZ 1g, once per i.v. just before HoLEP/TUEB). However, the study title is 1-day vs. 2-day. Cefazolin is a cephalosporin antibiotic and is time-dependent. Therefore, 1-day (two or three doses per a day) and single-dose are different dosages.\n\nSecond, most studies select a 3-day regimen as a comparator. Why did you choose 2-day? The reason for comparing the 2-day regimen in this study should be more clear.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "4603",
"date": "30 Apr 2019",
"name": "Katsumi Shigemura",
"role": "Author Response",
"response": "First, one-day dosing and single-dose dosing should be distinguished. If you look at your protocol, it's single-dose (CEZ 1g, once per i.v. just before HoLEP/TUEB). However, the study title is 1-day vs. 2-day. Cefazolin is a cephalosporin antibiotic and is time-dependent. Therefore, 1-day (two or three doses per a day) and single-dose are different dosages. (Amendment)Thanks, our 1-day protocol means single-dose protocol. Second, most studies select a 3-day regimen as a comparator. Why did you choose 2-day? The reason for comparing the 2-day regimen in this study should be more clear. (Amendment)Thanks, but JUA guideline recommend single-dose or dose withing 48 hours (ref: Yamamoto S, Shigemura K, Kiyota H, Wada K, Hayami H, Yasuda M, Takahashi S, Ishikawa K, Hamasuna R, Arakawa S, Matsumoto T; Japanese Research Group for UTI.Essential Japanese guidelines for the prevention of perioperative infections in the urological field: 2015 edition. Int J Urol. 2016 Oct;23(10):814-824.)."
},
{
"c_id": "4618",
"date": "09 May 2019",
"name": "Katsumi Shigemura",
"role": "Author Response",
"response": "And my previous comment (single dose) were reflected in the text (See in many places including title) Next comment (most studies select a 3-day regimen as a comparator. Why did you choose 2-day? The reason for comparing the 2-day regimen in this study should be more clear.): We reflected on the text (See in 4) HoLEP in Japan in Discussion)."
},
{
"c_id": "4620",
"date": "09 May 2019",
"name": "Katsumi Shigemura",
"role": "Author Response",
"response": "I have posted new version of manuscript."
}
]
},
{
"id": "47977",
"date": "02 May 2019",
"name": "Florian M.E. Wagenlehner",
"expertise": [
"Reviewer Expertise infections of the urogenital tract"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 submitted a protocol studying two different prophylactic regimens in patients undergoing Holmium Laser enucleation of the prostate. The study is randomized.\nThe topic of the study is worth while studying, as there are relatively little data especially in laser enucleation of the prostate.\nThe study design is well described and set up, including the important in- and exclusion criteria. There is one exclusion criterium leucocyturia, which should be reconsidered, as especially patients with large prostates, might have elevated leucocytes in their urine, and those patients would not be included in the study, although holmium laser enucleation is especially done in patients with large prostates.\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": "4617",
"date": "09 May 2019",
"name": "Katsumi Shigemura",
"role": "Author Response",
"response": "Thanks, we added the patients with leucocyturia and reflected on the text (See in Eligibility criteria).We revised as same way in another part (See in Screening test)."
}
]
}
] | 1
|
https://f1000research.com/articles/8-161
|
https://f1000research.com/articles/7-416/v1
|
29 Mar 18
|
{
"type": "Software Tool Article",
"title": "Gene Annotation Easy Viewer (GAEV): Integrating KEGG’s Gene Function Annotations and Associated Molecular Pathways",
"authors": [
"Trung Huynh",
"Sen Xu",
"Trung Huynh"
],
"abstract": "We developed a Gene Annotation Easy Viewer (GAEV) that integrates the gene annotation data from the KEGG (Kyoto Encyclopedia of Genes and Genomes) Automatic Annotation Server. GAEV generates an easy-to-read table that summarizes the query gene name, the KO (KEGG Orthology) number, name of gene orthologs, functional definition of the ortholog, and the functional pathways that query gene has been mapped to. Via links to KEGG pathway maps, users can directly examine the interaction between gene products involved in the same molecular pathway. We provide a usage example by annotating the newly published freshwater microcrustacean Daphnia pulex genome. This gene-centered view of gene function and pathways will greatly facilitate the genome annotation of non-model species and metagenomics data. GAEV runs on a Windows or Linux system equipped with Python 3 and provides easy accessibility to users with no prior Unix command line experience.",
"keywords": [
"molecular pathway",
"Daphnia",
"genome annotation",
"visualization",
"homologous genes"
],
"content": "Introduction\n\nIn our efforts to de novo assemble a draft genome, describing the biological function of computationally annotated genes and the molecular pathways formed by these genes’ products is critical for identifying the genetic basis of the various unique biological attributes (e.g., physiology, life history, behavior) of the species in question. Computational search against DNA/protein databases, e.g., NCBI Blast (Boratyn et al., 2013), UniProt (Bateman et al., 2017), InterPro (Finn et al., 2017), based on homology and protein domain information using computational tools, such as Blast (Camacho et al., 2009), InterProScan (Jones et al., 2014), and Hmmer (Mistry et al., 2013), can make predictions for individual gene functions. In contrast, delineating the molecular pathways encoded by the entire suite of genes of a single species is a much more challenging task, especially for non-model species. To this extent, mapping genes to the molecular pathways derived from intensively studied model organisms provides an entry point for addressing this need.\n\nFor mapping genes into known molecular pathways, the Kyoto Encyclopedia of Genes and Genomes (KEGG) provide comprehensive web services (Kanehisa et al., 2017; Kanehisa & Goto, 2000; Kanehisa et al., 2016a). KEGG is an integrated database for biological interpretation of genome sequences. The molecular function of genes is classified using ortholog groups, i.e., KEGG Orthology (KO). KEGG also contains KEGG pathways, BRITE hierarchies, and KEGG modules, all of which are networks of KO nodes. It is possible to annotate the molecular functions of a set of genes from complete/partial genome assembly or metagenomics dataset and their encoded molecular pathways using KEGG automatic annotation services that are provided through webservers BlastKOALA and GhostKOALA (Kanehisa et al., 2016b). For a non-model species, we can use KAAS (KEGG Automatic Annotation Server) web services to annotate the complete or random set of genes to describe their molecular function and map them into identified molecular pathways. The annotation results consist of KO numbers for each gene, genes mapped to KEGG pathway database, and genes mapped to BRITE. Nonetheless, the resulting complete set of pathways and BRITE hierarchy can only be viewed through the temporary URL provided by KEGG, which are only available for several days after the analyses are completed. Although these results are organized through either curated KEGG pathways or BRITE hierarchy, KAAS does not provide an integrative gene-centered view of gene function and pathways, i.e., the complete summary of gene function and all associated molecular pathways for each gene.\n\nAs can be envisioned, integrating the gene function annotation based on KEGG orthology and KEGG pathways can provide an efficient way to characterize both the predicted genes and associated pathways for a newly assembled genome or metagenomics dataset. Despite numerous computational packages for retrieving KEGG pathways using the API interface provided by KEGG database (e.g., Moutselos et al., 2009; Wrzodek et al., 2011), none of these packages to our best knowledge allows us to reconstruct the complete set of molecular pathways contained in a newly assembled genome. To provide a means to utilizing the highly informative resources at KEGG for annotating genomic sequences and molecular pathways for non-model species, we have developed a Gene Annotation Easy Viewer (GAEV) for integrating results of KEGG orthology annotation and KEGG pathways mapping using KEGG API tools in both Windows and Linux environment. GAEV is implemented in Python 3 and can be used as an independent package.\n\n\nMethods\n\nAssuming that the KEGG ortholog number is known for a single gene, the KO information can be retrieved from KEGG database by utilizing KEGG REST-style API. GAEV uses the ‘get’ operation of the KEGG API to extract data on the gene and linked pathways of every K number provided in the input file. The data extracted from KEGG database are stored in data files that can be loaded into GAEV to skip the data extraction step (Figure 1).\n\nOnce data extraction from KEGG’s database is complete and the data file is generated, GAEV helps the user handle and visualize the data by exporting the data as a table in an HTML file. GAEV populates the table with the user defined gene ID provided in the input file and the associated K number provided in the input file, as well as the gene name, definition, and linked pathways that have been retrieved from the KEGG database. The linked pathway map URLs that highlight identified genes in the genome assembly are created using the following formula: http://www.kegg.jp/kegg-bin/show_pathway?map=[mapno]&multi_query=%23bfffbf%0d%0a[k-num1]+ %23bfffbf%0d%0a[k-num2]+... %23bfffbf%0d%0a[k-num_interest]+%23[node_color],%23[font_color].\n\nIn the above URL, [mapno] represents the pathway accession number. [k-num{1,2,3…}] represents the K number for each gene in the pathway that is present in the provided genome assembly, and [k-num_interest] represents the K number of the focal gene that will be highlighted with a unique color. [node_color] and [font_color] represent the desired color of the focal gene’s node and font on the pathway map, respectively. By default, the node color of the focal gene is dark red, whereas the node color of other genes in the same pathway that are present in the genome assembly is light green.\n\n\nUse cases\n\nThe most up-to-date version of this software can be downloaded at https://github.com/UtaDaphniaLab/kegg_path_generator. This software requires Python 3 or newer to run. It is recommended that this software be used as a standalone program simply by double clicking on GAEV.py or by using the ‘python 3 GAEV.py’ command.\n\nWe analyze the newly published Daphnia pulex genome (Ye et al., 2017) to demonstrate the usage of our package. The required input file for our package contains two columns. The first column contains the gene names, whereas the second column represents the KO (KEGG orthology) numbers (Figure 2, Supplementary File 1). The KO numbers for the entire set of genes can be obtained through KEGG Automatic Annotation Server. Briefly, users can provide the query protein sequences in a fasta file and use one of the provided search algorithms (e.g, Blast, GhostX, GhostZ) to assign KO numbers to each of queried genes. At the end of this analysis, the user will receive via email a link to the result page, where the query result can be downloaded. The downloaded query result can be directly used as input file for our package even when some genes are not provided a KO number (which will be automatically excluded from further analysis)\n\nWith the obtained input file, the annotation analysis can be started by simply running GAEV.py and following the instructions of the menus. The first menu provides the option of using the obtained input file to extract data from KEGG or skipping the data extraction step by loading a pre-generated data file. Next, GAEV will prompt the user for the location of the input or data file. Both absolute and relative paths are accepted, but it is recommended that the GAEV.py file be placed in the same folder as the input or data file, so that the relative path can be easily used. After the data extraction from KEGG’s servers is completed, a data file will be created, which can be repeatedly used for making different pathway tables. The next several menus guide the user through the process of customizing the output table. The user has the options to apply filters so that GAEV only outputs a table using genes with a specific keyword in its definition or linked pathways.\n\nThe output file is an html file that can be opened in any internet browser (for example see Supplementary File 2). The results are organized in three different sections. The first section is the Genes and Linked Pathways, where for each query gene the molecular function based on KO and relevant pathways are listed. For each gene, its associated pathway(s) contains a link to the corresponding pathway page on KEGG website, where this specific gene is colored in red and all the identified genes from the genome assembly are colored in green. The other two sections contain a list of the pathways sorted by the number of identified genes and by alphabetic order, respectively. These two sections provide a pathway-centered view of the functions of the annotated genome.\n\n\nConclusions\n\nThe integrative annotation approach implemented in our package GAEV draws upon resources available at KEGG and provides an efficient way to explore the molecular pathways embodied in a draft genome. The integration of the generated html file with KEGG web services provides an intuitive interface to explore specific molecular pathways, with all the identified KEGG homologs highlighted in the pathway map. This type of information is essential to initial exploration of non-model organisms’ genomes to understand the conservation of specific pathways compared to established model systems. For example, if we examine the circadian rhythm pathway in the Daphnia genome, we see strong conservation between Daphnia and Drosophila, with only 1 gene (i.e., Vri ) in this pathway missing an identified homolog in the Daphnia assembly (Figure 3). Further efforts can be dedicated to verifying the absence of Vri gene in Daphnia genome. The strong conservation of the circadian pathway can greatly aid future efforts in using the freshwater microcrustacean Daphnia to understand the internal clock of aquatic organisms in response to aquatic environments.\n\nIn principle, GAEV can be used for visualizing functions and pathways for gene sets of any scale, ranging from genome-wide data to subsets of genes in a genome. For example, we can use GAEV to visualize the pathways that differentially expressed genes are involved in. Often the large number of differentially expressed genes from RNA-seq experiments prevents clear cataloging of these genes and molecular pathways. Analyzing the genes of interest using our package can provide a quick, integrative view of the genes and affected pathways.\n\nIn summary, with a user-friendly design (e.g., no requirement of UNIX command line experience) in mind, we have developed GAEV to provide a fast, easily accessible summary for KEGG gene annotation results. We expect that GAEV will find its use in many bioinformatic analyses, especially those involving non-model species.\n\n\nData and software availability\n\nSoftware source code available from: https://github.com/UtaDaphniaLab/Gene_Annotation_Easy_Viewer\n\nArchived source code as at time of publication: https://zenodo.org/record/1186291#.WpbWVa6nGUk\n\nLicense: This software is licensed under the MIT license",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is supported by start-up funds from University of Texas at Arlington to SX.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank M. Snyman for testing the software.\n\n\nSupplementary Files\n\nSupplementary File 1. Example input file https://raw.githubusercontent.com/UtaDaphniaLab/Gene_Annotation_Easy_Viewer/master/gene_annotation_easy_viewer/example_input.txt\n\nSupplementary File 2. Example output file https://raw.githubusercontent.com/UtaDaphniaLab/Gene_Annotation_Easy_Viewer/master/gene_annotation_easy_viewer/Example_Output.html\n\n\nReferences\n\nBateman A, Martin MJ, O'Donovan C, et al.: UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017; 45(D1): D158–D169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoratyn GM, Camacho C, Cooper PS, et al.: BLAST: a more efficient report with usability improvements. Nucleic Acids Res. 2013; 41(Web Server issue): W29–W33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCamacho C, Coulouris G, Avagyan V, et al.: BLAST+: architecture and applications. BMC Bioinformatics. 2009; 10: 421. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinn RD, Attwood TK, Babbitt PC, et al.: InterPro in 2017-beyond protein family and domain annotations. Nucleic Acids Res. 2017; 45(D1): D190–D199. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones P, Binns D, Chang HY, et al.: InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014; 30(9): 1236–1240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Furumichi M, Tanabe M, et al.: KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017; 45(D1): D353–D361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Sato Y, Kawashima M, et al.: KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016a; 44(D1): D457–D462. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Sato Y, Morishima K: BlastKOALA and GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome Sequences. J Mol Biol. 2016b; 428(4): 726–731. PubMed Abstract | Publisher Full Text\n\nMistry J, Finn RD, Eddy SR, et al.: Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 2013; 41(12): e121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoutselos K, Kanaris I, Chatziioannou A, et al.: KEGGconverter: a tool for the in-silico modelling of metabolic networks of the KEGG Pathways database. BMC Bioinformatics. 2009; 10: 324. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWrzodek C, Dräger A, Zell A: KEGGtranslator: visualizing and converting the KEGG PATHWAY database to various formats. Bioinformatics. 2011; 27(16): 2314–2315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYe Z, Xu S, Spitze K, et al.: A New Reference Genome Assembly for the Microcrustacean Daphnia pulex. G3 (Bethesda). 2017; 7(5): 1405–1416. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32636",
"date": "16 Apr 2018",
"name": "Fragiskos N Kolisis",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this article present a new tool, compiled as a single Python script, that aids in visualizing the gene annotation analysis results of the webserver-based KEGG services. The function of this tool is to generate an html-based view of the genes analyzed by KEGG tools and the appropriate pathways with which they are linked. The html view provides also an overview of the total existing pathways per associated genes, a task which can be useful for whole genome and metagenome annotation queries.\nThe authors provide a thorough explanation about how the tool works and communicates with KEGG API by generating the appropriate links and exporting the generated data. The authors also provide some test datasets which can be used to generate the results mentioned in the manuscript.\nNevertheless this reviewer considers this endeavour to have already been covered by other bioinformatic tools with which a comparison would be necessary to underline the importance of the new tool. For example tools like MEGAN can provide a thorough investigation of the existing KEGG pathways in a genome/metagenome (although by using an older and not commercial version of the KEGG database). Furthermore MinPath can be also used in combination with KEGG generated datasets in order to provide a similar pathway reconstruction analysis. Maybe the authors could elaborate a little bit more about what makes their tool more suitable than these already published tools.\nMoreover during the Conclusions section the authors do not seem to explain the methodology in order to examine the differences between Daphnia and Drosophila and how that (and similar analyses) can be achieved solely (or more intuitively) by exploiting this particular tool.\nIn general this tool seems like a good addition to a bioinformatic pipeline for genomic or metagenomic analysis but this reviewer thinks that the author must emphasize more on the differences and/or improvements regarding similar tools.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3674",
"date": "28 Jun 2018",
"name": "Trung Huynh",
"role": "Author Response",
"response": "Dear Dr. Kolisis, Thanks very much for your comments on our manuscript. Please see below for how we revised our manuscript to address your concerns. 1. Nevertheless this reviewer considers this endeavour to have already been covered by other bioinformatic tools with which a comparison would be necessary to underline the importance of the new tool. For example tools like MEGAN can provide a thorough investigation of the existing KEGG pathways in a genome/metagenome (although by using an older and not commercial version of the KEGG database). Furthermore MinPath can be also used in combination with KEGG generated datasets in order to provide a similar pathway reconstruction analysis. Response: Our goal with our new tool is to provide a gene-centric view of molecular pathways, where each gene is accompanied by all the pathways where this gene is predicted to play a role. This is different from the purposes of MEGAN and Minpath. We emphasized this idea in the last paragraph of Introduction and drew comparison with MEGAN and Minpath. 2. Moreover during the Conclusions section the authors do not seem to explain the methodology in order to examine the differences between Daphnia and Drosophila and how that (and similar analyses) can be achieved solely (or more intuitively) by exploiting this particular tool. Response: This example of Daphnia circadian pathway is to demonstrate using this tool for initial exploration of non-model organisms’ genomes to understand the conservation of specific pathways compared to established model systems (i.e., Drosophila). The Drosophila circadian pathway is provided through KEGG database. Users can directly examine their interested pathways from the results of GAEV (click on the link in the generated html file) and view the pathways and mapped genes on KEGG website. We provide a brief explanation of how to technically view the pathway on KEGG server in Discussion."
}
]
},
{
"id": "32642",
"date": "02 May 2018",
"name": "Tonia S. Schwartz",
"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 review of the manuscript “Gene Annotation Easy Viewer (GAEV): Integrating KEGG’s Gene Function Annotations and Associated Molecular Pathways” by Huynh and Xu. The authors present a method via a python script that can take KEGG IDs and output three tables of useful annotation information in the context of the molecular pathways.\n\nThe manuscript is well written and is easy to read. I envision that the tool developed will be useful, although see the comments below. With corrections I think this manuscript will be a good contribution to the resources available to researchers working in genomics/transcriptomics in non-model organisms.\n\nMajor Comments Although I can start the python script running using python3, the example_input.txt file cannot be found when I enter the relative path or the absolute path. This is despite the input file being in the same folder as the script. My bioinformatics technician also encountered the same problem. Thus it seems there is an error in the script that would need to be corrected prior to acceptance of the manuscript.\n\nMinor Comments This tool is presented as accessible and user-friendly, and the authors use the example from Ye et al. 2017 of “the query protein sequences in a fasta file”. But a protein sequence file is not provided with that paper (that I could find). Thus there an assumed knowledge gap that your reader needs to be able to use this tool as you describe. I suggest you provide additional information on how users can go from a genome with predicted genes (i.e. a .gtf or .gff3 file) to obtain those query sequences for input into the KEGG.\n\nYou may also want to mention that researchers working with non-model organisms could start with a de novo transcriptome or as an input file.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3675",
"date": "28 Jun 2018",
"name": "Trung Huynh",
"role": "Author Response",
"response": "Dear Dr. Schwartz, Thanks very much for your comments on our manuscript. Please see below for how we revised our manuscript to address your concerns. 1. Although I can start the python script running using python3, the example_input.txt file cannot be found when I enter the relative path or the absolute path. This is despite the input file being in the same folder as the script. My bioinformatics technician also encountered the same problem. Thus it seems there is an error in the script that would need to be corrected prior to acceptance of the manuscript. Response: We have found the bug in our code that prevented the script from finding files on Linux and Mac OS properly. This bug has been fixed as of version 1.1.1. 2. This tool is presented as accessible and user-friendly, and the authors use the example from Ye et al. 2017 of “the query protein sequences in a fasta file”. But a protein sequence file is not provided with that paper (that I could find). Thus there an assumed knowledge gap that your reader needs to be able to use this tool as you describe. I suggest you provide additional information on how users can go from a genome with predicted genes (i.e. a .gtf or .gff3 file) to obtain those query sequences for input into the KEGG. Response: We provide a fasta file of Daphnia protein sequence through the github page for GAEV. Moreover, in the manuscript we recommend users to use tools such as gff2sequence to create query sequences using information from gtf/gff files. See 2nd paragraph of Use Cases. 3. You may also want to mention that researchers working with non-model organisms could start with a de novo transcriptome or as an input file. Response: Addressed accordingly. See 2nd paragraph of Use Cases."
}
]
}
] | 1
|
https://f1000research.com/articles/7-416
|
https://f1000research.com/articles/8-614/v1
|
03 May 19
|
{
"type": "Research Article",
"title": "The health, economic, and social effects of cannabis use in Thailand",
"authors": [
"Sujitta Ritmontree",
"Manop Kanato",
"Poonrut Leyatikul",
"Sujitta Ritmontree",
"Poonrut Leyatikul"
],
"abstract": "Background: Controversy surrounds the harm and benefit of cannabis use. Further research on the impact of cannabis might guide the government in developing appropriate policies. This research aims to examine the health, economic, and social effects of cannabis use in Thailand. Methods: From a prospective cohort of 261 cannabis users in Kalasin province, Thailand, we followed 45 cannabis users over 1 year as part of an in-depth study. Quantitative and qualitative data on the health, social, and economic consequences of use were gathered. In-depth interviews, participant observation by researchers during home visits , and self-report instruments were utilized. To supplement the cannabis users’ data, we also collected data from 10 health personnel, 16 community leaders, and 480 laypeople.\n\nResults: Our results indicate that cannabis use causes health problems. We determined the disability-adjusted life years of cannabis users and found a total loss of 120.09 years and a mean loss of 0.78 years. The possible economic impacts of cannabis treatment include medical expenses, loss of revenue for both cannabis users and their caregivers, and costs due to law enforcement and possible lawsuits. The economic costs measured during the study period totaled 1,561,460 baht, of which 1,347,950 was attributed to the costs of law enforcement and legal prosecution. However, we found no costs due to accidental losses. Cannabis does appear to not cause community conflict or crime. Despite this, cannabis use remains a social problem, and has been associated with outbreaks of illegal drug use. Conclusions: This study showed that using cannabis can harm users, their family, and society as a whole. This should be essential for policy advocacy.",
"keywords": [
"cannabis",
"law enforcement",
"Thailand",
"disability-adjusted life years",
"medical expenses",
"crime",
"illegal drug use"
],
"content": "Introduction\n\nCannabis is the most commonly used illegal drug worldwide, with the fastest growing rate of use1. This may be due to growing research on the medical uses of cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC), both of which are found in cannabis plants and can be used to treat various medical diseases, including glaucoma2,3 and epilepsy4. In addition, cannabis can be used to treat pain and reduce nausea in patients with cancer receiving chemotherapy or to increase the appetite of HIV patients5,6.\n\nHowever, THC also binds to the brain’s cannabinoid receptors, interfering with the body’s natural cannabinoid system, resulting in possible euphoria, anxiety, hallucinations, or delusions7. For example, cannabis users are at least four times as likely to get in an accident while driving8,9. In addition, among employed workers, those using illicit drugs (including cannabis) are more likely to be fired10.\n\nCannabis has been recognized as a food ingredient and traditional herb throughout the history of Thailand, although it has been outlawed since 193411. Although cannabis is illegal, the number of cannabis users is increasing throughout the country12. Increasingly, they are calling for the legalization of cannabis. Revision of such national policies, however, requires a considerable evidence base that should be taken into account. Thus, the health, economic, and social impacts of using cannabis should be explored. Such data could be of considerable benefit to policymakers as well as academics.\n\n\nMethods\n\nThis was a prospective cohort study design. Kalasin province in the northeast of Thailand was selected as the study site because it is a major area of cannabis production.\n\nA total of 261 cannabis users registered as patients in hospitals. Registered cannabis users in 2014 who settled in Kalasin province, a province chosen randomly from those with the greatest cannabis production in Thailand, were identified as the target population for this study, and were approached to participate in person at an appointment. All 261 agreed and gave their written consent to participate in a study on the burden of disease in April 2015. Of these, 58 cannabis users agreed and gave their written consent to participate in an in-depth study. These individuals gave their private telephone number to the research team and allowed the research team to visit them at home in April 2015. After 1 year, 13 cannabis users moved to other provinces or had lost contact with the research team. Thus, 45 cannabis users remained in April 2016. These 45 individuals lived in 16 communities in 10 districts in Kalasin province.\n\nBesides cannabis users, 16 community leaders were recruited to provide information about community demographics and drug use. In addition, 480 laypeople from 16 communities (30 each of both sexes in various age groups) agreed to answer the Thai Addiction Stigma Scale13 (for comparison with the cannabis users). All participants were recruited in April to May 2016.\n\nIn this study, investigators and research assistants were essential. One investigator had a PhD and over 20 years of experience in drug research, one had a PhD and 10 years of experience in drug research, and one had a master’s degree with 5 years of experience in qualitative research. Assistance was provided by health officers who have worked with target communities for over 10 years.\n\nA psychiatric nurse with a master’s degree and over 20 years of experience working with drug addicts, developed three in-depth interview guidelines (for healthcare personnel, community leaders, and cannabis users). Diaries were created for cannabis users to self-report signs/symptoms, healthcare activities, healthcare and related expenditures, and cannabis-related expenditures. The Thai Addiction Stigma Scale (content validity index = 0.97, Cronbach’s alpha = 0.77)13 was used to gather data from the laypeople. This scale had a total score ranging from 16 to 120 and comprised 5 subscales: familiarity, risk perception, fear, social distance, and community response. Furthermore, data on burden of disease among cannabis users were gathered using the interview guidelines developed by Loeiyood14.\n\nIn April 2015, data on medical history, cannabis use patterns, and personal characteristics were collected from cannabis users though in-depth interviews. A total of 261 cannabis users who gave their consent were interviewed at their home, without anyone else present, after their regular appointment at a hospital. At each appointment, cannabis users provided 30–60 minutes for the research team to interview them. When we did not reach data saturation for a particular participant, we asked participants to take part in another interview at their next appointment and repeated this until the data were saturated (ranging from 1 to 3 times). We did not use voice or video recording or take photographs of the interviews or users. However, notes were taken during the interview. For the 58 cannabis users who consented to the in-depth study, the research team visited them at home for another private interview and participant observation during daily activities on a monthly basis, half a day each. Again, recording devices were not used. Diaries were given to each cannabis user to enable them to record data on the symptoms and expenditures of cannabis use. This diary was exchanged with a researcher once per month based on appointments until April 2016. During the monthly appointments, cannabis users were asked to clarify what they had recorded in the diary and were interviewed in more detail. During the fieldwork period, we interviewed 10 healthcare personnel and 16 community leaders. The Thai Addiction Stigma Scale was administered to the laypeople in early 2016. On cannabis users’ last appointment, around April 2016, they were interviewed about the burden of disease and their medical history in the past year.\n\nData from cannabis users’ diaries and interviews were triangulated with data from the health personnel and community leaders. Content analysis was employed for the interview data, while the health events in the diaries were tallied. To evaluate the burden of disease, we calculated users’ disability-adjusted life years (DALYs) from their 1-year medical history. The direct economic costs, both public (e.g., hospital expenses for – medication, supplies, admission) and private (e.g., food, transportation, and others), of cannabis use were tallied. Furthermore, we calculated the economic losses based on individual non-working hours/days. The costs incurred from other cannabis-related events were also tallied.\n\nOnce data were collected from the participants, the researchers used data coding to categorized information. Data analysis was conducted by considering the essence of the content of the health, economic, and social effects of cannabis use in Thailand.\n\nDuring the data collection stage, participants provided their written informed consent. Personal identifiers (names, full addresses) were stripped from the dataset. This research project was approved by the Human Research Ethical Committee of Khon Kaen University (HE 571473) based on the principles of the Declaration of Helsinki and the Good Clinical Practice standards of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).\n\n\nResults\n\nThe 261 cannabis users we initially selected made 2,698 visits to healthcare facilities in the past 12 months due to illness. Furthermore, 150 out of these 261 users visited the hospital due to cannabis-related illness. Approximately 150 users reported psychiatric problems and 3 experienced multi-drug use. A total of 14 users had road accidents, 6 users were injured by others, and 1 user made a suicide attempt.\n\nOf the 45 participants who completed the in-depth study, all were male and had an average age of 43.51 years. The majority were married (55.53%) and had primary degrees (53.33%), and 37.77% worked as farmers. For most respondents, alcohol was their first drug (53.33%), and they later moved to cannabis. The average age at which participants first used cannabis was 19.02 years. The primary method of cannabis use was bamboo pipes. Participants had used cannabis for an average of 26.86 years. All of them perceived that they were addicted to cannabis. The most common frequency of use was three times per day (42.22%), and the average amount was about 1 g per use.\n\nWhen researchers followed up with 45 participants after one year, they examined the health, economic, and social effects of cannabis. While none of the users died over the study period, there was a considerable loss of DALYs, at 120.09, with a mean loss of 0.78. Interestingly, among the three users of multiple drugs, cannabis was not the gateway—they had used other drugs before cannabis\n\nAlthough a number of cannabis users had received treatment for illness over the study year, they generally perceived themselves to be of good health. Of the 45 respondents, 3 reported that they had a preexisting medical condition (one had diabetes and two had epilepsy). One participant told researchers:\n\n“My mother and brother are diabetic. 15 years ago, I went to the hospital and the doctors told me that I was diabetic. An elderly man told me that cannabis can be used to treat diabetes. I tried cannabis and have used it since then. When I went to the hospital last month, the doctors said that I had normal blood sugar.”\n\nOverall, 89.77% of participants viewed themselves as in good health. For example, one respondent said:\n\n“I never get sick. I check on my health every year. I do not have diabetes or blood pressure issues.”\n\nIn addition, 87.33% users reported that they did not believe they were abusing drugs, despite their continuous cannabis use. For example, respondents said:\n\n“I can repay everyone. People who use cannabis alone will be healthier. However, if cannabis is used in combination with other drugs like alcohol, then people might be exposed to certain health risks.”\n\n“Most old people in the past, they smoked cannabis, and did not see anyone as crazy or schizophrenic. In contrast, it’s good for smokers...good health and longevity.”\n\nOf the 45 cannabis users, 17 reported contact with law enforcement since using cannabis is illegal in Thailand. Using data from the self-report diary, we analyzed the private costs of cannabis use, and found that economic losses tended to result from law enforcement—both direct costs (e.g., fines, lawyer fees, transportation) and indirect costs (for both the self and relatives, e.g., production losses, litigation, and accidental losses such as compensation for accident and injury and related costs).\n\nThe total medical costs for the cannabis users, including the treatment of addiction, amounted to 142,560 baht (4,537 USD), with an average of 3,168 baht (101 USD) per case. Meanwhile, the total of loss of income, other than prosecution amounted to 32,500 baht (1035 USD), with average of 723 bath (23 USD) per case.\n\nThe total costs related to law enforcement and prosecution litigation amounted to 1,445,330 baht (44,267 USD), with an average of 85,019 baht (2,604 USD) per case (range: 12.3–5997.1 USD). Of these total costs, direct costs for litigation (e.g., fines, lawyer fees, bailout) accounted for 1,230,000 baht (39,145 USD), or about 85.1% of all private costs, with an average of 72,353 baht (2,303 USD) per case (range: 0–5,360 USD). The indirect cost accounted for 14.9% of the total costs, and included loss of income during prosecution (82,266 baht or 2,519.6 USD) and caregiver expenses (133,064 baht or 4,075.5 USD), which included transportation/food/accommodation costs during prosecution (102,900 baht or 3,151.6 USD) and loss of caregiver income during prosecution (30,614 baht or 923.9 USD). In addition, the loss of income due to prosecution process was 38,450 baht (1212 USD), an average of 854 baht (27 USD) per case.\n\nInterestingly, we found no costs associated with accidents or property destruction due to drug-related accidents. This may be because when individuals use cannabis, they are careful to keep to themselves or in small groups. Cannabis users do not drive and are often not social while under the influence, as demonstrated by the following responses:\n\n“Since using cannabis, I have never had an accident. I have never heard of anyone using cannabis having an accident. When you smoke cannabis, it puts you in a relaxing, calm mood and you are careful to smoke it by yourself…”\n\n“Do not connect accidents with cannabis use. Cannabis does not cause impulsive moods and thoughts like amphetamines or alcohol.”\n\nSocial impact in this study was defined as stigma related to cannabis. The 45 cannabis users lived in 16 communities. Within these 16 communities, the addiction stigma score of the 480 laypeople was analyzed. We found a mean stigma score of 84.23 with an SD of 13.44 (95% CI [83.15, 85.31]) and a range of 50 to 111 score. This indicates a moderate level of addiction.\n\nAddiction stigma reflects the negative perception of drug use among community members, and scale scores reflect the degree to which community members attribute various problems in their communities to drug users, including cannabis users. Cannabis users, on the other hand, perceived that cannabis use itself did not cause these social problems, nor did it have an impact on their work or the likelihood of crime, as shown by the following responses:\n\n“I am a hairdresser. Every day, when I smoke cannabis in the morning, I want to do good work and I do it. After work, I smoke in the evening, then go to bed, never disturbing anyone.”\n\n“I have heard rumors that cannabis users are aggressive and assault other people. But no, cannabis puts you in a good mood. I do not want to argue with anyone. It is difficult to find the time to smoke, and it is rare to have a chance to smell the smoke. If you know which family members use cannabis, you will see that these family members do not quarrel. Unless maybe the quarrel was about the use of illegal cannabis, the fear of being arrested, or the fear that others knew they were using drugs.”\n\n“The villagers know who uses cannabis, and they are not offended by it. Cannabis users do not fight and annoy people. But we think that people in the community hate us because it’s illegal. The community thinks that people who use drugs are bad people. The cannabis community has been branded as a drug community . . . if cannabis were legal like cigarettes, the community would not mind. I would not have come to such a dark place.”\n\n\nDiscussion\n\nThe study analyzed the effects of cannabis use and found that cannabis is not a gateway to the use of other additive drugs. These results are in contrast to those of previous studies15.\n\nParticipants that reported using cannabis medically might not use it over the long-term, but still reported a change in their perceived health status. Although this shift may be gradual, it reflects the findings of Lev-Ran et al., who conducted a literature review of 14 studies and found that cannabis users have a 17% lower risk of developing depression than do non-users; in people who use large amounts of cannabis this number increases to a 62% lower risk of depression16.\n\nOur study showed that cannabis use has a notable economic impact in terms of the government’s incurred costs regarding medical treatment, loss of income (for the user and caregiver), and law enforcement and prosecution costs for cannabis-related offenses. Our findings of unemployment of cannabis use are similar to those from a previous study. Past-year job loss preceded past-moth cannabis use, among those who were not using cannabis previously. Moreover, the prior cannabis use presented as a risk factor for unemployment10. In contrast, our findings differ from those of previous studies in that we did not find any costs associated with accidents. In the United States, for example, in 2006, more than one third of motorists who suffer a fatal road accident used cannabis either before or during their drive9. However, our results indicate that cannabis users are self-conscious of their impairment and do not use the drug before or during a drive.\n\n\nConclusions\n\nCannabis use led to a pronounced disease burden and considerable economic losses. However, it does not appear to lead to violence or crime within either the family or community. Despite this, the associated stigma of cannabis use might harm the community. Once the community has labelled drugs as an epidemic, the community begin surveilling the drugs much more intensively, thereby disrupting the normal lives of villagers.\n\nThis research admittedly has limitations. This study only included males, and the study period only covered 1 year. In addition, the findings might not be applicable to studies of cannabis use in other locations.\n\n\nData availability\n\nRaw datasets have not been made available at the request of the ethics committee in order to maintain participant confidentiality. Access to the complete raw data can be obtained upon request and with the permission of Ethics Committee of Khon Kaen University (www.ekku.ac.th); since all data were collected in Thai, all data including quotes are only available in Thai. Anyone wishing to access the data should first contact the corresponding author who will facilitate contact with the ethical review board (Contact email: manopkanato@gmail.com). Access will be granted to researchers that wish to use the data for grant applications or similar.",
"appendix": "Grant information\n\nThe authors would like to thank the Office of the Narcotics Control Board (ONCB75/2558) for funding support of this research project.\n\n\nAcknowledgements\n\nGrateful acknowledgement is made to the ISAN Academic Network, Khon Kaen University, for generous support for facilities.\n\n\nReferences\n\nUnited Nations Office on Drugs and Crime: World Drug Report 2013. Vienna: UNODOC; 2013. Reference Source\n\nMerritt JC, Crawford WJ, Alexander PC, et al.: Effect of marihuana on intraocular and blood pressure in glaucoma. Ophthalmology. 1980; 87(3): 222–8. PubMed Abstract | Publisher Full Text\n\nMerritt JC, Olsen JL, Armstrong JR, et al.: Topical delta 9-tetrahydrocannabinol in hypertensive glaucomas. J Pharm Pharmacol. 1981; 33(1): 40–1. PubMed Abstract | Publisher Full Text\n\nCunha JM, Carlini EA, Pereira AE, et al.: Chronic administration of cannabidiol to healthy volunteers and epileptic patients. Pharmacology. 1980; 21(3): 175–85. PubMed Abstract | Publisher Full Text\n\nMechoulam R, Parker LA, Gallily R: Cannabidiol: an overview of some pharmacological aspects. J Clin Pharmacol. 2002; 42(S1): 11S–19S. PubMed Abstract | Publisher Full Text\n\nCarlini EA: The good and the bad effects of (-) trans-delta-9-tetrahydrocannabinol (Delta9-THC) on humans. Toxicon. 2004; 44(4): 461–7. PubMed Abstract | Publisher Full Text\n\nRadhakrishnan R, Wilkinson ST, D’Souza DC: Gone to Pot - A Review of the Association between Cannabis and Psychosis. Front Psychiatry. 2014; 5: 54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAsbridge M, Mann R, Cusimano MD, et al.: Cannabis and traffic collision risk: findings from a case-crossover study of injured drivers presenting to emergency departments. Int J Public Health. 2014; 59(2): 395–404. PubMed Abstract | Publisher Full Text\n\nMadras BK: Update of Cannabis and Its Medical Use. Boston, MA: Department of Psychiatry, Harvard Medical School; 2015. Reference Source\n\nCompton WM, Gfroerer J, Conway KP, et al.: Unemployment and substance outcomes in the United States 2002-2010. Drug Alcohol Depend. 2014; 142: 350–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanato M, Leyatikul P: Evaluation of Cannabis decriminalization. Bangkok: Office of the Narcotics Control Board; 2015.\n\nKanato M: Cannabis situation in the years 2002-2011 (in Thai). In: Thaikha K, Areesantichai J, Saingam D, et al., editors. The Synthesis of the Situation of Drugs in the Year 2002-2012. Bangkok: Office of the Narcotics Control Board; 2013; 1–14.\n\nKanato M, Leyatikul P: Thai Addiction Stigma Scale. Commun Health Develop Q. 2014; 2(2): 1–18.\n\nLoeiyood J: Lost Years and Economic Impact Society of Drug Use Khon Kaen (in Thai). Khon Kaen: Graduate School, Khon Kaen University; 2017.\n\nFergusson DM, Boden JM, Horwood LJ: Cannabis use and other illicit drug use: testing the cannabis gateway hypothesis. Addiction. 2006; 101(4): 556–69. PubMed Abstract | Publisher Full Text\n\nLev-Ran S, Roerecke M, Le Foll B, et al.: The association between cannabis use and depression: a systematic review and meta-analysis of longitudinal studies. Psychol Med. 2014; 44(4): 797–810. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "53644",
"date": "14 Oct 2019",
"name": "Jurgen Rehm",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe following points should be clarified before I would consider this article to be “scientifically sound”.\nCan we please give details for the following statement: Although cannabis is illegal, the number of cannabis users is increasing throughout the country12. Please indicate also how solid the underlying data are. To my knowledge, 0.2% of the Thai general population used cannabis in the last year (Angkurawaranon et al., 20181), and it would be very hard to be sure about increases in use.\n\nThe original sampling is not at all clear. What is a “registered user” in Thailand? Registered where? Please indicate exactly this process and what potential biases it may introduce. It is also not clear if these people were medical cannabis users or not.\n\nThe biggest problem seems to me that it is not at all clear for which group the results are representative or typical? Less than 20% of the original sample was re-interviewed, and it is not even clear what the original sample stood for. So the discussion needs to focus on this question way better. Who were the people who consented to follow-up and why?\n\nThen, the role of stigma, which may be the most important part of the study, should be clarified. The used scale needs to be described in greater detail, and the results need to be better contextualized.\n\nNo causal language should be used. The authors report some perceived associations, and this should be described as such.\n\nWith respect to the gateway drug, if I am not mistaken, the conclusions are based on N=3 cases. Much more caution in the interpretation is necessary.\n\nThe authors speak about cannabis-related disease. Please define, what disease groups were considered as cannabis related.\n\nGiven all of the above, the authors should make a detailed limitations section in the discussion and based on this state, what conclusions can be drawn from the study with what uncertainty.\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": "60540",
"date": "10 Mar 2020",
"name": "Jakob Manthey",
"expertise": [
"Reviewer Expertise Epidemiology",
"public health",
"cannabis and alcohol use and disorders."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall: This is an important contribution for the discussion around the regulation of cannabis in Thailand. However, the paper has several major problems and should be thoroughly revised.\nPoint-by-point:\n\nThe abstract uses causal language (“Cannabis does appear to not cause community conflict or crime”), which is inappropriate given the study design and sample. \"cannabis use […] has been associated with outbreaks of illegal drug use” is absolutely not a finding of this study and should be removed altogether.\n\nI am not sure if cannabis use still has the steepest increases globally or if cocaine is leading. In any way, the most recent UNODC World Drug Report should be cited. The second sentence is not logically connected to the first, as medical use is legal in many countries and thus not driving illegal use rates. I would recommend to distinguish between recreational and medical use instead. “In addition, among employed workers, those using illicit drugs (including cannabis) are more likely to be fired”. I imagine this is more due to the legal status rather than attributable to cannabis use.\n\nIt should be stated whether Kalasin is renowned for illegal or legal, i.e. medicinal, cannabis cultivation. It is unclear what kind of patients were included in the study. Were they admitted to hospital because of cannabis use problems? What kind of diagnoses did they meet? The term “cannabis users” seems inappropriate, at least for the smaller sample of 45 persons, as they all perceive to be addicted to cannabis. It is unclear how many of the original sample have been asked to participate in the in-depth study. Refusal rates should be reported. “Laypeople” should be defined. I suppose this means a general population sample of non-users? It is not specified how healthcare personnel was recruited. \"symptoms […] of cannabis use” is incorrect and leads to a medicalization and stigmatization of use per se. See also comment above if the study refers to users or persons with CUD. The content of the diary should be specified in more detail and the length of assessment is currently missing. It is not clear to me how exactly the burden of disease, i.e. DALYs, were calculated. An example and reference for calculation should be given. It is not clear to me how exactly economic losses were calculated. What was the reference for calculating losses (e.g. no illness/always working)? I suggest to align terminology with the health economics literature, referring to direct (e.g. medical) and indirect (e.g. productivity losses) costs. Also, state that intangible costs were not assessed. I understand that a wealth of information was collected in the study and it is hard to summarize all data analyses but this section is really slim and insufficient, making it impossible to reproduce this study. I don’t understand what data were collected from community leaders and healthcare professionals. How were they analysed?\n\nGenerally, I am missing estimates of uncertainty for all results. Further, I would recommend to add descriptive tables for each section (demographics, health, economics, social). An average of 1g per use occasion is quite unusual for Western countries. If available, please state the average THC content in available cannabis products in Thailand. A “mean loss of 0.78” is not clear and should be specified. Interestingly, …” This should be removed as it is judgemental and actually, before the background of evidence regarding gateway drugs, not surprising. As extensive data on medical records were collected, I would expect more data presented in the paper, at least in a form of a descriptive table. For the economic impact section, the corresponding time frame and the specific sample (all 261 or 45 participants) should be explicitly mentioned. It should be reported how many of the persons had how many legal problems/encounters in which time frame. This would help to contextualize the “economic losses”. If baht are converted to USD, the year of date of conversion should be mentioned. The stigma results cannot be interpreted if no reference was given (e.g., mean point, range, results for other conditions/risk factors). Also, how can a stigma scale be an indicator for addiction? This should be removed altogether: “This indicates a moderate level of addiction.”\n\n\"The study analyzed the effects of cannabis use and found that cannabis is not a gateway to the use of other additive drugs”. No, this was not the aim of the study, nor was it really investigated. This sentence is misleading and should be removed. Also, this finding (if it was a real finding) is not in contrast to a large body of research on this topic. Citing the findings from Lev-Ran regarding risk of depression among cannabis users seems out of context. It is not clear why this study was cited here. Instead, results of the study should be discussed in context of the literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-614
|
https://f1000research.com/articles/6-2060/v1
|
29 Nov 17
|
{
"type": "Method Article",
"title": "META-pipe cloud setup and execution",
"authors": [
"Aleksandr Agafonov",
"Kimmo Mattila",
"Cuong Duong Tuan",
"Lars Tiede",
"Inge Alexander Raknes",
"Lars Ailo Bongo",
"Aleksandr Agafonov",
"Kimmo Mattila",
"Cuong Duong Tuan",
"Lars Tiede",
"Inge Alexander Raknes"
],
"abstract": "META-pipe is a complete service for the analysis of marine metagenomic data. It provides assembly of high-throughput sequence data, functional annotation of predicted genes, and taxonomic profiling. The functional annotation is computationally demanding and is therefore currently run on a high-performance computing cluster in Norway. However, additional compute resources are necessary to open the service to all ELIXIR users. We describe our approach for setting up and executing the functional analysis of META-pipe on additional academic and commercial clouds. Our goal is to provide a powerful analysis service that is easy to use and to maintain. Our design therefore uses a distributed architecture where we combine central servers with multiple distributed backends that execute the computationally intensive jobs. We believe our experiences developing and operating META-pipe provides a useful model for others that plan to provide a portal based data analysis service in ELIXIR and other organizations with geographically distributed compute and storage resources.",
"keywords": [
"ELIXIR",
"Portability",
"META-pipe",
"OpenStack",
"EGI Federated Cloud",
"Amazon Web Services",
"AAI federation",
"Apache Spark"
],
"content": "Introduction\n\nELIXIR was established to unite European life science resources. It has 21 member states and more than 180 research organizations that each take responsibility for an analysis service, database, software tool, training material, or provide cloud storage and compute resources. For example, one of the deliveries from ELIXIR Norway are marine metagenomics analysis services, whereas ELIXIR Finland provides cloud storage and compute resources. An ELIXIR user from Portugal may therefore use a service maintained in Norway run on resources in Finland. In this paper, we describe our approach for setting up distributed execution of such analyses.\n\nMETA-pipe1 is a complete workflow for the analysis of marine metagenomic data. It provides assembly of high-throughput sequence data, functional annotation of predicted genes, and taxonomic profiling. We provide META-pipe as an Analysis-as-a-Service for Norwegian and Finnish ELIXIR users. However, additional compute resources are necessary to open the service to all ELIXIR users. Users log into the META-pipe web application where they can upload data to analyze, select tool parameters, start analyses, and download analysis results. The functional annotation is computationally demanding and must therefore be run on a high-performance computing (HPC) cluster or a compute cloud. Job execution is handled by the META-pipe backend, such that resource allocation, parallel execution, and fault handling is hidden from the user.\n\nMETA-pipe has a distributed architecture with three central servers and geographically distributed execution managers (Figure 1). We have currently four META-pipe execution managers: (i) the Sigma2 Stallo supercomputer in Tromsø, which is a Norwegian academic HPC; (ii) the CSC cPouta OpenStack based Infrastructure-as-a-Service cloud, Finland; (iii) the CESNET-MetaCloud OpenNebula cloud that supports the open cloud computing interface, Czech Republic; and (iv) the commercial Amazon EMR cloud service. cPouta is an ELIXIR compute service. CESNET-MetaCloud is part of the EGI Federated Cloud.\n\nThe authorization server, which is integrated with the ELIXIR AAI, enables login for Elixir users. The storage server stores all META-pipe input, output and provenance data. The job server schedules and maintains submitted analysis jobs. The jobs are implemented as Spark programs that are executed by an execution manager running in an execution environment. There can be multiple execution managers distributed over many HPC clusters and clouds.\n\nAn important design goal for the META-pipe backend is to make the execution mangers portable. In addition, we have taken care to make setup and maintenance of the execution mangers easy. We achieve these goals since our backend is designed such that all state is maintained at the central servers. We also use the widely available Apache Spark framework to execute the pipeline analyses. The execution managers are stateless and the jobs are idempotent. In addition, we have implemented tools that make it easy to setup and administer the execution mangers. In this paper, we describe these tools and their use. We make the following contributions:\n\n1. We demonstrate the use of geographically distributed compute resources for life science data analysis.\n\n2. We describe the design and implementation of cloud setup tools for our analysis service.\n\n3. We describe our experiences developing and operating the META-pipe analysis service.\n\nWe designed our analysis service to be powerful, easy to use, and easy to maintain. We believe our work provides a useful model for others that plan to provide a portal based data analysis service in ELIXIR and other organizations with geographically distributed compute and storage resources.\n\n\nMethods\n\nThe META-pipe is deployed as shown in Figure 2. META-pipe is implemented as a Spark program and requires Spark v1.6.1 or v1.6.2. Spark and META-pipe executable require Scala v2.10.6. In addition, the META-pipe execution environment requires the Java v1.8 OpenJDK. Here we describe how META-pipe is setup on the cPouta cloud. The setup on other clouds may differ as described below. Additional details, including instructions for using the cluster setup tool are in the META-pipe cloud setup design document.\n\nEnd-users run analyses using the META-pipe web app. The web app is integrated with ELIXIR AAI, so users can authenticate using their home institution username and password. Resource providers use the cluster setup tool to setup an execution manager, on, for example, the cPouta OpenStack cloud, which executes analysis jobs. The execution manger, pipeline, and dependencies are all read from an artifacts server. META-pipe developers use git to maintain the code. Our GitLab is integrated with Jenkins that compiles and runs integration tests and pushes new META-pipe versions to the artifacts server. META-pipe administrators administer all jobs using the META-pipe Job manger interface.\n\nA META-pipe execution environment has three types of virtual machines (VMs):\n\nBastion VM: acts as the gateway machine used for cluster management and gateway to an initiated cluster.\n\nCluster Master VM: acts as Spark Master, NFS Server, and it runs the main META-pipe executable.\n\nCluster Worker VMs: act as Spark Workers, NFS Clients, and the runners of parallelized tasks of META-pipe jobs.\n\nIn addition, META-pipe requires the use of NFS-shared storage used by the worker VMs to read and write temporary computation data including intermediate result files. The master VM contains a NFS-server that serves the access to the storage. The worker VMs are NFS-clients that have full read-write access to the shared storage. The NFS server also has the Spark and Scala installations. The NFS storage can be either a Master VM internal volume, or a virtual volume attached to the Master. We typically use the latter, since it makes deployment easier and it allows META-pipe volume caching (as described below). We have, however, not compared the performance of these two approaches.\n\nThe META-pipe executable is downloaded from our artifacts server. The executable is a jar file that contains the Spark job, and it is submitted as a Spark job from the Master VM. The executable jar does not contain 3rd party tools, tool libraries, and the reference databases used by the tools. These dependencies must be downloaded from the artifacts server. Each worker VM must have access to the META-pipe dependencies, so they are typically stored locally on each cluster VM. If there are not enough volume resources, NFS storage is used.\n\nAfter setting up the Spark, a simple parallelized script should be used to test that Spark and META-pipe is setup correctly. We first test Spark by submitting a parallelized version of prime number counter, wait for all Workers to be done, and then ensure that there were no error messages and that the result is correct.\n\nTo validate the correctness of META-pipe installation, we use the built-in validation procedure in the META-pipe executable. This procedure will check the state of all tools required and their dependencies that are required for META-pipe execution. It will also check that the tools do not return errors.\n\nAfter initialization, the submitted executable will listen for, and run, new jobs until the spark-submit is stopped. The jobs are submitted to the central META-pipe job server that checks the tag in the job and submits it to a specific META-pipe executable. The executable downloads the input data from the META-pipe storage server to a data structure and launches the META-pipe job using the spark-submit command. When the job is completed, the executor uploads the output datasets to the META-pipe storage server, which are then accessible by the user on META-pipe portal. After the execution of each tool in the pipeline, the intermediate output datasets are also uploaded to the storage server, so that if the job fails, it can be restarted from the last successful pipeline step.\n\nTo setup the META-pipe execution manager on cPouta we created an execution manager setup tool that setups the virtual machines, storage, Spark, and META-pipe as described above. It is implemented as a command line tool written in Java, with some parts implemented in Bash, Ansible and Python. The tool’s requirements, usage information, as well as more detailed technical information is in our design document.\n\nWe have optimized cluster provision by caching virtual volumes with the META-pipe execution manager and dependencies. To avoid downloading, unpacking and preparing META-pipe files for each new cluster instance, we store these in a virtual volume the first time a cluster is setup. This volume is used as the storage of prepared META-pipe files (a cache), which is used to create volumes for cluster VMs in later cluster provision. This reduces the time to create a cluster from 30 minutes to 10 minutes.\n\nTo setup the META-pipe execution manager on the CESNET-MetaCloud cloud we adapted the cPouta tool to create a tool that uses the Open Cloud Computing Interface (OCCI). It is therefore compatible with all EGI Federated Clouds, since they all support OCCI. The tool is a rOCCI Client implemented in Python and ansible that uses X509 VOMS certificates. It implements a Terraform OCCI plugin. Additional details are in https://github.com/cduongt/mmg-cluster-setup-CESNET.\n\nThe manager of the server must provide a contextualization file and Terraform configuration file that define the technical features of the virtual cluster. When the launching command is issued the tool first builds the virtual cluster to the given endpoint and then automatically installs the software components and reference datasets to the new virtual cluster as described above.\n\nThe end users submit analysis tasks from the META-pipe web app to the META-pipe backend running in EGI Federated cloud. The end users do not need certificates, Virtual Organization membership or the tools required to launch the META-pipe backend. Instead, the end users just authenticate to the META-pipe web interface using ELIXIR AAI.\n\nTo setup the META-pipe execution manager on Amazon Web Services (AWS), we use the AWS Elastic MapReduce (EMR) console and a custom cluster boot-time script. We use AWS’s EMR managed framework since it natively supports Spark. The cluster setup is therefore simpler than on OpenStack and OpenNebula, but it is not as configurable. For instance, EMR clusters always use the YARN resource allocator and cannot be configured to use Spark’s “standalone” mode instead, which we use on the other platforms. This has not been a big problem in practice, but it constitutes an uncontrollable variable when optimizing the execution for various cloud platforms. A detailed description of the setup is in https://gitlab.com/uit-sfb/metapipe-on-aws. We plan to implement a setup tool like the OpenStack and EGI Federated Cloud tools described above. For automation of EMR cluster setup, AWS offers a comprehensive API and CLI, and CloudFormation.\n\nFor META-pipe, we make AWS EMR clusters on demand. Our clusters use spot instances with VM flavors that provide the best cost-performance for META-pipe (currently we use c4.4xlarge). An EMR cluster boots up with a compatible version of Spark provisioned by Amazon. Our automatically started boot-time script then provisions META-pipe tools and dependencies from an S3 bucket. The whole cluster creation process takes about 10 minutes. Similar to other execution environments, the cluster is idle until we start the META-pipe execution manager on the cluster's master node. The execution manager continually fetches jobs submitted using the META-pipe web app and submits them to Spark on the cluster. We must terminate the cluster ourselves, either by a script through the AWS API or manually in the EMR console.\n\n\nUse cases\n\nHere we describe a use case where the computationally demanding parts of META-pipe are setup to execute on a cloud resource provided for a new user community. The new user group first applies for computational resources from its partner clouds, and then utilize these resources easily through the interface provided by the META-pipe web application. The tools described here makes it easy for a resource provider to administer the META-pipe job executions, and the use of standardized technologies and protocols ensure compatibility and portability of the META-pipe analysis backend across clouds. Below we describe the execution of analysis jobs from the point of view from META-pipe end users, compute resource providers, and META-pipe service providers. We have also described these use cases in an ELIXIR webinar (November 2016) about the ELIXIR compute platform.\n\nThe new user community, represented for example by their national ELIXIR node, have applied and received compute resources from an academic cloud provider. One of the academic users has a marine metagenomics dataset they want to analyze:\n\n1. The user logs into the META-pipe web application using their home institution credentials. The login page is the single sign on provided by ELIXIR AAI. The datasets are typically up to a few GB in size and they can quickly be uploaded using the browser.\n\n2. The user uploads their dataset to be analyzed, and possibly changes some of the analysis parameters. This is done in the META-pipe web app.\n\n3. The user tags the cloud to use for the analysis and submits the job for analysis using the web app GUI.\n\n4. Once computations finish, the data is returned to the portal, and the user can download the enriched results for further analysis or visualization using separate tools such as Krona2, Artemis3, or METAREP4.\n\nThe resource provider must setup the META-pipe execution manager that executes the Spark job that implements the analysis. In addition, the resource provider must test and maintain the execution manager. The tools described in the previous section simplifies this task.\n\nThe first time the META-pipe backend is setup on a cloud environment, the resource administrator needs to edit a configuration file that defines the virtual cluster to be created. After that the administrator runs the following commands in the tool:\n\n1. create-env: to set up the environment and META-pipe files caching volume that will be used in create-cluster.\n\n2. create-cluster: provision cluster resources, install and configure the execution manager (Spark and NFS), install META-pipe tools, dependencies, and reference databases on the provisioned cluster VMs, and test the setup.\n\nTo setup a cluster as second time, only step 2 is run. It will use cached META-pipe volumes created previously.\n\nThen to accept META-pipe jobs from the job server:\n\n3. sw-launch: start Spark and the server that listens for new META-pipe jobs to execute.\n\nTo stop accepting new jobs:\n\n4. sw-kill: stop all META-pipe related processes on all cluster VMs.\n\nTo free the allocated resources:\n\n5. remove-cluster: remove the cluster and keep the environment for future use.\n\n6. remove-env: remove the environment, including cached volumes.\n\nStep 6 is only done when the resource is not intended to be used for META-pipe jobs anymore. Steps 2–5 may be automated.\n\nThe META-pipe team providing the service do not need to make any changes to the central services since the new execution manager is authorized using the META-pipe authorization server, and since the end-user specifies the tag for the new execution manager.\n\nIn April 2017 we used META-pipe in a metagenomics course organized by the Finnish ELIXIR node (https://www.csc.fi/web/training/-/metagenomics also described in the report “EGI-Engage D6.15 Demonstrator for ELIXIR workflows implemented in the EGI Federated cloud“). We setup two META-pipe execution environments. The main execution environment was running in the cPouta cloud environment at CSC, and a backup execution environment that was running in EGI Federated Cloud (CESNET-MetaCloud). These META-pipe backends were set up by the course organizers and so the students did not need to any technical preparations to use the cloud services. Instead, they only needed to define one extra parameter in the web interface to guide their analysis tasks to a specific external META-pipe backend. 42 students participated to the metagenomics course and successfully used these temporary execution environments through the META-pipe web interface without any interference.\n\n\nDiscussion\n\nBioinformatics pipelines can be specified for portable execution in either a popular bioinformatics pipeline (workflow) manager, such as Galaxy5 or Chipster6, or in a standardized language, such as the Common Workflow Language7, that is supported by many pipeline managers including Galaxy and Toil8. META-pipe is implemented in Apache Spark. Spark is widely used for big data processing, and it is supported natively in Amazon Web Services, Microsoft Azure, and Google Cloud Platform.\n\nAnother approach for portable bioinformatics tool execution is to package the tools as containers. Repositories such as BioContainers provides many tools. Several pipeline mangers support containers, including Nextflow9, Toil, Pachyderm, and Luigi10. META-pipe does not use containers, since our artifacts server and the ansible scripts used by the setup tools take care of META-pipe dependencies. In addition, even when using containers there is a need to setup container orchestration for parallel execution on distributed resources. Systems such as Kubernetes and Docker Swarm can be used to orchestrate containers. The META-pipe execution manager use Spark for orchestration.\n\nThe EBI Cloud Portal enables execution of pipelines on cloud resources. Users can sign on using ELIXIR AAI, add their applications, pipelines as virtual machine images, and configure cloud compute and storage resources. We attempt to hide these details to the end users - commercial solutions include platform such as Illumina BaseSpace, where developers can provide apps for analysis on AWS of data uploaded to BaseSpace. Currently, most of the provided apps are single tools instead of complete pipelines, such as META-pipe.\n\nAn important limitation of our approach is that we do not handle resource allocation for end users. Instead users must contact a service provider in their country (or be added to an EGI based VO) to allocate resources and setup an execution environment. This is not something all users know how to do, and it is unnecessarily complicated for small projects. There are four possible solutions. First, for small projects an ELXIR node may provide the computation resources for all users. Second, an ELIXIR node may provide computation resources for all their users. Third, federated cloud resources can be used through EGI Federated Cloud or the ELIXIR federated cloud testbed. Long term usage of federated approach requires that access guarantees (SLAs, OLAs) are arranged. Fourth, the users can allocate and pay for resources from a commercial cloud provider. Such pay-by-use is especially easy for industry users.\n\nWe do not provide a service for predicting resource usage for META-pipe jobs. However, we believe that we can make good estimates based on the input file size. We are also currently evaluating and optimizing META-pipe job execution. An important part of such optimization is to choose the most cost efficient virtual machine flavors and storage solutions on a cloud.\n\n\nConclusion\n\nWe have described our approach for setting up and executing the functional analysis of META-pipe on academic and commercial clouds. To make our analysis service easy to use and to maintain, we use a distributed architecture where we combine central servers with multiple distributed backends that execute the computationally intensive jobs. A key issue in ELIXIR is to obtain computing resources for META-pipe end users. It is still to be decided how resources will be obtained, managed and allocated to the individual end users. For all solutions, an up-to date and highly automatized tool for launching a META-pipe execution environment is needed. We will continue improving the META-pipe backend and to add support for additional resource providers.\n\n\nData and software availability\n\nNo data is needed to use the cloud setup tools.\n\nThe code for the three setup tools are open source at:\n\nCSC cPouta OpenStack cloud: https://gitlab.com/uit-sfb/METApipe-cPouta-cloud-setup.\n\nCESNET-MetaCloud OpenNebula: https://github.com/cduongt/mmg-cluster-setup-CESNET.\n\nAmazon Web Services EMR: https://gitlab.com/uit-sfb/metapipe-on-aws.\n\nArchived code at time of publication for all: http://doi.org/10.5281/zenodo.105380711. All use the MIT license. The above repositories include user guides.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by ELIXIR, The Research Council of Norway (project number 270675), EGI-Engage, and UiT The Arctic University of Norway. ELIXIR received funding from the European Union’s Horizon 2020 research and innovation program (ELIXIR- EXCELERATE, grant agreement no 676559). The EGI-Engage project is co-funded by the European Union (EU) Horizon 2020 program under Grant number 654142.\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\nRobertsen EM, Kahlke T, Raknes IA, et al.: META-pipe - Pipeline Annotation, Analysis and Visualization of Marine Metagenomic Sequence Data. ArXiv160404103 Cs. 2016. Reference Source\n\nOndov BD, Bergman NH, Phillippy AM: Interactive metagenomic visualization in a Web browser. BMC Bioinformatics. 2011; 12: 385. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarver T, Harris SR, Berriman M, et al.: Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics. 2012; 28(4): 464–469. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoll J, Rusch DB, Tanenbaum DM, et al.: METAREP: JCVI metagenomics reports--an open source tool for high-performance comparative metagenomics. Bioinformatics. 2010; 26(20): 2631–2632. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAfgan E, Baker D, van den Beek M, et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016; 44(W1): W3–W10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKallio MA, Tuimala JT, Hupponen T, et al.: Chipster: user-friendly analysis software for microarray and other high-throughput data. BMC Genomics. 2011; 12: 507. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmstutz P, Crusoe MR, Tijanić N, et al.: Common Workflow Language, v1.0. Publisher Full Text\n\nVivian J, Rao AA, Nothaft FA, et al.: Toil enables reproducible, open source, big biomedical data analyses. Nat Biotechnol. 2017; 35(4): 314–316. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDi Tommaso P, Chatzou M, Floden EW, et al.: Nextflow enables reproducible computational workflows. Nat Biotechnol. 2017; 35(4): 316–319. PubMed Abstract | Publisher Full Text\n\nSchulz WL, Durant T, Siddon AJ, et al.: Use of application containers and workflows for genomic data analysis. J Pathol Inform. 2016; 7(1): 53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgafonov A, Mattila K, Tuan CD, et al.: META-pipe Cloud Setup and Execution (Version Tag: Zenodo-F1000). Zenodo. 2017. Data Source"
}
|
[
{
"id": "28995",
"date": "15 Dec 2017",
"name": "Olivier Collin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes the setup of a multi-cloud architecture that can support the execution of META-pipe jobs for marine metagenomics data analysis.\nIt is intended for a technically inclined audience and focuses on the different configuration steps required for the setup of a complex computing architecture involving several clouds in several countries.\nThe purpose is to be able to open the META-pipe service to a large number of ELIXIR users. Several cloud middleware have been configured (Openstack, OpenNebula, Amazon).\n\nThe description of the infrastructure is clear and additional links to github repositories or online documents allow the reader to fetch extra technical information.\n\nRemarks :\n\nIn the introduction, the authors indicate that the design goal is to make execution mangers (sic) portable and that the setup and maintenance of execution mangers (sic) is easy. The authors state that these goals are achieved because all state is maintained on central servers. This section is not completely clear and should be rewritten to highlight the advantages of the central servers architecture. Emphasizing the fact that the META-pipe environment will be pushed from a central server to the execution clouds could clarify things.\n\nIn the chapter concerning the META-pipe executables and dependencies (page 4), it could be interesting to better describe these dependencies. These dependencies are downloaded from the artifact server but no technical information is given about this server (for example : location, size of the downloads). Are the reference databases provided by the artifact server ?\n\nThe infrastructure is articulated around a central server hosting the configuration of META-pipe. This creates a single point of failure. It could be interesting to describe the actions that are taken in order to have a more resilient system.\n\nIn the META-pipe job execution paragraph, the input data is downloaded to a data structure (page 5, col 1, line 18). What is this data structure ?\n\nTypos:\n\nPage 3 / Col 2 / Line 4, 5, 11 : manager/manger\nPage 3 / Col 2 / Line 27 : executables instead of executable\n\nPage 3 / Col 2 / Line 29 : is set up\n\nPage 5 / Col 1 / Line 3 : is set up\n\nPage 5 / Col 1 / Line 27 : to set up\n\nPage 5 / Col 2 / Line 42 : are set up\n\nPage 5 / Col 2 / Line 45 : utilizes\n\nPage 6 / Col 1 / Line 13 : its dataset\n\nPage 6 / Col 1 / Line 23 : set up\n\nPage 6 / Col 1 / Line 28 : set up\n\nPage 6 / Col 1 / Line 38 : To set up a cluster for a second time\n\nPage 6 / Col 2 / Line 19 : We set up\n\nPage 6 / Col 2 / Line 24 : did not need to : Missing word\n\nPage 6 / Col 2 / Line : several pipeline mangers : managers ?\n\nPage 7 / Col 1 / Line 19 : ELIXIR\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3350",
"date": "18 Jan 2018",
"name": "Lars Ailo Bongo",
"role": "Author Response",
"response": "Thank you for your remarks. We have made the following changes in our revised version: We improved the description of the centralized server and the execution environments in the introduction. We added additional details about the dependencies and the artifacts server in “META-pipe executable and dependencies” We now specify that the data is downloaded into a Spark RDD. We have fixed the typos. We agree that centralized servers create a single-point-of-failure, but at the same time it simplifies the implementation of the backend. We are not a stage at the project yet where we believe the improved availability justifies prioritizing this issue."
}
]
},
{
"id": "29076",
"date": "22 Dec 2017",
"name": "Kang Ning",
"expertise": [
"Reviewer Expertise Microbiome"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes the setup of a multi-cloud architecture that can support the execution of META-pipe jobs for marine metagenomic data analysis. And META-pipe provides assembly of high-throughput sequence data, functional annotation of predicted genes, and taxonomic profiling.\n\nRemarks:\nThe introduction of META-pipe backend architecture and deployment is detailed and clear\n\nIt achieves the goal which is to provide a powerful analysis service that is easy to use and to maintain since META-pipe has web app and GUI that the users just set parameters and then they can make analysis of data.\n\nIn the chapter concerning resource provider (page 6), “The tools described in the previous section simplifies this task“ needs more details like names of the tools since there are some tools referred in the previous section. It would be easy to understand.\n\nThere is less example to indicate tasks in META-pipe can be easy and fast. It would be better to give some data like running time and volume to state. Giving other projects differed from this as ‘ control group ’and setting characters to make comparisons will be more persuasive.\n\nThere are some spelling mistakes like ‘manger’.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3349",
"date": "18 Jan 2018",
"name": "Lars Ailo Bongo",
"role": "Author Response",
"response": "Thank you for your remarks. We have specified that the tools used by the resource provider are the execution manager setup tools. Performance and scalability evaluation of META-pipe on different execution environments is important, but outside the scope of this paper. Here we describe the work done before and after executing pipeline jobs. This approach can be used for other bioinformatics pipelines (implemented as Spark jobs). A performance evaluation would require a detailed explanation of the META-pipe tools. To make this point clearer we have added a line in “cPouta Open Stack setup” about META-pipe job execution time being several hours (compared to 10-30 minutes of setup time)."
}
]
}
] | 1
|
https://f1000research.com/articles/6-2060
|
https://f1000research.com/articles/8-612/v1
|
02 May 19
|
{
"type": "Software Tool Article",
"title": "Optimized functional annotation of ChIP-seq data",
"authors": [
"Bohdan B. Khomtchouk",
"William C. Koehler",
"Derek J. Van Booven",
"Claes Wahlestedt",
"William C. Koehler",
"Derek J. Van Booven",
"Claes Wahlestedt"
],
"abstract": "Different ChIP-seq peak callers often produce different output results from the same input. Since different peak callers are known to produce differentially enriched peaks with a large variance in peak length distribution and total peak count, accurately annotating peak lists with their nearest genes can be an arduous process. Functional genomic annotation of histone modification ChIP-seq data can be a particularly challenging task, as chromatin marks that have inherently broad peaks with a diffuse range of signal enrichment (e.g., H3K9me1, H3K27me3) differ significantly from narrow peaks that exhibit a compact and localized enrichment pattern (e.g., H3K4me3, H3K9ac). In addition, varying degrees of tissue-dependent broadness of an epigenetic mark can make it difficult to accurately and reliably link sequencing data to biological function. Thus, there exists an unmet need to develop a software program that can precisely tailor the computational analysis of a ChIP-seq dataset to the specific peak coordinates of the data and its surrounding genomic features. geneXtendeR optimizes the functional annotation of ChIP-seq peaks by exploring relative differences in annotating ChIP-seq peak sets to variable-length gene bodies. In contrast to prior techniques, geneXtendeR considers peak annotations beyond just the closest gene, allowing users to investigate peak summary statistics for the first-closest gene, second-closest gene, ..., nth-closest gene whilst ranking the output according to biologically relevant events and iteratively comparing the fidelity of peak-to-gene overlap across a user-defined range of upstream and downstream extensions on the original boundaries of each gene's coordinates. We tested geneXtendeR on 547 human transcription factor ChIP-seq ENCODE datasets and 198 human histone modification ChIP-seq ENCODE datasets, providing the analysis results as case studies. The geneXtendeR R/Bioconductor package (including detailed introductory vignettes) is available under the GPL-3 Open Source license and is freely available to download from Bioconductor at: https://bioconductor.org/packages/geneXtendeR/",
"keywords": [
"ChIP-seq",
"functional annotation",
"epigenetics"
],
"content": "Introduction\n\nThe field of epigenetic research studies the process by which heritable changes in gene expression occur without underlying alterations in the DNA sequence. Epigenetics plays a key role in human biology, and dysregulation in epigenetic processes is associated with the pathogenesis of cancer and many other diseases. Epigenetic mechanisms have been demonstrated to be necessary for biological programs that are important for a variety of health and disease outcomes. Understanding the impact of epigenetic architecture on the accessibility of gene promoters and its effect on gene expression patterns is therefore critical for linking chromatin biology to clinical indications. One way to measure such events involves investigating histone modifications, namely post-translational modifications to histones (referred to as chromatin marks) that regulate gene expression by organizing the genome into active regions of euchromatin, where DNA is accessible for transcription, or inactive heterochromatin regions, where DNA is more compact and less accessible for transcription1.\n\nChromatin marks come in a variety of different shapes and sizes, ranging from the extremely broad to the extremely narrow2–6. This spectrum depends on a number of biological factors ranging from qualitative characteristics such as tissue-type7 to temporal aspects such as developmental stage8. Depending on the peak caller used, computational factors such as the variance observed in peak coordinate positions (peak start, peak end) – both in terms of length distribution of peaks as well as the total number of peaks called – is an issue that persists even when samples are run at identical default parameter values9,10. This variance becomes a factor when annotating peak lists genome-wide with their nearest genes as peaks can be shifted in genomic position (towards 5’ or 3’ end) or be of different lengths, depending on the peak caller employed. In total, the combined effect of these factors exerts a unique influence over the functional annotation and understanding of genomic variability, which ultimately complicates the study of epigenetic regulation of biological function.\n\nPrior software in the chromatin immunoprecipitation-sequencing (ChIP-seq) functional annotation arena (e.g., ANNOVAR11, GREAT12, PAVIS13, ChIPpeakAnno14, ChIPseeker15, annotatr16, HOMER17, and BEDTools18) has focused exclusively on distance-minimizing algorithms between peaks and the transcriptional start site (TSS) regions of their nearest genes. In contrast, geneXtendeR significantly expands this definition to include n-dimensional annotation, whereby a user can investigate second-closest, third-closest, . . . , nth -closest genes to any given peak (or set of peaks), thereby focusing on and prioritizing the biology over simply the raw numbers (in base pairs). Detailed expositions of these new methods and their implications on the interpretation of results from data analyses are presented as case studies in the geneXtendeR package vignette.\n\ngeneXtendeR19 makes functional annotation of ChIP-seq data more robust and precise, regardless of peak variability attributable to parameter tuning or peak caller algorithmic differences. Since different ChIP-seq peak callers produce differentially enriched peaks with large variance in peak length distribution and total peak count, annotating peak lists with their nearest genes can often be a noisy process where an adjacent second or third-closest gene may constitute a more viable biological candidate, e.g., during cases of linked genes that are located close to each other. As such, the goal of geneXtendeR is to robustly link differentially enriched peaks with their respective genes, thereby aiding experimental follow-up and validation in designing primers for a set of prospective gene candidates during qPCR.\n\n\nMethods\n\nThe key algorithm in the geneXtendeR R/Bioconductor package19 is the extension algorithm, implemented in the C programming language for performance and efficiency. The process of “extending\" refers to performing sequential iterative gene-feature overlaps after adding to the gene-span a user-specified region upstream of the start of the gene model and a fixed (500 bp) region downstream of the gene, resulting in assigning to a gene the features that do not physically overlap with it but are sufficiently close. This process is repeated multiple times across a range of extension parameters set by the user and a series of visualizations are returned as output to help users hone in on the optimal functional annotation. This is in contrast to most past and present epigenetic analyses, in both ChIP-seq20 and ATAC-seq21 studies, that assign gene body definitions (e.g., assigning a default 2 kbp as the cutoff for gene-proximal peaks) ad hoc before mapping the peaks to genomic features. Figure 1 shows why such a practice may be limiting.\n\nLarge-scale computational geneXtendeR analysis using hg19 reference genome of 198 histone modification and 547 transcription factor ChIP-seq datasets from ENCODE. To make data directly comparable to each other, the y-axis represents a normalized count of peak clusters (number of peak clusters in a specific interval divided by the total number of peak clusters across all 0-10 kbp intervals for a given chromatin mark or TF), where a peak cluster is defined as a genomic locus harboring at least 5 overlapping peaks. The x-axis, which is segmented into 20 discrete regions (“1\" = 0-500 bp interval, “2\" = 500-1000 bp interval, ..., “20\" = 9500-10000 bp interval), represents a genomic distance (in bp) of the closest protein-coding gene to each respective peak cluster. A steady decline in peak cluster count at further upstream intervals is detected for all (broad and narrow) chromatin marks as well as transcription factors, i.e., peak clusters do not congregate proximally within any specific region of intervals (e.g., 0-2000 bp) of their respective protein-coding genes, as there is a large number of peak clusters that reside further upstream of their nearest gene. For instance, in the 9500-10000 bp interval alone, there are 1043 peak clusters for the H2AFZ chromatin mark, 569 peak clusters for the H3K4me1 chromatin mark, and 716 peak clusters across all transcription factor ChIP-seq datasets. However, there are certainly exceptions like the H3K9me1 chromatin mark, which has only 1 peak cluster in the 7000-7500 bp interval (see the big dip at x-axis=15 in the right-hand panel) and only 7 peak clusters in the 9500-10000 bp interval (see S1_Appendix and S2_Appendix for reproducible code and data).\n\nFrom a performance standpoint, the extension algorithm is optimized to handle the computational complexity inherent to performing compute-intensive n-dimensional annotation. This ultimately aids in efficiently capturing cis-regulatory and proximal-promoter element relationships between ChIP-seq peaks and the genes they are (dys-)regulating, as described in further detail in the vignette. All of geneXtendeR’s source code is implemented in the C and R programming languages and shipped within a standalone R/Bioconductor package release that is publicly available for download from either Bioconductor or GitHub. Within its codebase, geneXtendeR leverages the AnnotationDbi22, BiocStyle23, data.table24, dplyr25, GO.db26, networkD327, RColorBrewer28, rtracklayer29, SnowballC30, testthat31, tm32, and wordcloud33 libraries.\n\nFigure 2 summarizes the key steps of a sample workflow. For an end-to-end example of a comprehensive biological workflow and case-study, please refer to the vignette. An earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/082347).\n\nSample biological workflow using geneXtendeR in combination with existing statistical software to evaluate the role of ChIP-seq peak significance during functional annotation tasks (see description of hotspotPlot() function in package vignette). It is not uncommon for significant peaks to be located thousands of base pairs away from their nearest genes, suggesting that sequences under these respective peaks may further be extracted and analyzed for the presence of known regulatory elements or repeats (e.g., using software programs like TRANSFAC, MEME/JASPAR, or RepeatMasker) or for investigating potential enhancer effects.\n\n\nResults\n\nFirst, we tested geneXtendeR19 on all publicly available transcription factor and histone modification ChIP-seq datasets in ENCODE. After downloading and analyzing data from the ENCODE ChIP-seq Experiment Matrix (hg19)34, our large-scale analysis (Figure 1) indicated that ChIP-seq peaks do not concentrate within any specific upstream extension (e.g., 2000 bp) of their nearest protein-coding genes. This observation that ChIP-seq peaks drop off gradually with genomic distance from the start of a gene (first exon) suggests that there is no good general guideline cutoff for capturing proximal histone modifications (e.g., prior studies20,21 have used 2000 bp) or transcription factor binding peaks. There are still hundreds of peak clusters that reside in proximal promoter regions that are 2000–3000 bp away from their nearest protein-coding genes and in distal regions beyond 3 kbp, making ad-hoc decisions like 2 kbp cutoffs too general to be of broad utility across specific use cases. When applying geneXtendeR to both proximal and distal transcription factor (TF) binding peaks for all cell types, we observed some cell type-dependent and TF-dependent peak aggregation dynamics in intervals ranging from 0 to 10 kbp (Figure 3). Similarly, examining distal peaks in representative plots of different chromatin marks in different cell types indicated that peaks indeed aggregate in a cell type and chromatin mark-dependent manner (Figure 4). S1_Appendix35 and S2_Appendix36 provide downloads to the complete compendium of all proximal/distal datasets analyzed from ENCODE.\n\nRunning geneXtendeR on 547 human transcription factor (TF) ChIP-seq datasets obtained from ENCODE shows that many peaks tend to reside within 500 bp upstream of their respective protein-coding genes yet, depending on the identity of the transcription factor (e.g., EP300) and the specific cell type (e.g., K562), there may be more or less peaks located further upstream and, therefore, a generalized upstream cutoff is not applicable.\n\nRunning geneXtendeR on 198 human histone modification ChIP-seq distal peak datasets obtained from ENCODE reveals that most distal peaks are not congregating within any specific upstream region of their respective protein-coding genes (here we define “distal” as only those peaks that are more than 2000 bp away from their nearest gene). Additional comprehensive analyses (see S1_Appendix and S2_Appendix35,36) were run for proximal peaks (≤ 2000 bp) as well as the complete set of peaks (proximal + distal) from all 198 histone modification ChIP-seq datasets, and similar patterns were observed.\n\nWe then focused our attention on using geneXtendeR to perform an end-to-end analysis of a published histone modification ChIP-seq dataset37 deposited in the Gene Expression Omnibus under accession number GSE83979. At the peak-calling stage (Figure 2) we ran two different peak callers (SICER38 and CisGenome39) producing two highly variable peak length profiles even at default run parameters (Supplementary Figure 1). Despite the stark difference in peak profiles, geneXtendeR consistently identified the same top two gene candidates, highlighting its utility for robust functional annotation even in the face of extreme peak variability. Details are discussed in the package vignette.\n\nWe followed up this computational analysis by performing n-dimensional annotation of the GSE83979 dataset to provide an expanded view of the gene neighborhood around each individual peak – effectively annotating every peak n times (once for the closest gene, once for the second-closest gene, etc.) and grouping the results into a tabular summary format. We show in the vignette how the second-closest gene may be a preferable candidate for experimental follow-up/validation, especially if the first-closest gene is putative/predicted, while the second-closest gene is known to play a role in a similar biological process based on previously published literature.\n\n\nDiscussion\n\nThe cell-type and TF/chromatin mark-specific complexity apparent in Figure 3 and Figure 4 motivated the design and implementation of user-friendly functions that can calculate ratios of statistically significant peaks to total peaks in various genomic intervals (see hotspotPlot() documentation in geneXtendeR vignette). Similarly, users can transform peaks into merged peaks (see peaksMerge()). geneXtendeR also allows users to explore gene ontology differences at various extensions (see diffGO()) as interactive network graphics (see makeNetwork()) or word clouds (see makeWordCloud()). Furthermore, users can investigate mean (average) peak lengths within any genomic interval (see meanPeakLengthPlot()), showing how average peak broadness can change at different upstream extensions, or examine the variance of peak lengths within a specific genomic interval (see peakLengthBoxplot()). It is also possible to examine unique genes and their associated ChIP-seq peaks between any two upstream extension levels (see distinct()). For example, Figure 5 displays all unique genes (and their respective gene ontologies) that are associated with peaks located between 2–3 kbp across the genome. geneXtendeR also allows users to examine the distribution of peak lengths across the entire peak set (see allPeakLengths()), a function that is useful for visualizing the length distribution of all peaks from a peak caller. These functions (and more) are all explored in detail within the package vignette. After a user has explored the peak coordinates data using these functions to determine the optimal alignment of peaks to a GTF file, the peaks file can be functionally annotated with the annotate() function or one of its counterparts (gene_annotate() or annotate_n()) for n-dimensional annotation.\n\nAll unique genes (and their respective gene ontologies (GO)) that are associated with peaks located in promoter-proximal regions between 2–3 kbp genome-wide. Put another way, these are all gene-GO pairs associated with peaks that are distinct between 2000 and 3000 bp upstream extensions across the genome. Orange color denotes gene names, purple color denotes GO terms. A user can hover the mouse cursor over any given node to display its respective label directly within RStudio. Likewise, users can dynamically drag and re-organize the spatial orientation of nodes, as well as zoom-in and out of them for visual clarity.\n\nWe have successfully applied geneXtendeR19 during the analysis of a histone modification ChIP-seq study investigating the neuroepigenetics of alcohol addiction40, where geneXtendeR was used to determine an optimal upstream extension cutoff for H3K9me1 enrichment (a commonly studied broad peak) in rat brain tissue based on line plots of both significant peaks and total peaks. This analysis helped us to identify, functionally annotate, and experimentally validate synaptotagmin 1 (Syt1) as a key mediator in alcohol addiction and dependence40. This analysis is explored in detail in the package vignette. Taken together, geneXtendeR’s functions are designed to be used as an integral part of a broader biological workflow (Figure 2).\n\n\nConclusions\n\nWe present an R/Bioconductor package, geneXtendeR19, that goes beyond the typical nearest-to-gene analyses commonplace to most standard computational ChIP-seq workflows. geneXtendeR offers n-dimensional functional annotation and the ability to investigate the effect of variable-length gene bodies when mapping peaks to genomic features, thereby serving as a next-generation model of peak annotation to nearby features in modern bioinformatics workflows. geneXtendeR therefore represents a critical first step towards tailoring the functional annotation of a ChIP-seq peak dataset according to the details of the peak coordinates (chromosome number, peak start position, peak end position) and their surrounding genomic features.\n\n\nData availability\n\nA variety of different publicly available datasets were used to test geneXtendeR. From ENCODE, a large-scale computational analysis using the hg19 reference genome was performed on 198 histone modification and 547 transcription factor ChIP-seq datasets. These transcription factor and histone modification ChIP-seq datasets in ENCODE are publicly available.\n\nIn addition, geneXtendeR was tested on a histone modification ChIP-seq dataset37 deposited in the Gene Expression Omnibus under accession number GSE83979.\n\nZenodo: S1 Appendix. geneXtendeR analysis on 547 human TF ChIP-seq ENCODE datasets. https://doi.org/10.5281/zenodo.264670235\n\nZenodo: S2 Appendix. geneXtendeR analysis on 198 human histone modification ChIP-seq ENCODE datasets. https://doi.org/10.5281/zenodo.264670736\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSoftware available from: https://bioconductor.org/packages/geneXtendeR/\n\nSource code available from: https://github.com/Bohdan-Khomtchouk/geneXtendeR\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.264669619\n\nLicense: GNU General Public License-3",
"appendix": "Author contributions\n\n\n\nBBK conceived the study, designed the algorithms, implemented the code, performed the analyses, and wrote the manuscript. WCK and DJVB assisted with implementation and analysis. CW supervised the study.\n\n\nGrant information\n\nThis work was supported by the American Heart Association (AHA) Postdoctoral Fellowship grant #18POST34030375 (Khomtchouk). This work was also partially supported by the Stanford Training Program in Aging Research grant (NIH/NIA T32-AG0047126) and the Army Research Office (ARO), National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a – both awarded to BBK from 2014–2018. The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Heart Association, National Institutes of Health, or Department of Defense.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\n\n\nViolin plot showing the differences in peak length distributions of the same ChIP-seq data (available through the Gene Expression Omnibus database, accession identifier GSE83979) analyzed with two separate peak callers (SICER and CisGenome) – despite significant differences in peak lengths generated by the two callers (i.e., peak variability), geneXtendeR’s gene_annotate() function can still robustly call top gene candidates consistently, as explained in the geneXtendeR package vignette.\n\n\nReferences\n\nAbcam: Histone modifications: a guide. Reference Source\n\nSquazzo SL, O’Geen H, Komashko VM, et al.: Suz12 binds to silenced regions of the genome in a cell-type-specific manner. Genome Res. 2006; 16(7): 890–900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPepke S, Wold B, Mortazavi A, et al.: Computation for ChIP-seq and RNA-seq studies. Nat Methods. 2009; 6(11 Suppl): S22–S32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandt SG, Marinov GK, Kundaje A, et al.: ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 2012; 22(9): 1813–1831. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKellis M, Wold B, Snyder MP, et al.: Defining functional DNA elements in the human genome. Proc Natl Acad Sci U S A. 2014; 111(17): 6131–6138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeinig M, Colomé-Tatché M, Taudt A, et al.: histoneHMM: Differential analysis of histone modifications with broad genomic footprints. BMC Bioinformatics. 2015; 16: 60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRintisch C, Heinig M, Bauerfeind A, et al.: Natural variation of histone modification and its impact on gene expression in the rat genome. Genome Res. 2014; 24(6): 942–953. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHa M, Ng DW, Li WH, et al.: Coordinated histone modifications are associated with gene expression variation within and between species. Genome Res. 2011; 21(4): 590–598. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoohy H, Down TA, Spivakov M, et al.: Correction: A Comparison of Peak Callers Used for DNase-Seq Data. PLoS One. 2014; 9(8): e105136. Publisher Full Text | Free Full Text\n\nThomas R, Thomas S, Holloway AK, et al.: Features that define the best ChIP-seq peak calling algorithms. Brief Bioinform. 2017; 18(3): 441–450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang K, Li M, Hakonarson H: ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010; 38(16): e164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcLean CY, Bristor D, Hiller M, et al.: GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol. 2010; 28(5): 495–501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang W, Loganantharaj R, Schroeder B, et al.: PAVIS: a tool for Peak Annotation and Visualization. Bioinformatics. 2013; 29(23): 3097–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu L, Gazin C, Lawson N, et al.: ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics. 2010; 11(1): 237. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu G, Wang LG, He QY: ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015; 31(14): 2382–3. PubMed Abstract | Publisher Full Text\n\nCavalcante RG, Sartor MA: annotatr: genomic regions in context. Bioinformatics. 2017; 33(15): 2381–2383. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeinz S, Benner C, Spann N, et al.: Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 2010; 38(4): 576–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhomtchouk B, Koehler W: Bohdan-Khomtchouk/geneXtendeR: Optimized Functional Annotation Of ChIP-seq Data (Version 1.8.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2646696\n\nMaze I, Feng J, Wilkinson MB, et al.: Cocaine dynamically regulates heterochromatin and repetitive element unsilencing in nucleus accumbens. Proc Natl Acad Sci U S A. 2011; 108(7): 3035–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang J, Zibetti C, Shang P, et al.: ATAC-Seq analysis reveals a widespread decrease of chromatin accessibility in age-related macular degeneration. Nat Commun. 2018; 9: 1364. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPagès H, Carlson M, Falcon S, et al.: AnnotationDbi: Annotation Database Interface. R package version 1.42.1. 2018.\n\nOleś A, Morgan M, Huber W: BiocStyle: Standard styles for vignettes and other Bioconductor documents. R package version 2.8.2. 2018. Reference Source\n\nDowle M, Srinivasan A: data.table: Extension of ‘data.frame‘. R package version 1.11.4. 2018.\n\nWickham H, François R, Henry L, et al.: dplyr: A Grammar of Data Manipulation. R package version 0.7.6. 2018.\n\nCarlson M: GO.db: A set of annotation maps describing the entire Gene Ontology. R package version 3.6.0. 2018.\n\nAllaire JJ, Gandrud C, Russell K, et al.: networkD3: D3 JavaScript Network Graphs from R. R package version 0.4. 2017. Reference Source\n\nNeuwirth E: RColorBrewer: ColorBrewer Palettes. R package version 1.1.2. 2014. Reference Source\n\nLawrence M, Gentleman R, Carey V: rtracklayer: an R package for interfacing with genome browsers. Bioinformatics. 2009; 25(14): 1841–1842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBouchet-Valat M: SnowballC: Snowball stemmers based on the C libstemmer UTF-8 library. R package version 0.5.1. 2014. Reference Source\n\nWickham H: testthat: Get Started with Testing. R J. 2011; 3(1): 5–10. Publisher Full Text\n\nFeinerer I, Hornik K, Meyer D: Text Mining Infrastructure in R. J Stat Softw. 2008; 25(5): 1–54. Publisher Full Text\n\nFellows I: wordcloud: Word Clouds. R package version 2.5. 2014.\n\nhttps://genome.ucsc.edu/encode/dataMatrix/encodeChipMatrixHuman.html\n\nKhomtchouk B: Bohdan-Khomtchouk/ENCODE_TF_geneXtendeR_analysis: ENCODE_TF_geneXtendeR_analysis (Version v1.0) [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2646702\n\nKhomtchouk B: Bohdan-Khomtchouk/ENCODE_histone_geneXtendeR_analysis: ENCODE_histone_geneXtendeR_analysis (Version v1.0) [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2646707\n\nGidlöf O, Johnstone AL, Bader K, et al.: Ischemic Preconditioning Confers Epigenetic Repression of Mtor and Induction of Autophagy Through G9a-Dependent H3K9 Dimethylation. J Am Heart Assoc. 2016; 5(12): pii: e004076. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZang C, Schones DE, Zeng C, et al.: A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics. 2009; 25(15): 1952–1958. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJi H, Jiang H, Ma W, et al.: An integrated software system for analyzing ChIP-chip and ChIP-seq data. Nat Biotechnol. 2008; 26(11): 1293–1300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarbier E, Johnstone AL, Khomtchouk BB, et al.: Dependence-induced increase of alcohol self-administration and compulsive drinking mediated by the histone methyltransferase PRDM2. Mol Psychiatry. 2017; 22(12): 1746–1758. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "48487",
"date": "03 Jun 2019",
"name": "Vincent J. Carey",
"expertise": [
"Reviewer Expertise Biostatistics",
"computational biology",
"clinical trials",
"epidemiology",
"statistical computing."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall this is a clearly written paper, although I would take issue with the term \"Optimized\" in the title. What is \"optimal functional annotation\"? The abstract includes the phrase \"precisely tailor the computational analysis of a ChIP-seq dataset to the specific peak coordinates of the data and its surrounding genomic features\". This is a complicated objective and can be unpacked in many ways, specifically with respect to \"computational analysis\".\nWhat the system brings to analysis of ChIP-seq data seems to be tunability and inclusiveness, in the important area of combinatorics of binding events and of histone modifications. (\"Inclusiveness\" pertains to allowing inspection of the order of proximities.) Can these be related to the term \"Optimized\" in the title?\nFootnote 35 gives reference to http://www.doi.org/10.5281/zenodo.2646702 which throws a \"Bad Gateway\" error.\nFigure 1: Oscillations in a few traces in the left and right panels are probably artifactual. More stable estimates of the relationship between \"normalized peak cluster count\" and distance from TSS could be obtained using overlapping sliding windows.\nI do not find Figure 2 particularly illuminating. The relationship of geneXtendeR and differential expression-oriented packages is not clear and is not described in the caption. It might be more informative to schematize the data structures for peak sets and how they lead to multi-sample hypothesis testing (e.g., with edgeR) or ontology/network inference.\nFigure 3 is difficult to parse. Somehow a comparative interpretation is desirable, but all 4 panels look qualitatively similar. The y-axis ranges are different and perhaps log rescaling would be useful. Are the plotted points estimates, and if so, are uncertainty intervals of interest?\nFigure 4 is similarly challenging. Are the oscillations seen after the jumps statistically meaningful?The y axis is labelled \"differences\". This is not explained in the caption.\nFigure S1 should employ a logarithmic axis.\nIf we try example(makeNetwork) and then example(makeWordCloud) in the same session, a rat GTF is downloaded twice. BiocFileCache can be used to simplify user interactions with servers if these are needed.\nIn fact, the GTF file used by this package is available to Bioconductor users with the AnnotationHub package.\n\n> ah = AnnotationHub::AnnotationHub() > AnnotationHub::query(ah, c(\"gtf\", \"rattus\", \"84\")) AnnotationHub with 3 records # snapshotDate(): 2019-05-02 # $dataprovider: Ensembl # $species: Rattus norvegicus # $rdataclass: GRanges # additional mcols(): taxonomyid, genome, description, #\n\ncoordinate_1_based, maintainer, rdatadateadded, preparerclass, tags, #\n\nrdatapath, sourceurl, sourcetype # retrieve records with, e.g., 'object[[\"AH50914\"]]'\n\ntitle\n\nAH50914 | Rattus_norvegicus.Rnor_6.0.84.abinitio.gtf\n\nAH50915 | Rattus_norvegicus.Rnor_6.0.84.chr.gtf\n\nAH50916 | Rattus_norvegicus.Rnor_6.0.84.gtf\n\nIf we use\n\n> z = ah[[\"AH50916\"]] downloading 1 resources retrieving 1 resource\n\n|==========================================================| 100%\nloading from cache\n\n‘AH50916 : 57654’ Importing File into R ..\nwe have a cached version of the required annotation. Any package code requiring this GTF information can use\nAnnotationHub::AnnotationHub()[[\"AH50916\"]]\n\nto get it. Thus:\n\n> AnnotationHub::AnnotationHub()[[\"AH50916\"]]\nsnapshotDate(): 2019-05-02 downloading 0 resources loading from cache\n\n‘AH50916 : 57654’ Importing File into R .. GRanges object with 750896 ranges and 24 metadata columns:\n\nseqnames\n\nranges strand |\n\nsource\n\ntype\n\nscore\n\n<Rle>\n\n<IRanges> <Rle> | <factor>\n\n<factor> <numeric>\n\n[1]\n\n1 396700-409676\n\n+ | ensembl\n\ngene\n\n<NA>\n\n[2]\n\n1 396700-409676\n\n+ | ensembl transcript\n\n<NA> …\nThe peaks Input function writes a file \"peaks.txt\" to the current working directory! This is very poor form and could destroy user data. The function does not even include an option to write the file elsewhere. This content is then regarded as globally accessible to functions like make Network.\nIn summary the paper describes a number of utilities of potential interest, but essential statistical considerations should be enhanced. Downstream work such as network construction is entirely dependent on a fixed set of peak addresses, but the addresses must be associated with false discovery rates and/or boundary uncertainties. The discussion starts with \"mark-specific complexity\" apparent in Figures 3 and 4 but it is not clear that \"complexity\" is the right concept here. Different factors have different effects in different contexts, and distance to nearby gene is one component of context. To the extent that the paper gives users a mechanism for \"determining the optimal alignment of peaks to a GTF file\", I feel it is the concept of optimality raised here, and not the various functions that support \"exploration\", that should be detailed clearly in the paper. The optimization process should not be referred to the vignette. Once this optimality concept is stated precisely, the roles of the various functions can be usefully highlighted.\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? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "48667",
"date": "04 Jun 2019",
"name": "Ruslan I. Sadreyev",
"expertise": [
"Reviewer Expertise Bioinformatics",
"epigenetics",
"epigenomics",
"chromatin remodeling",
"chromatin structure"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Khomtchouk et al introduces geneXtendeR, a new tool for annotating ChIP-seq peaks, and more specifically, the peaks that show differential enrichment between experimental conditions, by providing possible links to genes in the genomic vicinity of each peak. The main novelty of this method is the \"extension\" algorithm for assigning possible cis-regulated genes to each peak, which provides additional flexibility in terms of the cutoff of the distance form a gene and includes the genes that are not the closest to the peak. The biological intuition behind this approach is sound and based on the well-known facts that (a) the binding loci of regulatory proteins and the regions of enrichment of chromatin marks that are involved in the regulation of gene activity often do not conform to standard cutoffs of distance to the transcription start site or gene body (confirmed in Fig. 1,3,4), and (b) enhancers and other regulatory elements often affect the activity of a gene that is not the closest to this element.\nThe manuscript provides a general justification of the approach and an overall description of the method itself. However, one part that can definitely be improved is the description of the results that the user can expect and a clear explanation, with examples, of how the user can interpret these results and generate specific biological hypotheses about the involvement of a protein or chromatin mark in the regulation of a specific gene or a group of genes. It seems that reference 40 includes an example of application of this method to a specific experimental dataset, and Fig. 5 is an example of functional annotation of promoters, but a clearly described user case showing the input, output, interpretation, and biological conclusions would be important. Another part that is unclear to me is how the user should interpret the multiple sets of 1st, 2nd, 3rd etc closest gene for a specific peak set. How one should approach selecting the most relevant gene set among these multiple options? An informative example may help clarify this point.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "48490",
"date": "10 Jun 2019",
"name": "Michael Lawrence",
"expertise": [
"Reviewer Expertise Genomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article presents a new tool for finding the k-nearest neighbouring genes for a set of ChIP-seq peaks or other type of genomic feature. The idea is not particularly novel (it sounds a lot like bedtools closest -k, contrary to what the paper says), but the tool appears to be useful. Most of my concerns are around the organization of the article and how it describes the software.\n\nThe Introduction is well written and makes a good case for the tool. It could also incorporate some of the figures and data-driven arguments that come later (or those could go into the Discussion).\n\nIt is strange how the Methods section begins with Implementation (what about abstractly describing the method?) but even stranger how the Implementation section includes arguments for why the method is important (e.g., Figure 1).\n\nThe Results spends too much time arguing for why finding the k-nearest points is abstractly useful (there being no obvious cut-off). The paper would be strengthened by describing some interesting biological results, such as a meaningful/validated regulatory relationship not discovered by less flexible tools. Maybe these are described in the vignettes but it would be good to highlight them here. The actual examples can stay in the vignettes, but it would be nice to have the salient features described in this section, rather than Discussion. Since “optimized” is emphasized in the title, it would be good to have some details on performance here.\n\nThe Discussion should focus on limitations of the tool, potential integration points, and other topics that transcend the tool and method. It seems that the alcohol dataset belongs in Results.\n\nI’m not sure I agree that this is a “critical first step” when there are many tools that find the closest gene; this one just finds the n-closest.\n\nI wonder whether it would have been simpler (if a bit less efficient) to just find all genes within a wide margin of the peaks and then restrict those to the closest ‘k’. The iterative overlap finding, implemented in C, sounds complicated. I’m also concerned about the package having so many dependencies, including both dplyr and data.table in addition to Bioconductor.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-612
|
https://f1000research.com/articles/8-239/v1
|
01 Mar 19
|
{
"type": "Research Article",
"title": "Determinants of knowledge, attitude and practice in patients with both type 2 diabetes and chronic kidney disease in Fiji",
"authors": [
"Mohammed Alvis Zibran",
"Masoud Mohammadnezhad",
"Mohammed Alvis Zibran"
],
"abstract": "Background: In Fiji, Type 2 diabetes mellitus (T2DM) and Chronic kidney disease (CKD) are amongst the top four causes of premature mortality, disability and death. This study aims to identify the determinants of knowledge, attitude and practice (KAP) in T2DM patients with CKD in Fiji in 2018. Methods: A cross-sectional study was conducted at Sigatoka Sub-divisional Hospital (SSH) in Fiji in July-August, 2018 using a self-structured questionnaire to test KAP of 225 patients. The inclusion criteria were confirmed T2DM patients (Fijian citizens) with CKD, aged 30 years or above and attending Special Out-Patient's Department (SOPD) at SSH. Independent t-test and ANOVA was used to test differences between demographic variable and practice score while non-parametric tests were used for knowledge and attitude. Spearman correlation and multiple linear regressions were also done. All the tests were set at 5% level of significance. Results: The mean KAP level was high: knowledge, 23.3 (SD±3.25); attitude, 23.1 (SD±2.73) and practice, 7.1 (SD±2.04). A high level of knowledge was seen in those with university-level education (p<0.001), unemployed (p=0.05) and high average monthly income (p=0.03). Those aged 61-70 years had a 0.53-point lower attitude score (p=0.05) than other age categories, while those >70 years had a 1.78-point lower attitude score (p=0.01) than other age categories. Fijians of Indian descent (FID) had lower attitude (p=0.002) and higher practice (p=0.001) scores. Conclusion: Patients with both T2DM and CKD at SSH have high levels of KAP. Those with higher levels of education, the unemployed and those with high monthly income had higher knowledge, FID had low attitude but high practice scores, and the higher age category had lower attitude scores. The study identified high-risk groups for low KAP, which can become the focus of future public health intervention.",
"keywords": [
"Knowledge",
"Attitude",
"Practice",
"Type 2 Diabetes Mellitus",
"Chronic Kidney Disease",
"Determinants",
"Fiji."
],
"content": "Introduction\n\nType 2 diabetes mellitus (T2DM) is characterized by fasting blood glucose of more than 7 mmol/L or random blood sugar of more than 11 mmol/L in the presence of symptoms of increase thirst/hunger, frequent urination and weight loss (O’Neil et al., 2012). T2DM is the seventh-leading cause of death globally (Zheng et al., 2018), and various complications arise as a consequence of this disease—one of the major ones being chronic kidney disease (CKD) (Idris et al., 2018; Yakush Williams, 2017). CKD, the ninth-leading cause of deaths globally (Rifkin et al., 2012), is defined as estimated glomerular filtration rate (eGFR) of less than 60 ml/min for at least 3 months (Bouchard et al., 2010). Fiji’s STEP-wise Surveillance Report (STEPS) of 2011 showed that 15.6% of the population had raised fasting blood sugar (Snowdon & Tukana, 2011). This is quite worrying, since T2DM is the major cause of CKD, accounting for 44% of all cases and hence a rise in T2DM will lead to a greater burden of CKD in Fiji (Atkins & Zimmet, 2010). On that same note, T2DM and CKD were found to be among the top four causes of premature deaths and death and disability combined in Fiji (IHME, 2016). The final stage of CKD is very costly since it requires renal dialysis optimally three times a week and renal transplant eventually whereby cost of each dialysis session in Fiji can range from $USD70 to USD120 (Consumer Council of Fiji, 2017). Apart from these costs, the healthcare expenses related to management of these medical conditions are borne by the government of Fiji and hence trimming these expenses via addressing these 2 diseases from a public health perspective would be beneficial (MoHMS, 2015).\n\nThe knowledge, attitude and practice (KAP) survey, which uses questionnaires to gather information from the patients on specific aspects of certain conditions (Kaliyaperumal, 2004), is a useful tool for assessing and improving control of patient’s disease, delaying associated complications (Ghannadi et al., 2016), influencing better health policy (Stanifer et al., 2015) and increasing awareness for disease prevention. Various factors have been linked to level of KAP in T2DM patients with CKD; for instance female gender has been linked to poor knowledge but good attitude (Yusoff et al., 2016) while males have been shown to have good practice (Stanifer et al., 2016). Similarly, employed participants were found to have higher knowledge while married ones had high attitude and practice (Mutiso et al., 2011; Thirsk et al., 2014; Yusoff et al., 2016). Consequently, a KAP survey of individuals with T2DM and CKD will provide an insight on the current status of the level of KAP and its determinants in a referral hospital in Fiji- Sigatoka Sub-divisional Hospital (SSH), which can be utilized to inform public health programs and help target high-risk cases to improve awareness, promote self-control in patients and reduce or delay complications from T2DM and CKD (Ghannadi et al., 2016).\n\nSince, there have been no studies on KAP of T2DM patients with CKD in Fiji, this study aimed to identify the determinants of KAP in T2DM patients with CKD in Fiji in 2018. The specific objectives were to identify the level of each of the aspects of KAP and to investigate for significant links between the level of KAP and the socio-demographical features in T2DM patients with CKD in Fiji in 2018.\n\n\nMethods\n\nThis research applied a cross-sectional, quantitative design to identify determinants of KAP in patients with T2DM and CKD at SSH from 1st July 2018 to 31st August 2018. The inclusion criteria for the study sample were patients with confirmed T2DM plus CKD, attending in the waiting area in front of the Special Out-Patient’s Department (SOPD) clinic at SSH, citizen of Fiji, age ≥30 years and most importantly they had to agree to participate in the study. The exclusion criteria were patients with CKD but not T2DM, patients with any illness that jeopardizes their mental ability to participate and those who were not interested in taking part in the study.\n\nThe study was conducted at SSH’s SOPD clinic. SSH is a Sub-Divisional Hospital in the Western Division of Fiji and it is a secondary-level hospital which provides general outpatient services, SOPD, inpatient services, maternity, child-health, eye-care, laboratory tests, radiological examinations and pharmacy. It is the only hospital in Nadroga/Navosa Sub-division and it accepts primary referrals from its Health-Centers, while it refers cases to its tertiary hospital—Lautoka Hospital.\n\nPurposive sampling was used, which included all the patients who attended SOPD at SSH during the study period and satisfied the inclusion and exclusion criteria. Potential sources of bias were addressed by attempting to reduce selection bias by allowing all eligible participants to be part of the study while ensuring all criteria for inclusion/exclusion are adhered to. Every participant who qualified under the study criteria was eligible for the study. As a result, from a total of 265 patients who satisfied the inclusion/exclusion criteria, a sample size of 225 was finally selected to participate in this study.\n\nThe data collection tool was the KAP questionnaire (Mohammadnezhad, 2019), which had been developed by reviewing the literature and using other similar questionnaires that have been used previously, like the CKD Screening Index (Khalil & Abdalrahim, 2014) and KAP questionnaire developed by Stanifer et al. (2015). This questionnaire was divided into two sections: Section A contained general information on 7 factors and Section B measured the KAP aspect of T2DM patients with CKD. For the knowledge component, each item was given a score of “2” for a correct answer, “1” for “do not know” and “0” for incorrect response. Hence, the total scoring range for this section of 15 questions was 0–30 for each participant. Those with a score of 0–15 were considered as “low level of knowledge”, 16–22 as “medium level of knowledge” and 23 and over as “high level of knowledge” (Kim, 2008; Tekanene et al., 2018). For the attitude component, each item was given a score of “2” for a positive attitude, “1” for “neutral” and “0” for negative attitude. Hence, the total scoring range for this section of 15 questions was 0–30 for each participant. Those with a score of 0–15 were considered as “low level of attitude”, 16–22 as “medium level of attitude” and 23 and over as “high level of attitude” (Kim et al., 2009; Lincoln et al., 2018). For the practice component, each item was given a score of “1” for a positive practice and “0” for negative practice. Hence, the total scoring range for this section of 10 questions was 0–10 for each participant. Those with a score of less than 5 were considered as “low level of practice” and 5 or over as “high level of practice” (Kim et al., 2008; Tekanene et al., 2018).\n\nBefore collecting the data, face validity was assessed among 10 volunteer T2DM patients with CKD who were attending the SOPD clinic at SSH and satisfied the inclusion/exclusion criteria (5 males and 5 females) to assess whether the questionnaire was legible, clear, simple, easy and understandable. The content validity was also conducted among three experts (research supervisor, co-supervisor and a Medical Registrar from Lautoka Hospital) to decide whether the content of the questionnaire met the objective of the study or not. Apart from the English version, the questionnaire (plus the information sheet and consent form (Mohammadnezhad, 2019)) was translated into two other languages (Hindi and iTaukei) by a bi-lingual translator and then cross-translated to ensure the contents of the original questionnaire matched the translated version.\n\nAfter providing the information sheets, those who were eligible and consented for the study were given the questionnaire to either fill and return on the same day or return it later before the due-date by dropping it in the specially marked box in SOPD. The illiterate participants were assisted by the research assistant who provided non-bias support in filling the questionnaire on their behalf.\n\nAll the questionnaires received by 31st August 2018 were used for analysis, while the rest were classified as non-responders. The information from the questionnaire was entered in Microsoft Excel Data Sheet for cleaning and coding after which it was transferred to SPSS Version 25. The continuous variables were analyzed and expressed as means and standard deviation while the categorical variables were displayed as counts and percentages in a frequency distribution table. The Kolmogorov-Smirnov test was used to assess the normality for continuous variables. The tests of baseline differences in demographic characteristics and practice scores were done using independent t-test and ANOVA. Apart from comparing gender with KAP, all other comparisons were made using non-parametric tests to check the differences between demographic characteristics and knowledge and attitude respectively. Multiple linear regression analysis was conducted to see which independent variables were significant predictors of the dependent variable. P<0.05 was considered to indicate statistical significance.\n\nEthical approval for this study was obtained from the Fiji National University College Health Research Ethics Committee (CHREC) and the Fiji National Health Research Ethics and Review Committee (FNHRERC) – approval number 2018.128.W.D. Each participant provided their written informed consent to take part in this study.\n\n\nResults\n\nThe study sample for this research comprised of 225 participants aged 38–92 years (mean=58.6, SD=9.99). Majority of the participants were in the age range of 46–60 years (52.4%) and there was almost an equal number of male (48.9%) and female (51.1%) participants in this study. In terms of ethnicity, there was almost an equal number of iTaukei (48.9%) and FID (48.4%). In total, 48% of the study subjects had secondary-level education, 38% of the participants were employed, 80.4% were married and 67.1% of the participants had an average monthly income of <$400 (see Table 1).\n\nTable 2 displays the overall scores for the participant’s KAP. The highest score for knowledge was 30, while the mean score was 23.3 (SD ±3.25), which shows that the overall knowledge was high. For the attitude component, the highest score was 28 while the mean was 23.1 (±2.73) which show that the overall attitude was high. The highest score for practice was 10 while the mean was 7.1 (±2.04), which shows that the overall practice was also high.\n\nTable 3 shows that there was a significant association between level of education and knowledge (p<0.001), employment status and knowledge (p=0.05), average monthly income and knowledge (p=0.03), ethnicity and attitude (p=0.002) and ethnicity and practice (p=0.001).\n\n*Kruskal-Wallis test. **Parametric test. ***Non-parametric test.\n\nTable 4 shows that none of the independent variables were a significant predictor for overall knowledge score. All the independent variables could predict only 6.7% of the total knowledge scores (R2 = 0.129, adjusted R2 = 0.067). On the other hand, age categories of 61–70 (t = -0.664, p = 0.05) and >70 (t = -1.653, p = 0.01) and Ethnicity (FID: t = -3.287, p = 0.001) were significant predictors of overall attitude score. Those aged 61–70 years had a 0.53-point lower attitude score compared to other age categories (with other variables constant) while those aged >70 years had 1.78-point lower attitude score compared to other age categories (with other variables constant). Similarly, FID had a 1.5-point lower attitude score compared to other ethnic groups (holding other variables constant). All independent variables could predict only 2.9% of the total attitude scores (R2 = 0.094, adjusted R2 = 0.029). Finally, ethnicity (FID: t = 3.714, p < 0.001) was the only significant predictor of overall practice score. FID had a 1.03-point higher practice score compared to other ethnicities (holding other variables constant). All independent variables could predict only 6.1% of the total practice scores (R2 = 0.123, adjusted R2 = 0.061).\n\n\nDiscussion\n\nThis research had sought to identify the determinants of KAP towards causes, prevention, diagnosis, treatment and management in T2DM patients with CKD at SSH in 2018. Those aged 61–70 years had a 0.53-point lower attitude score (p=0.05) compared to other age categories, while those aged >70 years had 1.78 points lower attitude score (p=0.01) compared to those in other age categories. On the contrary, those aged >30 years were associated with having a good attitude in another study (Yusoff et al., 2016). White et al. (2008) noted that people aged less than 60 years had better knowledge of kidney disease but nil association with attitude was mentioned. Similarly, those aged >60 years were associated with having poor knowledge of renal impairment in a study from Malaysia (White et al., 2008). On that same note, age less than 60 was linked to high knowledge score in studies from Tanzania (Stanifer et al., 2016), USA (Li et al., 2014) and Iran (Roomizadeh et al., 2014). Conversely, older age was linked to higher knowledge in another article from USA (Ryder et al., 2013) while it was associated with high practice scores in the Jordan study (Khalil & Abdalrahim, 2014).\n\nAge is an independent variable that has been linked to various diseases in multiple studies; for instance, a rise in age is directly linked with an increased risk of cardiac events (Canto et al., 2012). Age has also been associated with KAP of diabetes, but there are few studies which have tested and found significant links between age and KAP in individuals with diabetic kidney disease (DKD) (Islam et al., 2014).\n\nIn terms of ethnicity, the current research showed that FID had significantly lower attitude scores but higher practice scores than iTaukei and Fijians of others Descent (FoD). This finding is evident in one of the aspects of practice at the daily SOPD clinics at SSH, since in Fiji’s SOPD clinics, the majority of the patients attending the clinic are FID, while iTaukei patients usually default their booked clinics and thus end up in making the majority of the numbers for NCD-related admissions. Kazley et al. (2015) had found poor knowledge scores in the African Americans but there was no mention of attitude or practice (Kazley et al., 2015).\n\nThe link between ethnicity and health-related KAP is extremely important in the Pacific setting as they are usually culturally influenced and tend to prioritize behaviors practiced by their ancestors over the past generations—this means that if their ancestors had certain attitude or practice regarding a health-issue, the Pacific people are inclined to follow the same. Culture seems to play an integral part in lots of decision-making in these PICs, as preferences are governed by the ethnic roots of most of the Pacific Islanders (Ryan et al., 2010). Subsequently, understanding and identifying the specific ethnic group with low KAP in SSH would help in tailoring the suitable health campaigns which are culturally appropriate and effective. Thus, this research shows that the iTaukei ethnicity will need to be considered while drafting and designing future public health preventive programs, as these are the individuals who need assistance with KAP towards DKD at SSH.\n\nThe level of education had the strongest link with knowledge scores of the participants at SSH in 2018 whereby those with the highest level of education had higher mean knowledge scores. This result did not come as a surprise as it makes sense that the higher the level of education, the more knowledge a person will have. However, high education levels do not necessarily equate to high levels of attitude and practice (Sa’adeh et al., 2018). White et al. (2008) had also reported higher knowledge in subjects with higher education which was supported by Stanifer et al. (2016); Khalil & Abdalrahim (2014) and Yusoff et al. (2016).\n\nHealth literacy deals with an individual’s ability to obtain, read, process and understand health-related information to make applicable health decisions (Jain & Green, 2016; Van den Broucke, 2014). The influence of health literacy on health-related decision-making helps to explain the link between knowledge and level of education as shown by this current study in SSH. As the participants of this study with higher levels of education are likely to have a better ability to comprehend medical information given to them, it seems likely that their knowledge scores would be relatively greater compared to those who have lower level of education (thus lower health literacy). The real challenge of the health sector lies in this finding, since it means that health information must be translated into the simplest terms (free of medical jargon) and made available in the widest accessible form, so as to reach the greater subset of the population who lack higher education.\n\nSurprisingly, unemployed subjects were found to have significantly higher level of knowledge in this study, although were not associated with better attitude and practice. Stanifer et al. (2016) had found similar link between unemployed participants and knowledge (Stanifer et al., 2016). On the contrary, Yusoff et al. (2016) concluded that employed people had higher knowledge and attitude which was supported by Li et al. (2014) and Ryder et al. (2013). These are lot of conflicting information regarding employment and level of knowledge and perhaps future studies of DKD patients locally, regionally or internationally could clarify the doubts.\n\nRuhm (2005) found that health-related prevention behaviors were higher in unemployed people. This analysis was made from a Behavioral Risk Factor Surveillance System (BRFSS) in USA, and could probably explain the findings of the current research whereby unemployed participants had higher knowledge levels (Ruhm, 2005). However, the attitude and practice levels could not be linked with unemployment in SSH in 2018 and thus it is difficult to use the BRFSS solely as it deals with behaviors rather than knowledge alone.\n\nPatients with T2DM and CKD at SSH in 2018 with high average monthly income had significantly higher knowledge levels regarding DKD, although the link to attitude and practice was insignificant. Similarly, Yusoff et al. (2016) found poor knowledge in those with low family income, while Khalil & Abdalrahim (2014) had showed that subjects with high monthly income had high practice scores. Income is one of the key pillars of socioeconomic status (SES)—the other two being occupation and education. Therefore, it makes sense if participants with high monthly income have high knowledge since the interplay between the social gradient and health literacy is quite predictable (Diamond et al., 2011; Quinlan et al., 2013).\n\nFiscella (2016) stated that a person’s behavior is limited by their SES and hence their health-seeking behavior will change if their access to resources is increased (Fiscella, 2016). This means that the socio-economic disparities of participants with lower monthly income at SSH will need to be tackled by primary healthcare workers to influence KAP. This is not an easy task and thus may involve health and policy-making at the operational level to take into account the issues of health equality and equity.\n\nThe strengths of the study are that this is probably the first study done in Fiji to focus on the KAP of patients with T2DM and CKD. The baseline demographical information showed almost equal representation of sample in terms of gender, ethnicity and employment status, and thus the biasness is substantially reduced. The availability of the survey tool in three languages enabled the collection of data from all the ethnic groups (FID, iTaukei and FoD).\n\nThe limitations of this study are that the sample size was very small and the lack of generalizability of the results to all the population. Due to the questionnaire being self-answered by the participants, there is also a high chance of errors or misrepresentation of information.\n\n\nConclusion\n\nThis quantitative, cross-sectional study showed that the study participants have an overall high level of KAP, with average values of 23.3 (SD±3.25), 23.1 (SD±2.73) and 7.1 (SD±2.04), respectively. Those with higher level of education, unemployed and high monthly income had higher knowledge while FID had lower attitude scores but high practice scores, while the higher age category had lower attitude scores.\n\nConsequently, this research was able to identify high-risk groups with low levels of KAP, towards whom public health interventions can be targeted in future. The results of this study can enable informed public-health policies to be made to target the specific groups with low KAP and consequently increase their KAP through well-planned, appropriate and tailored strategies that suit the identified groups. Consequently, health promotion activities are vital in improving KAP, and it is recommended for interventional studies to be conducted using the results of this study among patients to measure the effectiveness of health promotion intervention.\n\n\nData availability\n\nOpen Science Framework: T2DM among CKD patients. https://doi.org/10.17605/OSF.IO/A25GD (Mohammadnezhad, 2019). Raw data are included in the indicated file.\n\nOpen Science Framework: T2DM among CKD patients. https://doi.org/10.17605/OSF.IO/A25GD (Mohammadnezhad, 2019).\n\nThis project contains the following extended data:\n\nQuestionnaire.pdf (questionnaire in each language).\n\nConsent Forms.pdf (in each language).\n\nInformation sheet.pdf (information for participants in each language).\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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe are grateful to the Sub-Divisional Medical-Officer of Sigatoka Sub-divisional Hospital, Dr Amos Zibran, for agreeing to utilize the hospital’s Special Outpatient Department for the research. We thank Mrs. Sabiha Khan for her advisory input and encouragement.\n\n\nReferences\n\nAtkins RC, Zimmet P: Diabetic kidney disease: act now or pay later. Nephrol Dial Transplant. 2010; 25(2): 331–333. PubMed Abstract | Publisher Full Text\n\nBouchard J, Macedo E, Soroko S, et al.: Comparison of methods for estimating glomerular filtration rate in critically ill patients with acute kidney injury. Nephrol Dial Transplant. 2010; 25(1): 102–107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCanto JG, Rogers WJ, Goldberg RJ, et al.: Association of age and sex with myocardial infarction symptom presentation and in-hospital mortality. JAMA. 2012; 307(8): 813–822. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConsumer Council of Fiji: Costs of Kidney Dialysis. Retrieved February 19, 2018, 2017. Reference Source\n\nDiamond C, Saintonge S, August P, et al.: The development of building wellness™, a youth health literacy program. J Health Commun. 2011; 16(Suppl 3): 103–118. PubMed Abstract | Publisher Full Text\n\nFiscella K: Relationships Between Income, Health Behaviors, and Life Expectancy. JAMA. 2016; 316(8): 880. PubMed Abstract | Publisher Full Text\n\nGhannadi S, Amouzegar A, Amiri P, et al.: Evaluating the Effect of Knowledge, Attitude, and Practice on Self-Management in Type 2 Diabetic Patients on Dialysis. J Diabetes Res. 2016; 2016: 3730875. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIdris I, Tohid H, Muhammad NA, et al.: Anaemia among primary care patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD): a multicentred cross-sectional study. BMJ Open. 2018; 8(12): e025125. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIHME: Country Profile - Fiji. Retrieved February 10, 2018, from Institute of Health Metrics and Evaluation, 2016. Reference Source\n\nIslam FM, Chakrabarti R, Dirani M, et al.: Knowledge, attitudes and practice of diabetes in rural Bangladesh: the Bangladesh Population based Diabetes and Eye Study (BPDES). Plos One. 2014; 9(10): e110368. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain D, Green JA: Health literacy in kidney disease: Review of the literature and implications for clinical practice. World J Nephrol. 2016; 5(2): 147–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaliyaperumal K: Guideline for conducting a knowledge, attitude and practice (KAP) study. AECS illumination. 2004. Reference Source\n\nKazley AS, Johnson E, Simpson K, et al.: African American patient knowledge of kidney disease: A qualitative study of those with advanced chronic kidney disease. Chronic Illn. 2015; 11(4): 245–55. PubMed Abstract | Publisher Full Text\n\nKhalil A, Abdalrahim M: Knowledge, attitudes, and practices towards prevention and early detection of chronic kidney disease. Int Nurs Rev. 2014; 61(2): 237–45. PubMed Abstract | Publisher Full Text\n\nKim SS: Predictors of short-term smoking cessation among Korean American men. Public Health Nurs. 2008; 25(6): 516–25. PubMed Abstract | Publisher Full Text\n\nKim SS, Gulick EE, Nam KA, et al.: Psychometric properties of the alcohol use disorders identification test: a Korean version. Arch Psychiatr Nurs. 2008; 22(4): 190–9. PubMed Abstract | Publisher Full Text\n\nKim SS, Kim SH, Gulick EE: Cross-cultural validation of a smoking abstinence self-efficacy scale in Korean American men. Issues Ment Health Nurs. 2009; 30(2): 122–30. PubMed Abstract | Publisher Full Text\n\nLi C, Wen X-J, Pavkov ME, et al.: Awareness of kidney disease among US adults: Findings from the 2011 behavioral risk factor surveillance system. Am J Nephrol. 2014; 39(4): 306–313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLincoln J, Mohammadnezhad M, Khan S: Knowledge, Attitudes, and Practices of Family Planning Among Women of Reproductive Age in Suva, Fiji in 2017. J Women's Health Care. 2018; 7(3): 431. Publisher Full Text\n\nMohammadnezhad M: T2DM among CKD patients. 2019. http://www.doi.org/10.17605/OSF.IO/A25GD\n\nMoHMS: Diabetes. Ministry of Health and Medical Services. Retrieved February 10, 2018. 2015. Reference Source\n\nMutiso J, Kayima J, Amayo EO: Knowledge, attitudes and practices on measures to retard disease progression among chronic kidney disease patients at Kenyatta National Hospital. University of Nairobi. 2011. Reference Source\n\nO’Neil PM, Smith SR, Weissman NJ, et al.: Randomized placebo-controlled clinical trial of lorcaserin for weight loss in type 2 diabetes mellitus: the BLOOM-DM study. Obesity (Silver Spring). 2012; 20(7): 1426–36. PubMed Abstract | Publisher Full Text\n\nQuinlan P, Price KO, Magid SK, et al.: The relationship among health literacy, health knowledge, and adherence to treatment in patients with rheumatoid arthritis. HSS J. 2013; 9(1): 42–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRifkin DE, Coca SG, Kalantar-Zadeh K: Does AKI truly lead to CKD? J Am Soc Nephrol. 2012; 23(6): 979–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoomizadeh P, Taheri D, Abedini A, et al.: Limited knowledge of chronic kidney disease and its main risk factors among Iranian community: an appeal for promoting national public health education programs. Int J Health Policy Manag. 2014; 2(4): 161–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuhm CJ: Healthy living in hard times. J Health Econ. 2005; 24(2): 341–363. PubMed Abstract | Publisher Full Text\n\nRyan D, Talemaitoga A, Fa’amoe A, et al.: Best health outcomes for Pacific peoples: Practice implications. Medical Council of New Zealand. 2010. Reference Source\n\nRyder PT, Coy K, Ohmit A, et al.: Knowledge, Attitudes, Behaviors, and Beliefs about Chronic Kidney Disease in Indiana’s Minority Communities: A Community-Based Survey. 2013. Reference Source\n\nSa’adeh HH, Darwazeh RN, Khalil AA, et al.: Knowledge, attitudes and practices of hypertensive patients towards prevention and early detection of chronic kidney disease: a cross sectional study from Palestine. Clin Hypertens. 2018; 24(1): 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSnowdon W, Tukana I: Fiji - NCD Risk Factors STEPS REPORT 2011. Suva: Ministry of Health and Medical Services. 2011. Reference Source\n\nStanifer JW, Karia F, Voils CI, et al.: Development and validation of a cross-cultural knowledge, attitudes, and practices survey instrument for chronic kidney disease in a Swahili-speaking population. PLoS One. 2015; 10(3): e0121722. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStanifer JW, Turner EL, Egger JR, et al.: Knowledge, Attitudes, and Practices Associated with Chronic Kidney Disease in Northern Tanzania: A Community-Based Study. PLoS One. 2016; 11(6): e0156336. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTekanene M, Mohammadnezhad M, Khan S: Knowledge, Attitude and Practice (KAP) study towards type 2 diabetes mellitus among adults who attend public health clinics in South Tarawa, Kiribati in 2017. Fiji National University, The School of Public Health and Primary Care, College of Medicine, Nursing, and Health Sciences. 2018.\n\nThirsk LM, Moore SG, Keyko K: Influences on clinical reasoning in family and psychosocial interventions in nursing practice with patients and their families living with chronic kidney disease. J Adv Nurs. 2014; 70(9): 2117–2127. PubMed Abstract | Publisher Full Text\n\nVan den Broucke S: Health literacy: a critical concept for public health. Arch Public Health. 2014; 72(1): 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite SL, Polkinghorne KR, Cass A, et al.: Limited knowledge of kidney disease in a survey of AusDiab study participants. Med J Aust. 2008; 188(4): 204–208. PubMed Abstract\n\nYakush Williams JK: Management Strategies for Patients with Diabetic Kidney Disease and Chronic Kidney Disease in Diabetes. Nurs Clin North Am. 2017; 52(4): 575–587. PubMed Abstract | Publisher Full Text\n\nYusoff DM, Yusof J, Kueh YC: Knowledge, Attitude And Practices Of The Risk For Chronic Kidney Disease Among Patients In A Tertiary Teaching Hospital. The Malaysian Journal of Nursing. 2016; 8(2): 3–11. Reference Source\n\nZheng Y, Ley SH, Hu FB: Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat Rev Endocrinol. 2018; 14(2): 88–98. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "45137",
"date": "27 Mar 2019",
"name": "Gade Waqa",
"expertise": [
"Reviewer Expertise NCD risk factors",
"health behavioral changes",
"policy analysis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors utilized a self-structured questionnaire to test KAP of 225 patients at Sigatoka Sub-divisional Hospital in Fiji, and used independent t-test and ANOVA to test the differences between demographic variable and practical score while non-parametric tests were used for knowledge and attitude which allowed guidance to promote effective public health interventions to high risk groups. The power of using the survey tool in three languages was demonstrated through the high response rate, although the sample size was very small that lack the generalization of the results to all the population.\nIt would be interesting in the discussion to compare this findings to any Fiji findings with other low income countries, including in the region if that is available.\nClarification is needed regarding definitions of the three ethnic groups since the results are discussed using the different ethnicity category, as usually data for Fiji is based on three groupings.\nThe method and discussion both mention the problem of poor knowledge and education level affecting data, yet the issue is not really explored as to its possible impact on the outcomes of the analysis. What are the implications of this? This should be discussed in the discussion section.\nThe discussion focuses only on ethnic differences with KAP, would it be possible to also discuss the gender differences with KAP? It is interesting that many aspects of lifestyle are better in females than males according to STEPS 2002, and this should be discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4555",
"date": "16 Apr 2019",
"name": "Masoud Mohammadnezhad",
"role": "Author Response",
"response": "Thanks for your time and comments. I would like to respond to your comments as below: No other similar KAP studies on T2DM patients with CKD was found that was done in other countries in the Western Pacific Region The 3 ethnic groups used are the I-Taukei, Fijians of Indian Descent and Fijian of Others Descent. Since the inclusion criteria included Fiji citizens only, the above 3 groups of people would qualify for the study. Poor education level affecting data has not been discussed in detail as this was not the main objective of this study Gender differences in KAP did not show any significant differences and hence was not discussed."
}
]
},
{
"id": "45138",
"date": "02 Apr 2019",
"name": "Barbara M. Daly",
"expertise": [
"Reviewer Expertise Quantitative research",
"Management of diabetes",
"gestational diabetes (screening and long term outcomes)"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral feedback This paper reports on knowledge, attitudes and practice or behaviours of diabetes patients with chronic kidney disease (CKD) surveyed while attending an out-patients clinic in Suva, Fiji in 2018. Despite patients having a major complication of diabetes, indicating poor control of HbA1c, hypertension and tobacco use, overall patients displayed a good knowledge, attitude and behavioural practice toward diabetes. This may reflect increased knowledge and practice in long-term diabetes patients who have to manage a serious complication or it may reflect a disconnect between knowledge and good self-management.\n\nOverall, authors identify factors that relate to knowledge, attitudes and practice that may influence health behaviour and outcomes in diabetes patients with CKD. However, authors need to carefully edit this manuscript, greatly reduce the word count by deleting repetitive themes, ensure results are clear, review reference groups for multiple linear regression analyses and adhere to the usual academic writing style. Specific suggestions to improve the paper are outlined below.\n\nTitle – suggest 'Determinants of knowledge, attitude and practice in patients with type 2 diabetes and chronic kidney disease in Fiji'\n\nAbstract: Methods\nOnly refer to Fiji and elaborate later, only include the year and self-administered questionnaire. You need to include the number of patients / total who came to the clinical and number in study. Avoid most abbreviations in the abstract. Remove unnecessary descriptions: name of hospital (Fijian citizens, special out-patients department, (Suva, Fiji is sufficient). Remove most of the statistical information and leave this to methods section.\n\nResults\n\nOutline numbers in study Response rate – this is very important Results: Out of what score i.e. 23.3 / 30. Can you qualify ‘unemployed’. For example, are these mostly students, mothers are they collecting an unemployment benefit, are they actively seeking work. As it stands this large group of participants do not typically resemble ‘unemployed people’ (around 5% in developed countries at the moment. Explain why so many people in the study were ‘unemployed’. This will help you contextualise unemployed group surveyed.\n\nConclusion\nSummarise the main outcomes are rather than KAP so the reader fully understands this and avoid just repeating the results.\nIntroduction\nPlease give some more details about diabetes in Fiji – almost all have type 2 I believe. What proportion have CKD (what proportion are on dialysis and is this available to al. What is the mortality rate for patients with CKD. If you cannot find national data can you look at the data from the clinic? If not you need to suggest that this is done in a ‘recommendation section’ in the discussion. It is important to give readers a sense of the seriousness of the epidemic in Pacific countries.\nGrammar\nRemove first sentence Delete all emotive language i.e. ‘this is quite worrying’ instead compare 15.6% with other populations. Paragraph two – check description. When was this developed and is it considered the best tool to test these attributes. Is there evidence to suggest that patients who score highly are more likely to have fewer complications? Has this been used before in high-risk patients (diabetes and CKD)? Paragraph 3 – change beginning to: This is the first study in Fiji …\n\nMethods\nSample – it is important to describe this. What number of patients registered at the clinical with diabetes and CKD? Look at your sample does this represent the gender, ages, ethnicity, education etc. of the total outpatient clinic – this is what you need to discuss regarding selection bias. If Polynesian Fijians are less likely to come for appointments, are they underrepresented? Also comment on this in the limitation section especially if you cannot get these details about the total cohort of patients registered. If you cannot get data on the total cohort i.e. to check gender, age and ethnicity then you need to discuss whether the patients sampled are representative of all patients registered or enrolled in the clinic. Are all patients in Suva able to register – what about rural based patients?\n\nUnemployed – I suggest a different definition. It important you give more information about ‘unemployed’ because your finding do not fit the usual picture. A third of your sample are unemployed. How was this defined i.e. on a benefit, actively looking for work and what is the unemployment rate in Fiji. I wonder if a large proportion of these participants are students or recent graduates i.e. well educated but not employed. Are better-off family members or partners supporting some of them? If many are in the younger age categories this might be the case so you could have a look at the data. Alternatively, are they too sick to work? If Fiji has a very high unemployment rate then you need to explain this so that the reader knows that being unemployed is different to the usual characteristics of most unemployed people receiving a benefit. If there is a strong other characteristic such as recent university graduate then call this something else i.e. currently seeking employment or something that better defines them. It is very confusing for readers as this group are atypical. Are they largely mothers who have not returned to the workforce?\n\nPlease briefly define how ‘knowledge, attitude and practice’ were measured and explain the main focus of the knowledge questionnaire i.e. mostly about diabetes and HbA1c control and consider including a couple of key questions. What were the main ‘attitude questions’ i.e. positive attitude, negative (explain to the reader) and similarly with practice i.e. include behaviours measured such as smoking. Consider given a couple of examples to illustrate the focus.\n\nBring the information about the questionnaire into this section. Also, cover the history of the questionnaire. Remove unnecessary text i.e. SPOD – only include descriptions if this is necessary. Remove ‘most importantly’ – no one will participate if they don’t want to. Did they sign a consent form or give oral consent. We know you cannot make patients participant. What did you do to encourage patients to participate i.e. what measures to increase participation? Review exclusion criteria and avoid repeating ideas. What was mental ability i.e. an inability to complete the questionnaire i.e. be specific. It seems to me as if all patients with type 2 and CKD were invited to participate. However XX patients were unable to ‘read’, write or complete the questionnaire with assistance and were therefore excluded from the study. (I think only people who were incapable of completing the questionnaire were excluded so make this clear (this is good as most people were able to participate). Para 2 – review and delete as most of this is repetition Para 3 – describe accurately i.e. all patients who attended … see above most of this is repetition. A cross-sectional study is quantitative not convenient. Describe the cohort of patients eligible, were patients recruited every day during the study period. If all patients were invited, provided they could complete the questionnaire, then state this. Move comment on bias to the discussion under strengths and limitations. Para 4 – briefly state the total score in the abstract as this is important. Para 5 – Reduce this paragraph i.e. … the questionnaire was piloted by… to ensure face validity. Content validity was assessed by ?? an expert panel… (you do not need all the details that you would include in a thesis). Data collection: reduce details and write succinctly. I suggest you change ‘illiterate’ to ‘unable to complete the questionnaire on their own’ i.e. they may need glasses to read! Data analysis – how many questionnaires were handed out how many returned?\n\nResults\n\nTitles – You have far too many titles so need to reduce the number.\n‘Participant’ demographic characteristics. Factors related to knowledge etc.\n\nPara 1\nAvoid repeating all data from the tables – summarise i.e. over half the patients were aged 46-60 years. Avoid repeating phrases i.e. participant in this study Define intake (indigenous or Polynesian – what do people prefer) Do not use FID (think about the international reader) Fijian Indian / Fijian of Indian decent or the official term or what people prefer.\n\nPara 2 and 3\nAvoid repeating results. Summarise the main points.\n\nTable 4\nGo through and review all your reference groups – these should be groups with a reasonable number of people not extreme groups with few numbers i.e. use 46-60 for age, level of education use secondary, use employed, use married, income either <400 or 4-800. Abbreviations should be listed under the tables B = beta coefficient\n\nPara 4\nSummarise the main points i.e. what factors were associated with knowledge … what were negatively associated. Similarly for attitude and practice Summarise overall findings – were any factors positively associated with all three attributes, two or none. Adjust text after you change the reference groups and reanalysis this data.\n\nDiscussion\nNew Para 1\nAlways write a summary of main findings a little like the above paragraph and use knowledge, attitude and practice. Readers need to be able to easily understand what was measured (avoid KAP in the main summaries). Are there any trends (see Para 4 above). You did not measure prevention. All of these patient were already diagnosed and have a major complication (CKD). Be specific to this group of patients sampled. You cannot generalise your results to other diabetes patients.\n\nNew para 2\nComparing main results with other study findings. Typically cite the studies with same findings then studies with opposite findings. Avoid informal phrases i.e. ‘another article from the USA’ …\n\nOld Para 2\nDelete first sentence this is irrelevant. Focus on Type 2 diabetes with major complications i.e. CKD. CKD is related to time since diagnosis and exposure to risk factors and other factors such as genetics, ethnicity.\n\nPara 3\nMove this to the methods section and describe the total cohort of patients with type 2 and CKD registered at the outpatients clinic. Delete all judgemental statements. Include barriers to good health care facility in Fiji and cite evidence.\n\nInclude a recommendation section at the end. Include recommendations to reduce barriers to healthcare i.e. mobile clinic for rural-based patients, funding so they can attend clinic, free transport (buses to clinic), convenient time for appointments. Think about why patients do not come to appointments.\n\nPara 4\nReview this para. You are being very judgemental and have very few references. Please appreciate that all Pacific populations have been influenced by unhealthy western lifestyle changes. When you think about it if Pacific populations continued the behaviour of their ancestors they would be fit, lean, non-smoking and very physically active. Consider how could the Government could enact healthy lifestyle changes i.e. tax on sugar, fat and tobacco? Avoid comparing ethnic groups unless you have good evidence. Focus on your findings on knowledge, my understanding is that this was not to ethnicity!\n\nPara 5\n\nMove this into comparisons with other studies above.\n\nPara 6\nYou did not measure health literacy you cannot bring this into your findings. Focus on the associations you found. Remember that you found a good mean level of knowledge and yet these patients had progressive complications for diabetes. Knowledge does not always equate to better management. Highlight that despite good self-reported ‘knowledge’ demonstrated in the survey these were high-risk patients, with a major complication and are at high risk for further poor outcomes. Can you comment on what changes need to occur in Fiji to reduce the epidemic of people developing diabetes and complications?\nPara 7\nReview definition of ‘unemployed’. This usually refers to people receiving an unemployment benefit and looking for work. A third of the sample are ‘unemployed’ so it is very different from the usual unemployed group in developed countries. Again focus on your results and comparative studies. Avoid discussing other issues that you did not examine in your survey.\n\nPara 8\nThis is not clear. Do not bring in other questionnaires but refer to other studies that support your findings or contrast and try to explain why there might be differences between your sample and other populations. Unemployment in the US is very different from in Fiji i.e. <5% are unemployed in the US.\n\nLimitations\nDiscuss the questionnaire. Is it an ideal questionnaire to test knowledge, attitudes and practice in the high-risk group of patients who already have CKD. Are other populations where this questionnaire has been tested the same as people registered in the outpatients in Suva? If not how might the questionnaire have been adapted. Were there knowledge, attitude and practice questions about CKD and management of risk factors? My understanding is that the questionnaire focused on Hba1c. I believe there were very few questions about other risk factors such a hypertension, smoking and cholesterol which are very important for good management and to avoid further renal dysfunction. Point this out as a limitation of the survey tool or questionnaire. Recommend that the questionnaire may need updating to include all risk factors for complications and adapted for those with progressive serious complications. This tool may not be a good measure of healthy behaviour in diabetes patients with CKD i.e. has it been used in this group before i.e. very high-risk group of people with type 2. This is important for other researchers to consider. You must compare you sample with the total cohort of high risk patients in the clinic not just those in the sample (age ethnicity and gender). If your sample differs from the whole cohort then it is not representative of the total cohort. You must be specific when discussing bias and why (which way will it increase bias) i.e. selection, information and confounding. Be clear which potential bias you are referring to. You must say why the sample size was small i.e. would there be greater differences or significant differences if the sample was larger. Why do you say this is not generalizable? Provide you asked all diabetes patients with CKD over the study period it will be representative of those high risk patients during the study period provided equal proportions of people not come to their clinic appointments. If this was the case you need to state this provided you have data on this. Comment on self-reported responses and the implications of this i.e. information bias toward ‘politically correct’ responses. Because this is not random error information bias in this case information bias would be in the positive direction. No clinical indices were measured. For example you do not report Hba1c so you cannot report on associations between knowledge attitude, behaviours and risk factors.\nNew Recommendation paragraph\nSee suggestions above. Mostly to reduce barriers to health care, support for patients to improve lifestyles and to better manage risk factors for complications. Most important how to improve healthy behaviours in this high risk patients group – they need intensive health care provision.\n\nConclusion\n\nSummarise the main findings. Delete all data and avoid using KAP. Move the last paragraph up to the first under conclusion and focus on the main findings. You cannot make most of these statements as you do not know what those strategies are. I suggest you finish on how on further research is necessary to identify strategies to improve management and outcomes for these high risk patients.\n\nEditing required\nDelete all emotive language and lay descriptions i.e. on the other hand, it did not come as a surprise, this is not an easy task, it makes sense… check the whole document and delete all of these phrases. You need to write in standard academic language. Check all abbreviations and avoid non-standard abbreviations. Be consistent with people in the study either use ‘participants or patients – not subjects! Check titles and avoid unnecessary descriptors i.e. type two diabetes with CKF (we know this is the patient group). Avoid repeating participants in study. You often have this twice in one sentence or paragraph. Check capitalisation i.e. ethnicity Most authors now use ‘among’ rather than amongst. Abbreviations should be listed under the tables ‘B’ = beta coefficient. Delete all judgemental statements.\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": "4556",
"date": "16 Apr 2019",
"name": "Masoud Mohammadnezhad",
"role": "Author Response",
"response": "Thanks for your time and comments. Your comments were very useful and we tried to revise our paper based on your valuable comments. Kindly pleas find blow our response to your comments. Abstract It is better to clearly mention the setting of the study The number mentioned (225) is based on the 2 months period of study for total eligible sample size – our sample size is not based on the total number of attendance The abbreviations are based on the first repeat in the article and it already was reviewed by the journal editors They all are study inclusion/exclusion criteria which need to be mentioned in the study method A brief description of the tests used for this study is mentioned and we followed the word limitation of the journal Results Number of participants added Sentence added to abstract about response rate Score added Employment status was categorized only based on yes/no and further details were not asked Conclusion Conclusion corrected as per reviewer’s comments Introduction Because of limited information, we have included all data that we could access and hence we do not have information about types of Diabetes in Fiji and the CKD dialysis and mortality rates in Fiji Clinic data has not been audited and thus cannot be used for this study Grammar We have to define the terms used in this study Emotive language changed as per reviewer comments Since this is a quantitative KAP survey, a KAP questionnaire which has been developed from 2 KAP questionnaires (with Cronbach’s alpha of more than 0.7), this is the best tool for the KAP measurement. We are not doing a study to check association between KAP score and its link to complications of T2DM Changed as per reviewer Method The hospital used for this study is a referral hospital and thus it receives patients from all settings, ethnicities, ages, gender, etc. This study showed that the ethnic distribution was almost equal in the 2 major ethnicities thus chance of biasness is limited. The questionnaire did not ask details about employment status. This study did not look at the younger age categories thus they could not be recent graduates. The participants are attending SOPD clinic and usually are older age groups. More info about the KAP questions added. The information details were provided. Questionnaire history already mentioned in introduction SOPD needs to be mentioned as abbreviation of the clinic. Most-importantly removed. The details of recruitment is mentioned in data collection process and all patients were provided consent forms and information sheets. No incentives was provided for participation but the potential benefit of participation was provided in info sheet and hence they were happy to help researcher with the study. Changed as per reviewer comments Para 2 – since this is a description of study setting, it cannot be deleted entirely. Para 3 – corrected Corrected as per comments Para 4 – already added total score Para 5 – corrected Data collection – corrected Data analysis – corrected Results Titles – corrected Para 1 – corrected Para 2 and 3 – these are main finding hence need to be explained in text. Table 4 – reference groups have been already identified and do not affect results. Abbreviation added Para 4 – Main points summarized Discussion New para 1 added New para 2 – not added but old para 1 revised Old para 2 – first sentence not removed since it is used to explain the paragraph. Diabetic kidney disease is used interchangably for T2DM patients with CKD. Para 3 – not moved since it is used to discuss the burden of CKD at SSH. The total cohort of patients with type 2 and CKD registered at the outpatients clinic at SSH is not available as there is no proper registration done. The comparison of FID is not a judgmental statement but others changed. Recommendation added in the end. Para 4 – reviewed. Judgmental statements altered. Government statements added. Ethnicity used to show determinants of KAP hence it is used in discussion. Para 5 – comparison is done in this paragraph since it follows the original pattern of discussion. Health literacy mentioned to elaborate on the link between patient’s ability to understand/comprehend information and their level of knowledge as a consequence. However, knowledge part added as per reviewer comments. Fiji’s changes added as per comments. Para 7 – unemployment details were not asked further in the main questionnaire hence cannot change it now. Other issues have been discussed despite not being examined in this study since they help to connect the points that have been identified in this research. Para 8 – The other questionnaire is a reference to discuss the topic in the paragraph. Sample differences have been explained in the latter paragraphs. Limitation The questionnaire was ideal as its validity and reliability have been mentioned. The other settings where KAP questionnaire have been tested are developing countries similar to Fiji. The KAP questions looked at both T2DM and CKD. The other risk factors were not included in the questionnaire since the focus was only T2DM and its link to CKD. Additionally, T2DM is the major cause of CKD thus this link was tested in the questionnaire. Other behavioral risk factors were not deliberated. It is difficult to compare my sample with the other cohorts in the clinic since there is no register to show the registered cases of high risk. The paper-based record keeping used in the clinic used for this study does not have any specific book to record all its cases by diagnosis and so forth. Biasness explained Sample size explanation added Generalizability portion edited Information bias added as per comment Comment added to limitation Conclusion Main findings summarized. Changes made as per comments Last paragraph moved to first Statements revised Changes made Recommendation New paragraph on recommendation added"
},
{
"c_id": "4597",
"date": "01 May 2019",
"name": "Masoud Mohammadnezhad",
"role": "Author Response",
"response": "Thanks you for your comments and suggestions. We corrected as per your comments. We also changed the sentence structure as you advised."
}
]
}
] | 1
|
https://f1000research.com/articles/8-239
|
https://f1000research.com/articles/8-348/v1
|
28 Mar 19
|
{
"type": "Research Note",
"title": "Pollen tube contents from failed fertilization contribute to seed coat initiation in Arabidopsis",
"authors": [
"Xiaoyan Liu",
"Parakash Babu Adhikari",
"Ryushiro D. Kasahara",
"Xiaoyan Liu",
"Parakash Babu Adhikari"
],
"abstract": "Plant seeds are essential for human beings, constituting 70% of carbohydrate resources worldwide; examples include rice, wheat, and corn. In angiosperms, fertilization of the egg by a sperm cell is required for seed formation; therefore, fertilization failure results in no seed formation, except in the special case of apomixis. Initially, plants produce many pollen grains inside the anthers; once the pollen grain is deposited onto the top of the pistil, the pollen tube elongates until it reaches the ovule. Generally, only one pollen tube is inserted into the ovule; however, we previously found that if fertilization by the first pollen tube fails, a second pollen tube could rescue fertilization via the so-called fertilization recovery system (FRS). Our previous reports also demonstrated that failed fertilization results in pollen tube-dependent ovule enlargement morphology (POEM), enlarged seeds, and partial seed coat formation if the pollen tube releases the pollen tube contents into the ovule. However, we have not determined whether all the ovules enlarge or produce seed coats if an ovule accepts the pollen tube contents. Therefore, we conducted a partial seed coat formation experiment taking into account both the FRS and POEM phenomena. Notably, the ratios of failed fertilization and the ovules with partial seed coats matched, indicating that all ovules initiate seed coat formation if the fertilization fails but the pollen tube contents enter the ovule. In addition, we confirmed that the agl62 mutant , defective in early endosperm formation, showed seed coat initiation with and without fertilization, indicating that for a normal seed coat initiation, fertilization is not required; however, for the completion of normal seed coat formation, both normal fertilization and endosperm formation are required. Further molecular evidence is required to understand these phenomena because very few factors related to FRS and POEM have been identified.",
"keywords": [
"Arabidopsis",
"Pollen tube",
"fertilization recovery system",
"pollen tube-dependent ovule enlargement morphology",
"GCS1",
"pollen tube contents",
"Seed coat initiation",
"AGL62"
],
"content": "Introduction\n\nIn angiosperms, seed formation begins with pollination1,2. Once the pollen grain lands on the stigma at the top of the pistil, pollen tubes from the grain elongate toward the ovule. Fusion of the two gametophytes is required for seed formation. The male gametophyte is the pollen grain and the female gametophyte is the embryo sac3. Immediately after arrival at the ovules, the pollen tube bursts and the pollen tube contents (PTC) are released to the female gametophyte4.\n\nIn a previous study, we reported that once the ovule accepts the PTC inside the female gametophyte, it begins enlargement and seed coat formation, irrespective of fertilization5,6. We named this phenomenon pollen tube-dependent ovule enlargement morphology (POEM). We also reported that if fertilization of the ovule fails, a partial seed coat is still produced, even though a complete seed coat cannot be formed. However, we have not confirmed whether all the ovules have the partial seed coat phenotype when ovule fertilization fails but PTC is accepted. To address this question, statistical experiments were conducted that included the fertilization recovery system (FRS), where a second pollen tube rescues the fertilization if fertilization by the first pollen tube fails, which we previously identified7,8. We reported that the seed formation ratio of gcs1/+ mutants9–11 was approximately 65%; the remaining mutants were unable to produce seeds because fertilization of these ovules failed. Therefore, matching of the ratio of the ovules with the partial seed coat phenotype to the seed abortion ratio suggests that there was fertilization failure for all ovules with a partial seed coat. We also conducted experiments to determine whether agl62 mutants12 had the partial seed coat phenotype. In agl62 seeds, the endosperm cellularizes prematurely, indicating that AGL62 is required for suppression of cellularization during the syncytial phase. During seed development, AGL62 is exclusively expressed in the endosperm. Because agl62 mutants have an abnormal endosperm phenotype after central cell fertilization, these mutants are ideal for investigating the relationship between endosperm formation and seed coat initiation and formation.\n\n\nMethods\n\nArabidopsis thaliana ecotype Columbia (Col-0) plants were used as the wild-type (WT) plants. Test cross experiments were conducted in gcs1/+9–11, agl62/+12, and WT plants. Seeds were sterilized with 5% sodium hypochlorite containing 0.5% Triton X-100 and germinated on plates containing 0.5× Murashige and Skoog salts (pH 5.7) (Wako Pure Chemical), 2% sucrose, Gamborg’s B5 vitamin solution (Sigma), and 0.3% Gelrite (Wako Pure Chemical) in a growth chamber at 21.5°C under 24 h of light after cold treatment (4°C) for 2 days. Next, 10-day-old seedlings were transferred to Metro-Mix 350 soil (Sun Gro) and grown at 21.5°C under 24 h of light.\n\nFor staining the silique tissue, the WT flowers were emasculated at stage 12c13 and pollinated with gcs1/+ pollen grains. For agl62 experiments, the agl62 mutant flowers were emasculated at stage 12c and pollinated with WT, gcs1/+, and agl62 pollen grains. The siliques were collected at 3 days after pollination (DAP).\n\nFor vanillin staining, the ovules were manually dissected from the ovaries and mounted on slides in 1% (wt/vol) vanillin (4-hydroxy-3-methoxybenzaldehyde; Sigma) in 6 N HCl solution. Slides were analyzed after 20 min of incubation. Samples were analyzed with a Leica DM2500 microscope using differential interference contrast optics. Images were recorded with a Leica DFC 300 FX digital camera at a magnification of 5×, 10× and 20×. The microscopic protocols followed were as previously described5.\n\n\nResults and discussion\n\nFirst, the WT plants as both the female and male parent (Figure 1A) were crossed and the silique after vanillin staining at 3DAP was observed. The ratio of full seed coat formation was 98.7±1.2% (mean ± SD; n=10 pistils), which was consistent with our previous WT fertilization data7,8. By contrast, when the WT plants as the female parent and gcs1/+ as the male parent were crossed, the ratio of full seed coat formation was 68.7±5.8% (n=10 pistils) and the ratio of partial seed coat formation was 32.2±6.5% (n=10 pistils), which also was consistent with our previous gcs1/+ fertilization data. These data suggest that all successfully fertilized ovules produce a full seed coat and all unfertilized but PTC accepted ovules produce a partial seed coat.\n\n(A) Wild-type (WT) silique crossed with WT pollen and stained with vanillin. Almost all ovules were stained. Bar: 300µm. (B) Representative image of whole seed coat staining. Bar: 50µm. (C) WT silique crossed with gcs1 pollen and stained with vanillin. Several ovules had partial seed coat (arrowhead) staining. Bar: 300µm. (D) Representative image of partial seed coat staining. Bar: 50 µm. (E) Comparison of seed formation ratio and vanillin staining ratio. ♀WT♂WT seed formation ratio (SF) indicates that a WT silique was crossed with WT pollen and the seed formation ratio was calculated. ♀WT♂WT vanillin stained in whole seed coat (VSW) indicates that a WT silique was crossed with WT pollen and the whole seed coat staining ratio was calculated. ♀agl62♂WT VSW indicates that an agl62/+ silique was crossed with WT pollen and the whole seed coat staining ratio was calculated. ♀WT♂gcs1 SF indicates that a WT silique was crossed with gcs1/+ pollen and the seed formation ratio was calculated. ♀WT♂gcs1 VSW indicates that a WT silique was crossed with gcs1/+ pollen and the whole seed coat staining ratio was calculated. ♀agl62♂gcs1 VSW indicates that an agl62/+ silique was crossed with gcs1/+ pollen and the whole seed coat staining ratio was calculated. ♀WT♂gcs1 seed abortion ratio (SA) indicates that a WT silique was crossed by gcs1/+ pollen and the seed abortion ratio was calculated. ♀WT♂gcs1 (vanillin stained in partial seed coat) indicates that a WT silique was crossed with gcs1/+ pollen and the partial seed coat staining ratio was calculated. ♀agl62♂gcs1 VSP indicates that an agl62/+ silique was crossed with gcs1/+ pollen and the partial seed coat staining ratio was calculated. ♀agl62♂ agl62 VSW indicates that an agl62/+ silique was crossed with agl62/+ pollen and the whole seed coat staining ratio was calculated. ♀agl62♂agl62 VSP indicates that an agl62/+ silique was crossed with agl62/+ pollen and the partial seed coat staining ratio was calculated. (F) A ♀agl62♂agl62 VSP ovule. The arrowhead indicates the vanillin-stained zone. Bar: 50 µm. For normal seed coat initiation, fertilization is not required; however, for completion of normal seed coat formation, both normal fertilization and normal endosperm formation are required.\n\nBecause the agl62 mutant had an abnormal and arrested endosperm formation phenotype after fertilization, this mutant was ideal for investigating the relationship between endosperm formation and seed coat initiation and formation. The agl62/+ plants as the female parent and the WT as the male parent were crossed (Figure 1) and the silique after vanillin staining at 3DAP was observed. The ratio of full seed coat formation was 97.6±2.1% (n=10 pistils), which was consistent with our previous WT fertilization data. By contrast, when agl62/+ plants as the female parent and agl62/+ as the male parent were crossed, the ratio of full seed coat formation was 74.7±3.9% (n=10 pistils) and the ratio of partial seed coat formation was 25.2±5.4% (n=10 pistils), which was consistent with our previous agl62 data12. These results suggest that normal endosperm formation is required for completion of seed coat formation, irrespective of fertilization. When agl62/+ plants as the female parent and gcs1/+ as the male parent were crossed, the ratio of full seed coat formation was 66.9±6.2% (n=10 pistils) and the ratio of partial seed coat formation was 33.2±5.9% (n=10 pistils), which also was consistent with our previous gcs1/+ fertilization data. These results suggest that agl62/+ abnormal endosperm prevents normal seed coat formation, but these ovules still produce a partial seed coat because these ovules had accepted the PTC. In summary, for normal seed coat initiation, fertilization is not required; however, for completion of normal seed coat formation, both normal fertilization and normal endosperm formation are required.\n\n\nData availability\n\nOpen Science Framework: Vanillin staining project. https://doi.org/10.17605/OSF.IO/6U73H14.\n\nThis project contains the following underlying data:\n\n# of seeds data (Sheet2 contains the number of seeds stained out of the total number of seeds; Sheet1 the data summary used to produce Figure 1E)\n\nagl61-3v.tif (raw image of stained agl62/+ seeds)\n\ngcs1 vaniline.tif (raw image of stained gcs1/+ seeds)\n\nWT vaniline.tif (raw image of stained wild-type seeds)\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 work was supported by start-up funds from the School of Life Sciences, Fujian Agriculture and Forestry University (Grant #: 114-712018008) and the FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Shaowei Zhu and Xiaoyan Wu for technical assistance.\n\n\nReferences\n\nMaheshwari P: An Introduction to the Embryology of Angiosperms. (New York: McGraw-Hill). 1950. Publisher Full Text\n\nKasahara RD: The Regulation of Sperm Cells Delivery to the Embryo Sac. Pollination in Plants. 2018. Publisher Full Text\n\nDrews GN, Koltunow AM: The female gametophyte. Arabidopsis Book. 2011; 9: e0155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamamura Y, Saito C, Awai C, et al.: Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr Biol. 2011; 21(6): 497–502. PubMed Abstract | Publisher Full Text\n\nKasahara RD, Notaguchi M, Nagahara S, et al.: Pollen tube contents initiate ovule enlargement and enhance seed coat development without fertilization. Sci Adv. 2016; 2(10): e1600554. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKasahara RD, Notaguchi M, Honma Y: Discovery of pollen tube-dependent ovule enlargement morphology phenomenon, a new step in plant reproduction. Commun Integr Biol. 2017; 10(4): e1338989. Publisher Full Text | Free Full Text\n\nKasahara RD, Maruyama D, Hamamura Y, et al.: Fertilization recovery after defective sperm cell release in Arabidopsis. Curr Biol. 2012; 22(12): 1084–1089. PubMed Abstract | Publisher Full Text\n\nKasahara RD, Maruyama D, Higashiyama T: Fertilization recovery system is dependent on the number of pollen grains for efficient reproduction in plants. Plant Signal Behav. 2013; 8(4): e23690. PubMed Abstract | Publisher Full Text\n\nMori T, Kuroiwa H, Higashiyama T, et al.: GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization. Nat Cell Biol. 2006; 8(1): 64–71. PubMed Abstract | Publisher Full Text\n\nvon Besser K, Frank AC, Johnson MA, et al.: Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization. Development. 2006; 133(23): 4761–4769. PubMed Abstract | Publisher Full Text\n\nNagahara S, Takeuchi H, Higashiyama T: Generation of a homozygous fertilization-defective gcs1 mutant by heat-inducible removal of a rescue gene. Plant Reprod. 2015; 28(1): 33–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang IH, Steffen JG, Portereiko MF, et al.: The AGL62 MADS domain protein regulates cellularization during endosperm development in Arabidopsis. Plant Cell. 2008; 20(3): 635–647. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChristensen CA, King EJ, Jordan JR, et al.: Megagametogenesis in Arabidopsis wild type and the Gf mutant. Sexual Plant Reproduction. 1997; 10(1): 49–64. Publisher Full Text\n\nKasahara R: Vanillin staining project. 2019. http://www.doi.org/10.17605/OSF.IO/6U73H"
}
|
[
{
"id": "46381",
"date": "08 Apr 2019",
"name": "Tomoko Igawa",
"expertise": [
"Reviewer Expertise Gamete imaging during doubler fertilization in flowering plants",
"cytological and morphological analysis of sexual plant reproduction processes",
"molecular biology focusing on proteins regulating fertilization"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNormal seed formation required for the birth of the progeny is a critical phenomenon for plant production and breeding field. This research note reports that the involvements of the fertilization and the normal endosperm development in seed coat formation. First, the involvement of the pollen tube contents was analyzed showing the reproducibility of their previous report. Also, it was indicated that normal endosperm development is required for successive seed coat formation, using agl62 mutant.\n\nIntroduction\nAuthors described constructively what they wanted to clarify. The experimental design and the statistical analysis performed in this study were enough to examine the questions.\n\nResults and Discussion\nIn the present state, the descriptions and the graph (Fig.1E) were confusing to understand the results. How about using different colors for each SF, VSF, AS, and VSP. Also, if the graph-bars obtained with the same female parent-genotype are grouped and indicated, it would be helpful for easy comparing the results. As a conclusion, the authors claimed that \"for completion of normal seed formation, normal endosperm formation is required in addition to normal fertilization.\" In the previous studies, the aborted seed caused by the single fertilization of the central cell seemed to have developed the seed coat (kokopelli, gex2, dmp9 mutants, etc...). These studies are supportive of the authors' claim, but the endosperm of these mutants was not normal as the wild type seed. Therefore, how about describing \"normal endosperm 'development' is required for seed coat formation.\" As a minor point, authors describe the previous study of agl62 (Kang et al., 2008)1 as 'our previous....' but no same authors in this paper.\nOverall, the content of this research note is logically written. If the points described above are re-considered, it would be improved.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4550",
"date": "09 Apr 2019",
"name": "Kasahara Ryushiro",
"role": "Author Response",
"response": "Dear Dr. Igawa, Thank you very much for your critical comments. I will improve the paper based on your comments as soon as possible. Best regard, Ryushiro Dora Kasahara"
},
{
"c_id": "4563",
"date": "30 Apr 2019",
"name": "Kasahara Ryushiro",
"role": "Author Response",
"response": "Dear Dr. Igawa,I have finished revising based on your suggestions.In the present state, the descriptions and the graph (Fig.1E) were confusing to understand the results. How about using different colors for each SF, VSF, AS, and VSP. Also, if the graph-bars obtained with the same female parent-genotype are grouped and indicated, it would be helpful for easy comparing the results.I have submitted a new version of the Fig.1. Please take a look.As a conclusion, the authors claimed that \"for completion of normal seed formation, normal endosperm formation is required in addition to normal fertilization.\" In the previous studies, the aborted seed caused by the single fertilization of the central cell seemed to have developed the seed coat (kokopelli, gex2, dmp9 mutants, etc...). These studies are supportive of the authors' claim, but the endosperm of these mutants was not normal as the wild type seed. Therefore, how about describing \"normal endosperm 'development' is required for seed coat formation.\"I changed the word to normal endosperm development.As a minor point, authors describe the previous study of agl62 (Kang et al., 2008)1 as 'our previous....' but no same authors in this paper.I removed \"our\". from the text.Again, thank you very much for your critical comments.Best regards,Ryushiro Dora Kasahara"
}
]
},
{
"id": "47023",
"date": "11 Apr 2019",
"name": "Nobutaka Mitsuda",
"expertise": [
"Reviewer Expertise plant biotechnology",
"transcription factor",
"reproductive development",
"cell wall",
"seed",
"cuticle"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 showed basically the confirmation of the previous report of the POEM paper. However, because this report showed that the relationships between pollen tube contents and seed coat formation statistically, including the data for agl62 mutants, they clearly confirmed that almost all ovules started seed coat formation without fertilization but just required for the pollen tube contents. I believe this report is a sort of important discoveries in the plant science field and will contribute to further research for seed coat formation in angiosperms.\n\nIntroduction:\nAt the introduction part, it is clear what they need to understand and what sorts of approach were taken. I believe their approach conducted here is adequate and leads to a neat statistics for seed coat initiation and completion study.\n\nResults and Discussion:\nI agree with the first reviewer’s comment. Authors could improve the paper if they change the colors for Fig. 1. However, I think this change is not mandatory because if authors change the color dramatically, it could be too showy. I thought current style looks fair enough. I also agree with the first reviewer's comment that because the study of agl62 was not the authors study, \"our previous agl62 data\" sounds strange. But this is just a very minor correction to be done.\n\nData availability:\nI think the data title agl61-3v. tif is wrong. Title should be \"agl62-3v\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4564",
"date": "11 Apr 2019",
"name": "Kasahara Ryushiro",
"role": "Author Response",
"response": "Dear Dr. Mitsuda,Thank you very much for your critical comments.We will improve our paper based on your suggestion.Again, thank you very much.Ryushiro Dora Kasahara"
}
]
},
{
"id": "46516",
"date": "15 Apr 2019",
"name": "Tomokazu Kawashima",
"expertise": [
"Reviewer Expertise Sexual plant reproduction",
"cellular dynamics",
"molecular biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes that the pollen tube content is sufficient to partially initiate seed coat without fertilization in Arabidopsis. This work appears the extension and further clarification of the previously published work (Kasahara et al., Science Advances 20161, and Roszak and Kohler, PNAS, 20112). During plant fertilization, many cellular processes are happening, and it is important to integrate the identified phenomena together for comprehensive understanding of plant sexual reproduction. The authors studied the relationships among FRS, POEM, seed coat initiation, and endosperm development, and I think the work reported in this paper is an important piece of information in sexual plant reproduction.\nI have a few suggestions:\nThe first paragraph of the introduction, the author stated that \"gametophytes\" fuse. I believe the authors mean \"gametes\". From the authors previous work in Science Advances, 20161), it is clear that PTC initiates seed coat development (transcriptionally and anatomically). It is important to state these details in the introduction to remind the readers. Along with this, please describe what vanillin staining detects (proanthocyanidin accumulation) and why this can be used for seed coat visualization. In Roszak and Kohler, 20112, the authors reported that not fis2 and mea, but fie and msi1 autonomously developing seeds differentiate seed coat, suggesting that fertilization is not required for seed coat initiation. The authors also described seed coat phenotypes in agl62. Please refer this publication and highlight and/or discuss new findings/hypotheses in this paper, then this paper will be more impactful. As other reviewers suggested, Figure 1E is difficult to dissect information. I believe \"vamiline\" in the Y-axis label should be \"vanillin\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4573",
"date": "16 Apr 2019",
"name": "Kasahara Ryushiro",
"role": "Author Response",
"response": "Dear Dr. Tomokazu Kawashima, Thank you very much for your critical comments. We will improve our paper based on your suggestion. Again, thank you very much. Ryushiro Dora Kasahara"
},
{
"c_id": "4574",
"date": "16 Apr 2019",
"name": "Kasahara Ryushiro",
"role": "Author Response",
"response": "Dear Dr. Tomokazu Kawashima,Thank you very much for your critical comments.We will improve our paper based on your suggestion.Again, thank you very much.Ryushiro Dora Kasahara"
}
]
}
] | 1
|
https://f1000research.com/articles/8-348
|
https://f1000research.com/articles/8-605/v1
|
30 Apr 19
|
{
"type": "Research Article",
"title": "Trends in Medicare base payment rates for hospital discharges, 2009-2018",
"authors": [
"Zachary Rapp",
"Ryan Egeland",
"Frank S. David",
"Zachary Rapp",
"Ryan Egeland"
],
"abstract": "Background: Under the inpatient prospective payment system (IPPS), Medicare assigns hospital discharges to medical severity-adjusted diagnosis related groups (MS-DRGs), and determines a fixed payment amount for each discharge based on its MS-DRG that is adjusted annually based on providers’ reported costs. Trends in these capitated reimbursement rates may affect the incentive for manufacturers to develop drugs and devices used in inpatient care, as well as the predilection of customers (hospitals) to purchase them. Methods: In this descriptive study, we analyzed the growth of inflation-adjusted MS-DRG base payment rates to acute care hospitals from 2009 to 2018. Results: Inflation-adjusted base reimbursement rates for MS-DRGs in continuous existence from 2009 to 2018 (N=211) had a median best-fit compound annual growth rate (CAGR) of -0.26%. Medical MS-DRGs exhibited a more negative median best-fit CAGR than surgical ones. Among surgical MS-DRGs, those related to musculoskeletal diagnoses had a larger median best-fit CAGR that was statistically significant compared with those associated with digestive or infectious disease diagnoses. Conclusions: The majority of MS-DRG base payment rates for inpatient discharges have failed to keep pace with inflation, and this negative growth is more pronounced in certain clinical areas. The relationship between these reimbursement trends and decision-making by manufacturers and hospitals warrants further study.",
"keywords": [
"Diagnosis related group",
"DRG",
"Medicare",
"inpatient",
"hospital",
"reimbursement",
"payment"
],
"content": "Introduction\n\nThe Centers for Medicare and Medicaid Services (CMS) reimburses most inpatient medical and surgical discharges in the U.S. under a capitated framework known as the inpatient prospective payment system (IPPS)1. Upon discharge, a patient is assigned to a medical severity diagnosis-related group (MS-DRG) based on the nature of the patient’s reason for hospitalization as well as any associated complications or comorbidities. Thus, providers are paid a fixed (capitated) rate for inpatient discharges, irrespective of the intensity of the services provided or the specific consumables (drugs or devices) utilized. This stands in contrast to ambulatory settings, where most products and services are reimbursed individually on a line-item basis.\n\nCMS adjusts the payment rates for MS-DRGs annually2. Each MS-DRG is assigned a weight based on providers’ submitted costs, which is intended to reflect the average resources expended to care for a patient in that MS-DRG, independent of region- and institution-specific factors. CMS also defines standardized base payment rates for labor, non-labor, and capital costs, which are also adjusted annually. These weights and standardized rates are used, together with institution-, region-, and case-specific modifiers, to determine a particular hospital’s reimbursement for a discharge under a particular MS-DRG.\n\nHospital administrators use Medicare base payment rates as a significant input into their decision to adopt new products for inpatient care3, and these rates may also influence manufacturers’ investment activities across various clinical areas. However, we have not observed in the literature a comprehensive examination across MS-DRGs of how these rates have changed over time. In this descriptive study, we analyzed base payment rates from 2009 to 2018 to explore the extent to which reimbursement for individual MS-DRGs has (or has not) kept pace with inflation during this period.\n\n\nMethods\n\nWe identified all MS-DRGs in continuous existence from fiscal year 2009 (the first full year after the transition to severity-based coding and cost-based reimbursement was completed4) to fiscal year 2018, and assigned them to major diagnostic categories (MDCs), using CMS definitions and annual IPPS “Final Rule” data tables (e.g., these data from fiscal year [FY]2018). For years in which correction notices were issued, we used the latest (corrected) data. In order to focus on MS-DRGs with the largest clinical and budgetary relevance, we limited our analysis to those with over $100 million in total Medicare spending in 2015 (per CMS data). Our final analysis set was comprised of 211 MS-DRGs (of the 761 MS-DRGs in existence in 2019), distributed across 21 MDCs (including “pre-MDC” and unassigned/unclassified). Our complete data file is available as Extended data.\n\nFor each year, we used available data provided by CMS to construct a “base reimbursement rate” for each MS-DRG, adapting methods described elsewhere5. We first obtained the weight of each MS-DRG from that year’s Final Rule tables (e.g., Table 5 in the FY2018 document). We then calculated the sum of the standardized labor- and non-labor-related amounts (Table 1A of the FY2018 document, “meaningful use” data), plus the capital amount (Table 1D of the FY2018 document) for that year. We then multiplied this total dollar amount by the MS-DRG’s weight to obtain a value for the base reimbursement rate in nominal dollars.\n\nWe adjusted each MS-DRG’s base reimbursement rate from nominal dollars to constant (January 2009) dollars by using the CPI-U calculator provided by the Bureau of Labor Statistics.\n\nFor each MS-DRG, we determined the “best fit” compound annual growth rate (CAGR) (r in the best-fit exponential line equation [y = a(1 + r)x]), which we refer to as the “best-fit CAGR” in the remainder of this article. Compared with standard CAGR calculations, which use just the starting and ending reimbursement rates, this approach takes into account all of the observed values over the time period. We also calculated each MS-DRG’s “expected” reimbursement rate by year, based on the derived slope and intercept of its best-fit line, in order to calculate the coefficient of determination (R2) between the calculated best-fit exponential equation values and the observed reimbursement rates.\n\nWe examined differences in best-fit CAGR between subsets of MS-DRGs with the Kruskal-Wallis test (on MDCs containing five or more MS-DRGs), and confirmed statistically significant results with post hoc pairwise comparisons (Dunn test with Bonferroni correction). All mathematical calculations and statistical analyses were performed using Microsoft Excel (see here and here).\n\n\nResults\n\nFrom FY2009 to FY2018, inflation-adjusted MS-DRG base reimbursement rates had a median best-fit CAGR of -0.26% (interquartile range [IQR], -0.89% to 0.47%) (Table 1)a. Over the analyzed time period, 59.2% (125/211) of MS-DRGs had negative best-fit CAGRs. In bivariate models, the variability in the best-fit CAGRs of individual MS-DRGs was not explained by either the 2015 Medicare spending level or 2018 inflation-adjusted base reimbursement rate (Figure 1; R2 < 0.02 for each).\n\nMajor diagnostic categories (MDCs) containing fewer than five medical severity diagnosis-related groups (MS-DRGs) are not independently reported or analyzed.\n\nIQR, interquartile range.\n\nEach circle represents one medical severity diagnosis-related group. Best-fit line with R2 is shown in each scatter plot.\n\nMedical MS-DRGs exhibited a more negative median best-fit CAGR than surgical ones (-0.57% vs. -0.14%; adjusted H=5.631, df=1, p=0.018 via Kruskal-Wallace). A total of 66% of medical MS-DRGs exhibited negative best-fit CAGRs (71/108), compared with 52% (54/103) of surgical ones.\n\nIn total, 11 MDCs were comprised of five or more MS-DRGs, and thus were suitable for further analysis. We found a statistically significant difference in median best-fit CAGRs between MDCs (adjusted H=63.128, df=10, p=9.23 × 10-10 via Kruskal-Wallace). Upon pairwise analysis (Dunn with Bonferroni correction), we determined that the best-fit CAGRs of MS-DRGs for musculoskeletal diagnoses (MDC 08) were statistically significantly larger than that of MS-DRGs for nervous, respiratory, digestive, or infectious diseases (MDCs 01, 04, 06, and 18, respectively) (Table 2).\n\nCorrected P values for post hoc pairwise comparisons of growth rates for best-fit compound annual growth rates by MDC. Statistically significant results (P<0.05) indicated in italics.\n\nOne plausible explanation for differences in median best-fit CAGRs could be the relative fraction of surgical MS-DRGs (which have higher growth) in different therapeutic areas; indeed, the musculoskeletal MDC is substantially enriched for surgical MS-DRGs (25/29 (86%), compared with 103/211 (49%) in the overall sample). Thus, we re-analyzed median best-fit CAGRs across MDCs after first separating medical MS-DRGs from surgical ones. There were eight MDCs comprised of 89 surgical MS-DRGs, and seven MDCs comprised of 84 medical MS-DRGs, suitable for analysis (i.e., comprised of five or more MS-DRGs).\n\nLooking only at surgical MS-DRGs, we found a statistically significant difference in median best-fit CAGRs between MDCs (adjusted H=32.465, df=7, p=3.3 × 10-5 by Kruskal-Wallace), and on pairwise analysis, we confirmed that the median best-fit CAGR for surgical MS-DRGs for musculoskeletal diagnoses (MDC 08) was statistically significantly larger than that for digestive and infectious ones (MDCs 06 and 18, respectively) (Table 3). Looking only at medical MS-DRGs, we failed to identify a statistically significant difference in median best-fit CAGRs between MDCs (adjusted H=11.129, df=6, p=0.084 by Kruskal-Wallace).\n\nCorrected P values for post hoc pairwise comparisons of growth rates for best-fit compound annual growth rates by MDC. Statistically significant results (P<0.05) indicated in italics.\n\n\nDiscussion\n\nUnder the IPPS, Medicare annually adjusts base payment rates for inpatient discharges based on providers’ reported costs. In this descriptive study, we observed that the majority of MS-DRG base payment rates have failed to keep pace with inflation. We also observed that reimbursement rates for medical discharges have declined more rapidly than those for surgical ones, and within surgery, reimbursement for orthopedics-related discharges has grown more rapidly than reimbursement for gastrointestinal or infectious disease discharges.\n\nThe IPPS is intended to contain health care spending by encouraging hospitals to reduce costs and increase efficiency, and it has been largely effective at achieving that aim6. That success is a double-edged sword, however. Capitated payments encourage hospitals to adopt new technologies that boost efficiency or directly enable overall cost savings, and discourage them from purchasing those that accomplish neither of these aims; however, that latter group may include products that improve patient care in ways that are not clearly reflected on institutions’ financial balance sheets, while increasing hospitals’ costs7–9. The extent to which declining reimbursement rates for many MS-DRGs further exacerbates these behaviors warrants further study.\n\nFederal programs and policies exist to help offset expenses that exceed MS-DRG payments, but available evidence suggests their impact is limited10. Medicare’s new technology add-on program (NTAP) is only available for products that meet stringent criteria for economic and clinical impact, and only 19 drugs and devices in total were approved for supplemental payments through this mechanism from 2001 to 201511. Thus, in many cases, particularly for technologies with an intermediate level of added cost and benefit, increased expenses for inpatient discharges are borne by providers without additional compensation beyond that derived from the MS-DRG reimbursement formula.\n\nIn addition to affecting hospitals’ behavior, declining reimbursement rates may reduce the economic incentive to develop new drugs or devices for inpatient care. Numerous empirical economic studies (including those cited here and here) in the pharmaceutical industry have shown a positive relationship between R&D spending (and output) and market opportunity size12–15. As noted above, this phenomenon may be particularly significant for new products that provide benefits to patients and hospitals that are difficult to quantify, as well as those that fail to meet the clinical and financial criteria to quality for add-on payments. Further work, building on the descriptive analysis presented here, is warranted to examine in more detail the relationship between new product development for inpatient care and reimbursement growth rates.\n\nAn important consideration regarding the interpretation of MS-DRG payment trends relates to the distinction between gross (top-line) reimbursement, which is measured in this study, and net (bottom line) profitability. It is entirely possible that some MS-DRGs with flat or declining reimbursement can still be highly profitable compared to others with increasing payment levels, if hospital costs can be disproportionately reduced. In theory, profitability, not reimbursement, should dictate the attractiveness of developing and purchasing drugs and devices for inpatient use under a given MS-DRG. In practice, however, we believe payment levels do, in fact, influence the perceptions—and, ultimately, the behaviors—of manufacturers and hospitals. Anecdotal evidence suggests that hospitals rarely have insight into the profitability of specific inpatient procedures (illustrated here and here), and often base purchasing decisions on payment levels16. Similarly, in our professional experience with medical device executives, we have found that calculations of reimbursement rate trends like the ones performed here are a key component of investment decisions around developing and commercializing inpatient care products.\n\nThis work has some additional limitations. First, the base reimbursement rates for each MS-DRG we determined do not precisely reflect the amounts that particular institutions receive, due to adjustments for factors like low socioeconomic status in a hospital’s catchment area and the institution’s involvement in graduate medical education. Thus, a specific hospital may experience different MS-DRG reimbursement rates and growth trends than the ones calculated here. Second, reimbursement rates by private payers may not follow exactly the same trends as those described here. Non-Federal payers typically reimburse at rates above Medicare17, but trends for individual MS-DRGs have not been comprehensively examined. Third, since 2015, some Medicare providers have opted to participate in bundled payment programs for inpatient and post-hospitalization care in some clinical areas instead of the IPPS, so their reimbursement rates for some MS-DRGs may not be accurately reflected by the data analyzed here. This is particularly relevant for surgical MS-DRGs for musculoskeletal discharges (MDC 08), for which we observed larger reimbursement growth than other surgical MS-DRGs, because this MDC includes many procedures that are now covered by bundled payments. In summary, although the data presented here are generally applicable, the experience of a particular hospital depends on factors specific to its payer mix, geography, and other factors.\n\nNotwithstanding its limitations, our analysis is the first to calculate trends in inflation-adjusted Medicare base reimbursement rates for inpatient discharges across individual MS-DRGs. We show here that since 2009, these reimbursement rates have declined for a substantial fraction of MS-DRGs, particularly for medical discharges as compared with surgical ones, and that reimbursement for musculoskeletal discharges appears to have been relatively spared compared with some other surgical domains. To the extent that manufacturers and hospitals use these rates to guide their investment decisions, the IPPS may unintentionally promote or disincentivize innovation and commercial uptake in specific clinical areas. The relationship between these reimbursement trends, new product development, and market adoption in inpatient care warrants further study.\n\n\nData availability\n\nMS-DRG weights, standardized reimbursement amounts, and 2015 total Medicare payments by MS-DRG were obtained from Final Rule tables for 2009-2018 from the website of the Center for Medicare and Medicaid Services (CMS).\n\nThese data are publicly available from CMS, without any restrictions.\n\nOpen Science Framework: MS-DRG data and analysis. https://doi.org/10.17605/OSF.IO/FKMYA.\n\nThis project contains Supplemental File 1: “MS-DRG data and analysis”\n\nExtended data 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Julie Lin for helpful comments, and Emily Villas for assistance with data collection.\n\n\nFootnotes\n\naExamination of R2 values across all MS-DRGs suggested that for most of them, the calculated best-fit CAGR explained a sizable fraction of the observed variability in reimbursement rates (mean R2 0.58, standard deviation 0.32).\n\n\nReferences\n\nPayments to hospitals for inpatient hospital services. 42 U.S.C. § 1395ww. Reference Source\n\nDepartment of Health and Human Services, Office of Evaluation and Inspections: Medicare hospital prospective payment system: How DRG rates are calculated and updated. August 2001. OEI-09-00-00200. accessed March 26, 2019. Reference Source\n\nEgeland RD, Rapp Z, David FS: From innovation to market adoption in the operating room: The \"CFO as customer\". Surgery. 2017; 162(3): 477–482. PubMed Abstract | Publisher Full Text\n\nMedicare program; proposed changes to the hospital inpatient prospective payment systems and fiscal year 2009 rates, 42 Fed. Reg. 23528. (April 30, 2008). Reference Source\n\nKrinsky S, Ryan AM, Mijanovich T, et al.: Variation in Payment Rates under Medicare's Inpatient Prospective Payment System. Health Serv Res. 2017; 52(2): 676–696. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMedicare Payment Advisory Commission (MedPAC): Report to Congress: Medicare and the health care delivery system. 2019. accessed April 5, 2019. Reference Source\n\nSorenson C, Drummond M, Torbica A, et al.: The role of hospital payments in the adoption of new medical technologies: an international survey of current practice. Health Econ Policy Law. 2015; 10(2): 133–159. PubMed Abstract | Publisher Full Text\n\nFreedman S: Health insurance and hospital technology adoption. Adv Health Econ and Health Serv Res. 2012; 23: 177–198. PubMed Abstract | Publisher Full Text\n\nScheller-Kreinsen D, Quentin W, Busse R: DRG-based hospital payment systems and technological innovation in 12 European countries. Value Health. 2011; 14(8): 1166–1172. PubMed Abstract | Publisher Full Text\n\nClyde A, Bockstedt L, Farkas JA, et al.: Experience with Medicare’s new technology add-on payment program. Health Aff (Millwood). 2008; 27(6): 1632–1641. PubMed Abstract | Publisher Full Text\n\nHernandez J, Machacz SF, Robinson JC: US hospital payment adjustments for innovative technology lag behind those in Germany, France, and Japan. Health Aff (Millwood). 2015; 34(2): 261–270. PubMed Abstract | Publisher Full Text\n\nAcemoglu D, Linn J: Market size in innovation: Theory and evidence from the pharmaceutical industry. Quart J Econ. 2004; 119(3): 1049–1090. Publisher Full Text\n\nBlume-Kohout ME, Sood N: Market size and innovation: Effects of Medicare Part D on pharmaceutical research and development. J Public Econ. 2013; 97: 327–336. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuBois P, Mouzon O, Scott-Morton F, et al.: Market size and pharmaceutical innovation. RAND J Econ. 2015; 46: 844–871. Publisher Full Text\n\nBruen B, Docteur E, Lopert R, et al.: The impact of reimbursement policies and practices on healthcare technology innovation. Office of the Assistant Secretary for Planning and Evaluation. U.S. Department of Health and Human Services. 2016. accessed November 14, 2018. Reference Source\n\nEgeland RD, Rapp Z, David FS: From innovation to market adoption in the operating room: The \"CFO as customer\". Surgery. 2017; 162(3): 477–482. PubMed Abstract | Publisher Full Text\n\nMaeda J: An analysis of hospital prices for commercial and Medicare Advantage plans. Congressional Budget Office. 2017. accessed March 8, 2019. Reference Source"
}
|
[
{
"id": "54919",
"date": "22 Nov 2019",
"name": "Allen Dobson",
"expertise": [
"Reviewer Expertise Evaluating Medicare’s various PPS policies"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nQ-1)\nThere are a few technical terminology errors that should be corrected. MS-DRG weights are based on relative resource use (costs) for each MS-DRG. Overall provider costs are never used; either in weight calculations or in charging the base payment amounts one year to the next unless the system is recalibrated (setting payments to costs) over time. MS-DRG weights are not “capitated” amounts for a given person; they are prospective payment amounts set for each case as defined by individual MS-DRGs. MS-DRGs are Medicare-Severity – DRGs, not Medical severity – DRGs, and as I say below, CMS does not use CPI-U as its inflation adjustor for healthcare costs. It uses the hospital market bracket (MB) for this purpose.\nQ-2)\nThe study design would be more accurate if the hospital MB were used, as opposed to the CPI-U, as the study price deflector. The hospital MB was designed in this purpose and is used by CMS exclusively in its payment system design and calibration. The paper notes that surplus financial capacity comes from profits (margins), not absolute payment amounts. Low payments that are profitable, are more conducive for product development than higher paid MS-DRGs that are big losers in terms of margin (profits).\nAlong these lines, I did not see in the paper a mention of payment “compression”. That is, high cost cases tend to be underpaid and low-cost cases are overpaid in case-mix-based payment systems. This would seem to be important to understanding the relative payment growth in case types across time. That is, high cost cases that are underpaid initially may set better payment rules over time.\nQ-4/6)\nThe analysis indicates that CMS IPPS (inpatient prospective payment) payments lag behind the payment rates one year to the next. This is not in and of itself news. It is well known that MB and productivity cuts, Medicare DSH payment reductions, and hospital quality penalties reduce the rate of increase in IPPS case payments well below MB growth rates. The distinction between MS-DRG types (Medical vs. Surgical) is somewhat novel and worth presenting, but some information for why this happens would be useful. An example of the payment dollars involved over 2 to 3 years might also be useful.\nThe focus on underpayments thwarting innovation and new product development and diffusion is likely directionally correct, but as the paper notes, this is more an issue of MS-DRG profitability relative to costs, as opposed to absolute payment amounts. I would argue that device manufacturers know which MS-DRGs are profitable and which are not. I have worked with a few larger companies and they know which MS-DRGs by hospital are profitable. Admittedly, smaller companies do not have the Medicare cost report skills to make these calculations. Additionally, many hospitals also know which services are profitable (e.g. cardiology and orthopedics). But, nevertheless, as payments fall relative to costs over time the downward pressure on product innovation and diffusion must intensify as the paper states.\nThe hospital outpatient side of the payment equation must be somewhat less harmful to product innovation to the extent that APGs are based on relative input costs on a more or less real time basis. The paper notes this, but does not indicate that OP PPS bundles clinically comparable procedures within the same APG. Thus, high cost procedures in low payment ARGs are less well paid.\nAs noted, the CPI-U is not the measure of inflation used by CMS for hospital input factor price inflation. CMS uses the hospital market bracket developed specifically for hospital payments. More properly the paper should use MB not CPI-U as its price deflector. Some might argue this is a critical flaw in the paper. I would have thought the differences between payments and inflation would have been larger then reported.\nThat said, the study contention that CMS IPPS does not keep up with inflation is undoubtedly directionally correct. And the study’s distinction between medical and surgical cases is also likely directionally correct, unless the CMS MB and CPI-U price medical and surgical service inputs differently. I would guess this is unlikely.\nNote: Providers’ reported costs should be reported as providers reported relative MS-DRG costs. A given providers’ absolute MS-DRG costs are not covered under CMS- IPPS by design.\nThe notion of product value (e.g. some services may reduce overall healthcare costs – (e.g. drugs may reduce hospital costs) is well taken, but this is more of a coverage issue then an issue that will be explicitly accounted for in a payment system. CMS IPPS covers average production costs with no regard for internal MS-DRG overall system efficiency. If a product is covered, it is paid for.\nGiven the finding that Medical MS-DRGs are paid relatively less well over time, as compared to surgical MS-DRGs, one would expect either that medical MS-DRG are more efficiently provided over time (better medicines, diagnoses, and therapies) or cost accounting in the Medicare Cost Report undervalues Medicaid MS-DRG relative cost over time. The CMS IPPS is not developed to favor one MS-DRG type over another. So it may be that the Medical MS-DRGs worse position in keeping up with inflation reflects productivity growth, not declining profits. If so, the paper’s conclusion is challenged.\nSummary\nI was the research director for Medicare when IPPS was developed and implemented in 1983. Device manufacturers frequently came unto CMS and recited the arguments presented in this paper. Over the years, I have not heard that CMS IPPS has stalled innovation. Probably because Academic Medical Centers have been relatively well paid under IPPS. The arguments against IPPS are more frequently related to quality; hence the CMS emphasis on quality measures and penalties as it develops its prospective payment systems. Admittedly, CMS has developed a series of adjustments for new technology. However, these mostly provide price supports for new services and technologies that are not well priced as products come to the market. After volume increases and costs are better known, the relative MS-DRG prices for new technology should be as fair as prices for older technologies.\nIn the end, prospective payment systems put pressure on resource use within each MS-DRG. This forces device manufacturers to set their prices with some care and set their prices with consideration of medical value. While CMS does not price to value, each hospital can pay for value within a given MS-DRG. These effects may be as important, if not intentional, as the “underpricing” presented in the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "59104",
"date": "18 Feb 2020",
"name": "Keith A. Joiner",
"expertise": [
"Reviewer Expertise Health economics",
"health care payment reform."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n19th March 2020 - This report was updated at the request of the reviewer, as they wished to include further information in their review.\n\nThe authors do a careful and well-informed analysis of the CAGR for base MS-DRG over the period from 2009-2018. While the overall trend of a 0.26% decrease in CAGR over this time frame is useful information, the comparison across MDCs provides more insight, and potentially actionable information.\n\nHaving said this, the author’s implication that such results have or could have substantial impacts on hospital decision making seem exaggerated. This is particularly true in the current environment of CMS metrics for hospitals, where hospitals can lose up to 6% of total Medicare revenues annually through the hospital readmissions reduction program (HRRP), the hospital acquired conditions program (HAC), and the hospital value-based purchasing program (VBP). Similarly, hospital can earn bonuses if they are top performers in these categories. In other words, the financial implications and consequences of these programs far outstrip small decreases (or increases) in base MS-DRG rates, and as such have a much greater impact on hospital decision making.\nAs more specifically related to the data in this manuscript, the case mix index for hospitals has been steadily rising over the time period of this study. This indicates that, despite small declines in the base MS-DRG, the patient mix at hospitals is increasing in complexity, and accordingly in reimbursements. This is occurring through two mechanisms. Disproportionately more patients with higher base MS-DRG are being hospitalized. In addition, the mix of admitted patients with MS-DRG weights with either complicating conditions (CC) or major complicating conditions (MCC) is increasing. Both mechanisms are more than offsetting the small declines in base MS-DRG. Hospital decisions on investment or resource allocation as related to MS-DRG will be based on the patient mix, and not on changes in base MS-DRG.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-605
|
https://f1000research.com/articles/8-265/v1
|
07 Mar 19
|
{
"type": "Research Article",
"title": "Fast and accurate differential transcript usage by testing equivalence class counts",
"authors": [
"Marek Cmero",
"Nadia M. Davidson",
"Alicia Oshlack",
"Marek Cmero"
],
"abstract": "Background: RNA sequencing has enabled high-throughput and fine-grained quantitative analyses of the transcriptome. While differential gene expression is the most widely used application of this technology, RNA-seq data also has the resolution to infer differential transcript usage (DTU), which can elucidate the role of different transcript isoforms between experimental conditions, cell types or tissues. DTU has typically been inferred from exon-count data, which has issues with assigning reads unambiguously to counting bins, and requires alignment of reads to the genome. Recently, approaches have emerged that use transcript quantifications estimates directly for DTU. Transcript counts can be inferred from 'pseudo' or lightweight aligners, which are significantly faster than traditional genome alignment. However, recent evaluations show lower sensitivity in DTU analysis. Transcript abundances are estimated from equivalence classes (ECs), which determine the transcripts that any given read is compatible with. Recent work has proposed performing differential expression testing directly on equivalence class read counts (ECs). Methods: Here we demonstrate that ECs can be used effectively with existing count-based methods for detecting DTU. We evaluate this approach on simulated human and drosophila data, as well as on a real dataset through subset testing. Results: We find that ECs counts have similar sensitivity and false discovery rates as exon-level counts but can be generated in a fraction of the time through the use of pseudo-aligners. Conclusions: We posit that equivalence class read counts are a natural unit on which to perform many types of analysis.",
"keywords": [
"RNA-seq",
"differential transcript usage",
"equivalence class",
"transcript compatibility class",
"pseudo-alignment",
"DEXSeq",
"Salmon",
"Kallisto"
],
"content": "Introduction\n\nRNA sequencing with short-read sequencing technologies (RNA-seq) has been used for over a decade for exploring the transcriptome. While differential gene expression is one of the most widely used applications of this data, significantly higher resolution can be achieved by using the data to explore the multiple transcripts expressed from each gene locus. In particular, it has been shown that each gene can have multiple isoforms, sometimes with distinct functions, and the dominant transcript can be different across samples1. Therefore, one important analysis task is to look for differential transcript usage (DTU) between samples.\n\nDTU can be inferred through differential exon usage, where the proportions of RNA-Seq fragments aligning to each exon change relative to each other between biological groups. Anders et al.2 showed that exon counts could be used to test for differential exon usage with a generalized linear model that accounts for biological variability. However, counting fragments across exons is not ideal because many fragments will align across multiple exons, making their assignment to an individual exon ambiguous. Moreover, individual exons often need to be partitioned into multiple disjoint counting bins when exon lengths differ between transcripts. Typically, there will be more counting bins than transcripts, resulting in lower power to detect differences between samples.\n\nAn alternative to using exon counts for testing DTU is to perform tests directly on estimated transcript abundances3. Recently, fast and accurate methods for quantifying gene expression at the transcript level have been developed4,5. These methods use transcript annotations that include multiple known transcript sequences for each gene as a reference for the alignment. The expression levels of individual transcripts can be estimated from pseudo-aligned reads that are compatible with transcripts associated with a specific gene6. Transcript abundance estimates can be used as an alternative starting measure for DTU testing, which has been shown to perform comparably with state-of-the-art methods3. In addition, pseudo-alignment is significantly faster than methods that map to a genome. However, in the most comprehensive comparison using simulated data, exon-count based methods were shown to have slightly better performance compared with methods that first estimate transcript abundances3.\n\nConceptually, quantification by lightweight or ‘pseudo’ alignment begins by using a transcript annotation as a reference and then assigns each read as ‘compatible’ with one or more transcripts that are a close alignment to the read4. Because different transcripts of the same gene share large amounts of sequence, many reads are compatible with several transcripts. Reads are therefore assigned to an equivalence class, or transcript compatibility class, which reflects the combination of transcripts compatible with the read sequence (Figure 1). For the purposes of this work, we consider an equivalence class to be defined as in Bray et al.4, i.e. any fragments that are pseudo-aligned to the same set of transcripts are considered to be part of the same equivalence class. Figure 1 shows a toy example of a gene with three different transcripts. Depending on its sequence, a read can align to all three transcripts, only two of the transcripts or just one transcript. These different combinations result in four possible equivalence classes, containing read counts, for this gene.\n\nThe example shows a gene consisting of six exons (Ex1-6) and three transcripts (t1-3) resulting in four equivalence classes (EC1-4). t1 is predominantly expressed in condition 1 (S1), whereas t3 is predominantly expressed in condition 2 (S2). The DTU is evident as a change in the relative counts for EC2, EC3 and EC4 between conditions. The pipelines for the three alternative methods for detecting DTU are shown: quantification of transcript expression followed by DTU testing, assignment of read counts to equivalence classes followed by testing of equivalence class counts (DECU) and assignment of read counts to exons followed by differential exon counts (DEU). Genes that are detected to have DECU or DEU are inferred to have DTU. The transcript quantification table in the left-most column is example data only, and is not based on real inference.\n\nRecently, equivalence classes have been used for clustering single-cells7,8 and Yi and colleagues have recently introduced direct differential testing on equivalence classes in a catch-all method to identify genes that display any transcript-level phenomena such as cancellation (isoform switching), domination (high abundance isoform(s) that mask transcript-level differences) and collapsing (multiple transcripts exhibiting small changes in the same direction)9. Here we focus on the case of isoform switching using methods originally designed for testing exon read counts. We evaluate the appropriateness of equivalence class read counts as an alternative choice for quantification compared to exon- and transcript-level quantification. We propose that DTU can be more accurately detected using equivalence class counts directly, rather than using these counts to first estimate individual transcript abundances before performing DTU. Soneson et al. applied a conceptually similar method with MISO10 by defining counting bins as combinations isoforms and counting according to isoform compatibility3. In our scenario, count-based DTU testing procedures such as DEXSeq are applied directly to equivalence classes generated from fast lightweight aligners, such as Salmon and Kallisto. DTU testing on equivalence class counts is not only fast but also bypasses inherent uncertainty in directly estimating transcript abundances before statistical testing.\n\nWe evaluate the performance of DTU testing on equivalence class read counts using real and simulated data, and show that the approach yields higher sensitivity and lower false discovery rates than estimating counts from transcript abundances, and performs faster with accuracies similar or better than counting across exons.\n\n\nResults\n\nHere we propose an alternative pipeline for performing DTU and evaluate its performance using simulated and real datasets23. The method we propose is to first perform alignment with a lightweight aligner and extract equivalence class (EC or transcript compatibility) counts. These EC counts are assigned to genes using the annotation of the transcripts matching to the EC. Next, each gene is tested for DTU between conditions using a count based statistical testing method where exon counts are replaced with EC counts (Figure 1). Significant genes can then be interpreted to have a difference between the relative abundance of transcripts of that gene between conditional groups. In evaluating the EC approach, we used Salmon for pseudo-alignment and DEXSeq for differential testing. We then compared DTU results against the alternative quantification and counting approaches, also using DEXSeq for testing (see Methods). It should be noted that we are not attempting to evaluate the statistical testing method (DEXSeq) in relation to other methods, as this has been done previously in several papers3,9.\n\nThe datasets we used to evaluate performance were simulated data from human and drosophila from Soneson et al.3 and biological data from Bottomly et al.11. Each of the Soneson datasets consisted of two sample groups, each with three replicates, where 1000 genes were randomly selected to have DTU such that the expression levels of the two most abundant transcripts were switched. The Bottomly dataset contains 10 and 11 replicates each from two mouse strains that were used to call truth and then were subsampled to three replicates in the testing scenarios.\n\nThe number of counting bins used for DTU detection has an impact on sensitivity. More bins leads to lower average counts per bin and therefore lower statistical power per bin and more multiple testing correction. We therefore examined the number of ECs, transcripts and exons present in each dataset. Although the theoretical number of ECs from a set of transcripts can be calculated from the annotation and has the potential to be large, not all combinations of transcripts exist or are expressed. The number of equivalence classes calculated from pseudo-alignment depends on the experimental data as only ECs with reads assigned to them are reported. We compared the number of transcripts and exons in the three datasets (with at least one read) to the number of ECs. In both the simulated human and drosophila datasets, as well as in the Bottomly mouse data, the number of ECs is greater than the number of transcripts, but substantially fewer than the number of exons, indicating that there might be more power for testing DTU using ECs, compared to exon counts (Figure 2a).\n\n(a) The number of transcripts, equivalence classes and exons per gene, where each feature has at least one associated read. (b) The density of the log2 of the variance of counts over the mean for each feature (calculated per condition).\n\nIn addition, we found that the variability of counts across replicates calculated from ECs was lower than that from estimated transcript abundances across all three data sets (Figure 2b). Count variability of ECs was on average closer to the exon count variability distribution than ECs. For instance, the Bottomly data had an average log2 variance to mean ratio of -2.249 and -1.519 in exons and ECs respectively, compared to 0.115 in transcripts. The simulated data followed a similar pattern. Supplementary Figure 121 shows the dispersion-mean trends, again demonstrating lower dispersion in ECs compared to transcript abundance estimates. We hypothesise that the greater dispersion observed for transcript data arises from the abundance estimation step used by pseudo-aligners to infer transcript counts. Due to the lower dispersion, we anticipate that ECs yield greater power for DTU compared to transcript abundance estimates.\n\nSeveral methods were previously tested on the simulated data from Soneson et al.3; DEXSeq’s default counting pipeline and featureCounts were shown to perform best. We recalculated exon counts using DEXSeq’s counting pipeline (as recommended by Soneson et al., we excluded region of genes that overlapped on the same strand in the input annotation) and ran Salmon5 to obtain both transcript abundance estimates and equivalence class counts. All other comparison results were obtained from Soneson et al.3. For the simulated datasets, we found that ECs had the highest sensitivity in both the drosophila and human datasets (Figure 3a) with a TPR of 0.697 and 0.739 respectively (FDR < 0.05). However, ECs also had a slightly higher FDR than exon-counting methods.\n\n(a) The equivalence class method compared to other state-of-the-art methods on simulated data described in Soneson et al.3. (b) The ability of the equivalence class, transcript and exon-based methods to recreate the results of a full comparison (10 vs. 11) of the Bottomly data, using only a (randomly selected) subset of samples (3 vs. 3) across 20 iterations. The union of all genes called as significant across all three methods is used to calculate the FDR, and the intersect (genes called by all three methods) is used for the TPR. Full results (union, intersect and each method’s individual truth set) is shown in Supplementary Figure 3.\n\nWe next tested the performance of the EC method on a biological dataset from Bottomly et al. We tested the complete RNA-seq dataset (10 vs. 11) for DTU using DEXseq on counts generated from transcript abundance estimates, exons and ECs. To calculate the FDR, we considered the set of 'true' DTU genes to be the union of all genes called significant (FDR < 0.05) across the three methods. To calculate the TPR, the intersect of genes called by all methods was used. Supplementary Figure 221 shows the number of significant genes and overlap between all three methods. ECs called the highest number of genes with significant DTU (1485 genes, in contrast to the 748 and 391 genes called significant by the transcript and exon-based methods respectively). Similar to the FDR experiments described in Pimentel et al.12, we randomly selected three samples per condition and performed DTU using all three methods and repeated this for 20 iterations. Figure 3b shows the results. EC-based testing performed the best, with a mean FDR of 0.305 across all iterations (compared to a mean FDR of 0.569 and 0.373 for the transcript and exon-based methods respectively). The mean TPR was also slightly higher for ECs at 0.544, compared to exons at 0.539 and 0.36 for the transcript-based method. Results for all three combinations of the ‘truth gene’ sets (union, intersect and individual) are shown in Supplementary Figure 321. The EC-based method had consistently lower FDR, which is also illustrated by the rank-order plot (Supplementary Figure 421), showing the number of false positives present in the top 500 FDR-ranked genes. In terms of the TPR, ECs performed better than transcripts, but worse than exons when using the union of all methods as the truth set. In the Bottomly analysis, Salmon was used as a representative method for transcript abundance estimation. We also performed the analysis with Kallisto, which gave results consistent with Salmon (Supplementary Figure 521).\n\nWhile the performance of EC counts in term of sensitivity and FDR are only slightly better than exons level counts, another advantage of using ECs for analysis is the speed of alignment. The process can be broken down into workflow components that include alignment of sequenced reads, quantification and testing. Table 1 shows the compute times for all three methods on all three datasets broken down into workflow components. For the exon counting method, STAR was used for the alignment of reads to the genome (see Methods). In every case, the transcript quantification method had the fastest total run time followed by ECs and then exons. The difference was mainly driven by the speed of using pseudo alignment for transcript and EC quantification, indicating that for larger datasets the speed of analysis will be significantly faster for our proposed EC based method compared with traditional exon counting methods. A small amount of extra time was also needed for the the EC method for matching EC counts to genes. In addition, DEXSeq generally runs more slowly with larger numbers of counting bins, which is the case for ECs compared with transcripts and improved scalability of DTU approaches is likely to narrow this performance gap. The speed of featurecounts over DEXseq’s counting significantly improved run times for the exon-based method; however, the total run times still lagged behind the psuedo-alignment methods. We also note that the transcript-abundance inference stage performed by pseudo-aligners is not necessary for EC-based DTU testing, making salmon slightly faster to run when quantification is skipped (Table 1).\n\nCompute times shown in hh:mm:ss for the simulated data (101 bp paired-end) and Bottomly (76 bp single-end) read data, with each sample aligned and quantified in serial with access to 256GB RAM and 8 cores per sample, and post-quantification steps performed on count data from all samples from each batch in a single run with 256GB RAM and 8 cores. The alignment and quantification steps show the total time taken for all samples (i.e. the serial runtime). The drosophila and human samples contained approximately 25M and 40M reads respectively, and the Bottomly samples contained approximately 16M reads. Exons counts were quantified using DEXSeq-count (ds) and featureCounts (fc).\n\nWe also considered peak RAM usage (shown in Supplementary Table 121), and alignment was found to use the most RAM. Overall, methods utilising pseudo alignment required significantly lower memory compared with traditional alignment. For the most RAM intensive dataset, the human simulation, exon counting required 29 GB compared to 10 GB for ECs and 5 GB for estimated transcript abundances.\n\n\nDiscussion\n\nDTU detection has previously been approached by either testing for changes to the read counts across exons or changes in the relative abundance of transcripts. These approaches are intuitive but are not necessarily optimal for short read data analysis. In particular, individual exons are not necessarily the optimal unit of isoform quantification as there are often many more exons than transcripts. In addition, transcript quantification can be difficult because read assignment is ambiguous. Fortunately, transcript quantification methods generate equivalence class counts as a forestep to estimating abundances. We propose that equivalence classes are the optimal unit for performing count based differential testing. Equivalence class counts benefit from the advantages of both exon and transcript counts: they can be generated quickly through pseudo-alignment, there are fewer expressed than exons, and they retain the low variance between replicates seen in exon counts compared to transcripts abundances.\n\nHere we evaluated the use of equivalence classes as the counting unit for differential transcript usage. We used two simulated datasets from drosophila and human and one biological dataset from mouse. Our results suggest that equivalence class counts provide equal or better accuracy in DTU detection compared to exon counts or estimated transcript abundances. We also found the analysis was quick to run and we provide code to convert pseudo alignments into gene level EC annotations.\n\nThe ECs used in our evaluation are defined using only the set of transcripts for which reads are compatible. Extensions to this model have been proposed that incorporate read-level information, such as fragment length, to more accurately calculate the probability of a read arising from a given transcript13. Although, we do not consider probability-based equivalence classes in this work, incorporating this information for DTU deserves exploration in future work. In addition, EC counts may be calculated from full read alignment rather than pseudo-alignment14,15, which has the potential to improve accuracy further. In this work, we limited our investigation to comparing the best counting metric preceding DTU statistical testing, using DEXSeq as a representative method. Evaluation of statistical testing methods for DTU is outside the scope of this manuscript and would require further work.\n\nOne limitation of using equivalence classes is in the interpretation of the results. Although we can detect DTU at the gene-level, it is not simple to determine which isoforms have changed abundance without further work. We propose that superTranscripts16, which are a method for visualising the transcriptome, could be used for interpretation. Alternatively, transcript abundances, which are generated together with ECs, can still be used to provide insight into the isoform switching.\n\nFinally, in this work, we have focused on differential transcript usage, but EC counts have the potential to be useful in a range of other expression analysis. EC counts have already been applied to areas such as clustering and dimensionality reduction7, gene-level differential expression9, single-cell transcriptomics7,8 and fusion detection20. We foresee that equivalence classes could serve as a base unit of measurement in many other types of analyses.\n\n\nMethods\n\nWe detected sequence content bias in the Bottomly RNA-seq data using FastQC v0.11.4, and therefore performed trimming using Trimmomatic14 0.35, using recommended parameters. The simulated Soneson data was not trimmed.\n\nTo obtain transcript abundance counts, Salmon5 v0.13.0 (development version) was run on the drosophila, human and Bottomly datasets in quant mode using default parameters. To obtain EC counts, the --dumpEq argument was used, as well as the --skipQuant to skip the quantification step. Kallisto4 0.43.0 was run in pseudo mode with the --batch argument to run all samples simultaneously. Fragment length and standard deviation were estimated from all reads of a single sample from the Bottomly data (SRR099223). Equivalence classes were then matched between samples and compiled into a matrix using the python scripts (create_salmon_ec_count_matrix.py and create_kallisto_ec_count_matrix.py), available on GitHub and archived on Zenodo22. Equivalence classes mapping to more than a single gene were removed. No other filtering was performed on any of the data types.\n\nTo perform the exon-based counts, raw reads were first aligned using STAR15 v2.5.2a, then the DEXSeq-count annotation was prepared excluding overlapping exon-parts, from different genes, on the same strand (--aggregate=’no’). DEXSeq-count was then run using default parameters. The same genome and transcriptome references for drosophila and human were used as in Soneson et al.3, with only protein-coding transcripts considered for the Salmon index. For the Bottomly data, we used the NCBIM37 mm9 mouse genome and Ensembl release 67 transcriptome. Non-protein-coding transcripts were filtered out, as with the Soneson transcriptome reference. DEXSeq v1.26 was used to run all DTU analyses.\n\nAn earlier version of this article can be found on bioRxiv (DOI: https://doi.org/10.1101/501106).\n\n\nData availability\n\nThe Soneson et al.3 drosophila and human simulation data was obtained from ArrayExpress repository, accession number E-MTAB-3766.\n\nTruth data was obtained from http://imlspenticton.uzh.ch/robinson_lab/splicing_comparison/, files diff_splicing_comparison_drosophila.zip and diff_splicing_comparison_human.zip.\n\nThe Bottomly et al.8 dataset was obtained from the NCBI Sequence Read Archive, accession number SRP004777.\n\nZenodo: Supplementary Material for \"Fast and accurate differential transcript usage by testing equivalence class counts\". https://doi.org/10.5281/zenodo.256154621. The following extended data are available:\n\nSupplementary Figure 1: Shows the dispersion versus mean normalised counts for all features across the three data sets, generated using DEXSeq’s ‘plotDispEsts’ function. As described in Love et al., the red line shows the fitted dispersion-mean trend, the blue dots indicate the shrunken dispersion estimates, and the blue circles indicate outliers not shrunk towards the prior.\n\nSupplementary Figure 2: Shows the significant genes (FDR < 0.05) shared between the methods, obtained from DEXSeq run on the full Bottomly et al. data set for each feature.\n\nSupplementary Figure 3: Shows the ability of the three methods to recreate the results of a full comparison (10 vs. 11) of the Bottomly et al. data using random subsets of 3 vs. 3 samples across 20 iterations. The lines between the plots join data points from the same iteration. Each row uses a different ‘truth’ set: union is the set of genes called significant by any method, intersect is the set of genes called significant by all methods, and individual is the set of genes called significant by that method only.\n\nSupplementary Figure 4: The number of false positives versus each gene’s rank (by FDR) for one iteration (3 vs. 3) of the Bottomly subset tests for the top 500 genes. The union of significant genes across all methods was used as the truth set.\n\nSupplementary Figure 5: Kallisto versus Salmon’s performance on the Bottomly subset testing experiments, using each method’s significant genes from the full (10 vs. 11) run as the truth set for calculating both metrics.\n\nSupplementary Table 1: Maximum RAM usage for each job in GB. Each task was run as specified in the compute times table in the main paper (Table 1).\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nPipeline used to reproduce the quantification data generated in this paper:https://github.com/Oshlack/ec-dtu-pipe.\n\nArchived source code at time of publication:https://doi.org/10.5281/zenodo.256759623.\n\nSource code to run the analyses and generate the paper figures:https://github.com/Oshlack/ec-dtu-paper.\n\nArchived source code at time of publication:https://doi.org/10.5281/zenodo.256154922.\n\nLicense: MIT license.",
"appendix": "Grant information\n\nThis work was supported by NHMRC project grant number APP1140626 to AO and ND.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank Rob Patro for discussions on using equivalence classes in salmon, for providing us with a version to bypass transcript quantification and feedback on our manuscript. We would also like to acknowledge members of the twitter community who provided constructive feedback on the first version of this manuscript.\n\n\nReferences\n\nGonzàlez-Porta M, Frankish A, Rung J, et al.: Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene. Genome Biol. 2013; 14(7): R70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnders S, Reyes A, Huber W: Detecting differential usage of exons from RNA-seq data. Genome Res. 2012; 22(10): 2008–2017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSoneson C, Matthes KL, Nowicka M, et al.: Isoform prefiltering improves performance of count-based methods for analysis of differential transcript usage. Genome Biol. 2016; 17(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBray NL, Pimentel H, Melsted P, et al.: Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016; 34(5): 525–527. PubMed Abstract | Publisher Full Text\n\nPatro R, Duggal G, Love MI, et al.: Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14(4): 417–419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatro R, Mount SM, Kingsford C: Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms. Nat Biotechnol. 2014; 32(5): 462–464. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNtranos V, Kamath GM, Zhang JM, et al.: Fast and accurate single-cell RNA-seq analysis by clustering of transcript-compatibility counts. Genome Biol. 2016; 17(1): 112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNtranos V, Yi L, Melsted P, et al.: A discriminative learning approach to differential expression analysis for single-cell RNA-seq. Nat Methods. 2019; 16(2): 163–166. PubMed Abstract | Publisher Full Text\n\nYi L, Pimentel H, Bray NL, et al.: Gene-level differential analysis at transcript-level resolution. Genome Biol. 2018; 19(1): 53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatz Y, Wang ET, Airoldi EM, et al.: Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods. 2010; 7(12): 1009–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBottomly D, Walter NA, Hunter JE, et al.: Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays. PLoS One. 2011; 6(3): e17820. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPimentel H, Bray NL, Puente S, et al.: Differential analysis of RNA-seq incorporating quantification uncertainty. Nat Methods. 2017; 14(7): 687–690. PubMed Abstract | Publisher Full Text\n\nZakeri M, Srivastava A, Almodaresi F, et al.: Improved data-driven likelihood factorizations for transcript abundance estimation. Bioinformatics. 2017; 33(14): i142–i151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavidson NM, Oshlack A: Corset: enabling differential gene expression analysis for de novo assembled transcriptomes. Genome Biol. 2014; 15(7): 410. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYi L, Liu L, Melsted P, et al.: A direct comparison of genome alignment and transcriptome pseudoalignment. BioRxiv. 2018. Publisher Full Text\n\nDavidson NM, Hawkins ADK, Oshlack A: SuperTranscripts: a data driven reference for analysis and visualisation of transcriptomes. Genome Biol. 2017; 18(1): 148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarber M, Grabherr MG, Guttman M, et al.: Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods. 2011; 8(6): 469–477. PubMed Abstract | Publisher Full Text\n\nDobin A, Davis CA, Schlesinger F, et al.: STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1): 15–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMelsted P, Ntranos V, Pachter L: The Barcode, UMI, Set format and BUStools. BioaRxiv. 2018. Publisher Full Text\n\nVu TN, Deng W, Trac QT, et al.: A fast detection of fusion genes from paired-end RNA-seq data. BMC Genomics. 2018; 19(1): 786. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCmero M, Davidson N, Oshlack A: Supplementary Material for \"Fast and accurate differential transcript usage by testing equivalence class counts\" (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2561546\n\nCmero M: Oshlack/ec-dtu-paper: f1000 submission (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2561550\n\nCmero M: Oshlack/ec-dtu-pipe: f1000 submission (Version v0.1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2567597"
}
|
[
{
"id": "45465",
"date": "20 Mar 2019",
"name": "Kristoffer Vitting-Seerup",
"expertise": [
"Reviewer Expertise Bioinformatics with a focus of analysis of transcripts from RNA-seq data"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary In the manuscript “Fast and accurate differential transcript usage by testing equivalence class counts” by Cmero et al suggest to use the ability of modern lightweight RNA-seq aligners to produce transcript compatibility counts (TCC) in combination with standard tools designed for differential transcript usage (DTU). Although the idea, as described in the introduction of the article, have been partly touched on by previous publications from the Pachter Lab, the approach used in this manuscript is novel since it describes a direct DTU analysis whereas the previous publications only inferred DTU indirectly. In this manuscript Cmero et al compares a TCC based DTU workflow against at transcript based and an exon based workflow using both simulated and real data reaching the conclusion that a TCC based workflow is superior – a novel and important finding. The manuscript is overall well presented and the analysis approach is state-of-the art. Unfortunately the analysis is not quite extensive enough and it suffers from a few major technical problems which together with a general lack of clarity in the writing means the manuscript requires major revisions.\n\nMajor comments:\nThe authors should also evaluate on the simulated data from Love et al 20181 to test the effect of:\nA different simulation scheme (since the FDRs are so high for Soneson et al data). Investigate the stability of the results using different number of replicates\n\nAll analysis presented is performed on unfiltered data which is problematic. Firstly it does not reflect typical RNA-seq analysis workflows which always include a step which filters out lowly expressed features before continued analysis. Furthermore, and more problematically, the lack of expression filtering will affect all analysis presented since many lowly or zero expressed features will be analyzed thereby skewing the global comparison due to the difference in the proportion of low/zero in the different datasets/pipelines2. Therefore, the authors should include (or replace the current analysis with) an analysis based on dataset which have been pre-filtered for expression. For inspiration of expression filtering1,2, the edgeR::filterByExpr() function or use the classical 1 TPM cutoff. Naturally the 3 dataset should be also filtered to be comparable (same transcripts/genes tested with all methods). To ensure correct quantification and to make the genome based (STAR) and lightweight based (Salmon) analysis comparable the Salmon index should be build from all transcripts and subsequently (after quantification) the data should be reduced to only protein coding genes. This is necessary to ensure that reads mapping to both protein coding genes and lncRNAs are correctly quantified (and are quantified in a manner comparable to the genome based approach). The manuscript is in general not concise enough. Throughout, the manuscript is very hard to follow which workflow is referred to and the order in which workflows they are presented is not logical (e.g. starting a section with explain about the alternative workflow does not make sense). Figures contain data never mention or used. Especially the discussion falls short of the mark as major parts are either repetitive non-informative.\nMinor comments:\nGenerally:\nThe authors should use “transcript compatibility counts” (TCC) (aka not “equivalence class read counts” (EC) and derivations thereof) since TCC is the terminology used in the field when ECs are used for quantification3.\n\nTitle:\nThe title seems to lack a word after such as “analysis” or “testing” after “differential transcript usage”\n\nAbstract:\nIn the sentence “However, recent evaluations show lower sensitivity in DTU analysis” I guess the authors mean compared to exon-level analysis but this needs to be specified. The conclusion is to broad. The authors investigate DTU but conclude about “many” analysis. Such a sentence should probably be saved for a review paper.\n\nIntroduction:\nIn addition to exon and transcript based analysis approaches the authors also need to mention analysis of individual splice events (via tools such as SUPPA2 and rMATS) as well as the types of analysis which groups multiple features together (such as Leafcutter and MAJIQ) to make clear that there are 4 different approaches (with TCC based approach being a fifth (or a deviation of transcript based)). I do not require the authors to also compared the TCC based approach to the two omitted workflows – but they should be mentioned in the introduction for completeness. I think it could be beneficial to refer more the lower part of Figure 1 in the Introduction since it very clearly present the 3 different workflows in question? The drawbacks of pseudo/quasi alignment should be mentioned/discussed either in the introduction or discussion. In the sentence “Depending on its sequence, a read can align to all three transcripts, only two of the transcripts or just one transcript. These different combinations result in four possible equivalence classes, containing read counts, for this gene” the last statement is wrong. There are 6 possible (the authors omit uniquely t2 and uniquely t3). This should either be mention or it should be highlighted the example reads in Figure 1 give rise to 4 possibilities. The authors should provide a reference 3 for the term “transcript compatibility count”. The authors should also discuss the ideas presented in Ntranos et al 20194 in the discussion of the Yi et al 2018 paper. Specifically the “catch-it-all” and “any transcript-level phenomena” part of the sentence in Cmero et al: “Yi and colleagues have recently introduced direct differential testing on equivalence classes in a catch-all method to identify genes that display any transcript-level phenomena” needs to be changed as aggregation of DTE p-values cannot identify isoform switches if the gene expression is also changing (as discussed in detail in Ntranos et al 2019) – hence the need for methods specifically designed for DTU detection and thereby also the need for the workflow presented by the authors Cmero et al. For Figure 1: Could it be beneficial to divide Figure 1 into A and B referring to respectively TCC and analysis pipelines?\n\nMethods:\nIt needs to be described in detail how the fragment length and standard deviation were estimated from the Bottomly data since it is single end data. The actual values should also be reported for reproducibility. Is there a particular reason why Salmon/Kallisto was not run with the bias correction algorithms? Since the authors have to rerun salmon anyway (see major comments) it might be beneficial to update to Salmon v0.13.1 and also use the “--validateMappings” option. Please state the parameters used with Trimmomatic for reproducibility. Please provide info on how the transcript-level counts was obtained (and specify if any scaling was done with e.g. tximport). Please also indicate how the exon/transcript level analysis was summarized to gene-level for each of the 3 workflows. Please provide the unfiltered salmon quantification results (the “quant.sf” files) from the Bottomly et al data as supplementary files to facilitate reproducibility. Please provide details of how the STAR mapped data was converted to DEXSeq ready counts (currently only implied in the result section).\n\nResults:\nFrom the first paragraph in results it is not clear that you are actually doing all 3 types of analysis and comparing them. And starting with mentioning the “alternative approach” is not reader friendly. For references to previous DTU benchmarking please also cite Love et al 20181. For the “Fewer equivalence classes are expressed than exons” analysis it is unclear whether it is the number of exons or disjointed exon bins necessary for a standard DEXSeq workflow (due to alternative 3’ and 5’ splice sites) which are quantified. Figure 2: Was any pseudocounts or transformation used to calculate the normalized cross replicate variance (Log2(var / mean))? Figure 3: Please add “A” and “B” to the figure in accordance with the figure legend. Figure 3A:\nWhat is visualized is not explained in figure legend. It is currently not possible to distinguish between the different methods on the plot. Please provide zoom in versions of the plot to enhance visual comparison. From the point of this paper (comparing the 3 workflows depicted in the lower half of figure 1) it is very strange that multiple exon-based workflows as well as the result of a MISO based workflow (which is never discussed) are also shown. Would it not make more sense to only show exon-based workflow used and omit the MISO based workflow? Furthermore since the supplementary figures show that Salmon and Kallisto produce the same results why not only show one of them?\n\nFigure 3B:\nPlease report which FDR cutoff was used to call significance. Please also report the result analysis on the feature level (transcript/exon) and not just for the gene-level. The authors should discuss the much larger variance in FDR for TCC and exon based approaches as well as the generally large FDR values (gussing the target value was 0.05)\n\nDiscussion:\n“We propose that equivalence classes are the optimal unit for performing count based differential testing” is to broad a claim since this article is about DTU analysis. Save it for a review :-). Please refer to sashimi plots in addition to superTranscripts – they had the visualization idea first.\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?\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": "4582",
"date": "29 Apr 2019",
"name": "Alicia Oshlack",
"role": "Author Response",
"response": "Thank you for taking the time to review our paper and for the helpful suggestions. Major comments: We ran our EC-based method, as well as the transcript and exon-based methods, through DTU analysis on the simulated data from Love et al.[1]. EC-based results can be seen to be on par with transcript-based results (Supplementary Figure 8). As we note in the revised paper, the simulations were based on baseline abundances derived using Salmon, which may have favoured Salmon-derived transcript quantifications in the downstream analysis. Together with the Soneson simulation, this indicates that ECCs perform as well as the best method regardless of the assumptions and biases in the simulated datasets. Regarding filtering, we note that DEXSeq automatically filters out zero-count features and low-count data. In order to show the effects of basic filtering on the EC, transcript and exon-based approaches, we present Supplementary Figure 6. This figure shows that filtering performs slightly better in controlling FDR per approach. Importantly, ECC-based DTU still out-performs transcript-based DTU in both the drosophila and human data. We agree with the reviewer’s comment that the pseudo-alignment index should be built on all transcripts and only subsequently filtered. The Soneson simulation data, however, is restricted to protein-coding genes only. All downstream results obtained from the Soneson data were run on references containing protein-coding genes only, therefore we opted to keep references consistent with the EC-based approach for optimal fairness. As we also compared the Bottomly data with the Soneson data in Figure 2, we opted to take the same approach with the Bottomly data. We have noted this decision in the methods. We used the whole transcript index without gene filtering for the Love data. We have updated the manuscript for conciseness, and updated labels figures and captions to improve clarity. Minor comments: General, title and abstract: We have opted to retain the use of ‘equivalence class counts’, noting that both ‘transcript compatibility counts’ and ‘equivalence class counts’ are used in the literature. We have updated the manuscript title for clarity. We have addressed the points regarding the abstract. Introduction: We have now cited transcript-assembly and spliced-in DTU approaches. We now discuss some of the limitations of pseudo-alignment in the Discussion. We opted to show four equivalence classes in Figure 1 for simplicity. We have noted the possibility of ECs containing solely t2 and t3 in the main text. We have added a reference for the term ‘transcript compatibility counts’. We have corrected the discussion on ideas presented in Ntranos et al.[2] We have added (a) and (b) labels for Figures 1-3 Methods: Fragment lengths and standard deviations were estimated directly from the read lengths (as these varied between reads due to trimming). The length and standard deviation values have been added to the methods section. Salmon/Kallisto were run with default arguments (apart from returning equivalence class counts) in order to run the software more-or-less ‘out of the box’ without parameter tuning, which may take focus away from the conceptual advance of using equivalence classes. Additionally, --validateMappings can be seen as a further optimisation to EC-derivation. Trimmomatic parameters have been added to the methods section. STAR was run with default parameters, which has also been added to the methods section. Tximport with ‘scaledTPM’ scaling was used to obtain transcript abundances from Salmon. This is now reflected in the methods. DEXSeq’s perGeneQValue function was used to obtain gene-level significance values. This is now reflected in the methods. Salmon’s “quant.sf” files are available in the ec-dtu-paper github repository. This is now reflected in the methods. We have clarified how exon counts are obtained from STAR counts. Results: We have revised the first paragraph for clarity. We now cite Love et al.[1] in reference to DTU method benchmarking. The “Fewer equivalence classes are expressed than exons” analysis considers exon counting bins. This has now been clarified in the main text. Cross-replicate log2(var / mean) calculations were performed on CPM-transformed and lightly filtered data. This is now reflected in the methods section. Figure 3 We have described the figure in greater detail in the caption. Supplementary Figure 6 has been added, which uses zoomed-in axes and shows results for ECC, exon and transcript counts. We show MISO as the way the method was used in Soneson et al. is conceptually similar and have removed feaureCounts and kallisto results to remove clutter. FDR cutoff is now stated in the figure legend. Reporting the results on the feature level is not feasible as truth data is not available at the feature level. Additionally, equivalence classes do not map cleanly to features, which would make it difficult to asses the truth of features even if exon and transcript-level truth were available. For the Bottomly replication data, we now note the FDR variance of the ECC and exon-count based methods, indicating that this may be the result of substructure in the data. Importantly, FDR is lower in all iterations but one for ECCs compared with transcript counts. Discussion: We have addressed the suggestions for the discussion. References:[1] Love, M. I., Soneson, C., & Patro, R. (2018). Swimming downstream: statistical analysis of differential transcript usage following Salmon quantification. F1000Research, 7, 952. https://doi.org/10.12688/f1000research.15398.1[2] Ntranos, V., Yi, L., Melsted, P., & Pachter, L. (2019). A discriminative learning approach to differential expression analysis for single-cell RNA-seq. Nature Methods, 16(February), 1. https://doi.org/10.1038/s41592-018-0303-9"
}
]
},
{
"id": "45466",
"date": "25 Mar 2019",
"name": "Alejandro Reyes",
"expertise": [
"Reviewer Expertise Bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCmero, Davidson and Oshlack propose a novel approach to use RNA-seq data to test for differences in transcript usage between conditions. Instead of using exon-level or transcript-level counts, the authors propose using equivalence class counts (ECCs) resulting from pseudo-aligning/quasi-mapping to reference transcriptomes as input to existing methods to test for differences in exon usage. Using both simulated and real datasets, the authors show that using ECCs is comparable to using exon-level counts in terms of false discovery rates and true positive rates. They show that the ECC approach is computationally more efficient, although its results are more difficult to interpret. The analyses are reproducible and available through Github.\n\nThe manuscript is well written and easy to follow. The whole idea is straightforward and very clever.\n\nBelow are two suggestions for improving the implementation of the software:\nAlthough some python scripts are available, they need better documentation and examples with toy datasets. From the code in the Github repository, it is not clear what steps one should follow to use the ECC approach for DTU. I would suggest writing a Bioconductor-like vignette that explains how to run kallisto/salmon with the parameters to get equivalence classes, how to use the python scripts to generate the equivalence class matrices, and how to transform these matrices into objects from the DEXSeq, DRIMseq and similar packages. As the authors acknowledge, a strong limitation of the ECC approach is result interpretation, which could be improved by visualizing the ECC equivalence classes. The interpretation of the ECC approach would be much easier if the authors provide code to plot transcripts and ECC classes of a gene (as it is done in the cartoon of Figure 1) linked with the counts of each equivalence class for each sample.\nMinor points:\nIt would be helpful for the reader if the authors improved figure labels and figure legends. For example, in Figure 3a, rather than just referring to the paper by Soneson et al.1, I would suggest to describe what each point represents, what each axis is and how the metrics shown were defined. In the introduction, the authors say “Typically, there will be more counting bins than transcripts, resulting in lower power to detect differences between samples.” Could the authors either explain further this statement or cite a reference that explains it? I understand the logic behind defining a “truth set” of genes with DTU in the analysis of the real data. However, the real number of true positives is likely larger and thus the resulting metric is not strictly a true positive rate. Perhaps it would be more accurate to call it differently (see for example, Norton et al.2).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4583",
"date": "29 Apr 2019",
"name": "Alicia Oshlack",
"role": "Author Response",
"response": "Thank you for taking the time to review our paper and for the helpful suggestions. Major comments: We have created a step-by-step Bioconductor-style vignette to allow users to easily reproduce ECC-based DTU testing with a toy data set. We include instructions for running each step manually, as well as an automated analysis using the ec-dtu-pipe pipeline we have developed. The vignette can be found here, which we note in the paper. Figure 1 in the original paper shows a highly simplified version of how ECs can be derived from a small set of transcripts and exons. In reality, genes have on average many more transcripts, exons and, consequently, equivalence classes. Furthermore, ECs may be disjoint (not connected by intervening sequence) or require a junction. As ECs are determined by kmers, creating a direct mapping between ECs and the genome is challenging. Given the complexity of ECs, even a clean mapping between EC and genome position may be difficult to interpret. Given these limitations, we have instead opted to include a simple visualisation option, similar to DEXSeq, plotting EC names and their relative log counts across conditions, per gene. Such a visualisation example can be found in Supplementary Figure 7 (note the large number of ECs present in this gene). The function to create these plots (plot_ec_usage) is found in the ec-dtu-paper repository (and is referenced in the vignette) will also print all significant ECs of the gene, and their associated transcripts. In the example, one of the significant ECs has a single associated transcript, making DTU inference relatively straight-forward. Minor comments: We have added explanatory text in Figure 3a to explain the FDR/TPR plots and their respective FDR cutoffs. We have also added (a) and (b) labels for Figures 1-3. We have further explained the sentence about how the number of counting bins affects power. We have also added Supplementary Table 2 to illustrate this point, which shows the average number of exons and transcripts per gene for the Ensembl human gene reference. We have rename the TPR to ‘Fraction recalled’ (also in Supplementary Figure 3) to indicate that the metric does not strictly measure false positive rate."
}
]
},
{
"id": "45467",
"date": "12 Apr 2019",
"name": "Leonardo Collado-Torres",
"expertise": [
"Reviewer Expertise RNA-seq",
"Bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript the authors Marek Cmero, Nadia M. Davidson and Alicia Oshlack describe in detail their proposed approach for identifying genes with differential transcript usage (DTU, particularly isoform switching) using equivalence classes obtained through pseudo-alignment methods such as Salmon and Kallisto. By doing so, the authors leverage the computational advantages of pseudo-alignment methods, particularly speed and RAM requirements, together with statistical methods initially developed for differential exon usage (mainly DEXSeq) to identify genes with DTU events at a comparable (or even lower) error rates than exon based analyses which are more precise than transcript-level analyses. That is, their proposed method is fast, has low computational requirements (measured by RAM usage), and has error rates comparable if not better than state of the art alternatives. If time and computational resources are not limiting factors, the method the authors propose still gains an advantage over exon based methods by taking advantage of the nature of the human and mouse transcriptomes where genes can have more exons than transcripts, thus leading to power gains by their method. However, as presented their method also relies on a correct annotation of the transcriptome since un-annotated isoforms that involve new exons or new exon boundaries could potentially affect the results.\nNevertheless, I think that it should be possible to apply their method in combination with others in order to minimize this issue. Overall the authors of this manuscript did an excellent job explaining their new method, comparing against earlier work, and explaining the different implications of their work. I look forward to their future software for applying this method as https://github.com/Oshlack/ec-dtu-paper has all the foundations for making an R/Bioconductor package.\nMinor points\nFigures 2 and 3 are missing labels for each sub-panel. For example, the legend for Figure 2 talks about (a) and (b) and while one can assume that the top panel is (a), it's best to be explicit about this type of information. Figure 2 top panel. Maybe show the data points in case the boxplots mask some information about the distribution. See here for some code by Rafael Irizarry or here for longer code examples that I wrote. If it looks like a bell-shaped distribution, then I think that it could be okay to simply mention that in the text (in the case that the figure has many points and you prefer not to include it). From Figure 3 bottom panel, I can see that you already plotted the points in that case. Figure 2 bottom panel. This also has some code for showing density plots with little bars in the bottom for the observed points. Page 5, bottom left. \"Count variability of ECs was on average closer to the exon count variability distribution than ECs.\" is incorrect. I believe that it should read \"Count variability of ECs was on average closer to the exon count variability distribution than transcripts\". Figure 3, top panel. I can't distinguish the colors between `featurecounts_flat` and `salmon`. Figure 3, top panel. I don't know what the dotted lines represent: maybe FDR 0.01, 0.05 and 0.1? Figure 3, top panel. You might want to consider annotating with text the highest TPR point for each dataset which is quoted in the text in page 5 right side. I appreciate that Figure 3 (top panel) shows the full range, but maybe it would be useful to have a zoomed-in version in the supplementary material in order to see the differences more clearly. Maybe have a ylim from 0.5 to 0.8, and an xlim from 0 to 0.6 (or something like that). Page 6, bottom left. \"ECs called the highest number of genes with significant DTU (1485 genes, in contrast to the 748 and 391 genes called significant by the transcript and exon-based methods respectively).\" That sentence is incorrect based on Supplementary Figure 2. The numbers for genes with significant DTU match for the transcript and exon based methods, but they don't for the EC based method since 228 + 204 + 147 + 96 = 675. This numerical change affects the conclusions drawn from Supplementary Figure 2. Page 6, bottom right. Were all samples from the full bottomly dataset used in any of the 20 iterations? Or were there some samples that were used in many of the replications? With 20 iterations I guess that there's a small chance that some samples were under-represented or over-represented in the iterations. Figure 3, bottom panel. I really liked the lines you show in Supplementary Figure 3 to identify the different comparable replicates. The lines helped me visualize that the ranks were consistent across replicates since the lines rarely intersect each other. I suggest mentioning those lines in the caption for Figure 3 where you refer to Supplementary Figure 3, or maybe even swapping the panels from Figure 3 (bottom) for the equivalent ones from Supplementary Figure 3 (no need to change Figure S3 in that case, that is, it's okay to repeat the panels). Page 7, right side. Typo \"psuedo-\" instead of \"pseudo-\". Page 8, left side. \"We also found the analysis was quick to run and we provide code to convert [...]\". I highly recommend including the URL here for the code or mention in a parenthesis in which section of the paper can one find the link to the code. Page 8, right side. I recommend also citing the Bottomly et al paper when you mention that the data was downloaded from SRR099223. You already cite the paper in other parts of your manuscript. From the link to the bioRxiv pre-print I was able to find tweets citing the pre-print and have to agree with this tweet saying \"this type of stuff is what the field needs\". Here, I didn't find the actual versions of the packages used. I suggest including the output of \"options(width = 120); sessioninfo::session_info()\" somewhere in that repository. I think that you don't need to call gc() manually in your function calls here. Normally R takes care of it. Since I see here that 8 cores were used for your method, I'm curious now looking at Supplementary Table 1 if the RAM presented there is by thread (core) or by process, and if so, how many cores were used for the other steps.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4584",
"date": "29 Apr 2019",
"name": "Alicia Oshlack",
"role": "Author Response",
"response": "Thank you for taking the time to review our paper and for the helpful suggestions. Minor points: We have fixed the issues with Figures 2 and 3, and have added a description of the dotted lines in Figure 3a. We now use more distinguishable colour-palettes in all cases where colours identify data points. Supplementary Figure 6 has been added (showing filtered vs. unfiltered results), and uses zoomed-in axes. This plot contains the original results for ECC, exon and transcript counts, which should now be easier to distinguish. We agree with the reviewer that boxplots can mask information. However, due to the discrete nature of the data, combined with the log-scale, results in a stepwise artefact. Please see Soneson et al.[1] Supplementary Figure 3 for an example. We have therefore opted to retain boxplots. We note that the source code for generating these plots is available in the ec-dtu-paper github repository should readers want to inspect the raw data. We also like the suggestion of the density ridges, however, due to the high number of data points (>1 million), this did not add any additional information to the visualisation. Instead of annotating TPR points for clarity on the plots in Figure 3a, we have added Supplementary Figure 6, which contains the same data points for ECs, transcripts and exons, as well as their respective performance using filtered features. We inspected the random samples selected for the Bottomly analyses (we have provided random seeds in the R markdown notebook) and noted that all samples were used at least one time. We have added code to the paper R markdown notebook to show sample usage across iterations. As is apparent in Supplementary Figure 3, the usage of particular samples is less important relative to the performance ranks observed of the method types across the iterations. The number of significant genes found, reflected in Supplementary Figure 2, has been corrected in the main text. We now mention the lines between replications in Figure 3’s caption. We have added all suggested links, references and fixed the typos pointed out in the paper. Session info has been added to the main paper, and we have removed the gc() statements from the code. Supplementary Table 1 lists RAM by process; this has been clarified in the caption. References:[1] Soneson, C., Matthes, K. L., Nowicka, M., Law, C. W., & Robinson, M. D. (2016). Isoform prefiltering improves performance of count-based methods for analysis of differential transcript usage. Genome Biology, 17(1), 1–15. https://doi.org/10.1186/s13059-015-0862-3"
}
]
}
] | 1
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https://f1000research.com/articles/8-265
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https://f1000research.com/articles/8-53/v1
|
14 Jan 19
|
{
"type": "Study Protocol",
"title": "Costs, cost-effectiveness and detection rate of glaucoma screening in cataract camps in urban India: A study protocol",
"authors": [
"Shalinder Sabherwal",
"Denny John",
"Suneeta Dubey",
"Saptarshi Mukherjee",
"Geetha R. Menon",
"Atanu Majumdar",
"Suneeta Dubey",
"Saptarshi Mukherjee",
"Geetha R. Menon",
"Atanu Majumdar"
],
"abstract": "India has an estimated 12 million people affected with glaucoma; however, no organised screening programme exists. Cases are usually detected opportunistically. This study documents the protocol for detecting glaucoma in suspects in cataract camps conducted by Shroff Charity Eye Hospital in North India. We report a prospective study design from patients attending cataract camps where glaucoma screening will be integrated. The eligible population for glaucoma screening is non-cataract patients. Patients will undergo glaucoma screening by a trained optometrist using a pre-determined glaucoma screening algorithm. Specific diagnostic cut-off points will be used to identify glaucoma suspects. Suspected patients will be referred to the main hospital for confirmatory diagnosis and treatment. This group will be compared to a cohort of patients arriving from cataract camps conducted by the institute in similar areas and undergoing examination in the hospital. The third arm of the study includes patients arriving directly to the hospital for the first time. Cost data will be captured from both the screening components of cataract-only and glaucoma screening-integrated camps for screening invitation and screening costs. For all three arms, examination and treatment costs will be captured using bottom-up costing methods at the hospital. Detection rates will be calculated by dividing the number of new cases identified during the study by total number of cases examined. Median, average and range of costs across the three arms will be calculated for cost comparisons. Finally, cost-effectiveness analysis will be conducted comparing cost per case detected across the three arms. This is the first such study conducted in India. The study protocol will be useful for researchers and practitioners for conducting similar economic evaluation studies in their context. The protocol publication will be a good step to ensure transparency of methods of reporting of economic evaluation studies in LMICs.",
"keywords": [
"screening",
"glaucoma",
"costs",
"cost-effectiveness",
"screening",
"detection rate"
],
"content": "Introduction\n\nGlaucoma is the second commonest cause of blindness in the world1. There are estimated 12 million people affected by glaucoma in India2. The two main types of glaucoma are angle-closure glaucoma (ACG) and open-angle glaucoma (OAG). It is estimated that about 70% of OAG and 80% of ACG cases occur in developing nations3. In India it is estimated that primary OAG (POAG) affects around 6.48 million people and primary ACG (PACG) affects around 2.54 million2. In India, it is estimated that among the 40+ age group, every eighth person could be at risk of, or, suffering from glaucoma2. As there is no organised screening for glaucoma, opportunistic case finding is the commonest method for case detection. It has been shown that around 90% of the glaucoma remains undiagnosed in both rural and urban populations in India4,5. Another study found that only 50% of people with glaucoma ever visited an ophthalmologist6. Thus, stressing the need of a more effective screening program\n\nThe use of intraocular pressure (IOP), field testing and optic disc findings for diagnosis of glaucoma has been stressed for effective screening program for identifying glaucoma in the community. It has been shown that none of them individually have positive predictive value or the sensitivity to be used for community-based screening. These tests when used together have much better sensitivity to diagnose early glaucoma.\n\nIn most of the developing countries, there is a shortage of ophthalmologists. In India, the availability of ophthalmologists in rural setting is low7. In order for an effective screening program to function there is need for including other ophthalmic personnel such as optometrists and ophthalmic assistants. It has been shown that trained ophthalmic assistants can be effective in detecting glaucoma in the community8 Thus, the equipment used should be selected according to the cadre involved in screening.\n\nIOP is an important risk factor for developing glaucoma. Although used alone it has a sensitivity of only 47.1%, and specificity of 92.4% if a cut-off of more than 21 mmHg is used for diagnosing POAG9. Tonometers based on rebound technology have been found to have accuracy similar to applanation tonometry10. An optic disc examination for the cup to disc ratio is used to diagnose glaucoma. Direct ophthalmoscope is relatively inexpensive and portable equipment which has been used in the community setting11. It’s sensitivity has been shown to be around 59%12. Frequency doubling technology (FDT) perimetry has emerged as a quick and inexpensive alternative. The sensitivity of the test has been shown to be around 50% in some studies13 as compared to more than 95% for automated perimetry14. Gonioscopy is considered gold standard for identifying eyes at risk of angle closure but it requires clinical expertise to conduct and interpret the test. Thus, it is not an appropriate screening test15. Van Herick test for peripheral anterior chamber depth (ACD) has been shown to have a sensitivity of 91% for detecting shallow chambers16 and of 61.9% in detecting occludable angles17.\n\nWHO Vision 2020 has prioritised interventions for five causes of avoidable blindness18. Cataract being the commonest cause of avoidable blindness in India has been prioritised by NPCB. There are regular cataract screening camps organised by both government and non-government service providers. Within these camps the focus is to examine the maximum number of patients over 50 years of age. This research protocol aims to study the costs, cost-effectiveness and detection rate of glaucoma cases in cataract screening camps in North India.\n\n\nMethods\n\nDr. Shroff’s Charity Eye Hospital (SCEH), based in New Delhi, is a tertiary referral center providing general and subspeciality services and training. SCEH conducts one-day cataract screening clinics in villages closer to the city of New Delhi. This is made possible through the financial support of private donors. These programs target patients who have not sought out care due to limited or non-existent local eye care facilities, financial constraints, or in most case a lack of awareness regarding eye-care. In anticipation of the one-day screening clinics, local organizers are responsible for advertising the clinic (e.g. through posters, flyers, or door-to-door visits), managing volunteers and procuring an appropriate facility. The cost of transportation of patients to SCEH is borne by hospital These camps are managed by Program Manager (Outreach) based at SCEH with regards to planning, monitoring, and strategy.\n\nIn our data of the camps conducted by the outreach team in Delhi, between June 2016 and May 2017, out of 8283 population of ≥40 years age screened, only 0.25% (n=21) were identified as glaucoma suspects. This is well below the prevalence (1.6-3.5%) found in the rural and urban studies in India19. Model based studies of community screening for glaucoma in rural and urban India has been shown to be cost-effective [20, 21]. The rationale of this study is to analyse the cost and detection rate of detecting glaucoma in cataract camps. If found to be cost effective, this model can be scaled up for detecting glaucoma suspects from cataract screening camps and referring them for further investigations and management.\n\nIn addition to the original team for the outreach camp, one of the trained optometrists, a vision technician for handling FDT and a counsellor would travel for the interventional camps.\n\nAll people of age ≥40 years, not detected as having a cataract would undergo screening for glaucoma by trained optometrist using additional equipment. Those detected with cataracts are not included in the study as they would have undergone glaucoma screening as a routine before being advised surgery. Those included in the study would have already undergone vision testing and refraction if indicated. The registration number given at the start of the camp would be recorded by the counsellor. There would be two stations for glaucoma screening. The trained optometrist would carry out disc examination, ACD examination and IOP recording. At the second station FDT would be carried out. Any result beyond cut-off and requiring further intervention would be flagged off. After that the person screened would be received by the counsellor. The test used for screening would be disc examination, FDT and IOP using Icare rebound perimetry.\n\nTraining. There will be two optometrists trained for the study. The optometrists selected would already be carrying out refractions, basic history taking and slit-lamp examination. The curriculum would pertain to glaucoma-related history-taking and slit-lamp examination. Specifically, for the study the following training would be included for various test to be conducted in the study\n\nThe optometrists would have explained to them the need to calibrate before starting measurements and explained the process of calibration. The principles of avoiding injury while recording IOP would be discussed. The cut-offs being used in the study would be stressed upon\n\nThe optometrists would be trained to use direct ophthalmoscope and focus on the disc. They would be exposed to patients and photographs with a variety of disc findings.\n\nThe concept of the Van Hericks test would be explained. Although they would be already using a slit-lamp, they would be trained to use a hand-held slit lamp. The cut-offs to be used would be explained. The optometrists would be maintaining a log-book to record their findings, and these would be checked and signed by a glaucoma consultant or a senior fellow.\n\nTo carry out a field test with FDT, the optometrists would be trained in handling the equipment, giving instructions to the patients and commenting on the printouts keeping the cut-offs of the study in mind.\n\nThe training would be conducted in a classroom with visual presentations for theory. A senior glaucoma fellow ophthalmologist would be involved to take classes with a problem-based approach. Theory class would be scheduled for 1 hour in every working day for 2 weeks. A total of 12 hours would be spent in theory classes. The topics to be covered are mentioned in Table 1.\n\nJunior glaucoma consultant would be involved in imparting practical training. In total, 30 h would be taken to develop their practical skills for each trainee. A log book record would be maintained for the patients examined. The criteria used for assessing the level of training are highlighted in Table 2.\n\nIOP, intraocular pressure; ONH, optic nerve head.\n\n\nValidation\n\nThis would be carried out after the training is complete. A total of 30 cases each would be given to both optometrists for recording IOP, ACD and C/D ratio. These would be either new cases or they would be masked to previous records. A senior optometrist would independently record IOP and a glaucoma consultant would examine for ACD and C/D ratio. Both the senior optometrist and glaucoma consultant would be masked to the trainee optometrists’ findings. Sensitivity and specificity would be calculated keeping the trainers as gold standard. If not found to be within an acceptable range (85% sensitivity, and 90% specificity), the training would be repeated for the concerned trainee in the identified area of weakness.\n\nOnce found acceptable in all aspects, on a separate day, 20 patients would be assigned to both the trainees for calculating inter-observer variation. The cut-off to be used for labelling as glaucoma suspect at the camp site in our study would be based on parameters mentioned in Table 3.\n\nIn FDT perimetry reporting, one abnormal spot in the central field or more than one spot in the peripheral field would be considered below the cut-off for normal in this test . Any patient with normal disc examination but abnormal FDT findings will not be labelled as suspected glaucoma but will be advised to undergo detailed visual field analysis in the hospital.\n\nRecords of each person screened would be entered Those requiring further investigations would be counselled about glaucoma and importance of early detection. They would be offered a free drop to the base hospital. Those not agreeing to travel on the same day would be given a contact number, in case they would like to report later. Those not reporting within a week would be given a reminder call.\n\n\nConventional detection\n\nIn the case of India, currently no organized community-screening program specifically for glaucoma detection exists. At present, detection of glaucoma is through ‘opportunistic case finding’ of patients who present themselves to various eye clinics in the country for various ophthalmic complaints [22].\n\nThe opportunistic case finding of patients presenting in SCEH would be the comparator group for our study. The inclusion criteria would be patients ≥40 years of age and belonging to the low-paid category in the hospital to match the socioeconomic status. People with prior history of glaucoma screening in community or clinic or already diagnosed with glaucoma would be excluded from the study. After presentation to SCEH, patients would undergo glaucoma consultation at the hospital.\n\nFor those travelling to the hospital, transport and investigations in the hospitals would be offered free of cost. Overnight stay if required would also be provided free. A record would be made of the tests, number of persons transported and number staying overnight for cost analysis.\n\nIn the hospital, a detailed workup would be done for the glaucoma suspects, so as to confirm or rule out the diagnosis of glaucoma with the following tests: refraction, applanation tonometry, gonioscopy, automated visual field analysis and nerve fibre analysis. Patients will be classified as normal or with OH, probable glaucoma, or glaucoma as per the criteria in Table 4. After the investigations, they would be examined by a glaucoma consultant masked to the source of patient sent. Classification will be done using the criteria mentioned in Table 4. During their time at the hospital the patients/care-givers would be administered a questionnaire about glaucoma awareness (Annexure 1, extended data)20. After the investigations, they would be examined by a glaucoma consultant, who would make a diagnosis of glaucoma, glaucoma suspect, angle closure disease or impending angle closure requiring intervention, using the criteria mentioned in Table 4. The patient requiring intervention would be counselled and others would be discharged. Those requiring surgery would be offered surgery at fixed rates meant for camp patients needing non-cataract surgery. The patients staying overnight would be offered a free drop and records would be maintained.\n\n\nAim and objectives\n\nThe aim of the study is to measure the cost, cost-effectiveness and detection rate of glaucoma screening in cataract camps in rural India conducted by SCEH. The comparator group would be patients who are undergoing assessment for cataract in other such camps conducted by SCEH and diagnosed with glaucoma on their presentation to the SCEH hospital. The comparator will also include patients reporting directly to SCEH for cataract and/or glaucoma assessment.\n\nThe specific objectives of the study are:\n\na) To measure the detection rate measured by dividing the number of new cases identified during the study by the total number of cases examined.\n\nb) To compare the costs of setting up and implementing glaucoma screening in cataract camps.\n\nc) To estimate the direct and indirect costs of the intervention to the patients undergoing glaucoma screening and subsequent treatment in the camps.\n\nd) To estimate the cost-effectiveness based on cost per case detected of glaucoma screening in comparison with opportunistic case finding in hospitals from non-screened populations.\n\nA prospective cohort study of glaucoma detection and cost assessment of the glaucoma screening conducted in the community. The total and incremental costs of the intervention prospectively from a societal perspective, measuring programme, provider and household costs.\n\nThe camps will be held in urban slums in and around the national capital region of Delhi in North India. Figure 1 and Figure 2 depict the flow of patients, that will be maintained in the routine camps and intervention camps, respectively.\n\nIOP, intraocular pressure; ACD, anterior chamber depth; FDT, frequency doubling technology.\n\nFigure 1 shows the patient flow in a routine cataract camp, whereas, in Figure 2, the patient flow in camps which constituted interventional arm is depicted.\n\nA. Intervention arm: From intervention cataract camps\n\nAll people examined by ophthalmologist with torch and ophthalmoscope.\n\nPatients aged ≥40 years not having cataract and diagnosed as normal at this step would be included in this arm.\n\nIncluded patients would undergo ophthalmoscopy, AC depth examination, FDT and IOP measurement with rebound tonometry.\n\nIf identified as neither glaucoma or suspected glaucoma (criteria as per Table 3), they would be discharged from the camp after being labelled as normal.\n\nIf labelled as suspected cases (criteria as per Table 3), referred to hospital.\n\nIn hospital, these referred cases will undergo the following examinations:\n\nUndergo complete clinical examination with gonioscopy and 90D examination for disc by a glaucoma specialist.\n\nDetailed visual field examination.\n\nIf normal, discharged.\n\nIf diagnosed with glaucoma (criteria as per Table 4), advised appropriate treatment (YAG PI, surgery offered free or prescribed medication).\n\nB. Non-intervention arm: Routine cataract camps\n\nThose ≥40 year old identified from routine cataract camps without any extra equipment or trained optometrist for glaucoma diagnosis:\n\nPeople attending camps undergo routine tests by ophthalmologist with torch and ophthalmoscope.\n\nIf diagnosed as having cataract by the ophthalmologist, the subject will undergo IOP with Schiötz tonometry.\n\nGlaucoma suspects (Table 4) in this arm would come from routine cataract camps if\n\n1. detected by ophthalmologist in the camp as suspected glaucoma\n\n2. if picked up as cataract from camps, brought for cataract surgery but diagnosed to be having glaucoma after examination in the hospital\n\nUndergo tests similar as intervention group in hospital:\n\nUndergo complete clinical examination with gonioscopy and 90D examination for disc by a glaucoma specialist\n\nDetailed visual field examination\n\nIf normal, the subject is discharged\n\nIf diagnosed glaucoma--- advised appropriate treatment\n\nc. Hospital arm: patients presenting to comprehensive semi-private OPD (with similar socio-economic status to camp patients) (Figure 3)\n\nPatients suspected glaucoma for the first time will undergo the following diagnostic tests:\n\nExamination including gonioscopy with glaucoma specialist\n\nVisual field test\n\nThis figure has been reproduced with permission of John (2011)21.\n\nIf diagnosed as glaucoma (Table 4), appropriate treatment is advised.\n\nThe detection rate or yield will be measured by dividing the number of new cases identified during the study by the total number of cases examined. However, various other indicators will also be estimated through the study, which are expected to throw light on the efficacies of the FDT setting at the intervention camps in comparison with normal cataract camps and conventional detection setting at the hospital. Therefore, the sample sizes are estimated for different components of the study separately, based on the desired accuracy of the indicators at different level. For an accuracy of ±0.1 with a confidence interval of 95% and assuming a rate of around 50% positive predictive value (PPV) and negative predictive value (NPV), the sample size was estimated to be 97 suspects as diagnosed by the additional setting.\n\nThe sample size mentioned above is applicable for both FDT (used in camps and hospital set-up B) and conventional detection setting (triage) at the hospital set-up A. This sample sizes are estimated using the following formula24:\n\n\n\nWhere\n\nα is such that (1- α) is the probability of confidence interval\n\nZ1-α/2 is defined as probability {|X|< Z1-α/2 | X~ N(0,1)} = (1- α)\n\nd = desired accuracy of the estimate. In other words, length of the confidence interval is 2d.\n\nSince the individuals not suspected of having glaucoma in the intervention camps will not be referred to the hospital for confirmatory diagnosis as per the study protocol, the sample for normal population will be captured by FDT setting at the hospital set-up B. We assume that only 75% post-screening follow-up compliance rates, i.e. people suspected of having glaucoma, referred from the intervention camps turning up for confirmatory diagnosis. However, the confirmatory diagnosis test can be conducted on all those diagnosed as suspected of having glaucoma in the hospital arm as they will be present in the hospital itself. It has been assumed, for the purpose of estimation, that the rate of detection of people suspected of having glaucoma would not exceed 12% in the community. We further assume that it can be, in reality, as low as 10%. The number of screenings in the intervention, non-intervention and hospital arm should be adjusted accordingly in order to capture the required minimum sample sizes for people suspected of having glaucoma (required for estimating PPV) and normal (required for estimating NPV) populations. Although the FDT setting in the non-intervention arm will also capture a few suspected glaucoma cases, its primary objective is to identify normal individuals to be referred for confirmatory diagnosis, the target for capturing suspects at the intervention camps is reduced. Conventional detection setting in the the Hospital arm will identify both suspects and normal for confirmatory diagnosis.\n\nGiven the above assumptions on (i) post-screening follow-up compliance rates, (ii) prevalent suspect rates for FDT setting and conventional detection setting and (iii) required minimum sample sizes for estimating PPV and NPV, the number of screenings required for different components are estimated as per Table 5.\n\n*Conventional detection setting at hospital would identify many more suspects and normal than what is required to estimate its PPV and NPV. But we need only 97 from each category (suspect and normal). NA, not applicable.\n\nAll people of age ≥40 years, not detected as having cataract who would consent for screening for glaucoma would be included. A trained optometrist and vision technician would conduct various screening tests, such as disc examination, ACD depth, IOP reading, and FDT. Any result beyond cut-off points as mentioned in Table 3 would be flagged as suspected glaucoma cases and would be received by counselor. For those travelling to the hospital, transport and investigations in the hospitals would be offered free of cost. Overnight stay if required would also be provided free.\n\nIn the hospital, a detailed workup would be done for the glaucoma suspects. For glaucoma, they would undergo applanation tonometry, gonioscopy, automated visual field analysis and nerve fibre analysis. After the investigations, they would be examined by a glaucoma consultant, who would be masked to the study arm to which patient belongs. A diagnosis of glaucoma, suspected glaucoma, angle closure disease or impending angle closure requiring intervention will be made. The patient requiring intervention would be counselled and others would be discharged. Those requiring surgery would be offered surgery at fixed rates meant for camp patients needing non-cataract surgery. The patients staying overnight would be offered free transport both to the hospital from the camp and back to the camp site after discharge. Records would be maintained of all patients offered free transport.\n\nA prospective observational study of 1000 adults ≥40 years age attending for glaucoma screening in cataract screening camps will be conducted. These adults would be recruited over an 18-month period across camps being conducted in villages near Delhi. The study is ongoing and recruitment is expected to be completed by December 2019.\n\nThe comparator group would be an equal number of adults of similar age who are screened in cataract camps in other nearby similar areas by SCEH and are identified as glaucoma suspects and as glaucoma-positive during their visit to the hospital. Additional comparator would also be patients >40 years age of similar socio-economic backgrounds who visit the hospital for the first time as routine or referral patients for eye check-up and are identified as glaucoma suspects and glaucoma positive.\n\nThe primary outcome of the screening algorithm would be the detection rate of the screening tests. The detection rate will be calculated by dividing the number of new cases identified during the study by the total number of cases examined [28].\n\nWe will use a step-down costing methodology for capturing costs of screening camps, whereby program costs will be entered into a customized tool created in MS Excel (Annexure 4, extended data)20. Cost data will be regularly entered into the tool for a period of 3 months to reflect any changes in the cost structure of the screening program. Using a step-down method, the main worksheets for entering data allocate costs to one of the following categories: training, pre-screening and screening.\n\nThe costs will be calculated by adding the unit costs of all resources used in the different activities occurring the screening process. This process includes all activities and procedures performed to detect cases with glaucoma from the invitation to participate in the screening campaign to the moment when the ophthalmologist establishes the diagnosis in the hospital. We will use the concept of activity-based costing for the screening program, but for the hospital we will use a standardized cost-accounting system. For the activity-based costing, the costs inputs will include screening invitation, screening costs (health professionals, devices, screening venue etc.). Personnel costs will be calculated from the total cost of each professional participating in a certain activity according to the time specifically dedicated to that particular activity. For example, optometrists dedicated 5–6 hours of their daily work time to the screening and only that part of their full salary will be considered as a cost. For ophthalmologists conducting different tasks (training, examination or test interpretation), only the fraction of their work time dedicated to the specific task involved in the glaucoma diagnostic process will be added.\n\nAll diagnostic equipment with a usage life of more than a year will be considered as capital equipment. The annual and monthly rental value of the capital equipment, and screening clinic costs per year/month will be calculated as per methods for assessing costs of glaucoma screening in India in other studies [20, 21]. All other diagnostic equipment will be considered as non-capital and will be considered with a depreciation rate of 100% in the first year itself.\n\nKey information interviews with Program Manager (Outreach) will be conducted in identifying any donated goods requiring reevaluation and in allocating joint costs between program components. The allocation of joint costs to program components and activities, will also be informed by monthly sheets of screening camps.\n\nThe fixed costs at the hospital (electricity, water, gas, maintenance, and security) will be estimated from the annual structural costs of the facility divided by the number of patients seen and multiplied by the number of patients evaluated in the hospital in both screening and conventional arms. The structural costs at the hospital (glaucoma consultation) will be calculated from the hospital cost-accounting system.\n\nAll costs captured from accounting statements, such as fixed costs, salaries, etc., will be converted to economic costs. This means that capital costs will be annualized over their expected useful life, and any donated goods or volunteer time appearing as zero costs in the accounting data, or not appearing in the accounting data in any form, will be added to the cost sheets and assigned their current market value. Costs to the community would be captured using a structured questionnaire and will include direct medical (consultation, medicines etc.), direct non-medical (travel), and indirect costs (wage loss of patient) (See Annexure 2, extended data)20.\n\nFor the cost-effectiveness analysis, the cost per case detected would be the primary effectiveness outcome measure, in comparison with conventional detection, i.e. opportunistic case finding in hospitals.\n\nThe costs for the screening program will be based on the study sample and may vary significantly depending on the size of the target population and other factors. Hence, a sensitivity analysis will be performed using mean of detection rates from other studies in similar developing country settings.\n\nThe costs will also be recalculated for the community of 1 million subjects in order to generalize the findings to India as a whole. Costs will be presented in 2019 prices in Indian rupees and United States dollars (USD). Since the study period is less than 12 months, costs will not be adjusted for inflation, nor will discounting be considered for costs and outcomes.\n\nData entry will be conducted by a single data entry operator at the research unit of SCEH. One of the co-authors (AM) will be reviewing the data entry to check for any discrepancies including any data entry errors from the data entry form. The data will be stored in a desktop computer with access to the data entry operator, and co-auth or (AM). Once the data entry is completed, and cleaned, the data sheet will be transferred to laptops of co-author (AM & DJ) for further analysis. After the analysis these data sheets will be destroyed in these laptops and the data sheet would be available only with the desktop present at research unit of SCEH.\n\nThe patients being screened would be undergoing standard investigative procedures and no experimental investigation or procedure would be carried out. Informed consent would be taken before administering the questionnaire regarding cost incurred by patient/relative for screening and hospital examination (Extended data, Annexure 3)20. The identity of the person would not be disclosed, and no identifiable data would be shared or published. The identifiable data stored in database would be password protected.\n\nThe study received ethical approval from Dr Shroff’s Charity Eye Hospital-Ethics Committee (study reference number SCEHEC/2018/02) on 13.02.18.\n\nThe study results will be submitted to a suitable peer-review publication within 6 months of study period (i.e. patients entering camps and then arriving at hospital) with an acceptable sample size as described in relevant section above. Additionally, the results will also be presented in suitable national/international conferences based on resources available for participation.\n\n\nConclusion\n\nThis paper constitutes the first published protocol for the cost and cost effectiveness of a glaucoma screening program in a low-middle-income country. The protocol, which will adhere to internationally recognized guidelines for conducting and reporting economic evaluation studies, serves to heighten the transparency of the economic evaluation and planned analyses. The findings from this study will inform funding organizations, eye care institutions and policymakers about the relative value of glaucoma screening in the community. The evidence will contribute significantly to the scarce evidence regarding the cost-effectiveness of community screening for glaucoma in reducing blindness in LMICs.\n\n\nData availability\n\nExtended data for this study, described below, are available on figshare. DOI: https://doi.org/10.6084/m9.figshare.750388720.\n\nAnnexure 1. Questionnaire concerning glaucoma awareness. English and Hindi versions are given.\n\nAnnexure 2. Patient expenses data form.\n\nAnnexure 3. Patient informed consent form.\n\nAnnexure 4. Customised Excel tool template.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPascolini D, Mariotti SP: Global estimates of visual impairment: 2010. Br J Ophthalmol. 2012; 96(5): 614–8. PubMed Abstract | Publisher Full Text\n\nGeorge R, Ve RS, Vijaya L: Glaucoma in India: estimated burden of disease. J Glaucoma. 2010; 19(6): 391–7. PubMed Abstract | Publisher Full Text\n\nThylefors B, Négrel AD: The global impact of glaucoma. Bull World Health Organ. 1994; 72(3): 323–6. PubMed Abstract | Free Full Text\n\nDandona L, Dandona R, Srinivas M, et al.: Open-angle glaucoma in an urban population in southern India: the Andhra Pradesh eye disease study. Ophthalmology. 2000; 107(9): 1702–9. PubMed Abstract | Publisher Full Text\n\nJacob A, Thomas R, Koshi SP, et al.: Prevalence of primary glaucoma in an urban south Indian population. Indian J Ophthalmol. 1998; 46(2): 81–6. PubMed Abstract\n\nRamakrishnan R, Nirmalan PK, Krishnadas R, et al.: Glaucoma in a rural population of southern India: the Aravind comprehensive eye survey. Ophthalmology. 2003; 110(8): 1484–90. PubMed Abstract | Publisher Full Text\n\nThomas R, Paul P, Rao GN, et al.: Present status of eye care in India. Surv Ophthalmol. 2005; 50(1): 85–101. PubMed Abstract | Publisher Full Text\n\nThapa SS, Kelley KH, Rens GV, et al.: A novel approach to glaucoma screening and education in Nepal. BMC Ophthalmol. 2008; 8(1): 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTielsch JM, Sommer A, Katz J, et al.: Racial variations in the prevalence of primary open-angle glaucoma. The Baltimore Eye Survey. JAMA. 1991; 266(3): 369–74. PubMed Abstract | Publisher Full Text\n\nIliev ME, Goldblum D, Katsoulis K, et al.: Comparison of rebound tonometry with Goldmann applanation tonometry and correlation with central corneal thickness. Br J Ophthalmol. 2006; 90(7): 833–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNduaguba C, Lee RK: Glaucoma screening: current trends, economic issues, technology, and challenges. Curr Opin Ophthalmol. 2006; 17(2): 142–52. PubMed Abstract | Publisher Full Text\n\nQuigley HA, Katz J, Derick RJ, et al.: An evaluation of optic disc and nerve fiber layer examinations in monitoring progression of early glaucoma damage. Ophthalmology. 1992; 99(1): 19–28. PubMed Abstract | Publisher Full Text\n\nIwase A, Tomidokoro A, Araie M, et al.: Performance of frequency-doubling technology perimetry in a population-based prevalence survey of glaucoma: the Tajimi study. Ophthalmology. 2007; 114(1): 27–32. PubMed Abstract | Publisher Full Text\n\nKatz J, Sommer A, Gaasterland DE, et al.: Comparison of analytic algorithms for detecting glaucomatous visual field loss. Arch Ophthalmol. 1991; 109(12): 1684–9. PubMed Abstract | Publisher Full Text\n\nParikh R, Mathai A, Parikh S, et al.: Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol. 2008; 56(1): 45–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlsbirk PH: Limbal and axial chamber depth variations. A population study in Eskimos. Acta Ophthalmologica. 1986; 64(6): 593–600. PubMed Abstract | Publisher Full Text\n\nThomas R, George T, Braganza A, et al.: The flashlight test and van Herick's test are poor predictors for occludable angles. Aust N Z J Ophthalmol. 1996; 24(3): 251–6. PubMed Abstract | Publisher Full Text\n\nPizzarello L, Abiose A, Ffytche T, et al.: VISION 2020: The Right to Sight: a global initiative to eliminate avoidable blindness. Arch Ophthalmol. 2004; 122(4): 615–20. PubMed Abstract | Publisher Full Text\n\nVijaya L, George R, Baskaran M, et al.: Prevalence of primary open-angle glaucoma in an urban south Indian population and comparison with a rural population. The Chennai Glaucoma Study. Ophthalmology. 2008; 115(4): 648–54.e1. PubMed Abstract | Publisher Full Text\n\nSabherwal S: extended data for protocol. figshare. Fileset. 2018. http://www.doi.org/10.6084/m9.figshare.7503887.v1\n\nAllingham RR: Shields Textbook of Glaucoma. In: 6th, editor. 176. Publisher Full Text\n\nFoster PJ, Buhrmann R, Quigley HA, et al.: The definition and classification of glaucoma in prevalence surveys. Br J Ophthalmol. 2002; 86(2): 238–42. Publisher Full Text\n\nHodapp E: The asymptomatic patient with elevated pressure. Clinical decisions in glaucoma. 1993. Reference Source\n\nMurthy MN: Sampling Theory and Methods. 2nd ed, 1967. Reference Source"
}
|
[
{
"id": "43006",
"date": "30 Jan 2019",
"name": "Anil Gumber",
"expertise": [
"Reviewer Expertise Health economics",
"Indian health care system and financing"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have made editing comments on the PDF file.\nThe protocol is well written.\nSome editing comments e.g. for a couple of tables the title is misleading. Also at some places the population considered for screening mentioned as over 40 years whereas in most places including in tables/figures >= 40. This needs to be corrected.\nAt several places abbreviations are used, so it would be useful to provide a glossary at the end of the paper. Also, the term \"Icare\" is used without defining it properly.\nIn the introduction paragraph, the authors have not provided difference in two types of glaucoma and why one's share is greater then other in India and whether their share is consistent with other LMICs.\nIn the methods section there are problems in calculating various costs at the organisation level as well as at the patient level. For patient level, the out-of-pocket expenditures on medical equipment/aids has not been considered due to glaucoma care.\nThe training cost to field staff has been considered but their validation of enhanced skills for correct detection of glaucoma screening has not been elaborated.\nAt organisation level, there is not enough clarity about calculating the unit cost and portioning of staff time used for screening. Multiple activities of staff and multi-usages of fixed cost items such as equipment, furniture and buildings as well as variable cost items (e.g. drugs and consumables) has not clearly been spelt out. Salary cost does not include the employer contribution to pension, earned/casual leave etc. and thus staff cost to the organisation turned out to be 40-60% higher than salary alone. The overhead cost (e.g. admin and management - usually 29% of medical personnel cost) has not been fully explained. Repairs and maintenance of equipment and depreciation for equipment/furniture/vehicle are not explained. Most importantly, the capital cost in terms of building and land (rental value) has not been considered at all.\nThe opportunity cost of buildings and equipment used in the production of services, we need to have an estimate of the costs involved and to make assumptions about both the length of time that the ‘investment’ will be tied up in the service, and the rate of return on that investment. In public health services ‘long-run marginal opportunity cost’ is focused, which is the cost of supporting one extra patient while recognising the financial implications of necessary expansion to the service. In the UK, a buildings’ useful life is considered 60 years thus the annuitizing rate is 2.5% (i.e. converting a capital investment into the annual equivalent cost for the period over which the investment is expected to last).\nIn terms of cost and cost-effective analysis, the paper has not provided enough details including methods of smoothing the costs data. The long-term benefits of early detection of glaucoma through screening has not been elaborated and modelled. It is important to bring into cost-effectiveness framework by quantifying the contribution of screening and prevention in reducing the disease related morbidity, blindness and premature mortality.\nThe inclusion of above comments will definitely improve the quality of the protocol.\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": "4419",
"date": "12 Feb 2019",
"name": "shalinder sabherwal",
"role": "Author Response",
"response": "Response to Reviewer 1 Comment: The protocol is well written.Response: Much thanks for this commentComment: Some editing comments e.g. for a couple of tables the title is misleading. Also at some places the population considered for screening mentioned as over 40 years whereas in most places including in tables/figures >= 40. This needs to be corrected.Response: We have corrected the tables. We have also used > 40 years uniformly across all sections of the manuscript.Comment: At several places abbreviations are used, so it would be useful to provide a glossary at the end of the paper. Also, the term \"Icare\" is used without defining it properly.Response: We have added a section on Glossary of terms at the end of the paper. Details of Icare is now inserted as foot note.Comment: In the introduction paragraph, the authors have not provided difference in two types of glaucoma and why one's share is greater then other in India and whether their share is consistent with other LMICs.Response: We have now inserted these sections in the introductory paragraphs.Comment: In the methods section there are problems in calculating various costs at the organisation level as well as at the patient level. For patient level, the out-of-pocket expenditures on medical equipment/aids has not been considered due to glaucoma care.Response: We have detailed section of organisational costs. Patient costs including out-of-pocket expenditure including drugs have been inserted in Annexure 2. Comment: The training cost to field staff has been considered but their validation of enhanced skills for correct detection of glaucoma screening has not been elaborated.Response: Validation of skills is mentioned in the training section of the manuscript.Comment: At organisation level, there is not enough clarity about calculating the unit cost and portioning of staff time used for screening. Multiple activities of staff and multi-usages of fixed cost items such as equipment, furniture and buildings as well as variable cost items (e.g. drugs and consumables) has not clearly been spelt out. Salary cost does not include the employer contribution to pension, earned/casual leave etc. and thus staff cost to the organisation turned out to be 40-60% higher than salary alone. The overhead cost (e.g. admin and management - usually 29% of medical personnel cost) has not been fully explained. Repairs and maintenance of equipment and depreciation for equipment/furniture/vehicle are not explained. Most importantly, the capital cost in terms of building and land (rental value) has not been considered at all.Response: We have mentioned screening time per patient section which details the time of optometrist and receptionist time per patient for screening is now inserted. We have also added sections to capture salary, fringe benefit of optometrist, receptionist and helper. Overhead cost of 29% has been inserted. Annual maintenance costs of diagnostic equipment at 10% of annual rental value has been considered. Rental value of screening clinic as per local information has been inserted.Comment: The opportunity cost of buildings and equipment used in the production of services, we need to have an estimate of the costs involved and to make assumptions about both the length of time that the ‘investment’ will be tied up in the service, and the rate of return on that investment. In public health services ‘long-run marginal opportunity cost’ is focused, which is the cost of supporting one extra patient while recognising the financial implications of necessary expansion to the service. In the UK, a buildings’ useful life is considered 60 years thus the annuitizing rate is 2.5% (i.e. converting a capital investment into the annual equivalent cost for the period over which the investment is expected to last).Response: We have used opportunity cost of buildings and equipment as per standards in Indian settings, including life-cycle and discounting considerations.Comment: In terms of cost and cost-effective analysis, the paper has not provided enough details including methods of smoothing the costs data.Response: Smoothing of costs data has been considered using annualised costs, including simultaneous equation method for overhead cost distribution. Comment: The long-term benefits of early detection of glaucoma through screening has not been elaborated and modelled. It is important to bring into cost-effectiveness framework by quantifying the contribution of screening and prevention in reducing the disease related morbidity, blindness and premature mortality.Response: Our objective of the study is to capture the cost per detection rate across the intervention and comparator arms. We have not considered the long-term benefits of early detection of glaucoma and modeling as 2 studies in urban and rural India using decision analytical model have been considered by John & Parikh (2017), and John & Parikh (2018), and mentioned in references. However, as a follow-up we might consider building a Markov model based on study results to estimate the long-term benefits of glaucoma screening in India. The inclusion of above comments will definitely improve the quality of the protocol."
}
]
}
] | 1
|
https://f1000research.com/articles/8-53
|
https://f1000research.com/articles/8-563/v1
|
26 Apr 19
|
{
"type": "Systematic Review",
"title": "Diverse mechanisms and treatment strategies to confront fatigue in multiple sclerosis: A systematic review",
"authors": [
"Sumanth Khadke",
"tehmina siddique",
"tehmina siddique"
],
"abstract": "Background: Firm conclusions about the applicability of treatment methods other than pharmacotherapy in treating fatigue in multiple sclerosis (MS) remain elusive. Our objective is to synthesize and review the epidemiological literature systematically and find an effective therapeutic plan for fatigue. The effect of individual treatment and combined treatment strategies are studied. Methods: An electronic database search included EBSCO, PubMed, SCIENCE DIRECT and Scopus from January 1, 2013, to September 30, 2018. Search terms used are “Fatigue AND Multiple sclerosis AND therapy”. The articles included in the study are open access, published in last five years, not restricted to region and language. The search included randomized controlled trials (RCTs), observational studies, and systematic reviews. Results: We included 13 systematic reviews, 10 RCTs and 7 observational studies. A Cochrane review on 3206 patients showed exercise therapy to have a positive effect on fatigue in RRMS patients. The EPOC trial showed switching interferon therapy or glatiramer to fingolimod showed improved fatigue levels. The FACETS trial showed incorporating behavioral therapy to ongoing recommended therapy is beneficial. Few observational studies demonstrated that fatigue is influenced by pain, mood problems, and depression. Conclusions: The diverse pathology of fatigue related to MS is important in understanding and quantifying the role of each causal factor. Evidence reveals a positive effect on fatigue levels of RRMS patients with regular CBT and exercise-based combination therapy. Progressive forms of the disease have the worst prognosis. Individually aerobic exercises, behavioral therapy and pharmacotherapy have positive effects. A modified amalgamation of the same is a better hope for MS patients.",
"keywords": [
"Multiple sclerosis",
"fatigue",
"cognitive behavioral therapy",
"combined therapy",
"fatigue in MS."
],
"content": "Introduction\n\n“The idea that the brain can change its own structure and function through thought and activity is, I believe, the most important alteration in our view of the brain since we sketched out its basic anatomy and the workings of its basic component, the neuron.” – Norman Doidge.\n\nFatigue is a major symptom of multiple sclerosis (MS), which can lead to the difficulty in the carrying out the daily errands and lowers the quality of life; it is prevalent in 80% of patients and hinders the quality of life in nearly 70%1. Fatigue is disabling as it causes problems in daily life necessitating the need for a caregiver, causes embarrassment at workplaces where timebound work is, employment issues that can lead to premature retirement1. Drugs used to treat MS are categorized as oral drugs, injectables, and infusions. Oral drugs include fingolimod, dimethyl fumarate, teriflunomide, and cladribine; injectables include INFβ1a/1b, daclizumab, and glatiramer acetate; infusions include natalizumab, alemtuzumab, and ocrelizumab2. Even upon arrival of new efficacious drugs which can halt the progression of the disease, fatigue remains the most troublesome symptom of patients, giving rise to forms of alternate treatment. This is a systematic review concerning how well pharmacological and non-pharmacological interventions influence fatigue levels in MS patients when compared to healthy adults.\n\nMS is a chronic neurodegenerative disease characterized by disseminated plaque like sclerotic lesions distributed in space and time. They are seen in both grey and white matter of CNS. MS is affecting 2,000,000 people worldwide and 400,000 people in the United States per year. The annual economic burden of the disease in the United States is approximately 10 billion dollars per year1. The 2015 statistics revealed MS disability-adjusted life year (DALY) count was 1234 (1033 to 1437) per 100,000 population, increase in DALY since 1990 to 2015 was 42.4% (31.8 to 57.3%) and age-standardized rate per 100 000 is 17 (14 to 20) per 100 0003. The epidemiological basis of MS is based on genetic and environmental risk factors4. Although we do not have the most recent data on widespread MS investigation, it is estimated that the numbers can be alarmingly higher than the previous records.\n\nDistribution of disease burden according to a survey in 2013 is shown in Figure 1.\n\nThis shows that the disease has a high prevalence in cold countries especially The United States of America and Canada. ©MSIF 2013; reproduced with permission.\n\nMS is characterised by autoreactive T cells like CD4+T cells in the perivascular space and CD8+T cells invading neural parenchyma causing damage to the myelin. Acute sclerosing plaques are due to astrocyte and microglial activation. Microglia clear the dysfunctional synapses that exhibit classical complement proteins C1q and C3. This clearing process can be pathologic if aberrant activation of astrocytes occurs, causing increased complement expression in synapses, resulting in increased degeneration. Neuronal changes, like ballooning of the cell and eccentric nucleus with increased amounts of phosphorylated neurofilaments in the grey matter, are signs of anterograde or retrograde degeneration which could be after effects of axonal disruption in white matter1. The oligodendrocyte precursor cells comprise 5% of CNS cells; they express a proteoglycan called NG2 and can differentiate into mature oligodendrocyte. They also participate in immune reactions by responding to inflammatory cytokines hence limiting our strategy to promote the differentiation of precursor cells to mature oligodendrocytes1. The genome-wide differences present in DNA methylation dictate the susceptibility of damage to oligodendrocytes5. Neuroinflammatory mediators such as INF gamma, TNFα and ILβ promote synaptopathy, demyelination and axonal loss5. This implies that if the inflammatory milieu is stopped, hence the subsequent progression of the disease.\n\nThere are four types of MS, relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS) and primary relapsing MS (PRMS). Initially, the disease starts as RRMS and then progresses to SPMS. The disease occurs most commonly in those aged 20–50 years. It occurs more commonly in females than in males, as seen in other autoimmune conditions. The prognosis of the disease depends on the age of presentation and number of exacerbations or relapses of the disease since the initial presentation6. Actively demyelinating lesions in the background of inflammation causing blood-brain barrier dysfunction as seen in RRMS7. Biomarkers of the disease include fetuin-A, nitric oxide synthase and osteopontin8. Symptoms of MS include fatigue, visual problems, cognitive problem, dizziness, gait problem, sensory symptoms, sleep and sexual dysfunction9,10.\n\nThe review describes fatigue treatment in MS using pharmacotherapy, exercise therapy and behavioral therapy in the last five years and their efficacy in treatment.\n\n\nMethods\n\nThis review was conducted according to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement, using the methodology described in Cochrane Handbook for Systematic review of interventions.\n\nThe following electronic databases were searched for articles published from the database on September 30, 2018: EBSCO, PubMed, SCIENCE DIRECT and Scopus databases were searched from January 1, 2013, to September 30, 2018. The search strategy included following words “Fatigue and Multiple sclerosis” OR “multiple sclerosis” OR “exercise in MS” OR “pharmacotherapy in MS” OR “Cognitive behavioural therapy and MS”.\n\nAll abstracts identified by this search were independently screened by title and abstract by S.K. and T.S. Duplicates were removed by screening based on title of the article and author name. All relevant full-text articles were evaluated for eligibility against the inclusion criteria. Any dispute which arose was solved by mutual consensus. As the scope of the article was limited to systematic review, additional analysis such as sub-group analysis and meta-regression was not done.\n\nThe data was extracted independently by two authors S.K and T.S. We collected data from the included randomized controlled trials (RCTs) regarding characteristics of patients, baseline data, expanded disability status scale scores, duration of disease and treatment and outcomes in the study. The changes in fatigue according to different scales was noted in outcomes. We also collected data from systematic reviews in the form of the population included, intervention carried out, comparatives and outcomes of the review with their analytical results on a data extraction sheet. The data was compared and reported while scripting of the discussion. The data of systematic reviews has been exposed to quality analysis using AMSTAR grading shown in Table 1.\n\nThe articles included in the study are open access and not restricted by region or language. The selection included Randomized controlled trials, observational studies, and systematic reviews. We also included studies which has patients with clinically diagnosed MS and patients >18 years old with fatigue as their presenting complaint. We included studies which reported on patients with both primary and secondary MS. We excluded articles about neuroplasticity in diseases other than MS22–29. We excluded articles which focussed on non-motor aspects of MS or where experimental studies30–42 opinion article43–45, updates46 Letters47 study protocols48 and extended abstracts49. A list of excluded studies is available as Extended data50.\n\nIncluded studies were independently rated by S.K. and T.S. using the Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. The rating process followed the description in the Cochrane Handbook for Systematic Review of Interventions (part 2:8.5.1) using RevMan version 5.1. Any disagreements during the process was solved by mutual discussions.\n\nThe quality of the identified studies was appraised using AMSTAR guidelines51.\n\n\nResults\n\nWe identified 1343 articles from the database search using Scopus, Science Direct, EBSCO and Pub med with no additional articles from other sources (Figure 2). We found 1203 articles to be remaining after removal of duplicates. We excluded 1131 publications based on title and abstract and date of publication. We had 72 full-text articles assessed for eligibility of which 42 articles were excluded among which we excluded articles which related to cognitive changes52–65. We included 10 RCT, 7 observational reviews and 13 systematic reviews1,11–21,66 for the study. A flow diagram is shown in Figure 2.\n\nThe study characteristics and summary of systematic reviews is elaborated in Table 2. The study characteristics and summary of RCT is presented in Table 3. The study characteristics and summary of observational studies are depicted in Table 4.\n\nEPHPP, Effective Public Health Practice Project; Cochrane RoB tool, Cochrane risk of bias tool.\n\nTAU, treatment as usual.\n\nA Cochrane review showed exercise therapy to have a significant positive effect on fatigue in RRMS patients [standard mean deviation (SMD) -0.53, 95% confidence interval (CI) -0.73 to -0.33; P-value <0.01] but there was significant heterogeneity [I2>58%] among the trails compared. A few studies showed exercise improved walking speed with 10-minute walking test showing mean difference [MD] reduction in walking time of 1.76 s; [95% (CI), 2.47 to 1.06; P<0.001]66. Another study comes in support of the use of exercise which shows that pooled Effect size was 0.57 (95%CI: 0.10–1.04, P = 0.02)19. These findings suggest that exercise can help to reduce fatigue in MS patients. A study by Taylor et al. mentions a study showing exercise worsening fatigue in MS (P<0.05)17.\n\nAmantadine2,11 is anti-parkinsonian medication that gives an inconsistent improvement in 20–40% of patients over the short term. Yang et al. showed that amantadine might be the most effective drug for treating MS fatigue: SMD and CI were −1.09 [−1.30 to −0.87], and the z-score was 9.75 [P < 0.00001]; however, there was a high variation in number size of patients, causing heterogeneity to be 91%11. The two most effective drugs in treatment are natalizumab and alemtuzumab, but they cause progressive multifocal leukoencephalopathy (PML) due to John Cunningham virus and autoimmune diseases of thyroid along with thrombocytopenic purpura with immune glomerulonephritis respectively. A 6-month study in 2016, ECTRIMS showed no increase in mortality. Ocrelizumab, the first drug effective to slow down PPMS and which targets B cells in RRMS and PPMS, is in a phase 3 trial2. A counter drug in SPMS is still to be discovered as IFNβ1b has not shown efficacy in American SPMS trials. Hence trials should be performed with combination therapy including ocrelizumab and IFNβ1b to counter SPMS, which has a poor prognosis2.\n\nCognitive behavioural therapy [CBT] can help reducing fatigue in MS (pooled SMD = −0.71, 95% CI: −1.05 to −0.37, P = 0.77) as compared to active controls1. Supporting studies also show a positive effect [SMD] = −0.47;95% [CI] = −0.88; −0.06; I2 = 73%]. A long-term positive effect of CBT [SMD = −0.30; CI −0.51; −0.08; I2 = 0%] is also shown but had limited number of studies14. Thus, CBT shows a positive effect on fatigue in MS. Practices like yoga show some effect compared to usual care [SMD = 20.52; 95% CI = 21.02 to 20.02; p = 0.04] but fail to prove better than exercise therapy [SMD = 0.03; 95% CI = 20.24 to 0.30; p = 0.83]23.\n\nTrials based on pharmacotherapy have shown that a change to new drugs like oral fingolimod was beneficial to many patients for fatigue in MS as shown by EPOC trial. The TSQM Global satisfaction scores were superior after the switch from intravenous disease-modifying therapy iDMT to oral fingolimod [p<0.001]67. Aerobic training exercises were delivered in ambulatory MS patient which showed improvements. This view was supported by the TREFAMS-AT trial (p<0.014). The non-fatigue related outcomes such anxiety, depression, and cognition showed improvement in the certain trials, which explains the dynamic connections with fatigue as a symptom67,68.\n\nExercise therapy is a potential treatment modality, and when combined with education therapy it can cause behavior modification in many patients. This view was supported by the STEP IT UP and FACETS trial. It was able to prove that mobility was increased in intervention groups through the intervention time was relatively short (10 weeks)68–70.\n\nThe chronicity of symptoms in MS has a tremendous impact on the probability to show improvement to any therapy. It will be difficult to expect a positive change in a patient who has suffered chronic fatigue when compared to fatigue of new onset in MS patients. A study showed that multi-disciplinary rehabilitation on chronic fatigue patients was not effective in bringing the fatigue levels to a significant low that could be appreciated subjectively71,72.\n\nAll criteria were judged as low, high or unclear risk of bias. In summary, most of the studies had a low risk of bias. The risk of bias graph is show in Figure 3 and Figure 4. Calkwood et al.67, had high risk of bias as it lacks random sequence generation and allocation concealment. Calkwood et al.67, Thomas et al.70, failed to fulfil blinding of participants and outcomes in their respective studies, which were thus prone to performance and detection bias. It was unclear in a few studies whether allocation concealment and blinding of participants was carried out in studies like Heine et al.71 and Rietberg et al.72.\n\nAs a result of heterogeneity among studies due to different study designs taken into consideration and a smaller number of participants in various studies owing to loss of follow up and the pathogenicity of the disease, a meta-analysis was not carried out.\n\n\nDiscussion\n\nThe primary outcomes in most of the trials used MSIS, FSS and CIS-20R scales69–71. MSIS is a subjective scale based on a patient experiencing fatigue. CIS-20R subscale measures fatigue severity. FSS measures the impact of fatigue on normal functioning. The changes measured on any scale should be accompanied by a change in FSS scale to make it clinically meaningful to adopt as a standard measure for generalizability. Not every severely fatigued patient (in most cases of advanced MS) will give expected results on standard exercising protocol. It is an arguable viewpoint leading to reverse causality whether exercise therapy is worsening fatigue levels in MS patients as supported by a systematic review by Taylor et al.17.\n\nConsidering the therapeutic interventions for MS-related fatigue, a variety of exercise methods (pilates85, calisthenics80, Tai Chi82 and aerobic) of exercises have gained attention. Numerous studies have shed light on the efficiency of exercise for the PwMS, almost all studies designated the exercise as a remarkable factor in improving the fatigue and its related distress in MS84. Certain observational studies have been conducted to find out if the cause of fatigue in PwMS is the physical activity instead of neural demyelination and lack of neuroplasticity. One cross-sectional study ruled out the possibility of fatigue associated with the physical activity instead they found a strong association between the mental health and fatigue84.\n\nFatigue in MS can be due to depression, which intercedes the association between neuronal issues and physical conditions84. Pharmaceutical interventions like melatonin supplementation have been effective to treat the fatigue related to MS79. Melatonin can act as anti-inflammatory and immunomodulatory drug that does not cross the blood-brain barrier. The anti-oxidative effect can be used to treat MS patients as they have high oxidative stress owing to elevated levels of plasma lipid peroxide and activated nitrosative-oxidative pathways79.\n\nAn observational retrospective study showed that switching from interferon-β to glatiramer improved work productivity, activity impairment and health-related quality of life [HRQoL] and fatigue. Transcranial magnetic stimulation [TMS] is an innovative way to record neurophysiological responses by measuring corticospinal-neuromuscular pathway excitability. The persistent excitation of brain neurons plays an important role in progressive forms of the disease83.\n\nPlasticity is a functional reorganization of neurons carried out through anatomical reorganization and axonal sprouting with synaptogenesis. Physical training is known to induce compensatory plasticity and increases activity-dependent synaptic potentiation. Exercise is known to cause an increase in endocannabinoid signalling86.\n\nThe summary of included observational studies can be found in Table 4.\n\n\nConclusion\n\nThe diversity of pathological phenomena involved in fatigue related to MS is a major concern in understanding and quantifying the role of each causal factor. Our study has found a significant positive effect on fatigue levels of RRMS patients with regular CBT and exercise-based combination therapy. These results were not supported in case of PPMS or SPMS patients due to the aggressive nature of the disease. Emphasizing the clinical significance of combinational therapy which can be prescribed in MS, yet this does not undermine the need for statistical analysis and correlation. Future research should focus on improving the quality of life of progressive forms of MS. Factors responsible for a high drop-out rate should be studied and correlated with morbidity and mortality rates. We believe an amalgamation of sound mental health, physical health, and pharmacological health shall tone down or blunt the effect of fatigue in the daily life of MS patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: DIVERSE MECHANISMS AND TREATMENT STRATEGIES TO CONFRONT FATIGUE IN MULTIPLE SCLEROSIS -A SYSTEMATIC REVIEW. https://doi.org/10.17605/OSF.IO/W5DA450\n\nThis project contains references for all excluded studies.\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nOpen Science Framework: PRISMA checklist for study “Diverse mechanisms and treatment strategies to confront fatigue in multiple sclerosis: A systematic review”. https://doi.org/10.17605/OSF.IO/W5DA4",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPhyo AZZ, Demaneuf T, De Livera AM, et al.: The Efficacy of Psychological Interventions for Managing Fatigue in People With Multiple Sclerosis: A Systematic Review and Meta-Analysis. Front Neurol. 2018; 9: 149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSedal L, Winkel A, Laing J, et al.: Current concepts in multiple sclerosis therapy. Degener Neurol Neuromuscul Dis. 2017; 7: 109–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGBD 2015 Neurological Disorders Collaborator Group, Feigin VL, Abajobir AA, et al.: Global, regional, and national burden of neurological disorders during 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Neurol. 2017; 16(11): 877–897. 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}
|
[
{
"id": "48607",
"date": "20 May 2019",
"name": "Andrew Soundy",
"expertise": [
"Reviewer Expertise Methodology and publications in this area including one cited by the authors."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, I think more time is needed to explain what you did and show the reader that a quality process has been undertaken. Some points below are critical for me.\n\nAbstract As a reader I am not sure how you integrated and weighted findings from different designs e.g., you mention two random trials in the results EPOC and FACETS – yet review evidence would include more accurate results and you have identified 13 reviews?\n\nIntroduction Does the reader need to know about the different drugs for treatments of MS or the main point which is fatigue remains Not sure of the need for Figure – given the focus and aim of this work\n\nThere is no overview of why a review or what type of review is needed within this section?\n\nMethods There is no protocol If you are using Cochrane then you would include systematic reviews in your synthesis – you would ideally aim to get a meta-analysis done of past empirical research. So the design is questionable. As your review is mixed methods review requiring aspects which are needed for mixed methods design including a decision around the point of integration of data. If you used Cochrane as a basis your key words would have likely increased and the number of databases and search methods would be different from what you state Your methods has no detail on synthesis as point in abstract is made\n\nResults Page 5 Think about the how you lay out the information – currently for me there is limited information which is hard to understand how and why you have presented it like this. Think about how you integrate the quality assessment onto findings\n\nDiscussion Without clarity from above – I can't assess the discussion currently\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? No",
"responses": []
},
{
"id": "48171",
"date": "18 Jun 2019",
"name": "Tomas Kalincik",
"expertise": [
"Reviewer Expertise multiple sclerosis",
"neuroimmunology",
"biostatistics",
"clinical outcomes research"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article aims at providing a systematic review of literature on the treatment of fatigue in multiple sclerosis. This is an important topic, as management of MS-related fatigue is a difficult subject and the results achieved in clinical practice are often frustrating to clinicians and patients alike. Unfortunately, the review falls short of its goal, providing only a superficial summary of the identified literature and a discussion based on a handful of selected anecdotes. The review would benefit from a more systematic approach to the topic.\n\nMajor comments: The systematic review only included open access articles. This is potentially a significant limitation. How may otherwise eligible articles were excluded on these grounds? The information summarised in the Introduction is selective and only remotely related to this article. The referencing style in the Introduction is suboptimal, with only limited number of indirect references cited. The summary given in the Introduction, paragraph 5 is unnecessary. Most of this information is not immediately relevant to this manuscript and the section is not appropriately referenced. Similarly, the information shown in paragraph 6 is patchy and poorly referenced. The authors cite the old phenotypic classification of MS. Instead, the 2013 revisions of the classification of MS phenotypes (Lublin et al., Neurology 2014[Ref-1]) should be used.\nDuring screening, the authors have excluded a large number of identified articles. This does not impact negatively on the quality of the systematic review but it highlights that the search terms employed were very broad.\nThe Results are hard to follow. The effect of different interventions on physical performance and fatigue are presented intermittently. In fact, the most of the section on the results of meta-analyses focuses on the effects of treatments on physical performance, with only a very brief mention of the reported effect on fatigue. The references of the individual results are scattered and often given without the appropriate context (for instance “6-month study in 2016, ECTRIMS showed no increase in mortality.” The summary of the safety issues mentioned for some of the presented interventions is not balanced. The authors erroneously state that there is no evidence of therapeutic effect in SPMS, whereas siponimod has in fact shown effect on slowing progression of disability in a phase 3 trial (EXPAND). There is some duplication of the reported results, as some of the clinical trials were also included in the meta-analyses. This is unavoidable, but the amount of overlap between the meta-analyses and the trials reported individually in this review should be reported. An improvement in 20-40% of patients treated with amantadine is referenced – it is unclear which domain and in what test this improvement involves. Similarly, it is unclear what is meant by the statement ‘The two most effective drugs in treatment are natalizumab and alemtuzumab…”. Does this relate to their effect on relapses/disability/MRI or fatigue? The results of the individual randomised controlled trials should be summarised not only with p values but, most importantly, with the measures of effect size (both point and interval estimates). The authors mention an issue of the efficacy of interventions in the context of the duration of the symptoms. This should be expanded upon and provided with more supporting evidence if this theme is to be retained within the article.\nThe manuscript would benefit from a stylistic review (a few examples – Introduction, paragraph 2, lines 6-7; Introduction, paragraph 3, lines 12-16; Introduction, paragraph 5, last line; Introduction, paragraph 5, lines 10-12; Methods, Inclusion/Exclusion criteria, lines 9-11; Figure 2 etc.). The authors should also use terminology more accurately – e.g. ’widespread MS investigation’, ‘disease burden’, ‘experimental studies’. The formulation “MS is affecting 2,000,000 people worldwide and 400,000 people in the United States per year” implies that the cited information represents incidence, whereas the numbers given are keeping with MS prevalence. The Discussion is not systematically structured and would benefit from more thorough, systematic evaluation of the mentioned topics. These include the present anecdotes related to the link between depression and fatigue, use of melatonin, TMS and plasticity. An in-depth discussion of the summarised literature, including critical appraisal of the presented studies and meta-analyses, is needed.\nThe opening statement of the Discussion provides a perspective on relevant measurable outcomes in the tests of MS-related fatigue. This discussion should be linked to the results presented and the magnitude of the impact of the studied interventions on FSS should be evaluated.\nThe authors have phrased the conclusion in the fashion of an original study (“Our study has found a significant positive effect…”). However, it should be remembered that this article is a systematic review of literature, in which no novel results were generated. The Conclusion section should use the language of a review. In this context, references to ‘significant’ findings are not appropriate.\nThe following proposed conclusions are not supported by the results of the reported trials, including the following:\nThe suggestion that CBT and exercise-based therapies are not associated with better control of fatigue due to a more aggressive nature of PPMS and SPMS relative to RRMS The proposed need for combination therapy for MS, which is also being implied as an accessible option (please note that combination immunotherapy is not approved for use in MS)\nThe sentence containing “…should focus on improving the quality of life of progressive forms of MS” objectifies patients and should be rephrased.\n\nMinor comments: Table 1: should provide explanation of the AMSTAR items in a footnote. Table 2: Minor inconsistencies are present (such as n-acetyl carnitine vs. L-carnitine; the format of presenting control groups)\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? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-563
|
https://f1000research.com/articles/8-344/v1
|
27 Mar 19
|
{
"type": "Research Article",
"title": "Burden of drug-resistant pulmonary tuberculosis in Pakistani children: A cross-sectional study",
"authors": [
"Ghulam Shabbir Laghari",
"Zahid Hussain",
"Lavina Khemani",
"Syed Zohaib Maroof Hussain",
"Uzair Yaqoob",
"Ghulam Shabbir Laghari",
"Zahid Hussain",
"Lavina Khemani",
"Syed Zohaib Maroof Hussain"
],
"abstract": "Introduction: The incidence of drug-resistant tuberculosis (TB) is rapidly increasing worldwide. Children in high TB burden countries are rapidly being reported to be affected by multidrug-resistant TB (resistant to isoniazid and rifampicin). The aim of this study is to evaluate the pattern of drug sensitivity among children suffering with TB. Methods: Known cases of pulmonary TB, with sputum smear positive even after two months of compliance to 1st line anti-tuberculous therapy were included after gaining informed consent. Specimens used for drug sensitivity analysis were either sputum or bronchoalveolar lavage. Patient age, gender, history of TB contact, and duration of treatment were also recorded. Data was entered and analyzed using SPSS v.22. Results: There were 32 male (64%) and 18 female (36%) children in the study. Their mean age was 12.84 ± 2.54 years. History of household TB contact was positive in 29 (58%) children. Among 1st line anti-tuberculous therapy, ethambutol and streptomycin were most sensitive (n=44; 88%). Rifampin was least sensitive (n=17; 34%). There were 32 (64%) children with multidrug-resistant tuberculosis (MDR-TB). A positive history of household TB contact (either resistant or non-resistant) was seen to have a statistically significant impact on incidence of MDR-TB (p value=0.03) Conclusion: Pediatric drug-resistant TB is a rising concern. Awareness programs on national and international levels are needed to educate the general population regarding the importance of preventing TB household contact, especially amongst children.",
"keywords": [
"pediatric tuberculosis",
"multi-drug resistant tuberculosis",
"isoniazid",
"rifampicin",
"household T contact",
"Pakistan"
],
"content": "Introduction\n\nGlobally, there are approximately 67 million children suffering from Mycobacterium tuberculosis (MTB) infection. The incidence of isoniazid (INH) mono-resistance has been estimated to be 5 million, and 2 million for combined isoniazid and rifampicin (RIF) resistance. In 2014 alone, an estimated 850,000 children developed pulmonary tuberculosis with 25,000 multidrug-resistant cases1. The statistics surged drastically, and in 2017 1 million new cases of paediatric TB were reported2. Adding to the current poor trajectory there have also been reports of extensive drug resistance in paediatric pulmonary tuberculosis, with almost 100,000 such cases reported1.\n\nIn TB patients, drug resistance results from spontaneous genetic mutations in the MTB genome. The risk of genetic mutation increases with increasing bacterial load, explaining why resistance is more commonly seen in adult cavitary TB, which has large bacilli load. In children the more common reasons of drug resistance are transmission of a resistant bacillus and previous treatment with anti-tuberculous therapy (ATT). Other factors that predispose to drug-resistant TB include inappropriate drug regimens, monotherapy, and drug non-adherence3.\n\nPakistan is among the top 20 TB-endemic countries, which share 84% of global TB burden and 87% of MDR-TB burden, according to the World Health Organization (WHO)2. Though, there have been various studies highlighting the incidence of MDR-TB in Pakistani adults4, and some studies also included children; we couldn’t find any study from Pakistan that discussed the incidence of paediatric MDR-TB in particular. The aim of this study is to assess the pattern of sensitivity to 1st line and 2nd line ATT among Pakistani children (≤18 years).\n\n\nMethods\n\nA prospective, cross-sectional study was conducted from 1st July 2018 to 31st Dec 2018 in the Department of Paediatrics, Civil Hospital, Jamshoro. Known cases of pulmonary tuberculosis being followed up at the outpatient TB clinic were recruited. The inclusion criteria included children <18 years with a working diagnosis of pulmonary TB who had been taking 1st line ATT for two months but still had sputum smears (or sputum culture) positive for MTB. For children less than five years old, informed consent was taken from their parents/guardians. For children of age five years or above, informed consent from the parents/guardians and assent from the children was taken. Children who had become negative for MTB on sputum smear or culture with 1st line ATT, indicating response to these drugs, were not included. Children who were sputum positive but also non-compliant to their medications (compliance checked from their TB dosage card) were also excluded. Follow up patients in the TB clinic whose parents/guardians did not consent or children older than 5 who did not assent to participate were also excluded.\n\nFor culture and sensitivity, either sputum sample was utilized or bronchoalveolar lavage specimen (in cases of no sputum production). The samples were not specifically taken for this research, but were a part of their standard management, hence, no additional burden was placed on the participants of the study. Mycobacterium was isolated from the specimens by using Lowenstein-Jensen medium and Mycobacterium Growth Indicator Tube (MGIT) medium (Becton Dickinson, Franklin Lakes, NJ, USA). BACTEC NAP test (Becton Dickinson) was then performed on the isolated mycobacterium to differentiate MTB from other mycobacteria. Drug susceptibility testing was then done using an agar proportion method on enriched Middle brook 7H10 medium (BBL Microbiology Systems, Cockeysville, MD, USA) following the standard laboratory protocols of the Civil Hospital, Jamshoro. Concentrations used for every drug was: isoniazid (INH) 0.2μg/ml, rifampicin (RIF) 1μg/ml, ethambutol (EMB) 5μg/ml, and streptomycin (SM) 2μg/ml and 10μg/ml. For pyrazinamide (PZA) sensitivity, BACTEC 7H12 medium was used with pH 6.0, at 100μg/ml (BACTEC PZA test medium, Becton Dickinson). Strains which were not susceptible to INH and RIF were termed as MDR strains. MDR-TB strains were then tested for susceptibility to 2nd anti-tuberculosis agents: capreomycin 10μg/ml, ofloxacin 2μg/ml, ethionamide 5μg/ml, and kanamycin 6 μg/ml.\n\nA brief questionnaire (See Extended data5) was generated which included patient demographics such as age, gender, history of TB contact, and duration of treatment. Data was entered and analyzed using SPSS Version 22.0. Armonk, NY: IBM Corp. Mean ± standard deviation (SD) was calculated for continuous variables such as age and duration of treatment. Frequency and percentages were calculated for all other variables including drug sensitivity.\n\n\nResults\n\nThe study was completed by 50 children. There were 32 male (64%) and 18 female (36%) children in the study. Their mean age was 12.84 ± 2.54 years with the youngest child being 7 and the oldest 18. The demographic profile of these patients is shown in Table 1 (data at patient level is available as Underlying data5).\n\nTB – Tuberculosis\n\nThe sensitivity pattern of 1st line ATT is shown in Table 2. Ethambutol and streptomycin were most sensitive (n=44; 88%). RIF was least sensitive (n=17; 34%). There were 32 (64%) children with combined sensitivity to INH and RIF and 18 (36%) children were multidrug-resistant i.e., combined resistance to INH and RIF. Other than MDR cases, and among the first line drugs used alone, RIF showed the highest isolated resistance (n=33; 66%).\n\nMDR- multidrug-resistant\n\nOf the 18 MDR cases, 10 (55.6%) were boys and 8 (44.4%) were girls. Their mean age was 14.01 ± 1.50 years with the youngest of aged 12 and oldest aged 15.\n\nThe sensitivity pattern of second-line line ATT is shown in Table 3. Kanamycin and capreomycin showed 100% sensitivity. Ethionamide was sensitive in 47 (94%) children and ofloxacin was sensitive in 38 (76%) children.\n\nA positive history of household TB contact (either resistant or non-resistant) was seen to have a statistically significant impact on incidence of MDR-TB as seen in Table 4.\n\n\nDiscussion\n\nThe incidence of drug-resistant TB among children is a global health concern. Public health specialists must pay keen attention to this issue in order to prevent unnecessary mortalities. Pakistan is already a high TB burden country. Poor detection, diagnosis, and management of TB, along with unchecked household contact of children with tuberculosis patients, has markedly contributed to the rising incidence of MDR-TB among both adults and children in Pakistan6. This study reported 66% of children to be mono-resistant to RIF, 36% to be MDR, and although no case of extended drug resistance was seen, 24% of children tested positive for fluoroquinolone resistance.\n\nComparatively, in a Pakistani study conducted in 2010–14, of all the MDR-TB cases in the study, only 1.6% were aged 0–144. In another survey from 2013–14, household contacts of 209 diagnosed cases of MDR-TB were screened. It was seen that 378 of 1463 contacts (26%) were children aged 0–15. Of these, 11 children were symptomatic for TB, were tested, and 4 cases of TB were diagnosed from these children, all of which were MDR7. This study highly reinforces the impact of household TB contact on the development of MDR-TB in children, which has also been highlighted in our study. In another study, with 62% individuals resistant to all first-line agents, ofloxacin resistance was among 52.7%; which is relatively low in the current study (24%)8.\n\nThis study highlights the prevailing situation of anti-tuberculosis resistance in Pakistani children and their predisposing factors. It emphasises the need to protect the children from TB infected persons. This study has its limitations too. It was based in one institute only which is in the rural part of Pakistan. The study cannot be generalised to the national status of Pakistan. Multi-center studies all across Pakistan must be conducted to completely understand the current status of anti-tuberculosis drugs resistance in Pakistan among both children as well as adults. Studies should also be conducted to evaluate disease outcome in these patients.\n\n\nConclusion\n\nDrug-resistant TB, especially in the pediatric population, is a public health concern. Awareness programs on national and international levels are needed to educate the masses regarding importance of preventing TB household contact especially among children. Long term studies should be conducted to study the prognosis of children with MDR-TB and deduce strategies to prevent drug resistance.\n\n\nEthical approval and consent to participate\n\nThe study was assessed and approved by the Institutional Review Board of Civil Hospital, Jamshoro (IERB: 18-679) with informed consent taken from all participants.\n\n\nData availability\n\nFigshare: Burden of drug-resistant pulmonary tuberculosis in Pakistani children. https://doi.org/10.6084/m9.figshare.7823741.v45\n\nThis project contains the following underlying data:\n\nPTB.sav (Antibiotic sensitivity analysis data)\n\nData Dictionary.spv (Data dictionary for underlying data)\n\nFigshare: Burden of drug-resistant pulmonary tuberculosis in Pakistani children. https://doi.org/10.6084/m9.figshare.7823741.v45\n\nThis project contains the following extended data:\n\nQuestionnaire.docx (Study questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nDodd PJ, Sismanidis C, Seddon JA: Global burden of drug-resistant tuberculosis in children: a mathematical modelling study. Lancet Infect Dis. 2016; 16(10): 1193–201. PubMed Abstract | Publisher Full Text\n\nGlobal Tuberculosis Control. [Internet]. World Health Organization; 2018 [cited 2 March 2019]. Reference Source\n\nShah I: Multidrug-resistant tuberculosis in children. Pediatr Infect Dis J. 2012; 31(9): 970–2. PubMed Abstract | Publisher Full Text\n\nAkhtar AM, Arif MA, Kanwal S, et al.: Prevalence and drug resistance pattern of MDR TB in retreatment cases of Punjab, Pakistan. J Pak Med Assoc. 2016; 66(8): 989–3. PubMed Abstract\n\nYaqoob U: Burden of drug-resistant pulmonary tuberculosis in Pakistani children. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7823741.v4\n\nAhmad AM, Akhtar S, Hasan R, et al.: Risk factors for multidrug-resistant tuberculosis in urban Pakistan: a multicenter case-control study. Int J Mycobacteriol. 2012; 1(3): 137–42. PubMed Abstract | Publisher Full Text\n\nQadeer E, Fatima R, Haq MU, et al.: Yield of facility-based verbal screening amongst household contacts of patients with multi-drug resistant tuberculosis in Pakistan. J Clin Tuberculosis Mycobacterial Dis. 2017; 7: 22–7. Publisher Full Text\n\nAhmad N, Javaid A, Sulaiman SA, et al.: Resistance patterns, prevalence, and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients. Braz J Infect Dis. 2016; 20(1): 41–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "46354",
"date": "11 Apr 2019",
"name": "H Simon Schaaf",
"expertise": [
"Reviewer Expertise Childhood tuberculosis with specific interest in drug-resistant 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\nData on the burden or incidence of drug-resistant tuberculosis (DR-TB) in children is sparse, as bacteriological confirmation of tuberculosis in children is challenging. This study did not set out to determine the incidence of DR-TB in Pakistani children in their setting, but rather to evaluate DR-TB in children (0-<18 years) who still had a positive smear microscopy for acid-fast bacilli or cultures for Mycobacterium tuberculosis positive after 2 months of adherent first-line antituberculosis treatment. The authors found a very high rate of drug resistance in this highly selected patient group (previously treated, mainly adolescents), it is difficult to extrapolate this data to other settings even in the same country. The reviewer has the following comments: Major comments:\nAbstract: The way in which the drug susceptibility test (DST) results are presented is confusing and the abstract data regarding MDR-TB is different from the manuscript (32 cases in abstract vs 18 in manuscript text/table). I suggest change results to: rifampicin resistance was highest at 33/50 (66%), and resistance to streptomycin and ethambutol were the lowest (6/50; 12%). Please provide the correct data for MDR-TB. Abstract conclusion: These conclusions cannot be made from this study. This is a biased study of highly selected, mainly older children for DST. What could be said is that with the selected method used to identify mainly older children with drug resistance, the yield for drug-resistant TB was high. Introduction 1st paragraph: There is a clear difference between TB infection and TB disease. In this paragraph, these two entities are confused. It should clearly state that the numbers presented refer to infection and not disease, e.g. it is estimated that 5 million children are infected with INH mono-resistant M. tuberculosis strains, 2 million with MDR strains and (in the last line) 100,000 are infected with XDR strains. These are not TB cases! Also, there was not 1 million child TB cases “reported” – this was only estimated – only about a third of this number were reported cases. Introduction 2nd paragraph: “Children” in this study covers a wide age range from 0-<18 years. Especially in young children <5-10 years of age (before they develop cavitary adult type disease) transmission of drug-resistant strains is the main reason for DR-TB. Once they become adolescents, with adult-type disease, incorrect prescribing and/or poor adherence to medication become much more common reasons for developing DR-TB. Previous treatment in children returning with DR-TB is often not because of developing resistance, but because DR-TB was not diagnosed at initial presentation. In young children, because of the low bacillary load, a two-month negative culture does not always mean a good response – they may fail later during treatment or relapse after completion of first-line treatment. This is likely one of the reasons why so few young children were identified in this study. Methods: In resource limited settings, plans sometimes need to be made to identify the highest risk group(s) for DR-TB, which the authors likely did in this study. Unfortunately the highly selected group of children studied means that nothing can be said about the incidence of DR-TB in this community – other than maybe that DR-TB is unlikely to be higher than this study’s findings in this setting. It is unfortunate that children with poor compliance to treatment who were still positive (AFB smear or culture) after two months were excluded, as this group has a high risk of developing resistance in the first two months of treatment. The authors should define “non-compliant” to medication. It would also be helpful for the readers to place the studied cases and DR-TB cases identified during this study into context if they knew how many children in total were on TB treatment at the time of the study in this setting, how many parents/children did not consent/assent to the study and how many were excluded due to non-compliance. Are these numbers available? Results: Presenting the results in the text as “most/least sensitive” and “combined sensitivity” is confusing – it even confused the authors themselves (see abstract vs text). It is actually not necessary to repeat results that are presented in the table in the text again. An interesting observation to the reviewer is the very high rate of rifampicin mono/poly resistance – if the MDR-TB numbers are correct, 15 (30%) of these children had rifampicin mono/poly resistance. Do the authors have an explanation for this? This also has implications both for treatment and preventive therapy in contacts, as INH should still be effective. It would also be interesting to know how many of the MDR-TB cases had additional resistance to ofloxacin (PreXDR-TB) Discussion: As mentioned above, this study cannot be used to determine DR-TB in the children of Pakistan or even in this setting, as the study method of highly selected children (or rather, adolescents) does not at all represent the TB population. This data cannot be compared to data of more general drug resistance surveys. This should be mentioned as study limitations. What it does show is that in a carefully selected high risk group of adolescents not responding well to TB treatment after two months, the rate of DR-TB is very high. However, it would be far more appropriate to do DST on all children with bacteriologically confirmed TB before starting any TB treatment.\nMinor comments:\nAbstract: - Line 2: suggest changing “rapidly” to “increasingly”. - Line 3: regarding definition - add: (Mycobacterium tuberculosis resistant to…). - Line 6: suggest: “…smear positive for acid-fast bacilli after…”. - Line 8: suggest: “drug susceptibility testing”. - Line 12 (Results): Start with total number of children in study: “Fifty children, 32 male (64%) … were included. Methods: - Page 3, 2nd column, line 16: suggest change “not susceptible” to “resistant”. - Further, the authors use both “sensitive/sensitivity” and “susceptible/susceptibility” in the manuscript – for consistency suggest change all to “susceptible/susceptibility”. Results: - Table 1: Do the authors know the concordance of child and adult source cases’ M. tuberculosis strains DST results in the source case/child contact pairs? Discussion: - Page 4, 1st column, line 5: suggest: “…, along with child household contacts of MDR-TB cases not being screened and managed appropriately,…” - Page 4, 1st column, line 9: not RIF mono-resistant but resistant to RIF. - Page 4, 1st column, line 10: extensive drug resistance.\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? No",
"responses": [
{
"c_id": "4565",
"date": "11 Apr 2019",
"name": "Uzair Yaqoob",
"role": "Author Response",
"response": "Thank you so much for this great and comprehensive review, we will surely consider all comments and upload the updated versions with editing done as much as we can."
},
{
"c_id": "4580",
"date": "23 Apr 2019",
"name": "Uzair Yaqoob",
"role": "Author Response",
"response": "Response to comments is following, a newer version has been uploaded. the reviewer will soon see the newer version.Major comments Done Done Done Rightly said, non-compliant has been defined We were only allowed to access patients and data at the time of the study and currently we cannot access hospital data. Done Done Done Minor comments: Done Done, sensitive/sensitivity looked more appropriate esp in tables Authors are not aware of this Done "
}
]
}
] | 1
|
https://f1000research.com/articles/8-344
|
https://f1000research.com/articles/8-539/v1
|
24 Apr 19
|
{
"type": "Method Article",
"title": "Two novel strategies to assess in vivo meiotic protein expression in Arabidopsis thaliana",
"authors": [
"Niels van Tol",
"Martijn Rolloos",
"Paul J.J. Hooykaas",
"Bert J. van der Zaal",
"Niels van Tol",
"Martijn Rolloos",
"Paul J.J. Hooykaas"
],
"abstract": "For studies on key meiotic processes such as crossover formation and genome haploidization, the availability of portable promoter sequences for effector protein expression in meiocytes is of great importance. In this study, we present two novel strategies to facilitate screening for such promoter elements. The first strategy was based on expression of a previously constructed GFP-tagged zinc finger protein for visualization of the pericentromeric regions of chromosomes in meiocytes. Here, we show that expression of this reporter protein under control of different promoters allowed for the visualization of fluorescence foci in meiocytes, demonstrating that this is a useful tool for such purposes. The second reporter system was based on the visualization of cytotoxicity triggered by expression of the Agrobacterium tumefaciens virulence protein VirD5. We show that constitutive expression of VirD5 is lethal, but when driven by meiotic promoters led to reduced fertility with normal vegetative growth. We show that both strategies offer useful tools for the assessment of meiotic effector protein expression, especially when combined with available gene expression data sets.",
"keywords": [
"Arabidopsis",
"meiosis",
"gene expression",
"promoter",
"meiocytes",
"zinc finger",
"fluorescence reporter",
"negative selection marker"
],
"content": "Introduction\n\nThe cell division process of meiosis generates haploid gametes for fertilization. Cells that are primed for meiosis (meiocytes) undergo two consecutive rounds of chromosome segregation. The first division (meiosis I) results in separation of the homologous chromosomes1, which is facilitated by the formation of physical connections (chiasmata) at double stranded break sites generated by transesterases, such as SPO11-12,3 and SPO11-24 in the model plant species Arabidopsis thaliana. Resolution of these junctions can lead to the reciprocal exchange of parts of chromosome arms, a process known as meiotic crossing over5. The second round of division (meiosis II), results in separation of the sister chromatids1, finally yielding four haploid daughter cells from a diploid meiocyte. In Arabidopsis, male and female meiosis are separated within the flower and take place in the anthers and carpels, respectively. Male meiocytes are organized as column-like structures (referred to in this study as male meiocyte columns)6,7, and upon completion of meiosis give rise to four haploid pollen cells that are physically attached to each other as structures known as tetrads. Maturation of tetrads eventually leads to the release of individual pollen cells.\n\nThe process of meiosis is of great interest to plant breeders for different reasons. Most importantly, crossover events are the driving force behind genetic variation in plants5,8,9. Research on crossover frequency has thus received a considerable amount of attention in literature (e.g. 10–12). To facilitate the engineering of crossover-related events in meiocytes without affecting non-meiotic tissue, there is interest in identifying gene promoters that allow for effector activity during meiosis. Although there is a vast amount of literature available on the genetics and cytogenetics of plant meiosis1, it has been challenging to identify DNA sequences that can be used to express proteins of interest preferably in meiotic cells.\n\nRelatively recent studies on the male meiocyte transcriptome6,13 have provided cues for the identification of genes that are more or less specifically expressed during male meiosis. The upstream regions of these genes (generally representing the gene control region14) are mostly referred to as ‘promoters’. Such promoters can be regarded as portable elements for the assembly of gene expression cassettes that can mediate meiotic effector protein expression. Accurately quantifying meiotic transgene expression, however, is subject to a number of practical limitations. Firstly, relative expression levels do not necessarily correlate with promoter activity, which is determined by endogenous RNA metabolism as well (e.g. 15). Secondly, the isolation of meiocytes for quantitative gene expression analysis is technically very challenging and laborious, and could easily result in contamination of RT-qPCR or transcriptome data with non-meiocyte transcripts. Qualitative assessment of promoter activity using fluorescence microscopy is an attractive possibility. However, the dynamic cytoplasmic and nuclear states of meiocytes make it difficult to determine the exact localization of fluorescence, and to distinguish it from autofluorescence.\n\nIn this study, we have investigated two novel strategies to screen for portable promoter elements to express effector proteins specifically or at high levels in meiocytes. The first strategy was based on expressing a previously constructed GFP-tagged zinc finger protein (1803F-GFP) to visualize pericentromeric fluorescence foci in meiocytes. Here, we show that 1803F-GFP expression driven by the promoter of the Arabidopsis RPS5A gene (pRPS5A) which is active in vegetative16–18 and meristematic tissue18, resulted in clear GFP foci in male meiocytes. We also selected and cloned 14 promoter sequences that could be of interest for expression of transgenes in meiocytes based on published transcriptome data, and assessed their activities in meiocytes using the 1803F-GFP reporter system. As a second strategy, we assembled constructs encoding the highly cytotoxic Agrobacterium tumefaciens virulence protein VirD5, which was recently shown to disturb chromosome segregation in plant cells by interacting with the kinetochores of chromosomes19. We reasoned that, due its mode of cytotoxicity, VirD5 could be used as a novel negative selection marker to assess effector protein expression in meiocytes. Here, we show that the VirD5 reporter system can be used to obtain in planta cues about promoter strength and specificity. Altogether, our data show that both strategies can provide valuable in vivo information regarding promoter activity in meiocytes.\n\n\nResults\n\nTo investigate the activity and specificity of promoters of interest to drive effector protein expression in meiocytes, we first cloned the promoters of a selection of genes which are involved in meiosis or highly expressed in meiocytes. In total, we cloned the promoter sequences of 14 genes based on the two available studies on the male meiocyte transcriptome6,13. An overview of these 14 genes is provided in Table 1. In brief, we firstly selected five genes with important roles in meiosis (ZYP1A, SPO11-2, NBS1, SPO11-1, MUS81) that are expressed in male meiocytes6,13, and rather specifically so based on meiocyte/anther and meiocyte/seedling expression ratios6 (Table 1). In addition, we selected four genes (ASK1, DMC1, SMC1 and MS5) which are involved in meiosis and are expressed in male meiocytes6,13, but not specifically so (Table 1). Secondly, we selected four genes (At1g27710, At5g25980, At5g26622, At5g42530) which are the most highly expressed genes in male meiocytes (13 and personal communications with the authors). Finally, we selected the promoter of the gene At4g40020, which has been described to be expressed specifically during meiosis I7.\n\nThe genes are listed in locus ID order.\n\nPreviously, we have described a GFP-based reporter to monitor chromosomes in vivo in vegetative tissue of Arabidopsis plants20. This construct encoded a fusion between the fluorescent protein GFP and an array of three zinc fingers (3F) targeting a cognate 9 bp DNA sequence14 in the highly conserved pericentromeric 178 bp repeats which make up a large portion of the pericentromeric DNA in Arabidopsis21. Expression of this fusion protein, which is referred to here as ‘1803F-GFP’, can result in distinctive GFP signals that visualize the pericentromeric regions of chromosomes or the cytological chromocenters20,22. These signals are particularly easy to observe in non-green tissues, such as roots22. An important benefit of the formation of foci is that the GFP signal is strongly concentrated, making discrimination from autofluorescence much easier compared to the detection of diffuse GFP signal. In addition, as the 178 bp repeat regions are strongly conserved and present in the pericentromeric regions of all five Arabidopsis chromosomes, the 1803F-GFP reporter system also allows for ploidy determination. For these reasons, we considered the 1803F-GFP system a suitable read-out for meiotic protein expression. An overview of the 1803F-GFP strategy is provided in Figure 1.\n\nA promoter of interest (promoter of interest) is cloned into the binary vector pRF 1803F:(GGGGS)3:GFP Kana (e.g. as a PCR-amplified SalI-NotI fragment). The promoter drives expression of a fusion protein consisting of three zinc fingers (ZF1, ZF2 and ZF3) together recognizing 9 bp of DNA which are unique to a major fraction of the 178 bp pericentromeric repeats, fused to the open reading frame of GFP (GFP), spaced by a flexible linker peptide (Linker), and with an N-terminal nuclear localization signal (NLS) and FLAG tag (FLAG). Transcription termination is under control of the NOS terminator sequence (tNOS). The construct is introduced into Arabidopsis plants as a T-DNA with left and right border sequences (LB and RB, respectively), through Agrobacterium tumefaciens-mediated transformation. Primary transformants are selected for by kanamycin resistance due to NPTII expression. Expression of 1803F-GFP under control of the promoter of interest can subsequently be visualized as nuclear GFP foci by microscopy.\n\nPreviously, we have used the 1803F-GFP reporter system20,22 under control of the promoter of the Arabidopsis RPS5A gene (pRPS5A) in the binary vector pRF 1803F:GFP Kana. For this study, we slightly modified the backbone of pRF 1803F:GFP Kana to accommodate the introduction of other promoter fragments. In addition, we introduced a new, flexible hydrophilic (GGGGS)3 linker23,24 to possibly enhance the read-out of the 1803F-GFP system. This newly generated binary vector was named pRF 1803F:(GGGGS)3:GFP Kana. More details about the construction of pRF 1803F:(GGGGS)3:GFP Kana are provided in the Methods section. An overview of the DNA sequence and the key components of pRF 1803F:(GGGS)3:GFP Kana is provided in Figure 2, and the amino acid sequence encoded by the 1803F:(GGGGS)3:GFP open reading frame is provided in Figure 3.\n\nThe fusion construct consists of the 1803F encoding sequence (bold blue font) flanked by SfiI sites, fused to the eGFP encoding sequence (bold green font) through a flexible linker (bold orange font), tagged with a FLAG tag and a nuclear localization signal (NLS) at the 5´ end. Other relevant unique restriction sites are also indicated. Expression of the fusion construct is under control of a promoter of interest, which can be inserted at the indicated position, and of the NOS terminator sequence (bold grey font).\n\nThe fusion protein consists of 1803F (consisting of three ZFs; ZF1 in blue, ZF2 in red and ZF3 in purple font, respectively) fused to GFP (bold green font) through a flexible linker peptide consisting of a trimer of the aa sequence GGGGS, and tagged with a FLAG tag and a nuclear localization signal (NLS) at the N-terminus (398 amino acids in total).\n\nPromoters of interest can easily be introduced into the pRF 1803F:(GGGGS)3:GFP Kana backbone, for instance by ligating SalI-NotI fragments of PCR-amplified promoter sequences into SalI-NotI digested pRF 1803F-GFP Kana, thereby directly placing the 1803F-GFP open reading frame under their control. The binary vector is also compatible with other cloning strategies, e.g. as described in this study for pSPO11 (further details described in the Methods section).\n\nTo investigate the 1803F-GFP system as a novel tool to visualize protein expression in vivo in meiocytes, we firstly verified whether the nuclear GFP foci distinct for 1803F-GFP binding to pericentromeric regions (Figure 1) could be observed in root tip cells (diploid; 2n=10) of transgenic plants harboring the pRPS5A::1803F:(GGGGS)3:GFP construct. Nuclei of root tip cells could easily be visualized as expected, and in most cases contained ten distinct GFP foci in a full Z-stack of images, corresponding to the diploid (2n=10) number of chromosomes (Figure 4A). The foci could be examined directly by zooming in on confocal microscopy images and did not require further image analysis.\n\n(A) Confocal fluorescence microscopy images of a root tip (T2 generation) expressing 1803F-GFP under control of pRPS5A. Highlighted is a nucleus in which 10 distinct GFP foci corresponding to the diploid number of chromosomes (2n=10) are visible. Scale bar represents 10 μM. A contrasted black and white (‘Binary’) version of the GFP image is included to clearly mark GFP-fluorescent chromocenters with red arrows. (B) Confocal fluorescence microscopy images of male meiocytes of wild-type Col-0 plants, primary transformants (T1 generation) expressing 1803F-GFP under control of the RPS5A promoter (pRPS5A::1803F-GFP), and primary transformants expressing 1803F-GFP under control of the GAL4-based two-step expression system (p2S-RPS5A::1803F-GFP). Images taken with the RFP channel are presented as negative controls for autofluorescence. Scale bars represent 20 μM. (C) False color images of nuclei of meiocytes harboring p2S-RPS5A::1803F-GFP (n=8 presented). Below every nucleus the number of counted GFP foci is presented. A diploid somatic cell (2n=10) is provided as a control. (D) DAPI-stained spread of wild-type Col-0 flower bud tissue. Highlighted are a diploid cell (2n=10) and a haploid cell (n=5). Scale bar represents 10 μM.\n\nTo assess the 1803F-GFP system in meiocytes, we subsequently isolated male meiocyte columns from wild-type plants and from transgenic plants harboring pRPS5A::1803F:(GGGGS)3:GFP, and examined them by confocal fluorescence microscopy. Male meiocytes from transgenic plants harboring pRPS5A::1803F:(GGGGS)3:GFP clearly displayed GFP foci corresponding to 1803F-GFP fusions binding to pericentromeres (Figure 4B), while meiocytes isolated from wild-type Col-0 plants did not (Figure 4B). These observations demonstrated that the 1803F-GFP reporter system provides for a clear read-out for meiotic effector protein expression, and that pRPS5A is active during meiosis, which is a novel finding. To corroborate these observations, we constructed a GAL4-based two-step (2S) variant of the pRPS5A expression cassette, in which pRPS5A drives the expression of GAL4-VP16, which in turn can transactivate a flanking 4xUAS-minimal35S::1803F:(GGGGS)3:GFP reporter gene (p2S-RPS5A::1803F:(GGGGS)3:GFP in short). This 2S construction, which was based on an earlier published enhancer trap approach25,26, was expected to provide for an essentially similar expression pattern as in pRPS5A:: 1803F:(GGGGS)3:GFP reporter lines, but, due to the fact that GAL4-VP16 is a potent activator of multiple UAS-driven gene expression, would have higher overall strength. We subsequently isolated male meiocyte columns from transgenic plants harboring the two-step p2S-RPS5A:: 1803F:(GGGGS)3:GFP variant, and examined them by confocal microscopy. Nuclei of meiocytes harboring p2S-RPS5A::1803F:(GGGGS)3:GFP indeed displayed clear GFP foci which now had a slightly higher signal intensity (Figure 4B), and displayed less background GFP fluorescence than meiocytes from transgenic plants harboring pRPS5A:: 1803F:(GGGGS)3:GFP. These data showed that the 2S expression cassette is also a suitable reporter for meiotic activity of promoters of interest.\n\nWe further investigated the resolution of the 1803F-GFP system by assessing the number of visible chromosomes in the nuclei of meiocytes in male meiocyte columns (Figure 4C). When examining 1803F-GFP foci in the surrounding somatic tissue, the number of chromosomes per cell was always ten (2n=10). This dropped to approximately five GFP foci in meiocytes of transgenic plants harboring p2S-RPS5A::1803F:(GGGGS)3:GFP (Figure 4C), confirming that these cells were either haploid, or that chromocenters were in close proximity. In this case, it also has to be noted that the raw images did not require any further processing besides zooming in on nuclei. Hence, the 1803F-GFP reporter system combined with confocal microscopy had sufficient resolution to visualize chromosomes in vivo in meiocytes using the 2S expression cassette. To further corroborate this, we compared images taken from meiocytes expressing 1803F-GFP to confocal images of chromosome spreads which were prepared from flower bud tissue (Figure 4D). We found that images of DAPI-stained spreads of flower bud cells (Figure 4D) had a resolution which in terms of visibility of chromocenters was very similar to images obtained through squashing of 1803F-GFP expressing flower buds (Figure 4C), although the latter were captured in vivo without requiring any tissue fixation and staining. These observations indicated that the 1803F-GFP system had sufficient resolution to match the standard of the best available cytological images.\n\nTo further investigate the 1803F-GFP system as a read-out for meiotic effector protein expression, the cloned promoter sequences of meiotic genes (Table 2) were each ligated into binary vector pRF 1803F-GFP Kana, thereby replacing pRPS5A. Arabidopsis Col-0 plants were transformed with these fusion constructs using the floral dip method. Male meiocyte columns were then isolated from young flower buds of kanamycin-resistant primary transformants and examined by confocal fluorescence microscopy, as was done for pRPS5A. As controls, we also examined tetrads and pollen, and other tissues of flower buds which were released during the isolation of meiocytes. An overview of the results of the confocal fluorescence microscopy is provided in Table 2. Confocal microscopy images of tissues in which fluorescence foci were observed, are provided in Figure 5.\n\nFlower buds from on average five independent primary transformants (n=5) were examined. The promoters are listed in locus ID order. Positive fluorescence signals are indicated in grey.\n\nConfocal images of male and female meiocytes (male meiocyte columns and ovule primordia, respectively) are presented, along with tetrads and pollen. For the sake of clarity of the figure only the cases where GFP signals could be observed are presented. Images of all four tissue types in Col-0 are presented as a negative control, with white arrows indicating the location in case of low background fluorescence. Scale bars represent 20 μM.\n\nUsing the 1803F-GFP system, we could show that the activity of the promoters of SPO11-1, SMC1, MS5 and p5g26622 indeed resulted in GFP foci in male meiocytes (Table 2 and Figure 5). For pSPO11-1 and p5g26622 we did not observe foci in any other tissues of the flower bud (Table 2), indicating that these promoters are indeed specifically active during meiosis. In the cases of seven other promoters (pZYP1A, pSPO11-2, pNBS1, pDMC1, pMUS81, p4g40020, and p5g42530) we could not detect GFP foci in any tissue type (Table 2), while transcriptome data for five of these genes (ZYP1A, SPO11-2, NBS1, DMC1, and MUS81) are indicative of rather specific expression during meiosis (Table 2)6. These observations suggested that the expression levels of these genes in meiocytes are too low to be detected using the 1803F-GFP system as a reporter, which is in accordance with their low expression values in meiocytes (Table 2)6,13. For three promoters (p1g27710, pTGG2, and p5g42530) we did not observe expression in meiocytes, tetrads, or pollen, but noted GFP signals in other tissues in the flower bud, such as the tapetum, epithelium, and vascular cells (Table 2; Figure 5), suggesting that these promoters have very low activities in meiocytes and are not at all meiosis specific.\n\nAn overview of promoter activities described in this study which either confirm findings described in literature or are completely novel is presented in Figure 6.\n\nPresented are the most clear examples of 1803F-GFP expression in meiocytes and pollen under control pRPS5A, the GAL4-based two-step expression system (p2S-RPS5A::1803F-GFP) and of the meiotically active promoters verified in this study. Fluorescence intensity and size of the confocal images was optimized to qualitatively show the nuclear foci representing 1803F-GFP fusions binding to pericentromeric regions. Therefore, no scale bars are shown.\n\nAs a second strategy to assess the strength and specificity of meiotic promoter activity, we assembled reporter constructs with the Agrobacterium tumefaciens virulence gene virD5. Recently, the virulence protein VirD5 was described as being cytotoxic to plant cells by binding to kinetochores through an interaction with the protein Spt4, thereby disturbing mitotic cell division and leading to cell death and aneuploidy19. Transgenic plants expressing VirD5 under control of an inducible promoter could be obtained, but these died upon induction of expression19. In the present study, we hypothesized that VirD5 could also be cytotoxic to meiotic cells, and that plants expressing VirD5 under control of a meiotic promoter would grow normally, but would show partial or full sterility due to abortion of meiosis. In that manner, VirD5 expression might act as a dominant negative indicator of meiotic protein expression. We chose for VirD5 over other dominant negative selection marker systems because it specifically interferes with chromosome segregation and is thus likely to be particularly toxic to actively dividing cells, such as meiocytes. A schematic overview of this approach is presented in Figure 7. To establish a VirD5 based reporter system we cloned the virD5 open reading frame (ORF) from Agrobacterium tumefaciens strain LBA110027 and generated fusion constructs for a selection of nine different cloned meiotic promoters of genes for which both expression data and specificity data were available6,13 (Table 1).\n\nA promoter of interest (Promoter of interest) is introduced into the binary vector pVirD5 Kana (e.g. as a PCR-amplified SalI-NotI fragment). The promoter then drives expression of the virD5 open reading frame with an N-terminal FLAG-tag (FLAG) and nuclear localization signal (NLS), under control of the NOS terminator sequence (tNOS). The construct is introduced into Arabidopsis plants as a T-DNA with left and right border sequences (LB and RB, respectively), through Agrobacterium tumefaciens-mediated floral dip transformation into wild-type Col-0 plants. Primary transformants (T1 generation) are selected as kanamycin-resistant individuals due to expression of the NPTII gene. Depending on the strength and tissue specificity of the activity of the promoter of interest, the viability, growth and fertility of the primary transformants can be affected. Possible examples of these effects are illustrated. In case of meiosis-specific activity, vegetative and floral development are unaffected, but the plants are sterile.\n\nWild-type Col-0 plants were transformed with VirD5 expression constructs under control of the nine cloned promoters using the floral dip method. Viable primary transformants could be obtained at normal transformation frequencies with all of the promoters except for pASK1 and pSMC1 (Table 3), which did not yield any viable primary transformants (Table 3), indicating that these promoters are not at all meiosis-specific and already active in embryos and seedlings. In case of pASK1 this is in accordance with already observed vegetative ASK1 expression patterns (Table 1), but was unexpected for pSMC1, which is, more or less, a meiosis-specific promoter based on available gene expression data (Table 1). Viable, fully developed primary transformants could be obtained at high frequencies for the other seven promoters, indicating that none of them were very active in vegetative tissue.\n\nThe promoters are listed in locus ID order.\n\n* Primary transformants could be obtained at low frequencies, but died when transferred to soil\n\n** Based on Yang et al. (2011)\n\nWe further noted that VirD5 expression under control of pZYP1A, pSPO11-1 and pSPO11-2 did not impact vegetative development and flowering, but did lead to strongly reduced fertility (Table 3). Phenotyping VirD5 expressing primary transformants thus provided experimental evidence that these promoters are largely inactive during vegetative development and therefore might indeed be meiosis specific. In the case of pZYP1A this would be in accordance with a meiocyte/seedling expression ratio of 17.0 (Table 1 and Table 3), indicating meiosis-specific expression. Furthermore, these data indicated that both pSPO11-1 and pSPO11-2 could be more meiosis-specific than might be expected based on the meiocyte/seedling expression ratios (Table 1). We decided to further analyze the effects of pZYP1A and pSPO11-1 in more detail, because these primary transformants were the most viable and at the same time displayed the most reduced fertility, indicating meiosis specificity.\n\nTo further assess meiosis specificity of pZYP1A and pSPO11-1 we stained siliques of representative primary transformants with cottonblue-lactophenol to examine the quality of seed set and the viability of the embryos. Whereas siliques of control plants expressing 1803F-GFP under control of pRPS5A exhibited normal silique development with all positions in the siliques producing viable seeds (Figure 8A), siliques of transformants expressing VirD5 under control of pZYP1A and pSPO11 were mostly much shorter and also contained many fewer viable seeds (Figure 8A). As the flowers did not show any obvious defects, these observations indicated that VirD5 is indeed cytotoxic to meiocytes or to their precursor cells, leading to substantially reduced fertility. In the case of pSPO11-1, loss of fertility was accompanied by substantial heterogeneity in seed size among the T2 seed pool (Figure 8B). Remarkably, most small seeds still germinated and finally produced normal plants. It has to be noted, however, that even though expression of VirD5 under control of pSPO11-1 severely affected fertility, it never completely abolished seed set, indicating that pSPO11-1, even though meiosis-specific, is not a very strong promoter. As an alternative possibility, it might be that interference with meiotic chromosome segregation by VirD5 could still allow for the generation of a small fraction of balanced gametes with 5 chromosomes, just as observed for spo11-1 mutants28. To further address this, we constructed a 2S expression construct (placing expression of GAL4-VP16 under control of pSPO11-1, and transactivating a flanking 4xUAS-minimal35S::virD5 reporter construct) to artificially enhance the activity of pSPO11-1, as we observed for the p2S-RPS5A construct (Figure 8B). Primary transformants expressing VirD5 under control of this 2S construct were only slightly hampered in vegetative rosette development but were fully sterile compared to control plants (Figure 8C). This again corroborated that pSPO11-1 is likely to be a very meiosis-specific promoter with at most low activity in other tissues. To further investigate the cause of the reduced fertility observed in plants harboring pSPO11-1 and pZYP1A, we analyzed pollen production and pollen viability in five different plant lines for each construct. Staining of stage 12 flower buds29,30 showed that pollen production as well as viability were indistinguishable from wild-type plants. Hence, using the promoters mentioned, VirD5 expression did not distort the development of cells prior to the formation of pollen, neither of pollen development itself. It might have been that female gametogenesis was partially distorted, but further experimental exploration of this possibility has not yet been investigated.\n\n(A) Cotton blue staining of siliques of primary transformants expressing VirD5 under control of pZYP1A and pSPO11. Two stained and unstained siliques, respectively, are presented for three individual primary transformants. Siliques of primary transformants harboring the construct pRPS5A::1803F-GFP are presented as a negative control. Scale bars represent 2 mm. (B) Photos of seeds of three independent primary transformants expressing VirD5 under control of pSPO11. Seeds of a representative primary transformant harboring pRPS5A::1803F-GFP are presented as a negative control. The seeds were spread on grid paper, with each square representing 1 mm2. (C) Photos of primary transformants expressing VirD5 under control of the GAL4-based two-step expression system (p2S-RPS5A::1803F-GFP). Primary transformants harboring the construct pRPS5A::1803F-GFP are presented as a control. Scale bar represents 10 mm.\n\nComplete raw imaging data are available on Figshare31.\n\n\nDiscussion\n\nHere, we have described two novel strategies to assess the suitability of portable promoter elements to drive effector protein expression in meiocytes. The first strategy, based on expression of 1803F-GFP fusions under control of promoters of interest, has proven to be a suitable method to investigate such activity in meiocytes in vivo without the requirement of tissue lysis, fixation or staining. The second strategy, based on expression of the cytotoxic Agrobacterium tumefaciens virulence protein VirD5, proved to be an additional and partly complementary tool to gather in planta cues regarding promoter strength and specificity, especially when combined with available gene expression data sets.\n\nThe 1803F-GFP strategy allows for the relatively simple introduction of PCR-amplified promoter fragments into the binary vector pRF 1803F-GFP Kana. Visualization of GFP signal in meiocytes can then be performed directly in the T1 generation after floral dip transformation. In addition, an important advantage of this approach over other available approaches is that promoter activity can be studied in vivo without the requirement of disruptive procedures such as lysis, fixation or staining. Finally, the formation of GFP foci localized to the pericentromeric regions of all five chromosomes allows for the discrimination of true GFP signals from autofluorescence as well as for ploidy determination. The 1803F-GFP system could also be combined with other techniques (e.g. chromosome spreading) to draw further conclusions with respect to expression levels or cytology. Of course, just as for any other reporter of gene expression, claims regarding promoter specificity should be interpreted with care. GFP signals in meiocytes could in theory also be due to inheritance of 1803F-GFP transcripts or proteins from progenitor cells.\n\nIn the present study we could not detect GFP foci when 1803F-GFP expression was driven by the promoters of ZYP1A, SPO11-2, NBS1, DMC1 and MUS81 (Table 2). These genes are by their function known to be expressed during early prophase I of meiosis I1, but their mRNA levels are relatively low in meiocytes (Table 1). It could therefore be that low and subphase-specific promoter activity is difficult to detect with the 1803F-GFP system. In addition, it is known that meiocytes progress through prophase I of meiosis I relatively quickly32, making the isolation of meiocytes columns which are in a specific subphase of prophase I a very challenging task. The usefulness of the 1803F-GFP reporter system is therefore to some extent limited by the ability to isolate meiocytes in the correct meiotic subphase of interest.\n\nThe VirD5 reporter system in principle allows for the screening of promoter elements with tissue-type specific activities without the requirement of any a priori knowledge. Valuable biological cues regarding promoter strength and specificity can already be gathered by phenotyping primary transformants expressing VirD5 under control of a promoter element. In addition, when gene expression data sets are available, the VirD5 reporter system can be used to further verify promoter activity in a tissue type of interest, or to confirm that expression patterns are indeed limited to a specific tissue type. It must be realized that expression of VirD5 in cells that support reproductive tissues could also lead to sterility. Further investigation of promoter activity might thus be required when VirD5 expression is observed to trigger sterility.\n\nThe strategy of using VirD5 expression as a dominant negative indicator for cell ablation offers an interesting alternative to the use of other negative selection markers, because it specifically interferes with chromosome segregation19. Its expression is therefore only detrimental to dividing cells, and less likely to be toxic to other types of cells when expressed at low levels. By comparison, the protein synthesis inhibiting diphtheria toxin from the pathogenic bacterium Corynebacterium diphteriae, which is widely used as a potent negative selection marker (e.g. 33–35), is highly toxic to all cells undergoing mRNA translation and therefore lethal to every cell type, also when expressed at low levels. The VirD5 strategy might therefore offer an interesting alternative by excluding lethality due to biologically insignificant levels of transgene expression.\n\n\nConclusions\n\nAltogether the data presented here demonstrated that effector protein expression in meiocytes can be visualized in vivo using the 1803F-GFP reporter system, which facilitates the assessment of meiotic promoter activity. The promoters investigated in this study were selected primarily based on published studies on male meiosis6,13, but for future studies the activity and strength of any promoter could in principle be assessed in meiocytes (or other tissue types of interest) using the 1803F-GFP reporter system. In addition, we showed that expression of the Agrobacterium tumefaciens virulence protein VirD5 can be used as a novel negative selection marker and can also provide for experimental cues on tissue specificity and strength of cloned promoter fragments of interest. Combining the 1803F-GFP and VirD5 reporter systems with available gene expression data sets could greatly facilitate the identification of portable promoter elements with desired tissue specificity and/or strength.\n\n\nMethods\n\nAll plants in this study were grown on soil in a climate-controlled growth chamber at a constant temperature of 20°C, 70% relative humidity, a light intensity of approximately 150 μmol m-2 s-1 of photosynthetically active radiation from SPAR tubes, and at a 16 h photoperiod. All transgenic plants were generated in the Col-0 background.\n\nThe selected promoter sequences (Table 1) were PCR-amplified from genomic DNA of Col-0 plants isolated with the CTAB protocol36, using nested PCR with a gene-specific forward primer (Table 4) and four overlapping reverse primers (three gene-specific and one universal reverse primer; Table 4). The PCR conditions are listed in Table 5. Where possible, PCR primers were designed to amplify the full genomic region between the ATG start codon of the gene of interest and the first or last exon of the adjacent gene (depending on the ORF of that gene lying on the positive or negative strand). Due to a lack of suitable primer target sites, the cloned intergenic regions were 300 bp shorter for At1g75950 (ASK1) and 170 bp shorter for At3g54670 (SMC1). For At1g22260 (ZYP1A), the cloned 2102 bp fragment reached up to At1g22275 (ZYP1B) and included the complete At1g22270 (SMO2) sequence. The DMC1 gene (At3g22880) is separated by 7280 bp from the neighboring At3g22886 (MIR167A) sequence. In this case, we amplified a 3123 bp upstream sequence which includes an 1881 bp LIMPET1 transposable element. The amplified sequence is 177 bp longer than the reportedly active Col-0 sequence used in a previous study7 but might be effectively shorter than the ~3200 bp upstream sequence from the Landsberg erecta ecotype which does not contain the LIMPET1 element37. The forward primer and the universal reverse primer were added to the PCR mix at the final concentration advised by the manufacturer of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Reverse primers 3, 2, and 1 were added to 1/5, 1/10, and 1/10 of the final concentration advised by the manufacturer, respectively. In this manner, relatively large PCR products of the promoter sequences fused to a FLAG tag and a nuclear localization signal (NLS) at the 3´ end, flanked by SalI and NotI restriction sites (5´ and 3´ ends, respectively, except for the promoter sequence of SPO11-1, which was flanked by XhoI and NotI sites) could be generated in a single PCR reaction. Although the FLAG tag did not serve a direct purpose in the present study, it can be useful for immunological purposes such as pulldown of meiocyte proteins interacting with FLAG-tagged proteins of interest. The PCR products were ligated into the pJET Blunt cloning vector using the CloneJet PCR Cloning Kit (Thermo Fisher Scientific) and their identity was confirmed by Sanger sequencing (Macrogen Europe).\n\nThe genes are listed in locus ID order.\n\nFor the construction of pRF 1803F:(GGGGS):GFP Kana, the previously published binary vector pRF EAR Kana16 was taken as a cloning scaffold. The oligo DNA fragments ‘Kpn EAR Sac FW’ and ‘Kpn EAR Sac RV’ (Table 6) were annealed and ligated into KpnI and SacI digested pRF EAR Kana, thereby removing the KpnI site and introducing (among others) XhoI and SpeI sites into the backbone. Subsequently, oligo DNA sequences encoding the (GGGGS)3 flexible linker (GGGGS FW and RV; Table 6) were annealed and ligated into the backbone between the XhoI and SpeI sites, yielding the plasmid pRF (GGGGS)3 Kana. The sequence encoding eGFP was PCR amplified from the plasmid pART738, and subsequently ligated between the former KpnI site and the SacI site, yielding the plasmid pRF (GGGGS)3:GFP Kana. The 1803F encoding sequence20,22 was ligated as a SfiI fragment into SfiI digested pRF (GGGGS)3:GFP Kana, finally yielding the binary vector pRF 1803F:(GGGGS)3:GFP Kana.\n\nThe cloned promoter sequences were digested from pJET with SalI and NotI (or XhoI and NotI in the case of pSPO11-1), and were ligated into SalI and NotI digested pRF 1803F:GFP Kana (containing pRPS5A)20. For the construction of a GAL4-based two-step (“2S”) expression system (pRPS5A or any other promoter of choice driving the expression of GAL4-VP16, which transactivates a flanking 4xUAS-minimal35S::1803F:GFP reporter construct), a 1471 bp DNA sequence with key elements essentially as described previously25,26, but directly compatible with our vector systems was synthesized (BaseClear, Leiden, The Netherlands; Figure 9), and subsequently ligated as an EagI fragment into pRF 1803F:GFP Kana predigested with NotI.\n\nFor the amplification of the virD5 ORF, genomic DNA of Agrobacterium tumefaciens strain LBA1100 harboring the disarmed octopine-type Ti plasmid pTiB627 was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions, except that lysis of the cells was performed in TES (10 mM Tris HCl pH 8.0, 50 mM EDTA, 50 mM NaCl) containing 400 μg/ml lysozyme. The virD5 ORF followed by its TGA stop codon and 54 bp of its downstream non-coding sequence was PCR amplified from the genomic DNA of LBA1100 using the forward and reverse primer combination VirD5 NotI FW (5´- ATTAGCGGCCGCTGACAGGAAAGTCGAAAGTTCAC-3´; NotI site underlined) and VirD5 SpeI RV (5´- TAATACTAGTTATCAACCAGCGATCGATGC-3´; SpeI site underlined). The resulting PCR product was ligated into the pJET Blunt cloning vector using the CloneJet PCR Cloning Kit (Thermo Fisher Scientific) and sequenced by Sanger sequencing (Macrogen Europe). Subsequently, the virD5 ORF was ligated as a NotI-SpeI fragment into pRF 1803F:GFP Kana derivatives described above harboring the cloned promoter sequences of MUS81, NBS1, SMC1, SPO11-1, SPO11-2 and ZYP1A, respectively. The relevant restriction sites are depicted in Figure 2. This procedure deleted the 1803F coding domain as well as the adjacent sequence encoding the flexible linker, replacing it by the VirD5 coding region with its own TGA stop codon. Due to the presence of a SpeI site in the cloned promoter sequences of ASK1 and MS5, a similar cloning strategy was followed, except that the virD5 ORF was PCR amplified using VirD5 NotI FW combined with the reverse primer VirD5 SalI RV (5´-TAATGTCGACTATCAACCAGCGATCGATGC-3´; SalI site underlined), and was ligated as a NotI-SalI fragment into NotI-XhoI digested binary vectors harboring the cloned promoter sequences of ASK1 and MS5, respectively. Additionally, in these cases, the virD5 ORF was followed by its TGA stop codon and 54 bp of its downstream non-coding sequence. As a small but functionally irrelevant difference with the other constructs, the short sequence encoding the flexible linker now remained downstream of the virD5 ORF (see position of restriction sites depicted in Figure 2).\n\nBinary vectors were mobilized to the Agrobacterium tumefaciens strain AGL1 through triparental mating39. Col-0 plants were transformed with the constructs using the floral dip method40. The seed pools resulting from floral dip transformation were sterilized and stratified at 4°C for 3-4 days, after which they were plated on MA medium containing 35 μg/ml kanamycin. After one to two weeks of growth on selection medium, kanamycin-resistant seedlings were transferred to soil.\n\nYoung, unopened flower buds6 were harvested from the primary transformants harboring promoter::1803F-GFP constructs. For the isolation of male meiocyte columns, anthers were sectioned from the flower buds and placed in a drop of physiological buffer solution (0.9% w/v NaCl, Tris buffered to pH 7.0) on microscope slides. Male meiocyte columns were then released by gently applying slight pressure on the cover slide, thereby also releasing tetrads and pollen into the buffer solution. Chromosome spreads of Col-0 flower tissue were performed as described previously41,42, and subsequently stained with 4’,6-Diamidino-2-phenylindole (DAPI). Confocal fluorescence microscopy was performed using a Zeiss LSM5 exciter (Zeiss, München, Germany) with Illuminator HXP 120V and a standard photomultiplier tube (PMT) detector (maximal gain of 1250). Excitation of the tissue was performed at a wavelength of 488 nm. Red fluorescence was collected as a negative control with a 560 nm long-pass filter; GFP fluorescence was collected with a 505-530 nm band-pass filter, with a master gain of 678 ± 66 (mean ± standard deviation). Images were collected as Z-stacks with a pixel dwell time of 1.7 ± 1.4 s (average ± standard deviation). All images were collected with the same laser power.\n\nThe Z-stack confocal images (.lsm files) were transformed into 3D projections using the ‘3D project’ function of the program Fiji (Image J version 1.48f). Representative projections were compressed to 2D images, and brightness of the images was slightly enhanced using Adobe Photoshop CC 2018 (19.1.5 release) for the sake of clarity of the figures. This was done in the same way for all images. For the false color images presented in Figure 6 an overlay of the GFP channel and bright field channel was created in Adobe Photoshop, and opacity was adjusted to visualize the GFP signal inside of the tissue. The images were then merged, inverted and the intensity of black pixels was enhanced to qualitatively visualize foci of fluorescence.\n\nSiliques of primary transformants harboring pRPS5A::1803F-GFP, pZYP1A::virD5 and pSPO11::virD5 constructs were harvested in Carnoy’s fixative (60% ethanol, 30% chloroform and 10% glacial acetic acid, all v/v) and stored at 4 °C until further use. Siliques were then stained with cottonblue-lactophenol/lactophenol as described previously43. The primary transformants were subsequently allowed to further set seeds (T2), which were collected. Seed quality and homogeneity were assessed by collecting images of T2 seeds on grid paper using a LeicaMZ16FA stereomicroscope (Leica, Wetzlar, Germany).\n\n\nData availability\n\nFigshare: Two novel strategies to assess in vivo meiotic protein expression in Arabidopsis thaliana. https://doi.org/10.6084/m9.figshare.780837831.\n\nThis project contains raw confocal microscopy images of meiocytes (and other flower bud tissues) isolated from transgenic plants expressing 1803F-GFP under control of the different promoter elements described in this study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nPlant materials and bacterial strains are available from the corresponding author upon reasonable request.",
"appendix": "Grant information\n\nThis research was supported by a partnership between Rijk Zwaan and the Dutch Technology Foundation STW (partnership project number 12427), which is the applied science division of NWO, and the Technology Programme of the Ministry of Economic Affairs, Agriculture and Innovation.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Yinxiang Wang and Prof. Dr. Hong Ma (State Key Laboratory of Genetic Engineering, Institute of Plant Biology, Fudan University, Shanghai, China) for personal communications about promoters of genes described in Yang et al., 201113. We would like to thank Prof. Dr. Hans de Jong (Wageningen UR) for helpful discussions. We would like to thank Shuai Shao for extracting genomic DNA from Agrobacterium strain LBA1100, and Dr. Reza Roushan for help with pollen analysis.\n\n\nReferences\n\nMercier R, Mézard C, Jenczewski E, et al.: The molecular biology of meiosis in plants. Annu Rev Plant Biol. 2015; 66: 297–327. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nLindhout BI, Meckel T, van der Zaal BJ: Zinc finger-mediated live cell imaging in Arabidopsis roots. Methods Mol Biol. 2010; 649: 383–98. PubMed Abstract | Publisher Full Text\n\nSimon L, Voisin M, Tatout C, et al.: Structure and Function of Centromeric and Pericentromeric Heterochromatin in Arabidopsis thaliana. Front Plant Sci. 2015; 6: 1049. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLindhout BI, Fransz P, Tessadori F, et al.: Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins. Nucleic Acids Res. 2007; 35(16): e107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuston JS, Levinson D, Mudgett-Hunter M, et al.: Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988; 85(16): 5879–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCook JM, Charlesworth A: Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins. Protein Eng Des Sel. 2017; 30(4): 313–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngineer CB, Fitzsimmons KC, Schmuke JJ, et al.: Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis. Bmc Plant Biol. 2005; 5: 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWaki T, Miyashima S, Nakanishi M, et al.: A GAL4-based targeted activation tagging system in Arabidopsis thaliana. Plant J. 2013; 73(3): 357–67. PubMed Abstract | Publisher Full Text\n\nBeijersbergen A, Dulk-Ras AD, Schilperoort RA, et al.: Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens. Science. 1992; 256(5061): 1324–7. PubMed Abstract | Publisher Full Text\n\nGrelon M, Vezon D, Gendrot G, et al.: AtSPO11-1 is necessary for efficient meiotic recombination in plants. Embo J. 2001; 20(3): 589–600. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss, Slovin JP, Chen C: A simplified method for differential staining of aborted and non-aborted pollen grains. Int J Plant Biol. 2010; 1(2): 66–9. Publisher Full Text\n\nSmyth DR, Bowman JL, Meyerowitz EM: Early flower development in Arabidopsis. Plant Cell. 1990; 2(8): 755–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Tol N, Rolloos M, Hooykaas PJJ, et al.: Two novel strategies to assess in vivo meiotic protein expression in Arabidopsis thaliana. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7808378\n\nStronghill PE, Azimi W, Hasenkampf CA: A novel method to follow meiotic progression in Arabidopsis using confocal microscopy and 5-ethynyl-2'-deoxyuridine labeling. Plant Methods. 2014; 10(1): 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollier RJ: Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century. Toxicon. 2001; 39(11): 1793–803. PubMed Abstract | Publisher Full Text\n\nWeijers D, Van Hamburg JP, Van Rijn E, et al.: Diphtheria toxin-mediated cell ablation reveals interregional communication during Arabidopsis seed development. Plant Physiol. 2003; 133(4): 1882–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuerineau F, Sorensen AM, Fenby N, et al.: Temperature sensitive diphtheria toxin confers conditional male-sterility in Arabidopsis thaliana. Plant Biotechnol J. 2003; 1(1): 33–42. PubMed Abstract | Publisher Full Text\n\nRichards E, Reichardt M, Rogers S: Preparation of genomic DNA from plant tissue. Curr Protoc Mol Biol. 2001; Chapter 2:Unit2.3. PubMed Abstract | Publisher Full Text\n\nKlimyuk VI, Jones JD: AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression. Plant J. 1997; 11(1): 1–14. PubMed Abstract | Publisher Full Text\n\nPeiter E, Maathuis FJ, Mills LN, et al.: The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement. Nature. 2005; 434(7031): 404–8. PubMed Abstract | Publisher Full Text\n\nLindhout BI, Pinas JE, Hooykaas PJJ, et al.: Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis. Plant J. 2006; 48(3): 475–83. PubMed Abstract | Publisher Full Text\n\nClough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16(6): 735–43. PubMed Abstract | Publisher Full Text\n\nRoss KJ, Fransz P, Jones GH: A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Res. 1996; 4(7): 507–16. PubMed Abstract | Publisher Full Text\n\nZhong XB, Hans de Jong J, Zabel P: Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH). Chromosome Res. 1996; 4(1): 24–8. PubMed Abstract | Publisher Full Text\n\nHowden R, Park SK, Moore JM, et al.: Selection of T-DNA-tagged male and female gametophytic mutants by segregation distortion in Arabidopsis. Genetics. 1998; 149(2): 621–31. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "48670",
"date": "20 May 2019",
"name": "Christophe Lambing",
"expertise": [
"Reviewer Expertise meiosis",
"plant reproduction",
"cytogenetics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 from Niels van Tol et al. presents two strategies to assess promoter activity in Arabidopsis meiosis. The identification of promoters with expression specific to meiosis is of interest to the field. However, some limitations in the techniques used and in the experimental designs impaired the conclusions drawn by the authors.\n\nSpecific comments:\n\nPage 7, Figure 4B: What is the rational to use RFP signal as control of autofluorescence for the GFP signal? These two signals have different excitation and emission wavelengths and the RFP signal will not provide any information on how much autofluorescence passing through the GFP filter is detected. A wild type plant without GFP protein is the best control for GFP autofluorescence.\n\nPage 7, Figure 4A and 4B: It is unclear to me whether the cells labelled with GFP are meiocytes or not. It is also unclear at what stage of meiosis the cells are. It is important to use a chromatin dye here, e.g. DAPI. – see below for more comments on this\n\nPage 7, Figure 4C: It is not apparent that we have cells with 10 and 5 chromocenters. It seems to me that there is a range of chromocenters from 3 to 10 – this would support the idea that the number of chromocenters doesn’t necessary reflect the ploidy content but rather reflects specific stages in the cell cycle.\n\nPage 7, Figure 4D: On the 2 zoom-in images (2n=10 and n=5), it seems to me that none of the two cells presented are meiocytes.\n\nPage 8 - “this dropped to approximately five GFP foci in meiocytes” - I don’t understand the idea that cells with less chromocenters are meiocytes. The interest of using this technique is to test for promoter activity in meiocytes. However, the authors have not fully demonstrated that they can identify meiocytes in their assay.\n\nThe authors seem to suggest that the number of chromocenters could help differentiate between meiocytes and somatic cells but the authors are not referring to any paper to support their claim. The authors then suggest that a cell with fewer than 10 chromocenters is either “haploid or that chromocenters were in close proximity”. The authors didn’t comment on the latter possibility and its implication with meiosis, which makes their explanation unclear. The former possibility is that cells are haploid. On the images in figure 4B, the cells seem widely spaced out, ruling out that cells are at dyad-tetrad stage and so if the cells are haploids, there are post-meiosis and the technique is no longer suitable to assess for promoter activity in meiocytes.\n\nMore worrying, the authors show two cells on the the 2 zoom-in images (2n=10 and n=5) in figure 4D. In figure 4D, both cells are somatic cells – the change in chromocenter number likely reflects a change in the somatic cell cycles but there is no meiocyte and no post-meiotic cell on the 2 zoom-in images provided here. There is also no evidence that one of the two cell has reduced ploidy content.\n\nSince the technique described by the authors is intended to test promoter activity in meiocytes, it is thus essential to accurately differentiate meiocytes from somatic cells. Unfortunately, Figure 4 shows no clear evidence for presence of meiocytes. I suggest the authors to include a chromatin dye - meiotic chromatin is very distinctive and more structured (with formation of the meiotic chromosome axis) that somatic chromatin.\n\nPage 10 and Table 2: It is surprising that most promoters from known meiotic genes give no signal. What meiotic stages were used for the analysis? How are the data comparable and how can we accurately assess the sensitivity of the technique if we cannot clearly state the stage of meiosis? This can easily be resolved by adding a chromatin dye to the tissue preparation. If some of the preparation had immature germ cells then the data presented in Table 2 would be inaccurate and not showing promoter activity in meiosis. It is important that the authors address this major point of concern and demonstrate that the analysis was done on meiotic cells at the same stage (ideally prophase I since the authors used promoters from protein-coding genes important in prophase I of meiosis). Chromatin-stained prophase I of meiosis are very distinctive from later stages of meiosis or from somatic cells and the analysis will be more straightforward.\n\nFigure 6: I don’t see the rational for not adding a scale bar for each image. The entire figure is unclear and there is no explanation in the text to help me understand the figure. The data presented here seems to have limited interest and doesn’t add more support to the conclusions drawn by the authors and therefore the figure could be removed.\n\nPage 11. The VirD5 expression system is supposed to test promoter activity in meiosis – as suggested throughout the text and in the title. However, in my opinion, this system tests promoter activity during plant life cycle (reproductive) but it will not give indication on cell specificity. The authors acknowledged this limitation in the discussion (p14). The system is based on the reduction of plant fertility, but many reasons could cause this phenotype other than defects in meiosis: defect in the tapetum, post meiosis defects, pollen or ovule viability defects…and therefore, I would describe the system as a method to test promoter activity in floral tissues, irrespective of the cell type.\n\nPage 11 “unexpected for pSMC1, which is more or less a meiosis specific promoter based on available gene expression data”. This is not entirely accurate. SMC1 is a well characterised core component of the cohesin ring complex and is expressed in every cells. There are substantial published data studying smc1 mutant phenotype showing pleiotropic effects. This raises some concerns on the validity to use the expression data for the work presented here.\n\nTable 3: Comparing ratio of expression is not informative – the authors should report values of expression for meiocytes and somatic cells separately. What is the definition of reduced fertility? The authors need more clarity here. Is it a quantitative or a qualitative assessment of plant fertility?\n\nPage 12 “ quality of seeds” - what does this mean? The authors should use more technical words.\n\nPage 12 “ all positions in the silique … seeds”. The authors should use a more quantitative methodology. The observations should be better described for the all section on “VirD5 expression under control of meiosis specific promoters pZYP1A and pSPO11-1” and the claims made by the authors lack scientific support.\n\nI suggest the authors to count the number of seed-set per silique\n\n“As the flowers did not show any obvious…” The authors cannot exclude an effect downstream meiosis. A lot of meiotic promoters are active in pollen and because the analyses of pZYP1 and pSPO11-1 specificity in Figure 5 and Table 2 are inconclusive, we cannot rule out this possibility “pSPO11-1 is likely to be a very meiosis-specific promoter with at most …” This is a vague statement with no scientific support. What about the implication of other cell types in the reproductive system?\n\n“Pollen production as well as viability were indistinguishable from wild type plants…”. This data suggests that male meiosis is normal, meaning that this technique is not suited to specifically assess gene expression in meiocytes. “It might have been that female gametogenesis was partially distorted but further experimental …” This is quite elusive. The authors are claiming to develop a technique to test for promoter activity in meiosis specifically. Unfortunately, it seems that the VirD5 system isn’t specific to male meiosis. The authors should investigate female meiosis if they want to demonstrate that their technique can actually be used to test for meiotic promoter activity.\n\nFigure 8 is vague. The technique used for Figure 8A is arbitrary. The authors could analyse the data using a quantitative methodology, e.g. seed set per silique, silique length, variation between samples and plants, statistical test... Figure 8B: I don’t understand what the interest is to present this figure. If the authors want to show that the seeds have different sizes, it is not apparent on the photos. Figure 8C: It seems that the plants on the left panel with virD5 have slightly retarded growth compared to the plants on the right with the GFP system. The authors should comment on this observation, which may suggest that pSPO11-1 isn’t meiotic specific.\n\nPage 13 “ We have described two novel strategies to … expression in meiocytes” The authors need to provide more scientific evidence to support this claim for the reasons mentioned above, or re-phrase their claim - e.g. virD5 system could be used to assess floral expression specific while the GFP-based method could be used in complement to assess for cell type specificity\n\nPage 14 “ GFP foci localized to the pericentromeric … ploidy determination” – This statement is not entirely accurate. Centromeric clustering can reduce GFP centromeric foci without changing ploidy level.\n\nPage 14”GFP signals in meiocytes could … progenitor cells” – This sentence is not clear and need some clarifications.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No",
"responses": []
},
{
"id": "48966",
"date": "11 Jun 2019",
"name": "Christine Mézard",
"expertise": [
"Reviewer Expertise Meiosis",
"Recombination",
"molecular genetics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, van Tol and co-authors present a method to test the specificity of promoters to be expressed during meiosis with two different methods. Both are based on published transcriptomic data. Promoters supposed to be expressed in meiosis are used in two different experimental systems. One aims to address GFP at centromeres of meiotic cells/. The other is based on the expression of an Agrobacterium toxic protein VirD5 that should kill plant cells if expressed during plant development and renders plant sterile if it is only expressed during gametogenesis. In principle, both strategies are very interesting. However, experiments need more controls to obtain any conclusions.\n\nIs the rationale for developing the new method (or application) clearly explained? The authors could also mention that the need of meiotic specific promoters is a prerequisite to (i) study the function of genes that are essential to cell cycle genes (ii) to inactivate some genes by RNAi or other methods that are ubiquitously expressed but essential to meiosis (iii) to overexpress genes selectively during meiosis….\n\nIs the description of the method technically sound? There is just one detail. The cloning is supposed to remove the FLAG based on Figure 2 because the Sal1 and Not 1 sites are on each side of the FLAG. Hovewer, on Figure 1 the FLAG is kept after the cloning of the promoters\n\nAre sufficient details provided to allow replication of the method development and its use by others? If any results are presented, are all the source data underlying the results available to ensure full reproducibility? In general, cytological experiments suffer of a lack of appropriate controls. As mentioned by the other reviewer, RFP cannot be used as a control in Figure 4B. Where are the scale bars in all the figures ? It is known that GFP loses its fluorescence in meiotic columns after a few minutes. Details are needed to be sure that control cells and tested cells are visualized in the same conditions. The absence of fluorescence does not mean that the construct is not expressed. It could be expressed for a short period of time. It is very important to visualized meiotic stages by adding either DAPI or propidium iodide. Another method would be to fix the cells and make spreads to be sure of the meiotic stage. In Figure 4C, the absence of DNA staining makes impossible to determine the stage of meiosis. And the assumption of having 5 dots in a meiocytes is wrong. There are stages in which meiocytes have 10 dots, others where thy have 5 dots and they are not haploid (Armstrong et al, JCS, 20011). In Figure 4D, it is difficult to know which cells are shown. They are no meiotic spreads for sure. Moreover, with the pictures provided it is almost impossible to count the dots. Again in Figure 5, it is always very difficult to determine the stages of meiotic cells. For the VIr5 experiments, we fully agree with the first reviewer, the sterility should be quantified by counting seed sets. For the conclusions, when expression is not detected in any cell types, the absence of expression cannot be relied to the weakness of the promoters. The first control would be to be sure that the fragment containing the promoter is functional. For that, complementation would be the best way to be sure of the functionality of the promoter. One of the best example is DMC1 promoter which has been used several times to express different constructs (example in Yang et al, Plant Physiol, 20082). The lack of expression could mean that the authors have missed the stage of expression and the same explanation could be given for other promoters. As the meiotic stages are not mentioned in this study, it is hard to know if meiosis has been fully explored. Finally, as mentioned by the other reviewer, SMC1 is known to be expressed outside meiosis (Schubert et al., 20093). This ways that transcriptomic data on meiocytes should be used with precautions.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No",
"responses": []
},
{
"id": "51289",
"date": "25 Jul 2019",
"name": "Monica Pradillo",
"expertise": [
"Reviewer Expertise Meiosis",
"Arabidopsis",
"cytogenetics",
"Homologous Recombination"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 method article, van Tol et al. explain two techniques to determine the activity of potential meiosis-specific promoters. They applied both to test 14 promoter sequences that were selected because they correspond to genes with a meiotic function or with high expression levels in meiocytes. The first technique is based on the use of a GFP reporter system to visually detect the chromocenters and the second involves the negative selection marker VirD5, which interacts with kinetochores and disturbs cell division. Both strategies might provide important tools for meiosis studies.\nHowever, I agree with the other reviewers and consider some of the results presented are not completely clear.\nIn the first paragraph of the introduction there are some statements that need to be revised. The authors state “female meiosis takes place in carpels”: it is more accurate to say that it occurs in the ovary. Later in the text “…upon completion of meiosis (male meiocytes) give rise to four haploid pollen cells”: please consider that after telophase II the PMC gives rise to a tetrad of four microspores. After that, each microspore undergoes two subsequent mitotic divisions to produce one vegetative cell and two sperm cells in the mature pollen grain. At the end of the paragraph, “four haploid pollen cells that are physically attached to each other”: please consider that in dicotyledonous male meiocytes cytokinesis occurs when both meiotic divisions are finished. Do the authors refer to the fact that cytokinesis has not yet taken place at the end of meiosis, and hence the physical connection?\nThe interpretation of the “meiocytes” shown in Figure 4 based on a smaller number of chromocentres is not correct, I agree with the other reviewers on this. The authors should consider that the nuclei of most cells exhibit a chromocenter pattern ranging from 4 to 10 (mean ∼8) (Fransz et al., 20021; Del Prete et al., 20142). The comment in the text about Figure 4C: “confirming that these cells were either haploid, or that chromocenters were in close proximity” should be more specific, as the authors should know whether the images correspond to meiocytes in the first or second meiotic division.\nIn general, there is no appropriate quantification for the results about the GFP patterns displayed by the different promoters. Five plants (according to the caption in Table 2) have been analyzed. Did they present the same phenotype? Some variability would be expected because it is possible that more than one transgene is introduced during the transformation with Agrobacterium, plants are analyzed in heterozygous condition (T1), and transgenes have been introduced in different regions (with a different epigenetic regulation) of the genome. The way of presenting the images in Figure 5 is quite confusing. In addition to taking into account the cell type, images should be grouped considering the genetic backgrounds to which they belong.\nRegarding the strategy based on VirD5 expression, the “unexpected” result for pSMC1 is actually logical, since SMC1 is indispensable for sister chromatid cohesion, chromosome condensation and DNA repair, not only in meiotic cells (Lam et al., 20053).\nSeed quantification in siliques of transformants expressing VirD5 under control of pZYP1A and pSPO11 is necessary. In addition, a cytological analysis would be useful to determine the effect of the presence of VirD5 on meiotic chromosome segregation. As the authors state “interference with meiotic chromosome segregation by VirD5 could still allow for the generation of a small fraction of balanced gametes with 5 chromosomes”. This could easily be confirmed with a simple cytological analysis. Aneuploidies (which are tolerated in Arabidopsis) could be the cause of the different size of the seeds in transformants expressing VirD5 under control of pSPO11. Was this difference in seed size observed in the case of pZYP1A?\nIn conclusion, the results of the two strategies are only coincident for only one of the 14 promoters analyzed (pSPO11-1). Using the 1803F-GFP system, the activity of pSPO11-1 resulted in GFP foci in male meiocytes and VirD5 expression under control of this promoter did not affect vegetative development, but produced a reduction in the seed set. In summary, the strategies presented in this paper could be interesting in determining the meiotic activity of the promoters. However, it would be necessary to improve the experimentation associated with more detailed cytological observations, proper analyses and quantitative analyses of the different observed parameters (GFP foci, seed set content) in several plants. Nevertheless, these methodologies should be combined with information about tissue-specific expression levels and functional analyses of the genes tested.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-539
|
https://f1000research.com/articles/8-537/v1
|
24 Apr 19
|
{
"type": "Systematic Review",
"title": "Outcome and safety of upper pole versus non-upper pole single puncture PCNL for staghorn stones: a systematic review and meta-analysis",
"authors": [
"Steven Gunawan",
"Ponco Birowo",
"Nur Rasyid",
"Widi Atmoko",
"Steven Gunawan",
"Ponco Birowo",
"Widi Atmoko"
],
"abstract": "Background: Staghorn stones are mostly treated by percutaneous nephrolithotomy (PCNL), either with an upper-pole (UP) or non-upper (lower- or middle-) pole (NP) approach. NP access has a lower risk of bleeding and thoracic complications but may not be sufficient for complete stone clearance. UP access is advocated as the preferred approach, because of direct access to the collecting system. However, it is associated with a higher complications rate, including pneumothorax and hydrothorax, and a higher risk of bleeding. This meta-analysis aimed to describe the outcomes and safety of PCNL for staghorn stones using UP and NP approaches. Methods: A systematic literature review was conducted using several databases such as: PubMed; EBSCO; Science Direct; Cochrane and Google Scholar. Data from all selected articles were extracted by two independent reviewers. Relevant parameters explored using Review Manager V5.3. Results: Five comparative studies of staghorn stones involving 384 renal units were analyzed; 176 cases used the UP approach and 208 the NP approach. There was no significant difference in stone-free rate between these approaches, with 74.4% undergoing the UP approach and 71.1% the NP approach considered stone-free (OR: 1.55; 95% CI: 0.92-2.63; P=0.10). The rate of thoracic complications (hydrothorax and pneumothorax) did not differ significantly (OR: 3.14; 95% CI: 0.63-15.62; P=0.16). However, we noted that 5 of 176 patients that underwent the UP approach experienced thoracic complications. The incidence of post-procedural fever and sepsis is similar (OR: 1.18; 95% CI: 0.52-2.64; P=0.69). Neither post-procedural urine leakage (OR: 2.03; 95% CI: 0.70-5.85; P=0.19) nor requirement of blood transfusions (OR: 0.49; 95% CI: 0.14-1.76; P=0.27) differed significantly. Conclusion: PCNL with UP access for staghorn stone has a similar stone-free rate to the NP approach. Thoracic complication rate which was believed to be higher in the UP group is also deemed similar with NP access.",
"keywords": [
"percutaneous nephrolithotomy",
"pcnl",
"upper pole access",
"lower pole access",
"staghorn stones"
],
"content": "Introduction\n\nCurrently, percutaneous nephrolithotomy (PCNL) remains the mainstay of treatment of all type of renal calculi, with high a success rate and stone-free rate (SFR). The complication rate was notably low in this PCNL procedures compared with another procedure to treat any kind of renal stones. Thus, in the past few decades, nearly all open surgery for treating renal stones, whether simple stones or complex staghorn stones, have been changed to this minimally invasive procedure1.\n\nFor complex renal stones and staghorn stones, PCNL is the preferred surgical modality2. Modifications to the original technique aimed to increase both efficacy and safety of PCNL procedures to treat patients with large and complex renal stones. There are three approaches to perform PCNL for renal stones: lower pole (LP) access, middle pole (MP) access and upper pole (UP) access. The traditional LP access has been proved as the safest approach for renal collecting system access, with a lower risk of bleeding and other thorax-related complications (either hydrothorax or pneumothorax)3. However, on the other hand, this LP approach may not be sufficient for complete stone clearance in patients with complex or staghorn renal stones as well as proximal ureteral stones4,5. Due to this reason, UP access is advocated as the preferred PCNL approach for complex and staghorn renal stones. PCNL with UP access is considered to allow a higher stone-free rate due to its direct access to the intrarenal collecting system, fewer punctures and less manipulative trauma compared to LP access. However, this UP approach has a notable deficiency. UP access is believed to be associated with a higher complication rate, which is mainly related to thoracic and abdominal complications, particularly when the puncture is done above the 11th rib. We designed this meta-analysis to systematically describe the outcomes and complications of PCNL for staghorn stones in upper and non-upper pole (lower and middle) approach.\n\n\nMethods\n\nA systematic literature review was performed in August to September 2018 using several electronic databases such as: PubMed; EBSCO; Science Direct; and Cochrane to identify any relevant studies. The keywords used for this searching process were (percutaneous nephrolithotomy OR percutaneous nephrolithotomies OR pcnl) AND (lower pole puncture OR lower pole access) AND (upper pole puncture OR upper pole access OR supracostal puncture) AND (nephrolithiasis OR urinary calculi OR renal stone OR complex urinary calculi OR staghorn stone). All keywords used were searched for their respective MeSH thesaurus (Table 1). This data searching process was not limited by date of publication, and we only included full-text articles in English. Article selection was done according to the search strategy recommended by PRISMA (a completed PRISMA checklist is available on Open Science Framework6). Only studies comparing UP access PCNL and LP access PCNL for complex and staghorn renal stones were assessed for further analysis. Participants were men and women above 18 years old with staghorn stones. Studies with paediatric subjects, patients with congenital kidney anomalies, patients with bleeding diathesis and patients with non-staghorn kidney stones were excluded from this review. PCNL procedures requiring multiple access also excluded from this study. Data from all selected articles were extracted independently by two reviewers. Any disagreements were solved by consensus. Relevant parameters were explored using Review Manager V5.3.\n\nThis review used all comparative studies of UP access PCNL compared to NP access (either LP or MP access) PCNL for patients with staghorn stones. Only full-text studies were included. Unpublished articles and abstracts were excluded from the study.\n\nInterventions used in this study was single access UP approach PCNL compared to single-access NP approach PCNL. Type of differences of lithotripsy technique, anesthesia procedures and either pre or postoperative medications were not analyzed, which was considered as a limitation of this study.\n\nThe primary outcome of intervention was the SFR in each group. We also analyzed perioperative and postoperative outcome, operation duration, hospital length of stay, and hemoglobin decrement. Complications rate for both groups including, pneumothorax or hydrothorax, blood transfusion requirement, postoperative fever or sepsis; and persistent urinary leakage were noted.\n\nThis study used Cochrane Risk of Bias assessment tools to assess interventional study’s quality. These assessments were done by two authors independently. Quantitative synthesis of included studies was done using Review Manager 5.3. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated for binary variables. Heterogeneity of studies was assessed using χ2 and I2. Fixed-effect models were used for homogenous data, and random effects analysis was considered for heterogeneous data. Forest plots were used to present meta-analysis results.\n\n\nResults\n\nThree prospective and two retrospective comparative studies involving 384 renal units with staghorn stones (Figure 1 contains a flow diagram), with 176 cases done using the UP approach and the other 208 cases using either lower or middle pole approaches. In this present time there was no randomized controlled study that comparing upper and lower pole approach PCNL for staghorn stone. Study characteristics are shown in Table 2.\n\nQuality of the studies is shown in Table 3. Three of five had 8 stars2,7,8, one study9 had 7 stars, and one had 6 stars.\n\n* median (range)\n\n** mean (range)\n\nAll of 384 patients whom underwent PCNL procedures were included in the analysis of stone-free rate. A total of 131 out of 176 staghorn stones patients treated with UP approach PCNL were stone-free (74.4%), compared to 148 from 208 staghorn patients (71.1%) in NP approach PCNL (OR: 1.55; 95% CI: 0.92-2.63; P=0.10) (Figure 2). Because of homogenous data (I2= 0% and P=0.78) we performed fixed effect measure for this quantitative analysis.\n\nThree7,8,10 out of the four studies2,7,8,10 demonstrated longer operative duration in those who underwent UP access than in NP access. The longest median operative duration was found in those who underwent MP/LP access in the study conducted by Netto et al.7, which was 139.1 minutes (15.0–36.0), whereas the shortest duration was in the UP access group in the study conducted by Aron et al.10, which was 48.0 minutes (35.0–60.0).\n\nThree studies2,7,8 demonstrated similar mean or median LOS between both groups. Singh et al.8 found the longest mean LOS (4.74 ± 1.33 vs. 4.69 ± 1.32 days in the UP and LP access group, respectively) among the other studies, while the shortest median LOS was in the study conducted by Blum et al.2 (1 [1-21] vs. 1 [1.0-35.0] days in the UP and LP access group, respectively).\n\nAmong the 176 cases of UP access PCNL, 5 (2.8%) had either hydrothorax or pneumothorax. None of the patients in the NP access group experienced this complication. In spite of this data, our quantitative analysis for this subgroup noted that there was no significant difference of thoracic complications rate between patients that underwent UP and those undergoing NP (OR: 3.14; 95% CI: 0.63-15.62; P=0.16) (Figure 3). Because of non-heterogenous (I2= 0% and P=0.71) data, we used fixed-effect analysis.\n\nIn total, 14 of the 141 patients in the UP access PCNL group (9.9%) experienced either fever or sepsis, while 13 of the 98 NP access group (6.5%) experience the same condition postoperatively. From Figure 4 we can see that there was no significant difference in incidence fever or sepsis between these two groups of patients (OR: 1.18; 95% CI: 0.52-2.64; P=0.69). We used fixed-effect analysis to analyze this homogenous data (I2= 0% and P=0.93).\n\nThree studies2,8,10 demonstrated similar median LOS between both groups. The highest hemoglobin decrement was noticed in the study conducted by Aron et al.10. They found that the patient whom underwent UP- and LP-access PCNL had median hemoglobin decrement of 6.0 (4.0-8.0) and 6.5 (4.0-8.0), respectively.\n\nA total of 18 out of the 180 patients in both groups included in this subgroup analysis underwent blood transfusion after PCNL procedure. Of the 59 patients that underwent UP access PCNL, 3 required a blood transfusion (5.1%), while 15 of 121 patients from NP access procedure required post-procedural blood transfusions (12.4%). Although from this review we can see that NP has a notably higher requirement of blood transfusions, meta-analysis found that there was similar rate of blood transfusions requirement from both groups of patients (OR: 0.49; 95% CI: 0.14-1.76; P=0.27) (Figure 5). Because of homogenous data (I2= 0% and P=0.32), we performed fixed-effect analysis.\n\nA total of 21 patients out of 143 patients in both groups included in this subgroup analysis experienced persistent urinary leakage after the PCNL procedure. In total, 6 of 29 patients (20.7%) from UP access PCNL and 15 of 114 patients (13.1%) from the NP-access procedure experienced persistent urinary leakage. Although from this review we can see that the UP-access group has a notably higher urinary leakage incidence, meta-analysis found that there was similar rate of this complication from both groups of patients (OR: 2.03; 95% CI: 0.70-5.85; P=0.19). Due to heterogeneous data (I2= 62% and P=0.10), we applied random-effect model (Figure 6).\n\nThere was a similar SFR between the UP and NP approaches, with 74.4% and 71.1% of PCNL procedures with UP and NP access, respectively, were considered successful after a single procedure (OR: 1.55; 95% CI: 0.92-2.63; P=0.10). Incidence of thoracic complications such as hydrothorax and pneumothorax were also similar between the two groups (OR: 3.14; 95% CI: 0.63-15.62; P=0.16). However, we noted that 5 of 176 patients with upper pole approach experienced thoracic complications. On the contrary, none of the patients with LP approach had these events. The incidence of post-procedural fever and sepsis is similar between these two groups of patients (OR: 1.18; 95% CI: 0.52-2.64; P=0.69). We also found that neither post-procedural urine leakage (OR: 2.03; 95% CI: 0.70-5.85; P=0.19) nor requirement of transfusions (OR: 0.49; 95% CI: 0.14-1.76; P=0.27) differs significantly between these two approaches.\n\n\nDiscussion\n\nFor patients with staghorn renal calculi, PCNL is the preferred modality of treatment. A successful PCNL procedure requires optimal placement of the percutaneous renal access, thus providing good access for intrarenal stone clearance procedure11. Currently UP access and LP access are the two favored approaches to performed PCNL in patients with staghorn calculi2. The current consensus among endourologist is that a prone position PCNL procedure, which done through posterior calyx approach, will give highly limited access to the upper calyx. Because of this reason, upper calyx approach was the preferred procedure to treat large and complex staghorn stones. Upper calyx access provides direct access down to most renal calyxes and the ureter, and most of middle calyces can be accessed with the UP calyx approach, with the usage of flexible nephroscope. Previous literature show that the usage of UP calyx approach in patients with staghorn renal calculi will result in satisfying SFRs, less access punctures in PCNL, and less renal tissue trauma due to minimal intrarenal manipulation compared to LP access7,12–14. Mostly, the UP calyx approach is done in a supracostal fashion, which results in markedly higher rates of thoracic and other complication, such as pneumothorax and hydrothorax as well as higher risk of bleeding, especially with punctures access that made above the 11th rib7,12–20. However, despite the high risk of complications explained above, evidence suggests the use of the UP approach, as the higher SFR and better access to multiple calyces overshadows the drawbacks of this procedure7,14,21. Nevertheless, this approach should be limited to cases when there is no other available alternative. Performing LP access maybe result in decreased risk of complications, but a complete stone-free condition in some complex upper calyx calculi is limited because of limitations to the LP approach of upper calyx access22.\n\nTo completely eliminate calculi with PCNL, good access is mandatory. Ideally, PCNL access tract should provide the shortest and most straight access to mostly of the renal stones. Calculi in a single inferior calyx can be easily removed through a single LP calyx access tract. However, for complex and staghorn calculi occupying several calices in the lower pole, access through the superior calyx is thought to be beneficial10. In the present meta-analysis, of 5 studies involving 384 renal units, we did not find a significant difference in SFRs between these approaches, with SFRs of 74.4% for the UP approach and 71.1% for the NP approach (OR: 1.55; 95% CI: 0.92-2.63; P=0.10). This suggests that in staghorn calculi patients, SFRs for the UP access approach is similar to those for the NP access approach. This result is probably different from other studies12,13, maybe because we analyzed single access PCNL in patients with staghorn calculi only, while the majority of other studies rarely analyzed staghorn stone or single access PCNL alone. The advantage of UP access is its naturally direct access to the intrarenal collecting system and to the upper ureter23. Because of the linear position of the upper pole with the ureteropelvic junction, intraoperatively the PCNL wire will enter into ureter in the majority of cases. Excellent visualization of the superior calyx, pelvis, and the anterior and posterior inferior calyces of the kidney will be provided by the straight tract along the long axis of the kidney. This condition will make it easier to move and manipulate the nephroscope and forceps thus will reduces the possibility of intraoperative bleeding11. PCNL access through UP calyx is one of the most important way of ensuring better renal calculi clearance. This access can be done with either supracostal or infracostal approaches23. LP calyx is located in the lateral and anterior portion of kidney, meanwhile UP calyx is located in the medial posterior portion of the kidney. From this anatomical mapping, we can see that in patient placed in prone position the puncture access along with the kidney axis that performed from the posterior area is more effective and useful compared to anterior approach. Lower pole access approach has limited accessibility, particularly compared to the UP approach, to the visualize complete intrarenal collecting system in prone-position patients. A PCNL access through the lower pole calyx will form a more acute angle compared to upper pole calyx access due to the anatomical position of the kidney that has been stated above. Nevertheless, in some circumstances that PCNL in UP access is difficult to achieve, than a more lateral fashion of lower calyx access can also facilitate a wider angle of intrarenal access22.\n\nThe most common reason to perform LP approach is to minimize complications caused by the UP approach2. However, although studies have shown that UP is associated with intrathoracic complications, we found that the rate of thoracic complications (hydrothorax and pneumothorax) did not differ between both groups (OR: 3.14; 95% CI: 0.63-15.62; P=0.16). Although previous studies demonstrated an increasing risks of intrathoracic and other complications associated with an upper pole access24,25, in recent years these kind of complications have decreased exponentially in adult patients whom undergo PCNL for renal calculi23,24. Furthermore, even if these thoracic complications did occur, the majority of patients that experienced these complications will recover either spontaneously or by simple intervention with minimal future comorbidity26.\n\nWith these advantages, UP access is usually used as the preferred approach in selected patients with complex or staghorn renal calculi which need more than one intrarenal access. It has also been suggested that UP access is associated with increased bleeding. However, higher intraoperative bleeding in PCNL with UP access has been reported previously11. But in this present meta-analysis, we did not find an increased requirement of blood transfusions in those whom undergoing PCNL with UP approaches (OR: 0.49; 95% CI: 0.14-1.76; P=0.27). Oner et al.27 performed 1750 PCNL procedures and found that upper calyx access is associated with a lower blood transfusion requirement and easier guidewire placement to the ureter, easier tract dilatation, and more comfortable manipulation. This suggests that UP access should be used if it facilitates stone clearance.\n\nThis meta-analysis found that there was no significant difference in terms of SFR and complications between staghorn calculi patients those underwent single UP-access PCNL and single NP-access PCNL. However, this study only included data with single PCNL access and there are only a few studies included in the present analysis. We suggest further multicenter randomized controlled trials are required to compare safety and efficacy of UP-access and NP-access PCNL for staghorn calculi patients.\n\n\nConclusions\n\nThe present analysis found that PCNL with single UP access has similar SFR and complication rates compared to single NP approach PCNL. Further randomized clinical trials are required to compare safety and efficacy of UP-access and NP-access PCNL.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: PRISMA checklist for “The outcome and safety of upper pole vs non-upper pole single puncture PCNL for staghorn stones: a meta-analysis”. https://doi.org/10.17605/OSF.IO/DEU6P6.",
"appendix": "Grant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nPreminger GM, Assimos DG, Lingeman JE: Chapter 1: AUA guideline on management of staghorn calculi: diagnosis and treatment recommendations. J Urol. 2005; 173(6): 1991–2000. PubMed Abstract | Publisher Full Text\n\nBlum KA, Parkhomenko E, Thai J, et al.: A contemporary lower pole approach for complete staghorn calculi: outcomes and efficacy. World J Urol. 2018; 36(9): 1461–1467. PubMed Abstract | Publisher Full Text\n\nde la Rosette JJ, Zuazu JR, Tsakiris P, et al.: Prognostic factors and percutaneous nephrolithotomy morbidity: a multivariate analysis of a contemporary series using the Clavien classification. J Urol. 2008; 180(6): 2489–93. PubMed Abstract | Publisher Full Text\n\nMuslumanoglu AY, Tefekli A, Karadag MA, et al.: Impact of percutaneous access point number and location on complication and success rates in percutaneous nephrolithotomy. Urol Int. 2006; 77(4): 340–6. PubMed Abstract | Publisher Full Text\n\nBreda A, Ogunyemi O, Leppert JT, et al.: Flexible ureteroscopy and laser lithotripsy for single intrarenal stones 2 cm or greater--is this the new frontier? J Urol. 2008; 179(3): 981–4. PubMed Abstract | Publisher Full Text\n\nGunawan S, Birowo P, Rasyid N, et al.: The outcome and safety of upper pole vs non-upper pole single puncture PCNL for staghorn stones: a meta-analysis. 2019. http://www.doi.org/10.17605/OSF.IO/DEU6P\n\nNetto NR Jr, Ikonomidis J, Ikari O, et al.: Comparative study of percutaneous access for staghorn calculi. Urology. 2005; 65(4): 659–62. PubMed Abstract | Publisher Full Text\n\nSingh R, Kankalia S, Sabale V, et al.: Comparative evaluation of upper versus lower calyceal approach in percutaneous nephrolithotomy for managing complex renal calculi. Urol Ann. 2015; 7(1): 31–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong C, Leveillee RJ: Single upper-pole percutaneous access for treatment of > or = 5-cm complex branched staghorn calculi: is shockwave lithotripsy necessary? J Endourol. 2002; 16(7): 477–81. PubMed Abstract | Publisher Full Text\n\nAron M, Goel R, Kesarwani PK, et al.: Upper pole access for complex lower pole renal calculi. BJU Int. 2004; 94(6): 849–52. PubMed Abstract | Publisher Full Text\n\nOner S, Karagozlu Akgul A, et al.: Upper pole access is safe and effective for pediatric percutaneous nephrolithotomy. J Pediatr Urol. 2018; 14(2): 183.e1–183.e8. PubMed Abstract | Publisher Full Text\n\nShah HN, Hegde SS, Shah JN, et al.: Safety and efficacy of supracostal access in tubeless percutaneous nephrolithotomy. J Endourol. 2006; 20(12): 1016–21. PubMed Abstract | Publisher Full Text\n\nSukumar S, Nair B, Kumar PG, et al.: Supracostal access for percutaneous nephrolithotomy: less morbid, more effective. Int Urol Nephrol. 2008; 40(2): 263–7. PubMed Abstract | Publisher Full Text\n\nMunver R, Delvecchio FC, Newman GE, et al.: Critical analysis of supracostal access for percutaneous renal surgery. J Urol. 2001; 166(4): 1242–6. PubMed Abstract | Publisher Full Text\n\nEl-Nahas AR, Shokeir AA, El-Assmy AM, et al.: Post-percutaneous nephrolithotomy extensive hemorrhage: a study of risk factors. J Urol. 2007; 177(2): 576–9. PubMed Abstract | Publisher Full Text\n\nHopper KD, Yakes WF: The posterior intercostal approach for percutaneous renal procedures: risk of puncturing the lung, spleen, and liver as determined by CT. AJR Am J Roentgenol. 1990; 154(1): 115–7. PubMed Abstract | Publisher Full Text\n\nStening SG, Bourne S: Supracostal percutaneous nephrolithotomy for upper pole caliceal calculi. J Endourol. 1998; 12(4): 359–62. PubMed Abstract | Publisher Full Text\n\nGupta M, Bellman GC, Smith AD: Massive hemorrhage from renal vein injury during percutaneous renal surgery: endourological management. J Urol. 1997; 157(3): 795–7. PubMed Abstract | Publisher Full Text\n\nKukreja R, Desai M, Patel S, et al.: Factors affecting blood loss during percutaneous nephrolithotomy: prospective study. J Endourol. 2004; 18(8): 715–22. PubMed Abstract | Publisher Full Text\n\nOlvera-Posada D, Tailly T, Alenezi H, et al.: Risk Factors for Postoperative Complications of Percutaneous Nephrolithotomy at a Tertiary Referral Center. J Urol. 2015; 194(6): 1646–51. PubMed Abstract | Publisher Full Text\n\nLam HS, Lingeman JE, Barron M, et al.: Staghorn calculi: analysis of treatment results between initial percutaneous nephrostolithotomy and extracorporeal shock wave lithotripsy monotherapy with reference to surface area. J Urol. 1992; 147(5): 1219–25. PubMed Abstract | Publisher Full Text\n\nSofer M, Giusti G, Proietti S, et al.: Upper Calyx Approachability through a Lower Calyx Access for Prone Versus Supine Percutaneous Nephrolithotomy. J Urol. 2016; 195(2): 377–82. PubMed Abstract | Publisher Full Text\n\nLojanapiwat B, Prasopsuk S: Upper-pole access for percutaneous nephrolithotomy: comparison of supracostal and infracostal approaches. J Endourol. 2006; 20(7): 491–4. PubMed Abstract | Publisher Full Text\n\nGolijanin D, Katz R, Verstandig A, et al.: The supracostal percutaneous nephrostomy for treatment of staghorn and complex kidney stones. J Endourol. 1998; 12(5): 403–5. PubMed Abstract | Publisher Full Text\n\nFuchs EF, Forsyth MJ: Supracostal approach for percutaneous ultrasonic lithotripsy. Urol Clin North Am. 1990; 17(1): 99–102. PubMed Abstract\n\nAnand A, Kumar R, Dogra PN, et al.: Safety and efficacy of a superior caliceal puncture in pediatric percutaneous nephrolithotomy. J Endourol. 2010; 24(11): 1725–8. PubMed Abstract | Publisher Full Text\n\nOner S, Okumus MM, Demirbas M, et al.: Factors Influencing Complications of Percutaneous Nephrolithotomy: A Single-Center Study. Urol J. 2015; 12(5): 2317–23. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "47631",
"date": "29 Apr 2019",
"name": "Guohua Zeng",
"expertise": [
"Reviewer Expertise Treatment of urinary stones."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aimed to compare the effect and safety of upper pole and non-upper pole single puncture PCNL for treating staghorn stones through a meta-analysis. Five comparative studies were included in this meta-analysis, and the authors found that these two approaches have similar SFR and complication rate. This study is of interest, but I still have some major concerns about it.\nThis study aimed to compare the upper pole and non-upper pole approaches, but the searching keywords only contained “lower pole” and “upper pole”, so how about the middle pole?\n\nIn the Methods section, the authors did not describe how they extracted the data from eligible studies, and how did the authors deal with the missing data?\n\nThe Cochrane Risk of Bias assessment tools are used to assess the quality of RCTs, but the included studies in this meta-analysis were not RCTs; in addition, the contents of Table 3 seemed to be the Newcastle-Ottawa Scale, but not Cochrane Risk of Bias assessment tools; please explain it.\n\nIn the statistical analysis parts, the authors should define the details about the existence of heterogeneity, such as the I2 > 50% and P < 0.10.\n\nIn the flow diagram of study selection, the detailed number of excluded studies should also be indicated with the specific reasons.\n\nIn Table 2 of characteristics of studies, detailed study designs of included studies should be indicated, such as RCT, cohort or case-control studies, but not only the description of retrospective or prospective.\n\nTable 2 should describe the baseline characteristics of all eligible studies, such as the study population, study design, gender of patients, stone size, follow-up time, and so on; but not the list of study outcomes. This is because we should first make sure the baseline characteristics of each study were comparable, then we can pool the outcomes.\n\nIn this study, only binary variables were pooled, and the continuous variables were compared using mean and SD. Actually, the continuous variables could also be pooled using RevMen.\n\nIn Figure 2, the left side of the forest plot should be “Favours non-upper pole”, but not “Favours upper pole”.\n\nIs the definition of SFR the same in all included studies? And what’s the definition of SFR in this meta-analysis? What’s the time of SFR - one month or three months after operation?\n\nThe authors only compared the SFR and complication rate in this meta-analysis, how about the operation time and hospital stay time? These factors could also impact the recovery of patients.\n\nThe limitations of this meta-analysis should be pointed out in the Discussion section.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": []
},
{
"id": "72823",
"date": "06 Nov 2020",
"name": "Charles U. Nottingham",
"expertise": [
"Reviewer Expertise Medical and surgical management of nephrolithiasis. Medical and surgical management of LUTS due to enlarged prostate."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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:\nThe phrasing of the first sentence has awkward phrasing and should be reworded.\n\nAuthors should clarify that the published data actually demonstrating these complications of UP access is actually very limited, although we in the urology community continue to restate this.\nMethods:\nFirst paragraph of the Methods section states that only UP and LP access locations were compared, but later in this section and elsewhere in the manuscript it states that NP includes but UP and MP. Please clarify if MP access locations were included.\nResults:\n\"Hemoglobin decrement\" section states \"similar median LOS\", but probably should read \"similar median hemoglobin decrement\". Please clarify.\nDiscussion:\nFirst paragraph states that the prone position with posterior calyx \"will give highly limited access\" to the upper calyx. Did you mean to state that it gives optimal access, given that the next sentence states that UP is \"preferred\"?\n\nAuthors cite references 7 and 12-20 to describe rates of UP pneumothorax and hydrothorax in published series. However this is a large range of variability of these complications. It would be helpful for the authors to state this range in the reported rates of these complications to the reader, and they should highlight this high variability.\n\nThe authors state in the Introduction and Discussion that UP has higher reported rates of bleeding than LP, but they also cite examples of LP access having higher rates of bleeding. The authors should correct this phrasing to at least state that there is variability in reports of bleeding complications for LP vs UP. The authors should provide potential explanations for bleeding complication with certain types of access such as the potentially higher likelihood of liver or splenic injury with LP access.\n\nDo the authors think it is UP vs NP, or supracostal vs subcostal, which is the true determinant of thoracic/pleural complications? See Lang Reference.\n\nThe authors need to review more of the limitations of this study. Keep in mind that many if not all of these studies included will have a surgeon selection bias regarding who received UP vs NP punctures. The surgeon will make a judgement as to the optimal access intraoperatively based on experience and impression of the patient's anatomy, and this likely has a significant impact on the complication rate. Things such as this should be acknowledged.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-537
|
https://f1000research.com/articles/8-532/v1
|
23 Apr 19
|
{
"type": "Software Tool Article",
"title": "pysradb: A Python package to query next-generation sequencing metadata and data from NCBI Sequence Read Archive",
"authors": [
"Saket Choudhary"
],
"abstract": "The NCBI Sequence Read Archive (SRA) is the primary archive of next-generation sequencing datasets. SRA makes metadata and raw sequencing data available to the research community to encourage reproducibility and to provide avenues for testing novel hypotheses on publicly available data. However, methods to programmatically access this data are limited. We introduce the Python package, pysradb, which provides a collection of command line methods to query and download metadata and data from SRA, utilizing the curated metadata database available through the SRAdb project. We demonstrate the utility of pysradb on multiple use cases for searching and downloading SRA datasets. It is available freely at https://github.com/saketkc/pysradb.",
"keywords": [
"bioinformatics",
"metadata",
"SRA",
"NGS",
"NCBI",
"GEO"
],
"content": "Introduction\n\nSeveral projects have made efforts to analyze and publish summaries of DNA-1 and RNA-seq2,3 datasets. Obtaining metadata and raw data from the NCBI Sequence Read Archive (SRA)4 is often the first step towards re-analyzing public next-generation sequencing datasets in order to compare them to private data or test a novel hypothesis. The NCBI SRA toolkit5 provides utility methods to download raw sequencing data, while the metadata can be obtained by querying the website or through the Entrez efetch command line utility6. Most workflows analyzing public data rely on first searching for relevant keywords in the metadata either through the command line utility or the website, gathering relevant sample(s) of interest and then downloading these. A more streamlined workflow can enable the performance of all these steps at once.\n\nIn order to make querying both metadata and data more precise and robust, the SRAdb7 project provides a frequently updated SQLite database containing all the metadata parsed from SRA. SRAdb tracks the five main data objects in SRA’s metadata: submission, study, sample, experiment and run. These are mapped to five different relational database tables that are made available in the SQLite file. The metadata semantics in the file remain as they are on SRA. The accompanying package, SRAdb8, made available in the R programming language9, provides a convenient framework to handle metadata queries and raw data downloads by utilizing the SQLite database. Though powerful, SRAdb requires the end user to be familiar with the R programming language and does not provide a command-line interface for querying or downloading operations.\n\nThe pysradb package10 builds upon the principles of SRAdb, providing a simple and user-friendly commandline interface for querying metadata and downloading datasets from SRA. It obviates the need for the user to be familiar with any programming language for querying and downloading datasets from SRA. Additionally, it provides utility functions that will further help a user perform more granular queries, which are often required when dealing with multiple datasets on a large scale. By enabling both metadata search and download operations at the command-line, pysradb aims to bridge the gap in seamlessly retrieving public sequencing datasets and the associated metadata.\n\npysradb10 is written in Python11 and is currently developed on GitHub under the open-source BSD 3-Clause License. To simplify the installation procedure for the end-user, it is also available for download through PyPI and bioconda12.\n\n\nMethods\n\npysradb10 is implemented in Python and uses pandas13 for data frame based operations. Since downloading datasets can often take a long time, pysradb displays progress for long haul tasks using tqdm14. The metadata information is read in the form of an SQLite15 database, made available by SRAdb7.\n\nEach sub-command of pysradb contains a self-contained help string that describes its purpose and usage example. The help text can be accessed by passing the ‘–help’ flag. There is also additional documentation available for the sub-commands on the project’s website. We also provide example Jupyter16 notebooks that demonstrate the functionality of the Python API.\n\npysradb’s development primarily occurred on GitHub and the code is tested continuously using Travis CI webhook. This monitors all incoming pull requests and commits to the master branch. The testing happens on Python version 3.5, 3.6, and 3.7 on an Ubuntu 16.04 LTS virtual machine, while testing webhooks on the bioconda channel provide additional testing on Mac-based systems. Nevertheless, pysradb should run on most Unix derivatives.\n\npysradb10 can be run on either Linux- or Mac-based operating systems. It supports Python 3.5, 3.6 and 3.7. Requiring just two additional dependencies, pysradb can be easily installed using either a pip- or conda-based package manager via the bioconda12 channel.\n\nAn earlier version of this article can be found on bioRxiv https://doi.org/10.1101/578500\n\n\nUse cases\n\npysradb10 provides a chain of sub-commands for retrieving metadata, converting one accession to other and downloading. Each sub-command is designed to perform a single operation by default, while additional operations can be performed by passing additional flags. In the following section we demonstrate some of the use cases of these sub-commands.\n\npysradb uses SRAmetadb.sqlite, a SQLite file produced and made available by SRAdb7 project. The file itself can be downloaded using pysradb as:\n\n\n\nThe SRAmetadb.sqlite file is required for all other operations supported by pysradb. This file is required for all the sub-commands to function. By default, pysradb assumes that the file is located in the current working directory. Alternatively, it can supplied using the ‘–db path/to/SRAmetadb.sqlite’ argument. The SRAmetadb.sqlite is available at: https://s3.amazonaws.com/starbuck1/sradb/SRAmetadb.sqlite.gz or alternatively at https://gbnci-abcc.ncifcrf.gov/backup/SRAmetadb.sqlite.gz. The examples here were run using SRAmetadb.sqlite with schema version 1.0 and creation timestamp 2019-01-25 00:38:19.\n\nConsider a case where a user is looking for Ribo-seq17 public datasets on SRA. These datasets will often have ‘ribosome profiling’ appearing in the abstract or sample description. We can search for such projects using the ‘search’ sub-command:\n\n\n\nThe results here list all relevant ‘ribosome profiling’ projects.\n\nEach SRA project (accession prefix ‘SRP’) on SRA consists of single or multiple experiments (accession prefix ‘SRX’) which are sequenced as single or multiple runs (accession prefix ‘SRR’). Each experiment is carried out on an individual biological sample (accession prefix ‘SRS’).\n\npysradb metadata can be used to obtain all the experiment, sample, and run accessions associated with a SRA project as:\n\n\n\nHowever, this information by itself is often incomplete. We require detailed metadata associated with each sample to perform any downstream analysis. For example, the assays used for different samples and the corresponding treatment conditions. This can be done by supplying the ‘–desc’ flag:\n\n\n\nThis can be further expanded to reveal the data in ‘sample_attribute’ column into separate columns via ‘–expand’ flag. This is most useful for samples that have associated treatment or cell type metadata available.\n\n\n\nAny SRA project might consist of experiments involving multiple assay types. The assay associated with any project can be obtained by providing –assay flag:\n\n\n\nThe Gene Expression Omnibus database (GEO)18 is the NCBI data repository for functional genomics data.\n\nIt accepts array and sequence-based data from gene profiling experiments. For sequence-based data, the corresponding raw files are deposited to the SRA. GEO assigns a dataset accession (accession prefix ‘GSE’) that is linked to the corresponding accession on the SRA (accession prefix ‘SRP’). It is often necessary to interpolate between the two accessions. gse-to-srp sub-command allows converting GSE to SRP:\n\n\n\nIt can be further expanded to obtain the corresponding experiment and run accessions:\n\n\n\nAny GEO study (accession prefix ‘GSE’) will involve a collection of experiments (accession prefix ‘GSM’). We can obtain an entire list of experiments corresponding to the study using the gse-to-gsm sub-command from pysradb:\n\n\n\nHowever, a list of GSM accessions is not useful if one is performing any downstream analysis, which essentially requires more detailed information about the metadata associated with each experiment. This relevant metadata associated with each sample can be obtained by providing gse-to-gsm additional flags:\n\n\n\nThe metadata information can then be parsed from the sample_attribute column. To obtain more structured metadata, we can use an additional flag ‘–expand’:\n\n\n\ngsm-to-srr allows conversion from GEO experiments (accession prefix ‘GSM’) to SRA runs (accession prefix ‘SRR’):\n\n\n\npysradb enables seemless downloads from SRA. It organizes the downloaded data following the NCBI hiererachy: ‘SRP => SRX => SRR’ of storing data. Each ‘SRP’ (project) has multiple ‘SRX’ (experiments) and each ‘SRX’ in turn has multiple ‘SRR’ (runs). Multiple projects can be downloaded at once using the download sub-command:\n\n\n\ndownload also allows Unix pipes-based inputs. Consider our previous example of the project SRP000941 with different assays. However, we want to be able to download only ‘RNA-seq’ samples. We can do this by subsetting the metadata output for only ‘RNA-seq’ samples:\n\n\n\nThis will only download the ‘RNA-seq’ samples from the project.\n\n\nSummary\n\npysradb10 provides a command-line interface to query metadata and download sequencing datasets from the SRA. It enables seamless retrieval of metadata and conversion between different accessions. pysradb is written in Python 3 and is available on Linux and Mac OS. The source code is hosted on GitHub and licensed under BSD 3-clause license. It is available for installation through PyPI and bioconda.\n\n\nData availability\n\nDataset from DDBJ Sequence Read Archive, Accession number DRP003075: https://identifiers.org/ insdc.sra/DRP003075\n\nDataset from EMBL-EBI Sequence Read Archive, Accession number ERP013565: https://identifiers. org/insdc.sra/ERP013565\n\nDataset from Gene Expression Omnibus, Accession number GSE24355: https://identifiers.org/geo/ GSE24355\n\nDataset from Gene Expression Omnibus, Accession number GSE25842: https://identifiers.org/geo/ GSE25842\n\nDataset from Gene Expression Omnibus, Accession number GSE100007: https://identifiers.org/ geo/GSE10000719\n\nDataset from Gene Expression Omnibus, Accession number GSE41637: https://identifiers.org/geo/ GSE4163720\n\nDataset from NCBI Sequence Read Archive, Accession number SRP010679: https://identifiers.org/ insdc.sra/SRP01067921\n\nDataset from NCBI Sequence Read Archive, Accession number SRP000941: https://identifiers.org/ insdc.sra/SRP00094122\n\n\nSoftware availability\n\nSoftware available from: https://pypi.org/project/pysradb/.\n\nSource code available from: https://github.com/saketkc/pysradb.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.257944610.\n\nLicense: BSD 3-Clause\n\n\nAuthor endorsement\n\nDr. Luiz O. Penalva confirms that the author has an appropriate level of expertise to conduct this research, and confirms that the submission is of an acceptable scientific standard. Dr. Luiz O. Penalva declares they have no competing interests. Affiliation: UT Health San Antonio, Children’s Cancer Research Institute, San Antonio, Texas, 78229, USA",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe author thanks Amal Thomas, Meng Zhou, Rishvanth Prabakar, Wenzheng Li, and Xiaojing Ji at the University of Southern California (USC) and Shweta Ramdas at the University of Pennsylvania for helpful discussions and comments on the software and manuscript. The author acknowledges support from the USC Provost Graduate Research Fellowship.\n\n\nReferences\n\nMacArthur DG, Balasubramanian S, Frankish A, et al.: A systematic survey of loss-of-function variants in human protein-coding genes. Science. 2012; 335(6070): 823–828. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLachmann A, Torre D, Keenan AB, et al.: Massive mining of publicly available RNA-seq data from human and mouse. Nat Commun. 2018; 9(1): 1366. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollado-Torres L, Nellore A, Kammers K, et al.: Reproducible RNA-seq analysis using recount2. Nat Biotechnol. 2017; 35(4): 319–321. PubMed Abstract | Publisher Full Text\n\nLeinonen R, Sugawara H, Shumway M, et al.: The sequence read archive. Nucleic Acids Res. 2011; 39(Database issue): D19–D21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSRA Toolkit Development Team: Sra toolkit. 2018; [Online; accessed 10-December-2018]. Reference Source\n\nKans J: Entrez direct: E-utilities on the unix command line. 2018. Reference Source\n\nZhu Y, Stephens RM, Meltzer PS, et al.: SRAdb: query and use public next-generation sequencing data from within R. BMC Bioinformatics. 2013; 14(1): 19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu J, Davis S: Bioconductor:sradb. 2018. Publisher Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Reference Source\n\nChoudhary S: saketkc/pysradb v0.9.0. 2019. http://www.doi.org/10.5281/zenodo.2579446\n\nvan Rossum G, Drake FL: The Python Language Reference Manual. Network Theory Ltd., 2011, ISBN 1906966141, 9781906966140. Reference Source\n\nGrüning B, Dale R, Sjödin A, et al.: Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods. 2018; 15(7): 475–476. PubMed Abstract | Publisher Full Text\n\nMcKinney W: Data structures for statistical computing in python. In Stéfan van der Walt and Jarrod Millman, editors, Proceedings of the 9th Python in Science Conference. 2010; 51–56. Reference Source\n\nda Costa-Luis C, Stephen L, Mary H, et al.: tqdm/tqdm: tqdm v4.20.0 stable. 2018. Publisher Full Text\n\nSqlite home page. 2018; [Online; accessed 10-December-2018]. Reference Source\n\nKluyver T, Ragan-Kelley B, Pérez F, et al.: Jupyter notebooks - a publishing format for reproducible computational workflows. In F. Loizides and B. Schmidt, editors, Positioning and Power in Academic Publishing: Players, Agents and Agendas. IOS Press, 2016; 87–90. Publisher Full Text\n\nIngolia NT, Ghaemmaghami S, Newman JR, et al.: Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; 324(5924): 218–223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarrett T, Wilhite SE, Ledoux P, et al.: NCBI GEO: archive for functional genomics data sets--update. Nucleic Acids Res. 2013; 41(Database issue): D991–D995. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlair JD, Hockemeyer D, Doudna JA, et al.: Widespread Translational Remodeling during Human Neuronal Differentiation. Cell Rep. 2017; 21(7): 2005–2016. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerkin J, Russell C, Chen P, et al.: Evolutionary dynamics of gene and isoform regulation in Mammalian tissues. Science. 2012; 338(6114): 1593–1599. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHsieh AC, Liu Y, Edlind MP, et al.: The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature. 2012; 485(7396): 55–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchultz MD, He Y, Whitaker JW, et al.: Human body epigenome maps reveal noncanonical DNA methylation variation. Nature. 2015; 523(7559): 212–6. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "47560",
"date": "26 Apr 2019",
"name": "Simon Andrews",
"expertise": [
"Reviewer Expertise bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPysradb is a command line utility, written in python, which provides an easy scriptable interface for querying metadata and datasets from the SRA database.\nThe authors correctly point out that interacting with GEO/SRA through the official site and APIs can be slow and frustrating, and thus having a tool which can make this process more streamlined is of great value.\nBoth the paper and the documentation for the tool are well written and easy to understand. The tool provides a series of individual operations performing queries, translations or downloads and these can be run individually or chained together through pipes (which is really nice!).\nThe tool requires an initial download of the sqlite database from the SRAdb project. Whilst I can see how this then makes all subsequent operations quick, it does mean that you have to download a >2GB file (which expands to >30GB), taking 30+ mins before you can do anything with the program. It presumably also means that you need to re-download this file every time there is an update to the data in GEO otherwise your searches are likely to be out of date. On our site at least, people are often getting data for papers which have just been released so this is going to entail a lot of waiting for this file to download. It would be great if there was a way to point to a publicly accessible SQL server to do queries without having to do the local download, and then providing the option of pulling a local copy if you need greater performance. Also having a way to do incremental updates to this file instead of re-downloading the whole thing would be nice. Neither of these is a deal breaker, but they mightn't be too hard to implement?\nThe individual tools all worked as described, with the exception of the issues listed at the bottom, and the experience was generally very good with the tool.\nOne frustrating limitation is that the piping support is not univeral throughout the tool. You can pipe into the download command, but not, for example, into the metadata command. Being able to chain operations such as:\npysradb gse-to-srp GSE24355 | pysradb metadata | pysra download\n..or\npysradb search '\"oocyte development\"' | head | pysradb metadata\n..would be really nice and presumably not too hard to support?\nThe downloading side of the tool is very useful and probably the part which is hardest to achieve in the main SRA site. Whilst this worked as described there are some aspects of the way it works which make it a little frustrating. Firstly, it downloads SRA files, which hardly anyone wants - having a way to get the fastq files directly would be a really useful addition rather than having to run fastq-dump manually afterwards. It also downloads into a structured set of folders, which makes sense, but for large downloads means your files are scattered through multiple folders which makes life harder when you want to process them. Even the --out-dir option doesn't mean the files are in that directory, but just that it's used as a basename. For the names of the files it would be nicer to have a name which incorporated the relevant SRR/SRX ids and maybe the user submitted sample name so that you can actually have a meaningful and complete name from the file. For example, the types of filenames generated by SRA explorer (https://ewels.github.io/sra-explorer/) are a nice compromise between being predictable, unique and yet informative at the same time.\nIf I'm being really picky I'd also quibble a bit at the choice of some of the defaults in the API. For example, I can't see why the --desc and --expand options aren't the default for the metadata sub-program - give me everything in a nice format and let me cut that down if I don't need everything.\n\nOverall this tool is really nice and will be useful for a lot of people. With a small amount of refinement this is likely to become part of our standard toolbox.\n-----------------------\nMinor points to address:\n1) The API seems to have changed since the paper was written. The option to download the metadata is now pysradb metadb, and not pysradb srametadb. This is wrong in both the paper and the Jupyter notebook example on the github page and should be changed. It might be nice to allow srametadb as a fallback if people have been using the old name?\n2) Some of the documentation is incomplete. For example the quickstart documentation at https://saket-choudhary.me/pysradb/quickstart.html#the-full-list-of-possible-pysradb-operations doesn't list the search operation so I couldn't look up the options I had for that.\n3) The piping option is great, but on my system generates a crash if there is too much output (possibly more than the pipe buffer can hold?). Submitted as bug #7\n4) The metadata command line in the paper is broken. You need double dashes before the options, so --desc rather than -desc. This seems to happen elsewhere as well and might just be an auto-format problem.\n5) If using wget to download the progress bar for downloading doesn't work. The data comes down but the progress stays at 0%.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "47516",
"date": "07 May 2019",
"name": "Ryan K. Dale",
"expertise": [
"Reviewer Expertise bioinformatics",
"genomics",
"chromatin",
"gene expression throughout 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\nPysradb is a Python package that extends the existing SRAdb R package by exposing command-line functionality such that metadata queries can be piped to other command-line tools.\n\nOverall this is a well-written tool with good documentation and tests. As-is, it is very useful and my comments here are only aimed at making the tool more useful.\n\nDownloading: It would be really nice to be able to download fastqs, perhaps using SRAdb's strategy of downloading them from EBI.\n\nWhen trying to run the example from the docs, \"pysradb download -p SRP000941\", I got a 550 HTTP error. Upon closer inspection, some of the SRAs in this entry are no longer available, but it causes the whole download to fail. I'm not quite sure of the underlying cause—this seems to be a mismatch between what's in SRA and the metadata database even though I just downloaded a fresh database with a timestamp of a week ago.\n\nTwo suggestions for making the download more robust. The first is to move on if there are any failures, reporting any failures at the end to stderr. The second is to provide a --list option that only prints the table with URLs such that the user can extract the URLs as needed (for example, to allocate to separate nodes on a cluster where failures can be handled by other mechanisms). With the current set of arguments, the only way to do the latter is to interactively answer \"N\" to the question of whether to continue downloading.\n\nIt could be useful to just download individual SRRs; I do not see that functionality available.\n\nSearching: An explanation of the search syntax would be helpful (e.g., SQL syntax, which allows wildcards but not regular expressions). More complex examples would be good here. Given the heterogeneity of user-entered metadata in SRA, it would be convenient to use regular expressions when searching (which appears to be possible with Python and sqlite3 using a callback function).\n\nI would prefer a nicer-formatted \"no results found\" printed to stderr rather than RuntimeWarning and UserWarning.\n\nOther: An interface to check the timestamp from the metaInfo table of the database would be helpful.\n\nThis may be a personal preference, but I would rather have output be tab-separated instead of requiring additional `tr -s \" \" | cut -d \" \"` commands (or awk) to do any sort of manipulation on the command line.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-532
|
https://f1000research.com/articles/8-531/v1
|
23 Apr 19
|
{
"type": "Research Article",
"title": "A pilot study on assessing the gap between nurses’ task performances and knowledge pertaining the same with reference to “ I COUGH” initiative- a call for promoting patient ‘care bundle’ assignments in low-income nations",
"authors": [
"Sunil Munakomi",
"Sangam Shrestha",
"Anita Luitel",
"Sangam Shrestha",
"Anita Luitel"
],
"abstract": "Background: The health sector in low-income nations has been crippled owing to low resources, lack of trained staff and a scarcity of effective health-related reforms. Amidst such a scenario, implementation of patient-centered care bundle approaches could help reprise the autonomy and standards of care for healthcare providers as well as safeguard patient safety. Methods: We sought to determine the gap between task performance and the underlying knowledge pertaining the same among nurses from intensive and high dependency neurosurgical units within three hospitals in Nepal through a questionnaire-based approach focusing on task assignments to prevent pulmonary complications among their patients and scoring them with references to\n\nthe variables of ‘I COUGH’, a similar patient care bundle initiative. Results: There is a gross discrepancy between the patterns of task performance and the knowledge regarding the rationale behind the same tasks among nurses working in critical care neurosurgical units. In reference to I COUGH, nurses had below 50% knowledge on interventions aimed to prevent pulmonary complications among their patients, irrespective of the level of experience attained in the units. Furthermore, none of them had complete knowledge regarding all components of effective chest physiotherapy. Conclusion: There is the utmost need for the implementation of patient-focused care bundle approaches in upraising the health delivery standards, especially in low-income nations. Such initiatives can promote autonomy amongst healthcare professionals on patient care as well as assuring better patient outcomes by minimizing complications.",
"keywords": [
"Knowledge",
"task performance",
"care bundle"
],
"content": "Introduction\n\nAmong surgical specialties worldwide, there is alarmingly high incidence of post-operative pulmonary complications, being reported as high as 40%, and leading to 30 days post-surgery mortality for almost 20%1. These complications also lead to an increase in average hospital stay by 8 days and thereby increase in average hospital costs by almost 55%1 Therefore, there is a substantial need to produce a care bundle based on an enhanced recovery protocol within these surgical specialties1.\n\nIn context of low-income nations like Nepal, with a per capita income of below US $700, the current health sector situation is even more alarming. The limited resources available coupled with political turmoil further exacerbates the issue2. It reportedly has only 16.7 intensive care unit (ICU) beds per million of its population3. Even in the capital city, only 7.2 ICU beds equipped to provide backup ventilation are available per 100,000 of the population. Moreover, there is a significant shortage of effective manpower, with a nurse patient ratio of >1:4 during night shifts4. The present nurse to population ratio in Nepal is approximately 11 nurses per 10,000 of the population (with 50 considered to be optimum)5. Recruitment of nurses is low in national hospitals with many opting to go abroad owing to higher salaries, improved facilities and relatively better scope for career building opportunity. This accounts for the temporary employment pattern prevalent in our hospitals; with one study showing up to 50 nurses leaving their hospital within a single calendar year. Moreover, lack of in-service education and timely reforms of the existing nursing guidelines have further affected the nursing profession5.\n\nThe I COUGH program is a postoperative pulmonary care bundle approach aimed at reducing the incidence of postoperative pneumonia and unplanned intubation among patients6. This acronym incorporates six variables namely 1) Incentive spirometry, 2) Cough/ chest physiotherapy, 3) Oral care, 4) Understanding, 5) Get out of bed and 6) Head end elevation6.\n\nSuch initiative not only helped promote patient’s health but also in improving performance standards and thereby their autonomy1. Therefore, currently upmost emphasis is being given for promoting such performance appraisal methods1. There is upmost need for bridging the gap between nurses’ performance and knowledge of the underlying reasons for the tasks they perform through the application of care bundle approaches such as the I COUGH initiative6.\n\nWe performed a pilot study to identify significant discrepancy between the knowledge of and performance of relevant tasks among health care providers in the critical care setting up in regards to the I COUGH initiative, and thereby call for initiation of care bundle approaches in our context as well.\n\n\nMethods\n\nA questionnaire based observational analytical study was carried out aiming for maximum inclusion of all nurses practicing in the intensive care and the high dependency neurosurgical units from three major teaching hospitals of Kathmandu University namely 1) Nobel Medical College, Biratnagar 2) College of Medical Sciences, Chitwan and 3) National Institute of Neurosurgery and Allied Health Sciences, Kathmandu were enrolled for our study. We initially confirmed the practice of inclusions of all the variables listed within the I COUGH initiative among the daily assigned nursing task assignments in all these three hospitals. We then opted to determine the level of discrepancy between their patterns of work performance with the underlying knowledge governing the same. The authors requested the management team of each institution to make provisions for maximum attendance of their nurses from their intensive and the high dependency care units in our short educational programme. Prior to beginning the educational course, the authors asked all of the participating 101 nurses to list in numerical order major components of their daily tasks aimed at reducing the risk of pulmonary complications among their patients. They were also advised to tabulate different components of effective chest physiotherapy. Their answers were recorded by a researcher, marked, tabulated and scored out of a total score of six with one score allocated for each of the six variables within the already validated I COUGH protocol. In order to reduce possible bias from early knowledge of the test, the authors performed the test in a single group session the same day of obtaining permission from the respective hospital administrations. The answer session was followed by a short educational class conducted by the researchers describing the importance of application of patient care bundle approach measures such as I COUGH in safeguarding their patients’ lives.\n\nThe acquisition of data was done for score of each of the six variables of I COUGH program for each of the participating 101 nurses from the three hospitals. The results were analyzed and tabulated with the help of Windows Excel version 2007 for our result analysis. Frequency analysis was performed on data gathered from nurses.\n\nThe study was approved by the Institutional Review Committee (IRC) of Nobel Medical College and Teaching Hospital (NMCTH) (approval number 259/2019). Permission for conducting the research was obtained from each of the hospitals that were included in the study.\n\n\nResults\n\nA total of 101 nurses working in the intensive care and high dependency neurosurgical units were enrolled for our study within three different teaching hospitals under Kathmandu University. The average scores obtained by the nurses from three hospitals were below 50% scores (37.83%, 40.83% and 42.33% respectively) in terms of their knowledge while comparing to the I COUGH strategy items (Figure 1). The average scores of the nurses were 2.27, 2.45 and 2.54 for CMS, NINAS and NMCTH respectively.\n\nNMCTH - Nobel Medical College and Teaching Hospital, CMS - College of Medical Sciences, Chitwan, NINAS - National Institute of Neurosurgery and Allied Health Sciences.\n\nIn terms of individual variables of I COUGH, Cough and Head end elevations were mentioned by most number of nurses whereas Oral hygiene and Understanding aspects were mentioned by the least number of nurses (Figure 2).\n\nNMCTH - Nobel Medical College and Teaching Hospital, CMS - College of Medical Sciences, Chitwan, NINAS - National Institute of Neurosurgery and Allied Health Sciences.\n\nCough was mentioned the most (96.42%, 97% and 100% respectively) whereas oral hygiene was mentioned the least (Figure 3 and Figure 4).\n\nNMCTH - Nobel Medical College and Teaching Hospital, CMS - College of Medical Sciences, Chitwan, NINAS - National Institute of Neurosurgery and Allied Health Sciences.\n\nNMCTH - Nobel Medical College and Teaching Hospital, CMS - College of Medical Sciences, Chitwan, NINAS - National Institute of Neurosurgery and Allied Health Sciences.\n\nIn terms of individual variables, Incentive spirometry was mentioned by most nurses from College of Medical Sciences whereas Cough was mentioned the highest from nurses of Nobel Medical College and National institute of Neurosurgery and Allied Sciences institutions (Figure 5).\n\nNMCTH - Nobel Medical College and Teaching Hospital, CMS - College of Medical Sciences, Chitwan, NINAS - National Institute of Neurosurgery and Allied Health Sciences.\n\nDespite naming Cough correctly by most nurses from all three institutions, paradoxically none of the nurses correctly mentioned of all the essential components of Chest physiotherapy.\n\n\nDiscussion\n\nVAP in associated in 9–27% of all mechanically ventilated patients7. One study showed incidence of 25 VAPs per 100 ventilated patients, thereby protracting ICU stays and increasing the risk of mortality among them8. The provision of dedicated physiotherapists in critical care is still in its infancy in our context9.\n\nNurses play pivotal role by standing in the frontline of the health surveillance systems and thereby are supposed to detect, make inferences and then implement the correct course of action during any adverse events in the ICU set up. This is undermined however, by the lack of trained staff. Moreover, there are frequent interruptions during in their assigned work schedule owing to factors such as unplanned emergency procedures, assisting for critical events, disjointed and missing medical supply etc. This increases the risks of some omissions in their care delivery amidst such stacking of cognitive and physical loads. However, merely increasing the number of nurses in a patient care unit may not be a panacea if the environment in which they work is not conducive to clinical decision making. There should be task organization skill simulations to improve their ability to manage such high cognitive managerial tasks10.\n\nThe importance of effective clinical leadership in ensuring safe and efficient care has been reiterated time and again. Nurses need to gain autonomy over their own practicing behavior in order to improve their patients’ clinical outcome11. Effective model study should be framed to upraise the standards of such pivotal role playing the front-line staffs12,13. Future shortages of nurses may be unpreventable, but making provisions for effective interdisciplinary teamwork, coupled with pivotal approaches for determining quality insurance and promoting safe environments for patients, could help mitigate their harmful impacts14.\n\nA similar study carried out to assess the knowledge of nurses regarding chest physiotherapy found that though they had good results in their clinical performances, with regards to clinical knowledge encompassing the same, they ironically performed poor15. Almost 67% of them were not aware about indication of postural drainage while 76% were not aware about definition of percussion and vibration15. Similar discrepancy with regards to task performance and the knowledge regarding underlying rationales for the same have been seen in our study as well. Our study also revealed that despite understanding that chest physiotherapy is an essential strategy to minimizing pulmonary complications, none of the nurses were able to correctly outline different components of the same namely positioning, percussion, vibration, squeezing and finally suctioning16,17.\n\nAmidst the scenario of having an extreme shortage of effective manpower in the health sector on the one hand and the prevalence of huge discrepancy between knowledge of the tasks being performed among the healthcare practitioners on the other; the application of care bundle approaches could promote the notion of “effective tasking through minimal manpower”. This can help improve the standards of the health care providers as well as safeguard patient’s health and safety.\n\nThere are certain limitations to our study. Foremost being inclusion of nurses from Neurosurgical units of only three teaching hospitals of our country. One of the prevailing limiting factors in carrying out such observational study is the risk of coherent bias in the results due prior knowledge of such study among the probable study groups from their peers who already participated in the study. However, the results can further be validated through its multicentric application throughout national and international hospitals with inclusions of nurses from other surgical subspecialties as well as those practicing in the general incentive care units. There is also pivotal need in assessing the effect of such patient focused care bundle approaches in minimizing complications among the patients following its application. The fundamental rational of this study is to promote inclusions of patient care bundle approaches in managing patients to safeguard our patients’ health.\n\n\nConclusion\n\nThe initiation of patient focused care bundle approaches is imperative to improving health delivery standards especially in low income nations. Such initiatives can promote health care provider autonomy on patient care, as well as assure better patient outcomes by minimizing complications.\n\n\nData availability\n\nOpen Science Framework: I COUGH PROJECT. https://doi.org/10.17605/OSF.IO/4U8C718\n\nThis project contains the following underlying data:\n\nI COUGH DATA F1000.xlsx (Nurses scores for I COUGH and chest physiotherapy awareness)\n\nOpen Science Framework: I COUGH PROJECT. https://doi.org/10.17605/OSF.IO/4U8C718\n\nThis project contains the following extended data:\n\nQUESTIONNAIRE.docx (Study questionnaire)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGriffiths SV, Conway DH, POPC-CB Investigators, et al.: What are the optimum components in a care bundle aimed at reducing post-operative pulmonary complications in high-risk patients? Perioper Med (Lond). 2018; 7: 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAcharya SP: Critical care medicine in Nepal: where are we? Int Health. 2013; 5(2): 92–5. PubMed Abstract | Publisher Full Text\n\nMurthy S, Leligdowicz A, Adhikari NK: Intensive care unit capacity in low-income countries: a systematic review. PLoS One. 2015; 10(1): e0116949. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShrestha RR, Vaidya PR, Bajracharya GR: A survey of adult intensive care units in Kathmandu Valley. Postgraduate Med J Nat Acad Med Sci. 2011; 11(2): 1–7. Reference Source\n\nPaudel K: Report on status of nurses in Nepal. Nepal Health Research Council, 2010; 123456789. Reference Source\n\nCassidy MR, Rosenkranz P, McCabe K, et al.: I COUGH: reducing postoperative pulmonary complications with a multidisciplinary patient care program. JAMA Surg. 2013; 148(8): 740–745. PubMed Abstract | Publisher Full Text\n\nKalanuria AA, Ziai W, Mirski M: Ventilator-associated pneumonia in the ICU. Crit Care. 2014; 18(2): 208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMishra DR, Shah N, Shah DS: Incidence and Outcome of Ventilator Associated Pneumonia in ICU of a Tertiary Care Hospital in Nepal. JNMA J Nepal Med Assoc. 2017; 56(207): 304–8. PubMed Abstract | Publisher Full Text\n\nBaidya S, Acharya RS, Coppieters MW: Physiotherapy practice patterns in Intensive Care Units of Nepal: A multicenter survey. Indian J Crit Care Med. 2016; 20(2): 84–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPotter P, Wolf L, Boxerman S, et al.: An Analysis of Nurses' Cognitive Work: A New Perspective for Understanding Medical Errors. In: Henriksen K, Battles JB, Marks ES, et al. editors. Advances in Patient Safety: From Research to Implementation. (Research Findings). Rockville (MD): Agency for Healthcare Research and Quality (US); 2005; 1. PubMed Abstract\n\nKieft RA, de Brouwer BB, Francke AL, et al.: How nurses and their work environment affect patient experiences of the quality of care: a qualitative study. BMC Health Serv Res. 2014; 14: 249. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeedleman J, Hassmiller S: The role of nurses in improving hospital quality and efficiency: real-world results. Health Aff (Millwood). 2009; 28(4): w625–33. PubMed Abstract | Publisher Full Text\n\nClarke SP, Donaldson NE: Nurse Staffing and Patient Care Quality and Safety. In: Hughes RG, editor. Patient Safety and Quality: An Evidence-Based Handbook for Nurses. Rockville, MD: Agency for Healthcare Research and Quality; 2008. PubMed Abstract\n\nBuerhaus PI, Donelan K, Ulrich BT, et al.: Impact of the nurse shortage on hospital patient care: comparative perspectives. Health Aff (Millwood). 2007; 26(3): 853–862. PubMed Abstract | Publisher Full Text\n\nKheder M: Assessment of Nurses Knowledge & Practice Regarding Chest Physiotherapy in Elmek Nimer University Hospital. master degree in medical surgical nursing. shendi university, faculty of post graduate studies and scientific research 2016; 28–30. Reference Source\n\nVan der Schans CP: Conventional chest physical therapy for obstructive lung disease. Respir Care. 2007; 52(9): 1198–206; discussion 1206–9. PubMed Abstract\n\nCiesla ND: Chest physical therapy for patients in the intensive care unit. Phys Ther. 1996; 76(6): 609–625. PubMed Abstract | Publisher Full Text\n\nMunakomi S: I COUGH PROJECT. 2019. http://www.doi.org/10.17605/OSF.IO/4U8C7"
}
|
[
{
"id": "49482",
"date": "17 Jun 2019",
"name": "Enrique Castro-Sánchez",
"expertise": [
"Reviewer Expertise infection prevention and control",
"antimicrobial stewardship",
"health literacy",
"nursing"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear authors,\n\nMany thanks for the opportunity to review your manuscript, reporting on the concordance between Nepali nurses’ knowledge and performance of clinical interventions included in the ICOUGH program targeting postoperative complications. The following comments and suggestions aim to clarify and improve the paper for the readers.\n\nIntroduction I thought that the content was meandering from one topic to other. For example, the first paragraph focused on the prevalence of surgical specialities, but then the following paragraph touched upon the Nepalese human resources crisis, particularly on nurses (I would suggest not to use ‘manpower’ and talk about ‘human resources’). I think that it would have been very useful to learn about the prevalence of surgical complications in Nepal or at least, the 3 hospitals sampled.\n\nFurther, the ICOUGH program is mentioned, but would be useful to offer 1-2 sentences regarding any potential improvement obtained by ICOUGH. Additionally, I think that it would be necessary to expand a bit on each of the components, particularly what ‘oral care’, ‘understanding’ and ‘get out of bed’ entail. Finally, readers would benefit from any information about adaptation/cultural interpretation of the ICOUGH in Nepal.\n\nIt would be necessary as well to offer some evidence about knowledge-performance gaps related to bundles, and even how effective these have been or not. There would be enough room to include such details with a revision of the content related to nursing workforce, which is the lengthiest in the Introduction.\n\nMethods I could not really understand how the components of the ICOUGH initiative were corroborated in practice. The content mentions “We initially confirmed the practice of inclusions of all the variables listed within the I COUGH initiative among the daily assigned nursing task assignments in all these three hospitals”.\n\nAlso, the authors talk about ‘short educational programme’, and it would be necessary to add further details about what the programme included.\n\nThere is a mention to Ethical considerations, but there is no information about the selection, recruitment, consenting process, as well as the number of nurses invited to take part that led to the 101 responses.\n\nResults The authors could offer some further statistical measures, such as ranges, and if & are compared across centres, then it would be useful to consider whether to carry out a comparison of percentages analysis. Also, I feel that the text could include the % of the most frequently mentioned variables.\n\nI suggest that the graphs avoid using 3D figures, which do not really offer much. Figure 1 should have axes and axes titles, and I am not sure about how well the figures facilitate comparison. Figure 2 should also include the missing axis title and remove the 120% if proportion of nurses. Each of the letters of ICOUGH should be defined.\n\nI suggest that Figure 3 removes the stacked results and presents 3 bars for each hospital, and same for Figure 5. This graph, additionally, seems not to have results for each of the hospitals…\n\nDiscussion The 1st para introduces data unrelated to the study or the topic, by talking about physiotherapists.\n\nThe authors then shift the debate towards lack of staff but then also towards the need to ensure clinical leadership. I don’t understand the sentence “Nurses need to gain autonomy over their own practicing behavior in order to improve their patients’ clinical outcome” – what does “gaining autonomy” mean?\n\nThe text is also confusing here: “There should be task organization skill simulations to improve their ability to manage such high cognitive managerial tasks” (what does “task organization skill simulations” mean?). Equally, this paragraph is not clear “Future shortages of nurses may be unpreventable, but making provisions for effec-tive interdisciplinary teamwork, coupled with pivotal approaches for determining quality insurance and promoting safe environments for patients, could help mitigate their harmful impacts.” Finally, the sentence “the application of care bundle approaches could promote the notion of “effective tasking through minimal manpower”. Needs referencing, and particularly the notion of “effective tasking”, particularly as task-based nursing is now and outdated approach to care.\n\nConclusion Could the authors offer some more evidence that “Such initiatives [patient focused care bundle approaches] can promote health care provider autonomy on patient care”?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "65131",
"date": "01 Jul 2020",
"name": "Dibya Sharma",
"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\nIntroduction:\n1st paragraph – Citation number can be provided once at the end of the paragraph instead of repeating it a number of times. Similar correction can be done in 6th and 7th paragraph.\n\nMethods:\nIs it a observational analytical or descriptive cross sectional study? I think the design adopted is descriptive cross sectional study. How did you do the scoring in percentage and average scoring (2.27, 2.45 & 2,54), please discuss about the scoring part in detail. Data acquisition: Is the data shown in the table? I think it’s better to write “frequency distribution is shown in the bar diagram and line graph.\"\n\nResults:\nPlease write the sample size n = 101 in all the figures. Figure no. 2: No need to keep percentage in the 2 decimal like 120.00%, 100.00%, 80.00% etc. instead it can be written as 120%, 100%, 80%, etc. I think there is repetition of the same data in line graph (Figure 2) and bar diagram (Figure 5)…can keep only of one those.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "65133",
"date": "03 Jul 2020",
"name": "Valarmathi Selvaraj",
"expertise": [
"Reviewer Expertise Nursing health 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\nThanks for the opportunity to review the articles titled ‘A pilot study on assessing the gap between nurses’ task performances and knowledge pertaining the same with reference to “I COUGH” initiative- a call for promoting patient ‘care bundle’ assignments in low-income nations’. The concept behind this research is novel and impressive for me.\nIntroduction:\nThere should be more info related to the background aspects of this research. The authors need to mention the pulmonary complication occurring after the surgery. Here the authors need to cite the articles related to ‘I COUGH’ programme. The authors need to explain about Care bundle approach method.\n\nMethodology:\nThe I COUGH protocol has to be mentioned in detail mentioning all the six components to enable readers other than nurses to understand and interpret.\n\nThe study design needs to be explained under this section. What do the authors mean by ‘Analytical study?’. It needs explanation. The authors have mentioned 101 nurses were advised to tabulate different components of effective chest physiotherapy. The authors mentioned only about ‘C’ and the remaining other five components such as 'Oral care', 'Understanding', 'Get out of bed' and 'Head end elevation'. The methodology should be written in detail mentioning duration of study, how many samples the authors have collected in a day, and details on the short educational activity. Overall, it will be nice to divide the methodology into subsections to improve understanding.\n\nResults:\nIn the results part the authors have mentioned clearly about nurses knowledge but the authors never mention the knowledge of nurses in the methodology. This needs rewriting. Figure 3 and Figure 4 have the same headings and need to be modified. The authors need to perform inferential statistics with the results. In the results the authors have mentioned about level of understanding but in the discussion, in a few places the authors have mentioned ‘knowledge’. It is confusing and hence needs to be rewritten.\n\nDiscussion:\nIn the first paragraph the authors did not mention what VAP is. This needs expansion and a probable explanation. The ‘task organization skill simulations’ need more elaborations.\n\nConclusion:\nI would suggest the authors to mention about how to improve the health care standard. Conclusion should be written based on the objective. Hence it can be rewritten.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": []
},
{
"id": "65132",
"date": "13 Jul 2020",
"name": "Nirmala Pradhan",
"expertise": [
"Reviewer Expertise Psychiatric 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\n1. Abstract: It is ok, but the result is focused only on knowledge.\n2. Methodology:\nHow was the sample size (101) calculated? On what basis? Since the population of this study is Nurses, you need to define it. Did the nurses selected in all three settings have similar experience and educational level? Brief description of the knowledge questionnaire must be mentioned. How it was interpreted? Need to mentioned the reliability of the tool.\n\n3. Result:\nSocio-demographic profile is not highlighted. Add on this. Figure 1 there is no index. Its not understandable [Color code used]. If you have observed the Nurse’s task performance, nowhere its mentioned. It will be nice to include this aspect as your study is looking for practice gap. You can include like frequency of observation while caring the post-operative patients. Was it a participatory or non- participatory observation? How did you over-come the biases? Statistical analysis was extremely basic. Only descriptive analysis was used. You could have used inferential statistical analysis consulting the statistician.\n\n4. Ethical consideration: How did you maintain the confidentiality and privacy of the nurses at the time of data collection?\n5. In your title you’ve mentioned that there is a gap in Practice and also mentioned to find the gap. I see that your second objective is un-answered. Please include it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-531
|
https://f1000research.com/articles/8-287/v1
|
14 Mar 19
|
{
"type": "Software Tool Article",
"title": "3D based on 2D: Calculating helix angles and stacking patterns using forgi 2.0, an RNA Python library centered on secondary structure elements.",
"authors": [
"Bernhard C. Thiel",
"Irene K. Beckmann",
"Peter Kerpedjiev",
"Ivo L. Hofacker",
"Bernhard C. Thiel",
"Irene K. Beckmann",
"Peter Kerpedjiev"
],
"abstract": "We present forgi, a Python library to analyze the tertiary structure of RNA secondary structure elements. Our representation of an RNA molecule is centered on secondary structure elements (stems, bulges and loops). By fitting a cylinder to the helix axis, these elements are carried over into a coarse-grained 3D structure representation. Integration with Biopython allows for handling of all-atom 3D information. forgi can deal with a variety of file formats including dotbracket strings, PDB and MMCIF files. We can handle modified residues, missing residues, cofold and multifold structures as well as nucleotide numbers starting at arbitrary positions. We apply this library to the study of stacking helices in junctions and pseudo knots and investigate how far stacking helices in solved experimental structures can divert from coaxial geometries.",
"keywords": [
"RNA",
"Python",
"RNA tertiary structure",
"RNA secondary structure",
"coaxial stacking",
"pseudo knots"
],
"content": "Introduction\n\nIn RNA 3D structure prediction, knowledge-based potentials are commonly used, especially for coarse-grained approaches that are suitable for larger RNA molecules1. The creation of such potentials requires knowledge extraction from solved RNA structures, usually taken from the Protein Data Bank (PDB)2. PDB files and their newer replacement, MMCIF files, contain atomic coordinates and additional information in the header fields, but do not contain any base-pairing annotations. To extract information about base pairs and their types3, dedicated software like MC-Annotate4, RNAView5, FR3D6, DSSR7 or the RNApdbee web server8 is required. Due to the hierarchical organization of the RNA energy landscape9, it is often most convenient to treat secondary and tertiary structure of RNA molecules separately and predict tertiary structures given a secondary structure10.\n\nFor knowledge extraction from RNA-containing PDB files, it is highly desirable to have a software library at hand, which understands the semantics of RNA secondary structures and makes tasks like iterating over loops of a certain type straightforward. Ideally, such a library should be written in an easily accessible scripting language, be well documented and tested and should be available under an open-source license.\n\nLibraries other than the forgi library presented here only partly fill these needs. The Vienna RNA package11 can be used to predict secondary structures from sequence, including advanced features like G-Quadruplex prediction and incorporation of SHAPE data. It provides Python and Perl bindings, but deals exclusively with secondary structure. Biopython12,13 is useful for dealing with RNA sequence data and can be used to load RNA 3D structures, but has no dedicated support for RNA secondary structure. PyCogent14, a library for genomic biology, has extensive support for nucleic acid sequences, but contains only a lightweight class for RNA secondary structures and code for pseudo knot removal15. A stand-alone version of the latter was included into the forgi library under the terms of the GNU General Public License 3.0. More specialized libraries include modeRNA16 (homology modeling, Python) and the FR3D suite6 (RNA motif search, Matlab). Here, we present the forgi library17, which is centered on RNA secondary structure elements (such as stems, bulges and loops) and makes them usable for 3D structure analysis. forgi aims at providing a high level API for many common operations, but can be easily extended with new functionality. The flexibility of providing an open source library in a scripting language is a clear benefit over other programs that are only distributed as binaries. While not restricted to work with PDB or MMCIF files, forgi shines especially where 3D information is analyzed in the context of its secondary structure environment.\n\n\nMethods\n\nThe forgi library17 is strongly object-oriented, but takes advantage of module-level Python functions where appropriate. The core object representing the secondary structure is the BulgeGraph object, which holds a Sequence instance for the primary sequence. To include secondary structure based 3D coordinates, the BulgeGraph’s subclass, CoarseGrainRNA is available. For all-atom 3D analysis, the forgi library has a built-in integration with Biopython.\n\nWe will briefly describe the three main data structures that hold the sequence, secondary structure and the tertiary structure representation of an RNA.\n\nPrimary structure: The Sequence class. Each BulgeGraph holds a Sequence object. Since forgi supports loading of data from PDB files (see below), this Sequence object has to account for many special cases arising from experimental considerations, which will be detailed in the following paragraphs. There are two numbering schemes commonly used for sequences: 1-based indexing and indexing based on an external reference. In particular, many sequences in structural experiments use “1” to dedicate the first residue of a biological macro-molecule, whereas the first residue actually used in the experiment can be upstream of the functional RNA (leading to negative indices) or after the start of the biological unit (leading to an index above 1). The latter is especially common if fragments of larger RNA molecules like the ribosome are studied. Finally, experimenters might decide to insert nucleotides in the middle of a molecule. In order not to affect the numbering of subsequent residues, these inserted residues get the same number as the previous one, followed by a letter (called insertion code).\n\nTo handle both kinds of indexing, the Sequence class distinguishes indices by type. Integer indices always refer to 1-based indexing, while tuples compatible to the indices used in Biopython’s PDB module are interpreted as the second kind of indices.\n\nFor many applications, it is necessary to restrict the RNA alphabet to 4 letters (i.e. 4 residue types), “G”, “C”, “A” and “U”. However, in the cell many RNA molecules are post-transcriptionally modified at certain positions. Many modifications, including the methylation of OH or NH2 groups and A to I editing, have been implicated with a variety of biological functions18. During parsing of PDB files, we automatically convert such modified residues to the unmodified parent, but in addition store the modification as an annotation in the Sequence object. The corresponding unmodified parents for 3-letter codes of common modified residues were obtained from PDBeChem19 (http://www.ebi.ac.uk/pdbe-srv/pdbechem/) and the forgi library has the ability to query this database on the fly if it encounters a new 3-letter code.\n\nFinally, many experimental 3D structures do not contain coordinates for all residues present in the experiment. The forgi Sequence class stores two version of the sequence, with and without missing residues.\n\nThe secondary structure of an RNA is internally represented as a graph, the Bulge Graph, where secondary structure elements (stems, single-stranded regions, interior loops and hairpin loops) form the nodes. Whenever these elements are adjacent along the backbone, they are connected by an edge in this graph. During the Bulge Graph creation, each node gets a unique name such as “s0” for the first stem or “h0” for the first hairpin. The concept of the Bulge Graph, illustrated in Figure 1, has been described previously in more detail20 and is related to the independently developed RAG (RNA as Graph) approach21.\n\nSecondary structure elements (stems, bulges and single-stranded regions) are nodes connected by edges. The sequence is shown around the Bulge Graph.\n\nforgi supports element-based transformations of the Bulge Graph, such as condensing the secondary structure to an representation similar to RNAshapes22, which we use to classify pseudo knots, for example (see below).\n\nThe BulgeGraph object allows for easy identification, selection and classification of structural domains, such as multi loops, helices consisting of multiple stems and bulges (termed “rods” in forgi), and pseudo knots.\n\nThe CoarseGrainRNA class holds 3D structures. 3D structures are loaded from PDB or MMCIF files using Biopython’s PDB module13. In order to assign a secondary structure to the RNA, forgi can call either MC-Annotate4 (Linux only, no MMCIF-support, binary available at http://major.iric.ca/MajorLabEn/MC-Tools.html) or DSSR7 (binary available after free registration at http://forum.x3dna.org). As an alternative, forgi has a built-in heuristic for the detection of canonical base pairs and GU wobble pairs. This heuristic is based on distances along the hydrogen bonds and the coplanarity of the bases, and is not intended to compete with the power of more specialized tools since it will fail in some edge cases, e.g. involving modified residues or residues with missing atoms. This heuristic is a useful fallback, if the above mentioned programs are not available.\n\nDuring loading of the RNA, a helix axis is assigned to stems as described previously20. For each stem, we store start and end coordinates of the helix axis as well as two twist vectors that point towards the minor groove at the beginning and the end of the stem. Similarly, start and end coordinates for bulges, loops and single-stranded regions are stored and can be accessed using the element’s name.\n\nforgi 2.0 now fully supports co- and multi-fold structures and can load multiple chains that are connected by base pairs into a single CoarseGrainRNA object, while chains not connected by any base pair are loaded into separate objects.\n\nUsing Biopython’s KD-Tree implementation (Bio.PDB.NeighborSearch), a list of residues within 6 angstrom from a non-RNA C or N atom is obtained. These residues are considered protein-/ligand interacting. Knowledge of interacting residues is particularly useful to avoid biases in statistics about structural features of bare RNA.\n\nA cleaned version of the PDB as Biopython chains is stored in the CoarseGrainRNA’s chains attribute. This cleaned version has the names of modified residues converted to their canonical parent and non-RNA molecules removed.\n\nforgi17 is compatible with Python 2.7, 3.5 and 3.6, should run on all operating systems where its dependencies are available and has been tested on Linux, Mac and Windows. It makes heavy use of the NumPy23 library to speed-up array-based calculations and also depends on SciPy24, NetworkX25, Biopython12,13, pandas26,27 and appdirs, all available via the Python Package Index (PyPi) or Anaconda.\n\nHelpful utility scripts. forgi comes with the two very useful scripts: rnaConvert.py can be used to convert between many common file formats for RNA structures, including the Vienna format and fasta variants, the bpseq format, the connectivity table (ct) format, MMCIF format and PDB files. visualize_rna.py can be used to display a coarse-grained representation of an RNA’s secondary structure in PyMol alongside the all atom structure from PDB files, producing visualizations like those in Figure 3 and Figure 5.\n\nAnalysis of stacking geometries. To illustrate how the forgi library makes secondary structure elements usable for 3D structure analysis, we used it to analyze the stacking of adjacent helices in multi loops and pseudo knots in a representative set of RNA 3D structures28 (version 3.36, available at http://rna.bgsu.edu/rna3dhub/nrlist/).\n\nWhile loading these 3D structures into forgi, DSSR7 (Version v1.7.1-2017nov01) was called to obtain the secondary structure and nucleotide level reference stacking annotations. We count a pair of connected helices as stacking, if at least one nucleotide of the first helix’s closing/opening base pair is in a continuous stack with at least one nucleotide of the second helix’s opening/closing base pair. This allows for any number of stacking nucleotides between the stems (not necessarily connected via the backbone) and for bulged out nucleotides which do not contribute to the stack. Our definition of stacking is more relaxed than stricter criteria used elsewhere29.\n\nFor each pair of adjacent stems within a junction, we used forgi to calculate a number of properties, such as the angle between the stem vectors, the separation vector between the stems’ ends and an offset calculated as distance between two rays that start at the helix end and extend the helix axis.\n\nThe following code is a simplified example how forgi can be used to calculate such properties:\n\n\n\n\n\n\n\n\n\n\n\nWe then used pandas26,27, Matplotlib30 and a custom library (https://github.com/Bernhard10/filterAndView) to analyze and visualize the collected data. The results were collected for different classes of RNA independently. This was necessary, because the representative sets of RNA structures contain, by design, homologs of the same molecule in multiple species.\n\nFor the classification of pseudo knots, Reidys’ concept31 based on the definition of the mathematical genus is used. Classes of pseudo knots are defined based on their shadow representations, which contain only crossing base pairs and only one base pair each. On the level of these shadows, only four distinct classes exhibit genus 1, two of which are well known: the H-type pseudo knot and the kissing hairpin. pseudo knots with higher genus contain, among others, the case where a genus 1 pseudo knot is nested within another pseudo knot.\n\nIn combination with the forgi library, we wrote a tool that is able to convert structures to their shadow representation, identify and classify the pseudo knots within the structure and describe their helix arrangement in the 3D structure. This tool, called pseudoknot_analyzer.py, is distributed with the forgi library in the folder “examples”. We used it to gather statistics about simple H-type and kissing hairpin pseudo knots. Figure 4a, b illustrates how we measured the angle between stems in pseudo knots via vector directions. In kissing hairpins the angles α and β restrict the possible values of the angle γ. We also include the representative structure of an intermolecular kissing hairpin interaction (lacking the green connection in Figure 4b) in our analysis. In this special case α and β are indistinguishable and were assigned arbitrarily.\n\nWe used the helix-centered representation of the 3D structure to analyze the geometry of coaxially stacking helices. The relative geometry of two cylinders in 3D space can be described by five parameters: A separation vector (three parameters) and two angles for the relative orientation in 3D space. In Figure 2 and Figure 4, we show the single angle calculated between the vectors along the helix axes, as a proxy for these parameters.\n\n(a) Angles close to 0° mean parallel stems whereas angles close to 180° correspond to potentially stacking stems. Angle distributions in (b) tRNAs and (c) ribosomal RNA are shown as a histogram mapped onto a circular plot illustrating the angles. The (inner) blue bars are instances where DSSR detects stacking on the atom-level scale. The orange bars start at the top of the blue bars and indicate geometries where DSSR does not detect stacking.\n\nGreen cylinders were fitted to stems, blue cylinders represent hairpins and the pink cylinder the unpaired nucleotides at the 3’ end. Red cylinders connect stems and indicate single-stranded connections between these stems (multi loop segments). Left: The stems of the anticodon arm (top) and the D-arm stack (according to DSSR) despite being at an angle of 143°. Right: View along the axis of the anticodon arm’s stem. The red nucleotide is the unpaired multi loop segment which mediates stacking to the stem of the D-arm. This illustration was generated using PyMol33 via the visualize_rna.py wrapper in forgi.\n\n(a) and (b) show the vector directions used for the angle measurement. The distributions of the angles between adjacent stems building c) an H-type pseudo knot or (d) a kissing hairpin are shown as histograms mapped to a circle. Furthermore, the distribution of angles between the regular stems of kissing hairpin pseudo knots is shown in the second panel of (d). Like mentioned in Figure 2, the angles in c) measured between stacking stems (detected via DSSR) are colored in blue. Geometries without stacking are colored in orange. The color scheme in (d) (shades of orange, blue and green) refer to the respective vectors/measured angles (α, β, γ) in the same color scheme as in (b). Blue bars stand for stacking between the two related stems, whereas orange ones for non-stacking stems. Dark green bars in the second kissing hairpin associated histogram show angles measured between a intramolecular interaction (kissing hairpin pseudo knot), whereas light green bars stand for angles measured between two RNA chains (kissing hairpin interaction).\n\nExamples of coaxial stacking within (a) an H-type pseudo knot (PDB id 2XD0) and (b-d) the three major structural families of kissing hairpins (PDB ids 5KPY, 4FRN and 1ZCI, from left to right) In all four representations the green cylinders were fitted to stems, the turquoise and pink cylinder represent the unpaired nucleotides at the 5’ and 3’ end respectively. Red cylinders connect stems and indicate single-stranded connections between these stems (pseudo knot or multi loop segments). The light-colored stems in (b)–(d) represent the regular stems of kissing hairpins, whereas the kissing stem is colored yellow. Note that panel (d) (PDB id 1ZCI) shows a kissing hairpin interaction between two RNA chains. Below the 3-dimensional representation of the kissing hairpins, we show a schematic sketch of the structural family’s helix arrangement. These illustrations were generated using PyMol33 via the visualize_rna.py wrapper in forgi.\n\nThe distribution of angles between adjacent stems in tRNAs multi loops (see Figure 2a) shows the expected bimodal distribution with one mode slightly below 90° and another mode between 160° and 170°, which fits to the known helix arrangement in the L-shaped tRNA. As confirmed by comparison to annotations with DSSR, the second peak is almost exclusively due to stacking helices, even at angles as small as 140°. In Figure 3 we show an example of such a coaxial stack that has been strongly bent (possibly by the tRNA synthetase) without completely breaking the stack or affecting the canonical tRNA secondary structure.\n\nThe second family of RNA molecules with lots of data available is ribosomal RNA. Here we find that the angle of adjacent stems in multi loops is almost uniformly distributed above 20° until a peak from 135° to 170°, where DSSR annotates roughly half of the geometries as stacking (see Figure 2c). 7% of the stacking geometries and 67% of the non-stacking geometries with an angle above 140° also have an offset above 10Å between the extended stem axes.\n\nAdditionally we analyzed the angle distribution between the stems forming simple H-type pseudo knots or kissing hairpins. Most angles measured within an H-type pseudo knot are between 120° and 180° (see Figure 4c). In this range, 35 out of 67 instances correspond to stacking. One example within this range is shown in Figure 5a, which represents a processed non-coding RNA, which regulates a bacterial antiviral system (PDB id 2XD034).\n\nThe distribution illustrated in Figure 4d shows mainly values between 130° and 180° for the angles β and especially α. One additional angle measurement between the two regular stems (see Figure 4d, angle γ) shows one peak at about 20° and one at about 65°. Within the class of kissing hairpins we often find coaxial stacking, but most of the time only one of the two regular stems stacks with the kissing stem in the middle. With the help of the coarse grained representation we were able to divide the class of kissing hairpins into three different structural families (see Figure 5b-d).\n\nThe first family is especially common among (A-)riboswitches. Here the two regular stems are oriented almost parallel (γ below to 32°) with the second one (counting from the 5’ end) stacking onto the kissing stem. The angles α and β are above 130°. One example is the structure with PDB id 5KPY35 shown in Figure 5b. Here, the link from the first regular stem stacks onto it and interacts with the kissing stem via base multiplets and A-Minor interactions. This way, the roughly parallel orientation of all 3 stems is stabilized by stacking and base-pairing interactions. Interestingly, the kissing stem is more parallel to the first stem than to the second stem, onto which it stacks directly (α > β).\n\nThe second structural family is shown in Figure 5c. Here the two regular stems are close to 90°, whereas the kissing stem is along the arc between them. This conformation is found in several groups of non coding RNA including some 23S ribosomal RNA and the cobalamin riboswitch regulatory element (PDB id 4FRN36) shown in Figure 5c.\n\nThe two families as well as some conformations in between are observable within intramolecular pseudo knots. As mentioned, forgi is also able to model multiple RNA chains that interact with each other forming an intermolecular complex. One example is the dimerization initiation site of the HIV type 1 (PDB id 1ZCI37), illustrated in Figure 5d and being the only example of the third family of kissing hairpin pseudo knots. Here the kissing hairpin interaction shows a nearly perfect coaxial stacking between all three involved stems. In this case β is close to 0°, because stacking takes place at the other side of the kissing stem and thus the complementary angle is close to 180°.\n\n\nDiscussion\n\nUsing the forgi library17 we have analyzed the relative orientation of helices in junctions and pseudo knots. While stacking between adjacent helices is common, we found that their orientation often deviates significantly from co-linearity, suggesting that junctions introduce flexibility into the RNA structure while still maintaining the benefit of stacking interactions. Previous studies that assign coaxial geometries based on manual inspection38 miss this deviation from coaxiality that becomes apparent once you actually calculate the helix axis. Our approach allowed us to perform this analysis on the scale of stems, as opposed to the smaller all-atom scale used by the program DSSR and in previous surveys39 or the level of networks of (noncanonical) base pairs38.\n\nConversely, we found that out of 1257 pairs of adjacent stems in ribosomal RNA structures with an angle above 140°, only roughly half (608) are annotated as stacking by DSSR. This becomes clear if we consider that the angle between stems is only a proxy for a 5-dimensional orientation parameter. In particular, a large offset means that stem axes with an angle close to 180° can be parallel without being coaxially stacked. Indeed, it is not uncommon that all three stems in a 3-way junction are roughly parallel, with two stems forming a coaxial stack and the third having a higher offset40.\n\nThere is growing interest in predicting pseudo knots in RNA structures as they are involved in a variety of biological functions41. The forgi library allows us to easily identify pseudo knots in RNA 3D structures and gather statistics on the frequency of pseudo knot types, sizes, and composition. Kissing hairpins formed between two RNA molecules are known to often form continuous stacks between all three helices, such as the pseudo knot in Figure 5d. In contrast to these intermolecular kissing hairpins, we find that stacking between all three helices is seldom possible in intramolecular pseudo knots. Instead we find that the limited length of all loop segments makes it nearly impossible to form coaxial stacking of all three stems in one line in a single RNA chain. Thus, in the main families of kissing hairpin conformations, the regular (outer) stems are oriented parallelly or almost perpendicular with the kissing helix stacking onto at most one of the two regular stems. These structures are often further stabilized by base triplets like those found in Figure 5b.\n\nSimilar to stacking conformations in multi loops, many stacking helices in pseudo knots form angles below 180°. A deviation of the coaxiality between stems can have multiple reasons such as a higher flexibility of short helices within a pseudo knot as well as strain from short stem connections. But there are also structures that show more classical coaxial stacking like PDB id 2XD0, see Figure 5a.\n\nResults and challenges of the application of forgi to the analysis of pseudo knots are described in more detail in the thesis by Beckmann42.\n\nVarying additional parameters like the minimal number of base pairs required to count an interaction as a helix allow for a better understanding about the principles of the formation of three-dimensional RNA structures. The insight in the world of pseudo knots and multi loops presented here can support the improvement of the prediction of RNA structures and help identify unrealistic multi-loop conformations predicted by current RNA structures prediction tools. We are now in the process of implementing additional features for the RNA 3D structure prediction program Ernwin20 based on our findings about stacking and pseudo knots.\n\n\nConclusions\n\nforgi17 is a multi-purpose Python library for dealing with RNA on the levels of sequence, secondary structure and tertiary structure. By providing our code as a Python library, we give users of our tool more flexibility than a single executable program could provide. Furthermore, our code is fully open source, giving researchers the possibility to comprehend and where needed extend the inner workings of the code.\n\nforgi is well documented, with a tutorial available at https://ViennaRNA.github.io/forgi/ and a complete API documentation following the Python standard of docstrings parsable by Sphinx. It contains an extensive automatic test suite (unit and integration tests) and has been optimized for speed, maintainability and usefulness.\n\n\nData availability\n\nThe PDB ids analyzed were taken from version 3.36 of the representative sets of RNA 3D structures28 at a cutoff of 4 Å, http://rna.bgsu.edu/rna3dhub/nrlist/release/3.36/4.0A.\n\nOpen Science Framework: 3D based on 2D: forgi 2.0 Extended Data. https://doi.org/10.17605/OSF.IO/HDJRU43. This project contains the following extended data files:\n\nassignment_of_classes_to_pdbids.csv: Assignment of PDB ids to RNA type. These annotations were generated in a semi-manual way.\n\nmultiloop_angles.csv: Angles and offsets measured in PDB structures for multi loops, generated with the script describe_rna.py, which is available in the examples folder of forgi.\n\npseudoknot_angles.tsv: Angles measured in pseudoknots, generated with pseudoknot_analyzer.py, which is available in the examples folder of forgi.\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSoftware available from: https://pypi.org/project/forgi/ (pip install forgi) and Bioconda.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.258287017.\n\nLicense: GNU General Public License 3.0.",
"appendix": "Grant information\n\nThis work was partly funded by the Austrian science fund (FWF) projects F 43 “RNA regulation of the transcriptome” and I 2874 “Prediction of RNA-RNA interactions” and Doctoral College W 1207 “RNA Biology”.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank all contributors, who submitted a pull-request or filed an issue in the forgi repository on github. We thank Roman Ochsenreiter for proofreading, Gregor Entzian for useful inputs about many quirks in the PDB format and for proofreading and Lorena Pantano for creating the first version of a Bioconda recipe for forgi.\n\n\nReferences\n\nDawson WK, Maciejczyk M, Jankowska EJ, et al.: Coarse-grained modeling of RNA 3D structure. Methods. 2016; 103: 138–156. 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Master’s thesis, University of Vienna, 2018. Reference Source\n\nThiel BC, Beckmann IK, Kerpedjiev P, et al.: 3D based on 2D: Forgi 2.0 Extended Data. 2019. https://www.doi.org/10.17605/OSF.IO/HDJRU"
}
|
[
{
"id": "45753",
"date": "27 Mar 2019",
"name": "Marta Szachniuk",
"expertise": [
"Reviewer Expertise Structural bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is written in a clear and concise way. Illustrations perfectly complement the content. I have only 3 comments to the authors and I would like them to be taken into account in the revision:\nFor unknown reasons, the authors introduced a new spelling of the name pseudoknot. Throughout the whole article, they use spelling with space: \"pseudo knots\". I haven't met such a form before. The authors of the article also used the term \"pseudoknot\" and not \"pseudo knot\" in their previous works. So, the spelling should be corrected. On page 3, the authors write about FR3D suite, but they do not cite the paper about the tool. Please, include the appropriate citation here. As the paper subject is calculating helix angles and stacking parameters, I would expect to see the formulas used to calculate these data (or - at least - the pseudocode showing how they are calculated). The lack of such details is serious negligence.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4529",
"date": "11 Apr 2019",
"name": "Bernhard Thiel",
"role": "Author Response",
"response": "Thank you for the constructive comments.We have now included the exact formulas for the helix angles as requested. In particular this makes clear what sign we use for the helix vectors.The FR3D reference was present in our LaTeX source and the resulting HTML version, but got lost in the pdf conversion. This has been fixed in the revised version."
}
]
},
{
"id": "45731",
"date": "27 Mar 2019",
"name": "Jerome Waldispühl",
"expertise": [
"Reviewer Expertise RNA bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a new version of the forgi software, a python library to analyze the tertiary structure of secondary structure elements in RNA. It takes as input almost any format of 2D or 3D information.\nThis manuscript describes a new method for easily obtaining the angles and stacking patterns in a multi-branched loop and a pseudoknot/kissing hairpin. The authors validate their results by testing the software on known pseudo-knots and junctions, and obtain results that are consistent with the state-of-the-art knowledge of RNA local structure organization.\nThe authors highlight significant improvements compared to previously published packages and specifically all-atom based methods. For instance, a deviation from coaxiality is very common but can be easily missed if a manual inspection is performed without doing computations.\nThe software is clearly presented, well-validated, freely available and open source. The results presented are significant and the tool will be very useful to the RNA bioinformatics community.\nAlthough, this manuscript does not discuss extensively the analysis of the results, the latter is included in the first author’s thesis, and freely accessible as further reading. ` Minor remarks\nPage 4: ‘’with and without missing residues’’, it is easy to imagine how the graph without the missing residue is, but how are the ‘’missing residues’’ added? (i.e. are you inferring which residues are missing, and if yes, how?) Page 5: The authors mention a ‘’cleaned version’’ of the PDB. Is the only ‘’cleaning’’ performed is the conversion of the residue names? Page 5: It would be helpful to clarify what is a ‘’simplified example’’ of the code? What sort of simplification was applied to it? Is the real code significantly more complicated to use than the example? If it is not, the authors might want to consider rewording this as it appears to undermine the real simplicity of use of forgi (which is very user friendly).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4530",
"date": "11 Apr 2019",
"name": "Bernhard Thiel",
"role": "Author Response",
"response": "Thank you for the constructive comments.Our PDB and CIF parsers extract the information about missing residues from the files. For this purpose we have patched the biopython PDB parsing routine; this has already been merged into the biopython source. Following your input, we have revised the text to reflect this.Yes, the cleaned version only has modified residues renamed and non-rna molecules removed. We hope this is now clear from the revised text.We have expanded the sample code to compute the proper direction of helices, thus it is now fully functional and no longer \"simplified\".We have also added the code used to calculate the offset."
}
]
}
] | 1
|
https://f1000research.com/articles/8-287
|
https://f1000research.com/articles/8-509/v1
|
17 Apr 19
|
{
"type": "Case Report",
"title": "Case Report: Culture negative cutaneous tuberculosis",
"authors": [
"Maria Qadri",
"Qurban Hussain Sheikh",
"Mir Tahir Hussain Talpur",
"Uzair Yaqoob",
"Khalil Ullah Shabbir",
"Maria Qadri",
"Qurban Hussain Sheikh",
"Mir Tahir Hussain Talpur",
"Khalil Ullah Shabbir"
],
"abstract": "Cutaneous tuberculosis (TB) can present in a number of ways, making it difficult to diagnose. It most commonly presents as scrofuloderma, which commonly affects the supra-clavicular region, axilla and the cervical region. All the different presentations of cutaneous TB should be known to clinicians, in order to diagnose it early. The objective of this article is to describe a case of scrofuloderma presenting with different cutaneous lesions at the same time, which were culture negative. We present a 23-year-old male with no known co-morbidities, presenting to us with fever and multiple swellings on the body. Cultures of pus and blood were negative for TB; GeneXpert detected the microorganism. Cutaneous TB, although a rare disease with wide spectrum of cutaneous lesions, should be considered in differential diagnosis of cold abscesses and nodules, especially of the head and neck region.",
"keywords": [
"Tuberculosis",
"Scrofuloderma",
"Cutaneous Tuberculosis",
"Cold Abscesses"
],
"content": "Introduction\n\nTuberculosis (TB) is a systemic illness caused by a rod-shaped bacillus, Mycobacterium tuberculosis. It is usually pulmonary. Cutaneous TB constitutes 1–2% of extrapulmonary TB and 0.15% of skin diseases; this is significant, considering the overall prevalence of TB is 2% per year1–4.\n\nCutaneous TB can present in a number of ways clinically, during histopathology as well as in treatment response, making it difficult to diagnose. These cases should be recognized early for timely and accurate management4,5. Cutaneous TB most commonly presents as Tuberculosis cutis colliquativa (also known as scrofuloderma), a type of cutaneous TB presenting with cold abscesses and commonly affecting the supraclavicular region, axilla, and the cervical region6. Lupus vulgaris (LV) is another less common manifestation of TB7. Some rarer ones include inguinal scrofuloderma, ulcerative type of LV, and acute military cutaneous TB3. Cutaneous TB is usually confined to the skin but can be multifocal8.\n\nHere, we describe a case of scrofuloderma presenting with different cutaneous lesions at the same time, which were culture negative for TB.\n\n\nCase presentation\n\nA 23-year-old male laborer, with no known co-morbidities was admitted to Jinnah Postgraduate Medical Centre, Karachi, Pakistan in March 2018, with complaints of fever for 18 months and multiple swellings on different parts of the body for 12 months.\n\nAccording to the patient, he had developed a fever, which was gradual in onset, low grade (100oF), intermittent, occurring mostly in the evening and relieved by taking Paracetamol. The fever was not associated with rigors or chills. After six months of fever, the patient noticed multiple swellings of variable size on different parts of body, which included the right lower back, left lower base of neck, upper part of middle chest, front of right ear and upper surface of right foot. The largest swelling was over the back. Swelling was gradual in onset, increasing in size, ranging from lemon size (upper surface of foot) to melon size (abdomen), associated with discharge from left lower neck and chest swelling. Discharge was yellowish in color with no blood in it and was not associated with itching or pain.\n\nThe patient had a history of undocumented weight loss for one year and using antipyretics and proton pump inhibitors (Omeprazole, 20mg once daily). The rest of the history was unremarkable.\n\nOn general physical examination, the patient was average height and thinly built, cooperative, with visible parotid swelling on the right side, and lying comfortably on the bed. His vitals were all normal. There was no pallor, icterus, cyanosis, clubbing, koilonychias, splinter hemorrhages, edema, or ear discharge. Oral and thyroid examination was normal.\n\nBilateral anterior cervical and inguinal lymph nodes were palpable, firm in consistency, non-tender, matted, mobile, with no underlying erythema or discharging sinus, not adherent to overlying skin or any underlying structure. Size ranged from 2cm to 4.5cm. Parotid swelling was noted, non-tender, soft in consistency, size ranging from 5cm to 7cm, mobile over underlying structure, with no overlying erythema, sinus or discharge. There was a round, well demarcated lesion on the chest, ulcerated, 3 inches in size, reddish color with a yellowish crust. Right lumbar swelling measured ~3×16cm, soft, non-tender, non-discharging and without any gibbus. There was a scar noted over the left supra-clavicular region, above and parallel to the clavicle. Figure 1 provides images of the patient’s swellings. There was no hyperpigmentation or petechiae noted. Bone tenderness was absent. Systemic examination was found to be unremarkable.\n\nA round, well demarcated lesion on the chest, ulcerated, 3 inches in size, reddish color and with yellowish crust. Right lumbar swelling measured about 13×16cm, soft, non-tender, non-discharging and without any gibbus. A swelling was also noted on the right foot.\n\nThe patient’s laboratory test results are presented in Table 1. Viral markers and HIV testing were negative, while pus for gram staining and Acid-Fast Bacillus staining was also negative. Chest X-ray was unremarkable.\n\nOn ultrasound of the abdomen, the liver was 14cm and spleen was 11.7cm in size with normal borders. A psoas abscess was found measuring 11×11.9cm with septations and calcifications. Fine-needle aspiration cytology of the cheek swelling showed necrotizing inflammation. Fungal stain was negative and skin split test for Leishmania donovani bodies was also negative. Mycobacterium tuberculosis was detected after GeneXpert analysis of the pus from the psoas abscess, which had previously shown no growth on culture. Blood culture was also negative.\n\nComputed tomography of chest with contrast showed multiple fluid collections at right supraclavicular region, anterior chest wall and along with bilateral psoas abscess formation predominantly on right side.\n\nInitially, a differential diagnosis of deep fungal infection (actinomycosis), cutaneous leishmaniasis, lymphoma and TB was made. After GeneXpert, HIV serology, and fungal stain were performed, cutaneous tuberculosis (scrofuloderma) was the final diagnosis and anti-tuberculous therapy was started. After consulting the Dermatological Department, Lupus vulgaris was also ruled out. A standard regimen of four drugs, i.e. Isoniazid (5 mg/kg), Rifampin (10 mg/kg), Ethambutol (20 mg/kg), and Pyrazinamide (24 mg/kg), in combined form, i.e. Tablet Myrin-P-Forte 4 × daily, was given for two months and converted to the combination of Isoniazid and Rifampin in combined form, i.e. Tablet Rifna 4 × daily, for a further 8 months. The patient was discharged after 8 days and kept on regular follow-up.\n\n\nDiscussion\n\nThe incidence of TB is increasing worldwide, which may be due to the growing number of HIV cases and resistance to antituberculous drugs4. Immunocompromised individuals have a greater propensity to get this disease. Cutaneous TB is either transmitted via a hematogenous route or direct extension from a primary focus, which is elsewhere in the body, usually pulmonary. However, primary infection can also occur by direct introduction of the microbe into the skin or mucosa of a susceptible individual by trauma or injury4.\n\nIn our case, no primary source was found in any organs and there was no history of TB contact. Moreover, there was no reason to identify the patient as immunocompromised.\n\nCutaneous TB rarely involves the region of head and neck, especially lymph nodes, larynx, oropharynx, salivary glands, nose, ear, skin, and paranasal sinuses9. Our patient had bilateral cervical and inguinal lymphadenopathy, parotid swelling, an ulcerated lesion on the chest, a right lumbar swelling, and a scar on the left supra-clavicular region. This distribution also favored our final diagnosis of scrofuloderma.\n\nDue to the different type of lesions, correct diagnosis may be delayed because investigations fail to detect TB, as in our case where cultures were negative and TB was only detected by GeneXpert10.\n\n\nConclusion\n\nOur case illustrates that scrofuloderma, although a rare disease with a wide spectrum of cutaneous lesions and higher rates of false negative investigations, should still be considered in differential diagnosis of cold abscesses and nodules, especially of the head and neck region.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nTuberculosis Key Facts. World Health Organization. 2018; Accessed: August 28, 2017. Reference Source\n\nSethi A: Tuberculosis and infections with atypical Mycobacteria. In: Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffel DJ, editors. Fitzpatrick’s Dermatology in General Medicine. 8th ed. New York, NY: McGraw Hill Medical; 2012; 3164–3189. Reference Source\n\nSantos JB, Figueiredo AR, Ferraz CE, et al.: Cutaneous tuberculosis: epidemiologic, etiopathogenic and clinical aspects - part I. An Bras Dermatol. 2014; 89(2): 219–228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPacheco C, Silva E, Miranda J, et al.: Cutaneous tuberculosis as metastatic tuberculous abscess. J Bras Pneumol. 2015; 41(2): 200–202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGunawan H, Achdiat PA, Hindritiani R, et al.: Various cutaneous tuberculosis with rare clinical manifestations: A case series. Int J Mycobacteriol. 2018; 7(3): 288–291. PubMed Abstract | Publisher Full Text\n\nHo SC: Cutaneous tuberculosis: Clinical features, diagnosis, and management. HK Dermatol Venereol Bull. 2003; 11: 130–138. Reference Source\n\nPai VV, Naveen KN, Athanikar SB, et al.: A clinico-histopathological study of lupus vulgaris: A 3 year experience at a tertiary care centre. Indian Dermatol Online J. 2014; 5(4): 461–465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmraoui N, Krich S, Meziane M, et al.: Cutaneous tuberculosis revealing multifocal tuberculosis in immunocompetent patients. Int J Mycobacteriol. 2015; 4(3): 255–257. PubMed Abstract | Publisher Full Text\n\nBruzgielewicz A, Rzepakowska A, Osuch-Wójcikewicz E, et al.: Tuberculosis of the head and neck - epidemiological and clinical presentation. Arch Med Sci. 2014; 10(6): 1160–1166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaase O, von Thomsen AJ, Zillikens D, et al.: Recurrent abscesses of the neck: scrofuloderma. JAMA Dermatol. 2014; 150(8): 909–910. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "48990",
"date": "04 Jun 2019",
"name": "Vanessa Lucília Silveira de Medeiros",
"expertise": [
"Reviewer Expertise Tuberculosisis",
"leishmaniasis",
"psoriasis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nScrofuloderma itself is not uncommon in countries endemic for TB. The main reason for the presentation of the case is the multiplicity of abscesses, a rare presentation of the scrofuloderma. Some sentences need to be better built and emphasize this point: “a type of cutaneous TB presenting with solitary or few cold abscesses and commonly affecting the supraclavicular region, axilla, and the cervical region6. Here, we describe a case of scrofuloderma with an uncommon presentation (presenting ) with many cutaneous lesions at the same time, which were culture negative for TB7.\"\n\nThe authors contradict themselves when writing in the introduction “Cutaneous TB most commonly presents as Tuberculosis cutis colliquativa (also known as scrofuloderma), a type of cutaneous TB presenting with cold abscesses and commonly affecting the supraclavicular region” and then in the discussion “Cutaneous TB rarely involves the region of head and neck, especially lymph nodes, larynx, oropharynx, salivary glands, nose, ear, skin, and paranasal sinuses”. These sentences should be rewritten or remove the phrase from the discussion.\n\nRewrite the phrase \"Our case illustrates that scrofuloderma, although a rare disease with a wide spectrum of cutaneous lesions and higher rates of false negative investigations, should still be considered in differential diagnosis of cold abscesses and nodules, especially of the head and neck region…” There is no “wide spectrum of cutaneous lesions” but rather different locations of lesions, some rare as the lesion of the chest and foot.\n\nClarify the phrase “After GeneXpert, HIV serology, and fungal stain were performed, cutaneous tuberculosis (scrofuloderma) was the final diagnosis and anti-tuberculous therapy was started.\" Did you investigate HIV, fungus only after the TB diagnosis? There was no emphasis in the text that HIV serology was negative.\nThe following phrases should be deleted from the text:\n\n“Cutaneous TB can present in a number of ways clinically, during histopathology as well as in treatment response, making it difficult to diagnose. These cases should be recognized early for timely and accurate management4,5\". “Lupus vulgaris (LV) is another less common manifestation of TB7”. “After consulting the Dermatological Department, Lupus vulgaris was also ruled out.“ “Moreover, there was no reason to identify the patient as immunocompromised.” “Some rarer ones include inguinal scrofuloderma, ulcerative type of LV, and acute military cutaneous TB3 . Cutaneous TB is usually confined to the skin but can be multifocal8”.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "52560",
"date": "06 Sep 2019",
"name": "Rita V. Vora",
"expertise": [
"Reviewer Expertise Dermatology and Leprosy."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author wants to discuss cutaneous tuberculosis, but the case is not fitting in any spectrum of cutaneous tuberculosis.\n\nIrrelevant case history has been mentioned without diagnostic features of cutaneous tuberculosis.\n\nHistopathology of any lesions has not been mentioned.\n\nThere is more description of cold abscess rather than cutaneous tuberculosis.\n\nIdeally culture for AFB is positive in hardly 5% cases of cutaneous tuberculosis. So what is the meaning of given title and conclusion?\n\nThe case looks like extrapulmonary tuberculosis, rather than cutaneous tuberculosis.\n\nThe morphology of cutaneous lesions is not fitting in description of cutaneous tuberculosis.\nSo, with this title and description, this manuscript is not acceptable for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-509
|
https://f1000research.com/articles/8-507/v1
|
17 Apr 19
|
{
"type": "Case Report",
"title": "Case Report: Favoring biventricular repair beyond the conventional boundaries with primary arterial switch operation in a young adult",
"authors": [
"Sumit Agasty",
"Sangdup Tsering",
"P Ramesh Menon",
"Sandeep Chauhan",
"Akshay Kumar Bisoi",
"Sumit Agasty",
"Sangdup Tsering",
"Sandeep Chauhan",
"Akshay Kumar Bisoi"
],
"abstract": "Transposition of great arteries (TGA) presents in neonates or in infancy. We report a case of TGA with ventricular septal defect (VSD) and pulmonary stenosis (PS) in an adult male patient of 23 years age. Arterial switch operation with VSD closure and neo-aortic valve replacement was done. The patient recovered well in the post-operative period. In adult patients, conversion from atrial to arterial switch has been widely reported, both directly and after prior pulmonary artery banding in two stages, but primary arterial switch for TGA has not been reported previously. In this patient there was a benefit of having a large VSD and severe PS.",
"keywords": [
"Grown Up Congenital Heart disease",
"arterial switch operation"
],
"content": "Introduction\n\nTransposition of great arteries (TGA) presents in neonates or in infancy1. Recently, we managed an adult patient with cyanotic congenital heart disease [TGA with sub pulmonic ventricular septal defect (VSD) with severe pulmonary stenosis (PS)] with complete biventricular repair by performing an arterial switch operation.\n\n\nCase report\n\nA 23-year-old man presented with complaints of bluish discoloration of skin and nails from childhood, associated with frequent attacks of pneumonia and dyspnea on exertion, off and on from school going age. He was deeply cyanosed and had grade 3 clubbing. Pulse oximetry showed 76% saturation in room air, not improving on oxygen supplementation. Auscultation revealed an ejection systolic murmur over the upper left sternal border, and normal heart sounds. Bilateral vesicular breath sounds were heard equally, and the abdomen was soft with no palpable organomegaly.\n\nEchocardiography revealed ventriculoarterial discordance: aorta arising from right ventricle (RV) and pulmonary artery (PA) from left ventricle (LV); aorta found to the right and anterior to PA; evidence of severe valvular PS (gradient-83 mm Hg), with large sub-pulmonic VSD; normal biventricular function. The initial plan of management was for Rastelli procedure with valved conduit, and the patient was planned for cardiac catheterization study which suggested 92% PA saturation and 66% femoral artery saturation, PA pressure of 73/31 mm Hg and PVRI of 7.9 wood units, pulmonary flow Qp 4.3, systemic flow Qs 5.3, Qp/Qs-0.8. LV angiography suggested large S/A routable VSD, no additional VSD, and confluent PAs. Prior to surgery, an echocardiogram was repeated and the patient reassessed: VSD was found to be non-routable, with normal biventricular function. Therefore, the patient was planned for arterial switch operation (ASO) with VSD closure and pulmonary valve replacement.\n\nSurgical correction was performed via median sternotomy, on cardiopulmonary bypass using high ascending aortic and bicaval cannulation under mild hypothermia. Cold blood cardioplegia (Delnido) with no topical cooling was used.\n\nPeroperative findings were as follows: aorta was present anterior to and to the right of PA and arising from RV (anterior ventricle) and PA from LV (posterior ventricle); moderate sized sub pulmonic VSD; pulmonic valve was bicuspid with calcification; no atrial septal defect (ASD).\n\nThe PAs were dissected up to the lung hila after division of the ductus arteriosus. The aorta was cross-clamped, cardioplegia given to the aortic root, and the aorta transected. Coronary buttons were harvested from the aortic sinuses together with a variable amount of sinus wall using minimum dissection. The main PA was transected 3 to 4 mm below the bifurcation. Bicuspid and calcific cusps excised and valved sized and seated with 17 MM SJM Regent valve. One of the pulmonary valve cusps had a thrombus, which was completely removed. This was causing an outlet obstruction for the systemic ventricle (future LV) at the valvular level. The coronary buttons were re-implanted into the neo-aortic sinus rather than above the sino-tubular junction. Trans aortic PTFE patch VSD closure was done. Neo-aortic anastomosis was completed after LeCompte maneuver. Right atrium opened to check for ASD, with no ASD was present. Autogenous fixed pericardial patch reconstruction of the neo-pulmonary root was done. Aortic cross clamp was released, root vented and neo-pulmonary anastomosis was carried out during rewarming phase. Patient was weaned off from bypass. Aortic decannulation was done after giving protamine (single dose of 1.5mg/kg in order to neutralize Heparin given at 1mg/kg).\n\nPostoperative recovery was uneventful in the ICU. Inotropes (Dobutamine at 10mic/kg/min slowly tapered and stopped within 24hours), antibiotics (Inj. Cefotaxime 1g twice a day for first 24hrs postoperatively) and supplements (nutrition) were followed as per unit policy. On the third postoperative day, with active mobilization, the patient had complaints of occasional dizziness and headache. Detailed ENT and neurological evaluation showed no anatomical substrate for his symptoms. His symptoms gradually subsided over a period of few days.\n\n\nDiscussion\n\nThe conventional age boundaries for cardiac surgery for complex cyanotic cases have been pushed previously2. We report a singular experience of a 23-year-old young adult with congenital TGA (with VSD and PS) who successfully underwent a complete repair – ASO with transaortic VSD closure with neo-aortic valve replacement.\n\nWhereas most case reports of ASO in adults3–5 prior to ours have reported cases of converting and atrial switch to arterial switch operation after prior LV retraining as a two stage ASO, we report a successful primary arterial switch in this adult patient.\n\nMassimo et al. in 20003 reported a case (Table 1) with moderate PA hypertension and preserved LV function operated for ASO. In their case, moderate pulmonary hypertension played a role in preserving some degree of LV hypertrophy. Di Donato6 reviewed the literature on ASO in older children and adults post PA banding. The longer the morphological LV works at low pulmonary pressure, the lower is the capability of inducing LV hypertrophy by PA banding. Hence the mature myocardium may have poor response to pressure overload.\n\nPA, pulmonary artery.\n\nOur patient had good LV morphology and function due to the presence of two favorable factors – a large VSD and presence of severe PS that provided afterload to morphologic LV, which is otherwise not achieved in cases without severe PS. Thus, these two factors ensured that a primary ASO with VSD closure and neo-aortic valve replacement was adequate in this young man.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the report of his case.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMartins P, Castela E: Transposition of the great arteries. Orphanet J Rare Dis. 2008; 3: 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBisoi AK, Sharma P, Chauhan S, et al.: Primary arterial switch operation in children presenting late with d-transposition of great arteries and intact ventricular septum. When is it too late for a primary arterial switch operation? Eur J Cardiothorac Surg. 2010; 38(6): 707–13. PubMed Abstract | Publisher Full Text\n\nPadalino MA, Stellin G, Brawn WJ, et al.: Arterial switch operation after left ventricular retraining in the adult. Ann Thorac Surg. 2000; 70(5): 1753–1757. PubMed Abstract | Publisher Full Text\n\nCochrane AD, Karl TR, Mee RB, et al.: Staged conversion to arterial switch for late failure of the systemic right ventricle. Ann Thorac Surg. 1993; 56(4): 854–61; discussion 861–2. PubMed Abstract | Publisher Full Text\n\nde Jong PL, Bogers AJ, Witsenburg M, et al.: Arterial switch for pulmonary venous obstruction complicating Mustard procedure. Ann Thorac Surg. 1995; 59(4): 1005–7. PubMed Abstract | Publisher Full Text\n\nDi Donato RM, Fujii AM, Jonas RA, et al.: Age-dependent ventricular response to pressure overload. Considerations for the arterial switch operation. J Thorac Cardiovasc Surg. 1992; 104(3): 713–22. PubMed Abstract"
}
|
[
{
"id": "47423",
"date": "04 May 2020",
"name": "Marc M. Anders",
"expertise": [
"Reviewer Expertise congenital heart disease",
"mechanical support",
"ecmo",
"pediatric heart failure",
"anticoagulation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author should expand on the preop cath findings, the degree and location of the ps. pulsoximetry findings in the context of the pda - as noted later.\n\nThe perioperative monitoring especially in a patient with severe PHT should be highlighted and discussed in further detail.\n\nThe discussion should refer to the other case reports too.\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? Partly",
"responses": []
},
{
"id": "79357",
"date": "12 Feb 2021",
"name": "Ali Hosseinsabet",
"expertise": [
"Reviewer Expertise echocardiography"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUnfortunately the catherization and echocardiographic data presented is incomplete and without more explanations.\n\nI expected that you present more clinical data after surgery and follow up data. In addition, echocardiography movies and pictures and catheterization figures is required.\n\nYour discussion should be more detailed regarding the messages of this case and importance and novelty of this case lessons should be learned.\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? 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/8-507
|
https://f1000research.com/articles/7-702/v1
|
05 Jun 18
|
{
"type": "Research Note",
"title": "Investigation of gut microbiome association with inflammatory bowel disease and depression: a machine learning approach",
"authors": [
"Pedro Morell Miranda",
"Francesca Bertolini",
"Haja N. Kadarmideen",
"Pedro Morell Miranda",
"Francesca Bertolini"
],
"abstract": "Background: Inflammatory bowel disease (IBD) is a group of chronic diseases related to inflammatory processes in the digestive tract generally associated with an immune response to an altered gut microbiome in genetically predisposed subjects. For years, both researchers and clinicians have been reporting increased rates of anxiety and depression disorders in IBD, and these disorders have also been linked to an altered microbiome. However, the underlying pathophysiological mechanisms of comorbidity are poorly understood at the gut microbiome level. Methods: Metagenomic and metatranscriptomic data were retrieved from the Inflammatory Bowel Disease Multi-Omics Database. Samples from 70 individuals that had answered to a self-reported depression and anxiety questionnaire were selected and classified by their IBD diagnosis and their questionnaire results, creating six different groups. The cross-validation random forest algorithm was used in 90% of the individuals (training set) to retain the most important species involved in discriminating the samples without losing predictive power. The validation set that represented the remaining 10% of the samples equally distributed across the six groups was used to train a random forest using only the species selected in order to evaluate their predictive power. Results: A total of 24 species were identified as the most informative in discriminating the 6 groups. Several of these species were frequently described in dysbiosis cases, such as species from the genus Bacteroides and Faecalibacterium prausnitzii. Despite the different compositions among the groups, no common patterns were found between samples classified as depressed. However, distinct taxonomic profiles within patients of IBD depending on their depression status were detected. Conclusions: The machine learning approach is a promising approach for investigating the role of microbiome in IBD and depression. Abundance and functional changes in these species suggest that depression should be considered as a factor in future research on IBD.",
"keywords": [
"Inflammatory Bowel Disease",
"Depression",
"Microbiome",
"Machine Learning",
"Random Forest",
"Metagenomic",
"Metatranscriptomic."
],
"content": "Introduction\n\nIncreased depression rates have been frequently reported on patients with inflammatory bowel disease (IBD) (Graff et al., 2009), which is a big concern from a clinical standpoint, since increased levels of stress and anxiety are major drivers of IBD relapse and severity (Mawdsley & Rampton, 2006). Both IBD and depression are heavily influenced by the gut microbiome structure, which controls anti-inflammatory processes and permeability in the gut, and communicates with the brain by a complex and close relationship with the Autonomous Nervous System that is known as the brain-gut axis (Foster & McVey Neufeld, 2013; Luna & Foster, 2015).\n\nAltered microbiomes can have big impacts on the health and development of both the gut and brain, and alterations in the ecology of this microbiome, a process known as dysbiosis, have been separately linked to both depression and IBD (Kaur et al., 2011; Rogers et al., 2016). However, little is known about the role of the microbiome in the two diseases.\n\nThe availability of the large amount of data derived from the recent explosion in metagenomics and metatranscriptomics provides unique opportunities for investigation. However, it is sometimes difficult to identify informative species. Recently, machine learning algorithms have been successfully applied because they allow the identification of patterns in situations where large, multi-dimensional and heterogeneous datasets are available.\n\nAmong the several machine learning approaches available, random forest is an algorithm used for classification and regression based on an ensemble that builds a population of decision tree classifiers, such that the result of a prediction from a given set of features is the most frequent result from the different trees of the “forest” (Breiman, 2001). This is an efficient and generalist algorithm that has already been applied in several metagenomic investigations in human diseases, such as IBS (Saulnier et al., 2011).\n\nThe aim of this work was to apply the random forest approach to identify the microbiome species that may be mostly involved in IBD and depression outcomes and that are responsible for the most relevant changes in the population structure between IBD, depression and patients comorbid for both conditions, and to provide insights on how the microbiome is involved in this comorbidity.\n\n\nMethods\n\nThe datasets used for the analyses were retrieved from the Inflammatory Bowel Disease Multi-Omics Database (IBDMDB) (Schirmer et al., 2018), which is part of the Integrative Human Microbiome Project (NIH HMP Working Group et al., 2009). The IBDMDB database contains a wide array of omics data (e.g., 16S and shotgun metagenomic, metatranscriptomic, proteomic and host genomes) of 132 individuals classified by IBD diagnostic in ulcerative colitis, Crohn’s disease and controls. Participants provided bi-weekly stool samples at five hospitals in the United States. Metagenomic and metatranscriptomic data was processed as described in Schirmer et al., 2018 (Abubucker et al., 2012; Truong et al., 2015)\n\nFrom this dataset, the 70 unique participants who answered an additional self-reported depression and anxiety questionnaire during registration (the answers to which are listed in the HMP2 metadata, column EC to EL) were selected. As the questionnaire model was not specified, only individuals with raw scores over 6 on this test was considered as showing “signs of depression”. To calculate the raw scores, a severity scale was generated, with the following scores: 0, never; 1, rarely; 2, sometimes; 3, often; 4, always. The scores were then summed to give a final total. In the case of individuals undergoing multiple tests, the lower score was used. We selected a low threshold in order to be able to identify putative dysbiotic individuals that were not experiencing severe depression symptoms. All the others were classified as “no sign of depression”. The combination between the test and the IBD diagnosis divided the dataset in six groups: Crohn’s disease with no detectable sign of depression (CD; n=15), Crohn’s disease with signs of depression (CDD; n=20), ulcerative colitis with no sign of depression (UC; n=4), ulcerative colitis with signs of depression (UCD, n=11), signs of depression but no inflammation (nonIBDD; n=7) and the control group: no inflammation/no depression (nonIBD; n=13).\n\nFor each of the six groups, abundance matrices of the metagenomic data, metatranscriptomic data, and the combination of metagenomics and metatranscriptomics were used for random forest classification. Each of the datasets was divided randomly into a training set (90% of the individuals) and a validation set (10% of the individuals). Random forest analysis were performed using the library Scikit-learn 0.19.1 (Pedregosa et al., 2011) on the training sets to identify the most important species involved in discriminating the samples without losing predicting power. A 1000-fold cross-validation for the combined dataset, and 500-fold for metagenomic and metatranscriptomic data, considering one model for each iteration was performed and only the most important species in the construction of this model was retained. Only models with a precision classification >80% were considered, and among the considered models, only species that appeared more than once were selected. Afterwards, the validation sets were run with the selected species only to measure the possible loss of predictive capability and computed the area under the receiver operating characteristic (auROC) curve for the prediction of the validation set classes as a performance metric.\n\nIn order to assess the significance of the differences between the abundances of the selected species, we performed a one-way ANOVA (Scipy 1.0.0, Jones et al., 2001) with a Tukey’s honest significant difference (HSD) post-hoc test. This test makes pair-wise comparisons between the different means to see which classes are different. For clarity, confidence intervals for Tukey’s HSD test can be found in Supplementary Materials (Supplementary Figure 1 and Supplementary Figure 2).\n\nThe functional activity of the selected species was retrieved from the HUMAnN metatranscriptomic analyses described above. Only the pathways in which the selected species are involved and those that were different between the groups from the ANOVA test were selected and the correlation between these species was calculated using Spearman’s correlation coefficient. A significance level of 0.05 was applied for all statistical tests.\n\n\nResults and discussion\n\nThe random forest cross-validation selection of the most informative species showed a combined list of 24 species, as can be seen in Figure 1. The validation models for DNA, RNA and the combined dataset shows micro-averaged auROC values of 0.96, 0.91 and 0.99, respectively (Supplementary Figure 3–Supplementary Figure 5). This metrics highlight the performance of the model that, even with a reduced subset of species, has not lost predictive power.\n\nAll species exhibited differences in at least one group in a one-way ANOVA (alpha=0.05, Supplementary Table 1), and no significant differences were found between DNA and RNA abundances for these species (Supplementary Table 2).\n\nThe analyses showed an increase in the number of species from the genus Bacteroides in dysbiotic groups compared with the control (nonIBD) (Figure 2), as has been reported in other dysbiotic samples (Bloom et al., 2011), with the exception of Bacteroides dorei, which is more abundant in nonIBD than in any other group. Aside from Bacteroides dorei, nonIBD samples had a higher abundance of Alistipes shahii and Ruminococcus bromii, while a typical species associated with nonIBD, Faecalibacterium prausnitzii, was significantly decreased in nonIBDD and CD.\n\nDNA (A) and RNA (B) taxonomic abundances for the selected species. Abundances were quantified by the relative abundances of their sequences, and for each level they should sum to 1 (including unclassified sequences).\n\nBoth of the Crohn’s disease-related groups (CD and CDD) showed higher abundances of Bacteroides ovatus and Bacteroides uniformis. However, CD samples exhibited higher abundances for several specific species, including Bacteroides xylanisolvens, Parasutterella excrementihominis and Bacteroides fragilis, compared with CDD, but decreased abundance of Faecalibacterium prausnitzii, which did not differ significantly in abundance between nonIBD and CDD groups.\n\nUlcerative colitis samples had the most distinctive microbiome profile. Several species, including Burkholderiales bacterium 1_1_47, Bacteroides eggerthii and Bacteroides finegoldii were characteristic of this group, and absent in the others, except for B. finegoldii, which was also present in a lower abundance in nonIBD samples. Only UCD samples exhibited an increased abundance of Bacteroides fragilis, Bacteroides vulgatus and Haemophilus pittmaniae, this last species being almost exclusive to the UCD group.\n\nThe nonIBDD was the group with the highest number of changes in microbiome diversity when compared with its non-depressed counterpart (Table 1). However, most of those changes followed a similar pattern in other dysbiotic groups.\n\nIncreases/decreases shown are statistically significant.\n\nA notable change was observed in Faecalibacterium prausnitzii, which was present in almost the same abundances in nonIBD, UCD and CDD samples, and a high variability in UC while being significantly lower in CD and nonIBDD (Supplementary Table 3 and Supplementary Table 4). This is particularly interesting, since this species is considered to have anti-inflammatory activity. It seems counterintuitive to find a depleted population of one of the species most associated in the literature with a healthy microbiome compared to an IBD one in a group that doesn’t show any inflammatory process. However, Parabacteroides goldsteinii was increased in nonIBDD and was depleted in all IBD groups in comparison with control samples. The Parabacteroides genre have been associated previously with anti-inflammatory activity (Neff et al., 2016; Schirmer et al., 2016), so the increase in abundance of this bacteria may explain why the nonIBDD microbiome is not associated with inflammation in the gut.\n\nOther than Parabacteroides goldsteinii, nonIBDD samples did not contain other characteristic groups, and, more notably, none of the selected species was specific for depressed or non-depressed phenotypes.\n\nRegarding the functional activity of these species, seven pathways that were more abundant in dysbiotic groups than in nonIBD were identified (Supplementary Figure 1) and were correlated between each other and inversely correlated with most of the others (Supplementary Figure 2 and Supplementary Table 5). Those pathways are folate transformations II, N10-formyl-tetrahydrofolate biosynthesis, de novo L-ornithine biosynthesis, superpathway of pyridoxal 5’phosphate biosynthesis and salvage, phosphopantothenate biosynthesis I, preQ0 biosynthesis and queuosine biosynthesis. Folate (vitamin B9) and pyroxidal 5’-phosphate (vitamin B6) deficiencies have been linked both to depression (Coppen & Bolander-Gouaille, 2005; Hvas et al., 2004; Mitchell et al., 2014), as they are key for the synthesis of several neurotransmitters, and IBD (Pan et al., 2017; Yakut et al., 2010), although this association is not well understood and does not seem to be evidence of causation. Increased levels of L-ornithine derivatives have also been linked to depression (Zheng et al., 2010). However, even if nonIBDD have the highest activity for almost all of these pathways, CD and UC were also significantly increased, while functional activity in CDD was generally lower and non-significant in some pathways. Moreover, UCD did not differ from nonIBD in any of them.\n\nThis difference in functional activity again highlights the lack of a concrete pattern of gut microbiome abundance between depressed groups.\n\n\nConclusions\n\nThe random forest approach was able to successfully identify informative changes in abundance at the species level, revealing specific patterns for the depressed and non-depressed groups without losing predictive power. This work provided, to our knowledge for the first time, an overview about the difference in the bacterial communities of patients with signs of depression and the combination with depression and inflammatory bowel disease. Our findings suggest a complex landscape of microbiome interactions, both at population structure and functional activity levels. However, the results showed that there are distinct taxonomic profiles within patients of IBD depending on their depression status, providing further input for future investigations.\n\n\nData availability\n\nThe datasets used for the analyses were retrieved from the Inflammatory Bowel Disease Multi-Omics Database (IBDMDB) (Schirmer et al., 2018), a part of the Integrative Human Microbiome Project (NIH HMP Working Group et al., 2009).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAuthors thank funding from The Danish Independent Research Council (Technology and Production) grant number DFF – 6111-00471. P.M.M. thanks funding from Technical University of Denmark, DTU Bioinformatics for his research assistantship to conduct this study.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Figure 1. Relative abundances of the pathways that showed significant differences between groups (alpha= 0.05).\n\nClick here to access the data.\n\nSupplementary Figure 2. Correlation between the different pathways contributed by the selected species. Color gradient shows positive (red) or negative (blue) correlation.\n\nClick here to access the data.\n\nSupplementary Figure 3. Receiver operating characteristic curves for the validation model with combined metagenomic and metatranscriptomic data.\n\nClick here to access the data.\n\nSupplementary Figure 4. Receiver operating characteristic curves for the validation model with metagenomic data.\n\nClick here to access the data.\n\nSupplementary Figure 5. Receiver operating characteristic curves for the validation model with metatranscriptomic data.\n\nClick here to access the data.\n\nSupplementary Table 1. ANOVA results for each of the selected species in metagenomic and metatranscriptomic data sets.\n\nClick here to access the data.\n\nSupplementary Table 2. A t-test was used to assess the difference between DNA and RNA abundances per species and a nested column per group.\n\nClick here to access the data.\n\nSupplementary Table 3. Tukey’s honest significant difference test for the metagenomic data. Results are organized by species with two nested columns, confidence intervals at 0.95 and the decision. Each row represents a pair-wise comparison.\n\nClick here to access the data.\n\nSupplementary Table 4. Tukey’s honest significant difference test for the metatranscriptomic data. Results are organized by species with two nested columns, confidence intervals at 0.95 and the decision. Each row represents a pair-wise comparison.\n\nClick here to access the data.\n\nSupplementary Table 5. Tukey’s honest significant difference test for the pathways correlated pathways. Results are organized by species with two nested columns, confidence intervals at 0.95 and the decision. Each row represents a pair-wise comparison.\n\nClick here to access the data.\n\n\nReferences\n\nAbubucker S, Segata N, Goll J, et al.: Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS Comput Biol. 2012; 8(6): e1002358. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBloom SM, Bijanki VN, Nava GM, et al.: Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease. Cell Host Microbe. 2011; 9(5): 390–403. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreiman L: Random Forests. Mach Learn. 2001; 45(1): 5–32. Publisher Full Text\n\nCoppen A, Bolander-Gouaille C: Treatment of depression: time to consider folic acid and vitamin B12. J Psychopharmacol. 2005; 19(1): 59–65. PubMed Abstract | Publisher Full Text\n\nFoster JA, McVey Neufeld KA: Gut-brain axis: how the microbiome influences anxiety and depression. Trends Neurosci. 2013; 36(5): 305–312. PubMed Abstract | Publisher Full Text\n\nGraff LA, Walker JR, Bernstein CN: Depression and anxiety in inflammatory bowel disease: a review of comorbidity and management. Inflamm Bowel Dis. 2009; 15(7): 1105–1118. PubMed Abstract | Publisher Full Text\n\nHvas AM, Juul S, Bech P, et al.: Vitamin B6 level is associated with symptoms of depression. Psychother Psychosom. 2004; 73(6): 340–343. PubMed Abstract | Publisher Full Text\n\nJones E, Oliphant T, Peterson P: {SciPy}: open source scientific tools for {Python}. 2001. Reference Source\n\nKaur N, Chen CC, Luther J, et al.: Intestinal dysbiosis in inflammatory bowel disease. Gut Microbes. 2011; 2(4): 211–216. PubMed Abstract | Publisher Full Text\n\nLuna RA, Foster JA: Gut brain axis: diet microbiota interactions and implications for modulation of anxiety and depression. Curr Opin Biotechnol. 2015; 32: 35–41. PubMed Abstract | Publisher Full Text\n\nMawdsley JE, Rampton DS: The role of psychological stress in inflammatory bowel disease. Neuroimmunomodulation. 2006; 13(5–6): 327–336. PubMed Abstract | Publisher Full Text\n\nMitchell ES, Conus N, Kaput J: B vitamin polymorphisms and behavior: evidence of associations with neurodevelopment, depression, schizophrenia, bipolar disorder and cognitive decline. Neurosci Biobehav Rev. 2014; 47: 307–320. PubMed Abstract | Publisher Full Text\n\nNeff CP, Rhodes ME, Arnolds KL, et al.: Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties. Cell Host Microbe. 2016; 20(4): 535–547. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNIH HMP Working Group, Peterson J, Garges S, et al.: The NIH Human Microbiome Project. Genome Res. 2009; 19(12): 2317–2323. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan Y, Liu Y, Guo H, et al.: Associations between Folate and Vitamin B12 Levels and Inflammatory Bowel Disease: A Meta-Analysis. Nutrients. 2017; 9(4): pii: E382. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: Machine Learning in Python. J Mach Learn Res. 2011; 12: 2825–2830. Reference Source\n\nRogers GB, Keating DJ, Young RL, et al.: From gut dysbiosis to altered brain function and mental illness: mechanisms and pathways. Mol Psychiatry. 2016; 21(6): 738–748. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaulnier DM, Riehle K, Mistretta TA, et al.: Gastrointestinal microbiome signatures of pediatric patients with irritable bowel syndrome. Gastroenterology. 2011; 141(5): 1782–1791. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchirmer M, Franzosa EA, Lloyd-Price J, et al.: Dynamics of metatranscription in the inflammatory bowel disease gut microbiome. Nat Microbiol. 2018; 3(3): 337–346. PubMed Abstract | Publisher Full Text\n\nSchirmer M, Smeekens SP, Vlamakis H, et al.: Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity. Cell. 2016; 167(4): 1125–1136.e8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTruong DT, Franzosa EA, Tickle TL, et al.: MetaPhlAn2 for enhanced metagenomic taxonomic profiling. Nat Methods. 2015; 12(10): 902–903. PubMed Abstract | Publisher Full Text\n\nYakut M, Ustün Y, Kabaçam G, et al.: Serum vitamin B12 and folate status in patients with inflammatory bowel diseases. Eur J Intern Med. 2010; 21(4): 320–323. PubMed Abstract | Publisher Full Text\n\nZheng S, Yu M, Lu X, et al.: Urinary metabonomic study on biochemical changes in chronic unpredictable mild stress model of depression. Clin Chim Acta. 2010; 411(3–4): 204–209. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "35101",
"date": "25 Jun 2018",
"name": "Yasir Suhail",
"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\nOverview\nThe paper's central thesis of the correlation, or putative causative mechanism of the gut microbiome on IBD and depression, is important. The application of machine learning techniques may be suitable because of the high dimensional structure of the data and lack of knowledge of a simple causal relationship between the microbiome and IBD/depression.\nI have a number of concerns and suggestions about the article in its present form. Most of these concern the presentation of the data, methods, and results. Addressing these concerns will help bring the article into a shape where readers can better evaluate the results and contribution of this study.\nPresentation of the methods and results\nThe article should explicitly specify the dimensions and form of the data/features that were fed to the random forest algorithm. Were they the estimates of each species abundance in each individual from the respective RNA and DNA sequences? Was it derived from ribosomal seqeunces only? How many species were included? What was the total dimension of this data set? Does it correspond to a specific table in the IBDMDB resource?\n\nSince the intended audience may be researchers generally interested in IBD or metagenomics who are not experts in random forests, algorithmic details should be provided in terminology that is common to the broader field. N-fold cross-validation generally refers to using 1 out of N data samples as a hold out test sample. However, the article mentions 1000-fold and 500-fold cross-validation for a data-set with 70 individuals. Does this correspond to individual re-sampled bagging or feature bagging in the random forest? A more explicit explanation will make this comprehensible to more readers.\n\nThe supplementary figures and tables may have been mixed/corrupted. S. Figures 1 and 2 are described as corresponding to pathway analysis but are actually ROC curves. SF4 and 5 are supposed to be ROC curves but probably show pathway results. The resolution is too low to make out the text. The method of calculating the pathway abundances should also be described somewhere. Is it the total number of reads corresponding to genes in the pathway, does it depend on the species abundances or any other parameters?\n\nSome of the ambiguity in the analysis may be removed by providing the code for any pre-processing, random forest analysis, feature selection, and pathway abundance analysis etc.\n\nInterpretation and Conclusion\nA machine learning algorithm can build an accurate prediction system, or generate hypotheses about the mechanisms at play or provide some other insight into the process. Here, I see two possible results of the ML analysis:\nThe prediction accuracy can be a measure of the amount of information contained in the microbiome about the diseases. Alternatively, how predictive is the gut microbiome, and does this imply evidence for the causative effect of the microbiome on the disease? These would be comparatively harder claims to make, and would probably require a few more calculations.\n\nThe random forests are used to arrive at the most important features (bacterial species) affecting bowel disease and depression. I think this is the main claim/result of the analysis. In this case, how much more does information does ML give us compared to simply finding the species whose abundance is most different between the disease and non-disease states (in terms of fold-change or p-value). For the case of the multi-class problem, ANOVA can provide p-values for the non-random abundances in the different classes of patients. The article describes the results of such t-tests and ANOVA results. A sufficient and logical argument for the ML approach supported by any relevant calculations will strengthen the case for this analysis.\n\nOverall, I feel the discussion of the possible role of some of the species and metabolic pathways etiology of the disease is the most interesting for biologists and clinicians. The article is important in this regard and further development of this discussion can only add to its strength.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3984",
"date": "17 Sep 2018",
"name": "Pedro Morell Miranda",
"role": "Author Response",
"response": "The precision metric was used for each Cross Validation model, so each precision metric was calculated using the test split for that iteration from the original train dataset. The validation dataset was only used to measure the final metrics of the model trained only with the features selected by the Cross Validation method. Regarding the \"only species that appeared more than once were selected\" statement, it refers to the fact that, from the list of selected features, we saw a few cases of species present only in all the samples of only one individual, which in some cases can end being considered as quite characteristic by the model as each patient contributed with several samples. Setting this condition allowed us to avoid those \"one hit wonders\" that were not descriptive of the group and appeared only because of the longitudinal experimental design."
}
]
},
{
"id": "37362",
"date": "04 Oct 2018",
"name": "Manikandan Narayanan",
"expertise": [
"Reviewer Expertise Computational biology",
"bioinformatics",
"systems biology",
"gene networks",
"disease-disease interactions",
"multi-tissue genomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview process: The open review model calls for a special reviewing strategy since previous reviews/comments are already available. I read the paper and formed my independent opinions before looking at the previous reviews. Then I wrote this report to express my opinions in the context of the previous reviewer's opinion.\nSummary: The authors attempt to unravel the complex interaction between IBD and depression that is potentially mediated by the gut microbiome. More specifically, they identify a subset of (24) microbial species that could discriminate between various single-disease and co-morbid cases of IBD and depression, and discuss the potential roles of these species and associated functional pathways in co-morbidity in the context of prior literature. Their methodology involves applying a combination of a machine learning approach (building a random forest to predict disease labels from species abundances and feature selection of informative species in the final model) and performing statistical tests (ANOVA-based test to identify which of the 24 selected/informative species differ between pairwise groups of interest such as the IBD with vs. without depression groups).\n\nStrengths: The authors use a systematic data-driven approach to address an important question of which microbial species and which functional pathways are involved in the \"microbiome-brain-gut axis\" (or specifically which species/pathways discriminate between different groups of individuals such as IBD disease with vs. without depression). The idea of using a random forest to select informative species with predictive power and then doing all subsequent analysis with the selected species is an interesting strategy to deal with the heterogeneity in the data and small sample size per strata/group (though I've some concerns with this approach/idea as mentioned below, which may be addressed with some additional analysis). The nice summary of key results in Table 1 (of species whose abundances have changed between IBD with vs. without depression) and the related discussion of identified species in the context of prior literature on single/comorbid cases of IBD/depression would be of immediate interest to researchers in this field.\n\nConcerns: I agree with the previous reviewer's concerns/feedback on improving presentation (such as by adding key details on data dimensions and cross-validation folds to the text) and improving interpretation (such as by comparing the 24 selected species to whatever species would be detected from applying a classic t-test or ANOVA test to all species rather than just the 24 species selected in the random forest analysis).\nI now provide my feedback/comments not already covered by the previous reviewer below.\n\nStatistical concerns due to small sample size and potentially confounding covariates: Though the overall population is a decent sample size of 70 individuals, the per-strata or per-group sample sizes are low to moderate, with some groups like UC having only 4 patients!! The authors are aware of this issue and use a random forest feature selection to tackle the heterogeneity in sparse human population data. But I am not fully convinced that a machine learning model can recover from insufficient sample sizes as low as n=4. One way to address this issue could be to merge together UC with CD and then label them as a single IBD group and then study this group with/without signs of depression. If the conclusions are similar before/after this merging of UC with CD, then the authors may mention that this additional test yielded similar results and keep the current results in the paper as is.\n\nAnother issue with heterogeneous and sparse human data is that covariates such as age, gender, BMI, genotype, etc. are more likely to confound the association of gut microbiota with disease status. Are these covariates available from the original cohort (I would assume so since the authors say that even host genomes are available)? Importantly, if available, are these covariates matched between the different groups being compared here? If not matched, is the data adjusted for these covariates and how do the results change before/after this adjustment? Providing such information would be critical to readers to properly interpret the detected microbiota associations with IBD/depression.\n\nThe all-relevant vs. minimal-informative set of species: Please provide clarification on the text on whether the 24 species is a minimal, non-redundant set of species that has predictive power to classify the disease labels, or whether it includes all the relevant species that is associated with disease labels (or whether it is somewhere in-between in this spectrum). In other words, is any other species other than these 24 species associated with disease status? While a minimal set is sufficient to build a predictive model and simplifies further interpretation, the all-relevant set would be useful to understand the comprehensive role of all species and the overall mechanisms involved, as explained nicely in the Introduction of this paper1 on the Boruta feature selection package.\n\nBased on the details provided on random forest based feature selection, the reported results may be closer to the all-relevant than a minimal-informative set, but the requirement of a species to be present in at least 2 models to be selected as an informative species is somewhat ad hoc (i.e., why not 3 or 4 models as a cutoff) and makes it unclear on whether all relevant species are selected. A more systematic way would be to assess the statistical significance of each species' association using a \"wrapper method\" around the random forest, such as the shuffling-based Boruta feature selection package (which is also used in the Saulnier et al. 2011 paper that the authors have cited). An alternative could be one of the methods in the paper \"Statistical interpretation of machine learning-based feature importance scores for biomarker discovery\"2.\n\nThe context from prior literature that the authors already provide could be strengthened even further by connecting them to the brain-gut-microbiome axis reviewed in some recent articles3,4.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-702
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https://f1000research.com/articles/7-1346/v1
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29 Aug 18
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{
"type": "Software Tool Article",
"title": "MSF: Modulated Sub-graph Finder",
"authors": [
"Mariam R. Farman",
"Ivo L. Hofacker",
"Fabian Amman",
"Mariam R. Farman",
"Fabian Amman"
],
"abstract": "High throughput techniques such as RNA-seq or microarray analysis have proven to be invaluable for the characterization of global transcriptional gene activity changes due to external stimuli or diseases. Differential gene expression analysis (DGEA) is the first step in the course of data interpretation, typically producing lists of dozens to thousands of differentially expressed genes. To further guide the interpretation of these lists, different pathway analysis approaches have been developed. These tools typically rely on the classification of genes into sets of genes, such as pathways, based on the interactions between the genes and their function in a common biological process. Regardless of technical differences, these methods do not properly account for cross talk between different pathways and rely on binary separation into differentially expressed gene and unaffected genes based on an arbitrarily set p-value cut-off. To overcome this limitation, we developed a novel approach to identify concertedly modulated sub-graphs in the global cell signaling network, based on the DGEA results of all genes tested. Thereby, expression patterns of genes are integrated according to the topology of their interactions and allow potentially to read the flow of information from the perturbation source to the effectors. The described software, named Modulated Sub-graph Finder (MSF) is freely available at https: //github.com/Modulated-Subgraph-Finder/MSF.",
"keywords": [
"Differential gene expression analysis",
"pathway analysis",
"combining p-value",
"cell signalling network"
],
"content": "Introduction\n\nHigh throughput sequencing techniques have been widely used to yield differentially expressed genes (DEG) (Malone & Oliver, 2011). To this end, changes in transcript abundance are measured, e.g. by next generation sequencing techniques, and interpreted as an indicator of differential expression of genes. DEGs can be used to gain insights into the mechanism underlying differences between conditions of samples, such as healthy versus diseased. Differential gene expression analysis (DGEA) informs about the magnitude of expression changes between the conditions which are often expressed as fold change, sign of fold change and the confidence level of observing an authentic change, often expressed as p-value. These DEGs information is further interpreted to extract meaningful biological insights. For example, genes that could be involved in the response to a particular stimuli or maybe the cause of a disease. To this end, pathway-based analysis has become an important tool to further interpret the results of a DGEA and to acquire understandings of the perturbations in a biological system. Biological pathways are sets of genes and their interactions forming a functional unit. DEGs help to identify pathways or networks that may be altered during a change of condition providing important information about diseases and its treatment process (Khatri et al., 2012). Pathway-based methods use predefined pathways or networks such as KEGG (Kanehisa & Goto, 2000) and Reactome (Fabregat et al., 2018), the expression measurements of the genes obtained from DGEA in combination with statistical methods and algorithms to identify specifically modulated pathways and processes (García-Campos et al., 2015).\n\nWell established resources for pathway annotation are KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa & Goto, 2000) and Reactome (Fabregat et al., 2018). KEGG pathways is a branch of KEGG database that hosts a collection of manually drawn pathway maps representing the molecular interaction, reaction and relation networks of cellular functions. Similarly, Reactome is an open-source, manually curated, peer-reviewed database for signaling and metabolic molecules with their interactions formed into different biological pathways for nineteen species (Fabregat et al., 2018). Both provide predefined pathways which are sets of genes and their interactions categorized into functional units. Starting from a gene interaction network, genes are labeled according to their role in a specific biological process. In this sense a particular gene can be assigned to different pathways. E.g., the human gene STAT1 is associated with 24 different pathways in the pathway annotation curated by KEGG and in 12 different pathways in the Reactome database. Although carefully produced, the assignment of genes to those predefined pathway units can be considered to be subjective to some degree and suffers from observational bias (Schnoes et al., 2013).\n\nExisting pathway-based analysis approaches use different research designs, which can be categorized into ORA (Over-representation analysis), FCS (Functional class scoring) and pathway topology based methods. All of which aim to find a subset of genes, e.g., significantly differentially expressed genes, genes associated with a certain pathway more often than expected given the total set of examined genes, e.g. the whole genome background. ORA is the first and the most basic method of pathway analysis (García-Campos et al., 2015). It uses a DEG list with user defined cut-off for the log-fold change and pvalue (most commonly using absolute log-fold change ≥ 2 and p-value ≤ 0.05). Subsequently, sets of genes associated with annotated pathways are tested for being over-represented in the set of DEGs. To this end, hypergeometric distribution, chi-square tests, binomial probability or the Fisher’s exact test are used. Thereby the information of the topology of genes in the pathways is neglected (Bayerlová et al., 2015). Furthermore, ORA assumes that the biological pathways are independent of each other and ignores the fact that they cross-talk and overlap (García-Campos et al., 2015; Khatri et al., 2012).\n\nUnlike ORA, FCS has no artificial cut-off to define a DEG list. FCS works in three steps. First it calculates the gene-level statistics including correlation of molecular measurements using differential expression of individual genes, ANOVA, t-test and Z-score. In the second step the statistics of individual genes in a pathway are transformed to an individual pathway-level statistic commonly using Kolmogorov-Smirnov statistic, mean or median. Finally the statistical significance of the pathwaylevel statistics is assessed. Although FCS covers some of the limitations from ORA, it still ignores the topology of genes in a pathway, cross-talk and overlap of the pathways (García-Campos et al., 2015; Khatri et al., 2012). Pathway topology based methods are similar to FCS except that they consider the topology of each gene during the gene-level statistics but still don’t aim to link different functional pathways (Khatri et al., 2012).\n\nOn these grounds we propose a novel approach to make use of the rich gene and protein interaction annotation resources available to gain additional functional insights from basic DGEA. To this, we start with the presupposition that expression of neighboring genes within a functional pathway are not independent from each other. Rather, they are often regulating each others expression or are part of the same regulon (Michalak, 2008). We understand that the categorization of links between genes into labeled pathways is often an arbitrary one, given the extensive cross talk between different pathways. Although these categories have proven to be useful in many situations, they force a certain perspective onto the interpretation of novel data. Based on these two principles, we aim to find sub-graphs of connected genes within cell signaling network which exhibit as a whole significant differential expression changes. This approach differs in two main aspects from common pathway analysis tools. First, it does not aim to identify functional pathways enriched in differentially expressed genes, but detects sub-graphs or branches in a network graph (potentially spanning more than one functionally grouped pathway) which is coherently modulated. Second, it aims to improve the DGEA on the gene level, by collecting the information of neighboring genes, which as a whole might exhibit prominent enough signal to be called, again as a whole, significantly modulated.\n\nAs input, information on functional links between genes provided by e.g. KEGG or Reactome and information on the differential expression status of single genes resulting from a DGEA, are required. As a result the analysis returns sub-graphs and their joint confidence scores, reflecting how the perturbation is migrated through the network. Furthermore, the entry points of perturbation in the networks and overlap with conventional pathway categories are returned. The output is prepared in a directed adjacency file, convenient for visualization, e.g., with StringApp (Morris et al., 2018), available as a Cytoscape plug-in (Shannon et al., 2003).\n\nAll of this can be helpful to understand the cause and effect of a stimulus and might inform about potential points of intervention. The proposed algorithm was implemented as a java program, which was named Modulated Sub-graph Finder (abbreviated MSF). MSF can help transform the information obtained from DGEA into comprehensible knowledge of signal transduction of genes and thereby being a valuable complement to existing pathway based methods. MSF is freely accessible from GitHub https://github.com/Modulated-Subgraph-Finder/MSF.\n\n\nMethods\n\nMSF is developed as a novel heuristic approach to find concertedly modulated sub-graphs in networks of biological interactions. MSF does not use predefined gene sets grouped into functional units, but rather relies purely on the network of interacting genes. The input network consists of nodes corresponding to genes and edges representing interactions. Furthermore it utilizes comprehensive results from a differential gene expression analysis to discover the sub-graphs, or modules, which are as a whole modulated.\n\nMSF uses the individual gene’s p-values generated from the DGEA. The p-value expresses the probability that the null hypothesis of unmodified gene expression can’t be rejected for a given statistical model. To find significantly modulated sub-graphs individual p-values of the vicinal genes in the global network are combined into a single combined p-value, using a statistical method for combining dependent p-values described by Hartung (Hartung, 1998). Hartung’s method uses the inverse of standard normal distribution function, individual gene p-values are first transformed to their corresponding normal score. Then using these normal scores, the correlation between genes is calculated, the normal scores and correlation are applied to the inverse normal function to calculate the combined p-value for all genes examined, namely the examined sub-graph. The combined p-value of a sub-graph will express the significance of all genes in the sub-graph being modulated together. Thereby, the information from the different genes are used as, although not independent, replicated measurement of the behavior of the whole sub-graph. This potentially increases the power to detect also significant sub-graphs consisting of genes which are not significant on there own.\n\nTo reduce the complexity to score all possible connected sub-graphs MSF applies a four step heuristic as described in the following. The proceeding identification of modulated sub-graphs from a network by MSF is presented as a flowchart diagram (Figure 1).\n\nInitial modulated sub-graphs. MSF constructs the first sub-graph starting with the genes associated with the lowest (most significant) p-value deduced from the DGEA. From this seed it tries to extend the sub-graph by adding directly neighboring genes, starting with the most significant one. A single combined p-value is calculated for the extended sub-graph. If the combined p-value is smaller than the p-value of the original sub-graph, the extended sub-graph is accepted. This step is iteratively repeated until no further extension is accepted. In this case the process starts over with all remaining genes not yet in a significantly modulated sub-graph. This step identifies all simple sub-graphs that are modulated in the whole network.\n\nExtending modulated sub-graphs. In the next step, we check if any of the initial modulated sub-graphs could further be extended by adding more than one gene at a time. This is done by testing all possible extension paths up to N (default 2) genes at all nodes in the sub-graph. Again, this step is iteratively repeated until no further genes are added to the significant differentially expressed sub-graphs. This step bridges small gaps of genes without a clear differential signal in the DGEA.\n\nMerging modulated sub-graphs. After detection and extension of the modulated subgraphs, they are tested if combined sub-graphs score better than on their own. The merging of the sub-graphs is done by depth first search traversal from the first sub-graph to the second sub-graph. If the two sub-graphs merge with the connector of at most N genes (default 1 gene) and the combined p-value of the merged sub-graph including the bridging genes in between is less than the individual p-values of the two sub-graphs, the two sub-graphs are merged together to one big modulated sub-graph. This step is repeated iteratively until no sub-graphs could be merged.\n\nFinding sources & sinks. In a last post processing step MSF identifies the trigger points of the modulated sub-graphs. These trigger genes are the sources of the sub-graphs with only outgoing edges. These genes can be interpreted as the possible entry points of perturbation from where the stimulus causes downstream effects. In the same spirit the most downstream genes of the modulated sub-graph are identified and defined as sinks. Sinks can be interpreted as the effectors where the integrated information within the signal transduction network is set to action. Due to circular loops not all sub-graphs are guaranteed to have sources or sinks.\n\nOperation. MSF requires Java version 8 and JDK 1.8. The few package dependencies are already been added to the release. MSF runs fast on a standard laptop computer and so it has normal system requirements. To run MSF, the user must provide two text files, one containing the DGEA and the second one containing the interactions in an adjacency format file. Example files and a detailed tutorial to use MSF has been provided on github https://github. com/Modulated-Subgraph-Finder/MSF.\n\n\nResults\n\nTo demonstrate its usefulness, MSF is applied to an RNA-seq data set of primary human monocytederived macrophages (MDMs) infected with Ebola virus (GSE84188) (Olejnik et al., 2017). Ebola Virus (EBOV) belongs to the Filoviridea family; filamentous, enveloped and single stranded RNA viruses. EBOV causes hemorrhagic fever in humans, inducing the host innate and adaptive immune response to be unable to control virus infection (Prins et al., 2009). Until now, there are no approved antiviral drugs for the treatment of Ebola virus infection (Konde et al., 2017; Rhein et al., 2015). The initial targets of EBOV are the macrophages and dendritic immune cells (Falasca et al., 2015; Rhein et al., 2015). EBOV inhibits the critical innate immune response of the host, which includes the activation of alpha/beta interferon (IFN-α/β) (Cárdenas et al., 2006; Konde et al., 2017; Prins et al., 2009). It has been proposed that IFNα/β should be tested against Ebola for its antiviral activity through clinical trials (Konde et al., 2017). Ebola infection data was selected to test the approach because it has been well recognized for the last several decades, and vast literature is available for the pathogenesis of Ebola. Thereby facilitating the verification of the results of MSF with the vast literature present on Ebola infection. Especially, the detection of IFN-α/β as point of action for the virus, could be considered as a basic indicator of the correctness and usefulness of the approach.\n\nEBOV infection count data was downloaded from Gene Expression Omnibus (GEO) (GSE84188). Differential gene expression analysis was performed on the count data with edgeR package (version 3.4.2) (Robinson et al., 2010). The DEG analysis results generated by edgeR were used as input for MSF. Directed cell signaling interactions were filtered from Reactome Functional interactions (FIs) Version 2016 (Wu et al., 2016), which was used as a second input for MSF.\n\nThe EBOV infection experiments describe the course of infection at three time-points 6, 24 and 48 hour post infection (hpi). For the earliest time point at 6 hpi, three large modulated sub-graphs were identified with 41, 107, and 18 genes were identified, as well as two with less than 4 genes. The modulated sub-graphs consist predominantly of cytokines, chemokines (CXCL10, CCL8) and Interleukin genes (IL6, IL27, IL23). IFNB1 and IFNA1 were both identified as two of the possible sources in one of the sub-graph identified with 18 genes. Most of the sources identified by MSF were type I interferon induced genes (Supplementary material 6H). At 24 hpi eight modulated sub-graphs were identified with four main sub-graphs consisting of 27, 101, 134 and 194 genes, others being smaller than 10 genes. IFNB1 and IFNA1 were identified as the two sources out of the total sources. For the last time-point 48 hpi, six modulated sub-graphs were identified. Three of the sub-graphs were less than nine genes and main sub-graphs had 122, 191 and 202 genes. IFNB1 was identified as one of the sources in the most significantly modulated sub-graph (Supplementary).\n\nAs stated earlier IFN-α/β was reported to be one of the target genes of Ebola infection. We were able to successfully identify IFNA1 as a source in all three time-points and INFB1 in two of the time-points. Although IFNA1 and IFNB1 were already among the most significant gene in the DGEA during the later time points, MSF was also able to detect them as a source in the very early time-point when the genes were not significant based on the individual DGEA alone. Identifying the possible sources will reduce the search space for potential target genes and can help the biologist as the starting point of clinical testing for drugs and vaccines against an infection.\n\nTable 1 compares the results of MSF, namely the number of detected sub-modules and their total genes numbers, to a simple analysis of mapping significantly modulated genes from the DGEA to the network and joining neighbors to modules. The numbers indicate that MSF detects less but larger sub-modules, applying its statistical test. Furthermore, the dependency of the results from the p-value cutoff choice is demonstrated for the DGEA, which is avoided for MSF altogether.\n\nhpi - hours post infection.\n\nThree main modulated sub-graphs identified by MSF at 6 hpi are shown in Figure 2. The gene based graphs on the right hand side, represent the immediate output of the MSF-analysis, visualized by StringApp (Morris et al., 2018) in Cytoscape (Shannon et al., 2003). Each node represents a gene part of a modulated sub-graph, whereby the associated colors code the functional annotation deduced from KEGG Pathways. The cross-talk between the pathways and also the multiple employment of many genes is evident. The more schematic drawing on the right side represents the effortlessly deduced flow of information between the sensors and effectors in this particular example.\n\nThe node coloring is associated to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways referring to the colors in the legend. The graph edges are from Reactome. Important genes to EBOV infection as from literature are enlarged in the graph.\n\nIn more detail, sub-graph 1 (top) shows how the activation of toll-like receptor, cytokine, chemokine and jakstat genes lead via TNF into apoptosis. The next significant sub-graph (sub-graph 2: middle) reveals how information from the Extra-cellular matrix (ECM) receptor, which are reported to interact with Ebola glycoprotein (GP) (Veljkovic et al., 2015), chemokines and cytokines, and cytosolic DNA sensing, is integrated into again modulation of apoptosis pathway. Eventually, sub-graph 3 (bottom) demonstrate how INFA1 and INFB1 modulate once more, via only a few intermediate steps, the apoptotic response of the cell.\n\nThis show cast example demonstrates with how little effort complex data can be interpreted, help to apprehend the dynamics of the underlying processes and suggest testable hypothesis and potential points of intervention.\n\nA potential concern is how noise in the gene expression measurements affects our analysis. To assess the robustness and stability of our method, we therefore added Poisson distributed noise to the read counts of the three time-points data set, used above. Then DGEA was carried out on the disturbed data with the same parameters as for the native data using edgeR, followed by analysis with MSF. This procedure was carried out 100 times. Every time the genes from the modulated sub-graphs identified from noisy data were compared to the genes of sub-graphs identified from the native data. This was also done for the DEG obtained for each run, using three different cutoffs of FDR 0.01, 0.05 and 0.1. The robustness of MSF and the DEG analysis for the time-point 6, 24, and 48 hpi are shown in Figure 3. The procedure for how data noise was modeled can be considered as rather stringent, which is already reflected by the limited recall rate in the edgeR based DGEA, between 68 % (6 hpi) and 93 % (24 hpi). For MSF-analysis the observed median recall rates lay between 71 % (6 hpi) and 84 % (48 hpi). The better performance of the pure DGEA can be explained by the fact that disturbed p-values do not change the results for DGEA as long as the p-value does not rise above the chosen cutoff value. In contrast, MSF is sensitive to p-value changes across the whole range of possible values.\n\nGene enrichment analysis was performed using Reactome Analyze data tool (Fabregat et al., 2018). Reactome’s over-representation analysis tests whether certain Reactome pathways are enriched by the list of genes submitted. Genes from MSF identified sub-graphs for each time-point were analyzed for gene enrichment analysis. For comparison the DEG results from edgeR were filtered using three different cut offs of adjusted p-value 0.01, 0.05 and 0.1. This three subsets of DEG list were used for gene enrichment analysis. The compression of MSF and the three subset DEG list is shown in Figure 4.\n\nAll the different toll-like receptor signaling pathways identified from Modulated Sub-graph Finder (MSF) identified genes are marked.\n\nThe comparison shows most of the pathways known from literature to be dis-regulated by Ebola infection are enriched in both the enrichment analysis. Toll-Like receptor signaling pathway when interacts with EBOV glycoprotein (GP), it triggers the activation of cytokines (Olejnik et al., 2017). Toll-like receptor pathway is expected to be dis-regulated in the early stage of infection, this pathway was not identified as significantly dis-regulated when p-value cut off DEG lists were analyzed for enrichment. Ten toll-like receptor cascades were identified as dis-regulated from gene enrichment analysis of MSF identified genes, not a single one of these pathways was shown to be dis-regulated in DEG cut off lists. Since MSF considers the complete DEG results, even the weak signal at the earliest time-point was detected; for example Toll-like receptor signaling.\n\n\nDiscussion\n\nClassic pathway analysis tools aim to detect in lists of significantly deregulated genes enriched associations with pathway genes categorized by their biological function and their interactions. Thereby, depending on the tool, the internal pathway topology is considered or neglected all together. The here presented tool, MSF, employs a different approach, by aiming to detect sub-graphs in whole gene regulatory networks which are significantly deregulated in a concerted manner. To this end, neighboring genes in the user provided network are tested for jointly common regulation. Exploiting that each gene’s abundance, although not independent from its neighbors, is measured repeatedly on its own, sensitivity can be increased by our applied p-value meta-analysis, namely Hartung’s method. This potentially enables to call nonsignificant modulated genes based on the DGEA, to be convincingly part of a deregulated gene group. Furthermore, it allows to identify connected sub-graphs, representing the propagation of gene regulation perturbation in the input network. A better understanding of this propagation, especially the critical spots such as sensors, effectors, or intermediate bottlenecks and hubs, facilitates the projection of potential intervention points, e.g., for drug development. Since MSF only uses interaction information in gene regulation networks, but not the functional grouping of the genes into functional pathways, it is especially adapted to discover so called cross-talk between such pathways.\n\n\nConclusions\n\nMSF is a fast, robust and easy to use tool to find concertedly modulated sub-graphs in a given network. Its implementation in JAVA enables its use across many operating systems. It requires as input the results from a differential gene expression analysis in the appropriate file format. So far the raw output from edgeR (Robinson et al., 2010) and DESeq2 (Love et al., 2014) are supported. Furthermore, a gene network has to be provided in the format of a directed adjacency list.\n\n\nData availability\n\nThe Ebola infection RNA-seq data set analyzed during the current study are available in the GEO repository (GSE84188) (Olejnik et al., 2017). The cell signaling network file used is from Reactome Functional interactions (FIs) Version 2016 (Wu et al., 2016).\n\n\nSoftware availability\n\nSource code is available from GitHub: https://github.com/Modulated-Subgraph-Finder/MSF\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.1400242 (Farman, 2018).\n\nSoftware license: MIT license.",
"appendix": "Grant information\n\nThis work was funded by the FWF (“Fonds zur Förderung der wissenschaftlichen Forschung”) within the project Internationalen Kooperationsprojektes - Intl cooperation Project (Joint Project - Lead Agency Verfahren) [I 1988-B22]. The grant was assigned to ILH. FA was funded by the Austrian Science Fund (FWF) [SFB F43].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary material is available form GitHub: https://github.com/Modulated-Subgraph-Finder/MSF\n\n\nReferences\n\nBayerlová M, Jung K, Kramer F, et al.: Comparative study on gene set and pathway topology-based enrichment methods. BMC Bioinformatics. 2015; 16(1): 334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCárdenas WB, Loo YM, Gale M Jr, et al.: Ebola virus vp35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling. J Virol. 2006; 80(11): 5168–5178. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFabregat A, Jupe S, Matthews L, et al.: The Reactome Pathway Knowledgebase. Nucleic Acids Res. 2018; 46(D1): D649–D655. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFalasca L, Agrati C, Petrosillo N, et al.: Molecular mechanisms of Ebola virus pathogenesis: focus on cell death. Cell Death Differ. 2015; 22(8): 1250–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarman M: Modulated-Subgraph-Finder/MSF V.1 (Version V.1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1400242\n\nGarcía-Campos MA, Espinal-Enríquez J, Hernández-Lemus E: Pathway Analysis: State of the Art. Front Physiol. 2015; 6: 383. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHartung J: A note on combining dependent tests of significance. Technical report, Technical Report, SFB 475: Komplexitätsreduktion in Multivariaten Datenstrukturen, Universität Dortmund, 1998. Reference Source\n\nKanehisa M, Goto S: Kegg: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhatri P, Sirota M, Butte AJ: Ten years of pathway analysis: current approaches and outstanding challenges. PLoS Comput Biol. 2012; 8(2): e1002375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKonde MK, Baker DP, Traore FA, et al.: Interferon β-1a for the treatment of Ebola virus disease: A historically controlled, single-arm proof-of-concept trial. PLoS One. 2017; 12(2): e0169255. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalone JH, Oliver B: Microarrays, deep sequencing and the true measure of the transcriptome. BMC Biol. 2011; 9(1): 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMichalak P: Coexpression, coregulation, and cofunctionality of neighboring genes in eukaryotic genomes. Genomics. 2008; 91(3): 243–248. PubMed Abstract | Publisher Full Text\n\nMorris J, Jensen LJ, Doncheva NT: stringApp 1.3.0. [Online; accessed 19-Februar-2018]. 2018. Reference Source\n\nOlejnik J, Forero A, Deflubé LR, et al.: Ebolaviruses Associated with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages. J Virol. 2017; 91(11): pii: e00179-17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrins KC, Cárdenas WB, Basler CF: Ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases IKKepsilon and TBK-1. J Virol. 2009; 83(7): 3069–3077. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRhein BA, Powers LS, Rogers K, et al.: Interferon-γ Inhibits Ebola Virus Infection. PLoS Pathog. 2015; 11(11): e1005263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edger: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchnoes AM, Ream DC, Thorman AW, et al.: Biases in the experimental annotations of protein function and their effect on our understanding of protein function space. PLoS Comput Biol. 2013; 9(5): e1003063. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeljkovic V, Glisic S, Muller CP, et al.: In silico analysis suggests interaction between Ebola virus and the extracellular matrix. Front Microbiol. 2015; 6: 135. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu G, Feng X, Stein L: Reactome FIs. [Online; Version 2016]. 2016. Reference Source"
}
|
[
{
"id": "39844",
"date": "06 Nov 2018",
"name": "Guanming Wu",
"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\nIn this manuscript, Farman et al described an approach to search for network modules based on p-values collected from differential gene expression analysis to address pathway crosstalk, which cannot be addressed in conventional pathway enrichment analysis approaches. During the past decade, many network module-based approaches have been developed to understand the functions of genes collected from differential gene expression analysis and other omics approaches (for a review, see Mitra et al, 20131). Though the network approach described here has some innovative ideas (e.g. searching for sources and sinks in subgraphs), however, the authors introduce their approach in the context of pathway analysis, without mentioning these previously published similar approaches, let alone comparing their approach to others. Also, it is worthy of mentioning that the described approach in this manuscript is very similar to jActiveModule (Ideker et al, 20122), the first of this kind, widely used for network-based data analysis.\n\nThe manuscript used the Ebola virus (EBOV) time course gene expression data set to show case MSF, trying to demonstrate the validity and usefulness of the approach. Indeed, the authors found that IFNA1 and IFNB1 are source genes in subgraphs across multiple time points, as reported by literature references. However, the authors have not discussed other genes in the found subgraphs, though the whole lists of them are provided in their GitHub site. IFNA1 and IFNB1 are among other source genes. The authors should develop a statistic approach to evaluate p-values and FDRs for subgraphs and individual source genes, therefore, providing users a way to choose the most significant genes for unknown phenotypes or biological processes. The current way to showcase the usefulness of the approach is not stringent and may not be useful if too many genes are collected in the subgraphs.\n\nThe authors compared MSF results with raw gene lists based on p-value cutoffs (Table 1). However, Table 1 is not a fair comparison. Only the largest subgraphs are listed for edgeR + MSF, while all subgraphs are listed for raw gene lists (e.g. for 6 hpi, 3 for edgeR + MSF, 39 for edgeR (p-value <= 0.1)). For a fair comparison, all subgraphs should be listed for edgeR + MSF too.\n\nSection “Comparison to Reactome pathway analysis tool” and Figure 4 compare results produced by Reactome pathway enrichment analysis for different gene lists. The whole section is confusing. First, the section title is misleading: the comparison is for results generated from Reactome pathway analysis tool, not to that tool. Second, I cannot see too much value in this setting using different adjusted p-value cutoffs for gene lists, probably one (e.g 0.05) should be enough, to reduce the clutter in Figure 4. Third, the authors want to point out MSF can enrich Toll-like receptor signaling pathway, but not the raw gene lists. However, such a comparison has not clearly indicated in Figure 4. The set comparisons include too many gene lists. Fourth, the authors should point out what p-value or FDR cutoff value used to choose pathways from the Reactome analysis tool. It is not correct to choose all pathways listed in that tool for comparison. Finally, where “Ten” toll-like receptor cascade pathways come from?\n\nSearching for sources/targets in individual MSF subgraphs based on the directions in the Rectome FI network and then drawing the schematic diagrams as illustrated in Figure 2 are interesting. It will be better to show directions in the Cytoscape network view (the right-side networks in Figure 2). The schematic diagrams in Figure 2 are interesting, but may dramatically simplify things occurring inside cells. The Reactome FI network provides functional relationships among genes or proteins, which are not necessary gene regulatory relationships. The authors should point this out in the manuscript.\n\nFinally, the writing of this manuscript is under question. The authors should really read their manuscript much more carefully. There are far too many typos, wrong uses of punctuations, and grammar errors. For examples, “the Filoviridea family; filamentous” should be “the Filoviridea family: filamentous”; “pathogenesis of Ebola. Thereby facilitating” should be “pathogenesis of Ebola, thereby, facilitating”; “among the most significant gene in the DGEA” should be “among the most significant genes in the DGEA”, and many others.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "4490",
"date": "22 Mar 2019",
"name": "Mariam R Farman",
"role": "Author Response",
"response": "First of all, we would like to say thank you for your valuable review and your critical comments. Thank you very much for making us aware of the paper by Mitra et al and work by Ideker et al. Frankly, these papers skipped our attention. Meanwhile, the review and the tool JactiveModules has been cited in the paper. Using the same expression data and same interaction file we compared the results from MSF and JactiveModules. Although the methods to find the overall score for the modules is similar, there are differences in the sub-graphs identified. The differences seen in the modules identified by the two methods are because MSF starts building the sub-graphs from one gene, incorporating and combining the p-value of the next gene, with the check that the combined p-value of new sub-graph should be better than the original. On the other hand jActiveModules first transforms all the gene’s p-values to z-scores and tries to find connected sets of genes with unexpectedly high levels of differential expression, in this case high z-scores. And then the overall score of the sub-network is calculated by combining the z-scores of the genes. Then using simulated annealing jActiveModules tries to find the highest scoring modules. Given the observed differences and our focus on the flow of perturbation, namely sinks and sources, we think that our contribution is not redundant to the previous work. To strengthen even further our perspective we also worked on the software itself, which now also scores the sources according to their reliability and the potential impact onto the modified sub-module. Since we believe that this improved the usability of the software critically, we would like to thank the reviewers particularly for point us to this. Again, we thank the reviewer for the valuable input to evaluate the source genes. We acted on this suggestion by amending the software. We have now incorporated an impact score for each source gene, which expresses the percentage of genes in the sub-module which are downstream of the particular source. This should be helpful to prioritize different sources of one sub-module. MSF also performes a t-test for each source gene, testing if the p-values ofthe downstream genes are different from the upstream genes. This would help to see if the source identified indeed marks the border between two different regulation regimes. The table (Table 1) has been modified. It shows number of MSF identified sub-graphs with the number of genes in it. When we apply different cut-offs to p-values of genes in the MSF identified sub-graph, it shows how they break from larger interpretable sub-graphs to smaller, less interpretable sub-graphs also consisting of single genes. Section “Comparison to Reactome pathway analysis tool” has been modified. Figure 5 (previously figure 4) setup has been changed to one cut-off only, i.e. 0.05. Ten Toll-like receptor cascades were seen to be enriched from the genes in MSF identified sub-graphs that did not appear in the cut-off gene list. Since MSF uses no cut-off, the sub-graphs identified had genes from Toll-like receptor cascades even when their signal was weak. Figure 5 caption modified for better understanding. A cut-off of 0.05 was used to choose pathways from Reactome pathway analysis for both MSF and cut-off gene list. The nine Toll-like receptor cascades have been mentioned in the manuscript, tenth being Toll-like receptor itself. Directions have been added to the output of MSF that could be easily imported into Cytoscape. In addition, source impact score and log-fold sign for each individual gene can also be imported into Cytoscape. Functional relationship provided by Reactome FI has been mentioned. The schematic diagram, meant to give our take on the interpretation of the raw MSF output, was removed since we agree on the oversimplification criticism. The writing of the manuscript has been carefully checked and improved."
}
]
},
{
"id": "40009",
"date": "12 Nov 2018",
"name": "Stefanie Widder",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a novel method for finding groups of genes that concomitantly change their expression profile upon signals and conditional changes. As opposed to standing tools in the field that rely on predefined functional classification, this approach is based on topology of the global interaction network and enables an unbiased identification of larger functional blocks highlighting pathway cross-talk. It furthermore includes a topological method for identifying source and sink pathways that provides a prediction of process causality. The latter is particularly useful for hypothesis generation and add-on experimental validation far beyond the field of biomedicine.\nThe paper is structured into the presentation of the algorithm, a biomedical use-case with validating background information and a slim benchmarking against differential expression analysis with different cut-offs.\nWhile the method clearly fills an existing gap in high-throughput gene expression analysis and is very elegantly reasoned, I would like to raise a number of comments with regards to writing and benchmarking.\n\nIntroduction:\nThe main line of argumentation gets lost sometimes, in particular in paragraph 1. The narrative would furthermore benefit from actual examples instead of repeating a general statement of ‘changed conditions’. Sometimes, the same argument is repeated in differently phrased sentences.\n\nParagraph 2: Suppress ‘be subjective to some degree’.\n\nParagraph 4: Better highlight and delimit the novelty of the presented approach.\n\nOverall, shorter sentences benefit the reader.\n\nMethods:\nThe context of Hartung’s method is described nicely, yet the actual way how the individual p-values are combined to result in a single measure is omitted. Please include as this information provides instructive benefit to the reader.\n\nResults:\nCase Study:\nParagraph 1: Readability would profit from focusing the background information, e.g. 'Ebola infection data was (->were) selected (...)' - rephrase into a half-sentence.\n\nWording: Until now->currently.\nRobustness:\nThe recall-comparison (MSF to differential expression analysis) is weakening the proposed method, because no increase in recall for MSF can be achieved. It would be informative to see also precision stats in comparison. Generally, one would expect more robust statistics on larger subgraphs (MSF vs. DEG groups).\n\nAlso, I am not entirely convinced by adding noise to real and thus – already - noisy data. A small, artificial mock example with detailed known outcome (+/- noise) might be more suitable and supportive.\nFigure 4:\nThis figure is very difficult to follow. I suggest to i) enhance the label sizes and texts, with particular emphasis on delimiting MSF and DEG, ii) improve the figure caption text including fundamental information of what is shown.\n\nGithub material:\nImportantly - provide an example directory that contains complete examples cases (+*all* files) including the presented Ebola case + options and outcome to enable a swift recapitulation of the tool for the new user.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4491",
"date": "22 Mar 2019",
"name": "Mariam R Farman",
"role": "Author Response",
"response": "Thank you for your helpful comments about our manuscript. Paragraph 1 has been rephrased, “changed conditions” has been replaced with example of treated verse healthy example. Paragraph 2 modified accordingly. The novelty of MSF is provided in paragraph 5. Firstly, MSF does not use the predefined sets of genes to find the modulated sub-graphs but starts building the sub-graph by using the information from DGEA and interactions from whole cell signaling network. Second MSF considers the signal of the neighbouring genes to find significance of the modulated sub-graph. We revisited the overall text and tried to emphasis more on clear readability. More details about Hartung’s methods are provided in the appropriate section. The case study is rephrased and wording modified. The reviewer is correct about the recall-comparison weakening the proposed method. We have been very strict with testing the robustness of the method by adding extra noise to already noisy data. We expected the method to be more robust than DGEA but unfortunately that was not the case. Since for cut-off based DGEA robustness it does not matter if a few genes p-values go up or down as long as they are below the chosen cut-off. In contrast, for MSF the robustness analysis showed that it makes a difference for the sub-graphs identified. The reviewer is correct again to expect more robustness in the larger sub-graphs which is shown at later time-point 48 hpi that has larger sub-graphs than the other two time-points. If you look at the robustness of MSF alone it varies from 71 % (6 hpi) and 84 % (48 hpi) , we agree that the numbers are not outstanding but we consider them to be reasonable. Since our assumption that a post analysis with MSF can not only gain insights into the pathway modulation but also improve the DGEA for single genes, using their neighbours information did not hold, we removed that aspect to improve readability. We agree that the data is already noisy even before adding noise. Again, we have been very strict to test the robustness of the method,. As mentioned above the larger the sub-graphs the more robust they are, we are not sure that having a small mock example would elaborate more. Figure 5 (previously figure 4) set-up has been changed to one cut-off only i.e. 0.05. Figure label size and texts enhanced. Figure caption modified. Unfortunately we can not provide the Ebola count files since they belong to Olejnik et al. A tutorial is provided to reproduce the results from the Ebola infection data, with details on how to obtain the raw data used from the third party source (GSE84188). MSF output files are also provided in the supplementary material."
}
]
},
{
"id": "40208",
"date": "29 Nov 2018",
"name": "Haibo Liu",
"expertise": [
"Reviewer Expertise bioinformatics and computational biology",
"transcriptomics and systems 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\nIn this manuscript, the authors reported their newly developed tools, MSF, for interpreting gene lists from differential gene expression analysis. Their tool differs from existing pathway analysis tools: (1) it can identify concertedly modulated sub-graphs from user-provided gene networks, thus it can accounting for cross-talk between pathways; (2) it can potentially infer the flow of biological information in response to a perturbation from source to sink. Like the gene set enrichment analysis (GSEA), no arbitrary p-vale threshold is set to dichotomize whole gene lists before applying MSF analysis, instead all genes from DGEA are ordered by p-values from the smallest to the largest. An algorithm similar to the widely used network propagation algorithm is used for subgraph initialization and extension. The authors applied their tools to analyze an RNA-seq dataset from an Ebola virus infection experiment and showed their tool outperform the other tools. They concluded this tool, fast, robust and easy-to-use, is a good supplement to existing pathway-based analysis tools. However, the overall writing is very problematic and there are quite a few issues needing to be fixed. See the details listed as follows.\n\nFirst of all, the code for the tool is not available via Github. I carefully checked multiple times the Github repository: https://github.com/Modulated-Subgraph-Finder/MSF, however I can't find the ModulatedSubPathFinder.jar file, which is the Java implementation of the proposed tool, MSF. There are too many grammar issues and writing issues. Just mentioned a few, in the first paragraph of Introduction, \"mechanism\" in Line 6 should be plural, while \"stimuli\" in Line 14 should be \"stimulus\", \"maybe\" in Line 15 should be \"may be\". Careful proof-reading is strongly recommended. The authors have a few misconceptions. For example, they treated \"effectors\" and \"sinks\" equally. In my opinion, effectors include sources, intermediate genes and sinks, i.e., all genes responding to perturbations. The authors think of the significance of statistical tests in the form of p-values as a confidence level of observing an authentic expression change. This might not be correct. Besides small p-values, the magnitude of fold changes is also important metric of authentic expression change. By the way, the fold change of gene expression is always non-negative. The expression of \"sign of fold change\" is not meaningful. Only log-transformed fold change is signed. The flow of information/idea is not fluent in many places. For example, at the end of the first paragraph of Introduction, the authors mentioned the KEGG and Reactome Pathways. Then at the beginning of the second paragraph, they gave a detailed description of the two pathway databases, which might be unnecessary and disrupted the flow to set up the stage to introduce why their tool is necessary and useful. Some information about how their tool was implemented given in the last paragraph of Introduction should be moved to the Implementation section of Methods. Paragraph 3 under the section of “Case Study”, the DEGA results might better be described immediately after the second sentence of Paragraph 2. Similarly, some information in Conclusion should be move to the section of “Implementation”. The title for the section of \"Initial modulated sub-graphs” should be “Initializing modulated sub-graphs “. Under this section, “starting with the most significant one” should be “starting with the next most significant one”. “… not yet in a significantly modulated …” should be “…not yet in the significantly modulated …”. Under the section of “Extending modulated sub-graphs”, it is not clear how the sub-graphs are extended by adding “MORE THAN ONE” gene at a time. If doing this way, there are infinite possibilities. The criterion to accept or reject added genes is not clear. Under the section of “Merging modulated sub-graphs”, the authors mentioned that “After detection and extension of the modulated sub-graphs, they are tested if combined subgraphs SCORE better than on their own.” At this point, no merging has been done yet, how are the combined subgraphs tested? What is the SCORE used here? How can a depth-first search traverse from the FIRST sub-graph to the SECOND sub-graph before they are merged (Aren’t the subgraphs not necessarily connected?)? Under the section of “Finding sources & sinks”, “circular loops” should be just “loop”. Paragraph 2 Under “Case Study”, some details about edgeR-based DEGA are missing. How the directed cell signaling interactions ere filtered from the Reaction FIs? Based on what? The directions of edges in right panel of Figure 2 should be showed, because the MSF can generate directed subgraphs. In the last paragraph of the section of “Modulated sub-graphs at 6 hpi”, “This show cast example…” should be “This show case example…”. The Robustness test seemed to show that the MSF is not robust enough to extra noise. What is the authors’ conclusion and explanation? The authors compared the results from their tool to those from the Reactome pathway analysis tool and demonstrated better performance of their tool. Can they also compare the results from their tool to those from the GSEA tools, which don’t set arbitrary cutoff beforehand? The latter comparison might be more convincing. In Discussion, what are “intermediate bottlenecks”? In Conclusion, the authors claimed their tool is “fast, robust and easy-to-use”. However, they did provide evidence to show their tool is fast. The robustness of their tool is not apparent. Issues with figures: all legend titles are too long. In Figure 1, Texts in the flow chart symbols are not well-written. There are inconsistent case issues; “initial” should be “initializing”; symbol for condition test (“checked all interaction?”) should be diamond, not hexagon. Question marks might be added to condition test for readability. In the legend to Figure 1, what does “without exhaustively testing all connected subgraphs” mean? Does it the output from MSF analysis might not be comprehensive? Legends to Figure 3 and 4 are poorly written. Is the Toll-like receptor signaling one of the 149 shared pathways in Figure 4. It is not clearly described in the legend. Limitations of their tool: In their implementation, the fold changes and direction of changes are not taken into account. If a resulting subgraph contains both down-regulated genes and up-regulated genes, how should the users interpret it? The authors didn’t test the sensitivity and specificity of their tool in this manuscript.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "4492",
"date": "22 Mar 2019",
"name": "Mariam R Farman",
"role": "Author Response",
"response": "Thank you very much for your suggestions. So far the ModulatedSubPathFinder.jar was only available under the release tag on git hub (https://github.com/MariamFarman/Modulated-SubGraph-Finder/releases). Meanwhile we added the full source code to git hub. The writing of the manuscript has been carefully checked and improved, especially the examples mentioned by the reviewers. We agree with the reviewer that we used the word “effector” not very cautiously. Therefore, we changed it accordingly. The reviewer is again right to consider the magnitude of the fold change as an important metric of the system behaviour. We consciously ignore it since it is not straight forward to include it in our model, and we consider that the p-value at least partly reflect the magnitude of fold change since it expresses the probability that the observed fold change differs from 1. The suggestions are taken into account and the text was modified accordingly. The details about KEGG and Reactome were considered necessary since later the information from both the databases would be used to showcase the results of MSF. Text modified. The use of extending sub-graph here is if the sub-graph could not be extending any more by one gene because the direct neighbouring genes have high p-values. To avoid producing fragmented sub-graphs, we try to, instead of single genes, append branches of up to three genes simultaneously. Thereby the accumulated signal of the whole branch can compensate for single unfavourable genes. Since we limit the procedure to branches of length three, the number of possibilities to be tested is limited. Again, a branch is only accepted if the overall score of the sub-module is better after extension. Details about criterion of rejection and acceptance of extension was added to the text. The wording has been changed for better understanding of the paragraph describing merging of sub-graphs. The score to pass merging is that the combined p-value of the sub-graph after merging two sub-graphs (including connector genes) is smaller than the individual p-values of the two sub-graphs. The depth first search traverse is used to find connectors between the two sub-graphs to merge them. Text modified. Details about edgeR were added to the text. The interaction file downloaded from Reactome is filtered for only direct interactions . The tutorial on how the file was filtered is also provided on MSF git hub page. The directions have been added to the figure and as a output for MSF to import in Cytoscape for easy visualization for the user. Text modified. While developing the algorithm we believed MSF would not only give insights into the network modulation but could also be used to increase robustness of DGEA of single genes by using additional information from their neighbours. Our analysis disproved this assumption, which we wanted to communicate with this robustness analysis. Given the comments by several reviewers we adapted the corresponding paragraph, omitting the comparison to the DGEA but showing only the overall robustness of our tool which is, after applying strong noise, still reasonable with a median recall rate of 71 to 84%. MSF analysis comparison with GSEA analysis added. Although GSEA was able to find pathways known from literature to be dis-regulated during Ebola infection, it could not show the cross-talk between the different pathways like MSF does. By intermediate bottlenecks we actually meant a gene that is actually connecting a number of sources with a number of sinks and thereby a potential point of intervention if one would like to uncouple the input stimuli from the downstream effects. As stated above the reviewer is right about the robustness, the text has been modified accordingly. The figure legends have been rewritten for better understanding. Figure 1 chart symbols edited, symbols shapes modified as suggested. With the phrase “Without exhaustively testing all connected sub-graphs” we intended to say that not all possible connectors to merge the graphs are tested but sub-graphs are connected with the first connector that passes the threshold. In figure 5, Toll-like receptor signaling is actually in the 164 shared pathways between different time-points of MSF. We agree and are aware that magnitude and direction of the fold changes are important. On the magnitude we commented already further above. For the direction, we would like to mention that its interpretation is not straight forward without further information of the type of interaction between the genes, which is not always available. To illustrate, the up-regulation of an inhibitor and the down regulation of an activator can have the same effect on the network."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1346
|
https://f1000research.com/articles/8-458/v1
|
12 Apr 19
|
{
"type": "Research Article",
"title": "Evaluating the retention of skills in postgraduate physician students following a theoretical-practical course in Advanced Cardiovascular Life Support",
"authors": [
"Juan Pablo Holguín Carvajal",
"Rodrigo Alejandro Robalino Guerrero",
"Carla Marina Salgado Castillo",
"Luis René Buitrón Andrade",
"Carla Patricia Zamora Rosero",
"María Fernanda Salgado Castillo",
"Juan Pablo Holguín Carvajal",
"Rodrigo Alejandro Robalino Guerrero",
"Carla Marina Salgado Castillo",
"Luis René Buitrón Andrade",
"Carla Patricia Zamora Rosero"
],
"abstract": "Background: For every minute CPR is delayed, the probability of survival decreases by up to 10%. For this reason, guidelines recommend routine CPR training for health care providers to improve their performance and patient results. The objective of the present study was to evaluate the retention capacity of postgraduate students of Critical Areas of the Pontificia Universidad Católica del Ecuador following a theoretical-practical course in Advanced Cardiovascular Life Support (ACLS). Methods: A total of 140 students were recruited and divided into three groups according to studied subject: Emergency Medicine and Disasters, Anesthesiology, and Critical Medicine. A theoretical-practical course was carried out, and theoretical and practical skills were assessed immediately and subsequently one month after ACLS training. For statistical analysis, measures of central tendency, one-way ANOVA, T-test and ANCOVA were used. Results: Scores for the immediate theoretical exam were 58.6% immediately after the intervention vs 40% 30 days after the intervention; in the immediate practical exam this was 77% vs 35.7%, respectively. No statistically significant difference was found between the three groups for the initial practical examination; however, for the evaluation 30 days after training a significant difference was found between Anesthesiology and the other two postgraduate studies. Conclusions: Knowledge and practical skills in ACLS of postgraduate physicians of Critical Areas deteriorate 30 days after training, especially in practical skills compared with theoretical knowledge. The results of this research indicate that it is necessary to carry out update courses more frequently, in order to keep knowledge and skills at a level that guarantees adequate care to the patient to reduce potential risk of death or disability.",
"keywords": [
"Advanced Cardiovascular Life Support",
"ACLS",
"Cardiopulmonary Resuscitation",
"Critical Areas"
],
"content": "Introduction\n\nDeaths related with cardiovascular disease occur at earlier ages in developing countries. In Latin America and the Caribbean, men are at risk of premature death as a result of this disease, although in recent years the rate has increased in women1. People who are victims of cardiovascular disease face difficulties due to the disability of the disease, which affects their families and the economy. According to the World Health Organization, the mortality rate in the Americas in 2016 with regard to ischemic heart diseases, cerebrovascular diseases and diabetes mellitus was 63.1, 35.2 and 33.5, respectively, out of 1000,0001. Between the years 2010 and 2013, ischemic heart diseases were the leading cause of death in the Americas (10.99%), followed by cerebrovascular diseases (6.70%) and diabetes mellitus (5.49%)2. Just in Ecuador, the number of deaths from heart disease in 2010 was 11992 (51.68% in men and 48.32% in women)3.\n\nMortality from arterial hypertension, cerebrovascular disease and diabetes mellitus has increased in general4; therefore, it has been found that adequate knowledge in cardiopulmonary resuscitation (CPR) is essential in order to reduce mortality caused by these conditions. The early onset of CPR and defibrillation are important and necessary for the reduction of morbidity-mortality in patients with cardiorespiratory arrest. For every minute CPR is delayed, the probability of survival decreases by up to 10%. For this reason, guidelines recommend routine CPR training for health care providers to improve their performance and patient results5,6.\n\nIn the present study, the retention capacity of the Advanced Cardiovascular Life Support (ACLS) course was evaluated in postgraduate students of Critical Areas of the Pontificia Universidad Católica del Ecuador.\n\n\nMethods\n\nThe study was conducted between January and February of 2017 at the Pontificia Universidad Católica del Ecuador, Quito-Ecuador. A total of 219 students from critical areas (Emergencies and Disasters, Intensive Care and Anesthesiology) were involved in the study. Taking a heterogeneity of 50%, with a margin of error of 5% and a level of confidence of 95%, the sample was 140 students. For the selection of the sample, randomized sampling was carried out. Groups of 7 people were organized for each evaluator in a random manner, with a total of 14 participants per day, ending at the 10th day with the 140 participants. Eligibility criteria: post-graduate students from the critical areas who wished to participate in the study, and who were not on duty at time of the intervention.\n\nThe students were identified and invited to the courses from the university \"Pontificia Universidad Católica del Ecuador\" database (postgraduate students from critical areas). Every student has to pass these tests at least once during their studies according to the University's requirements. The participation in the study was voluntary.\n\nInformed written consent was obtained from the participants prior to the study start.\n\nA theoretical-practical ACLS course was provided to postgraduate-students of Medicine at Pontificia Universidad Católica of Ecuador according to 2015 Guidelines ACLS Course. A theoretical and practical post-test was provided to the participants according to the 2015 RCP guidelines. The instructors were certified in ACLS and Basic Life Support (BLS) with experience in the practice, teaching and performance evaluation.\n\nThe participants had a theoretical assessment in the first 10 minutes (see Extended data7) to determine prior knowledge in ACLS, then for 1 hour 30 minutes a theoretical intervention based on Adult Cardiac Arrest Algorithm-2015 (Update of the American Heart Association (AHA) was performed with the participants.\n\nLater an practical intervention, where simulated practical cases were performed, was provided in order to develop the skills required to administer high-quality cardiopulmonary resuscitation together with the adequate recognition of defibrillable and non-defibrillable rhythms, according to the 2015 AHA guidelines.\n\nTheoretical standardized tools were used for evaluating the course received. All the tests used the same scenarios, AHA's Skill Assessment List (practices), and private simulation environments, using mannequins and tools.\n\nThe exam results were expressed in percentage according to the ACLS book for instructors8, that states that 84% must be reached for the theoretical exam; and all the steps of the checklist must be met to pass the practical exam. The checklist to evaluate the practical test is available in Extended data9.\n\nAfter 30 days, the theoretical and practical evaluation was carried out once again for each of the participants.\n\nCohen Kappa index was initially performed to determine the concordance and standardization of the knowledge taught in the intervention. The result was 0828, which indicates a high agreement between the two instructors10; therefore, it was possible to continue with the study.\n\nThe average results of the first evaluation were compared with the average results of the second evaluation (30 days after intervention). Central tendency and dispersion tests were performed, as well as Student’s t-test, one-way ANOVA and ANCOVA. The statistical analysis was carried out using the SPSS software version 24.0.\n\n\nResults\n\nThe characteristics of the participants are detailed in Underlying data11. There were statistically significant differences in the test scores between the subject groups for the first theoretical test immediately after the ACLS course. It was observed that these differences were between the Emergencies and Disasters and Anesthesiology groups (p< 0.05), as well as between the Critical Medicine and the Anesthesiology groups (p = 0.027). No statistical difference was seen between the Emergencies and Disasters and Critical Medicine groups. Likewise, for the second theoretical test score (30 days after the ACLS course), a statistical difference was seen between the Emergencies and Disasters and Anesthesiology groups (p < 0.05), and the Critical Medicine and Anesthesiology groups (p= 0.020), but not between the Emergencies and Disasters and Critical Medicine groups (p= 0.539).\n\nThere was no statistically significant differences in the first practical examination between the groups when evaluating their practical skills acquired immediately after the intervention of ACLS (p = 0.066). However, in the second practical examination assessed after 30 days of the initial intervention, statistically significant differences were identified. When conducting the post-hoc test through Scheffe, it was observed that there were differences in the level of practical skills between the Emergencies and Disasters and Anesthesiology groups (p= 0.04), as well as between the Critical Medicine and Anesthesiology groups (p= 0.013). A statistically significant difference was not observed between the Emergencies and Disasters and Critical Medicine groups (p= 0.994).\n\nFor all students for the theoretical examination, there was a decrease in the result (Table 1) between the test carried out immediately after the intervention (mean = 83.43) and 30 days after the intervention (mean= 76.21) (p< 0.05). In the practical examination, a statistically significant decrease was found in the result between the test carried out immediately after the intervention (mean = 93.81) and 30 days after the intervention (mean = 75.71) (p< 0.05).\n\nFinally, a covariance analysis (ANCOVA) was performed for the results of the theoretical and practical examinations after 30 days of the intervention on ACLS, in order to investigate whether the relationship between the variables found is maintained when controlling the effect (introducing them as covariates) (Underlying data7).\n\nSignificant variables for scores in the theoretical test after 30 days were: age and sex of the participant, stress, year of the postgraduate course, validity of the certificate in ACLS, number of times that the person used the information from the ACLS course during clinical practice, if they were instructors of ACLS, and regularity of the studies. No statistically significant differences were found in relation to rest during the previous night in the results of written examination. Unlike the results of the theoretical examination, no statistically significant differences were found in the participant's post-shift status in the 30-day practical evaluation.\n\n\nDiscussion\n\nThere are international organizations, such as the American Heart Association, which have developed cardiopulmonary resuscitation treatment programs; at Pontificia Universidad Católica del Ecuador, it is considered essential in undergraduate with Basic Life Support (BLS) program and postgraduate (BLS and ACLS Advanced Cardiovascular Life Support) training. However, these are no courses carried out in health centers where these students receive their daily training, instead these are organized by scientific companies.\n\nThe present study is the first study, to the best of our knowledge, carried out in Ecuador that compares the retention of theoretical and practical knowledge of postgraduate students in Critical Areas courses, including Emergencies and Disasters, Anesthesiology and Critical Medicine. These students are the ones who have the highest possibility of facing situations that require advanced cardiovascular care.\n\nThe findings of this study are consistent with previous literature that has shown that the knowledge and practical skills of health providers have been reduced before the recommended training interval (2 years). The average in which this reduction is observed is between 6 months to 1 year10,12; however, there are not many previous studies that indicate the behavior between 1 to 6 months.\n\nIn the present study, the participants' theoretical evaluations showed an average of 83.43% immediately after the intervention, and 76.21% in the 30-day post intervention evaluation. The practical examination showed an average of 93.81% immediately after, and 75.71% at 30 days; therefore, it is concluded that practical skills have more declination than theoretical knowledge, which agrees with the literature, which indicate that practical skills are more affected over the time12–16, and also that the results of theoretical examinations are not good predictors of practical evaluation results17. In this study, the most impaired practical skills were: treatment according to the rhythm of arrest, ensure scene, perform aftercare and adequate recognition of arrest rhythms. These findings suggest that appropriate intervals and re-training strategies should be differentiated between knowledge and skills, with reinforcement of the latter.\n\nComparing the three postgraduate groups in Critical Areas, it was found that the Emergency and Disaster and Critical Medicine groups did not have statistically significant differences in the knowledge of both written and practical evaluations within 30 days of the intervention. However, it was observed that there is a difference between the Anesthesiology group with the other two groups (p < 0.05). Similar results were observed in the study carried out by Botha et al. (2012) in which the participants of Emergency Medicine had a better overall performance in the evaluations with respect to the participants of Anesthesiology, taking into account that they both belong to Critical Areas18. The reason for having less retention in this group can be explained by several factors, including the low incidence of patients with cardiorespiratory arrest in operating rooms, where anesthesiologists perform their daily work. In a study conducted by An et al. (2011) in Pittsburgh, USA, 23 cases of intraoperative cardiac arrest of a total of 218274 patients were found, resulting in a prevalence of 1.1 cases for 1000019. Hence, the application of the Adult Cardiac Arrest Algorithm-2015 Update of the American Heart Association for Advanced Cardiovascular Life Support (ACLS) will be very infrequent and will not be enough to maintain knowledge and skills. Conversely in Department of Emergencies and Intensive Care Unit, 42% of all hospital cardiac arrests are recorded in these departments according to a study conducted in Uganda20; in another study carried out in the USA 59% of these events were reported in these departments21.\n\nIn conducting covariance analysis in this study, a statistically significant relationship was found between time of the last training course of ACLS with satisfactory results in theoretical and practical evaluations, which means that the more recent the intervention, the better results there will be. This agrees with the results of a study conducted by Jensen et al. (2009), which found that declination of knowledge in ACLS was lower when courses were conducted at 0 and 6 months, with evaluations at 6 months and 12 months, with average scores of 73, 85 and 82%, respectively22.\n\nIn relation to the years of clinical practice, it was observed in the present study that this factor influenced the evaluation results (p< 0.05). In the study carried out by Yang et al. (2012), it is mentioned that the participants who had at least half a year of clinical practice, obtained better results than those that had less time13.\n\nOn the contrary, there was statistically significant difference between the results and level of stress a student had. This contrasts with the study of Júnior et al. (2002), which shows that stress significantly affects the performance of the participant, and sleep time did not affect the results23.\n\nThe study has several limitations. Since the qualification of the evaluations do not affect the student's final performance in the post-graduate degree, the written examination grade may not reflect the participant's knowledge. Since the research was held at Pontificia Universidad Católica of Ecuador, it is difficult to generalize the study, taking into account that in this institution it is compulsory for graduate students to perform training in ACLS; however, in most universities in Ecuador, these are not conducted, so the level of previous knowledge will vary depending on the situation.\n\n\nConclusions\n\nCardiovascular diseases are included in the first 10 causes of mortality in Ecuador, which means it is necessary to perform training in the management of cardiorespiratory arrest.\n\nKnowledge gained from courses in ACLS of post-graduate studies in Emergencies and Disasters, Anesthesiology and Critical Medicine deteriorate after 30 days in advanced cardiovascular life support. It should be emphasized that practical skills are the most affected compared with theoretical skills.\n\nThe results of this research indicate that it is necessary to carry out courses more frequently, in order to keep knowledge and skills at a level that guarantees that adequate care to patients is provided, reducing potential risk of death or disability.\n\n\nData availability\n\nFigshare: Raw data for each participant. https://doi.org/10.6084/m9.figshare.7965020.v111.\n\nFigshare: Data for Evaluation of the retention of skills in advanced cardiovascular life support (ACLS), following a theoretical-practical intervention in postgraduate physician students of critical areas. https://doi.org/10.6084/m9.figshare.7844999.v17.\n\nThis project contains the following aggregated underlying data:\n\n- Characteristics of the participants\n\n- Performance of the participants\n\n- Results of the evaluation\n\n- Practical skills\n\n- ANCOVA written\n\n- ANCOVA practical\n\nFigshare: Survey used in Evaluation of the retention of skills in advanced cardiovascular life support (ACLS), following a theoretical-practical intervention in postgraduate physician students of critical areas. https://doi.org/10.6084/m9.figshare.7848245.v19.\n\nFigshare: Checklist for practical exam. https://doi.org/10.6084/m9.figshare.7940606.v124.\n\nAll data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nOMS, OPS: Indicadores Básicos 2016. 2016; 9. Reference Source\n\nOMS, OPS: Leading causes of deaths. Paho. org. [cited 2016 Nov 3]. Reference Source\n\nINEC: Egresos Hospitalarios 2010/Anuario de Estadísticas Vitales: Nacimientos y Defunciones. Año 2010/Defunciones años desde 2004 al 2010. 2010.\n\nMsp: Datos esenciales de Salud: una mirada a la decada 200-2010. Minist Salud Pública. 2012; 1–60. Reference Source\n\nHunziker S, Johansson AC, Tschan F, et al.: Teamwork and leadership in cardiopulmonary resuscitation. J Am Coll Cardiol. 2011; 57(24): 2381–8. PubMed Abstract | Publisher Full Text\n\nBhanji F, Donoghue AJ, Wolff MS, et al.: Part 14: Education: 2015 American Heart Association Guidelines Update for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation. 2015; 132(18 Suppl 2): S561–73. PubMed Abstract | Publisher Full Text\n\nSalgado Castillo MF: Data for EVALUATION OF THE RETENTION OF SKILLS IN ADVANCED CARDIOVASCULAR LIFE SUPPORT (ACLS), FOLLOWING A THEORETICAL-PRACTICAL INTERVENTION IN POSTGRADUATE PHYSICIAN STUDENTS OF CRITICAL AREAS. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7844999.v1\n\nLibro del instructor de SVCA/ACLS en versión electrónica. American Hearth Association. Dallas, USA. ISBN: 978-1-61669-539-2. Reference Source\n\nSalgado Castillo MF: Survey used in EVALUATION OF THE RETENTION OF SKILLS IN ADVANCED CARDIOVASCULAR LIFE SUPPORT (ACLS), FOLLOWING A THEORETICAL-PRACTICAL INTERVENTION IN POSTGRADUATE PHYSICIAN STUDENTS OF CRITICAL AREAS. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7848245.v1\n\nBuitrón Andrade LR: Estudios de Concordancia y coeficiente de correlación interclase. In: Herramientas en Epidemiología. Primera Ed. Quito: Pontificia Universidad Católica del Ecuador; 2016; 75–82.\n\nSalgado Castillo MF: Raw data for each participant. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7965020.v1\n\nSmith KK, Gilcreast D, Pierce K: Evaluation of staff's retention of ACLS and BLS skills. Resuscitation. 2008; 78(1): 59–65. PubMed Abstract | Publisher Full Text\n\nYang CW, Yen ZS, McGowan JE, et al.: A systematic review of retention of adult advanced life support knowledge and skills in healthcare providers. Resuscitation. 2012; 83(9): 1055–60. PubMed Abstract | Publisher Full Text\n\nBurkhardt JN, Glick JE, Terndrup TE: Effect of prior cardiopulmonary resuscitation knowledge on compression performance by hospital providers. West J Emerg Med. 2014; 15(4): 404–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams NM: Advanced life support training and assessment: A literature review. Australas Emerg Nurs J. 2011; 14(4): 240–5. Publisher Full Text\n\nSutton RM, Nadkarni V, Abella BS: \"Putting it all together\" to improve resuscitation quality. Emerg Med Clin North Am. 2012; 30(1): 105–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodgers DL, Bhanji F, Mckee BR: Written evaluation is not a predictor for skills performance in an Advanced Cardiovascular Life Support course. Resuscitation. 2010; 81(4): 453–6. PubMed Abstract | Publisher Full Text\n\nBotha L, Geyser MM, Engelbrecht A: Knowledge of cardiopulmonary resuscitation of clinicians at a South African tertiary hospital. South African Fam Pract. 2012; 54(5): 447–54. Publisher Full Text\n\nAn JX, Zhang LM, Sullivan EA, et al.: Intraoperative cardiac arrest during anesthesia: a retrospective study of 218,274 anesthetics undergoing non-cardiac surgery. Chin Med J (Engl). 2011; 124(2): 227–32. PubMed Abstract | Publisher Full Text\n\nOcen D, Kalungi S, Ejoku J, et al.: Prevalence, outcomes and factors associated with adult in hospital cardiac arrests in a low-income country tertiary hospital: a prospective observational study. BMC Emerg Med. 2015; 15: 23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeberdy MA, Kaye W, Ornato JP, et al.: Cardiopulmonary resuscitation of adults in the hospital: a report of 14720 cardiac arrests from the National Registry of Cardiopulmonary Resuscitation. Resuscitation. 2003; 58(3): 297–308. PubMed Abstract | Publisher Full Text\n\nJensen ML, Lippert F, Hesselfeldt R, et al.: The significance of clinical experience on learning outcome from resuscitation training-a randomised controlled study. Resuscitation. 2009; 80(2): 238–43. PubMed Abstract | Publisher Full Text\n\nLima E Jr, Knopfholz J, Menini CM: Stress during ACLS courses: is it important for learning skills? Arq Bras Cardiol. 2002; 79(6): 589–92, 585–8. PubMed Abstract | Publisher Full Text\n\nSalgado Castillo MF: Checklist for practical exam. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7940606.v1"
}
|
[
{
"id": "52300",
"date": "13 Aug 2019",
"name": "William D. Grant",
"expertise": [
"Reviewer Expertise Statistics",
"clinical research design and implementation",
"emergency medicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe reference that delay of CPR leads to 10% per minute reduction in survival is from a 10 year old reference. Given the changes in CPR administration and training over the past 10 years (such as no breath CPR) is there a more up-to-date figure?\n\nThe details of what specifically was examined are limited? What were the specific score parameters for the theoretical and for the practical sections?\n\nAs there is no global agreement as to the standards of acceptability of homogeneity and the fact that the values are almost always determined post hoc, and the admission that this is the first study of its type, what standard was used to determine that 50% was acceptable and how was this tested to determine if it was met at the end of the study?\n\nSample size is usually determined as the basis of the smallest intended group for analysis not for over-all sample size. Given that the individual critical areas samples are small this should be addressed in the limitations.\n\nThe study is actually a repeated measures study and should be addressed as such.\n\nLittle demographic data is presented in the text but the reader is referred to the available data to do their own determination. Table 1 of the study should be a demographics table by the three groups.\n\nStatistical results presentations are undergoing evolution with less emphasis being placed on p values (see recent NEJM announcements). Each statistical finding should be presented with the statistic which provides information on the likelihood of observing a specific value using a specific analysis and should include a 96% Confidence Interval estimate for each difference noted. These two pieces of information provide different information. One is the likelihood of an observed value and the other is an estimate of the magnitude of the difference between the groups. For example: \"...the difference between the groups was 15.7 [t=3.67,df 2, p<0.05 95%CI +/- 3.6],....\"\n\nTable 1 should include N's for each group especially if the numbers changed from post test to follow-up.\n\nThere is information on the field of learning and practice effect including that learning occurs in plateau fashion. Students show learning growth, reach a plateau even while practising, then gain again in step increase manner. So it is not just the frequency of practice but also the tracking of students' skills to identify when they have reached a plateau and need to be pushed through it to reach proficiency.\n\nThe study focuses on pre-post-follow-up differences. Unless it was missing in reading there was no measurement against a minimally acceptable standard. For example in Table 1 Emergencies and Disasters had a post practical score of 79%. Does this mean that their score is so bad that they should be prevented from administering CPR? What is the acceptable standard?\n\nThank you for the opportunity to review this article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "52804",
"date": "02 Sep 2019",
"name": "Eduardo Kattan",
"expertise": [
"Reviewer Expertise Intensive Care Medicine",
"Anesthesiology",
"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\nI appreciate the opportunity to revise this manuscript. In brief, the authors have performed a theoretical and practical course on CPR in postgraduate students of critical areas in Ecuador.\n\nAfter finishing the course, they tested one month later the retention of students both in the theoretical and practical aspects delivered during the course. There is a significant decrease in cognitive and skills performance, and it is further analysed according to each speciality training.\n\nI believe this is a well-written manuscript, with clear objectives, adequate methodology and a developed discussion that addresses the relevant topics involved in CPR training and simulation training in general.\n\nI have a few comments, including:\nConsider including the following updated reference for delays in CPR: Bircher NG, Chan PS, Xu Y; et al. Delays in Cardiopulmonary Resuscitation, Defibrillation, and Epinephrine Administration All Decrease Survival in In-hospital Cardiac Arrest. Anesthesiology. 2019 Mar;130(3):414-422. doi: 10.1097/ALN.0000000000002563.1\n\nTable 1 should include N° of participants in each group, plus the standard deviation of each measurement. Even though results are expressed as a percentage, they are an average of a score of the study group, as presented in the table’s title. In this sense, a dispersion value is needed to better understand the sample's distribution.\n\nWhen mentioning the use of Students’ t-test, did you refer to both independent and paired measures test? Considering that the comparison between groups are independent groups but intra-group comparison is a repeated measure test? If so, it should be specified in the methods section.\n\nWhat happened with the students that failed to meet the minimum passing score? Are there remedial sessions or new tests performed?\n\nAfter this study, will there be any change of conduct in your institution? What would you recommend for future versions of the course? Include more training sessions to the program? Shorten the gap between training sessions? Specific targeting of the groups that performed poorly?\n\nI believe this is an interesting study, that allows to explore more in-depth the fine details of training of a complex and lifesaving skill like CPR. Indeed, this data allows to improve training programs and optimise the learning of medical trainees.\n\nCongratulations to the authors for the work done.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "52805",
"date": "09 Sep 2019",
"name": "Marcia A. Corvetto",
"expertise": [
"Reviewer Expertise Simulation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI really appreciate the opportunity to review this manuscript. The feedback written have been done with respect to the authors and with the aim to improve the manuscript. I hope to have a good reception by them. Simulation research is just starting in Latin America, so I congratulate the authors for the excellent work that they have done. The manuscript initially looks interesting, well written and well referenced. But reading it more deeply, few concerns arises. The majority of my concerns revolve around how the manuscript was written and to have more information about methods and stats.\n\nMethods:\nDid the authors perform an ethical committee submission? This should be stated in this section.\n\nLine 5: “Taking a heterogeneity of 50%, with a margin of error of 5% and a level of confidence of 95%, the sample was 140 students”. In order to have the statistical details all together in one part of the manuscript, I would move this part to stats.\n\nLn 7: “For the selection of the sample, randomized sampling was carried out”.I don´t understand completely this part. How did the authors the randomization process?. The randomization was performed to assign the instructor for the assessment? I would expect a deeper explanation about how the authors did the selection of participants.\n\nLn 8: “Groups of 7 people were organized for each evaluator in a random manner, with a total of 14 participants per day, ending at the 10th day with the 140 participants”.I would explain the training first and then the assessment. From my perspective methods should be written in order to be clearer for the reader.\n\nLn 41: “Theoretical standardized tools were used for evaluating the course received”.I understand that AHA assessments are standardized. But as a reader I would expect a deeper explanation about the assessment. Did the authors videotape the assessment sessions? Did they do the assessment by direct observation? Did the authors used 2 observers for the assessment?\n\nLn 50: “After 30 days, the theoretical and practical evaluation was carried out once again for each of the participants. Again, I would expect a deeper explanation about the assessment. 1 versus 2 observers? Direct observation versus videotaped? If videotaped, blinded assessment regarding first and second evaluation? Agreement calculation?\nData analysis:\nI would state here how the authors did the sample size calculation. What difference did the authors used to calculate the sample size?\n\nLn 1: “Cohen Kappa index was initially performed to determine the concordance and standardization of the knowledge taught in the intervention”. I am not clear about what type of agreement did the authors calculated with Cohen Kappa?\n\nLn 3: “The result was 0828, which indicates a high agreement between the two instructors; therefore, it was possible to continue with the study”.This should be explained in results. I would use a point in the number 0.828.\n\nLn 8: “Central tendency and dispersion tests were performed, as well as Student’s t-test, one-way ANOVA and ANCOVA”. Regarding Students’ t-test. Did the author used independent or paired measures test?\nResults:\nLn 2: “There were statistically significant differences between the subject groups for the first theoretical test immediately after the ACLS course. It was observed that these differences were between the Emergencies and Disasters and Anesthesiology groups (p< 0.05), as well as between the Critical Medicine and the Anesthesiology groups (p = 0.027). No statistical difference was seen between the Emergencies and Disasters and Critical Medicine groups”. It’s difficult for me to understand the differences. Maybe to add a table would be better for the reader. The same for the second theoretical assessment.\n\nLn 16: “There was no statistically significant differences in the first practical examination between the groups when evaluating their practical skills acquired immediately after the intervention of ACLS (p = 0.066)”. Again, maybe add a table. I would add the test that the authors did to perform the comparisons between groups.\n\nTable 1: Please include the number of participants per group. Additionally, a dispersion measure should be state for each line.\n\nDiscussion:\nLn 36: “These findings suggest that appropriate intervals and re-training strategies should be differentiated between knowledge and skills, with reinforcement of the latter”. I would discuss the idea of rethink how powerful is the intervention. I understand that ACLS protocols have been done succesfully for years. However, the mission of educators and researchers is to questioning how we are doing it, supported by this interesting new data. Maybe a 1 day course is not enough training to achieve strong competencies. A good score immediately after the course, could be explained by repetition and doesn’t assure a good achievement of competencies. Previous literature supports the idea of skill decay. But 1 month is so short from a practical perspective, that we should rethink how we should train these competencies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "52806",
"date": "13 Sep 2019",
"name": "Paweł Więch",
"expertise": [
"Reviewer Expertise Emergency medicine and nursing",
"body composition",
"telemedicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 am grateful for the opportunity to review the manuscript presented to me. I hope that the comments in the review would be helpful in deciding whether to index the manuscript in your journal. I believe the paper is worth considering for indexing, however requires major revision.\nGeneral comments\nAbstract:\nThe abbreviation CPR should be explained the first time it is used in the text. Scores for the immediate theoretical exam were 58.6% immediately after the intervention vs 40% 30 days after the intervention – in my opinion better will be: Scores for the immediate theoretical exam were 58.6% immediately after the intervention vs 40% thirty days after the intervention.\nInroduction:\n\nA few sentences of the introduction about the need and effectiveness of resuscitation in the study group are missing. Why the study was conducted specifically among medical students? It should be explained.\nMethodology:\nYou used …students of Medicine at Pontificia Universidad Católica of Ecuador … two times : in Participants and study setting section and Theoretical-practical course section – its more than enough. Mannequins and tools should be thoroughly described. I need much more information about your author's survey questionnaire. After 30 days, the theoretical and practical evaluation was carried out once again for each of the participants – in my opinion most of the available studies in the above scope analyse a longer period of time than 1 month. Whether pilot studies were carried out? What was the statistically significant? What was the ethical consideration?\nResults:\n… When conducting the post-hoc test through Scheffe…I did not see this information in the data analysis section. Only one Table presenting the data is insufficient.\nDiscussion:\n…Basic Life Support (BLS) program and postgraduate (BLS and ACLS Advanced Cardiovascular Life Support) training… the abbreviations were already explained in the beginning of the article. …however, there are not many previous studies that indicate the behavior between 1 to 6 months … - we need to citations.\nConclusion:\nShould be in 1-2 sentence.\nReferences:\nThe record of citations should be standardised. In your references I see many lacks , eg. 15. Burkhardt JN, Glick JE, Terndrup TE: Effect of prior cardiopulmonary resuscitation knowledge on compression performance by hospital providers. West J Emerg Med. 2014; 15(4): 404–8. Vs. 7. Salgado Castillo MF: Data for EVALUATION OF THE RETENTION OF SKILLS IN ADVANCED CARDIOVASCULAR LIFE SUPPORT (ACLS), FOLLOWING A THEORETICAL-PRACTICAL INTERVENTION IN POSTGRADUATE PHYSICIAN STUDENTS OF CRITICAL AREAS. figshare. Dataset. 2019. I did not see similar studies form last 2-3 years. I recommend some new one1.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-458
|
https://f1000research.com/articles/8-457/v1
|
12 Apr 19
|
{
"type": "Review",
"title": "Searching basic units in memory traces: associative memory cells",
"authors": [
"Jin-Hui Wang"
],
"abstract": "The acquisition of associated signals is commonly seen in life. The integrative storage of these exogenous and endogenous signals is essential for cognition, emotion and behaviors. In terms of basic units of memory traces or engrams, associative memory cells are recruited in the brain during learning, cognition and emotional reactions. The recruitment and refinement of associative memory cells facilitate the retrieval of memory-relevant events and the learning of reorganized unitary signals that have been acquired. The recruitment of associative memory cells is fulfilled by generating mutual synapse innervations among them in coactivated brain regions. Their axons innervate downstream neurons convergently and divergently to recruit secondary associative memory cells. Mutual synapse innervations among associative memory cells confer the integrative storage and reciprocal retrieval of associated signals. Their convergent synapse innervations to secondary associative memory cells endorse integrative cognition. Their divergent innervations to secondary associative memory cells grant multiple applications of associated signals. Associative memory cells in memory traces are defined to be nerve cells that are able to encode multiple learned signals and receive synapse innervations carrying these signals. An impairment in the recruitment and refinement of associative memory cells will lead to the memory deficit associated with neurological diseases and psychological disorders. This review presents a comprehensive diagram for the recruitment and refinement of associative memory cells for memory-relevant events in a lifetime.",
"keywords": [
"Associative memory cell",
"synapse",
"neuron",
"learning",
"memory trace",
"cognition",
"brain"
],
"content": "Introduction\n\nAssociative learning stands for a process, in which multiple exogenous signals, such as information, experiences and knowledge, are jointly acquired by sensory systems. Associative memory is termed as the integrative storage of these associated signals in the brain, which is indicated by memory retrievals (recall and representation) on the basis of speech, writing, gesture, countenance and emotion reactions. Associative learning and memory is a very common approach of signal storage for cognitions in life1–6 since the acquisition of new unitary signals or the reorganized learning of previously acquired unitary signals are fulfilled in the integrative manner7. In learning processes, the associated exogenous signals come from sensory organs and reside into sensory cortices through cross-modal and intramodal manners7,8. The coactivation of sensory cortical neurons makes their axon projections and synapse innervations mutually, in which these neurons are recruited to be primary associative memory cells for the integrative storage and reciprocal retrieval of multiple exogenous signals9–11. In the meantime, these primary associative memory cells project their axons and make synapse innervations convergently onto their downstream neurons in certain brain areas for the integrative storage of endogenous associated signals that are essential to logical reasoning, associative thinking and other integrative cognitions. Their axons also divergently innervate the neurons in various brain areas that are relevant to cognition, emotion and behaviors for the storage of endogenous signals in many places and the participation in multiple memory-relevant events7,12. As cognitive processes and emotional reactions can be recalled, these neurons that memorize endogenous signals are named as secondary associative memory cells7,8. In other words, when the neurons encoding one of associated signals are activated, they can attract axon projection and synapse innervations from the coactivated neurons encoding another of associated signals. After associative memory cells are recruited from the coactivated neurons by receiving the innervation of multiple synapse inputs, their subsequent activities will change their excitability and synapse functions. The recruitment and refinement in the population of associative memory cells constitute a principle of activity together, connection together, strengthening together and coordination together7,8. Therefore, associative memory cells are nerve cells that are able to encode multiple signals which have been associatively acquired, as well as receive axon projection and synapse innervations that carry these signals. Currently, two kinds of associative memory cells have been detected experimentally, which carry out the activities of searching basic units of memory traces. Comprehensive cellular architectures underlying associative memory in the different periods of the lifespan are yet to be revealed to better understand memory-related physiology and psychology in the brain. This review focuses on current advances in associative memory cells as basic units of memory trace or engrams, an expansion on our previous review7.\n\n\nAssociative learning and memory\n\nLearning is defined as the acquisition of new information, knowledge and experiences, which may be new unitary signals or the reorganized unitary signals that are learned previously. Signal acquisition can be classified into associative and non-associative styles1,6. Associative learning stands for the joint acquisition of multiple signals that can be sensory signals or sensory signals plus rewards after behavior operations are achieved. Their joint acquisitions are characterized as the association of new signals with an innate signal or a formerly learned signal, as well as the reorganized association of unitary signals that have been previously learned, such as the reorganization of letters into different words and phrases7. Associative memory to these signals appears emerged if these associated signals can be retrieved reciprocally by cues or recalled by automatic conversion among sensory modalities. Two physiognomies of associative memory are the integrative storage and distinguishable retrieval of associated signals11. On the other hand, non-associative learning is termed as the acquisition of a given sensory signal, in which repeated stimulations lead to habituation or sensitization to this sensory signal1. It is thought that non-associative learning is not involved in acquiring new knowledge and experiences, except repetitive activations in a sensory system for its upregulation or downregulation by the sensitization or desensitization of sensory receptor and cortical neurons. In this regard, it may fall into the term “review” instead of learning. Therefore, the acquisition and storage of almost all of the new signals require them be associated with a signal that has been stored in the brain for the facilitation of their memory7.\n\nIn associative learning, multiple featured signals in each object or an environment are detected by different modalities from sensory receptors to cerebral cortices. These cross-modal signals are integrated for their associative storage. For instance, a fruit is detected by the olfactory system for aromatic odor, the visual system for shape and color, the taste system for sweetness, the auditory system for name and the somatosensory system for its surface sign. After they are jointly memorized, a signal induces the recall of its associated signals, or the other way around. The recall can even be done by automatic signal conversion among different modalities, e.g., image signals are recalled by verbal signals with no additional cues. In addition, multiple signals with identical features can be associatively detected through a single modality, i.e., one type of sensory receptors and its projected cortex. These signals are associatively acquired in an intramodal manner and primarily stored in one of sensory cortices. Therefore, memory traces imprint the joint storage and distinguishable retrieval of multiple associated signals. Primary associative memory cells that encode multiple signals based on the synapses from innate inputs and new innervations from coactivated brain areas have been detected for the integrative storage of associated signals9–11,13–17.\n\nIn addition, logical reasoning, associative thinking, computation and integrative imagination based on those exogenous associated signals that have been stored in primary associative memory cells of sensory cortices may lead to the secondary integrations of those signals for their storage and representation, i.e., secondary associative memory that is essential for cognition, emotions and behaviors at the high orders under the consciousness condition7,8. Although the memory occurs presumably in the prefrontal cortex, hippocampus and amygdala18–30, these studies still do not reveal whether the memory in their data is secondary to the information storage in primary associative memory cells from sensory cortices. Currently, secondary associative memory cells are detected in the motor cortex, the prefrontal cortex and the hippocampus, which reside in the downstream of sensory cortices and receive synapse innervation from primary associative memory cells12,31,32.\n\nVarious memory patterns are classified in psychology, such as explicit versus implicit memory, declarative versus nondeclarative memory and episodic versus semantic memory in memory contents as well as sensory versus short-term or long-term memory in temporal feature6,33. Declarative memory, i.e., explicit memory, refers to stored information that can be stated consciously, including episodic memory (specific processes and their contexts) and semantic memory (generalized knowledge and concepts). Non-declarative memory, or implicit memory, denotes the operations of various skills and procedures without the need of consciousness. In fact, there is no clear border line between explicit and implicit memory. The procedures and skills operated in the implicit memory can be consciously stated. The specific processes and their contexts after repetitive practices can be executed effortlessly. In the field of neuroscience, the classifications of memory formation in the brain are based on the combination of the location of information storage with the memory of featured signals, for instance, spatial memory in the hippocampus, emotional memory in the amygdala, perceptual memory in sensory cortices and prospective, attentive and working memory in the frontal cortex6. Moreover, it has been suggested that memory formation is classified based on cellular mechanisms, such as the different types of associative memory cells in the neural circuit for memory, i.e., memory trace or engram, and the sources of memorized signals from cross-modal versus intramodal sensory systems or exogenous versus endogenous resources7,8.\n\nAssociative memory to exogenous signals from external environments refers to the integration and storage of associated signals that are inputted from cross-modal or intra-modal sensory modalities. Associative memory to endogenous signals refers to the integration and the storage of associated signals that originate from sensory cortices and regenerate during logical reasoning and associative thinking in cognition- and emotion-relevant brain areas. Intramodal associative memory is related to the integration and storage of associated signals inputted from a single modality, such as a sensory modality or a brain area involved in cognition, emotion or behavior. Cross-modal associative memory is termed as the integration and storage of associated signals that come from different sensory modalities or brain regions related to cognitions, emotions or behaviors7,8.\n\nTo better understand these memory patterns and their correspondent mechanisms, we should figure out the basic units in memory traces or engrams that conduct the integration and storage of associated signals, constitute the foundation of cognitions (logical reasoning, associative thinking, computation, imagination and so on), achieve the integration and storage of endogenous signals generated from associative thinking and logical reasoning, and control the future presentation of stored associative signals. How the memory is formed in different modalities and encoded under the different states of consciousness, attention and psychological motion remains to be elucidated. Therefore, the comprehensive view of cellular mechanisms underlying associative memory should be established like an effort to see individual trees as well as the forest.\n\n\nThe highlights of memory-relevant events for searching memory cells\n\nThe mechanism underlying learning and memory has been systemically studied for more than one century6,34–36. Many observations and concepts have been proved to be solid, however, data inconsistencies and indication controversy still block our clear vision to abstract cellular architectures and molecular profiles. Major reasons for this vagueness may be due to lack of the reliable standards to uncover memory cells in neural circuits (the basic units of memory trace or engram) that encode specific signals stored, to identify molecules resided in memory cells specifically for their recruitment, as well as to validate behaviors specifically initiated by memory cells. In order to set up reliable criteria for judging whether memory cell ensembles or memory traces being recruited are correlated to memory formation and retrievals, the changes at the levels of molecules, neurons and behaviors in learning and memory should be precisely estimated.\n\nIn the study of memory retrieval, the stimulus-induced or cue-induced expression of specific behaviors that have been presented during learning events and memory retrievals are better used to denote the persistent presence of memory traces. The use of this strategy may have the following shortfalls. Behaviors, perceptions and cognitions are quickly developed postnatally37,38. Postnatal developments in perception and cognition versus behavior are not parallel in aspects of their patterns and contents39–41. The number of arm/body language patterns is much lower than the number of memory contents as well as the number of verbal language patterns. Although the number of memory contents is matched with the number of verbal language patterns, one arm/body language pattern may represent several memory contents. For instance, the thumb-up gesture usually represents memory contents relevant to all positive events. Moreover, patterns and varieties in sensory input signals, memory contents, cognitive processes or emotional reactions are much enriched in comparison with behavioral patterns that are presented by common output pathways, i.e., all of these signals, contents and processes are expressed by behavioral output patterns and pathways in the limit number. For instance, the “OK” gesture is used to express appropriate sensory stimulations, good perception, successful memory retrieval and other good cognitions. In other words, behaviors may not well present the retrievals of specific memory contents, except for verbal language. This limitation of behavioral presentation to memory and cognition may be an issue in the study of memory retrieval based on behaviors in animals. For instance, body freeze and involuntary/voluntary shaking used to signify fear memory can be induced by extreme fear, anxiety, emotional reactions (e.g., anger and fighting) and physiological processes (e.g., hypothermia and hypoglycemia). Furthermore, the brain in matured human beings and animals is highly wired, and its different regions are interconnected42. Stimulations to potential memory traces by electrical, optogenetic or chemogenetic approaches being given to a location of the brain may indirectly activate other areas connected with this location to induce memory-related behaviors indirectly or behaviors across or similarly to memory retrieval, i.e., the replay of “memory-related behaviors” may not be directly or realistically controlled by memory traces.\n\nThe learning process generally includes the associative acquisition of simple signals or unitary signals and the reorganized acquisition of these unitary signals. At young age, language learning includes letters and words, and knowledge learning is mainly definition and concept. After the primary learning of unitary signals, the advanced learning moves forward to more complicated concepts by the reorganization of unitary signals, i.e., the acquisition of sentences and articles by the association of letters and words in language, as well as the acquisition of principles and theories by the association of definition and concepts in knowledge. Unlike verbal presentation, arm/body behaviors are not obviously upregulated to the complicated version for expressing the advanced language and knowledge, such that similar behaviors in different postnatal periods may represent different contents and knowledge. In other words, memory retrievals represented by similar arm/body behaviors likely include different contents. Taken together, we assume that the retrievals of stored signals by the replay of similar behaviors may be changeable in their contents spatially and temporally, i.e., behavior replays are unreliable, except for the reoccurrence of cues-induced behaviors.\n\nAt the cellular level, the basic units in the brain are neurons and glia cells. It is important to figure out new features of those neurons that have been recruited as memory cells for storing specific signals, in order to map their working principles during memory formation and retrieval. In addition to their conventional natures, such as innate synapse input, synapse transmission, neuron excitability and excitation outputs, memory cells theoretically encode the newly learned signals and receive new synapse inputs that carry these newly learned signals. As the most common style of learning and memory is associative in nature, i.e., the integrative storage of associated signals, associative memory cells recruited should encode both innate signals and new signals, as well as receive new axon projection and synapse innervations in addition to the innate input. In this regard, the detection of new synapse innervations and multiple signal encoding by recording approaches (cell electrophysiology and imaging) is critically important in reporting the finding of memory traces. Moreover, learning and memory involve the memory of unitary signals in the young and the memory of complicated signals, i.e., reorganized unitary signals, in the later period of development. Individual associative memory cells presumably encode multiple unitary signals, and their assemblies work together to store unitary signals in different reorganizations. In other words, ensembles of associative memory cells store advanced knowledge contents in specific spatial and temporal patterns7.\n\nIn the principle of cell physiology, neuronal excitation is driven by synapse inputs and neuronal excitability is controlled by spiking threshold43,44. The patterns and frequencies of neuronal spikes are influenced by synaptic transmission and spiking threshold, i.e., there is a proportional correlation between the intensity of neuronal activities and the strength of synapse inputs, but not the nature of input contents. Similarly, activity patterns and spiking frequencies of memory cells in memory traces can denote their activity strength but not memory contents, such that the replay of certain neuronal activity patterns, such as spontaneous sharp-wave ripple, indicates the reemergence of neuronal activity strength without the necessary implication of memory features and contents being encoded. Cues-induced neuronal activity may reflect the retrieval of memory contents. Learning-cues should be used to track the distribution of associative memory cells in the different grades of memory traces.\n\nIn order to figure out the features of memory cells and their working principle during memory retrieval, we expect to reveal the featured molecules in associative memory cells to label these memory cells. Based on analyses above, the formation of memory cells recruited from neurons involves axon projection and synapse innervation, two nonspecific processes in neurons. In this regard, the elucidation of molecular markers for memory cells is challenge. Recently, immediate early genes have been used to label memory cells45,46, which depends on a proposal that activated memory cells express immediate early genes47,48. Unfortunately, the expression of immediate early genes is proportional to the strength of neuronal activities which are not specific for memory cells. In this regard, the neurons with combo features in the labeling of immediate early genes, the innervation of new synapses and the encoding of new/innate signals are better termed as engrams.\n\nIn summary, neurons that meet all of the following criteria, such as cues-induced behaviors, cues-induced replay of neuronal activities, new synapse innervations and active molecule labeling can be defined as memory cells. Strategies to find out memory traces that meet these criteria are discussed below.\n\n\nStrategies used to search basic units of memory traces\n\nTwo issues are important to clearly address the cellular and molecular mechanisms underlying associative memory formation and retrieval, i.e., animal models and strategies for searching cell assemblies in memory traces or engrams. Based on the studies of learning and memory over centuries, we summarize the animal models and strategies that have been used.\n\nAs associative learning of multiple signals is the most common approach of signal acquisition in life, the mechanisms underlying the integrative storage of these associated signals should be addressed by using appropriate animal models featured by association. A few animal models have been used to the study of associative learning and memory, such as classical conditioning that includes Pavlov’s conditioned reflex, eyeblink conditioning and fear conditioning in rodents and withdraw reflex in Aplysia, as well as operant conditioning that includes various types of reward memory (e.g., operation plus reward and place plus reward) in mammalians45,49–63. In these models, a stimulus is unconditioned, whereas another stimulus is conditioned. However, in human beings, the memory of associated signals occurs by the signal inducing the recall of its associated signals, or the other way around. This reciprocal retrieval of associated signals constitutes the basis of associative thinking, logical reasoning, computation and imagination in forward and backward manners. It seems to us that these animal conditioning models do not signify whether the air-puffing to the cornea or the electric shocks to the feet is able to induce the recall of sound signal after the onset of eyelid-blinking conditioning or fear conditioning. That is, these conditioning models may not be ideally used to study associative memory. Moreover, electrical shocks may activate the whole brain by spreading electrical current in the body, so that the association is not region-specific in the brain7,8. Compared with electrical stimulations used in the study of fear memory, physical and psychological stresses in social interactions are closer to real life situations64–66. Recently, an animal model has been introduced to study associative memory in that the association of whisker and olfactory stimulations in mice leads to odorant-induced whisker motion and whisker-induced olfactory responses, a typical example of reciprocal retrieval of associated signals9–11,15,16,67.\n\nIn terms of strategies to study associative learning and memory, theoretical analyses and experimentation in vivo are used68–72. Theologists in the field of learning and memory focus on drawing potential units for information storage in the brain, such as memory traces, engrams and cell assemblies. Experimenters make efforts to figure out molecular substrates and cellular architectures for memory formation. In order to prove causal relationships between newly formed neuron substrates and memory behaviors, three criteria should be met. The emergence of new substrates and architectures is parallel to memory formation. The downregulation of newly formed substrates and architectures substantially reduces memory formation through the approaches of surgical ablations to brain tissues, pharmacological blockades to neuronal activities and genetic knockout/mutagenesis to molecules in nerve cells or synapses. The upregulation of these newly emerged substrates and architectures significantly facilitate memory formation through the approaches of pharmacological, electrical or optogenetic stimulations to nerve cells and gene overexpression in neurons and synapses7–9,73.\n\nIn addition to the term “memory traces” for information storage coined by the ancient Greeks, theoretical terms “engram and ecphory” have been suggested by Richard Semon74, a renowned theologist in the field of learning and memory. Engram and ecphory correspond to memory traces and memory retrievals, respectively71,75. In addition, his view on the relevance of memory retrievals says that the interaction between the stored engram and retrieval cues may generate new engrams. As long as an engram-awakening stimulus is similar to an original stimulus, this incomplete retrieval cue is sufficient to retrieve the stored engram. Awakening the originally stored engram may generate a new engram related to this event. The old retrieved engrams and new engrams become associated through contiguity to strengthen original memory. Moreover, the simultaneous retrieval of multiple engrams with similar contents and their subsequent associations, i.e., resonance among engrams, would provide the basis for the complicated cognitive processes, such as abstraction, generalization and knowledge formation76. This theory may be the first to hypothesize that awakening engrams is dynamic and use-dependent. Although the engram termed by Simon lacks experimental evidence during that period, his frameworks about engrams are consistent with the features of memory activities. For instance, more representations lead to deeper memory, and the repeated simultaneous recalls of similar memory contents induce them to be summarized, conceptualized and generalized. In brief, his work has led to the consensus of memory traces or engrams as the basis of information storage.\n\nDonald Hebb, another well-known theologist, describes memory traces or engrams to be cell assemblies as the basis of memory behaviors. According to his and Penfield’s observation that the destruction of large amounts of cerebral cortices in human beings produces little effect on memory77,78, as well as Lashley’s experiments that the ablation of widespread cortices in animals does not induce parallel changes in memory behaviors79,80, he has proposed the term “cell assemblies” that are the widely distributed neural substrates for memory. Each cell ensemble is a group of interconnected cells in that their interconnections are formed during their simultaneous activities81,82. Since these cells are interconnected, the activity in this circuit is maintained briefly after the event, i.e., short-term memory. Activities recurring for a sufficient duration within this cell ensemble can induce growth or metabolic change that strengthens those interconnections among assembly cells, such that short-term memory is converted into longer-term memory82. The strengthening of connections between presynaptic and postsynaptic nerve cells in their simultaneous activities confers these neurons the property of firing together and strengthening together, which has been hypothesized as being a neuron connection. The strengthening of neuron connections has been shown in long-term potentiation of synaptic transmission83,84. The high number of interconnections among cells may allow the entire ensemble to be activated if a subset of cells is activated by the process of pattern completion that induces memory retrieval. As Hebb’s cell assembly is widely distributed in and across brain areas, destruction in a small proportion of cells may not lead to catastrophic memory traces, or graceful network degradation, which may account for Lashley’s experimental results. In summary, Hebb’s theory has overlapped multiple spatial scales from the integrated synaptic strengthening (a microscale level) to cell assembly formation (a mesoscale level).\n\nThe computational simulation of neuronal substrates for learning and memory has been used to deliver the theoretical model of memory traces, in which the data for modeling is based on experimental results. In the study of neuronal and synaptic architectures for memory traces and memory related behaviors, there are clear indications that show the involvement of neuronal ensemble and synaptic plasticity in processes of learning and memory in spite of a lack of evidences about synapses, neurons and their plasticity specifically correlated to memory formation76,85–89.\n\nIn summary, the study of memory formation by theoretical models has generated great frameworks that can provide useful guideline for addressing cellular mechanisms underlying learning and memory. However, these hypotheses about memory traces (or engrams) and cell assemblies have not indicated any insight about the integrative storage of associated signals and need be proved by experimentation. In experimental studies about learning and memory, three strategies can be used to confirm causal relationships between memory traces (cell assemblies) and memory-related behaviors. Memory traces should be detected during memory formation and cue-induced memory retrievals. The downregulation of memory cell assemblies can restrain memory-relevant behaviors. The upregulation of memory cell assemblies can facilitate memory-relevant behaviors7–9,73. There are two usual methods to track memory traces (engram) or cell assemblies, i.e., the detection of memory cells during learning and memory and the activation of memory cells to retrieve memory-relevant behaviors. The detection of memory cells involves observing their responses to memory cues by electrophysiological recording and two-photon calcium imaging and localizing their distribution by AAV-carried fluorescent neural tracing after memory formation. The activation of memory cells can be done by electrical, pharmacological, optogenetic or chemogenetic stimulations to induce the emergence of memory-relevant behaviors7. It is noteworthy that memory traces are widely distributed in the brain and brain areas are interconnected. These stimulations may lead to the antegrade and retrograde activation of neural pathways. The indirect activation of memory traces is unable to localize primary versus secondary allocations for memory formation.\n\nNeuronal activities are indicated by electrical signals generated on the cell membrane and calcium signals raised in cells, such that the recording of electrical signals and the imaging of intracellular calcium dynamics can be applied to track cell assemblies relevant to memory formation and retrieval, i.e., the functional detection of memory traces90. The electrophysiological recordings by electrodes or electrode array have been used to detect the replays of neurons in the hippocampus, visual/auditory cortices, the amygdala and ventral tegmental areas under different conditions, such as retrieval cues, wakefulness and sleep state30,91–104. For example, coordinate interactions from the hippocampus to the prefrontal cortex and associative cortices, including parietal and midline areas but not primary areas, are involved in spatial memory tasks105,106. The cortical-hippocampal-cortical circuit is critical for memory consolidation107. Hippocampal assemblies trigger neuronal activities in the ventral striatum during the replay of place-reward information55. The acquisition of associative memory in the hippocampus initiates a gradual-to-stable encoding process in the medial prefrontal cortex without continued trainings29. Emotional memory is reactivated in the hippocampus-amygdala system during the sleeping state108. These data from this functional study are supported by anatomical evidence within the hippocampus, the prefrontal cortex and the thalamic nucleus109.\n\nRecently, two-photon cell calcium imaging in vivo110–112 has been used to detect memory traces or memory cell assemblies in cerebral cortices. For instance, the gradual emergence of neuronal activity relevant to spatial memory in the retrosplenial cortex, which is the major recipient of hippocampus, depends on the intact hippocampus. Indirect connections between the retrosplenial cortex and the hippocampus indicate hippocampal influence polysynaptically within the neocortex, i.e., widely distributed memory traces in the hippocampus and cerebral cortices113. Repetitive motor learning induces the formation of dendritic spines in vivo114. Associative memory cells developed in response to retrieval cues are detected in primary sensory cortices and the prefrontal cortex9,11,12,32. Thus, memory traces or cell assemblies can be tracked by electrophysiological and imaging recordings based on their activities in response to retrieval cues and during memory-relevant behaviors.\n\nImportantly, the data above support the functional presence of memory traces or cell assemblies, which is better validated by morphological traces, i.e., their morphology and distribution should be quantified and localized. Two methods can be used for this purpose: the trace of their synapse innervations from axon inputs that are carrying the learned signals, as well as the labeling of these cell assemblies by molecules specifically relevant to memory. In the study of associative memory cells, fluorescent expression mediated by adeno-associated virus (AAV) vectors in neurons and their axons has been done by injecting AAVs, tagged with genes of fluorescent proteins, in the source side of predicted memory traces and by detecting axon terminals and their target on associative memory cells9,13,15. These associative memory cells receive both innate and new synapse innervations. It is noteworthy that the combination of tracing new synapse contacts and labeling memory assemblies with memory-relevant molecules would be an ideal way to denote memory cell assemblies.\n\nNeuronal activities may lead to a change in certain molecules115–117, so that learning and memory recruit the neurons to be memory cells presumably by molecular substrates. The labeling of memory cells by these molecules can be applied to indicate the allocation of memory cell assemblies, based on the facts that the stimulation of neurons couples with the expression of immediate early genes48 and their expression in dendrites is regulated by synapse activity47. For instance, immediate early gene Arc is specifically linked to the neural encoding process118. Immediate early genes are widely expressed in the brain after fear memory and the number of labeled cells is positively correlated to fear memory behaviors45,46. It seems that there is an association between the expression of immediate early genes and the active strength of memory cells. The detection of immediate early gene expression is usually used to label the engrams, so that their morphologies and functions can be studied112,119–122. However, the upregulated expression of immediate early genes is also associated with neuron hyperactivity, such as seizure discharge in epilepsy123–126 and neuron toxicity in the brain ischemia127–129. In these regards, immediate early genes may be suitable for identifying all of the neurons that are highly active. Genes and proteins specifically linked to memory cell assemblies and their memory contents remain to be explored68,130.\n\nAs the retrieval of memory-specific behavior is presumably based on memory traces that are formed during memory formation, the activation of memory cell assemblies to induce the emergence of memory-relevant behaviors should be included in the study of memory formation and retrievals. The use of this strategy is based on the positive correlation between memory cell assemblies and memory formation/retrieval, i.e., if some neurons are memory cells that store specific memory content, the activation of these cells by electrical, pharmacological and optogenetic methods should induce the representation of memory-relevant behaviors. Electrical stimulations to memory traces in the brain were given by Penfield who expected to localize the source of epilepsy131. Stimulations to the temporal lobe in wakeful epileptic patients induced their memory recalls, i.e., engrams were detected in this cortical area132,133. Pharmacological stimulation has been used to activate the serotonin or norepinephrine system to examine the facilitation of memory formation by the transmitters successfully134,135. Recently, optogenetic stimulation has been used to activate memory engrams, which mediate fear memory and false memory22,136–139. Therefore, these results support the positive correlation between memory traces and memory-related behaviors. It is noteworthy that the direct optogenetic activation of neurons without increases of synaptic strength and dendritic spine density leads to memory retrievals140, implying nonspecific neuron activation. As pointed above, the wide distribution of memory traces in the brain and the interconnection among brain areas may result in the stimulations being antegrade and retrograde activations of neural pathways. The indirect activation of memory traces is unable to localize primary versus secondary allocations for memory formation.\n\nSimilarly, if behaviors related to specific memory content depend on memory traces that are formed during memory formation, the downregulation of certain molecules critical for memory cell assemblies by pharmacological blocking, gene knockout or optogenetic methods should prevent or attenuate the formation and emergence of memory-relevant behaviors, which is commonly used to address the causal relationships among molecular substrates, cellular architectures and memory formation. The first use of surgical ablation to search the distribution of memory traces or engrams was done by Lashley. Although he failed to localize memory traces, his studies imply the wide distribution of memory traces in the cerebral brain79,80,141. The following studies indicate that the removal of the temporary lobe in human beings leads to the loss of recent memory due to the impairment of the hippocampus78,142–144. In the study of memory traces using pharmacological reagents, recent memory can be blocked by using intracerebral injection of puromycin145,146. These studies reveal a causal relationship of memory traces in wide brain areas to memory formation and retrieval, although memory traces specific for content-related behaviors remain to be tracked and localized. With the advanced molecular biology, the downregulation of gene expressions by gene knockout147 and optogenetics148,149 have been successfully used to find negative correlations among molecules, memory cells and behaviors. These studies provide strong evidence for the causal relationships among molecular substrates, cellular architectures and memory formation.\n\nThe advantages and disadvantages of these strategies and approaches have to be evaluated and validated. In logical analyses, parallel changes, negative correlations and positive correlations between functions and changeable factors should be met in order to ensure the causal relationship. Studies involving manipulations of molecules and cells causing changes to memory-relevant behaviors in these three criteria should be combinedly used to identify memory traces formed after learning. Consistent results are expected to reach the conclusion. However, inconsistent results may occur in these studies. For instance, silencing and stimulating the patriate cortex lead to inconsistent results in memory retrieval. Parietal lesions do not normally yield severe episodic-memory deficits, whereas parietal activations are seen frequently in the function-neuroimaging studies of episodic memory150. These two categories of evidence suggest that the answer to the puzzle requires us to distinguish the contributions of dorsal versus ventral parietal regions and the influence of top-down versus bottom-up attention on memory. The features of memory traces or engrams based on these studies include the following. Memory traces encode the trained signals, receive synapse inputs and undergo synaptic plasticity35,69–71. The activation of memory traces evokes strong memory retrieval. Memory events are upregulated by norepinephrine and serotonin. How memory traces memorize multiple associatively learned signals needs to be addressed by observing cells in memory traces that encode the associated signals.\n\n\nAssociative memory cells as basic units of memory traces\n\nAssociative learning includes the acquisition of associated signals that are basic features of various objects, knowledge and experience as well as the acquisition of the complicated signals that are reorganized from those basic featured signals in intramodal or cross-modal manner. Associative memory stands for the integrative storage and the distinguishable retrieval of these associated signals in neurons. Associative memory cells are presumably basic units to fulfill these processes during associative learning and memory by encoding multiple associated signals as well as receiving innate and new synapse innervations in the cerebral brain7,8. The integrative ability of associative memory cells indicates that activity-dependent synaptic plasticity in a single neural pathway, such as long-term potentiation and depression of synaptic transmission83,151,152 and activity-dependent neuronal plasticity43,153–155, may not be directly involved in the integrative storage of multiple associated signals, though this plasticity may influence memory retrievals7,8.\n\nIn terms of the location of information storage, memory traces appear to be widely distributed in the brain, such as the hippocampus, amygdala, motor cortex, sensory cortices and associative cortices3,12,21,22,25,26,28,30,106,156–160. Memory contents reside hypothetically in cell assemblies by the strengthening of neurons’ interconnection that is triggered by their correlated activity in information acquisition81. These studies do not explain why cell assemblies are widely distributed and how plasticity at synapses and neurons coordinately integrate associated signals for their storage in primary and secondary manners, i.e., the characteristics and working principle of these neurons that coordinately encode associative memory7,8. Neuronal and synaptic plasticity cannot interpret memory patterns, e.g., explicit versus implicit memory, declarative versus non-declarative memory, episodic versus semantic memory and memory transformation among these patterns33, the temporal features of associative memory as well as the contribution of associative memory to cognitive processes, e.g., associative thinking and logical reasoning. How endogenous signals generated in associative thinking and logical reasoning are memorized for future representation remains unknown. How memory is encoded under different consciousness states needs to be addressed. The natures of these cell assemblies, the patterns of their connection strengthening and the coordination of their encoding memory need to be examined in a comprehensive manner.\n\nAssociative memory cells that encode multiple associated signals as well as receive innate and new synapse inputs have been detected to be recruited by the coactivation of cortical neurons7–9,11,67. The coactivation of sensory cortices evokes their mutual synapse innervations, and recruits associative memory cells to integrate and encode associative signals10,11,16. Based on mutual innervations among associative memory cells9,11,15, the associations of sensory signals for their integrative storage make each signal induce the recall of its associated signals in a reciprocal manner. In the meantime, these primary associative memory cells in the sensory cortices send their axonal projections toward brain areas relevant to cognitions, emotions and behaviors, and undergo synaptic convergence with individual neurons in these areas during logical reasoning and associative thinking to recruit them as secondary associative memory cells7,8,12. In this regard, mutual synapse innervations among primary associative memory cells in sensory cortices and their innervations to secondary associative memory cells in brain areas related to cognition, emotion and behavior constitute the basic cellular architecture for the reciprocal recall of associated signals, the automatic conversion of associated signals during their recalls and cognition at the high orders7,8 (Figure 1). In addition to the learning of associated signals from cross-modal sensory cortices, the acquisition of associated signals can be achieved in one of intramodal sensory cortices, such as the association of letters or words in the auditory cortex, the association of unitary images in the visual cortex, and so on. The recruitment and features of these associative memory cells are described below.\n\nThree groups of primary associative memory cells (blue, green and yellow) in sensory cortices are synaptically innervated. Three groups of secondary associative memory cells (blue, red and pink) in brain areas relevant to cognition, emotion and behaviors are synaptically innervated. Mutual synapse innervations among associative memory cells in each group are intramodal, and mutual synapse innervations among three groups of associative memory cells are cross-modal. The axons of primary associative memory cells convergently and broadly innervate secondary associative memory cells, whose axons project back to primary associative memory cells. All neurons possess innate synapse innervations (yellow axons). The synapse innervations among the functional corresponding groups of primary and secondary associative memory cells are labeled by bigger presynaptic boutons.\n\nAssociative memory cells recruited in sensory cortices: Associative learning by paring whisker, odor and tail stimuli in mice leads to reciprocal responses induced by each of these signals, such as odorant-induced whisker motion, odorant-induced tail withdraw, tail-induced whisker motion, tail-induced olfaction response, whisker-induced olfaction response and whisker-induced tail withdraw9,11,13,15. Their barrel cortical neurons are able to encode new odor and tail signals alongside innate whisker signal as well as receive new synapse innervations from the piriform and S1-tail cortices besides innate inputs from the thalamus9,17. Their piriform cortical neurons encode new whisker signal and innate odor signal, as well as receive new synapse innervations from the barrel cortex alongside innate input from the olfactory bulb15. In other words, a portion of the sensory cortical neurons in mice after associative learning become able to encode associated signals as well as receive new synapse inputs based on their mutual innervations alongside innate input, which are named as associative memory cells10,11,13,16. These associative memory cells have been assured to include glutamatergic neurons, GABAergic neurons and astrocytes9–11,13,15–17. Thus, the coactivation or simultaneous activity of sensory cortices can trigger the new synaptogenesis for mutual synapse innervations and the recruitment of associative memory cells for the storage of associated signals. The association of cross-modal sensory signals may occur among all of sensory cortices, such as visual signal with auditory, olfactory, taste and somatosensory signals; auditory signal with visual, olfactory, taste and somatosensory signals; and so on, i.e., primary associative memory cells can be recruited in auditory, visual, olfactory, gustatory and somatosensory cortices by their mutual synapse innervations7,8 (Figure 2 and Figure 3).\n\nAssociative learning and memory include the acquisition of associated signals, the integration and storage of exogenous signals, the integration and storage of endogenous signals, as well as memory retrieval through behavioral presentation. Associative memory cells (AMCs) are classified into primary AMCs (pAMCs) in sensory cortices, including visual, auditory, olfactory, gustatory and somatosensory cortices for the integrative storage of exogenous associated signals, as well as secondary AMCs (sAMCs) in brain areas related to cognitive processes (logical reasoning, associative thinking, computation, imagination, concept, judgement conclusion, decision and so on in the prefrontal cortex), emotional reactions (fear, aversion, happiness, angry and so on in the amygdala, ventral tegmental area (VTA) and nucleus accumbens (NAc)), sensation integration (understanding and perception in association cortices) as well as spatial localization in the hippocampus. pAMCs are mutually connected through cross-modal and intramodal synapse innervations for the integrative storage and the reciprocal retrieval of associated signals. The axons of pAMCs convergently innervate onto sAMCs for cognition, emotion and spatial localization. sAMCs are mutually connected through their synapse innervations for the integration of cognition, emotion, perception, localization and so on. All of these primary and secondary associative memory cells will send their axons toward brain areas relevant to behaviors (language, gesture and countenance in motion cortices) and their coordination (the systems for maintaining internal environment, e.g., the hypothalamus to control autonomic nerves and hormones). Cross-modal associative memory cells are recruited by mutual innervations among sensory cortices or between cognition- and emotion-relevant brain areas. Intramodal associative memory cells are recruited by mutual innervations among the neurons in single-modality sensory cortex, cognition brain area or emotion brain area. In addition to the activation by innate input and new synapse innervation from the coactivated brain regions to integrate and encode associated signals, associative memory cells are activated by the arousal system, including the ascending reticular activating pathway in the brain stem and thalamus as well as the ascending activating pathways from the cholinergic nuclei, midbrain raphe nuclei, locus coeruleus and substance nigra that release acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE) and dopamine (DA), respectively, which can maintain wakefulness, permit normal consciousness as well as grant specific alertness and attention. In addition, associative memory cells are regulated by hormones that are released from the hypothalamus-pituitary-glands. The upregulations of AMC number and activity strength can facilitate memory to be impressive, or vice versa. The function downregulation of motion-relevant brain regions leads to the inability of memory retrieval and presentation.\n\nA) A basic neural circuit (memory trace) for associative memory includes primary associative memory cells (pAMC) and secondary associative memory cells (sAMC). Each of these primary associative memory cells receives synapse innervations from the innate inputs (their colors correspondent to those of the cell bodies), the input from the arousal system (dark red) as well as the mutual synapse innervations among them (i.e., from other primary associative memory cells). These primary associative memory cells send their axons convergently to secondary associative memory cells (green) and make synapse innervations. All of these associative memory cells send their axons to memory output neurons (MON). B) The relationships between the excitation state of associative memory cells and the strength of memory formation/retrievals. The excitation state of such associative memory cells is influenced by the number and the function state of their synapse inputs and by their own excitability. If the excitability of associative memory cells rises, their relationship curve (dark red) shifts towards the left (yellow) and the efficiency of learning and memory increases. C) denotes the relationship between different associative memory cells and their excitation levels. If the threshold to fire spikes (excitation) decreases, the relative excitation levels of associative memory cells increase, as well as more neurons will be coactivated for the recruitment and refinement of associative memory cells in memory formation and retrieval.\n\nAssociative memory cells recruited through the coactivation of sensory cortices are diversified in their encoding abilities and contents. Some cells encode all associated signals (full associative memory cells) and others encode two or more signals (incomplete associative memory cells), e.g., triple, two or one of odor, whisker and tail signals9. If neurons are activated together and wired together, the coactivated strengths among these sensory cortical neurons may be different based on their variable excitability44. Neurons that encode one signal are called new memory cells or innate memory cells9,11. The recruitment of diversified populations of associative memory cells in their encoding ability dissects complicated events, objects or images into simple unitary signals for their storage, future retrievals in different patterns and the reorganization of unitary signals in future associative learning7. In addition, the repeated coactivations of these sensory cortical neurons can facilitate the recruitment of full associative memory cells from incomplete associative memory cells, as well as the formation of more en passant synapses among their mutual innervations, so that the number and the activity strength of associative memory cells are upregulated32. The proportional relationship among associative memory efficiency, associative memory cells and their plasticity9,11,13,14,161,162 indicates an activity-dependent positive cycle between the recruitment and refinement of associative memory cells7.\n\nA feature of associative memory cells is the mutual axon projections and synapse innervations for encoding multiple associated signals. The molecules potentially responsible for axon growth and synapse formation are likely substrates underlying the recruitment of associative memory cells. Current studies indicate that antagomirs for microRNA-324 and microRNA-133a, through influencing Ttbk1 and Tet3 expression, attenuate associative memory, new synapse innervation and associative memory cell formation9,73. The downregulation of miRNA-342 expression and the upregulation of Nlgn3 and Nrxn1 expression are coupled with the recruitment and refinement of associative memory cells15,17. These genes and proteins are related to axon prolongation and synapse formation. Thus, the recruitment of synapse innervations and associative memory cells may be based on a chain reaction from intensive neuronal spikes to microRNA-regulated genes and proteins that specifically manage axon prolongation and synapse formation9,13,73. In addition, the inhibition of sensory cortices blocks associative memory11,13 and the injection of microRNA antagomirs into sensory cortices lowers the strength of associative memory and the recruitment of new synapse innervations and associative memory cells9,73. Therefore, the primary location to encode associative memory is likely in the sensory cortices, where mutual synapse innervations and primary associative memory cells are recruited7,8.\n\nThe pair-encoding neurons that encode two signals, similar to the encoding property of associative memory cells, have been detected in the animal visual cortex in vivo2,104. These pair-encoding neurons in intramodal cortices may work for the integrative memory of the associated signals inputted from a single sensory modality, such as associated photon beams in images to the visual system, associated odor signals to the olfactory system, associated letters and words to the auditory system and so on (Figure 2). It should be emphasized that the morphological evidence about mutual synapse innervations among the pair-encoding neurons in single modality cortices remains to be indicated.\n\nAs nerve cells, associative memory cells recruited in sensory cortices have specific features for associative memory and general features for neurons, in which specific features are used as criteria for identifying whether the neurons detected are associative memory cells. As their coactivation via the synchronous activity of cortical neurons triggers their mutual synapse innervations and recruits them as associative memory cells, the specific features of associative memory cells include the following7,8. Associative memory cells receive new synapse innervations from coactivated sensory cortical neurons for their mutual connections alongside innate sensory input. Associative memory cells encode new and innate associated signals for their integrative storage. Their axons convergently project to and synapse onto the neurons in brain areas relevant to cognitive processes, emotional reaction and behaviors. Their recruitment is controlled by microRNA-regulated genes and proteins that manage axon projections and synapse formations9,13,73. The mutual synapse innervations among associative memory cells allow the reciprocal recall of associated signals and the conversion of signal retrieval among different modalities, e.g., image signals are presented through verbal language, verbal signals in stories are presented by visual diagrams. Their synapse convergences onto downstream neurons and the activation of associative memory cells permit logical reasoning, associative thinking, computing and so on. In general, for neuron and function outcome, the number and the function state of associative memory cells influence memory strength and maintenance. The number of associative memory cells is affected by their mutual synapse innervations under the induction of coactivation strength and repetitive coactivations as well as by developmental stages9,11. The functional state of associative memory cells is influenced by the strength of innate and new synapse inputs, their ability to convert synaptic analogue signals into digital spikes, as well as their ability to output spikes44,163–165. In addition, glutamatergic associative memory cells will suppress the activity of other neurons through GABAergic associative memory cells and lateral inhibition to have themselves to be dominantly active for memory retrieval16,17.\n\nIn summary, synapse innervations to associative memory cells determine the specificity of memory content. The number and functional state of associative memory cells as well as the connection and activity strengths in their synapse inputs and axon output partners influence the power and persistence of memory and retrievals9,14,73,162. For instance, barrel cortical neurons receive new synapse innervations from the piriform cortex after associative learning alongside innate inputs from the thalamus. Synapse activities in the pathway of odor signal will drive barrel cortical neurons toward spiking threshold under the basal activity of thalamic inputs. Once the spiking threshold reaches, their spikes activate downstream motor cortical neurons for odorant-induced whisker motion. With these associative memory cells in sensory cortices9,11,73, their axon-innervated downstream neurons are able to encode these associated signals12,18,23,27,29,31,166. The stimulations to any of these areas in neural circuits from sensory cortices to behavior- and emotion-related brain nuclei induce memory representation21,22,25,26,28,30. It is noteworthy that there are around ten thousand types of proteins in living cells167, which is much less than unit signals remembered in life, such as words, unitary images, odorants, and so on. As more than ten billion neurons reside in the central nervous system, those neurons with synapse interconnection, i.e., associative memory cells should be the basic units for memory traces, instead of the possibility of a specific protein for the given memory content.\n\nAssociative memory cells in cognition-, emotion- and behavior-relevant brain areas: In addition to primary associative memory cells in sensory cortices to integrate exogenous signals, secondary associative memory cells that integrate and store endogenous signals may be recruited in cognition, emotion and behaviors8. The contents, processes and outcomes generated from logical reasoning and associative thinking can be remembered. Emotional reactions to various stimulations and operations can be recalled. All of these specific events in mind may be generated based on the associative storage of learnt exogenous signals in sensory cortices, such as images, stories, tastes and odors, and can be memorized in brain areas relevant to cognition, emotion or behaviors in the integrative manner for subsequent recalls. In terms of cellular substrates, the reorganized association of the stored signals in the sensory cortices may make primary associative memory cells to strengthen their mutual synapse innervations and convergent innervation on downstream neurons as well as to receive feedback synapse innervations during cognitive processes and emotional reactions. These downstream neurons become able to encode the associated signals and are recruited to be secondary associative memory cells that memorize specific contents generated in associative thinking and logical reasoning12,32. The feedforward and feedback interaction among primary and secondary associative memory cells make associative thinking and logical reasoning with the inclusion of sensory origins7,8 (Figure 1 and Figure 2).\n\nIn terms of brain areas to produce secondary associative memory7, prefrontal cortical neurons demonstrate a sustained activity after pair stimulations27,29. Cue-response neurons in the inferotemporal cortex are detected after associative learning23. Neurons in response to conditioned and unconditioned stimulations and their response transformation are seen in the amygdala168. Neurons in the hippocampus and amygdala are involved in contextual fear memory169. Memory cell assemblies for temporal signals are overlapped and recorded in the hippocampus18. The activation of engram cells in the amygdala or the hippocampus is sufficient to induce fear responses21,22. These data imply that memory cells are generated in the prefrontal cortex, hippocampus, amygdala and associative cortices for memory retrievals26,170. Whether these memory cells are synaptically innervated by primary associative memory cells in sensory cortices remains to be examined.\n\nAfter associative learning by pairing whisker, odor and tail signals, neurons that encode three signals are detected in the motor cortex, prefrontal cortex and hippocampus12,31,32, in addition to the barrel and piriform cortices9,11,15. The responses of the neurons in the prefrontal cortex, the hippocampus and the motor cortex to the signals are attenuated by inhibiting barrel or piriform cortical functions. Their responses and plasticity are sustained in the barrel cortex for long-term and are decayed in the motor cortex after the pair training ends. Individual neurons in the prefrontal cortex, motor cortex and hippocampus receive synapse innervations from the coactivated sensory cortices after paired stimulations12,31,32. These results provide functional and morphological evidences for the recruitment of secondary associative memory cells in the prefrontal cortex, the hippocampus and motor cortex through their coactivity with primary associative memory cells in sensory cortices8.\n\nWhether memory cells in the downstream of sensory cortices undergo cross-modal connections, similar to primary associative memory cells8, appears indicated by recent studies. The pathway from the ventral hippocampus to the nucleus accumbens is involved in social memory24. Engrams in the prefrontal cortex emerge after receiving inputs from the hippocampus and amygdala in contextual fear memory20. Axon projection from the prefrontal cortex and hippocampus to the amygdala is formed during fear memory171. The pathway from the prefrontal cortex to the striatum plays a crucial role in reward memory25.\n\nThe characteristics of secondary associative memory cells in cognition- and emotion-related brain areas and association cortices are listed below. They receive new synapse innervations convergently from primary associative memory cells in coactivated sensory cortices in cognitive processes and emotion reaction. They encode endogenous associated signals from sensory cortices for the integrative storage. The association of cognition events and emotion reactions induces mutual synapse innervation among these secondary associative memory cells. Their axons project to memory-output cells in behavior-related brain areas for memory representation by language, countenance, gesture and writing. The number of secondary associative memory cells is influenced by mutual synapse innervation evoked by coactivation strength and repetitive coactivations during cognition as well as by development stage. The function state of secondary associative memory cells is influenced by synapse input, their ability to convert synaptic analogue signals into digital spikes and their ability to output spikes that drive memory-output cells. Synapse innervations to secondary associative memory cells determine the specificity of memory contents during cognitions and emotions. The number and excitability of secondary associative memory cells as well as their connection and activity strengths set up the persistence and power of memory formation and retrievals. Activations to secondary associative memory cells permit the rehearsal of associative thinking, logical reasoning and emotional reactions. It is pointed out that the outputs of secondary associative memory cells innervate brain areas, such as the hypothalamus and extrapyramidal system, to influence sympathetic/parasympathetic balance, temperature set-point, food ingestion and hormones to be involved in emotional reactions and behaviors.\n\nAssociative memory cells detected in cerebral cortices include glutamatergic neurons, GABAergic neurons and astrocytes9–11,13,15–17. The connections between glutamatergic neurons and GABAergic neurons is mutually upregulated after memory formation15,17. These data indicate that all of these memory cells constitute the basic units to store specific associated signals. The activation of glutamatergic associative memory cells will cause them to be excited and their neighboring neurons to be inhibited by GABAergic associative memory cells and lateral inhibition, so that the memory of associated signals is maintained in a contrasting manner. In the meantime, these glutamatergic associative memory cells can limit themselves so not to become over-excited through GABAergic associative memory cells and recurrent inhibition7. In terms of interactions among associative memory neurons and astrocytes, the working load of associative memory neurons can be supported by associative memory astrocytes, which transfer nutrients and waste products between neurons and blood vessels7,9,11.\n\nIn addition to associative memory cells in cross-modal sensory cortices or among cognition- and emotion-related brain areas, associative memory cells can be located in intramodal cortices, such as associated photon beams in images to the visual system2,104, associated odor signals to the olfactory system, associated letters and words to the auditory system and so on (Figure 2 and Figure 3). Neuronal afferent pathways for associated signals in a single sensory modality may innervate multiple groups of neurons, in which neurons in each group encode one of these associated signals. For instance, different groups of auditory cortical neurons receive neural afferents carrying different frequency sounds in a point-by-point manner and each group of neurons encodes one of specific frequency sounds. The different visual cortical neurons receive synapse innervations from different retina cone cells in a point-by-point manner. The coactivation of the neurons that encode different intramodal signals can induce their mutual synapse innervations, such that associative memory cells in a single modality of the sensory cortices are recruited. The associative memory cells in a given sensory cortex are recruited to memorize intramodal signals with different features, strengths and locations of input signals. With associative memory cells in intramodal sensory cortices, intramodal memory to associated signals is formed, e.g., image one induces image two recall, odor one induces odor two recall and word one induces word two recall, or the other way around7. It is noteworthy that there is the time delay among intramodal signals, in which activity persistence in different sets of neurons in a given sensory cortex may grant the partially temporal overlap of their coactivity to recruit intramodal associative memory cells. The different proportions, activity strengths and connections of the intramodal associative memory cells are responsible for the storage and retrieval of intramodal signals with different features90. Intramodal associative memory cells may also be recruited within one of brain areas relevant to cognitions and emotions8.\n\nIn terms of the relationship between primary and secondary associative memory cells in memory traces and their role in memory-related processes, our proposed model is given as follows. Basic architectures for their working together include mutual synapse innervations among primary associative memory cells in the sensory cortices and their axon terminations onto secondary associative memory cells in brain areas relevant to cognitions, emotions and behaviors. Each set of primary associative memory cells connects one set of secondary associative memory cells reciprocally, whose functions are closely related (Figure 1). The axons from all of these associative memory cells terminate to motor neurons for memory output (memory output cell) and innate reflex (Figure 2). Mutual synapse innervations among primary associative memory cells constitute the interaction circuits for the reciprocal retrieval of associated signals by each of the sensory cues, as well as the automatic conversion retrieval of associated signals among different modalities9,11,15,17. The convergent synapse innervations from primary associative memory cells to secondary associative memory cells (Figure 1) confer logical reasoning, associative thinking and other integrative cognition induced by one of cues12. For instance, one of secondary associative memory cells is convergently innervated by three sets of primary associative memory cells that carry three kinds of signals, which maintain basic activities in this secondary associative memory cell. When an input cue activates three sets of primary associative memory cells by their mutual synapse innervations, these primary associative memory cells can convergently activate this secondary associative memory cell, in addition to its activation through the dominantly innate chain from one set of primary associative memory cells onto one set of secondary associative memory cells. In other words, three kinds of signals triggered by one of these cues drive this secondary associative memory cell to achieve the integration of three associated signals for associative thinking and logical reasoning. This integration is also facilitated by mutual synapse innervations among secondary associative cells that contribute to interactions of the higher order cognition and emotions. The divergent synapse innervations from primary associative memory cells to secondary associative memory cells (Figure 1) mean that associated signals are stored in several brain areas for long-term maintenance with less chance of being lost, as well as being used for different cognitive processes and emotional reactions12. In addition to this feedforward innervation from primary to secondary associative memory cells, there may be a feedback connection from secondary associative memory cells to primary associative memory cells, by which the learnt exogenous signals will automatically initiate cognition and emotions as well as endogenous signals from cognitive events and emotional reactions which usually contain sensory signal sources7 and (Figure 1 and Figure 2).\n\n\nThe refinement of associative memory cells\n\nCell assemblies formed by connection strengthening through their correlated activities, especially the coincidence activity of presynaptic and postsynaptic cells, presumably work for learning and memory81. This hypothesis is well matched by synaptic and neuronal plasticity69,172–174, e.g., long-term potentiation and depression in synaptic transmission83,152 or neuronal activity27,155. Many studies about synapse and neuron plasticity were not carried out in memory cells despite brain areas presumably relevant to memory. Synaptic plasticity in a given neuronal pathway does not reveal how multiple signals are integrated and encoded in associative memory cells. These uncertainties raise the issue of how these data about plasticity are written in the profile of cellular mechanisms underlying associative memory. Based on current studies, there are two forms of plasticity in associative memory cells, i.e., the refinement during their recruitment for them to coordinate with each other and the refinement induced by cues to recall specific signals, both of which are activity-dependent based on coactivation among neurons9,11,13,15,17,32,73, i.e., recruitment-related refinement and activity-dependent plasticity.\n\nIn the recruitment of associative memory cells from cortical neurons through their coactivation and mutual synapse innervations, the number of excitatory synapses and the transmission strength at each of these synapses on glutamatergic and GABAergic neurons are enhanced; the output of glutamatergic neurons is enhanced and the output of GABAergic neurons is weakened15,17,161,162,166. In addition, the active intrinsic property of glutamatergic associative memory cells is upregulated and the excitability of GABAergic associative memory cells is downregulated15,17,161,162. Mutual synapse innervations among the associative memory cells are increased15,17. Increases in the driving force from excitatory synapses and in the excitability of memory cells, as well as decreases in the driving force from inhibitory synapses, shift the balance of these cortical neurons between excitation and inhibition towards excitation. Their high activity can attract more synapse innervations, recruit more glutamatergic/GABAergic associative memory cells, promote their functional state to an optimal level for information storage and facilitate the activation of these associative memory cells for retrieval of the associated signals11,13,14,17. The increased number and function of excitatory synapse inputs in associative memory cells strengthen the encoding ability and precision44,164,165 for efficient memory formation and precise retrieval. If excitatory associative memory cells are over active, they can activate neighboring inhibitory neurons to prevent hyperactivity through recurrent negative feedback43,44,175.\n\nThere are two forms of neuronal excitation plasticity to interpret how neuronal refinements are involved in the formation and the retrieval of associative memory, i.e., the downregulation of threshold potential to fire spikes and the upregulation of spiking ability to fire more sequential spikes. The intensive activity of cortical neurons by high frequency stimulus, similar to neuronal coactivation during associative learning, shifts spike threshold potential toward the resting membrane potential, so that the firing of neuronal spikes is facilitated155. The intensive neuronal activity also upregulates the capacity to fire sequential spikes27,69. Both mechanisms elevate the neuronal capability to encode digital spikes, which strengthens a chain reaction from spikes to microRNA-regulated expression of genes and proteins that facilitate the recruitments of new synapse innervations and associative memory cells9,11,15,17,73 as well as the retrieval of the associated signals176. These changes have been detected in associative memory cells15,17,162. Thus, plasticity in neuronal excitability may play one of central roles in learning and memory, which is reiterated by a current review177.\n\nIn the study of memory traces or cell assemblies, synaptic potentiation has been detected at engram cells in slices of the prefrontal cortex, the hippocampus and the amygdala140 and the excitation enhancement of B51 neurons is isolated from Aplysia178. In the study, by using cues to sensory inputs in vivo, activity-dependent potentiation in response to associated signals was evoked at input pathways in the active group of primary and secondary associative memory cells, and activity-dependent conversion from silent into active neural pathways in response to associated signals was initiated in the inactive group of associative memory cells7,12,32. This activity-dependent upregulation in response to associative signals in the given group of associative memory cells may allow them to become more excited than their neighboring neurons and to be highly sensitive to the excitatory driving force from sensory cues, such that more associative memory cells are recruited by their increased mutual synapse innervations in response to all associated signals15,17. Furthermore, activity-dependent potentiation in response to associated signals can be induced through homosynaptic and heterosynaptic pathways32, which facilitates the reciprocal recall and logical reasoning of those associated signals. Activity-dependent potentiation at associative memory cells in response to the associated signals inputted through new synapses may be mechanistically caused by the enhancement of individual synapses and/or the conversion of inactive or silent synapses into functional synapses179,180, since new mutual synaptic innervations have been formed among these associative memory cells7,9,11,12,15,17,32,73. In terms of function impacts, activity-dependent potentiation at primary associative memory cells may facilitate the memory retrieval of exogenous associated signals. Activity-dependent potentiation at secondary associative memory cells facilitates the memory retrieval of endogenous signals generated during cognitive processes and emotional reaction. Thus, the spontaneous or cue-induced recalls of these signals are emerged for the rehearsal of cognitions and emotional pulses. Recruitment-related neural potentiation and activity-dependent neural potentiation are supported by the fact that the enhancement of neuronal excitability is multi-grade in nature155.\n\nRecruitments of primary associative memory cells in sensory cortices and of secondary associative memory cells in cognition/emotion-relevant brain areas endorse the specificity of the storage of associated signals8,9,11,13,15,17,73. The number and function state of associative memory cells influence the strength and maintenance of specific memory as well as the efficiency of memory retrieval9,13,14,73. Structural and functional plasticity at subcellular compartments of associative memory cell influences whether they sensitively integrate associated signals, precisely memorize these signals and efficiently trigger their target neurons for memory retrievals15,17. The maintenance of activity-dependent refinement at associative memory cells supports the period for them to be sensitive to the cue for memory retrieval. It is emphasized that both recruitment and refinement of associative memory cells depend on their simultaneous activity9,11,13,15,17. The activities of associative memory cells as central point comprise coactivity-dependent positive cycle in their recruitment and refinement, i.e., activity together, mutual innervation together and strengthening together. Highly active neurons while receiving associated signals are recruited as associative memory cells and are functionally upregulated. The upregulated population and function state of associative memory cells during repeated learning processes recruit more associative memory cells and upregulate their active state further7. Activity-dependent positive cycle in the recruitment and refinement of associative memory cells, which is based on the function compatibility between neuronal partners163, can interpret realistic practices under conditions of normal consciousness and well attention, i.e., the more learning times is, the more associative memory cell recruitment/refinement is, and the more impressive memory is. It should be pointed that associative memory cells fall into the active group of neurons in the brain, but active neurons labeled by non-specific immediate early genes may not be memory cells.\n\nIn terms of the functional states of primary and secondary associative memory cells influenced by synapse inputs, the number and strength of the inputted synapses are proportional to the excitation levels of these associative memory cells9,14,73,161,162. The increase of synapse inputs that carry specific memory content and their upregulation from receiving repeated cues drive associative memory cells to become more excitable for the retrieval of this specific memory and the full recruitment of memory cells. The increased activity of synapse inputs from the arousal system boosts associative memory cells to become more excitable for the retrieval of memory contents stored in a nonspecific manner. Moreover, the increase of excitability or the decrease of spiking threshold in associative memory cells will make them be easily activated for the retrieval of memory contents nonspecifically and the recruitment of more associative memory cells15,17. A theoretical illustration of associative memory cells driven by synapses and neuronal excitability is given in Figure 3.\n\nThe neurons in the central nervous system that are dominantly recruited as associative memory cells needs to be determined. Based on the principle that the simultaneous coactivation of cortical neurons and the activity-dependent positive cycle between the recruitment and refinement of associative memory cells are the primary driving force for the neurons being recruited as associative memory cells7, we assume that the neurons with high levels of excitation and synapse inputs are preferentially are recruited as associative memory cells. In other words, the cortical neurons, which possess a lower spiking threshold caused by their activities, as well as stronger synapse inputs driven by attention calls from previously learned relevant associated signals carried by the synapses formed in those events or by the consciousness levels maintained by the arousal system plus memory, are favorably recruited as associative memory cells. These dominantly active neurons are always recruited to be associative memory cells at the first grade, their activation and recruitment trigger the neighboring neurons through their synapse connections to be more active and become associative memory cells in the second grade, and so on. This preferential grading of the recruitment of associative memory cells leads to a time sequence for groups of cortical neurons to be recruited as associative memory cells when multiple associated signals are exposed to learners sequentially, such as words by words in sentences or articles and images by images in visual or video views7.\n\nThere are a few interesting observations about the recruitment and refinement of associative memory cells. The establishment of associative memory follows a development change, i.e., memory formation shows initial increase and then decrease with aging11. Synapse and neuron plasticity mature during postnatal development155,180. These studies indicate the dominant roles of recruitment versus refinement of associative memory cells in memory formation and retrieval in different developmental stages. The activity-dependent recruitment of associative memory cells may play the dominant role in associative memory during early and young age, while the activity-dependent refinement of associative memory cells works dominantly after these stages. The knowledge learned in young age is in the form of relatively simple unitary signals whereas the knowledge learned in matured age is complicated in the form of reorganized unitary signals. In this regard, associative memory cells recruited in young age store unitary signals, and associative memory cells refined in matured age work by learning the reorganized unitary signals7.\n\n\nAssociative memory cells are modulated by transmitters and hormones\n\nIn addition to new and innate synapse innervations on primary associative memory cells, their convergent and divergent innervations on secondary associative memory cells and reciprocal synapse innervations among them (Figure 1), such associative memory cells may receive synaptic innervations from the arousal system, including the ascending reticular activating pathway181,182 and the ascending activating pathway from the neuronal axons of the locus coeruleus, the midbrain raphe nuclei, the cholinergic nuclei and substance nigra183–186. The arousal system widely innervates the neurons in cerebral brain to maintain wakefulness and to permit consciousness through their released acetylcholine, serotonin, norepinephrine and dopamine. It has been proposed that this arousal system, under the conditions of alter and rewards, supports the coactivation of cortical neurons for their recruitment to be associative memory cells, as well as maintains the basal activity of primary and secondary associative memory cells7 (Figure 2). This proposal is supported by recent studies that memory formation and retrievals are upregulated by acetylcholine, norepinephrine, serotonin and dopamine134,135,187–193, although these studies are not focused on associative memory cells. In addition, there is a coordinated strengthening effect of serotonin and norepinephrine on associative memory cells to raise the efficiency of associative learning and memory32. Serotonin increases neuron responses to synaptic inputs194,195, and dopaminergic neurons enhances synaptic bouton formation196, indicating that these neurotransmitters act on synapses and neurons to facilitate memory formation.\n\nIn addition to neurotransmitters, hormones may influence the recruitment and refinement of associative memory cells. It has been found that estrogen upregulates the dendritic spines on hippocampal neurons197–199, luteinizing hormone downregulates cognitive processes and spine density200, and gonadotropin-releasing hormone regulates spine density201. Moreover, estrogen and luteinizing hormone can upregulate associative learning, but this upregulation is attenuated by their combined applications202. These data indicate that monoamine transmitters and hormones modulate learning and memory. The targets of these molecules on memory cells need to be addressed.\n\n\nAssociative memory cells in physiology and psychology\n\nAssociative memory cells are essential for memory formation, memory retrieval, cognitions and emotional reactions9,11,12,15,31,73,161,162. The features and activity principles of associative memory cells can be applied to construct a working map (Figure 1 and Figure 3) relevant to associative memory by cross-modal or intramodal manners, which includes the efficiency of associative learning, the integrative storage of multiple signals, the strength and preservation of associative memory, the efficiency of memory retrieval, the transformation of simple to complex information storage, the temporal sequence of learning and memory to multiple signals, the correlation of associating memory to cognitive process and emotional reactions, and so on. The features and working principles of associative memory cells also assist with interpreting memory patterns, e.g., declarative (explicit) versus nondeclarative memory (implicit), episodic versus semantic memory and transformation between such patterns under the conditions of consciousness and attentions.\n\nThe simultaneous activity of the neurons among different brain areas is essential for recruiting new synapse innervations and associative memory cells. The coactivity of sensory cortical neurons by cross-modal or intramodal manners induces their mutual synapse innervations, so that these neurons become able to encode multiple associated signals, i.e., these neurons are recruited as associative memory cells9,11,15,17. The coactivation of these primary associative memory cells also drives their axon prolongation and convergent synapse innervations onto the neurons in cognition and/or emotion-relevant brain areas, recruiting them as secondary associative memory cells in logical reasoning and associative thinking7,12,31,32,166. These associative memory cells based on their synapse inputs and mutual synapse innervations constitute memories specific to associated signals. The activity-dependent positive cycle in the recruitment and refinement of associative memory cells recruits more associative memory cells to enhance memory strength and maintenance7. These data provide new insights for memory formation, suggesting that mutual synapse innervations among primary associative memory cells endorse a reciprocal retrieval of associated signals and that secondary associative memory cells based on synapse convergences from primary associative memory cells function in associative thinking and logical reasoning. These results in activity together, connection together and strengthening together also upgrade a hypothesis by Hebb that the repeated coactivation of interconnected cells evokes the strengthening of neural wire to form cell assemblies for memory81.\n\nAssociative memory formed by the association of multiple signals from cross-modal sensory modalities is commonly seen in life, such as the association of visual and auditory signals. Memory retrievals can be achieved by the automatic conversion of visual signals into verbal signals, or other way around, in addition to the retrieval reciprocally induced by either of the associated signals7. For instance, images in movies or videos can be recalled and represented by verbal styles. The contents in verbal stories can be recalled as diagrams. Primary associative memory cells, by mutual synapse innervations among cross-modal sensory cortices, may contribute to the reciprocally induced retrieval and the automatic conversional retrieval of associated signals among cross-modal sensory modalities. Similarly, the intramodal association of multiple signals is commonly seen, such as different objects in single view and different words in single sentence. Primary associative memory cells by mutual synapse innervations7 and pair-encoding2,104 in the single sensory cortex endorse memory retrieval in a picture-by-picture or word-by-word manner. It is noteworthy that signals in the visual system and the auditory system are usually complicated. An image consists of numerous photon beams with various light strengths and colors. Each sentence consists of many words and letters. Physiologically, the images that consist of numerous photon beams with different spatial distributions and light strengths are detected by different cone cells in the retina, which transmit these photon signals through visual nerves to visual cortical neurons in a point-by-point manner. Sound wave frequencies from words and letters are detected by hair cells in different segments on the cochlea base membrane, where hair cells are stimulated and their electrical signals are transmitted via auditory nerves to auditory cortical neurons in a point-by-point manner203. How these unitary signals included in an image or a sentence are reintegrated and memorized in cerebral cortices is largely unknown7.\n\nIn line with the principle of activity together, connection together and strengthening together7, the coactivations of auditory cortical neurons, which receive synapse inputs from hair cells on cochlea base membrane and encode words or letters with different sound frequencies in early life, induce mutual synapse innervations among these neurons to recruit intramodal primary associative memory cells that store these unitary sound signals. As cortical neurons possess a few folds of differences in their excitability44,204, it may be postulated that the neurons with the highest excitatory state are dominantly activated. The afterdischarge of the neurons initially activated by the first letter or word coincides with the discharge of the neurons activated by the second ones, the afterdischarge of the neurons for the second letters or words coincides with the discharge of neurons for the third ones, and so on. The coactivation of these neurons evokes their mutual synapse innervations, which may constitute the integrative storage of letters in a given word or words in a given sentence. In repeated learning of this sentence or word, this group of auditory cortical associative memory cells is strengthened in their mutual synapse innervations and activities. The recruitment and refinement in this group of auditory cortical associative memory cells confer the consolidated memory of this word or sentence for subsequent retrievals. In the subsequent lifespan, sound signals to the auditory system become complicated, which are often the reorganization of unitary sound signals including letters and words. The learning of these reorganized unitary signals will strengthen their correspondent associative memory cells that have stored unitary sound signals via their synapse innervations and excitability, in order to encode these newly listened words and sentences, which will be preferentially activated in memory retrievals. In addition, glutamatergic associative memory cells suppress the activity of other neurons through GABAergic associative memory cells and lateral inhibition to have themselves activated preferentially for memory retrievals16,17.\n\nSimilarly, the coactivation of visual cortical neurons that receive point-by-point synapse innervations from retina cone cells in early life evokes mutual synapse innervations among these neurons in order to recruit intramodal associative memory cells that store unitary signals (photon beams with different intensity and color) in visual images. There is a proportional relationship between neural activity strength and stimulus intensity17, so the neurons receiving the stronger light are more active. In line with the principle of neurons becoming active together, connecting together and strengthening together7, mutual synapse innervations among these strongly active neurons will be dominant. These active neurons are recruited to become a group of intramodal primary associative memory cells to fulfill the integrative storage of strong light beams in given visual images. In the meantime, the axons of these associative memory cells may project to visual association cortices205,206 and make convergent synapse innervation onto their neurons to recruit secondary associative memory cells32. This process of transferring from primary to secondary associative memory cells fulfills a transferring of image signals, especially strong photon beams, into the integrative storage at the secondary level as well as allows primary associative memory cells in the visual cortex to be able to receive new signals. As the neurons in the visual cortex correspond to the retina cone cells in a point-by-point manner, intramodal primary associative memory cells in the visual cortex receive major and minor synapse innervations based on their activity strength stimulated by signals from cone cells. Secondary associative memory cells in visual association cortices mainly receive convergent synapse innervations from active primary associative memory cells with major synapse innervations and active synapses converted from silent ones, such that major features in images are the integrative storage and subsequent retrieval. Our suggestions are supported by a recent report that visual association areas are recruited during memory formation207. In subsequent associative learning, based on the reorganization of unitary signals in various new images, the portion of associative memory cells reactivated by those reorganized unitary signals will be integrated together through the conversion of inactive/silent synapses into active synapses among them to fulfill the integrative storage of new associated signals180.\n\nIn practice, intramodal and cross-modal associative learning and memory occur simultaneously, especially the association of visual and auditory signals. For instance, unitary signals in visual images are associated to verbal signals during social activities, such as family activities, personal communications and classroom studies, in which each feature of a visual image is given clear definition by words or sentences. During social interactions, numerous associations are formed between unitary signals from the visual modality and words/phrases from the auditory modality. These associations at the unitary level will confer the learning of complicated information based on the reorganization of these unitary signals and the reorganized integration of associative memory cells. After cross-modal associative learning, individuals are able to fulfill the reciprocal recall of the associated signals, i.e., a signal evokes the recall of its associated signals, or other way around, as well as the automatic recall of signals through one modality that have been learned through another modality, i.e., the view of images is converted into verbal recall, or other way around7. There are two mechanisms underlying these processes. In early life, the learning of associated signals from two or more modalities coactivates sensory cortical neurons in these modalities and induces mutual synapse innervations among them. Visual cortical neurons that encode unitary signals in the image mutually innervate with auditory cortical neurons that encode words or phrases, e.g., the neurons for unitary signals in an image “lemon” connect the neurons that encode words “lemon”, “yellow” and “oval”, based on their coactivation in initial learning7. Numerous associations between unitary visual signals and auditory signals in social activities induce mutual synapse innervations between visual and auditory cortical neurons to form thousands and thousands of cell-pairs, primary cross-modal associative memory cells. Their active states grant memory retrievals. The accumulation of these associative memory cells that encode pairs of unitary signals will confer the learning of complicated signals based on the reorganization of these unitary signals. In postnatal development, the capabilities of axon growth and synapse formation are gradually attenuated11. The learning of complicated signals during aging may utilize another mechanism for memory, i.e., the coactivity-dependent upregulation of associative memory cells in their excitability and mutually innervated synapses7,17,32 or the activity-dependent conversion of inactive synapses into active synapses180. As long as their function upregulations are maintained at a sufficient high level, these complicated signals can be retrieved automatically and/or by cues.\n\nThrough the coactivation-induced mutual synapse innervation for recruiting associative memory cells and coactivation-induced functional upregulation among associative memory cells, individuals can gradually memorize associated signals from unitary to reorganized unitary, i.e., the transformation of simple to complicated information storage, in a topic-related manner8. Initially, the associations of simple images in the different intramodal features with words based on letters activate visual and auditory cortical neurons, respectively. With their mutual synapse innervation, intramodal and cross-modal associative memory cells are recruited including AMCs for pictures, letters as well as for picture and word. The repeated activations of these associative memory cells through practices will induce their activity-dependent plasticity and recruit more associative memory cells, i.e., coactivity-dependent positive cycle in the recruitment and refinement of associative memory cell. The first grade of associative memory cells is formed7. With the accumulation of associative memory cells to store unitary signals, they become recruitment-ready neurons to be associative memory cells that encode complicated associative signals. The complicated visual and auditory signals can be associatively learned through activating the first grade of associative memory cells in visual and auditory cortices. Their mutual synapse innervation and activity upregulation lead to the formation of the second grade of associative memory cells that encode complicated images and sentences organized from unitary signals. Thus, numerous groups of the first and second grades of associative memory cells are accumulatively recruited in lifespan learning. In advanced learning, multiple grades of associative memory cells are recruited to encode more complicated signals. When the different groups and grades of associative memory cells are accumulated, subsequent learning may be based on their activity-dependent function upregulation, which makes them to be easily activated for quickly memory. Reading book or looking images induces the intensive activities in some groups of associative memory cells that encode these sentences and images, which leads to activity-dependent function upregulation at these associative memory cells. Their low threshold potential to fire spikes and active synapse inputs to drive these associative memory cells permit the cues dominantly to reactivate them for the recalls of images and sentences, and even the spontaneous activation of these cells to drive secondary associative memory cells for free associative thinking. The activity of these associative memory cells will lead to memory presentation by behaviors if they successfully drive the activation of memory-presentation neurons in the motor cortex7.\n\nIt is noteworthy that the complicated signals can also be dissected and memorized through the formation of associative memory cells that are able to encode multiple signals9. The complicated signals are composed of numerous unitary signals, which can be detected through the dissections by different sensory systems and intramodal sensory neurons. While learning these complicated signals, associative memory cells are recruited to integrate multiple simple signals, based on the random association of these unitary signals to induce mutual synapse innervations among their correspondent recruitment-ready neurons. These associative memory cells with different integrative ability to associated signals are recruited and the activation of portions of these associative memory cells leads to the selective recall of these complicated signals7.\n\nThere are three resources of synaptic inputs to drive and maintain the activities of associative memory cells, including new synaptic innervations from coactivated brain areas, innate synaptic inputs formed during development, and synaptic inputs from the arousal system. The latter two synapse-driving forces activate the neurons ready to be recruited as new associative memory cells. The ascending reticular activating pathway from the brain stem and the thalamus receives various sensory inputs and widely innervates the entire cerebral brain to permit the wakefulness and consciousness181,182,208. The ascending pathways from neuronal axons in the cholinergic nuclei, midbrain raphe nuclei and locus coeruleus innervate the forebrain to keep alertness and consciousness by releasing acetylcholine, serotonin and norepinephrine183,184,186. This arousal system maintains the basal activity of associative memory cells, and confers them to integrate innate and new synaptic inputs specifically and to memorize associated signals. This arousal system may also activate recruitment-ready neurons to influence the efficiencies of associative learning, of associative memory cells to facilitate memory retrievals as well as of primary and secondary associative memory cells to permit the association of memory with cognitive process and emotional reactions.\n\nLearning efficiency is influenced by neuronal excitability, synapse responsiveness and neurons ready to be recruited7. Neurons ready to be recruited for storing new associated signals may be those cells that have been able to encode the storage of previous learnt signals from specific synapse innervations. These stored signals may be closely relevant to those associated signals that will be learned, and these ready recruited neurons can be activated by giving topic cues in the attention call. The number of the ready recruited neurons influences how the information is acquired and memorized easily as well as how the complicated signals can be efficiently learnt. That is a reason why the efficiency of associative learning is influenced by a fact whether individuals are knowledgeable in the topic to be learnt. In addition, the cortical neurons are diversified in their synapse inputs and intrinsic property44, and the neurons with more synapse inputs and lower threshold potential are easily activated to fire spikes for high learning efficiency11,162, which triggers the chain reaction of intensive spikes and microRNA expression changes for axon prolongation and synapse innervations9,73. Thus, activity-dependent upregulations in neuron excitability and synapse innervations facilitate the recruitment of associative memory cells to influence learning efficiency.\n\nThe efficiency of memory retrievals is influenced by the number and function state of associative memory cells as well as the coactivity-dependent positive cycle between recruitments and refinements of associative memory cells7. Under the conditions of normal consciousness and alertness, the recruited number of associative memory cells is positively proportional to the activated associative memory cells in memory retrieval, so that the efficiency of memory retrieval would be consistent to the efficiency of associative learning11,162, the functional state of associative memory cells affects how they are easily activated in memory retrievals17, and the coactivity-dependent positive cycle between the recruitment and refinement of associative memory cells will add more associative memory cells into memory traces. Therefore, the efficiency of memory retrieval would be high under the conditions of normal consciousness and alertness. Whether the stored information can be successfully retrieved also depends on the function state of memory-output cells, since the function downregulation of memory execution cells in the motor cortex leads to the inability of memory retrieval (i.e., memory extinction) though primary associative memory cells are well-maintained in the normal function14,31. Thus, the high number and the active intrinsic property of associative memory cells in memory traces as well as the coactivity-dependent positive cycle of their recruitment and refinement lead to automatic memory retrieval after repeated learning and thinking without the need of cues.\n\nIn the transformation from exogenous signals to endogenous signals and their integrative memories7,12,31,32,166, the efficiency to correlate associative memory with cognitive processes and emotional reactions is a critical issue. In this process, the interactions between primary and secondary associative memory cells by their mutual synapse innervations (Figure 1) as well as the number and functional state of these associative memory cells should be taken into account during logical reasoning and associative thinking7. Thus, cellular processes involved in the efficiency for the learning, storage and retrieval of exogenous associated signals may similarly work for the transformation of exogenous-to-endogenous signals.\n\nIn terms of the relationships between associative memory cells and memory patterns, such as declarative or explicit memory versus nondeclarative or implicit memory, episodic memory versus semantic memory as well as the transformation between these patterns, our interpretations are below. In spite of these psychological classifications, there is no clear border line to separate them. Declarative memory is an intentional remember with clear state under consciousness, while nondeclarative memory is an effortless remember with no conscious awareness6,33. In fact, implicit memory is formed in individuals by paying attention when they initially learn these processes and operations. With long-term practice to be skilled, the expression of such processes and operations are not necessarily to be fulfilled with conscious effort. Based on coactivity-dependent positive cycle in the recruitment and refinement of associative memory cells, the repeated coactivations of primary and secondary associative memory cells can recruit more associative memory cells and upregulate their function state7,11,15,17,32,166, as well as strengthen synapse connections from associative memory cells to memory-output cells in the motor cortex14,31, so that explicit memory can be converted into implicit memory. In other words, there may be the reverse relationship between the number and upregulation of associative memory cells and the requirement of consciousness, a homeostasis for memory retrieval. Implicit memory based on more associative memory cells that are easily activated is supported by phenomena that it can usually be expressed spontaneously. In explicit memory, episodic memory in individual events can be converted into semantic memory after the repeated associative thinking and logical reasoning strengthen associative memory cells that have stored a common signal of the events through central synapse innervations or place associative memory cells that have stored those events with similar topics together to reorganize them into a group of memory cells for the general concepts and to convergently innervate on another grade of associative memory cells in an abstraction manner7.\n\nConsciousness is the combinational state of wakefulness and memory for individuals to aware and identify themselves and objects in the environment209. The normal consciousness may be based on the basal activation of associative memory cells by the arousal system and the specific activation of associative memory cells from their associated inputs triggered by sensory cues7. Thus, the number and functional state of associative memory cells are proportional to the state of consciousness. The combination of consciousness and a specific alert constitutes the attention, in which a specific group of associative memory cells is activated for memory retrieval as well as the alert-relevant recruitment-ready neurons are coactivated for learning alert-relevant signals. Once individuals are under consciousness, they have two forms of logical reasoning and associative thinking, i.e., critical versus creative. The critical thinking activates more recruited secondary associative memory cells for the evaluation, while creative thinking may generate newer secondary associative memory cells for inspiration7.\n\nThe awareness state can be classified into consciousness and unconsciousness. The sleeping can be fell into unconsciousness (slow wave sleeping) and incomplete consciousness (fast wave sleeping)209. How do different groups of associative memory cells work together during fast wave sleeping or dreaming? Dreams are often accompanied by highly activities in electronic encephalograph and behaviors, such as rapid eye movement, muscle twitch and active respiration/heat beat, indicating high activity in the forebrain. In the meantime, associative memory cells for specific events, which have been frequently thought in daytime, are activated. Associative memory cells that are intensively activated in daytime lead to the coactivity-dependent positive cycle of their recruitment and upregulation. So, these events are playbacks. As the reverse relationship between the upregulation of associative memory cells and the requirement of consciousness, associative memory cells with large population and upregulated function due to repeated learning and thinking can be activated under incomplete consciousness condition, such that playback events are incompletely identical to realistic ones7. As the playbacks can be recalled and stated, associative thinking and logical reasoning (the integration of endogenous signals) based on primary and secondary associative memory cells can be fulfilled under incomplete consciousness8. This viewpoint is granted by an observation that temporal sequences of place cell activities in a novel spatial experience are detected during the resting or sleeping period preceding the experience. This preplay occurs in the disjunction to sequences of replay in a familiar experience. These results suggest that internal neuronal dynamics during resting or sleep organize cellular assemblies into temporal sequences that contribute to encode a relevant novel experience in the future210.\n\nFurthermore, images, odors, tastes and events are presented by word-based language in associative thinking and logical reasoning. In initial learning, the sensations, perceptions and events are associated to their correspondent word descriptions, such that associative memory cells for encoding these processes and word descriptions have been recruited. Once these processes are recalled in the sequential playbacks, their word descriptions in these associative memory cells are initiated to substitute the complicated images and events, which is the requirement of speeding up memory retrieval and cognition. The substitution of words to images and events is realized based on the recruitment of more associative memory cells and their upregulations in coactivity-dependent positive cycle manner by repeated practices. However, if words and these processes are associated improperly, the corrections of these associations are difficult because of the presences of these recruited synapse innervations, associative memory cells and their circuits7.\n\n\nAssociative memory cells in pathology\n\nThe integrative storage and the reciprocal retrieval of the associated signals are critical for the bidirectional alertness and prediction in the life. Based on primary and secondary associative memory cells as well as their multi-grade integrations7, one signal will induce the recall of its associated signals, or the other way around, as well as the signals learned from one modality are recalled through the conversion into another modality. Individuals are able to fulfill logical reasoning and associative thinking as well as to predict future events in forward and backward manners. Furthermore, associative memory cells in each of the coactivated brain regions encode the associated innate signal and newly learnt signal, as well as each of the associated signals is stored in multiple brain areas, which largely reduces the chance of memory loss8,11. The storage of multiple signals in an associative memory cell strengthens the efficiency of memory retrieval9. The storage of multiple signals in a cortical area and the recall of one signal triggered by multiple signals will enable these individuals to strengthen their abilities in memory retrieval and well-organized cognitions. In these regards, the deficit of associative memory cells in their morphology, functions and local environment declines memory retrievals and cognitions as well, which are usually associated with neurological diseases and psychiatric disorders.\n\nIt is widely accepted that the normal consciousness and well attention are important for memory formation33,211,212, which can be explained by associative memory cells and their features7. With the arousal system to maintain wakefulness and the activation of recruitment-ready neurons by the topic cues in attention call, their activation and activity make them to be able to encode the associated signals. These recruited associative memory cells under the wakefulness condition will grant individuals to identify themselves and environmental objects, which constitute consciousness. On the other hand, the consciousness based on wakefulness and memory supports the activation and activity of associative memory cells to execute activity-dependent positive cycle in their refinement and recruitment, so that more associative memory cells are recruited and impressive memory is formed in the mind. Therefore, the deficit of associative memory cells will make consciousness to be obscure.\n\nPsychological disorders, such as anxiety, depression and even schizophrenia, are accompanied by unusual memory143,213. For instance, fear memory induced by acute stress is often associated with anxiety202,214. Stimulations of engram cells through an optogenetic approach in the hippocampus activate fear memory recall and anxiety22. Memory regarding the outcomes of chronic mild stresses are associated with depression-like behaviors215,216. On the other hand, the activation of positive memory traces by optogenetic methods in the amygdala suppresses depression-like behaviors139. These data indicate that the formation of associative memory cells induced by different patterns of abnormal stimulations can lead to psychological disorders, i.e., acute severe stresses recruit associative memory cells relevant to fear memory and anxiety, and chronic mild stresses recruit associative memory cells related to negative memory and depression214,215.\n\nThe proper coactivation of active neurons makes them to be recruited as associative memory cells7,11, and the activity-dependent upregulation of associative memory cells facilitates the integrative storage of associated signals7,14,17,161,162. These processes constitute the coactivity-dependent positive cycle in the recruitment and refinement of associative memory cells, such that more associative memory cells will be recruited. However, the further upregulation of associative memory cells, such as the dysfunction of GABAergic neurons in schizophrenia and epilepsy217,218, allows associative memory cells to be overly and widely activated. The overly upregulation of associative memory cells in sensory cortices will lead to hallucination. The overly upregulation of associative memory cells in cognition- and emotion-related brain areas leads to illusion7.\n\nThe efficiency of learning and memory decays in age-relevant manner219,220. There is a bell-shaped pattern in the efficiency of associative learning and memory11. In terms of cell mechanisms, synaptic potentiation matures during postnatal development180, and neuronal excitability in cortical neurons is upregulated until a plateau level at postnatal weeks 3–4155, which matches dynamical changes in associative memory well11. Neural plasticity and associative memory cell recruitment in postnatal development constitute the coactivity-dependent positive cycle in the recruitment and refinement of associative memory cells, such that more associative memory cells are recruited to increase the efficiency of learning and memory161,162. In older mammalians, the accumulations of insoluble β-amyloid and phosphorylated tau-proteins in the brain influence axon prolongations and synapse formations9,73 to suppress the recruitment and upregulation of the associative memory cells, to silence active associative memory cells and/or to deteriorate those recruited associative memory cells for the memory deficit7,8,221. On the other hand, the activity of associative memory cells can strengthen the coactivity-dependent positive cycle in the recruitment and refinement of associative memory cells, which prevents the conversion of soluble β-amyloid into its insoluble form and promotes the clearance of β-amyloids by associative memory astrocytes7,11. A current report supports this point in that light and sound stimulations coordinately reduce the accumulation of β-amyloid222.\n\nIn age-related neurodegeneration, such as Alzheimer’s disease, insoluble β-amyloid may be accumulated differently in various brain areas. For instance, the optogenetic activation of engram cells, which have a lack of increased synaptic strength and dendritic spines under protein synthesis inhibition-induced amnesia, leads to memory retrievals140. The optogenetic activation of hippocampal engram cells leads to memory retrievals in mice, though they show the amnesia under the condition of using natural recall cues in the transgenic mouse model of early Alzheimer’s disease137. In addition to the indication about the wide distribution of memory traces for signal storage and retrieval, these results suggest that areas involved in natural memory retrieval are dominantly impaired by the deposition of β-amyloid, rather than memory trace cells, as well as that areas responsible for memory retrieval are not specific for a given memory. In this regard, synapse connections from associative memory cells to memory-output cells should be strengthened in the early stage of Alzheimer’s disease7,14,31.\n\nIn terms of memory maintenance versus extinction, the recruitment and refinement of associative memory cells are not significantly declined, but the activity of memory-output neurons in the motor cortex is lowered14,31. The sustained presence of associative memory cells as well as the recruitment of more associative memory cells in repeated brain activities confer memorized signals to be retrieved in lifespan, in which the information can be retrieved as long as their innervations onto memory-output neurons successfully drive the latter to be functionally active. It is noteworthy that memory retrievals show different patterns in spontaneous, cue-induced and realistic object-triggered manner with the ages. For instance, spontaneous retrievals often occur in child stage or brain excitation, the cue-induced retrievals usually occur in young and adult, the real object-induced retrievals occur in senior individuals. In addition, when the brain is highly excited in many areas, such as euphoria perception, extreme fear and strong stimulations, more associative memory cells are recruited through their mutual innervations, so that impressive memory and spontaneous recalls to these experiences are generated in lifespan14,31. It is difficult to remove the newly formed synapse innervation and the recruited associative memory cells to relieve fear memory and addiction. Alternative ways are the avoidance of fear stimuli and the induction of happiness in order to rebalance these two states and weaken fear memory, since the lack of uses in neural circuits related to fear memory, especially from associative memory cells to memory-output neurons, may drive them to be functional silence. In the brains of individuals with history of substance abuse or addiction, primary and secondary associative memory cells relevant to these events are recruited in large amount and in extensive areas under euphoria condition, leading to potential relapses in their lifetime8. Strategies to weaken the addiction in these individuals include the avoidance of the environment cues associated with substance abuse to reduce the output of the relevant associative memory cells, as well as the establishment of alternative happiness to recruit associative memory cells that innervate memory-output cells through competition with the innervations from addiction memory cells, so that the rebalance of these two states strengthens memory-output pathway for happiness7.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "Grant information\n\nThis study is funded by National Key R&D Program of China (2016YFC1307100) and Natural Science Foundation China (81671071) to JHW.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nAs numerous papers related to learning and memory are published, author apologizes for not being able to list all references in the review of associative memory cells. Author thanks to Ms. Shan Cui for drawing partial diagrams.\n\nThis review is updated more than 75% in the difference from our previous review7\n\n\nReferences\n\nByrne JH: Cellular analysis of associative learning. Physiol Rev. 1987; 67(2): 329–439. PubMed Abstract | Publisher Full Text\n\nAlbright TD: On the perception of probable things: neural substrates of associative memory, imagery, and perception. Neuron. 2012; 74(2): 227–45. 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}
|
[
{
"id": "47192",
"date": "18 Apr 2019",
"name": "Ru-Rong Ji",
"expertise": [
"Reviewer Expertise Neuroscience."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely and well written review on associative memory from an expert. The author has been working on this topic for many years, and this extensive review is a very nice summary of the field. All three figures are well presented. I also have a few comments:\nThe manuscript will be greatly improved by including a table or a chart to show historic perspective (e.g., milestones) of the concept development by different groups.\n\nIt is interesting to discuss specific miRNAs involved in this process. It will help the readers if the author can include an illustration or a figure to show how specific miRNAs regulate associative memory via specific molecular targets. Overall, discussion on molecular mechanisms of associative memory could be strengthened.\n\nPeople often say “no pain, no gain”1. Is this related to associative memory?\n\nThe author discussed some pathological conditions. After anesthesia, there is cognitive decline, such as delirium, especially postoperative delirium in elderly. Is this a related pathological condition?\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": "47193",
"date": "23 Apr 2019",
"name": "Jian-Guo Chen",
"expertise": [
"Reviewer Expertise Neuropsychopharmacology",
"anxiety and depression"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Wang has systemically reviewed cellular units of engrams, presumably associative memory cells. In addition to the history and research approach about engrams, author presents major features of associative memory cells at primary and secondary levels, as well as their impacts in psychology and pathology. The concepts about associative memory cells and their circuits are renewing our understanding to various patterns of learning and memory. This is one of the best reviews in the topic of memory traces, which I would like to approve its publication in F1000Research without delay. I have a few suggestions for the author to move forward in future study:\nThree diagrams assume neural circuits for associative memory cells to execute memory formation and retrieval, which are mainly based on the studies by author’s group in sensory modalities including somatosensory and olfactory cortices, as well as their downstream regions, the prefrontal cortex and hippocampus. In order to have these diagrams to be generalized truth, the author should also conduct his studies in other areas, such as the visual cortex and auditory cortex.\n\nMemories to visual images are mostly complicated in the integration of spatial and temporal information, in comparison with other modalities. Although the author has assumed potential processes for the integration and memory of visual images, I feel that the hypothesized diagram is not perfect and remains to be experimentally tested.\n\nThe author should design experiments to examine how associative memory cells become dysfunctional under pathological conditions in relevance to memory deficits or amnesia, and are influenced by abnormal internal environments, such as Alzheimer’s disease and Parkinson’s disease.\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": "47195",
"date": "08 May 2019",
"name": "Hongxin Dong",
"expertise": [
"Reviewer Expertise Translational research on neuropsychiatric disorders",
"in particular",
"Alzheimer's disease."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the review, Dr. Wang has significantly expanded discussion on associative memory cells in the brain from the molecular and cellular levels to functional outcomes. The proposed model for learning and memory was supported by work from the author’s own laboratory, as well as from accumulated literature. This is a timely update that will help reveal novel mechanisms in learning and memory formation and loss, which may help to advance understanding of memory deficits in neurodegenerative disorders, such as Alzheimer’s disease.\nIn addition to general comments above, the following some suggestions that the author may consider for the future studies.\nThe author has simply discussed the possible relationship between associative memory cells and affective disorders such as anxiety and major depression. It sounds interesting but lacks experimental support. I would suggest the author to give more literature view or conduct some proof-and-concept experiments on this aspect the future.\n\nAlthough the author intends to establish that basic units of memory traces, or engrams, are associative memory cells named by author’s laboratory. It would be more supportive if the author makes the systemic and comprehensive comparisons between cell assemblies in memory traces and associative memory cells. It seems to me that associative memory cells encode the storage of multiple signals, which has not been emphasized by the studies of engrams. As the author states that almost all of learning activities are associative learning, the units to memorize particular associative signals are called as associative memory cells is straight forward. Has the author thought to make a mutual agreement in that associative memory cells and engrams are presenting the same cell ensemble in relevance to memory formation?\n\nThe theory of associative memory cells may apply in psychology, although numerous terms and theoretical models about learning and memory have been suggested in this field. Hopefully the author and the team would provide more evidence and information to support this theory.\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/8-457
|
https://f1000research.com/articles/8-447/v1
|
11 Apr 19
|
{
"type": "Research Article",
"title": "Assessment of magnesium sulphate usage in pre-eclamptic and eclamptic women in Omdurman Maternity Hospital, 2017: A cross-sectional study",
"authors": [
"Bashir Alsiddig Yousef",
"Alaa Abdulmoniem Merghani",
"Walaa Salah Abdulla",
"Rasha Rifaat Binni",
"Alaa Abdulmoniem Merghani",
"Walaa Salah Abdulla",
"Rasha Rifaat Binni"
],
"abstract": "Background: Eclampsia, the end phenomena of the preeclampsia spectrum, appears as new onset grand mal seizures in women with preeclampsia. Recently, the World Health Organization has recommended magnesium sulphate (MgSo4) as the main drug of choice to treat/prevent eclampsia. However, in Sudan no real assessment data about MgSo4 efficacy, benefits and risks in preeclamptic and eclamptic women has been reported. Methods: A cross-sectional hospital-based study was conducted between January and April 2017 at Omdurman Maternity Hospital using a checklist. Data was collected from the medical records of 130 preeclamptic/eclamptic pregnant women, including: age, blood pressure, protein urea appearance, admission diagnosis, reason for using MgSo4, policy of MgSo4 administration, outcomes after using MgSo4, side effects of using MgSo4, and MgSo4 toxicity recording. After data collection, IBM SPSS (version 21) was used to analyze the data. Results: Out of 130 recruited women, 78% were diagnosed with preeclampsia and 22% were diagnosed with eclampsia. Magnesium sulphate was indicated as a prophylactic in 88 patients and as treatment in 42 patients. Interestingly, only 9 patients had uncontrolled- recurrent seizures after using magnesium sulphate and only one patient developed drug related toxicity. Conclusion: After MgSO4 administration, the majority of patients (121; 93.1%) had controlled seizures and only one patient developed MgSO4 toxicity (respiratory paralysis). Therefore, MgSo4represents an effective and safe drug of choice used to treat/prevent eclampsia in Sudan.",
"keywords": [
"Pre-eclampsia",
"eclampsia",
"pregnancy",
"Magnesium Sulphate"
],
"content": "Introduction\n\nPreeclampsia/eclampsia is a pregnancy-specific disease with multisystem physiological disorder, which may lead to increased risk of maternal and perinatal mortality and morbidity1. Regarding etiology, no clear cause has been defined, but some reports indicate that oxidative stress and placental defects during early pregnancy may represent the main causes2. Preeclampsia is defined as pregnancy-related systemic disorder, which is associated with incidence new onset of hypertension as well as proteinuria3. Eclampsia is described as the beginning of grand mal seizure symptoms in preeclamptic women4. Epidemiologically, around 50,000–60,000 pregnant women die due to preeclampsia/eclampsia each year, which represents around one-quarter of maternal deaths in Latin America, and approximately one-tenth of maternal deaths in Africa and Asia5,6.\n\nMagnesium sulphate (MgSO4) is one of the main interventions used to decrease the morbidity and mortality from preeclampsia/eclampsia. In 2011, MgSO4 was been strongly suggested by the World Health Organization (WHO) to be the drug of choice for both treatment and prevention of eclampsia7. Currently, the WHO and other international organizations recommend two MgSO4 prophylactic protocols for eclampsia. The first one is the Pritchard regimen, in which MgSO4 is administered intramuscularly, while it is injected intravenously in the second protocol named as Zuspan regimen8. MgSO4 might be toxic in some conditions, and the toxicity depends on the serum magnesium levels, for example: loss of the knee jerk may develop at 3.5–5 mmol/l; respiratory paralysis may occur at 5–6.5 mmol/l, cardiac conduction is altered at higher than 7.5 mmol/l9. Meanwhile, it can lead to death if the serum magnesium exceeds 12.5 mmol due to cardiac arrest9.\n\nIn Sudan, preeclampsia/eclampsia is considered as one of the main factors leading to an increase in morbidity and mortality among pregnant women2. There is not enough assessment data reported about MgSO4 efficacy, benefits and risks in pre-eclamptic and eclamptic women in this setting. Thus, the present study was carried out to assess MgSO4 usage in preeclamptic and eclamptic women in Khartoum, Sudan.\n\n\nMethods\n\nA cross-sectional pilot hospital-based study was conducted between January and April 2017 at Omdurman Maternity Hospital (Khartoum, Sudan) using a checklist, which was completed by the researcher. Data was collected from patient medical records.\n\nEligibility criterion was as follows: women who were pregnant and had preeclampsia/eclampsia. The total number of preeclamptic/eclamptic patients admitted during the time period was 130 patients. Therefore, the data records of 130 preeclamptic/eclamptic pregnant women were included in this study.\n\nThe checklist (Extended data10) collected the following data variables from records: age; blood pressure; protein urea appearance; admission diagnosis (eclampsia/pre-eclampsia); reason of using MgSo4 (prophylactic in pre-eclampsia or treatment in eclampsia); MgSo4 administration policy (loading dose and maintenance dose); outcomes after MgSo4 therapy; monitoring of MgSo4 side effects (BP, pulse rate, respiratory rate, pulse oximeter, urine output, deep tendon patellar reflexes); Mgso4 toxicity recording.\n\nMisdiagnosis of admitted patients might be a source of bias. This was addressed as follows.\n\nAs per hospital policy, any patient who was admitted with blood BP≥ 140/90 and protein urea the following criteria were measured and only patients who have one or more of the following criteria were diagnosed and admitted as pre-eclamptic patients: refractory oliguria (<500 cc over 24 h), renal function compromise (minimal criterion would be a rise in serum creatinine of 1 mg/dl above baseline), persistent right upper quadrant or epigastric pain or both, persistent headache, scotomata or blurred vision, shortness of breath with reduced oxygen saturation or pulmonary edema, thrombocytopenia (platelets <100,000/cu.mm), hemolysis (based on peripheral smear analysis or increased bilirubin), impaired liver function of unclear etiology, and estimated fetal weight below 5th percentile for gestational age are important parameters to be noted.\n\nPatients who were admitted with blood BP≥ 140/90, protein urea and grand mal seizures were diagnosed as eclamptic patients.\n\nAfter data collection, IBM SPSS Statistics software (version 21) was used to descriptively analyze the data.\n\nEthical clearance (FPEC-06-2018) was obtained from the Ethical Committee of the University of Khartoum, Faculty of Pharmacy. Official agreement from the general manager and the medical directors of the hospital preceded the conduction of this study. After records were as being eligible for the study, the patients were contacted for their consent to use the records in this study. Since the majority of the patients were illiterate, oral informed consent was preferred to written informed consent.\n\n\nResults\n\nAs shown in Figure 1A, 62 participants were 25–34 years (~48%). About 34% of participants were 15–24 years and only 18% were 35–45 years old. Regarding admitting diagnosis, the majority (78%) were diagnosed with preeclampsia, while 22% were diagnosed with eclampsia (Figure 1B).\n\nGraphs showing the distribution of the study population according to (A) age; (B) admitting diagnosis; (C) MgSO4 indication; (D) administration of loading dose of MgSO4; (E) administration of maintenance dose of MgSO4\n\nEvery patient recruited to the study was followed up while they received MgSo4 therapy. During follow-up some of the preeclamptic women developed eclampsia; therefore, the ratio of preeclampsia:eclampsia became 88:42 instead of 101:29.\n\nMgSO4 was used as a prophylactic agent in 88 preeclamptic women and as treatment for 42 eclamptic women, as shown in Figure 1C. Moreover, magnesium sulphate was administered as a loading dose in 51 (39.2%) patients, while most treated patients (98.5%) were administered a maintenance dose of MgSO4 (Figure 1D and E).\n\nTo avoid practitioner-related errors, patients’ vital signs and parameters must be monitored frequently during MgSO4 administration11. In this study, only vital signs (Pulse Rate, Blood Pressure, Respiratory Rate, and Pulse Oximeter) were checked ten minutes after starting loading dose (LD). At the end of LD, 4 hourly checks were performed during maintenance dose (Figure 2A). Other important parameters, such as urine output and deep tendon patellar reflexes, were checked in 42% and 25% of patients, respectively.\n\n(A) Parameters monitored after MgSO4 administration; (B) distribution of study population according to seizure outcome after administration of MgSO4.\n\nRegarding outcomes after administration of MgSO4, the majority of patients (121; 93.1%) had controlled seizures (Figure 2B). Only one patient developed MgSO4 toxicity (respiratory paralysis).\n\n\nDiscussion\n\nBeing pregnant at age 35 or more is one of the highest risks of developing preeclampsia/eclampsia12, however, in the present study the 25–34 year age group was the largest age group of women with preeclampsia/eclampsia. MgSO4 is considered as the golden choice for treatment and prevention of eclampsia, as endorsed by the WHO7. Therefore, it is no wonder that MgSO4 has been used to control every admitted eclamptic/preeclamptic case included in the present study.\n\nRegarding the administration protocol, MgSO4 must be given by either Pritchard or Zuspan regimen. In Pritchard regimen, a loading dose of 4 g is injected intravenously, then a maintenance dose will be started immediately by intramuscular injections of 10 g followed by 5 g 6 times/day. In Zuspan regimen, 4 g dose is given a loading dose, then maintenance dose by using 1 to 2 g/h controlled infusion pump13. In present study, poor practice is observed as 61% of admitted patients didn’t receive loading doses and two patients didn’t receive maintenance dose.\n\nSome reports indicate that the rate of eclamptic seizures was recorded as being lower than 35% of all recruited cases after using MgSO4, while in present study the rate of uncontrolled-recurrent seizures was only 7% after using MgSO414. In this study, no patient died as a result of using MgSO4 whereas some literature indicates that “if MgSO4 was universally prescribed for all pre-eclamptic pregnant women, the death from magnesium toxicity may be more than that from seizures”14. On the other hand, the prevalence of MgSO4 toxicity is very mild and only one toxicity case had been reported worldwide to date15.\n\n\nConclusion\n\nAlthough practitioners in the present study did not properly adhere to MgSo4 protocol, the successful output of its usage as a treatment for eclampsia and prophylaxis for pre-eclampsia was shown by the majority of patients (93.1%) only having controlled seizures, with only one patient developing toxicity.\n\nPractitioners’ adherence to MgSo4 protocol should be assured in the future to increase the percentage of the well-controlled patients.\n\n\nData availability\n\nOpen Science Framework: Assessment of Magnesium Sulphate Usage in Pre-eclampsic and Eclampsic Women in Omdurman Maternity Hospital, 2017, https://doi.org/10.17605/OSF.IO/YV76K10.\n\nChecklist used in the present study: https://doi.org/10.17605/OSF.IO/YV76K10.\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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbalos E, Cuesta C, Grosso AL, et al.: Global and regional estimates of preeclampsia and eclampsia: a systematic review. Eur J Obstet Gynecol Reprod Biol. 2013; 170(1): 1–7. PubMed Abstract | Publisher Full Text\n\nElmugabil A, Hamdan HZ, Elsheikh AE, et al.: Serum Calcium, Magnesium, Zinc and Copper Levels in Sudanese Women with Preeclampsia. PLoS One. 2016; 11(12): e0167495. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupte S, Wagh G: Preeclampsia-eclampsia. J Obstet Gynaecol India. 2014; 64(1): 4–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchenone MH, Miller D, Samson JE, et al.: Eclampsia characteristics and outcomes: a comparison of two eras. J Pregnancy. 2013; 2013: 826045. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGhulmiyyah L, Sibai B: Maternal mortality from preeclampsia/eclampsia. Semin Perinatol. WB Saunders. 2012; 36(1): 56–59. PubMed Abstract | Publisher Full Text\n\nSay L, Chou D, Gemmill A, et al.: Global causes of maternal death: a WHO systematic analysis. Lancet Glob Health. 2014; 2(6): e323–33. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: WHO Recommendations for Prevention and Treatment of Pre-Eclampsia and Eclampsia. 2011. PubMed Abstract\n\nNHS National Institute for Health and Clinical Excellence (NICE): Hypertension in pregnancy: diagnosis and management. NICE. 2010. Reference Source\n\nTukur J: The use of magnesium sulphate for the treatment of severe pre-eclampsia and eclampsia. Ann Afr Med. 2009; 8(2): 76–80. PubMed Abstract | Publisher Full Text\n\nSalah W: Assessment of Magnesium Sulphate Usage in Pre-Eclampsic and Eclampsic Women in Omdurman Maternity Hospital, 2017. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/YV76K\n\nGrissinger M: Preventing magnesium toxicity in obstetrics. Pharmacy and Therapeutics. 2009; 34(8): 403. Free Full Text\n\nUzan J, Carbonnel M, Piconne O, et al.: Pre-eclampsia: pathophysiology, diagnosis, and management. Vasc Health Risk Manag. 2011; 7: 467–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu JF, Nightingale CH: Magnesium sulfate in eclampsia and pre-eclampsia: pharmacokinetic principles. Clin Pharmacokinet. 2000; 38(4): 305–314. PubMed Abstract | Publisher Full Text\n\nSibai BM: The magpie trial. Lancet. 2002; 360(9342): 1329. PubMed Abstract | Publisher Full Text\n\nOkusanya BO, Oladapo OT, Long Q, et al.: Clinical pharmacokinetic properties of magnesium sulphate in women with pre-eclampsia and eclampsia. BJOG. 2016; 123(3): 356–366. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "49423",
"date": "25 Jun 2019",
"name": "Girija N. Wagh",
"expertise": [
"Reviewer Expertise Maternal medicine and high-risk obstetrics",
"infertility and laparoscopic 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 study is a genuine and ethical presentation of the practices in the Omdurman Maternity Hospital in Sudan and has proved the efficacy of the magnesium sulfate in the treatment of preeclampsia and eclampsia. Magnesium sulfate is a valuable therapy in the management and prevention of eclampsia1. But many parts of the world obstetricians continue to have reservations of using magnesium sulfate due to the fear of apparent toxicity. As the evidence is coming up and the experience of the use of the drug is increasing, magnesium sulfate is proving to be an effective therapy for seizure prevention and control with the added advantage of fetal neuroprotection and pain alleviation. The fear among the physicians is a result of inadequate knowledge, and infrastructure and lack of organization and this are beautifully depicted in a recent study in Brazil2.\nThe current publication in Sudan has scientifically designed and conducted the study and has come up with valuable information on the magnesium level and the significant advantages of using magnesium sulfate. This study is important as the blood levels of magnesium have been studied and correlated. In practice actually, it is not essential to determine the blood levels of magnesium. Clinical signs if studied well and closely monitored are enough to ensure the correct delivery of the drug at the right dose. Also, it is important to keep the calcium gluconate3 injection handy as an antidote to excessive levels of magnesium sulfate. Magnesium sulfate especially is of benefit during labor management to prevent eclampsia4.\nI would also suggest the center to take up the universal and optimum delivery of magnesium sulfate as a quality improvement parameter and this would go a long way in reducing the perinatal adverse outcomes in the association of gestosis (preeclampsia).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "51556",
"date": "30 Jul 2019",
"name": "Melania Maria Ramos Amorim",
"expertise": [
"Reviewer Expertise Obstetrics and Gynecology",
"High-risk pregnancy",
"Hypertension in Pregnancy",
"Evidence-based Medicine",
"Prenatal",
"Chilbirth and Postpartum Care",
"Maternal Mortality",
"Abortion",
"Reprodutive Rights"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough the use of magnesium sulfate in preeclampsia and eclampsia is indeed a very important issue, the study is very small, it was conducted in a single center, the objectives are not clearly defined, the sample size has not been calculated, the data collection was performed for only 4 months and the study fails to add any new finding to the current state of the art. If we consider for example the outcome \"seizure recurrence\", which occurred at 7% of patients, considering a confidence level of 95% and an absolute precision of 2%, a sample size of 625 patients would be required.\nThe effectiveness of magnesium sulfate for seizure prevention and control in patients with preeclampsia and eclampsia has already been demonstrated in solid randomized controlled trials and systematic reviews available from the Cochrane Library, so an observational study would have to provide important additional information to be considered valid. With unclear objectives and inconsistent methods, without adequate presentation of the analysis variables, this is an important concern.\nI also question a fact that seems very strange to me, only 39% of the patients had the loading dose of magnesium sulfate administered. How is this possible? A maintenance dose can only be done without an loading dose. If this was a prospective study it is a very serious and ethically unjustified mistake because this problem being identified the authors would have to warn the clinical staff and provide the patient with the best available treatment.\nThe study also does not follow STROBE recommendations.\nTherefore, I do not recommend its indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-447
|
https://f1000research.com/articles/8-446/v1
|
11 Apr 19
|
{
"type": "Method Article",
"title": "A Bioconductor workflow for the Bayesian analysis of spatial proteomics",
"authors": [
"Oliver M. Crook",
"Lisa M. Breckels",
"Kathryn S. Lilley",
"Paul D.W. Kirk",
"Laurent Gatto",
"Oliver M. Crook",
"Lisa M. Breckels",
"Kathryn S. Lilley",
"Paul D.W. Kirk"
],
"abstract": "Knowledge of the subcellular location of a protein gives valuable insight into its function. The field of spatial proteomics has become increasingly popular due to improved multiplexing capabilities in high-throughput mass spectrometry, which have made it possible to systematically localise thousands of proteins per experiment. In parallel with these experimental advances, improved methods for analysing spatial proteomics data have also been developed. In this workflow, we demonstrate using `pRoloc` for the Bayesian analysis of spatial proteomics data. We detail the software infrastructure and then provide step-by-step guidance of the analysis, including setting up a pipeline, assessing convergence, and interpreting downstream results. In several places we provide additional details on Bayesian analysis to provide users with a holistic view of Bayesian analysis for spatial proteomics data.",
"keywords": [
"pRoloc",
"pRolocdata",
"proteomics",
"Bayesian",
"software",
"Bioconductor",
"spatial proteomics",
"machine learning"
],
"content": "Introduction\n\nDetermining the the spatial subcellular distribution of proteins enables novel insight into protein function1. Many proteins function within a single location within the cell; however, it is estimated that up to half of the proteome is thought to reside in multiple locations, with some of these undergoing dynamic relocalisation2. These phenomena lead to variability and uncertainty in robustly assigning proteins to a unique localisation. Functional compartmentalisation of proteins allows the cell to control biomolecular pathways and biochemical processes within the cell. Therefore, proteins with multiple localisations may have multiple functional roles3. Machine learning algorithms that fail to quantify uncertainty are unable to draw deeper insight into understanding cell biology from mass spectrometry (MS)-based spatial proteomics experiments. Hence, quantifying uncertainty allows us to make rigorous assessments of protein subcellular localisation and multi-localisation.\n\nFor proteins to carry out their functional role they must be localised to the correct subcellular compartment, ensuring the biochemical conditions for desired molecular interactions are met4. Many pathologies, including cancer and obesity are characterised by protein mis-localisations5–14. High-throughput spatial proteomics technologies have seen rapid improvement over the last decade and now a single experiment can provide spatial information on thousands of proteins at once15–18. As a result of these spatial proteomics technologies many biological systems have been characterised2,15,17,19–21. The popularity of such methods is now evident with many new studies in recent years17,22–29.\n\nBayesian approaches to machine learning and statistics can provide more insight, by providing uncertainty quantification30. In a parametric Bayesian setting, a parametric model is proposed, along with a statement about our prior beliefs of the model parameters. Bayes’ theorem tells us how to update the prior distribution of the parameters to obtain the posterior distribution of the parameters after observing the data. It is the posterior distribution which quantifies the uncertainty in the parameters. This contrasts from a maximum-likelihood approach where we obtain only a point estimate of the parameters.\n\nAdopting a Bayesian framework for data analysis, though of much interest to experimentalists, can be challenging. Once we have specified a probabilistic model, computational approaches are typically used to obtain the posterior distribution upon observation of the data. These algorithms can have parameters that require tuning and a variety of settings, hindering their practical use by those not familiar with Bayesian methodology. Even once the algorithms have been correctly set-up, assessments of convergence and guidance on how to interpret the results are often sparse. This workflow presents a Bayesian analysis of spatial proteomics to elucidate the process for practitioners. Our workflow also provides a template for others interested in designing tools for the biological community which rely on Bayesian inference.\n\nOur model for the data is the t-augmented Gaussian mixture (TAGM) model proposed in 1. Crook et al.1 provide a detailed description of the model, rigorous comparisons and testing on many spatial proteomics datasets, including a case study in which a hyperLOPIT experiment is performed on mouse pluripotent stem cells17,31. Revisiting these details is not the purpose of this computational protocol; rather we present how to correctly use the software and provide step-by-step guidance for interpreting the results.\n\nIn brief, the TAGM model posits that each annotated sub-cellular niche can be modelled using a Gaussian distribution. Thus the full complement of proteins within the cell is captured as a mixture of Gaussians. The highly dynamic nature of the cell means that many proteins are not well captured by any of these multivariate Gaussian distributions, and thus the model also includes an outlier component, which is mathematically described as a multivariate student’s t distribution. The heavy tails of the t distribution allow it to better capture dispersed proteins.\n\nThere are two approaches to perform inference in the TAGM model. The first, which we refer to as TAGM MAP, allows us to obtain maximum a posteriori estimates of posterior localisation probabilities; that is, the modal posterior probability that a protein localises to that class. This approach uses the expectation-maximisation (EM) algorithm to perform inference32. Whilst this is a interpretable summary of the TAGM model, it only provides point estimates. For a richer analysis, we also present a Markov-chain Monte-Carlo (MCMC) method to perform fully Bayesian inference in our model, allowing us to obtain full posterior localisation distributions. This method is referred to as TAGM MCMC throughout the text.\n\nThis workflow begins with a brief review of some of the basic features of mass spectrometry-based spatial proteomics data, including our state-of-the-art computational infrastructure and bespoke software suite. We then present each method in turn, detailing how to obtain high quality results. We provide an extended discussion of the TAGM MCMC method to highlight some of the challenges that may arise when applying this method. This includes how to assess convergence of MCMC methods, as well as methods for manipulating the output. We then take the processed output and explain how to interpret the results, as well as providing some tools for visualisation. We conclude with some remarks and directions for the future. Source code for this workflow, including code used to generate tables and figures, is available on GitHub33\n\n\nGetting started and infrastructure\n\nIn this workflow, we are using version 1.23.2 of pRoloc34. The package pRoloc contains algorithms and methods for analysing spatial proteomics data, building on the MSnSet structure provided in MSnbase. The pRolocdata package provides many annotated datasets from a variety of species and experimental procedures. The following code chunks install and load the suite of packages require for the analysis.\n\n\n\n\n\n\n\n\n\n\n\nWe assume that we have a MS-based spatial proteomics dataset contained in a MSnSet structure. For information on how to import data, perform basic data processing, quality control, supervised machine learning and transfer learning we refer the reader to 35. Here, we start by loading a spatial proteomics dataset on mouse E14TG2a embryonic stem cells36. The LOPIT protocol15,37 was used and the normalised intensity of proteins from eight iTRAQ 8-plex labelled fraction are provided. The methods provided here are independent of labelling procedure, fractionation process or workflow. Examples of valid experimental protocols are LOPIT37, hyperLOPIT17,31, label-free methods such as PCP16, and when fractionation is perform by differential centrifugation18,38.\n\nIn the code chunk below, we load the aforementioned dataset. The printout demonstrates that this experiment quantified 2031 proteins over 8 fractions.\n\n\n\n\n\nIn Figure 1, we can visualise the mouse stem cell dataset use the plot2D function. We observe that some of the organelle classes overlap and this is a typical feature of biological datasets. Thus, it is vital to perform uncertainty quantification when analysing biological data.\n\n\n\n\nMethods: TAGM MAP\n\nWe can use maximum a posteriori (MAP) estimation to perform Bayesian parameter estimation for our model. The maximum a posteriori estimate is the mode of the posterior distribution and can be used to provide a point estimate summary of the posterior localisation probabilities. In contrast to TAGM MCMC (see later), it does not provide samples from the posterior distribution, however it allows for faster inference by using an extended version of the expectation-maximisation (EM) algorithm. The EM algorithm iterates between an expectation step and a maximisation step. This allows us to find parameters which maximise the logarithm of the posterior, in the presence of latent (unobserved) variables. The EM algorithm is guaranteed to converge to a local mode. The code chunk below executes the tagmMapTrain function for a default of 100 iterations. We use the default priors for simplicity and convenience, however they can be changed, which we explain in a later section. The output is an object of class MAPParams, that captures the details of the TAGM MAP model.\n\n\n\n\n\n\n\n\n\nThe previous code chunk outputs a message concerning data collinearity. This is because the covariance matrix of the data has become ill-conditioned and as a result the inversion of this matrix becomes unstable with floating point arithmetic. This can lead to the failure of standard matrix algorithms upon which our method depends. In this case, it is standard practice to add a small multiple of the identity to stabilise this matrix. The printed message is a statement that this operation has been performed for these data.\n\nThe results of the modelling can be visualised with the plotEllipse function on Figure 2. The outer ellipse contains 99% of the total probability whilst the middle and inner ellipses contain 95% and 90% of the probability respectively. The centres of the clusters are represented by black circumpunct (circled dot). We can also plot the model in other principal components. The code chunk below plots the probability ellipses along the first and second, as well as the fourth principal component. The user can change the components visualised by altering the dims argument.\n\n\n\nThe EM algorithm is iterative; that is, the algorithm iterates between an expectation step and a maximisation step until the value of the log-posterior does not change32. This fact can be used to assess the convergence of the EM algorithm. The value of the log-posterior at each iteration can be accessed with the logPosteriors function on the MAPParams object. The code chuck below plots the log posterior at each iteration and we see on Figure 3 the algorithm rapidly plateaus and so we have achieved convergence. If convergence has not been reached during this time, we suggest to increase the number of iterations by changing the parameter numIter in the tagmMapTrain method. In practice, it is not unexpected to observe small fluctuations due to numerical errors and this should not concern users.\n\n\n\nThe code chuck below uses the mappars object generated above, along with the E14RG2aR dataset, to classify the proteins of unknown localisation using tagmPredict function. The results of running tagmPredict are appended to the fData columns of the MSnSet.\n\n\n\nThe new feature variables that are generated are:\n\ntagm.map.allocation: the TAGM MAP predictions for the most probable protein sub-cellular allocation.\n\n\n\n\n\ntagm.map.probability: the posterior probability for the protein sub-cellular allocations.\n\n\n\n\n\ntagm.map.outlier: the posterior probability for that protein to belong to the outlier component rather than any annotated component.\n\n\n\n\n\nWe can visualise the results by scaling the pointer according the posterior localisation probabilities. To do this we extract the MAP localisation probabilities from the feature columns of the the MSnSet and pass these to the plot2D function (Figure 4).\n\n\n\nThe TAGM MAP method is easy to use and it is simple to check convergence, however it is limited in that it can only provide point estimates of the posterior localisation distributions. To obtain the full posterior distributions and therefore a rich analysis of the data, we use Markov-Chain Monte-Carlo methods. In our particular case, we use a collapsed Gibbs sampler39.\n\n\nMethods: TAGM MCMC a brief overview\n\nThe TAGM MCMC method allows a fully Bayesian analysis of spatial proteomics datasets. It employs a collapsed Gibbs sampler to sample from the posterior distribution of localisation probablities, providing a rich analysis of the data. This section demonstrates the advantage of taking a Bayesian approach and the biological information that can be extracted from this analysis.\n\nFor those unfamiliar with Bayesian methodology, some of the key ideas for a more complete understanding are as follows. Firstly, MCMC based inference contrasts with MAP based inference in that it samples from the posterior distribution of localisation probabilities. Hence, we do not just have a single estimate for each quantity but a distribution of estimates. MCMC methods are a large class of algorithms used to sample from a probability distribution, in our case the posterior distribution of the parameters40. Once we have sampled from the posterior distribution, we can estimate the mean of the posterior distribution by simply taking the mean of the samples. In a similar fashion, we can obtain estimates of other summaries of the posterior distribution.\n\nA schematic of MCMC sampling is provided in Figure 5 to aid understanding. Proteins, coloured blue, are visualised along two variables of the data. Probability ellipses representing contours of a probability distribution matching the distribution of the proteins are overlaid. We now wish to obtain samples from this distribution. The MCMC algorithm is initialised with a starting location, then at each iteration a new value is proposed. These proposed values are either accepted or rejected (according to a carefully computed acceptance probability) and over many iterations the algorithm converges and produces samples from the desired distribution. Samples from the mean of this distribution are coloured in red in the schematic figure. A large portion of the earlier samples may not reflect the true distribution, because the MCMC sampler has yet to converge. These early samples are usually discarded and this is referred to as burn-in. The next state of the algorithm depends on its current state and this leads to auto-correlation in the samples. To suppress this auto-correlation, we only retain every rth sample. This is known as thinning. The details of burn-in and thinning are further explained in later sections.\n\nProteins are coloured in blue and probability ellipses are overlaid representing contours of a probability distribution matching the distribution of the proteins. MCMC samples from the mean of this distribution are then coloured in red.\n\nThe TAGM MCMC method is computationally intensive and requires at least modest processing power. Leaving the MCMC algorithm to run overnight on a modern desktop is usually sufficient, however this, of course, depends on the particular dataset being analysed. For guidance: it should not be expected that the analysis will finish in just a couple of hours on a medium specification laptop, for example.\n\nTo demonstrate the class structure and expected outputs of the TAGM MCMC method, we run a brief analysis on a subset (400 randomly chosen proteins) of the tan2009r1 dataset from the pRolocdata, purely for illustration. This is to provide a bare bones analysis of these data without being held back by computational requirements. We perform a complete demonstration and provide precise details of the analysis of the stem cell dataset considered above in the next section.\n\n\n\nThe first step is to run a few MCMC chains (below we use only 2 chains) for a few iterations (we specify 3 iterations in the below code, but typically we would suggest in the order of tens of thousands; see for example the algorithms default settings by typing ?tagmMcmcTrain) using the tagmMcmcTrain function. This function will generate a object of class MCMCParams.\n\n\n\n\n\nInformation for each MCMC chain is contained within the chains slot. If needed, this information can be accessed manually. The function tagmMcmcProcess processes the MCMCParams object and populates the summary slot.\n\n\n\n\n\nThe summary slot has now been populated to include basic summaries of the MCMC chains, such as organelle allocations and localisation probabilities. Protein information can be appended to the feature columns of the MSnSet by using the tagmPredict function, which extracts the required information from the summary slot of the MCMCParams object.\n\n\n\nWe can now access new variables:\n\ntagm.mcmc.allocation: the TAGM MCMC prediction for the most likely protein sub-cellular annotation.\n\n\n\n\n\ntagm.mcmc.probability: the mean posterior probability for the protein sub-cellular allocations.\n\n\n\n\n\nWe can also access other useful summaries of the MCMC methods:\n\ntagm.mcmc.outlier the posterior probability for the protein to belong to the outlier component.\n\ntagm.mcmc.probability.lowerquantile and tagm.mcmc.probability.upperquantile are the lower and upper boundaries to the equi-tailed 95% credible interval of tagm.mcmc.probability.\n\ntagm.mcmc.mean.shannon a Monte-Carlo averaged Shannon entropy, which is a measure of uncertainty in the allocations.\n\n\nMethods: TAGM MCMC the details\n\nThis section explains how to manually manipulate the MCMC output of the TAGM model. In the code chunk below, we load a pre-computed TAGM MCMC model. The data file e14tagm.rda is available online1 and is not directly loaded into this package due to its size. The file itself if around 500mb, which is too large to directly load into a package.\n\n\n\nThe following code, which is not evaluated dynamically, was used to produce the tagmE14 MCMCParams object. We run the MCMC algorithm for 20,000 iterations with 10,000 iterations discarded for burn-in. We then thin the chain by 20. We ran 6 chains in parallel and so we obtain 500 samples for each of the 6 chains, totalling 3,000 samples. The resulting file is assumed to be in our working directory.\n\n\n\nManually inspecting the object, we see that it is a MCMCParams object with 6 chains.\n\n\n\n\n\nAssessing whether or not an MCMC algorithm has converged is challenging. Assessing and diagnosing convergence is an active area of research and throughout the 1990s many approaches were proposed41–44. We provide a more detailed exploration of this issue, but readers should bare in mind that the methods provided below are diagnostics and cannot guarantee convergence. We direct readers to several important works in the literature discussing the assessment of convergence. Users that do not assess convergence and base their downstream analysis on unconverged chains are likely to obtain poor quality results.\n\nWe first assess convergence using a parallel chains approach. We find producing multiple chains is benificial not only for computational advantages but also for analysis of convergence of our chains.\n\n\n\n\n\nThe following code chunks set up a manual convergence diagnostic check. We make use of objects and methods in the package coda to perform this analysis45. Our function below automatically coerces our objects into coda for ease of analysis. We first calculate the total number of outliers at each iteration of each chain and, if the algorithm has converged, this number should be the same (or very similar) across all 6 chains.\n\n\n\nWe can observe this from the trace plots and histograms for each MCMC chain (Figure 6). Unconverged chains should be discarded from downstream analysis.\n\n\n\nChains 3, 5 and 6 are centred around an average of 153, with rapid back and forth oscillations. Chain 2 should be immediately discarded, since it has a large jump in the chain with clearly skewed histogram. The other two chains oscillate differently with contrasting quantiles to the 3 chains (3, 5 and 6) that agree with one another, suggesting these chains have yet to converge. We can use the coda package to produce summaries of our chains. Here is the coda summary for the third chain.\n\n\n\n\n\nSo far, our analysis appears promising. Three of our chains are centred around an average of 153 outliers and there is no observed monotonicity in our output. However, for a more rigorous and unbiased analysis of convergence we can calculate the Gelman diagnostic using the coda package42,44. This statistic is often referred to as R^ or the potential scale reduction factor. The idea of the Gelman diagnostics is to compare the inter and intra chain variances. The ratio of these quantities should be close to one. A more detailed and in depth discussion can be found in the references. The coda package also reports the 95% upper confidence interval of the R^ statistic. In this case, our samples are approximately normally distributed (see histograms on the right in Figure 6). The coda package allows for transformations to improve normality of the data, and in some cases we set the transform argument to apply log transformation. Gelman and Rubin42 suggest that chains with R^ value of less than 1.2 are likely to have converged.\n\n\n\n\n\n\n\n\n\n\n\n\n\nIn all cases, we see that the Gelman diagnostic for convergence is < 1.2. However, the upper confidence interval is 1.32 when all chains are used; 1.31 when chain 2 is removed and when chains 1, 2 and 4 are removed the upper confidence interval is 1.01 indicating that the MCMC algorithm for chains 3,5 and 6 might have converged.\n\nWe can also look at the Gelman diagnostics statistics for groups or pairs of chains. The first line below computes the Gelman diagnostic across the first three chains, whereas the second calculates the diagnostic between chain 3 and chain 5.\n\n\n\n\n\n\n\n\n\nTo assess another summary statistic, we can look at the mean component allocation at each iteration of the MCMC algorithm and as before we produce trace plots of this quantity (Figure 7).\n\n\n\n\n\nAs before we can produce summaries of the data.\n\n\n\n\n\nWe can already observe that there are some slight difference between these chains which raises suspicion that some of the chains may not have converged. For example each chain appears to be centred around 5.7, but chains 2 and 4 have clear jumps in the their trace plots. For a more quantitative analysis, we again apply the Gelman diagnostics to these summaries.\n\n\n\n\n\nThe above values are close to 1 and so we there are no significant difference between the chains. As observed previously, chains 2 and 4 look quite different from the other chains and so we recalculate the diagnostic excluding these chains. The computed Gelman diagnostic below suggest that chains 3, 5 and 6 have converged and that we should discard chains 1, 2 and 4 from further analysis.\n\n\n\n\n\nFor a further check, we can look at the mean outlier probability at each iteration of the MCMC algorithm and again computing the Gelman diagnostics between chains 4, 5 and 6. An R^ statistics of 1 is indicative of convergence, since it is less than the recommend value of 1.2.\n\n\n\n\n\nAlong with the Gelman diagnostic, which uses parallel chains, we can also apply a single chain analysis using the Geweke diagnostic41. The Geweke diagnostic tests to see whether the mean calculated from the first 10% of iterations is significantly different from the mean calculated from the last 50% of iterations. If they are significantly different, at say a level 0.01, then this is evidence that particular chains have not converged. The following code chunk calculates the Geweke diagnostic for each chain on the summarising quantities we have previously computed.\n\n\n\n\n\n\n\n\n\n\n\n\n\nThe first test suggests chain 2 has not converged, since the p-value is less than 10−10 suggesting that the mean in the first 10% of iterations is significantly different from those in the final 50%. Moreover, the second test and third tests also suggest that chain 2 has not converged. Furthermore, for the second test chain 4 has a marginally small p-value, providing further evidence that this chain is of low quality. These convergence diagnostics are not limited to the quantities we have computed here and further diagnostics can be performed on any summary of the data.\n\nAn important question to consider is whether removing an early portion of the chain might lead to an improvement of the convergence diagnostics. This might be particularly relevant if a chain converges some iterations after our orginally specified burn-in. For example, let us take the second Geweke test above, which suggested chains 2 and 4 had not converged and see if discarding the initial 10% of the chain improves the statistic. The function below removes 50 samples, known as burn-in, from the beginning of each chain and the output shows that we now have 450 samples in each chain. In practice, as 2 chains are sufficient for good posterior estimates and convergence we could simply discard chains 2 and 4 and proceed with downstream analysis with the remaining chains.\n\n\n\n\n\n\n\n\n\nThe following function recomputes the number of outliers in each chain at each iteration of each Markov-chain.\n\n\n\nThe code chuck below computes the Geweke diagnostic for this new truncated chain and demonstrates that chain 4 has an improved Geweke diagnostic, whilst chain 2 does not. Thus, in practice, it maybe useful to remove iterations from the beginning of the chain. However, as chain 4 did not pass the Gelman diagnostics we still discard it from downstream analysis.\n\n\n\n\n\nHaving made an assessment of convergence, we decide to discard chains 1,2 and 4 from any further analysis. The code chunk below removes these chains and creates a new object to store the converged chains.\n\n\n\nThe MCMCParams object can be large and therefore if we have a large number of samples we may want to subsample our chain, known as thinning, to reduce the number of samples. Thinning also has another purpose. We may desire independent samples from our posterior distribution but the MCMC algorithm produces autocorrelated samples. Thinning can be applied to reduce the auto-correlation between samples. The code chuck below, which is not evaluated, demonstrates retaining every 5th iteration. Recall that we thinned by 20 when we first ran the MCMC algorithm.\n\n\n\nWe initially ran 6 chains and, after having made an assessment of convergence, we decided to discard 3 of the chains. We desire to make inference using samples from all 3 chains, since this leads to better posterior estimates. In their current class structure all the chains are stored separately, so the following function pools all sample for all chains together to make a single longer chain with all samplers. Pooling a mixture of converged and unconverged chains is likely to lead to poor quality results so should be done with care.\n\n\n\n\n\n\n\n\n\nTo populate the summary slot of the converged and pooled chain, we can use the tagmMcmcProcess function. As we can see from the object below a summary is now available. The information now available in the summary slot was detailed in the previous section. We note that if there is more than 1 chain in the MCMCParams object then the chains are automatically pooled to compute the summaries.\n\n\n\n\n\nTo create new feature columns in the MSnSet and append the summary information, we apply the tagmPredict function. The probJoint argument indicates whether or not to add probabilistic information for all organelles for all proteins, rather than just the information for the most probable organelle. The outlier probabilities are also returned by default, but users can change this using the probOutlier argument.\n\nBayesian analysis requires users to specify prior information about the parameters. This may appear to be a challenging task; however, good default options are often possible. Should expert information be available for any of these priors then the users should provide this, otherwise we have found that the default choices work well in practice. The priors also provide regularisation and shrinkage to avoid overfitting. Given enough data the likelihood overwhelms the prior and the influence of the prior is weak.\n\nWe place a normal inverse-Wishart prior on the parameters of the mutivariate normal mixture components. The normal inverse-Wishart prior has 4 hyperparameters that must be specified. These are: the prior mean mu0 expressing the prior location of each organelle; a prior shrinkage lambda0, which is a scalar expressing uncertainty in the prior mean; the prior degrees of freedom nu0; and a scale prior S0 on the covariance. Together, nu0 and S0 specify the prior variability on organelle covariances. The same prior distribution is assumed for the parameters of all mutivariate normal mixture components.\n\nThe default options for these are based on the choice recommended by46. The prior mean mu0 is set to be the mean of the data. lambda0 is set to be 0.01 meaning some uncertainty in the covariance is propagated to the mean, increasing lambda0 increases shrinkage towards the prior. nu0 is set to the number of feature variables plus 2, which is the smallest integer value that ensures a finite covariance matrix. The prior scale matrix S0 is set to\n\n\n\nand represents a diffuse prior on the covariance. Another good choice which is often used is a constant multiple of the identity matrix. The prior for the Dirichlet distribution concentration parameters beta0 is set to 1 for each organelle. Another reasonable choice would be the non-informative Jeffery’s prior for the Dirichlet hyperparameter, which sets beta0 to 0.5 for each organelle. The prior weight for the outlier detection class is a ℬ (u, v) distribution. The default for u = 2 and the default for v = 10. This represents the reasonable belief that uu+v=16 proteins a priori might be an outlier and we believe is unlikely that more than 50% of proteins are outliers. Decreasing the value of v, represents more uncertainty about the number of protein that are outliers.\n\nNow that we have a single pooled chain of samples from a converged MCMC algorithm, we can begin to analyse the results. Preliminary analysis includes visualising the allocated organelle and localisation probability of each protein to its most probable organelle, as shown on Figure 8.\n\n\n\nWe can visualise other summaries of the data including a Monte-Carlo averaged Shannon entropy, as shown in Figure 8 on the right. This is a measure of uncertainty and proteins with greater Shannon entropy have more uncertainty in their localisation. We observe global patterns of uncertainty, particularly in areas where organelle boundaries overlap. There are also regions of low uncertainty indicating little doubt about the localisation of these proteins.\n\nWe are also interested in the relationship between localisation probability to the most probable class and the Shannon entropy (Figure 9). Even though the two quantities are evidently correlated there is still considerable spread. Thus it is important to base inference not only on localisation probability but also a measure of uncertainty, for example the Shannon entropy. Proteins with low Shannon entropy have low uncertainty in their localisation, whilst those with higher Shannon entropy have uncertain localisation. Since multi-localised protein have uncertain localisation to a single subcellular niche, exploring the Shannon can aid in identifying multi-localised proteins.\n\n\n\nAside from global visualisation of the data, we can also interrogate each individual protein. As illustrated on Figure 10, we can obtain the full posterior distribution of localisation probabilities for each protein from the e14Tagm_converged_pooled object. We can use the plot generic on the MCMCParams object to obtain a violin plot of the localisation distribution. Simply providing the name of the protein in the second argument produces the plot for that protein. The solute carrier transporter protein E9QMX3, also referred to as Slc15a1, is most probably localised to plasma membrane in line with its role as a transmembrane transporter but also shows some uncertainty, potentially also localising to other comparments. The first violin plot visualises this uncertainty. The protein Q3V1Z5 is a supposed constitute of the 40S ribosome and has poor UniProt annotation with evidence only at the transcript level. From the plot below is is clear that Q3V1Z5 is a ribosomal associated protein, but it previous localisation has only been computational inferred and here we provide experimental evidence of a ribosomal annotation. Thus, quantifying uncertainty recovers important additional annotations.\n\n\n\n\nDiscussion\n\nThe Bayesian analysis of biological data is of clear interest to many because of its ability to provide richer information about the experimental results. A fully Bayesian analysis differs from other machine learning approaches, since it can quantify the uncertainty in our inferences. Furthermore, we use a generative model to explicitly describe the data, which makes inferences more interpretable compared to the less interpretable outputs of black-box classifiers such as, for example, support vector machines (SVM).\n\nBayesian analysis is often characterised by its provision of a (posterior) probability distribution over the biological parameters of interest, as opposed to single point estimate of these parameters. In the case that is presented in this workflow, a Bayesian analysis “computes” a posterior probability distribution over the protein localisation probabilities. These probability distributions can then be rigorously interrogated for greater biological insight; in addition, it may allow us to ask additional questions about the data, such as whether a protein might be multi-localised.\n\nDespite the wealth of information a Bayesian analysis can provide, the uptake amongst cell biologists is still low. This is because a Bayesian analysis presents a new set of challenges and little practical guidance exists regarding how to address these challenges. Bayesian analyses often rely on computatinally intensive approaches such as Markov-chain Monte-Carlo (MCMC) and a practical understanding of these algorithms and the interpretation of their output is a key barrier to their use. A Bayesian analysis usually consists of three broad steps:\n\n(1) Data pre-processing and algorithmic implementation, (2) assessing algorithmic convergence and (3) summarising and visualising the results. This workflow provides a set of tools to simplify these steps and provides step-by-step guidance in the context of the analysis of spatial proteomics data.\n\nWe have provided a workflow for the Bayesian analysis of spatial proteomics using the pRoloc and MSnbase software. We have demonstrated, in a step-by-step fashion, the challenges and advantages associated with taking a Bayesian approach to data analysis. We hope this workflow will help spatial proteomics practitioners to apply our methods and will motivate others to create detailed documentation for the Bayesian analysis of biological data.\n\n\nSession information\n\nBelow, we provide a summary of all packages and versions used to generate this document.\n\nThe source of this document, including the code necessary to reproduce the analyses and figures is available in a public manuscript repository on GitHub47.\n\n\nData availability\n\nThe data used in this workflow was first published in Breckels et al. (2016)36 and is available in the pRolocdata package.\n\n\nSoftware availability\n\nComputational workflow for this study available from: https://github.com/ococrook/TAGMworkflow47\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.259371233\n\nLicense: CC BY 4.0",
"appendix": "Grant information\n\nPDWK was supported by the MRC (project reference MC_UU_00002/10). LMB was supported by a BBSRC Tools and Resources Development grant (Award BB/N023129/1) and a Wellcome Trust Technology Development Grant (Grant number 108441/Z/15/Z). OMC is a Wellcome Trust Mathematical Genomics and Medicine student supported financially by the School of Clinical Medicine, University of Cambridge.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nFootnotes\n\n1 https://drive.google.com/open?id=1zozntDhE6YZ-q8wjtQ-lxZ66EEszOGYi\n\n\nReferences\n\nCrook OM, Mulvey CM, Kirk PDW, et al.: A Bayesian mixture modelling approach for spatial proteomics. PLoS Comput Biol. 2018; 14(11): e1006516. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThul PJ, Åkesson L, Wiking M, et al.: A subcellular map of the human proteome. Science. 2017; 356(6340): pii: eaal3321. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nShin SJ, Smith JA, Rezniczek GA, et al.: Unexpected gain of function for the scaffolding protein plectin due to mislocalization in pancreatic cancer. Proc Natl Acad Sci U S A. 2013; 110(48): 19414–19419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiljee JE, Wang Y, Bernard AA, et al.: Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity. Nat Genet. 2018; 50(2): 180–185. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDunkley TP, Hester S, Shadforth IP, et al.: Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci U S A. 2006; 103(17): 6518–6523. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFoster LJ, de Hoog CL, Zhang Y, et al.: A mammalian organelle map by protein correlation profiling. Cell. 2006; 125(1): 187–199. PubMed Abstract | Publisher Full Text\n\nChristoforou A, Mulvey CM, Breckels LM, et al.: A draft map of the mouse pluripotent stem cell spatial proteome. Nat Commun. 2016; 7: 8992. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGeladaki A, Kočevar Britovšek N, Breckels LM, et al.: Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics. Nat Commun. 2019; 10(1): 331. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan DJ, Dvinge H, Christoforou A, et al.: Mapping organelle proteins and protein complexes in Drosophila melanogaster. J Proteome Res. 2009; 8(6): 2667–2678. PubMed Abstract | Publisher Full Text\n\nHall SL, Hester S, Griffin JL, et al.: The organelle proteome of the DT40 lymphocyte cell line. Mol Cell Proteomics. 2009; 8(6): 1295–1305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreckels LM, Gatto L, Christoforou A, et al.: The effect of organelle discovery upon sub-cellular protein localisation. J Proteomics. 2013; 88: 129–140. PubMed Abstract | Publisher Full Text\n\nBeltran PM, Mathias RA, Cristea IM: A Portrait of the Human Organelle Proteome In Space and Time during Cytomegalovirus Infection. Cell Syst. 2016; 3(4): 361–373.e6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJadot M, Boonen M, Thirion J, et al.: Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome. Mol Cell Proteomics. 2017; 16(2): 194–212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nItzhak DN, Davies C, Tyanova S, et al.: A Mass Spectrometry-Based Approach for Mapping Protein Subcellular Localization Reveals the Spatial Proteome of Mouse Primary Neurons. Cell Rep. 2017; 20(11): 2706–2718. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMendes M, Peláez-García A, López-Lucendo M, et al.: Mapping the Spatial Proteome of Metastatic Cells in Colorectal Cancer. Proteomics. 2017; 17(19): 1700094. PubMed Abstract | Publisher Full Text\n\nHirst J, Itzhak DN, Antrobus R, et al.: Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval. PLoS Biol. 2018; 16(1): e2004411. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavies AK, Itzhak DN, Edgar JR, et al.: AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A. Nat Commun. 2018; 9(1): 3958. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrre LM, Vesterlund M, Pan Y, et al.: SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization. Mol Cell. 2019; 73(1): 166–182.e7. PubMed Abstract | Publisher Full Text\n\nNightingale DJ, Geladaki A, Breckels LM, et al.: The subcellular organisation of Saccharomyces cerevisiae. Curr Opin Chem Biol. 2019; 48: 86–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGelman A, Carlin JB, Stern HS, et al.: Bayesian Data Analysis. Chapman & Hall, London, 1995. Reference Source\n\nMulvey CM, Breckels LM, Geladaki A, et al.: Using hyperLOPIT to perform high-resolution mapping of the spatial proteome. Nat Protoc. 2017; 12(6): 1110–1135. PubMed Abstract | Publisher Full Text\n\nDempster AP, Laird NM, Rubin DB: Maximum likelihood from incomplete data via the em algorithm. J Roy Stat Soc B Met. 1977; 39(1): 1–38. Reference Source\n\nCrook OM, Gatto L, Kirk PDW, et al.: ococrook/tagmworkflow: F1000 submission. 2019. http://www.doi.org/10.5281/zenodo.2593712\n\nGatto L, Breckels LM, Wieczorek S, et al.: Mass-spectrometry-based spatial proteomics data analysis using pRoloc and pRolocdata. Bioinformatics. 2014; 30(9): 1322–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreckels LM, Mulvey CM, Lilley KS, et al.: A Bioconductor workflow for processing and analysing spatial proteomics data [version 2; peer review: 2 approved]. F1000Res. 2016; 5: 2926. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreckels LM, Holden SB, Wojnar D, et al.: Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics. PLoS Comput Biol. 2016; 12(5): e1004920. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDunkley TP, Watson R, Griffin JL, et al.: Localization of organelle proteins by isotope tagging (LOPIT). Mol Cell Proteomics. 2004; 3(11): 1128–1134. PubMed Abstract | Publisher Full Text\n\nItzhak DN, Tyanova S, Cox J, et al.: Global, quantitative and dynamic mapping of protein subcellular localization. eLife. 2016; 5: pii: e16950. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith AFM, Roberts GO: Bayesian computation via the gibbs sampler and related markov chain monte carlo methods. J Roy Stat Soc B Met. 1993; 55(1): 3–23. Publisher Full Text\n\nGilks WR, Richardson S, Spiegelhalter D: Markov chain Monte Carlo in practice. Chapman and Hall/CRC, 1995. Reference Source\n\nGeweke J: Evaluating the accuracy of sampling-based approaches to the calculation of posterior moments. BAYESIAN STATISTICS. 1992. Reference Source\n\nGelman A, Rubin DB: Inference from iterative simulation using multiple sequences. Stat Sci. 1992; 7(4): 457–472. Publisher Full Text\n\nRoberts GO, Smith AFM: Simple conditions for the convergence of the gibbs sampler and metropolis-hastings algorithms. Stoch Process Their Appl. 1994; 49(2): 207–216. Publisher Full Text\n\nBrooks SP, Gelman A: General methods for monitoring convergence of iterative simulations. J Comput Graph Stat. 1998; 7(4): 434–455. Publisher Full Text\n\nPlummer M, Best N, Cowles K, et al.: Coda: Convergence diagnosis and output analysis for mcmc. R News. 2006; 6(1): 7–11. Reference Source\n\nFraley C, Raftery AE: Bayesian regularization for normal mixture estimation and model-based clustering. Technical report, Washington Univ Seattle Dept of Statistics, 2005. Reference Source\n\nCrook OM, Gatto L: A bioconductor workflow for the bayesian analysis of spatial proteomics. 2019. Reference Source"
}
|
[
{
"id": "47079",
"date": "30 Apr 2019",
"name": "Pierre M. Jean Beltran",
"expertise": [
"Reviewer Expertise My area of expertise is in interaction",
"global",
"and organelle proteomics analysis. My work includes application of experimental",
"statistical",
"and computational methods for the study of disease using a proteomics approach."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe spatial proteomics field has seen increased popularity over the past few years through development of experimental, statistical, and computational methodologies. In this Method Article, Crook OM and colleagues present a bioinformatics workflow for the analysis of spatial proteomics data using a set of Bayesian analysis tools. This work is a useful guide for biologists that wish to properly apply and diagnose a Markov-chain Monte-Carlo (MCMC) inference procedure using the pRoloc package.\nThe article is timely and relevant given the increasing number of biologists acquiring programming skills yet lacking extensive expertise in this area of statistics and modeling. In particular, the authors did a good job explaining basic terminology and rationale for the diagnostics necessary during MCMC inference. The methodology is clearly explained, all the code is provided, and the packages and datasets are available through Bioconductor, making them easily accessible and reproducible.\nHowever, I believe this article lacks in background information and details for those readers that are not experts in the spatial proteomics field. In addition, the value of applying the more resource-intensive MCMC method compared to the expectation-maximisation (EM) algorithm is not clear from the data presented. I recommend this article for indexing given that the following issues are resolved.\nMajor comments\nCurrently, there is no direct comparison of the results provided by the EM and MCMC algorithms. Given that MCMC is computationally intensive and requires additional diagnostics, the reader needs to be convinced that there is a tangible benefit from using the MCMC approach. A discussion of scenarios in which the EM algorithm would be preferable over MCMC would be useful for the reader. For example, can MCMC fail to converge? If so, what kind of interpretation can be obtained by looking at the point-estimates from the EM algorithm instead? Additional background information and references would be useful, as this article seems to assume that the reader is familiar with spatial proteomics data sets. For example, the author can explain the organelle MS-proteomics data sets, the use of organelle markers to train the model, and the prediction of unannotated proteins from the model fitted on the organelle markers. In addition, I would suggest pointing the reader to the review by Gatto L et al. 20141 in MCP, or another similar review as a primer. Page 20 – The authors claim that lower Shannon entropy is an indication of low uncertainty in localization and therefore can aid in identifying multi-localized proteins. This claim should be backed up by some examples or evidence from the literature that agree with the data presented.\nMinor comments\nFigure 1 – Additional details should be provided in the figure legend. Is this plot only showing organelle markers? A short description of the outlier component and how the analyst can interpret this component would be particularly helpful. Page 15 – It is not clear why the Gelman diagnostic performed on mean allocation of all chains helps discriminate chains 1, 2, and 4. The upper C.I. is already near to 1 (1.01) for this example. I observed several small writing mistakes; the article should be proof-read before publication. Some examples are indicated below. Page 15 – sentence ending with “… Gelman diagnostics between chains 4, 5, 6”. However, the code shows that the diagnostic was performed on chains 3, 5, 6. Page 21 – “From the plot below is is…” should read “it is”.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "47076",
"date": "01 May 2019",
"name": "Susan Holmes",
"expertise": [
"Reviewer Expertise Multivariate statistics",
"Bayesian methods",
"MCMC generation of posterior samples",
"data visualization",
"microbiome studies involving NGS and metabolomics."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides an important advance in the study of spatial proteomics. Through the use of Monte Carlo sampling the authors are able to provide evaluations of uncertainty by using the T-augmented Gaussian mixture model proposed by Crook et al. The authors have been careful to provide all the code and the relevant data are included in the package 'pRolocdata'.\nAlthough the focus is on the spatial localisation of proteins, the authors only study the probabilities of correct localizations and do not show a spatial plot of the probabilities so one does not see clearly how the probabilities vary spatially.\nFirst Issue: PCA plots\na) Ratios:\nThere are a few corrections needed to the figures that show PCA plots. It is probably not worth showing the percentages of variance up to two decimal digits, however it is important to respect the axes relative scale and units. For instance, since the ratio of variances in Figure 1 is 40:25 the plot should be rectangular, this is more egregious in Figures 2, especially the right side panel. I suggest making the figures on top of each other so they are not narrow (For a reference see Chapter 7 of the book1 that explains why usually PCA plots should be rectangular and not square).\nb) Construction of ellipses on the PCA plots in Figure 2 is very confusing. These cannot be the posterior distributions and their construction presupposes there is a relevant multivariate normal distribution which I do not think is justified here.\nSecond Issue: Bayesian computations\na) Priors\nIn the \"Aside\" subsection on priors, I suggest exploring the possibility of using prior data to construct a prior as in practical situations this is often how pragmatic Bayesians think about building priors. I think it is worth supporting the statement that the posterior is overwhelmed by the data with a small example. This could be done following the type of process recommended by Betancourt who gives very good examples of the effect of priors through the use of posterior predictive distributions (see here https://betanalpha.github.io/assets/case_studies/principled_bayesian_workflow.html#24_model_adequacy).\nb) Convergence diagnostics\nThe use of trace plots and Gelman and Rubin's Rhat can be quite confusing and can only show non-convergence, the current version puts too much emphasis on a few short runs. The authors have shown some chains that have not converged but do not mention that the contemporary literature is much more careful of how small the Rhat should be, see the preprint by Vats,D and Knudson, C 20182 and the discussions by Dan Simpson and Andrew Gelman for instance (https://statmodeling.stat.columbia.edu/2019/03/19/maybe-its-time-to-let-the-old-ways-die-or-we-broke-r-hat-so-now-we-have-to-fix-it/), the preprint is on the arXiv3.\nThird Issue:\nVisualization of results: Figure 8 does not show the uncertainty in an intuitive way. It is very hard to differentiate the size of the dots on the left hand plot and the dots give the impression of a circular posterior distribution which is incorrect. The reference Ren et al, 20174 contains several examples of posterior confidence contours that communicate uncertainty in a more precise way which enables the comparisons of uncertainty across different locations in the PCA. It would also be illuminating if the authors could make a plot with uncertainty contours as a function of the underlying spatial coordinates.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "47080",
"date": "09 May 2019",
"name": "Angus Lamond",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe subcellular distribution of the proteome is a very important determinant of cell function and therefore the ability to analyse subcellular protein localisation is vital for studying cell biology and regulation. Consequently, technical developments that facilitate the analysis of how the proteome is distributed between cell compartments are of great value to both the proteomics and molecular cell biology research communities. Bioconductor is also a fantastic source of tools that can be used to analyse ‘omics’ datasets, hence further additions to this toolbox are always welcome and of value. Here the authors provide a detailed workflow in Bioconductor that enables the convenient Bayesian analysis of spatial proteomics data. The explanations of the models provided by the authors are user friendly and allow also non-bioinformaticians to understand clearly the mechanics of the different methods. The suggestions provided relating to default parameters for the models are also very welcome. Overall, I view this as a timely and very useful contribution for the community that will be well received and of genuine value. I note below a few specific points that the authors should address.\nSpecific Points:\nI was initially unable to load either pRoloc or pRolocdata on R and therefore could not follow any of the examples provided. I had to upgrade to a new version of R to resolve this issue. The authors should specify the R version compatibility required.\n\nLine 1: Remove one ‘the’\n\n3rd page 5 paragraph, second sentence is disconnected.\n\nExample dataset uses iTRAQ, which means the data will have false positives all across\n\nThe authors should explain/justify their assertion that their Method is independent of the isotope labelling method because TMT and iTRAQ are expected to provide different challenges?\n\nIt would be helpful for the Gelman diagnostic to suggest minimum a number of chains for MCMC - Suggestion for an ideal number of chains for a medium range desktop computer would be useful too\n\n“Trace for Chain 2 & 4 have clear jumps” This is for figure 7 - This sounds imprecise; hard to spot and replicate. Also, I do not see the “clear jumps” the authors refer to – can this be clarified?\n\nSuggesting default priors for the Bayesian analysis is very useful. However, it would be good to show the visual consequences of changing S0 from beta0 of 1 to beta0 of 0.5\n\nThe suggestion by the authors that it is likely that 16.7% of proteins are outliers needs to be justified.\n\nThe use of Shannon entropy to focus on proteins with multiple localisations is an interesting approach that could be discussed further.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-446
|
https://f1000research.com/articles/8-104/v1
|
25 Jan 19
|
{
"type": "Research Note",
"title": "The study of protein recruitment to UV-induced DNA lesions can be distorted by photoconversion of DNA dyes like Hoechst or DAPI",
"authors": [
"Verena Hurst",
"Susan M. Gasser",
"Verena Hurst"
],
"abstract": "A common approach used to assess DNA repair factor binding in mammalian cells is to induce DNA damage with a UV laser and follow the movement of GFP-tagged proteins to the site of damage. Often these measurements are performed in the presence of the blue DNA intercalating dye Hoechst or DAPI, which is used to label nuclear DNA. A UV-induced switch of Hoechst and DAPI from a blue-light to a green-light emitter will give a false positive signal at the site of damage. Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results.",
"keywords": [
"Photoconversion",
"Hoechst",
"DAPI",
"UV laser",
"DNA repair"
],
"content": "Abbreviations\n\nDAPI: 4', 6-diamidino-2-phenylindole; UV: ultraviolet light; U2OS: human bone osteosarcoma epithelial cells; YOYO-1: tetracationic homodimer of Oxazole Yellow; GFP: Green fluorescent protein; 53BP1: Tumor suppressor p53-binding protein 1; XRCC1: X-ray repair cross-complementing protein 1; FEN-1: Flap endonuclease 1; PARP-1: Poly [ADP-ribose] polymerase 1; Ku70: 5'-deoxyribose-5-phosphate lyase Ku70/X-ray repair cross-complementing protein 6, MDC1: Mediator of DNA damage checkpoint 1; SMARCA5: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5\n\n\nIntroduction\n\nA variety of DNA binding dyes, such as DAPI, Hoechst, Vybrant DyeCycle Violet and YOYO-1, can change their optical properties upon exposure to light. Whereas the induction of YOYO-1 blinking is an intended change that is exploited for super resolution microscopy1, the photoconversion of DAPI, Hoechst or Vybrant Dye Cycle Violet during multicolor fluorescence microscopy is unexpected and can lead to false-positive signals.\n\nUpon UV exposure or a low pH, the emission spectra of DAPI, Hoechst and Vybrant DyeCycle Violet shift from the blue to the green wavelength with detectable signals in the yellow and orange2–4. This shift makes the signal indistinguishable from the emission of other standardly used fluorescent proteins such as GFP. An experimenter expecting the DNA dyes to emit in the blue can misinterpret the green signal as that arising from another probe in the sample. This risk has been raised previously3,5,6, yet the artefact is rarely controlled for.\n\nWith respect to these findings, a microscopic setup like the one used to study the localization of repair proteins to a UV laser-induced zone of DNA damage can be particularly problematic. Very commonly, cell nuclei are stained with DAPI or Hoechst and a restricted part of the nucleus is exposed to a strong UV laser. The protein of interest is detected in the green channel thanks either to its fusion to GFP or else through an antibody labelled with a green light-emitting fluorophore. Unfortunately, photoconversion of the DNA dye is usually not checked7–12. Here will illustrate the problem and suggest necessary controls.\n\n\nResults\n\nTo study the recruitment of a potential DNA damage related protein, we made use of a previously established setup in which cell nuclei are sensitized with Hoechst, DNA damage is induced with a UV laser, and the recruitment of a protein of interest is measured over time by fluorescence microscopy. Unexpectedly, cells stained with Hoechst that did not express any GFP-tagged protein showed a similar increase in the green channel at the UV damage site, as cells expressing the GFP-tagged protein (Figure 1). The detected increase in signal was not due to protein recruitment to the damage site, since there was no GFP-tagged protein in the cell. Moreover, in cells expressing the GFP-tagged protein that were not stained with Hoechst, there was no increase in signal intensity at the UV damage site. This demonstrates conclusively that the increase in fluorescence in the green channel was a false-positive result. Raw images are available on figshare13.\n\n\nDiscussion\n\nWe illustrate here that one should avoid exposing DAPI or Hoechst to a strong UV laser if one is imaging green light emitting probes such as GFP or a secondary antibody coupled to fluorescein/Alexa488. This is because photoconverted Hoechst and DAPI emit strongly in the same channel. As an alternative nuclear marker, we suggest employing a fluorescently tagged protein that localizes at the nuclear periphery and does not interfere with the experimental process.\n\nIf Hoechst is employed as a sensitizing agent, we suggest using the minimum dye concentration and laser power necessary and to combine it with probes/secondary antibodies of a color that is well separable from photoconverted DAPI/Hoechst. For instance, far red emission is compatible with photoconverted DAPI/Hoechst3. Yet, quantitation of the signal of the investigated protein requires normalization to a background control that is obtained by performing the laser experiment on DAPI/Hoechst-stained but otherwise native cells and acquiring signal with the same channel and exposure conditions, as used for the experimental probe. However, there is evidence showing that DNA sensitization prior to laser exposure is not required: DNA repair proteins such as 53BP114–16, XRCC115, FEN-115, PARP-115, Ku7015, MDC116, and SMARCA516 are recruited to sites of damage without previous sensitization by Hoechst.\n\nWe note that in addition to particular situations in which one induces damage, the photoconversion of DAPI can occur during standard dual color microscopy6. To minimize artefacts one should be careful about the order in which dyes are observed6, and visualize the green channel prior to exposing to short-wave light.\n\n\nMethods\n\nU2OS cells (a gift from Prof. Primo Leo Schaer, Department of Biomedicine, University of Basel) were incubated with 1.5 µg/ml Hoechst 33342 (Thermo Fisher Scientific, H1399) for at least 30 minutes prior to photoconversion. Photoconversion was induced with a VisiFRAP module (Visitron) mounted on the backport of the microscope and equipped with a 405 nm laser (Toptica, illumination power at the objective 12.8 mW). Confocal images were acquired with an Olympus IX81 microscope equipped with a PlanApo 100x/1.45 TIRFM oil objective, a CSU-X1 scan-head (Yokogawa), an Evolve 512 EMCCD camera (Photometrics), a 491nm laser (Cobolt Calypso 100), a 488/568 dichroic (Semrock Di01-T488/568-13x15x0.5), a band-pass 525/40 emission filter (Semrock FF01-525/40-25) and controlled with the Visiview Software (Visitron). Images in Figure 1 show maximum intensity projections of stacks13 covering 7 µm.\n\n\nData availability\n\nRaw images of the stacks taken during this study are available on figshare. DOI: https://doi.org/10.6084/m9.figshare.758396013.\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\nMedia\n\nThe three available avi files, C1 green, C2 blue and composite, represent a time series of maximum intensity projections showing the UV-induced emission change of the DNA intercalating dye Hoechst from the blue to the green region of the visible spectrum. Under live conditions, a Hoechst-stained cell nucleus was irradiated with 405 nm UV laser light along a predefined pattern (#). A time series of image stacks was acquired (25 equally spaced time points over 65s, stacks covering 7-µm sample depth) in two channels (C1 “green”: 491/525 nm, C2 “blue”: 405/450 nm). DOI: https://doi.org/10.6084/m9.figshare.758396013.",
"appendix": "Grant information\n\nThis study was funded by the Swiss National Science Foundation and Novartis Research Foundation.\n\n\nReferences\n\nFlors C: Photoswitching of monomeric and dimeric DNA-intercalating cyanine dyes for super-resolution microscopy applications. Photochem Photobiol Sci. 2010; 9(5): 643–8. PubMed Abstract | Publisher Full Text\n\nBarooah N, Mohanty J, Pal H, et al.: pH and temperature dependent relaxation dynamics of Hoechst-33258: a time resolved fluorescence study. Photochem Photobiol Sci. 2011; 10(1): 35–41. PubMed Abstract | Publisher Full Text\n\nZurek-Biesiada D, Kedracka-Krok S, Dobrucki JW: UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry A. 2013; 83(5): 441–51. PubMed Abstract | Publisher Full Text\n\nZurek-Biesiada D, Szczurek AT, Prakash K, et al.: Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe. Data Brief. 2016; 7: 157–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJež M, Bas T, Veber M, et al.: The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem. Histochem Cell Biol. 2013; 139(1): 195–204. PubMed Abstract | Publisher Full Text\n\nPiterburg M, Panet H, Weiss A: Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation. J Microsc. 2012; 246(1): 89–95. PubMed Abstract | Publisher Full Text\n\nAndrin C, McDonald D, Attwood KM, et al.: A requirement for polymerized actin in DNA double-strand break repair. Nucleus. 2012; 3(4): 384–95. PubMed Abstract | Publisher Full Text\n\nBaldeyron C, Soria G, Roche D, et al.: HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair. J Cell Biol. 2011; 193(1): 81–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDantas TJ, Daly OM, Conroy PC, et al.: Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair. PLoS One. 2013; 8(7): e68487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDinant C, de Jager M, Essers J, et al.: Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. J Cell Sci. 2007; 120(Pt 15): 2731–40. PubMed Abstract | Publisher Full Text\n\nIsmail IH, Davidson R, Gagné JP, et al.: Germline mutations in BAP1 impair its function in DNA double-strand break repair. Cancer Res. 2014; 74(16): 4282–94. PubMed Abstract | Publisher Full Text\n\nSustáčková G, Kozubek S, Stixová L, et al.: Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin. J Cell Physiol. 2012; 227(5): 1838–50. PubMed Abstract | Publisher Full Text\n\nHurst V, Gasser S: Photoconversion of Hoechst. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7583960.v1\n\nCarvalho S, Vítor AC, Sridhara SC, et al.: SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint. eLife. 2014; 3: e02482. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKong X, Mohanty SK, Stephens J, et al.: Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells. Nucleic Acids Res. 2009; 37(9): e68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmeenk G, Wiegant WW, Marteijn JA, et al.: Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling. J Cell Sci. 2013; 126(Pt 4): 889–903. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "43632",
"date": "29 Jan 2019",
"name": "Vincent Dion",
"expertise": [
"Reviewer Expertise DNA repair",
"chromatin",
"expanded trinucleotide repeat 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 short report by Hurst and Gasser exposes an important experimental detail that is often, but not always, overlooked: Hoechst-sensitized cells irradiated with UV light produce a signal in the same region of the spectrum as GFP. Consequently, without the proper controls, it may lead to false positives. That is, that it may be falsely concluded that GFP-tagged proteins are recruited to laser-induced damage.\nIn the discussion, they point out potential controls to that would prevent an experimenter to make this mistake. Specifically, they propose leaving out Hoechst or DAPI altogether, using the appropriate control cells that do not express the GFP-tagged protein of interest, using another nuclear marker, such as a protein localizing to the nuclear periphery, or using a different fluorescent protein. Those are all good ways of getting around the reported problem. I would also add the use of a UVC source (Dabin et al., 20181).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4526",
"date": "11 Apr 2019",
"name": "Verena Hurst",
"role": "Author Response",
"response": "Thank you for your comments! In version 2 we have added a section discussing the different types of damage generated at different UV/VIS wavelengths."
}
]
},
{
"id": "43603",
"date": "11 Feb 2019",
"name": "Sophie E. Polo",
"expertise": [
"Reviewer Expertise epigenetics",
"UV damage repair",
"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\nIn this research note, Hurst & Gasser highlight Hoechst photoconversion as a potential caveat in UV laser damage experiments. The UV-induced switch of Hoechst (or DAPI) from blue to green may indeed give a false positive signal in the green channel.\nThe following points need to be addressed:\n\nThe authors refer to a “strong UV laser”. It is not clear which conditions are required for DAPI/Hoechst photoconversion (laser power, exposure time) and how they compare to classical exposure times for DAPI visualization and to UV laser settings for laser damage induction. It is important to clarify this point to determine if we face a marginal or a general problem. The authors should be more specific when referring to UV laser or UV light. They should specify “UVA”, as other UV wavelengths such as UVC, also used to introduce local DNA damage (Dinant et al.,20071), may not have the same effect. They could suggest using BrdU instead of Hoechst to pre-sensitize cells to UVA light as done in a number of studies (e.g. Lukas et al.,20032). The authors illustrate the problem with GFP-tagged proteins but the issue would be similar with YFP-tagged proteins. They explain that far-red emission is compatible with photoconverted DAPI/Hoechst. How about red emission? There are 55 files on figshare, which seems excessive, and they are difficult to navigate through so in the end it is not very useful. Result section: “cells expressing the GFP-tagged protein (Figure 1)”. These cells are not shown on the figure and which GFP-tagged protein is it?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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": "4527",
"date": "11 Apr 2019",
"name": "Verena Hurst",
"role": "Author Response",
"response": "Thank you very much for your comments! Version 2 of this research note accommodates the changes that you suggested unless stated otherwise. The authors refer to a “strong UV laser”. It is not clear which conditions are required for DAPI/Hoechst photoconversion (laser power, exposure time) and how they compare to classical exposure times for DAPI visualization and to UV laser settings for laser damage induction. It is important to clarify this point to determine if we face a marginal or a general problem. Indeed, we use higher laser power than used in most studies in order to demonstrate the photoconversion effect. The purpose of this research note is to increase the awareness of the phenomenon and highlight that a negative control is particularly essential in this experimental setup. Even small amounts of photoconverted dye can change image quantitation and may alter apparent recruitment dynamics. We do think that this effect is particularly relevant to the study of uncharacterized proteins. Explorative studies may tempt the researcher to vary laser power and exposure time because the degree of damage required for protein recruitment is not known. This bears the risk of a false positive result. A preventive measure is to generate the damage without using Hoechst.We now cite a study in which photoconversion was detected and minimized, indicating the relevance of this note.A recent report on photoconversion in standard multiple color microscopy applications specifies exposure times for photoconversion1. The authors should be more specific when referring to UV laser or UV light. They should specify “UVA”, as other UV wavelengths such as UVC, also used to introduce local DNA damage (Dinant et al.,20071), may not have the same effect. We added a section on the type of damage generated +-sensitizing agents and cited Dinant et al. They could suggest using BrdU instead of Hoechst to pre-sensitize cells to UVA light as done in a number of studies (e.g. Lukas et al.,20032). Now we mention BrdU as a sensitizing agent and specify which type of damage is generated in its presence (double strand breaks 2). However, we favor omission of sensitizing agents altogether. The authors illustrate the problem with GFP-tagged proteins but the issue would be similar with YFP-tagged proteins. They explain that far-red emission is compatible with photoconverted DAPI/Hoechst. How about red emission? We have used GFP in our setup and therefore mainly discuss GFP. However, according to the publications we cited the effect may apply to YFP and other proteins with emission in the orange and near red as well 1,3. This is now mentioned. There are 55 files on figshare, which seems excessive, and they are difficult to navigate through so in the end it is not very useful. We were asked to deposit the raw data on figshare. As mentioned in our note we also deposited avi files of timelapse MIPs in both channels as well as a channel merge. These files are are easy to handle. Result section: “cells expressing the GFP-tagged protein (Figure 1)”. These cells are not shown on the figure and which GFP-tagged protein is it? We changed the position of the reference to Fig. 1 in this sentence to avoid implying that we will show cells expressing a GFP-tagged protein.Rather than naming the protein we want to highlight that this type of false-positive result can apply to any GFP-tagged protein tested, which is not recruited to the damage site. References1) Karg, T. J. & Golic, K. G. Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation. Chromosoma 127, 235-245, doi:10.1007/s00412-017-0654-5 (2018).2) Ferrando-May, E. et al. Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling. Front Genet 4, 135, doi:10.3389/fgene.2013.00135 (2013).3) Zurek-Biesiada, D., Kedracka-Krok, S. & Dobrucki, J. W. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry A 83, 441-451, doi:10.1002/cyto.a.22260 (2013)."
}
]
},
{
"id": "43604",
"date": "26 Feb 2019",
"name": "Jerzy Dobrucki",
"expertise": [
"Reviewer Expertise Cell Biophysics",
"DNA repair",
"microscopy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper “The study of protein recruitment…” by Hurst and Gasser is a very useful and timely technical report. It touches upon an important but often overlooked methodological aspect of studies of recruitment of repair factors to DNA lesions. When unnoticed, photoconversion of UV-excited DNA fluorescent probes can constitute a pitfall leading to incorrect interpretation of imaging data.\n\nGeneral comments:\n\n“UV-induced lesions” In the title and the text of this report the authors use a term “UV-induced DNA lesions”. The use of this term is misleading, since UV-induced lesions are generally defined as pyrimidine dimers and photoproducts. However, the authors refer to DNA lesions inflicted by 405nm light (which is not UV, see below) in the presence of a DNA-bound fluorescent dye Hoechst, and this leads to induction of a host of various lesions, not only “UV-induced”. When Hoechst 33342 has been introduced into live cells and exposed to 405nm focused laser light, it is (marginally) excited and acts as a photosensitizer. This leads to induction of oxidative damage and DNA breaks, i.e. not typical UV-induced lesions.\n\n“UV laser” The term ‘UV laser’ is used throughout the manuscript. This term appears incorrect, since the authors used a laser emitting 405nm wavelength light. By definition, 405 nm is visible light (and indeed it is readily visible by human eye, as opposed to UVA, UVB or UVC). I suggest to refer to the Toptica laser, which the authors used, as ‘blue laser”, as in the paper [Kong et al. 2009] which the authors cite.\n\nHoechst photosensitization vs DNA labeling Fluorescent DNA probe Hoechst is described in two disguises in this paper – as a photosensitizer and as a counterstain. These two roles and the ensuing problems are not clearly distinguished and explained in the paper. If Hoechst or DAPI are added to live cells prior to microirradiation with a focused beam of light of 405nm wavelength, the dye acts as a photosensitizer (as correctly stated in Results). However, in the Abstract the authors state that “the measurements are performed in the presence of the blue intercalating dye Hoechst or DAPI which is used to label nuclear DNA”. This statement appears incorrect for two reasons: 1. Hoechst is usually added to live cells in order to photosensitise and yield massive, readily detectable damage, not to just label DNA (if Hoechst were to be used as a label to mark DNA, it could be added after microirradiation); 2. Hoechst and DAPI are not DNA intercalators, they are rather minor groove binders (the mode of binding is complex and depends on a number of factors, including the type of DNA and a DNA/dye ratio).\n\nMinor comments:\n\nIt is unclear what the authors mean by referring to photoconversion of Hoechst, DAPI and VybrantDyeCycle and, in the same sentence and context, to blinking of YoYo described as “a change in optical properties”. What change of optical properties of YoYo do the authors have in mind?\n\nThe authors state (in Discussion): “As an alternative nuclear marker, we suggest employing a fluorescently tagged protein that localizes at the nuclear periphery and does not interfere with the experimental process.”\n\nWhy use such a complicated approach? It is much simpler to detect a transmitted light image, preferably using phase contrast or Nomarski interference contrast, and mark the outline of the nucleus in image overlay.\n\nThe authors state: “However, there is evidence showing that DNA sensitization prior to laser exposure is not required:...” This is true, but again the way the authors put it is somewhat confusing and they refer to research in which UVA as well as visible light was used. Indeed, photosensitisation by exogenous DNA-binding compounds is never required – regardless of the type of light, UV or VIS. Moreover, adding a photosensitizer to the experimental system influences the type of damage. Thus, I suggest to distinguish two cases:\nusing UV-excited dyes and UV or near-UV (405nm) light, and using dyes excited by visible light and visible light excitation.\n\nIn the case (1) the exciting light (UV or 405) will induce typical UV damage (PP, PD). Adding a photosensitizer prior to microirradiation will result in photodynamic effect, and cause induction of more types of lesions, and more extensive damage. There is vast literature about the action of UV alone and photodynamic effect type I and II. Some reference to this field of knowledge should be made in this paper.\n\nContrary to general belief, in the case (2) the exciting visible light alone, without any exogenous photosensitisers, will also induce DNA damage – single- and double-strand breaks, and recruitment of repair factors (Solarczyk et al., 2012, DNA Repair1).\n\nIn summary, indeed various types of DNA damage can be induced without adding photosensitisers to cells prior to microirradiation, not only when UV, or 405 nm light is used, but also when visible light is applied. I suggest to clarify these facts in the paper.\n\nA few inaccurate statements should be straightened out:\n“A common approach used to assess DNA repair factor binding in mammalian cells is to induce DNA damage with a UV laser and follow the movement of GFP-tagged proteins to the site of damage”. To be precise, induction of (local) DNA damage by a focused laser beam is used to detect recruitment of repair factors, but not their binding per se. “Movement of GFP-tagged proteins to the site of damage”. Movement of GFP-tagged proteins is not detected (this can be done by FCS) – only local increase of a concentration of a fusion protein is detected, and this arises from recruitment.\n\nThe authors state: „exposing DAPI or Hoechst to a strong UV laser” - light intensities and doses of energy delivered to the exposed region of the nucleus (area?) should be given.\n\nFull information (dose) is lacking but it appears that the authors used an excessive power of 405nm light (12.8 mW at objective). DNA damage, especially in the presence of a photosensitizer, can be expected when using only microJ of energy (microW in the laser beam, seconds of exposure). This means that the photoconversion the authors describe most likely would have been less prominent, had a lower intensity and dose of energy been used. Applying excessive energy during microirradiation leads to such an extensive damage that relevant physiological studies may be impossible (Note that some images in the paper [Kong et al.] show microirradiation tracks in phase contrast images; the power which was used in this study was very high). I suggest that the authors state that the intensities (and doses of energy) they used may have been too high to induce DNA damage on the level encountered under typically encountered physiological conditions.\n\nTypo: “Here will illustrate the problem and suggest necessary controls.” Here we will.\n\nHope this helps.\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? 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": "4528",
"date": "11 Apr 2019",
"name": "Verena Hurst",
"role": "Author Response",
"response": "A=Authors, R=ReviewerA: Thank you for sharing your expertise. Your comments helped to significantly increase the quality of the article. R: “UV-induced lesions”In the title and the text of this report the authors use a term “UV-induced DNA lesions”. The use of this term is misleading, since UV-induced lesions are generally defined as pyrimidine dimers and photoproducts. However, the authors refer to DNA lesions inflicted by 405nm light (which is not UV, see below) in the presence of a DNA-bound fluorescent dye Hoechst, and this leads to induction of a host of various lesions, not only “UV-induced”. When Hoechst 33342 has been introduced into live cells and exposed to 405nm focused laser light, it is (marginally) excited and acts as a photosensitizer. This leads to induction of oxidative damage and DNA breaks, i.e. not typical UV-induced lesions.A: We added information on the types of damage generated at different wavelengths +- photosensitizing agents. R:“UV laser”The term ‘UV laser’ is used throughout the manuscript. This term appears incorrect, since the authors used a laser emitting 405nm wavelength light. By definition, 405 nm is visible light (and indeed it is readily visible by human eye, as opposed to UVA, UVB or UVC). I suggest to refer to the Toptica laser, which the authors used, as ‘blue laser”, as in the paper [Kong et al. 2009] which the authors cite.A: You are right. We corrected that. R: Hoechst photosensitization vs DNA labelingFluorescent DNA probe Hoechst is described in two disguises in this paper – as a photosensitizer and as a counterstain. These two roles and the ensuing problems are not clearly distinguished and explained in the paper. If Hoechst or DAPI are added to live cells prior to microirradiation with a focused beam of light of 405nm wavelength, the dye acts as a photosensitizer (as correctly stated in Results). However, in the Abstract the authors state that “the measurements are performed in the presence of the blue intercalating dye Hoechst or DAPI which is used to label nuclear DNA”. This statement appears incorrect for two reasons: 1. Hoechst is usually added to live cells in order to photosensitise and yield massive, readily detectable damage, not to just label DNA (if Hoechst were to be used as a label to mark DNA, it could be added after microirradiation); 2. Hoechst and DAPI are not DNA intercalators, they are rather minor groove binders (the mode of binding is complex and depends on a number of factors, including the type of DNA and a DNA/dye ratio).A: We corrected and clarified that point. Actually using Hoechst helps identify the placement of the laser beam. R: Minor comments:It is unclear what the authors mean by referring to photoconversion of Hoechst, DAPI and VybrantDyeCycle and, in the same sentence and context, to blinking of YoYo described as “a change in optical properties”. What change of optical properties of YoYo do the authors have in mind?A: In order to focus on Hoechst and DAPI we removed the comments on the other two dyes. R: The authors state (in Discussion): “As an alternative nuclear marker, we suggest employing a fluorescently tagged protein that localizes at the nuclear periphery and does not interfere with the experimental process.” Why use such a complicated approach? It is much simpler to detect a transmitted light image, preferably using phase contrast or Nomarski interference contrast, and mark the outline of the nucleus in image overlay.A: We and other labs standardly use fluorescent labels of the nuclear envelope without complications. We are not sure transmitted light images are compatible with rapid time lapse imaging since filters have to be changed but we mentioned this possibility in the text. R: The authors state: “However, there is evidence showing that DNA sensitization prior to laser exposure is not required:...” This is true, but again the way the authors put it is somewhat confusing and they refer to research in which UVA as well as visible light was used. Indeed, photosensitisation by exogenous DNA-binding compounds is never required – regardless of the type of light, UV or VIS. Moreover, adding a photosensitizer to the experimental system influences the type of damage. Thus, I suggest to distinguish two cases: using UV-excited dyes and UV or near-UV (405nm) light, and using dyes excited by visible light and visible light excitation. In the case (1) the exciting light (UV or 405) will induce typical UV damage (PP, PD). Adding a photosensitizer prior to microirradiation will result in photodynamic effect, and cause induction of more types of lesions, and more extensive damage. There is vast literature about the action of UV alone and photodynamic effect type I and II. Some reference to this field of knowledge should be made in this paper.A: We have added a section discussing the types of damage generated under different conditions.R: Contrary to general belief, in the case (2) the exciting visible light alone, without any exogenous photosensitisers, will also induce DNA damage – single- and double-strand breaks, and recruitment of repair factors (Solarczyk et al., 2012, DNA Repair1).A: We noticed this phenomenon in our own experiments, and have now commented upon this in the text and have cited the reference suggested.R: In summary, indeed various types of DNA damage can be induced without adding photosensitisers to cells prior to microirradiation, not only when UV, or 405 nm light is used, but also when visible light is applied. I suggest to clarify these facts in the paper. A few inaccurate statements should be straightened out: “A common approach used to assess DNA repair factor binding in mammalian cells is to induce DNA damage with a UV laser and follow the movement of GFP-tagged proteins to the site of damage”. To be precise, induction of (local) DNA damage by a focused laser beam is used to detect recruitment of repair factors, but not their binding per se. A: True. Corrected. R: “Movement of GFP-tagged proteins to the site of damage”. Movement of GFP-tagged proteins is not detected (this can be done by FCS) – only local increase of a concentration of a fusion protein is detected, and this arises from recruitment. A: Corrected.R: The authors state:„exposing DAPI or Hoechst to a strong UV laser” - light intensities and doses of energy delivered to the exposed region of the nucleus (area?) should be given.Full information (dose) is lacking but it appears that the authors used an excessive power of 405nm light (12.8 mW at objective). DNA damage, especially in the presence of a photosensitizer, can be expected when using only microJ of energy (microW in the laser beam, seconds of exposure). This means that the photoconversion the authors describe most likely would have been less prominent, had a lower intensity and dose of energy been used. Applying excessive energy during microirradiation leads to such an extensive damage that relevant physiological studies may be impossible (Note that some images in the paper [Kong et al.] show microirradiation tracks in phase contrast images; the power which was used in this study was very high). I suggest that the authors state that the intensities (and doses of energy) they used may have been too high to induce DNA damage on the level encountered under typically encountered physiological conditions.A: See response to Anna Fortuny, who raised this issue as well. Now we state that we use high laser power in order to demonstrate the effect and provide further information on the laser conditions. Furthermore, we refer to a study detecting and minimizing such an effect in their setup. R: Typo:“Here will illustrate the problem and suggest necessary controls.” Here we will.A: Corrected. R: Hope this helps.A: Yes. Thank you!"
}
]
}
] | 1
|
https://f1000research.com/articles/8-104
|
https://f1000research.com/articles/8-168/v1
|
07 Feb 19
|
{
"type": "Research Article",
"title": "Apoptosis induction on human breast cancer T47D cell line by extracts of Ancorina sp.",
"authors": [
"Woro Anindito Sri Tunjung",
"Puspa Restu Sayekti",
"Puspa Restu Sayekti"
],
"abstract": "Background: Breast cancer is the second leading cause of death in women. Alternative medicine with high efficacy is needed for breast cancer treatments, for example induction of apoptosis using natural products. It has been found that many natural apoptosis-inducing compounds are isolated from marine sponge. The objective of this study is to analyze the ability of extracts of the sponge Ancorina sp. to induce apoptosis on human breast cancer T47D cell line and find out its mechanism. Methods: T47D cells were treated with crude extracts of methanol, dichloromethane:methanol (1:1) and dichloromethane Ancorina sp. for 24 h, and doxorubicin was used as a positive control. Methods used for this study were MTT assay to examine cell viability and determine IC50 of the three extracts, while the percentage of apoptosis and caspase-3 were investigated by flow cytometry. Results: IC50 values of methanol, dichloromethane:methanol (1:1), and dichloromethane extract were 84.25, 121.45, and 99.85μg/mL respectively. The percentages of apoptotic cells after treatment with methanol, dichloromethane:methanol (1:1), and dichloromethane extracts were 88.68, 27.54 and 53.63% respectively, whereas the percentage of caspase-3 was 77.87, 12.66 and 12.97%, respectively. Conclusions: These results revealed that all extracts of Ancorina sp. have strong or moderate cytotoxicity and have the ability to induce apoptosis on T47D human breast cancer cell line. However, methanol crude extract has high efficacy to induce apoptosis through caspase-3 activation compared to the other extracts. Hence methanol extract warrants further investigation as a natural medicine for human breast cancer.",
"keywords": [
"Ancorina sp.",
"cytotoxicity",
"apoptosis",
"caspase-3",
"breast cancer"
],
"content": "Introduction\n\nBreast cancer is the second leading cause of death in women after cervical cancer. In 2016 breast cancer cases have occurred in 40 per 100,000 women in Indonesia1. Medical treatment for breast cancer is currently widely applied2. However, medical treatment can cause side effects, namely the death of healthy cells surrounding cancer cells3. Alternative methods of breast cancer treatment with reduced side effects are needed, such as treatments using natural anticancer agents3.\n\nThere are many cancer treatment methods such as anti-angiogenesis therapy4, cell cycle inhibitors5, and photodynamic therapy6. Induction of apoptosis is the most common approach in cancer therapy because apoptosis has specific abilities to kill certain cells7. One characteristic of cancer cell is loss of ability for apoptosis8. The ability of apoptosis to kill abnormal cells can prevent the occurrence of cancer growth9. Induction of apoptosis occurs through three apoptotic-signaling pathways:extrinsic, intrinsic and perforin/granzyme pathways. Apoptosis path activation is marked by the activation of caspases. Caspase is found in normal cells as an inactive zymogen (procaspase). Active caspase activates other caspases, forming the 'caspase cascade'. Activation of caspase 8 and 9 will cause activation of caspase-3 as a downstream effector, which induces apoptosis10.\n\nPrevious studies found many natural apoptosis-inducing compounds isolated from marine sponge that can be developed as natural medicine11. Fraction of Negombata magnifica sponge is able to induce apoptosis in hepatocellular carcinoma12. Sponge extract of Haliclona sp. able to increase the percentage of apoptosis and significantly increase the expression of apoptotic gene p53, p21, caspase-8, and caspase-3 in A549 lung cancer cells13.\n\nNatural anticancer agents are usually extracted by a particular solvent. Different solvents cause different effects on the disease. Some previous researchers have isolated sponge bioactive compounds using both polar and non-polar solvents. For example, cytotoxic compounds have been successfully isolated from sponge Dactylospongia elegans and Pachychalina alcaloidifera using methanol14,15. Organic compounds have been successfully isolated from the sponge Condrosia reniformes, Tethya rubra, Tethya ignis, Mycale angulosa and Dysidea avara as a drug therapy for Chagas disease using acetone solvents16. Terpenoids have been successfully isolated from sponge Iricina sp. and Spongia sp. using ethanol solvent17. Anticancer compounds have been successfully isolated from Petrosia sp., Jaspis sp. and heterogeneous Pericharax using dichloromethane:methanol (1:1)18. Some studies also mention that sponge bioactive compounds, antiviral, antimicrobial, antifungal, and anticancer compounds, have been successfully isolated with methanol19–21, ethanol22, dichloromethane and combination of dichloromethane:methanol (1:1)23–26.\n\nThe objective of this study is to determine the cytotoxicity of Ancorina sp. extract in breast cancer T47D cells and measure extract-induced apoptosis through activation of caspase-3. In this study we use three solvents: methanol (polar), dichloromethane (non-polar) and mixture of both solvents to determine the most effective solvent. Furthermore this study used T47D cells as a model for breast cancer cells because T47D cells are able to express caspase-3, which is an effector of apoptotic induction27.\n\n\nMethods\n\nAncorina sp. were collected from Wedi Ombo Beach, Gunungkidul, Yogyakarta, Indonesia. Samples were washed to remove debris and residual salt. Samples were transferred to the laboratory in methanol, dichloromethane and dichloromethane:methanol (1:1) under cool condition.\n\nFresh samples were crushed in a blender in methanol, dichloromethane and dichloromethane methanol (1:1) then macerated for 24 hours. The samples were filtered and the residue was re-extracted for two times. The total filtrate was then evaporated to obtain crude extract paste.\n\nWe used T47D cells obtained from Integrated Laboratory of Research and Testing, Universitas Gadjah Mada (LPPT UGM).\n\nThe cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2% penicillin streptomycin and 0.5% Fungizone. Cells were harvested after reaching 80% confluence using 0.25% Trypsin-EDTA. Cells were cultured in 96-well microplates (1 × 103 cells/well) in 100 μL RPMI and incubated at 37°C with 5% CO2 overnight.\n\nDoxorubicin at 5 µg/mL was used as the positive control whereas T47D cells cultured in medium was used as the negative control and cells cultured in 0.5% DMSO in medium was used as the solvent blank.\n\nCytotoxicity was assessed using the MTT assay. After the cells were incubated for 24 h with the serial dilution from 15.68 to 250 µg/mL, 0.5% MTT solution was added and the cells were incubated for 4 h followed by addition of stopper reagent (10% SDS in 0.1 N HCl). The optical density (OD) was measured at 550 nm using Microplate Reader BIO-RAD 680XR. MTT data was then analyzed by Probit to determine the IC50 value. IC50 of each extract is used for FACS experiment.\n\nBriefly, T47D cells were seeded in 6-well microplates in 3×103μL RPMI. In total, 1×106 cells were treated by IC50 concentrations of three extracts or doxorubicin for 24 h. Cells were stained by Annexin V-PI Biolegend for apoptosis test and by BD Cytofix / Cytoperm™ for caspase-3 activation test. The sample was measured using flow cytometer BD FACSCalibur™. Flowcytometry output by BD FACSCalibur™ was shown in four quadrants. The first quadrant contains normal living cells population that respond negatively to Annexin V-FITC and propidium iodide (PI). Second quadrant contains early apoptotic cells populations that respond positively to Annexin V-FITC. Third quadrant contains the late apoptotic cells population which responds positively to Annexin V-FITC and Propidium Iodide (PI). Whereas in the fourth quadrant contains a population of necrotic cells that respond negatively to Annexin V-FITC and respond positively to PI28.\n\nThe IC50 value was determined by Probit analysis. IC50 value and percentage of apoptosis are further analyzed by one-way ANOVA and Tukey’s test at 5% significance level using IBM SPSS Statistic 23.0 program. P < 0.05 indicated statistical significance.\n\n\nResults\n\nThe cell viability of T47D cells after methanol, dichloromethane and dichloromethane: methanol (1:1) extracts treatment are presented in Figure 1. The concentration of extracts reduced the viability of investigated cells by 50% (IC50), which has been reported in Table 1.\n\nMethanol (M), Dichloromethane:Methanol (D:M) and Dichloromethane (D).\n\nNote: different letter showed the significant difference at the 0.05 level\n\nAll Ancorina sp. extracts inhibited the proliferation of cancer cells in a dose dependent manner. The higher concentration of extract caused the lower percentage of T47D cell viability. All extracts were cytotoxic to T47D cells. IC50 value of methanol was significantly different to dichloromethane:methanol but wasn’t significantly different to dichloromethane.\n\nWe analyzed cell death qualitatively by examining cell morphological change and quantitively by flow cytometry using Annexin-V after 24 h incubation of extracts.\n\nCell morphology after treatment can be seen in Figure 2. Morphology of T47D cell showed that methanol extract caused most cells population to undergo death (approximately more than 70%), while dichloromethane extract resulted in almost half cell population deaths. The combination of methanol and dichloromethane (1:1) extract causes fewer cell deaths (<50%).\n\nControl (A), Methanol (B), Dichloromethane:Methanol (C), Dichloromethane (D), Doxorubicin (E) and DMSO (F). Arrow shows dead cells.\n\nThe control (DMSO) cell did not cause cell death but after doxorubicin and extract treatment most cells undergo death. This data supports the cytotoxicity assay that the Ancorina sp. extracts successfully induced cell death.\n\nDetection of apoptosis marker after treatment by extracts can be seen in Figure 3.\n\nCell control (A), doxorubicin (B), methanol (C), dichloromethane:methanol (1:1) (D), and dichloromethane (E). R1 (normal cells), R2 (early apoptosis cells), R3 (late apoptotic cells), and R4 (necrosis cells).\n\nAll Ancorina sp. extracts increase the percentage of apoptotic cells compared to control cells (Figure 3). The highest percentage of apoptosis was obtained in the methanol group (88.68%), which was even higher than doxorubicin as a positive control (75.74%) (Table 2).\n\nNote: different letter showed the significant difference at the 0.05 level. Statistical analysis is focused to percentage of apoptosis among group.\n\nThe three extracts showed the same pattern with doxorubicin, i.e. a high percentage of apoptotic cell while the percentage of necrotic cells is low (Table 2).\n\nWe further investigated the apoptotic mechanism by examining the percentage of caspase-3. Detection of caspase-3 can be seen in Figure 4, while percentage of caspase-3 activation and correlation between percentage of apoptosis and caspase-3 activation can be seen in Table 3 and Figure 5, respectively.\n\nNegative control (A), Doxorubicin (B), Methanol (C), Dichloromethane:Methanol (D) and Dichloromethane (E).\n\nDoxorubicin (Dox), Methanol (M), Dichloromethane:Methanol (1:1) (D:M), Dichloromethane (D), and negative control (control).\n\nThe highest percentage of caspase-3 was detected with methanol extract, which almost equaled doxorubicin, while the value of the other extracts was lower (Table 3).\n\nThe three extracts have a positive correlation between percentage of apoptosis and caspase-3. Although dichloromethane showed lower percentage of apoptosis and caspase-3, but they still have strong cytotoxicity (99.85 µg/mL), which shows potency as natural anticancer agents.\n\n\nDiscussion\n\nSponges are highly diverse in Indonesia. In particular, encrusting sponges abundantly live in Gunung Kidul, Yogyakarta. Marine sponges produce some secondary metabolites, which can be used as antiviral22, antimicrobial22,23, antifungal22, and anticancer drugs17,24,29. The cell adhesion and immune system in sponge allow the different forms of the body plan30. When encrusting sponges grow together, sponges can survive by producing chemicals to kill fast dividing cells from the neighboring sponges. This ability of the chemicals can be used for chemotherapy since the basis of chemotherapy treatments is to disturb cancer cell growth31.\n\nSponge Ancorina sp. is a member of family Ancorinidae, which contains bioactive compounds such as ancorinoside BD, penazetidine A (Penares sollasi), ecionines A & B (Ecionemia sp.) and Iso malabaricane triterpenes (Rhabdastrella globostellata)32. Ancorinoside is a MT1-matrix metalloproteinase inhibitor in the development and metastasis of tumor cells33, whereas Penazetidine A strongly inhibits PKC-β1 activity in tumor cells with IC50 value 0.3 μg / mL34.\n\nEcionines A (biemnadin) and B (meridine) are anticancer compounds for many cancer cells, including bladder cancer cells33. Further, Iso malabaricane triterpenes were also found to be anticancer after testing on three types of cancer cells, namely L5178Y (mouse lymphoma), HeLa (human cervical carcinoma), and PC-12 (pheochromocytoma in mice)35. Ancorina sp. is a source of bioactive compounds such as ancorinoside A Mg salt, ancorinolates AC, bis-ancorinolate B, ancorinazole, indolo [3,2-a] carbazole, and (+) - 7-bromotrypargine36–38. These previous data show the high potency of Ancorinidae to be used as natural anticancer agents; hence this study is focused on the potency of Ancorina sp. as an anticancer agent and its mechanism, which is possibly through apoptosis induction.\n\nCytotoxicity is categorized into three levels by IC50 extract values. Very strong cytotoxicity has IC50 less than 10 μg / mL, strong cytotoxicity has IC50 values between 10 -100 μg/mL, and moderate cytotoxicity has IC50 values between 100 - 500 μg/mL39. According to these ranges, IC50 of the methanol and dichloromethane extracts in the present study had strong cytotoxic ability, while dichloromethane:methanol (1:1) extract belonged to moderate cytotoxicity. Ancorina sp. extracts have greater value of IC50 compared with the study34, which mentioned penazetidine A, a bioactive compound of marine sponge and highly inhibits PKC-β1 activity in tumor cells with lower IC50 of 0.3 μg/mL. This difference is due to the non-fractionated extract of our sponge, so that unsorted bioactive compounds possibly affect the cytotoxicity ability of extracts34.\n\nBioactive compounds from natural products depend on solvents. Based on the polarity of solvents, in the present study, Ancorina sp. extracts with polar solvent (methanol) lead to a higher apoptosis than non-polar (dichloromethane) or combination. These results are supported by a previous study that showed some compounds of Ancorinidae, such as ancorinoside BD, penazetidine A, echionines A and B and isomalabaricane triterpenes, are polar compounds that have anti-tumor and anti-cancer characteristics32. Interestingly, some studies in sponge also show same phenomenon such as cytotoxic compounds from sponge Dactylospongia elegans and Pachychalina alcaloidifera has been isolated using polar solvent methanol14,15. Terpenoids from sponge Iricina sp. and Spongia sp. have been isolated using polar solvent ethanol14,15,17. Bioactive compounds of sponge, both antiviral, antimicrobial, antifungal, and anticancer compounds have been successfully isolated by polar solvent as methanol19–21 and ethanol22. Considering all extracts in this study have low necrosis values (Table 2), they are safe to be used as medicine. Therefore, further studies are needed to find out the specific compounds of Ancorina sp. extracts.\n\nApoptosis can be triggered by extrinsic stimulation through death receptors on cell surfaces, such as TNFα (Tumor Necrosis Factor-α), Fas receptor (CD95 / APO1) and TRAIL (TNF related to ligand-inducing apoptosis) or by intrinsic stimulation through mitochondrial signaling pathways. In these two main pathways, activation of cysteine aspartyl proteases or caspase can produce mitochondrial permeabilization membrane, chromatin condensation and DNA fragmentation. These events stimulate the cells that are undergo apoptosis and lead to a distinctive cell morphology, such as the appearance of pyknosis, chromatin condensation, nucleus fragmentation, and apoptotic body formation, but organelles are still intact40. This can be seen in the present study in Figure 2.\n\nApoptotic pathways commonly occur by the activation of caspase-3, which is the effector of intrinsic, extrinsic and perforin pathways41. Caspase-3 is a key protease that is activated during the early stages of apoptosis. Caspase-3 is proteolytically active, cuts and activates other caspases, as well as relevant targets such as targets in the cytoplasm (D4-GDI and Bcl-23) and nucleus (poly (ADP-ribose) polymerase; PARP1)42.\n\nIn the present study, the highest percentage of caspase-3 was detected in methanol extract, which almost equal to doxorubicin, while the other extracts was lower (Table 3). Doxorubicin as a commercial drug in chemotherapy revealed a high percentage of apoptotic cells and caspase-3 activation. Among Ancorina sp. treatment groups, methanolic extract showed the highest percentage of both apoptosis and caspase-3. Interestingly, the methanolic extract showed a higher percentage than doxorubicin, and revealed its great potency to be used as a cancer medicine (Table 3).\n\nThe three extracts in this study have a positive trend between percentage of apoptosis and caspase-3 activation. Although dichloromethane showed a lower percentage of apoptosis and caspase-3, they had strong cytotoxicity (99.85 µg/mL) which shows potential as natural anticancer agents. It is possible that anticancer mechanism of dichloromethane and mixture of dichloromethane and methanol (1:1) excludes caspase-3 activation as effector caspases. Another pathway, such as caspase-6 or 7, can also induce apoptosis in T47D breast cancer cells27. More investigation is needed to elucidate the anticancer mechanism of these extracts.\n\n\nConclusions\n\nAll extracts of Ancorina sp. have strong or moderate cytotoxicity and have the ability to induce apoptosis in T47D human breast cancer cell line.\n\n\nData availability\n\nOpen Science Framework: Apoptosis induction on human breast cancer T47D cell line by extracts of Ancorina sp., https://doi.org/10.17605/OSF.IO/AEJ9643\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 work was financially supported by Penelitian Dasar Unggulan Perguruan Tinggi No: 24/UN1/DITLIT/DIT-LIT/LT/2018 from Indonesia Ministry of Research, Technology and Higher education (to W.A.S.T).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe acknowledge Nur Faizah, Rudi Nirwantoro, and Ratih Aryasari for the fruitful discussions and helping in sponge sampling and identification.\n\n\nReferences\n\nIARC: International Agency for Research on Cancer (IARC) Official Website. [Online]. 2018; [Accessed 20 Apr. 2018]. Reference Source\n\nKawabe T: G2 checkpoint abrogators as anticancer drugs. Mol Cancer Ther. 2004; 3(4): 513–519. PubMed Abstract\n\nMaione P, Rossi A, Airoma G, et al.: The role of targeted therapy in non-small cell lung cancer. Crit Rev Oncol Hematol. 2004; 51(1): 29–44. PubMed Abstract | Publisher Full Text\n\nVasudev NS, Reynolds AR: Anti-angiogenic therapy for cancer: current progress, unresolved questions and future directions. Angiogenesis. 2014; 17(3): 471–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDickson MA, Schwartz GK: Development of cell-cycle inhibitors for cancer therapy. Curr Oncol. 2009; 16(2): 36–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPłonka J, Latocha M: [Photodynamic therapy in the treatment of breast cancer]. Pol Merkur Lekarski. 2012; 33(195): 173–5. PubMed Abstract\n\nGerl R, Vaux DL: Apoptosis in the development and treatment of cancer. Carcinogenesis. 2005; 26(2): 263–270. PubMed Abstract | Publisher Full Text\n\nMohammad RM, Muqbil I, Lowe L, et al.: Broad targeting of resistance to apoptosis in cancer. Semin Cancer Biol. 2015; 35(Suppl): S78–S103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRossi D, Gaidano G: Messengers of cell death: apoptotic signaling in health and disease. Haematologica. 2003; 88(2): 212–218. PubMed Abstract\n\nWürstle ML, Laussmann MA, Rehm M: The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp Cell Res. 2012; 318(11): 1213–1220. PubMed Abstract | Publisher Full Text\n\nEssack M, Bajic VB, Archer JA: Recently confirmed apoptosis-inducing lead compounds isolated from marine sponge of potential relevance in cancer treatment. Mar Drugs. 2011; 9(9): 1580–1606. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRady HM, Hassan AZ, Salem SM, et al.: Induction of apoptosis and cell cycle arrest by Negombata magnifica sponge in hepatocellular carcinoma. Med Chem Res. 2016; 25(3): 456–465. Publisher Full Text\n\nBae W, Lim HK, Kim KM, et al.: Apoptosis-Inducing Activity of Marine Sponge Haliclona sp. Extracts Collected from Kosrae in Non small Cell Lung Cancer A549 Cells. Evid Based Complement Alternat Med. 2015; 2015: 717959. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEbada SS, De Voogd N, Kalscheuer R, et al.: Cytotoxic drimane meroterpenoids from the Indonesian marine sponge Dactylospongia elegans. Phytochem Lett. 2017; 22: 154–158. Publisher Full Text\n\nCavalcanti BC, Sombra CM, de Oliveira JH, et al.: Cytotoxicity and genotoxicity of ingenamine G isolated from the Brazilian marine sponge Pachychalina alcaloidifera. Comp Biochem Physiol C Toxicol Pharmacol. 2008; 147(4): 409–415. PubMed Abstract | Publisher Full Text\n\nCarreira J, Paula D, Cristina V, et al.: Trypanocidal activity of organic extracts from the Brazilian and Spanish marine sponges. Rev Bras Farmacogn. 2015; 25(6): 651–656. Publisher Full Text\n\nAbdjul DB, Yamazaki H, Kanno S, et al.: FuranoTerpenes, new types of protein tyrosine phosphatase 1B inhibitors, from two Indonesian marine sponges, Ircinia and Spongia spp. Bioorg Med Chem Lett. 2017; 27(5): 1159–1161. PubMed Abstract | Publisher Full Text\n\nBeedesseea G, Ramanjoolooa A, Aubertc G, et al.: Cytotoxic activities of hexane, ethyl acetate and butanol extracts of marine sponges from Mauritian Waters on human cancer cell lines. Environ Toxicol Pharmacol. 2012; 34(2): 397–408. PubMed Abstract | Publisher Full Text\n\nWillemsen PR: The Screening of Sponge Extracts for Antifouling Activity using a Bioassay with Laboratory-reared Cyprid Larvae of the Barnacle Balanus Amphitrite. Int Biodeter Biodegr. 1994; 34(3–4): 361–373. Publisher Full Text\n\nKumar MS, Pandita NS, Pal AK: LC-MS/MS as a tool for identification of bioactive compounds in marine sponge Spongosorites halichondriodes (Dendy 1905). Toxicon. 2012; 60(6): 1135–1147. PubMed Abstract | Publisher Full Text\n\nShaala LA, Youssef DTA, Badr JM, et al.: Bioactive alkaloids from the Red Sea marine Verongid sponge Pseudoceratina arabica. Tetrahedron. 2015; 71(41): 7837–7841. Publisher Full Text\n\nYu HB, Gu BB, Wang SP, et al.: New diterpenoids from the marine sponge Dactylospongia elegans. Tetrahedron. 2017; 73(47): 6657–6661. Publisher Full Text\n\nSkariyachan S, G Rao A, Patil MR, et al.: Antimicrobial potential of metabolites extracted from bacterial symbionts associated with marine sponges in coastal area of Gulf of Mannar Biosphere, India. Lett Appl Microbiol. 2014; 58(3): 231–41. PubMed Abstract | Publisher Full Text\n\nBedesee G, Ramanjooloo A, Aubert G, et al.: Ethyl acetate extract of the Mauritian sponge Jaspis sp. induces cell arrest in human promyelocytic leukemia cells. Environ Toxicol Pharmacol. 2013; 36(1): 58–65. PubMed Abstract | Publisher Full Text\n\nMahdian D, Iranshahy M, Shakeri A, et al.: Cytotoxicity evaluation of extracts and fractions of five marine sponges from the Persian Gulf and HPLC fingerprint analysis of cytotoxic extracts. Asian Pac J Trop Biomed. 2015; 5(11): 896–901. Publisher Full Text\n\nBeesoo R, Bhagooli R, Neergheen-Bhujun VS, et al.: Antibacterial and antibiotic potentiating activities of tropical marine sponge extracts. Comp Biochem Physiol C Toxicol Pharmacol. 2017; 196: 81–90. PubMed Abstract | Publisher Full Text\n\nMooney M, Al-sakkaf KA, Brown BL, et al.: Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells. Br J Cancer. 2002; 87(8): 909–917. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJi Y, Yu M, Qi Z, et al.: Study on apoptosis effect of human breast cancer cell MCF-7 induced by lycorine hydrochloride via death receptor pathway. Saudi Pharm J. 2017; 25(4): 633–637. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSu JH, Chang W, Chen H, et al.: 10-acetylirciformonin B, a sponge furanoterpenoid, induces DNA damage and apoptosis in leukemia cells. Molecules. 2012; 17(10): 11839–48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWiens M, Krasko A, Perovic S, et al.: Caspase-mediated apoptosis in sponges: Cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa. Biochim Biophys Acta. 2003; 1593(2–3): 179–189. PubMed Abstract | Publisher Full Text\n\nKustrin SA, Morton D, Kettle C: Structural Characteristic of Bioactive Marine Natural Products. In: Kim SK, ed. Marine Biomaterials: Characterization, Isolation and Applications. CRC Press Taylor & Francis Group. 2013; 177. Reference Source\n\nFujita M, Nakao Y, Matsunaga S, et al.: Ancorinosides B–D, inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP), from the marine Sponge Penares sollasi Thiele. Tetrahedron. 2001; 57(7): 1229–1234. Publisher Full Text\n\nBarnes EC, Said NABM, Williams ED, et al.: Ecionines A and B, two new cytotoxic pyridoacridine alkaloids from the Australian marine sponge, Ecionemia geodides. Tetrahedron. 2010; 66: 283–287. Publisher Full Text\n\nNagle DG, Zhou Y, Mora FD, et al.: Mechanism targeted discovery of antitumor marine natural products. Curr Med Chem. 2004; 11(13): 1725–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFouad MA, Debbab A, Wray V, et al.: New bioactive alkaloids from the marine sponge Stylissa sp. Tetrahedron. 2012; 68: 10176–10. Publisher Full Text\n\nOhta E, Ohta S, Ikegami S: Ancorinoside A Mg salt from the marine sponge, Ancorina sp., which specifically inhibits blastulation of starfish embryos. Tetrahedron. 2001; 57(22): 4699–4703. Publisher Full Text\n\nMeragelman KM, West LM, Northcote PT, et al.: Unusual sulfamate indoles and a novel indolo[3,2-a]carbazole from Ancorina sp. J Org Chem. 2002; 67(19): 6671–7. PubMed Abstract | Publisher Full Text\n\nDavis RA, Duffy S, Avery VM, et al.: (+)-7-Bromotrypargine: an antimalarial β-carboline from the Australian marine sponge Ancorina sp. Tetrahedron Lett. 2010; 51: 583–585. Publisher Full Text\n\nWeerapreeyakul NN, Barusrux S, Thitimetharoch T, et al.: Evaluation of the anticancer potential of six herbs against a hepatoma cell line. Chin Med. 2012; 7(1): 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNikoletopoulou V, Markaki M, Palikaras K, et al.: Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta. 2013; 1833(12): 3448–3459. PubMed Abstract | Publisher Full Text\n\nBali R, Chandra A, Verma R: Apoptosis in normal oral tissues and odontogenesis. Eur J Gen Dent. 2013; 2(3): 195–198. Publisher Full Text\n\nAffar EB, Germain M, Winstall E, et al.: Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis. J Biol Chem. 2001; 276(4): 2935–42. PubMed Abstract | Publisher Full Text\n\nTunjung WAS: Apoptosis Induction on Human Breast Cancer T47D Cell Line by Extracts of Ancorina Sp. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/AEJ96"
}
|
[
{
"id": "44186",
"date": "26 Feb 2019",
"name": "Sutiman Bambang Sumitro",
"expertise": [
"Reviewer Expertise Herbal medicine",
"complexity and Nano Biological approaches"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPage 3:\n\nMethods: Extraction:\nStatement: “Fresh samples were crushed in a blender in methanol, dichloromethane and dichloromethane methanol (1:1) then macerated for 24 hours. The samples were filtered and the residue then was re-extracted for two times. The total filtrate was then evaporated to obtain crude extract paste.”\n\nQuestions:\nWhat is used to filter the extract? What is used to evaporate? What degree of temperature is used to evaporate? What is the type of the tool?\n\nStatement: “The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2% penicillin streptomycin and 0.5% Fungi zone. Cells were harvested after reaching 80% confluence using 0.25% Trypsin-EDTA. Cells were cultured in 96-well micro plates (1 × 103 cells/well) in 100 μL RPMI and incubated at 37°C with 5% CO2 overnight”.\n\nSuggestion: It's better to explain what DMSO, MTT, etc., stand for.\n\nPage 4:\n\nResults: Figure 1. Breast cancer T47D cell viability after treatment with extracts of Ancorina sp. Methanol (M), Dichloromethane: Methanol (D:M) and Dichloromethane (D).\n\nQuestions:\nWhat is the unit of the cell viability? The log concentration (?). Add the scaling line next to the numbers.\n\nPage 5:\nTable 1. IC50 values of Ancorina sp. extracts. Treatments IC50 value (μg/mL) Methanol\n\n84.25a ± 9.52 Dichloromethane:methanol (1:1)\n\n121.45 b ± 10.11 Dichloromethane\n\n99.85ab ± 11.79\nNote: different letters showed the significant difference at the 0.05 levels.\n\nQuestions:\nHow is the IC50 calculated? Why do dichloromethane (non-polar solvent) and methanol indicate moderate cytotoxicity, while on the other hand, methanol solvent (polar solvent) indicates the highest cytotoxicity?\n\nFigure 2. Cell morphology of breast cancer T47D cells after treatment with extracts of Ancorina sp. Control (A), Methanol (B), Dichloromethane:Methanol (C), Dichloromethane (D), Doxorubicin (E) and DMSO (F). Arrow shows dead cells.\nQuestions:\nWhat are the morphological differences among the treatments? This needs to be explained. Is it only to show dead and living cells? If this is the case one figure is enough. The figures were taken using what kind of microscope? Add the microscopic scale on the figure.\n\nPage 7:\nFigure 4. Detection of caspase-3 activation in breast cancer T47D cells after treatment with extracts of Ancorina sp. Negative control (A), Doxorubicin (B), Methanol (C), Dichloromethane: Methanol (D) and Dichloromethane.\nQuestion: What is R1?\n\nPage 8:\nFigure 5. Correlation between percentage of apoptosis and caspase 3 activation in breast cancer T47D cells after treatment with extracts of Ancorina sp. Doxorubicin (Dox), Methanol (M), Dichloromethane: Methanol (1:1) (D:M), Dichloromethane (D), and negative control (control).\n\nSuggestion: The figure is not clear. Add the scaling line next to the numbers.\n\nPage 8:\nAncorina sp. is a source of bioactive compounds such as ancorinoside A Mg salt, ancorinolates AC, bis-ancorinolate B, ancorinazole, indolo [3,2-a] carbazole, and (+) - 7-bromotryparg.\n\nSuggestion: Mention the characteristics of these compounds e.g. the level of their polarity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "44188",
"date": "21 Mar 2019",
"name": "Richard Luke Daniels",
"expertise": [
"Reviewer Expertise Cell physiology",
"Cell signaling"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the cytotoxic effects of extracts from a marine sponge. The authors provide evidence that extracts were cytotoxic, and that methanol-extracts were the most cytotoxic (as measured by MTT assay). Additional studies showed that the mechanism of cell death is likely through apoptosis (as measured by examining Annexin V/PI staining and caspase 3 activity via flow cytometry). These are novel results that contribute to the search for anti-cancer agents, and it is this reviewer’s opinion that indexing is warranted. There are a few minor modifications that I would suggest making before finalizing this manuscript.\nMethods: It would be helpful to include additional details about the methods, such as:\nspecific extraction methods. the method in which the IC50 was calculated.\n\nFigure 1: This reviewer agrees that the MTT data supports the idea that these various extracts are cytotoxic. In Figure 1, it seems that the y-axis is the percentage of cells that are healthy relative to controls (?), but the units are not given. It would be helpful in Figure 1 to:\ngive the units for the y-axis. provide some explanation of the dose that is given in each treatment (currently given in the methods). know the number of trials this experiment represents. What is the n, what do the error bars represent? (Standard error? Standard deviation?)\nIt might also be beneficial to do an ANOVA to better understand whether differences among these experimental treatments might be statistically significant (in other words, are the dose-dependent changes in cell viabilities statistically different from each other?). I do not consider this to be essential for indexing.\nFigure 2: This reviewer agrees that these treatment compounds seem to negatively impact cells (based on their morphology). It would be beneficial to:\ndescribe the differences that were observed for each treatment condition, as there seems to be some variation (C vs. E for example).\n\nFigure 3 & 4: It appears that the flow cytometry experiments were repeated a number of times, as implied by the variability that is reported in Tables 2 & 3 (+/- values). It would be helpful to:\ngive the number of times the experiment repeated. state what this variability represents (standard error? Standard deviation?).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-168
|
https://f1000research.com/articles/8-167/v1
|
07 Feb 19
|
{
"type": "Research Article",
"title": "Evaluation of retention of knowledge, skill and competency of health workers one year after completion of the Helping Babies Breathe training program in South Sudan",
"authors": [
"Christopher Vunni Draiko",
"Khemika Yamarat",
"Alessio Panza",
"Judith Draleru",
"Khemika Yamarat",
"Alessio Panza",
"Judith Draleru"
],
"abstract": "Introduction: The aim of the study is to evaluate the long-term retention of knowledge, skill and competency of health workers who completed Helping Babies Breathe (HBB) training and effect on newborn mortality Methods: The study employed pre-post-interventions study and participants were selected based on their previous training on HBB protocols. Health workers were assessed for knowledge, skill and competency pre, post training in March, and 3months in June 2017 and 1-year post implementation in September 2018. Health workers were scored on knowledge, skill and competency. The mean score was obtained and repeated ANOVA, chi-squared test and Pearson’s test was used to compare pre- and post-intervention and one-year implementation. Retention of health worker’s knowledge, skill and competency was assessed using the HBB questionnaires, checklist, practical skill and drills, and were scored on knowledge, skill and competency. The scores were computed into percentages, mean scores and mean differences, and compared against scores at 3 months and 1 year. Impact on management of newborn asphyxia was conducted using a review of delivery registry at maternity and children ward scores were group into percentages, averages means, computed using the Chi-square test. Results: There were 53 health workers evaluated; 29 were in the intervention and 24 in the control hospital. There was marked decline in the knowledge (84% to 69.4% p=0.001), skills, (94.6% to 77%, p=0.001), competency for simple resuscitation (88.5% to 76.4%, p=0.36) and complex resuscitation (83.3% to 76.9%, p=0.001) in intervention hospitals. Health workers in the control had good retention and improvement of knowledge (50.6% to 61.2%, p= 0.004), skills (40.3% to 56.5% p=0.004), competency for simple resuscitation (38.0% to 53.1% p=0.001) and complex resuscitation (33.1% to 53.4% p=0.001). Conclusions: Health workers in the control hospital had improvement in retention of their knowledge, skill and competency. Newborn mortality decreased in both hospitals.",
"keywords": [
"Knowledge",
"skill",
"competency",
"retention",
"neonatal resuscitation",
"Education"
],
"content": "Introduction\n\nOf the 200,000 children born annually in South Sudan, an estimated 40% die in the first month of life1. Newborn mortality contributes to 39% of all deaths of under-fives in South Sudan. The decline of newborn mortality rate in South Sudan has remained slower than the global average rate for the last decade2,3. The highest risk to newborn life is within the first day of life. Death within this period accounts for almost 5% of newborn deaths4,5. Globally and in South Sudan, the main direct causes of newborn deaths are usually infection-related complications (26%), intrapartum complications (24%) including birth asphyxia, preterm delivery (34%), and congenital abnormalities (9%). In South Sudan, 20% of newborn deaths are associated with birth asphyxia6,7.\n\nThe transition from intrauterine to extrauterine life requires initiation of breathing which is critical physiological changes required for newborn survival. Research indicates that the majority of newborns initiate breathing within 30 minutes and an estimated 10% breathe when they receive drying and stimulations from health workers, 3% of newborns require positive pressure (PPV), while another 2% need ventilation and intubation offered by knowledgeable and skilled health workers8,9. Training of health of health workers on newborn resuscitation and provision of effective and timely resuscitation could prevent a good number of newborn with asphyxia and subsequently improve their survival rate.\n\nVarious studies on the effectiveness of newborn resuscitation training programs such as HBB showed increased knowledge, skill and competency after training which was sometimes retained for 1 year. Evaluation of newborn resuscitation training has shown an immediate increased in knowledge, practical skills and competency with improved newborn outcome10,11. Although training has been carried out, there has been limited rigorous evaluation of retention of knowledge, skill, competency and newborn outcome in low-resource and post-conflict settings like South Sudan.\n\nThe outcome of training depends on the extent to which knowledge, skill and competency of health workers is retained, alongside the ability to use the learned skills, knowledge and application at appropriate time. However, the retention of knowledge and skills, and their application to improve newborn outcomes depends on several factors, such as conditions at the clinical environment, regular supervision, settings, opportunity to practice, availability of supplies and appropriate equipment9.\n\nTraining of health workers appropriately, provision of refresher training and support and ensuring good training environment facilitates the long-term retention of knowledge, skill and competency among health workers in low resource settings12,13. In South Sudan, nurses and midwives manage normal delivery and birth asphyxia is not recognized early enough. Medical doctors are often involved in the late stages of the management of birth asphyxia due to critical shortages, even in a major hospital setting. The objective of study is to evaluate retention of knowledge, skill and competency of the health workers and the impacts on newborn mortality after one year of implementation.\n\n\nMethods\n\nThe trial has been registered with Pan African Clinical Registry with registration number PACTR20170800246922514. The study TREND statement is available as supporting document. The original aim of the study was intended to measure only effectiveness of improving health workers, knowledge, skill and competency. The measurement of newborn mortality was conceived later leading to late registration.\n\nCalculation of sample size was based on the ability to detect a 20% increase in knowledge, practical skill and competency and 20% reduction in newborn mortality with an error of 0.05, 20% and dropout rate of 50%. The study used this value as our references for the sample size calculation. Using G*Power version 3.1, we determined a sample size of 74 participants in each arm to account for losses, but due to the ongoing conflict the actual participate for both arms was less than estimated sample size\n\nThe following selection criteria were applied. Medical officers/doctors, nurses, midwives, maternal child health officers, community health workers and clinical officers working and practicing in maternity, operating theater and children ward; health providers self-reporting that they provide routine care services at delivery and neonatal unit or departments; and Health workers willing to be available for data collection and during the period of study.\n\nThe health workers were identified and recruited from maternity wards, newborn operating theater and children ward of Juba Training Hospital (intervention site) and Wau Teaching Hospital (control). After the completion of the recruitment process, an invitation was sent to those health workers who met the inclusion criteria to participate in the study. Written informed consent was obtained from the health workers. All newborn delivered in maternity, newborn unit and operating theatre who met the inclusion criteria were included in the study.\n\nData on the pre and post training on knowledge, practical skills of the health workers and records of newborn asphyxia and deaths both in intervention and control group was collected from the area of practice, maternity ward, operating theatre and newborn unit.\n\nHealth workers recruitment commenced on February 1st and ended in 25th February 2017. Training intervention was 27th–28th February 2017. The intervention commenced in March and follow-up started on June 1st and ended on 30th June 2017. The 1-year evaluation was conducted in August 2018.\n\nOur study objectives were as follows. To assess change in health worker knowledge, psychomotor skills, competency of the health workers regarding managing neonates with birth asphyxia after training intervention, and the ratio of perinatal mortality due to asphyxia of hospital admission within 24 hours of birth after training interventions. Meanwhile we hypothesized that HBB training would result in 20% increase in knowledge, skill and subsequent reduction of early newborn mortality among newborn born with asphyxia.\n\nThe main primary outcome was any improved knowledge, skill and competency of the health workers on HBB protocol. The secondary outcome was the ratio of early newborn reduction with asphyxia within 24 hours.\n\nData was collected by use of a questionnaire with 17 multiple choice questions to assess the knowledge of health workers on the HBB protocol, a seven-step checklist for bag-and-mask skill assessment, a five-step checklist for preparation at birth skill checks, a 13-step checklist with simulation for the first Objective Structural Clinical Examination (OSCE), a self-observation questionnaires consisting of a 25-step checklist maternity and newborn register for recording delivery, and neonatal a registration form15.\n\nThe validity of the HBB instruments was determined by expert reviews and opinions from Chulalongkorn University, college of Public Health Sciences. The study used HBB instruments for assessing the knowledge, practical skill and competency of the health workers was a standard HBB instrument tool which has been validated and used in other low setting countries where HBB and skills evaluation have been conducted11.\n\nThe retention evaluation of skill was conducted following, pre- and post-test study design conducted in Juba and Wau Teaching hospitals in Republic of South Sudan. The pre- and post-training assessment was completed in March 2017 and the 3-month assessment was done on knowledge, skill competency and newborn mortality in June 2017. Assessment at 1 year post-intervention to determine retention of knowledge skill, competency and review of neonatal mortality was conducted in September 2018. During evaluation, health workers who were available and received previous HBB training in both hospitals were approached and interviewed according to the set exclusion and inclusion criteria. The Study was approved by South Sudan Ethical Committee, Ministry of Health in Juba, South Sudan and reviewed by Chulalongkorn University, College of Public Health Sciences Bangkok, and Ethics Review committees of Juba Teaching Hospital and Wau Teaching Hospital. Informed consent, both written and verbal, was obtained from the health workers before the training intervention. Informed consent was sought from each of the health workers available at the time of evaluation. Additionally, verbal approval was sought from the mothers with newborn asphyxia by the health worker.\n\nIn December 2016, four midwives went through the HBB trainers of trainer’s (TOT) workshop and become research assistants and facilitators. Training of midwives as TOTs covered teaching methodology, neonatal resuscitation using HBB model, evaluation skills and practical skill training. The second part of TOT involved conducting HBB simulation drills, preparation of births, newborn routine care, the golden minute and ventilation of newborn. The last component of TOT program was facilitation and coaching skills using American Pediatric Association model for HBB.\n\nCompletion of TOT course for facilitators was followed by recruitment of health workers in the Juba Teaching Hospital (intervention) and Wau Teaching hospitals (Control) to participate in the HBB study. The health workers in the control hospital were not trained on HBB protocol during the implementation period. However, plans are underway to provide training after final evaluation. The two hospitals were located 300 km apart and in different regions. Both are national teaching hospitals that train health workers. Health workers were selected from maternity and newborn wards and the antenatal clinic (ANC). All health workers working in maternity, neonatal ward and the ANC were approached to participate in the study. Informed consent written in Arabic and English were read and explained to the health workers, who signed a copy before their full participations.\n\nPre-training assessment was conducted before the commencement of training on demographics, previous knowledge and exposure to HBB training, knowledge, skill and competency, and also review the birth registry for number of births, neonatal asphyxia and mortality for the past year. The survey used a multiple-choice questionnaire15 and simulated environment using Bag and Mask checklist for psychomotor skill and OSCEA&B11,15 for competency at pre-intervention, immediately after intervention, 3 months post-intervention and 1 year post-intervention.\n\nThe initial training program covered preparation for birth, routine care, the golden minute and ventilation of newborn using bag and mask techniques. Individual training involved conducting simulation drills on mannequins (mama and baby NeoNatalie). Immediate feedback on performance of the health workers and lectures by the facilitators. The health workers were allowed to practice a number of drills, demonstration and return demonstration using mannequins. The demonstration was accompanied with an explanation on how to perform tasks to help newborn breath at birth during the study, individual health workers were assessed on their knowledge, skills and competency on neonatal resuscitation using HBB protocol, pre-training, immediately after training, at 3 months and at 1 year to determine short- and long-term retention of knowledge, skill and competency. A 20-minute written multiple-choice test was administered to the participants before and after the training to evaluate knowledge. Skills and competency assessment for the health workers were evaluated through clinical simulation using the NeoNatalie newborn simulator. The trainees were directed and observed as they perform the task on the mannequin. OSCE A&B were administered to test the performance and competency of the trainee health workers on HBB. OSCE A consists of 13 items while OSCE B consist of 18 items indicating the key component of what the trainee must learned and practice. Each item in objective OSCE A and B were scored 1 when correctly done and score 0 for not correctly done. A passing score of 80% was required to complete the course successfully. In each training session, health workers were asked to evaluate the training using a questionnaire with a five-point Likert scale15.\n\nThe HBB training included trainers and trainees reviewing training modules and practical sessions on newborn asphyxia, routine care and ventilation. Health workers practiced on the mannequin and were provided with equipment, and each of the practice sessions on the mannequin was rated by the facilitators, who were senior midwives and trained as master trainers. The mannequin which presented mother and newborn was placed on the resuscitation table and each situation presenting the condition of the baby and mother is controlled with the health workers present caring for the mother and newborn. The facilitator (rater) was responsible to ensure participants completed the skill-based checklist for assessing the performance of the health workers during the practical drill. The participant performed the drills in either Arabic or English, communication channels commonly used in South Sudan. The task performed by the health workers were explained and verbalized to the midwife rater as the health workers conducted the newborn resuscitation drill.\n\nEach topic was introduced by the main facilitators followed by demonstration and return demonstration by the participants. This was followed by self-directed and self-assessment exercise using the learner work book. The training comprised of six classroom hours and two hours for practical. A series of procedures surrounding births were reviewed through practical exercises under the supervision of the trainers. Scenarios reproducing routine care and neonatal resuscitation at birth were performed on a NeoNatalie new born simulator. During the training, one simulator, a stethoscope, resuscitator and suction device were made available for every two trainees. The intervention was delivered to a group 20 health workers each. Training was provided to the intervention group in Juba Teaching hospital. The HBB training intervention course was facilitated by two senior midwives who were trained as research assistants (master trainer) and assisted by the principal investigator and the research assistants. The newborn resuscitation was conducted by the health workers in various unit selected. The intervention was delivered at the main hospital training hall and the observation was at maternity, newborn unit and operating theatre. Two training session was conducted to allow adequate time for facilitators and participants to learn and practice. The ratio of one facilitator and six participants was maintained to support participants to learn in pairs as standard protocol indicated in the training. The duration lasted for 2 days. The training intervention was delivered in 6 hours each for two days and practical seasons was 2o minutes. Observation of health workers practicing resuscitation was observed throughout the 3 month period by the research assistants. Bag and mask drill was delivered every morning 5 days a week for 3 months. Health workers were provided breakfast and lunch during the two days training interventions and support supervision during morning drill of mask and bags.\n\nCompleting tasks such as preparation for ventilation, assessing the newborn, conducting ventilation using bag and mask, conducting stimulations and referral of the newborn was directly performed on the mannequin and scored by the experienced rater. Raters score health workers one on skill and competency when the task is correctly completed and zero when not correctly completed. Health workers must have obtained 80% or above to be considered competent in all the three aspect of knowledge, skill and competency to help newborns breathe.\n\nPost-training and intervention assessment was done immediately, and at 3 months using the simulated HBB knowledge-, skill- and competency-based checklist. Assessment were categorized into knowledge, practical skills and competency. The sum of the scores were calculated by determining the degree of completion of each task in knowledge, skill and competency, the total score for each task was 80% for knowledge, skill and competency respectively.\n\nAll health workers who completed the initial HBB training and were present 1 year later were asked to participate in HBB knowledge, skill drills and competency assessment to determine long term retention of their knowledge, skill and competency. The retention of knowledge, skill and competency assessment used the same check list for assessment and scoring at the initial training. During 1-year evaluation, each individual health workers was assigned knowledge, skill and competency score presented as percentage of correctly performed items at 1-year evaluation. Health workers were asked to complete the same five-point Likert scale survey as well their confidence in HBB practice.\n\nDuring the interventions, health workers were assessed for simple resuscitation using Objective Structural Clinical Examination (OSCE A) which was made up of 13 observation steps consisting of scripted information on preparation for birth, drying the baby thoroughly, ability to recognize the baby is not breathing, positioning of the head and clearing the airway, evaluation of the breathing, clamping or tying and cutting the cord, position skin to skin and communication with mother. The successful completion requires a total score of 10 correct of 13 steps and the health worker being observed must include the Dries thoroughly, Recognizes Baby is not crying and positioning and clearing the airway. OSCE A is a performance assessment of preparation for birth and routine newborn care, and a learner must perform = 80% (10 of 13 steps) correctly to pass, including three essential steps. OSCE A is a performance assessment of preparation for birth and routine newborn care, and a learner must perform = 80% (10 of 13 steps) correctly to pass, including three essential steps.\n\nWe conducted assessment of health workers for complex neonatal resuscitation using Objective Structural Clinical Examination (OSCE B) was made up of 18 scripted scenario on preparation for birth, drying of the baby, recognize the baby is not breathing, ventilation at 40 breathes per minutes (30–50) acceptable, looking for chest movement, evaluate breathing, call for help, improve ventilation thorough, repositioning the head, reapplication of mask, clear secretion, open mouth slightly and squeezing the bad hardly. The 18 items reflects the key components of the training course for newborn survival. Each item was scored 1 if carried out correctly and any partial or incorrect action was scored zero. Similar to OSCE A, OSCE B was a performance assessment of a complex resuscitation scenario that requires bag-mask ventilation, and a learner must perform 14 of the 18 steps correctly to be evaluated to have the competency to help newborn with asphyxia to breathe.\n\nData on neonatal mortality was collected from the hospital register at the delivery room, operating theater and neonatal unit admission book pre- and post-implementation for June 2017–June 2018. In the pre-implementation phase, hospital monthly record was used to collect the number of deliveries, neonates with breathing problems; neonates resuscitated using HBB protocols and the perinatal mortality due to asphyxia outcome immediately post-training and end of intervention. For easy follow-up, a simple form was designed by the research team to track deliveries, use of HBB protocol for newborn with birth asphyxia and the outcome of the resuscitation within 24 hours. The mean ratio of the perinatal death was used to determine the outcome/changes of the intervention within the period of four months. The review of hospital registry and form was approved by the South Sudan ethical review board and the hospital ethics committee.\n\nThe mean score and the significant level within and between groups were tested using repeated ANOVA and Chi squared test in the three performance areas of knowledge, skill and competency. Newborn mortality was determined and tested with Pearson chi squared test. The statistical analyses were performed using SPSS version 20 and p<_0.05 was considered statically significant.\n\n\nResults\n\nIn total, 70 health workers completed pre-training course and 67 were evaluated at 3 months. Of the health workers who original completed the training and were evaluated at 3 months, 53 were available for the one year were evaluated for HBB knowledge, skill and competency. A total of 58.6% of the health workers in intervention and 66.7% in the control spent their full time working in maternity (delivery unit). The health worker in average attends 115 deliveries and managed 7.7 asphyxia cases in 1 year. The Table 1 shows demographic characteristics of the health workers after 1 year of implementation.\n\nAs shown in the Figure 2, the status of knowledge scores among the health workers in the intervention group declined from 84.5% at 3 months to 69.4%, with a mean difference of 69.4±18.8(−15.0 (−22.7–7.4)) in the 17 domains that was assessed after 3 months and at one year after training. The changes in scores of knowledge measure at 1 year was statistically significant (p<0.05) (Table 2). Meanwhile the assessment established that knowledge scores increased for health workers in the control group from 50% to 61%, with a mean difference of 61.2±20.3 (10.5 (0.54–20.6)). The changes in the knowledge among the control were significant (Table 2).\n\nScores expressed as mean difference.*Significant level at 0.005 at 3 months and one year after training. Pa-Value within intervention group tested by repeated ANOVA ,0.001 and 0.001 between immediate post intervention and 3 months follow up **No baseline conducted for control group for bag and mask, OSCEA&B.\n\nSkills retention among the health workers were assessed using the seven domains of determining skill applied at pre-training, immediately post-training and at 3 months post-training assessment for both groups. The 1-year mean skill scores declined from 94.5% to 77% from 3 months’ assessment. The decline is visible across all the seven domains: i) Check equipment; ii) select the correct mask; iii) apply the mask to make a firm seal; iv) ventilate at 40 breaths; v) Look at chest movement; vi) improve ventilation if the chest does not move; vii) reapply mask and reposition, clear secretions and squeeze the bag. Mean differences of 77.0±21.8 (−17.5(−27.2–−7.8) were found. the result showed a marked increase in the mean scores among the control group (Table 2). The mean skill score increased from 40.3% to 56.5%. The mean differences in skills was 56.5±25 (16.2 (1.2–31.2) which was statically significant (p<0.05).\n\nThe competency of the health workers declined among the intervention (Juba Teaching Hospital) and greatly increased in the control (Wau Teaching Hospital) after one-year evaluation when compared with the 3 months’ post implementation. There was significant decrease in competency between the 3 months and 1 year of training (88.6±8.6 to 76.4±13.6) (mean difference decline of −12.2 (−18.3–−6.1 p<0.001). The mean decreased between 3-months and 1-year after training evaluation for the retention of was significant. The status of retention of competency among the health workers in control showed significant increase from 38.0±9.1 to 53.9±11.8 (mean difference 15.1 (8.7–21.4 p< 0.001)) (Table 2).\n\nEvaluation of retention status of health worker’s competency in both groups after one year of post implementation assessment has shown that there was marked decreased among the intervention while control has shown significant increased. In the intervention, the mean competency score decreased from 90.4±8.6 to 76.9±11.6 (mean difference −13.6 (−19.8–−7.4; p<0.001) whilst the control had significant increase in their competency for complex from 32.4±8.3 to53.4±21.7 (mean difference 21.0 (10.9–31.0); p<0.01).\n\nA review of records in the hospital registry and forms from June 2017 to June 2018 found out that 6,072 live births were registered in the maternity and newborn wards and ANC. A total of 4,887 of the total live birth were recorded in the intervention hospital and 1,210 in the control hospital. All births were reported to be assisted by health workers trained in the HBB. A 1-year post-training evaluation found that there was a significant increase in the number of newborns with asphyxia being resuscitated in both hospitals. More newborns in intervention hospital received neonatal support (resuscitation). 98.4% of the newborn received support compared to 1.6% in control group at 3 months. Meanwhile (86.7%) compared to the control (48.4%) newborn received neonatal resuscitation support from health workers at assessment after 1 year. The support given to newborn by health workers was statically significant (p<0.050; Table 3). Review of data on early mortality due to asphyxia shows that mortality decreased from 30.7% at 3 months to 17.9% at 1 year in the intervention hospital which is significant (p<0.05). There was also significant reduction of early newborn mortality in the control hospital. Mortality reduced from 69.2% at 3 months to 49.4% at 1 year after implementation (p<0.05). The result showed that more newborn deaths occurred within 24 hours of birth in both hospitals in comparison to deaths that occurred after 24 hours up to 1 year (Table 3).\n\nTested by Pearson Chi square test 2×2 sided significance for birth asphyxia, newborn death within and after 24 hours before and after implementation.\n\n\nDiscussion\n\nThis study attempted to evaluate the retention of knowledge, skills, competency and impact of modified HBB at Public Tertiary Hospital in South Sudan. The status of knowledge, skill and competency among the health workers in the intervention and control was evaluated after 1 year and compared with the status at 3 months. The objective of the training was to build the knowledge, skills and competency of health workers. More focus was placed on enhancing skill and competency through hands on practice and avenue for continued peer-to-peer simulation refresher training, supervision and mentoring. The evaluation at 1-year post-training showed a significant decline in the retention of knowledge, skills and competency among the health workers. The causes of considerable decline in the retention of knowledge, skill and competency were not clear among the health workers and this was consistent in all the domains evaluated and further evaluation is needed to further determine causes for the decline.\n\nOur assumption was that training in the modified HBB could lead to longer retention of the acquired knowledge, skill and competency. Review on the general status of the implementation of HBB protocol conducted through review of newborn records, conducting drills and observing health workers resuscitate newborn with asphyxia and interactions with health workers in the intervention hospital revealed that the setup of HBB simulation corners at the hospital that provides opportunity for the health workers to have peer-to-peer clinical practice using mannequins, bag and mask was not available, and space was limited by the ongoing major hospital renovation. Furthermore, health workers reported a lack of support from experienced mentors assigned during training, as most of them move out of the hospital for better-paying jobs, probably contributing to low retention of knowledge, skill and competency. More feedback from health workers suggested that they had little time to link theory to practice due to the high work load resulting from high staff attrition. The intervention hospital was purely supported by government, such that it lacks most of the necessities for practical applications of the HBB interventions.\n\nSurprisingly, evaluation of Health worker’s knowledge, skill and competency in the control hospital showed a significant increase after 1 year despite having considerable gap in all the domains of knowledge, skill and competency during the pre, post and 3 months assessment. We undertook extensive review to understand activities leading to increased knowledge after 1 year of training. Our review and interaction with health workers indicated that, health workers were supported and the hospital maternity, newborn and ANC unit is entirely managed by non-governmental organizations (NGOs). Health workers were provided opportunity to learn through the introduction of online HBB models. Although practical training has not yet been conducted, emphasis was placed on HBB as one of the key actions to reduce newborn mortality. Health workers had better opportunity to learn and understand the concepts and rationale from the mentors who were placed in the hospital by the NGO. The good environment could have contributed to increased knowledge after 1 year.\n\nOur evaluation documented the challenges of resuscitation in developing countries with high newborn mortality. The decline in performance in retention of knowledge, skill and competency of the health workers trained in HBB brings in issues to what requires to be done to ensure health workers retention of acquired knowledge, skills and competency in HBB in tertiary hospitals and other local primary health care centers. The majority of the health workers in the intervention facilities had limited opportunity to perform appropriate interventions to resuscitate a newborn with asphyxia in comparison with the control hospital that could have resulted in significant changes observed in the results.\n\nOur study is relevant as it documented what happens to knowledge, skills and competency in terms of retention by the health workers trained and provided a chance to practice in the hospital. This study demonstrated that knowledge, skill and competency tends to disappears with time and refresher training is necessary for the health workers to keep abreast with skills and knowledge.\n\nThere are few evaluation reports that document the retention of knowledge, skill and competency after training for a period of 1–2 years. In Tanzania, report indicates significant improvement in knowledge. However, retention of skills remained poor in comparison to the knowledge16. In similar evaluation of the retention of knowledge and skill among the Canadian Doctors and Nurses, there was better retention of knowledge than skills. The skills of nurses and doctors dropped upon evaluation at 6 months after training.\n\nIn terms of the long-term effect of HBB training on reduction of newborn mortality, our study documented a significant increase in the number of newborns assisted to breathe by the health workers in both hospitals. The rate of neonatal mortality due to asphyxia reduced from 30.7% to 17.9% in the intervention hospital, and from 69.2% to 49.4% at the same period. It is interesting to document a significant reduction in the number of newborns in the control hospital although no formal training was known to have been provided. The results documented pointed out that reduction of mortality does not only depend on learned skill and developed competency, but also on the availability of resources. However, the relationship between mortality and resources was not evaluated. Despite the small sample size, our evaluation showed a similar result to a large randomized study conducted in Tanzania to determine neonatal outcome after health worker receiving HBB training where newborn mortality within 24 hours and stillbirth reduced significantly17. Although, evaluation studies were conducted at global level, still few evaluation studies focused on determining impact of modified resuscitation training on newborn outcomes. Therefore, more evaluation is recommended particularly, in post-conflict settings.\n\nThe strength of the study is that the majority of the health workers were nurses and midwives who had training on newborns and spent their full time in the maternity and children ward; therefore, they represented a population that typically encounters and manages birth asphyxia as they occur in hospital setting. The documentation of knowledge, skill, competency and birth asphyxia during the pre-training, immediately post-training and at 3 months post-training provided an opportunity to evaluate the same health workers available at the hospital at the time of assessment.\n\nThe result of the study could have limited by relying on the documentation of newborns in maternity and children ward of both hospitals to determine newborn outcome after pre-training, immediately post-training and at 3 months post-training where trained staff were available to help with documentation. There still exists a large gap in the documentation of medical records and keeping and data use among the health workers, clerks and managers in the hospitals and health facilities in South Sudan.\n\n\nConclusion\n\nThe evaluation of retention of knowledge, skill and competency and it impact on newborn mortality at 1-year after HBB training could be first in South Sudan, a country emerging from war that has low-skilled health workers. The evaluation has shown mixed results, with health workers in the intervention hospital showing marked decline in knowledge, skills and competency, while encouraging increase in the level of knowledge, skill and competency in the control group. The decline in knowledge, skill and competency scores and the level of increase among the groups were consistent. Although the reason for the observed gap is not clear, it could be attributed to limited opportunity to practice, lack of support and high attrition rate among the health workers in interventions and whereas, the control group had opportunity to learn from mentors, participate in online studies and availability of resources for practicing their skills. A further review is recommended to understand the various factors that affects acquisition and retention of knowledge, skill and competency on newborn resuscitation in developing countries. Despite the changes in level of retention of knowledge, skills and competency, there has been significant decrease in newborn mortality in both intervention and control groups.\n\n\nData availability\n\nData concerning newborn mortality and the performance of health workers are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/3NTJB15.\n\nConsent forms and assessment tools used in this study are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/3NTJB15.\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\nReporting guidelines\n\nA completed TREND statement is available on OSF15.",
"appendix": "Grant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe acknowledge the Government of South Sudan, Ministry of health, Directorate of Reproductive of Health, Juba and Wau teaching Hospital administration, state Ministry of health for their continued support in making the evaluation successful. Our appreciation goes to the health workers (medical doctors, nurses, midwives) in Juba and Wau teaching Hospital for the contribution through their time in accomplishing the result documented in the study. Finally, our Research team for their tireless time through training, simulation rating, data analysis and production of the manuscript. Thank you all.\n\n\nReferences\n\nCountdown Coverage Writing Group: Countdown to 2015 for maternal, newborn, and child survival. 2015.\n\nGoldenberg RL, McClure EM, Bann CM: The relationship of intrapartum and antepartum stillbirth rates to measures of obstetric care in developed and developing countries. Acta Obstet Gynecol Scand. 2007; 86(11): 1303–9. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: World health statistics. 2010. Reference Source\n\nWorld Health Organization: Making pregnancy safer: the critical role of the skilled attendant. A joint statement by WHO, ICM and FIGO. Geneva: World Health Organization; 2005. Reference Source\n\nCarlo WA, Wright LL, Chomba E, et al.: Educational impact of the neonatal resuscitation program in low-risk delivery centers in a developing country. J Pediatr. 2009; 154(4): 504–508.e5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLawn JE, Lee AC, Kinney M, et al.: Two million intrapartum-related stillbirths and neonatal deaths: where, why, and what can be done? Int J Gynaecol Obstet. 2009; 107 Suppl 1: S5–18, S19. PubMed Abstract | Publisher Full Text\n\nSinghal N, Lockyer J, Fidler H, et al.: Helping Babies Breathe: global neonatal resuscitation program development and formative educational evaluation. Resuscitation. 2012; 83(1): 90–6. PubMed Abstract | Publisher Full Text\n\nLawn JE, Kerber K, Enweronu-Laryea C, et al.: 3.6 million neonatal deaths--what is progressing and what is not? Semin Perinatol. 2010; 34(6): 371–386. PubMed Abstract | Publisher Full Text\n\nTrevisanuto D, Ferrarese P, Cavicchioli P, et al.: Knowledge gained by pediatric residents after neonatal resuscitation program courses. Paediatr Anaesth. 2005; 15(11): 944–7. PubMed Abstract | Publisher Full Text\n\nSave the Children: Ending Newborn Deaths. London, UK: Author. 2014. Reference Source\n\nAmerican Academy of Pediatrics Helping Babies Breathe. 2014. Reference Source\n\nUNICEF: Committing to Child Survival: A Promise Renewed – Progress Report 2013. New York, 2013. Reference Source\n\nOpiyo N, English M: In-service training for health professionals to improve care of the seriously ill newborn or child in low and middle-income countries (Review). Cochrane Database Syst Rev. 2010; 14(4): CD007071. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan African Clinical Trial Registry (PACTR). South Africa, PACTR201708002469225, 2017. Reference Source\n\nVunni C: Evaluation of Retention of Knowledge, Skill, Competency of Health Workers One Year After Completion of Helping Babies Breathe Training Program in South Sudan. 2019. http://www.doi.org/10.17605/OSF.IO/3NTJB\n\nMsemo G, Massawe A, Mmbando D, et al.: Newborn mortality and fresh stillbirth rates in Tanzania after helping babies breathe training. Pediatrics. 2013; 131(2): e353–60. PubMed Abstract | Publisher Full Text\n\nKaczorowski J, Levitt C, Hammond M, et al.: Retention of neonatal resuscitation skills and knowledge: a randomized controlled trial. Fam Med. 1998; 30(10): 705–711. PubMed Abstract"
}
|
[
{
"id": "44166",
"date": "22 Feb 2019",
"name": "Brett D. Nelson",
"expertise": [
"Reviewer Expertise Newborn medicine",
"Helping Babies Breathe",
"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\nI congratulate and thank the authors for their important work on newborn health in South Sudan. Having worked on various HBB projects, and having worked for a couple of years in South Sudan, I really enjoyed reading your article.\nThe paper is great. However, I would like to humbly provide a strong recommendation that the current abstract please be updated by the authors. The abstract's Results and Conclusion sections currently seem to suggest that the Control group faired better than the Intervention group and had better retention than the Intervention group. (E.g., \"Conclusions: Health workers in the control hospital had improvement in retention of their knowledge, skill and competency. Newborn mortality decreased in both hospitals.\")\nRespectfully, I think this could be inadvertently misleading to many readers. While the Control group's scores did increase slightly (and statistically significantly, for the possible reasons discussed in the paper), the Control group's scores were always much lower than the Intervention group's scores (since the Controls didn't receive the training yet). So I would argue that the Control group never showed any significant knowledge or skills in HBB and, therefore, never \"retained\" this knowledge and skills. Because they hadn't yet been formally taught the knowledge and skills (again, because they were Controls), they never had the knowledge and skills to lose and never had the ability to \"retain\" the HBB skills.\nMeanwhile, the Intervention group did acquire substantial knowledge and skills following the successful training and lost some knowledge and skills over time, which we've seen in other HBB studies -- unless active measures are taken to retain knowledge and skills via on-the-job training, daily practice, refresher training, supervisory visits, etc. In short, I think the small improvement in the Control group's comparatively low scores should not be the primary focus of the abstract's Results and Conclusion sections, and I think it may be inadvertently misleading to readers to simply conclude that \"Health workers in the control hospital had improvement in RETENTION of their knowledge, skill and competency\" (my emphasis added). This could give the mis-perception that the Controls did better than the Intervention group.\nI hope you don't mind the suggestion, and hopefully this might be some helpful feedback to further strengthen your paper. And again, I congratulate and thank the authors for their important 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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "44164",
"date": "12 Mar 2019",
"name": "Beena D. Kamath-Rayne",
"expertise": [
"Reviewer Expertise neonatal resuscitation",
"Helping Babies Breathe",
"educational outcomes"
],
"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\nLong term retention of neonatal resuscitation skills is an important concept that must be studied as there are many unknown factors that could potentially affect competency and skills retention. Educational efficiency is an important component of the Formula for Survival, which denotes potential survival being a product of medical science x educational efficiency x local implementation. The authors are to be commended to undertaking a study that specifically evaluates retention of skills in this population in South Sudan. However, while the topic is important, the way in which these authors have presented their data and results needs to be improved.\n\nMy interpretation of the study design is that in the intervention hospital, HBB training occurred, and then workshop participants were evaluated pre/post and then at 3 and 12 months with the knowledge and skills-based assessments associated with HBB. In the control hospital, no HBB training was offered. Furthermore, in the intervention hospital, bag-mask ventilation drills were used to augment retention of skills over time.\nThe manuscript could use some copyediting to fix basic grammatical, spelling and word choice mistakes. There is also some confusion about the secondary outcome of neonatal mortality, which the authors refer to as neonatal mortality due to birth asphyxia. In the abstract, for example, this is only called neonatal mortality. If this is asphyxia-related mortality only the authors could do a better job of determining how this specifically was assessed as the cause of death rather that something else. Also, some babies, like premature babies, can have both asphyxia and prematurity as contributors to their deaths. How was this sorted out?\n\nFurther detail on the methods/results would be helpful to improve the readers' interpretation of the investigators' findings. With the delivery room observations and calculation of neonatal mortality, was it known whether the individual leading the resuscitation had been trained in HBB? Given the frequent turnover in such settings, it would be important to know whether the resuscitator was in the study protocol or not. It would also be helpful to know how many individuals in each hospital did not consent to the study; what proportion of health providers potentially eligible for the study in each hospital who take care of newborns were in the study?\nWhat types of health workers were included in the study, and did they have any previous exposure to neonatal resuscitation or simulation? What individuals refused to be in the study or were lost to follow-up? Were they predominantly one type of health provider? Tabangin et al.1 showed that doctors performed better in simulated evaluations (OSCE B) due to having had previous experience with neonatal resuscitation and simulation training, which allowed their performance to improve at a greater level than nurses, who had not had prior experience.\nHow was it known that the daily bag-mask ventilation drills were performed, and that the intervention group received the intervention? The CONSORT diagram was difficult to follow as the numbers did not appear to add up correctly.\nThe authors could be much more rigorous in their literature search to support their arguments. Because of their lack of a complete literature review, the study is not able to fully analyze their results within the context of the previously studies and their nuances. I believe the study was done with HBB 1st Edition materials, however, the correct citation was not reported; there is a publication date of 2014 which would be incorrect for either the 1st or 2nd Edition. This is important to distinguish because the 2nd Edition has greater emphasis about an ongoing system for practice, an improved OSCE, and more standardization about giving feedback and debriefing after the OSCES. References 10 and 11 do not support their points regarding an immediate increase in knowledge and skills showing improvements in neonatal outcome. Some of the most important papers regarding retention of skills both in the short and long term after HBB training are not cited. These are referenced below.\n\nThe findings that the skills declined in the intervention group does not fit with what would be expected nor with what has been seen in previous studies. It also does not fit with neonatal mortality decreasing if the skills are not being performed as well. It would be helpful for the authors to hypothesize why they had such markedly different findings from all the other studies that have been published. It also underscores the importance of knowing whether the intervention such as the ongoing practice and drills were being completed as desired.\n\nIn regards to the greater numbers of babies being \"assisted to breathe\" during the study period - which the authors also referred to as \"resuscitation\" - could they be more specific about what this resuscitation entailed? Some studies have shown that fewer babies require bag-mask ventilation after HBB because more babies are being stimulated appropriately and respond to that alone. If they are referring to bag-mask ventilation, can they comment on whether the preceding steps to bag-mask ventilation were appropriately performed or not, or the health providers just went right to bag-mask ventilation?\nWhile the authors are to be applauded for their efforts on this study, major revisions are required before this study can be considered worthy for indexing. I am concerned that their studies are framed in a dangerous way, to suggest that ongoing practice is not beneficial for retention of skills, that goes against both common sense and what has been shown in other studies. The authors need to provide some justification for these findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-167
|
https://f1000research.com/articles/8-423/v1
|
10 Apr 19
|
{
"type": "Case Report",
"title": "Case Report: Partial nephrectomy in primary renal sarcoma presenting as Wunderlich syndrome; a rare tumour with rare presentation managed atypically",
"authors": [
"Ramanitharan Manikandan",
"Ketan Mehra",
"Lalgudi Narayanan Dorairajan",
"Rajesh Nachiappa Ganesh",
"Sreerag K. Sreenivasan",
"Rajeev Kumar",
"Ramanitharan Manikandan",
"Lalgudi Narayanan Dorairajan",
"Rajesh Nachiappa Ganesh",
"Sreerag K. Sreenivasan",
"Rajeev Kumar"
],
"abstract": "Spontaneous retroperitoneal haemorrhage also called Wunderlich Syndrome (WS) may be caused by various aetiologies. One of the most common causes is renal tumour. Renal sarcoma is a rare cause of WS, and renal sarcoma in itself is a rare entity. In the era of nephron-sparing surgery, optimum management of primary renal sarcoma remains a dilemma as there are limited number of cases available in the literature. Nevertheless, radical nephrectomy remains the recommended treatment, keeping in mind the aggressiveness of the tumour. We report a case of primary undifferentiated renal sarcoma, which presented as WS, and which was managed by partial nephrectomy.",
"keywords": [
"Renal Sarcoma",
"Wunderlich syndrome",
"Partial nephrectomy",
"Undifferentiated sarcoma."
],
"content": "Introduction\n\nWunderlich syndrome (WS) is a life-threatening medical emergency defined as spontaneous non-traumatic bleeding confined to the perinephric region1. It is often characterised by Lenk’s triad consisting of acute flank pain, flank mass and hypovolemic shock2. Various aetiologies, including benign and malignant conditions attributable to renal and extra-renal origin, have been reported to cause WS in the literature3. We report a rare presentation of WS in a young woman secondary to an underlying renal sarcoma, which was managed by robotic assisted laparoscopic partial nephrectomy. Furthermore, this report aims to address the difficulties in preoperative diagnosis and concerns of partial nephrectomy in the setting of renal sarcoma.\n\n\nCase report\n\nA 21-year-old woman presented to the emergency department with a history of severe left flank pain and giddiness. She did not report any history of trauma, but had a past history of hypertension. The patient’s pulse rate was 96/min (reference range: 60–80/minute), blood pressure was 90/60 mmHg (reference range: 120/80 mmHg), haemoglobin was 5 gm% (reference range: 12–15 gm%), and serum creatinine was 1.8 mg/dL (reference range: 0.5–1.1 mg/dL). Ultrasonogram revealed a large left perirenal hematoma of size 8x6 cm. Non contrast CT scan suggested an 8 cm lesion in the left kidney without any other information about the aetiology. The patient received blood transfusions and intravenous fluids.\n\nAs the patient was stable, she was managed conservatively for the resolution of the hematoma to facilitate better surgical planning. After two weeks, the patient underwent MRI in which the hematoma had decreased in size to 5 cm, but the underlying cause could not be ascertained (Figure 1). Keeping in mind that a renal tumour, either benign or malignant, could be the cause of haemorrhage, the patient underwent robotic assisted laparoscopic left partial nephrectomy with guidance of intra-operative ultrasonography to delineate the lesion margins (Figure 2A). The warm ischemia time was 25 min with a blood loss of about 200 ml. The patient was discharged on day three with normal serum creatinine.\n\n(A) Intraoperative image during partial nephrectomy of the left kidney; (B) gross morphology of the resected tumour.\n\nMacroscopically, the tumour was 5 cm in largest dimension, fleshy with yellow-brown appearance possibly due to haemorrhage (Figure 2B). Microscopically, the tumour cells appeared small, round to oval shaped, with scant cytoplasm vesicular chromatin and tiny nucleoli with extensive haemorrhagic areas with hemosiderin laden macrophages. The tumour cells were about 5mm away from the resected margins. The tumour cells were positive for vimentin, BCL2 and FL1, and negative for Pan CK, CK7, CD10, CD31, S100, HMB45, SMA, ER, PR and WT-1, suggestive of “primary undifferentiated renal sarcoma” with hematoma (Figure 3 and Figure 4).\n\n(A) Small round blue tumour cells arranged in sheets and nests infiltrating the adjacent stroma. Numerous hemosiderin laden macrophages are seen at the interface. No viable renal parenchyma is preserved, which is entirely replaced by dense fibrosis. Haematoxylin and eosin stain, x40; (B) Tumour cell morphology at higher magnification with high nuclear cytoplasmic ratio, inconspicuous cytoplasm and occasional mitoses. Haematoxylin and eosin stain, x400; (C) Tumour with adjacent bluish immature myxoid connective tissue. Haematoxylin and eosin stain, x400.\n\n(A) Tumour cells with strong diffuse nuclear expression for FLI1. Immunohistochemistry with DAB counterstain, DAKO monoclonal antibody, x400; (B) Negative staining of tumour cells with CD99. Immunohistochemistry with DAB counterstain, DAKO monoclonal antibody, x400; (C) Strong diffuse staining of tumour cells for Vimentin. Immunohistochemistry with DAB counterstain, DAKO monoclonal antibody, x400; (D) Strong cytoplasmic and Golgi expression of WT1 and no nuclear expression in tumour cells. Immunohistochemistry with DAB counterstain, DAKO monoclonal antibody, x400; (E) Tumour cells showing strong cytoplasmic expression for Bcl2. Immunohistochemistry with DAB counterstain, DAKO monoclonal antibody, x400.\n\nSince there are no definite guidelines regarding the role of partial nephrectomy in the background of renal sarcoma, we offered completion radical nephrectomy taking into account the young age of the patient. But the patient refused radical nephrectomy. Observation with regular follow-up was chosen by the patient as the best course of action. At the last follow-up of 12 months, there is no evidence of recurrence on contrast enhanced computerised tomography scan (Figure 5).\n\n\nDiscussion\n\nIn 1856, Wunderlich first described the condition of spontaneous renal bleeding with dissection of blood into the sub capsular and/or perinephric spaces1. In a series of 165 patients with WS, Zhang et al. observed that renal neoplasm was the most common cause, with angiomyolipoma being the most common neoplasm4. There are only scant reports of renal sarcomas presenting with primarily as WS5,6.\n\nSarcomas of the kidney constitute a heterogeneous group of rare neoplasms with aggressive clinical course and account for < 1% of renal cancers1. Sarcomas usually present at a young age with a large tumour size. Renal sarcomas present with similar clinical and radiologic features as renal cell carcinoma and are rarely suspected pre-operatively7. The 5-year overall survival rate for localised renal sarcoma is 46% in comparison to 8% for metastatic disease8. As this tumour is rare, appropriate guidelines for the management and follow-up have not yet been established.\n\nMoreira et al. performed a study with 489 patients of primary renal sarcoma. Primary treatment modality data was available for 367 patients; 210 (57%) underwent surgery, 51 (14%) underwent only radiation, 46 (13%) had both radiation and surgery, and 60 (16%) received neither radiation or surgery. The authors reported that surgery as the modality of treatment had a lower cancer specific mortality rate as compared to other measures. Nephron Sparing Surgery (NSS) or radical nephrectomy was not segregated in their study8. The role of NSS in renal sarcomas is controversial. Wang et al. reported NSS in a patient with sarcoma with a disease-free interval of 42 months5. Cocuzza et al. suggested that renal sarcomas with a diameter of 5 cm may be considered for NSS9. Satoh et al. reported a case of cystic renal leiomyosarcoma, which was managed by partial nephrectomy with a normal follow up of 44 months10. As long as the surgical margins are negative after NSS, it would be unnecessary to perform salvage radical nephrectomy in patients presumed to be renal cell carcinoma pre-operatively, but ultimately turns out to be a sarcoma in the final histology.\n\nOur case presented as spontaneous renal haemorrhage at a young age. All three imaging modalities (ultrasonography, CT and MRI) were unable to delineate the cause of haemorrhage. In view of renal tumours being the most common cause, partial nephrectomy, through a minimal invasive approach, was accomplished. The final histopathology suggested it was a primary renal undifferentiated sarcoma. We were able to manage a case of renal sarcoma with nephron sparing surgery without disease recurrence on follow up. But due to lack of substantial literature, intense follow-up will be needed.\n\n\nConclusion\n\nRenal sarcoma presenting as Wunderlich syndrome is a rare phenomenon, which can mask its preoperative diagnosis. Due to scarcity of cases, optimum management of renal sarcoma is still debatable. Keeping in mind lack of evidence and aggressive nature of primary renal sarcoma, radical nephrectomy seems to be the optimum treatment. In the era of nephron sparing surgery, partial nephrectomy can be offered to patients of primary renal sarcoma with negative surgical margins.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAlbi G, Del Campo L, Tagarro D: Wünderlich's syndrome: causes, diagnosis and radiological management. Clin Radiol. 2002; 57(9): 840–5. PubMed Abstract | Publisher Full Text\n\nBaishya RK, Dhawan DR, Sabnis RB, et al.: Spontaneous subcapsular renal hematoma: A case report and review of literature. Urol Ann. 2011; 3(1): 44–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMolina Escudero R, Castillo OA: Spontaneous retroperitoneal haemorrhage of renal origin (Wunderlich syndrome): analysis of 8 cases. Arch Esp Urol. 2013; 66(10): 925–9. PubMed Abstract\n\nZhang JQ, Fielding JR, Zou KH: Etiology of spontaneous perirenal hemorrhage: a meta-analysis. J Urol. 2002; 167(4): 1593–6. PubMed Abstract | Publisher Full Text\n\nWang X, Xu R, Yan L, et al.: Adult renal sarcoma: Clinical features and survival in a series of patients treated at a high-volume institution. Urology. 2011; 77(4): 836–41. PubMed Abstract | Publisher Full Text\n\nKim JW, Kim JY, Ahn ST, et al.: Spontaneous perirenal hemorrhage (Wunderlich syndrome): An analysis of 28 cases. Am J Emerg Med. 2019; 37(1): 45–7. PubMed Abstract | Publisher Full Text\n\nWang Z, Zhong Z, Zhu L, et al.: Primary synovial sarcoma of the kidney: A case report. Oncol Lett. 2015; 10(6): 3542–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoreira DM, Gershman B, Thompson RH, et al.: Clinicopathologic characteristics and survival for adult renal sarcoma: A population-based study. Urol Oncol. 2015; 33(12): 505.e15–20. PubMed Abstract | Publisher Full Text\n\nCocuzza M, Arap S, Lucon AM, et al.: Renal leiomyosarcoma treated with partial nephrectomy. Clinics (Sao Paulo). 2005; 60(4): 345–6. PubMed Abstract | Publisher Full Text\n\nSatoh Y, Kakinoki H, Tokuda Y, et al.: Cystic renal leiomyosarcoma treated with partial nephrectomy. UroToday Int J. 2010; 3(4). Publisher Full Text"
}
|
[
{
"id": "46984",
"date": "30 Apr 2019",
"name": "Nikhil Khattar",
"expertise": [
"Reviewer Expertise Reconstructive Urology",
"urinary Incontinence",
"stricture urethra",
"female and functional urology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case report.\nIt will be good if the authors can provide the NCCT picture at initial presentation with hematoma alongside the already provided MRI images.\nThe authors should detail the follow up plan with duration considering the patients young age and propensity of renal sarcomas to recur even after 13-15 years1.\nAre there any preoperative hints to suspect a sarcoma on imaging or with biomarkers published in the literature? A word about the same should find a place in the manuscript.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "46982",
"date": "08 Jul 2019",
"name": "Bipinchandra Pal",
"expertise": [
"Reviewer Expertise Uro-Oncology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLittle more clarification is needed in the radiological findings. How was a plan decided for partial in presence of haematoma? Was a serum creatinine repeated before taking the case for surgery? Initially it was 1.8 mg% and after 3 days of surgery it was normal. Better to write the value. What is the follow up schedule?\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": "50127",
"date": "22 Jul 2019",
"name": "M. Hammad Ather",
"expertise": [
"Reviewer Expertise Urological oncologist (Bladder",
"prostate and kidney cancer)",
"Endourology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 have described a case of young woman presenting with spontaneous rupture of a renal sarcoma managed by RA partial nephrectomy. The management was unconventional as the standard approach to such a case is to manage conservatively as they did in the first few days, waiting for the resolution of hematoma and stabilization of the kidney function. Once the serum creatinine is normal she can be imaged either with contrast enhanced CT or MRU or contrast enhanced MRI. If the diagnosis is still uncertain a percutaneous biopsy and followed by definitive managment. The definitive managment, as the authors also pointed out is radical nephrectomy.\nThe differential diagnoses included anaplastic sarcoma of the kidney (ASK), anaplastic Wilms tumor, mesenchymal chondrosarcoma, sarcomatoid renal cell carcinoma, clear cell sarcoma of the kidney, rhabdoid tumor of the kidney, congenital mesoblastic nephroma, and synovial sarcoma. The managment is highly dependent of the histology(Chen and Liao. Ci Ji Yi Xue Za Zhi. 2019 Apr-Jun;31(2):129-1321)\nMy suggestions to the authors are:\nJustify the indication of RA partial prior to establishing a diagnosis in a stable patient.\n\nDiscuss the histopathological aspects of the tumor and how to differentiate various types of sarcoma.\n\nHow the authors intend to follow up patient considering her young age. Is there a role of re biopsy of the tumor bed, for how long should the CT contrast or MRI scan be done. Are there any recommendations for just doing ultrasound if the CT or MRI are normal after first year.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-423
|
https://f1000research.com/articles/8-83/v1
|
21 Jan 19
|
{
"type": "Research Article",
"title": "A qualitative exploration of factors affecting mothers of malnourished children under 5 years old in Kiribati",
"authors": [
"Antje Reiher",
"Masoud Mohammadnezhad",
"Antje Reiher"
],
"abstract": "Background: In Kiribati, malnutrition is the leading cause of death for children aged less than 5 years. The purpose of this study was to explore contributing factors among mothers of malnourished children under 5 years old in Kiribati. Methods: This qualitative study was conducted in an urban area of South Tarawa among mothers of malnourished children aged less than 5 years in 11 public health centers in 2016. The study included 9 focus group discussions, with a sub sample of 3 to 4 in each group, having a total of 35 participants. Using a semi-structured questionnaire, data was collected and thematic analysis was applied to analysis the data. Results: Seven main themes were identified including; knowledge, behaviors, perceived severity, perceived benefits to action, perceived barriers and cultural related issues. These encompassed a variety of reasons which could explain the malnutrition in children of those particular mothers. Conclusion: In order to tackle malnutrition in Kiribati, it is crucial to identify the main factors that are hindering this preventable disease. This study provides information essential to enhanced decision making, health care delivery planning and has policy implications for the improvement of quality of health care in Kiribati.",
"keywords": [
"Mothers",
"Malnutrition",
"Children under 5 years",
"Qualitative study",
"Kiribati"
],
"content": "Introduction\n\nMalnutrition remains a major problem not only in developing countries, but worldwide affecting mostly children under 5 years old1,2. The first 1,000 days of the child’s life affected by poor nutrition can lead to stunted growth, which is a permanent result and is related with impaired cognitive ability and reduced school and work performance2–6. The consequences of malnutrition are varied and include: increased susceptibility to infection, impaired child development, increased mortality rate and individuals will function in substandard ways7,8. The World Health Organization (WHO) revealed that globally, childhood malnutrition accounts for approximately 35% of all deaths among children under the age of 5 years9. A report on malnutrition has stated that severe malnourished children have a higher risk of death from ordinary childhood illness such as diarrhea, pneumonia10.\n\nMalnutrition amongst children under 5 years old is a major public health issue, particularly on the main island of South Tarawa in the Republic of Kiribati with nearly 15% of the children underweight and with considerable gaps in immunization11. Globally, Kiribati contributed to 28% of all under 5 deaths, with 15% of under 5 year old due to severe malnutrition12.\n\nUnited Nations Children’s Fund (UNICEF) statistics show that the total population of Kiribati under 5 was 11,000, of which there are 6,934 children aged 0-4 on South Tarawa alone - this is almost 14% of the total population13. Kiribati faces challenges to improve its nation’s health-care as malnutrition and diarrhea remain major problems11. The 2009 Demographic health survey reported 23% of children are underweight or severely underweight14. This figure positioned Kiribati above the WHO threshold (10%), making the prevalence of underweight children a significant public health issue. More children are underweight amongst the poorer households than in wealthier ones15. Therefore, it is important for the health system to detect malnutrition at an early stage for planning and implementing timely interventions at the community level.\n\nLloyd stated that globally, mothers are charged with the task of feeding and providing care for their children, irrespective of the environment or resources available to them16. It is important to recognize that many interventions to improve child health and nutritional status depend on someone’s behavior, frequently the mother17. It is vital to study the knowledge, attitude and practice (KAP) of mothers regarding breastfeeding, complementary feeding, dietary practices and immunization, as these factors have a huge impact on young children’s health. Therefore, mothers as the primary caretakers for young children need to be knowledgeable in looking after their children, especially earlier in life.\n\nConsequently, this study aims to identify the KAP of mothers of malnourished children (under 5 years old) with respect to contributing factors to malnutrition (breastfeeding, complementary feeding, dietary practice and immunization) on South Tarawa in Kiribati.\n\n\nMethods\n\nA qualitative study design was conducted for almost 8 weeks from 21st of December 2015 to 12th of February 2016, to determine the level of mother’s KAP on breastfeeding, weaning, dietary and immunization on South Tarawa (capital island of Kiribati). Tarawa is the capital island of Kiribati and is divided into two parts, North and South Tarawa, with South Tarawa being the only part of Kiribati considered ‘urban’. The focus group discussion (FGD) method was used which allowed participants to discuss problems in a group setting and allowed more detailed responses.\n\nThe FGD was held at nine different public health clinics on South Tarawa during working hours, as ‘saturation’ was reached after nine FGDs.\n\nThe inclusion criteria were all women with children under 5 years old who were identified as being malnourished and were admitted to the Pediatric wards of selected public health clinics within the period of 1st January to 31st May 2015. The study participants had to be Kiribati citizens (self-identified) and their children’s names must be registered under the public health clinics. The exclusion criteria were mothers who didn’t meet the study inclusion criteria and were not willing to participate in the FGD, mothers or children who were not residents on South Tarawa and mothers with children aged more than 5 years old. Critical stage children that were admitted to the Pediatric ward were also not included.\n\nA non-probability purposive sampling method was used to recruit participants for nine FGDs. Each group contained 3 to 4 participants, with a total sample size of 35 participating mothers.\n\nA semi-structured questionnaire was used in this study whereby there were two open-ended questions allocated for each of the factors (breastfeeding, complementary feeding, dietary practice and immunization). Prior to collecting the data, the questionnaire was validated using two group discussions, during child health care visits. The participants who met the inclusion criteria were selected and given the questionnaire to answer to ensure the questions were understandable and readable for face validity. Content validity was also used whereby three academic experts (assistant professors and lecturers at the Public Health at Fiji National University) assessed the construction in question and checked whether the responses by the person answering the questions were affected by other factors. Once done, their comments were revised accordingly and a new version of the questionnaire was then distributed among the participants.\n\nMothers that were interested to participate in the study then signed a consent form to proceed with the discussion. The places and time of the discussions were chosen by the researcher that was convenient to the participants. All participants sat together in an isolated room in each public health clinic, in a circle, so the main researcher could have equal access with the voice recording. The researcher, who had previous experiences in conducting FGDs, and was bilingual, was present and available to translate the questions from English to Kiribati language for participants to have a clear understanding of the questions raised during discussion. The discussions were in the local language (Kiribati) and both audio recording and note taking were used.\n\nThe discussion began informally with the greetings and personal instructions from the researcher. When all participants were settled and ready to start, the researcher led the discussion by starting with a question for each topic on breastfeeding, complementary feeding, dietary practice and immunization. The participants in each group mingled well with each other so they communicated freely with one another. Each FGD had an average duration of 60 minutes. All conversations during the FGDs were conducted in Kiribati language so that participants were able to talk freely without any restrictions in expressing their opinions.\n\nQuestions were as follows:\n\nBreastfeeding:\n\n1. What are your perceptions on breastfeeding?\n\n2. What problems you faced while breastfeeding and how did you overcome it?\n\nWeaning:\n\n1. How do you know it is right to start weaning and how do you approach it?\n\n2. What foods do you give your baby when weaning, home-made foods or store-bought foods and why?\n\nDiet:\n\n1. What are the obstacles you (mothers) you face in making recommended diet for your children?\n\n2. As your baby gets older what do you think will be the key things that you will be concerned about diet wise?\n\nImmunization:\n\n1. What are your perceptions on immunization? Why are children immunized?\n\n2. What are obstacles to immunizing your children and why?\n\nThe researcher transcribed all digital recordings of the focus groups in Kiribati language, then translated these transcripts into English language. Transcripts and field notes were compared to ensure that no information was misinterpreted or omitted.\n\nData were then analyzed manually, by the author, to discover inter-rater coding dependability. After the first review of the data, a categorizing method was developed by using thematic analysis based on the study objectives. Then the researcher identified themes and sub-themes developed from the data analysis and lastly produced a final thematic index to code the data. The researcher listened to the recordings and read the written notes numerous times to understand the participants’ views and ideas they expressed through their own words. Data were then interpreted and conclusions were drawn based on coding summaries and contextual field notes provided by direct quotes from participants. Codes were then grouped into themes which can be defined as repeated features or patterns.\n\nThe ethical approval for this study was received from the Kiribati Ministry of Health after the research proposal was approved by Fiji National University’s College Health Research Ethics Committee (CHREC).\n\n\nResults\n\nSeven main themes were identified: knowledge, behaviors, perceived severity, perceived benefit to action, perceived barriers, cues to action and cultural related issues.\n\nKnowledge and understanding from experiences is another way to carry out things in our daily lives. When mothers were asked about when was the right time to start complementary feeding, only two groups mentioned that complementary feeding should be started when the child is refusing to breastfeed.\n\nI start complementary feeding because my child refuses to breastfeed and crying most of time and when I start feeding him I knew that he want to eat more than to breastfeed, so I think that is a good sign for me to start giving complementary food to my child. (FGD 5).\n\nFour different groups were not sure about the right age to start complementary feeding. One mother from one group said that 1 year old is the right age to start complementary feeding, whereas others said it was 2 years old, 3 years old and 4 years old.\n\nMothers provided different ranges of knowledge when asked about their perception on breastfeeding. Most of the groups (6 groups) stated that breastfeeding will prevent disease to their child and make the child healthier (5 groups). One participant said that during antenatal and postnatal clinic, she always got advised by the nurses about the importance of breastfeeding;\n\nI always breastfeed all my children because I know it is good to prevent them from disease but also because I do not want to risk my child life and to blame myself for it as I already know the importance of breastfeeding from the nurse. (FGD 4)\n\nWhen mothers were asked about their perception on immunization, a few of the groups (4 groups) stated that immunization was free of charge. One participant mentioned that immunization is important to her child and it is a service that is provided so you do not have to spend money on it:\n\nI understand that immunization does not cost any money and nurses were willing to visit us at home when you did not come to take our child for immunization. It is the best opportunity for our children and we supposed to make use of it. (FGD 9)\n\nMothers had different knowledge when asked about the key things on diet when their child becomes older. Most of the groups (7 groups) reported that more solid food and a balanced diet should be introduced when their child is older. They are more knowledgeable on the types of food to give their children because they were given a pamphlet during the child health clinic to guide them with feeding their child and from their past experiences from previous children. One mother pointed out that nurses play a major role in assisting mothers to know how to provide the proper food to their children:\n\nI am very thankful that nurses are very concern about our children and to make sure we are giving them the right food. Health education they give us with pamphlet to take home is of good benefit to our children but it is then over to us mothers on how to practice this at home is a different story. (FGD 3)\n\nMany of the FDGs reported that forgetfulness was continuously the reason for not immunizing their children on schedule. This was because they were not concerned about the effect of not being immunized would have on the children. They thought it was alright to miss their child’s vaccines and when they remember next time they will go and attend immunization clinic.\n\nSometimes I forgot to take my child for injection because when I was given an appointment card and when I get home I just leave it lying anywhere and then I forgot the time to come back to the clinic. I will find another time that suit me to go to the clinic and take my child for injection when I remember. (FGD 1)\n\nMothers had different behaviors when asked about breastfeeding. Most groups (5 groups) stated when they were faced with breast milk insufficiency the only option is to give formula milk straight away. One participant claims that because she does not want her child to get hungry and cry she had no choice but to give formula milk.\n\nEven though I understand the importance of breastfeeding, I cannot stand to see my child crying and because I understand that there is a formula milk sold for babies, my only option is to buy it and give to my baby. (FGD 8)\n\nIn regards to immunization, most of the mothers (7 groups) had poor behaviors in getting their child immunized with forgetfulness as one of their obstacles in fulfilling a good immunization record. One participant admitted that forgetfulness is a common excuse they will provide for not attending to their child immunization.\n\nI am always worried of what to say to the nurse the next time I visited the clinic for my child immunization, but because I was not ready to take my child for immunization at the certain booked times. I always make excuse and tell the nurse that I forgot about the immunization appointment given to me. I experienced that the only way to stop the nurse from getting upset with us for not attending immunization is forgetfulness. (FGD 3)\n\nWhen mothers responded to a question on what obstacles they faced in immunizing their children, most of the focus groups (5 groups) stated that mothers worried that their child will get a fever after receiving an immunization. One mother explained that they do not want to take their child for immunization because they feel sorry for their child and they do not want to stay up and cool sponge the child when he/she gets a fever:\n\nI do not mind taking my child for injection because I know that it is important for him but the problem is that I am not accepting the facts that I do not have to sleep well and look after my child when he gets a fever. (FGD 2)\n\nOut of the nine FGDs, most groups (6 groups) believed that breastfeeding prevented disease in the child. Most mothers continue to breastfeed their children because they are advised during antenatal and postnatal clinics that breastfeeding is important for their children because it will protect them from infectious diseases. They had the knowledge that breastfeeding is critical, especially during their child’s young age. In one group, the mother shared her experience and compared her situation with one of her siblings that did not breastfeed her children.\n\nBreastfeeding is good for the child because it protecting the child from infectious disease. I experienced this because my younger sister never breastfeed all her two children and these children are always sick when there is an outbreak of diarrhea they get it and having a recurrent episode of pneumonia as well. I feel sorry for these children. (FGD 1)\n\nWhen they were asked about their perception on immunization and why their child is immunized, all nine FGDs mentioned that immunization is preventing disease to young children. However, mothers had problems getting the child to their appointment for immunization because they were too lazy to walk to the health clinic. They are expecting the nurse to visit them at home to give immunization injections. One of the mothers in a group explained that they continue to immunize their child because they know the importance and benefits of it to their children.\n\nI just knew that after the completion of our injection, I just understand that the injection really protected them from getting the diseases. But the big issue is how to get there, that is why we cannot complete our child’s injection on schedule. I think it is related with our poor attitude of laziness to get to a far place. (FGD 3)\n\nChildren are giving injection to make a child healthy and prevent them from disease and also prevent them from getting blindness. (FGD 6)\n\nImmunization is good for my child because it prevents disease and also when your child get the disease it not as critical and it just hit your child and gone. (FG5)\n\nIn responding to the question about their perception on breastfeeding, four focus groups said that breastfeeding is convenient and enhances the child’s learning ability. They explained that breast milk, food for their baby, is always available at any time, and they do not have to do much work to prepare it for their child. Mothers also mentioned that breastfeeding enhances the child’s learning ability, as it is good for the child’s brain. They understand that a breastfed child is more intelligent in school compared to someone that was not breastfed. One participant stated that when she goes somewhere with her baby she does not have to worry about taking a lot of things as breast milk is always available and convenient:\n\nI really agree that breastfeeding is available and convenient for our baby, I do not like carrying so many things plus my child when I went to visit my families, that is why I always breastfeed all my children. (FGD 3)\n\nAmong the nine FGDs it was found that there were barriers to action, such as one’s belief in the physical and psychological costs of the advised behavior. When mothers were asked about problems they faced while breastfeeding many groups reported that they were having problems with breast milk insufficiency. One participant said that she is not getting enough breast milk when she breastfed her child and she got advice from the nurse to start giving formula milk while continuing with breastfeeding.\n\nI gave birth in the hospital and my breast milk is not enough so the nurse advised me to give formula milk and to continue with to breast feed milk because my child is born prematurely. (FGD 7)\n\nTwo focus groups reported that breast abscess, the child refusing, and no breast milk were problems they faced while breastfeeding. One participant mentioned that she had a breast abscess in one breast and she knew that she was not giving as much breast milk to her child as when she was giving both breasts to feed her child.\n\nI did not breastfeed my child well because I had breast abscess so only 1 breast to give to my child, this always happens when I got pregnant. Therefore, I knew that I did not give enough breast milk to my child, but I keep breastfeed with only 1 breast. (FGD 5)\n\nMost of the mothers (6 groups) mentioned that breast milk insufficiency is another barrier they are facing with breastfeeding. One participant from two different groups stated that they did not have breast milk at all after delivery and they felt bad for their child because she was always crying.\n\nI am not sure what happen but I keep breastfeeding my child after birth and I was unable to produce milk, my child is always crying and my mother give me local medicine to get the breast milk out but still not coming out, so I started to give formula milk as I feel sorry for my baby. (FGD 7)\n\nMothers are facing obstacles to immunizing their children. A few of the groups indicated that these obstacles included a family problem, loss of their child immunization card and moving to a new place. They stated that when they are fighting or arguing with their husband it affects everything and they are not prioritizing what is best for their child. Another view is that when they lose their immunization card, they are scared to attend immunization clinic as the nurse will scold them for losing an important card so they stay home. When mothers move to a new place and they are not sure where to take their child for immunizations, they miss taking them for immunization.\n\nThere is one tough old nurse in the health clinic that everyone is always scared of. One time I lost my child immunization card and she told me off that I cannot take responsibility and look after my child cards and from that time I felt embarrassed because she scolded at me in front of other people so I prefer to stay home with my child after that. (FGD 9)\n\nThe public health nurses’ poor attitude contributed a lot to the barriers for mothers to attend immunization clinics. Two different responses from mothers said that once they miss their child immunization they will not go again. This happens because nurses are not welcoming or encouraging mothers to attend a regular immunization clinic. One participant said that nurses should be politer when talking to mothers because they are causing mothers to be reluctant about getting their child’s immunizations.\n\nI wish to have a good nurse in our clinic that can accept our failures and encourage us to bring our child to immunization clinic more often without any restrictions. (FGD 3)\n\nOne participant had stated she missed taking her child for immunization because of advice from other people, especially old people. She was told that there is no point of getting immunizations because the other generations did not receive any immunization and they are still healthy and surviving till now.\n\nI was told from my grandfather that they is no such thing as immunization, and I should not be taking my child for immunization. It is giving lots of bad things to the child such as pain, fever and there is no use. (FGD 2)\n\nIn responding to a question about “what are the obstacles they are facing in making a recommended diet for their children?” most groups (5 groups) stated that they cannot give proper food to their children because of peer group advice. Most mothers said that rumors from other mothers in the community affected the way we feed our children. One participant said they were advised to control the food you give to your child while they are young because when they grow up it is hard to control them.\n\nI always had this advice in mind that I should give less food to my child and not to feed them lots of protein foods because they will grow fat in their young age and it will not be healthy for them when they get older. Therefore, I tried to limit the food I give to my children because of the advice from other mothers. (FGD 3)\n\nAccording to the mothers’ FGD, five groups stated that children should be given complementary foods when the mother is pregnant again or if the child is having diarrhea after breastfeeding. It is common that when the mother is pregnant the grandmother makes sure the child is not breastfed again. One of the mothers shared her experience with her child when she got pregnant again.\n\nI started giving complementary feeding to my child when I fall pregnant again and my mother advised me to stop breastfeeding otherwise my child is weak or not as active. I knew it is our culture so she took my child away from every time she saw me breastfeeding and she is very upset with me, I knew that our mothers are very controlling especially when to do with their grandchildren. (FGD 5)\n\nAnother issue with culture is that people are living in extended families and the appropriate food that should be given to young children was affected by it. When asked about “obstacles faced in making a recommended diet for their children”, three focus groups described that they cannot give special food to their young children because there are so many people living in one home. One of the respondents claimed that they feel sorry for their young children because it is not fair for them to be treated the same way as adults.\n\nI always feel bad and wish that one time will be stayed alone with our children in our house, but it is hard because we do not want our relatives to think that we are not welcoming them to our home and they might be talking bad about us. It is so hard but our young children are suffering a lot from this. (FGD 4)\n\n\nDiscussion\n\nOur study showed that mothers were not aware of a balanced diet and most of them were ignorant on what a balanced diet is that should be given to their child. To prevent unhealthy weight gain among children it is necessary to reduce total fat to less than 30% of total energy intake18. Mothers need to enhance their knowledge on diet and understand the proper diet or balanced diet to be given to their children and what portions should be given to a grown-up child. It is vital that mothers have a different way of thinking towards the right types of food to give to their children. “A low level of maternal education and a lack of knowledge on good childcare practices means children do not receive optimal nutrition and care”19. Even when mothers do know about the importance of breastfeeding, complementary feeding, balanced diet, and immunization, the lack of progress is more complex. These factors are the role of a mother.\n\nHowever, health workers should consider that lack of awareness is not the only barrier; habit, socio-cultural constraints, and social norms make it difficult to change20. During child health care clinics, health professionals should conduct health awareness to enhance mother’s knowledge on a healthy, balanced diet through different stages in the child’s life. According to Briscoe and Aboud, in order to change the practice of mothers or caregivers, an active learning approach is considered for the implication that a trained worker needs to demonstrate and caregivers need to practice21. This strategy is more effective than reading information alone.\n\nMother’s perception of breastfeeding in the present FGDs showed that many of the groups knew breastfeeding was helping to prevent disease and make the child healthier. Nurses were the major sources of information during ante-natal clinic. As mentioned by the WHO, breastfeeding is a natural way of providing young infants with the nutrients they need for healthy growth and development22. The information mothers receive during antenatal care might encourage them to breastfeed their children longer.\n\nThe complementary feeding time stated by mothers was when the child refused to breastfeed and the recommended age for complementary feeding varied between 1 to 4 years. It is not recommended to begin weaning before 6 months of age, but it is recommended to begin adding complementary foods at 6 months because breastfeeding alone will no longer fully meet the nutritional needs of the child11. Therefore, the type and timing of weaning foods introduced in an infant’s diet have a great impact on the child’s nutritional status23.\n\nAs recommended by the WHO, infants should be exclusively breastfed for the first 6 months of life to achieve optimal growth, development and health11. Subsequently, to meet their changing nutritional requirements, infants should be given nutritionally sufficient and safe complementary foods, while breastfeeding remains for up to 2 years of age or beyond22. In a different study by Berra among 240 mothers of under 5 children residing in Nekemte town, Ethiopia, it is stated that most mothers practice early weaning because of insufficient breast milk24. Mothers had different practices and reasons for weaning, which might indicate that their knowledge on weaning is not adequate and needs to be targeted by health professional awareness programs.\n\nMothers claimed that immunizations had two perceived severities which cause mothers to not take their child for injection: pain and fever. Mothers had bad experiences after taking their child for immunization when they got swelling at the injection site and the baby cried a lot. These bad experiences from poor practice of health workers contributed to the mother not taking their child for immunization. Health workers should be encouraged and updated on standard procedures to give injections to avoid poor practice as it showed a poor impact on children immunization records. Additionally, health workers, especially in the public health clinic, should reassure mothers about the side effects of immunization and inform them, prior to injection, that the fever will disappear in one or two days and it shows that the immunization given is working properly.\n\nIn this present study, mothers’ perceived benefits towards breastfeeding were that they knew and understood that breastfeeding is beneficial to their children. A similar study by Andrade et al. was conducted in a health center in South Brazil which stated the reason the mothers comply to vaccination was because it helped prevent the child from getting various diseases25.\n\nWhen dealing with food, most mothers perceived benefits to homemade foods because of its freshness and being healthy and part of a balanced diet, as they can add their green leaves to it. Their good perception is not related to what they practice at home, though. They may be struggling to get healthy food for their children due to living in extended families with only one or two people being employed. The government could help find ways to involve people with planting green leaves and vegetables for their families, especially the young children for their growth and development. A similar study in rural Africa found that inadequacy of resources is perceived to be another contributing factor to under-nutrition and hence the intervention dealt with resources that were available and any constraint in accessing to them for future planning26.\n\nIn a setting such as Kiribati, culture dominates and hence things could be changed in the opposite direction which could be negative or positive. In the present study, the breastfeeding and complementary feeding of children was affected by the Kiribati culture. When the mother is pregnant again the grandmother is involved with breastfeeding cessation and introducing alternative milk to the child. The local thought is that when the mother is pregnant her milk is not good and it makes the child develop diarrhea, from which the child can then become very sick. This is a similar finding to a study by Kakute et al., which identified cultural factors influencing decisions of mothers in feeding their children27.\n\nIn a similar study by Abubakar et al., which was conducted in Kilifi District, on the Kenyan coast, a total of ten FGDs were undertaken with eight to ten mothers in each group. The result reported that in all FGDs, mothers were constrained in their ability to provide optimal care due to the social setting in which they raise their children. One common theme mentioned was living in large households where everything was shared with members of the household and those visiting26.\n\nIn this study, older people are a higher priority when it comes to food than the younger ones. If there is good food, it is kept aside for the older people to eat. The thinking is that they are weak and respected so they offer what is best for them. This cultural practice is common in Kiribati settings and it is a huge contributor to a child’s feeding. These cultures are passed on from generation to generation and it is very hard to change this thinking, but there are ways to try and educate them and slowly, in a manner that they will accept, so that the older people are still shown the necessary respect needed in their culture. The intervention for culture is to involve older people in health talks, not only mothers but also the father and grandparents, as they are the people that need to support the mothers and children. It is best to involve them and have them be part of health promotion and to try and change the way of thinking about certain thing, especially with child’s feedings.\n\n\nConclusion\n\nThe themes raised from the mother’s responses were knowledge, behaviors, perceived severity, perceived benefits, perceived barriers, cues to action and cultural related issues. It is confirmed from this study that in order to efficiently and effectively decrease the incidence of malnutrition among children residing on South Tarawa, mother’s knowledge need to be enhanced, attitudes need to be changed, as well as developing practices to have a very clear role and understanding on breastfeeding, weaning, diet and immunization. This could be fulfilled with the assistance from health professionals, the ministry of health, community and different stakeholders, for a bright future for the young generation of Kiribati who are the future assets of tomorrow. The cultural related issues mostly had an impact on dietary, as they are not prioritizing children’s feedings as important due to men and old people benefitting more from special food.\n\nParticipants recognized the benefits of a healthy diet and the positive effects on children. However, due to their status in the family, work and domestic responsibilities along with limited financial resources, healthy and adequate maternal nutrition is not a priority. Improving education for young adolescent girls is key to improving women’s status in society and in their family. Furthermore, educating women offers them the skills to think freely, be involved in the workforce and practice eating habits that benefit not only themselves, but also their future children.\n\n\nEthical statement\n\nThe ethical approval for this study was received from the Kiribati Ministry of Health after the research proposal was approved by Fiji National University’s College Health Research Ethics Committee (CHREC). All participants provided written informed consent to participate in the study.\n\n\nData availability\n\nAs the transcripts of the interviews encompass identifiable information and therefore the data are not openly provided. Limited de-identified transcripts of the interviews performed with the mothers (in Kiribati and English) are available on request from the corresponding author (masoud.m@fnu.ac.fj).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe are extremely grateful to the participating women who gave so freely of their time and without their support this research would not have been possible. We would also like to extend our gratefulness to all nurses and village nurses in the public health clinics for their time in informing mothers to attend the interview.\n\n\nReferences\n\nRice AL, Sacco L, Hyder A, et al.: Malnutrition as an underlying cause of childhood deaths associated with infectious diseases in developing countries. Bull World Health Organ. 2000; 78(10): 1207–21. PubMed Abstract | Free Full Text\n\nBlack RE, Victora CG, Walker SP, et al.: Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet. 2013; 382(9890): 427–51. PubMed Abstract | Publisher Full Text\n\nHoddinott J, Alderman H, Behrman JR, et al.: The economic rationale for investing in stunting reduction. Matern Child Nutr. 2013; 9 Suppl 2: 69–82. PubMed Abstract | Publisher Full Text\n\nRuel MT, Alderman H; Maternal and Child Nutrition Study Group: Nutrition-sensitive interventions and programmes: how can they help to accelerate progress in improving maternal and child nutrition? Lancet. 2013; 382(9891): 536–51. PubMed Abstract | Publisher Full Text\n\nGrantham-McGregor SM, Fernald LC, Kagawa RM, et al.: Effects of integrated child development and nutrition interventions on child development and nutritional status. Ann N Y Acad Sci. 2014; 1308(1): 11–32. PubMed Abstract | Publisher Full Text\n\nBritto PR, Lye SJ, Proulx K, et al.: Nurturing care: promoting early childhood development. Lancet. 2017; 389(10064): 91–102. PubMed Abstract | Publisher Full Text\n\nRodríguez L, Cervantes E, Ortiz R: Malnutrition and gastrointestinal and respiratory infections in children: a public health problem. Int J Environ Res Public Health. 2011; 8(4): 1174–1205. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHien NN, Kam S: Nutritional status and the characteristics related to malnutrition in children under five years of age in Nghean, Vietnam. J Prev Med Public Health. 2008; 41(4): 232–40. PubMed Abstract | Publisher Full Text\n\nWorld Health Organisation: Global Database on Child Growth and Malnutrition. 2016. Reference Source\n\nSalam RA, Das JK, Bhutta ZA: Current issues and priorities in childhood nutrition, growth, and infections. J Nutr. 2015; 145(5):1116S–1122S. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKiribati National Statistics Office: Kiribati Demographic and Health Survey. 2010. Reference Source\n\nDepartment of Foreign Affairs and Trade: Kiribati Program Poverty Assessment. 2014. Reference Source\n\nUNICEF. (2013b): Kiribati. 2013. Reference Source\n\nKiribati Health Information System: Leading cause of Death for children under 5 years. Ministry of Health. Kiribati. 2014.\n\nUnited Nations International Children's Emergency Fund: Improving child nutrition: the achievable imperative for global progress. New York: UNICEF. 2013. Reference Source\n\nLloyd A: Maternal knowledge, attitudes and practices and health outcomes of their preschool-age children in urban and rural Karnataka, India. 2009. Reference Source\n\nBrown K, Dewey K, Allen L: Complementary feeding of young children in developing countries: a review of current scientific knowledge. 1998. Reference Source\n\nHooper L, Abdelhamid A, Moore HJ, et al.: Effect of reducing total fat intake on body weight: systematic review and meta-analysis of randomised controlled trials and cohort studies. BMJ. 2012; 345: e7666. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUnited Nations International Children's Emergency Fund: Changing lives a Portrait of Children in Malawi. 2010. Reference Source\n\nAffleck W, Pelto G: Caregivers' responses to an intervention to improve young child feeding behaviors in rural Bangladesh: a mixed method study of the facilitators and barriers to change. Soc Sci Med. 2012; 75(4): 651–8. PubMed Abstract | Publisher Full Text\n\nBriscoe C, Aboud F: Behaviour change communication targeting four health behaviours in developing countries: a review of change techniques. Soc Sci Med. 2012; 75(4): 612–21. PubMed Abstract | Publisher Full Text\n\nWorld Health Organisation: Breastfeeding. 2016. Reference Source\n\nChoudhari S, Mudey A, Joge U, et al.: Weaning and supplementary practicesimpressions from a rural community. Indian J of Maternal and Child Health. 2012; 14(1): 1–9.\n\nBerra WG: Knowledge, Perception and Practice of Mothers/Caretakers and Family’s regarding Child Nutrition (under 5 years of age) in Nekemte Town, Ethiopia. Sci Technol Arts Res J. 2013; 2(4): 78–86. Publisher Full Text\n\nAndrade DRS, Lorenzini E, Silva EF: Mothers’ knowledge regarding the vaccination program and factors which lead to delays in infant vaccination. Cogitare Enferm. 2014; 19(1): 96–102. Reference Source\n\nAbubakar A, Holding P, Mwangome M, et al.: Maternal perceptions of factors contributing to severe under-nutrition among children in a rural African setting. Rural and remote health. 2011; 11(1): 1423. PubMed Abstract | Free Full Text\n\nKakute PN, Ngum J, Mitchell P, et al.: Cultural barriers to exclusive breastfeeding by mothers in a rural area of Cameroon, Africa. Journal of midwifery & women’s health. 2005; 50(4): 324–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "43341",
"date": "18 Feb 2019",
"name": "John F. Smith",
"expertise": [
"Reviewer Expertise Public health/developing countries",
"qualitative research",
"social determinants of health",
"health development",
"health promotion"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis modest research project is on a topical and critical issue for many in a wide range of developing countries, effecting not only the developmental trajectory of individual children themselves but also their potential future contributions to their societies.\nThe paper is clearly written and very readable throughout. The Method section is clear and replicable. Themes from qualitative data analysis are succintly expressed in the Results.\n\nThe Introduction emphasizes the mother's primary responsibility for care/feeding of children and identifying maternal KAPs in respect to child malnourishment. Not downplaying this, but there are also other broader system factors (determinants) that often influence mothers' health/capacity to provide nourishment for small children, e.g., work/life balance for the mother--does she, by necessity, have to leave the infant in care of others while working during the day?, food security? quality and quantity of good food for the mother's to underpin quality breastfeeding, economic factors impinging on access to good food for the mother etc. Some of these are alluded to in the results under later themes.\n\nThere appears to have been little in the FGD process to prompt for understandings of these broader social/economic/cultural variables role in malnourishment. Some allusion to these does come up in the Discussion section but study would have been stronger if these addressed directly via FGD data collection phase. KAP studies typically produce recommendations for strengthening individual's knowledge, behaviour, practices etc via health education, literacy interventions, decontextualised from important broader social/economic, cultural structural variables outside any one individual's capacity to change and that require different intervention approaches. A broader health development/social determinants - driven approach may be needed rather than focusing on individuals skills knowledge etc.\nNotwithstanding the above still a useful, albeit modest, study on this important topic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4544",
"date": "15 May 2019",
"name": "Masoud Mohammadnezhad",
"role": "Author Response",
"response": "We really appreciate your time for reviewing our paper. We tried to cover your comments. We reported all the themes were emerged in this study however we also believe there are other factors influencing children malnutrition especially in small Pacific countries such Kiribati. Some recommendation about considering cultural believes, role of economic status, and role of government were already provided and other recommendation on broader health development/social determinants has been added to the conclusion section."
}
]
},
{
"id": "43343",
"date": "27 Feb 2019",
"name": "Sari Andajani",
"expertise": [
"Reviewer Expertise Public health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is an original work. The study rationale is sound, the underdeveloped introduction has made it hard for the reviewers to see how this study differ to the previous ones. As findings and discussion are suggestive of the use of Transtheoretical Theory, it needs to be included in the introduction chapter. Findings are presented as series of quotes and at a few points were confusing for readers. I suggest rework of the finding sections, and give signposting to each section. For example for Behavioural factor, states clearly that behaviour factors including mothers' time restriction, conflict of interest, or not keeping up scheduled immunization. Very interesting and specific cultural context was presented in the finding and discussion, unfortunately it has not been described earlier in the study sample. I suggest to add a small Pará on participants context explaining who there are, family, structure, and other relevant context which may support or hinder immunization. A couple of notes on the methods section re 'saturation\" nature, purpose of saturation shall be operationalised in a way that is consistent with the research question and theoretical position (in this case seems like the Transtheoretical theory).\nRe: Inter rater reliability to establish consistency of findings. In Qualitative research do not make explicit this concept, however implicitly they can provide descriptions of the procedures for carrying out the analysis. Needs to be referenced and mentioned, saturation to which study question?\nRe: inter-rater reliability, in a qualitative research it is implicitly mention as a process of which are followed by both of the authors. For example meetings of two researchers' to discuss and negotiate agreements and disagreements about coding. Or codings were brainstormed to put the first order statement from respondents and agree them.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4545",
"date": "15 May 2019",
"name": "Masoud Mohammadnezhad",
"role": "Author Response",
"response": "We really appreciate all your time for reviewing our paper. We tried to cover your comments. There was no any study conducted in Kiribati earlier so it was difficult to foreseen what model or theory will be developed. The themes were emerged in this study are unique. Based on the results of the study, the main elements of Health belief Model (HBM) was developed so we added a sentence in “Discussion” to highlight the importance of developing an intervention using HBM. For the behavioral factor- The main behavioral factor was “forgetfulness” which can be due to time restriction or not keeping up scheduled immunization. We added a paragraph in “introduction” about cultural context of Kiribati people. Information about data saturation is added. For inter-rater coding dependability - the process of emerging codes has been explained in the methodology section. All has been done by the main researcher by reading and re-reading transcripts to identify themes and sub-themes developed from the data analysis and lastly produced a final thematic index to code the data."
}
]
},
{
"id": "43339",
"date": "04 Mar 2019",
"name": "Diana H Arabiat",
"expertise": [
"Reviewer Expertise Qualitative research",
"child health and development",
"multicultural research."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis topic is both timely and relevant, since most of children death takes place in the developing world, and there is tremendous need to educate parents about balanced nutrition in children and when to seek medical help to prevent serious sequelae. Moreover, more information about Kiribati is also welcome in the recent literature, since this society is sorely underrepresented.\nThe followings are suggestions for manuscript revision:\nTitle The study topic is more related with ‘Nutritional knowledge, attitude and dietary practices of mothers of malnourished children in Kiribati’, so the authors should consider revising the study title.\n\nAbstract The abstract needs editing and reconstruction, in particular with relation to the study aim listed under background. The authors stated exploring factors contributing to malnourishment in children aged below 5 years old, yet the study did not explore demographic or socio-economic factors in mothers that may contribute to malnourishment in children. I would suggest revising the aim into exploring nutritional knowledge, attitude and dietary practices of mothers with malnourished children under 5 years old. The study aim should be listed in a constant way in abstract, methods and discussion. As with regard to the results, I would suggest revising and editing the themes accordingly to the major theme of nutritional knowledge, attitude and dietary practices and then to include related subthemes (e.g., breastfeeding practices, complementary feeding’ …etc) under the major themes listed in the study.\n\nMethods It is unusual to use a semi-structured questionnaire for a qualitative design, so I wonder if the author intended to describe the interview guide used for promoting the FG discussion around dietary practices. If so, this section needs to be revised accordingly. Further information about who facilitate the FG discussion, in what language need to be added. In page 4, ‘isolated room’ should be changed into ‘private room’. The section related with semi-structured questionnaire needs to be deleted and an example of the questions used in the interview guide can be included within the text. Questions related with immunization is not directly related with the study aim, yet it can be added as a theme emerged or noticed among mothers of malnourished children.\nResults I found the presenting themes confused and confusing. There is a need first to elaborate on the thematic analysis process used in transcribing the FGS and who and how those themes were generated. This should be added as a heading named ‘Analysis of data’ in the methods section. Another section should be added under results and this should include the socio-demographic characteristic of the sample.\n\nSecond, it is important to refine the major key themes emerged in the data analysis along with the subthemes and selected quotations this can be also presented under a table. Significantly, the authors listed attitudes and practices related with immunisation, yet they did not link it directly with mothers’ knowledge or perception of malnourishment in child. Failing to highlight this relation will risk the overall quality of study.\nThird, findings related with perceived barriers to action can be listed nicely as a subtheme for the major theme of ‘dietary practices’. The authors can refine their findings presentation to list beliefs related with breastmilk insufficiency as a contributing factors that negatively impact child’s health and wellbeing in Kiribati. The authors needs to rewrite and present this section before it can be ready for final publication.\nDiscussion The first sentence in the discussion needs editing and I would encourage authors to change “were ignorant” with “they lacked needed knowledge on”. In addition, elaborating finding related with Kiribati culture, in term of respect to the elders and feeding the food leftover for children may help in understanding some of the factors contributing to malnourishment in children, in particular if the food leftover were low quality in terms of nutritional values (e.g., no protein or iron source as red meat or chicken).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4546",
"date": "15 May 2019",
"name": "Masoud Mohammadnezhad",
"role": "Author Response",
"response": "We really appreciate your time for reviewing our paper. We tried to cover your comments. Title: Thanks for your suggestion. This study focuses on behavioral contributing factors so we added “behavioral” in the title. Abstract: This study focuses on behavioral contributing factors (not socio-demographic factors) so we added “behavioral” in the aim of the study in the “introduction” section. Methodology: For the qualitative study it is common to use a “semi-structured questionnaire” to collect data. We added “open ended” to the questionnaire. The one who facilitated FGs and the language was used to collect data is clearly mentioned in the methodology section. “Private room” is replaced. The section related to the semi-structured questionnaire has been added based on the journal editor suggestion. The section related with “immunization” is directly related to study aim which has been clearly mentioned in the last paragraph of “Introduction” section. It is clearly mentioned that “Consequently, this study aims to identify the KAP of mothers of malnourished children (under 5 years old) with respect to contributing factors to malnutrition (breastfeeding, complementary feeding, dietary practice and immunization) on South Tarawa in Kiribati”. Results: There is a separate section as “Analysis of data” called “Data management and analysis” in the paper and we clearly mentioned the analysis process used in transcribing the FGs and how those themes were generated. The information related to socio-demographic characteristic of the sample is included. As this study focused on behavioral contributing factors we listed 7 themes were emerged in this sturdy. As we mentioned “perceived barriers to action” is one of seventh key theme were emerged (not as sub-theme for dietary practices) in our study and has been presented separately. Discussion: The sentence is changed based on reviewer comment. The sentence about the effect of leftover food is also added."
}
]
}
] | 1
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https://f1000research.com/articles/8-83
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https://f1000research.com/articles/8-235/v1
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28 Feb 19
|
{
"type": "Research Article",
"title": "Prevalence and diversity of Salmonella isolated from layer farms in central Ecuador",
"authors": [
"Gabriela A. Salazar",
"Ricardo Guerrero-López",
"Liliana Lalaleo",
"Diana Avilés-Esquivel",
"Christian Vinueza-Burgos",
"William Calero-Cáceres",
"Gabriela A. Salazar",
"Ricardo Guerrero-López",
"Liliana Lalaleo",
"Diana Avilés-Esquivel",
"Christian Vinueza-Burgos"
],
"abstract": "Background: Given the considerable role played by Salmonella in the incidence of food poisoning around the world, surveillance of this infection is prioritized by both food producers and health care authorities. Data remains insufficient concerning the prevalence of Salmonella in poultry systems in Ecuador and in Latin America in general. Methods: In this study we evaluated the prevalence and diversity of Salmonella serovars in samples taken from 21 layer farms and backyard layers in central Ecuador during August-November 2017. Salmonella was isolated following standardized methods (ISO 6579) and the serovar determination was carried out by PCR. Results: A significant presence of Salmonella was detected, with an incidence of 76% (95% confidence interval (CI): 58–94) in farms, 33% (95%CI: 13–53) in pooled cloacal swabs from layer hens, 33% (95%CI: 12–55) on feed samples, and 10% (95%CI: 0–22) in backyard layer feces from traditional local markets. The dominant serovars detected were S. Infantis and S. Typhimurium. Conclusions: This study forms a basis for further surveillance of Salmonella serovars in layer farms in central Ecuador.",
"keywords": [
"Salmonella",
"Layer Poultry",
"Ecuador",
"Serovars."
],
"content": "Introduction\n\nThe genus Salmonella is considered a leading cause of foodborne illnesses around the world (WHO, 2017). These bacteria are among the most significant agents of food and water poisoning in the United States and Europe (Bäumler et al., 2000; Callejón et al., 2015; Varma et al., 2005). Globally, it is estimated that 93.8 million cases of Salmonella-related gastroenteritis occur annually, resulting in 155,000 deaths (Majowicz et al., 2010). In Ecuador, typhoid, together with paratyphoid fever, causes around 1,500 hospitalizations per year, while non-typhoidal salmonellosis leads to more than 2,000 hospitalizations over the same period. These infections have accounted for approximately 25% of total reported gastrointestinal illnesses in recent years (MSP, 2018). Salmonella represents a complex and diverse genus, but only a small number of serovars are involved in human infections (Issenhuth-Jeanjean et al., 2014; Thiennimitr et al., 2012).\n\nSalmonella detection and the investigation of foodborne outbreaks is of maximum importance to public health, which has resulted in the establishment of epidemiological surveillance programs in many developed countries (EFSA, 2013). These measures provide information about endemic Salmonella-serovar patterns, outbreaks, temporal trends and the monitoring of control actions (CDC, 2018). The principal reservoir of S. enterica is the intestinal tract of livestock, representing one of the main sources of infection for humans (Antunes et al., 2016). Despite this epidemiological and economic importance, in South America there is a paucity of information concerning Salmonella in poultry systems (Alexandre et al., 2000; Donado-Godoy et al., 2012). Research conducted in 2016 pertaining to broiler chicken farming in Ecuador indicated the presence of Salmonella serotypes S. Infantis, S. Enteritidis and S. Corvallis (Vinueza-Burgos et al., 2016). However, among layer hens, no information about the prevalence or diversity of Salmonella has been reported. The purpose of this study was to assess the prevalence and diversity of Salmonella bacteria present in layer farms in central Ecuador.\n\n\nMethods\n\nSamples were collected between August–November 2017 from different layer farms in central Ecuador (Tungurahua and Cotopaxi provinces), which accounts for around 60% of egg production in the country. A total of 21 farms (>1,000 birds) in Latacunga, Cevallos, Quero and Ambato (all Ecuador) were sampled, based on their willingness to provide verbal consent for this study (verbal consent was obtained over written consent owing to the farmers’ reluctance to sign their names, as they perceived this could be used to identify them). Further details for each site can be found in Dataset 1 (Calero-Cáceres, 2019a)). One laying hen house per farm was selected. The following samples were collected in each house: 21 pooled cloacal swabs (10 cloacal swabs per pool); 21 manure drag swabs (environmental swabs, 1 per business); 21 caecum content samples (1 layer per farm). To evaluate the potential risk from contaminated feed, 18 composite samples were taken from farmyards (18 of the 21 farms consented verbally to having these samples taken). Additionally, 21 fecal samples from backyard layers were sampled in traditional local markets. All samples were transported in an icebox at 3–5°C within 2 hours of collection for bacterial isolation. The experiment was performed under supervision of the ethical committee of the Faculty of Agricultural Sciences, Universidad Técnica de Ambato.\n\nSalmonella was isolated following standardized methods (ISO 6579) (ISO, 2017). The samples were pre-enriched in buffered peptone water (Oxoid, Basingstoke, England) and then incubated at 37±1°C for 18 h ± 2 h. Next, Rappaport Vassiliadis Soy Broth (RVS Broth) (Merck Millipore, Darmstadt, Germany) was used a selective medium, being inoculated with the pre-enriched culture and incubated at 41.5±1°C for 24±3 h. One loopful of the selective enrichment medium was streaked onto xylose lysine deoxycholate agar (XLD agar) (Becton Dickinson GmbH, Heidelberg, Germany) and incubated at 37±1°C for 24±3 h. Presumptive Salmonella isolates (identified as red/yellow colonies with a black center) were purified in Mac Conkey agar (Merck, Darmstadt, Germany) and incubated at 37±1°C for 24 h. Isolates were Gram stained and the following biochemical tests were performed for confirmation the genus Salmonella: a catalase test using 30% hydrogen peroxide (Merck Millipore, Darmstadt, Germany); triple sugar iron agar test (TSI) (Becton Dickinson GmbH, Heidelberg, Germany), Simmons citrate agar test (Merck, Darmstadt, Germany), Christensen urea agar test (Britania Lab., Buenos Aires, Argentina), and indole reaction using tryptone water (Merck, Darmstadt, Germany) and Kovac’s reagent (Sigma Aldrich, St. Louis, USA). One isolate per positive sample was selected and cryopreserved using overnight growth in LB broth (Sigma Aldrich, St. Louis, USA) supplemented with 30% glycerol (Merck Millipore, Darmstadt, Germany) and maintained at -80°C until analysis.\n\nPCR assays were performed to identify the genes under specific conditions (Table 2). DNA was extracted from overnight cultures in Casein-Peptone Soymeal-Peptone Broth (Merck, Darmstadt, Germany) as described by Muniesa et al. (2004). Approximately ≈50 ng/reaction resulting from the thermal shock of bacterial suspensions (>107 UFC/ml) diluted in sterile ddH20 was used as template for the PCR reactions. The following reference Salmonella strains were used as positive controls for the six serovars under investigation: S. Enteritidis UNIETAR 1, S. Gallinarum b. Gallinarum NCTC 13346, S. Gallinarum b. Pullorum ATCC 19945®, S. Typhi ATCC® 19430, S. Typhimurium ATCC® 26930 and S. Infantis UNIETAR 3CT7.\n\nThe reaction mixture contained: 12.5 µl of DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Massachusetts, USA), 0.5 µl of each primer (30 µM), 9 µl of nuclease-free water (Thermo Fisher Scientific, Massachusetts, USA), and 2.5 µl of crude DNA were used. PCRs were performed with an Applied Biosystems SimplyAmp Thermal Cycler (Thermo Fisher Scientific, Massachusetts, USA). A total of 10 µl of each PCR product were analyzed by agarose gel electrophoresis and stained using Sybr® Safe DNA Gel Stain (Invitrogen, Carlsbad, USA).\n\n\nResults\n\nOverall, 31 out of 34 isolates which showed phenotypic characteristics in accordance with Salmonella were confirmed by PCR as S. enterica (bcfC gene amplification) (Table 1). Of the 21 farms, 16 (76%, 95% confidence interval (CI): 58–94) showed the presence of Salmonella on environmental surfaces, and Salmonella bacteria were isolated in 7 pooled cloacal swabs (33%, 95%CI: 13–53) and in 6 of 18 composite feed samples (33%, 95%CI: 12–55). Feces from backyard layers showed the presence of Salmonella in 2 of 21 samples (10%, 95%CI: 0–22). Dataset 1 shows the location of each sample and the presence or absence of Salmonella (Calero-Cáceres, 2019a)\n\nRegarding to the presence of individual serovars (Figure 1), the highest occurrence was of S. Infantis, detected in 58% (18/31) of isolates, followed by S. Typhimurium, present in 32% of samples (10/31) (Supplementary Figure 1 (Calero-Cáceres, 2019c)). The serovar of 10% of Salmonella isolates (3/31) were unable to be serotyped by the panel of tests. In poultry feed samples, S. Infantis was present in 100% (6/6), while in S. enterica isolated from manure drag swabs, S. Infantis was detected in 50% (8/8) and S. Typhimurium in 50% (8/8). In backyard poultry feces from local markets, S. Infantis was detected in 100% of positive samples (2/2). The most heterogeneous diversity of Salmonella serotypes was observed in pooled cloacal swabs, with 2 S. Infantis, 2 S. Typhimurium and 3 S. enterica consisting of serovars not covered within the panel. Dataset 1 lists the identity of the serovar taken from each sampling location (Calero-Cáceres, 2019a). Dataset 2 shows PCR gels for confirmation of Salmonella serovars (Calero-Cáceres, 2019b).\n\n\nDiscussion\n\nThe overall results showed a significant presence of Salmonella in layer farms in the central Ecuador region, one of the main sources of egg production in the country, with an incidence of more than 76% of the evaluated farms. This result is similar to that reported in Colombia (65%) (Donado-Godoy et al., 2012), and considerably higher than the presence of Salmonella in broiler chicken farms in northern Ecuador (15.9%) (Vinueza-Burgos et al., 2016).\n\nThe notable presence of Salmonella in poultry feed suggests that this may constitute a potential reservoir of this bacteria for poultry systems in the evaluated region, and consequently, a potential route of infection and colonization of poultry and subsequent entry into the food chain (Fink-Gremmels, 2012; Jones, 2011). In recent research conducted in an integrated poultry farm in Ecuador, 4.1% (8/194) of samples were positive for Salmonella, particularly in animal-based feed (Villagómez Estrada et al., 2017). Therefore, the accurate detection of this pathogen in feed is necessary in order to identify the critical points of contamination (facilities, raw materials, transport, storage, producers), that one may apply effective measures to reduce the risk of transmission.\n\nThe finding of only S. Infantis in poultry feed may be attributed to the high level of persistence of this serovar over time in poultry feed, which in turn results in its considerable presence in Ecuadorian broiler chicken (Andino et al., 2014; Vinueza-Burgos et al., 2016). Although the origin of this serovar in feed it is not well defined, studies have pointed to cross-contamination with feces, persistent contamination of storage bins and surfaces, and poor ingredient selection as main causes of feed contamination with Salmonella (Davies & Wray, 1997; Huss et al., 2018; Laban et al., 2014). Genomic tools, such as multilocus sequence typing, BOX-PCR, (GTG)5-PCR or whole-genome sequencing may help to identify the origin of feed contamination in a larger study.\n\nDrag swabs revealed the presence of two different serovars in the sampled poultry farms: Infantis and Typhimurium. These non-typhoidal S. enterica serovars are commonly associated with poultry systems and are linked to outbreaks of foodborne illness (Anderson et al., 2016; Pui et al., 2011). Serovars vary in their persistence over time and geographic distribution around the world (Hendriksen et al., 2011), making further studies desirable in order to evaluate the variations in serovar persistence in the locations sampled in this research.\n\nIn backyard layer feces sampled at local markets, S. Infantis alone was detected (2/2), but further evaluation of Salmonella serovars in backyard flocks is recommended in order to improve surveillance of this potential source of salmonellosis (Behravesh et al., 2014). In pooled cloacal swabs, the serovars detected were Infantis (2/7), Typhimurium (2/7) and unidentified serovars (3/7). Infantis and Typhimurium serovars are commonly detected in poultry around the world (Foley & Lynne, 2008; Foley et al., 2011). Complementary analysis by serotyping the unclassified serovars is necessary in order to identify Salmonella bacteria not covered by the panel.\n\nData remains insufficient concerning the prevalence of Salmonella in poultry systems in Ecuador and in Latin America in general. The coordination of similar future studies may provide a starting point for surveillance of zoonotic bacteria within a defined public health area, leading to an improvement in policies and safe practices in the food industry. Such studies may reduce the risk of infection and establish protocols for corrective measures to be implemented in key upstream points of the chain as indicated by the data.\n\nAt the same time, the intervention of public health authorities may be required in order to ensure the participation of a fully representative range of poultry businesses in future research. This study depended on the voluntary consent of farm owners to providing samples, which may have led to selection bias. In order to establish a thorough program of surveillance, all potential sources of Salmonella infection in the region need to be made accessible to researchers.\n\nThese findings show a significant presence of Salmonella in layer farms in the central zone of Ecuador. The predominant serovars are S. Infantis and S. Typhimurium, typified by PCR. The principal source of infection could be related with poultry feed. From a public health perspective, it is necessary to establish adequate surveillance of Salmonella, including protocols covering biosecurity practices, antibiotic usage and random sampling programs.\n\n\nData availability\n\nFigshare: Dataset 1. Origin, biochemical tests, PCR results and serovar of Salmonella isolates. https://doi.org/10.6084/m9.figshare.7726163.v2 (Calero-Cáceres, 2019a)\n\nFigshare: Dataset 2. PCR results of bcfC, fliC and M.SinI genes. https://doi.org/10.6084/m9.figshare.7726217.v2 (Calero-Cáceres, 2019b)\n\nFigshare: Supplementary figure 1. Multiplex PCR amplification of serovar-specific genes. a) bcfC and mSinI fragments (S. Infantis), b) bcfC and fliC fragments (S. Typhimurium).\n\nhttps://doi.org/10.6084/m9.figshare.7732934.v1 (Calero-Cáceres, 2019c.)\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 study was supported by the Dirección de Investigación y Desarrollo DIDE-Universidad Técnica de Ambato (Project 1568-CU-P-2017).\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\nAgron PG, Walker RL, Kinde H, et al.: Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar enteritidis. Appl Environ Microbiol. 2001; 67(11): 4984–4991. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlexandre SM, Pozo MC, González GV, et al.: Detección de Salmonella enteritidis en muestras de productos avícolas de consumo humano en la Región Metropolitana. Revista Médica de Chile. 2000; 128(10): 1075–1083. Publisher Full Text\n\nAnderson TC, Nguyen TA, Adams JK, et al.: Multistate outbreak of human Salmonella Typhimurium infections linked to live poultry from agricultural feed stores and mail-order hatcheries, United States 2013. One Health. 2016; 2: 144–149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndino A, Pendleton S, Zhang N, et al.: Survival of Salmonella enterica in poultry feed is strain dependent. Poult Sci. 2014; 93(2): 441–447. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAntunes P, Mourão J, Campos J, et al.: Salmonellosis: the role of poultry meat. Clin Microbiol Infect. 2016; 22(2): 110–121. PubMed Abstract | Publisher Full Text\n\nBäumler AJ, Hargis BM, Tsolis RM: Tracing the origins of Salmonella outbreaks. Science. 2000; 287(5450): 50–52. PubMed Abstract | Publisher Full Text\n\nBehravesh CB, Brinson D, Hopkins BA, et al.: Backyard poultry flocks and salmonellosis: a recurring, yet preventable public health challenge. Clin Infect Dis. 2014; 58(10): 1432–1438. PubMed Abstract | Publisher Full Text\n\nCallejón RM, Rodríguez-Naranjo MI, Ubeda C, et al.: Reported foodborne outbreaks due to fresh produce in the United States and European Union: trends and causes. Foodborne Pathog Dis. 2015; 12(1): 32–38. PubMed Abstract | Publisher Full Text\n\nCalero-Cáceres W: Dataset 1. Origin, biochemical tests, PCR results and serovar of Salmonella isolates. figshare. Dataset. 2019a. http://www.doi.org/10.6084/m9.figshare.7726163.v2\n\nCalero-Cáceres W: Dataset 2. PCR results of bcfC, fliC and M.SinI genes. figshare. Figure. 2019b. http://www.doi.org/10.6084/m9.figshare.7726217.v2\n\nCalero-Cáceres W: Supplementary figure 1. figshare. Figure. 2019c. http://www.doi.org/10.6084/m9.figshare.7732934.v1\n\nCDC: Salmonella Atlas. Reports and Publications. Salmonella. CDC [Online]. [Accessed: 7 February 2019]. 2018. Reference Source\n\nDavies RH, Wray C: Distribution of Salmonella contamination in ten animal feedmills. 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PubMed Abstract | Free Full Text\n\nThiennimitr P, Winter SE, Bäumler AJ: Salmonella, the host and its microbiota. Curr Opin Microbiol. 2012; 15(1): 108–114. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVarma JK, Greene KD, Ovitt J, et al.: Hospitalization and antimicrobial resistance in Salmonella outbreaks, 1984-2002. Emerg Infect Dis. 2005; 11(6): 943–946. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVillagómez Estrada S, Logacho Pilataxi M, Vinueza Burgos C: Presencia y Resistencia a los Antimicrobianos de serovariedades de Salmonella enterica aisladas en una empresa avícola integrada del Ecuador. Rev Ecuat Med Cienc Biol. 2017; 38(1). Publisher Full Text\n\nVinueza-Burgos C, Cevallos M, Ron-Garrido L, et al.: Prevalence and Diversity of Salmonella Serotypes in Ecuadorian Broilers at Slaughter Age. PLoS One. 2016; 11(7): e0159567. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Salmonella. WHO. [Online]. [Accessed: 7 February 2019]. 2017. 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}
|
[
{
"id": "45084",
"date": "11 Mar 2019",
"name": "Tania Gaviria Cantín",
"expertise": [
"Reviewer Expertise Molecular microbiology",
"bacterial genetics",
"molecular biology",
"genetic 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\nTitle I consider that the title is not totally in agreement with the study. This could be modified at “Samonella diversity isolated from ... “ without the word “Prevalence” because the study is not a follow-up of previously reported cases in those same conditions\nIntroduction In the fourth line you would change the word “poisoning” by contamination At the end correct one “and” that is in italics When you say \"among the layer hens, no information about prevalence or diversity of Salmonella has been reported\", you could specify or support if that lack of information is only in Ecuador or at a South American or global level. In the same way, it could important to include some epidemiological data of salmonelosis in Ecuador.\nMethodology I think that is not necessary to do DNA extraction to perform PCR, because Salmonella an is gram negative bacteria and during the start of the PCR where the temperature rises to 90 ° C it is possible to destroy cellular wall and release the DNA. However, it does not affect the results, taking into account that the procedure has been carried out correctly avoiding cross contamination. On the other hand, in order to reproduce the methodology of this study, the program used for PCR should be indicated. The concentration of primers (30 uM) is the final or initial concentration? It would be useful to indicate why those particular genes were used as molecular markers for the identification of the species and the serovars\nResults You can ignore the phrase \"amplification of the bcfC gene\" and leave the table 1 referenced because it is a bit redundant Regarding statistical analysis and sample number in representation or not, I prefer not to give many opinions because I'm not qualified enough for that. However, it would be important that the methodology explain in detail how the statistical analysis was performed and the calculation of the incidence. In order of reference, table 1 of results should be table 2 and vice versa Regarding table 1 (positive Salmonella samples ...), it could be a little more consistent with what they explain in the methodology because it took me a bit to understand it, taking into account the classification of columns in terms of location, samples, etc.\nDiscussion I believe that at some point it can be said that the regulations of Ecuador in relation to the health and control of layer farms and local markets and explain how the work is contributing to improve local control, taking into account that this layer farms produce around 60% of egg production in the country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? Yes",
"responses": []
},
{
"id": "45085",
"date": "21 Mar 2019",
"name": "Eloy Gonzalez-Gustavson",
"expertise": [
"Reviewer Expertise Epidemiology",
"biostatistics",
"environmental microbiology",
"parasitology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe scientific names in the manuscript need to be reviewed.\nThe author use incorrectly the terms prevalence and incidence as synonyms.\nIncidence is defined as the presence of new cases of an illness over a period of time. The manuscript does not correspond to an incidence study.\nI would like to see more information in the manuscript about the design to consider the results as prevalence. I have not found details about the representativeness of the sampling (sample size to get a prevalence) or if they were obtained randomly. If the method does not satisfy the requirement to be considered as prevalence, I recommend replacing the term prevalence by frequency, presence or proportion.\nThe confidence interval in one case was incorrectly estimated. \"Feces from backyard layers showed the presence of Salmonella in 2 of 21 samples (10%, 95%CI: 0–22)\" This values have to be calculated based on binomial distribution. Actually in the manuscript, the confidence interval was estimated based on normal or t distribution and 0 was included as lower interval.\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": []
}
] | 1
|
https://f1000research.com/articles/8-235
|
https://f1000research.com/articles/8-416/v1
|
09 Apr 19
|
{
"type": "Research Article",
"title": "Anaesthesia for total hip and knee replacement: A review of patient education materials available online",
"authors": [
"Rebecca Marshall",
"Eoghan Pomeroy",
"Catriona McKendry",
"Michael Gilmartin",
"Paula McQuail",
"Mark Johnson",
"Eoghan Pomeroy",
"Catriona McKendry",
"Michael Gilmartin",
"Paula McQuail",
"Mark Johnson"
],
"abstract": "Background: Patients frequently consult the internet for health information. Our aim was to perform an Internet-based readability and quality control study using recognised quality scoring systems to assess the patient information available online relating to anaesthesia for total hip and knee replacement surgery. Methods: Online patient information relating to anaesthesia for total hip and knee replacement was identified using Google, Bing and Yahoo with search terms ‘hip replacement anaesthetic’, ‘knee replacement anaesthetic.’ Readability was assessed using Flesch Reading Ease (FRE), Flesch-Kincaid grade level (FKGL) and Gunning Fog Index (GFI). Quality was assessed using DISCERN instrument, Health On the Net Foundation seal, and Information Standard mark. Results: 32 websites were analysed. 25% were HONcode certified, 15.6% had the Information Standard. Mean FRE was 55.2±12.8. Mean FKGL was 8.6±1.9. Six websites (18.8%) had the recommended 6th-grade readability level. Mean of 10.4±2.6 years of formal education was required to read the websites. Websites with Information Standard were easier to read: FKGL (6.2 vs. 9, P < 0.001), GFI (8.8 vs. 10.7, P = 0.04), FRE score (64.2 vs. 9, P = 0.02). Mean DISCERN score was low: 40.3 ± 13. Conclusions: Overall, most websites were poor quality with reading levels too high for the target audience. Information Standard NHS quality mark was associated with improved readability, however along with HONcode were not found to have a statistically significant correlation with quality. Based on this study, we would encourage healthcare professionals to be judicious in the websites they recommend to patients, and to consider both the readability and quality of the information provided.",
"keywords": [
"Anaesthesia",
"Quality",
"Readability",
"Total knee replacement",
"Total hip replacement",
"Internet",
"Patient information."
],
"content": "Introduction\n\nTotal hip replacement (THR) and total knee replacement (TKR) are proven interventions for patients with advanced arthritis, and are among the most common elective surgical procedures carried out in the UK and Ireland1. National Joint Registry data for both the UK and Ireland reveals that close to 200,000 total hip and knee replacements are performed each year2,3. Demand for both THR and TKR is set to increase dramatically in the coming decades due to changing demographics and an ageing population, with studies suggesting the demand for TKR in the United States will grow by 673% by 20304–6. Anaesthesia can play a significant role in reducing perioperative morbidity, and in an increasingly complex patient population it is important that patients are given accurate and up to date information about the various anaesthetic techniques used7.\n\nInternet use is increasing worldwide, with 85% of adults in the United States using the Internet8. In the UK, the Oxford Internet Survey group stated that in 2013, 78% of people used the Internet and, of these, up to 71% sought health related information9. Patient education materials (PEM) can be beneficial for assisting patients in the informed consent procedure, by explaining indications, risks, benefits and alternatives10. A recent online poll revealed that up to 90% of patients who access the Internet for their health information believe it to be accurate, and over 60% reported that it impacted their medical decision making11. However, the Internet is a completely unregulated source susceptible to provider bias and has the capacity to negatively influence consumer health outcomes12. Implementing and enforcing standards is very difficult, and health information available has been shown to be of poor quality and largely unreliable8,11,13,14. It is important for doctors to be aware of the information available to patients on the Internet and to understand confusion surrounding such information. As well as supplying high quality accurate health information, the readability of the website must be suitable for the target audience. Several medical organisations , including the National Institute of Health (NIH), and the American Medical Association (AMA) recommend that all PEM should be written at or below sixth grade level (reading age 11-12 years) in order to be effectively understood by the general public15. However many previous studies have shown that a significant proportion of health information websites are written above this recommended level, suggesting that it may be beyond the comprehension of a substantial proportion of the patient population accessing it 16–18.\n\nOver the past number of years there has been several studies assessing both the quality and readability of PEM available on the Internet across all medical specialties, including general and regional anaesthesia for labour and pain procedures19–22. We also already know that orthopaedic patients research their conditions extensively online11,13,23,24, but to our knowledge none of these studies have looked at the availability of high quality health information relating to anaesthesia for common surgical procedures. Therefore, our aim was to assess the readability and quality of patient information available on the Internet relating to anaesthesia for both TKR and THR.\n\n\nMethods\n\nAccording to the policy activities that constitute research at the institute in question, this work met criteria exempt from ethics review.\n\nOn 26/09/2017 the search terms ‘hip replacement anaesthetic’ and ‘knee replacement anaesthetic’ were entered into the top three most commonly used search engines for 2017: Google, Bing and Yahoo. Most Internet users do not go beyond the first three pages of returned searches22, so we only included those websites in the analysis; 27 sites for each of the above terms on both Google and Bing (9 websites per page) and 30 for each on Yahoo (10 websites per page), giving a total of 168. Websites were then excluded from further analysis if they were not PEM, if they were written in a language other than English, if they were inaccessible, or in a non-written format, i.e. video. Duplicate websites were also excluded. In total 32 unique websites were identified for examination, as shown in Table 1.\n\nWebsites listed by search engine, authorship group and the presence or absence of the HONcode and/or Information Standard NHS quality marks. NPO, Not for profit organisations.\n\nWebsite authorship was determined independently by close examination by the first two authors (R.M, E.P.) and each one was placed in one of the following categories: 1) physician, author or authors were individual or group physicians with no university or research group; 2) academic, author or authors were affiliated with a university or research group; 3) commercial site, author or authors were marketing a product related to the subject; 4) commercial/physician, author or authors were individual or group physicians also marketing a product related to the subject; 6) Government/Not for profit organisations (NPO), author or authors were affiliated with a government or registered charity; 7) media-related, author or authors affiliated with the media; and 7) social/discussion, to reflect the growing trend of the use of these modalities to distribute information12.\n\nThe readability of a text is determined as the education level a person completed to understand the written material, based on the US reading grade level10. We assessed the readability of each website using three validated, commonly used readability assessment tools: the Flesch Reading Ease (FRE) score, the Flesch-Kincaid grade level (FKGL), and the Gunning Fog Index (GFI). The FRE score generates a score between 0–100, using the formula: 206.835 − 1.015(total words/total sentences) − 84.6(total syllables/total words). It is based on the total words, syllables and sentences in a written passage and a score <60 considers the document to be difficult to read by the general public8,19. The FKGL corresponds to the US reading grade level and is calculated using the formula: 0.39(total words/total sentences) + 11.8(total syllables/total words) − 15.59. The GFI is calculated using the formula: 0.4[(words/sentences) + 100(complex words/words), and estimates the number of years of formal education required to read a passage of text8. Readability scores for all PEM websites were generated using an online readability calculator.\n\nThe quality of each website was assessed by the two authors using the DISCERN instrument, and according to the presence or absence of the Health On the Net (HON) Foundation seal and the Information Standard mark. The DISCERN instrument is a validated rating tool of the quality of health information developed by the NHS Executive Research and Development Programme. It consists of 15 key questions plus an overall quality rating and generates a score between 80 (highest) and 16 (lowest), a lower score being reflective of a website that is of poor quality information on treatment choices25. The HON Foundation criteria was developed in 1995 by a non-profit, non-governmental organisation, accredited to the Economic and Social Council of the United Nations, in an attempt to improve the quality of internet-based health information. The HON Foundation provides a code of conduct seal for websites that meet its quality and reliability standards8,14. The Information Standard quality mark was established by the UK Department of Health to help patients and the public make informed choices about their lifestyle, condition and options for treatment and care. It is a certification scheme for health and social care information, which indicates that an organisation is a reliable source of health and social care information.\n\nCalculations were performed using SPSS version 23 (SPSS, Inc., Armonk, NY). Mean scores and standard deviation are presented for normally distributed variables. Median values and standard deviation are presented for non-normally distributed data. One-way ANOVA/Independent samples Kruskal-Wallis Test were used as appropriate in intergroup comparisons. In comparisons between certified and noncertified groups, independent samples T test/Mann Whitney U test were applied as appropriate. Significance was set at P < 0.05 for all studies.\n\n\nResults\n\nOut of the 168 initial search results, 32 were analysed further as per the previously described exclusion criteria (Figure 1). Each website was categorised according to authorship; seven were academic, seven were commercial/physician, five were discussion/social media related, four were physician, four were Government/NPO related, four were commercial and one was media-related (Figure 2). Only 8/32 sites (25%) were HONcode certified and 5/32 (15.6%) had the Information Standard quality mark.\n\n27 sites for each of the above terms on both Google and Bing (9 websites per page) and 30 for each on Yahoo (10 websites per page), giving a total of 168. Websites were then excluded from further analysis if they were not PEM, if they were written in a language other than English, if they were inaccessible, or in a non-written format i.e. video. Duplicate websites were also excluded. In total 32 unique websites were analysed further.\n\nSeven were academic, seven were commercial/physician, five were discussion/social media related, four were physician, four were Government/NPO related, four were commercial and one was media-related.\n\nThe readability of each website was assessed using three validated commonly used readability assessment tools: the Flesch Reading Ease (FRE) score, the Flesch-Kincaid grade level (FKGL), and the Gunning Fog Index (GFI). Table 2 summarises the study’s readability and quality scores. The overall mean readability scores indicated that the websites as a group were difficult to read. The mean FRE score was 55.2 ±12.8, with social/discussion networks associated with both the minimum (3.3) and maximum (74.2) FRE scores. The mean FKGL score was 8.6 ±1.9, with only six websites (18.8%) having the recommended readability level of sixth-grade or less (Figure 3). Overall, a mean of 10.4 years (mean GFI 10.4 ±2.6) of formal education was required to read the websites included in this study. Commercial websites had the highest mean GFI (11.7 ±2.3), while social/discussion networks had the lowest (8.3±3.9).\n\nOverall results for each scoring system, presented as mean ±standard deviation for normally distributed data and median ±standard deviation for non-normally distributed data (FRE Score). FRE, Flesch Reading ease; FKGL, Flesch-Kincaid Grade Level; GFI, Gunning Fox Index; DISCERN, DISCERN instrument.\n\nThe line denotes the recommended readability level of the sixth-grade level with a minority of websites falling at or below this level.\n\nOrganisations that have achieved the Information Standard quality mark were as a group easier to read. This was seen across all three readability assessment tools. Member websites had a significantly lower mean FKGL (6.2 vs. 9, P < 0.001) and GFI (8.8 vs. 10.7, P = 0.04) and a significantly higher median FRE score (64.2 vs. 9, P = 0.02) than non-member websites. There was no difference in FKGL (8.5 vs. 8.6, P = 0.78), GFI (10.8 vs. 10.3, P = 0.92) or FRE (51.2 vs. 56.4, P = 0.31) between HONcode certified and noncertified groups.\n\n\nDISCERN Instrument\n\nThe DISCERN instrument was used to assess each website. Overall, the mean DISCERN score was low, 40.3 ± 13 out of a maximum of 80. Three of the top five scoring websites were of academic authorship with one being of physician authorship and one Government/NPO. Eight websites (25%) scored 51 or above, representing good quality, with academic authorship associated with both the single highest DISCERN score (61) and the highest mean DISCERN score across all groups (49.7). Six websites (18.75%) scored less than 26 points, representing very poor quality with extensive shortcomings. Average DISCERN scores by authorship groups are shown in Figure 4. Neither HONcode nor the presence of the Information Standard quality mark was associated with a higher mean DISCERN score (P=0.08 and P=0.7, respectively). Academic and physician-related websites achieved significantly higher mean DISCERN scores than social/discussion networks (P = 0.005 and P = 0.032, respectively; Figure 5).\n\nFrom highest (61) to lowest (20) mean DISCERN score. Error bars denote 95% confidence interval.\n\nSignificantly higher for academic (P = 0.005) and physician-related (P = 0.032) websites versus the mean DISCERN score for social/discussion networks.\n\n\nDiscussion\n\nThere can be no doubt that the Internet has changed the manner in which patients access information. Traditionally, information was passed from doctor to patient in a single direction and decisions were made under the paternalistic guidance of the doctor. In the Internet era, in which information is immediately available, this flow of information is no longer appropriate, nor is it acceptable to patients. More and more, patients are accessing this medical information online and using it to make decisions regarding their own healthcare8,18. However, the reliability and suitability of these online patient education materials is increasingly being called into question8,13,14,16–18. The majority of Internet users start their search with a search engine, and most do not trawl beyond the websites from the first three pages returned22. The aim of this study was to assess both the quality and the readability of Internet information relating to anaesthesia for total hip replacement and total knee replacement, using three validated tools to assess readability (FRE, GFI, FKGL) and the DISCERN instrument to assess quality of information obtained. We also looked for websites that displayed HONcode certification or had received the Information Standard NHS quality mark.\n\nOur results show that 81.2% of the websites assessed were above the recommended sixth-grade readability level for PEM. This should be a concern for healthcare providers; many patients will have a limited understanding of the health information available to them online, and thus even those patient education materials that may be of good quality may not be understood. This could have an adverse effect on the informed consent and decision-making process. These findings echo multiple studies over the last decade, suggesting that producing information at an appropriate readability level is still a challenge for healthcare providers10,18,19,21. Encouragingly, our study found that websites that had been awarded the Information Standard NHS quality mark were statistically significantly more likely to achieve the appropriate readability level, suggesting that these are the websites that healthcare providers should be recommending to our patients.\n\nThe quality of the websites was assessed using the DISCERN instrument. It is important to note that the DISCERN instrument does not take into account the readability of the material, and thus when recommending websites to patients, healthcare providers should seek websites which are of both high quality and appropriate readability. In our study, only a small number of the websites analysed (25%) scored highly using the DISCERN instrument, which indicates that most PEM related to anaesthesia for TKR and THR available on the Internet are of poor quality. Websites were more likely to achieve high DISCERN scores if the authors were physicians, affiliated with academic institutions or government agencies. Again, this highlights the importance of healthcare professionals directing patients towards more reliable and appropriate PEM.\n\nOne of the most significant and disappointing findings from our study relates to the presence or absence of the HONcode seal on websites. Although the HONcode seal indicates that a website has met certain quality and reliability standards, our study did not find that HONcode certified websites achieved higher readability or quality standards than those without the HONcode seal. The Information Standard quality mark was introduced to help patients make informed choices about their condition and options for treatment. In our study, we found that although there was a statistically significant relationship between the presence of the Information Standard quality mark and appropriate reading levels, there was no correlation between this quality mark and quality of information using the DISCERN instrument.\n\nA number of limitations to this study are recognised. We performed our online search in one country, and only analysed PEM from websites written in the English language. The search was limited to the first three pages of returned websites, as it has been shown previously that the general public usually don’t pursue beyond this22. We acknowledge that comprehension of healthcare information is not solely related to readability of text and that other factors, i.e. videos and visual tools, can contribute greatly to a patient’s understanding. The examination of such materials was beyond the scope of this study and previous studies have also acknowledged this limitation8,16. While readability indices have been validated in the literature, there is no general consensus on which index is best and each one uses a different formula to calculate readability. Scores by different indices may vary substantially. It should also be noted that although there is a large volume of material available to guide users when appraising websites using the DISCERN instrument, there is still the potential for variability among raters, which is a limitation not present when assessing readability.\n\nIn conclusion, we aimed to assess the quality and readability of information available online regarding anaesthesia for total knee replacement and total hip replacement. Overall, we found that most of the websites were of poor quality and many had reading levels which were too high for the target audience. These findings echo many other studies that examine online information relating to healthcare8,11,13,16,17,19,20. We found that while the Information Standard NHS quality mark was associated with improved readability, neither the quality mark nor the HONcode were found to have a statistically significant correlation with quality of material. Based on this study we would encourage doctors and other healthcare professionals to be judicious in the websites they recommend to patients, and to consider both the readability and the quality of the information provided.\n\n\nData availability\n\nFigshare. WorkbookFinalPEM.xlsx. Data figures relating to an internet-based readability and quality control study using recognised quality scoring systems to assess the patient information available online for anaesthesia for total hip and knee replacement surgery. https://doi.org/10.6084/m9.figshare.7940753.v126.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCulliford D, Maskell J, Judge A, et al.: Future projections of total hip and knee arthroplasty in the UK: results from the UK Clinical Practice Research Datalink. Osteoarthritis Cartilage. 2015; 23(4): 594–600. PubMed Abstract | Publisher Full Text\n\nO’Neill BJ, Nugent M, Cashman JP, et al.: The Irish National Joint Registry: where are we now? Ir J Med Sci. 2014; 183(1): 77–83. PubMed Abstract | Publisher Full Text\n\nThe NJR Editorial Board: 2017 14th Annual Report: National Joint Registry for England, Wales, Northern Ireland and the Isle of Man. Surgical data to 31 December 2016. National Joint Registry; 2017. Reference Source\n\nGalbraith JG, Fenelon C, Gibbons J, et al.: Enhanced recovery in lower limb arthroplasty in the Irish setting. Ir J Med Sci. 2017; 186(3): 687–91. PubMed Abstract | Publisher Full Text\n\nJohnson RL, Kopp SL, Burkle CM, et al.: Neuraxial vs general anaesthesia for total hip and total knee arthroplasty: a systematic review of comparative-effectiveness research. Br J Anaesth. 2016; 116(2): 163–76. PubMed Abstract | Publisher Full Text\n\nPatel A, Pavlou G, Mújica-Mota RE, et al.: The epidemiology of revision total knee and hip arthroplasty in England and Wales: a comparative analysis with projections for the United States. A study using the National Joint Registry dataset. Bone Joint J. 2015; 97–B(8): 1076–81. PubMed Abstract | Publisher Full Text\n\nGrant CRK, Checketts MR: Analgesia for primary hip and knee arthroplasty: the role of regional anaesthesia. Continuing Education in Anaesthesia, Critical Care Pain. 2008; 8(2): 56–61. Publisher Full Text\n\nO’Neill SC, Baker JF, Fitzgerald C, et al.: Cauda equina syndrome: assessing the readability and quality of patient information on the Internet. Spine (Phila Pa 1976). 2014; 39(10): E645–9. PubMed Abstract | Publisher Full Text\n\nDutton W, Blank G, Groselj D: Cultures of the Internet: The Internet in Britain. Oxford Internet Survey 2013 Report. [Internet]. Oxford: Oxford Internet Institute; 2013 [cited 2017 Oct 26]. Reference Source\n\nDe Oliveira GS Jr, Jung M, Mccaffery KJ, et al.: Readability evaluation of Internet-based patient education materials related to the anesthesiology field. J Clin Anesth. 2015; 27(5): 401–5. PubMed Abstract | Publisher Full Text\n\nNason GJ, Baker JF, Byrne DP, et al.: Scoliosis-specific information on the internet: has the \"information highway\" led to better information provision? Spine (Phila Pa 1976). 2012; 37(21): E1364–9. PubMed Abstract | Publisher Full Text\n\nMurray E, Lo B, Pollack L, et al.: The impact of health information on the Internet on health care and the physician-patient relationship: national U.S. survey among 1.050 U.S. physicians. J Med Internet Res. 2003; 5(3): e17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCassidy JT, Baker JF: Orthopaedic Patient Information on the World Wide Web: An Essential Review. J Bone Joint Surg Am. 2016; 98(4): 325–38. PubMed Abstract | Publisher Full Text\n\nElhassan Y, Sheridan G, Nassiri M, et al.: Discectomy-related information on the internet: does the quality follow the surge? Spine (Phila Pa 1976). 2015; 40(2): 121–5. PubMed Abstract | Publisher Full Text\n\nWeiss BD: Health literacy and patient safety: Help patients understand. Manual for clinicians [Internet]. 2nd ed. Chicago: American Medical Association Foundation; 2007 [cited 2017 Oct 27]. Reference Source\n\nPatel SK, Gordon EJ, Wong CA, et al.: Readability, Content, and Quality Assessment of Web-Based Patient Education Materials Addressing Neuraxial Labor Analgesia. Anesth Analg. 2015; 121(5): 1295–300. PubMed Abstract | Publisher Full Text\n\nKumar G, Howard SK, Kou A, et al.: Availability and Readability of Online Patient Education Materials Regarding Regional Anesthesia Techniques for Perioperative Pain Management. Pain Med. 2017; 18(10): 2027–32. PubMed Abstract | Publisher Full Text\n\nTallgren M, Bäcklund M: Patient information about general anaesthesia on the internet. Anaesthesia. 2009; 64(4): 408–15. PubMed Abstract | Publisher Full Text\n\nLynch NP, Lang B, Angelov S, et al.: Breast reconstruction post mastectomy- Let's Google it. Accessibility, readability and quality of online information. Breast. 2017; 32: 126–9. PubMed Abstract | Publisher Full Text\n\nBailey MA, Coughlin PA, Sohrabi S, et al.: Quality and readability of online patient information for abdominal aortic aneurysms. J Vasc Surg. 2012; 56(1): 21–6. PubMed Abstract | Publisher Full Text\n\nRitchie L, Tornari C, Patel PM, et al.: Glue ear: how good is the information on the World Wide Web? J Laryngol Otol. 2016; 130(2): 157–61. PubMed Abstract | Publisher Full Text\n\nHirsch M, Aggarwal S, Barker C, et al.: Googling endometriosis: a systematic review of information available on the Internet. Am J Obstet Gynecol. 2017; 216(5): 451–458.e1. PubMed Abstract | Publisher Full Text\n\nCrozier-Shaw G, Queally JM, Quinlan JF: Metal-on-Metal Total Hip Arthroplasty: Quality of Online Patient Information. Orthopedics. 2017; 40(2): e262–8. PubMed Abstract | Publisher Full Text\n\nMathur S, Shanti N, Brkaric M, et al.: Surfing for scoliosis: the quality of information available on the Internet. Spine (Phila Pa 1976). 2005; 30(23): 2695–700. PubMed Abstract | Publisher Full Text\n\nCharnock D, Shepperd S: The DISCERN Handbook: Quality criteria for consumer health information on treatment choices [Internet]. Oxford: Radcliffe Medical Press; 1998 [cited 2017 Oct 28]. Reference Source\n\nMarshall R, Pomeroy E: WorkbookFinalPEM.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7940753.v1"
}
|
[
{
"id": "58795",
"date": "31 Jan 2020",
"name": "Thomas W Wainwright",
"expertise": [
"Reviewer Expertise orthopaedic surgery",
"physiotherapy",
"rehabilitation",
"enhanced recovery after surgery",
"quality improvement"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well-written analysis of online patient education materials on anaesthesia for total hip and knee replacement.\nWe commend the authors for investigating this relatively under-researched but important topic.\nOverall, they have presented a valuable addition to the literature-base. We do, however have the following suggestions which we believe would strengthen the contribution of this article to healthcare professionals looking to create or direct patients to relevant educational materials online.\n\n1. Whilst DISCERN is an appropriate tool for assessing the quality of written information about treatment choices, we invite the authors to elaborate on the following points:\nQuestions 1-8 of the DISCERN tool addresses the reliability of the publication, and questions 9 to 15 focus on the specific details on the information on treatment choices. The final component of the DISCERN tool is an overall quality score. It may be valuable to the reader for the authors to present the results from each criterion of the DISCERN score (for example, sources of information, aims, relevance, support with shared-decision making etc.), or at least each section (reliability, treatment options, overall quality), so they have specific details on what needs improving in the future design of online patient education materials.\n\nThree of the top five scoring websites were of academic authorship. Is it possible for the authors to provide some specific examples from these websites which led to a “high-quality” DISCERN score? Demonstrating examples of exemplar education materials may guide healthcare professionals in the design of high-quality educational materials in the future.\n\nThe quality of each website was assessed by two authors using the DISCERN instrument. Were there any discrepancies or disagreement between the two authors whilst evaluating the websites with the DISCERN tool? How were these discrepancies resolved?\n\n2. We commend the authors for conducting three, validated readability tests. The authors found the Information Standard NHS quality mark was associated with improved readability; however along with HONcode were not found to have a significant correlation with quality. We would be interested to know if the “written passages” selected for the readability tests were all taken from the same section of the websites included in this study to reduce variability.\n\n3. Search engines: Were the searches conducted on browsers with an empty cache? If not – choosing to include the first three pages of results only would likely create an element of selection bias, whereby the websites are ones familiar to the authors. Hence, the sample chosen in this study may not represent the search of a layperson.\n\n4. Finally, we must acknowledge that the search for patient education materials was conducted in September 2017. As the content available to patients online is continuously changing, the results of this search have likely been updated, or replaced, with new materials. It may be useful to re-run the search, so that the published results are as relevant as possible to current practice.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5271",
"date": "02 Mar 2020",
"name": "Mark Johnson",
"role": "Author Response",
"response": "Thank you Thomas and Louise for your time and effort in reviewing this paper."
}
]
},
{
"id": "74794",
"date": "07 Dec 2020",
"name": "John McNamara",
"expertise": [
"Reviewer Expertise Regional anaesthesia",
"Cardiac anaesthesia"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting paper.\nThe use of online health information resources is clearly increasing and will continue to do so. This point is well made by the authors, and appropriate references are well cited.\nThis research is timely, in an era when we are increasingly aware of the potential for spread of misinformation online. The medical profession has long been aware of the potential pitfalls of misguided internet searches for health information. It is interesting to determine the prominence of low quality information. The approach of using multiple internet search engines is pragmatic and makes the results of the article applicable. Perhaps inclusion of the term “anaesthesia”, as well as “anaesthetic”, in the search terms would have captured additional relevant websites.\nOverall the article is well-written and easy to read, and describes a topical issue which is not frequently addressed by the medical profession, despite the potential for positive impact on the health of our patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/8-416
|
https://f1000research.com/articles/7-906/v1
|
25 Jun 18
|
{
"type": "Systematic Review",
"title": "Psychological factors contributing to parenting styles: A systematic review",
"authors": [
"Zahra Vafaeenejad",
"Fourozan Elyasi",
"Mahmood Moosazadeh",
"Zohreh Shahhosseini",
"Zahra Vafaeenejad",
"Fourozan Elyasi",
"Mahmood Moosazadeh"
],
"abstract": "Background: The set of strategies used by parents to put their children’s behaviors under control are called parenting styles, which can be influenced by numerous factors including socio-economic variables, cultural differences, personal characteristics, and psychological factors. These factors can differently contribute to parenting style. Thus, the purpose of this systematic review was to examine psychological factors affecting parenting style. Methods: This study was a comprehensive literature review using the keywords of parenting styles, psychological factors, and parenting to search the databases of Google Scholar, PubMed, Scopus, Web of Science, and Scientific Information Database. In this respect, 416 articles were extracted. 368 articles were removed after reviewing their abstracts and full text and eventually 48 articles were selected to elicit the required data. Results: Our findings were classified under two categories: factors related to parents (mental health status, self-efficacy, parenting stress, perfectionism, personality traits, childhood trauma, marital satisfaction, parents’ attachment style, perceived parenting style, and substance abuse); and those related to children (child developmental and mental disability, child temperament, and anxiety). Conclusions: Considering the multiple psychological factors that affect parenting style, it is recommended to include an assessment of parent-child psychological status in family health programs in order to identify the needs for health-oriented care and take steps towards the development of parenting skills.",
"keywords": [
"Parenting Styles",
"Psychological Factors",
"Parenting"
],
"content": "Introduction\n\nParenting styles refer to the set of strategies adopted by parents to control the behaviors of their children1. It has two models: positive parenting styles, i.e. authoritative parenting style; and negative parenting styles that are authoritarian, permissive, and neglectful2,3. In this respect, positive parenting styles are accompanied by encouraging outcomes for children such as optimism, self-esteem, and social-emotional development4–6, while negative parenting can lead to emotional disorders, behavioral problems7, aggression8, as well as child anxiety9. Although, previous review studies have investigated different factors contributing to raising children and child maltreatment, including socio economic factors8,10, and parent and child characteristics8,11–16, less is known about psychological factors that contribute to parenting style. The objective of this systematic review was to conduct a comprehensive literature review on the psychological factors affecting parenting styles.\n\n\nMethods\n\nIn this review, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was used as a guideline17. See Supplementary File 1 for the PRISMA checklist.\n\nConsidering the \"P\" component of PICO (Population of interest, intervention, control, outcome) criteria and FINER (Feasibility, Interesting, Novel, Ethical, Relevant) criteria, the research question was developed as below:18,19.\n\nWhat are the psychological factors contributing to parenting styles?\n\nAccording to the research question in this study, a search was carried out in the databases of Google Scholar, PubMed, Scopus, Web of Science, and Scientific Information Database (a Persian database). In this regard, the required articles were retrieved based on the use of medical subject headings, text words, and related keywords. The search strategy was as follows:\n\n(“Psychosocial Factors” OR Factors OR Determination OR Psychology) AND (“Child Rearing OR Child Rearing Styles” OR Parenting OR “Parent-Child Relations OR Parent-Child Relationship” OR “Parenting Styles”).\n\nEligibility criteria. All indexed and non-indexed original cross-sectional, longitudinal or review studies, in English or Persian, that meet the inclusion criteria, addressed the research question, reported parenting styles in at least one of the parents were retrieved, irrespective of the types of parenting style, recruitment method and instruments used for the assessment of parenting style. Studies that reported on the results of clinical trials were excluded from our review. 416 articles published within February 1984 and April 2017 were extracted. The search time lasted for four weeks between January 23rd and February 23rd in 2018.\n\nStudy selection. After removing duplicate articles (191), those remaining were examined in two stages. Firstly, the titles and a summary of all the remaining articles were independently reviewed by two authors (ZS and ZV). At this stage, 120 articles were excluded from the study.\n\nSecondly, the full texts of all the remaining articles were examined and the items not referring to psychological factors in spite of attention to the factors related to parenting styles were excluded. Additionally, the reference lists of the selected articles were reviewed for more articles. Finally, 48 articles were used. Figure 1 illustrates the study flow.\n\nTwo authors (ZH and ZV) independently extracted basic study information (author's name, title and year of publication, sample characteristics, type of study and outcomes such as: parenting styles, parent's behavior, parent child interaction, family interaction) for all included papers using a predefined evidence table shell. Third author (FE) reviewed the evidence tables for accuracy and completeness. The final evidence table is presented in Table 1. After selecting the final articles, the researchers carefully examined all the relevant articles, extracted the data, and then organized the information needed for the present study. The results of the literature review led to categorization of the contents on psychological factors contributing to parenting styles into several categories as presented in the Results section.\n\nCritical appraisal checklists were used to evaluate the quality of the studies. Checklists were adapted from the Newcastle–Ottawa quality assessment scale20 to assess three broad perspective of each study: the selection of the study groups, the comparability of the groups, and the ascertainment of either the exposure for case-control studies or the outcome of interest for cohort studies and cross-sectional studies. This checklist includes 8 questions for case control studies and cohort studies with maximum 9 score. For cross-sectional studies, this checklist includes 6 questions with maximum 7 score. Ottawa quality assessment scale has established content validity and inter-rater reliability21,22. We used The HE QAT to assess the methodological quality of all included reviews as well. The HE QAT assesses 10 criteria to measure the extent to which the methodological approach of a review guarded against bias with maximum 10 score23. In this review, studies that received ≥ 5 score from Newcastle–Ottawa scale and The HE QAT were included20,23.\n\nThe authors assumed ethical considerations and general standards of publication including avoidance of plagiarism as well as multiple and simultaneous submissions and respect for the intellectual property rights of studies.\n\n\nResults\n\nThe quality assessment of all studies presented in Table 1 is included in Supplementary File 2. The review of the literature led to the categorization of psychological factors affecting parenting styles as factors related to parents and those to children.\n\nA summary of included studies are presented in Table 1.\n\nMental health status. Parents’ mental health status is often directly correlated with parenting styles. As can be seen, parents affected with psychological distress may treat their own children with hostility and rejection. Such parents may adopt harsh disciplinary rules and probably make use of physical punishment10,24. In this regard, it has been shown that a history of major depressive disorders is inversely correlated with authoritative parenting styles and it is positively correlated with authoritarian parenting style24. Moreover, depressed parents do not show proper sentiments or emotions towards their children or their feelings about parenting responsibilities are assumed negative25. These parents may have low self-esteem, reduced self-efficacy, negative emotions, more anger and distress, as well as negative worthlessness to themselves or negative attitudes towards their parenting abilities11,26,27, which have an impact on the trust between parents and children12,28,29. On the other hand, mothers suffering from bipolar disorder are likely to adopt an avoidant and insecure attachment style towards their children and show more anger in their interactions with family members13. Anxiety can be also a stressor with undesirable effects on a healthy coping system and parents’ compliance problems and finally create negative parenting. Such parents may use harassment of their children as a first choice of parenting12, or parents’ interactions with children and their parenting may be accompanied by excessive control and rejection30,31.\n\nOne of the serious problems in the domain of parents’ mental health affecting parenting can be schizophrenia. In this regard, it has been shown that children that have schizophrenic parents grow up with many environmental stressors, such as parental dysfunction. Schizophrenia also has a significant effect on the ability to maintain a close and reciprocal relationship and this issue has an impact on parenting capacity. It has been observed that mothers of schizophrenic women are more remote, insensitive and it is likely to be correlated with less parenting skills32. In addition, such parents may be less involved with their children and they cannot create a positive emotional atmosphere for them13.\n\nSelf-efficacy. Parents with higher self-efficacy are endowed with more self-confidence in order to achieve effective parenting skills and competence and they are also likely to have more success with positive parenting. Parental self-efficacy may affect parenting satisfaction and such an impact on coping ability can be positive. These parents may proactively make efforts in problematic situations, such as lack of social support or presence of economic problems, to reduce the negative effects of these problems on their children. In contrast, parents with lower levels of self-efficacy may not be able to adopt positive parenting strategies26.\n\nParenting stress. One of the factors associated with parents’ characteristics is parenting stress. Parenting stress arises when parenting demands exceed the actual resources available to parents that permit them to succeed in parenting. Accordingly, parents with higher parenting stress are more rejectionists and less protective33. Greater parenting stress tends to use more punishment and less affection toward children34. Stresses affecting parenting also include child-rearing stress as well as a sense of being restrained due to the presence of children35. It has been also observed that parents with parenting stress adopt authoritarian parenting styles34. Parenting stress can similarly give parents anxiety and emotional distress and cause irritability and hostile behaviors by creating negative feelings. These parents may easily react with psychological aggression and physical punishment in the case of misbehavior by children34.\n\nPerfectionism. Perfectionism is a parental characteristic and also a personality trait. Accordingly, perfectionist parents try to be perfect and unflawed. They are extremely critical of themselves and their behaviors. These parents similarly consider wishes and goals they could not reach for themselves for their own children and apply their own standards to them36. Moreover, these parents may show their love to their children when children act in accordance with parents expectations. In order to maintain their self-esteem, they also put more pressure on their children to avoid failures, characterizing authoritarian parenting styles. Furthermore, perfectionist parents have high expectations of their children and these parental characteristics can result in authoritative parenting styles if they are responsive to their children37.\n\nPersonality traits. Parental personality traits are among the most important factors influencing parenting styles38,39. According to the existing literature, the personality traits of extraversion, conscientiousness, agreeableness, and openness to experience can be accompanied by greater intimacy in parenting styles8,39,40 and a neurotic personality trait can be seen in less intimate parents. Giving a smaller amount of autonomy to children is also related to authoritarian parenting styles8,41,42. Parents with agreeable personality traits, due to their ability to obtain more social support and avoid social conflicts, generally are less likely to develop depression43,44. Agreeable parents also try to have flexible and child-centered parenting. Parents who are open to new experiences have emotionally stable personality traits and enjoy new experiences using their imagination and participate in a wide range of mental and experiential endeavors; therefore, this personality trait may be associated with positive parenting since having a child is a new experience44. As well, parents who are conscientiousness are disciplined and they are individuals with good parenting roles. Their children also accept them as an appropriate model45. Moreover, extraverted individuals have positive emotional states and feel good about themselves and the world; and ultimately neurotic-psychotic parents have much more adverse and negative emotions44,46.\n\nChildhood trauma. The history of physical, sexual, or emotional abuse among parents in their childhood is considered as a risk factor leading to negative parenting styles27,47.\n\nIn this respect, perceived childhood maltreatment towards parents can have an effect on creating interpersonal problems including interactions with their own children. It is also a risk factor for subsequent emotional defects, which can result in a series of interpersonal difficulties such as distrust, uncertainty, and avoidance of intimate relationships. Also, there is a relationship between physical and emotional abuse in childhood and adverse outcomes for parents such as less parenting competence, more parenting stress, reduced use of effective parenting styles, parental hostility, use of physical punishment, and neglect towards children. In other words, a history of maltreatment can create a lasting environment during the development of children that can last until adulthood. Moreover, it has been observed that mothers with sexual abuse history in childhood may suffer from greater parenting stress, which can lead to diminished empathy with their own children27,48.\n\nMarital satisfaction. Among the parental characteristics contributing to parenting styles is marital satisfaction49. In this respect, parents with satisfactory marital relationships may have positive behaviors with their children. Conversely, when parents are dissatisfied with marital relationships, negative emotions and behaviors can be transferred through parent-child interactions24. Marital conflict as a stressor can affect couples and increase their anger. Consequently, this anger can spread to children and decrease affection towards them15,50. It has been argued that marital maladjustment can lead to an increase in instability in socio-emotional domains in families resulting in ineffective and inconsistent parenting practices by parents51.\n\nParents’ attachment style. Parental characteristics including their attachment style and family conditions in the past such as stress or supportive relationships in their immediate family can determine their parenting styles52,53. People with secure attachment styles towards their own parents consider their relationships, whether positive or negative, clear, consistent and coherent. These parents have more intimate parenting style and they are responsive to their children53. However, parents with insecure or anxious attachment in their childhood can have pervasive anger as well as lower intimacy and participation in their current relationships with their children53,54. These problems can have long-term consequences in mental health and interpersonal relationships in terms of parenting12 or some parents showing more anger towards their own parents may make special efforts to create positive relationships with their own children53.\n\nPerceived parenting style. Individuals that have loving and responsive childhood with no severe restrictions on them are endowed with healthy socio-emotional development; they also have high self-esteem and internalized control55. As the result of emotional security, behavioral independence and social competence created in them can lead to the formation of a healthy personality and personal maturity and these people can rely more on others. Eventually, these individuals have active interactions as well as more intimacy and acceptance towards their children in the future and ultimately adopt a positive parenting style8. In contrast, there are parents with harsh parenting during their own childhood who may treat their children strictly and believe in using more physical punishment for their children as their parents believed56,57.\n\nSubstance abuse. Substance abuse is considered as a factor affecting parenting14. Substance abuse is also recognized as a risk factor for maltreatment of children and may cause the use of violence47. Marital problems as well as psychological disorders of substance-abusing individuals are related to poor parenting14.\n\nDevelopmental and mental disabilities. Illnesses and disabilities of children can cause emotional distress in parents, which may lead to psychopathology, such as more anxiety, in both parents. This mental disorder can also result in negative and inappropriate parenting styles58. For example, children with disabilities such as Down’s syndrome may have more behavioral problems than children without this disability, and their parents overprotect them which can lead to improper parenting. On the other hand, the siblings of these children may be cared for and controlled less than children that have no disability59.When parents cannot deal with emotional difficulties and control child temperament because of too much stress, they cannot have positive parenting styles, especially the ability to respond appropriately using a suitable approach towards their children58. Although it is demonstrated that if parents perceive the cause of their children’s difficult emotional temperaments, it is possible that earning necessary skills to address these problems can reduce stress in parents and create a more positive parenting style58.\n\nChild temperament. Child temperament such as negative emotions, maladjustment, and anger can make it difficult to care for children. It can also undermine parents’ performance particularly in childhood and their behavior may become more hostile lacking love and affection26,60. Parents of children with a difficult temperament also have higher parenting stress and psychological problems, such as feeling negative about their parenting. Some characteristics seen in children, such as hyperactivity and inability to establish suitable social relationships, are similarly considered among their temperament characteristics and can have an adverse effect on parent-child relationships61. In addition, shyness is among the characteristics associated with child temperament. Thus, children with behavioral inhibition and social fearfulness are restrained and their tolerance threshold is different62. Thus, parents show more intimate behaviors towards children who have more social interactions and they are more likely to adopt much more authoritative parenting styles31. Finally, parents with children with higher emotional intelligence can establish a better relationship with them and they may also adopt positive parenting styles63.\n\nAnxiety. Anxiety disorder in children may lead to the adoption of a negative parenting style, such as more control. For example, a study revealed that parenting was significantly correlated with children’s anxiety disorder. Such a disorder, regardless of the level of anxiety in parents, is associated with a less intimate relationship with children. Moreover, children’s anxiety also causes mothers to have overprotection for their own children16,30. As well, parents having children affected with anxiety disorder may give them less independence and show not as much of acceptance and love to them14.\n\n\nDiscussion\n\nThis systematic review was an attempt to examine a range of psychological factors related to parenting styles to offer a useful collection that considers parent-child characteristics. The results of this study showed that studies that identify effective psychological factors for parenting styles were related. Consequently, increased self-efficacy and reduced parenting stress as well as lower depression and anxiety in parents could lead to the adoption of more appropriate strategies12,24,26,34. Moreover, dimensions of perfectionism in parents and parental personality traits could effect parenting styles37,39,40. The range of parents’ psychological disturbances such as depression and anxiety could also affect parental dysfunction, leading to child maltreatment; and consequently parents’ psychopathology could increase the likelihood of inappropriate and ineffective parenting14,24.\n\nParenting is also influenced by the characteristics of parents’ personality traits40. These characteristics are created in the process of parents’ growth and development8. Therefore, parents with greater agreeableness, extraversion, who are conscientiousness and open to new experiences, with lower neuroticism, are more intimate, organized, and stable and they are also more responsive to their children40,41.\n\nThe history of parental evolution and the way parents have interacted with their own parents can also influence how they behave with their children in the future56. If parents have been mentally disturbed in these previous relationships, their parenting ability can be adversely affected27,48. Parents experiencing love in their childhood and having a secure attachment to their parents can show more positive parenting in adulthood for their children, while insecure attachments may be a risk factor for future parenting and reduce their positive parenting capacities53,54. Studies have also shown how childhood trauma as well as physical, sexual, and emotional abuse during childhood can shape parenting styles in the future. The experience of these injuries can similarly lead to emotional and social impairment and disturb parent-child interactions, and consequently make parents adopt negative parenting styles27,48.\n\nThe results of this study indicated that parents having satisfactory and supportive marital relationships were more sensitive and responsive to their child’s needs34,49. In addition, psychological factors such as depression and parenting stress can affect other types of family relationships, such as marital and parent-child relationships24,34,35. Moreover, substance abuse was recognized as a risk factor for exercising violence against children47.\n\nIn general, the findings suggested that children’s psychological characteristics such as developmental and mental disabilities, temperament, social fearfulness and shyness, attachment, anxiety, and emotional intelligence should be considered in determining the factors contributing to parenting styles. These factors may also bring about psychological problems in parents such as negative feelings about parenting or even lead to challenging behaviors in children or mental health problems in children or parents, which in turn can have an effect on parenting styles30,59,63.\n\nIn conclusion, considering these multiple psychological factors influencing parenting styles, we recommended including parent-child psychological status assessment in family health programs in order to identify the needs for health-oriented care and take steps towards the development of parenting skills among parents.\n\nAlthough this study examined the psychological factors contributing to parenting styles, the impact of couples’ psychological characteristics on each other’s parenting styles was not elucidated. Therefore, future research can shed light on the psychological characteristics of couples interacting with each other as well as the effects on their parenting styles. Despite these limitations it seems the result of this study can be used in the development and implementation of family health intervention programs. Also clinicians, psychologists, psychiatrists, and counselors may consider the psychological factors affecting parenting styles reported in this review for further interventions; the assessment of parent-child mental health status, as well as positive parenting education and in this way help with positive parent-child interactions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nStudent Research Committee of Mazandaran University of Medical Sciences for their financial support of this project (code number, 2952).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: PRISMA checklist.\n\nClick here to access the data.\n\nSupplementary File 2: Quality assessment of all articles.\n\nClick here to access the data.\n\n\nReferences\n\nSmetana JG: Parenting styles and beliefs about parental authority. New Directions for Child Adol Develop. 1994; 1994(66): 21–36. Publisher Full Text\n\nHaghighi M, Khalilzadeh R: A Survey on relationship between marital satisfaction and parenting styles. J Urmia Nurs Midwifery. 2011; 10(1): 21–26. 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}
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[
{
"id": "35469",
"date": "02 Aug 2018",
"name": "Maryam Zamanian",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn my view this paper is valuable and suitable for indexing after doing the comments\nThe methodological quality of the study is fair but I think it should be evaluated by an expert of Psychiatry in terms of psychology concepts, classifications of Psychological characteristics and other issues. Maybe the category of “Psychological factors relating to children” is not needed. The section of “These factors can differently contribute to parenting style. Thus,” is unnecessary in abstract section. The introduction section of manuscript needs more clarification on parenting styles and factors contributing to raising children and the importance of . The discussion section just has repeated the results, while it should compare the results with those from other studies, and provide possible reasons to explain the results with additional clarifications. Moreover it should be written in order of most to least important. The conclusions section is just one recommendation, not the real conclusion. It should be written more powerful and as a real conclusion of research which re-states the main points in a new concise way. It should be worked to improve the quality of the English grammar throughout the manuscript.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": [
{
"c_id": "4524",
"date": "09 Apr 2019",
"name": "Zohreh Shahhosseini",
"role": "Author Response",
"response": "In my view this paper is valuable and suitable for indexing after doing the comments The methodological quality of the study is fair but I think it should be evaluated by an expert of Psychiatry in terms of psychology concepts, classifications of Psychological characteristics and other issues. Maybe the category of “Psychological factors relating to children” is not needed. Response: Some child psychologic factors affecting parenting styles so “Psychological factors relating to children” did not delete. The section of “These factors can differently contribute to parenting style. Thus,” is unnecessary in abstract section. Response: Introduction section was revised in page 1, paragraph 2, line 3 The introduction section of manuscript needs more clarification on parenting styles and factors contributing to raising children and the importance of . Response: Introduction section was revised in page 2, paragraph 1, line 1-15 and in page 2, paragraph 2, line 4-6 The discussion section just has repeated the results, while it should compare the results with those from other studies, and provide possible reasons to explain the results with additional clarifications. Moreover it should be written in order of most to least important. Response: The Discussion section was revised in page 11, paragraph 1, line 1-5 and in, paragraph 2, line 8-10 and page 12, paragraph 1, line 7- 11 The conclusions section is just one recommendation, not the real conclusion. It should be written more powerful and as a real conclusion of research which re-states the main points in a new concise way. Response: The conclusions section was revised in page 12, paragraph 1, line 6-7 and in, It should be worked to improve the quality of the English grammar throughout the manuscript. Response: It was revised."
}
]
},
{
"id": "44614",
"date": "04 Mar 2019",
"name": "Weiqiao Fan",
"expertise": [
"Reviewer Expertise personality theory and assessment",
"career assessment"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of the manuscript is extremely important and interesting. The manuscript provided a summary for factors of parenting styles. However, the manuscript did not establish a clear research gap and a clear research purpose. The author(s) did not provide logic rationales for the research. What’s more, there is not a logical introduction to deduce the research questions. In other words, more attention should be paid for the major issues. Therefore, I do not think the manuscript should be indexed.\nThere are some specific comments to the manuscripts, which may help the author(s) to improve the work.\nThere are many models for parenting styles in the literature. Different model may relate with different factors. However, the author(s) did not touch this issue. Or rather, this is not a logical introduction to this paper. Accordingly, this manuscript cannot have a significant contribution in theory or practice. The definition of “parenting styles” in the first sentence is not liberal. And it cannot reflect the main meaning of parenting style in literature. In Introduction section, the concepts and theory are not clear. Authors should clearly describe parenting styles, the factors and models. Meanwhile, the research questions and significance of the study should also be clearly stated.\n\nI cannot see a thorough review for “review of parenting style” in literature. This should be the foundation of the current manuscript. In Method section, each subsection is well defined with clear headings, but in “search strategy” section, I find it difficult to understand why the retrieved articles were based on medical subject headings.\n\nAfter results, the author(s) should explain the results and compare the results to those previous review works in the literature, rather than simply repeating the results. The author(s) should also add some sections as contribution and implication section to discuss the contributions of the manuscript in theory at least. The authors should reorganize the paragraphs based on the importance of factors or some other logics. The writing and grammar should be improved, and the manuscript is limited by the lack of clarity in the writing.\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? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": [
{
"c_id": "4525",
"date": "09 Apr 2019",
"name": "Zohreh Shahhosseini",
"role": "Author Response",
"response": "The topic of the manuscript is extremely important and interesting. The manuscript provided a summary for factors of parenting styles. However, the manuscript did not establish a clear research gap and a clear research purpose. The author(s) did not provide logic rationales for the research. What’s more, there is not a logical introduction to deduce the research questions. In other words, more attention should be paid for the major issues. Therefore, I do not think the manuscript should be indexed. There are some specific comments to the manuscripts, which may help the author(s) to improve the work. There are many models for parenting styles in the literature. Different model may relate with different factors. However, the author(s) did not touch this issue. Or rather, this is not a logical introduction to this paper. Accordingly, this manuscript cannot have a significant contribution in theory or practice. Response: Introduction section was revised in page 2, paragraph 1, line 1-15 The definition of “parenting styles” in the first sentence is not liberal. And it cannot reflect the main meaning of parenting style in literature. Response: Introduction section was revised in page 2, paragraph 1, line 1-3 In Introduction section, the concepts and theory are not clear. Authors should clearly describe parenting styles, the factors and models. Meanwhile, the research questions and significance of the study should also be clearly stated. Response: Introduction section was revised in page 2, paragraph 1, line 1-15 and in page 2, paragraph 2, line 4-6 I cannot see a thorough review for “review of parenting style” in literature. This should be the foundation of the current manuscript. Response: Introduction section was revised in page 2, paragraph 1, line 3-15 In Method section, each subsection is well defined with clear headings, but in “search strategy” section, I find it difficult to understand why the retrieved articles were based on medical subject headings. Medical Subject Headings is a comprehensive controlled vocabulary for the purpose of indexing journal articles and books in the life sciences; it serves as a thesaurus that facilitates searching. After results, the author(s) should explain the results and compare the results to those previous review works in the literature, rather than simply repeating the results. Response: Discussion section was revised in page 11, paragraph 1, line 1-5 and in, paragraph 2, line 8-10 and page 12, paragraph 1, line 7- 11 The author(s) should also add some sections as contribution and implication section to discuss the contributions of the manuscript in theory at least. Response: Implication section was add in page 12, paragraph 3, line 1-2 The authors should reorganize the paragraphs based on the importance of factors or some other logics. Response: Introduction section was revised in page 5-9 The writing and grammar should be improved, and the manuscript is limited by the lack of clarity in the writing. Response: It was revised."
}
]
}
] | 1
|
https://f1000research.com/articles/7-906
|
https://f1000research.com/articles/8-70/v1
|
18 Jan 19
|
{
"type": "Research Article",
"title": "Anti-infective potential of hydroalcoholic extract of Punica granatum peel against gram-negative bacterial pathogens",
"authors": [
"Chinmayi Joshi",
"Pooja Patel",
"Vijay Kothari",
"Chinmayi Joshi",
"Pooja Patel"
],
"abstract": "Background: Punica granatum extracts have been prescribed in traditional medicine for management of a variety of disease conditions including microbial infections. Generation of scientific evidence for validation of P. granatum peel extract’s anti-pathogenic efficacy is required. Methods: Hydroalcoholic extract of P. granatum peel (PGPE), prepared by microwave assisted extraction method was evaluated for its quorum-modulatory potential against two different human-pathogenic bacteria viz. Chromobacterium violaceum and Pseudomonas aeruginosa. Results: This extract was able to modulate in vitro production of quorum sensing-regulated pigments in both these test bacteria at ≥5 μg/ml. Virulence traits of P. aeruginosa like haemolytic activity, and biofilm formation were negatively affected by the test extract, and it also made P. aeruginosa more susceptible to lysis by human serum. Antibiotic susceptibility of both test bacteria was modulated owing to pre-treatment with PGPE. Exposure of these test pathogens to PGPE (≥0.5 μg/ml) effectively reduced their virulence towards the nematode Caenorhabditis elegans. Repeated subculturing of P. aeruginosa on PGPE-supplemented growth medium did not induce resistance to PGPE in this notorious pathogen, and this extract was also found to exert a post-extract effect on P. aeruginosa. Individual constituent phytocompounds of PGPE were found to be less efficacious than the whole extract. PGPE seemed to interfere with the signal-response machinery of P. aeruginosa and C. violaceum. PGPE also exhibited notable prebiotic potential by promoting growth of probiotic strains- Bifidobacterium bifidum and Lactobacillus plantarum at ≤50 μg/ml. Conclusions: This study indicates PGPE to be an effective antipathogenic and prebiotic preparation, and validates its therapeutic use mentioned in traditional medicine. This study also emphasizes the need for testing any bioactive extract at broadest possible concentration range, particularly in vivo, so that an accurate picture of dose-response relationship can emerge.",
"keywords": [
"Punica granatum peel",
"Quorum Sensing",
"Post Extract Effect",
"Prebiotic",
"Microwave Assisted Extraction"
],
"content": "Introduction\n\nAntimicrobial resistance (AMR) has been recognized as a global problem. Globalization and international travel has increased the vulnerability of any country to infections prevalent in other countries. Rapid spread of resistant factors across different species of pathogenic bacteria, particularly through horizontal gene transfer permitting promiscuous exchange of genetic material, presents a global threat to public health. It is estimated that every 5 minutes one child on the earth dies because of bacterial resistance to antibiotics (ESRC Report, 2014). One of the most problematic features of AMR is that resistance to a new antimicrobial can begin simultaneous to its development, meaning that simply developing new bactericidal antibiotics is not the panacea.\n\nHitherto, antimicrobial therapy has largely remained dependent on conventional bactericidal antibiotics. However, owing to their killing effect, such antibiotics pose considerably strong selection pressure on the pathogen populations to evolve resistant phenotypes. This necessitates thinking of alternative approaches for handling microbial infections, which asks for identifying novel targets, other than those (e.g. cell wall synthesis, nucleic acid/protein synthesis, cell membrane) targeted by current antibiotics (Beceiro et al., 2013; Fair & Tor, 2014; Kohanski et al., 2010). Among such possible novel targets, quorum sensing (QS) has attracted considerable attention from the researchers working in the area of AMR. QS is a mechanism of chemical signalling (via acyl homoserine lactones (AHL) in gram-negative bacteria, and autoinducing peptides (AIP) in gram-positive bacteria) based process, which allows the bacterial population to modulate its behaviour in relation to its cell density (Moore et al., 2015). Though a large part of the bacterial genome (including the genes coding for virulence) is regulated by QS, it is not that essential for their survival as an individual cell, hence quorum-modulatory agents can be expected to exert their anti-infective/antipathogenic effect on susceptible bacterial populations, without necessarily putting a strong selection pressure on them.\n\nThough the modern medicine has almost always focused on search of purified active molecules as therapeutic leads, knowledge of Indian/Chinese/Arabian traditional medicine makes us aware of a variety of plant extracts and inorganic formulations prescribed for management of infections. One such widely mentioned item in these ancient systems of complementary and alternative medicine, including the Indian system Ayurved, is Punica granatum L. extracts. This plant belongs to the family Lythraceae (Punicaceae), and the common English name is pomegranate. Different parts of this plant have been applied in traditional medicine to treat a variety of health problems. A peep into literature with particular focus on its peel reveals that in Egyptian culture, common ailments such as inflammation, diarrhea, intestinal worms, cough, and infertility were treated by exploiting pomegranate peel extract (Ismail et al., 2012). P. granatum has been indicated in traditional Indian and Iranian medicine for its antimicrobial potential, for treatment of throat infections (e.g. sore throat caused by bacterial infection), diarrhea, wound healing, etc. (Hosein Farzaei et al., 2014). In Charak Samhita (Sutrsthana) pomegranate has been mentioned as an important ingredient of a Yavagu formulation for treatment of dysentery. In India, Tunisia, and Guatemala, decoction of dried peels of P. granatum is employed externally as well as internally as an astringent and germicide, and utilized for treating aphthae and diarrhoea. In Ayurvedic medicine, this plant described as ‘dadim’ (its Sanskrit name), is considered as a ‘blood purifier’ and suggested to cure parasitic infections (Colombo et al., 2013). A search in the IMPPAT database (https://cb.imsc.res.in/imppat/basicsearch/therapeutics) using the search term “Punica granatum” yields 75 different therapeutic uses, of which multiple conditions (vaginal trichomoniasis, pharyngitis, periodontitis, paracoccodioidomycosis, malaria, leshminasis, Klebseiella Pneumonia, Helicobacter pylori infection, gonorrhea, gingivitis, genital herpes, dysentery, diarrhea, conjunctivitis, cholera, etc.) for which P. granatum is indicated can involve microbial infections. Ethnobotanical use of P. granatum for treatment of diarrhoea has been documented from South Africa too (Mathabe et al., 2006). Mexican traditional medicine also mentions utility of P. granatum in treatment of gastrointestinal disorders (Alanis et al., 2005). Based on Iranian traditional medicinal practice, Hajimahmoodi et al. 2011 demonstrated peel extracts of eight cultivars of pomegranate to be effective against Helicobacter pylori. One ethnomedical practice reported from the Paliyar tribe from Tamilnadu in India involves taking internally, dried fruit coat of P. granatum after grinding and mixing with water, to treat stomach ache and diarrhoea (Duraipandiyan et al., 2006). Ayurvedic pharmacopeia of India mentions formulations containing rind of P. granatum to be useful in conditions such as fever, dysentery, bacteremia, and infections of oral cavity (Khare, 2004). Decoction containing P. granatum peel is used for gastrointestinal benefit in Algeria too (Gharzouli et al., 1999). Despite their wide use in medicinal texts of various cultures, efficacy of pomegranate extracts needs to be validated through appropriate experiments for their wider acceptance in the modern world.\n\nThis study aimed at investigating the possible anti-infective potential of pomegranate peel extract against two gram-negative bacteria, Pseudomonas aeruginosa and Chromobacterium violaceum. P. aeruginosa is amongst the most notorious bacterial pathogens, associated with chronic and acute urinary and respiratory tract infections, and its carbapenem-resistant phenotype has been listed as a pathogen of ‘critical’ priority against which new antimicrobials are urgently warranted (WHO report, 2017). C. violaceum is widely used in QS studies and is also being viewed as an emerging pathogen (Kothari et al., 2017). WHO advises for future research to focus on the development of new antibiotics specifically against drug-resistant gram-negative bacteria (WHO report, 2017). In both gram-negative bacteria selected in this study, pigment production is a trait, which is reported to be under control (largely but not fully) of their QS machinery, and this was used as a marker trait by us while assaying effect of our extract on these bacteria. QS-regulated pigments produced by these bacteria are: pyoverdine and pyocyanin in P. aeruginosa (Adonizio et al., 2007; Lee & Zhang, 2015), and violacein in C. violaceum (Vasavi et al., 2013).\n\n\nMethods\n\nAll the growth media/ media ingredients/ assay reagents, and antibiotics were procured from Himedia, unless specified otherwise. TLC plates, and all organic solvents used in this study were procured from Merck. Whatman paper, and bacteriological filters were from Axiva (Haryana). Catechin was from Sigma Aldrich (USA). Triton X-100 was from CDH (New Delhi). All experiments were performed with the same set of reagents.\n\nC. violaceum (MTCC 2656), and Lactobacillus plantarum (MTCC 2621), were procured from MTCC (Microbial Type Culture Collection, Chandigarh), whereas Bifidobacterium bifidum (NCDC 255) was procured from NCDC (National Collection of Dairy Cultures), NDRI (National Dairy Research Institute, Karnal). C. violaceum was grown in nutrient broth (HiMedia, Mumbai), L. plantarum was grown in Lactobacillus MRS medium (HiMedia, Mumbai), and B. bifidum was grown on MRS with 0.05% cysteine. Incubation temperature and time for C. violaceum, L. plantarum, and B. bifidum was 37°C, and 22–24 h. Antibiotic susceptibility profile of the bacterial strains used in this study was generated using the antibiotic discs-Dodeca Universal – I, Dodeca G - III – Plus, and Icosa Universal -2 (HiMedia, Mumbai). C. violaceum was found to be resistant to cefadroxil (30 µg), ampicillin (10 µg), cloxacillin (1 µg), and penicillin (10 µg). Culture of P. aeruginosa was obtained from Microbiology Department, M.G. Science Institute, Ahmedabad. Pseudomonas agar (HiMedia, Mumbai) was used for the maintenance of the culture. Strain of P. aeruginosa was found to be resistant to amoxicillin (30 µg), cefadroxil (30 µg), ampicillin (10 µg), cloxacillin (1 µg), penicillin (10 µg), chloramphenicol (30 µg), cefixime (5 µg), clindamycin (2 µg), and nitrofurantoin (300 µg).\n\nPeels of P. granatum were procured from the fruits purchased from local market in the city of Ahmedabad. Plant material (ref no: GU/Bot/29/4/2015) was authenticated for its unambiguous identity by Dr Archana Mankad (Deparment of Botany, Gujarat University, Ahmedabad). The plant name was checked against http://www.theplantlist.org on April 20, 2018. Collected peels were shade-dried, before being used for extract preparation. We also analysed another pomegranate fruit extract as ‘Pomella’ by Pharmanza Herbal Pvt. Ltd., which contained not less than 30% punicalagin\n\nA hydroalcoholic extract of the plant material was prepared in 50% ethanol using the microwave-assisted extraction (MAE) method (Kothari et al., 2009). Dry peel powder (total 5 g; 1 g in each vessel) was soaked into the solvent in a ratio of 1:50, and subjected to microwave heating (Electrolux EM30EC90SS) at 720 W. Total heating time was 120 seconds, with intermittent cooling. This was followed by centrifugation (at 10,000 rpm for 15 min.), and filtration with Whatman paper #1 (Axiva, Haryana). Solvent was evaporated from the filtered extract and then the dried extract was reconstituted in dimethyl sulfoxide [DMSO (absolute); Merck] for broth dilution assay. Reconstituted extract was stored under refrigeration for further use. Extraction efficiency was calculated as percentage weight of the starting dried plant material, and its value was 42% w/w.\n\nAssessment of QS-regulated pigment production by test pathogens in presence or absence of the test formulation, was done using broth dilution assay (Patel et al., 2017). Organisms were challenged with different concentrations (0.5-500 μg/ml) of P. granatum peel extract (PGPE). Nutrient broth or Pseudomonas broth (peptic digest of animal tissue 20 g/l, potassium sulphate 10 g/l, magnesium chloride 1.4 g/l, pH 7.0 ± 0.2) was used as a growth medium. Inoculum standardized to 0.5 McFarland turbidity standard was added at 10% v/v, to the media supplemented with required concentration of PGPE, followed by incubation at appropriate temp for each organism. Appropriate vehicle control containing DMSO was also included in the experiment, along with abiotic control (containing extract and growth medium, but no inoculum). Catechin (50 μg/ml; Sigma Aldrich, USA) was used as positive control, since it is already known QS inhibitor (Nazzaro et al., 2013; Vandeputte et al., 2010).\n\nAt the end of the incubation, bacterial growth was quantified at 764 nm (Joshi et al., 2016). This was followed by pigment extraction and quantification, as per the method described below for each of the pigments. Purity of each of the extracted pigment was confirmed by running a UV-vis scan (Agilent Cary 60 UV-visible spectrophotometer). Appearance of single major peak (at the λmax reported in literature) was taken as indication of purity.\n\nExtraction of violacein from C. violaceum culture was executed as described by Choo et al. (2006). A total of 1 ml of the culture broth was centrifuged (Eppendorf 5417 R) at 15,300g for 10 min at room temperature, and the resulting supernatant was discarded. The remaining cell pellet was resuspended into 1 ml of DMSO, and vortexed, followed by centrifugation at 15,300g for 10 min. The purple coloured violacein was extracted in the supernatant; OD was measured at 585 nm. Violacein unit was calculated as OD585/OD764. This parameter was calculated to nullify the effect of any change in cell density on pigment production.\n\nExtraction of pyoverdine and pyocyanin from P. aeruginosa culture was achieved as described in El-Fouly et al. (2015) and Unni et al. (2014), respectively. A total of 1 ml of the culture broth was mixed with chloroform (Merck, Mumbai) in 2:1 proportion followed by centrifugation at 12,000 rpm (13,520g; REMI CPR-24 Plus) for 10 min. This resulted in formation of two immiscible layers. OD of the upper water-soluble phase containing yellow-green fluorescent pigment pyoverdine was measured at 405 nm. Pyoverdine Unit was calculated as OD405/OD764.\n\nThe lower chloroform layer containing pyocyanin was mixed with 0.1 N HCl (Merck; at the rate of 20%v/v), resulting in a colour change from blue to pink. Absorbance of this pyocyanin in acidic form was measured at 520 nm. Pyocyanin Unit was calculated as OD520/OD764.\n\nAHL extraction: AHL was extracted from the bacterial culture broth as described by Chang et al. (2014). OD of overnight grown bacterial culture was standardized to 1.00 at 764 nm. It was centrifuged at 5000g for 5 min. Cell free supernatant was filter sterilized using 0.45 µm filter (Axiva, Haryana), and was mixed with equal volume of acidified ethyl acetate [0.1% formic acid (Merck) in Ethyl acetate (Merck)]. Ethyl acetate layer was collected, and evaporated at 55°C, followed by reconstitution of the dried crystals in 100 µl phosphate buffer saline (pH 6.8). Identity of thus extracted AHL was confirmed by thin-layer chromatography (TLC). Rf value of purified AHL from C. violaceum culture, while performing TLC [Methanol (60): Water (40); TLC Silica gel 60 F254 plates; Merck] McClean et al., (1997) was found to be 0.70, near to that (0.68) reported for N-hexonyl homoserine lactone (C6-HSL) (Shaw et al., 1997). TLC of AHL extracted from P. aeruginosa resulted in three spots corresponding to Rf values of 0.43, 0.68, and 0.92 near to those (0.41, 0.68, 0.84) for C8-HSL, C6-HSL, and C4-HSL respectively.\n\nBacterial culture growing in presence of test formulation was supplemented with 2%v/v AHL after 6 hours of incubation, and at the end of a total 24-hour incubation pigments were extracted from AHL-supplemented experimental tubes, as well as AHL-non-supplemented control tubes. If the QS-regulated pigment production is found inhibited in both these tubes in comparison to bacteria growing in absence of extract as well as AHL, then the effect of test extract was interpreted as signal-response inhibitor, because if the test formulation would have acted as a signal-supply inhibitor, then exogenous supply of AHL should restore pigment formation by the bacteria.\n\nThis assay was done as described by Neun et al. (2015). OD764 of overnight grown culture was standardized to 1.00. Cell free supernatant was prepared by centrifugation at 15,300 g for 10 min. A total of 10 µl human blood (collected in heparinized vial) was incubated with this cell free supernatant for 2 h at 37°C, followed by centrifugation at 800 g for 15 min; 1% Triton X-100 (CDH, New Delhi) was used as a positive control. Phosphate buffer saline was used as a negative control. OD of the supernatant was read at 540 nm, to quantify the amount of haemoglobin released.\n\nThis assay was performed as described by Ferro et al. (2016). Serum was separated by centrifuging blood at 1,500 rpm (800g) for 10 min. Bacterial culture grown in media with and without PGPE was centrifuged, and the cell pellet was reconstituted in PBS, so that the resulting suspension attains OD764=1. A total of 200 µl of this bacterial suspension from control or experimental tubes was mixed with 740 µl of PBS and 60 µl of serum. After incubation for 24 h at 37°C, absorbance was read at 764 nm. DMSO -treated cells (0.5%v/v) suspended in PBS served as control, against which OD of the PGPE-treated cells (serum-exposed) was compared. Tubes containing bacterial cells exposed neither to DMSO nor serum were also included in the experimental set-up, to nullify any interference from autolysis.\n\nHuman blood was obtained from the authors, who each gave their written informed consent. The use of this blood was approved by the Institutional Ethics Committee of the Institute of Science, Nirma University (approval no: EC/NU/18/IS/4).\n\nThe OD764 of the culture was adjusted to 1.00. Next, 400 µl of phosphate buffer was added into a 2 ml vial followed by 400 µl H2O2. To this 200 µl of the bacterial culture was added, and the mixture was incubated for 10 min. Then 10 µM of sodium azide was added to stop the reaction (Iwase et al., 2013), followed by centrifugation at 12,000 rpm for 10 min. OD of the supernatant was measured at 240 nm to quantify remaining H2O2 (Weydert & Cullen, 2010), with phosphate buffer as blank. Disappearance of the substrate H2O2 was taken as the measure of the catalase activity.\n\nIn this assay, the control and experimental groups contained nine test tubes. In each group, three subgroups were made. First subgroup of three test tubes in the experimental group contained Pseudomonas broth supplemented with PGPE, whereas remaining six tubes contained Pseudomonas broth with no PGPE on first day of experiment. All these tubes were inoculated with inoculum (10%v/v) standardized to 0.5 McFarland turbidity standard (making the total volume in the tube 1 ml), followed by incubation at 37°C for 24 h under static condition, which resulted in formation of biofilm as a ring on walls of the glass tubes. This biofilm was quantified by crystal violet assay (Patel et al., 2013), preceded by quantification of bacterial cell density and pigment. Content from the remaining six test tubes from rest of the two subgroups were discarded following cell density and pigment estimation, and then the biofilms remaining on inner surface of these tubes were washed with phosphate buffer saline (PBS; pH 7) to remove loosely attached cells. Now, 2 ml of minimal media (Sucrose 15 g/l, K2HPO4 5.0 g/l, NH4Cl 2 g/l, NaCl 1 g/l, MgSO4 0.1 g/l, yeast extract 0.1 g/l, pH 7.4±0.2) containing PGPE, was added into each of these tubes, so as to cover the biofilm completely, and tubes incubated for 24 h at 37°C. At the end of incubation, one subgroup of 3 tubes was subjected to crystal violet assay to know whether any eradication of the pre-formed biofilm has occurred under the influence of PGPE, and the last subgroup of 3 tubes was subjected to viability assessment through MTT assay. For the crystal violet assay, the biofilm- containing tubes (after discarding the inside liquid) were washed with PBS in order to remove all non-adherent (planktonic) bacteria, and air-dried for 15 min. Then, each of the washed tubes was stained with 1.5 ml of 0.4% aqueous crystal violet solution for 30 min. Afterwards, each tube was washed twice with 2 ml sterile distilled water and immediately de-stained with 1500 µl of 95% ethanol. After 45 min of de-staining, 1 ml of de-staining solution was transferred into separate tubes, and read at 580 nm. For the MTT assay (Trafny et al., 2013), the biofilm-containing tubes (after discarding the inside liquid) were washed with PBS in order to remove all non-adherent (planktonic) bacteria, and air-dried for 15 min. Then 900 µl of minimal media was added into each tube, followed by addition of 100 µl of 0.3% MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, HiMedia]. After 2 h incubation at 37°C, resulting purple formazan derivatives were dissolved in DMSO and measured at 560 nm.\n\nBacterial surface hydrophobicity was measured using the bacterial adhesion to hydrocarbon (BATH) assay as described in Hui & Dykes (2012). P. aeruginosa culture was collected at stationary phase and pelleted by centrifugation (NF800R, NUVE, Belgium) at 7,000 rpm for 10 min. This pellet was washed twice in phosphate buffer saline (PBS; pH 7.4), and then resuspended in PBS with PGPE (25 and 50 μg/ml) to OD764= 1.00. Same procedure was repeated with P. aeruginosa culture without PGPE, as a control. Each bacterial suspension was then incubated for 1 h at room temperature. A 2-ml sample of each suspension was collected and absorbance (A) at 764 nm was measured, using PBS as the blank. Next, 1 ml xylene (HiMedia, Mumbai) was added to the 2-ml cell suspension, and this mixture was vortexed for 2 min. The phases were then allowed to separate for 1 h. The absorbance of the aqueous phase (A0) was again determined. Results were expressed as % attachment to xylene = (1-A/A0) × 100.\n\nAfter in vitro assessment of QS modulatory (QSM) property of the test formulation, effect of this PGPE on antibiotic susceptibility of the test pathogen was investigated. The bacterial cells pre-treated with PGPE were subsequently challenged with sub-MIC concentration of antibiotic. All the antibiotics were procured from HiMedia, Mumbai. Names and concentrations of the antibiotics used can be seen in Figure 1c, Figure 2c, Figure 5c and Figure 7e.\n\nIn vivo efficacy of the PGPE was evaluated using the nematode worm Caenorhabditis elegans as the model host, using the method described by Eng & Nathan (2015) with some modification. This worm was maintained on Nematode Growing Medium (NGM; 3 g/l NaCl, 2.5 g/l peptone, 1 M CaCl2, 1 M MgSO4, 5 mg/ml cholesterol, 1 M phosphate buffer of pH 6, 17 g/l agar-agar) with Escherichia coli OP50 (procured from LabTIE B.V., JR Rosmalen, the Netherlands) as the feed. Worm population to be used for the in vivo assay was kept on NGM plates not seeded with E. coli OP50 for three days, before being challenged with the test pathogen.\n\nTest bacterium was incubated with the PGPE for 22–24h. Following incubation, OD764 of the culture suspension was equalized to that of the DMSO control. 100 µl of this bacterial suspension was mixed with 900 µl of the M9 buffer containing 10 worms (L3–L4 stage). This experiment was performed in 24-well (sterile, non-treated) polystyrene plates (HiMedia), and incubation was carried out at 22°C. Number of live vs. lead worms was counted everyday till five days by putting the plate (with lid) under light microscope (4X). Standard antibiotic- (ampicillin 500 µg/ml; gentamicin 0.1 µg/ml) and catechin- (50 µg/ml) treated bacterial suspension were used as positive control. Straight worms were considered to be dead. On last day of the experiment, when plates could be opened, their death was also confirmed by touching them with a straight wire, wherein no movement was taken as confirmation of death.\n\nP. aeruginosa was subcultured on PGPE (500 μg/ml)-containing agar medium 10 times, and this PGPE-habituated culture was then challenged with PGPE (500 μg/ml) in liquid media for broth dilution assay, wherein bacterial cell density and pigment production were quantified as detailed in preceding sections. Virulence of this PGPE-habituated culture towards C. elegans was also assessed using the same methodology as described above under the heading ‘In vivo assay’.\n\nChromatographic fingerprint of PGPE was generated through HPLC (Shimandzu Lab Solutions) by running the extract through a C18 column (Shimandzu) for 75 min. Mobile phase consisted of a mixture of trichloroacetic acid (0.05%, w/v) in water, and trichloroacetic acid (0.05%, w/v) in methanol, wherein proportion of latter was varied over a gradient of 10–80%. Flow rate was 1 ml/min. Detection wavelength of the UV detector was set at 258 nm. Punicalagin A and punicalagin B (both at 100 ppm; Sigma Aldrich) were used as standard compounds.\n\nStructures of the pure compounds were downloaded from PubChem: catechin (PubChem CID: 73160), gallic acid (GA) (PubChem CID: 370), quercetin (PubChem CID: 5280343), ellagic acid (PubChem CID: 5281855), cinnamic acid (PubChem CID: 444539), and chlorogenic acid (PubChem CID: 1794427). Structure of the bacterial target proteins was downloaded from the Protein Data Bank (PDB): CviR of C. violaceum (PDB ID 3QP4), LasR (PDB ID 2UV0), and PqsR (PDB ID 4JVC) of P. aeruginosa. AutoDock Vina (Trott & Olson, 2010) was used for docking studies, Discovery Studio Visualizer 4.0.1 was used to study protein-ligand interactions, Pymol viewer (version v1.3r1) was used to convert protein-ligand docked structures from .pdbqt to .pdb format, and MglTools was used to prepare protein and ligand files for docking.\n\nAll the in vitro experiments were performed in triplicate, and measurements are reported as mean ± standard deviation (SD) of three independent experiments. In vivo experiments were run in four replicates. Statistical significance of the data was evaluated by applying t-test using Microsoft Excel. p values ≤0.05 were considered to be statistically significant.\n\n\nResults\n\nC. violaceum was challenged with the PGPE at 0.5–500 μg/ml (Figure 1A). PGPE had a negative effect on bacterial growth only at concentrations ≤25 μg/ml; however, an increase in concentration could not inhibit growth to any greater extent. In fact, statistically all the concentrations in the range 50–500 μg/ml had equivalent effect on C. violaceum growth. On the other hand, production of the QS-regulated pigment violacein was sensitive to PGPE at lesser concentrations (i.e. at ≤5 μg/ml), and the inhibitory effect increased with increase in concentration of the extract. PGPE can be said to exert a purely QSM effect at 5–10 μg/ml, as at these concentrations, it could inhibit pigment production significantly without inhibiting C. violaceum growth.\n\n‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and violacein production of C. violaceum. (A) Effect of PGPE on growth and QS- regulated violacein production in C. violaceum: Bacterial growth was measured as OD764; OD of violacein was measured at 585 nm, and Violacein Unit was calculated as the ratio OD585/OD764 (an indication of violacein production per unit of growth); Catechin (50 µg/ml) did not exert any effect on growth of C. violaceum, but inhibited violacein production by 47.69±0.03%. (B) PGPE acts as a signal-response inhibitor against C. violaceum. (C) PGPE-pre-treatment enhances susceptibility of C. violaceum to different antibiotics. (D) PGPE-treatment attenuates virulence of C. violaceum towards C. elegans: Catechin (50 μg/ml) and ampicillin (500 μg/ml) employed as positive controls conferred 100% protection on worm population, and pre-treatment of bacteria with PGPE at concentrations (10, 25, 50 and 100 μg/ml) other than those shown in figure allowed 75%, 77.5%, 80%, and 75% worm survival, respectively; DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and PGPE at tested concentrations showed no toxicity towards the worm. *p<0.05, **p<0.01, ***p<0.001; AS, antibiotic susceptibility; QS, quorum sensing; PGPE, Punica granatum peel extract.\n\nOnce the QS-inhibitory potential of PGPE against C. violaceum was confirmed, we experimented to know whether it acts as a signal-supply inhibitor or signal-response inhibitor. To know this, we added AHL (2% v/v; 6 h post-inoculation) into quorum inhibited culture of C. violaceum growing in the presence of PGPE (10–100 μg/ml). Following AHL (signal) augmentation, the quorum inhibitory effect of PGPE on violacein production was not reversed (Figure 1B), indicating that PGPE acts as a signal-response inhibitor.\n\nWhen PGPE-treated C. violaceum culture was subsequently challenged with sub-MIC concentrations of four antibiotics belonging to four different classes, extract pre-treatment was found to enhance susceptibility of this bacterium to all these antibiotics. However, pre-treatment with higher (25 μg/ml) PGPE concentration was found to be no better than the lesser (10 μg/ml) concentration, with respect to making bacteria more susceptible to given antibiotic, except in the case of ampicillin (Figure 1C).\n\nPGPE was found to curb haemolytic potential of C. violaceum in a dose-dependent fashion, wherein this extract at 100 μg/ml could reduce haemolysis by ~36% (Table 1). This extract could also reduce the catalase activity of C. violaceum marginally; however, the effect on this oxidative stress response enzyme activity did not increase with increase in PGPE concentration (Table 1).\n\n*p<0.05, **p<0.01, ***p<0.001; Catalase assay was done by monitoring disappearance of H2O2 at 240 nm; Hemoglobin concentration was measured at OD540; DMSO in ‘vehicle control’ tube had no effect on catalase and haemolytic activity of any of the three bacteria; Chloramphenicol (0.5 μg/ml) enhanced catalase activity of C. violaceum by 11.23%*.± 0.01; Tetracycline (0.5 μg/ml) inhibited catalase activity of P. aeruginosa by 21.51%* ± 0.02.\n\nIn vitro demonstration of the QSM potential of PGPE was followed by assessment of its anti-infective potential in vivo, employing C. elegans as the model host, wherein PGPE was found to confer survival benefit on C. elegans upon challenge with C. violaceum at all the concentrations (0.5–100 μg/ml) tested (Figure 1D). Notably, even the concentrations (0.5–2.5 μg/ml) having no significant in vitro effect on C. violaceum growth and pigment production, were found to be effective in vivo. Maximum in vivo benefit was obtained at 5 μg/ml PGPE concentration, and worm survival percentage at any of the higher concentrations were statistically no better than that obtained at 5 μg/ml. Onset of death in the worm population was also delayed by 2 days (i.e. death started on first day-post infection in control wells, as against on third day in experimental wells) when the challenger bacteria were pre-treated with PGPE. Movement of the surviving worm in ‘vehicle control’ wells was slowed down, whereas worms surviving in the ‘experimental’ wells showed normal movement. Further, worms in the experimental well corresponding to 100 μg/ml PGPE could also generate progeny worms, which were not observed in any other well.\n\nP. aeruginosa was challenged with PGPE at 0.5–500 μg/ml, wherein growth of this bacterium got notably inhibited 50 μg/ml onwards. Pyocyanin production was affected negatively from 5 μg/ml onwards in a concentration-dependent manner, whereas pyoverdine production was enhanced from 2.5 μg/ml onwards in a largely concentration-dependent fashion except at 500 μg/ml (Figure 2A). When AHL was added exogenously to the quorum-modulated P. aeruginosa culture, the inhibitory effect of PGPE on pyocyanin and its stimulatory effect on pyoverdine production was not reversed (Figure 2B) indicating that PGPE acts as a signal-response inhibitor against this pathogen.\n\n‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and pigment production of P. aeruginosa. (A) Effect of PGPE on growth and QS-regulated pigment production in P. aeruginosa: Bacterial growth was measured as OD764; OD of pyoverdine was measured at 405 nm, and Pyoverdine Unit was calculated as the ratio OD405/OD764 (an indication of pyoverdine production per unit of growth), Pyocyanin Unit was calculated as the ratio OD520/OD764 (an indication of pyocyanin production per unit of growth); Catechin (50 µg/ml) inhibited pyoverdine and pyocyanin production by 17.13±0.06% and 23.65±0.04% respectively, without affecting the bacterial growth. (B) PGPE acts as a signal-response inhibitor against P. aeruginosa. (C) P. aeruginosa challenged with antibiotics following pre-treatment with PGPE. (D) PGPE modulates susceptibility of P. aeruginosa to lysis by human serum: ‘Control’ in serum-dependent lysis assay was PGPE-unexposed cells of P. aeruginosa incubated with human serum. (E) Effect of PGPE on P. aeruginosa biofilm formation, eradication, and viability. Crystal violet assay was performed to measure biofilm formation, and biofilm eradication, followed by the measurement of OD at 580 nm; Cell viability in biofilm was estimated through MTT assay, wherein OD was measured at 560 nm. (F) PGPE-treatment reduces the virulence of P. aeruginosa towards C. elegans: Catechin (50 μg/ml) and gentamicin (0.1 μg/ml) employed as a positive controls conferred 100% and 80% protection on worm population respectively. Pre-treatment of bacteria with PGPE at concentrations (2.5, 5, 10, 25, and 100 μg/ml) other than shown in figure allowed 73.75, 75, 77.5, 77.5, and 75% worm survival respectively; DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and PGPE at tested concentrations showed no toxicity towards the worm. (G) Effect of PGPE on P. aeruginosa growth, Pyoverdine Unit, and Pyocyanin Unit remained unaltered after repeated exposure to PGPE (500 μg/ml). *p<0.05, **p<0.01, ***p<0.001. AS, antibiotic susceptibility; QS, quorum sensing; PGPE, Punica granatum peel extract.\n\nEffect of PGPE pre-treatment on antibiotic susceptibility of P. aeruginosa was also investigated. However, no major changes were observed in bacterial susceptibility to four different test antibiotics, when challenged with these antibiotics following PGPE exposure (Figure 2C). This extract was not found to alter catalase activity much; however, haemolytic potential of P. aeruginosa was notably affected 25 μg/ml onwards (Table 1). PGPE at 50 μg/ml could enhance (by 13.55%; p=0.03) susceptibility of this bacterial pathogen to lysis by human serum (Figure 2D).\n\nPGPE was able to reduce P. aeruginosa biofilm formation up to 17-30%, when tested at 10–50 μg/ml (Figure 2E). When this extract was applied on pre-formed biofilm, it could eradicate the biofilm by ~15–19%; and biofilm viability (metabolic activity as measured by MTT assay) was reduced by ~13–22%. However, whether this observed reduction in viability is due to lesser number of cells, or some other reason, remains to be investigated. Reduced biofilm formation by P. aeruginosa in presence of PGPE may partly be due to this extract’s ability to reduce CSH marginally, which is an important determinant of biofilm forming ability in bacteria (Hui and Dykes, 2012). Xylene adherence of P. aeruginosa treated with PGPE at 25 and 50 μg/ml was found to be reduced by 14.19% (p<0.05; % adherence=44.74) and 7.82% (p<0.05; % adherence =38.37) respectively, against 30.55% adherence of control.\n\nIn vivo assay did confirm the anti-infective potential of PGPE against P. aeruginosa. At concentrations as low as 0.5–1 μg/ml, it could support overall survival of the worm population up to 56-71% in face of P. aeruginosa challenge, as against only 12.5% worm survival in control population. Higher PGPE concentrations did not offer any better protection to C. elegans against P. aeruginosa (Figure 2F). Notably, in vitro experiments showed PGPE to have no effect on P. aeruginosa growth below 5 μg/ml, and no effect on its QS-regulated pigments below 2.5 μg/ml; whereas PGPE at 0.5–1 μg/ml was able to confer significant survival benefit on C. elegans against P. aeruginosa challenge. Onset of death in worm population was also delayed by 1–3 days, when P. aeruginosa was exposed to PGPE before being allowed to infect C. elegans.\n\nAfter confirming the anti-infective potential of PGPE against P. aeruginosa, we investigated whether this bacterium can develop resistance to PGPE upon repeated exposure to this extract. We sub-cultured P. aeruginosa on PGPE (500 μg/ml) containing agar medium 10 times, and this PGPE-habituated culture was then challenged with PGPE (500 μg/ml) in liquid media for broth dilution assay, wherein effect of PGPE on growth and pigment production in P. aeruginosa was not found to be much different than that on P. aeruginosa with no previous PGPE exposure (Figure 2G). Similarly, there was no difference in the virulence of P. aeruginosa towards C. elegans, irrespective of whether it was previously habituated to PGPE. (Figure 2F).\n\nWith P. aeruginosa, we did an additional experiment to investigate whether the daughter cells of PGPE-exposed P. aeruginosa bear any attenuation of their virulence owing to their parent cells being exposed to PGPE. When the PGPE-exposed P. aeruginosa was subsequently subcultured on PGPE-free media, pigment production in cells obtained after first such subculturing was still altered in a pattern similar to the PGPE-exposed parent culture (Figure 3A). Though QS-regulated pigment production remained deviated from ‘control’ upon second subculturing too, it did not follow the same pattern, as observed with cells obtained after first subculturing on PGPE-free media. With each subculturing, virulence of P. aeruginosa towards C. elegans approached nearer to that of ‘control’, but it still did not got equivalent to ‘control’ (Figure 3B), clearly suggesting that effect of PGPE-treatment was not completely absent even from the progeny cells (who never were directly exposed to PGPE), whose parent cells received single PGPE-exposure. This phenomenon of long-lasting effect after single-time exposure to the antimicrobial agent is described as ‘post-antimicrobial effect’. Here, in case of a plant extract, we prefer to call it post-extract effect (PEE), which refers to the persistent suppression of one or more bacterial traits (e.g. growth/virulence, etc.) after one-time-exposure to antimicrobial agents, and may last for many hours depending on the concentration of test agent and the susceptibility of the target bacterium (Pfaller et al., 2004; Ramadan et al., 1995; Ramanuj et al., 2012). During actual animal/human infections, such phenomenon can be of high significance, as though the infectious bacteria may multiply inside the host system, their progenies may not have the virulence at par with the parent cells.\n\n‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and pigment production of P. aeruginosa. (A) Effect of PGPE on growth and QS-regulated pigment production in P. aeruginosa after subculturing of cells in PGPE-free media. Bacterial growth was measured as OD764; OD of pyoverdine was measured at 405 nm, and Pyoverdine Unit was calculated as the ratio OD405/OD764 (an indication of pyoverdine production per unit of growth), Pyocyanin Unit was calculated as the ratio OD520/OD764 (an indication of pyocyanin production per unit of growth). (B) PGPE-treatment reduces the virulence of P. aeruginosa towards C. elegans even after subculturing of cells in PGPE-free media. Catechin (50 μg/ml) and gentamicin (0.1 μg/ml) employed as a positive controls conferred 100% and 80% protection on worm population respectively; DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and PGPE at tested concentrations showed no toxicity towards the worm. *p<0.05, **p<0.01, ***p<0.001; AS, antibiotic susceptibility; QS, quorum sensing; PGPE, Punica granatum peel extract.\n\nOnce in vitro and in vivo anti-pathogenic potential of PGPE was confirmed, we proceeded to chromatographic fingerprinting of this extract. The resultant chromatogram is shown in Figure 4. Since punicalagin is one of the most widely reported bioactive metabolite from P. granatum, we quantified its amount in our extract, which was found to be present at 6.6%. Earlier we had analyzed another pomegranate fruit extract marketed as ‘Pomella’ by Pharmanza Herbal Pvt. Ltd., which contained not less than 30% punicalagin for its QS-modulatory potential (extended data, Figure S1A) (Joshi et al., 2018). Though there were no major differences with respect to the in vitro effect on growth and pyocyanin production by P. aeruginosa, pyoverdine production was affected much more by Pomella than PGPE. However, in vivo efficacy of PGPE was bit better than that of Pomella; PGPE at 0.5-50 μg/ml allowed 56–80% worm survival, as against 55–65% worm survival supported by ‘Pomella’ at identical concentrations (extended data, Figure S1B) (Joshi et al., 2018). From this, we may speculate punicalagin not to be the major constituent responsible for P. granatum extract’s anti-pathogenic activity. Against C. violaceum too, except at 250 μg/ml, effect of PGPE on violacein production was statistically not different from that of Pomella, with latter exerting a bit more inhibitory effect on growth of this bacterium (extended data, Figure S1C) (Joshi et al., 2018). However, role of punicalagins cannot be completely ruled out, as they are known to be metabolized inside human system to urolithins, and latter has been reported to be capable of exerting anti-QS effect (Giménez-Bastida et al., 2012). Larrosa et al. (2010) suggested the possibility of urolithin-A being the most active anti-inflammatory compound derived from pomegranate ingestion in healthy subjects.\n\nPeak 11 and 14 occupied 24.61% and 27.48% of total area, which corresponds to Punicalagin A and B, respectively. PGPE, Punica granatum peel extract.\n\nOther than punicalagin, pomegranate peel extracts have been reported to contain pyrogallol, catechin, rutin, cinnamic acid, benzoic acid, chlorogenic acid, acacetin, ferulic acid, kampferol, genistein (Ali et al., 2014), coumaric acid (Khan et al., 2017) tannins (punicalin, pedunculagin, GA and ellagic acid) (Ismail et al., 2012; Pagliarulo et al., 2016), and quercetin (Saxena et al., 2017). To gain more insight, we docked all these reported compounds against LuxR analogues of C. violaceum and P. aeruginosa, against which our extract was found to act as signal-response inhibitor. Results of this molecular docking exercise are presented in Table 3. The amino acid residues involved in this on-screen ligand-receptor binding, for compounds used in wet-lab study, are shown in extended data, Table S1 (Joshi et al., 2018).\n\nPure compounds against C. violaceum. Against the LuxR analogue of C. violaceum, docking scores higher than that of its natural ligand (C6-HSL) were obtained for acacetin, catechin, and quercetin. However, for further in vitro experiments, we did not set any cut-off value with respect to docking score, instead we experimented with all those pure compounds from those listed in Table 2, which were available in our lab viz. catechin, quercetin, chlorogenic acid, cinnamic acid, and GA. Cinnamic acid had no effect on C. violaceum growth and pigment production in the concentration range 5–100 μg/ml, except 26% reduction in violacein production at 100 μg/ml (Figure 5A). Hence all subsequent experiments with cinnamic acid were performed at 100 μg/ml. Cinnamic acid was able to enhance C. violaceum susceptibility to streptomycin by 11.94%, but had no effect on its susceptibility to cephalexin and tetracycline (Figure 5C). Haemolytic potential and catalase activity of C. violaceum were curbed to a smaller (8.47% and 2.68%, respectively), albeit statistically significant extent (Table 3). This phenylpropanoid metabolite was also able to confer some survival benefit (24% lesser worm death) on C. elegans (Figure 5D). Cinnamic acid induced violacein inhibition was not reversed upon exogenous supply of AHL (extended data, Figure S2) (Joshi et al., 2018), indicating its mode of QS-inhibition to be signal-response inhibition.\n\n#C6-HSL autoinducer of C. violaceum, C4-HSL autoinducer of Las QS system of P. aeruginosa, and PQS quinolone signal of PQS system of P. aeruginosa were used for docking as control.\n\n*p<0.05, **p<0.01, ***p<0.001. †Mean ± SD. Catalase assay was done by monitoring disappearance of H2O2 at 240 nm; Hemoglobin concentration was measured at OD540; DMSO in ‘vehicle control’ tube had no effect on catalase and haemolytic activity of any of the three bacteria; Chloramphenicol (0.5 μg/mL) enhanced catalase activity of C. violaceum by 11.23%*. ± 0.01; Tetracycline (0.5 μg/mL) inhibited catalase activity of P. aeruginosa by 21.51%* ± 0.02.\n\n‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and violacein production of C. violaceum. (A) Effect of cinnamic acid on growth and violacein production in C. violaceum. Bacterial growth was measured as OD764; OD of violacein was measured at 585 nm, and Violacein Unit was calculated as the ratio OD585/OD764 (an indication of violacein production per unit of growth). (B) Effect of chlorogenic acid on growth and violacein production in C. violaceum. Bacterial growth was measured as OD764; OD of violacein was measured at 585 nm, and violacein unit was calculated as the ratio OD585/OD764 (an indication of violacein production per unit of growth). (C) Pre-treatment of C. violaceum with chlorogenic acid and cinnamic acid enhances its susceptibility to streptomycin and cephalexin. (D) Cinnamic acid, chlorogenic acid, and catechin reduced virulence of C. violaceum towards C. elegans. Catechin (50 μg/ml) and ampicillin (500 μg/ml) employed as positive controls conferred 100% protection on worm population. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and compounds at tested concentrations showed no toxicity towards the worm. *p<0.05, **p<0.01, ***p<0.001. AS, antibiotic susceptibility; QS, quorum sensing.\n\nChlorogenic acid could not affect C. violaceum growth and pigment production at any of the test concentrations (5–100 μg/ml), except 16.21% inhibition of violacein production at 75 μg/ml (Figure 5B). Notably, violacein production was not affected at higher concentration (i.e. 100 μg/ml). Hence, all subsequent experiments with chlorogenic acid were performed at 75 μg/ml. At this concentration chlorogenic acid could make C. violaceum 8.91% more susceptible to streptomycin; however, it did not alter this bacterium’s susceptibility to cephalexin and tetracycline (Figure 5C). Chlorogenic acid was also able to inhibit catalase activity and haemolytic activity of C. violaceum by 3.84% and 10.16% respectively (Table 3). It could also offer a sizable (49%) survival benefit to C. elegans, when challenged with C. violaceum (Figure 5D). AHL augmentation of the C. violaceum culture experiencing chlorogenic acid induced QS inhibition, did not result in reversal of this inhibition (extended data, Figure S2) (Joshi et al., 2018), indicating this plant metabolite to act as a signal-response inhibitor.\n\nGA was tested at 10-200 μg/ml for its potential quorum modulatory action against C. violaceum, and it was found to inhibit the growth of this bacterium in a dose-dependent fashion (Figure 6A). Violacein production was negatively affected from 100 μg/ml onwards, and this inhibition was reversed upon external AHL supply (Figure 6B). Since GA was indicated to act as a signal-supply inhibitor, we docked it against the LuxI analogue of C. violaceum i.e. CviI, the enzyme responsible for AHL synthesis, wherein the docking score was found to be -6.9 kcal/mol. At all test concentrations GA was able to induce catalase activity marginally.\n\nBacterial growth was measured as OD764; OD of violacein was measured at 585 nm, and Violacein Unit was calculated as the ratio OD585/OD764 (an indication of violacein production per unit of growth) ‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and violacein production of C. violaceum. (A) Effect of gallic acid on growth and violacein production in C. violaceum. (B) GA acts as a signal-supply inhibitor. (C) Effect of quercetin on growth and violacein production in C. violaceum. (D) Quercetin acts as a signal-response inhibitor. (E) Effect of catechin on growth and violacein production in C. violaceum. (F) Catechin acts as a signal-response inhibitor. *p<0.05, **p<0.01, ***p<0.001. QS, quorum sensing; GA, gallic acid.\n\nWhen C. violaceum was challenged with quercetin (10–200 μg/ml), its growth was negatively affected, and violacein production even more so. Concentration of 150 μg/ml quercetin was able to achieve almost complete inhibition of violacein production (Figure 6C), and this inhibition was not reversible in response to exogenous AHL augmentation (Figure 6D), indicating quercetin to act as a signal-response inhibitor. This observation also matches with the docking score (-8.5 Kcal/mol) of quercetin against CviR, which is second highest amongst all the docked compounds. Quercetin was also able to curb the catalase activity of C. violaceum, maximum inhibition being observed at 150 μg/ml.\n\nCatechin had no effect on C. violaceum growth till 200 μg/ml, whereas its quorum-inhibitory effect started from 10 μg/ml onwards. Hence, catechin can be said to have a purely quorum-inhibitory effect on C. violaceum (Figure 6E), and its inhibitory effect on violacein production was not reversed upon AHL augmentation, indicating it to be acting as a signal-response inhibitor (Figure 6F). This compound could notably reduce haemolytic potential of C. violaceum, and also had little but statistically significant effect on catalase activity (Table 3). Irrespective of its presence in PGPE, we employed catechin in all our QS experiments as a positive control, since it is a known QS inhibitor (Vandeputte et al., 2010). Information on its in vivo efficacy has been provided in respective figure legends (Figure 1D, Figure 2F, Figure 5D).\n\nPure compounds against P. aeruginosa. With respect to docking score against lasR receptor of P. aeruginosa, catechin, querectin and genistein exhibited highest affinity towards this receptor; whereas punicalagin, punicalin, rutin, and pedunculagin exhibited maximum affinity for the pqsR receptor of this bacterium. Preliminary in vitro experiments revealed cinnamic acid, chlorogenic acid, and catechin to exert their maximum effect on P. aeruginosa QS-regulated pigment production at different concentrations. Data for the most effective concentration for each of these three compounds is presented in Figure 7A–C. Production of both pigments was affected the most by chlorogenic acid, and this compound was also able to make P. aeruginosa more susceptible to cephalexin (Figure 7E); however, it could not alter this bacterium’s susceptibility to ofloxacin and gentamicin. With respect to antibiotic susceptibility result obtained with cinnamic acid (Figure 7E) were identical to those with chlorogenic acid. AHL augmentation was able to reverse effect of chlorogenic as well as cinnamic acid on production of both the QS-regulated pigments of P. aeruginosa (Figure 7D), suggesting these compounds to disturb the QS machinery of this pathogen by acting as signal-supply inhibitors. Both these compounds were able to negate haemolytic ability of P. aeruginosa notably (19.11%; p =0.04 and 35.29%; p= 8.31363E-05 reduction respectively by cinnamic acid and chlorogenic acid), whereas catalase activity was enhanced to a marginal (1.47%) extent only by chlorogenic acid. In vivo efficacy of chlorogenic acid was found to be better than that of cinnamic acid (Figure 7F).\n\nBacterial growth was measured as OD764; OD of pyoverdine was measured at 405 nm, and Pyoverdine Unit was calculated as the ratio OD405/OD764 (an indication of pyoverdine production per unit of growth), Pyocyanin Unit was calculated as the ratio OD520/OD764 (an indication of pyocyanin production per unit of growth). ‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and pigment production of P. aeruginosa. (A) Effect of cinnamic acid on growth and QS-regulated pigment production in P. aeruginosa. (B) Effect of chlorogenic acid on growth and QS-regulated pigment production in P. aeruginosa. (C) Effect of catechin on growth and QS-regulated pigment production in P. aeruginosa. (D) Cinnamic acid and chlorogenic acid act as signal-supply inhibitors against P. aeruginosa, whereas catechin acts as a signal-response inhibitor. (E) P. aeruginosa receiving pre-incubation with cinnamic acid or chlorogenic acid becomes more susceptible to cephalexin. (F) Pre-treatment of P. aeruginosa with cinnamic acid or chlorogenic acid reduces its virulence towards C. elegans: Catechin (50 μg/ml) and gentamicin (0.1 μg/ml) employed as positive controls conferred 100% and 80% protection on worm population respectively; DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and compounds at tested concentrations showed no toxicity towards the worm. *p<0.05, **p<0.01, ***p<0.001; AS, Antibiotic susceptibility; QS, Quorum sensing.\n\nCatechin could reduce production of pyocyanin and pyoverdine both, in P. aeruginosa, without affecting its growth (Figure 7C). However, there was not much difference in the effects exerted by different concentrations of catechin. For example, the effect on pyoverdine production exerted by all concentrations in the range 10–100 μg/ml was statistically same; and so was the case for the effect exerted by catechin on pyocyanin production in the concentration range of 100-200 μg/ml. Catalase and haemolytic activity of P. aeruginosa were both negatively affected by catechin (Table 3). Catechin seemed to act as a signal-response inhibitor against P. aeruginosa (Figure 7D).\n\nGA and quercetin were tested against P. aeruginosa at 10–200 μg/ml. Both these compounds were able to inhibit the bacterial growth, and this growth inhibitory effect was more profound in case of quercetin. Production of both the QS-regulated pigments in P. aeruginosa was enhanced by GA. Quercetin had a negative effect on pyocyanin production, whereas pyoverdine production was enhanced by it from 10 μg/ml onwards. Catalase activity remained unaffected in GA-treated P. aeruginosa culture, whereas it was affected to a minor extent in quercetin-treated culture. Both of these plant compounds were able to reduce biofilm-forming capacity of P. aeruginosa notably (Figure 8A, B).\n\n‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and pigment production of P. aeruginosa; Bacterial growth was measured as OD764; OD of pyoverdine was measured at 405 nm, and Pyoverdine Unit was calculated as the ratio OD405/OD764 (an indication of pyoverdine production per unit of growth), Pyocyanin Unit was calculated as the ratio OD520/OD764 (an indication of pyocyanin production per unit of growth); Crystal violet assay was performed to measure biofilm formation, and biofilm eradication, followed by the measurement of OD at 580 nm. (A) Effect of GA on growth and QS-regulated pigment production in P. aeruginosa. (B) Effect of quercetin on growth and QS-regulated pigment production in P. aeruginosa. *p<0.05, **p<0.01, ***p<0.001; AS, antibiotic susceptibility; QS, quorum sensing; GA, gallic acid.\n\nGrowth promoting effect of PGPE on probiotic strains. PGPE was able to enhance growth of L. plantarum and B. bifidum at 10–50 μg/ml. Higher concentration (100 μg/ml) did not seem to promote their growth further (Figure 9).\n\nBacterial growth was measured as OD660. *p<0.05, **p<0.01, ***p<0.001. PGPE: Punica granatum peel extract.\n\n\nDiscussion\n\nOur in vitro and in vivo experiments indicated hydroalcoholic extract of P. granatum peel to possess appreciable antipathogenic activity against both the test bacteria. Antimicrobial activity of pomegranate extracts against bacteria, fungi, and plasmodium has previously been reported by many researchers (Rahmani et al., 2017). Other researchers have reported P. granatum extracts to possess antimicrobial and/or anti-QS effect. However almost all of them have shown the effective concentrations to be much higher than what we report for PGPE in the present study. Ethanolic extracts of pomegranate were shown to inhibit methicillin-resistant S. aureus isolates at 200–400 μg/ml (Voravuthikunchai & Kitpipit, 2005). A high tannin pomegranate extract was reported to be effective against MRSA at 1–5 mg/ml (Su et al., 2012). Helicobacter pyroli isolates were shown to be inhibited by pomegranate extracts at 50–100 μg disc-1. Pomegranate peel extract was reported to inhibit Salmonella typhimurium and Salmonella aureus on meat surfaces, at 250 μg/ml (Tayel et al., 2012). Effective concentrations reported in our study are also lower than the quorum-modulatory concentrations reported by Yang et al., (2016). They reported tannin-rich fraction from pomegranate rind (TFPR) caused a reduction (~50-70%) in C. violaceum pigmentation at 0.312–0.625 mg/ml, whereas comparable pigment inhibition was achieved by our extract at 50–150 μg/ml (Figure 1A). Oh et al. (2015) reported pomegranate peel extract to inhibit bacterial growth at 3–12 mg/ml. Our extract could inhibit violacein production by 78.26% at 10 times lesser concentration (200 μg/ml; Figure 1A) than the concentration (2 mg/ml) they reported for their extract to achieve a comparable (78.5%) violacein inhibition in C. violaceum. Inhibition of biofilm formation of human pathogens by P. granatum extract at <40 μg/ml reported by Bakkiyaraj et al. (2013) matches with our results of the present study (Figure 2E). Nuamsetti et al. (2012) reported pomegranate extracts to exert antibacterial activity against four different bacteria with MIC values reaching 103.6-207 mg/ml.\n\nMuch evidence for antimicrobial action of pomegranate extracts has come from in vitro assays, whose confirmation through in vivo assays is necessary (Howell & D’Souza, 2013). Dazal et al. (2015) reported ethanolic extract of P. granatum pericarp to inhibit QS in P. aeruginosa at 219.78–2197.80 μg/ml; whereas our extract could modulate production of both QS-regulated pigments from 5 μg/ml onwards in vitro; notable in vivo efficacy was observed at 0.5 μg/ml onwards. An ethanolic extract of pomegranate exhibited MICs of 0.49–1.95 mg/ml and MBCs of 1.95–3.91 mg/ml against E. coli O157:H7. Compared to these relatively higher effective concentrations, PGPE reported in the present study exhibited notable (>50% worm survival, as against 12.5–35% in control wells) in vivo efficacy at lower (0.5–5 μg/ml) concentrations (Figure 1D and Figure 2F).\n\nHigher anti-infective efficacy shown by our extract reported in this study may be attributed to the extraction method employed by us i.e. MAE. This method is known to be capable of fast extraction of phenolics Proestos & Komaitis, (2008), and also suitable for heat-labile phytoconstituents (Gupta et al., 2012). The fact that same extract when prepared using different extraction methods, can vary with respect to its efficacy, has previously also been emphasized by us (Kothari et al., 2012).\n\nThis study has found PGPE to be an effective quorum modulator against two gram-negative bacterial pathogens. An ideal quorum-modulator is expected to have no or minimal effect on bacterial growth, and thus to exert lesser selection pressure on the target pathogenic population. PGPE reported in this study was observed to exert a purely quorum-modulatory effect against C. violaceum till 10 μg/ml, and P. aeruginosa till 2.5 μg/ml. It is generally expected that owing to lesser effect on bacterial growth, bacterial populations are likely to develop resistance to quorum-targeting antimicrobials at a slower pace. Further, in the case of plant extracts, there is always a possibility of multiple phytocompounds being simultaneously responsible for the observed antipathogenic effect, and they may be targeting multiple cellular components in the target bacteria, hence the susceptible pathogen population may find it difficult to develop resistance against them. Precisely this was observed in our study with P. aeruginosa, wherein even repeated exposure of this bacterium to PGPE did not induce resistance, and even the PGPE-habituated P. aeruginosa remained susceptible to this extract, in vitro (Figure 2G) as well as in vivo (Figure 2F).\n\nOne of the frequently observed problem with conventional antibiotic treatments is that besides killing the target pathogen, they simultaneously disturb the normal human microbiota, which may lead to the condition of dysbiosis. Hence, it is desirable from an anti-infective preparation that it should not have any negative effect on the normal microbiota organisms. This study has found PGPE to promote growth of two bacteria (L. plantarum and B. bifidum), which are part of normal human gut biota (Figure 9). From these results, PGPE can be said to have appreciable prebiotic potential. Li et al. (2015) had also indicated pomegranate whole fruit extract to work as a potential prebiotic, with beneficial effect on Bifidiobacterium and Lactobacillus. Pomegranate extracts can be expected to exert meaningful prebiotic and anti-infective effect in vivo in human body, as they are rich in ellagitanins, which are hydrolysed in the gut to ellagic acid, which further is metabolized by the colon microbiota to form urolithin A and urolithin B. These urolothins at micromolar concentrations were indicated to be potent in vivo QS-inhibitors by Giménez-Bastida et al. (2012). Prebiotic preparations with bifidogenic property, besides improving gastrointestinal function, can also find applications in management of conditions like depression and psychological distress (Messaoudi et al., 2011). Overall GI tract benefits expected from P. granatum extracts, as indicated in ethnomedicinal records (Colombo et al., 2013; Hollebeeck et al., 2012) can be in part due to its prebiotic potential, which makes use of pomegranate in management of inflammatory bowel disease appear relevant.\n\nOur results regarding effect of PGPE on haemolytic activity of both the test bacteria, and its effect on P. aeruginosa susceptibility to human serum also indicate the high probability of this extract to be of therapeutic relevance. Haemolysis has been considered as an important virulence factor, and infection by haemolytic bacteria may lead to severe anaemia (Orf & Cunnington, 2015). By reducing the haemolytic activity of the pathogens, PGPE-like extracts can significantly reduce iron-availability for the pathogens, as lysis of red blood cells is one of the effective strategies for many pathogens to ensure sufficient supply of iron, necessary for their survival inside host. Enhanced susceptibility of P. aeruginosa, under influence of PGPE, to lysis by human serum is also of clinical significance, as this can aid the clearance of pathogenic bacteria by human body, while facing bacteremia. This may represent an additional possible mechanism by which PGPE may confer in vivo protection against bacterial infection.\n\nAny quorum-inhibitory substance may act either by targeting the signal-synthesis machinery, sequestering the already synthesized signals, or by interfering with the signal-reception (Zhang & Li, 2016). Though different phytocompounds of a given crude extract may have different mode of action, as a whole extract PGPE was found to act as a signal-response inhibitor against C. violaceum (Figure 1B) and P. aeruginosa (Figure 2B). Among the pure compounds tested in this study, except GA, remaining four compounds seemed to act as a signal-response inhibitor against C. violaceum. Notably, cinnamic acid and chlorogenic acid were found to behave as signal-supply inhibitors against P. aeruginosa, and as signal-response inhibitor against C. violaceum. This suggests that same plant compound may act differently against different bacteria. Here this fact becomes even more interesting, given both C. violaceum and P. aeruginosa are gram-negative bacteria, and a heavy overlap is suggested to be there among the QS machinery of different members of gram-negative bacterial group (Papenfort & Bassler, 2016). Since PGPE contains a mixture of signal-supply and -response inhibitors, it can be difficult for the susceptible bacteria to develop simultaneous resistance to both mechanisms (i.e. against the whole extract). Compared to this, it may be easier for the bacteria to develop resistance against single-molecule antibiotics with bactericidal action.\n\nAt 0.5-1 μg/ml PGPE showed in vitro effect neither on growth, nor on pigment production in any of the test bacteria. In case of C. violaceum, any in vitro effect was not observed till 2.5 μg/ml. However, all these concentrations that were ‘not effective’ in vitro were able to significantly reduce virulence of these bacterial pathogens towards C. elegans (Figure 1D and Figure 2F). This fact highlights the necessity of assaying any test extract at broadest possible concentration range in vivo, including at least one level below the lowest effective in vitro concentration. Usually in vitro screens are performed at a broader concentration range to identify the most effective concentration(s), and in vivo assays are conducted at this narrowed concentration range; though logical, this approach carries an inherent risk of missing out some concentrations meaningful in vivo. A simple explanation which can be proposed for this apparent mismatch between in vitro and in vivo results is that, in vitro we look for effect of test extract on a limited number of parameters (e.g. only two parameters, cell density and pigment production, in current study), whereas in vivo assays provide the test extract an opportunity to interact with multiple targets in host as well as pathogen. We may not be able to study all these targets, but the overall effect can be detected in terms of survival benefit of the model host. Even the magnitude of difference in in vitro effects of different concentrations, may not always match with their in vivo effect. For example, in vitro pyoverdine by P. aeruginosa was overproduced 2.51-fold more at 50 μg/ml that that at 25 μg/ml PGPE; but there was no significant difference in the in vivo efficacy of these two concentrations. On the same line, in vitro effect of PGPE at 25-100 μg/ml on C. violaceum pigment production was 42-56%, whereas in vivo effect of these concentrations fell within a narrower range of 75-80% (i.e. a 5% in vivo difference for 14% in vitro difference in efficacy values).\n\nResults of this study also suggest that while screening natural/synthetic compounds for their possible effect on bacterial QS, what we should observe for is ‘quorum modulation’ and not just ‘quorum inhibition’. This is particularly evident from the effect of PGPE on pyoverdine production in P. aeruginosa, wherein this virulence factor production is not inhibited by PGPE, but promoted to quite notable extent, and this pyoverdine-enhancing (modulatory) effect of PGPE does not prevent it from being effective in vivo. Similar enhancement in pigment production by Serratia marcescens challenged with an anti-infective quorum-modulatory polyherbal Panchvalkal formulation was reported by us earlier (Patel et al., 2017), and the formulation was effective in vivo.\n\nWhile investigating the effect of PGPE and/or its constituent phytocompounds against the two gram-negative bacteria in this study, the dose-response relationship was not always found to be linear. Since dose-response relationships can be described by various models (Calabrese, 2004), we made an effort to see, which model fits best to results obtained in this study. For example, effect of PGPE on C. violaceum growth did not follow a linear pattern. Concentrations till 2.5 μg/ml affected neither cell density nor pigment production (Figure 1A); this phenomenon can be said to fit in the threshold model, wherein the biological effect is not observed until the threshold concentration is crossed. This concept of threshold model also seems applicable while describing effect of PGPE on P. aeruginosa cell density and pigment production, as none of the parameters seem to be affected until the threshold concentration of 1 μg/ml was not crossed (Figure 2A). At higher concentrations, the effect on pyoverdine production seemed to somewhat follow an inverted U-shaped hormetic model. Whether such a pattern would have continued at still higher concentrations, cannot be predicted without actually performing experiment at those concentrations. Similar analysis with respect to pure compounds revealed that effect of GA on C. violaceum cell density and pigment production (Figure 6A) can be said to follow the linear non-threshold model, within the tested concentration range. The term ‘hormesis’ is used in general to describe a biphasic dose response (Mattson, 2008), and this concept can be relevant while describing ‘adaptive stress response’ in bacteria challenged with some antimicrobial/quorum modulatory agent. Hormetic response can be said to occur (or not to occur) within the concentration range actually tested during a particular experiment, but it need not to be extrapolated into the realm of uncertain i.e. concentrations not really tried, on either side of the test concentration range.\n\nOverall, the whole extract seemed to be more efficacious than any of the pure compounds tested. For example, at 10 μg/ml, PGPE affected pyoverdine production in P. aeruginosa much more than either GA or quercetin. Such synergistic action of the pomegranate constituents, apparently being superior to that of its individual constituents, has been indicated in literature (Jurenka, 2008). Among the many constituents of PGPE, GA in this study was found to act as a signal-supply inhibitor against C. violaceum, whereas all other compounds as well as the whole extract were found to act as signal-response inhibitor against this bacterium. There may be few other unknown compounds present in this extract, which may either inhibit AHL synthesis, sequester synthesized AHLs, or bind to the LuxR component. It is likely to be difficult for the bacteria to develop complete resistance against any such poly-component preparation, as different compounds will target different components of the bacterial QS machinery. Anand et al. (2013) has also indicated the combination of LuxR non-competitive inhibitors and LuxI inhibitors, as a robust therapeutic strategy to act multiplicatively across a broad parameter range.\n\nIn silico docking exercise indicated both, quercetin and, catechin to have affinity for CviR more than its natural ligand C6-HSL (Table 2), i.e. a high probability of them being capable of acting as signal-response inhibitor. In vitro experiments validated this possibility (Figure 6D, F). Though the docking scores of both these phytochemicals against CviR were identical, in vitro violacein production was inhibited bit more by quercetin. Gallic acid, which showed second lowest affinity (-5.9 kcal/mol) for CviR among all docked phytocompounds, was also not able to act as signal-response inhibitor, instead it acted as signal-supply inhibitor against C. violaceum, and its docking score against the signal synthesizing enzyme AHL synthase was also better i.e. (-7.9 kcal/mol). Cinnamic acid displayed lowest in silico binding affinity against P. aeruginosa receptor protein PqsR (Table 2), and this corroborates well with the fact revealed from in vitro experiment that it acts as a signal-supply inhibitor (Figure 7D). Though the binding affinity of catechin and quercetin both, against P. aeruginosa protein LasR was identical as per docking score, indicating a high probability of them acting as a signal-response inhibitor with respect to pyoverdine, whose production is controlled by the las system, and in vitro experiments did find catechin to act as a signal-response inhibitor (Figure 7D); pyoverdine production was affected much more by quercetin than by catechin. If we focus on docking scores of only those five compounds, with which in vitro experiments were also conducted in this study, against both the P. aeruginosa receptors, quercetin gives an impression of being more effective than GA, and in vitro experiments also show quercetin to have more effect on pigment production by this bacterium, than GA (Figure 8A and 8B). Similarly docking scores of catechin against both P. aeruginosa receptor proteins were better than that of cinnamic acid (Table 2), and in vivo assay also found catechin to be more efficacious than cinnamic acid (Figure 7F). Overall there seems to be an empirical match between the in silico and wet-lab experiments.\n\nThis study has found PGPE to be an effective quorum-modulator against two different antibiotic resistant strains of gram-negative bacteria. In general, it is more challenging to find antimicrobials effective against gram-negative bacteria, than against gram-positive bacteria, mainly for the reason that the former possess an additional barrier in form of outer membrane, posing extra-challenge to the intracellular entry of any antimicrobial. Besides affecting QS-regulated pigment production in the test bacteria, this extract could also effectively modulate other traits, including catalase activity, haemolytic activity, biofilm formation, susceptibility of bacteria to antibiotics and to lysis by human-serum factors. Though researchers have reported the antimicrobial potential of P. granatum extracts (Al-Zoreky et al., 2009; Choi et al., 2011; Prashanth et al., 2001) or nanoparticles derived from it (Devanesan et al., 2018), reports describing in vivo anti-infective efficacy of PGPE with focus on its quorum modulatory potential are still warranted. This extract was not found to inhibit growth of test bacteria too heavily, at effective quorum-modulatory concentrations, and this is what is expected from a ‘good’ quorum-modulator (i.e. having no or minimal effect on growth of the test bacterium, and exerting no great selection pressure on the pathogen population).\n\nFurther studies regarding elucidation of the molecular mechanism associated with such appreciable anti-infective activity of PGPE is warranted, as that will give us a better insight into its mode of action. This study also highlights the importance of using different phytocompounds in combination (e.g. as a whole extract; concept of synergy), rather than always focusing on the single-active-molecule approach. Results reported here can be said to be supportive of the concept of polyherbalism (Parasuraman et al., 2014) widely employed by the practitioners of complementary and alternative medicine.\n\n\nData availability\n\nAll raw data containing the underlying data behind each figure and table are available on figshare. DOI: https://doi.org/10.6084/m9.figshare.7471979 (Joshi et al., 2018).\n\nExtended data are available on figshare. DOI: https://doi.org/10.6084/m9.figshare.7471979 (Joshi et al., 2018).\n\nFigure S1. Antipathogenic effect of Pomella on P. aeruginosa.\n\nFigure S2. Quorum-inhibitory effect of cinnamic and chlorogenic acid on violacein production in C. violaceum is not reversed following augmentation of the quorum-inhibited culture by AHL\n\nTable S1. Binding affinity of different phytocompounds when docked against LuxR homologue of test pathogens.\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 work received no extramural funding, and was fully supported by our departmental research budget (Institute of Science, Nirma University).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nAuthors thank NERF (Nirma Education and Research Foundation), Ahmedabad for financial and infrastructural support; Viridis Biopharma Pvt. Ltd., Mumbai for HPLC analysis of PGPE; Pharmanza Herbal Pvt. Ltd. for consenting to mention data on their product ‘Pomella’; Dr. Archana Mankad, Deparment of Botany, Gujarat University for authentication of plant material; and Microbiology Department, M.G. Science Institute, Ahmedabad for providing culture of P. aeruginosa.\n\n\nReferences\n\nAdonizio A, Kong KF, Mathee K: Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. 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PubMed Abstract | Publisher Full Text\n\nMathabe MC, Nikolova RV, Lall N, et al.: Antibacterial activities of medicinal plants used for the treatment of diarrhoea in Limpopo Province, South Africa. J Ethnopharmacol. 2006; 105(1–2): 286–293. PubMed Abstract | Publisher Full Text\n\nMattson MP: Hormesis defined. Ageing Res Rev. 2008; 7(1): 1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcClean KH, Winson MK, Fish L, et al.: Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology. 1997; 143(Pt 12): 3703–3711. PubMed Abstract | Publisher Full Text\n\nMessaoudi M, Lalonde R, Violle N, et al.: Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects. Br J Nutr. 2011; 105(5): 755–764. PubMed Abstract | Publisher Full Text\n\nMoore JD, Rossi FM, Welsh MA, et al.: A Comparative Analysis of Synthetic Quorum Sensing Modulators in Pseudomonas aeruginosa: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design. J Am Chem Soc. 2015; 137(46): 14626–14639. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNazzaro F, Fratianni F, Coppola R: Quorum sensing and phytochemicals. Int J Mol Sci. 2013; 14(6): 12607–12619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeun BW, Ilinskaya AN, Dobrovolskaia MA: Analysis of hemolytic properties of nanoparticles. NCL method ITA-1 Version 1.2, Nanotechnology Characterization Laboratory, Frederick, MD. 2015. Reference Source\n\nNuamsetti T, Dechayuenyong P, Tantipaibulvut S: Antibacterial activity of pomegranate fruit peels and arils. Sci Asia. 2012; 38(3): 319–322. Publisher Full Text\n\nOh SK, Chang HJ, Chun HS, et al.: Pomegranate (Punica granatum L.) peel extract inhibits quorum sensing and biofilm formation potential in Yersinia enterocolitica. Microbiol Biotechnol Lett. 2015; 43–4: 357–366. Publisher Full Text\n\nOrf K, Cunnington AJ: Infection-related hemolysis and susceptibility to Gram-negative bacterial co-infection. Front Microbiol. 2015; 6: 666. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPagliarulo C, De Vito V, Picariello G, et al.: Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli. Food Chem. 2016; 190: 824–831. PubMed Abstract | Publisher Full Text\n\nPapenfort K, Bassler BL: Quorum sensing signal-response systems in Gram-negative bacteria. Nat Rev Microbiol. 2016; 14(9): 576–588. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParasuraman S, Thing GS, Dhanaraj SA: Polyherbal formulation: Concept of ayurveda. Pharmacogn Rev. 2014; 8(16): 73–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatel I, Patel V, Thakkar A, et al.: Tamarindus indica (Cesalpiniaceae), and Syzygium cumini (Myrtaceae) seed extracts can kill multidrug resistant Streptococcus mutans in Biofilm. J Nat Remed. 2013; 13(2): 81–94. Reference Source\n\nPatel P, Joshi C, Palep H, et al.: Anti-infective potential of a quorum modulatory polyherbal extract (Panchvalkal) against certain pathogenic bacteria. J Ayurveda Integr Med. 2017. Publisher Full Text\n\nPfaller MA, Sheehan DJ, Rex JH: Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clin Microbiol Rev. 2004; 17(2): 268–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrashanth D, Asha MK, Amit A: Antibacterial activity of Punica granatum. Fitoterapia. 2001; 72(2): 171–173. PubMed Abstract | Publisher Full Text\n\nProestos C, Komaltis M: Application of microwave-assisted extraction to the fast extraction of plant phenolic compounds. LWT Food Sci Technol. 2008; 41(4): 652–659. Publisher Full Text\n\nRahmani AH, Alsahli MA, Almatroodi SA: Active constituents of Pomegranates (Punica granatum) as potential candidates in the management of health through modulation of biological activities. Pharmacogn J. 2017; 9(5): 689–695. Publisher Full Text\n\nRamadan MA, Tawfik AF, Shibl AM, et al.: Post-antibiotic effect of azithromycin and erythromycin on streptococcal susceptibility to phagocytosis. J Med Microbiol. 1995; 42(5): 362–366. PubMed Abstract | Publisher Full Text\n\nRamanuj K, Bachani P, Kothari V: In vitro antimicrobial activity of certain plant products / seed extracts against multidrug resistant Propionibacterium acnes, Malassezia furfur, and aflatoxin producing Aspergillus flavus. Res Pharm. 2012; 2(3): 22–31. Reference Source\n\n[Report]: Anti-microbial resistance: setting the social science agenda. Report of an ESRC Working Group. 2014; 1. Reference Source\n\n[Report]: Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. World Health Organization Report. 2017. Reference Source\n\nSaxena R, Sharma R, Nandy BC: Chromatographic determination of phenolic profile from Punica granatum fruit peels. Int Res J Pharm. 2017; 8(1): 61–65. Publisher Full Text\n\nShaw PD, Ping G, Daly SL, et al.: Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci U S A. 1997; 94(12): 6036–6041. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSu X, Howell AB, D’Souza DH: Antibacterial effects of plant-derived extracts on methicillin-resistant Staphylococcus aureus. Foodborne Pathog Dis. 2012; 9(6): 573–578. PubMed Abstract | Publisher Full Text\n\nTayel AA, El-Tras WF, Moussa SH, et al.: Surface decontamination and quality enhancement in meat steaks using plant extracts as natural biopreservatives. Foodborne Pathog Dis. 2012; 9(8): 755–761. PubMed Abstract | Publisher Full Text\n\nTrafny EA, Lewandowski R, Zawistowska-Marciniak I, et al.: Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids. World J Microbiol Biotechnol. 2013; 29(9): 1635–1643. 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 | Free Full Text\n\nUnni K, Priji P, Geoffroy V, et al.: Pseudomonas aeruginosa BUP2—A novel strain isolated from malabari goat produces Type 2 pyoverdine. Adv Biosci Biotechnol. 2014; 5(11): 874–885. Publisher Full Text\n\nVandeputte OM, Kiendrebeogo M, Rajaonson S, et al.: Identification of Catechin as one of the flavonoids from Combretum albiflorum bark extract that reduces the production of quorum-sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1. Appl Environ Microbiol. 2010; 76(1): 243–253. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVasavi HS, Arun AB, Rekha PD: Inhibition of quorum sensing in Chromobacterium violaceum by Syzygium cumini L. and Pimenta dioica L. Asian Pac J Trop Biomed. 2013; 3(12): 954–959. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVoravuthikunchai SP, Kitpipit L: Activity of medicinal plant extracts against hospital isolates of methicillin-resistant Staphylococcus aureus. Clinic Microbiol Infect. 2005; 11(6): 510–512. PubMed Abstract | Publisher Full Text\n\nWeydert CJ, Cullen JJ: Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc. 2010; 5(1): 51–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang Q, Wang L, Gao J, et al.: Tannin-Rich Fraction from Pomegranate Rind Inhibits Quorum Sensing in Chromobacterium violaceum and Biofilm Formation in Escherichia coli. Foodborne Pathog Dis. 2016; 13(1): 28–35. PubMed Abstract | Publisher Full Text\n\nZhang W, Li C: Exploiting Quorum Sensing Interfering Strategies in Gram-Negative Bacteria for the Enhancement of Environmental Applications. Front Microbiol. 2016; 6: 1535. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "43899",
"date": "19 Feb 2019",
"name": "Saif Hameed",
"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\nIn this study, the authors analyze an “Anti-infective potential of hydroalcoholic extract of Punica granatum peel against gram-negative bacterial pathogens” with various experiments QS, biofilm hemolytic activity, catalase activity, surface hydrophobicity. Authors also performed in-vivo activity with C. elegans model organism and also analyzed with docking tool. While this study is comprehensive and important for better understanding of gram negative bacteria (C. violaceum and P. aeruginosa) physiology and pathogenesis, experiments are enough and paper sounds good but requires minor corrections with respect to the overall presentation of the manuscript.\nComment 1: In the introduction, the ESRC reference should be changed as it is from 2014.\nComment 2: Brief description about AMR and how bacteria develop these mechanism would be better.\n\nComment 3: Broth dilution assay: Give the proper reference of MIC determination method and how they defined MIC for example (MIC50, MIC80 or MIC90) or complete at the end point of experiment. Better to put the reference of MIC according to CLSI guidelines.\nComment 4: Measurement of bacterial growth and pigment production: better to merge this sentence with above and keep the pigment production in separate heading.\n\nComment 5: In vivo assay: last day of the experiment, when plates could be opened, their death was also confirmed by touching them with a straight wire, wherein no movement was taken as confirmation of death. Please mention what metal was used.\n\nComment 6: In Results section:\nFigure 1A: is not clear and difficult to understand. The values shown in boxes or brackets - what are these values defining? Are they necessary to show? If yes, better to show in table form. Similarly for Fig 1C. Figures 1D: Better to show C. elegans survival graph along with their pictures (Fig. 1D 2F, 3B). Figure 2: very overcrowded and messy. Is it necessary to show 5 figures A,B,C,D,E,F in a single panel? If there is any figure limitation then show only relevant ones and put rest as supplementary figures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "43897",
"date": "21 Feb 2019",
"name": "Sisir Nandi",
"expertise": [
"Reviewer Expertise Synthesis of bioactive molecules",
"drug design",
"discovery of lead and molecular modeling."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChinmayi and co-workers studied hydroalcoholic extract of P. granatum peel (PGPE), prepared by microwave assisted extraction method for its quorum-modulatory potential against two different human-pathogenic bacteria viz. Chromobacterium violaceum and Pseudomonas aeruginosa. This is a very significant work to produce antimicrobials. The following minor comments should be considered before acceptance of this article.\n\n\"Though the modern medicine has almost always focused on search of purified active molecules as therapeutic leads, knowledge of Indian/Chinese/Arabian traditional medicine makes us aware of a variety of plant extracts and inorganic formulations prescribed for management of infections\". Cite the references. \"One such widely mentioned item in these ancient systems of complementary and alternative medicine, including the Indian system Ayurved, is Punica granatum L. extracts\". Cite the references. \"In India, Tunisia, and Guatemala, decoction of driedpeels of P. granatum is employed externally as well as internally as an astringent and germicide, and utilized for treating aphthae and diarrhoea\". Cite the references. \"Klebseiella Pneumonia\" Spelling mistake is there. It should be corrected as klebsiella pneumonia. \"Incubation temperature and time for C. violaceum, L. plantarum, and B. bifidum was 37°C, and 22–24 h\". Cite the references. \"Antibiotic susceptibility profile of the bacterial strains used in this study was generated using the antibiotic discs- Dodeca Universal – I, Dodeca G - III – Plus, and Icosa Universal -2 (HiMedia, Mumbai)\". Cite the references. \"The plant name was checked against http://www.theplantlist.org on April 20, 2018. Collected peels were shade-dried, before being used for extract preparation. Biological activity of the plant fruit even depends on the time and season of productions\". This fruit is generally produced in season in the Northern Hemisphere from September to February and in the Southern Hemisphere from March to May. Please comment on this. Authors are being also suggested to carry out the same experiment using the Peels of P. granatum were procured from different times. (optional) \"Purity of each of the extracted pigment was confirmed by running a UV-vis scan (Agilent Cary 60 UV-visible spectrophotometer). Appearance of single major peak (at the λmaxx reported in literature) was taken as indication of purity\". Cite reference.\n\nFig 4 is not clear and acceptable in the present form. Please prepare a high resolution figure as per the authors instruction. Why did the author do molecular docking study? It should be clearly mentioned in the methods section along with references1,2. Docking score is not the ultimatum measurement of potency or binding affinity of the ligand. I suggest giving the figures of mode of ligand-receptor interactions and discussing the crucial interacting amino acids of the protein. \"One ethnomedical practice reported from the Paliyar tribe from Tamilnadu in India involves taking internally, dried fruit coat of P. granatum after grinding and mixing with water, to treat stomach ache and diarrhoea (Duraipandiyan et al., 2006)\". Correct the typographical error of Tamilnadu. The present version contains many grammatical and typographical errors. Authors are suggested to do these corrections. \"Inoculum standardized to 0.5 McFarland turbidity standard was added at 10% v/v, to the media supplemented with required concentration of PGPE, followed by incubation at appropriate temp for each organism\". Correct the spelling error of temp. Separate conclusion should be given based on experimental and in-silico 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? No",
"responses": []
},
{
"id": "43898",
"date": "25 Feb 2019",
"name": "Shilpa Deshpande Kaistha",
"expertise": [
"Reviewer Expertise Antimicrobial resistance and biocontrol strategies"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study provides comprehensive experimental evidence for the activity of Punica granatum peel extract in modulating pathogenicity of Pseudomonas aeruginosa and quorum sensing pigment producing Chromobacterium violaceum. The effect of PGPE on various aspect of pathogenicity related to antibiotic sensitivity, biofilm formation, surface hydrophobicity, quorum sensing, hemolysin & catalase production and in vivo C. elegans infection model together make a comprehensive and elegant study of the herbal extract activity. The effect of concentration dependent PGPE treatment on various parameters and subsequent generations of the pathogens make for an interesting read. It is recommended that the article be indexed with some minor improvements as suggested below:\nIntroduction: At several places, relevant references may be cited.\nMaterial and Methods:\nPigment production can be separate heading and subheadings for each pigment, to make clear. Growth measurement can be included in the write up. Details of Chloramphenicol enhanced catalase maybe provided.\nResults:\nThe quality of all the figures requires significant improvement. The details of significant values maybe kept in the figures to avoid crowding and enhance understanding. Font size of Figures, for example Figure 2C, needs to be clarified. The standard deviations are not visible in most of the figures. A brief summary of comprehensive results at the end of each section will help the reader grasp the crux of the experiment without having to go through the written text regarding working concentrations of the extracts etc.\nDiscussion:\nRequires a section wherein the findings are succinctly concluded regarding the findings for Ps. aeruginosa and C. violaceum. Please include in Discussion why does the PGPE enhance probiotic activity while showing anti-infective activity against the pathogens studied.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-70
|
https://f1000research.com/articles/8-396/v1
|
08 Apr 19
|
{
"type": "Research Article",
"title": "Consumer perspectives on reproductive health after using the Jakpros smart mobile health application: A cross-sectional, qualitative study",
"authors": [
"Budi Wiweko",
"Aida Riyanti",
"Shanty Olivia",
"Muhammad Priangga",
"Vita Silvana",
"Aruan Lewis Putro",
"Yohanes Satrya Wibawa",
"Ilonna Putri Pertiwi",
"Sarah Chairani Zakirah",
"Achmad Kemal Harzif",
"Gita Pratama",
"R. Muharam",
"Andon Hestiantoro",
"Aida Riyanti",
"Shanty Olivia",
"Muhammad Priangga",
"Vita Silvana",
"Aruan Lewis Putro",
"Yohanes Satrya Wibawa",
"Ilonna Putri Pertiwi",
"Sarah Chairani Zakirah",
"Achmad Kemal Harzif",
"Gita Pratama",
"R. Muharam",
"Andon Hestiantoro"
],
"abstract": "Background: Good reproductive health is important for individuals and also for the development of children. Knowledge plays an important role in women’s reproduction health. Our study examined women’s perspectives and knowledge of reproductive health after using the free Jakarta Reproduction Sehat (Jakpros) application (app) on a daily basis for two weeks. Methods: Our study used a cross-sectional, qualitative design. The sample consisted of 12 participants from two sub-district general hospitals in Jakarta. Participants were chosen using purposive sampling and consisted of women of reproductive age who had completed previous questionnaires on their use of Jakpros. We used descriptive analytics and a qualitative method. Data were collected by direct observation in small focus group discussions. Results: Participants said that after they used the Jakpros app, they were more aware of their reproductive health. Their knowledge increased after counselling combined with Jakpros usage. They also said that the features in the app made it easier to contact their doctor and to access their nearest hospital. Conclusion: Jakpros is a convenient way to access reliable reproductive health information.",
"keywords": [
"Jakarta Reproduksi Sehat",
"Jakpros",
"Mobile Application",
"Reproductive Health",
"Social Media"
],
"content": "Expression of Concern\n\nSince publication, the F1000Research editorial office has been informed of concerns regarding suspected duplicate publication of this article. We have contacted the authors and all editorial activity has been suspended pending further investigation. Further action will be dependent on the outcome of these discussions.\n\n\nIntroduction\n\nGood reproductive health is important for individuals and also for the development of children. Reproductive health is, or should be, a serious concern for every woman. A nation’s mortality and morbidity are dependent on the reproductive health of its citizens. Knowledge plays an important role in women’s reproduction health. For example, it informs them on how and when to seek health services1.\n\nMobile health applications (apps) are rapidly proliferating and are widely used around the world to access health information2. In 2018, more than 50% of people worldwide had at least one mobile health app on their device3. There are more than 100,000 smartphone health apps available today. The main goal of internet-based health apps is ready accessibility4. Mobile health apps have a number of benefits: they enable fast, easy searches of almost unlimited health information2; they bring doctors and patients closer together; and they provide health information at any time5.\n\nThe increasing popularity of mobile health apps is due to a number of things. Firstly, people are more comfortable sharing their experiences, expressing emotions, and exchanging ideas on specific topics within a like-minded community. Secondly, people are curious and want more information about their health experiences6. Mobile health apps have been successful in reducing health costs. Preventive action by internet disseminated information is much more efficient than manual methods such as brochures, leaflets or posters7. Technology has the potential to alleviate time management issues involved in counselling, treatment waits, and registration7.\n\nOne limitation of mobile health apps is that, due to the lack of quality controls on the internet, they vary widely in quality and accuracy. Providing good-quality, evidence-based health information to patients is an important goal6.\n\nDue to the increasing popularity of mobile health apps, the Jakarta Reproduction Sehat (Jakpros) smart mobile app was developed in 2018 (Figure 1). This free app is specifically designed to provide women with a convenient way to improve reproductive health knowledge. Jakpros has a section that shows the nearest hospitals from patients to help them access medical care. Other features include an educational page with general information on reproductive health, which is divided into several sections (e.g. contraception, high-risk pregnancy, cancer). Furthermore, the Jakpros application has a question and answer service supported by obstetrics and gynecology doctors (Figure 2B).\n\n(A) Jakpros log-in page; (B) Jakpros news page.\n\n(A) Educational Page Feature; (B) Questions and Answers Section.\n\nCurrently, the Jakpros app is used in several subdistrict hospitals in Jakarta. The aim of the present study was to examine perspectives and knowledge of reproductive health issues in women who used Jakpros as part of their daily activity. Our results will help us improve the usage and quality of the Jakpros app for women in the community.\n\n\nMethods\n\nThis study follows on from our past quantitative research8. In the previous study, participants used the Jakpros smart mobile app for 2 weeks. During this time, they read the educational page feature to enrich their knowledge (Figure 2A). They could also consult their healthcare providers about their complaints or for health information (Figure 2B). We saw an improvement in health knowledge using pre- and post-test questionnaires. In the current study, we explored participants’ opinions and detailed perspectives on reproductive health and the Jakpros application. We used a qualitative method to analyze the main topic.\n\nWe explored the different perspectives and behaviors of participants in detail. We adopted phenomenology study as our basic paradigm. The goals of this paradigm are to evaluate participants’ experience to be as factual as possible. Furthermore, it describes the different perspectives of the participants9.\n\nParticipants were recruited in May 2018. All participants had participated in a previous quantitative study of Jakpros. We recruited participants from two sub-district hospitals in Jakarta: Tanjung Priok Subdistrict Hospital and Tanah Abang Sub-district Hospital. We used purposive sampling of women with the following criteria:\n\nThey had previously participated in our quantitative research.\n\nThey had used the Jakpros application for 2 weeks.\n\nThey lived in Jakarta.\n\nThey provided written informed consent to participate.\n\nOur study method was focus group discussion and direct observation during these discussions. The study consisted of two groups of six participants interviewed on different days. Discussions were held in the hospital hall. Our research team for the discussions consisted of two obstetrics and gynecology specialists (AR and SO) and one general practitioner (IPP).\n\nThe participants were instructed to use the Jakpros app for two weeks on a daily basis. Information on nutrition during pregnancy, contraception and family planning, cervical cancer, and high-risk pregnancy were given to participants in the Jakpros app, such as.\n\nThe list of questions was prepared by our obstetrics and gynecology specialist from Universitas Indonesia. Each participant was interviewed within 10 minutes using a list of questions.\n\nThe reproductive section of the discussion used the same questions as our previous questionnaire8 by modifying the quantitative questions into qualitative questions. For this study, we were able to record participants’ answers and their perspectives too. Participants were asked about their reproductive knowledge in areas such as cancer, cervical cancer, pregnancy care, contraception, and high-risk pregnancy. We asked these questions to measure participants’ knowledge after using Jakpros for 2 weeks. Next, we focused on participants’ use of the Jakpros application. They shared their personal experiences of using it in their day-to-day lives. Participants were asked about obstacles and benefits of using the application.\n\nIn the discussion, we asked questions and participants took turns to answer them. We also facilitated the discussion. We emphasized that there were no wrong or right answers, we did not offer opinions and took a neutral stance to answers. We tried to make the discussion as relaxed as possible so participants would enjoy it. Each discussion lasted one hour, we audio-recorded all of the discussion and documented it with photos. For data analysis, we transcribed the data from audio-recorder into narrative document.\n\n\nResults\n\nThe discussion forum was conducted with 12 participants from two sub-district hospitals in Jakarta. Most of them were homemakers, 24–38 years old with high school diplomas as their highest educational qualification. All had participated in our previous quantitative study, which included a post-test questionnaire. This current study was a qualitative study, with results obtained using focused discussion.\n\nOur first question was about cancer in reproductive health, and the discussion of perspectives went into considerable depth. Early identification and treatment of cancer improves outcomes, so awareness can be life-saving. Answers were promising; participants knew about several types of cancer in women and their initial symptoms, and the etiology of cervical cancer and that it is preventable by HPV vaccination. Some of them knew about the type of examination for detection of malignancy, and treatments such as radiotherapy or hysterectomy in cervical cancer. They understood about the importance of hygiene in cervical cancer prevention, and that more sexual partners increases the risk of cervical cancer.\n\n“Cervical cancer is cancer in the neck of uterus. I know other types of cancer in women such as ovarian cancer. For the signs, bleeding is a sign for cervical cancer and bump in breast for breast cancer. I heard food can be an etiology for cervical cancer. A blood test is for detecting cervical cancer. I heard too for cervical cancer treatment, the doctor will do cryotherapy, but I do not know what it is and doctor will do surgery too. To prevent it, we have to maintain good genital hygiene such as changing pads regularly during periods and getting HPV vaccination. I think people who are sexually active have to have further examinations for early detection and if the result is positive, the treatment will be done earlier.”\n\n“The breast cancer shows as a bump in the breast and changes in the skin surface like an orange skin. I’ve heard about USG for detecting ovarian cancer and, if positive, the doctor will do radiotherapy.”\n\n“I think bloody pee is a sign of cervical cancer, but I am not sure. It’s caused by HPV virus. Causes can be genetic and a large number of sexual partners. Detection is by pap smear examination. One of type of treatment is chemotherapy. My friend did it before, she had cervical cancer and now she has passed away. Preventable measures are good hygiene and being faithful to your sexual partner. I heard about HPV vaccine, you will do it three times and you can get hepatitis B vaccine too. If people are sexually active, they have to do routine pap smears or IVA tests at least once a year.”\n\n“The important things for prevention are don’t ever change your sexual partner, be faithful and keep good hygiene. Get your pap smear test after marriage.”\n\n“Bleeding is an early sign of uterus neck cancer. I do not know the difference between cervical cancer or uterus neck cancer. But I know IVA is an early test to detect malignancy. If you are sexually active, you should get the test so you can be treated early if you have it.”\n\nThe second question was about anemia and nutrition in pregnancy. Anemia and nutrition are related, and this information is covered in the Jakpros education section. Most participants did not know the initial symptoms of anemia, or the main nutrition components recommended during pregnancy, but most participants knew the main symptoms of anemia. They knew about tests and treatment for anemia, but there was less understanding of the importance of iron in food. Surprisingly, just one participant noted that postpartum hemorrhage can have anemia as a complication. Their answers about nutrition showed only basic knowledge.\n\n“Sign of anemia are dizziness and weakness. Anemia is decreased of red blood cells which can be measured through blood test. So the doctor will give blood booster supplement. If you get anemia during your pregnancy, you will need a blood transfusion after birth. I know components of nutrition are carbohydrate, calcium, iron, fat, vegetable and vitamins. Those are so important in pregnancy. Calcium for fetal bone. Protein for fetal brain, carbohydrate for fetal weight and iron for mother blood. I am not sure, but I think you have to take folic acid, iron and calcium supplement once in daily during trimester 2 and 3.”\n\n“Anemia is decreased iron in your body. I eat meat regularly to prevent it. Bleeding after birth can cause anemia which is a serious side effect. I know folic acid important for your baby’s brain.”\n\n“If your blood is decreased you will be dizzy. The reason you feel it is because your nutrition is low. Good nutrition is important.”\n\n“Anemia can be prevented by eating food that contains iron. A baby’s weight will be low because of it.”\n\nAnemia is lack of blood, we need vitamins.”\n\n“During anemia, the sign is nausea.”\n\nWe continued the discussion by asking about contraception and family planning. Knowledge on contraception and family planning are crucial for all couples. Although two or three participants declined to answer or participate in discussion of previous questions, most answered this question. This appeared to reflect their knowledge of or interest in these topics. They answered the question and shared their personal experience. Most participants were sexually active and knew about contraception. They knew what its main goals were, and how various contraception types functioned, and what their side effects were. They were also confident in their ability to choose contraception. In most cases, their husbands and doctors were involved in their choice of contraception. Participants felt that they knew their bodies better than other people and considered what was best for their bodies when they chose contraception.\n\n“Type of contraception are condom, pill, IUD and injection. I have used the pill and it made me fat. Pills and injections will make you gain weight. For injections, you must have regular injections monthly or every three months. I was lazy and I choose an IUD. It can be used for five years. I had a USG check before choose the IUD and I involved my husband in my choice. Now, I use an IUD for contraception”\n\n“The aim of contraception is to space pregnancies. I used not to use contraception. I was pregnant 1.5 years ago and I am two months pregnant right now.”\n\n“The only contraception for preventing STD is condoms. I know that side effects of the pill and injections is bleeding. I talked to my husband before I choose contraception. Our convenience is important.”\n\n“I used contraception for spacing my pregnancies. Types of contraception are the calendar method (the traditional method), pills, condoms, and surgery. Condoms can prevent STD, the pill causes bleeding. IUDs will make you bleed too, but not gain weight. If you are careless, you shouldn’t use the pill or injection. I saw my doctor before I chose contraception. Now I use natural methods such as the calendar method and coitus interruptus. My husband and I can work together.”\n\n“I take a pill a day. I always remember it. It depends on your body, just you know it well.”\n\n“IUD can be used for five years. It works for me.”\n\nThis was the last question about reproductive health. In this section, we asked about the signs of high-risk pregnancy. We wanted to see how aware participants were of its symptoms. They seemed to know early signs, but not much beyond that. They knew it affected both the fetus and themselves. For this question, participants did not describe their experiences. They offered fewer comments than for the previous question. We assumed that they had never been in that situation, or that they did not know much about this topic. But we considered it positive that they were aware of early signs of risk.\n\n“High-risk pregnancy is if the mother has a history of asthma, untreatable nausea and bleeding. We have to take vitamins regularly and control every month to prevent it.”\n\n“If mother has diabetes, the baby will be overweight, and it is not healthy.”\n\n“Hypertension in pregnancy causes side effects such as seizure and the death of fetus. Headache is an early sign. You can check your blood pressure.”\n\n“An example of high risk is a mother with asthma. People have to have bed rest in pregnancy and not get too tired.”\n\n“Vaginal discharge is serious sign and might lead to preterm delivery.”\n\n“Diabetes in pregnancy is high risk. Be aware.”\n\nOur last question asked about participants’ experiences of the Jakpros application – what were benefits or obstacles for daily use. Participants shared opinions and stories. They were positive about the Jakpros application and its benefits for them, such as increased knowledge.\n\n“I used it to get knowledge about reproductive health. My purpose was to get updated news. So I routinely read the educational page. It would be better if they posted it every day or weekly, so I could be better updated.”\n\n“I checked my doctor’s schedule through Jakpros. It made easier to access information, and I didn’t have to call the hospital to ask. I can check my pregnancy estimate by inputting the dates of my last period. Unfortunately, it is not connected to direct registration and is not available in all Jakarta hospitals.”\n\n“Jakpros has increased my knowledge. I totally trust it because I get it from trusted sources. The question and answer section helped me a lot. Sometimes I feel too shy or lazy to go to a doctor just to ask something. Now I can access information anywhere.”\n\n“I can ask a doctor questions without hesitation and shame. I wish the doctors could be online 24 hours a day, and give faster responses.”\n\n\nDiscussion\n\nIn this study, we explored participants’ knowledge and perspectives of reproductive health and experiences of the Jakpros application after using it on a daily basis for two weeks. Firstly, they could recognize the early signs of cancer or something wrong with their bodies (e.g. bleeding, bumps in breast). Furthermore, for cancer, they knew about basic diagnostic examinations and treatment. Our results were the qualitative study from our previous quantitative study, which found that women are likely to inform themselves of signs and risk factors for cancer in reproductive systems. They were alert to its signs and risk factors10. According to another previous study, patients search the internet for further information about their symptoms and rely on this information to make decisions about their health11. In our study, we found that participants understood about cervical cancer prevention. They told us that to prevent it people have to maintain good genital hygiene, not change sexual partners, and have regular HPV vaccinations. They agreed that sexually active or married people should have routine examinations routinely too for prevention and early detection. In a previous study, social media has been found to be an effective platform for increasing knowledge and awareness of cancer12.\n\nWhen we asked about contraception, we found that participants were more confident and willing to speak. They could explain what contraception was used for, and also knew about the benefits and side effects of various types of contraception. They involved their husbands and doctors in making their decisions about contraception. They perceived contraception as a way of controlling pregnancy. They told us that family planning was important and required the cooperation of their husbands and consideration of their own bodies and needs. They depended on their social networks for information in making their decisions about contraception. The participants said they were more comfortable obtaining information from their doctors after they read information in Jakpros application. However, they were also interested in further discussion on social media.\n\nFor the discussion on pregnancy, we asked participants about anemia, nutrition, and high-risk pregnancy. They knew the definition of anemia, and its early signs, examination, treatment and prevention. They agreed that anemia in pregnancy is a serious condition. Also, they said that nutrition was important in pregnancy, and they could name the nutritional components how they function in pregnancy. They were aware of early signs of high-risk pregnancy. They considered that to prevent pregnancy becoming high risk, women should have regular antenatal care visits and keep a healthy lifestyle during pregnancy. In our past study we found that pregnant women tend to use mobile health applications – for example, to network within communities of people who have the same condition as them. Pregnancy can be stressful, and women can get reassurance by sharing their experiences in online discussion groups. Women also appreciate the convenience of being able to access advice and information any time13.\n\nThe increasing use of digital devices impacts community expectations for accessing health information. All participants agreed that Jakpros provided a service that helped them access information. Our previous study showed that people like to get information as fast as possible and from wherever they are8. Most of our respondents felt that going to the doctor required too much effort to go to doctor13.\n\nPrevious studies have described how poor-quality information online can lead to health consumers making ill informed decisions6 Our study showed that patients did not hesitate to get information from Jakpros, and they felt this platform delivered reliable medical information. In particular, they appreciated the free advice from doctors about their reproductive problems.\n\nIn response to participant comments, there are a number of things we could do to improve the quality of the app. Firstly, to update the educational page more regularly. Secondly, to improve doctor responses to questions, ideally within 24 hours. Lastly, we hope to extend the information on doctor availability from a few sub-district hospitals to all hospitals in Jakarta.\n\nIn our previous quantitative study, we found that user knowledge increased after two weeks’ use. This current study demonstrated users’ perspectives on reproductive health after using the app. In addition to basic knowledge of reproductive health, their level of awareness had increased, as was observed in the previous quantitative study8. The knowledge and awareness provided by Jakpros can help inform women’s important decisions on their reproductive health.\n\nLimitations of this study include the lack of perspective study of the consumers as the references of our qualitative and previous quantitative study of Jakpros application, and the short time allocated to the previous quantitative study.\n\n\nConclusions\n\nRespondents felt that Jakpros was a helpful application for daily use. Future development of this application is still needed to improve its features. Our aim for Jakpros is that it will one day be a platform for women to access reliable health information that they can apply in their lives.\n\n\nEthical approval\n\nThis study was approved by the Ethics Committee of the Faculty of Medicine Universitas Indonesia, Jakarta (Committee reference number: 1326/UN2.F1/ETIK/2018).\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Supplementary Files for Jakpros_F1000. https://doi.org/10.7910/DVN/KMF8BG14\n\nThe project contains the following underlying data files:\n\n- Transkrip Wawancara Pasien NEW – blind name: Transcripts from the focus group discussions\n\nHarvard Dataverse: Replication Data for: Supplementary Files for Jakpros_F1000. https://doi.org/10.7910/DVN/KMF8BG14\n\nThe project contains the following extended data files:\n\n- Wawancara Kualitatif Pasien 1: Interview guidelines\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 research publication is supported by the United States Agency for International Development (USAID) through the Sustainable Higher Education Research Alliance (SHERA) Program for University of Indonesia’s Scientific Modeling, Application, Research, and Training for City-centered Innovation and Technology (SMART CITY) Project, Grant AID-497-A-1600004, Sub Grant IIE00000078-UI-1.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank Ann Seward, ELS, of AuthorAID, for English-language assistance with an early draft of the manuscript.\n\n\nReferences\n\nHaque M, Hossain S, Rumana Ahmed K, et al.: A Comparative Study on Knowledge about Reproductive Health among Urban and Rural Women of Bangladesh. J Family Reprod Health. 2015; 9(1): 35–40. PubMed Abstract | Free Full Text\n\nElfaki AO, Alotaibi M: The role of M-health applications in the fight against Alzheimer's: current and future directions. Mhealth. 2018; 4: 32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu C, Hu Y, Xie J, et al.: The Use of Mobile Health Applications to Improve Patient Experience: Cross-Sectional Study in Chinese Public Hospitals. JMIR Mhealth Uhealth. 2018; 6(5): e126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKayyali R, Peletidi A, Ismail M, et al.: Awareness and Use of mHealth Apps: A Study from England. Pharmacy (Basel). 2017; 5(2): pii: E33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEytan T, Benabio J, Golla V, et al.: Social media and the health system. Perm J. 2011; 15(1): 71–4. PubMed Abstract | Free Full Text\n\nDe Martino I, D'Apolito R, McLawhorn AS, et al.: Social media for patients: benefits and drawbacks. Curr Rev Musculoskelet Med. 2017; 10(1): 141–145. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantoso HYD, Supriyana S, Bahiyatun B, et al.: Android Application Model of \"Suami Siaga Plus\" as an Innovation in Birth Preparedness and Complication Readiness (BP/CR) Intervention. J Family Reprod Health. 2017; 11(1): 30–36. PubMed Abstract | Free Full Text\n\nWiweko B, Riyanti A, Olivia S, et al.: Jakpros: Reproductive Health Education Application for Pregnant Women. IEEE Xplore Digital Library. 2018. Publisher Full Text\n\nHasbiansyah O: Pendekatan Fenomenologi: Pengantar Praktik Penelitian dalam Ilmu Sosial dan Komunikasi. DIKTI. 2005; 56. Reference Source\n\nMukama T, Ndejjo R, Musabyimana A, et al.: Women's knowledge and attitudes towards cervical cancer prevention: a cross sectional study in Eastern Uganda. BMC Womens Health. 2017; 17(1): 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan SS, Goonawardene N: Internet Health Information Seeking and the Patient-Physician Relationship: A Systematic Review. J Med Internet Res. 2017; 19(1): e9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLenoir P, Moulahi B, Azé J, et al.: Raising Awareness About Cervical Cancer Using Twitter: Content Analysis of the 2015 #SmearForSmear Campaign. J Med Internet Res. 2017; 19(10): e344. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYee L, Simon M: The role of the social network in contraceptive decision-making among young, African American and Latina women. J Adolesc Health. 2010; 47(4): 374–380. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPohan S: Replication Data for: Supplementary Files for Jakpros_F1000. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/KMF8BG"
}
|
[
{
"id": "52067",
"date": "06 Aug 2019",
"name": "Kristian Almstrup",
"expertise": [
"Reviewer Expertise Reproduction",
"endocrinology",
"genomics"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Wiweko et al. submitted to F1000Research investigates how the users of the Jakpros app can help users to get a better knowledge of their reproductive health.\n\nAlthough the manuscript appears well-written and is submitted to a scientific research journal as a research article, this study does not appear to be a well-conducted scientific study.\n\nThe data and collection of data appear highly biased for the following reasons: Four of the authors have been involved in the development of the app, the study population is small and appear highly selective, there is no baseline interview (only post-use interviews), the questionnaires have not been validated, and the results are based on group interviews where participants easily can influence the opinion of each other.\n\nFurthermore, the authors have published on the Jakpros app before and in the Wiweko et al. (20191) paper, which is not cited in this manuscript - the title, list of authors, study population, cited references, as well as several whole paragraphs of the text, appear to be the same.\n\nTaken together, this manuscript appears to be a copy of an earlier paper and the study does not adhere to good scientific practise.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-396
|
https://f1000research.com/articles/7-1860/v1
|
28 Nov 18
|
{
"type": "Method Article",
"title": "False positives in trans-eQTL and co-expression analyses arising from RNA-sequencing alignment errors",
"authors": [
"Ashis Saha",
"Alexis Battle"
],
"abstract": "Sequence similarity among distinct genomic regions can lead to errors in alignment of short reads from next-generation sequencing. While this is well known, the downstream consequences of misalignment have not been fully characterized. We assessed the potential for incorrect alignment of RNA-sequencing reads to cause false positives in both gene expression quantitative trait locus (eQTL) and co-expression analyses. Trans-eQTLs identified from human RNA-sequencing studies appeared to be particularly affected by this phenomenon, even when only uniquely aligned reads are considered. Over 75\\% of trans-eQTLs using a standard pipeline occurred between regions of sequence similarity and therefore could be due to alignment errors. Further, associations due to mapping errors are likely to misleadingly replicate between studies. To help address this problem, we quantified the potential for \"cross-mapping'' to occur between every pair of annotated genes in the human genome. Such cross-mapping data can be used to filter or flag potential false positives in both trans-eQTL and co-expression analyses. Such filtering substantially alters the detection of significant associations and can have an impact on the assessment of false discovery rate, functional enrichment, and replication for RNA-sequencing association studies.",
"keywords": [
"Mappability",
"Cross-mappability",
"Co-expression",
"Trans-eQTL",
"RNA-sequencing",
"Alignment"
],
"content": "Introduction\n\nSequence similarity among distinct genomic regions makes alignment of short sequencing reads difficult1,2. Genomes, including the human genome, contain diverse classes of elements with sequence similarity across regions, ranging from large segmental duplications to pseudogenes to transposable elements. Alignment-based quantification of genomic phenotypes such as gene expression or epigenetic signal is less reliable for such regions3–6.\n\nDespite attention to the importance of alignment errors, the full range of consequences is not always considered in downstream analyses. Here, we focus on evidence that sequence similarity between pairs of genes and resulting alignment errors between them may lead to false positives in association studies from RNA-sequencing (RNA-seq) data, specifically in expression quantitative trait locus (eQTL) and co-expression analyses. eQTL studies, revealing associations between genetic variants and gene expression levels, have contributed to a greater understanding of gene regulation and genetics of complex traits7–9. Trans-eQTLs, where the genetic variant is distant or on a different chromosome from the associated gene, are of particular interest, but have proven challenging to identify in human data due to power, confounders, small effect sizes, and other challenges10,11. Given that a trans-eQTL analysis performs genome-wide tests, it is more prone to be affected by systematic errors between genomic regions than a cis-eQTL analysis where only variants close to the target gene are considered. Here, we discuss the impact of alignment errors on RNA-seq association studies. Figure 1A illustrates a cartoon example, where all reads truly originate from transcripts of Gene A, but due to sequence similarity between Gene A and Gene B, some of the reads incorrectly map to Gene B, causing it to erroneously appear to be expressed in the sample. The number of reads misaligned to Gene B across samples may be directly proportional to the number of reads for Gene A, or may be determined by genetic variation creating sequence mismatches with the correct region. In either case, spurious associations can then arise. In Figure 1A, the two genes incorrectly appear to be co-expressed. In addition, a variant associated with expression of Gene A may also appear to be associated with Gene B, giving rise of a false positive trans-eQTL. We note that such errors are not entirely mitigated by filtering multi-mapped reads—some alignment errors may remain between similar regions even among uniquely aligned reads due to genetic variation, errors in the reference genome, and other complications.\n\nA) Some of the reads generated from Gene A are incorrectly mapped to Gene B because of sequence similarity between the genes, leading to false positive co-expression. Consequently, a variant which is a true cis-eQTL of Gene A appears as a false positive trans-eQTL of Gene B. B) We align the ambiguous (orange) k-mers (75-mers from exons and 36-mers from UTRs) from Gene A to the reference genome using Bowtie and count how many k-mers from Gene A map to each other gene to compute cross-mappability. Here, the number beside each ambiguous (orange) k-mer represents the identifier for the ambiguous k-mer based on its position in Gene A.\n\nPrevious studies have shown that uniqueness of sequence in genomic regions should be considered in an analysis of sequencing data4,5,12. Karimzadeh et al. showed that a differential methylation analysis can identify false signals due to poor mappability5. We have previously filtered trans-eQTLs based on sequence similarity as part of the Genotype-Tissue Expression (GTEx) project10 and the Depression Genes and Networks (DGN) study13. Pickrell et al.14 also suggested that the most significant distant eQTL in their RNA-seq study was likely an artifact arising due to sequencing reads originating from a gene near the SNP mapping to another distant gene. Related effects were also discussed in greater depth for microarrays, where probes intended for one gene may cross-hybridize to other genes11,15. In microarray studies, one could identify and replace probes displaying poor specificity, but in RNA-seq, any region of sequence similarity between genes can cause alignment errors. Previous studies have not presented a systematic analysis of alignment-related false positives in RNA-seq association testing.\n\nHere, we report the prevalence of potential false positives in trans-eQTL and co-expression analyses arising from alignment errors. We present a method to assess the potential for mapping error between pairs of genes, which can then be used to filter or flag associations that could arise from these errors. We introduce a new metric, “cross-mappability”, representing the extent to which reads from one gene may be mapped to another gene. Using gene expression data from GTEx10 and DGN13, we demonstrate the impact of misalignment on both trans-eQTL detection and co-expression analysis in real data. Notably, we show that over 75% of trans-eQTLs detected in any GTEx tissue using a naive pipeline are potential false positives, emphasizing that it is critical to consider these errors. To support future studies, we have published codes in Github16 and also made cross-mappability resources publicly available for the human genome (hg19 and GRCh38)17.\n\n\nMethods\n\nWe developed a new metric, cross-mappability, to quantify the potential for incorrect read alignment where reads originating from one gene may incorrectly map to another gene. Based on annotated transcripts for each gene, we evaluated k-mers from exonic and untranslated regions (UTRs) of the reference genome that serve as a proxy for reads in an RNA-seq experiment. We defined cross-mappability from Gene A to Gene B, crossmap(A, B), as the number of Gene A’s k-mers whose alignment, allowing mismatches, start within exonic or untranslated regions of Gene B.\n\nThough cross-mappability is a straightforward metric, its computation is non-trivial due to the size of the genome. We followed a systematic approach to compute genome-wide cross-mappabilities in practice. Inspired by Derrien et al.4, we define mappability of a k-mer as 1Ck, where Ck is the number of positions where the k-mer maps to the genome with a tolerance of up to 2 mismatches. We computed exon- and UTR-mappability of a gene as the average mappability of all k-mers in exonic regions and untranslated regions, respectively. Then, mappability of a gene is computed as the weighted average of its exon- and UTR-mappability, weights being proportional to the total length of exonic regions and UTRs, respectively. Importantly, we only have to compute cross-mappability from genes with mappability < 1, as no k-mer from a gene with mappability = 1 will map to other regions of the genome (i.e. these will all result in cross-mappability of 0). Moreover, we need to consider only k-mers with mappability < 1 from a gene, as uniquely mapped k-mers will not map to other genes. So, we align all such k-mers from exonic and untranslated regions of each gene to the reference genome using Bowtie v1.2.218, tolerating up to 2 mismatches, and then count the number of k-mers whose alignment start within exonic or untranslated regions of every other gene to compute cross-mappability with each gene genome-wide (Figure 1B).\n\nThe length k may be tuned to match particular read length or alignment method. Here, if the value of k is not mentioned for k-mers, the default value of k is 75 for exons and 36 for UTRs. We used a smaller k for UTRs than for exons because UTRs are generally shorter than exons. Notably, mappability of a gene and cross-mappability to/from a gene is undetermined if all the exons of the gene are shorter than 75 bp and all the UTRs are shorter than 36 bp.\n\nWe computed genome-wide mappability and cross-mappability for human genome hg19 using annotations from Gencode v1919. 26,200 (out of 57,820) genes had at least one k-mer cross-mapping to/from another gene. There were 31,167,448 gene pairs (0.93%) that were cross-mappable (cross-mappability > 0). Supplementary Figure 1A shows the cross-mappability distribution. We found that 2.45–4.92% of gene pairs expressed and quantified in five tissues of the GTEx v7 data were cross-mappable (Supplementary Figure 1B). We also computed the same set of resources for human genome GRCh38 using annotations from Gencode v26, all of which are publicly available17.\n\nWe downloaded fully processed, filtered and normalized gene expression data used in GTEx eQTL analysis from the GTEx portal (www.gtexportal.org). For this study, we focused on gene expression data from 5 tissues: whole blood, skeletal muscle, thyroid, sun-exposed skin, and testis. We also obtained covariates including 3 genotype PCs representing ancestry, sex, genotyping platform, and PEER factors20 as released in GTEx v7. Notably, GTEx quantified gene expression levels using uniquely mapped reads only. We downloaded genotype data from GTEx release v7 from dbGaP (accession number: phs000424.v7.p2).\n\nWe also collected genotype, processed RNA-seq, and covariate data for the DGN cohort, which is available through the National Institute of Mental Health (NIMH) Center for Collaborative Genomic Studies on Mental Disorders. Latent factors inferred from the expression data have already been regressed out of the processed DGN data to address hidden confounders, as described in 13. Gene symbols were mapped to Ensembl gene ids using Gencode v19.\n\nWe downloaded the list of trans-eQTLs in 33 cancer types detected by PancanQTL21 from http://bioinfo.life.hust.edu.cn/PancanQTL. For consistency with our study, we used trans-eQTLs where the variant and the gene were on different chromosomes, and the gene symbols were mapped to unique Ensembl gene ids according to Gencode v19.\n\nFor trans-eQTL analysis, we selected autosomal variants with MAF ≥ 0.05 that did not fall in a repeat region as annotated by the UCSC RepeatMasker track22. We tested trans-eQTL association for each inter-chromosomal variant-gene pair using Matrix-eQTL’s linear model test23. For GTEx, three genotype PCs, genotyping platform, sex, and PEER covariates estimated by GTEx were used as covariates in Matrix-eQTL. We computed the false discovery rate using the Benjamini-Hochberg method within each tissue. The covariates used for trans-eQTL replication in DGN were three genotype PCs, sex and age, as the expression data already had latent factors regressed out.\n\nWe quantified co-expression of a pair of genes as the absolute Pearson correlation (|r|) between expression levels of the genes across all available samples. For GTEx, we regressed out all covariates including PEER factors before co-expression analysis. For DGN, we used the corrected data which also regresses out latent factors.\n\n\nResults\n\nTo investigate the effects of alignment errors on trans-eQTL detection, we performed a standard trans-eQTL analysis using data from the GTEx project for five human tissues. For this study, we categorized an eQTL as “cis” if the variant is within 1Mb of the transcription start site (TSS) of the gene, and “trans” if they are on different chromosomes, approximating the regions where cis and trans mechanisms are likely to occur. We call a trans-eQTL “cross-mappable” if any gene within 1Mb of the identified trans-eQTL variant cross-maps to the trans-eQTL target gene. The cross-mappable trans-eQTLs represent suspicious hits that could potentially arise simply due to alignment errors, although cross-mappability does not definitively establish that any individual trans-eQTL is a false positive.\n\nWe identified 19,348 unique trans-eQTLs (variant-gene pairs) at FDR ≤ 0.05 from five tissues corresponding to 14,785 unique SNPs and 1,419 unique genes. Notably, a large majority (75.14%) of these statistically significant trans-eQTLs were cross-mappable. Furthermore, the cross-mappable eQTLs tended to be the most highly significant (ordered by increasing p-value, Figure 2A). In GTEx tissues, 90.8–97.3% of top 1000 trans-eQTLs were cross-mappable, compared to a background rate of 19.1–25.6% (based on all tested variant-gene pairs). The fraction of cross-mappable trans-eQTLs is very high even when we restrict our analysis to protein-coding genes or to genes with mappability ≥ 0.8 (Supplementary Figure 2A–C).\n\nWe observed a similar pattern in the trans-eQTLs reported from RNA-seq data of 33 cancer types21 (Supplementary Figure 2D). We also observed that randomly selected variant-gene pairs susceptible to cross-mapping yield more trans-eQTLs than randomly selected pairs with no cross-mapping potential (Supplementary Figure 3). Overall, the high fraction of cross-mappable eQTLs among the top associations in multiple tissues and multiple datasets indicates that alignment errors could be a major source of artifacts, dominating legitimate trans-eQTLs. It is also important to note that filtering such prevalent potential false-positives necessitates re-assessing FDR. For example, while 4,809 trans-eQTLs with no evidence of cross-mapping (corresponding to 969 unique genes) were among the 19,348 hits from the original scan of GTEx, only 2,456 (corresponding to 228 unique genes) would appear significant if FDR were reassessed after filtering cross-mapping hits.\n\nA) Fraction of cross-mappable trans-eQTLs among the top significant variant-gene pairs (ordered by increasing FDR) in each tissue (color). Each dotted horizontal line represents the background cross-mappable rate in a given tissue. B) An example of likely false positive trans association between the variant chr5:149826526 and the gene RP11- 343H5.4. The coverages (reads per million, RPM) of the trans-eGene RP11-343H5.4 (top) and its cross-mapping gene RPS14 (bottom) in Thyroid are shown along with their exons and UTRs (black lines below the coverage plot), and mappability of 75-mers. The regions of mappability less than 1.0 have sequence similar between the two genes.\n\nWhen we further analyzed the composition of the 19,348 significant naive trans-eQTLs, we observed a majority (>70%) of cross-mappable eQTLs corresponded to pseudogene targets. The non-cross-mappable eQTLs contained far fewer pseudogene targets (30%, Supplementary Figure 4). Likewise, we observed that more than 85% of eQTLs corresponding to pseudogenes were cross-mappable. Due to sequence similarity between pseudogenes and their corresponding parent genes, this is not surprising and could be due to alignment errors. One simple preventative measure in trans-eQTL studies would be to simply exclude pseudogenes entirely. However, 42.4% of eQTLs corresponding to protein-coding genes were also cross-mappable, which still exceeded expectation, and the top hits remained enriched for cross-mapping errors as noted above.\n\nWe investigated one GTEx trans-eQTL in greater detail for illustration – variant: chr5:149826526 and gene: RP11-343H5.4 (ENSG00000224114) – which was significant in each of 5 GTEx tissues. RP11-343H5.4 is a pseudogene on chromosome 1. In the coverage plots of the gene, we noticed that reads were aligned to only a fraction of the exonic region of the gene; if the gene were truly expressed, we would expect reads being mapped across the whole exon (Figure 2B). Notably, RP11-343H5.4 is cross-mappable with RPS14 (ENSG00000164587), a protein-coding gene in chromosome 5 near the putative trans-eQTL variant. There was also a cis-association between the variant and RPS14. k-mers from RPS14 indeed map to the region within RP11-343H5.4, where we observed a non-zero number of reads. Interestingly, in this case, read mapping appears to be genotype-dependent - the variant at chr5:149826526 alters sequence such that it would lead to reads from RPS14 uniquely, but likely incorrectly, mapping to RP11-343H5.4.\n\nFinally, we found that cross-mappable eQTLs, which we believe to be enriched for false-positives, are highly replicable between datasets. This misleading replication occurs because it is driven by the underlying sequence of the genome, and similar alignment errors frequently occur regardless of tissue and study. We showed this by measuring the replication between the significant trans-eQTLs detected at FDR ≤ 0.05 from whole blood from GTEx and whole blood data from the DGN study13. To avoid the effects of linkage disequilibrium, we tested for trans-association in DGN only for the best variant per GTEx trans-eQTL gene (with the lowest p-value in GTEx), where both the variant and the gene were present in the DGN data. At FDR ≤ 0.05, only 10.71% (3 out of 28) non-cross-mappable trans-eQTLs were replicated in DGN while 31.25% (5 out of 16) cross-mappable trans-eQTLs were replicated. The Q-Q plot in Figure 3A shows that cross-mappable trans-eQTLs were more likely to be replicated compared to non-cross-mappable ones. We observed the same phenomenon when we attempted to replicate significant trans-eQTLs detected from one GTEx tissue in other GTEx tissues. On average, 63.0% (range: 50.3–70.2%) and 16.3% (range: 7.6–25.1%) of cross-mappable and non-cross-mappable trans-eQTLs, respectively, were replicated (Figure 3B). This suggests that replication of a trans-eQTL does not necessarily indicate a true positive. Overall, we suggest that regardless of replication, cross-mappable trans-eQTLs require further investigation to establish that they arise from biological regulation rather than alignment artifacts.\n\n(A) Q-Q plot, replication p-values from DGN for variant-gene pairs discovered in GTEx Whole Blood, grouped by cross-mappability. (B) The fraction of significant eQTLs in each GTEx tissue (row) replicated in another tissue (column) at FDR ≤ 0.05, for cross-mappable eQTLs (left) and not cross-mappable eQTLs (right).\n\nNext, we evaluated evidence that alignment errors between genes can cause spurious correlation between gene expression levels (co-expression). If alignment errors did not affect co-expression analysis, we would expect that the distribution of pairwise correlation between cross-mappable genes would not deviate from that between non-cross-mappable genes. To test this, we used the gene expression data in five tissues from GTEx v7 after correction for covariates and latent confounders (see Methods). For each tissue, we selected a random set of 10,000 non-cross-mappable gene pairs and a random set of 10,000 cross-mappable gene pairs chosen with probability proportional to their cross-mappabilities (sampling probability proportional to cross-mappability ensures sampling from the whole cross-mappability range, as opposed to just from the massive number of low cross-mappability pairs). Then we computed the absolute Pearson correlation (|r|) between expression levels of the genes in each randomly selected pair. We found that expression levels of cross-mappable genes were more correlated than expression levels of non-cross-mappable genes (median p across tissues ≤ 4.7 × 10−5, Wilcoxon rank-sum test, Figure 4A). The difference was more significant when uncorrected data were used (median p ≤ 1.3 × 10−74, Supplementary Figure 5). We also observed that the correlation coefficient tends to increase with increasing levels of cross-mappability between genes (Supplementary Figure 6), indicating a high rate of false co-expression in the most highly cross-mappable genes. The increased correlation between cross-mappable genes was observed even after discounting genes from same gene family (Supplementary Figure 7), somewhat alleviating concerns that our observations were due to exclusively true functional relationships. We observed a similar pattern using data from an independent RNA-seq study, DGN (Supplementary Figure 8).\n\n(A) Comparison of co-expression between randomly drawn pairs of cross-mappable genes and not cross-mappable genes. Each violin plot shows the distribution of the absolute Pearson correlation (y-axis) between corrected gene expression levels of randomly drawn 10,000 gene pairs in a tissue (color). P-value of the Wilcoxon test to determine whether cross-mappable genes are more correlated than not cross-mappable genes in each tissue is shown in the legend. (B) Fraction of top co-expressed genes that are cross-mappable and thus potential false positives.\n\nTo demonstrate the impact of this pattern on a realistic genome-wide co-expression analysis, we evaluated how many of the top-most correlated gene pairs in each GTEx tissue suffer from cross-mappability. We observed that cross-mappable pairs of genes are over-represented among the top hits, with gene pairs ordered by the absolute Pearson correlation after excluding pairs of genes whose genomic coordinates actually overlap (Figure 4B, Supplementary Figure 9). Overall, the impact of cross-mappability on co-expression appears to be less than on trans-eQTL analysis, but the phenomenon may still require consideration when examining specific co-expressed gene pairs or enrichment patterns.\n\n\nDiscussion\n\nMisalignment of short sequencing reads has the potential to induce false positives in association studies. For RNA-seq, both trans-eQTL and co-expression analyses are susceptible to these artifacts, related to false positives in microarray analysis due to probe cross-hybridization. This is readily apparent from the enrichment of processed pseudogenes among the top hits for such association studies, but misalignment can affect protein-coding genes as well. Our results demonstrate that trans-eQTL associations in a standard pipeline are dominated by potential false-positives due to sequence similarity and replication rates between studies may be artificially inflated due to this pattern. Additionally, genes with sequence similarity display more correlated expression levels, and mapping errors should be considered in co-expression analysis as well.\n\nOur results do not imply that all instances of co-expression or trans-eQTL associations arising from genes with sequence similarity are in fact false positives. Genes with sequence similarity also sometimes have true functional relationships. Pseudogene transcripts may interact with coding transcripts, and some associations with pseudogene expression may reflect true regulatory relationships24. Furthermore, the background (random) rate of sequence similarity between any two regions in the human genome is above zero; that is, a hit may occur between regions of sequence similarity by chance, even when no actual misalignment of reads has taken place. However, we believe the exceedingly high fraction of cross-mappable regions among trans-eQTLs from a naive analysis warrants suspicion that these hits are predominantly false positives. Researchers should consider their particular application and tolerance for false negatives and false positives when applying filters targeting alignment errors. Other information, such as base-level coverage plots and outside functional information can help disambiguate particular cases of interest.\n\nExtensions, modifications, and other approaches related to this problem should also be considered. First, specifics of study design, and in particular sequencing read length, should be taken into account when using our data to filter potential false positives. If read length is much shorter or longer than our k-mer setting, our existing data may be insufficient and new mappability and cross-mappability estimates should be derived. In the initial resource provided, we used k-mer alignment to the genome, which does not directly handle splice junctions in transcriptomic data (and also limits appropriate k-mer length even for studies with longer reads). Alignment to the transcriptome or splice-aware alignment may offer future improvements, but computational cost and inaccuracies due to incorrect annotation will have to be evaluated. Our observations and methods may be relevant to analyses of other functional genomic data as well, including detection of interactions from HI-C, and detection of associations with data types such as ATAC-seq or ChIP-seq. Other approaches, such as filtering reads themselves before quantification can also be applied if raw reads rather than quantified data are available and tractable12. Our evaluation provides evidence that misalignment of reads should be considered as a potential source of false positives in association studies, particularly for trans-eQTL analysis. The resources we provide can be used directly to filter potential false positives, or the ideas presented may be tuned and adapted to new studies and data types.\n\n\nData availability\n\nPre-computed cross-mappability resources for human genomes (hg19 and GRCh38) are available on figshare, DOI: 10.6084/m9.figshare.c.4297352.v317. GTEx (v7) expression and covariate data are available from www.gtexportal.org. GTEx (v7) genotype data are available from dbGap (accession number: phs000424.v7.p2). DGN data are available by application through NIMH. Other data, including annotations and intermediate results, required to reproduce analyses in the manuscript are available on figshare, DOI: 10.6084/m9.figshare.7309625.v225.\n\nSupplementary Figure 1–Supplementary Figure 9 are available on figshare, DOI: 10.6084/m9.figshare.7359539.v126.\n\nSupplementary Figure 1. Cross-mappability statistics. (A) Distribution of cross-mappability between cross-mappable pairs of genes, restricted to gene pairs with cross-mappability > 0, using Gencode v19 annotations on human genome hg19. (B) Background percentage of cross-mappable gene pairs between all available expressed genes in GTEx data, categorized by tissue. For both panels, directed gene pairs were used; i.e., (Gene A, Gene B) and (Gene B, Gene A) pairs were considered different.\n\nSupplementary Figure 2. Cross-mappability among top trans-eQTLs. Detected (A) using protein-coding genes in GTEx, (B) using genes with mappability ≥ 0.8 in GTEx, (C) using protein-coding genes with mappability ≥ 0.8 in GTEx, and D) by PancanQTL where unique eQTLs were ordered by lowest p-value across all cancer types.\n\nSupplementary Figure 3. Large number of trans-eQTLs among random cross-mappable gene pairs. We tested for trans-eQTLs taking the same number of random variant-gene pairs in 3 different categories: 1) Not cross-mappable, 2) Cross-mappable, and 3) Cross-mappable (Top). In the first category, we randomly selected 1,000 not cross-mappable gene pairs (g1, g2) where g1 and g2 were on different chromosome and there was at least one variant near g1 (within 1Mb of the TSS of g1), then selected the best cis-variant s (with lowest p-value) for g1, and finally tested for trans-association between s and g2. Variant-gene pairs for other two categories were selected in a similar way as the first category except that the gene pairs were cross-mappable (crossmap(g1, g2) > 0) in the second category, and highly cross-mappable (among top 10,000 cross-mappable pairs) in the third category. The above plot shows the number of significant trans-eQTLs (y-axis) detected at a given FDR (x-axis) in each category (line marker) in each tissue (color).\n\nSupplementary Figure 4. Composition of trans-eQTLs. (A) Representation of gene types among trans-eQTL target genes, categorized by cross-mappability. (B) Proportion of cross-mappable eQTLs categorized by gene type. Only the four most frequent gene types in trans-eQTL hits are shown.\n\nSupplementary Figure 5. Correlation between random gene pairs using uncorrected data. Each violin plot shows the distribution of absolute Pearson correlation (y-axis) between uncorrected gene expression levels of 10,000 randomly drawn gene pairs in a tissue (color). The p-value of the Wilcoxon test to determine whether cross-mappable genes are more correlated than not cross-mappable genes in each tissue is shown in the legend.\n\nSupplementary Figure 6. Correlation between random gene pairs increases with cross-mappability. Gene pairs available in each tissue were categorized into 22 groups (x-axis) based on quantiles. A quantile group \"q1 − q2(n)\" represents gene pairs of (q1 * 100, q2 * 100]- th percentile of cross-mappability with a total of n pairs. In order to visualize the impact of the highest range of cross-mappability, the rightmost nine quantile groups were selected in such a way that each contains about a certain number of pairs: (from right) 2,000, 2,000, 2,000, 2,000, 2,000, 5,000, 10,000, 25,000, 50,000. The leftmost quantile group \"0\" represents gene pairs which are not cross-mappable. From each group, 1,000 gene pairs were randomly selected where the probability of drawing a pair was proportional to its cross-mappability. Each violin plot shows the distribution of absolute Pearson correlation (y-axis) between corrected expressions of the genes in each pair.\n\nSupplementary Figure 7. Increased correlation between cross-mappable genes is not exclusively due to sequence similarity between genes from same gene family. Here, two genes in the same HGNC gene family were artificially excluded from cross-mappable pairs. We computed the absolute Pearson correlation between gene pairs within different groups as described in Figure 4A and Supplementary Figure 6. Note: gene family information was downloaded from www.genenames.org. A–B) Comparison of co-expression between 10,000 randomly drawn pairs of cross-mappable and not cross-mappable genes in Muscle – Skeletal (A) and Whole Blood (B). C-D) Random correlation between genes in Muscle – Skeletal (C) and Whole Blood (D).\n\nSupplementary Figure 8. Co-expression analysis using gene expression data from DGN. A) Comparison of co-expression between 10,000 randomly drawn cross-mappable and non-cross-mappable gene pairs. B) Random correlation between genes in DGN increases with cross-mappability.\n\nSupplementary Figure 9. Fraction of gene pairs with cross-mappability ≥ 100 among the top co-expressed genes, categorized by GTEx tissues.\n\n\nSoftware availability\n\nGitHub repository to compute cross-mappability: https://github.com/battle-lab/crossmap.\n\nArchived code at time of publication: https://doi.org/10.5281/zenodo.149185216.\n\nLicense: GPL-3.\n\nGitHub repository to replicate analyses in the manuscript: https://github.com/battle-lab/crossmap_analysis.\n\nArchived code at time of publication: https://doi.org/10.5281/zenodo.149186927.\n\nLicense: GPL-3.",
"appendix": "Grant information\n\nA.B. is supported by NIH grant 1R01MH109905, NIH grant R01HG008150 (NHGRI; Non-Coding Variants Program), and NIH grant R01MH101814 (NIH Common Fund; GTEx Program).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Sara Mostafavi, Xiaowei Zhu, and Barbara Engelhardt for discussions and feedback. We thank Brian Jo for running k-mer mappability for hg38. We also thank François Aguet for the generating the coverage plots. We also thank Yuan He and Princy Parsana for reviewing codes. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health. Additional funds were provided by the NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Donors were enrolled at Biospecimen Source Sites funded by NCI\\SAIC-Frederick, Inc. (SAIC-F) subcontracts to the National Disease Research Interchange (10XS170), Roswell Park Cancer Institute (10XS171), and Science Care, Inc. (X10S172). The Laboratory, Data Analysis, and Coordinating Center (LDACC) was funded through a contract (HHSN268201000029C) to The Broad Institute, Inc. Biorepository operations were funded through an SAIC-F subcontract to Van Andel Institute (10ST1035). Additional data repository and project management were provided by SAIC-F (HHSN261200800001E). The Brain Bank was supported by a supplements to University of Miami grants DA006227 & DA033684 and to contract N01MH000028. Statistical Methods development grants were made to the University of Geneva (MH090941 & MH101814), the University of Chicago (MH090951, MH090937, MH101820, MH101825), the University of North Carolina - Chapel Hill (MH090936 & MH101819), Harvard University (MH090948), Stanford University (MH101782), Washington University St Louis (MH101810), and the University of Pennsylvania (MH101822). The genotype data used for this manuscript were obtained from dbGaP accession number phs000424.v7.p2.\n\n\nReferences\n\nKahles A, Behr J, Rätsch G: MMR: a tool for read multi-mapper resolution. Bioinformatics. 2016; 32(5): 770–772. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson NR, Yeoh JM, Coruh C, et al.: Improved Placement of Multi-mapping Small RNAs. G3 (Bethesda). 2016; 6(7): 2103–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobert C, Watson M: Errors in RNA-Seq quantification affect genes of relevance to human disease. Genome Biol. 2015; 16(1): 177. 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Genome Res. 2014; 24(1): 14–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPickrell JK, Marioni JC, Pai AA, et al.: Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature. 2010; 464(7289): 768–772. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReilly C, Raghavan A, Bohjanen P: Global assessment of cross-hybridization for oligonucleotide arrays. J Biomol Tech. 2006; 17(2): 163–72. PubMed Abstract | Free Full Text\n\nSaha A, Battle A: battle-lab/crossmap: Github repository to compute cross-mappability (release 1.1). 2018. http://www.doi.org/10.5281/zenodo.1491852\n\nSaha A, Battle A: Pre-computed cross-mappability resources for human genomes (hg19 and grch38). 2018. http://www.doi.org/10.6084/m9.figshare.c.4297352.v3\n\nLangmead B, Trapnell C, Pop M, et al.: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3): R25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarrow J, Frankish A, Gonzalez JM, et al.: GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012; 22(9): 1760–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStegle O, Parts P, Piipari M, et al.: Using probabilistic estimation of expression residuals (PEER) to obtain increased power and interpretability of gene expression analyses. Nat Protoc. 2012; 7(3): 500–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGong J, Mei S, Liu C, et al.: PancanQTL: systematic identification of cis-eQTLs and trans-eQTLs in 33 cancer types. Nucleic Acids Res. 2018; 46(D1): D971–D976. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCasper J, Zweig AS, Villarreal C, et al.: The UCSC Genome Browser database: 2018 update. Nucleic Acids Res. 2017; 46(D1): D762–D769. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShabalin AA: Matrix eQTL: ultra fast eQTL analysis via large matrix operations. Bioinformatics. 2012; 28(10): 1353–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPink RC, Wicks K, Caley DP, et al.: Pseudogenes: pseudo-functional or key regulators in health and disease? RNA. 2011; 17(5): 792–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaha A, Battle A: Data required to analyze effects of cross-mappability in trans-eqtl and co-expression studies. 2018. http://www.doi.org/10.6084/m9.figshare.7309625.v2\n\nSaha A, Battle A: False positives in trans-eqtl and co-expression analyses arising from rna-sequencing alignment errors (supplementary). 2018. http://www.doi.org/10.6084/m9.figshare.7359539.v1\n\nSaha A, Battle A: battle-lab/crossmap_analysis: Github repository to analyze effects of cross-mappability in trans-eqtl and co-expression studies (release 1.3). 2018. http://www.doi.org/10.5281/zenodo.1491869"
}
|
[
{
"id": "41234",
"date": "12 Dec 2018",
"name": "Michael I. Love",
"expertise": [
"Reviewer Expertise Developer of methods for estimating gene and transcript expression",
"and statistical testing of expression across samples."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 detailed analysis of how false positives arise in trans-eQTL and co-expression analyses, due to cross-mapping of reads among genes with sequence homology. They present a cross-mappability metric, and provide pre-computed cross-mappability for two human Gencode annotations (v19 and v26). They also provide software for efficiently computing cross-mappability available at a GitHub link. The command line software has detailed instructions online. The software requires genome FASTA files, a GTF file, a mappability bedgraph or bigwig file, Bowtie (v1) and an index, and a few R packages (data.table, intervals, argparser, stats).\nThe report provides an important warning to the research community, and the pre-computed cross-mappability and the software will be a valuable resource for groups studying trans-eQTL and co-expression and making use of unique read counts for gene expression quantification.\nOne of the key points of the article is noting that the cross-mappable and likely spurious trans-eQTLs are highly replicable between datasets, because \"it is driven by the underlying sequence of the genome, and similar alignment errors frequently occur regardless of tissue and study.\" This logic also extends to co-expression and gene networks built on gene-gene expression correlations. Another key point was that filtering out of cross-mappable trans-eQTLs necessitates re-assessment of FDR, as the highly significant cross-mappable trans-eQTLs bring down the nominal FDR for other eQTLs.\nMy main comment on the article is regarding details of the gene expression quantification.\nThe authors note that,\n\n\"The number of reads misaligned to Gene B across samples may be directly proportional to the number of reads for Gene A, or may be determined by genetic variation creating sequence mismatches with the correct region....We note that such errors are not entirely mitigated by filtering multi-mapped reads—some alignment errors may remain between similar regions even among uniquely aligned reads due to genetic variation, errors in the reference genome, and other complications.\"\nHow would the analysis change if an expectation maximization (EM) approach were used for quantifying transcript and gene expression, where a latent variable is used for the origin of each read or pair of reads (both across isoforms and gene loci)? Such methods may resolve issues as shown in Figure 1A, because the observed coverage and expression of only gene A may give a higher likelihood than the observed coverage and expression of gene A and gene B. However the degree to which these methods may offer an improvement with respect to the cross-mappability issue depends on the distribution of genetic variation and errors in the reads, and on potential errors in the reference genome/transcriptome and on incomplete gene annotations. Therefore, it would be critical to perform the trans-eQTL analysis with regards to cross-mappability, when an EM algorithm, or a similar method that resolves multi-mapping reads, is used to quantify gene expression. Methods such as RSEM, Kallisto, or Salmon may be used to quantify gene and transcript abundance, which use EM or variational Bayes EM to resolve multi-mapping reads (as well as reads consistent with multiple isoforms of a gene) (disclosure: I am a co-author of the Salmon method).\nThe authors note that:\n\"Alignment to the transcriptome or splice-aware alignment may offer future improvements, but computational cost and inaccuracies due to incorrect annotation will have to be evaluated.\"\nOne potential solution which may alleviate both the issue of cross-mappability and incorrect annotation, would be to use an isoform discovery method to detect and characterize novel isoforms in a particular dataset (large datasets of rare tissues or sequencing of RNA from populations which have been under-represented in previous studies may very well discover novel isoforms), and then to use an EM or similar method to quantify expression.\nThe details about how the GTEx (v7) and DGN data were quantified is missing, although these details are critical for understanding how broad the conclusions of the analysis may be. Gene expression was quantified in GTEx v7 using RNA-SeQC v1.1.8 which does not use an EM approach to resolve multi-mapping reads. According to the Methods section of Battle, et al (2014), for the DGN dataset, HTSeq was used to quantify gene expression, which also does not use an EM approach. I would recommend to add such quantification details of the datasets to this article.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4552",
"date": "11 Apr 2019",
"name": "Ashis Saha",
"role": "Author Response",
"response": "Thank you for reviewing our paper. Please see our responses inline in bold and italic font following your comments in standard font. The authors present a detailed analysis of how false positives arise in trans-eQTL and co-expression analyses, due to cross-mapping of reads among genes with sequence homology. They present a cross-mappability metric, and provide pre-computed cross-mappability for two human Gencode annotations (v19 and v26). They also provide software for efficiently computing cross-mappability available at a GitHub link. The command line software has detailed instructions online. The software requires genome FASTA files, a GTF file, a mappability bedgraph or bigwig file, Bowtie (v1) and an index, and a few R packages (data.table, intervals, argparser, stats). The report provides an important warning to the research community, and the pre-computed cross-mappability and the software will be a valuable resource for groups studying trans-eQTL and co-expression and making use of unique read counts for gene expression quantification. One of the key points of the article is noting that the cross-mappable and likely spurious trans-eQTLs are highly replicable between datasets, because \"it is driven by the underlying sequence of the genome, and similar alignment errors frequently occur regardless of tissue and study.\" This logic also extends to co-expression and gene networks built on gene-gene expression correlations. Another key point was that filtering out of cross-mappable trans-eQTLs necessitates re-assessment of FDR, as the highly significant cross-mappable trans-eQTLs bring down the nominal FDR for other eQTLs. >> Thank you for nicely summarizing the contribution of our work. My main comment on the article is regarding details of the gene expression quantification. The authors note that, \"The number of reads misaligned to Gene B across samples may be directly proportional to the number of reads for Gene A, or may be determined by genetic variation creating sequence mismatches with the correct region....We note that such errors are not entirely mitigated by filtering multi-mapped reads—some alignment errors may remain between similar regions even among uniquely aligned reads due to genetic variation, errors in the reference genome, and other complications.\" How would the analysis change if an expectation maximization (EM) approach were used for quantifying transcript and gene expression, where a latent variable is used for the origin of each read or pair of reads (both across isoforms and gene loci)? Such methods may resolve issues as shown in Figure 1A, because the observed coverage and expression of only gene A may give a higher likelihood than the observed coverage and expression of gene A and gene B. Howeverthe degree to which these methods may offer an improvement with respect to the cross-mappability issue depends on the distribution of genetic variation and errors in the reads, and on potential errors in the reference genome/transcriptome and on incomplete gene annotations. Therefore, it would be critical to perform the trans-eQTL analysis with regards to cross-mappability, when an EM algorithm, or a similar method that resolves multi-mapping reads, is used to quantify gene expression. Methods such as RSEM, Kallisto, or Salmon may be used to quantify gene and transcript abundance, which use EM or variational Bayes EM to resolve multi-mapping reads (as well as reads consistent with multiple isoforms of a gene) (disclosure: I am a co-author of the Salmon method). >> We appreciate the importance of this concern. We agree that EM-based quantification in principle has the potential to help address the cross-mappability issue to some extent. We performed additional analyses using RSEM-quantified gene expressions and we still observed a high rate of potential false positives in our analyses, particularly for trans-eQTLs. We updated the manuscript along with a supplementary figure (Supplementary Figure 11). \"We also note that utilization of improved alignment and quantification methods to generate gene expression data may also be helpful to avoid false positives. For example, quantification of gene expression levels using RSEM[28], an expectation maximization based quantification tool, results in a smaller fraction of false positive trans-eQTLs (60.17%) than that using RNA-SeQC (75.14%). However, potential false positives due to cross-mappability still remain abundant in both trans-eQTL and co-expression studies (Supplementary Figure 11).\" We also added a brief description of the quantification pipelines of GTEx v7 and DGN in the revised manuscript. The authors note that: \"Alignment to the transcriptome or splice-aware alignment may offer future improvements, but computational cost and inaccuracies due to incorrect annotation will have to be evaluated.\" One potential solution which may alleviate both the issue of cross-mappability and incorrect annotation, would be to use an isoform discovery method to detect and characterize novel isoforms in a particular dataset (large datasets of rare tissues or sequencing of RNA from populations which have been under-represented in previous studies may very well discover novel isoforms), and then to use an EM or similar method to quantify expression. >> This is an interesting idea. For the current analysis, we plan to leave the approach as specified, but would like to explore this in the future. We note, however, that EM-based approaches still do not address many cross-mappability errors in general, as shown in response to the previous question. The details about how the GTEx (v7) and DGN data were quantified is missing, although these details are critical for understanding how broad the conclusions of the analysis may be. Gene expression was quantified in GTEx v7 using RNA-SeQC v1.1.8 which does not use an EM approach to resolve multi-mapping reads. According to the Methods section of Battle, et al (2014), for the DGN dataset, HTSeq was used to quantify gene expression, which also does not use an EM approach. I would recommend to add such quantification details of the datasets to this article. >> We appreciate your concern. GTEx v7 used only uniquely mapped reads, but did not use an EM approach in the original analysis, though we have added the RSEM version for the revision as well. To make the manuscript self-contained, we briefly described the gene expression quantification pipeline of both GTEx v7 and DGN in the manuscript."
}
]
},
{
"id": "41237",
"date": "21 Dec 2018",
"name": "Rob Patro",
"expertise": [
"Reviewer Expertise I design and develop methods and algorithms for processing high-throughput sequencing data",
"with some specific focus on methods for gene and transcript quantification and de novo transcriptome 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\nIn this manuscript, Saha and Battle describe how errors in the genomic alignment of RNA-seq data can confound specific downstream analyses. Specifically, the focus on the discovery of trans-eQTLs and on co-expression analysis. Surprisingly, they find that, when a \"naive\" pipeline is used for trans-eQTL discovery, up to 75% of the trans-eQTL events detected may be false positives resulting from cross-mappability (the type of alignment error they discuss and characterize). In addition to describing this phenomenon, and demonstrating its effect on trans-eQTL and co-expression analysis, the authors also propose a new \"cross-mappability\" score, which allows one to map out which genes in a reference are likely to suffer from the types of spurious alignments, and subsequently, spurious correlations, that are described. The idea of cross-mappability seems a useful and logical extension of the mappability concept, where one is instead interested in which pairs or groups of genes share homologous sequence. The authors also provide pre-computed cross-mappability scores for hg19 and GRCh38.\n\nThe paper is well-written, and the issues that the authors raise are important ones. It suggests that researchers should be cautious in interpreting the results of eQTL and co-expression analyses, and, importantly, provides them with tools to reassess their data and help control for the strong potential confounding factor of cross-mappability. I believe this is an important contribution.\n\nMy main questions and comments about the manuscript concern cross-mappability scores, and the alignment errors that lead to the observed problems.\n\nThough the authors only explore the effect of alignment errors on eQTL and co-expression analysis, it seems that these types of issues could affect most analyses involving spliced-mapping of RNA-seq data to the reference genome. Specifically, the type of alignment errors illustrated in Figure 1 (A) would affect even basic expression quantification, let-alone co-expression analysis. This is particularly true for reads where this effect persists even when one considers only reads aligned uniquely by the tool. What would cause the aligner to return only a single (incorrect) locus for the read when multiple equally-good alignments should exist? Are the alignments contiguous at one locus but spliced at the other, or is the manner in which the read would align to the \"true\" and \"spurious\" locus different? Interestingly, it seems as though the cross-mappability map could act as a sort of homology table 1 that might even be useful for correcting these spurious alignments, or at least suggesting the true locus as an equally-good match. Given that cross mappability is computed by mapping specific k-mers back to the genome (allowing up to 2 mismatches) using Bowtie, how does it deal with accounting for k-mers that span splicing junctions? It seems to me that the specific case where reads map to pseudogenes rather than what is presumed to be the true (protein-coding) locus of the read could be explained by regions of the genome that are contiguous (un-spliced) in the pseudogene, but which span a splicing junction in the protein coding gene. If the cross-mappability score doesn't account for the cross-mappability of k-mer that may span splice junctions, then it seems it might miss such important cases. However, given that the score is computed assuming some known annotation, it would be possible to explicitly extract appropriately-sized contexts around each known splicing junction, and to include them into the reference that Bowtie maps against when computing the cross-mappability scores. How would the cross-mappability scores change if they also accounted for junction-spanning k-mers rather than just genomically contiguous k-mers? Related to the above point, but thinking in the other direction, might the cross-mappability scores be too \"conservative\" in some cases? Specifically, the scores are computed using k-mers that are the length of relatively short reads for exons, and k-mers that are much shorter than typical reads for UTRs. However, the ambiguity of these sequences individually may not be sufficient to induce a false alignment when the reads being processed are paired-end reads. In that case, one would expect misalignments to require that there be sequence ambiguity supporting both ends of the sequencing read in order for the read to align, concordantly, to the wrong locus. The GTEx data that was analyzed relies on spliced alignment to the genome using STAR, and I presume the same tools were used to analyse the DGN cohort data (is this correct?). It would be enlightening to see if and how results would change if the reads were instead aligned using HISAT2. I am aware, for example, that HISAT2 takes special measures to avoid aligning reads to pseudogenes when similar quality alignments to transcripts of other biotypes are available (personal communication with Daehwan Kim). This may have some effect on the type and magnitude of false alignments you see (though, certainly, will not account for all of them). Likewise, it would be very interesting to see how the analyses differ if alignment is done directly to the transcriptome using, e.g., Bowtie2 ( the authors already mention this possibility in the discussion section). I agree with Mike Love's suggestion that it seems important to explore the extent to which this effect may be mitigated by adopting more accurate methods for gene expression quantification.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4553",
"date": "11 Apr 2019",
"name": "Ashis Saha",
"role": "Author Response",
"response": "Thank you for reviewing our paper. Please see our responses inline in bold and italic font following your comments in standard font. In this manuscript, Saha and Battle describe how errors in the genomic alignment of RNA-seq data can confound specific downstream analyses. Specifically, the focus on the discovery of trans-eQTLs and on co-expression analysis. Surprisingly, they find that, when a \"naive\" pipeline is used for trans-eQTL discovery, up to 75% of the trans-eQTL events detected may be false positives resulting from cross-mappability (the type of alignment error they discuss and characterize). In addition to describing this phenomenon, and demonstrating its effect on trans-eQTL and co-expression analysis, the authors also propose a new \"cross-mappability\" score, which allows one to map out which genes in a reference are likely to suffer from the types of spurious alignments, and subsequently, spurious correlations, that are described. The idea of cross-mappability seems a useful and logical extension of the mappability concept, where one is instead interested in which pairs or groups of genes share homologous sequence. The authors also provide pre-computed cross-mappability scores for hg19 and GRCh38. The paper is well-written, and the issues that the authors raise are important ones. It suggests that researchers should be cautious in interpreting the results of eQTL and co-expression analyses, and, importantly, provides them with tools to reassess their data and help control for the strong potential confounding factor of cross-mappability. I believe this is an important contribution. >> Thank you, we are appreciate your comments on our contribution. My main questions and comments about the manuscript concern cross-mappability scores, and the alignment errors that lead to the observed problems. ● Though the authors only explore the effect of alignment errors on eQTL and co-expression analysis, it seems that these types of issues could affect most analyses involving spliced-mapping of RNA-seq data to the reference genome. Specifically, the type of alignment errors illustrated in Figure 1 (A) would affect even basic expression quantification, let-alone co-expression analysis. This is particularly true for reads where this effect persists even when one considers only reads aligned uniquely by the tool. What would cause the aligner to return only a single (incorrect) locus for the read when multiple equally-good alignments should exist? Are the alignments contiguous at one locus but spliced at the other, or is the manner in which the read would align to the \"true\" and \"spurious\" locus different? Interestingly, it seems as though the cross-mappability map could act as a sort of homology table 1 that might even be useful for correcting these spurious alignments, or at least suggesting the true locus as an equally-good match. >> Thank you for this comment. We agree that alignment errors potentially affect quantification of expression including splicing-aware quantifications. We observed cross-mapping errors even when only uniquely-mapped reads were used in the quantification (for e.g., in GTEx v7). There could be several reasons an aligner returns only a single incorrect locus for the read when multiple equally-good alignments should exist including genetic variation, errors in the reference genome, incorrect annotations, ambiguity due to splicing, and sequencing errors. It is difficult to assess the relative contribution of each of these. We briefly mentioned the issues in the Introduction section of the manuscript. \"some alignment errors may remain between similar regions even among uniquely aligned reads due to genetic variation, errors in the reference genome, and other complications.\" Regarding the pattern of alignment errors in \"true\" and \"spurious\" loci, we manually inspected suspected false positives, but did not observe any clear pattern of alignment errors. We observed multiple classes of errors including cases where alignment is contiguous at one locus (either true or spurious) but spliced at the other locus, and cases where alignment is contiguous at both loci. Finally, it is a nice idea to see if alignment methods could directly utilize our cross-mappability resources or related approaches. We would be interested to follow up on this. ● Given that cross mappability is computed by mapping specific k-mers back to the genome (allowing up to 2 mismatches) using Bowtie, how does it deal with accounting for k-mers that span splicing junctions? It seems to me that the specific case where reads map to pseudogenes rather than what is presumed to be the true (protein-coding) locus of the read could be explained by regions of the genome that are contiguous (un-spliced) in the pseudogene, but which span a splicing junction in the protein coding gene. If the cross-mappability score doesn't account for the cross-mappability of k-mer that may span splice junctions, then it seems it might miss such important cases. However, given that the score is computed assuming some known annotation, it would be possible to explicitly extract appropriately-sized contexts around each known splicing junction, and to include them into the reference that Bowtie maps against when computing the cross-mappability scores. How would the cross-mappability scores change if they also accounted for junction-spanning k-mers rather than just genomically contiguous k-mers? >> Thanks for raising this important issue. We did not account for k-mers spanning splice junctions and thus our current approach might miss some cross-mapping cases. However, we expect that for many genes with reasonable length exons, some k-mers that fall completely within the exon will also cross-map, and the genes will be identified as cross-mapping by our method in this case, but exceptions to this will also occur. Cross-mappability scores would sometimes increase (but not decrease) if junction-spanning k-mers are considered, and we would need to consider whether these are also sometimes spurious. We mention in the manuscript that alignment to the transcriptome or splice-aware alignment might offer future improvements, but the computational cost and potential for some inaccuracies due to incorrect annotation would also have to be evaluated. In future work, we plan to extend cross-mappability scores using splice-aware alignments. One challenge here is to find all alignments of a k-mer using a spliced aligner. Spliced aligners are generally optimized to find the best alignment, and it is not as fast to find all alignments. Our current plan is to use STAR aligner (instead of Bowtie in the current setting) with a high number of multiple alignments allowed per read. In addition, we have to modify the k-mer generation process to include exon-exon junction spanning k-mers. Our plan is to evaluate every k-mer spanning exon-exon junctions of the annotated transcripts from a gene, along with the k-mers from the current collapsed gene model. As the k-mer generation process and the alignment process are going to be changed, we can no longer use the GEM Library to compute mappability of each k-mer; we will have to implement this part as well. We leave the implementation of this, and evaluation, as future work, but agree it could offer improvements. ● Related to the above point, but thinking in the other direction, might the cross-mappability scores be too \"conservative\" in some cases? Specifically, the scores are computed using k-mers that are the length of relatively short reads for exons, and k-mers that are much shorter than typical reads for UTRs. However, the ambiguity of these sequences individually may not be sufficient to induce a false alignment when the reads being processed are paired-end reads. In that case, one would expect misalignments to require that there be sequence ambiguity supporting both ends of the sequencing read in order for the read to align, concordantly, to the wrong locus. >> This is a good observation. Yes, depending on the goal of the analysis, cross-mappability scores may be conservative in some situations. In a simple case, the value of k we used may be too short for some studies. One can easily recompute cross-mappabilities with appropriate k for the sequencing protocol, using our released code. Also, as you mentioned in the comment, misalignment of paired-end reads would require sequence ambiguity in both ends. Thus, our method may conservatively suggest two genes are cross-mappable, when paired-end reads would mostly or completely resolve the ambiguity. To compute cross-mappability incorporating pair-ended k-mers, we would need to model the distribution of fragment lengths in addition to splice junctions, which we have not yet addressed here. For simplicity and usability of our resources across different studies with different sequencing methods, we treat k-mers like single-ended reads. In general, small cross-mappability scores (where only a few k-mers overlap between genes) may not be sufficient to introduce alignment errors. One can easily filter gene pairs more stringently by thresholding the cross-mappability scores to higher numbers, which may weakly approximate requiring both ends of reads to align. Importantly, we do not claim that all instances of co-expression or trans-associations between cross-mappable pairs are actually false positives, some sequence similarity may occur between genes where a true hit also exists. One should consider the baseline rate of cross-mapping compared with that among observed hits, along with parameters of the study that may necessitate a specialized analysis. ● The GTEx data that was analyzed relies on spliced alignment to the genome using STAR, and I presume the same tools were used to analyse the DGN cohort data (is this correct?). It would be enlightening to see if and how results would change if the reads were instead aligned using HISAT2. I am aware, for example, that HISAT2 takes special measures to avoid aligning reads to pseudogenes when similar quality alignments to transcripts of other biotypes are available (personal communication with Daehwan Kim). This may have some effect on the type and magnitude of false alignments you see (though, certainly, will not account for all of them). Likewise, it would be very interesting to see how the analyses differ if alignment is done directly to the transcriptome using, e.g., Bowtie2 ( the authors already mention this possibility in the discussion section). >> Good points. Firstly, GTEx and DGN used different alignment and quantification tools. While GTEx v7 used STAR for alignment and RNA-SeQC for quantification, DGN used TopHat for alignment and HTSeq for quantification. False positives are observed for multiple tools. Secondly, we definitely agree avoiding pseudogenes (using --avoid-pseudogene option in HISAT2 or using other tools) would have an effect on the number of false positives. Another option is to simply not map trans-eQTLs for pseudogenes (or other biotypes) at all, which some studies in fact do. As mentioned in the manuscript that 42.4% of eQTLs corresponding to protein-coding genes were also cross-mappable, compared to 75.14% of all eQTLs). This is lower than for pseudogenes, but still above the expected background level, giving us an idea of the impact such a change would have. Of course, most researchers use public datasets without re-aligning them, so our resource shouldn’t rely on a specific aligner or parameters. Finally, we agree that alignment to the transcriptome would be an important avenue for future research. ● I agree with Mike Love's suggestion that it seems important to explore the extent to which this effect may be mitigated by adopting more accurate methods for gene expression quantification. >> We agree. We performed additional analyses using RSEM-quantified gene expressions and we still observed potential false positives in our analyses. Please see our response to Mike Love for details along with Supplementary Figure 11."
}
]
},
{
"id": "41238",
"date": "27 Dec 2018",
"name": "Aaron R. Quinlan",
"expertise": [
"Reviewer Expertise Human genomics",
"computational genomics",
"structural variation",
"genome evolution"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMost assays that leverage high-throughput DNA sequencing ultimately involve counting molecules that align to genome loci. The observed counts are, in turn, used as proxies for some underlying cellular phenomenon. If the process of sequence alignment is fundamentally biased, so too will be the observations and potential conclusions drawn.\n\nIn this manuscript, Saha and Battle explore the important role of sequence similarity between genes (\"cross-mapping\") on the spurious detection of trans eQTLs and gene co-expression. In particular, they argue that up to 75% of trans e-QTLs detected with standard methods could be spurious owing to systematic mapping (and thus both downstream read-counting and subsequent expression measures) artifacts caused by sequence similarity. This manuscript is well-structured, easy to read, and provides yet another warning that properly accounting for mapping and alignment artifacts is critical genomic research. The approach and analyses are sound overall. However, there are a few area that would benefit from clarification, and some technical aspects that warrant greater detail and perhaps further analysis.\nChoice of k-mer size. While the choice of k=75 for exons and k=36 for UTRs is certainly reasonable, one wonders whether these choices provide the greatest power to detect and quantify cross-mappability. Were analyses conducted to choose these k-mer sizes empirically? It would also be nice to add methods describing from which gene models the k-mers were derived. Furthermore, 5' UTRs are, on average, only slightly smaller than the typical exon, and 3' UTRs are actually larger on average (https://gist.github.com/arq5x/0d44bae195fc6260984ee01fd253712c). Therefore, it is not clear that k=36 for UTRs versus k=75 for exons is well-justified.\n\nWhen reading the manuscript, one naturally wonders whether the effects revealed by cross-mappability could be detected via existing \"mappability\" scores from ENCODE and others. It would helpful to have an explicit discussion and/or analysis of how this metric differs.\n\nBy my reading and interpretation of Figure 1B, k-mers spanning exon/exon junctions were not modeled. This is perhaps critical given the results describing an enrichment of cross-mappable eQTLs corresponding to processed pseudogenes.\n\nEchoing the point raised by Rob Patro, it is important to understand how the gene expression qualification was done for GTeX and discuss how these methods impact cross-mappability analysis.\n\nMappability is defined as 1/C_k where C_k is the number of genomic loci to which the k-mer aligns with <= 2 mismatches. Given sequencing errors, polymorphism, and the fact that RNA-seq is actually cDNA-seq, one might be concerned that allowing more mismatches (and/or INDEL errors) would more properly model cross-mappability. While perhaps a minor concern, it would be nice to know how much the scores and resulting cross-mappability table differ with tolerance for larger edit distances.\n\nIn the results describing an enrichment of spurious trans eQTLs associated with cross-mapping pseudogenes, it is unclear whether the authors are referring to all types of pseudogenes or solely processed pseudogenes. Crossmapping could arise from both \"classic\" (i.e., duplicated owing to NAHR) and \"processed\" (i.e., mature RNAs duplicated/inserted into the genome). However, the effect of excluding k-mers that span exon/exon boundaries from the cross-mappability would have a differential effect on these two types of pseudogenes. It would be helpful to be explicit in which types of pseudogenes are being discussed.\n\nSNPs were excluded from evaluation if they overlapped annotations in the RepeatMasker track. I would argue that SNPs should (and typically would be) excluded if they lie within segmental duplications. Such regions also harbor genes with a high degree of paralogy (because of the ancestral duplication) and if excluded, may have a substantial impact on the results. For example, I suspect many \"classic\" pseudogenes would also be excluded were SNPs in segmental duplications excluded. While I have never conducted a trans eQTL analysis, I suspect that removal of segdups is standard practice owing to the inherently high paralogy between duplicated regions.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4554",
"date": "11 Apr 2019",
"name": "Ashis Saha",
"role": "Author Response",
"response": "Thank you for reviewing our paper. Please see our responses inline in bold and italic font following your comments in standard font. Most assays that leverage high-throughput DNA sequencing ultimately involve counting molecules that align to genome loci. The observed counts are, in turn, used as proxies for some underlying cellular phenomenon. If the process of sequence alignment is fundamentally biased, so too will be the observations and potential conclusions drawn. In this manuscript, Saha and Battle explore the important role of sequence similarity between genes (\"cross-mapping\") on the spurious detection of trans eQTLs and gene co-expression. In particular, they argue that up to 75% of trans e-QTLs detected with standard methods could be spurious owing to systematic mapping (and thus both downstream read-counting and subsequent expression measures) artifacts caused by sequence similarity. This manuscript is well-structured, easy to read, and provides yet another warning that properly accounting for mapping and alignment artifacts is critical genomic research. The approach and analyses are sound overall. However, there are a few area that would benefit from clarification, and some technical aspects that warrant greater detail and perhaps further analysis. >> Thank you for your positive assessment of our approach. 1. Choice of k-mer size. While the choice of k=75 for exons and k=36 for UTRs is certainly reasonable, one wonders whether these choices provide the greatest power to detect and quantify cross-mappability. Were analyses conducted to choose these k-mer sizes empirically? It would also be nice to add methods describing from which gene models the k-mers were derived. Furthermore, 5' UTRs are, on average, only slightly smaller than the typical exon, and 3' UTRs are actually larger on average (https://gist.github.com/arq5x/0d44bae195fc6260984ee01fd253712c). Therefore, it is not clear that k=36 for UTRs versus k=75 for exons is well-justified. >> We appreciate this consideration. To alleviate such concerns regarding the value of k, we have provided options to select appropriate k for exons or UTRs in our code for users that would prefer a different setting. Our choice of k=75 was originally guided by the RNA-seq read-length of the GTEx data, and a reduced length for UTRs based on manual investigation of our top hits suggesting that k=75 for UTRs left too many questionable instances unfiltered. However, because there is no gold standard for which trans-eQTLs are truly false positives, we did not think of a way to tune this more closely. To provide further insight, we have also now computed cross-mappability using 75-mers from both exons and UTRs and shared these resources publicly. We also shared resources with 50-mers from exons and 36-mers from UTRs. Using 75-mers from both exons and UTRs, 67.2% of unique trans-eQTLs appeared as cross-mappable, compared to 75.1% when 75-mers from exons and 36-mers from UTRs were used. We believe it is likely missing some false-positives at this larger k, but we cannot prove this for certain. Cross-mappable eQTLs still tend to be the most highly significant. We have added discussion of the issue in the manuscript along with Supplementary Figure 10A. “Researchers should carefully choose settings according to the study design and goals. Genome version and gene annotations can be directly matched, but other parameter choices such as k and the maximum number of mismatches allowed in alignment may affect the detection of false positives. Small values of k will produce more conservative cross-mappability scores, but large k may not correctly handle small exons or UTRs. For example, if 75-mers (instead of 36-mers) were used from UTRs, a smaller proportion of trans-eQTLs (67.2% instead of 75.14%) would appear as cross-mappable in GTEx, although cross-mappable trans-eQTLs would still tend to be most highly significant (Supplementary Figure 10A)” We also clarified that we used a collapsed gene model to generate k-mers where overlapped exons and overlapped UTRs were merged to form exonic and UTR regions, respectively. A locus that overlaps both an annotated UTR and an annotated exon will be considered as both. 2. When reading the manuscript, one naturally wonders whether the effects revealed by cross-mappability could be detected via existing \"mappability\" scores from ENCODE and others. It would helpful to have an explicit discussion and/or analysis of how this metric differs. >> Thank you for raising the point. Existing \"mappability\" scores (described by either Derrien et al. or Karimzadeh et al.) correspond to a single genomic region (e.g., gene). In contrast, our \"cross-mappability\" score corresponds to a pair of genes. While mappability score tells about how unique the sequences of a gene (or a region) is, it does not talk about how similar the sequences of a given pair of genes are. A gene with mappability=1 (mappability = 1 for every k-mer of the gene) will have cross-mappability = 0 from/to any other gene (we directly used this property to optimize our computation of cross-mappability genome-wide). However, cross-mappability between two genes with mappability < 1 cannot be revealed by mappability. We now described the difference between these two metrics in the revised manuscript. Genes with mappability < 1 may indeed cross-map with other genes, or may have sequence similarity with non-transcribed regions of the genome, in which case they would not appear cross-mappable in our analysis. \"Notably, existing mappability scores [4, 5] correspond to a single region (or gene) describing uniqueness of the sequences of the region in the genome, our cross-mappability score corresponds to a pair of genes describing similarity between the sequences of the genes.\" 3. By my reading and interpretation of Figure 1B, k-mers spanning exon/exon junctions were not modeled. This is perhaps critical given the results describing an enrichment of cross-mappable eQTLs corresponding to processed pseudogenes. >> We appreciate your concern. We do think accounting for k-mers spanning exon-exon junctions would offer improvements of cross-mappability, and we plan to investigate this further. Please see our response to Rob Patro for details. 4. Echoing the point raised by Rob Patro, it is important to understand how the gene expression qualification was done for GTeX and discuss how these methods impact cross-mappability analysis. >> We agree. We performed additional analyses using RSEM-quantified gene expressions and we still observed potential false positives in our analyses. Please see our response to Mike Love for details along with new Supplementary Figure 11. 5. Mappability is defined as 1/C_k where C_k is the number of genomic loci to which the k-mer aligns with <= 2 mismatches. Given sequencing errors, polymorphism, and the fact that RNA-seq is actually cDNA-seq, one might be concerned that allowing more mismatches (and/or INDEL errors) would more properly model cross-mappability. While perhaps a minor concern, it would be nice to know how much the scores and resulting cross-mappability table differ with tolerance for larger edit distances. >> This is a good point. The number of mismatches is a configurable parameter of our code, and one can compute cross-mappabilities with any desired value. The cross-mappability scores may increase if the allowed number of mismatches is increased. We have now also added an additional analysis with <=3 mismatches (Bowtie v1 allows max 3 mismatches) along with new Supplementary Figure 10B. Maximum 3 mismatches results in a slightly higher percentage of cross-mappable trans-eQTLs than maximum 2 mismatches (76.1% vs. 75.14%). “Similarly, increasing the number of mismatches allowed in k-mer alignment results in an increased number of cross-mappable trans-eQTLs (Supplementary Figure 10B)” 6. In the results describing an enrichment of spurious trans eQTLs associated with cross-mapping pseudogenes, it is unclear whether the authors are referring to all types of pseudogenes or solely processed pseudogenes. Crossmapping could arise from both \"classic\" (i.e., duplicated owing to NAHR) and \"processed\" (i.e., mature RNAs duplicated/inserted into the genome). However, the effect of excluding k-mers that span exon/exon boundaries from the cross-mappability would have a differential effect on these two types of pseudogenes. It would be helpful to be explicit in which types of pseudogenes are being discussed. >> This is a good suggestion. It would be nice to see how different types of pseudogenes contribute to cross-mappability. Gencode v19 annotation does not distinguish among different types of pseudogenes, so we mapped the types of pseudogenes from Gencode v26 annotation, which should be sufficiently accurate for aggregate characterization. Our results show that processed pseudogenes are most common source of cross-mappability, while other pseudogenes do also contribute to cross-mappability. We added an extra plot in Supplementary Figure 4 showing the representation of different types of pseudogenes in trans-eQTLs with pseudogene targets. 7. SNPs were excluded from evaluation if they overlapped annotations in the RepeatMasker track. I would argue that SNPs should (and typically would be) excluded if they lie within segmental duplications. Such regions also harbor genes with a high degree of paralogy (because of the ancestral duplication) and if excluded, may have a substantial impact on the results. For example, I suspect many \"classic\" pseudogenes would also be excluded were SNPs in segmental duplications excluded. While I have never conducted a trans eQTL analysis, I suspect that removal of segdups is standard practice owing to the inherently high paralogy between duplicated regions. >> This is a good point. We do mask SNPs based on RepeatMasker, but this only covers a subset of segdups. In general for our false positives, the SNP is actually not within a segdup itself (segdup SNP eQTLs display a slightly higher rate of cross-mappability than background - 83% vs 75%) . The classic pattern is a SNP in the cis-region of one gene appearing associated with a distant region similar to the cis-gene (potentially a segdup in some cases), but the region of similarity does not include the SNP locus. Excluding pseudogenes and/or segdups from candidate genes in a trans-eQTL study is certainly one option. Making SNPs or excluding genes in segdups does not appear to be standard practice to the best of our knowledge, nor does it cover every likely false positive we observe, but is a change worth considering to standard trans-eQTL pipelines."
}
]
}
] | 1
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https://f1000research.com/articles/7-1860
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https://f1000research.com/articles/7-1933/v1
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14 Dec 18
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{
"type": "Research Article",
"title": "Transcription factor binding site clusters identify target genes with similar tissue-wide expression and buffer against mutations",
"authors": [
"Ruipeng Lu",
"Peter K. Rogan",
"Ruipeng Lu"
],
"abstract": "Background: The distribution and composition of cis-regulatory modules composed of transcription factor (TF) binding site (TFBS) clusters in promoters substantially determine gene expression patterns and TF targets. TF knockdown experiments have revealed that TF binding profiles and gene expression levels are correlated. We use TFBS features within accessible promoter intervals to predict genes with similar tissue-wide expression patterns and TF targets. Methods: Genes with correlated expression patterns across 53 tissues and TF targets were respectively identified from Bray-Curtis Similarity and TF knockdown experiments. Corresponding promoter sequences were reduced to DNase I-accessible intervals; TFBSs were then identified within these intervals using information theory-based position weight matrices for each TF (iPWMs) and clustered. Features from information-dense TFBS clusters predicted these genes with machine learning classifiers, which were evaluated for accuracy, specificity and sensitivity. Mutations in TFBSs were analyzed to in silico examine their impact on cluster densities and the regulatory states of target genes. Results: We initially chose the glucocorticoid receptor gene (NR3C1), whose regulation has been extensively studied, to test this approach. SLC25A32 and TANK were found to exhibit the most similar expression patterns to NR3C1. A Decision Tree classifier exhibited the largest area under the Receiver Operating Characteristic (ROC) curve in detecting such genes. Target gene prediction was confirmed using siRNA knockdown of TFs, which was found to be more accurate than those predicted after CRISPR/CAS9 inactivation. In-silico mutation analyses of TFBSs also revealed that one or more information-dense TFBS clusters in promoters are required for accurate target gene prediction. Conclusions: Machine learning based on TFBS information density, organization, and chromatin accessibility accurately identifies gene targets with comparable tissue-wide expression patterns. Multiple information-dense TFBS clusters in promoters appear to protect promoters from effects of deleterious binding site mutations in a single TFBS that would otherwise alter regulation of these genes.",
"keywords": [
"Transcription factors",
"position-specific scoring matrices",
"chromatin",
"binding sites",
"gene expression profiles",
"Bray-Curtis similarity",
"mutation",
"machine learning",
"information theory"
],
"content": "Introduction\n\nThe distinctive organization and combination of transcription factor binding sites (TFBSs) and regulatory modules in promoters dictates specific expression patterns within a set of genes1. Clustering of multiple adjacent binding sites for the same TF (homotypic clusters) and for different TFs (heterotypic clusters) defines cis-regulatory modules (CRMs) in human gene promoters. Experimental studies have shown that these clusters can reinforce (and in some instances, amplify) the impact of individual TFBSs on gene expression through increasing binding affinities, facilitated diffusion mechanisms and funnel effects2. Because tissue-specific TF-TF interactions in TFBS clusters are prevalent, these features can assist in identifying correct target genes by altering binding specificities of individual TFs3. Previously, we derived iPWMs from ChIP-seq data that can accurately detect TFBSs and quantify their strengths by computing associated Ri values (Rate of Shannon information transmission for an individual sequence4), with Rsequence being the average of Ri values of all binding site sequences and representing the average binding strength of the TF3. Furthermore, information density-based clustering (IDBC) can effectively identify functional TF clusters by taking into account both the spatial organization (i.e. intersite distances) and information density (i.e. Ri values) of TFBSs5.\n\nTF binding profiles, either derived from in vivo ChIP-seq peaks6–8 or computationally detected binding sites and CRMs9, have been shown to be predictive of absolute gene expression levels using a variety of tissue-specific machine learning classifiers and regression models. Because signal strengths of ChIP-seq peaks are not strictly proportional to TFBS strengths3, representing TF binding strengths by ChIP-seq signals may not be appropriate; nevertheless, both achieved similar accuracy10. CRMs have been formed by combining two or three adjacent TFBSs9, which is inflexible, as it arbitrarily limits the number of binding sites contained in a module, and does not consider differences between information densities of different CRMs. Chromatin structure (e.g. histone modification (HM) and DNase I hypersensitive sites (DHSs)) were also found to be statistically redundant with TF binding in explaining tissue-specific mRNA transcript abundance at a genome-wide level7,8,11,12, which was attributed to the heterogeneous distribution of HMs across chromatin domains8. Combining these two types of data explained the largest fraction of variance in gene expression levels in multiple cell lines7,8, suggesting that either contributes unique information to gene expression that cannot be compensated for by the other.\n\nThe number of genes directly bound by a TF significantly exceeds the number of differentially expressed (DE) genes whose expression levels significantly change upon knockdown of the TF. Only a small subset of direct target genes whose promoters overlap ChIP-seq peaks were DE after individually knocking 59 TFs down using small interfering RNAs (siRNAs) in the GM19238 cell line13. Using these knockdown data on 8,872 genes as the gold standard, correlation between TFBS counts and gene expression levels across 10 different cell lines was more predictive of DE targets than setting a minimum threshold on TFBS counts14. Their TFBS counts were defined as the number of ChIP-seq peaks overlapping the promoter, though it was unknown how many binding sites were present in these peaks; positives might not be direct targets in the TF regulatory cascade, as the promoters of these targets were not intersected with ChIP-seq peaks. By perturbing gene expression with CAS9-directed clustered regularly interspaced short palindromic repeats (CRISPR) of 10 different TF genes in K562 cells, the regulatory effects of each TF on 22,046 genes were dissected by single cell RNA sequencing with a regularized linear computational model15; this accurately revealed DE targets and new functions of individual TFs, some of which were likely regulated through direct interactions at TFBSs in their corresponding promoters. Machine learning classifiers have also been applied in a small number of gene instances to predict targets of a single TF using features extracted from n-grams derived from consensus binding sequences16, or from TFBSs and homotypic binding site clusters5.\n\nTo investigate whether the distribution and composition of CRMs in promoters substantially determines gene expression profiles of direct TF targets, we developed a general machine learning framework that predicts which genes have similar tissue-wide expression profiles to a given gene and predicts DE direct TF targets by combining information theory-based TF binding profiles with DHSs. Upon filtering of accessible promoter intervals based on the locations of DHSs, features designed to capture the spatial distribution and information composition of CRMs were extracted from clusters of iPWM-detected TFBSs identified by IDBC. Though not all direct targets regulated by multiple TFs share a common tissue-wide expression profile, this framework provides insight into the transcriptional program of genes with similar profiles by dissecting their cis-regulatory element organization and strengths. We identify genes with comparable tissue-wide expression profiles by application of Bray-Curtis similarity17. Using transcriptome data generated by CRISPR-15 and siRNA-based13 TF knockdowns, we predicted DE TF target genes that are simultaneously direct targets whose promoters overlap tissue-specific ChIP-seq peaks.\n\n\nMethods\n\nTo identify genes with similar tissue-wide expression patterns, we formally define tissue-wide gene expression profiles and pairwise similarity measures between profiles of different genes. A general machine learning framework relates features extracted from the organization of TFBSs in these genes to their tissue-wide expression patterns. Since protein-coding (PC) sequences represent the most widely studied and best understood component of the human genome18, positives and negatives for deriving machine learning classifiers for predicting DE direct TF target genes that encode proteins (TF targets for short below) were obtained from CRISPR- and siRNA-generated knockdown data (see below).\n\nThe Genotype-Tissue Expression (GTEx, version 6p) Project measured expression levels in 53 tissues, each of which is represented by different numbers of individuals (5 to 564). For each tissue population, the median expression value is given in RPKM (Reads Per Kilobase of transcript per Million mapped reads) for each gene19. Data are available on Zenodo20. To capture the tissue-wide overall expression pattern of a gene instead of within a single tissue, the tissue-wide expression profile of a gene was defined as its median RPKM across GTEx tissues, which is described by a 53 element vector (Equation 1). Note that different isoforms whose expression patterns may significantly differ from each other cannot be distinguished by this approach.\n\n\n\nwhere EPA is the tissue-wide expression profile of Gene A, MEV1A is the median expression value of Gene A in Tissue 1, MEV2A is the median expression value of Gene A in Tissue 2, etc.\n\nTo discover other genes whose tissue-wide expression profiles are similar to a given gene, we computed the Bray-Curtis Similarity (Equation 2) between the tissue-wide expression profiles of gene pairs. Compared to other similarity metrics (Table 1, Example 1, Additional file 121), the application of this function is justified by desirable properties, including: 1) maintaining bounds between 0 and 1, 2) achieving the maximal similarity value 1 if and only if two vectors are identical, and 3) larger values having a larger impact on the resultant similarity value.\n\n† The symbols √ and ×, respectively, indicate that the similarity metric satisfies and does not satisfy the property. ‡The interval in each cell indicates the range in which the result computed by the similarity metric lies.\n\n\n\nExample 1. Assume that Genes A,B,C,D,E,F respectively have the following GTEx expression profiles across two tissues: EPA = [1,1], EPB =[2,2], EPC = [3,3], EPD = [1,2], EPE = [1,99], EPF = [1,100]. The ground-truth similarity relationships that we can intuitively infer include sim(EPC, EPA) < sim(EPC, EPB) < 1, and sim(EPA, EPD) < sim(EPE, EPF) < 1. Only the results computed by the Bray-Curtis Similarity are completely concordant with these ground-truth relationships (Table 2).\n\nThe framework for identifying genes whose tissue-wide expression profiles most resemble a particular gene is shown in Figure 1A, B. The genomic locations of all DHSs in 95 cell types generated by the ENCODE project[22; hg38 assembly] were selected for known promoters23, then 94 iPWMs exhibiting primary binding motifs for 82 TFs3 were used to detect TFBSs within the overlapping intervals. Data are available on Zenodo20. When detecting heterotypic TFBS clusters with the IDBC algorithm, a minimum threshold 0.1 * Rsequence was specified for the Ri values of TFBSs in order to remove weak binding sites that were more likely to be false positive TFBSs3.\n\nRed and blue contents are respectively specific to prediction of genes with similar tissue-wide expression profiles and prediction of TF targets. (A) An overview of the machine learning framework. The steps enclosed in the dashed rectangle vary across prediction of genes with similar tissue-wide expression profiles and TF targets. The step with a dash-dotted border that intersects promoters with DHSs is a variant of the primary approach. In the IDBC algorithm (Additional file 121), the parameter I is the minimum threshold on the total information contents of TFBS clusters. In prediction of genes with similar tissue-wide expression profiles, the minimum value was 939, which was the sum of mean information contents (Rsequence values) of all 94 iPWMs; in prediction of direct targets, this value was the Rsequence value of the single iPWM used to detect TFBSs. The parameter d is the radius of initial clusters in base pairs, whose value, 25, was determined empirically. The performance of seven different classifiers were evaluated with statistics (accuracy, sensitivity and specificity) (Additional file 121). (B) Obtaining of the positives and negatives for identifying genes with similar tissue-wide expression profiles to a given gene (Additional file 221). (C) Obtaining of the positives and negatives for predicting target genes of seven TFs using the CRISPR-generated perturbation data in K562 cells (Additional file 321). (D) Obtaining of the positives and negatives for predicting target genes of 11 TFs using the siRNA-generated knockdown data in GM19238 cells (Additional file 421).\n\nThe information density-related features (Additional file 121) derived from each TFBS cluster included: 1) The distance between this cluster and the transcription start site (TSS); 2) The length of this cluster; 3) The information content of this cluster (i.e. the sum of Ri values of all TFBSs in this cluster); 4) The number of binding sites of each TF within this cluster; 5) The number of strong binding sites (Ri > Rsequence) of each TF within this cluster; 6) The sum of Ri values of binding sites of each TF within this cluster; 7) The sum of Ri values of strong binding sites (Ri > Rsequence) of each TF within this cluster.\n\nFor a gene, each of Features 1-3 was defined as a vector whose size equals the number of clusters in the promoter; thus, the entire vector could be input into a classifier. If two genes contained different numbers of clusters, the maximum number of clusters among all instances was determined, and null clusters were added at the 5’ end of promoters with fewer clusters, enabling all instances to have the same cluster count. Using all instances as training data, machine learning classifiers with default parameter values in MATLAB were used to generate ROC curves (Additional file 121).\n\nUsing gene expression in the CRISPR-based perturbations. Dixit et al. performed CRISPR-based perturbation experiments using multiple guide RNAs for each of ten TFs in K562 cells, resulting in a regulatory matrix of coefficients that indicate the effect of each guide RNA on each of 22,046 genes15. Data are available on Zenodo20. The coefficient of a guide RNA on a gene is defined as the log10(fold change in gene expression level)15. Among these ten TFs, we have previously derived iPWMs exhibiting primary binding motifs for seven (EGR1, ELF1, ELK1, ETS1, GABPA, IRF1, YY1)3. Therefore, the framework for predicting TF targets in the K562 cell line (Figure 1A and 1C) was applied to these TFs. The criteria for defining a positive (i.e. a target gene), of a TF were:\n\n1) The fold change in the expression level of this gene for each guide RNA of the TF exceeds (or is less than) 1, eliminating those genes exhibiting both increased and decreased expression levels for different guide RNAs, and maximizing the possibility that the gene was downregulated (or upregulated) by the TF (Additional file 121). and\n\n2) The average fold change in the expression level of this gene for all guide RNAs of the TF exceeds the threshold 𝜀 (or is less than 1/𝜀), and\n\n3) The promoter interval (10 kb) upstream of a TSS of this gene overlaps a ChIP-seq peak of the TF in the K562 cell line.\n\nIf the coefficients of all guide RNAs of the TF for a gene are zero, the gene was defined as a negative. As the threshold ε increases, the number of positives strictly decreases; as ε decreases, we have increasingly lower confidence in the fact that the positives were indeed differentially expressed because of the TF perturbation. To achieve a balance, we evaluated three different values (i.e. 1.01, 1.05 and 1.1) for ε (Additional file 121). For each TF, all ENCODE ChIP-seq peak datasets from the K562 cell line were merged to determine positives. Data are available on Zenodo20. To make the numbers of negatives and positives equal to avoid imbalanced datasets that significantly compromise the classifier performance26, the Bray-Curtis function was applied to compute the similarity values in the tissue-wide expression profile between all negatives and the positive with the largest average coefficient, then the negatives with the smallest values were selected (Figure 1C).\n\nThe DHSs in the K562 cell line were intersected with known promoters. Data are available on Zenodo20. Because TFs may exhibit tissue-specific sequence preferences due to different sets of target genes and binding sites in different tissues3, the iPWMs of EGR1, ELK1, ELF1, GABPA, IRF1, YY1 from the K562 cell line were used to most accurately detect binding sites; for ETS1, we used the only available iPWM from the GM12878 cell line3. Six features (Features 1-5 and 7) were derived from each homotypic cluster (i.e. Feature 6 became identical to Feature 3, because only binding sites from a single TF were used) (Figure 1A). The results of 10 rounds of 10-fold cross validation were averaged to more accurately evaluate the predictive power of the classifier.\n\nUsing gene expression in the siRNA-based knockdown. In the GM19238 cell line, 59 TFs were individually knocked down using siRNAs, and significant changes in the expression levels of 8,872 genes were indicated according to their corresponding P-values13. Data are available on Zenodo20. In these cases, the P-value of a gene for a TF is the probability of observing the change in the expression level of this gene under the null hypothesis of no differential expression after TF knockdown; thus, the larger the change in the expression level, the smaller the P-value and the more likely this gene is differentially expressed. They also indicated whether the promoters of these genes display evidence of binding to TFs by intersecting with ChIP-seq peaks in the GM12838 cell line. Among these 59 TFs, we have previously derived accurate iPWMs exhibiting primary binding motifs for 11 (BATF, JUND, NFE2L1, PAX5, POU2F2, RELA, RXRA, SP1, TCF12, USF1, YY1)3. Therefore, the framework for predicting TF targets in the GM19238 cell line (Figure 1A, D) was applied to these 11 TFs.\n\nWe defined a positive (i.e. a target gene) for a TF, if the P-value of this gene for the TF was ≤ 0.01, and the promoter interval (10 kb) upstream of a TSS of this gene overlapped a ChIP-seq peak of the TF in the GM12878 cell line. All other genes with P-values > 0.01 were considered to exhibit insufficient evidence of being TF targets, i.e. these were considered negatives or non-targets.\n\nThe DHSs in the GM19238 cell line mapped from the hg19 genome assembly were first remapped to the hg38 assembly using liftOver prior to being intersected with known promoters27. Data are available on Zenodo20. Aside from RELA, RXRA and NFE2L1, the iPWMs of TFs from the GM12878 cell line were used to detect binding sites. For RELA, the iPWM from the GM19099 cell line was used; for RXRA and NFE2L1, the only available iPWMs were respectively derived from HepG2 and K562 cells and were applied. Although the knockdown was performed in GM19238, GM12878 and GM19099 are also lymphoblastic cell lines, with GM19099 and GM19238 both being derived from Yorubans. For this analysis, the iPWMs derived in GM12878 and GM19099 were more appropriate sources of accessible TFBSs than those from HepG2 and K562, since GM12878 and GM19099 are of the same tissue type and are thus more likely comparable to GM19238 than HepG2 and K562. Similarly, the results of 10 rounds of 10-fold cross validation were averaged to more accurately evaluate the predictive power of the classifier.\n\nTo better understand the significance of individual binding sites for information-dense clusters and the regulatory state of direct targets, we evaluated the effects of sequence changes that altered the Ri values of these sites on cluster formation and whether a gene was predicted to be a TF target. Mutations were sequentially introduced into the strongest binding sites in TFBS clusters of the EGR1 target gene, MCM7, to determine the threshold for cluster formation after disappearing clusters disabled induction of MCM7 expression. For one target gene of each TF from the CRISPR-generated perturbation data, effects of naturally occurring TFBS variants present in dbSNP28 were also evaluated to explore aspects of TFBS organization that enabled both clusters and promoter activity to be resilient to binding site mutations. This was done by analyzing whether the occurrence of individual or multiple single nucleotide polymorphisms (SNPs) lead to the loss of binding sites and the corresponding clusters, and resulted in changes in the predictions for these targets.\n\n\nResults\n\nTo confirm that the Bray-Curtis Similarity can indeed effectively measure how akin the tissue-wide expression profiles of two genes are to one another, Equation 2 was applied to compute the similarity values between the tissue-wide expression profiles of the glucocorticoid receptor (GR or NR3C1) gene and all other 18,812 PC genes. NR3C1 is an extensively characterized TF with many known direct target genes29. As a constitutively expressed TF activated by glucocorticoid ligands, the protein can mediate the up-regulation of anti-inflammatory genes by binding of homodimers to glucocorticoid response elements and down-regulation of proinflammatory genes by complexing with other activating TFs (e.g. NFKB and AP1) and eliminating their ability to bind targets29. NR3C1 can bind its own promoter forming an auto-regulatory loop, which also contains functional binding sites of 11 other TFs (e.g. SP1, YY1, IRF1, NFKB) whose iPWMs have been developed and/or mutual interactions have been described previously3,29. However, since the tissue-wide expression profile of NR3C1 comprises all different splicing and translational isoforms (e.g. GRα-A to GRα-D, GRβ, GRγ, GRδ), the tissue-specific expression patterns of these isoforms are indistinguishable (e.g. levels of the GRα-C isoforms are significantly higher in the pancreas and colon, whereas levels of GRα-D are highest in spleen and lungs)29. SLC25A32 and TANK have the greatest similarity in expression to NR3C1 (0.880 and 0.877 respectively), which is evident based on their overall similar expression patterns across the 53 tissues (Figure 2).\n\nVisualization of the expression values (in RPKM) of these genes across 53 tissues from GTEx. For each gene, the colored rectangle belonging to each tissue indicates the valid RPKM of all samples in the tissue, the black horizontal bar in the rectangle indicates the median RPKM, the hollow circles indicate the RPKM of the samples considered as outliers, and the grey vertical bar indicates the sampling error. By comparing the pictures, the overall expression patterns of the three genes across the 53 tissues resemble each other (e.g. all three genes exhibit the highest expression levels in lymphocytes and the lowest levels in brain tissues).\n\nIn prediction of genes with similar tissue-wide expression profiles to NR3C1, we generated ROC curves to compare the performance of different classifiers (Naïve Bayes, Decision Tree (DT), Random Forest and Support Vector Machines (SVM) with four different kernels), under two scenarios depending on whether promoter sequences were first intersected with DHSs (Figure 3). DT exhibited the largest AUC (area under the curve) under both scenarios, and was one of two most stable classifiers (i.e. ΔAUC < 0.01), with the other being the SVM with RBF kernel. Inclusion of DHS information significantly improved AUC values of the other classifiers with the exception of Naïve Bayes, and in many instances, all TFBSs in a contiguous DHS interval formed a single binding site cluster.\n\n(A) ROC curves and AUC of seven classifiers without intersecting promoters with DHSs. (B) ROC curves and AUC of seven classifiers after intersecting promoters with DHSs. The Decision Tree classifier exhibited the largest AUC under both scenarios, and inclusion of DHS information significantly improved other classifiers’ AUC except for Naïve Bayes.\n\nSince the DT classifier performed the best in distinguishing genes with NR3C1-like tissue-wide expression profiles from others, we further used this classifier type to predict TF targets respectively based on the CRISPR-15 and siRNA-generated13 perturbation data, and assessed its performance with 10 rounds of 10-fold cross validation. To validate that using all six machine learning features more comprehensively capture the distribution and composition of CRMs in the promoter, all of the features, except for TFBS counts, were removed. The classifier performance decreased, except for CRISPR-perturbed GABPA, IRF1 and YY1 after inclusion of DHS information (Additional file 521).\n\nOn the CRISPR-generated knockdown data, after eliminating TFBSs in inaccessible promoter intervals, i.e. those excluded from tissue-specific DHSs, the DT classifier predicted TF targets with greater sensitivity and specificity (Table 3). Specifically, predictions for TFs: EGR1, ELK1, ELF1, ETS1, GABPA, and IRF1 were more accurate than for YY1, which itself represses or activates a wide range of promoters by binding to sites overlapping the TSS (Table 3). Accordingly, the perturbation data indicated that YY1 has ~4-22 times more PC targets in the K562 cell line than the other TFs (ε = 1.05), and its binding has a more significant impact on the expression levels of target genes (for YY1, the ratio of the PC target counts at ε = 1.1 vs ε = 1.01 was 0.334, which significantly exceeded those of the other TFs (0.017-0.082); Additional file 321). This is concordant with our previous finding that YY1 extensively interacts with 11 cofactors (e.g. DNA-binding IRF9 and TEAD2; non-DNA-binding DDX20 and PYGO2) in K562 cells, consistent with a central role in specifying erythroid-specific lineage development3.\n\n†The average performance of 10 rounds of 10-fold cross validation when setting ε to 1.05 is indicated. The CRISPR-generated knockdown data were obtained from Dixit et al.15.\n\nDespite a high accuracy of target recognition, sensitivity did not exceed specificity except for IRF1 (Table 3), due to a relatively large number of false negative genes. We find that promoters of most TF targets contain accessible, likely functional binding sites that significantly are correlated with changes in gene expression levels. By contrast, promoters of non-targets contain either no accessible binding sites at all, or accessible, but non-functional sites. The fact that these false negatives were erroneously predicted to non-targets was attributable to the inability of the classifier to distinguish between likely functional binding sites in their promoters and non-functional ones in non-targets in some cases. In vivo co-regulation mediated by interacting cofactors, which was excluded by the classifier, assisted in distinguishing these non-functional sites that do not significantly affect gene expression13.\n\nAs the threshold ε increased, the accuracy of the classifier for all the TFs monotonically increased as expected (Figure 4). For a gene to be defined as a DE target of a TF, the average fold change in its expression level for all guide RNAs that downregulated the TF were required to reach the minimum threshold ε. Upon TF knockdown, ε is inversely correlated with the number of target genes, but positively correlated with fold changes in their corresponding expression levels. In general, more significantly DE genes have been associated with a higher number of TFBSs in their promoters13. Thus, at greater ε, there are larger differences in the values of machine learning features derived from TFBS clusters between direct targets and non-targets.\n\nEach accuracy value was averaged from 10 rounds of 10-fold cross validation, when the minimum threshold 𝜀 on the average fold change in gene expression levels under all guide RNAs of the TF took three different values 1.01, 1.05 and 1.1. As 𝜺 increased, accuracy for all seven TFs monotonically increased.\n\nWith the siRNA-generated knockdown data, the performance of the DT classifier was compared to the approach inferring DE targets by correlating TF binding with gene expression levels across ten cell types14. In this correlation-based approach, three measures (i.e. the absolute Pearson correlation coefficient (PCC), the absolute Spearman correlation coefficient (SCC), and the absolute combined angle ratio statistic (CARS)), whose performance was evaluated with precision-recall curves, were alternatively used to compute a correlation score between the number of ChIP-seq peaks overlapping the promoter and gene expression values. Genes predicted to be DE targets had scores above the threshold resulting in a 1.5-fold increase compared to the background precision (i.e. the DE target count / 8,872). For example, in the case of YY1, which was the only TF analyzed by both approaches, the performance of the DT classifier was 0.98 (precision) and 0.55 (recall) after including DHS information (Table 4). This classifier outperformed all three correlation measures (PCC: 0.467 and 0.003; SCC: 0.467 and 0.006; CARS: 0.467 and 0.003 directly obtained from 14), even though the correlation-based approach used a less stringent P-value threshold (0.05) for defining differential expression of likely non-direct targets, and intersected ChIP-seq peaks over shorter 5kb promoter intervals upstream of the TSS. Three reasons explain why the correlation-based approach exhibited lower recall, including: 1) it did not use machine learning classifiers, 2) its larger P-value threshold (0.05) generated a larger number of positives and, 3) positives also include DE targets that cannot be directly bound.\n\n†The average performance of 10 rounds of 10-fold cross validation is indicated. The siRNA-generated knockdown data were obtained from Cusanovich et al.13.\n\nTo determine how many TF targets have similar tissue-wide expression profiles, we intersected the set of targets with the set of 500 PC genes with the most similar tissue-wide expression profiles for each TF (Table 5, Additional file 621). The TFs PAX5 and POU2F2 are primarily expressed in B cells, and their respective targets IL21R and CD86 are also B cell-specific, which accounts for the high similarity in the tissue-wide expression profile between them. There are respectively 21 and 7 nuclear mitochondrial genes (e.g. MRPL9 and MRPS10, which are subunits of mitochondrial ribosomes) in the intersections for YY1 in the K562 and GM19238 cell lines30. Previous studies reported that YY1 upregulates a large number of mitochondrial genes by complexing with PGC-1α in C2C12 cells31, and genes involved in the mitochondrial respiratory chain in K562 cells15, which is consistent with the idea that YY1 may broadly regulate mitochondrial function (within all 53 tissues in addition to the erythrocyte, lymphocyte and skeletal muscle cell lines).\n\n§The rank of each target in the list of similar genes in the descending order of Bray-Curtis similarity values is shown in the brackets immediately following the target.\n\nBetween 0.4%–25% of genes with similar tissue-wide expression profiles to the TFs are actually their targets (Table 5); the majority are non-targets whose promoters contain non-functional binding sites that are distinguished from targets by their lack of co-regulation by corresponding cofactors. For YY1 and EGR1, we validated this hypothesis by contrasting the flanking cofactor binding site distributions and strengths in the promoters of the most similarly expressed target genes (YY1: MRPL9, BAZ1B; EGR1: CANX, NPM1) and non-target genes (YY1: ADNP, RNF25; EGR1: GUCY2F, AWAT1). In the promoters of these target genes, strong and intermediate recognition sites for TFs: SP1, KLF1, CEBPB formed heterotypic clusters with adjacent YY1 sites; as well TFBSs of SP1, KLF1, and NFY were frequently present adjacent to EGR1 binding sites (Additional file 721). These patterns contrasted with the enrichment of CTCF and ETV6 binding sites in gene promoters of YY1 and EGR1 non-targets (Additional file 721). Previous studies have reported that KLF1 is essential for terminal erythroid differentiation and maturation32, direct physical interactions between YY1 and the constitutive activator SP1 synergistically induce transcription33, the activating CEBPB promotes differentiation and suppresses proliferation of K562 cells by binding the promoter of the G-CSFR gene encoding a hematopoietin receptor34, EGR1 and SP1 synergistically cooperate at adjacent non-overlapping sites on EGR1 promoter but compete binding at overlapping sites35; whereas occupied CTCF binding sites often function as an insulator blocking the effects of cis-acting elements and preventing gene activation by mediating long-range DNA loops to alter topological chromatin structure36,37, and ETV6, a member of the ETS family, is a transcriptional repressor required for bone marrow hematopoiesis and associated with leukemia development38.\n\nBecause the promoters of direct target genes contain multiple binding site clusters, we hypothesized that this organization could stabilize gene expression against the effect of mutations in individual binding sites; in other words, the other clusters might be able to compensate for the loss of a cluster destroyed by mutations, so that the mutated promoters would still be capable of effectively regulating gene transcription upon TF binding. First, we examined whether introducing artificial variants into binding sites in silico in the promoter of the target gene MCM7 of EGR1 changed the classifier output (Figure 5). Specifically, in the K562 cell line, MCM7 is upregulated by EGR1. Knockdown of MCM7 has an anti-proliferative and pro-apoptotic effect on K562 cells39 and the loss of EGR1 increases leukemia initiating cells40, which suggests that EGR1 may act as a tumor suppressor in K562 cells through the MCM7 pathway.\n\nThis figure depicts the effect of a mutation in each EGR1 binding site cluster of the MCM7 promoter on the expression level of MCM7, which is a target of the TF EGR1. The strongest binding site in each cluster were abolished by a single nucleotide variant. Upon loss of all three clusters, only weak binding sites remained and EGR1 was predicted to no longer be able to effectively regulate MCM7 expression. Multiple clusters in the promoters of TF targets confer robustness against mutations within individual binding sites that define these clusters.\n\nFirst, the strongest binding site (at position chr7:100103347[hg38], - strand, Ri = 12.0 bits) in the promoter was eliminated by a G->A mutation, resulting in the loss of Cluster 1, which consists of two sites (the other site at chr7:100103339, -, 4.3 bits). The other two clusters comprising weaker binding sites of intermediate strength (chr7:100102252, +, 7.0 bits; chr7:100102244, +, 1.3 bits; chr7:100101980, +, 8.9 bits; chr7:100101977, +, 2.2 bits; chr7:100101984, +, 1.9 bits) were still predicted to compensate for this mutation, enabling the promoter to maintain capability of inducing MCM7 expression (Figure 5). It is well known that adjacent clustered sites, which themselves may not be strong enough to individually bind TFs and activate transcription, can stabilize each other’s binding2. The weaker sites flanking a strong binding site in a cluster can direct the TF molecule to the strong site and extend the period of the molecule physically associating with the strong site, which is termed, the funnel effect2. In this example, Clusters 2 and Cluster 3 were also respectively removed by G->T and C->G mutations abolishing the strongest site in either cluster, which altered the prediction, that is, EGR1 should lose the capability to induce MCM7 transcription (Figure 5). The remaining four sparse weak sites do not form a cluster and cannot completely supplant the disrupted strong sites.\n\nFurther, we examined the predicted impacts of known natural SNPs on binding site strengths, clusters and the regulatory state of the promoter for a direct target of each of the seven TFs from the CRISPR-generated perturbation data (Table 6). Often a single SNP (e.g. rs996639427 of EGR1) can affect the strengths of multiple binding sites (Table 6). Apart from SNPs that are predicted to abolish binding (Figure 5), leaky variants that merely weaken TF binding are common (Table 6). Binding stabilization between adjacent sites and the funnel effect enable CRMs comprised of information-dense clusters to be robust to mutations in individual binding sites2,41. In this way, neither mutations that abolish TFBSs nor leaky SNPs in flanking weak sites would be expected to destroy clusters (e.g. rs1030185383 and rs5874306 of IRF1), whereas SNPs with large reductions in Ri values of central strong sites are more likely to abolish clusters (e.g. rs865922947, rs946037930, rs917218063 and rs928017336 of YY1) (Table 6). More generally, the presence of multiple clusters enables promoters to be effectively resilient to the effects of binding site mutations; only the complete abolishment of all clusters resulting from the simultaneous occurrence of multiple SNPs should be able to transform the promoter to be unresponsive to TF binding to residual weak sites (e.g. rs997328042, rs1020720126 and rs185306857 of GABPA) (Table 6). Furthermore, a relatively small number of SNPs that strengthen TF binding and eventually reinforce the regulatory effect of the TF are also present in these cases (e.g. rs887888062 of EGR1 and rs751263172 of ELF1) (Table 6), suggesting that, in addition to deleterious mutations, potentially benign variants may also be found in promoters, consistent with the expectations of neutral theory42.\n\n§All coordinates are based on the hg38 genome assembly. A bold italic letter in a binding site sequence indicates the base where a SNP occurs. For each normal and variant binding site sequence, the genome coordinate of its most 5’-end base and its Ri value are indicated. The negative Ri value of a variant binding site sequence implies this site is abolished. The SNPs strengthening binding sites and corresponding variant binding site sequences are underlined.\n\n‡The impact on whether the occurrence of a single SNP resulted in the disappearance of the cluster containing it is shown; ‘Abolished’ indicates that the cluster is eliminated by the existence of the variant allele.\n\n†After a single SNP occurred or multiple SNPs simultaneously occurred, the classifier produced a new prediction on whether the TF is still capable of significantly affecting gene expression via the variant promoter.\n\n\nDiscussion\n\nIn this study, the Bray-Curtis similarity function was initially shown (for the NR3C1 gene) to measure the relatedness of overall expression patterns between genes across a diverse set of tissues. A machine learning framework distinguished Bray-Curtis function-defined similar from dissimilar genes based on the distribution, strengths and compositions of TFBS clusters in accessible promoters, which can substantially account for the corresponding gene expression patterns. Using knockdown data as the gold standard, the combinatorial use of TF binding profiles and chromatin accessibility was also demonstrated to be predictive of TF targets. A binding site comparison confirmed that coregulatory cofactors can be used to distinguish between functional sites in targets and non-functional ones in non-targets. Furthermore, mutation analyses on binding sites of targets demonstrated that the existence of both multiple TFBSs in a cluster and multiple information-dense clusters in a promoter enables both the cluster and the promoter to be resilient to binding site mutations.\n\nThe DT classifier improved after intersecting promoters with DHSs in prediction of TF targets with the CRISPR-generated knockdown data (Table 3). This intersection eliminated noisy binding sites that are inaccessible to TF proteins in promoters; specifically, it widened discrepancies in feature vectors between positives and negatives. If the 10 kb promoter of a gene instance does not overlap DHSs, its feature vector will only consist of 0; the percentages of negatives whose promoters do not overlap DHSs considerably exceeded those of positives (Additional file 821), which led to an excess of negatives with feature vectors containing only 0 after intersection. This explains why these negatives are not DE targets of the TFs in the K562 and GM19238 cell lines, because their entire promoters are not open to TF molecules; other regulatory regions besides the proximal promoters (e.g. intronic enhancers43) still enable the TFs to effectively control the expression of the positives with inaccessible promoters. The relatively poor performance of the classifier on YY1 (Table 3) is attributable to its smaller percentage of negatives with inaccessible promoters (Additional file 821) and the larger number of functional binding sites in the K562 cell line.\n\nOur in-silico mutation analyses revealed that some deleterious TFBS mutations could be compensated for by other information-dense clusters in the same promoter2; thus, predicting the effects of mutations in individual binding sites might not be sufficient to interpret downstream effects without considering their context. Though compensatory clusters may maintain gene expression, the promoter will provide lower levels of activity than the wild-type promoter could, which is a recipe for achieving natural phenotypic diversity41. Few published studies in molecular diagnostics have specifically examined the effects of naturally occurring variants within clustered TFBSs; thus, IDBC-based machine learning provides an alternative computational approach to predict deleterious mutations that actually impact (i.e. repress or abolish) transcription of target genes and result in abnormal phenotypes, and to simultaneously minimize false positive calls of TFBS mutations that individually have little or no impact.\n\nApart from these TFs, the Bray-Curtis Similarity can be directly applied to identify the ground-truth genes with overall similar tissue-wide expression patterns to any other gene whose expression profile is known. Further studies could investigate the biological significance underlying the phenomenon that all these genes share a common expression pattern, including the similarity between other regulatory regions besides proximal promoters in terms of TFBSs and epigenetic markers. This machine learning framework can also be applied to predict target genes for other TFs and in other cell lines, depending on the availability of corresponding knockdown data.\n\nThere are a number of limitations of our approach. The Bray-Curtis function seems unable to accurately measure the similarity between the tissue-wide expression profiles of a gene (e.g. MIR23A) without any detectable mRNA in any of the 53 tissues analyzed and genes (e.g. the ubiquitously expressed NR3C1 and stomach-specific PGA3) that are expressed in at least one tissue. Intuitively, in terms of expression patterns PGA3 is more similar to MIR23A than NR3C1; however, the Bray-Curtis similarity values indicate that both PGA3 and NR3C1 bear no similarity to MIR23A (i.e. simBray-Curtis (NR3C1, MIR23A) = simBray-Curtis (PGA3, MIR23A) = 0). Another possible limitation in classifier performance in the prediction of genes with similar tissue-wide expression profiles is that only binding sites of 82 TFs were analyzed due to a lack of available iPWMs for other TFs, given that 2000-3000 sequence-specific DNA-binding TFs are estimated to be encoded in the human genome44. For example, four TFs (CREB, MYB, NF1, GRF1) were previously reported to bind the promoter of the NR3C1 gene to activate or repress its expression; however, their iPWMs exhibiting known primary motifs could not be successfully derived from ChIP-seq data3,29. Regarding the CRISPR-generated knockdown data used here, positives were inferred to be direct targets by intersecting their promoters with corresponding ChIP-seq peaks, which may not be completely accurate, due to the presence of noise peaks that do not contain true TFBSs3,45. Small fold changes in the expression levels of DE targets could arise from compromised efficiency of knockdowns as a result of suboptimal guide RNAs or the limitations of perturbing only a single allele of the TF46. Finally, the framework developed here only takes into account the 10 kb interval proximal to the TSS, and would not therefore capture long range enhancer effects beyond this distance; by contrast, correlation based approaches have successfully incorporated multiple definitions of promoter length14.\n\n\nConclusions\n\nThe Bray-Curtis function is able to effectively quantify the similarity between tissue-wide gene expression profiles. By analysis of information theory-based TF binding profiles that captured the spatial distribution and information contents of TFBS clusters, ChIP-seq and chromatin accessibility data, we described a machine learning framework that distinguished tissue-wide expression profiles of similar vs dissimilar genes and identified TF target genes. Functional binding sites in target genes that significantly alter expression levels upon direct binding are at least partially distinguished by TF-cofactor coregulation from non-functional sites in non-targets. Finally, in-silico mutation analyses demonstrated that the presence of multiple information-dense clusters in the promoter, as a protective mechanism, reduces deleterious mutations that can significantly alter the regulatory state and expression level of the gene.\n\nAn earlier version this article is available from bioRxiv: https://doi.org/10.1101/28326747.\n\n\nData availability\n\nThe median RPKM, TSS coordinate, DNase I hypersensitivity and ChIP-seq data were respectively obtained from the GTEx Analysis V6p release (www.gtexportal.org), Ensembl Biomart (www.ensembl.org) and ENCODE (www.encodeproject.org). The CRISPR- and siRNA-generated knockdown data were obtained from the supplementary information files of Dixit et al.15 and Cusanovich et al.13. The source datasets generated and/or analysed by this framework, along with sample results and compiled software are available from Zenodo. DOI: https://doi.org/10.5281/zenodo.170742320.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nAdditional files are available from Zenodo. DOI: https://doi.org/10.5281/zenodo.169828121.\n\nAdditional file 1. The mathematical definitions of the four other similarity metrics, the workflow of the IDBC algorithm, an example feature vector, the mathematical definitions of five statistical variables to measure classifier performance, the default parameter values of classifiers in MATLAB, and histograms visualizing the first two criteria for selecting positives from the CRISPR-generated knockdown data.\n\nAdditional file 2: The lists of positives and negatives in the machine learning classifiers to predict genes with similar tissue-wide expression profiles.\n\nAdditional file 3: The lists of positives and negatives in the DT classifier to predict TF targets based on the CRISPR-generated knockdown data.\n\nAdditional file 4: The lists of positives and negatives in the DT classifier to predict DE direct targets based on the siRNA-generated knockdown data.\n\nAdditional file 5: The performance of the DT classifier using only TFBS counts.\n\nAdditional file 6: The list of the most similar 500 PC genes to each TF in terms of tissue-wide expression profiles, and the intersection of these 500 genes and target genes of the TF.\n\nAdditional file 7: Cofactor binding sites adjacent to YY1 and EGR1 sites in the promoters of their targets and non-targets.\n\nAdditional file 8: The percentages of positives and negatives whose promoters do not overlap DHSs for the CRISPR-perturbed TFs.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSource code implementing the machine learning framework available at: https://bitbucket.org/cytognomix/information-dense-transcription-factor-binding-site-clusters/.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.189205148.\n\nLicense: GNU General Public License 3.0.",
"appendix": "Grant information\n\nNatural Sciences and Engineering Research Council of Canada Discovery Grant [RGPIN-2015-06290]; Canada Foundation for Innovation; Canada Research Chairs; Cytognomix Inc. Compute Canada and Shared Hierarchical Academic Research Computing Network (SHARCNET) provided high performance computing and storage facilities. Funding for open access charge: University of Western Ontario and the Natural Sciences and Engineering Research Council.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Ben Shirley and Eliseos Mucaki for constructive comments on the paper.\n\n\nReferences\n\nHosseinpour B, Bakhtiarizadeh MR, Khosravi P, et al.: Predicting distinct organization of transcription factor binding sites on the promoter regions: a new genome-based approach to expand human embryonic stem cell regulatory network. Gene. 2013; 531(2): 212–9. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu R, Rogan PK: Information-dense transcription factor binding site clusters identify target genes with similar tissue-wide expression profiles and serve as a buffer against mutations. bioRxiv. 2018; 283267. Publisher Full Text\n\nLu R, Rogan PK: Information dense transcription factor binding site clusters identify target genes with similar tissue-wide expression profiles and buffer against mutations - source code. Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1892051"
}
|
[
{
"id": "42458",
"date": "09 Jan 2019",
"name": "Daphne Ezer",
"expertise": [
"Reviewer Expertise bioinformatics",
"transcriptional regulation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 that this is an interesting paper that should be published. Often, gene expression patterns are clustered and TF binding sites are used as features to build a classifier for identifying the cluster to which those genes belong or similar schemes such as clustering TFs and gene expression together- see Clements et al1 and Berman et al2. However, a biologist may want to identify what TFs regulate a specific gene of interest. They could then use the 'Bray-Curtis Similarity' index to find a set of genes whose expression pattern is similar to their gene of interest. Then, they can use the pipeline presented here to identify features that are predictive of this kind of gene expression pattern.\n\nThey also create a scheme to test how different combinations of features from different experiments influence their predictions.\nI think that the text would garner much more interest if it focused more on the research questions that are being addressed. The method details are discussed in depth, but the big picture is hard to find amidst the details.\n\nMain points:\nOne of the main things that bothers me about the Bray-Curtis Similarity metric is that it seems to assume that tissues are independently sampled. However. we see from Fig.1 that there are many brain samples that seem to (at least in the three genes shown) have similar gene expression values. Is there a lot of covariance between gene expression values in pairs of tissues? If so, is there a way to adjust this metric to acknowledge stratification of the tissues. I don't think that the whole paper needs to be re-written with a new metric, but it would be nice if the authors address this directly. Another issue I have is with this metric is that it uses RPKM gene expression values in the Bray-Curtis Similarity metric. Imagine that two genes have extremely high gene expression values in some tissue (like the brain) and low values in another tissue (like the pancreas). However, one of these genes is always expressed at 10 times the level of the other gene. Lets say that a third gene has almost no change of gene expression value across the tissues but has a mean RPKM that is similar to the first gene's mean RPKM. Would the Bray-Curtis SImilarity metric say that gene 1 and 2 are more similar? Or gene 1 and 3? If gene 1 and 3, then this might be resolved by comparing z-scores or otherwise normalising gene expression values across tissues. Every time a machine learning method is named, it should be clear: (1) what are the input features (ii) what are the labels -- i.e. what is being classified (iii) what is the cross-validation or training-testing-validation scheme. Since there are so many machine learning things being done, it is hard sometimes to make sense of what is being done in each specific case.\n\nFor the method described in (B) in Fig 1: Does it necessarily make sense to compare the 'most similar' to the 'least similar'? Genes that have exactly the opposite gene expression pattern to the one you are interested might be tightly regulated by a different set of TFs. You might be picking up this signal instead of the one you care about! This might be an even bigger issue since you are using raw gene expression values-- genes that are very highly or very lowly expressed in all tissues might always come up in your negative set. Biologists don't just want good classifiers, they want feature selection! Can you show the Gini scores of the features in a supplemental table?\nSmaller changes:\nIs all the code for generating every figure available online? Let's help make research reproducible. If you have 10 values (from cross validation), why don't you show them all in Figure 4 so we can evaluate the spread. \"Our in-silico mutation analyses revealed that some deleterious TFBS mutations could be compensated for by other information-dense clusters in the same promoter2; thus, predicting the effects of mutations in individual binding sites might not be sufficient to interpret downstream effects without considering their context.\" This is something that me and my collaborators have recently studied3. Don't feel pressure to add this citation-- I just thought it would be interesting for you to read! (Also, thanks for discussing IDBC in this paper-- I hadn't heard of it before but it would be relevant to my research.) It would be great if you discussed how Bray-Curtis is used in other fields in the Discussion.\n\nBetter subsection names in the results section - emphasizing the biological conclusions rather than what was done.\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": "4506",
"date": "08 Apr 2019",
"name": "Peter Rogan",
"role": "Author Response",
"response": "Comment 1: One of the main things that bothers me about the Bray-Curtis Similarity metric is that it seems to assume that tissues are independently sampled. However. we see from Fig.1 that there are many brain samples that seem to (at least in the three genes shown) have similar gene expression values. Is there a lot of covariance between gene expression values in pairs of tissues? If so, is there a way to adjust this metric to acknowledge stratification of the tissues. I don't think that the whole paper needs to be re-written with a new metric, but it would be nice if the authors address this directly. Response: ① There are 13 brain tissues among all the 53 tissues investigated by GTEx. Admittedly, genes tend to exhibit closer expression values between some of these brain tissues; for example, seen from Figure 2, the NR3C1 gene has close, low expression values in multiple brain tissues (Amygdala, Anterior cingulate cortex (BA24), Caudate (basal ganglia), etc). To investigate how much this covariance can influence similarity values between tissue-wide expression profiles of genes computed by the Bray-Curtis function, using the brain tissues as an example, we retained only one brain tissue and removed all other brain tissues at one time, and recomputed the Bray-Curtis similarity values between NR3C1 and all other protein-coding genes. Thus, there are 13 variant cases due to the presence of 13 brain tissues. Then we compared the resultant set of 500 most similar genes in each variant case to that when all 53 tissues were used (given in Additional file 2). All of the top 100 most similar genes when using all 53 tissues were among the top 500 genes in every variant case. The top 200 genes when using all 53 tissues differed by 0-3 genes from the top 500 genes in variant cases. The top 500 genes when using all 53 tissues differed by approximately 22% (112-117) from top 500 genes in variant cases. This suggests that the increased number of brain tissues does not significantly influence the results of the Bray-Curtis metric for the most similar genes but does affect results at lower similarity threshold. Especially, in all 14 cases (i.e. the 13 variant cases and using all 53 tissues), the three most similar genes to NR3C1 are the same (SLC25A32, TANK, CDC27). Therefore, this covariance between these brain tissues is not a dominant factor in identifying genes with similar tissue-wide expression profiles to a particular gene using the Bray-Curtis Function. ② On the other hand, the situation is also present that a gene has closer expression values between two tissues from two different organs, than between two more similar tissues from the same organ. For example, both the Cerebellar Hemisphere (CH) tissue and the Amygdala tissue are from the brain; the Visceral Adipose (VA) tissue and the Adrenal Gland (AG) tissue are not. Seen from Figure 2, for the NR3C1 gene’s expression, there is a larger difference between CH and Amygdala. Instead, CH is closer to VA, whereas Amygdala is closer to AG. Despite well established developmental lineages for these tissues, we prefer not to make assumptions regarding the covariance in expression values between similar tissues from the same organ. The null hypothesis should not discriminate between tissues or weight them differently, without explicit prior information about tissue-specific expression, which is what we are trying to measure. For this reason, when computing similarity values between tissue-wide expression profiles of genes using the Bray-Curtis Function or other metrics, it may be more appropriate to assign the same weight to every tissue and treat every tissue equally. Comment 2: Another issue I have is with this metric is that it uses RPKM gene expression values in the Bray-Curtis Similarity metric. Imagine that two genes have extremely high gene expression values in some tissue (like the brain) and low values in another tissue (like the pancreas). However, one of these genes is always expressed at 10 times the level of the other gene. Lets say that a third gene has almost no change of gene expression value across the tissues but has a mean RPKM that is similar to the first gene's mean RPKM. Would the Bray-Curtis SImilarity metric say that gene 1 and 2 are more similar? Or gene 1 and 3? If gene 1 and 3, then this might be resolved by comparing z-scores or otherwise normalising gene expression values across tissues. Response: Gene 1 and 3 will be more similar according to Bray-Curtis Similarity. The inference is as follows: Assume that there are two tissues t1 and t2. The expression values of Gene 1 in the two tissues are [a, b] (b>>a>0), the expression values of Gene 2 are [10a, 10b], and the expression values of Gene 3 are [(a+b)/2, (a+b)/2]. Then the Bray-Curtis similarity value between Gene 1 and Gene 2 is: simBC (G1,G2) = 2/11. The Bray-Curtis similarity value between Gene 1 and Gene 3 is: simBC (G1,G3) = (3a+b)/(2a+2b). Thus, simBC (G1,G3) > simBC (G1,G2). However, this is not unreasonable. In other words, this is not a problem, thus it does not need to be resolved. There is no ground-truth relationship between simBC (G1,G2) and simBC (G1,G3). The reason is described below. When measuring the similarity between two vectors, there are two factors to be considered: 1. the sizes of the vectors (i.e. the distance between the two vectors), 2. the directions of the vectors (i.e. the angle between the two vectors). In this context of measuring similarity between tissue-wide expression values of genes (each gene is mapped to a vector), both factors matter. It is stated in the Methods section that Bray-Curtis Similarity satisfies three desirable properties. In fact, Property 2 (achieving the maximal similarity value 1 if and only if two vectors are identical) ensures Factor 1 to be considered, and Property 3 (larger values having a larger impact on the resultant similarity value) ensures Factor 2 to be considered. Thus, Table 1 shows that Bray-Curtis Similarity is more appropriate than the other five metrics, which is exactly due to the fact it takes both factors into account. In contrast, Euclidean Similarity does not take vectors’ directions into account; Cosine Similarity, Pearson Correlation and Spearman Correlation do not take the sizes of the vectors into account. Thus, to be able to infer a ground-truth similarity relationship, on both Factor 1 and Factor 2 the intuitive comparison results must be concordant. In Example 1 (see Additional File 1), the angle between Gene A and Gene C is identical to that between Gene B and Gene C, and the distance between A and C is larger than that between B and C; thus, sim (A,C) < sim (B,C). Similarly, the distance between A and D is identical to that between E and F, and the angle between A and D is larger than that between E and F; thus, sim(A,D) < sim(E,F). But in this case, the distance between Gene 1 and Gene 2, i.e. 9*sqrt(a2 + b2), is larger than that between Gene 1 and Gene 3, i.e. (b-a)/sqrt(2), but the angle between Gene 1 and Gene 2 (i.e. 0) is smaller than that between Gene 1 and Gene 3 (>0). Thus, a ground-truth similarity relationship is unable to be inferred. Thus, the result given by Bray-Curtis Similarity, i.e.simBC (G1,G3) > simBC (G1,G2), is not unreasonable. Comment 3: Every time a machine learning method is named, it should be clear: (1) what are the input features (ii) what are the labels -- i.e. what is being classified (iii) what is the cross-validation or training-testing-validation scheme. Since there are so many machine learning things being done, it is hard sometimes to make sense of what is being done in each specific case. Response: In the module to predict genes with similar tissue-wide expression profiles to a particular gene, to make these points clearer, the following changes were made: (i) Using the red color Figure 1A shows that seven features were derived from TFBS clusters. In addition, in the legend of Figure 1A, the following sentence was added “The seven ML features derived from TFBS clusters were described in the Methods section.” The second paragraph of the second subsection of the Methods section details the seven features; its first sentence was revised to “The seven information density-related ML features derived from each TFBS cluster included …” (ii) The first sentence of the ‘Prediction of genes with similar tissue-wide expression profiles” subsection of the Methods section was revised’ to ‘The framework for predicting whether the tissue-wide expression profile of a gene resembles a particular gene is indicated in Figure 1A, B.”, so that it is clear that the two labels are ‘similar to the particular gene’ and ‘dissimilar to the particular gene’. In addition, Figure 1B also indicates that 500 most similar genes and 500 most dissimilar genes were selected as positives and negatives. (iii) The last step (‘Performance evaluation’) of Figure 1A was revised to ‘Performance evaluation (ROC curve/10-fold cross validation)’; the red color indicates that ROC curves were used to validate the classifiers in this module. In addition, the last sentence of the legend of Figure 1A was revised to “The performance of ML classifiers was evaluated with ROC curves and 10-fold cross validation”. The last sentence of the second subsection of the Methods section was revised to “This allowed all genes to be used as training data for ML classifiers. Default parameter values for these classifiers were used to generate ROC curves in MATLAB”, and also the first sentence of the corresponding second subsection of the Results section was revised to “Several ML classifiers (Naïve Bayes, Decision Tree (DT), Random Forest and Support Vector Machines (SVM) with four different kernels) were evaluated to determine how well TFBS-related features could predict genes with tissue-wide expression profiles similar to NR3C1. Their performance were compared using ROC curves…”. Thus, it is now clear that ROC curves were used to validate the classifiers and all instances were used as training data (i.e. there were no test sets). In the module to predict TF target genes, to make these points clearer, the following changes were made: (i) Using the blue color Figure 1A shows that six features were derived from TFBS clusters. Also, the penultimate sentence of the last paragraph of the “Using gene expression in the CRISPR-based perturbations” subsection of the Methods section states “Six features (Features 1-5 and 7) were derived from each homotypic cluster (i.e. Feature 6 became identical to Feature 3, because only binding sites from a single TF were used) (Figure 1A).”. Combining with the detailed descriptions about what the features are in the second paragraph of the previous subsection, it is clear that these six features (Features 1-5 and 7) were used. (ii) The last sentence of the first paragraph of the ‘Using gene expression in the CRISPR-based perturbations” subsection of the Methods section was revised to “We defined a positive TF target gene in a cell line as:…” And the sentence in the fifth paragraph was revised to “If the coefficients of all guide RNAs of the TF for a gene are zero, the gene was defined as a negative (i.e. a non-target gene).” Thus it is clear that the two labels are “TF target genes” and “non-target genes”. Combining with the last sentence of the first paragraph of the Methods section, ‘Since protein-coding (PC) sequences represent the most widely studied and best understood component of the human genome [19], positives and negatives for deriving machine learning classifiers for predicting DE direct TF target genes that encode proteins (TF targets, below) were obtained from CRISPR- and siRNA-generated knockdown data’, it is clear that the ‘target genes’ here stands for ‘PC, direct, DE target genes’. (iii) As stated above, the last step (‘Performance evaluation’) of Figure 1A was revised to ‘Performance evaluation (ROC curve/10-fold cross validation)’; the blue color indicates that 10- fold cross validations were used to validate the classifiers in this module. In addition, the last sentence of the legend of Figure 1A was revised to “The performance of ML classifiers was evaluated with ROC curves and 10-fold cross validation”. The last sentence of the last paragraph of the “Using gene expression in the CRISPR-based perturbations” subsection of the Methods section was revised to “The results of 10 rounds of 10-fold cross validation were averaged to evaluate the accuracy of the classifier.” Thus it is clear that the validation scheme is 10-fold cross validation. Comment 4: For the method described in (B) in Fig 1: Does it necessarily make sense to compare the 'most similar' to the 'least similar'? Genes that have exactly the opposite gene expression pattern to the one you are interested might be tightly regulated by a different set of TFs. You might be picking up this signal instead of the one you care about! This might be an even bigger issue since you are using raw gene expression values-- genes that are very highly or very lowly expressed in all tissues might always come up in your negative set.: Response: Yes, it makes sense. As stated in the above response to Comment 3, in this module to predict genes with similar tissue-wide expression profiles to a particular gene, the two labels are ‘similar to the given gene’ and ‘dissimilar to the given gene’. Therefore, it makes the most sense that the most similar genes were selected as positives and the most dissimilar genes were selected as negatives. The first sentence of the last paragraph of the Background section, “…the distribution and composition of cis-regulatory modules in promoters substantially determines gene expression profiles…, is exactly the underlying rationale why this machine learning framework is able to distinguish between “similar genes” and “dissimilar genes”. In other words, “similar genes” and “dissimilar genes” have different expression patterns, presumably because they have different organizations and compositions (i.e. different TF sets) of TFBSs in their promoters. Therefore, the potential fact that “similar genes” and “dissimilar genes” are regulated by different TF sets was exactly what we expected to see and validate. Comment 5: Biologists don't just want good classifiers, they want feature selection! Can you show the Gini scores of the features in a supplemental table?: Response: In the module to predict TF target genes based on CRISPR- and siRNA-generated knockdown data, to assess the relative importance of the six features to the Decision Tree classifiers, we computed their Gini importance values, which are added to Additional file 5. For the seven CRISPR-perturbed TFs in the K562 cell line, the cluster-level Features 1-3, especially Feature 3 capturing the information density of TFBS clusters, have the largest contribution to the classifiers’ predictive power. By contrast, for the 11 siRNA-perturbed TFs in the GM19238 cell line, the binding site-level Feature 5 capturing the spatial distribution of strong TFBSs has the largest contribution. Accordingly, in this revision, this observation is described in the second and third sentences of the first paragraph of the third subsection of the Results section. Comment 6: Is all the code for generating every figure available online? Let's help make research reproducible. Response: As stated in the Software availability section, the code that implemented this machine learning framework and produced all the results has been deposited at https://bitbucket.org/cytognomix/information-dense-transcription-factor-binding-site-clusters/src and https://doi.org/10.5281/zenodo.1892051. The input of the code to derive the figures is directly taken from the output of the ML framework code. There are no intermediate steps or variable required to generate the figures in MATLAB. The code for generating the figures is used to visualize the results. in this revision, we also deposited the MATLAB code for generating all the figures at https://bitbucket.org/cytognomix/information-dense-transcription-factor-binding-site-clusters/src Comment 7: If you have 10 values (from cross validation), why don't you show them all in Figure 4 so we can evaluate the spread. Response: To avoid making Figure 4 too complicated, in this revision, the accuracy values of all individual rounds of 10-fold cross validations in prediction of TF target genes were added to Additional file 5. Accordingly, the legends of Table 2, Figure 4 and Table 3 were revised to indicate this. Comment 8: \"Our in-silico mutation analyses revealed that some deleterious TFBS mutations could be compensated for by other information-dense clusters in the same promoter(2); thus, predicting the effects of mutations in individual binding sites might not be sufficient to interpret downstream effects without considering their context.\" This is something that me and my collaborators have recently studied3. Don't feel pressure to add this citation-- I just thought it would be interesting for you to read! (Also, thanks for discussing IDBC in this paper-- I hadn't heard of it before but it would be relevant to my research.) Response: In this revision, this publication has now been referenced (number 47) in the manuscript. Comment 9: It would be great if you discussed how Bray-Curtis is used in other fields in the Discussion. Response:The following sentences were added to the penultimate paragraph, “Previous applications of this index include: a) measurement of the ecological transfer of species abundance from dense to sparse plots 48 and comparative difference analyses of species quantities between reference and algorithm-derived metagenomic sample mixtures (https://precision.fda.gov/challenges/3/view/results). b) improvement of friend recommendation in geosocial networks by using it to compare users’ movement history 49, 50 ..” Comment 10: Better subsection names in the results section - emphasizing the biological conclusions rather than what was done. Response: In the Results section of this revision, the title of each subsection was revised as follows, now summarizing the primary conclusion from this subsection. The title of the first subsection was revised from ‘Similarity between GTEx tissue-wide expression profiles of genes’ to ‘The Bray-Curtis Function can accurately quantify the similarity between tissue-wide gene expression profiles’. The title of the second subsection was revised from ‘Prediction of genes with similar GTEx tissue-wide expression profiles’ to ‘The Decision Tree classifier performed best in prediction of genes with similar tissue-wide expression profiles’. The title of the third subsection was revised from ‘Prediction of TF targets” to ‘The Decision Tree classifier was predictive of TF target genes’. The title of the fourth subsection was revised from ‘Intersection of genes with similar tissue-wide expression profiles and TF targets’ to ‘Some TF target genes also display similar tissue-wide expression profiles to the TFs, themselves. The title of the fourth subsection was revised from ‘Mutation analyses on promoters of direct targets’ to ‘Transcription factor binding site clusters buffer against expression changes from mutations in single sites’."
}
]
},
{
"id": "42457",
"date": "08 Feb 2019",
"name": "Nicolae Radu Zabet",
"expertise": [
"Reviewer Expertise genomics",
"chromatin biology",
"transcription regulation",
"bioinformatics",
"statistical models"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Ru and Rogan use Bray-Curtis Similarity and several machine-learning algorithms to identify genes that have similar expression patterns. They use transcription factor binding sites within promoter regions and DNA accessibility data to train their models. This is a very important question and the authors propose an interesting mechanistic approach to address it. Nevertheless, there are several limitations that need to be addressed.\n\nSpecific comments:\n\nWhile the grammar is at a good level, the way the information is presented makes the text very difficult to read. Some sentences are very long and there are many notations. One suggestion is to move some of the less important parts in the Supplementary Material. On page 3 in the introduction, the authors claim that signal strength of ChIP-seq peaks are not strictly proportional to TF binding strength. This is not always true and we showed in1 that in fact the number of TF molecules controls the height of the ChIP-seq peak. On page 5, it is not clear why the authors talk of Features 1-3, since it seemed they had 7 features. The way the machine learning information is presented should be improved. The authors test Naïve Bayes, Decision Tree, Random Forest and SVM. I was wondering if they consider also Neural Networks. They don’t need to implement that now, but they should at least mention what was behind their selection for the machine-learning algorithms. One of the main findings is that DNA accessibility improves predictions, because it masks potential TF binding sites. This is something that was previously showed in the context of TF binding to the genome by us and other scientists (e.g. References 1,2,3). Figure 4 needs re-plotting (e.g. x axis labels do not fit the figure). In the discussion, none of the statements are referred back to any of the figures in the results section. This makes the reading difficult. The lower performance for YY1 needs to be better explained. The authors claim that this could be explained by lower percentage of negatives in inaccessible promoters. Are there other examples of TFs displaying similar features? What is their performance? One of the main limitations of the manuscript is that the authors use only 82 TFs and claim that there are no iPWM for others. Have they tried to use MotifDB (https://bioconductor.org/packages/release/bioc/html/MotifDb.html), which has approximately 1000 PWMs for human TFs? When talking about the accuracy of the ChIP-seq signal, they could also reference this paper4.\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": "4505",
"date": "08 Apr 2019",
"name": "Peter Rogan",
"role": "Author Response",
"response": "Comment 1: While the grammar is at a good level, the way the information is presented makes the text very difficult to read. Some sentences are very long and there are many notations. One suggestion is to move some of the less important parts in the Supplementary Material. Response: The manuscript has been extensively edited to improve clarity of the presentation. Sentence lengths have been reduced. Duplicate terms and text have been eliminated. All abbreviations have been defined. Two paragraphs have been moved to the Supplementary Methods. The revised manuscript has been shortened by 400 words and approximately 2 pages. Comment 2: On page 3 in the introduction, the authors claim that signal strength of ChIP-seq peaks are not strictly proportional to TF binding strength. This is not always true and we showed in1 that in fact the number of TF molecules controls the height of the ChIP-seq peak. Response: In (1), it was discovered that signal strengths of ChIP-seq peaks are not strictly proportional to strengths (Ri values) of the strongest TFBSs contained in the peaks. The finding in (2) provides a complementary explanation about the determinants of signal strengths of ChIP-seq peaks, which is that ‘the number of TF molecules controls the height of the ChIP-seq peak’. Therefore, this sentence is revised to “Because signal strengths of ChIP-seq peaks are not strictly proportional to strengths of the contained strongest TFBSs and are instead controlled by TFBS counts [3, 10], representing…”. Comment 3: On page 5, it is not clear why the authors talk of Features 1-3, since it seemed they had 7 features. The way the machine learning information is presented should be improved. Response: In this sentence, we would like to make it easier for readers to understand the generation of classifier predictors, by explaining how the seven high-level features were transformed to low-level predictors that were directly input into the classifiers. Therefore, this sentence was revised to “Each of the Features 1-3 was defined in a gene as a vector, whose size equals the number of clusters in the gene promoter; each cluster was mapped to a single value in the vector. In Features 4-7, each cluster itself was mapped to a vector corresponding to binding sites for 82 TFs (Additional file 1).” Also, Section 5 of Additional file 1 gives a specific example about the predictor vector of a gene instance. Comment 4: The authors test Naïve Bayes, Decision Tree, Random Forest and SVM. I was wondering if they consider also Neural Networks. They don’t need to implement that now, but they should at least mention what was behind their selection for the machine-learning algorithms. Response: We did not select Neural Networks due to two considerations. First, it requires much more data to train than traditional machine learning algorithms, as in at least thousands if not millions of labeled samples (3). In this study the numbers of both positives (i.e. protein-coding genes with similar tissue-wide expression profiles to NR3C1) and negatives (i.e. dissimilar genes) are 500, which is insufficient to apply Neural Networks. Second, it is more computationally expensive than traditional algorithms (4). Comment 5: One of the main findings is that DNA accessibility improves predictions, because it masks potential TF binding sites. This is something that was previously showed in the context of TF binding to the genome by us and other scientists (e.g. References 1,2,3). Response: Accordingly, in this revision, the last sentence of the second subsection of the Results section was revised to “Consistent with previous findings (2, 5, 6), inclusion of DHS information significantly improved AUC values of the other classifiers with the exception of Naïve Bayes.”. And in the second paragraph of the Discussion section, the second sentence was revised to “This intersection eliminated noisy binding sites that are inaccessible to TF proteins in promoters (2, 5, 6),…” Comment 6: Figure 4 needs re-plotting (e.g. x axis labels do not fit the figure). Response: In this revision, Figure 4 was replotted to fix this issue. Comment 7: In the discussion, none of the statements are referred back to any of the figures in the results section. This makes the reading difficult. Response: In the first paragraph of the Discussion section of this revision, references to the figures in the Results section were added to the following sentences, “In this study, the Bray-Curtis similarity function was initially shown (for the NR3C1 gene) to measure the relatedness of overall expression patterns between genes across a diverse set of tissues (Figure 2). A ML framework distinguished similar from dissimilar genes based on the distribution, strengths and compositions of TFBS clusters in accessible promoters, which can substantially account for the corresponding gene expression patterns (Figures 1 & 3). Using gene expression knockdown data, the combinatorial use of TF binding profiles and chromatin accessibility was also demonstrated to be predictive of TF targets (Figure 4, Tables 2 & 3). A binding site comparison confirmed that coregulatory cofactors can be used to distinguish between functional sites in targets and non-functional ones in non-targets. Furthermore, in silico mutation analyses on binding sites of targets suggested that the existence of both multiple TFBSs in a cluster and multiple information-dense clusters in the same promoter enables both the cluster and the promoter to be resilient to mutations in individual TFBS (Figure 5, Table 5).” In the third paragraph, references to the figures in the Results section were added to the following sentence, “Mutation analyses revealed that some deleterious TFBS mutations could be compensated for by other information-dense clusters in the same promoter (Figure 5, Table 5)” Comment 8: The lower performance for YY1 needs to be better explained. The authors claim that this could be explained by lower percentage of negatives in inaccessible promoters. Are there other examples of TFs displaying similar features? What is their performance? Response: In this sentence, all the seven CRISPR-perturbed TFs were split into two sets; one consisting of only YY1, the other consisting of the remaining six TFs. This sentence was comparing the performances of the Decision Tree classifiers on these two TF sets. Seen from Table 3, the classifier’s performance on YY1 was markedly lower than that on the other six TFs after intersecting promoters with DHS sites, which is due to the fact that YY1 has a smaller percentage of negatives with inaccessible promoters. To make this clearer, in this revision, this sentence was revised to “Compared to the other six TFs, the poorer performance of the classifier on YY1 (Table 2) is attributable to …” Comment 9: One of the main limitations of the manuscript is that the authors use only 82 TFs and claim that there are no iPWM for others. Have they tried to use MotifDB (https://bioconductor.org/packages/release/bioc/html/MotifDb.html), which has approximately 1000 PWMs for human TFs? Response: We selected to use these 94 iPWMs of 82 TFs that were derived from ENCODE ChIP-seq datasets using entropy minimization in (1), since we want to ensure the high quality of the iPWMs used in the analyses. Compared to the MotifDB PWMs, the reliability and accuracy of these iPWMs were extensively and independently validated using four methods, including detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. These iPWMs were used to scan for 803 experimentally confirmed TFBSs, and there was complete concordance between these true binding sites and those detected with the iPWMs, both in terms of their locations and relative strengths (1). And these iPWMs were further used to explain the experimentally observed effects of 153 SNPs on binding site strengths, based on the changes in the Ri values. For 130 SNPs (∼85.0%), the predictions of the iPWMs and the experimental observations are completely concordant; for 16 SNPs (∼10.5%), the predicted and observed experimental findings are concordant, but the extents of these changes differ (e.g. TF binding is predicted to only be weakened, but binding was completely abolished in experiments); for only 7 SNPs (∼4.6%), the predicted and observed experimental changes were discordant. The PWMs in MotifDB are not information theory-based (i.e. not iPWMs). Instead, they are given in the form of count matrices or frequency matrices. The performance of the iPWMs that used in the present study has been shown to outperform other PWM-based approaches for binding site detection and quantification (7). Comment 10: When talking about the accuracy of the ChIP-seq signal, they could also reference this paper4. Response: In this revision, the reference was added to the following sentence in the last paragraph of the Discussion section, “Regarding the CRISPR-generated knockdown data, positives were inferred to be direct targets by intersecting their promoters with corresponding ChIP-seq peaks. This may not be completely accurate, due to the presence of noise peaks that do not contain true TFBSs 3, 50, 51.” References: 1. Lu,R., Mucaki,E.J. and Rogan,P.K. (2017) Discovery and validation of information theory-based transcription factor and cofactor binding site motifs. Nucleic Acids Res., 45, e27. 2. Zabet,N.R. and Adryan,B. (2015) Estimating binding properties of transcription factors from genome-wide binding profiles. Nucleic Acids Res., 43, 84–94. 3. Zhang,Y. and Yang,Q. (2017) A Survey on Multi-Task Learning. ArXiv170708114 Cs. 4. Ghodsi,Z., Gu,T. and Garg,S. (2017) SafetyNets: Verifiable execution of deep neural networks on an untrusted cloud. In Advances in Neural Information Processing Systems.Vol. 2017-December, pp. 4673–4682. 5. Kaplan,T., Li,X.-Y., Sabo,P.J., Thomas,S., Stamatoyannopoulos,J.A., Biggin,M.D. and Eisen,M.B. (2011) Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development. PLoS Genet., 7, e1001290. 6. Simicevic,J., Schmid,A.W., Gilardoni,P.A., Zoller,B., Raghav,S.K., Krier,I., Gubelmann,C., Lisacek,F., Naef,F., Moniatte,M., et al. (2013) Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics. Nat. Methods, 10, 570–576. 7. Erill I and O’Neill MC. (2009) A reexamination of information theory-based methods for DNA binding site identification. BMC Bioinformatics, 10, 57."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1933
|
https://f1000research.com/articles/8-388/v1
|
05 Apr 19
|
{
"type": "Research Article",
"title": "Diagnostic value of urea, creatinine and blood parameters in patients with pneumonia diagnosed with chronic obstructive pulmonary disease",
"authors": [
"Seha Akduman"
],
"abstract": "Background: This study aimed to investigate the diagnostic value of urea, creatinine and other blood parameters in patients with pneumonia diagnosed with chronic obstructive pulmonary disease (COPD) for the first time. Methods: In this retrospective study, patients who had been diagnosed with COPD for the first time and were diagnosed with pneumonia were included. A total of 193 patients were divided into three groups as COPD + pneumonia (n=123), COPD (n=36) and pneumonia (n=34). Results: In total, 59 women (48.0%) and 64 men (52.0%) from the COPD + pneumonia group, 13 women (36.1%) and 23 men (63.9%) from the COPD group, 21 women (61.8%) and 13 men (38.2%) from the pneumonia group were assessed. The mean age of the COPD + pneumonia group was 69.58±13.62, 66.28±12.55 for the COPD group and 53.97±19.72 for the pneumonia group. The highest values of C-reactive protein (CRP), urea, creatinine, white blood cells (WBC), neutrophils, eosinophils and hemoglobin were the highest in COPD + pneumonia group. CRP levels were significantly different between COPD + pneumonia group (p<0.05). The parameters urea, WBC and neutrophils were significantly different between COPD + pneumonia group and pneumonia group (p<0.05). There was a statistically significant difference between COPD and pneumonia groups in terms of neutrophils and eosinophils values (p<0.05). According to the results of receiver operating characteristic analysis, the diagnostic value of the urea parameter in determining the COPD + pneumonia group was not statistically significant (p>0.05). On the other hand, the diagnostic value of CRP, WBC and neutrophils values were statistically significant (p<0.05). Conclusions: Elevation in WBC and neutrophil values in patients diagnosed with pneumonia have an important role in diagnosis of COPD.",
"keywords": [
"COPD",
"Pneumonia",
"urea",
"creatinine",
"blood count"
],
"content": "Introduction\n\nPneumonia has been known since late 1800s, and has been recognized as a major cause of death1. Pneumonia is one of the most common causes of mortality in children under 5 years, and common in South Asia and sub-Saharan Africa; it is a form of acute respiratory tract infection2. The annual incidence of community-acquired pneumonia (CAP) ranges from 5 to 11 per 1000 persons in the United States. It is common in the winter. There are several risk factors associated with CAP, such as neurologic and gastrointestinal abnormalities, male gender, multilobar involvement, high fever, etc.3. CAP is an infection of the pulmonary parenchyma by causative microorganisms4. Streptococcus pneumonia is the leading cause of bacterial pneumonia, causing 30-50% of childhood pneumonia in developing countries5. In diagnosis of the disease, chest X-rays, complete blood count and electrolyte count tests are used5. In treatment of pneumonia, medication and antibiotic therapy are commonly used. A 6-month observation is recommended by the UK National Institute for Health and Care Excellence. In addition, it is important to examine environmental conditions as well as given medication or therapy6. It is reported that smoking, chronic lung diseases, heart disease and diabetes increase the prevalence of disease7.\n\nChronic obstructive pulmonary disease (COPD) is another common lung disease, characterized by a slow and debilitating progression8. It causes changes in the lungs with airflow restrictions and physical symptoms such as dyspnoea9. Obstructive bronchiolitis and emphysema may represent comorbidities of COPD10,11. There is evidence that inhaled corticosteroids, a treatment for COPD, and increase risk of pneumonia12. In addition, previous research has shown that exacerbation of COPD and pneumonia in COPD has different clinical and analytical properties, although mechanisms are similar12,13.\n\nThis study aimed to investigate the diagnostic value of urea, creatinine and some blood parameters in patients with pneumonia that had been diagnosed with COPD for the first time.\n\n\nMethods\n\nIn this retrospective study, patients who had been diagnosed with COPD for the first time and were diagnosed with Pneumonia and who were admitted to Kadıköy Medicana Hospital, Istanbul, Turkey, between 12 October 2017 and 12 October 2018 were included in the study. A sample of convenience was used, with a voluntary patient consent form. The eligibility criteria were as follows:\n\n— Previously diagnosed with pneumonia,\n\n— First diagnosed with COPD,\n\n— Without a history of clinical intervention in patient epicrisis,\n\n— Without a malignant disorder\n\n— Completed patient consent form\n\nA total of 123 patients with pneumonia among 160 patients who met inclusion criteria were included in the study. A total of 193 patients were divided into three groups as COPD + pneumonia (n = 123), COPD (n = 36) and pneumonia (n = 34). In the second group of patients diagnosed with COPD, the patients who were not diagnosed with pneumonia were designated as the first control group, and patients diagnosed with pneumonia and patients who were not diagnosed with COPD was the second control group. Each patient provided written informed consent for their clinical details to be used when admitted; the hospital management granted approval for this study.\n\nThe variables assessed in this study were age, gender, CRP, Urea, Creatine, WBC, NEU, EOS and HGB, which were taken from patient records with approval.\n\nThe binary and ordinal data were described by frequency analysis and the other measurement parameters by mean and standard deviation values. Kolmogorov-Smirnov analysis was performed to assess normality distribution of the data before the difference analysis. Independent samples t-test was used for the difference of normal distribution between WBC and hemoglobin data. The Mann-Whitney U-test was used to analyze the difference between paired groups (COPD + pneumonia vs COPD only; COPD + pneumonia vs pneumonia only; COPD only vs pneumonia only) of CRP, urea, creatinine, neutrophils and eosinophils parameters which did not conform to normal distribution. Receiver operating characteristics (ROC) analysis was used for CRP, urea, WBC and NEU. All analyses were performed in SPSS 17.0 for Windows, and performed at 95% confidence interval.\n\n\nResults\n\nThe distribution of gender, age and some clinical parameters of the case groups included in the study are shown in Table 1.\n\nCOPD, chronic obstructive pulmonary disease; CRP, C-reactive protein; WBC, white blood cells; NEU, neutrophils; EOS, eosinophils; HGB, hemoglobin.\n\nIn total, there were 59 women (48.0%) and 64 men (52.0%) in the COPD + pneumonia group, 13 women (36.1%) and 23 men (63.9%) in the COPD group, and 21 women (61.8%) and 13 men (38.2%) in the pneumonia group included in the study. The mean age of patients was 70±14 in the COPD + pneumonia group, 66±13 in the COPD group and 54±20 in the pneumonia group. The highest values of CRP, urea, creatinine, WBC, neutrophils, eosinophils and hemoglobin were observed in the COPD + pneumonia group. The results of the differential analysis of the clinical parameters in the study are presented in Table 2. Raw values are available on Open Science Framework14.\n\naMann-Whitney U-test; bindependent samples t-test. Group 1, COPD+pneumonia; group 2, COPD only; group 3, pneumonia only. Figures in bold represent statistically significant differences. CRP, C-reactive protein; WBC, white blood cells; NEU, neutrophils; EOS, eosinophils; HGB, hemoglobin.\n\nAccording to the results of the difference analysis, there was a statistically significant difference between CRP levels of the COPD + pneumonia and COPD groups (p<0.05). The parameters urea, WBC and neutrophils were significantly different between the COPD + pneumonia and pneumonia groups (p<0.05). There was a statistically significant difference between COPD and pneumonia groups in terms of neutrophils and eosinophils values (p<0.05). The results of ROC analysis of CRP, urea, WBC and neutrophils variables were given as follows.\n\nAccording to the results of the ROC analysis (Figure 1), the diagnostic value of the urea parameter in determining the COPD + pneumonia group was not statistically significant (p>0.05). On the other hand, the diagnostic value of CRP, WBC and neutrophils values were statistically significant (p<0.05). The distribution of areas under the curve according to ROC analysis is given in Table 3.\n\nCRP, C-reactive protein; WBC, white blood cells; NEU, neutrophil.\n\nThe area under the curve was the highest for the CRP value.\n\n\nDiscussion\n\nPneumonia15–17 and COPD18–20 are two diseases that are the focus of considerable research into respiratory complaints. In addition, there are studies reporting the distribution of these diseases in different demographic groups21–26. In our study, pneumonia was more common in women, whereas COPD was more common in men. In both groups, the gender distributions were similar, although males made up the majority. However, the mean age of patients was the highest in COPD + pneumonia patients.\n\nIn patients with COPD and pneumonia, the clinical condition of the patients is poorer than for patients with one or the other conditions, and it is suggested that two diseases trigger each other25–34. In our study, urea and creatinine levels were higher in patients with pneumonia who were diagnosed with COPD for the first time. WBC and neutrophil values were statistically higher among patients with COPD than in patients with only pneumonia. The difference in urea, WBC and neutrophil values was not significant between the COPD and COPD + pneumonia groups. It can be said that the increase in these three parameters may have diagnostic implications for COPD in patients with pneumonia.\n\n\nConclusion\n\nAccording to the results of this study, a history of pneumonia aggravates the clinical course of patients with COPD for the first time. On the other hand, the increase in CRP, WBC and neutrophil values in patients diagnosed with pneumonia but not COPD may give the first indication of a possible development of COPD.\n\nAccording to the results of ROC analysis, CRP is among the most important determinants of patients with the first time diagnosed with COPD and diagnosed with pneumonia. On the other hand, WBC and neutrophil elevation in patients with previously diagnosed pneumonia may also be an important factor in the diagnosis of COPD.\n\n\nData availability\n\nOpen Science Framework: Diagnostic value of urea, creatinine and blood parameters in patients with pneumonia diagnosed with chronic obstructive pulmonary disease. https://doi.org/10.17605/OSF.IO/Y4V8T14.\n\nThis project contains the demographic and clinical variables of patients measured in this study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in funding supporting this work.\n\n\nReferences\n\nMusher DM, Thorner AR: Community-acquired pneumonia. N Engl J Med. 2014; 371(17): 1619–28. PubMed Abstract | Publisher Full Text\n\nTong N: Priority Medicines for Europe and the World \"A Public Health Approach to Innovation. Background Paper 6.22, Pneumonia, 2013. Reference Source\n\nWatkins RR, Lemonovich TL: Diagnosis and management of community-acquired pneumonia in adults. Am Fam Physician. 2011; 83(11): 1299–1306. PubMed Abstract\n\nMackenzie G: The definition and classification of pneumonia. Pneumonia (Nathan). 2016; 8: 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZafar MZ: A Case Study: Pneumonia. Occup Med Health Aff. 2016; 4: 4. Publisher Full Text\n\nNICE: Pneumonia in adults: diagnosis and management. Clinical guideline Published: 3 December 2014. Reference Source\n\nChung Y, Morgan L: Pneumonia Who is at risk in your practice? MedicineToday. 2015; 16(8): 35–42. Reference Source\n\nLanders A, Wiseman R, Pitama S, et al.: Severe COPD and the transition to a palliative approach. Breathe (Sheff). 2017; 13(4): 310–316. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOHTAC COPD Collaborative: Chronic obstructive pulmonary disease (COPD) evidentiary framework. Ont Health Technol Assess Ser. 2012; 12(2): 1–97. PubMed Abstract | Free Full Text\n\nLaraeau SC, Fahy B, Meek P, et al.: Chronic Obstructive Pulmonary Disease (COPD). Am J Respir Crit Care Med. 2019; 199(1): P1–P2. PubMed Abstract | Publisher Full Text\n\nProsser TR, Bolmeier SG: Chronic Obstructive Pulmonary Disease. Pharmacotheraphy Self-Assesment Program. 6th Edition. 2008. Reference Source\n\nRestrepo MI, Sibila O, Anzueto A: Pneumonia in Patients with Chronic Obstructive Pulmonary Disease. Tuberc Respir Dis (Seoul). 2018; 81(3): 187–197. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoixeda R, Baca S, Elias L, et al.: Pneumonia as Comorbidity in Chronic Obstructive Pulmonary Disease (COPD). Differences Between Acute Exacerbation of COPD and Pneumonia in Patients With COPD. Arch Bronconeumol. 2014; 50(12): 514–520. PubMed Abstract | Publisher Full Text\n\nAkduman S: Diagnostic value of urea, creatinine and blood parameters in patients with pneumonia diagnosed with chronic obstructive pulmonary disease. 2019. http://www.doi.org/10.17605/OSF.IO/Y4V8T\n\nRuiz M, Ewig S, Marcos MA, et al.: Etiology of community-acquired pneumonia: impact of age comorbidity, and severity. Am J Respir Crit Care Med. 1999; 160(2): 397–405. PubMed Abstract | Publisher Full Text\n\nFarr BM, Bartlett CL, Wadsworth J, et al.: Risk factors for community-acquired pneumonia diagnosed upon hospital admission. British Thoracic Society Pneumonia Study Group. Respir Med. 2000; 94(10): 954–63. PubMed Abstract | Publisher Full Text\n\nCommunity-acquired pneumonia in adults in British hospitals in 1982-1983: a survey of aetiology, mortality, prognostic factors and outcome. The British Thoracic Society and the Public Health Laboratory Service. Q J Med. 1987; 62(239): 195–220. PubMed Abstract | Publisher Full Text\n\nShukla SD, Muller HK, Latham R, et al.: Platelet-activating factor receptor (PAFr) is upregulated in small airways and alveoli of smokers and COPD patients. Respirology. 2016; 21(3): 504–10. PubMed Abstract | Publisher Full Text\n\nErnst P, Gonzalez AV, Brassard P, et al.: Inhaled corticosteroid use in chronic obstructive pulmonary disease and the risk of hospitalization for pneumonia. Am J Respir Crit Care Med. 2007; 176(2): 162–6. PubMed Abstract | Publisher Full Text\n\nMackay AJ, Hurst JR: COPD Exacerbations: Causes, prevention, and treatment. Med Clin North Am. 2012; 96(4): 789–809. PubMed Abstract | Publisher Full Text\n\nLim WS, Lewis S, Macfarlane JT: Severity prediction rules in community acquired pneumonia: a validation study. Thorax. 2000; 55(3): 219–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeemungal TA, Donaldson GC, Paul EA, et al.: Effect of exacerbation on quality of life in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1998; 157(5 Pt 1): 1418–22. PubMed Abstract | Publisher Full Text\n\nArancibia F, Bauer TT, Ewig S, et al.: Community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk, and prognosis. Arch Intern Med. 2002; 162(16): 1849–58. PubMed Abstract | Publisher Full Text\n\nNiederman MS, Mandell LA, Anzueto A, et al.: Guidelines for the management of adults with community-acquired pneumonia: diagnosis, assessment of severity, antimicrobial therapy, and prevention. Am J Respir Crit Care Med. 2001; 163(7): 1730–54. PubMed Abstract | Publisher Full Text\n\nJusto D, Lachmi S, Saar N, et al.: C-reactive protein velocity following antibiotics in patients with chronic obstructive pulmonary disease exacerbation and community acquired pneumonia. Eur J Intern Med. 2009; 20(5): 518–21. PubMed Abstract | Publisher Full Text\n\nBafhadel M, Clark TW, Reid C, et al.: Procalcitonin and C-reactive protein in hospitalized adult patients with community-acquired pneumonia or exacerbation of asthma or COPD. Chest. 2011; 139(6): 1410–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPifarré R, Falguera M, Vicente-de-Vera C, et al.: Characteristics of community acquired pneumonia in patients with chronic obstructive pulmonary disease. Respir Med. 2007; 101(10): 2139–44. PubMed Abstract | Publisher Full Text\n\nJeong SW, Lee JH, Choi KJ, et al.: Comparisons of clinical characteristics and outcomes in COPD patients hospitalized with community-acquired pneumonia and acute exacerbation. Zhonghua Jie He He Hu Xi Za Zhi. 2010; 69(1): 31–8. Publisher Full Text\n\nBestall J, Paul E, Garrod R, et al.: Usefulness of the Medical Research Council (MRC) dyspnoea scale as a measure of disability in patients with chronic obstructive pulmonary disease. Thorax. 1999; 54(7): 581–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaubin C, Parienti JJ, Fradin S, et al.: Procalcitonin levels and bacterial aetiology among COPD patients admitted to the ICU with severe pneumonia: a prospective cohort study. BMC Infect Dis. 2009; 9: 157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLin SH, Perng DW, Chen CP, et al.: Increased risk of community-acquired pneumonia in COPD patients with comorbid cardiovascular disease. Int J Chron Obstruct Pulmon Dis. 2016; 11(1): 3051–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuissa S, Patenaude V, Lapi F, et al.: Inhaled corticosteroids in COPD and the risk of serious pneumonia. Thorax. 2013; 68(11): 1029–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGómez-Junyent J, Garcia-Vidal C, Viasus D, et al.: Clinical Features, Etiology and Outcomes of Community-Acquired Pneumonia in Patients with Chronic Obstructive Pulmonary Disease. PLoS One. 2014; 9(8): e105854. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalverley PMA, Stockley RA, Seemungal TAR, et al.: Reported Pneumonia in Patients With COPD: findings from the INSPIRE study. Chest. 2011; 139(3): 505–512. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "55807",
"date": "30 Oct 2019",
"name": "Hakan Tanrıverdi",
"expertise": [
"Reviewer Expertise copd",
"ıcu",
"infection"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 population is enough but the control group is to small. Maybe a power is needed.\n\nIn the abstract section the authors wrote \"According to the results of receiver operating characteristic analysis, the diagnostic value of the urea parameter in determining the COPD + pneumonia group was not statistically significant (p>0.05). ROC curves gives the sensitivity and specificity and cut off values not the difference between two variables. I think they mean the AUC value. So the article should be revised by an statistican specialist.\n\nThere is no information about the study population whether they are outpatients or hospitalized patients. All the patient are CAP?\n\nAlso in the eligibility criteria, I did not understand the meaning of:\n- Previously diagnosed with pneumonia, (did you mean: diagnosed with pneumonia?)\n- First diagnosed with COPD, (did you mean: new diagnosed COPD?)\n- Without a history of clinical intervention in patient epicrisis? The authors should explain what did they mean and clearly explain the including and excluding criteria.\n\nAre the all COPD patients are stable or in acute exacerbation?\n\nIn table 1: authors give the descriptive values of study groups but the p values is absent. In the table 2 comparing the paired group there is no information about mean age, gender\n\nIn table 2 when compared the paired groups, interestingly there is no difference between COPD group and COPD with pneumonia and pneumonia without COPD. How can you explain these results?\n\nThe discussion section too small and it is similar with the result section. The authors should detail in the discussion section and discuss the results, not repeat the results\n\nI am not a native speaker but the article should revised for language.\nAs a result according to me: The article is not well written, well designed and well discussed. So it is not eligible for indexing without a major revision.\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?\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": "55810",
"date": "09 Dec 2019",
"name": "Elena Titova",
"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 retrospective study is aimed “to investigate the diagnostic value of urea, creatinine and some blood parameters in patients with pneumonia that had been diagnosed with COPD for the first time”.\nI would like to make following comments:\nIntroduction:\nArticle starts with the sentence “Pneumonia has been known since late 1800s, and has been recognized as a major cause and death” with reference 1.\nI absolutely agree with the author that pneumonia remains a significant cause of mortality worldwide.\n\nBut the original text (reference 1) was written as follows: “Long recognized as a major cause of death, pneumonia has been studied intensively since the late 1800s “\nPneumonia was first described by Hippocrates (460-370 BC), pathological features were made 22 centuries later in 1819 by Laennec, while Rokitansky in 1842 was the first to differentiate lobar and bronchopneumonia (ref.: G. Mackenzie, Pneumonia (Nathan), 2016; 8:141).\nThe author mentioned that annual incidence of CAP in the United States ranges from 5 to 11 per 1000 persons.\n\nIt should be natural to include a reference here because the numbers can vary depending on the source.\nCould the data on patients with pneumonia taken from the Turkish Thoracic Society CAP database (TURCAP) be more interesting/relevant compared to the data from USA?\nThe author wrote that “Obstructive bronchiolitis and emphysema may be represent comorbidities of COPD”. Maybe the author can formulate this sentence in a different way.\nThe chronic airflow limitation that is characteristic of COPD is caused by a mixture of small airway disease (e.g. obstructive bronchiolitis) and parenchymal destruction (emphysema), the relative contribution of which vary from person to person (GOLD COPD, 2019)\nI would like the author to clarify the choice of urea, creatinine, white blood cells, neutrophills, eosinophils and haemoglobin as the parameters are of interest in his research.\n\nMethods:\nPatients in the study get diagnosis COPD for the first time. Spirometry is required to make the COPD diagnosis.\n\nBasic characteristic (FEV1, FEV1/FVC) in patients with COPD should be listed.\nIt should be explained to readers what the criteria “without a history of clinical intervention in patient epicrisis” means.\n\nIt would be nice to know when blood samples in the study were taken (at admission? after admission?\n\nIt is important to describe clinical status of patients with COPD patients. Did the patients have the COPD exacerbation or not; what symptoms did the patients have?\n\nResults, conclusion and discussion:\n\nI would like to propose that the Results, Conclusion and Discussion parts of this research should be rewritten with necessary details.\nThis article has a great potential for improvement and needs major revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-388
|
https://f1000research.com/articles/8-386/v1
|
05 Apr 19
|
{
"type": "Research Article",
"title": "High Frequency Jet Ventilation during stereotactic ablation of liver tumours: an observational study on blood gas analysis as a measure of lung function during general anaesthesia",
"authors": [
"Karolina Galmén",
"Jan G Jakobsson",
"Jacob Freedman",
"Piotr Harbut",
"Jan G Jakobsson",
"Jacob Freedman",
"Piotr Harbut"
],
"abstract": "Background: Stereotactic ablation of tumours in solid organs is a promising curative procedure in clinical oncology. The technique demands minimal target organ movements to optimise tumour destruction and prevent injury to surrounding tissues. High frequency jet ventilation (HFJV) is a novel option during these procedures, reducing the respiratory-associated movements of the liver. The effects of HFJV via endotracheal catheter on gas exchange during liver tumour ablation is not well studied. Methods: The aim of this explorative study was to assess lung function and the effects on blood gas and lactate during HFJV in patients undergoing stereotactic liver ablation. Blood gases were analysed in 25 patients scheduled for stereotactic liver ablation under general anaesthesia pre-induction, every 15 minutes during HFJV and following extubation in the recovery room. The HFJV was set at fixed settings. Results: None of the patients developed hypoxia or signs of increased lactate production but a great variation in PaO2/FiO2 ratio was found; from 13.1 to 71.3. An increase in mean PaCO2 was observed, from a baseline of 5.0 to a peak of 7.1 at 30 minutes (p <0.001) and a decrease was found in median pH, from a baseline of 7.44 to 7.31 at 15 minutes (p=0.03). We could not see any clear association between a decrease in PaO2/FiO2 ratio and PaCO2 elevation. Conclusions: HFJV during general anaesthesia in patients undergoing stereotactic liver ablation is feasible and it did not cause hypoxemia or signs of increased lactate production. A reversible mild to moderate impairment of gas exchange was found during HFJV.",
"keywords": [
"High-Frequency Jet Ventilation",
"Blood Gas Analysis",
"Anesthesia",
"General",
"Liver Neoplasms",
"Stereotaxic Techniques",
"Surgery",
"Computer-Assisted/methods"
],
"content": "Introduction\n\nStereotactic ablation of tumours in solid organs is a promising, dynamically developing and potentially curative procedure in clinical oncology. High precision in targeting malignant lesions with the use of image fusion for pre-interventional diagnostic imaging is highly dependent on immobilisation of the operation field. Respiratory movements cause diaphragmatic shifts, displacing abdominal organs during stereotactic-guided procedures. Biro et al.1 observed that breathing-related liver motion decreased from 20mm to 5mm using high frequency jet ventilation (HFJV) as compared to conventional ventilation.\n\nHFJV is an attractive novel ventilatory strategy, minimizing movements in proximity to the lungs and keeping the abdominal and extraperitoneal target organ immobilised2. The surgical ablation procedure usually requires at least 30 to 45 minutes to be performed and as the patient is anesthetised before ablation begins and a CT-scan is performed before the procedure can start, HFJV will thus be applied for up to at least an hour not uncommonly in elderly ASA Class 2-3 patients (ASA Class: American Association of Anesthesiologists Classification, ASA 2 being a patient with mild systemic disease and ASA 3 being a patient with severe systemic disease). The effects on oxygenation, carbon dioxide elimination, pH and lactate formation are not well-studied in this patient category during HFJV with the jet catheter placed through the endotracheal tube during liver ablation.\n\nThe primary aim of this explorative observational study was to study changes over time in arterial oxygenation, carbon dioxide elimination, pH and lactate formation in adult patients undergoing liver ablation under general anaesthesia who are ventilated with HFJV. The secondary aim was to describe the PaO2/FiO2 ratio and its relation to PaCO2 during HFJV.\n\n\nMethods\n\nThis study conforms to the standard of the Declaration of Helsinki and the study was approved by the Regional Ethics Committee in Stockholm (Dnr 2016/1124-32, June 7th, 2016) and the local Radiation Protection Committee at Danderyd University Hospital (Project number 2016-1, June 1st, 2016). Written informed consent for participation and publication was obtained from all subjects.\n\nTwenty-five consecutive patients (age>=50 years) with primary or secondary malignant liver tumours accepted for elective stereotactic ablation were included in this study after obtaining their written and oral informed consent. Exclusion criteria applied before inclusion of the 25 patients were pregnancy, recent pneumothorax or severe, poorly controlled lung disease. Patient demographics are presented in Table 1. The study was conducted October-December 2017 at Danderyd University Hospital, Stockholm, Sweden.\n\nStudy population demographics.\n\n* number of patients in each ASA-classification group\n\n** 8 patients with previous smoking habits\n\n***2 patients with mild asthma, 2 patients with lung metastasis, 1 patient with pulmonary hypertension and Sjogren’s disease, 1 patient with earlier postoperative pulmonary embolism.\n\nAll patients had total intravenous anaesthesia based on propofol (Propofol-Lipuro®, B. Braun Melsungen AG, Melsungen, Germany) 3–12 mg/kg/min and remifentanil (Ultiva®, GlaxoSmithKline AB, Solna, Sweden) 0,05–2 mikrgr/kg/min for induction and maintenance. Neuromuscular block was achieved by IV injection of rocuronium (Esmeron®, MSD, Haarlem, Netherlands) 0,6 mg/kg at induction.\n\nThe patients were preoxygenated with FiO2 of 0.8 at induction and intubated with an endotracheal tube (ETT) one size larger than the standard (i.e. size 8 for women and size 9 for men) to create sufficient space for the jet cannula and allow passive exhalation. An alveolar recruitment maneuver was performed following endotracheal intubation and confirmation of correct ETT placement and, subsequently, initiation of conventional ventilation was carried out while preparing for the HFJV.\n\nThe HFJV was performed with a Monsson III device (Acutronic, Switzerland). A jet cannula (Laserjet 40, double lumen jet catheter acc Biro, Acutronic Medical System AG, Hirzel, Germany) was put through the ETT. The jet ventilator was initiated with standardised settings on the jet ventilator; driving pressure (DP) between 1.2-1.4 bar, frequency of 220 min-1, oxygen 80% throughout the procedure. The ventilator had pre-set limits for ventilatory pressures at which ventilation is aborted and an alarm sound is activated.\n\nThe following protocol for a raise in PaCO2 was used. When PaCO2 rises above 10 kPa, DP is increased. If this does not lead to an acceptable level of PaCO2, the frequency is decreased. If still not a satisfactory PaCO2, HFJV is aborted and conventional ventilation is started. All these steps are carried out in close communication with the surgeon.\n\nMonitoring consisted of 3-lead ECG, oxygen saturation via pulse oximetry on a finger, invasive blood pressure via an arterial line in radial artery and level of muscle relaxation through train of four (TOF), recorded every 5 minutes in accordance with routine practice.\n\nAll patients had an arterial line inserted in accordance with routine practice for patients having HFJV. In 19 patients it was placed before anaesthesia and in 6 patients right after induction. Blood gas and lactate were sampled at baseline, after the start of HFJV, every 15 minutes (15’, 30’ and 45’) during the procedure and when extubated in the recovery room. No further follow-up was done; the study protocol ended with the postoperative blood gas analysis.\n\nBlood gases were analysed with a standard ABL 90 FLEX (Radiometer Medical Aps, Brönshöj, Denmark) in a standard blood gas syringe and was analysed immediately after the blood was taken from the patient (approximately 1–3 minutes).\n\nPaCO2 was categorised in three groups; <6 kPa, 6-8 kPa and >8 kPa. PaO2/FiO2 ratio was categorised in three groups; <20, 20-40 and >40.\n\nThis is an observational study thus; no power analysis or statistical plan has been conducted and we planned for 25 patients to target the primary outcomes. Data is presented as mean and SD for normal distributed data and median and range for non-normal distributed data. Data was tested for normality with the Shapiro-Wilk test. Repeated measures one-way ANOVA was used on normally distributed data and repeated measures ANOVA on ranks was used when data was non-normally distributed. The Bonferroni or Tukey Test was used in all pairwise multiple comparison procedures. Missing data; calculations are made on available data only. A p<0.05 was considered statistically significant. All statistical tests were conducted with SigmaPlot (version 14, Software Inc., San Jose, California, USA).\n\n\nResults\n\nAll 25 patients had an uncomplicated perioperative course. Anaesthesia, surgery and early recovery was uneventful apart from one patient who experienced a minor pneumothorax associated with the surgical procedure and was left with no further intervention. Changes in the HFJV settings per the protocol for increasing PaCO2 had to be initiated in one patient. HFJV did not need to be discontinued in any patients. One patient had a DP of 1,6 during the whole procedure. In one patient, oxygen was raised from 80% to 100% after 5 min of HFJV as saturation dropped to 93%. Saturation was then normalised.\n\nA total number of 131 blood gases were analysed in this study.\n\nMean PaO2 at baseline was 11.6 kPa (SD 2.3). All patients had a PaO2 above the normal lower limit of 8 kPa except for one patient (7.9 kPa). The PaO2 increased during HFJV and all patients had perioperative PaO2>10 kPa (see Table 2). An inter-individual variation was seen in oxygenation during HFJV and the PaO2/FiO2 ratio significantly decreased but was restored at recovery (see Figure 1). During the stay in the recovery room, three patients required supplementary oxygen and in two cases values were not accessible. All remaining 20 patients breathing room air had a PaO2 above the normal lower limit of 8 kPa (see Table 2).\n\nPaO2/FiO2 ratio at baseline, during high frequency jet ventilation and after extubation in the recovery room. An inter-individual variation was seen in oxygenation during HFJV and the PaO2/FiO2 ratio significantly decreased but was restored at recovery.\n\nPaO2 at baseline, during high frequency jet ventilation (HFJV) and after extubation in the recovery room. No hypoxemia was seen during HFJV. At recovery 3 patients required additional oxygen.\n\nMean PaCO2 at baseline was 5.0 (SD 0.4). None of the patients had a PaCO2 above the normal limit of 6 kPa. A significant raise was seen in mean PaCO2 from baseline to t=1st CT; 5.0 to 6.1 kPa (p=0.003). The mean PaCO2 value increased further to peak mean PaCO2 7.1 kPa at 45’ (see Figure 2). The number of patients with a PaCO2 >6 kPa was 14/24 at t=1st CT, 15/24 at t=15’, 18/22 at t=30’, 15/19 patients at t=45’. Mean PaCO2 during recovery (5,6 kPa) was not significantly different from the mean baseline. Five out of 23 patients had a PaCO2 value >6 kPa at recovery, the highest value being 8.03 kPa.\n\nPaCO2 at baseline, during high frequency jet ventilation and after extubation in the recovery room. A significant raise was seen in mean PaCO2 from baseline to 1st CT. Five out of 23 patients had a PaCO2 value >6 kPa at recovery, the highest value being 8.03 kPa.\n\nMedian pH decreased from baseline 7.44 to 7.31 at t=15’ (p=0.03). There was a small further drop in pH but with no statistical difference between time points (see Figure 3). Fourteen out of 24 patients had a pH <7.35 at t=1st CT, 18/24 patients at t =15’, 17/22 at t =30’ and 14/19 at t=45’. Median pH during recovery (7.37) was not significantly different from the median baseline. Four out of 23 patients had a pH<7.35 at recovery, with the lowest value being 7.24 (see Figure 3).\n\nAll lactate values were within normal range during HFJV. One patient had a minor increase (2.3 mmol L-1) during recovery.\n\nThere was no clear correlation between decrease in PaO2/FiO2 ration and the PaCO2 increase (see Figure 4). Ten out of 16 blood gas analyses (62%) with a PaCO2>8 had a PaO2/FiO2 ratio of >40. Thirteen out of 39 (33%) of the blood gas analyses with normal PaCO2 had PaO2/FiO2 ratio <40.\n\npH at baseline, during high frequency jet ventilation and after extubation in the recovery room. There was a significant drop in pH from baseline during HFJV. Four out of 23 patients had a pH<7.35 at recovery, with the lowest value being 7.24.\n\nPlotted blood gas pairs of PaO2/FiO2 ration vs PaCO2 in the 25 patients studied. No clear correlation could be seen.\n\n\nDiscussion\n\nThere is sparse information about arterial blood gas changes during HFJV during abdominal surgery. No previous study has explicitly assessed oxygenation and carbon dioxide elimination during CT-guided stereotactic liver ablation with the jet ventilation catheter placed inside an ETT. Our study showed that this technique, high frequency jet ventilation by a catheter placed through the ETT, maintained adequate oxygenation in all patients. The gas exchange, the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2), decreased and varied considerably. The carbon dioxide tension increased and pH decreased reasonably as an effect of the CO2 retention. Furthermore, the impairment in oxygenation, decrease in PaO2/FiO2 ratio and CO2 elimination was found somewhat surprisingly not to correlate, showing a scattered pattern. There was also only modest residual impairment of gas exchange during early recovery.\n\nHigh frequency ventilation is not new3. However, most HFJV use is for airway interventions, for example during a bronchoscopy or laryngoscopy. This may not entirely translate to its use in abdominal surgery, in this case during puncture of the liver under CT-guidance, with the jet catheter placed through an endotracheal in patients who are under general anaesthesia and receiving muscle relaxants. The HFJV catheter in our study was inserted into the endotracheal tube and thus the explicit effective inspired oxygen fraction cannot be defined. The PaO2/FiO2 findings may thus not be entirely true. The open system may also impact the expired pressure as well as gas elimination. Pause pressure was measured via the tip of the jet cannula, providing the pressure at tracheal level between the jet inspirations. This is not a fully representative pressure for the whole lung but may still be seen as an indicator for built-up auto positive end expiratory pressure (PEEP). The HFJV technique is dependent on passive outflow, contrary to high frequency oscillation that possibly has a more active exhalation. There is indeed, with HFJV, an obvious risk of carbon dioxide retention as well as a potential for a build-up of intrinsic PEEP and subsequent risk for barotrauma.\n\nAll patients had total intravenous anaesthesia because of the open airway system and all had muscle relaxation to promote the stereotactic liver ablation, both factors that impact on lung function4. Age and supine position are also factors known to impair the lung function during anaesthesia5. In the present study a FiO2 of 0.8 was used during the entire procedure except for one patient who had a FiO2 of 1.0. High oxygen fraction may cause rapid absorption atelectasis and anaesthesia per se causes loss of muscle tone and reduced lung volume, factors having a negative effect on ventilation/perfusion matching6. It should also be acknowledged that preoxygenation was performed before induction, thus further promoting lung collapse when the high oxygen fraction is maintained7. Several of the patients had a history of pulmonary disease, further impairing ventilation/perfusion matching during anaesthesia8. The oxygenation as well as the arterial carbon dioxide tension was somewhat surprisingly restored by the arterial blood gas measurement in the recovery room. Lung recruitment manoeuvre is standard procedure following intubation but not during emergencies.\n\nBickel et al.9 found that HFJV during laparoscopy in ASA 1-2 patients lessened the cardiovascular effects of pneumoperitoneum as compared to conventional ventilation. They only briefly commented on the effects on blood gases. Contrary to the present findings, they state that PaO2, PaCO2 and pH was similar during all phases and between the study groups. Explicit values were, however, not presented. They used the same type of HFJV apparatus, with an average ventilator frequency of 150 cycles/min. In an earlier study, transtracheal HFJV was started with 100% oxygen at 30 to 35 pounds per square inch of driving pressure (equals 2.1-2.4 bar), 100 cycles per minute and an I:E ratio of 25%. An increase in CO2 was noted at 10 minutes, similar to the present findings10. There is also a risk for hypercapnia and/or hypoxia when HFJV is used during airway surgery as shown by Fernandez-Bustamante et al.11. Their study included 316 patients who underwent an interventional rigid bronchoscopy under general anaesthesia and HFJV. The most common complications were hypoxia, hypercapnia and hemodynamic instability. Sutterlin et al.12 found that increasing frequency raised arterial carbon dioxide, possibly by reducing the gas elimination time. The carbon dioxide and pH normalised after awakening. It would indeed be of value to be able to monitor the inspired alveolar oxygen pressure and carbon dioxide tension as well as PEEP during HFJV.\n\nAlternative lung ventilation modes could be considered during liver tumour ablation instead of HFJV. Various forms of high flow systems for apnoeic oxygenation have recently gained interest, including THRIVE (transnasal humidified rapid-insufflation ventilatory exchange). Several reports show the feasibility of THRIVE during general anaesthesia in the difficult airway situation13 and in laryngeal surgery14. In the difficult airway situation, THRIVE is a way of prolonging the apnoeic time until a definitive airway is secured. Whether this technique could have a place in anaesthetic management for stereotactic liver ablation needs to be investigated.\n\nThere are several limitations that should be considered. We have not included any control group having conventional ventilation as this would jeopardise intervention safety and accuracy, increasing the risk for bleeding and tissue damage. As pointed out, only one HFJV setting was used. Pressure and frequency was not adjusted, except in one patient when pressure was slightly adjusted according to a raise in PaCO2. Likewise, a fixed oxygen fraction was used throughout the procedure and, as mentioned, oxygen concentration reaching the alveoli and PEEP levels cannot be assessed. The focus of our study was gas exchange during HFJV, following arterial blood gases under steady state jet ventilation. We did not include any further follow-up after the recovery phase.\n\n\nConclusion\n\nThis study shows the feasibility of using HFJV through an ETT with an FiO2 of 0.8 during liver tumour ablation. It did not cause any hypoxia or increase in lactate. It was associated with mild to moderate impairment in gas exchange that was restored during early recovery.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for; High Frequency Jet Ventilation during stereotactic ablation of liver tumours – an observational study on blood gas analysis as a measure of lung function during general anaesthesia. https://doi.org/10.7910/DVN/MXHFWW15\n\nThis project contains the following underlying data:\n\n- Bloood gas HFJV F1000_data_190401.tab (Blood gas data associated to HFJV)\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\nThe study has been supported by funds from Stockholm county council/ Stockholms Läns Landsting (ALF).\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\nBiro P, Spahn DR, Pfammatter T: High-frequency jet ventilation for minimizing breathing-related liver motion during percutaneous radiofrequency ablation of multiple hepatic tumours. Br J Anaesth. 2009; 102(5): 650–653. PubMed Abstract | Publisher Full Text\n\nAbderhalden S, Biro P, Hechelhammer L, et al.: CT-guided navigation of percutaneous hepatic and renal radiofrequency ablation under high-frequency jet ventilation: feasibility study. J Vasc Interv Radiol. 2011; 22(9): 1275–1278. PubMed Abstract | Publisher Full Text\n\nGalmén K, Harbut P, Freedman J, et al.: High frequency jet ventilation for motion management during ablation procedures, a narrative review. Acta Anaesthesiol Scand. 2017; 61(9): 1066–1074. PubMed Abstract | Publisher Full Text\n\nTokics L, Hedenstierna G, Strandberg A, et al.: Lung collapse and gas exchange during general anesthesia: effects of spontaneous breathing, muscle paralysis, and positive end-expiratory pressure. Anesthesiology. 1987; 66(2): 157–167. PubMed Abstract | Publisher Full Text\n\nGunnarsson L, Tokics L, Gustavsson H, et al.: Influence of age on atelectasis formation and gas exchange impairment during general anaesthesia. Br J Anaesth. 1991; 66(4): 423–432. PubMed Abstract | Publisher Full Text\n\nHedenstierna G, Edmark L: Effects of anesthesia on the respiratory system. Best Pract Res Clin Anaesthesiol. 2015; 29(3): 273–284. PubMed Abstract | Publisher Full Text\n\nReber A, Engberg G, Wegenius G, et al.: Lung aeration. The effect of pre-oxygenation and hyperoxygenation during total intravenous anaesthesia. Anaesthesia. 1996; 51(8): 733–737. PubMed Abstract | Publisher Full Text\n\nHedenstierna G, Rothen HU: Respiratory function during anesthesia: effects on gas exchange. Compr Physiol. 2012; 2(1): 69–96. PubMed Abstract | Publisher Full Text\n\nBickel A, Trossman A, Kukuev I, et al.: The effects of high-frequency jet ventilation (HFJV) on pneumoperitoneum-induced cardiovascular changes during laparoscopic surgery. Surg Endosc. 2011; 25(11): 3518–24. PubMed Abstract | Publisher Full Text\n\nNakatsuka M, MacLeod AD: Hemodynamic and respiratory effects of transtracheal high-frequency jet ventilation during difficult intubation. J Clin Anesth. 1992; 4(4): 321–324. PubMed Abstract | Publisher Full Text\n\nFernandez-Bustamante A, Ibañez V, Alfaro JJ, et al.: High-frequency jet ventilation in interventional bronchoscopy: factors with predictive value on high-frequency jet ventilation complications. J Clin Anesth. 2006; 18(5): 349–356. PubMed Abstract | Publisher Full Text\n\nSütterlin R, Priori R, Larsson A, et al.: Frequency dependence of lung volume changes during superimposed high-frequency jet ventilation and high-frequency jet ventilation. Br J Anaesth. 2014; 112(1): 141–149. PubMed Abstract | Publisher Full Text\n\nPatel A, Nouraei SA: Transnasal Humidified Rapid-Insufflation Ventilatory Exchange (THRIVE): a physiological method of increasing apnoea time in patients with difficult airways. Anaesthesia. 2015; 70(3): 323–329. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGustafsson IM, Lodenius Å, Tunelli J, et al.: Apnoeic oxygenation in adults under general anaesthesia using Transnasal Humidified Rapid-Insufflation Ventilatory Exchange (THRIVE) - a physiological study. Br J Anaesth. 2017; 118(4): 610–617. PubMed Abstract | Publisher Full Text\n\nJakobsson J: Replication Data for; High Frequency Jet Ventilation during stereotactic ablation of liver tumours – an observational study on blood gas analysis as a measure of lung function during general anaesthesia. Harvard Dataverse, V1, UNF: 6:r3eyhTywgvSVkMJAGyD4hg== [fileUNF]. 2019. http://www.doi.org/10.7910/DVN/MXHFWW"
}
|
[
{
"id": "49490",
"date": "03 Jul 2019",
"name": "Marzenna Podhorska-Okolow",
"expertise": [
"Reviewer Expertise oncology",
"ophthalmology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHigh frequency jet ventilation (HFJV) is an alternative method to conventional ventilation which is known to minimize movement of the thorax and abdomen what finally could facilitate surgical procedures. The aim of the study of the revised manuscript was to analyze usefulness, efficacy and safety of HFJV during stereotactic ablation of liver tumors with particular emphasis on gas exchange. The study was performed on 25 patients with malignant liver tumors. In conclusions, authors demonstrated possibility and benefits of HFJV’s usage during the liver’s surgical procedures. However, influence of some disadvantages on patients’ gas exchange was also critically discussed. In general the work presents interesting results and, in my opinion, could be accepted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "46791",
"date": "18 Sep 2019",
"name": "Astrid Bergmann",
"expertise": [
"Reviewer Expertise Anaesthesia in cardiac and thoracic 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 authors address the question if high frequency jet ventilation (HFJV) affects blood gases when applied during sterotactic ablation of liver tumours. This procedure has evolved as an auspicious treatment in the field of hepatic oncology. It is crucial to minimize the movement of the liver to establish optimal conditions for the surgeon throughout the intervention. Hence, HFJV has evolved as a promising method. No data are available so far on the effects of HFJV on gas exchange during stereotactic ablation of liver tumours. Therefore, 25 patients have been involved in this observational study. Blood gases were assessed before induction and every 15 minutes during HFJV and finally in the recovery area after extubation. No hypoxia could be observed in any of the patients even though paO2/FIO2 showed a wide range. PaCO2 increased during HFJV and returned to baseline levels after extubation. pH decreased due to CO2 retention during HFJV. There was no correlation between paO2/FIO2 and CO2. The authors conclude that HFJV does not cause hypoxaemia during stereotactic liver ablation under general anaesthesia but that gas exchange is slightly impaired during the procedure.\n\nThis article makes an important contribution to the anaesthetic practice during stereotactic ablation of liver tumours. Working conditions for the surgeon can be optimised by HFJV without compromising the patient's gas exchange and metabolic status.\nThe manuscript is well written and attractive. The abstract fulfils its purpose in giving a clear overview of the whole study. The background of the intervention as well as the importance in the oncological field and the problems associated with ventilation are clearly pictured in the introduction. A sound description of the aims of the study are also provided. The study population, anaesthetic methods and blood gas analysis are soundly described in the methods section. The presentation of the results is clear and straightforward with well-designed graphs and tables to illustrate the outcome parameters. Every result is discussed and literature in the field is cited and compared with the findings of this study. A perspective for future studies is given and the promising conclusion is drawn that HFJV is feasible to use during liver tumour ablation without causing hypoxia or an increase in lactate although a moderate impairment in gas exchange was observed during the procedure.\nAll in all, I think that this paper is an important addition to the literature with a well-thought-out protocol, clear results and a promising conclusion. It is scientifically sound and should be accepted for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "46793",
"date": "23 Sep 2019",
"name": "Göran Hedenstierna",
"expertise": [
"Reviewer Expertise Respiratory physiology",
"in particular gas exchange and lung morphology during anesthesia and in acute lung injury."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHFJV causes minor movement of liver tissue and is thus advantageous in liver ablation, compared to standard mechanical ventilation. This study shows that gas exchange can be maintained at a reasonable level for an hour-long anethesia/surgery. A successive fall in PaO2 was seen but in no patient was hypoxemia seen. Similarly, PaCO2 rose during the hour-long observation period, but not to any critical level. The authors found no correlation between fall in PaO2 and increase in PaCO2. Thus, hypoventilation was not the major or only cause of impaired oxygenation. FIO2 was in most patients 0.8, but in an open system, as here, the alveolar FO2 may be different and also vary between patients. The authors discuss a certain degree of auto-PEEP but have not been able to make measurements of it. Atelectasis may be reduced with increase in auto-PEEP, as inferred by the authors. One may speculate in varying distance between catheter tip and carina with effects on ventilation of each lung. Impairment in lung perfusion, possibly reflected by decrease in cardiac output, might be an additional cause of varying oxygenation. However, all these comments are speculations and need not be in a paper. On the whole, the study is well performed in a simple and straightforward way. Results are clear and show a reasonably maintained gas exchange under the studied conditions. My only minor criticism is that the ventilatory technique is mentioned in different parts of the paper and could be brought together in the Methods 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": []
}
] | 1
|
https://f1000research.com/articles/8-386
|
https://f1000research.com/articles/8-381/v1
|
04 Apr 19
|
{
"type": "Research Note",
"title": "Perception of the risk of medicinal marijuana in postgraduate medical residents",
"authors": [
"Sandra Castillo-Guzmán",
"Dionicio Palacios Ríos",
"Teresa Adriana Nava Obregón",
"Daniela Alejandra Becerril Gaitàn",
"Keren Daniela Juangorena García",
"Danya Carolina Domínguez Romero",
"Misael Jerónimo Reyes Rodríguez",
"Dionicio Palacios Ríos",
"Teresa Adriana Nava Obregón",
"Daniela Alejandra Becerril Gaitàn",
"Keren Daniela Juangorena García",
"Danya Carolina Domínguez Romero",
"Misael Jerónimo Reyes Rodríguez"
],
"abstract": "Background: Drugs can often cause adverse reactions, and the perception of the risk of prescription drugs could influence prescription behaviour. Methods: This was a prospective cross-sectional study of the perception of postgraduate physicians in training of the risk of using medical marijuana, comparing it with their perception of paracetamol and sedatives. A visual analogue scale with ranges from 0 (no risk) to 10 (totally risky) was used. Results: A total of 197 postgraduate students were evaluated; 48 women and 149 men took part, with a mean age 27.8 years. Among the different specialties, there was a difference with regard to the perception of medical marijuana and paracetamol and all perceived a greater risk with sedatives. There was no evidence of a risk perception of marijuana in relation to factors such as alcohol consumption and smoking. Conclusions: There is a difference in the perception of risk of medical marijuana and paracetamol with this perception being greater with sedatives. Regarding specialties, the perception of risk was greater for medical marijuana in general surgery than in urology.",
"keywords": [
"Risk perception",
"marijuana",
"méxico",
"postgraduate medical residents."
],
"content": "Introduction\n\nMultiple therapeutic uses have been proposed for marijuana; however, in Mexico, the legalization of its therapeutic and recreational use is currently in the process of being accepted1. Cannabinoids have been used for the management of certain symptoms, such as nausea and vomiting induced by chemotherapy2, appetite stimulation in patients with HIV/AIDS3, chronic pain2, spasticity induced by paraplegia or multiple sclerosis, and anxiety, sleep, psychosis, glaucoma and Tourette syndrome4.\n\nA systematic review and meta-analysis published by Whiting et al.4 in 2015 evaluated the benefits of using cannabinoids. They found moderate-quality evidence for the treatment of chronic pain and spasticity and low-quality evidence regarding nausea and vomiting due to chemotherapy, weight gain in HIV infection, sleep disorders and Tourette syndrome. However, cannabinoids were associated with an increased risk of adverse effects in the short term.\n\nThe risk perceived by doctors in training as specialists is important because they could be responsible for their prescription in the future. Their perception may be similar to the fear of prescribing morphine or underestimating the effect of anti-inflammatory drugs and paracetamol.\n\nContinuing with the research on the risk perception toward drugs, the objective of this study was to assess the perception of risk to medicinal marijuana in postgraduate medical residents of an academic hospital and compare this with the risk perception of paracetamol and sedatives.\n\n\nMethods\n\nThis was a cross-sectional study that was carried out during May 2017 in the Dr. Jose E. Gonzalez University Hospital in Monterrey, Mexico, an academic hospital that cares for uninsured patients from three north-eastern states in Mexico. The objective was to evaluate the perception of the use of medical marijuana in postgraduate medical residents. The study protocol was previously approved by the Ethics Committee of the Universidad Autonoma de Nuevo Leon, registration number PL17-00134. We used a convenience sample of residents in postgraduate training from diverse specialties in our institution. A total of 197 medical residents from 8 medical specialties were included (anesthesiology, 34; general surgery, 34; traumatology, 20; neurosurgery, 15; urology, 14; otorhinolaryngology, 11; gynecology, 29; and internal medicine, 40). Participants were recruited and assessed in-person in offices, operating room and halls of the hospital. Individuals were assessed to determine if they met the inclusion criteria (postgraduate students in training in a medical specialty of both sexes, regardless of age, who accepted to participate in the study). Only those who did not wish to participate were excluded. After accepting, information about the study was provided and informed consent was obtained. Verbal consent was used because of the low-risk of the study.\n\nA survey consisting of two sections was applied (the Spanish and English versions of the survey are available on Figshare5). Section 1 included general data collection questions such as age, sex, marital status, city of residence, place of birth, and time living in the city. Also, current health problems, smoking and active alcohol consumption, level of education, name of the specialty they were training in, the current semester of study. Economic income and the participant’s name, as optional data, were asked. Section 2 consisted of a 10-centimeter long line that represented the level of risk that they felt a drug could have. The respondent had to mark with an X the level of risk, considering the left side as being completely safe and the right as completely unsafe. The drugs evaluated were paracetamol, medical marijuana and sedatives. Text regarding the three drugs was arranged in three different ways in each questionnaire to avoid influencing the respondent’s decision; afterwards, the median response in centimetres was considered.\n\nStatistical analysis was performed with the SPSS statistical program, version 20. Descriptive statistics of the total sample were calculated. Risk perception was reported as median with interquartile ranges. The Kruskal-Wallis test followed by a Dunn post hoc test was used to determine the difference among the different medical services. The Mann-Witney U-test was used to compare the perception of the three drugs between smokers and non-smokers and between alcohol consumers and non-consumers.\n\n\nResults\n\nA total of 197 postgraduate medical students were evaluated; there were 48 women and 149 men with a mean age of 27.8 years (range 25 to 32). The percentage of surveyed participants according to the year of residence was 26.9% for the first year, 29.4% for the second year and 43.7% for >3 years of training. The frequency of self-reported smoking was 24.4% while for self-reported drinking it was 81.2%.\n\nOverall, the risk perception of medical marijuana was 3 cm (IQ range = 1–5 cm) which we consider as risk perception of medium grade; for paracetamol it was 1 cm (IQ range = 1–2) which we consider as a risk of low grade, and for sedatives, 5 cm (IQ range= 4–8 cm) which we consider as a risk perception of high grade. There was a significant difference in the risk perception among the three drugs studied (p<0.01).\n\nThe risk perception according to specialty shows that all specialties studied have a greater risk perception to sedatives than to the other drugs studied (p<0.01). In this sense, the drug with the lowest risk perception was paracetamol (Table 1). Among the specialties, general surgery had the highest risk perception to medical marijuana while urology had the lowest risk perception (p<0.01). In the case of paracetamol, general surgery residents also had the highest risk perception (2 cm), while that rest of the specialties had lower risk perception (from 1 to 1.5 cm) (p= 0.04). In the case of sedatives, there no were a significant difference in risk perception between the residents of each specialty (Table 2).\n\nValues are given as median (IQ range).\n\n*Alcohol user vs non-alcohol user. †Smoker vs non-smoker.\n\nBoth groups, smokers and non-smokers, had a greater risk perception of sedatives than other drugs. The risk perception of medical marijuana (p=0.11), paracetamol (p= 0.37) and sedatives (p= 0.59) was not statistically different between smokers and non-smokers (Table 2). In the case of self-reported alcohol consumption, sedatives also had the greatest risk perception in both alcohol users and non-alcohol users. There was no significant difference between groups in the risk perceived for medicinal marijuana, paracetamol and sedatives.\n\n\nDiscussion\n\nIn this study, we evaluated the risk perception of medical marijuana in medical residents of several specialties in Monterrey, Mexico. To our knowledge, this is the first study in medical residents. Overall, the risk perception of medical marijuana was 3 cm. We consider that the perceived risk to medical marijuana is medium, since medical marijuana, in reality, is still under study for the treatment of some diseases; therefore, its use should be cautious. The low risk perception may also suggest that medical residents have a favourable attitude towards medicinal marijuana and thus, a willingness to recommend it. The latter should be evaluated in other studies.\n\nWe previously evaluated the risk perception toward medicinal marijuana in undergraduate medical students6. The risk perception of the use of paracetamol, marijuana and sedatives is the same in graduate students and undergraduate students. However, in this previous study, the risk perception of medical marijuana in undergraduate students was different between smokers and non-smokers and between alcohol and non-alcohol users.\n\nOne of our limitations is that only the degree of risk perception was assessed; however, the degree of knowledge, which is an objective of future research, should be contemplated by other studies. It is worth mentioning that the lack of knowledge of drugs has been associated with a negative attitude to prescribing them, as in the case of opioids (opiophobia)7,8.\n\nThere is evidence of knowledge of the potential risks of medical marijuana and recommendations have been made for medical marijuana use in patient care in the United States9. In Mexico there are no studies that have evaluated the knowledge and experience in the use of medical marijuana in patient care nor a legal basis for it.\n\n\nConclusions\n\nRisk perception to medical marijuana in medical residents of Monterrey, Mexico is 3 cm (IQ range = 1-5 cm) which we consider as a risk perception of medium grade. Among the different specialties, there was a difference with regard to the perception of risk of medical marijuana. There was no difference between smokers and alcohol users in risk perception. The degree of knowledge toward medicinal marijuana is an objective of future research in medical students and postgraduate medical residents.\n\n\nData availability\n\nFigshare: Raw data and survey of risk perception to medical marijuana of medical residents. https://doi.org/10.6084/m9.figshare.7742723.v15.\n\nUnderlying data for this study are available in file “Raw date Risk perception of medical marijuana in Postgraduate medical residents.xlsx”.\n\nFigshare: Raw data and survey of risk perception to medical marijuana of medical residents. https://doi.org/10.6084/m9.figshare.7742723.v15.\n\nThe following extended data are available:\n\nSurvey Risk perception of Medicinal marijuana Mexico.pdf (English translation of questionnaire).\n\nSurvey Risk perception of Medicinal marijuana Mexico.pdf (Original Spanish-language questionnaire).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Sergio Lozano-Rodriguez, M.D. for his help in translating and editing the manuscript, and Braulio H. Velasco-Sepulveda and Omar González Santiago for their help with statistical analysis.\n\n\nReferences\n\nArellanes FJV, Mendívil EAS: La mariguana en México: implicaciones biológicas y sociales en su legalización y regulación [Marijuana in Mexico: biological and social implications in its legalization and regulation]. RIIAD. 2018; 4(1): 36–52. Publisher Full Text\n\nWilkie G, Sakr B, Rizack T: Medical Marijuana Use in Oncology: A Review. JAMA Oncol. 2016; 2(5): 670–675. PubMed Abstract | Publisher Full Text\n\nFride E, Bregman T, Kirkham TC: Endocannabinoids and food intake: newborn suckling and appetite regulation in adulthood. Exp Biol Med (Maywood). 2005; 230(4): 225–34. PubMed Abstract | Publisher Full Text\n\nWhiting PF, Wolff RF, Deshpande S, et al.: Cannabinoids for Medical Use: A Systematic Review and Meta-analysis. JAMA. 2015; 313(24): 2456–2473. PubMed Abstract | Publisher Full Text\n\nCastillo-Guzman S: Raw data and survey of risk perception to medical marijuana of medical residents. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7742723.v1\n\nCastillo-Guzmán S, Palacios-Ríos D, Nava-Obregón TA, et al.: Risk perception of medicinal marijuana in medical students from northeast Mexico [version 1; peer review: 1 not approved]. F1000Research. 2017; 6: 1802. Publisher Full Text\n\nGer LP, Ho ST, Wang JJ: Physicians' knowledge and attitudes toward the use of analgesics for cancer pain management: a survey of two medical centers in Taiwan. J Pain Symptom Manage. 2000; 20(5): 335–44. PubMed Abstract | Publisher Full Text\n\nWeinstein SM, Laux LF, Thornby JI, et al.: Physicians' attitudes toward pain and the use of opioid analgesics: results of a survey from the Texas Cancer Pain Initiative. South Med J. 2000; 93(5): 479–87. PubMed Abstract\n\nChaudhry HJ, Hengerer AS, Snyder GB: Medical Board Expectations for Physicians Recommending Marijuana. JAMA. 2016; 316(6): 577–578. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "115460",
"date": "09 Feb 2022",
"name": "Julie Dupouy",
"expertise": [
"Reviewer Expertise Primary care",
"substance use disorders"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Castillo-Guzman et al., reports the perception of the risk of medicinal marijuana in postgraduate medical residents in training in one hospital. My first comment is that the need to investigate this topic is not clearly indicated in the introduction. I think that if the authors want to consider medical cannabis as one analgesic drug they have to discuss deeply the efficacy of medical cannabis and cannabinoids. In the meta analysis in the BMJ about chronic pain1, medical cannabis had small effect on pain, and some could argue, as the authors of this meta-analysis, that the clinical significance of some positive results found in this meta-analysis was not achieved. Adverse drug reactions were important in the a recent Cochrane review2. Data are even more difficult to synthesize in psychiatric disorders (Black et al., 20193 and McKee et al., 20194) and are prone to be discussed.\nThis survey was approved by an Ethic Committee. Nevertheless, I was surprised that data about name (even if optional), income, city of residence, marital status, medical illness, etc. were asked in the questionnaire. This seems to be an ethical concern if questionnaires were not anonymous and were answered face-to-face. In addition, completing the questionnaires face-to-face may influence the results, even more if the person in charge of administering the questionnaire was a physician known by the resident.\nI did not find any explanation in the paper of what hypothesis sustain the comparison between specialties? Another question I had was why not to ask students about marijuana self-use in addition to alcohol and tobacco self-use?\nThere are other limits to discuss in this paper: the monocentric aspect of the survey, the low response rate, the fact that few specialties were represented and not medical ones (except internal medicine). These limits should be discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "126852",
"date": "23 Mar 2022",
"name": "Wangjun Qin",
"expertise": [
"Reviewer Expertise Clinical pharmacy in pain 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\nThis article tells us a risk perception of medium grade for medical marijuana in medical residents in one hospital of Monterrey, which is interesting for researchers from other counties. Marijuana is used in Europe and America more than that in Asia. Thus, it is supposed there are significant variations among medical residents from these countries. Even so, the present study provides a baseline in Monterrey and a reference for other countries and regions.\nThere are some limits for the present version. First, the authors did not describe the way to select the participants recruited and readers may wonder whether a random sample was used. Second, the risk perception for medical marijuana may associated with the medical knowledge. However, the difference in risk perception among yeas of residence is not analyzed. Third, the face-to-face questionnaires may influence the results of the participants.\nOverall the article is useful in providing an understanding the risk perception for medical marijuana in medical residents and its influence factors.\n\nIs the work 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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-381
|
https://f1000research.com/articles/7-1388/v1
|
03 Sep 18
|
{
"type": "Clinical Practice Article",
"title": "Early hyaluronidase use in preventing skin necrosis after treatment with dermal fillers: Report of two cases",
"authors": [
"Francesco Ciancio",
"Maria Stella Tarico",
"Giuseppe Giudice",
"Rosario Emanuele Perrotta",
"Maria Stella Tarico",
"Giuseppe Giudice",
"Rosario Emanuele Perrotta"
],
"abstract": "Injection of dermal fillers, like hyaluronic acid (HA), is a safe procedure, with few and transient side effects such as erythema, bruising and swelling etc. The aim of this report is to provide our protocol for the early treatment of necrotic complications after facial treatment with dermal fillers. We present two cases of skin suffering of the face after dermal infiltration of HA, treated successfully with our early protocol. Our protocol includes the early infiltration of hyaluronidase in the treated areas. We start with infiltration of hyaluronidase distributed over the area to be treated through micro-injections with dosage 40 IU per cm2. Our protocol includes the use of systemic corticosteroids for 4 days, anti-aggregation therapy, oral antibiotic, topical cream with nitric oxide and compresses with gauze and warm water. In the skin complications after dermal filler treatment, marked pain and characteristic reticulated erythema in the skin distribution of the affected vessels is often developed. Due to the implementation of our protocol in these patients, we managed to avoid an irreversible necrotic complication of the face in both cases. In this report, our protocol was compared with results published in the literature and allowed us to avoid complications such as skin necrosis with permanent damage.",
"keywords": [
"dermal filler",
"skin necrosis",
"filler complications",
"vascular complications"
],
"content": "Introduction\n\nThe use of dermal fillers is increasingly required in cosmetic surgery1. Injection of dermal fillers is a safe procedure, with few and transient side effects. However, cases of skin necrosis have been reported, including with involvement of vision and ocular globe2. The cause is an impediment of the blood supply by compression and/or obstruction of the vessel(s) with filler material, and/ or direct injury to the vessel3,4. Several therapeutic approaches have been described5–7.\n\nThe aim of this report is to present our protocol for the early treatment of vascular complications after facial rejuvenation with dermal fillers in order to avoid skin necrosis. We present two cases of vessel damage and skin suffering of the face after dermal infiltration of hyaluronic acid (HA), which occurred in 2017 and was treated successfully with our protocol.\n\n\nProtocol\n\nInfiltration of hyaluronidase is performed firstly, at a deep dermal level, and is distributed over the area to be treated through micro-injections at a dosage of 40 IU per cm2. The distribution is homogeneous except for nodular areas in which a double dose is infiltrated (in any case, at least 150 IU of hyaluronidase in 1th infiltration is used). The lithic enzyme must be infiltrated at the dermal level, homogeneously distributed, in order that from the point of infiltration it is distributed to the vessels by diffusion. The rational use of hyaluronidases is to breakdown HA particles and allow reabsorption. It is well known that hyaluronidases are inactivated by the immune system8, so the treatment must be repeated to obtain an adequate concentration. In our clinical practice, we use the maximum dosage (40IU per cm2) for three consecutive days. The areas to be treated are established on the basis of clinical signs of damage/ischemia distribution for which, in selected cases, maintenance doses (40 UI/cm2) can be applied in the areas most affected. The treatment is repeated after a few hours if the ischemic area shows no improvement with double daily doses but, in all cases, never extended beyond 72h.\n\nSystemic corticosteroids for four days, oral salicylic acid 100 mg, antibiotic therapy, topical cream with nitric oxide, and compresses with gauze and warm water are also recommended in this protocol. In the following days, the evolution of clinical signs should be monitored and therapy continued if needed.\n\n\nCase 1\n\nIn June 2017 a 36-year-old female patient was admitted for treatment of infiltration of HA-based dermal fillers. The patient had received treatments with dermal fillers in the past without adverse reactions. Immediately after the dermal filler procedure, the treated areas appeared in good condition without signs of skin suffering. Three days later, at a follow-up examination, the left treated area appeared cyanotic and swollen despite the patient not complaining of discomfort. The skin appeared erythematous with distribution along the left nasolabial folds up to the lateral nasal wall, and the capillary refill time appeared slow or absent (Figure 1). Consequently, treatment with the protocol, as stated above, was performed immediately. We used 40UI of hyaluronidase per cm2, two times a day for 3 days. The patient received acetylsalicylic acid 100 mg / 24h for 10 days, prednisone 25mg / 24h for 4 days, levofloxacin 500mg / 24h for 4 days, topical cream with nitric oxide 2 times a day and compresses with gauze and warm 3 times a day.\n\nThree days after dermal filler treatment, erythematous and blister formation was observed along the nasolabial vessels. Part of the erythema extends to the middle of the nose.\n\nNecrotic complications of the face were avoided in this patient (Figure 2).\n\nLeft panel: 7 days after first treatment; middle panel: after 15 days; right panel: clinical check after 45 days. It should be noted that there is no scarring in the final image.\n\n\nCase 2\n\nIn August 2017 a 45-year-old woman was treated with HA to fill the region of nasolabial folds. In the past the patient had received similar treatments without adverse reactions. At the clinical check after three days, the patient shows signs of skin suffering. Compared to Case 1 the erythematous area was smaller with distribution retained to the medial region of the cheek (Figure 3). Treatment with the protocol, as stated above, was performed immediately and 40UI/cm2 of hyaluronidase was injected every 12 h per 2 days, after only 1 dose for the third day. Systemic corticosteroids, antiplatelet therapy, antibiotic therapy and local topics were used according to protocol, as expressed in Case 1.\n\nThere is an erythematous halo, blisters and livedo reticularis in the middle third of the left cheek.\n\nNecrotic complications of the face were avoided in this patient (Figure 4).\n\nLeft panel: the erythematous lesion has decreased in intensity after 7 days from treatment; middle panel: after 12 days; right panel: after 45 days. The green dots in the picture are the result of a damaged camera, we have not modified the image.\n\n\nDiscussion\n\nDamage after dermal fillers can lead to severe consequences, such as skin necrosis, and the involvement of ocular, nerve and muscle structures2. Skin necrosis is the most significant complications after dermal filler. The incidence of vascular damage after use of fillers has been estimated at 3–9 per 10,000 for HA products, but the true incidence of this complication is unknown9,10. The skin necrosis after dermal fillers injection is an emergency, the best treatment is often the quickest. The gold standard is prompt injection of hyaluronidase with a dose of 40 IU per cm2 of affected area. Some authors have described oral antiplatelet drugs, like cardioaspirin while some have cited the use of vasodilator drugs. Today again there is no international standard protocol for the treatment of these complications. In our cases, the patients manifested skin problems probably from filler emboli or direct destruction of the vessels during needle manoeuvres. Interestingly our patients did not experience any pain or discomfort during and after the HA dermal filler procedure. In the literature, some manoeuvres have been reported to reduce the risk of vascular damage, such as aspiration during infiltration11, low pressure injection, continuously moving the needle or cannula while injecting, injection of small quantities (maximum 0.1 mL of filler per pass)12, observing skin changes during the immediate post-injection phase, and excellent knowledge of anatomy13–15. Typically in areas with terminal vascular circulation, the cutaneous vessels suffering may be more likely; in our cases it is one of the most vascularized areas of the face, the nasolabial folds15.\n\nIn this kind of complication, an early treatment is the best choice. The gold standard is prompt injection of hyaluronidase16.\n\nDe Lorenzi presents a protocol with high doses of hyaluronidase8, where the dosage of hyaluronidases is quantified on the basis of the regions of the face affected (for example: for the glabellar region 500 IU of hyaluronidase). We agree with the De Lorenzi that the use of elevated quantities of hyaluronides is required for the treatment of adverse events of vascular filler; however, we believe that a distribution per cm2 is more precise. More treatments has been described but there is no international standard protocol for the treatment of these complications.\n\nAs expressed by various Societies of Plastic and Aesthetic Surgery, to minimize the incidence of this type of damage it is essential to contact qualified and trained medical personnel, who follow modern international protocols17–19.\n\nThe strengths of our protocol is certainly the result with no residual mark of skin suffering that has been seen in our cases, while the major limitation is the necessary execution of the protocol within 72h from the damage.\n\n\nConclusion\n\nToday, the best solution for vascular damage is to categorically avoid dermal fillers treatments with non-medical and untrained personnel. This report aims to offer present a protocol for the early treatment of vascular damage after dermal fillers. Our early-implementation protocol has been compared with results presented in the literature and allowed us to avoid complications such as skin necrosis with permanent damage20,21.\n\n\nConsent\n\nWritten informed consent was obtained from the patients for the publication of this case report and any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAmerican Society of Plastic Surgeons: 2012 plastic surgery procedural statistics. Reference Source\n\nSalval A, Ciancio F, Margara A, et al.: Impending Facial Skin Necrosis and Ocular Involvement After Dermal Filler Injection: A Case Report. Aesthetic Plast Surg. 2017; 41(5): 1198–1201. PubMed Abstract | Publisher Full Text\n\nKim DY, Eom JS, Kim JY: Temporary Blindness After an Anterior Chamber Cosmetic Filler Injection. Aesthetic Plast Surg. 2015; 39(3): 428–430. PubMed Abstract | Publisher Full Text\n\nSchanz S, Schippert W, Ulmer A, et al.: Arterial embolization caused by injection of hyaluronic acid (Restylane). Br J Dermatol. 2002; 146(5): 928–9. PubMed Abstract | Publisher Full Text\n\nUrdiales-Gálvez F, Delgado NE, Figueiredo V, et al.: Treatment of Soft Tissue Filler Complications: Expert Consensus Recommendations. Aesthetic Plast Surg. 2018; 42(2): 498–510. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVanaman M, Fabi SG, Carruthers J: Complications in the Cosmetic Dermatology Patient: A Review and Our Experience (Part 1). Dermatol Surg. 2016; 42(1): 1–11. PubMed Abstract | Publisher Full Text\n\nFitzgerald R, Bertucci V, Sykes JM, et al.: Adverse Reactions to Injectable Fillers. Facial Plast Surg. 2016; 32(5): 532–555. PubMed Abstract | Publisher Full Text\n\nDeLorenzi C: New High Dose Pulsed Hyaluronidase Protocol for Hyaluronic Acid Filler Vascular Adverse Events. Aesthet Surg J. 2017; 37(7): 814–825. PubMed Abstract | Publisher Full Text\n\nBeleznay K, Humphrey S, Carruthers JD, et al.: Vascular compromise from soft tissue augmentation: experience with 12 cases and recommendations for optimal outcomes. J Clin Aesthet Dermatol. 2014; 7(9): 37–43. PubMed Abstract | Free Full Text\n\nRzany B, DeLorenzi C: Understanding, Avoiding, and Managing Severe Filler Complications. Plast Reconstr Surg. 2015; 136(5 Suppl): 196S–203S. PubMed Abstract | Publisher Full Text\n\nCasabona G: Blood Aspiration Test for Cosmetic Fillers to Prevent Accidental Intravascular Injection in the Face. Dermatol Surg. 2015; 41(7): 841–847. PubMed Abstract | Publisher Full Text\n\nColeman SR: Avoidance of arterial occlusion from injection of soft tissue fillers. Aesthet Surg J. 2002; 22(6): 555–557. PubMed Abstract | Publisher Full Text\n\nGoodman GJ, Roberts S, Callan P: Experience and Management of Intravascular Injection with Facial Fillers: Results of a Multinational Survey of Experienced Injectors. Aesthetic Plast Surg. 2016; 40(4): 549–555. PubMed Abstract | Publisher Full Text\n\nLazzeri D, Agostini T, Figus M, et al.: Blindness following cosmetic injections of the face. Plast Reconstr Surg. 2012; 129(4): 995–1012. PubMed Abstract | Publisher Full Text\n\nde Maio M, DeBoulle K, Braz A, et al.: Facial Assessment and Injection Guide for Botulinum Toxin and Injectable Hyaluronic Acid Fillers: Focus on the Midface. Plast Reconstr Surg. 2017; 140(4): 540e–550e. PubMed Abstract | Publisher Full Text\n\nCavallini M, Antonioli B, Gazzola R, et al.: Hyaluronidases for treating complications by hyaluronic acid dermal fillers: Evaluation of the effects on cell cultures and human skin. Eur J Plast Surg. 2013; 36(8): 477–484. Publisher Full Text\n\nThe British Association of Aesthetic Plastic Surgeons: BAAPS Consumer Safety Guidelines. Accessed February 2, 2016. Reference Source\n\nThe American Society for Aesthetic Plastic Surgery: Patient Safety Guidelines by ASAPS. Reference Source\n\nColombo G, Caregnato P, Stifanese R, et al.: A vast intranasal filler-induced granulomatous reaction: a case report. Aesthetic Plast Surg. 2010; 34(5): 660–663. PubMed Abstract | Publisher Full Text\n\nFeller-Heppt G, Haneke E, Heppt MV: Diagnosis and management of filler adverse effects: an algorithm. Facial Plast Surg. 2014; 30(6): 647–55. PubMed Abstract | Publisher Full Text\n\nLo Russo G, Spolveri F, Ciancio F, et al.: Mendeley: an easy way to manage, share, and synchronize papers and citations. Plast Reconstr Surg. 2013; 131(6): 946e–7e. PubMed Abstract"
}
|
[
{
"id": "38457",
"date": "03 Oct 2018",
"name": "Nicolò Bertozzi",
"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\nVery good written article about the author's experience with the use of hyaluronidase in preventing skin necrosis after dermal filler infiltration. Their protocol is clearly stated and the pictures are very representative of the clinical course of the two patients treated. Discussion could be more complete by citing further authors. Thanks for your effort.\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": "41307",
"date": "04 Dec 2018",
"name": "Gottfried Lemperle",
"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\nSorry to say: but these 2 cases are not representative for an outcome of any therapy; they would have been solved by themselves, i.e. by Nature within one week. Check your own publication (below) or google images: much worse superficial necroses and infections healed without obvious scarring. - My PC contains at least 10 serious cases from Brazil and elsewhere, where cosmeticians and even hairdressers are allowed to inject dermal fillers.\n\nThe reason for skin necrosis in the face is always intra-arterial injection: therefore, its prevention is using a cannula or better: moving the needle or a cannula back and forth during injection. The facial artery can easily be palpated from intraorally just above the mucosa and way beneath the nasolabial fold. To inject HA soo deeply shows the lack of knowledge of the anatomical layers of the face.\n\nHyaluronidase is a logical therapy (and the present Gold Standard, I know) - if it could be injected into the embolized artery. DeLorenzi´s experiments on blocked dead arteries in hyaluronidase are not convincing that this enzyme dissipates through the arterial wall from the outside. I just reviewed a manuscript from Beijing where similar experiments in a living rabbit ear showed slight recovery of the necrosis only when hyaluronidase was injected intra-arterially within the first 4 hours. It is difficult to demonstrate an effect of any therapy in emergencies.\n\nTo make it short, as long as there is no proof that hyaluronidase enters blocked arteries from the outside (why should it?) we should stick to an immediate physical procedure to try to spread the intra-arterial HA by retrograde scratching with a fingernail through the oral mucosa and emptying it thereby - or massive massaging of the blocked area in all directions hoping to distribute the HA to other branches. I have not done it - but it sounds logical to me.\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? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? No",
"responses": [
{
"c_id": "4306",
"date": "17 Dec 2018",
"name": "Francesco Ciancio",
"role": "Author Response",
"response": "It is necessary to clarify about the review of Gottfried Lemperle. We agree on the use of blunt cannulas and on infiltration techniques with continuous moviments, small quantities of filler and anything else procedure to reduce the risks of ischemic damage from filler infiltration.The reviewer affirms that “these 2 cases are not representative for an outcome of any therapy; they would have been solved by themselves, i.e. by Nature within one week” we do not agree with him.The signs of vascular complications after dermal fillers are described in the literature 1. In fact, the early signs are blisters (3 days), erithema, livedo reticularis (up to a few days) and then crusting, necrosis (after days 6), slough, and finally healing by secondary intention − a process that may take six weeks or more. Therefore, in our clinical opinion, the photos shown in the paper are indicative of early signs of vascular compromise from dermal fillers and require integrated early treatment. The presumption that the scar results would be the same even without treatment must be scientifically proven.We do not agree when he says \"Delorenzi's Experiments on Online Blonde Dead Arteries in Hyaluronidase Are Are not Convincing This Enzyme Dissipates Through The Arterial Wall From The Outside ... to Make It Short, As Long As there is not Proof That Hyaluronidase Enters Blocked Arteries from the Outside (Why Whyu Shted it?)\"2.In literature there are many papers that state the effectiveness of the use od hyaluronidases in the subcutaneous tissue, near the affected area. The skin damage may result from extrinsic obstruction of the vessels so the necrosis may also occur secondary to local edema or to occlusion of adjacent vessels, secondary to the hydrophilic properties of the product, motivated the extravascular use of hyaluronidases. Use of this enzyme around the vessels is finalized to reduce the extrinsic vessel’s obstruction by hyaluronic acid. Several studies showed that the injection of hyaluronidase, throughout the area around the vascular occlusion point, promote its intravascular penetration and facilitate removal of the HA that is obstructing the vessel3-4-5. De Lorenzi's experiment on sections of arteries and facial veins appears interesting and the concept of \"flooding\" the affected area deserves respect. Until there is no contrary evidence, it is a logical and appropriate rational. So we are in disagree with the reviewer. Urdiales-Gálvez F, Delgado NE, Figueiredo V, et al.: Treatment of Soft Tissue Filler Complications: Expert Consensus Recommendations. Aesthetic Plast Surg. 2018; 42(2): 498–510.
DeLorenzi C: Transarterial degradation of hyaluronic acid filler by hyaluronidase.Dermatol Surg. 2014; 40 (8): 832-41 DeLorenzi C: New High Dose Pulsed Hyaluronidase Protocol for Hyaluronic Acid Filler Vascular Adverse Events.Aesthet Surg J. 2017; 37 (7): 814-825 Hirsch RJ, Cohen JL, Carruthers JD (2007) Successful manage- ment of an unusual presentation of impending necrosis following a hyaluronic acid injection embolus and a proposed algorithm for management with hyaluronidase. Dermatol Surg 33(3):357–360 Bachmann F, Erdmann R, Hartmann V, Wiest L, Rzany B (2009) The spectrum of adverse reactions after treatment with injectable fillers in the glabellar region: results from the Injectable Filler Safety Study. Dermatol Surg 35(Suppl 2):1629–1634"
}
]
},
{
"id": "45979",
"date": "01 Apr 2019",
"name": "Robert J. Besaw",
"expertise": [
"Reviewer Expertise Clinical trials",
"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\nCiancio and colleagues present a new protocol for preventing skin necrosis for patients undergoing treatment of dermal fillers. As the use of dermal fillers become increasingly common, this could offer a new approach to mitigating potential serious adverse events.\nThis study offers an interesting new proof of concept with two cases that underwent the protocol. Ideally, the authors will next present a larger observational study to further support their concept and demonstrate the benefits of this regimen.\n\nTo make sure that others would be able to duplicate such a procedure, it's important to be specific. The authors should take the time to explain in more detail why each of the components is included in the regimen, i.e. what each piece is adding. Also, would recommend detailing when systemic corticosteroids should begin (prior to rest of the regimen? after?). If doses/timing is all done on a case-by-case basis, then this must be stated.\n\nOverall, nice outcomes in two cases from an interesting protocol. Will require a larger study to demonstrate if this protocol warrants more widespread uptake.\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? Partly",
"responses": [
{
"c_id": "4531",
"date": "03 Apr 2019",
"name": "Francesco Ciancio",
"role": "Author Response",
"response": "Revision:As recommended by the Review we added (in red) details of the dosage on the presented protocol, we have included the rationale of use for each treatment. We have added a paragraph in the section of the Protocol indicating the times of use of the drugs and when to begin treatment with each category of drugs."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1388
|
https://f1000research.com/articles/7-1956/v1
|
19 Dec 18
|
{
"type": "Research Article",
"title": "Correction of gene model annotations improves isoform abundance estimates: the example of ketohexokinase (Khk)",
"authors": [
"Christophe D. Chabbert",
"Tanja Eberhart",
"Ilaria Guccini",
"Wilhelm Krek",
"Werner J. Kovacs",
"Tanja Eberhart",
"Ilaria Guccini",
"Wilhelm Krek"
],
"abstract": "Next generation sequencing protocols such as RNA-seq have made the genome wide characterization of the transcriptome a crucial part of many research projects in biology. Analyses of the resulting data provide key information on gene expression and in certain cases on exon or isoform usage. The emergence of transcript quantification software such as Salmon has enabled researchers to efficiently estimate isoform and gene expressions across the genome while tremendously reducing the necessary computational power. Although overall gene expression estimations were shown to be accurate, isoform expression quantifications appear to be a more challenging task. Low expression levels and uneven or insufficient coverage were reported as potential explanations for inconsistent estimates. Here, through the example of the ketohexokinase (Khk) gene in mouse, we demonstrate that the use of an incorrect gene annotation can also result in erroneous isoform quantification results. Manual correction of the input Khk gene model provided a much more accurate estimation of relative Khk isoform expression when compared to quantitative PCR (qPCR measurements). In particular, removal of an unexpressed retained intron and a proper adjustment of the 5’ and 3’ untranslated regions both had a strong impact on the correction of erroneous estimates. Finally, we observed a better concordance in isoform quantification between datasets and sequencing strategies when relying on the newly generated Khk annotations. These results highlight the importance of accurate gene models and annotations for correct isoform quantification and reassert the need for orthogonal methods of estimation of isoform expression to confirm important findings.",
"keywords": [
"RNA-seq",
"quantification",
"gene expression",
"transcriptomics",
"Khk",
"Salmon",
"alternative splicing"
],
"content": "Introduction\n\nAccurate measurement of mRNA expression levels is a crucial component in many modern biological studies. Common and standardized techniques such as reverse transcription real-time quantitative PCR (RT-qPCR) have remained limited in throughput, only allowing measurements for a handful of genes at a time. The emergence of Next Generation Sequencing (NGS) based protocols such as RNA-seq has overcome this limitation and enabled researchers to profile mRNA expression at the genome wide level1–3. While such experiments are now routinely performed, the subsequent bioinformatics analysis and data interpretation still pose computational challenges. As sequencing reads are currently much shorter (usually 100bp – 150bp) than most isoforms, tailored approaches are necessary to study complex events such as splicing or isoform usage switch. In addition, low number of replicates per condition together with a high dynamic range in expression levels across the genome require appropriate statistical frameworks4–8.\n\nOne common approach consists in identifying significant changes in expression levels between two or more experimental conditions using gene-level counts9. Such counts are usually obtained from the alignment of sequencing reads to a reference genome or transcriptome when available, and a subsequent counting step during which reads are assigned to annotated genes based on their mapping locations. In the absence of a large number of biological replicates, the following statistical analysis usually requires a reliable estimation of count dispersions for each gene4,6–8. Statistical tools such as edgeR6, DESeq24 or limma5 offer a panel of solutions to this problem and have been shown to perform equally well in settings where few biological replicates are available10. Nevertheless, despite such progress in the differential gene expression (DGE) analysis, investigating changes of splice variants and the methods for their quantification continue to be an active field of research. Being able to accurately measure such changes is all the more crucial since they are connected to various biological processes and pathologies and may be used as biomarkers and therapy targets11.\n\nIndeed, although early approaches have followed a framework similar to gene level studies and used exon level counts to implement differential exon usage analysis8,12–14, results from such studies provide valuable insights but often remain difficult to interpret given the complexity of mammalian transcriptional units. In addition, the recent development of alignment-free transcript quantification methods has provided the possibility to quantify each individual transcript15–19. This task is often impossible to complete using traditional count approaches given the usually high level of sequence overlap between isoforms of the same gene20. Such approaches are computationally much less demanding and faster than alignment-based methods15. Moreover, they have been shown to overcome the difficulty of handling multi-mapped reads which can create biased results in count-based analysis21. Although transcript quantification estimates may be used to improve gene-level inference in DGE20, testing for changes in isoform usage between conditions remains a challenging task with most approaches focusing on junction and exon read counts rather than transcript quantifications themselves. DESeq2-tximport22, sleuth23 and DRIMSeq24 do make use of such quantifications, with DESeq2-tximport and sleuth incorporating estimates of inferential variances obtained during the quantification step. In contrast, DRIMSeq relies on a Dirichlet-multinomial model to estimate relative transcript usage and test for differential transcript usage.\n\nRegardless of the method used, several studies have now reported limitations and pitfalls associated with transcript abundance estimations20,25. Systematic errors in estimation may stem from sample-specific GC content biases26, which should be accounted for when comparing conditions. In addition, a systematic assessment of quantification performance on simulated datasets also revealed a weaker accuracy in transcript abundance estimates when compared to gene abundance estimates20. To explain such discrepancies, it has been suggested that certain transcript abundances cannot be reliably estimated from the data, in particular in cases where coverage is lacking in genomic regions allowing a distinction between transcripts20. To our knowledge, no systematic evaluation of the impact of these configurations has been conducted and detailed reports of such examples in real datasets are still missing.\n\nAdditionally, quantification tools rely on an input reference transcriptome to compute quantifications with the exception of Cufflinks17, casper27 and FlipFlop28, which may be used to assemble de-novo transcripts. These tools are therefore limited to current gene models made available in databases such as Ensembl or RefSeq and it is unclear whether these models properly recapitulate the actual complexity of transcriptional units. An assessment of the impact of erroneous or incomplete annotations on transcript quantifications is still missing. Additionally, meticulous examination of the concordance between transcript quantification and mRNA isoforms measured using of gene-tailored experimental method has not been undertaken.\n\nIn this study, we focus on the murine ketohexokinase (Khk) gene to better understand and evaluate the impact of genomic annotation on transcript quantifications. Khk, also known as fructokinase, is the first rate-limiting enzyme in the fructose metabolic pathway and catalyzes the conversion of fructose and ATP to fructose 1-phosphate (F1P) and ADP, respectively. Previous studies have shown that this gene predominantly expresses two usually exclusive isoforms, KhkA and KhkC that are generated via the specific excision of exon 3C and 3A, respectively29 (Figure 1A). With a greater affinity for fructose30, KhkC is thought to be responsible for the functional role of this gene in metabolism, in particular in liver where it is highly expressed31. Epidemiological and animal studies implicate overconsumption of fructose in the development of nonalcoholic fatty liver disease. While the physiological substrate of KhkA is unknown, several studies have highlighted the importance of Khk isoforms choice in the development of clear cell renal cell carcinoma (ccRCC), hepatocellular carcinoma (HCC)32, and pathological cardiac hypertrophy33. Given the clear importance of Khk isoforms expression in several disease settings, it is therefore crucial to accurately quantify these variants in order to understand relevant biological mechanisms.\n\nA – Khk gene model provided by the Ensembl and Genecode. Genomic coordinates are indicated on the top ribbon. Usual isoform names are mentioned next to the Ensembl ids. Murine Khk is thought to express 4 main isoforms, all protein coding (KhkA, KhkC and ENSMUST00000201571.3 and ENSMUST00000031053.14 termed Khk.Skip and KhkA.C for this study) and one isoform with a retained intron (Khk.RI). B – Normalized Khk expression levels across tissues in mouse (data from Li et al., 201734) using gene count tables as input. The liver, kidney and small intestine are clearly expressing Khk mRNA at higher levels compared with other tissues. C – Relative exon coverage of the Khk gene. Normalized exon counts are indicated in the central panel, while normalized gene expression counts are plotted to the right. Exons colored in black were called as differentially used across all considered tissues (adjusted p value < 0.001). KhkC and KhkA expression are mutually exclusive in each tissue, with KhkC strongly expressed in liver and kidney while KhkA is expressed in heart, lung and spleen.\n\nBy re-processing publicly available RNA-seq data34,35, we confirm that Khk isoforms are differentially expressed in various mouse tissues. Using DRIMSeq proportion estimations, we show that quantification of these isoforms as output by Salmon is biased by the presence of an annotated retained intron that is expressed at very low levels. We also highlight the importance of correct 3’ and 5’ UTR annotation to improve transcript quantification estimates and validate our computational findings by RT-qPCR. Finally, through the comparison of various datasets, we illustrate the importance of using correct annotations to avoid the emergence of discrepancies between library preparation protocols and datasets.\n\n\nResults\n\nIn order to assess tissue-specific expression patterns of Khk in mouse, we downloaded RNA-seq data generated from 14 different mouse tissues34. The availability of 4 biological replicates (2 males and 2 females) per tissue enabled us to conduct a differential gene expression analysis using standard, gene-count based analytical workflows. As previously described in gene specific studies36, we identified a strong tissue specific expression of Khk (adjusted p-value of 0 from DESeq2), with significantly higher expression levels in the liver, small intestine and kidney when compared to other tissues (Figure 1B; Extended Data Figure 1 A and B; Underlying data: Table 1). Interestingly, gender did not impact the overall Khk expression levels. We also sought to evaluate changes in relative exon usage for this gene using junctionSeq14, including both exon and junction counts in our analysis. Therefore, we selected five tissues (liver, spleen, lung, heart, kidney) with known variations in Khk isoform usage31–33,36 and different levels of overall expression. We clearly identified a preferential inclusion of exon 3C (Ensembl ID ENSMUSE00000186455) in liver and kidney, as previously described, while exon 3A (Ensembl ID ENSMUSE00001361691) is preferentially retained in heart, spleen and lung (p value < 0.001) (Figure 1C). This trend was also reflected in junctions spanning these exons (Underlying data: Table 2). Variations in 5’ UTR were also observed, most likely resulting from alternative transcription start site usage in liver and kidney. In summary, using traditional count-based methods, we confirmed the tissue-specificity of Khk expression and identified exons preferentially retained in some tissues.\n\nAs count-based methods might not always reflect the full complexity of splicing patterns and isoform diversity, we sought to quantify relative proportions of Khk isoforms in each tissue to fully capture the complexity of its expression patterns. The Genecode annotation together with the Salmon15 quantifier were used to obtain these estimations (Underlying data: Table 3). Interestingly, the quantification results showed that the annotated retained intron (Khk.RI) accounts for more than 15% of expressed isoforms in 8 tissues (Figure 2A). This trend was consistently observed in DRIMSeq24 proportion estimates (Figure 2A; Underlying data: Table 3) and the raw TPM (transcripts per million) values output by Salmon (Extended data: Figure 2). Nevertheless, examination of the coverage tracks generated from the alignment of the reads to the reference genome showed hardly any detectable expression of the transcript (Figure 2B; Extended data: Figure 3). This observation was also supported by the differential exon usage analysis which clearly revealed very low expression levels for the only Khk.RI-specific exonic region (Figure 1B; Extended data: Figure 4A). To experimentally confirm the absence of Khk.RI expression, we designed PCR primers amplifying fragments specific to this transcript (see Extended data: Figure 4B; Extended data: Table 1 for a list of primers used in the study) and used RT-qPCR and semiquantitative RT-PCR to measure its expression levels. Khk.RI could hardly be detected using this sensitive method (Extended data: Figure 4C and 4D) and comparison with the expression levels in a Khk-A/C-/- mouse model showed that it is hardly expressed in heart, kidney, liver, lung and spleen, whereas a product could be amplified using genomic DNA as template (Figure 2C). This experimental validation further demonstrates that the contribution of Khk.RI to the overall Khk expression level was overestimated during the quantification step. Taken together, these results suggest that the presence of non-expressed transcripts in the “raw” gene annotation may result in erroneous detection by a transcript quantification software.\n\nA – Estimated relative Khk isoform expression across all tissues in mouse. Proportions were output by DRIMseq using Salmon quantification as an input. B – RNA-seq coverage tracks and sashimi plots highlighting the absence of Khk.RI expression in Thymus, Spleen and Bone Marrow samples. Data obtained from all biological replicates were merged prior to plotting. C – Products derived from semiquantitative RT-PCR analysis on cDNAs prepared from total RNA of different mouse organs using the primers specific for Khk.RI. Genomic DNA isolated from the liver was used as positive and RNA isolated from organs of KHK-A/C knockout mice as negative control. (n = 5 and products from two representative mice are shown)\n\nSince the current genomic annotation did not reflect Khk isoform expression and introduced biases in quantifications, we manually removed the Khk.RI transcript (Ensembl ID ENSMUST00000200978.1) prior to the quantification step. Despite this adjustment, inspection of the junction reads and coverage tracks revealed discrepancies between quantification estimates and expected results for several tissues (Figure 3A and 3B). An example may be found examining the heart coverage tracks: while it is quite clear that the KhkC-specific exon 3C (Ensembl ID ENSMUSE00000186455) is hardly captured in comparison with exon3A (Ensembl ID ENSMUSE00001361691), quantification estimates yielded a score of 39.9% and 16.7% for KhkC and KhkA respectively (Underlying data: Table 3). Similarly, KhkC estimates were inflated in lung and spleen (14.9% and 18.6% to be compared with the absence of coverage of exon 3C), as were Khk.Skip estimates (32%) in liver (Underlying data: Table 3). Since such quantifications are relying on the reference transcripts provided during the indexing step15, we reasoned that incorrect transcript models might be the cause of the observed discrepancies and therefore compared coverage tracks and isoform models. We identified annotated differences in 5’ and 3’ end annotations between all isoforms which were not reflected on our coverage tracks (Figure 1C and Figure 3B). To investigate the impact of these variations on transcript quantifications, we manually updated Khk isoform annotations to provide an identical 3’ end to all isoforms (Underlying data: File 1 and 2) and re-estimated isoform proportions (Figure 3A, middle panel). Despite this adjustment, inspection of the proportion estimates still revealed erroneous estimations of isoform expression in particular in the case of liver where KhkA was detected in levels similar to KhkC. Finally, we manually modified Khk isoforms to provide an identical 5’ and 3’ end to all listed isoforms (Underlying data: File 3). Transcript quantification performed using this updated annotation yielded more concordant results when compared to coverage tracks and in light of previous reports, in particular for tissues such as liver31, small intestine37, heart33, spleen and lung (Figure 3A and 3B; Extended data: Figure 5). Interestingly, a substantial fraction of isoforms detected in kidney were still attributed to the Khk.Skip isoform while both junction count analysis and single gene studies reported a prevalence of KhkC36.\n\nA - Estimated relative Khk isoform expression across all tissues in mouse, using three different genomic annotations: a naive annotation after removal of the Khk.RI transcript, an annotation without Khk.RI and with adjusted 3’ UTR and an annotation without Khk.RI and adjusted 3’ and 5’ UTR. The choice of annotation greatly impacts the quantification results. B – RNA-seq coverage track and sashimi plots illustrating the predominance of different isoforms in spleen, lung, liver, heart and kidney. C – RT-qPCR analysis of total Khk expression in different mouse organs. Values are expressed as fold-change compared to the expression levels obtained for the heart, which was arbitrarily defined as 1. β-actin was used as the invariant reference gene. Data are mean ± SD (n = 5). D - RT-qPCR analysis of total Khk, KhkA, and KhkC expression in different mouse organs. Values are expressed as fold-change compared to the expression levels obtained for total Khk, which was arbitrarily defined as 1. Data are mean ± SD (n = 5).\n\nTo confirm the biological relevance of our newly estimated proportions, we designed primer pairs to specifically target Khk isoforms (see Extended data: Figure 6A and 6B; Extended data: Table 1) and evaluated their relative expression using RT-qPCR. The expression of total Khk was ~60-fold higher in liver and kidney compared to heart, lung, and spleen (Figure 3C), confirming previously described findings36. While we did not manage to achieve a reliable quantitative evaluation of Khk.Skip and KhkA.C expression alone, we accurately measured the expression of (KhkC + KhkA.C) and (KhkA + KhkA.C) in five mouse tissues and normalized it to the total Khk expression (Figure 3C and 3D). We thereby confirmed a strong prevalence of (KhkA + KhkA.C) expression in heart while (KhkC + KhkA.C) accounted for most of the expression measured in kidney and liver (Figure 3D). Therefore, we concluded that KhkA.C levels of expression were much lower than KhkA and KhkC in these three tissues and that the proportion estimates derived from our adjusted genomic annotation reflected the RT-qPCR measurements. The isoform measurements in lung and spleen highlighted low levels of KhkA, KhkC and KhkA.C compared to total Khk expression, strongly suggesting the prevalence of Khk.Skip expression, as observed on coverage tracks (Figure 3B) and in the proportion estimates derived from the updated genomic annotation (Figure 3A).\n\nAltogether, these findings demonstrate that UTR and more generally 5’ and 3’ end annotation may greatly influence transcript quantification results. The resulting biases lead to the identification of isoforms that can hardly be detected in biological samples, therefore highlighting the importance of inspecting results for any given gene of interest.\n\nWe next investigated whether such discrepancies could be observed when using different sequencing library strategies. To do so, we artificially created a single-end dataset by removing one read for each tissue sample. We also created a short-read dataset by trimming the remaining reads to only retain the first 50bp. In both cases, we aligned reads to the reference genome and independently quantified transcript expressions using Salmon as previously described. Regardless of the sequencing strategy considered, we observed a similar overestimation of the Khk.RI fraction as well as discrepancies between proportion estimates and junction counts (Extended data: Figure 7A and 7B). Both differences were corrected using the aforementioned manually curated annotations. We then compared proportion estimates between datasets for each annotation. Quite strikingly, we noted a much better agreement in transcript estimates using our manually modified annotation (Figure 4A–C). While the use of the “raw” annotation only resulted in a 0.56 correlation (Pearson) between estimates from the paired-end and 50bp single-end libraries, the use of an updated annotation resulted in a 0.97 correlation between both platforms. This trend was also observed between paired-end and single-end and between single-end and 50bp single-end respectively (Extended data: Figure 8A-F). To further evaluate the impact of annotations on estimate concordance across datasets, we downloaded RNA-seq data from another study profiling mRNA expression across 13 tissues35. We quantified isoform usage for each tissue and compared those estimates with the ones from the 50bp single-end dataset from Li et al. 201734 in order to avoid biases due to differences in read length. In total, 8 tissues could be compared between both studies. Quite strikingly, the agreement between both datasets was not high (Pearson correlation 0.72) when considering fractions derived using the original annotation (Figure 4D–F). While the removal of Khk.RI did further hinder reproducibility between both datasets, we unexpectedly observed a much stronger consistency of proportion estimates when using the manually updated annotation (Figure 4F, Pearson correlation 0.91). These results therefore further underscore the importance of appropriate annotations during transcript isoform quantifications. The use of erroneous gene models may further increase discrepancies between sequencing libraries and datasets as exemplified in the case of the Khk gene.\n\nA – Comparison of relative Khk isoforms expression estimates between the full length paired-end dataset from Li et al. 201734 and the short read, single-end dataset. The estimates were generated using the naive Ensembl annotation. B – Comparison of relative Khk isoforms expression estimates between the full length paired-end dataset from Li et al., 201734 and the short read, single-end dataset. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed. C – Comparison of relative Khk isoforms expression estimates between the full length paired-end dataset from Li et al., 201734 and the short read, single-end dataset. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed and the 5’ and 3’ UTR of other transcripts were all adjusted. D – Comparison of relative Khk isoforms expression estimates between the Li et al., 201734 dataset and the Söllner et al., 201735 dataset, using single-end, short (50bp) reads in each case. The estimates were generated using the naive Ensembl annotation. E – Comparison of relative Khk isoforms expression estimates between the Li et al., 201734 dataset and the Söllner et al. 20135 dataset, using single-end, short (50bp) reads in each case. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed. F – Comparison of relative Khk isoforms expression estimates between the Li et al., 201734 dataset and the Söllner et al. dataset, using single-end, short (50bp) reads in each case. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed and the 5’ and 3’ UTR of other transcripts were all adjusted.\n\n\nDiscussion\n\nGene and transcript quantifications are essential steps in many genomic studies where the characterization of gene expression patterns is of biological relevance. The recent development of quasi-mapping methods such as Salmon15 has drastically improved the computational speed of these quantifications steps while relying on reduced computational power. However, unlike more traditional and alignment-based methods, they strongly rely on the provided genomic annotation. Through the example of Khk gene expression in mouse, we describe the importance of using a properly curated annotation to avoid biases and erroneous isoform proportion estimates. We show that the inclusion of an annotated, yet not detected retained intron (Khk.RI) was sufficient to wrongly predict isoform usage in several tissues. We also found that differences in 5’ and 3’ end annotations may result in inaccurate transcript quantifications. Manual adjustment of such differences resulted in a better agreement between isoform proportion estimates, coverage tracks inspections, junction counts and qPCR results in at least 3 tissues. Finally, comparison of these estimates across different datasets and sequencing library strategies revealed that the use of a corrected annotation strongly improves the reproducibility of estimations between each dataset.\n\nThe use of gene or exon level counts to assess differences in gene expression or exon usage between conditions has been described as a robust method by several independent studies8. Recent reports20 have also emphasized the reliability of gene-level quantification estimates and their biological relevance. It was therefore reassuring to observe a very good agreement between both methods when assessing the tissue specificity of Khk expression in this dataset. Identification of higher levels of expression in liver, small intestine and kidneys reflects previously described findings36. We thereby provide further evidence of the reliability of gene quantification, albeit at the single gene level. Additionally, the variations observed in expression levels across tissues together with the previously reported alternative splicing events make Khk an ideal gene to study performance of bioinformatics tools.\n\nWe identified strong discrepancies between proportion estimates and coverage tracks when quantifying Khk isoforms while relying on the original Genecode/Ensembl annotation. In particular, the annotated retained intron Khk.RI (ENSMUST00000200978.1) was identified as a predominantly expressed isoform in several tissues while hardly any read could be mapped to the genomic region specific to this isoform. qPCR validations confirmed the extremely low levels of Khk.RI in 5 mouse tissues. Previous work relying mostly on simulated data showed that in the case of lowly expressed genes, transcript-level estimates lack accuracy20. However, through the instance of Khk, we provide a concrete example of a strong overestimation in transcript quantification and show that this is not limited to tissues with very low expression of the corresponding gene.\n\nDifferential exon usage analysis clearly revealed, as expected, a preferential usage of either exon 3A (Ensembl ID ENSMUSE00001361691) or 3C (Ensembl ID ENSMUSE00000186455) in various tissues36. The discovery of a potential change in 5’ transcription start site, while not previously described for Khk, further underscores the importance of alternative start and termination sites in transcript isoform diversification in mammals38. Interestingly, inspection of the usage of other exons revealed that these new start sites are most likely specific to the retention of exon 3A or 3C, therefore suggesting that the determination of the isoform choice for Khk could be achieved solely by considering junction and exon counts in this region.\n\nThis idiosyncrasy was further confirmed by the various isoform proportion estimations performed using updated annotations of the Khk gene. Estimations reflected experimental measurements, junction counts and coverage tracks only when both 5’ and 3’ end annotations were harmonized across all annotated isoforms. Importantly, when using the naive Gencode annotation, Salmon and DRIMseq failed to reliably quantify isoform proportions. Such misestimations are likely to be observed in other genes for which current annotations are either limited or inaccurately reflect experimental measurements.\n\nDuring the preparation of this manuscript, a preprint from Soneson et al. reported a similar observation and proposed the creation of a new index to flag such problematic genes39. While the current manuscript strongly emphasizes the role of 3’ UTRs in the emergence of estimation biases, we could pinpoint at least one example where 5’ UTRs play a similar role in the issue. The use of the JCC (Junction Coverage Compatibility) score introduced by Soneson et al. will be greatly useful to prevent misinterpretation of transcriptomics studies in the future but will tie quantifications to the results of computationally demanding alignment methods. Improvement of current genomic annotations might ultimately offer an alternative as they will allow for the sole use of fast quantification algorithms.\n\nUsing an additional dataset from Söllner et al., 201735 and in-silico single-end and short single-end datasets from Li, B et al. 201734, we show that such updated annotations have the potential to reduce discrepancies between methods and experiments. While this is only exemplified at the level of the Khk gene, it is very likely that other instances will emerge as new metrics such as JCC will enable scientists to flag problematic genes. This study exclusively focuses on the use of Salmon as a quantification software and DRIMSeq to estimate relative proportions, in particular as recent reports have suggested that most quantification pipelines might perform similarly25. However, it will be interesting to assess whether similar estimation biases to the ones reported here may arise with different tools. Results from Soneson et al. indicate that this might be the case, at least for a group of human genes39.\n\nFinally, the results presented in our study will provide a valuable resource to the scientific community investigating the role of fructose metabolism and Khk in mammals. As each Khk isoform might harbor different functions, the complete mapping of their usage across tissues will help further pinpoint their role in different biological contexts. Our analysis was conducted on mouse tissues but further exploration of the results presented in Reyes et al. 201738 also showed that exons 3A and 3C of KHK are selectively included in human tissues, across individuals. Refining our understanding of these expression patterns in human will be critical in particular as the KHK protein product has already been identified as a promising target in the treatment of non-alcoholic steato-hepatitis (NASH)40. This study provides important background information to improve the results of the transcriptomic work that might therefore be necessary in the future.\n\n\nMethods\n\nKhkA/C-/- mice, which are of C57BL/6 background and are lacking both ketohexokinase-A and ketohexokinase-C, were obtained from R. Johnson (University of Colorado). C57BL/6J and Khk-A/C-/- mice were housed in a pathogen-free facility at the ETH Phenomics Center (EPIC) under standard conditions (12 h light and 12 h dark cycle) with free access to food and water. 3 female and 2 male C57BL/6J mice and 2 male Khk-A/C-/- mice were euthanized with CO2 at the age of 6 weeks and heart, kidney, liver, lung, and spleen were subsequently removed and shock-frozen in liquid nitrogen. The mice did not suffer during the euthanasia with CO2; the mice were placed into a chamber that contained room air and then CO2 was gradually introduced with no more than 6 psi to displace at least 20% of the chamber volume per minute. All protocols for animal use and experiments were reviewed and approved by the Veterinary Office of Zurich (Switzerland).\n\nTotal RNA was prepared from frozen tissues with RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with DNase I to remove traces of DNA. First-strand complementary DNA (cDNA) was synthesized with random hexamer primers using the High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368813; Applied Biosystems). Quantitative reverse transcription PCR (RT-qPCR) was performed on a Roche LightCycler 480 in duplicates using 10 ng cDNA and the 2x KAPA SYBR FAST qPCR Master Mix LC480 (Sigma). Thermal cycling was carried out with a 5 min denaturation step at 95 °C, followed by 45 three-step cycles: 10 sec at 95 °C, 10 sec at 60 °C, and 10 sec at 72 °C. Finally, melt curve analysis was carried out to confirm the specific amplification of a target gene and absence of primer dimers. Relative mRNA amount was calculated using the comparative threshold cycle (CT) method. β-actin was used as the invariant reference gene. The PCR amplification efficiency of RT-qPCR primer sets was determined with serial dilutions of liver cDNAs and was similar for all primer sets. Semiquantitative RT-PCR was performed using Phusion High-Fidelity DNA Polymerase (New England Biolabs) and the following 3-step amplification protocol: 30 sec at 98 °C (denaturation), 30 cycles of 10 sec at 98 °C, 30 sec at 63 °C, and 40 sec at 72 °C, and a final elongation step for 5 min at 72 °C. PCR products were evaluated after gel-electrophoresis. Primer sequences are listed in Extended data: Table 1.\n\nA fasta file containing all manually modified Khk transcripts was created. All exonic sequences used to modify the gene model were downloaded from the Ensembl website. A fasta file containing nucleotide sequences of all transcripts from the Genecode M14 annotation was loaded into R using the Biostrings v 2.46.0 package. All original Khk transcripts were removed from the DNAStringSet object and the updated transcripts were then added. The resulting annotation was written to a fasta file to generate Salmon indexes (see following sections for more details).\n\nFastq files from Li et al., 201734 and Söllner et al., 201735 were downloaded from SRA using the sra-tools software v2.7.041. As they only provided two replicates (instead of 4), we excluded the testis and ovary samples from Li et al., 201734. Additionally, due to very low library complexities, we also excluded the pancreatic samples from Söllner et al., 201735. Reads were aligned to the M14_GRCm38.p5 reference genome using STAR 2.4.2a42 together with the Genecode M14 annotation (STAR was run with default parameters). The search for novel junctions was allowed during the mapping step. Gene, exon and junction level read counts were generated using the QoRTs software v1.2.4243 after excluding reads with multiple alignments (MAPK score less than 255). All workflows were orchestrated using Snakemake v 3.13.344.\n\nSalmon 0.9.115 was used for transcript quantifications. Indexes were built from each fasta files using the default quasi-mapping mode and a k-mer of length 31 as recommended in the software documentation. Transcript isoforms were quantified using the default VEBM algorithm. Library types were inferred by the software. Sequence and GC bias corrections were performed during each quantification (--seqBias and –gcBias options) and 100 bootstraps were run to estimate the variance of abundance estimates.\n\nGene count tables were loaded into R (v 3.4.1) as a DESeq24 object to conduct differential gene expression analysis. For the purpose of differential gene expression analysis, we only retained features labelled as “gene” in the Gencode annotation and of type protein_coding, antisense, sense_intronic, 3prime_overlapping_ncRNA, sense_overlapping or non_coding in order to exclude transcription products requiring specific library preparations to be accurately measured. Genes with very low counts were excluded from downstream analysis: the threshold was set at 50 mapped reads across all samples, corresponding to less than one read per sample on average. Estimated size factors were used to correct for differences in library size. Following the standard DESeq2 workflow45, changes in gene expression were modelled using a variable accounting for differences in tissue of origin for each sample. A Likelihood Ratio Test (“LRT” option in DESeq2) was performed to compare this model to a reduced model consisting of only an intercept. Results were extracted with the DESeq2 results function and multiple testing correction was performed using the Benjamini Hochberg procedure46.\n\nExon and junction count tables from QoRTs were loaded in R (v 3.4.1) using the junctionSeq (v 1.8.0) package14. Changes in exon usage were modelled using the tissue of origin as a main variable. Size factors and dispersions were estimated using default parameters (options “byGenes” and “advanced” respectively, see the JunctionSeq vignette for more details). Dispersion function fits, test for differential usage and estimation of effect sizes were also run using default parameters. Final test results were extracted using the writeCompleteResults function. Feature-level p values were adjusted using the Benjamini Hochberg procedure46. Only features with an adjusted p-value below 0.05 were retained.\n\nTranscript isoform quantifications from Salmon were loaded into R (v 3.5.1) using the tximport package20. Relative isoform proportions and differential transcript usage were then modelled using a Dirichlet-multinomial model as described in Nowicka et al., 201624 and implemented in the DRIMSeq package (v 1.6.0). Model precisions were estimated by the dmPrecision function run with default parameters, except for the search grid ranges which were set to -15 and 15. For each sample, feature proportions were then computed using the dmFit function (default parameters).\n\nGenomic annotations and coverage tracks were plotted using the Gviz package (v 1.22.3)47. Data from all available biological replicates were pooled together prior to plotting each track. Results from differential exon and junction usage were visualized using junctionSeq. Other plots were generated with the ggplot2 package.\n\n\nData availability\n\nFastq files from Li et al., 201734 are available from: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA375882\n\nFastq files from Söllner et al., 201735 are available from: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6081/\n\nScripts used to generate the analysis presented in this paper are freely and publicly available on Github: https://github.com/chbtchris/Khk_quantifications (Archived scripts: http://doi.org/10.5281/zenodo.197507348).\n\nAll underlying data are available at: https://doi.org/10.17605/OSF.IO/9E3BD49. Data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication). Files available are as follows:\n\nTable 1: Normalized Khk counts for each sample (and therefore tissue) considered.\n\nTable 2: Normalized counts for each exonic regions and tissue group output by junctionSeq.\n\nTable 3: Relative Khk isoform expression across all tissues using the different genomic annotations.\n\nFile 1: Fasta file containing the sequences of Khk transcript isoforms downloaded from Genecode.\n\nFile 2: Fasta file containing the sequences of Khk transcript isoforms after removal of the Khk.RI isoform and adjustment of the 3’ UTR for all remaining transcripts.\n\nFile 3: Fasta file containing the sequences of Khk transcript isoforms after removal of the Khk.RI isoform and adjustment of the 5’ and 3’ UTR for all remaining transcripts.\n\nAll extended data are available at: https://doi.org/10.17605/OSF.IO/9E3BD49. Data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication). Files available are as follows:\n\nTable 1: List of qPCR primers used in this study\n\nFigure 1: A - Normalized Khk expression levels across tissues in mouse (data from Li et al. 201734) using transcript abundance estimates from Salmon as input. Similar to observations made using raw counts, the liver, kidney and small intestine are clearly expressing Khk mRNA at higher levels compared to other tissues. B – Comparison of gene expression estimates using count tables and transcript abundance estimates.\n\nFigure 2: Transcript Per Million (TPM) values for each annotated isoform of the Khk gene as returned by Salmon. The naive Ensembl annotation was used to estimate the abundances.\n\nFigure 3: RNA-seq coverage tracks and sashimi plots highlighting the absence of Khk.RI expression in all the tissues investigated in this study. Data obtained from all biological replicates were merged prior to plotting.\n\nFigure 4: A – Normalized fractions of reads mapped to all exonic region overlapping the Khk.RI transcript. E12, which is the only Khk.RI specific region, clearly show a reduced coverage when compared to other regions. Exonic regions were determined using the junctionSeq package and their associated genomic coordinates are available in Supplementary Table 2. B – Schematic representing the location of Khk.RI specific primer targets. C – Amplification plots of RT-qPCR analysis of Khk.RI expression in different mouse organs (n = 5 mice). Note that the Ct or threshold cycle value at which the fluorescence generated within a reaction crosses the threshold, a numerical value assigned for each run reflecting a statistically significant point above the calculated baseline, is very high (> 40) and can be considered as noise. D – Melt curves from RT-qPCR analysis of Khk.RI expression in different mouse organs.\n\nFigure 5: RNA-seq coverage track and sashimi plots illustrating the predominance of different isoforms in each tissue inspected in this study. Data obtained from all biological replicates were merged prior to plotting.\n\nFigure 6: A – Schematic representing the location of Khk.A, KhkC, KhkA.C and total Khk specific primer targets. B – Amplification plots of RT-qPCR analysis of total Khk, KhkC, and KhkA expression in different mouse organs (n = 5 mice). C – Melt curves from RT-qPCR analysis of total Khk, KhkC, and KhkA expression in different mouse organs.\n\nFigure 7: A - Estimated relative Khk isoform expression across all tissues in mouse, using the single-end dataset created from Li et al., 201734. Three different genomic annotations were considered: a naive annotation, a naive annotation after removal of the Khk.RI transcript, and an annotation without Khk.RI and with adjusted 3’ and 5’ UTR. The choice of annotation greatly impacts the quantification results. B - Estimated relative Khk isoform expression across all tissues in mouse, using the 50bp single-end dataset created from Li et al., 201734. Three different genomic annotations were considered: a naive annotation, a naive annotation after removal of the Khk.RI transcript, and an annotation without Khk.RI and with adjusted 3’ and 5’ UTR. The choice of annotation greatly impacts the quantification results.\n\nFigure 8: A – Comparison of relative Khk isoforms expression estimates between the full length paired-end dataset from Li et al., 201734 and the full length, single-end dataset. The estimates were generated using the naive Ensembl annotation. B – Comparison of relative Khk isoforms expression estimates between the full length paired-end dataset from Li et al., 201734 and the full length, single-end dataset. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed. C – Comparison of relative Khk isoforms expression estimates between the full length paired-end dataset from Li et al., 201734 and the full length, single-end dataset. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed and the 5’ and 3’ UTR of other transcripts were all adjusted. D – Comparison of relative Khk isoforms expression estimates between the full length single-end dataset generated from Li et al., 201734 and the short read, single-end dataset. The estimates were generated using the naive Ensembl annotation. E – Comparison of relative Khk isoforms expression estimates between the full length single-end dataset generated from Li et al., 201734 and the short read, single-end dataset. The estimates were generated using the modified Ensembl annotation where the Khk.RI transcript has been removed. F – Comparison of relative Khk isoforms expression estimates between the full length single-end dataset generated from Li et al., 201734 and the short read, single-end dataset. The estimates were generated using the modified Ensembl annotations where the Khk.RI transcript has been removed and the 5’ and 3’ UTR of other transcripts were adjusted.",
"appendix": "Grant information\n\nThis work was supported in part by a donation from Dr. Walter and Edith Fischli to W.K.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the whole Krek Laboratory for useful discussions and comments on this manuscript. We also would like to thank Malgorzata Nowicka for her help in getting the DRIMSeq package to run. Finally, we would like to thank Alejandro Reyes for helpful discussions.\n\n\nReferences\n\nCloonan N, Forrest AR, Kolle G, et al.: Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods. 2008; 5(7): 613–619. PubMed Abstract | Publisher Full Text\n\nMiyoshi T, Kanoh J, Saito M, et al.: Fission yeast Pot1-Tpp1 protects telomeres and regulates telomere length. Science. 2008; 320(5881): 1341–1344. 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}
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[
{
"id": "42141",
"date": "08 Jan 2019",
"name": "Patrick K. Kimes",
"expertise": [
"Reviewer Expertise Bioinformatics",
"Statistical Genomics",
"Statistical 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\nUsing the example of murine ketohexokinase (Khk), the authors present an analysis of the limitations of a current alignment-free approach (Salmon) to isoform-level quantification using RNA-seq data. In particular, using publicly available data sets, primarily from the Mouse BodyMap project 1, the authors show that transcript quantification using Salmon is highly sensitive to the specified reference transcriptome. These results are corroborated using qRT-PCR measurements of relative isoform abundance across several mouse tissues included in the study.\n\nThe authors apply two approaches to modify the commonly used GENCODE annotations for Khk to obtain improved estimates of isoform abundance using Salmon. First, the authors consider removing an annotated transcript, Khk.RI, with low support in unique exonic regions. Second, the authors modify the original annotations by trimming all 5' and 3' differences between isoforms. The authors show that with these two procedures, the resulting isoform estimates are more consistent across library protocols and across experiments - comparing against data from a separate study of mouse tissues 2. The advantage of removing weakly supported isoforms is in line with previous work which showed the benefits of prefiltering isoforms for false discovery rate control in studies of differential transcript usage 3.\nThe conclusion is the need for \"manual curation\" of transcript annotations to improve quantification by current alignment-free approaches, and no general approaches are provided for improving all annotations. This is fine, as providing general guidelines (aside from careful review of isoform annotations) does not appear to be the purpose of this article.\nOverall, the paper provides an interesting and detailed analysis of a single gene that complements the growing literature on challenges in isoform-level quantification with RNA-seq. However, a few issues should be addressed before the paper and the analysis can be considered complete. These issues and a few other minor points are described below.\nMajor Issues\nIt appears that all gene-level analyses were carried out using gene count tables obtained using the QoRTs software, presumably based on the original (\"naive\") GENCODE transcript annotations. However, as one of the references cited by the authors points out 4, isoform-level quantification provides improved estimates of gene expression over simple count-based approaches. I am curious to see how the Salmon-based estimates (using tximport) of gene-level expression 1) compare with the QoRTs count tables, and 2) change with modifications to the transcript annotations. While the modified annotations appear to improve isoform-level estimates, it is also important to verify that they do not reduce the accuracy of gene-level estimates. This would further confirm the \"reliability of gene expression\", as described by the authors. In the analysis of Salmon-based isoform quantification results, the authors claim that \"inspection of the junction reads and coverage tracks revealed discrepancies between quantification estimates and expected results for several tissues.\" How were the expected results determined? From the text, this appears to be based on visual inspection of the coverage plots. However, per-base coverage can be tricky to visually interpret due to biases in RNA-seq data, e.g. sequence specific bias and fragment-level GC bias 5. Ideally, the definition of \"expected results\" should be made more concrete, e.g. using a metric such as the expected bias-corrected junction coverage as in Soneson et al., (2018) 6 or quantification of exon coverage as in Figure 1C. Regardless, the wording should be updated to more clearly state what constitutes \"expected results\" and how they were determined. While the authors claim that \"differences in 5' and 3' end annotations between all isoforms ... were not reflected on our coverage tracks,\" this does not appear to be true. In fact, there appears to be a clear difference in TSS coverage between tissues (Figure 3B). Notably, differences in 5' coverage appear to correspond to differences in coverage of the Khk.Skip splice junction (in agreement with GENCODE annotations). Additionally, clear TSS coverage differences were also observed and noted in the exon-level count analysis (Figure 1C), further supporting the presence and importance of TSS differences. The problem appears to be a mis-annotation of the 5' start site for the KhkC isoform (see Figure 1C where liver and kidney, two tissues with high KhkC preference according to RT-qPCR analysis, show significantly lower coverage in alternate start sites E01-E03). In light of these observations, the decision to completely trim all 5' and 3' differences seems rather extreme, and referring to the trimmed annotations as a \"correction\" of the gene models may not be accurate. Especially since such a trimming would result in the complete loss of the differential 5' behavior discovered earlier in the manuscript. This is particularly important as others have shown that start and termination site differences are the primary sources of isoform differences across human tissues 7. More justification and careful discussion of the limitations of this general trimming procedure are needed. Rather than a \"correction\", this appears to be a particularly aggressive modification that works in this setting because all isoforms differ by more than just the trimmed 5' and 3' regions (and not because \"differences were not reflected on our coverage tracks\", as stated in the text). In modifying the GENCODE annotations, the authors use two steps. First, the removal of unexpressed transcript annotations, and second, the trimming of 5' and 3' differences. Figure 4D-F shows that simply removing the Khk.RI isoform from the annotations actually reduces the agreement between the two data sets, while additionally trimming 5' and 3' ends appears to greatly improve agreement. Have the authors considered how estimates change by applying the 5' and 3' trimming procedure without removing the Khk.RI transcript from the annotations? From the current analysis, it is unclear how much removing Khk.RI is improving the final result (with trimming), because these changes are not additive. While details are included for the Khk A/C-/- negative control mice, details on the non-control mouse tissue samples used for RT-PCR and qRT-PCR analyses are missing. How were these samples obtained and matched with the publicly available Mouse BodyMap RNA-seq data? This should be described in the Methods section.\n\nMinor Issues\nThe authors claim that alignment-free transcript quantification methods have \"provided the possibility to quantify each individual transcript\", and that \"this task is often impossible to complete using traditional count approaches.\" However, several methods for transcript quantification predate alignment-free methods, including some referenced later in the same section (Cufflinks, casper, FlipFlop), among others (RSEM, eXpress). It is also unclear what is meant by \"often impossible.\" The wording in this section should be clarified. It is unclear what \"one common approach\" is referencing at the beginning of the second paragraph describing differential gene expression (DGE) analysis. This is particularly jarring as the previous paragraph ends with noting the challenges of studying \"complex events such as splicing or isoform usage switch\". DGE analysis does not address these questions. The authors claim that \"DESeq2-tximport and sleuth [incorporate] estimates of inferential variances obtained during the quantification\". While this is true of sleuth, I do not believe this is true of DESeq2-tximport. If it is, an appropriate reference should be added (none of the currently cited references describe this feature). Additionally, the reference linked to DESeq2-tximport (reference 22 in the article) 8 describes benchmarking several DGE methods to scRNA-seq data, and is a primary source for neither DESeq2 nor tximport. The more relevant reference would seem to be (reference 20 in the article) 4 which describes the tximport software package. It is unclear what is being referred to by \"these configurations\", when claiming \"no systematic evaluation of the impact of these configurations has been conducted.\" The figure referenced in \".. hardly any detectable expression of the transcript (Figure 2B ...\", should be \"Figure 2C\". It would also be helpful if the exon ID from the figure (E012) was also included in the text for easier reference. Description and axes of Figure 3C,D should be corrected to reflect the fact that KhkA and KhkC estimates are actually (KhkA + KhkA.C) and (KhkC + KhkA.C) estimates, as described in the text. In the Results section, \"junctionSeq\" should be stylized \"JunctionSeq\" for consistency. Throughout, \"Genecode\" is probably meant to be \"GENCODE\" (or \"Gencode\").\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "4488",
"date": "03 Apr 2019",
"name": "Christophe Chabbert",
"role": "Author Response",
"response": "We thank all the reviewers for their insightful comments and suggestions which have helped improve the quality of the manuscript. The main changes are summarized in the “Amendments from Version 1” section describing the new version of the manuscript. We will address the specific comments in this section. Major Point 1Thank you for making this suggestion. In order to address this point, we have used tximport to compute Khk expression levels from salmon estimates generated using the naive and all modified annotations (including the new one added to this version, see major comment 4). A comparison between the naïve annotation estimates and QoRTs counts is provided in the extended data, Figure 1B. A comparison between estimates from all tested annotations is now presented in the extended data, Figure 7 A and B. Examination of the tissue expression patterns of Khk (Panel A) and the correlation between gene level estimates clearly show a very good agreement between all annotations, thereby suggesting that the modifications have little impact on these estimates. We also verified that the very high tissue specificity of Khk in every case (adjusted p-value of 0 with DESeq2, following the similar analytical framework as described in the first version of the manuscript).In order to clarify this point, we have added the following statement in the result section: “These results were all confirmed using the gene level estimates based on Salmon quantifications (Extended Data Figure 1A and B). In particular, patterns of Khk expression across tissues were highly concordant between count-based and Salmon estimates.” “Finally, we evaluated whether the changes brought to the Khk transcript annotations affected the estimations of overall Khk expression levels. Gene level estimates obtained using quantifications based on the 3 modified annotations were strongly correlated (Pearson, r > 0.99) with estimates derived from the naïve GENCODE annotation (Extended data: Figure 7A). In addition, the high tissue specificity of Khk expression was observed in all cases, with identical expression patterns between annotations (Figure 1A, Extended data: Figure 7B).” Major Point 2The expected results were determined using an inspection of the coverage tracks, the normalized exon coverage results obtained QoRTs and DEXSeq and previous reports from single gene studies. We have modified the wording in this section: “Despite this adjustment, inspection of the junction reads, coverage tracks, and normalised exon and junction counts derived from QoRTs (Figure 1C) revealed discrepancies between quantification estimates and results derived from alignment-based methods (Figure 3A and 3B). These quantification estimates also did not reflect the observations made by previous reports focusing on the characterisation of Khk expression patterns36. An example of such discrepancy may be found examining the heart coverage tracks” Major Point 3Thank you for pointing to this lack of clarity in the manuscript. It is indeed not the objective of this manuscript to suggest that a trimming or a standard homogenisation of the UTRs of all annotated transcripts of a gene will improve quantification results. Such an update of the annotation is possible because all Khk isoforms can be detected unambiguously just by considering the exclusion or inclusion levels of exon 3A and 3C (as partially stated in the discussion section). We have now made this clearer in the result section as well and explicitly mentioned this idiosyncrasy of the Khk gene model. We have also expanded the discussion section to elaborate on the limitations of this method for cases where UTRs are the main source of variability between isoforms of the same gene.Finally, we would like to emphasize that the term correction is never used in this result section. In the title and abstract of this work, it refers primarily to the removal of the Khk.RI which is indeed a correction as this transcript expression levels remain below the threshold of detectability in the highly sensitive RT-qPCR assays. The newly modified result section now reads as follows: “Since such quantifications are relying on the reference transcripts provided during the indexing step 15 , we reasoned that incorrect transcript models might be the cause of the observed discrepancies and therefore compared coverage tracks, normalized exon counts and isoform models. We identified annotated differences in 3’ end annotations between all isoforms which were not reflected on our coverage tracks (Figure 1C and Figure 3B). As Khk isoforms can be identified unambiguously based on the exclusion patterns of the exons 3A and 3C and regardless of differences in UTRs, we could investigate the impact of these UTR variations on transcript quantifications. We therefore manually updated Khk isoform annotations to provide an identical 3’ end to all isoforms (Underlying data: File 1 and 2) and re-estimated isoform proportions (Figure 3A, middle panel). Despite this adjustment, inspection of the proportion estimates still revealed erroneous estimations of isoform expression in particular in the case of liver where KhkA was detected in levels similar to KhkC. Further examination of the results revealed that, while some differences in 5’ end coverage in the dataset were concordant with the current gene annotation, they were not always reflected in the gene model (Figure 1C and Figure 3A, Heart, Lung and Spleen 5’ UTR coverage). Following a similar approach to the one described earlier for 3’ UTRs, we finally manually modified Khk isoforms to provide an identical 5’ and 3’ end to all listed isoforms (Underlying data: File 3).” The updated discussion section reads as follows: “However, systematic harmonization of all UTRs across annotated transcripts might not be a general approach, especially in cases when such differences are reflecting tissue specific expression patterns” Major Point 4To address this point, we have generated an additional annotation where Khk.RI is retained while all other Khk annotated transcripts have identical 5’ and 3’ UTRs. This annotation was used to estimate Khk isoform proportions in the Li et al (using the paired end and 50bp single-end data) and the Söllner et al datasets. We observed that implementing this new UTR model was not sufficient to remove the Khk.RI estimation bias in the paired-end data (updated Figure 3A). This bias was still particularly important in the bone marrow and spleen datasets where Khk.RI was estimated to account for more that 20% of the overall gene expression.Comparison of the estimates between the paired-end dataset and the 50bp dataset derived from Li et al showed that this modification was however more beneficial than the removal of Khk.RI (Pearson correlation of 0.86 as opposed to 0.79 when Khk.RI is removed). Nevertheless, the best agreement between both libraries was still obtained after removal of Khk.RI and extension of the UTRs (Pearson correlation of 0.97).Finally, comparison of the estimates of the Li et al and Söllner et al datasets revealed that extension of the UTRs (Pearson correlation of 0.73) was more beneficial than Khk.RI removal (Pearson correlation of 0.46) but provided hardly any improvement over the naïve GENCODE annotations (Pearson correlation of 0.73). The complete set of modifications including Khk.RI removal and homogenization of UTR was required to reach higher concordance between both datasets (Pearson correlation 0.91).Overall, these observations suggest that both modifications are indeed needed to improve Khk transcript quantification results in the datasets considered in this study.We have updated main Figure 3, main Figure 4 and the underlying data table 3 to report these findings. The result and discussion section of the article have also been modified accordingly: “Additionally, we computed the relative Khk isoform usage using a new annotation with identical 5’ and 3’ end for all isoforms except Khk.RI which was retained as such in the gene model (Figure 3A). This modification was not sufficient to remove Khk.RI estimation biases, with the retained intron predicted to erroneously account for 20% of the overall gene expression in bone marrow or spleen. We therefore confirmed the impact and importance of both Khk.RI and UTRs annotations on isoform expression estimates for that gene.” “Both the removal of Khk.RI and UTR adjustments were necessary to reach this concordance between profiling methods.” Major Point 5ll mice used in this study, C57BL/6J control and KhkA/C -/- mice, were treated in a similar fashion and tissue were harvested on the same day. Additionally, RNA extraction, cDNA synthesis and gene expression analysis via RT-qPCR and semiquantitative RT-PCR were performed within the same experimental run. Differences in mRNA expression for Khk and Khk isoforms were evaluated via normalization to the internal reference gene β-actin for each sample. The wording in the methods section of the article has been modified accordingly:“C57BL/6J were obtained from The Jackson Laboratory while KhkA/C -/- mice, which are of C57BL/6 background and are lacking both ketohexokinase-A and ketohexokinase-C, were obtained from R. Johnson (University of Colorado) and used as negative control. All mice were housed in a pathogen-free facility at the ETH Phenomics Center (EPIC) under standard conditions (12 h light and 12 h dark cycle) with free access to food and water.”“Total RNA from C57BL/6J and KhkA/C-/- mice was prepared from frozen tissues with RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with DNase I to remove traces of DNA.” Minor Point 1This sentence is indeed confusing and misleading and has therefore been removed from the introduction. Minor Point 2We have modified the start of the second paragraph to make the statement clearer: “One common approach to analyse RNA-seq datasets consists in identifying significant changes in expression levels between two or more experimental conditions using gene-level counts” Minor point 3Indeed, DESeq2-tximport cannot incorporate estimates of inferential variances. We have therefore updated the statement and corrected the reference, which should be the publication describing the tximport package. Minor point 4We have corrected the sentence to hopefully clarify its meaning: “To our knowledge, no systematic evaluation of the impact of the presence of low coverage on such key regions has been conducted and detailed reports of such examples in real datasets are still missing.” Minor point 5We have updated the figure reference in the main text and referenced the exon E012. Minor point 6, 7, 8Thank you for taking the time to highlight these discrepancies. We have corrected the wording in the Figure and the changed the “JunctionSeq” and “GENCODE” formatting in the main text."
}
]
},
{
"id": "42139",
"date": "16 Jan 2019",
"name": "Stephen N Floor",
"expertise": [
"Reviewer Expertise gene expression",
"computational biology",
"RNA 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\nChabbert and colleagues did intricate work to measure how erroneous gene annotations without careful curation can lead to errors in gene expression analysis. This is a recurring problem in analysis of gene expression that has recently started to be addressed. In this work, they used a mouse gene (ketohexokinase) as a model to analyze publicly available data employing current alignment-free approaches to transcript quantification. The authors bring a real problem to the table and presents a good alternative as to how to proceed before analysis. Thanks for the deep study on how isoform annotations can affect gene expression quantification.\n\nComments –\nIn Figure 2, is it possible to show the same coverage tracks and assayed tissues (2B vs 2C)? What does the R1 isoform look like in the spleen? Do the authors have any theories about why the software is misusing the retained intron information during quantification? Why might correcting 3’ UTRs fix the problem? Would manually curating the 3’ UTR for each isoform make a difference? Same question for the 5’ UTR. A lot of times, the difference in isoforms is in transcription start sites, which have been observed to relate to downstream transcript processing events including splicing and poly-A site selection. Do the authors have any theories about how to generalize this approach for studies about many genes? How do the annotations for Khk change between databases? Would a unified annotation set using data from beyond Ensembl improve isoform quantification?\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": "4495",
"date": "03 Apr 2019",
"name": "Christophe Chabbert",
"role": "Author Response",
"response": "We thank all the reviewers for their insightful comments and suggestions which have helped improve the quality of the manuscript. The main changes are summarized in the “Amendments from Version 1” section describing the new version of the manuscript. We will address the specific comments in this section.Point 1:The coverage tracks for all available tissues are shown either in Figure 2 or in the Extended Data, Figure 3 (accessible via this link - please make sure to expand the explorer tree on the left to make all files visible).In this study we focused on identifying an example of gene model configuration that might be problematic. Although identifying which aspects of the quantification algorithms are causing this issue is definitely of interest, this question is beyond the scope of this study. Nevertheless, we invite the reviewers to consult the paper from Soneson et al as it provides additional insights into this aspect and explores other parameters, such as the choice of quantification method. We are hoping that the bioinformatics community will be able to identify the root cause of these biases. Point 2:This study focuses on providing an example of quantification bias and on highlighting some aspects of a gene model that have a strong impact on this bias. Identifying the algorithmic cause of the problem would be very interesting but is unfortunately beyond the scope of the work reported here. In the example of Khk, we have shown in this paper that the use of homogenous 3’ UTRs and 5’ UTRs across transcripts has helped overcome the quantification issue. Additional work will be required to determine whether that would be the case for all problematic genes and but this is also not part of the current study.As pointed out, some reports have indeed shown that changes in 5’ or 3’ end are an important source of isoform diversity, which might complicate the interpretation of quantification results. As we mentioned in the abstract and in the discussion, it will be important to always thoroughly confirm such findings derived from transcript quantification studies. In addition, it is important to note that standard RNA-seq protocols are usually not suited to identify such changes in 5’ or 3’ end usage. Indeed, it is recommended to use protocols such as CAGE or 3’T-fill capture that can unambiguously identify transcription start or end. Therefore, such cases should be handled with an adequate experimental setting rather than standard RNA-seq strategies. Point 3:As previously commented in Points 1 and 2, our study did not aim at providing a generalized method that identifies problematic genes such as Khk or to provide a universal solution to the issue. The study from Soneson et al introduces the JCC index, which should be very helpful to identify problematic genes. In addition, in order to reduce the risk of making erroneous conclusions from large data analyses, we recommend confirming important findings using orthogonal methods (as stated in the abstract) such as count based approaches or well-targeted low throughput experiments such as qPCR for example. Point 4:In mouse, Khk annotations differ between Ensembl and RefSeq (please see the RefSeq annotation here, curated transcript NM_). When focusing on the curated transcripts, the most striking differences with the GENCODE annotation are the lack of Khk.RI, Khk.Skip and identical 5’ and 3’ UTRs. This would therefore be an equivalent situation to the one presented in this paper but without the Khk.Skip transcript available for quantification. This would therefore result in erroneous predictions as well. To comment on this point, we have added the following statement in the discussion section of the paper: “It is also important to note that the curated RefSeq Khk gene model differs from the GENCODE as it is missing Khk.RI, Khk.Skip and the 3’ and 5’ UTRs of all curated transcripts are identical. While this would be a close configuration to the optimal annotation presented in this study, the absence of Khk.Skip in the gene model would result in erroneous quantifications as well.” While a consolidated annotation based on transcript model derived from large datasets might be an appealing idea, recent results from Soneson et al showed that in practice, this did not improve transcript estimate accuracy for problematic genes. We updated the discussion section of the paper in order to elaborate on this matter: “Improvement of current genomic annotations might ultimately offer an alternative as they will allow for the sole use of fast quantification algorithms. This might partially be achieved using transcript catalogues obtained from large scale studies such as CHESS even though Soneson et al reported very little to no improvement in their JCC scores using these new annotations.”"
}
]
},
{
"id": "42140",
"date": "18 Jan 2019",
"name": "Magnus Rattray",
"expertise": [
"Reviewer Expertise Bayesian inference",
"probabilistic models",
"genomic data analysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChabbert et al. provide an interesting study of the effect of gene model annotation on isoform abundance estimation from RNA-Seq data. They take a gene with 5 annotated alternative transcripts and look at expression over several tissues. They explore how well a transcript-based estimation method (Salmon) and differential transcript usage method (DRIMSeq) does when considering additional evidence from visualisation of read coverage and experimental validation of alternative transcripts by qPCR. They demonstrate that the inferred transcript abundance using the standard model is inconsistent with read coverage (e.g. an inferred highly expressed transcript has very low read density or junction read evidence for its only unique exon) and they show that better results are obtained when the model is modified by hand in a number of ways. They also show that through using their handcrafted gene model, consistency is improved when the dataset is changed to single-ended and shorter reads, which I found to be an interesting experiment. They also look at concordance across different RNA-Seq datasets measuring the same tissues and find the modification of UTRs in the gene model to improve concordance.\n\nOverall I found this to be an interesting and useful contribution. Recent work (cited by the authors) has looked at metrics to identify discordance of the type observed here but nevertheless this careful study of one gene model is a useful contribution, especially give the experimental validation using qPCR. I think the data will be of great interest to methodology developers grappling with this problem although I guess long-read datasets may provide evidence for more genes in future studies than this kind of qPCR approach.\n\nMajor comments:\n\nAs far as I can understand, in the end two kinds of change were made to the annotated gene model at the same time. A transcript Khk.RI was removed (this was tried first) and then both the UTRs were modified to be the same for all isoforms (first 3’ and then 5’) which means that new isoforms are added to the gene model and some were removed. The removal of a transcript strongly affecting results is arguably more problematic for transcript quantification methods because we would often expect the set of isoforms to contain unexpressed isoforms in a specific dataset. The other issue (an incomplete gene model) is already known to be problematic 1. However, I was not sure whether the UTR issue could potentially make the Khk.RI isoform issue worse. Therefore, I think it would be helpful to check whether Khk.RI inclusion remains problematic after making the UTR changes to the other isoforms. If adding an unexpressed isoform to the gene model can cause such an issue then this is a problem even when the gene model is correct. Also, there is a statement later on that “the removal of Khk.RI did further hinder reproducibility between both datasets” which seems to contradict the idea that removing this isoform is a good idea. I think this sentence is also a bit unclear and could do with some more explanation.\n\nThe modifications to the gene model in the end involves creating a new set of isoforms by combining existing exons and UTRs in a new configuration. Algorithms exist to do this in a data-driven way, e.g. the FlipFlop algorithm is designed to learn a set of isoforms from data by searching over all exon combinations using sparse inference on a splicing graph. Another example is StringTie and there may be later ones I'm not aware of. I think it would be useful to attempt to apply an algorithm of this type to this dataset to see how it would perform as this may provide a computational way to do something similar to what has been done manually in this example. Alternatively, these methods may fail and that would also be of interest.\n\nMinor comments:\nPage 3 - “the recent development of alignment-free….has provided the possibility to quantify each individual transcript”. Methods that predate Salmon were already available for transcript quantification e.g. RSEM, eXpress, bitseq etc. I don't think we should confuse the speed-up introduced in Salmon and Kallisto with the transcript-level inference model introduced by RSEM and I think RSEM could have been used in this paper with similar conclusions. It would be good to know whether gene expression estimates based on the different gene models are performing differently, i.e. estimating gene expression by summing up isoform expression rather than counting. This has been discussed in the literature 2 and this issue could be explored in this dataset.\n\nTypos:\nPage 7 - “on estimate concordance” should be estimated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4496",
"date": "03 Apr 2019",
"name": "Christophe Chabbert",
"role": "Author Response",
"response": "We thank all the reviewers for their insightful comments and suggestions which have helped improve the quality of the manuscript. The main changes are summarized in the “Amendments from Version 1” section describing the new version of the manuscript. We will address the specific comments in this section. Major Point 1 We have subjected the gene model annotation to two rounds of modifications to assess the impact of Khk.RI and UTRs on relative Khk isoform expression estimates. To assess the importance of the order in which these modifications happen, we have generated an additional annotation where Khk.RI is retained and all other Khk annotated transcripts have identical 5’ and 3’ UTRs. This annotation was used to estimate Khk isoform proportions in the Li et al (using the paired end and 50bp single-end data) and the Sollner et al datasets. We observed that implementing this new UTR model was not sufficient to remove the Khk.RI estimation bias in the paired-end data (updated Figure 3A). This bias was still particularly important in the bone marrow and spleen datasets where Khk.RI was estimated to account for more that 20% of the overall gene expression.Comparison of the estimates between the paired-end dataset and the 50bp dataset derived from Li et al showed that this modification was however more beneficial than the removal of Khk.RI (Pearson correlation of 0.86 as opposed to 0.79 when Khk.RI is removed). Nevertheless, the best agreement between both libraries was still obtained after removal of Khk.RI and extension of the UTRs (Pearson correlation of 0.97).Finally, comparison of the estimates of the Li et al and Sollner et al datasets revealed that the extension of the UTRs (Pearson correlation of 0.73) was more beneficial than Khk.RI removal (Pearson correlation of 0.46) but provided hardly any improvement over the naïve GENCODE annotations (Pearson correlation of 0.73). The complete set of modifications including Khk.RI removal and homogenization of the UTR was required to reach higher concordance between both datasets (Pearson correlation 0.91).Overall, these observations suggest that both modifications are indeed needed to improve Khk transcript quantification results in the datasets considered in this study.We have updated main Figure 3, main Figure 4 and the underlying data table 3 to report these findings. The result and discussion section of the article have also been modified accordingly: “Additionally, we computed the relative Khk isoform usage using a new annotation with identical 5’ and 3’ end for all isoforms except Khk.RI which was retained as such in the gene model (Figure 3A). This modification was not sufficient to remove Khk.RI estimation biases, with the retained intron predicted to erroneously account for 20% of the overall gene expression in bone marrow or spleen. We therefore confirmed the impact and importance of both Khk.RI and UTRs annotations on isoform expression estimates for that gene.” “Both the removal of Khk.RI and UTR adjustments were necessary to reach this concordance between profiling methods.” Finally, we have modified the mentioned statement to emphasise the importance of performing both modifications on the concordance between estimates: “While the removal of Khk.RI without an adjustment of the UTR did further hinder reproducibility between both datasets, we observed a much stronger consistency of proportion estimates when using the manually updated annotation” Major Point 2 Following this recommendation, we used StringTie as a complementary method to estimate transcript abundance across all tissues, with and without the reference annotation from GENCODE. Estimations output using the reference annotation still showed that the Khk.RI isoform still accounts for at least 15% of expressed isoform in 9 tissues. The results are presented in the Extended data, Figure 10A and the quantification results stored in the underlying data table 4. We also obtained StringTie quantifications without using a supporting genomic annotation, thereby allowing for the assembly of transcript by relying only on the current dataset. After merging the discovered transcript, we examined the resulting annotation for the Khk gene and compared it with the current GENCODE gene model (Extended data, Figure 10B). Quite interestingly, no corresponding Khk.RI isoform could be detected but Khk.A, Khk.C and Khk.Skip were all identified, albeit with UTR regions differing from the GENCODE annotation. No KhkA.C isoform could be detected while junction counts and coverage tracks indicate that it is expressed in low levels in certain tissues (liver or small intestine for example).Taken together, these results suggest that, when relying on current annotations, StringTie is subject to similar biases as Salmon (in accordance with the observations made in Soneson et al) when it comes to the erroneous detection of the Khk.RI unexpressed transcript. When relying on transcript assembly, StringTie does provide different UTRs from the GENCODE annotation but fails to detect the KhkAxC isoform. As it is beyond the scope of this study to deliver a computational solution to what was achieved manually and at the single gene level, we have reported our observations but will not evaluate the feasibility of the scalability of an improved StringTie approach to tackle this problem.We have modified the discussion section of the article as follows: “To complement our main findings, we estimated transcripts abundance in the Li et al dataset using StringTie, as an example of tool relying on the construction of a splicing graph to quantify isoforms. When using the GENCODE annotation as a guide for quantification, we found that StringTie overestimated the Khk.RI expression in a fashion similar to Salmon. This is in concordance with the report from Soneson et al39. In addition, we used StringTie without any supporting annotation to enable transcript assembly from the alignments. Inspection of the resulting newly assembled transcripts showed that Khk.RI could not be detected (Extended Data, Figure 10). Nevertheless, KhkA.C was also not identified in the dataset while junction counts clearly indicate that it could be detected in a handful of tissues, albeit with low expression levels. We therefore suggest that the issue reported here in the case of Salmon might be commonly found across quantification software, and it will be interesting to assess whether similar biases may arise with more tools. Results from Soneson et al. strongly indicate that this is the case, at least for a group of human genes 39 .” The method section has also been updated to document the parameters and settings used for the StringTie quantifications:“In order to quantify transcript using stringTie, alignments to the reference genome were first re-generated as described in the “RNA-seq data processing and alignment” section with the additional STAR flag “--outSAMstrandField intronMotif”. Quantifications were then performed with and without the GENCODE annotation used as a guide (-G option in stringTie). All other parameters were set to default values. Newly assembled transcripts were then merged using the transcript merge mode (--merge) with default parameters.” Minor Point 1 This is true and we have updated this section of the introduction to make sure this distinction is made clear. The section is now written as follows: “In addition, the recent development of alignment-free transcript quantification methods has provided the possibility to efficiently and rapidly quantify each individual transcript 15– 19 . Such approaches are indeed computationally much less demanding and faster than alignment-based methods 15 . Moreover, they have been shown to overcome the difficulty of handling multi-mapped reads, which can create biased results in count-based analysis 21” Minor Point 2 This is a good suggestion and following this recommendation and the major point 1 raised by Patrick Kimes, we have used tximport to obtain gene level estimates based on Salmon transcript quantifications from 3 of the modified annotations and the original GENCODE annotation. The results are now presented in the Extended Data, Figure 7. We found the gene level estimates to be consistent across all annotations and tissues and the patterns of expression across tissues to be conserved. In particular, Khk was found to be mostly expressed in liver, kidney and small intestine and that its expression was strongly tissue specific (adjusted p-value of 0 using the LRM from DESeq2 as described in the first version of the article). We have consequently added the following paragraph in the result section: “Finally, we evaluated whether the changes brought to the Khk transcript annotations affected the estimations of overall Khk expression levels. Gene level estimates obtained using quantifications based on the 3 modified annotations were strongly correlated (Pearson, r > 0.99) with estimates derived from the naïve GENCODE annotation (Extended data: Figure 7A). In addition, the high tissue specificity of Khk expression was observed in all cases, with expression patterns identical between annotations (Figure 1A, Extended data: Figure 7B).”"
}
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}
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https://f1000research.com/articles/7-1956
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https://f1000research.com/articles/8-371/v1
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03 Apr 19
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{
"type": "Research Article",
"title": "Evaluation of patient satisfaction and shade matching of Vita Suprinity versus lithium disilicate (E-max) ceramic crowns in the esthetic zone: a randomized controlled clinical trial",
"authors": [
"Sameh Abou-Steit",
"Jylan ElGuindy",
"Amina Zaki",
"Jylan ElGuindy",
"Amina Zaki"
],
"abstract": "Background: Optical impairments of teeth in the esthetic zone constitute a problem for many dentists and patients as several studies concluded that patients were dissatisfied of their dental appearance because of their restorations' color. The purpose of this study was to assess patient satisfaction combined with shade matching of VITA SUPRINITY versus lithium disilicate (IPS e-max CAD) all-ceramic crowns in esthetic zone. Methods: 26 patients with teeth problems indicated for full coverage restoration on one tooth in the esthetic zone (from upper central incisor to 1st premolar) were randomized into 2 equal groups (n=13) for which different CAD-CAM ceramic materials were used; (VITA SUPRINITY) a zirconia reinforced lithium silicate ceramic and (IPS e.max CAD) lithium disilicate glass ceramic. The crowns were fabricated to match the shade of the contra-lateral/ adjacent tooth and assessed using the modified USPHS criteria. Also patient satisfaction was assessed. The data obtained by evaluating each assessment criteria were statistically analyzed using the Chi-square test that was performed in categorical data. Results: According to the modified USPHS criteria, 100% of the patients were Alpha score with 0% for Bravo, Charlie and Delta scores in both groups. While according to the visual analogue scale (VAS), 100% of the patients were satisfied while 0% were dissatisfied by the restoration in both groups. Conclusions: The results for both materials in terms of patient satisfaction and shade matching were Alpha. This indicated that both materials are clinically accepted to be used as full coverage restorations for excellent esthetic outcome. Registration: NCT0284611.",
"keywords": [
"E.max CAD",
"VITA SUPRINITY",
"shade matching",
"patient satisfaction",
"ceramic restorations",
"esthetic dentistry."
],
"content": "Introduction\n\nIn dentistry, accurate and predictable shade matching between natural teeth and restorative materials represents a challenge for clinicians and laboratory technicians. For decades, visual shade matching was most commonly used for the shade selection in the dental clinics as it is easy and does not require expensive equipment1,2. Several studies reported that the color of the shade tabs is uniformly distributed and the shade guide enables accurate shade matching with the natural teeth3,4. Other studies stated that the number of correct matches might increase if the Vitapan 3D-Master shade guide is used4–6. They also suggested the use of Vitapan 3D-Master along with shade mapping, as in this study, to allow the exclusion of the effect of operators’ experience on shade-matching process and to ensure correct placement of different shade effects and characterizations4–6.\n\nHowever, many factors may negatively affect the quality of shade matching clinically, including tooth texture, lighting conditions, the surroundings and background7, as well as the subjective nature of human color observation, through color blindness or color perception defects8,9. Differences in gender10, variations in experience of the evaluators, lighting conditions, metamerism and eye fatigue that could affect proper shade selection11. Additionally, factors such as tooth dehydration as result of prolonged clinical procedures12 and color alteration of shade tabs after chemical disinfection can cast a significant effect on the final shade matching13–15.\n\nTherefore, in order to eliminate the confounders during the color matching process, instrumental methods have been introduced16. Greater correlation in several studies have been found between both the objective (spectrophotometer) and the subjective (visual) shade selection methods with respect to color dimension of value or lightness, followed by hue and finally chroma17–19. It was concluded that combination of both methods should be used for a successful esthetic outcome. While other studies found that using a spectrophotometer for shade matching is more accurate than using a conventional shade guide20–24. In other studies, instrumental color matching was suggested as a supplementary tool for a better esthetic outcome18,19,22.\n\nIn addition, the final color of all-ceramic restorations is affected by different factors including the porcelain layering technique25, dental ceramic type, ceramic brand26, veneering ceramic thickness27–29, and firing temperature and conditions30–32. Moreover, clinical parameters, including type33, color34, and thickness35,36 of cement can also influence the final result37.\n\nTherefore, the esthetic needs of the patients during treatment should be taken into consideration, otherwise patient satisfaction will not be achieved, which has a significant effect on patient self-perception and quality of life38,39. A survey looking at patient satisfaction with esthetic treatment found that the restoration color was the primary cause for patient dissatisfaction, as 89.3% of patients were not satisfied of their esthetic treatment because of the color of their restoration40. Other studies found that tooth color and gender also affect patient satisfaction, with females more dissatisfied than males41,42.\n\nNewly introduced ceramic systems with improved optical properties may lead to improve clinical shade matching and patient satisfaction. VITA SUPRINITY, introduced by VITA Zahnfabrik, is a glass ceramic material enriched with approximately 10% zirconia by weight. This very fine homogenous dual microstructure resulted in high flexural strength while simultaneously providing a high percentage of glassy matrices. These structural effects provide the ceramic with good optical and polishing properties and allow delivery of restorations with excellent esthetics43,44.\n\nLithium disilicate ceramic (such as IPS e.max CAD, manufactured by Ivoclear Vivadent) is considered one of the more esthetic ceramics due to the glass matrix embedded with needle-like lithium disilicate crystals that results in reduction of internal scattering of the light as it passes through the material. There are also other optical properties for better mimicking of adjacent natural teeth, which include the chameleon effect45,46.\n\nTherefore, this study was conducted to evaluate patient satisfaction according to the visual analogue scale (VAS) and shade-matching of VITA SUPRINITY versus (IPS e-max CAD) lithium disilicate all ceramic crowns in esthetic zone using the modified United States Public Health Service (USPHS) criteria in a prospective controlled randomized clinical trial. The null hypothesis of the study was that there would be difference in patient satisfaction and shade matching of VITA SUPRINITY if compared with lithium disilicate ceramic crown used in the esthetic zone.\n\n\nMethods\n\nThis study was approved by the Research Ethics Committee of the Faculty of Dentistry (approval no: 1082016). Written informed consent regarding treatment sequence, publishing of their images and results was obtained from all participants.\n\nThis trial was registered at the ClinicalTrials.gov registry under registration number NCT02846116 on July 27, 2016.\n\nThis study was a double blind randomized controlled clinical trial with a 1:1 allocation ratio. This article was written in concordance with the CONSORT checklist; a completed checklist is available on Open Science Framework47.\n\nAll participants were recruited from the outpatient clinic of the Department of Fixed Prosthodontics of Faculty of Dentistry, Cairo University, Cairo, Egypt. A face-to-face participants’ selection was performed according to the patients’ need for a full coverage restoration on a tooth in esthetic zone. A total of 26 participants were recruited for this study during the time from July 2017 till September 2017. This study was completed by May 2018. Full medical and dental history were obtained from all participants. ACONSORT flow diagram is available as extended data47.\n\nInclusion criteria. (1) Age ranging between 20 and 60 years old; (2) patients psychologically and physically able to tolerate the treatment procedures; (3) patients with no periodontal or pulpal diseases with acceptable restorations; (4) patients with teeth in esthetic zone indicated for all-ceramic crowns (e.g. mild-to-moderate discoloration, coronal fracture where partial coverage is not indicated); (5) patients with endodontic treated teeth requiring all-ceramic crowns.\n\nExclusion criteria. (1) Lack of patient motivation; (2) patients with psychiatric problems or unrealistic expectations; (3) patients with parafunctional habits; (4) teeth with increased incisal translucency; (5) severe discolored teeth; (6) no opposite occluding dentition.\n\nA total of 26 crowns (13 in each group) was sufficient with 80% power and at 5% significance. The sample size was calculated using G*power Version 3.0.1048\n\nRandomization was carried out using computerized sequence generation (https://www.randomizer.org/) in the Center of Evidence Based Dentistry, Cairo University.\n\nParticipants were divided into two groups (A and B) according to the ceramic material used. Each participant received a sealed opaque envelope with their randomized number.\n\nNumber for each member in each group was written by indispensable pen on large white paper sheet. The sheet was folded eight times and saved inside opaque well sealed envelope.\n\nThe candidate under supervision was responsible for providing allocation generation and dividing patients into two groups and save it in the envelopes in secured place until the date of performing procedure.\n\nThe trial participants and outcome assessors were blinded throughout the whole procedures (double blind), as the dentist practitioner (S.A.) was responsible for all clinical procedures.\n\nTwo crown systems (IPS e.max CAD and VITA SUPRINITY ceramics) were selected for this study (Table 1). All treatment procedures were performed by the same clinician (S.A.). Scaling and polishing were performed for each patient before shade selection in order to remove any dental plaque, calculus and staining, which will affect the accurate shade selection. The tooth color was recorded visually using VITA 3D-Master shade guide system (VITA, Zahnfabrik, Germany) in accordance to the contra-lateral/adjacent tooth under different light conditions: natural day light, incandescent light and color-corrected light (using Smile Lite, Smile Line, Switzerland) to avoid metamerism. This was done with the help of three prosthodontists, who had performed Ishihara's test to determine deficiency in color vision49. Their results showed no color blindness.\n\nVisual shade selection was carried out within a standardized environment at midday, when incident daylight is most balanced with the visible light spectrum50. While the patients were positioned on dental chair so that his/her mouth was at the same level and 40 cm apart from observers’ eyes. All bright colors were removed from the field of view (as makeup, tinted eye glasses) and a neutral gray patient bib masked clothing colors. Upper and lower teeth were separated with the tongue retracted. Both tooth (the contra-lateral/adjacent tooth) and shade tab were placed in the same plane with neutral gray background (Flexipalette Color Match, Smile line, Switzerland). Shade selections were done in three areas of the tooth (incisal, middle and cervical third) to match the subject's maxillary contra-lateral/adjacent tooth5,51. (Figure 1)\n\nThe color-corrected light, with a color correlated temperature of 5,500K, 1,500 lux at a distance of approximately 10 cm and a color rendering index (CRI) of 92, was used as a confirmatory procedure. The evaluators were asked to stand opposite the targeted tooth so that shade matching was performed from the see-thru rectangular window (Figure 2)52–54.\n\nShade matching was also confirmed with Vita Easyshade V spectrophotometer (VITA, Zahnfabrik, Germany). It was calibrated before starting each measurement. As stated by the manufacturer, the measurement was acknowledged when three consecutive, identical readings were generated using tooth area measurement mode for shade selection (Figure 3)22. Vita Easyshade V was used as a confirmatory tool for better shade selection by the operator only and wasn’t used for the clinical evaluation or assessment procedure of the final restorations.\n\nPreparation of teeth for full coverage restoration were performed with smooth, round contours and line-angles, chamfer finish lines of 1 mm in diameter with round internal angles, and incisal reduction of 2 mm46,55,56. The shade of the prepared abutment tooth was recorded visually using the IPS Natural Die Material shade guide (Ivoclar Vivadent) under natural day light and color corrected light in order to fabricate a die mimicking the oral situation for optimum desired final esthetic results (Figure 4)46,57.\n\nVinyl polysiloxane elastomeric impressions (Honigum impression material, DMG, Germany) were made, and provisional restorations (Structur 2 SC, VOCO, Germany) were cemented with non-eugenol provisional cement (RelyX Temp NE, 3MESPE, USA)55.\n\nThe construction of the all-ceramic crowns was performed using CAD/CAM Cerec InLab MC X5 milling machine, with Cerec 15.0.0 software. The fitting surfaces of the all-ceramic crowns were treated and silanated according to the manufacturer's instruction and abutment teeth were prepared where self-etch adhesive protocol was obeyed using adhesive resin cement (Bifix-QM, VOCO, Germany) (Figure 5)58,59.\n\n(A) Frontal view. (B) Palatal view.\n\nPatients were given a handheld mirror to view their teeth and definitive restoration in the clinic and evaluated their own restoration57. Patients were asked to fill in patient satisfaction chart for either satisfied or dissatisfied.\n\nIn addition, restorations were evaluated by three prosthodontists from the Department of Fixed Prosthodontics, Cairo University. A few minutes before the procedure, these evaluators entered the clinic in order to adapt to the environmental lighting conditions51,54,57,59–61. Evaluation was performed with the same sequence used for initial shade-matching (visually using VITA 3D-Master shade guide system (VITA, Zahnfabrik, Germany) in accordance to the contra-lateral/adjacent tooth under different light conditions (natural day light, incandescent light and color-corrected light). To avoid eye fatigue, evaluators were asked to rest their eyes by temporarily closing them or looking at a neutral grey zone every 10 seconds5,18. Evaluators were asked to fill in a clinical assessment chart with each patient number.\n\nThe two groups of patients were assessed using the Visual Analogue Scale (VAS)40,42,57 which is binary and documented in chart including number of satisfied and unsatisfied.\n\nThe two groups were assessed using the modified United States public health service (USPHS) criteria by the three evaluators61. Alpha (Excellent), Bravo (Acceptable), Charlie (Acceptable but modifications needed) and Delta (Unacceptable).\n\nThe results were analyzed using Graph Pad Instat (Graph Pad, Inc.) software for windows. A value of P≤0.05 was considered statistically significant. Data obtained by evaluating each assessment criteria were statistically analyzed using the Chi-square test that was performed in categorical data.\n\n\nResults\n\nThe patient satisfaction associated with restorations for both groups is highlighted in Table 2 and Figure 6. Raw data are available on Open Science Framework47.\n\nAccording to the visual analogue scale (VAS), 100% of patients were satisfied while 0% were dissatisfied by the restoration in both groups. This was statistically non-significant as verified by chi square test (p>0.05). Table 2 and Figure 6. No harms were recorded.\n\nRegarding shade matching, no statistical difference was found between the two tested groups (p >0.05). According to the modified USPHS criteria, 100% of the patients were Alpha score with 0% for Bravo, Charlie and Delta scores in both groups (Table 3 and Figure 7).\n\n\nDiscussion\n\nConcerning shade matching results, all patients recorded no color difference between their restorations and adjacent/contra-lateral teeth which might be attributed to the strict and meticulous shade matching protocol followed in respect to the staining procedure in accordance to each material used. In addition to the similarity in microstructure of both materials being lithium silicate-based glass-ceramics. This was in accordance with several studies that stated that in order to achieve a tooth-colored restoration, two different steps are required: select the proper shade using a shade guide and/or an electronic shade-taking device, and reproduce this shade with a suitable dental material37,63.\n\nConcerning IPS e.max CAD all-ceramic crowns, Fasbinder et al.64 stated that no color change was noted for CAD/CAM lithium disilicate crowns after 2 years of service. Rauch et al.65 reported USPHS alpha scores for all monolithic lithium disilicate crowns in the posterior zone upon evaluation after 6 and 10 years. Contradicting to our results those found by Chaiyabutr et al.46, who found color variation in CAD/CAM lithium disilicate glass-ceramic restorations. They explained that this may be due to the optical characteristics of the material, which allows the underlying tooth shade stump to affect the final color of the crown. They reported that the final color of 1 mm ceramic thickness in the cervical area combined with low-translucency ceramic blocks may be affected by intense discoloration of underlying abutment tooth color. This was avoided in our current study, as severely discolored abutments were among our exclusion criteria.\n\nRinke et al.66 found the excellent esthetic properties of VITA SUPRINITY may be related to the homogenous dual microstructure, consisting of very fine lithium disilicate and lithium metasilicate crystals with average size of 0.5–0.7 μm which are four to eight times smaller than lithium disilicate crystallites. The result is very fine microstructure that provides a higher ratio of glass matrix/zirconium oxide in solution.\n\nHowever, contradicting our results, Jirajariyavej et al.29 found that the different ceramic materials affected the final esthetic outcome of the restoration. This color difference may be due to difference in translucency between the silicate-based glass-ceramic materials, with the IPS e.max CAD being more translucent than VITA SUPRINITY.\n\nConcerning patient satisfaction results, all the patients were satisfied with their restorations. Additionally, there was no statistically significant difference between the two groups. This might be attributed to the meticulous shade matching protocol and laboratory procedure followed in this study. Ballard et al.55 found that patients reporting high satisfaction ratings may have been affected by the high value of the restoration. Al-Wahadni et al.67 found that patients’ satisfaction increased when the restorations were received in an academic institution, denoting that patients’ confidence in the school or good relationship with the dental student may have elevated their opinions of the care received.\n\nContradicting our results are those by Shah et al.41 who found the overall rating of patient satisfaction was acceptable (USPHS Bravo and Charlie scores). They explained that patient level of education might affected the results. Well-educated patients tended to be more satisfied with their tooth color compared to patients with primary level of education. Samorodnitzky-Naveh et al.40 found discrepancies between overall satisfaction with tooth appearance and satisfaction with tooth color. They explained that age might have a significant effect on patient satisfaction, as the selected young cohort of subjects might have been excessively sensitive about the appearance of their teeth. This was avoided in our study by selecting large age range between 20 and 60 years old.\n\nFinally, the hypothesis was rejected as no statistical difference was found between the two tested groups (IPS e.max CAD and VITA SUPRINITY).\n\nMore clinical studies are required with prolonged follow-up periods to evaluate long-term esthetic clinical performance of the materials along with patient satisfaction in order to be used in different situations for better esthetic outcome.\n\nSpectrophotometer (Vita Easyshade V) was used as clinical confirmatory tool only in this study by the operator and not for final outcome assessment. Further studies utilizing spectrophotometer as a main assessment tool are required.\n\n\nConclusions\n\nVITA SUPRINITY and IPS e.max CAD full coverage restorations revealed excellent patient satisfaction and color matching. This indicates that both materials can be recommended as full coverage restorations in clinical situations for optimum esthetic outcome. The choice depends on the dentist's preference.\n\n\nData availability\n\nOpen Science Framework: Evaluation of Patient Satisfaction and Shade Matching of Vita Suprinity Versus Lithium Disilicate (E-max) Ceramic Crowns in Esthetic Zone (Randomized Controlled Clinical Trial). https://doi.org/10.17605/OSF.IO/ZH6SC47.\n\nUnderlying data for this study are available in file “Raw data.xlsx”.\n\nOpen Science Framework: Evaluation of Patient Satisfaction and Shade Matching of Vita Suprinity Versus Lithium Disilicate (E-max) Ceramic Crowns in Esthetic Zone (Randomized Controlled Clinical Trial). https://doi.org/10.17605/OSF.IO/ZH6SC47.\n\nThis project contains the following extended data:\n\nResearchRandomizer.csv\n\nCONSORT flow diagram of experimental stages and group distribution.docx\n\nTrial protocol.doc\n\n\nReporting guidelines\n\nOpen Science Framework: CONSORT checklist for article “Evaluation of patient satisfaction and shade matching of Vita Suprinity versus lithium disilicate (E-max) ceramic crowns in esthetic zone: a randomized controlled clinical trial”. https://doi.org/10.17605/OSF.IO/ZH6SC67.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
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PubMed Abstract | Publisher Full Text\n\nAbou-Steit S, Al-Guindy J, Zaki A: Evaluation of Patient Satisfaction and Shade Matching of Vita Suprinity Versus Lithium Disilicate (E-max) Ceramic Crowns in Esthetic Zone (Randomized Controlled Clinical Trial). 2019. http://www.doi.org/10.17605/OSF.IO/ZH6SC\n\nTaskonak B, Sertgöz A: Two-year clinical evaluation of lithia-disilicate-based all-ceramic crowns and fixed partial dentures. Dent Mater. 2006; 22(11): 1008–13. PubMed Abstract | Publisher Full Text\n\nParavina RD: Techniques for improvement of clinical shade matching procedures. Ph.D. dissertation, University of Nis School of Medicine, Nis, Serbia, 2000.\n\nLi Q, Wang YN: Comparison of shade matching by visual observation and an intraoral dental colorimeter. J Oral Rehabil. 2007; 34(11): 848–54. PubMed Abstract | Publisher Full Text\n\nFairchild MD, Reniff L: Time course of chromatic adaptation for color-appearance judgments. J Opt Soc Am A Opt Image Sci Vis. 1995; 12(5): 824–33. PubMed Abstract | Publisher Full Text\n\nLagouvardos PE, Fougia AG, Diamantopoulou SA, et al.: Repeatability and interdevice reliability of two portable color selection devices in matching and measuring tooth color. J Prosthet Dent. 2009; 101(1): 40–5. PubMed Abstract | Publisher Full Text\n\nJoiner A: Tooth colour: a review of the literature. J Dent. 2004; 32 Suppl 1: 3–12. PubMed Abstract | Publisher Full Text\n\nClary JA, Ontiveros JC, Cron SG, et al.: Influence of light source, polarization, education, and training on shade matching quality. J Prosthet Dent. 2016; 116(1): 91–7. PubMed Abstract | Publisher Full Text\n\nEtman MK, Woolford MJ: Three-year clinical evaluation of two ceramic crown systems: a preliminary study. J Prosthet Dent. 2010; 103(2): 80–90. PubMed Abstract | Publisher Full Text\n\nRam HK, Shah RJ, Agrawal HS: Evaluation of three different tooth preparation techniques for metal ceramic crowns by comparing preparation depths: An in vitro study. J Indian Prosthodont Soc. 2015; 15(2): 162–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBallard E, Metz MJ, Harris BT, et al.: Satisfaction of Dental Students, Faculty, and Patients with Tooth Shade-Matching Using a Spectrophotometer. J Dent Educ. 2017; 81(5): 545–53. PubMed Abstract | Publisher Full Text\n\nWang F, Takahashi H, Iwasaki N: Translucency of dental ceramics with different thicknesses. J Prosthet Dent. 2013; 110(1): 14–20. PubMed Abstract | Publisher Full Text\n\nDavidowitz G, Kotick PG: The use of CAD/CAM in dentistry. Dent Clin North Am. 2011; 55(3): 559–70. PubMed Abstract | Publisher Full Text\n\nRistic I, Stankovic S, Paravina RD: Influence of Color Education and Training on Shade Matching Skills. J Esthet Restor Dent. 2016; 28(5): 287–94. PubMed Abstract | Publisher Full Text\n\nJoshi R, Acharya J: Shade Matching Ability of Dental Students Using Two Visual Light Sources. Nepal Med Coll J. 2017; 19(1): 24–6. Reference Source\n\nBatson ER, Cooper LF, Duqum I, et al.: Clinical outcomes of three different crown systems with CAD/CAM technology. J Prosthet Dent. 2014; 112(4): 770–777. PubMed Abstract | Publisher Full Text\n\nBagis B, Turgut S: Optical properties of current ceramics systems for laminate veneers. J Dent. 2013; 41 Suppl 3: e24–30. PubMed Abstract | Publisher Full Text\n\nFasbinder DJ, Dennison JB, Heys D, et al.: A clinical evaluation of chairside lithium disilicate CAD/CAM crowns: a two-year report. J Am Dent Assoc. 2010; 141 Suppl 2: 10S–4S. PubMed Abstract | Publisher Full Text\n\nRauch A, Reich S, Schierz O: Chair-side generated posterior monolithic lithium disilicate crowns: clinical survival after 6 years. Clin Oral Investig. 2017; 21(6): 2083–9. PubMed Abstract | Publisher Full Text\n\nRinke S, Rödiger M, Ziebolz D, et al.: Fabrication of Zirconia-Reinforced Lithium Silicate Ceramic Restorations Using a Complete Digital Workflow. Case Rep Dent. 2015; 2015: 162178. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAl-Wahadni A, Ajlouni R, Al-Omari Q, et al.: Shade-match perception of porcelain-fused-to-metal restorations: a comparison between dentist and patient. J Am Dent Assoc. 2002; 133(9): 1220–5. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "69488",
"date": "28 Aug 2020",
"name": "Omid Savabi",
"expertise": [
"Reviewer Expertise I am a Prosthodontist"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 compares two kinds of LiSi glass-ceramics CAD/CAM materials.\n\nIt is a well-written article and the methods are valid.\n\nI wonder that all the patients score their restorations 10 out of 10 or if they only score it satisfactory.\n\nIt would be useful if you explain the USPHS criteria for color matching.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "69491",
"date": "24 Sep 2020",
"name": "Hidehiko Watanabe",
"expertise": [
"Reviewer Expertise dental materials",
"ceramics",
"composites",
"color property",
"bonding",
"dental 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\nIt is hard to find a good clinical study in these days mainly due to the cost. I know this is not a large extensive study, but it is still informative for clinicians.\nI have some questions/comments for the authors.\nWhat is the primary purpose of this study? Why did those two materials need to be compared? In this study design, it looks like lithium disilicate serves as a control, but Suprinity is not a new material anymore. Are there any reasons to use Sprinity instead of emax in esthetic cases? If those materials provide a similar level of esthetics, what would be the benefit for clinicians? The authors mentioned that the selection of each material is a dentists’ preference, but what are the differences between them in terms of clinical use?\n\nIn the introduction, the authors mentioned several variables regarding shade matching procedures such as the types of shade guides, lighting conditions, background, spectrophotometer, and so on. However, almost none of these aspects are directly related to the variables in this study since they compared two different materials in the same conditions. Since the authors conducted very meticulous shade taking procedures, it would have been possible to add one or two more variables in terms of shade taking procedures. Those can be Easyshade and human eyes, experienced or unexperienced shade takers, or different light settings.\n\nWhat is the reason for excluding Easy shade for the final results? I am not saying you have to use them, but I would like to know the rationale for that in the discussion.\n\nThe reason for the inclusion criteria of endodontic treated teeth should have been presented. What was wrong with including a vital tooth?\n\nDid a technician get involved in the crown fabrication procedures? Did they conduct staining and glazing? The authors have to clarify it.\n\nWhat translucency types of the blocks did the authors use, and how many of each? HT, MT, or LT?\n\nThe example FINAL clinical photos are so small compared to others, although I know we can magnify them, but the quality gets worse. They should be larger and, the authors should have indicated which material they used in that particular photo. The authors should have presented both Sprinity and emax cases.\n\nIn the first paragraph of the discussion, “In addition to the similarity in microstructure of both materials being lithium silicate-based glass-ceramics.” is an incomplete sentence.\n\nIn the second paragraph, “Concerning IPS e.max CAD all-ceramic crowns, Fasbinder et al.64 stated that no color change was noted for CAD/CAM lithium disilicate crowns after 2 years of service. Rauch et al.65 reported USPHS alpha scores for all monolithic lithium disilicate crowns in the posterior zone upon evaluation after 6 and 10 years.” is nothing to do with this study. The authors did not investigate the color stability.\n\nIn the third paragraph, “Rinke et al.66 found the excellent esthetic properties of VITA SUPRINITY may be related to the homogenous dual microstructure, consisting of very fine lithium disilicate and lithium metasilicate crystals with average size of 0.5–0.7 μm which are four to eight times smaller than lithium disilicate crystallites. The result is very fine microstructure that provides a higher ratio of glass matrix/zirconium oxide in solution.” This part describes the findings of the previous study, but there is no sentence that connects the findings in this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-371
|
https://f1000research.com/articles/7-1359/v1
|
30 Aug 18
|
{
"type": "Research Article",
"title": "Virologic failure and HIV drug resistance on simplified, dolutegravir-based maintenance therapy: Systematic review and meta-analysis",
"authors": [
"Gilles Wandeler",
"Marta Buzzi",
"Nanina Anderegg",
"Delphine Sculier",
"Charles Béguelin",
"Matthias Egger",
"Alexandra Calmy",
"Marta Buzzi",
"Nanina Anderegg",
"Delphine Sculier",
"Charles Béguelin",
"Matthias Egger",
"Alexandra Calmy"
],
"abstract": "Background: Dolutegravir-containing maintenance therapy is a promising simplification strategy for virologically suppressed HIV-infected individuals. However, most of the available data to inform this strategy come from small, uncontrolled studies. We estimated the proportion of HIV-infected patients experiencing virological failure (VF) and developing drug resistance on dolutegravir (DTG)-based maintenance therapy. Methods: We searched Medline, Embase, Cochrane Central, Web of Science, and conference abstracts for studies assessing VF on DTG-based maintenance therapy. Studies including ≥5 adults with an undetectable viral load on antiretroviral therapy (ART) who switched to a DTG-based mono- or dual therapy were included. Pooled proportions of VF were estimated using random-intercept logistic meta-regression and acquired drug resistance mutations described for each strategy. Results: Of 1719 studies considered, 21 met our selection criteria, including seven interventional and 14 observational studies. Eight studies including 251 patients assessed VF on DTG monotherapy and fourteen studies including 1670 participants VF on dual therapy. The participant’s median age ranged from 43 to 63 years, their median nadir CD4 count from 90 to 399 cells/µl, and 27.6% were female. The proportion of participants experiencing VF on DTG-monotherapy was 3.6% (95% confidence interval [CI] 1.9-6.7) at 24 weeks and 8.9% (95% CI 4.7-16.2) at 48 weeks. Resistance mutations developed in seven (3.6%) participants on DTG-monotherapy. Among patients on dual therapy, ten (0.7%, 95% CI 0.4-1.3) experienced VF by 48 weeks and none developed resistance to DTG. In adjusted analyses, VF at 24 weeks was less likely on dual therapy than on monotherapy (adjusted odds ratio: 0.10, 95% CI 0.03-0.30). Conclusions: Whereas VF is relatively common on DTG maintenance monotherapy, DTG-based dual therapy appears to be a promising simplification strategy for individuals with a suppressed HIV viral load on triple-ART.",
"keywords": [
"Dolutegravir",
"simplified therapy",
"HIV",
"meta-analysis"
],
"content": "Introduction\n\nThe concept of combination antiretroviral therapy (ART) for the treatment of HIV infection was established twenty years ago, when the results of the first studies evaluating protease inhibitor-based regimens were published1. In recent years, several strategies of treatment optimization and simplification gained interest, with the objectives of improving quality of life, minimizing ART-related toxicity and drug-drug interactions (DDI), as well as reducing health-related costs. So far, ART de-escalation from three to one (mono-) or two drugs (dual-) therapies has mainly been evaluated in virologically suppressed patients. The first simplified maintenance strategy studied included a boosted protease inhibitor (bPI), with the hope that the high genetic barrier to resistance would help achieve durable virological suppression. In a meta-analysis including ten studies, bPI monotherapy was found to be inferior to triple ART for the maintenance of viral suppression2, but non-inferior with regards to loss of future treatment options3. In contrast, dual therapy with bPI and lamivudine (3TC) was found to be non-inferior to triple ART4–6 and is now recognized as a valid switch strategy by current HIV treatment guidelines in selected situations7. However, bPI-based maintenance strategies are not widely applicable because of cost, toxicity and DDI.\n\nDue to its interesting pharmacokinetic profile, good tolerability and high barrier to resistance, dolutegravir (DTG), a new integrase strand transfer inhibitor (InSTI), has attracted much interest for its use in simplified treatment regimens. While preliminary analyses of a Dutch DTG monotherapy simplification trial seemed encouraging at 24 weeks, rates of virological failures increased significantly by week 48, suggesting a sub-optimal potency of this regimen8. On the other hand, several studies evaluating DTG-based dual therapy with either 3TC or rilpivirine (RPV), showed a high virological efficacy9–12. However, most reports were from small, observational cohort studies, with the exception of one DTG-RPV industry-sponsored randomized controlled trial (RCT)11.\n\nWe performed a systematic review of the literature and a meta-analysis to provide precise estimates of the rate of virological failure (VF) and drug resistance in patients switched to a DTG-based maintenance mono- or dual therapy, and to clarify which drugs or combinations should be evaluated in further studies and implemented in clinical practice.\n\n\nMethods\n\nThe protocol for this systematic review was written and registered with the International Prospective Register of Systematic Reviews (PROSPERO registration number CRD42017070045)13. The reporting of the review followed the PRISMA guidelines14 (Supplementary File 1).\n\nWe searched Medline, EMBASE, Cochrane Central and Web of Science, as well as abstracts of major HIV conferences (CROI, AIDS, HIV Glasgow, AFRAVIH, IAS and EACS between 2013 and 2017) on 4. January 2018 for studies assessing the proportion of individuals developing VF on DTG-based maintenance therapy. In Medline we combined free text words and medical subject headings (MESH) describing the study population and the outcome (Supplementary File 2). This search strategy was adapted for the other databases. We considered RCTs, single-arm clinical trials, cohort studies, and case-series that included at least five HIV-infected adults (≥18 years) on DTG-based simplified therapy. No language restrictions were applied. Studies had to report on virological outcomes of patients who switched to a DTG monotherapy or dual therapy after having an undetectable VL on triple ART. We excluded studies that only reported in vitro data and those selecting participants based on the outcome during DTG-based maintenance therapy. Two investigators (MB and GW) independently selected studies based on titles and abstracts, and, in a second step, based on the full text of potentially eligible articles. Discrepancies in study selection were resolved through discussions with a third investigator (AC).\n\nThe following data were extracted independently for each study by two reviewers (GW and MB), using a standardized spreadsheet: bibliographic details, study design, inclusion and exclusion criteria, definitions of outcomes, country, number of participants and their main demographic and clinical characteristics, including duration since HIV diagnosis, ART history, immunological status (CD4 cell count at switch and nadir) and virological parameters (HIV RNA peak and at baseline, HIV-DNA at baseline and changes during the study, VF as defined by the study, and the presence at drug resistance at switch). Again, discrepancies in data extracted were resolved through discussions with a third investigator (AC).\n\nA checklist for the assessment of risk of bias was designed to ensure data quality assessment for each study was included. The form for RCTs included information on the sequence generation, allocation concealment, blinding (participants, personnel and outcome assessor), incomplete outcome data, selective outcome reporting and other sources of bias. The methodological components of the randomized trials were assessed by two independent authors and classified as high, low or unclear risk of bias, as recommended by the Cochrane collaboration15. For observational studies it was not appropriate to use the ROBINS-I tool16, as we only considered data from the group of patients on simplified, maintenance therapy. Thus, we assessed the population characteristics and missing outcome data for each study.\n\nWe described the study design as well as the demographic and clinical characteristics of the population from each study by type of maintenance therapy (DTG-based monotherapy or dual therapy). Pooled proportions of VF and treatment failure (VF or departure from simplified strategy due to toxicity, loss to follow-up, patient’s or physician’s decision), and 95% confidence intervals (CI) were estimated using random intercept logistic meta-regression. These analyses were performed separately at 24 weeks and 48 weeks after the switch from triple ART to maintenance therapy. For all models, statistical evidence for heterogeneity between studies was assessed using the tau-squared statistics17. We evaluated the association between type of maintenance strategy and VF using random intercept logistic meta-regression (binomial-normal) models. All models were adjusted for potential confounders, including age (median or mean), sex (proportion of female participants) and study type (interventional or observational). Furthermore, the proportion of participants acquiring new drug resistance mutations was assessed for each treatment strategy and the mutations described in detail. Statistical analyses were conducted in STATA version 14.1 (StataCorp, Texas, USA) and R version 3.2.3 (R Core Team, Vienna, Austria).\n\nThe funder of the study had no role in the design, data collection, data analysis, data interpretation of the results or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.\n\n\nResults\n\nOf 1719 single studies identified, 63 remained potentially eligible after the screening of titles and abstracts. Of these, 21 studies, including four RCTs, three single-arm clinical trials and 14 observational studies met our inclusion criteria8,11,12,18–35 (Figure 1). A description of the main study characteristics by type of maintenance strategy is given in Table 1. Eight studies (two from France, two from The Netherlands, one from Germany, one from Switzerland and two from Spain) including 251 patients assessed the switch to DTG monotherapy and 14 (five from Italy, four from France, three for Spain, one from US, and one multi-country study), including 1670 participants, the switch to DTG-based dual therapy. Dual therapy consisted of DTG + 3TC (seven studies) or RPV (four studies) or atazanavir (ATV, two studies) or darunavir (DRV, one study). Overall, 14 studies allowed the inclusion of patients with previous virological failure, including five monotherapy studies18–20,22–24. In one study, patients with previous InSTI failure were also included12. Nineteen studies assessed virological outcomes at six months of maintenance therapy, whereas ten of them additionally showed outcomes at one year8,11,12,24,27,29–33,35. Two studies assessed virological outcomes only at 48 weeks25,27. Median (or mean) age of participants included in the studies varied from 43 years11 to 63 years24 and 27.6% of them were female. 16 studies reported on the median nadir CD4 cell count, which ranged from 90 cells/µl12 to 399 cells/µl29.\n\nAbbreviations: VF: virologic failure, ART: antiretroviral therapy, DTG: dolutegravir, 3TC: lamivudine, FTC: emtricitabine, RPV: rilpivirine, ATV: atazanavir, InSTI: integrase stand transfert inhibitor, F: France, NL: The Netherlands, D: Germany, E: Spain, CH: Switzerland, I: Italy, USA: United States of America, O: observational study; I: interventional study\n\n£: no abnormal standard biological parameter\n\nAll RCTs were open-label non-inferiority trials8,18,35, of which one was a single center trial18 and three were multicenter trials8,11,35. They reported adequate generation of random allocation sequences and allocation concealment. Three single-arm trials were included, of which two included less than 10 patients21,24,29. All interventional studies adequately addressed incomplete outcome data: proportions of drop-outs were low and outcome data were missing for less than 20% of participants in all studies. Five of seven trials reported on virological outcomes at both time-points of interest for this study (24 and 48 weeks)8,11,24,29,35. There was no evidence of selective reporting in any of the studies. In each of the 14 observational studies included in this review, the main demographic and clinical characteristics of the study populations were similar and patients were followed for 24 weeks in most studies. Among the observational studies, the majority did not report detailed inclusion and exclusion criteria. Five observational studies reported virological outcomes at both time-points12,30–33. Amplification for drug resistance testing was successful for 19 of the 27 (70%) patients with VF. Finally, patient retention was over 90% in all 14 cohort studies.\n\nThe pooled estimate of the proportion of participants who experienced a VF on DTG-based monotherapy was 3.6% (95% CI 1.9-6.7) at 24 weeks and 8.9% (95% CI 4.7-16.2) at 48 weeks (Figure 2). The high proportion of treatment failures among patients on monotherapy at 48 weeks was driven by the two studies from the Netherlands, which observed between 8 and 20% of VF8,24. Among patients on dual therapy, an estimated 0.4% (95% CI 0.2-0.9) experienced a VF at 24 weeks and 0.7% (95% CI 0.4-1.3) at 48 weeks. Independently of the combination used (DTG/3TC, DTG/RPV, DTG/ATV or DTG/DRV), 11 of 14 studies evaluating the effectiveness of dual therapy had less than 1% of patients developing VF. Compared to patients on monotherapy, those on dual therapy were less likely to experience VF by 24 weeks (odds ratio [OR] 0.10, 95% CI 0.03-0.32, p<0.001) and 48 weeks (OR 0.07, 95% CI 0.03-0.18, p<0.001). In analyses adjusted for study type (interventional or observational), age (median or mean) and sex (proportion of female participants), the OR for VF at 24 weeks and 48 weeks were very similar to the unadjusted estimates (aOR 0.10, 95% CI 0.03-0.30 for 24 weeks and aOR 0.06, 95% CI 0.01-0.30 for 48 weeks, respectively). The only variable that contributed to explaining the between-study heterogeneity in both the 24 and 48-week analyses was treatment strategy. When including this variable, the tau-squared were reduced from 1.17 (95% CI 0.33-2.19) to 0.00 (95% CI 0.00-1.11) in the 24 week analysis and from 1.37 (95% CI 0.54-2.15) to 0.00 (95% CI 0.00-1.00) in the 48 week analysis. The inclusion of other variables did not impact the estimates of tau-squared.\n\nTreatment failure occurred in 5.2% (2.0–12.9) of patients at 24 weeks and 12.3% (4.5–29.4) at 48 weeks on DTG-monotherapy, whereas this outcome was observed in 2.8% (1.4–5.7) of patients at 24 weeks and 6.5% (4.3–9.6) at 48 weeks on DTG-based dual therapy. At 24 weeks, patients on dual therapy tended to be less likely to experience treatment failure compared to those on monotherapy (aOR 0.52, 95% CI 0.15-1.85). Due to multi-collinearity in the model, we were not able to report on multivariable analyses comparing treatment failure between mono and dual therapy at 48 weeks.\n\nAcquired resistance mutations to InSTI developed in 9/251 (3.6%) participants on DTG-based monotherapy, which corresponded to 56% of the cases of VF (Table 2). Three individuals developed the Q148R or Q148H mutation in combination with other resistance mutations, conferring high-level resistance to DTG20,22. These three patients did not have a history of previous VF and had a suppressed HIV viral load for several years before switching to DTG-monotherapy. No InSTI resistance mutations developed in patients on dual therapy. Of 962 patients on RPV/DTG, only one developed a major drug resistance mutation to non-nucleoside reverse transcriptase inhibitors (K101E). No resistance was observed in plasma among 237 individuals on DTG/3TC.\n\n*bold: InSTI resistance\n\n**in 7% of integrated DNA in PBMC\n\n*** in ≤1.5% of integrated DNA in PBMC\n\n£ in integrated DNA in PBMC\n\n\nDiscussion\n\nWe performed a comprehensive systematic review of studies that reported on VF among patients switched to DTG-based maintenance therapy. Our meta-analysis shows that DTG-based dual therapy is successful in sustaining virological control in ART-experienced HIV-infected patients: only 12 of 1670 (0.7%) experienced a VF and none of them developed resistance mutations to DTG. On the contrary, 16 of 251 (6.4%) individuals switched to DTG monotherapy had a VF, of which more than one-half developed resistance to DTG. Although the proportion of patients experiencing a confirmed viral rebound on DTG-monotherapy does not seem to be higher than in patients on PI-monotherapy, the risk of losing future treatment options is higher with DTG-monotherapy3. Overall, our findings suggest that DTG-based monotherapy is not an appropriate simplification strategy and that further studies are urgently needed to confirm the long-term efficacy of DTG-based dual therapy.\n\nDTG-based dual therapy is a promising simplification strategy, especially when combined with 3TC or emtricitabine (FTC, both compounds referred to as XTC), as the likelihood of developing toxicity events and DDI on such regimens is very low. No drug resistance mutations to DTG developed among more than 1600 patients on dual therapy followed for 24 to 48 weeks and only one had a resistance mutation to another drug class. Although based on very few patients, the results seemed to be independent of previous virological failures. For instance, no virological failures were noted among patients on DTG/3TC despite the presence of a 184V mutation at the time of simplification in several studies. The impact of the latter mutation on viral fitness has been extensively described and could also potentially explain the improved treatment outcomes in these patients compared to those switched to DTG-monotherapy without any previous failures. Interestingly, similar observations were made for bPI-based regimens, for which efficacy was improved when 3TC was added, despite the presence of the 184V mutation36. Although the proportion of patients experiencing a confirmed viral rebound on DTG-monotherapy does not seem to be higher than in patients on PI-monotherapy, their chances of losing future treatment options is higher than reported in most PI-monotherapy trials\n\nWe also report on estimates of treatment failure, which includes other reasons for treatment interruptions, such as toxicity or loss to follow-up. In our meta-analysis, the proportion of patients experiencing this combined outcome was more than twice as high among patients on monotherapy compared to those on DTG-based dual therapy. Although this outcome is important in evaluating the clinical efficacy of a novel ART strategy, our capacity to analyze this outcome in detail was limited by the missing information on the specific reasons for treatment interruptions in many studies and by the small number of events, especially at 48 weeks of therapy.\n\nOf all simplification strategies evaluated to date, the DTG/XTC combination could be the one most readily accessible for patients in low- and middle-income countries: both DTG and XTC are available and prequalified by stringent regulatory authorities in generic formulations. In order to be widely implemented, the efficacy of this dual combination should first be evaluated in large studies among different patient populations. The results from the studies included in our meta-analysis are mainly based on selected populations of HIV-infected individuals from European cohorts, and are not generalizable. Furthermore, long-term data are needed, as most treatment failures occurred after the first 24 weeks in several monotherapy studies. Recently, results from the only study which assessed 96-week outcomes with this regimen to date were reported: among 27 ART-experienced individuals with previous VF, DTG/3TC was 100% efficacious virologically12. However, despite these encouraging results, data from larger studies are needed. In addition, more data on the activity of DTG-based simplified regimens in compartments other than blood are needed. Letendre et al. showed that DTG achieved therapeutic concentrations in the central nervous system (CNS), with a CNS penetration effectiveness score of four37. However, these results were based on a very small sample of patients and data from individuals on simplified, DTG-based therapies are lacking.\n\nAs a wealth of data on the efficacy of DTG-maintenance strategies from small studies are being disseminated at a fast pace, this systematic review is the first analysis to provide comparative estimates of virological failure between DTG-based monotherapy and dual therapy. More than 1700 studies were screened, including abstracts from all important HIV conferences in the past years. As our meta-analysis included studies with diverse study designs and populations, it could be argued that the comparison of studies with such differences might be problematic. However, the estimates of VF were very similar across studies, especially in the DTG-based dual therapy arm. This finding highlights the potency of this combination, even in the presence of previous drug resistance mutations or multiple co-morbidities. Unfortunately, only studies including low numbers of patients reported outcomes from individuals on DTG-monotherapy, and data on dual therapy was dominated by one large study that assessed the efficacy of the DTG/RPV combination. As a consequence, the comparison of DTG-monotherapy vs. DTG/XTC, which would have been the most interesting one, was not possible. Furthermore, the lack of availability of individual data from the different studies precluded the analysis of risk factors of VF in the different simplification regimens. As most studies were observational, it is possible that the investigators mainly included patients with good adherence, which may have limited the generalizability of their findings. Finally, our results might have slightly under-estimated the proportion of patients with VF as individuals who were lost to follow-up might have experienced this outcome without them being accounted for. However, our treatment failure estimates showed that even when other reasons of treatment failure were considered, DTG-based dual therapy was superior to monotherapy.\n\nIn summary, DTG-based dual maintenance therapy seems to be a promising simplification strategy with high virological efficacy and low potential for DDI and toxicity. Such a treatment regimen could be an interesting alternative to classical triple-ART in selected patients. Furthermore, dual therapy might be a cost-effective global ART strategy38. A number of large prospective studies evaluating the efficacy of DTG-based dual therapy are under way and will inform its potential implementation at a large scale. In addition to the studies on maintenance therapy39,40, clinical trials are also assessing the efficacy of DTG/XTC in treatment-naïve patients41. Furthermore, it will be critical to evaluate the efficacy of DTG-XTC dual therapy in specific sub-groups such as pregnant and breast-feeding women, adolescents, patients with previous failure to standard triple regimens and harboring the M184V resistance mutation, as well as in patients with HIV associated neurocognitive disorder and tuberculosis coinfection.\n\n\nData availability\n\nF1000Research: Dataset 1. Dolutegravir meta-analysis summary data., 10.5256/f1000research.15995.d21572442",
"appendix": "Grant information\n\nThis study was supported by the Swiss National Science Foundation (Ambizione-PROSPER fellowship PZ00P3_154730 to GW, grant 32FP30-174281 to ME, and grant 33IC30_166819 to AC).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Completed PRISMA checklist.\n\nClick here to access the data.\n\nSupplementary File 2: Search strategy for Medline.\n\nClick here to access the data.\n\n\nReferences\n\nGulick RM, Mellors JW, Havlir D, et al.: Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. N Engl J Med. 1997; 337(11): 734–9. PubMed Abstract | Publisher Full Text\n\nMathis S, Khanlari B, Pulido F, et al.: Effectiveness of protease inhibitor monotherapy versus combination antiretroviral maintenance therapy: a meta-analysis. PLoS One. 2011; 6(7): e22003. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaton NI, Stöhr W, Arenas-Pinto A, et al.: Protease inhibitor monotherapy for long-term management of HIV infection: a randomised, controlled, open-label, non-inferiority trial. Lancet HIV. 2015; 2(10): e417–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPulido F, Ribera E, Lagarde M, et al.: Non-inferiority of dual-therapy (DT) with darunavir/ritonavir (DRV/r) plus 3TC versus triple-therapy (TT) with DRV/r plus TDF/FTC or ABC/3TC for maintenance of viral suppression: 48-week results of the DUAL-GESIDA 8014 trial - abstract n°: 0331. HIV Glasgow Conference. Glasgow, 23–26 October 2016. 2016. Reference Source\n\nArribas JR, Girard PM, Landman R, et al.: Dual treatment with lopinavir-ritonavir plus lamivudine versus triple treatment with lopinavir-ritonavir plus lamivudine or emtricitabine and a second nucleos(t)ide reverse transcriptase inhibitor for maintenance of HIV-1 viral suppression (OLE): a randomised, open-label, non-inferiority trial. Lancet Infect Dis. 2015; 15(7): 785–92. PubMed Abstract | Publisher Full Text\n\nPerez-Molina JA, Rubio R, Rivero A, et al.: Dual treatment with atazanavir-ritonavir plus lamivudine versus triple treatment with atazanavir-ritonavir plus two nucleos(t)ides in virologically stable patients with HIV-1 (SALT): 48 week results from a randomised, open-label, non-inferiority trial. Lancet Infect Dis. 2015; 15(7): 775–84. PubMed Abstract | Publisher Full Text\n\nEACS Guidelines version 9.0. 2017. Reference Source\n\nWijting I, Rokx C, Boucher C, et al.: Dolutegravir as maintenance monotherapy for HIV (DOMONO): a phase 2, randomised non-inferiority trial. Lancet HIV. 2017; 4(12): e547–e554. PubMed Abstract | Publisher Full Text\n\nCahn P, Rolón MJ, Figueroa MI, et al.: Dolutegravir-lamivudine as initial therapy in HIV-1 infected, ARV-naive patients, 48-week results of the PADDLE (Pilot Antiretroviral Design with Dolutegravir LamivudinE) study. J Int AIDS Soc. 2017; 20(1): 21678. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCharpentier C, Peytavin G, Lê M, et al.: High virological suppression rates regardless to the genotypic susceptibility score after switching to a dolutegravir-based regimen: W48 results in a prospective cohor. Abstract number: MOPEB0316. International AIDS Society (IAS) conference. Paris, 23–26 July 2017. 2017. Reference Source\n\nLlibre JM, Hung CC, Brinson C, et al.: Efficacy, safety, and tolerability of dolutegravir-rilpivirine for the maintenance of virological suppression in adults with HIV-1: phase 3, randomised, non-inferiority SWORD-1 and SWORD-2 studies. Lancet. 2018; 391(10123): 839–49. PubMed Abstract | Publisher Full Text\n\nReynes J, Meftah N, Tuaillon E, et al.: Dual regimen with dolutegravir and lamivudine maintains virologic suppression even in heavily treatment experienced HIV-infected patients: 96 weeks results from maintenance DOLULAM study. Abstract n°: MOPEB0322. International AIDS Society (IAS) conference. Paris, 23–26 July 2017. 2017. Reference Source\n\nBooth A, Clarke M, Dooley G, et al.: The nuts and bolts of PROSPERO: an international prospective register of systematic reviews. Syst Rev. 2012; 1: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHiggins JP, Altman DG, Gøtzsche PC, et al.: The Cochrane Collaboration's tool for assessing risk of bias in randomised trials. BMJ. 2011; 343: d5928. PubMed Abstract | Publisher Full Text | Free 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. 2016; 355: i4919. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlanco JL, Rojas J, Paredes R, et al.: Dolutegravir-based maintenance monotherapy versus dual therapy with lamivudine: a planned 24 week analysis of the DOLAM randomized clinical trial. J Antimicrob Chemother. 2018; 73(7): 1965–1971. PubMed Abstract | Publisher Full Text\n\nGubavu C, Prazuck T, Niang M, et al.: Dolutegravir-based monotherapy or dual therapy maintains a high proportion of viral suppression even in highly experienced HIV-1-infected patients. J Antimicrob Chemother. 2016; 71(4): 1046–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatlama C, Soulié C, Caby F, et al.: Dolutegravir as monotherapy in HIV-1-infected individuals with suppressed HIV viraemia. J Antimicrob Chemother. 2016; 71(9): 2646–50. PubMed Abstract | Publisher Full Text\n\nSculier D, Doco-Lecompte T, Yerly S, et al.: Stable HIV-1 reservoirs on dolutegravir maintenance monotherapy: the MONODO study. HIV Med. 2018; 19(8): 572–577. PubMed Abstract | Publisher Full Text\n\nOldenbuettel C, Wolf E, Ritter A, et al.: Dolutegravir monotherapy as treatment de-escalation in HIV-infected adults with virological control: DoluMono cohort results. Antivir Ther. 2017; 22(2): 169–72. PubMed Abstract | Publisher Full Text\n\nRojas J, Blanco JL, Marcos MA, et al.: Dolutegravir monotherapy in HIV-infected patients with sustained viral suppression. J Antimicrob Chemother. 2016; 71(7): 1975–81. PubMed Abstract | Publisher Full Text\n\nRokx C, Schurink CA, Boucher CA, et al.: Dolutegravir as maintenance monotherapy: first experiences in HIV-1 patients. J Antimicrob Chemother. 2016; 71(6): 1632–6. PubMed Abstract | Publisher Full Text\n\nBonijoly T CA, Cheret A, Cotte L, et al.: Week-48 efficacy and safety of dolutegravir + rilpivirine dual therapy as switch strategy in a real-life cohort study. Abstract n°: PE9/15 European AIDS Clinical Society (EACS) conference, Milano, 25–27 October 2017. 2017.\n\nBorghetti A, Baldin G, Ciccullo A, et al.: Virological control and metabolic improvement in HIV-infected, virologically suppressed patients switching to lamivudine/dolutegravir dual therapy. J Antimicrob Chemother. 2016; 71(8): 2359–61. PubMed Abstract | Publisher Full Text\n\nCastagna AGR, Rusconi S, Riva A, et al.: Durability and Safety of a Dual Antiretroviral Regimen with Dolutegravir and Unboosted Atazanavir in HIV-1 Infected Patients with Virological Suppression. Abstract n°: PE9/63. European AIDS Clinical Society (EACS) conference, Milano, 25–27 October 2017. 2017.\n\nGantner P, Cuzin L, Allavena C, et al.: Efficacy and safety of dolutegravir and rilpivirine dual therapy as a simplification strategy: a cohort study. HIV Med. 2017; 18(9): 704–8. PubMed Abstract | Publisher Full Text\n\nJoly V, Burdet C, Landman R, et al.: Promising Results of Lamivudine + DolutegravirMaintenance Therapy in ANRS 167 Lamidol Trial. Abstract number: 458. Conference on Retrovirus and Opportunistic Infections (CROI), Seattle, 13–16 February 2017. 2017. Reference Source\n\nMaggiolo F, Gulminetti R, Pagnucco L, et al.: Lamivudine/dolutegravir dual therapy in HIV-infected, virologically suppressed patients. BMC Infect Dis. 2017; 17(1): 215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaggiolo FGR, Pagnucco L, Digaetano M, et al.: Durability of dolutegravir + lamivudine as simplification cART in patients with suppressed HIV-RNA. Abstract number: PE9/49. European AIDS Clinical Society (EACS) conference, Milano, 25–27 October 2017. 2017.\n\nNavarro JSJ, Silva A, Burgos J, et al.: Efficacy of Once-daily Dolutegravir Plus Boosted-Darunavir as a Switch Strategy in HIV-infected Heavily-treated Patients. Abstract n°: PE9/93. European AIDS Clinical Society (EACS) conference, Milano, 25–27 October 2017. 2017.\n\nRevuelta-Herrero JL, Chamorro-de-Vega E, Rodríguez-González CG, et al.: Effectiveness, Safety, and Costs of a Treatment Switch to Dolutegravir Plus Rilpivirine Dual Therapy in Treatment-Experienced HIV Patients. Ann Pharmacother. 2018; 52(1): 11–8. PubMed Abstract | Publisher Full Text\n\nRiva AP RS, Bonora S, Cattelan M, et al.: Dolutegravir and unboosted atazanavir: a dual NRTI‐ and booster‐free antiretroviral regimen simplification in HIV-1 infected patients with viral suppression. Abstract number: P090. HIV Glasgow Conference, Glasgow, 23–26 October 2016. 2016.\n\nTaiwo BO, Marconi VC, Berzins B, et al.: Dolutegravir Plus Lamivudine Maintains Human Immunodeficiency Virus-1 Suppression Through Week 48 in a Pilot Randomized Trial. Clin Infect Dis. 2018; 66(11): 1794–1797. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCiaffi L, Koulla-Shiro S, Sawadogo AB, et al.: Boosted protease inhibitor monotherapy versus boosted protease inhibitor plus lamivudine dual therapy as second-line maintenance treatment for HIV-1-infected patients in sub-Saharan Africa (ANRS12 286/MOBIDIP): a multicentre, randomised, parallel, open-label, superiority trial. Lancet HIV. 2017; 4(9): e384–e92. PubMed Abstract | Publisher Full Text\n\nLetendre SL, Mills AM, Tashima KT, et al.: ING116070: a study of the pharmacokinetics and antiviral activity of dolutegravir in cerebrospinal fluid in HIV-1-infected, antiretroviral therapy-naive subjects. Clin Infect Dis. 2014; 59(7): 1032–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGirouard MP, Sax PE, Parker RA, et al.: The Cost-effectiveness and Budget Impact of 2-Drug Dolutegravir-Lamivudine Regimens for the Treatment of HIV Infection in the United States. Clin Infect Dis. 2016; 62(6): 784–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCamly A BM, Sculier D, Wandeler G: Evaluation of a simplified strategy for the long-term management of HIV infection: a non-inferiority, randomized, controlled, open-label clinical trial: The Simpl'HIV Trial. NCT03160105.\n\nE. M. Dolutegravir-based Simplification Strategies: DOLAM. EudraCT 2015-000274-35.\n\nTaiwo BO, Zheng L, Stefanescu A, et al.: ACTG A5353: A Pilot Study of Dolutegravir Plus Lamivudine for Initial Treatment of Human Immunodeficiency Virus-1 (HIV-1)-infected Participants With HIV-1 RNA <500000 Copies/mL. Clin Infect Dis. 2018; 66(11): 1689–1697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWandeler G, Buzzi M, Anderegg N, et al.: Dataset 1 in: Virologic failure and HIV drug resistance on simplified, dolutegravir-based maintenance therapy: Systematic review and meta-analysis. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.15995.d215724"
}
|
[
{
"id": "37755",
"date": "03 Sep 2018",
"name": "Esteban Martinez",
"expertise": [
"Reviewer Expertise Antiretroviral therapy strategies",
"complications of HIV infection and its 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 paper compared the efficacy of dolutegravir-based monotherapy vs dual therapy using the methodology of systematic review and meta-analysis. The topic is of great interest as many different studies with these simplication strategies have been done. Although dolutegravir monotherapy is not recommended at present due to the risk of virological failure with development of resistance mutations and dolutegravir dual therapy seems a promising strategy with recent evidence from large clinical trials, this systematic review and meta-analysis is timely because there are almost no direct comparisons between dolutegravir-based monotherapy vs dual therapy. The design and the methods (including PRISMA reporting) are adequate, as they are the interpretation of results. It is interesting that not only monotherapy was inferior to dual therapy but the difference resulted highly increased from 24 weeks to 48 weeks of follow-up, thus indicating that the risk of failure with the monotherapy strategy may greatly increase after the initial 24 weeks of follow-up. This is remarkable as many exploratory studies had 24-week results only.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4394",
"date": "03 Apr 2019",
"name": "Gilles Wandeler",
"role": "Author Response",
"response": "No comments needing to be addressed"
}
]
},
{
"id": "37757",
"date": "10 Sep 2018",
"name": "Laurent Hocqueloux",
"expertise": [
"Reviewer Expertise Monotherapy and dual therapy",
"HIV reservoirs",
"primary-infection",
"post-treatment control."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWandeler and colleagues provide here an excellent and comprehensive review on DTG-based maintenance therapy, even though this review will lack the latest communications on the topic (at IAS 2018). Nevertheless, the conclusions they make are in accordance with what is currently admitted by experts in the field and worldwide guidelines: DTG monotherapy lead to an unacceptable virologic failure (VF) rate (because of >50% of emerging mutations to the class) whereas dual therapy has an excellent efficacy and no VF with mutations to the class.\n\nI only have minor comments or questions: Results:\n(page 4) Can the authors provide any data on the impact of CD4 nadir on VF during monotherapy (as described by Wijting in the DOMONO trial)? (page 8) Authors should give more explicit conclusion on this paragraph “The only variable that contributed to explaining the between-study heterogeneity in both the 24 and 48-week analyses was treatment strategy. When including this variable, the tau-squared were reduced from 1.17 (95% CI 0.33-2.19) to 0.00 (95% CI 0.00-1.11) in the 24 week analysis and from 1.37 (95% CI 0.54-2.15) to 0.00 (95% CI 0.00-1.00) in the 48 week analysis. The inclusion of other variables did not impact the estimates of tau-squared.”\nDiscussion:\n(page 9) Please provide some references (at least one) for the impact of M184V on viral fitness (this one is of interest for DTG-based regimen: doi: 10.1097/QAD.0000000000001191) (page 9) I think authors should provide some data on VF (%, emerging mutations) during switch from a triple therapy to another one (in order to have an “historical comparator” for dual therapy) (page 10, “In addition, more data on the activity of DTG-based simplified regimens in compartments other than blood are needed.”) There are some references for mono- or dual-therapy in the genital tract (Hocqueloux et al.1 and Gianella et al.2)and CNS (Doco Lecompte et al.3) (page 10) Authors should cite recent reports (all communicated at the IAS 2018 in Amsterdam) confirming their conclusions, even though they cannot include them in the analyses: two randomized-controlled clinical trials on DTG monotherapy (Braun et al.4 and Hocqueloux et al.5), the extended follow-up of the SWORD trials at week 100 (Aboud et al.6) and results of the GEMINI trials (Cahn et al.7 ).\nReferences:\n(references 25 and 28) I think two references (Gantner and Bonijoly) are duplicates (as they are based on the analyze of the same database; Bonijoly et al. have included more patients / with longer duration of follow-up than Gantner).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4393",
"date": "03 Apr 2019",
"name": "Gilles Wandeler",
"role": "Author Response",
"response": "(page 4) Can the authors provide any data on the impact of CD4 nadir on VF during monotherapy (as described by Wijting in the DOMONO trial)? Unfortunately, we do not have these data from most of the studies. (page 8) Authors should give more explicit conclusion on this paragraph “The only variable that contributed to explaining the between-study heterogeneity in both the 24 and 48-week analyses was treatment strategy. When including this variable, the tau-squared were reduced from 1.17 (95% CI 0.33-2.19) to 0.00 (95% CI 0.00-1.11) in the 24 week analysis and from 1.37 (95% CI 0.54-2.15) to 0.00 (95% CI 0.00-1.00) in the 48 week analysis. The inclusion of other variables did not impact the estimates of tau-squared.” The first sentence says explicitly that only the treatment strategy was found to contribute to heterogeneity, and we are not sure how to make the conclusion more explicit. The second sentence quantifies the statement. (page 9) Please provide some references (at least one) for the impact of M184V on viral fitness (this one is of interest for DTG-based regimen: doi: 10.1097/QAD.0000000000001191) We included references in the new version of the paper (see 2nd paragraph in the discussion section). (page 9) I think authors should provide some data on VF (%, emerging mutations) during switch from a triple therapy to another one (in order to have an “historical comparator” for dual therapy) We added a comment in the 1st paragraph of the discussion section. « We performed a comprehensive systematic review of studies that reported on VF among patients switched to DTG-based maintenance therapy. Our meta-analysis shows that DTG-based dual therapy is successful in sustaining virological control in ART-experienced HIV-infected patients: only 12 of 1670 (0.7%) experienced a VF and none of them developed resistance mutations to DTG. For comparison, recent trials testing InSTI-based triple maintenance combinations showed a similar virological failure rate (ref ci dessous 1-4). On the contrary, 16 of 251 (6.4%) individuals switched to DTG monotherapy had a VF, of which more than one-half developed resistance to DTG. » (page 10) “In addition, more data on the activity of DTG-based simplified regimens in compartments other than blood are needed.” There are some references for mono- or dual-therapy in the genital tract (Hocqueloux et al.1 and Gianella et al.2) and CNS (Doco Lecompte et al.3) We modified the text in the new version of the paper (see 4th paragraph of the discussion section). « In addition, more data on the activity of DTG-based simplified regimens in compartments other than blood are needed. Letendre et al. Showed that DTG achieved therapeutic concentrations in the central nervous system (CNS), with a CNS penetration effectiveness score of four. In the MONODO study, all patients had an undetectable plasma HIV viral load at week 24 on DTG maintenance monotherapy, whereas only one had a detectable viral load in the cerebrospinal fluid (ref 21 by Sculier et al). Moreover, levels of HIV-1 RNA in the genital tract on DTG-monotherapy (nouvelle ref 1 ) and under DTG-3TC (nouvelle ref 2) were comparable to those under standard cART. However, these results were based on a very small sample of patients and data from individuals on simplified, DTG-based therapies are lacking. » (page 10) Authors should cite recent reports (all communicated at the IAS 2018 in Amsterdam) confirming their conclusions, even though they cannot include them in the analyses: two randomized-controlled clinical trials on DTG monotherapy (Braun et al.4 and Hocqueloux et al.5), the extended follow-up of the SWORD trials at week 100 (Aboud et al.6) and results of the GEMINI trials (Cahn et al.7). In accordance with the pre-specified analysis plan described in our study protocol, we decided not to include these results as they had not been presented at the time of the last version of our analyses. References: (references 25 and 28) I think two references (Gantner and Bonijoly) are duplicates (as they are based on the analyze of the same database; Bonijoly et al. have included more patients / with longer duration of follow-up than Gantner). We agree with the reviewer. However, as one study reported 24 weeks and the other 48 weeks outcomes, we decided to keep both estimates. Furthermore, we did not combine them as the study populations were not exactly the same."
}
]
},
{
"id": "37756",
"date": "10 Sep 2018",
"name": "Omar Sued",
"expertise": [
"Reviewer Expertise HIV clinical trials"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is extremely important, the conclusion is very relevant for informing the clinical practice and the timing is perfect. Please see below some comments to improve this excellent systematic review\nNote that reference 9 is for naive patients, and not for switching. REPRODUCIBILITY: Risk of bias checklist is not available. It could be good idea to include as supplementary material a sample of the checklist of assessment of bias. The number of reviewed studies is 21, but the authors reported 8 for monotherapy and 14 for dual therapy without clarifying that some are focused on both strategies. Similarly, in figure 2, the name of the author is followed by the (n) of participants. This makes the reader think this is the reference. Please consider to add the reference number in this place, and an additional column for \"n\" Regarding the phrase \"These three patients did not have a history of previous VF and had a suppressed HIV viral load for several years before switching to DTG-monotherapy\". Please comment if patients experiencing INSTI resistance in monotherapy were previously exposed to INSTI. Please review Table 2, because some INSTI mutations are not in bold, and the $ symbol is not explained in notes In discussion you mention \"For instance, no virological failures were noted among patients on DTG/3TC despite the presence of a 184V mutation at the time of simplification in several studies.\" but you are not showing this result. Given the potential clinical importance of this issue, consider show how many participants had this mutation and show the outcomes. In the discussion, these phrases look similar:\n\nFirst Paragraph: \"Although the proportion of patients experiencing a confirmed viral rebound on DTG-monotherapy does not seem to be higher than in patients on PI-monotherapy, the risk of losing future treatment options is higher with DTG-monotherapy\" Second Paragraph \"Although the proportion of patients experiencing a confirmed viral rebound on DTG-monotherapy does not seem to be higher than in patients on PI-monotherapy, their chances of losing future treatment options is higher than reported in most PI-monotherapy trials\"\n\nWhy to mention DTG-XTC if no study was presented with FTC?. I would suggest to stick to the presented data, therefore to discuss about 3TC.\n\nThe phrase \"In addition to the studies on maintenance therapy39,40, clinical trials are also assessing the efficacy of DTG/XTC in treatment-naïve patients\" should be updated based on the results of GEMINI1&2. It could be good to try to explain the higher rate of failure in those three trials in dual therapy (Blanco, and Navarro). Please check the failure rate in figure 2 for Taiwo (44 patients with failure 2.7%).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4392",
"date": "03 Apr 2019",
"name": "Gilles Wandeler",
"role": "Author Response",
"response": "Note that reference 9 is for naive patients, and not for switching. We agree with the comment. However, we prefer to keep this unchanged as we used the reference in a generic sentence. REPRODUCIBILITY: Risk of bias checklist is not available. It could be good idea to include as supplementary material a sample of the checklist of assessment of bias. We used the Cochrane Collaboration list to assess risk of bias among the two RCT included in the meta-analysis. For observational studies, it was not appropriate to use the ROBINS-I tool, as we only considered data from the group of patients maintenance therapy. Thus, we assessed the population characteristics and missing outcome data for each study. This is explained in detail in the methods section of the published manuscript. The number of reviewed studies is 21, but the authors reported 8 for monotherapy and 14 for dual therapy without clarifying that some are focused on both strategies. We explained this in the text (see data analysis section). Similarly, in figure 2, the name of the author is followed by the (n) of participants. This makes the reader think this is the reference. Please consider to add the reference number in this place, and an additional column for \"n\" The reference is indicated differently in the text and we decided to keep the figure 2 unchanged. Regarding the phrase (page 9) \"These three patients did not have a history of previous VF and had a suppressed HIV viral load for several years before switching to DTG-monotherapy\". Please comment if patients experiencing INSTI resistance in monotherapy were previously exposed to INSTI. We included this information in the new version of the paper (see drug resistance section). Please review Table 2, because some INSTI mutations are not in bold, and the $ symbol is not explained in notes We adapted the table 2 in the new version of the paper. In discussion you mention \"For instance, no virological failures were noted among patients on DTG/3TC despite the presence of a 184V mutation at the time of simplification in several studies.\" but you are not showing this result. Given the potential clinical importance of this issue, consider show how many participants had this mutation and show the outcomes. As this information was missing from most studies, we described cases where reported but could not formally evaluate the association between specific mutations and treatment failure. In the discussion, these phrases look similar: First Paragraph: \"Although the proportion of patients experiencing a confirmed viral rebound on DTG-monotherapy does not seem to be higher than in patients on PI-monotherapy, the risk of losing future treatment options is higher with DTG-monotherapy\" Second Paragraph \"Although the proportion of patients experiencing a confirmed viral rebound on DTG-monotherapy does not seem to be higher than in patients on PI-monotherapy, their chances of losing future treatment options is higher than reported in most PI-monotherapy trials\" Adapted in the text (doubled sentence cancelled from the 2nd paragraph of the discussion section). Why to mention DTG-XTC if no study was presented with FTC?. I would suggest to stick to the presented data, therefore to discuss about 3TC. Adapted in the new version of the paper (see discussion section): XTC canceled and FTC used in reference to study using FTC. The phrase \"In addition to the studies on maintenance therapy39,40, clinical trials are also assessing the efficacy of DTG/XTC in treatment-naïve patients\" should be updated based on the results of GEMINI1&2. Adapted in the new version of the paper (see discussion section) It could be good to try to explain the higher rate of failure in those three trials in dual therapy (Blanco, and Navarro). This is an important comment. However, available data within these studies did not allow us to make specific conclusions. Blanco dual: 3.45% VF at W24. Bonijoly: 1.49% VF at W48. Navarro: 3.70% of VF at W48. Taiwo: 2.27% VF at W 24 and W48 All others dual < 1% of VF both at 24 and 48 weeks. Please check the failure rate in figure 2 for Taiwo (44 patients with failure 2.7%). We checked again data and did not find any error in the figure. Only one of the 44 patients exposed to dual therapy had a rel VF (1/44 0 2.7%). with the FDA snapshot algorithm 2 other patients (one lost to FUP and one switching therapy because AE) were considered as failure by authors at W24."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1359
|
https://f1000research.com/articles/8-38/v1
|
09 Jan 19
|
{
"type": "Research Note",
"title": "A simple formula for enumerating comparisons in trials and network meta-analysis",
"authors": [
"Farhad Shokraneh",
"Clive E. Adams",
"Clive E. Adams"
],
"abstract": "We present use of a simple formula to calculate the number of pairwise comparisons of interventions within a single trial or network meta-analyses. We used the data from our previous network meta-analysis to build a study-based register and enumerated the direct pairwise comparisons from the trials therein. We then compared this with the number of comparisons predicted by use of the formula and finally with the reported number of comparisons (indirect or direct) within the network meta-analysis. A total of 133 trials included in the network generated 163 comparisons (16 unique direct comparisons for 8 interventions). The formula predicted an expected 28 indirect or direct comparisons and this is the number that were indeed reported. The formula produces an accurate enumeration of the potential comparisons within a single trial or network meta-analysis. Its use could help transparency of reporting should a shortfall occur between comparisons actually used and the potential total.",
"keywords": [
"Pairwise Comparisons",
"Study-Based Registers",
"Clinical Trials",
"Randomised Controlled Trials",
"Network Meta-Analysis",
"Systematic Reviews"
],
"content": "Introduction\n\nThe pairwise comparisons reported within each randomized controlled trial are being documented in study-based registers1. This lends itself to accurate indexing and enumeration of these comparisons within the studies and then subsequent supply of immediate, highly sensitive and highly specific search results to those wishing to investigate one or more particular comparisons within systematic reviews and meta-analyses or overviews and network meta-analysis1,2.\n\nTo gain a perspective on the absolute effectiveness of a treatment it is ideal to compare all the existing medications with placebo and for relative effects with each other in pairwise comparison trials. However, some of pairwise comparisons of the medications have not been tested within trials at all. Finally, even if some of the possible pairwise comparisons have been directly tested within trials not all may be eligible for inclusion in a network meta-analysis3. This leaves a gap between the research has been done and the research that should or could have been undertaken and finding this highlights gaps in the fair testing of treatments4.\n\nA two-arm trial will generate one pairwise comparison. A three-arm trial, however. will generate three, and a six-arm study, 15 pairwise comparisons. It is easy to lose track of how many comparisons one study can generate. This is more likely when it comes to the many direct, indirect or mixed comparisons within a network. This paper describes a simple formula for enumerating the possible number of comparisons within a single trial or planned network meta-analysis in advance.\n\n\nMethods\n\nThe formula below solves this where n is the number of arms in a single study or network and N is the number of pairwise comparisons:\n\n\n\nWhere n > 0;\n\nn is a natural number;\n\nThen every intervention is compared to every other intervention except itself so: n*(n-1);\n\nBecause N is a bidirectional comparison (X vs. Y = Y vs. X) so: (n*(n-1))/2;\n\nN is a triangular number.\n\nA visual proof of a network of five interventions and (5*(5-1))/2=10 pairwise comparisons is presented in Figure 1.\n\nAdding any new intervention to the trial or network will create n-1 new pairwise comparisons. For example, where there are 6 arms in a trial—or 6 nodes in network meta-analysis—there will be (6*(6-1))/2=15 comparisons; adding a new intervention (6+1=7) will create 7-1=6 new pairwise direct comparisons in an individual trial and 6 direct or indirect comparisons in a network meta-analysis. Although this formula has been used for other purposes such as Metcalfe’s law in telecommunication, its use in the current context is novel.\n\nWe used the open data5 from our previously published network meta-analysis6 to re-create and enumerate the comparisons within the network. Using the direct comparisons reported in the trials within the network, we applied the formula and then compared the number of potential or expected comparisons (formula-derived) and the actual or observed number reported within the network analysis.\n\n\nResults\n\nWe built a small study-based register based—thus avoiding the pitfall of multiple counting—containing all 133 included studies in our previous network meta-analysis6,7. These trials reported comparisons from 8 interventions. Using our formula, 8 interventions should create 28 unique comparisons: (8*(8-1))/2=28 (Figure 2).\n\nOnly 16 out of 28 comparisons have been directly compared in trials (green lines).\n\nWe extracted the separate intervention arms from the open data to re-create the direct comparisons from within trials. There trials had either two or three arms so each study could create either two or three comparisons. As a result the 133 studies had 163 comparisons, the majority of which were duplicated. After removing these duplicates, this created 16 unique direct comparisons with between 1 and 47 studies per comparison for 8 interventions (Table 1). These 16 observed comparisons are 57% of the 28 expected by use of the formula above.\n\n* Amphetamines include Lisdexamfetamine.\n\nAmong five networks reported in the final paper, the number of comparisons in these five network meta-analyses, however, varies from 6 (for 3 networks) to 11 (for 1 network) and 13 (for 1 network) (Figure 3). As visualized in Figure 3, only 21.42% to 46.42% of comparisons were eligible for pairwise meta-analysis (Table 2).\n\n(Dark lines are eligible comparisons for pairwise meta-analysis, added dotted blue lines show indirect comparisons). This image has been modified from Cotese et al. 20186 under Creative Commons Attribution License (CC BY).\n\n* There are five networks in Figure 3 and each has 6, 11, or 13 eligible comparisons. Three out of 16 comparisons from trials have not been included in any of five network plots.\n\nFrom Figure 3 we can calculate that about 42% of comparisons expected though use of the formula have not been tested directly in trials. This is a direct evidence-gap. The number of missing comparisons varies between nine out of 15 in three networks with six interventions, 17 out of 28 in one network with eight interventions, and 15 out of 28 in another network with eight interventions (Figure 3). However, all 28 comparisons expected by use of the formula were utilized and reported within the network meta-analysis. It is possible that some of the comparisons predicted by the formula would have been deemed ineligible—either by adherence to a network review protocol or though post hoc exclusions—but this was not the case in this particular review (Figure 4).\n\n\nDiscussion\n\nThis formula can be employed when estimating the total number of comparisons (direct and indirect combined) theoretically possible within a proposed network meta-analysis. It would be possible that there would sometimes be a discrepancy between the number of comparisons theoretically possible and those actually employed within any given network meta-analysis. The formula would highlight this for researchers and readers and, before and after analyses, facilitate descriptions of why particular comparisons have not been included.\n\n\nConclusion\n\nThe formula produces an accurate enumeration of the potential comparisons within a single trial or network meta-analysis.\n\nAny shortfall between the full potential of the data and the actual number of comparisons within a network meta-analysis should be possible to explain through reference to pre-stipulated eligibility criteria or post hoc exclusions.\n\n\nData availability\n\nThe data analyzed in the present study have been published previously6,7.",
"appendix": "Grant information\n\nThe authors declared that no grants were involved in funding this work.\n\n\nReferences\n\nShokraneh F, Adams CE: Study-based registers of randomized controlled trials: Starting a systematic review with data extraction or meta-analysis. BioImpacts. 2017; 7(4): 209–217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShokraneh F, Adams CE: Gallstone, snake venom and witchcraft for schizophrenia: the challenges of classifying [schizophrenia] trials. Evidence-Based Medicine. 2018; 23(Suppl. 1): A18. Publisher Full Text\n\nLi T, Puhan MA, Vedula SS, et al.: Network meta-analysis-highly attractive but more methodological research is needed. BMC Med. 2011; 9: 79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEvans I, Thornton H, Chalmers I, et al.: Testing Treatments: Better Research for Better Healthcare. Pinter and Martin Publishers. 2011. PubMed Abstract\n\nCipriani A: Cortese el al_LancetPsyc 2018_OPEN DATA. Mendeley Data. 2018; v1. Publisher Full Text\n\nCortese S, Adamo N, Del Giovane C, et al.: Comparative efficacy and tolerability of medications for attention-deficit hyperactivity disorder in children, adolescents, and adults: a systematic review and network meta-analysis. Lancet Psychiatry. 2018; 5(9): 727–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCortese S, Adamo N, Mohr-Jensen C, et al.: Comparative efficacy and tolerability of pharmacological interventions for attention-deficit/hyperactivity disorder in children, adolescents and adults: protocol for a systematic review and network meta-analysis. BMJ Open. 2017; 7(1): e013967. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "43427",
"date": "13 Feb 2019",
"name": "Julie Broderick",
"expertise": [
"Reviewer Expertise Systematic reviews",
"overview methodology",
"physical activity in chronic disease"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn my opinion, this is a novel approach which allows the quantification of comparators in trials and network meta-analyses. Following on from this, the comparators which are possible versus how many were reported can be discussed in further depth. This formula has great applicability in terms of network meta-analysis methodology which is still very much a developing area. Some very minor points;\n\nPlease add a table which demonstrates the use of the formula to quantify how many comparators are possible for trials from 2 interventions up to 10. Pg 3, paragraph with the heading 'Reported comparisons within the trials' - line 2 change 'there to 'the'. Explanation of Figue 4 in text is not clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4513",
"date": "03 Apr 2019",
"name": "Farhad Shokraneh",
"role": "Author Response",
"response": "Dear Dr Julie Broderick, Thanks for spending your time for reviewing our work and commenting on it. In the following lines, we replied to your comments and made changes to the paper to cover your suggestions. COMMENT: In my opinion, this is a novel approach which allows the quantification of comparators in trials and network meta-analyses. Following on from this, the comparators which are possible versus how many were reported can be discussed in further depth. This formula has great applicability in terms of network meta-analysis methodology which is still very much a developing area. Some very minor points; please add a table which demonstrates the use of the formula to quantify how many comparators are possible for trials from 2 interventions up to 10.REPLY: Thanks for your positive comment. We added a single visual proof for this formula to show how it works. The networks of 2 to 10 interventions will create networks in shapes of line, triangle, rectangle, pentagon, hexagon, heptagon, octagon, and decagon.CHANGE: We added: “The networks of 2 to 10 interventions will create networks in shapes of line, triangle, rectangle, pentagon, hexagon, heptagon, octagon, and decagon”. COMMENT: Pg 3, paragraph with the heading 'Reported comparisons within the trials' - line 2 change 'there' to 'the'.REPLY: Thank you and sorry for this typo.CHANGE: We change ‘there’ to ‘the’. COMMENT: Explanation of Figure 4 in text is not clear. REPLY: We added a few sentences in the text to clarify it.CHANGE: “This diagram shows that only some of the comparisons from trials in study-based register could be included in pairwise meta-analysis. In addition, the number of comparisons in network meta-analysis (calculated by formula) is larger and inclusive of all the comparisons in the network of interventions and includes all the possible unique comparisons even if the comparisons are not in trials or in pairwise meta-analysis”. Thanks again for your valuable comments. Best Regards,Farhad Shokraneh"
}
]
},
{
"id": "44117",
"date": "04 Mar 2019",
"name": "G. Mustafa Soomro",
"expertise": [
"Reviewer Expertise Systematic reviews and meta-analysis and secondary data analysis."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful paper for demonstrating and discussing how an established formula could be used for calculating all possible pairs of comparisons for interventions in a network meta-analysis. Thus, reviewers would be able to find out how many potential comparisons have not been carried out.\n\nI have some minor suggestions numbered from 1) to 4):\nIn the abstract the following sentence should be amended for clarity perhaps as follows: “A total of 133 trials included in the network generated 163 comparisons (16 unique direct comparisons for 8 interventions)”. Amendment: “A total of 133 trials of 8 interventions were selected which included 163 comparisons. The network of these showed 16 unique direct comparisons.”. On page 2, it says that N is a triangular number. Either this point is not relevant, or why being a triangular number is important should be described. I think they should say that the formula they have used is an established formula from combinatorics for calculating number of pairs for a number of items in a set. On page 3 under “Comparisons in network meta-analysis plots”, line 2 and line 11 “though” should be changed to \"through\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4514",
"date": "03 Apr 2019",
"name": "Farhad Shokraneh",
"role": "Author Response",
"response": "Dear Dr G. Mustafa Soomro, Thanks for spending your time for reviewing our work and commenting on it. In the following lines, we replied to your comments and made changes to the paper to cover your suggestions. COMMENT: This is a useful paper for demonstrating and discussing how an established formula could be used for calculating all possible pairs of comparisons for interventions in a network meta-analysis. Thus, reviewers would be able to find out how many potential comparisons have not been carried out. I have some minor suggestions: In the abstract the following sentence should be amended for clarity perhaps as follows: “A total of 133 trials included in the network generated 163 comparisons (16 unique direct comparisons for 8 interventions)”. Amendment: “A total of 133 trials of 8 interventions were selected which included 163 comparisons. The network of these showed 16 unique direct comparisons.”REPLY: Thank you. We revised the sentence as suggested.CHANGE: “A total of 133 trials of 8 interventions were selected which included 163 comparisons. The network of these showed 16 unique direct comparisons”. COMMENT: On page 2, it says that N is a triangular number. Either this point is not relevant, or why being a triangular number is important should be described.REPLY: We agree. We deleted this part.CHANGE: “N is a triangular number” was deleted. COMMENT: I think they should say that the formula they have used is an established formula from combinatorics for calculating number of pairs for a number of items in a set.REPLY: Thank you for adding this. We agree and we added your suggestion in the text right after formula.CHANGE: “This is an established formula from combinatorics for calculating number of pairs for a number of items in a set”. COMMENT: On page 3 under “Comparisons in network meta-analysis plots”, line 2 and line 11 “though” should be changed to \"through\".REPLY: Thank you for detecting these errors. We collected both.CHANGE: we replaced ‘though’ with ‘through’. Thanks again for your valuable comments. Best Regards,Farhad Shokraneh"
}
]
}
] | 1
|
https://f1000research.com/articles/8-38
|
https://f1000research.com/articles/7-905/v1
|
25 Jun 18
|
{
"type": "Research Article",
"title": "Dietary patterns and their association with the components of metabolic syndrome: A cross-sectional study of adults from northeast Thailand",
"authors": [
"Pornpimon Chupanit",
"Benja Muktabhant",
"Frank Peter Schelp",
"Pornpimon Chupanit",
"Frank Peter Schelp"
],
"abstract": "Background: Nutritional transition influences a shift in eating behaviour that is associated with a rise in the prevalence of non-communicable diseases (NCDs). Metabolic syndrome (MetS) comprises a set of NCD risk factors. This study aimed to investigate dietary patterns and to determine the relationship between dietary patterns and MetS and its components. Methods: A cross-sectional study was conducted among 468 healthy adults aged 35–60 years who were residents of a semi-urban district of one of the central provinces in the northeast of Thailand. A factor analysis identified dietary patterns based on the consumption of 21 food groups, which were assessed by using a semi-quantitative food frequency questionnaire. MetS was identified by using the harmonized criteria that were stipulated by six leading international organisations. The association between dietary patterns and MetS and its components were evaluated by multiple logistic regressions. The confounding factors adjusted in the model were age, sex, smoking status, physical activity, and medication intake. Results: Two dietary patterns were identified: a traditional pattern characterised by high intakes of sticky rice and animal source foods; a mixed pattern included high intakes of white rice and a variety of food groups. The two dietary patterns did not show any association with MetS. Participants in the highest tertile of the traditional pattern was significantly related to high triglycerides (adjusted OR = 1.74, 95% CI: 1.10–2.88), in comparison to those from the lowest tertile, whereas participants in the highest tertile of the mixed pattern was inversely associated with abdominal obesity (adjusted OR= 0.49, 95% CI: 0.30–0.81) than those in the lowest tertile. Conclusions: Adherence to a traditional dietary pattern among the northeast Thai adults, in the context of nutrition transition, was associated with high triglyceride levels while the mixed dietary pattern was inversely related to abdominal obesity.",
"keywords": [
"dietary patterns",
"metabolic syndrome",
"factor analysis",
"the components of metabolic syndrome",
"the northeast",
"Thailand",
"nutritional transition",
"traditional"
],
"content": "Introduction\n\nPublic health in most low- and middle-income countries is challenged, in addition to infectious diseases, by chronic or non-communicable diseases (NCDs)1. This situation coincides with economic development, demographic transition, and epidemiological changes in a population, which leads to a shift in dietary behaviours and physical activity, also known as nutrition transition2. The underlying dietary changes have been highlighted for Asia, including Thailand3. A high consumption of red meat, processed meat, and refined sugar has increased in high- and low-income countries substantially. The changes in dietary intake are aggravated by hyper-palatable processed food high in fat, salt, or sugar4. As an after-effect, NCDs such as cardiovascular diseases, especially type 2 diabetes mellitus (T2DM), have emerged as a pressing health problem because obesity and T2DM are closely related5. Thailand, as a high middle-income country, was not spared from these developments and faced a rapidly increasing T2DM prevalence; the prevalence of T2DM among Thai adults has risen from 2.3% in 19916 to 8.0% in 20157. Thailand is divided into four major regions, and the northeast seems to have a higher prevalence of T2DM than other regions8. Particular regional dietary habits might be linked to the nutritional status and subsequently, to health risks such as T2DM. Sticky or glutinous rice is a staple food and more popular in the north-eastern region, whereas ordinary rice is a favourite in the other regions of Thailand4. Metabolic syndrome (MetS), a cluster of metabolic abnormalities, such as impaired blood glucose, dyslipidemia, abdominal obesity, and high blood pressure, is known as a major precursor to T2DM and cardiovascular disease (CVD)9,10 A systematic review study reported that the prevalence of MetS ranged from 11% to 40% among adults in countries of the Asia-Pacific region11. The fourth National Health Examination Survey in Thailand, in 2009, reported that the prevalence of MetS was 23.2%12, which is somewhat higher than in other Asian countries13.\n\nIn nutritional epidemiology, the analysis of dietary patterns is based on the concept of the overall food consumption and this approach was used to determine the relationship between diet and the risk of chronic diseases14,15. However, dietary patterns cannot be measured directly, thus the use of statistical methods to identify dietary patterns is necessary. Factor analysis based on intercorrelations between dietary items is the predominant method to identify dietary patterns16. Several studies of dietary pattern analysis found that dietary patterns characterized by a high intake of meat, high-fat foods and refined carbohydrates were positively associated with an increase in risk of MetS and its components17–19, whereas a dietary pattern characterized by a high consumption of whole grains, fish, vegetables, and fruits was found to be protective against MetS and its components20,21. One previous investigation in Thailand identified a dietary pattern consisting of a high intake of carbohydrate in the form of sticky rice, fermented fish, chili paste and bamboo shoots that was associated with MetS and its individual components17. However, the studies to determine dietary patterns in the Thai population are limited. It has remained unclear whether the traditional Thai diet or a changed pattern resulting from the nutrition transition is associated with the risk of MetS. The objective of this study was to identify dietary patterns derived from factor analysis and to determine the association between dietary patterns and MetS and its components.\n\n\nMethods\n\nThe investigation was conducted as a cross-sectional study based on the STROBE cross sectional reporting guidelines22. Thai adults aged 30 to 60 years living in Nam Phong district of the Khon Kaen province were invited to take part in the study. Khon Kaen is one of the central provinces in the northeastern region of Thailand, and Nam Phong district is located close to the provincial municipality and considered a semi-urban location. A two-stage cluster random sampling was conducted to select study participants. Random villages were selected from 12 sub-districts and then each study participant from one household was recruited by using a simple random sampling method. The required sample size was estimated by using a formula for multiple logistic regression23 and the desired power of 80% was used to detect a statistically significant difference. A minimum required sample size of 416 was obtained, with 15% increase to allow for potential non-responders. Therefore, the total number of samples was 478. However, only 468 samples were remained in this study due to the exclusion criteria. Subjects were excluded if they were diagnosed with cardiovascular disease, thyroid disease, cancer and other serious health conditions as well as if their total daily energy intake was <500 kcal/day or ≥5000 kcal/day. Six participants were excluded due to cardiovascular disease, thyroid disease, cancer and other serious health conditions. Another four participants were excluded because of an implausible total energy intake. The final study population was 468 participants that included 326 women and 142 men. The study protocol was approved by the Ethics Committee of Khon Kaen University for Human Research (HE552143). After informing all participants about the details of the study, they signed an informed written consent.\n\nAll participants received an appointment date to be interviewed about demographic and health information at the local primary health care center at sub-district level by trained staff. By questionnaire the following information was obtained: age; sex; smoking status; marital status; educational level; monthly income; family history of a first-degree relative with DM, hypertension (HT), and dyslipidemia; medical history and medication use for DM, HT, or dyslipidemia (Part 1 and 2 – Supplementary File 1). Physical activity (PA) was assessed by using the Global Physical Activity Questionnaire (GPAQ)24. Levels of PA were classified as low, moderate, and vigorous according to the guidelines (Part 3 – Supplementary File 1). Blood pressure (BP) was measured in a sitting position using a digital sphygmomanometer (Omron Model HEM-711, Japan) by trained staffs at the local health care center after participants had rested for 5 minutes. A repeated measurement of BP was taken, and the average value was recorded.\n\nThis procedure was conducted at the local primary health care center. Anthropometric measurement was done by trained staff. Weight and height were measured by using a calibrated digital scale and a portable stadiometer while the participants were dressed in light clothing and without shoes. Waist circumference (WC) was assessed in centimeters on a horizontal plane midway between the lower end of the rib cage and the top of the iliac crest while the individual was standing. The measurements were taken twice to the nearest 0.1 cm, and the average of the values was adopted.\n\nBlood samples were collected by a registered nurse in the morning after fasting overnight for at least 8 hours. Fasting blood glucose (FBG), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) was determined at the laboratory of the Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, by using an automatic analyser (Roche Cobas Mira-S Analyzer) in accordance with the manufacturer’s instructions for the use of reagents and operating the equipment. Fasting blood glucose was quantified by the glucose oxidase method25. The levels of TG and HDL-C were measured by enzymatic methods26.\n\nA semi-quantitative food frequency questionnaire (semi-FFQ) was used by trained assistants in a face-to-face interview at the local primary health care center to determine the participant’s food consumption (Part 4 – Supplementary File 1). The participants were asked about the frequency of consumption and the portion size of foods during the previous month. The picture of food sizes and kitchen equipment (e.g., cup, glass, spoon, table spoon, and ladle) were used for estimating the portion size of foods. The amount of food consumption was calculated into grams per day per person. The calculation of energy and nutrient intakes was done by applying the INMUCAL-N Version 2.0 computer software (provided by the Institute of Nutrition, Mahidol University based on the Thai food composition tables)27. The testing of the validity and reliability of the semi-FFQ was described in detail previously28. Briefly, semi-FFQ was validated with repeated 24-hour dietary recall. The Pearson’s correlation of energy and nutrient intake between two methods varied from 0.7 – 0.9.\n\nBefore the analysis of dietary patterns, a total of 104 food items in a semi-FFQ were categorized into 21 food groups based on the similarity of the nutrient profiles and local food groups specific to the northeast of Thailand for instance insects, freshwater animals (frogs, pond snails, and small shrimps), and internal organs of animals (Table S1; Supplementary File 2). Grain and grain products are the staple food of Thai cuisine, so this group was classified into three subgroups such as white rice, sticky rice, noodles and bread. Some food items had to be listed as a single food item and could not be included into a food group because of its unique nature such as eggs, milk and yogurt, seafood, energy drinks, and alcohol.\n\nThe factor analysis through principal component analysis (PCA) was used to distinguish the dietary patterns. The principal component analysis (PCA) combines correlated food groups into factors that represent dietary patterns of the population being studied16,29. The varimax option (orthogonal rotation) was used to assemble food groups into dietary patterns. Food groups were considered a significant component of dietary pattern if they had a factor loading of > 0.3. A number of dietary patterns were retained based on eigenvalues of > 2.0 and a break point in the scree plot as well as the interpretability of each pattern. Dietary patterns were named based on the interpretation of the feature of food grouping. All participants received factor scores for each dietary pattern by summing up the intake of food groups weighted by their factor loadings. The scores of each dietary pattern were categorized into tertiles to indicate the levels of food consumption.\n\nThe MetS was identified according to the harmonized criteria agreed upon from six organisations, namely the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity30. Participants with three or more of the five components were included in the group of MetS. The components of MetS are the following: (1) waist circumference ≥ 90 cm in men or ≥ 80 cm in women, (2) fasting blood glucose (FBG) ≥100 mg/dL or treated diabetes, (3) triglycerides ≥150 mg/dL, (4) low high-density lipoprotein cholesterol (HDL-C) < 40 mg/dL in men and <50 mg/dL in women, (5) systolic blood pressure (SBP) ≥130 mmHg or diastolic blood pressure (DBP) ≥85 mmHg or treated hypertension.\n\nCategorical variables were given as numbers and percentages, and continuous variables were reported as mean ± SD. To test the differences in distribution or means of the characteristic variables of study participants, the chi-square tests for categorical variables and Student’s t-test for continuous variables were used. The magnitude of the association between dietary patterns and MetS and its components was determined by multiple logistic regression analysis, using backward elimination. The confounding factors adjusted in the model were age, sex, smoking status, physical activity, and medication intake. The overall trend of odds ratios (ORs) across tertiles of dietary pattern scores was assessed by using the Mantel-Haenszel chi-square test. The lowest or first tertile score of each dietary pattern was considered to be the reference. Statistical significance was considered at p-value ≤0.05. All statistical analyses were done by using STATA version 11 (Stata Corp, College Station, TX).\n\n\nResults\n\nCharacteristics of the study participants are presented in Table 1. Females, at 69.7%, were in the majority compared with 30.3% of males. The age range of women and men was not significantly different, with a mean of 49.1 ± 6.4 years. A higher proportion of females were obese (50.9%) than men (36.6%). Most of the participants were married and had finished elementary school. Men’s income exceeded that of women. Physical activity seemed to be on a vigorous level (53.2%; 68.3% in men and 46.6% in women). Only a few women smoked (1.0%), but 64.1% of the men did so. Within the group of women, 49.7% indicated that a first-degree relative had a history of diabetes, hypertension or dyslipidemia, but the proportion (36.6%) was less frequent for men. Only a minority of study participants, less than 10%, regularly took medicine for diabetes, high blood pressure, or dyslipidemia. Energy intake of men was significantly above that of women, but the intake of defined nutrients such as carbohydrate, protein, fat, fiber, cholesterol, and sugar did not differ between the sexes.\n\nDM: diabetes mellitus; HT: hypertension\n\n†Medication use for treating type 2 diabetes mellitus or high blood pressure or dyslipidemia\n\n* p-value< 0.001; **p-value < 0.05 by using Student’s t-test.\n\nThe distribution of the components of MetS within the group of participants without MetS and those with MetS are given in Table 2. The proportion of individual components of MetS varies between the two groups as given in the table. Of the 248 participants with abdominal obesity, 62.1% falls into the group of MetS, but a considerable proportion of those with this component, 37.9%, were not in the MetS group. Likewise, the proportion of participants without MetS but with FBG above the cut-off point was18.6%; for low HDL-C, it was 45.6%; for high blood pressure was 32.4%; and for high triglyceride was 31.3%.\n\nAbdominal obesity (waist circumference ≥ 90 cm in men or ≥ 80 cm in women); high blood pressure (SBP ≥ 130; DBP ≥ 85 mmHg or HT treatment); high fasting blood glucose (FBG ≥100 mg/dL or DM treatment); high triglyceride (TG ≥ 150 mg/dL) low HDL-C (HDL-C <40 mg/dL in male; <50 mg/dL in women\n\nTwo dietary patterns, named traditional and mixed, were identified by using factor analysis (Table 3). The two patterns together explained 26.9% of the total variance. The variance for the mixed pattern was 14.2% and the variance for the traditional pattern was 12.7%. The mixed pattern was characterized by the consumption of a variety of foods consisting of fruits, noodles and bread, milk and yogurt, soybean and soybean products, vegetables, eggs, sweet beverages, bakery and snacks, processed meat, legumes and nuts, seafood, and white rice.The traditional pattern included a high intake of sticky rice and animal source foods consisting of internal organs of animals, freshwater animals (frogs, pond snails, small shrimps), poultry, processed meat (Thai sausage, fermented pork sausage, hot dog sausage, and pork cracking), insects, red meat, fish, and seafood as well as energy drinks. Alcohol was not included into both the mixed pattern and traditional pattern because of a very low factor loading.\n\nFactor loading less than 0.30 are not displayed.\n\nThe distribution of age, sex, MetS and the five components of MetS according to tertile scores of the two dietary patterns are presented in Table 4. The proportions of men and women were significantly different according to tertiles of each dietary pattern. Participants in the highest tertile of mixed pattern were women more than men. Participants with MetS did not differ in the highest tertile of each dietary pattern score comparing to the lowest tertile score. The proportions of those participants in the second and third tertile score who were found to consume the traditional dietary pattern were significantly associated with high triglyceride levels in comparison with those falling into the first tertile. A reversed feature evolved for low HDL-C in those preferring the traditional pattern. The proportion within the tertiles of the mixed pattern did not differ significantly as far as the five components of MetS were concerned.\n\nT: tertiles of dietary pattern scores, BP: blood pressure; FBG: fasting blood glucose; TG: triglyceride; HDL-C: high density lipoprotein cholesterol\n\nTable 5 shows the relationship of the mixed and traditional dietary patterns with MetS and each component of MetS after applying a multiple logistic regression, controlling for other covariates. The lowest tertile of each dietary pattern was considered as reference. By using this approach, neither the mixed nor the traditional pattern was associated with MetS. Comparing the lowest tertile score of the mixed and traditional patterns with the highest tertile score, the mixed pattern was inversely associated with abdominal obesity (adjusted OR=0.49, 95% CI: 0.30–0.81). Increasing consumption of the traditional pattern was likely to be associated with high TG (adjusted OR= 1.74, 95% CI: 1.10–2.88).\n\nBP: blood pressure;FBG: fasting blood glucose; TG: triglyceride; HDL-C: high density lipoprotein cholesterol; T: tertiles of dietary pattern scores\n\n*All models were adjusted for age, sex, smoking status, level of physical activity, and medication use.\n\n\nDiscussion\n\nTwo prominent dietary patterns of this cross-sectional study among northeast Thai people in the context of nutritional transition were generated by using the factor analysis, namely, the mixed pattern and the traditional pattern. A positive association was found between the traditional pattern and high TG, whereas a negative association was observed between the mixed pattern and abdominal obesity.\n\nThe traditional pattern as assessed through this study showed that a distinctive pattern still follows a traditional way of food intake such as eating more sticky rice and animal source foods. The consumption of freshwater animals (frogs, pond snails, and small shrimps), internal organs of animals, and insects of various kinds have long been recognised as ingredients of the northeast diet4. Following the classification of a diet as traditional, sticky rice as a staple food can be rightly considered a traditional food. Previous studies reported that an ethnic-specific diet, characterized by high consumption of rice, legumes, and vegetables, was positively related to MetS and its components (low HDL-C)31, while another study did not find the association with MetS and its components32. The traditional pattern in this study, although not associated with MetS, nevertheless was significantly associated with high triglycerides. This finding might be explained by the intake of high amounts of sticky rice in the traditional pattern. Sticky rice (Oryzaglutinosa) has a high glycemic index33 and stimulates high serum triglyceride levels34. Recently, the Fourth Thai National Health Examination Survey found that “carbohydrate dietary pattern” characterized by consumption of sticky rice, fermented fish, chili paste and bamboo shoots was associated with the risk of MetS and its components such as high TG and low HDL-C17. A study from the Korean National Health and Nutrition Examination Survey also showed that the high intake of rice-oriented pattern was significantly correlated with a higher prevalence of high TG and low HDL-C35. Traditional patterns in this study not only included a high intake of sticky rice but also a high intake of animal source foods. Meat is an important source of total visible and invisible fat intake, particularly saturated fat. Several studies showed that dietary saturated fat consumption is associated with the risk of CVD due to increasing plasma lipid and lipoprotein36,37. However, the intake of several animal source foods in this pattern, comprising insects, freshwater animals (frogs, pond snails, small shrimps) and fish, are good sources of protein and are typically low in fat. This may explain the lack of association between the traditional pattern and the MetS, but this pattern was related to high TG.\n\nThe mixed pattern was characterized by a consumption of a wide variety of food items long known to be part of the Thai cuisine (such as white rice, fruits, and vegetables), mixed with food items marketed by the food industry (such as noodles of various brands and bread, milk and yogurt, bakery and snacks, sweet beverages, and soybean and soybean products). A previous study indicated that a dietary pattern consisting of a variety of foods (such as grains and starches, vegetables and fruits, fish and seafood, meat, poultry, eggs, legumes, and dairy products) was inversely associated with the incidence of MetS among Korean adults38. Fruits, milk and yogurt, soybean and soybean products and vegetables, major foods characterising the mixed pattern, seem to constitute a beneficial diet39,40. Fruits and vegetables constitute a high-fibre diet, which is associated with a decreased risk of obesity40, potentially accounting for some of the association between the mixed pattern and decreased abdominal obesity in this study. The meta-analysis of randomized controlled trials (RCTs) showed that the consumption of dairy products may have a modest benefit in facilitating weight loss in short-term or energy-restricted RCTs39.\n\nAs evident from Table 2, a rather high proportion of participants had one or two components of MetS (such as low HDL-C, abdominal obesity, high BP, and high TG), but they were not included in the MetS group. Thus, MetS as such could not be statistically associated with two dietary patterns, but some components of MetS, significant statistically, could be linked to dietary patterns. High triglycerides were significantly linked to the consumption of the traditional pattern, whereas participants with no abdominal obesity related to the mixed pattern. Interpreting the statistics with caution, the mixed pattern appears to be more beneficial for health than the traditional pattern. Fruits and vegetables have long been constituents of the overall Thai dietary intake, but white rice is still the staple food throughout the country4 and should not be considered unhealthy. What appears to be less beneficial in the traditional pattern is the intake of glutinous rice (Oryzaglutinosa) with a high glycemic index33, which stimulates high serum triglyceride levels36. Hypothetically, elevated triglyceride levels as linked to the traditional pattern here indicates the risk for consumers of this pattern to develop T2DM, and the mixed pattern might to a certain extent prevent consumers of this dietary intake from acquiring T2DM. High triglyceride blood levels have been found to be independent risk factors pointing toward the development of diabetes41–43. A promising approach for further studies would be to include metabolic variables closely linked to T2DM, such as insulin resistance, found to occur in hypertriglyceridemia and seem to be a risk factor for T2DM44.\n\nSome limitations of this study should be considered. Dietary patterns by using factor analysis involves subjective decisions for instant the consolidation of food items into food groups, the number of dietary patterns to be retained and the identified dietary patterns could be different in case of the subjects due to different ethnicity or culture14. The other limitation of the cross-sectional study is not optimal for assessing causal relationship because the investigator measures the outcome and the exposures at a single period time.\n\n\nConclusion\n\nTwo different dietary patterns could be recognized; the traditional- and the mixed pattern. These dietary patterns were not found to be associated with MetS. Adherence to the traditional pattern among the northeast Thai adults, in the context of nutrition transition, was associated with high triglyceride levels and seemed to be less beneficial for the mixed pattern, which was inversely related to abdominal obesity. Our findings show that the consumption of a variety of foods may be beneficial for health outcomes. Further studies on a larger sample population, as part of a prospective study, will provide important insights into the associations of dietary patterns and the metabolic syndrome.\n\n\nData availability\n\nDataset 1: Raw data gathered from the questionnaire 10.5256/f1000research.15075.d20724545",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Research Group on Prevention and Control of Diabetes Mellitus in the Northeast of Thailand from the Khon Kaen University.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors also are grateful thank to all participants for their contributions to this work.\n\n\nSupplementary materials\n\nSupplementary File 1: Questionnaire used to interview participants. The questionnaire comprises of 4 part (1) General information, (2) Health information, (3) Global Physical Activity Questionnaire (GPAQ), (4) Food intake assessment by a semi-quantitative food frequency questionnaire.\n\nClick here to access the data.\n\nSupplementary File 2: Table S1 - Food lists of 20 food groups in the study.\n\nClick here to access the data.\n\n\nReferences\n\nWorld Health Organisation: Preventing chronic diseases: a vital investment. Geneva: World Health Organisation Press. 2005. Reference Source\n\nPopkin BM: Nutrition Transition and the Global Diabetes Epidemic. Curr Diab Rep. 2015; 15(9): 64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKelly M: The Nutrition Transition in Developing Asia: Dietary Change, Drivers and Health Impacts. In: Jackson P, Spiess WEL, Sultana F, editors. Eating, Drinking: Surviving: The International Year of Global Understanding - IYGU. Cham: Springer International Publishing; 2016; 83–90. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong Y, Joung H: A traditional Korean dietary pattern and metabolic syndrome abnormalities. Nutr Metab Cardiovasc Dis. 2012; 22(5): 456–62. PubMed Abstract | Publisher Full Text\n\nChen YJ, Sun FH, Wong SH, et al.: Glycemic index and glycemic load of selected Chinese traditional foods. World J Gastroenterol. 2010; 16(12): 1512–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParks EJ, Hellerstein MK: Carbohydrate-induced hypertriacylglycerolemia: historical perspective and review of biological mechanisms. Am J Clin Nutr. 2000; 71(2): 412–33. PubMed Abstract | Publisher Full Text\n\nSong SJ, Lee JE, Paik HY, et al.: Dietary patterns based on carbohydrate nutrition are associated with the risk for diabetes and dyslipidemia. Nutr Res Pract. 2012; 6(4): 349–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChiu S, Williams PT, Krauss RM: Effects of a very high saturated fat diet on LDL particles in adults with atherogenic dyslipidemia: A randomized controlled trial. PLoS One. 2017; 12(2): e0170664. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiri-Tarino PW, Sun Q, Hu FB, et al.: Saturated fatty acids and risk of coronary heart disease: modulation by replacement nutrients. Curr Atheroscler Rep. 2010; 12(6): 384–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaik I, Lee M, Jun NR, et al.: A healthy dietary pattern consisting of a variety of food choices is inversely associated with the development of metabolic syndrome. Nutr Res Pract. 2013; 7(3): 233–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen M, Pan A, Malik VS, et al.: Effects of dairy intake on body weight and fat: a meta-analysis of randomized controlled trials. Am J Clin Nutr. 2012; 96(4): 735–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwingshackl L, Hoffmann G, Kalle-Uhlmann T, et al.: Fruit and Vegetable Consumption and Changes in Anthropometric Variables in Adult Populations: A Systematic Review and Meta-Analysis of Prospective Cohort Studies. PLoS One. 2015; 10(10): e0140846. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe S, Wang S, Chen X, et al.: Higher ratio of triglyceride to high-density lipoprotein cholesterol may predispose to diabetes mellitus: 15-year prospective study in a general population. Metabolism. 2012; 61(1): 30–6. PubMed Abstract | Publisher Full Text\n\nRiediger ND, Clark K, Lukianchuk V, et al.: Fasting triglycerides as a predictor of incident diabetes, insulin resistance and β-cell function in a Canadian First Nation. Int J Circumpolar Health. 2017; 76(1): 1310444. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcLaughlin T, Abbasi F, Cheal K, et al.: Use of metabolic markers to identify overweight individuals who are insulin resistant. Ann Intern Med. 2003; 139(10): 802–9. PubMed Abstract | Publisher Full Text\n\nEckardt K, Taube A, Eckel J: Obesity-associated insulin resistance in skeletal muscle: role of lipid accumulation and physical inactivity. Rev Endocr Metab Disord. 2011; 12(3): 163–72. PubMed Abstract | Publisher Full Text\n\nChupanit P, Muktabhant B, Schelp FP: Dataset 1 in: Dietary patterns and their association with the components of metabolic syndrome: A cross-sectional study of adults from northeast Thailand. F1000Research. 2018. Data Source"
}
|
[
{
"id": "35562",
"date": "17 Jul 2018",
"name": "Nipa Rojroongwasinkul",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn \"Abstract\", session \"Methods\", the study was conducted among 468 healthy adults but some participants in study are not healthy, please write only 468 adults. In Table 1, focus at dietary intake, the result show the statistical significant difference of total energy intake between men and women. Do you test the difference of the other nutrients intake such as carbohydrate, protein, fat, fiber, cholesterol, and sugar between men and women? According to the distribution of these nutrients are skewed (not normal distribution), using Student’s t-test may not be appropriate test. For sure, testing normality of these nutrients, if the nutrient is not normal distribution the appropriate statistical testing is Mann-Whitney U Test (nonparametric statistic). Please show the prevalence of MetS separate by sex.\nThe mixed pattern was characterized by the consumption of a variety of foods (factor loading >0.3) consisting of\nFruits (F=0.575 ) noodles and bread (F=0.553) milk and yogurt (F=0.542) soybean and soybean products (F=0.524) vegetables (F=0.48) eggs (F=0.455) sweet beverages (F=0.434) bakery and snacks (F=0.378) processed meat (F=0.376) legumes and nuts (F=0.37) seafood (F=0.372) white rice (F=0.362)\nThe traditional pattern included a high intake of rice (sticky & white rice) and animal source foods (factor loading >0.3) consisting of\nsticky rice (F=0.603) internal organs of animals (F=0.566) poultry (F=0.499) freshwater animals (frogs, pond snails, small shrimps) (F=0.477) white rice (F= -0.467) processed meat (Thai sausage, fermented pork sausage, hot dog sausage, and pork cracking) (F=0.453) insects (F=0.427) energy drinks (F=0.411) red meat (F=0.366) fish (F=0.311) seafood (F=0.304)\nThe 2 pattern that authors suggested, in each pattern some variable are shown in both pattern (processed meat) and the variables in each pattern are not clear cut. I checked from your data and found that the participants in your study who consumed both sticky rice and white rice are 85% (398 from 468) so the dietary pattern in this study is not follow the traditional way of food intake that eating more sticky rice but they consumed both rice. From theory of factor analysis, the greater the loading, the more the variable is a pure measure of the factor. Comrey and Lee (1992) suggest that loadings in excess of .71 (50% overlapping variance) are considered excellent, .63 (40% overlapping variance) are considered very good, .55 (30% overlapping variance) are considered good, .45 (20% overlapping variance) are considered fair, and .32 (10% overlapping variance) are considered poor. [Cited in Tabachnick BG and Fidell LS. Using multivariate statistics. 6th ed. New Jersey: Pearson Education, Inc.: 2013.] If we decided to use a loading of .45 (20% variance overlap between variable and factor). With the use of the .45 cut, we will get the variables in each factor are as follow:\nFactor 1: Healthier pattern\n\nFruits (F=0.575 ) noodles and bread (F=0.553) milk and yogurt (F=0.542) 4. soybean and soybean products (F=0.524)\n\nvegetables (F=0.48) eggs (F=0.455)\n\nFactor 2: Rice and meat pattern\nsticky rice (F=0.603) internal organs of animals (F=0.566) poultry (F=0.499) freshwater animals (frogs, pond snails, small shrimps) (F=0.477)\n\nwhite rice (F= -0.467)\n\nprocessed meat (Thai sausage, fermented pork sausage, hot dog sausage, and pork cracking) (F=0.453)\n\nPlease consider to assign the name of the new factors I just try to assign the name but the name may be not appropriate. From the new factors that we got, please re-run the analysis and discuss the results accordingly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4464",
"date": "03 Apr 2019",
"name": "Pornpimon Chupanit",
"role": "Author Response",
"response": "1) In \"Abstract\", session \"Methods\", the study was conducted among 468 healthy adults but some participants in study are not healthy, please write only 468 adults. answer: We had already revised. 2) In Table 1, focus at dietary intake, the result show the statistical significant difference of total energy intake between men and women. Do you test the difference of the other nutrients intake such as carbohydrate, protein, fat, fiber, cholesterol, and sugar between men and women? According to the distribution of these nutrients are skewed (not normal distribution), using Student’s t-test may not be appropriate test. For sure, testing normality of these nutrients, if the nutrient is not normal distribution the appropriate statistical testing is Mann-Whitney U Test (nonparametric statistic). answer: As suggested, the difference between groups has been tested now also by non-parametric statistics.We used the Mann-Whitney U Test for total energy carbohydrate protein and fat (showed in Table 1). 3) Please show the prevalence of MetS separate by sex. answer: We added the prevalence of MetSin Table 1. 4) The 2 pattern that authors suggested, in each pattern some variable are shown in both pattern (processed meat) and the variables in each pattern are not clear cut. I checked from your data and found that the participants in your study who consumed both sticky rice and white rice are 85% (398 from 468) so the dietary pattern in this study is not follow the traditional way of food intake that eating more sticky rice but they consumed both rice. From theory of factor analysis, the greater the loading, the more the variable is a pure measure of the factor. Comrey and Lee (1992) suggest that loadings in excess of .71 (50% overlapping variance) are considered excellent, .63 (40% overlapping variance) are considered very good, .55 (30% overlapping variance) are considered good, .45 (20% overlapping variance) are considered fair, and .32 (10% overlapping variance) are considered poor. [Cited in Tabachnick BG and Fidell LS. Using multivariate statistics. 6th ed. New Jersey: Pearson Education, Inc.: 2013.] If we decided to use a loading of .45 (20% variance overlap between variable and factor). answer: In the international literature for dietary pattern studies this loading factor is frequently used and seems to be appropriate1-6.So for instance Castro MA. et al. reported that to use ≥ 0.25 for factor loading was an acceptable to look into dietary patterns, so factor loading > 0.3 as use in our study should be appropriate. In a normal life situation it is unlikely that the majority of groups of the ordinary population sticks to a very fixed diet, especially not if the objective of the study was to test the dietary intake in the situation of the ‘nutritional transition’ which is mentioned in the abstract, the introduction and the discussion. Higher loading factors will ‘squeeze’ the data into ‘unnatural fixed groups’ representing those refusing to or not being influenced by the ‘transition’. Transition means that the majority of those tested might not stick on a daily basis to a very fixed dietary intake. One and the other food items will appear in both groups but with different loading factors as for instance for sticky rice and white rice (see table 3). It is mention that the majority of participants (85%) consumed both sticky rice and white rice. It is argued by the referee that the dietary pattern in this study doesn’t follow the traditional way of food intake in that that both kinds of rice is consumed. In Fact this result should be expected in the situation of a transition of dietary intake. The loading factor for sticky rice consumed for the here so called ‘traditional diet’ with a 0.6 exceeds the loading factor for sticky rice for the mixed group with -0.323 by far. A similar result was achieved for white rice where the traditional group had a loading factor of -0.467 much lower that the mixed group with 0.362. So it can be concluded that the the traditional groups still have a strong tendency to follow the traditional way of consumption but already started to alternate between both kinds of rice. 1. Aekplakorn W, Satheannoppakao W, Putwatana P, Taneepanichskul S, Kessomboon P, Chongsuvivatwong V, et al. Dietary pattern and metabolic syndrome in thai adults. J Nutr Metab. 2015;2015:468759. 2. Naja F, Nasreddine L, Itani L, Adra N, Sibai AM, Hwalla N. Association between dietary patterns and the risk of metabolic syndrome among Lebanese adults. Eur J Nutr. 2013;52(1):97-105. 3. Panagiotakos DB, Pitsavos C, Skoumas Y, Stefanadis C. The Association between Food Patterns and the Metabolic Syndrome Using Principal Components Analysis: The ATTICA Study. Journal of the American Dietetic Association. 2007;107(6):979-87. 4. Suliga E, Kozieł D, Cieśla E, Głuszek S. Association between dietary patterns and metabolic syndrome in individuals with normal weight: a cross-sectional study. Nutrition Journal. 2015;14(1):55. 5. Woo HD, Shin A, Kim J. Dietary patterns of Korean adults and the prevalence of metabolic syndrome: a cross-sectional study. PLoS One. 2014;9(11):e111593. 6. Castro MA, Baltar VT, Selem SS, Marchioni DM, Fisberg RM. Empirically derived dietary patterns: interpretability and construct validity according to different factor rotation methods. Cad Saude Publica. 2015;31(2):298-310."
}
]
},
{
"id": "36423",
"date": "19 Sep 2018",
"name": "Sakda Pruenglampoo",
"expertise": [
"Reviewer Expertise Food chemistry",
"nutrition",
"epidemiology and 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\nIt may be concluded as the following:\n\n1. Clear research objectives. 2. The study design is appropriate for the objectives. However, it might be replaced by “Analytical cross sectional study design . 3. The criteria which used in this study for outcome variables were suitable. However it should add some more details about the accuracy of the methods/instruments for measuring anthropometric and biochemical measurements and also the components of the metabolic syndrome. 4 .The methods used for food intake assessment (Semi-FFQ, the picture of food sizes and kitchen equipment and INMUCAL-N version 2) sound reasonable for the study. 5. Factor analysis used to distinguish the dietary pattern of the subjects has an advantage that it can reduce the number of variables , by combing two or more variables into single factor. However, it may have some limitations which the authors of this study have mentioned already. 6.The methods of analysis and the statistical methods employed were suitable for the types of variables (nominal versus ordinal versus continuous) in this study. 7.Regarding discussion part, it might be useful for the readers if the authors mention in briefly about possible sources of bias which might be occurred and how to control or to decrease the effect of the bias in the study.\n\n8. In conclusion part, sample population in a prospective study should include all part of Thailand (north, north- eastern, central and south).\n9. Other comments for minor changes: (See PDF) 9.1 Abstract: A cross-sectional study should be changed to An analytical cross-sectional study. 9.2 Introduction: Check grammar for writing through the chapter of introduction. 9.3 Methods: Put the meaning of STROBE in parentheses (The Strengthening the Reporting of Observational Studies in Epidemiology) and check grammar for writing through the chapter of methods. 9.4 Results: Table 3 should have foot note to explain that factor loading less than 0.30 were not displayed and the factor loading shown in the table did not consider the signs of ±). Table 4 should have foot note to state the statistical methods for testing P value. 9.5 Discussion: Check grammar for writing through the chapter of discussion. 9.6 Conclusion: Check grammar for writing through the chapter of conclusion. 9.7 References: Check format and completeness of the references.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nAre all 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/7-905
|
https://f1000research.com/articles/8-360/v1
|
02 Apr 19
|
{
"type": "Research Article",
"title": "Enhanced conventional method is as precise as navigation for distal femur resection during total knee replacement: a randomized controlled trial",
"authors": [
"Rohan Bhimani",
"Fardeen Bhimani",
"Rohan Bir Singh",
"Preeti Singh",
"Fardeen Bhimani",
"Rohan Bir Singh",
"Preeti Singh"
],
"abstract": "Introduction: The purpose of this prospective study was to determine the accuracy of distal femoral cut and femoral component placement in the coronal plane with the enhanced conventional technique when compared to computer navigation during total knee replacement (TKR). Methods: In total, 475 total knee arthroplasties (TKA) were analyzed (200 optimized conventional TKAs and 275 navigated TKAs) for postoperative mechanical alignment or hip-knee-ankle angle and femoral component coronal alignment and compared between the two groups Results: Mean femoral component coronal alignment was not significantly different (p=0.35) when navigation and enhanced conventional groups were compared. There was no significant difference in the mean femoral component coronal alignment between knees with a valgus correction angle (VCA) <5° (p=0.28), knees with VCA 5°-7° (p=0.48) and knees with >7° (p=0.09). No significant difference was noted in the mean femoral component coronal alignment between knees with varus deformity <10° (p=0.19), varus deformity 10°-20° (p=0.72) and valgus deformity (p=0.35). Conclusions: Using the enhanced conventional technique in each patient to perform distal femoral cut during total knee arthroplasty can help achieve the coronal alignment of the femoral component comparable to navigation technique. Registration: UMIN-CTR ID UMIN000036204.",
"keywords": [
"distal femur",
"total knee arthroplasty",
"valgus cut",
"computer navigated arthroplasty",
"alignment",
"conventional knee arthroplasty"
],
"content": "\n\nSince publication the authors have alerted the F1000Research editorial office to disagreements regarding author contributions to the study and the interpretation of results. All editorial activity has been suspended pending further investigation. Further action will be dependent on the outcome of these discussions.\n\n\nIntroduction\n\nThe survival of implants used during total knee replacement (TKR) is influenced by many factors, such as precision of bone cuts, accurate positioning of the component, and soft tissue balancing during surgery1–3. Malpositioning of the component during the surgery can result in early failure of TKR due to early polyethylene wear and aseptic loosening4–7. Throughout TKR, the goal is to align within ±3° of neutral with regard to their mechanical axis, both femoral and tibial components, to aid equal dispersal of forces across the implant post-operatively. As the anatomical and mechanical axes of the femur are not coincident, a distal femoral cut is accomplished by resecting the distal femur perpendicular to the coronal femoral mechanical axis using the valgus correction angle (VCA) which is equivalent to the angle formed between the mechanical and anatomical axis of the femur8–9.\n\nThe different techniques of performing the distal femoral cut using the VCA include taking a 5°–7° fixed VCA for all cases, measuring VCA on preoperative scanogram (i.e. measuring angle between the mechanical and anatomical axis on a full-length, standing, hip-to-ankle radiograph), and with a computer-assisted navigation system. Regular practice for most surgeons is to reference the distal femoral cut using an intramedullary rod for the anatomical axis using a fixed VCA range of 5°–7°. Nevertheless, numerous studies have shown a wide variation in VCA in patients undergoing TKR, where it can range between 2°–13° and use of a fixed VCA range can result in miscalculation in distal femoral cut and malalignment of the femoral component10,11. Thus, it is suggested that VCA should be modified in each patient by measuring it on scanogram. Computer navigation aids the surgeon in femoral component alignment perpendicular to the mechanical axis in the coronal plane without depending on the VCA and to overcome any disparity between the anatomical and mechanical axis of femur. Previous studies found that modified VCA results in more precise positioning of the femoral component in the coronal plane in comparison to utilizing a fixed distal VCA10–13. However, these past studies are unclear on the precision of femoral component placement using the VCA technique modified in respect to each patient when compared to computer navigation during TKR.\n\nTo our knowledge, no study has been undertaken which aimed to determine the precision of cut for distal femur and femoral component placement in the coronal plane with the enhanced conventional technique when compared to computer navigation system during TKR. We formulated an enhanced conventional technique to execute the distal valgus cut by modifying VCA in respect to each patient using scanogram and confirming the distal femoral coronal alignment cut using radio-opaque indicator for the centre of the head of femur using an image intensifier and extramedullary guide rod. Therefore, the aim of our study is to determine the precise cut for distal femur and femoral implant positioning in the coronal plane with the enhanced conventional technique when compared to computer navigation during TKR. We hypothesised that the enhanced conventional technique will be as precise as computer navigation in carrying out the distal cut for femur during TKR.\n\n\nMethods\n\nWe prospectively included 475 consecutive, primary TKRs from August 2016 to July 2017. The inclusion criteria included patients who underwent primary TKR for tricompartmental knee arthritis and provided their informed consent for participation in this study. The exclusion criteria included patients with inappropriate quality of pre- or post-operative radiographs, patients whose radiographs were unavailable for evaluation and patients who were lost in follow-up. All surgeries were performed by a single surgeon. We randomized 475 patients into enhanced conventional or navigated TKR group. The randomization was performed on the day of the surgery from a set of two envelopes with computer navigation and conventional technique mentioned in each of them. The envelope was picked randomly by a junior resident on the day of the surgery and it was conveyed to the surgeon on the type of technique (conventional vs. computer navigation) selected for the patient. There were no changes in methods after commencement of the trial. Out of 475 TKRs, 200 patients underwent total knee arthroplasties in the enhanced conventional group and 275 patients underwent total knee arthroplasties in the computer navigation group. There were 455 varus knees and 20 valgus knees in the study. The sample size was calculated to give a 95% confidence interval and 4.5% margin of error. This gave us a sample size of 464. We decided to include 475 patients to allow for any dropouts. However, there were no dropouts.\n\nThe study was approved by the Ethics Committee of Hinduja Healthcare Surgicals (Approval Number: HHS/16-17/EC/091) and conducted according to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all patients.\n\nWeight-bearing, full-length, hip-to-ankle radiographs (scanogram) were obtained before and within 4 weeks after surgery. The postoperative scanograms were all acquired using standard radiographic techniques with both patellae facing forward, knees in complete extension, and both feet pointing forwards to avoid malrotation of the limb during scanogram. All scanograms were screened by one of the authors for any malrotation in the coronal plane, which made it inappropriate for the study. Excessive rotation in the coronal plane was measured on radiographs not only by the position of the lesser trochanter and head of the fibula, but also the position of the patella in terms of central, medial or lateral positioning. Digital pictures of the scanogram were utilized for measurement of several radiographic factors using ImageJ image processing and analysis software (version 1.41, U.S. National Institute of Health). Scanogram factors measured included pre- and post-operative coronal limb alignment also known as hip-knee-ankle (HKA) angle described as the angle between the femoral mechanical axis (i.e. line joining the center of the femoral head and center of the knee joint) and the tibial mechanical axis (i.e. line joining the center of the knee joint and center of the ankle joint), the distal femoral valgus correction angle (VCA), defined as the angle between the femoral anatomical and mechanical axis (i.e. the mid-medullary axis of the distal diaphysis of the femur), and the post-operative femoral component coronal alignment (described as the medial angle between the femoral mechanical axis and the line tangential to the prosthetic femoral condyles)8. All measurements on scanogram were performed independently by two authors who were unaware of the technique used by the operating surgeon.\n\nAll surgeries were carried out with a tourniquet and after the cementing of the implant, the tourniquet was deflated and haemostasis was achieved by electrocoagulation. A polyethylene liner then was inserted followed by layered closure of the wound. There was an interval of approximately 40 to 50 minutes between tourniquet deflation and conclusion of surgery. The tourniquet was deflated when the cement hardened. A standard medial parapatellar arthrotomy approach was used in all cases. A cemented, posterior-stabilized, fixed-bearing implant, PFC®Sigma series of total knee prostheses (DePuy Orthopaedics Inc, Warsaw, IN, USA) with patellar resurfacing was performed in all patients. The cutting blocks used for carrying out all bony resection in both the groups were identical. The purpose was to achieve a coronal mechanical axis of the limb in the range of 180° ±3° and place the femoral component perpendicular to the femoral mechanical axis ±1°. The amount of soft tissue release was based on the amount of soft tissue tightness assessed using spacer and an appropriate amount of soft tissue release was allowed.\n\nIn the computer navigation-assisted total knee group, registration of the anatomic landmarks was performed in standard fashion using the Ci Navigation System with accompanying software (Brain Lab, Munich, Germany). The coronal mechanical axis of the lower limb in the coronal plane was derived by navigation using the center of femoral head, the center of the intercondylar notch, centre of the tibial plateau and the center of the ankle plafond. The navigation system arrays also aided in positioning the cutting blocks and verified and quantified the distal femoral cut.\n\nIn the enhanced conventional TKR group, preoperative scanograms were used to measure VCA in each knee which in turn assisted to customize the coronal plane of distal femoral resection in each patient. Therefore, if the VCA on the scanogram was measured, for example as 7°, the distal femoral resection guide was set at 7° valgus, so as to accomplish a distal femoral cut perpendicular to the femoral mechanical axis. After the patient was under anaesthesia, an image intensifier was used to recognize the centre of the head of femur and an adhesive radio-opaque marker was placed in the groin over the approximate center of the head of femur. Intra-operatively, an extra-medullary rod was attached to the cutting block to determine the accuracy of the distal femoral resection with reference to the previously placed femoral head centre served by the radio-opaque marker.\n\nStatistical analysis was performed using Stata® 8.2 software. Data from the two groups of patients were compared using Student’s t-test and Fisher exact test. The significance level used for all tests was p ≤ 0.05.\n\n\nResults\n\nThere was no significant difference between demographic parameters in the conventional and navigation groups (Table 1). The mean femoral component coronal alignment was not significantly different (p=0.35) when navigation and optimized conventional groups were compared (Table 2). No harms were observed during this study. Raw data are available on Harvard Dataverse14.\n\nAll values presented as mean ± standard deviation.\n\nBMI, body mass index; n, number of knees.\n\nAll values presented as mean ± standard deviation.\n\nVCA, distal femur valgus correction angle; HKA, hip-knee-ankle;\n\nWhen knees in the two groups were compared based on pre-operative VCA (Table 3) there was no significant difference in the mean femoral component coronal alignment between knees with VCA <5° (p=0.28), knees with VCA 5°–7° (p=0.48) and knees with >7° (p=0.09). Similarly, when knees in the two groups were compared based on their preoperative HKA angles (Table 4), there was no significant difference in the mean femoral component coronal alignment between knees with varus deformity <10° (p=0.19), varus deformity 10°–20° (p=0.72) and valgus deformity (p=0.35). However, knees with varus deformity >20° showed significant (p=0.003) differences in the mean femoral component coronal alignment between the two groups.\n\nAll values presented as mean ± standard deviation.\n\nAll values presented as mean ± standard deviation.\n\nThe restoration of the coronal mechanical alignment was significantly more accurate (p <0.0001) in terms of mean postoperative HKA angle in the navigation group than the optimized conventional group.\n\n\nDiscussion\n\nThe key outcome of this study was that the mean femoral component coronal alignment was not significantly different when enhanced conventional and navigation groups were compared. Thus, our study found an enhanced conventional technique using modified VCA in each patient and the extramedullary guide rod is as accurate as computer navigation in performing distal cut for the femur during TKR.\n\nIn a meta-analysis of 29 studies comparing computer-assisted TKR to conventional TKR, Mason et al.15 found that 90.4% of computer-assisted TKRs had femoral component positioning within 2° of perpendicular to the mechanical axis, as opposed to 65.9% in the conventional total knee arthroplasty group. However, the majority of such studies in the literature8–10 comparing conventional and navigated TKRs used a fixed VCA range of 5°–7° and have not modified VCA in each patient during total knee arthroplasty, which may be the reason for accurateness of conventional technique in distal femoral resection being inferior to computer navigation method. Palanisami et al.12 in a prospective study described substantial enhancement in both femoral component placement and postoperative alignment when VCA was modified in each patient when matched to taking a fixed VCA of 5° in patients with moderate and severe varus deformity. Shi et al.13, in a randomized prospective study, stated that 83.6% of knees with femoral component alignment within ±3° of the mechanical axis of femur were in the customized VCA group compared to 39.4% in the knees with fixed VCA group.\n\nThe VCA demonstrates extensive deviation among knees, especially those accompanied with extra-articular deformities such as excessive coronal bowing of the femur or significant varus deformities8,11. Using a fixed VCA range of 5°–7° in these patients can cause major inaccuracies in distal femoral resection and final femoral implant coronal alignment. In a study on Chinese patients, Yau et al.10 stated that result if a routine VCA of 5°, 6°, or 7° was chosen then it would result in a planning error of greater than 2° in at least 31%, 31%, and 34% of the limbs with femoral bowing, respectively. Even though the enhanced conventional technique by customized VCA in each patient can help attain precise distal femoral cuts during total knee arthroplasty, the use of an extra-medullary guide rod instead of an intramedullary rod is essential. An intra-medullary rod may be deceptive in patients with significant femoral bowing or an extra-articular abnormality; for instance, a fracture malunion in the distal femur may lead to malalignment of the intramedullary guide rod and distal femoral cutting block. The precision of the extramedullary guide rod is improved by determining the center of the femur head in advance using image intensifier. However, one downside of the enhanced conventional technique is a slight increase in the overall surgical time due to the use of an image intensifier to locate the centre of the femoral head after the patient has been anaesthetized. On the other hand, computer navigation can avoid any extra-articular deformity in the distal femur and aids in directly aligning the distal femur cutting block perpendicular to the coronal mechanical axis of the femur, thus decreasing the chances of an erroneous cut. Furthermore, the majority of conventional distal femur cutting guides have a maximum VCA setting of 9°, beyond which the surgeon may have to lateralise the entry point, increasing the chances of cutting error. In such cases, using computer navigation or the described enhanced conventional method can help improve precision of femoral component placement.\n\nThere are some limitations to our study. As the objective of this study was to compare the accuracy of locating the femoral component in the coronal plane on scanogram, there is no clinical follow-up data regarding functional scores or revision rates. Future studies based on clinical outcome and implant survival between the two follow-up groups may be needed. This study involved measuring various parameters on a scanogram, which is predisposed to errors in measurement. Nonetheless, the measurements of limb and component alignment on full length hip-to-ankle radiographs (scanogram) have been reported to be reliable and reproducible with good intra- and inter-observer correlation16.\n\n\nConclusion\n\nUsing the enhanced conventional technique in each patient to perform distal femoral cut during total knee arthroplasty can help achieve the coronal alignment of the femoral component comparable to navigation technique.\n\n\nData availability\n\nHarvard Dataverse: Enhanced conventional method is as precise as navigation for distal femur resection during total knee replacement. https://doi.org/10.7910/DVN/DKOK6714.\n\nThis project contains de-identified raw data on pre- and post-operative knee angles, age and body mass index.\n\nHarvard Dataverse: CONSORT checklist for article “Enhanced conventional method is as precise as navigation for distal femur resection during total knee replacement: a randomized controlled trial”. https://doi.org/10.7910/DVN/DKOK6714.\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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nDyrhovden GS, Fenstad AM, Furnes O, et al.: Survivorship and relative risk of revision in computer-navigated versus conventional total knee replacement at 8-year follow-up. Acta Orthop. 2016; 87(6): 592–599. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJasper LL, Jones CA, Mollins J, et al.: Risk factors for revision of total knee arthroplasty: a scoping review. BMC Musculoskelet Disord. 2016; 17: 182. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBabazadeh S, Stoney JD, Lim K, et al.: The relevance of ligament balancing in total knee arthroplasty: how important is it? A systematic review of the literature. Orthop Rev (Pavia). 2009; 1(2): e26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThiele K, Perka C, Matziolis G, et al.: Current failure mechanisms after knee arthroplasty have changed: polyethylene wear is less common in revision surgery. J Bone Joint Surg Am. 2015; 97(9): 715–720. PubMed Abstract | Publisher Full Text\n\nValkering KP, Breugem SJ, van den Bekerom MP, et al.: Effect of rotational alignment on outcome of total knee arthroplasty. Acta Orthop. 2015; 86(4): 432–439. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSrivastava A, Lee GY, Steklov N, et al.: Effect of tibial component varus on wear in total knee arthroplasty. Knee. 2012; 19(5): 560–563. PubMed Abstract | Publisher Full Text\n\nRitter MA, Davis KE, Meding JB, et al.: The effect of alignment and BMI on failure of total knee replacement. J Bone Joint Surg Am. 2011; 93(17): 1588–1596. PubMed Abstract | Publisher Full Text\n\nLee CY, Huang TW, Peng KT, et al.: Variability of distal femoral valgus resection angle in patients with end-stage osteoarthritis and genu varum deformity: Radiographic study in an ethnic Asian population. Biomed J. 2015; 38(4): 350–355. PubMed Abstract | Publisher Full Text\n\nDeakin AH, Basanagoudar PL, Nunag P, et al.: Natural distribution of the femoral mechanical-anatomical angle in an osteoarthritic population and its relevance to total knee arthroplasty. Knee. 2012; 19(2): 120–123. PubMed Abstract | Publisher Full Text\n\nYau WP, Chiu KY, Tang WM, et al.: Coronal bowing of the femur and tibia in Chinese: its incidence and effects on total knee arthroplasty planning. J Orthop Surg (Hong Kong). 2007; 15(1): 32–36. PubMed Abstract | Publisher Full Text\n\nNam D, Vajapey S, Haynes JA, et al.: Does Use of a Variable Distal Femur Resection Angle Improve Radiographic Alignment in Primary Total Knee Arthroplasty? J Arthroplasty. 2016; 31(9 Suppl): 91–96. PubMed Abstract | Publisher Full Text\n\nPalanisami D, Iyyampillai G, Shanmugam S, et al.: Individualised distal femoral cut improves femoral component placement and limb alignment during total knee replacement in knees with moderate and severe varus deformity. Int Orthop. 2016; 40(10): 2049–2054. PubMed Abstract | Publisher Full Text\n\nShi X, Li H, Zhou Z, et al.: Comparison of Postoperative Alignment Using Fixed vs Individual Valgus Correction Angle in Primary Total Knee Arthroplasty With Lateral Bowing Femur. J Arthroplasty. 2016; 31(5): 976–983. PubMed Abstract | Publisher Full Text\n\nSingh R: Enhanced conventional method is as precise as navigation for distal femur resection during total knee replacement. Harvard Dataverse, V2. 2019. http://www.doi.org/10.7910/DVN/DKOK67\n\nMason JB, Fehring TK, Estok R, et al.: Meta-analysis of alignment outcomes in computer-assisted total knee arthroplasty surgery. J Arthroplasty. 2007; 22(8): 1097–1106. PubMed Abstract | Publisher Full Text\n\nSkyttä ET, Haapamäki V, Koivikko M, et al.: Reliability of the hip-to-ankle radiograph in determining the knee and implant alignment after total knee arthroplasty. Acta Orthop Belg. 2011; 77(3): 329–335. PubMed Abstract"
}
|
[
{
"id": "46620",
"date": "17 Apr 2019",
"name": "Robert Wen-Wei Hsu",
"expertise": [
"Reviewer Expertise TKA.THR. Arthroscopy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 concise draft with clear, specific aims and somewhat reasonable conclusions. The authors conducted an RCT to study the specific radiographic outcomes between the “enhanced conventional” and “computer assisted” TKAs. However, some points related to the article can be raised for discussion. These were described in the following:\n\nTwo terminologies, “optimized conventional” and “enhanced conventional” methods, were stated inside the article. What was the difference?\n\nThe “enhanced conventional method” was not clearly defined, please address it.\n\nThe “Radio-opaque marker” method was used before and abandoned later. During the TKA procedures, the lower limb was frequently moved. The accuracy of femoral head center is uncertain, especially in the obese patients. The stability of marker locations were in question. That is why this method is not popular in current TKA practice. Was there any modification in current method comparing previous recants?\n\n“BMI” might have some influence on this “enhanced conventional method outcome, please discuss it.\n\nThe use of fluoroscopy with potential radiation is another disadvantage with this method.\n\nThe overall alignment of lower limbs. Including MA and FVA in the coronal plane are not the only factors which might affect the final ”functional outcomes”. The impact of this \"enhanced conventional method” on future outcome is still unknown.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-360
|
https://f1000research.com/articles/8-87/v1
|
22 Jan 19
|
{
"type": "Opinion Article",
"title": "Open laboratory notebooks: good for science, good for society, good for scientists",
"authors": [
"Matthieu Schapira",
"The Open Lab Notebook Consortium",
"Rachel J. Harding"
],
"abstract": "The fundamental goal of the growing open science movement is to increase the efficiency of the global scientific community and accelerate progress and discoveries for the common good. Central to this principle is the rapid disclosure of research outputs in open-access peer-reviewed journals and on pre-print servers. The next bold step in this direction is open laboratory notebooks, where research scientists share their research — including detailed protocols, negative and positive results — online and in near-real-time to synergize with their peers. Here, we highlight the benefits of open lab notebooks to science, society and scientists, and discuss the challenges that this nascent movement is facing. We also present the implementation and progress of our own initiative at openlabnotebooks.org, with more than 20 active contributors after one year of operation.",
"keywords": [
"open lab notebooks",
"open science",
"peer-review",
"preprints",
"publishing",
"science communication"
],
"content": "Introduction\n\nThe function of the scientific peer-reviewed system is to provide greater confidence that published research is scientifically sound. This system is widely accepted as the best available, although imperfect (as peer reviewers may miss technical flaws or be biased)1, to guide the global scientific community towards progress. Peer-reviewed publishing is also used by research scientists, funders and institutions as a mechanism to claim ownership of their discoveries. As a result, the community widely believes that findings should be kept secret until they are published in a peer-reviewed journal. This tradition of secrecy, which protects the scientist as opposed to the science, has been transmitted from mentor to trainee for centuries (Galileo kept his discoveries to himself until they were published). In the life sciences, this belief can reach near-mystical levels2. The peer-review and publication process grew in an era where communication was largely in paper format. Today, in the age of instant communication, one would imagine there should be more efficient ways to operate.\n\n\nOpen lab notebooks: good for science and society\n\nWe believe that open laboratory notebooks, where research scientists record their work online and in near-real time, are an efficient way to disseminate data before it is published in peer-reviewed journals, and has several advantages over the traditional “release after publication” system3. First, making the data accessible within weeks rather than keeping it hidden for years means that others will be able to build upon the research, and avoid spending time and resources on redundant experiments4. Second, open lab notebooks should include detailed protocols that can be reproduced, which is often not the case in peer-reviewed publications5,6. Third, negative data, which are almost never disclosed in the current publishing system but are provided in open lab notebooks, can sometime provide important insight7,8. Fourth, open lab notebooks offer a space for anyone to comment on experimental records. This allows experts to provide insight, but also to flag technically unsound experiments, thereby reducing the potential for flawed science to appear in peer-reviewed journals and in pre-print media. Open lab notebooks can therefore help save time, resources, and knowledge. If adopted by many, they should lead to a more synergistic way to do science and to more efficient use of public funds.\n\n\nGood for scientists\n\nMany believe that openly sharing work online will limit career opportunities. We argue that open lab notebooks have compensating advantages that are good for scientists. To succeed in academia, one must get funding, assert primacy over discoveries, be known in a field of research and be able to present work and ideas clearly and convincingly. Open lab notebooks can help in all aspects.\n\nFirst, funding agencies are seeing the open science movement as a long lasting and far-reaching shift for the best, and are increasingly supportive of efforts to embrace open science principles. For instance, the symposium set to launch openlabnotebooks.org was entirely sponsored by the Wellcome Trust and the Canadian Institute of Health Research, and senior representatives from the Gates Foundation and the Chan-Zuckerberg Initiative were also in attendance (https://www.thesgc.org/open-lab-notebooks-2018). The NIH’s National Institute on Aging dedicated an entire session to open science at their 2018 Alzheimer’s research summit (https://www.nia.nih.gov/research/nih-ad-summit-2018-program-agenda), as did the 2018 Enroll-HD congress of the CHDI Huntington’s Disease Foundation (https://www.enroll-hd.org/enroll-hd-congress-2018/). The Wellcome Trust has recently launched the Wellcome Open Research publishing platform and Open Research Fund. Grant applications that highlight the use of open lab notebooks are being viewed positively. For example, Huntington’s disease (HD) research funders such as the CHDI Foundation, the Huntington Society of Canada and the Huntington Society of America, have all generously funded studies of HD biochemistry at the SGC Toronto.\n\nSecond, results in open lab notebook are date-stamped, thus claiming temporal priority of the data. Indeed, public repositories such as Zenodo9 add a date-stamp to depositions, and assign a citable DOI to open lab notebook records (detailed below).\n\nThird, early career scientists can use their open notebooks to connect with their peers and with experts in the field, start new collaborations and build their own network. Fourth, the use of open lab notebooks provides opportunity to present work clearly and concisely to both experts and non-experts. This is an important skill to master in order to write convincing grant applications. Fifth, junior scientists will also find their open lab notebook a good medium to showcase their technical skills and scientific insight, and may find it useful to add a link in their resume when applying for their next position. Finally, many will find a personal satisfaction in embracing open science and FAIR data principles10.\n\n\nImplementation of an open lab notebook platform\n\nFollowing our prediction that open lab notebooks should be good for science and good for scientists, and after a 2-year pilot study where Rachel Harding, a post-doctoral fellow at the Structural Genomics Consortium (SGC) shared her work on Huntington’s disease at labscribbles.com (https://www.vox.com/2016/3/3/11148452/science-blog), we launched openlabnotebooks.org in January 2018, where 12 scientists from the SGC started reporting their work live, online11,12. Each post is composed of two documents. (1) A detailed and rigorous experimental record, including all data and protocols, which experts can evaluate, comment on or build upon (Figure 1); (2) a blog, aimed at the non-specialist that explains in simple terms the motivation and rational for the experiment, summarizes results – positive and negative – and outlines next steps (Figure 2). The blogs, posted at openlabnotebooks.org, are managed by a webserver downloaded from wordpress.org and link to the experimental records, which are deposited at Zenodo (zenodo.org), but can also be made available from other public repositories, such as GitHub (github.com) or Figshare (figshare.com). The Zenodo repository enables sharing research outputs from across all fields of research, creation and curation of complete digital repositories, flexible licensing with controlled degree of openness and safe storage of the data for the future in the same cloud infrastructure as CERN's own LHC research data. While the experimental details posted at Zenodo are important scientifically, the blog written in layman’s term can be used to engage with scientists that may have a complementary set of expertise for future collaborations as well as other stakeholders in the research process, including patient groups, a dimension that most in academia are missing.\n\nA citable DOI is automatically generated (right-middle panel), and the number of visits and downloads provided (top right).\n\nThe ultimate goal of this open lab notebook initiative is not only to increase the impact of our work but also, along with precursors in the field such as Open Source Malaria (http://opensourcemalaria.org/) and other isolated open lab notebook efforts, to inspire others to follow, and contribute to the creation of a new open science movement in the life sciences. While it is too early to judge the success of this initiative, the number of contributing scientists and institutions is steadily increasing (Figure 3). While only one scientist was contributing in November 2017, 23 scientists from six institutions (University of Toronto, University of Oxford, University of North Carolina, University of Leicester, the Karolinska Institute in Sweden and University of Montpellier in France) are recording their work at openlabnotebooks.org as of December 2018.\n\nThe Number of (a) scientists and (b) institutions actively contributing to openlabnotebooks.org. (c) The average number of unique visits for each experimental record.\n\nAs importantly, impact is also increasing, judging by the average number of views per experimental record calculated from statistical data available at Zenodo.org (Figure 3). Some reports raised a considerable interest. For instance, the crystal structure of USP5 in complex with small molecule fragments has 821 unique views and 324 unique downloads as of December 201813. If the initiative is successful, we anticipate that within three to five years, usage metrics are comparable at openlabnotebooks.org and bioRxiv, the preprint server for biology.\n\nData posted at openlabnotebooks.org are raising interest in academic groups, but also in the industry. For instance, a notebook contributor was directly contacted by a big pharmaceutical company to further discuss the results that he had shared online, and a big biotech company asked permission to another contributor to include their data in a presentation at a public scientific meeting. Some of the research reported at openlabnotebooks.org is of direct relevance to patient groups. For instance, four scientists record their results on testing chemical inhibitors of the kinase ALK2, a potential therapeutic target for the treatment of the pediatric brain tumor diffuse intrinsic pontine glioma (DIPG), and the heterotopic ossification disorder fibrodysplasia ossificans progressive (FOP)14,15. The compounds, developed by the open science biotech company M4KPharma, are still in pre-clinical phase of development but should ultimately lead to clinical trials for these incurable diseases16. Scientists working on projects with a clear path to the clinic are eager to share their enthusiasm and commitment with patient groups (sometimes using social media to announce their latest open notebook post) who, in turn, follow their work.\n\n\nThe challenges of open lab notebooks\n\nThree antagonizing points that inhibit scientists from starting their own open lab notebook are the fear of being scooped, the inability to report collaborative work when collaborators want to keep data secret, and the concern that an open notebook will take time away from an already overburdened schedule17. The language barrier for non-native English speakers, and the availability of open lab notebook solutions can also be challenging. It is indeed likely that maintaining an open lab notebook increases the chances of being scooped, but it is too early at this point to know whether this effect is minor or significant. Paradoxically, and given the territorial nature of the current frameworks for funding and managing scientific research, entries in one’s open lab notebook may mark one’s area very effectively, especially in a conceivable future when funding trusts and councils start looking into them. We would argue that most, if not all, scientists get scooped during their career, and that open lab notebooks serve as a safety net for early career scientists who have a citable record of their work if they ever get scooped. Obtaining permission from collaborators to report collaborative work in open lab notebooks can be challenging. We believe that the best way to avoid such a situation is to clearly state at the outset of a collaboration the intention to adopt open science principles18. Scientists are more likely to agree if presented with the idea well in advance. The time invested in practicing clear, concise and engaging scientific writing is not lost on one’s career. After some practice, maintaining an open lab notebook should not take more time than using a regular lab notebook.\n\n\nFuture directions and conclusion\n\nOpen lab notebooks represent a major departure from current practices in science (especially biomedical sciences) and hold a mix of promises and risks. As the community producing these lab notebooks is increasing, there is an opportunity to move beyond ideology and anecdotal data to evidence-based policy design. In the spirit of openness, we call on colleagues from both the life science and the social sciences communities to conduct systematic evaluation of the benefits and downsides of open lab notebooks. It will be important to compare several parameters on a yearly basis. These may include the frequency of research being scooped among scientists disclosing their work in open lab notebooks versus a less open reference group; the frequency of new collaborations; the frequency of comments and ideas received by the authors of open notebooks; and instances where open lab notebooks were essential for compliance with funder or institutional requirements. More difficult to assess will be issues such as recognition, career progression, speeding up research, and impact on reproducibility, but they could all be addressed with appropriate questionnaires and data analytics.\n\nOur goal is to see the number of open lab notebooks increase exponentially over the coming years. Future implementation of novel features, such as the ability to search for experiments containing compounds with specific chemical templates, is expected to extend the reach of the platform to medicinal and computational chemists. Indexing of open lab notebooks by popular search engines such as Google Scholar (which already indexes pre-prints and other non-peer-reviewed documents) would increase the visibility and impact of open notebooks. Importantly, open lab notebook data deposited at Zenodo.org is already searchable with Google’s Dataset search engine. To further encourage scientists to break free from the tradition of secrecy that has been passed on for generations, a cultural change needs to be supported at institutional and governmental levels. Funding bodies are starting to define and enforce open science publication practices19. Similarly, universities could take a more proactive role, for instance by including adhesion to open-access principles as an evaluation criteria for career advancement20. Indeed, while strong incentives described above already exist for junior scientists to start their own open lab notebook, the benefit to their PIs who already have established a professional network and don’t need to showcase their skills is not always as clear. As long as scientists are not convinced that open science is good for them, Science 2.0 will have to wait.\n\n\nData availability\n\nNo data are associated with this article",
"appendix": "Grant information\n\nThe Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and The Wellcome Trust [106169/ZZ14/Z]. RJH is a recipient of the HDSA Berman/Topper HD Career Development Fellowship. PR is funded by the Wellcome Trust Leicester ISSF award reference 204801/Z/16/Z and the Leicester Institute of Chemical and Structural Biology (LISCB).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank the following principal investigators whose group members are contributing to openlabnotebooks.org: Cheryl Arrowsmith, Dalia Barsyte, Paul Brennan, Alex Bullock, David Drewry, Susanne Gräslund, Brian Marsden, Dave Morris, Panagiotis Prinos, Frank Von Delft, Tim Willson, and Wyatt Yue.\n\nThe Open Lab Notebook Consortium is made up of Roslin Adamson, Jose Brandao-Neto, Elizabeth J. Brown, Antoine Claessens, David Damerell, David Dilworth, Thomas Durcan, Benjamin J. Eduful, Aled M. Edwards, Opher Gileadi, Jolene Caifeng Ho, Leonidas Koukouflis, Tobias Krojer, Genna M. Luciani, Sabrina Mackinnon, Mandeep Mann, Carolyn Marks, Sean O’Byrne, Alfredo Picado, Pietro Roversi, Louisa Temme, Eleanor Williams, Jong Fu Wong, Wen Yih Aw.\n\n\nReferences\n\nSmith R: Peer review: a flawed process at the heart of science and journals. J R Soc Med. 2006; 99(4): 178–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nResnik DB: Openness versus Secrecy in Scientific Research Abstract. Episteme (Edinb). 2006; 2(3): 135–147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWoelfle M, Olliaro P, Todd MH: Open science is a research accelerator. Nat Chem. 2011; 3(10): 745–8. PubMed Abstract | Publisher Full Text\n\nPowell K: Does it take too long to publish research? Nature. 2016; 530(7589): 148–51. PubMed Abstract | Publisher Full Text\n\nWallach JD, Boyack KW, Ioannidis JPA: Reproducible research practices, transparency, and open access data in the biomedical literature, 2015–2017. PLoS Biol. 2018; 16(11): e2006930. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReality check on reproducibility. Nature. 2016; 533(7604): 437. PubMed Abstract | Publisher Full Text\n\nMlinari A, Horvat M, Šupak Smolčić V: Dealing with the positive publication bias: Why you should really publish your negative results. Biochem Med (Zagreb). 2017; 27(3): 030201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarroll HA, Toumpakari Z, Johnson L, et al.: The perceived feasibility of methods to reduce publication bias. PLoS One. 2017; 12(10): e0186472. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNielsen LH: Sharing your data and software on Zenodo. 2017.\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOpen notebooks galore: The Structural Genomics Consortium. eLife. [Accessed: 24-Dec-2018] 2018. Reference Source\n\nSchapira M: Open Lab Notebooks to increase impact and accelerate discovery. Research Data at Springer Nature. [Accessed: 24-Dec-2018] 2018. Reference Source\n\nMann M, Harding R, Ravichandran M, et al.: Co-crystal structures of USP5 Zf-UBD and weak binding compounds. Zenodo. 2018. Publisher Full Text\n\nvan Dinther M, Visser N, de Gorter DJ, et al.: ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation. J Bone Miner Res. 2010; 25(6): 1208–1215. PubMed Abstract | Publisher Full Text\n\nTaylor KR, Vinci M, Bullock AN, et al.: ACVR1 Mutations in DIPG: lessons learned from FOP. Cancer Res. 2014; 74(17): 4565–4570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan MR, Roberts OG, Edwards AM: Ideation and implementation of an open science drug discovery business model – M4K Pharma. Wellcome Open Res. 2018; 3: 154. Publisher Full Text\n\nRobertson MN, Ylioja PM, Williamson AE, et al.: Open source drug discovery - a limited tutorial. Parasitology. 2014; 141(1): 148–157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMasum H, Rao A, Good BM, et al.: Ten simple rules for cultivating open science and collaborative R&D. PLoS Comput Biol. 2013; 9(9): e1003244. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElse H: Radical open-access plan could spell end to journal subscriptions. Nature. 2018; 561(7721): 17–18. PubMed Abstract | Publisher Full Text\n\nAlperin JP, Fischman GE, McKiernan EC, et al.: How significant are the public dimensions of faculty work in review, promotion, and tenure documents? 2018. Publisher Full Text"
}
|
[
{
"id": "43417",
"date": "06 Feb 2019",
"name": "Matthew H. Todd",
"expertise": [
"Reviewer Expertise Open source drug discovery",
"organic and medicinal chemistry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion piece is on a timely, important topic and is clearly and engagingly written. Anecdotally, we find that many of our colleagues in science are unaware that open lab notebooks exist. This article will help.\nThe authors identify several important advantages and challenges associated with the near-immediate deposition of results into the public domain, online. They use examples from their own research to highlight the possibilities.\nThe refereeing team behind this review are seasoned users of open lab notebooks, and so are in a good position to judge the piece. We judge it to have cleared peer review from our perspective, once the following comments and suggestions have been acted upon. There are a number, which should be read not as criticism but as testament to our shared enthusiasm for this subject and its importance in the future of research.\n1) Secrecy. In the introduction, reasons are suggested for why scientists may keep results secret. We would suggest that there are two important reasons that are not explicitly mentioned: i) that the scientist may want to patent something, and ii) that the scientist cannot be bothered to work out how to release research using atypical means. The first point is alluded to where mention is made of ownership, and the second point is alluded to by the mention of \"paper\" but we would argue these two factors are significant enough that they should be made explicit.\n2) Careers. We'd be interested in whether there is a justification for the statement \"Many believe that openly sharing work online will limit career opportunities.\" If there is none, then perhaps rephrase this more as a possibility?\n3) Grants. The statement \"Grant applications that highlight the use of open lab notebooks are being viewed positively\" may be true (one hopes it is), but the evidence presented doesn't support that statement (the grants may have been funded because the science was so good, regardless of the dissemination plan), so again, this probably needs to be made more aspirational.\n4) Errors. The authors need to address what happens if an experiment is recorded containing a mistake, and a mistake that might propagate via an ungrounded conclusion. Is there a danger in leading colleagues down the wrong path? Is there a danger to the reputation of young scientists? Would incorrect conclusions, if indexed by search engines, lead to literature pollution that might be hard to correct?\n5) Machines. One must make the assumption that few people will ever read open lab notebooks, just as few people read regular lab notebooks. More needs to be made of this, since a) it is essential, rather than just desirable, that the notebook is searched and indexed by e.g. Google, and b) the entries need, ideally, to be machine readable. Could the authors comment on this - i.e. how Googleable the contents are (beyond the Dataset Search they mention) and what can be done to ensure that the entries can be understood by machines?\n6) Permanence. There are many web links in the article. This is an academic publication, which is intended to last forever. It's likely those URLs will not last forever. Is there a way that the pages pointed to can be archived somehow? An important, relevant example: this manuscript does not refer to one of the pioneers of open lab notebooks - indeed he coined the phrase \"open notebook science\" - Jean-Claude Bradley. Bradley's blog can still be accessed but the wiki that used to house all the raw data has gone. In publishing our most recent paper that made use of open lab notebooks1, we took pains to archive the lab notebooks on a repository to mitigate potential loss by external providers, and to back up web pages (as PDFs) to similar places. Can the authors address the two issues here: i) should we link to web pages in academic articles without backing them up? 2) How are the lab notebooks backed up and archived?\n7) Time Stamps. The authors mention \"results in open lab notebook are date-stamped.\" It would be interesting to know whether the authors have looked into whether the date stamps are \"convincing\" from a legal standpoint. Would they, for example be sufficient to claim priority in a legal challenge? Is there an issue with the fact that ELN pages can be edited, over an extended period of time, or is it sufficient to ensure that the page has a robust revision history?\n8) Scooping. A scooper could claim that they were simply unaware of the open scientist's work. This seems a reasonable defense. What needs to happen for ignorance of an open lab notebook not to be a defense against ignorance of prior art?\n9) Citations. We are cited twice (thank you), but there are two other relevant papers the authors might be interested in citing should these be deemed by them to be appropriate: i) an extensive discussion on the use cases of an open source ELN called Labtrove 2 pioneered by Jeremy Frey's team at Southampton, and ii) a large medchem project by Open Source Malaria which was conducted entirely using open lab notebooks 1 and which may be instructive in terms of how to publish a paper based on open notebook work (also described informally here.\n10) Examples of Inputs. Are there any examples the authors can point to where the open ELN has helped the research, e.g. where suggestions have been made, and acted upon, to help the science?\n11) Examples of ELNs. Are there other examples of open lab notebooks being used in biomedical research? Is this, in fact, still a highly niche activity?\n12) Language. The language barrier is mentioned, but we're not sure this is relevant to this paper. Language is already a barrier in the current system, though English is essentially a lingua franca across science.\n13) Licence. What is the license that covers the authors' i) blog posts and ii) notebooks? To what extent is this an important choice? For example, can others take and re-use the content without restriction, or only for non-commercial purposes?\n14) Raw Data. The authors mention that the ELN entries include \"all data\". This is an important feature of open ELNs that distinguishes the practice from a great deal of open science in which highlights might be discussed, or blogged about, but without the attendant raw data. We investigated briefly to see how much of the raw data could be accessed in the authors' cases. We have noticed that a fair number of the lab notebook entries recorded by the team in Zenodo contains PDF/Word-style summaries of data, rather than the data themselves. This limits re-use of the data. Could the authors comment on the central importance of the availability of all raw data?\nExamples:\nThis blog post mentions performing synthesis of analogues based on lit procedures but there’s no link to an ELN. Same with this page. And this. This page has a link (in the 2nd paragraph) to a (structure?) data file but no backlink from the data back to the blog page, risking orphaning of content? This blog page does have a link to the ELN. There is more experimental detail, but there is no raw characterization data. Also the backlink to the blog page is broken. Many biology entries contain pictures of purifications and for the most part, the ELN pages only have a docx or pdf there. Should there be any raw data available for these kinds of pages, or are docx and PDF enough?\n\n15) Identifiers. It is mentioned that is is desirable to have \"the ability to search for experiments containing compounds with specific chemical templates\". We agree. Some ELNs already allow this (e.g. Chemotion 3 and C6H6 4). We have found a stop-gap is the manual inclusion of chemical strings such as SMILES, InChI. Can the authors comment on whether similar things can be included in order to allow machines to understand the biology contained within the entries, e.g. UniProt numbers?\n16) Metrics re Access. Is there a way of distilling out access by non-team people, or even access by the author of the page who may have accessed multiple times during the editing of a page?\n17) Ownership. When a researcher moves on, does that researcher have any responsibility towards the ELN? e.g. if a mistake is found, who needs to correct it? Is it the responsibility of the PI to act as future curator?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "4510",
"date": "02 Apr 2019",
"name": "Matthieu Schapira",
"role": "Author Response",
"response": "1) Secrecy. In the introduction, reasons are suggested for why scientists may keep results secret. We would suggest that there are two important reasons that are not explicitly mentioned: i) that the scientist may want to patent something, and ii) that the scientist cannot be bothered to work out how to release research using atypical means. The first point is alluded to where mention is made of ownership, and the second point is alluded to by the mention of \"paper\" but we would argue these two factors are significant enough that they should be made explicit. Points well taken. The following statement was added to the Introduction “…and can be compounded by constraints associated with patent protection procedures or the absence of clear mechanism to make one’s data publicly available.” 2) Careers. We'd be interested in whether there is a justification for the statement \"Many believe that openly sharing work online will limit career opportunities.\" If there is none, then perhaps rephrase this more as a possibility? This was not clear. The sentence was replaced as follows: “Many believe that the chances of getting scooped before one publishes their work in a peer-reviewed journal increase when openly sharing their work online [9]” 3) Grants. The statement \"Grant applications that highlight the use of open lab notebooks are being viewed positively\" may be true (one hopes it is), but the evidence presented doesn't support that statement (the grants may have been funded because the science was so good, regardless of the dissemination plan), so again, this probably needs to be made more aspirational. This was revised as follows: “Our personal observations seem to indicate that grant applications highlighting the use of open lab notebooks are being viewed positively.” 4) Errors. The authors need to address what happens if an experiment is recorded containing a mistake, and a mistake that might propagate via an ungrounded conclusion. Is there a danger in leading colleagues down the wrong path? Is there a danger to the reputation of young scientists? Would incorrect conclusions, if indexed by search engines, lead to literature pollution that might be hard to correct? We agree that this important point was missing. The following was added to the section “The challenges of open lab notebooks”: Open notebooks being published before peer-review, there is a risk that dubious experiments, erroneous analysis or misinterpretations find their way on open platforms, get amplified over the internet and mislead colleague scientists, patient groups or other communities. Once they become indexed by popular search engines, open lab notebooks could become a source of pollution of the scientific (and non-scientific) literature. This risk, which is not limited to open notebooks but extends to the increasing number of Journals that adopt a post-publication peer-review mechanism, is real and serious. We believe that the best way to mitigate this risk is for open notebooks to provide a platform for open comments. In principle, this could be an even stronger quality control than the current peer-review system in place in most scientific journals, as the number of “open reviewers” for any given report is limitless. At the moment, we find that very few comments are posted at openlabnotebooks.org, a platform that is only a year old, but we see that comments are mainstream, and sometimes turn into healthy discussions at Open Source Malaria, a pioneer in the field. 5) Machines. One must make the assumption that few people will ever read open lab notebooks, just as few people read regular lab notebooks. More needs to be made of this, since a) it is essential, rather than just desirable, that the notebook is searched and indexed by e.g. Google, and b) the entries need, ideally, to be machine readable. Could the authors comment on this - i.e. how Googleable the contents are (beyond the Dataset Search they mention) and what can be done to ensure that the entries can be understood by machines? We believe that in the life sciences, most would find the constraint of formatting their notebook in a standardized language an unacceptable burden. Open lab notebooks should be indexable by Google or other search engines as free text records. Chemists would benefit from the integration of cheminformatics functionality, such as fingerprint-based substructure or similarity searches. These tools are openly provided by RDKit, and we expect that they will soon be integrated into open lab notebook platforms. 6) Permanence. There are many web links in the article. This is an academic publication, which is intended to last forever. It's likely those URLs will not last forever. Is there a way that the pages pointed to can be archived somehow? An important, relevant example: this manuscript does not refer to one of the pioneers of open lab notebooks - indeed he coined the phrase \"open notebook science\" - Jean-Claude Bradley. Bradley's blog can still be accessed but the wiki that used to house all the raw data has gone. In publishing our most recent paper that made use of open lab notebooks1, we took pains to archive the lab notebooks on a repository to mitigate potential loss by external providers, and to back up web pages (as PDFs) to similar places. Can the authors address the two issues here: i) should we link to web pages in academic articles without backing them up? 2) How are the lab notebooks backed up and archived? We appreciate this concern. We have multiple layers of backup for the openlabnotebooks.org web-site including snapshots, replication, and off-site tape storage. We also export all blog posts automatically at the end of each week to the following GitHub repository https://github.com/thesgc/static-openlabnotebooks. The Information Technology Services at University of Toronto is now also maintaining an archive of the website at https://wayback.archive-it.org/6473/*/https:/opennotebook.thesgc.org/, which is updated quarterly. The data posted on Zenodo is also backed-up, as specified at https://about.zenodo.org/infrastructure/. Archiving each website that one cites is an impressive commitment to the permanence of one’s work. We assume that consent should first be obtained to archive someone else’s research output. We are not planning to follow this path at the moment. The following statement was added to the article: “The blogs, posted at openlabnotebooks.org, are managed by a webserver downloaded from wordpress.org, archived weekly to GitHub (repository https://github.com/thesgc/static-openlabnotebooks), quarterly to archive.org (https://wayback.archive-it.org/6473/*/https:/opennotebook.thesgc.org/), and link…” We also added the following section: “Open laboratory notebooks need to guarantee that the data will remain accessible, in order to avoid the fate suffered by the pioneer open notebook of Jean-Claude Bradley, which is still accessible while its associated raw data wiki is not. Zenodo is strongly committed to preserving the data it archives. CERN has existed since 1954 and has an experimental program defined for the next 20+ years. Each file copy has two replicas located on different disk servers. In the highly unlikely event that Zenodo closes operations, they guarantee migration of all content to other suitable repositories, and since all uploads have DOIs, citations and links to Zenodo resources (including data) will not be affected.” 7) Time Stamps. The authors mention \"results in open lab notebook are date-stamped.\" It would be interesting to know whether the authors have looked into whether the date stamps are \"convincing\" from a legal standpoint. Would they, for example be sufficient to claim priority in a legal challenge? Is there an issue with the fact that ELN pages can be edited, over an extended period of time, or is it sufficient to ensure that the page has a robust revision history? We believe that whether a particular electronic date stamp is sufficient to prove the date of disclosure would depend on how well the system was configured and how good the records were logged. Website businesses do depend on date stamping to establish when a particular change to their terms & conditions was made, for example. Our understanding is that the system implemented at Zenodo clearly indicates when a record was posted and when it was edited, which should be sufficient evidence for open notebooks to count as prior art. To clarify the date-stamp/versioning mechanism in place at Zenodo, we added the statement “once a record has been published, it can no longer be modified, but revised versions can be appended if necessary” 8) Scooping. A scooper could claim that they were simply unaware of the open scientist's work. This seems a reasonable defense. What needs to happen for ignorance of an open lab notebook not to be a defense against ignorance of prior art? We believe that scientists learn to live with the potential and reality of being scooped. Plagiarism is another problem, but we believe that it is more an academic conduct issue than a legal issue. If materials & methods and results published in a date-stamped open notebook are later authored by another scientist in a peer-reviewed journal, this should in principle raise reputational problem for that person. 9) Citations. We are cited twice (thank you), but there are two other relevant papers the authors might be interested in citing should these be deemed by them to be appropriate: i) an extensive discussion on the use cases of an open source ELN called Labtrove 2 pioneered by Jeremy Frey's team at Southampton, and ii) a large medchem project by Open Source Malaria which was conducted entirely using open lab notebooks 1 and which may be instructive in terms of how to publish a paper based on open notebook work (also described informally here. We added a citation to the LabTrove paper in the section “Implementation of an open lab notebook platform”, and to the Open Source Malaria paper just above the “Future directions and conclusion” section. 10) Examples of Inputs. Are there any examples the authors can point to where the open ELN has helped the research, e.g. where suggestions have been made, and acted upon, to help the science? Yes. Examples include researchers from a pharmaceutical company that contacted a scientist to further discuss his results, a big biotech company that kindly asked permission to include data from an open notebook at a conference talk, and academics interested in specific chemical templates included in an open report. Rachel Harding’s open notebook has had input from a number of readers including Professor Ray Truant (McMaster University) and Professor Jeff Carroll (Western Washington University), both of whom have initiated discussions about the research questions Dr. Harding is pursuing and ultimately lead to collaborative experimental design and analysis of data. 11) Examples of ELNs. Are there other examples of open lab notebooks being used in biomedical research? Is this, in fact, still a highly niche activity? Open lab notebooks are far from mainstream and the number of active examples remains small. However, we enjoy following contributors of the Open Notebook Science Network http://onsnetwork.org/ amongst others. 12) Language. The language barrier is mentioned, but we're not sure this is relevant to this paper. Language is already a barrier in the current system, though English is essentially a lingua franca across science. We agree, but we have examples of scientists who do not feel confident enough in English to use our platform. 13) Licence. What is the license that covers the authors' i) blog posts and ii) notebooks? To what extent is this an important choice? For example, can others take and re-use the content without restriction, or only for non-commercial purposes? We currently license all of our open notebook blog posts and Zenodo deposits using a Creative Commons Attribution license. This allows our work to be shared and reused as widely as possible whilst asking for attribution of the original work only. More complex licenses may put off readers of the notebooks from reusing the content, which is in opposition of the open lab notebook ethos. 14) Raw Data. The authors mention that the ELN entries include \"all data\". This is an important feature of open ELNs that distinguishes the practice from a great deal of open science in which highlights might be discussed, or blogged about, but without the attendant raw data. We investigated briefly to see how much of the raw data could be accessed in the authors' cases. We have noticed that a fair number of the lab notebook entries recorded by the team in Zenodo contains PDF/Word-style summaries of data, rather than the data themselves. This limits re-use of the data. Could the authors comment on the central importance of the availability of all raw data? Examples: This blog post mentions performing synthesis of analogues based on lit procedures but there’s no link to an ELN. Same with this page. And this. This page has a link (in the 2nd paragraph) to a (structure?) data file but no backlink from the data back to the blog page, risking orphaning of content? This blog page does have a link to the ELN. There is more experimental detail, but there is no raw characterization data. Also the backlink to the blog page is broken. Many biology entries contain pictures of purifications and for the most part, the ELN pages only have a docx or pdf there. Should there be any raw data available for these kinds of pages, or are docx and PDF enough? We agree that all raw data should be included in open notebook reports, and we encourage scientists using openlabnotebooks.org to adhere to this principle. We are also aware that enforcing virtuous habits can put-off new users, and are opting for a more permissive approach, where we prefer to educate in best practices rather than imposing them. 15) Identifiers. It is mentioned that is desirable to have \"the ability to search for experiments containing compounds with specific chemical templates\". We agree. Some ELNs already allow this (e.g. Chemotion 3 and C6H6 4). We have found a stop-gap is the manual inclusion of chemical strings such as SMILES, InChI. Can the authors comment on whether similar things can be included in order to allow machines to understand the biology contained within the entries, e.g. UniProt numbers? Algorithm could easily be integrated in an notebook platform to interpret UniProt numbers. For machine to understand the biology associated with a UniProt number, if there is such a thing, is beyond our expertise. 16) Metrics re Access. Is there a way of distilling out access by non-team people, or even access by the author of the page who may have accessed multiple times during the editing of a page? The access metrics are directly generated by Zenodo, provides the number of visits, downloads, unique visits and unique downloads. Even if the author revisits or downloads multiple times his or her record, it would count as a single “unique event”. 17) Ownership. When a researcher moves on, does that researcher have any responsibility towards the ELN? e.g. if a mistake is found, who needs to correct it? Is it the responsibility of the PI to act as future curator? All records posted at openlabnotebooks.org and Zenodo remain indefinitely accessible. Our position is that the labnotebook is owned by the scientist who wrote it."
}
]
},
{
"id": "43420",
"date": "04 Mar 2019",
"name": "Lara M. Mangravite",
"expertise": [
"Reviewer Expertise open science",
"bioinformatics",
"pharmaceutical 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\nThe opinion piece on Open Laboratory Notebooks written by Dr.s Schapira, Harding, and the Open Lab Notebook Consortium provides a thoughtful commentary on an interesting and relevant topic. The Open Labnotebook provides an interesting and potentially powerful approach to support transition of scientific communication beyond traditional peer-reviewed scholarly publications. As the authors note, the availability of digital media provides a much wider array of approaches to support meaningful research dissemination – and to promote scientific reuse and collaboration.\n\nThe authors speak primarily from their own experience – and, because the use of open lab notebooks is quite new, there is often not a lot of data to back up their comments. As such, it was hard to understand what ideas were well supported and what ideas were in need of evaluation. Along those lines, the authors do not put their work into context of the broader field – I do not have a clear sense of who else is using an open labnotebook approach, whether their experiences are similar or different, and what portions of the scientific community were finding open labnotebooks useful. This context would help in synthesizing the ideas in the manuscript.\n\nSecondly, it would be enormously helpful to understand how the authors anticipate that open labnotebooks would operate in the context of the current scientific communication framework. Do you expect labnotebooks to replace peer-review journals or to complement them? How might a researcher convey the overarching hypothesis that they are looking to address through the set of experiments run across a series of notebook entries? It seems that neither the labnotebook approach nor the existing publication approach are sufficient to fully convey scientific output by themselves. I would very much enjoy understanding the authors’ perspective on this.\n\nAlong these same lines, I am curious as to how notebook readers will incorporate this form of communication into their workflow. It will have to be useful – and easy – in order to gain the desired level of adoption. I appreciated the inclusion of the notebook use statistics – it would be helpful to understand how users found these posts, what kind of users were attracted, and how they responded to this medium as a form of scientific discourse.\n\nThe role of the gatekeeper was also not addressed. Open LabNotebooks are reliant on a series of technical platforms. These platforms will have an impact on the overall success based on their sustainability models, their archiving approaches, and their need for financial viability. Is there a cost to using the system – and, if not, how is it funded? Are the content fully owned by the authors and do they remain under full control of the authors? What are the responsibilities of the platforms and how might their choices impact the approach?\n\nFinally, the authors have a tremendous opportunity to ground some of this theoretical work in their own practical application. It would be useful to understand how your scientists’ experiences compared to those were anticipated. For example, one frequent concern heard by researchers is one of time – they are worried that use of the system will require an additional burden in an already over-committed schedule. How much time do the current users spend on their posts? Did this reduce over time? This same approach can be used to address the anticipate benefits.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "4511",
"date": "02 Apr 2019",
"name": "Matthieu Schapira",
"role": "Author Response",
"response": "The authors speak primarily from their own experience – and, because the use of open lab notebooks is quite new, there is often not a lot of data to back up their comments. As such, it was hard to understand what ideas were well supported and what ideas were in need of evaluation. Along those lines, the authors do not put their work into context of the broader field – I do not have a clear sense of who else is using an open labnotebook approach, whether their experiences are similar or different, and what portions of the scientific community were finding open labnotebooks useful. This context would help in synthesizing the ideas in the manuscript. Open lab notebooks remain a niche of scholarly communication and the number of active examples is limited. An additional section has been added in “Implementation of an open lab notebook platform” describing the brief history and status of the open lab notebook community: “Open lab notebooks have been pioneered and championed by a number of practitioners but remain a niche activity in the scientific community. Jean-Claude Bradley first coined the term “open notebook science” in 2006 and his definition of this method of scholarly communication have laid the foundations for our own efforts [12]. In addition to the notebooks of individual researchers following Bradley’s template, open notebook examples now include collective efforts from the Open Lab Notebook Network (http://onsnetwork.org) and Open Source Malaria (http://opensourcemalaria.org). However, the open lab notebook community remains small, the practice is not consistently defined or implemented and the impact of these efforts in the field have not been systematically evaluated.” Secondly, it would be enormously helpful to understand how the authors anticipate that open labnotebooks would operate in the context of the current scientific communication framework. Do you expect labnotebooks to replace peer-review journals or to complement them? How might a researcher convey the overarching hypothesis that they are looking to address through the set of experiments run across a series of notebook entries? It seems that neither the labnotebook approach nor the existing publication approach are sufficient to fully convey scientific output by themselves. I would very much enjoy understanding the authors’ perspective on this. We see open lab notebooks as a mechanism for scientists to share their work before they publish it in peer-reviewed journals. As such, they are complementary to peer-reviewed publication. This is specified in the article as follows: “We believe that open laboratory notebooks, where research scientists record their work online and in near-real time, are an efficient way to disseminate data before it is published in peer-reviewed journals, and …” Along these same lines, I am curious as to how notebook readers will incorporate this form of communication into their workflow. It will have to be useful – and easy – in order to gain the desired level of adoption. I appreciated the inclusion of the notebook use statistics – it would be helpful to understand how users found these posts, what kind of users were attracted, and how they responded to this medium as a form of scientific discourse. We agree that much is to be gained from knowing the demographics and motivations of open lab notebook readers. The readers input in the well populated comment section of the platform Open Source Malaria strongly suggest that most readers are scientists in the field, sharing common goals and interests. The role of the gatekeeper was also not addressed. Open LabNotebooks are reliant on a series of technical platforms. These platforms will have an impact on the overall success based on their sustainability models, their archiving approaches, and their need for financial viability. Is there a cost to using the system – and, if not, how is it funded? Are the content fully owned by the authors and do they remain under full control of the authors? What are the responsibilities of the platforms and how might their choices impact the approach? The server hosting openlabnotebooks.org is maintained by the Structural Genomics Consortium at University of Oxford. It relies on Wordpress, which is an open source platform, and requires limited maintenance. Consequently, the service is free for both contributors and readers. Zenodo is hosted by the CERN and is also free. The issue of permanence, sustainability, archiving and ownership are important ones and are addressed in our response to Reviewer 1 (sections #6 and #17) and discussed in the revised version of the manuscript. Finally, the authors have a tremendous opportunity to ground some of this theoretical work in their own practical application. It would be useful to understand how your scientists’ experiences compared to those were anticipated. For example, one frequent concern heard by researchers is one of time – they are worried that use of the system will require an additional burden in an already over-committed schedule. How much time do the current users spend on their posts? Did this reduce over time? This same approach can be used to address the anticipate benefits. When initiating this project, prospective open notebookers were surveyed regarding their concerns, questions and attitudes about running their own open notebook projects which highlighted many of the concerns you raise, in particular, the time burden. We are planning to conduct a follow-up survey once the number of scientists joining our platform increases."
}
]
}
] | 1
|
https://f1000research.com/articles/8-87
|
https://f1000research.com/articles/8-22/v1
|
07 Jan 19
|
{
"type": "Opinion Article",
"title": "Climate change, migration and health systems resilience: Need for interdisciplinary research",
"authors": [
"Valéry Ridde",
"Tarik Benmarhnia",
"Emmanuel Bonnet",
"Carol Bottger",
"Patrick Cloos",
"Christian Dagenais",
"Manuela De Allegri",
"Ariadna Nebot",
"Ludovic Queuille",
"Malabika Sarker",
"Tarik Benmarhnia",
"Emmanuel Bonnet",
"Carol Bottger",
"Patrick Cloos",
"Christian Dagenais",
"Manuela De Allegri",
"Ariadna Nebot",
"Ludovic Queuille",
"Malabika Sarker"
],
"abstract": "Climate change is one of today's major challenges, among the causes of population movements and international migration. Climate migrants impact health systems and how they respond and adapt to their needs and patterns. But to date, the resilience of health systems in the context of climate change has been little explored. The purpose of this article is to show the importance of studying, from an interdisciplinary perspective, the relationships between climate change, migration, and the resilience of health systems. Resilience is an old concept, notably in the field of psychology, and is increasingly applied to the study of health systems. Yet, no research has analysed the resilience of health systems in the context of climate change. While universal health coverage is a major international goal, little research has to date focused on the existing links between climate, migration, health systems and resilience. We propose an interdisciplinary approach relying on the concept of health system resilience to study adaptive and transformative strategies to articulate climate change, migration and health systems.",
"keywords": [
"Climate Change",
"Migrations",
"Health Systems",
"Resilience",
"Interdisciplinary"
],
"content": "Introduction\n\n“Four thousand migrants arrive in Dhaka, the capital of Bangladesh due to various ‘push’ factors including frequent natural disasters”1. Indeed, environmental changes due to climate change are projected to cause substantial increases in population movement, within and between countries, in the coming decades. Haiti faces a similar situation according to a 2008 report estimating that 100,000 people had moved for climate change reasons from rural areas to the capital Port-au-Prince2. Environmental changes (e.g. drought, soil erosion, extreme weather events, etc.) lead to substantial impact on health, economic and political dimensions at the population level, including influencing migration patterns and may result in adverse health outcomes, both for displaced and for host populations, as we will discuss in more detail later3–5. Consistently, the World Health Organization (WHO) identifies climate change as a defining challenge of the 21st century; and considers it an emerging priority for the public health community to ensure protection against its health impact6,7. In 2015, The Rockefeller Foundation and The Lancet published the report of the Commission on Planetary Health8 and the UN Sustainable Development Goal 13 calls for “urgent action to combat climate change and its impacts\".\n\nFor this article, we conducted an heuristic literature review on climate change, migration and health systems. As a result of a peer-reviewed article search in the PUBMED database using climate change, health systems, and migrants as keywords, only 10 results published between 1994 and 2017 were identified. Of these, six (60%) were written in the past decade and included: two opinion papers, two study reviews, one qualitative study, and one protocol for a review that will be completed in 2018.\n\nIn this article, we describe and discuss the fundamental role that health care systems resilience can play in this regard and we identify interdisciplinary research as key to better understand the existing linkages between climate change, migration and health systems and how to build more resilient health systems. We also propose some questions and axes to orient future research proposals.\n\n\nClimate migrants and health systems\n\nClimate change can be translated into a wide range of environmental degradations, including hurricanes9, rising sea levels, and/or reduced rainfall in drylands and water scarcity10. Populations confronted by climate change consequences such as exposure to hazards, loss in land productivity, absence of habitability, and/or shortage of food/energy/water security may have difficulties to subsist in a given area11. Climate change consequences compounded by socio-economic pressures and/or political instability, increase propensity to migrate. Although evidence is still missing to prove this association, environmental factors are increasingly influencing an already complex pattern of human mobility. A recent paper suggests “a statistically significant relationship between fluctuations in asylum applications and weather anomalies”12. Climate migrants may be forced to leave their homes due to rapid-onset disasters, such as flooding and hurricanes (as in Haiti and Bangladesh for example)1,2,13,14.\n\nNowadays, there is no conceptual consensus on the notions of environmental refugee or climate change migrants yet, or the more rarely used terms ecomigrants or environmentally displaced persons15,16. Since 2007, the International Organization for Migration (IOM) has defined environmental migrants as “persons or groups of persons who, for compelling reasons of sudden or progressive change in the environment that adversely affects their lives or living conditions, are obliged to leave their habitual homes, or choose to do so, either temporarily or permanently, and who move either within their country or abroad”17. Others suggest restricting the definition to victims of extreme weather, drought/water scarcity, and sea-level rise and excluding the effects of the spread of tropical diseases16. The estimation that is most widely accepted is that over 200 million persons will be displaced globally by 2050 because of climate change13,15,18, and according to the last Lancet Countdown “the total number of people vulnerable to migration might increase to 1 billion by the end of the century without significant further action on climate change”5.\n\nClimate-related migrants may or may not perceive how climate change influences and has an impact on their health needs and social patterns. For example, in Burkina Faso, the close relationship between climate change and flooding is not always fully perceived by the Burkina population suffering from it, as documented by the authors of this manuscript in previous studies (Box 1) However, climate-related migrants experience difficulties or face challenges similar to those of refugees fleeing war and/or political persecution: overcrowded settlements, unsanitary conditions, poor nutritional status, unsafety, inequity and limited access to health services1,2,19,20. In addition, environmental change migrant population is usually the most vulnerable as well because migration is often expensive and climate change factors can easily lie on the top of other strong socio-economic factors. For example, Haiti and Bangladesh were respectively ranked 3rd and 6th globally in the Long-Term Climate Risk Index (CRI) from 1995 to 201421, while their health systems’ performances were ranked by the WHO in 2000 as 138th and 88th, respectively, out of 191 countries22. The very recent Global Climate Risk Index 2018 confirms these two places for Haiti and Bangladesh but also shows that several African countries (Mozambique, Malawi, Ghana, Madagascar) are very affected and have little research on climate migrants.23.\n\nA recent survey of Sahelian floods in Ouagadougou, Burkina Faso24, reveals that climate change is not seen by the population as responsible for the floods. They consider that the responsibility lies more with the authorities who did not act to maintain the water supply facilities. The links with climate change do not seem to be perceived by the citizens of Ouagadougou. In the meantime, they also report changes in overwintering dates, an increase in extreme rainfall incidence and precipitation variability. There are several documented direct and indirect health impacts associated with such patterns such as water-borne and vector borne diseases or food security10,25,26. These patterns in regards to the change in precipitation regimes with increases in the frequency of extreme wet and dry years are known to be intensified in the context of climate change27. It thus appears that, while the impacts of climate change on precipitation regimes are already observed by local populations, their perceptions about potential links still need to be enhanced.\n\nIn parallel, some individuals might be escaping slow-onset disasters, such as rising sea levels and declining agricultural yields; their migration patterns may be more similar to those of rural–urban migrants, and they might experience many similar obstacles and barriers to their health as well28. From the literature, it can be seen that some health related challenges may be identical: 1) Re-emergence of infectious diseases and geographical migration of diseases29: migrants spatially re-distribute infections from endemic areas to new populations; they are also exposed to new diseases due to unsanitary living conditions. 2) Reduced access to healthcare services: mass migration applies population pressure exceeding local health and social services capacity; perceptions of long wait times, confusing administrative procedures, or discrimination also impede health system access for migrants30. 3) Disrupted social support networks contribute to adverse mental health outcomes31, higher risk of violence, and spread of STIs, including HIV infection. Migrants are often seen as potential security challenges for countries18,32. Niger is one example for conducting more research to understand the phenomenon of infection diseases and migration, but at the same time for the health system to better adapt (Box 2)\n\nNiger, and it’s Agadez region, has long been known as a crossroads for the regional transhumance and immigration to the North. Agadez is one of the driest regions of the country with a very low and irregular rainfall level and therefore it’s classed as a hypo-endemic region of malaria33. In 2016, Agadez region reported 55411 malaria cases, of which the age groups of adults aged 25 over and children aged from 1 to 4 being 37% and 20%, respectively. These data contrast with the other countries where adults aged 25 and over account for only 17.4% and children aged 1 to 4 for 42.6%34. In fact, this is not an isolated case because the data for the last 6 years show the same relationship. This may be explained in part by the irregularity of malaria transmission, which leads to a loss of immunity to malaria by the population35. But it is also necessary to take into account that people that travel through this region are mostly young adults. One hypothesis could be that several cases reported as indigenous cases are, in fact, exported cases that have very different profiles (Plasmodium falciparum strain, drug resistance, associated pathology, behaviour toward the illness, etc.). Niger’s malaria control programs must adapt to these challenges.\n\nHowever, the lack of consensus of climate change migrant definition makes research on its health needs and patterns still difficult.\n\n\nClimate change in the global health context\n\nWith its inclusion in Goal 3 of The Sustainable Development Agenda, the concept of Universal Health Coverage (UHC) has obtained consensus from the international community36. UHC, regarded as the third global health transition37, or, according to former WHO director Margaret Chan, “the most effective concept that public health can offer”, aims at ensuring access to good quality care while limiting the impoverishment of people as a result of their illness38. In September 2015, the Director of WHO/PAHO for the Americas, Carissa F. Etienne, stated that “we must all cooperate to reduce those factors that are contributing to climate change and to mitigate its health effects.” Consecutively, in September 2017, the new WHO Director-General has set UHC as his greatest challenge and highlighted at the UN General Assembly on Migration Health in New York City that “health systems must be sensitive to the needs of migrants.” The direct and indirect effects of climate change on population health and disease development are now well discussed5,39, but there is still little literature on the health effects of migration (within and between countries) influenced by natural disasters and droughts exacerbated by climate change5. In addition, the role of the health care system as a social determinant of health40 and its capacity to protect populations affected by climate change was recently identified by the WHO6 and the Canadian Public Health Association41.\n\nHealth systems (and health professionals) suffer the shocks provoked from climate change and migration42,43. These shocks can be the direct consequence of climate change (floods, heat waves, hurricanes, etc) or indirect effects, i.e. the influx of patients suffering from diseases whose emergence or abnormally high frequency is due to climate change44. Therefore, health care systems need to adapt to population migrations (in and across countries) due to climate changes by taking into account the effects of both phenomena on the epidemiology of diseases45 (e.g. dengue vs malaria) and, more globally, by identifying and responding to specific social (e.g. social acceptability of migrants)46 and health problems (e.g. mental health) in this context. In this sense, there is a very close link between UHC and emergency preparedness, as the WHO has just pointed out calling for “a mutual reinforcement of emergency preparedness and health systems strengthening strategies”. Health security must also be achieved through good health systems preparedness for the disasters caused by climate change47. The capacity of health systems and their actors to prepare for and adapt to these climate-related shocks is known as resilience.\n\n\nHealth systems resilience in the climate change context\n\nResilience is a longstanding concept in the disciplines of life sciences, psychology (Box 3) and climate change48, but it is relative new to the study of health systems43,49,50. Health system are compounded of both hardware (structure; organization; technology; resourcing) and software (values; norms; actors; relationships) components, and their resilience require to be measured and understand accordingly51.\n\nAccording to the Merriam Webster dictionary, the first use of the term resilience dates back to 1807. It was then used in physics about the ability of materials to resist shocks or regain their original shape after being compressed or deformed52. During the 1970s, in community psychiatry, we look at the phenomenon of so-called \"invulnerable\" children who, in the confrontation of stress and adversity, do not develop psychological disorders. In 1979, the child psychiatrist Michael Rutter uses the term resilience to describe these children he is studying to understand what are the protective factors that allow them to cope with stress53,54. His work has notably helped to identify social support as one of the main protective factors. The definition of resilience used today to study the capacity of health systems to cope with the consequences of climate change is consistent with this work. The Intergovernmental Panel on Climate Change: “The capacity of social, economic, and environmental systems to cope with a hazardous event or trend or disturbance, responding or reorganizing in ways that maintain their essential function, identity, and structure, while also maintaining the capacity for adaptation, learning, and transformation”55.\n\nIn 2015, the WHO proposed an operational framework to build climate resilient health systems within the context of climate change42, but the scientific and empirical basis for its production is unclear, and the issue of population migration is not mentioned\n\nRecently, an article has developed a non-normative index for assessing the resilience of health systems, but its validation has not yet been completed56.\n\nThe 2017 and 2018 Lancet Countdown paper series suggests some indicators (at least: 2.1, 2.4, 2.6; 2.7, 2.8, 3.9) (Box 4), which could be useful to understand the link between climate change and health system resilience, even though the concept of health system resilience adoption is still limited and “does not capture the quality or effectiveness of efforts”, as it was said for the 2017 report5,57 neither the resilience of health staff (at its already important brain drain) nor community is taken into account. Health systems’ resilience cannot be evaluated only in terms of infrastructures. In contrast, from a more holistic and fundamental research perspective, several recent articles propose conceptual frameworks49,50,56 that suggest analysing the five main dimensions of a resilient system: awareness, diversity, self-regulation, integration, and adaptiveness50.\n\nIndicator 2.1: National adaptation plans for health\n\nIndicator 2.4: Climate change adaptation to vulnerabilities from mosquito-borne diseases\n\nIndicator 2.6: National assessments of climate change impacts, vulnerability, and adaptation for health\n\nIndicator 2.7: Spending on adaptation for health and health-related activities\n\nIndicator 2.8: health adaptation funding from global climate financing mechanisms\n\nIndicator 3.9: Health-care sector emissions\n\nIn addition, according to the Sendai Framework (2015-2030) adopted at the Third United Nations World Conference on Disaster Risk Reduction in March 2015, it is essential “to enhance the resilience of national health systems”58 but very little attention has been paid to the role of the health system in responding to climate change and its resilience42,43,59. One of the major global health journals (Health Policy and Planning) just released in November 2017 the first, to our knowledge, supplement issue about “Resilient and Responsive Health Systems”60. But none of the 11 articles addressed climate change.\n\nAs seen and cited, migration and climate connections has followed similar research progress, typically by a different group of scholars, on understanding migration’s health dimensions45. In the same way, migration, climate, population’s health and resilience of health systems are usually analysed as separated components through disciplines and approaches in silos. Research on the intersection between all these components is still very scarce. Consequently, there are gaps and a predominant compartmented analysis on the existing links between all of them. In contrast, interdisciplinary means a certain level of integration of knowledge, methods and/or ideas to construct and analyse the problematic under study61,62. Hence, interdisciplinary research can lead to the understanding of the links between migration and health require mixed methods63, and the collaboration of environmental, health and social sciences, in order to inform strategies and interventions to protect population health. “By learning from other researchers one increases the possibilities of creative solutions”64.\n\n\nFor interdisciplinary research\n\nClimate change is one of the main challenges of our century, having the potential to trigger important changes in population health also by forcing migration. The role of health systems in the context of targeting universal health coverage may be central to address these challenges.\n\nAs exposed in this manuscript, the research on the intersection between climate change, health systems, and migrants is still very scarce. Because of its complexity, we need to move from a multidisciplinary to an interdisciplinary approach64 to understand the multiple pathways that link migration driven by climate change and population’s health.\n\nWe believe there is a need for an interdisciplinary approach relying on the concept of resilience to help to study adaptive strategies in both places of origin and destination. Further investments should be made to unravel the link between climate change, migration, and health system resilience. We propose a series of interdisciplinary research questions to provide initial guidance in this direction (Box 5). We suggest in Table 1 and Figure 1 a first summarization attempt of the challenges triggered by climate change for the resilience of health systems.\n\n• How is the concept of climate migrant delineated?\n\n• What conceptual frameworks can support research on health systems’ resilience to climate change?\n\n• In what ways are the health systems resilient to climate change-related migration?\n\n• What role does climate change play in population movements and what are the health impacts?\n\n• How do people displaced by climate change have access to health systems?\n\n• How to promote health systems’ preparedness and resilience in the face of climate change?\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "Grant information\n\nPart of this paper has been done thanks to CIHR-funded Research Chair in Applied Public Health (CPP-137901) hold by VR.\n\n\nAcknowledgements\n\nCo-authors of this manuscript obtain permission to thank to Donna Riley who translated and edited a first version of this article, to Nathalie C. Tan for some literature review, to Aline Philibert for helpful comments and to Esther Mc Sween Cadieux for the Figure 1.\n\n\nReferences\n\nCharlesworth E, Ahmed I: Factors Pertaining to Building Resilience in Urban Slum Settlements of Dhaka, Bangladesh. Melbourne, VIC: Architects Without Frontiers (AWF); 2012; 18. Reference Source\n\nVerner D: Labor Markets in Rural and Urban Haiti Based on the First Household Survey for Haiti. World Bank. (POLICY RESEARCH WORKING PAPER 4574); 2008; 29. Reference Source\n\nLaczko F, editor: Migration, environment and climate change: assessing the evidence. Geneva: Internat. Organization for Migration; 2009; 441. Reference Source\n\nWarn E, Adamo SB: The Impact of Climate Change: Migration and Cities in South America. World Meteorological Organization. 2015; [cited 2017 Sep 7]. Reference Source\n\nWatts N, Amann M, Ayeb-Karlsson S, et al.: The Lancet Countdown on health and climate change: from 25 years of inaction to a global transformation for public health. Lancet. 2018; 391(10120): 581–630, [cited 2017 Nov 1]. PubMed Abstract | Publisher Full Text\n\nWHO: Protecting health from climate change connecting science, policy, and people. Copenhagen, Denmark: World Health Organization Regional Office for Europe; 2009. Reference Source\n\nPapworth A, Maslin M, Randalls S: Is climate change the greatest threat to global health?: Commentary. Geogr J. 2015; 181(4): 413–22. Publisher Full Text\n\nWhitmee S, Haines A, Beyrer C, et al.: Safeguarding human health in the Anthropocene epoch: report of The Rockefeller Foundation-Lancet Commission on planetary health. Lancet. 2015; 386(10007): 1973–2028. PubMed Abstract | Publisher Full Text\n\nMcLeman RA, Hunter LM: Migration in the context of vulnerability and adaptation to climate change: insights from analogues. Wiley Interdiscip Rev Clim Change. 2010; 1(3): 450–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStanke C, Kerac M, Prudhomme C, et al.: Health effects of drought: a systematic review of the evidence. PLoS Curr. 2013; 5: pii: ecurrents.dis.7a2cee9e980f91ad7697b570bcc4b004. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMora C, Spirandelli D, Franklin EC, et al.: Broad threat to humanity from cumulative climate hazards intensified by greenhouse gas emissions. Nat Clim Chang. 2018; 8(12): 1062–71. Publisher Full Text\n\nMissirian A, Schlenker W: Asylum applications respond to temperature fluctuations. Science. 2017; 358(6370): 1610–4. PubMed Abstract | Publisher Full Text\n\nMcMichael C, Barnett J, McMichael AJ: An ill wind? Climate change, migration, and health. 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Procedia - Social and Behavioral Sciences. 2016; 218: 202–13. Publisher Full Text\n\nKreft S, Eckstein D, Dorsch L, et al.: Global climate risk index 2016. Who Suffers Most From Extreme Weather Events? Weather-related Loss Events in 2014 and 1995 to 2014. Berlin: Germanwatch e.V; 2016; 32. Reference Source\n\nWHO: The World Health Report 2000 - Health Systems: Improving Performance. 2000. Reference Source\n\nKreft S, Eckstein D, Dorsch L, et al.: Global climate risk index 2016. Who Suffers Most From Extreme Weather Events? Weather-related Loss Events in 2014 and 1995 to 2014. Berlin: Germanwatch e.V. 2017; 32. Reference Source\n\nHeaney AK, Winter SJ: Climate-driven migration: an exploratory case study of Maasai health perceptions and help-seeking behaviors. Int J Public Health. 2016; 61(6): 641–9. PubMed Abstract | Publisher Full Text\n\nBonnet E, Amalric M, Nikiema A, et al.: Connaissances des inondations par les ouagalais. Ouagadougou, Burkina Faso: IRD. 2017; 4. Reference Source\n\nSena A, Ebi KL, Freitas C, et al.: Indicators to measure risk of disaster associated with drought: Implications for the health sector. Zia A, editor. PLoS One. 2017; 12(7): e0181394. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevy K, Woster AP, Goldstein RS, et al.: Untangling the Impacts of Climate Change on Waterborne Diseases: a Systematic Review of Relationships between Diarrheal Diseases and Temperature, Rainfall, Flooding, and Drought. Environ Sci Technol. 2016; 50(10): 4905–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPolade SD, Gershunov A, Cayan DR, et al.: Precipitation in a warming world: Assessing projected hydro-climate changes in California and other Mediterranean climate regions. Sci Rep. 2017 [cited 2017 Dec 17]; 7(1): 10783. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcMichael C: Climate change-related migration and infectious disease. Virulence. 2015; 6(6): 548–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChase LE, Cleveland J, Beatson J, et al.: The gap between entitlement and access to healthcare: An analysis of \"candidacy\" in the help-seeking trajectories of asylum seekers in Montreal. Soc Sci Med. 2017; 182: 52–9. PubMed Abstract | Publisher Full Text\n\nTorres JM, Casey JA: The centrality of social ties to climate migration and mental health. BMC Public Health. 2017; 17(1): 600. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarcelin LH, Cela T, Shultz JM: Haiti and the politics of governance and community responses to Hurricane Matthew. Disaster Health. 2016; 3(4): 151–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPetkova EP, Ebi KL, Culp D, et al.: Climate Change and Health on the U.S. Gulf Coast: Public Health Adaptation is Needed to Address Future Risks. Int J Environ Res Public Health. 2015; 12(8): 9342–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJulvez J, Develoux M, Mounkaila A, et al.: [Diversity of malaria in the Sahelo-Saharan region. A review apropos of the status in Niger, West Africa]. Ann Soc Belg Med Trop. 1992; 72(3): 163–77. PubMed Abstract\n\nDirection des Statistiques du Ministère de la Santé Publique: Annuaire des statistiques sanitaires du Niger année 2016. 2017. Reference Source\n\nDoudou MH, Mahamadou A, Ouba I, et al.: A refined estimate of the malaria burden in Niger. Malar J. 2012 [cited 2017 Nov 14]; 11(1): 89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobert E, Lemoine A, Ridde V: Que cache le consensus des acteurs de la santé mondiale au sujet de la couverture sanitaire universelle? Une analyse fondée sur l’approche par les droits. Can J Dev Stud. 2017; 38(2): 199–215. Publisher Full Text\n\nRodin J, de Ferranti D: Universal health coverage: the third global health transition? Lancet. 2012; 380(9845): 861–2. PubMed Abstract | Publisher Full Text\n\nWHO: Arguing for universal health coverage. Geneva: World Health Organization. 2013; 39. Reference Source\n\nWatts N, Adger WN, Agnolucci P, et al.: Health and climate change: policy responses to protect public health. Lancet. 2015; 386(10006): 1861–914. PubMed Abstract | Publisher Full Text\n\nEvans RG, Barer ML, Marmor TR: Why are some people healthy and others not?: the determinants of health of populations. New York: A. de Gruyter; (Social institutions and social change). 1994; xix: 378. Reference Source\n\nCPHA: Global Change and Public Health: Addressing the Ecological Determinants of Health. Ottawa: Canadian Public Health Association Discussion Document. 2015; 36. Reference Source\n\nWHO: Operational framework for building climate resilient health systems. Switzerland: World Health Organization. 2015; 47. Reference Source\n\nWitter S, Hunter B: Resilience of health systems during and after crises – what does it mean and how can it be enhanced? London: ReBUILD Consortium. 2017; 4. Reference Source\n\nVerner G, Schütte S, Knop J, et al.: Health in climate change research from 1990 to 2014: positive trend, but still underperforming. Glob Health Action. 2016; 9(1): 30723. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan W: Migration as a mediator of climate-related infectious disease risk [Internet]. 2018; 6. Reference Source\n\nWitter S, Wurie H, Chandiwana P, et al.: How do health workers experience and cope with shocks? Learning from four fragile and conflict-affected health systems in Uganda, Sierra Leone, Zimbabwe and Cambodia. Health Policy Plan. 2017; 32(Suppl 3): iii3–13. PubMed Abstract | Publisher Full Text\n\nSchmets G, Hanssen O, Soucat A: Interconnectedness of UHC and health security. Background paper developed for the 9th Global Meeting of Heads of WHO offices. Geneva, Switzerland: WHO. 2017; 5. Reference Source\n\nTanner T, Bahadur A, Moench M: Challenges for resilience policy and practice. London: Overseas Development Institute. 2017; 25. Reference Source\n\nGilson L, Barasa E, Nxumalo N, et al.: Everyday resilience in district health systems: emerging insights from the front lines in Kenya and South Africa. BMJ Glob Health. 2017; 2(2): e000224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKruk ME, Myers M, Varpilah ST, et al.: What is a resilient health system? Lessons from Ebola. Lancet. 2015; 385(9980): 1910–2. PubMed Abstract | Publisher Full Text\n\nGilson L, editor: Systems research. A methodology reader. Alliance for Health Policy and Systems Research. World Health Organization. 2012; 472. Reference Source\n\nReid R, Botterill LC: The Multiple Meanings of ‘Resilience’: An Overview of the Literature. Aust J Publ Admin. 2013; 72(1): 31–40. Publisher Full Text\n\nRutter M: Resilience in the face of adversity. Protective factors and resistance to psychiatric disorder. Br J Psychiatry. 1985; 147(6): 598–611. PubMed Abstract | Publisher Full Text\n\nRutter M: Protective factors in children’s responses to stress and disadvantage. In: Primary prevention in psychopathology: Social competence in children. Hanover. University Press of New England. 1979; 8: 49–74.\n\nIPCC: Annex II: Glossary. In: Climate Change 2014: Impacts, Adaptation, and Vulnerability Part B: Regional Aspects Contribution of Working Group II to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press. 2014; 1757–76. Reference Source\n\nKruk ME, Ling EJ, Bitton A, et al.: Building resilient health systems: a proposal for a resilience index. BMJ. 2017; 357: j2323. PubMed Abstract | Publisher Full Text\n\nWatts N, Amann M, Arnell N, et al.: The 2018 report of the Lancet Countdown on health and climate change: shaping the health of nations for centuries to come. Lancet. 2018; 392(10163): 2479–2514. PubMed Abstract | Publisher Full Text\n\nUNISRD: Sendai Framework for Disaster Risk Reduction 2015–2030. Geneva, Switzerland: UNISRD. 2015; 38. Reference Source\n\nRidde V, Ramel P: The migrant crisis and health systems: Hygeia instead of Panacea. Lancet Public Health. 2017; 2(10): e447. PubMed Abstract | Publisher Full Text\n\nMills A: Resilient and responsive health systems in a changing world. Health Policy Plan. 2017; 32(suppl_3): iii1–2. PubMed Abstract | Publisher Full Text\n\nCloos P: Racialization, Between Power and Knowledge: A Postcolonial Reading of Public Health as a Discursive Practice1. Journal of Critical Race Inquiry. 2011; 1(2): 57–76. Reference Source\n\nRobert E, Ridde V: Quatre principes de recherche pour comprendre les défis des systèmes de santé des pays à faible et moyen revenu. Can J Public Health. 2016; 107(4–5): e362–e365. 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}
|
[
{
"id": "42732",
"date": "04 Feb 2019",
"name": "Lucy Gilson",
"expertise": [
"Reviewer Expertise Health policy and systems research"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important paper on a vital topic. It provides useful directions for future research around climate change, migration and health system resilience. Nonetheless, the overall argumentation of the paper is not fully clear – and so it is difficult fully to judge the use of evidence and assess the conclusions.\n\nThe broad argument seems to be intended as:\nClimate change and migration are inter-linked and have negative health consequences (‘climate migrants and health systems’). Health systems are vital to tackling public health challenges such as those of climate change and migration (‘climate change in the global health context’). Whilst there is increasing focus on health system resilience, this has not yet included concern for climate change or migration (‘health systems resilience in the climate change context’). There is a need for ‘interdisciplinary research’ on climate change, health systems and migrants (‘for interdisciplinary research’).\n\nHowever, whilst the last section presents a case for interdisciplinary research, the earlier sections essentially work towards the conclusion that climate change, migration and health systems are interlinked. In addition, although there is reference to the point that current research is conducted in silos with little consideration of the intersection between these terrains on p.5, this point is not clearly argued previously in the paper.\n\nIn supporting the final step of the paper’s argument (point 4 above), I suggest, then, that there would be value in strengthening the argument around the current silo-based nature of research in these domains as well as discussing further why and how interdisciplinary research is valuable for this area of work. I propose placing both these sets of issues in the section ‘for interdisciplinary research’ (some are currently in the previous section). There may also be value in clarifying that in this context ‘disciplines’ are, I think, equated to areas of work (climate change, migration, health systems) as opposed to e.g. sociology, anthropology, clinical science etc. And then I suggest it would be helpful to: expand on the point that ‘interdisciplinary’ means ‘a certain level of integration of knowledge, methods and/or ideas’ (p.5), and to discuss more than the need for mixed methods; clarify why an interdisciplinary approach is better than a multidisciplinary one for this work (p.5); and deepen the point about the value of the focus on resilience and adaptive strategies in supporting interdisciplinary research (p.5) – as well as explaining more of the detail of Table 1 and Figure 1. (As an aside, in Table 1 I would propose there would be value in thinking about health system software as more than staff training, essentially; relationships among staff within the system and with the public also matter, for example).\n\nIn terms of the earlier sections of the paper I was not sure why the first section is titled ‘climate migrants and health systems’, as the focus is on health challenges rather than health systems. I also found that the logic and structure of the sections ‘climate change in the global health context’, and ‘health systems resilience in the climate change context’ made it difficult to follow the argument within them. In ‘climate change in the global health context’, this might be because the very tight referencing practice has overshadowed the argument. In ‘health systems resilience in the climate change context’, the linkage between the different points presented is not very clear (i.e. the argument connecting them).\n\nSome other minor points for review in p.3:\nWhat is an heuristic literature review? How are the 10 papers that were identified in the PubMed search used in the text, or is the point here that only 10 papers were identified? At the first mention, briefly clarify the significance of the Lancet Countdown for this paper.\nOne final comment: given that this is a very closely argued piece, there would be value in some really close copy editing – as, for example, missing words in sentences, long sentences, and sentences that are phrased quite clumsily, hinder understanding.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "4578",
"date": "18 Apr 2019",
"name": "Ariadna Nebot",
"role": "Author Response",
"response": "Answers to Lucy Gilson's comments, who approved with reservations This is an important paper on a vital topic. It provides useful directions for future research around climate change, migration and health system resilience. Nonetheless, the overall argumentation of the paper is not fully clear – and so it is difficult fully to judge the use of evidence and assess the conclusions. The broad argument seems to be intended as:Climate change and migration are inter-linked and have negative health consequences (‘climate migrants and health systems’).Health systems are vital to tackling public health challenges such as those of climate change and migration (‘climate change in the global health context’).Whilst there is increasing focus on health system resilience, this has not yet included concern for climate change or migration (‘health systems resilience in the climate change context’).There is a need for ‘interdisciplinary research’ on climate change, health systems and migrants (‘for interdisciplinary research’). Thank you for this summary which indeed corresponds to the approach we adopted in our paper. However, whilst the last section presents a case for interdisciplinary research, the earlier sections essentially work towards the conclusion that climate change, migration and health systems are interlinked. In addition, although there is reference to the point that current research is conducted in silos with little consideration of the intersection between these terrains on p.5, this point is not clearly argued previously in the paper. In supporting the final step of the paper’s argument (point 4 above), I suggest, then, that there would be value in strengthening the argument around the current silo-based nature of research in these domains as well as discussing further why and how interdisciplinary research is valuable for this area of work. I propose placing both these sets of issues in the section ‘for interdisciplinary research’ (some are currently in the previous section). There may also be value in clarifying that in this context ‘disciplines’ are, I think, equated to areas of work (climate change, migration, health systems) as opposed to e.g. sociology, anthropology, clinical science etc. And then I suggest it would be helpful to: expand on the point that ‘interdisciplinary’ means ‘a certain level of integration of knowledge, methods and/or ideas’ (p.5), and to discuss more than the need for mixed methods; clarify why an interdisciplinary approach is better than a multidisciplinary one for this work (p.5); and deepen the point about the value of the focus on resilience and adaptive strategies in supporting interdisciplinary research (p.5) Thanks for this very pertinent comment. Following your recommendations, we have reviewed each section and introduced at the end of section 1, section 2 and section 4 how interdisciplinarity may be useful to address the current gaps regarding the elements we describe about climate change, migration and health systems resilience.We have also included a few sentences to describe the importance of distinguishing interdisciplinary from multidisciplinary. As well as explaining more of the detail of Table 1 and Figure 1. (As an aside, in Table 1 I would propose there would be value in thinking about health system software as more than staff training, essentially; relationships among staff within the system and with the public also matter, for example). A presentation and explanation have been provided in the article now. In terms of the earlier sections of the paper I was not sure why the first section is titled ‘climate migrants and health systems’, as the focus is on health challenges rather than health systems. The subtitle of this section has been changed to \"climate migrants and health challenges\". I also found that the logic and structure of the sections ‘climate change in the global health context’, and ‘health systems resilience in the climate change context’ made it difficult to follow the argument within them. In ‘climate change in the global health context’, this might be because the very tight referencing practice has overshadowed the argument. We have reviewed the flow of this section, and the subtitle of this section has been changed to \"climate migrants and health systems\". In ‘health systems resilience in the climate change context’, the linkage between the different points presented is not very clear (i.e. the argument connecting them). We have reviewed the flow of this section and changed its subtitle to emphasize the need to continue research on this concept, which is still a little too vague. The last section has been moved to become the first section on the need for interdisciplinarity. Some other minor points for review in p.3:What is an heuristic literature review? A review of the non-systematic literature but which only includes useful articles on the subject and to develop our arguments. We made this clear in the correction. How are the 10 papers that were identified in the PubMed search used in the text, or is the point here that only 10 papers were identified? Yes, the point is that only 10 articles have been published, which shows how little is still written on the subject and the most relevant are cited in our article At the first mention, briefly clarify the significance of the Lancet Countdown for this paper. Thanks for this suggestion. We have clarified this significance in the text (p.7) One final comment: given that this is a very closely argued piece, there would be value in some really close copy editing – as, for example, missing words in sentences, long sentences, and sentences that are phrased quite clumsily, hinder understanding. We had the latest version revised by a scientific editor, Donna Riley. However, for this re-submission, we have asked an additional native English speaker (Lara Schwarz) to review and edit the whole text."
}
]
},
{
"id": "42728",
"date": "11 Feb 2019",
"name": "Katharina Waha",
"expertise": [
"Reviewer Expertise Climate change impact 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 article calls for further research on climate change, migration and health system resilience and an interdisciplinary approach but does not present a convincing line of reasoning. Often the individual paragraphs seem unconnected.\nFor example, in the section on “Climate migrants and health systems” I am not sure what the authors are trying to do or say. It’s a loose collection of thoughts to me at the moment. The first two paragraphs are about general impacts of climate change and migration and the authors are careful to not link them directly which is good. The next paragraph is about perception of migrants which is interesting and then the authors move into the Burkina Faso example where it is not clear if people have migrated at all.\nI think the topic is interesting and very relevant and one thing that might help to structure the article better, is to clarify whether the authors are interested in the effects of climate change on an individual’s health (migrants) or a country’s health system or both. This seems to be mixed up in the article. Table 1 and Figure 1 seems to sort of help with that, but they are not integrated in the text at all, they are just added at the end but should be central to the paper.\n\nOther comments:\n“The estimation that is most widely accepted is that over 200 million persons will be displaced globally by 2050 because of climate change13,15,18”. Inappropriate use of literature. McMichael et al. (2012) (13) actually says that 200 million is the figure most widely accepted and refers to Myers (2002) as the source of that figure, but also says that the empirical basis has been questioned. This is an important consideration that needs to be added here. The other two references are not needed then, except if they are given to support the notion of ‘most widely accepted’ in which case I would expect more studies.\n\nBox 1: Conclusion in the last sentence about perceptions of local populations needing to be enhanced does not follow from previous paragraphs. The authors would have to establish a disconnect between perceptions of climate change and flooding and results from a detection and attribution study in order to conclude that.\n\nBox 4 and the resilience section: These indicators seem to be for resilience and adaptation planning, not just for resilience. The concept of resilience seems to be important in the article but only got mentioned once in the second last section and there it gets mixed up with adaptation indicators. The Lancet Countdown Report gives some of them as \"Adaptation Planning and Resilience for Health Indicators\". Can you strengthen this part and explain better why this is an important consideration?\n\n“The role of health systems in the context of targeting universal health coverage may be central to address these challenges.” The authors speak about universal health coverage only once before and do not give any reason for this conclusion.\n\nHow are health needs and health system resilience different between “climate change migrants” and other migrants that e.g. flee war? The authors state that they “face challenges similar to those of refugees fleeing war and/or political persecution” and “might experience many similar obstacles and barriers to their health as well”, so why the need to study this topic separately?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No",
"responses": [
{
"c_id": "4579",
"date": "18 Apr 2019",
"name": "Ariadna Nebot",
"role": "Author Response",
"response": "Answers to Katharina Waha who approved with reservations The article calls for further research on climate change, migration and health system resilience and an interdisciplinary approach but does not present a convincing line of reasoning. Often the individual paragraphs seem unconnected. Thank you for your feedback. In light of Lucy Gilson's comments, we have tried to reorganize some sections of the article. We hope that this will make more sense. For example, in the section on “Climate migrants and health systems” I am not sure what the authors are trying to do or say. It’s a loose collection of thoughts to me at the moment. The first two paragraphs are about general impacts of climate change and migration and the authors are careful to not link them directly which is good. The next paragraph is about perception of migrants which is interesting and then the authors move into the Burkina Faso example where it is not clear if people have migrated at all. We have renamed this first section to better show that its objective is to set the scene for the relationship between climate migrants and health. We have made it clear that the people from Burkina Faso affected by Box 1 have been displaced by flooding, thank you for the comment. I think the topic is interesting and very relevant and one thing that might help to structure the article better, is to clarify whether the authors are interested in the effects of climate change on an individual’s health (migrants) or a country’s health system or both. This seems to be mixed up in the article. Table 1 and Figure 1 seems to sort of help with that, but they are not integrated in the text at all, they are just added at the end but should be central to the paper. We have tried to restructure the article to better show that what interests us is not so much the link between climate migrants and health but rather the link between climate migrants and health system because it has not yet been much addressed by research. We have added text to better integrate and explain Table 1 and Figure 1. Other comments:“The estimation that is most widely accepted is that over 200 million persons will be displaced globally by 2050 because of climate change13,15,18”. Inappropriate use of literature. McMichael et al. (2012) (13) actually says that 200 million is the figure most widely accepted and refers to Myers (2002) as the source of that figure, but also says that the empirical basis has been questioned. This is an important consideration that needs to be added here. The other two references are not needed then, except if they are given to support the notion of ‘most widely accepted’ in which case I would expect more studies. Thanks for this very relevant comment. Accordingly, and in order to avoid confusion, we have replaced the figure with empirical basis questioned by the following sentence: “the simple fact is that nobody really knows with any certainty what climate change will mean for human population distribution. Current estimates range between 25 million and 1 billion people by 2050.” (Brown 2008) and we have also deleted the 2 references (15, 18); not necessary in supporting the argument anymore. Box 1: Conclusion in the last sentence about perceptions of local populations needing to be enhanced does not follow from previous paragraphs. The authors would have to establish a disconnect between perceptions of climate change and flooding and results from a detection and attribution study in order to conclude that. Yes, thanks for this suggestion. We have just deleted the last sentence in order to clarify the disconnection suggested by the reviewer. Box 4 and the resilience section: These indicators seem to be for resilience and adaptation planning, not just for resilience. The concept of resilience seems to be important in the article but only got mentioned once in the second last section and there it gets mixed up with adaptation indicators. The Lancet Countdown Report gives some of them as \"Adaptation Planning and Resilience for Health Indicators\". Can you strengthen this part and explain better why this is an important consideration? Yes, thanks for this comment. It is correct that the Lancet Countdown indicator’s section 2 refers to “adaptation, planning and resilience for health” and not only to resilience. The reason is of course that adaptation and resilience are directly related and this section 2 aims to put at the front the adaptation efforts to promote and achieve community resilience, as we can see in p.13 of the 2018 Lancet Countdown’s report: “With the observed and future health impacts of climate change becoming increasingly evident, and emission trajectories committing the world to further warming, accelerated adaptation interventions are needed to safeguard populations’ health. As the 2030 agenda shows, 45 strategies to improve community resilience are often linked to poverty reduction and broader socioeconomic development imperatives, creating the possibility of no regret scenarios”. However, in this same p.13, it is said that, although the 2018 Lancet Countdown report counts on improved indicators for this section, the community resilience is still few explored and that collected data give more insights in adaptation than in resilience:“The health sector should be at the forefront of adaptation efforts, ensuring health systems, hospitals, and clinics remain anchors of community resilience. This underrecognised, yet growing area of practice, is the focus of this section.”The data are incomplete, providing more insight into adaptation than resilience, and predominantly allow for process-based indicators.” Therefore, the authors of this paper considered some of these Lancet Countdown indicators as a good example to visibilise the still reductionist and uni-disicipline approach of how resilience is interpreted In order to make this intention more explicit; we have added these two-lines in p 7. “The role of health systems in the context of targeting universal health coverage may be central to address these challenges.” The authors speak about universal health coverage only once before and do not give any reason for this conclusion. We have added some clarifications to this sentence, p 9. How are health needs and health system resilience different between “climate change migrants” and other migrants that e.g. flee war? The authors state that they “face challenges similar to those of refugees fleeing war and/or political persecution” and “might experience many similar obstacles and barriers to their health as well”, so why the need to study this topic separately? Thanks for this comment. According to what we could find in the literature (and therefore, what is already documented) climate migrants health needs may share similar patterns to refugees and/or to rural-urban migrants (P.4). However, in this same paragraph, we also mention the additional vulnerability that this category of population may have: “In addition, environmental change migrant population is usually the most vulnerable as well because migration is often expensive and climate change factors can easily lie on the top of other strong socio-economic factor.” Considering this ‘additional vulnerability’, the author’s underlying hypothesis may be that climate migrants health needs and health system resilience may be slightly different. However, the non-integrated disciplines that can be looking at that doesn’t allow to further explore this specificities. We have modified the last statement of this paragraph in order to strengthen this idea: “However, the lack of consensus of climate change migrant suggests how the same phenomenon is defined from different and non-integrated disciplines and, therefore, how climate change migrant health needs and patterns are still scarcely documented. “"
}
]
}
] | 1
|
https://f1000research.com/articles/8-22
|
https://f1000research.com/articles/7-1728/v1
|
31 Oct 18
|
{
"type": "Opinion Article",
"title": "Three more steps toward better science",
"authors": [
"Jose D. Perezgonzalez"
],
"abstract": "Science has striven to do better since its inception and has given us good philosophies, methodologies and statistical tools that, in their own way, do reasonably well for purpose. Unfortunately, progress has also been marred by warring among different perspectives, leading to troubles such as the current reproducibility crises. Here I wish to propose that science could do better with more resilient structures, more useful methodological tutorials, and clearer signaling regarding how much we can trust what it produces.",
"keywords": [
"philosophy of science",
"methodology",
"statistics"
],
"content": "\n\nScience has striven to do better since its inception. For example, empiricism was sought as an alternative mode of learning as early as the XVI Century (Ball, 2012); XIX Century researchers sought a less subjective approach to learning from data via frequentist statistics, which progressively displaced Bayesian inference (Gigerenzer et al., 1989); in the XX Century, seeking a better way of establishing causation, Fisher (e.g., 1954) popularized a consistent framework of experimental design and frequentist inference based on small samples; Neyman & Pearson (e.g., 1928) expanded on Fisher’s statistical innovations to bring about more control of research power; Jeffreys (e.g., 1961) countered with a more nuanced approach toward evidential support for hypotheses via his Bayes factor; Cohen (1988) veered the focus away from significance testing and toward practical importance with his seminal work on effect sizes and power analyses; Mayo (e.g., 2018) is nowadays popularizing a framework based on severity testing for better frequentist inference; and computational advancements are giving full Bayesian inference a new opportunity to claw back the territory lost since the XX Century (McGrayne, 2012).\n\nSuch historical drive has given us good tools for purpose, including philosophies and methodologies, as well as statistical tools for exploratory data analyses, data testing, hypothesis testing, and replication research. The path has not been easy, with a lot of effort gone onto warring among different philosophies, methodologies, and statistical approaches, and leading to troubles such as the current reproducibility crises (e.g., Fanelli, 2018).\n\nStill, most approaches have been put forth and defended on the common goal of bettering science and, in their own way, all do so reasonably well. For example, Table 1 summarizes results obtained using different testing approaches, all concluding with similar inferences. Therefore, the real “enemy” is not what makes for better science but what makes for worse science: namely, problems with methodological control, with the misunderstanding and misuse of statistics, and with unsupported conclusions (i.e., with ethical concerns and with the use of scientific methods in a pseudoscientific manner; Perezgonzalez & Frías-Navarro, 2018).\n\nNotes. Based on data from Vincent (2018; Perezgonzalez & Vincent, 2018). Case: tests are one-tailed (1t) or two-tailed (2t). Cohen’s d: exploratory tests assessing effect sizes against Cohen d = 0.5 (i.e., the sample size—n1= 23; n2 = 23—was sensitive to d ≥ 0.5; Perezgonzalez, 2017). p: p-values from independent t-tests (Fisher’s approach, e.g., 1954). Decision: frequentist decision—noH0 = reject H0; H0 = no decision—based on level of significance = 0.05 (e.g., Perezgonzalez, 2015). SEV: severity tests (e.g., Mayo, 1996). BF: Bayes Factors with alternative model based on a Cauchy distribution (e.g., Rouder et al., 2009). Evidence: Bayesian evidence in favor of the null model (M0) or the alternative model (M1; e.g., Wagenmakers et al., 2018). The effect sizes of Cases II, IV, and VI had signs opposite to those expected (therefore, the high p’s); Cases III, V, and VII are two-tailed tests of Cases II, IV, and VI (thus, the similar d’s). Only Case V may lead a Jeffreysian to an inference contrary to those of frequentists; most likely, they would refrain from inferring support based on anecdotal posterior probabilities (e.g., Jarosz & Wiley, 2014).\n\nSuch enemy will be difficult to defeat. On first impression, science seems to suffer the fate of the ‘tragedy of the commons’, the ‘free-rider dilemma’ being, perhaps, its most specific affliction (Fisher, 2008). A recent book by Taleb (2018) on asymmetry sheds some light on the gaming element of science, namely on its misuse of analytical models, agency problems, asymmetric information sharing, and the rationality of the enterprise. Taleb also proposes three solutions that we could expand upon to provide a synergic path for how to go about bettering science (Perezgonzalez, 2018).\n\nFirstly, there is a need to make ‘scientific structures’ more resilient, for them to deliver the outcomes they were set up for: widespread accessibility and quality control. For example, open access publishing is nowadays countering the paywall limitations of traditional scientific publishing and its bias toward novel research with significant results, thus addressing important academic and social backlashes (Kelly, 2018; Schiltz, 2018). Unfortunately, it has also motivated the rise of predatory journals catering for the same pool of conscientious researchers. To counter the explosion of these predatory journals some idiosyncratic blacklists (e.g., the defunct Beall’s list) and organizational whitelists (e.g., Directory of Open Access Journals) have been created, albeit with mix success. Meanwhile, pre-print servers are challenging the entry costs of open access journals, thus making widespread communication more resilient but with the drawback of lacking good quality control.\n\nQuality control itself has received more attention of lately, with some journals becoming more transparent about who peer-reviews, while platforms such as Publons.com provide peer-review services and credit, including access to peer-reviews when allowed. Among quality-control structures is worth mentioning F1000Research, a publication platform that sits at the fringe of a paid preprint and a fully transparent peer-reviewed open access journal. This seems a more resilient structure worthy of emulation and improvement.\n\nPerhaps more importantly, a new need is becoming imperative: To find an effective solution to the indexing and curation of the ever expanding universe of research outputs. We do have, for example, Altmetric.com, albeit it is too geared toward scoring research outputs. Instead, what we need is an integrated solution to the indexing of both an output and all related content relevant to it, including post-publication reviews, comments in blogs and preprint servers, retraction notices, and the like. We also need a good solution to curating the entire spectrum of research outputs, moving from a plethora of stand-alone manuscripts toward mega-content organized as, for example, research topics.\n\nSecondly, ‘minority movements’ do have an impact on science via creating the above new structures (e.g., open access, pre-print archives…), but also by improving on legacy ones (e.g., post-publication review sites such as PubPeer.com). Paramount among such movements have been those calling for Open Science (e.g., Banks et al., 2018) and research ethics (e.g., Committee on Publications Ethics, RetractionWatch.com).\n\nMinority movements also have an impact on other aspects of science, from calls toward a better use of frequentist statistics (Perezgonzalez, 2015) to the outright banning of p-values (Trafimow & Marks, 2015), to the alternative use of Bayesian statistics (Wagenmakers et al., 2018b) or mixed approaches (Perezgonzalez & Frías-Navarro, 2018). Because of the intrinsic social dynamics of minority groups, the polarization of inter-group attitudes and consequential external warring are not only unsurprising but also expected. Yet, as alternative scientific approaches mostly have a different research focus, science has been less productive than it could be because more effort has been put into warring between factions than into clearly explaining what each provides to the advancement of science. This has allowed specific methodological knowledge to be too much textbook-based, thereby more aligned with editorial concerns than with the advancement of science (Gigerenzer, 2004), or to be polarized by the intrinsic dynamics of minority groups. Thus, what we presently need are good tutorials on the purpose of each approach and on how to effectively use them for such purpose; preferably, tutorials which are independently created rather than centrally edited, so as to provide a diversity of options able to address the same topic from different perspectives and to cater to different stakeholders (e.g., researchers, reviewers, and readers; novice and experts; technically-focused, philosophically-aware, as well as practitioners; etc). Such diversity will also allow for progressively developing optimal tutorials that minimize steep learning curves, capture methodological errors, and avoid philosophical and interpretive misconceptions.\n\nFinally, there is the need to signal how much ‘soul is in the game’ in each piece of published research. The pre-registration movement is achieving this via badges; most journals require authors to signal adherence to ethical principles via the corresponding disclaimers; some journals actively signal their peer-reviewing by naming peer-reviewers—e.g., Frontiersin.com, F1000Research.com—or by allowing open access to peer-reviews—e.g., via Publons.com. What we are presently lacking is good signaling to address methodological concerns and the avoidance of pseudoscience. That is, for authors to signal that they have followed, for example, Fisher’s approach to data testing, or Neyman-Pearson’s approach, or Mayo’s severity approach, or Jeffreys’s approach, or a full Bayesian approach; in brief, for them to signal when their research is compliant with the requisites of any of those approaches. The purpose of this signaling is to prevent what Farrington (1961, p. 311) already denounced, that “. . . there is no human knowledge which cannot lose its scientific character when [we] forget the conditions under which it originated, the questions which it answered, and the function it was created to serve”. This signaling could work in a manner similar to when authors specify a creative commons license for an open-access document: for a particular manuscript researchers could signal the specific methodological approach followed. This, of course, calls for negotiating the appropriate standards and for hosting them for quick referencing both by prospective authors and their peers.\n\nIn brief, following from the ideas of Taleb (2018), science could do better with more resilient structures, with more useful methodological tutorials, and with good signaling regarding how much we can trust what it produces. Thus my overall recommendation: let’s veer the focus from warring and onto improving our structures, tutorials and signals.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "Grant information\n\nThe author(s) declared that no grant(s) were involved in supporting this work.\n\n\nReferences\n\nBall P: Curiosity: How Science became interested in everything. Chicago, IL: The University of Chicago Press. 2012. Reference Source\n\nBanks GC, Field JG, Oswald FL, et al.: Answers to 18 questions about Open Science practices. J Bus Psychol. 2018; 1–14. Publisher Full Text\n\nCohen J: Statistical power analysis for the behavioral sciences. (2nd ed.). New York, NY: Psychology Press. 1988. Publisher Full Text\n\nFanelli D: Opinion: Is science really facing a reproducibility crisis, and do we need it to? Proc Natl Acad Sci U S A. 2018; 115(11): 2628–2631. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarrington B: Greek Science: Its meaning for us. (Rev. Ed). Middlesex, UK: Penguin Books. 1961. Reference Source\n\nFisher L: Rock, paper, scissors: Game theory in everyday life. New York, NY: Basic Books. 2008. Reference Source\n\nFisher RA: Statistical methods for research workers. (12th ed.). Edinburgh, UK: Oliver and Boyd. 1954. Reference Source\n\nGigerenzer G: Mindless statistics. J Soc Econ. 2004; 33(5): 587–606. Publisher Full Text\n\nGigerenzer G, Swijtink Z, Porter T, et al.: The empire of chance: How probability changed science and everyday life. Cambridge, UK: Cambridge University Press. 1989. Publisher Full Text\n\nJarosz AF, Wiley J: What are the odds? A practical guide to computing and reporting Bayes Factors. J Problem Solving. 2014; 7(1): 2. Publisher Full Text\n\nJeffreys H: Theory of probability. (3rd ed.). Oxford, UK: Oxford University Press. 1961. Reference Source\n\nKelly E: EU research chief’s next act: changing the future of academic publishing. Science Business. 2018. Reference Source\n\nMayo DG: Error and the growth of experimental knowledge. Chicago, IL: The University of Chicago Press. 1996. Reference Source\n\nMayo DG: Statistical inference as severe testing: How to get beyond the statistics wars. Cambridge, UK: Cambridge University Press. 2018. Publisher Full Text\n\nMcGrayne SB: The theory that would not die: How Bayes' rule cracked the enigma code, hunted down Russian submarines, and emerged triumphant from two centuries of controversy. New Haven, CT: Yale University Press. 2012. Reference Source\n\nNeyman J, Pearson ES: On the use and interpretation of certain test criteria for purposes of statistical inference: part I. Biometrika. 1928; 20A(1/2): 175–240. Publisher Full Text\n\nPerezgonzalez JD: Fisher, Neyman-Pearson or NHST? A tutorial for teaching data testing. Front Psychol. 2015; 6: 223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerezgonzalez JD: Statistical sensitiveness for the behavioural sciences. PsyArxiv. 2017. Publisher Full Text\n\nPerezgonzalez JD: Book review: Skin in the game. Front Psychol. 2018; 9: 1640. Publisher Full Text | Free Full Text\n\nPerezgonzalez JD, Frías-Navarro MD: Retract p < 0.005 and propose using JASP, instead [version 2; referees: 3 approved]. F1000Res. 2018; 6: 2122. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerezgonzalez JD, Vincent N: Análisis paralelos frecuentista-bayesianos con JASP [Parallel frecuentist-Bayesian data analysis with JASP]. IIn Frías-Navarro D, García-Pérez F, Pascual-Llobell J. (Eds.), Investigación en Psicología: diseño y análisis (ch.5). Valencia, Spain: Palmero ediciones. 2018.\n\nRouder JN, Speckman PL, Sun D, et al.: Bayesian t tests for accepting and rejecting the null hypothesis. Psychon Bull Rev. 2009; 16(2): 225–237. PubMed Abstract | Publisher Full Text\n\nSchiltz M: Science without publication paywalls: Coalition S for the realisation of full and immediate Open Access. Front Neurosci. 2018; 12: 656. Publisher Full Text\n\nTaleb NN: Skin in the game: Hidden asymmetries in daily life. New York, NY: Random House. 2018. Reference Source\n\nTrafimow D, Marks M: Editorial. Basic Appl Soc Psych. 2015; 37(1): 1–2. Publisher Full Text\n\nVincent N: Situational awareness of pilots in the cruise. Unpublished Master’s thesis, Massey University, New Zealand. 2018.\n\nWagenmakers EJ, Love J, Marsman M, et al.: Bayesian inference for psychology. Part II: Example applications with JASP. Psychon Bull Rev. 2018; 25(1): 58–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWagenmakers EJ, Marsman M, Jamil T, et al.: Bayesian inference for psychology. Part I: Theoretical advantages and practical ramifications. Psychon Bull Rev. 2018b; 25(1): 35–57. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "44361",
"date": "18 Feb 2019",
"name": "Ben van Calster",
"expertise": [
"Reviewer Expertise Methodology",
"(medical) 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 opinion piece, so I reviewed it as such. The paper discusses a few possible directions for improving the scientific process. I largely agree with the expressed opinions. That said, I think that the text remains quite general and vague at times. For example, with respect to the second issue, what do you mean with ‘tutorials which are independently created rather than centrally edited’? About the third issue: what is your specific suggestion? That researchers should better frame their work before starting it (as to what methodology and statistical approach they will use), and adhere to that when writing the report? This is unclear.\n\nFurther comments:\nIn the abstract, the statement ‘warring among different perspective’ may need a bit more clarity. The paragraph where the second issue is explained, could start with ‘Secondly’. (The first issue is introduced with ‘firstly’, the third issue with ‘finally’.) Table 1: too concise in my opinion. On what N are the p-valus based? Are the Cohen’s d values observed ones? A bit more information on severity tests might be useful. Perhaps columns that belong together (p and Decision; BF and Evidence; if I’m correct) may be put together more clearly. The paragraph starting with ‘such enemy will be difficult to defeat’ is quite vague. E.g. what do you mean with terms like ‘tragedy of the commons’, ‘asymmetric information sharing’, ‘rationality of the enterprise’, and others? Pre-print servers (line 2 of p3): do you refer to repositories like arXiv? Regarding scientific structures and peer-review: my issue is that peer review is very important (I am afraid that reviewers are sometimes the only people who check the contents of a study report), but it remains a largely uncredited – almost secret – endeavor. Don’t you agree that it still needs to be made more official, e.g. that universities do not only demand their researchers to publish papers, but also to deliver decent peer review reports? For example, when I started my tenure track, the university told me what they expected in terms of publication output, grants, and PhD guidance, but they said nothing about peer review, and they never evaluated me on these terms. A decent peer review should almost count as much as publishing a paper? 'Factions' (bottom of first column on p3): what is that? Would the STRATOS initiative (www.stratos-initiative.org), coordinated by Willi Sauerbrei from Freiburg, be in line with the second issue? It brings together experts on different methodological topics in the context of observational studies, with the aim to provide guidance on how to address these topics. (I am a member of this initiative.)\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "4508",
"date": "01 Apr 2019",
"name": "Jose Perezgonzalez",
"role": "Author Response",
"response": "Thank you very much for you review (and apologies for the long response time). This is an opinion piece, so I reviewed it as such. The paper discusses a few possible directions for improving the scientific process. I largely agree with the expressed opinions. That said, I think that the text remains quite general and vague at times. For example, with respect to the second issue, what do you mean with ‘tutorials which are independently created rather than centrally edited’? About the third issue: what is your specific suggestion? That researchers should better frame their work before starting it (as to what methodology and statistical approach they will use), and adhere to that when writing the report? This is unclear. I agree that the text is “general and vague at times”, albeit this is namely because I don’t know in which form the recommendations will eventually be implemented. Independently created tutorials calls for multiple and diverse works to be done by independent authors or groups of authors, as opposed to them being ‘centrally edited’ by a publisher (e.g., of textbooks on methods, or statistics). The noted sentence follows from an earlier assertion, that “methodological knowledge [is] too much textbook-based, thereby more aligned with editorial concerns than with the advancement of science (Gigerenzer, 2004)”. At the time, I also found it difficult to write it better without repeating ‘independently / independent authors’, and ‘centrally edited / textbook editors’. I have attempted it with synonyms here (albeit it may read a bit ‘forced’). It now reads “…tutorials which are independently created by unfettered authors rather than centrally abridged by textbook editors…” The third issue is a bit simpler than framing our work a priori (although it may help with this, as well). It is more like framing our work for publication. E.g., those who have followed a Fisherian approach, would say so but also write in a manner that is consistent with such approach, as the assumptions and inferential process are different to those following a Neyman-Pearson approach, or a Jeffreysian approach, etc. If no approach is clear or if they got mixed up in the process of doing the research, then no standard ought to be indicated (it would be misleading, otherwise). The recommendation is more or less the following: In the same way we may decide to release a document under a particular creative commons license (or none), a license which we need to specify and which binds the document to it; we could also release a research report under a particular research standard (or none), and we shall specify such standard so that peer-reviewers can assess the manuscript as per compliance with those standards, and readers can understand the results in reference to such standards. This also means the standards need to be negotiated, approved and hosted as for facilitating quick referencing for the aforementioned peer-reviewers and readers (and, of course, authors). Further comments: In the abstract, the statement ‘warring among different perspective’ may need a bit more clarity. The paragraph where the second issue is explained, could start with ‘Secondly’. (The first issue is introduced with ‘firstly’, the third issue with ‘finally’.) I have re- written the abstract, substituting ‘warring’ by “historical clashes among different perspectives, typically between frequentists and Bayesians”. The second issue actually starts with ‘secondly’, when I introduced the idea of ‘minority movements’. Table 1: too concise in my opinion. On what N are the p-valus based? Are the Cohen’s d values observed ones? A bit more information on severity tests might be useful. Perhaps columns that belong together (p and Decision; BF and Evidence; if I’m correct) may be put together more clearly. I have added a new column to Table 1, now describing the test, degrees of freedom, and test statistic. I also noted explicitly that Cohen’s d describes observed effects. The columns in the Table follow the suggested grouping: p-values and 'frequentist decision' Severity statistics (which is also a frequentist approach) Bayes factors and ‘Bayesian inference’ I also clarified a bit more severity testing. The paragraph starting with ‘such enemy will be difficult to defeat’ is quite vague. E.g. what do you mean with terms like ‘tragedy of the commons’, ‘asymmetric information sharing’, ‘rationality of the enterprise’, and others? All those constructs are found in the book by Taleb (2018). The paragraph is meant to quickly brush over them as a quick introduction, as the really relevant constructs (also Taleb’s) follow: ‘scientific structures’, ‘minority movements’, and ‘soul in the game’. Pre-print servers (line 2 of p3): do you refer to repositories like arXiv? Both, actually, as they often have such a dual role: either as a final repository or as a repository of manuscripts prior to them being sent for publication elsewhere (nowadays, many journals accept the latter, as long as they are in repositories / preprint servers). I have nonetheless added the concept ‘online repositories’ to the text. Regarding scientific structures and peer-review: my issue is that peer review is very important (I am afraid that reviewers are sometimes the only people who check the contents of a study report), but it remains a largely uncredited – almost secret – endeavor. Don’t you agree that it still needs to be made more official, e.g. that universities do not only demand their researchers to publish papers, but also to deliver decent peer review reports? For example, when I started my tenure track, the university told me what they expected in terms of publication output, grants, and PhD guidance, but they said nothing about peer review, and they never evaluated me on these terms. A decent peer review should almost count as much as publishing a paper? I fully agree. In fact, I think that a decent peer-review should count as much as publishing a paper and academics could, ideally, gain tenure and the like on peer-reviewing alone, not just on teaching or researching, as this allows for good, committed peer-reviewers and increased quality standards (the problem is how to define ‘decent’!). That’s why I added an entry on ‘quality control’ under recommendation 1 for more resilient scientific structures. However, it will be very difficult to know who has peer-reviewed what and with what quality. This means that all comes down to trust: the academic on the tenure track would claim to have done ‘x’ reviews for ‘y’ journals…but it will be difficult to prove. Thus far, I know of platforms such as Publons, PubPeer, FrontiersIn, and F1000Research, which would somewhat “reward” reviewers and/or publish peer-reviews. Of these, Publons actually rewards peer-reviewing and allows for generating some statistics in the form of percentiles and graphs. But reviews are only displayed depending on particular journal permissions. On the other hand, F1000Research does actually publish reviews and give them a doi. But there is no statistical summary of any form for reviewers. Thus, independently acknowledging / rewarding peer-review will be difficult unless the action of peer-reviewing have been somewhat independently ‘vetted’ (Publons have such control…but I am not too sure how well it works) or peer reviews are openly displayed (F1000Research). I don’t’ see Universities caring for a career in peer-reviewing any time soon, though. 'Factions' (bottom of first column on p3): what is that? Factions follow the “war” theme of Mayo’s latest book, consistent with similar concepts in the paragraph (enemies, polarization, warring, etc.). I have repeated Mayo’s reference in this paragraph. Would the STRATOS initiative (www.stratos-initiative.org), coordinated by Willi Sauerbrei from Freiburg, be in line with the second issue? It brings together experts on different methodological topics in the context of observational studies, with the aim to provide guidance on how to address these topics. (I am a member of this initiative.) Yes… I think that you have almost ‘pulled the rug from under my feet’ here. The STRATOS initiative is pretty much in line with that point. I have added the following reference to the manuscript (“—see also the STRATOS Initiative, already working toward a similar goal, www.stratos-initiative.org” )."
}
]
},
{
"id": "44358",
"date": "14 Mar 2019",
"name": "Lincoln J. Colling",
"expertise": [
"Reviewer Expertise Philosophy of Statistics",
"Philosophy of Cognitive Science",
"Neuroscience"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this paper could benefit from a minor revision.\n\nSome of the statements in the paper are a little vague. Most of the \"meat\" of the paper comes in the penultimate paper. It might be worth separating out and clearly stating the specific recommendations.\n\nTable 1 presented SEV values. However, SEV is always with respect to a specific inference and an observation (an observation). For example, for an observation of e.g., mu = 17, the inference mu1 = 12 and the inference mu1 = 14 would have different SEV values, but Table 1 gives no indication of what inference is being made (I assume the inference is the same as the observed result, but this should at least be indicated somewhere). It also appears that Table 1 presented the results of t-tests. I would be inclined to include a test statistic (or an N or both - with an N the reader could calculate the test stat, or with a test stat the reader could calculate the N). I think it is almost essential, because the sample size is one of the key determinants of when the various inferential frameworks actually come apart. I think in its current form Table 1 paints a slightly misleading picture.\n\nOn Page 3, Para 1: It may be worth discussing \"overlay journals\". These exist in physics and computing, but recently an overlay journal in neuroscience has also been launch (Neuroscience, Behaviour, Data and Theory).\nPage 3, Para 5: There are some examples of these tutorial style papers: 1. Four reasons to prefer Bayesian analyses over significance testing, Dienes et al.1 and 2. Statistical Inference and the Replication Crisis, Colling et al.2.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "4509",
"date": "01 Apr 2019",
"name": "Jose Perezgonzalez",
"role": "Author Response",
"response": "Thank you very much for your review and useful pointers. I think this paper could benefit from a minor revision. Some of the statements in the paper are a little vague. Most of the \"meat\" of the paper comes in the penultimate paper. It might be worth separating out and clearly stating the specific recommendations. I agree that the text is a little vague at times, albeit this is namely for the reason of lack of effective control on how the recommendations will eventually be implemented. For example, I can foresee the benefit of tutorials, in general (recommendation 2), but I cannot phantom whether there is a perfect tutorial to satisfy everyone (multiple and diverse tutorials may be needed for a “market-type” selection to kick-in, thus signalling helpful from less helpful ones). Actually, it is the third recommendation (in the penultimate paragraph) the one I see affected the least by variability in our imagination, as it calls for specific standards similar to standards already existing elsewhere (which is also why I placed it last, so to somewhat finish the commentary with a clearer, less vague, recommendation). It is for those reasons that I find it difficult to make the remaining recommendations more specific, and had to resort to the use of the conventional keywords “firstly”, “secondly”, and “finally” to somewhat constrain them to particular sections in the manuscript. Still so, I also aimed to put the recommendations more clearly towards the end of their sections: better indexing and curation as Recommendation 1 (this implies software-based indexing and curation, but I have little idea whether such software will work well); tutorials (but, again, I am not sure how well the idea will work; nonetheless, I added a correction linking to the STRATOS Initiative, at www.stratos-initiative.org, which may be one of the ways forward; other could be a methodology-based overlay journal, as you mentioned). Table 1 presented SEV values. However, SEV is always with respect to a specific inference and an observation (an observation). For example, for an observation of e.g., mu = 17, the inference mu1 = 12 and the inference mu1 = 14 would have different SEV values, but Table 1 gives no indication of what inference is being made (I assume the inference is the same as the observed result, but this should at least be indicated somewhere). It also appears that Table 1 presented the results of t-tests. I would be inclined to include a test statistic (or an N or both - with an N the reader could calculate the test stat, or with a test stat the reader could calculate the N). I think it is almost essential, because the sample size is one of the key determinants of when the various inferential frameworks actually come apart. I think in its current form Table 1 paints a slightly misleading picture. I have added a new column with the t-test statistics and corresponding degrees of freedom. I have also extended the note on Severity to clarify it and also give a quick pointer for assessment, as “SEV: severity tests based on the observed effects (severity is strong if greater than 0.80; e.g., Mayo, 1996)” On Page 3, Para 1: It may be worth discussing \"overlay journals\". These exist in physics and computing, but recently an overlay journal in neuroscience has also been launch (Neuroscience, Behaviour, Data and Theory). Thanks for the pointer. I wasn’t aware of overlay journals. But they certainly are a pretty good solution. I have added the following statement to paragraph 1, page 3: “—although overlay journals are taking care of the later drawback”. Page 3, Para 5: There are some examples of these tutorial style papers: 1. Four reasons to prefer Bayesian analyses over significance testing, Dienes et al. and 2. Statistical Inference and the Replication Crisis, Colling et al. I am aware of several tutorials; I even have one of my own. However, I thought it would be not too good an idea to name them as the recommendation focuses on the idea of generating them; thus pointing to some (which may or may not be useful, in hindsight) seems distracting. I have, however, added a new entry to an initiative that seems to be working on the same idea, the STRATOS Initiative)."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1728
|
https://f1000research.com/articles/8-42/v1
|
10 Jan 19
|
{
"type": "Software Tool Article",
"title": "Expanding the Orthologous Matrix (OMA) programmatic interfaces: REST API and the OmaDB packages for R and Python",
"authors": [
"Klara Kaleb",
"Alex Warwick Vesztrocy",
"Adrian M. Altenhoff",
"Christophe Dessimoz",
"Klara Kaleb",
"Alex Warwick Vesztrocy",
"Adrian M. Altenhoff"
],
"abstract": "The Orthologous Matrix (OMA) is a well-established resource to identify orthologs among many genomes. Here, we present two recent additions to its programmatic interface, namely a REST API, and user-friendly R and Python packages called OmaDB. These should further facilitate the incorporation of OMA data into computational scripts and pipelines. The REST API can be freely accessed at https://omabrowser.org/api. The R OmaDB package is available as part of Bioconductor at http://bioconductor.org/packages/OmaDB/, and the omadb Python package is available from the Python Package Index (PyPI) at https://pypi.org/project/omadb/.",
"keywords": [
"orthologs",
"paralogs",
"hierarchical orthologous groups",
"comparative genomics",
"orthologous matrix",
"oma",
"API",
"R",
"python",
"REST",
"bioconductor"
],
"content": "Introduction\n\nOrthologs are pairs of protein coding genes that have common ancestry and have diverged due to speciation events1. The detection of orthologs is of fundamental importance in many fields in biology, such as comparative genomics, as it allows us to propagate existing biological knowledge to ever growing newly sequenced data2,3.\n\nThe Orthologous Matrix (OMA) is a method and resource for the inference of orthologs among complete genomes4. The OMA database (https://omabrowser.org) features broad scope and size with currently over 2,100 species from all three domains of life.\n\nThe OMA browser has supported multiple ways of exporting the underlying data from its beginning. Users can download data either via bulk archives or interactively through the browser—using where possible standard file formats, such as FASTA, OrthoXML5, or PhyloXML6. For programmatic access, early OMA database releases offered an Application Programming Interface (API) in the form of the Simple Object Access Protocol (SOAP). However, the complexity and limited adoption of SOAP has prompted us to recently switch to the simpler, faster, and more widely used Representational State Transfer (REST) protocol for the OMA API4. Here, we provide a description of this new OMA REST API.\n\nFurthermore, the R environment is widely used in bioinformatics due to its flexibility as a high-level scripting language, statistical capabilities, and numerous bioinformatics libraries. In particular, the Bioconductor open source framework contains over 2,000 packages to facilitate either access to or manipulation of biological data7. This motivated us to develop the OmaDB Bioconductor package which provides a more idiomatic and user-friendly access to OMA data in R implemented on top of the REST API.\n\nFinally, to also enable Python users to easily interact with the database, we have developed a similar package in that language, compliant with the conventions and with support of typical complementary Python packages as outlined below.\n\n\nMethods\n\nWe start by describing the OMA REST API, before moving on to detail the OmaDB Bioconductor package, and finally outline the omadb Python package.\n\nThe REST framework is an API architectural style that is based on URLs and HTTP protocol methods. It was designed to be stateless and thus is context independent. That is, it does not save data internally between the HTTP requests which minimises server-side application state, thus easing parallelism. The combination of the HTTP and JSON data formats makes it particularly suitable for web applications and easily supported by most programming languages.\n\nSince the backend of the OMA browser is almost fully based on Python and its frontend is supported by the Django web framework8, we have opted to use the Django Rest Framework (DRF) to implement a REST API in our latest release4. Most API calls require querying the OMA database, stored in HDF59, using a custom Python library (“pyoma”). The query results are serialised in the format requested by the user — typically JSON.\n\nMost data available through the OMA browser is now also accessible via the API. This includes individual genes and their attributes such as protein or cDNA sequences, cross-references, pairwise orthologs, hierarchical orthologous groups10, as well as species trees and the corresponding taxonomy. The API documentation as well as the interactive interface can be found at https://omabrowser.org/api/docs (Figure 1).\n\nTo facilitate simplified access to the API and downstream analyses in the R environment, we have also developed an API wrapper package in R, now available in Bioconductor7 (http://bioconductor.org/packages/OmaDB/). This allowed for abstraction of the server interface, eliminating the need to know structure of the database or the URL endpoints to access the required data.\n\nThe package consists of a collection of functions that import OMA data into R friendly objects, namely S3 objects and data frames—depending on the query supplied. Due to the volume of the data available, some selected object attributes are at first given as URL endpoints. However, these are automatically loaded upon accession. OmaDB also facilitates further downstream analyses with other Bioconductor packages, such as GO enrichment analysis with topGO11, sequence analysis with BioStrings12, phylogenetic analyses using ggtree13 or gene locus analyses with the help of GenomicRanges14.\n\nThe open source code is hosted at https://github.com/DessimozLab/OmaDB/. The package requires R version >= 3.6 and Bioconductor version >= 3.9, as well as a stable internet connection.\n\nPackage Installation\n\n\n\nFor Python users, we provide an analogous package also named omadb. Results are supplied to users as a hybrid attribute-dictionary object. As such, both attribute and key-based access is possible. Where the URL of a further API call is listed in a response, this has been designed to be automatically requested for the user.\n\nFor data that can be represented as a table, the pandas package15 is supported. HOGs can be analysed or displayed using the pyham library16. Trees are retrievable as DendroPy17 or ETE318 Tree objects. Gene Ontology enrichment analyses are possible through the use of the goatools package19.\n\nThe open source code is hosted at https://github.com/DessimozLab/pyomadb/. The package requires Python >=3.6, as well as a stable internet connection. It is also available to download from PyPI, installable using pip.\n\nPackage Installation\n\n\n\n\nResults\n\nWe provide six illustrative examples in R. The first shows a direct call to the REST API, while the other five showcase the OmaDB R library. These examples are also available as a Jupyter notebook20 as part of the OmaDB R code repository. We have also provided analogous examples in Python, also in the form of a Jupyter notebook, included in its code repository—with the exception of Example 6, which uses a package only available in R.\n\nOne way to access the API is to directly send a request using httr21 in R. This approach requires the user to know the URL of the API endpoint, as well as the URL of the API function of interest. Some additional processing steps of the resultant response is usually needed. A simple example to retrieve information on the P53_RAT protein is provided below.\n\n\n\nBelow is a simple workflow using the OmaDB package to annotate a given protein sequence, using the mapSequence() function.\n\n\n\nIn this example, the sequence mapping identified one target sequence. From the seq_annotation object further information can be obtained as follows:\n\n\n\nThus, our sequence is human lactotransferrin (also known as lactoferrin). Lactotransferrin is one of four subfamilies of transferrins in mammals22.\n\nTo investigate the evolutionary history of genes more precisely, we turn to Hierarchical Orthologous Groups (HOGs)—sets of genes which have descended from a single common ancestral gene within a taxonomic range of interest10. For an introduction to HOGs, we refer the interested reader to the following short video: https://youtu.be/5p5x5gxzhZA.\n\nBy knowing the ID of the HOG to which our sequence belongs, we can obtain a list of all the HOG members (i.e. all genes in the HOG), as follows:\n\n\n\nNote that it is also possible to access information on a HOG using the ID of one of its members. Therefore the below will produce the same output.\n\n\n\nWe can easily retrieve the Gene Ontology (GO) terms23 that are associated to each of the members using OmaDB.\n\n\n\nThe resultant list of GO terms per gene is in the “geneID2GO” format by default, which is used by the topGO11 package.\n\nTo compare the function of lactotransferrins with their paralogous counterparts, we can retrieve a background set consisting of all members of the transferring HOG defined at the root of the eukaryotes\n\n\n\nWe can now construct a topGO object using the getTopGO function as seen below. Note that the background set of terms is set by getTopGO to all terms appearing in the list of annotations. This may not be appropriate in all cases—the choice of background set requires careful consideration24.\n\n\n\nAs the output in Table 1 indicates, several enriched terms in the mammalian lactotransferrin are related to bone formation, consistent with previous reports in the literature (e.g. 25). So is the role of lactotransferrin in antimicrobial activity (e.g. 26).\n\nThe taxonomic data obtained using the OmaDB package can easily be plugged into ggtree13 for phylogenetic tree visualisation. First, the tree is obtained using the getTaxonomy() function. In this example, the tree is rooted at the Hominoidea taxonomic level. The default format of the object returned is newick.\n\n\n\nThe resultant object can directly be used to build a phylogenetic tree using the ggtree package as below:\n\n\n\nThe tree can be further annotated using species silhouettes from PhyloPic (http://phylopic.org/). This functionality is already enabled within the ggtree package and just requires obtaining the relevant image codes. The workflow to produce Figure 2 is below.\n\n\n\n\n\nTo obtain all orthologous pairs between two genomes, we can use the getGenomePairs() function. To limit server load, the resultant response is paginated and by default only returns the first page, capped at 100 entries. This is easily adjustable by setting the ‘per_page’ parameter to either the number of orthologs required or simply to ‘all’.\n\nIn this example, we compare the distribution of PAM distances (Point accepted mutations; 27) between orthologs of two species-pairs, namely human-dog and human-mouse. First, we request the required data:\n\n\n\nWe can then bind the two resultant data frames and plot the results (Figure 3), as so:\n\n\n\nThe two-sample Kolmogorov-Smirnov test can be performed on the two distributions, using the command:\n\n\n\nThis returns p-value < 2.2e-16. The median distance between dog and human is shorter than that of mouse and human (8.8 vs. 11.8). This is consistent with previous observations that the rodent has a longer branch than humans and carnivores, in part due to their shorter generation time28.\n\nAlthough the OMA database currently analyses over 2,100 genomes, many more have been sequenced, and the gap keeps on widening. It is nevertheless possible to use OMA to infer the function of custom protein sequences through a fast approximate search against all sequences in OMA4.\n\n\n\nThis results in 54 GO annotations. By comparison, this sequence has merely 15 GO annotations in UniProt-GOA29 — all of which are also predicted by this method in OMA.\n\nWe go back to the lactotransferrin gene family from Example 2. We can use OmaDB in conjunction with the BgeeDB Bioconductor package30 to retrieve expression data from the Bgee database31 as follows.\n\n\n\nAmong the tissues in which lactotransferrin is expressed according to Bgee (Table 2), we note the bone marrow and the palpebral conjunctiva (the eyelid inner surface). This is consistent with the aforementioned involvement of lactotransferrin in bone formation and anti-microbial activity.\n\nFurther tutorials on the OmaDB package can be found in the accompanying vignettes:\n\n\n\n\nDiscussion and outlook\n\nOrthology is used for various purposes, such as species tree inference, gene evolution dynamic, or protein function prediction. The retrieval of orthologs is thus typically just the starting point of a larger analysis. Therefore, this overhaul and expansion of the OMA programmatic interface will facilitate the incorporation of OMA data in such larger analyses.\n\nOur R package will continue to be maintained in line with the biannual Bioconductor releases. Further work to improve the package includes improvement in performance. For example, the responses are currently fully loaded into an R object of choice which, depending on the response size, may create some time lag in the response. We will also continue to update the package and API to incorporate new functionalities of OMA, such as support for local synteny which is currently under development.\n\nLikewise, we will also maintain and further develop the Python package. In particular, we will explore the possibility of further integration with the BioPython library32.\n\nMore generally, in OMA we will keep supporting the various ways of accessing the underlying data, including the interactive web browser and flat files in a variety of formats. The REST API is also complemented by a new SPARQL interface that enables highly specific queries, as well as federated queries over multiple resources4. However, the query language is more complex.\n\nWe very much welcome feedback and questions from the community. We also highly appreciate contributions to the code in the form of pull requests. Our preferred channel for support is the BioStar website33, where we monitor all posts with keyword “oma”.\n\n\nSoftware availability\n\nPlease note that this manuscript uses version 2.0 of the OmaDB R package, which is in the development version of Bioconductor (v.3.9). Until the release of Bioconductor v.3.9 in Spring 2019, there are two possible ways of installing it:\n\n1) Install the development version of R (v.3.6) — required for Bioconductor v.3.9 — and install OmaDB using the command:\n\n\n\n2) Install OmaDB 2.0 directly from the github repo using the devtools R package:\n\n\n\nREST API available from: https://omabrowser.org/api\n\nDocumentation available from: https://omabrowser.org/api/docs\n\nR OmaDB package available from: http://bioconductor.org/packages/OmaDB/\n\nSource code available from: https://github.com/DessimozLab/OmaDB/\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.253025334\n\nLicense: GPL-2\n\nomadb Python package available from: https://pypi.org/project/omadb/\n\nSource code available from: https://github.com/DessimozLab/pyomadb/\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.253025035\n\nLicense: LGPL-3",
"appendix": "Grant information\n\nWe acknowledge support by Swiss National Science Foundation grant 150654, UK BBSRC grant BB/M015009/1, the Swiss State Secretariat for Education, Research and Innovation (SERI), as well as a UCL Genetics, Evolution and Environment Departmental Summer Bursary (to KK).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Natasha Glover for helpful feedback on the manuscript, and Frédéric Bastian for help on the example involving BgeeDB.\n\n\nReferences\n\nFitch WM: Distinguishing homologous from analogous proteins. Syst Zool. 1970; 19(2): 99–113. PubMed Abstract | Publisher Full Text\n\nSonnhammer EL, Gabaldón T, Sousa da Silva AW, et al.: Big data and other challenges in the quest for orthologs. Bioinformatics. 2014; 30(21): 2993–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nForslund K, Pereira C, Capella-Gutierrez S, et al.: Gearing up to handle the mosaic nature of life in the quest for orthologs. Bioinformatics. 2017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAltenhoff AM, Glover NM, Train CM, et al.: The OMA orthology database in 2018: retrieving evolutionary relationships among all domains of life through richer web and programmatic interfaces. Nucleic Acids Res. 2018; 46(D1): D477–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmitt T, Messina DN, Schreiber F, et al.: Letter to the editor: SeqXML and OrthoXML: standards for sequence and orthology information. Brief Bioinform. 2011; 12(5): 485–8. PubMed Abstract | Publisher Full Text\n\nHan MV, Zmasek CM: phyloXML: XML for evolutionary biology and comparative genomics. BMC Bioinformatics. 2009; 10: 356. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDjango Software Foundation. Django. [cited 2018]. Reference Source\n\nFolk M, Heber G, Koziol Q, et al.: An overview of the HDF5 technology suite and its applications. Proceedings of the EDBT. 2011. Publisher Full Text\n\nAltenhoff AM, Gil M, Gonnet GH, et al.: Inferring hierarchical orthologous groups from orthologous gene pairs. PLoS One. 2013; 8(1): e53786. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlexa A, Rahnenfuhrer J: topGO: Enrichment analysis for Gene Ontology. R package version 2.28.0. Bioconductor. 2016.\n\nPagès H, Aboyoun P, Gentleman R, et al.: Biostrings: Efficient manipulation of biological strings. R Package Version. 2017; 2(0). Reference Source\n\nYu G, Smith DK, Zhu H, et al.: ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. McInerny G editor. Methods Ecol Evol. 2017; 8(1): 28–36. Publisher Full Text\n\nLawrence M, Huber W, Pagès H, et al.: Software for computing and annotating genomic ranges. PLoS Comput Biol. 2013; 9(8): e1003118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcKinney W: pandas: a foundational Python library for data analysis and statistics. Python for High Performance and Scientific Computing. 2011; 1–9. Reference Source\n\nTrain C-M, Pignatelli M, Altenhoff A, et al.: iHam & pyHam: visualizing and processing hierarchical orthologous groups. Bioinformatics. 2018. PubMed Abstract | Publisher Full Text\n\nSukumaran J, Holder MT: DendroPy: a Python library for phylogenetic computing. Bioinformatics. 2010; 26(12): 1569–71. PubMed Abstract | Publisher Full Text\n\nHuerta-Cepas J, Serra F, Bork P: ETE 3: Reconstruction, Analysis, and Visualization of Phylogenomic Data. Mol Biol Evol. 2016; 33(6): 1635–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKlopfenstein DV, Zhang L, Pedersen BS, et al.: GOATOOLS: A Python library for Gene Ontology analyses. Sci Rep. 2018; 8(1): 10872. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKluyver T, Ragan-Kelley B, Pérez F, et al.: Jupyter Notebooks-a publishing format for reproducible computational workflows. In: ELPUB. 2016; 87–90. Publisher Full Text\n\nWickham H: httr: Tools for Working with URLs and HTTP. 2018. Reference Source\n\nLambert LA, Perri H, Meehan TJ: Evolution of duplications in the transferrin family of proteins. Comp Biochem Physiol B Biochem Mol Biol. 2005; 140(1): 11–25. PubMed Abstract | Publisher Full Text\n\nAshburner M, Ball CA, Blake JA, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25(1): 25–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaudet P, Dessimoz C: Gene Ontology: Pitfalls, Biases, and Remedies. Methods Mol Biol. In: Dessimoz C, Škunca N, editors. The Gene Ontology Handbook. New York, NY: Springer New York; 2017; 189–205. PubMed Abstract | Publisher Full Text\n\nNaot D, Grey A, Reid IR, et al.: Lactoferrin--a novel bone growth factor. Clin Med Res. 2005; 3(2): 93–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrsi N: The antimicrobial activity of lactoferrin: current status and perspectives. Biometals. 2004; 17(3): 189–96. PubMed Abstract | Publisher Full Text\n\nDayhoff MO, Schwartz RM, Orcutt BC: A model of evolutionary change in proteins. In: Atlas of Protein Sequence and Structure. 1978; 345–52. Reference Source\n\nEasteal S: Generation time and the rate of molecular evolution. Mol Biol Evol. 1985; 2(5): 450–3. PubMed Abstract | Publisher Full Text\n\nHuntley RP, Sawford T, Mutowo-Meullenet P, et al.: The GOA database: gene Ontology annotation updates for 2015. Nucleic Acids Res. 2015; 43(Database issue): D1057–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKomljenovic A, Roux J, Wollbrett J, et al.: BgeeDB, an R package for retrieval of curated expression datasets and for gene list expression localization enrichment tests [version 2; referees: 2 approved, 1 approved with reservations]. F1000Res. 2016; 5: 2748. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBastian F, Parmentier G, Roux J, et al.: Bgee: Integrating and Comparing Heterogeneous Transcriptome Data Among Species. In: Data Integration in the Life Sciences. (Lecture Notes in Computer Science). Springer Berlin Heidelberg. 2008; 124–31. Publisher Full Text\n\nCock PJ, Antao T, Chang JT, et al.: Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009; 25(11): 1422–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParnell LD, Lindenbaum P, Shameer K, et al.: BioStar: an online question & answer resource for the bioinformatics community. PLoS Comput Biol. 2011; 7(10): e1002216. PubMed Abstract | Publisher Full Text | Free Full Text\n\nklarakaleb, Altenhoff A, bioc-gitadmin, et al.: DessimozLab/OmaDB: v1.99.1 (Version 1.99.1). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2530253\n\nAlex WV, Altenhoff A: DessimozLab/pyomadb: v2.0.0 (Version 2.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2530250"
}
|
[
{
"id": "42912",
"date": "05 Feb 2019",
"name": "Bastian Greshake Tzovaras",
"expertise": [
"Reviewer Expertise bioinformatics",
"evolutionary biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a new REST API as an interface to the well-established Orthologous Matrix database. As identifying and evaluating orthologs is a central step in many biological analyses, an easy way to query the over 2,100 species in OMA is highly valuable. To further facilitate querying the data through their API, the authors present packages for R and Python. The API is well documented on the OMA website and the R package comes with vignettes describing different use cases. The manuscript presented here focuses on the OmaDB R-package and showcases some of its functions.\nBeing somewhat \"ahead of it's time\", the R package as described in the manuscript requires both the development version of R (v3.6) and Bioconductor (v3.9). The package installation instructions at the beginning of the manuscript only glances over it, more complete instructions are only found in the Software availability section at the end. We recommend including more explicit warnings/instructions about the required versions at the beginning, otherwise potential users might be confused when trying to follow along with the examples given in the manuscript (As happened to us and it took us some time to figure out what's going on).\nWhile the Python package is not extensively discussed in this manuscript, the authors provide a Binder that can be used to reproduce the same analyses using Python. We recommend putting a link to it (https://mybinder.org/v2/gh/DessimozLab/pyomadb/master?filepath=examples%2Fpyomadb-examples.ipynb) in the manuscript, to help users with taking up the Python library.\nWe welcome the switch from the SOAP API to a more modern REST implementation and the provided packages to interface with the API will be valuable for a lot of researchers working with orthologs.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4499",
"date": "29 Mar 2019",
"name": "Christophe Dessimoz",
"role": "Author Response",
"response": "Being somewhat \"ahead of its time\", the R package as described in the manuscript requires both the development version of R (v3.6) and Bioconductor (v3.9). The package installation instructions at the beginning of the manuscript only glances over it, more complete instructions are only found in the Software availability section at the end. We recommend including more explicit warnings/instructions about the required versions at the beginning, otherwise potential users might be confused when trying to follow along with the examples given in the manuscript (As happened to us and it took us some time to figure out what's going on). RESPONSE: We agree that this might cause confusion and we have now updated the OmaDB package section in Methods to mention explicitly that the package version used in the manuscript is 2.0, which until April 2019 is in the development version of Bioconductor. We also point the readers to the Software Availability section where further instructions for package installation are provided. We are hesitant to amend the package installation instructions at the beginning of the manuscript to reflect this as it will change shortly. While the Python package is not extensively discussed in this manuscript, the authors provide a Binder that can be used to reproduce the same analyses using Python. We recommend putting a link to it (https://mybinder.org/v2/gh/DessimozLab/pyomadb/master?filepath=examples%2Fpyomadb-examples.ipynb) in the manuscript, to help users with taking up the Python library. RESPONSE: This has now been added."
}
]
},
{
"id": "42908",
"date": "11 Feb 2019",
"name": "Laurent Gatto",
"expertise": [
"Reviewer Expertise Computational biology and bioinformatics",
"research software",
"reproducible research",
"omics."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKaleb et al. present two packages, namely R/Bioconductor OmoDB and python omadb, that allow users to query and use data from the Orthologous Matrix database. The article is well written and the authors provide 6 examples that convincingly demonstrate the usefulness and reach of their work.\nI have a couple of comments and suggestions below, presented in chronological order. The only serious one is a request for the authors to describe the outputs in their examples a bit more (see below for details), to facilitate the adoption for users that wouldn't be familiar with R.\nIn https://omabrowser.org/api/docs, the pagination example has a typo. The genomes should be replaced with genome:\n``` $ \"https://omabrowser.org/api/genomes/?page=2\" HTTP/1.1 404 Not Found Server: nginx Date: Mon, 11 Feb 2019 06:04:30 GMT Content-Type: text/html; charset=utf-8 Connection: keep-alive X-Frame-Options: SAMEORIGIN Vary: Cookie Set-Cookie: __utmmobile=d41d8cd98f00b204e9800998ecf8427e; expires=Wed, 10-Feb-2021 06:04:30 UTC; Path=/ Set-Cookie: sessionid=9zb42ljib7apkubml1e1t742i5p6f3a6; expires=Mon, 25-Feb-2019 06:04:30 GMT; HttpOnly; Max-Age=1209600; Path=/\n$ curl -I \"https://omabrowser.org/api/genome/?page=2\" HTTP/1.1 200 OK Server: nginx Date: Mon, 11 Feb 2019 06:04:32 GMT Content-Type: application/json Connection: keep-alive Link: ; rel=\"first\", ; rel=\"prev\", ; rel=\"next\", ; rel=\"last\" X-Total-Count: 2198 Vary: Accept, Cookie Allow: GET, HEAD, OPTIONS X-Frame-Options: SAMEORIGIN Set-Cookie: __utmmobile=d41d8cd98f00b204e9800998ecf8427e; expires=Wed, 10-Feb-2021 06:04:32 UTC; Path=/ Set-Cookie: sessionid=9h5n3ouuwvh4ock9dz3q4yiprmb8iw0d; expires=Mon, 25-Feb-2019 06:04:32 GMT; HttpOnly; Max-Age=1209600; Path=/ Strict-Transport-Security: max-age=15768000 ```\nIn the introduction, the authors explain that 'Most data available through the OMA browser is now accessible via the API'. I think it would be useful to know what data isn't available and whether the browser and REST API would ever be equivalent in terms of data served. This might be partly addressed later, in the discussion, where the authors mention 'support for local synteny'. Some additional details would be useful to redirect users to the appropriate interface. Similarly, it would be useful to know if the R and python packages provide access to the same data, or if differences also exist there. I didn't see mention of the R and python packages on the OmaDB web page. This would be a useful addition for visitors. In the Bioconductor package section, the authors explain that data is provided in 'R friendly objects, namely S3 objects and data frames'. I would suggest to rephrase this and only refer to objects, as S4 objects are also returned and the nature of the technical class system is probably not necessary in the frame of this document. Regarding the R package, I would suggest to add URL and BugReports fields in the packages DESCRIPTION file. This helps users find the GitHub repository and report issues. I also noted that in the 'getting started' vignette, it looks like some section a missing a space after the section markup. I have send a pull request fixing these and some other minor issue. Note that the html and R version of the vignette shouldn't be included in the package source. In the python package section, the authors mention that this package is also named 'omadb'. I would argue that the packages have different names, as programming languages are case sensitive and suggest to drop the also to avoid any confusion. In the first sentence of the result section, authors should replace R library by R package, as they are referring to their package, not the location where the package is being installed (the library). In general, it would be very useful for the authors to describe the different outputs they have. I am not expecting the authors to provide full details of the REST API responses, but describing how the results match the text would be important. For example, in example 1, they only show how to produce the `response_content_list` response. Here, it would be useful to explain that this R list directly maps the REST json message, and point to the specific documentation entry point. Such an explanation motivates the example in the text and helps users, that aren't familiar with REST, to understand the relation between the server and the package. Similarly in example 2, the authors create the `seq_annotation` variable and mention that only one target sequence was identified. Here, it would be useful to show that `length(seq_annotation$targets)` is equal to 1, to back their claim, to indicate how users can verify the number of targets, and motivate the use of the first list index in later code chunks. Still in example 2, the authors query and extract the hog members. These data are however already present in the first output of that example, under `seq_annotation$targets[[1]]$oma_hog_members`. It would be useful to explain why the authors send a second query to obtain that data and clarify whether `oma_hog_members` is always equivalent to calling `getHOG` and `getProtein`. When trying to reproduce the code, I first failed to run the code chunks calling `getProtein`. Later, the authors clarify the software requirements in more details. It would however be useful to briefly mention, early on in the Results section, what version was used for the examples. In example 5, I would suggest to update to new function name, as `getAnnotations` is expected to be deprecated in the next release, especially as the new version of the package is anyway required for the `getProtein` function.\n``` > myAnnotations <- getAnnotation(mysterySeq) Warning message: 'getAnnotation' is deprecated. Use 'annotateSequence' instead. See help(\"Deprecated\") ```\nAnother example where an explanation of the output is important is example 5. The authors call `myAnnotation <- getAnnotations(mysterySeq)` and then refer to 54 GO annotation results. In repeating their analysis, I obtain a data frame with 55 observations (see below). It is this unclear whether I have a different result, if one observation should be dropped, or if my output is completely wrong (was I even expecting a data frame?).\n\n``` > dim(myAnnotations) [1] 55 13 ```\nIn general, given the nature of the package, i.e. that it accesses an online repository that is (or can be) updated regularly, results may change, this also explaining why I may have different results.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4500",
"date": "29 Mar 2019",
"name": "Christophe Dessimoz",
"role": "Author Response",
"response": "In https://omabrowser.org/api/docs, the pagination example has a typo. The genomes should be replaced with genome. RESPONSE: Thanks for spotting this typo. It has been fixed. In the introduction, the authors explain that 'Most data available through the OMA browser is now accessible via the API'. I think it would be useful to know what data isn't available and whether the browser and REST API would ever be equivalent in terms of data served. This might be partly addressed later, in the discussion, where the authors mention 'support for local synteny'. Some additional details would be useful to redirect users to the appropriate interface. Similarly, it would be useful to know if the R and python packages provide access to the same data, or if differences also exist there. RESPONSE: As mentioned in the Discussion, the package currently lacks availability of the data on local synteny, mainly due to the complexity of the data representation in the API format. We aim to bridge this gap in the next release. We have now amended the Methods section to reflect the difference between OMA browser and the API more explicitly, as well as the discussion to reassure the users the API and OMA browser will be kept in sync with further OMA developments. Both R and Python packages use the same API and supply the same data. I didn't see mention of the R and python packages on the OmaDB web page. This would be a useful addition for visitors. RESPONSE: We already had a link to the R package in the /api/docs, but we have now also included link directly from the \"compute\" menu. In the Bioconductor package section, the authors explain that data is provided in 'R friendly objects, namely S3 objects and data frames'. I would suggest to rephrase this and only refer to objects, as S4 objects are also returned and the nature of the technical class system is probably not necessary in the frame of this document. RESPONSE: This has now been amended. Regarding the R package, I would suggest to add URL and BugReports fields in the packages DESCRIPTION file. This helps users find the GitHub repository and report issues. I also noted that in the 'getting started' vignette, it looks like some section a missing a space after the section markup. I have send a pull request fixing these and some other minor issue. RESPONSE: Thank you for the pull request, this has now been merged with OmaDB version 2.0. Note that the html and R version of the vignette shouldn't be included in the package source. RESPONSE: This has now been amended. In the python package section, the authors mention that this package is also named 'omadb'. I would argue that the packages have different names, as programming languages are case sensitive and suggest to drop the also to avoid any confusion. RESPONSE: Amended as requested. In the first sentence of the result section, authors should replace R library by R package, as they are referring to their package, not the location where the package is being installed (the library). RESPONSE: This has now been amended. In general, it would be very useful for the authors to describe the different outputs they have. I am not expecting the authors to provide full details of the REST API responses, but describing how the results match the text would be important. For example, in example 1, they only show how to produce the `response_content_list` response. Here, it would be useful to explain that this R list directly maps the REST json message, and point to the specific documentation entry point. Such an explanation motivates the example in the text and helps users, that aren't familiar with REST, to understand the relation between the server and the package. RESPONSE: We agree, and further information on the output generated in the manuscript has now been added to example 1. Similarly in example 2, the authors create the `seq_annotation` variable and mention that only one target sequence was identified. Here, it would be useful to show that `length(seq_annotation$targets)` is equal to 1, to back their claim, to indicate how users can verify the number of targets, and motivate the use of the first list index in later code chunks. RESPONSE: This has now been updated. Still in example 2, the authors query and extract the hog members. These data are however already present in the first output of that example, under `seq_annotation$targets[[1]]$oma_hog_members`. It would be useful to explain why the authors send a second query to obtain that data and clarify whether `oma_hog_members` is always equivalent to calling `getHOG` and `getProtein`. RESPONSE: It is true that the hog members can also be directly accessed via the oma_hog_members attribute. However, the members are only loaded once the attribute is accessed, so we do not add unnecessary requests. Even more importantly, if we would load the hog members via the oma_hog_members attribute, it is not obvious for which taxonomic level the members are loaded. We therefore prefer to keep the current slightly more verbose way to access the data. When trying to reproduce the code, I first failed to run the code chunks calling `getProtein`. Later, the authors clarify the software requirements in more details. It would however be useful to briefly mention, early on in the Results section, what version was used for the examples. RESPONSE: We agree that this can be confusing, and we have now amended the Methods and the Results section to explicitly mention the usage of OmaDB v2.0 for the examples. In example 5, I would suggest to update to new function name, as `getAnnotations` is expected to be deprecated in the next release, especially as the new version of the package is anyway required for the `getProtein` function. RESPONSE: This was a mistake on our side and has now been amended. Another example where an explanation of the output is important is example 5. The authors call `myAnnotation <- getAnnotations(mysterySeq)` and then refer to 54 GO annotation results. In repeating their analysis, I obtain a data frame with 55 observations (see below). It is this unclear whether I have a different result, if one observation should be dropped, or if my output is completely wrong (was I even expecting a data frame?). In general, given the nature of the package, i.e. that it accesses an online repository that is (or can be) updated regularly, results may change, this also explaining why I may have different results. RESPONSE: We can confirm that this is due to the December 2018 OMA release, where there are indeed 55 results returned for that particular query. The fact that the results may vary due to the continued updates of the OMA database has now been explicitly mentioned in the beginning of the methods section of the manuscript. We have also added what version of the database was used to generate the examples in the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/8-42
|
https://f1000research.com/articles/8-43/v1
|
10 Jan 19
|
{
"type": "Research Article",
"title": "EEG correlation at a distance: A re-analysis of two studies using a machine learning approach",
"authors": [
"Marco Bilucaglia",
"Luciano Pederzoli",
"William Giroldini",
"Elena Prati",
"Patrizio E. Tressoldi",
"Marco Bilucaglia",
"Luciano Pederzoli",
"William Giroldini",
"Elena Prati"
],
"abstract": "Background: In this paper, data from two studies relative to the relationship between the electroencephalogram (EEG) activities of two isolated and physically separated subjects were re-analyzed using machine-learning algorithms. The first dataset comprises the data of 25 pairs of participants where one member of each pair was stimulated with a visual and an auditory 500 Hz signals of 1 second duration. The second dataset consisted of the data of 20 pairs of participants where one member of each pair received visual and auditory stimulation lasting 1 second duration with on-off modulation at 10, 12, and 14 Hz. Methods and Results: Applying a ‘linear discriminant classifier’ to the first dataset, it was possible to correctly classify 50.74% of the EEG activity of non-stimulated participants, correlated to the remote sensorial stimulation of the distant partner. In the second dataset, the percentage of correctly classified EEG activity in the non-stimulated partners was 51.17%, 50.45% and 51.91%, respectively, for the 10, 12, and 14 Hz stimulations, with respect the condition of no stimulation in the distant partner. Conclusions: The analysis of EEG activity using machine-learning algorithms has produced advances in the study of the connection between the EEG activities of the stimulated partner and the isolated distant partner, opening new insight into the possibility to devise practical application for non-conventional “mental telecommunications” between physically and sensorially separated participants.",
"keywords": [
"EEG",
"correlation at distance",
"machine learning",
"linear discrimination analysis."
],
"content": "Introduction\n\nThe test of whether or not the brain activities of two people who are only linked mentally – and with absolutely no other form of conventional communication – are correlated is a small research field that has existed for about 50 years (see Table S1 in (Giroldini et al., 2016).\n\nDespite accumulating evidence in favor of this correlation, this phenomenon is still considered controversial because of the proposed theories to explain it, no single theory is widely accepted. This correlation excludes the possibility of it originating from information received via the sense organs, from any direct link, even internet connections (e.g. (Jiang et al., 2018; Lee et al., 2017)) or from the electroencephalogram (EEG) activity of the stimulated partner of the pair, given their physical distance and isolation from each other.\n\nWe are left with postulating a type of quantum-like connection based on the distant mental connection between each partner (Galli Carminati et al., 2017; Walach & Römer 2011; Walach et al., 2016), even if this connection operates on a neurophysiological level.\n\nThe most commonly used method in this field of research is that of isolating the two partners, ensuring that there is no chance of sensorially obtaining information in the usual way, and the synchronous parallel recording of their respective neurophysiological activities. Therefore, one partner is stimulated at non-regular intervals with visual and/or auditory stimuli which can be structured, e.g. images, or non-structured, such as arrangements of black and white squares, or short duration sounds at a specific frequency. The choice to present the stimulations at irregular intervals, possibly also randomly, is important because it reduces neurophysiological autoactivation due to the expectation of stimulation.\n\nAt the end of the stimulation stage, each partner’s recorded neurophysiological parameters are compared. If the non-stimulated partner’s neurophysiological output shows activity that is simultaneous with the activity in the stimulated partner, and this activity is statistically greater than during the non-stimulated periods, we can confirm that it derives from a mental connection, or at least from a means that is different from conventional electromagnetic transmission.\n\nRegarding the type of correlation between the partners’ neural activities, a further step with respect to simply proving its existence is determining whether it is aspecific or specific; this means whether the type of observed activity – for example in the activity of the partner receiving specific sensorial stimulations, such as a visual stimulation with an on-off modulation at 10 Hz and 14 Hz – is also seen in the mentally connected partner, or if the activation is undifferentiated, for example at 10 Hz.\n\nAs would be expected, in the correlated activities of the two partners, the neurophysiological activity of the non-stimulated partner is always smaller, and it could be that classical analysis methods are unable to separate the activity signal from the background.\n\nBeginning in the last few years, thanks to the development of artificial intelligence algorithms, different machine-learning algorithms have been applied to the analysis of EEG data (Chai et al., 2017; de Carvalho et al., 2017; Lotte et al., 2007; Stober et al., 2016). Among their many advantages is the possibility of simultaneously analyzing many variables that can be associated with a given event in both a linear and non-linear way, revealing coactivity configurations that would otherwise be difficult to see with other techniques. For an introduction to this type of algorithms see (Lantz, 2015).\n\nIn this study we re-analysed, using machine-learning techniques, the data of two studies aiming to more precisely identify the neurophysiological parameters of the EEG activity of non-stimulated partners correlated to those produced by their partners given specific sensorial stimulations.\n\nThe first dataset was obtained from the study of (Giroldini et al., 2016). In this study, conducted using 25 pairs of participants, the stimulated partner was given a series of 128 visual and auditory stimulations of 1 second duration at 500 Hz, separated by random intervals of non-stimulation lasting from 4 to 6 seconds. Each partner’s EEG activity was simultaneously recorded using 14-channel equipment.\n\nThe second dataset was obtained testing 20 pairs of participants, by (Giroldini et al., 2018). In this study the stimulated partner was given random sequences of 32 visual and auditory stimulations of 1 second duration modulated at 10, 12, or 14 Hz, while the EEG activities of each partner were simultaneously recorded.\n\n\nMethods\n\nParticipants. Six Italian Caucasian healthy adults were chosen for the experiment, comprised of five men and one woman, with an average age of 35.5 years (SD = 8.3).\n\nThey were selected from the members of EvanLab, the private laboratory involved in this study. The criteria for their voluntary inclusion were their mutual friendship (> 10 years), and their experience in being able to maintain prolonged focused concentration, a product of their familiarity with meditation and other practices requiring control of mental activities.\n\nAll together they contributed 25 different pairs of data.\n\nThe data were collected over three non-consecutive days. The dataset (available from: http://dx.doi.org/10.6084/m9.figshare.1466876 (Tressoldi, 2016)) includes details of pairings and the EEG data from a 14 EEG channels system.\n\nStatement of ethics. The use of experimental subjects is in accordance with ethical guidelines as outlined in the Declaration of Helsinki, and the study has been approved by the Ethical Committee of the University of Padova’s Department of General Psychology (prot. n. 63, 2012). Before taking part in the experiment, each subject gave his/her informed consent in writing after having read a description of the experiment.\n\nProcedure. Each stimulated subject received 128 audio-visual stimulations (AVS). The auditory stimulus was composed of a 500 Hz sinusoid applied through 32 Ohm Parrot ZIK® earphones at a volume of about 80 dB. The visual stimulation was from high intensity red LEDs in a 4×4 arrangement placed approximately one meter from the subject being stimulated. The subject kept his/her eyes closed because the light could easily be detected through the eyelids.\n\nAdditionally, for each subject (no matter whether stimulated or not) 128 “surrogated” stimuli were drawn (nAVS). Their onsets were placed exactly 3s before the AVS onsets in order to be discriminated from the nAVS by our classification algorithm.\n\nAll preprocessing steps were done in MatLab using the EEGLab toolbox (Delorme & Makeig, 2004).\n\nAfter filtering with a zero-phase FIR band-pass filter (2~40Hz), an ICA (Independent Component Analysis) was performed using a SOBI algorithm that, according to a previous study (Urigüen & Garcia-Zapirain, 2015), exhibits the best performance in the detection of the artifacts.\n\nICs (Independent Components) corresponding to artifacts were automatically identified and rejected using the MARA algorithm (Winkler et al., 2011) that is based on a set of spatial, temporal and statistical features. Finally, a CAR (Common Average Reference) was performed.\n\nGiven that the type of stimulation produced a P300-like wave, we followed the common procedures used with Brain Computer Interfaces based on P300 (Hoffmann et al., 2008). Data were first epoched with length of a 1s after the stimulus onset, then a sixth order Butterworth bandpass filter (1 ~ 12Hz) was applied and downsampled to 32Hz. Epoch lengths were thus lowered to 64 samples located at time points:\n\n\n\nSamples were then amplitude-scaled according to the maximum and the minimum value inside each epoch, according to the equation:\n\n\n\nFinally, scaled samples were concatenated to form a feature vector v, with a dimensionality of 65 × 14 = 910:\n\n\n\nwhere xk(tj) is the sample at time point tj from channel k.\n\nFeature selection. Reduction of the feature dimensionality is crucial to improve classification performances. A simple method for feature selection is based on the ranking of the Biserial Correlation Coefficients (Müller et al., 2008). Considering a two-class dataset D (with just one numerical feature), the Biserial Correlation Coefficient r2 for the feature is defined as:\n\n\n\nwhere DA, DB are the subsets of the dataset D composed by instances that belong to class A, B. m(.) and s(.) are, respectively, the sample mean and standard deviation operators. r2 score is a measure of the “discriminative power” of the feature, reflecting how much of its “variance” is “explained” by the class affiliation.\n\nAfter summing the coefficients of each feature and sorting the scores in descendent order, we selected the features of which the added score came to 95% of the total score.\n\nFor each stimulated and non-stimulated group of participants, the scalp distributions of r2 coefficients (expressed as percentage, normalized to the total score) were drawn. Since each channel is associated with at most 65 features (as well as 65 r2 coefficients), coefficients (one coefficient for each channel) are calculated as mean value.\n\nClassification. As a classifier, we chose a Linear Discriminant (LD) classifier and the estimation of the classification accuracy was performed 10 times (to compensate the random variability of the estimated accuracy) by a 10-fold stratified cross-validation scheme.\n\nWe choose LD because, according to the literature (Lotte et al., 2007), it has a very low computational requirement, is simple to use and generally provides good results (i.e. robustness to overfitting).\n\nSince the feature selection is performed using information from the class labels, in order to avoid the overfitting and to train the classifier in a “fair way”, feature selection was performed at each step within the cross-validation.\n\nIn addition to the LD classifier, at each step we classify our data using a Random classifier (which gets a prediction uniformly distributed between the classes) that serves as a “baseline” to evaluate the LD classifier performance. The random classification is simply obtained randomly assigning (with uniform probability p=1/n) to each instance of the test, one of the class {C_1,C_2,…,C_n }, where n is the number of classes to be predicted.\n\nAt each cross-validation step, we calculated the mean accuracy and, finally, we calculated the mean and standard deviation of these mean accuracies.\n\nThe syntax in MatLab code is available here: https://doi.org/10.6084/m9.figshare.5132647.v6 (Tressoldi et al., 2018)\n\nThe dataset is composed of 14 channels EEG data obtained from 20 pairs of participants (available from: https://doi.org/10.6084/m9.figshare.5132647.v6 (Tressoldi et al., 2018)).\n\nParticipants. Five adults, two women and three men with an average age of 38.3 years (SD = 7.5), took part in this study. The selection criteria were their experience in mind control techniques (mainly meditation) and their mutual friendship, which we consider as essential pre-requisites for an adequate “mental and emotional connection” between the pairs. Each participant took turns in being both the stimulated partner and the non-stimulated partner with each of the others, making a total of 20 pair combinations.\n\nStatement of ethics. The use of experimental subjects is in accordance with ethical guidelines as outlined in the Declaration of Helsinki, and the study has been approved by the Ethical Committee of the University of Padova’s Department of General Psychology (prot. n. 63, 2012). Before taking part in the experiment, each subject gave his/her informed consent in writing after having read a description of the experiment\n\nThis sample size of 20 pairs of participants was estimated by setting the following parameters: statistical power = .80, a one-tailed Type I error = .05 and an expected effect size d = .5 for a one sample t-test (difference from a constant = 0: http://powerandsamplesize.com/Calculators/Test-l-Mean/1-Sample-1-Sided).\n\nStimuli. Visual stimuli consisted of red light flashes at 10, 12, 14 Hz (32 stimuli for each frequency) lasting 1 s with a minimum inter-stimulus interval of 4 s, while audio stimuli consisted of a 900 Hz sine tone, modulated on-off at the flashing frequencies.\n\nThe audio modulation was performed on a 900 Hz sinusoidal carrier and applied through 32 ohm impedance earphones at a volume of about 80 dB.\n\nThe interval between the three blocks was randomly varied at between 40 and 90 seconds.\n\nThe visual stimuli were provided by an array of 16 red LEDs positioned about 30 cm from the stimulated partner’s closed eyes.\n\nThe three frequency blocks were presented randomly without repetition of the same frequency.\n\nAdditionally, for each subject (regardless of whether stimulated or not) the onsets of 32 “surrogated” stimuli (0 Hz) were drawn from random positions of the entire recording: the first and the last 10 s were discarded, maintaining the same minimum inter-stimulus distance.\n\nPreprocessing. All pre-processing steps were done as for the first dataset. Data were epoched with a 2s window after each stimulus onset. From each epoch and from channel, the PSD (Power Spectral Density) was estimated with a Welch’s periodogram (1s long Hamming window with 50% of overlap) and spectral bins were normalized to the maximum value of the PSD.\n\nFollowing the feature extraction procedure normally used with Brain-Computer-Interfaces systems based on SSVEP (Müller-Putz et al., 2005), we concatenated spectral bins corresponding to both stimulus frequencies and their first harmonics: F = {10, 20; 12, 24; 14, 28} Hz.\n\nThe feature vector v, with a dimensionality of 6 × 14 = 84, is thus defined as:\n\n\n\nwhere pk (f) is the spectral power at frequency fj ∈ F from channel k.\n\nFeature selection. The 4-class (10, 12, 14, 0) prediction problem was decomposed in 6 different 2-class prediction problems, creating 6 different datasets containing all the possible couples of the 4 classes: {10, 12}, {10, 14}, {10, 0}, {12, 14}, {12, 0} and {14, 0}.\n\nFeature selection was based on the ranking of the Biserial Correlation Coefficients as with the first dataset.\n\nClassification. As with the first dataset, we used a Linear Discriminant (LD) classifier compared with a Random classifier. Again, we performed a 10-fold stratified cross-validation scheme, with feature selection inside each cross-validation step.\n\n\nResults\n\nFigure 1 and Figure 2 show the scalp distributions of r2 coefficients (expressed as percentage, normalized to the total score) for the stimulated and not-stimulated participants, respectively.\n\nAs it is shown in Figure 1, the class-discrimination (AVS vs nAVS) in the stimulated participants derives mostly from central and left frontal and right temporal electrodes, while in the non-stimulated participants their class is best discriminated by features from the right temporal and left occipital electrodes.\n\nThe percentages of the classification accuracy are reported in Table 1 and Table 2 for the stimulated and the non-stimulated participants, respectively.\n\nLD: linear discriminant classifier; Random: random classifier.\n\nInferential statistics and parameters estimation. For the inferential statistics, we chose the comparison of percentage of correct discrimination between the stimulation and non-stimulation conditions, comparing each with the percentage obtained with the Random Classifier using a one-tailed paired t-test and a comparison using the Bayes Factor calculation using the default Cauchy prior value of .707. As a parameter estimation, we chose the measurement of Cohen’s effect size d and the relative confidence interval of 95%, all one-directional. All statistical analyses were conducted using Jasp software (Jasp Team, 2018).\n\nResults are presented in Table 3 below, while data from the Bayes Factor Robustness Check are shown in the Extended data.\n\nComment. While results for the stimulated participants were as expected, those observed from non-stimulated participants were interesting. Indeed, even if the correct discrimination percentage is not particularly high, all the inferential statistics and parameter estimates indicate the presence of an EEG signal corresponding to the stimulation periods. Furthermore, the origins of these differences (see Figure 2) correspond to channels T8 and O1, which correspond to brain areas involved in processing auditory and visual information respectively.\n\nFigure 3 and Figure 4 show the scalp distributions of the mean r2 coefficients (expressed as percentage, normalized to the total score) for each 2-class problem for each group (stimulated and non-stimulated pairs).\n\nTop left: 14 Hz vs 0 Hz; top centre: 12 Hz vs 0 Hz; top right: 12 Hz vs 14 Hz; bottom left: 10 Hz vs 0 Hz; bottom centre: 10 Hz vs 14 Hz; bottom right; 10 Hz vs 12 Hz.\n\nTop left: 14 Hz vs 0 Hz; top centre: 12 Hz vs 0 Hz; top right: 12 Hz vs 14 Hz; bottom left: 10 Hz vs 0 Hz; bottom centre: 10 Hz vs 14 Hz; bottom right; 10 Hz vs 12 Hz.\n\nComment. As shown in Figure 3, in the stimulated participants the EEG activity generated by the three frequencies of stimulation is best distinguished from the 0 Hz class (non-stimulation) by the right occipital and temporal regions’ features.\n\nIn the non-stimulated participants (Figure 4), the 10 Hz and 14 Hz are best distinguished from the 0 Hz class by frontal and frontocentral regions features.\n\nPercentages of classification accuracy. The percentages of correct classification for each comparison for each of the 10 cross-validation iterations and their descriptive statistics observed in the stimulated participants are presented in Table 4.\n\nInferential statistics and parameters estimation. Similarly to the first dataset, for the inferential statistics we chose the comparison with the percentage of LD classifier discrimination between the three stimulation conditions at 10 Hz, 12 Hz, and 14 Hz, and the non-stimulation (0 Hz), comparing each with the percentages obtained from the Random Classifier using a paired one-tailed t-test and a comparison by way of the Bayes Factor calculation using the default Cauchy prior value of .707. As a parameter estimate, we chose Cohen’s d and the relative confidence interval of 95%, all one-directional. These results are presented in Table 5, while the Bayes Factor Robustness Check data are shown in the Extended data.\n\nComment. As seen from the means, the percentage of correct classification of EEG activity between the non-stimulation and stimulation periods with the three frequency values is always greater than 70%, as confirmed by the inferential statistics results.\n\nThe percentages of correct classification of each comparison for each of the 10 cross-validation interactions and their descriptive statistics observed in the non-stimulated participants are presented in Table 6.\n\nInferential statistics and parameters estimation. Results of inferential statistics and parameter estimates are shown in Table 7.\n\nComment. The most interesting result is that the activity related to at least two of the three stimulation frequencies, 10 Hz and 14 Hz, is classified above 50% with respect to the non-stimulation condition, even if in a more evident way for 14Hz. The fact that there are no differences between the 12Hz and the non-stimulation condition suggests that the base rhythm in non-stimulated participants is predominantly at 12Hz. Moreover, since the direct comparison between the activity corresponding to 10Hz and 14Hz, does not show differences with respect to the random condition (Mean = 50.2; SD = .75; Mean = 50.3; SD = 1.79), it suggests that in the non-stimulated participants, the EEG activity corresponding to these two frequencies either above or below 12Hz.\n\n\nDiscussion\n\nThe machine-learning re-analyses of data from two studies relative to the distant correlation between EEG activity of participant pairs who were sensorially stimulated and participants who were connected to them only mentally, not only confirmed the existence of a correlation, as shown in the two studies from which they were taken, but also pointed out the degree of classification between stimulated and non-stimulated conditions depended on the stimulation frequency used.\n\nIn the first dataset, the stimulation of EEG activity with luminous and auditory stimuli at 500Hz produced a slight perturbation also in that of the non-stimulated partners which allowed to distinguish it from the non-stimulation condition with a modest 50.74%.\n\nIn the second dataset, however, stimulation with frequencies from 10Hz and 14Hz, allowed to better distinguish this type of related perturbation, with percentages of 51.17% and 51.91% respectively, while for 12Hz stimulation, the discrimination was similar to the random classifier one.\n\nEven though, with hindsight, the choice to use frequencies that are within or close to the Alpha activity was not optimal, given that this was the dominant EEG activity in non-stimulated participants, the evidence that these are distinguishable even in the EEG activity of non-stimulated participants opens interesting possibilities, should this research path be continued in terms of practical applications.\n\nInformation regarding the potential neural sources of these signals obtained from this study must also be validated with confirmatory studies and also with more sophisticated equipment than those used by us.\n\nFrom a theoretical point of view, while we can safely reject the theory that the observed activity in non-stimulated participants is due to a traditional sensorial transmission (albeit weak) from the stimulated participants’ EEG activity, we as yet do not have enough information to determine if this correlation – which does not necessarily involve transmission – has the same characteristics as that seen in quantum mechanics. Indeed, to determine this it would, first of all, entail closing a series of loopholes, as was done in quantum mechanics to reject the theory that distant connection used the laws of classical physics (Giustina et al., 2015; Shalm et al., 2015). For example, the loophole showing the impossibility of communication at the speed of light between participant pairs would need closing.\n\nPending a better understanding of the laws regulating the correlation between the EEG activity of two mentally-connected participants, we believe that data obtained so far in this field, including that of other researchers, clearly show that if classification algorithms of signals down to the level of single participants are perfected, along with an online instead of the offline classification, the road towards practical use of mental telecommunication can be opened, even if for now it is based on binary symbolic codes.\n\nMental telecommunication, not just those based on electromagnetic waves but also those of the future based on quantum technologies, have the advantage of not requiring repeaters or security measures and would certainly be beneficial from an economical point of view.\n\n\nData availability\n\nFirst dataset: Figshare: EEG correlates of social interaction at distance, http://dx.doi.org/10.6084/m9.figshare.1466876 (Tressoldi, 2016).\n\nSecond dataset: Figshare: Brain-to-Brain interaction at a distance: a global or differential relationship? https://doi.org/10.6084/m9.figshare.5132647.v7 (Tressoldi et al., 2018)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nDataset 1: Stimulated participants. Bayes Factor Robustness Check; Non-stimulated participants: Bayes Factor Robustness Check\n\nDataset 2: Non-stimulated participants. Bayes Factor Robustness Check: 10 Hz vs 0 Hz; Bayes Factor Robustness Check: 14 Hz vs 0 Hz\n\nBoth datasets are available on Figshare: Brain-to-Brain interaction at a distance: a global or differential relationship? https://doi.org/10.6084/m9.figshare.5132647.v7 (Tressoldi et al., 2018)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nChai R, Ling SH, San PP, et al.: Improving EEG-Based Driver Fatigue Classification Using Sparse-Deep Belief Networks. Front Neurosci. 2017; 11: 103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Carvalho SR, Filho IC, de Resende DC, et al.: A Novel Procedure for Classification of Early Human Actions from EEG Signals. In 2017 Brazilian Conference on Intelligent Systems (BRACIS). IEEE. 2017; 240–245. Publisher Full Text\n\nDelorme A, Makeig S: EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods. 2004; 134(1): 9–21. PubMed Abstract | Publisher Full Text\n\nGalli Carminati G, Martin F, Carminati F: A Very Simple Quantum Model of Mind and Matter. NeuroQuantology. 2017; 15(2). Publisher Full Text\n\nGiroldini W, Pederzoli L, Bilucaglia M, et al.: EEG correlates of social interaction at distance [version 5; referees: 2 approved]. F1000Res. 2016; 4: 457. Publisher Full Text\n\nGiroldini W, Pederzoli L, Bilucaglia M, et al.: Exploring the Brain-to-Brain interaction at a distance: a global or differential relationship? 2018. Publisher Full Text\n\nGiustina M, Versteegh MA, Wengerowsky S, et al.: Significant-Loophole-Free Test of Bell’s Theorem with Entangled Photons. Phys Rev Lett. 2015; 115(25): 250401. PubMed Abstract | Publisher Full Text\n\nHoffmann U, Vesin JM, Ebrahimi T, et al.: An efficient P300-based brain-computer interface for disabled subjects. J Neurosci Methods. 2008; 167(1): 115–125. PubMed Abstract | Publisher Full Text\n\nJASP Team: JASP (Version 0.9.2.0) [Computer software]. 2018. Reference Source\n\nJiang L, Stocco A, Losey DM, et al.: BrainNet: A Multi-Person Brain-to-Brain Interface for Direct Collaboration Between Brains. ArXiv. 2018. Reference Source\n\nLantz B: Machine learning with R: discover how to build machine learning algorithms, prepare data, and dig deep into data prediction techniques with R. Packt Publishing. 2015. Reference Source\n\nLee W, Kim S, Kim B, et al.: Non-invasive transmission of sensorimotor information in humans using an EEG/focused ultrasound brain-to-brain interface. PLoS One. 2017; 12(6): e0178476. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLotte F, Congedo M, Lécuyer A, et al.: A review of classification algorithms for EEG-based brain-computer interfaces. J Neural Eng. 2007; 4(2): R1–R13. PubMed Abstract | Publisher Full Text\n\nMüller-Putz GR, Scherer R, Brauneis C, et al.: Steady-state visual evoked potential (SSVEP)-based communication: impact of harmonic frequency components. J Neural Eng. 2005; 2(4): 123–130. PubMed Abstract | Publisher Full Text\n\nMüller KR, Tangermann M, Dornhege G, et al.: Machine learning for real-time single-trial EEG-analysis: from brain–computer interfacing to mental state monitoring. J Neurosci Methods. 2008; 167(1): 82–90. PubMed Abstract | Publisher Full Text\n\nShalm LK, Meyer-Scott E, Christensen BG, et al.: Strong Loophole-Free Test of Local Realism. Phys Rev Lett. 2015; 115(25): 250402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStober S, Sternin A, Owen AM, et al.: Deep Feature Learning for EEG Recordings. ArXiv. 2016. Reference Source\n\nTressoldi P: EEG correlates of social interaction at distance. figshare. Fileset. 2016. http://www.doi.org/10.6084/m9.figshare.1466876.v8\n\nTressoldi P, Pederzoli L, Bilucaglia M, et al.: Brain-to-Brain interaction at a distance: a global or differential relationship? figshare. Fileset. 2018. http://www.doi.org/10.6084/m9.figshare.5132647.v7\n\nUrigüen JA, Garcia-Zapirain B: EEG artifact removal-state-of-the-art and guidelines. J Neural Eng. 2015; 12(3): 031001. PubMed Abstract | Publisher Full Text\n\nWalach H, Römer H: Generalized Entanglement - A Nonreductive Option for a Phenomenologically Dualist and Ontologically Monist View of Consciousness. In Studies in Neuroscience, Consciousness and Spirituality. Berlin, Heidelberg: Springer Science Business Media. 2011; 81–95. Publisher Full Text\n\nWalach H, Tressoldi P, Pederzoli L: Mental, behavioural and physiological nonlocal correlations within the Generalized Quantum Theory framework. Axiomathes. 2016; 26(3): 313–328. Publisher Full Text\n\nWinkler I, Haufe S, Tangermann M: Automatic classification of artifactual ICA-components for artifact removal in EEG signals. Behav Brain Funct. 2011; 7(1): 30. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "42945",
"date": "30 Jan 2019",
"name": "Arnaud Delorme",
"expertise": [
"Reviewer Expertise EEG 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\nThis article intends to show that machine learning methods might be able to pick on subtle EEG activity and perform classification above chance level and show true EEG correlation at a distance. The article is well written and clear.\nI was able to download the data and reproduce the results of experiment 2. I am not sure about the first experiment as the processing script for that experiment seems to be missing. The data processing, machine learning and cross-validation scheme seemed straightforward and appropriate. It is still possible that by trying many machine learning approaches, one could obtain positive results just by chance. This should be mentioned in the discussion as a potential limitation.\n\nThe most interesting result is that participants who are not exposed to stimuli nevertheless have a response to them that the classifier can pick up above chance expectations. One important caveat I have seen in the experimental design of experiment 2 is that the stimuli are not randomly intermixed. Instead there is a sequence of one stimulus type and then another one, etc. As subjects perform the experiment, their EEG changes (for example they might get tired as the experiment progresses which could affect their alpha brain waves). So one might have more alpha for one stimulus type simply because a given stimulus type is presented first. The classifier can then pick up on that. The sequence of stimuli should have been randomized for that reason. In other words, since stimuli of a given type are grouped, the classifier might simply pick up on the fact that some epochs belong to the beginning of the experiments, and some others belong to the end.\nIf my assessment is correct, I would remove the second experiment from the manuscript. Once I get the processing script for the first experiment, I would be able to assess potential pitfalls.\nAbstract: the abstract should mention statistical 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?\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": "4388",
"date": "31 Jan 2019",
"name": "Patrizio Tressoldi",
"role": "Author Response",
"response": "Sorry for missing the syntax for reproducing our machine learning analyses on the first dataset. We have just uploaded it \"MachineLearningSyntaxExperiment1\" on https://doi.org/10.6084/m9.figshare.5132647.v8We are also pleased you were able to reproduce our analyses on the dataset 2 and appreciated their appropriateness.As to your main concerns that the results observed in the participants who were not exposed to stimuli, were due to \"alpha activity of one stimulus type simply because a given stimulus type is presented first and they might get tired as the experiment progresses which could affect their alpha brain waves\", we consider this interpretation disputable given the following reasons: The stimulus types (10, 12, 14 Hz) were presented randomly. How is it possible to obtain a fair good level of correct classification of all three stimulus types with respect to the non-stimulation periods in the stimulated participants and in two out of three stimulus types in the non-stimulated participants? Let us suppose an almost equal distribution of the initial presentation of the three stimulus types among the 20 pairs of participants, how is it possible to discriminate their corresponding EEG activity with respect to the non-stimulation one if based on just the type of the first stimulus? In the non-stimulated participants we observed differences with respect to the non-stimulation periods only for the 10 and 14 Hz, more pronounced for the last one (see percentage of classification, effect size and Bayes Factor values). How is it possible to justify these differences postulating a common tiring effect?"
}
]
},
{
"id": "42948",
"date": "21 Mar 2019",
"name": "Schubert R. Carvalho",
"expertise": [
"Reviewer Expertise Machine Learning",
"Deep Learning",
"Brain-Computer Interface"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presented in this paper is challenging and can be fascinating; it aims to verify whether or not two participants are only connected mentally without using sensory organs. To check this connection, the authors decided to use a machine learning approach. They used a linear classifier and framed the problem as a classification task. The methodology was validated on two public datasets that both used EEG signals as a measure for brain activity. The methodology is simple to grasp and understand. The main steps of the methodology can be reproduced. However, it seems the authors get lost in the results section.\n\nThe study design can be improved to verify mental telecommunication (whether or not two brains are connected). Figures 1 and 2 show the stimulated and non-stimulated brains of the participants. One good comparison, was showing the differences between the brains at rest. If the goal is to verify if two brains are connected, one observation that is missing is the brain at rest vs. brain at stimulus for both groups. For example, if the two brains (two groups) are connected, then the classification between both should be random. The results shown in table 1 and 2 sound obvious. Table 1 shows the accuracy of the stimulated and non-stimulated brain (stimulated participants group), the classes show different patterns of activity, as measured by the high classification accuracy compared to the data of table 2. However, the comparison that is missing is between the data of the stimulated and non-stimulated subjects during the stimulation period. So, if the classification accuracy is at random, it suggests a brain connection. Mental telecommunication is the primary goal of this paper after all.\nThe same principle of analysis was done for dataset 2 (Results section: Second dataset). It is not possible to conclude mental telecommunication from this analysis, as shown in table 4. So, one interesting experiment that could be done to verify mental telecommunication would be to analyze the non-stimulated group data before and during the stimulation, to see if the mental states are separable as in the stimulated group. If this analysis is in the paper, it is not clear.\n\nIn the second paragraph of the discussion section, the authors argue that a modest classification accuracy of 50.74% allowed to distinguish it from the non-stimulation condition. From a machine learning point of view, this is considered a random classification. Moreover, this study (as it is in the moment) did not show evidence (machine learning results) for mental telecommunication — the same for the third paragraph (second dataset).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "4503",
"date": "25 Mar 2019",
"name": "Patrizio Tressoldi",
"role": "Author Response",
"response": "\"The study design can be improved to verify mental telecommunication (whether or not two brains are connected). Figures 1 and 2 show the stimulated and non-stimulated brains of the participants. One good comparison, was showing the differences between the brains at rest. If the goal is to verify if two brains are connected, one observation that is missing is the brain at rest vs. brain at stimulus for both groups. For example, if the two brains (two groups) are connected, then the classification between both should be random.\"Reply: what Figures 1 and 2 show, is precisely the spatial distribution of the features (extracted with the method described in the “Features selection” section) ranked by their discriminative power (r2 coefficients) for the stimulation versus the non-stimulation periods, in the stimulated (Figure 1) and the non-stimulated participants (Figure 2). The same information is presented in the Figures 3 and 4 with respect to the second dataset.We now have clarified this in the Figures’ captions.\"The results shown in table 1 and 2 sound obvious. Table 1 shows the accuracy of the stimulated and non-stimulated brain (stimulated participants group), the classes show different patterns of activity, as measured by the high classification accuracy compared to the data of table 2. However, the comparison that is missing is between the data of the stimulated and non-stimulated subjects during the stimulation period. So, if the classification accuracy is at random, it suggests a brain connection. Mental telecommunication is the primary goal of this paper after all.\"Reply: your suggestion that a random classification accuracy comparing the data of the stimulated and non-stimulated subjects during the stimulation period, should be the critical proof of a “brain-to-brain” connection at distance should be valid postulating a similar EEG activity in the stimulated and non-stimulated subjects. In the new paragraph “Experimental hypotheses” on page 5, we now have clarified that this effect is not possible because there cannot be a transmission or connection of the “physical” characteristics of the EEG activity trigger by the visual-auditory stimulations, between the stimulated and non-stimulated subjects, but only a still undefined type of information which weakly affect the EEG activity of the non-stimulated subjects.\"The same principle of analysis was done for dataset 2 (Results section: Second dataset). It is not possible to conclude mental telecommunication from this analysis, as shown in table 4. So, one interesting experiment that could be done to verify mental telecommunication would be to analyze the non-stimulated group data before and during the stimulation, to see if the mental states are separable as in the stimulated group. If this analysis is in the paper, it is not clear.\"Reply: we now have clarified that the data and the statistics reported in Tables are precisely those related to the comparison between the periods of stimulation and the interstimulus (no-stimulation) periods.\"In the second paragraph of the discussion section, the authors argue that a modest classification accuracy of 50.74% allowed to distinguish it from the non-stimulation condition. From a machine learning point of view, this is considered a random classification. Moreover, this study (as it is in the moment) did not show evidence (machine learning results) for mental telecommunication — the same for the third paragraph (second dataset).\"Reply: we agree that the marginal 0.74% difference, even if statistically different from the random classification, cannot be considered reliable (now added in the discussion).However, the differences observed in the second dataset, even if very modest according to the standards used in the machine learning community, it is sufficient to support the hypothesis that the EEG activity of the non-stimulated participants is in some way related to those of the stimulated one. As commented in the “Discussion” section, we think that this hypothesis can be further supported improving our machine learning algorithms. This is our optimistic opinion of course."
}
]
}
] | 1
|
https://f1000research.com/articles/8-43
|
https://f1000research.com/articles/8-9/v1
|
03 Jan 19
|
{
"type": "Research Article",
"title": "Economic valuation from direct use of mangrove forest restoration in Balikpapan Bay, East Kalimantan, Indonesia",
"authors": [
"Abubakar M. Lahjie",
"Bagus Nouval",
"Annisa Abubakar Lahjie",
"Yosep Ruslim",
"Rochadi Kristiningrum",
"Bagus Nouval",
"Annisa Abubakar Lahjie",
"Rochadi Kristiningrum"
],
"abstract": "Background: The mangrove forests in Balikpapan Bay, Indonesia, have been used as a source of livelihood for local community more than 150 years. Since the natural products of the mangrove forest, such as wood and seafood, are not able to meet the economic needs of the local community, some areas have been converted into brackish water ponds with traditional aquaculture systems. The growth of brackish water ponds over the last five decades has been identified as the main cause of ecosystem destruction. However, the mangrove ecosystem has been restored naturally through tidal action and seeds falling from mangrove trees. Methods: This study focused on the mangrove tree species Rhizophora apiculata, with ages ranging from 3 to 40 years. Initially, the study site (area, 1 ha) was plotted. The study sample size included 30% of the local population, chosen by systematic random sampling. The data collection was undertaken as follows: 1) measurement of the diameter and height of mangrove trees; 2) observation of local fish auctions; and 3) interviewing of fishers and local communities regarding the direct use of the natural products of the mangrove ecosystem. Results: It is suggested that the total income from wood production is IDR 742,425,000 year-1 or US $0.933 person-1 day-1. Furthermore, the total income from fishing is IDR 1,019,056,640 year-1 or US $1.28 person-1 day-1. Pre-thinning income level for wood harvesting is still low. The income difference between wood production and fishing resulted in the rate of overfishing reaching 37.3%. The highest observed wood production was reached at the age of 25 years, and the highest value of mean annual increment (MAI) is 5.39 m3 ha-1 at the age of 40 years. Conclusions: This study showed that tree thinning, ranging from 90 to 350 trees ha-1, can increase the value of MAI by around 24.5%.",
"keywords": [
"economic valuation",
"ecosystem services",
"direct use",
"mangrove restoration"
],
"content": "Introduction\n\nMangrove forest are one of the most productive ecosystems worldwide and are located in the brackish water zone of sub-tropical and tropical coastal regions. The ecosystem benefits provided by mangrove forests are not only limited to the provision of habitats for numerous kinds of seafood (including fishes, crustaceans, and molluscs), but also assist with nutrient recycling and soil conservation through sediment trapping1. Furthermore, the economic value of mangrove ecosystems, in terms of wood and seafood production, has provided a major source of income for local communities in coastal areas2. In tropical regions, some mangrove forests have largely disappeared3–6. The main factors that have caused the destruction of mangrove forests include urban development, development of brackish water ponds, freshwater flows diversion, over-cutting of trees for wood, and development by the local community such as brackish water ponds7–9. The destruction of mangrove forests has been the major cause of ecosystem loss in developing countries, and it is predicted that mangrove forests will disappear over the next 100 years in sub-tropical and tropical regions10.\n\nThere has been increasing awareness among government and local communities of the important role of natural ecosystems in protecting against floods, the reduction of coastal erosion and riverbanks, and in water quality management7,11,12. As a consequence, mangrove restoration programs have been proposed in coastal areas where local ecological knowledge has been adopted13. However, these restoration programs have not always been successful14,15. The failure of restoration programs has caused economic losses of millions of dollars. For example, mangrove restoration in the Philippines has only achieved a 10–20% long-term survival rate for reintroduced species due to inappropriate habitat selection16,17.\n\nES were first defined as the natural functions, consisting of the combination of soil, animals, plants, water, and air, that provide various benefits to society and thereby enhance quality of life for people18,19. This paper, in line with the Millennium Ecosystem Assessment20, defines ES as goods and services provided by ecosystems and their contributions to the sustenance of human well-being. The benefits of ES for agricultural production (food crops, seafood, medicine, and building materials), maintaining biodiversity (soil production, waste assimilation, and sources of clean water), public policy (microclimate and disease prevention), and intangible aesthetic and cultural benefits (education and recreational projects) are widely acknowledged in the literature21–27. Furthermore, the Millennium Ecosystem Assessment Board28 categories ES into four groups of services: 1) provisioning (food, freshwater, fuel, wood, and fibre); 2) regulating (disease prevention, water purification, and climate and flood regulation); 3) cultural (aesthetic, spiritual, education, and recreational); and 4) supporting (nutrient cycling, soil formation, and primary production). Although stakeholder groups, such as economists and local communities, depend significantly on the existence of ES for their wellbeing, they often neglect the important of ES. Thus, the value of ES is difficult to estimate29.\n\nThe purposes of this study are to: 1) analyses the production of wood of the Rhizophora apiculata; 2) identify the age of trees reached the highest increments of wood of the Rhizophora apiculata; 3) measure the highest value of mean annual increments (MAI) of the Rhizophora apiculata; and 4) analyse the economic valuation of direct used from the natural productions of mangrove forest in Balikpapan Bay.\n\n\nMethods\n\nBalikpapan Bay is a strategic port in the province of East Kalimantan (Figure 1). The bay area has 16,000 ha of water, and 156.836 ha of land. Balikpapan Bay has rapidly gained prominence domestically as one of the leading ports of Indonesia. Balikpapan City is now the primary trade and industry centre for mining, fishing, plantation, and forestry in East Kalimantan. With 3.2% population growth per year from 2010 to 2015, around 720,000 people live in Balikpapan. An estimated 108,200 people live in 54 sub-watersheds (more 22 villages) that drain into the bay. As consequence, development in Balikpapan Bay has caused significant ecosystem destruction—approximately 47.6% of the mangrove ecosystem has been lost, and mangrove forests have decreased by around 12.5% in last 15 years. Specifically, mangrove forests decreased from 19,428 ha in 2002 to 17,000 ha in 2017. Furthermore, there has been large-scale habitat destruction that has impacted some species on the endangered species lists, including proboscis monkey (Nasalis larvatus), dolphin (Orcaella brevirostris), saltwater crocodile (Crocodylus porosus), and dugong (Dugong dugon).\n\nThis figure has been reproduced with kind permission from Muliadi and co-authors31; Lahjie and co-authors32).\n\nFour systematically selected 50 m x 50 m plots are established in the study sites from which the increment of wood of the Rhizophora apiculata were examined. In particular, in the study sites, each two sampling plots located in different forest monitoring sites (300 m apart) are identified to measure the tree height and diameter for prior and after tree thinning treatment. The sample of this study are selected from 30% of tree population. The method of low thinning was adopted to remove trees which are below two cm from the average of tree diameter.\n\nThe principal stated that the rate of return earned on investment should be expressed in the nominal price of Indonesian currency (IDR) (including inflation). Thus, the rate of return is widely known as the nominal rate of return or the nominal interest rate. The principal was proposed by Klemperer30 as the following equation:\n\n\n\nWhere i is the inflated or nominal interest rate, In is the inflated or current (IDR) value in year n, n is the number of years in investment period, V0 is the initial value at the start of an investment period.\n\nThis study used both direct and indirect approaches to measure the incremental growth of mangrove tree from 2001 to 2018, with trees ranging in age between 3 to 20 years.\n\nFor the direct approach methods, this study carried out the following. 1) Measured the diameter and height of mangrove trees to examined biophysical condition of mangrove trees. 2) Observed local fish auctions. 3) Interviewed fishermen and local community members in person at their home in the village and the coastal zone when they catch fishes. Those included must have lived with their family in the community of Kariangau village for more than three decades and made a living as the fisherman for 15 years. This study eliminated fishermen who are categoried as new members of community in Kariangau village, who typically have lived in the village for less than 5 years and work as factory workers in Balikpapan bay, meaning catching fish is not their main income source (under 20% of total income). The topic of interview focuses on the direct use of the natural products of the mangrove ecosystems (e.g., wood and seafood). 4) Undertaken a review of the literature relating to the description of mangrove ecosystem, the restoration of mangroves, and the natural products of mangrove ecosystems in Balikpapan Bay.\n\nThree kinds of observations were made at the fish auction: 1) identify fish species offered; 2) identify the total of sales and the price per kilogram; 3) examine the mechanism of fish auction. The period of observation for sea food production, from fish catch to fish offered in the auction, has been done from April to November 2017.\n\nThe data is collected through interview and questionnaire with fisherman in Balikpapan bay, which adopted the technique of accidental and snowball sampling. A copy of the questionnaire is provided on OSF. The period when interviews were conducted was from April to November 2017. The population of fisherman located in the coastal zone of Kariangau village are 40 fishermen. The selected sample is 30 of 40 fishermen population (75%), since they are active fishermen during the interview period. For the indirect approach, government documents regarding the strategic plan in the management of coastal zone in Balikpapan bay has been used as references to present the condition of mangrove forests in Balikpapan bay.\n\nThe area utilised for fisheries in mangrove forest covers 300 ha and is located on the border area between Kariangau Village and Batu Ampar Village. The area utilized for fisheries is located at 01° 12′ 50.5″ S, 116° 49′ 26.8″ E. In this study, an exchange rate of US$ 1 = IDR 13,300 has been used, with data supplied by the Indonesian central bank, Bank Indonesia (2018, February).\n\nThis study was approved by the general research of Mulawarman University ethics board (approval number 208-41/KL/2017), and each participant gave their written informed consent.\n\nThis study used Microsoft Excel to perform calculations and generate graphs. As Van Gardingen33 argued, there are two proxies that can be adopted to measure the wood production of Rhizophora apiculata. The initial proxy for the wood production of Rhizophora apiculata, mean annual increment (MAI), is formulated using the total standing volume divided by tree age. The second proxy, period annual increment (PAI), is the absolute difference between total standing volume at age t and age t-1, scaled by the time interval between each measurement age. Both MAI and PAI have been used as proxies for wood production by Lahjie32 and Winarni34. The MAI formula is represented as:\n\n\n\nWhere MAI is the mean annual increment, Vt is the total standing volume at age t, and t is the tree age.\n\nThe PAI formula is represented as:\n\n\n\nWhere PAI is the period annual increment, Vt is the total standing volume at age t, Vt-1 is the total standing volume at age t-1, and t is the time interval between each measurement age.\n\nIn examining a single statistical series of R (range) with involved N (sample), the optimal class interval (C) could be approximated using the formula proposed by Sturges35. The formula represents the class interval for measures of the means, dispersion, coefficient of variation, and skewness of the frequency distribution. This can provide the proper distribution into classes for entire numbers, which are powers of two, by a series of binomial coefficients. The Sturges formula is:\n\n\n\nTo analyse the net income of fishers, this study collects data from the population of 40 households (164 people) whose livelihood depends on the natural productivity of the mangrove forests in Balikpapan Bay. Using accidental and snowball sampling method, 30 household (123 people) were further selected for inclusion in the dataset (see Table 1, below).\n\nNet income class, million IDR; median income, million IDR; total net income (million) IDR; total net income US$ person-1 day-1; IDR, Indonesian rupiah. 1 US$ = IDR 13,300.\n\n\nResult and Discussion\n\nAll raw data generated in this study are available on OSF37.\n\nTable 1 shows the result of the net income of the fishers based on six class of net income in Balikpapan bay. Table 1 shows that the fishers in Balikpapan Bay have varied net household incomes, which can be divided into six classes. The variability in net household income was influenced by fishing boat capacity, quality of equipment, and number of available working days per year for fishing. The majority of fishers (40 people) had net annual incomes ranging from IDR 25 million to IDR 28 million, with a total net annual income of IDR 212 million. The median net annual income was IDR 26.5 million. The net income per person per day was IDR 17,024 (or US$1.28). The median of the lowest net income class (9 people) was IDR 14.5 million with a total net income of IDR 43.50 million. The net income per person per day was IDR 13,170 (or US$0.99). The median of the highest net income class (12 people) was IDR 34.5 million with a total net income of IDR 138 million. The net income per person per day was IDR 31,525 (or US$ 2.37).\n\nAs Nurmanaf36 observed, the levels of household income ranged from the first class, which was categorised as low income with a net household income of under US$1 per person per day, to the sixth class, which was categorised as high income with a net household income of more than US$1.7 per person per day. It can be concluded that 7% of fishers in Balikpapan Bay were categorised as having low level household income, 83% had a middle level household income, and 10% had a high level household income. In this study, it was calculated that the potential higher income that can be made from fishing, compared with harvesting wood, has resulted in the rate of overfishing reaching 37.3%.\n\nTable 2 and Figure 2 shows the relationship between net household income, cost of operation, and nominal rate of return (NRR) per month for fishers in Balikpapan Bay, as shown in table below. Table 2 and Figure 2 show that the total of cost operation that produce highest rate of NRR (11.4%) is IDR 10 million with a net benefit of IDR 26.5 million. This rate is high, compared to the short-term rate of return from the Indonesian Central Bank through the credit card bank rate of 3.5% per month.\n\nNet household income years-1 (million) IDR; cost years-1, (million) IDR; NRR, nominal rate of return, %.\n\nTable 3 and Figure 3 shown the wood potential of mangrove forests for Rhizophora apiculata ranging in age from 3 to 35 years, as determined by the simulation described. Table 3 shows that the highest growth increment of wood production was reached at the age of 25 years, and the highest value of MAI was 5.39 m3 per hectare.\n\nThe number of mangrove trees decline gradually because of natural plant death.\n\nYear, Age of trees; N, Population of Mangrove (trees ha-1); D, Tree Diameter (cm); H, Branch-free Height (m); F, Trees Form Factor; TV, Total Volume (m3 ha-1); MAI, Mean Annual Increment (m3 ha-1 year-1); CAI, Current Annual Increment (m3 ha-1 year-1).\n\nThe restoration of mangrove forests in Balikpapan Bay produced a MAI for Rhizophora apiculate of 185.07 m3 h-1 for trees aged 40 years due to the high density of the mangrove trees. This result shows that the restoration of mangrove forests may produce a high value of ecosystem services for local communities. However, the optimal ecosystem service value will likely be achieved when the mangrove forests produces a MAI of 229.50 m3 h-1 for trees aged 40 years. Therefore, it is necessary to adopt tree thinning or tree clearing practices, thinning forest plots by 20% to 40% (Table 4).\n\nYear, Age of trees; N, population of mangrove (trees ha-1); D, tree diameter (cm); H, branch-free height (m); F, trees form factor; TV, total volume (m3 ha-1); MAI, mean annual increment (m3 ha-1 year-1); CAI, current annual increment (m3 ha-1 year-1).\n\nWhen measuring the benefits of ecosystem services, this study assumed that the net income per person income before tree thinning was less than US$1, while the net income per person after tree thinning was more than US$2. It is this low, pre-thinning income level for wood harvesting that has led to over-fishing, since fishing currently offers a higher income. Furthermore, the table shows that although the MAI for of 25-year-old trees was higher than that of trees aged 40 years (6.71 and 5.74, respectively), the total volume of 40-year-old trees was higher than that of 25-year-old trees (229.50 and 167.77, respectively).\n\n\nConclusions\n\nThis study has provided some important conclusions regarding the restoration of mangrove forest in Balikpapan Bay. The study sampled 40 households (164 people) whose livelihood depends on the natural productivity of the mangrove forests in Balikpapan Bay, and additional 30 households (123 people) that were chosen via the accidental and snowball sampling method. The study showed that the total income from wood products is IDR 742,425,000 year-1 or US $ 0.933 person-1 day-1 (300 ha × 98.99 × IDR 500,000, over 20 years). Furthermore, the total income from fishing is IDR 1,019,056,640 or US $ 1.28 person-1 day-1. Wood production provides a higher income to local community higher than fishing. Specifically, the income differences between wood production and fishing resulted in the rate of overfishing reaching 37.3%. The highest wood production was observed with 25-year-old trees, and the highest value of MAI was 5.39 m3 ha-1 for 40 years old trees. The results also suggest that tree thinning ranging, from 90 to 350 trees ha-1, can increase the MAI value by around 24.5%.\n\n\nData availability\n\nRaw data associated with this study, including all de-identified questionnaires and raw data from thinning experiments, are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/C6HFK37.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank Dharman, a local public figure, for his valuable information. We are also grateful to Umbar Sujoko for his help in creating the map of study site.\n\n\nReferences\n\nBarbier EB, Hacker SD, Kennedy C, et al.: The value of estuarine and coastal ecosystem services. Ecol Monogr. 2011; 81(2): 169–93. Publisher Full Text\n\nFood and Agriculture Organization of the United Nations: The world’s mangroves 1980-2005. FAO Forestry Paper 1, 2007; 1–6. Reference Source\n\nBlasco F, Aizpuru M, Gers C: Depletion of the mangroves of Continental Asia. Wetl Ecol Manag. 2001; 9(3): 255–66. Publisher Full Text\n\nGiri C, Ochieng E, Tieszen LL, et al.: Status and distribution of mangrove forests of the world using earth observation satellite data. Glob Ecol Biogeogr. 2011; 20(1): 154–9. Publisher Full Text\n\nMijan-Uddin SM, Hoque AT, Abdullah SA: The changing landscape of mangroves in Bangladesh compared to four other countries in tropical regions. J Forestry Res. 2014; 25(3): 605–611. Publisher Full Text\n\nOkpiliya FI, Udida AA, Oka P: Effect of Timber Resource Processing on the Edibe-Edibe Creek in Calabar South Local Government Area of Cross River State, Nigeria. International Journal of Physical and Human Geography. 2013; (1): 10–17. Reference Source\n\nSaenger P, Hegerl EJ, Davie JDS: Global status of mangrove ecosystems. Environmentalist. 1983; 3(3): 1–88. Reference Source\n\nValiela I, Bowen JL, York JK: Mangrove Forests: One of the World's Threatened Major Tropical Environments: At least 35% of the area of mangrove forests has been lost in the past two decades, losses that exceed those for tropical rain forests and coral reefs, two other well-known threatened environments. Bioscience. 2001; 51(10): 807–815. Publisher Full Text\n\nAlongi DM: Present State and Future of the World's Mangrove Forests. Environ Conserv. 2002; 29(03): 331–349. Publisher Full Text\n\nDuke NC, Meynecke JO, Dittmann S, et al.: A world without mangroves? Science. 2007; 317(5834): 41–42. PubMed Abstract | Publisher Full Text\n\nEwel KC, Twilley RR, Ong JE: Different Kinds of Mangrove Forests Provide Different Goods and Services. Global Ecol Biogeogr. 1998; 7(1): 83–94. Publisher Full Text\n\nMoberg F, Ronnback P: Ecosystem services of the tropical seascape: Interactions, substitutions and restoration. Ocean Coast Manag. 2003; 46(1–2): 27–46. Publisher Full Text\n\nLahjie AM, Isminarti, Simarangkir BD: Community forest management: Comparison of simulated production and financial returns from agarwood, tengkawang and rubber trees in West Kutai, Indonesia. Biodiversitas. 2018; 19(1): 126–133. Publisher Full Text\n\nElster C: Reasons for reforestation success and failure with three mangrove species in Colombia. For Ecol Manage. 2000; 131(1–3): 201–214. Publisher Full Text\n\nLewis RR: Ecological engineering for successful management and restoration of mangrove forests. Ecol Eng. 2005; 24(4): 403–418. Publisher Full Text\n\nPrimavera JH, Esteban JMA: A review of mangrove rehabilitation in the Philippines: successes, failures and future prospects. Wetl Ecol Manag. 2008; 16(5): 345–358. Publisher Full Text\n\nSamson MS, Rollon RN: Growth performance of planted mangroves in the Philippines: revisiting forest management strategies. Ambio. 2008; 37(4): 234–240. PubMed Abstract | Publisher Full Text\n\nKing RT: Wildlife and man. New York Conservationist, 1966; 20: 8–11.\n\nHelliwell DR: Valuation of wildlife resources. Regional Stud. 1969; 3(1): 41–47. Publisher Full Text\n\nMillennium Ecosystem Assessment Board: Ecosystems And Human Well-being. A Framework For Assessment. Washington, DC, 2005. Reference Source\n\nBengtsson J: Which species? What kind of diversity? Which Ecosystem function? Some Problems in studies of relations between biodiversity and ecosystem function. Appl Soil Ecol. 1998; 10(3): 191–199. Publisher Full Text\n\nDaily GC: Valuing And Safeguarding Earth’s Life Support Systems. Island Press, Washington, DC, 1997; 365–374. Reference Source\n\nCostanza R, Folke C: Valuing Ecosystem Services With Efficiency, Fairness And Sustainability As Goals. Island Press, Washington, DC, 1997; 49–70. Reference Source\n\nKing DM, Wainger LA, Bartoldus C, et al.: Expanding Wetland assessment procedures: linking indices of wetland function with services and values. Wetland Research Program, Washington, DC, USA. 2000. Reference Source\n\nDe Groot RS, Wilson MA, Boumans RMJ: A Typology for the classification, description and valuation of ecosystem functions, goods and services. Ecol Econ. 2002; 41(3): 393–408. Publisher Full Text\n\nBanzhaf BA: What Are Ecosystem Services? Resources For the Future. 2007.\n\nWallace KJ: Classification Of ecosystem services: problems and solutions. Biol Conserv. 2007; 139(3–4): 235–246. Publisher Full Text\n\nMillennium Ecosystem Assessment Board: Ecosystems And Human Well-being: Current State And Trends. Island Press, Washington, 2005; 1. Reference Source\n\nDaily GC, Alexander S, Ehrlich PR, et al.: Ecosystem Service: Benefits Supplied To Human Societies By Natural Ecosystems. Island Press. 1997. Reference Source\n\nKlemperer WD: Forest resource economics and finance. McGraw-Hill Inc. 2003. Reference Source\n\nMuliadi M, Lahjie AM, Simarangkir BDAS, et al.: Bioeconomic and environmental valuation of dipterocarp estate forest based on local wisdom in Kutai Kartanegara, Indonesia. Biodiversitas. 2017; 18(1): 401–408. Publisher Full Text\n\nLahjie AM, Simarangkir BDAS, Kristiningrum R, et al.: Financial analysis of dipterocarp log production and rubber production in the forest and land rehabilitation program of Sekolaq Muliaq, West Kutai District, Indonesia. Biodiversitas. 2018; 19(3): 757–766. Publisher Full Text\n\nVan Gardingen PR, McLeish MJ, Philips PD, et al.: Financial and ecological analysis of management options for logged-over dipterocarp forests in Indonesia Borneo. For Ecol Manag. 2003; 183(1–3): 1–29. Publisher Full Text\n\nWinarni B, Lahjie AM, Simarangkir BDAS, et al.: Tengkawang cultivation model in community forest using agroforestry systems in West Kalimantan, Indonesia. Biodiversitas. 2018; 18(2): 765–772. Publisher Full Text\n\nSturges HA: The Choice of a Class Interval. J Am Stat Assoc. 2012; 21(153): 65–66. Publisher Full Text\n\nNouval B, Lahjie AM, Ruslim Y, et al.: Economic Valuation from Direct Use of Mangrove Forest Restoration in Balikpapan Bay, East Kalimantan, Indonesia. OSF. 2018. http://www.doi.org/10.17605/OSF.IO/C6HFK\n\nNurmanaf AR: Rural Household Income and Expenditure and Its Relation to Poverty Rate. Soca (Socio-Economic of Agriculture And Agribusiness). 2014; 1–12."
}
|
[
{
"id": "42539",
"date": "29 Jan 2019",
"name": "William L. Hargrove",
"expertise": [
"Reviewer Expertise Environmental Science",
"Water Quantity and Quality"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 focuses on mangrove forests, an important ecosystem in coastal areas. The study focuses on the economic value of wood production and also of fish production in brackish water ponds, which, along with overharvesting, contribute to the destruction of the mangrove ecosystem. Nonetheless some mangrove ecosystems have been restored through natural reseeding. The study provides interesting economic data on the value of mangrove forests. The impacts of thinning and also of restoring mangrove are assessed in economic terms, demonstrating that thinning can increase the economic value by about 25%. The economic value of fishing is also quantified. The greater economic value of fishing as measured in the daily income also contributes to overfishing.\n\nMy criticism of this paper is that the authors also discuss \"ecosystem services\", but seem to equate ecosystem services with the economic value of the wood or fish harvested. I consider ecosystem services to include other impacts related to water quality, habitat for flora and fauna, and other environmental impacts that are difficult to monetize. These are not really considered, only the economic value of wood production. Thus the authors should limit their discussion to the “economic services” of mangrove forests, and their contribution to the livelihoods of the local people. This, of course, does not diminish the importance of the mangrove forests as an economic resource, but evaluation of ecosystem services would require a different approach to evaluating all the environmental benefits of mangrove forests.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4406",
"date": "12 Mar 2019",
"name": "Bagus Nouval",
"role": "Author Response",
"response": "I agree with the suggestion from the referee. Since assessing ecosystem services provided ecological, sociocultural and economic human benefits, and monetary are essential, this research focused on examining the direct use of the natural products of the mangrove ecosystem in order to meet economic needs of the local community in Balikpapan bay, Indonesia. Identifying various mangrove ecosystem services, including wood production and fishing, which is consumed and sold at the local auction by local community leads this research to adopt economic value. Previous studies have adopted the economic value to evaluate ecosystem services. Therefore, the definition of ecosystem services in our manuscript will be limited on the economic services of mangrove ecosystem."
}
]
},
{
"id": "43850",
"date": "06 Feb 2019",
"name": "Roni Bawole",
"expertise": [
"Reviewer Expertise Coastal 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\nIntroduction:\nThe concepts of ES and VE should be introduced briefly. Please explain what they mean and how they are defined related to mangrove forests in order to assess fishery activities.\n\nWhat is the state of the knowledge of this research? From what I see I have found this research only reports what has been done by the project. So I suggest that the authors should be able to convey the novelty of this research. What's different about VE in Kalimantan from VE in Indonesia or elsewhere in the world?\n\nMaterials and methods:\nPlease give more information about fishers, for example of their activities. Do they do one day fishing or not, and whether being fishermen is their main job, like many fishermen in Indonesia? This time is allocated for fishing for one week, month or year.\n\nDiscussion:\n\nThe authors should also be able to discuss the relationship of mangroves (Rizophora apiculata) with fisheries production based on location. Does low area abundance provide low fisheries production in areas with Rizaphora? This is seen from the EV approach.\n\nConclusions:\nConclusions should be corrected or revised. I see that the current conclusion is still a summary of the methods and results and discussion. Summarize what has been learned and why it is interesting and useful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4417",
"date": "12 Feb 2019",
"name": "Bagus Nouval",
"role": "Author Response",
"response": "Introduction: 1. The definition of ES has been described in the manuscript (refer to p.3), but we will add the definition of EV in the manuscript. Economic valuation (EV) is a quantitative assessment that is able to present the different range of values based on the specific method used, but it may allow fishermen to make decisions according to utilized goods and services provided by mangrove ecosystems.2. The study is novel in that it examines the economic benefits of mangrove ecosystems. Since the mangrove is in the primary trade and industry centers, there is a conflict of interest in land-used between huge companies (e.g., mining, shipping, and cement) and local communities that are economically dependent (e.g., fishermen).Materials and methods:We already presented the criteria of fishermen activities in the manuscript at the section of data collection (refer to p. 4).Discussion:The relationship of mangrove trees (Rizophora apiculata) and fishermen production is already discussed in the manuscript at the section of result and discussion (refer to p.5). The economic value produced by fishing is higher than harvesting wood of mangrove trees which is caused by overfishing. Conclusions:Since the mangrove is located in the primary trade and industry centers, there is a conflict of interest in land-used between huge companies (e.g., mining, shipping, and cement) and local communities that are economically dependent (e.g., fisherman). This issue becomes an interesting topic to be analyzed."
}
]
}
] | 1
|
https://f1000research.com/articles/8-9
|
https://f1000research.com/articles/8-10/v1
|
03 Jan 19
|
{
"type": "Research Article",
"title": "Selection and regeneration of purple sweet potato calli against drought stress simulated by polyethylene glycol",
"authors": [
"Widi Sunaryo",
"Darnaningsih Darnaningsih",
"Nurhasanah Nurhasanah",
"Darnaningsih Darnaningsih",
"Nurhasanah Nurhasanah"
],
"abstract": "Background: Water shortage due to natural and/or technical drought stress, widespread throughout Sumatra, Java, Sulawesi and Kalimantan islands, significantly reduces crop production. The development of varieties tolerant to drought stress is important since it is more effective rather than improving irrigation infrastructure to increase the sweet potato productivity. Methods: Selection and regeneration experiments assessing purple sweet potato callus tolerance of drought stress, simulated by polyethylene glycol (PEG), were conducted to generate new variant plants tolerant of drought stress. Sterile explants (leaf and petiole) generated from previous in vitro culture were inoculated to the Murishage and Skoog (MS) medium containing plant growth regulator combination as treatments to induce calli. The calli were then transferred to half-MS medium containing 0, 5, 10, 15 and 20% PEG as selection agent for drought tolerance. The surviving calli were regenerated in the MS medium containing 0, 0.5, 1 or 1.5 mg l-1 6-benzylaminopurine (BAP). The callus formation, growth and survivability during in vitro culture were measured. Results: Calli were successfully formed in almost all media containing 2,4-Dichlorophenoxyacetic acid (2,4-D ) with the concentration of 1, 2, 3 and 4 mg l-1 and BAP (concentration: 0.5 and 1 mg l-1), but the medium of MS + 2 mg l-1 2,4-D + 0.5 mg l-1 BAP resulted in the highest number of induced calli per treatment (mean=11.36), with the percentage of responsive explants standing at around 96%. The higher the concentration of PEG, the lower the number of surviving calli. At 20% PEG, only 54.42% calli survived. There were five plants successfully regenerated from the survived calli at 20% PEG, using MS medium containing 1.5 mg l-1 BAP. Conclusions: The experiment has successfully produced putative drought-tolerant plants by callus screening using PEG as drought-tolerance-selecting agent in purple sweet potato.",
"keywords": [
"Callus formation",
"purple sweet potato",
"drought tolerance",
"Polyethylene Glycol",
"in vitro selection"
],
"content": "Introduction\n\nDrought stress is a major limiting factor in increasing the production of several important crops in a large number of regions in Indonesia. The effect of drought stress on plant growth is largely determined by the amount of stress the plant is exposed to and the growth phase the plant was in during drought stress. In addition, drought stress causes inhibition of plant growth and plant roots1,2 and as a consequence, plants will grow slowly3 and their productivity is severely reduced4. Development of cultivated plants tolerant to drought stress, including sweet potato, is an important approach to solving water shortage issues5.\n\nIn vitro selection is a breeding strategy widely used to produce new variant plants that are resistant or tolerant to disease, herbicides or extreme environmental stresses, including drought stress6. This method selects genetic variation arising from tissue culture processes especially produced from natural or artificial mutation7. The rapid multiplication of undifferentiated cells during callus formation increases the possibility of natural mutation due to rapid cell division8. Such genetic changes are subsequently selected as useful traits in breeding programs.\n\nPolyethylene glycol (PEG) solution can be used as drought-tolerant selecting agent in soybean9 and other plants. PEG is able to control the decrease of water potential homogeneously, therefore it can mimic the potential of groundwater10. The long-term use of PEG will not cause cell damage, because PEG has molecular weight of more than 6000 g/mol that cannot be absorbed into plant tissues11. This research attempted to produce new variant of purple sweet potato tolerant to drought stress via in vitro selection using PEG as a selection agent.\n\n\nMethods\n\nYoung leaves and petioles from previous in vitro-generated plants grown using standard Murishage and Skoog (MS) medium12 were used as explants. The intact leaf were sliced into 1 cm2 of lamina and 1 cm length of petiole. The explants were then inoculated to the MS containing plant growth regulator of 2,4-dichlorophenoxyacetic acid (2,4-D) (Merck, Cat. No.31518, Germany) combined with 6-benzylaminopurine (BAP, Merck, Cat. No. B3408, Germany) to induce calli. The explants were grown and placed at a sterilized 600 ml bottle containing around 20 ml solidified medium for a month at the culture room at a temperature of 25±2°C. The composition of the treatments were Z1 (MS + 0 mg L-1 2,4-D + 0 mg L-1 BAP), Z2 (MS + 1 mg L-1 2,4-D1 + 0.5 mg L-1 BAP), Z3 (MS + 2 mg L-1 2,4-D1 + 0.5 mg L-1 BAP), Z4 (MS + 2 mg L-1 2,4-D1 + 1 mg L-1 BAP), Z5 (MS + 3 mg L-1 2,4-D1 + 0.5 mg L-1 BAP), Z6 (MS + 3 mg L-1 2,4-D1 + 1 mg L-1 BAP), Z7 (MS + 4 mg L-1 2,4-D1 + 0.5 mg L-1 BAP), and Z8 (MS + 4 mg L-1 2,4-D1 + 1 mg L-1 BAP). All treatments were replicated five times. In total there were two types of explants x eight treatments x five replication x five explants per replication/bottle = 400 experiment units.\n\nThe green, fresh and compact calli produced at the first experiment were selected and transferred to MS medium with half the usual level of nutrients containing 0, 5, 10, 15 and 20% PEG as selection agent medium of drought tolerance. All treatments were replicated three times. In total there were five treatments x three replications x four explants per replication/bottle = 60 experiment units. The surviving calli in the PEG selection-agent medium were regenerated in MS medium containing 0, 0.5, 1 or 1.5 mg L-1 BAP to induce shoots and roots. The growth variables during in vitro culture, such as the number of calli induced per treatment, percentage of responsive explants to induce calli, percentage of surviving calli under drought stress simulated by polyethylene glycol, and number of regenerated plants, were observed.\n\n\nResults and Discussion\n\nAll media applied in the experiment could successfully induce callus formation except the basal medium containing no growth regulator (control). The existence of 2,4-D and BAP in various concentration combination was able to induce callus formation but the number of callus per explant and the percentage of responsive explant were varied (Table 1, Figure 1A, B). The highest number of induced calli per treatment was observed using the Z3 treatment medium. These results were observed using both leaf and petiole (Figure 1A, B). Callus formation was successfully induced in sweet potato using both explants13,14. The use of 2,4-D to induce callus formation is common13,15. Callus formation can be induced by other plant growth regulators such as zeatin, or 1-naphthaleneacetic acid combined with gibberellin (GA3)14,16. These calli emerged and was developed from the mesophyll cells in the leaf and protoplast from the stem or petiole13,14.\n\nValues shown are mean ± standard deviation unless indicated.\n\nMS, Murishage and Skoog medium; BAP, 6-benzylaminopurine.\n\n(A) Callus induction from leaf explants (black arrow shows irresponsive explant; green arrow shows the responsive explant). (B) Callus induction from petiole explants (black arrow shows unresponsive explant; green arrow shows the responsive explant). (C) Callus selection for drought tolerant using polyethylene glycol (black arrow shows dead explant; green arrow shows the survived explant). (D) Callus regeneration (green arrow shows the initiated shoot from the callus).\n\nThe fresh, green and compact calli produced in the first experiment was transferred to the drought stressed medium simulated by MS medium containing different concentration of PEG to discover the putative drought tolerant calli of purple sweet potato. At least one callus survived in all PEG-containing medium, but the survival rate was varied (Table 2, Figure 1C). The higher the concentration of the PEG in the medium the lower survival rate of the calli in the selection medium. This indicates that drought stress caused by PEG simulation affects callus survivability and can be used as drought-tolerant selection medium, as also reported in other crop plants such as soybean9,17–20, tobacco21, grass22, rice23 and chili24. The putative drought-tolerant plants are screened from regenerated plants derived from the survived calli in the highest concentration of PEG. There was no significant different response between calli derived from the leaf and petiole (Table 2).\n\nThe surviving calli grown in medium containing 40% PEG were incubated in the regeneration medium to initiate shoot and/or root. The only medium MS containing 1.5 mg L-1 BA can successfully induce shoot and root growth (Table 3, Figure 1D). This indicates that the calli exposed with high concentration of PEG are still viable to regenerate into whole plant. The results increase the possibility to get a new purple sweet potato variant tolerant to drought stress as also reported in soybean19,20.\n\nMS, Murishage and Skoog medium; BAP, 6-benzylaminopurine.\n\nThe raw data for calli and explant growth are available on OSF25.\n\n\nConclusions and outlook\n\nInitial production of putative drought tolerant plants by callus screening using PEG as drought tolerant-selecting agent in purple sweet potato was successfully done in this experiment. Callus was successfully induced in MS medium containing many different combinations of 2,4-D and BAP. Drought-tolerant screening using PEG is generally effective since there was a strong indication that the increase of PEG concentration caused the reduction of the callus survivability. However, the high percentage of surviving calluses at the highest concentration of PEG (40%) may indicate that the drought stress applied in the experiment was not apparently sufficient to induce cell mutation. Therefore, a future experiment using higher concentrations of PEG could provide more tolerant calli and increase genetic mutations, especially with regards to drought tolerance. Genetic evaluation and field experiments using water shortage treatment experiments will be the next investigation to clarify the genetic mutations involved and the stability of the drought-tolerance characteristics.\n\n\nData availability\n\nThe raw data on the number of calli formed per bottle for different treatment conditions and subsequent explant growth are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/DEGVY25.",
"appendix": "Grant information\n\nThe authors declare that no grants were involved in funding this work.\n\n\nReferences\n\nHamim: Drought tolerance of soybean: morphology and physiology approach. [M.Sc.-Thesis]. Bogor Agricultural University, Bogor. [Indonesian]. 1995.\n\nJones HG: Plants and microclimate: a quantitative approach to environmental plant physiology. second edition. Cambridge University Press, Cambridge. 1992. Reference Source\n\nHassan NS, Shaaban LD, Hashem ESA, et al.: In vitro selection for water stress tolerant callus line of Helianthus annus L. Cv. Myak. Int J Agri Biol. 2004; 6(1): 1–18. Reference Source\n\nArvin MJ, Donnelly DJ: Screening potato cultivars and wild species to abiotic stresses using an electrolyte leakage bioassay. J Agric Sci Technol. 2008; 10: 33–42. Reference Source\n\nTuinstra MR, Ejeta G, Goldbrough P: Evaluation of near-isogenic sorghum lines contrasting for QTL markers associated with drought tolerance. Crop Sci. 1998; 38(3): 835–842. Publisher Full Text\n\nIgnacimuthu S: Plant Biotecnology. Science Publishers, inc. 1997; 284.\n\nLarkin PJ, Scowcroft WR: Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor Appl Genet. 1981; 60(4): 197–214. PubMed Abstract | Publisher Full Text\n\nPhillips GC, Hubstenberger JF, Hansen EE: Plant regeneration from callus and cell suspension cultures by somatic embryogenesis. In: Gamborg OL, Phillips GC (eds). Plant cell, tissue and organ culture. Springer Lab Manual. Springer, Berlin, Heidelberg. 1995. Publisher Full Text\n\nSunaryo W, Widoretno W, Aswidinnoor H, et al.: Efectiveness of poly-ethylene glycol .PEG to characterize the soybean genotype responses against drought stress. Frontier. 2005; 19(2): 29–35. [Indonesian].\n\nMichel BE, Kauffmann MR: The osmotic potential of polyethylene glycol 6000. Plant Physiol. 1973; 57(5): 914–916. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHardegree SP, Emmerich WE: Seed Germination Response of Four Southwestern Range Grasses to Equilibration at Subgermination Matric-Potentials. Agron J. 1992; 84(6): 994–998. Publisher Full Text\n\nMurashige T, Skoog F: A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol Plant. 1962; 15(3): 473–497. Publisher Full Text\n\nMurata T, Hoshino K, Miyaji Y: Callus formation and plant regeneration from petiole protoplast of sweet potato, Ipomoea batatas (L.) LAM. Japanese J Breed. 1987; 37(3): 291–298. Publisher Full Text\n\nYoshinaga M, Komaki K: Formation of calli and adventitious roots from mesophyll- and embryogenic callus-derived protoplasts of sweet potato, Ipomoea batatas (L.) Lam. Bulletin of the Kyushu National Agricultural Experiment Station. 1993; 28(1): 17–28. Reference Source\n\nRahman MM, Sultana RS: Sweet potato (Ipomoea batatas L.): suspension culture for cell aggregation. J Plant Sci Res. 2017; 4(2): 1–5. Reference Source\n\nBendif H, Adouni K, Boudjeniba M: The effect of growth regulators and explants on callus induction in four cultivars of potato (Solanum tuberosum L.). J Biores Val. 2017; 02(1): 34–41. Reference Source\n\nWidoretno W, Guharja E, Ilyas S, et al.: Effectiveness of polyethylene glycol for evaluating response of soybean at germination stage against drought stress. Hayati. 2002; 9(2): 33–36.\n\nWidoretno W, Megia R, Sudarsono, et al.: Responses of soybean somatic embryos to polyethylene glycol and its uses for in vitro selection of soybean against drought stress. Hayati. 2003; 10(4): 134–139.\n\nWidoretno W, Arumingtyas EL, Basuki N, et al.: Drought resistance selection on soybean somaclonal variants. J Basic Appl Sci Res. 2012; 2(8): 7994–7997. Reference Source\n\nSunaryo W, Widoretno W, Nurhasanah, et al.: Drought tolerance selection of soybean lines generated from somatic embryogenesis using osmotic stress simulation of poly-ethylene glycol (PEG). Nus Biosci. 2016; 8(1): 45–54. Publisher Full Text\n\nKrishnasany V, Irullapan I: Germination response to water stress in the seeds of hot pepper and eggplant genotypes. In Proceedings: Adaptation of food crops to temperature and water Stress. AVRDC, Taipei. 1993. Reference Source\n\nMullayhey JJ, West SH, Cornell JA: Effects of simulated drought by polyethylene glycol on bahiagrass germination. Seed Sci Tech. 1996; 24: 219–224. Reference Source\n\nPerez-Molphe-Balch E, Gidekel M, Seguro-Nieto M, et al.: Effects of water stress on plant growth and root proteins in three cultivars of rice (Oryza sativa) with different levels of drought tolerance. Physiol Plant. 1996; 96(2): 284–290. Publisher Full Text\n\nKomori T, Myers PN, Yamada S, et al.: Comparative study of the Nicotiana species with respect to water deficit tolerance during early growth. Euphytica. 2000; 116(2): 121–130. Publisher Full Text\n\nSunaryo W, Nurhasanah: Raw Data Drought Tolerant for Sweet Potato-Revision. OSF. 2018. http://www.doi.org/10.17605/OSF.IO/DEGVY"
}
|
[
{
"id": "42525",
"date": "21 Jan 2019",
"name": "Yosep Seran Mau",
"expertise": [
"Reviewer Expertise Plant breeding and plant protection"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general, the article is sufficient in both presentation and content, and provides new findings in the field. However, several of the following concerns, as parts of my review to the article, may need to be clarified by the authors:\n\nDoes it cite the current literature?\nPartly, as only about 25% of the references are within the last 10 years.\n\nIs the statistical analysis and its interpretation appropriate?\nPartly, as there is inconsistency between the method and the results (analysis). In the method, the highest PEG concentration is 20% but in the results (Table 3) the highest PEG concentration is 40%, which is not consistent.\n\nAre all the source data underlying the results available to ensure full reproducibility?\n\nPartly, in Table 3, there are four calli survived in the regeneration stage using K3 medium. If the 4 calli from petiole were all survived, then there must be 16 calli that have been regenerated in this stage, assuming that the same number of calli (four calli) was assigned into each growth medium. This does not match with the method and the results in Table 2, where only 57% calli (about 7 calli) survived the 20% PEG. 16 calli are needed for the four growth medium treatments in the regeneration stage. This needs to be clarified.\n\nAre the conclusions drawn adequately supported by the results?\nPartly, for the following reasons:\n1) Inconsistency in the highest PEG concentration employed: 20% in the method and 40% in the results and conclusions.\n2) More than 50% calli survived at the highest PEG concentration may indicate mild to moderate stress intensity, thus, its effectiveness as in vitro selection of drought tolerance needs to be improved.\n3) There were only five calli survived in the regeneration stage (Table 3), which is very low in number as a starting genetic material for in vivo selection of drought-tolerant purple sweet potato clones.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4483",
"date": "25 Mar 2019",
"name": "Widi Sunaryo",
"role": "Author Response",
"response": "Honourable reviewersThank you very much for the valuable comments in improving the scientific and writing quality for our manuscripts. We have carefully read and paid much attention for all comments, and directly made the required revisions according to the suggestions. Our responses concerning the comments from the reviewer are: Thank you very much for the \"Approved with reservations\" status, we will make revisions as suggested. We added 9 more recent pieces of literature (within the last 10 years) in the new version of the manuscript. We have made revisions for the wrongly written (40% PEG) and replaced with the true PEG concentration (20%) in all the text (in the new version of the manuscript). The survived calli in PEG medium (20%) at petiole explants were 57%. Each survived calli subsequently can be separated/divided into some smaller calli. This will provide enough calli for the next stage of the experiment (regeneration initiation). In the PEG selection medium, even in the highest concentration of PEG, the survived calli were still growing and larger. That is why the number of calli was still sufficient for the next stage of experiment. Table 3 shows the number of plantlets (regenerated plants) and in the regeneration experiment. There was no selection (PEG selection) anymore. Concerning this comment: \"More than 50% calli survived at the highest PEG concentration may indicate mild to moderate stress intensity, thus, its effectiveness as in vitro selection of drought tolerance needs to be improved“. We do agree with this suggestion, and we have put this suggestion in the conclusion and outlook part. Since we have only 5 regenerated plants in the regeneration experiment, we will propagate (vegetatively) the plantlets to get a sufficient number of plant materials for the in vivo experiment. The in vitro propagation procedure for purple sweet potato is established and can be carried out very easily and fast. Thank you very much for the comments. If there are still any mistakes or corrections, we are open for the next revision and improvement."
}
]
}
] | 1
|
https://f1000research.com/articles/8-10
|
https://f1000research.com/articles/7-1521/v1
|
21 Sep 18
|
{
"type": "Method Article",
"title": "Improved inference of chromosome conformation from images of labeled loci",
"authors": [
"Brian C. Ross",
"James C. Costello"
],
"abstract": "We previously published a method that infers chromosome conformation from images of fluorescently-tagged genomic loci, for the case when there are many loci labeled with each distinguishable color. Here we build on our previous work and improve the reconstruction algorithm to address previous limitations. We show that these improvements 1) increase the reconstruction accuracy and 2) allow the method to be used on large-scale problems involving several hundred labeled loci. Simulations indicate that full-chromosome reconstructions at 1/2 Mb resolution are possible using existing labeling and imaging technologies. The updated reconstruction code and the script files used for this paper are available at: https://github.com/heltilda/align3d.",
"keywords": [
"chromosome",
"conformation",
"reconstruction",
"fluorescence",
"genetic",
"loci"
],
"content": "Introduction\n\nMeasurement of in vivo chromosome conformation is a major unsolved problem in structural biology despite its known biological importance1. The present state-of-art is either to obtain indirect information about conformations using 3C-derived methods which measure DNA-DNA contacts (typically in a cell-averaged population)2, or else to directly measure the cellular locations of individual chromosomal loci in single cells by microscopy3. The major limitation of direct localization is one of throughput: only ~ 3 – 5 labeled loci can be uniquely identified ‘by color’ in a standard microscope image, whereas a whole-chromosome reconstruction would involve labeling and identifying hundreds or thousands of loci. Several research efforts aim to remove this limitation using either experimental or computational approaches. The experimental approaches aim to allow an increased number of labels that can be distinguished in an image4–6. Alternatively, computational methods are being developed to infer the identity of each label if it cannot be uniquely identified in an image7–9, by using the known label positions along the DNA contour. Here we focus on the computational inference method called align3d8, and present improvements to allow high-quality, chromosome-scale conformational reconstructions.\n\nFirst, we briefly describe the align3d algorithm. Using a) the genomic locations and colors of labeled loci and b) the spatial locations and colors of spots in a microscope image, together with a relation tying the genomic distance between two loci to their average spatial displacement, this method constructs a table of ‘mapping probabilities’ p(L → s) for a given labeled genomic locus L having produced spot s in the microscope image. Each mapping probability p(L → s) is calculated by dividing the summed statistical weights of conformations where locus L maps to spot s, which we term a mapping partition function and denote ZL→s, by the full partition function Z that is the summed weight of all conformations. A proper calculation of ZL→s and Z would consider all conformations having no more than one locus at any given spot in the image1, similar to a traveling salesman tour10, but this exact calculation is intractable for large problems. Instead, align3d counts all conformations for which adjacent loci do not overlap at the same spot (see Figure 1), using a variant of the forward-backward algorithm11 that can propagate between non-adjacent layers. This is a major source of error as the vast majority of conformations contributing to the partition function overlap at non-adjacent loci, and one consequence is that the normalization of mapping probabilities makes no sense for a non-overlapping conformation, as ∑L p(L → s) can exceed 100% for certain spots. To recover from this error, align3d assigns a penalty to each spot and iteratively adjusts these penalties until the spot normalization is sensible. Although somewhat ad hoc, use of spot penalties recovers significant information about medium-sized conformations (∼ 30 labeled loci), although larger simulated experiments (∼ 300 loci) have convergence problems due to the cost function plateauing at very small or large values of the spot penalties.\n\nThe final step is to use the mapping probabilities to construct the range of likely conformations compatible with the microscope image. Uncertainty in the conformation results from inaccuracy or uncertainty in the mapping probabilities due to three factors: inaccuracy in the DNA model (the relation between genomic and spatial distance), error in estimating the partition functions, and the inherent uncertainty in the data even with a perfect reconstruction algorithm. Improvements to the DNA model will require direct measurements of in vivo conformations, which have not yet been made. Here we focus on improving the partition function estimate, using two different strategies. First, we give an efficient method for optimizing the spot penalties when there are hundreds of spots in the image. Next, we provide formulas for the partition functions which allow them to be estimated to arbitrarily high accuracy (given enough computational time), without using spot penalties or any optimization. As we show using simulations, these two methods used individually or in tandem permit confident, chromosome-scale conformational reconstructions using existing experiments.\n\nSchematic showing one legal and one illegal conformation passing through spots A, B and C. align3d counts both legal and overlapping conformations in estimating the partition Z (although it is able to prevent adjacent loci from overlapping).\n\n\nMethods\n\nFirst we provide a method for efficiently optimizing the spot penalties regardless of the number of labeled loci. This rule guarantees that a) the rate of missing spots is as expected, and b) the mapping probabilities are properly normalized. Let qs denote the penalty attached to spot s; then the update rule for that spot penalty is:\n\n\n\nwhere N is the number of loci, P(s) = ∑L p(L → s) is the total probability of mapping any locus to spot s, and pfn(c) is the estimated rate of missing spots having color c. The justification for this rule is given in Appendix 1 (Supplementary File 1).\n\nWe can also update a penalty q−c that is associated with missing spots of color c. This gives a faster way to enforce a desired missing spot rate because there are fewer q− penalties than q penalties. An update to q−c is equivalent to a reverse update to all qs for spots s of color c, so the update rule is:\n\n\n\nTypically, we first optimize the q− parameters to achieve a target missing spot rate, then optimize the q parameters to enforce P(s) ≤ 1 while maintaining the missing spot rate. In either case, we apply Equation 1 or Equation 2 to bring the q or q− parameters close to their final values. When the cost function stops improving, we switch to the steepest-descent algorithms used in Ross and Wiggins, 20128 to polish q or q−.\n\nNext, we give two exact formulas for the partition functions ZL→s and the full partition function Z that determine our locus-to-spot mapping probabilities. We focus on the full partition function Z since the formulas for ZL→s are identical. The largest term in each formula, which we denote Z˜0 (or Z˜0opt when spot penalty optimization is used), is the original estimate from Ross and Wiggins, 20128 calculated using a variant of the forward-backward algorithm11. Additional terms are computed in the same way, except that certain loci are constrained to map to certain spots. All of the constraints we will apply are illegal constraints, in that they force multiple loci to overlap at some spot in the image; therefore these terms only count illegal conformations that we would like to remove from the baseline calculation. By computing these terms and subtracting them from Z˜0 we eliminate the overlapping conformations and improve the calculation. It turns out that this process erroneously subtracts conformations with multiple overlaps more than once and thus we have to add back in higher-order corrections (i.e. partition functions having multiple constrained spots). Repeating this logic leads to exact formulas for Z taking the form of series expansions, which are dominated by the lowest-order terms as those have the fewest restrictions on conformational overlaps. Figure 2A illustrates an example of such a series expansion, where each parenthetical subscript (X Y . . . )s on a term label denotes an illegal constraint forcing loci X, Y, . . . to overlap at spot s when that term is calculated. We use this notation throughout.\n\nA. Schematic showing terms in a series expansion, in a case where series 1 and series 2 have the same terms. The full series gives the exact partition function for the 4-locus experiment shown where only 2 spots appeared in the image (due to a high rate of missing spots). Cartoons show only the constrained loci for each term (so for example each term includes the illegal conformation visiting spots s → t → s → t ). B. An illegal conformation for which loci A, C and E overlap at spots s, and loci F and H overlap at spot t. Series expansion 1 includes this conformation in terms Z˜0, Z˜(ACE)s, Z˜(FH)t, and Z˜(ACE)s(FH)t. Series expansion 2 includes this conformation in the same terms with the addition of Z˜(AC)s, Z˜(AE)s, Z˜(CE)s, Z˜(AC)s(FH)t, Z˜(AE)s(FH)t, and Z˜(CE)s(FH)t.\n\nThere are two ways we might remove conformations containing overlapping loci, leading us to two different series expansions for the true partition function Z. Suppose that we are calculating the term Z˜(AC... )s whose single illegal constraint forces loci A, C, . . . to overlap at spot s. One option is to forbid any of the other unconstrained loci from also mapping to spot s, since spot s is already overused. This leads to series expansion 1. Alternatively, allowing further overlaps with spot s from the unconstrained loci gives us series expansion 2. Figure 2B illustrates the differences between the two series.\n\nEach of the two series expansions is a weighted sum over all possible illegally-constrained terms having two properties: 1) each locus and each spot appear at most once in the indices, and 2) two or more loci map to each constrained spot. To be formal, we use Ω to represent the set of all possible illegal constraints: each element of Ω consists of a set of two or more non-adjacent loci and a single spot where they are forced to overlap. Each expansion thus takes the form\n\n\n\nwhere Zϕ is zero if any two constraints share a locus or spot. We will choose the integer weights wϕ so as to cancel out the overlapping conformations. By symmetry arguments, the weighting factor should not depend on the identities of the loci or spots, but rather only by the number of constrained spots nϕ, and the number of loci nkϕ involved in each kth constraint. For example, w(ACE)s(BD)t is determined by nϕ = 2, n1ϕ = 3 and n2ϕ = 2.\n\nHere we specify each series expansion by giving a formula for the weights wϕ in terms of nϕ and the various niϕ . We also explain how to select an appropriate set of terms ψ when there are too many terms to evaluate. Our selection prohibits any legal or overlapping conformation from contributing a negative weight to the partition function estimate, thereby guaranteeing positive mapping probabilities and allowing use of the reconstruction-quality metrics given in Ross and Wiggins, 20128. Derivations of the coefficient formulas and the term-selection criteria for each series expansion appear in Appendix 2 (Supplementary File 1).\n\nSeries expansion 1 For series expansion 1, we do not allow the unconstrained loci to map to spots that were used in constraints. Then the weights wϕ are given by:\n\n\n\nTo select terms for a series approximation, we first choose a set of illegal constraints ψ to disallow, then include all series terms Zϕ containing only those constraints: i.e. ϕ ⊆ ψ. This guarantees non-negative mapping probabilities. In order to efficiently evaluate the largest terms, we recommend selecting the Nψ constraints having the highest product of mapping probabilities in the baseline calculation Z˜0 (or Z˜0opt if spot penalties will be used). For example, we would include (AC)s if p(A → s) · p(C → s) is sufficiently large.\n\nSeries expansion 2 For series expansion 2, the unconstrained loci are allowed to map to spots that were used in constraints. Then the weights wϕ are given by:\n\n\n\nTo select terms for a series approximation, we first choose a set of Nψ single-locus-to-spot mappings Ψ, then include all terms Zϕ whose illegal constraints use only mappings in Ψ. For example, the constraint (AC)s would be included if Ψ ⊆ {A → s, C → s}. In order to select the largest terms, we recommend building Ψ from the Nψ largest mapping probabilities calculated from Z˜0 or Z˜0opt .\n\n\nResults\n\nWe tested the improved align3d method by generating random chromosome conformations using our software tool wormulator (version 1.1), and simulating the process of error-prone labeling, imaging and finally producing the locus-to-spot mapping probabilities. We considered three scenarios for our simulations. 1) The ‘Toy’ scenario involves 10 genomic loci, where each locus is labeled using one of 3 colors. For these simple problems the partition function can be calculated exactly. 2) Our simulated Experiment 1 uses standard DNA labeling methods and traditional 3-color microscopy to label 30 loci with 3 colors, thus interrogating a significant fraction of a chromosome contour. 3) Our simulated Experiment 2 labels 300 loci across a chromosome-length contour. The reconstruction of Experiment 2 is made possible by using the Oligopaints labeling technique4 to label in 20 different colors.\n\nFor each scenario, we randomly generated 100 conformations using a wormlike chain model (packing density = 0.3 kb/nm, persistence length = 300 kb, as suggested by the measurements of Trask, Pinkel and van den Engh, 198912); applied a random labeling at a mean density of 1 locus per megabase; and simulated experimental error: 100/200-nm Gaussian localization error in xy/z, a 10% rate of missing labels, and a 10% rate of nonspecifically-bound labels. A typical simulated experiment from the Toy scenario is shown in Figure 3A.\n\nA. Randomly generated and labeled chromosome contour with simulated experimental error: localization error (lines offsetting spots from the labeled genomic loci) and missing labels (open circles). This example lacks nonspecifically-bound labels (floating spots). B. Spot mapping probabilities calculated using both the largest series term Z˜0 (grey circles), and the exact Z that can be computed using 2210 series terms (blue circles). The dotted red line connects the true locus-to-spot mappings, which are used to calculate the unrecovered information. I(Z˜0) = 0.835 bits/locus and I(Z) = 0.54 bits/locus. C. Unrecovered information I and entropy S (left panel) and log Z (right panel) versus the number of terms used in the series expansions.\n\nFor each simulated conformation, we fed the label positions and colors together with the simulated 3D images into the align3d algorithm to produce locus-to-spot mapping probabilities. For example, the experiment shown in Figure 3A produced the mapping probabilities shown graphically in Figure 3B using circles, where the size of each circle indicates probability magnitude. Here grey circles show the mapping probabilities computed from Z˜0 with no use of spot penalties, and blue circles show those same probabilities computed using the exact Z. This example shows how excluding high-weight and heavily-overlapping conformations reduces and improves the partition function estimate (see Figure 3C) and concentrates the probability mass into the ‘true’ locus-to-spot mappings (shown connected by the dotted red line in Figure 3B).\n\nOur reconstruction quality metric is the amount of unrecovered information from the mapping probabilities, defined as I = – 〈log p(Li → si)〉i where the average 〈 . 〉 is taken over the set of true locus-to-spot mappings (Li, si). The ideal case of I → 0 implies a perfect reconstruction with no mistakes and zero uncertainty, but in practice I is always positive. In a real experiment where the true mappings are not known, we use a proxy for unrecovered information that we term entropy, defined as S = − 〈p(Li → sj) log p(Li → sj)〉ij whose average is taken over all locus-to-spot mappings, not just the correct mappings. The goal is to have S ≈ I so that a real experiment will have an accurate estimate of the reconstruction performance. The left-hand panel of Figure 3C shows how I and S depend on the accuracy of the calculation for the simple example shown, using either of the two series expansions and varying the number of terms from 1 (simply Z˜0) to 2210 which is the full set of terms for either series and thus computes Z exactly. Entropy generally overestimates the amount of unrecovered information (see Supplementary Figure S1 and Supplementary Figure S2, Supplementary File 1), because the large mapping probabilities should be even larger, and the small ones even smaller, than their assigned values (see Supplementary Figure S3, Supplementary File 1). We believe this miscalibration is mostly caused by align3d overestimating the missing spot rate.\n\nValidation of Equation 1–Equation 4. We first validated each of the two series expansions by comparing them against exact partition function calculations for the simulated Toy experiments. In all cases, both series expansions, when taken to their maximum number of terms, exactly reproduced the partition function calculations obtained by direct enumeration over all possible non-overlapping conformations. This test validates Equation 3 and Equation 4. We also verified that both series expansions could be used in conjunction with spot penalty optimization (Equation 1 and Equation 2), both by numerically validating the cost function gradient calculation and by testing for convergence on these small problems.\n\nImproved optimization allows large-scale reconstructions. First, we tested whether the iterative spot-penalty optimization rules given by Equation 1 and Equation 2 could work on large-scale problems such as those of Experiment 2, where the old gradient descent optimizer in align3d had difficulty8. The results are shown in Figure 4, which compares the number of iterative steps required to converge the q− (missing-spot penalty) and q (spot penalty) parameters without/with use of our improved optimization rules (labeled ‘old’/‘new’ respectively in the legend). Since the spot penalties q are optimized for probability normalization only after q− parameters have been optimized to achieve a desired missing spot frequency, we only attempted to optimize the q parameters for simulations where q− converged. There were two results from this experiment. First, more attempts to optimize the q− and q parameters successfully converged when using the new optimization rules in conjunction with gradient descent, as indicated by the greater volume of the ‘new’ histogram and the correspondingly larger numbers shown in the legends. Secondly, of the trials that did converge, our new method required significantly fewer iterations and thus less computation time than the old method, as indicated by the relative skews of the distributions.\n\nEach panel compares the number of iterations required to achieve convergence using the old (purple) versus new (yellow) optimization methods. Only trials that successfully converged are counted, so the histograms are not normalized relative to each other. The first number in parentheses of each legend entry shows the number of converged trials, and the second number shows the total number of trials. Note that the second numbers in the right-hand panel equal the first numbers in the left-hand panel, since we required convergence in q− in order to attempt optimization of the q parameters.\n\nUse of more colors dramatically improves reconstructions. Our most striking result is that simulations of the ambitious Experiment 2 produce far better results than even the Toy scenario, despite the fact that these simulations have more loci per color than either the Toy scenario or Experiment 1. This can be seen in the amount of unrecovered information I shown in the simulation-averaged plots of Figure 5A. Thus a push to 20-color labeling could prove critical for genomic reconstruction at the chromosome scale and beyond. At the end of this section we revisit Experiment 2, in order to assess the reconstruction quality when analyzing more realistic DNA contours having tighter confinement.\n\nA. Median unrecovered information I as a function of the number of terms used in each series expansion, without using spot penalty optimization (solid lines) versus with optimization (dotted lines), and over the three simulation scenarios (panels left-to-right). Each curve was derived from the 100 individual curves corresponding to the 100 simulations in each scenario using a simple point-by-point median average. B. Percentile distribution of the difference between the unrecovered information using series 2 minus the unrecovered information using series 1; the fact that this difference quickly drops below zero in nearly all individual simulations shows that series 2 recovers more information in the first few terms than does series 1.\n\nSeries expansion 2 outperforms series expansion 1. Next, we compared the convergence properties of our two expansions on the three scenarios of simulated experiments. Figure 5A gives a sense of how the amount of unrecovered information varies with the number of terms taken in each series, without (solid lines) and with (dotted lines) the use of spot penalties. Each of the 3 panels summarizes all 100 simulated experiments of that scenario, and each experiment in that scenario shows a unique relationship between information recovery and number of series terms computed. Representative curves of individual experiments in each scenario are shown in Supplementary Figure S1 (Supplementary File 1). In order to summarize these very dissimilar curves, Figure 5A shows a median average of all 100 individual experimental curves taken at each data point. Note that this averaging process does not necessarily preserve the shape of the curves from typical individual simulations.\n\nIn order to directly compare the two series expansions, we plotted their difference in unrecovered information I2 − I1 versus the number of series terms in Figure 5B. In this case, we plotted the full distribution showing the median (50th percentile) as well as the 10th, 25th, 75th and 90th percentile curves. These plots show directly that series 2 almost always outperforms series 1 when only a few terms can be evaluated. The reason is that the terms in series 2 are larger in magnitude owing to their looser constraints, and thus remove the extraneous part of the partition function more quickly than the terms of series 1 (see Supplementary Figure S1 and Supplementary Figure S4, Supplementary File 1). Based on these results, we recommend using series expansion 2 in all situations where the partition function cannot be evaluated exactly.\n\nSpot penalty optimization is the most efficient way to recover information. Spot penalty optimization is an iterative process where each iterative step requires the evaluation of some number of series terms. An optimization requiring t iterations thus multiplies computation time by a factor of t relative to the simple evaluation of the series. Alternatively, one could spend the extra computation time on taking the series to a higher order without spot penalty optimization. Figure 6A plots the unrecovered information when a) taking series 2 to a certain order without optimization, versus b) using spot penalty optimization on only the first term yielding Z˜0opt . The dotted line in each panel shows the median number of terms requiring the same computation time as Z˜0opt . The Toy scenario shows that, if the series expansion is carried deep enough, it becomes more accurate than Z˜0opt : in other words the difference I−I0opt becomes negative. However, for the practical scenarios of Experiments 1 and 2 this crossover point requires taking more terms than would be needed to match the computational cost of calculating Z˜0opt (the dotted line). Based on these results, we recommend always performing spot penalty optimization, especially for larger reconstructions.\n\nA. Comparison of unrecovered information using series expansions without iteration, denoted I, to the unrecovered information obtained by optimizing spot penalties using only the first series term, denoted I0opt , over three experimental situations. Vertical dotted lines indicate the median number of series terms computable with the same computational time as was required to obtain I0opt . For Experiments 1 and 2 the difference I−I0opt is typically positive at the intersection of the dotted line, indicating that spot penalty optimization method is the more efficient way of recovering information. B. Comparison of unrecovered information using spot-penalty optimization in conjunction with multiple series terms versus optimization of Z˜0 alone, showing the added benefit of including more terms in the series.\n\nSeries expansions can improve optimization information recovery. Although spot penalty optimization is the most efficient way to recover information, that process alone can only extract a certain fraction of the recoverable information: once the cost function is zero, optimization can proceed no further despite the problem not having been solved exactly. At this point, the only way forward is to go higher in the order of series terms used; we can still apply spot penalties to this sum of terms and iteratively optimize them as before using Equation 1 and Equation 2. Figure 6B plots the difference in unrecovered information when applying spot penalty optimization between a) a variable number of terms in series expansion 2, and b) only Z˜0 (the first series term). This figure shows that including additional series terms in the optimization improves the information recovery, albeit at a slow rate (especially for large problems).\n\n20-color labeling leads to near-perfect reconstructions. As shown in Figure 5A, the unrecovered information for the whole-chromosome Experiment 2 averages around 0.2 bits per locus, implying near perfect mapping probabilities. However, because these results were based on randomly-generated unconfined conformations, they do not establish whether such good information recovery is possible with real chromosomes which are likely to be more compact. To test Experiment 2 on realistic chromosome conformations, we generated four plausible conformations of human chromosome 4 by running the GEM software package13 on the smoothed human Hi-C data set provided by Yaffe and Tanay, 201114 and using a 3D spline interpolation to increase the resolution from 1 Mb to 100 kb. These conformations were then virtually labeled at 300 randomly-selected loci with simulated experimental error as before (100/200 nm localization error in xy/z, 10% missing- and extra-spot rates). Mapping probabilites were reconstructed by taking series expansion 2 to the lowest order that included at least 1000 terms, then applying and optimizing spot penalties. Compared with the random-walk conformations used to test the Experiment 2 scenario, these reconstructions did somewhat worse (∼ 0.25 versus ∼ 0.2 bits of unrecovered information per locus) owing to fact that physical confinement of chromosomes increases the density of competing spots in the image.\n\nDespite the drop in performance, 0.25 bits of unrecovered information per locus is still an extremely strong reconstruction, implying that the correct locus-to-spot mappings are assigned p-values averaging around 2−0.25 ≈ 84%. Starting from such accurate and confident mapping probabilities, one can infer a reasonable conformation simply by assigning each locus to the spot to which it maps with the highest probability (or calling a missing spot if 1−∑s′ pL→s′ > any pL → s), and drawing a line in the image that connects these spots in genomic order. The conformations produced by this simple rule are shown in Figure 7: the correct conformation is shown with a blue line and errors in the inferred conformation are shown in red. The reconstructed conformations are ∼ 90% accurate as determined by an alignment between the true and inferred spot sequences traveling along the DNA contour (40, 33, 26, 33 mismatches + indels for the 4 respective experiments). Most mistakes are of a sort that does not change the large-scale structure. For example, one common error is to erroneously skip one or more spots in the image (since at present align3d overestimates the missing-spot rate), thus ‘looping out’ a small part of the conformation and effectively lowering the resolution.\n\nGrey shaded lines indicate the underlying DNA contours; blue lines trace the ideal reconstructed contours given the simulated labeling (shown in small panels at upper right); red lines show our reconstructed contours where they deviate from the ideal contours. Captions indicate the amount of unrecovered information per locus after/before the reconstruction process.\n\n\nDiscussion\n\nWe have developed and evaluated two improvements to the align3d method for reconstructing chromosome structure. Both of these improve the partition function estimates that determine the locus-to-spot mapping probabilities, which can provide the basis for (probabilistic) reconstructed conformations. The first improvement is a more robust spot-penalty optimizer that allows for large-scale reconstructions involving hundreds of labeled loci, such as will be needed to uncover whole-chromosome conformations. The second improvement is two series expansion formulas for the partition functions, which in principle allow the mapping probabilities to be solved to arbitrary accuracy within the limitations of the experiment and the underlying DNA model. In practice, the series approach is difficult for two reasons: 1) there are a huge number of terms in each series expansion, and 2) the lowest-order approximation Z˜0 overestimates Z by many orders of magnitude, unlike other series expansions where the initial approximation is close to the final answer. Despite the difficulties, the series formulas that we give offer some way forward to improve on the original estimate Z˜0opt . Of the two formulas, we recommend using series expansion 2, which has the larger-magnitude terms and thus recovers the most information when only a few terms can be evaluated.\n\nOur problem of finding likely (i.e. low-free-energy) DNA conformations passing through a set of imaged spots is similar to the well-known traveling salesman problem (TSP), in which a salesman must find the shortest route connecting a set of cities. Somewhat more closely related is a generalization of the TSP called the time-dependent traveling salesman problem (TDTSP)10, where the intercity distances change every step on the tour; this is analogous to our situation where the free energy needed to thread DNA between two spots depends not only on their separation but also on the length of DNA used to connect them. In our case, the presence of missing and extra spots generalizes our problem still further: in the TDTSP analogy the salesman would be allowed to skip stops and cities for a penalty. Our main finding is that the partition function of this generalized TDTSP (which encompasses traditional TSP and TDTSP problems) can be expressed as a sum of terms computable using a (modified) forward-backward algorithm. This result should also apply to other route-finding applications, and might provide a fresh approach since research has historically focused on estimating the single optimal solution rather than the partition function.\n\nFrom a genomic standpoint, our most exciting result is that the combination of our computational improvements together with 20-color labeling technology gives almost perfectly-accurate reconstructions. Out of ~ 4 bits per locus of uncertainty inherent in the reconstruction problem, our method recovers about 3.75 bits, despite somewhat overestimating the missing-spot rate in these simulations. Such confident mapping probabilities allow for the direct construction of individual conformations that are about 90% accurate. We want to emphasize that our reconstructions used model parameters that were correctly calibrated, but not necessarily ‘correct’ in terms of details that an experimenter would not know. For example, because the conformations were generated using a wormlike chain DNA model but align3d assumes a Gaussian chain model, the underlying DNA model used in the reconstruction is wrong in a sense, just as it will be using in vivo data where the underlying DNA model is unknown. However, we did set the Gaussian decay constant to correctly match the persistence length as that can be measured experimentally. Additionally, the correct average rates of missing and extra spots over all experiments were provided to the analysis, but using those align3d still had to estimate the actual number of missing spots per color in each experiment. The robustness of the analysis to experimental unknowns gives evidence that reconstructions using real-world experimental data will be of similar quality to those in our simulations, and if so then direct measurement of chromosome conformations is possible today with current technology.\n\n\nData availability\n\nThe simulated conformations and labelings used to generate the plots in this paper, together with the output of the align3d analysis, can be found at: https://github.com/heltilda/align3d/blob/master/seriesExpansions/a3dRawData.zip\n\n\nSoftware availability\n\nResults in this paper were generated using version 1.1 of align3d, built using version 1.1 of Cicada scripting language. Simulated conformations and labelings were generated using version 1.1 of wormulator.\n\nAll source files used in preparing this paper are available from the GitHub page for this paper: https://github.com/heltilda/align3d/tree/master/seriesExpansions.\n\nLicense: GPL 3.0\n\nArchived code at time of publication:\n\nalign3d: http://doi.org/10.5281/zenodo.141150115\n\nLicense: GPL 3.0\n\nwormulator: http://doi.org/10.5281/zenodo.141150316\n\nLicense: GPL 3.0\n\nCicada scripting language: http://doi.org/10.5281/zenodo.141150517\n\nLicense: MIT License",
"appendix": "Grant information\n\nFunding was provided by the Boettcher Foundation (J.C.C.), National Institutes of Health [2T15LM009451 to B.C.R.], and a Cancer League of Colorado grant (J.C.C. and B.C.R.).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors want to thank Rani Powers and Jenny Mae Samson for helping review the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: align3dSupplement.pdf. File containing three appendices giving the derivations of the equations used in this text (Appendix 1 and Appendix 2), and the supplemental figures (Appendix 3).\n\nClick here to access the data\n\n\nFootnotes\n\n1 1Depending on how the experiment is done, two spots of the same color sufficiently close in the image may appear as a single spot where the conformation self-overlaps. We prefer to treat this scenario as a missing-spot measurement error rather than relax the one-spot-per-locus rule. If the spots have been properly localized, then the underlying conformation visits any given spot once at most\n\n\nReferences\n\nDekker J, Belmont AS, Guttman M, et al.: The 4D nucleome project. Nature. 2017; 549(7671): 219–226. PubMed Abstract | Publisher Full Text | Free Full Text\n\nImakaev MV, Fudenberg G, Mirny LA: Modeling chromosomes: Beyond pretty pictures. FEBS Lett. 2015; 589(20 Pt A): 3031–3036. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKocanova S, Goiffon I, Bystricky K: 3D FISH to analyse gene domain-specific chromatin re-modeling in human cancer cell lines. Methods. 2018; 142: 3–15. PubMed Abstract | Publisher Full Text\n\nWang S, Su JH, Beliveau BJ, et al.: Spatial organization of chromatin domains and compartments in single chromosomes. Science. 2016; 353(6299): 598–602. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakei T, Shah S, Harvey S, et al.: Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH. Biophys J. 2017; 112(9): 1773–1776. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMa H, Tu LC, Naseri A, et al.: Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016; 34(5): 528–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLowenstein MG, Goddard TD, Sedat JW: Long-range interphase chromosome organization in Drosophila: a study using color barcoded fluorescence in situ hybridization and structural clustering analysis. Mol Biol Cell. 2004; 15(12): 5678–5692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss BC, Wiggins PA: Measuring chromosome conformation with degenerate labels. Phys Rev E Stat Nonlin Soft Matter Phys. 2012; 86(1 Pt 1): 011918. PubMed Abstract | Publisher Full Text\n\nBarton C, Morganella S, Oedegaard O, et al.: Chromotrace: Reconstruction of 3d chromosome configurations by super-resolution microscopy. bioRxiv. 2017; 115436. Publisher Full Text\n\nGouveia L, Voß S: A classification of formulations for the (time-dependent) traveling salesman problem. Eur J Oper Res. 1995; 83(1): 69–82. Publisher Full Text\n\nBaum L: An inequality and associated maximization technique in statistical estimation of probabilistic functions of a markov process. Inequalities. 1972; 3: 1–8. Reference Source\n\nTrask B, Pinkel D, van den Engh G: The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics. 1989; 5(4): 710–717. PubMed Abstract | Publisher Full Text\n\nZhu G, Deng W, Hu H, et al.: Reconstructing spatial organizations of chromosomes through manifold learning. Nucleic Acids Res. 2018; 46(8): e50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYaffe E, Tanay A: Probabilistic modeling of Hi-C contact maps eliminates systematic biases to characterize global chromosomal architecture. Nat Genet. 2011; 43(11): 1059–65. PubMed Abstract | Publisher Full Text\n\nheltilda: heltilda/align3d: align3d incorporating series formulas, improved spot penalty optimization (Version 1.1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1411501\n\nheltilda: heltilda/wormulator: wormulator version for paper (Version 1.1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1411503\n\nheltilda: heltilda/cicada: Cicada version used in F1000Research paper (Version 1.1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1411505"
}
|
[
{
"id": "38643",
"date": "07 Nov 2018",
"name": "Ewan Birney",
"expertise": [
"Reviewer Expertise genomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper the authors update and build on a method they have previously published known as ‘align3d’. This method attempts to infer the chromosome conformation based on images of fluorescently tagged genomic loci. The authors claim that this updated method increases the accuracy of the inferred conformation as well as allowing the method to run on larger instances of the problem. They then go on to demonstrate where the method allows for the near perfect reconstruction of larger scale, simulated, labelled images. We believe that the article is worthy of indexing on the condition that some minor issues, outlined below, are addressed.\n\nIn the introduction the authors mention a couple of other methods attempting to resolve similar problems. I think that this section should be expanded as there is no critical comparison of how this method compares to each of those mentioned. In particular the computational methods should be compared and contrasted so it is clear to the reader how this method differs from others.\n\nWhilst reviewing this paper we were unable to access the supplementary data. This must be made available before the paper can be indexed. Some things the authors could include in the supplementary section that would be useful from the computational perspective would be the type of series expansion being used and any information on how quickly the series expansion converge to the original formula. The authors have experimentally checked on the convergence properties but sometimes it's quite simple to determine theoretically how quickly some approximation converges. This information could be useful in determining better expansions and would explain more concretely why they get some of the results they see.\n\nIn the experimental section of the paper the authors generate three different types of simulation that they denote 'Toy', 'Experiment 1' and 'Experiment 2'. In the discussion of the results the authors make the following comment:\n\n‘Use of more colors dramatically improves reconstructions. Our most striking result is that simulations of the ambitious Experiment 2 produce far better results than even the Toy scenario, despite the fact that these simulations have more loci per color than either the Toy scenario or Experiment 1. This can be seen in the amount of unrecovered information shown in the simulation-averaged plots of Figure 5A. Thus a push to 20-color labeling could prove critical for genomic reconstruction at the chromosome scale and beyond. At the end of this section we revisit Experiment 2, in order to assess the reconstruction quality when analyzing more realistic DNA contours having tighter confinement.’\n\nThe authors should make some attempt to explain this situation. Actually if you increase the number of colours and also increase the number of loci with the same colour then you would not obviously assume that the problem should be harder. It very much depends on how each is increased within proportion to each other.\n\nAn increase in the number of unique colours available should lead to the problem being exponentially easier as you are effectively exponentially decreasing the ambiguity in the data set. Should you also increase the number of loci labelled with the same colour then you wouldn't expect the problem to become harder unless that increase was large enough to outweigh the effects of the increase in the number of available colours. In this sense it could be argued that many instances of the 'Toy' example are fundamentally more challenging than the (on the face of it) more complicated 'Experiment 2'. This should somehow be addressed by the authors.\n\nAlso in the discussion of the experimental results the authors note that '20-color labelling leads to near-perfect reconstructions.' This result is consistent with our results reported by Barton et al. (20171). It would be good to mention this as although the computational methods are different, the simulations are generated in different ways and the resolution simulated is different, both methods suggest that if ~20 colours are available then near perfect reconstruction is possible. The authors should also point out the similarity of the number of colours needed in both their method and ours.\n\nThe differences in computational methodology yet similarity in the numbers of colours needed for near perfect reconstruction perhaps suggests to me that both methods are in some sense 'naive'. There must exist a minimum number of colours required for a certain average reconstruction performance (with the appropriate caveats) but we would be surprised if it was as high as 20. It could be interesting to see the authors add some discussion about this connection and any insight they might have into it.\n\nFinally there have been a number of different attempts to simulate super resolved images of the type used in this and other computational methods. If the authors can use this data as input or the data can easily be coerced into an appropriate format for this method then the paper would be much stronger with the addition of results of using the method against these datasets. In this way the authors can clearly demonstrate that the method they propose is not simply good on their own simulated data, but also performs robustly on other independently generated simulations.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "4477",
"date": "13 Mar 2019",
"name": "Brian Ross",
"role": "Author Response",
"response": "We wish to thank Reviewers 1 for their many helpful comments and insights. Based on these comments and those of Reviewers 2, we have made some changes to our analysis, updated our results (particularly those shown in Figure 7) and the content of the main paper, and added a new appendix.We apologize for the problems Reviewers 1 had in accessing the Supplemental Material. The material was uploaded and available (to us), but the 'Appendix x' links lead nowhere in the published version. These seemingly dead links have been removed.Reviewers 1 suggested that in the Introduction we compare our align3d method to the other published method that we are aware of (ChromoTrace) to highlight their differences. We agree that this is indeed a useful addition, and so we have added several sentences to the Introduction (2nd paragraph) contrasting the two algorithms. We are not experts on ChromoTrace, and if we have mischaracterized it in some way we apologize and hope the reviewers will correct us.Reviewers 1 also inquired about the exact series expansion formulas we used. The expansion formulas are in the Methods section of the main text, not the Supplemental material. To make this clearer we have added an equation number to the series definition preceding the coefficient formulas (this is the new Equation 3), and referenced that equation explicitly in the two coefficient formulas (which are now Equations 4-5). Thus the series definitions are fully in the main body of the paper, and only their derivations are in Appendix 2.One technical detail is that our original code could not use our series expansions in conjunction with the preexisting capability to 'fix' certain loci to map to certain spots in the image, in order to obtain mapping probabilities that are conditional on the fixed loci. This has been addressed in the new version of the code. This oversight did not affect the results shown in the paper, but it did require us to add a explanatory paragraph to the end of Appendix 2.Reviewers 1 asked about the finding that our simulated Experiment 2 reconstructions came out much better than the Experiment 1 reconstructions, despite having more labeled loci of a given color. We have added several sentences to the Results section (\"Use of more colors dramatically improves reconstructions\" section) explaining that we believe that it is the spatial density of labeled loci rather than the absolute number that determines the reconstruction quality. Reviewers 1 noticed the same finding in Barton et al. (2018); we have added this citation. We have not systematically tested performance as a function of the number of colors; we chose 20 simply based on the fact that 10 sequential hybridizations is reasonable for our planned experiment based on conversations with our collaborator (Wang et al., 2016, demonstrate 17 rounds). Since we haven't noticed a plateau in reconstruction performance versus number of colors, as evidenced by the fact that the 20-color reconstructions still have some uncertainty, we do not see a reason to go towards fewer colors.A final question raised by Reviewers 1 concerned the issue of superresolution in the simulated images. Since our spots are presumed well-separated (based on the data of Wang et al., 2016) we believe we can get super-resolved spot localization without having to use special microscopes or fluorophores, and without having to resolve individual fluorophores. Thus the superresolution comes for free on normal images at the scale we consider here. We have added text explaining this (new final paragraph of Results), and also a second set of conformational reconstructions to Figure 7 showing explicitly the benefit of superresolving the spot locations. If we were to push to higher-genomic-resolution labeling (say, 10s-100s kb locus separation; current simulations are at ~600 kb) then we would indeed need superresolution microscopes, but since those are not the experiments simulated here we did not try to simulate those images. In fact this is why we chose to label these simulations at the 600 kb resolution.Although we were not able to increase the Hi-C inferred resolution, we did discover that we had misinterpreted the scale of the Hi-C-derived conformations of Figure 7, thus underestimating the relative magnitude of microscope error in these simulations. Our new plots have corrected this error. Owing to the larger microscope error our new reconstruction quality is somewhat worse as measured by our information metric. To compensate we improved our script that estimates a likely conformation from our output (mapping p-values), and as a result these likely conformations are roughly of the same quality as before. We also added a parallel set of superresolution reconstructions to this figure, in order to show explicitly the benefit of reducing microscope error.References:Barton, C., Morganella, S., Oedegaard, O., Alexander, S., Ries, J., Fitzgerald, T., ... & Birney, E. (2018). Chromotrace: reconstruction of 3D chromosome configurations by super-resolution microscopy. bioRxiv, 115436.Wang, S., Su, J. H., Beliveau, B. J., Bintu, B., Moffitt, J. R., Wu, C. T., & Zhuang, X. (2016). Spatial organization of chromatin domains and compartments in single chromosomes. Science, 353(6299), 598-602.Zhu, G., Deng, W., Hu, H., Ma, R., Zhang, S., Yang, J., ... & Zeng, J. (2018). Reconstructing spatial organizations of chromosomes through manifold learning. Nucleic acids research, 46(8), e50-e50."
}
]
},
{
"id": "40510",
"date": "10 Dec 2018",
"name": "Marco Cosentino Lagomarsino",
"expertise": [
"Reviewer Expertise Statistical Physics",
"Quantitative Biology",
"Chromosome Organization"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript “Improved inference of chromosome conformation from images of labeled loci\", Ross and Costello present a computational inference method to reconstruct genome conformation from measurements of the positions of m labelled loci of known coordinates with n<<m colored fluorescent foci. The presented tool is a new version of their computational tool \"align3d\" with multiple improvements. The tool has the aim of inferring the polymer conformation of chromosomes in-vivo, starting from images of fluorescently-tagged genomic loci, where each color tags different loci at the same time.\nThe authors provide a test of the algorithm with data that are generated computationally, in a simple (short polymers) or more complex (longer polymers, more colors) setting. Finally they provide a simplified (and limited - see point 5 below) test using data that are derived from empirical data.\nThe question appears interesting due to its experimental motivation, although probably the method is not yet close to something with concrete applicability to experimental data. We think that this could develop into a useful tool for the global effort of understanding the chromosome conformation of organisms in-vivo. As physicists, we are concerned with some aspects related to the representation (modelling) of the polymer and the experimental situation. Our observations might be useful for the authors or for other scientists that intend to analyse this kind of experimental data.\n\nThe authors state that the inaccuracy of the DNA (conformation) model, i.e. how the physical distance of two loci scales with arclength distance along the genomic coordinate is a major factor of error (more precisely, this is a conditional distribution of distances given distance along the chain). They further state that nothing is known about this. However, this is not really the case, as both Hi-C and FiSH experiments with labelled loci give information about these quantities (Lagomarsino et al., 20151 and Fudenberg and Mirny, 20122).\nIn particular, the assumption that the polymer is a Gaussian chain seems very restrictive. A much less restrictive (though still limited) assumption would be that this scaling relation is a tuneable power-law. This assumption is particularly interesting because in this case the scaling law relating physical distance to distance along the genome is related to the contact probability measured in Hi-C data (Fudenberg and Mirny, 20122). Indeed, in this scenario the contact probability (sometimes called “P(s)”, where s is the arclength distance) and the connection between genomic distance and typical spatial distance R(s) are related by a scaling (Polovnikov et al., 20183). Thus Hi-C data could be used to directly constrain the inference, or to compare with the results.\nIn this last scenario one could use the inference to learn the scaling from data. It seems quite reasonable to us that this scaling should be one of the main observables to infer from the data. Imposing this scaling appears like imposing a specific behaviour on the configurations that we are attempting to infer. In this regard, one big question is whether the observable “scaling of physical distance with arclength distance” can be inferred from the data without making the problem under-determined. We would like to stimulate the authors to spend some words to address this question.\nAs we suggest above, there are multiple possible approaches to this practical issue, such as the use of the observable quantity “P(s)”, the contact probability measured with Hi-C, or the use of an ansatz, such as a power law (Marie-Nelly et al., 20144), accompanied by a procedure to optimize the parameters.\n\nThe authors’ main hypothesis is that only one locus can map to each identified spot in the image, and, for this reason, the solution proposed is a heuristic method to solve the traveling salesman problem for the polymer on those loci. We observe that this might be a good practical assumption but it is not necessarily a good one for the chromosome, and for polymers in general. Polymers can have loops, even randomly. The definition of those loops depends on the resolution of observation (which experimentally will be limited by diffraction). The frequency of loops in chromosomes depends on important physical and biological parameters such as active looping (Fudenberg et al., 20165), the presence of different solvent phases and the balance between steric and other kinds of interactions (Scolari and Lagomarsino, 20156) as well as from steps of the experimental protocols (Scolari et al., 20187). Hi-C experiments, measure loops and quantify their specific and generic properties. In terms of the genomic distance, it has been shown that at small distances the chromosomes are very compact, and the amount of this compaction varies widely across conditions (Lazar-Stefanita et al., 20178 and Muller et al., 20189) even for the same organism. For increasingly longer distances, generally, the probability of making a loop normally decreases monotonically with genomic distance. Thus, we think that the authors’ approach should be applicable to an increasing number of cases by increasing the scale of observation and modelling, under the condition that the relation that ties the genomic distance to the three-dimensional distance is chosen correctly.\n\nThe algorithm is focused on a single chain conformation and does not exploit ensembles. Typically in such experiments one expects to have fairly low precision of localisation, but almost arbitrarily large amount of realisations (different cells). Each will be different but will also have common properties, and relaxing the question could make the inference process much easier. After all, inferring precisely a single configuration is not so relevant, because it will change in time due to natural fluctuations of the system. It is more useful (and well defined) to infer some ensemble properties (at fixed conditions for the cells such as time and phase into the cell cycle), and then quantify the cell-to-cell diversity with respect to such average behavior.\n\nThese images will come from microscopy and they will likely be 2D projections, or have lower resolution in the z direction. The authors do not address this issue (and in general the issue of resolution seems underestimated), but we expect it to be quite important in any concrete situation.\n\nIn regard to the final example, we notice that the data is binned at 1mb and then interpolated at 100kb with a spline, we wonder if this resolution improvement introduces any alterations in the reconstructed conformations of the polymer. For this reason, it seems reasonable to perform a more thoughtful statistical analysis with different levels of interpolation to support this choice.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "4478",
"date": "13 Mar 2019",
"name": "Brian Ross",
"role": "Author Response",
"response": "We wish to thank Reviewers 2 for their many helpful comments and insights. To address their comments as well as several concerns of our own, we have made a number of changes to our analysis, our results and the content of the main paper, and added a new appendix. The changes made to the code required us to regenerate all 9 figures that show results, although only Figure 7 changed significantly. We have also updated our GitHub repository containing all our code and example data.Reviewers 2 pointed out that the we were too strong in our language stating that 'nothing is known' about the DNA model. We agree and we have removed this wording from the last paragraph in the Introduction. They also suggest the use of contact probabilities as a basis for inferring the distance function used in the model. This would be possible, but there is actually a direct measurement of the distance function (Wang et al., 2016) which we have decided to use instead. Wang et al. found a 1/3 scaling exponent between L and R at the relevant length range, which we incorporated into our model for analyzing the Hi-C reconstruction (but not the random chains, whose exponent is 1/2) and then reran the results shown in Figure 7. The results did not change very much because most adjacent loci are close enough that R = k*L, i.e. the exponent is 1. The reviewers point out this could be due to our spline interpolation used to increase the resolution of the conformation. Unfortunately we were not able to directly infer higher-resolution conformations because the Hi-C data set recommended by Zhu et al., 2018 (the Hi-C inference method called 'GEM') is binned at 1 Mb resolution, and this sets the resolution of the GEM conformations. We do not believe this is a problem for 2 reasons: 1) our average label spacing is ~2/3 Mb, not far below the 1 Mb GEM conformation subunit length, and 2) the inferred conformations seem to have a persistence length somewhat above the length of a subunit, although we cannot rule out that this might change with a different bin density. We agree that it would be ideal to have a higher-resolution structure (although bin sampling would become an issue using this Hi-C data set), but we suspect that the errors in Hi-C inferences probably overwhelm the resolution issue.We want to point out that our use of a Gaussian chain for reconstruction (but not for producing the DNA chains) is not incompatible with the scaling relations mentioned by Reviewer 2, because these scaling relations determine average spatial separation R of two loci based on their genomic separation L, but not the form of the distribution p(r|R). We have chosen to model p(r|R) with a Gaussian (partly for convenience since it is easy to factor in localization error, but partly for other reasons; see below) having = R^2, but R is in turn calculated as R = L^power. In our earlier draft this power was fixed at 1/2 for distances above a persistence length, but as Reviewers 2 pointed out recent experimental data show exponents of 1/3 - 1/5. To address this issue, we generalized our program to accept more general DNA models consisting of different power laws at different inter-locus distance regimes, and our new results use exponents of either 1/2 and 1/3 for long DNA segments, depending on the simulated experiment. To make this clearer, we have added a new paragraph to the Results section (2nd paragraph) explaining the model selection in our simulations, as well as how a model would be chosen in a real experiment.In our initial submission we claimed that a systematic error seen in the mapping probabilities was due to overestimation of the missing-spot rate. Since then we have both fixed the missing-spot rate estimation and made major progress in figuring out the real cause of the error, which we explain in detail in a new Appendix 3 and refer to in several places in the text. The error comes from the fact that the Gaussian chain model used for reconstruction differs significantly from the wormlike chain model used to generate our simulated contours. While Reviewers 2 were concerned that the use of a 'wrong' model would skew the results, we believe that the opposite interpretation is more accurate: the fact that we obtain high-quality results even when the reconstruction model differs from the model used to produce the conformations shows that our approach is robust to model error. Appendix 3 justifies this intuition, by showing that model error causes our results to appear less certain than they are, but does not cause reconstruction errors if the reconstruction model is less sharply-peaked than the true model. This is the other justification for using the Gaussian chain model, which is quite permissive of unexpected behavior that we may find given that the true in-vivo DNA model may behave unexpectedly sometimes which may be very difficult to measure exactly in calibration experiments. We have also added a new 3rd paragraph to the Discussion explaining this.We agree with Reviewers 2 that loops certainly can happen and, to the extent that they can be distinguished by microscopy, our algorithm is certainly capable of finding looped conformations (even if the loop is over 2 adjacent loci -- our Gaussian model peaks at r = 0). If two loci of the same color happen to overlap in a microscope image, one may be missed -- this is considered a missing-spot or false-negative error, as mentioned in the footnote in the Introduction. Since our algorithm is capable of handling both false negatives and false positives (extra unbound spots), we do not anticipate loops to be a problem. If there are many points of overlap coming from an identical color sequence (e.g. if two copies of the same chromosome overlap) then the reconstruction fragments can become fragmented, with ambiguity as to which piece goes with which other piece -- we have added a brief note about this to the Discussion section.Reviewers 2 point out that align3d is a single-cell method, not an ensemble method, and we completely agree. We believe that aggregating single-cell conformations will give many interesting insights that one could not get by aggregating, for example, pairwise distances. Our method should be seen as one possible means of obtaining these cell conformations.Finally, Reviewers 2 raise the issue of resolution in the z dimension: we certainly do consider localization error in z, both in generating the spot localizations (which have z error as well as x/y error) and in the reconstructions (where the errors in x/y/z are required inputs). In all simulations we set the z localization error higher than x/y error (200 vs 100 nm in normal resolution, 50 vs 30 nm in superresolution), reflecting the fact that axial resolution is worse in most setups. We have updated the main text to more explicitly give the localization error in the various simulations.References:Wang, S., Su, J. H., Beliveau, B. J., Bintu, B., Moffitt, J. R., Wu, C. T., & Zhuang, X. (2016). Spatial organization of chromatin domains and compartments in single chromosomes. Science, 353(6299), 598-602.Zhu, G., Deng, W., Hu, H., Ma, R., Zhang, S., Yang, J., ... & Zeng, J. (2018). Reconstructing spatial organizations of chromosomes through manifold learning. Nucleic acids research, 46(8), e50-e50."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1521
|
https://f1000research.com/articles/8-337/v1
|
26 Mar 19
|
{
"type": "Research Article",
"title": "Influenza virus infection of well-differentiated human airway epithelial cells by infectious aerosols: insights into the earliest stages of infection",
"authors": [
"Claire M. Smith",
"Dani Do Hyang Lee",
"Hemant Kulkarni",
"Priya Radhakrishnan",
"Robert Hirst",
"Andrew Easton",
"Chris O'Callaghan",
"Dani Do Hyang Lee",
"Hemant Kulkarni",
"Priya Radhakrishnan",
"Robert Hirst",
"Andrew Easton",
"Chris O'Callaghan"
],
"abstract": "Background: Influenza virus is a major human pathogen, yet surprisingly little data is available on the earliest stage of infection. We have developed a novel method to study natural transmission influenza infection by aerosol and to observe the effects of early infection on the ciliated airway epithelium using high-speed video microscopy. Methods: Primary human ciliated epithelial cultures were infected with influenza A (H1N1), delivered either by aerosol or by liquid immersion. Cells were stained for viral antigens and the level of inflammatory mediators, and the number of motile ciliated cells and ciliary beat frequency and pattern was measured. Results: Infection by aerosol and liquid inoculums of influenza virus was shown to be trophic for ciliated cells. Infection by both methods also led to a significant decrease in the number of cells with motile cilia over the first 24 hours; however, the ciliary beat frequency and beat pattern of the remaining cilia was maintained over 24 hours. Conclusions: Influenza virus aerosols readily infect human ciliated nasal epithelial cells resulting in early loss of motile ciliated cells. Delivery of the virus by aerosol elicited an anti-inflammatory Th2 response, which was distinct from cells exposed to virus by liquid immersion delivery. This suggests our aerosol model may provide a more clinically relevant model for studying the early effects of influenza infection.",
"keywords": [
"influenza",
"cilia",
"airway epithelial cells"
],
"content": "Introduction\n\nInfluenza viruses cause annual epidemics that are associated with considerable morbidity and mortality1,2. The infectivity of influenza virus depends on the proteolytic cleavage of the haemagglutinin (HA) protein by host serine proteases3. The proteases are thought to be secreted by differentiated airway epithelial cells that are considered to be the main target for human influenza virus4–6. However, the cytopathogenesis of influenza virus, particularly in the earliest stages of infection, remains largely unknown.\n\nWe have previously used human ciliated epithelial cells in culture to evaluate the effects of bacteria and viruses7–11, observing ciliary function in real time using high-speed video photography. Work using ciliated epithelial models to investigate other viruses has provided significant insight into the pathophysiology of infection4–6,12–14. For example, respiratory syncytial virus (RSV) has been shown to be trophic for human ciliated cells10,14, inducing ciliary dyskinesia10, enhancing bacterial attachment10,15, and producing an inflammatory response early in the infectious process11.\n\nThe primary aim of this study was to evaluate the effect of influenza virus infection, either by aerosol or by liquid immersion, on ciliary function and inflammation of differentiated primary human nasal epithelial cells.\n\n\nMethods\n\nHuman nasal epithelial cells were obtained from healthy control subjects (n=9). Subjects had not experienced a symptomatic upper respiratory tract infection in the preceding 6 weeks. Primary ciliated epithelium was obtained by brushing the inferior nasal turbinate and cultured to air-liquid interface as previously described10.\n\nType-2 alveolar basal epithelial cells (A549) cells (American Type Culture Collection (ATCC), Manassas, VA) were grown in RPMI medium (Gibco) with 10% heat-inactivated foetal calf serum (Sigma), 100 U/ml penicillin, 10 μg/ml streptomycin (pen/strep) and fungizone. Madin-Darby canine kidney (MDCK) (ATCC, Manassas, VA) cells were grown to in Dulbecco's modified eagle medium (DMEM) with 10% foetal calf serum, and pen/strep as above.\n\nHuman influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) was grown in fertilized chicken eggs. Eggs were infected at day 9, incubated at 37°C for 2 days and virus harvested in the allantoic fluid at day 11. Allantoic fluid was harvested from uninfected chicken eggs for use as a negative control. Virus stocks were titred by plaque assay using MDCK cells grown to 90% confluence in 96-well dishes. Cells were washed with PBS and infected with serial dilutions of the virus in DMEM for 1 h at 37°C. The inoculum was removed and cells were incubated with 200 μl DMEM (medium containing 1.4% BSA, 2 μg/ml of trypsin and 1x penicillin/streptomycin antibiotics (VX15140122, Fisher)) at 37°C, 5% CO2 for 2-3 days. Virus plaques were visualized by staining with 1:200 mouse H36-4.5-2 anti-HA antibodies (in-house monoclonal provided by A. Easton) and an Alexa-594 labelled secondary anti-mouse antibody diluted 1:250 (A-11062, Invitrogen, UK).\n\nInfluenza aerosols were generated from a 1 ml suspension containing 1010 plaque-forming units (PFU)/ml using a Pari-Therm nebuliser (PARI Respiratory Equipment Inc, Midlothian, VA, USA). This was connected to a viable impactor (6-stage Microbial sampler, Westech Scientific Instruments, Upper Stondon, Bedfordshire, UK) to collect and fractionate the aerosolised influenza virus particles by aerodynamic size (Figure 2A) as described previously16. Particles were impacted into separate Petri dishes containing 20 ml of medium (RPMI Media 1640, Life Technologies, Grand Island, NY, USA) with added penicillin (50 μg/ml), streptomycin (50 μg/ml) and Fungizone (1 μg/ml).\n\nQuantification of viable influenza virus in the fractionated aerosolized particles was performed using MDCK cells seeded into 96 well plates. Triplicate wells were exposed to 200 μl of a log dilution series of the impacted air sample at 37°C in 5% CO2 for 2 hours. The sample was removed using two washes with DMEM and incubated at 37°C for 48 hours. Influenza virus was detected as above by direct immune-fluorescent staining. The number of immune-fluorescent plaques per well were counted and the total PFU in each sample calculated.\n\nThis was performed using the traditional method of liquid immersion cells with fluid containing virus or using our bespoke aerosolisation system (Figure 1). Frozen aliquots of influenza virus were thawed immediately prior to use and diluted in BEBM basal medium (Lonza, CC3171) to 1x105 PFU/ml. Prior to infection, the basolateral culture medium was removed and the apical surfaces of ALI cultures were rinsed with medium. Next, 200 µl of viral suspension (multiplicity of infection (MOI) of ~0.1 (based on an estimate of 5x105cells/ALI well)) in BEBM was directly applied to the apical surface for 1 hour and then removed. We also delivered the virus using a bespoke nebulisation system (Figure 1A), which allows aerosols to be delivered to the surface of ciliated cells in culture under direct vision using an inverted microscope (Figure 1B). We generated influenza aerosols from suspensions containing doses of 105, 106 or 107 PFU/ml using the Pari-Therm nebuliser. The nebulised virus was then delivered directly to the Transwell inserts via a static-free stainless-steel pipe to negate the effect of static charge on the aerosolised particles generated. Delivery to the cells in culture was via a stainless-steel t-piece at the end of the steel pipe. Both ends were clear to both reduce any pressure effects on the ciliated cultures and to allow real-time observation by high-speed video photography. The entire system was encased in a heated (37°C) Perspex chamber, to mimic body temperature and to prevent the spread of the biological agents. The Pari-Therm was chosen as it warms the aerosols to approximately 32°C thus reducing the possible negative cellular effects of cold aerosol landing on the cells. Cultures were exposed to influenza aerosols for 1 minute. The nebuliser was then switched off and the cultures left for 1 hour. All cultures were maintained at 37°C. Control wells received nebulised allantoic fluid without virus in BEBM. Cells were then washed three times with BEBM to remove unbound virus and the infections were allowed to continue for up to 24 hours. At this time the apical surface of the cells was rinsed and the wash was stored at -80°C for plaque assay and cytokine analyses. Cells were fixed overnight at 4°C with 4% paraformaldehyde in phosphate buffered saline (PBS) for immunostaining.\n\n(A) Photograph and (B) diagram of the cell culture aerosolisation system. Aerosol generated in the Pari-Therm by compressed gas is heated by an internal heater to 32°C and transported via aluminium tubing, to reduce static attraction of particles, to cell cultures grown at an air-liquid interface. Vents at the top and base of the ‘t-piece’ were incorporated to reduce the pressure effect of the compressed gas used to power the nebuliser on the cells in culture. The open top vent allowed continual observation, with high-speed video imaging, during nebulisation. The cells were contained in an inner chamber heated to 37°C to prevent leakage of biological aerosols in to the atmosphere. The nebuliser and inner chamber were surrounded by an outer Perspex chamber, heated to 37°C to help stabilise experimental temperature and to prevent cooling of the aerosolised aerosol as it passed from the nebuliser to the cells in culture.\n\n(A) A549 cells infected with aerosolised influenza virus. Cells were stained with an antibody specific for influenza virus HA antigens (red). Nuclei were stained using Hoescht (blue). No influenza virus was found after 24 hours in the control wells. (B) Quantification of the amount (PFU/ml) of viable influenza particles collected by the different stages of the six-stage viable impactor (Westech) used in this study. The impactor consists of six stages allowing fractionation of the particles into the following aerodynamic size ranges: 7 µm and above (Stage 1); 4.7-7 µm (Stage 2); 3.3-4.7 µm (Stage 3); 2.1-3.3 µm (Stage 4); 1.1-2.1 µm (Stage 5); 0.65-1.1 µm (Stage 6). The nebuliser was loaded with a dose of 2 ml of 2×104 HAU/ml ~ 1010 influenza A/PR/8 virus particles and run for 10 minutes. Viable virus was quantified by plaque assay using MDCK cells. All data the mean average of n=3 impactor runs. (C) Efficiency of liquid immersion system (LIS) verses aerosol (ALI) flu delivery on to Transwell inserts containing MDCK cultures grown at an air-liquid interface. Cells were stained with an antibody specific for influenza virus HA antigens (red) and the whole well was imaged for infected cells, a reduction in fluorescence indicates a reduction in the number of infected cells. These images indicate a 10-fold loss in viral progeny when virus was aerosolised compared to liquid immersion infections (n=3). Arrows indicate infected cells. (D) The viral titre (PFU/ml) of apical supernatants collected from MDCK cells infected with aerosolised or liquid immersion preparations of influenza A/PR/8 virus for 24 hours (n=3). Clear squares represent the aerosol method, filled squares represent the liquid immersion method. Values represent the mean (± SD).\n\nCultures were placed in an incubation chamber at 37°C for 30 minutes and were observed via an inverted microscope system (Nikon TU1000, UK). Beating cilia were recorded using a digital high-speed video camera (Lake Image Systems, USA) at a rate of 250 frames per second using an x40 objective as previously described17. Ciliary dyskinesia was defined as ciliated cells that displayed uncoordinated motile cilia or those that beat with a stiff, flickering or twitching motion. The dyskinesia index was calculated as the percentage of dyskinetic ciliated cells relative to the total number of motile ciliated cells.\n\nChemokines and cytokines were measured using a 96-well multispot assay (Meso Scale Discovery, Maryland, USA) as described previously10. The lower limit of detection was 1 pg/ml.\n\nCells were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C. Following fixation, cells were washed three times with PBS, blocked with 3% (w/v) BSA in PBS for 10 minutes, and washed again three times with PBS. All subsequent antibody incubations were carried out in PBS containing 1% (w/v) BSA. Reagents used for immunofluorescence in this study were anti-influenza virus HA mouse monoclonal antibody H36-4.5-2 (40 µg/ml) as above and 1:50 anti-β-tubulin III rabbit monoclonal antibody (ab52623, Abcam). Subsequent incubations were performed as described previously10. High resolution optical sections were obtained using a Leica TCS SP5 confocal microscope using a 63x oil objective (numerical aperture, 1.4). Images acquired by confocal microscopy were rendered by Imaris Software v7.2 (Bitplane AG).\n\nImages obtained using a 20x objective were used to quantify the level of anti-β-tubulin III staining by mean fluorescence intensity using NIS Elements Software (Nikon Instruments, Kingston, UK).\n\nStatistical analysis was performed using GraphPad Prism 5 (GraphPad, San Diego, CA, USA). Any difference in the ciliary activity observed for control and influenza virus was determined using paired t-tests. Within-group comparisons of the magnitude of chemokine/cytokine release were conducted using a Wilcoxon signed ranks test. Between-groups comparisons were performed using the Mann–Whitney U-test.\n\nEthical approval was obtained from the University of Leicester Committee for Research Ethics (Leicester, UK). All adult subjects provided informed written consent.\n\nWork with embryonated hen’s eggs was approved by the University of Warwick Animal Welfare and Ethical Review Body carried out under a licence approved by the UK Home Office as required by the Animal Scientific Procedures Act (1986).\n\n\nResults\n\nThe Pari-therm nebuliser generated infectious influenza virus aerosol particles that ranged from 0.65 to >7 µm in size (Figure 2B). A substantial proportion (84%) of infectious influenza virus was contained in particles less than 4.7 µm, a particle size where inhalation into the lower respiratory tract is likely. The total number of infectious influenza virus particles produced by the nebuliser was 2x109 PFU indicating an approximate 5-fold loss in viral viability or infectivity from the inoculum (1010 PFU). Raw PFU data are available on Figshare18\n\nWe found that nebulising 1×108 PFU for 1 minute resulted in 105 PFU landing on the membrane growth area of an empty Transwell (no cells). This aerosol inoculum (MOI of approximately 0.2) was shown to result in approximately 20% of infected epithelial cells being virus-positive after 24 h. In separate wells, cells were infected via liquid immersion using a viral inoculum of MOI of 0.2. This was to confirm that any effects were due to the amount of virus landing on the cells, rather than any difference in the ability of the virus to infect (Figure 2C). This data showed that the nebuliser was required to contain a virus preparation that was ten-fold higher than that delivered in the immersion system. This was reflected in the amount of virus released by these infected cells into the apical fluid after 24 h infection (Figure 2D).\n\nEffect of influenza virus infection on ciliary function. We found that influenza virus infection significantly reduced the proportion of epithelial cells with motile cilia (Table 1). Using the standard liquid immersion method, the number (median (IQR)) of ciliated epithelial cells per field significantly reduced (P<0.05) as early as 18 hours post-influenza virus infection (20 (11-39) cells/4.2 mm2) compared to the uninfected controls (27 (27-37) cells/4.2 mm2). After 24 hours infection this reduced to less than half (16 (11-29) cells/4.2 mm2) that detected in the uninfected controls (33 (27-40) cells/4.2 mm2). Despite this reduction in the numbers of cells with motile cilia, the ciliary beat frequency of the motile cilia on other ciliated cells in culture was unaffected by influenza virus infection over the 24-hour study period. The median (IQR) ciliary beat frequency of motile cilia on influenza virus infected nasal ciliated cells was indistinguishable after 24 hours (14.7 (9.7-15.2) Hz) from the uninfected controls (14.0 (11.7-15.7) Hz) (P=0.87). The cilia that remained motile following influenza virus infection showed no evidence of ciliary dyskinesia. The median (IQR) dyskinesia index was the same after 24 hours (0% (0%-5%)) as the uninfected controls (0% (0%-0%)) (P=0.87). Raw data on ciliary activity are available on Figshare19.\n\nData expressed as median (IQR).\n\n*Motility index, number of motile ciliated cells per sample area of ~4200 µm2 (n=2-9 donors). †Dyskinesia index, percentage of dyskinetically beating cilia amongst all cilia examined n=3 donors. ‡ Significant difference from time point 0 (P<0.05) using a Wilcoxon signed ranks test. nd, not done; p.i., post-infection.\n\nInfection of ciliated epithelial cells by aerosolised influenza virus results in similar cytopathology as standard method. Human ciliated airway epithelial cells infected with aerosolised influenza A virus resulted in similar levels of cytopathology (cells rounding, lifting off and tight junctions detected) as those immersed in the same concentration of influenza virus. The number (median (IQR)) of motile ciliated epithelial cells per field reduced two-fold to 7.8 (4.0-10.4) cells/4.2 mm2) compared to the pre-infection level (14.0 (5.2-22.8) cells/4.2 mm2). The ciliary beat frequency (CBF) of the remaining motile ciliated epithelial cells exposed to aerosolised inoculum of influenza A virus (16.6 (15.4-17.6) Hz) remained unchanged from pre-infection level (15.8 (15.2-16.3) Hz) and the uninfected control of 15.5 (13.1-17.3) Hz. These values do not account for cilia on cells that have become static.\n\nThe concentration of inflammatory mediators detected in the apical fluid of cell cultures subjected to the aerosol delivery of the control and influenza virus preparations resulted in significantly lower levels of inflammatory mediators after 24 hours compared to the liquid immersion system (Table 2). In addition to lower concentrations, the two systems produced a different cytokine response to influenza virus infection. Among the ten cytokines measured, influenza virus infection by liquid immersion caused a significant (P<0.05) downregulation of IL-6 secretion to a median (IQR) of 333 pg/ml (107-1304) from 548 pg/ml (418-1525) in control uninfected cells. However, the aerosol delivery of influenza virus resulted in an upregulation of IL-6 to 1297 pg/ml (1107-1371) from 968 pg/ml (700-1235) in control uninfected cells. The same trend was seen with another anti-inflammatory cytokine IL-13, which showed significant upregulation in the aerosol group from the control 50 pg/ml (48-53) to 65 pg/ml (55-65) following infection, compared to a downregulation in the liquid immersion group from 70 pg/ml (65-76) to 55 pg/ml (22-76), respectively.\n\n*Significant difference from control at same time point (P<0.05) using a Wilcoxon signed ranks test. nd, not detected\n\nAmong the nine chemokines measured, ciliated epithelial cells exposed to a liquid immersion inoculum of influenza virus resulted in a significant upregulation of CCL22 from a median (IQR) uninfected control of 2,0545 pg/ml (9986-23181) to 22812 pg/ml (20,342-30,853) (Table 2), whereas exposure to a aerosolised inoculum and control solution resulted in undetectable levels of this chemokine. MCP-1 and MCP-4 both showed a significant (P<0.05) increase following aerosolised delivery, but this was not significantly altered in the group exposed to the liquid immersion inoculum. However, both delivery systems show similar CXCL8 and CXCL10 responses to influenza virus, where production increased approximately 1.5-fold and 6.5-fold, respectively. In the liquid immersion delivery system, median (IQR) CXCL8 increased from 594 pg/ml (506-989) in the uninfected control cells to 887 pg/ml (692-1190). The aerosol delivery system resulted in much higher concentrations of CXCL8 from 65,523 pg/ml (44,759-86,287) in the uninfected control cells to 109,039 pg/ml (86,701-113,242) compared to the liquid immersion system. CXCL10 increased from 698 pg/ml (433-974) in the uninfected control cells to 4140 pg/ml (823-19884). MIP-1β, Eotaxin-3, TARC and MDC were unable to be detected in the apical fluids of cells exposed to aerosolised medium or virus, but liquid immersion the membrane with medium alone caused levels to exceed 30 pg/ml, 3500 pg/ml, 290 pg/ml and 20,000 pg/ml, respectively. Raw data from measurement of inflammatory mediators are available on Figshare20.\n\nThe distribution of influenza HA antigen on human ciliated epithelial cells. We have previously shown that RSV preferentially infects ciliated cells and this infection progresses with viral antigen being displayed on the cell surface, leading to viral antigen moving into the ciliary shaft after 24 h1,2. To determine whether influenza virus follows a similar pattern of viral antigen spread we used immunofluorescent staining to detect influenza virus haemagglutinin (HA) and ciliary (β-tubulin III) antigens on infected cells. We found a high level of influenza virus HA antigen accumulated on the distal ends of the cilia (ciliary tips) (Figure 3A, panel A) and the apical cell surface of ciliated cells (Figure 3A, panel B). We observed the co-localisation of influenza virus and anti-β-tubulin III, indicating virus HA antigen is displayed on the ciliary axoneme (Figure 3A, panel C). Notably, we found that cells either displayed viral antigens only on the ciliary tips or the full length of the ciliary axoneme. In cells where the apical membrane (cell surface) showed evidence of viral antigen, antigen was always present over the full length of the ciliary axoneme suggesting spread of antigen from the tip to the base of the cilium. Raw mean fluorescence intensity values are available on Figshare\n\n(A) Immunolocalisation of influenza virus HA protein and cilia proteins on the surface of infected ciliated epithelial cells grown at air-liquid interface. Panel A: ciliated cell displaying viral antigen on the ciliary tips but not on the shaft of the cilia and not on the apical cell surface (virus indicated by the arrow a). Panel B: influenza virus infected ciliated epithelial cells 24 h post-infection showing viral antigen displayed on the surface of the cell (red) and the ciliary axoneme (yellow in merge). Panel C: Z-projection ofinfluenza virus infected ciliated epithelial cells 24 h post infection showing viral antigen displayed on the ciliary shaft (yellow in merge).Panel D: denuded ciliated cells displaying viral antigen on the surface of the cell and fragments of ciliary β-tubulin III staining (indicated by arrow b). Cells were co-stained with antibodies against β-tubulin III to detect the axonemal microtubules (FITC) and an antibody specific for influenza virus HA antigens (Alexa-594). Nuclei were stained using Hoescht (blue in merge). Merged images are shown where green indicates β-tubulin III protein, red indicates influenza HA and yellow indicates areas of influenza-tubulin antigen co-localisation. All images show the maximum intensity projection. Scale bars, 5 µm. (C) Quantification of tubulin staining on the surface of control (uninfected) and influenza virus infected ciliated epithelial cell cultures grown at air-liquid interface. Values represent the mean (and SD) fluorescence intensity of tubulin staining from 10 images (approx. 200 cells per image). **P<0.01 vs control.\n\nA number of cells were seen with partial and in some cases almost complete loss of cilia (Figure 3A, panel D). In these cells fragments of β-tubulin III staining was seen the surface of the cell indicating that the cilia may have been lost from infected ciliated cells. To determine whether cilia were lost as a result of influenza infection, we compared the level of anti-β-tubulin III, a marker of cilia, (as measured by fluorescence intensity) in control and infected cultures. This showed that influenza infection resulted in significantly (P<0.01) less anti-β-tubulin III staining compared to uninfected cultures (Figure 3C).\n\n\nDiscussion\n\nTo our knowledge, this study shows for the first time that human ciliated epithelial cells from the upper airway are a target of influenza virus infection and suggests infection is primarily seen in ciliated cells. Previous studies have indicated ciliated cell tropism by influenza virus in the lower airway of humans and ferrets4–6. Our study demonstrated that not only are nasal ciliated cells also a target, but early influenza virus infection causes a significant decrease in the number of cells with motile cilia. At 24 hours after influenza infection the number of motile cilia present in the epithelial cultures reduced to under half that seen in uninfected control cells. Such loss could be due to cilia being cleaved from the cell surface, cells being destroyed or cells undergoing apoptosis21 or the ciliated cells being shed from the surface. Confocal images suggested loss of cilia from the apical surface of ciliated cells was at least in part responsible for the reduction in cilia. Infected cells with no detectable cilia had evidence of small amounts of anti-β-tubulin III staining at their surface, suggesting cilia had been cleaved, supporting the indication of tropism of influenza for ciliated cells4–6. To further confirm the loss of ciliary axonemes following influenza infection we compared the levels of anti-β-tubulin III staining in infected and control wells. The levels were significantly reduced in infected wells, again supporting our finding that the number of cilia had decreased. These results are consistent with findings of reduced mucociliary transport in tracheal cultures and the tracheas in animals infected with influenza virus12,22,23. It is possible that in vivo, this rapid reduction in the number of motile ciliated cells may reduce mucociliary clearance early in the infective process, predisposing the respiratory tract to colonisation and infection by bacteria.\n\nIncreased ciliary dyskinesia is a common finding during infection of ciliated cells with bacteria and viruses such as RSV,6,10,24. However, influenza virus infection did not increase ciliary dyskinesia or reduce the ciliary beat frequency of the cilia that remained motile. Longer time courses are needed to determine the degree of further ciliary loss and to determine if ciliary function is affected.\n\nWe also found that influenza virus interacts with the ciliary tips and ciliary axonemes. In a number of images in our study influenza appeared to be present on the tips of cilia without the underlying cell being infected indicating that the virus may initially bind to the ciliary tip. On other cells, the ciliary tips and the top part of the ciliated shaft showed presence of antigen, with no antigen present on the rest of the ciliary membrane or cells surface, suggesting that virus may use the cilium as an entry point to the cell. Previous reports suggest that influenza virus budding occurs at the tips of the microvilli rather than the cilia themselves13. Some cells showed antigen on the ciliary membrane and on the cell surface. However, no cells showed antigen on the cell surface without antigen being present on the full length of the ciliary membrane. As discussed above, cells without cilia where antigen was present also showed staining with anti-β-tubulin III, suggesting they had previously been covered by cilia. These findings suggest a novel infective route by which influenza binds to the ciliary tips and enter the cell at that point with infection spreading down into the cell resulting in ciliary loss. The initial attachment to the ciliary tips rather than the cell surface, is consistent with the work of Button and colleagues25 who have shown that particles greater than 40 nm are unable to penetrate between cilia to reach the surface of the ciliated cells. Influenza virus particles are between 80 to 120 nm in diameter26 and would not be expected to easily penetrate this barrier. It will be important to investigate if other strains and subtypes of influenza virus show differences in their tropism for ciliated epithelial cells.\n\nInfluenza virus is thought to be spread by aerosols made when infected individuals cough, sneeze or talk27. However, all human and in vitro studies to date have delivered infectious virus to subjects in solution. We aimed to determine whether there were any differences in the response of ciliated airway epithelial cells to influenza virus delivered in suspension compared to the aerosolised form. We first validated the method so that the cells received the same dose whether delivered by aerosol or by liquid immersion. We found that the infectious nature of the virus was not altered by aerosolisation and, accounting for virus loss, we detected the same level of cytopathology and cell tropism as we reported using the liquid immersion method. However, we observed a significant difference in the type of inflammatory response. In the liquid immersion system, we found that influenza virus infection resulted in a significant downregulation of the anti-inflammatory cytokine IL-6 compared to the uninfected control cells. Notably, this was not accompanied by significant upregulation of pro-inflammatory mediators and no other cytokine tested showed any change in levels. Using the aerosol delivery method, we found that another anti-inflammatory cytokine IL-13 significantly increased. This was accompanied by an increase in IL-6, although this increase was not statistically significant, most likely due to a type two error associated with the low number of repeats. This data indicates that aerosol delivery of influenza virus may lead to an anti-inflammatory Th2 cytokine response\n\nWe also detected a huge difference between the aerosol and liquid immersion methods in regard to the baseline concentrations of all nine chemokines we tested, with some up to 20,000 times higher in one method than the other. In all but one chemokine tested (CXCL8), liquid immersion the epithelium with medium resulted in much higher concentration of chemokine production after 24 hours compared to the aerosol delivery method. MIP-1β, Eotaxin-3, TARC and MDC were not detected in the apical fluids of cells exposed to aerosolised medium or virus, but was detected when cells were infected by liquid immersion the membrane. This could indicate that liquid immersion the membrane removes or dilutes key feedback mechanisms that exist to regulate expression of these chemokines (or the carrier fluid in greater amounts is more toxic or liquid immersion means they are no longer exposed to air).\n\nDespite these differences in the baseline levels of chemokines, the response to infection in both models followed the same trend. Both increased secretion of the interferon gamma-induced protein 10 (CXCL10) and the neutrophil recruitment chemokine CXCL8 following influenza virus infection. This is consistent with our previous findings of viral infection in this model10 and suggests that the response of the infected epithelium is to recruit neutrophils to aid the host antiviral response. Although we did not detect a significant change in CXCL8 secretion using the aerosol system, our data analysis were limited by the number of biological repeats (n=3) and the natural variation in response we have previously reported from different donors10.\n\nIn summary, we have presented evidence that the influenza virus is tropic to ciliated cells of the upper respiratory tract and interacts directly with the tips and ciliary axoneme of motile cilia. Subsequent infection of the cell results in loss of cilia. We also show that the route of infection (via large or small droplet size) can impact on the type of inflammatory response produced by the cells within the first 24 hours. Further investigations using our unique aerosol delivery system should begin to address the mechanisms behind this.\n\n\nData availability\n\nFigshare: Ciliary activity (raw data for ciliary beat frequency/motility). https://doi.org/10.6084/m9.figshare.780380619.\n\nFigshare: Confocal dataset (raw confocal z-stack of infected epithelial cells). https://doi.org/10.6084/m9.figshare.780355728.\n\nFigshare: MFI of B tubulin with influenza infection (mean fluorescence intensity values for β-tubulin). https://doi.org/10.6084/m9.figshare.780354829.\n\nFigshare: chemokine/cytokine/NO multispot assay (raw data for chemokine/cytokine/NO assay). https://doi.org/10.6084/m9.figshare.780354520.\n\nFigshare: Viable Impactor results (raw pfu/ml of nebulised influenza A virus). https://doi.org/10.6084/m9.figshare.780353918.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThis work was supported by grants from Action Medical Research (SP4499 and SP4118). This research was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.\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\nThompson WW, Shay DK, Weintraub E, et al.: Influenza-associated hospitalizations in the United States. JAMA. 2004; 292(11): 1333–40. PubMed Abstract | Publisher Full Text\n\nThompson WW, Shay DK, Weintraub E, et al.: Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA. 2003; 289(2): 179–86. PubMed Abstract | Publisher Full Text\n\nZhirnov OP, Ikizler MR, Wright PF: Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases. J Virol. 2002; 76(17): 8682–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZeng H, Goldsmith CS, Maines TR, et al.: Tropism and infectivity of influenza virus, including highly pathogenic avian H5N1 virus, in ferret tracheal differentiated primary epithelial cell cultures. J Virol. 2013; 87(5): 2597–607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Riel D, Munster VJ, de Wit E, et al.: Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals. Am J Pathol. 2007; 171(4): 1215–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThompson CI, Barclay WS, Zambon MC, et al.: Infection of human airway epithelium by human and avian strains of influenza a virus. J Virol. 2006; 80(16): 8060–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHirst RA, Gosai B, Rutman A, et al.: Streptococcus pneumoniae damages the ciliated ependyma of the brain during meningitis. Infect Immun. 2003; 71(10): 6095–100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith CM, Fadaee-Shohada MJ, Sawhney R, et al.: Ciliated cultures from patients with primary ciliary dyskinesia do not produce nitric oxide or inducible nitric oxide synthase during early infection. Chest. 2013; 144(5): 1671–1676. PubMed Abstract | Publisher Full Text\n\nChilvers MA, McKean M, Rutman A, et al.: The effects of coronavirus on human nasal ciliated respiratory epithelium. Eur Respir J. 2001; 18(6): 965–70. PubMed Abstract | Publisher Full Text\n\nSmith CM, Kulkarni H, Radhakrishnan P, et al.: Ciliary dyskinesia is an early feature of respiratory syncytial virus infection. Eur Respir J. 2013; 43(2): 485–96. PubMed Abstract | Publisher Full Text\n\nSmith CM, Sandrini S, Datta S, et al.: Respiratory syncytial virus increases the virulence of Streptococcus pneumoniae by binding to penicillin binding protein 1a. A new paradigm in respiratory infection. Am J Respir Crit Care Med. 2014; 190(2): 196–207. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoke CH Jr, Hopkins JA, Meiklejohn G, et al.: Comparison of several wild-type influenza-viruses in the ferret tracheal organ-culture system. Rev Infect Dis. 1979; 1(6): 946–54. PubMed Abstract | Publisher Full Text\n\nKolesnikova L, Heck S, Matrosovich T, et al.: Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells. J Gen Virol. 2013; 94(Pt 5): 971–6. PubMed Abstract | Publisher Full Text\n\nZhang L, Peeples ME, Boucher RC, et al.: Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol. 2002; 76(11): 5654–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAvadhanula V, Rodriguez CA, Devincenzo JP, et al.: Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner. J Virol. 2006; 80(4): 1629–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKulkarni H, Smith CM, Lee Ddo H, et al.: Evidence of Respiratory Syncytial Virus Spread by Aerosol. Time to Revisit Infection Control Strategies? Am J Respir Crit Care Med. 2016; 194(3): 308–16. PubMed Abstract | Publisher Full Text\n\nChilvers MA, O'Callaghan C: Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods. Thorax. 2000; 55(4): 314–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith C: Viable Impactor results. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7803539.v1\n\nSmith C: Ciliary activity. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7803806.v1\n\nSmith C: chemokine/cytokine/NO multispot assay. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7803545.v1\n\nMorris SJ, Price GE, Barnett JM, et al.: Role of neuraminidase in influenza virus-induced apoptosis. J Gen Virol. 1999; 80(Pt 1): 137–46. PubMed Abstract | Publisher Full Text\n\nPittet LA, Hall-Stoodley L, Rutkowski MR, et al.: Influenza virus infection decreases tracheal mucociliary velocity and clearance of Streptococcus pneumoniae. Am J Respir Cell Mol Biol. 2010; 42(4): 450–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark K, Bakaletz LO, Coticchia JM, et al.: Effect of influenza A virus on ciliary activity and dye transport function in the chinchilla eustachian tube. Ann Otol Rhinol Laryngol. 1993; 102(7): 551–8. PubMed Abstract | Publisher Full Text\n\nFreestone PP, Hirst RA, Sandrini SM, et al.: Pseudomonas aeruginosa-catecholamine inotrope interactions: a contributory factor in the development of ventilator-associated pneumonia? Chest. 2012; 142(5): 1200–10. PubMed Abstract | Publisher Full Text\n\nButton B, Cai LH, Ehre C, et al.: A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science. 2012; 337(6097): 937–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCouch RB: Orthomyxoviruses. In: Baron S, editor. Medical Microbiology. 4th edition ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. PubMed Abstract\n\nTellier R: Aerosol transmission of influenza A virus: a review of new studies. J R Soc Interface. 2009; 6 Suppl 6: S783–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith C: confocal dataset. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7803557.v1\n\nSmith C: MFI of B tubulin with influenza infection. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7803548.v1"
}
|
[
{
"id": "46273",
"date": "11 Apr 2019",
"name": "Gemma L Saint",
"expertise": [
"Reviewer Expertise Bronchiolitis",
"RSV",
"RhV",
"NTM infection in CF"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Smith et al. explores the earliest stage of influenza infection on epithelial cells, specifically ciliated airway epithelium cells. They use striking confocal microscopy and high-speed video microscopy to visualise influenza-cilia interaction. In addition, they present a novel method of inoculating airway cells with influenza, which aims to better replicate presumed in vivo virus transmission. The lower level of cytokine responses in the non-infected control samples, using the aerosol method compared to liquid immersion, hint that this method may reduce responses elicited from immersion rather than infection itself. I anticipate this novel inoculation method will be invaluable in further studies.\n\nThe report is well written, and all data are explained in sufficient detail and presented well. All source data is accounted for and accessible to the reader.\n\nI have answered ‘partly’ to the question - are the conclusions drawn adequately supported by the results – due to one point that I think requires some clarification if possible.\n\nI note the motility index for the aerosol method (both control and aerosol) was considerably lower than those of the liquid method. Is this normal variation for the ALI cultures? In addition, I note that for the controls of the aerosol model the motility index significantly reduces from time point 0 to 24hrs. This is not addressed in the manuscript and the results section only references the motility of the cells from the liquid method. If I have understood correctly, the aerosol method does not show a change in motility index compared to the control as both show a reduction? Also could the authors explain why the dyskinesia index was not done for time point 0 for the aerosol method?\n\nThere is just one minor point which is the following sentence, which I think just needs amending for clarity. “This could indicate that liquid immersion the membrane removes or dilutes key feedback mechanisms that exist to regulate expression of these chemokines (or the carrier fluid in greater amounts is more toxic or liquid immersion means they are no longer exposed to air).”\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "49307",
"date": "10 Jun 2019",
"name": "Kirsty R Short",
"expertise": [
"Reviewer Expertise influenza virus"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Smith et al, looks at the interactions between influenza virus and ciliated epithelial cells. Specifically, they compare infection and cytokine production after PR8/34 is delivered by immersion or aerosol. Overall, the article is well written and data is presented clearly. The infection via aerosol developed herein is a highly useful method and could be employed by other researchers in a variety of different contexts, including trying to better understand influenza virus transmission. Overall, I just have two minor points:\nThe authors use PR8/34 in this study and describe it as a ‘human influenza virus’. Whilst originally this was a human influenza virus strain, it has since been passaged over 100 times in mice and is more accurately described as ‘mouse adapted influenza’. Can the authors explain the choice of virus? It would be fantastic to see some of these data repeated with a real human influenza virus strain. At a bare minimum, it should be acknowledged that PR8/34 is a suboptiminal virus strain to use for studies with human cells.\nThe authors state that “the total number of infectious influenza virus particles produced by the nebuliser was 2x109 PFU indicating an approximate 5-fold loss in viral viability” yet in the discussion they state that “the infectious nature of the virus was not altered by aerosolisation”. Can the authors please clarify? I would hypothesise that the process of aerosolisation resulted in more non-infectious virus particle which, in turn, stimulated a differential pro-inflammatory response in the ciliated epithelial cells. Can the authors please 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-337
|
https://f1000research.com/articles/8-336/v1
|
26 Mar 19
|
{
"type": "Case Report",
"title": "Case Report: Paradoxical acrodermatitis of Hallopeau-like eruption following anti-IL-17 therapy",
"authors": [
"Giulia Tadiotto Cicogna",
"Francesco Messina",
"Linda Nalotto",
"Serena Szekely",
"Mauro Alaibac",
"Giulia Tadiotto Cicogna",
"Francesco Messina",
"Linda Nalotto",
"Serena Szekely"
],
"abstract": "Psoriasis is a chronic immune-mediated inflammatory disease. Up to 40% of patients with psoriasis may develop psoriatic arthritis. Currently, interleukin (IL)-17/IL-23 pathways are identified as key factors in the immunopathogenesis of both conditions. Here we describe the case of a patient who developed psoriasiform skin lesions 10 months after the initiation of anti-IL17 therapy for psoriatic arthritis. The underlying disease had responded well to the therapy, but the patient developed a striking pustular eruption at the fingers with nail involvement, onycholysis, yellow discoloration, and subungual keratosis. Clinical and histological findings were consistent with an acrodermatitis continua of Hallopeau-like eruption. Skin lesions subsided after discontinuation of the responsible anti-IL17 agent. The interpretation of this paradoxical side effect of biological therapies remains unclear but may relate to an unbalanced inflammatory cytokine response induced by the inhibition of TNF activity. It is likely that patients, who are genetically prone, may respond exaggeratedly to a cytokine imbalance. The identification of this kind of patient, in the future, could be useful in order to choose the correct therapy.",
"keywords": [
"IL-17",
"psoriasis",
"Acrodermatitis continua of Hallopeau",
"paradoxical reaction"
],
"content": "Introduction\n\nPsoriasis is an immune-mediated inflammatory disease characterized by a chronic course and a systemic involvement. Numerous cytokines are involved in the pathogenesis of psoriasis, but the interleukin (IL)-23/17 axis has been identified as one of the key pathways1. Up to 40% of patients with psoriasis may develop psoriatic arthritis (PsA). Both conditions share common pathogenic mechanisms2. Among the therapies that can be used for both skin and joint manifestations, anti-TNF agents and new biologics targeting IL-17 have shown impressive efficacy3. We report the case of a patient who presented with an acrodermatitis continua of Hallopeau (ACH)-like paradoxical reaction to anti-IL17 therapy for PsA.\n\n\nCase report\n\nA 52-year-old-woman affected by PsA presented to our Dermatology Unit complaining of a painful eruption of pustules with scaling and tender swelling on the fingers of both hands (Figure 1), which had begun one month before. Her medical history revealed concurrent PsA in complete remission after 10 months of the, the anti-IL-17 drug, secukinumab, at the dosage of 150mg every 4 weeks. The patient did not suffer from any other relevant disease and there was no family history of psoriasis.\n\nA skin biopsy was taken, and the subsequent histopathological examination showed a stratified squamous epithelium with parakeratosis, hyperkeratosis and irregular elongation of the rete ridges of the epidermis with some lymphocytes and subcorneal collections of neutrophils forming spongiform pustules of Kogoj. This result, together with clinical features and negative results of multiple cultures confirmed our suspect of an ACH-like eruption4.\n\nIt was then decided to stop anti-IL-17 therapy. A subsequent treatment plan comprised topical clobetasol propionate, once daily, and acitretin 10mg daily.\n\nAt the follow-up visit 2 months later, the lesions had visibly regressed and the patient referred a 70% reduction in symptoms measured with Dermatology Life Quality Index questionnaire. The interruption of secukinumab notwithstanding, PsA showed no recrudescence.\n\n\nDiscussion\n\nThe natural history of psoriasis has been modified in the last years by new biologic agents that have allowed specific targeting of key cytokines such as TNF alpha, IL-12, IL-23 and IL-17.\n\nTNF alpha inhibitors are the oldest, and therefore most studied, biologic drugs that have been introduced in the therapy of psoriasis. By inhibiting the whole pathway, TNF alpha blockers determine a stronger alteration of cytokine network5. This has been associated not only with impressive efficacy, but also remarkable side effects, such as infections, autoimmune diseases, lymphomas and cutaneous adverse events, mainly represented by paradoxical psoriasis6.\n\nIn the last years, new biologic therapies targeting cytokines situated downstream to TNF alpha, such as ustekinumab, secukinumab and, more recently, ixekizumab have shown even higher efficacy due to their selectivity in blocking cytokines specific to the pathogenic pathways. Also for this drug, although uncommon, important adverse reactions such as opportunistic infections, can be observed7.\n\nIn the literature, one case of paradoxical psoriasic reaction has recently been described in association to secukinumab8. In our case report we have observed an unexpected case of paradoxical ACH-like eruption, which to the best of our knowledge, has not been described in literature as an adverse event of secukinumab.\n\nIt is possible that secukinumab, by blocking IL-17, has induced a rearrangement of the cytokine pattern in this patient, determining a paradoxical increase of other pathogenic molecules, such as TNF alpha. This hypothesis has already been suggested for paradoxical reactions to ustekinumab, which exerts a blockade of IL12 and IL239. On the other hand, is also possible that the inhibition of IL17 induced a negative feedback in the IL23-IL17 axis, thus determining an increase of IL23 which, in turn, stimulated Th17 cells to produce other cytokines, such as IL-22, which also exerts proliferation and activation of keratinocytes. Activated keratinocytes could induce the chemotaxis of neutrophils (IL-8 etc), causing the clinical presentation that we have observed in our patient10. This is the first case report describing ACH-like lesions induced by secukinumab; therefore few studies are available in the literature elucidating the pathogenesis of this paradoxical reaction. Hence, our experience needs to be reinforced by further investigations.\n\nIt is likely that our patient was genetically prone to respond exaggeratedly to a cytokine imbalance. The distinction of such patients could be useful in the future, in order to predict this type of adverse reaction and, therefore, suggesting them an alternative to biologic therapies.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nTsai Y, Tsai T: Anti-interleukin and interleukin therapies for psoriasis: current evidence and clinical usefulness. Ther Adv Musculoskelet Dis. 2017; 9(11): 277–294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMease PJ, Armstrong AW: Managing patients with psoriatic disease: the diagnosis and pharmacologic treatment of psoriatic arthritis in patients with psoriasis. Drugs. 2014; 74(4): 423–441. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGan EY, Chong WS, Tey HL: Therapeutic strategies in psoriasis patients with psoriatic arthritis: focus on new agents. BioDrug. 2013; 27(4): 359–373. PubMed Abstract | Publisher Full Text\n\nSehgal VN, Verma P, Sharma S, et al.: Acrodermatitis continua of Hallopeau: evolution of treatment options. Int J Dermatol. 2011; 50(10): 1195–1211. PubMed Abstract | Publisher Full Text\n\nChiricozzi A, Romanelli P, Volpe E, et al.: Scanning the Immunopathogenesis of Psoriasis. Int J Mol Sci. 2018; 19(1): pii: E179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeluso R, Cafaro G, Di Minno A, et al.: Side effects of TNF-α blockers in patients with psoriatic arthritis: evidences from literature studies. Clin Rheumatol. 2013; 32(6): 743–753. PubMed Abstract | Publisher Full Text\n\nGarcia-Valladares I, Cuchacovich R, Espinoza LR: Comparative assessment of biologics in treatment of psoriasis: drug design and clinical effectiveness of ustekinumab. Drug Des Devel Ther. 2011; 5: 41–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoshina D, Haga N, Furuya K, et al.: Paradoxical localized exacerbation of psoriatic eruptions triggered by secukinumab. Clin Exp Dermatol. 2018; 43(6): 718–719. PubMed Abstract | Publisher Full Text\n\nWenk KS, Claros JM, Ehrlich A: Flare of pustular psoriasis after initiating ustekinumab therapy. J Dermatolog Treat. 2012; 23(3): 212–214. PubMed Abstract | Publisher Full Text\n\nPuig L: Paradoxical Reactions: Anti-Tumor Necrosis Factor Alpha Agents, Ustekinumab, Secukinumab, Ixekizumab, and Others. Curr Probl Dermatol. 2018; 53: 49–63. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "48170",
"date": "15 May 2019",
"name": "Archana Gopalakrishnan",
"expertise": [
"Reviewer Expertise Immunology of infected diseases",
"Immune response to tuberculosis and influenza",
"TLR signaling",
"inflammation",
"host directed therapy and vaccine mediated immunity."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nParadoxical effects of anti-cytokine therapy is a very important area that the authors have highlighted in this case study. As has been well documented, the use of therapies, such as anti-TNF, although groundbreaking does raise a multitude of issues - including higher risk to infections - specifically tuberculosis. The authors have documented well that the patient who was on anti-IL-17 therapy did not suffer from any infections, an important advantage of using secukinumab.\nSome of the points that could have been elaborated:\nWould decreasing the dosage of the drug (from 150mg every 4 weeks) been able to reduce the ACH like eruption along with treating PsA? It would be useful if the authors could reference successful cases, if any documented, where the drug has been functional at lower doses. A cytokine imbalance leading to an increase in TNF during secukinumab treatment has been suggested - is there corroborating evidence that implies this? If blood samples from the patient has been saved, can systemic levels of TNF be assayed for? The authors raised an excellent point about underlying genetics contributing to the potential cytokine imbalance. This might be a very crucial finding and if so, could the authors highlight the nature of the polymorphisms to look for in future cases?\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": "5286",
"date": "09 Mar 2020",
"name": "Mauro Alaibac",
"role": "Author Response",
"response": "Dear Reviewer,Thank you for the very careful review of our paper. Please find below a point by point response to your constructive comments Would decreasing the dosage of the drug (from 150mg every 4 weeks) been able to reduce the ACH like eruption along with treating PsA? It would be useful if the authors could reference successful cases, if any documented, where the drug has been functional at lower doses. We are not able to confirm if a reduced dosage is more effective in suppressing the ACH like eruption than the discontinuation of the treatment as there are no documented cases appeared in the literature concerning this issue. 2. A cytokine imbalance leading to an increase in TNF during secukinumab treatment has been suggested - is there corroborating evidence that implies this? If blood samples from the patient has been saved, can systemic levels of TNF be assayed for? We have not saved a blood sample from the patient, but evaluation of systemic levels of TNF- alpha in patients under treatment with secukinumab could be an interesting investigation which could corroborate our hypothesis. 3 The authors raised an excellent point about underlying genetics contributing to the potential cytokine imbalance. This might be a very crucial finding and if so, could the authors highlight the nature of the polymorphisms to look for in future cases? There is a large heterogeneity of inflammatory networks driving psoriasis and this make difficult to identify a specific polymorphism which could be responsible for the potential cytokine imbalance."
}
]
},
{
"id": "51595",
"date": "05 Aug 2019",
"name": "Melinda J. Gooderham",
"expertise": [
"Reviewer Expertise Psoriatic disease",
"clinical trials",
"real world experience"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case of a 'paradoxical' reaction to a biologic agent, secukinumab, which is used to treat psoriatic disease. In this case, acrodermatitis of Hallopeau developed in this patient being treated successfully with secukinumab for psoriatic arthritis. It is important to report these rare events as we build our knowledge of real-world experience of the use of biologic agents in psoriatic disease.\nHowever, this is not the first report of this type of reaction to secukinumab in the literature. Sladden et al.1 reported a similar reaction of fingertip and nail psoriatic disease (ACH-like) after treatment of plaque psoriasis with secukinumab. The similarity in the presentation of these cases is quite interesting and should be noted. Similar to the story of 'paradoxical' psoriasis/psoriasiform eruption with TNF antagonists, as we use more IL-17 inhibitors in the real world, we can better categorize these unexpected eruptions and further research can help to determine the underlying pathophysiological relationship.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? 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": "5287",
"date": "09 Mar 2020",
"name": "Mauro Alaibac",
"role": "Author Response",
"response": "Dear Reviewer,Thank for your thoughtful and constructive comments. We do agree with you that similarly to TNF-alpha, IL-17 may show unexpected \"paradoxical\" eruptions and further experimental and clinical observations are necessary to determine the underlying pathophysiology driving this reaction."
}
]
}
] | 1
|
https://f1000research.com/articles/8-336
|
https://f1000research.com/articles/8-335/v1
|
26 Mar 19
|
{
"type": "Research Article",
"title": "The effect of a one-time 15-minute guided meditation (Isha Kriya) on stress and mood disturbances among operating room professionals: a prospective interventional pilot study",
"authors": [
"Valluvan Rangasamy",
"Ammu Thampi Susheela",
"Ariel Mueller",
"Tracy F. H. Chang",
"Senthilkumar Sadhasivam",
"Balachundhar Subramaniam",
"Valluvan Rangasamy",
"Ammu Thampi Susheela",
"Ariel Mueller",
"Tracy F. H. Chang",
"Senthilkumar Sadhasivam"
],
"abstract": "Background: Operating room professionals are exposed to high levels of stress and burnout. Besides affecting the individual, it can compromise patient safety and quality of care as well. Meditation practice is getting recognized for its ability to improve wellness among various populations, including healthcare providers. Methods: Baseline stress levels of perioperative healthcare providers were measured via an online survey using a Perceived Stress Scale (PSS) questionnaire. An in-person meditation workshop was demonstrated during surgical grand rounds and an international anesthesia conference using a 15-minute guided Isha Kriya meditation. The participants were then surveyed for mood changes before and after meditation using a Profile of Mood States (POMS) questionnaire. Results: Surgeons and anesthesiologists were found to have higher median (interquartile range) Perceived Stress Scores as compared to nurses respectively (17 [12, 20] and 17 [12, 21] vs 14 [9, 19]; P = 0.01). Total mood disturbances were found to be significantly reduced after meditation in both the surgical grand rounds (pre-meditation median [IQR] 99 [85, 112] vs 87 [80, 93] post-meditation; P < 0.0001) and anesthesia conference cohorts (pre-meditation 92 [86, 106] vs 87 [81, 92] post-meditation; P < 0.0001). Conclusions: Isha Kriya, a guided meditation, is easy to learn and takes less than 15 minutes to complete. This meditation technique improves mood changes and negative emotions among operating room professionals and could be used as a potential tool for improving wellness.",
"keywords": [
"meditation",
"stress",
"burnout",
"healthcare providers",
"anesthesia",
"surgery",
"operating room professionals"
],
"content": "Introduction\n\nWellness is not just merely an absence of a disease, it encompasses physical, mental and social well-being1. Stress associated with work, when left unaddressed, results in burnout. Around 30–60% of healthcare providers suffer from burnout and 400 physicians die by suicide every year2. Work environment (work overload, insufficient reward), demographic variables (early career, lack of life partner or children), and personality traits (low confidence, overachieving, impatience, perfectionist) can contribute to stress and burnout2.\n\nOperating rooms are highly stressful environments involving complex, respectful, and essential interactions. Surgeons, anesthesiologists and nurses can develop health problems, social issues and substance use due to stress and burnout3,4. Employee disengagement is estimated to cost around 450 billion USD per year, and illness-related absences are common in burned out employees5. Despite the critical need, no effective treatment options and well-described organizational approach exists for wellness among healthcare providers6.\n\nWorld Health Organization (WHO) emphasizes the need for workplace health interventions aiming to improve the wellness of employees1. However, there is a lack of high-quality trials with simple and potentially effective interventions. Isha Kriya (IK) is a 15-minute, simple guided meditation tool employing thought, breathing, and awareness5. We hypothesized that operating room professionals experience significant stress, and IK meditation would decrease their mood disturbances. In this pilot study, we sought to measure the a) stress levels among surgeons, nurses and anesthesiologists, and b) mood changes following a one-time IK meditation session.\n\n\nMethods\n\nThis prospective interventional pilot study was conducted after Institutional Review Board approval (Beth Israel Deaconess Medical Center, Boston, MA, US. Protocols 2017P000657, 2018P000200, 2018P000585) with an electronic consent. Written informed consent was waived by our IRB. This manuscript adheres to the applicable Consolidated Standards of Reporting Trials (CONSORT) statement for pilot and feasibility trials7,8.\n\nAn online survey with Perceived Stress Scale (PSS) questionnaire (available as extended data9) was used to assess the stress levels among the operating room professionals at a single academic teaching hospital. This 10-item questionnaire measures an individual’s feeling about a stress factor over the past 30 days10,11. Anesthesiologists were surveyed in February 2018, and other operating room professionals including nurses and surgeons were surveyed in August 2018. The survey was conducted anonymously and willing participants could complete using Research Electronic Data Capture (REDCap 8.10.6; Vanderbilt University) with three weekly automatic reminders for incomplete surveys.\n\nFollowing this survey, a 15-minute guided IK meditation (guide available as extended data12) was demonstrated during a) surgical grand rounds at our institution (September 2018) and b) the Annual Meeting of American Society of Anesthesiologists (ASA) wellness workshop (October 2018). The Profile of Mood States (POMS) [Revised Version 15.2]13 questionnaire (available as extended data14) was used to assess the mood changes before and after meditation. POMS is a psychological rating scale assessing transient and distinct mood states. In both the settings, willing participants could complete the survey anonymously either on paper or by using a link directly in REDCap.\n\nDescriptive data were presented as median (interquartile range) or frequencies and proportions. Normality was assessed using Shapiro-Wilk test. Differences between cohorts were assessed using Wilcoxon-Mann-Whitney or chi-square test, as appropriate. With small cell sizes, Fisher’s Exact test was used. No sample size calculations were done in this pilot trial. All willing members in the specific settings were included in the study.\n\nOur primary outcome, the mood changes with meditation, was assessed using separate Wilcoxon signed rank sum tests. Bonferroni correction was used to keep the familywise error rate at 0.05. P values were considered statistically significant when < 0.025 (0.05 / 2 tests). Analysis of secondary outcomes including positive and negative subscales with their respective components were deemed exploratory and P values < 0.05 were considered statistically significant. All data were analyzed using SAS 9.4 (SAS Institute Inc., Cary, NC).\n\n\nResults\n\nTable 1 displays the demographic information from the PSS survey. Of the total 910 participants, 362 (39.8%) completed the survey, including 101 (27.9%) anesthesiologists, 61 surgeons (16.9%) and 151 (41.7%) nurses. Respondents were primarily female (65.8%), white (73.1%), non-Hispanic/Latino (87.6%) and 30–44 years old (38%). One-third (32.8%) of respondents had more than 15 years of work experience. Nurses reported more years of work experience than the other respondents (47.7% vs 23% and 22.2%; P < 0.0001). Raw data from this survey are available on Figshare15.\n\nValues are presented as number (%) or median (quartile 1, quartile 3).\n\nSurgeons and anesthesiologists had higher median [IQR] perceived stress scores than nurses (17 [12, 20] and 17 [11.5, 21] vs 13.5 [9, 19]; P = 0.01). Table 2 compares the perceived stress scores of based on the clinical role. Surgery residents (17 [15, 19]) and fellows (18.5 [14, 20]) had higher stress scores than attending physicians (17.5 [10, 20]), although this difference was not significant (P = 0.12). A similar relationship was observed among residents and fellows in anesthesia (P = 0.71).\n\n*Values are presented as median (quartile 1, quartile 3).\n\nDemographic data for participants who completed the POMS survey were listed in Table 3. A total of 50 surgeons participated in IK meditation during surgical grand rounds and 28 (56%) completed the POMS questionnaire. Raw data from this questionnaire are available on Fighsare16. Similarly, 52 anesthesiologists attending the ASA conference participated in the meditation workshop and 44 (85%) completed the questionnaire. Respondents from the anesthesia conference were significantly older, more likely to be attending physicians and had more experience as compared to those from surgical grand rounds (all P values < 0.0001). A significantly higher number of anesthesia conference respondents reported meditating (P = 0.03) or performing aerobic exercise (P = 0.002) three to four times a week as compared to the surgical respondents at our institution. Only 7% of surgical and 27% of anesthesia conference respondents practiced meditation routinely.\n\nValues are presented as number (%).\n\nMood changes before and after IK meditation during surgical grand rounds and anesthesia conference were presented in Table 4 and Figure 1. In surgical grand rounds, total mood disturbances (TMD) were significantly reduced after meditation (pre vs post meditation: median [IQR] 99 [85, 112] vs 87 [80, 93]; P < 0.0001). A similar reduction was observed with all negative subscales (19 [7, 32] vs 7 [2, 31]; P < 0.0001), including tension, anger, fatigue, confusion and depression (all statistically significant; P-values < 0.003). No significant change was observed for positive subscales (22 [17, 26] vs 22 [18, 27]; P = 0.94), including esteem-related affect and vigor.\n\nValues are presented as median (quartile 1, quartile 3). Note: In order to account for multiple testing of our primary outcome, total mood disturbance, p-values for the primary outcome are significant if the p-value is < 0.025 (= 0.05 / 2). All other secondary outcomes including individual subscales are considered exploratory, therefore p-values < 0.05 are considered statistically significant. Significant values are denoted as * in the table above.\n\nEach of the boxplots represents the total mood disturbance before and after a single guided meditation session (Isha Kriya) among all respondents, including those at both the surgical grand rounds and the anesthesia conference.\n\nConsistent with the results from surgical respondents, TMD during anesthesia conference significantly reduced after meditation (92 [86, 106] vs 87 [81, 92]; P < 0.0001). Negative subscales showed a significant reduction after meditation (14 [11, 23] vs 4 [1, 9]; P < 0.0001). Individual subscales such as tension, anger, fatigue, confusion, and depression were also reduced significantly (all P values < 0.0001). No significant changes were seen for positive subscales.\n\n\nDiscussion\n\nThis study showed two important findings: a) high levels of stress among operating room professionals and b) one-time 15-minute guided IK meditation significantly reduced TMD, including negative subscales such as tension, anger, fatigue, depression and confusion.\n\nStress levels among physicians in this study were higher than those levels reported in the general population10. In a 2011 national survey, physicians had higher levels of stress and burnout than any other working population in the United States17. Excessive workload, administrative burdens, decreased meaning from work, and difficulty in managing personal life were some of the prominent reasons suggesting a significant workplace contribution for burnout.\n\nThe higher stress levels found among surgical residents and fellows in training than attending physicians were consistent with a nationwide survey from France that showed a 40% incidence of severe burnout in surgery residents18. Those authors insisted on further research and interventions to prevent any devastating effects among residents. In this study, we explored the effectiveness of guided IK meditation and found a significant improvement in mood changes.\n\nMindfulness and meditation techniques were utilized for the wellness of healthcare providers19. The estimated cost of burnout among physicians in Canada was found as $213 million20. Harvard business review reports that Johnson & Johnson saved around $250 million on health care costs by implementing in-house wellness programs21. In this study, after a 15-minute, simple, guided IK meditation, participants reported a reduction in mood disturbances. It is encouraging to compare these results with previous research where healthcare professionals reported widespread benefits after meditation programs19. However, the participants in those studies were from various parts of healthcare system, where the levels of stress and burnout could be different.\n\nThe operating room environment is unique, and conflicts may arise due to a difference in information, opinion, experience and interests. IK meditation could be used as a simple tool to de-identify thoughts and bodily sensations.\n\nOur study has several limitations. Although this study utilized anonymous surveys, participation bias becomes unavoidable. The participation was kept entirely voluntary, and self-selection bias could limit extrapolation of the observed results. Furthermore, our sample size was relatively small, which may have implications in the generalization of these results. To our knowledge, this study was the first to use a simple, one-time, 15-minute guided IK meditation for improving wellness among operating room professionals. We used a validated PSS and POMS questionnaire to observe the perceived stress level as well as mood changes after meditation.\n\nIn conclusion, our findings suggest that IK, a 15-minute guided meditation technique, could improve mood changes among operating room professionals. Stress levels observed among physicians emphasize the need for strategies aiming to improve wellness. Moreover, randomized trials and research observing long term effect of meditation practice compliance and positive provider wellbeing that will eventually translate to better patient outcomes.\n\n\nData availability\n\nFigshare: Perceived Stress Scale (PSS) Survey.xlsx. https://doi.org/10.6084/m9.figshare.780872315.\n\nFigshare: Profile of Mood States (POMS) survey before and after Isha Kriya meditation.xlsx. https://doi.org/10.6084/m9.figshare.780872916.\n\nFigshare: Perceived Stress Scale (PSS) survey questionnaire. https://doi.org/10.6084/m9.figshare.78087479.\n\nFigshare: Instructions and details of IshaKriya meditation session. https://doi.org/10.6084/m9.figshare.780874112.\n\nFigshare: Profile of Mood States (POMS) questionnaire https://doi.org/10.6084/m9.figshare.780875614.\n\nFigshare: CONSORT checklist, extension for Pilot and Feasibility Trials for study “The effect of a one-time 15-minute guided meditation (Isha Kriya) on stress and mood disturbances among operating room professionals: a prospective interventional pilot study”. https://doi.org/10.6084/m9.figshare.78087358.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declare that no grants were involved in supporting this work.\n\n\nReferences\n\nAnon: Healthy workplaces: a WHO global model for action. WHO. [Accessed October 28, 2018]. Reference Source\n\nDzau VJ, Kirch DG, Nasca TJ: WHO | Healthy workplaces: a WHO global model for action. N Engl J Med. 2018; 378(4): 312–314. PubMed Abstract | Publisher Full Text\n\nBalch CM, Shanafelt TD, Sloan JA, et al.: Distress and career satisfaction among 14 surgical specialties, comparing academic and private practice settings. Ann Surg. 2011; 254(4): 558–568. PubMed Abstract | Publisher Full Text\n\nHyman SA, Shotwell MS, Michaels DR, et al.: A Survey Evaluating Burnout, Health Status, Depression, Reported Alcohol and Substance Use, and Social Support of Anesthesiologists. Anesth Analg. 2017; 125(6): 2009–2018. PubMed Abstract | Publisher Full Text\n\nAnon: Isha Kriya Yoga - Free Online Guided Meditation Video By Sadhguru. [Accessed November 11, 2018]. Reference Source\n\nRomani M, Ashkar K: Burnout among physicians. Libyan J Med. 2014; 9: 23556. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEldridge SM, Chan CL, Campbell MJ, et al.: CONSORT 2010 statement: extension to randomised pilot and feasibility trials. BMJ. 2016; 355: i5239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRangasamy V: CONSORT checklist. figshare. Paper. 2019. http://www.doi.org/10.6084/m9.figshare.7808735.v1\n\nRangasamy V: Perceived Stress Scale (PSS) survey questionnaire. figshare. 2019. Paper. http://www.doi.org/10.6084/m9.figshare.7808747.v1\n\nCohen S, Kamarck T, Mermelstein R: A global measure of perceived stress. J Health Soc Behav. 1983; 24(4): 385–396. PubMed Abstract | Publisher Full Text\n\nTaylor JM: Psychometric analysis of the Ten-Item Perceived Stress Scale. Psychol Assess. 2015; 27(1): 90–101. PubMed Abstract | Publisher Full Text\n\nRangasamy V: Instructions and details of IshaKriya meditation session. figshare. Paper. 2019. http://www.doi.org/10.6084/m9.figshare.7808741.v1\n\nGrove JR, Prapavessis H: Preliminary evidence for the reliability and validity of an abbreviated profile of mood states. Int J Sport Psychol. 1992; 23(2): 93–109. Reference Source\n\nRangasamy V: Profile of Mood States (POMS) questionnaire. figshare. 2019. Paper. http://www.doi.org/10.6084/m9.figshare.7808756.v1\n\nRangasamy V: Perceived Stress Scale (PSS) Survey.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7808723.v1\n\nRangasamy V: Profile of Mood States (POMS) survey before and after Isha Kriya meditation.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7808729.v1\n\nShanafelt TD, Boone S, Tan L, et al.: Burnout and satisfaction with work-life balance among US physicians relative to the general US population. Arch Intern Med. 2012; 172(18): 1377–1385. PubMed Abstract | Publisher Full Text\n\nFaivre G, Kielwasser H, Bourgeois M, et al.: Burnout syndrome in orthopaedic and trauma surgery residents in France: A nationwide survey. Orthop Traumatol Surg Res. 2018; 104(8): 1291–1295. PubMed Abstract | Publisher Full Text\n\nLynch J, Prihodova L, Dunne PJ, et al.: Mantra meditation programme for emergency department staff: a qualitative study. BMJ Open. 2018; 8(9): e020685. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDewa CS, Jacobs P, Thanh NX, et al.: An estimate of the cost of burnout on early retirement and reduction in clinical hours of practicing physicians in Canada. BMC Health Serv Res. 2014; 14: 254. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerry LL, Mirabito AM, Baun WB: What’s the Hard Return on Employee Wellness Programs? [Accessed October 29, 2018]. Reference Source"
}
|
[
{
"id": "63520",
"date": "26 May 2020",
"name": "Akshay Anand",
"expertise": [
"Reviewer Expertise Yoga",
"Neurodegeneration",
"Amyotrophic Lateral Sclerosis",
"Retinal injury",
"Age-related macular degeneration"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled, ‘The effect of a one-time 15-minute guided meditation (Isha Kriya) on stress and mood disturbances among operating room professionals: a prospective interventional pilot study’ reports an interesting finding and is well written. Following comments will further help in improving the manuscript.\n\nComments\nOut of the 362 participants who completed the PSS, the data is presented for 313 participants including 101 anesthesiologists, 61 surgeons and 151 nurses. Who were the remaining 49 participants? Why were they excluded from the study?\n\nIn Table 1, the total number of participants in each column i.e. surgeons, anaesthesiologists and nurses under the categories/parameters such as age, race, ethnicity, clinical role and years of service does not match with number of participant i.e. 101 anesthesiologists, 61 surgeons and 151 nurses included in the study. For example, the sum of participants under surgeon column and race parameter is 70, which is more than the 61 surgeons who had completed the PSS.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "64639",
"date": "30 Jun 2020",
"name": "Salvatore Zaffina",
"expertise": [
"Reviewer Expertise Occupational medicine",
"Workplace health promotion",
"Public Health",
"Work related stress"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of the article is the use of meditation as intervention for the health and wellness promotion of healthcare professionals of operating room. Stress levels observed among physicians emphasize the need for strategies aiming to improve wellness.\nMeditation practice is getting recognized for its ability to improve wellness among various populations, including healthcare providers. Before the intervention the researchers provide a first measurement of stress of healthcare professionals of operating room. An in-person meditation workshop was demonstrated during surgical grand rounds and an international anesthesia conference using a 15-minute guided Isha Kriya meditation. The participants were then surveyed for mood changes before and after meditation using a Profile of Mood States questionnaire. The results of the pre-post measurements showed an improvement of mood changes and negative emotions.\n\nThe study is interesting and well conducted. The reviewer suggests some integrations and corrections described in the following comments.\n\nIn the abstract the Perceived Stress Scores of nurses presented a different value from that reported in the result section (14 instead of 13.5). In the abstract and all the manuscript change P<0.0001 with p<0.001 without spaces and P with p.\nIn the abstract sections and methods briefly describe the hospital where the study was conducted.\n\nIn the second line of results, the total number of participants in the three categories is 313 and not 362 (101 anesthesiologists, 61 surgeons and 151 nurses). Re-calculate the percentage by category and the resulting values of the other variables.\n\nIn the discussion on page 7 at the beginning of the first column, the authors should briefly compare their findings with some recently published studies on the use of mindfulness in healthcare professionals.\nPérez-Fuentes MDC, Gázquez Linares JJ, Molero Jurado MDM et al. The mediating role of cognitive and affective empathy in the relationship of mindfulness with engagement in nursing (2020)1\nLa Torre G, Raffone A, Peruzzo M et al. Yoga and Mindfulness as a Tool for Influencing Affectivity, Anxiety, Mental Health, and Stress among Healthcare Workers: Results of a Single-Arm Clinical Trial (2020) 2\nDucar DM, Penberthy JK, Schorling JB et al. Mindfulness for healthcare providers fosters professional quality of life and mindful attention among emergency medical technicians (2020) 3\nKlein A, Taieb O, Xavier S, Baubet T, Reyre A. The benefits of mindfulness-based interventions on burnout among health professionals: A systematic review. (2020) 4\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-335
|
https://f1000research.com/articles/2-82/v1
|
11 Mar 13
|
{
"type": "Method Article",
"title": "Solid state fluorescence of proteins in high throughput mode and its applications",
"authors": [
"Saurabh Gautam",
"Munishwar N Gupta",
"Saurabh Gautam"
],
"abstract": "A simple method to determine fluorescence emission spectra of proteins in solid state is described. The available commercial accessories can only accommodate solid samples and hence do not allow a direct comparison between fluorescence spectra of a sample in solution and solid state form. Such comparisons are valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). The commercially available accessories also do not allow working in a high throughput mode. We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples. This method works with a 96-well plate format. It enables the comparison of fluorescence spectra of a sample in a solid state with solution spectra, using comparable quantities of protein. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form and the lyophilized powder form. α-Chymotrypsin in solution showed a λmax of 335 nm while a high-activity preparation of the same enzyme for non-aqueous media, known as enzyme precipitated and rinsed with propanol (EPRP), showed an increase in the intensity of the fluorescence emission spectra. However, there was a small red shift of 2 nm (λmax of 337 nm) in contrast to lyophilized powder which showed a λmax of 328 nm. This is due to a difference in the tertiary structure of the protein as well as the microenvironment of aromatic residues between the two preparations. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. The relative fluorescence intensity of lyophilized GFP powder was decreased significantly to 17% as compared to GFP in solution, and showed a red shift of 4 nm in the emission λmax. It was found that fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP was significantly diminished. This indicated the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.",
"keywords": [
"Fluorescence spectroscopy is a powerful tool to study protein structure1–3. Measurement of the fluorescence of proteins",
"when the latter is present in the solid state",
"is useful in several different contexts. Solid state fluorescence has a number of uses including in protein assays with protein electrophoresis samples4",
"enzyme immobilization5",
"microscopy6",
"detecting changes in protein tertiary structure upon lyophilization7",
"sensors and microarrays8",
"9 and characterizing solid waste10. Of these",
"fluorescence measurement of lyophilized samples is itself valuable for a variety of different kinds of studies. Enzyme catalysis in low water media is often carried out with lyophilized enzyme powders11–14. Only recently",
"circular dichroism (CD) of α-chymotrypsin “dried” (bulk water removed) with different methods has been reported with the help of a special accessory for recording CD spectra of solid samples as suspensions15."
],
"content": "Introduction\n\nFluorescence spectroscopy is a powerful tool to study protein structure1–3. Measurement of the fluorescence of proteins, when the latter is present in the solid state, is useful in several different contexts. Solid state fluorescence has a number of uses including in protein assays with protein electrophoresis samples4, enzyme immobilization5, microscopy6, detecting changes in protein tertiary structure upon lyophilization7, sensors and microarrays8,9 and characterizing solid waste10. Of these, fluorescence measurement of lyophilized samples is itself valuable for a variety of different kinds of studies. Enzyme catalysis in low water media is often carried out with lyophilized enzyme powders11–14. Only recently, circular dichroism (CD) of α-chymotrypsin “dried” (bulk water removed) with different methods has been reported with the help of a special accessory for recording CD spectra of solid samples as suspensions15.\n\nSome commercially available accessories for spectrofluorimeters allow recording the fluorescence emission spectra of the solid samples7,16–19. These available commercial accessories can only accommodate solid samples and hence do not allow a direct comparison between fluorescence spectra of a sample in solution and solid state form. These accessories also do not allow working in a high throughput mode.\n\nWe describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory. This method works with a 96-well plate format. It enables the comparison of fluorescence spectra of a sample in a solid state with solution spectra, using comparable quantities of protein. It was found that, just like spectra recorded with these commercial accessories, the spectra of lyophilized powders obtained by our method showed a blue shift of λmax (as compared to the solution spectra). After this validation, the method was used for two specific applications. In the first case, the method was used for assessing the tertiary structure of “dried” α-chymotrypsin. It was also used to track changes in fluorescence spectra of green fluorescent protein (GFP) when it is dried. While the former application is relevant to non-aqueous enzymology, the latter provides some insight into fluorescence resonance energy transfer (FRET) between tryptophan of GFP (Trp57) and its cyclic chromophore20,21.\n\nThese illustrative examples show that the valuable information provided by fluorescence emission spectroscopy about conformational changes in proteins upon drying can be obtained in a simple manner by anybody with a fluorescence-based microplate reader.\n\n\nMaterials\n\nAmpicillin, bovine serum albumin (BSA, cat. no. A7030), α-chymotrypsin (protease from bovine pancreas, cat. no. C4129), lysozyme (from chicken egg white, cat. No. L6876), phenylmethanesulfonylfluoride (PMSF) and n-propanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl β-D-thiogalactopyranoside (IPTG) and LB medium were obtained from Himedia Laboratories (Mumbai, India). TLL (Thermomyces lanuginosus lipase) was a kind gift from Novozymes (Denmark). Candida rugosa lipase was a gift from Amano Enzyme Inc. (Nagoya, Japan). Ninety-six well polystyrene microplates were obtained from Porvair Sciences (Leatherhead, UK). All other chemicals used were of analytical grade. All the proteins used were >95% pure on SDS-PAGE.\n\n\nOverexpression and isolation of GFP\n\nThe plasmid pGFPuv expressing recombinant GFP was transformed into E. coli BL21 (DE3)22. A single colony was picked and inoculated into 5 mL LB medium containing 100 μg mL-1 ampicillin. In total, 1% of primary inoculum was transferred into 1 L fresh LB broth (amp+) and grown at 37°C with shaking at 200 rpm until absorbance at 600 nm reached 0.8. Induction was carried out by adding 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) (final concentration). The culture was further grown under similar conditions for 12 h. The cells were harvested by centrifugation at 8000g for 10 min at 4°C. GFP was isolated from E. coli cells by sonication in 50 mM phosphate buffer, pH 7.5, containing 2 M NaCl and 100 μM phenylmethanesulfonylfluoride (PMSF), three times with 15 s pulses on ice, and centrifugation at 9000g for 10 min at 4°C. The supernatant thus obtained was used as a crude extract for GFP and further purified to homogeneity (as shown by single band on SDS-PAGE) by immobilized metal affinity chromatography using nickel-alginate beads as described earlier22.\n\n\nLyophilization\n\nLyophilization of all the proteins was carried out on a freeze dryer from Allied Frost (New Delhi, India). Proteins were dialyzed against buffer (10 mM Tris-HCl, pH 7.0 for BSA, TLL, lysozyme, CRL and α-chymotrypsin; and 10 mM phosphate buffer, pH 7.5 for GFP) and were frozen at -70°C for 1 h before lyophilization.\n\n\nPreparation of enzyme precipitated and rinsed with propanol (EPRP) of α-chymotrypsin\n\nEnzyme precipitated and rinsed with propanol (EPRP) of α-chymotrypsin was prepared as described previously15. A total of 4 mg of α-chymotrypsin was dissolved in 400 µL of 10 mM Tris-HCl buffer, pH 7.8. Enzyme solution was then added drop wise to 4 ml of n-propanol with stirring at 4°C. After addition, the suspension was stirred for 30 min at 4°C. The suspension was then centrifuged at 5000g for 10 min at 4°C, and the precipitate was rinsed three times with dry and chilled n-propanol.\n\n\nFluorescence measurements\n\nAll fluorescence spectra were recorded on a Cary Eclipse, Varian spectrofluorimeter (Varian Inc., Mulgrave, Victoria, Australia) at 25°C by using the microtitre plate reader accessory for reading 96-well microplates. The typical protein concentration of proteins used for fluorescence measurements in solution was 2 mg/mL in a total volume of 200 µL. Proteins were lyophilized at the same concentration and same amount of protein was used for solid state fluorescence measurements. The fluorescence emission spectra were recorded from 300 nm to 400 nm upon excitation at 280 nm2. For GFP, the fluorescence emission spectra were recorded from 450 nm to 600 nm upon excitation at 395 nm23. The excitation and emission slit widths were kept at 2 nm and 5 nm, respectively. All fluorescence spectra were normalized and corrected for background contributions including the buffer.\n\n\nEstimation of protein concentration\n\nProtein concentration was estimated by the dye binding method using bovine serum albumin as the standard protein24.\n\n\nResults and discussion\n\nThe method developed here consists of simply placing the lyophilized powder of the protein in the well of 96-well microplate. The fluorescence spectra were recorded on a standard Varian microplate reader. The λmax excitation known for the protein solutions were used for solid samples as well. Intrinsic fluorescence emission spectra of four different commercial proteins were obtained after lyophilization from the aqueous buffer and compared with the spectra of the respective protein in the aqueous buffer solution (Figure 1). The amount of protein in each solution was the same as was used for obtaining the lyophilized powders. In all the cases there was a blue shift in emission λmax (Table 1) and an increase in the intensity of the emission spectra of the lyophilized proteins as compared to the protein in aqueous solution. It is important to note that a similar blue shift in the λmax have been reported by Ramachander et al7. while comparing the solid state and solution state fluorescence spectra of four therapeutic proteins (the identities of the proteins were not disclosed by these authors). These workers had used a special accessory (called a solid state holder set up) for the Cary Eclipse spectrofluorimeter. The blue shift in the lyophilized state reflects that the environment of intrinsic fluorophores is more non-polar. This is expected due to the removal of water. The small differences in the extent of the blue shift (Table 1) in case of the four proteins presumably originate from the differences in the microenvironments of tryptophan in the folded structure of each of the proteins. To start with, when in solution, the microenvironments of tryptophan are expected to be different between different proteins.\n\nProtein in aqueous buffer (10 mM Tris-HCl, pH 7.0) (—) and lyophilized protein powder (- -). All these fluorescence emission spectra were recorded with excitation at 280 nm using excitation and emission slit widths of 2 nm and 5 nm, respectively.\n\naBSA = Bovine serum albumin.\n\nbTLL = Thermomyces lanuginosus lipase.\n\ncCRL = Candida rugosa lipase.\n\nFigure 2 shows the fluorescence emission spectra of α-chymotrypsin in solution and in the solid state. Native α-chymotrypsin in aqueous buffer showed emission λmax of 335 nm while upon lyophilization it was blue shifted to 328 nm with an increase in the intensity. This is likely again due to the non-polar environment of the aromatic residues. It has been shown that lyophilized preparations of α-chymotrypsin show poor esterification/transesterification activity in low water media containing organic solvents25. Low activities of lyophilized powders in such media have been explained due to structural changes which occur upon lyophilization14. “Dry” preparations obtained by precipitation of α-chymotrypsin from its aqueous solution by addition of water miscible organic solvents are known to show much better activities in low water media15,26,27. Recently, Solanki et al15. found that changes in the CD spectra upon “drying” correlated well with catalytic activities in low water media for various α-chymotrypsin preparations. A high activity preparation of α-chymotrypsin for low water media (EPRP)15 showed a very small red shift in the emission λmax to 337 nm with an increase in the intensity of the emission spectra, in contrast to the lyophilized protein which showed a blue shift. This further highlights that the lyophilized protein is different from the high activity preparation (EPRP) in terms of the tertiary structure, demonstrating that the simple fluorescence method proposed here can successfully monitor changes in the tertiary structure of different types of formulations of solid proteins.\n\nα-Chymotrypsin in aqueous buffer (10 mM Tris-HCl, pH 7.0) (black curve), lyophilized α-chymotrypsin powder (blue dashed curve) and solid sample of enzyme precipitated and rinsed with propanol (EPRP) of α-chymotrypsin (red curve). All these fluorescence emission spectra were recorded with excitation at 280 nm using excitation and emission slit widths of 2 nm and 5 nm, respectively.\n\nTo further examine the application of this new method, we recorded the fluorescence spectra of the lyophilized formulation of recombinant GFP. In this case, the high intrinsic fluorescence of GFP due to the cyclic moiety present in the protein, which is very sensitive to changes in the structure of protein23, was studied (Figure 3). The lyophilized protein showed a considerable decrease in the intensity of the fluorescence emission spectra to 17% as compared to that of GFP in solution. The emission λmax was also slightly red shifted upon lyophilization (4 nm, from 508 nm to 512 nm). These changes (in fluorescence intensity and shift of λmax emission) were opposite to what was observed with other proteins (Figure 1). Visser et al20. have shown that the fluorescence of the cyclic chromophore in GFP results from the energy transfer from the tryptophan. Figure 4 shows that the energy transfer between the tryptophan and the cyclic chromophore is much less in the lyophilized form. It is noteworthy that the change in the emission intensity due to tryptophan residues (at ~340 nm) was observed in GFP (Figure 4), just as for the other proteins (Figure 1). Both changes reflect how the microenvironment affects the emission fluorescence of the unique chromophore of GFP and could be due to the degradation of this cyclic chromophore upon lyophilization.\n\nGFP in aqueous buffer (50 mM PBS) (solid line) and lyophilized powder of GFP (dashed line). These fluorescence emission spectra were recorded with excitation at 395 nm using excitation and emission slit widths of 2 nm and 5 nm, respectively.\n\nGFP in aqueous buffer (50 mM PBS) (solid line) and lyophilized powder of GFP (dashed line). These fluorescence emission spectra were recorded with excitation at 295 nm using excitation and emission slit widths of 2 nm and 5 nm, respectively.\n\n\nConclusion\n\nA simple method of placing the dry protein powder in a 96-well microplate enables the generation of fluorescence spectra of a protein in the solid state. As the fluorescence spectra of the solution can also be recorded in an identical fashion, the exact comparison between the solution and solid state spectra is possible.",
"appendix": "Author contributions\n\nMNG designed the study. MNG and SG participated in the interpretation of data and the writing of the manuscript. SG carried out the experimental work. Both authors approved the submission of the final manuscript.\n\n\nCompeting interests\n\nNo relevant competing interests where disclosed.\n\n\nGrant information\n\nThis work was funded by research support from the Department of Biotechnology (DBT) [Grant number: BT/PR14103/BRB/10/808/2010] and the Department of Science and Technology (DST) [Grant number: SERB/F/1776/2011–2012], Government of India. Financial support was also provided by the Council of Scientific and Industrial Research to SG in the form of a Junior Research Fellowship.\n\n\nAcknowledgments\n\nThe financial support provided by the Council of Scientific and Industrial Research to SG in the form of a Junior Research Fellowship is gratefully acknowledged.\n\n\nReferences\n\nRoyer CA: In: Shirley BA (ed) Protein stability and folding. Humana Press, Totowa, New Jersey (1995).\n\nLakowicz JR: Principles of fluorescence spectroscopy. 3rd edn., Springer, New York. (2009).\n\nBrand L, Johnson ML: Methods in Enzymology. Academic Press, San Diego (2008); 150.\n\nAgnew BJ, Murray D, Patton WF: A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents. Electrophoresis. (2004); 25: 2478–2485.\n\nTorres-Salas P, del Monte-Martinez A, Cutiño-Avila B, et al:Immobilized biocatalysts: novel approaches and tools for binding enzymes to supports. Adv Mater. (2011); 23: 5275–5282.\n\nLower BH, Yongsunthon R, Vellano FP 3rd, et al:Simultaneous force and fluorescence measurements of a protein that forms a bond between a living bacterium and a solid surface. J Bacteriol. (2005); 187: 2127–2137.\n\nRamachander R, Jiang Y, Li C, et al:Solid state fluorescence of lyophilized proteins. Anal Biochem. (2008); 376: 173–182.\n\nKostov Y, Albano CR, Rao G: All solid-state GFP sensor. Biotechnol Bioeng. (2000); 70: 473–477.\n\nLeppänen A, Cummings RD: Fluorescence-based solid-phase assays to study glycan-binding protein interactions with glycoconjugates. Methods Enzymol. (2010); 478: 241–264.\n\nMuller M, Milori DM, Déléris S, et al:Solid-phase fluorescence spectroscopy to characterize organic wastes. Waste Manag. (2011); 31: 1916–1923.\n\nMattiasson B, Adlercreutz P: Tailoring the microenvironment of enzymes in water-poor systems. Trends Biotechnol. (1991); 9: 394–398.\n\nGupta MN: Enzyme function in organic solvents. Eur J Biochem. (1992); 203: 25–32.\n\nAnthonsen T, Sjursens BJ: In: Gupta MN (ed) Methods in non-aqueous enzymology. Birkhauser Verlag, Basel (2000).\n\nLee M, Dordick J: Enzyme activation for nonaqueous media. Curr Opin Biotechnol. (2002); 13: 376–384.\n\nSolanki K, Gupta MN, Halling PJ: Examining structure-activity correlations of some high activity enzyme preparations for low water media. Bioresour Technol. (2012); 115: 147–151.\n\nDesie G, Boens N, De Schryver FC: Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin. Biochemistry. (1986); 25: 8301–8308.\n\nSharma VK, Kalonia DS: Steady-state tryptophan fluorescence spectroscopy study to probe tertiary structure of proteins in solid powders. J Pharm Sci. (2003); 92: 890–899.\n\nVarian Cary Eclipse Fluorescence Spectrophotometer. [cited March 2013], Available from: http://www.chem.agilent.com/Library/brochures/Cary-Eclipse_FLR-brochure.pdf. 16pp.\n\nHoriba Jobin Yvon Florescence Technical Note FL-6: Fluorescence in Small or Solid Samples. [Cited March 2013] available at from: http://www.horiba.com/fileadmin/uploads/Scientific/Documents/Fluorescence/Small_Samples_FL-6.pdf. 2pp.\n\nVisser NV, Borst JW, Hink MA, et al:Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins. Biophys Chem. (2005); 116: 207–212.\n\nJames NG, Ross JA, Stefl M, et al:Applications of Phasor Plots to in Vitro Protein Studies. Anal Biochem. (2011); 410: 70–76.\n\nDalal S, Raghava S, Gupta MN: Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media. J Biochem Eng. (2008); 42: 301–307.\n\nTsien RY: The green fluorescent protein. Annu Rev Biochem. (1998); 67: 509–544.\n\nBradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. (1976); 72: 248–254.\n\nTriantafyllou AO, Wehtje E, Adlercreutz P, et al:Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media. Biotechnol Bioeng. (1995); 45: 406–414.\n\nRoy I, Gupta MN: Preparation of highly active alpha-chymotrypsin for catalysis in organic media. Bioorg Med Chem Lett. (2004); 14: 2191–2193.\n\nMajumder AB, Singh B, Gupta MN: Diastereoselective synthesis of (R)-(alkyl)-beta-D-galactopyranoside by using beta-galactosidase (Aspergillus oryzae) in low-water media. Bioorg Med Chem Lett. (2008); 18: 124–128."
}
|
[
{
"id": "834",
"date": "13 Mar 2013",
"name": "Vincent Rotello",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe use of solid state fluorescence to provide protein quality control is a modest technical advance. This study looks at both fluorescence peak position and intensity. The latter value, however, will be affected by light scattering that is both particle size and morphology dependent. I would suggest focusing on the peak position as this will provide a much better metric.",
"responses": [
{
"c_id": "393",
"date": "15 Mar 2013",
"name": "Munishwar N Gupta",
"role": "Author Response",
"response": "1. The work is not aimed at suggesting that fluorescence emission spectroscopy should be applied to solid samples of proteins instead of using their solutions. On the other hand, as discussed in the manuscript, there are many investigations (enzyme immobilization on solid surfaces, enzyme powders in low water enzymology) wherein structural characterization would be valuable with the solid state samples. The availability of some commercial accessories fulfills this need. So, we offer a way of measuring fluorescence spectra in a 96-well format without any additional accessories.2. The 96-well plates used were black from all sides except from the top. So, the emission takes place along the same path direction as the exciting radiation. This minimizes scattered light and distortion of the spectra. This results in the spectra which are of reasonable quality as seen in our raw data. 3. The references provided in the manuscript to the earlier work carried out with commercial accessories described differences between the solution spectra and the solid state spectra and list changes in both intensity and λmax shift. In order to validate our method, we wanted to point out that both/similar changes occur with our method as well. However, as Table 1 of our manuscript shows, we have focused on the peak position. In the amended manuscript we will point out that intensities can be affected by many variables and that one should rely more upon the λmax position. These explanations will be added to our amended manuscript."
}
]
},
{
"id": "838",
"date": "15 Mar 2013",
"name": "Vladimir Uversky",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe suggested technique for the analysis of solid state fluorescence of proteins is simple and can find multiple applications. Although the authors analyzed both fluorescence intensity and λmax of protein powders and corresponding protein solutions, the applicability of fluorescence intensity is questionable. In fact, fluorescence intensity depends on a wide range of factors and cannot be easily interpreted, especially if samples are in different aggregated states (solid versus solution). Therefore, only λmax should be taken as a parameter for analysis since this characteristic has understandable physical grounds.",
"responses": [
{
"c_id": "392",
"date": "15 Mar 2013",
"name": "Munishwar N Gupta",
"role": "Author Response",
"response": "The references provided in the manuscript to the earlier work carried out with commercial accessories described differences between the solution spectra and the solid state spectra and list changes in both intensity and λmax shift. In order to validate our method, we wanted to point out that both/similar changes occur with our method as well. However, as Table 1 of our manuscript shows, we have focused on the peak position. The amended manuscript can point out that intensities can be affected by many variables and one should rely more upon the λmax position. These explanations can be added in the amended manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/2-82
|
https://f1000research.com/articles/8-334/v1
|
25 Mar 19
|
{
"type": "Research Article",
"title": "The common patterns of abundance: the log series and Zipf's law",
"authors": [
"Steven A. Frank"
],
"abstract": "In a language corpus, the probability that a word occurs n times is often proportional to 1/n2. Assigning rank, s, to words according to their abundance, log s vs log n typically has a slope of minus one. That simple Zipf's law pattern also arises in the population sizes of cities, the sizes of corporations, and other patterns of abundance. By contrast, for the abundances of different biological species, the probability of a population of size n is typically proportional to 1/n, declining exponentially for larger n, the log series pattern. This article shows that the differing patterns of Zipf's law and the log series arise as the opposing endpoints of a more general theory. The general theory follows from the generic form of all probability patterns as a consequence of conserved average values and the associated invariances of scale. To understand the common patterns of abundance, the generic form of probability distributions plus the conserved average abundance is sufficient. The general theory includes cases that are between the Zipf and log series endpoints, providing a broad framework for analyzing widely observed abundance patterns.",
"keywords": [
"scaling patterns",
"ecology",
"demography",
"linguistics",
"probability theory"
],
"content": "Introduction\n\nA few simple patterns recur in nature. Adding up random processes often leads to the bell-shaped normal distribution. Death and other failures typically follow the extreme value distributions.\n\nThose simple patterns recur under widely varying conditions. Something fundamental must set the relations between pattern and underlying process. To understand the common patterns of nature, we must know what fundamentally constrains the forms that we see.\n\nWithout that general understanding, we will often reach for unnecessarily detailed and complex models of process to explain what is in fact some structural property that influences the invariant form of observed pattern.\n\nWe already understand that the central limit theorem explains the widely observed normal distribution1. Similar limit theorems explain why failure often follows the extreme value pattern2,3.\n\nThe puzzles set by other commonly observed patterns remain unsolved. Each of those puzzles poses a challenge. The solutions will likely broaden our general understanding of what causes pattern. Such insight will help greatly in the big data analyses that play an increasingly important role in modern science.\n\nZipf’s law is one of the great unsolved puzzles of invariant pattern. The frequency of word usage4, the sizes of cities5,6, and the sizes of corporations7 have the same shape. On a log-log plot of rank versus abundance, the slope is minus one. For cities, the largest city would have a rank of one, the second largest city a rank of two, and so on. Abundance is population size.\n\nThe abundance of species is another great unsolved puzzle of invariant pattern. In an ecological community, the probability that a species has a population size of n individuals is proportional to pn/n, the log series pattern8. Communities differ only in their average population size, described by the parameter, p. Actual data vary, but most often fit closely to the log series9.\n\nIn this article, I show that Zipf’s law and the log series arise as the opposing endpoints of a more general theory. That theory provides insight into the particular puzzles of Zipf’s law and species abundances. The analysis also suggests deeper insights that will help to unify understanding of commonly observed patterns.\n\n\nTheory\n\nThe argument begins with the invariances that define alternative probability patterns10,11. To analyze the invariances of a probability distribution, note that we can write almost any probability distribution, qz, as\n\nNow consider the consequences if the average of some value over the distribution qz is conserved. That constraint causes the probability pattern to be invariant to a multiplicative stretching (or shrinking), Tz ↦ bTz, because\n\nConserved total probability and conserved average value cause the probability pattern to be invariant to an affine transformation of the Tz scale, Tz ↦ a + bTz, in which “affine” means both shift and stretch.\n\nThe affine invariance of probability patterns with respect to Tz induces significant structure on the form of Tz and the associated form of probability patterns. Understanding that structure provides insight into probability patterns and the processes that generate them10,12,13.\n\nIn particular, Frank & Smith12 showed that the invariance of probability patterns to affine transformation, Tz ↦ a + bTz, implies that Tz satisfies the differential equation\n\nTurning now to the log series and Zipf’s law, the relation n = er between observed pattern, n, and process, r, plays a central role. Here, r represents the total of all proportional processes acting on abundance. A proportional process simply means that the number of individuals or entities affected by the process increases in proportion to the number currently present, n.\n\nThe sum of all of the proportional processes acting on abundance over some period of time is\n\nHere, m(t) is a proportional process acting at time t to change abundance. The value of r = log n is the total of the m values over the total time, τ. For simplicity, I assume n0 = 1.\n\nProportional processes are often discussed in terms of population growth5,14. However, many different processes act individually on the members of a population. If the number of individuals affected increases in proportion to population size, then the process is a proportional process.\n\nGrowth and other proportional processes often lead to an approximate power law, qn ≈ kn–ρ. However, the exponent of a growth process does not necessarily match the values observed in the log series and Zipf’s law. We need both the power law aspect of proportional process and something further to get the specific forms of those widely observed abundance distributions. That something further arises from conserved quantities and their associated invariances.\n\nThe log series and Zipf’s law follow as special cases of the generic probability pattern in Equation 3. To analyze abundance, focus on the process scale by letting the variable of interest be z ≡ r, with the key scaling simply the process variable itself, w(r) = r. Then Equation 3 becomes\n\nThe value of k always adjusts to satisfy the constraint of invariant total probability, and the value of λ always adjusts to satisfy the constraint of invariant average value.\n\nFor β = 1, we obtain the log series distribution\n\nreplacing n – 1 by n in the exponential term which, because of affine invariance, describe the same probability pattern. The log series is often written with e–λ = p, and thus qn = kpn/n. One typically observes discrete values n = 1, 2, . . . . The Supplemental material for this article15 shows the relation between discrete and continuous distributions16 and the domain of the variables. The continuous analysis here is sufficient to understand pattern.\n\nFor β → 0, we have (nβ – 1)/β → log n, which yields\n\nFor any average abundance that is finite and not small, λ → 1, which is Zipf’s law.\n\nEquation 5 provides a general expression for abundance distributions. The log series and Zipf’s law set the endpoints of β = 1 and β → 0. We can understand the differences between abundance distributions in terms of the parameter β by writing the distribution in the generic form of Equation 1, with the defining affine invariant scale\n\nThis scale expresses the invariances that define the pattern. At the Zipf’s law endpoint, β → 0, the scale becomes 2 log n = 2r, when satisfying the constraint that the average abundance, 〈n〉, is sufficiently large.\n\nIn this case, with affine invariant scale Tn = 2r, neither addition nor multiplication of process value, r ↦ a + br, alters the pattern. We could have started with this affine invariance, and derived the probability pattern from the invariance properties10,11.\n\nFor the log series endpoint, β = 1, the affine invariant scale is\n\nThe dominant aspect of the scale changes with n. For small abundances, the logarithmic scale r = log n dominates, and for large abundances, the linear scale n = er dominates. Many common probability patterns change their scaling with magnitude13,17.\n\nFor log series patterns, the dominance of scale at small magnitude by r corresponds to affine invariance with respect to r. At larger abundances, the dominance by the effectively linear scale, n, corresponds to invariance to a shift in process r ↦ a + r, but not to a multiplication of process, r ↦ br, because ebr = nb is a power transformation of abundance. Linear scales are not invariant to power transformations. Once again, we could have derived the pattern from the invariances.\n\nIn Equation 8, intermediate values of β combine aspects of Zipf’s law and the log series. The closer β is to one of the endpoints, the more the invariance characteristics of that endpoint dominate pattern.\n\n\nConclusion\n\nThis analysis shows how two great and seemingly unconnected puzzles solve very simply in terms of a single continuum between alternative invariances. This approach reveals the simple invariant structure of many common probability patterns.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Supplemental Material for “The common patterns of abundance: the log series and Zipf’s law”. https://doi.org/10.5281/zenodo.259789515.",
"appendix": "Grant information\n\nThe Donald Bren Foundation supports my research.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nI completed this work while on sabbatical in the Theoretical Biology group of the Institute for Integrative Biology at Eidgenössische Technische Hochschule (ETH) Zürich.\n\nA previous version of this article is available on arXiv: https://arxiv.org/abs/1812.09662\n\n\nReferences\n\nFischer H: A History of the Central Limit Theorem: From Classical to Modern Probability Theory. Springer, New York, 2011. Publisher Full Text\n\nKotz S, Nadarajah S: Extreme Value Distributions: Theory and Applications. World Scientific, Singapore, 2000. Publisher Full Text\n\nColes S: An Introduction to Statistical Modeling of Extreme Values. Springer, New York, 2001. Publisher Full Text\n\nZipf GK: The Psycho-biology of Language. Houghton Mifflin, Boston, 1935. Reference Source\n\nGabaix X: Zipf’s law for cities: an explanation. Q J Econ. 1999; 114(3): 739–767. Reference Source\n\nArshad S, Hu S, Ashraf BN: Zipf’s law and city size distribution: a survey of the literature and future research agenda. Physica A: Stat Mech Appl. 2018; 492: 75–92. Publisher Full Text\n\nAxtell RL: Zipf distribution of U.S. firm sizes. Science. 2001; 293(5536): 1818–1820. PubMed Abstract | Publisher Full Text\n\nFisher RA, Corbet AS, Williams CB: The relation between the number of species and the number of individuals in a random sample of an animal population. J Anim Ecol. 1943; 12(1): 42–58. Publisher Full Text\n\nBaldridge E, Harris DJ, Xiao X, et al.: An extensive comparison of species-abundance distribution models. PeerJ. 2016; 4: e2823. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrank SA: Common probability patterns arise from simple invariances. Entropy. 2016; 18(5): 192. Publisher Full Text\n\nFrank SA: Measurement invariance explains the universal law of generalization for psychological perception. Proc Natl Acad Sci U S A. 2018; 115(39): 9803–9806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrank SA, Smith E: A simple derivation and classification of common probability distributions based on information symmetry and measurement scale. J Evol Biol. 2011; 24(3): 469–484. PubMed Abstract | Publisher Full Text\n\nFrank SA: How to read probability distributions as statements about process. Entropy. 2014; 16: 6059–6098. Publisher Full Text\n\nGibrat R: Les Inégalités Économiques. Librairie du Recueil Sirey, Paris. 1931. Reference Source\n\nFrank SA: Supplemental Material for “The common patterns of abundance: the log series and Zipf’s law”. 2019. http://www.doi.org/10.5281/zenodo.2597895\n\nAu C, Tam J: Transforming variables using the Dirac generalized function. Am Stat. 1999; 53(3): 270–272. Publisher Full Text\n\nFrank SA: The invariances of power law size distributions [version 2; peer review: 2 approved]. F1000Res. 2016; 5: 2074. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "46232",
"date": "15 Apr 2019",
"name": "Luís M. A. Bettencourt",
"expertise": [
"Reviewer Expertise Population Dynamics",
"Dynamical Systems",
"Ecology and Evolution",
"Statistical Mechanics",
"Complex Systems."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 approaches the origins of two particularly important distributions describing abundances in biological and social populations from the perspective of mathematical invariances of their mathematical forms.\n\nThe author shows in this way that Fisher’s log series distribution and Zipf’s “law” can arise in different limits of the same parameter, characterizing a family of affine transformations that includes translations and scale transformations of growth rates.\n\nThe mathematical derivation is clear and elegant, so that the manuscript makes an important contribution to formal models deriving these abundance distributions.\n\nWhat I think would improve the manuscript is greater contact with other methods for the derivation of these same distributions of abundance and an expanded discussion of limits.\n\nSpecifically:\n\nThe relationship between population dynamics and invariances of the abundance (or rate) distributions could be made a little more explicit: Population dynamics models (in analogy to other dynamical systems) are mappings, tracing explicit variable transformation over time, such as changes of “position” (translations, r-> a + r), or dilations (r -> b r). Asking for invariances of distributions under these dynamical transformations is the usual way to derive the distributions as steady state abundances. Power laws, such as Zipf’s law, are invariant under (stochastic) dilations, for example, while Fisher’s log series are invariant under other simple types of population dynamics (as in Volkov et at1). I’d appreciate a bit more discussion bridging these two approaches.\n\nAs, the author shows the derivation of Zipf’s law requires not only a parameter choice (beta -> 0) but also the limit of the average abundance -> infinity. Without the latter, the power law exponent won’t be Zipf’s. In dynamical derivations of Zipf’s law one asks instead that geometric random motion of the population abundances, is subjected to a (“reflecting”) boundary condition for small population sizes that stops them from getting too small, as in [5]. Under what circumstances are these two additional requirements (besides transformational invariances under multiplicative growth) equivalent? They seem to have a different character as one is a limit, while the other a boundary condition—is the limiting condition on the average the most general condition?\n\nIt would be interesting to describe the conditions (in terms of beta and any limits or time dependence on averages) for deriving the third distribution often invoked to describe the same abundances, the log-normal, in terms of the reasoning about invariances advanced here. This is discussed to some extent in previous work by the author, in reference [12]. I think its inclusion and discussion would benefit the current manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4588",
"date": "23 Apr 2019",
"name": "Steven Frank",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I appreciate the thoughtful comments from Luis Bettencourt. The review and my reply are part of the final published version. So I will use this space to develop my comments, leaving the original manuscript unchanged. I quote the first few words of each reviewer comment in bold, and then follow with my reply.The relationship between population dynamics and invariances of the abundance (or rate) distributions could be made a little more explicit: ...I agree that connecting dynamic models of process to the invariances that set pattern is the great missing piece in this work. Ultimately, an invariance perspective can only achieve its full usefulness if one can compare and empirically test alternative hypotheses about mechanistic processes. That might, for example, require one to identify the particular aspect within a mechanistic set of processes that ultimately defines the invariance that dominates pattern. Then, by comparing different mechanistic models, one can reduce that comparison to the contrast between alternative component processes that dominate invariance, and so develop a more focused empirical test. I am not yet certain how to make these connections between abstract invariances and mechanistic dynamical models in the most meaningful way, so I have refrained from writing about these issues. This is a primary topic for future work.As, the author shows the derivation of Zipf’s law requires not only a parameter choice ...First, one just needs average abundance to be not small to get an exponent that is essentially equivalent to Zipf’s law and sufficient for empirical comparison. Second, I agree that it would be useful to consider the relations between boundary conditions in dynamics and simplified invariance models. I don’t know the answer. It would be a useful topic for future work.It would be interesting to describe the conditions (in terms of beta and any limits or time dependence on averages) for deriving ...I have a new unpublished manuscript that unifies the log series, Zipf’s law, and the lognormal. A single additional invariance leads to a unified distribution that includes all of those distributions as special cases and also some intermediate forms that commonly arise in certain empirical examples. Thus, I agree with the importance of this comment, but withhold further details until I can complete my new work."
}
]
},
{
"id": "46236",
"date": "17 Apr 2019",
"name": "Jose Lobo",
"expertise": [
"Reviewer Expertise Urban economics",
"growth models",
"analysis of statistical distributions as models of 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\nThe manuscript addresses the relationship between two probability distributions that, although originated in specific research domains, have gone on to be widely used as representations of growth processes.\n\nThe mathematical derivations are clear.\n\nThe conclusion that \"two great and seemingly unconnected puzzles solve very simply in terms of a single continuum between alternative invariances. This approach reveals the simple invariant structure of many common probability patterns.\" clearly follows from the exposition and is a useful contribution.\n\nThe usefulness and scope of the conclusion would be strengthened if the author considered another distribution which arises often in the explorations of growth processes: the log normal.\n\nIt would also strengthen the usefulness of the manuscript if the author were to expand on \"this approach reveals the simple invariant structure of many common probability patterns.\", in particular recapitulating what is the invariant structure.\n\nHaving connected two widely used distributions, what sort of research questions can now be addressed? How can the invariant structure linking two distributions be used in contexts other than Zipf's law or species distributions?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4589",
"date": "23 Apr 2019",
"name": "Steven Frank",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I appreciate the thoughtful comments from Jose Lobo. The review and my reply are part of the final published version. So I will use this space to develop my comments, leaving the original manuscript unchanged. I quote the first few words of each reviewer comment in bold, and then follow with my reply.The usefulness and scope of the conclusion would be strengthened if the author considered another distribution which arises often in the explorations of growth processes: the log normal.Copying my reply to Luis Bettencourt’s comment on the same topic: I have a new unpublished manuscript that unifies the log series, Zipf’s law, and the lognormal. A single additional invariance leads to a unified distribution that includes all of those distributions as special cases and also some intermediate forms that commonly arise in certain empirical examples. Thus, I agree with the importance of this comment, but withhold further details until I can complete my new work.It would also strengthen the usefulness of the manuscript if the author were to expand on \"this approach ...\"I agree that the current manuscript is rather terse about this issue. However, I have written several prior manuscripts that extensively develop this aspect, cited in the current publication. I prefer to keep the current manuscript short and focused on the new aspect related to the log series and Zipf’s law, and refer to earlier publications for the background.Having connected two widely used distributions, what sort of research questions ...I think the key advance will not come until more can be said about linking dynamic models to the abstract invariant structures. Copying my reply to Luis Bettencourt’s review: I agree that connecting dynamic models of process to the invariances that set pattern is the great missing piece in this work. Ultimately, an invariance perspective can only achieve its full usefulness if one can compare and empirically test alternative hypotheses about mechanistic processes. That might, for example, require one to identify the particular aspect within a mechanistic set of processes that ultimately defines the invariance that dominates pattern. Then, by comparing different mechanistic models, one can reduce that comparison to the contrast between alternative component processes that dominate invariance, and so develop a more focused empirical test. I am not yet certain how to make these connections between abstract invariances and mechanistic dynamical models in the most meaningful way, so I have refrained from writing about these issues. This is a primary topic for future work."
}
]
},
{
"id": "46235",
"date": "17 Apr 2019",
"name": "Scott E. Page",
"expertise": [
"Reviewer Expertise Complexity"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 found this article to be fascinating and elucidating but also a bit frustrating to read. The central claim of the article is that one can construct a family of distributions such that Zipf's law and species abundance are the endpoints of a more general process.\nFor that result to be interesting, the result has to be for a general process and not for a family of distributions.\n\nThe latter is easy. I just say, \"here is a family of distributions, f(x) = x^{-a}” and then say that at one endpoint a=1 I have a species area law and at the other endpoint a=2, I get Zipf's law.\n\nThe contribution of the article lies in convincing us that the paper has done something other than an elaborate change of variables that simply restates that result through obfuscation.\nSo what does the paper do? The paper shows that if we restrict attention to probability patterns (by the way, it would be nice if \"pattern\" were formally defined) that are invariant to affine transformations then we have a specific form given by equation (3).\nGiven the form in equation (3), the author then claims that n represents pattern and r represents process. This needs to be elucidated.\n\nFor the main result, once we have invariance to affine transformation we get the differential equation with\ndT_z/dw = \\alpha + \\beta T_z\nFrom here, why doesn't it just follow that if \\beta = 0, we have something that's going to fall off with a common invariant scale and for \\beta = 1 the invariant scale changes with n.\nThe conclusion of the paper needs to be expanded. As a reader, I need a richer explanation for how the approach \"reveals the simple invariant structure\" of common probability distributions. In the conclusion, we should be given more intuition for how the holding the average abundance constant drives the results. Also, it would be nice to have more insight into what would cause a system to have more or fewer proportional processes acting on it.\nQuibble: Why isn't r a function of tau—the period of time?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "4590",
"date": "23 Apr 2019",
"name": "Steven Frank",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I appreciate the thoughtful comments from Scott Page. The review and my reply are part of the final published version. So, I will use this space to develop my comments, leaving the original manuscript unchanged. I quote the first few words of each reviewer comment in bold, and then follow with my reply.For that result to be interesting, the result has to be for a general process and not for a family of distributions.I partially agree. It is very interesting to understand how a general process relates to a family of distributions. I am currently pursuing that by studying the species abundance problem in ecology in more detail as an example. In my new work, I show how various well known general processes, such as neutral theory, relate to an abstract family of distributions characterized by simple invariances. My new work will show a much simpler way in which to understand the relations between process and pattern in ecology than is currently the case in the literature of that subject. I think that new work will help a bit with regard to this question, because the log series and Zipf’s law are special cases of a broader family of distributions that also includes the lognormal. The current article is a step on the way to the more ambitious study. However, I also partially disagree. Identifying the invariant structure of probability patterns is by itself useful. It guides one in more focused projects and provides a way to understand what is expected and what is not. Further, one can specify a general process that leads to a Gaussian distribution, but that process will not be a full understanding of the Gaussian distribution, just one instance of a process that associates with that pattern. There are other general processes that are distinct but also converge to the Gaussian. So, we need to understand both the different types of general process and the abstract aspects of the Gaussian that unify all conforming general processes. The current article is entirely on the abstract side. That is a necessary but not sufficient component. I agree that more needs to be done and am working on that in the ecology application mentioned above. I hope to contribute along those lines in my future work.So what does the paper do? The paper shows that if we restrict attention to probability patterns (by the way, it would be nice if \"pattern\" were formally defined) ...No widely agreed definition of “pattern” exists, which is interesting. I believe that “pattern” and “invariance” are the same thing, but that remains an open issue.Given the form in equation (3), the author then claims that n represents pattern and ...My use of n as pattern simply describes what people have typically measured and reported as a pattern. In other words, people have measured population sizes and reported those data as a pattern. My claim that r corresponds to process reflects the general agreement that populations change by birth, death, migration, and other processes that act multiplicatively, again a description of the common understanding. For example, the neutral theory in ecology, which has become a dominant approach to the study of ecological process, is about demographic processes that act proportionally or multiplicatively. So, both by intuition and by consensus, I have adopted r as associated with process.From here, why doesn't it just follow that if \\beta = 0, we have something that's going to fall off with a common invariant scale and for \\beta = 1 the invariant scale changes with n.I do not understand the question. The point of the differential equation is that we can find a new scale, w, that is shift invariant but not stretch invariant with respect to probability pattern. As \\beta goes to zero, w becomes both shift and stretch invariant (affine invariant). It turns out that \\beta is a sort of curvature that describes departure from stretch (multiplicative) invariance. As an observation, working with w has turned out to provide a key way in which to unify diverse probability distributions within a single common system, suggesting an invariant structure that unifies commonly observed probability patterns. That was the topic of several of my prior publications. Here, I was just using some of the prior insight to try and understand the nature of the log series and Zipf’s law and perhaps something about how those distributions arise. I will expand on that in a future manuscript, see next comment.The conclusion of the paper needs to be expanded. As a reader, I need a richer explanation ...First, my prior publications discuss invariant structure and its consequences in an abstract way. But I think that is not the issue that is being asked about here. So, second, I am currently finishing a new manuscript that extends this current article in several ways. My new manuscript focuses on invariance in ecological pattern. By emphasizing a particular application and its associated literature, I am able more explicitly to get at some of the issues that are too vague in the current manuscript. For example, I will consider directly the role of average values by relating maximum entropy and invariance approaches explicitly in the context of ecological applications. I think this will help some. Many of the other comments raised by the reviewers also come up in the new work, suggesting that there are some obvious missing steps here that need further attention. These issues cannot be resolved in a few paragraphs, so I am going to wait until I finish the new work before trying to address these problems. I apologize for putting off thoughtful and important comments, but I need more time to complete the new work before I can give good answers.Why isn't r a function of tau—the period of time?It is. Whether that matters depends on the particular question. One aspect is that, as long as tau is taken as a fixed value, such as generation time in a discrete generation model of populations, then one can take r directly without concern about varying tau. Alternatively, if one has reason to consider tau as varying, then it may matter for certain aspects. I have not looked into that. I agree that it would be worthwhile to understand this issue more clearly."
}
]
},
{
"id": "46234",
"date": "29 Apr 2019",
"name": "Neil McRoberts",
"expertise": [
"Reviewer Expertise Quantitative biology",
"decision-making",
"epidemiology",
"science for policy support and analysis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper continues a series of investigations by Prof Frank into general explanations for the presence of common patterns in the world. Previous work has examined underlying reasons for common probability distributions across a range of types of observation and common size distributions. Here, Steve Frank turns to rank abundance measures.\nThere are two central pieces to the analysis presented in the paper. The first derives from E.T. Jaynes' work on the information content of probability distributions and the derivation of probability distributions by considering information constraints on the observed quantity. The second is drawn from Frank's own work (with Eric Smith) on the invariance of the emergent probability distributions to certain scalings of variables, in particular the affine transformation. The same invariance to affine transformation lies at the heart of Frank's previous analyses.\n\nFor this reviewer, the importance of the paper lies not so much in the technical results (which flow directly as entailments of the algebra given the premises established in equation 1, and the arguments preceding equation 2, but more in the wider issues it raises about observation in general. In particular, as with the previous papers on related subjects which Frank has written, this paper gives a principled way to derive observed, generic, relationships about rank and abundance that are independent of the physical details of the system being investigated. One possible response to this kind of result is to see it as a consequence of the way that the argument is set up in the premises but I think the way the various parameters are connected with physical properties (albeit it in a generic and hence somewhat abstract way) shows that there is something more fundamental at work here than mathematical slight of hand. Imagine, for example, how strange (to us, here and now) the universe would seem if the informational constraints on probability distributions were not invariant to affine transformation; if, for example, the Poisson distribution described random counts of small numbers of small things, but not small numbers of large things. So, in the same way that understanding what it means for small random counts to follow a Poisson distribution adds to understanding a set of data, it is not a trivial thing (nor a piece of pure phenomenology) for researchers to be able use the general relationship that Frank has derived here, to understand observed rank abundance relationships in terms of the scale at which underlying processes expressing measurable phenomena.\n\nIn a paper written in such a telegraphic style, Frank does a decent job of connecting the derived relationships with physical examples, but I suspect that anyone who hasn't been following the development of this area of work over the preceding publications, or who isn't familiar with the connections between information and probability, will find the paper concise to the point of abruptness. I hope that Frank plans a synthesis and review of the specific examples that have been published at some later date; although it could be argued that the general synthesis was laid out in earlier papers and the more recent work is unpacking specific case studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-334
|
https://f1000research.com/articles/8-333/v1
|
25 Mar 19
|
{
"type": "Case Report",
"title": "Case Report: Takayasu arteritis in a male patient",
"authors": [
"Syed Mohammad Mazhar Uddin",
"Aatera Haq",
"Zara Haq",
"Uzair Yaqoob",
"Osama Mohiuddin",
"Anosh Aslam Khan",
"Syed Mohammad Mazhar Uddin",
"Aatera Haq",
"Zara Haq",
"Osama Mohiuddin",
"Anosh Aslam Khan"
],
"abstract": "Takayasu arteritis (TA) is a type of primary systemic vasculitis mainly affecting the medium and large arteries. The signs and symptoms are due to systemic inflammation or ischemia of an organ or limb and include angiodynia, claudication, peripheral pulselessness, murmurs, ischemic stroke, myocardial infarction and severe systemic arterial hypertension. The disease tends to affect more women than men. Here we present a case of TA in a 22-year-old male patient. Our patient presented with complaints of aphasia and right-sided weakness, with on-and-off symptoms of malaise, generalized weakness, unilateral headache, fatigue and shortness of breath lasting two years. Color Doppler ultrasound was sufficient for a diagnosis of TA, after which we started the patient on medical treatment and also consulted the department of vascular surgery. Overall, this case report highlights the importance of screening for TA in male patients so that the diagnosis is not overlooked, and also adds more data to the limited literature on male patients.",
"keywords": [
"Takayasu Arteritis",
"Autoimmune",
"autoimmune in males",
"vascular disease"
],
"content": "Introduction\n\nTakayasu arteritis (TA), also known as “pulseless disease”, is a type of primary systemic vasculitis affecting medium and large arteries, including the aorta and its branches, as well as the pulmonary and coronary arteries1. It is a chronic inflammatory disease of unknown origin characterized by granulomatous vasculitis, leading to thickening, dilatation, stenosis, and/or aneurysm formation of the involved vessels1,2. Furthermore, the signs and symptoms exist due to systemic inflammation or ischemia of an organ or limb, and encompass angiodynia, claudication, peripheral pulselessness, murmurs, ischemic stroke, myocardial infarction, severe systemic arterial hypertension, etc1. TA tends to affect females more than males, with 80% of patients being female. However, the female-to-male ratio varies, from 9:1 in Japan and 6.9:1 in Mexico to 1.2:1 in Israel3. Furthermore, TA is associated with significant morbidity and can be life threatening. Around 20% of patients experience monophasic and self-limited disease, whereas others can have a progressive or relapsing/remitting disease. Moreover, the overall 10-year survival rate for this disease is approximately 90% which can be reduced in the presence of major complications4. Here we present a case of TA in a 22-year-old male.\n\n\nCase report\n\nA 22-year-old male college student, belonging to Interior Sindh province, with no known history of comorbid presented to the emergency department of civil hospital, Karachi, Pakistan, on 3rd January 2019, with an insidious onset of aphasia and right-sided weakness for six days. According to the patient’s brother, he was in his usual state of health six days back when he developed an altered level of consciousness, which led them to bring him to hospital. In the hospital, the patient regained consciousness on the day of admission but was unable to speak and move his right side of the body. There was no additional history of fever, fits, nausea, vomiting, urinary incontinence, fecal incontinence, as well as any visual symptoms. However, the patient had complained of malaise, generalized weakness, shortness of breath and fatigue for the last two years. Furthermore, the patient also complained of intermittent unilateral headache which was not relieved by over-the-counter pain medications. The patient had no other significant past medical, surgical and family history. On arrival, his blood pressure was 140/90 in the left arm but non-recordable in the right arm, pulse was 60 beats/minute (only recordable in the left arm), and temperature 36.7°C and respiratory rate 22 breaths/minute. On examination, his general physical, respiratory and abdominal examinations were unremarkable. However, central nervous system examination revealed problems, with aphasia in speech. Motor examination on the right side revealed reduced power in both upper and lower limbs as well as upgoing plantars on the right lower limb. The rest of the central nervous examination was normal. His cardiovascular examination showed muffled heart sounds, and no murmurs were present, but he was positive for bilateral carotid bruit.\n\nBased on the history and examination, we ordered pertinent laboratory tests along with other specific tests. When his baseline laboratory values were ordered, complete blood count (CBC) showed hemoglobin (Hb) of 12.3 g/dL (normal: 11.1–14.5 g/dL), mean corpuscular volume of 77.5 fL (normal, 76–96 fL), WBC count of 7.5×109/L (normal: 4–10×109/L) and platelets were 180×109/L (normal, 150–400×109/L); however, C-reactive protein was 140 mg/L (normal <3 mg/L) and erythrocyte sedimentation rate was 80 mm/hour (normal, 0–22 mm/hour). As the patient had intermittent shortness of breath, we also performed a chest x-ray and echocardiography, both of which were normal. The patient’s coronary and renal angiogram was also normal. As the carotid bruit was audible bilaterally, we also performed carotid artery color Doppler imaging (evaluating the common carotid artery (CCA), internal carotid artery (ICA), external carotid artery (ECA) and vertebral artery (VA)). Findings showed diffuse homogenous intimal thickness involving the bilateral CCA, ICA, and ECA, demonstrating macaroni sign, causing marked luminal narrowing. Other findings include minimal flow and reduced peak systolic velocity (PSV) in the right CCA and ICA on power Doppler and no flow on color and power Doppler images in the right ECA. The right VA was dilated with normal flow and no flow was seen in the PSV, left proximal and mid CCA on power Doppler images (suggestive of complete occlusion). However, left ICA and ECA showed minimal flow and reduced PSV on power Doppler (possibly due to collateral supply), left VA shows no flow (suggestive of complete occlusion) and there was turbulent flow in the aortic arch. Overall, on the basis of clinical manifestations and carotid artery Doppler findings, a diagnosis of TA was made. We started the patient on oral prednisone (60 mg) once daily on a tapered basis and consulted the department of vascular surgery for a possible surgical intervention. The patient had an endarterectomy two weeks after admission which led to an improvement in his symptoms, after which he was discharged on 5th February 2019, and no follow-up has since been conducted.\n\n\nDiscussion\n\nTA can be seen in a broad geographical area, but it is mainly found in Asia and Africa. The nature of the disease is autoimmune, involving arterial walls resulting in panarteritis1,3. According to the American Rheumatological society, for diagnosing TA, out of the following six clinical findings, three are compulsory: (i) Onset before 40 years; (ii) Claudication of the extremities; (iii) Decrease in the brachial pulse in one or both arms; (iv) Difference of 10 mmHg or more in blood pressure measured in both arms; (v) audible bruit on auscultation of the aorta or subclavian artery; and (6) narrowing at the aorta or its primary branches on arteriogram2,3. Our patient met at least five of the above criteria. He was a 22-year-old male, and literature2,5 supports the fact that it is common in second and third decades. Moreover, in adults, almost 80% of the patients are women2.\n\nThe disease progresses in two phases; the early active phase that lasts weeks to months, giving constitutional symptoms and can have relapse and remission, and the late chronic phase which is caused by arterial stenosis along with ischemia and occlusion of organs. The clinical illustrations can be different based on the location of arterial lesions (Table 1)2,3.\n\nOur patient presented primarily with symptoms of the aortic branches, such as malaise, absent pulse on the right upper extremity and headache.\n\nA diagnosis of TA is primarily based on clinical and radiological findings, as the results of biopsy are nonspecific as the histopathology may imitate other types of vasculitis6. Suspected TA always warrants prompt vascular imaging, enabling earlier diagnosis and further decreasing the risk to the patient. Although angiography was considered to be the standard method for diagnosis of TA, it has been replaced by computed tomography angiography or angiography or magnetic resonance angiography2. Furthermore, literature has shown that ultrasound with color Doppler flow imaging and angiography are highly useful for detecting and determining the severity of the disease (except for right brachiocephalic artery)2,3,6. However, in our setting, due to limited resources, we only conducted carotid artery Doppler imaging, and the findings were sufficient to achieve a diagnosis of TA. As far as treatment is concerned, immunosuppressants such as prednisone and/or methotrexate can lead to significant improvement3. Cyclophosphamide is reserved for treatment-resistant cases; a prior study revealed that steroids and cyclophosphamide are effective in the early acute phase when surgical management is not considered6. However, in the presence of symptomatic occlusive lesions (fibrotic phase), procedures such as bypass grafts, patch angioplasty, endarterectomy, percutaneous transluminal angioplasty, or stent placement should be considered3,6. Furthermore, despite the ongoing medical treatment, 50% of patients with TA progress to a stage that requires one or more surgical procedures6. In our case, we started the patient on 60 mg prednisone orally daily, but due to the stenotic lesions, department of vascular surgery was also consulted for a possible endarterectomy.\n\n\nConclusion\n\nOverall, TA is a rare disease (especially in men) that is both diagnostically and therapeutically challenging to physicians. Early diagnosis is very important in this debilitating disease, in order to improve the outcomes. As far as the role of patient gender and prognosis is concerned, the literature is sparse, and more studies should be conducted. Furthermore, although medical treatment is considered as the mainstay for TA, it is imperative to apprehend both the indications and the available options of surgical interventions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.",
"appendix": "Grant information\n\nThe author(s) declare that no grants were involved in supporting this work.\n\n\nReferences\n\nMont'Alverne AR, Paula LE, Shinjo SK: Features of the onset of Takayasu's arteritis according to gender. Arq Bras Cardiol. 2013; 101(4): 359–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLalhmachhuani PC, Daimei SL, Ralte RL, et al.: A case of takayasu arteritis in elderly male patient. Indian Journal of Medical Research and Pharmaceutical Sciences. 2017; 4(12). Publisher Full Text\n\nKhan MAM, Banoo H: A case report of takayasu’s arteritis. Med Today. 2012; 24(2): 79–81. Publisher Full Text\n\nTakayasu Arteritis. 2018; Accessed: March 16, 2019. Reference Source\n\nKothari S: Takayasu's arteritis in children - a review. Images Paediatr Cardiol. 2001; 3(4): 4–23. PubMed Abstract | Free Full Text\n\nNazzal MD, Agko M, Zingale K, et al.: A unique presentation of Takayasu's arteritis in a 39-year-old male with chest pain, vertigo, and blindness. J Vasc Surg. 2011; 54(2): 529–32. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "48509",
"date": "17 May 2019",
"name": "Samuel Katsuyuki Shinjo",
"expertise": [
"Reviewer Expertise Inflammatory myopathies",
"systemic vasculitis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease give us more information about a possible involvement of thoracic and abdominal aortic and its main branches. Was the patient treated only with prednisone? Table 1. Please correct the sentence (\"Temporal arteritis\"). Discussion: should be more explored; There is no relevance to describe the \"TA classification criteria\" in the Discussion; the relevance of the present case report should be more explored throughout the text.\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": []
},
{
"id": "75658",
"date": "07 Jan 2021",
"name": "Enrico Tombetti",
"expertise": [
"Reviewer Expertise Vasculitis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 report a young male with neurological symptoms that was diagnosed of TA.\nThe neurological presentation is interesting, but I have some comments.\nMajor\nTreatment: the patients opt for single therapy with steroids. This is unusual, since TA has frequently a polyphasic course, as the authors disclose. Considering the risk of recurrences and the severity of presentation, I would have used upfront combination therapy with steroids & immunosuppressive agents (e.g.: methotrexate). If the authors wants to presents this case, they should discuss better therapy, explain why they have not considered immunosuppressive agents, and acknowledge that this would not have been the preferred therapeutic choice in most centers.\n\nDisease characterisation. I agree that with US data there is enough evidence to diagnose TA. However, I think that this patient deserve further disease characterisation, given the severity of presentation and considering that the patient underwent endoarterectomy, I would have studied him with antio-MRI or angio-CT. Has this been done? If yes, I would describe findings. if not, this should be discussed highlighting that optimal management should include, if possible CT or MR, unless in case of burn-out mild disease.\n\nOutcome of management: The authors simply report that the symptoms have improved. This is too vague. I would add a) postoperative results on carotid disease, b) clinical outcomes, and which symptoms have improved and the degree of improvement. In the case of follow-up data, discussion of the outcome may be performed at follow-up.\n\nFollow-up. I wonder if the authors have already performed the first follow-up assessment or not. If yes, please describe results. Otherwise, please describe how you plan to follow the patient.\nMinor\nPast medical history should be better described.\n\nThe authors correctly avoided to perform surgery without medical therapy. This point deserves further discussion in the light of literature data.\n\n\"However, central nervous system examination revealed problems\" this sentence should be rephrased.\n\n\"with normal flow and no flow was seen in the PSV,\" I cannot understand this sentence. In general US findings are difficult to be read, and I suggests to rephrase this section.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
},
{
"id": "76799",
"date": "07 Jan 2021",
"name": "Tetsuro Yokokawa",
"expertise": [
"Reviewer Expertise Cardiovascular 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\nAuthors demonstrated a young male patient of Takayasu arteritis. This is a rare case report diagnosed by using ultrasound imaging. This reviewer thinks this report has lack of novelty and have some concerns as shown below.\nSome patients with Takayasu arteritis are male. Ultrasound imaging is usual examination for the diagnosis for vascular diseases. What is the novelty of this case report?\n\nTakayasu arteritis should be diagnosed with imaging for inflammation such as positron emission tomography (PET). PET imaging is useful to monitor of immunosuppressive treatment. Did this patient undergo PET?\n\nAuthors should show the ultrasound images as Figures.\n\nAuthors should show laboratory data including liver and kidney function in a new Table.\n\nThis patient had elevated C-reactive protein. How did authors exclude infectious diseases?\n\nThis patient underwent endarterectomy. Was the pathological finding from the endarterectomy sample compatible for Takayasu arteritis? Please provide the pathological image as a Figure.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-333
|
https://f1000research.com/articles/8-182/v1
|
14 Feb 19
|
{
"type": "Case Report",
"title": "Case Report: Unprovoked venous thromboembolism in a young adult",
"authors": [
"Jeyhan Dhabhar",
"Varshil Mehta",
"Nimit Desai",
"Sameer Dawoodi",
"Sojib Bin Zaman",
"Jeyhan Dhabhar",
"Nimit Desai",
"Sameer Dawoodi"
],
"abstract": "A 24-year-old male was presented to us with sudden onset of chest pain and dyspnea for the past one hour. There was no history of calf pain, trauma, surgery, prolonged immobilization, long-haul air travel, bleeding diathesis or any other co-morbidity. The patient denied any addiction history. The Electrocardiogram showed tachycardia with S1Q3T3 pattern. The left arterio-venous Doppler study was suggestive of a thrombus in popliteal vein and sapheno-popliteal junction. The CT-Pulmonary Angiogram scan was suggestive of a massive pulmonary thromboembolism. The patient was thrombolysed with Intravenous Alteplase immediately and was put on tab Rivaroxaban for maintenance. He was later discharged after being stable. Unprovoked venous thromboembolism (VTE) is very rare and has the potential to lead to pulmonary embolism which could be disastrous, especially in young adults. We present such a case where unprovoked VTE was diagnosed and treated. This case suggests that high clinical suspicion is the key for the diagnosis of acute pulmonary embolism, especially in the absence of history suggestive of deep vein thrombosis.",
"keywords": [
"Pulmonary Embolism",
"Idiopathic",
"Thromboembolism"
],
"content": "Introduction\n\nVenous thromboembolism (VTE) consists of pulmonary embolism (PE) and deep vein thrombosis (DVT). It is one of the leading causes of cardiovascular disability and impaired quality of life. It also causes major long-term complications, which include recurrent VTE, chronic thromboembolic pulmonary hypertension and post-thrombotic syndrome1.\n\nPE is said to be the third leading cause for cardiovascular mortality (after myocardial infarction and stroke). It leads to 100 000 deaths annually. It is not only the leading preventable cause of death in admitted patients1 but also the leading cause of deaths in mothers during pregnancy in developed countries2.\n\nIdiopathic or unprovoked VTE is defined as “any VTE in the absence of an identifiable predisposing factor”3. Taking into consideration that unprovoked VTE is very rare, especially in the young adults, we put forward a case report of VTE in a 24-year-old man.\n\n\nCase report\n\nA 24-year-old man was brought to the emergency department of a hospital, by his office-colleagues, complaining of sudden onset of chest pain and dyspnea at rest, for the last one hour. It was not associated with sweating, palpitations, cough, hemoptysis, syncope, giddiness, leg pain, pedal edema, fever, rash, or any bleeding manifestations.\n\nHistory of calf pain, trauma, surgery, prolonged immobilization, long-haul air travel, bleeding diathesis or any other co-morbidity was not reported by the patient. The patient also denied any addiction history. Family history was found to be insignificant.\n\nOn admission, the patient’s heart rate was 114/min, and blood pressure was 106/90 mmHg. His respiratory rate was 22/min, and O2 saturation rate was 82% at room air. BMI was 20.76 kg/m2. There was no murmur or gallop on cardiovascular examination. Air entry was reduced in the left infra-axillary region. Electrocardiogram (ECG) showed tachycardia with S1Q3T3 pattern, and chest X-ray was suggestive of obliteration of left costo-phrenic angle. The D-Dimer (17.31 ug/ml) was elevated, 34 times above the normal upper limit (0.5 ug/ml).\n\nCT-Pulmonary Angiogram (Figure 1) was suggestive of a massive pulmonary thromboembolism. The pulmonary trunk was dilated to ~30 mm. There was a non-lumen occluding circumferential filling defect in the main pulmonary trunk, with maximum thickness of 4.5 mm. A large partial-lumen occluding filling defect was noted in the left main pulmonary artery, which was extending further into the hilar branch, occluding the lumen completely. Another larger complete lumen occluding filling defect was noted in the right main pulmonary artery. These filling defects were extending into the segmental and sub-segmental branches of the lateral segment of the right middle, lingual and bilateral lower lobe. The RV: LV ratio was 2:1. All four pulmonary veins were normal, and there was no evidence of mediastinal pathology.\n\nOn admission, the patient also underwent a bilateral arteriovenous Doppler study, which was suggestive of a partially-lumen- occluding thrombus in the proximal part of left popliteal vein and a completely lumen-occluding thrombus at the left saphenopopliteal junction, approximately 14 cm long. The veins of both legs showed arterialized waveforms.\n\nOn 2D echocardiography, right atrium and right ventricle was mildly dilated with grade I, tricuspid regurgitation (TR) and pulmonary arterial systolic pressure by TR jet was 55 mmHg suggestive of moderate pulmonary artery hypertension. No regional wall motion abnormality was observed and left ventricular ejection fraction was 60%.\n\nThe coagulation profile was within normal limits. All other blood investigations i.e. hemogram, serum electrolytes, renal and liver function tests were within normal range. The patient had mild hyperuricemia with serum uric acid level being 7.4 mg/dL (Normal – 3.5 – 7.2 mg/dL). Cardiac enzymes (Creatine PhosphoKinase-MB and Troponin T) were mildly elevated.\n\nAlthough it was a clear case of massive VTE, the underlying etiology of such an event could not be extrapolated. Since the patient was a 24-year-old man, without any risk factors or comorbidities, the final diagnosis of unprovoked VTE was made.\n\nThe patient was thrombolysed with Injection Alteplase infusion (100mg IV, over two hours) with Injection Enoxaparin 60 mg given subcutaneously every 12 hours. After which, the patient developed hypotension which was treated with inotropic support. Although he had tachycardia post-thrombolysis for the next two days, his blood pressure returned to normal on the third day. 48-hours after giving thrombolytic treatment, the left lower limb venous doppler was done which was suggestive of a partial-lumen occluding the thrombus in popliteal vein extending from saphenopopliteal junction to mid-leg approximately 10 cm long. The CT-pulmonary angiogram was not repeated (post-thrombolysis), due to financial constraints; however, the patient improved drastically. He was shifted to general ward on day 4 post admission. Tab Rivaroxaban 15 mg (12 hourly, orally) was prescribed for the next three weeks. The patient was discharged successfully on the 15th day of admission. One week after discharge, the patient was advised Tab. Rivaroxaban 20 mg, once daily.\n\n\nDiscussion\n\nVTE in a young male patient is life-threatening, especially if it is unprovoked. The most common and important ECG finding in a patient with PE is sinus tachycardia and the presence of S1Q3T3 pattern in the ECG. Presence of these findings shortened the time for diagnosis of acute PE in our patient and the management after that4.\n\nLiterature suggests that the risk of early death among patients with symptomatic PE is 18-fold higher when compared with the patients having DVT alone5. Had this patient presented with sinus tachycardia alone, he would have undergone a battery of investigations, and prompt thrombolysis would have been delayed. Furthermore, the incidence rate of VTE is about 1.5 per 1,000 person-years while appearance of DVT is twice as common6.\n\nSince our patient did not have any clinical features suggestive of DVT, having a Doppler study suggestive of DVT, was more of an additional finding, confirming VTE in this patient. A young man with sudden chest pain and dyspnea without any leg pain and who is otherwise healthy, is likely to be mismanaged, as suspicion of pulmonary or venous thrombo-embolism is quite low.\n\nBefore attempting to use the Wells criteria, a point-score based on clinical features and the likelihood of diagnoses other than PE, the clinician must first have a suspicion of the diagnosis before attempting to apply the Wells criteria7. On admission, the Wells score for PE was 4.5 with a moderate probability of having a PE for the present patient based on the history and clinical features. The findings on CT Pulmonary Angiogram scan and ECG, increased the possibility of a PE, hence 3 points and 1.5 points were added for presence of tachycardia in our patient’s total score. The well’s score for DVT was 0, as there was no history of cancer, surgery, immobilization, calf swelling, superficial veins, lower limb swelling, tenderness, paralysis or previous history of DVT. Hence high clinical suspicion remains the key in diagnosing PE, especially in the absence of DVT.\n\nA case of unprovoked VTE, especially acute PE, needs to be extensively worked up, to prevent recurrence. Apart from history, family members need to be screened for coagulopathy or asymptomatic VTE, which was not done in this case. Although, coagulation profile, i.e. International Normalised Ratio (INR), prothrombin time, and activated partial thromboplastin time (APTT), was normal in our patient, it is advisable to do a thrombophilia profile. The thrombophilia profile will help to anticipate the tendency to develop pathological clotting or thrombotic disorders and consists of namely, factor V Leiden mutation, prothrombin gene mutation, protein C resistance, anti-beta 2 glycoprotein, anticardiolipin antibodies, protein S activity and serum homocysteine levels8. Due to the huge cost of these investigations, it becomes difficult to run such investigations while dealing with a case of acute PE in hospital. Thrombophilia profile is not indicated for the patients who are on anticoagulation therapy. Our patient gave a negative consent for doing a thrombophilia profile, hence the procedure was not done.\n\nInj. Alteplase is indicated as a fibrinolytic agent for the lysis of the clot seen in acute massive PE9. Massive PE is caused by obstruction (more than 50% of the cross-sectional area) of the pulmonary arterial tree, leading to an acute and subsequently severe cardiopulmonary failure due to right ventricular overload10.\n\n\nConclusion\n\nSilent VTE can develop into PE which may be unrecognized for a long time before the clinical features develop. High clinical suspicion is the key, for the diagnosis of acute PE in young patients, especially in the absence of history suggestive of DVT. In the absence of hemodynamic instability, the decision of fibrinolysis, in acute massive PE, largely depends on CT-Pulmonary Angiogram.\n\n\nConsent\n\nWritten informed consent was taken from the patient and hospital authority to publish this case.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nMedkrux Research Group.\n\n\nReferences\n\nJerjes-Sánchez C: Mechanisms of thrombosis. In: Thrombolysis in Pulmonary Embolism. Springer, Berlin. 2015; 1–17. Publisher Full Text\n\nKonstantinides SV, Barco S, Lankeit M, et al.: Management of Pulmonary Embolism: An Update. J Am Coll Cardiol. 2016; 67(2): 976–990. PubMed Abstract | Publisher Full Text\n\nIorio A, Kearon C, Filippucci E, et al.: Risk of recurrence after a first episode of symptomatic venous thromboembolism provoked by a transient risk factor: a systematic review. Arch Intern Med. 2010; 170(19): 1710–6. PubMed Abstract | Publisher Full Text\n\nTapson VF: Acute pulmonary embolism. N Engl J Med. 2008; 358(10): 1037–52. PubMed Abstract | Publisher Full Text\n\nHeit JA, Silverstein MD, Mohr DN, et al.: Predictors of survival after deep vein thrombosis and pulmonary embolism: a population-based, cohort study. Arch Intern Med. 1999; 159(5): 445–453. PubMed Abstract | Publisher Full Text\n\nNaess IA, Christiansen SC, Romundstad P, et al.: Incidence and mortality of venous thrombosis: a population-based study. J Thromb Haemost. 2007; 5(4): 692–9. PubMed Abstract | Publisher Full Text\n\nWells PS, Anderson DR, Rodger M, et al.: Excluding pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to the emergency department by using a simple clinical model and d-dimer. Ann Intern Med. 2001; 135(2): 98–107. PubMed Abstract | Publisher Full Text\n\nVenous thromboembolic diseases: diagnosis, management and thrombophilia testing. Clinical guideline [CG144]. Published on 27 June 2012, Last updated on November 2015. Reference Source\n\nMartin C, Sobolewski K, Bridgeman P, et al.: Systemic Thrombolysis for Pulmonary Embolism: A Review. P T. 2016; 41(12): 770–775. PubMed Abstract | Free Full Text\n\nSadeghi A, Brevetti GR, Kim S, et al.: Acute massive pulmonary embolism: role of the cardiac surgeon. Tex Heart Inst J. 2005; 32(3): 430–433. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "44458",
"date": "15 Feb 2019",
"name": "Shakti A. Goel",
"expertise": [
"Reviewer Expertise Medicine",
"Surgery",
"Orthopaedics",
"Spine",
"Innovation",
"Technoinnovations"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case report which discusses a rare phenomenon of unprovoked venous thromboembolism and its management. I would congratulate the authors for their work. However, there are a few concerns:\nThough the authors have mentioned that there was no history of long air travel, it will be helpful to know if the patient had any history of long car travel before the event precipitated. It has been noted that DVT is commonly encountered in patients who undergo long car travel. Such information, if added, shall make the physicians more aware.\nDid the physicians/authors conduct any clinical examination to test DVT. Were Homan's or Mosses sign performed. If yes, what were the results. If not, it will be helpful to know the reason for not performing them. A little detail on clinical examination of the patient should also be added to the manuscript besides the symptoms and findings.\nIt will also be helpful to know the Trop I values, if they were assessed by the authors.\nThe authors have mentioned that patient was kept on Rivaroxaban. Were there any side effects that were encountered. It will also be helpful if the authors could mention about the follow-up period of the patient in the manuscript.\nWas any assessment done in the post discharge period. Was there any change in vessel size? Any adaptive remodeling seen with the treatment using Rivaroxaban .\nWas the patient satisfied with the treatment? Any satisfaction index which was assessed. Authors may add it if found to be appropriate. They could use the attached reference for the same.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "4422",
"date": "15 Feb 2019",
"name": "Sojib Bin Zaman",
"role": "Author Response",
"response": "Thank you Dr for reviewing our paper and taking your time out for the same.Please find my replies to your queries. There was no history of prolonged air or car travel. Also, the detailed examination was performed and was deemed unremarkable; hence was not mentioned here (including Homan's and Moses sign). Trop I level test are not available in our center, hence was not done, However, we did Trop T and CPK MB, which were normal. The patient had followed up in our center for the first three months, following which relocated to Uttar Pradesh, India (his hometown). He experienced no side effects of Rivaroxaban. At three months, post discharge, a lower limb doppler was repeated which showed more than 50% reduction in size of the obstructing thrombus as compared to the previous one done at admission. Post three months of discharge, the patient has not returned back yet and has been lost to follow up. The patient was most satisfied with the treatment and the recovery he'd made, but this was not recorded in a form of any official questionnaire (but he had mentioned this verbally); hence not mentioned here. Hopefully, these comments will satisfy the queries. If you have any more queries, please feel free to post and we shall be happy to reply them."
},
{
"c_id": "4431",
"date": "19 Feb 2019",
"name": "Shakti A. Goel",
"role": "Reviewer Response",
"response": "I had an opportunity to go through the review/reply and believe that the authors have addressed the comments meticulously. The article can be accepted once these details have been added to the article. This article will certainly be very helpful for the physicians around the globe especially in low to middle income countries."
}
]
},
{
"id": "45517",
"date": "18 Mar 2019",
"name": "Himel Mondal",
"expertise": [
"Reviewer Expertise Cardiovascular physiology",
"Exercise physiology",
"Public health",
"Medical 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\nOverall evaluation This is an interesting case report of venous thromboembolism in a young adult male without any predisposing factors. The authors described the case in detail with relevant and recent literature references. However, there is a need of a minor revision to enrich the manuscript. The author may revise the manuscript keeping in following points in mind:\nTitle As this is a case of a “young adult male,” authors may add the word “male” in the title of the manuscript.\nAbstract The patient presented with chest pain and dyspnoea. The second line of the “Abstract” described that the patient did not have any “calf pain.” Authors could add some cardiological history. They could also add a glimpse of clinical examination (especially heart rate and rhythm) rather than starting with “ECG showed tachycardia…”\nKeywords Authors may add more keywords according to MeSH for wider dissemination of the article.\nIntroduction PE causes “100000 deaths” annually. Is it global or regional data? Please specify.\nCase report Authors may add the time when the patient was brought to the hospital, along with the nature of his works in previous 12 hours, if the information is available. What was the rhythm of the pulse in clinical examination? If the data is available, the authors may add it.\nDiscussion Discussion is adequate and well written.\nConclusion Written well and it is within the scope of the article.\nReferences References are relevant and recent.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "4494",
"date": "25 Mar 2019",
"name": "Sojib Bin Zaman",
"role": "Author Response",
"response": "Thank you for your reviews and approving it. Changes we made: In title: We have added \"male\" in the title. Abstract: We have also added heart rate and bp in the abstract. Introduction: It leads to 100 000 deaths annually globally The heart rhythm was normal and hence we didn't add it. Hopefully, we have made adequate changes. Once again thank you for reviewing for us. Sojib."
}
]
}
] | 1
|
https://f1000research.com/articles/8-182
|
https://f1000research.com/articles/8-12/v1
|
03 Jan 19
|
{
"type": "Research Note",
"title": "Anti-infective efficacy of Psidium guajava L. leaves against certain pathogenic bacteria",
"authors": [
"Pooja Patel",
"Chinmayi Joshi",
"Tannaz Birdi",
"Vijay Kothari",
"Pooja Patel",
"Chinmayi Joshi",
"Tannaz Birdi"
],
"abstract": "Water extracts of Psidium guajava leaves prepared by three different extraction methods were compared with respect to their anti-infective activity against Pseudomonas aeruginosa and Staphylococcus aureus in the nematode host Caenorhabditis elegans. The water extract prepared by Microwave Assisted Extraction method was found to have better anti-infective activity, and its activity was further compared with hydroalcoholic extract prepared using the same extraction method against five different pathogenic bacteria. Both these extracts could attenuate virulence of P. aeruginosa, S. aureus, Serratia marcescens, and Chromobacterium violaceum, towards C. elegans. Anti-infective efficacy of P. guajava leaf extract seems partly to stem from its quorum-modulatory property, as it could modulate production of quorum sensing-regulated pigments in all the susceptible bacteria.",
"keywords": [
"Guava leaf",
"Microwave Assisted Extraction (MAE)",
"Caenorhabditis elegans",
"Quorum Sensing (QS)",
"Antimicrobial Resistance (AMR)",
"Anti-virulence"
],
"content": "Introduction\n\nGiven the heavy global burden of infectious diseases, it is imperative to discover novel pharmaceutical assets for combating antimicrobial resistance, with particular focus on antibiotic-resistant bacterial pathogens recently listed by the World Health Organization as of high/ critical priority (Tacconelli et al., 2018). Since the antibiotic pipeline lacks new mechanisms against resistant bacteria, particularly gram-negative bacteria (see here for more information), it is necessary to look for new antibiotics as well as non-antibiotic approaches to tackle bacterial infections.\n\nA reverse pharmacology approach (Raut et al., 2017) of investigating plant extracts, particularly those employed in documented or folklore traditional medicine, for their potential anti-pathogenic efficacy may pave the way for discovery and development of novel antimicrobial molecules/ formulations. We undertook the current study to investigate anti-infective potential of one such plant extract, Psidium guajava L. (common name- guava; Family- Myrtaceae) leaf extract, against five different pathogenic bacteria. This plant has traditionally been used for treatment of various gastrointestinal problems including diarrhea and dysentery (Birdi et al., 2010), which are caused usually due to microbial infections.\n\n\nMethods\n\nShade dried mature guava leaves of Sardar variety, one of the five common Indian varieties were used. The leaves were collected in September 2014 from Shirwal, Satara district, Maharashtra, India. The dried leaves were stored in a sealed plastic bag at 25°C. A voucher specimen was deposited at Naoroji Godrej Centre for Plant Research (NGCPR, Shirwal) under herbarium number NGCPR 712.\n\nPathogenic bacteria used in this study (Dataset 1: Extended data) included Staphylococcus aureus (MTCC 737); beta-lactamase producing multidrug resistant strains of Chromobacterium violaceum (MTCC 2656) and Serratia marcescens (MTCC 97); multidrug resistant Pseudomonas aeruginosa; and Streptococcus pyogenes (MTCC 1924). Resistance to three or more antibiotics during antibiotic susceptibility profiling (Dataset 1) was taken as the criteria for tagging any organism as ‘multidrug resistant’. P. aeruginosa was sourced from our internal culture collection. All other cultures were procured from MTCC (Microbial Type Culture Collection, Chandigarh, India).\n\nIn order to identify the best possible extraction method with respect to the desired biological activity, we extracted the powder of the dried leaves in water using three different extraction methods: Decoction, Microwave Assisted Extraction (MAE), and Vacuum Assisted Extraction (VAE). Protocols employed for each extraction method are described below:\n\n\nDecoction\n\nDecoction of guava leaves was prepared in accordance to the traditional method described in the Ayurvedic texts (Thakkur, 1979). 1 g of the plant material was boiled in 16 mL double distilled water, till the volume was reduced to 4 mL.\n\n\nMicrowave Assisted Extraction (MAE) (Kothari et al., 2009)\n\n1 g of leaf powder was soaked into 16 mL of water or 50% ethanol, and subjected to microwave heating (Electrolux EM30EC90SS) at 720 W. Total extraction duration was 140 s, of which first heating was for 40 s, and subsequent two heating cycles of 10 s each. Intermittent cooling period between any two heating cycles was kept 40 s. Liquid volume at the end of extraction was 4 mL.\n\n\nVacuum Assisted Extraction (VAE) (Wang et al., 2014)\n\n1 g of dry leaf powder was mixed with 16 mL of water. Vacuum pump (MEDICA INSTRUMENT Mfg. Co.) was attached to the vessel containing plant material and solvent, and the working pressure was set at 7.36 psi (15 In. Hg). Total duration of heating was 20 min, of which for 15 min the system was at 65°C (at which boiling started). Extraction was stopped when liquid volume was reduced to 4 mL.\n\nExtraction performed by methods described above, was followed by macro-filtration using nylon strainer followed by centrifugation (at 10,000 rpm for 15 min; Remi BZCI-8729), and filtration with Whatman paper # 1 (Axiva, Haryana). After this filtration, solvent was evaporated from the extract. For bioassay, extracts was reconstituted in absolute DMSO (Merck, Mumbai). Reconstituted extracts were collected in sterile flat bottom glass vials (15 mL, Merck, Mumbai) covered with aluminum foil, and protected from light to avoid photo-oxidation of light-sensitive compounds. The internal surface of vial cap was also wrapped with aluminum foil to avoid leaching of vial cap material (Houghton & Raman, 1998). Reconstituted extract was stored under refrigeration for further use. Extraction efficiency was calculated as percentage weight of the starting dried plant material.\n\nExtraction efficiency obtained with these methods was 6.30%, 5.80%, and 6.0% respectively. All the extracts were reconstituted in dimethylsulfoxide (DMSO, Merck) upon drying, and stored under refrigeration (4-8º C) till further use.\n\nIn vivo efficacy of these water extracts against Pseudomonas aeruginosa, and Staphylococcus aureus was tested in the nematode host Caenorhabditis elegans, wherein the extract prepared by MAE had better anti-infective activity. Therefore, the extract prepared by MAE was compared with its hydroalcoholic extract prepared using the same method. Extraction efficiency obtained for the latter case was 2.0%.\n\nIn vivo efficacy of the guava leaf extract (GLE) was evaluated using the nematode worm Caenorhabditis elegans as the model host, employing the method described by Eng & Nathan, (2015) with some modification. C. elegans was maintained on Nematode Growing Medium (NGM) which consisted of 3 g/L NaCl, 2.5 g/L peptone, 1 M CaCl2, 1 M MgSO4, 5 mg/mL cholesterol, 1 M phosphate buffer of pH 6, 17 g/L agar-agar with E. coli OP50 (procured from LabTIE B.V., JR Rosmalen, the Netherlands) as the feed. The worm population to be used for the in vivo assay was kept on NGM plates not seeded with E. coli OP50 for three days, before being challenged with the test pathogen.\n\nPathogenic bacteria was incubated with GLE for 22-24h (48 h in case of S. marcescens and S. aureus) at 37°C (28°C for S. marcescens). Following incubation, OD764 of the culture suspension was equalized to that of the DMSO control. 100 μL of this bacterial suspension was mixed with 900 μL of the M9 buffer containing 10 worms (L3-L4 stage). This experiment was performed in 24-well (sterile, non-treated) polystyrene plates (HiMediaTPG24), and incubation was carried out at 22°C. Number of live vs. lead worms was counted daily for five days by putting the plate (with lid) under light microscope (4X). Standard antibiotic- and catechin- treated bacterial suspension were used as positive control. Straight worms were considered to be dead, Plates were gently tapped to confirm lack of movement in the dead-looking worms. On the last day of the experiment, when plates could be opened, their death was reconfirmed by touching them with a straight wire, wherein no movement was taken as confirmation of death.\n\nValues reported are means of four independent experiments, whose statistical significance was assessed using t-test performed through Microsoft Excel (2013). P values ≤0.05 were considered to be statistically significant.\n\n\nResults\n\nGLE prepared by three different methods were compared, at three different concentrations, for their anti-infective activity against P. aeruginosa and S. aureus (Figure 1; Dataset 1: Underlying data). At 50 µg/mL, GLE prepared by MAE proved superior to that prepared by decoction or VAE method, with respect to its ability to attenuate P. aeruginosa’s virulence towards C. elegans. At 0.5 µg/mL, extract prepared by decoction method registered least activity against this bacterium. At the same concentration, against S. aureus, extract prepared by VAE displayed the least activity. Based on these results, we concluded MAE as a better extraction method, and then extracted guava leaves using this method in water as well as water:alcohol (1:1) mixture. Both of these extracts prepared using MAE were then assayed for their anti-infective potential against five different pathogenic bacteria.\n\nComparison of in vivo anti-infective efficacy of P. guajava leaf extracts prepared by three different extraction methods, against P. aeruginosa (A–C), and S. aureus (D–F). Catechin (50 μg/mL) and gentamicin (0.1 μg/mL) employed as positive controls conferred 100% and 80% protection on the worm population, respectively. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans. DMSO (0.5%v/v) and GLE at tested concentrations showed no toxicity towards C. elegans. MAE: Microwave Assisted Extraction; VAE: Vacuum Assisted Extraction; GLE: Guava Leaf Extract\n\nBoth water as well as the hydroalcoholic extract of guava leaves could attenuate virulence of all the test pathogens (except S. pyogenes) towards C. elegans (Figure 2; Dataset 1: Underlying data). Both these extracts exhibited statistically similar anti-pathogenic efficacy against all susceptible bacteria, but the hydroalcoholic extract exhibited 10-15% better activity against S. aureus than the water extract. Despite the lowest extraction yield among all extracts reported in this study, the hydroalcoholic GLE was found to possess the highest (at par with water extract against all gram-negative pathogens) anti-pathogenic activity. Critical importance of choice of most appropriate extraction method and solvent for preparation of bioactive extracts has earlier been also emphasized by us (Gupta et al., 2012; Kothari et al., 2012), and others (Ngo et al., 2017; Sasidharan et al., 2011).\n\nFigures A-E shows data against P. aeruginosa, S. aureus, S. marcescens, C. violaceum and S. pyogenes respectively. Catechin (50 μg/mL) employed as a positive control conferred 100% protection on worm population against all the pathogenic bacteria except S. pyogenes. Against S. pyogenes, catechin could not offer any protection to host worms. Gentamicin (0.1 μg/mL) allowed survival of worm population to the extent of 80% in face of P. aeruginosa, S. aureus, or S. pyogenes challenge; and 100% against the remaining two pathogens. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacteria towards C. elegans. DMSO (0.5%v/v) and GLE at tested concentrations showed no toxicity towards C. elegans.\n\nTo have some insight into the mode of action of GLE, we incubated all the five test bacteria with GLE to investigate whether it affects bacterial growth and/or quorum-sensing (QS) regulated pigment production (a marker trait). Bacterial cell density and pigment production were quantified as earlier described by us (Joshi et al., 2016; Patel et al., 2018). At least one concentration of GLE was found to modulate pigment production in all the four pigmented bacteria (Figure 3; Dataset 1: Underlying data). This extract did not inhibit bacterial growth heavily, and hence can be expected to exert lesser selection pressure on susceptible bacterial populations.\n\n(A) P. aeruginosa (B) S. aureus (C) S. marcescens (D) C. violaceum (E) S. pyogenes. Bacterial cell density and pigment production were quantified as earlier described by us (Joshi et al., 2016). Bacterial growth was measured as OD764 for the four pigmented bacteria, while for S. pyogenes OD660 was used. OD of pyoverdine was measured at 405 nm, and that of pyocyanin at 520 nm; Pyoverdine Unit was calculated as the ratio OD405/OD764 (an indication of pyoverdine production per unit of growth); Pyocyanin Unit was calculated as the ratio OD520/OD764 (an indication of pyocyanin production per unit of growth. OD of staphyloxanthin was measured at 450 nm, and Staphyloxanthin Unit was calculated as the ratio OD450/OD764 (an indication of staphyloxanthin production per unit of growth). OD of prodigiosin was measured at 535 nm, and Prodigiosin Unit was calculated as the ratio OD535/OD764 (an indication of prodigiosin production per unit of growth). OD of violacein was measured at 585 nm, and Violacein Unit was calculated as the ratio OD585/OD764 (an indication of violacein production per unit of growth). QS: Quorum sensing\n\n\nConclusion\n\nResults of the present study validate the traditional use of guava leaves for medicinal purposes and suggests one of the possible mechanisms through which it exerts its anti-infective activity, i.e. its ability to interfere with the bacterial QS machinery. Further investigation regarding GLE’s effect on pathogenic bacteria at the whole transcriptome level is warranted to unravel the molecular mechanisms underlying its anti-pathogenic efficacy.\n\n\nData availability\n\n\n\n\nUnderlying data\n\nF1000Research: Raw data for Figure 1–Figure 3 showing the anti-infective efficacy of Psidium guajava L. leaves against pathogenic bacteria., https://doi.org/10.5256/f1000research.17500.d230522 (Patel et al., 2018a).\n\n\nExtended data\n\nF1000Research: Details of organisms used in this study including antibiogram., https://doi.org/10.5256/f1000research.17500.d230521 (Patel et al., 2018b).",
"appendix": "Grant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nAuthors thank Nirma Education and Research Foundation (NERF), Ahmedabad for financial and infrastructural support.\n\n\nReferences\n\nBirdi T, Daswani P, Brijesh S, et al.: Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea. BMC Complement Altern Med. 2010; 10(1): 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEng SA, Nathan S: Curcumin rescues Caenorhabditis elegans from a Burkholderia pseudomallei infection. Front Microbiol. 2015; 6: 290. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta A, Naraniwal M, Kothari V: Modern extraction methods for preparation of bioactive plant extracts. International Journal of Applied and Natural Sciences. 2012; 1(1): 8–26. Reference Source\n\nHoughton P, Raman A: Laboratory Handbook for the Fractionation of Natural Extracts. Chapman and Hall: London, UK. 2016; 18.\n\nJoshi C, Kothari V, Patel P: Importance of selecting appropriate wavelength, while quantifying growth and production of quorum sensing regulated pigments in bacteria. Recent Pat Biotechnol. 2016; 10(2): 145–152. PubMed Abstract | Publisher Full Text\n\nKothari V, Gupta A, Naraniwal M: Comparative study of various methods for extraction of antioxidant and antibacterial compounds from plant seeds. Journal of Natural Remedies. 2012; 12(2): 162–173. Reference Source\n\nKothari V, Punjabi A, Gupta S: Optimization of microwave-assisted extraction of Annona squamosa seeds. The Icfai University Journal of Life Sciences. 2009; 3(1): 55–60. Reference Source\n\nNgo TV, Scarlett CJ, Bowyer MC, et al.: Impact of different extraction solvents on bioactive compounds and antioxidant capacity from the root of Salacia chinensis. L. J Food Quality. 2017; 2017. Publisher Full Text\n\nPatel P, Joshi C, Palep H, et al.: Anti-infective potential of a quorum modulatory polyherbal extract (Panchvalkal) against certain pathogenic bacteria. J Ayurveda Integr Med. 2018. Publisher Full Text\n\nPatel P, Joshi C, Birdi T, et al.: Anti-infective efficacy of Psidium guajava L. leaves against certain pathogenic bacteria. F1000Research. 2018a. https://www.doi.org/10.5256/f1000research.17500.d230522\n\nPatel P, Joshi C, Birdi T, et al.: Anti-infective efficacy of Psidium guajava L. leaves against certain pathogenic bacteria. F1000Research. 2018b. https://www.doi.org/10.5256/f1000research.17500.d230521\n\nRaut AA, Chorghade MS, Vaidya ADB: Reverse Pharmacology. In: Innovative Approaches in Drug Discovery (Eds: Patwardhan B and Chaguturu R). 2017; 89–126. Publisher Full Text\n\nSasidharan S, Chen Y, Saravanan D, et al.: Extraction, isolation and characterization of bioactive compounds from plants' extracts. Afr J Tradit Complement Altern Med. 2011; 8(1): 1–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTacconelli E, Carrara E, Savoldi A, et al.: Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018; 18(3): 318–327. PubMed Abstract | Publisher Full Text\n\nThakkur CG: Introduction to ayurveda: Basic Indian medicine. Jamnagar: Gulakunverba Ayurvedic Society, 1976; 2.\n\nWang YQ, Wu ZF, Ke G, et al.: An effective vacuum assisted extraction method for the optimization of labdane diterpenoids from Andrographis paniculata by response surface methodology. Molecules. 2014; 20(1): 430–45. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "42519",
"date": "21 Jan 2019",
"name": "Virupakshi Soppina",
"expertise": [
"Reviewer Expertise Cell and molecular biology",
"biochemistry",
"C. elegans",
"biophysics",
"fluorescent microscopy",
"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\nPatel et al. study the anti-infective properties of Psidium guajava leaf extract, against five different pathogenic bacteria. They use C. elegans as a model system to study the anti-infective efficiency of P. guajava leaf extract formulated from three different extraction methods. Overall this is an exciting paper, validates the traditional use of guava leaves for medicinal purposes and also a possible mechanism. The topic is important, and this paper adds something new to the pharmacology field.\n\nThe key finding of this study is that the water and hydroalcoholic extracts prepared using microwave-assisted extraction method could successfully attenuate the virulence of different pathogenic bacteria and also exhibit anti-infective property towards C. elegans. I do not have any significant concerns or comments on the manuscript. However, there are some minor comments to improve the manuscript readability and understand the experiments.\nThe manuscript needs a more relevant background to understand the significance of the manuscript. It would be useful to state why the authors have specifically used three extraction methods that are used in the paper over several extraction methods available in the field. Under In vivo assay for anti-infective activity section, the sentence ‘Pathogenic bacteria were incubated with GLE for 22-24h (48h in case of S. marcescens and S. aureus) at 37°C (28°C for S. marcescens). Following incubation, OD764 of the culture suspension was equalized to that of the DMSO control.’ is highly confusing so please rewrite with precise details. The sentence ‘Number of live vs. lead worms was counted daily for five days by putting the plate’ should be written as ‘Number of live vs. dead worms was counted daily for five days by putting the plate.’ In Figure 1B, please use consistent symbol shapes for each data set. Graphs in Figure 2 are too small and crowded (it is difficult to appreciate the results), so please consider increasing the size of graphs or using different symbol shapes or think of presenting the data in bar graph format. Please include the data for positive controls [catechin (50 μg/mL) and gentamicin (0.1 μg/mL)] in Figure 1 and 2. Please provide scientific background for using catechin and gentamicin as positive controls. The sentence ‘At least one concentration of GLE was found to modulate pigment production in all the four pigmented bacteria (Figure 3; Dataset 1: Underlying data). This extract did not inhibit bacterial growth heavily, and hence can be expected to exert lesser selection pressure on susceptible bacterial populations.’ is difficult to understand so please consider rewriting with clear statements. The results of Figure 3 need more discussion in further details in the context of published literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4484",
"date": "19 Mar 2019",
"name": "Vijay Kothari",
"role": "Author Response",
"response": "We thank both the referees for devoting their time in reviewing our manuscript. Our comment-wise response to referee-1’s comments is as under: Comment 1: A line has been added in ‘Introduction’ telling the significance of such studies aimed at validating the traditional medicine claims. Comment 2: Basis of selection of these three extraction method has been added in the ‘Methods’ section under subheading ‘Extraction’. Comments 3,4, and 9: Sentences have been rewritten to correct spelling mistake, and add clarity. Comment 5: Error regarding symbol shape has been corrected in the revised version of Figure-1. Comment 6: To avoid the crowded appearance of Figure-2, in the revised version, we have divided all the five parts A-E into two separate graphs, one for water extract, and another for hydroalcoholic extract. Comment 7: Data for positive controls has already been there in legends of Figure 1-2. Adding separate lines for them in graph will again make the figures crowded. Comment 8: Scientific background for selection of positive controls has been added under the heading “In vivo assay for anti-infective activity”. Comment 10: Relevant content has been added discussing the results of Figure-3, citing appropriate references. Since this is a short ‘Research Note’, we have focused more on presenting our results, and refrained from adding too much content for ‘Discussion’."
}
]
},
{
"id": "42508",
"date": "25 Feb 2019",
"name": "Vivekananda Mandal",
"expertise": [
"Reviewer Expertise ethnopharmacology",
"extraction and purification of natural products"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this short study, the authors have studied anti-pathogenic potential of P. gujava leaves, which is an important plant in traditional medicine. It is good to see that among test bacteria, authors have included multi-drug resistant/beta-lactamase producing gram-negative bacteria, as it is difficult to find 'hits' against gram-negative bacteria in general. Their idea of comparing the same leaf extract prepared using different extraction methods also seems to be logical, as choice of the most appropriate extraction method is very much crucial while assessing the biological activity of plant extracts. It can have a significant bearing on the final results.\nThey have found MAE to be a good method. MAE has earlier been also reported by various groups to be an efficient extraction method, particularly for fast extraction of plant phenolic compounds. Further, they have used the worm C. elegans as the model host for their test pathogens. This worm is a good choice for generating useful preliminary data on in vivo efficacy of potential anti-pathogenic extracts/ formulations.\nIn the case of some bacteria like P. aeruginosa, there is an overlap among virulence factors (e.g. pyocyanin) responsible for damaging the human cells and those killing the worm. They have also compared the GLE prepared in water vs. that prepared in water + alcohol, and have emphasized the importance of choice of most appropriate extraction method and solvent for preparation of bioactive extracts.\nTheir in vitro experiments have provided a good clue on one of the possible ways regarding mode of action of GLE i.e. QS interference. QS in recent years has been reported by many research groups to be a target worth pursuing, in search of novel antimicrobials. Raw data submitted by the authors also seem to be in good shape, and in line with their findings reported in main text.\nOverall, this seems to be an okay study, and can be approved for indexing without any major changes. However, in future the authors should try to come up with a full-length report describing molecular mechanisms at the genome/transcriptome level explaining the mechanistic basis of GLE's anti-pathogenic efficacy.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-12
|
https://f1000research.com/articles/8-329/v1
|
25 Mar 19
|
{
"type": "Research Article",
"title": "Growth evaluation of several types of energy crops from tropical shrubs species",
"authors": [
"Dwi Susanto",
"Auliana Auliana",
"Rudianto Amirta",
"Auliana Auliana",
"Rudianto Amirta"
],
"abstract": "Background: Few species of tropical shrubs potentially produce biomass to replace fossil fuels for heat production and electricity. The aims of this study were to determine the growth and nutrient status of leaves of several types of energy crops from tropical shrub species with NPK fertilizer application. Methods: Randomized block design was used with ten replications of four levels of fertilizer treatment: T0 = 40 g, T1 = 80 g, T2 = 120 g and T4 = 160 g per plant. Results: The results indicated that fertilization increased plant growth and the quantity of nutrients in leaves. The plants accumulated a lot of potassium, followed by nitrogen and phosphorus. The species of tropical shrubs with the best growth were Vernonia amygdalina, Calliandra calothyrsus and Gliricidia sepium, which are all potentially cultivated as sustainable energy crops. Conclusions: Serious attention must be paid to the availability of soil nutrients in order to sustain the cultivation of these plants.",
"keywords": [
"Pioneer species",
"Tropical shrubs",
"Energy crops"
],
"content": "Introduction\n\nThe Indonesian government has issued a national energy policy to encourage the development of alternative energy. This policy targets the replacement of diesel and gasoline with biodiesel and bio-ethanol by 5% in Indonesia by 2025. The Government commissioned the Ministry of Forestry to play an active role in the development of biomass production. The use of raw material biomass to replace fossil fuels for heat and electricity production is prioritized, including permits for the utilization of plantations in the form of unproductive areas and permits for natural forest utilization1,2.\n\nDifferent types of forest plants and lignocellulose weeds have the potential to be developed as biomass feedstock for electricity production. Woody shrub species, such as Vernonia amygdalina Delile, Piper aduncum L., Gliricidia sepium (Jacq.) Kunth ex Walp., Calliandra calothyrsus Meissner., Bridelia tomentosa Blume, Vitex pinnata L., Vernonia arborea Buch.-Ham. and Bauhinia purpurea var. corneri de Wit., are suitable for use as sustainable raw materials for electrical energy3. This type of bush plant is a pioneer species that can easily be grown in secondary forests and open lands that have formerly been cultivated and logged. Energy crops are defined as cultivated plants that are developed and grown specifically for fuel and are rapidly growing, resistant to pests and droughts and can quickly be harvested, so they could have competitive prices if used as fuel4.\n\nWood is a renewable resource as it is sustainable and its supply will always be available. Meanwhile, cellulotic bio-fuel is produced from non-food (feed) stocks that play a critical role in reducing dependence on oil imports5. Woody biomass is produced, among others, by establishing plantations of energy crops. Coppice is a tree process regenerated with new shoots from the stumps that have been harvested and the Short Rotation Coppice (SRC) from hardwoods is more promising in generating biomass for bio-energy because it consists of fast-growing tree species and high-yield planted varieties (5000-15000 stems per ha), harvested in a two to six year rotation cycle.\n\nThis study focused on the growth and nutrient status of leaves of several types of energy crops from tropical shrub species with NPK fertilizer application, as a first step in the preparation of ready-to-plant if they are cultivated in the future as raw materials for biomass for renewable electricity.\n\n\nMethods\n\nThis study was conducted from January to September 2018 in a secondary forest located at Suka Damai Village, Muara Badak Sub-district, Kutai Kartanegara District, East Kalimantan province, Indonesia (00°17’ to 18°2” S latitude and 117°14’ to 14°39.5” E longitude). The wet season varies from 9 to 12 months and the dry season varies from 0 to 3 months. The average monthly temperature is 27.5°C and average air humidity is 82%6.\n\nSeedling plants (Vernonia amygdalina, Piper aduncum, Gliricidia sepium, Symplocos fasciculata, Vitex pinnata, Bauhinia purpurea, Melastoma malabathricum, and Calliandra calothyrsus) were taken from the Mulawarman University Botanical Garden. Seedlings are included in polyethylene bags and maintained for 3 months. Those with uniform height are selected before being planted in the research plot.\n\nComplete randomized block design (CRBD) was used as the experimental design. There were 8 plant species and their growth responses were evaluated based on 5 treatments using NPK fertilizer (commercial by YARA International ASA, Oslo, Norway; 16% N, 16% P2O5, 16% K2O5, 1.5% MgO, and 5% CaO). Five groups in five plots (each plant species): T0: with no fertilizer as control group (0 g/plant); T1: supplemented with NPK 40 g/plant; T2: supplemented with NPK 80 g/plant; T3: supplemented with NPK 120 g/plant; T4: supplemented with NPK 160 g/plant. The fertilizer treatments were conducted 2 weeks after sowing. There were 10 sample plants (as replication) for each treatment, a total of 50 plants for each species (divided into 5 experimental plots). Therefore, in total, there were 40 experimental plots, each plant was separated 1 m in length within each plot while it was separated 3 m in length between each plot (Figure 1).\n\nAll data were recorded after 5 months of planting. Stem height was measured by retractable tape measure (Shiro, Japan), basal stem diameter was measured by Vernier digital caliper (Mitutoyo, Japan), and the number of leaves and branches of each plant were counted.\n\nAll 10 replications of each treatment were pooled into one composited plant sample. Plant materials were analyzed at Forest Soil Science Laboratory, Faculty of Forestry, Mulawarman University to determine the total nitrogen, phosphorus, potassium, calcium and magnesium concentration. Total nitrogen was estimated using the Kjeldahl method7. Briefly, leaves were extracted using the wet destruction method using concentrated H2SO4 (MERCK, Germany). The extract was distilled and added to 20 ml of 0.05 N NaOH (MERCK, Germany).\n\nTo measure the elements of P, K, Ca and Mg, the plant materials were extracted using high pressure digestion method at a temperature of 1800 C for 10 hours with HNO3 65% (MERCK Milipore, Germany) as a reductant. The calorimetric technique used nitric acid-molybdate-vanadate (MERCK, Germany) as a coloring agent and it was determined by spectrophotometer (GENESYS™20 Visible Spectrophotometer, Thermo Scientific™, Thermo Electron North America LLC, 14-385-445) at a wavelength 470 nm. Meanwhile potassium, calcium and magnesium concentration were measured by Atomic Absorption Spectrophotometer (Trace 1800, Aurora Biomed, Canada) at wavelengths of 766.5, 489.5 and 245.2 nm, respectively5.\n\nThe plant growth data were expressed as mean ± standard error. The data were subjected to ANOVA, followed by Duncan’s Multiple Range Test (DMRT) to evaluated significant differences among the groups of treatment. All analysis was done using SPSS 22 (SPSS Inc. USA) and all significant tests were set at p≤0.05. The data of leaves’ nutrient concentration were analyzed descriptively.\n\n\nResults\n\nThe first part was the result of planting several types of tropical shrubs, including height growth, stem diameter, number of branches and leaves, while second part included the results of the analysis of nutrient content that was accumulated on the leaves of several types of tropical shrubs, namely nitrogen, phosphorus, potassium, calcium and magnesium.\n\nBased on Table 1, the best stem height was measured from 67.00 cm to 320.50 cm in V. pinnata and V.amygdalina, respectively, where both results were observed with T4 treatment. The best stem diameter ranged from 0.68 cm to 1.60 cm in B. purpurea and P. aduncum, respectively. The B. purpurea result was observed in T4 treatment, whereas the P. aduncum result was in T1 treatment. The highest leaf number ranged from 34.10 in B. purpurea to 271.50 in V.amygdalina. The first result from B. purpurea was observed in T1 treatment while the second from V. amygdalina was in T4 treatment. The highest branch number varied from 2.90 in B. purpurea to 12.00 in V. amygdalina. Similar to the leaves, B. purpurea result was observed in T1 treatment while V. amygdalina was different, because it was obtained from only T2 treatment. Other results on several plant species also show highest numbers however they are not significant within each species.\n\nT0 = 0 g (control), T1 = 40 g, T2 = 80 g, T3 = 120 g and T4 = 160 g of NPK. Number followed by the same letter in the same column show no significant difference in the DMRT test (p=0.05) after analysis by ANOVA.\n\nThe results show that fertilization treatment affects the growth of superior energy tropical shrub plants. In plants V. amygdalina, P. aduncum, V. pinnata, C. callothyrsus, S. fasciculata and G. sepium, the best growth was found in T4 treatment, whereas in M. malabathricum and B. purpurea, the best growth was obtained by T1 treatment. This indicates that the response of plants varies according to the fertilization treatment. Stem height and stem diameter of the various plants after 5 months of planting are presented in Figure 2.\n\n(A) Height and (B) diameter comparison of tropical shrub stems 5 months after planting measured from all fertilizer applications.\n\nAccording to Figure 2, the highest stem height was observed in V. amygdalina while the lowest was in V. pinnata, S. fasciculata and B. purpurea. Moreover, the large stem diameter was measured in P. aduncum, while the lowest was in V. pinnata and B. purpurea.\n\nThe accumulation of nutrients in the leaves of the tropical shrub plants varied widely. The highest nitrogenous nutrients accumulated in the leaves of M. malabatricum, C. calothyrsus and V. pinnata, while the highest phosphorus and potassium nutrients accumulated in the leaves of V. amygdalina, P. aduncum and G. sepium. Calcium nutrients accumulated the most in the leaves of P. aduncum, G. sepium and S. fasciculata. On the other hand, the highest accumulation of magnesium nutrients occurred in the leaves of B. purpurea, V. amiqdalina and G. sepium (Figure 3). Full data of the effect of fertilizer applications on tropical shrubs growth (height, diameter, and leaf and branch number) and leaves’ nutrient concentration (N, P, K, Ca and Mg) are available8.\n\nNutrient accumulation in leaves of tropical shrubs 5 months after planting measured from all fertilizer applications (A. Nitrogen, B. Phosphorus, C. Potassium, D. Calcium, and E. Magnesium).\n\n\nDiscussion\n\nIn this study, the growth and accumulation of nutrients in the leaves of tropical shrub plants varied greatly. The tropical shrubs that grew the best were V. amygdalina, G. sepium and C. calothyrsus, which accumulated mostly phosphorus and potassium nutrients in the leaves, while nitrogen, calcium and magnesium were least accumulated. These plants responded to NPK fertilizer up to 160 g per plant, resulting in best growth and high production of biomass, showing their suitable as raw material for biomass to create electricity. A previous study has shown that V. amygdalina provides 2.25 MWh, G. sepium 2.08 MWh and C. calothyrsus 2.01 MWh per ton of dry biomass3. As reported by Susanto and Amirta, fast-growing pioneer species such as M. gigantea absorbs the most potassium reaching 35% in every ton of plant biomass6,9. For V. amygdalina, growth parameters are positively correlated to rainfall, relative humidity and cloud cover10. On the other hand, G. sepium can be harvested at a residual height of 70 cm, with better agronomic characteristics and chemical composition occurring in the fall11. C. calothyrsus also has good growth in planting plots in previous research12, and it was previously reported that mycorrhizae such as Glomus sp. and Acaulospora sp. have significant influence on its height. In Colombia, planted fallows using C. calothyrsus have an additional benefit of producing large quantities of wood for household use13. Based on the growth and nutrient analysis in the present study, we believe that these tree plants species can be developed widely to support a sustainable supply of biomass feedstock for the green electricity program in Indonesia.\n\nThe growth of five plants species, namely M. malabatricum, P. aduncum, V. pinnata, B. purpurea, S. fasciculata was lower (less than half) than the three plants species discussed earlier (Table 1). In the present study, several plant species that grew slower actually accumulated more nutrients of nitrogen and phosphorus in the leaves, such as S. fasciculata, P. aduncum, V. pinnata, B. purpurea and M. malabatricum, P. aduncum and S. fasciculata also accumulated a large amount of potassium and calcium nutrients in their leaves. Moreover B. purpurea accumulated magnesium mostly in its leaves (Figure 3). For M. malabathricum, the availability of phosphorus and aluminum in the rhizosphere increases its growth, it can also adapt to low soil pH14 and absorb heavy metals in contaminated soils15. On the other hand, the mean foliar aluminum concentration in wild plants of M. malabathricum had positive correlation with foliar calcium, total nitrogen, calcium and magnesium concentrations within this species16 while Symplocos sp. mean foliar aluminum concentrations were detected at 4107 (±1474 mg kg-1) and 4290 (±4025 mg kg-1) for seedlings and saplings, respectively17. P. aduncum can be propagated with seeds and shoot cuttings18 and can accumulate large amounts of potassium, as previously reported19; at 23 months, it had accumulated 222 kg N, 50 kg P, 686 kg K, 255 kg Ca, 75 kg Mg, and 24 kg S ha− 1. More than half of the P, K, Ca and Mg nutrients were found in the stem (wood). Its leaf litter is significant and becomes an easily decomposable source of potassium, but G. sepium’s leaf litter contains much nitrogen18 while B. purpurea, which is a light-demanding tree, only grew 25% in a shady house with full sunlight20. Therefore, each tropical shrubs species in this study varies in accumulating plant nutrients N, P, K, Ca and Mg in their leaves.\n\nBased on this study, it is necessary to pay attention to the fast developing plant type, especially for tropical shrubs that have the potential as raw materials for biomass energy. Many beneficial nutrients also accumulated mostly in the leaves. This accumulation reflects the nutrient requirements of those plants, which will be cultivated as energy raw materials; therefore at the initial stage, the demand for plant fertilizer can be predicted. The carrying capacity of soil nutrients requires serious attention for the sustainment of plant cultivation. These wood shrubs species (V. amygdalina, G. sepium and C. calothyrsus) were also able to re-grow naturally by generation more than single shoots on their coppice trees. The scheme of Short Rotation Coppices (SRC) was an effort to achieve forest energy plantation using fast growing trees and wood shrubs species for aiming for a sustainable cycle3.\n\n\nConclusion\n\nOf the eight types of tropical shrubs in this study, three species, namely V. amygdalina, G. sepium and C. calothyrsus, had the best growth and could potentially be developed as Energy Crops. The most accumulated nutrients in the leaves of these three species of plants are phosphorus and potassium.\n\n\nData availability\n\nOpen Science Framework: Growth Evaluation of Several Types of Energy Crops from Tropical Shrubs Species, https://doi.org/10.17605/OSF.IO/3G8FH8.\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 work was financially supported by the Grant of Mulawarman University Research of Excellent Program The Development of Four Universities as the Center of Excellent for Nation Competitiveness provided by the Directorate General of Research and Development, the Ministry of Research, Technology, and Higher Education of Indonesia (2248/UN17.11/PL/2018).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank the head of Suka Damai village for providing permission to conduct the field works and thanks to laboratory technician of Forest Soil Science Laboratory, Faculty of Forestry, Mulawarman University for plant material analyzing. We are grateful to our students for their help and contributions during the field work.\n\n\nReferences\n\nFirman M, Tuanakota A, Rahmansyah A: Action Plan for Alternative Energy Development Based on Nyamplung (Calophyllum inophyllum) Plants 2010-2014. Indonesian Ministry of Forestry. 2009.\n\nAmirta R, Nafitri SI, Wulandari R, et al.: Comparative characterization of Macaranga species collected from Secondary forests in East Kalimantan for biorefinery of unutilized fast growing wood. Biodiversitas. 2016; 17(1): 116–123. Publisher Full Text\n\nAmirta R, Angi EM, Ananto BR, et al.: Plant Diversity and Energy Potency of Community Forest in East Kalimantan, Indonesia: Searching for fast growing wood species for energy production. Nusantara Biosci. 2016; 8(1): 22–31. Publisher Full Text\n\nKamm J: A new class of plants for a biofuel feedstock energy crop. Appl Biochem Biotechnol. 2004; 113–116: 55–70. PubMed Abstract | Publisher Full Text\n\nGalik CS, Abt R, Yun W: Forest Biomass Supply in the Southeastern United States-Implications for Industrial Round wood and Bioenergy Production. J Forest. 2009; 107(2): 69–77. Reference Source\n\nSusanto D, Mulyati S, Purnomo H, et al.: Growth, biomass production and nutrient accumulation of Macaranga gigantea in responds to NPK fertilizer application. Nusantara Biosci. 2017; 9(3): 330–337. Publisher Full Text\n\nYoshida S, Forno DA, Cook JH, et al.: Laboratory Manual for Physiological Studies of Rice. 3rd ed. The International Rice Research Institute, Los Banos, Laguna. 1976; 14–16. Reference Source\n\nSusanto D, Auliana, Amirta R: Growth Evaluation of Several Types of Energy Crops from Tropical Shrubs Species. 2019. http://www.doi.org/10.17605/OSF.IO/3G8FH\n\nSusanto D, Amirta R: Nutrient distribution in soil and above ground biomass of Macaranga gigantea five years after planting. Asian J For. 2018; 2(1): 12–19. Publisher Full Text\n\nAkachuku CO: Growth of Bitter leaf (Vernonia amygdalina, Del. Compositeae) and the Nutritive Values of its Processed and Unprocessed Leaves. Discov Innovat. 2001; 13(3): 227–233. Publisher Full Text\n\nSilva SF, Carneiro MS, Edvan RL, et al.: Agronomic characteristics and chemical composition of Gliricidia sepium grown under different residual heights in different seasons. Cien Inv Agr. 2017; 44(1): 35–42. Publisher Full Text\n\nSebuliba E, Nyeko P, Majaliwa M, et al.: Enhanced growth of multipurpose Calliandra (Calliandra calothyrsus) using arbuscular mycorrhiza fungi in Uganda. ScientificWorldJournal. 2012; 2012: 830357. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarios E, Cobo JG: Plant growth, biomass production and nutrient accumulation by slash/mulch agroforestry systems in tropical hillsides of Colombia. Agroforest Syst. 2004; 60(3): 255–265. Publisher Full Text\n\nWatanabe T, Osaki M: Role of organic acids in aluminum accumulation and plant growth in Melastoma malabathricum. Tree Physiol. 2002; 22(11): 785–92. PubMed Abstract | Publisher Full Text\n\nNur-Nazirah PM, Abdu A, Jusop S: Potentiality of Melastoma malabathricum as Phytoremediators of soil contaminated with sewage sludge. Sci Agric. 2018; 75(1): 27–35. Publisher Full Text\n\nKhairil M, Burslem DFRP: Controls on foliar aluminium accumulation among populations of the tropical shrub Melastoma malabathricum L. (Melastomataceae). Tree Physiol. 2018; 38(11): 1752–1760. PubMed Abstract | Publisher Full Text\n\nSchmitt M, Watanabe T, Jansen S: The effects of aluminium on plant growth in a temperate and deciduous aluminium accumulating species. AoB Plants. 2016; 8: pii: plw065. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSusanto D, Sudrajat, Suwinarti W, et al.: Seed germination and cuttings growth of Piper aduncum. IOP Conf. Series: Earth and Environmental Science, 2018; 144(2018): 012018. Publisher Full Text\n\nHartemink AE, O’Sullivan JN: Leaf litter decomposition of Piper aduncum, Gliricidia sepium and Imperata cylindrica in the humid lowlands of Papua New Guinea. Plant Soil. 2001; 230(1): 115–124. Publisher Full Text\n\nCai ZQ, Poorter L, Cao KF, et al.: Seedling growth strategies in Bauhinia species: comparing lianas and trees. Ann Bot. 2008; 100(4): 831–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "46151",
"date": "31 Oct 2019",
"name": "M. Khairil",
"expertise": [
"Reviewer Expertise Ecology",
"Botany",
"Biodiversity and Plant Eco-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\nThe abstract needs to have a clear result of the metal elements instead of using 'a lot'.\n\nIntroduction: I would suggest the authors add some description or mechanism of plants producing energy or electricity. This is optional.\n\nThe method is fine.\n\nResults: the author analyses the nutrient concentration in the leaves. How about the stem and roots? Is there any specific reason to not include the nutrient concentrations in the stem and roots?\n\nHow about the biomass of each plant part?\n\nThe conclusion is fine.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "127967",
"date": "08 Apr 2022",
"name": "Naqib Ullah Khan",
"expertise": [
"Reviewer Expertise Crop Breeding and 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\nI have gone through the article entitled “Growth evaluation of several types of energy crops from tropical shrubs species” and found it fit for indexing after minor revision. It is a good study, paper is well written, however, there are quite a few grammatical mistakes. It would be better if the language and grammar were checked throughout the text.\nPaper can be Approved after the incorporation of all comments given below:\nThe title is good and interesting for readers.\n\nRewrite the appropriate abstract, lots of similar work is done on this study. What is new in your paper?\n\nOverall, the introduction is written poorly. It seems to review the literature. The author failed to justify the need for his research by giving logical arguments. He has cited old references with well-established facts.\n\nNeeds to add some more recent references. I suggested some references below:\nFitmawati, Desti, Juliantari E, Novela D, Kapli H (2022)1. Abduova AA, Kupriyanov OA, Yessengeldi A, Kupriyanov AN, Sataev MI, Moshkalov BM (2022)2. Krishna T.P.A., S. Antony Ceasar, T. Maharajan, M. Ramakrishnan, V. Duraipandiyan, N.A. Al-Dhabi and S. Ignacimuthu. 2017. Haq, I.U., M. Riaz, A. Nawaz, A.U. Rehman, H. Mukhtar and Q.U.A. Syed. 20203. Asad, W., T. Kiran, F. Saleem, S. Siddiqui and S.A. Rasool. 20214. Nan, L.L. and Q.E. Guo. 20215. Bauyrzhan, T., K. Natalya, I. Zarina, K. Andrey, K. Мeruert, A. Кarime and B. Aliya. 20226.\n\nThe author analyzed the nutrient concentration only in the leaves. Did not give about stem and roots? It is recommended to write a few lines. Also write about the biomass of each plant part.\n\nThe entire discussion section is lacking an in-depth look at what the driving factors for the results were. Most of this section appears to be a literature review of what others have done and does not draw strong arguments to the data presented. This is in direct contradiction with several statements. This section needs a complete rewrite.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-329
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https://f1000research.com/articles/7-1976/v1
|
24 Dec 18
|
{
"type": "Software Tool Article",
"title": "Radtools: R utilities for smooth navigation of medical image data",
"authors": [
"Pamela H. Russell",
"Debashis Ghosh",
"Debashis Ghosh"
],
"abstract": "The radiology community has adopted several widely used standards for medical image files, including the popular DICOM (Digital Imaging and Communication in Medicine) and NIfTI (Neuroimaging Informatics Technology Initiative) standards. These file formats include image intensities as well as potentially extensive metadata. The NIfTI standard specifies a particular set of header fields describing the image and minimal information about the scan. DICOM headers can include any of >4,000 available metadata attributes spanning a variety of topics. NIfTI files contain all slices for an image series, while DICOM files capture single slices and image series are typically organized into a directory. Each DICOM file contains metadata for the image series as well as the individual image slice.\n\nThe programming environment R is popular for data analysis due to its free and open code, active ecosystem of tools and users, and excellent system of contributed packages. Currently, many published radiological image analyses are performed with proprietary software or custom unpublished scripts. However, R is increasing in popularity in this area due to several packages for processing and analysis of image files. While these R packages handle image import and processing, no existing package makes image metadata conveniently accessible. Extracting image metadata, combining across slices, and converting to useful formats can be prohibitively cumbersome, especially for DICOM files.\n\nWe present radtools, an R package for smooth navigation of medical image data. Radtools makes the problem of extracting image metadata trivially simple, providing simple functions to explore and return information in familiar R data structures. Radtools also facilitates extraction of image data and viewing of image slices. The package is freely available under the MIT license at https://github.com/pamelarussell/radtools and is easily installable from the Comprehensive R Archive Network (https://cran.r-project.org/package=radtools).",
"keywords": [
"Medical imaging",
"DICOM",
"NIfTI",
"R package"
],
"content": "Introduction\n\nMedical image analysis often lies at the boundary of research and the clinic, presenting challenges in both domains. Institutional and privacy concerns can compete with the objective of open data for research purposes. In particular, it remains standard practice to perform analysis with proprietary software or unpublished scripts. Additionally, the majority of imaging studies do not make image data publically available due to patient privacy requirements. These complex challenges can present barriers for scientists working in the image analysis domain.\n\nIn recent years, a small but growing number of open source computational tools have been developed to process and analyze medical images, promoting sharing of code; some of the most widely adopted are described in 1–3. To address the issue of availability of public image data, our group previously developed TCIApathfinder4, an open source R package to simplify access to the thousands of publicly available images in The Cancer Imaging Archive5. Here, we present radtools6, an open source R package that lowers barriers to image analysis by simplifying the extraction of image properties and complex header information. Although several excellent image processing and analysis packages exist for the R environment2,7–10, none currently offers special functionality for convenient presentation of image metadata. Radtools6 implements a layer of processing to convert image metadata to familiar R data structures, eliminating the need for specialized knowledge and custom scripts to parse image files.\n\nRadtools6 supports the two most common medical image formats, DICOM (Digital Imaging and Communication in Medicine)11 and NIfTI-1 (Neuroimaging Informatics Technology Initiative)12. The industry standard DICOM format combines a header and two-dimensional image data into one file, so that an image acquisition typically produces multiple DICOM files. DICOM header fields consist of a “tag” that identifies the attribute, followed by the attribute value. There is no fixed size for a DICOM header; any number of thousands of possible attributes may be included. Each DICOM file for an acquisition contains its own header; many attributes will be constant across image slices. NIfTI-1 format was developed primarily for multidimensional imaging data as an improvement over the previous ANALYZE format13. NIfTI-1 combines header information and the entire multidimensional image acquisition into either a single file or two files (one header file and one image file). Unlike DICOM, NIfTI-1 specifies a particular set of required header attributes, and the header conforms to a fixed size with an option to add extended header information. Radtools6 provides simple functions to explore and return image properties and header data from both image formats in familiar R data structures. Radtools6 also facilitates extraction of image data and viewing of image slices.\n\n\nMethods\n\nRadtools6 is provided as a package (extension to the language) for the programming language R. The package is hosted on the Comprehensive R Archive Network (CRAN), and can be installed into the user’s local R environment with the command ‘install.packages(“radtools”)’. The package is loaded into an R session or script with the command ‘library(radtools)’. Radtools consists of a collection of functions that can be called within R scripts or interactively from the R console. Package usage is documented in a vignette that can be viewed on the GitHub page (https://github.com/pamelarussell/radtools), the CRAN page (https://cran.r-project.org/package=radtools), or from the R console with the command ‘browseVignettes(“radtools”)’. The package reference manual provides documentation of each individual function and is available on the CRAN page.\n\nThe only system requirement is a working installation of R version ≥3.4.0. The radtools workflow consists of calling radtools functions from the R console or within R scripts.\n\n\nUse cases\n\nRadtools6 can extract image properties and header data from any valid DICOM or NIfTI-1 file. Image datasets are loaded with the `read_dicom` and `read_nifti1` functions. Several generic functions extract attributes from either data type, including `img_dimensions`, `num_slices`, `header_fields`, which reports the set of header fields present, and `header_value`, which returns the value(s) of a particular attribute. Additionally, functions are provided to specifically address one format or the other. All header data present in a DICOM acquisition can be extracted into a matrix, where rows are attributes and columns are slices, with the `dicom_metadata_matrix` function. As most DICOM headers contain numerous attributes and many of these are constant across all slices, the `dicom_constant_header_values` function produces a named list of common attributes across slices. NIfTI-specific functions include `nifti1_num_dim`, which returns the number of dimensions, and `nifti1_header_values`, which returns a named list of all metadata attributes for the image.\n\nThe image itself can be extracted as a multidimensional matrix of intensities for either file format with `img_data_to_mat`. Image slices can be visualized with `view_slice`.\n\nFinally, functions are provided to explore aspects of the DICOM standard itself. The functions `dicom_all_valid_header_tags`, `dicom_all_valid_header_names`, and `dicom_all_valid_header_keywords` return complete lists of valid DICOM header attributes. The functions `dicom_search_header_names` and `dicom_search_header_keywords` return attributes matching a search term.\n\n\nConclusions\n\nRadtools6 fills a specific need in the existing ecosystem of R packages for image processing and analysis: namely, the need for smooth extraction of image metadata. The package will accelerate workflow development and provide researchers with easy access to attributes that they may not have otherwise considered using. The inclusion of the package on CRAN, along with clear documentation, make it trivially simple for R users to obtain and begin using radtools.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nSoftware availability\n\nRadtools can be installed with the R command “install.packages(“radtools”).\n\nRadtools is available from CRAN: https://cran.r-project.org/package=radtools.\n\nSource code available from: https://github.com/pamelarussell/radtools.\n\nArchived source code at time of publication: http://dx.doi.org/10.5281/zenodo.147709313.\n\nLicense: MIT License.",
"appendix": "Grant information\n\nThis work has been supported by the Grohne-Stapp Endowed Chair for Cancer Research (University of Colorado Cancer Center).\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\nvan Griethuysen JJM, Fedorov A, Parmar C, et al.: Computational Radiomics System to Decode the Radiographic Phenotype. Cancer Res. 2017; 77(21): e104–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhitcher B, Schmid V, Thorton A: Working with the DICOM and NIfTI Data Standards in R. J Stat Softw. Articles. 2011; 44(6): 1–29. Publisher Full Text\n\nFedorov A, Beichel R, Kalpathy-Cramer J, et al.: 3D Slicer as an image computing platform for the Quantitative Imaging Network. Magn Reson Imaging. 2012; 30(9): 1323–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRussell P, Fountain K, Wolverton D, et al.: TCIApathfinder: An R Client for the Cancer Imaging Archive REST API. Cancer Res. 2018; 78(15): 4424–6. PubMed Abstract | Publisher Full Text\n\nClark K, Vendt B, Smith K, et al.: The Cancer Imaging Archive (TCIA): maintaining and operating a public information repository. J Digit Imaging. 2013; 26(6): 1045–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRussell P: pamelarussell/radtools: 1.0.1 (Version v1.0.1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1477093\n\nGet Images Out of DICOM Format Quickly [R package divest version 0.7.1]. [cited 2018 Nov 6]. Reference Source\n\nClayden JD, Maniega SM, Storkey AJ, et al.: TractoR: Magnetic Resonance Imaging and Tractography with R. J Stat Softw. Articles. 2011; 44(8): 1–18. Publisher Full Text\n\nFast R and C++ Access to NIfTI Images [R package RNifti version 0.10.0]. [cited 2018 Nov 6]. Reference Source\n\nBordier C, Dojat M, Micheaux P: Temporal and Spatial Independent Component Analysis for fMRI Data Sets Embedded in the AnalyzeFMRI R Package. J Stat Softw. Articles. 2011; 44(9): 1–24. Publisher Full Text\n\nDICOM Standard. [cited 2018 Nov 5]. Reference Source\n\nJenkinson M: NIfTI-1 Data Format — Neuroimaging Informatics Technology Initiative. 2005. [cited 2018 Nov 5]. Reference Source\n\nFormatAnalyze - MRC CBU Imaging Wiki. [cited 2018 Nov 6]. Reference Source"
}
|
[
{
"id": "42221",
"date": "07 Jan 2019",
"name": "Andrey Fedorov",
"expertise": [
"Reviewer Expertise medical image computing",
"imaging informatics",
"applications of DICOM for implementing FAIR principles in medical image computing"
],
"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 new R package developed to support the use of DICOM and NIfTI files from the R environment. The authors rightfully discuss the popularity of R and the need to support image processing tasks in this environment. The argument for development of the proposed package, radtools, is that \"[...] no existing package makes image metadata conveniently accessible. Extracting image metadata, combining across slices, and converting to useful formats can be prohibitively cumbersome, especially for DICOM\". The resulting package is available from CRAN, and this reviewer confirmed its installation and basic functions.\nThe major issues that need to be addressed to make the article sound are the following:\nJustification of the development of a new package for working with DICOM, or with NIfTI, is not sufficient. No details are provided about how the functionality was tested, and about the capabilities and limitations of the package in terms of supporting specific DICOM objects. Related to 2), no details are provided about how the DICOM files are handled \"under the hood\", i.e., whether all IO functionality was implemented from scratch, or the package is using some other DICOM libraries.\nThrough the text, the authors reference other R packages for similar tasks, and most notably oro.dicom and oro.nifti 1. Those packages have been around for quite a long time, are broadly used, based on citations of the corresponding articles, and arguably provide the functionality of the proposed new package (loading data in the aforementioned formats, examination of the attributes, visualization of the images), plus more (e.g., writing of the NIfTI data).\nDICOM is a complex standard, with a lot of ways information can be stored. For example, there are different methods to encode the content (transfer syntax), different character sets that can be used, private attributes. Therefore, often the quality of a DICOM implementation is defined to a large degree by the data that was used to test the implementation. The quality is also usually improved over time with the usage of the implementation. The proposed package is not accompanied by any details about what types of DICOM objects are supported, what was tested and how. Given it is a new package with a short development and usage history, one has to make a very strong argument for introducing such new tools in presence of existing alternatives.\nOther suggestions:\nThe discussion of the DICOM objects is an oversimplification, which is reflected in the implementation of the functionality. The standard defines various types of objects that can be serialized as files, but those objects are not limited to images. As an example, DICOM defines Structured Reporting object, which will not have PixelData. The proposed package fails to read such object. The authors can find sample SR objects in the familiar to them TCIA (e.g., see QIN-HEADNECK collection). \"smooth\" is a subjective qualifier that is redundant in the title and the text.\n\nI will be happy to reconsider this article after the authors address the above concerns. But my current opinion is that oro.dicom and oro.nifti set a rather high bar for any new implementation of similar functionality.\n\nIs the rationale for developing the new software tool clearly explained? No\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? 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? No",
"responses": []
},
{
"id": "42220",
"date": "07 Jan 2019",
"name": "Volker Schmid",
"expertise": [
"Reviewer Expertise statistical analysis of medical images"
],
"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 R package radtools. The aim of the R package is \"smooth navigation of medical image data\".\nThe idea to provide functions which appear \"smooth\" for the end user is of great importance. However, the functions provides in the package do not seem to be of much (additional) benefit to the end user. Functions for reading NIfTI and DICOM images are wrappers around function in the packages oro.nifti and oro.dicom; visualisations of medical images are realised in oro.nifti. Only the functions for exploring DICOM headers are genuinely original. This is of course an important part of working with (DICOM) images.\nFrom my understanding, F1000Research requires software tools articles to contain examples of the use of the tools. This manuscript does not contain any examples. An example (or two examples) would not only strengthen the manuscript, but could/would also show the benefit of the R package itself.\n\nIs the rationale for developing the new software tool clearly explained? No\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? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-1976
|
https://f1000research.com/articles/8-324/v1
|
22 Mar 19
|
{
"type": "Research Article",
"title": "Interaction of N-3-oxododecanoyl homoserine lactone with transcriptional regulator LasR of Pseudomonas aeruginosa: Insights from molecular docking and dynamics simulations",
"authors": [
"Hovakim Grabski",
"Lernik Hunanyan",
"Susanna Tiratsuyan",
"Hrachik Vardapetyan",
"Lernik Hunanyan",
"Susanna Tiratsuyan",
"Hrachik Vardapetyan"
],
"abstract": "Background: In 2017, the World Health Organization announced a list of the most dangerous superbugs. Among them is Pseudomonas aeruginosa, an opportunistic human pathogen with high levels of resistance to antibiotics that is listed as one of the ‘ESKAPE’ pathogens, which are the leading cause of nosocomial infections. A major issue is that it mostly affects vulnerable patients such as those suffering from AIDS, cystic fibrosis, cancer and severe burns. P. aeruginosa creates and inhabits surface-associated biofilms which increase resistance to antibiotics and host immune responses and contribute to the ineffectiveness of current antibacterial treatments. It is therefore imperative to find new antibacterial treatment strategies against P. aeruginosa. The LasR protein is a major transcriptional activator of P. aeruginosa and plays a pivotal role in biofilm formation and the activation of many virulence genes, although detailed characteristics of the LasR protein are not currently known. In the present study, we aimed to analyse the molecular properties of the LasR protein as well as its interactions with the signalling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Methods: We used a combination of molecular docking, molecular dynamics (MD) simulations and machine learning techniques to study the interaction of the LasR protein with the 3OC12-HSL ligand. We assessed conformational changes occurring upon their interaction and analysed the molecular details of their binding. Results: A new possible interaction site for 3OC12-HSL and LasR was found, involving conserved residues from the ligand binding domain (LBD), beta turns in the short linker region (SLR) and the DNA-binding domain (DBD). This interaction is referred to as the LBD-SLR-DBD bridge or ‘the bridge’ interaction. Conclusions: This study may enable future experimental studies to detect the interaction of signalling molecules with “the bridge” of the LasR protein and suggests a potential new interaction site to assist antibacterial drug design.",
"keywords": [
"Pseudomonas aeruginosa",
"transcriptional regulator",
"LasR",
"antibiotic resistant",
"ESKAPE",
"3OC12-HSL",
"homology modelling",
"molecular docking",
"molecular dynamics",
"machine learning techniques"
],
"content": "Abbreviations\n\nPDB, Protein Data Bank; MD, molecular dynamics; PCA, principal component analysis; 3OC12-HSL, N-3-oxododecanoyl homoserine lactone; AI, autoinducer; SLR, short linker region; BLAST, Basic local alignment search tool; DBI, David-Bouldin index; pSF, pseudo-F statistic; IQS, integrated quorum sensing; QS, quorum sensing; AHL, acyl homoserine lactone; HCN, hydrogen cyanide; HTH, helix-turn-helix; MSA, multiple sequence alignment; RMSD, root-mean-square-deviation; LBD, ligand binding domain; DBD, DNA binding domain\n\n\nIntroduction\n\nPseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, monoflagellated, obligate aerobe1,2. This species of bacteria is one of the ‘ESKAPE’ pathogens, which includes six bacterial pathogens which are commonly associated with antimicrobial resistance and are the leading cause of nosocomial infections throughout the world3. It can be found in many diverse environments such as in soil, plants and hospitals1,4. P. aeruginosa is an opportunistic human pathogen because it rarely infects healthy people, mostly affecting patients suffering from AIDS, cystic fibrosis, cancer and burns2,5. Most of the deaths caused by cystic fibrosis are due to this pathogen1,6. The pathogenicity of P. aeruginosa is due to virulence factors such as: the synthesis of proteases, hemolysins, exotoxin A, pyocyanin, hydrogen cyanide (HCN) and rhamnolipids; the possession secretion systems types one (T1SS), two (T2SS), three (T3SS), four (T4SS)7, five (T5SS), and six (T6SS)8; the formation of biofilms7,9.\n\nBiofilm formation is a common characteristic of many bacteria as the bacterial population density increases in the body during pathology. These bacteria possess a system of regulating gene expression in response to population density, called quorum sensing (QS), that uses hormone-like molecules called autoinducers (AIs) which accumulate in the extracellular matrix. When a threshold is reached, the AIs bind to their cognate receptors and then a response regulator modulates the expression of QS virulence genes which regulate adaptation, colonization, antibiotic resistance, plasmid conjugation and other bacterial processes.\n\nPseudomonas aeruginosa has four QS systems10. The type one system is regulated by LuxI/LuxR-type proteins. AIs diffuse freely across the bacterial membrane and bind to the transcriptional activator LuxR. Type two and three systems are also LuxI/LuxR-type systems, the first LasI that produces 3OC12-HSL and the second, RhlI that synthesizes C4-HSL; both acyl homoserine lactones (AHL) which regulate virulence and biofilm formation. A fourth integrated QS (IQS) system has been characterized very recently and the genes involved in IQS synthesis are non-ribosomal peptide synthase genes of the ambBCDE operon. The transcriptional regulator of IqsR is the IQS receptor11.\n\nQS regulates the expression of virulence factors such as HCN. Exposure to HCN can lead to neuronal necrosis through the inhibition of cytochrome c oxidase, the terminal component of the aerobic respiratory chain12,13. In P. aeruginosa, the hcnABC operon is responsible for HCN biosynthesis by the enzyme HCN synthase14. Three transcriptional regulators (LasR, ANR and Rh1R15) control the transcription of the hcnABC gene cluster14 although it has been proposed that LasR is the crucial activator of hcnABC genes following mutagenesis experiments16.\n\nThe transcriptional activator protein LasR regulates target gene expression by recognizing a conserved DNA sequence termed a lux box16,17. LasR has two domains: 1) a ligand binding domain at the N-terminus (LBD); 2) a DNA-binding domain at the C-terminus (DBD)18. LasR has a DNA-binding helix-turn-helix (HTH) motif19. Binding of the AI 3-O-C12HSL stabilizes LasR and promotes its dimerization. After that, the resulting LasR-AI homodimer complex binds to the promoter of the target DNA and induces gene transcription18.\n\nDuring colonization and invasion, both the pathogen and the host are exposed to molecules released by each other, such as bacterial AIs or host stress hormones and cytokines. The mechanisms and receptors that might be involved in cross-talk between microbial pathogens and their hosts are not yet well described. LuxR homologue studies have demonstrated that LuxR-type activators are homodimers and consist of two domains. These two functional domains are joined by a short linker region20,21.\n\nThere remains a need for a further understanding of the LasR monomer because to date there is no detailed information about LasR monomer interactions. Hence, we analysed the molecular details of the interactions of 3OC12-HSL with the LasR protein. So far, this is the first report that shows that 3OC12-HSL can interact with conserved amino acid residues from the ligand binding domain (LBD), beta turns in the short linker region (SLR) and the DNA-binding domain of LasR. For simplification, we refer to this region as the LBD-SLR-DBD bridge or “the bridge”. This could mean that modulation of the transcriptional regulator LasR is more nuanced than previously thought. This study may be utilized for the development of new therapeutic agents against P. aeruginosa targeting both the LBD and the LBD-SLR-DBD bridge of LasR in order to inhibit the expression of virulence genes.\n\n\nMethods\n\nFigure 1. Flow chart providing an overview of the methodology used in this study.\n\nThis part of the methodology was based on the work by Chowdhury et al. to reproduce the full structure of LasR protein1. The amino acid sequence of the LasR protein of P. aeruginosa was obtained from UniprotKB22 (Uniprot ID: P25084). The crystal structure of the LasR protein LBD (amino acid residues 7 to 169) from P. aeruginosa was acquired from the Protein Data Bank23 (PDB ID: 3IX3). However, the entire structure of the LasR protein was required in order to have a better understanding of its molecular properties.\n\nFor this reason, the amino acid sequence (Uniprot ID: P25084) was used to search the PDB databank using BLASTp24 in order to identify suitable templates for homology modelling. The crystal structure of QscR bound to 3OC12-HSL was found to be the best fit based on sequence identity, query coverage, and E-value from the BLAST results (PDB ID: 3SZT)20. The full list of similar structures obtained from the BLAST search are shown in Table 1.\n\nAs the current available crystal structures of LasR only contain the ligand binding domain18, we needed to reconstruct the full structure of LasR with the missing DNA-binding domain (DBD). The HHPred web server25 was used for homology modelling of the missing LasR DBD as was performed by Chowdhury et al.1. The four templates used for homology modelling of the LasR DBD have the PDB identifiers 3SZT, 3IX3, 1FSE and 3ULQ. The experimental structure of LasR18 and these templates were used to reconstruct the final model of LasR that contains both the LBD and the DBD. The final reconstructed model of the LasR protein was verified using PROCHECK25 (Figures S1 and S2, Extended data26), Verify3D26 and Ramachandran plots. More than 96.65% of the amino acid residues in the final reconstructed model had a 3D–2D score > 0.2 as indicated by Verify3D (Figure S3, see Extended data26). This score indicates a suitable computational model27. The Ramachandran plot (Figure S1, see Extended data26) revealed that no amino acid residues were located in the disallowed regions. The ProSA28 value of this model was -6.69 which suggests that the quality of the homology model is high28. Although the obtained model is slightly different from that obtained by Chowdhury et al.1 it is still viable.\n\nThe analysis of docking conformations and trajectories was performed using custom Python scripts (see Extended data26). These scripts use pandas v0.8.129, scikit-learn v0.18.230 and MDTraj v1.931 libraries. pandas29 facilitates data manipulation and analysis. In particular, it offers data structures and operations for manipulating numerical tables and time series. scikit-learn30 is a machine-learning library, which provides a set of common algorithms to Python users through a consistent interface. The MDTraj Python library31 facilitates the combination of machine learning libraries such as scikit-learn for trajectory analysis. MDTraj serves as a bridge, connecting molecular dynamics (MD) data with statistics and graphics libraries developed for general data science users.\n\nDiagrams of the interaction of 3OC12-HSL with LasR were obtained using LigPlot+ v.1.432 using default parameters, which automatically generates 2D diagrams of ligand–protein interactions from 3D coordinates by loading the coordinate files in pdb format. Plot visualization was carried out using matplotlib v2.0.132,33 with seaborn v0.8.134. The seaborn library aims to make visualization a central part of exploring and understanding data. It also provides a concise, high-level interface for drawing statistical graphics. Figures and videos were prepared with PyMOL v1.9.0.035, VMD 1.9.336 and UCSF Chimera v1.11.237. VMD36 is often used for this purpose as it has the ability to read trajectory files created during simulations in formats produced by many different software packages. PyMOL35 and UCSF Chimera37 are also commonly used by researchers and produce high quality images but are typically less straightforward to use for viewing and making videos of the trajectories.\n\nThe analysis protocol was similar to the work of Wolf et al.38 and Zamora et al.39, using PCA and cluster analysis modules within scikit-learn v0.18.2. Principal component analysis (PCA) is an unsupervised statistical technique that is often used to make data easy to explore and visualize. PCA tries to explain the maximum amount of variance with the fewest number of principal components40. The process of applying PCA to a protein trajectory is called essential dynamics (ED)41,42. PCA analysis, performed using Cartesian coordinates, has become an important tool for the examination of conformational changes. Default parameters within the scikit-learn library were used. Cluster analysis is another unsupervised technique that tries to identify structures within data. It is a data exploration tool for dividing a multivariate dataset into groups. Clustering algorithms can be grouped into partitional and hierarchical clustering approaches43. kmeans algorithm44,45 was used for molecular docking analysis. Agglomerative clustering using Ward’s linkage46 was used for molecule dynamics trajectory analysis with the connectivity matrix built on the basis of the k-Neighbors graph30. Default parameters within the scikit-learn library were used.\n\nMolecular dynamics (MD) simulations of all systems were conducted with the GROMACS suite, version 5.1.247, utilizing the Amber ff99SB-ILDN force field48, which exhibits considerably better agreement with NMR data than other forcefields. In all cases, short-range non-bonded interactions were cut off at 1.4 nm. The Particle mesh Ewald (PME) method49,50 was used for the calculation of long-range electrostatics.\n\nLasR monomer simulation. In order to generate the starting structure of the LasR monomer before docking, the Las R molecule was placed in a dodecahedron box of TIP3P water51. After that, 100 mM NaCl was added, including neutralizing counterions. Following two minimizations by the method of steepest descent algorithm, the LasR monomer was equilibrated in two stages. The first stage involved simulating for 200 picoseconds under a constant volume (NVT) ensemble. The second stage involved simulating for 200 picoseconds under a constant pressure (NpT), maintaining pressure isotropically at 1.0 bar. Production simulation was conducted for 100 nanoseconds in the absence of any restraints. The temperature was sustained at 300 K using the v-rescale52 algorithm. For isotropic regulation of the pressure, the Parrinello-Rahman barostat53 was used. This combination of thermostat and barostat use ensures that an adequate NpT ensemble is sampled. Finally, the trajectory of the LasR monomer simulation was used for the calculation of chemical shifts. SPARTA+ v2.9054 and SHIFTX2 v1.1055 were used to predict the chemical shifts of the protein backbone atoms with the help of the MDTraj wrapper functions31. Both programs have been proven to be remarkably accurate, especially for predicting 13C shifts54. This was used to validate whether the force field is appropriate for further investigation. The final extracted structure that was used for docking and MD simulations was also verified with Gaia56 from the Chiron webserver57, which compares the intrinsic structural properties of in silico protein models to high resolution crystal structures.\n\nMolecular dynamics simulations using docking poses. The 3D model of 3OC12-HSL was obtained from PubChem (CID: 127864) (Figure 2)58. It has been shown that docking has its limitations59. For this reason, after finishing the molecular docking simulations of 3OC12-HSL with LasR, the docking poses were extracted to perform molecular dynamics (MD) simulations. The ligand parameters for the molecular dynamics simulations were calculated for the General Amber Force Field (GAFF)60 using the ACPYPE tool61 with AM1-BCC partial charges62 and generation of a GAFF topology. A time step of 2 femtoseconds was used during heating and the production run and coordinates were recorded every 1 picosecond. Three simulations of 300 nanoseconds were performed. Further details about the simulation protocol can be found in ‘LasR monomer simulation’ section.\n\nTo build the LasR–ligand complex, the ligand 3OC12-HSL was docked with the LasR monomer using AutoDock Vina v1.1.263. However, AutoDock Vina currently uses several simplifications that affect the obtained results. The most notable simplification, as the creators note63, is the use of a rigid receptor. The amount of computation used during a docking experiment is determined by a parameter called ‘exhaustiveness’ in AutoDock Vina63,64. However, the default exhaustiveness value is 8 and the creators claim that increasing this value leads to an exponential increase in the probability of finding the energy minima63,64. The conformational space of the whole protein was searched using grid box dimensions 60×62×48Å. The following exhaustiveness values were tested in this study: 8, 16, 32, 64, 128, 256, 512, 1024, 2048 and 4096. The ligand and target structures containing hydrogen atoms were prepared using the AutoDockTools65 from MGLTools v1.5.6.\n\nPrincipal component analysis (PCA)40 and cluster analysis using k-means clustering44,45 was performed (Figures S4 and S5, Extended data26). The results demonstrated that the number of interaction sites does not change for exhaustiveness values from 1024 to 4096. An exhaustiveness value of 1024 was chosen as it provides good results, good speed and thorough sampling of the docked configurations. The exhaustiveness value was set to 1024 and the maximum number of binding modes to generate was set to 20. After that, 100 independent docking calculations were carried out with random initial seeds. The target was taken from the molecular dynamics simulation of the LasR monomer. Water molecules were removed from the structure.\n\nWe also performed another set of docking simulations using AutoDock Vina v1.1.263, rDock v2013.166 and FlexAID v2.4867. We performed 10 docking simulations using each program. Each individual run was set to generate 20 docked poses. For AutoDock Vina, we performed one docking run for each exhaustiveness value: 8, 16, 32, 64, 128, 256, 512, 1024, 2048 and 4096.\n\nDocking was also performed using the rDock program66 which was previously found to be successful in predicting the binding mode for the CCDC-Astex set of 85 diverse protein–ligand complexes in approximately 80% of cases66. A cavity with a radius of 36 Å was set and centred on the LasR monomer to cover the whole protein surface and define the docking volume. AutoDock Vina and rDock were chosen based on the performance of these academic programs68.\n\nFlexAID (parameters are available in Extended data26) was also used as, unlike other programs, it is robust against increasing structural variability67. FlexAID performs better than AutoDock Vina in all scenarios against non-native complex structures63. FlexAID uses a soft and smooth scoring function based on contact surfaces which is different from Autodock Vina and rDock. FlexAID was run using default parameters.\n\ng_mmpbsa v1.669 was used for the evaluation of binding energies. It was also used for the estimation of the energy contribution of each residue to the binding energy. g_mmpbsa calculates the relative binding energy rather than the absolute binding energy, so the entropy contribution (T∆S) is not evaluated69. From the two simulations with length of 300 nanoseconds, we cut out 10 nanoseconds with an interval of every 120 picoseconds for the calculation of binding energies. g_mmpbsa uses the MM-PBSA approach, which has grown to be one of the most widely used methods to compute interaction energies and is often employed to study biomolecular complexes65.\n\nTo find out whether 3OC12-HSLs interact with conserved amino acid residues, we performed multiple sequence alignment (MSA) using the R msa package v1.4.570. ClustalW71, Clustal Omega72 and MUSCLE73 within the msa package were used to carry out multiple sequence alignments. ClustalW and MUSCLE are commonly and widely used, while Clustal Omega is a more recent and powerful method that is used when aligning a large number of sequences72. We performed multiple sequence alignment of the LasR protein and closest homologue sequences, based on the work by Chowdhury et al.1. The consensus threshold parameter in msa was set to 75, the rest was set to default. Table 2 shows the sequences used for sequence alignment.\n\nWhen docking homology models, it is preferable to have experimental evidence suggesting the general interaction site (within ~10 Å). Representative structures from molecular dynamics simulations were used for protein-protein docking using ClusPro v2.074. ClusPro was chosen because it gave equivalent results to the best human predictor group according to the latest CAPRI experiments carried out in 201368.\n\nFrom the experimental X-ray data, it was found that ‘Trp152’, ‘Lys155’ and ‘Asp156’ play an important role during dimerization. The distance between ‘Trp152’ from chain A and ‘Asp156’ from chain B of the crystallographic structure was 0.276 nm and the distance between ‘Asp156’ from chain A and ‘Lys155’ from chain B was 0.279nm. These residues were used as attraction constraints.\n\n\nResults\n\nWe performed a simulation run of 100 nanoseconds using a standard MD protocol for the assessment of conformational changes of the LasR monomer. The overall stability of the molecule was assessed using the root-mean-square-deviation (RMSD) of the protein atoms. RMSD was calculated with reference to the initial frame through the time of the MD run (Figure S6, Extended data26). Another suitable measurement for the stability of the LasR monomer structure is the radius of gyration, which also shows the structure is stable (Figure S7, Extended data26).\n\nDuring the examination of MD trajectories, principal component analysis (PCA)40 is usually used for the visualization of the motion of the system (Figure 3a). Generally, in order to capture more than 70% of the variance in the trajectory data, the first handful of components are sufficient39. PCA can uncover the fundamental movements contained in an MD trajectory, however, it does not group the snapshots into different clusters43. This can be accomplished by clusterization of the PCA data.\n\nLasR monomer simulation analysis a) Simulation trajectory projected onto its first two principal components. The black scale indicates the time progression from 0 ns (white) to 100 ns (black) b) Clustering results of ward-linkage algorithm formed by first two PCs c) Colour-coded RMSD of the simulation obtained from Ward-linkage cluster analysis.\n\nCluster analysis was performed on the LasR monomer simulation for the selection of an initial starting structure to study docking. Identifying a distinct, representative structure of the cluster allows blind docking to be performed on the whole structure. It should be also noted that cluster analysis allows the evaluation of the frequent conformations of LasR. For the clustering analysis, we chose agglomerative clustering using Ward’s linkage46. This algorithm appears to be the most appropriate as it is deterministic, allowing for reproducibility of the resulting clusters, thus minimizing the amount of bias.\n\nWe performed several rounds of agglomerative clustering using Ward’s linkage. The accuracy of the clustering was assessed with the help of the Davies–Bouldin index (DBI)75, Dunn index76, silhouette score77 and Calinski-Harabasz pseudo-F statistic (pSF)78 metrics. An optimal number of clusters were chosen, simultaneously accounting for low DBI, high silhouette, high Dunn index and high pSF values.\n\nThe distribution of clusters over the simulation is visualized in Figure 3b and the four clusters are: cluster 4 (black) at the beginning of the simulation (after equilibration), cluster 2 (green) in the middle, cluster 1 (light green) and cluster 3 (dark blue) at the end. The clusterization defined by the first two principal components (Figure 3b) provides a coherent picture and it is also supported by good DBI, Dunn index, silhouette score and pSF values (Figure S8, Extended data26). Figure 3c shows clearly that the simulation has converged.\n\nFor validation of the molecular dynamics simulation quality, theoretical NMR shifts were calculated using Sparta+51 and ShiftX252. For the NMR shifts calculation, snapshots from cluster 3 (Figure 3b) were used because of their stability. We compared NMR values of C-alpha atoms from the experimental NMR of the LBD with theoretical NMR values of the LBD from our molecular dynamics simulations (BMRB: 6271)18. Unfortunately, the current available NMR information only includes the 173 amino acid residues of the LBD. In Figure 4, one can see that simulated NMR shift values from the two programs are close to the experimental data, thus demonstrating the quality of the MD simulation of the LasR monomer.\n\nIt should be noted this protein becomes partially soluble when the ligand interacts with it18. The differences between the theoretical chemical shifts and experimental values could be attributed to: 1) the theoretical model does not involve the interaction with the ligand 2) the experimental NMR only includes information about the LBD and not the DBD, which can affect the LBD.\n\nAfter that, the representative structure (Figure 5c) was extracted from cluster 3 (Figure 3c) to study docking. The representative structure of the LasR system and its differences from the homology modelled structure (Figure 5b) are visible in Figure 5c. Secondary structure analysis of the representative structure was performed using PDBSum79 (Figure S9, Extended data26). Finally, the extracted centroid structure was validated using Gaia56 (Figure S10, Extended data26), which also shows the model is of a high quality. Therefore, in this study, a model has been developed to include the dynamics of the full-length LasR molecule (residues 1 to 239) and it has been shown that the dynamics of the complete C-terminal region of LasR modulate the N-terminal region, as will be discussed later.\n\n3D LasR protein structures: a) Crystal structure of the N-terminal LBD of LasR protein b) Full structure of the protein with the missing DBD, which was modelled using homology modelling c) Representative structure after 100ns MD run. Images generated with UCSF Chimera36.\n\nIn this study, a molecular docking approach was used to inspect the possible binding modes of 3OC12-HSL with the LasR monomer. PCA and cluster analysis were performed on docking data (Figure 6a, b) and the results show three binding sites. Cluster 2 corresponds to experimental data, while cluster 1 and cluster 3 do not (Figure 6c). These results clearly support the findings of Bottomley et al.18, who also demonstrated that 3OC12-HSL binds to the LBD.\n\nAnalysis of Autodock Vina docking results a) Silhouette plot analysis of clusterization quality b) Clustering results using k-means algorithm formed by first two PCs c) 3D visualization of the analysed docking data with their representative structures and clusters.\n\nWe performed several rounds of k-means clustering. The accuracy of the cluster analysis was evaluated using the DBI75, Dunn index76, silhouette score77 and pSF78 metrics (Figure S11, see Extended data26). An optimal number of clusters were chosen for docking analysis, simultaneously accounting for low DBI, high Dunn, high silhouette and high pSF values. We generated 2000 docked poses and performed representative structure extraction for use in MD simulations of the LasR 3OC12-HSL binding sites. The representative structures were produced by identifying the centroid conformations of the clusters using the following algorithm:\n\n1. Compute all pairwise root mean square deviations (RMSDs) between the conformations.\n\n2. Transform the distances into similarity scores and they are calculated as\n\n\n\nWhere sij is the pairwise similarity, dij is the pairwise distance, and dscale is the standard deviation of the values of d to make the computation scale invariant.\n\n3. Then the centroid is defined with β=1 as\n\n\n\nRepresentative structures from each cluster were extracted for further use in MD simulations. The binding energy of the representative structure of cluster 1 is -5.4 kcal/mol and the mean binding affinity for the whole cluster is -5.257 ±0.233 kcal/mol. Cluster 1 contains 839 docked poses from a total 2000 (41.95%). For cluster 2, the binding affinity of the representative structure is -5.1 kcal/mol and the mean for the whole cluster is -5.593 ±0.386 kcal/mol. These results correspond to the experimental binding site data18. Cluster 2 contains 864 docked poses from a total of 2000 (43.2%). For cluster 3, the representative structure features the highest binding affinity of -5.7 kcal/mol and the mean binding affinity for the whole cluster is -5.264 ±0.27 kcal/mol. Cluster 3 contains 297 docked poses from 2000 (14.85%) which does not correspond to the experimental binding site.\n\nIt has been suggested that molecular docking is not appropriate for the prediction of binding affinity or binding poses of protein-ligand complexes. However, docking analysis can still provide important information regarding potential binding sites and the resulting poses can be used for MD simulations59,80.\n\nWe also ran another set of simulations to corroborate the blind docking with other molecular docking software, including AutoDock Vina63, rDock66 and FlexAID67 (Figure 7). All three programs, which each use different algorithms, suggest that there are two potential binding sites. One is within the LBD, which corresponds to experimental data18, and the other is within the ‘bridge’. PCA of the results from these docking programs was performed for easier comprehension (Figure S12, Extended data26).\n\nRed circles - binding interactions that are characteristic to all programs a) Autodock Vina b) rDock c) FlexAid.\n\nIn total, 900 nanoseconds of MD simulations have been performed and used for the analysis of 3OC12-HSL interaction with the LasR monomer. The representative structures were taken from docking results (Figure 6c) and used as starting points for MD simulations with LasR. Simulations were conducted for a sufficient time to allow the positions of 3OC12-HSL to reach equilibrium in the LasR molecule.\n\nThe stability of the molecule was evaluated by calculating the RMSD of the backbone atoms. RMSD was calculated with reference to the initial snapshot. Figure 8a shows that Simulation 2 and 3 experience a substantial RMSD deviation from the initial starting point. Simulation 2 corresponds to cluster 2 in the docking simulations, while Simulation 3 corresponds to cluster 3 (Figure 8a). For Simulation 1, which corresponds to cluster 1, the 3OC12-HSL molecule did not fixate and reach equilibrium and therefore no further analysis was performed (Figure 8a) for this simulation. Simulation 2 shows that after 230 nanoseconds, the structure becomes stable. For Simulation 3, the structure changes its conformation at 100 nanoseconds and becomes stable between 260 nanoseconds and 300 nanoseconds. Simulation 2 and 3 represent the binding sites predicted by all docking programs from Figure 7. Calculation of the root-mean-square fluctuation (RMSF) was used to evaluate the flexibility of the LasR monomer. The average per-residue RMSF was calculated for each simulation run (Figure 8b).\n\nAnalysis for 3 independent runs of 300 ns using representative structures from 3OC12-HSL docking poses as starting points a) RMSD evolution b) RMSF of Cα-atoms of LasR for each individual run.\n\nIt is visible in Figure 8b that the residues from 165 to 176, which correspond to beta turns in the SLR of LasR, are highly mobile. Simulations 2 and 3 show that 3OC12-HSL has two binding modes, one with the LBD (Figure 9a), which corresponds to experimental data and one with the LBD-SLR-DBD bridge of LasR (Figure 9d).\n\nVisualizations of the interactions of 3OC12-HSL with LasR. Conformations taken from centroids from cluster analysis a) 3OC12-HSL with LasR LBD b) Insertion of 3OC12-HSL in LBD of LasR c) Superimposition of the modelled interaction (red) of 3OC12-HSL with LasR LBD and crystallographic structure (green) d) 3OC12-HSL with the “bridge” of LasR e) Putative hydrogen bonds of 3OC12-HSL with Lys182 and Leu177.\n\nPCA and cluster analysis were performed on simulation 2. Hydrogen bond analysis was also performed, based on cut-offs for distance and angles according to the Wernet-Nilson criteria81 using MDTraj31. During the simulation, 3OC12-HSL forms hydrogen bonds with Tyr56, Ser129 and Trp60, which is in agreement with experimental data18 (Figure S14, Extended data26). Over the course of simulation 2, 3OC12-HSL also establishes a large number of hydrophobic contacts with the amino acid side chains of the LasR LBD (Figure 9b). This phenomenon is not unexpected, given the large hydrophobic surface area of the LasR LBD and the low solubility of 3OC12-HSL in water. 3OC12-HSL has hydrophobic interactions mainly with amino acids from helix 3, helix 5 and sheet 1 side chains. RMSD analysis of the conformations of LasR with and without 3OC12-HSL bound to the LBD gives a value of 7.027 Å between the conformations (Figure S16, Extended data26). RMSD of the modelled full structure of LasR with ligand superimposed onto the experimentally obtained tertiary structure of LBD of LasR with ligand gives a value of 1.692 Å, where RMSD <2 Å in relation to the experimentally studied structure (Figure 9c). Residues that participate in hydrophobic interactions and hydrogen bonding are shown in Figure 9e and Figure S18 and 19 (Extended data26).\n\nThe second binding mode involves the interaction of 3OC12-HSL with the LBD-SLR-DBD bridge. PCA, cluster and hydrogen bond analysis were also performed on simulation 3. Over the course of simulation 3, 3OC12-HSL establishes hydrogen bonds and a large number of hydrophobic contacts with amino acid side chains in the beta turns of the LasR SLR. In simulation 3, 3OC12-HSL forms hydrogen bonds mainly with the ‘Lys182’ and ‘Leu177’ beta turn residues. RMSD analysis of the two conformations of the LasR monomer and when LasR is bound to 3OC12-HSL via the DBD gives a value of 1.677 Å (Figure S20, see Extended data26). Residues that participate in hydrogen bonding and hydrophobic interactions are shown in Figure S16. Complete data sets for MD simulations are available as extended data (Figures S13-S2026).\n\nIn order to analyse the binding sites in detail, MM-PBSA69 binding energy calculations were performed for each binding site based on the MD trajectories. The binding energy calculations demonstrated that the interaction of the ligand with the LBD-SLR-DBD bridge is not competitive with its interaction with the LBD (Table 3). Analysis of the energy terms demonstrated that only polar solvation energy contributes positively. However, for the interaction with the “the bridge”, the electrostatic interaction energy is 3.6 times higher than the energy for the LBD interaction.\n\nThis suggests that a combination of Van der Waals, electrostatic interaction and non-polar solvation energy contribute to the stability of the LasR-3OC12-HSL binding complex. To assess which amino acids are involved, we performed analysis of the energy contribution of residues to binding for both simulations obtained from MM-PBSA calculations using g_mmpbsa. Sequence alignment was performed using the R msa package70. ClustalW71, Clustal Omega72 and Muscle73 algorithms were used for sequence alignment. Full alignments using these algorithms are available as extended data26. Analysis suggested that LasR residues Tyr64, Tyr56, Trp88, Leu36 and Trp60 contribute the most to binding with 3OC12-HSL (Figure 10a), with a binding energy of approximately -128.578 kJ/mol (Table 3).\n\nEnergy contributions of residues and multiple sequence alignments demonstrating the conserved amino acid residues of LasR that interact with 3OC12-HSL: a) All amino acids that interact with LBD b) Conserved amino acids that interact with LBD. Blue boxes and arrows point the amino acid residues that interact with 3OC12-HSL c) All amino acids that interact with the “bridge” d) Conserved amino acids that interact with “bridge”. Black boxes and arrows point the residues that interact with 3OC12-HSL.\n\nEnergy calculations to assess residue interactions and sequence alignment show that 3OC12-HSL interacts with highly conserved amino acid Trp60, which agrees with experimental data82. The interaction also involves nine other conserved amino acids; Tyr64, Asp73, Pro74, Val76, Phe102, Phe103, Ala105 and Gly113 (Figure 10b). In total, 16 amino acids that contribute to the interaction have over 75% conservation.\n\nAnalysis suggested that ligand residues Leu236, Leu177, Val176, Phe219, Lys182 and Trp19 contribute the most (Figure 10c) to the newly described binding state involving ‘the bridge’ of LasR, with a binding energy of approximately -166.456 kJ/mol (Table 3). The analysis of residue sequence alignment shows that 3OC12-HSL interacts with highly conserved amino acids such as Leu236, Leu177, Phe219 and Trp19 (Figure 10d). The 3OC12-HSL interacts with 12 fully conserved amino acids in this case. In total, 16 amino acids participate in the interaction, where amino acid conservation is more than 75%. In this new binding site, Trp19 and Asp156 of the LBD, Leu177 of the SLR and Arg180, Glu181, Glu196, Lys218, Arg224 and Lys236 of the DBD of LasR participate in the interactions with 3OC12-HSL. A lactone head group interacts with the conserved Trp19 of the LBD.\n\nIt is known that LasR binds to the corresponding promoter as a dimer1 after interacting with the autoinducer. Thus, monomeric LasR–3OC12-HSL complexes were subjected to dimerization as an additional experiment. The protein docking experiments using structures from MD runs were performed using ClusPro v2.074,83–85 because of its success in the CAPRI (Critical Assessment of Predicted Interactions) scoring experiment. We used the centroid conformations of the LasR bridge-3OC12-HSL and LasR LBD-3OC12-HSL complexes from MD simulations. Then we performed protein docking using protein conformations without the ligands and used constraints based on the residue distances between monomers from the experimental data18. For the selection of the model, we used an approach recommended by the authors of ClusPro74, which suggests finding the most populated clusters. For LasR bridge-3OC12-HSL complex docking, the top model contained 80 members and the scores by ClusPro for the docking model were -951.4 for the centre and -1332.0 and for the lowest energy region, suggesting a favourable binding mode (Figure 11a). For the LBD LasR-3OC12-HSL complex, the top model contained 122 members and the scores for the docking model were -1440.7 for the centre and -1517.9 for the lowest energy region (Figure 11b). Both docking structures feature negative energy and this could suggest the possibility of multiple docking conformations. It is possible that the multiple binding modes of 30C12-HSL imply that LasR has multiple dimerization interfaces like QscR (Figure 11c)20, which is a post-translational repressor of LasR. In the absence of 3OC12-HSL, it forms a heterodimer with LasR. It appears this protein may have a much more delicate regulation than was initially thought.\n\nLasR monomer protein docking results and crystallographic dimer structure of quorum sensing control repressor QscR bound to 3OC12-HSL a) using centroid conformation from the LBD LasR-3OC12-HSL complex b) using centroid conformation from the “bridge”-3OC12-HSL complex c) crystallographic dimer structure of QscR, which is a homologue of LasR.\n\n\nDiscussion\n\nPseudomonosa aeruginosa is one of the ESKAPE pathogens3, which are the leading cause of nosocomial infections throughout the world. However, the molecular mechanisms of the LasR protein in regulating quorum sensing machinery have not been fully elucidated. LasR has been problematic to structurally analyse for the last decade because of the insolubility of the full protein18. All of the available crystallographic structures contain only the LBD and lack the DBD. The lack of a full crystal structure has prevented us from furthering our understanding of LasR as knowledge of its full 3D structure is vital. It is therefore very important to study the interaction of the autoinducer with the full structure of the LasR protein. Adequate knowledge of this interaction would provide the opportunity to conduct improved structure-based drug discovery studies.\n\nResults from our analysis show that the interaction of 3OC12-HSL with LasR has two binding modes. The results were cross-checked using multiple docking programs, molecular dynamics simulations and machine learning techniques. The 3OC12-HSL has a long hydrophobic tail and is therefore capable of binding to both the LBD and the “bridge” of LasR. These results suggest that both the C-terminal and the N- terminal regions of LasR interact with 3OC12-HSL. The binding of 3OC12-HSL provokes conformational transitions in the structure of LasR. Binding energy analysis showed that the “bridge” does not compete with LBD. An MM-PBSA approach was used for the calculation of relative binding energy69, although this finding needs confirmation through further research. The calculation of absolute binding energy could give more reliable results, though this type of calculation is more time consuming62. The results of our in silico experiments for the interaction of 3OC12-HSL with the LasR LBD is comparable with the results of other authors18,82, although currently there are no studies investigating the DBD.\n\nThis work is connected to recent experimental studies concerning LasR regulation. It should be noted that the LasR quorum-sensing system of P. aeruginosa is regulated by pre-quorum and post-quorum regulating systems86. The first system is controlled by post-translational repressors QscR, QslA and QteE20,86–89. In the absence of 3OC12-HSL, they can each form a heterodimer with LasR. Structural and functional studies of bacterial QS anti-activators revealed three different modes of action for LuxR-type transcriptional regulation: the destruction of the LBD dimerization interface in P. aeruginosa, the occupation of the AHL binding pocket and binding to the DBD11. All these interactions indirectly change the conformation of the DBD, thus allosterically preventing DNA binding. It was previously shown that QslA can bind to LasR and thereby inhibit its DNA-binding capability86. Moreover, it has been shown that QslA supresses formation of the LasR homodimer complex by occupying the same surface area that is used for dimerization88,89.\n\nIt has also been shown that QscR interacts with 3OC12-HSL20. QscR has multiple dimerization interfaces and possesses both an LBD and DBD20. The heterodimer QslA-LasR affects the dimerization interface and prevents receptor activation and DNA promoter binding88. According to one of the mechanisms, the anti-activator QslA blocks the regions that are necessary for transcription factors multimerization15. Moreover, 3OC12-HSL does not overcome the repression of LasR exerted by QslA. This indicates that 3OC12-HSL interacts with helixes that form part of these dimerization interfaces. It may be that the multiple binding modes of 30C12-HSL imply that LasR has multiple dimerization interfaces like QslA and QscR.\n\nThere is also another aspect to be discussed. One major strategy for LasR inhibition is the development of small-molecule antagonists that mimic the native autoinducer and inactivate LasR. However, the mechanism by which they inactivate LasR is unknown90. It has been suggested that LasR interactions with these antagonists promote a robust, unnatural fold of LasR that does not permit DNA binding90. Further research studying flavonoids as potential inhibitors shows that they do not bind in the ligand-binding pocket of LasR91. It has also been shown that they prevent LasR from binding to promoter DNA, but do not stop dimerization91. Unfortunately, in this study it was not possible to assay the LasR DBD due to insufficient protein yields91. The author suggest that the AI and flavonoid inhibitors can simultaneously bind to LasR and therefore postulate that flavonoids do not use the canonical AI binding site. We suggest that flavonoids and other inhibitors may interact with the bridge, thereby preventing LasR from binding to promoter DNA.\n\nOur model might explain the molecular mechanisms proposed by recent experimental studies involving the study of flavonoids and other inhibitors which suggest the possibility of another binding site90,91. It may also explain why computational drug design has not been successful in targeting this protein. However, the lack of a full, experimentally-derived structure for LasR is problematic and requires further experimental and computational research. We expect that our molecular insights on LasR can shed more light on this protein and assist in the development of new treatments against P. aeruginosa.\n\n\nConclusion\n\nFrom the simulations, it appears that the AI ligand 3OC12-HSL can bind to both the LBD and the “bridge” of the transcriptional regulator LasR. The interaction with the “bridge” is a novel site. Binding energy analysis shows that the interaction of 3OC12-HSL with the “bridge” and its interaction with the LBD is not competitive. Conserved amino acids such as Leu236, Phe219, Leu177, Lys182 and Trp19 contribute the most during the interaction with “bridge”. This could suggest that the interaction of 3OC12-HSL with the “bridge” is necessary for the DNA binding capability of LasR. One possible explanation for these multiple binding sites is that LasR may have multiple dimerization interfaces. This study may reveal new insights into the interactions of the native 3OC12-HSL ligand with transcriptional regulator LasR in P. aeruginosa. Results from this study may aid future drug development endeavours.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Interaction of N-3-oxododecanoyl homoserine lactone with transcriptional regulator LasR of Pseudomonas aeruginosa: Insights from molecular docking and dynamics simulations. https://doi.org/10.5281/zenodo.258655926\n\nThis project contains the following extended data:\n\n- AdditionalFile1_SupportingInformation.pdf (Figures S1-20)\n\n- AdditionalFile2_ClustalW.pdf (Full sequence alignment of LasR using ClustalW algorithm)\n\n- AdditionalFile3_ClustalOmega.pdf (Full sequence alignment of LasR using Clustal Omega algorithm)\n\n- AdditionalFile4_Muscle.pdf (Full sequence alignment of LasR using Muscle algorithm)\n\n- HSL_dock.zip (Molecular docking simulations input and output for AutoDock Vina)\n\n- HSL_exhaustiveness.zip (Molecular docking simulations input and output for the determination of exhaustiveness (AutoDock Vina))\n\n- LasR_HSL_multidock.zip (Molecular docking simulations input and output for AutoDock Vina, rDock and FlexAID)\n\n- MD100ns.tar.gz (Molecular dynamics simulation data of LasR protein for 100 nanoseconds)\n\n- HSL_LasR_simulation_1.zip (1st MD simulations data for 3OC12-HSL with LasR) HSL_LasR_simulation_2.zip (2nd MD simulation data for 3OC12-HSL with LasR)\n\n- HSL_LasR_simulation_3.zip (3rd MD simulation data for 3OC12-HSL with LasR)\n\n- scripts.zip (Scripts that were used for the analysis of the data based on the molmolpy set of scripts92)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe authors gratefully acknowledge financial support by the Russian-Armenian University [NIR 25/15].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Yerevan Physics Institute for providing time on the cluster for the molecular dynamics simulations.\n\n\nReferences\n\nChowdhury N, Bagchi A: Molecular insight into the activity of LasR protein from Pseudomonas aeruginosa in the regulation of virulence gene expression by this organism. Gene. 2016; 580(1): 80–7. PubMed Abstract | Publisher Full Text\n\nBotzenhart K, Döring G: Ecology and Epidemiology of Pseudomonas aeruginosa. In: Campa M, Bendinelli M, Friedman H, editors. Pseudomonas Aeruginosa Opportunistic Pathog, Boston, MA: Springer US; 1993; 1–18. Publisher Full Text\n\nPendleton JN, Gorman SP, Gilmore BF: Clinical relevance of the ESKAPE pathogens. 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}
|
[
{
"id": "63083",
"date": "22 May 2020",
"name": "Jon Paczkowski",
"expertise": [
"Reviewer Expertise X-ray crystallography",
"biochemistry",
"signal transduction",
"and bacterial cell-cell communication."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, Grabski et al. use molecular docking techniques to define a second binding site for the LasR autoinducer molecule, 3OC12-HSL. The researchers use several computational platforms to confirm that 3OC12-HSL binds to a \"bridge\" domain that links the ligand binding domain and the DNA binding domain in LasR.\nI am no expert in computational modeling or docking. I have some familiarity with the tools used in this study and I do not find them particularly compelling in \"this new finding\", especially calling what they do \"machine learning.\" Otherwise, the methods were quite detailed and well-written.\nThe authors have some factual errors in their introduction. They seem to imply that there are three LuxI/R QS systems in Pseudomonas aeruginosa. That is not the case. They entirely omit the PQS system in their introduction. Furthermore, it is not entirely clear why they focus on HCN in the introduction. They never come back to it and it is not the focus of the study. It seems irrelevant.\nDuring the introduction they mention that LasR requires its AI to fold, function, and dimerize. Next, they state they're interested in studying LasR in the monomeric state. There is no evidence that LasR exists as a stable monomer on its own, let alone plays any functional role.\nInexperience with docking simulations aside, it's particularly intriguing to discover that there might be a second AI binding site. The statistics seem fine. Docking a hydrophobic molecule with a protein that contains a hydrophobic patch will result in a positive result. That doesn't necessarily mean that the interaction occurs in nature. Furthermore, the primary binding site for 3OC12-HSL is a mixture of hydrophilic interactions with the lactone head group and hydrophobic interactions with the acyl tail. The residues defined as the second binding site do not appear particularly hydrophilic to account for stabilizing the lactone head group. How do the authors account for this discrepancy? Additionally, there is no hydrophobic \"core\" that stabilizes the tail in its entirety. Simply put, I think there is too much solvent exposed surface on 3OC12-HSL for this binding to occur. Can the authors compare the canonical binding site to the secondary binding site in this regard?\nFurthermore, what role does this second binding site even play? LasR has been purified in the presence of excess 3OC12-HSL and the ratio is 1 LasR:1 AI. How do the authors reconcile this? There needs to be additional biochemical or cell-based assays performed to a) show that this second binding site actually exists and is not an artifact of the limitations of molecular docking and b) determine what its potential role is.\nIt would be an interesting exercise if the authors performed similar docking experiments with other LuxR-type proteins with their partner AI. Would they get the same results?\nWhile I am sure the data are sound, I am not convinced of its relevance or importance in the context of LasR signaling. There needs to be some other data to prove that the second binding site is biologically relevant.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "92461",
"date": "20 Oct 2021",
"name": "Faizul Azam",
"expertise": [
"Reviewer Expertise Drug design and discovery",
"medicinal chemistry",
"molecular docking",
"molecular dynamics."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled \"Interaction of N-3-oxododecanoyl homoserine lactone with transcriptional regulator LasR of Pseudomonas aeruginosa: Insights from molecular docking and dynamics simulations [version 1]\" employs several computational techniques to study the interactions of N-3-oxododecanoyl homoserine lactone with transcriptional regulator LasR protein.\nThe manuscript is well designed and robust experimental protocols were implemented. However, following issues must be rectified:\nRecent citations from 2021, 2020 and recent years should be included in the introduction/discussion sections.\n\nIn the methods section, Analysis of docking conformations and trajectories should be separated. So, the docking and MD simulation should not be mixed.\n\nPCA analysis of docking poses? Please provide more details.\n\nIn Figures, please remove the background color.\n\nIn total, how many snapshots were included in mm/pbsa binding energy calculations?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "97391",
"date": "25 Oct 2021",
"name": "Kapil Dave",
"expertise": [
"Reviewer Expertise Protein misfolding and aggregation",
"Molecular dynamics simulations",
"Protein-protein interactions",
"ultrafast-spectroscopy",
"Molecular Biophysics",
"Cellular Biophysics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, authors have employed computational methods (Docking and MD simulations) to study the interaction of autoinducer molecule, 3OC12-HSL, and LasR protein (Pseudomonas aeruginosa). The study revealed a second interaction site \"bridge\" for 3OC12-HSL and LasR protein. This bridge interaction involves the ligand-binding domain (LBD), beta turns in the short linker region (SLR), and the DNA-binding domain (DBD). The in-silico experiments are well defined with sufficient details provided for the computational methods. Overall, the data support the conclusion made in this manuscript. However, the authors should address the comments and concerns below.\nThe authors need to expand the introduction to include the PQS system. Authors should also include recent references from work conducted by Bagchi et al.\n\nAuthors mentioned their LasR protein model (with LBD and DBD domains) was different from that previously reported model by Chowdhury et al. Authors should briefly discuss the prime reasons that resulted in differences between their and previously reported LasR model.\n\nThe manuscript demonstrated Cluster 2 and 3 (15% of total structures) as the binding interactions from various molecular docking programs in Figure 7. However, Cluster 1, which constituted 41.95% of the total structure as reported in the docking analysis, was not observed in any other molecular docking software results. Figure 7a (Autodock Vina) does show cluster 1 interactions. The authors should provide clarification regarding these findings.\n\nIn Table 3 binding energies numbers are reported past 3 decimal places, is that accurate? If not authors should report numbers with significant decimal places.\n\nPage 13 the reference to Figure 11a and Figure 11b are not consistent with the figure's caption. Please correct the manuscript text or figure caption.\n\nThe authors should discuss the tendency of LasR to form heterodimer when 3OC12-HSL is interacting at LBD vs the bridge site. What implications an additional interacting site can have on the binding of the LasR complex to the target DNA?\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-324
|
https://f1000research.com/articles/8-320/v1
|
22 Mar 19
|
{
"type": "Research Article",
"title": "First draft genome assembly and identification of SNPs from hilsa shad (Tenualosa ilisha) of the Bay of Bengal",
"authors": [
"Md. Bazlur Rahman Mollah",
"Mohd Golam Quader Khan",
"Md Shahidul Islam",
"Md Samsul Alam",
"Mohd Golam Quader Khan",
"Md Shahidul Islam"
],
"abstract": "Background: Hilsa shad (Tenualosa ilisha), a widely distributed migratory fish, contributes substantially to the economy of Bangladesh. The harvest of hilsa from inland waters has been fluctuating due to anthropological and climate change-induced degradation of the riverine habitats. The whole genome sequence of this valuable fish could provide genomic tools for sustainable harvest, conservation and productivity cycle maintenance. Here, we report the first draft genome of T. ilisha from the Bay of Bengal, the largest reservoir of the migratory fish. Methods: A live specimen of T. ilisha was collected from the Bay of Bengal. The whole genome sequencing was performed by the Illumina HiSeqX platform (2 × 150 paired end configuration). We assembled the short reads using SOAPdenovo2 genome assembler and predicted protein coding genes by AUGUSTUS. The completeness of the T. ilisha genome assembly was evaluated by BUSCO (Benchmarking Universal Single Copy Orthologs). We identified single nucleotide polymorphisms (SNPs) by calling them directly from unassembled sequence reads using discoSnp++. Results: We assembled the draft genome of 710.28 Mb having an N50 scaffold length of 64157 bp and GC content of 42.95%. A total of 37,450 protein coding genes were predicted of which 29,339 (78.34%) were annotated with other vertebrate genomes. We also identified 792,939 isolated SNPs with transversion:transition ratio of 1:1.8. The BUSCO evaluation showed 78.1% completeness of this genome. Conclusions: The genomic data generated in this study could be used as a reference to identify genes associated with physiological and ecological adaptations, population connectivity, and migration behaviour of this biologically and economically important anadromous fish species of the Clupeidae family.",
"keywords": [
"Hilsa",
"anadromous",
"Bay of Bengal",
"whole genome",
"SNP"
],
"content": "Introduction\n\nHilsa shad (Tenualosa ilisha) is a migratory fish of the Clupeidae family. It is distributed from the South China Sea and through the Bay of Bengal to the Persian Gulf. The riverine habitats of this fish include the Padma, Jamuna, Meghna, Karnaphuli and the coastal rivers of Bangladesh, the Tigris and Euphrates of Iran and Iraq, the Indus of Pakistan, the rivers of Eastern and Western India and the Irrawaddy of Myanmar (Freyhof, 2014; Pillay & Rosa, 1963). Ecologically, three different types of hilsa shad have been recognized in Bangladesh waters such as anadromous, potamodromous and marine (Milton, 2010).\n\nHilsa is the most popular and economically important food fish in Bangladesh, contributing 12% of the total fish production and 1.15% of GDP. Of its world catch, 60% amounting to 0.5 million metric tons, comes from Bangladesh (DoF, 2018). Though the overall production of hilsa increased over the years, gross decline in productions were evident from inland waters. A number of factors such as overexploitation, siltation in river beds, decrease in water flow from upstream and fragmentation of the rivers are attributed to this fluctuation in productivity (Ahsan et al., 2014). To enhance hilsa production, programs like the establishment of sanctuaries, restrictions on the use of fishing equipment and a ban on fishing in certain periods of the year to protect parent and juvenile fish have been initiated. It is, however, very important that the management activities be matched with the biological features of the fish for their effectiveness. Inconclusive information about the management units and the level of connectivity amongst them is considered as the major constraint in formulating appropriate hilsa management plans.\n\nThere are controversies regarding the number of hilsa stocks in Bangladesh waters. Studies involving morphological and genetic analyses using allozyme, Random Amplification of Polymorphic DNA (RAPD) and mtDNA-restriction fragment length polymorphism (RFLP) markers proved to be insufficient in resolving the stock disputes of this species (Ahmed et al., 2004; Mazumder & Alam, 2009; Salini et al., 2004). DNA markers derived from whole genome sequencing are more efficient to define management units, quantify the extent of adaptive divergence and connectivity among stocks, and to perform mixed-stock analysis (Martinez Barrio et al., 2016; Figueras et al., 2016; Machado et al., 2018). Single nucleotide polymorphic (SNP) markers allow whole genome coverage and high levels of automation. Conventionally, SNP markers are developed by comparing nucleotide sequences with a reference genome. Recent advancement in generating SNPs from reference-free whole genome sequences accelerated identification of SNPs from non-model organisms. Although hilsa is a very important fish biologically and economically, it lacks a reference genome and genomic resources, imposing a severe bottleneck to understand its physiological and ecological requirements. Therefore, we performed whole genome sequencing (Mollah et al., 2018), constructed a draft genome assembly and identified SNPs from T. ilisha of the Bay of Bengal.\n\n\nMethods\n\nWe captured ten T. ilisha specimens from the seashore of the Bay of Bengal (21.981753 N 90.305556 E) (Figure 1). All efforts were made to ameliorate harm to the fish by using a seine net of appropriate mesh size (20 mm) to avoid any physical injuries and suffocation. Due to the nature of this species, the fish died immediately after taking them out of the water. Dorsal and caudal fin tissues were immediately clipped on board from a dead female fish (560.42g) and preserved in 96% ethanol. The fish were handled according to the guidelines of the Animal Welfare and Ethical Committee (AWEC) of Bangladesh Agricultural University. The genomic DNA was isolated using the standard phenol:chlorofom:isoamyl alcohol method (Sambrook & Russell, 2001). DNA purity was evaluated using a NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific, cat # ND-2000) and 0.8% agarose gel electrophoresis. DNA was quantified using Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, Cat. # Q32851) and used to systematically generate the whole genome sequence data (Figure 2).\n\nPhotograph of a T. ilisha specimen (a) and a map of Bangladesh showing the sampling site (21.981753 N 90.305556 E) (b).\n\nSchematic diagram illustrating the methodology of whole genome sequencing, de novo assembly and identification of SNPs in T. ilisha from the Bay of Bengal.\n\nFor sequencing, a PCR-free DNA library was prepared using the Illumina TruSeq DNA PCR-free Library Preparation Kit (Cat. # 20015963), following the manufacturer’s recommendations (Illumina, CA, USA). The library was fragmented, size was selected following the 350 bp insert size scheme and validated using TapeStation (2100 Bioanalyzer,Agilent Technologies, CA, USA). The DNA library was quantified using a Qubit 2.0 Fluorometer as well as by real time PCR (ABI 7500, Applied Biosystems, CA, USA) using the KAPA Library Quantification Kit (Cat. # KK4824) following the manufacturer’s standard protocol with the primer pair Primer 1: 5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3'. The PCR condition was followed as initial denaturation at 95°C for 5 min followed by 35 cycles (denaturation at 95°C for 30 sec, annealing/extension/data acquisition at 60°C for 45 sec) and melt curve analysis at 65 – 95°C. Sequencing was performed on the Illumina HiSeqX instrument according to the manufacturer’s instructions. The library was sequenced using a 2× 150 paired-end (PE) configuration (GENEWIZ, LLC. South Plainfield, NJ, USA).\n\nThe raw reads were filtered based on quality and length using Trimmomatic-0.32 (Bolger et al., 2014) after evaluating with FastQC v. 0.11.8 (Andrews, 2018) as follows: i) removal of adaptor sequences; ii) removal of read pairs from either ends when the base quality was <20; iii) trimming low quality fragments at both ends of the reads within a window size of 4 bp and an average quality threshold of 15; iv) removal of read pairs having <75 nucleotides. Jellyfish v. 2.2.6 (Marçais & Kingsford, 2011) was used to obtain a separate frequency distribution of three different high occurring kmers (21, 31 and 33) in the raw HiSeq sequence reads, and the histograms were uploaded to GenomeScope for estimating genome size, repeat content, repeat length, unique length and heterozygosity following kmer-based statistical approaches (Vurture et al., 2017).\n\nWe assembled the short reads using SOAPdenovo2 genome assembler (Luo et al., 2012), developed specifically for use with next-generation short-read sequences. SOAPdenovo2 uses the de Bruijn graph algorithm. We tested several kmers to assemble the T. ilisha genome and finally selected the assembly with a kmer of 33. The completeness of the T. ilisha genome assembly was evaluated by BUSCO (Benchmarking Universal Single Copy Orthologs) (Simão et al., 2015). For BUSCO analysis (-m geno –sp zebrafish settings), the genome was searched against the Actinopterygii database (actinopterygii_odb9), which was constructed from 20 fish species consisting of 4584 orthologs. AUGUSTUS ab initio gene prediction was performed to predict protein-coding genes. The protein sequences of fish species and other vertebrates, including Rhincodon typus, Cyprinus carpio, Takifugu rubripes, Salmo salar, Mus musculus and Homo sapiens, were downloaded from the NCBI non-redundant protein sequences (nr) database (Table 3) and aligned against the T. ilisha genome using BLASTP (Altschul et al., 1997).\n\nWe used discoSnp++ v2.2.10 (Uricaru et al., 2015) with default parameters for reference-free detection of isolated SNPs (SNPs not flanked by other SNPs, Indels or structural variants) by calling SNPs directly from sequence reads without a reference genome. This method identifies isolated SNPs from the sequences of two homologous chromosomes within a single individual.\n\n\nResults and discussion\n\nThe estimated haploid genome size of T. ilisha ranged from 649.48 to 660.73 Mb. We observed heterozygosity and a repeat peak (Figure 3), with an estimated heterozygosity of 0.579 to 0.660% and repeats of 8.30 to 13.57% (Table 1). We assembled the draft genome of 710.28 Mb, having an N50 scaffold length of 64157 bp and a GC content of ~43% (Table 2). The whole genome assembly of a notable Clupeid fish, the Atlantic herring, based on short reads (170 bp to 20 kb inserts) was 808 Mb with a scaffold N50 of 1.84 Mb and GC content of 44%, with repetitive elements making up 31% of the assembly (Martinez Barrio et al., 2016). The genome size of another important Clupeid fish, the European sardine (Sardina pilchardus), was estimated to be 655-850 Mb (Machado et al., 2018) and 935-950Mb (Louro et al., 2018).\n\nDataset show the fit of the GenomeScope model (black) based on 33-kmers in Illumina HiSeq sequence reads, max kmer coverage at 300× (a) and 10000× coverage (b).\n\n1Kmers are unique subsequences of a sequence of length k. The estimated genome size varies according to kmer value. The estimated haploid genome lengths obtained from kmer 31 and kmer 33 are very close.\n\nThe assembled T. ilisha genome was searched for BUSCO analysis against the Actinopterygii database, consisting of 4,584 orthologs constructed from 20 fish species. We found 3,578 complete (C: 78.1%), 3,456 complete and single-copy (S: 75.4%), 122 complete and duplicated (D: 2.7%), 351 fragmented (F: 7.7%) and 655 missing BUSCOs (M: 14.2%). These results suggest higher completeness of the T. ilisha genome assembly of the Bay of Bengal. The BUSCO analysis of its closely related species, the European sardine, showed 84% genome completeness (Louro et al., 2018). The completeness of genome assembly may depend on the sequencing platform used. For example, 92.3% BUSCO completeness was obtained using only the Illumina reads compared to 94.2% completeness from the Illumina + Nanopore reads in the Murray cod (Maccullochella peelii) (Austin et al., 2017).\n\nAUGUSTUS ab initio gene prediction was performed to predict protein-coding genes. We found 37,450 protein coding genes from the assembled T. ilisha genome (Mollah et al., 2019a). To annotate the proteins, predicted amino acid sequences of T. ilisha were aligned against the NCBI non-redundant protein sequences (nr) database of other vertebrates (Table 3) using BLASTP. Among the five vertebrate genomes compared, a minimum of 70.71% genes of T. ilisha was annotated by Mus musculus and a maximum of 78.34% by Salmo salar (Table 3). The numbers of predicted protein coding genes in two other important Clupeids, the Atlantic herring and the European sardine, were 23,336 and 29,408, respectively (Martinez Barrio et al., 2016; Louro et al., 2018).\n\nWe identified a total of 792,939 isolated SNPs in T. ilisha genome, of which 510,251 were transitions and 282,688 were transversions (Table 4) (Mollah et al., 2019b). We also detected 155,574 indels ranging in sizes from 1 to 60 nucleotides (Figure 4). A total of 5.3 million raw SNPs in the Atlantic stock and 5.2 million SNPs in the Baltic stock of the Atlantic herring were detected by Feng et al. (2017). In contrast, Louro et al. (2018) identified a total of 2.3 million filtered heterozygous SNPs in the European sardine. Since there is no high quality reference genome available in T. ilisha, we used discoSNP++ because of its nobility and efficiency in detection of SNPs from unassembled genome sequences. Uricaru et al. (2015) genotyped 384 SNPs out of a total of 312,088 discoSNP++ predicted SNPs, of which 368 (95.8%) were accurately validated in the tick Ixodes ricinus.\n\nThe indel values ranged from 1 to 60 nucleotides. It shows that the frequency of indels decreased with the increase in size.\n\n\nConclusions\n\nWe report here the first de novo genome of T. ilisha from the Bay of Bengal. The assembled genome can be used as a reference for genetic studies of T. ilisha and related species. The SNPs generated could provide a valuable resource for resolving stock disputes and phylogenetic or adaptation investigation of the Clupeidae family.\n\n\nData Availability\n\nTenualosa ilisha collected from Bay of Bengal, Accession number SAMN07556897: https://www.ncbi.nlm.nih.gov/biosample/?term=SAMN07556897\n\nTenualosa ilisha whole genome sequencing and assembly, Accession number SRP116260: https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP116260\n\nTenualosa ilisha whole genome shotgun sequencing project, Accession number SCED00000000: https://www.ncbi.nlm.nih.gov/nuccore/SCED00000000\n\nZenodo: Amino acid sequences of the proteins predicted from the whole genome of hilsa shad (Tenualosa ilisha) of the Bay of Bengal. https://doi.org/10.5281/ZENODO.2539223 (Mollah et al., 2019a)\n\nZenodo: Single Nucleotide Polymorphisms (SNPs) identified from the whole genome sequences of hilsa shad (Tenualosa ilisha) of the Bay of Bengal. https://doi.org/10.5281/ZENODO.2538155 (Mollah et al., 2019b)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Bangladesh Fisheries Research Institute (BFRI) for assistance in sample collection. We also acknowledge Bangladesh Agricultural University Research System (BAURES) for institutional support and BdREN (Bangladesh Research and Education Network), University Grants Commission of Bangladesh, for sharing high performance computing infrastructure.\n\n\nReferences\n\nAhmed A, Islam M, Azam M, et al.: RFLP analysis of the mtDNA D-loop region in Hilsa shad (Tenualosa ilisha) population from Bangladesh. Indian J Fish. 2004; 51(1): 25–31. Reference Source\n\nAhsan DA, Naser MN, Bhaumik U, et al.: Migration, spawning patterns and conservation of Hilsa Shad (Tenualosa ilisha) in Bangladesh and India. 1st ed. Academic Foundation, New Delhi, India, 2014. Reference Source\n\nAltschul SF, Madden TL, Schäffer AA, 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\nAndrews S: FastQC -A quality control tool for high throughput sequence data. [WWW Document]. 2018. Reference Source\n\nAustin CM, Tan MH, Harrisson KA, et al.: De novo genome assembly and annotation of Australia's largest freshwater fish, the Murray cod (Maccullochella peelii), from Illumina and Nanopore sequencing read. GigaScience. 2017; 6(8): 1–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoF: National Fish-Week 18-24 July, 2018 Compendium. 2018. Reference Source\n\nFeng C, Pettersson M, Lamichhaney S, et al.: Moderate nucleotide diversity in the Atlantic herring is associated with a low mutation rate. eLife. 2017; 6: pii: e23907. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFigueras A, Robledo D, Corvelo A, et al.: Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes): a fish adapted to demersal life. DNA Res. 2016; 23(3): 181–192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreyhof J: The IUCN Red List of Threatened Species 2014. International Union for Conservation of Nature - IUCN. 2014. Reference Source\n\nLouro B, De Moro G, Garcia CM, et al.: A haplotype-resolved draft genome of the European sardine (Sardina pilchardus). bioRxiv. 2018; 441774. Publisher Full Text\n\nLuo R, Liu B, Xie Y, et al.: SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience. 2012; 1(1): 18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMachado AM, Felício M, Fonseca E, et al.: A resource for sustainable management: De novo assembly and annotation of the liver transcriptome of the Atlantic chub mackerel, Scomber colias. Data Brief. 2018; 18: 276–284. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarçais G, Kingsford C: A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011; 27(6): 764–770. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartinez Barrio A, Lamichhaney S, Fan G, et al.: The genetic basis for ecological adaptation of the Atlantic herring revealed by genome sequencing. eLife. 2016; 5: pii: e12081. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMazumder SK, Alam MS: High levels of genetic variability and differentiation in hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes) populations revealed by PCR-RFLP analysis of the mitochondrial DNA D-loop region. Genet Mol Biol. 2009; 32(1): 190–196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilton DA: Status of hilsa (Tenualosa ilisha) management in the Bay of Bengal: an assessment of population risk and data gaps for more effective regional management. Bay of Bengal Large Marine Ecosystem Project (BOBLME), Phuket, Thailand. 2010. Reference Source\n\nMollah MB, Khan MG, Islam MS, et al.: First Draft Genome Sequence of Anadromous Hilsa Shad (Tenualosa ilisha) and Development of Genomic Resources for Conservation. In: Plant and Animal Genome Conference XXVI. San Diego, 2018; P0330. Reference Source\n\nMollah MBR, Khan MGQ, Islam MS, et al.: Amino acid sequences of the proteins predicted from the whole genome of hilsa shad (Tenualosa ilisha) of the Bay of Bengal. 2019a. http://www.doi.org/10.5281/ZENODO.2539223\n\nMollah MBR, Khan MGQ, Islam MS, et al.: Single Nucleotide Polymorphisms (SNPs) identified from the whole genome sequences of hilsa shad (Tenualosa ilisha) of the Bay of Bengal. 2019b. http://www.doi.org/10.5281/ZENODO.2538155\n\nPillay SR, Rosa JH: Synopsis of biological data on hilsa, Hilsa ilisha (Hamilton) 1882. FAO fisheries biology Synopsis 25. 1963.\n\nSalini JP, Milton DA, Rahman MJ, et al.: Allozyme and morphological variation throughout the geographic range of the tropical shad, hilsa Tenualosa ilisha. Fish Res. 2004; 66(1): 53–69. Publisher Full Text\n\nSambrook JF, Russell DW, (Eds.): Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York. 2001. Reference Source\n\nSimão FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31(19): 3210–3212. PubMed Abstract | Publisher Full Text\n\nUricaru R, Rizk G, Lacroix V, et al.: Reference-free detection of isolated SNPs. Nucleic Acids Res. 2015; 43(2): e11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVurture GW, Sedlazeck FJ, Nattestad M, et al.: GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics. 2017; 33(14): 2202–2204. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "47164",
"date": "29 Apr 2019",
"name": "Joel A. Malek",
"expertise": [
"Reviewer Expertise Genomics",
"genome sequencing",
"assembly and annotation."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a new reference genome for the hilsa shad fish of significant commercial importance in the Bay of Bengal. The methods they use are standard and the resulting completeness analysis by BUSCO showed approximately 78% of core genes present. While it would be ideal to have added libraries for scaffolding the assembly (reads from 5-20kb inserts, or 10X Genomics reads, or Pacific Bioscience), this initial assembly will certainly allow some general comparisons and the building of genomic resources to better study the fish.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "47105",
"date": "21 May 2019",
"name": "Yuzine B. Esa",
"expertise": [
"Reviewer Expertise Fish 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\nOverall, the manuscript was well written and contains all relevant information in accordance with its scope.\nProbably, it would be better if Table 3 is being presented in a figure form (e.g. Venn diagram), showing overlapping regions of genes between species.\nA linkage map (e.g using zebrafish as reference genome) and gene synteny analyses would be another interesting aspect that can be included.\nIt would be more interesting if the genome of male and female of this species can be compared and analysed, since the fish is known as protandrous hermaphrodite.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "53826",
"date": "01 Oct 2019",
"name": "Shotaro Hirase",
"expertise": [
"Reviewer Expertise Population genomics."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors constructed a draft genome assembly by whole genome sequencing and identified SNPs from Tenualosa ilisha of the Bay of Bengal. This paper is well written, and that the analysis methods are generally appropriate. I have several suggestions to improve this manuscript as follows:\nAuthors described that “We captured ten T. ilisha specimens from the seashore of the Bay of Bengal” in the Method section. But it is confusing because one individual was used finally in this study. Please revise this point.\n\nAuthors said that three different ecological types of hilsa shad have been recognized in Bangladesh. What kind of ecotype is the individual used for genome assembly expected to have? This information may be important for future studies focusing genomic bases of the ecotype divergence.\n\nAuthors described that “These results suggest higher completeness of the T. ilisha genome assembly of the Bay of Bengal.” It is a difficult problem whether it is higher completeness or not, but it seems that there are too many missing genes to say higher completeness.\n\nSNP typing was performed with a reference-free method. However, I think that it would be better to detect SNP using the reference constructed newly in this study (mapping and SNP calling by samtools etc). The SNP information (e.g. vcf file) based on the constructed reference genome should be useful for readers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-320
|
https://f1000research.com/articles/8-319/v1
|
22 Mar 19
|
{
"type": "Case Report",
"title": "Case Report: Presentation of an unusual cause of carpal tunnel syndrome with accompanying literature review",
"authors": [
"Paul Patiniott",
"Matheesha Herath",
"Peter Riddell",
"Matheesha Herath",
"Peter Riddell"
],
"abstract": "Background: Carpal tunnel syndrome (CTS) is a condition seen commonly in clinical practice; high-flow arteriovenous malformations (AVM) can be a rare but important cause. Case Report: We discuss a case of a patient who had developed left CTS in the fifth decade of life as the result of a progressively enlarging congenital peripheral AVM affecting his left upper limb. This case illustrates the clinical challenges encountered in the surgical and interventional management of this complex issue. Discussion:High-flow AVMs affecting the extremities may be comprised of a convoluted network of vessels in high-flow, low-resistance systems that often recur despite intervention. Conclusion: Peripheral AVM affecting the hand can be a rare and therapeutically challenging cause of carpal tunnel syndrome that warrants multidisciplinary team discussion.",
"keywords": [
"surgery",
"neuropathy",
"arteriovenous",
"congenital",
"malformation",
"interventional"
],
"content": "Introduction\n\nCarpal tunnel syndrome (CTS) is a condition commonly encountered by physicians and surgeons alike, with the prevalence in the general population ranging from 3–5%1 and accounting for up to 90% of all entrapment neuropathies2. The finding of a congenital arteriovenous malformation (AVM) is a much less common occurrence in clinical practice, with some studies suggesting a prevalence of 1 in 100,000, with the majority of these occurring in the head and neck, with peripheral limb AVMs rarer still3. AVMs have a tendency to grow progressively larger with age, only becoming symptomatic when associated with haemodynamic instability or compression of surrounding structures4.\n\nMedline, PubMed, Ovid, WorldCat, ProQuest and Access Medicine databases were accessed with the input of the following search terms: arteriovenous malformation; arteriovenous malformation upper limb; arteriovenous malformation flexor retinaculum; compressive lesion upper limb. This initial search yielded 1290 results to which the following filters were applied: Peer reviewed; English Language; date within 25 years, which narrowed the results to 675. To further refine the search, of these articles, only those containing the following terms were included: carpal tunnel syndrome; carpal tunnel; median neuropathy; flexor retinaculum, which resulted in a total of 57 articles all of which were reviewed. Of these articles, 8 were identified as being similar.\n\nThe most similar case published in the literature was presented by Krishnamoorthy and colleagues who described a case of an 18-year-old man with persistent median artery and causing compression of the carpal tunnel with the median artery itself behaving as the main feeding vessel to the AVM5. Although the pathology described was similar, the lesion was less complex than the one reported in our case and accordingly, attracted a different approach to the management. The remaining 7 cases shared some similarity with respect to the aetiology of symptoms6–10 or the underlying pathology11,12; however, in each of these cases, the lesions described were not as progressive or involved as that which was observed in our patient.\n\n\nCase report\n\nOur patient was a 58-year-old man who had migrated to Australia from South Sudan. He was left hand dominant with no known medical comorbidities or past surgical history. He had been referred to our general surgical outpatients clinic by his general practitioner with a nerve conduction test that had confirmed the presence of a severe left sided CTS.\n\nThe patient provided us with a 12 month history of transient paraesthesia in the distribution of the median nerve with sparing of the palmar cutaneous region. He reported that his left hand had been pulsatile his whole life and despite being ungainly was, until recently, asymptomatic. On inspection, his symptomatic left hand was 50% larger in comparison to his right. On palpation, thrills were appreciated in association with the multiple aneurysmal bulges. We hypothesised that an AVM was present deep to the flexor retinaculum and had caused some degree of compression to the median nerve. We subsequently arranged for him to undergo CT angiography, which confirmed our suspicions. (Figure 1)\n\nIn an attempt to relieve his symptoms we arranged for the patient to undergo angiography with endovascular embolisation. Multiple large aneurysmal sacs were coiled and feeding vessels embolised; however, the extent of the abnormal vasculature in this instance was considerable and definitive treatment was unable to be established without incurring a significant risk of ischaemia to the patient’s hand.\n\nImmediately post-intervention, the patient did not report any difference in his symptoms and this remained to be the case until 6 months post-procedure. At this visit it was observed that his AVM had significantly reduced in size macroscopically and the patient reported improvement in his symptoms. This change correlated with a volumetric reduction on repeat CT angiogram; it was postulated that this could be attributed to a delayed fibrosis following embolisation (Figure 2).\n\nUnfortunately, this improvement was only transient and at 9 months post procedure his AVM had again spontaneously reverted to its original pre-intervention dimensions.\n\nThe patient’s case was subsequently discussed at several multidisciplinary meetings, eventually a high-risk surgical procedure involving resection of the AVM with multiple vascular graft reconstruction was offered to the patient. However, he ultimately decided to continue living with his symptoms rather than risk compromising his hand function or viability.\n\n\nDiscussion\n\nDirect compression of the median nerve by a congenital AVM within the flexor retinaculum is a particularly unlikely cause of carpal tunnel syndrome, made even more remarkable by the fact that the patient in this case only became symptomatic in the fifth decade of life.\n\nLow-flow AVMs may be clinically less concerning as they are more amenable to surgical or endovascular intervention and appear to have lower rates of recurrence when compared to high-flow AVMs.\n\nMost AVMs involing the extremities feature low-resistance niduses between the arteries and veins. Rosen and colleagues suggest that in the management of high-flow vascular malformations if these niduses are not adequately controlled, then attempts at embolising or coiling of the proximal feeding vessels are likely to fail as new collateral feeders will invariably develop to replace them13, such as was the experience with our patient.\n\n\nConclusion\n\nPeripheral limb high-flow AVM can be a rare and therapeutically challenging cause of carpal tunnel syndrome. It is a complex issue which warrants multidisciplinary team discussion, as complications of treatment and recurrence must be carefully weighed against the likelihood of improvement on a case-by-case basis.\n\n\nConsent\n\nFormal written consent for publication was obtained from the patient prior to the writing of the case report.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors of this case report would like to acknowledge that the images for this case report were made available to the authors with permission from the patient by the Department of Radiology at Flinders Medical Centre, Bedford Park, South Australia.\n\n\nReferences\n\nAtroshi I, Gummesson C, Johnsson R, et al.: Prevalence of carpal tunnel syndrome in a general population. JAMA. 1999; 282(2): 153–8. PubMed Abstract | Publisher Full Text\n\nIbrahim I, Khan WS, Goddard N, et al.: Carpal tunnel syndrome: a review of the recent literature. Open Orthop J. 2012; 6(1): 69–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNassiri N, Cirillo-Penn NC, Thomas J: Evaluation and management of congenital peripheral arteriovenous malformations. J Vasc Surg. 2015; 62(6): 1667–76. PubMed Abstract | Publisher Full Text\n\nMulliken JB, Glowacki J: Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg. 1982; 69(3): 412–22. PubMed Abstract | Publisher Full Text\n\nKrishnamoorthy L, Murison MS, Sykes PJ: Arteriovenous malformation of the forearm as a result of a persistent median artery. J Hand Surg Br. 1998; 23(6): 820–1. PubMed Abstract | Publisher Full Text\n\nHariri A, Cohen G, Masmejean EH: Venous malformation involving median nerve causing acute carpal tunnel syndrome. J Hand Surg Eur Vol. 2011; 36(5): 431–2. PubMed Abstract | Publisher Full Text\n\nAydin A: Carpal Tunnel Syndrome Caused by Intraneural Lipoma of the Median Nerve and Arteriovenous Malformation. J Cutan Aesthet Surg. 2018; 11(1): 29–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStavros K, Paik D, Motiwala R, et al.: Median nerve penetration by a persistent median artery and vein mimicking carpal tunnel syndrome. Muscle Nerve. 2016; 53(3): 485–7. PubMed Abstract | Publisher Full Text\n\nNogueira A, Pena C, Martinez MJ, et al.: Hyperostotic macrodactyly and lipofibromatous hamartoma of the median nerve associated with carpal tunnel syndrome. Chir Main. 1999; 18(4): 261–71. PubMed Abstract | Publisher Full Text\n\nGonzález Porto SA, González Rodríguez A, Midón Míguez J: Intraneural Venous Malformations of the Median Nerve. Arch Plast Surg. 2016; 43(4): 371–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZiadeh MJ, Richardson JK: Arnold-Chiari malformation with syrinx presenting as carpal tunnel syndrome: a case report. Arch Phys Med Rehabil. 2004; 85(1): 158–61. PubMed Abstract | Publisher Full Text\n\nSierakowski A, Tare M, Maan Z: Co-existing neuroma of the palmar cutaneous branch of the median nerve and an arterio-venous malformation after open carpal tunnel decompression. J Plast Reconstr Aesthet Surg. 2012; 65(4): 541–2. PubMed Abstract | Publisher Full Text\n\nRosen RJ, Nassiri N, Drury JE: Interventional management of high-flow vascular malformations. Tech Vasc Interv Radiol. 2013; 16(1): 22–38. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "51209",
"date": "19 Aug 2019",
"name": "Su Rak Eo",
"expertise": [
"Reviewer Expertise hand surgery"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease add clinical photos of this patient - preop, and post procedural views. Describe the AVM more in detail in shape, size, and characteristics.\nThis patients seemed to have persistent symptoms according to author's report. Please describe that in the discussion section.\n\nDescribe how the embolization was performed more in detail.\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? Yes",
"responses": [
{
"c_id": "4983",
"date": "22 Oct 2019",
"name": "Paul Patiniott",
"role": "Author Response",
"response": "Dear Dr Su Rak Eo, Thank you for your review and valuable feedback relating to our case report on an unusual case of carpal tunnel syndrome. I agree that the article can be improved by including additional pre and post operative clinical photographs, further describing the AVM in addition to elaborating on the technical / procedural details. We are grateful for your expert peer-review of our literature review and case study. Best wishes, Dr Paul Patiniott Surgical Registrar, SA Health, South Australia"
}
]
},
{
"id": "79381",
"date": "15 Feb 2021",
"name": "Guy Rubin",
"expertise": [
"Reviewer Expertise hand surgery"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a rare case report. I think the literature review should be more elaborate. There were 8 papers with similar cases. It is interesting to the reader to get some more information. Maybe a table that can summarize important information from each paper. The discussion section should also be more informative and compare this case and what we know so far about similar cases.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "79399",
"date": "16 Feb 2021",
"name": "Carlos Henrique Fernandes",
"expertise": [
"Reviewer Expertise Hand surgery",
"wrist arthroscopy",
"carpal tunnel syndrome",
"congenital hand deformities",
"fractures",
"tendon",
"nerve."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCongratulations for the authors, very interesting and challenging case.\nI have a few considerations about the case that I believe will be useful for the readers of the journal.\nIn the text the authors described , \"On inspection, his symptomatic left hand was 50% larger in comparison to his right”. Reviewer: I believe that a photo of the patients hands are very important for a good comprehension.\nIn the text the authors described that the patient was undergo CT angiography, which confirmed their suspicions. Reviewer: Why the choice by CT angiography? I think that the authors can include a discussion about the methods of diagnose. What are the differential diagnosis? Please, include in the discussion.\nA photo to demonstrate the hand volumetric reduction is necessary too.\nAuthors: The patient’s case was subsequently discussed at several multidisciplinary meetings, eventually a high-risk surgical procedure involving resection of the AVM with multiple vascular graft reconstruction was offered to the patient. Reviewer: A question, a simple decompression of the carpal tunnel could improve the patient symptoms? What are the possible consequences? Please, include in the discussion section.\nAuthors: However, he ultimately decided to continue living with his symptoms rather than risk compromising his hand function or viability. Reviewer: What the authors think about the patient future? What will occur with the patient? What the authors will do when the patient return? I Believe the possibilities can be included in the discussion\nI believe that the conclusion can be limited to main purpose of the research, describe the AVM as a possible cause of median nerve compression inside of carpal tunnel.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-319
|
https://f1000research.com/articles/8-318/v1
|
21 Mar 19
|
{
"type": "Data Note",
"title": "Whole genome sequence and genome-wide distributed single nucleotide polymorphisms (SNPs) of the Black Bengal goat",
"authors": [
"Md. Bazlur Rahman Mollah",
"Md. Shamsul Alam Bhuiyan",
"M.A.M. Yahia Khandoker",
"Md. Abdul Jalil",
"Gautam Kumar Deb",
"Md. Panir Choudhury",
"Nure Hasni Desha",
"Md. Shamsul Alam Bhuiyan",
"M.A.M. Yahia Khandoker",
"Md. Abdul Jalil",
"Gautam Kumar Deb",
"Md. Panir Choudhury",
"Nure Hasni Desha"
],
"abstract": "The Black Bengal goat (BBG) is a dwarf sized heritage goat (Capra hircus) breed from Bangladesh, and is well known for its high fertility, excellent meat and skin quality. Here we present the first whole genome sequence and genome-wide distributed single nucleotide polymorphisms (SNPs) of the BBG. A total of 833,469,900 raw reads consisting of 125,020,485,000 bases were obtained by sequencing one male BBG sample. The reads were aligned to the San Clemente and the Yunnan black goat genome which resulted in 98.65% (properly paired, 94.81%) and 98.50% (properly paired, 97.10%) of the reads aligning, respectively. Notably, the estimated sequencing coverages were 48.22X and 44.28X compared to published San Clemente and the Yunnan black goat genomes respectively. On the other hand, a total of 9,497,875 high quality SNPs (Q ≥ 20) along with 1,023,359 indels, and 8,746,849 high quality SNPs along with 842,706 indels were identified in BBG against the San Clemente and Yunnan black goat genomes respectively. The dataset is publicly available from NCBI BioSample (SAMN10391846), Sequence Read Archive (SRR8182317, SRR8549413 and SRR8549904), with BioProject ID PRJNA504436. These data might be useful genomic resources in conducting genome wide association studies, identification of quantitative trait loci (QTLs) and functional genomic analysis of the Black Bengal goat.",
"keywords": [
"Black Bengal goat",
"whole genome sequence",
"short reads",
"SNP"
],
"content": "Introduction\n\nThe Black Bengal goat (BBG) is a small-sized breed of goat (Capra hircus) distributed throughout Bangladesh, West Bengal, Bihar, and Orissa regions of northeastern India. The predominant coat color of this breed is black but it is also found in brown, white and gray (Jalil et al., 2018). It is a heritage goat breed of Bangladesh, and well known for its high fertility, excellent meat and skin quality. This animal is a source of high quality meat, milk, and leather, and contributes substantially to the economy of Bangladesh (Amin et al., 2000; Faruque et al., 2017). The BBG is reported to have originated from wild goat, also known as the bezoar or Pasang (Capra aegagrus) (Herre & Röhrs, 1990), having introgressed genes from the markhor (Capra falconeri). Inheritance of genetic materials from the goats from the Southern region of China to the BBG has been hypothesized, given the historical cultural and geographical connection between South China and the Bengal across the South-Eastern offshoot of the Tibetan plateau (Nozawa, 1991). Despite its economic importance, no large-scale genomic resource is available to date for this goat breed. Here we used the Illumina HiSeq sequencing platform to sequence the whole genome of the BBG, generated short reads and identified high quality genome wide distributed single nucleotide polymorphisms (SNPs). These data might provide useful insight for conducting genome wide association studies, the identification of quantitative trait loci (QTLs) and functional genomic analysis of the Black Bengal goat.\n\n\nMethods\n\nThe experimental goat was reared at Bangladesh Livestock Research Institute (BLRI) goat farm under semi-intensive management system including slatted floor, well ventilated open sided house attached to pasture. All efforts were made to ameliorate harm to the animal. A small piece of ear tissue from an adult (30 months old) pedigreed goat (BioSample SAMN10391846) was collected by ear punching using a sterilized tissue puncher following local anesthesia (Lidocaine hydrochloride, 2%) on the right ear and immediately frozen into liquid nitrogen. Prior to ear punching, the goat was handled calmly with great care by a trained animal operator to prevent distress and injury to the animal and the handler. The tissue punching site was finally treated with antiseptic cream (Cetrimide, 0.5% and chlorhexidine digluconate 0.1%). All the animal procedure conformed the guidelines of the AWEC (Animal Welfare and Ethical Committee) of Bangladesh Agricultural University.\n\nThe tissue was finely ground by Micro Pestle (Sigma-Aldrich cat # SIAL501ZZ0), and high molecular weight DNA was extracted from the fresh frozen tissue using the Phenol:chloroform:isoamyl alcohol method (Sambrook & Russell, 2001). DNA purity was evaluated by NanoDrop 1000 Spectrophotometer (Life Technologies, CA, USA) and 0.8% agarose gel electrophoresis. DNA quantity was quantified using Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay Kit (Life Technologies, CA, USA cat # Q32851). DNA was fragmented by acoustic disruption using Covaris S220 ultrasonicator and then underwent end repair, detailing, adapter ligation and purification (NEBNext UltraII DNA library Prep Kit cat # E7645S) following manufacturer instructions. The purified DNA was further selected for the right size before PCR amplification for library construction. The preliminary quantification and dilution of the library was performed using Qubit 2.0 Fluorometer, and, then Agilent 2100 Bioanalyzer was used to determine the insert size and nucleic acid concentration of the resulting library. The effective concentration of each sample in the library mixture was determined by qPCR (ABI 7500, Applied Biosystems, CA, USA) using the KAPA Library Quantification Kit (Cat. # KK4824) following the manufacturer’s standard protocol with the primer pair Primer 1: 5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3'. The PCR conditions were as follow: an initial denaturation at 95°C for 5 min followed by 35 cycles (denaturation at 95°C for 30 sec, annealing/extension/data acquisition at 60°C for 45 sec) and melt curve analysis at 65 – 95°C before sequencing to ensure the accuracy of the sample concentration.\n\nSequencing was performed on the Illumina system (HiseqX) according to manufacturer’s instructions. The samples were sequenced using a 2 × 150 paired-end (PE) configuration (GENEWIZ, Suzhou, China) using Illumina Truseq SBS Kit v4 (cat # FC-401-4003) in high output mode. Base calling was achieved with the sequencer built-in software Real-Time Analysis (RTA) (v1.5.15.1), which performs real-time conversion of the four fluorescent signals obtained from CCD (charge-coupled device) to binary base call (BCL) data. BCL data were then converted to fastq files using bcl2fastq (v2.17, Illumina). Data demultiplexing was then performed simultaneously based on index information.\n\nPrimary analysis was performed using the sequencer’s built-in software HCS (v3.4.0) to determine whether the read can pass the chastity filter based on the signal quality of the first 25 cycles. If the read had no more than 2 out of the 25 cycles with chastity values below 0.6, the read was called PF (Pass Filter). PF clusters converted by bcl2fastq were called PF data and stored in FASTQ format. The raw data were filtered to remove adapter sequences, PE reads having Q scores of < 20 and N composition of >10%. After primary cleaning of reads, mitochondrial genomes were removed. Then the remaining high quality, contamination-free reads were aligned to both the San Clemente (GCA_001704415.1) and the Yunnan black goat genome (GCA_000317765.2) separately using Bowtie2 (v2.3.4.3) (Langmead & Salzberg, 2012). Samtools (v1.9) (Li et al., 2009) was used to convert the resulting SAM sequence alignment files to BAM format, followed by sorting, indexing and quality filtering. BCFtools (v1.9) (Narasimhan et al., 2016) was used to call and filter the variants.\n\nA total of 833,469,900 raw reads consisting of 125,020,485,000 bases were obtained by sequencing of one male BBG sample (BioSample SAMN10391846). After the QC, a total of 812,209,030 reads containing 118,911,538,136 bases were kept which was 97.45% of the total raw reads. After quality filtration and removal of the mitochondrial genome, the reads were aligned to the San Clemente and the Yunnan black goat genome which resulted in 98.65% (properly paired, 94.81%) and 98.50% (properly paired, 97.10%) of the reads aligning, respectively. Additionally, a total of 9,497,875 high quality SNPs (Q ≥ 20) along with 1,023,359 indels were identified in BBG versus the San Clemente genome (See underlying data (Mollah et al., 2019a)). Similarly 8,746,849 high quality (Q ≥ 20) SNPs along with 842,706 indels were identified BBG versus the Yunnan black goat genome genome (See underlying data (Mollah et al., 2019b)). The transition and transversion ratio was 2.27 and 2.29 in BBG against the San Clemente and the Yunnan black goat respectively.\n\n\nData availability\n\nCapra hircus isolate:Black Bengal Goat breed:Black Bengal Goat (goat). BioProject PRJNA504436:\n\nhttps://www.ncbi.nlm.nih.gov/bioproject/PRJNA504436\n\nThis project contains the following underlying data:\n\nSRR8549904 (Alignment of BBG whole genome sequences with Yunnan Black goat)\n\nSRR8549413 (Alignment of BBG whole genome sequence with San Clemente goat)\n\nSRR8182317 (WGS of Black Bengal Goat: Adult male ear tissue)\n\nFigshare: Genome-wide distributed SNPs identified in the Black Bengal Goat versus the San Clemente genome. https://doi.org/10.6084/m9.figshare.7708010.v1 (Mollah et al., 2019a)\n\nThis project contains the following underlying data:\n\nBBG_aln_SanClemente.bam.calls.q20.bcf (SNP data identified in the Black Bengal Goat versus the San Clemente genome)\n\nBBG_aln_SanClemente.bam.calls.q20.bcf.stats (output file from analysis of .bcf file with bcftools)\n\nFigshare: Genome-wide distributed SNPs identified in the Black Bengal goat versus the Yunnan Black goat genome. https://doi.org/10.6084/m9.figshare.7707929.v1 (Mollah et al., 2019b)\n\nThis project contains the following underlying data:\n\nBBG_aln_Yunnan.bam.calls.q20.bcf (SNP data identified in the Black Bengal Goat versus the Yunnan Black goat genome)\n\nBBG_aln_Yunnan.bam.calls.q20.bcf.stats (output file from analysis of .bcf file with bcftools)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThis work was supported partly by Bangladesh Agricultural University Research System (BAURES) [2018/671].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Bangladesh Livestock Research Institute (BLRI) for providing pedigreed Black Bengal goat for sample collection. We also acknowledge the BdREN (Bangladesh Research and Education Network, UGC) for sharing high performance computing infrastructure.\n\n\nReferences\n\nAmin MR, Husain SS, Islam AB: Evaluation of Black Bengal goats and their cross with the Jamunapari breed for carcass characteristics. Small Rumin Res. 2000; 38(3): 211–215. PubMed Abstract | Publisher Full Text\n\nFaruque M, Choudhury M, Ritchil C, et al.: Assessment of performance and livelihood generated through community based goat production in Bangladesh. SAARC J Agric. 2017; 14(2): 12–19. Publisher Full Text\n\nHerre W, Röhrs M: Haustiere - zoologisch gesechen. 2nd ed. Springer Sepktrum, Heidelberg. 1990. Publisher Full Text\n\nJalil M, Choudhury M, Kabir M, et al.: Morphometric characterization of Black Bengal Goat under farming condition in Bangladesh. Asian J Med Biol Res. 2018; 4(1): 95–104. Publisher Full Text\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4): 357–359. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMollah MBR, Bhuiyan MSA, Khandoker MAMY, et al.: Genome-wide distributed SNPs identified in the Black Bengal Goat versus the San Clemente genome. figshare. Fileset. 2019a. http://www.doi.org/10.6084/m9.figshare.7708010.v1\n\nMollah MBR, Bhuiyan MSA, Khandoker MAMY, et al.: Genome-wide distributed SNPs identified in the Black Bengal goat versus the Yunnan Black goat genome. figshare. Fileset. 2019b. http://www.doi.org/10.6084/m9.figshare.7707929.v1\n\nNarasimhan V, Danecek P, Scally A, et al.: BCFtools/RoH: a hidden Markov model approach for detecting autozygosity from next-generation sequencing data. Bioinformatics. 2016; 32(11): 1749–1751. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNozawa K: Domestication and history of goats. In: Maijala K. (Ed.), World Animal Science: Disciplinary Approach. Elsevier, Amsterdam. 1991; 391–404.\n\nSambrook JF, Russell DW: Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York. 2001. Reference Source"
}
|
[
{
"id": "46140",
"date": "10 Apr 2019",
"name": "Jason Miller",
"expertise": [
"Reviewer Expertise Genome assembly"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWith reference genome assemblies already available for the goat species Capra hircus, this manuscript announces a set of whole-genome shotgun reads from one individual of a breed that had yet to be sequenced. The data size is small, constituting less than one single run of an Illumina HiSeq X. The data were enhanced by mapping and SNP calling and those data are also made available. The manuscript is presented as a Data Note for which the content and format seem appropriate.\nThe abstract and introduction provide possibly excessive background on the goat breed. The text includes “dwarf sized”, “small-sized”, “high fertility”, “excellent meat and skin quality”, “high quality meat”, “contributes substantially to the economy”, and “Despite its economic importance.” Even with citations, these descriptions are imprecise for a scientific journal. The introduction could reference one respectable study of this goat’s importance. It should address the need for a genomic explanation for certain traits, with references if possible.\n\nTwo genomic references were used for SNP discovery and at least one mitochondrial genome sequence was used for filtering reads. The manuscript provides accessions for both genomic sequences but none for the mitochondria. The manuscript should provide literature citations for all of these reference sequences.\n\nThe introduction should briefly review previous goat sequencing projects, e.g. the Yunnan goat (Dong et al., 20131), the domestic goat (Bickhart et al., 20172), and some appropriate goat mitochondria study (e.g. Colli et al., 20153).\n\nThe text should explain that one library was prepared from one tissue sample and then multiplexed for sequencing with several (unrelated?) libraries. The text only hints at this by using the words “mixture” and “demultiplexing”. It is critical to understand that the reads were multiplexed since otherwise the reported number of reads seems low for a HiSeq run.\n\nThe English grammar could be polished in a few places: “transition and transversion ratio” should say “transition to transversion”; “were as follow:” should say “as follows”; “sequencer built-in software” should say “sequencer’s built-in software”. The Illumina instrument name seems to be spelled HiSeq X, not HiseqX.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
},
{
"id": "46138",
"date": "24 Apr 2019",
"name": "Almas A. Gheyas",
"expertise": [
"Reviewer Expertise Genomics",
"Bioinformatic analysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is mostly well written and provides sufficient background about the importance of the Black Bengal goat as a rationale for generating the genomic data. There are, however, several places in the Methods section, which would require further information and/or clarification. I highlight the points below:\n\n“DNA was fragmented by acoustic disruption using Covaris S220 ultrasonicator and then underwent end repair, detailing...”:\n\nBy “detailing”, do you mean dA-tailing?\n\n“The purified DNA was further selected for the right size before PCR amplification for library construction.”\n\nWhat was considered the “right size” and how were the fragments of desired size range selected?\n\n“The effective concentration of each sample in the library mixture…”.\n\nSince the sequencing was performed on a single BBG sample, the authors need to clarify that it was sequenced in a multiplex with other unrelated samples.\n\n“If the read had no more than 2 out of the 25 cycles with chastity values below 0.6...”.\n\nDo you mean 2 bases out of 25 cycles?\n\n“The raw data were filtered to remove adapter sequences, of < 20... “\nDoes this Q score represent average value per read?\n\n“After primary cleaning of reads, mitochondrial genomes were removed.”\n\nPlease clarify why and how the mitochondrial genome was removed?\n\nThe study mentions using Bowtie2 for mapping sequence reads against reference genomes but does not provide any detail about parameters used for mapping. If the default setting was used, that needs to be mentioned. I am also wondering if any post-alignment processing (e.g. marking duplicate reads, indel realignment etc.) was applied. These processing steps are important as these will affect the variant calling and false discovery rate.\n\nThe authors mention about variant calling with BCFtools but do not provide any details about parameters used for calling/filtering variants. The only filtration criteria mentioned is Q>20 and the filtered set has been called “high quality”. I am not convinced if calling variants from a single individual with Q>20 can be called “high quality” without performing further validation or without providing any information about false discovery rate. Besides I am wondering if the same criteria were used for calling/filtering both SNPs and indels? Please clarify this. I would also suggest the authors to remove the words “high quality” as in my opinion these are only a preliminary set of variables, which would require further filtration and validation before being used in further studies.\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": []
}
] | 1
|
https://f1000research.com/articles/8-318
|
https://f1000research.com/articles/8-315/v1
|
21 Mar 19
|
{
"type": "Research Article",
"title": "Using pens as an incentive for trial recruitment of older adults: An embedded randomised controlled trial",
"authors": [
"Katie Whiteside",
"Lydia Flett",
"Alex S. Mitchell",
"Caroline Fairhurst",
"Sarah Cockayne",
"Sara Rodgers",
"David J. Torgerson",
"OTIS Study Group",
"Lydia Flett",
"Alex S. Mitchell",
"Caroline Fairhurst",
"Sarah Cockayne",
"Sara Rodgers",
"David J. Torgerson"
],
"abstract": "Background: Meeting recruitment targets for randomised controlled trials is challenging. This trial evaluated the effectiveness of including a pen within the trial invitation pack on the recruitment of older adults into a randomised controlled trial. Methods: This trial was embedded within the Occupational Therapist Intervention Study, a falls-prevention randomised controlled trial. Potential participants (n = 1862), who were posted an invitation pack from two General Practitioner practices, were randomised to either not receive a pen (n = 1295) or receive a pen (n = 648) with their invitation pack, using a 2:1 ratio. The primary outcome was the likelihood of being randomised, and therefore fully recruited, to the host trial. To be randomised to the host trial, participants had to: return a consent form and screening form; be eligible on their screening form; and return a baseline questionnaire and a monthly falls calendar. Secondary outcomes were: the likelihood of returning (and time to return) a screening form; being eligible for the host trial; and remaining in the trial for at least 3 months. Results: The likelihood of being randomised to the host trial did not differ between the pen group (4.5%) and no pen group (4.3%; odds ratio 1.04; 95% confidence interval: 0.65 to 1.67; p = 0.86). There were marginal differences in secondary outcomes in favour of the pen group, particularly in screening form return rates, though these differences were not statistically significant. Conclusion: Pens may not be an effective incentive for the recruitment of older adults into randomised controlled trials, though future trials are required. Registration: ISRCTN22202133; SWAT 37.",
"keywords": [
"Randomised Controlled Trial",
"Embedded Trial",
"Recruitment",
"Incentive",
"Pen"
],
"content": "Introduction\n\nRandomised controlled trials (RCTs) are vital in establishing the effectiveness of interventions. However, recruitment into RCTs remains a substantial challenge1, with only around 55% of healthcare RCTs achieving their recruitment target and about 32% having to extend their recruitment period2,3. This can lead to underpowered trials that fail to find relevant group differences as statistically significant, as well as delayed results and increased costs due to recruitment extensions1. Despite this, there is currently a lack of evidence to inform researchers of how recruitment into RCTs might best be improved1, though this has been identified as a high priority to address4. Therefore, it is crucial that potential strategies to improve recruitment are robustly evaluated by embedding RCTs evaluating such strategies into real ‘host’ RCTs5.\n\nOne strategy to improve recruitment is the use of incentives. Based on the principle of reciprocity, receiving incentives is hypothesised to encourage individuals to respond to the positive behaviour in a positive way6,7. Both monetary and non-monetary incentives are frequently used by clinical trials units in the UK to support recruitment, despite a lack of evidence of their impact8. Some evidence suggests that monetary incentives improve RCT recruitment rates1; however, this strategy is expensive and ethically controversial9. In contrast, non-monetary incentives, such as providing pens, are cheaper and more ethically sound10.\n\nThe use of pens as an incentive is an especially appealing strategy for RCTs that utilise large-scale database recruitment. This method involves distributing invitation packs to individuals identified as potentially eligible for a trial from database searches (e.g. General Practitioner [GP] records) and is particularly suitable for recruiting participants with chronic conditions and for recruitment into RCTs that evaluate public health interventions11. Database recruitment is minimally labour intensive, inexpensive, and associated with improved recruitment rates compared to opportunistic recruitment11. Nevertheless, it would be valuable to explore how this strategy could be more efficient, as recruitment yield can still be low. This can especially be the case when recruiting older adults, a population faced with numerous barriers to trial participation, such as reduced mobility, a lack of trust and understanding of trials, and the belief that participation would be too burdensome12,13. For example, an RCT evaluating a podiatry intervention for falls prevention in older adults randomised just 2.7% of those approached via database recruitment into the trial14.\n\nThe inclusion of pens within trial invitation packs may not only improve recruitment rates through encouraging reciprocal positive behaviour, but the convenience of a pen being readily available may prompt rapid completion and return of trial documentation15. Despite this, no previous RCTs have evaluated the impact of distributing pens within invitation packs on recruitment into RCTs. However, a trial evaluating recruitment into a questionnaire survey reported that providing a study-branded pen within the invitation pack, to individuals who had previously not responded, improved response rates16.\n\nSome relevant trials have explored whether providing pens improves response rates to postal questionnaires, though these have yielded mixed findings. A previous trial found that sending a pen with a postal questionnaire to consultants did not improve response rates15. However, other trials have reported that providing a study-branded pen or pencil was a cost-effective strategy which improved follow-up questionnaire response rates16,17. Similarly, a UK-based embedded RCT, evaluating an osteoporosis screening programme, found that including pens with postal questionnaires led to a marginal increase in response rates, a reduction in the number of reminders required, and a reduction in time to return the questionnaire10. While the effects reported in this trial were all very small, the provision of pens was considered cost-effective due to their low cost. Given these promising results, it would be valuable for further embedded RCTs to evaluate whether these findings generalise to improvements in trial recruitment when a pen is included within the invitation pack.\n\nIn this paper we describe an embedded RCT (or ‘study within a trial’ [SWAT]) designed to evaluate the effectiveness of including a pen within the trial invitation pack on the recruitment of older adults, identified from GP database searches, into the Occupational Therapist Intervention Study (OTIS)18. Specifically, this RCT evaluated the impact of providing pens on subsequent recruitment rates into the OTIS trial, as well as return rates of recruitment documentation, eligibility of respondents, and the retention of participants. This trial not only helped to address the lack of RCTs on the use of pens as an incentive for trial recruitment, but also explored how to further improve the efficiency of database recruitment, focusing specifically on older adults, who can be particularly challenging to recruit.\n\n\nMethods\n\nThis two-arm RCT was embedded within OTIS, which is a UK-based modified cohort RCT. The protocol for the OTIS trial has been published previously18. In brief, the OTIS trial aimed to assess whether home environmental assessment and modification, led by an occupational therapist (OT), could reduce risk of falling among community dwelling, older adults at elevated risk of falling. Approval for the OTIS trial and this embedded trial was granted by the National Health Service West of Scotland Research Ethics Committee 3; the University of York, Department of Health Sciences Research Governance Committee; and the Health Research Authority. This embedded trial was registered with the ISRCTN registry as part of the host trial registration (ISRCTN22202133; date registered: 20th June 2016) and was also registered with the Northern Ireland Hub for Trials Methodology Research SWAT Repository (SWAT 37; date registered: 20th February 2016).\n\nOne of the main recruitment methods for the OTIS trial was GP mail-outs. We embedded this trial in mail-outs from two UK-based GP practices. Within these mail-outs, men and women identified as potentially eligible (i.e. aged over 65 years and community dwelling) in database searches were posted a trial invitation pack. These packs included an invitation letter, a participant information sheet, consent form, screening form, and a pre-paid return envelope. Participants allocated to the intervention group of this embedded trial also received a York Trials Unit branded pen in their invitation pack. The control participants did not receive a pen in their invitation pack. Recipients of an invitation pack were asked to return a completed consent form and screening form if they were willing to take part in the OTIS trial.\n\nTo be eligible for the OTIS trial, participants had to be: over 65 years, community dwelling, currently able to walk 10 feet (with a walking aid if needed), willing and able to provide informed consent and to receive an OT home visit, and must not have had an OT assessment in the previous 12 months or be on the waiting list for one. Additionally, participants had to have one of the following risk factors for falling: have had at least one fall in the past 12 months; or report that they worry about falling at least some of the time. Participants who were eligible except for fulfilling a risk factor for falling were contacted again 4 to 6 months later for rescreening.\n\nEligible participants were then posted a baseline questionnaire to complete along with an Age UK falls prevention advice leaflet, and monthly falls calendars to return at the start of each month with details of any falls they had during the previous month (for up to 12 months after randomisation). Once participants had returned their completed baseline questionnaire and at least one falls calendar, they become eligible to be randomised into the OTIS trial to either receive an OT home visit or usual care.\n\nRecruitment of embedded trial participants into the host trial commenced in May 2017 and follow-up for this embedded trial ended in May 2018.\n\nAs is typical for an embedded trial, a formal sample size calculation was not carried out. The sample size was constrained by the number of invitation packs distributed via GP mail-outs during the time-period in which this embedded trial took place. Allocation to either the intervention ‘pen’ arm (to receive a York Trials Unit branded pen with the invitation pack) or the control ‘no pen’ arm (to receive the invitation pack with no pen) was achieved using block randomisation stratified by GP practice. We used a 2:1 allocation ratio, in favour of the no pen arm. Unique participant identification numbers for each invitation pack, prepared for the two GP practice mail-outs involved in this embedded trial, were randomised within three blocks. A single randomisation block, the size of the full mail-out, was used for the first GP practice mail-out and two blocks, of roughly equal size, were used for the second GP practice mail-out. Generation of the allocation sequence was undertaken by the OTIS trial statistician, who was not involved with production of the invitation packs, using Stata version 1319.\n\nThe primary outcome was the proportion of embedded trial participants who were randomised into the OTIS main trial. Secondary outcomes were:\n\na) proportion of participants who returned a screening form;\n\nb) time to return screening form;\n\nc) proportion of participants who were initially 'pending' in terms of their eligibility on initial screening (i.e. fulfilled all eligibility criteria apart from a risk factor for falling);\n\nd) proportion of participants who were eligible on initial screening;\n\ne) proportion of participants who remained in the trial at three months post randomisation (defined as returning at least the first three months’ worth of falls calendars post-randomisation).\n\nData were analysed on an intention-to-treat basis using two-sided tests at the 5% significance level. Categorical data were compared using logistic regression models and time to response data were analysed using a Cox proportional hazards model. All models adjusted for the GP site the invitation packs were mailed out from. Additionally, the logistic regression model used to analyse trial retention adjusted for the OTIS trial group allocation (usual care or intervention). The odds ratio (OR) or hazard ratio (HR) from each model associated with the pen embedded trial allocation is presented along with the corresponding 95% confidence interval (CI) and p-value. All analyses were conducted using Stata version 1520.\n\n\nResults\n\nWe randomised 1943 participants into this embedded trial (648 to receive a pen with their invitation pack; 1295 to not receive a pen); however, 81 of these invitation packs were not sent out (pen arm = 28; no pen arm = 53) and were excluded from this analysis (Figure 1). Therefore, we included 1862 participants in this analysis (pen arm = 620, 33.3%; no pen arm = 1242, 66.7%). Of these participants, 919 (49.4%) were posted an invitation pack from GP practice 1 and 943 (50.6%) were posted a pack from GP practice 2. Raw data are available on Open Science Framework21.\n\nOf the 1862 embedded trial participants, 82 (4.4%) were randomised into the OTIS trial (pen: 28/620 [4.5%]; no pen: 54/1242 [4.3%]; difference of 0.17%; 95% CI of difference: -1.82% to 2.16%). The two groups did not significantly differ in their likelihood of being randomised into the OTIS trial (OR 1.04; 95% CI: 0.65 to 1.67; p = 0.86).\n\nIn total, 233 (12.5%) of the 1862 embedded trial participants returned a screening form (pen: 88/620 [14.2%]; no pen: 145/1242 [11.7%]). The two groups did not significantly differ in their likelihood of returning a screening form (OR 1.25; 95% CI: 0.94 to 1.67; p = 0.12).\n\nFor the 233 screening forms returned, the median time to return was 22 days (interquartile range [IQR]: 17 to 29) in the pen arm and 20 days (IQR: 17 to 28 days) in the no pen arm. There was no statistically significant difference in the time to respond between the two arms (HR 1.23; 95% CI: 0.94 to 1.60; p = 0.13). As the response rate was less than 50%, the median time to return a screening form could not be calculated from Kaplan-Meier survival estimates, so the 10th percentile survival times were estimated instead. It took 26 days (95% CI: 24 to 39) in the pen arm, and 39 days (95% CI: 25 to 119) in the no pen arm, for 10% of the mailed screening forms to be returned.\n\nOf the 1862 embedded trial participants, 86 (4.6%) were initially ‘pending’ in their eligibility for the OTIS trial, whereby they met all eligibility criteria apart from a risk factor for falling (pen: 34/620 [5.5%]; no pen: 52/1242 [4.2%]). The two groups did not significantly differ in their likelihood of having an eligibility status of ‘pending’ on initial screening (OR 1.33; 95% CI: 0.85 to 2.07; p = 0.21).\n\nIn total, 113 (6.1%) of the 1862 embedded trial participants were eligible for the OTIS trial on their initial screening form (pen: 40/620 [6.5%]; no pen: 73/1242 [5.9%]). The two groups did not significantly differ in their likelihood of being fully eligible on initial screening (OR 1.11; 95% CI: 0.74 to 1.65; p = 0.62).\n\nOf the 1862 embedded trial participants, 76 (4.1%) remained in the OTIS trial 3 months post-randomisation (pen: 27/620 [4.4%]; no pen: 49/1242 [3.9%]). There was no statistically significant difference in the number of embedded trial participants remaining in the OTIS trial 3 months post-randomisation between the two groups (pen: 27/28 [96.4%]; no pen: 49/54 [90.7%]; OR 2.63; 95% CI: 0.29 to 24.1; p = 0.39). This analysis adjusted for OTIS trial group allocation and therefore the sample size (n = 82) reflected the number of embedded trial participants randomised into the OTIS trial.\n\n\nDiscussion\n\nThis embedded RCT evaluated the effectiveness of including a non-monetary incentive, in the form of a York Trials Unit branded pen, within invitation packs mailed out from GP practices on the recruitment of older adults into the OTIS trial. The absolute difference in the percentage of embedded trial participants randomised to the OTIS trial was 0.17% (4.5% in the pen arm, 4.3% in the no pen arm) and was not statistically significant, which suggests that providing a pen within trial invitation packs was not an effective incentive to improve recruitment of older adults into the host RCT.\n\nWhether providing pens as a recruitment incentive is cost-effective remains uncertain. Based on the randomisation rate of 4.3% (or 43 per 1,000) achieved in this embedded trial using standard invitation packs, and given that the printing, packaging, and postage costs for each standard pack was £2.53, it costs £2,530 to send 1,000 standard packs to recruit 43 participants into the host trial, or £58.84 per participant. The pens cost £0.32 each, so it costs an additional £320 per 1,000 packs distributed with a pen. For this price, approximately five participants could be recruited using standard packs; therefore, including a pen would need to increase the percentage of eligible participants randomised by 0.5% (or 5 per 1,000) to be cost-effective. If the point estimate reported here (0.17%) is the true difference, providing pens would not be cost-effective. However, if the upper 95% confidence limit of the difference (2.16%) is the true difference, providing pens would likely be cost-effective. Consequently, additional trials are needed to evaluate this recruitment strategy. Furthermore, as the cost of pens could be reduced if a non-branded style were used, further trials could additionally evaluate the effectiveness of the branding.\n\nWithin this embedded RCT, the provision of pens was not associated with a significant difference in any of the secondary outcomes, though all results favoured the pen arm. Providing a pen in the invitation pack resulted in a small increase in screening form return rates (absolute difference of 2.5%). There were also trends for those who received a pen to return their screening form more quickly and to be more likely to remain in the OTIS trial for at least 3 months after being randomised (96.4% vs. 90.7%). These results suggest that including a pen in trial invitation packs may marginally boost the return of trial recruitment documentation among older adults, a population that can be particularly difficult to recruit12,13, and may have benefits on trial retention. While improvements in screening form response rates did not translate to improvements in randomisation rates in this trial, it is possible that it may do in other trials, particularly those with broader eligibility criteria. This further highlights the need for future trials to evaluate pens as a recruitment incentive.\n\nThis embedded RCT adds to the limited and mixed literature on the provision of pens on response rates to trial documentation, with some previous trials showing an effect10,16,17 and others showing no effect15. While previous trials have considered the impact of providing pens on questionnaire return rates, this trial was the first to evaluate the inclusion of a pen within the trial invitation pack on RCT recruitment.\n\nThis embedded trial was limited by only involving two GP practice mail-outs and focusing on older adults. It would be beneficial for future embedded trials, within large-scale RCTs utilising database recruitment, to involve a greater number of mail-outs to further evaluate this question. Further research should also explore the impact of providing pens on the recruitment of different participant populations (e.g. different age groups). Meta-analysis could then be used to explore the effectiveness of including pens within trial invitation packs and whether this varies depending on participant demographics.\n\n\nConclusions\n\nProviding a pen within trial invitation packs had marginally beneficial effects on screening forms return rates and retention within this embedded trial, though did not improve randomisation rates of older adults into the host RCT. Further embedded trials are necessary to evaluate whether providing pens in invitation packs is a cost-effective incentive for trial recruitment.\n\n\nData availability\n\nOpen Science Framework: Underlying data and CONSORT checklist for using pens as an incentive for trial recruitment of older adults: An embedded randomised controlled trial. https://doi.org/10.17605/OSF.IO/6FMGC21. This project contains the following underlying data files:\n\nDataset1_OTIS_pensubstudy_F1000_data.csv (raw data in CSV format)\n\nDataset1_OTIS_pensubstudy_F1000_data.sav (raw data in SAV format)\n\nDataset1_OTIS_pensubstudy_F1000_variable_key.csv (definition or abbreviations in dataset)\n\nOpen Science Framework: CONSORT checklist for “Using pens as an incentive for trial recruitment of older adults: An embedded randomised controlled trial”. https://doi.org/10.17605/OSF.IO/6FMGC21\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 study was funded by the National Institute for Health Research (NIHR) Health Technology Assessment (HTA) Programme (Programme grant number 14/49/149). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. The University of York is the study sponsor and has legal responsibility for the initiation and management of the trial (sponsor representative: Dr Michael Barber, Research and Enterprise Directorate, University of York, Ron Cooke Hub, Heslington, York, UK, YO10 5GE).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank the GP practices that participated in this embedded trial: Falkland Surgery, Great Yarmouth, UK; Lighthouse Medical Practice, Eastbourne, UK. The authors would also like to thank the embedded trial participants who returned trial recruitment documentation.\n\nThis paper was written by the authors on behalf of the OTIS Study Group, which is made up of the following individuals: Sophie Boyes (York Teaching Hospital NHS Foundation Trust); Sarah Cockayne (University of York); Belen Corbacho (University of York); Shelley Crossland (Leicestershire Partnership NHS Trust); Avril Drummond (University of Nottingham); Caroline Fairhurst (University of York); Simon Gilbody (University of York); Catherine Hewitt (University of York); Sarah E Lamb (University of Oxford); Jennifer McCaffery (University of York); Alison Pighills (Mackay Base Hospital; Mackay Australia and James Cook University); Clare Relton (University of Sheffield); Sara Rodgers (University of York); and David J. Torgerson (University of York). With the exception of Jennifer McCaffery, who supported coordination and data collection for the host OTIS trial and this embedded trial, all members of the OTIS Study Group were co-applicants on the grant for the host OTIS trial and were involved in designing the study.\n\nThe authors acknowledge Adwoa Parker (University of York) for pre-registering this embedded trial with the Northern Ireland Hub for Trials Methodology Research SWAT Repository (SWAT 37).\n\n\nReferences\n\nTreweek S, Pitkethly M, Cook J, et al.: Strategies to improve recruitment to randomised trials. Cochrane Database Syst Rev. 2018; 2: MR000013. PubMed Abstract | Publisher Full Text\n\nSully BG, Julious SA, Nicholl J: A reinvestigation of recruitment to randomised, controlled, multicenter trials: a review of trials funded by two UK funding agencies. Trials. 2013; 14: 166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWalters SJ, Bonacho Dos Anjos Henriques-Cadby I, Bortolami O, et al.: Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open. 2017; 7(3): e015276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTudur Smith C, Hickey H, Clarke M, et al.: The trials methodological research agenda: results from a priority setting exercise. Trials. 2014; 15: 32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadurasinghe VW, Sandra Eldridge on behalf of MRC START Group and Gordon Forbes on behalf of the START Expert Consensus Group: Guidelines for reporting embedded recruitment trials. Trials. 2016; 17: 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFehr E, Gächter S: Fairness and retaliation: The economics of reciprocity. J Econ Perspect. 2000; 14(3): 159–81. Publisher Full Text\n\nFehr E, Falk A: Psychological foundations of incentives. Eur Econ Rev. 2002; 46(4–5): 687–724. Publisher Full Text\n\nBower P, Brueton V, Gamble C, et al.: Interventions to improve recruitment and retention in clinical trials: a survey and workshop to assess current practice and future priorities. Trials. 2014; 15: 399. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPermuth-Wey J, Borenstein AR: Financial remuneration for clinical and behavioral research participation: ethical and practical considerations. Ann Epidemiol. 2009; 19(4): 280–5. PubMed Abstract | Publisher Full Text\n\nBell K, Clark L, Fairhurst C, et al.: Enclosing a pen reduced time to response to questionnaire mailings. J Clin Epidemiol. 2016; 74: 144–50. PubMed Abstract | Publisher Full Text\n\nStuardi T, Cox H, Torgerson DJ: Database recruitment: a solution to poor recruitment in randomized trials? Fam Pract. 2011; 28(3): 329–33. PubMed Abstract | Publisher Full Text\n\nRidda I, MacIntyre CR, Lindley RI, et al.: Difficulties in recruiting older people in clinical trials: an examination of barriers and solutions. Vaccine. 2010; 28(4): 901–6. PubMed Abstract | Publisher Full Text\n\nPiantadosi C, Chapman IM, Naganathan V, et al.: Recruiting older people at nutritional risk for clinical trials: what have we learned? BMC Res Notes. 2015; 8: 151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCockayne S, Rodgers S, Green L, et al.: Clinical effectiveness and cost-effectiveness of a multifaceted podiatry intervention for falls prevention in older people: a multicentre cohort randomised controlled trial (the REducing Falls with ORthoses and a Multifaceted podiatry intervention trial). Health Technol Assess. 2017; 21(24): 1–198. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark TJ, Khan KS, Gupta JK: Provision of pen along with questionnaire does not increase the response rate to a postal survey: a randomised controlled trial. J Epidemiol Community Health. 2001; 55(8): 595–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite E, Carney PA, Kolar AS: Increasing response to mailed questionnaires by including a pencil/pen. Am J Epidemiol. 2005; 162(3): 261–6. PubMed Abstract | Publisher Full Text\n\nSharp L, Cochran C, Cotton SC, et al.: Enclosing a pen with a postal questionnaire can significantly increase the response rate. J Clin Epidemiol. 2006; 59(7): 747–54. PubMed Abstract | Publisher Full Text\n\nCockayne S, Pighills A, Adamson J, et al.: Can occupational therapist-led home environmental assessment prevent falls in older people? A modified cohort randomised controlled trial protocol. BMJ Open. 2018; 8(9): e022488. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStataCorp: Stata Statistical Software: Release 13. College Station, TX: StataCorp LP, 2013.\n\nStataCorp: Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC, 2017.\n\nWhiteside K, Flett L, Mitchell A, et al.: Underlying data and CONSORT checklist for using pens as an incentive for trial recruitment of older adults: An embedded randomised controlled trial. 2019. http://www.doi.org/10.17605/OSF.IO/6FMGC"
}
|
[
{
"id": "46146",
"date": "27 Mar 2019",
"name": "Daniel Hind",
"expertise": [
"Reviewer Expertise 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\nThis article reports the findings of a study within a trial (SWaT), a methodological design currently promoted by at least one major funder within the UK. Many SWaTs, including this one, are aimed at improving participant recruitment to RCTs, the principal source of study failure.\nThe study is well conducted and, in almost all regards, suitable to be indexed as is. I have only one observation, which regards the conclusion that more trials are warranted. It is not obvious to this reader that the background or results support this conclusion.\nIn the background, the authors cite, as a warrant for this trial, Clark et al.'s paper (Clark et al., 20011) in which a pen did not improve the response rate of a survey. In White et al.’s study (White et al., 20052) the opposite was the case. In this study, the inclusion of a pen did not increase participant recruitment. 'The best of three' - the pen loses.\nSo, if we're going to do this on a purely empirical basis, it would be useful to know at what point we will decide that the pen is ineffective and White's success can be put down to the play of chance. What are the 'stop-go' criteria?\nOn the other hand, if we look at this more mechanistically, we could ask why an incentive which is insufficient to motivate people in this population group would be any more likely to motivate people in other population groups. Is there some plausible link between returning your form more quickly and consenting to study entry? It's not obvious to me that there is.\nOtherwise, the report is a useful addition to the growing evidence for what works in the conduct of trials and I recommend it to be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4557",
"date": "10 Apr 2019",
"name": "Katie Whiteside",
"role": "Author Response",
"response": "We would like to thank the reviewer for taking the time to review our paper. In this response, we have addressed the reviewer’s reservations regarding our conclusion that more trials are required to evaluate pens as a trial recruitment incentive.The reviewer referenced trials by Clark et al.1 and White et al.2 to support his suggestion that further studies may not be required in addition to our trial. However, these trials did not evaluate pens as an incentive for recruitment into randomised controlled trials and therefore cannot be used to make solid conclusions regarding the use of pens in this way. As outlined in the introduction to our paper, both Clark et al. and White et al. evaluated pens as an incentive for the completion and return of postal questionnaires. While White et al. did additionally evaluate pens as a recruitment incentive, participants were being recruited into a questionnaire survey, rather than a randomised controlled trial.The reviewer seemed to imply that only White et al.2 found that providing pens was an effective incentive for the return of trial documentation. As referenced in our paper, Bell et al.3 also found that providing a pen with a postal questionnaire improved response rates and reduced the number of reminders required. The positive effects shown in these trials were part of our rationale for exploring whether similar effects can be found when providing pens within trial invitation packs.The reviewer commented on the effect on screening form return time and questioned why this is indicative of pens as an effective recruitment incentive. Given the large proportion of trials that have to extend their recruitment period4, we believe that an incentive that could speed up the return of recruitment documents is useful. Nevertheless, the effect on screening form return time was very small in our trial and, in our discussion, we actually focused more on the finding that providing a pen in the invitation pack resulted in a small increase in screening form return rates (absolute difference of 2.5%). We noted that while improvements in screening form return rates did not translate to improvements in randomisation rates in this trial, it is possible that it may do so in other trials, particularly those with broader eligibility criteria. Indeed, if the difference in randomisation rates was the same as the difference we found in screening form return rates, providing pens would have been a cost-effective incentive.We gave further justification for the need for more trials in the cost-effectiveness discussion paragraph, in which we noted that the cost-effectiveness of the incentive is uncertain. As part of this paragraph, we suggested evaluating the use of cheaper, non-branded pens as a further avenue for research. Providing cheaper pens would mean that the impact on randomisation rates required for the incentive to be cost-effective would be reduced.Finally, the reviewer questioned what the ‘stop-go’ criteria should be for evaluating pens as a trial recruitment incentive. Ideally, further trials would be conducted to evaluate pens as a recruitment incentive for different trials and meta-analysis would then be used to systematically review the impact of providing pens. References: Clark TJ, Khan KS, Gupta JK: Provision of pen along with questionnaire does not increase the response rate to a postal survey: a randomised controlled trial. J Epidemiol Community Health. 2001; 55(8): 595–6. White E, Carney PA, Kolar AS: Increasing response to mailed questionnaires by including a pencil/pen. Am J Epidemiol. 2005; 162(3): 261–6. Bell K, Clark L, Fairhurst C, et al.: Enclosing a pen reduced time to response to questionnaire mailings. J Clin Epidemiol. 2016; 74: 144–50. Walters SJ, Bonacho Dos Anjos Henriques-Cadby I, Bortolami O, et al.: Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open. 2017; 7(3): e015276"
}
]
},
{
"id": "46144",
"date": "01 Apr 2019",
"name": "Nicola Mills",
"expertise": [
"Reviewer Expertise Optimising recruitment to RCTs"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports findings from a randomised study within a host RCT assessing pens within trial invite packs as an incentive for recruitment. Recruitment to RCTs is often problematic and there is little evidence on effective methods to optimise recruitment, so research in this field is worthy. A Study within a Trial (SWAT), as undertaken by this study group, is a potentially effective and efficient way of generating robust evidence to redress the gap. The work is clearly presented, drawing upon evidence from key literature, and the methodology appears sound.\nI’m not wholly convinced by the authors' conclusion that more trials are needed given that this was a study with robust methodology undertaken in a population who ‘may’ be less mobile and therefore appreciative of a pen to hand, and given the existing literature already around pens as an incentive to participate in research. I would like to have seen a wider discussion on the use of incentives and initiatives more generally to optimise recruitment, with consideration of the reason for their findings and direction for future research. Overall though, a focus on strategies to optimise recruitment to RCTs to build on the evidence base should be encouraged and I therefore recommend this article be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4558",
"date": "10 Apr 2019",
"name": "Katie Whiteside",
"role": "Author Response",
"response": "We would like to thank the reviewer for taking the time to review our paper. Regarding the reviewer’s reservations for our conclusion that more trials are required to evaluate pens as a trial recruitment incentive, please refer to our response to Reviewer Report 11 as this was considered here.References Hind D: Referee Report For: Using pens as an incentive for trial recruitment of older adults: An embedded randomised controlled trial [version 1]. F1000Research. 2019; 8: 315."
}
]
}
] | 1
|
https://f1000research.com/articles/8-315
|
https://f1000research.com/articles/8-313/v1
|
20 Mar 19
|
{
"type": "Research Article",
"title": "In silico docking analysis of CCL28 (C-C motif chemokine ligand 28) and astragalin as the potential inhibitor of rheumatoid arthritis",
"authors": [
"Sadaf Noor",
"Syeda Tahira Qousain",
"Syed Aun Muhammad",
"Sadaf Noor",
"Syeda Tahira Qousain"
],
"abstract": "Background: Rheumatoid arthritis is an inflammatory and chronic disease of the joints affecting 1% of the world’s population. Women are three times more likely to be affected than men. Many drugs are being used for the treatment of rheumatoid arthritis but they often have severe side effects. C-C motif chemokine ligand 28 (CCL28) recruits leukocytes and other proinflammatory factors to the site of joint inflammation. The purpose of the present research is the computational evaluation of astragalin, a natural flavonoid extracted from black tea, as an inhibitor of CCL28 by in silico docking.\n\nMethods: The three-dimensional structure of CCL28 to act as a molecular target was obtained from the Protein Data Bank (PDB ID: 6CWS). The quality of the CCL28 structure was assessed using Phyre2 and Molecular Operating Environment (MOE) software was used for binding affinity analysis. Astragalin served as a ligand for docking and naproxen, a known drug for rheumatoid arthritis, was used as a standard for comparison. Results: In molecular docking, astragalin showed significant binding affinity with the CCL28 target molecule, with a binding energy of -5.40 kcal/mol, in comparison with naproxen which has a binding energy of -4.87 kcal/mol. Astragalin has strong binding affinity for CCL28 as compared to standard naproxen. Conclusion: This study revealed that astragalin could have the potential to serve as an inhibitor of CCL28 for the treatment of rheumatoid arthritis.",
"keywords": [
"Rheumatoid arthritis",
"CCL28 inhibition",
"Molecular docking",
"Astragalin",
"Binding affinity"
],
"content": "Introduction\n\nRheumatoid arthritis (RA) is an autoimmune inflammatory disease of synovial joints1. The prevalence of RA is about 1% worldwide and women are three times more likely to be affected than men. The onset of RA is mostly seen in people aged 30–50 but can occur at any age2,3. In the past two decades, research has advanced understanding of RA pathogenesis which has revolutionized RA treatment and led to the development of new therapeutic agents4. Non-steroidal anti-inflammatory drugs (NSAIDs) and disease-modifying anti-rheumatic drugs (DMARDs) are groups of drugs commonly used to control and alter the progression of RA4,5. However, these medications cannot completely cure RA, are expensive and have some notable side effects. Therefore, the development of new therapeutics for RA with fewer side effects is required.\n\nAstragalin (Kaempferol 3-O-beta-D-glucoside) is a flavonoid found in black tea, alcoholic beverages such as red wine and Cuscuta plants. Preliminary studies have shown it has potential pharmacological characteristics such as anti-inflammatory and antioxidant properties6,7. Computational analysis is an important technique for determining the potential of astragalin in inhibiting C-C motif chemokine ligand 28 (CCL28). Virtual screening tools provide an easy way to identify therapeutic targets suitable for multiple ligands8.\n\nCCL28, a mucosa-associated epithelial chemokine, is found in the epithelial cells of many tissues such as the lungs, guts and salivary glands. Studies have reported the involvement of CCL28 in RA angiogenesis, a process that occurs early in RA pathogenesis9. RA angiogenesis is fostered by the interaction of pro-inflammatory cytokines and chemokines. The ligation of CCL28 to its receptor CCR10 is involved in B- and T-cell trafficking in RA pathogenesis10.\n\nThe ability of astragalin to bind and inhibit CCL28 has not yet been reported. Therefore, in this paper we will describe the potential inhibition of the CCL28 target by an astragalin ligand using computational and bioinformatics tools.\n\n\nMethods\n\nThe three-dimensional crystal structure of the target protein CCL28 (PDB ID: 6CWS)11 was downloaded from the Protein Data Bank12 in PDB format using a similar method to that carried out by Esther et al.13\n\nThe two-dimensional chemical structure of astragalin was accessed from the PubChem database (PubChem CID: 5282102) and drawn using Accelrys Draw 4.1 software14 with default parameters. The resulting MOL file of the ligand was converted into PDB format by Accelrys Discovery Studio 4.1 software15 using default parameters. The chemical structure of astragalin is shown in Figure 1. We used naproxen as standard drug which is being used against arthritis. The chemical structure of this molecule was taken from the PubChem database (PubChem CID: 156391).\n\nPDB coordinates of target and ligand proteins were optimized by energy minimization, 3D protonation and stable conformation parameters of Molecular Operating Environment (MOE) software16 (version 2014.0901) and files were saved in MDB format.\n\nThe quality of the CCL28 predicted protein structure was estimated using the Phyre2 (Protein Homology/analogY Recognition Engine, version 2.0) online web server17. The ProQ2 quality assessment and Ramachandran plot was applied for identifying the structural conformation of the proteins using the default parameters of the tool.\n\nWe computed and analysed the binding sites of target protein using the default parameters of the MOE software16.\n\nA computational ligand-target docking approach was used to determine structural complexes of CCL28 with astragalin in order to understand the structural basis of these protein targets specificity. Finally, docking was carried out by MOE based on RMSD and E-score. The energy of the interaction of these derivatives with the protein targets is assigned “grid point”. At each step of the simulation, the energy of the interaction of ligand and protein was evaluated using atomic affinity potentials computed on a grid. Molecular docking was performed to analyse the binding of the astragalin ligand with the active sites of the CCL28 target protein. The optimized ligand was docked with the target using MOE software16 with default parameters as described by Esther et al.13\n\n\nResults\n\nThe quality of the CCL28 predicted protein structure was estimated by the Phyre2 web server17. Phyre2 provided a 3D structure of the target protein and the dimensions of the 3D structure in Å were X=48.506, Y=53.506 and Z=38.552. Analysis of the secondary structure of the target protein provided by Phyre2 revealed high confidence in the predicted structure, indicated by red regions in Figure 2, suggesting that the protein structure quality is high. The sequence profile graph shows that M residues are highly favourable, L and V residues are moderately favourable and remaining residues are less favourable. The ProQ2 quality assessment showed the quality of protein target and could be considered as \"good\" model indicating less mutation sites in the protein structure (Figure 3). The Ramachandran plot shows that approximately 98% amino acid residues of the target protein model exist in the most favoured region whereas <2% exist in the disallowed region. This Ramachandran plot representing the backbone conformation of CCL28 amino acid residues is shown in Figure 4.\n\nThe sequence alignment of protein molecule shows high confidence, indicated by red colour, while blue indicates low confidence. 49% of the structure is in alpha helices, 11% is in beta-strands and 13% is in transmembrane helices.\n\nRed colour indicates “Good” regions while blue represents “Bad” regions of the structure. The quality of model indicated the minimal disordered region (shown by broken lines).\n\nFor a “good” protein model there must be ≥90% amino acid residues in the most favoured region or <2% in the disallowed region of the plot.\n\nBinding sites of the target protein were analysed using MOE software16. Analysis revealed that His 99 and Thr 101 residues were present in a pocket of the target protein. The spatial orientation of the astragalin ligand in the binding pocket of the target is shown in Figure 5. In silico molecular docking using MOE software16 resulted in the successful docking of target CCL28 with the astragalin ligand. The binding energy of astragalin with CCL28 is -5.40 kcal/mol, as compared to standard naproxen which has a binding energy of -4.87 kcal/mol. A comparison of the binding energies and root-mean-square deviation (RMSD) values of astragalin and standard naproxen with the CCL28 target is shown in Table 1 and Table 2 respectively. Results show that astragalin has strong binding affinity for the CCL28 target molecule as compared to naproxen. The ligand interaction module of MOE16 was used to view ligand interactions. Figure 6 shows astragalin interacting with CCL28 via hydrogen bonding with the His 99 and Thr 101 residues.\n\n\nDiscussion\n\nThe CCL28 protein is involved in the angiogenesis of rheumatoid arthritis as it recruits leukocytes to the site of joint inflammation. Inhibition of the CCL28 protein blocks the migration of leukocytes and other proinflammatory factors and this serves as a promising target for arthritis18.\n\nMethotrexate and leflunomide are DMARDs used for rheumatoid arthritis treatment. In a few patients, serious liver damage due to methotrexate has been observed. Similarly, leflunomide also exhibits certain side effects19. These side effects have increased the need for the development of safe alternative, treatment approaches. Current research is focused on the development of bioactive molecules from natural resources which don’t have side effects20. Astragalin isolated from the leaves of black and green tea possesses potential therapeutic effects including anti-inflammatory, antioxidant and anticarcinogenic activities. It may have the ability to inhibit the production of inflammatory mediators such as IL-1b, IL-6 and TNF-alpha19.\n\nAstragalin acts as an inhibitor and prevents the binding of CCL28 to its receptor CCR10 which is a primary step for the recruitment of leukocytes and formation of blood vessels inside inflamed joints of RA patients21.\n\nIn silico docking studies of astragalin with CCL28 were carried out using MOE software16. Lowest binding energy and root mean square values (RMSD) from docking studies indicate a strong interaction between ligand and target. In silico molecular docking showed successful binding of CCL28 with the astragalin ligand. The binding energy of astragalin with CCL28 is -5.40 kcal/mol, a lower binding energy than that of naproxen with CCL28 which is 4.87 kcal/mol. These docking results suggest that astragalin can act as a potential inhibitor for CCL28.\n\n\nConclusion\n\nMolecular docking results of astragalin with CCL28 showed that this ligand has strong binding affinity for CCL28. Therefore, astragalin may act as an important inhibitor of CCL28 to treat rheumatoid arthritis in the future.\n\n\nData availability\n\nStructure of CCL28 from Protein Data Bank, Accession number 6CWS: https://identifiers.org/pdb/6CWS.\n\nStructure of naproxen from PubChem Database, Accession number 156391: https://identifiers.org/pubchem.compound/156391\n\nStructure of astragalin from PubChem Database, Accession number 5282102: https://identifiers.org/pubchem.compound/5282102",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBarhamain AS, Magliah RF, Shaheen MH, et al.: The journey of rheumatoid arthritis patients: a review of reported lag times from the onset of symptoms. Open Access Rheumatol. 2017; 9(1): 139–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrennan-Olsen SL, Cook S, Leech MT, et al.: Prevalence of arthritis according to age, sex and socioeconomic status in six low and middle income countries: analysis of data from the World Health Organization study on global AGEing and adult health (SAGE) Wave 1. BMC Musculoskelet Disord. 2017; 18(1): 271. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRudan I, Sidhu S, Papana A, et al.: Prevalence of rheumatoid arthritis in low- and middle-income countries: A systematic review and analysis. J Glob Health. 2015; 5(1): 010409. PubMed Abstract | Free Full Text\n\nHussain W, Noorwali A, Janoudi N, et al.: From Symptoms to Diagnosis: An Observational Study of the Journey of Rheumatoid Arthritis Patients in Saudi Arabia. Oman Med J. 2016; 31(1): 29–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKahlenberg JM, Fox DA: Advances in the medical treatment of rheumatoid arthritis. Hand Clin. 2011; 27(1): 11–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiaz A, Rasul A, Hussain G, et al.: Astragalin: A Bioactive Phytochemical with Potential Therapeutic Activities. Adv Pharmacol Sci. 2018; 2018(1): 9794625. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCho IH, Choi YJ, Gong JH, et al.: Astragalin inhibits autophagy-associated airway epithelial fibrosis. Respir Res. 2015; 16(1): 51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou W, Cai JF, Yuan F, et al.: In silico targeting of interleukin-6 by natural compounds. Bangladesh J Pharmacol. 2014; 9(3): 371–76. Publisher Full Text\n\nLazarus NH, Kunkel EJ, Johnston B, et al.: A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts. J Immunol. 2003; 170(7): 3799–805. PubMed Abstract | Publisher Full Text\n\nChen Z, Kim SJ, Essani AB, et al.: Characterising the expression and function of CCL28 and its corresponding receptor, CCR10, in RA pathogenesis. Ann Rheum Dis. 2015; 74(10): 1898–906. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomas MA, He J, Peterson FC, et al.: The Solution Structure of CCL28 Reveals Structural Lability that Does Not Constrain Antifungal Activity. J Mol Biol. 2018; 430(18 Pt B): 3266–3282. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerman HM, Westbrook J, Feng Z, et al.: The Protein Data Bank. Nucleic Acids Research. 2000; 28: 235–242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEsther MYJ, Subramaniyan V, Kumar AP, et al.: Molecular docking, ADMET analysis and dynamics approach to potent natural inhibitors against sex hormone binding globulin in male infertility. Pharmacogn J. 2017; 9(6s): 35–43. Publisher Full Text\n\nDassault Systèmes BIOVIA: Accelrys Draw, version 4.1. San Diego: Dassault Systèmes, 2012. Reference Source\n\nDassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016.\n\nMolecular Operating Environment (MOE), 2013.08; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2019.\n\nKelley LA, Mezulis S, Yates CM, et al.: The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6): 845–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzekanecz Z, Koch AE: Successes and failures of chemokine-pathway targeting in rheumatoid arthritis. Nat Rev Rheumatol. 2016; 12(1): 5–13. PubMed Abstract | Publisher Full Text\n\nBartold PM, Marshall RI, Haynes DR: Periodontitis and rheumatoid arthritis: a review. J Periodontol. 2005; 76(11 Suppl): 2066–74. PubMed Abstract | Publisher Full Text\n\nCarvalho CdS, Andrade LEC, Keusseyan SP, et al.: Study of advanced rheumatoid arthritis. Rev Bras Eng Bioméd. 2014; 30(1): 54–63. Publisher Full Text\n\nJia Q, Wang T, Wang X, et al.: Astragalin Suppresses Inflammatory Responses and Bone Destruction in Mice With Collagen-Induced Arthritis and in Human Fibroblast-Like Synoviocytes. Front Pharmacol. 2019; 10(1): 94–06. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "50253",
"date": "22 Jul 2019",
"name": "Ihsan Ul Haq",
"expertise": [
"Reviewer Expertise Pharmaceutical Sciences",
"Biochemistry."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes, the current work is accurately presented including the recent literature. Authors have discussed about the recent updates and potential application of Astragalin. The role of C-C motif chemokine ligand 28 (CCL28) has been cited explaining the recruitment of leukocytes and other proinflammatory factors at the site of joint inflammation and arthritis.\n\nIs the study design appropriate and is the work technically sound? Yes, authors have designed in silico study properly and focused on computational evaluation of astragalin as inhibitor of CCL28 for treatment of rheumatoid arthritis.\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes, the detail has been provided to replicate the steps of study. The quality valuation of CCL28 was estimated and Molecular Operating Environment software was used for binding affinity analysis. Along with astragalin, naproxen was used as standard drug.\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, the source data underlying the results are available.\n\nAre the conclusions drawn adequately supported by the results? Yes, the conclusion is supported by the results revealing that astragalin could have potential to serve as inhibitor of CCL28 for the treatment of rheumatoid arthritis. Binding energy of astragalin with its target is -5.40 kcal/mol is very low in comparison to standard naproxen which has -4.87 kcal/mol binding energy. Docking results proved that astragalin can act as potential inhibitor for CCL28 enzyme.\nHowever, some minor comments are recommended.\nAdd some pharmacological aspects of astragalin in \"Introduction\" section.\n\nBetter to combine Figure 5 and 6.\n\nImprove the resolution of the figures.\n\nProof reading is required.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "78162",
"date": "09 Feb 2021",
"name": "Sagarika Biswas",
"expertise": [
"Reviewer Expertise Proteomics",
"immune proteomics",
"bioinformatics",
"metabolomics",
"arthritis",
"Coronary artery disease"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the Introduction section:\n\nInformation about RA angiogenesis and early RA pathogenesis may be provided.\n\nWhat is the chemical formula of astragalin and what are its biological function?\n\nWhat are the diseases in which the effect of astragalin is already reported?\n\nThe information of other ligands that are reported to be inhibited by astragalin and hence can be potential inhibitors of other inflammatory diseases may also be provided.\n\nIn the Method section:\n\nAuthor used various software such as Accelrys Draw 4.1 software, Accelrys Discovery Studio 4.1 software, Molecular Operating Environment (MOE) software. However, why particularly was this software used? Information is missing. The importance of using each of these softwares used for the present study may be provided.\n\nIn the Results section:\n\nThe statement “Analysis of the secondary structure of the target protein provided by Phyre revealed high confidence in the predicted structure” is unclear. It may be explained elaborately. How did the author arrive for a high confidence state in the predicted structure and what about a low confidence state? What are the criteria of a high confidence state? The analysis of results with respect to the figure may be explained elaborately.\n\nAlso, the analysis ProQ2 quality assessment may be provided. Based on this analysis how the protein quality is decided to be good. Ref for ProQ2 quality assessment may also be provided.\n\nFurther, the in silico molecular docking study using MOE software resulted in the successful docking. What are the criteria of successful docking? How has a successful docking state has been resulted? Also, why has naproxen been used as standard? What is the other standard recommended to be used in general in RA?\n\nThe in-silico validation could have been done for better results.\n\nIn the discussion section:\n\nThe results could have been discussed more elaborately for better impact.\n\nIt is not clear from the study why only one ligand is targeted. Other ligands could have been also studied and comparative analysis could have been provided.\n\nWhy and how astragalin is specifically targeted to inhibit C-C motif chemokine ligand 28 only?\n\nThe conclusion of the study could have been stronger:\nThe ref may be provided for the following lines: “The CCL28 protein is involved in the angiogenesis of rheumatoid arthritis as it recruits leukocytes to the site of joint inflammation”. “In a few patients, serious liver damage due to methotrexate has been observed”. “Astragalin isolated from the leaves of black and green tea ….. activities”. “Methotrexate and leflunomide are DMARDs used for rheumatoid arthritis treatment”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-313
|
https://f1000research.com/articles/7-742/v1
|
14 Jun 18
|
{
"type": "Opinion Article",
"title": "Recommendations for the packaging and containerizing of bioinformatics software",
"authors": [
"Bjorn Gruening",
"Olivier Sallou",
"Pablo Moreno",
"Felipe da Veiga Leprevost",
"Hervé Ménager",
"Dan Søndergaard",
"Hannes Röst",
"Timo Sachsenberg",
"Brian O'Connor",
"Fábio Madeira",
"Victoria Dominguez Del Angel",
"Michael R. Crusoe",
"Susheel Varma",
"Daniel Blankenberg",
"Rafael C. Jimenez",
"BioContainers Community",
"Yasset Perez-Riverol",
"Bjorn Gruening",
"Olivier Sallou",
"Pablo Moreno",
"Felipe da Veiga Leprevost",
"Hervé Ménager",
"Dan Søndergaard",
"Hannes Röst",
"Timo Sachsenberg",
"Brian O'Connor",
"Fábio Madeira",
"Victoria Dominguez Del Angel",
"Michael R. Crusoe",
"Susheel Varma",
"Daniel Blankenberg",
"Rafael C. Jimenez"
],
"abstract": "Software Containers are changing the way scientists and researchers develop, deploy and exchange scientific software. They allow labs of all sizes to easily install bioinformatics software, maintain multiple versions of the same software and combine tools into powerful analysis pipelines. However, containers and software packages should be produced under certain rules and standards in order to be reusable, compatible and easy to integrate into pipelines and analysis workflows. Here, we presented a set of recommendations developed by the BioContainers Community to produce standardized bioinformatics packages and containers. These recommendations provide practical guidelines to make bioinformatics software more discoverable, reusable and transparent. They are aimed to guide developers, organisations, journals and funders to increase the quality and sustainability of research software.",
"keywords": [
"containers and packages",
"best practices bioinformatics",
"reproducibility"
],
"content": "Introduction\n\nThe ability to reproduce the results of a scientific study is a cornerstone of the scientific method, and yet remains one of the most significant challenges in modern science. Evidence from multiple authors suggest that reproducibility in biomedical research is lower than 85%1, with 90% of researchers asserting a reproducibility crisis within science2. One major obstacle to reproducible science is the accurate and complete reporting of all experimental and computational steps required to obtain the described results. The ability to accurately reproduce bioinformatics analysis, including data handling and statistical downstream processing frequently poses significant challenges—even when performed by the original authors of a study3,4.\n\nIn addition, reproducing computational analysis by other researchers requires the deployment of the bioinformatic software at a different site. Previous publications have highlighted the importance of openness and availability of tools, software, scripts and data3,5, and focused on three central premises for reproducible bioinformatics software deployment: (i) documenting the version of all software, (ii) open source availability of the source code and all custom software, (iii) adopting a license and complying with third-party dependency licenses3,6.\n\nHowever, even if source code and data are published in a public repository as Supplementary material to a paper, the source code may have non-obvious dependencies on other software, configuration options, operating systems and other subtleties that hamper re-usability7. Building, installing, and deploying scientific software often requires additional information missing in the published manuscript or the accompanying documentation. Additionally, workflows and pipelines commonly combine software developed by different teams and groups, adding another layer of complexity and introducing challenges such as compatibility and management of dependencies, running serial and parallel processes and working with a broad variety of software types and user-defined parameters. Software containers have emerged as a powerful technology to address primary dependency issues and enable distributing and deploying scientific software in a runnable state8.\n\nSoftware packages are collections of computer programs along with metadata, such as dependencies, descriptions, and versions, required for distribution and deployment. Package management systems are used to search for software, resolve dependencies, and then install the requisite dependencies and software. The most common package managers exist at the level of the operating system, such as yum, apt, and pacman. These allow for the installation of software on a system-wide basis. Containers constitute lightweight software components and libraries that can be quickly packaged, are designed to run anywhere8, and are useful and essential tools to leverage bioinformatics software reproducibility. Package managers are often used to create the execution environment within containers. Conda packages and Docker/Singularity containers are well-known technologies that have already gained traction in the field of bioinformatics9. By May 2018, the BioContainers8, and Bioconda10 communities have released more than 4000 public containers, facilitating the development of complex and reproducible workflows and pipelines11,12.\n\nThis manuscript describes a core set of recommendations and guidelines to improve the quality and sustainability of research software based on software packages and containers. It provides easy-to-implement recommendations that encourage the adoption of packaging (e.g. Conda) and container (e.g. Docker, Singularity) technologies in bioinformatics and software development for research. It provides recommendations about making research software and its source code more reproducible, deployable, reusable, transparent and more compatible with other tools and software. In this manuscript, software is broadly defined to include command line tools, graphical user interfaces, application program interfaces (APIs), infrastructure scripts and software packages (e.g. R packages).\n\n\nRecommendations\n\nA software package is self-contained software including all the dependency libraries and packages necessary to execute the software. Some of the most popular and well-known package management systems are operating system-level, such as apt or yum, are language-specific resources, such as pip/PyPI, CPAN, or CRAN, or are third-party package managers such as Zero Install, Homebrew, and Conda. When choosing a package manager, it is important to select one that is cross-platform (e.g. works on various Linux distributions and MacOS), allows multiple versions of each package, does not require administrative nor elevated privileges to use and, ideally, is not restricted to a single programming language. Being cross-platform enhances reusability, providing for multiple versions enables reproducibility, and allowing user-based installation and multiple development languages simplifies usability.\n\nConda, the most popular package manager in research software, quickly installs, runs and updates packages and their dependencies. It handles dependencies for many languages, such as C, C++, R, Java, Perl, and Python. It works cross-platform and does not require special permissions for installation of itself or requested packages. Conda supports the creation of individual and unique execution environments and allows multiple versions of packages to be installed in a user-declared fashion.\n\nThe field of computational biology has developed an active community around Bioconda10. Bioconda is a channel for the Conda package manager specialised in bioinformatics software. You can create a Conda package by defining a BioConda recipe (Box 1). This recipe includes enough information about the dependencies, the license and fundamental metadata to find, retrieve and use the package. When a recipe is added to Bioconda, it is automatically built into a usable package, tested and made available as a Docker container via Quay.io and as a Singularity container13, and is displayed in the BioContainers registry8.\n\npackage:\n\nname: deeptools\n\nversion: '3.0.2'\n\nsource:\n\nfn: deepTools-3.0.2.tar.gz\n\nurl:\n\nhttps://files.pythonhosted.org/packages/21/63/095615a9338c824dcc1496a302d04267c674175f0081e1ee2f897f33539f/deepTools-3.0.2.tar.gz\n\nmd5: 4553d9c828ba4b5b93ca387917649281\n\nbuild:\n\nnumber: 0\n\nrequirements:\n\nbuild:\n\n- python\n\n- setuptools\n\n- gcc\n\nrun:\n\n- python\n\n- pybigwig >=0.2.3\n\n- numpy >=1.9.0\n\n- scipy >=0.17.0\n\n- matplotlib >=2.1.1\n\n- pysam >=0.14.0\n\n- py2bit >=0.2.0\n\n- plotly >=1.9.0\n\n- pandas\n\ntest:\n\nimports:\n\n- deeptools\n\ncommands:\n\n- bamCompare –version\n\nabout:\n\nhome: https://github.com/fidelram/deepTools\n\nlicense: GPL3\n\nsummary: A set of user-friendly tools for normalisation and visualisation of deep-sequencing data\n\nextra:\n\nidentifiers:\n\n- biotools:deeptools\n\n- doi:10.1093/nar/gkw257\n\nMicroservice and modular architectures14 provide a way of breaking large software projects down into smaller, independent and loosely coupled modules. These software applications can be viewed as a suite of independently deployable, small, modular components in which each tool runs a unique process and communicates through a well-defined, lightweight mechanism to serve a specific task14. Each of these independent modules is referred to as a container. A container is essentially an encapsulated and immutable version of an application, coupled with the bare-minimum operating system components (e.g. dependencies) required for execution8.\n\nContainers should be defined to be as granular as possible, with the premise of one tool, one container. Each container should encapsulate only one piece of software that performs a unique task with a well-defined goal (e.g. sequence aligner, mass spectra identification). This recommendation, one tool, one container, should be implemented carefully, keeping containers as modular and scoped in functionality as possible. Developers may use their judgement to compose a layered container based on other containerised tools. Here, we strongly recommend that the modular composition of these tools should also be exposed as a single modular tool - still abiding by \"one tool, one container\".\n\nThe tool or software wrapped inside the container should be fixed explicitly to a defined version through the mechanism available by the package manager used (Box 2). The version used for this main software should be included in both the metadata of the container (for ease of identification) and the container tag. The tag and metadata of the container should also include a versioning number for the container itself, meaning that the tag could look like a version of the tool or version of the container. The container version, which does not track the tool changes but the container revision, should follow semantic versioning to signal its backward compatibility.\n\nThe metadata contains the license of the software.\n\nFROM biocontainers/biocontainers:v1.0.0_cv4\n\nLABEL base_image=“biocontainers:v1.0.0_cv4”\n\nLABEL version=“3”\n\nLABEL software=“Comet”\n\nLABEL software.version=“2016012”\n\nLABEL about.summary=“an open source tandem mass spectrometry sequence database search tool”\n\nLABEL about.home=http://comet-ms.sourceforge.net\n\nLABEL about.documentation=http://comet-ms.sourceforge.net/parameters/parameters_2016010\n\nLABEL about.license_file=http://comet-ms.sourceforge.net\n\nLABEL about.license=“SPDX:Apache-2.0”\n\nLABEL extra.identifiers.biotools=“comet”\n\nLABEL about.tags=“Proteomics”\n\nLABEL maintainer=“Felipe da Veiga Leprevost <felipe@leprevost.com.br>”\n\nUSER biodocker\n\nRUN ZIP=comet_binaries_2016012.zip && wget https://github.com/BioDocker/software-archive/releases/download/Comet/$ZIP-O/tmp/$ZIP&&unzip/tmp/$ZIP-d/home/biodocker/bin/Comet/&&chmod-R 755/home/biodocker/bin/Comet/*&&rm/tmp/$ZIP\n\nRUN mv/home/biodocker/bin/Comet/comet_binaries_2016012/comet.2016012.linux.exe/home/biodocker/bin/Comet/comet\n\nENV PATH /home/biodocker/bin/Comet:$PATH\n\nWORKDIR /data/\n\nIf a copy is done via git clone or equivalent, a specific commit or a tagged git version should be specified, never a branch only. Cloning a branch (master, develop, etc.) will always use the latest source code in that branch making impossible to reproduce the build process since the different source code will be built as soon as the branch is updated by the software authors. Upstream authors should be asked to create a stable version of their software with reasonable guarantees that the specified version works as advertise including passing all automated tests (Recommendation 7)—this will often be a release version. Any patches added on top of the officially released source code should be highlighted.\n\nFor projects that practice agile software development (including continuous integration) where each version is stable, tested and works as advertised, the SVN or git identifier can be used as the tool version for the container—possibly with the addition of a date in YYYYMMDD format to easily identify newer versions from older versions. Please note that depending on the used source code management system (git, hg, svn, cvs) it is possible to remove entire commits or rewrite the commit history of a project. Therefore, the safest way is to use a release tarball and can be archived separately, as Bioconda and BioContainers are doing.\n\nIt is a well-known feature of Docker that the entry point of the container can be over-written by definition (e.g., ENTRYPOINT [ /bin/ping]). The ENTRYPOINT specifies a command that will always be executed when the container starts. Even when the ENTRYPOINT helps the user to get a default behaviour for a tool, it is generally not recommended because of reproducibility concerns of the implicit hidden execution point. By explicitly executing the tool by its executable inside the container (using the container as an environment and not as a fat binary merely through its ENTRYPOINT) the user (e.g. workflow) can recognise and trace the tool that is used within the container.\n\n4.1. Relevant tools and software should be executable in the PATH. If for some reason the container needs to expose more than a single executable or script (for instance, EMBOSS or OpenMS11 or other packages with many executables), these should always be executable and be available in the container's default PATH. This will, almost always, be the case by default for everything installed via package managers (dpkg, yum, pip, etc.), but if you are adding tailored made scripts or installing by source, take care to add the executables to the PATH. This allows the container to be used as an environment or to specify alternative commands to the main ENTRYPOINT easily (Recommendation 4).\n\nAs containers are frequently pushed and pulled (uploaded and downloaded) to/from container registries over the internet, their size matters. There are multiple ways to reduce the size of your container during builds, the most efficient way is to have two different containers: one for building the app and the second container for deploying the app (which will be the one users will download). While you may need multiple libraries, source code and dependencies for building the app, you should only include the bare necessities in the deployment container, which will actually run the app. Some general guidelines that can be followed are listed below (see Supplementary File 1):\n\nAvoid installing \"recommended\" packages in apt based systems in your deployed container.\n\nDo not keep build tools in the deployed image (this includes compilers and development libraries). You can install these tools in the build image.\n\nUse a lightweight base image for your deployed container, such as Alpine. Only use a more mainstream image such as Ubuntu or CentOS if absolutely required for your application to run.\n\nData can dramatically increase the size of the container (Recommendation 5), thereby reducing the capability to share, deploy and deposit it in public registries. In order to implement tests during the building and deployment steps, we recommend downloading or cloning the data from public data repositories and deleting it after the testing is finished. This mechanism is similar to the one stated in Recommendation 5 for retrieving source and binaries.\n\nMany bioinformatics tools require access to large reference datasets to perform meaningful analysis. These reference datasets should also not be included within the container but should be stored in a user-configurable location and retrieved either on-demand during runtime, or as part of a setup process. Not only does storing reference datasets outside of the container reduce the size of the container, but other tools that require access to the same reference data will be able to directly access the data without additional overhead. It is also recommended that datasets themselves are versioned and all downloaded files are verified using secure cryptographic hashes.\n\nIf others want to build your container locally, want to rebuild it later on with an updated base image, want to integrate it to a continuous integration system or for many other reasons, users might want to test that the built container still serves the function for which it was initially intended. For this, it is useful to add some functional testing logic to the container (in the form of a bash script for instance) in a standard location (here we propose a file called “runTest.sh”, executable and in the path), which includes all the logic for:\n\nInstalling any packages that might be needed for testing, such as wget for instance to retrieve example files for the run.\n\nObtaining sample files for testing, which might be for instance an example data set from a reference archive.\n\nRunning the software that the container wraps with that data to produce an output inside the container.\n\nComparing the generated output and exit with an error code if the comparison is not successful.\n\nThe file containing testing logic is not meant to be executed during container build time, so the retrieved data and packages do not increase the size of the container when it is built. However, because the testing file is inside the container, any user who has built the container or downloaded the container image can check that the container is working as intended by the author by executing “runTest.sh” inside the container.\n\nWhen adding software or data in a container, always check the license of the resource being added. A free-to-use license is not always free to distribute or copy. The license must always be explicitly defined in your Docker labels. For some licenses, the license file needs to be shipped within the container and with the software. If a license is not specified, you should ask the upstream author to provide a license5,15.\n\nBiomedical research and bioinformatics demands more efforts to make bioinformatics software and data more findable (discoverable), accessible, interoperable, and reusable (FAIR principles)16. Leveraging those principles, we recommend to the bioinformatics community and software developers to make their containers and packages more findable. To make your package available, we recommend the following steps:\n\nAnnotate packages and containers with metadata that allows users (e.g. biologists and bioinformaticians) to find them.\n\nMake packages and containers available. We recommend developers to make the recipe of how to build a container available for others, including i) the source code or binaries of the original tools; ii) the configuration settings and test data.\n\nRegister packages and container in existing bioinformatics registries helping users and services to find them.\n\nRegistries such as BioContainers8 and bio.tools17 collaborate with each other by exchanging metadata and information using different APIs and a common identifier system.\n\nDeposit the built container image in a public container registry, such as Docker Hub, Quay.io or a publicly available and well supported institutional registry for container images.\n\nWhile containers strive to make research reproducible and transparent, it is equally essential that the process of creating and building containers themselves is transparent and reproducible. Many Docker containers do not provide an associated Dockerfile, which would allow an independent party to reproduce and verify the container build independently. Other build procedures rely on the presence of specific web resources, download binary files from the internet or can only be built with in-house resources that are not available to the public.\n\nWith BioConda and BioContainers every recipe is available and the mechanisms to create and build it. The Biocontainers registry provides a view to each recipe. Our recommendation is to provide clear documented steps on how to generate all the binaries directly from the source code; if it is possible, engage with one of these two open-source communities to make your recipe available. Adding documentation to BioContainer and Conda recipes will allow the author as well as users to understand the build process and modify it their needs. If a particular resource may not be readily available or consists of a binary file, provide further instructions on how to re-create this resource (e.g. a link to a second recipe that creates the resource).\n\nUsability and discoverability are crucial for packaged containers. If your tool provides a help “-h”, “--help” or “?” message, consider providing this as the default command, “CMD” in case of a Dockerfile. If your tool does not provide a default usage message, consider providing this information in an ancillary “README.md” message. Your tool’s help or usage message is a useful place to provide a list of commands in logical groups, along with each command, giving a brief description, defaults, required arguments, and options.\n\n\nConclusions\n\nThis manuscript promotes and encourages the adoption of package and container technologies to improve the quality and reusability of research software. The recommendations share a set of core views that are summarised below:\n\nSimplicity: the encapsulated software should not be a complex environment of dependencies, tools and scripts.\n\nMaintainability: the more software included in the container, the harder it is to maintain it, especially when the software comes from different sources.\n\nSustainability: the developers of the software should be engaged or made aware of supporting the sustainability of the container.\n\nReusability: a tool container should be safe to reuse by any other workflow component or task through its access interface.\n\nInteroperability: different tools should be easy to connect and exchange information.\n\nUser’s acceptability: a tool container should perform a specific atomic task, so it is easier to check and use.\n\nSize: containers should be as small as possible. Smaller containers are much quicker to download and therefore they can be distributed to different machines much quicker.\n\nTransparency: containers should be transparent in how they are built, which tasks they are designed to perform and how the build process can be reproduced.\n\nAs with many tools, a learning curve lays ahead, but several basic yet powerful features are accessible even to the beginner and may be applied to many different use-cases. For users involved in scientific research and bioinformatics interested in this topic without experience working with software packages or containers, we recommend exploration and engagement with the BioContainers initiative.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was partially supported by ELIXIR-EXCELERATE. ELIXIR-EXCELERATE is funded by the European Commission within the Research Infrastructures programme of Horizon 2020, grant agreement numbers 676559. The BioContainers workshop (Paris 2017) and the BioContainers community that developed these recommendations are supported by the ELIXIR Tools platform.\n\n\nSupplementary material\n\nSupplementary File 1. Additional guidelines to make your container smaller.\n\nClick here to access the data.\n\n\nReferences\n\nMacleod MR, Michie S, Roberts I, et al.: Biomedical research: increasing value, reducing waste. Lancet. 2014; 383(9912): 101–4. PubMed Abstract | Publisher Full Text\n\nBaker M: 1,500 scientists lift the lid on reproducibility. Nature. 2016; 533(7604): 452–4. PubMed Abstract | Publisher Full Text\n\nSandve GK, Nekrutenko A, Taylor J, et al.: Ten simple rules for reproducible computational research. PLoS Comput Biol. 2013; 9(10): e1003285. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrüning B, Chilton J, Köster J, et al.: The backbone of research reproducibility-sustainable and flexible tool deployment. F1000Res. In Bioinformatics Open Source Conference 2017; 2017. Publisher Full Text\n\nPerez-Riverol Y, Gatto L, Wang R, et al.: Ten Simple Rules for Taking Advantage of Git and GitHub. PLoS Comput Biol. 2016; 12(7): e1004947. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiménez RC, Kuzak M, Alhamdoosh M, et al.: Four simple recommendations to encourage best practices in research software [version 1; referees: 3 approved]. F1000Res. 2017; 6: pii: ELIXIR-876. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoettiger C: An introduction to Docker for reproducible research. ACM SIGOPS Operating Systems Review. 2015; 49(1): 71–9. Publisher Full Text\n\nda Veiga Leprevost F, Grüning BA, Alves Aflitos S, et al.: BioContainers: an open-source and community-driven framework for software standardization. Bioinformatics. 2017; 33(16): 2580–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNekrutenko A; Team G, Goecks J, et al.: Biology Needs Evolutionary Software Tools: Let's Build Them Right. Mol Biol Evol. 2018; 35(6): 1372–5. PubMed Abstract | Publisher Full Text\n\nGrüning B, Dale R, Sjödin A, et al.: Bioconda: A sustainable and comprehensive software distribution for the life sciences. bioRxiv. 2017; 207092. Publisher Full Text\n\nPfeuffer J, Sachsenberg T, Alka O, et al.: OpenMS - A platform for reproducible analysis of mass spectrometry data. J Biotechnol. 2017; 261: 142–8. PubMed Abstract | Publisher Full Text\n\nAfgan E, Baker D, Batut B, et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018. PubMed Abstract | Publisher Full Text\n\nKurtzer GM, Sochat V, Bauer MW: Singularity: Scientific containers for mobility of compute. PLoS One. 2017; 12(5): e0177459. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalalaie A, Heydarnoori A, Jamshidi P: Microservices architecture enables devops: Migration to a cloud-native architecture. IEEE Software. 2016; 33(3): 42–52. Publisher Full Text\n\nLeprevost Fda V, Barbosa VC, Francisco EL, et al.: On best practices in the development of bioinformatics software. Front Genet. 2014; 5: 199. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTyler J, Frith GH: Primary drug abuse among women: a national study. Drug Alcohol Depend. 1981; 8(4): 279–86. PubMed Abstract | Publisher Full Text\n\nIson J, Rapacki K, Ménager H, et al.: Tools and data services registry: a community effort to document bioinformatics resources. Nucleic Acids Res. 2016; 44(D1): D38–47. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "35062",
"date": "16 Jul 2018",
"name": "Ka Yee Yeung",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors addressed the reproducibility issue in bioinformatics research. While many factors can be attributed to this issue, the paper focused on the tools and software packages. With more research being done and more tools in Bioinformatics made available as packages and complex workflows, this work is a good reminder of the importance of writing well-written products that the community can use and replicate.\nThe manuscript provided sound advice in building and maintaining these tools. It also presented easy to follow sample and template of the solutions. The guidelines are itemized with a clear explanation of the necessity of the rules. Not only for bioinformatics community, but the guidelines are also general enough to serve as good practices for general use in writing software packages. Recommendations include choosing a cross-platform package manager, encapsulating a single tool in each container, reducing the package size, keeping data outside the container, providing testing logic, checking licensing issues, and providing usage messages.\nAs a demonstration, the authors scoped the samples and templates to BioConda and BioContainer, with claims that these software packages are the most popular among bioinformatics community. The reviewers would like to request a reference for the statement “Conda, the most popular package manager in research software”. The example is concise and easy to follow. However, the sample does not strictly reflect what the guidelines proposed in the manuscript. For instance, the manuscript recommended the readers to use Alpine for the base image for Docker containers, the example template in “Box 2” used another pre-built Docker Image instead. The reviewers would like to request the authors to explain or revise the example.\nAdditionally, the manuscript suggested to break down each package or containers as an atomic task to enhance modularity in Guideline #2. However, the authors did not discuss the crucial issue of how to assemble these different packages into a single workflow. Especially, as the authors also noted, bioinformatics workflows often consist of complex pipelines. Coverage on how parameters should be organized would also be useful (e.g., using environment variables versus a text file or command line parameters).\nIn addition, recommendation #4 (avoid entry point) assumed that the tools would always be command-line based; this may not always be the case. Please elaborate when handling GUI based containerized tools.\nLastly, we failed to replicate the provided sample script for Comet software with error `Syntax error - can't find = in \"open\". Must be of the form: name=value` on Docker for Mac: Version 18.03.1-ce-mac65 (24312). Please double check.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "4244",
"date": "20 Nov 2018",
"name": "olivier sallou",
"role": "Author Response",
"response": "Hi, regarding cCnda reference as \"the most popular\", I agree the should be rephrased as \"a popular\" Regarding Alpine base image, you suppose you refer to \"Use a lightweight base image for your deployed container, such as Alpine. Only use a more mainstream image such as Ubuntu or CentOS if absolutely required for your application to run.\" Biocontainers (Dockerfile based) makes indeed use of an other image (Ubuntu based) to allow user to exec bash/vim/... operations in containers and easily extend them. If not needed (oout of biocontainers scope), Alpine is recommended to limit container size and scope. This sentence should be rephrased to explain this difference. Workflow composition is not addressed in the scope of this article as we are tied to the one container = one tool paradigm (though not being too strict on this...) Workflow tools (nextflow, cwl based or others) will execute 1 task per tool, each tool being one container. But maybe that, indeed, it would be worth to explain users the such tools are needed for workflow execution in containers context. Recommendation #4 about entry point still applies for GUI based tools. A GUI tool is just a cmd line tool that \"pops up\" a frontend. Only difference is the need to mount some x11 directorties with hosts for Docker but Docker usage/tutorial is out of the scope of this article. About Comet example, there are some quotes errors in text (text editor related) and a few missing space. We will fix this, thanks for pointing to that error"
}
]
},
{
"id": "36273",
"date": "02 Aug 2018",
"name": "Monther Alhamdoosh",
"expertise": [
"Reviewer Expertise Bioinformatics",
"Translational Bioinformatics",
"Drug Discovery",
"Biomarkers Discovery",
"Research Software Development"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGruening et al. addressed a very important issue in the field of bioinformatics, which is the reproducibility of research analytics. The authors clearly highlighted the importance of software packaging in containers in order to enable fast deployment and building of bioinformatics workflows and analytics. Namely, they focused on the importance of documenting software versions, the availability of source codes and the need to adopt a license when using third-party software. Extending the definition of a software package to include command line tools, GUIs, APIs, scripts and specialized software packages is a very good idea although I have some reservations (see below). The authors proposed eleven recommendations to improve software packaging and containerization in bioinformatics. They also present some of the best practices as part of their recommendations.\n\nI have a few comments on this manuscript that authors would probably like to address in their revised version.\n\nMajor comments ----------------------\nIt would be helpful if you could provide a figure on how BioContainers, BioConda and Docker talk to each other in light of these recommendations. Probably, this figure from your GitHub can be adopted https://github.com/BioContainers/specs/blob/master/imgs//workflow.png\n\nIt would be helpful to show an example of how to make use of these packaged and containerized objects.\n\nSome weblinks need to be fixed on the BioContainers website, e.g., http://biocontainers.pro/docs/developer-manual/deploy-dockerhub/, to enable new users to adopt these recommendations.\n\nMinor comments ----------------------\nBioconductor R packages usually released in versions following milestone releases of Bioconductor. Why are they distributed in separate containers? How do you make sure that people will not use different versions from different releases?\n\nThe use of the words \"computational biology\" and \"bioinformatics\" interchangeably is confusing. I would recommend using one of these two words.\n\nThe purpose of the first recommendation is not clear. It might need to be pronounced further.\n\nThe concepts of the metadata and tag are not explained clearly. This might make it difficult for people not familiar with containers to understand Recommendation 3.\n\nOverall, the manuscript introduces a great way of standardising bioinformatics research and empowering reproducibility of data analytics in the biomedical field.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "4243",
"date": "20 Nov 2018",
"name": "olivier sallou",
"role": "Author Response",
"response": "Hi, thanks for your comments. Regarding usage examples, we tried to avoid explaining Docker concepts as it does not focus on Docker only. Indeed containers will work with any container image compliant image (rkt, singularity), and would certainly need further explanations on what are Docker volumes etc.... We expect user to understand the basis of container usage. Regarding web links, you're right, some examples in documentation need to be fixed About R packages, they could be in a single container, but this would create a huge container to manipulate and difficult to maintain (and which packages should be put?). If user really needs a container with multiple R packages, he can create one based on an existing container and add what is necessary. We focus on programs (R for example) and not libraries (r-xxx lib) as it is not possible to provide a container matching all user needs. Some libraries are provided either with automatic package injection from other sources or for the need of a few users. If a user need a workflow/composition of multiple *tools*, user should use scripting of container enabled workflow tools that will execute one task = one tool = one container, and avoid putting all workflow tool in a single container."
}
]
}
] | 1
|
https://f1000research.com/articles/7-742
|
https://f1000research.com/articles/7-1839/v1
|
22 Nov 18
|
{
"type": "Research Article",
"title": "Phytochemical, antioxidant and antimicrobial properties of Litsea angulata extracts",
"authors": [
"Harlinda Kuspradini",
"Indah Wulandari",
"Agmi Sinta Putri",
"Sabeti Yulis Tiya",
"Irawan Wijaya Kusuma",
"Indah Wulandari",
"Agmi Sinta Putri",
"Sabeti Yulis Tiya",
"Irawan Wijaya Kusuma"
],
"abstract": "Background: Litsea angulata is a plant species belonging to Lauraceae family that is distributed throughout Indonesia, Malaysia, and New Guinea. The seeds have been traditionally used by local people in Kalimantan, Indonesia for the treatment of boils; however, there is no information about the potency of its branch, bark and leaves yet. This study aimed to determine the antioxidant, antimicrobial activity as well as the phytochemical constituent of Litsea angulata branch, bark, and leaves. Methods: Extraction was performed by successive maceration method using n-hexane, ethyl acetate, and ethanol solvent. Antioxidant activity was evaluated by DPPH radical scavenging assay. The antimicrobial activity using the 96 well-plate microdilution broth method against Staphylococcus aureus and Streptococcus mutans. Results: Based on the phytochemical analysis, it showed that extract of L. angulata contains alkaloids, flavonoids, tannins, terpenoids, and coumarin. The results showed that all extracts of plant samples displayed the ability to inhibit DPPH free radical formation and all tested microorganisms. Conclusions: L. angulata contains secondary metabolites such as alkaloids, flavonoids, tannins, terpenoids, carotenoids, and coumarin. The antioxidant activity on different plant extracts was a range as very strong to weak capacity. All extracts in this study could inhibit the growth of S. aureus and S. mutans.",
"keywords": [
"Litsea angulata",
"maceration",
"phytochemical",
"antioxidant",
"antimicrobial"
],
"content": "Introduction\n\nMany plants species from the genus Litsea are a potential source of biologically active compounds and are used as traditional medicines such as antispasmodic, wound healing, relieving rheumatism and cold1,2. There is little information about the potency of Litsea angulata species. L. angulata, belonging to the Lauraceae family, which can be found in East Kalimantan, Indonesia, and to our knowledge, data are limited on its biological activities. Therefore the present study aimed to assess the phytochemical constituents, antioxidant, and antimicrobial of different plant part of L. angulata.\n\n\nMethods\n\nThe plant material was obtained from Education Forest Laboratory of Forestry Faculty, Mulawarman University, East Kalimantan, Indonesia. Three different plant parts of L. angulata (bark, branch, and leaves) were separated, ground and extracted. The successive maceration extraction method was adopted from Sruthi and Indira3, with the solvents modified. About 50 g of the air-dried powder of each plant material was extracted individually with one of the following solvents: n-hexane, ethyl acetate, and 96% ethanol. The extracts were filtered and concentrated under vacuum using a rotary evaporator until the solvent was completely evaporated. In total, nine different extracts were produced, with each solvent used to produce extract from each plant part (branch, n-hexane; branch, ethyl acetate; branch, ethanol; bark, n-hexane; bark, ethyl acetate; bark, ethanol; leaves, n-hexane; leaves, ethyl acetate, and leaves, ethanol extracts).\n\nA total of 60 mg of each extract were dissolved individually in 1 ml solvent that used for extraction and the solutions were used to test for qualitative phytochemical tests. The tests were done according to the standard procedures described into literature by Kokate4, Senthilmurugan5, Harborne6 to detect the following bioactive compounds: alkaloids, flavonoids, saponins, tannins, terpenoids, steroids, carotenoids, and coumarin.\n\nThe DPPH assay was performed as described by Kuspradini et al.7. Various concentrations of samples of each extract (12.5, 25, 50 and 100 ppm) in 96% ethanol were added with DPPH. After 20 minutes, the absorbance of the resulting solution and the blank were recorded. Ascorbic acid was used as a positive control. The absorbance was recorded spectrophotometrically at a wavelength of 517 nm. DPPH free radical scavenging activity was stated as % inhibition = (1 – Absorbance of sample/Absorbance of control) × 100. To inhibitory activity, half-maximal inhibitory concentration (IC50) values were calculated.\n\nThe minimum inhibitory concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the samples were assessed against Staphylococcus aureus and Streptococcus mutans using the 96-well microdilution and solid medium, respectively. The bacterial concentration in the inoculum was standardized at 0.5 McFarland turbidity scale, equivalent to 108 CFU ml–1. The method of MIC was adopted from the method outlined by Mohsenipour and Hassanshahian, with modifications8. A stock solution was prepared by dissolving 5 mg extracts in 1 ml of 40% ethanol. A total of 50 µl stock solution were serially diluted twofold in 40% ethanol to achieve the range of test concentrations (1250, 625, 312.5 and 156.25 ppm), which were added to wells of a 96-well microplate. Next, 100 µl sterile nutrient broth culture medium (NB) and 50 µl of the culture of the respective organism were added into each well. The inoculated microplates were incubated at 37°C for 24 h.\n\nAt 1 hour before the end of incubation, the bacterial growth was confirmed by adding 0.01% solution of 2,3,5-triphenyl tetrazolium chloride (TTC, Merck, Germany) (50 µl) and the plate was incubated for another hour. The viable bacterial cells reduced the yellow TTC to pink. The inhibition of growth was visually detected when the solution in the well remained clear after incubation with TTC. Positive controls (bacteria + NB + chloramphenicol), negative controls (bacteria and NB), vehicle controls (bacteria + NB + solvent), and media controls (NB) were included in each test. MBC was determined by inoculating the assay from the wells showing no microbial growth onto the surface of nutrient agar medium on the petri dish. The petri dishes were incubated for 24 h at 37°C and subjected to visual inspection. MBC was considered as the lowest concentration where there was no resumption of bacterial growth.\n\nAll experiments were conducted three times. Regression analysis was used to calculate IC50 values of antioxidant. All statistical analyses used Microsoft Excel 2010 software.\n\n\nResults\n\nThe result of phytochemical screening showed that L. angulata contain alkaloids, flavonoids, tannins, terpenoids, carotenoids and coumarin (Table 1). It can be shown that the ethanolic extract of the L. angulata showed more number of secondary metabolites when compared with other extracts.\n\nAll extracts could inhibit DPPH radical scavenging activity (Table 2). The IC50 values with regards to different used solvents and plant parts were, in increasing order, as follows: leaves, ethanol; bark, ethanol; branch, ethanol; branch, ethyl acetate; bark, ethyl acetate; branch, n hexane; leaves and bark, n hexane. Raw absorbance data from which IC50 values were calculated are shown in Dataset 19.\n\nAll extracts could inhibit the growth of S. mutans and S. aureus and showed the MIC value at 156.25 ppm concentration (Table 3). The MBC value could not detect in the range of 156.25–1250 ppm concentration. It is indicated that the MBC value in this study was higher than 1250 ppm.\n\n\nDiscussion\n\nPlant extracts have been reported to have numerous biological activities due to their phytochemical contents, which contribute significantly towards the antioxidant and antimicrobial activities such as flavonoids, tannins, and terpenoids10–12. The solubility or insolubility of the active compound(s) in the solvent used for extraction can caused the differential effects on antioxidant and antibacterial13,14. According to Blois15 sample which had an IC50 value more than 150 ppm was a weak antioxidant, 101–150 ppm was a medium antioxidant, 50–100 ppm indicated as a strong antioxidant, while lower than 50 ppm was a very strong antioxidant. Antimicrobials are considered as bactericidal if the MBC is not more than four times higher than the MIC, and MBC value is always equal or higher than MIC16.\n\n\nConclusions\n\nL. angulata can be used as a source of natural antioxidant to prevent damage associated with DPPH free radicals and antibacterial to inhibit the growth of S. mutans and S. aureus bacteria.\n\n\nData availability\n\nDataset 1. Raw data associated with this study. Data include the absorbance values obtained from the DPPH scavenging assay, and the resultant IC50 values generated. DOI: https://doi.org/10.5256/f1000research.16620.d2236779.",
"appendix": "Grant information\n\nIslamic Development Banking Grant and Ministry of Research, Technology, and Higher Education of the Republic of Indonesia (grant number: 448/UN.17.45/DL/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\nZhang SY, Guo Q, Gao XL, et al.: [A phytochemical and pharmacological advance on medicinal plant Litsea cubeba (Lauraceae)]. Zhongguo Zhong Yao Za Zhi. 2014; 39(5): 769–76. PubMed Abstract\n\nRobinson CB, Haque T, Uddin MZ, et al.: Propagation, antibacterial activity and phytochemical profiles of Litsea glutinosa (Lour.). Dhaka Univ J Biol Sci. 2014; 23(2): 165–171. Publisher Full Text\n\nSruthi DR, Indira G: A comparative evaluation of maceration, soxhlation and ultra sound assisted extraction for the phytochemical screening of the leaves of Nephelium lappaceum. L. (Sapindaceae). J Pharmacogn Phytochem. 2016; 5(5): 386–389. Reference Source\n\nKokate CK: Pharmacognosy 16th Edn. (Mumbai India : Niali Prakasham). 2001.\n\nSenthilmurugan GB, Vasanthe B, Suresh K: Screening and antibacterial activity analysis of some important medicinal plants. International Journal of Innovation and Applied Studies. 2013; 2(2): 146–152. Reference Source\n\nHarborne JB: Phytochemical method: modern method guidelines of plants analysis (Bandung: ITB) [Indonesia]. 1987.\n\nKuspradini H, Rosiarto AM, Putri AS, et al.: Antioxidant and toxicity properties of anthocyanin extracted from red flower of four tropical shrubs. Nusantara Bioscience. 2016; 8(2): 135–140. Publisher Full Text\n\nMohsenipour Z, Hassanshahian M: Antibacterial Activity of Euphorbia hebecarpa Alcoholic Extracts Against Six Human Pathogenic Bacteria in Planktonic and Biofilm Forms. Jundishapur J Microbiol. 2016; 9(6): e34701. PubMed Abstract | Free Full Text\n\nKuspradini H, Wulandari I, Putri AS, et al.: Dataset 1 in: Phytochemical, antioxidant and antimicrobial properties of Litsea angulata extracts. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16620.d223677\n\nLay MM, Karsani SA, Mohajer S, et al.: Phytochemical constituents, nutritional values, phenolics, flavonols, flavonoids, antioxidant and cytotoxicity studies on Phaleria macrocarpa (Scheff.) Boerl fruits. BMC Complement Altern Med. 2014; 14: 152. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOliver CC, Blumberg J: Are there age related changes in flavonoid bioavailability? Phytochemicals aging and health. New York: Taylor Francis Group; 2008.\n\nRivière C, Hong VN, Pieters L, et al.: Polyphenols isolated from antiradical extracts of Mallotus metcalfianus. Phytochemistry. 2009; 70(1): 86–94. PubMed Abstract | Publisher Full Text\n\nLinthoingambi W, Mutum SS: Antimicrobial activities of different solvent extracts of Tithonia diversifolia (Hemsely) A. Gray. Asian J Plant Sci Res. 2013; 3(5): 50–54. Reference Source\n\nWidyawati PS, Budianta TDW, Kusuma FA, et al.: Difference of Solvent Polarity To Phytochemical Content and Antioxidant Activity of Pluchea indicia Less Leaves Extracts. International Journal of Pharmacognosy and Phytochemical Research. 2014; 6(4): 850–855. Reference Source\n\nBlois MS: Antioxidant determination by the use of stable free radicals. Nature. 1958; 181(4617): 1199–2000. Publisher Full Text\n\nLevison ME: Pharmacodynamics of antimicrobial drugs. Infect Dis Clin North Am. 2004; 18(3): 451–465, vii. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "41787",
"date": "07 Jan 2019",
"name": "Mariateresa Cristani",
"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\nI like the concept of this manuscript, and it could be interesting, but I think it needs some additional work. The only DPPH test is not enough to demonstrate the antioxidant activity and it is a easy test. Phytochemical screening is already known so it would be interesting at least HPLC analysis. The discussion could be a bit deeper explaining the benefits of extracts and comparing better which extracts is more useful and why. It would have been interesting to have information on the pedoclimatic characteristic of the place where the plant are harvested. The description of experimental part is for the most part good and clear. The written English needs to be better. The following reference which appeared recently in Natural Products Research, 20181 should be added.\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": "41035",
"date": "15 Jan 2019",
"name": "Morina Adfa",
"expertise": [
"Reviewer Expertise Natural product chemistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nManuscripts entitled Phytochemicals, antioxidants and antimicrobial properties Litsea angulata extract has been written well. Revise in the keyword Litsea angulata in italic format, add your information where the species (Litsea angulata) was determined. Please make sure the alkaloids contained in n-hexane extract because positive false in phytochemical tested are often reported. Authors wrote that all statistical analyses used Microsoft Excel 2010 software, but in the results and discussion, we cannot find the data with statistical analysis. The authors may add the data with statistical analysis or they can explain in the discussion because in the method they used the statistical analysis for data analysis. The authors may add the data with statistical analysis or they can explain in the discussion because in the method they used the statistical analysis for data analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "43839",
"date": "31 Jan 2019",
"name": "Sarifah Nurjanah",
"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 an interesting manuscript containing new information on the potential of Litsea angulata. The paper is well written and provides valuable data. However, there are some suggestions to improve.\n1. Introduction:\nNot only include the reason for exploring the potential of Litsea angulate, but also why should it be seen from each part of the material (bark, branch and leaves), is there any previous research that each part of the plant has different active ingredients? It should also be written the reasons for using Staphylococcus aureus and Streptococcus mutans.\n2. Results:\nAn increase of IC50 value in antioxidant activity written in succession leaves, ethanol; bark, ethanol; ethanol branch; branch, ethyl acetate; bark, ethyl acetate; branch, n hexane; leaves and bark, n hexane; it should be bark, ethyl acetate; leaves, ethanol; bark, ethanol; ethanol branch; bark, ethyl acetate; branch, ethyl acetate; bark, n hexane; leaves ethyl acetate; branch and leaves n hexane. In the written method there are several controls used, namely positive control, negative control, vehicle control and control machines, but in the results only positive controls are written, what are the other controls?\n3. Discussion:\nNeed clarification as to why ethanol can extract more active components such as tannin, carotenoid and coumarin compared to n hexane and ethyl acetate. Is there a relationship between antioxidant properties and antibacterial with component ingredients (phytochemical assessment results).\n4. Conclusion:\nIn conclusion section, only conclude about antioxidant activity and antibacterial activity. It should be mentioned also the conclusion of phytochemical assessment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-1839
|
https://f1000research.com/articles/8-306/v1
|
19 Mar 19
|
{
"type": "Research Article",
"title": "Usefulness of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio as a predictor of disease-free survival in breast cancer: A cross-sectional study",
"authors": [
"Yaala S. Al-Bairmany",
"Adil S. Aqabi",
"Farah H. Al-Hasnawi",
"Alaa S. Al-Aawad",
"Adil S. Aqabi",
"Farah H. Al-Hasnawi",
"Alaa S. Al-Aawad"
],
"abstract": "Background: The relationship between neutrophil-lymphocyte ratio (NLR) with outcome is a complex issue. A high NLR reflects systemic inflammation. This study aimed to estimate the relationship between NLR, and platelet-lymphocyte ratio (PLR) in disease-free survival (DFS). Methods: This was a cross-sectional study in which we reviewed the patient files of 102 patients with breast cancer treated at the Babylon Oncology Center from January 2009 to September 2014, who had follow-up for at least 36 months. The following data were collected from patient files: age, diagnosis date, date of recurrence and/or metastasis, follow-up, histological tumor type, tumor size, node metastasis stage, histological differentiation degree, estrogen and/or progesterone receptor expression, HER2 neu status, and metastasis site. Results: The mean age of patients was 50.4 ± 11.7 years and lowest period of follow up was 40 months. Longest DFS was 62 months, with 5 years DFS in 52.5% of patients. Stage N0 was associated with a significantly higher DFS compared to stage N1. Isolated local recurrence was seen in 15% of patients and combined local recurrences with distant metastasis was observed 37%. NLR had the highest discrimination ability to predict recurrence and distant metastasis. Conclusion: An increase in NLR was associated with poor DFS, and it can therefore be a predictive and prognostic factor. NLR’s established prediction model warrants further investigation.",
"keywords": [
"Disease Free Survival",
"Neutrophil-lymphocyte ratio",
"Platelet lymphocyte ratio",
"Breast cancer"
],
"content": "Introduction\n\nWorldwide, breast cancer is the most diagnosed cancer type among women1. Animal models suggest “immune-editing” in which activation of immune mechanisms control the tumor, but over time lead to the selection of tumor cells that escape the immune pressure and grow progressively2. Most tumor antigens identify as non-mutated self-antigens. Tumors are heterogeneous, and the antigens on cells of one tumor are variable, even within the same patient, so the down-regulation of major histocompatibility complex molecules and other components of the antigen-presentation process can occur1. Tumors also do not express the ligands recognized by innate immune cells that microbes express or the co-stimulatory ligands necessary to stimulate adaptive T cells2. The expression of Fas ligand by some tumor cells help to maintain a state of immune privilege that induce apoptosis. Tumor cells lead to the release of many cytokines and soluble factors, such as prostaglandin E2, that are not conducive to antitumor immunity. Cancer-associated factors have been shown to inhibit the production and stimulatory capacity of tumor cells3. T-helper cell responses skewed toward a Th2 phenotype, lead to inhibition of the Th1 response and cellular immunity that mediates tumor rejection4.\n\nThe evidence of the relationship between inflammation and cancers prognosis has increased in past years, especially gastrointestinal tumors and non-small-cell lung cancers, and even breast cancers. The systemic inflammatory responses may mimic biochemical or hematological markers, such as raised C-reactive protein and the elevation of white blood cells, neutrophils, and platelets. Elevation of neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) in breast cancer patients is not well studied in Iraq. This relationship is a complex and multifactorial process that is still poorly understood, but a high NLR may reflect systemic inflammation in enhancing angiogenesis, tumor growth and development of metastasis. Therefore, this study aimed to estimate the relationship between NLR, PLR and disease-free survival (DFS) in breast cancer patients in Iraq.\n\n\nMethods\n\nThis is a cross-sectional retrospective study. Breast cancer patients who were treated at Babylon Oncology Center, Baghdad, Iraq from January 2009 to September 2014 were included in this study. The data was collected between May 2017 and April 2018.\n\nInclusion criteria: Tumor stage up to stage IIA (T1-T2, N0/ T1, N1); follow-up for at least 36 months; availability of pretreatment complete blood count (CBC) with differential count.\n\nExclusion criteria: Locally advanced and metastatic breast cancer (T3-T4, N1-N3, M1); neoadjuvant treated patients; patients with data lacking for follow-up and pretreatment CBC with differential count; known causes of neutrophilia (those who already have this disease such as infections, inflammation, burns, heart attack, and drugs, e.g. steroids).\n\nThe following data were collected from patient files: age, diagnosis date, date of recurrence and/or metastasis, follow-up, histological tumor type, tumor size, node metastasis stage, histological differentiation degree, estrogen and/or progesterone receptor expression, HER2 neu status, and metastasis site.\n\nThe statistical analysis used the following tests: Anderson darling test, a statistical test of whether a given sample of data is drawn from a given probability distribution; Kaplan–Meier analysis, a non-parametric statistic used to estimate the survival function from lifetime data, which was used to assess DFS; hazard ratio (HR), the ratio of the hazard rates corresponding to the conditions described by two levels of an explanatory variable; ROC curve, a performance measurement for classification of problems at various thresholds settings; sensitivity analysis for test quality; and specificity analysis for test extension. SPSS 20.0 software package was used to for statistical analysis. P-value of <0.05 was considered significant. Patients who had missing data for variables were excluded from the analysis.\n\nThe Medical Ethical Committee of The Iraqi Board for Medical Specializations approved this study [CODE: 2015; date, 24-09-2013]. Participant consent was waived by the committee, since only patient files were reviewed.\n\n\nResults\n\nOut of a total of 1167 case files only 102 patients fit the eligibility criteria and completed the study, with a mean age of 50.4 ± 11.7 years (range, 23–75 years, the rest of the disease characteristic illustrated in (Table 1). Local recurrence had the lowest rate, while combined local and distant recurrence had the highest rate (Table 1). The median DFS was 62 months with 5 years DFS in 52.5% patients, patients with stage N0 had a significantly higher DFS compared to stage N1, and those patients with a positive hormonal status have a significantly better DFS compared to those with a negative hormonal status (Table 2; Figures 1A–D). NLR had the highest discrimination ability to predict recurrence (since AUC between 0.7 – 0.79), while the rest of the variables show poor discrimination ability, as illustrated in Table 3 and Table 4 and Figures 1E and F. NLR fair specificity (76.9%) with lower sensitivity (62.2%), with optimal cut point of >2.194 to predict all recurrence. NLR also showed similar predictability for distant metastasis, while for local recurrence NLR had poor ability to predict local recurrence (Table 5 and Table 6; Figure 1G and Figure 2). Table 7 shows uni- and multivariate analysis of predictors of DFS with a significant p- value (p=0.007) for NLR, which means that it is an independent risk factor (Figure 3).\n\nA: DFS for all patients, B: median DFS (MDFS) by T staging, C: MDFS by N staging, D: MDFS by hormonal status, E: NLR MDFS by months, F: PLR MDFS by months, G: Kaplan–Meier estimator of DFS using NLR cut point (2.194).\n\nThe linear trend for each level of NLR and PLR was used (-3. -1, +1, +3)\n\nAUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value.\n\nNLR, neutrophil-lymphocyte ratio\n\n\nDiscussion\n\nThis is the first study to show an association between high NLR and poor prognosis in Iraqi breast cancer patients. A higher NLR independently reflected a higher risk of local recurrence and distant metastasis in women with early breast cancer with a cutoff point of 2.194.\n\nWe found significant differences in DFS (16 months) according to our NLR cut off point with significant p-value (p=0.004). The role of lymphocytes in cancer is exemplified by the strong association between high densities of tumor-infiltrating lymphocytes and better responses to both cytotoxic treatments and outcome in patients5,6. Two meta-analysis studies have confirmed the association between elevated NLR and poor prognosis for breast cancer7,8; however, studies about these values are rare and not done in Iraq. In this study, we validated the usefulness of high NLR in early stage breast cancer up to stage IIA and to estimate DFS for at least 36 months follow-up.\n\nIn our univariate and multivariate analysis, we found hazard ratio (HR) for NLR 2.5 with significant p-value (p=0.007), while in the univariate analysis the nodal status and hormonal status were significant as dependent prognostic factors. Comparing our result with Ethier et al., a meta-analysis which comprised patients with reported HRs for DFS, and included only non-metastatic cases8, our result has the same significance with good sample size and follow-up period; however, we couldn't calculate overall survival (OS). Although another study shown that PLR was not related with DFS or OS in women9, in our study PLR was neither sensitive nor specific with non-significant p-value, so it will not considered as a prognostic index.\n\n\nConclusion\n\nThe role of NLR is a prognostic marker and elevated NLR is correlated with poor DFS in early breast cancer patients.\n\n\nData availability\n\nZenodo: Neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in early stage breast cancer as predictor of disease-free survival. https://doi.org/10.5281/zenodo.253112410.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMonica M, Harold JB, Jay RH: Malignant Tumors of the Breast. In: DeVita VT, Lawrence TS, Rosenbergs SA (editors): Devita, Hellman, and Rosenberg’s Cancer Principles and Practice of Oncology. 10th edt. Wolters Kluwer Health / Lippincott Williams & Wilkins. USA. Philadelphia. 2016; 1117–1128.\n\nDunn GP, Bruce AT, Ikeda H, et al.: Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol. 2002; 3(11): 991–998. PubMed Abstract | Publisher Full Text\n\nAlmand B, Resser JR, Lindman B, et al.: Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res. 2000; 6(5): 1755–1766. PubMed Abstract\n\nRayman P, Wesa AK, Richmond AL, et al.: Effect of renal cell carcinomas on the development of type 1 T-cell responses. Clin Cancer Res. 2004; 10(18 Pt 2): 6360S–6366S. PubMed Abstract | Publisher Full Text\n\nLoi S, Sirtaine N, Piette F, et al.: Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. J Clin Oncol. 2013; 31(7): 860–867. PubMed Abstract | Publisher Full Text\n\nTempleton AJ, McNamara MG, Šeruga B, et al.: Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: a systematic review and meta-analysis. J Natl Cancer Inst. 2014; 106(6): dju124. PubMed Abstract | Publisher Full Text\n\nXin-Ji Z, Yong-Gang L, Xiao-Jun S, et al.: The prognostic role of neutrophils to lymphocytes ratio and platelet count in gastric cancer: A meta-analysis. Int J Surg. 2015; 21: 84–91. PubMed Abstract | Publisher Full Text\n\nEthier JL, Desautels D, Templeton A, et al.: Prognostic role of neutrophil-to-lymphocyte ratio in breast cancer: a systematic review and meta-analysis. Breast Cancer Res. 2017; 19(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCihan YB, Arslan A, Cetindag MF, et al.: Lack of prognostic value of blood parameters in patients receiving adjuvant radiotherapy for breast cancer. Asian Pac J Cancer Prev. 2014; 15(10): 4225–4231. PubMed Abstract | Publisher Full Text\n\nAl-Bairmany YS: neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in early stage breast cancer as predictor of disease free survival [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2531124"
}
|
[
{
"id": "48267",
"date": "10 May 2019",
"name": "Shelly Kaushik",
"expertise": [
"Reviewer Expertise Cancer Biology",
"treatment resistance",
"tumor recurrence",
"cell biology",
"molecular biology",
"biochemistry",
"breast cancer",
"brain cancer",
"mechanobiology",
"tissue tension"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, Al-Bairmany et al attempt to explore the effect of neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) on Disease Free Survival (DFS) in patients with breast cancer. In this retrospective study, the authors find that high NLR correlates with poor DFS, suggesting its use as a prognostic marker in breast cancer. While the study attempts to address an important question, that of a clinically unmet challenge of finding robust biomarkers that can predict/prognosticate breast cancer survival, the manuscript is poorly written and referenced, the results are inadequately presented with not enough information in either the methods, figure legends or the plots to derive and interpret the information and results. While the authors claim that the prognostic role of NLR is controversial, they do not discuss the controversy adequately either in the introduction or discussion. For specific comments, please refer to the point-by-point review below.\nMany Grammatical errors, choice of words and awkward sentence structure make it very difficult to read through and review and manuscript, starting from the abstract. We advise the authors to kindly revise the manuscript thoroughly and correct the language such that the reviewers can focus on critiquing the data rather than the linguistic shortcomings of the manuscript. Additionally, several references are missing (as highlighted in the comments below) making It difficult to get a solid understanding of the background through cross-referencing. Also, a lot of these references are outdated and the field has progressed profoundly in the last few years. The authors need to thoroughly review recent literature.\nAbstract:\n\nPlease amend sentence to: “the relationship between neutrophil-lymphocyte ration (NLR) and outcome is controversial”. Also, do the authors mean Survival outcome? If yes, please clarify.\n\nPlease amend to: “this study aims to explore the relationship between NLR…”. Also, the authors mention PLR in the same sentence. However, unlike NLR, they do not clarify why the relationship between PLR and “outcome” (DFS?) is important. They need to introduce PLR alongside NLR.\n\nWhat does “lowest period of follow-up” mean? 40 months was the shortest time between diagnosis and follow-up?\nIntroduction:\n“Animal models suggest “immune-editing” in which activation of immune mechanisms control the tumor, but over time lead to the selection of tumor cells that escape the immune pressure and grow progressively.” I am not sure if the authors here are trying to define immune-editing, suggesting that immune-editing has been shown in animal models or implicating immune-editing as a therapeutic option for when tumor cells evade the immune system?\n\n“Tumors are heterogeneous, and the antigens on cells of one tumor are variable, even within the same patient, so the down-regulation of major histocompatibility complex molecules and other components of the antigen-presentation process can occur.” I am not sure how tumor heterogeneity in terms of antigen variability can contribute to down-regulation of the major histocompatibility complex? Are the authors trying to say that cells can express variable antigens even within the same tumor and that some of these antigens can be immune-suppressive through their down-regulation of the MHC?\n\nHow can the expression of Fas ligand by tumor cells, while maintaining immune privilege, also induce apoptosis? Are the authors suggesting that the apoptosis is induced in the surveilling immune cells? Also, the authors need to provide a reference for this claim.\n\nThe authors need to provide reference for “Tumor cells lead to the release if many cytokines…antitumor immunity”\n\nThe authors cite Rayman et al to talk about tumor cells skewing the immune response to Th2 phenotype. This a 2004 paper and the field has since shifted to suggest that the contribution of these phenotypes to cancer is fluid and controversial. We urge the authors to review the most recent literature on the subject and state the facts with the most recent/accurate references.\n\nPlease provide reference for “the evidence of the relationship between inflammation and cancer prognosis….even breast cancers”.\n\nThe authors need to include more information on other studies that have done the NLR and PLR analysis in breast or other cancers and what these results are.\n\nResults:\n“Local recurrence had the lowest rate”? Are the authors talking about rate of incidence? The term \"Lowest rate\" is vague.\n\nAll panels of Figure 1 need p-values. How do we know if the survival is significant between different groups if the appropriate statistical analysis has not been depicted in the figures or at least mentioned in the legends?\n\nFigure 1B: Is this correct that patients with stage T1 tumors with less aggressive tumors have worse prognosis than stage 2, which is more aggressive? Please explain this outcome.\n\nFigures 1E-F: What are Q1-Q4? Please make this clear in the figure and the legend. Without knowing what these are how can we interpret the robustness of the data or appreciate the differences in survival?\n\nFigure 1G: Why is the red curve cut short? One would expect a tail with patients that have an NLR of greater than 2.194?\n\nAlso, how was the cutoff value of 2.194 determined? This information is missing from Methods.\n\nNeutrophil-lymphocyte ratio or NLR: were these derived from blood samples or biopsies? As demonstrated in other cancers such as brain tumors,the presence of neutrophils in the blood versus in the tumor can predict response to treatment. The authors need to mention in their methods where the neutrophils, lymphocytes and platelets are derived from (blood, biopsy etc) and explain how they were quantified.\n\nAlso, the patients who were included in the study, are they treated in any way i.e. surgery, radiation and chemotherapy? The therapeutic strategy also affects DFS and as such should be explicitly stated by the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "63525",
"date": "29 Jun 2020",
"name": "Hutcha Sriplung",
"expertise": [
"Reviewer Expertise Cancer 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 title, objective, and abstract don’t say that the analysis is limited to stage I up to IIA diseases. If you find the NLR and PLR are not homogeneous to stage III of the disease, it is better to stratify analysis and clearly show the difference in survival according to the stage, or clearly state why you chose stages I - IIA in your study.\n\nIn the first paragraph of the result, is the proportion of exclusion from the analysis too big? Please give the reasons why you excluded them. I think you walked through steps of case selection, and many cases probably were not in your inclusion criteria, but you counted in 1167 files.\n\nAm I right that you recruited the cohort and measured all the predictors in the past? And then you followed them up for 36 months and saw the results at the end of the follow up when the event occurred or at the end of the follow-up time.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-306
|
https://f1000research.com/articles/8-305/v1
|
19 Mar 19
|
{
"type": "Research Article",
"title": "Clinical outcomes of zirconia-reinforced lithium silicate partial coverage crowns compared to lithium disilicate partial coverage crowns. A randomized controlled split-mouth clinical study",
"authors": [
"Hanaa Nassar",
"Carl H Halim",
"Hesham A Katamish",
"Carl H Halim",
"Hesham A Katamish"
],
"abstract": "Background: Despite the fact that preliminary clinical results of conservative partial coverage restorations (PCRs) are promising, the clinical behavior of different PCR ceramic materials is rarely investigated in clinical trials. This study aimed to evaluate the clinical outcomes of partial coverage restorations (PCR) fabricated with zirconia-reinforced lithium silicate ceramic system compared to partial coverage restorations fabricated with lithium disilicate ceramic system. Methods: 46 vital premolars and molars of 14 patients were restored with PCRs (23 Vita Suprinity and 23 IPS e.max CAD). PCRs were CAD/CAM fabricated in the lab and adhesively luted with dual-polymerizing resin cement (Duolink. BISCO, USA). Clinical evaluation of PCRs was performed according to the Modified United States Public Health Service (USPHS) at baseline, 6 and 12 months post-insertion. Absolute failure was demonstrated by Kaplan-Meier survival rate analysis. Results: After 12 months observation, all PCRs of both ceramic groups demonstrated 100% survival rate. Non-significant decrease in Alpha ratings for marginal adaptation (p = 0.1560) and marginal discoloration (p = 0.6078) in e-max group. While in the Suprinity group, PCRs demonstrated 100% Alpha ratings for marginal adaptation and only one Bravo rating (p= 0.3625) for marginal discoloration after 12 month observation. Conclusions: Both Vita-Suprinity and e.max CAD partial coverage restorations are considered reliable treatment options for restoring larger defects in posterior dentition. Trial registration: ClinicalTrials.gov NCT02861729 04/08/2016",
"keywords": [
"Partial coverage restorations",
"posterior teeth",
"ceramic",
"Zirconia reinforced lithium silicate",
"lithium disilicate",
"CAD/CAM"
],
"content": "Introduction\n\nHigh survival rates, fracture resistance and proper marginal integrity of CAD/CAM partial coverage restorations (PCRs) were reported in studies simulating 5-year clinical service1–4.\n\nHowever, clinical behavior of PCRs utilizing morphology driven preparation design was never assessed in randomized clinical trials5–11.\n\nFurthermore, long-term clinical studies have shown that bulk fracture and marginal deterioration of PCRs has a direct correlation to the use of brittle ceramic materials, such as feldspathic and leucite-based ceramics12–15, which encouraged researchers to use higher strength lithium disilicate glass ceramic in such restorations16–21. Although some clinical studies tested the performance of lithium disilicate PCRs, no randomized clinical trial tried to compare between lithium disilicate ceramic material and the newly introduced zirconia-reinforced lithium silicate ceramic material in posterior partial coverage22–24.\n\nThe aim of this randomized controlled split-mouth clinical study was to evaluate the clinical outcomes of zirconia-reinforced lithium silicate (Vita Suprinity) and lithium disilicate (IPS e.max CAD) partial coverage restorations. The null hypothesis was that there would be no difference between the two ceramic materials over 12 months.\n\n\nMethods\n\nThis study was approved by Ethics Committee of Faculty of Oral and Dental Medicine in October 2016 (Approval number: 03102016).\n\nWritten informed consent for all the study procedural steps and publication of their clinical results and images were obtained from the patients.\n\nThis trial was registered with ClinicalTrials.gov under trial number NCT02861729 on the 04/08/2016.\n\nThis study was a double blinded, split-mouth randomized controlled clinical trial, with an allocation ratio of 1:1.\n\nThis article was written in concordance with the CONSORT checklist 2010 (see Reporting guidelines).\n\nAll patients were recruited from the outpatient clinic of the Department of Fixed Prosthodontics, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt. Between May 2017 and June 2017, a total of 14 adult patients (8 females and 6 males) were included in this study after fulfilling all inclusion criteria. A total of 46 premolars and molars (20 maxillary and 26 mandibular) were restored in this study, according to split-mouth design; at least two restorations (one of each ceramic material) were placed in each patient.\n\nInclusion criteria:\n\nAdult patient aged 18–50 years old. Patient with good oral hygiene (papillary bleeding index (PBI < 35%).\n\nTeeth: vital, with large carious lesions/defective restorations and teeth in occlusion.\n\nExclusion criteria:\n\nPatient with severe systemic disorder, smokers, xerostomia or buxism\n\nTeeth: non-vital, endodontically treated, mobile or periodontally affected teeth.\n\nBased on the previous paper by Guess et al. 200915, the probability of surface roughness among interventions is 0.48. If the true probability among controls is 0.11, it was estimated that a total of 46 samples (n= 23 of each ceramic material group) would be required to reject the null hypothesis that the exposure rates for case and controls are equal with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis is 0.05. Sample size was calculated using G* Power program, version 3.0.10.\n\nA random sequence was generated by computer software (http://www.randomizer.org/) in the Center of Evidence Based Dentistry, Cairo University. The table was kept with the assistant supervisor (CHH). Participants received numbered papers each contains a number from 1 to 2 representing ceramic material and a letter R or L representing the side where PCR will be placed on folded paper placed in sealed opaque envelops. The patient selected the ceramic material for the first tooth randomly, and then the following tooth received the alternate ceramic material according to the split-mouth design.\n\nAll clinical steps were performed by one operator (HN) and laboratory steps by one technician.\n\n\nFirst visit (teeth preparation)\n\nA new cavity preparation design; morphology driven preparation (MDP) design was selected for this study. In this design, preparations were guided by the anatomical and structural morphology of the teeth25–27.\n\nInterior walls were prepared with 6–10°divergence, well-defined margins and rounded inside angles. Inter-proximal box was prepared with 1–1.2mm butt-joint, all obtained with medium-grit 80μm diamond truncated conical bur (4137-856-025, Microdont, USA). (Figure 1)\n\nOcclusal reduction of 1.5–2mm was performed with egg-shaped football bur (3118-368-023, Microdont, USA), and verified with silicon index. The outer axial walls with inclined planes were prepared with hollow chamfer margin obtained with round bur (1014-801-014, Microdont, USA). (Figure 2)\n\nAll preparation was finished with fine finishing bur (4137F-856-025, Microdont,USA).\n\nAll undercuts were blocked with Herculite™ ultra-flow composite (Kerr-Germany, Catalogue no.: 2201-35392).\n\nFull arch Vinylpolysiloxane (EliteHD+. Zermack-Germany, Catalogue no.: F121007 - 2016-05) impression and interocclusal records (Occlufast. Zermack-Germany, Catalogue no.: F121009 - 2016-05) were taken. Provisional restorations were fabricated with Structure-2 bis-composite (Voco-Germany, Catalogue no.: VC 84 001479 GB 0918 V) and cemented with temporary cement (Dentotemp-Itena. France, Catalogue no.: K03330 9).\n\n\nLaboratory fabrication\n\nMaster models were poured with type IV dental stone (FujiRock-EP, GC-Belgium, Catalogue no.: 890366), then scanned with an extraoral scanner (Identica-blue. Medit, England). Final PCRs were designed using CAD/CAM software (Exocad-DentalCAD, Exocad GmbH-Slovenia) and milled with 5-axis machine (CAM5-S1impression.Vhf, Ammerbuch-Germany) of Suprinity (VITA-ZahnfabrikH. Rauter-GmbH-Germany, Catalogue no.: 2002E – 0114 (X.) S Version (02)) and e.maxCAD (Ivoclar-Vivadent, Schaan-Liechtenstein, Catalogue no.: 721198/e/2018-11) blocks. After staining, PCRs crystallization was done in ceramic furnace (Programat-P310) according to manufacturer instructions.\n\n\nSecond visit (PCRs try-in and bonding)\n\nDefinitive PCR try-in was performed to confirm the restoration proper seating, marginal integrity, shade matching and proper occlusal and proximal contacts.\n\nAll PCRs bonding steps were performed under rubber-dam isolation.\n\nThe internal surfaces of PCRs were etched for 20 second with 9.5% hydrofluoric acid (BISCO-USA, Catalogue no.: E-5702EP), rinsed, air dried, then Bis-silane (BISCO-USA, Catalogue no.: B-2221P) was applied, left for 60 seconds and air dried.\n\nEnamel margins of the preparations were etched with 37% phosphoric acid (BISCO-USA) for 30 seconds, rinsed and air dried. All-bond universal adhesive was applied, air thinned, and cured for 20 seconds (Elipar™, 3MESPE, USA, Catalogue no.: 70-2013-0430-3-B). Duolink adhesive resin cement (BISCO-USA, Catalogue no: A-19010P), was applied to fitting surface; restoration was seated with gentle pressure, glycerin barrier was applied to margins (Deox.Ultradent-USA, Catalogue no: 238), then light curing was performed for 40 seconds (Elipar™, 3MESPE, USA, Catalogue no.: 70-2013-0430-3-B).\n\nResidual cement was removed and occlusion was carefully checked.\n\nThe PCRs were assessed for clinical outcomes by an independent outcome assessors according to the modified United States Public Health Service (USPHS) criteria28–30; at baseline, 6 and 12 months post-treatment. PCRs were visually inspected with mirror, probe and dental floss; all changes were recorded and photographed31.\n\nPrimary outcome: survival rate\n\nFor survival rate, only Alpha ratings were considered success.\n\nAbsolute failure was defined by loss of retention, fracture, crack development which required a replacement of the entire restoration, secondary caries or endodontic complications32–35 (Table 1).\n\nSecondary outcomes: marginal adaptation and marginal discoloration\n\nAlpha and Bravo scores were considered success, while PCRs rated Charlie or Delta were considered failure15,16,32,35.\n\nThis study was a double-blinded study; both patient and outcomes’ assessors were blinded to the assigned PCR material for each tooth throughout all preparation and clinical evaluation steps. However, the operator wasn’t blinded for purpose of lab communication and ceramic material construction steps.\n\nThe blinded assessors were asked to fill a chart for each outcome with the number corresponding to each patient without knowing the PCR material allocated to each side of the mouth for each participant. The template for clinical assessment chart can be found with the trial protocol (Extended data36).\n\nThe results were analyzed using IBM SPSS, version 21 (SPSS, Chicago, IL, USA). Chi square test was performed for categorical data, a value of P < 0.05 was considered statistically significant. Sample size (n=23/group) was large enough to detect significant effects and perform pair-wise comparisons with a satisfactory level of power set at 80% and a 95% confidence level.\n\n\nResults\n\nAll 14 patients (8 females and 6 males) attended 6 and 12 month follow-up. A total of 46 PCRs were fabricated in this study. A patient flow diagram is available as part of the Reporting guidelines section.\n\nSurvival rates on Kaplan Meier survival curve are provided in (Table 1) and (Figure 3). After 12 months, all PCRs of both groups remained in situ, with a survival-rate of 100% (P=0.8254).\n\nFor criteria marginal adaptation, e-max CAD group showed a statistically non-significant decrease in Alpha ratings to 95.65% (p=0.1560), while Vita Suprinity group maintained 100% Alpha scores after 12 months (Table 2, Figure 4 and Underlying data36).\n\nns; non-significant (p >0.05)\n\nFor marginal discoloration, e-max CAD group showed a non-significant decrease in Alpha ratings to 95.65 % (p = 0.1560) after 6 months and 87 % ( p=0.6078) after 12 months (Table 3 and Figure 5).\n\nns; non-significant (p >0.05)\n\nBravo ratings of 13% for discoloration and palpable marginal ditching were recorded in e-max CAD group after 12 month (Figures 6–Figure 8), while 4.35% Bravo rating were recorded in the Vita Suprinity group after 12 months.\n\n\nDiscussion\n\nThis study aimed to compare the clinical performance of Vita Suprinity and e-max CAD partial coverage restorations in a prospective double-blinded split-mouth design. Selection biases can be avoided in split-mouth studies as the patient acts as their own control, in this way direct comparison of two ceramic materials can be performed37–39.\n\nCompared to full coverage restorations, posterior partial coverage restoration utilizing more tooth structure conservation concept has the potential to reinforce and protect tooth structure, preserve enamel, and safeguard pulp vitality while achieving the desired aesthetic results5,6. Overtime, various ceramic materials have been developed for restoring posterior teeth7–9. The excellent combination of high mechanical strength and optical properties of lithium disilicate glass ceramic material made it the gold standard for comparison of new monolithic ceramic materials9,11,12. In our study CAD/CAM lithium disilicate ceramic material (IPS e.max CAD) was selected as the control to compare the clinical outcomes of the newly introduced zirconia-reinforced lithium silicate ceramic material (ZLS) (VITA Suprinity). Reinforced with about 10% zirconium dioxide, ZLS belongs to a new generation of CAD/CAM ceramic that combines positive mechanical characteristics of zirconia with glass-ceramic aesthetic appearance22. Still all findings regarding this new material are either laboratory or initial clinical experience findings22–24. Moreover, the indication for this material should be chosen with strict observation of the material-specific processing instructions regarding the necessary minimum wall thickness and required adhesive luting22,23. All of these findings make it crucial to conduct randomized clinical studies to verify the clinical performance of this new material.\n\nIn this study, a novel tooth preparation design; MDPT was selected. According to Venezian M25, this preparation design aims to minimize the loss of healthy tooth tissue and reduce the areas of dentin exposure. A hollow chamfer margin was created on the outer surface of the preparation to optimize the cutting of enamel prisms, thereby bonding and color blending at the transitional zone between tooth and restoration are enhanced25–27(Figure 1), (Figure 2).\n\nRegarding our results, Kaplan Meier analysis was used for survival assessment during the observational period of 12 months32–35; both Suprinity and e.max-CAD had a survival rate of 100%. All restorations remained in situ and in good function.\n\nComparison of our results to similar clinical studies regarding Suprinity is limited due to the novelty of the material22,23.\n\nClinical studies on e.max PCRs reported 98.99–100% survival rates over periods of 1–5 years15,17,40–43. In a long-term evaluation study, Guess et al.16 reported 100% survival rates for e.max PCRs with only evidences of relative failures as small repairable chipping after 8 years, but none of PCRs were fractured or de-bonded.\n\nIn our study, none of PCRs were de-bonded after 12 months. Other studies reported de-bonding of PCRs as one of the common causes of failure13,14. In those studies, de-bonding mainly was associated with endodontically treated teeth which were among the exclusion criteria in this study.\n\nFor marginal adaptation, all PCRs were rated Alpha at base-line and after 6 months. However; after 12 months, palpable margin ditching resulted in Bravo ratings for one of the e.max-CAD PCRs (4.35%). Marginal deterioration might be attributed to degradation of cement due to fatigue in the oral cavity15,16.\n\nSuprinity PCRs sustained Alpha rating after 12 months, which can be attributed to the higher marginal quality and fatigue resistance of zirconia lithium silicate over lithium disilicate as reported by Preis et al.23,43.\n\nNevertheless, results by Elsaka and Elnaghy24 were in disagreement with our results as they reported lower brittleness index of e.max CAD compared to Vita Suprinity, and consequently according to the parameters determined by Boccaccini44 and Chaysuwan et al.45; e.max CAD might show lower marginal chipping rates than Suprinity24.\n\nMarginal discoloration of e.max PCRs has been reported by Guess et al.15,16 and Santos et al.17 as the most common clinical finding occurring in 37.5% of PCRs after 7 years.\n\nFor marginal discoloration; three of e.max CAD (13%) and one Vita Suprinity (4.35%) restorations showed yellowish marginal staining (Bravo) after 12 months. Still both materials showed clinically acceptable margins.\n\nThe null hypothesis for this study was accepted as there was no statistically significant difference in clinical outcomes of the two tested ceramic materials.\n\nThis study is randomized clinical trial conducted on relatively big sample size patients, in real clinical settings and was conducted efficiently. This is the first study to compare the clinical performance of e.max CAD and Vita Suprinity partial coverage restorations utilizing a novel preparation design (MDP). Our present study proposes a more conservative and efficient alternative to full coverage restorations for treatment of decayed, vital posterior teeth with high survival rates and excellent marginal quality.\n\nThe following limitations should be considered: The morphology driven preparation technique is a new design that wasn’t tested in previous randomized clinical trials before, reliability of the new design irrespective of the ceramic material used needs to be investigated in further clinical trials.\n\nThe short follow-up period was one of our study limitations, although no significant differences were found between the two materials, there was notable differences regarding marginal discoloration, thus longer term clinical trials are required to investigate the clinical performance of these ceramic materials.\n\n\nConclusion\n\nBoth Vita-Suprinity and e.max CAD partial coverage restorations are considered reliable treatment options for restoring larger defects in posterior dentition.\n\n\nData availability\n\nOpen Science Framework: Clinical Outcomes of Zirconia-reinforced Lithium Silicate Partial Coverage Crowns Compared to Lithium Disilicate Partial Coverage Crowns. A Randomized Controlled Split-mouth Clinical Study. https://doi.org/10.17605/OSF.IO/UNGCJ36\n\nThis project contains the following underlying data:\n\nResults Data\n\n◦ KM curves.xlsx (Kaplein-Meier curves for crown survival)\n\n◦ Results raw.xlsx (Performance data for partial coverage crowns used)\n\nOpen Science Framework: Clinical Outcomes of Zirconia-reinforced Lithium Silicate Partial Coverage Crowns Compared to Lithium Disilicate Partial Coverage Crowns. A Randomized Controlled Split-mouth Clinical Study. https://doi.org/10.17605/OSF.IO/UNGCJ36\n\nThis project contains the following extended data:\n\nStudy Settings\n\n◦ Grouping.xlsx (Patient grouping information)\n\n◦ ResearchRandomizer.csv (Randomizer results)\n\nSupplementary files\n\n◦ Trial protocol.docx (Trial protocol with copies of all forms used for data collection)\n\nOpen Science Framework: CONSORT checklist and flow diagram for ‘Clinical outcomes of zirconia-reinforced lithium silicate partial coverage crowns compared to lithium disilicate partial coverage crowns. A randomized controlled split-mouth clinical study’ https://doi.org/10.17605/OSF.IO/UNGCJ36\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patients.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nStappert CF, Chitmongkolsuk S, Silva NR, et al.: Effect of mouth-motion fatigue and thermal cycling on the marginal accuracy of partial coverage restorations made of various dental materials. Dent Mater. 2008; 24(9): 1248–1257. PubMed Abstract | Publisher Full Text\n\nStappert CF, Guess PC, Chitmongkolsuk S, et al.: All-ceramic partial coverage restorations on natural molars. Masticatory fatigue loading and fracture resistance. Am J Dent. 2007; 20(1): 21–26. PubMed Abstract\n\nManhart J, Chen H, Hamm G, et al.: Buonocore Memorial Lecture. Review of the clinical survival of direct and indirect restorations in posterior teeth of the permanent dentition. Oper Dent. 2004; 29(5): 481–508. PubMed Abstract\n\nLima FF, Neto CF, Rubo JH, et al.: Marginal adaptation of CAD-CAM onlays: Influence of preparation design and impression technique. 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}
|
[
{
"id": "50631",
"date": "11 May 2020",
"name": "Oliver Schierz",
"expertise": [
"Reviewer Expertise Prosthetic dentistry",
"clinical studies"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article contains 12 month results of a randomized clinical trial in partial crowns using two different dental ceramics.\n\nThe title does match the content of the publication.\n\nThe keywords are appropriate.\n\nThe introduction is very short.\n\nThe paper is linguistically sufficiently written and structured. Sample size calculation is insufficient, calculating for surface roughness, a parameter not reported and potentially secondary aim at best. This caused a severe miscalculation in number of patients needed to answer the scientific question, reflected by insignificant p-values in the results.\n\nAccording to material and methods each participant receives 2 crowns, one of ZLS and one LiS. When 14 participants are included this should result in 28 crowns, not 46. This difference should be explained and is probably due to more than one pair of partial crowns in each participant.\n\nThe time frame of 12 months follow-up is much too short to find differences in respect that known clinical survival within 5 years is up 98 percent.\n\nA report about the distribution of included restorations in premolars and molars is missing.\n\nWith the current number of participants the scientific value is low.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "66779",
"date": "03 Aug 2020",
"name": "Samah Saker",
"expertise": [
"Reviewer Expertise Prosthetic dentistry",
"Dental Biomaterial",
"Clinical Trials."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review this research article addressing a very important topic, clinical outcome of all ceramic partial coverage restorations, and the article is well-written. I agree with the authors that this study has a number of strengths (a randomized, split mouth study). However, I have some concerns about the design and the interpretation of the results that I believe should be addressed to make the paper stronger.\nThe title It could be better to consider the following title for your study: Clinical outcome of CAD/CAM all-ceramic partial-coverage restorations: A Prospective clinical split-mouth study1.\nAbstract\nRegarding the abstract section it’s informative.\nIntroduction section\nIs short and the rationale of the study is not well presented, kindly add a short review about the previous clinical studies about all ceramic partial coverage restoration focusing in the preparation design, the cause of the use such design, and why in your study you use morphology driven preparation from biomechanical prospective.\n\nMethod section\nRegarding to tooth preparation, Kindly specify the following; - Cavity depth, cavity width (BL) in both premolar and molar. - The buccal and lingual extension of the interproximal box walls. - Regarding to the sentence, all undercuts were blocked……kindly, explain.\n\nRegarding to clinical outcome; - Each restoration was examined by one or two assessors? Kindly specify. - Although, 12 months of clinical service is too short period to assess the survival rate of the restoration as mentioned by the authors in the study limitation section, the finding could be considered as a preliminary finding.\n\nResults section\nKindly, add a table of restoration distribution, for example, how many molars, premolars, and their frequency distribution.\n\nIs there any patient drop out during the evaluation period or not, kindly add this part.\n\nDiscussion section\nYour null hypothesis is accepted or rejected? And why? Kindly discuss.\n\nThe second paragraph in the discussion section, is fitted best to your introduction section, as it describe your study rationale.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-305
|
https://f1000research.com/articles/7-1864/v1
|
29 Nov 18
|
{
"type": "Study Protocol",
"title": "Protocol: New approaches to managing the social deficits of Turner Syndrome using the PEERS program",
"authors": [
"Jeanne Wolstencroft",
"William Mandy",
"David Skuse",
"William Mandy",
"David Skuse"
],
"abstract": "Turner Syndrome (TS) is a sex chromosome aneuploidy (45,X) associated with social skill difficulties. Recent clinical care guidelines recommend that the Program for the Education and Enrichment of Relational Skills (PEERS) social skills intervention programme be trialled in this population. PEERS has been successfully used in adolescents with autism spectrum conditions without intellectual disabilities. The PEERS program will be piloted with adolescents and young women with TS aged 16-20 using an uncontrolled study trial with a multiple-case series design. The program will be delivered face to face and online. The assessment battery is designed to measure social skills comprehensively from diverse informants (parent, teacher young person). It includes measures of social performance, social knowledge and social cognition. Parents and young people taking part in the intervention will also feedback on the acceptability and feasibility of the pilot. The outcomes of this small scale pilot (n=6-10) will be used to adapt the programme based on feedback and estimate the sample for a future randomised controlled trial.",
"keywords": [
"social skills training",
"social skills",
"peers",
"turner syndrome",
"sex chromosome aneuploidy"
],
"content": "Introduction\n\nTurner Syndrome (45,X; TS) is one of the most common sex chromosome aneuploidies, with an incidence of 1 in 2500 female births1. TS is associated with a variety of morbidities affecting nearly every bodily system, including skeletal abnormalities such as short stature, dysmorphic features, hearing difficulties, infertility, cardiac abnormalities, diabetes and thyroid problems. These difficulties have been well characterized in the literature (see Gravholt et al. 20172 for the most recent review) and require clinical monitoring across the lifespan.\n\nTS females have social difficulties throughout childhood, but these become more apparent in adolescence when socialisation becomes more complex3. Social deficits are exemplified by difficulties integrating within social groups, with poor deciphering and processing of social cues3. Previous research has shown TS is associated with specific deficits in social cognitive competence, especially forming and maintaining peer relationships4.\n\nSome of the social deficits observed in TS are reminiscent of difficulties associated with Autism Spectrum Disorders (ASD). Although systematic evaluations of the mental health of young women with TS have never been conducted, research studies have found an association with ASD5–9, anxiety disorders, depression and low self-esteem10–13.\n\nSocial skills deficits are known to have a significant impact on academic, adaptive and psychological functioning14–17, and are likely to have a substantial impact on the wellbeing of girls and women with TS across the lifespan3. At present, psychosocial intervention research with young women with TS is scarce; only one intervention targeting self-esteem in adults aged 18–30 has been documented in the literature10. The latest TS Clinical Care Guidelines recommend that a social skills training intervention should be trialled in this population2. They suggest using the Program for the Education and Enrichment of Relational Skills (PEERS) developed for children with ASD18. There is good evidence for the efficacy of PEERS when delivered with children and young adults with ASD without intellectual disabilities19–24. This pilot project will be the first to examine the feasibility and acceptability of the PEERS Protocol in adolescents with TS.\n\n\nProtocol\n\nThe main objectives of the study are:\n\n1) To pilot the PEERS intervention in adolescents with TS;\n\n2) Assess its feasibility and acceptability to families.\n\nWe hypothesise social skills training will improve social competence with peers and may produce secondary improvements in social cognition, self-esteem and anxiety (social and generalised).\n\nWe will be employing an uncontrolled trial design. To maximise the clinical reliability of the trial we will use a systematic multiple-case series design with case tracking. All participants will be matched for age, degree of social impairment, intellectual ability and hormone therapy treatment.\n\nA sample size of 6–10 girls and their parents will be invited to take part in the study - this is the group size recommended by the PEERS intervention manual. At present the effect size for this intervention in girls with TS is unknown. This pilot will serve as the basis to estimate the intervention’s effect size and sample size for a future randomised control trial.\n\nParticipants will be recruited from the Social Skills and Relationships in Turner Syndrome Study (SOAR), which recruits children and young women with TS from the Turner Syndrome Support Society, the NHS Great Ormond Street Hospital and the NHS University College London Hospitals.\n\nThe SOAR study is conducting online mental health and social cognition questionnaires with 200 girls and young women with TS and their parents. A subset of families from this large cohort that meet the trial’s inclusion criteria will be invited to take part in the intervention study.\n\nInclusion criteria for the intervention include: 1, a confirmed diagnosis of TS; 2, age 16–20 years; 3, significant social skills difficulties (screened for in the SOAR online questionnaires).\n\nThe exclusion criteria for the intervention include: 1, severe difficulties with hearing or vision; 2, intellectual disability (VIQ<70); 3, concurrent participation in other psychological treatment.\n\nThe UCLA PEERS for Adolescents is a manualized treatment program that consists of 14 90 min sessions18. The program runs two concurrent groups, one for the adolescents and one for parents. At the end of each session the two groups are reunited for review and questions. Between sessions the adolescent group are given homework tasks, which they are to complete with the help of their parent who is trained to support them as their social coach. Parents are provided with concise handouts for each session, which include an overview of the lesson material and the homework.\n\nThe adolescent group sessions are structured to provide didactic instruction as well as social skill rehearsal. The parent sessions mirror the adolescent sessions and provide a space for the parents to problem-solve any difficulties they may have encountered the previous week. The didactic lessons provide instruction on (a) conversational skills; (b) electronic forms of communication; (c) developing friendship networks and finding sources of friends; (d) appropriate use of humour; (e) peer entry strategies; (f) peer exit strategies; (g) organizing get-togethers with friends; (h) handling teasing and embarrassing feedback; and (i) resolving arguments with friends18.\n\nThe adolescents and parents will attend separate concurrent sessions led by a certified PEERS Instructor. Three face to face sessions will take place in London at the start, middle and end of the program. All other sessions will be conducted online using a virtual meeting room. The face to face sessions will deliver two PEERS lessons, whereas the weekly online sessions will deliver one lesson. Research assistants (graduate or undergraduate psychology students) will monitor treatment fidelity, assist with role-playing demonstrations, and provide social coaching with performance feedback during behavioural rehearsal exercises. All research assistants will be trained and supervised throughout the intervention.\n\nParticipants will complete assessments at different time points throughout the study. The study will last 9 months in total, including a 3 month baseline, 3 months of intervention and a 3 month follow-up period. The screening measures will be delivered at T=0, the baseline assessments will be delivered at T=12 weeks and the post intervention assessments will be delivered at T=20 weeks. The primary outcome measure will be delivered at regular intervals of 4 weeks throughout the course of the study (see Table 1).\n\nInformants for each assessment are included in brackets (P – Parent; T – Teacher; YP – Young Person). Assessment acronyms: BAI – Beck’s Anxiety Inventory; IAQ – Intervention Acceptability Questionnaire; PEERS – Program for Education and Enrichment of Relational Skills; PEERS QPQ – PEERS Quality of Play Questionnaire; PEERS TASSK-R – PEERS Test of Adolescent Social Skills Knowledge; RSE – Rosenberg Self-esteem Scale; SCP – Social Competence with Peers; SDQ – Strengths and Difficulties Questionnaire; SRS – Social Responsiveness Scale; SASI - Schedules for the Assessment of Social Intelligence; SWS – Spence Social Worries Scale; WAIS-Wechsler Adult Intelligence Scale.\n\nDevelopment and Wellbeing Assessment (DAWBA): The DAWBA will be used to collect information on the child’s behavioural adjustment and mental health. The DAWBA has been used both in UK national and international surveys25–28. The DAWBA data will be reviewed by a psychiatrist in accordance with the ICD-10/DSM-V diagnostic criteria. This methodology has been used successfully to gather data of high quality by parental online report. The DAWBA is available in 26 languages. The DAWBA will be completed online by parents.\n\nStrengths and Difficulties Questionnaire (SDQ): The SDQ is a brief behavioural screening questionnaire29. The SDQ includes scales that measure emotional symptoms, conduct problems, hyperactivity/inattention difficulties, peer relationship problems and prosocial behaviour. The first four scales are combined to create a total difficulties score. An additional impact scale measures the impact of this composite score on daily life. It has been validated for use in children aged 4–17 in UK National studies of psychological adjustment, and a new form for 18+ years old has recently been developed. It will be completed online by the adolescents, parents and teachers.\n\nSocial Responsiveness Scale (SRS): The SRS measures the severity of autistic traits and the instrument has convergent validity with other ASD diagnostic tools30,31. The SRS subscales measure Social Awareness, Social Cognition, Social Communication, Social Motivation, and Restricted Interests and Repetitive Behaviour. The SRS will be administered online to parents and teachers.\n\nHealth Questionnaire (HQ): The questionnaire was developed by the UCLH Turner Syndrome Life Course Project to record information about physical health, health care, education, social life, physical activity and relationships32. The self-report version of the questionnaire will be completed by adolescents.\n\nSchedules for the Assessment of Social Intelligence (SASI): The SASI is a socio-cognitive assessment that measures facial expression recognition, face recognition memory, gaze-monitoring and theory of mind. The SASI is sensitive to subtle deficits in social cognition and has been shown to have excellent reliability and validity33. Adolescents will be asked to complete the SASI online.\n\nWechsler Adult Intelligence Scale - Fourth UK Edition (WAIS-IV UK): The WAIS-IV is an IQ test which measures verbal comprehension, perceptual reasoning, working memory and processing speed. It has been widely used and validated34. It will be administered to adolescents in person.\n\nPEERS Screener: The PEERS Screener Questionnaire assesses the participant’s willingness to take part in the PEERS intervention18. It will be administered to parents and adolescents over the phone.\n\nSocial Competence with Peers (SCP): The SCP assesses the consequences of young people’s interactions with peers, such as the existence and duration of friendships or social invitations35. A modified version of the SCP will be used to adapt the tool for use in young adults. The adolescent group and the parent group will be asked to complete the SCP at regular intervals (every 4 weeks) from baseline to follow-up. Teachers will be asked to complete the SCP at baseline, post-intervention and follow-up.\n\nStrengths and Difficulties Questionnaire (SDQ): Described previously. It will be completed by the young people, parents and teachers at baseline and post-intervention.\n\nSocial Responsiveness Scale (SRS): Described previously. It will be completed online by parents and teachers at baseline and post-intervention.\n\nSpence Social Worries Scale (SWS): The Spence Social Worries Scale is a psychological questionnaire designed to identify symptoms of social phobia and other forms of anxiety, in children and adolescents. The parent and teacher forms are reported to have excellent internal validity35. It will be completed online by the adolescents, parents and teachers at baseline and post-intervention.\n\nSchedules for the Assessment of Social Intelligence (SASI): Described previously. It will be administered online to the adolescent at baseline and post-intervention.\n\nPEERS Test of Adolescent Social Skills Knowledge (TASSK-R): The TASSK-R is a questionnaire designed to evaluate what the participants have learned from the intervention18. This is the only outcome measure to evaluate changes in social knowledge. It will be administered to the adolescents at baseline and post-intervention.\n\nPEERS Quality of Play Questionnaire (QPQ): The QPQ is designed to evaluate the quality of young people’s socialization and frequency of get-togethers18. It will completed online by the parents at baseline and post-intervention.\n\nRosenberg Self-esteem Scale (RSE): The RSE scale is assesses global self-esteem36. It will completed online by the adolescent at baseline and post-intervention.\n\nBeck’s Anxiety Inventory (BAI): This scale is a self-report measure used for measuring the severity of anxiety in children and adults37. It will be completed online by the parent and adolescent groups at baseline and post-intervention.\n\nCamouflaging measure (CAT-Q): The CAT-Q measures camouflaging (e.g. strategies to mask or compensate autistic characteristics) behaviour in social situations. It is comprised of 25 items and has high internal reliability in autistic adults. Its subscales measure compensation, masking and assimilation38. The CAT-Q will be completed by adolescents.\n\nIntervention Acceptability Questionnaire (IAQ): The IAQ has been developed for the study to assess parent and adolescent satisfaction with the intervention (Supplementary File 1). It will be completed by the parent and adolescent groups once the intervention has ended.\n\nThe occurrence of missing data will be reported for each questionnaire and study time point. Participant intervention adherence, planned absences and study dropouts will be recorded and reported. When possible the causes for missing data, absences or dropout will be reported. Families that miss sessions will be caught up over the phone or conference call before the next session.\n\nAdverse events will be recorded.\n\nThe primary outcome measure (SCP questionnaire)16 will be analysed using visual analysis and multi-level modelling to track individual participant changes over 9 months from baseline to follow up39.\n\nThe secondary outcome measures will be analysed for pre-post differences. Data will be analysed using SPSS version 22 statistical software. It is likely that we will be underpowered to detect any significant statistical differences between the pre and post intervention scores; therefore effect sizes (Cohen’s d) will also be calculated. The parent, teacher and adolescent responses to the questionnaires will also be compared to investigate the consistencies between different informants.\n\nWe anticipate that adolescent informants will report the greatest positive changes compared to other informants. We also anticipate that the adolescents will report greater improvements on the social knowledge on the TASSK-R, than on the social performance on the SCP or SDQ (prosocial or peer scale) and social cognition on the SASI. We also expect to see secondary improvements on adolescent self-reports of anxiety on the BAI raw score, social anxiety on the SWS raw total score and self-esteem on the RSE raw total score. We expect to see an increase in camouflaging on the CAT-Q on all the subscales.\n\nIn line with previous social skills intervention research we anticipate that positive changes in social performance will be noted by the parents, but that schoolteachers will not observe a change post intervention on the SRS, SDQ and SWS. Specifically we expect parents to report improvements in the SWS total raw score, as well as improvements on the SDQ raw prosocial scale and peer difficulties scale, and improvements on the SRS social communication scale and repetitive and ritualised behaviours scale.\n\nThe acceptability of the intervention to families will be assessed using the IAQ. Descriptive statistics will be used to summarise the responses alongside a qualitative summary of the open text answers. We expect that most families will report having positive experiences of the PEERS programme. However, in consideration of the substantial time commitment involved, we predict that adherence will be on average 80% and that up to two participating families may dropout.\n\n\nEthics and dissemination\n\nAll participants (young people aged 16–20 and their parent) will give written informed consent prior to entry to the SOAR study. The study has been approved by the West London GTAC Ethics Committee (IRAS: 219817).\n\nThe results of the study will be disseminated at the Turner Syndrome Support Society conference, the study website, at international research conferences and in research articles published in peer-reviewed journals.\n\n\nDiscussion\n\nThis is the first study to pilot a social skills training program with adolescents and young women with TS. Given the PEERS program’s success with teenagers on the spectrum, it is anticipated that young women with TS will also benefit from taking part.\n\nThis pilot study has been designed to take an approach of high internal validity. This approach is appropriate given that it is a feasibility pilot conducted with a small number of participants (n=6–10), however the disadvantage of the approach is that the study has low external validity, which reduces the generalizability of the findings. This study will need to be replicated with young people with different social skills profiles, intellectual ability and hormone treatment status.\n\nTo our knowledge this will also be the first trial of PEERS delivered online and offline. TS is a rare genetic disorder and the delivery of the full program face to face would have resulted in many families being excluded due to geographical constraints. The program’s acceptability to families will be assessed and this feedback will be used to inform future replications of the intervention. Should the combination of online and offline prove successful, this will enable the to program to be made more widely available.\n\nWhen assessing social skills it is important to employ a range of assessment tools, which assess different domains of social skills (social knowledge, performance and cognition), as well as a variety of informants40,41. Meta-analyses of social skills intervention studies show that parents and young people report changes in social skills after taking part in social skills interventions. However, these improvements are rarely reported by teachers41,42. There is a trend for young people to overestimate the changes in their social skills compared to other informants41,42. However, a recent meta-analysis of the young person self-report measures suggests that the improvements relate to changes in their social knowledge rather than their social performance41.\n\nThe assessment battery has been designed to measure changes in social skills, in the domains of social performance, social knowledge and social cognition. These outcomes will be reported on by the parents, teachers and the young people themselves. Teachers and parents will be asked to report on changes in social performance through questionnaires. The young people will complete questionnaires which measure social performance and social knowledge, as well as an online task to measure changes in social cognition. The maintenance of any potential treatment gains in social performance will be assessed by the parent report at a 3 month follow-up.\n\nIt is likely that the adolescent and parent reports will be prone to expectancy biases43. They may exaggerate treatment effects due to their investment in taking part in the intervention. Using external observers (such as teachers or blinded study administrative assessors) is essential to help understand these biases and assess whether changes in performance generalise to other settings41,44. Unfortunately, due to the small scale of this project, assessments by external observers will not be feasible.\n\nMeta-analyses of social skills interventions for children on the autistic spectrum using the SRS have shown that the largest treatment gains are made in the social communication and repetitive and ritualised behaviours scale21. The changes in repetitive and ritualised behaviours may be mediated by reductions in anxiety or increases in social awareness21,45. The majority of the participants included in the meta-analyses were adolescent males, therefore it remains to be seen whether these patterns of improvement will be replicated in females with TS.\n\nThis study will also use a novel measure of social camouflaging46. Social camouflaging is a strategy adopted by people on the spectrum to manage social situations. It has been likened to wearing a ‘social mask’, where the individual puts on ‘their best self’46. Camouflaging typically involves masking and compensating for social deficits46–48. This might involve consciously performing a range of non-verbal cues such as making eye contact during conversations and imitating facial expressions and gestures, or following learnt social scripts such as using pre-prepared jokes or comments49. Recent research suggests that females are better at camouflaging than males48,50. We anticipate that the intervention will help the participants become more aware of their camouflaging and help them to camouflage more effectively if they choose to use it as a strategy.\n\n\nConclusion\n\nThis will be the first social skills training programme trialled with adolescents and young women with TS. Should the trial prove successful, the initial results will be used to inform the sample size for a future randomised controlled trial. Additionally, neither research trials using the PEERS program exclusively in girls, nor trials delivering PEERS online have been published. Therefore, this trial may have a broader impact on the development of treatment strategies for both for young women that experience social skills difficulties (including those on the autistic spectrum), but also for broadening access to treatment by using technology.\n\n\nData availability\n\nNo data are associated with the article.",
"appendix": "Grant information\n\nThis work was supported by BRC Infrastructure and Child Health Research Charitable Incorporated Organisation.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Young Person Intervention Acceptability Questionnaire; Parent Intervention Acceptability Questionnaire.\n\nClick here to access the data.\n\n\nReferences\n\nJacobs P, Dalton P, James R, et al.: Turner syndrome: a cytogenetic and molecular study. Ann Hum Genet. 1997; 61(6): 471–83. PubMed Abstract\n\nGravholt CH, Andersen NH, Conway GS, et al.: Clinical practice guidelines for the care of girls and women with Turner syndrome: proceedings from the 2016 Cincinnati International Turner Syndrome Meeting. Eur J Endocrinol. 2017; [cited 2018 Jun 18]; 177(3): G1–G70. PubMed Abstract | Publisher Full Text\n\nSkuse DH: Turner - Know your body! Gravholt C, editor. Gothenburg: Novo Nordisk; 2009; 200–217.\n\nHong DS, Dunkin B, Reiss AL: Psychosocial functioning and social cognitive processing in girls with Turner syndrome. J Dev Behav Pediatr. 2011; [cited 2018 Jun 18]; 32(7): 512–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnickmeyer RC: Turner syndrome: advances in understanding altered cognition, brain structure and function. Curr Opin Neurol. 2012; 25(2): 144–9. PubMed Abstract | Publisher Full Text\n\nSaad K, Abdelrahman AA, Abdel-Raheem YF, et al.: Turner syndrome: review of clinical, neuropsychiatric, and EEG status: an experience of tertiary center. Acta Neurol Belg. 2014; 114(1): 1–9. PubMed Abstract | Publisher Full Text\n\nSkuse DH, James RS, Bishop DV, et al.: Evidence from Turner’s syndrome of an imprinted X-linked locus affecting cognitive function. Nature. 1997; 387(6634): 705–8. PubMed Abstract | Publisher Full Text\n\nKesler SR: Turner syndrome. Child Adolesc Psychiatr Clin N Am. 2007; 16(3): 709–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCreswell CS, Skuse DH: Autism in association with Turner syndrome: Genetic implications for male vulnerability to pervasive developmental disorders. Neurocase. 1999; [cited 2018 Jun 27]; 5(6): 511–8. Publisher Full Text\n\nChadwick PM, Smyth A, Liao LM: Improving self-esteem in women diagnosed with Turner syndrome: results of a pilot intervention. J Pediatr Adolesc Gynecol. 2014; [cited 2018 Jun 27]; 27(3): 129–32. PubMed Abstract | Publisher Full Text\n\nSchmidt PJ, Cardoso GM, Ross JL, et al.: Shyness, social anxiety, and impaired self-esteem in Turner syndrome and premature ovarian failure. JAMA. 2006; [cited 2018 Jun 18]; 295(12): 1374–6. PubMed Abstract | Publisher Full Text\n\nReimann GE, Bernad Perman MM, Ho PS, et al.: Psychosocial Characteristics of Women with a Delayed Diagnosis of Turner Syndrome. J Pediatr. 2018; [cited 2018 Jun 14]; 199: 206–211. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCardoso G, Daly R, Haq NA, et al.: Current and lifetime psychiatric illness in women with Turner syndrome. Gynecol Endocrinol. 2004; [cited 2018 Jun 18]; 19(6): 313–9. PubMed Abstract | Publisher Full Text\n\nElliott SN, Malecki CK, Demaray MK: New Directions in Social Skills Assessment and Intervention for Elementary and Middle School Students. Exceptionality. 2001; [cited 2018 Jun 27]; 9(1–2): 19–32. Publisher Full Text\n\nRoff M, Sells SB, Golden M: Social Adjustment and Personality Development in Children. University of Minnesota Press; 1972; [cited 2018 Jun 27]; 217. Reference Source\n\nSpence SH: Social Skills Training with Children and Young People: Theory, Evidence and Practice. Child Adolesc Ment Health. 2003; [cited 2018 Jun 27]; 8(2): 84–96. Publisher Full Text\n\nCoie J, Terry R, Lenox K, et al.: Childhood peer rejection and aggression as predictors of stable patterns of adolescent disorder. Dev Psychopathol. 1995; [cited 2018 Jun 27]; 7(4): 697–713. Publisher Full Text\n\nLaugeson EA, Frankel F: Social skills for teenagers with developmental and autism spectrum disorders: The PEERS treatment manual. Routledge. 2011. Reference Source\n\nLaugeson EA, Frankel F, Mogil C, et al.: Parent-assisted social skills training to improve friendships in teens with autism spectrum disorders. J Autism Dev Disord. 2009; 39(4): 596–606. PubMed Abstract | Publisher Full Text\n\nLaugeson EA, Gantman A, Kapp SK, et al.: A Randomized Controlled Trial to Improve Social Skills in Young Adults with Autism Spectrum Disorder: The UCLA PEERS® Program. J Autism Dev Disord. 2015; 45(12): 3978–89. PubMed Abstract | Publisher Full Text\n\nWolstencroft J, Robinson L, Srinivasan R, et al.: A Systematic Review of Group Social Skills Interventions, and Meta-analysis of Outcomes, for Children with High Functioning ASD. J Autism Dev Disord. 2018; 48(7): 2293–2307. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchohl KA, Van Hecke AV, Carson AM, et al.: A replication and extension of the PEERS intervention: examining effects on social skills and social anxiety in adolescents with autism spectrum disorders. J Autism Dev Disord. 2014; 44(3): 532–45. PubMed Abstract | Publisher Full Text\n\nAmundson E, Boman UW, Barrenas ML, et al.: Social Responsiveness Scale, (SRS-2)(Western Psychological Services, Torrance, CA). J Autism Dev Disord. 5th ed. 2012; 19(1): 629–34.\n\nGantman A, Kapp SK, Orenski K, et al.: Social skills training for young adults with high-functioning autism spectrum disorders: a randomized controlled pilot study. J Autism Dev Disord. 2012; [cited 2018 Jun 27]; 42(6): 1094–103. PubMed Abstract | Publisher Full Text\n\nFord T, Goodman R, Meltzer H: The British Child and Adolescent Mental Health Survey 1999: the prevalence of DSM-IV disorders. J Am Acad Child Adolesc Psychiatry. 2003; [cited 2018 Jun 27]; 42(10): 1203–11. PubMed Abstract | Publisher Full Text\n\nGreen H, McGinnity Á, Meltzer H, et al.: Mental health of children and young people in Great Britain, 2004. 2004. Reference Source\n\nHeiervang E, Stormark KM, Lundervold AJ, et al.: Psychiatric disorders in Norwegian 8- to 10-year-olds: an epidemiological survey of prevalence, risk factors, and service use. J Am Acad Child Adolesc Psychiatry. 2007; [cited 2018 Jun 27]; 46(4): 438–47. PubMed Abstract | Publisher Full Text\n\nEmerson E, Hatton C: Mental health of children and adolescents with intellectual disabilities in Britain. Br J Psychiatry. 2007; [cited 2018 Jun 27]; 191: 493–9. PubMed Abstract | Publisher Full Text\n\nGoodman A, Lamping DL, Ploubidis GB: When to use broader internalising and externalising subscales instead of the hypothesised five subscales on the strengths and difficulties questionnaire (SDQ): data from British parents, teachers and children. J Abnorm Child Psychol. 2010; [cited 2018 Jun 27]; 38(8): 1179–91. PubMed Abstract | Publisher Full Text\n\nConstantino J, Gruber C: Social responsiveness scale (SRS). 2012; [cited 2018 Jun 27]. Reference Source\n\nConstantino J, Gruber C: Social responsiveness scale (SRS): Western Psychological Services Los Angeles. 2007; [cited 2018 Jun 27].\n\nCameron-Pimblett A, La Rosa C, King TFJ, et al.: The Turner syndrome life course project: Karyotype-phenotype analyses across the lifespan. Clin Endocrinol (Oxf). 2017; [cited 2018 Jun 29]; 87(5): 532–8. PubMed Abstract | Publisher Full Text\n\nSkuse D, Lawrence K, Tang J: Measuring social-cognitive functions in children with somatotropic axis dysfunction. Horm Res. 2005; [cited 2018 Jun 27]; 64 Suppl 3: 73–82. PubMed Abstract | Publisher Full Text\n\nWechsler D, Coalson DL, Railford SE: WAIS-IV. Wechsler Adult Intelligence Scale: Fourth Edition. Fourth. San Antonio: TXNCS Pearson; 2008. Reference Source\n\nSpence SH: Social skills training: Enhancing social competence with children and adolescents. Nfer-Nelson; 1995. Reference Source\n\nRosenberg M: Society and the adolescent self-image. Princeton: NJ Princeton University Press; 1965. Reference Source\n\nBeck AT, Steer RA, Ball R, et al.: Use of the Beck Anxiety and Depression Inventories for Primary Care with Medical Outpatients. Assessment. 1997; [cited 2018 Jun 27]; 4(3): 211–9. PubMed Abstract | Publisher Full Text\n\nHull L, Petrides KV, Mandy W: Does Intention Lead to Success? Social Camouflaging in Autistic Girls. In: INSAR 2018 Annual Meeting. Rotterdam; 2018; [cited 2018 Jun 6]. Reference Source\n\nSmith JD: Single-case experimental designs: a systematic review of published research and current standards. Psychol Methods. 2012; [cited 2018 Jun 27]; 17(4): 510–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReichow B, Barton EE, Boyd BA, et al.: Early intensive behavioral intervention (EIBI) for young children with autism spectrum disorders (ASD). Cochrane Database Syst Rev. 2012; 10: CD009260. PubMed Abstract | Publisher Full Text\n\nGates JA, Kang E, Lerner MD: Efficacy of group social skills interventions for youth with autism spectrum disorder: A systematic review and meta-analysis. Clin Psychol Rev. 2017; [cited 2018 Jun 27]; 52: 164–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaat AJ, Lecavalier L: Group-based social skills treatment: a methodological review. Res Autism Spectr Disord. 2014; 8(1): 15–24. Publisher Full Text\n\nMcMahon CM, Lerner MD, Britton N: Group-based social skills interventions for adolescents with higher-functioning autism spectrum disorder: a review and looking to the future. Adolesc Health Med Ther. 2013; 2013(4): 23–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams White S, Keonig K, Scahill L: Social skills development in children with autism spectrum disorders: a review of the intervention research. J Autism Dev Disord. 2007; 37(10): 1858–68. PubMed Abstract | Publisher Full Text\n\nRodgers J, Glod M, Connolly B, et al.: The relationship between anxiety and repetitive behaviours in autism spectrum disorder. J Autism Dev Disord. 2012; 42(11): 2404–9. PubMed Abstract | Publisher Full Text\n\nHull L, Petrides KV, Allison C, et al.: “Putting on My Best Normal”: Social Camouflaging in Adults with Autism Spectrum Conditions. J Autism Dev Disord. 2017; [cited 2018 Jun 27]; 47(8): 2519–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAttwood T: The complete guide to Asperger’s syndrome. Jessica Kingsley Publishers; 2006; 397. Reference Source\n\nLai MC, Lombardo MV, Ruigrok AN, et al.: Quantifying and exploring camouflaging in men and women with autism. Autism. 2017; 21(6): 690–702. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLai MC, Baron-Cohen S: Identifying the lost generation of adults with autism spectrum conditions. Lancet Psychiatry. 2015; 2(11): 1013–27. PubMed Abstract | Publisher Full Text\n\nDean M, Harwood R, Kasari C: The art of camouflage: Gender differences in the social behaviors of girls and boys with autism spectrum disorder. Autism. 2017; [cited 2018 Jun 4]; 21(6): 678–89. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "41256",
"date": "27 Dec 2018",
"name": "David E. Sandberg",
"expertise": [
"Reviewer Expertise I am a pediatric psychologist involved in clinical care and research focusing on people born with disorders/differences of sex development (DSD): TS is classified as a “sex chromosome DSD”. I served as co-lead for the neurocognition and behaviour section of the updated 2017 clinical practice guidelines for DSD the investigators refer to in the Introduction to their proposal. I have been funded by the US National Institutes of Health for methods development and intervention studies in the area of DSD. I was also a member of the writing group for the Consensus Statement on DSD."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study protocol describes a pilot project that examines the effectiveness of a social skills training program – originally developed for youth on the autism syndrome spectrum – applied to the social skills deficits of adolescent and young women with Turner syndrome (TS)(45,X). The neurocognitive profile of girls and women with TS has been extremely well documented and has repeatedly been shown to be associated with deficits in social cognition and skills. This aspect of the TS phenotype is likely a significant factor accounting for the gap between educational attainment in this population (shown to exceed to population norms), and their occupational status and measures of independence from family caregivers. Women with TS have also been shown to exhibit both delays and arrest in psychosexual milestones which are more than likely linked to the characteristic social behaviour phenotype associated with this karyotype1.\n\nThe investigators should be commended for proposing to adopt a proven efficacious and effective intervention for social skills deficits to potentially modify the behavioural phenotype in TS. The PEERS program is well-suited to the task because of the similarities in social skills deficits in high-functioning ASD and TS. Work on this topic is long overdue.\n\nThe rationale for the pilot study is well described, although the authors have possibly overstated, in the Introduction, the lack of “systematic evaluations of the mental health of young women with TS…”. In fact, there are multiple studies assessing both the psychiatric status and psychosocial/sexual adaptation of this population. What has been sorely missing are psychosocial interventions to potentially ameliorate deficits, and the proposed study is directed precisely toward this objective.\n\nThere are the following elements I found missing from the protocol or require further consideration:\np.2 Study Design - it’s unclear what the following refers to: “All participants will be matched for age, degree of social impairment, intellectual ability and hormone therapy treatment.” Each participant will serve as their own control, so I don’t understand the “matching” piece. p. 2. Participant inclusion and exclusion criteria – details are not provided regarding the SOAR questionnaire screening for eligibility based on social skills deficits. Will recruitment be restricted to girls/women with a 45,X karyotype or will those with a variant, including chromosomal mosaicism, be eligible? The Discussion notes that self and parent reports are prone to bias because of expectations regarding the intervention and note that a remedy to overestimating the benefits can come from employing external observers. The investigators justify not employing external observers because of the small scale of this project. However, one could turn that argument around by questioning whether it would be worthwhile to pursue a full-scale trial of PEERS in TS if the effects observed in the pilot are driven by biased reports.\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": "4438",
"date": "18 Mar 2019",
"name": "Jeanne Wolstencroft",
"role": "Author Response",
"response": "Dear David Sandberg, Thank you for your comments. We are encouraged to hear that you are convinced of the value of using the PEERS protocol with young women with TS. We have addressed your concerns in turn below: 1.Participants in the treatment group were chosen because they have similar degrees of social impairment and intellectual ability. We have clarified the wording around the ‘matching of participants’ in the protocol v2. You are correct, they will act as their own controls. 2.Motivation to take part in the intervention is assessed using the PEERS screener interview (Laugeson et al., 2009). It was essential to ensure that the young people and their parents were motivated to take part in the social group in order to minimize potential attrition over the 12 week intervention period. Their social deficits were assessed using a combination of clinical judgement and screening questionnaires: Social Aptitude Scale (SAS): The SAS is presented as part of the Development and Wellbeing Assessment’s autism module. The SAS is a ten item parent-report measure which assesses social understanding and social ability (as opposed to peer interaction deficits) (Liddle et al., 2008). Strengths and Difficulties Questionnaire (SDQ): The SDQ is a brief behavioural screening questionnaire (Goodman et al., 2010). The SDQ includes scales that measure emotional symptoms, conduct problems, hyperactivity/inattention difficulties, peer relationship problems and prosocial behaviour. 3.Recruitment will not be restricted to young women with a monosomic non-mosaic 45,X karyotype. This has now been clarified in the protocol v2. 4.Objectively evaluating the outcome of social skills interventions presents a number of challenges. We agree that expectancy biases are likely to occur. Some outcome measures focus only on self-assessment of social performance or social knowledge. Questionnaires that have been designed for parents, teacher or adult observer respondents, may lack ecological validity. To our knowledge, the outcome of PEERS has not yet been measured by peer-ratings of change. We are currently testing a novel methodology that could address this deficiency. Currently, we do have some potentially objective measures of change. We obtain teacher ratings of social behaviour, parent reports of changes in the TS girl’s social relationships, and individual increases in social knowledge. As we have indicated, the intervention is designed to take account of individual differences. Accordingly, outcomes are diverse. There will be variability within the sample, and it is unlikely that treatment benefits will be captured by standardized questionnaires alone. Jeanne Wolstencroft References: Goodman, A., Lamping, D. L., & Ploubidis, G. B. (2010). When to use broader internalising and externalising subscales instead of the hypothesised five subscales on the Strengths and Difficulties Questionnaire (SDQ): data from British parents, teachers and children. Journal of abnormal child psychology, 38(8), 1179-1191. Laugeson, E. A., Frankel, F., Mogil, C., & Dillon, A. R. (2009). Parent-assisted social skills training to improve friendships in teens with autism spectrum disorders. Journal of autism and developmental disorders, 39(4), 596-606. Liddle, E. B., Batty, M. J., & Goodman, R. (2009). The social aptitudes scale: an initial validation. Social psychiatry and psychiatric epidemiology, 44(6), 508."
}
]
},
{
"id": "41839",
"date": "15 Jan 2019",
"name": "Claus H. Gravholt",
"expertise": [
"Reviewer Expertise Turner syndrome. Other sex chromosome abnormalities. Endocrinology",
"epidemiology",
"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\nWolstencroft et al presents a pilot protocol designed to improve the social cognition of adolescents with Turner syndrome (TS) based on the PEERS program. Overall, this study is very interesting and timely, given that many with TS have a social skills deficit.\n\nI have some comments:\nDesign and sample size: uncontrolled, with the aim of including 6-10 TS. The uncontrolled design is acceptable in a pilot trial. I’m more worried about the rather low n. There is a large variability, perhaps even larger than among normal females, in the presentation of females with TS and that may not be captured satisfactorily with a n of 6-10. However, one could ask if it is at all necessary to perform a pilot study, given that this program has shown to be a success in other study groups? Inclusion criteria: the inclusion criteria are rather strict, and I think that the authors will end up excluding a rather large proportion of females with TS, which is a pity. Many females that in their youth may not present with social skills difficulties, will actually do this at a later age, and I think it would be interesting to have some of these females included as well. Can females with hearing difficulties, but treated with a hearing aid, be included? The intervention program, PEERS, is certainly very relevant. The scales used to monitor effect seem relevant. The primary and secondary outcomes are relevantly described. The intervention program seems rather massive with multiple scales and 12 times 90 minutes interventions. Have the authors considered how this will affect the participation rate in the study? I guess they must have contemplated this. Are there experience from other groups of patients? The authors expect 2 family dropouts – and if the inclusion ends at 6 families, that would then leave 4 families – hardly enough to call it a pilot study? Conclusively, if this pilot study proves successful, it will be a welcome addition to the program of care established by excellence center for TS around the world.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "4439",
"date": "18 Mar 2019",
"name": "Jeanne Wolstencroft",
"role": "Author Response",
"response": "Dear Claus Gravholt, Thank you for your comments. We are encouraged to hear that you are convinced of the value of using the PEERS protocol with young women with TS. We have addressed your concerns in turn below: 1.As you correctly point out, there is large variability between young women with TS. Our pilot will only recruit young women experiencing difficulties with friendships who wish to improve their social skills, but within this group there will still be a substantial amount of variability. We are currently conducting a survey of mental health and social skills difficulties in TS (SOAR Study). Our preliminary findings indicate that girls and women with TS have significantly more peer interaction problems when compared to population female norms as measured by the Strengths and Difficulties Questionnaire (Goodman et al., 2010). On our measure of autistic symptomatology (SRS-2; Constantino et al., 2012) 40% of young women with TS scored in the normal range, 14.5% in the mild range, 17.1% in the moderate range and 27.4% in the severely impaired range (SOAR study unpublished findings; n=117). Our sample for the PEERS pilot is representative of this range of social skills difficulties; among our enrolled participants, scores lie in the normal to abnormal range on the Strengths and Difficulties subscale for quality of peer-interactions and they also range from normal to severely impaired on the SRS-2. We believe it is necessary to conduct a pilot of the PEERS program with young women with TS before conducting a full-scale trial because the program was initially developed to treat adolescent boys with autism. The emerging literature on young women with autism shows clearly that women with social communication impairments face different challenges to those experienced by young men with autistic traits. We will therefore need to adapt the content of the program. For example, some of the lessons focus on issues such as ‘good sportsmanship’ and ‘appropriate uses of humour’; females with TS would not regard these skills as being of core relevance to their social adaptation. Additionally, there is no precedent for delivering social skills training online, therefore there is a need to pilot the acceptability of virtual meeting rooms and to adapt the behavioural rehearsal components of the training to an online environment. 2.You are correct in noting that many girls with TS have impaired adaptation to the social environment that manifests most obviously once they enter adolescence. We have found that social difficulties emerge and intensify over that period, from the time of entry into secondary education to early adulthood. Therefore, our pilot study’s age range (16-20 years) is designed to help young women at a time when their social difficulties are emerging, and they are becoming aware of them. We are not excluding anyone on the grounds of impaired hearing, if that problem is being successfully managed. Two young women who wear hearing aids are currently enrolled in the pilot study. Unfortunately, the program would need to be substantially modified in order to accommodate those with more profound hearing impairments. The inclusion criteria have been clarified in the protocol v2. 5. As originally devised, the PEERS program required there to be no more than 8-10 participants in the treatment group. The program is very intensive and is characterized by a focus on individual needs as well as on group dynamics. Several staff are required on site to manage the child/parent groups. By adapting the program to be delivered from an online platform we aim to increase its acceptability to participants and to reduce the associated costs. Randomized control trials of PEERS have reported attrition rates of 7-13% (Schohl et al.,2014; Laugeson et al., 2015), hence our prediction that 1-2 participants out of 10 may drop out. However, we are now three-quarters of the way through the pilot trial and we have not yet had any dropouts. Best wishes, Jeanne Wolstencroft References: Constantino, J. N., & Gruber, C. P. (2012). Social responsiveness scale–second edition (SRS-2). Torrance: Western Psychological Services. Goodman, A., Lamping, D. L., & Ploubidis, G. B. (2010). When to use broader internalising and externalising subscales instead of the hypothesised five subscales on the Strengths and Difficulties Questionnaire (SDQ): data from British parents, teachers and children. Journal of abnormal child psychology, 38(8), 1179-1191. Laugeson, E. A., Gantman, A., Kapp, S. K., Orenski, K., & Ellingsen, R. (2015). A randomized controlled trial to improve social skills in young adults with autism spectrum disorder: The UCLA PEERS® program. Journal of autism and developmental disorders, 45(12), 3978-3989. Schohl, K. A., Van Hecke, A. V., Carson, A. M., Dolan, B., Karst, J., & Stevens, S. (2014). A replication and extension of the PEERS intervention: examining effects on social skills and social anxiety in adolescents with autism spectrum disorders. Journal of Autism and Developmental Disorders, 44(3), 532-545."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1864
|
https://f1000research.com/articles/8-301/v1
|
18 Mar 19
|
{
"type": "Research Note",
"title": "Comparative antimicrobial studies on plant species known as ’Pasak Bumi': Eurycoma longifolia Jack., Rennelia elliptica Korth. and Trivalvaria macrophylla miq.",
"authors": [
"Harlinda Kuspradini",
"Sisilia Silau",
"Supartini Supartini",
"Enih Rosamah",
"ICTROPS",
"Sisilia Silau",
"Supartini Supartini",
"Enih Rosamah"
],
"abstract": "Pasak Bumi is a local name for a medicinal plant in Kalimantan, Indonesia. It is a famous medicinal plant and commonly used in traditional medicine as an aphrodisiac, as well as in the treatment of malaria. Pasak Bumi is a commercial name for Eurycoma longifolia (Simaroubaceae) plant species. Besides Eurycoma longifolia there are two other plant species also known locally as Pasak Bumi, Rennelia elliptica (Rubiaceae) and Trivalvaria macrophylla (Annonaceae). This study was performed to investigate the antimicrobial activities of the different species of Pasak Bumi and its total phenol contents. The antimicrobial activity of the ethanol extract was determined using the Agar Well Diffusion method at various concentrations while the phenol content was determined by the Folin - Ciocalteu method. The results of the ethanol extract from the different root showed that the T. macrophylla had the highest phenol content, and the highest activity index (AI) was found in the E. longifolia (0.96 at 1000 µg concentration). The results of this study show that the three different Pasak Bumi have potential as antimicrobials against oral pathogen; 1 yeast: Candida albicans, and 3 bacterias: Staphylococcus aureus, Streptococcus mutans, and Streptococcus sobrinus.",
"keywords": [
"Pasak Bumi",
"Eurycoma longifolia",
"Trivalvaria macrophylla",
"Rennelia elliptica",
"antimicrobial activity"
],
"content": "Introduction\n\nPasak Bumi is a plant used in traditional medicinal that grows in the tropical forests of Kalimantan of Indonesia. It is used by the local people as an aphrodisiac, for postpartum treatment, fever, and malaria1,2. In Central Kalimantan, there are three different plant species on Pasak Bumi; Yellow Pasak Bumi (Eurycoma longifolia Jack., Simaroubaceae), Red Pasak Bumi (Rennelia elliptica, Rubiaceae) and Black Pasak Bumi (Trivalvaria macrophylla, Rubiaceae)3. Previous research of Pasak Bumi (E. longifolia Jack) from different regions has shown activity in inhibiting the growth of microbes, however, research on the other species of Pasak Bumi such as Rennelia elliptica and Trivalvaria macrophylla are still limited. From the above information, the research aim is to compare the inhibition activities of the three different plants against one yeast: Candida albicans, and three bacterias: Staphylococcus aureus, Streptococcus mutans, and Streptococcus sobrinus. The research was also designed to extend our knowledge and help us explore the antimicrobial activities of the three different plant species.\n\n\nMethods\n\nOne kilogram of each plant was excavated and harvested from Katingan district, Central Kalimantan. The root was chopped and separated from its stem and leaves. The roots were sliced into small sections with a knife and allowed to dry under shade. The dried samples were crushed into powder using an electric blender. Once crushed, 50 grams of each powder of the plant root was weighed using a digital balance (Mettler Toledo, Mettler-Tokyo Group). Furthermore, the powder was extracted using successive maceration with the following solvents: n-hexane, ethyl acetate, and 96% ethanol. The ethanol filtrate was evaporated under a vacuum rotary evaporator (Eyela, N-N series) at 35°C until dry and used for the present study (Figure 1).\n\nThe total phenolic content was determined spectrophotometrically (UV Mini 1240 Shimadzu) in accordance to the Folin-Ciocalteu method4. The sample solution was prepared by dissolving the dry extracts (2 mg) in 100 μl DMSO and 900 μl of distilled water. The reaction mixture was made by mixing 200 µl of the extract from sample solution (200 μg/mL), 300 μl of distilled water, 250 μl of 10% Folin-Ciocalteu reagent (Merck Millipore, CAS No. 109001) and 1250 μl of 7.5% sodium carbonate. After a 90 minutes incubation at room temperature, the absorbance was determined spectrophotometrically at 760 nm. Gallic acid (Wako, CAS No. 5995-86-8) was used as a reference standard for plotting a calibration curve (concentration range: 2 to 10 μg/mL). The total phenolic content was expressed as a Gallic Acid Equivalent (GAE)/mg extract, using a standard calibration graph.\n\nFour pathogenic microbial strains; C. albicans (CA), S. aureus (SA), S. mutans (SM) and S. sobrinus (SS) from the Forest Product Chemistry Laboratory’s culture collections, were used for the present study. The in vitro activity was screened using the agar well diffusion method in Nutrient Agar medium5,6. The extracts of each plant at a concentration of 10 mg/ml in 40% ethanol were prepared, and an aliquot of the test solution was put in to get a final concentration of 100, 250, 500, and 1000 μg/well. It was then placed on the inoculated nutrient agar plates and incubated for ±18–24 h at 37˚C. Ten μg/well of chloramphenicol (PT. Indofarma, Tbk., Indonesia) and 40% ethanol were employed as a positive and negative control. After incubation, the diameter of the inhibition zones was measured by a ruler. The experiment was performed in triplicate. The antimicrobial index (AI) was calculated using the formula6,7: Activity index (AI) = Inhibition Zone of the sample/Inhibition Zone of chloramphenicol.\n\n\nStatistical analysis\n\nAll experiments were conducted three times. Regression analysis was used to make a calibration curve and calculate the total phenol content. All statistical analyses used Microsoft Excel 2010 software.\n\n\nResults\n\nThe total phenolic contents were calculated using the following linear equation based on the calibration curve of gallic acid: y = 0.0667x + 0.009; R2 = 0.9948, where y is absorbance and x is amount of gallic acid in µg (Table 1). T. macrophylla root extract obtaining higher total phenolic content in comparison to E. longifolia and R. elliptica. The extracts exhibited dose-dependent antimicrobial activities (Figure 2), and the results indicated that the in vitro antimicrobial activity of the T. macrophylla, E. longifolia, and R. elliptica extracts were ranked in the following order; SS>SM>SA>CA; SA>SM>SS>CA; and SS>SA>CA>SM, respectively. The highest activity was found in E. longifolia against S. aureus, with a maximum AI value (0.96) at 1000 μg/well concentration while the lowest activity at all concentration was found in R. elliptica extracts.\n\n\nDiscussion\n\nPlant extracts with a high AI value indicates that the extracts have good antimicrobial activity against the selected pathogens6. The inhibitory activity of E. longifolia root extracts was in agreement with previous literature, it could inhibit S. aureus8–10 and C. albicans11. R. elliptica was found to be able to inhibit the growth of C. albicans and S. aureus, which is contrary to a previous study where it was found to be inactive12; however, there was no information about the extraction method for R. elliptica and the concentration used on that study. So far there have been no reports of the T. macrophylla being antimicrobial, but in this study T. macrophylla has proven to be an inhibitor for the growth of S. aureus, S. mutans, S. sobrinus and C. albicans. This is believed to be the first report to explore and compare the antimicrobial potentials of the three different Pasak Bumi plants. The antimicrobial activity may be attributed to the high content of the phenols present. Phenolic compound such as gallic acid can causes irreversible changes (such as charge, intra and extracellular permeability, and physicochemical properties) in the properties of microbial membranes, with consequent leakage of essential intracellular constituents13. E. longifolia possess a higher antimicrobial activity than T. macrophylla, but its phenolic content was lower than T. macrophylla. E. longifolia extract might contain more non-phenolic compounds, or possess phenolic compounds that contain a higher number of active groups than the other extract. The interactions between chemical compounds (phenolic and non-phenolic compounds) might also be responsible for the antimicrobial effects14.\n\n\nConclusions\n\nThe present study performed in vitro studies of antimicrobial properties of three different Pasak Bumi (E. longifolia Jack, R. elliptica and T. macrophylla) on oral pathogens which gave positive results and different degree of activity.\n\n\nData availability\n\nUnderlying data is available form Open Science Framework\n\nOSF: Dataset 1. Pasak Bumi root extract, https://doi.org/10.17605/OSF.IO/Q6X7R15\n\nLicense: CC0 1.0 Universal",
"appendix": "Grant information\n\nThis work was supported by the Forest Product Chemistry Laboratory, Forestry Faculty of Mulawarman University 2017/2018 and DIPA B2P2EHD 2017.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors gratefully acknowledge the Dipterocarps Forest Ecosystem Research and Development Center, Samarinda, East Kalimantan and members of the Laboratory of Forest Products Chemistry, Mulawarman University.\n\n\nReferences\n\nYusro F, Mariani Y, Diba F, et al.: Inventory of medicinal plants for fever used by four dayak sub ethnic in West Kalimantan, Indonesia. Kuroshio Science. 2014; 8(1): 33–38. Reference Source\n\nOsman CP, Ismail NH: A review on the chemistry and pharmacology of Rennellia elliptica Korth. Indonesian Journal of Tropical and Infectious Disease. 2017; 6(6): 131–140. Publisher Full Text\n\nSupartini S: Teknik pemanenan akar pasak bumi secara tradisional. Prosiding ekspose hasil-hasil penelitian Balai Besar Litbang Ekosistem Hutan Dipterokarpa, 165–174.\n\nSingleton VL, Orthofer R, Lamuela-Raventos RM: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Meth Enzymol. 1999; 299: 152–178. Publisher Full Text\n\nBalouiri M, Sadiki M, Ibnsouda SK: Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal. 2016; 6(2): 71–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuspradini H, Putri AS, Mitsunaga T: Chemical composition, antibacterial and antioxidant activities of essential oils of Dryobalanops lanceolata Burck. Leaf. Res J Med Plants. 2018; 12(1): 19–25. Publisher Full Text\n\nSridhar N, Duggirala SL, Puchchakayala G: Antimicrobial activity of ethanolic extracts of Justicia neesii. Bangladesh J Pharmacol. 2014; 9(4): 624–627. Publisher Full Text\n\nDanial M, Saghal G, Mubbarakh SA, et al.: Antibacterial studies on in vivo plant parts of medicinally important Eurycoma longifolia (Tongkat Ali). Pak J Bot. 2013; 45(5): 1693–700. Reference Source\n\nKhanam Z, Wen CS, Bhat IUH: Phytochemical screening and antimicrobial activity of root and stem extracts of wild Eurycoma longifolia Jack (Tongkat Ali). Journal of King Saud University – Science. 2015; 27(1): 23–30. Publisher Full Text\n\nFaisal GG, Zakaria SM, Najmuldeen GF: In vitro antibacterial activity of Eurycoma longifolia Jack (Tongkat Ali) root extract. The International Medical Journal Malaysia. 2015; 14(1): 77–81. Reference Source\n\nFaisal GG, Zakaria SM, Najmuldeen GF, et al.: Antifungal activity of Eurycoma longifolia Jack (Tongkat Ali) root extract. J Int Dent Med Res. 2016; 9(1): 70–74. Reference Source\n\nPyla R, Kim TJ, Silva JL, et al.: Enhanced antimicrobial activity of starch-based film impregnated with thermally processed tannic acid, a strong antioxidant. Int J Food Microbiol. 2010; 137(2–3): 154–160. PubMed Abstract | Publisher Full Text\n\nBorges A, Ferreira C, Saavedra MJ, et al.: Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microb Drug Resist. 2013; 19(4): 256–265. PubMed Abstract | Publisher Full Text\n\nRachdiati H, Zakariya NA: Ethnobotanical survey phytochemical and antimicrobial screening on Temiar community at Kg. Husin, Jalong Tinggi, Sungai Siput (U), Perak West Malaysia. Der Pharma Chemica. 2018; 10(1): 26–29. Reference Source\n\nKuspradini H: Pasak Bumi root extract. 2019. http://www.doi.org/10.17605/OSF.IO/Q6X7R"
}
|
[
{
"id": "45864",
"date": "07 May 2019",
"name": "Sarifah Nurjanah",
"expertise": [
"Reviewer Expertise process engineering",
"essential oil",
"microbiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting manuscript describing the antimicrobial activity of tree species plants known as Pasak Bumi (Eurycoma longifolia, Rennelia elliptica and Trivalvaria macrophylla) against bacteria: Staphylococcus aureus, Streptococcus mutans and Streptococcus sobrinus and yeast: Candida albicans. The study proved that Pasak Bumi not only can be used as an aphrodisiac, but also as a postpartum treatment for fever and malaria. It also has the potential as an antimicrobial agent. The paper is well written and structured, but there are some suggestions as follows:\n\nIntroduction:\nIn line 8, clarify the references of research on Pasak Bumi that have been done (references number 8, 9, 10 and 11).\n\nIn line 11 the authors said that there is no research on R. elliptica yet. This is not in accordance with what is written on the Discussion line 5-9 which states that there were studies on the antimicrobial activity of R. elliptica against C. albicans and S. aureus. So, it should be explained in the Introduction that there were antimicrobial studies on R. elliptica as well as E. longifolia.\n\nMethods:\nThe authors used 760 nm wavelengths on the spectrometer in determining phenol content (used the Folin-Ciocalteu method), what is the reason for the use of these wavelengths? Do you use the results of other research or do you have your own tests? We recommend that you mention the basis used. Based on several studies there were also 750 nm (Rollando and Monica, 20183) or 765 nm (Pourmorad et al., 20064) used.\n\nIt is not mentioned how long the maceration process was carried out for. We recommend that you write down how long the maceration process was for each solution (n-hexana, ethyl acetate and ethanol).\n\nIn determining the total phenol content, what DMSO stands for should be stated. Is it dimethyl sulfoxide?\n\nDiscussion:\nThe phenol component is thought to be a component that is responsible for antimicrobial properties. Although, the result showed that T. macrophylla contains higher phenol than E. longifolia but did not show higher antimicrobial activity. For this reason, it is better to find out its chemical composition to determine the components that affect antimicrobial activity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "58440",
"date": "05 Feb 2020",
"name": "Lalit Sharma",
"expertise": [
"Reviewer Expertise Microbial Infections",
"Natural Products"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presents an interesting study in which the antimicrobial activity of three plant species Eurycoma longifolia Jack., Rennelia elliptica Korth. and Trivalvaria macrophylla miq. was investigated.\nWeakness of the manuscript\nIntroduction\nIntroduction part is very poor. The rationale of the need of comparative antimicrobial study of these plant species should have been elaborated.\n\nNo literature about the microbial infections has been cited. Authors needs to add some information about the microbial infections and about the role of herbal plants in treating microbial infections.\n\nAuthors must include the literature about the chief constituents present in these plant species.\n\nAuthors reported that Pasak Bumi is a local plant in Kalimantan, Indonesia. Authors must include other geographical regions where this plant grows.\n\nAuthors must include some information that why only Candida albicans yeast and Staphylococcus aureus, Streptococcus mutans, and Streptococcus sobrinus bacterial strains were taken for the study?\nMethods\nFor identification of the phytoconstituents present in these plant species authors must have included the preliminary phytochemical screening, (a qualitative identification) or must have reported the literature on the phytochemical screening of these plants.\n\nWhy 40% ethanol was used to prepare different concentrations of the plant extracts?\n\nIn figure 1 authors have reported that ±50 gram of sample powder was taken for further maceration with n-hexane. Is there any variations in the initial weight of the powder? Why symbol ± is added? Is there any standard deviation or SEM?\nResults The activity index (Fig. 2) shows very little difference in concentration 500 and 1000 µg/well. As the concentration 1000 µg/well is double of the 500 µg/well so the difference in activity index should have been more. This suggests that something is wrong with the assays and/or with the presentation of the data. Authors need to analyze and discuss this point critically.\n\nSuggestion/corrections in addition to the above\nGrammatical errors/Corrections\nEg. In conclusion The present study performed in vitro studies of antimicrobial properties of three different Pasak Bumi (E. longifolia Jack, R. elliptica and T. macrophylla) on oral pathogens which gave positive results and different degree of activity.\nThe above statement needs to be revised “The present study performed in vitro studies…………?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-301
|
https://f1000research.com/articles/7-1821/v1
|
20 Nov 18
|
{
"type": "Research Article",
"title": "Prevalence and associated risk factors of HIV in prisons in Balochistan, Pakistan: a cross-sectional study",
"authors": [
"Muhammad Dawood Khan",
"Ahmad Wali",
"Razia Fatima",
"Aashifa Yaqoob",
"Shoaib Aziz",
"Ahmad Wali",
"Razia Fatima",
"Aashifa Yaqoob",
"Shoaib Aziz"
],
"abstract": "Background: The prevalence of HIV is 0.1% in Pakistan, with epidemicity in high-risk groups. The infection is on verge of transmission from key populations to the general population through people who inject drugs and sexual transmission. Prisoners are vulnerable to infectious diseases like HIV. This study was conducted in four prisons in Balochistan. Pakistan to determine the prevalence of HIV and associated risk factors. Methods: This cross sectional study was conducted from March to June 2018, in the prisons of Balochistan. WHO-approved rapid diagnostic kits were used for determining the prevalence of HIV and structured interviews were conducted for the assessment of risk factors. Results: Out of 2084 screened prisoners, 33 (1.6%) were found to be positive. A subset of 104 interviews were analysed for risk factors of HIV. Among HIV-infected prisoners 68.8% (OR 4.48; 95% CI 1.41-14.2) had extramarital sex, 43.8% (OR 2.09 95% CI 0.69-6.28) had homosexual experience, and 50% had history of needle sharing (OR 43; 95% CI 7.77-237). About 94% (OR 16.42; 95% CI 2.09-129.81) of prisoners had history of drug addiction of any type while 50% (OR 13; 95% CI 2.82-60.01) of those infected with HIV had a history of using injectable drugs. Around 75% of HIV-infected prisoners had spent 1-5 years in prison, and 25% had spent more than 10 years. Conclusion: The high prevalence of HIV in prisons of Balochistan demands that preventive and treatment strategies should be designed and implemented carefully, allowing early diagnosis and treatment initiation to minimize the spread of infection among the prisons and ultimately their onward transmission into the community.",
"keywords": [
"Prison",
"HIV",
"AIDS",
"prevalence",
"risk-factors."
],
"content": "Introduction\n\nGlobally; about 36.7 million people were living with HIV. HIV infection causes AIDS and is epidemic throughout the world. In 2016; about 1.8 million were reported as newly infected and 1 million died from AIDS related illnesses in the same period (http://www.unaids.org). In Pakistan, an estimated 0.13 million peoples are infected with HIV, out of which only 17% are registered with the AIDS Control programme and 54% of total registered people currently living with HIV/AIDS are getting treatment from antiretroviral therapy (ART) centres (http://www.nacp.gov.pk). The prevalence rate of HIV is 0.1% in Pakistan (http://www.unaids.org). AIDS in Pakistan was limited and epidemic among key populations over the decades, but the latest evidence suggests that there is a shift from key populations through transmission via needle-sharing by injecting drug users (IDUs) to sexual transmission via multiple sexual partners, and further transmission to their intimate partners is increasing1. According to Integrated Biological and Behavioural Survey (IBBS), HIV prevalence was 38.4% among people who inject drugs (PWIDs), about 7.1% among transgender (TG), 3.5% among men who have sex with men and 2.2% among female sex workers1–3.\n\nPrisons are the easily approachable venues for health interventions. Unfortunately, prisoners are among the most marginalized and restricted populations, whose access to health services, interventions and surveys is very limited4. Prisoners are one of the most vulnerable populations for acquiring infectious diseases, especially HIV, because people from diverse backgrounds, communities and with diverse risk factors are kept together for varying time5. The prevalence of HIV is alarmingly increasing in PWIDs and an evidence through IBBS indicated that 38.5% of PWIDs were arrested in past 12 months, which makes the other prisoners also vulnerable as PWIDs have access to drugs in the prisons2. A study of HIV-positive prisoners in five jails in Sindh, Pakistan indicated that 12.3% of prisoners in the jails had ever used drugs by injection in the jails6.\n\nThe revised Pakistan AIDS Strategy III, developed in 2017 by the National AIDS Control Program (NACP) to guide Pakistan’s overall national response for HIV and AIDS till 2020, through focused interventions with set targets, costs, roles and responsibilities, has emphasized on adopting precision targeting of groups (including prisoners) to achieve the required level of impact on the epidemic1. The Balochistan AIDS Control Program (BACP) conducted awareness and screening of prisoners in Central Prison Gaddani in 2017 and found that 27 (6.85%) out of 394 prisoners were positive for HIV. Most of the studies from Pakistan have found that HIV is prevalent at about 2% in prisoners, which is much higher than the prevalence in the general population4,5,7–13.\n\nThe availability of limited data across the different prisons of the province and high prevalence of HIV found in Gaddani central jail warranted evidence to be produced by conducting a detailed study in other prisons of Balochistan as well. The study aimed to assess the prevalence and risk factors of HIV in prisoners of four major prisons in Balochistan.\n\n\nMethods\n\nA descriptive cross-sectional study was carried out from March to June 2018 in four major prisons of Balochistan: Quetta, Gaddani, Mach and Loralai. Balochistan is the most widely spread and less populated province of Pakistan with the population of 12.3 million (http://www.pbs.gov.pk). Area wise it is the largest province and covers 347,190 km2 and comprises of 33 districts (http://www.balochistan.gov.pk). The province is bordered by Afghanistan in the north and north-west, Iran in the south-west, Khyber Pakhtunkhwa province in the north-east and Punjab and Sindh provinces in the east.\n\nBACP is a vertical program of the health department, providing preventive and curative services to high risk population in the province. The program has two treatment centres (ART Centres) located in one of the largest tertiary care hospitals (Bolan Medical Complex Hospital) at the provincial capital Quetta, and District Headquarter (DHQ) hospital of Kech, in the city of Turbat. The program is also running 30 screening centres in DHQ Hospitals. Patients with HIV are referred to ART centres for registration and treatment provision from all the screening centres and other health care facilities of the province.\n\nSpecific settings. There are 11 prisons in Balochistan, of which three are central prisons and remaining eight are district jails14. Jails are meant to keep the prisoners who are under trial or imprisoned for short durations, whereas prisons are meant to keep the prisoners who are imprisoned for long duration. In Balochistan, jails may serve as prisons and prisons may serve as jails due to limited capacity for prisoners and long distances from the trial court. The four prisons were selected on the basis of number of prisoners and type of jail, as these prisons caters majority of the prisoners from Balochistan.\n\nThe provincial program started 6-month-long service delivery packages (SDPs) in four selected prisons of Balochistan from March to August 2018. In these SDPs, screening as well as treatment and preventive services were provided to jail prisoners.\n\nThe study included all the prisoners enrolled at the time of screening irrespective of their status or duration of imprisonment, age, sex, marital status, education status, history of drug addiction, place of origin and employment status. Prisoners were screened for HIV after obtaining written informed consent. Blood specimen collection and screening were conducted in the health facility of jails. Interviews were conducted for assessment of risk factors with HIV. The prisoners found positive on screening were linked with treatment centres in order to commence treatment procedures.\n\nData was collected by a team of trained data collectors that was already involved in screening and provision of SDPs in the mentioned prisons.\n\nScreening was conducted through World Health Organization (WHO)-approved rapid diagnostic kits for HIV Screening (Alere Determine HIV-1/2 Ag/Ab Combo, with sensitivity >99%) provided by the Balochistan AIDS Control Program. The HIV status of HIV-positive prisoners was confirmed by initial confirmatory rapid diagnostic kits (Uni-Gold HIV with Specificity > 99%) and followed by confirmatory rapid diagnostic kits (SD Bioline HIV-1/2 3.0 with Sensitivity > 99%, Specificity > 98%)15.\n\nThe risk factors identification for HIV were assessed through structured a questionnaire developed, pretested and validated by United Nations Office on Drugs and Crime (UNODC), available on OSF19. The interviews primarily were not part of initial agreement between BACP and partner organization delivering the SDPs, but keeping in view the large number of HIV-positive prisoners found in Gaddani prison, they were added to assess the risk factors for HIV. For this purpose, interviews were conducted in special dedicated visits of the prisons. A total of 150 prisoners were approached to take part in the structured interview, out of which 135 prisoners gave their written informed consent to this aspect of the study. Out of 135 interviews conducted 104 were selected for final analysis while the remaining 31 were discarded due to lack of required information and incomplete filling. A total of 16 out of 104 (15.3%) interviewed prisoners were positive for HIV.\n\nThe screening and interview processes were randomly monitored by principal investigator to ensure the quality of data collection.\n\nThe outcome variables of the study included the total number of prisoners screened for HIV and the number and proportion of prisoners found positive for HIV. The presumptive exposure variables assessed were age, sex, education and occupation status of prisoners, type of imprisonment and time spent in imprisonment, ever been arrested for drug related offense (Y/N), marital status and extramarital sex status including homosexual sex status (Y/N), sharing needles, razors and blades (Y/N), history of drug addiction (Y/N) and injectable drug addiction (Y/N) and history of blood transfusion, surgery, dental procedure and tattooing (Y/N).\n\nData was double-entered into Epi-Data software (version 3.1) and analysed in Epi-Analysis (version 2.2.2.183 for analysis, EpiData Association, Odense, Denmark). Descriptive statistics were used to describe the data. Odds ratio (OR) with 95% confidence interval (CI) was calculated for possible presumptive variables.\n\nThe SDPs had already commenced in the selected prisons before the start of this study by BACP, with the permission of the Health Department of Balochistan and Home Department Government of Balochistan. Screening, health education sessions and collection of data was routinely done for the SDPs so the program data was utilized. The principal investigator was also the monitor of SDPs from BACP. Study and ethical approval was obtained from BACP (No. BACP: 01/2018-432) to collect and analyse the program data for the research purpose.\n\n\nResults\n\nA total of 2084 prisoners were screened, of whom 33 were found to be positive for HIV (Figure 1). All the HIV-positive prisoners were approached for assessment of risk factors but only 16 gave informed written consent for the interview.\n\n*Test 1 = Alere Determine HIV-1/2 Ag/Ab Combo for detection of Antigent/Antibody, **Test 2 = Uni-Gold HIV, **Test 3 = SD Bioline HIV-1/2 3.0.\n\nThe median age of the prisoners was 25 years (range 13–80 years, 94.23% in 15–45 years age group) years and 53 (50.9%) of them were married. The majority of them were uneducated (36.53%) or had been in education for less than 5 years (16.34%). Among them, 15% were unemployed and 8% were working on daily wages before the imprisonment. Around 93% of the prisoners were from Balochistan, 4% from other provinces and 3% from other countries (Table 1).\n\n*Employed includes prisoners who have worked both in public, private sector and/or self-employed.\n\nAround 75% HIV infected prisoners were convicted, 75% had spent less than five years, 25% had spent more than ten years in prisons 22% of total interviewed prisoners had been ever arrested for drug related offense (Table 2).\n\nExtramarital sex was reported by 11 (68.8%) out of 16 HIV-positive prisoners (OR 4.48; 95% CI 1.41–14.2). Homosexuality was reported by 7 (43.8%) HIV-positive prisoners (OR 2.09; 95% CI 0.69–6.28). About 50% of HIV positive prisoners had history of needle sharing (OR 43; 95% CI 7.77–237.87). Almost 94% of HIV positive prisoners had history of drug addiction of any type, of whom 50% had history of injectable drugs (OR 13; 95% CI 2.82–60.01). History of sharing razorblades and blood transfusion was 9.6% in HIV-positive prisoners; 12.5% had gone through any surgical procedure, 8.6% through any dental procedure and 6.7% had history of tattooing in the past 5 years (Table 3).\n\nOR, odds ratio; CI, confidence interval.\n\n\nDiscussion\n\nThis study was conducted in four major jails in Balochistan, Pakistan, and revealed that the HIV prevalence was 1.6%, which is much higher than the prevalence rate in general population in Pakistan3. A previous study conducted in Quetta jail indicates that the HIV prevalence was 0.5%16. A limitation of that study was generalizability to the whole province as the sample size was very small (only 200 prisoners were screened) and the prisoners of Quetta jail, although large in number, were mostly under trial, so the long-term sentenced prisoners were excluded from being assessed. The study findings are consistent with studies conducted in other provinces where the HIV prevalence ranged from 1 to 2.9%4,5,7,9,11,13.\n\nBased on our study findings, the majority of the HIV-positive prisoners belonged to the 15–45 years age group. The same age range was also found to be significant in other studies conducted in Pakistan511,13. Education status was also found as an important factor in HIV risk assessment, as a large majority of HIV positive prisoners (62.5%) were not educated or had been educated for less than 5 years, as found by another study conducted in Lahore11.\n\nThe prisoners who were single had a higher prevalence of HIV, as 62.5% of HIV-positive prisoners were unmarried. The findings are consistent with a study conducted in different jails of six cities in Sindh province13, Imprisonment status and duration of imprisonment was significant as 81% of HIV-positive prisoners were convicted and 25% prisoners had spent more than 10 years in prisons.\n\nA substantial proportion of HIV-infected prisoners were involved in behaviour considered to be presumptive risk factors associated with HIV; 68.8% were involved in extramarital sex and 43.8% had engaged in homosexual practices. A study of prisoners in Karachi found involvement of prisoners in homosexuality and sexual encounters with females to be 21.3% and 45.9%, respectively4. Moreover, drug addiction, especially of injectable drugs involving the sharing of needles, were also found to be present in significant majority of the HIV infected prisoners. Risk of acquiring of HIV increases in prisons as studies shows that 38.5% of PWID were arrested in past 12 months and around 12.3% of arrested PWID had access to injectable drugs in prisons2,17.\n\nThe young age of the prisoners, being less educated, having low socioeconomic status, addiction to injectable drugs, having multiple sex partners, sharing needles and involvement in drug related offense were notable findings of the study and consistent with studies conducted elsewhere in Pakistan4,5,7–13.\n\nPrison settings highly favour the spread of infectious disease as the prisoners from different sociodemographic backgrounds like low literacy levels and having pre-imprisonment risk factors such as drug use, extramarital sex encounters, etc. are forced to live together in confined space without having recreational facilities4. The prisoners are vulnerable to acquiring new infections in prisons due to overcrowding, poor medical facilities (especially diagnostic investigations) and the unavailability of harm reduction approaches such as the use of condoms or needle exchange program18. All these factors may not only lead to greater chances of spread of infections like HIV among the jails but can also be a major concern among the general population due to frequent flow of prisoners into the community4.\n\nAside from the many risk factors responsible for spread of infections in prison settings, prisons are still favourable venues for the screening and treatment of HIV, as the prisoners are confined to limited vicinity and easily approachable for diagnosis, treatment and health education. If the prisoners are approached within the prisons for diagnosis and treatment, this can help halt the spread of HIV to the general population and vice versa4.\n\nThe level of health services provided in prisons plays an important role in dealing with the associations of HIV-infected persons with the community. In a resource-constrained country like Pakistan, the chain of transmission can be broken by introducing urgent prevention efforts, including health education, initial screening at the time of entry of prisoners in prions, routine periodic screening of all the prisoners and treatment of infected prisoners in each prison so that its spread within the prisons and to the community after release of the prisoner could be minimized4. The presence of HIV-infected prisoners warrants that information and education programs are made accessible in the prisons to stop the spread of infectious diseases in the prisons, with particular emphasis on transmission through injectable drug use and high-risk sexual activities. Training health care providers to identify and treat those suspected of being, or know to be, infected with HIV and the provision of testing services may help the early diagnosis of infections and their treatment to reduce the spread of infection.\n\nBeing a comprehensive study covering four major jails of Balochistan province the study is strengthened by universal sampling for screening in which all the prisoners present during the study period were screened for HIV. The risk factors may vary in those prisoners who were not interviewed.\n\n\nConclusion\n\nThe high prevalence of HIV in prisons in Balochistan, compared with the non-prison population, indicates that preventive strategies should be designed and implemented carefully to minimize the spread of infection among the prisons and ultimately their linkages to the community. Early diagnosis of HIV infection through screening, initiation of treatment and addressing the stigma universally attached with the disease are effective ways of preventing the spread of infection.\n\n\nData availability\n\nRaw data for the present study, including demographic information and answers to the questionnaire, is available on OSF, DOI: https://doi.org/10.17605/OSF.IO/8Z4EJ19.\n\nThe questionnaire used in this study is available on OSF, DOI https://doi.org/10.17605/OSF.IO/8Z4EJ19.",
"appendix": "Grant information\n\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership led by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR). The training model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union, Paris, France) and Médecins Sans Frontières (MSF, Geneva, Switzerland). The specific SORT IT programme that resulted in this publication was implemented by the National Tuberculosis Control Programme of Pakistan, through the support of the Global Fund to Fight AIDS, Tuberculosis and Malaria (The Global Fund, Geneva, Switzerland). The publication fee was covered by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe Provincial AIDS Control Program, Balochistan, through its program Manager Dr. Noor Muhammad Qazi, supported the research by allowing the principal investigator to utilize the program data and resources.\n\n\nReferences\n\nNational AIDS Control Program Pakistan: Pakistan AIDS Strategy III (2015-2021). 2017.\n\nPakistan NACP 2nd Generation HIV Surveillance in Pakistan Round 5: Integrated Biological & Behavioral Surveillance in Pakistan. 2016. Reference Source\n\nPakistan NACP: AIDS Epidemic Modelling Exercise for Pakistan 2017. 2017. Reference Source\n\nKazi AM, Shah SA, Jenkins CA, et al.: Risk factors and prevalence of tuberculosis, human immunodeficiency virus, syphilis, hepatitis B virus, and hepatitis C virus among prisoners in Pakistan. Int J Infect Dis. 2010; 14 Suppl 3: e60–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIkram N, Firdus R, Tariq Baig PA: Screening of Jail Inmates for Hepatitis B,C and HIV Infections. J Rawalpindi Med Coll. 2011; 15(2): 79–81.\n\nBergenstrom A, Achakzai B, Furqan S, et al.: Drug-related HIV epidemic in Pakistan: a review of current situation and response and the way forward beyond 2015. Harm Reduct J. 2015; 12(1): 43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNafees M, Qasim A, Jafferi G, et al.: HIV infection, HIV/HCV and HIV/HBV co-infections among jail inmates of Lahore. Pak J Med Sci. 2011; 27(4): 837–41. Reference Source\n\nAli M, Nadeem M, Numan M, et al.: Thirty years of HIV in Pakistan: a systematic review of prevalence and current scenario. Future Virol. 2017; 12(10): 609–23. Publisher Full Text\n\nWaheed U, Satti HS, Arshad M, et al.: Epidemiology of HIV/AIDS and syphilis among high risk groups in Pakistan. Pak J Zool. 2017; 49(5): 1829–34. Publisher Full Text\n\nShah SSA, Ali M, Ahmad M, et al.: Screening of jail inmates for HIV and tuberculosis. Pak J Med Health Sci. 2013; 7(1): 172–5. Reference Source\n\nManzoor S, Tahir Z, Anjum A: Prevalence of HIV and Tuberculosis Among Jail Inmates in Lahore - Pakistan. Biomedica. 2009; 25: 36–41. Reference Source\n\nIqbal KJ, Arshad J, Muhammad A, et al.: Health status and imprisonment profile of jail inmates of district jail Rahim Yar Khan, Pakistan. Explor Anim Med Res. 2012; 2(2): 146–50. Reference Source\n\nSafdar S, Mehmood A, Abbas SQ: Short Report Prevalence of HIV / AIDS among jail inmates in Sindh. J Pak Med Assoc. 2009; 59(2): 111–112. Reference Source\n\nQureshi H, Iqbal R: Review of Health System in Prisons of Punjab, Pakistan. 2012. Reference Source\n\nPakistan Country Strategy for HIV Testing and Counseiling based on Situation and Response Analysis. 2012. Reference Source\n\nKakar N: Prevalence of human immunodeficiency virus in the inmates of district Jail Quetta, Balochistan. Pure Appl Biol. 2017; 6(1): 164–170. Publisher Full Text\n\nAkhtar S, Luby SP, Rahbar MH, et al.: HIV/AIDS knowledge, attitudes and beliefs based prediction models for practices in prison inmates, Sindh, Pakistan. Southeast Asian J Trop Med Public Health. 2001; 32(2): 351–61. PubMed Abstract\n\nBick JA: Infection Control in Jails and Prisons. Clin Infect Dis. 2007; 45(8): 1047–55. PubMed Abstract | Publisher Full Text\n\nKhan MD: Prevalence and Associated Risk Factors of HIV in Prisons in Balochistan, Pakistan: A Cross-Sectional Study. OSF. Web. 2018."
}
|
[
{
"id": "40898",
"date": "27 Dec 2018",
"name": "Quaid Saeed",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work quite clearly cites the current and latest literature regarding the HIV disease transmission dynamics in Pakistan and also cites other such studies conducting in different prisons of Pakistan. The study design is appropriate and the study has been conducted in the most difficult and remote prison in Pakistan giving good insight on conditions prevalent in such remote prison settings.\n\nThe study design was appropriate and a good number of prison inmates (2084) were selected. As with other similar studies the prevalence found was more than the one in the general population which warrants that urgent preventive measures should be taken to control spread of this infection in this closed population. In Pakistan, outbreaks of HIV have been reported in different jails of the country in the past. Sensational reports came in the media regarding outbreak of HIV in different prisons but no scientific studies were carried out to determine the reasons of this spread. This study is a good effort to highlight the prevalent conditions and risks for spread of HIV in prison settings.\n\nThe study outlines the risk factors associated with HIV transmission in this population as part of the general population but does not shed ample light on prevailing high-risk behaviours inside the prisons that may have led to transmission of this disease in the prisons. Thus, most of the readers would be interested whether high risk behaviours are prevalent in these prisons and whether drugs and injectable equipment is available in the prisons. Having said that it is not easy to divulge upon these areas since most prison authorities deny prevalence of such practices in their jurisdiction and do not allow such questions to be asked and explored by investigators. It is therefore conceivable that the of this study questionnaire must have been cleared and vetted by the jail authorities and therefore such information could not be explored during this study.\n\nAs far as the methods of analysis was concerned the details provided were quite useful for other investigators to replicate such a study. Details were provided on statistical tools applied and used.\n\nThe conclusion drawn are in line with the findings of the study. We lack data on prevalent behaviours of prison inmates in different prisons in Pakistan and such similar studies are needed to advocate and start prevention interventions in these settings in the country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "42869",
"date": "11 Feb 2019",
"name": "Pamela Valera",
"expertise": [
"Reviewer Expertise Prison Health",
"HIV/AIDS",
"and Prison 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\nThere is an urgent public health need to understand HIV/AIDS disease transmission globally especially in areas where war and conflict continues and resources to address communicable diseases are limited.\nThis paper is the first to my knowledge to shed light on a growing HIV epidemic especially in correctional facilities in different prisons and jails in Pakistan. The study design provides opportunities for future research to understand HIV/AIDS transmission in correctional facilities. However, I would have much appreciated more literature on the characteristics of offenders prior to incarceration.\nThe findings while not generalizable were instrumental in helping readers understand high-risk behaviors among people who inject drugs and people who engage in same-sex behaviors. Perhaps, the next step of this study might be to conduct a qualitative study exploring the lived experience of inmates living with HIV/AIDS in remote correctional settings in Pakistan\nThe conclusion points researchers in the right direction. We have a limited understanding of HIV prevalence and HIV risk-taking behaviors of offenders in Pakistan and other areas. We need to do more in understanding HIV prevalence in correctional settings, but also discuss opportunities for prevention intervention programs. Perhaps, a section on implications or future research in the conclusion section should be included.\nHere are potential articles to consider for this section:\n\nScott, D. P., Harzke, A. J., Mizwa, M. B., Pugh, M., & Ross, M. - Evaluation of an HIV peer education program in Texas prisons1. Solomon, L., Montague, B. T., Beckwith, C. G., Baillargeon, J., Costa, M., Dumont, D.,Rich, J. D. - Survey finds that many prisons and jails have room to improve HIV testing and coordination of postrelease treatment2. Valera, P., Chang, Y., & Lian, Z. - HIV risk inside US prisons: A systematic review of risk reduction interventions conducted in US prisons3.\n\nLastly, the manuscript would greatly benefit from proof-reading and addressing grammatical errors throughout the article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4461",
"date": "05 Mar 2019",
"name": "Muhammad Khan",
"role": "Author Response",
"response": "Dear Pamela ValeraThank you very much for your valuable feedback. I have tried to remove the grammatical mistakes from the manuscript and amend the conclusion section based on the references provided by you."
}
]
},
{
"id": "42871",
"date": "18 Feb 2019",
"name": "Laurent Getaz",
"expertise": [
"Reviewer Expertise Tropical medicine / Internal medicine / Public health / Health in detention"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes the prevalence of and risk factors associated with HIV in four prisons in Pakistan. These data are very important for prioritizing programs that are essential for HIV control in this vulnerable population. I propose a major revision of this manuscript.\nMethods section:\nHow was the HIV screening test proposed in the study population? Was the test optional or mandatory? If the test was not mandatory, what was the participation rate? How did you calculate the sample size, in particular for the number of participants who responded to the questionnaire? Please clarify the HIV diagnostic algorithm. For participants with a positive \"Alere Determine HIV test\" result, the HIV status \"was confirmed by initial confirmatory rapid diagnostic kits (Uni-Gold) and followed by confirmatory rapid diagnostic kits (SD Bioline)\". In the case of a positive \"Alere determine test\" result and negative \"Uni-Gold test\" result, did you process the SD Bioline test? How did you manage discordant results?\nDiscussion section:\n\"Education status was also found as an important factor in HIV risk assessment, as a large majority of HIV positive prisoners (62.5%) were not educated or had been educated for less than 5 years\". This statement is not true. In the results section, you should provide the results of a bivariate analysis to estimate the association between education level and HIV prevalence. The level of education is not associated with HIV (62.5% versus 51.1%, p=0.4, OR=1.6 (0.5-5.1)). \"The prisoners who were single had a higher prevalence of HIV, as 62.5% of HIV-positive prisoners were unmarried\". In the results section, you must also provide the results of a bivariate analysis to estimate the association between this variable and HIV. Marital status is not associated with infection (62.5% versus 46.6%, p=0.24). I believe an epidemiologist should be involved as a coauthor to validate the interpretation of the results. You should discuss the result in which a statistically significant association is identified in the study population and put it in perspective with the data from the literature. And when a generally recognized risk factor is not associated with infection in the study population, you should still discuss it. For example, in your study, \"extramarital sex status\" (OR-4.48, IC95% 1-4-14.2) is associated with HIV but not \"homosexual sex status\" (OR=2.1, IC95% 0.7-6.3). Also, you cannot state \"A substantial proportion of HIV-infected prisoners were involved in behaviour considered to be presumptive risk factors associated with HIV; 68.8% were involved in extramarital sex and 43.8% had engaged in homosexual practices\". I suggest you describe the limitations of the study. What potential biases do you suspect, and what are their expected impacts on the results?\nThe manuscript should be proofread by a native English speaker for spelling, grammar and punctuation, as it contains many errors throughout. Many phrases contain errors, which makes reading the manuscript difficult.\n\nIs the work 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": []
}
] | 1
|
https://f1000research.com/articles/7-1821
|
https://f1000research.com/articles/7-1232/v1
|
10 Aug 18
|
{
"type": "Research Note",
"title": "Pilot study of publicly available data to evaluate the relationship between forest fires and emergency department visits due to asthma in the state of California",
"authors": [
"Hayat Srour",
"Ruslan Fomenko",
"Joshua Baguley",
"Shandra Bellinger",
"Angel Jordan",
"Jennifer Sutton",
"Melany Santana",
"Armando Marull",
"Musheer Abdalhuk",
" Félix E. Rivera-Mariani",
"Hayat Srour",
"Ruslan Fomenko",
"Joshua Baguley",
"Shandra Bellinger",
"Angel Jordan",
"Jennifer Sutton",
"Melany Santana",
"Armando Marull",
"Musheer Abdalhuk"
],
"abstract": "The focus of this study was to determine the relationship between asthma-related emergency department (ED) visits and fires in the state of California. Publicly available data of ED visits due to asthma, as well as occurrence of forest fires in California from 2005 to 2015 were obtained, where the California counties were grouped by region: North, Coastal, Motherload, Central, and South. There were no statistical differences with regards to acres of forest burned, but statistically significant differences were found (although small) with regards to ED visits due to asthma attacks by region (Motherload higher than South region). When evaluating the relationship of ED visits due to asthma and acres of forest burned, forest fires barely explained the variability of emergency department visits (r2 = f 0.05, p<0.01). With aims to establish a connection between natural disasters and respiratory distress, we faced obstacles in data limitations and confounding variables. This paper serves as a pilot study supporting the need for further exploration of environmental, health, and socio-demographic variables that interplay when evaluating relationships of natural disasters and incidence of chronic diseases, such as asthma.",
"keywords": [
"asthma",
"forest fires",
"California",
"acres burned"
],
"content": "Introduction\n\nForest fires are devastating events causing widespread damage and contributing enormous environmental pollution1,2. According to the National Interagency Fire Center, in 2017 there were 9,988 fires within the state of California. While the immediate effects of forest fires are well known, the lingering implications are less apparent. As some of the main by-products of forest fires are CO2 and PM2.53,4, questions remain with regards to how forest fires contribute to the incidence of respiratory diseases.\n\nAsthma is a chronic respiratory illness characterized by the constriction of the bronchi, leading to difficult breathing and sometimes irreversible respiratory tissue remodeling5. According to the US Center of Disease and Control and Prevention, there is an asthma prevalence of 7.7% among the population in California. Because asthma symptoms can be exacerbated by airborne pollutants, such as CO2 and PM2.56,7, we hypothesized that an increase in acres of forest burned would be linked to increased emergency department (ED) visits due to asthma.\n\n\nMethods\n\nData was retrieved from the California Environmental Health Tracking Program. The inclusion criteria to extract the data included ED visits due to asthma, all ages, all races/ethnicities, and all genders.\n\nReported forest fires were collected from the Historical Wildfire Activity Statistics section (HWAS) of the California Department of Forestry and Fire Protection. Only fires greater than 300 acres were included in the final dataset; as per National’s Park Services terminology, fires above 300 acres burned are considered large fire events. For this data, during some years, some counties had missing values because they did not reach the 300 acres threshold.\n\nBoth datasets were inclusive to an 11-year period (January to December, 2005–2015).\n\nStatistical analysis was performed with R (version 3.5.0, https://www.r-project.org/). California’s counties were grouped into regions (Figure 1). Given the size of the state and no present guidelines for grouping of each county, we grouped counties by proximity into North, Coastal, Central, Motherload, and South regions. This classification was carried out within the R script included in Supplementary File 1. A pairwise t-test, with Bonferroni adjustment, was implemented to compare both the acres burned and the asthma rate, respectively, per region. A linear regression model, adjusted by year and region, was elaborated to evaluate the relationship between acres burned and ED asthma visits. p < 0.05 was considered statistically significant.\n\n\nResults\n\nEach county was assigned to a region based on geography and proximity. As existing maps were unable to distribute California’s counties evenly, we used a breakdown of regions (Figure 1). Table 1 provides the mean acres burned and mean asthma rates per region. The ED asthma visits rates were above 50% except for the South and Motherload regions. A pairwise t-test with Bonferroni adjustment did not detect statistically significant differences in acres burned between the regions (p = 0.38 to 1.0; Table 2).\n\nOn the contrary, a statistically significant difference (p = 0.003) was found between the South (48.8%) and the Motherload (43.0%) region with regards to asthma rates. A linear regression model, adjusting for years and region, was employed to evaluate the magnitude of relationship between acres burned and ED asthma visits (Figure 2). In this model, and although statistically significant (p = 0.005), forest fires only explained less than 5% of the variability of the ED asthma visits between the years 2006 to 2015 (r2 = 0.047). These results suggest that although regions within California may differ in ED asthma visits, forest fires did not explain these differences.\n\n\nDiscussion\n\nIn this study, data on forest fires within the state of California was compiled to address the potential relationship with ED asthma visits. As per our linear regression model, there was no link between the ED asthma visits and acres of forest burned. Future research should account for the additional variables related to asthma as well as forest fires.\n\nIn the current study, forest fires barely explained the ED visits from asthma during the years 2006 to 2015. It is possible that these ED asthma visits may have resulted from other stimuli, such as biological and non-biological indoor and outdoor air pollution besides forest fires8–10. Also, California participates in different programs that may reduce ED visits like the National Asthma Control Initiative, which was developed by the US National Institute of Health.\n\nInterestingly, asthma rates varied across all regions. The Motherload region had significantly lower mean asthma rates than the North region. This finding further warrants additional investigations into the asthma rates within states susceptible to natural disasters and diverse geographic and topography, such as the state of California. Future studies may shed light on the interplay of forest fires, environmental variables, and chronic respiratory diseases, such as asthma.\n\nLimitations came in several forms. First, fires are known to often cross county lines, which makes it difficult to assign appropriate values per county. Next, the Alpine county was not included in the dataset due to the lack of data relating to asthma. News coverage on forest fires may warn the population of areas to avoid, which potentially reduces the number of ED visits and could explain the decline in asthma rates as acres of forest burned increase (Figure 2) because some counties did not report forest fires above the 300 acres threshold. Also, the data on asthma rates was available by year, not by month, thus reducing our ability to match forest fires and asthma rates monthly. Finally, it is possible that ED asthma visits may also be modified by another variable; therefore, tracking the sale and consumption of asthma medication could provide more reliable information of asthma exacerbation rates as the result of forest fires.\n\nIn summary, findings from the current study warrant examination of additional variables that could potentially contribute to environmental air pollution, besides forest fires, and are also linked to exacerbations of asthma. These include sociodemographics, medication sales due to asthma, and others that could shed light on the activities that the people of California implement during forest fires events.\n\n\nData availability\n\nF1000Research Dataset 1: Forest fires acres burned and emergency department visits due to asthma for California, January-December 2005–2015. DOI, 10.5256/f1000research.15839.d21339711\n\nData for emergency department visits due to asthma: http://www.cehtp.org/page/asthma/query\n\nData for forest fires: http://www.fire.ca.gov/fire_protection/fire_protection_fire_info_redbooks\n\nThe full dataset is available as Dataset 1.\n\nR code for performing the analysis is available in Supplementary File 1.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: R code used for analysis of asthma and fires in California.\n\nClick here to access the data.\n\n\nReferences\n\nLiu JC, Mickley LJ, Sulprizio MP, et al.: Particulate Air Pollution from Wildfires in the Western US under Climate Change. Clim Change. 2016; 138(3): 655–666. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaoletti E, Bytnerowicz A, Andersen C, et al.: Impacts of air pollution and climate change on forest ecosystems--emerging research needs. ScientificWorldJournal. 2007; 7 Suppl 1: 1–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarvalho JA Jr, Amaral SS, Costa MAM, et al.: CO2 and CO emission rates from three forest fire controlled experiments in Western Amazonia. Atmos Environ. 2016; 135: 73–83. Publisher Full Text\n\nFujii Y, Tohno S, Amil N, et al.: Quantitative assessment of source contributions to PM2.5 on the west coast of Peninsular Malaysia to determine the burden of Indonesian peatland fire. Atmos Environ. 2017; 171: 111–117. Publisher Full Text\n\nHolgate ST: Pathogenesis of asthma. Clin Exp Allergy. Wiley Online Library. 2008; 38(6): 872–97. PubMed Abstract | Publisher Full Text\n\nGrover RS, Kumar R: Exhaled carbon monoxide levels: as a marker of clinical severity and control of asthma. J Asthma. 2008; 45(8): 677–680. PubMed Abstract | Publisher Full Text\n\nBurbank AJ, Sood AK, Kesic MJ, et al.: Environmental determinants of allergy and asthma in early life. J Allergy Clin Immunol. 2017; 140(1): 1–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreysse PN, Diette GB, Matsui EC, et al.: Indoor air pollution and asthma in children. Proc Am Thorac Soc. 2010; 7(2): 102–106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTzivian L: Outdoor air pollution and asthma in children. J Asthma. 2011; 48(5): 470–481. PubMed Abstract | Publisher Full Text\n\nCakmak S, Dales RE, Coates F: Does air pollution increase the effect of aeroallergens on hospitalization for asthma? J Allergy Clin Immunol. 2012; 129(1): 228–231. PubMed Abstract | Publisher Full Text\n\nSrour H, Fomenko R, Baguley J, et al.: Dataset 1 in: Pilot study of publicly available data to evaluate the relationship between forest fires and emergency department visits due to asthma in the state of California. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.15839.d213397"
}
|
[
{
"id": "38530",
"date": "21 Feb 2019",
"name": "Brian G. Oliver",
"expertise": [
"Reviewer Expertise respiratory diseases"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting pilot study examining the relationship between forest fires and visits to the hospital for asthma.\nThe main limitation of the study was the data available, in particular large forest fires of above 300 acres were included, and asthma treatment in emergency departments was only available as a yearly figure.\nThere are many studies which show that the rates of hospital admissions increases in the days/weeks following a woodland fire, so the analysis which was able to be undertaken in the present study may not be sensitive enough to detect increases immediately after fires. This perhaps could have been mitigated if there were several years in which there were no fires and and several with, or counties without any fires versus these with fires.\nAnother limitation of the study was the inability to be able to predict the \"normal\" and fire induced PM load in the townships near the fires and explore the impact that this had on admissions for asthma.\nI am not familiar with the geography of this region, but do for example people burn wood fires for heating in winter, what effect does this have.\nThis study could be improved by inclusion of a set of recommendations for the type of data that should be analysed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4462",
"date": "05 Mar 2019",
"name": "Felix Rivera-Mariani",
"role": "Author Response",
"response": "Reviewer: The main limitation of the study was the data available, in particular large forest fires of above 300 acres were included, and asthma treatment in emergency departments was only available as a yearly figure. We agree that the magnitude of data was a considerable limitation. For example, it prevented us from presenting visual representation in daily, weekly, or monthly intervals Reviewer: There are many studies which show that the rates of hospital admissions increases in the days/weeks following a woodland fire, so the analysis which was able to be undertaken in the present study may not be sensitive enough to detect increases immediately after fires. This perhaps could have been mitigated if there were several years in which there were no fires and and several with, or counties without any fires versus these with fires. We thank the reviewer for this comment. We proceeded to elaborate on possible explanations that would have affected the sensitivity of the statistical analysis. Reviewer: Another limitation of the study was the inability to be able to predict the \"normal\" and fire induced PM load in the townships near the fires and explore the impact that this had on admissions for asthma. We definitely agree with the reviewer on this point, and we consider this type of analysis. Due to page limitations and the pilot nature of the current study, we were not able to include additional predictive analysis to the current study. We would like to follow up on this in future studies of forest fires and respiratory diseases. Reviewer: I am not familiar with the geography of this region, but do for example people burn wood fires for heating in winter, what effect does this have. Thanks to the reviewer for bringing this point. We won’t be able to fully address how wood fires for heating in winter may have contributed to the incidence of forest fires. It is possible that the dataset included this cause of fire as “Miscellaneous or Undetermined” -- the other categories were arson, campfire, debris burning, electrical power, equipment use, railroad, smoking, and Vehicle. Reviewer: This study could be improved by inclusion of a set of recommendations for the type of data that should be analyzed. We thank the reviewer for this suggesting. We proceeded to update the limitation paragraph to include this recommendation."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1232
|
https://f1000research.com/articles/7-347/v1
|
21 Mar 18
|
{
"type": "Research Note",
"title": "Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division",
"authors": [
"Mark J. Hanemaaijer",
"Parker D. Houston",
"Travis C. Collier",
"Laura C. Norris",
"Abdrahamane Fofana",
"Gregory C. Lanzaro",
"Anthony J. Cornel",
"Yoosook Lee",
"Mark J. Hanemaaijer",
"Parker D. Houston",
"Travis C. Collier",
"Laura C. Norris",
"Abdrahamane Fofana",
"Gregory C. Lanzaro",
"Anthony J. Cornel"
],
"abstract": "Here we report the complete mitochondrial sequences of 70 individual field collected mosquito specimens from throughout Sub-Saharan Africa. We generated this dataset to identify species specific markers for the following Anopheles species and chromosomal forms: An. arabiensis, An. coluzzii (The Forest and Mopti chromosomal forms) and An. gambiae (The Bamako and Savannah chromosomal forms). The raw Illumina sequencing reads were mapped to the NC_002084 reference mitogenome sequence. A total of 783 single nucleotide polymorphisms (SNPs) were detected on the mitochondrial genome, of which 460 are singletons (58.7%). None of these SNPs are suitable as molecular markers to distinguish among An. arabiensis, An. coluzzii and An. gambiae or any of the chromosomal forms. The lack of species or chromosomal form specific markers is also reflected in the constructed phylogenetic tree, which shows no clear division among the operational taxonomic units considered here.",
"keywords": [
"Mitogenome",
"species identification",
"Africa",
"malaria vector",
"mosquitoes",
"Anopheles",
"single nucleotide polymorphisms",
"phylogenomics"
],
"content": "Introduction\n\nHistorically, mtDNA sequence has been used in taxonomy as a source of species diagnostic markers (Cronin et al. (1991); De Barba et al. (2014); Pegg et al. (2006)) or in population genetics and evolutionary studies (Fu et al. (2013); Harrison (1989); Llamas et al. (2016)). One advantage of using mitochondrial over nuclear DNA for such studies is that the mutation rate of mtDNA is about 10 times faster than nuclear DNA (Brown et al. (1979); Haag-Liautard et al. (2008)), hence amplifying the evolutionary trajectory of populations and species. In addition, mtDNA is easy to amplify, because there are more copies of mitochondrial DNA relative to nuclear DNA. Also, universal primers can be applied to a wide range of species. Widely used universal primers target the cytochrome b and cytochrome oxidase 1 genes (Tahir et al. (2016)), because both have conserved and highly variable regions. In addition to these, other genes as described in De Mandal et al. (2014), can also be used as markers. However, phylogenetic trees based on mtDNA can deviate from the ones that are derived from nuclear DNA (Phillips et al. (2013); Shaw (2002); Sota & Vogler, 2001).\n\nThe Anopheles gambiae species complex consists of eight morphologically identical species that can only be distinguished with molecular markers (Scott et al. (1993)) or, for some of the species, by cytological examination of polytene chromosomes (Green, 1972; Pombi et al., 2008). The currently used molecular markers are located within genomic islands of divergence located proximal to the centromeres (Lee et al. (2014); Turner et al. (2005)). Monitoring additional species-specific markers on mitochondrial DNA (mtDNA) could increase the ease of application and accuracy of species detection assays. In addition, mtDNA markers could enhance our understanding of divergence times among taxa within the complex.\n\nIn this study we wished to identify species-specific markers within the mtDNA for Anopheles arabiensis, An. coluzzii and An. gambiae, including among the chromosomal forms currently subsumed under the designations An. gambiae and An. coluzzii, with the goal of adding these to our existing Anopheles species detection assay (Lee et al. (2014)). We sequenced the whole mitogenomes of 70 individual mosquito specimens collected throughout Sub-Saharan Africa. The raw Illumina sequencing reads were mapped to the AgamP4 reference sequence, which included both nuclear and mitochondrial sequences. We explore the relationship among An. arabiensis, An. coluzzii, An. gambiae and four of the sub-specific chromosomal form mitogenome sequences.\n\n\nMethods\n\nAnopheles arabiensis raw Illumina sequencing reads were obtained from our previous study (Marsden et al. (2014)). These included 20 samples from three villages in Tanzania collected in 2012 (Lupiro ((-8.38000°N, 36.66912°W), Sagamaganga (-8.06781°N, 36.80207°W), and Minepa (-8.25700°N, 36.68163°W) in the Kilombero Valley) and 4 samples from Cameroon collected in 2005 (9.09957°N, 13.72292°W). The An. gambiae and An. coluzzii samples were collected as resting adults using mouth aspirators in Kela, Mali (11.88683°N, -8.44744°W) in 2012 and Mutengene, Cameroon (4.0994°N, 9.3081°W) in 2011. We subdivided the An. coluzzii specimen into the Forest and Mopti chromosomal forms. Similarly, we did this for the An. gambiae Savannah and Bamako chromosomal forms. We used the same definitions and methods to characterize the chromosomal forms as in Lanzaro & Lee, 2013.\n\nSequencing methods for An. arabiensis samples are as described in Marsden et al. (2014). In short, individually barcoded Illumina paired-end sequencing libraries, with insert sizes of 320-400 basepairs (bp) using NEXTflex Sequencing kits (NOVA-5144) and barcodes (NOVA-514102)(Bio Scientific, Austin, TX, USA), were sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) with 100-bp paired-end reads using twelve samples per lane. For the An. coluzzii and An. gambiae samples we used the same methods as described in Norris et al. (2015) and Main et al. (2015). For the latter species, libraries were created using the Nextera DNA Sample Preparation Kit (FC-121-1031) and TruSeq dual indexing barcodes (FC-121-103)(Illumina) and the samples were sequenced on an Illumina HiSeq2500 with 100-bp paired end reads.\n\nDe-multiplexed raw reads were trimmed using Trimmomatic (Bolger et al. (2014)) version 0.36 and mapped to the mitogenome reference sequence of An. gambiae (Genbank accession number = NC_002084 (Beard et al. (1993))). Freebayes (v1.0.1) (Garrison & Marth, 2013) was used for mitochondrial variant calling assuming single ploidy and without population prior. Mapping statistics were calculated using qualimap version 2.2 (Okonechnikov et al. (2016)) and the data is represented in Table 1. Following the recommendation of Crawford and Lazarro (Crawford & Lazzaro, 2012), we used a minimum depth of 8 to call variants for each individual. Between positions 1-13,470bp of the mitogenome, we obtained consistently high quality reads for all samples, which were used for further analysis. An AT-rich region located between 13,471 and 15,388 suffers from low or zero coverage for sequences generated with the Nextera library preparation kit. Therefore, we excluded these regions from further analysis. The Vcf2fasta program (Danecek et al. (2011)) was used to extract mitogenome sequences from vcf file to fasta format. Geneious version 10.1.3 was used for mitogenome alignments. The phylogenetic tree was generated using the Jukes-Cantor genetic distance model and Neighbor-Joining tree methods available in Geneious version 10.1.3. We used scikit-allel (v1.1.9), a software package for Python (Miles & Harding (2017)), to identify species specific markers.\n\nMapped reads indicates the reads that are mapped to the reference genome. Mean coverage indicates the average depth of reads on the mitochondrial DNA and standard deviation indicates the coverage deviation across the mitochondrial DNA.\n\n\nResults and Discussion\n\nWe identified a total of 783 single nucleotide polymorphisms (SNPs) over the entire mitogenome. The majority of these (58.7%) were singletons (found on one of the 70 mitogenomes). We did not identify any SNPs unique to the species or chromosomal forms (Table 2) and therefore conclude that mtDNA is not suitable for Anopheles gambiae complex species identification.\n\nThe lack of species-specific markers is also reflected in the phylogenetic tree (Figure 1). An. arabiensis, An. coluzzii and An. gambiae did not cluster separately, which is consistent with previous reports that compared mitochondrial genome sequence data from specimens originating from Kenya, Senegal and South Africa (Besansky et al. (1997)) and Burkina Faso, Cameroon, Kenya, Mali, South Africa, Tanzania and Zimbabwe (Fontaine et al. (2015), supplemental material).\n\nWith the exception of the outgroup, An. quadriannulatis, this analysis fails to reveal a clear division of the operational taxonomic units included in this analysis. Colors indicate the species or chromosomal form and numbers at the branches indicate the accuracy of the inferred branches on a scale of 0–100, where 100 represents the highest confidence.\n\nOf note, 36 of the samples that we used in our study originated from Kela (Mali). Kela is located near the village of Selinkenyi, where previous studies have shown a history of hybridization and introgression between An. gambiae and An. coluzzii (Lee et al. (2013); Main et al. (2015); Norris et al. (2015)) , which may have resulted in shared polymorphisms in their mitochondrial genomes. Shared polymorphisms in their mitochondrial genomes, where history has not been reported, also appeared to have occurred in Mutengene (Cameroon), where both An. gambiae and An. coluzzii occur sympatrically. Hybridization between either An. coluzzii or An. gambiae with An. arabiensis yields sterile males (Slotman et al. (2004)), but phylogenomic analysis of these species show patterns of introgression between all of them (Fontaine et al. (2015)). Our mitochondrial genome study does not provide conclusive evidence for hybridization and introgression among the taxa under study. However, our data suggest that this is a possibility.\n\n\nData availability\n\nAligned sequences were submitted to the National Center for Biotechnology Information (NCBI) Accession number: MG930826 - MG930896\n\nDataset 1. Aligned FASTA file of mitogenome samples 10.5256/f1000research.13807.d192892 (Hanemaaijer et al., 2018)",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe thank University of California - Irvine, Malaria Initiatives (UCIMI) for their support.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Michelle Sanford for her assistance in the field collection in Cameroon in 2011. We thank Clare Marsden for providing the raw data of An. arabiensis samples.\n\n\nReferences\n\nBeard CB, Hamm DM, Collins FH: The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects. Insect Mol Biol. 1993; 2(2): 103–124. PubMed Abstract | Publisher Full Text\n\nBesansky NJ, Lehmann T, Fahey GT, et al.: Patterns of mitochondrial variation within and between African malaria vectors, Anopheles gambiae and An. arabiensis, suggest extensive gene flow. Genetics. 1997; 147(4): 1817–1828. PubMed Abstract | Free Full Text\n\nBolger AM, Loshe M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrown WM, George M Jr, Wilson AC: Rapid evolution of animal mitochondrial DNA. Proc Natl Acad Sci U S A. 1979; 76(4): 1967–1971. PubMed Abstract | Free Full Text\n\nCrawford JE, Lazzaro BP: Assessing the accuracy and power of population genetic inference from low-pass next-generation sequencing data. Front Genet. 2012; 3: 66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCronin MA, Palmisciano DA, Vyse ER, et al.: Mitochondrial DNA in wildlife forensic science: species identification of tissues. Wildlife Soc B (1973-2006). 1991; 19(1): 94–105. Reference Source\n\nDanecek P, Auton A, Abecasis G, et al.: The variant call format and VCFtools. Bioinformatics. 2011; 27(15): 2156–2158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe Barba M, Adams JR, Goldberg CS, et al.: Molecular species identification for multiple carnivores. Conserv Genet Resour. 2014; 6(4): 821–824. Publisher Full Text\n\nDe Mandal S, Chhakchhuak L, Gurusubramanian G, et al.: Mitochondrial markers for identification and phylogenetic studies in insects–A Review. DNA Barcodes. 2014; 2(1). Publisher Full Text\n\nFontaine MC, Pease JB, Steele A, et al.: Mosquito genomics. Extensive introgression in a malaria vector species complex revealed by phylogenomics. Science. 2015; 347(6217): 1258524. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFu Q, Mittnik A, Johnson PLF, et al.: A revised timescale for human evolution based on ancient mitochondrial genomes. Curr Biol. 2013; 23(7): 553–559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarrison E, Marth G: Haplotype-based variant detection from short-read sequencing. arXiv: 1207.3907. Cornell University Library; 2013. Reference Source\n\nGreen CA: Cytological maps for the practical identification of females of the three freshwater species of the Anopheles gambiae complex. Ann Trop Med Parasitol. 1972; 66(1): 143–147. PubMed Abstract | Publisher Full Text\n\nHaag-Liautard C, Coffey N, Houle D, et al.: Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster. PLoS Biol. 2008; 6(8): e204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHanemaaijer MJ, Houston P, Collier TC, et al.: Dataset 1: Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division. F1000Research. 2018. Data Source\n\nHarrison RG: Animal mitochondrial DNA as a genetic marker in population and evolutionary biology. Trends Ecol Evol. 1989; 4(1): 6–11. PubMed Abstract | Publisher Full Text\n\nLanzaro GC, Lee Y: Speciation in Anopheles gambiae—The distribution of genetic polymorphism and patterns of reproductive isolation among natural populations. Anopheles mosquitoes-New insights into malaria vectors. InTech, 2013. Publisher Full Text\n\nLee Y, Marsden CD, Norris LC, et al.: Spatiotemporal dynamics of gene flow and hybrid fitness between the M and S forms of the malaria mosquito, Anopheles gambiae. Proc Natl Acad Sci U S A. 2013; 110(49): 19854–19859. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee Y, Marsden CD, Nieman C, et al.: A new multiplex SNP genotyping assay for detecting hybridization and introgression between the M and S molecular forms of Anopheles gambiae. Mol Ecol Resour. 2014; 14(2): 297–305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLlamas B, Fehren-Schmitz L, Valverde G, et al.: Ancient mitochondrial DNA provides high-resolution time scale of the peopling of the Americas. Sci Adv. 2016; 2(4): e1501385. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMain BJ, Lee Y, Collier TC, et al.: Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali. Mol Ecol. 2015; 24(20): 5145–5157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarsden CD, Lee Y, Kreppel K, et al.: Diversity, differentiation, and linkage disequilibrium: prospects for association mapping in the malaria vector Anopheles arabiensis. G3 (Bethesda). 2014; 4(1): 121–131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiles A, Harding N: cggh/scikit-allel: v1.1.8 (Version v1.1.8). Zenodo. 2017. Publisher Full Text\n\nNorris LC, Main BJ, Lee Y, et al.: Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets. Proc Natl Acad Sci U S A. 2015; 112(3): 815–820. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOkonechnikov K, Conesa A, García-Alcalde F: Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics. 2016; 32(2): 292–294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPegg GG, Sinclair B, Briskey L, et al.: MtDNA barcode identification of fish larvae in the southern Great Barrier Reef–Australia. Sci Mar. 2006; 70(S2): 7–12. Reference Source\n\nPhillips MJ, Haouchar D, Pratt RC, et al.: Inferring kangaroo phylogeny from incongruent nuclear and mitochondrial genes. PLoS One. 2013; 8(2): e57745. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPombi M, Caputo B, Simard F, et al.: Chromosomal plasticity and evolutionary potential in the malaria vector Anopheles gambiae sensu stricto: insights from three decades of rare paracentric inversions. BMC Evol Biol. 2008; 8(1): 309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScott JA, Brogdon WG, Collins FH: Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg. 1993; 49(4): 520–529. PubMed Abstract | Publisher Full Text\n\nShaw KL: Conflict between nuclear and mitochondrial DNA phylogenies of a recent species radiation: what mtDNA reveals and conceals about modes of speciation in Hawaiian crickets. Proc Natl Acad Sci U S A. 2002; 99(25): 16122–16127. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSlotman M, Della Torre A, Powell JR: The genetics of inviability and male sterility in hybrids between Anopheles gambiae and An. arabiensis. Genetics. 2004; 167(1): 275–287. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSota T, Vogler AP: Incongruence of mitochondrial and nuclear gene trees in the Carabid beetles Ohomopterus. Syst Biol. 2001; 50(1): 39–59. PubMed Abstract | Publisher Full Text\n\nTahir HM, Mehwish, Kanwal N, et al.: Genetic diversity in cytochrome c oxidase I gene of Anopheles mosquitoes. Mitochondrial DNA A DNA Mapp Seq Anal. 2016; 27(6): 4298–4301. PubMed Abstract | Publisher Full Text\n\nTurner TL, Hahn MW, Nuzhdin SV: Genomic islands of speciation in Anopheles gambiae. PLoS Biol. 2005; 3(9): e285. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32266",
"date": "30 Apr 2018",
"name": "Beniamino Caputo",
"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\nGeneral comments The present research note entitled:” Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division” is well analysed, reported and written. As already reported in previous study the submitted manuscript suggested the absence of any species-specific differences in the mitogenome of the three species examined. Although the manuscript is not innovative and the research is not based on any previous evidence, the present note confirms previous suggestions by examining the whole mitogenome of 70 specimens from field specimens and find the lack of species or chromosomal form specific markers.\n\nTitle and abstract Title and abstract are appropriate and summarize well the content of the article.\nIntroduction\nThe introduction gives a good description of the aims of the present study, although I would have added some references to previous studies performed on mtDNA of the examined species (for example Besansky 1997) and why you expected to obtain different results compared to previous studies.\nPlease revise also: “morphologically identical species that can only be distinguished with molecular markers” (Scott et al., 1993; Coetzee et al., 2013)\nThe currently used molecular markers are located within genomic islands of divergence located proximal to the centromeres (Lee et al. (2014); Turner et al. (2005)) please rephrase the citation and refer it only to detect genomic differences between An.gambiae e and An.coluzzii.\nPlease insert a sentence about chromosomal forms of An.gambiae.\nMethods\nPlease specified the method for collecting An. arabiensis as you already described for An.gambiae (e.g. indoor specimens, mouth aspirators, PSC collections).\nPlease insert a table with inversion polymorphism of chromosomal forms analyzed.\nPlease add the source of the An. quadriannulatus specimens you included in the phylogenetic analysis.\nResults Study design is well explained and results are given concisely.\nPlease add in Table 2 also the number of specimens you included for each species in the analysis.\nPlease add in Figure two an explanation of what “lineage” means for An. arabiensis specimens.\nPlease give results (also without table or figure) for each country separately.\nDiscussion Discussion is very concise but deals with most major points of interest. We would just suggest to explain better the conclusion on possible introgression (the more plausible hypothesis) between taxa and to evaluate other possible explanations for the absence of fixed differences between species (e.g. absence for divergent selection, or evolutionary characherestic of mitogenomes).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "33371",
"date": "14 May 2018",
"name": "Maria Anice Mureb Sallum",
"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 Phylogenetic analysis need to be improved, and the choice for NJ methods and JC model, justified in the article. There are several programs that have been largely employed for phylogenetic analysis, including for mitogenome data. The paper authored by Foster et al.1 contains useful information about analyses that have been carried out for inferring phylogenetic relationships within Anophelinae mosquitoes. I strongly suggest authors to verify how analyses were done.\n\nSample collection Authors - “The An. gambiae and An. coluzzii samples were collected as resting adults using mouth aspirators in Kela, Mali (11.88683°N, -8.44744°W) in 2012 and Mutengene, Cameroon (4.0994°N, 9.3081°W) in 2011.”\nComment - Can you please give more details the micro environment where your specimens of An. gambiae and An. coluzzii were resting?\n\nAuthors - “Similarly, we did this for the An. gambiae Savannah and Bamako chromosomal forms. We used the same definitions and methods to characterize the chromosomal forms as in Lanzaro & Lee, 2013.”\nComment - It is not clear to me if you examined the polytene chromosome of each specimen you identified as the Savannah, Bamako, Forest and Mopti forms. Please clarify.\n\nGenome sequencing Authors - “For the An. coluzzii and An. gambiae samples we used the same methods as described in Norris et al. (2015) and Main et al. (2015). For the latter species, libraries were created using the Nextera DNA Sample Preparation Kit (FC-121-1031) and TruSeq dual indexing barcodes (FC-121-103) (Illumina) and the samples were sequenced on an Illumina HiSeq2500 with 100-bp paired end reads.”\nComment - Please add a short sentence to clarify if you sequenced the whole genome and from the full sequence data you obtained the positions 1-13,470 of the mitogenome.\n\nData analysis Authors - “The phylogenetic tree was generated using the Jukes-Cantor genetic distance model and Neighbor-Joining tree methods available in Geneious version 10.1.3.”\nComment - Authors should clarify their choice for sequence analysis. The Geneious software has been developed for editing and aligning DNA / amino acid sequences. There are several softwares, which have been largely used to infer phylogenetic relationships. I suggest authors to refining and improving the phylogenetic analysis using appropriate programs and models that have been chosen for the mitogenome data you have at hand.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-347
|
https://f1000research.com/articles/8-292/v1
|
14 Mar 19
|
{
"type": "Research Article",
"title": "Association between +45T>G adiponectin polymorphism gene and type 2 diabetes mellitus and metabolic syndrome in a Venezuelan population",
"authors": [
"María Patricia Sánchez",
"Carem Prieto",
"Endrina Mujica",
"Kendry Vergara",
"Enifer Valencia",
"Eudymar Villalobos",
"Mayerlim Medina",
"Michael Parra",
"Rosanna D'Addosio",
"Kyle Hoedebecke",
"Johel E. Rodríguez",
"Valmore Bermudez",
"María Patricia Sánchez",
"Carem Prieto",
"Endrina Mujica",
"Kendry Vergara",
"Enifer Valencia",
"Eudymar Villalobos",
"Mayerlim Medina",
"Michael Parra",
"Rosanna D'Addosio",
"Johel E. Rodríguez",
"Valmore Bermudez"
],
"abstract": "Background: Adiponectin (ADIPOQ) is a hormone primarily synthesized by adipocytes and encoded by the ADIPOQ gene, which exerts anti-inflammatory, antiatheratogenic and insulin sensitizing functions. It has been shown that its plasma concentrations are decreased in individuals with metabolic syndrome (MS) and type 2 diabetes mellitus (DM2), which could be due to variations in the gene coding for this protein. The aim of this study was to detect the +45 T>G polymorphism of the ADIPOQ gene in subjects with DM2 and MS in Maracaibo municipality, Zulia state, Venezuela. Methods: A total of 90 subjects who attended the Center for Metabolic Endocrine Research \"Dr. Félix Gómez\" were enrolled for this study, 46 of which had MS-DM2 and 44 of which were healthy control individuals. Genomic DNA was extracted from blood samples and PCR-restriction fragment length polymorphism analysis was carried out for the promoter region of the ADIPOQ gene. Likewise, the +45 T> G polymorphism was identified and correlated with MS and DM2 in the studied population. Results: The most frequent allele in both groups was the T allele, and the predominant genotype was homozygous T/T (79%). Genotypes with heterozygous T/G and G/G homozygous polymorphism were more frequent in the control group than in the MS-DM2 group. Regarding the individuals with T/G and G/G genotypes, statistically significant lower mean values were found for fasting glucose, total cholesterol, triacylglycerides, abdominal circumference, and for the medians of systolic and diastolic blood pressure. Odds ratio were calculated for the presence or absence of MS and DM2. Conclusions: The results suggested that the presence of the G allele exerts a protective effect on the carrier individuals, thus avoiding the appearance of the aforementioned metabolic alterations.",
"keywords": [
"metabolic syndrome",
"type 2 diabetes mellitus",
"ADIPOQ gene",
"polymorphism",
"DNA"
],
"content": "Introduction\n\nAdiponectin contains 244 amino acids1 and this protein is expressed almost exclusively in white adipose tissues where it has anti-diabetic, anti-inflammatory, and anti-atherogenic properties in addition to cardioprotective effects2,3. It is considered one of the most abundantly secreted adipokines by the adipocyte, because its plasma concentrations in humans vary between 5–30 μg/ml, and although this values vary according to sex (generally higher in women)4, adiponectin represents 0.01% of total plasma proteins3. Unlike other known adipokines, its levels are decreased in insulin-resistance associated states, such as obesity, type 2 diabetes mellitus (DM2) and metabolic syndrome5.\n\nMetabolic syndrome (MS) is a complex of interrelated risk factors for the development of cardiovascular diseases and diabetes mellitus (DM)6. The prevalence of MS in the world is very high; 45% of the population over 50 years old and about 20% of the population under 50 years of age have MS7. Patients with MS have twice the risk of developing cardiovascular disease in 5 to 10 years after being diagnosed8, increasing the risk of myocardial infarction9.\n\nEpidemiological studies conducted in the U.S have shown that the general prevalence of MS is 24% in Caucasian populations, which increases according to age to more than 30% in people over 50 years of age and more than 40% in people over 60 years of age10. In a study carried out in Maracaibo, Venezuela, the prevalence of MS was 42.7%, presenting men at a higher risk when compared with women; as age increases, the risk also increases progressively. Similarly, diabetic individuals had up to 4 times higher risk of suffering from MS11.\n\nDiabetes mellitus is one of the main causes of morbidity and mortality, as well as an important cardiovascular risk factor and a poor prognosis in patients with established cardiovascular disease12. Its prevalence has drastically increased in the last 20 years and it is estimated that by the year 2030 the number of affected will exceed approximately 400 million people12, which translates into a 54% increase13.\n\nSome 422 million people suffer from diabetes in the world, totaling one out of every 11 people. It is estimated that in 2012, 1.5 million people died as a direct consequence of diabetes. According to WHO projections, diabetes will be the seventh greatest cause of mortality in 203014. In Venezuela, according to the 2011 Ministry of Public Power of Health, diabetes mellitus represents the fourth-leading cause of death. This data corresponds with WHO data. Furthermore, diabetes mortality projections remain high over the foreseeable future15. In an analysis conducted on a Venezuelan population (Maracaibo City, Zulia State) about the DM2 prevalence, it was found that 8.4% of the evaluated population had the disease, which is similar to that shown in our continent, in the Carmela study, which took in consideration seven cities in Latin America, evaluating more than 11,000 individuals aged between 25 and 64 years; this study reported a general prevalence of 7%, where the one shown in Mexico City stands out (8.9%) since is higher12.\n\nThe serum concentration of adiponectin is reduced in individuals with obesity, DM2, insulin resistance, obesity, dyslipidemia and coronary disease16,17. With an link to lower lipid oxidation, higher triglyceride content, and reduced levels of insulin-dependent signaling—reduced concentrations of adiponectin have been noted in the pathophysiology MS and DM218.\n\nThe ADIPOQ gene is located on chromosome 3q27, consists of 3 exons and is 15.8 kb long1. Genetic variants in ADIPOQ can lead to substantial changes in adiponectin levels, which provides convincing evidence that subtle alterations in the genetic code can modify the levels of this hormone, large enough to significantly affect the health, as mentioned before1. Several studies have determined that different polymorphisms (SNPs) have been located in the adiponectin gene, which can affect both the transcription and the activity of this hormone. Among the most prominent are the polymorphisms +45 T>G, +276 G>T, -11.377 C>G and -11.391 G>A19.\n\nStudies carried out in different populations, associated the +45T>G polymorphism of the ADIPOQ gene with the development of DM2, insulin resistance, obesity, coronary artery disease and hypoadiponectinemia19. Other studies reported that subjects with the T/G or G/G genotype at position 45 have a higher risk of developing DM2 when compared to those with T/T genotype20. Likewise, it has been observed that subjects who present the G allele of the +45T>G polymorphism have lower plasma levels of adiponectin19.\n\nDue to such significant findings, this study aimed to determine nucleotide alterations of the ADIPOQ gene, especially the +45T>G polymorphism and its relationship with DM2 and MS, in adult individuals of the Maracaibo municipality of Venezuela. The study aimed to identify cases of people carrying the alteration indicated in this gene, which could be related to DM2 and MS. In this way, the presence of the polymorphism would serve as a predictor for the development of MS and/or DM2 in the future16,21, guaranteeing the possibility that any person with a predisposing genotype can take opportune measures to prevent these diseases.\n\n\nMethods\n\nA total of 90 adult individuals, of which 46 had MS-DM2 and 44 were healthy controls, were enrolled in this study, which was carried out from January 2015 to January 2016. Participants were recruited through a mixed strategy. Some individuals were contacted by phone while in remote locations we made contact with the community leaders in order to verify the size and organization of each neighborhood since public records prove inaccurate. After this, we proceeded to draw sectors and blocks, and then, together with the community leaders, contact the members of the selected houses directly. These individuals participated in the Metabolic Syndrome Prevalence Project11 of the Metabolic Endocrine Research Center \"Dr. Félix Gómez\" (CIEM) of the Faculty of Medicine of University of Zulia performed from 2007 to 2008. The present study size was the same as the previous one. The participants signed an informed consent form, where they were explained everything related to the project and the handling of their samples and tests. This research was approved by the CIEM bioethics committee and complies with the Helsinki declaration. The studied subjects were chosen since they met the predetermined inclusion criteria and with the absence of selected exclusion criteria, depicted in Table 1. In order to make the diagnosis of MS and DM2, 2009 Consensus6 and ADA22 criteria was used respectively.\n\na Regarding Maracaibo, Venezuela these values are adjusted to the population11.\n\nWC, Waist circumference; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; HDLc, Cholesterol bound to high density lipoproteins; FG, fasting glucose; TG, triacylglycerides; DM2, type 2 diabetes mellitus; MS, metabolic syndrome.\n\nThe following variables were measured in the subjects:\n\n1. Waist circumference: taken with a graduated measure tape in centimeters at an equidistant point between the costal margin and the anterior superior iliac spine.\n\n2. Body mass index: estimated by dividing the kilograms of weight by the square of the height in square meters (BMI = kg/m2). A TANITA electronic scale was used to obtain the body weight. To measure the size, a height rod was used.\n\n3. Blood pressure: measured by the auscultatory method, for which a calibrated and properly validated sphygmomanometer was used.\n\n4. Levels of fasting glucose, total cholesterol, triacylglycerides and HDLc (cholesterol bound to high density lipoproteins): (previous fasting from 8 to 12 hours), for which 5 cm3 of blood was drawn from each individual obtained by antecubital venipuncture, which was placed in test tubes and centrifuged to 4000 rpm for 10 minutes. After this, the serum was extracted and placed in polypropylene test tubes for subsequent freezing at -70°C. The time between taking the sample and processing did not exceed three months. To determine the fasting glucose, an enzymatic-glucose-colorimetric enzyme kit (Glucose Liquicolor Ref. 10260300, Human Diagnostics) was used. For the quantification of total cholesterol, triacylglycerides and HDLc, commercial enzyme-colorimetric kits (Cholesterol Liquicolor Ref. 10028300; Triacylglicerides liquicolor mono Ref. 10724300; HDL Cholesterol liquicolor Ref. 10084300; all from Human Diagnostics) were used.\n\n5. Low-density lipoprotein bound to cholesterol (LDLc), calculated by the Friedelwald formula: (LDL-c = total cholesterol - (VLDL=c + HDL-c), where VLDL-c = triacylglycerides/5 (mg/dl) (provided that the triacylglycerides cannot exceed 400 mg/dl).\n\n6. Ultrasensitive CRP (usCRP): Turbidimetric method with latex for the quantitative determination of C Reactive Protein (CRP hs Turbitest AA Ref. 1683263, Wiener lab).\n\n7. Homeostatic model assessment of insulin resistance (HOMA-IR): estimated using HOMA with the following formula: HOMA-IR = fasting insulin (µIU/ml)×fasting glucose (mg/dl)/405, where ELISA methodology was used to determining fasting insulin (Insulin ELISA Ref. EIA2935 DRG International, Inc.)\n\nGenomic DNA extraction. A total of 5 ml peripheral blood (with EDTA as an anticoagulant) was obtained by venipuncture. For DNA extraction, the combined DNA-Salting out extraction technique was used, as described previously23, of the Molecular Genetics laboratory of the Medical Genetics Unit of the Faculty of Medicine was used.\n\nPCR amplification of 45 T>G polymorphism of the ADIPOQ gene. A 372 bp DNA fragment covering the region of interest was amplified by PCR, using this set of oligonucleotide primers: F5'-GAATGAGACTCTGCTGAGATGG-3' and R5'- TATCATGTGAGGAGTGCTTGGATG-3’ to amplify the region of interest (Figure 1). The amplification conditions were the following: 95°C for 6 min, followed by 35 cycles of 95°C for 1 min, 45 sec at 56°C and 45 sec at 72°C and a final extension at 72°C for 5 min24. For the amplification reactions, taq DNA polymerase from Promega was used.\n\nThe region of interest was amplified using this set of oligonucleotide primers: F5'-GAATGAGACTCTGCTGAGATGG-3' and R5'- TATCATGTGAGGAGTGCTTGGATG-3’.\n\nRFLP for the detection of the 45 T>G polymorphism of the ADIPOQ gene. The amplified fragment of 372bp was subjected to restriction analysis with the SmaI enzyme. A non-digested fragment (molecular weight, 372 bp) corresponded to the homozygous T/T genotype. The homozygous G/G genotype was evidenced by the presence of two fragments, one 219 bp and the other 153 bp. The product of the digestion of the heterozygous T/G genotype was evidenced by the presence of three fragments (372 bp, 219 bp and 153 bp), as shown in Figure 1.\n\nThe statistical package SPSS for Windows, version 21.0 was used. Allelic and genotypic frequencies were calculated for the total population evaluated and for the MS-DM2 and control groups. The χ2 test was used to compare frequencies and observed and expected values to evaluate the Hardy-Weinberg equilibrium (HWE). Means and medians were determined for biochemical, clinical and anthropometric characteristics accordingly. The normal distribution of the variables was verified by the Kolmorgov-Smirnov test. The comparison of the parametric variables with normal distribution was performed using Student's t-test, when 2 groups were compared and the Mann and Whitney U-test in those variables that had a non-normal distribution. A p-value <0.05 was considered statistically significant. Likewise, a binary logistic regression was carried out in order to obtain the odd ratio adjusted to a confidence interval of 95%, to estimate the relative risk of the variable under study. A p-value <0.05 was considered statistically significant.\n\n\nResults and discussion\n\nIn this study, the +45 T>G polymorphism of the ADIPOQ gene was associated with the development of MS and DM2 in adult individuals from Maracaibo City. No prior information was found on the association of that polymorphism of the ADIPOQ gene with individuals diagnosed with MS and DM2. Complete raw data are available on OSF25.\n\nClinical, biochemical and anthropometric characteristics of both groups (MS +DM2 and control patients) are shown in Table 2. Statistically significant differences were found when comparing the means and medians in almost all studied characteristics, with the exception of height and usCRP. Most of the parameters of MS and DM2 individuals were found to be higher than in healthy controls, with exception of HDL-c levels. In line with the findings of the present study, DM2 patients in Brazil, India and Russia18,26,27 showed similar results in clinical characteristics, such as BMI, HOMA-IR, fasting glucose, TC, TG, HDL-c, LDL-c, systolic blood pressure (SBP) and diastolic blood pressure (DBP). Moreover, Sahli et al.28 reported statistically significant differences in BMI, fasting glucose, TC, TG, HDL-c, LDL-c, DBP, and SBP in a Tunisian population. However, a DM2 Chinese Han population29, showed no significant difference when comparing height and usCRP in diabetic and healthy control groups. These differences could be related to the heterogeneity of the populations, due to the fact that they may have totally different lifestyles, including feeding, physical activity, environmental factors, and customs, among others.\n\nValues expressed as Median (interquartile range) unless indicated. *Variable expressed as logarithm. †Student’s t-test for independent samples. ‡Mann Whitney U-test. BMI, body mass index; WC, waist circumference; CRP, C-Reactive Protein; HOMA-IR, homeostatic model to estimate insulin resistance; FG, fasting glucose; TC, total cholesterol; TG, triacylglycerides; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP, diastolic blood pressure.\n\nTable 3 depicts the allelic and genotypic frequencies of the +45T>G polymorphism of the ADIPOQ gene, in patients with MS-DM2 and healthy control patients. The T/T genotype was found in greater frequency in the total population, although patients with MS-DM2 showed a greater frequency, as described by Sahli et al.28 in a Tunisian MS population. T/G and G/G genotypes were found in both groups but showed lower frequencies. The healthy controls showed a genotypic frequency of 27% and 5% for the T/G and G/G genotypes, respectively, which indicates that the homozygous T genotype is three times higher in healthy individuals. Other studies generated opposite results26,27,30,31, since the T/T genotype was found more frequently in healthy control patients than in DM2 ones.\n\nMS, metabolic syndrome; DM2, type 2 diabetes mellitus.\n\nRegarding the alleles, a higher frequency of the T allele was observed over the G allele in both study groups, the T allele being more frequent in the population with MS-DM2, where the Hardy-Weinberg balance χ2 = 5.99 is met, because the observed allele frequencies are as expected. Such findings relate to the MS population studied in Tunisia28 but differ with the type 2 diabetes mellitus individuals studied in Brazil, India and Ireland26,27,31 since the highest frequency of the T allele occurred in the healthy control group Table 4.\n\nMS, metabolic syndrome; DM2, type 2 diabetes mellitus.\n\nAs shown in Table 4, the population that differ the most from the one characterized in this study, turned out to be the Brazilian one26, although Brazil is near Venezuela, the studied subjects were of Japanese ancestry; this could be the reason of such genetic differences although the lifestyles and customs are similar in both countries.\n\nTable 5 shows that there was a significant difference among waist circumference, fasting glucose, total cholesterol, triacylglycerides, LDL-c, DBP and SBP, for the T/T and T/G + G/G genotypes regarding the total population. Those that had the presence of the G allele of the polymorphism exhibited lower values except for the usCRP in which those subjects that carried the T allele had lower levels in this parameter.\n\nValues expressed as median (interquartile range) unless indicated. *Variable logarithmically transformed for its normalization. †Student’s t-test for independent samples; ‡Mann-Whitney U-test. BMI, body mass index; CRP, C-reactive protein, HOMA-IR, homeostatic model to estimate insulin resistance, HDL-c, high-density cholesterol lipoproteins; LDL-c, low-density cholesterol lipoproteins; SBP, systolic blood pressure; DBP, diastolic blood pressure.\n\nOn the other hand, the control population (without MS-DM2) showed statistically significant difference regarding the DBP. In the MS-DM2, a significant difference was only observed in fasting insulin levels between groups. In addition, it was demonstrated that in these groups, the subjects who presented the G allele showed lower mean values than of those without this allele (homozygous T), except for the usCRP levels, which were higher in individuals with the G allele. Potapov et al.18 obtained contradictory results in their study in a DM2 Russian population, since the fasting glucose levels showed higher values for subjects carrying the G allele, and in control subjects, a statistically significant difference was only found in SBP, which was higher in those individuals who carried the G allele.\n\nSeveral studies have reported the behavior of several anthropometric, clinical and biochemical characteristics in their total populations concerning the +45 T>G polymorphism, finding opposite data that the one found during this study. In a non-diabetic female population in Greece16, fasting insulin and HOMA-IR levels were significantly lower in carriers of the rare +45G allele (T/G+G/G), which was also evidenced in Italian individuals with or without DM232, in non-diabetic individuals in Taiwan33, there were only significant differences in the BMI, with higher values for T allele carriers, unlike Punjab individuals34 who those that carried the G allele displayed higher BMI and WC values.\n\nAccording to Menzaghi et al.35, the biology of these associations with anthropometric, clinical and biochemical characteristics is still unclear, for example, it is mentioned in their research that studies in animal models show that adiponectin is a potent insulin activator and modulator, in addition to regulating energy homeostasis and glucose tolerance. This relationship has also been found between the adiponectin levels in rodents and humans, which suggests that the +45 T>G ADIPOQ polymorphism can exert its action by diminishing or enhancing the expression of adiponectin, which can increase or decrease body weight and insulin resistance. In other populations, this variant may be associated with DM2, obesity and MS, whereas in other association studies, insulin resistance and DM2 are a consequence of the presence of the T allele.\n\nAdditionally, a binary logistic regression model was used to determine the influence of the +45 T/G polymorphism of the ADIPOQ gene on MS-DM2, where the odds ratio showed no association of this polymorphism as a risk factor predisposing to MS-DM2. The presence of the rare allele is considered a protection factor (Table 6). These findings oppose those previously reported by Stumvoll et al.36 who studied non-diabetic Germans and recognized that the +45G allele was associated with higher IR indexes. This led to lower insulin sensitivity compared to T allele carriers while those subjects who carried the polymorphism could present a higher risk of obesity. The same phenomenon was described in diabetics and non-diabetics in Northern India34; however, no association between +45 T/G polymorphism and the development of DM2 was found in Koreans individuals with and without DM237. The G allele of the +45 T>G polymorphism of the ADIPOQ gene has been associated with endocrine-metabolic pathology in Chinese individuals with and without MS in a similar population, whereas in Tunisia no association of polymorphism with MS was found28,38.\n\nAlthough there is a lack of consistency among studies, there is evidence that the 45G allele of the ADIPOQ gene plays a protective role in MS and DM2, whereas the 45T allele behaves as a risk allele for the development of obesity and insulin resistance, which are both related to the development of MS and DM232,33,39, consistent with the findings in this study.\n\nThe +45 T>G polymorphism of the ADIPOQ gene is a silent mutation without its own biological effect, since it is a GGT to GGG substitution, which translates to Gly15Gly. Adiponectin’s role in insulin sensitivity and BMI, as well as its role in both MS and DM2 pathologies are associated with genetic alterations linked to the polymorphism-related disequilibrium. mRNA G allele transcripts vary, proving higher than those of T allele of heterozygous individuals. This suggests that promoter gene polymorphisms or other adiponectin gene regulatory elements could be involved in the decrease or increase of the expression of the studied adipokine, being able to affect the mRNA allowing the stability of the splice sites in the same, affecting the amount of circulating proteins28,30. It has also been observed that haplotypes that are in ligation unbalance with one or more functional variants can affect the levels of adiponectin in plasma, producing for example a change in the secondary structure of DNA, having a major influence on transcription, processing or translation; however, this is hypothetical, since the exact genetic mechanisms responsible for the expression of the specific allele have not been elucidated yet32.\n\n\nConclusions\n\nIn this study, biochemical, clinical and anthropometric characteristics corresponding to MS-DM2 were determined, and statistically significant differences were found between the means and medians of all the variables evaluated, except for height and usCRP. The genotype frequency of the +45 T/G polymorphism of the ADIPOQ gene, in the total population evaluated, was 79% for the homozygous T/T variant, 18% for the heterozygous T/G variant and 3% for the homozygous G/G variant. The allelic frequencies of the +45 T/G polymorphism were 0.88 for the T allele, while only 0.12 of the evaluated individuals exhibited the G allele.\n\nLikewise, it was found that there is a statistically significant difference between the means and medians of WC, fasting glucose, total cholesterol, triacylglycerides, LDL-c, DBP and SBP, between genotypes T/T and T/G with respect to the total population. In addition, the total population with the G allele showed lower mean values for these variables than those without this allele (homozygous T), except for the values of CRP and HDL-c, which were higher in individuals with the G allele. Overall, an association was found between the +45 T>G polymorphism of the ADIPOQ gene with MS-DM2. The results suggest that the presence of the G allele exerts a protective effect on carrier individuals, thus preventing the onset of MS and DM2.\n\n\nData availability\n\nOpen Science Framework: Association between +45T>G adiponectin polymorphism gene and type 2 diabetes mellitus and metabolic syndrome in a Venezuelan Population. https://doi.org/10.17605/OSF.IO/3HE6S25.\n\nThe project contains the following extended data:\n\nData Articulo.v3.xlsx (complete raw anthropometric data and ADIPOQ status).\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\nThe authors acknowledge the “Consejo de Desarrollo Científico, Humanístico y Tecnológico” (CONDES) of La Universidad del Zulia for the financial support of this project (Project No. CC-0429-16).\n\n\nReferences\n\nBreitfeld J, Stumvoll M, Kovacs P: Genetics of adiponectin. Biochimie. 2012; 94(10): 2157–63. PubMed Abstract | Publisher Full Text\n\nValero P, Souki A, Rodríguez NA, et al.: editors Valmore Bermúdez-Pirela Yaneth Herazo-Beltrán. Aspectos básicos en obesidad. Barranquilla: Ediciones Universidad Simón Bolívar; 2018. Reference Source\n\nNigro E, Scudiero O, Monaco ML, et al.: New insight into adiponectin role in obesity and obesity-related diseases. Biomed Res Int. 2014; 2014: 658913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFu Y: Adiponectin signaling and metabolic syndrome. Prog Mol Biol Transl Sci. 2014; 121: 293–319. 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}
|
[
{
"id": "47504",
"date": "07 May 2019",
"name": "Eman T Mehanna",
"expertise": [
"Reviewer Expertise Biochemistry and Molecular Biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors studied the relation of +45T>G adiponectin gene SNP and the parameters of type 2 diabetes mellitus and metabolic syndrome a Venezuelan population. They found that the rare G allele of this polymorphism may be protective against most of those parameters.\nComments:\nThe title should be modified to: (Association of +45T>G adiponectin gene polymorphism with type 2 diabetes mellitus and metabolic syndrome in a Venezuelan population). The language of the manuscript should be revised. Sometimes, the authors repeat sentences. For example, in the statistical analysis, the sentence (A p-value <0.05 was considered statistically significant) was repeated twice. I did not understand the purpose of Table 6. I think genotype and allele frequencies should have been compared in the study groups using chi square test to assess the relation of the polymorphism with the development of metabolic syndrome. In the legend of Figure 1, please define the genotype of each lane in the figure. Genotype TG is not clear at all due to the relatively faint bands. The conclusion is very long. I suggest summarizing it and mentioning only the important findings of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "47649",
"date": "08 May 2019",
"name": "Na Zhu",
"expertise": [
"Reviewer Expertise Bioinformatics",
"association study in human 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\nMajor concern:\nIn table 2, there is a significant age difference between case and control, it should be a covariate. The precision of the p-value is not precise, I suggest the author try other tools for the statistical test to get higher precision. In table 3, the allele frequency of this polymorphism varied among populations. The allele frequency of G in controls matched with the Latino population frequency in gnomAD. The allele frequency of G in cases is lower than gnomAD Latino population frequency but higher than African and Finnish. So, the ancestry is very critical for such analysis. How did the author decide the ancestry of each sample? Even the cases were completely Latino descents, as the cohort is small, the confidence interval of T’s AF is large, it could be possible to result in a higher frequency of T with p-value >0.001. In table 3, no association was present in the genotypic association or allelic association using a chi-square test. They also showed in table 6. In table 4, the frequency in Ireland case and controls cohort reversed with the author’s cohort, they also mentioned in the text, how did the author to explain it?\n\nMinor concern:\nIn the section “Frequency of ADIPOQ genotypes”, it said, “the homozygous T genotype is three times higher in healthy individuals”. How was it from? I saw three times heterozygous T/G, was it a typo? If yes, it was not significant at all. In table 2, I guess the age show median and interquartile range, then * should not be here. In table 4, there is a typo, G’s frequency 0.08 in healthy Ireland and Tunisia study. Several typos happened in table 5, the symbol is not consistent between case and control such as p-value in age, HOMA-R, and SBP. Table 5 has duplicated information as table 2, they may be able to optimize their presentation.\n\nOverall, I think the writing and the calculation are correct. However, the evidence they supplied cannot support their conclusions, such as no association between case and control and contrary finding among studies. +45T>G is a polymorphism, the presence of its frequency in case and control could occasionally happen for such a small cohort. It is not convincing to draw the conclusion that the phenotypic difference resulted from +45 T>G, this could be due to other common or rare variants.\n\nIs the work 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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "47149",
"date": "21 May 2019",
"name": "Tayebeh Pourmotabbed",
"expertise": [
"Reviewer Expertise Gene therapy",
"biochemistry",
"gene polymorphism studies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAdiponectin is a secretory protein and the most plentiful adipokine in circulation. It induces insulin sensitivity, has anti-inflammation and anti-atherogenic effects. Its low concentration is associated with an increase in body fat mass, insulin resistance and dyslipidemia. Adiponectin gene polymorphism has been shown to affect protein production and thus, its plasma concentration. In this manuscript, the authors have studied association between +45T>G polymorphism of the adiponectin gene (ADIPOQ) and risk of type 2 diabetes (DM2) and metabolic syndrome (MS) in subjects from Maracaibo municipality, Zulia state in Venezuela. Their data suggest that presence of the G allele at this position of adiponectin gene may protect the carrier individuals against MS and DM2, at least in this population.\n\nThe study is well thought of and well written, worth indexing. However, since low adiponectin level is associated with DM2 and MS and adiponectin gene polymorphism affects protein production; in the case of sample availability and technical support, it would be interesting to measure the level of adiponectin in the studied population. In addition, the presence of G allele may have an effect on stability of adiponectin RNA and thus the protein level, thus, determining the level of adiponectin RNA by semiquantitative PCR may shed some light on protective role of G allele.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-292
|
https://f1000research.com/articles/8-291/v1
|
14 Mar 19
|
{
"type": "Case Report",
"title": "Case Report: Successful embolisation of a ruptured splenic artery aneurysm in pregnancy",
"authors": [
"Tom Crawley-Smith"
],
"abstract": "Background: A case study of a successfully treated rupture of a splenic artery aneurysm during pregnancy, an exceedingly rare condition. The natural history, typical presentation, epidemiology, investigations and available treatments are discussed.\n\nCase: A multigravid 37-year-old presented with acute left upper quadrant pain in her twentieth week of pregnancy. After resuscitation and emergency imaging the patient was diagnosed with a ruptured splenic artery aneurysm. Remaining stable after initial resuscitation allowed for endovascular coiling of her aneurysm.\n\nDiscussion: The presentation of ruptured splenic artery aneurysm is rare. While pregnancy is a risk factor, it represents less than 0.1% of pregnancies. However, when it does present it is associated with a high maternal and foetal mortality rate. In this case the stability of the patient allowed for imaging to confirm the diagnosis and the provision of endovascular coiling of the aneurysm. On review of the literature the condition characteristically is diagnosed at laparotomy.",
"keywords": [
"Splenic artery aneurysm",
"pregnancy",
"endovascular coiling"
],
"content": "Abbreviations\n\nSAA, Splenic artery aneurysm; G6P3M1T1, Gravida 6 Para 3, 1 miscarriage, 1 termination; BIBA, Brought in by ambulance; LUQ, Left upper quadrant; PV, Per vaginal; SBP, Systolic blood pressure; FAST, Focussed assessment with sonography for trauma; ROTEM, Rotational thomboelastometry; QID, quarter in die/four times each day\n\n\nIntroduction\n\nSplenic artery aneurysm (SAA) is a rare diagnosis. Its high mortality and typically delayed diagnosis make it a surgical diagnosis not to be missed. While rare during pregnancy, rupture of a splenic artery aneurysm carries a maternal morbidity of 75% and neonatal mortality approaching 95%1. Within Australia recently there have been two prominent coroners’ reports originating from South Australia2 and Victoria3. While both coronial inquests recognised the disease’s rarity, it was commented that the high morbidity and mortality associated with the condition mandate a high clinical suspicion in the presentation of acute abdomen during pregnancy2,3.\n\nWhile the most common visceral aneurysm, splenic artery aneurysm is infrequently encountered. Within women of childbearing age it occurs in less than 0.1% of the population4, although true prevalence may be unknown, with many being asymptomatic5. It has a 4:1 female to male prevalence, and is typically seen at the 6th decade of life4–6. The risk factors include multiparity, angiodysplasia, atherosclerosis, polyarteritis nodosa, cirrhosis, portal hypertension and connective tissue disorders especially α1 anti-trypsin deficiency4–6. A five-year retrospective study conducted by vascular surgeons in Dallas, Texas, at one of the largest obstetric hospitals within the USA found no diagnoses of SAA for 67,000 births, with only 35 diagnosed cases during that time, none whom were pregnant4. 89% of female patients diagnosed had previously been pregnant however, with multiparity an increased risk factor4.\n\nThe state of pregnancy is a physiological and hormonal risk factor for developing SAA. Tremble and Hill’s classic proposal7 for the two causes of splenic aneurysm development, being initial weakness of the arterial wall compounded by increased systemic blood pressure, may be applied. During pregnancy, the hormones oestrogen, progresterone and relaxin cause weakness within the vessel wall8,9. Histologically this is described as subendothelial thickening, internal lamina fragmentation, medial myelodysplasia8,9 and increased acid glycosaminoglycans within the subintimal and medial layers10. While the wall is weakened it is vulnerable to the increased pressures associated with the increased cardiac output, circulating volume and portal hypertension associated with the gravid state8. Aortic aneurysm, cerebral aneurysm and ovarian aneurysm have also been noted within the literature to be associated with pregnancy and multiparity5.\n\nSplenic artery aneurysm may present incidentally during the screening ultrasounds of modern antenatal care, or with rupture5,8,9. The latter is classically described as a presentation of left upper quadrant pain, potentially with Kehr’s sign11 (pain radiating to left shoulder tip) with initial instability, a quiescent phase of bleeding into the lesser sac before further collapse. This double rupture phenomenon is only described in 20–30% of cases5 but is important clinically as it may provide a false reassurance to treating staff. The typical size of an aneurysm at rupture is typically above 2.5cm, although ruptures of aneurysms 0.5cm in size have been described. This makes the discovery of the incidental splenic artery aneurysm during antenatal scans problematic, particularly for those under 2cm in size. The diagnosis even at rupture can be a challenge, differentials which can present similarly including pulmonary embolus, amniotic embolism and placental abruption; due to the general instability of the presenting patient imaging may be a guide to treatment but should not interrupt prompt resuscitation. Many descriptions within the literature describe confirmation of the diagnosis at laparotomy and emergency caesarean section.\n\nSee Table 1 for a timeline of symptoms, diagnosis and intervention.\n\nThe patient who presented was a 37-year-old female with significant multigravid obstetric history. G6P3M1T1, she had an inert Mirena in situ at the time of her pregnancy and had had two prior presentations to emergency for threatened miscarriage during this pregnancy. She had a previous surgical history of laparoscopic cholecystectomy and Roux-en-Y gastric bypass for obesity, for which she had lost 50kg, current weight being 110kg.\n\nShe complained of two days of left upper quadrant (LUQ) pain, no fevers but sweats and vaginal spotting. There were no other reported symptoms of note.\n\nOn presentation to emergency with Queensland Ambulance Service she was diaphoretic, in obvious pain localising to the left upper quadrant. She was afebrile, with an initial systolic blood pressure of 80mmHg, which improved to 100-120mmHg with crystalloid resuscitation. Her abdomen was difficult to examine given her habitus, but she had localising peritonism to her LUQ.\n\nSimultaneous wide bore IV access was obtained while taking venous gas and formal bloods. A cross match was sent. A bedside FAST scan was positive. Obstetric and General surgical review was sought. After two litres of crystalloid resuscitation her blood pressure had stabilised. The initial haemoglobin had come back at 110g/L. A formal ultrasound was ordered within the resus bay. This revealed fluid within Morrison’s pouch and a likely splenic artery aneurysm.\n\nBecause the patient remained stable, the decision was made to perform a CT angiogram limited to the upper abdomen to limit radiation. The risks and benefits of this were discussed with the patient prior to ordering. This confirmed the diagnosis with a 30x18mm aneurysm arising from the distal splenic artery within the hilum with a small triangular beak anteriorly suspected to be the area of rupture.\n\nGiven ongoing haemodynamic stability, there was discussion between the Hepatobiliary consultant, Obstetricians and Intervention Radiology consultant of the risks and benefits of radiographic coiling compared to laparoscopic intervention. The outcome was that an attempt at coiling was made that night with as little radiation and contrast as could be allowed. Where possible single imaging shots were taken rather than continuous runs, with small aliquots of contrast administered in each run. The procedure was successful and the patient was returned to the ward.\n\nAfter observation for two days, a repeat ultrasound was performed. This showed no residual aneurysm. The patient complained of dysuria at day two and was put on oral cephalexin 250mg QID. She was discharged on day three on oral antibiotics with a plan to attend her obstetric clinic and a surgical clinic at 14 days.\n\n\nDiscussion\n\nWhile a rare presentation, rupture of a splenic artery aneurysm represents a life-threatening event for the patient and within an obstetric setting, her pregnancy. Prompt diagnosis and resuscitation in this case allowed for further evaluation with further imaging. The fact that the patient remained haemodynamically stable also allowed for the option for interventional radiological coiling. Considerations which were considered by the treating team were the risk to the foetus from radiation and contrast, combined with the potential delay of surgery in the event of a failed embolisation. The option of laparoscopic evaluation and potential progression to splenectomy was considered. This was to be the next approach in the event of a failed embolisation.\n\nEmbolisation of the spleen, while potentially less physiologically stressful than surgical intervention8 is not without risk of complication1. Potential risks included migration of the coil, distal infarction, abscess formation and further aneurysm rupture1,5. The decision to embolise also needs to consider the status of the patient, anatomical location of the lesion and availability of interventional radiology. In the event of instability, prompt surgical intervention, typically through caesarean laparotomy or laparoscopy with ligation of the splenic artery and splenectomy is usually the recommended action.\n\nThe mainstay treatment of SAA outside pregnancy is radiological coiling5,8. Introduction of coiling has drastically improved the morbidity and mortality of treatment of visceral aneurysms. Elective treatment should anticipate pregnancy in women of reproductive age with the criteria for intervention typically as a diameter >1cm5. Consideration of radiological coiling anticipates the anatomical location of the aneurysm with the goal of spleen preservation, which may not be possible with aneurysms affecting the splenic hilum. The discovery of a splenic artery aneurysm incidentally during pregnancy is worth considering. Typically, embolisation is recommend above 2cm10, with intervention ideally occurring in the 2nd trimester, both to minimise harm to the foetus and reduce the risk of rupture, the majority of ruptures being described in the 3rd trimester.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLang W, Strobel D, Beinder E, et al.: Surgery of a splenic artery aneurysm during pregnancy. Eur J Obstet Gynecol Reprod Biol. 2002; 102(2): 215–6. PubMed Abstract | Publisher Full Text\n\nCoroner of South Australia: Inquest 01.2015; 0597–2012. Reference Source\n\nCoroner of Victoria: Finding into death with inquest. CR2010/1093.\n\nNanez L, Knowles M, Modrall JG, et al.: Ruptured splenic artery aneurysms are exceedingly rare in pregnant women. J Vasc Surg. 2014; 60(6): 1520–3. PubMed Abstract | Publisher Full Text\n\nSadat U, Dar O, Walsh S, et al.: Splenic artery aneurysms in pregnancy--a systematic review. Int J Surg. 2008; 6(3): 261–5. PubMed Abstract | Publisher Full Text\n\nStanley JC, Fry WJ: Pathogenesis and clinical significance of splenic artery aneurysms. Surgery. 1974; 76(6): 898–909. PubMed Abstract\n\nTremble W, Hill J: Congestive splenomegaly(Banti’s disease) due to portal stenosis without hepatic cirrosis; aneurysm of the splenic artery. Arch Pathol Lab Med. 1942; 34: 423.\n\nAbbas MA, Stone WM, Fowl RJ, et al.: Splenic artery aneurysms: two decades experience at Mayo clinic. Ann Vasc Surg. 2002; 16(4): 442–9. PubMed Abstract | Publisher Full Text\n\nSelo-Ojeme DO, Welch CC: Review: spontaneous rupture of splenic artery aneurysm in pregnancy. Eur J Obstet Gynecol Reprod Biol. 2003; 109(2): 124–7. PubMed Abstract | Publisher Full Text\n\nMartinez E, Mendez A, Ablanedo P: Splenic artery aneurysms. Int Surg. 1986; 71: 95–9.\n\nKlimpel V: Stammt das “Kersche Zeichen” von Hans Kehr? Der Chirung. 2004; 75: 80–83."
}
|
[
{
"id": "58560",
"date": "04 Feb 2020",
"name": "Jamil Victor de Oliveira",
"expertise": [
"Reviewer Expertise Vascular 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\nThe text presents a disease that, although uncommon, is extremely relevant because it affects pregnant women. The text presents a good introduction and clinical description, but it suffers in the part of the intervention, which is poor, without images, without description of the procedure, doses of radiation, contrast, difficulties or technical facilities. In short, there is a lot described in these themes (which after all is the main reason for reading this article). I believe that, if these suggestions are implemented, it will become a rich article, which will be widely read.\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-291
|
https://f1000research.com/articles/8-286/v1
|
14 Mar 19
|
{
"type": "Opinion Article",
"title": "Improved control of non-communicable diseases (NCDs) requires an additional advanced concept for public health – a perspective from a middle-income country",
"authors": [
"Benja Muktabhant",
"Frank Peter Schelp",
"Ratthaphol Kraiklang",
"Pornpimon Chupanit",
"Pattara Sanchaisuriya",
"Benja Muktabhant",
"Ratthaphol Kraiklang",
"Pornpimon Chupanit",
"Pattara Sanchaisuriya"
],
"abstract": "A major consequence of all elements of the ‘epidemiological transition’ is the rapid emergence of non-communicable diseases (NCDs) in low- and middle-income countries. In contrast to the outcomes of the ‘Alma Ata Conference for Primary Health Care’, it has not yet been possible to introduce an equally powerful health policy for the prevention and control of NCDs. Major strategies so far are to advise individuals not to smoke and drink alcohol in excess. Additionally, ‘healthy’ nutrition and increased physical activity are also advocated. Policy for preventing and working against NCDs is now part of the Sustainable Development Goals, specifically target 3.4. So far, attempts to soften the influence of NCDs on the health of the people in low- and middle-income countries have been unsuccessful. It is argued here that additional concepts on how public health could operate against NCDs are needed. Major risk factors for NCDs interfere with and alter complex steps within the human metabolism. This paper explores how human metabolism works by assessing advances in molecular biology and research in genetics, epigenetics and gerontology. Recent developments in these scientific disciplines shed light on the complexity of how human health is maintained and diseases are invoked. Public health bodies should be aware, interested and possibly contribute to the aforementioned areas of interest, as far as NCDs are concerned, and translate major developments in a way, that could be useful in improving population health.",
"keywords": [
"public health",
"non-communicable diseases",
"sustainable development goals",
"primary health care",
"human metabolism",
"molecular epidemiology",
"epigenetic",
"geroscience",
"healthy aging"
],
"content": "Background\n\nIn recent decades, mortality due to non-communicable diseases (NCDs) have become priority health problems in countries in South and South-East Asia, as well as Latin America and Africa1. From the perspective of high-income countries2, this development did not fit well with infectious diseases, previously thought to be one of the issues for those countries3, which are predominantly located in the tropics and sub tropics. Many of these formerly so-called ‘developing countries’ still have low technical and financial resources. Their geographical location implies an environment favorable for infectious diseases. Although a number of infectious diseases are linked to NCDs4 at least to develop strategies to control the spread of infective agents, through various means such as hygienic measures, impregnated bed nets against malaria, antibiotics etc., is relatively easy in comparison to changing the ‘unhealthy behavior’ of individuals5. It is well accepted that ‘unhealthy behavior’ is the main underlying cause for the occurrence of NCDs.\n\nNCDs have been growing in high-income countries for much longer than in low and middle-income countries. In most high-income countries, the majority of the population lives an urban lifestyle, even if residing in rural areas. Under these circumstances’ public health bodies concentrate on how to finance health care and use mass media for health messages. Low- and middle-income countries base their health delivery systems on public health strategies focused on rural areas6, in which the majority of the population lives, while urban area lifestyles differ significantly from rural ones7. In rural areas it was and still is relatively easy for public health personal to approach the population directly.\n\nThe living conditions and the economic situation in many low- and middle-income countries have improved considerably in recent decades. However, public health initiatives of low- and middle-income countries cannot simply adopt the ways and means of high-income countries to cope with NCDs, but have to adjust public health policies to their own biological, socio-economic and cultural environments. It is argued in this communication that present strategies of public health, from low-, but in particular middle-income countries, should be reshaped by integrating developments and results in molecular biology, genetics and epigenetics as well as attempts to enable ‘healthy aging’ to facilitate the long-term control and prevention of NCDs.\n\n\nPrimary health care\n\nLow- and middle-income countries orientate the formulation of their health policy on recommendations from institutions of the United Nations. The World Health Organization (WHO) plays a crucial role in providing expertise, and assisting in setting targets scenarios for health policies of global importance. In 1978, WHO and United Nations International Children's Emergency Fund (UNICEF) were the leading UN agencies at the International Conference on Primary Health Care held in Alma-Ata, at that time the capital of Kazakhstan, then part of the Soviet Union. The major outcome of the conference was the Alma-Ata Declaration, which shaped health policies for decades worldwide. Key issues of primary health care (PHC) was the quest for a ‘comprehensive, universal, equitable and affordable health care service’ with the involvement of the community8. The original concept of ‘health for all by the year 2000’ was too ambitious9 and PHC in most low- and middle income countries tried to concentrate further on mother and child health care (MCH), undernutrition, hygiene, and prevention of infectious diseases10. Thirty years after the Alma Ata Declaration the usefulness of PHC was shown in the reduction of child mortality, increase in immunization and family planning for a number of countries. Especially Thailand was mentioned in achieving the highest average yearly reduction in mortality among the ‘under-fives’11.\n\nUnfortunately, the success of PHC was obscured by the emergence of AIDS and NCDs. Public health authorities started to face a ‘double burden’, providing care for infectious diseases but also facing the rapid increasing incidence and prevalence of NCDs1. The emergence of NCDs is often referred to as ‘the epidemiological transition’ including the ‘demographic-‘ and ‘nutritional transition’12. These developments were due to improved food security, prevention and advanced treatment of infectious diseases, as well as improvements in medical care as a whole and reduced fertility. Yet financial donors, who in many countries support the health system, were slow to acknowledge that NCDs were increasingly becoming a problem for poorer countries. This might be accounted for by the fact that controlling and treating infectious diseases, at least conceptually, is easier than preventing and caring for patients with NCDs. Communicable diseases are usually caused by one particular agent causing a disease with limited duration. Once the mode of infection and the life cycle of the infective agent are known, preventive and control measures can often be developed much more easily in comparison with NCDs. In contrast, NCDs have started to occur in low- and middle-income countries ‘with lethal consequences’, particularly in poorer countries where the health delivery system continued to focus heavily on infectious diseases, with little or no priority given to NCDs.\n\n\nSustainable Development Goals (SDG)\n\nThe pressure to pay adequate attention to the obvious problem which NCDs pose to low- and middle-income countries has finally become a political issue of global importance. The countries exposed to the phenomenon of the ‘epidemiological transition’ were well aware of the problem12. For instance at the start of the millennium NCDs were of major concern for the Thai Ministry of Public Health13. Initiated by Bangladesh and Caribbean Communities and supported by the International Diabetes Federation (IDF)14, it was finally decided that a ‘High Level Meeting’ on NCDs should be held September 19 to 20, 2011 in New York. Major additional driving forces for the meeting was the Global NCD Alliance and the ‘Lancet NCD Action’15. As far as prevention was concerned, the focus was laid on the so-called ‘best buys’: Improve tobacco control, eliminate tobacco use; reduce salt intake; promote diets low in fats and sugar; reduce alcohol consumption; increase physical activity; and treatment with generic drugs16.\n\nThe 2011 meeting was followed by a meeting in 2014 which established the most recent basis for a global policy known as the SDGs. Under target 3.4, the SDGs focus on cardiovascular diseases, diabetes, chronic obstructive pulmonary diseases and cancer17. The aim is to decrease premature death through NCDs by one-third up to 2030. The four main risk factors mentioned are smoking, ‘unhealthy’ diets, physical inactivity and ‘harmful’ use of alcohol. Governments and public health authorities are encouraged to enforce so called 16 ‘best buy’ strategies to reach the target.\n\n\nPoor prospects to reach SDG’s targets for controlling NCDs\n\nIn preparation of another UN High-Level-Meeting in 2018, it was realized that no progress in adequately dealing with NCDs was achieved and also could not be expected up to 2030, unless drastic action from governments was forthcoming. In preparation for the 2018 meeting the ‘Lancet Taskforce for NCDs and economics’ suggested to ‘use fiscal policies to regulate the prices and consumption of potential unhealthy products. The main targets are tobacco, alcohol, soft drinks and snacks18. However, the intention to stimulate governments to increase taxes in particularly on sugar and soft drinks failed and it was concluded that the ‘high-profile, somewhat risky and ultimately sobering test of the proposition that non-communicable disorders (NCDs) could become a new global health priority’. Feasible suggestions to governments were to increase taxes on tobacco, eliminate ‘trans fats’, work against consumption of salt, sugar and saturated fats and increase physical activity. Screening, counseling and therapy for heart disease and cervical cancer was outlined as well as immunization against hepatitis B.\n\n\nPresent strategies and problems of public health to work against NCDs\n\nThe disappointing overall results of the meeting, despite the forceful attempts of influential bodies such as the NCD Alliance and the Lancet Taskforce on NCDs18, should not have been unexpected. To increase taxes on very popular consumer products prompts counteractions of the commercial sector19. A telling example is how the US beverage manufacturers campaigned against cities willing to increase local taxes on sugar and soft drinks, by lobbying against statewide legislation via campaigns against ‘tax happy politicians’. Although tax increases were not planned for any grocery articles, except sugary beverages, the initiative to increase taxes on these items was finally abandoned20.\n\nDespite obvious constrains, the need and usefulness to work on health policies and agree on overall targets to improve world-wide developments cannot be questioned. It is the duty of governments and respective ministries to push forward towards the wellbeing of the populations under their responsibility. To work for prevention of NCDs remains a difficult task for governmental agencies, which already face difficulties providing an affordable health delivery system for those already suffering from NCDs. The main risk factors for a number of important NCDs are related to what is inhaled, as far as smoking is concerned, and ingested by mouth, in terms of alcohol and nutrients. People are generally more afraid of the more immediate and obvious effects of infectious diseases and hardly think about NCDs, which might or might not, affect them in the distant future21. To regulate behavior in authoritative ways creates resistance. The social sciences try their best to influence behavior based on well thought out but rather complex theories, such as the ‘Health Belief Model’, ‘Trans Theoretical Model’, and the ‘Theory of Planned Behavior’ and more recently by ‘Health Literacy’22–24.\n\n\nContemplating the complexity of human metabolism\n\nIt is possible that ‘health education’ could be more sustainable and more convincing, especially for educated, highly motivated, health conscious individuals, if the complexity of the human metabolism is given more attention by public health authorities with regards to health-related suggestions. This should also include the influence of genetic factors, the interaction of the environment on inherited factors, and the developments in the search for indicators measuring biological age, which might, in a positive way, trail chronological age. All this should be communicated in a way that is easily comprehensible by the general public. Governmental health authorities should consider, plan and monitor developments in health and diseases more efficiently, if the aforementioned issues are to be better known and understood. Those responsible for the improvement of the health of communities, on the professional and academic level, should be the link between medicine as well as molecular biology to public health, to turn recent advantages in epigenetic, and ‘gerosciences’ to the benefit of overall populations. This could be done without giving up the achievements of public health in following the strategies of PHC in the past, but could be even more constructive by integrating additional new concepts as it is mentioned here.\n\n\nPopulation based research in molecular epidemiology and other biomedical fields\n\nTo a certain extent, population-based research applying the tools of molecular epidemiology25, is already done of public health institutions and those representing other areas, such as medicine, nursing, social sciences and tropical medicine. In a subsequent communication the subject of molecular epidemiology and the use of biomarkers will be discussed more specifically. Additionally, we argue, increased attention should be given to the interaction of diseases such as diabetes and cancer26–28, genetic sciences, epigenetic features and geroscience.\n\nCarriers of certain genes are at risk of obesity29,30, and others develop diabetes mellitus and cancer31,32. Studies so far are more or less restricted to populations of high-income countries33. The importance of genetic factors for the manifestation of diabetes and cancer are not yet well known but should also be considered as potential important risk factors as well in addition to environmental risks especially for low- and middle-income countries.\n\nEpigenetic mechanisms are even more of importance. Nessa Carey, in her book ‘the Epigenetics Revolution’ (2012)34 mentioned that in the nineteenth century, Darwin and Mendel defined the area of evolution and genetics, Watson and Crick in the twentieth century the area of DNA, and in the twenty-first century ‘the new scientific discipline of epigenetics’ …[deconstructed]… ’so much of what we took as dogma and...[rebuild a]…’more complex, and…more beautiful fashion.’\n\nEpigenetics was originally defined as the interaction between genetics and environment. A more accurate definition refers to gene functionality which is not encoded into the DNA sequence, but can be hereditary. That is, the code of the DNA is not changed but the transcription from the code. One of the major control mechanisms related to epigenetics is linked to DNA methylation, for example the addition of a methyl group at the 5th carbon of the cytosine base at a CpG dinucleotide pair. Other mechanisms of epigenetic modification are chromatin remodeling and micro-RNAs modifying gene expression35. The important concept here is that the function of the gene is modified by environmental influences, especially in the field of nutrition36. Of particular interest for public health should be epigenetic features in embryogenesis and postnatal developments related to gestational diabetes, and efforts to prevent non-communicable diseases in later life or even in future generations. These aspects are of critical potential importance for mother and child health services37,38.\n\n\nGeroscience and healthy aging\n\nEpigenetic functions also play a major role in aging. The so called ‘epigenetic clock’39 received much attention in the attempt to differentiate the ‘biological age’ from the ‘chronological age’, an active research field in ‘geroscience’, formerly called ‘biogerontology’40. Research on biological aging tries to answer the question of why some people are already rapidly aging in their fifties, while others appear very young in mind and active far above their 80th birthday41,42. ‘Healthy aging’ is of eminent importance, since in the context of the demographic transition the elderly population is already increasing in low- and middle-income countries. The reasons for this include improved medical care, decrease in infectious diseases, better mother and child health care, better nutrition, hygiene, housing and education. All these combine to produce higher life expectancy43–45. While research in ‘geroscience’ manipulates the genetic settings of laboratory animals and the results are not yet of relevance for public health; preventive medicine, however, should work for ‘healthy aging’, and in this respect geroscience is of particular interest for public health. Increasing age, as such, is a risk factor for NCDs46. In future the topic ‘healthy aging’ will not only be of high relevance for high income countries but a global aspiration.\n\n\nSummary and conclusion\n\nPrevention and control of NCDs requires establishing among the population generally ‘healthy’ behaviors. Conservative public health strategies should be supplemented to promote longer lasting ‘intrinsic’ motivation for change into healthier life styles. This requires the complexity of human metabolism to be well communicated by health authorities and well understood by the recipients of the message. A precondition for this is that health authorities are more aware of developments in molecular biology, genetics and epigenetic features. In the near future it will be indispensable to work for ‘healthy aging’ by means of public health initiatives. It will be of mutual benefit for players in molecular biology, medicine, public health and governmental health authorities and other related fields to cooperate more closely. Cooperation doesn’t mean solely to support or join together in research projects and make use of the results for improving the health of the population, it also means a willingness for all involved to look beyond the narrow boundaries of their fields of interest.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "Grant information\n\nThis work was supported by the Research Division by the Khon Kaen University under the Excellence Centre for the ‘Research Group on Prevention and Control of Diabetes Mellitus in the Northeast of Thailand’.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors are grateful to Assoc. Prof. John F. Smith, Ph.D. from the Faculty of Public Health, Khon Kaen University, for revising the English of the manuscript.\n\n\nReferences\n\nBoutayeb A: The double burden of communicable and non-communicable diseases in developing countries. Trans R Soc Trop Med Hyg. 2006; 100(3): 191–9. PubMed Abstract | Publisher Full Text\n\nIllich I: Medical nemesis. The expropiration of health. New York: Pantheon Books; 1975. Reference Source\n\nWalsh JA: Disease problems in the Third World. Ann N Y Acad Sci. 1989; 569: 1–16. PubMed Abstract | Publisher Full Text\n\nO'Connor SM, Taylor CE, Hughes JM: Emerging infectious determinants of chronic diseases. Emerg Infect Dis. 2006; 12(7): 1051–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWillett WC, Koplan JP, Nugent R, et al.: Prevention of Chronic Disease by Means of Diet and Lifestyle Changes. In Jamisson DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A, Musgrove P, editor. Disease control priorities in developing countries. New York: International Bank for Reconstruction and Development; World Bank; Oxford University Press; 2006. PubMed Abstract\n\nStrasser R, Kam SM, Regalado SM: Rural Health Care Access and Policy in Developing Countries. Annu Rev Public Health. 2016; 37: 395–412. PubMed Abstract | Publisher Full Text\n\nWang'ombe JK: Public health crises of cities in developing countries. Soc Sci Med. 1995; 41(6): 857–62. PubMed Abstract | Publisher Full Text\n\nUnger JP, Killingsworth JR: Selective primary health care: a critical review of methods and results. Soc Sci Med. 1986; 22(10): 1001–13. PubMed Abstract | Publisher Full Text\n\nHall JJ, Taylor R: Health for all beyond 2000: the demise of the Alma-Ata Declaration and primary health care in developing countries. Med J Aust. 2003; 178(1): 17–20. PubMed Abstract\n\nWarren KS: The evolution of selective primary health care. Soc Sci Med. 1988; 26(9): 891–8. PubMed Abstract | Publisher Full Text\n\nRohde J, Cousens S, Chopra M, et al.: 30 years after Alma-Ata: has primary health care worked in countries? Lancet. 2008; 372(9642): 950–61. PubMed Abstract | Publisher Full Text\n\nPopkin BM: An overview on the nutrition transition and its health implications: the Bellagio meeting. Public Health Nutr. 2002; 5(1A): 93–103. PubMed Abstract | Publisher Full Text\n\nKosulwat V: The nutrition and health transition in Thailand. Public Health Nutr. 2002; 5(1A): 183–9. PubMed Abstract | Publisher Full Text\n\nColagiuri R, Keeling A: United Nations High Level Meeting on Non-Communicable Diseases: An unprecedented opportunity. Diabetes Res Clin Pract. 2011; 93(2): 137–8. PubMed Abstract | Publisher Full Text\n\nBeaglehole R, Bonita R, Alleyne G, et al.: UN High-Level Meeting on Non-Communicable Diseases: addressing four questions. Lancet. 2011; 378(9789): 449–55. PubMed Abstract | Publisher Full Text\n\nSacco RL, Smith SC, Holmes D, et al.: Accelerating progress on non-communicable diseases. Lancet. 2013; 382(9895): e4–5. PubMed Abstract | Publisher Full Text\n\nNugent R, Bertram MY, Jan S, et al.: Investing in non-communicable disease prevention and management to advance the Sustainable Development Goals. Lancet. 2018; 391(10134): 2029–35. PubMed Abstract | Publisher Full Text\n\nSassi F, Belloni A, Mirelman AJ, et al.: Equity impacts of price policies to promote healthy behaviours. Lancet. 2018; 391(10134): 2059–70. PubMed Abstract | Publisher Full Text\n\nCohen D: Will industry influence derail UN summit? BMJ. 2011; 343: d5328. PubMed Abstract | Publisher Full Text\n\nKamerow D: Under the cover of groceries: beating sugar taxes. BMJ. 2018; 363: k5111. PubMed Abstract | Publisher Full Text\n\nRosenberg C: Managed fear. Lancet. 2009; 373(9666): 802–3. PubMed Abstract | Publisher Full Text\n\nHealey B, Zimmermann RS Jr: The new worls of health promotion. Sudbury, Massachusetts Johns and Barlett Publishers, 2010. Reference Source\n\nJones CJ, Smith H, Llewellyn C: Evaluating the effectiveness of health belief model interventions in improving adherence: a systematic review. Health Psychol Rev. 2014; 8(3): 253–69. PubMed Abstract | Publisher Full Text\n\nSørensen K, Van den Broucke S, Fullam J, et al.: Health literacy and public health: a systematic review and integration of definitions and models. BMC Public Health. 2012; 12: 80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSlattery ML: The science and art of molecular epidemiology. J Epidemiol Community Health. 2002; 56(10): 728–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHabib SL, Rojna M: Diabetes and risk of cancer. ISRN Oncol. 2013; 2013: 583786. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShikata K, Ninomiya T, Kiyohara Y: Diabetes mellitus and cancer risk: review of the epidemiological evidence. Cancer Sci. 2013; 104(1): 9–14. PubMed Abstract | Publisher Full Text\n\nSun G, Kashyap SR: Cancer risk in type 2 diabetes mellitus: metabolic links and therapeutic considerations. J Nutr Metab. 2011; 2011: 708183. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClaussnitzer M, Dankel SN, Kim KH, et al.: FTO Obesity Variant Circuitry and Adipocyte Browning in Humans. N Engl J Med. 2015; 373(10): 895–907. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFawcett KA, Barroso I: The genetics of obesity: FTO leads the way. Trends Genet. 2010; 26(6): 266–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli O: Genetics of type 2 diabetes. World J Diabetes. 2013; 4(4): 114–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrank SA: Genetic predisposition to cancer - insights from population genetics. Nat Rev Genet. 2004; 5(10): 764–72. PubMed Abstract | Publisher Full Text\n\nLangenberg C, Lotta LA: Genomic insights into the causes of type 2 diabetes. Lancet. 2018; 391(10138): 2463–74. PubMed Abstract | Publisher Full Text\n\nCarez N: The epigenetics revolution. London: Icon Books LTD; 2012. Reference Source\n\nHartwell LH: Genetics. From Genes to Genomes. 4 ed. New York: McGraw-Hill; 2011; 200–31. Reference Source\n\nFradin D, Bougneres P: T2DM: Why Epigenetics? J Nutr Metab. 2011; 2011: 647514. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDrong AW, Lindgren CM, McCarthy MI: The genetic and epigenetic basis of type 2 diabetes and obesity. Clin Pharmacol Ther. 2012; 92(6): 707–15. PubMed Abstract | Publisher Full Text\n\nVeeraswamy S, Vijayam B, Gupta VK, et al.: Gestational diabetes: the public health relevance and approach. Diabetes Res Clin Pract. 2012; 97(3): 350–8. PubMed Abstract | Publisher Full Text\n\nHorvath S, Raj K: DNA methylation-based biomarkers and the epigenetic clock theory of ageing. Nat Rev Genet. 2018; 19(6): 371–84. PubMed Abstract | Publisher Full Text\n\nJylhävä J, Pedersen NL, Hägg S: Biological Age Predictors. EBioMedicine. 2017; 21: 29–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLowsky DJ, Olshansky SJ, Bhattacharya J, et al.: Heterogeneity in healthy aging. J Gerontol A Biol Sci Med Sci. 2014; 69(6): 640–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUnderwood E: The final countdown. Science. 2015; 350(6265): 1188–90. PubMed Abstract | Publisher Full Text\n\nRiley JC: Rising Life Expectancy. A Global History. Cambridge Univ. Press; 2001. Reference Source\n\nSchofieled R, Reher D, Bidau A: The decline of mortality in Europe. Oxford: Oxford, Univ. Press; 1991. Reference Source\n\nVaupel JW: Biodemography of human ageing. Nature. 2010; 464(7288): 536–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaeberlein M, Rabinovitch PS, Martin GM: Healthy aging: The ultimate preventative medicine. Science. 2015; 350(6265): 1191–3. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "45728",
"date": "17 Apr 2019",
"name": "Frank P. Mockenhaupt",
"expertise": [
"Reviewer Expertise Tropical Medicine",
"(molecular) 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\nThis is a thoughtful piece of opinion highlighting the need for additional efforts of Public Health systems in LMICs to achieve improved prevention and management of NCDs. The authors call for a better integration of various disciplines and consideration of epigentics and \"healthy ageing\".\nBeyond some editing, this paper can be indexed as it stands.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "48826",
"date": "06 Aug 2019",
"name": "Tanida Phatisena",
"expertise": [
"Reviewer Expertise Health Promotion",
"Health Behaviour",
"and Public Health."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article shows the opinions and the problems of NCD which is a rising trend. The authors analyse about the determinant of chronic disease thoroughly. This article is interesting because the authors show the main point of the factor of genetics which causes NCD. Therefore, health authorities should aware of developments in molecular biology and epigenetics for healthy aging.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-286
|
https://f1000research.com/articles/8-284/v1
|
13 Mar 19
|
{
"type": "Data Note",
"title": "A curated transcriptome dataset collection to investigate the blood transcriptional response to viral respiratory tract infection and vaccination.",
"authors": [
"Salim Bougarn",
"Sabri Boughorbel",
"Damien Chaussabel",
"Nico Marr",
"Sabri Boughorbel",
"Damien Chaussabel",
"Nico Marr"
],
"abstract": "The human immune defense mechanisms and factors associated with good versus poor health outcomes following viral respiratory tract infections (VRTI), as well as correlates of protection following vaccination against respiratory viruses, remain incompletely understood. To shed further light into these mechanisms, a number of systems-scale studies have been conducted to measure transcriptional changes in blood leukocytes of either naturally or experimentally infected individuals, or in individual’s post-vaccination. Here we are making available a public repository, for research investigators for interpretation, a collection of transcriptome datasets obtained from human whole blood and peripheral blood mononuclear cells (PBMC) to investigate the transcriptional responses following viral respiratory tract infection or vaccination against respiratory viruses. In total, Thirty one31 datasets, associated to viral respiratory tract infections and their related vaccination studies, were identified and retrieved from the NCBI Gene Expression Omnibus (GEO) and loaded in a custom web application designed for interactive query and visualization of integrated large-scale data. Quality control checks, using relevant biological markers, were performed. Multiple sample groupings and rank lists were created to facilitate dataset query and interpretation. Via this interface, users can generate web links to customized graphical views, which may be subsequently inserted into manuscripts to report novel findings. The GXB tool enables browsing of a single gene across projects, providing new perspectives on the role of a given molecule across biological systems in the diagnostic and prognostic following VRTI but also in identifying new correlates of protection. This dataset collection is available at: http://vri1.gxbsidra.org/dm3/geneBrowser/list.",
"keywords": [
"Transcriptomics",
"Bioinformatics",
"Software",
"Viral respiratory infection",
"Influenza viruses",
"Respiratory syncytial viruses (RSV)",
"Rhinoviruses",
"Whole Blood",
"PBMC."
],
"content": "Introduction\n\nViral respiratory tract infections (VRTI) are responsible for the majority hospitalizations among infants and the elderly. They are caused mainly by a heterogeneous group of viruses, including rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus (RSV), enteroviruses, coronaviruses, and certain strains of adenovirus1,2. Few antiviral therapies are currently approved and routinely used for VRTI. Most of these are specific inhibitors of influenza viruses3. Moreover, for most respiratory viruses, there is no licensed vaccine available4,5, with the exception of flu vaccines for which protection generally lasts only one flu season. Consequently, clinical management of individuals with VRTI is mostly restricted to supportive care5.\n\nAs clinical symptoms are often overlapping and are not specific for any of the viral species, it is difficult to establish a clinical diagnosis without laboratory testing1. Furthermore, clinical manifestations of VRTI are highly variable, ranging from asymptomatic infections or illness with mild symptoms (a common cold) to clinically severe disease with life-threatening complications, such as respiratory failure and in some cases may have a fatal outcome6. Infants, the elderly and patients with chronic lung or heart diseases in particular are at high risk7.\n\nThus, there is an evident need to better understand the molecular mechanisms underlying the disease pathogenesis, progression as well as severity of, and immunity against, VRTI among humans8. In this context, different large scale gene expression studies have been conducted using whole blood or peripheral blood mononuclear cells (PBMCs), to assess the human immune response to natural9–11 and experimental viral respiratory infections12,13; in particular, to influenza and RSV infections, and also to vaccination14–17.\n\nHere, we make available, through an interactive web application, a curated collection of datasets that were obtained from pediatric and adult patients with natural VRTI, volunteers with experimental exposition to respiratory viruses and also vaccinated volunteers. Transcriptomics datasets were obtained from whole blood and PBMCs.\n\nA total of 31 datasets were retrieved and selected from the NCBI Gene Expression Omnibus (GEO), a public repository of transcriptome profiles. The identified datasets are particularly relevant to our interest in understanding the pathobiology of VRTI and vaccination. As described in recent publications18,19, these datasets were loaded into a custom interactive web application, the Gene Expression Browser (GXB), which enables easy access to large datasets and interactive visualization of our dataset collection related to VRTI and vaccination against respiratory viruses. It also provides access to demographic and clinical information. Importantly, the user can customize data plots by adding multiple layers of parameters (e.g. age, gender, sample type, type of infection, type of vaccine, sample collection time), modify the sample ordering and genes, and generate links (mini URL) that can be shared via e-mail or used in publications. Therefore, we are providing here a resource enabling browsing of datasets relevant to blood transcriptional responses to VRTI and vaccination that offers a unique opportunity to identify host genes and their regulation that may be of diagnostic and/or prognostic value, or that may be tested as novel correlates of protection in subsequent studies. For example, a comparative approach of the transcriptional response signatures between experimentally infected and vaccinated individuals could be used to identify common mechanisms that define the poor health outcomes versus strong protection. The ability to pool, compare and analyze the immune responses to different infections and vaccines, in different individuals and at various age, offers a unique opportunity for a better understanding of the pathophysiology of VRTI.\n\n\nMethods\n\nA total of 120 datasets, potentially relevant to human immune responses to VRTI and vaccination, were identified in GEO using the following search query:\n\nHomo sapiens[Organism] AND ((“respiratory syncytial virus”[DESC] OR RSV[DESC] OR metapneumovirus[DESC] OR hMPV[DESC] OR influenza[DESC] OR parainfluenza[DESC] OR rhinovirus[DESC] OR rhinoviruses[DESC] OR adenovirus[DESC] OR adenoviruses[DESC] OR HAdV[DESC] OR coronaviruses[DESC] OR HCoV[DESC]) OR (vaccine OR vaccines OR vaccination) AND (blood[DESC] OR PBMC[DESC] OR PBMCs[DESC] OR lymphocyte[DESC] OR lymphocytes[DESC] OR “B cell”[DESC] OR “B cells”[DESC] OR “plasma cells”[DESC] OR “T cell”[DESC] OR “T cells”[DESC] OR Treg[DESC] OR Tregs[DESC] OR monocyte[DESC] OR monocytes[DESC] OR dendritic[DESC] OR DC[DESC] OR DCs[DESC] OR \"natural killer\"[DESC] OR NK[DESC] OR NKT[DESC] OR neutrophil[DESC] OR neutrophils[DESC]) AND (“Expression profiling by array”[gdsType] OR “Expression profiling by high throughput sequencing”[gdsType]).\n\nMost of retrieved datasets were generated from human blood and human PBMC, using Illumina or Affymetrix commercial platforms or RNA-sequencing. All the entries that were returned with this query were manually curated. The process involved reading all the descriptions available of the datasets, the study design and the GEO-linked article in pubmed. Finally, only studies using human whole blood and human PBMCs, associated with natural or experimental VRTI, or vaccination against VRTI, were retained for our dataset collection. For the retained datasets, if the platform used to generate the transcriptome profiles was not supported by GXB or if from an in vitro study, they were exlcluded from our dataset collection. Based on these criteria, 31 datasets were retained. These include datasets that were generated from whole blood or PBMCs of individuals who were either naturally (12) or experimentally infected (3) (with influenza viruses, RSV, Rhinovirus, Rotavirus) as well as from healthy, uninfected (age-matched) volunteers. The remaining 16 datasets were generated from whole blood or PBMCs of individuals who had received flu vaccines (Figure 1). The datasets that comprise our collection are listed in Table 1.\n\nThe pie chart indicates the type of studies carried out for the 31 datasets.\n\n*Gender information and/or Xist probe was not available. QC – quality control, ND – no data\n\nOnce the final selection had been made, each dataset was downloaded from GEO by using the SOFT file format. Then, the datasets were uploaded on the Gene Expression Browser (GXB), an interactive web application hosted on the Amazon Web Services cloud20. Information about samples and study design were also uploaded. The available samples were put into groups based on relevant study variables and genes were ranked according to the different groups comparisons. A detailed description of the GXB software tool is available from recent publications19–21. This software interface allows user to easily navigate and filter the dataset collection. A web tutorial can be easily accessed online. Annotation and functionality of the web software interface were described previously by our group18,19,21, and is reproduced here so that readers can use this article as a standalone resource. Briefly, datasets of interest can be quickly identified either by filtering on criteria from pre-defined sections on the left or by entering a query term in the search box at the top of the dataset navigation page. Clicking on one of the studies listed in the dataset navigation page opens a viewer designed to provide interactive browsing and graphic representations of large-scale data in an interpretable format. This interface is designed to present ranked gene lists and display expression results graphically in a context-rich environment. Selecting a gene from the rank ordered list on the left of the data-viewing interface will display its expression values graphically in the screen’s central panel. Directly above the graphical display drop down menus give users the ability: a) To change how the gene list is ranked - this allows the user to change the method used to rank the genes, or to only include genes that are selected for specific biological interest; b) To change sample grouping (Group Set button) - in some datasets, a user can switch between groups based on cell type to groups based on disease type, for example; c) To sort individual samples within a group based on associated categorical or continuous variables (e.g. gender or age); d) To toggle between the bar chart view and a box plot view, with expression values represented as a single point for each sample. Samples are split into the same groups whether displayed as a bar chart or box plot; e) To provide a color legend for the sample groups; f) To select categorical information that is to be overlaid at the bottom of the graph - for example, the user can display gender or smoking status in this manner; g) To provide a color legend for the categorical information overlaid at the bottom of the graph; h) To download the graph as a portable network graphics (png) image. Measurements have no intrinsic utility in absence of contextual information. It is this contextual information that makes the results of a study or experiment interpretable. It is therefore important to capture, integrate and display information that will give users the ability to interpret data and gain new insights from it. We have organized this information under different tabs directly above the graphical display. The tabs can be hidden to make more room for displaying the data plots, or revealed by clicking on the blue “show info panel” button on the top right corner of the display. Information about the gene selected from the list on the left side of the display is available under the “Gene” tab. Information about the study is available under the “Study” tab. Rolling the mouse cursor over a bar chart feature while displaying the “Sample” tab lists any clinical, demographic, or laboratory information available for the selected sample. Finally, the “Downloads” tab allows advanced users to retrieve the original dataset for analysis outside this tool. It also provides all available sample annotation data for use alongside the expression data in third party analysis software. Other functionalities are provided under the “Tools” drop-down menu located in the top right corner of the user interface. Some of the notable functionalities available through this menu include: a) Annotations, which provides access to all the ancillary information about the study, samples and dataset organized across different tabs; b) Cross-project view; which provides the ability for a given gene to browse through all available studies; c) Copy link, which generates a mini-URL encapsulating information about the display settings in use and that can be saved and shared with others (clicking on the envelope icon on the toolbar inserts the URL in an email message via the local email client); d) Chart options; which gives user the option to customize chart labels.\n\n\nQuality Control\n\nQuality control checks can be performed on the datasets loaded on GXB, for example by examining concordance of the gender-specific expression of the XIST gene in those datasets for which gender information was available as metadata. The XIST gene is essential for imprinted and random X-chromosome inactivation22 and therefore, expression is expected to be high in female and low in male samples. Respective hyperlinks are found in Table 1 allow you to visualize the XIST experession based on the gender information provided with the GEO submission. Figure 2 shows XIST gene expression in a representative dataset, along with gender information available that was recorded and made available in GEO.\n\nGene expression data were from whole blood of healthy adult volunteers before and after receiving either placebo (saline) injections, seasonal influenza (Fluzone) or pneumococcal (Pneumovax) vaccination.\n\n\nData availability\n\nAll datasets included in our curated collection are also available publically via the NCBI GEO website : https://www.ncbi.nlm.nih.gov/gds/; and are referenced throughout the manuscript by their GEO accession numbers (e.g. GSE17763). Signal files and sample description files can also be downloaded from the GXB tool under the “downloads” tab.",
"appendix": "Grant information\n\nAll the authors listed on this publication received support from the Qatar Foundation. Support for this project was provided by the Qatar National Research Fund [NPRP10-0205-170348].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank all the investigators who decided to make their datasets publically available by depositing them in GEO.\n\n\nReferences\n\nHodinka RL: Respiratory RNA Viruses. Microbiol Spectr. 2016; 4(4). PubMed Abstract | Publisher Full Text\n\nTan WC: Viruses in asthma exacerbations. Curr Opin Pulm Med. 2005; 11(1): 21–6. PubMed Abstract | Publisher Full Text\n\nIson MG: Clinical use of approved influenza antivirals: therapy and prophylaxis. Influenza Other Respir Viruses. 2013; 7Suppl 1: 7–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu T, Zhang C, Yu L, et al.: The preventive effect of vaccine prophylaxis on severe respiratory syncytial virus infection: A meta-analysis. Virol Sin. 2015; 30(5): 371–8. PubMed Abstract | Publisher Full Text\n\nCaballero MT, Polack FP, Stein RT: Viral bronchiolitis in young infants: new perspectives for management and treatment. J Pediatr (Rio J). 2017; 93Suppl 1: 75–83. PubMed Abstract | Publisher Full Text\n\nJong VL, Ahout IM, van den Ham HJ, et al.: Transcriptome assists prognosis of disease severity in respiratory syncytial virus infected infants. Sci Rep. 2016; 6: 36603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAckerson B, Tseng HF, Sy LS, et al.: Severe Morbidity and Mortality Associated With Respiratory Syncytial Virus Versus Influenza Infection in Hospitalized Older Adults. Clin Infect Dis. 2018. PubMed Abstract | Publisher Full Text\n\nMejias A, Dimo B, Suarez NM, et al.: Whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection. PLoS Med. 2013; 10(11): e1001549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParnell GP, McLean AS, Booth DR, et al.: A distinct influenza infection signature in the blood transcriptome of patients with severe community-acquired pneumonia. Crit Care. 2012; 16(4): R157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsuge M, Oka T, Yamashita N, et al.: Gene expression analysis in children with complex seizures due to influenza A(H1N1)pdm09 or rotavirus gastroenteritis. J Neurovirol. 2014; 20(1): 73–84. PubMed Abstract | Publisher Full Text\n\nZhai Y, Franco LM, Atmar RL, et al.: Host Transcriptional Response to Influenza and Other Acute Respiratory Viral Infections--A Prospective Cohort Study. PLoS Pathog. 2015; 11(6): e1004869. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWoods CW, McClain MT, Chen M, et al.: A host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza H1N1 or H3N2. PLoS One. 2013; 8(1): e52198. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaas AK, Chen M, Varkey J, et al.: Gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans. Cell Host Microbe. 2009; 6(3): 207–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParnell G, McLean A, Booth D, et al.: Aberrant cell cycle and apoptotic changes characterise severe influenza A infection--a meta-analysis of genomic signatures in circulating leukocytes. PLoS One. 2011; 6(3): e17186. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakaya HI, Hagan T, Duraisingham SS, et al.: Systems Analysis of Immunity to Influenza Vaccination across Multiple Years and in Diverse Populations Reveals Shared Molecular Signatures. Immunity. 2015; 43(6): 1186–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakaya HI, Wrammert J, Lee EK, et al.: Systems biology of vaccination for seasonal influenza in humans. Nat Immunol. 2011; 12(8): 786–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsang JS, Schwartzberg PL, Kotliarov Y, et al.: Global analyses of human immune variation reveal baseline predictors of postvaccination responses. Cell. 2014; 157(2): 499–513. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRinchai D, Boughorbel S, Presnell S, et al.: A curated compendium of monocyte transcriptome datasets of relevance to human monocyte immunobiology research [version 2; referees: 2 approved]. F1000Res. 2016; 5: 291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRahman M, Boughorbel S, Presnell S, et al.: A curated transcriptome dataset collection to investigate the functional programming of human hematopoietic cells in early life [version 1; referees: 2 approved]. F1000Res. 2016; 5: 414. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpeake C, Presnell S, Domico K, et al.: An interactive web application for the dissemination of human systems immunology data. J Transl Med. 2015; 13: 196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarr AK, Boughorbel S, Presnell S, et al.: A curated transcriptome dataset collection to investigate the development and differentiation of the human placenta and its associated pathologies [version 2; referees: 2 approved]. F1000Res. 2016; 5: 305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCerase A, Pintacuda G, Tattermusch A, et al.: Xist localization and function: new insights from multiple levels. Genome Biol. 2015; 16: 166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThakar J, Mohanty S, West AP, et al.: Aging-dependent alterations in gene expression and a mitochondrial signature of responsiveness to human influenza vaccination. Aging (Albany NY). 2015; 7(1): 38–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nObermoser G, Presnell S, Domico K, et al.: Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines. Immunity. 2013; 38(4): 831–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBermejo-Martin JF, Martin-Loeches I, Rello J, et al.: Host adaptive immunity deficiency in severe pandemic influenza. Crit Care. 2010; 14(5): R167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrand HK, Ahout IM, Ridder D, et al.: Olfactomedin 4 Serves as a Marker for Disease Severity in Pediatric Respiratory Syncytial Virus (RSV) Infection. PLoS One. 2015; 10(7): e0131927. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoang LT, Tolfvenstam T, Ooi EE, et al.: Patient-based transcriptome-wide analysis identify interferon and ubiquination pathways as potential predictors of influenza A disease severity. PLoS One. 2014; 9(11): e111640. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerdal JE, Mollnes TE, Wæhre T, et al.: Excessive innate immune response and mutant D222G/N in severe A (H1N1) pandemic influenza. J Infect. 2011; 63(4): 308–16. PubMed Abstract | Publisher Full Text\n\nFranco LM, Bucasas KL, Wells JM, et al.: Integrative genomic analysis of the human immune response to influenza vaccination. eLife. 2013; 2: e00299. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCao RG, Suarez NM, Obermoser G, et al.: Differences in antibody responses between trivalent inactivated influenza vaccine and live attenuated influenza vaccine correlate with the kinetics and magnitude of interferon signaling in children. J Infect Dis. 2014; 210(2): 224–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavenport EE, Antrobus RD, Lillie PJ, et al.: Transcriptomic profiling facilitates classification of response to influenza challenge. J Mol Med (Berl). 2015; 93(1): 105–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHerberg JA, Kaforou M, Gormley S, et al.: Transcriptomic profiling in childhood H1N1/09 influenza reveals reduced expression of protein synthesis genes. J Infect Dis. 2013; 208(10): 1664–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "45689",
"date": "26 Mar 2019",
"name": "Carlos Alberto Moreira-Filho",
"expertise": [
"Reviewer Expertise genomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Bougarn et al. describes the use of an interactive web application, GXB (Speake et al.(2015)1), for investigating the blood transcriptional response to viral respiratory tract infection and vaccination. A curated dataset collection was established for this purpose encompassing 31 datasets retrieved and selected from the NCBI Gene Expression Omnibus (GEO) public repository.\n\nThe rationale for creating the dataset collection and the methodology used in the paper are clearly described. The functionalities of the GXB tool are well explained to the reader, as well as the quality control procedures adopted by the authors. However, the GEO datasets do not always provide detailed information on virus genotypes. Distinct RSV genotypes induce very different PBMC transcriptional responses (see, for instance, Rodriguez-Fernandez et al. 20172; and Vieira et al. 20193). The authors could insert a comment on this issue in their paper.\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": "48849",
"date": "13 Jun 2019",
"name": "Benjamin M Tang",
"expertise": [
"Reviewer Expertise Genomics",
"immunology",
"viral respiratory infection"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper represents an extension of the authors' extensive work in this area - which aims to provide an accessible web-based platform for researchers to perform multi-cohort analysis of pooled transcriptomics data.\nThe Introduction outlines the scientific rationale to support the development of such a platform, which I mostly agree. There are a number of reasons why such a platform can assist researchers, including (1) help researchers to leverage on publicly available dataset to address each researcher's own research question, (2) obtaining a large sample size for analysis, which is otherwise not feasible by a single research group, (3) provide access to a diverse range of patient population and varying degree of disease severity, thereby generating more biological insights, which are not achievable by a single-centre study. For these reasons, the platform provided by these author represents an important contribution to the research community.\nTo further place this into perspective, one can estimate the likely cost (in time and money) to obtain the same analysis if such a platform is not available. To collect 31 transcriptomics datasets of viral respiratory infection (as outlined by this paper), a researcher need to perform an extensive literature review and database search. After all the datasets are collected, the researcher then need to bring in a bioinformatic team to perform a pooled analysis, which required advanced expertise due to the huge variability in technical platform, assay methods and normalization approach among different datasets. This bioinofrmatic work is extensive - if such work is outsourced to an external bioinformatician/company, the cost is in excessive of US$15,000 (our group has commissioned similar work in the past - hence our knowledge into the estimated cost). Here, the authors provides access to the analysis platform to the wider research community for free...an important step in democratising scientific data for the benefit of all researchers.\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/8-284
|
https://f1000research.com/articles/8-281/v1
|
12 Mar 19
|
{
"type": "Case Report",
"title": "Case Report: a novel chromosomal insertion, 46, XY, inv ins(18;2)(q11.2;q13q22), in a patient with infertility and mild intellectual disability",
"authors": [
"Murat Kaya",
"İlknur Suer",
"Şükrü Öztürk",
"Kıvanç ÇEFLE",
"Birsen Karaman",
"Şükrü Palanduz",
"İlknur Suer",
"Şükrü Öztürk",
"Kıvanç ÇEFLE",
"Birsen Karaman",
"Şükrü Palanduz"
],
"abstract": "Infertility is an important health problem affecting 15% of couples worldwide. Intellectual disability (ID) is characterized with significant impairment of intellectual function, adaptive daily life skills and social skills. Insertion is a rare chromosomal rearrangement causing infertility and ID. Here, we report a 39-year-old man presenting with primary infertility and mild ID. The patient’s spermiogram was consistent with azoospermia. Conventional cytogenetic analysis showed a novel inversion/insertion type of chromosomal aberration involving chromosomes 18 and 2: 46, XY, inv ins(18;2)(q11.2;q13q22). We carried out the array comparative genomic hybridization analysis to confirm the cytogenetic findings. Y micro-deletion analysis demonstrated that the AZF region as intact. We suggest that the novel insertion found in this case [46, XY, inv ins(18;2)(q11.2;q13q22)] may have caused infertility and mild ID in our patient. To the best of our knowledge, this chromosomal insertion has not previously been reported.",
"keywords": [
"Infertility",
"mild intellectual disability",
"insertion"
],
"content": "Introduction\n\nSeveral factors have been implicated in the pathogenesis of infertility, which are associated with both men and women. Infertility may also occur due to a combined etiology where both male and female factors are combined. In approximately 40% of the cases, the etiology is unclear1.\n\nBalanced chromosomal rearrangements (BCRs), such as autosomal reciprocal translocations, can be related to infertility. The frequency of BCRs in azoospermic men and oligozoospermic men is 0.6% and 1.7%, respectively2.\n\nIntellectual disability (ID) is characterized by significant impairment of intellectual function, adaptive daily life skills and social skills. ID is separated into five groups: mild, moderate, severe, profound and unable to classify3.\n\nID occurs most likely due to a genetic etiology including BCRs. ID is thought to affect about 1% of the population, of which 85% have mild ID. People with mild ID are slower nearly in all areas of intellectual development, social and daily life skills. These individuals can take care of themselves, learn basic skills associated to safety and health and they may acquire practical life skills4.\n\nIn this study, we define an azoospermic male with mild ID who was found to have a novel insertional chromosomal abnormality on conventional cytogenetic and molecular cytogenetic techniques.\n\n\nCase Report\n\nA 39-year-old man was referred to our clinic due to infertility. His height and weight were 175 cm and 82 kg, respectively. The patient left school when he was in the third grade of primary school because of learning issues. He was unable to read and write properly, and had deficits in intellectual ability like reasoning or problem solving. Currently, the patient was working as a cleaner in a factory. He was noted to have mild ID.\n\nOn physical examination, the patient had no dysmorphic features. He was married for 8 years; he and his wife were not consanguineous. His parents had two children and the family history of the patient was remarkable for a deceased brother at the age of 15 years, who had also ID (Figure 1). Since there was no history of spontaneous pregnancy during his marriage, the patient was considered to represent a case of primary infertility who had also mild ID. Sperm analysis showed complete azoospermia. In vitro fertilization was performed four times by testicular sperm extraction without success. Luteinizing hormone, follicular stimulating hormone and testosterone levels were compatible with hypergonadotropic hypogonadism (Table 1). Y micro-deletion analysis demonstrated that AZFa, AZFb and AZFc regions on the Y chromosome were intact. After conventional cytogenetic analysis, we performed array conventional cytogenetic technique (aCGH). Karyotype analysis could not be performed for the parents or the patient’s brother, since they were not alive.\n\nConventional cytogenetic technique: Peripheral blood lymphocytes were used for a 72-hour culture. Chromosome analysis was performed on phyto-haemagglutinin-induced peripheral blood lymphocytes. Metaphase plaques were analyzed using the GTG banding method at almost 500–550 band resolution.\n\naCGH: An Agilent SurePrint G3 CGH+SNP Microarray Kit (4x180K) was used for genetic analysis of the patient. Microarray data were analyzed using Feature Extraction and Agilent Cytogenomics v4.0.3.12 software. Log ratios between -0.5 – 0.5 and variations with less than 5 consecutive probes were excluded. Genomic positions were based on GRCh37/hg19 Homo sapiens assembly.\n\nConventional cytogenetic analysis revealed that the patient had an insertional translocation: 46, XY, inv ins(18;2)(q11.2;q13q22) (Figure 2). Array CGH did not show any deletion or duplication (Figure 3).\n\na) Patient’s GTG banded karyotype, b) Schematic of normal and derivative chromosomes 2 and 18 and breakpoints regions of chromosomes.\n\n\nDiscussion\n\nInsertions occur after at least three breaks on chromosomes5. Incidence of insertional chromosome translocation is nearly 1:80.000 live births6. Molecular mechanisms of chromosomal insertions are not well understood5. An individual with an inter-chromosomal insertion can transmit unblanced chromosomes to his/her child with a theoretical risk of 50% in each pregnancy6.\n\nInsertional translocations affecting gene function may cause abnormal phenotype in different ways. According to one hypothesis, the derivative chromosomes formed after insertional translocation can contain gene or genes with increased expression. A second hypothesis states that structure of a gene located at the breakpoint may be disturbed and as a result either loss or gain of function can occur. Lastly, genes located at the breaking segment can be deleted or duplicated which can give rise to gene expression abnormalities owing to position effects6. In addition to these genetic mechanisms, insertional translocations can also affect the genome by epigenetic means. For instance, some microRNAs may be located in the break regions. It is thought that nearly 60% of all human genes are regulated by microRNAs. Furthermore, many microRNAs are located in fragile regions of the genome and considered to have an important role in the pathogenesis of many diseases7.\n\nClassical chromosome analysis revealed that our patient carried an insertional translocation, 46, XY, inv ins(18;2)(q11.2;q13q22). To the best of our knowledge, this chromosomal insertion has not previously been reported. aCGH is used to detect genomic micro-deletions/duplications (copy number changes). However, aCGH failed to detect any micro-deletion or micro-duplication at the breakpoint regions of this insertional translocation [arr(1-22)×2,(XY)×1]. Although aCGH is a useful and a modern technique, it has some limitations. Mosaicism cannot be detected and BCR may be missed by this method. Furthermore, small deletions or duplications which are less than 10 kb can be difficult to detect with aCGH. Although conventional cytogenetic analysis is an old technique, it is still the cheapest and most useful method to obtain a general information about whole chromosomes rapidly8.\n\nLi and colleagues elucidated the insertional translocation 46,XY inv ins(18,7) (q22.1; q36.2q21.11) found in an azoospermic man with next generation sequencing (NGS). It was demonstrated that two disrupted genes, DPP6 and CACNA2D1, were at breakpoint sites of the chromosomes, suggesting they may be associated with azoospermia. Moreover, neither micro-deletions nor duplications were detected at these breakpoint regions9.\n\nBCRs detected by conventional karyotype analysis can be found to be unbalanced at the molecular level. We suggest that these chromosomal regions affected in our patient can be evaluated with advanced molecular techniques like NGS. Such an approach could unveil sequence abnormalities, which would potentially shed light on the molecular pathogenesis of azoospermia and/or ID.\n\n\nConsent\n\nInformed written consent for the publication of clinical details and images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPoongothai J, Gopenath TS, Manonayaki S: Genetics of human male infertility. Singapore Med J. 2009; 50(4): 336–47. PubMed Abstract\n\nCeylan GG, Ceylan C, Elyas H: Genetic anomalies in patients with severe oligozoospermia and azoospermia in eastern Turkey: a prospective study. Genet Mol Res. 2009; 8(3): 915–22. PubMed Abstract | Publisher Full Text\n\nKaufman L, Ayub M, Vincent JB: The genetic basis of non-syndromic intellectual disability: a review. J Neurodev Disord. 2010; 2(4): 182–209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTabet AC, Verloes A, Pilorge M, et al.: Complex nature of apparently balanced chromosomal rearrangements in patients with autism spectrum disorder. Mol Autism. 2015; 6: 19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGu S, Szafranski P, Akdemir ZC, et al.: Mechanisms for Complex Chromosomal Insertions. PLoS Genet. 2016; 12(11): e1006446. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang SH, Shaw C, Ou Z, et al.: Insertional translocation detected using FISH confirmation of array-comparative genomic hybridization (aCGH) results. Am J Med Genet A. 2010; 152A(5): 1111–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaratas OF, Guzel E, Suer I, et al.: miR-1 and miR-133b are differentially expressed in patients with recurrent prostate cancer. PLoS One. 2014; 9(6): e98675. PubMed Abstract | Publisher Full Text | Free Full Text\n\nToujani S, Dessen P, Ithzar N, et al.: High resolution genome-wide analysis of chromosomal alterations in Burkitt's lymphoma. PLoS One. 2009; 4(9): e7089. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi L, Chen H, Yin C, et al.: Mapping breakpoints of a familial chromosome insertion (18,7) (q22.1; q36.2q21.11) to DPP6 and CACNA2D1 genes in an azoospermic male. Gene. 2014; 547(1): 43–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "46870",
"date": "17 Apr 2019",
"name": "Murat Erdogan",
"expertise": [
"Reviewer Expertise medical genetics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInfertility and ID are important health problems. There are several reasons in the etiology of each of the two diseases, but genetic causes are more important. Among the genetic reasons, chromosomal rearrangements are remarkable. In this case, both infertility and mild-ID are seen. Genetic studies in this case indicate a chromosomal rearrangement that has not previously been in the literature. In the diagnostic pathway, physical examination and family history of the patient were first investigated. No dysmorphic features were observed in the patient. Then, routine laboratory tests were performed, and azoospermia was detected in the patient. In addition, according to the information obtained from patient, in vitro fertilization process failure was noted. Hypergonadotropic hypogonadism was also observed in the patient. According to the algorithm, genetic tests to be performed for the diagnosis of both infertility and ID were started with conventional cytogenetics and continued with aCGH. As a result, a balanced chromosomal translocation was detected in the patient. Insertional translocations affect various genetic mechanisms and disrupt gene functions. This type of translocation may lead to increased or decreased gene functions. These chromosomal translocations may also affect epigenetic mechanisms. If the affected genes in chromosomal fracture regions can be determined with more advanced methods, they will contribute to the etiology of infertility and ID.\nSome minor corrections may be required in this case. It is necessary to determine the number of areas studied during cytogenetic analysis, to indicate whether the change is in all areas, and the pedigree drawing shown in Figure 1 should be redrawn and numbered according to the basis principles. In order to clearly understand the fracture regions shown in Figure 2, it is useful to show to centromere subregions in the ideogram.\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": "48939",
"date": "13 Jun 2019",
"name": "Ender Karaca",
"expertise": [
"Reviewer Expertise Human genetics",
"cytogenetics",
"next generation sequencing",
"diagnostic genetics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMurat Kaya et al. report a male patient with mild intellectual disability and infertility. The authors used conventional G-banding, FISH and aCGH methods in order to investigate genetic etiology in the patient. They also provide the levels of luteinizing hormone, follicular stimulating hormone and testosterone, which were compatible with hypergonadotropic hypogonadism. Through their investigation they were able to detect an apparently balanced inverted inversion, 46,XY,ins(18;2)(q11.2;q22q13). A follow up aCGH analysis (using the platform Agilent aCGH+SNP Microarray, 4x180K) could not detect any unbalances at the break points involved in the insertion event. They also excluded Y micro-deletion via analyzing AZFa, AZFb and AZFc regions on the Y chromosome.\nOverall the case report is worth being indexed and clinicians and researchers who work on infertility and ID will benefit from it. However, it needs to be revised in order to correct some points and to improve the value of the presented clinical and genomic data. I explained my specific concerns below.\nTitle:\nPlease revise the cytogenetic nomenclature based on ISCN 2016. There should be no space after commas and you don’t need to use “inv”, you actually just have to change the order of the break points in chromosome 2 in the nomenclature according to the orientation of the inserted fragment. So the nomenclature will be: “46,XY,ins(18;2)(q11.2;q22q13)”.\n\nAbstract:\nThe abstract is well written, however the first 3 sentences could benefit from revising which would provide a better fluency. This revision could include the removal of the definition of ID, the second sentence of the abstract, as this definition is repeated a couple of times in the introduction. I’d sincerely recommend an entrance to the abstract: “Infertility and intellectual disability (ID) are important health problems affecting 15% of couples worldwide and 1-2% of the general population, respectively. Chromosomal aberrations including insertions are responsible for both infertility and ID in many cases.\" Of course, this is just a minor issue and the authors are free to consider or not.\n\nThe authors say in the abstract “We carried out the array comparative genomic hybridization analysis to confirm the cytogenetic findings”. How do they expect to confirm a balanced rearrangement with aCGH? Actually, in the discussion they say “aCGH is used to detect genomic micro-deletions/duplications (copy number changes). However, aCGH failed to detect any micro-deletion or micro-duplication at the breakpoint regions of this insertional translocation [arr(1-22)×2,(XY)×1].”, and this sounds like the aCGH method has been used to detect any potential copy number change at the break points, which is indeed a more appropriate reason to use aCGH method in this case. So, please revise the statement in the abstract according to your statement in the discussion.\n\nIntroduction:\nThe introduction would benefit from giving a little more detail from the background of both intellectual disability and infertility. I would rather revise the introduction in a way which will address the following questions:\n(Paragraph for infertility)\nWhat is the definition of infertility?\n\nWhat is the frequency of infertility in the general population (preferably providing male and female infertility ratios)?\n\nWhat type of genetic causes can lead to infertility (i.e. molecular, cytogenetic)?\n\nWhat is the frequency of BCRs in the etiology of infertility?\n\n(Paragraph for ID)\nWhat is the definition of ID?\n\nWhat is the frequency of ID in the general population?\n\nDo all IDs result from genetic causes?\n\nWhat type of genetic causes can lead to IDs (i.e. molecular, cytogenetic)?\n\nWhat is the frequency of BCRs in the etiology of ID?\n\n(Paragraph that provide literature summary for cases with both infertility and ID)\nAre there any cases in the literature representing with both infertility and ID? If so, what type of genetic cause(s) are reported in these patients? Is there any example for such a case (with both ID and infertility) who has BCRs?\n\nThe last paragraph will summarize your findings as in the current version. However, the authors need to correct the statement: \"In this study, we define an azoospermic male with mild ID who was found to have a novel insertional chromosomal abnormality on conventional cytogenetic and molecular cytogenetic techniques.\" as it means that they could detect the chromosomal rearrangement on aCGH too, which is not true.\n\nCase Report:\nID in the patient was classified as mild. Is there an IQ test score?\n\nIs there any medical record of an IQ test for the affected brother? Did he have mild ID too?\n\nDid his wife undergo any investigation for infertility? Please provide the clinical data - if available - whether the female infertility could be excluded or not.\n\nIt would be more informative for readers (especially for clinical genetics trainees) to provide images of the Y-microdeletion analysis, even in the extended data section.\n\nPlease correct \"array conventional cytogenetic technique\" to \"array comparative genomic hybridization\", which correctly corresponds to abbreviation \"aCGH\".\n\nPlease change \"phyto-haemagglutinin-induced\" to \"phytohaemagglutinin-induced\". You do not need to use dash within the word \"phytohaemagglutinin\".\n\nDiscussion:\nIn the second paragraph of the discussion, the authors mention about two hypotheses regarding the potential mechanisms by which insertions cause abnormality. Can they provide reference(s) for these two hypotheses?\n\nThe statement “For instance, some micro-RNAs may be located in the break regions” may cause confusion especially for clinicians who do not know the details of microRNA functions and the mechanisms that they use in the regulation of gene expression. The current sentence could mean either “micro-RNA coding genes” or “the genomic region that is a target for micro-RNA”, please clarify.\n\nCould the authors provide reference for the statement “It is thought that nearly 60% of all human genes are regulated by microRNAs”?\n\nIn the 3rd paragraph of the discussion, the authors claim that mosaicism cannot be detected by aCGH. Although aCGH has significant disadvantages with detecting low-level mosaicism, mosaicisms with the percentage over 20-25 (>20-25%) can be detected by most of the recent aCGH platforms. Thus, the statement \"mosaicism cannot be detected\" is misleading; please correct it.\n\nThe authors cite a report in which an inverted insertion “46,XY inv ins(18,7) (q22.1; q36.2q21.11)”. This nomenclature is wrong as well. Please consider ISCN 2016 as a reference for cytogenetic nomenclature. In this cited report, both DPP6 and CACNA2D1 are located on chromosome 7, while the overlapping chromosome that is involved in the rearrangement in both reports is chromosome 18. It would be interesting to know if there is any known or candidate gene for infertility and/or intellectual disability located in any of the 3 break points involved; 18q11.2, 2q13, or 2q22.\n\nIt seems that the most likely reason for infertility and ID in the reported male is the chromosomal rearrangement. However, it could be also possible that the given insertional translocation does not cause any of the ID or infertility in this patient. There may be a different mutation in a known gene which could be detected by sequencing methods. Even more, this insertion could be associated with just one of the phenotypes; infertility or ID; while a second mutation in another locus causes the other phenotype. So, NGS (WES or WGS) analysis could be helpful to further evaluate the break points as mentioned by the authors, moreover NGS could also identify a different locus that is not involved the current rearrangement, which could contribute to the phenotype of the patient. Please also discuss these etiologic possibilities in the discussion.\n\nFigure 1:\nThe pedigree looks like compressed from both sides. Please consider keeping the original square and circle shapes while you try to fit the image in the page.\n\nFigure 2:\nI want to thank to authors for this terrific G-banding.\n\nThis is also a very nice demo. It would be better to designate the bands where break points are located (2q13, 2q22, and 18q11.2). The red arrows are not necessary, since you labeled each chromosome (chromosome 2 vs derivative 2, etc.).\n\nFigure 3:\n\nRather than showing only entire chromosome plotting with very limited detail, you can also provide individual array plots of chromosome 2 and 18, which will be easy to evaluate.\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": []
}
] | 1
|
https://f1000research.com/articles/8-281
|
https://f1000research.com/articles/7-1522/v1
|
21 Sep 18
|
{
"type": "Software Tool Article",
"title": "scClustViz – Single-cell RNAseq cluster assessment and visualization",
"authors": [
"Brendan T. Innes",
"Gary D. Bader",
"Brendan T. Innes"
],
"abstract": "Single-cell RNA sequencing (scRNAseq) represents a new kind of microscope that can measure the transcriptome profiles of thousands of individual cells from complex cellular mixtures, such as in a tissue, in a single experiment. This technology is particularly valuable for characterization of tissue heterogeneity because it can be used to identify and classify all cell types in a tissue. This is generally done by clustering the data, based on the assumption that cells of a particular type share similar transcriptomes, distinct from other cell types in the tissue. However, nearly all clustering algorithms have tunable parameters which affect the number of clusters they will identify in data. The R Shiny software tool described here, scClustViz, provides a simple interactive graphical user interface for exploring scRNAseq data and assessing the biological relevance of clustering results. Given that cell types are expected to have distinct gene expression patterns, scClustViz uses differential gene expression between clusters as a metric for assessing the fit of a clustering result to the data at multiple cluster resolution levels. This helps select a clustering parameter for further analysis. scClustViz also provides interactive visualisation of: cluster-specific distributions of technical factors, such as predicted cell cycle stage and other metadata; cluster-wise gene expression statistics to simplify annotation of cell types and identification of cell type specific marker genes; and gene expression distributions over all cells and cell types. scClustViz provides an interactive interface for visualisation, assessment, and biological interpretation of cell-type classifications in scRNAseq experiments that can be easily added to existing analysis pipelines, enabling customization by bioinformaticians while enabling biologists to explore their results without the need for computational expertise. It is available at https://baderlab.github.io/scClustViz/.",
"keywords": [
"single-cell RNAseq",
"differential expression",
"functional analysis",
"interactive visualization",
"R Shiny",
"data sharing"
],
"content": "Introduction\n\nThe development of high-throughput single-cell RNA sequencing (scRNAseq) methods, including droplet-based (Klein et al., 2015; Macosko et al., 2015; Zheng et al., 2017) and multiplexed barcoding (Rosenberg et al., 2018) techniques, has led to a rapid increase in experiments aiming to map cell types within tissues and whole organisms (Ecker et al., 2017; Han et al., 2018; Regev et al., 2017; Saunders et al., 2018). The most common initial analysis of such scRNAseq data is clustering and annotation of cells into cell types based on their transcriptomes. Many workflows have been built and published around this use case (Kiselev et al., 2018; Lun et al., 2016; Sandrine, 2016; Satija, 2018), and many clustering algorithms exist to find cell type-associated structure in scRNAseq datasets (Li et al., 2017; Ntranos et al., 2016; Shao & Höfer, 2017; Xu & Su, 2015; Žurauskienė & Yau, 2016). This paper focuses on how to interpret the results of a scRNAseq clustering analysis performed by existing methods, specifically when it comes to selecting parameters for the clustering algorithm used and analysis of the results. This is implemented as an R Shiny software tool called scClustViz, which provides an interactive, web-based graphical user interface (GUI) for exploring scRNAseq data and assessing the biological relevance of clustering results.\n\nNearly all unsupervised classification (clustering) algorithms take a parameter that affects the number of classes or clusters found in the data. Selection of the appropriate resolution of the classifier heavily impacts the interpretation of scRNAseq data. An inappropriate number of clusters may result in missing rare but distinct cell types, or aberrantly identifying novel cell types that result from overfitting of the data. While there are general machine-learning-based methods for preventing overfitting, we propose a biology-based cluster assessment method; namely whether you could identify a given cluster-defined cell type in situ using imaging techniques based on marker genes identified, such as single molecule RNA fluorescence in situ hybridization (FISH). To identify marker genes and quantify the measurable transcriptomic difference between putative cell types given a clustering solution, scClustViz uses a standard differential expression test between clusters. If there are few differentially expressed genes between two clusters, then those clusters should not be distinguished from each other and over-clustering is likely. The researcher can then select a cluster solution that has sufficiently fine granularity, while still maintaining statistically separable expression of genes between putative cell types.\n\nOnce cell types are defined using the clustering method and parameters of choice, the researcher must then go through several data interpretation steps to assess and annotate these clusters and identify marker genes for follow-up experimentation. Before a final clustering result is chosen, it is important to assess the impact of technical factors on clustering. While that may have been done as part of the upstream workflow, it is helpful to see the cluster-wise distribution of technical factors such as library size, gene detection rates, and proportion of transcripts from the mitochondrial genome (Ilicic et al., 2016). To annotate cell types identified by the classifier, it is helpful to see the genes uniquely upregulated per cluster, as well as assess the gene expression distribution of canonical marker genes for expected cell types in the data. Finally, novel marker genes may be identified for a cell population of interest, which requires identifying genes that are both upregulated in the cluster in question and detected sparingly or not at all in all other clusters in the experiment.\n\nWe describe scClustViz, an R package that aids this common scRNAseq analysis workflow of identifying cell types and their marker genes from a heterogenous tissue sample. The package comprises two parts: a function to perform the differential gene expression testing between clusters for any set of clustering solutions generated by existing scRNAseq analysis workflows, and a R Shiny GUI that provides an interactive set of figures designed to help assess the clustering results, annotate cell types, and identify marker genes. The package was designed with transparency and modularity in mind to ease merging into existing workflows and sharing the results with collaborators and the public. This enables the tool to be of value to both experienced bioinformaticians developing workflows and bench scientists interpreting the results of a scRNAseq experiment.\n\n\nMethods\n\nWe propose a metric for assessing clustering solutions of scRNAseq data based on differential gene expression between clusters. We use the Wilcoxon rank-sum test to evaluate differential gene expression between clusters. This test was selected based on the rigorous differential expression methodology review carried out by Soneson and Robinson (Soneson & Robinson, 2018). In their testing, the Wilcoxon test had accuracy on par with that of the majority of methods tested (most methods were adequately accurate), and identified sets of differentially expressed genes similar to MAST (Finak et al., 2015) and limma (Ritchie et al., 2015), two popular alternatives. In terms of power and control of type I error rate, the Wilcoxon test was less powerful than more advanced methods, with a false discovery rate (FDR) more conservative than expected. However, unlike some more complicated tests, the Wilcoxon test is compatible with parallel processing of testing calculations to increase computation speed. Ultimately, the simplicity of the Wilcoxon test made it appealing for use in this tool, as it is understood by most users, is fast to compute and is available in base R.\n\nPrior to differential expression testing, expression ratios between genes from two cell types are calculated and reported in log base 2 (logGER, log gene expression ratio). Expression ratio calculations are one method used to filter the gene list prior to differential expression testing, thereby removing some of the multiple testing burden for the Wilcoxon test step. Given that scRNAseq gene expression data is expected to be sparse, and that some clusters may not contain any cells in which a certain gene was detected, it is necessary to add a pseudocount to the logGER calculations to prevent divide-by-zero errors and the resulting +/- Inf values. As exemplified in Figure 1, the choice of pseudocount impacts logGER results. A pseudocount of 1 is commonly used but tends to compress the magnitude of the ratio (Figure 1a). However, this does result in comparisons to zero being near the range of other logGER values. Using a small pseudocount such as 10-99, on the other hand, results in logGER values being very close to their true value, rather than suffering from the compression caused by the integer pseudocount. The only problem with this is that comparisons with zero result in very high magnitude logGER values, well outside the range of the rest of the results (Figure 1b). If zero counts of a transcript in a cell library truly represented that gene not being expressed at all in that cell (i.e. if high-throughput single-cell RNAseq experiments were exquisitely sensitive), then this wouldn’t be a problem, since the true expression ratio would be infinitely large. However, zero counts are better interpreted to mean that transcripts for the gene in question were not detected in that cell. Given the relatively poor sensitivity of current high-throughput scRNAseq technology on a per cell basis, this does not necessarily mean that the gene was not expressed. Thus, it would be better if logGER values for comparisons with zero were reasonably close in magnitude to the rest of the results. To accomplish this, we use a pseudocount representing the smallest possible “step” in the count-based data, set to the inverse of the number of cells in the data. This is sufficiently small as to not compress logGER magnitudes, while keeping comparisons with zero reasonably close to the range of potential logGER values.\n\nA. A scatter plot comparing true logGER (x-axis) with logGER calculated with various pseudocounts (y-axis). With a pseudocount of 1 (green squares), the magnitude of logGER is compressed at both ends relative to true logGER, while smaller pseudocounts are much closer to the true values (as denoted by the black line). A very small pseudocount (1e-99, orange circles) is far from the range of values when calculating a log gene-expression ratio where the gene is undetected in one cluster (dividing by zero). An alternative is to set the pseudocount to the smallest possible “step” in count-based data (1 / # of cells, light purple triangles) to prevent magnitude compression of logGER calculations while keeping division-by-zero values within the range of the data. B. Boxplots showing ranges of logGER values with various pseudocounts. With a very small pseudocount (1e-99, in orange), comparisons with zero yield values far from the range of the other data. Setting the pseudocount to the smallest possible “step” in count-based data (1 / # of cells, in light purple) ensures that the logGER values remain close to their true value, while comparisons with zero don’t yield logGER results too far outside the range of other logGER values.\n\nAn alternative to using gene expression ratio to quantify the magnitude of expression differences between clusters is to consider the difference in proportions of cells from each cluster in which the gene in question was detected (per cluster gene detection rate). The concept of detection rate in scRNAseq data stems from the low per-cell sensitivity and minimal amplification noise of droplet-based assays. Since there is a correlation between gene expression magnitude and per cluster gene detection rate, the detection rate may be a meaningful quantification of gene expression, and more suitable to use for identifying genes that uniquely “mark” certain cell populations. Further, the Wilcoxon rank-sum test tends to falsely identify genes as differentially expressed if they are detected at lower rates in the data (Soneson & Robinson, 2018), so filtering based on magnitude of detection rate differences will reduce the number of poorly detected genes tested. To test whether difference in detection rate (dDR) could be used as a filter prior to differential expression testing, its ability to predict significant differential expression (alpha < 0.01, Wilcoxon rank-sum test) was compared to that of a logGER filter. As shown in Figure 2, filtering using difference in detection rate had much better precision when predicting genes that were significantly differentially expressed in pairwise cluster comparisons in mouse cerebral cortex data collected by DropSeq or human liver data collected by 10X Genomics Chromium (MacParland et al., 2018; Yuzwa et al., 2017). Thus, scClustViz uses a 15% difference in detection rate as a default filter prior to differential expression testing between clusters. Given that this statistic may not be as effective when using more sensitive assays such as full-length scRNAseq, using logGER as the filter statistic is an optional parameter. Filtering with logGER is also used when comparing one cluster to the remaining cells in the data, as gene detection rates are less meaningful in such a typically heterogenous data set.\n\nUsing DropSeq data from embryonic day 13.5 from mouse cerebral cortex (green) and 10X Genomics Chromium data from the human liver (orange), a Wilcoxon rank-sum test was used to test the differences in gene expression for all genes between every pair of clusters. P-values less than or equal to 0.01 were considered significant. Difference in detection rate (dDR – dashed line) and gene expression ratio (logGER – solid line) were calculated for all genes between pairs of clusters, and the ability to predict significance differences in gene expression at different thresholds of logGER or dDR were used to generate precision-recall curves. Highlighted are potential thresholds for dDR or logGER.\n\nThree different comparisons are made during differential expression testing. First, marker genes for each cluster are determined by pairwise differential gene expression tests versus every other cluster. This is done by testing for differential gene expression for all genes above the dDR threshold in every combination of clusters, then finding genes that have a positive gene expression ratio and pass FDR threshold (default FDR < 1%) for a cluster in every comparison. This method is one of the two differential expression tests used in scClustViz to quantify cluster granularity. It ensures that there are marker genes for all clusters that are unique to each cluster, given all other clusters in the data.\n\nThe second way cluster granularity is quantified by scClustViz is by comparing each cluster to its nearest neighbouring cluster. By ensuring there is at least one positively differentially expressed gene (default FDR < 1%) between each set of neighbouring clusters, this metric enforces the requirement for having statistically separable clusters, which is less restrictive than requiring unique marker genes per cluster. Nearest neighbours are clusters with the fewest differentially expressed genes between them, as calculated above.\n\nFinally, scClustViz calculates differential gene expression between genes in each cluster versus the rest of the data combined. This is not used to assess clustering results but may be visualized by the user to identify distinguishing genes for that cluster, although this will only be valuable if there is enough heterogeneity in the data to identify differential genes. Though this represents an unbalanced comparison, the non-parametric nature of the Wilcoxon rank-sum test makes it robust to such imbalances.\n\nTo support quick display of the various figures in the user interface, other cluster-wise gene statistics are calculated. Detection rate (DR) is the proportion of cells in a cluster in which a given gene has a non-zero expression value. Mean detected transcript count (MDTC) is the mean of the normalized transcript counts for a gene in the cells of the cluster in which that gene was detected. And mean transcript count (MTC) is the mean normalized transcript count for a gene for all cells in the cluster. These are stored as a nested list object in R: a named list of cluster resolutions, containing a named list of clusters per resolution, containing a dataframe storing the cluster-wise gene statistics. This nested list format is also used to store each of the three differential expression test results. For comparison between a cluster and the rest of the data (DE vs. tissue), the dataframe stores log2 fold-change (logGER), p-value, and q-value (FDR) results for each gene per cluster that is determined to be positively differentially expressed (logGER > 1, FDR < 1%). For comparison between a cluster and each other cluster (marker genes), the dataframe stores log2 fold-change (logGER), difference in detection rate (dDR), and q-value for each cluster being compared to, with variable names indicating the cluster being compared to. For comparison with the neighbouring cluster, the relevant three variables from the marker genes are stored in the dataframe. Since all pairwise cluster comparisons are done once, they are also stored as a separate list of lists, and are accessed by the Shiny app when comparing clusters.\n\nThe package was built in R v3.5.0 (R Core Team, 2018). The R Shiny interactive web page generating tool (shiny v1.1.0) was used to generate the scClustViz user interface (Chang et al., 2018). Silhouette plots are generated using the R package cluster v2.0.7-1 (Maechler et al., 2018). Colour-split dots for plotting use code from the R package TeachingDemos v2.10 (Snow, 2016). Colour scales with transparency use the R packages scales v1.0.0, viridis v0.5.1, and RColorBrewer v1.1-2 (Garnier, 2018; Neuwirth, 2014; Wickham, 2017).\n\nThe scClustViz tool is available as an R package from GitHub. Usage details and example code is available on the website, but in short there are two steps involved prior to viewing data in the GUI: importing the data and running the differential gene expression testing. Each step is a single function call, and the output of these two steps is saved so that they only need to be run once. This allows the user to quickly view and easily share the results of their analysis following setup.\n\nThe first step is to import scRNAseq clustering output from an existing analysis pipeline. scClustViz takes the normalized gene expression matrix, cell-wise metadata, cluster assignments, and cell embeddings from dimensionality reduction methods (i.e. principal component analysis (PCA) and t-Distributed Stochastic Neighbor Embedding (tSNE)) from an existing scRNAseq analysis and stores them as a list in R. While there are some existing data structures designed to handle scRNAseq data, such as the Bioconductor SingleCellExperiment class (Lun & Risso, 2017), building this small custom list reduces the risk of unexpected inputs while minimizing both file size and dependencies on any particular data structure. There are currently two functions for passing existing data to scClustViz. The function readFromSeurat automatically pulls the relevant data from a Seurat data object generated using the analyses outlined in their tutorial (Butler et al., 2018). Alternatively, the readFromManual function enables the user to specify the relevant data from their analysis object of choice.\n\nThe second step is to run the differential expression testing. This is done using the function clustWiseDEtest, which takes as input the list generated in the first step, as well as optional arguments describing the state of the data and customizing testing thresholds. To calculate means of log-normalized data accurately, the function needs to know the log base and pseudocount used in the normalization. In most cases, gene expression data is transformed in log base 2, though Seurat uses the natural log. A pseudocount of 1 is generally used to avoid log-zero errors. As such, the function defaults to expecting log2-normalized data with a pseudocount of 1. The function also provides options for gene filter type and threshold prior to differential expression testing. As outlined above, difference in detection rate can be a better predictor of significant differential gene expression and is thus the default gene filtering method, but gene expression ratio can be used if desired. Thresholds for each method can also be set (defaulting to 0.15 for dDR and 1 for logGER). The false discovery rate threshold for determining significance can also be set, and defaults to 1%. Finally, since iteratively testing differential gene expression between clusters for many cluster solutions takes time, there is an option to stop testing cluster solutions once there are no longer statistically significant gene expression differences between the closest pair of nearest neighbouring clusters. This is determined by first sorting the clustering solutions in order of increasing number of clusters found, then testing them in order, stopping when no significant differential expression has been found between a pair of nearest neighbour clusters. If this option is selected, only cluster solutions tested will appear in the Shiny GUI. This has the potential to save time during setup.\n\nThe objects generated in the setup steps are saved to disk as a single compressed RData file prior to viewing them in the GUI. This is done to ensure that setup is a one-time process, and to simplify sharing of analyses. The function runShiny launches the R Shiny instance with the interactive data figures in the R integrated development environment (IDE) or a web browser. It loads the data from file, and has optional arguments to specify the annotation database and marker genes for expected cell types, as well as setting thresholds for differential expression testing. The latter arguments describing testing thresholds shouldn’t be necessary, as the parameters used in the differential testing step are saved and loaded as defaults by this function. The annotation database is used to find gene names to improve clarity of some figures and expects a Bioconductor AnnotationDbi object such as org.Mm.eg.db for mouse or org.Hs.eg.db for human. Finally, if passed a named list of canonical marker genes for expected cell types in the data, scClustViz will automatically generate cluster annotations (labels). This is done by assigning each cluster to the cell type with the top aggregate rank of gene expression for its marker genes. More in-depth and unbiased methods for assigning cell type identities to clustering results exist (Crow et al., 2018; Kiselev et al., 2017), so this is meant more as a convenience option for labelling purposes than a definitive automatic cluster annotation method.\n\nSystem requirements for this tool will depend heavily on the dataset in question, since the data will have to be loaded into memory, and the memory footprint of scRNAseq data depends on the number of cells being analysed. However, in all tests loading objects from Seurat into scClustViz, the saved objects after the setup and differential expression testing steps were smaller than the original Seurat object. It is thus safe to assume that scClustViz will run on the computer on which the dataset in question was analysed. For the data from the MouseCortex package, the largest dataset (E15, containing nearly 3000 cells) uses less than 1.2GB of memory.\n\n\nUse cases\n\nTo demonstrate the convenience of sharing analysed data with scClustViz, the MouseCortex package was built with data from a recent publication exploring the development of the mouse cerebral cortex using scRNAseq (Yuzwa et al., 2017). A tutorial for building similar R data packages calling scClustViz as the visualization tool can be found on the scClustViz website.\n\nThe MouseCortex package contains the four datasets published in the paper, and a wrapper function for runShiny that loads each dataset with the appropriate arguments. The embryonic day 17.5 dataset (opened by the command viewMouseCortex(“e17”)) will be used to demonstrate the purpose of the various figures in scClustViz and highlight its role in identifying a core gene set expressed in the neurogenic stem cell population of the cerebral cortex in the next sections. All figures from this point on were generated in the scClustViz Shiny app and saved using the “Save as PDF” buttons.\n\nThe first step in the post-clustering workflow is to assess the results of the various clustering parameterizations used. scClustViz uses a combination of differential gene expression between clusters and silhouette analysis for this. Differential gene expression is used as a metric in two ways: the number of positively differentially expressed genes between a cluster and its nearest neighbour, and the number of marker genes (positively differentially expressed vs. all other clusters in pairwise tests) per cluster. In Figure 3a, differential expression to the nearest neighbouring cluster is represented as a series of boxplots per cluster resolution, arranged on the x-axis to indicate the number of clusters in each boxplot. The highlighted boxplot indicates the currently selected cluster from the pulldown menu in the user interface. Both differential expression-based metrics can be visualized this way by switching the metric used, via the interface.\n\nA. Boxplots representing number of differentially expressed genes between neighbouring clusters for each cluster resolution. For each cluster at a specific resolution, the number of positively differentially expressed genes to its nearest neighbouring cluster is counted, and those counts are represented as a boxplot. The boxplots are arranged along the x-axis to reflect the number of clusters found at that resolution. Highlighted in red is the cluster resolution currently selected in the interface. A similar figure is also available for number of marker genes per cluster. B. Silhouette plot for the selected cluster resolution. A horizontal bar plot where each bar is a cell, grouped by cluster. Silhouette width represents the difference between mean distance to other cells within the cluster and mean distance to cells in the neighbouring cluster. Distance is Euclidean in reduced dimensional (generally PCA) space. Positive values indicate that the cell is closer to cells within its cluster.\n\nWhen a cluster resolution is selected, its silhouette plot is rendered to add another method of cluster assessment (Figure 3b). A silhouette plot is a horizontal bar plot where each bar is a cell, grouped by cluster. The width of each bar, referred to as silhouette width, represents the difference between mean distance to other cells within the cluster and mean distance to cells in the neighbouring cluster. Distance is Euclidean in the reduced dimensional space saved in the original analysis object (generally this is defined using PCA). Positive values indicate that the cell is closer to cells within its cluster. It is worth noting that the dimensions returned by methods such as PCA are not equally meaningful, since each explains a different proportion of the variance in the data, while Euclidean distance treats them all equally. This can be addressed by weighting the PCs by variance explained, a method implemented in newer versions of Seurat (Butler et al., 2018). To prevent unexpected results caused by assuming a PC weighting option in upstream analysis, the silhouette plot in scClustViz does not reweight PCs, so users are encouraged to consider this when interpreting this plot.\n\nOnce the user has chosen the appropriate cluster solution, they can click the “View clusters at this resolution” button to proceed to in-depth exploration and visualization of the results. They can also save this as the default resolution for future sessions. If a cluster resolution is saved as default, a file specifying the saved resolution will be generated in the same directory as the input data (or an optional output directory). Specifying a separate output directory is useful when the input data is part of a package, as in MouseCortex. If the same output directory is specified the next time the command is run, all saved data in that directory will be reloaded in the app.\n\nIn this section, the user can explore the dataset as a whole. The first panel, Figure 4a shows a two-dimensional representation of cells in gene expression space. This is generally a tSNE plot, but the user can use the 2D cell projection results of their choice by specifying the relevant data (i.e. UMAP) in readFromManual (van der Maaten & Hinton, 2008; McInnes & Healy, 2018). The cells are coloured by cluster and can be labelled by cluster number or automatically annotated with a predicted cell type based on known marker genes for expected cell types passed to runShiny. The user can select any cluster for downstream exploration by clicking on a cell from that cluster in this plot. This will highlight the cluster in other plots in the interface. Since we are interested in identifying marker genes for the precursor cell population, we may click on cluster 8 (purple) to select it for downstream analysis.\n\nA. A 2D projection of cells in gene expression space (frequently a tSNE plot) is coloured by cluster. Clusters can be labelled by number, or automatically annotated as seen here. B. An example of a metadata overlay on the cell projection. The library size (number of transcripts detected) per cell is represented by colour scale, where darker cells have larger library sizes. C. Metadata can be represented as a scatter plot. The relationship between gene detection rate (number of unique genes detected – y-axis) and library size (x-axis) is shown here. The cells from the selected cluster (cluster 8, cortical precursors) are highlighted in red. D. Categorical metadata is represented as a stacked bar plot showing the number of cells contributing to each category per cluster. This plot shows predicted cell cycle state, with G1 phase in green, G2/M in orange, and S phase in purple.\n\nThe distribution of various cellular metadata can be visualized in Figure 4b. Metadata is selected from a pulldown menu and is represented as colours on the cells in the 2D projection. In this manner the user can inspect the impact of technical artifacts such as gene detection rate, library size, or cell cycle stage on the clustering results. Numeric metadata can also be assessed as a scatter plot, where the axes can be defined by selecting from pulldown menus. Figure 4c shows the relationship between gene detection rate and library size per cell for both the dataset as a whole and the selected cluster. The cells from cluster 8, a cortical precursor cluster, were selected in the previous plot and are thus highlighted in red here. The cluster 8 cells are similar to other cells in the data, thus do not seem to be biased by the measures visualized in this plot. If this was not the case, we may want to consider confounding variables in the normalization process. For example, many authors have noted that gene detection rate is often strongly correlated with the first few principal components, and can thus influence clustering results (Finak et al., 2015; Risso et al., 2018). There are a few ways to handle this, from simply excluding those principal components, or explicitly normalizing for those factors when scaling the data (as implemented in Seurat), to including the offending technical variables as covariates in more complex dimensionality reduction (i.e. ZINB-WaVE) or differential expression testing (i.e. MAST) models. While those analyses are outside the scope of this tool, it is important to be able to visualize these technical factors in the analysed data to assess the efficacy of the chosen correction method.\n\nCategorical metadata is represented as a stacked bar plot in Figure 4d, as either absolute counts or relative proportions. Here we see that by E17.5 the cortical precursors of cluster 8 are not predicted to be actively in the cell cycle using the cyclone method (Scialdone et al., 2015). This fits expectations from known developmental biology, since neurogenesis is nearly complete by this stage, and the stem cell population that persists into adulthood is thought to enter quiescence around E15.5 (Fuentealba et al., 2015). For demonstration purposes, we will continue to focus on cluster 8, which is predicted to form the adult neurogenic stem cell population in the cerebral cortex. We will aim to identify marker genes for these cells.\n\nOnce the user is satisfied that their cluster solution is appropriate and unaffected by technical factors, the next step in data interpretation is to determine the cellular identity of each cluster by its gene expression. The three different differential expression tests done prior to running the visualization assist with this by highlighting the most informative genes in the dataset. In a sufficiently heterogeneous dataset, differential expression between a cluster and the rest of the data can be useful for identifying genes that uniquely define a cluster’s cellular identity. A more conservative form of this is the identification of marker genes – those genes that are significantly positively differentially expressed in all pairwise tests between a cluster and all other clusters. This highlights genes expected to be found at a significantly higher expression in this cluster than anywhere else in the data. Finally, there is the testing between each cluster and its nearest neighbour to highlight local differences in expression. Each of these sets of differentially expressed genes can be presented as a dot plot comparing clusters, as seen in Figure 5. A dot plot is a modified heatmap where each dot encodes both detection rate (by dot diameter) and average gene expression in detected cells (by dot colour) for a gene in a cluster. Here up to the top ten marker genes per cluster are shown, but both the type of differential expression test used to generate the gene set and the number of differentially expressed genes contributed per cluster can be adjusted using the interactive interface. At this point in the analysis, it is also possible to download any of these differential gene expression results as tab-separated value files for further analysis, by selecting the cluster of interest and differential expression type and clicking “Download gene list”. This may be of value if the user is using this platform to share the data online, or with those who would prefer not to use R for further analysis. In this dot plot, we can see the top 10 marker genes for our putatively quiescent cortical precursor cell population (cluster 8) include known marker genes for cortical radial precursors (Fabp7, Slc1a3, Vim, and Sox2), a known marker for adult neural stem cells (Dbi), as well as novel marker genes for this population (Mfge8, Ttyh1, Ddah1, and Ednrb) (Yuzwa et al., 2017). The dot plot format also shows us that while Ckb is significantly positively differentially expressed in cluster 8 relative to all other clusters, it is still quite highly expressed in all cells in the data, and thus would not be an optimal marker gene.\n\nA dot plot showing the relative expression of a subset of marker genes (x-axis) across all clusters (y-axis). A dot plot is a modified heatmap where each dot encodes both detection rate and average gene expression in detected cells for a gene in a cluster. Darker colour indicates higher average gene expression from the cells in which the gene was detected, and larger dot diameter indicates that the gene was detected in greater proportion of cells from the cluster. Cluster colours are indicated for reference on the left side of the plot. Cluster numbers are also indicated on the left side, along with the number of differentially expressed genes in each cluster. The genes included can be changed to reflect those differentially expressed per cluster when compared to the rest of the dataset as a whole (i.e. the tissue), the nearest neighbouring cluster, or marker genes unique to that cluster. The number of differentially expressed genes contributed per cluster can also be adjusted.\n\nTo more closely inspect the gene expression of an individual cluster, scClustViz presents gene expression data per cluster as a scatter plot with the proportion of cells from that cluster in which a gene is detected (more than zero transcript counts) on the x-axis, and mean normalized transcript count from cells in which the gene was detected on the y-axis, as seen in Figure 6a. This visualization method helps separate the contribution of zeros from the mean gene expression value, since like the dot plot it separates magnitude of gene expression from gene detection rate. It also highlights the strong relationship between magnitude of gene expression and likelihood of detection in droplet-based single-cell RNAseq data, since the trend goes from the plot’s bottom left (genes have low expression and are rarely detected) to top right (genes have high expression and are detected often). In this figure, the cortical precursor cluster 8 is shown, but the user can select the cluster shown from a pulldown menu in this panel as well. There are three ways to highlight various genes in this plot. First, the genes passed as known marker genes for expected cell types can be highlighted in colours corresponding to their cell type, if a marker gene list is defined by the user (Figure 6a). This figure indicates that this cluster was classified as cortical precursors based on the high relative expression of both Sox2 and Pax6, as well as Nes and Cux1 (markers for both cortical precursors and projection neurons). In Figure 6b, the plot shows differentially expressed genes, specifically the genes contributed by this cluster to the dot plot shown immediately above in the app (Figure 5). Thus, by changing the differential gene set or number of genes in the heatmap, the user can also adjust the genes highlighted in this scatter plot. Finally, the user can search for genes manually by entering a list of gene symbols or using a regular expression in the search box below the figure. To identify and compare gene expression for any point in this figure, the user can click on the point.\n\nA. A scatter plot representing gene expression in the highlighted cluster, the cortical precursor cluster 8. The x-axis represents the proportion of cells from that cluster in which a gene is detected (more than zero transcript counts), and the y-axis is the mean normalized transcript count from cells in which the gene was detected. The cell type marker genes are highlighted, indicating that this cluster was classified as cortical precursors based on the high relative expression of both Sox2 and Pax6, as well as Nes and Cux1 (markers for both cortical precursors and projection neurons). B. The same scatter plot is shown with the top 10 marker genes for cluster 8 highlighted, though the user can choose other differentially expressed gene sets from the heatmap, or search for genes of interest using the interface. The identity of any point can be determined by clicking on it in the interface. C. Boxplots comparing the expression of a gene of interest across all clusters. Clusters are arranged on the x-axis based on the cluster dendrogram generated for the dot plot above (Figure 5), and normalized transcript count for the gene of interest (Mfge8, in this case) is represented on the y-axis. The dots on each boxplot represent the individual data points, gene expression per cell. The red triangles indicate the rank of relative gene expression in that cluster, as indicated on the y-axis on the right side. This figure shows that Mfge8 may be a marker of cortical precursors. D. Gene expression overlaid on the cell projection. Gene expression is represented by a colour scale on the cells of the two-dimensional projection, where darker indicates higher expression. If multiple genes are selected, the maximum gene expression value per cell is shown. Clusters can be labelled by number or annotation. This figure shows the distribution of Mfge8 expression in the dataset.\n\nClicking on a point in the figure above will generate a series of boxplots comparing gene expression for the selected gene across all clusters (Figure 6c). Since the above scatter plot can be crowded, all genes near the clicked point are shown in a pulldown menu, so that the user can select their gene of interest. Alternatively, the gene(s) entered in the search box in the previous panel can be used to populate the pulldown list for selecting the gene of interest for this figure. By comparing gene expression across clusters, it is easier to assess the utility of putative marker genes. Here we see that Mfge8 is expressed nearly exclusively in cluster 8, with rare detection in any other clusters. This suggests that Mfge8 may be effective for identifying the cells of this cluster in situ. In fact, both fluorescence in situ hybridization for Mfge8 and immunohistochemistry for its protein lactadherin showed specificity for the cortical precursor cells in the embryonic mouse brain, as well as the B1 neural stem cells of the adult ventricular/subventricular zone (Yuzwa et al., 2017).\n\nFinally, the user can directly plot the expression of a gene or genes of interest on the tSNE plot to better visualize the distribution of gene expression in the dataset, as shown in Figure 6d. Genes are selected by entering gene symbols or a using a regular expression and selecting the matching gene symbols from a dropdown list. Gene expression is represented by a colour scale on the cells of the two-dimensional projection. If multiple genes are selected, the maximum gene expression value per cell is shown. This serves as another way of highlighting the specificity of Mfge8 for the cortical precursor cells in this dataset.\n\nThe final feature of scClustViz is the ability to generate MA plots comparing gene statistics for any two clusters, or any two sets of cells specified by the user (Figure 7a). MA plots (also known as Tukey’s mean-difference plot or Bland-Altman plot) show differences between samples comparing the log-ratio of gene expression between samples (y-axis) to the mean gene expression across those samples (x-axis). We take this one step further by giving the user the option of viewing the difference and average of all three gene statistics used in scClustViz: mean gene expression, mean detected gene expression, and detection rate. Furthermore, the user can manually select sets of cells to compare, and scClustViz will calculate differential gene expression statistics between the selected cells and the remaining cells in the data, and between sets of selected cells. Once the calculations are complete, the resulting comparison is represented as a separate “cluster solution” and can be explored in all the figures of scClustViz. These results can be saved to disk by clicking “Save this comparison to disk” when selecting it in the pulldown menu for cluster solution selection. Any saved comparisons will be loaded along with the data any time runShiny is run.\n\nA. An MA plot showing log-ratio of gene expression between cell sets on the y-axis, and average gene expression across sets on the x-axis. Set A here is a subset of cluster 5 with low library sizes, while set B is the subset of cluster 5 with high library sizes. Highlighted are the top differentially expressed genes upregulated in set A over set B (purple) and set B over set A (green). B. An MA-style plot showing difference in gene detection rate between set A and set B on the y-axis, and average gene detection rate across sets on the x-axis. The horizontal line is at zero difference in detection rate. Highlighted are genes from the mitochondrial genome, which are generally used as markers of damaged cells in single-cell RNAseq analyses.\n\nIn Figure 7 we’re investigating a potential technical artifact in the data, specifically the poor cohesion of cluster 5 as seen in the silhouette plot in Figure 3b. This poor cohesion could be due to the differences in library size within the cells of the cluster, as seen in Figure 4b. To investigate this, the cell selection tool in scClustViz was used to select the cells of cluster 5 with low library sizes (Set A) and those with high library sizes (Set B). After running the differential gene expression calculations, we can view the differentially expressed genes between the sets in the dot plot or MA-plot (Figure 7a). Set B seems to have more positively differentially expressed genes, which may be due to improved gene detection rate from higher library sizes. This can be seen in Figure 7b, where an MA-style plot showing difference in detection rate vs average detection rate across sets is shown. Most genes are more detected in the set with larger library sizes (set B), which might be expected, since more transcripts detected correlates with higher average transcript counts per gene. Clicking on a gene in this figure has the same functionality as the scatter plot in Figure 6; it will be selected for viewing in the boxplot above (Figure 6c). Using this, we noticed that genes from the mitochondrial genome were seemingly unaffected by the difference in library sizes, as they tended to fall near zero difference in detection rate. To highlight this, we searched for all genes from the mitochondrial genome using the search tool, which allowed us to highlight them here. If cells are damaged and leaking cytoplasm, they are likely to have smaller library sizes as they lose mRNA. However, since RNA from the mitochondrial genome is sequestered in a separate organelle, they are less likely to lose those transcripts (Ilicic et al., 2016). We can see evidence for this in the cells of cluster 5 with small library sizes, since the detection rate of their mitochondrial genes is unchanged. While this dataset was filtered to remove cells with higher than average mitochondrial gene transcript proportions, including that metric in the metadata would allow for tuning of the threshold used. Since these cells have both low library sizes and higher relative detection rate of mitochondrial transcripts, it is safe to assume they are damaged cells and remove them from the analysis.\n\n\nConclusion\n\nWe developed scClustViz to aid in the annotation of cell types and identification of marker genes from scRNAseq data. It provides both a metric for cluster assessment based on inter-cluster differential gene expression, as well as a convenient user interface for accomplishing this analysis and interpretation task. Using differential gene expression to assess clustering solutions ensures that the results are suited to addressing the relevant biological task of identifying cell types and their marker genes. The user interface is also focused specifically on this task by generating publication quality figures and providing analyses that help the user determine the appropriate number of clusters, identify cell types, and highlight genes unique to those cell types. There are other user interfaces available for the analysis of scRNAseq data (Rue-Albrecht et al., 2018; Zhu et al., 2017). However, scClustViz fills a niche between existing GUIs, which are either very user-friendly for non-technical users, at the cost of the ability to customize analysis, or very powerful and customizable, at the cost of providing a simple framework for accomplishing a common analysis task. The one-time setup step for scClustViz also simplifies data sharing, as it generates a file that can be shared for viewing by anyone using R. Data sharing can be made more user-friendly by building an R data package with a wrapper function calling scClustViz, as seen in the use case outlined in this paper. Building such a package is a quick process, and a tutorial is available on the scClustViz website. scClustViz is available at https://baderlab.github.io/scClustViz/ as free, open source software under the permissive MIT open source license.\n\n\nData availability\n\nThe example dataset used is available as an R package: https://github.com/BaderLab/MouseCortex\n\nArchived code at time of publication: http://doi.org/10.5281/zenodo.1411462 (Innes, 2018a)\n\nLicence: MIT\n\n\nSoftware availability\n\nscClustViz is available from: https://baderlab.github.io/scClustViz/\n\nSource code is available from GitHub: https://github.com/BaderLab/scClustViz\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.1411464 (Innes, 2018b)\n\nLicence: MIT",
"appendix": "Grant information\n\nThis research was undertaken thanks in part to funding provided to the University of Toronto Medicine by Design initiative, by the Canada First Research Excellence Fund.\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\nButler A, Hoffman P, Smibert P, et al.: Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018; 36(5): 411–420. PubMed Abstract | Publisher Full Text\n\nChang W, Cheng J, Allaire J, et al.: shiny: Web Application Framework for R. RStudio. 2018. Reference Source\n\nCrow M, Paul A, Ballouz S, et al.: Characterizing the replicability of cell types defined by single cell RNA-sequencing data using MetaNeighbor. Nat Commun. 2018; 9(1): 884. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEcker JR, Geschwind DH, Kriegstein AR, et al.: The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas. Neuron. 2017; 96(3): 542–557. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinak G, McDavid A, Yajima M, et al.: MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 2015; 16: 278. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFuentealba LC, Rompani SB, Parraguez JI, et al.: Embryonic Origin of Postnatal Neural Stem Cells. Cell. 2015; 161(7): 1644–1655. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarnier S: viridis: Default Color Maps from' ‘matplotlib’. R package. 2018. Reference Source\n\nHan X, Wang R, Zhou Y, et al.: Mapping the Mouse Cell Atlas by Microwell-Seq. Cell. 2018; 172(5): 1091–1107.e17. 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}
|
[
{
"id": "38629",
"date": "10 Oct 2018",
"name": "Martin Hemberg",
"expertise": [
"Reviewer Expertise My expertise is in computational biology and in particular on methods development for scRNA-seq. Thus",
"I feel qualified to evaluate this manuscript"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Innes and Bader present scClustViz, an R package for interactive assessment and visualization of unsupervised clustering methods for scRNA-se data. The topic is very timely as unsupervised clustering is one of the most important applications of scRNA-seq. Nevertheless, it is a challenging problem and despite several different software tools being available, it is still not possible to fully automate the process. Thus, having a method to facilitate this analysis that can be run on the output of other clustering methods is potentially very useful.\nMajor Comments:\n1. Installing scClustViz was straightforward and easy. However, I then had some issues running it. Using a Seurat object from another project where we are analyzing the data, I got the following error:\nDE_for_scClustViz <- clusterWiseDEtest(data_for_scClustViz,exponent=exp(1)) [1] “” [1] “” [1] “Calculating all DE stats for res.0.8” [1] “” [1] “Calculating cluster gene summary statistics” [1] “-- Gene detection rate per cluster --”\n\n|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 39s [1] “-- Mean detected gene expression per cluster --”\n\n|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 36s [1] “-- Mean gene expression per cluster --”\n\n|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 01m 24s [1] “” [1] “Calculating DE vs tissue with 16 clusters” [1] “-- logGER calculations --”\n\n|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 25m 39s [1] “-- Wilcoxon rank sum calculations --”\n\n|++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed = 11m 20s [1] “” [1] “Calculating marker DE with 120 combinations of clusters”\n\n|\n\n| 0 % ~calculating Error in intI(i, n = x@Dim[1], dn[[1]], give.dn = FALSE) : invalid character indexing\nIt may be that there are some issues with the Seurat object that I used as input, but the unspecific nature of the error message makes it very hard to troubleshoot.\nI then tried to run it using an SingleCellExperiment object. Here, the instructions were less clear and it required some fiddling around before I came up with the following lines of code for preparing the dataset, the FACS sorted lung data from the Tabula Muris:\nlogcounts(mySCE) <- log2(counts(mySCE) + 1) clusterAssignments <- grepl(\"^cell_type1\",colnames(colData(mySCE))) mySCE <- plotPCA(mySCE, return_SCE=T, draw_plot=F) mySCE <- plotTSNE(mySCE, return_SCE=T, draw_plot=F) data_for_scClustViz <- readFromManual(nge=logcounts(mySCE),\n\nmd=colData(mySCE)[,!clusterAssignments],\n\ncl=as.character(colData(mySCE)[,clusterAssignments]),\n\ndr_clust=reducedDim(mySCE,\"PCA\"),\n\ndr_viz=reducedDim(mySCE,\"tSNE\")) DE_for_scClustViz <- clusterWiseDEtest(data_for_scClustViz,\n\n# Stop once DE is lost between nearest neighbouring clusters\n\ntestAll=FALSE,\n\n# Normalized data is in log2 space\n\nexponent=2,\n\n# Pseudocount of 1 was added to log-normalized data\n\npseudocount=1,\n\n# False discovery rate threshold of 1%\n\nFDRthresh=0.01,\n\n# Use difference in detection rate to filter genes for testing\n\nthreshType=\"dDR\",\n\n# Genes with at least 15% detection rate difference will be tested\n\ndDRthresh=0.15\n\n)\nsave(data_for_scClustViz,DE_for_scClustViz,file=\"for_scClustViz.RData\")\nThis allowed me to get the shiny interface started, but there were several error messages appearing (most frequently \"object 'dr_viz' not found\") and I was not able to explore the different functionalities that had been highlighted in the manuscript. This may have been due to the fact that there was only one clustering present in the SingleCellExperiment object. However, if this is the case, then I think that the error messages should be more informative and scClustViz ought to do a better job of handling this special case. Thus, I was unable to explore the different functionalities that were highlighted in the manuscript.\n2. The multiple filters used by scClustViz is in general a good idea since it is not clear what is the best way of defining DE genes. However, filtering by changes in detection rate invalidates the multiple testing correction, genes could be filtered by expression level or detection rate but not difference in detection rate. The authors should comment on this complication\nMinor comments:\n1. Figure 3A plot would be better presented as a grouped scatterplot since the number of values per box is small. 2. Figure 4 D colours in legend do not match colours in figure.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4458",
"date": "12 Mar 2019",
"name": "Brendan Innes",
"role": "Author Response",
"response": "Drs. Andrews and Hemberg, Thank you very much for your careful and helpful comments on this manuscript and software. Your first major concern related to errors when trying to run the setup step. In the new version of scClustViz this has been addressed by using a formal S4 class object to store the results of the analysis. This object class includes built-in validity checking, so unexpected inputs are caught early and reported with meaningful error messages. In keeping with this, scClustViz now accesses data directly from SingleCellExperiment and Seurat S4 objects, which should reduce the number of unexpected inputs, as they have their own validity checking and return consistent data structures. Your second major concern was that filtering for difference in detection rate prior to differential expression testing invalidates the multiple testing correction, and you suggest filtering for detection rate instead. This is a very good point, since any filtering of hypotheses using a feature that correlates with the hypothesis being tested invalidates the assumption of uniform p-value distributions in FDR correction. We have addressed this by adopting the detection rate filter commonly used in the field, where genes must be detected above a certain rate (10% is our default) in at least one of the clusters being compared. This filtering method continues to protect against making comparisons with low-abundance genes that the Wilcoxon rank-sum tests may be biased towards. As a result, we have removed the section of the manuscript comparing the previous proposed filtering methods, and the previous figure 2. You also suggest using a grouped scatterplot instead of boxplot for Fig3a (now Fig2a). The reason we avoided a grouped scatterplot in this case was because some cluster solutions may result in the same number of clusters and thus overlap on the x-axis. This would make it challenging to display a grouped scatterplot without causing confusion. We’ve opted to compromise by showing the data points for the selected cluster solution only. Finally, you noted that the legend in Fig4d (now Fig3d) has an incorrect legend. We couldn’t identify the error, so if it persists we’d be happy to correct it. Thank you again for identifying important points to address. We hope these corrections will allow you to use the software without further difficulty."
}
]
},
{
"id": "38632",
"date": "10 Oct 2018",
"name": "Michael Steinbaugh",
"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\nStrengths\nInteractive Shiny interface. scClustViz enables researchers to work interactively with their single-cell RNA-seq data. The Shiny interface available in this package is excellent. I found it to be very intuitive and easy to use. Cluster identification and marker analysis is time consuming, and scClustViz helps accelerate these steps by enabling a user to quickly generate and save informative plots.\nCluster and marker visualizations. The suite of plots offered in this package are robust and informative. They all appear to render quickly, even for relatively large data sets. In particular, the silhouette plot approach (Figure 3B) is novel, and I like the ability to view the cluster and maker dimensional reduction plots side-by-side (Figure 4). Dot plots are a great way to identify cell-type specific markers, and the interactive tool available in the package works well (Figure 5).\n\nWeakness\nlogGER pseudocount calculations. The explanation of differential expression implementation needs improvement (see Methods section), and the functions are not documented in the text. The authors mention that they employ a small pseudocount calculation approach (e.g. using 10-99), which results in \"logGER values being very close to their true value\". More evidence is needed to support this claim, and I would like to see additional rationale as to why this is approach is preferable to other published methods that address dropout/zero count inflation (e.g. ZINB-WaVE, MAGIC)1-2.\nWilcoxon rank-sum test. Additionally, as mentioned in the text, the Wilcoxon rank-sum test isn't as powerful and doesn't control the false discovery rate as well as some other published methods3. In particular, edgeR, DESeq2, limma, scde, and MAST are validated differential expression callers in use in other single-cell analysis packages that are viable alternatives to Wilcoxon4-5-6-7-8-9. An option to use pre-calculated values inside the package would be a nice addition.\nReproducible code. While the interactivity provided by the Shiny interface is excellent and user-friendly for visualization, GUI-based data analysis is often difficult to reproduce. I would like to see scriptable, exported versions for all of the tools available in the package, so that a single-cell marker analysis can be run start-to-finish using scClustViz in a reproducible manner.\nRecommendations\nTake advantage of object-oriented programming. The authors mention in the text that scClustViz relies upon a \"small custom list\" of data generated using either the `readFromSeurat()` or `readFromManual()` functions, and that this approach \"reduces the risk of unexpected inputs\". I disagree with this statement, and recommend that the authors switch from an unstructured list to an S4 class object. Additionally, the paper doesn't describe what is stored in this list in detail. The S4 class system is documented in detail on the Bioconductor website, and is used by most popular single-cell RNA-seq analysis tools. S4 classes enable validity checks (see `validObject()`) and backwards compatibility support for legacy objects created with older versions of the package (see `updateObject()`).\n\nAdd native SingleCellExperiment support. The authors provide a function for importing data from Seurat (`readFromSeurat()`), but currently don't provide a simple coercion method for the popular `SingleCellExperiment` class container.\nAdd unit testing. I noticed that the package doesn't currently have any code coverage with unit tests. I strongly recommend adding these checks against a minimal dataset. In particular, the testthat package (http://testthat.r-lib.org) is recommended.\n\nImprove text labels. The gene marker labels on some plots are illegible because they are superimposed. The ggrepel package (https://cran.r-project.org/package=ggrepel) may help improve the legibility of plots with gene labels.\nTechnical issues\nUnable to launch Shiny browser in a remote R session. The example MouseCortex Shiny data package runs correctly on machines where a browser instance can be launched. I tested this on multiple local environments (Linux, macOS, Windows) and on a remote RStudio server. However, it fails to launch on some remote high-performance computing (HPC) environments from the R command line. In some cases this can potentially be fixed with `runApp(launch.browser = FALSE)`, but it's unclear to me whether a user can easily run the `viewMouseCortex(\"e11\")` example in a remote R session without RStudio. This may be an edge case, but many R users work primarily on remote environments, so it's worth mentioning this potential limitation in the text. Here is the error message that can occur:\n``` Listening on http://127.0.0.1:3899 xdg-open: no method available for opening 'http://127.0.0.1:3899' ```\nShiny console warnings. A number of warnings appear in my R console when running the example Shiny datasets. For reference, here are a few I can see in my log when viewing the MouseCortex example dataset:\n``` Warning: Error in if: argument is of length zero\n\n[No stack trace available]\nWarning: Error in tapply: arguments must have same length\n\n[No stack trace available]\nWarning: Error in switch: EXPR must be a length 1 vector\n\n[No stack trace available] ```\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\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": "4459",
"date": "12 Mar 2019",
"name": "Brendan Innes",
"role": "Author Response",
"response": "Dr. Steinbaugh, Thank you for your detailed review of our manuscript, and your helpful suggestions for improving the software. We address your comments below. logGER pseudocount calculations. The explanation of differential expression implementation needs improvement (see Methods section), and the functions are not documented in the text. We’ve clarified our differential expression testing methods. The authors mention that they employ a small pseudocount calculation approach (e.g. using 10-99), which results in \"logGER values being very close to their true value\". More evidence is needed to support this claim, and I would like to see additional rationale as to why this is approach is preferable to other published methods that address dropout/zero count inflation (e.g. ZINB-WaVE, MAGIC). Log gene ratio (also referred to as log fold change) is a common way of reporting effect size for differential gene abundance tests. These calculations are independent of the statistical test, but play a role in the interpretation of results, and we expressed a concern that the traditional method of calculating these ratios (using a pseudocount equal to 1) was underrepresenting the true magnitude of effect size due to the small abundances common to droplet-based scRNAseq data. Using a very small pseudocount (e.g. 10-99) is not appropriate either. Instead, we recommend using a pseudocount representing the smallest possible “step” in the count-based data, set to the reciprocal of the number of cells in the data. We generated a simulated dataset that more clearly represents the problem, comparing the three pseudocount options we discuss, with results plotted in Figure 1. The analysis used to generate the data is available as an R script installed with scClustViz and found in the R library path under scClustViz/paperFigs/Fig1.R. Wilcoxon rank-sum test. Additionally, as mentioned in the text, the Wilcoxon rank-sum test isn't as powerful and doesn't control the false discovery rate as well as some other published methods. In particular, edgeR, DESeq2, limma, scde, and MAST are validated differential expression callers in use in other single-cell analysis packages that are viable alternatives to Wilcoxon. An option to use pre-calculated values inside the package would be a nice addition. Great point. We’ve now included a simple way of passing results from other differential expression callers into the workflow (replacing default values in the scClustViz data object). Reproducible code. While the interactivity provided by the Shiny interface is excellent and user-friendly for visualization, GUI-based data analysis is often difficult to reproduce. I would like to see scriptable, exported versions for all of the tools available in the package, so that a single-cell marker analysis can be run start-to-finish using scClustViz in a reproducible manner. This is an excellent idea. scClustViz now exports all functions used for both calculation of the data presented in the Shiny interface, and those used to generate the figures available in the interface. Take advantage of object-oriented programming. The authors mention in the text that scClustViz relies upon a \"small custom list\" of data generated using either the `readFromSeurat()` or `readFromManual()` functions, and that this approach \"reduces the risk of unexpected inputs\". I disagree with this statement, and recommend that the authors switch from an unstructured list to an S4 class object. Additionally, the paper doesn't describe what is stored in this list in detail. The S4 class system is documented in detail on the Bioconductor website, and is used by most popular single-cell RNA-seq analysis tools. S4 classes enable validity checks (see `validObject()`) and backwards compatibility support for legacy objects created with older versions of the package (see `updateObject()`). This was a welcome suggestion and forms the basis for our major update to scClustViz. As outlined in the updated manuscript, the setup step prior to running the Shiny interface now interfaces with existing S4 objects of the SingleCellExperiment and Seurat classes, and stores results of its calculations in a custom S4 class “sCVdata”. This should make loading analyses into scClustViz simpler for the user, there is less opportunity for unexpected data formats, and error messages are now clearer. The one aspect of this suggestion we did not implement was the backwards compatibility support. If we change the class structure in the future, we will do so, but since Drs. Andrews and Hemberg identified a statistical error in our previous version, we opted to not support backwards compatibility to prevent the propagation of that error into users results going forward. Add native SingleCellExperiment support. The authors provide a function for importing data from Seurat (`readFromSeurat()`), but currently don't provide a simple coercion method for the popular `SingleCellExperiment` class container. scClustViz now interfaces with the SingleCellExperiment class. Add unit testing. I noticed that the package doesn't currently have any code coverage with unit tests. I strongly recommend adding these checks against a minimal dataset. In particular, the testthat package (http://testthat.r-lib.org) is recommended. Testing has been added for all functions performing calculations. Inspired by a recent blog post, we have also added integration of Travis CI and Codecov as in issue in our github tracker to incorporate in the near future. Improve text labels. The gene marker labels on some plots are illegible because they are superimposed. The ggrepel package (https://cran.r-project.org/package=ggrepel) may help improve the legibility of plots with gene labels. This was not implemented in the first version of scClustViz because all plots where gene labels are present are clickable, allowing the user to disambiguate overlapping labels. However, this doesn’t help when users export their figures for the purpose of static presentations, so this was a valuable suggestion. We’ve now developed a function (spreadLabels2) for base R graphics inspired by ggrepel and spreadLabels that attempts to eliminate label overlap while keeping labels as close to their data points as possible. Unable to launch Shiny browser in a remote R session. The example MouseCortex Shiny data package runs correctly on machines where a browser instance can be launched. I tested this on multiple local environments (Linux, macOS, Windows) and on a remote RStudio server. However, it fails to launch on some remote high-performance computing (HPC) environments from the R command line. In some cases this can potentially be fixed with `runApp(launch.browser = FALSE)`, but it's unclear to me whether a user can easily run the `viewMouseCortex(\"e11\")` example in a remote R session without RStudio. This may be an edge case, but many R users work primarily on remote environments, so it's worth mentioning this potential limitation in the text. An ellipsis argument to pass options to runApp is now included in the runShiny function (and wrapper functions calling it for published datasets), which may help the user troubleshoot in computing environments that don’t easily run Shiny apps. This is now mentioned in the system requirements section in the manuscript. Shiny console warnings. A number of warnings appear in my R console when running the example Shiny datasets. This seems to be a side-effect of Shiny’s real-time evaluation of functions. There are times when a function returns an error because an input it depends on is being calculated by another function. Shiny returns this as a warning, but once the calculation is complete, the dependent function can run error-free, so these warnings aren’t pertinent. This may be addressed by adding some internal checks to ensure dependent functions run only when their dependencies have been satisfied. We have added this to our github issue tracker to address in the future. Thank you again for all these valuable suggestions, which have improved the robustness and user-friendliness of scClustViz."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1522
|
https://f1000research.com/articles/8-279/v1
|
12 Mar 19
|
{
"type": "Research Note",
"title": "Mutations in Caenorhabditis elegans actin, which are equivalent to human cardiomyopathy mutations, cause abnormal actin aggregation in nematode striated muscle",
"authors": [
"Yuriko Hayashi",
"Kanako Ono",
"Shoichiro Ono",
"Yuriko Hayashi",
"Kanako Ono"
],
"abstract": "Actin is a central component of muscle contractile apparatuses, and a number of actin mutations cause diseases in skeletal, cardiac, and smooth muscles. However, many pathogenic actin mutations have not been characterized at cell biological and physiological levels. In this study, we tested whether the nematode Caenorhabditis elegans could be used to characterize properties of actin mutants in muscle cells in vivo. Two representative actin mutations, E99K and P164A, which cause hypertrophic cardiomyopathy in humans, are introduced in a muscle-specific C. elegans actin ACT-4 as E100K and P165A, respectively. When green fluorescent protein-tagged wild-type ACT-4 (GFP-ACT-4), is transgenically expressed in muscle at low levels as compared with endogenous actin, it is incorporated into sarcomeres without disturbing normal structures. GFP-ACT-4 variants with E100K and P165A are incorporated into sarcomeres, but also accumulated in abnormal aggregates, which have not been reported for equivalent actin mutations in previous studies. Muscle contractility, as determined by worm motility, is not apparently affected by expression of ACT-4 mutants. Our results suggest that C. elegans muscle is a useful model system to characterize abnormalities caused by actin mutations.",
"keywords": [
"actin",
"aggregates",
"cardiomyopathy",
"sarcomere",
"myofibrils"
],
"content": "Introduction\n\nActin is an essential component of the cytoskeleton in both muscle and non-muscle cells. A number of mutations in the six human actin genes cause a wide range of diseases in various tissues (Despond & Dawson, 2018; North & Laing, 2008; Rubenstein & Wen, 2014). In muscles, actin, together with myosin, generates contractile forces, and therefore, alterations in contractile and/or structural properties of actin can cause muscle malfunction. Mutations in skeletal muscle α-actin (ACTA1) cause congenital myopathies, including nemaline myopathy and intranuclear rod myopathy, in which skeletal muscle exhibits abnormal accumulations of sarcomeric components (Clarkson et al., 2004; Laing et al., 2009; North & Laing, 2008; Ono, 2010). Many of these cytoskeletal abnormalities can be reproduced by expression of mutant actins in cultured non-muscle or muscle cells (Bathe et al., 2007; Costa et al., 2004; Domazetovska et al., 2007; Vandamme et al., 2009a; Vandamme et al., 2009b) or in transgenic mice (Lindqvist et al., 2013; Ravenscroft et al., 2011). By contrast, mutations in cardiac α-actin (ACTC1) cause hypertrophic and dilated cardiomyopathies (Mogensen et al., 1999; Olson et al., 1998). Biochemical studies indicate that these cardiomyopathy mutations of actin alter its properties to generate contractile forces (Despond & Dawson, 2018). However, abnormalities in sarcomeric or cytoskeletal structures have not been reported when the mutant actins are expressed in cultured cells (Muller et al., 2012; Vang et al., 2005) or transgenic mice (Song et al., 2010; Song et al., 2011).\n\nIn this study, we used the nematode Caenorhabditis elegans as a model to examine effects of cardiomyopathy mutations in actin. The body wall muscle of C. elegans is obliquely striated muscle with a number of functional and structural similarities to vertebrate striated muscles (Ono, 2014). Four actin genes are expressed in C. elegans muscle (Files et al., 1983; Stone & Shaw, 1993), and they are 95% identical to human cardiac and skeletal muscle α-actins (Ono & Pruyne, 2012). Since all known residues that are mutated in human cardiomyopathies are conserved in C. elegans actins, we selected two representative hypertrophic cardiomyopathy mutations and tested whether these pathogenic mutations perturb the properties of actin in C. elegans muscle in vivo. We found that the mutant actins were incorporated into sarcomeres and also accumulated in abnormal aggregates, suggesting that C. elegans muscle is a unique model system to characterize pathogenic actin mutations.\n\n\nMethods\n\nWorms were cultured following standard methods (Stiernagle, 2006). Wild-type C. elegans strain N2 was obtained from the Caenorhabditis Genetics Center (Minneapolis, MN) and used in this study.\n\nAn expression vector for GFP-ACT-4(wild-type: WT) was constructed by inserting ACT-4 cDNA at the EcoRI-NheI sites of pPD118.20 (provided by Andrew Fire, Stanford University) in-frame with the 3’-end of the GFP coding sequence. Briefly, first-strand cDNAs were reverse-transcribed from total RNAs from the N2 strain using oligo-dT by a Maxima H- First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The ACT-4 cDNA with added EcoRI and NheI sites in the primer sequences was amplified from the pool of cDNAs by polymerase chain reaction using Pfu DNA polymerase (Agilent Technologies), digested with EcoRI and NheI, and ligated with pPD118.20 that had been cut with EcoRI and NheI. Expression vectors for GFP-ACT-4(E100K) and GFP-ACT-4(P165A) were generated by site-directed mutagenesis using a QuickChange Site-directed Mutagenesis Kit (Agilenet Technologies). Sequences of the inserts were verified by DNA sequencing. Transgenic nematodes were generated by microinjection of DNA vectors into the distal gonads as described previously (Mohri et al., 2006). Transgenic worms were selected by expression of GFP as observed by fluorescence microscopy, and the transgenes were maintained as extrachromosomal arrays. Strains used in this study are ON16, ktEx6[Pmyo-3::GFP::ACT-4(WT)]; ON209, ktEx154[Pmyo-3::GFP::ACT-4(E100K)]; and ON212, ktEx157[Pmyo-3::GFP::ACT-4(P165A)].\n\nTen adult worms were suspended in 15 µl SDS lysis buffer (2% SDS, 80 mM Tris-HCl, 5% β-mercaptoethanol, 15% glycerol, 0.05% bromophenol blue, pH 6.8), heated at 97°C for 2 min, homogenized briefly by sonication, heated again at 97°C for 2 min, and subjected to SDS-PAGE (12% acrylamide gel). The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membrane was blocked in 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and incubated for 1 hr with anti-actin mouse monoclonal antibody (C4, MB Biomedicals, catalog # 08691001; RRID:AB_2335127) at a 1:3000 dilution. The membrane was washed with PBS-T, treated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution) (Pierce/Thermo Scientific, catalog #31430) for 1 hr, and washed with PBS-T. The reactivity was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and exposure to X-ray films. Finally, the membrane was stained with 0.1% Coomassie Brilliant Blue R-250 (National Diagnostics) in 50% methanol and destained in a solution containing 10% acetic acid and 50% methanol to visualize total proteins (Welinder & Ekblad, 2011). The blots were scanned by an Epson Perfection V700 scanner at 300 dpi., and band intensity was quantified using ImageJ 1.47v.\n\nWorm motility was determined by counting swinging motions of worms for 30 seconds in M9 buffer as described (Epstein & Thomson, 1974; Ono et al., 1999).\n\nFixation and staining of worms with rhodamine-phalloidin were performed as described previously (Ono, 2001). GFP was observed by its own fluorescence. Specimens were observed by epifluorescence using a Nikon Eclipse TE2000 inverted microscope with a CFI Plan Fluor ELWD 40x (Dry; NA 0.60) objective. Images were captured by a SPOT RT monochrome CCD camera (Diagnostic Instruments) and processed by IPLab 4.0 imaging software (BD Biosciences) and Adobe Photoshop CS3.\n\nMolecular graphics in Figure 1A were generated using PyMol 2.1.0 (Schrödinger), and texts added using Adobe Photoshop CS3.\n\n(A) Structure of porcine cardiac α-actin (Risi et al., 2017) (Protein Data Bank accession number 5N0J) and C. elegans ACT-1 (Vorobiev et al., 2003) (Protein Data Bank accession number 1D4X). ACT-1 is also expressed in C. elegans muscle and differs from ACT-4 by only one amino acid. Mutated residues (E99 and P164 in porcine cardiac α-actin; E100 and P165 in C. elegans ACT-1) are shown in yellow. Actin subdomains 1-4 are labeled as SD1-SD4. Molecular graphics were generated by PyMol (Schrödinger). (B) Western blot analysis of expression levels of GFP-ACT-4 variants. Total worm lysates (10 worms each) from wild-type without a transgene (WT) or with transgenes expressing GFP-ACT-4 variants were analyzed by western blot using an anti-actin antibody (top). Coomassie Brilliant Blue staining of the membranes after chemiluminescence detection (bottom) was used to normalize protein loading. Positions of GFP-ACT-4 (70 kDa) and endogenous actin (42 kDa) are indicated on the right. Representative molecular weight markers in kDa are indicated on the left. For each transgenic strain, three independently prepared lysates (#1-3) were analyzed. (C) Quantitative analysis of the Western blot (Dataset 1). Band intensity in arbitrary units (AU) of GFP-ACT-4 was normalized to intensity of total protein staining by Coomassie Brilliant Blue (Welinder & Ekblad, 2011) and plotted on the graph. GFP-ACT-4(WT) and each GFP-ACT-4 mutant were compared on the same western blot, and no significant differences were found by Student’s t-test (ns) (n=3). (D-F) Localization patterns of GFP-ACT-4 (left) and F-actin (middle) in the C. elegans body wall muscle from worms expressing GFP-ACT-4(WT) (D), GFP-ACT-4(E100K) (E), and GFP-ACT-4(P165A) (F). Merged images (GFP in green and F-actin in red) are shown on the right. Bar, 20 µm. (G) Worm motility of each strain was examined by beating frequency (beats per 30 sec) (Dataset 2). The results were analyzed by one-way ANOVA (n=20): ns, not significant (p>0.05); *p<0.05 p<0.01; **p<0.01; and ***p<0.001. (H) Number of GFP-ACT-4 aggregates per cell was counted (Dataset 3). The results were analyzed by one-way ANOVA (n=30) and significant difference was found between the data for GFP-ACT-4(E100K) and GFP-ACT-4(P165A) (**p = 0.006).\n\nThe data used in Figure 1C were analyzed by Student’s t-test using SigmaPlot 14.0 (Systat Software, Inc.). The data used in Figure 1G were analyzed by one-way ANOVA with Turkey test using SigmaPlot 14.0. The data used in Figure 1H were analyzed by one-way ANOVA with pairwise multiple comparison using the Student-Newman-Keuls method using SigmaPlot 14.0.\n\n\nResults\n\nWe constructed an expression vector for GFP-tagged ACT-4, an actin isoform that is expressed in the body wall muscle (Stone & Shaw, 1993), under the control of the myo-3 promotor (Pmyo-3) (Okkema et al., 1993). The ACT-4 sequence was fused to the C-terminus of GFP with a 9-residue linker sequence (SPQALEFSS) to minimize the interference of actin function by GFP (Aizawa et al., 1997). We selected two missense mutations, E99K and P164A in human cardiac α-actin (Despond & Dawson, 2018; Olson et al., 2000), that dominantly cause hypertrophic cardiomyopathy. The E99K mutation weakens actin-myosin interaction (Bookwalter & Trybus, 2006) and increases the critical concentration of actin (Mundia et al., 2012). In a transgenic mouse model, E99K increases calcium sensitivity of the thin filaments and causes abnormal heart functions (Song et al., 2011). In contrast, the effect of P164A mutation remains unclear. Although P164A causes alteration in protein folding in vitro (Vang et al., 2005), an equivalent mutation in yeast actin does not change its basic biochemical properties (Wong et al., 2001). C. elegans ACT-4 is 95% identical in amino acid sequence to human cardiac α-actin, and E99 and P164 are conserved as E100 and P165, respectively (Figure 1A). Therefore, we introduced E100K and P165A mutations in GFP-ACT-4 and examined their effects on the sarcomeric structures in C. elegans body wall muscle.\n\nWe established at least three independent transgenic strains for each of the transgenes, GFP-ACT-4(wild-type: WT), GFP-ACT-4(E100K), and GFP-ACT-4(P165A), and examined expression levels of GFP-ACT-4 variants by western blot. We selected one strain each, which expressed the GFP-ACT-4 variants at similar levels (Figure 1B, C) for further analysis. Western blot analysis using anti-actin antibody showed that all the GFP-ACT-4 variants were expressed at much lower levels than endogenous actin in total worm lysates (Figure 1B). The level of GFP-ACT-4(WT) was roughly estimated by densitometry to be lower than 10% of that of total endogenous actin, although strong saturated signals for endogenous actin made precise quantification difficult. Considering that body wall muscle is the major tissue expressing actin as a sarcomeric component, the expression level of GFP-ACT-4 should be still much less than that of endogenous actin within the body wall muscle cells. Raw uncropped western blots, alongside all other raw data, are available on Figshare (Hayashi et al., 2019).\n\nGFP-ACT-4(WT) was incorporated into sarcomeres in body wall muscle cells (Figure 1D). Staining of F-actin in fixed animals with rhodamine-phalloidin showed a nearly identical localization pattern to GFP-ACT-4(WT) (Figure 1D). Motility of the worms expressing GFP-ACT-4(WT) (81.5 ± 7.5 beats/30 sec, n = 20), as determined by beating frequency in liquid, was slightly slower than that of wild-type worms with no transgene (94.8 ± 11 beats/30 sec, n = 20), suggesting that GFP-ACT-4(WT) has a weak negative effect on contractility of the body wall muscle.\n\nBoth GFP-ACT-4(E100K) and GFP-ACT-4(P165A) were incorporated into sarcomeres but also formed spherical aggregates in the cytoplasm of the body wall muscle cells (Figure 1E, F). Staining with rhodamine-phalloidin showed that sarcomeric organization of actin filaments were somewhat disorganized by expression of GFP-ACT-4(E100K) (Figure 1E) but not GFP-ACT-4(P165A) (Figure 1F). However, motility of the worms expressing GFP-ACT-4(E100K) or GFP-ACT-4(P165A) was not significantly different from that of wild-type worms (Figure 1G), suggesting that these actin mutants did not disturb muscle contractility. These aggregates resemble F-actin aggresomes induced by inhibitors of actin dynamics (Lázaro-Diéguez et al., 2008). However, the aggregates of GFP-ACT-4(E100K) or GFP-ACT-4(P165A) were not recognized by rhodamine-phalloidin, a specific probe for F-actin (Figure 1E, F). In addition, we could not detect these aggregates by immunofluorescence using anti-actin monoclonal or polyclonal antibodies, even after attempts to expose antigens using guanidine hydrochloride (Peränen et al., 1993) or microwave (Shi et al., 1991), suggesting that the mutant forms of actin were present in an inclusion-body-like state and not readily accessible to the actin probes. Such aggregates were not detected in worms expressing GFP-ACT-4(WT) (Figure 1D, H), while variable numbers (0 - 36 per cell) of aggregates were found in worms expressing GFP-ACT-4(E100K) or GFP-ACT-4(P165A) (Figure 1E, F). In randomly selected worms (n = 30), GFP-ACT-4(E100K) (median = 9.5 aggregates per cell) induced significantly more aggregates than GFP-ACT-4(P165A) (median = 6.0 aggregates per cell) (Figure 1H). These aggregates were randomly located in the cytoplasm but not within the nucleus. Thus, we conclude that the missense mutations in ACT-4 induced the formation of abnormal cytoplasmic aggregates in muscle cells.\n\n\nDiscussion\n\nFormation of actin aggregates by E99K (E100K in worm) or P164A (P165A in worm) mutation in actin has not been reported in human patients or other experimental systems. When cardiac α-actin mutants (E99K and P164A) are expressed in COS-7 cells, these actin mutants are not incorporated in the non-muscle actin cytoskeleton with no detectable aggregate formation (Vang et al., 2005). When E99K cardiac α-actin is expressed in the mouse heart, the mutant actin is incorporated in the cardiac thin filaments and causes disarray of cardiomyocytes but with no detectable aggregate formation (Song et al., 2011). Thus, effects of these actin mutations appear to be dependent on cellular contexts. Formation of actin aggregates by these actin mutations might be specific to the nematode muscle. We also cannot exclude the possibility that the aggregate formation is artificially enhanced by the GFP tag. Nonetheless, we were able to detect actin aggregates because of the GFP tag and might not have been able to detect the aggregates if a fluorescent tag was absent. Abnormal protein aggregates have been reported in idiopathic dilated cardiomyopathy (Gianni et al., 2010; Subramanian et al., 2015) and cardiomyopathies caused by mutations in desmin (McLendon & Robbins, 2011; Sanbe et al., 2004), filamin (Brodehl et al., 2016; Reinstein et al., 2016; Valdes-Mas et al., 2014), α-B-crystallin (Vicart et al., 1998), or phospholamban (Te Rijdt et al., 2016). Whether transient or stable protein aggregates are formed in actin-linked cardiomyopathies remains to be investigated. Our observations suggest that the C. elegans might be a relevant model system to study certain types of cardiomyopathies.\n\n\nData availability\n\nFigshare: Raw data - Mutations in Caenorhabditis elegans actin, which are equivalent to human cardiomyopathy mutations, cause abnormal aggregation in nematode striated muscle. https://doi.org/10.6084/m9.figshare.c.4424546 (Hayashi et al., 2019).\n\nThis collection contains the following underlying data:\n\nUncropped western blots\n\nUnprocessed microscopy images\n\nDataset 1–3 (containing western blot quantification, and raw data for worm motility and number of aggregates per cell)\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\nThe C. elegans strain N2 was provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). This work was supported by a grant from the National Institutes of Health (AR048615) to S.O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank Becky Diebold for her technical assistance.\n\n\nReferences\n\nAizawa H, Sameshima M, Yahara I: A green fluorescent protein-actin fusion protein dominantly inhibits cytokinesis, cell spreading, and locomotion in Dictyostelium. Cell Struct Funct. 1997; 22(3): 335–45. PubMed Abstract | Publisher Full Text\n\nBathe FS, Rommelaere H, Machesky LM: Phenotypes of myopathy-related actin mutants in differentiated C2C12 myotubes. BMC Cell Biol. 2007; 8: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBookwalter CS, Trybus KM: Functional consequences of a mutation in an expressed human alpha-cardiac actin at a site implicated in familial hypertrophic cardiomyopathy. J Biol Chem. 2006; 281(24): 16777–84. PubMed Abstract | Publisher Full Text\n\nBrodehl A, Ferrier RA, Hamilton SJ, et al.: Mutations in FLNC are Associated with Familial Restrictive Cardiomyopathy. Hum Mutat. 2016; 37(3): 269–279. PubMed Abstract | Publisher Full Text\n\nClarkson E, Costa CF, Machesky LM: Congenital myopathies: diseases of the actin cytoskeleton. J Pathol. 2004; 204(4): 407–17. PubMed Abstract | Publisher Full Text\n\nCosta CF, Rommelaere H, Waterschoot D, et al.: Myopathy mutations in alpha-skeletal-muscle actin cause a range of molecular defects. J Cell Sci. 2004; 117(Pt 15): 3367–77. PubMed Abstract | Publisher Full Text\n\nDespond EA, Dawson JF: Classifying Cardiac Actin Mutations Associated with Hypertrophic Cardiomyopathy. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nHayashi Y, Ono K, Ono S: Raw data - Mutations in Caenorhabditis elegans actin, which are equivalent to human cardiomyopathy mutations, cause abnormal aggregation in nematode striated muscle. figshare. Collection. 2019.\n\nLaing NG, Dye DE, Wallgren-Pettersson C, et al.: Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1). Hum Mutat. 2009; 30(9): 1267–1277. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLázaro-Diéguez F, Aguado C, Mato E, et al.: Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide. J Cell Sci. 2008; 121(Pt 9): 1415–1425. PubMed Abstract | Publisher Full Text\n\nLindqvist J, Cheng AJ, Renaud G, et al.: Distinct underlying mechanisms of limb and respiratory muscle fiber weaknesses in nemaline myopathy. J Neuropathol Exp Neurol. 2013; 72(6): 472–481. PubMed Abstract | Publisher Full Text\n\nMcLendon PM, Robbins J: Desmin-related cardiomyopathy: an unfolding story. Am J Physiol Heart Circ Physiol. 2011; 301(4): H1220–1228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMogensen J, Klausen IC, Pedersen AK, et al.: Alpha-cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy. J Clin Invest. 1999; 103(10): R39–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohri K, Ono K, Yu R, et al.: Enhancement of actin-depolymerizing factor/cofilin-dependent actin disassembly by actin-interacting protein 1 is required for organized actin filament assembly in the Caenorhabditis elegans body wall muscle. Mol Biol Cell. 2006; 17(5): 2190–2199. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuller M, Mazur AJ, Behrmann E, et al.: Functional characterization of the human a-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy. Cell Mol Life Sci. 2012; 69(20): 3457–3479. PubMed Abstract | Publisher Full Text\n\nMundia MM, Demers RW, Chow ML, et al.: Subdomain location of mutations in cardiac actin correlate with type of functional change. PLoS One. 2012; 7(5): e36821. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNorth KN, Laing NG: Skeletal muscle alpha-actin diseases. Adv Exp Med Biol. 2008; 642: 15–27. PubMed Abstract | Publisher Full Text\n\nOkkema PG, Harrison SW, Plunger V, et al.: Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics. 1993; 135(2): 385–404. PubMed Abstract | Free Full Text\n\nOlson TM, Doan TP, Kishimoto NY, et al.: Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2000; 32(9): 1687–1694. PubMed Abstract | Publisher Full Text\n\nOlson TM, Michels VV, Thibodeau SN, et al.: Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science. 1998; 280(5364): 750–752. PubMed Abstract | Publisher Full Text\n\nOno S: The Caenorhabditis elegans unc-78 gene encodes a homologue of actin-interacting protein 1 required for organized assembly of muscle actin filaments. J Cell Biol. 2001; 152(6): 1313–1319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOno S: Dynamic regulation of sarcomeric actin filaments in striated muscle. Cytoskeleton (Hoboken). 2010; 67(11): 677–692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOno S: Regulation of structure and function of sarcomeric actin filaments in striated muscle of the nematode Caenorhabditis elegans. Anat Rec (Hoboken). 2014; 297(9): 1548–1559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOno S, Baillie DL, Benian GM: UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle. J Cell Biol. 1999; 145(3): 491–502. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOno S, Pruyne D: Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans. Methods. 2012; 56(1): 11–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeränen J, Rikkonen M, Kääriäinen L: A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J Histochem Cytochem. 1993; 41(3): 447–454. PubMed Abstract | Publisher Full Text\n\nRavenscroft G, Jackaman C, Sewry CA, et al.: Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression. PLoS One. 2011; 6(12): e28699. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReinstein E, Gutierrez-Fernandez A, Tzur S, et al.: Congenital dilated cardiomyopathy caused by biallelic mutations in Filamin C. Eur J Hum Genet. 2016; 24(12): 1792–1796. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRisi C, Eisner J, Belknap B, et al.: Ca2+-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy. Proc Natl Acad Sci U S A. 2017; 114(26): 6782–6787. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRubenstein PA, Wen KK: Insights into the effects of disease-causing mutations in human actins. Cytoskeleton (Hoboken). 2014; 71(4): 211–229. PubMed Abstract | Publisher Full Text\n\nSanbe A, Osinska H, Saffitz JE, et al.: Desmin-related cardiomyopathy in transgenic mice: a cardiac amyloidosis. Proc Natl Acad Sci U S A. 2004; 101(27): 10132–10136. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShi SR, Key ME, Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39(6): 741–748. PubMed Abstract | Publisher Full Text\n\nSong W, Dyer E, Stuckey D, et al.: Investigation of a transgenic mouse model of familial dilated cardiomyopathy. J Mol Cell Cardiol. 2010; 49(3): 380–389. PubMed Abstract | Publisher Full Text\n\nSong W, Dyer E, Stuckey DJ, et al.: Molecular mechanism of the E99K mutation in cardiac actin (ACTC Gene) that causes apical hypertrophy in man and mouse. J Biol Chem. 2011; 286(31): 27582–27593. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStiernagle T: Maintenance of C. elegans. WormBook. 2006; 1–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStone S, Shaw JE: A Caenorhabditis elegans act-4::lacZ fusion: use as a transformation marker and analysis of tissue-specific expression. Gene. 1993; 131(2): 167–173. PubMed Abstract | Publisher Full Text\n\nSubramanian K, Gianni D, Balla C, et al.: Cofilin-2 phosphorylation and sequestration in myocardial aggregates: novel pathogenetic mechanisms for idiopathic dilated cardiomyopathy. J Am Coll Cardiol. 2015; 65(12): 1199–1214. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTe Rijdt WP, van Tintelen JP, Vink A, et al.: Phospholamban p.Arg14del cardiomyopathy is characterized by phospholamban aggregates, aggresomes, and autophagic degradation. Histopathology. 2016; 69(4): 542–50. PubMed Abstract | Publisher Full Text\n\nValdes-Mas R, Gutierrez-Fernandez A, Gomez J, et al.: Mutations in filamin C cause a new form of familial hypertrophic cardiomyopathy. Nat Commun. 2014; 5: 5326. PubMed Abstract | Publisher Full Text\n\nVandamme D, Lambert E, Waterschoot D, et al.: Phenotypes induced by NM causing alpha-skeletal muscle actin mutants in fibroblasts, Sol 8 myoblasts and myotubes. BMC Res Notes. 2009a; 2: 40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVandamme D, Rommelaere H, Lambert E, et al.: alpha-Skeletal muscle actin mutants causing different congenital myopathies induce similar cytoskeletal defects in cell line cultures. Cell Motil Cytoskeleton. 2009b; 66(4): 179–92. PubMed Abstract | Publisher Full Text\n\nVang S, Corydon TJ, Borglum AD, et al.: Actin mutations in hypertrophic and dilated cardiomyopathy cause inefficient protein folding and perturbed filament formation. FEBS J. 2005; 272(8): 2037–49. PubMed Abstract | Publisher Full Text\n\nVicart P, Caron A, Guicheney P, et al.: A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet. 1998; 20(1): 92–95. PubMed Abstract | Publisher Full Text\n\nVorobiev S, Strokopytov B, Drubin DG, et al.: The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism. Proc Natl Acad Sci U S A. 2003; 100(10): 5760–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWelinder C, Ekblad L: Coomassie staining as loading control in Western blot analysis. J Proteome Res. 2011; 10(3): 1416–9. PubMed Abstract | Publisher Full Text\n\nWong WW, Doyle TC, Cheung P, et al.: Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations. J Muscle Res Cell Motil. 2001; 22(8): 665–74. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "45623",
"date": "20 Mar 2019",
"name": "Michael Glotzer",
"expertise": [
"Reviewer Expertise Cell biology",
"RhoA regulation."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 short note describes the ability of two cardiomyopathy associated actin alleles to incorporate into body wall myosin filaments in C. elegans. The authors find that whereas transgenic, tagged wild-type control actin ACT-4 incorporates well into the muscle, the two mutant variants do so with lower efficiency and also accumulate as non-filamentous aggregates.\n\nThis report is technically sound and the conclusions are appropriately drawn from the evidence provided. In future studies it would be of interest to incorporate these mutations into the genome so that the relative dosage of the mutant protein would better reflect that seen in cardiomyopathy patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "45621",
"date": "20 Mar 2019",
"name": "Amy Shaub Maddox",
"expertise": [
"Reviewer Expertise contractility",
"cell division",
"development",
"C. elegans",
"cytoskeleton"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSeveral human muscle pathologies result from mutations in actin genes. To understand the cellular effects of such alleles, Hayashi and colleagues introduced the relevant mutations from human diseases into a muscle-enriched actin gene in C. elegans, an animal with a simple body plan and behavior. They found that mutant actins localize to muscle fibers but also form aggregates. At least at the low expression level examined, mutant actins did not cause animal movement defects.\n\nThis is a clear manuscript with interesting potential. However, before it is suitable to be indexed, several points need to be addressed.\n\nMajor point:\nThe authors should comment on the significant decrease in body bends exhibited by worms expressing GFP-ACT-4(WT). Could this relate to lower expression of this transgene – did this phenotype scale with expression level among the various isolates? Could it relate to the ability of the (lowly expressed) transgene to incorporate into muscle (measure GFP/F-actin staining ratio)? Arguably the relevant comparisons are between mutant and WT transgene, and there was a statistically significant difference in body bends for both these comparisons, though this was an increase. Are there known perturbations that lead to more beats per second than controls?\nMinor points:\nThe qualitative assay for aggregate formation is unlikely to have the dynamic range necessary to discern among conditions of different severity. Doing so would probably require a quantitative assay such as measuring the range of fluorescence intensity throughout regions of the image, or the proportion of the image occupied by pixels over some intensity threshold.\n\nThe E100K mutation appears to have severely disrupted muscle fiber organization – the 9-11 prominent F-actin bundles apparent in WT cells are replaced with many more partially-overlapping wispy structures. This effect should be related to the disarray noted by Song et al., 2011 (Discussion 3rd sentence). The degree of subcellular morphological perturbation could be quantified with image analysis. It is interesting that despite this dramatic change, animal mobility is normal.\n\nIt would be nice to note somewhere the origin of 3’ UTR (act-4).\n\n“Turkey” should be “Tukey”; “Promotor” should be “promoter”.\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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "45625",
"date": "01 Apr 2019",
"name": "James R. Bamburg",
"expertise": [
"Reviewer Expertise Cytoskeletal dynamics",
"especially actin 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 specific aim of this Research Note is to demonstrate that C. elegans can be used as a useful in vivo model to study mutations in actin associated with human myopathies, specifically in this case, cardiomyopathies. The authors have thus expressed only in the muscle tissue of C. elegans GFP-chimeras of wild type (WT) actin and two actin mutants (E100K and P165A) equivalent to human actin mutants E99K and P164A both of which are known to cause hypertrophic cardiomyopathy in humans. The authors then study by fluorescence microscopy the localization of the actin-GFP in the body wall muscle, the sarcomeric organization using fluorescent phalloidin to visualize F-actin, and worm motility. Their main findings are that all of the GFP-actin proteins incorporate into the sarcomere with only the E100K mutant causing sarcomeric disarray and that both mutant actins, but not WT, form GFP-positive aggregates that do not stain with fluorescent phalloidin or with an array of actin antibodies even after implementing antigen retrieval protocols. From that, the authors suggest the relevance of C. elegans to study actin-related cardiomyopathies. However, actin aggregates have not been detected in mouse heart from animals expressing the E99K mutant nor have aggregates been reported in human cardiomyopathies from subjects with either of the mutations used here. Thus, we are left to wonder, as indeed the authors have themselves, if the aggregates are specific to nematode muscle, if aggregate formation by the mutant actins is enhanced by the presence of the GFP-tag, and what other molecules are found in the aggregates that might provide a clue to their composition.\n\nAs the authors have clearly written in their introduction, there is a need for further characterization of pathogenic mutations and expanding the available organisms to study specific diseases is always welcomed, as every model comes with their benefits and caveats. In spite of this, and the fact that the manuscript is well written and self-critical, there are some limitations in the studies presented that prevent us from approving its acceptance at this stage.\n\nAlthough it is understandable why the authors choose to express the fluorescently tagged version of the actin mutants, expression of the untagged versions should also have been examined to see if the sarcomere disruption by the E100K occurs in the absence of the GFP tag. It is recognized that under these conditions it would not be possible to obtain ratios of the expressed transgene with the endogenous protein, but with a fluorescent protein expressed from a different promoter, it should be possible to obtain worms with the untagged actin mutants to determine its effect on sarcomere disruption. As recognized by the authors, the GFP tag could increase the propensity of the construct to oligomerize/aggregate. Whether this could be driven by GFP aggregation on its own is not clear. It has been reported that GFP linked via its C-terminus to a 16 amino acid \"degron\" peptide is not effectively degraded but forms aggregates very similar to the ones observed here when expressed in the body wall muscle of C. elegans (Link et al., 20061). Since the aggregates observed in the current study have not been found to contain actin, one has to wonder if some cleavage has occurred to give rise to the same type of GFP-aggregates observed by Link et al. Why these do not form with the GFP-WT actin is unknown, but might reflect its greater stability to proteolysis. It would be useful to know if the number of aggregates correlate with total GFP intensity in a muscle cell.\n\nAlso, the absolute level of the expressed proteins relative to endogenous actin is problematic. As recognized by the authors themselves, their western blot was made with whole organism lysate and the signal of endogenous actin was near saturation. Although the contribution of actin from regions outside the body wall muscle is thought by the author's to be low, if it were possible to isolate only the body wall muscle, the values would be more meaningful. For the results to mimic human disease pathology, the authors need to try and reproduce the ratio between mutant and endogenous actin. As the authors demonstrate decreased worm motility when overexpressing total WT GFP-actin, it is clear that the expression of exogenous actin must be kept low to avoid potential toxic effects, although in this case it is unclear if the decreased motility arises from the presence of the GFP-tag.\n\nWhile trying to explain the difference observed in their worm model with results from a previous paper (Song et al., 2011) in which no aggregates were found with the E99K mutant, the first explanation given is “Thus, effects of these actin mutations appear to be dependent on cellular context.” Such an explanation actually negates the main purpose of the paper. We would thus recommend to first try to resolve the issues that have been identified in this review, because, as interesting as the starting idea is, there are some flaws that render the results difficult to interpret.\n\nMinor questions:\nHow do the authors explain the lack of correlation between sarcomeric disarray and worm motility?\n\nDoes the expression of any of the constructs lead to an increased mortality or to a reduced lifespan?\n\nThe nature of the aggregates that contain the GFP should be explored in more detail. Are they phase separated protein clusters such as one finds in stress granules and which can be identified by rapid diffusion within the structure if photobleached, or are these structured solid aggregates that might be able to be isolated, such as those from human from idiopathic dilated cardiomyopathy? The reason for their lack of actin staining needs to be identified since right now the title of the manuscript that states \"human cardiomyopathy mutations, cause abnormal actin aggregation\" cannot be used since no actin in the aggregates has been detected.\n\nIs the work 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? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "45622",
"date": "02 Apr 2019",
"name": "Christophe Ampe",
"expertise": [
"Reviewer Expertise actin cytoskeleton"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports describe the expression of two mutant actins, associated with cardiac myopathy, in the body wall muscle of the model organism Caenorhabditis elegans. Expression of these mutants as N-terminally tagged GFP-variants at low to moderate expression levels (relative to the endogenous actin) disturbs the fine structure of body wall muscle and dot-like patterns of GFP-mutant protein are evident in contrast to the experiments with GFP-WT-ACT-4 (see also below).\nOn the whole the experiments are well conducted and properly documented. I have some suggestions regarding interpretation of the results which the authors should look into but this does not require further experimentation (i.e. only textual revision).\nExpression of WT GFP-ACT4 results in a negative effect on worm motility. This is mentioned by the authors in the results (Subcellular localization of GFP-ACT-4 variants and motility of worms). Such an observation is not unprecedented in mammalian cells and is basically due to the GFP—moiety possibly interfering with cellular functions. This was further investigated in Agbulut et al. (20071) and it was shown that GFP on its own interferes with myosin-based contractility which is potentially also the case here. In this respect should the authors not interpret the worm motility relative to the GFP-ACT-4 control rather than relative to WT worms? I realise it is more difficult to reconcile disturbed muscle structure (D-F) and the expected dominant-negative phenotype with increased worm motility (G) but the authors indicate the motility of the worms is significantly different between mutant and WT-GFP actin variants and this should be commented on in the results or the discussion section.\n\nIn the discussion the authors interpret their results solely in terms of cardiomyopathy based on the origin of the mutations. However the experimental system used is body wall muscle and interestingly the cellular phenotype is more reminiscent of nemaline myopathy (dotted patterns and irregular fibers). Functional differences between alpha-cardiac actin and alpha-skeletal muscle actin in human are unclear because the proteins are highly similar (only 4 amino acids different and these changes are even conservative) and differential phenotypes of disease mutants likely result from differential expression in the respective tissues (in agreement with the authors’ statement: “Thus, effects of these actin mutations appear to be dependent on cellular contexts”). These ACTC1 mutants do not cause aggregates in COS cells (Vang et al., 2005, mentioned in the discussion), therefore I suggest to also make the parallel with nemaline myopathy where dot-like patterns in cellular context of (fused) myoblasts are frequently documented.\n\nThe dots appear to be phalloidin-negative as judged by visual inspection (Figure 1D and 1E); perhaps this should be mentioned in the main text.\n\nOther minor points:\nIntroduction:\nIn the two sentences: “Many of these cytoskeletal abnormalities can be reproduced by expression of mutant actins in cultured non-muscle or muscle cells (Bathe et al., 2007; Costa et al., 2004; Domazetovska et al., 2007; Vandamme et al., 2009a; Vandamme et al., 2009b) or in transgenic mice (Lindqvist et al., 2013; Ravenscroft et al., 2011). By contrast, mutations in cardiac α-actin (ACTC1) cause hypertrophic and dilated cardiomyopathies (Mogensen et al., 1999; Olson et al., 1998).” a contrast is mentioned although the two items do not contrast each other.\nMethods:\nCorrect: Agilent. Figure 1A: upon printing (and in the 100% view option of PDF) these figures are difficult to interpret. I suggest to make them larger by rearranging this figure (quite some white space left and right).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": []
}
] | 1
|
https://f1000research.com/articles/8-279
|
https://f1000research.com/articles/7-1822/v1
|
20 Nov 18
|
{
"type": "Research Article",
"title": "Novel multiplex assay for profiling influenza antibodies in breast milk and serum of mother-infant pairs",
"authors": [
"Kirsi M. Järvinen",
"Jiong Wang",
"Antti E. Seppo",
"Martin Zand",
"Jiong Wang",
"Antti E. Seppo",
"Martin Zand"
],
"abstract": "Background: During early life, systemic protection to influenza is passively provided by transplacental transfer of IgG antibodies and oral and gastrointestinal mucosal protection via breast milk (BM) containing predominantly IgA. Immune imprinting, influenced by initial exposure of the infant immune system to influenza, has recently been recognized as an important determinant of future influenza immune responses. Methods: We utilized stored frozen BM from a prospective birth cohort to assess immune factors in human milk. The earliest available BM and a paired, timed serum sample was assessed from each of 7 mothers. Paired infant serum samples were assayed at up to three time points during the first 12 months of life, one prior to assumed disappearance of transplacentally transferred IgG, and one after. We utilized a novel multiplex assay to assess mothers’ and infants’ IgG and IgA antibodies in serum to a panel of 30 individual recombinant hemagglutinin (rHA) proteins of influenza virus strains and chimeric rHAs. We also characterized IgA and IgG antibody levels in breast milk providing mucosal protection. Results: Our pilot results, analyzing a small number of samples demonstrate the feasibility of this method for studying paired maternal-infant IgG and IgA anti-influenza immunity patterns. Unlike IgG antibodies, breast milk influenza virus HA-specific IgA antibody levels and patterns were mostly discordant compared to serum. As expected, there was a steady decay of infant influenza specific IgG levels by 6 to 8 months of age, which was not, however, comparable in all infants. In contrast, most of the infants showed an increase in IgA responses throughout the first year of life Conclusions: This new analytical method can be applied in a larger study to understand the impact of maternal imprinting on influenza immunity.",
"keywords": [
"breast milk",
"influenza",
"infants",
"protection",
"transplacental",
"antibody"
],
"content": "Introduction\n\nThe immune system in neonates and young infants is initially immature, without adequate protection against infections. The dogma is that during infancy, systemic protection is passively provided by transplacental transfer of IgG antibodies and oral and gastrointestinal mucosal protection via breast milk (BM) containing predominantly IgA and some IgG. However, the clinical protection provided by maternal influenza immunization or exposure varies by season and the corresponding match against circulating influenza strains. Therefore, maternal influenza exposure, whether through immunization or natural infection, provides maternal protection and has the potential to imprint the infant immune system and significantly impact infant morbidity and mortality, as recently comprehensively reviewed1.\n\nMaternal influenza immunization prior to or during pregnancy provides clinical protection with 70% efficiency in the infant2–5. Only one study has described IgA antibody levels in human milk to influenza6. Their results suggested that vaccination using a single strain of influenza A (A/New Caledonia/20/1999, H1N1) induced significantly higher IgA antibody levels than those seen in non-vaccinated, and those antibodies were positively correlated with viral neutralization. In addition, higher rates of exclusive breastfeeding in the first 6 months of life were associated with protection against febrile respiratory illness in the infants of vaccinated mothers, suggesting mucosal protection against influenza by BM antibodies. However, there is little data on how influenza antibody levels, or strain-specific antibody profiles, vary between mother’s serum, BM and infant serum. Data on the kinetic changes in anti-influenza IgG profiles between mother-infant pairs are also largely lacking.\n\nIn this pilot study utilizing a novel multiplex assay, we assessed infant immunity to various influenza strains reflecting maternal anti-influenza IgG levels and profiles in serum, as well as characterized IgA antibody responses in breast milk, which are distinct and reflect mucosal immunity. This new analytical method was applied to a small number of samples showing feasibility and patterns suggestive that a larger study needs to be done to understand the impact of maternal imprinting on influenza immunity.\n\n\nMethods\n\nWe utilized stored frozen human foremilk collected in the morning, from a prospective birth cohort recruited in 1997–2001 in Finland to assess immune factors in human milk7. The earliest available BM and a paired, timed serum sample was assessed from each of 7 mothers; ranging from 3 days to 2 months post-partum. Paired infant serum samples were assayed at up to three time points during the first 12 months of life, one prior to assumed disappearance of transplacentally transferred IgG, and one after. The samples collected in this cohort have been stored at -80°F with no recurrent freeze-thaw cycles. Aliquots have successfully been used in the past for measurement of serum and BM antibody levels with good antibody levels detected both for IgG and IgA7. These mothers were unvaccinated, as guidelines for maternal influenza immunization were not in place at the time samples were collected. None of the infants had been vaccinated to influenza. Clinical characteristics and timing of samples available are shown in Table 1. The study was approved by the institutional review boards of the Helsinki University Central Hospital, the City of Helsinki, and the University of Rochester Medical Center, Rochester, NY.\n\nnk, not known; BF, breastfeeding.\n\nWe have developed a multiplex assay (mPlex-Flu) that simultaneously measures absolute antibody concentrations against up to 50 influenza strains8. The mPlex-Flu assay has several advantages over the traditional hemagglutinin inhibition (HAI) titer assay: a linear readout over 4 logs, and high sensitivity. Our previous studies also showed that mPlex-Flu assay results highly consistent with the results from HAI and ELISA assays in human pre-and post-influenza vaccine study8. In the present study, a panel of 30 individual recombinant hemagglutinin (rHA) proteins of influenza virus strains and chimeric rHAs were used (see Table 2). This allowed us to estimate the specific anti-influenza IgG and IgA levels against H1, H2, H3 and Flu B seasonal influenza strains, as well as HA stalk specific antibodies using chimeric rHA (i.e. head from one influenza strain and stalk from another strain), cH5/1 and cH9/1 specific for group 1 (i.e. H1, H2, H5, H6) and cH4/7 and cH5/3 for group 2 (i.e. H3, H7) influenza strains, as previously described8.\n\nSeasonal Vaccine strains in Bold\n\nIn the present study, samples of maternal serum (diluted 1:500 for IgA and 1:5000 for IgG), infant serum (1:10) and BM (1:10) were diluted using PBS and incubated with rHA coupled Luminex beads (Luminex Corp, Austin, TX). IgG or IgA binding was detected with anti-human IgG or IgA specific secondary antibodies (SouthernBiotech, AL, Cat No 2040-09, 2050-09, respectively). Median fluorescence intensities (MFI) were measured using a MAGPIX multiplex reader (Luminex Co.,TX) and converted into absolute IgG concentrations (ng/mL) using a IgG standard curve generated with a human standard serum, which is a mixture of sera from four subjects containing high levels of anti-influenza HA IgG and IgA against multiple influenza strains8. Since serum IgA is monomeric, while BM secreted IgA (SIgA) is dimeric9, the standard curves of BM SIgA against influenza viruses are very different from that of serum standard curves generated from our human standard serum sample. We thus report the magnitude of BM IgA anti-influenza HA antibody levels in MFI units. For consistency, and to allow direct comparison, we also report IgG levels in MFI units. All data were analysis by Prism 7, and heatmap figures were generated by Mathematic 11.2.\n\n\nResults\n\nThis new analytical method was applied to a small number of samples showing feasibility and several interesting patterns. BM had a pattern of IgG reactivity very similar to maternal serum. Also, the levels and strain reactivity patterns of anti-influenza IgG in mother’s serum matched that of her infant, suggesting a robust transplacental transfer of antibodies. As expected, there was a steady decay of infant influenza specific IgG levels by 6 to 8 months of age (Figure 1A). This decay was, however, not comparable in all infants. Interestingly, mothers with highest anti-influenza HA IgG antibodies had infants with high initial anti-HA antibody maintained until 6 months of age (Pairs #3 and #7), compared to a mother with the lowest initial IgG (Pair #4), suggesting that initial levels attained transplacentally are directly associated with the rate of decline of passive systemic immunity. By the end of the first year, infant #1 maintained 6-month IgG antibody levels, which is likely due to new, natural exposure. Supplementary Figure 1 shows the heatmaps of IgG and IgA antibodies to influenza strains measured by multiplex array in paired mother’s serum (MS), breast milk (BM) and infant serum (IS) samples. Figure 2A shows the trajectory of IgG antibodies to selected individual strains.\n\nThe antibody levels against homologue and cross-reactive HA proteins from different influenza virus strains were evaluated by the mPlex-Flu assay using paired mother’s serum, breast milk (BM) and infant’s serum at different ages expressed as months. A. The IgG antibodies against individual HA of influenza virus strains. Maternal serum was diluted 1:5000, infant serum 1:10 and breast milk 1:10. The IgG antibodies against influenza virus HA were estimated using Phycoerythrin (PE)-conjugated anti-human IgG (γ chain specific) secondary antibodies (SouthernBiotech, AL) and shown as means of median fluorescence intensity (MFI) (n=3). The antibody titers (Log2(MFI+1) ) against individual rHA of influenza virus strains were plotted and connected by LOWESS curves. In the panel of IgG MFI units of infant serum samples, the gray is the area under HA antibody curvel of oldest sampling time point in the same subject. B. The IgA antibodies against individual HA of influenza virus strains. Maternal serum was diluted 1:500, infant serum 1:10 and breast milk 1:10. Then IgA antibodies were detected using PE-conjugated anti-human IgA (α chain specific) secondary antibodies (SouthernBiotech, AL) and shown as the mean of median fluorescence intensity (MFI) (n=3). The antibody titers (Log2(MFI+1) ) against individual rHA of influenza virus strains were plotted and connected by LOWESS curves. In the panel of Ig MFI units of infant serum samples, the gray is the area under the HA antibody curvel of the youngest time point in the same subject.\n\nA. IgG antibody levels against selected influenza virus HA were plotted over time during first year (infant age in months) for 7 infant subjects from Figure 1A data. The specific antibody concentration of IgG is shown as the means for triplicates (n=3). B. The IgA antibody levels against selected influenza virus HA in infant sera were plotted over time during first year for 7 infant subjects from Figure 1B data. The specific antibody levels of IgA are shown as the means for MFI units (n=3).\n\nUnlike with IgG antibodies, BM influenza virus HA-specific IgA antibody levels and patterns were mostly discordant compared to serum. Only some mother-infant pairs showed high a degree of concordance (Pairs #1 and #5). This may be due to the mucosal homing of IgA producing antibody-secreting cells to the mammary gland, resulting in a different antibody profile in breast milk from serum. Very low serum IgA antibodies in infants are consistent with the fact that IgA does not cross the placenta (Figure 1B). The pattern of IgA anti-HA antibody binding was largely similar to that of mother’s serum and milk IgG, and predominantly against H1, H3 and B influenza strains. As opposed to infant IgG responses, most of the infants (Pairs #1–4) showed an increase in IgA responses throughout the first year of life (Figure 1B), whereas no matching IgG antibody response was seen. This may be due to natural, mucosal exposure to influenza inducing local responses, possibly in the absence of a systemic infection inducing IgG antibodies. Figure 2B shows the trajectory of IgA antibodies to a few individual strains. Both anti-stalk group 1 (cH5/1, cH9/1) and group 2 (cH5/3 and cH4/7) IgG (Figure 1A) and IgA antibodies (Figure 1B), which can confer cross-strain immunity, were abundant in the early months in infant serum and BM, respectively.\n\n\nDiscussion\n\nOur pilot data suggest feasibility for measuring antibody responses to influenza strains in maternal breast milk and paired infant serum utilizing a novel multiplex assay to assess changes over time. We show an anticipated decline in IgG responses to influenza HA in the first 5 months of life reflecting waning passive transplacentally acquired immunity, whereas the systemic IgA response in infants appears to be relatively poor, consistent with no vertical transfer. This does not exclude the possibility that such young infants might have a response at mucosal surfaces, such as in saliva upon exposure. Throughout the first year, however, a small increase in IgA antibodies, but not IgG antibodies, is seen in these unvaccinated infants, possibly suggesting that the adaptive immune response to natural exposure, in the absence of systemic infection induces initially local IgA, but not IgG antibodies. We also show that while the IgG specificity patterns were rather similar between breast milk and maternal serum, the patterns for IgA specificity were distinct and more pronounced in BM than those seen in serum. These data suggest that during this time, breast milk IgA may indeed be an important means of providing mucosal protection, which closely reflects maternal mucosal exposure to a variety of influenza strains to benefit the infant. Our previous results comparing food-specific IgA in human milk and maternal serum have shown similarly marked differences between human milk and serum IgA antibody profiles7. This is likely reflecting the fact that IgA-producing cells in mammary gland originate in the gut- and bronchus-associated lymphoid tissue, which constitute an important defense mechanism of the newborn10.\n\nAlthough our study does not address the antibody profiles in vaccinated dyads, maternal influenza vaccination is recommended during pregnancy to induce infant post-partum passive immunity, and for infants after 6 months of age, although many choose to defer vaccination. As indicated by our pilot study, responses in mothers and infants are heterogeneous. At present, there is no robust literature or clinical method to optimize the vertical transfer of protective antibodies. Furthermore, the mechanisms of imprinting or maternal imprinting of the infant immune system are incompletely understood. Thus, there is a critical need for empirical data regarding maternal (serum, BM) and infant (serum) influenza-specific antibody levels over time to inform about maternal impact of influenza immunity, and to predict the individual window of infant susceptibility to influenza. Larger studies are required to further elucidate the interesting findings of this pilot study to aid in assessment of (maternal) imprinting of influenza immunity. Knowing the scope of passive immunity, both transplacental and that provided by BM, and when it vanishes, would allow for precision maternal-fetal and infant vaccination schedule design, also accounting for circulating influenza strains, seasonality, and vaccination status.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for the present study, https://doi.org/10.5256/f1000research.16717.d22413711, including the following files:\n\nInfluenza-specific IgA antibody data as MFI. The file contains the IgA antibody data expressed as MFI for a panel influenza strains generated by mPlex-Flu assay utilizing all breast milk and serum samples. (IgA_20160908_MFI.xlsx)\n\nInfluenza-specific IgG antibody data as MFI. The file contains the IgG antibody data expressed as MFI for a panel influenza strains generated by mPlex-Flu assay utilizing all breast milk and serum samples. (IgG_20160908_MFI.xlsx)\n\nComparison of influenza-specific IgA antibodies between paired samples. The MFI titer comparison of IgA antibody of maternal serum (MS) vs breast milk (BM) and infant’s serum (IS) over time using the Prism 7 software. (IgA version2018.pzfx)\n\nComparison of influenza-specific IgG antibodies between paired samples. The file contains MFI unit comparison of influenza-specific IgG antibodies of maternal serum (MS) vs breast milk (BM) and infant’s serum (IS) over time using the Prism 7 software. (IgG version2018.pzfx)\n\nProgram code for IgA heatmap. The Mathematica 2 program code for generation of the heatmap figure of IgA data of maternal serum (MS), breast milk (BM) and infant’s serum (IS) from mPlex-Flu assay. (IgA MFI Revised.nb)\n\nProgram code for IgG heatmap. The Mathematica 2 program code for generation of the heatmap figure of IgG data of maternal serum (MS), breast milk (BM) and infant’s serum (IS) from mPlex-Flu assay. (IgG MFI Revised.nb)",
"appendix": "Grant information\n\nThe project described was supported by Grant Number K08 AI091655 (KMJ), R21 AI138500 (MZ, JW) and R01AI129518 (MZ) from the National Institute of Allergy and Infectious Diseases, and UL1 TR002001 (MZ, JW) from the National Institute for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, the National Institute for Advancing Translational Sciences, or the National Institutes of Health.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Figure 1. Heatmaps of IgG and IgA antibodies to influenza strains measure by multiplex array in paired mother’s serum (MS), breast milk, (BM) and infant serum (IS) samples. The antibody levels against homologue and cross-reactive HA proteins from different influenza virus strains were evaluated by the mPlex-Flu assay using paired mother’s serum, breast milk (BM) and infant’s serum at different ages expressed as months. A. The IgG antibodies against individual HA of influenza virus strains. MS samples were diluted 1:5000, BM samples were diluted 1:10, and IS samples were diluted 1:10. The IgG antibodies against influenza virus HA were estimated using Phycoerythrin (PE)-conjugated anti-human IgG (γ chain specific) secondary antibodies (SouthernBiotech, AL) and shown as mean median fluorescence intensity (MFI) (n=3). B. The IgA antibodies against individual HA of influenza virus strains. MS samples were diluted 1:500, BM samples were diluted 1:10 and IS samples were 1:10 diluted. Then IgA antibodies were detected using PE-conjugated anti-human IgA (α chain specific) secondary antibodies (SouthernBiotech, AL) and shown as MFI unit also (n=3).\n\nClick here to access the data\n\n\nReferences\n\nMarchant A, Sadarangani M, Garand M, et al.: Maternal immunisation: collaborating with mother nature. Lancet Infect Dis. 2017; 17(7): e197–e208. PubMed Abstract | Publisher Full Text\n\nManske JM: Efficacy and effectiveness of maternal influenza vaccination during pregnancy: a review of the evidence. Matern Child Health J. 2014; 18(7): 1599–609. PubMed Abstract | Publisher Full Text\n\nZaman K, Roy E, Arifeen SE, et al.: Effectiveness of maternal influenza immunization in mothers and infants. N Engl J Med. 2008; 359(15): 1555–64. PubMed Abstract | Publisher Full Text\n\nMadhi SA, Cutland CL, Kuwanda L, et al.: Influenza vaccination of pregnant women and protection of their infants. N Engl J Med. 2014; 371(10): 918–31. PubMed Abstract | Publisher Full Text\n\nTapia MD, Sow SO, Tamboura B, et al.: Maternal immunisation with trivalent inactivated influenza vaccine for prevention of influenza in infants in Mali: a prospective, active-controlled, observer-blind, randomised phase 4 trial. Lancet Infect Dis. 2016; 16(9): 1026–35. PubMed Abstract | Publisher Full Text | Free Full Text\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; 8(8): e70867. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeppo AE, Savilahti EM, Berin MC, et al.: Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA? Clin Exp Allergy. 2017; 47(10): 1275–84. PubMed Abstract | Publisher Full Text\n\nWang J, Hilchey SP, Hyrien O, et al.: Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza. PLoS One. 2015; 10(6): e0129858. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFagarasan S, Kinoshita K, Muramatsu M, et al.: In situ class switching and differentiation to IgA-producing cells in the gut lamina propria. Nature. 2001; 413(6856): 639–43. PubMed Abstract | Publisher Full Text\n\nPeri BA, Theodore CM, Losonsky GA, et al.: Antibody content of rabbit milk and serum following inhalation or ingestion of respiratory syncytial virus and bovine serum albumin. Clin Exp Immunol. 1982; 48(1): 91–101. PubMed Abstract | Free Full Text\n\nJärvinen KM, Wang J, Seppo AE, et al.: Dataset 1 in: Novel multiplex assay for profiling influenza antibodies in breast milk and serum of mother-infant pairs. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16717.d224137"
}
|
[
{
"id": "41719",
"date": "11 Dec 2018",
"name": "Kirsty Le Doare",
"expertise": [
"Reviewer Expertise Vaccine immunity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting pilot study investigates the association between anti-influenza antibodies measured by Luminex in blood and breast milk.\nBreast milk is a much understudied body fluid. However, the numbers in this study and the fact that the timing of breast milk collection varies so greatly means that the results are merely descriptive. Breast milk changes throughout feeding, by time of day and by time of collection relative to feeds. Thus the sampling, although I understand the rationale of convenience, means that the results are not interpretable as they currently are described.\nIt would be useful to add more detail from the original study on timing of sample collection etc. How do you account for different rates of decline? IgG rate of decline is uniform across many pathogens and I am unsure why this should be any different in your study. What was the half life and how does it compare to the other studies you cite?\nCould the differences you see between pairs also be due to non-specific binding within your assay?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "4382",
"date": "28 Jan 2019",
"name": "Kirsi Jarvinen",
"role": "Author Response",
"response": "COMMENT: Breast milk is a much understudied body fluid. However, the numbers in this study and the fact that the timing of breast milk collection varies so greatly means that the results are merely descriptive. Breast milk changes throughout feeding, by time of day and by time of collection relative to feeds. Thus the sampling, although I understand the rationale of convenience, means that the results are not interpretable as they currently are described. REPLY: As mentioned under methods, we utilized stored frozen foremilk collected in the morning. The sample was not a convenience sample but taken according to the study protocol using strict criteria in a prospective manner. Specifically samples were foremilk (not mixed sampling or hindmilk) taken always between hours 8 and 11, and 1-2 hours after the last feeding. COMMENT: It would be useful to add more detail from the original study on timing of sample collection etc. How do you account for different rates of decline? IgG rate of decline is uniform across many pathogens and I am unsure why this should be any different in your study. What was the half life and how does it compare to the other studies you cite? REPLY: As part of the prospective study, breast milk and serum samples were collected during followup visits, and included colostrum and breast milk at 1 month, 3 months, 6 months, 9 months, and 12 months of duration of lactation. However, a few samples were never received due to missed visits or sample timing had to be moved due to illness or other difficulties in getting to the scheduled visits, and some samples have been used up in prior studies. Therefore, not all sample time points are available for all subjects. Regarding decline in IgG, we agree that the levels decline throughout the lactation, as seen in Fig 2a. Due to the small number of samples, no statistical modeling or halflife calculations have been done. COMMENT: Could the differences you see between pairs also be due to non-specific binding within your assay? REPLY: The mPlex-Flu assay is a novel assay to estimate the specific antibody binding to recombinant hemagglutinin (rHA) of multiple influenza virus strains. It has been shown to strongly correlate with all functional assays of influenza specific antibodies, such as hemagglutination inhibition (HAI) assay and influenza virus micro-neutralization assay (Wang J et al 2015 Ref 8, Wang J et al 2018 (new reference added #9)). In addition, in our previous study, mPlex-Flu assay had been shown to have excellent specificity for each influenza virus strains using the HA specific bind blocking in multiple plex assay (Ref 8), and we also used this method to confirmed the specificity of mPlex-Flu assay using human milk samples is similar to using human serum samples (New Supplementary Fig 1). These data suggest minimal non-specific binding between pair samples. As we have demonstrated before, there is cross-reactivity of anti-HA antibodies between antigenically similar influenza strains, which is directed against specific B cell epitopes with similar or homologous sequences and antigenic similarity. This type of cross reactivity, while it may be broad, is specific and based on shared epitopes."
}
]
},
{
"id": "43531",
"date": "04 Feb 2019",
"name": "David C Dallas",
"expertise": [
"Reviewer Expertise milk protein digestion and peptidomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this is a very interesting method and an interesting application. I have a few comments.\nPage 1, Methods: This should be “which can provide mucosal protection”.\nPage 3, Methods: Shouldn’t it be -80 Celsius? Not Fahrenheit. Page 3, Methods: Please specify how the dilutions used for each sample type were determined.\nPage 4, Table 2: Need to specify the meaning of the coloring of the text. It is not clear why some are colored and some are not. The table only says that “seasonal vaccine strains are in bold” but does not mention the colors.\nPage 5, Results, paragraph 2: How do you defined “concordance”? What are your thresholds for how similar it needs to be? Pair 4 (IgA titers) could also be argued to be concordant.\nPage 6, Figure 1 legend: curvel -> curve? Curvel is written twice—not sure if this is a word I’m not familiar with or a misspelling of “curve”. Page 6, Figure 1: The text is very small. Can you figure out how to enlarge please? Page 6, Figure 1 legend: MS abbreviation needs to be defined in the legend. Page 6, Figure 1 legend: “IS”, infant serum needs to be defined in legend. Page 6, Figure 1 legend: Need to explain why Log2 was selected for this display. Page 6, Figure 1 legend: Need to explain what “net” MFI refers to. What is the “net” part? Page 6, Figure 1 legend: Unclear why Log2(MFI + 1) is used. What is the + 1 for?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "4476",
"date": "12 Mar 2019",
"name": "Kirsi Jarvinen",
"role": "Author Response",
"response": "Overall, this is a very interesting method and an interesting application. I have a few comments. COMMENT: Page 1, Methods: This should be “which can provide mucosal protection”.REPLY: corrected.COMMENT: Page 3, Methods: Shouldn’t it be -80 Celsius? Not Fahrenheit.REPLY: corrected. COMMENT: Page 3, Methods: Please specify how the dilutions used for each sample type were determined.REPLY: Dilutions were determined by pilot testing to ensure IgG and IgA influenza virus-specific antibody binding within the mPlex-Flu assay range, i.e. the detectable range of the mPlex-Flu assay (the lower to the upper limits of quantification (LLOQ and ULOQ) concentrations for each influenza HA and each Ig isotype) (Wang et al 2018, ref #8). The pilot testing had been performed to confirm the dilution of samples enables all samples in this detectable range. Usually, the dilutions of samples are not laborious to be determined since mPlex-Flu can detect four Log10 scales.COMMENT: Page 4, Table 2: Need to specify the meaning of the coloring of the text. It is not clear why some are colored and some are not. The table only says that “seasonal vaccine strains are in bold” but does not mention the colors.REPLY: The color determines the subtype of influenza viruses. We added the notes under the table to make it clear.COMMENT: Page 5, Results, paragraph 2: How do you defined “concordance”? What are your thresholds for how similar it needs to be? Pair 4 (IgA titers) could also be argued to be concordant.REPLY: By visually comparing the patterns of antibody reactivity. We agree that #4 is also concordant and have revised the manuscript accordingly.COMMENT: Page 6, Figure 1 legend: curvel -> curve? Curvel is written twice—not sure if this is a word I’m not familiar with or a misspelling of “curve”.REPLY: should say curve. Corrected. COMMENT: Page 6, Figure 1: The text is very small. Can you figure out how to enlarge please?REPLY: We have increased the font on those figures. Thank you for pointing out. COMMENT: Page 6, Figure 1 legend: MS abbreviation needs to be defined in the legend.REPLY: Done COMMENT: Page 6, Figure 1 legend: “IS”, infant serum needs to be defined in legend.REPLY: Done COMMENT: Page 6, Figure 1 legend: Need to explain why Log2 was selected for this display.REPLY: Log2 is customarily used for antibody titers to indicate fold-change. COMMENT: Page 6, Figure 1 legend: Need to explain what “net” MFI refers to. What is the “net” part?REPLY: It should say MFI, not net MFI. We have removed “net”. COMMENT: Page 6, Figure 1 legend: Unclear why Log2(MFI + 1) is used. What is the + 1 for?REPLY: It should be Log2MFI. Corrected in the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1822
|
https://f1000research.com/articles/7-1031/v1
|
09 Jul 18
|
{
"type": "Research Article",
"title": "Comparison of the oxidative potential of primary (POA) and secondary (SOA) organic aerosols derived from α-pinene and gasoline engine exhaust precursors",
"authors": [
"Christopher Lovett",
"Mohamad Baasiri",
"Khairallah Atwi",
"Mohammad H. Sowlat",
"Farimah Shirmohammadi",
"Alan L. Shihadeh",
"Constantinos Sioutas",
"Mohamad Baasiri",
"Khairallah Atwi",
"Mohammad H. Sowlat",
"Farimah Shirmohammadi",
"Alan L. Shihadeh",
"Constantinos Sioutas"
],
"abstract": "Background: Primary (POA) and secondary (SOA) organic aerosols, deriving from both anthropogenic and biogenic sources, represent a major fraction of ambient particulate matter (PM) and play an important role in the etiology of respiratory and cardiovascular diseases, largely through systemic inflammation and cellular oxidative stress. The relative contributions of these species to the inhalation burden, however, are rather poorly characterized. In this study, we measured the in vitro oxidative stress response of alveolar macrophages exposed to primary and secondary PM derived from both anthropogenic and biogenic sources. Methods: POA and SOA were generated within an oxidation flow reactor (OFR) fed by pure, aerosolized α-pinene or gasoline engine exhaust, as representative emissions of biogenic and anthropogenic sources, respectively. The OFR utilized an ultraviolet (UV) lamp to achieve an equivalent atmospheric aging process of several days. Results: Anthropogenic SOA produced the greatest oxidative response (1900 ± 255 µg-Zymosan/mg-PM), followed by biogenic (α-pinene) SOA (1321 ± 542 µg-Zymosan/mg-PM), while anthropogenic POA produced the smallest response (51.4 ± 64.3 µg-Zymosan/mg-PM). Conclusions: These findings emphasize the importance of monitoring and controlling anthropogenic emissions in the urban atmosphere, while also taking into consideration spatial and seasonal differences in SOA composition. Local concentrations of biogenic and anthropogenic species contributing to the oxidative potential of ambient PM may vary widely, depending on the given region and time of year, due to factors such as surrounding vegetation, proximity to urban areas, and hours of daylight.",
"keywords": [
"Particulate Matter",
"SOA",
"Biogenic PM",
"Anthropogenic PM",
"Photochemical Aging"
],
"content": "Introduction\n\nA large fraction of ambient particulate matter (PM) in the urban atmosphere consists of a mixture of primary organic aerosols (POA), derived from anthropogenic and biogenic PM sources, as well as secondary organic aerosols (SOA) produced during the photo-oxidation of both types of POA (Baltensperger et al., 2005; Després et al., 2012). Urban PM can consist of up to 90% SOA, the majority originating from primary biogenic aerosols, including the monoterpene α-pinene, one of the largest components of primary biogenic PM worldwide (Hallquist et al., 2009; Seinfeld & Pankow, 2003).\n\nSeveral human health problems linked to ambient PM, including asthma, cardiovascular disease, and heart failure (Delfino et al., 2005; Dominici et al., 2006; Kim et al., 2013; Shah et al., 2013), are mediated largely by the cellular inflammatory response, including reactive oxygen species (ROS) formation (Li et al., 2003; Ray et al., 2012). Research investigating PM health effects has mostly focused on primary emissions, while studies of secondary PM effects are not as common. Some studies, however, report that both anthropogenic (Decesari et al., 2017; Saffari et al., 2015; Verma et al., 2014; Verma et al., 2015a; Verma et al., 2015b) and biogenic (Baltensperger et al., 2008; Gaschen et al., 2010; Rohr, 2013) SOA elicit greater adverse health effects than POA precursors.\n\nIn this study, we investigate the effects of photochemical oxidation on the oxidative potential of biogenic and anthropogenic PM. Samples of each PM type were collected before and after photochemical aging within a laboratory reaction chamber equipped with an ultraviolet lamp. The in vitro alveolar macrophage (AM) assay was used to quantify PM oxidative potential (Landreman et al., 2008; Li et al., 2008; Shafer et al., 2010).\n\n\nMethods\n\nPhotochemical oxidation of primary emissions occurred within a 64-liter stainless steel oxidation flow reactor (OFR) equipped with a single UV lamp (BHK Analamp, Model No. 82-9304-03) emitting radiation at 185 and 254 nm. Upstream of the PM sources, inlet air first passed through an activated carbon denuder and high-efficiency particulate air (HEPA) filter to remove all particles. Within the OFR, a warm, humid environment (22°C/60% RH) was maintained, allowing H2O to act as a source of hydroxyl radicals in the UV-catalyzed oxidation reactions, which resulted in SOA formation.\n\nThe biogenic sampling setup is depicted in Figure 1. Particle-free inlet air was introduced at a flow rate of 25 lpm. 0.5 lpm of this incoming air stream was diverted into a 250 ml Büchner flask containing a 15-ml glass vial of pure, reagent grade α-pinene. Three small holes in the vial cap allowed for diffusion of α-pinene vapors into the flask. The remaining 24.5 lpm flow of particle-free air proceeded through a humidifier (heated flask containing distilled water) and into the reactor, where it mixed with the α-pinene vapors, resulting in a dilution ratio of 50:1.\n\nThe anthropogenic sampling setup is depicted in Figure 2. Exhaust from a four-stroke single cylinder gasoline generator (Honda SHX1000, 49cc displacement, 8.0:1 compression ratio, operating at 3000 RPM) was drawn through a rotating disk dilutor (RDD; Testo Engineering, MD19-3E) operating at a dilution ratio of 50:1. 5 lpm of the diluted engine exhaust was diverted into the reaction chamber, where it mixed with 20 lpm of humidified, particle-fee air, resulting in a total dilution ratio of 250:1.\n\nParticles were collected downstream of the reaction chamber on Teflon and quartz filters. In the POA condition, PM was collected as the α-pinene or engine emissions passed through a dark OFR. In the SOA condition, the aerosol stream was sampled while a UV lamp was on, following a 90-minute reaction period.\n\nPrior to sampling, quartz filters were baked in a furnace oven at 500°C for 5 hours. Teflon filters were conditioned for 24 hours in a controlled environment (23°C and 46% relative humidity) before weighing. Teflon filters were weighed before and after sampling to determine the mass collected with an MT5 Microbalance (Mettler-Toledo Inc., Columbus, OH, USA). Mass collected on quartz filters was calculated based on the aerosol concentration (from Teflon filters) and sampling flow rate. After sampling, all filters were placed in petri dishes lined with baked aluminum foil, sealed with Teflon tape, and stored in a refrigerated environment until analysis.\n\nQuartz filters were analyzed for elemental carbon (EC) and organic carbon (OC) content by the National Institute for Occupational Safety and Health (NIOSH) Thermal Optical Transmission (TOT) Method 5040, using a flame ionization detector (FID) to quantify evolved carbon as CH4 (Birch & Cary, 1996; Peterson & Richards, 2002).\n\nThe alveolar macrophage (AM) in vitro assay was used to determine the oxidative potential of the Teflon filter PM samples. Alveolar macrophages obtained from the American Type Culture Collection (cell line NR8383, RRID: CVCL_4396) were maintained in Ham’s F12 medium (#11765-047, ThermoFisher, Waltham, MA, USA) supplemented with 2mM L-glutamine (GlutaMAX; #31765-035, ThermoFisher, Waltham, MA, USA), 1.176 g/L sodium bicarbonate, and 15% heat inactivated fetal bovine serum (FBS; #45000-734, VWR, Radnor, PA, USA). Cells were cultured in flasks and kept in an incubator at 37°C/5% CO2. Non-adherent cells were transferred to new flasks weekly. A floating cell concentration of approximately 4 × 105 cells/mL media was maintained. The macrophage cells were exposed to each type of PM sample for 2.5 hours, using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent probe to quantify the cellular formation of oxidative species. The non-fluorescent DCFH-DA acts by entering the cell, where it is de-acetylated by cellular enzymes to yield 2,7-dichlorodihydrofluorescein (DCFH), also non-fluorescent. DCFH is then oxidized by reactive species, generated during the cellular oxidative stress response to PM exposure, to form the highly fluorescent and detectable 2,7-dichlorofluorescein (DCF), which was quantified spectrophotometrically with a CytoFlour II automated fluorescence plate reader (PerSeptive Biosystems, Framingham, MA, USA) (Landreman et al., 2008; Shafer et al., 2010).\n\n\nResults\n\nEC/OC results are presented in Figure 3. EC was most abundant in engine POA (0.081 μg-EC/μg-PM), with no significant amount present in either engine or α-pinene SOA. Mass fractions of OC were higher than EC in all conditions (engine POA: 0.62 μg-OC/μg-PM, engine SOA: 0.54 μg-OC/μg-PM, α-pinene SOA: 0.54 μg-OC/μg-PM). Figure 4 presents oxidative potential results on a mass-fraction basis, standardized to Zymosan units (μg-Zymosan units/mg-PM). Mass fraction results reveal how the intrinsic PM toxicity as indexed by oxidative potential changes over time due to photochemical aging. The measured oxidative potential for engine POA was 51.4 (± 64.3) µg-Zymosan/mg-PM, and for engine SOA it was 1900 (± 255) µg-Zymosan/mg-PM, while for α-pinene SOA, the result was 1321 (± 542) µg-Zymosan/mg-PM (pure α-pinene was not assayed).\n\nError bars represent laboratory uncertainty values based on contributions of analytical error (standard deviation) and blank subtraction (standard deviation of at least three method blanks).\n\nError bars represent laboratory uncertainty values based on contributions of analytical error (standard deviation) and blank subtraction (standard deviation of at least three method blanks).\n\n\nSummary and conclusions\n\nThe findings of the current reaction chamber study indicate that both anthropogenic and biogenic SOA induce greater cellular oxidative stress than primary engine exhaust. This effect was found to be largest in response to engine exhaust SOA, thus implicating anthropogenic as the major contributor to adverse human health effects in urban environments, though the contribution of biogenic SOA can be quite significant in some geographical areas. Atmospheric aging of PM increases its intrinsic oxidative potential many fold, and thus photochemistry in a region that experiences abundant sunshine, long days, and/or stagnation of circulating air due to an inversion layer or some other reason, may increase the toxicity of PM over time.\n\n\nData availability\n\nThe following raw data sets are provided as comma separated values (.csv) files:\n\nDataset 1: Figure 3 EC-OC Raw Data 10.5256/f1000research.15445.d209280 (Lovett et al., 2018a)\n\nDataset 2: Figure 4 ROS Raw Data 10.5256/f1000research.15445.d209281 (Lovett et al., 2018b)",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported in part by the University of Southern California Viterbi Dean’s Ph.D. Fellowship, and by National Institutes of Health research grants [RF1-AG051521-01 and R21-AG050201-01A1].\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\nBaltensperger U, Dommen J, Alfarra MR, et al.: Combined determination of the chemical composition and of health effects of secondary organic aerosols: the POLYSOA project. J Aerosol Med Pulm Drug Deliv. 2008; 21(1): 145–154. PubMed Abstract | Publisher Full Text\n\nBaltensperger U, Kalberer M, Dommen J, et al.: Secondary organic aerosols from anthropogenic and biogenic precursors. Faraday Discuss. 2005; 130: 265–278; discussion 363–86, 519–24. PubMed Abstract | Publisher Full Text\n\nBirch ME, Cary RA: Elemental carbon-based method for monitoring occupational exposures to particulate diesel exhaust. Aerosol Sci Technol. 1996; 25(3): 221–241. Publisher Full Text\n\nDecesari S, Sowlat MH, Hasheminassab S, et al.: Enhanced toxicity of aerosol in fog conditions in the Po Valley, Italy. Atmos Chem Phys. 2017; 17(12): 7721–7731. Publisher Full Text\n\nDelfino RJ, Sioutas C, Malik S: Potential role of ultrafine particles in associations between airborne particle mass and cardiovascular health. Environ Health Perspect. 2005; 113(8): 934–46. PubMed Abstract | Free Full Text\n\nDesprés V, Huffman JA, Burrows SM, et al.: Primary biological aerosol particles in the atmosphere: a review. Tellus B Chem Phys Meteorol. 2012; 64(1): 15598, 1–58. Publisher Full Text\n\nDominici F, Peng RD, Bell ML, et al.: Fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases. JAMA. 2006; 295(10): 1127–1134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaschen A, Lang D, Kalberer M, et al.: Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles. Environ Sci Technol. 2010; 44(4): 1424–1430. PubMed Abstract | Publisher Full Text\n\nHallquist M, Wenger JC, Baltensperger U, et al.: The formation, properties and impact of secondary organic aerosol: current and emerging issues. Atmos Chem Phys. 2009; 9(14): 5155–5236. Publisher Full Text\n\nKim KH, Jahan SA, Kabir E: A review on human health perspective of air pollution with respect to allergies and asthma. Environ Int. 2013; 59: 41–52. PubMed Abstract | Publisher Full Text\n\nLandreman AP, Shafer MM, Hemming JC, et al.: A macrophage-based method for the assessment of the reactive oxygen species (ROS) activity of atmospheric particulate matter (PM) and application to routine (daily-24 h) aerosol monitoring studies. Aerosol Sci Technol. 2008; 42(11): 946–957. Publisher Full Text\n\nLi N, Sioutas C, Cho A, et al.: Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect. 2003; 111(4): 455–60. PubMed Abstract | Free Full Text\n\nLi N, Xia T, Nel AE: The role of oxidative stress in ambient particulate matter-induced lung diseases and its implications in the toxicity of engineered nanoparticles. Free Radic Biol Med. 2008; 44(9): 1689–1699. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLovett C, Baasiri M, Atwi K, et al.: Dataset 1 in: Comparison of the oxidative potential of primary (POA) and secondary organic aerosols (SOA) derived from α-pinene and gasoline engine exhaust precursors. F1000Research. 2018a. Data Source\n\nLovett C, Baasiri M, Atwi K, et al.: Dataset 2 in: Comparison of the oxidative potential of primary (POA) and secondary organic aerosols (SOA) derived from α-pinene and gasoline engine exhaust precursors. F1000Research. 2018b. Data Source\n\nPeterson MR, Richards MH: Thermal-optical transmittance analysis for organic, elemental, carbonate, total carbon, and OCX2 in PM2.5 by the EPA/NIOSH method. In Proceedings, Symposium on Air Quality Measurement Methods and Technology-2002. Air & Waste Management Association, Pittsburgh, PA. 2002; 83–1. Reference Source\n\nRay PD, Huang BW, Tsuji Y: Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal. 2012; 24(5): 981–990. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRohr AC: The health significance of gas- and particle-phase terpene oxidation products: a review. Environ Int. 2013; 60: 145–162. PubMed Abstract | Publisher Full Text\n\nSaffari A, Hasheminassab S, Wang D, et al.: Impact of primary and secondary organic sources on the oxidative potential of quasi-ultrafine particles (PM0.25) at three contrasting locations in the Los Angeles Basin. Atmos Environ. 2015; 120: 286–296. Publisher Full Text\n\nSeinfeld JH, Pankow JF: Organic atmospheric particulate material. Annu Rev Phys Chem. 2003; 54(1): 121–140. PubMed Abstract | Publisher Full Text\n\nShafer MM, Perkins DA, Antkiewicz DS, et al.: Reactive oxygen species activity and chemical speciation of size-fractionated atmospheric particulate matter from Lahore, Pakistan: an important role for transition metals. J Environ Monit. 2010; 12(3): 704–715. PubMed Abstract | Publisher Full Text\n\nShah AS, Langrish JP, Nair H, et al.: Global association of air pollution and heart failure: a systematic review and meta-analysis. Lancet. 2013; 382(9897): 1039–1048. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerma V, Fang T, Guo H, et al.: Reactive oxygen species associated with water-soluble PM2.5 in the southeastern United States: spatiotemporal trends and source apportionment. Atmos Chem Phys. 2014; 14(23): 12915–12930. Publisher Full Text\n\nVerma V, Fang T, Xu L, et al.: Organic aerosols associated with the generation of reactive oxygen species (ROS) by water-soluble PM2.5. Environ Sci Technol. 2015a; 49(7): 4646–4656. PubMed Abstract | Publisher Full Text\n\nVerma V, Wang Y, El-Afifi R, et al.: Fractionating ambient humic-like substances (HULIS) for their reactive oxygen species activity - Assessing the importance of quinones and atmospheric aging. Atmos Environ. 2015b; 120: 351–359. Publisher Full Text"
}
|
[
{
"id": "39486",
"date": "06 Nov 2018",
"name": "Barend L. van Drooge",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work of Lovett et al. presents interesting data on the possible inflammatory effects of SOA from traffic as well as biogenic (pinene) origin. The method setup is well designed, although the variables, such as conditions of relative humidity and temperature, could have been studied in different values. On the other hand, the results show that the biogenic SOA have similar high inflammatory effect in the test compared to traffic SOA, which is an important fact, and indicates that effects have been observed in real-world data. However, the study could have been more complete and higher quality if the researchers would have made an effort to detect and quantify the molecular chemical compounds that are present in the SOA fractions.\n\nThe work is suitable for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "43635",
"date": "25 Feb 2019",
"name": "Zhi Ning",
"expertise": [
"Reviewer Expertise Particle induced cellular oxidative stress",
"sensor development and application"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments:\nThe authors have investigated the oxidative potential of POA and SOA from two different sources namely alpha-pinene and gasoline engine exhaust. The experimental setup included an UV chamber (oxidation flow reactor), to mimic the sun light’s UV rays, to compare primary and secondary organic aerosol-induced radical generation under light and in dark. The comparison could contribute great value to the manuscript if additional parameters as listed below are included in it:\nPage 3: Right column: Line 4: The authors can address why they have selected only Hydroxyl radicals in the investigations. In some experiments, where UV rays are used to excite the organic aerosols, the elicitation of superoxide radicals is also possible.\n\nPage 3: Right column: Lines 28-30: The statement “aerosol stream was sampled while a UV lamp was on, following a 90-minute reaction period.” is not clear. Does that mean the whole sample streaming is done for a continuous 90 minutes? Was it the same for the aerosol sampling done in dark OFR?\n\nPage 3: Right column: Line 33: The information of the control sample needs to be included here.\n\nPage 4: Left column: Line 23: The analysis part has some information missing such as incubation time for cell growth, and are the same generation (life cycle) used for analysis?\n\nPage 4: Left column: Line 25: The cell exposure study has some basic information missing - PM dose, route of exposure (directly on filters or on PM extracts), number of times analysed (duplicate or triplicate). Please include for clarity.\n\nPage 5: Figure 4: The biogenic organic compounds (for example: the alpha-pinene) are believed to be more hydrophilic compared to engine exhaust organics. Please include a discussion if the water solubility of samples is also driving the difference in oxidative stress.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "4460",
"date": "05 Mar 2019",
"name": "Christopher Lovett",
"role": "Author Response",
"response": "Authors’ responses to specific comments of Dr. Zhi Ning:Comment 1: Page 3: Right column: Line 4: The authors can address why they have selected only Hydroxyl radicals in the investigations. In some experiments, where UV rays are used to excite the organic aerosols, the elicitation of superoxide radicals is also possible.Text Added:“While the production of superoxide radicals is possible, the major products of H2O oxidation generated in this type of OFR are hydroxyl radicals, especially at less than 80% relative humidity (Seinfeld & Pandis, 2016; Kamens et al., 2011; Jang et al., 2002).”Comment 2: Page 3: Right column: Lines 28-30: The statement “aerosol stream was sampled while a UV lamp was on, following a 90-minute reaction period.” is not clear. Does that mean the whole sample streaming is done for a continuous 90 minutes? Was it the same for the aerosol sampling done in dark OFR?Text Revision:“In the SOA condition, the aerosol stream was sampled while the UV lamp inside the reactor was on. During this condition, the sample stream was diverted for the first 90 minutes to ensure that the reactor was operating under steady-state conditions while samples were collected.” Comment 3: Page 3: Right column: Line 33: The information of the control sample needs to be included here.Authors’ Response:The only control samples were the filter blanks submitted to the laboratory for chemical and toxicological analyses as discussed in Section 2.3. Discussion of the control condition utilized in the macrophage assay (untreated cells) is included in the response to Comment 5. Comment 4: Page 4: Left column: Line 23: The analysis part has some information missing such as incubation time for cell growth, and are the same generation (life cycle) used for analysis?Text Added:“In preparation for PM exposures, the non-adherent fraction of macrophage cells was harvested from flasks and concentrated by centrifugation (750 RPM) for 5 minutes. The culture medium was removed, and the cell suspension was diluted to a concentration of 1,000 cells/μL. 100 μL aliquots of these suspended cells were then pipetted into each well of 96-well plates (100,000 cells/well) and incubated at 37°C/5% CO2 for 2 hours. Following the 2-hour incubation period, nearly all macrophages were adhered to each well bottom. During PM treatments, supernatant media was aspirated and replaced with 100 μL of sample extract or blank control solution in each well.” Comment 5: Page 4: Left column: Line 25: The cell exposure study has some basic information missing - PM dose, route of exposure (directly on filters or on PM extracts), number of times analysed (duplicate or triplicate). Please include for clarity.Text Added:“Each Teflon filter (PM sample or blank) was divided into two sections, one for the macrophage assay and one for chemical analysis. The filter halves used for the cell assay underwent sonication in 900 μL of purified water for approximately 16 hours at room temperature to extract the water-soluble components. These filters were then removed from the aqueous extracts, and 10x concentrated salts-glucose medium (SGM) was added to create buffered PM extract solutions for use in the PM treatments.”“Each treatment or control was run in triplicate (3 wells each). Additionally, several untreated and method blank controls were included on each 96-well plate. Zymosan was included as a positive control at a concentration of 0.125 mg-zymosan/mL in a buffered SGM solution. Results are reported as the increase in fluorescence due to sample treatments relative to the fluorescence observed in the untreated control condition.”Comment 6: Page 5: Figure 4: The biogenic organic compounds (for example: the alpha-pinene) are believed to be more hydrophilic compared to engine exhaust organics. Please include a discussion if the water solubility of samples is also driving the difference in oxidative stress.Text Added (to Summary and Conclusions):“While the macrophage assay is generally more sensitive to water-soluble components of PM, as compared to non-polar PM species, the large cellular oxidative stress response to SOA, especially anthropogenic SOA, cannot be attributed entirely to hydrophilicity. Biogenic organic compounds are generally more hydrophilic than organic products of anthropogenic processes such as combustion, yet greater ROS activity in response to anthropogenic PM was observed. The alveolar macrophage assay is considered an excellent model of inhalation toxicity, and increased ROS formation reliably indexes greater toxicity, whether ultimately due to increased bioavailability of harmful molecules or simply greater quantities of harmful PM species (Landreman et al., 2008). Thus, the results of this study clearly indicate that oxidized PM species, whether of biogenic or anthropogenic origin, are more toxic that primary PM, regardless of water-solubility, with the greatest toxicity resulting from anthropogenic SOA exposures.”"
}
]
}
] | 1
|
https://f1000research.com/articles/7-1031
|
https://f1000research.com/articles/8-275/v1
|
11 Mar 19
|
{
"type": "Research Article",
"title": "Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease",
"authors": [
"Serge Dionne",
"Sophie Restellini",
"Jamie Koenekoop",
"Pedro Salvador Escribano",
"Ciriaco A. Piccirillo",
"Patrick Charlebois",
"A. Sender Liberman",
"Barry Stein",
"Carl Frederic Duchatellier",
"Ernest Gerald Seidman",
"Serge Dionne",
"Sophie Restellini",
"Jamie Koenekoop",
"Pedro Salvador Escribano",
"Ciriaco A. Piccirillo",
"Patrick Charlebois",
"A. Sender Liberman",
"Barry Stein",
"Carl Frederic Duchatellier"
],
"abstract": "Background: Intestinal fibrosis represents a serious complication of inflammatory bowel diseases (IBD), often necessitating surgical resections. Myofibroblasts are primarily responsible for interstitial matrix accumulation in fibrotic diseases. However intestinal myofibroblasts (IMF) remain inadequately characterized. The aim was to examine fibroblast markers and fibrosis-associated gene expression in IMF isolated from resected intestine from IBD and control patients. As well as determining the effect of the fibrogenic cytokine TGFβ. Methods: Intestinal resections were obtained (n =35) from consenting patients undergoing elective surgery (2014-16). Primary cultures of IMF were isolated using DTT and EDTA and cultured. Viability and phenotypic characterization of IMF was carried out by flow cytometry and fluorescence microscopy. IMF (passages 3-8) were treated for 24 hours. Cytokines were quantified in IMF by real time PCR and in supernatants using the human pro-inflammatory cytokine panel Results: All markers and most fibrosis mediators studied were preferentially expressed by IMF compared to mucosal tissue. Metalloproteinases (MMP) 2 and 3, as well as their inhibitor TIMP1, are highly expressed by IMF. They also highly expressed inflammatory mediators, including IL-6, IL-8, CCL2 and PTGS2. Whereas mucosal expression of pro-inflammatory cytokines such as TNFα and IL-17 is increased in IBD, that of fibrosis mediators was not different. Fibrosis-related gene expression in IMF from IBD patients and controls was similar, but IMF from IBD expressed higher levels of several inflammatory genes. IMF from CD and UC had mostly similar expression profiles. TGFβ induced expression of fibrogenic genes αSMA, COL1A1, CTGF, FN1 and LOX. TGFβ-stimulated IMF released increased levels of IL-6, whereas IL-6, IL-8, as well as small amounts of IFN-γ and IL12p70 were produced following stimulation with IL-1β+IL-23. Conclusions: This study extends knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required.",
"keywords": [
"intestinal myofibroblasts",
"inflammatory bowel disease",
"fibrosis",
"crohn’s disease",
"ulcerative colitis",
"inflammation",
"cytokines gene expression"
],
"content": "Introduction\n\nThe two major forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are characterized by chronic and relapsing intestinal inflammation that develop as the result of an abnormal regulation of immune responses, likely directed at least in part, against the host commensal gut microbiota1–3. The prevalence of IBD is estimated to be over 3.6 million persons in North America and Europe, and the incidence is increasing in Asia and Africa4,5. Genetic studies have identified over 200 susceptibility loci for IBD, mostly shared between CD and UC6–8. These loci are enriched for pathways that interact with environmental factors to modulate intestinal homeostasis, including IL23R, ATG16L1, IRGM and NOD29.\n\nThe natural history and clinical course of IBD is extremely heterogeneous. Up to 16% of UC patients require a colectomy within 10 years, whereas about 50% of patients with CD develop complications such as strictures, fistulas, and abscesses that frequently require surgery within the same period10,11. Intestinal fibrosis is a common and potentially serious complication of CD, but not UC, that results from the reaction of intestinal tissue to the damage inflicted by chronic inflammation12. NOD2 has been long considered the most important genetic predictor of the evolution toward a fibrostenosing phenotype13,14. Fibrosis in CD is also associated with polymorphisms of the Jak2, ATG16L1, CX3CR1 and MMP3 genes14.\n\nAn improved understanding of the cellular and molecular mechanisms that underlie the pathogenesis of intestinal fibrosis is needed. The mechanisms are complex, dynamic, and likely involve multiple cell types and soluble factors12. Intestinal myofibroblasts (IMF) play important roles in inflammation and tissue remodeling15. The development of fibrosis results from an imbalance in extracellular matrix (ECM) deposition and degradation. Alpha-smooth muscle actin (α-SMA) positive myofibroblasts were identified as the primary cell type responsible for interstitial matrix accumulation in fibrotic diseases16,17. A hallmark of mesenchymal cell activation is the acquisition of a myofibroblast phenotype, whereby fibroblasts transform into myofibroblasts acquiring smooth muscle features, most notably the expression of α-SMA and synthesis of mesenchymal cell related matrix proteins. TGF-β1, a prototype of the TGF-β superfamily, is widely considered to be the major profibrogenic cytokine that is responsible for the myofibroblast differentiation and subsequent matrix synthesis16,18. Although the TGF-β/Smad pathway is considered a driving force of fibrosis, myofibroblasts are activated by numerous paracrine mediators in their environment that promote their production of ECM and proliferation including, TGF-β1, PDGF, CTGF, IGFI/II, bFGF and various interleukins: IL-1β, IL-6 and IL-1317,19.\n\nAlthough inflammation is necessary for fibrosis, recent evidence indicates that once initiated, fibrosis in CD can progress independently of inflammation17. Consequently, current anti-inflammatory treatments may not prevent fibrosis once excessive ECM deposition has commenced17,20. Matrix stiffness is capable of further activation of intestinal fibroblasts and can contribute to progression of fibrosis independently of inflammation21,22.\n\nThere is currently little information about the identity, abundance and characteristics of intestinal mesenchymal cells such as fibroblasts and IMF under normal and pathological conditions. In this study, we examined the expression of fibroblast and IMF molecular markers in the intestine from patients with CD, UC and from non-IBD control patients.\n\n\nMethods\n\nThe McGill University Health Center’s research ethics board approved the study design and consent forms. Intestinal resections were obtained (2014–2016) from patients undergoing elective intestinal surgery who voluntarily gave written informed consent to participate. Written informed consent for publication of the participants/patients’ details and/or their images was obtained from the participants/patients/parents/guardian/relative of the participant/patient Resected tissue was obtained from 15 CD and 6 UC patients, as well as from uninvolved surgical specimens (>5 cm from the tumor margin) of 14 control patients undergoing colectomy for carcinoma or polyps. The characteristics of the patient groups are shown in Table 1.\n\nCD: Crohn’s disease, IBD: inflammatory bowel disease, UC: ulcerative colitis\n\nPrimary cultures of IMF were isolated and cultured according to the method reported by Mahida et al.23. Briefly, tissue specimens were trimmed of fat and thoroughly washed. The mucosa was cut into 0.5 cm pieces and incubated in 1.5 mM DTT (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) in HBSS for 15 min to remove mucus. Tissue was washed and then incubated in HBSS containing 2 mM EDTA for 2 × 30 min in a shaker at 37°C.\n\nThe resulting mucosal samples denuded of epithelial cells were cultured at 37°C, in RPMI-1640 supplemented with 10% fetal calf serum (FCS) to allow myofibroblasts to migrate out, in order to establish primary cultures. Established colonies of IMF were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 1% non-essential amino acids (Gibco, Carlsbad, CA, USA), and 200 mM glutamine (Sigma). Experiments were performed on passages 3–8.\n\nCells obtained as above were prepared for flow cytometry as follows. After trypsinization, cells were counted and distributed in 1x106 per sample tubes. Cells were washed twice in PBS and blocked with Human BD Fc Block (BD Biosciences, Mississuagua, ON) according to the manufacturer’s instructions. Samples were then incubated with eBioscienceTM Fixable Viability Dye eFluor 450 (Life Technology Inc., Burlington, ON) for 30 min in ice, washed again in PBS and incubated in PBS containing the surface marker antibodies for 20 min in ice (Table 2). Samples were then permeabilized with BD Cytofix/Cytoperm Buffer (BD Biosciences) for 20 min on ice and then washed with BD Perm/Wash (BD Biosciences) and incubated with BD Perm/Wash Buffer containing intracellular marker antibodies for 20 min on ice (Table 2). Samples were washed with BD Perm/Wash and analyzed by flow cytometry using the BD LSRFortessa™ X-20 (BD Biosciences). Machine compensation was performed using unstained cells, viability dye stained cells and UltraComp ebeads (eBioscience) conjugated with each antibody. Dead cells and debris were gated out and purity of the cells was calculated on live cells. Fluorescence microscopy was performed with an Olympus IX71 microscope (Olympus Canada, Toronto, ON) equipped with an Olympus LUCPLFLN20X/0.45 lens.\n\nIn some experiments, cells were incubated with SMAD (5uM, x 2hr) and/or TGFα (3 ng/ml, overnight).\n\nIMF were plated on 6 well-plates and treated for 24 hours. The medium was collected and stored at -80°. Supernatants of the cultures were tested for IFNγ, TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70 and IL-13, using the human pro-inflammatory cytokine panel (Meso Scale Diagnostics, Rockville, MD).\n\nmRNA from IMF was isolated with RNeasy Mini Plus kit (Qiagen, Toronto, ON, Canada) according to the manufacturer’s instructions. cDNA was generated from 1 µg of total RNA using Transcriptor First Strand cDNA Synthesis kit (Sigma-Aldrich Canada Ltd). Real-time RT-PCR was performed by a “StepOnePLus” RT-PCR (Life Technologies, Burlington, ON) using PerfeCTa SYBR green Fast Mix (Quanta Biosciences, Beverly, MA). Primers were as described in Table 3.\n\nPrimers were ordered from Integrated DNA Technologies (Coralville, IA). Gene expression was standardized using GAPDH expression. Results were quantified using the ΔCt or 2ΔCt method.\n\nData were analyzed using GraphPad and presented as median. Statistical analysis of the results between groups was determined by the Mann-Whitney test. Significance was set as p<0.05.\n\n\nResults and discussion\n\nThe viability of IMF cells as determined by flow cytometry was 97.8% (n= 3/group; passages 3-8). No difference was observed between UC, CD and control groups. Immunofluroescence microscopy revealed that the IMF were aSMA+, CD90+, FSP1 +, and negative for hematopoietic and epithelial markers CD45 and EpCAM. Representative images are illustrated in Figure 1.\n\nA) Representative photomicrograph of colonic myofibroblasts isolated from a resection of a control patient. Cells have a fibroblast shape and express expressed a marker of myofibroblast, a-SMA (in green). B) IMF cultured from a colonic resection in a patient with ulcerative colitis. Cells also express CD 90 (in red).\n\nWe examined the gene expression of fibroblast markers in IMF cultures and compared their levels with those in the mucosa. The expression of fibroblast activation protein (FAP) was more than 200 times higher in IMF cultures than in mucosal tissue. Levels of Fibroblast specific protein 1 (FSP1, also known as S100A4) and those of the stromal marker CD90/Thy-1 were 5–8 times greater in IMF than in the mucosa. The myofibroblast marker αSMA in primary IMF cultures was 2-fold the mucosal level (Figure 2A). We then examined the expression of different mediators implicated in the fibrotic process. As illustrated in Figure 2A, IMF expressed high levels of type 1 collagen (COL1A1) and fibronectin 1 (FN1). Connective tissue growth factor (CTGF/CCN2), a matricellular protein recognized to promote matrix protein deposition and fibrogenesis, was similarly expressed by IMF and mucosa. CCN1, another matricellular protein, was preferentially expressed by IMF.\n\nGene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05, ** p< 0.005, *** p, 0.00\n\nFibrosis is a pathological condition characterized by the deposition of excessive or abnormal ECM components, including collagen type I. Metalloproteinases (MMP) are responsible for the degradation of ECM components and are thus play a central role in ECM remodeling. Their activity is controlled by tissue inhibitors of metalloproteinase (TIMP). MMP-2 and MMP-3 are highly expressed in IMF (over 100 times the mucosal levels). However, MMP-9 transcript levels were higher in the mucosa (p=0.05). IMF were found to be an important source of TIMP1, with expression largely exceeded that of the mucosa (Figure 2B). Accumulating evidence points toward the NAD(P)H oxidase of the Nox family and particularly Nox4 as the predominant enzyme source for ROS generation in fibrotic disease24. As shown in Figure 2B, IMF expressed significant levels of NOX4 transcripts.\n\nThe inflammatory potential of IMF was then investigated. IMF expressed high levels of pro-inflammatory cytokines IL-6 and IL-8, more than 40 and 30 times the mucosal levels, respectively (Figure 3). The chemokine CCL2 was also more expressed by IMF cultures than mucosal tissue. Transcripts of cytokines such as TNFα, IFNγ, IL-17A and IL-23A were undetectable in most IMF samples analyzed. PTGS2, also known as cyclooxygenase-2 or COX-2, is highly expressed by IMF, approximately 200 times the mucosa levels.\n\nGene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05, ** p< 0.005, *** p < 0.001\n\nWe then examined mRNA level of fibrosis related genes in the mucosa of IBD patients and of controls. No significant differences were found in fibroblast markers and fibrosis gene expression in mucosa from IBD patients compared to controls. MMP3, MMP9 and TIMP1 expression tended to be higher in IBD tissue, but the differences were not statistically different due to the large variation in IBD mucosa (data not shown). There was no clear expression profile related to the pathology except for TIMP1 which was more expressed in UC than in CD (Figure 4). Pro-inflammatory cytokine expression was greater in mucosa isolated from IBD patients. Expression of IL-17 and TNFα, but not IL-23A, were significantly greater in the intestine from IBD patients (Figure 4). Values for IL-6 and IL-8 were too dispersed to achieve significance.\n\nGene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05, ** p< 0.005\n\nExpression of fibroblast markers and fibrosis-related genes was not different between IMF generated from IBD and control patients (Figure 5). Increased CTGF expression by IBD IMF was marginally (significant p=0.053). FSP-1 expression was not different between IBD and control groups, but IMF from UC patients expressed lower transcript levels compared to those from controls (p=0.0357).\n\nGene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians.\n\nIMF from IBD resections expressed higher levels of IL-6 than those from controls (p=0.0357, Figure 6). IL-8 was nearly 100 times more expressed by IMF from IBD but due to the small number of samples the difference did not reach statistical significance. CCL2 expression was also 30 times more in IMFs from IBD patients. There was a trend towards higher PTGS2 expression in IMF from CD patients compared to those from controls and UC patients.\n\nGene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05\n\nTGFβ stimulation of IMF resulted in an increased expression of profibrotic genes COL1A1 and CTGF. CCN1 as well as FN1 expression were also upregulated following stimulation with TGFβ (Figure 7). Lysyl oxidase (LOX), a collagen modifying enzyme required for the cross-linking of collagen, was also induced. There was marked upregulation of αSMA of IMF. On the other hand, TGFβ down-regulated expression of fibroblast markers FSP1 and CD90. TGFβ slightly induced MMP9 and TIMP1 expression, although not significantly. No significant effect of TGFβ was observed on NOX4, IL-6, IL-8 and PTGS2 expression, although IL-6 expression was increased in 4 of 6 IMF cultures.\n\n* p< 0.05, ** p< 0.005, *** p < 0.001.\n\nTo assess the inflammatory properties of IMF, we determined their cytokine release following fibrogenic and inflammatory stimulation. IMF spontaneously released IL-6 and IL-8 (Figure 8a). TGFβ induced IL-6 release (Figure 8b). Stimulation with IL-1β+IL-23 increased IL-6 and IL-8 production. In addition, small amounts of IFNγ and IL-12p70 were released (Figure 8c).\n\nData are mean ± SEM from 3–5 different IMF cultures from IBD and control, respectively (A and B) and 2 IMF cultures from both IBD and control IMF cultures (C).\n\nFibrosis is a chronic, progressive process characterised by an excessive deposition of collagen and other ECM components. Intestinal fibrosis is a common complication of CD, an incurable disorder, forcing patients to undergo bowel resections over their lifetime. More than 40% of CD patients with ileal involvement will require one or more resections of strictures25. Although less common in UC, longstanding disease is believed to cause fibrosis resulting in altered bowel function26,27. In this study, isolation techniques used yielded multiple cell populations present in the intestinal lamina propria including: immune cells, epithelial cells, and MF. This permitted the establishment of primary IMF cultures with a high rate of viability. After the first passage, only MF remained, as identified by their unique phenotype (Figure 1).\n\nA variety of signals promote fibroblast differentiation into IMF and augment ECM expression. TGFβ1 represents the prototype of the profibrogenic mediators, with its unique ability to drive myofibroblast activation through both canonical and non-canonical signaling pathways leading to expression and deposition of ECM18.\n\nThere is little information about the identity, localization, and abundance of the different intestinal mesenchymal cell types. Various studies show that fibroblasts isolated from different tissues are morphologically and functionally heterogeneous subpopulations28. Several fibroblast markers have been described, but none of them is unique to fibroblasts, and not all fibroblasts express the proposed markers. This lack of specific markers has impeded the characterization of IMF and their putative precursors. Moreover, characterization of these markers under normal and pathological conditions is still lacking. This study aimed to determine the expression of several markers as well as fibrosis related mediators in IMF derived from the CD, UC and control patients.\n\nAmong the markers available to identify fibroblasts, Thy-1 (CD90), FAP and FSP1 (S100A4) are the most extensively studied15. Our results show that all three markers are highly expressed by IMF compared to the intestinal mucosa. IMF also express higher levels of the myofibroblast marker αSMA.\n\nHeterogeneous expression of surface receptor Thy-1 in fibroblasts from several tissues is well established. Normal lung fibroblasts express Thy-1, whereas myofibroblasts in the fibroblastic foci in idiopathic pulmonary fibrosis lack Thy-1 expression29. It has been shown that loss of Thy-1 in human lung fibroblasts induces a fibrogenic phenotype30. In contrast to lung fibroblasts, TGFβ up-regulated αSMA expression only in Thy-1+ myometrial and orbital fibroblasts31. These results show that the presence rather than the absence of CD90 apparently favors the appearance of a myofibroblast phenotype in response to TGFβ. In this study, we did not observe any difference in CD90 expression between IMF from IBD patients and controls.\n\nOur results reveal that IMF are also enriched in several mediators. COL1A1 and FN1 were highly expressed in IMF. However, expression of the fibrogenic gene CTGF, also known as CCN2, a key mediator of ECM production in pathological fibrotic conditions, was similar to that in the mucosa. This is likely because the epithelium is an important source of CTGF32. CCN1/CYR61 expression was augmented in IMF compared to the intestinal mucosa. To our knowledge, this is the first time that CCN1 expression is reported in IMF. CCN1 levels in parenchymal liver cells were relatively low compared to that in hepatic stellate cells and portal myofibroblasts33. The same study found that overexpressed CCN1 significantly inhibited production of collagen type I, attenuated TGFβ signaling and induced production of reactive oxygen species (ROS), leading to dose-dependent cellular senescence and apoptosis. On the contrary, CCN1 has been shown to augment TGF-β signaling and contribute to fibrogenic responses to lung injury34. Its role in intestinal fibrosis is still unknown.\n\nIt was recently demonstrated that CCN1 promotes mucosal healing in murine colitis. Mechanistically, CCN1 induced IL-6 in macrophages and fibroblasts and promoted intestinal epithelial healing35. IL-6 is produced by several cell types in the lamina propria. Our data indicates that IMF cultures spontaneously released IL-6 and IL-8. In addition, we found that both fibrogenic and inflammatory stimuli can up-regulate IL-6 production. IL-6 has been shown to induce production of collagen 1 and fibronectin in fibroblasts from normal lungs and in idiopathic pulmonary fibrosis. In vivo neutralization of IL-6 trans-signaling resulted in a reduction in pulmonary inflammation and fibrosis, associated with improvement in respiratory function36. Neutralization of autocrine IL-6 reversed STAT3 phosphorylation and normalized expression of TGFβ1 in structured intestinal muscle37.\n\n\nConclusions\n\nWe demonstrated that TGFβ induced several pro-fibrotic genes in IMF. Although IMF are reported to be activated and to express αSMA18,22,28, stimulation with TGFβ resulted in a 20-fold increase of αSMA expression. This is accompanied by upregulation of COL1A1, FN1 and CTGF. Interestingly, TGFβ increased expression of LOX, an enzyme required to modify collagen, a pre-requisite for the cross-linking of collagen. Inhibition of LOX has recently been reported to alleviate lung fibrosis by modulating the inflammatory response preceding myofibroblast accumulation38. NOX4 expression was also induced by TGFβ. NOX4 modulates TGFβ/SMAD-signaling via intracellular ROS production. Increased expression of NOX4 has been reported in idiopathic pulmonary fibrosis, suggesting its role in pathogenesis39. FSP1 and CD90 expression were decreased by TGFβ. αSMA expression by IMF and down-regulated by IFNγ40. IL-1 and TNFα induced loss of fibroblast Thy-1 surface expression in vitro29. IFNγ, in combination with TNFα, has been associated with the loss of pericryptal intestinal myofibroblasts41. Whether decreased FSP1 and CD90 expression by TGFβ has a functional consequence on IMF is currently unknown. A recent study provided evidence of HIF-1 dependent induction of Notch ligands associated with M1 macrophages42. In contrast to M2 macrophages, M1 cells activate Notch signaling pathway in epithelial cells. It was suggested that the prevalence of M2 over M1 macrophages in the mucosa of CD patients may mediate the diminished enterocyte differentiation and impaired mucosal regeneration observed in these patients42. The current study extends our knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required. A better understanding of the reciprocal regulation of macrophage phenotype and mucosal repair following intestinal damage will help to establish new approaches to CD therapy.\n\n\nData availability\n\nF1000Research: Dataset 1. The following raw data sets are provided as comma separated values (.csv) files:, https://doi.org/10.5256/f1000research.13906.d23172243.\n\nFigure 1 dataset on immunostaining of IMF primary cultures.\n\nFigure 2 dataset on the expression of fibrosis-related mediators and fibroblast markers by intestinal myofibroblasts (IMF) compared to intestinal mucosa levels in human bowel resections.\n\nFigure 3 dataset on the inflammation-related gene expression in intestinal myofibroblasts (IMF) and intestinal mucosa resections.\n\nFigure 4 dataset on the mucosal gene expression from control and inflammatory bowel disease patients.\n\nFigure 5 dataset on the fibrosis-related gene expression in intestinal myofibroblasts cultures obtained from resected bowel in control and inflammatory bowel disease patients.\n\nFigure 6 dataset on the inflammatory gene expression in intestinal myofibroblasts obtained from control and inflammatory bowel disease patients.\n\nFigure 7 dataset on the effect of TGFβ on fibrosis-related gene expression intestinal myofibroblasts obtained from control and inflammatory bowel disease patients.\n\nFigure 8 dataset on the effect of TGFβ and IL-1β+IL-23 on cytokine production by intestinal myofibroblasts obtained from control and inflammatory bowel disease patients.",
"appendix": "Grant information\n\nSupport for this research was in the form of funds provided to EG Seidman as a Tier 1 Canada Research Chair in immune mediated gastrointestinal disorders. PS Escobar was provided salary support through the Spanish government with a predoctoral training grant Resolución de la Presidencia de la Agencia Estatal de Investigación, por la que se conceden ayudas a la movilidad predoctoral para la realización de estancias breves en Centros de I+D, convocatoria 2016. S Restillini was provided salary support by the Government of Switzerland through her home institute Geneva University Hospital.\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\nAbraham C, Medzhitov R: Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology. 2011; 140(6): 1729–37. 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F1000Research. 2019. http://www.doi.org/10.5256/f1000research.13906.d231722"
}
|
[
{
"id": "48693",
"date": "24 May 2019",
"name": "Archana Gopalakrishnan",
"expertise": [
"Reviewer Expertise Innate immunity",
"tuberculosis induced immune response",
"macrophages",
"inflammation",
"flow cytometry",
"qRT-PCR",
"cytokines and chemokines."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSince fibrosis is a major complication in IBD patients and the published literature indicates a fibrogenic role of myofibroblasts, the authors have an important rationale for understanding the characteristics of myofibroblasts in the intestines (IMF). However, this study has introduced a number of variables, such as mucosal tissues versus IMF and IBD patient versus control groups that are confusing to the overall aim of the study. Some of the areas that can be better explained include:\nIt is unclear in the Results pertaining to Figures 2 and 3 from which of the four groups of patients the IMF cultures were characterized.\n\nWhen examining the inflammatory potential of IMF, an elevated level of IL-6 and IL-8 is reported, but not TNF and IL-17. It would be interesting to hear a speculation on why this is so.\n\nIMFs from control groups and IBD groups did not vary in their expression of fibrosis markers - doesn't this undermine the role of IMFs in IBDs, where fibrosis is a major complication?\n\nSeveral references have been made to the lung mucosal surface that differs significantly from the intestinal mucosa.\n\nIs the work 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? Yes",
"responses": []
},
{
"id": "49054",
"date": "12 Jun 2019",
"name": "Andrew Silver",
"expertise": [
"Reviewer Expertise Molecular genetic colorectal"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have presented an account of their study on characterising human myofibroblasts from IBD patients, both Crohn’s disease (CD) and ulcerative colitis. The authors are to be commended for obtaining human tissue from a good number of patients - the effort involved is significant. However, I do have some important comments on the manuscript, which I feel should be addressed so that the reader is better informed.\n\nComments:\nThere is insufficient detail on and phenotyping of the resection specimens. CD is a very diverse disease. There are a number of factors, e.g. site, active/inactive disease, presence of stricture etc., that can have a major impact on gene expression. The reader is unable to assess pathological features with respect to the expression data.\n\nFigure 1 is incomplete. Why only a control patient for panel A? Why CD90 for panel B and only one specimen from a UC patient? Why are so few cells in A showing a-SMA expression? Negative controls? Magnification? Use of overly? Make sure the legends fully explain the figure - see also comments below.\n\nI am unclear as to why the numbers of data points varies so often within (mucosa versus IMF) and between graphs (e.g., CCN1 Versus FN1) within a given Figure? The numbers appear very different to those indicated in the text. There could be a number of reasons why this has happened. The authors should explain this anomaly clearly along with any potential influence loss of data points has had on their analyses.\n\nThere are insufficient mRNA expression data points to do a sub-group (UC and CD) analysis. Given the nature and diversity of these diseases I would have focused on one or the other, especially given that fibrosis and subsequent stricture formation predominately affect CD patients. From the clinical table, it appears that seven Crohn’s patients had a structuring phenotype, how do markers of fibrosis in IMFs isolated from these patients compare with inflammatory IMFs for UC/CD controls? The authors could also compare their mRNA data to what is already published in the literature.\n\nmRNA levels of some inflammatory cytokines are enriched in IMFs over mucosa, how do the authors explain this, given IMFs are not the primary cytokine producing cells?\n\nData and text to justify the use of GADPH as the normaliser would be useful.\n\nFigures 7 and 8 are very difficult to follow because of a lack of information in the text and the legend. For example, the title for Figure 7 states, “gene expression intestinal myobroblasts (IMF) obtained from control and inflammatory bowel disease (IBD) patients” yet there is only a single bar for each gene? This should be explained in the legend and then numbers used should be given in the legend. The use of black plus, I think, two shades of grey which do not match the key complicate matter further. Once again it would be more clinically impactful to have compared response between different diseases and phenotypes. Currently numbers are too small to do this.\n\nIn Figure 8 have the cultures been pooled? Why are the numbers different between panels A/B and C? What were the criteria for selecting these particular cultures over others?\n\nThere is no clear explanation as to how the data presented in Figures 7 and 8 relate to the mRNA data given in earlier Figures. There is a lack of explanation as to why select the various cytokine combinations for stimulation experiments.\n\nOverall there is an absence of protein validation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-275
|
https://f1000research.com/articles/7-1145/v1
|
27 Jul 18
|
{
"type": "Software Tool Article",
"title": "EpiLog: A software for the logical modelling of epithelial dynamics",
"authors": [
"Pedro L. Varela",
"Camila V. Ramos",
"Pedro T. Monteiro",
"Claudine Chaouiya",
"Pedro L. Varela",
"Camila V. Ramos"
],
"abstract": "Cellular responses are governed by regulatory networks subject to external signals from surrounding cells and to other micro-environmental cues. The logical (Boolean or multi-valued) framework proved well suited to study such processes at the cellular level, by specifying qualitative models of involved signalling pathways and gene regulatory networks.\n\nHere, we describe and illustrate the main features of EpiLog, a computational tool that implements an extension of the logical framework to the tissue level. EpiLog defines a collection of hexagonal cells over a 2D grid, which embodies a mono-layer epithelium. Basically, it defines a cellular automaton in which cell behaviours are driven by associated logical models subject to external signals.\n\nEpiLog is freely available on the web at http://epilog-tool.org. It is implemented in Java (version ≥1.7 required) and the source code is provided at https://github.com/epilog-tool/epilog under a GNU General Public License v3.0.",
"keywords": [
"Logical modelling",
"Multicellular regulatory networks",
"Cellular automaton",
"Hexagonal grid"
],
"content": "Introduction\n\nPattern formation emerges from the interplay of interaction networks, at the cellular and multi-cellular levels1. To uncover the complex mechanisms at stake, computational modelling is very needed. In this context, cellular automaton approaches are particularly well suited2.\n\nOn the other hand, the logical formalism has proved efficient to explore cellular regulatory networks driving developmental processes (for a recent review on the logical modelling approach, see 3). The consideration of ensembles of communicating cells is often required to recapitulate observed patterns (e.g. 4,5).\n\nThis motivated the development of EpiLog, which implements an extension of the logical framework to hexagonal grids embodying simple epithelia.\n\nBriefly, a logical model assigns discrete (Boolean or multi-valued) variables to the regulatory components, and logical regulatory rules define the evolution of the variables depending on the variables associated with the component regulators. Input components embody cell receptors receiving external signals. Unlike internal components, inputs have no associated regulatory rules, and are generally maintained at constant values accounting for fixed environmental conditions. A model state is given by the values of the components, and the dynamics is generated according to specific updating schemes. In a synchronous update, a state has at most one successor state in which all the variables have been updated as prescribed by the regulatory rules. In contrast, the asynchronous update generates as many successors as the number of updated variables, yielding more complex and non-deterministic behaviours. Properties of the resulting dynamics relate to the model attractors (stable states or point attractors, and cyclical attractors), which can be associated with different phenotypes. Beyond identifying the attractors, a major question relates to reachability properties: given an initial condition, what is (are) the reachable attractor(s)?\n\nThe definition, analysis and simulation of logical models of cellular networks can be performed using one of the existing software tools6,7. Many of these support the exchange format SBML qual (Qualitative Models package for SBML)7,8.\n\nTo handle multi-cellular systems, Mendes et al. have established a compositional approach relying on a process algebra framework to assess stable states reachability in asynchronous dynamics9. This approach showed some limitations due to the huge size of the considered state spaces, and is thus limited to the analysis of models encompassing a reduced number of cells (up to a dozen cells). Nonetheless, in the cellular automaton framework considered here, we use the same approach to specify cell-cell communication through (logical) integration rules. These govern the evolution of cellular input components, depending on the values of internal components (representing e.g. secreted proteins) in neighbouring cells.\n\n\nMethods\n\nEpiLog is implemented in Java (requires Java 7 or higher), is freely available at http://epilog-tool.org, and launches a Graphical User Interface (GUI) based on JFC/Swing. It is provided as a single .jar file which can be launched from a graphical file manager by double clicking on it or through the command line as follows:\n\n\n\nwhere the flag --file can be used to optionally specify the file to be open. EpiLog .peps files represent projects containing multiple multicellular models, each with potentially distinct characteristics.\n\nAdditionally, an EpiLog model repository is provided at http://epilog-tool.org/models_repository where users can deposit their models by contacting support@epilog-tool.org. Each model page includes a title, a description, a taxon and process classification, model .peps file(s) and supporting paper(s).\n\nDevelopers can freely access the code repository at http://github.com/epilog-tool/epilog, clone it and extend the code. For dependency management of external libraries, EpiLog relies on Apache Maven. A particular library is the bioLQM Java toolkit (Logical Qualitative Models of biological regulatory networks), for the representation and manipulation of logical, cellular models, available at https://github.com/colomoto/bioLQM10.\n\nModel definition EpiLog defines projects that include models, called epithelia, each being specified by:\n\n1. The size of the hexagonal grid, as well as its border conditions; vertical and horizontal borders can be connected, or not leading to rectangular, cylindrical or toroidal grids;\n\n2. A (set of) logical model(s), each associated with cells of the grid; these cellular models can be defined in any tool supporting the SBML qual format8. EpiLog imports SBML files and identifies input components in these cellular models;\n\n3. The cellular input components; these are defined as constant positional inputs (e.g. morphogens produced by external sources) or evolving integration inputs; for the latter, logical integration rules specify which signals are received from (internal) components of neighbouring cells and how these signals are combined;\n\n4. An initial state defining the values of the internal components of all the cells.\n\nSimulation settings For each epithelium, simulation settings define the updating schemes for both the cellular and epithelium models. The default scheme is synchronous, that is to say, at each iteration, all the cellular models are considered for a synchronous update of their internal components. To overcome spurious oscillations generated by these synchronous updates (see Figure 1A and Supplementary File 1), EpiLog allows to specify:\n\n1. The cellular model update, with the definition of (synchronous) priority classes (increasing or decreasing) updates are gathered in ordered sets, allowing to account for different time scales for the mechanisms underlying component updates11;\n\n2. The epithelium update, with the definition of probabilistic updates of the cell states, inspired by N. Fatès’ alpha-asynchronism12: parameter α specifies the proportion of the cells randomly chosen for update.\n\n(A) An idealised Boolean model of lateral inhibition; the Green marker is induced by the Red marker secreted by the neighbouring cell, whereas Red is induced by low levels of Green in the same cell; the synchronous dynamics of this two cell model leads to a cyclic attractor (see Supplementary File 1). (B) Simulation in EpiLog, with the same cellular model over a square grid of 50 × 50 hexagonal cells under a synchronous update (Green induced if at least one contacting cell is Red), oscillations are due to the synchronous update of the cells, see Supplementary Movie 1. (C–F) Simulations under α-asynchrony (α = 0.25): (C) Stable pattern reached in 29 steps (Green induced if at least one contacting cell is Red), see Supplementary Movie 2; (D) Stable reverted pattern reached in 30 steps (Green induced if all contacting cells are Red); (E) Stable pattern reached in 63 steps (Green induced if at least 12 cells at distance up to 3 are Red); (F) Stable pattern reached in 70 steps, with the same setting as for panel E but considering a torus (i.e., no grid borders).\n\nModel perturbations The logical formalism permits to easily specify perturbations (e.g. knock-out or ectopic expression of cellular model components) by blocking the values of perturbed components. EpiLog includes this feature with the definition of component perturbations that can be applied to all the cells or to restricted regions of the grid.\n\nThe reader is invited to consult the documentation available at EpiLog web site to get further information (http://epilog-tool.org/documentation).\n\n\nUse cases\n\nAs a first illustration, we consider a very simple model, in which the cellular model includes two markers. When considering a juxtacrine signal (i.e., only contacting cells can communicate), this model accounts for the well-known Delta-Notch signalling, which implements a lateral inhibition process (see e.g. 13). The model is provided in Supplementary Model File 1. In Figure 1A, a two-cell model is shown, to illustrate interactions within and between the cells. Remaining panels of Figure 1 show EpiLog simulation results, illustrating the different patterns obtained when varying the updating mode, the integration rules and the border conditions.\n\nThe development of EpiLog was originally motivated by our second illustration, which relates to the dorsal appendage-forming regions of the Drosophila Melanogaster eggshell14 (see Figure 2). The logical model defined by Fauré et al. reproduces the patterning of the anterior follicular epithelium of the oocyte, defining the floor (Rho expressing cells) and roof (Br expressing cells) of the future appendages5. The model is provided in Supplementary Model File 2. EpiLog window is shown in Figure 2A, with the simulation panel displaying the stable pattern obtained with this model. The remaining panels C–G show different states of the grid, with panels F–G recapitulating patterns resulting from model perturbations (see Figures 6–8 in 5).\n\n(A) EpiLog main window with the stable pattern reached by a simulation starting with all the cells of the grid having their internal components at 0, and positional inputs defined as shown in panel C (phase 1). (B) Egg chamber with two dorsal respiratory appendages (DA); DA primordia are established as 2 regions on both sides of the oocyte midline, with follicle cells expressing Broad (Br in red), future roof of the DA, and cells expressing Rhomboid (Rho in blue), future floor of the DA. Establishment of these regions involve Gurken signalling (Grk, in orange) and Decapentaplegic signalling (Dpp, in violet). (C) Grk and Dpp gradient defined as positional inputs in EpiLog. (D) Stable pattern obtained with a simulation starting from the pattern displayed in panel A and without the Grk signal (phase 2), suggesting that Grk extinction is required to split the floor regions (see 5). (E) EpiLog simulation of a mild overexpression of Dpp. (F) EpiLog simulation of Pointed (Pnt, internal component) loss-of-function. (G) EpiLog simulation of Pnt gain-of-function clones. Note that in panels F–G, perturbed cellular models are indicated by bold borders in the grid.\n\n\nSummary\n\nTo the best of our knowledge, EpiLog is the first software tool for the definition, simulation and visualisation of qualitative, logical (Boolean and multivalued) models over hexagonal grids. It provides a graphical user interface and tools to conveniently support the study of epithelial pattern formation, relying on a logical framework.\n\nEpiLog is free, open source and implemented in Java for operating system independence. It relies on the SBML qual standard format to import cellular models.\n\n\nSoftware availability\n\nEpiLog v1.1 is available from: http://epilog-tool.org/downloads\n\nSource code available from: https://github.com/epilog-tool/epilog\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.132050315\n\nLicense: GNU General Public License v3.0\n\n\nAuthor information\n\nPV and CR developed the software. PTM acquired funding, developed the software and supervised the project. CC designed the project, acquired funding and supervised the project. PLV, CVR, PTM and CC wrote the article.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by national funds through Fundação para Ciência e a Tecnologia (FCT) with reference PTDC/BEX-BCB/0772/2014, UID/CEC/50021/2013 and IF/01333/2013. PLV was supported by grant PTDC/BEX-BCB/0772/2014 and PTDC/EIA-CCO/099229/2008. CVR was supported by PTDC/BEX-BCB/0772/2014 and IF/01333/2013/CP1204/CT0001. Furthermore, CC and CVR acknowledge support from the Fundação Calouste Gulbenkian.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors would like to thank Adrien Fauré who implemented a first Python prototype that inspired the development of EpiLog, and who provided insightful feedback. We are also grateful to Céline Hernandez for her useful comments.\n\n\nSupplementary material\n\nSupplementary File 1: Dynamics of the lateral inhibition model under synchronous and asynchronous updates.\n\nClick here to access the data.\n\nSupplementary Movie 1: Oscillatory behaviour of the lateral inhibition model under a synchronous update (see Figure 1 panel B).\n\nClick here to access the data.\n\nSupplementary Movie 2: Stable pattern of the lateral inhibition model under an α-asynchronous update (see Figure 1 panel C).\n\nClick here to access the data.\n\nSupplementary Model File 1: EpiLog file including variations of the lateral inhibition model as illustrated in Figure 1.\n\nClick here to access the data.\n\nSupplementary Model File 2: EpiLog file including variations of the DA model as illustrated in Figure 2.\n\nClick here to access the data.\n\n\nReferences\n\nKicheva A, Cohen M, Briscoe J: Developmental pattern formation: insights from physics and biology. Science. 2012; 338(6104): 210–2. PubMed Abstract | Publisher Full Text\n\nDeutsch A, Dormann S: Cellular automaton modeling of biological pattern formation: characterization, applications, and analysis. Birkhäuser Boston, 2005. Publisher Full Text\n\nAbou-Jaoudé W, Traynard P, Monteiro PT, et al.: Logical Modeling and Dynamical Analysis of Cellular Networks. Front Genet. 2016; 7: 94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSánchez L, Chaouiya C, Thieffry D: Segmenting the fly embryo: logical analysis of the role of the segment polarity cross-regulatory module. Int J Dev Biol. 2008; 52(8): 1059–75. PubMed Abstract | Publisher Full Text\n\nFauré A, Vreede BM, Sucena E, et al.: A discrete model of Drosophila eggshell patterning reveals cell-autonomous and juxtacrine effects. PLoS Comput Biol. 2014; 10(3): e1003527. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaldi A, Monteiro PT, Müssel C, et al.: Cooperative development of logical modelling standards and tools with CoLoMoTo. Bioinformatics. 2015; 31(7): 1154–9. PubMed Abstract | Publisher Full Text\n\nChaouiya C, Bérenguier D, Keating SM, et al.: SBML qualitative models: a model representation format and infrastructure to foster interactions between qualitative modelling formalisms and tools. BMC Syst Biol. 2013; 7: 135. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaouiya C, Keating SM, Berenguier D, et al.: The Systems Biology Markup Language (SBML) Level 3 Package: Qualitative Models, Version 1, Release 1. J Integr Bioinform. 2015; 12(2): 270. PubMed Abstract | Publisher Full Text\n\nMendes ND, Lang F, Le Cornec YS, et al.: Composition and abstraction of logical regulatory modules: application to multicellular systems. Bioinformatics. 2013; 29(6): 749–57. PubMed Abstract | Publisher Full Text\n\nNaldi A: bioLQM: a java library for the manipulation and conversion of Logical Qualitative Models of biological networks. bioRxiv. 2018; 287011. Publisher Full Text\n\nFauré A, Naldi A, Chaouiya C, et al.: Dynamical analysis of a generic Boolean model for the control of the mammalian cell cycle. Bioinformatics. 2006; 22(14): e124–131. PubMed Abstract | Publisher Full Text\n\nFatès N: A Guided Tour of Asynchronous Cellular Automata. In Cellular Automata and Discrete Complex Systems - 19th International Workshop, AUTOMATA 2013, Gießen, Germany, September 17–19, 2013. Proceedings. 2013; 15–30. Publisher Full Text\n\nCollier JR, Monk NA, Maini PK, et al.: Pattern formation by lateral inhibition with feedback: a mathematical model of delta-notch intercellular signalling. J Theor Biol. 1996; 183(4): 429–46. PubMed Abstract | Publisher Full Text\n\nPyrowolakis G, Veikkolainen V, Yakoby N, et al.: Gene regulation during Drosophila eggshell patterning. Proc Natl Acad Sci U S A. 2017; 114(23): 5808–5813. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVarela PL, Monteiro PT, Ramos CV: epilog-tool/epilog: EpiLog v1.1 (Version v1.1). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1320503"
}
|
[
{
"id": "37297",
"date": "20 Aug 2018",
"name": "Stefano Nichele",
"expertise": [
"Reviewer Expertise Artificial Life",
"Complex Systems",
"Evo-Devo",
"Cellular Automata"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes EpiLog, a software tool to model artificial development of epitelial tissue through cellular automata with hexagonal cells.\n\nEpiLog provides a high degree of customization through the graphical user interface. This includes: (a) model definition, such as CA size, logical models and initial states; (b) simulation settings, including updates (synchronous or asynchronous/probabilistic) and (c) perturbations on single cells or regions.\n\nThe article provides two simple use cases to introduce the user to the software. First, a model of lateral inhibition process is presented in FIg. 1. Second, a developmental model of Drosophila eggshell.\n\nMinor revisions:\nThe article would benefit by the following:\nbackground section with related work and description of CA/artificial development software tools, with brief description of similarities and differences;\n\na more advanced use case described in details (for the advanced reader).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4451",
"date": "11 Mar 2019",
"name": "Claudine Chaouiya",
"role": "Author Response",
"response": "We than you for the suggestions. We considered that a proper state of the art was beyond the scope of this application note. However, we now acknowledge the existence of two closely related tools, CoGNaC and PhysiBoSS. The DA formation is a real, advanced case study. The interested reader can refer to the original paper by Fauré et al., and can also download the model from EpiLog web site."
}
]
},
{
"id": "38246",
"date": "04 Oct 2018",
"name": "Alejandro F. Villaverde",
"expertise": [
"Reviewer Expertise David Henriques: dynamic modelling",
"bioinformatics",
"logic-based modelling",
"systems biology. Alejandro F Villaverde: dynamic modelling",
"system identification",
"systems biology",
"control theory."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes a new software tool for modelling epithelial dynamics in 2D. The software, called EpiLog, uses a logical framework that assigns discrete values to the model variables. The approach used for specifying the logical rules was previously presented in a paper from the same senior author (Mendes et al1).\nThe paper is well written, and the software is provided in a nice dedicated website, which also includes documentation and additional information. Furthermore, the source code is provided at GitHub.\nWe have a few comments and suggestions regarding both the methodological approach and the software implementation:\nIn regard to the method:\n1) It is stated that the published method can only handle up to a dozen cells. However, the graphical interface seems to describe more. Maybe the authors could elaborate on the limitations of the tool, as well as on its performance for different types of problems.\n2) It is not totally clear whether the approach used in this paper is exactly the same as the one published in (Mendes et al1). If it is not, and if there is no other published paper describing the method, an upgraded methods paper could be of interest. On the other hand, if there is another publication describing the method, it should be cited in the paper. It is also unclear if the method has been previously validated in epithelium (hexagonal) case. The authors may want to clarify these points in a revised version.\n3) We are aware that, being a software tool article, the present paper is probably not the best place for extended discussions about biological aspects. The following points may therefore be considered as suggestions for the content of an upgraded paper as described in the previous paragraph. Of course, they would also be welcome additions to an extended version of the present paper, if the authors deem them appropriate to address:\n3.1) It may be useful to describe how to define a new (toy) model and a grid from scratch.\n3.2) What type of cues/signals are considered?\n3.3) A real case study would make it easier to follow the pipeline.\n3.4) Some additional biological explanation or motivation of potential applications might make the paper more attractive.\nIn regard to the software:\n1) We tested EpiLog in Windows. We managed to load and simulate the Drosophila \"Eggshell Patterning\", but the software reported warnings/errors. Are these relevant?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4452",
"date": "11 Mar 2019",
"name": "Claudine Chaouiya",
"role": "Author Response",
"response": "We thank you for the comments and suggestions, and we briefly answer each point or the report below. 1) We believe the misunderstanding was due to the confusion with our mention to a previous work (Mendes at al 2013). This should now be clarified. 2) This should be clear in the revised text. The rationale to define cell-cell communication (i.e. the rules governing the behaviours of input components of cellular models) relies on the definitions presented in Mendes et al 2013. However, EpiLog provides a graphical 1 interface and a simulation platform, producing a single model trajectory, whereas Mendes et al. aimed at determining all the stable states reachable from a given initial condition and considering an asynchronous update. 3) 3.1 should now be addressed through the video that are available on EpiLog web site 3.2 integration inputs are now more explicit in the text (juxtacrine signals, or other secreted molecules influencing cells in the neighbourhood) 3.3 the DA formation is a real case study. The interested reader can refer to the original paper by Fauré et al., and can also download the model from EpiLog web site. 3.4 Indeed, we intend to include further arguments on biological applications in a forthcoming paper on lattice-based modelling approaches 4) Regarding the software: EpiLog relies on bioLQM, which in turn relies on JSBML libraries. The warning messages are being thrown by JSBML when loading a cellular model, but do not affect any of EpiLog’s behaviour."
}
]
},
{
"id": "37601",
"date": "10 Oct 2018",
"name": "Arnau Montagud",
"expertise": [
"Reviewer Expertise Systems Biology",
"Cancer",
"Boolean modelling",
"Multiscale modelling",
"Metabolic modelling",
"Data deconvolution"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 describes a software used for qualitative simulations of logical models in a hexagonal grid, called EpiLog. The paper is well written, even though some parts could be slightly expanded for a better comprehension. The software is easy to use for people with logical modelling background and is a visual way of exploring populations of Boolean models and their effect on neighbouring cells. The software is freely available, cross-platform, can use open standards of logical models and its code can be studied and forked in GitHub.\nNevertheless, the paper would benefit from some additions and clarifications:\nMajor:\nExpansion of introduction: The last sentence of first paragraph could be completed with some of the reasons cellular automaton are well suited. Also, the relationship of present paper and [1] could be better worded. It is unclear if authors used Mendes et al approach as is or if they improved Mendes et al approach and can work with more than a dozen cells. Addressing these would enhance the understanding of the work.\n\nDiscussion on the alpha parameter: The paper would benefit from a discussion on the use of “alpha-asynchronism” as logical modelling tools, until now, either used asynchronous and synchronous updates. Also, the paper presents results using alpha=1 (pure synchronous) or 0.25, but does not present results with alpha=0 (pure asynchronous). Presenting and discussing these (even in Supplementary) would allow users to have a better understanding on the effects of this parameter.\n\nDetailed tutorial: Even though the Documentation does a good job detailing the possibilities of the tool, the software users would benefit from a detailed discussion on the effect of changing spatial initial conditions, alpha parameter and, in general, the biological relevance of these. This supplementary file would also allow to expand on the discussion of Figure 2 that is absent from the paper (understandably being a software tool paper).\nMinor:\nComparison to other software: Even though this software is the first that includes hexagonal grid and Boolean models, naming some other software that can be used for similar projects (with their pros and cons) would give the reader a better sense on the current status of the field.\n\nIn Methods/Operation/Simulation settings, first sentence, authors say “For each epithelium, simulation settings define the updating schemes for both the cellular and epithelium models.” Does cellular model mean one cell and epithelium model the whole set of cells? As the phrase starts with epithelium, the current wording is a bit confusing.\n\nFigure 1 legend: “Stable pattern” is a bit confusing, maybe it would be better to use “pattern of stable states”.\n\nFigure 2 legend: Authors should describe “phase 1” and “phase 2” in the paper to understand what is the difference in panels C and D. In panel B, “DA” should be shown in the figure. In panel C, the colours for Grk and Dpp should be explained (even if they are described in previous panel).\n\nIn Supplementary text, in the last part of the last sentence, the Figure in main text you refer to is Figure 2, panels C-F.\n\nFollowing previous reviewers’ comments, I had no errors using EpiLog on Windows.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "4453",
"date": "11 Mar 2019",
"name": "Claudine Chaouiya",
"role": "Author Response",
"response": "We thank you for the detailed comments and suggestions. We have revised the manuscript accordingly, and we briefly answer your points below. We have revised the introduction, noting that cellular automata are a natural modelling choice to define local rules governing the behaviours of cells over a lattice. We also clarified the link between the framework as implemented in EpiLog and the work presented in Mendes et al 2013. We have included a supplementary text illustrating and discussing the use of the alpha-asynchronous update, and varying the values of alpha. This is done using the simple model of lateral inhibition. It is worth to note that a general statement of how the value of alpha impacts the dynamics (intermediate and final patterns) is hard to make, as such properties certainly depend on the model. Finally, alpha-asynchronism has been considered for cellular automata. The reviewer is right when noting that this update has not been employed for (cellular) logical models but, besides a theoretical interest, it is unclear if such an update would have any biological significance besides the fact that indeed a few cells may update their states simultaneously. Concerning your point on a detailed tutorial, in addition to the documentation,we now provide some video to illustrate the major steps in defining and simulating a model with EpiLog (available at https://www.youtube. com/channel/UCGGcpepqyYJkt7dhrhncQiQ, as indicated in the documentation). Results displayed in Figure 2 are thoroughly discussed in the original paper, Fauré et al. 2014. We now mention the two other software tools we are aware of that consider logical models in a multi-scale setting. We have clarified the terminology. In particular we have changed the legend of Fig 1, which now says ”stable state of the grid, referred to as stable pattern”. Indeed a stable pattern is defined by stable states of the cells, but not any combination of such cellular stable states remain stable in the multi-cellular model. Concerning Fig. 2, the requirement for the 2 phases is now shortly described in the main text. The legend has been modified as suggested, and dorsal appendage (DA) has been added in panel B."
}
]
}
] | 1
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https://f1000research.com/articles/7-1145
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https://f1000research.com/articles/8-273/v1
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11 Mar 19
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{
"type": "Research Article",
"title": "Analysis of transgenic zebrafish expressing the Lenz-Majewski syndrome gene PTDSS1 in skeletal cell lineages",
"authors": [
"Marian Seda",
"Emma Peskett",
"Charalambos Demetriou",
"Dale Bryant",
"Gudrun E. Moore",
"Philip Stanier",
"Dagan Jenkins",
"Marian Seda",
"Emma Peskett",
"Charalambos Demetriou",
"Dale Bryant",
"Gudrun E. Moore"
],
"abstract": "Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.",
"keywords": [
"Lenz-Majewski syndrome",
"Tol2-kit"
],
"content": "Introduction\n\nLenz-Majewski syndrome (LMS; MIM 151050) is a rare disease, characterized by a complex set of clinical features that are progressive and potentially life limiting. These include osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. The patients also have severe growth restriction and moderate to severe intellectual disability (Sousa et al., 2014). To date, only 16 affected individuals have been reported worldwide (Chrzanowska et al., 1989; Dateki et al., 2007; Gorlin & Whitey, 1983; Mizuguchi et al., 2015; Nishimura et al., 1997; Piard et al., 2018; Saraiva, 2000; Shoja et al., 2013; Sousa et al., 2014; Tamhankar et al., 2015; Wattanasirichaigoon et al., 2004; Whyte et al., 2015). We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene (Sousa et al., 2014). PTDSS1 encodes an enzyme, phosphatidylserine synthase 1 (PSS1), which catalyses the base-exchange reaction of serine for choline such that phosphatidylcholine is converted to phosphatidylserine (PS). PSS1 is in fact one of two enzymes (along with PSS2) involved in the production of PS, where in an alternative pathway, PSS2 catalyses a similar serine exchange reaction with phosphatidylethanolamine. PS is a quantitatively minor, but physiologically important phospholipid present in all mammalian cells (Vance et al., 2018). In addition to its role as a constituent of membranes, PS is also known to play important roles in other cellular pathways including apoptosis, cell signalling and mineralisation (Vance & Tasseva, 2013; Wu et al., 2008). Interestingly, despite complete loss of PSS1 in homozygous Ptdss1 knockout mice, which resulted in a significant reduction in PS synthase activity, the mice appeared morphologically normal, viable and fertile (Arikketh et al., 2008). Indeed, PS levels remained stable in most tissues, other than a modest reduction in the liver, presumably compensated for by PSS2 activity. In contrast, LMS was found to result from gain-of-function (GOF) mutations, leading to increased enzyme activity and blocking end-product inhibition of PSS1 (Sousa et al., 2014). Several of the reported missense mutations are recurrent in unrelated patients, with clustering of other mutations in close proximity, indicating a highly specific role for these particular amino acids and/or their functional domains (Piard et al., 2018; Sousa et al., 2014; Tamhankar et al., 2015).\n\nThe identification of genes that cause rare skeletal dysplasias and extreme bone mineral density phenotypes in humans, including osteosclerosis and craniotubular hyperostosis, along with analysis of vertebrate models of these conditions, has shed important insights into the mechanisms that regulate bone tissue homeostasis (Goltzman, 2002). Several key cell types are known to regulate bone formation and homeostasis and are conserved in zebrafish (Chatani et al., 2011; Dale & Topczewski, 2011; Eames et al., 2012; To et al., 2012). Chondrocytes are responsible for cartilage formation, which is composed primarily of collagen II/IX/XI, as well as proteoglycan and glycosaminoglycan, and serves as a scaffold within which osteoblasts and osteoclasts regulate bone mineralisation. Osteoblasts differentiate from mesenchymal stem cells through expression of factors that drive bone formation such as Runx2 and Osterix, through secretion of structural components of the bone matrix, enzymes and growth factors. The receptor ligand, RANKL, is expressed on the surface of osteoblasts, and binding of RANKL to its receptor, RANK, on the surface of monocytes stimulates their maturation into osteoclasts that resorb bone. This process is also negatively-regulated by the osteoblast-secreted decoy receptor, OPG, and so the RANK/RANKL/OPG signalling axis serves as a feedback mechanism to regulate bone turnover by osteoblasts and osteoclasts.\n\nHuman and zebrafish PTDSS1 orthologues share 78% amino acid identity. We previously showed that microinjection of physiologically high doses of RNA encoding human mutant forms of PTDSS1 found in LMS caused generalized embryo toxicity, including axial defects, eye loss and jaw cartilage patterning defects, whereas injection of wild-type RNA had no effect even at much higher doses (Sousa et al., 2014). The frequency of these defects correlated with RNA dose, thereby serving as a biochemical readout of LMS gain-of-function mutation activity. The abnormal embryos in these experiments did not survive beyond the larval free-feeding stage, and microinjected RNA is relatively unstable in zebrafish embryos, lasting for typically less than 3 days. In contrast, mineralized bone only emerges after 6 days in the zebrafish jaw and 2 weeks in the skull, and the skeleton is only fully formed after ~10 weeks (Eames et al., 2012; Mork & Crump, 2015; Schilling & Kimmel, 1997; Sharif et al., 2014). As such it is therefore not possible to investigate skeletal tissue development and homeostasis using transient RNA injections in zebrafish.\n\nIn this study, we used transpose-mediated transgenesis to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. We report multiple genomic integration sites for each of 8 different transgenes, with variation in the number of integrations between individuals. Despite the presence of multiple integration sites, we failed to detect gene expression at either the RNA or protein levels. All transgenic lines, however, exhibited incompletely penetrant mild scoliosis of the vertebrae, which was never observed in non-transgenic clutch mates. Taken together, these results indicate that PTDSS1 expression is either silenced to sub-detectable levels or inconsistent with formation of viable animals.\n\n\nMethods\n\nAdult and embryonic zebrafish (Danio rerio) embryos were obtained from a wild-type strain from the European Zebrafish Resource Centre and raised at 28.5°C in accordance with Home Office licence PPL 70/7892. (Westerfield, 1993). Our study adopted the ARRIVE guidelines. In various assays, we generated experimental animals from an incross of heterozygotes, and wild-type and mutant animals were compared. Animals for comparison were matched for age and size. Analyses were performed blinded to genotype. All efforts were made to minimize any suffering of animals through environmental enrichment of their habitat, avoiding overcrowding, and monitoring animals for signs of distress or discomfort on a daily basis. In situ hybridization was performed using standard techniques with RNA probes labelled with digoxigenin (Roche) and detected using NBT/BCIP (Sigma).\n\nThe Tol2 kit, plasmid construction and an explanation of the recombination events was as previously published (Kwan et al., 2007).\n\npDONRP4-P1r and pDONR221 were obtained as part of the Multisite Gateway Cloning kit (Invitrogen). The pDONR plasmids were maintained in ccdB-tolerant bacteria and grown in the presence of kanamycin and chloramphenicol. The WT human PTDSS1 ORF was cloned into pcDNA3.1 (Addgene) and mutagenesis was performed to introduce the c.1058A>G (p.Q353R) mutation (QuickChange II Site-Directed Mutagenesis Kit, Agilent Technologies). These clones were amplified using the primers shown in Table 1. The resulting fragment was included with pDONR221 in a BP recombination reaction to generate WT and mutant middle entry clones. To generate the 5’ entry clones, different promotors were amplified from zebrafish cDNA using the primers given in Table 1. BP recombination with pDONRP4-P1r was performed. The 3’ entry clone p3E mCherry IRES was synthesised by Genscript (full sequence available from (Seda et al., 2019)). Correct cloning was confirmed by Sanger sequencing using primers listed in Table 2.\n\nTo generate plasmids for injection into zebrafish embryos, Gateway LR reactions were performed according to the manufacturer's recommendations (Invitrogen). LR II Plus clonase was used and the reaction performed overnight at 25°C. 3ul of each reaction was transformed into Top10 competent cells (Invitrogen) and grown on ampicillin plates. Clear colonies were picked for miniprep and screening (Figure 1).\n\nMultisite Gateway© cloning was performed by combing a 5’ entry vector containing one of the four zebrafish promotors indicated, a middle entry vector encoding either wild-type (WT) or LMS mutant (Q353R) PTDSS1, and a 3’ entry vector encoding mCherry tagged with a nuclear localisation signal fused to a ribosome entry sequence (IRES), with a selectable Tol2 entry vector which also contained a separate cmclc2:GFP cistron for selection of animals with successful integration.\n\nOn the morning of the injection, Tol2 transposon DNA was mixed with an aliquot of Tol2 mRNA at a concentration of 12.5 ng/µL of both DNA and mRNA, diluted with RNase-free water as required. The injection volume was calibrated to inject 1–2 nL of DNA:RNA injection mix. Embryos were injected at the one cell stage using Picospritzer III (Parker Hannifin). Injected embryos were transferred to Petri dishes and incubated at 28–30°C. At the end of injection day, any dead or unhealthy embryos were removed.\n\nGFP and alizarin red images were captured using a Zeiss SteReo Lumar.V12 equipped with a Zeiss AxioCam HRc digital camera and Zeiss AxioVision Rel. 4.8 software.\n\nTo extract RNA and gDNA from the same zebrafish, the AllPrep® DNA/RNA Micro kit was used (Qiagen). Individual zebrafish (10 d.p.f) were lysed in 350ul buffer RLT plus using a micro tissue homogeniser and the protocol was followed. Genomic DNA was eluted in 50ul and RNA in 14ul.\n\nThe Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit was followed (Promega) using 12ul of RNA. Samples were incubated for 90 minutes at 37°C followed by 80°C for 10 minutes.\n\nqRT PCR was done using a T100 Thermal Cycler (BioRad) both on gDNA and cDNA for copy number and for gene expression analyses. DNA samples were diluted 1:10. Per sample 12.5ul SyBr Green (Applied Biosystems), 50ng each primer and 1.5ul water was added to 10ul of diluted DNA. Each sample was repeated in triplicate. The amplification parameters were: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. An internal control, EF1a, was run for each sample tested. All the primers used are shown in Table 3.\n\nA 6 well plate of Hela cells was transfected with 1ug plasmid DNA using Fugene (Promega) following the manufacturers protocol. The cells were incubated at 37°C for 48 hours before harvesting.\n\nCell pellets and zebrafish lysates were run on a western blot. Cell pellets were from a 6 well plate and zebrafish lysates contained 10 zebrafish from 3, 7 or 10 d.p.f. All samples were lysed in ice-cold NP-40 buffer (150 mM NaCl, 50 mM pH 8 Tris–HCl, 1% NP-40) containing 1× complete protease inhibitor cocktail (Roche) and 100 µM phenylmethanesulfonyl fluoride (Sigma). Insoluble contents were pelleted at 13 000g for 30 min at 4°C and the supernatant was prepared in Laemmli sample buffer (Bio-Rad) containing 50 mM dithiothreitol. Samples were heated at ~90°C for 10 minutes before being run on a 10% polyacrylamide gel made in house, along with a marker (Biorad 1610376). Proteins were transferred to a membrane using the Trans-Blot® Turbo™ Blotting System (Biorad) and blocked overnight at 4°C in 5% milk, prepared in PBS/0.1%Tween 20 (Sigma) (PBST). Blots were incubated with the following primary antibodies for 3 hours at room temperature in blocking buffer: mCherry (1:2000), mouse (Anti-mCherry antibody [1C51], Abcam, ab125096) or p44/42 MAPK (Erk1/2) (1:400) rabbit (cell signalling, 9102S).\n\nBlots were washed 4 x 5 minutes in PBST and incubated for 1 hour in PBST with the appropriate secondary antibody (1:5000). Blots were again washed 4 x 5 min in PBST and developed using Clarity Western ECL blotting substrate (BioRad) blots were visualised using the ChemiDoc imaging system (BioRad).\n\nThe protocol was performed at room temperature and each step was left overnight rolling. Adult zebrafish were fixed in 4% PFA/PBS followed by a rinse in tap water. The zebrafish were eviscerated and skinned, and then bleached in 1% KOH with 3% hydrogen peroxide. The following day the zebrafish were rinsed in tap water for 30 mins and subsequently 30ml saturated sodium tetraborate in 70ml water. The zebrafish were next stained with 1mg/ml alizarin red in 1% KOH. After 30 min rinse in tap water the zebrafish were cleared in 1% trypsin in 2% sodium tetraborate for several days. Once cleared the zebrafish were washed through a series of 1% KOH/100% glycerol solutions typically 20% final glycerol, 40% final glycerol and finally storage was in 70% final glycerol/30% alcohol (70%).\n\n\nResults\n\nIn total, 8 Tol2 vectors were produced, consisting of either the zebrafish beta-actin (ubiquitous expression), runx2 (osteoblast), ctsk (osteoclast) or col2a1a (chondrocyte) promoters fused upstream of the full length human PTDSS1 ORF encoding either the wildtype sequence or the p.Q353R variant. We chose to study the p.Q353R mutation because it has been found to occur independently in multiple affected families (Sousa et al., 2014). Since there is no existing information with which to predict whether tagged PTDSS1 is functional, we also included an mCherry cDNA sequence downstream of each promoter-PTDSS1 sequence separated by an internal ribosome entry site (IRES), which was itself downstream of the PTDSS1 stop codon to facilitate visualization of protein arising from this transgene. This entire cassette was expressed as a single cistron. In addition, the construct contained a GFP sequence driven by the heart specific promoter cmlc2 in order to identify successful integration of the transgene by visual inspection of the heart (Figure 1; Figure 2A). Each clone was Sanger sequenced to ensure sequence integrity.\n\nA) Example of a zebrafish with a GFP positive heart. B) Copy number analysis of integrated human PTDSS1, GFP and mCherry by q-PCR on gDNA isolated from individual GFP- fluorescing zebrafish. C) Comparison of DNA copy number (top panel) and RTq-PCR expression analysis (bottom panel) for individual zebrafish where DNA and RNA were extracted from the same samples. D) Comparison of endogenous zebrafish ptdss1 with human PTDSS1 showing significantly higher levels of endogenous transcripts over WT and mutant transgene transcripts (p=0.01 and 0.008 respectively).\n\nThe beta-actin Tol2 vectors were functionally tested by transient transfection into HeLa cells. Since antibodies reliably detecting PTDSS1 are not currently available, Western blotting using anti-mCherry antibody was therefore used to confirm expression from the β-actin promoter. Appropriate expression of mCherry was successfully detected in this way (Figure 3) indicating that a functional promoter was operating and that the IRES successfully initiated translation of the mCherry protein.\n\nA) Immunoblotting showing mCherry and ERK (control) in extracts from 3 d.p.f. transgenic zebrafish (either wild-type, Wt, or Q353R mutant, Mut) and non-transgenic (NT) zebrafish as well as HeLa cells transfected with the same beta-actin (BA) constructs. B) Similar western blot including transgenic zebrafish at 7 d.p.f and 14 d.p.f.\n\nAll 8 Tol2 vectors demonstrated successful integration with between 5.4% and 12.6% of embryos showing mosaic expression of GFP within the heart at 48 hours post-injection into one-cell stage zebrafish embryos. (Figure 2A, Table 4). We subsequently bred these transgenic zebrafish beyond F5, which generated animals with completely green hearts, demonstrating stable integration of the transgene. Genomic DNA was extracted from individual 10 dpf β-actin transgenic zebrafish and tested for copy number of human PTDSS1, GFP and mCherry using qPCR. We confirmed the presence of all three regions of the transgene with relative copy numbers ranging significantly. Further there was a strong correlation between the relative copy number assessed using PTDSS1, GFP or mCherry primer sets in each embryo, confirming that the transgene had most likely integrated in its entirety at each site within the genome (Figure 2B).\n\nNext, the expression of human PTDSS1 zebrafish was determined by qRT-PCR using cDNA extracted from the same individual zebrafish on which the relative number of integrations had been analysed at the gDNA level. We did not detect expression of human PTDSS1 in any of the 8 lines of transgenic zebrafish (Figure 2C). In contrast, we did detect expression of the endogenous zebrafish ptdss1a gene (Figure 2D). To further investigate transgene expression, whole mount in situ hybridization for human PTDSS1, GFP and mCherry was performed on 6 d.p.f beta-actin transgenic zebrafish and compared to non-transgenic controls. No specific expression of any of the probes was detectable (data not shown).\n\nTo investigate protein levels, protein lysates from transgenic zebrafish at different days post fertilization were analysed by western blotting. In each case, a negative control generated from non-transgenic zebrafish of the same age was used. The lysates from transient transfection of HeLa cells served as a positive control. No mCherry protein could be seen in any of the transgenic zebrafish tested, although it was clearly present in lysates from transfected HeLa cells (Figure 3). In conclusion, while we robustly detected successful integration of the entire and apparently functional construct for each of the 8 transgenes in multiple lines of zebrafish, no gene product could be detected.\n\nBecause LMS features prominent skeletal features, we performed alizarin red staining on 6-month-old transgenic zebrafish to investigate skeletal mineralisation of transgenic zebrafish (Figure 4A–D). We noted several prominent morphological defects that were never observed in non-transgenic animals of the same age, specifically sharp lateral bending of the caudal fin vertebrae and irregularities of the ribs, which were perfectly smooth in non-transgenic zebrafish (Figure 4E). These defects were present in all transgenic lines of zebrafish, affecting between 10–60% of animals, with no obvious relationship to the particular mutation or the promoter used.\n\nSpinal cord, ribs and fin rays in A) a non-transgenic zebrafish; B) a transgenic zebrafish showing spine/tail kink; C) a transgenic zebrafish with scoliosis. E) Graphical representation of the distribution of skeletal defects seen for each transgenic line tested.\n\n\nDiscussion\n\nZebrafish have generally proven to be a very useful tool for studying bone formation in vertebrates. Mineralization occurs in a predictable and stereotypic manner beginning at 3 dpf and craniofacial bones develop in a similar manner to those of higher vertebrates (Eames et al., 2012; Mork & Crump, 2015; Mackay et al., 2013; Schilling & Kimmel, 1997; Sharif et al., 2014). Previous overexpression studies following injection of PTDSS1 RNA showed no effects resulting from the wildtype sequence, while for the LMS mutant RNA, a marked effect on craniofacial structures such as widely spaced eyes, short jaw and a wide angle of Meckel’s cartilage was detected (Sousa et al., 2014). A limitation to this methodology is the ability to study the transition from cartilage to mature bone and beyond. Therefore, in this study we set out to create a series of constitutive transgenic zebrafish carrying wildtype and mutant human PTDSS1 under the control of different cell lineage-specific promoters. This included the β-actin promoter, which would constitutively express the human PTDSS1 in all transgene-containing cells and in a non-time dependant or differentiation-specific manner.\n\nOverall, we were satisfied that intact plasmids had been cloned and transduced successfully. This was based on several lines of evidence starting with Sanger sequence validation of the integrity of each construct. Despite all of the promoters used in the Tol2 constructs being generated from zebrafish-specific sequences, it was possible to test the β-actin constructs following transfection in HeLa cells. This led to strong expression of mCherry protein as detected by western blotting (Figure 3). This clearly indicated the presence of a functionally effective, zebrafish promoter sequence and the integrity of the IRES driving mCherry expression. Following transgenesis, we found a high percentage of the zebrafish injected with each construct to have GFP+ hearts (Figure 2A; Table 4), which showed that the cmlc2-GFP element of the construct integrated and was expressed. Furthermore, there was a high correlation for copy number of each different part of the transgene in individual zebrafish (Figure 2B). This not only indicated that the construct integrated in multiple copies in some zebrafish but also that in each case, the transgenes were very likely to be intact.\n\nWe also performed several other experiments including qRT-PCR and western blotting of mCherry (Figure 3). All methods failed to show any detectable RNA or protein expression of the transgene. Epifluorescence of mCherry, which we had hoped to serve as a live in vivo reporter of transgene expression, and also immunohistochemistry using an anti-mCherry antibody failed to produce a signal. For the latter we concentrated on mCherry detection since there are currently no good antibodies available for PTDSS1 and even if there were, data might have been compromised by cross-reaction with the endogenous zebrafish homologue. In contrast, mCherry transcripts and protein would be unique to the transgenic zebrafish and the available antibodies are well established, as demonstrated by the results obtained following HeLa cell transfection. Taken together, our data indicate that, in many zebrafish that we analysed, multiple integrations were successfully achieved, and that this resulted in limited transgene expression. We were able to demonstrate that the β-actin construct drove efficient gene and protein expression in transfection experiments using HeLa cells, which further shows that each independent element of the transgene is functional and suggests that this is not the explanation for failed transgene expression.\n\nThere are several possible explanations for the failed transgene expression. Formally, it is possible that the promoters are not active in zebrafish, however, this seems unlikely given that all four promoters have been shown to drive gene expression in zebrafish previously (Chatani et al., 2011; Dale & Topczewski, 2011; To et al., 2012). Another possibility is that the transgenes are silenced which could relate to the site of transgene insertion within the 3D genome and chromatin conformation. However, we showed many zebrafish to carry multiple transgene insertions, and so this explanation would require multiple independent silencing functions to be active, which seems unlikely. It is also possible that elevated expression of PTDSS1 is inconsistent with life, such that transgenic zebrafish expressing the transgene do not survive. It is unclear what the true explanation of these results is.\n\nPrevious studies have shown that PSS1 activity is normally under tight negative feedback regulation, while GOF mutations can attenuate this level of control (Sousa et al., 2014; Vance, 2018). In contrast, simple over-expression of WT PSS1 in human hepatoma cells was shown to increase PS synthesis although the overall quantity of PS was not increased (Stone & Vance, 1999). This was explained by two compensatory mechanisms, induction of PS decarboxylation and attenuation of PSS2 activity. Thus, PS homeostasis is maintained.\n\nWe also analysed adult zebrafish to see if any phenotype could be detected, particularly something attributable to mutant PTDSS1. In particular, we noted the variable occurrence of scoliosis, present in a proportion of the zebrafish. This was of particular interest since lumbar kyphoscoliosis was described in the LMS patient described by Majewski in 2000 (Majewski, 2000), although not specifically recorded for any of the other patients reported. While scoliosis is one of the most common naturally occurring malformation in zebrafish (Boswell & Ciruna, 2017), this usually occurs in the presence of pathogen infection and in very old zebrafish. By contrast, we never observe scoliosis in our aquatics facility at the ages reported here, and routine microbiological testing continues to exclude microbiological infections in our facility.\n\nOur initial experimental design included wild-type and mutant PTDSS1 for each of the four promoters used, with the intention that the wild-type transgene would serve as a negative control. We also anticipated that PTDSS1 expression would have selective effects in only a subset of the skeletal cell lineages under study and would additionally affect the ubiquitous β-actin mutant transgenic zebrafish. Contrary to these hypotheses, we observed skeletal abnormalities in all 8 transgenic lines. There are several possible explanations for this. Firstly, accurate cellular dosing of PTDSS1 could be important in all three skeletal lineages that we investigated, and that this is reflected by both silencing of the transgene and the appearance of specific skeletal defects in all transgenic lines. While transient expression of wild-type PTDSS1 did not produce embryonic defects in our previous study (Sousa et al., 2014), it is possible that long-term enhancement of PTDSS1 expression levels leads to cumulative defects with the bone. It is also possible that the bone malformations seen with alizarin red staining may be incidental findings, although we do note that similar defects were never seen in control non-transgenic animals. Whichever of these explanations is true, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model makes continued investigation of these transgenic lines impractical.\n\nIn conclusion, although the collective evidence suggests that the zebrafish have successfully integrated the human PTDSS1 WT or p.Q353R vectors respectively, we cannot visualize explicit RNA or protein expression or identify a specific phenotype in these zebrafish.\n\n\nData availability\n\nFigshare: Analysis of transgenic zebrafish expressing the Lenz-Majewski syndrome gene PTDSS1 in skeletal cell lineages. https://doi.org/10.6084/m9.figshare.7732328.v3 (Seda et al., 2019).\n\nThe project contains the following underlying data files:\n\n- Figure 1–Figure 3\n\n- Raw phenotype data\n\n- Raw qPCR data\n\n- P3E mCherry IRES (full sequence of the entry vector we used to cloning)\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 work was funded by a Medical Research Council Project Grant (MR/M004597/1) to PS, and a Medical Research Council New Investigator Research Grant (MR/L009978/1) and Action Medical Research Project Grant (GN2595) to DJ. PS is supported by Great Ormond Street Hospital Children’s Charity and the research was also supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.\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\nArikketh D, Nelson R, Vance JE: Defining the importance of phosphatidylserine synthase-1 (PSS1): unexpected viability of PSS1-deficient mice. J Biol Chem. 2008; 283(19): 12888–12897. PubMed Abstract | Publisher Full Text\n\nBoswell CW, Ciruna B: Understanding Idiopathic Scoliosis: A New Zebrafish School of Thought. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaraiva JM: Dysgenesis of corpus callosum in Lenz-Majewski hyperostotic dwarfism. Am J Med Genet. 2000; 91(3): 198–200. PubMed Abstract | Publisher Full Text\n\nSchilling TF, Kimmel CB: Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo. Development. 1997; 124(15): 2945–2960. PubMed Abstract\n\nSeda M, Peskett E, Demetriou C, et al.: Analysis of transgenic zebrafish expressing the Lenz-Majewski syndrome gene PTDSS1 in skeletal cell lineages.. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7732328.v3\n\nSharif F, de Bakker MA, Richardson MK: Osteoclast-like Cells in Early Zebrafish Embryos. Cell J. 2014; 16(2): 211–224. PubMed Abstract | Free Full Text\n\nShoja MM, Mortazavi MM, Ditty B, et al.: Lenz-Majewski syndrome associated with hydrocephalus and multiple congenital malformations. Biomd Int. 2013; 4: 45–52. Reference Source\n\nSousa SB, Jenkins D, Chanudet E, et al.: Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome. Nat Genet. 2014; 46(1): 70–76. PubMed Abstract | Publisher Full Text\n\nStone SJ, Vance JE: Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2. Biochem. J. 1999; 342(Pt 1): 57–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTamhankar PM, Vasudevan L, Bansal V, et al.: Lenz-Majewski syndrome: Report of a case with novel mutation in PTDSS1 gene. Eur J Med Genet. 2015; 58(8): 392–399. PubMed Abstract | Publisher Full Text\n\nTo TT, Witten PE, Renn J, et al.: Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model. Development. 2012; 139(1): 141–50. PubMed Abstract | Publisher Full Text\n\nVance JE: Historical perspective: phosphatidylserine and phosphatidylethanolamine from the 1800s to the present. J Lipid Res. 2018; 59(6): 923–944. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVance JE, Tasseva G: Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells. Biochim Biophys Acta. 2013; 1831(3): 543–554. PubMed Abstract | Publisher Full Text\n\nWattanasirichaigoon D, Visudtibhan A, Jaovisidha S, et al.: Expanding the phenotypic spectrum of Lenz-Majewski syndrome: facial palsy, cleft palate and hydrocephalus. Clin Dysmorphol. 2004; 13(3): 137–142. PubMed Abstract | Publisher Full Text\n\nWesterfield M: The zebrafish book. Eugene, OR: University of Oregon Press. 1993. Reference Source\n\nWhyte MP, Blythe A, McAlister WH, et al.: Lenz-Majewski hyperostotic dwarfism with hyperphosphoserinuria from a novel mutation in PTDSS1 encoding phosphatidylserine synthase 1. J Bone Miner Res. 2015; 30(4): 606–614. PubMed Abstract | Publisher Full Text\n\nWu LN, Genge BR, Wuthier RE: Analysis and molecular modeling of the formation, structure, and activity of the phosphatidylserine-calcium-phosphate complex associated with biomineralization. J Biol Chem. 2008; 283(7): 3827–3838. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "45567",
"date": "25 Mar 2019",
"name": "Jean-Marie Delalande",
"expertise": [
"Reviewer Expertise Embryology. zebrafish. Enteric nervous system."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this research article Seda et al., describes the use of zebrafish Tol-2 constructs to express wild type and mutant form of the human PTDSS1 gene. A mutation in this enzyme, causing gain of function, has been previously linked to Lenz-Majewski Syndrome (Sousa et al.,20141). Despite adequate design the construct didn’t deliver the predicted outcome.\nIt is clear from the material & methods, as well as control experiments, that the constructs were correctly designed and integrated. As show by figure 1 A&B. Tol-2-kit transgenesis is known to result in multiple insertions, which could have helped with this gain of function experiment, but also possibly could have driven the system beyond physiological limits and cause embryonic lethality. Using a bicistronic construct meant that the expression of the enzyme and the reporter gene were decoupled, which limited the usefulness of the reporter gene as indicator of transgene expression. As stated by the authors, a fusion protein might have resulted in the loss of enzymatic activity. However, the authors could mention this strategy in the discussion as some bifunctional enzyme-GFP fusions have been reported in the literature (Martin et al., 20092). Using the Tol2-kit, clone 455 \"pME-mCherry no stop\" might have been worth exploring. The authors should also discuss the fact that several articles mention issues using bicistronic IRES constructs, as it is likely to be the main issue here. Hennecke et al., state; “However, failures of bicistronic applications have been observed, although these were often not published” (20013). See also Mansha et al., 20124. Considering the results in this study, it is possible that the constructs produced unstable PTDSS1 mRNA. Multiple insertions combined with transient mRNA background expression of the transgene might explain the scoliosis phenotype (which is more prevalent in the osteoblast (ctsk) and chondroblast (col2a1a) specific promoters I figure 4E.).\n\nOverall because such results tend not to be published and issues with similar constructs might not have been previously reported. Therefore I recommend indexing of this article, as it will be useful to the scientific community in this field of research.\n\nMinor points:\n\nEnd of page 3: the methods part on in situ hybridization should be taken out as no in situresults are presented. Figure 2C: results are a bit puzzling and could be discussed further. Is the relative expression just minimal background expression? It seems strange that it is negatively correlated with the number of inserted copies. Figure 4: picture could be presented in a table clearly stating which fish are wild type and mutant.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "48264",
"date": "03 Jun 2019",
"name": "Carles Gaston-Massuet",
"expertise": [
"Reviewer Expertise Developmental genetics",
"tumour biology",
"stem cell biology",
"endocrinology ."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 in this article describes the use of zebrafish Tol-2 Kit transposase-system with Gateway constructs to express wild type and mutant form of the human PTDSS1 gene in zebra fish. The authors attempt correctly to generate a model Lenz-Majewski Syndrome previously shown to be caused by mutations in PTDSS1 gene.\n\nThe article is very well written and clear on the methods. The design of the constructs are well explained and correct, but the transgenic fish did not exhibit the predicted phenotypes. This is mainly due to the lack of expression of the transgene generated although integration of the transgene was achieved. The authors clearly explain several possibilities which could account for the lack of phenotype mainly as part of generating the transgenic fish. Although the results yield inconclusive results on the role of PTDSS1 in the Lenz-Majewski Syndrome in fish, I recommend the indexing of this article. I think that the lack of publications on technical challenges encountered in production of such transgenic lines will be useful for the zebra fish transgenic community that may encounter this type of difficulties in performing such studies.\n\nMinor comments:\nThe Tol2-Kit is a transposon-mediated whilst the authors refer to it as a transpose-mediated (abstract and thereafter). Transposon-mediated would be more accurate way to refer to this system. Figure 4 needs to be improved to make it easy to read for the none expert. Authors could add arrows to indicate the exact morphological abnormalities observed. Figure 4C: Which is the genotype of the transgenic fish, are both transgenics? I think the authors could improve the figure by adding more annotations and a clearer table of genotypes will help the general readability\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-273
|
https://f1000research.com/articles/8-5/v1
|
03 Jan 19
|
{
"type": "Review",
"title": "Nocebo as a source of bias in the assessment of treatment effect",
"authors": [
"Karolina Wartolowska"
],
"abstract": "The term nocebo refers to the worse outcomes or side effects experienced by patients as a result of their negative expectations regarding a treatment. It may distort estimates of treatment effectiveness and safety in both clinical trials and clinical practice; moreover, it may cause discontinuation of therapy or drop out from a trial. Nocebo effect is evoked by the information given to patients during a clinical consultation or during enrolment into a study, but information available from the media or the Internet may also play an important role. In research settings, a trial design may introduce bias from the nocebo effect. For example, if the non-treatment group is unblinded and aware that they are not receiving any treatment, their treatment expectations are not met, which results in worse outcomes, and subsequently, the problems that the trial was supposed to investigate may be enhanced in the non-treatment arm. Nocebo effect is common, and its magnitude may be large, but it receives less attention and research focus than the placebo effect. Unlike the placebo effect, which is usually taken into consideration while interpreting treatment results and controlled for in clinical trials, the nocebo effect is under-recognised by clinical researchers as well as clinicians. It is important to recognise and any potential nocebo effect must be considered while assessing the effect of treatment and should be minimised through careful choice and phrasing of treatment-related information given to the patients.",
"keywords": [
"Review (article)",
"Nocebo Effect",
"Placebo Group",
"Adverse Events in Clinical Trials",
"Randomised Clinical Trial (RCT)"
],
"content": "Introduction\n\nNocebo is often described as placebo’s evil twin, and it rarely gets discussed on its own. There is relatively little research on nocebo and this phenomenon is under-recognised in clinical practice or clinical trials, with many patients and healthcare professionals admitting that they are not aware of its existence (Berthelot et al., 2001).\n\nIn research, nocebo effect is defined as the adverse effect of a placebo intervention, for example placebo hyperalgesia, whereas in clinical or trial settings the term is used to describe the negative outcomes caused by negative expectations, such as lack of efficacy or harm from a drug or other intervention (Benedetti et al., 2007; Hahn, 1997; Häuser et al., 2012).\n\nNocebo can be easily evoked by verbal suggestion, such as negative information about the properties of the drug (Benedetti et al., 2007), or by conditioning (Klosterhalfen et al., 2009). Moreover, the magnitude of the effect is larger when it is caused by verbal suggestion and conditioning than by the verbal suggestion alone (Petersen et al., 2014).\n\nNocebo hyperalgesia is mediated by stress and anticipatory anxiety, which facilitate pain transmission (Bingel et al., 2011; Keltner et al., 2006). Nocebo is also associated with higher cortisol levels (Johansen et al., 2003) and with the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which controls reactions to stress. In addition to that, nocebo and HPA hyperactivity are reduced by anxiolytic drugs (Benedetti et al., 2006). Nocebo response is also associated with reduced activation of dopaminergic and opioidergic systems (Scott et al., 2008; Svedman et al., 2005) and with increased effects mediated by cholecystokinin (Benedetti et al., 1995).\n\n\nNocebo effect in clinical practice\n\nIn clinical settings, the nocebo effect manifests as a reduced response to treatment or the development of adverse events, which often result in non-adherence or discontinuation of treatment (Blasini et al., 2017).\n\nNocebo effect is caused by negative expectations about the outcomes of the treatment and negative emotions created during the patient-doctor communication (Häuser et al., 2012). These negative expectations are most often created unintentionally by the description of the treatment effects and side effects; either verbally during a consultation or as information on a drug leaflet (Benedetti et al., 2007; Tobert & Newman, 2016). Other sources of negative information include friends, family and other patients as well as news, internet, or social media (Crichton & Petrie, 2015). For example, patients in the countries where they are more likely to find websites about the side effects of statins are more likely to demonstrate statin intolerance (Khan et al., 2018). Nocebo effect may also be created by observing the symptoms, side effects, and behaviour of other patients undergoing the treatment (Colloca & Benedetti, 2009; Hahn, 1997; Świder & Bąbel, 2013).\n\nNocebo effect may be caused by dissatisfaction with the current or past treatment (Kessner et al., 2013). For example, patients often distrust generic drugs and believe they are less effective and more harmful than branded drugs (Al Ameri et al., 2011; Himmel et al., 2005). Switching to generic drugs may cause lower adherence (Labiner et al., 2010), worse outcomes, and more frequent adverse events (Häuser et al., 2012). Also, many doctors think that generic drugs are of lower quality (Heikkilä et al., 2007) and unintentional cues given by the doctors may make patients’ attitude even more negative and enhance the nocebo effect (Häuser et al., 2012).\n\nNot only the treatment but also a negative consultation may cause a nocebo effect. Patients expect a doctor to understand and recognise their problems (validation), give it a name (diagnosis), explain how it is going to progress (prognosis), and then to offer a treatment, usually in a form of a pill. If any of the elements of the consultation is negative, for example, a doctor dismisses patients’ complaints as being “all in their head”, patients may feel that their treatment needs were not met and their sickness was invalidated (Vangronsveld & Linton, 2012). This invalidation makes patients hopeless and angry (Häuser et al., 2012) and increases the nocebo effect (Barsky et al., 2002). Moreover, the negative effect of consultation may be stronger than the positive effects of consultation (Greville-Harris & Dieppe, 2015). It may persist for a long time (Blasini et al., 2017); although clinicians positive suggestions may reduce the effect of these negative messages (Crichton & Petrie, 2015).\n\nIn clinical settings, the nocebo effect is highly undesirable. Negative expectation can make the therapeutic intervention more painful, for example, an injection of an epidural analgesic can be made more painful when patients are warned that it would feel like a bee sting rather than told only that it would create a numbing sensation (Varelmann et al., 2010). Similarly, using the word “pain” rather than “cool sensation” in a description of a procedure may make this procedure painful (Lang et al., 2005).\n\nThe information about treatment is so crucial that it can interfere with the pharmacological effects of a drug. For example, pain ratings after the suggestion of hyperalgesia were higher than after the suggestion of analgesia, regardless whether they were accompanied by an application of analgesic cream or placebo (Aslaksen et al., 2015). Similarly, the efficacy of pharmacologically active drugs was greatly reduced when they were given with the contradictory information: bronchoconstrictors as reducing asthma and bronchodilators as provoking it (Luparello et al., 1970). In another study, information that the injection of a powerful opioidergic analgesic was stopped, reversed its analgesic effects despite the continued delivery of the drug (Bingel et al., 2011). Negative information may also evoke adverse events even after the delivery of an inert placebo substance. For example, nebulised saline evoked asthma attacks in patients with asthma if it was given with information that contained an irritant, while the same saline relieved these symptoms if it was presented as an active treatment (Luparello et al., 1968).\n\n\nNocebo effect in clinical trials\n\nIn trial settings, nocebo manifests as reduced improvement or increased frequency of adverse events, especially if they are subjective, not dose-dependent, and unrelated to the pharmacological properties of the drug; including adverse events after placebo. Patients’ withdrawal from a trial due to these adverse events is also considered to be a nocebo effect (Barsky et al., 2002; Blasini et al., 2017; Tobert & Newman, 2016). Nocebo effect in clinical trials is undesired. It may distort the results of the trial, for example, if patients do not improve sufficiently, it may be concluded that the tested treatment is not effective. On the other hand, if patients report many adverse events, the conclusions may be that the treatment is harmful and a trial may be terminated early. Moreover, if these adverse events lead to the withdrawal of many participants, the missing data may further complicate interpretation of such a trial (Mitsikostas et al., 2011).\n\nUnlike the placebo, the nocebo effect is under-recognised; it is rarely discussed in the context of clinical trials, and it may be not taken into the account while interpreting the results of a trial. The magnitude of nocebo effect (Petersen et al., 2014) and the percentage of patients in clinical trials who report adverse events as a result of the nocebo effect may be underestimated (Amanzio et al., 2009; Mitsikostas et al., 2011; Rief et al., 2006). For example, a meta-analysis of clinical trials of pharmacological treatments for neuropathic pain found that about 52.0% (95% CI: 35.7-67.9) of placebo-treated patients reported adverse events and 6.0% (95% CI: 4.5-8.0) withdrew from a trial due to these side effects (Papadopoulos & Mitsikostas, 2012).\n\nIn a clinical trial, like in a clinic, nocebo may be introduced by the information about the effects and side effects of the tested treatment that are described in the information letter or during the informed consent process. This information may bias the subsequent reporting and affect the trial outcomes, especially if these outcomes are based on patients’ report. For example, the frequency of reported gastrointestinal adverse events and the discontinuation rates due to these adverse events in a trial on aspirin were much lower in a centre that did not include information about possible gastrointestinal bleeds than in two centres that included this information (Cairns et al., 1985; Myers et al., 1987). These adverse events reported by trial participants but not caused by the pharmacological effects of the tested medication are referred to as the nocebo effect (Barsky et al., 2002). These symptoms are typically generalised and unspecific for example nausea, headaches, fatigue, or irritability. These symptoms are often not associated with any disease and commonly occur to healthy people not taking any medication (Eriksen & Ursin, 2004). For example, 77% of students responded that they had experienced at least one such a symptom in the previous three days (Reidenberg & Lowenthal, 1968). Patients participating in a trial may focus on their symptoms and may interpret normal physiological sensations or benign symptoms, that may usually get little attention, as side effects of the treatment (Barsky & Borus, 1999; Gurwitz et al., 2003; Rosenzweig et al., 1993).\n\nIn trial settings, about a quarter of patients taking placebo spontaneously report at least one side effect, and this figure increases when they are actively asked about side effects (Barsky et al., 2002; Rosenzweig et al., 1993). Furthermore, patients with negative expectations, are more likely to expect adverse events and misattribute them as related to the treatment (Barsky et al., 2002). Some of these symptoms are highly prevalent in populations in which the drug is prescribed, for example, headaches in women taking contraceptive pills (Grimes & Schulz, 2011) or muscle problems in older patients taking statins (Tobert & Newman, 2016). These “noise” symptoms may be misattributed to the treatment (Barsky et al., 2002; Grimes & Schulz, 2011; Tobert & Newman, 2016).\n\nNot all nocebo-related side events are “unspecific” (Rief et al., 2009). Some complaints may be disease-specific as patients may mistake symptoms of an underlying illness for treatment side effects (Fine & Johnston, 1993). Many adverse events reported by patients in the placebo group are typical for the treatment in the active arm (Amanzio et al., 2009; Barsky et al., 2002; Blasini et al., 2017; Rief et al., 2009). For example in the meta-analysis of trials on anti-migraine treatment, anorexia and problems with memory, which often occur in patients taking anti-epileptic drugs, were reported only in patients in the placebo arm of trials on anti-epileptic drugs (Amanzio et al., 2009). In another study, the rate of adverse events was much higher in the placebo arm of trials of tricyclic antidepressants than in trials on selective serotonin reuptake inhibitors, which reflects the side effect profile of these classes of drugs (Rief et al., 2009). These examples demonstrate that information about adverse effects of different classes of drugs causes expectations that may influence the experience of side effects and may bias clinical trial outcomes (Rief et al., 2009).\n\nSome of the elements of a trial’s design may evoke a nocebo effect. For example, random assignment to different treatment regimens means that patients are not given a choice, which may create a nocebo effect while having a choice increases the placebo effect (Bartley et al., 2016). Moreover, if the control consists of the patients on a waiting list or in a non-interventional group, patients randomised to this group are being left without any treatment. A non-interventional arm does not represent a natural history of disease because there is a double bias: not only these patients are not blinded, but also their treatment expectations are not met, because they are left without any treatment, which leads to the nocebo effect and either worsening of their symptoms or slower recovery.\n\nNocebo may distort the results of open-label trials, because not only is there information about possible adverse events but even the knowledge about the received treatment may affect the incidence of reported side events. For example, in a group of patients who knew they were taking atenolol and that erectile dysfunction may a possible side effect, the incidence of this particular side event was 31.2%, while in a group that was informed about the drug but not about the side effects the incidence was 15.6%, and if the group that was blinded and not told explicitly about this potential effect the incidence was only 3.1%. In the patients who reported this side effect, both Sildenafil or placebo were equally effective at curing it (Silvestri et al., 2003).\n\nThe bias caused by the nocebo effect is minimised in blinded randomised controlled trials (RCTs). In an RCT, bias is controlled by making the two compared groups differ only by the treatment allocation. Moreover, blinding of patients and assessors reduces placebo as well as nocebo bias, because the expectations are the same in both groups (Collins & MacMahon, 2007). An addition of a placebo control is useful not only to test whether the active treatment is more effective than placebo but also whether it is truly more harmful than placebo. Without a placebo control, all the side effects may be attributed to the active element of the treatment. For example, in a trial on statins, during the blinded and randomised phase, muscle-related symptoms were reported equally often in the active and the placebo arm, but during unblinded phase they were more frequent in patients receiving statins (Ganga et al., 2014; Gupta et al., 2017; Kashani et al., 2006). Moreover, patients with well-documented statin intolerance due to muscle symptoms usually tolerate a statin under double-blind conditions (Brown et al., 2016; Newman & Tobert, 2015).\n\n\nRecommendations and future directions\n\nUnlike improvement associated with placebo, there are no benefits related to nocebo and the nocebo effect so it has to be minimised by reducing the existing negative expectations or by preventing new ones (Tobert & Newman, 2016).\n\nNocebo effect can be prevented by careful phrasing of the information given to patients and by positive framing, for example, by focusing on chances of improvement, survival, being symptom-free, and of not developing side effects etc. (Crichton & Petrie, 2015). Similarly, some adverse events may not occur if they are not prompted; therefore, it may be beneficial not to inform the patients about potential adverse events that may be unrelated to the treatment or be of little clinical importance such as mild headaches or nausea (Tobert & Newman, 2016). However, it is crucial to warn patients about clinically important or potentially dangerous side effects caused by the pharmacological properties of a drug, for example, that, patients should not drive or operate heavy machinery after drugs that cause drowsiness. In a trial, it is also very important to record and include in the publication the exact content and phrasing of the information given to trial participants because it may have a substantial effect on the trial results.\n\nNocebo effect may be reduced by asking patients about their preconceptions and beliefs regarding a treatment. If patients beliefs are negative, for example, they think they are intolerant to the prescribed medicine, they will be more likely to report more side effects at the follow-up (Barsky et al., 2002), especially when starting new medications (Nestoriuc et al., 2010). Such patients will be also less likely to adhere to this treatment (Barsky et al., 2002), and may be more likely to stop taking this medication altogether (Nestoriuc et al., 2010). After a change of medication, patients with negative beliefs are likely to report even more adverse events than during the therapy with the original drug (Nestoriuc et al., 2010). Therefore, it is important to change the patient’s attitude before changing the medication. Moreover, it may be worth asking patients to agree to a re-challenge with a drug they claim they do not tolerate (Tobert & Newman, 2016) as having a choice is associated with better outcomes (Botti & Iyengar, 2004). It is also important not to leave the patient without treatment, as any type of treatment is better than staying on a waiting list (Khan et al., 2012).\n\n\nConclusions\n\nNocebo effect is always negative and unwanted, and it can easily be evoked by a careless word or unfortunate phrasing. Recognising the nocebo effect is important because it may make the treatment look ineffective or harmful. It may seem that there is no improvement or much less improvement than there should be. It may also seem that the treatment has many side effects and is not tolerated by the patient and lead to a change of treatment; however, patients who reported those unspecific complaints after one treatment are likely to report even worse symptoms after a change of treatment. Nocebo effect is also responsible for non-adherence to treatment and for discontinuation. When patients expect to feel worse or not improve, they treat every negative sensation as caused by the treatment, so they do not take the treatment regularly or stop it altogether, which, in turn, results in a subtherapeutic dose of medication and actual pharmacological consequences. Therefore, any potential nocebo effect must be recognised and minimised in the clinic and in clinical trials.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "Grant information\n\nThe author declares that no grants were involved in supporting this work.\n\n\nReferences\n\nAl Ameri MN, Whittaker C, Tucker A, et al.: A survey to determine the views of renal transplant patients on generic substitution in the UK. Transpl Int. 2011; 24(8): 770–779. PubMed Abstract | Publisher Full Text\n\nAmanzio M, Corazzini LL, Vase L, et al.: A systematic review of adverse events in placebo groups of anti-migraine clinical trials. Pain. 2009; 146(3): 261–269. PubMed Abstract | Publisher Full Text\n\nAslaksen PM, Zwarg ML, Eilertsen HI, et al.: Opposite effects of the same drug: reversal of topical analgesia by nocebo information. Pain. 2015; 156(1): 39–46. PubMed Abstract | Publisher Full Text\n\nBarsky AJ, Borus JF: Functional somatic syndromes. Ann Intern Med. 1999; 130(11): 910–921. PubMed Abstract | Publisher Full Text\n\nBarsky AJ, Saintfort R, Rogers MP, et al.: Nonspecific medication side effects and the nocebo phenomenon. 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PubMed Abstract | Publisher Full Text\n\nGurwitz JH, Field TS, Harrold LR, et al.: Incidence and preventability of adverse drug events among older persons in the ambulatory setting. JAMA. 2003; 289(9): 1107–1116. PubMed Abstract | Publisher Full Text\n\nHahn RA: The nocebo phenomenon: concept, evidence, and implications for public health. Prev Med. 1997; 26(5 Pt 1): 607–11. PubMed Abstract | Publisher Full Text\n\nHäuser W, Hansen E, Enck P: Nocebo phenomena in medicine: their relevance in everyday clinical practice. Dtsch Arztebl Int. 2012; 109(26): 459–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeikkilä R, Mäntyselkä P, Hartikainen-Herranen K, et al.: Customers’ and physicians’ opinions of and experiences with generic substitution during the first year in Finland. Health Policy. 2007; 82(3): 366–374. PubMed Abstract | Publisher Full Text\n\nHimmel W, Simmenroth-Nayda A, Niebling W, et al.: What do primary care patients think about generic drugs? Int J Clin Pharmacol Ther. 2005; 43(10): 472–479. PubMed Abstract | Publisher Full Text\n\nJohansen O, Brox J, Flaten MA: Placebo and Nocebo responses, cortisol, and circulating beta-endorphin. Psychosom Med. 2003; 65(5): 786–790. PubMed Abstract | Publisher Full Text\n\nKashani A, Phillips CO, Foody JM, et al.: Risks associated with statin therapy: a systematic overview of randomized clinical trials. Circulation. 2006; 114(25): 2788–2797. PubMed Abstract | Publisher Full Text\n\nKeltner JR, Furst A, Fan C, et al.: Isolating the Modulatory Effect of Expectation on Pain Transmission: A Functional Magnetic Resonance Imaging Study. J Neurosci. 2006; 26(16): 4437–4443. PubMed Abstract | Publisher Full Text\n\nKessner S, Wiech K, Forkmann K, et al.: The effect of treatment history on therapeutic outcome: an experimental approach. JAMA Intern Med. 2013; 173(15): 1468–1469. PubMed Abstract | Publisher Full Text\n\nKhan A, Faucett J, Lichtenberg P, et al.: A systematic review of comparative efficacy of treatments and controls for depression. PLoS One. 2012; 7(7): e41778. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhan S, Holbrook A, Shah BR: Does Googling lead to statin intolerance? Int J Cardiol. 2018; 262: 25–27. PubMed Abstract | Publisher Full Text\n\nKlosterhalfen S, Kellermann S, Braun S, et al.: Gender and the nocebo response following conditioning and expectancy. J Psychosom Res. 2009; 66(4): 323–328. PubMed Abstract | Publisher Full Text\n\nLabiner DM, Paradis PE, Manjunath R, et al.: Generic antiepileptic drugs and associated medical resource utilization in the United States. Neurology. 2010; 74(20): 1566–1574. PubMed Abstract | Publisher Full Text\n\nLang EV, Hatsiopoulou O, Koch T, et al.: Can words hurt? Patient-provider interactions during invasive procedures. Pain. 2005; 114(1–2): 303–309. PubMed Abstract | Publisher Full Text\n\nLuparello TJ, Leist N, Lourie CH, et al.: The interaction of psychologic stimuli and pharmacologic agents on airway reactivity in asthmatic subjects. Psychosom Med. 1970; 32(5): 509–514. PubMed Abstract | Publisher Full Text\n\nLuparello T, Lyons HA, Bleecker ER, et al.: Influences of suggestion on airway reactivity in asthmatic subjects. Psychosom Med. 1968; 30(6): 819–825. PubMed Abstract | Publisher Full Text\n\nMitsikostas DD, Mantonakis LI, Chalarakis NG: Nocebo is the enemy, not placebo. A meta-analysis of reported side effects after placebo treatment in headaches. Cephalalgia. 2011; 31(5): 550–561. PubMed Abstract | Publisher Full Text\n\nMyers MG, Cairns JA, Singer J: The consent form as a possible cause of side effects. Clin Pharmacol Ther. 1987; 42(3): 250–253. PubMed Abstract | Publisher Full Text\n\nNestoriuc Y, Orav JE, Liang MH, et al.: Prediction of nonspecific side effects in rheumatoid arthritis patients by beliefs about medicines. Arthritis Care Res (Hoboken). 2010; 62(2): 791–799. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNewman CB, Tobert JA: Statin intolerance: reconciling clinical trials and clinical experience. JAMA. 2015; 313(10): 1011–1012. PubMed Abstract | Publisher Full Text\n\nPapadopoulos D, Mitsikostas DD: A meta-analytic approach to estimating nocebo effects in neuropathic pain trials. J Neurol. 2012; 259(3): 436–447. PubMed Abstract | Publisher Full Text\n\nPetersen GL, Finnerup NB, Colloca L, et al.: The magnitude of nocebo effects in pain: a meta-analysis. Pain. 2014; 155(8): 1426–1434. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReidenberg MM, Lowenthal DT: Adverse nondrug reactions. N Engl J Med. 1968; 279(13): 678–9. PubMed Abstract | Publisher Full Text\n\nRief W, Avorn J, Barsky AJ: Medication-attributed adverse effects in placebo groups: implications for assessment of adverse effects. Arch Intern Med. 2006; 166(2): 155–160. PubMed Abstract | Publisher Full Text\n\nRief W, Nestoriuc Y, von Lilienfeld-Toal A, et al.: Differences in adverse effect reporting in placebo groups in SSRI and tricyclic antidepressant trials: a systematic review and meta-analysis. Drug Saf. 2009; 32(11): 1041–1056. PubMed Abstract | Publisher Full Text\n\nRosenzweig P, Brohier S, Zipfel A: The placebo effect in healthy volunteers: influence of experimental conditions on the adverse events profile during phase I studies. Clin Pharmacol Ther. 1993; 54(5): 578–583. PubMed Abstract | Publisher Full Text\n\nScott DJ, Stohler CS, Egnatuk CM, et al.: Placebo and nocebo effects are defined by opposite opioid and dopaminergic responses. Arch Gen Psychiatry. 2008; 65(2): 220–31. PubMed Abstract | Publisher Full Text\n\nSilvestri A, Galetta P, Cerquetani E, et al.: Report of erectile dysfunction after therapy with beta-blockers is related to patient knowledge of side effects and is reversed by placebo. Eur Heart J. 2003; 24(21): 1928–1932. PubMed Abstract | Publisher Full Text\n\nSvedman P, Ingvar M, Gordh T: \"Anxiebo\", placebo, and postoperative pain. BMC Anesthesiol. 2005; 5: 1–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nŚwider K, Bąbel P: The effect of the sex of a model on nocebo hyperalgesia induced by social observational learning. Pain. 2013; 154(8): 1312–7. PubMed Abstract | Publisher Full Text\n\nTobert JA, Newman CB: The nocebo effect in the context of statin intolerance. J Clin Lipidol. 2016; 10(4): 739–747. 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}
|
[
{
"id": "43476",
"date": "30 Jan 2019",
"name": "Paul Dieppe",
"expertise": [
"Reviewer Expertise Placebo",
"nocebo",
"wellbeing and healing research"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful review of a subject that, as the author says, deserves more attention than it gets. One of my concerns is that the abstract and opening sentences state that nocebo is a result of negative expectation, and yet, as mentioned later this is only one of a number of theories as to how the nocebo effect may be activated. Others include invalidation (mentioned briefly), conditioning (mentioned briefly) and activation of the fight or flight response (not mentioned at all).\n\nAnother issue for me is the contexts in which nocebo can be an issue. This article mentions clinical trials, experimental settings such as pain perception, and clinical practice. I would find it easier to navigate the article if there was a clearer differentiation between these very different contexts, perhaps with subheadings.\n\nIn relation to clinical practice, the article does not mention the fact that a consultation can make a patient’s disease or symptoms worse as a result of nocebo mechanisms.\n\nIn the context of clinical trials I think that more attention should be given to the fact that the consent procedure can make symptoms worse, as well as resulting in a reduced response or adverse events.\n\nI found the English a little clumsy in places.\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? Yes",
"responses": [
{
"c_id": "4465",
"date": "11 Mar 2019",
"name": "Karolina Wartolowska",
"role": "Author Response",
"response": "I would like to thank the Reviewer for their useful comments. I fully agree that expectations are only one of the mechanisms responsible for nocebo effect, but they are the one that is mentioned most frequently in the literature. Other mechanisms that may cause the nocebo effect, including stress or the “fight or flight” response, have also been discussed in this manuscript. I am grateful to the Reviewer for their comment on the lack of clarity regarding the subdivision of nocebo effect in clinical and trial contexts. In the revised version, additional subheadings have been added, and some paragraphs have been rearranged to follow the clinical/trial context subdivision followed by the possible causes and consequences of the nocebo effect. Hopefully, the new version of the manuscript is less clumsy and sufficiently highlights the role of conditioning, stress, consultation, and consent procedure in generating the nocebo effect."
}
]
},
{
"id": "42494",
"date": "04 Feb 2019",
"name": "Przemysław Bąbel",
"expertise": [
"Reviewer Expertise Pain",
"memory of pain",
"placebo and nocebo effects",
"learning mechanisms of pain and placebo effects"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written short summary of the studies on a very important issue which deserves much more attention than it actually gets. I have only two major concerns. First, the paper is focused mainly on verbal information as a source of the nocebo. Although I do agree that it is the most common source of the nocebo effect in clinical practice and clinical trials, two other sources are also important and their role should be discussed, i.e. previous experience (classical conditioning) and observation of other patients/participants of clinical trials (see for example 1). Especially, the role of those two additional sources should be included in the recommendations and conclusions sections of the paper.\nSecond, through the paper the nocebo effect is discussed mainly in terms of negative expectations, however, it is only one of the explanatory mechanisms of the nocebo effect. Although nocebo effects induced by verbal information and observational learning are usually mediated by expectations, there is growing evidence that the nocebo effect induced by classical conditioning may not always be mediated by expectations (see references 2-8). Thus, I would rather avoid discussing the nocebo effect as the result of sole negative expectancies as well as I would avoid defining it in terms of negative expectations.\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": "4466",
"date": "11 Mar 2019",
"name": "Karolina Wartolowska",
"role": "Author Response",
"response": "I would like to thank the Reviewer for a very constructive review. I agree with the Reviewer that the nocebo effect should not be defined only in terms of negative expectations regarding a treatment, especially, if the expectations are defined, in the narrow sense, as a set of beliefs about the treatment. However, the literature on nocebo uses the term “expectations” in a broader sense, being the negative state (conscious or subconscious) accompanying a treatment/therapeutic intervention. It comprises negative beliefs about treatment efficacy, negative emotions such as stress and anxiety, and anticipation and expectation of failure, lack of improvement or adverse effects. This state may be caused by previous bad experiences (either as a failed treatment or experimental classical conditioning), knowledge gained through experiences or information about treatment obtained from doctors, drug leaflets, media, other patients, by observing other patients or by learning from family and peers. As suggested by the Reviewer, the importance of classical conditioning and learning by observing others have been highlighted in the revised version of the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/8-5
|
https://f1000research.com/articles/7-1745/v1
|
05 Nov 18
|
{
"type": "Research Article",
"title": "Density artefacts at interfaces caused by multiple time-step effects in molecular dynamics simulations",
"authors": [
"Dominik Sidler",
"Marc Lehner",
"Simon Frasch",
"Michael Cristófol-Clough",
"Sereina Riniker",
"Dominik Sidler",
"Marc Lehner",
"Simon Frasch",
"Michael Cristófol-Clough"
],
"abstract": "Background: Molecular dynamics (MD) simulations have become an important tool to provide insight into molecular processes involving biomolecules such as proteins, DNA, carbohydrates and membranes. As these processes cover a wide range of time scales, multiple time-step integration methods are often employed to increase the speed of MD simulations. For example, in the twin-range (TR) scheme, the nonbonded forces within the long-range cutoff are split into a short-range contribution updated every time step (inner time step) and a less frequently updated mid-range contribution (outer time step). The presence of different time steps can, however, cause numerical artefacts. Methods: The effects of multiple time-step algorithms at interfaces between polar and apolar media are investigated with MD simulations. Such interfaces occur with biological membranes or proteins in solution. Results: In this work, it is shown that the TR splitting of the nonbonded forces leads to artificial density increases at interfaces. The presence of the observed artefacts was found to be independent of the interface shape and the thermostatting method used. It is further shown that integration with an impulse-wise reversible reference system propagation algorithm (RESPA) only shifts the occurrence of density artefacts towards larger outer time steps. Using a single-range (SR) treatment of the nonbonded interactions, on the other hand, resolves the density issue for pairlist-update periods of up to 40 fs. Conclusion: A SR scheme avoids numerical artefacts and offers an interesting alternative to TR RESPA with respect to performance optimization.",
"keywords": [
"Multiple Time-Step Integration",
"Molecular Dynamics",
"Resonance Artefacts",
"RESPA",
"Twin-range",
"Liquid Phase Interfaces"
],
"content": "Introduction\n\nTo describe the dynamic processes of biomolecules, such as proteins, DNA, carbohydrates and membranes, molecular dynamics (MD) simulations have been proven to be a powerful technique to provide insights at the atomic level. In MD simulations, physical processes that involve a wide range of time scales have to be considered appropriately. However, the accurate and efficient treatment of various time scales presents a notoriously difficult problem due to resonance artefacts arising from separation into distinct numerical subproblems1,2. Over the past decades, various methods have been proposed to mediate the issue and to speed up the simulation substantially. For example, fast vibrational modes (e.g. covalent bonds) can be removed completely by constraining the motion, e.g. using SHAKE3, SETTLE4, M-SHAKE5, LINCS6, or FLEXSHAKE7. Alternatively, multiple time-step (MTS) integration is an efficient approach to accommodate motions on different time scales. In the twin-range (TR) scheme8–10, the pairwise nonbonded forces finb acting on particle i are split into a short-range contribution from particles j within the cutoff Rs, and a mid-range contribution from particles j between Rs and the long-range cutoff Rl (see Figure 1),\n\nLeft: Schematic illustration of the twin-range (TR) setup with the inner cutoff Rs and the outer cutoff Rl. Interactions beyond Rl are not calculated explicitly. Middle: Schematic illustration of the planar layer systems. Right: Schematic illustration of the systems with a droplet in a water box.\n\n\n\nwith\n\n\n\n\n\nThe short-range forces finb,s are updated every time step ∆t (inner time step), while the mid-range forces finb,m are updated every nm-th time step, nm∆t with nm ≥ 1 (outer time step). Long-range electrostatic interactions beyond the cutoff radius Rl can be included using the (an)isotropic reaction-field methods (RF or ARF)11,12, or lattice-sum methods13,14. Although theoretically possible, the TR scheme is generally not used together with lattice-sum methods. Simulations with (A)RF typically use larger cutoffs Rl than simulations with PME (e.g. 1.4 nm for the GROMOS force field15 compared to 0.9 nm for the AMBER force field16), which in the past encouraged the use of a TR scheme to reduce the computational cost.\n\nIn practice, for each particle i two pairlists are generated, which track the neighbouring short-range and mid-range particles j. To be consistent, the period of updating both pairlists np ∆t should be equal to the period of updating the mid-range forces nm∆t. In the following, the TR scheme is defined by Rs < Rl and nm = np > 1, and the single-range (SR) scheme by Rs = Rl, nm = 1 and np ≥ 1, i.e. there is only one pairlist for SR.\n\nSplitting the forces into different contributions leaves some freedom of how to consider them in the integration. There are two main strategies for the inclusion of the outer contributions: (i) constant mid-range forces application (CFA) fim at every inner time step ∆t, or (ii) using a reversible reference system propagator algorithm (RESPA)8–10, which is based on the Trotter factorisation of the Liouville propagator17. In the case of RESPA, scaled mid-range forces nmfim are typically applied impulse-wise, i.e. only at every outer time step nm∆t. Note that in this work the leap-frog scheme18 is used for the integration of the equations of motion, which is similar19 to velocity-Verlet20 as typically employed with RESPA.\n\nAlthough computationally efficient, MTS algorithms have the disadvantage of introducing numerical artefacts. Due to nonlinearity, it is notoriously difficult to formulate general physical or chemical conditions, which favour or suppress the build-up of these undesired resonances. Instead, specialised thermostats have been proposed as a remedy, which suppress resonance artefacts from MTS integration, for example by imposing isokinetic constraints on every degree of freedom21–23, or by selectively targeting the high-frequency modes using a colored noise thermostat24. In practice, however, standard thermostats like weak-coupling25, Nosé-Hoover (chain)26–28, or stochastic thermostats29–31 remain widely used in combination with a moderate integration time-step difference between fast and slow motions, as this should limit resonance artefacts to an acceptable value.\n\nIn this work, artefacts due to MTS integration are investigated with the main focus on a water-chloroform layer system using different thermostats (Figure 1). This system represents a simplified version of a biological membrane, for which an accurate simulation is of high interest in biology. The impact of splitting the nonbonded interactions into a short-range and mid-range contribution on the density profile normal to the interface and the kinetic energy distribution is investigated. In addition, a planar system consisting of cyclohexane and water as well as corresponding droplet systems are investigated. These additional setups allow to assess the impact of different solvents as well as different interface geometries on the formation of MTS artefacts. Overall, the artefacts are larger than those observed for more well-behaved, homogeneous systems32,33.\n\n\nMethods\n\nFor all simulations, the GROMOS software package34 version 1.4.0 was used with the GROMOS 53A6 force field15. Periodic boundary conditions were applied. Newton’s equations of motion were integrated using the leap-frog scheme18 with an inner time step of 2 fs. Different outer time steps for the pairlist update or mid-range force evaluation were used (i.e. nm, np ∈ [1, 20]). If not indicated otherwise, the SR scheme corresponds to a midrange force evaluation every 2 fs (nm = 1) and a pairlist update every 10 fs (np = 5). The TR scheme typically corresponds to a mid-range force evaluation in combination with a pairlist update every 10 fs (nm = np = 5). If not stated otherwise, the CFA method was used for the mid-range force contributions. The inner cutoff radius Rs was set to 0.8 nm and the outer cutoff radius Rl to 1.4 nm. For all simulations with RF, a charge-group based pairlist algorithm and cutoff were used (each solvent molecule is a single charge group), whereas an atomic cutoff was used for the PME simulations. The center of mass motion was stopped every 1 ps. The coordinates were saved every 0.2 ps and the energies every 0.1 ps. Bond lengths were constrained using the SHAKE algorithm with a geometric tolerance of 10−4. Three different thermostats were tested, i.e. weak coupling25 (WC), Nosé-Hoover26,27 (NH,) and Nosé-Hoover chain28 (NHchain). Different temperature baths were used for different solvents. The coupling time τ was set to 0.1 ps for all three thermostatting methods. A chain length of three was employed for the NHchain thermostat. A standard homogeneous RF approach11 was used to mimic long-range electrostatic interactions beyond a charge-group based cutoff Rl . The dielectric permittivity was set to 78.5, which corresponds to the experimentally measured value of water35.\n\nThe MTS investigation was performed mainly using a water-chloroform layer, in combination with three auxiliary systems consisting of a water-cyclohexane layer, a chloroform sphere in a water box, and a cyclohexane sphere in a water box. An illustration of the setups is shown in Figure 1. With these additional systems, different geometries of the interface as well as different chemical compositions were tested. An overview of the simulated systems can be found in Table 1. Corresponding snapshots are provided in Figure S1 (Supplementary File 1).\n\nThe main system consisted of 2002 chloroform (CHCl3)36 molecules and 8129 SPC37 water molecules within an elongated rectangular box of 30.2 nm length in z-direction. Identical initial coordinates were used for all simulations, which were equilibrated for 1 ns using the SR scheme under NVT conditions. The simulation length of the production simulations was 1–4 ns, performed under NVT conditions. For the water-cyclohexane layer system, 6750 cyclohexane (C6H12) and 39’366 water molecules were used in an elongated rectangular box of 33.85 nm length in z-direction. This system was simulated under NVT as well as NPT conditions (using a Berendsen barostat25 with a coupling time τb = 0.5 ps). The droplet systems consisted of a droplet of about 3 nm radius, solvated in a box of 12.2 nm length. This setup led to a droplet with 847 CHCl3 molecules in 59’484 SPC water molecules, or a droplet with 619 C6H12 molecules solvated in 59’018 SPC water molecules, respectively. As the convergence towards the numerical artefacts was found to be very fast, all auxiliary systems were simulated without initial equilibration for 1 ns.\n\nFor the spatially resolved density analysis, a program was developed using the data structures provided by the GROMOS++ collection of programs38. The energy distributions were calculated by post-processing the output of the GROMOS++ program ene_ana accordingly. For the density calculations of the spherical interfaces, a slightly more sophisticated analysis approach was necessary. In order to obtain a radial density profile, the molecules were assigned to equidistant concentric spherical shells with respect to the center of geometry of the droplet. However, for the droplet it was difficult to define consistently a distance to the surface, because the interface may deviate substantially from its ideal spherical shape during simulation. In our analysis, the center of mass was chosen as an approximation of the center of geometry, which was then shifted (if necessary) in order to account for periodic boundary conditions, i.e. to ensure that the droplet remained at the center of the periodic box. Note that the volume of the spherical shells scales cubically with the radius, which increases the uncertainty of the estimator substantially close to the center of the droplet.\n\n\nResults & discussion\n\nThe density profile of the water-chloroform system was determined along the z-axis normal to the layer. As can be seen in Figure 2, the splitting of the forces into a short-range and a less frequently updated mid-range contribution (given in Equation (1)) using the TR CFA scheme with nm = np = 5 led to a significant density increase for chloroform close to the interface. The density increase was accompanied by a density decrease further away from the interface due to the constant volume conditions. The presence of an increase was found to be independent of the chosen thermostatting algorithm. An analysis of the density resonance pattern with respect to the outer time step nm∆t is shown in Figure 3. Note that the density artefacts are not perfectly symmetric with respect to the layer norm due to finite simulation time. It can be seen that the TR CFA scheme leads to large resonance artefacts beyond nm∆t ≈ 20 fs independent of the thermostatting method. On the other hand, evaluating the mid-range forces every step while keeping the pairlist update every fifth step (i.e. SR scheme with nm = 1 and np = 5) resolved the issue almost entirely for all three investigated thermostats (Figure 2 and Figure 3, data not shown for NH and NHchain). The minor differences between SR schemes with different update frequencies can only be seen in density difference plots (Figure 4) with respect to the SR reference run (nm = np = 1).\n\nFor the four setups with the twin-range (TR) scheme, the mid-range forces were updated with the same frequency as the pairlist (i.e. nm = np = 5). For the single-range (SR) scheme, the mid-range forces were evaluated every time step ∆t (i.e. nm = 1), whereas the pairlist was updated every 5th step (i.e. np = 5). The weak-coupling (WC), Nosé-Hoover (NH) or Nosé-Hoover chain (NHchain) thermostat was used.\n\nFor the single-range (SR) scheme, the mid-range forces were evaluated every time stepc ∆t (i.e. nm = 1). For the three setups with the twin-range (TR) scheme, the mid-range forces were updated with the same period as the pairlist (i.e. np = n). The TR scheme was applied in a constant (CFA) or impulse-wise (RESPA) manner. The weak-coupling (WC) or Nosé-Hoover (NH) thermostat was used. Note that the density range is chosen to visualize the major artefacts occurring for nm∆t > 15 fs. A visualisation of the minor artefacts around nm∆t ≈ 10 fs can be seen in Figure 4.\n\nThe Nosé-Hoover (NH) thermostat was used in all setups. The single-range (SR) scheme (red and blue lines) and the twin-range scheme with constant-force application (TR CFA, orange and green lines) and two different update frequencies (np = 5, 10) are compared. Running averages with a window of seven data points are shown.\n\nThese findings indicate that the observed density increase is mainly caused by resonance artefacts due to the CFA MTS integration. This argumentation is supported by the pairlist error analysis shown in Figure S2, Supplementary File 1. The change in the pairlist after 10 fs is only on the order of 1%, and, even more importantly, it scales sublinearly with nm. Thus, the error from the pairlist cannot explain the substantial density increases seen in Figure 3. Instead, resonance effects lead to the build-up of the observed density artefacts over time, until a local equilibrium configuration is reached at the interface. This was found to be accompanied by a local change in the diffusion of the molecules at the interface. The mean residence time with respect to the layer normal z shows that regions of high density imply a locally reduced diffusivity of the molecules and vice versa (Figure S3, Supplementary File 1).\n\nThe numerical artefacts stem likely from an increasing mismatch between the forces and the position of the atoms the forces are applied to. In line with this interpretation, the density artefacts were suppressed for a typical value of the outer time step nm∆t = 10 fs when the mid-range forces were applied impulse-wise (TR RESPA scheme, blue line in Figure 2), i.e. the direction of the forces and the atom positions matched at the time point when the forces were applied. However, the TR RESPA scheme does not eliminate the resonance artefacts but only shifts them to longer outer time steps in this context (Figure 3). Thus, the partitioning of the system into different time scales together with the choice of the time steps in the MTS integration remain delicate aspects of current MD implementations39, also for impulse-wise MTS algorithms1,2,40.\n\nOn the contrary, only small effects on the density profile could be observed for the SR scheme with pairlist update periods up to np∆t = 40 fs (Figure 3 and Figure 4). Due to the large cutoff Rl used here (i.e. 1.4 nm), the changes in the pairlist are small between updates and scale sub-linearly with nm (Figure S2, Supplementary File 1). This observation is in line with the study of Krieger et al.41, where they found a negligible energy drift when using a similarly low pair-list update frequency in a homogeneous system. A rough performance analysis of the run time showed that for a realistic setup approximately half of the computational performance loss due to evaluating the mid-range forces at every time step could be compensated by a less frequent update of the pairlist (Table 2).\n\nAll systems were simulated for 0.4 ns on 1 CPU with 4 cores. A time step of 2 fs was used. The pairlist was generated using a charge-group based algorithm.\n\nIn order to exclude that the observed artefacts are specific to the water-chloroform system, the densities at a water-cyclohexane interface in a layer system were investigated. The corresponding density difference plots with respect to a SR reference are given in Figure 4. They show a similar behaviour of the TR scheme compared to the one observed for the water-chloroform systems, with slightly less pronounced artefacts. In addition, imposing constant pressure (NPT) conditions in the simulation setup does not reduce the observed density artefacts (Figure 5).\n\nA semi-aniostropic pressure coupling was applied in x/y-direction using a Berendsen barostat, while the box length in z-direction remained fixed. A Nosé-Hoover (NH) thermostat was used in all setups. The single-range (SR) scheme (red, blue and orange lines) and the twin-range scheme with constant-force application (TR CFA, green and purple lines) and three different update frequencies (np = 1, 5, 10) are compared. Running averages with a window of seven data points are shown.\n\nIn addition to planar interfaces, systems with a droplet of chloroform or cyclohexane in a water box were investigated. As can be seen in the right panels of Figure 4, density artefacts occur also at curved interfaces. Note that the corresponding plots contain more noise at small radii compared to the planar setups due to surface fluctuations, which influence the position of the centre of mass used as a reference. In addition, the surface fluctuations also give rise to a broadening of interface in terms of densities compared to the planar setup as can be seen in Figure S4 (Supplementary File 1).\n\nThe impact of the chosen thermostatting algorithm as well as the MTS integrator, and the energy distributions was investigated for the water-chloroform layer system. The WC thermostat does not give a canonical distribution of the kinetic energy by construction42, whereas the NH and NHchain thermostats do. In Figure 6, the kinetic energy distributions within water and chloroform are shown for the different setups. For chloroform, the difference in the width of the distribution between WC and NH(chain) thermostats is clearly visible, whereas the average kinetic energy remains the same, despite the significant density gradient in this solvent. In water, on the other hand, a clear shift in the kinetic energy distribution towards a higher average value was observed for the TR CFA scheme compared to the SR scheme with the WC thermostat. This shift was less pronounced with the NHchain thermostat, and absent with the NH thermostat. These observations indicate that a temperature shift may occur due to MTS resonances. However, as the density increase occurred with all three thermostatting methods, but the temperature shift only with two of them, the latter cannot be the cause for the former. Therefore, one can conclude that the resonance artefacts caused by the TR scheme cannot be removed with thermostats that control the velocities globally. For this, one would have to control the velocities selectively, e.g. by imposing isokinetic constraints or restraints on each degree of freedom21–23.\n\nNote that a different number of molecules is simulated for chloroform (left) and water (right), which leads to a different width of the canonical distribution functions. For the twin-range (TR) scheme (solid lines), the mid-range forces were updated with the same period as the pairlist (i.e. nm = np = 5). For the single-range (SR) scheme (dashed lines), the mid-range forces were evaluated every time step Δt (i.e. nm = 1), whereas the pairlist was updated every 5th step (i.e. np = 5).\n\n\nConclusions\n\nThe effect of using the TR scheme with a constant force application (CFA) at solvent-solvent interfaces was investigated with layer and droplet systems of different composition (water-chloroform and water-cyclohexane). These systems represent simplified versions of a biological membrane and a protein in solution. The force splitting was found to lead to substantial MTS resonance artefacts at the interfaces. It could be shown that the presence of the artificial density increase at the interfaces is independent of the thermostatting method, the interface geometry as well as the chemical composition of the system although the magnitude could vary. In addition, the diffusivity of the solvent molecules at the interface changed due to the density artefacts.\n\nThe resonance effects likely stem from a mismatch between the forces and the position of the atoms the forces are applied to. This hypothesis is supported by the fact that the pairlist changes only in the order of 1 % between updates and that the TR scheme with RESPA (where the forces are applied impulse-wise) does not eliminate the numerical artefacts but shifts them to longer time steps. Based on these findings, the use of TR CFA is not recommended for heterogeneous systems. Instead, the SR scheme should be used, which did not show any significant density errors in the entire range of pairlist updates (analysed up to np∆t = 40 fs). Thus, the increase in computational cost for SR can be partially compensated by updating the pairlist less frequently (i.e. np∆t > 10 fs). In addition, using only a single pairlist reduces the complexity with respect to implementation, as the additional difficulties arising from efficient memory management of two (or more) pairlists can be avoided.\n\n\nData availability\n\nWe are unable to provide the output files from the simulations directly due to file size. We instead provide the input files that when used as input with the GROMOS software package will generate the same results -\n\nDataset 1: Zip containing simulation input files 10.5256/f1000research.16715.d22294943",
"appendix": "Grant information\n\nThe authors gratefully acknowledge financial support by the Swiss National Science Foundation [200021-178762] and by Eidgenössische Technische Hochschule (ETH) Zürich [ETH-34 17-2].\n\n\nAcknowledgements\n\nThe authors thank Philippe Hünenberger and Wilfred van Gunsteren for helpful discussions.\n\n\nSupplementary material\n\nSupplementary File 1: File containing the following supplementary figures:\n\nClick here to access data\n\nFigure S1: Snapshots of different simulation setups.\n\nFigure S2: Change in mid-range pairlist with respect to update frequency.\n\nFigure S3: Spatially resolved mean residence time.\n\nFigure S4: Droplet density profiles.\n\n\nReferences\n\nBishop TC, Skeel RD, Schulten K: Difficulties with multiple time stepping and fast multipole algorithm in molecular dynamics. J Comput Chem. 1997; 18(14): 1785–1791. Publisher Full Text\n\nMa Q, Izaguirre JA, Skeel RD: Verlet-I/r-RESPA/Impulse is limited by nonlinear instabilities. SIAM J Sci Comput. 2003; 24(6): 1951–1973. Publisher Full Text\n\nRyckaert JP, Ciccotti G, Berendsen HJ: Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys. 1977; 23(3): 327–341. Publisher Full Text\n\nMiyamoto S, Kollman PA: SETTLE: An analytical version of the SHAKE and RATTLE algorithm for rigid water models. J Comput Chem. 1992; 13(8): 952–962. Publisher Full Text\n\nKräutler V, van Gunsteren WF, Hünenberger PH: A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J Comput Chem. 2001; 22(5): 501–508. Publisher Full Text\n\nHess B, Bekker H, Berendsen HJC, et al.: LINCS: A linear constraint solver for molecular simulations. J Comput Chem. 1997; 18(12): 1463–1472. Publisher Full Text\n\nChristen M, van Gunsteren WF: An approximate but fast method to impose flexible distance constraints in molecular dynamics simulations. J Chem Phys. 2005; 122(14): 144106. PubMed Abstract | Publisher Full Text\n\nBerendsen HJ, Van Gunsteren WF, Zwinderman HR, et al.: Simulations of proteins in water. Ann N Y Acad Sci. 1986; 482(1): 269–286. PubMed Abstract | Publisher Full Text\n\nVan Gunsteren WF, Berendsen HJ, Geurtsen RG, et al.: A molecular dynamics computer simulation of an eight-base-pair DNA fragment in aqueous solution: comparison with experimental two-dimensional NMR data. Ann N Y Acad Sci. 1986; 482(1): 287–303. PubMed Abstract | Publisher Full Text\n\nvan Gunsteren WF, Berendsen HJ: Computer simulation of molecular dynamics: Methodology, applications, and perspectives in chemistry. Angew Chem Int Ed. 1990; 29(9): 992–1023. Publisher Full Text\n\nTironi IG, Sperb R, Smith PE, et al.: A generalized reaction field method for molecular dynamics simulations. J Chem Phys. 1995; 102: 5451–5459. Publisher Full Text\n\nSidler D, Frasch S, Cristòfol-Clough M, et al.: Anisotropic reaction field correction for long-range electrostatic interactions in molecular dynamics simulations. J Chem Phys. 2018; 148(23): 234105. PubMed Abstract | Publisher Full Text\n\nHockney RW, Eastwood JW: Computer Simulation Using Particles. McGraw-Hill, New York, NY, 1981. Reference Source\n\nDarden T, York D, Pedersen L: Particle mesh Ewald: An N log (N) method for Ewald sums in large systems. J Chem Phys. 1993; 98(12): 10089–10092. Publisher Full Text\n\nOostenbrink C, Villa A, Mark AE, et al.: A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J Comput Chem. 2004; 25(13): 1656–1676. PubMed Abstract | Publisher Full Text\n\nWang J, Cieplak P, Kollman PA: How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comput Chem. 2000; 21(12): 1049–1074. Publisher Full Text\n\nTuckerman M, Berne BJ, Martyna GJ: Reversible multiple time scale molecular dynamics. J Chem Phys. 1992; 97: 1990–2001. Publisher Full Text\n\nHockney RW: Potential calculation and some applications. Technical report, Langley Research Center, Hampton, Va., 1970. Reference Source\n\nTuckerman M, Berne BJ, Martyna GJ: Reply to comment on: Reversible multiple time scale molecular dynamics. J Chem Phys. 1993; 99: 2278–2279. Publisher Full Text\n\nSwope WC, Andersen HC, Berens PH, et al.: A computer simulation method for the calculation of equilibrium constants for the formation of physical clusters of molecules: Application to small water clusters. J Chem Phys. 1982; 76: 637–649. Publisher Full Text\n\nMinary P, Tuckerman M, Martyna GJ: Long time molecular dynamics for enhanced conformational sampling in biomolecular systems. Phys Rev Lett. 2004; 93(15): 150201. PubMed Abstract | Publisher Full Text\n\nLeimkuhler B, Margul DT, Tuckerman ME: Stochastic, resonance-free multiple time-step algorithm for molecular dynamics with very large time steps. Mol Phys. 2013; 111(22–23): 3579–3594. Publisher Full Text\n\nMargul DT, Tuckerman ME: A Stochastic, Resonance-Free Multiple Time-Step Algorithm for Polarizable Models That Permits Very Large Time Steps. J Chem Theory Comput. 2016; 12(5): 2170–2180. PubMed Abstract | Publisher Full Text\n\nMorrone JA, Markland TE, Ceriotti M, et al.: Efficient multiple time scale molecular dynamics: Using colored noise thermostats to stabilize resonances. J Chem Phys. 2011; 134(1): 014103. PubMed Abstract | Publisher Full Text\n\nBerendsen HJC, Postma JPM, van Gunsteren WF, et al.: Molecular dynamics with coupling to an external bath. J Chem Phys. 1984; 81(8): 3684–3690. Publisher Full Text\n\nNosé S: A unified formulation of the constant temperature molecular dynamics methods. J Chem Phys. 1984; 81(1): 511–519. Publisher Full Text\n\nHoover WG: Canonical dynamics: Equilibrium phase-space distributions. Phys Rev A Gen Phys. 1985; 31(3): 1695–1697. PubMed Abstract | Publisher Full Text\n\nMartyna GJ, Klein ML, Tuckerman M: Nosé–hoover chains: the canonical ensemble via continuous dynamics. J Chem Phys. 1992; 97(4): 2635–2643. Publisher Full Text\n\nSchneider T, Stoll E: Molecular-dynamics study of a three-dimensional one-component model for distortive phase transitions. Phys Rev B. 1978; 17(3): 1302. Publisher Full Text\n\nAndersen HC: Molecular dynamics simulations at constant pressure and/or temperature. J Chem Phys. 1980; 72(4): 2384–2393. Publisher Full Text\n\nBussi G, Donadio D, Parrinello M: Canonical sampling through velocity rescaling. J Chem Phys. 2007; 126(1): 014101. PubMed Abstract | Publisher Full Text\n\nSchlick T, Mandziuk M, Skeel RD, et al.: Nonlinear resonance artifacts in molecular dynamics simulations. J Chem Phys. 1998; 140(1): 1–29. Publisher Full Text\n\nSandu A, Schlick T: Masking resonance artifacts in force-splitting methods for biomolecular simulations by extrapolative Langevin dynamics. J Chem Phys. 1999; 151(1): 74–113. Publisher Full Text\n\nSchmid N, Christ CD, Christen M, et al.: Architecture, implementation and parallelisation of the GROMOS software for biomolecular simulation. Comput Phys Commun. 2012; 183(4): 890–903. Publisher Full Text\n\nHaynes WM: CRC Handbook of Chemistry and Physics. CRC Press, Boca Raton, FL, 2014. Reference Source\n\nTironi IG, van Gunsteren WF: A molecular dynamics simulation study of chloroform. Mol Phys. 1994; 83(2): 381–403. Publisher Full Text\n\nBerendsen HJC, Postma JPM, van Gunsteren WF, et al.: Interaction models for water in relation to protein hydration. In Intermolecular forces. Springer, 1981; 331–342. Publisher Full Text\n\nEichenberger AP, Allison JR, Dolenc J, et al.: GROMOS++ software for the analysis of biomolecular simulation trajectories. J Chem Theory Comput. 2011; 7(10): 3379–3390. Publisher Full Text\n\nReißer S, Poger D, Stroet M, et al.: Real Cost of Speed: The Effect of a Time-Saving Multiple-Time-Stepping Algorithm on the Accuracy of Molecular Dynamics Simulations. J Chem Theory Comput. 2017; 13(6): 2367–2372. PubMed Abstract | Publisher Full Text\n\nMorrone JA, Zhou R, Berne BJ: Molecular Dynamics with Multiple Time Scales: How to Avoid Pitfalls. J Chem Theory Comput. 2010; 6(6): 1798–1804. PubMed Abstract | Publisher Full Text\n\nKrieger E, Vriend G: New ways to boost molecular dynamics simulations. J Comput Chem. 2015; 36(13): 996–1007. PubMed Abstract | Publisher Full Text\n\nMorishita T: Fluctuation formulas in molecular-dynamics simulations with the weak coupling heat bath. J Chem Phys. 2000; 113(8): 2976. Publisher Full Text\n\nSidler D, Lehner M, Frasch S, et al.: Dataset 1 in: Density artefacts at interfaces caused by multiple time-step effects in molecular dynamics simulations. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16715.d222949"
}
|
[
{
"id": "40231",
"date": "23 Nov 2018",
"name": "Bojan Zagrovic",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the article \"Density artefacts at interfaces caused by multiple time-step effects in molecular dynamics simulations\" by Sidler et al., the authors investigate the possible artefacts introduced by multiple time-step (MTS) integration methods for molecular dynamics (MD) simulations with respect to the local density and diffusivity at simulated interfaces between polar and apolar media. The simulated systems include chloroform-water and cyclohexane-water mixtures, both arranged in either planar-layer or droplet conformations. For all simulated systems, the authors report artificial density increases at the interface of the two liquids, whereby the degree of the observed effect depends on the MTS algorithm used. The density artefacts are studied mainly in the NVT ensemble, but are shown to also appear in the NPT ensemble. Moreover, some of the investigated thermostats introduce a shift in the distribution of kinetic energies in the water component. Based on these findings, the authors recommend the usage of the single-range treatment of the non-bonded interactions with lower pairlist update frequencies, which are less sensitive with respect to the discussed artefacts as compared to the widely used twin-range schemes. The study makes a valuable contribution towards the improvement of the existing MD methods when it comes to both accuracy and computational performance. The observed artefacts are certainly pronounced and cannot be ignored. On the other hand, certain aspects of the study lack the necessary explanations or depth of exploration and thus have to be improved accordingly.\nMajor comments\n\nThe length of individual simulations is relatively short by state-of-the-art standards. This, of course, is not a major issue if the phenomena at hand converge comparatively quickly, as indeed implied by the authors. However, for completeness, the convergence should be quantitatively demonstrated. For all NVT simulations, the authors should perform preliminary runs of several 100 ps in the NPT ensemble for density equilibration, as well as several 100 ps of equilibration in the studied ensemble before the production runs. The overall density in the present NVT simulations is determined by the authors in the course of the setup of the simulation boxes. It is conceivable that the ideal simulated density would vary with respect to the integration method, time-step or thermostating method. As a consequence, some simulated NVT systems could exhibit too-low or too-high overall density, which on its own could introduce or at least influence some of the observed artefacts. The microscopic origin(s) of the density artefacts should be investigated in greater detail. In particular, an in-depth investigation of the molecular structure at the interfaces would be instructive. For example, are the relative orientations of the chloroform/cyclohexane molecules in the areas of higher density different from those in the bulk? How about water? If so, how can this be related to the algorithmic differences? This analysis should, inter alia, involve a decomposition of density profiles into chloroform (or cyclohexane) and water components, but also more elaborate structural analyses (for an example of such analysis, please refer to1). The density profiles in Figure 2 exhibit curious short-range fluctuation patterns, which even appear somewhat periodic. The authors should analyze the origin of these patterns and potentially link them with the main analysis at hand. The authors should analyze and comment more extensively on the asymmetric appearance of the TR-CFA-WC and TR-CFA-NH density profiles in Figure 3 with respect to the planar layers at high values of the pairlist update period. They comment that the asymmetry may be due to insufficient sampling, but given how extensive and relevant for the central argument of the paper it may be, it should be analyzed in more detail. In an ideal case (i.e. if largely symmetric) the density profiles could be averaged over the two sides for greater statistical power.\nMinor comments\nThe CFA and RESPA approaches need to be explained in greater detail in the introduction. This seems especially important since RESPA seems to be much less prone to density artefacts than CFA. The colors are referenced wrong in the caption of Figure 4. The caption of Figure 3 contains an \"n\" without an index. The authors should explicitly describe how the density was calculated even for planar systems (bin size, averaging volume etc.). While the advantages of updating the Pairlist less frequently are clearly illustrated, in the conclusions section the authors should also discuss in more detail the potential downsides of such a procedure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "4353",
"date": "10 Jan 2019",
"name": "Sereina Riniker",
"role": "Author Response",
"response": "Major comments: We have checked the convergence, which is reached within 100 ps (new Figure S6 in the Supporting Information), well below the simulation time. The individual solvent boxes were equilibrated in the NPT ensemble, before combining them in the layer system. In the absence of artefacts (e.g. with a single-range scheme), the density profiles remain perfectly constant in the NVT ensemble. We consider this to be a more suited starting configuration than the layer system equilibrated additionally in the NPT ensemble with the twin-range scheme, because it shows the full extent of the artefacts introduced by the latter. We thank the reviewers for this suggestion. We have investigated the orientation of the solvent molecules at the interface, which is affected by the artefacts from the MTS algorithm. The results are presented in the new Figure S5 in the Supporting Information and discussed in the text. A decomposition of the density profiles is shown in the new Figure S2. These \"fluctuations\" stem from the binning of the density profiles. We have changed Figure 2 to have a consistent bin size for all curves. These fluctuations arise mainly due to the finite box size. We have adapted the text accordingly. Minor comments: We have added additional explanations of the MTS algorithms with equations. We thank the reviewer for spotting this. We have corrected the caption of Figure 4 accordingly. We thank the reviewer for spotting this. We have corrected the caption of Figure 3 accordingly. We have added these details in the Methods section. Due to the new results with the stochastic dynamics thermostat, the conclusions have been adapted."
}
]
},
{
"id": "40235",
"date": "03 Dec 2018",
"name": "Nico. F. A. van der Vegt",
"expertise": [
"Reviewer Expertise molecular simulation",
"physical chemistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and well-conducted study on density artefacts observed in molecular dynamics simulations that use multiple time-step integration algorithms to study liquid-liquid interfacial systems. The authors nicely show how depending on choices made for pairlist updates and thermostatting algorithms twin-range schemes can lead to severe density artefacts at (water-chloroform and water-cyclohexane) liquid-liquid interfaces. It is shown that these density artefacts cannot be remedied when widely used thermostats (WC, NH, NH-chain) are used that control the velocities globally. Do the density artefacts observed in these systems disappear when a standard, stochastic thermostat is used instead?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4352",
"date": "10 Jan 2019",
"name": "Sereina Riniker",
"role": "Author Response",
"response": "We thank the reviewer for this suggestion. We have performed additional simulations with a stochastic thermostat, which show that the artifacts disappear with this thermostat (see updated Figures 2 and 3). We have adapted the figures, text and conclusions accordingly."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1745
|
https://f1000research.com/articles/8-267/v1
|
07 Mar 19
|
{
"type": "Research Article",
"title": "Efficacy of a silver colloidal gel against selected oral bacteria in vitro",
"authors": [
"Phat L. Tran",
"Keaton Luth",
"James Wang",
"Coby Ray",
"Anselm de Souza",
"Dilip Mehta",
"K. W. Moeller",
"C. D. Moeller",
"Ted W. Reid",
"Phat L. Tran",
"Keaton Luth",
"James Wang",
"Coby Ray",
"Anselm de Souza",
"Dilip Mehta",
"K. W. Moeller",
"C. D. Moeller"
],
"abstract": "Background: It is necessary to develop new strategies to protect against bacteria such as Streptococcus mutans, Streptococcus sanguis, and Streptococcus salivarius, which contribute to tooth decay and plaque formation. Our current study investigated the efficacy of a colloidal silver gel in inhibiting biofilm formation by these principal oral bacteria, in vitro. The aim of this study was to assess the efficacy of a colloidal silver gel formulation for inhibiting bacterial biofilm formation (Ag-gel) by the principal bacteria that cause plaque formation and tooth decay. Methods: The effect of Ag-gel on viability of S. mutans, S. sanguis, and S. salivarius was assessed by quantifying their colony forming units (CFU) in presence or absence of the test gel. The effect of this formulation on biofilm-forming ability of these bacteria was studied through scanning electron microscopy. Results: Using the CFU assays, over 6 logs of inhibition (100%) were found for S. mutans, S. sanguis, and S. salivarius for the Ag-gel-treated bacteria when compared with the control gel. In addition, the Ag-gel also inhibited biofilm formation by these three bacteria mixed together. These results were confirmed by scanning electron microscopy. Conclusions: The Ag-gel was effective in preventing biofilm formation by S. mutans, S. sanguis, and S. salivarius. This Ag-gel should be tested for the ability to block plaque formation in the mouth, through its use as a tooth paste.",
"keywords": [
"Silver",
"biofilm",
"dental plaque",
"dental caries"
],
"content": "Introduction\n\nProblems associated with maintenance of oral health are faced by many people throughout the world, irrespective of their age and gender. The most common oral problems amongst all are dental caries, bleeding gums (periodontal diseases) and oral cancers1. Over few decades, the severity and prevalence of dental caries, and oral cancer, which can be a fatal condition, have increased2,3.\n\nIn the US, caries were estimated to be five times as common as asthma and seven times as common as allergic rhinitis3. According to the World Health Organization (WHO), dental caries are caused due to high sugar consumption, which is also linked with being overweight and obesity4. The incidence of periodontal diseases is estimated to be about 20–50% of the global population5. There are two approaches for the management of caries: extraction and preventions. The primary treatment modality for these caries, though very painful, is extraction of carious teeth2,3. The routine prevention measures for dental caries are to maintain oral hygiene, involving the use of fluoride toothpaste and/or xylitol6,7. Looking at the currently prevailing painful treatment, there is a need for new products to be developed for the prevention of oral cavities. The pathological organisms responsible for these caries/ periodontal diseases are Streptococcus mutans, Streptococcus sanguis and Streptococcus salivarius.\n\nSince mouth washes and different tinctures have been found to be ineffective against dental biofilm formation8, finding novel products effective against cariogenic microbes like S. mutans is important. While Listerine®, has some antimicrobial activity, toothpastes such as Toss-K and Senquel-AD have no activity against four important dental caries pathogens9. Thus, the search continues for more effective agent(s)10. A novel product that is effective against biofilm formation would be an important contribution to chewing sticks, toothpastes or other dental products.\n\nS. mutans has the ability to adhere the enamel surface, produce acid metabolites, build glycogen reserves and to synthesize extracellular polysaccharides. Mutans streptococci create acidic environment creating a risk for formation of cavity. During the formation of dental plaque, S. mutans adhere to primary colonizers by cell to cell interaction which forms biofilm on the teeth which induces bacterial growth11.\n\nStreptococcus sanguis is normally found in the human oral cavity. Due to low cariogenicity, it forms a colony on tooth surface which gets aggregated by other oral bacteria and leads to maturation of dental plaque12. Another organism, Streptococcus salivarius belonging to the salivarius subspecies is found in oral cavity in humans a few hours after birth and remain there as the predominant inhabitant. All these organisms enhance caries formation and thus the progression of periodontal disease. The aforementioned treatment modalities are unsuccessful in controlling or killing these bacteria and hence in prevention of caries.\n\nSilver has been used since ancient times as antibacterial agent for various pathological elements. During the last century, the antimicrobial action of silver has been investigated13. Colloidal silver is observed to be less toxic than ionic silver and has good compatibility with human cells. Silver was found to be effective in dentine desensitizer and is used as root canal disinfectant14. Silver nanoparticles are also used in dental material depending on the type of material being used. For example, titanium samples are mainly soaked in AgNO3 solution for dental implants to avoid bacterial contamination15. The mechanism of action of silver compounds on carious tooth is to inhibit demineralization process and anti-bacterial effect by interfering with bacterial cell membrane, cytoplasmic enzyme and inhibition of DNA replication of bacteria14.\n\nOral health being a global concern, it is essential to develop strategies to prevent dental caries and plaque formation. This study aimed at investigating the efficacy of a colloidal silver gel in inhibiting biofilm formation in vitro by the principal oral bacteria, Streptococcus mutans, Streptococcus sanguis and Streptococcus salivarius.\n\n\nMethods\n\nStreptococcus salivarius strain ATCC® 13419TM, Streptococcus sanguis ATCC® 10556TM, and Streptococcus mutans strain ATCC® 35668TM were obtained from Remel (Lenexa, KS, USA). S. salivarius, S. sanguis, and S. mutans were routinely grown in Brain Heart Infusion (BHI, #53286, Sigma-Aldrich, St. Louis, MO), at 37°C for 24 h.\n\nColloidal silver in a gel form was obtained from Viridis BioPharma Pvt., Ltd. (Mumbai, India). It was tested by evenly spreading 0.5 g on a 6-mm blank paper disc (BD Diagnostic System, Sparks, US) inoculated with the bacteria listed in the above paragraph. We assessed the bacteria remaining on the disc by the CFU assays below.\n\nA total of three blank sterile (6 mm diameter) cellulose paper discs were placed onto individual LB agar plates. Approximately 1x103 CFUs of the test bacteria were inoculated onto each disc. In the biofilm mixture study, approximately 4x102 of each bacterium were combined together. Either no gel (untreated), Ag-gel, or placebo gel (Viridis gel without Ag), were placed over the discs inoculated with bacteria. The plates were incubated under micro-aerobic conditions, which were generated by placing the plates inside a gas jar containing an EZ GasPak (Catalog no. 260678, BD, Franklin Lakes, NJ, USA) at 37°C for 24 h. Following incubation, each cellulose disc was analyzed for the remaining viable bacteria by the CFU assay as previously described16. Each piece was carefully removed from the well, rinsed gently with sterile distilled H2O, and placed in a microcentrifuge tube containing 1 ml PBS. The tubes were placed in a water bath sonicator for 5 min to loosen the cells within the biofilm and then vigorously vortexed 3 times for 1 min to detach the cells. Suspended cells were serially diluted 10-fold in PBS, and 10-µl aliquots of each dilution were spotted onto BHI plates. The plates were incubated at 37°C for 24 h. In experiments where no bacteria were detected, the remaining 900 µl of undiluted samples were tested. Thus, the equation for back-calculating the bacterial concentration was CFU x dilution factor x 100, with the exception of the 100-µl sample which was calculated as CFU x 10. This means that the smallest number of bacteria that we could detect would be approximately 1 bacterium. All experiments were performed in triplicate.\n\nBiofilms formed on discs were prepared for SEM by standard techniques and the experiment was performed as previously described16–18. S. salivarius, S. sanguis, S. mutans or a mixture of S. salivarius, S. sanguis, and S. mutans biofilms were established on cellulose discs (with or without silver gel, as described above). After 24 h of incubation, each cellulose disc and any adherent bacteria were fixed with 2% (wt/vol) glutaraldehyde in filter-sterilized 0.05 M PBS (pH 7.4) at room temperature for 16 h and then rinsed three times for 15 min each in 0.05 M PBS. The fixed cellulose discs were then dehydrated in successive ethanol-water mixtures with increasing ethanol concentrations (20%, 40%, 60%, 80%, and 95% [vol/vol]) for 15 min each and then twice in absolute ethanol for 15 min. The ethanol-dehydrated samples were then placed in an absolute ethanol bath, which was placed in an EMS 850 critical point drier (Electron Microscopy Sciences, Hatfield, PA). The ethanol was replaced by successive additions of liquid carbon dioxide. Once the liquid CO2 had replaced the ethanol, the chamber was heated under pressure to reach the critical evaporation point of carbon dioxide. The chamber was then slowly vented of gaseous CO2 and the dry samples removed. The dried samples were affixed to aluminum mounts with double-sided carbon adhesive tape and sputter-coated with platinum and palladium to a thickness of 18 nm. Observations were performed at 5 to 7 kV with a scanning electron microscope (Hitachi S-570; Japan). Five fields of view at 5,000X-10,000X magnification were taken at randomly chosen areas from the optic surface of each sample. A biofilm-positive field was defined as being occupied by biofilm over at least half of the visible area.\n\nThe results of the CFU assays were analyzed with Prism® version 4.03 (GraphPad Software, San Diego, US) with 95% confidence intervals (CIs) of the difference. Comparisons of the in vitro biofilms formed on the cellulose discs with either Ag-gel dressings or Ag-free ones were analyzed by a two-tailed unpaired t-test to determine significant differences. All experiments were done in triplicate. The significance limit was P<0.05.\n\n\nResults\n\nThe results for the 24-h micro-aerobic in vitro studies using S. salivarius, S. sanguis, and S. mutans isolates, as well as the mixture of all three, are illustrated in Figure 1. As seen in Figure 1A–D, the cellulose discs that had no treatment, and those that were treated with the placebo gel, showed over 6 logs of bacterial growth in each case. However, the silver containing gel showed 100% inhibition (over 6 log of killing) in all cases with S. salivarius, S. sanguis, S. mutans, or the combination of all three strains, when compared with the control, as showed in Figure 1A–D. Raw data are available on OSF19.\n\n(A) S. salivarius, (B) S. sanguis, (C) S. mutans or (D) all three on untreated discs, discs treated with placebo gel or discs treated with colloidal silver gel.\n\nTo confirm results of CFU assay described in previous section, the biofilm formation of S. salivarius, S. sanguis, S. mutans or the mixture of all three strains was studied on cellulose discs by SEM. S. salivarius, S. sanguis, S. mutans and the mixture of all three strains, were inoculated onto the discs in the same manner as for the CFU biofilm assay. Untreated discs were coated with placebo gel only. As above the discs were incubated for 24 h under micro-aerobic conditions at 37°C. As seen in Figure 2, S. salivarius, S. sanguis, S. mutans and the mixture of all three strains, formed typical biofilms characterized by the presence of micro-colonies on the cellulose discs receiving no treatment, or treated with the placebo gel. However, no bacteria were seen on the cellulose discs treated with colloidal silver gel. These results confirm those obtained with the CFU assay. Raw SEM images are available on OSF19.\n\n(A) SEM analysis of S. salivarius, S. sanguis or S. mutans biofilm formation on untreated discs, discs treated with placebo gel or discs treated with colloidal silver gel. (B) SEM of a blank disc.\n\n\nDiscussion\n\nThere are over 700 different species that contribute to the formation of dental biofilm (plaque)9–11. Among these species, Streptococcus mutans, Streptococcus salavarius, and Streptococcus sanguinis are the main members of this plaque20–22. These bacteria are also considered to be the primary etiologic agents of human dental caries23–26. It is the interaction of S. mutans with other streptococci that is thought to be important in dental plaque formation.27. S. mutans can cause cariogenicigty by the production of glucosyltransferase enzymes that allows glucose from sucrose to be used for the synthesis of glucan, and have also been implicated in heart problems28,29. S. sanguinis is an another common organism in dental plaque, which can colonize dental cavities. An additional problem is that this organism is also often found in the bloodstream. This allows it to attach to heart valves, causing bacterial endocarditis22. Thus, S. sanguinis is a key agent in infective endocarditis21.\n\nSince S. salivarius is also part of the normal human flora, it can contaminate sterile body fluid. Thus, therapeutic interventions that disrupt the cells protecting the blood vessels can allow it to enter the blood stream and cause problems in areas such as the meninges and the cerebrospinal fluid.30–33. This results in a variety of infections such as meningitis and bacteraemia along with many other bacterial problems.27,30,34–39. Thus, these three bacterial species can not only work together to form plaque on teeth, but can play a major role in other medical problems in the body.\n\nThe objective of the current study was to evaluate the test Ag-gel for its efficacy to either control or annihilate the growth of these 3 organisms. The results present quantitative data of the antimicrobial effect on S. salivarius, S. sanguis, or S. mutans bacteria, of Ag-gel, layered on a cellulose disc which was inoculated with these bacteria or a combination of all three bacterial strains. The CFU assay results of in vitro studies using Ag-gel treated dressings showed over 6 log of killing (100%) for S. salivarius, S. sanguis, or S. mutans as compared with a control gel dressing containing no Ag-gel. Since biofilms adhere strongly to surfaces, the experiments were also studied by SEM. These SEM studies confirmed the CFU results with S. salivarius, S. sanguis, or S. mutans biofilms, in the presence of Ag-gel or placebo gel. As seen in Figure 2, mature biofilms formed in the presence of the placebo gel, but none in the presence of the Ag-gel.\n\nThe mixture of these three bacteria was also studied, since it is proposed that the combination of bacteria is more resistant to growth inhibition than the individual bacteria. Similar results to the individual bacteria, (>6-log kill rate, 100%) were obtained with the combination of the three bacteria by both the CFU assay and by SEM studies.\n\n\nConclusion\n\nAn Ag-gel was found to be capable of over 6 log (100%) inhibition of S. salivarius, S. sanguis, or S. mutans bacteria, or a mixture of all three bacteria forming biofilms on cellulose discs by CFU studies. These results were confirmed by SEM studies of biofilm formation by S. salivarius, S. sanguis, or S. mutans or a mixture of all three bacteria, where the Ag-gel dressing showed total inhibition of biofilm formation on cellulose discs. These results indicate that use of a colloidal silver gel is an effective way to inhibit the formation of biofilms by the most common bacteria implicated in oral plaque formation, and this gel stands good potential to be developed into an effective commercial dentifrice product.\n\n\nData availability\n\nRaw data for this study are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/AJNYU19.\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 study was funded by Viridis BioPharma Pvt. Ltd.\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\nLansdown AB: Silver I: Its antibacterial properties and mechanism of action. J Wound Care. 2002; 11(4): 125–30. PubMed Abstract | Publisher Full Text\n\nTredget EE, Shankowsky HA, Groeneveld A, et al.: A matched-pair, randomized study evaluating the efficacy and safety of Acticoat silver-coated dressing for the treatment of burn wounds. J Burn Care Rehabil. 1998; 19(6): 531–537. PubMed Abstract | Publisher Full Text\n\nWright JB, Lam K, Burrell RE: Wound management in an era of increasing bacterial antibiotic resistance: a role for topical silver treatment. Am J Infect Control. 1998; 26(6): 572–7. PubMed Abstract | Publisher Full Text\n\nYin HQ, Langford R, Burrell RE: Comparative evaluation of the antimicrobial activity of ACTICOAT antimicrobial barrier dressing. J Burn Care Rehabil. 1999; 20(3): 195–200. PubMed Abstract | Publisher Full Text\n\nThomas S, McCubbin P: A comparison of the antimicrobial effects of four silver-containing dressings on three organisms. J Wound Care. 2003; 12(3): 101–7. PubMed Abstract | Publisher Full Text\n\nThomas S, McCubbin P: An in vitro analysis of the antimicrobial properties of 10 silver-containing dressings. J Wound Care. 2003; 12(8): 305–8. PubMed Abstract | Publisher Full Text\n\nDunn K, Edwards-Jones V: The role of Acticoat with nanocrystalline silver in the management of burns. Burns. 2004; 30 Suppl: S1–9. PubMed Abstract | Publisher Full Text\n\nDa Silva NB, Alexandria AK, De Lima AL, et al.: In vitro antimicrobial activity of mouth washes and herbal products against dental biofilm-forming bacteria. 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PubMed Abstract | Publisher Full Text\n\nBraet F, De Zanger R, Wisse E: Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells. J Microsc. 1997; 186(Pt 1): 84–7. PubMed Abstract | Publisher Full Text\n\nMehta D: “Efficacy of a Silver Colloidal Gel against Selected Oral Bacteria in Vitro.” OSF. 2019. http://www.doi.org/10.17605/OSF.IO/AJNYU\n\nCaufield PW, Dasanayake AP, Li Y, et al.: Natural history of Streptococcus sanguinis in the oral cavity of infants: evidence for a discrete window of infectivity. Infect Immun. 2000; 68(7): 4018–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaik S, Senty L, Das S, et al.: Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis. Infect Immun. 2005; 73(9): 6064–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu P, Alves JM, Kitten T, et al.: Genome of the opportunistic pathogen Streptococcus sanguinis. J Bacteriol. 2007; 189(8): 3166–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamada S, Slade HD: Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev. 1980; 44(2): 331–84. PubMed Abstract | Free Full Text\n\nLoesche WJ: Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986; 50(4): 353–80. PubMed Abstract | Free Full Text\n\nDa Silva AC, Cruz Jdos S, Sampaio F, et al.: Detection of oral streptococci in dental biofilm from caries-active and caries-free children. Braz J Microbiol. 2008; 39(4): 648–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWescombe PA, Hale JD, Heng NC, et al.: Developing oral probiotics from Streptococcus salivarius. Future Microbiol. 2012; 7(12): 1355–71. PubMed Abstract | Publisher Full Text\n\nKreth J, Merritt J, Shi W, et al.: Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in the dental biofilm. J Bacteriol. 2005; 187(21): 7193–203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgawa A, Furukawa S, Fujita S, et al.: Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA. Appl Environ Microbiol. 2011; 77(5): 1572–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakano K, Inaba H, Nomura R, et al.: Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens. J Clin Microbiol. 2006; 44(9): 3313–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMegarbane B, Casetta A, Esvant H, et al.: Streptococcus salivarius acute meningitis with latent petromastoiditis. Scand J Infect Dis. 2000; 32(3): 322–3. PubMed Abstract | Publisher Full Text\n\nRafailidis PI, Prapas SN, Kasiakou SK, et al.: Effusive-constrictive calcific pericarditis associated with Streptococcus salivarius. Case report and review of the literature. Cardiol Rev. 2005; 13(3): 113–7. PubMed Abstract | Publisher Full Text\n\nLee TH, Hsueh PR, Yeh WC, et al.: Low frequency of bacteremia after endoscopic mucosal resection. Gastrointest Endosc. 2000; 52(2): 223–5. PubMed Abstract | Publisher Full Text\n\nCorredoira JC, Alonso MP, Garcia JF, et al.: Clinical characteristics and significance of Streptococcus salivarius bacteremia and Streptococcus bovis bacteremia: a prospective 16-year study. Eur J Clin Microbiol Infect Dis. 2005; 24(4): 250–255. PubMed Abstract | Publisher Full Text\n\nConte A, Chinello P, Civljak R, et al.: Streptococcus salivarius meningitis and sphenoid sinus mucocele. Case report and literature review. J Infect. 2006; 52(1): e27–30. PubMed Abstract | Publisher Full Text\n\nYaniv LG, Potasman I: Iatrogenic meningitis: an increasing role for resistant viridans streptococci? Case report and review of the last 20 years. Scand J Infect Dis. 2000; 32(6): 693–6. PubMed Abstract | Publisher Full Text\n\nTrautmann M, Lepper PM, Schmitz FJ: Three cases of bacterial meningitis after spinal and epidural anesthesia. Eur J Clin Microbiol Infect Dis. 2002; 21: 43–5. PubMed Abstract | Publisher Full Text\n\nBouhemad B, Dounas M, Mercier FJ, et al.: Bacterial meningitis following combined spinal-epidural analgesia for labour. Anaesthesia. 1998; 53: 292–5. PubMed Abstract | Publisher Full Text\n\nVeringa E, Van Belkum A, Schellekens H: Iatrogenic meningitis by Streptococcus salivarius following lumbar puncture. J Hosp Infect. 1995; 29: 316–8. PubMed Abstract | Publisher Full Text\n\nWisplinghoff H, Reinert RR, Cornely O, et al.: Molecular relationships and antimicrobial susceptibilities of viridans group streptococci isolated from blood of neutropenic cancer patients. J Clin Microbiol. 1999; 37: 1876–80. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "45449",
"date": "11 Mar 2019",
"name": "Vijay Kothari",
"expertise": [
"Reviewer Expertise Antimicrobial Resistance (AMR)",
"Bacterial biofilms",
"Antimicrobials",
"Traditional Medicine",
"Bioacoustics",
"Plant extracts",
"Quorum Sensing"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 has evaluated antibacterial efficacy of a colloidal silver gel against three important oral bacteria. In context of the currently available dentifrice products not being full effective in preventing the widespread problem of dental caries, need and research for new more effective dentifrices is justified. Their idea of assaying the test gel not only against individual bacteria, but also against the mixed-species is a logical one, as that represents real-life situations a bit more closely. In the recent past, maintenance of good oral health has been shown to be important for good cardiovascular health, healthy pregnancy, etc.\n\nThey may consider re-framing the last sentence of the third paragraph in 'Introduction'. They may also check for correct placement of all references; For e.g. references 2-3 have been cited in first paragraph in an oral health context, but their titles seem to be more relevant to wound management.\nWhile, I do not have any reservations in recommending this article in its present form for indexing, I suggest for following additional experiments for future studies by these authors:\nEffect of this gel on pre-formed biofilms of oral bacteria should be checked on two different parameters i.e. whether this gel can kill bacteria in biofilm, and whether it can eradicate the pre-formed biofilm with/without killing the bacteria in it. Results of current study indicate their gel to possess a bactericidal action. They may consider extending the incubation till 48-72 hours after transferring the gel-exposed bacteria onto fresh gel-free media, to confirm the true bactericidal effect, and absence of post-antibiotic effect (PAE). They may consider determination of MIC, MBC, and time required to kill. The latter is important as typical dentifrice products like mouthwashes/toothpaste get few seconds/minutes to exert their effect in the mouth. It will be interesting to investigate whether at sub-inhibitory concentrations their product can reduce lactic acid production by the Streptococci, which is a major virulence factor involved in demineralization of teeth. Whether sub-inhibitory concentrations can inhibit quorum-sensing in these bacteria? In the current study, cellulose discs were used as surface for biofilm formation. In future studies, they may consider some material similar to teeth-substance (e.g. calcium) as the surface for biofilm formation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "45453",
"date": "16 Apr 2019",
"name": "Mohan Kulkarni",
"expertise": [
"Reviewer Expertise Polymers",
"nanoparticles",
"drug delivery",
"patents."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCaries has been the one of the most prevalent dental conditions which needs effective treatment. Products currently in use such as mouthwashes recommended for oral hygiene are not very effective against dental biofilm formation.\nThis study evaluates the efficacy of colloidal silver gel formulations in inhibiting bacterial biofilm formation by S. mutans, S. sanguis, and S. salivarius which cause plaque formation. The choice of colloidal silver over ionic silver in view of its lower toxicity is well justified and so also the choice of bacteria for evaluating the efficacy of colloidal silver.\nThe effectiveness of colloidal silver has been demonstrated by quantifying colony forming units (CFU). Appropriate controls and statistical analyses have been provided to support the results. The results are further supported by scanning electron microscopy. The results show that no biofilms are formed in the presence of colloidal silver gel but do form in the control experiments carried out with placebo.\n\nSince the objective of the work is to develop a treatment for the prevention of caries, it would be desirable to optimize the concentration of colloidal silver in the gel in future. In addition to prevention of biofilm formation if colloidal silver is shown to be useful in disrupting biofilms already formed, it would enhance the scope of treatment.\nI recommend the indexing of the paper “Efficacy of a silver colloidal gel against selected oral bacteria in vitro”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "47334",
"date": "15 May 2019",
"name": "C S Srinandan",
"expertise": [
"Reviewer Expertise Biofilms."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Tran et al., is important, but preliminary report that shows colloidal silver to be effective against three Streptococcus species. Following are some of the concerns I have regarding this manuscript.\nThe authors state that “the Ag-gel should be tested for the ability to block plaque formation in the mouth, through its use as a toothpaste”. Let’s consider the following: (a) Oral microbiome is very rich and its composition is important for health status of an individual and (b) Literature says colloidal silver is toxic to the host cells.\n\nConsidering (a) and (b), using the Ag-gel as a toothpaste may not be a good idea as it is not specific towards the pathogens. Thus, I recommend the authors to measure the cost-benefit ratio and mention about the conditions or stages of oral disease at which it could possibly be used as a toothpaste.\nWhy only one concentration of 0.5g of colloidal silver used for the studies? Please provide the details for the figures in the legends, like (i) what is the sample size, (ii) what do the error bars indicate, and (iii) which statistical test was performed on the data. I fail to understand the biological relevance of negative data of CFU in Fig. 1. What happens to biofilm formation at lower concentrations of silver? As the previous reviewers commented, eradication of biofilms should also be tested. Therefore, this manuscript should accompany the Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) of colloidal silver. Determination of interaction of colloidal silver with antibiotics is also an important test that has to be performed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-267
|
https://f1000research.com/articles/8-266/v1
|
07 Mar 19
|
{
"type": "Research Article",
"title": "The impact of the open-access status on journal indices: a review of medical journals",
"authors": [
"Saif Aldeen AlRyalat",
"Mohammad Saleh",
"Mohammad Alaqraa",
"Alaa Alfukaha",
"Yara Alkayed",
"Maryann Abaza",
"Hadeel Abu Saa",
"Mohamed Alshamiry",
"Mohammad Saleh",
"Mohammad Alaqraa",
"Alaa Alfukaha",
"Yara Alkayed",
"Maryann Abaza",
"Hadeel Abu Saa",
"Mohamed Alshamiry"
],
"abstract": "Background: Over the past few decades, there has been an increase in the number of open access (OA) journals in almost all disciplines. This increase in OA journals was accompanied an increase in funding to support such movements. Medical fields are among the highest funded fields, which further promoted its journals to move toward OA publishing. Here, we aim to compare OA and non-OA journals in terms of citation metrics and other indices. Methods: We collected data on the included journals from Scopus Source List on 1st November 2018. We filtered the list for medical journals only. For each journal, we extracted data regarding citation metrics, scholarly output, and wither the journal is OA or non-OA. Results: On the 2017 Scopus list of journals, there was 5835 medical journals. Upon analyzing the difference between medical OA and non-OA journals, we found that OA journals had a significantly higher CiteScore (p< 0.001), percent cited (p< 0.001), and source normalized impact per paper (SNIP) (p< 0.001), whereas non-OA journals had higher scholarly output (p< 0.001). Among the five largest journal publishers, Springer Nature published the highest frequency of OA articles (31.5%), while Wiley-Blackwell had the lowest frequency among its medical journals (4.4%). Conclusion: Among medical journals, although non-OA journals still have higher output in terms of articles per year, OA journals have higher citation metrics.",
"keywords": [
"Open access",
"Journal",
"Medicine",
"Bibliometrics",
"Citation"
],
"content": "Introduction\n\nOpen access (OA) journals allow free (access to/availability of) academic articles, they enable any user to read, search, download, share, use them for indexing, print the full texts, or utilize them as data for software without being charged1. Over the past 20 years, there has been an increase in the number of OA medical journals. According to Web of Science, published OA articles as a proportion of total publications increased from 9.5% to 24% from 1998 to 2018. These OA journals provide an easily accessed source of information, a source that is accessible even for developing and low income countries2.\n\nBibliometric analysis are methods or applications used to measure the influence of authors or scientific papers, of which, citation analysis is the most commonly used methods3. Now several citation databases have become available, with the three largest being Web of Science, Scopus and PubMed. These databases record the number of times that a journal article has been cited by other papers4. The use of bibliometric analysis is becoming more popular to assess the performance of different aspects of the scholarly and scientific fields. Analysis can be at the level of the researchers themselves, journals, departments, universities, national organizations, and even entire nations5–8. There are several databases that can be used to perform the bibliometric analysis, with each database having its own characteristics; these include Google Scholar, Pubmed (Only biomedical citations), Scopus, and Web of Science4. According to the number, coverage, and quality of citations covered by the databases, Scopus has wide coverage of high quality journals, compared to high number of citations at the expense of quality for Google Scholar, and high quality at the expense of number of citations for Web of Science9–11.\n\nIt is claimed that the emergence of OA journals has led to better dissemination of knowledge with the additional benefit of more citations for the authors, although this is still a matter of debate12. In this study, we aim to study the OA status of medical journals and the impact of the open-access status on journal indices using the Scopus database.\n\n\nMethods\n\nWe collected data on the included journals from Scopus Source List on 1st November 2018 (see Underlying data13). We filtered the list for medical journals (which include all specialties in medicine, as per Scopus categorization).\n\nFor each journal, we extracted the following citation metrics: Citation count, Percent Cited, CiteScore, CiteScore Percentile, SCImago Journal Rank, Source Normalized Impact per Paper (SNIP), and SCImago Quartiles. Details about these metrics and how they are calculated can be found on Scopus website.\n\nMoreover, scholarly output is defined as sum of documents published in the serial title (e.g. 2017) in the 3 years prior to the year of the metric (e.g. 2014 – 16). Open access Journals covered by Scopus are indicated as Open Access if the journal is listed in the Directory of Open Access Journals (DOAJ) and/or the Directory of Open Access Scholarly Resources (ROAD).\n\nWe used SPSS version 22.0 (Chicago, USA) in our analysis. We used means (± standard deviation) to describe continuous variables (i.e. journal indices). We used counts (frequency) to describe other nominal variables (i.e. publishers and OA journals). We performed Mann-Whitney tests to analyze the difference between measurements and OA status, and we presented data as medians (25% to 75% quartiles). To analyze open access journals between radiology and medicine, we used the weighting cases function in SPSS and a Chi-square test. All underlying assumptions were met, unless otherwise indicated. A p value of 0.05 was considered as significant.\n\n\nResults\n\nIn the 2017 Scopus list of journals, there was 5835 medical journals. Regarding the 5 most common publishers, 890 (15.3%) journals were from Elsevier, 653 (11.2%) Springer Nature, 196 (6.8%) Taylor & Francis, 360 (6.2%) Wiley-Blackwell, and 304 (5.2%) Wolters Kluwer. 1293 (22.2%) journals were OA journals. Table 1 indicates the minimum, maximum, mean, and standard deviation of medical journal indices.\n\nSNIP: Source Normalized Impact per Paper; SJR: SCImago Journal Rank.\n\nUpon analyzing the difference between medical OA and non-OA journals, we found significant differences in the following indices:\n\nCiteScore (p< 0.001): with a median of 1.19 (25–75%: 0.53–2.21) for OA journals, and a median of 1.06 (25–75%: 0.26–2.18) for non-OA journals.\n\nScholarly output (p< 0.001): with a median of 157 (25–75%: 76–319.5) for OA, and a median of 205 (25–75%: 107–423) for non-OA journals.\n\nPercent cited (p< 0.001): with a median of 52% (25–75%: 32%-70%) for OA, and a median of 48% (25–75%: 19%–68%) for non-OA journals.\n\nSNIP (p< 0.001): with a median of 0.706 (25–75%: 0.370–1.023) for OA, and a median of 0.617 (25–75%: 0.176–1.013) for non-OA journals.\n\nUpon comparing open access journals between the 5 most common publishers, we found a significant difference (p< 0.001). Post-hoc analysis showed that Wiley-Blackwell has significantly lower number of open access journals 16 (4.4%) open access journals compared to others. Table 2 shows the open access status for the most common publishers.\n\n\nDiscussion\n\nOur study found that OA medical journals had significantly higher CiteScores, Percent cited and SNIP; which is consistent with a number of previous studies made across a variety of disciplines including philosophy, political science, engineering, mathematics, physics, computer science and agriculture; all of which concluded that open access publications have a greater research impact (higher citation rate) than non-open access publications12,14–16. On the other hand, in a randomized controlled trial conducted on 11 biological and medical journals, it was found that only 2 of these journals showed positive and significant OA effects. In addition, it was found that OA advantage is declining by about 7% per year, from 32% in 2004 to 11% in 200717. Chua et al. found that there was significantly more citations in OA articles than in non-OA articles within almost identical journals’ impact factor18. Moreover, comparing citations in OA and non-OA articles in the same journal showed significant citation privilege for OA publications in several studies. For example, for the Journal of Postgraduate Medicine a comparison of citations per 100 articles per year before and after the journal became open access showed an increase between 3 and 4.5 times in citations19. In a longitudinal study of a cohort of OA and non-OA articles, it was shown that OA articles are cited earlier, and almost 2 times more frequently than non-OA articles in the first 4–16 months after publication in the same journal20. Regardless of all the aforementioned findings, our study found that non-OA medical journals have significantly higher Scholarly Output which can be strongly linked to the fact that most non-OA medical journals have been established years before OA journals, which have only recently emerged21.\n\nWe found that the number of OA journals varied among publishers, with Whiley-Blackwell having the least, with only 16 journals (4.4%), and the most with Springer Nature (206, 31.5%). In a previous study that analyzed OA articles published by different publishers, regardless of the discipline, they found that Elsevier had the highest number of OA articles, followed by Springer Nature and Whiley-Blackwell22. A longitudinal study comparing hybrid open access articles between publishers found great variation depending on the discipline23. For instance, medicine is the discipline which most frequently publishes in hybrid OA23.\n\nOur study has potential limitations. In this study, we didn’t account for the effect of publishing OA articles in non-OA journals (hybrid journals), as “Gold” OA publishing (i.e. fully OA journals) relates to publication of articles that are freely available to view and these may occur in OA or hybrid journals. Moreover, future studies should consider analyzing specialties within medicine (e.g. oncology), where we believe there will be variations in the effect of OA publishing within these specialties.\n\n\nData availability\n\nHarvard Dataverse: Medical journals. https://doi.org/10.7910/DVN/YYUTGG13.\n\nThis project contains the following underlying data:\n\nMedical journals 2017 dataset.tab (Scopus search results from the 1st November 2018)\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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBudapest Open Access Initiative (BOAI). Interlending & Document Supply. 2002; 30(2). Publisher Full Text\n\nNwagwu WE, Ahmed A: Building open access in Africa. Int J Technol Manage. 2009; 45(1/2): 82–101. Publisher Full Text\n\nNigam A, Nigam PK: Citation Index and Impact factor. Indian J Dermatol Venereol Leprol. 2012; 78(4): 511–6. PubMed Abstract | Publisher Full Text\n\nAlRyalat SA, Malkawi LW, Momani SM: Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases. J Vis Exp (Pending Publication). 2019; e58494. Reference Source\n\nHirsch JE: An index to quantify an individual's scientific research output. Proc Natl Acad Sci U S A. 2005; 102(46): 16569–16572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdams J: The use of bibliometrics to measure research quality in UK higher education institutions. Arch Immunol Ther Exp (Warsz). 2009; 57(1): 19–32. PubMed Abstract | Publisher Full Text\n\nKinney AL: National scientific facilities and their science impact on nonbiomedical research. Proc Natl Acad Sci U S A. 2007; 104(46): 17943–17947. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKing DA: The scientific impact of nations. Nature. 2004; 430(6997): 311–316. PubMed Abstract | Publisher Full Text\n\nKulkarni AV, Aziz B, Shams I, et al.: Comparisons of citations in Web of Science, Scopus, and Google Scholar for articles published in general medical journals. JAMA. 2009; 302(10): 1092–6. PubMed Abstract | Publisher Full Text\n\nBurnham JF: Scopus database: a review. Biomed Digit Libr. 2006; 3: 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBar-Ilan J: Citations to the “Introduction to informetrics” indexed by WOS, Scopus and Google Scholar. Scientometrics. 2010; 82(3): 495–506. Publisher Full Text\n\nAntelman K: Do Open-Access Articles Have a Greater Research Impact? Coll Res Libr. 2004; 65(5): 372–382. Publisher Full Text\n\nAlRyalat SA: Medical journals. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/YYUTGG\n\nKousha K, Thelwall M, Rezaie S: Using the Web for research evaluation: The Integrated Online Impact indicator. J Informetr. 2010; 4(1): 124–135. Publisher Full Text\n\nLawrence S: Free online availability substantially increases a paper's impact. Nature. 2001; 411(6837): 521. PubMed Abstract | Publisher Full Text\n\nmcveigh. 2004.\n\nDavis PM, Lewenstein BV, Simon DH, et al.: Open access publishing, article downloads, and citations: randomised controlled trial. BMJ. 2008; 337: a568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChua SK, Qureshi AM, Krishnan V, et al.: The impact factor of an open access journal does not contribute to an article’s citations [version 1; referees: 2 approved]. F1000Res. 2017; 6: 208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSahu DK, Gogtay NJ, Bavdekar SB: Effect of open access on citation rates for a small biomedical journal. 2005. Reference Source\n\nEysenbach G: Citation advantage of open access articles. PLoS Biol. 2006; 4(5): e157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBjörk BC, Solomon D: Open access versus subscription journals: a comparison of scientific impact. BMC Med. 2012; 10(1): 73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPiwowar H, Priem J, Larivière V, et al.: The state of OA: a large-scale analysis of the prevalence and impact of Open Access articles. PeerJ. 2018; 6: e4375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaakso M, Björk B: Hybrid open access—A longitudinal study. J Informetr. 2016; 10(4): 919–932. Publisher Full Text"
}
|
[
{
"id": "45461",
"date": "12 Mar 2019",
"name": "Ernesto Roldan-Valadez",
"expertise": [
"Reviewer Expertise Bibliometrics",
"linear-mixed models",
"statistical methods",
"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\nThe authors have performed an interesting study comparing differences in selected metrics (CiteScore, percent cited, SNIP, scholarly output) between medical OA and non-OA journals. This study, however, presents some limitations that require significant revisions:\n\nAuthors are using a robust number of journals (5835 medical journals) that will allow the performance of parametric tests. I would like to see that they applied at least an ANOVA comparing three groups: OA, non-OA and OA articles in non-OA journals (hybrid journals). The hybrid group is fundamental, and they do not include it. The authors should mention to the reader the bias of this decision. Also in this point, it is essential the authors explain why, if previous studies have concluded that the Eigenfactorscore is the best predictor of citations1,2, they did not include this metric in their analyses? Please also explain to the readers, why a linear-mixed-model design analysis was not performed. It is necessary to mention references of recent articles3 citing the existing correlations between the selected bibliometrics (CiteScore vs SNIP, Citescore vs IF, etc.) with at least two purposes: that the authors justified why they did not include a correlation analysis in their study, and that the readers be aware of the limitations in the correlation analysis, and also how the medical speciality may influence the results. It would be desirable to present a subgroup analysis of the medical specialities with the higher number of citations (for example oncology) as an example of the expected variability within subspecialties. If you report in the methods section that you used the SCImago Quartiles, why not control the effect of this variable using ANCOVA O MANCOVA? For example, if the authors are using the data from 5835 medical journals, this data allows a more robust analysis besides descriptive statistics and Mann-Whitney tests.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "45458",
"date": "19 Mar 2019",
"name": "Suneet Sood",
"expertise": [
"Reviewer Expertise Surgery",
"Medical education",
"microflora"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 intends to measure journal indices for OA journals, and compare them with the indices of Non-OA journals. The authors used the Scopus database. Although the theme is not new, I believe that the large number of indices calculated by the authors is a useful contribution to the literature.\nDatabase: I am a little ambivalent about the Scopus database. It is an extensive one, but to my mind it has two characteristics that need to be kept in mind. First, Google Scholar has, in all probability, overtaken Scopus in the breadth of coverage of publications for indexing (though till about 8 years ago Google Scholar was inferior to Scopus). Secondly, GS is more lax in its selection of journals to index, and it is more than likely that several “poor quality” journals are included. The authors themselves make these observations. I submit that the so-called poor quality of the journals indexed by GS is not really a drawback, and inclusion of these journals is more likely to provide a realistic picture. That said, using the Scopus database is an acceptable choice, and the sampling is large.\nStatistics: The choice of statistical tests is acceptable. I think that choosing two groups (OA vs Non-OA) is adequate for the objective of this paper. Would a third group, hybrid, have helped? Possibly, but hybrid journals are extremely varied in the number of articles they allow for open access, and I doubt that one hybrid journal, allowing 10% of its articles for free access, can be compared with another that allows 25% access immediately and full access after 6 months. However, I was not able to duplicate the calculations of the authors. The authors state as follows: CiteScore median = 1.19 for OA journals, 1.06 for non-OA journals. However, I calculate 1.2750 and 1.16 respectively, though the differences are still statistically significant. I get different values for the other metrics as well. Perhaps the authors should consider rechecking the values with their statisticians.\nMethods: Although the methods are described with reasonable clarity, I was unsure of the period covered by the data collection. The data file suggests that the data was collected for journal issues published from 30 April 2018 onwards. Have I understood correctly? I would request the authors to provide this detail in the methods.\nResults: The authors have recorded data for SJR values but have not discussed the results. There may be no significant differences between the two groups (OA, NOA), but I believe that they must discuss the implication of this result. The median SJR for NOA papers is slightly (but not significantly) higher than that for OA papers. Is this because NOA papers have a higher “prestige”? (Compare with their comments for scholarly output.)\nRecommendation: The authors should consider: A. cross-checking the results, B. clarifying the time span that is covered by the study, and C. commenting on the SJR 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "46001",
"date": "03 Apr 2019",
"name": "Deeb N. Salem",
"expertise": [
"Reviewer Expertise Cardiology and QI"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 generally well done with respect to its method and purpose. My major concern is its purpose: assuming adequate methods, statistical analyses and result, what is the significance? If there are implications of the research or significance of the conclusions, they should be clearly stated. The article is dependent upon the accuracy and scope of the Scopus database. I wonder how that was chosen and whether Pub Med might have been consulted as well. The accuracy of the conclusions is no better than the comprehensiveness of Scorpus. Are articles that appear in print but are archived online included? Otherwise the study seems to have been conducted well (methodologically) and the results competently reported.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "45999",
"date": "03 Apr 2019",
"name": "Sung-Tae Hong",
"expertise": [
"Reviewer Expertise Tropical Medicine",
"Editing & Publishing"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comment: This article describes difference of journal citation metrics between OA and non-OA journals in medical scope. The subjected journals are 5835 medical journals which are indexed in the Scopus and compared indices are metrics data supplied by Scopus and SCImago. The data showed significantly higher citation indices in OA journals than those in non-OA journals. The conclusion is that OA medical journals are cited more. The conclusion is known already in OA journals of other disciplines but it is valuable to publish.\nThe conclusion is sound and positive as expected, but I think we have to think about other issues on this topic. OA means no financial barrier for readers, which can facilitate citations. But most of researchers in established institutions have no barrier to non-OA journals because their institutions are able to subscribe to the non-OA journals. For those researchers, non-OA journals are free. I think that is the main reason of the small difference of those indices between the two groups although the difference is statistically significant. The statistics must be supported by large number of subject journals. This point must be discussed.\n\nSpecific comments\nDefinition of OA: journals included in DOAJ or ROAD. There are more Scopus journals outside of the 2 directories. PubMed Central may add more OA journals. The core results are difference of the indices that must be presented in a Table. Tables 1 and 2 are supportive but not cores. For reader friendly writing, one Table is required for results described in the text. Table 2 analyzed total numbers of OA journals by publishers. It may be interesting for readers to summarize whether there are index differences by publishers. Add discussion on the difference of indices: CiteScore 0.19 vs. 1.06; Scholarly output median 157 vs. 205; Percent cited 52% vs. 48%; SNIP median 0.706 vs. 0.617. All are highly significant by p<0.001. The gaps are significant but not so big because there are many other factors of citation. We should approach more details of citation analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-266
|
https://f1000research.com/articles/8-259/v1
|
05 Mar 19
|
{
"type": "Research Article",
"title": "Endoscopic dacryocystorhinostomy with marsupialization of the lacrimal sac.",
"authors": [
"Erika Celis-Aguilar",
"Angel Castro-Urquizo",
"Lucero Escobar-Aispuro",
"José Alarid-Coronel",
"Nadia Villanueva-Ramos",
"Eugenia Nemiliztli Hernández-Castillo",
"Vicente Solórzano-Barrón",
"Rómulo Perdomo-Martinez",
"Angel Castro-Urquizo",
"Lucero Escobar-Aispuro",
"José Alarid-Coronel",
"Nadia Villanueva-Ramos",
"Eugenia Nemiliztli Hernández-Castillo",
"Vicente Solórzano-Barrón",
"Rómulo Perdomo-Martinez"
],
"abstract": "Objective: To analyze the efficacy of endoscopic dacryocystorhinostomy (DCR) with marsupialization of the lacrimal sac compared with other techniques of endoscopic dacryocystorhinostomy. Material and methods: Clinical chart review. Patients with lacrimal sac pathologies and endoscopic DCR with or without marsupialization of the lacrimal sac were included from 2011 to 2015. The outcome measurements were absence of ocular symptoms and permeability of the lacrimal sac. Results: A total of 24 patients were evaluated, 17 women and 7 men, average age was 47 years. Seven patients underwent DCR with marsupialization, 17 patients underwent other endoscopic techniques. Average follow-up was 18 months. The efficacy (absence of symptoms and permeability of the lacrimal sac) of the DCR technique with marsupialization was 71%, without significant difference compared to other techniques (p = 0.686). Conclusion: Similar results were found in the different types of endoscopic DCR techniques. More studies are needed to corroborate our results.",
"keywords": [
"dacryocystorhinostomy",
"marsupialization",
"lacrimal sac",
"endoscopy."
],
"content": "Introduction\n\nLacrimal disease manifests clinically as epiphora, recurrent conjunctivitis, or dacryocystitis, and it occurs most frequently in pediatric patients. Dacryocystorhinostomy (DCR) creates a low-pressure system by diverting tear flow through the lacrimal bone and an artificial opening. Toti first described external DCR in 1904, and Caldwell used an endonasal technique in 1893 that West modified in 19141–3.\n\nEndoscopic DCR is the surgical procedure of choice to treat saccular or post-saccular nasolacrimal obstruction; this technique has been gaining popularity, with high success rates (sustained ostium patency, symptom relief, or both) comparable with external DCR rates, primarily because of the technological advances of endoscopes and surgical instruments. Multiple modifications have been suggested regarding endoscopic DCR procedures, with pros and cons. Previous endoscopic DCR procedures included making a small opening in the lacrimal sac and removing the nasal and lacrimal mucosa; this procedure likely contributes to surgical failure because the small neoformed ostium is obstructed by the granulation tissue or synechia formed during the postoperative period1.\n\nCurrently, two techniques are used to perform endoscopic DCR: laser-assisted and \"cold steel\"; both can be performed with or without powered drilling equipment. The former technique is less effective, perhaps because of the size of the ostium and the laser heat that results in fibrosis and stenosis4.\n\nGenerally, the size of the ostium created during surgery is crucial to the procedure’s outcome. Therefore, the anatomical characteristics of the lacrimal sac should be evaluated to achieve complete exposure when approaching the sac intranasally5,6.\n\nMassegur et al. suggested a modification to the technique known as marsupialization of the lacrimal sac, which causes the flaps of the lacrimal mucosa to contact the nasal mucosa after the resection of the bone surrounding the sac, thereby incorporating the lacrimal sac in the lateral nasal wall1,3.\n\nThe current study describes the results of a DCR with lacrimal sac marsupialization compared with other endoscopic techniques.\n\n\nMethods\n\nA clinical chart review study was conducted in patients who presented with obstruction of the lacrimal route in their excretory portion and were submitted to endoscopic DCR. The inclusion criteria were any patient with obstruction of the lacrimal duct or sac that resulted in epiphora or lacrimal sac infection. Exclusion criteria were incomplete clinical information or lack of surgical data. This study was conducted at the Ophthalmology and Otorhinolaryngology clinic in a secondary care center, (Hospital Civil de Culiacán, Rosales, México), from November 2011 to September 2015. Data regarding age, gender, affected side, symptoms, relevant background for the condition (e.g., trauma, infection, and previous ocular surgery), operative experience and patient follow-up results, were retrospectively collected.\n\nA team of two otorhinolaryngologists and two certified ophthalmologists performed the surgical intervention using the following standardized technique with small individual variations.\n\nThe surgery was performed under general anesthesia. A topical decongestant was placed in the nasal cavity, and the lateral wall was infiltrated with 2 ml lidocaine with epinephrine at 2%. The surgery was guided using a 0° nasal endoscope. A scalpel was used to section a mucosal flap approximately 5–8 mm on top of the middle turbinate insertion in the lateral wall, extending the incision anteriorly by 8 mm. A vertical incision was made halfway up the middle turbinate. The flap was raised with a Freer elevator and hidden around the middle turbinate to avoid obstructing the dissection later. The frontal process of the maxilla was extracted or removed with a 90° Kerrison rongeur, until the medial and anterior wall of the lacrimal sac was exposed.\n\nTo perform the marsupialization, the wall of the medial lacrimal sac was incised vertically along its entire length and then horizontally in a \"cross-like shape\". The flaps of the lacrimal sac were exteriorized toward the lateral wall, leaving the lacrimal sac open (Figure 1). The superior and inferior canaliculi were canalized; then, a bicanalicular silicone probe was passed whose ends were knotted inside the nostril (Figure 1). A Gelfoam sponge with a dexamethasone patch was lightly squeezed into the exposed sac.\n\nMT, middle turbinate.\n\nOnce the lacrimal sac is exposed, a resection of the medial wall is performed with various surgical instruments, such as rongeurs, and/or Blakesly forceps. There is no intent to preserve the lacrimal sac. The superior and inferior canaliculi were canalized; then, a bicanalicular silicone probe was passed whose ends were knotted inside the nostril.\n\nA follow-up assessment of the patients was conducted. The results were measured subjectively based on improvements in the symptomatology (i.e., the absence of ocular symptoms and lacrimal sac permeability) compared with the preoperative conditions. Objective measures were conducted via endoscopic controls that enabled the observation of an open fistula (Figure 2).\n\nMT, middle turbinate.\n\nThe Research Committee at Hospital Civil de Culiacán approved this research (Comité de Investigación del Centro de Investigación y Docencia en Ciencias de la Salud, number: 278). Since this was a retrospective chart review and the clinical images were non-identifying, the ethics committee waived the need for participant consent.\n\nThe information was entered into a database using SPSS version 22 for Windows. Frequencies and percentages were calculated for the categorical variables. The numerical variables were evaluated considering the means, confidence intervals, minimums, and maximum. The qualitative variables were measured using frequencies. The continuous variables were compared with Student's t-test, whereas the categorical variables were compared with a chi-square test. A p-value of ≤0.05 was considered significant.\n\n\nResults\n\nDuring the study period, 24 endoscopic DCRs were performed on 17 women and 7 men with a mean age of 47.21 years (7–82 years). Of these patients, two patients presented with congenital disease, five suffered from traumatism, and one patient reported a history of eye surgery (Table 1).\n\nA total of seven patients (29.2%) underwent endoscopic DCR with lacrimal sac marsupialization. The remaining 17 patients underwent other endoscopic techniques. The follow-up period was 18 months (4–43 months). One patient received previous dacryointubation and two had previous dacryocystorhinostomies. Electric drilling equipment was used for five patients. Bone removal was performed via a Kerrison clamp for 19 patients. There were three patients lost to follow up.\n\nThe efficacy (i.e., the absence of symptoms and patent lacrimal sac) was 71% (n=5/7 patients) for the lacrimal sac marsupialization and 71% (n=10/17 patients) for the other endoscopic techniques. No significant differences were found in the surgical outcomes between the techniques (p=0.686). Raw data are available on figshare7.\n\nA total of seven patients presented with postoperative complications: six with infection and one with granuloma and infection (Table 2).\n\nA total of seven patients presented with postoperative complications: six with infection and one with granuloma and infection (Table 2). Patients with infection resolved with topical and oral antibiotics (typically a cephalosporin) and the one patient with granuloma resolved once it was removed the silastic tube. No long-term complications were reported in the study.\n\nThree of our patients were lost to follow up. Unfortunately, six cases reported no improvement, regardless of endoscopic technique.\n\n\nDiscussion\n\nExternal DCR became popular because of its high success rates. However, the endoscopic DCR described by McDonogh and Meiring in 19891,4,5 has been used more often because of the simplicity of its innocuous endonasal approach. In addition, it offers advantages over the external approach such as reduced surgical trauma and hemorrhage, the avoidance of facial scars, the maintenance of intact medial canthus structures, and a faster time to return to work3,5,6. Failures of up to 12% of patients have been reported8. The main causes of failure of endoscopic DCR have been attributed to failure to locate the lacrimal sac, insufficient osteotomy, granulation tissue, synechiae, and closure due to premature scarring, fibrosis, and osteogenesis1,5,8.\n\nThe additional advantages offered by endoscopic DCR are better visualization of intranasal structures, the avoidance of angular vein damage, the preservation of the pumping function of the nasolacrimal sac, the corroboration of the adequate site for nasolacrimal tube insertion, the better correction of errors, and the identification of surgical failures9.\n\nTo avoid or prevent the obstruction of the neoformed ostium, multiple techniques have been tried with several modifications (e.g., complete marsupialization of the lacrimal sac, i.e., the use of mucosal flaps after a wide resection of the bone that surrounds the sac)3,10. Massegur et al. proposed this modification in 2004, with surgical success ranging from 87 to 92%3. The present study used a similar technique, an endoscopic DCR with marsupialization of the mucosal flap sac and resection of the bone using Kerrison's rongeur. A mean follow-up time of 18 months was conducted. The other endoscopic techniques used in the study were partial resection of the lacrimal sac mucosa and maxillary line graft, using Blakesley forceps or Kerrison’s rongeur.\n\nA learning curve of the surgeons could explain similar results in both techniques. The first seven cases of DCR marsupialization are described in this case series.\n\nYigit et al. (2007) compared the results of external DCR (55 patients) to those of endoscopic DCR (48 patients) in 103 patients with chronic dacryocystitis. The evaluated results were considered as successful if the epiphora decreased, infections were reduced, or reflux from the canaliculus was absent during lacrimal irrigation. The patient management success rate was 69.9% for those undergoing external DCR, and it was 89.7% for those receiving endoscopic DCR. These results were evaluated based on a 1-year follow-up period11.\n\nLikewise, the use of a silastic tube has been a matter of debate. Grigori et al. (2008) examined 46 patients undergoing DCR via a prospective, randomized study: half with silastic tube insertion and the other half without a catheter. Success was defined as the absence of epiphora, decreased conjunctival discharge, and fewer infections. The success rate for the 46 patients was 89%; the success rates with and without the use of a silastic tube were 78% and 100%, respectively, a significant difference (p=0.049). The follow-up period was 6 months. In addition, the controversial use of a silastic catheter was demonstrated12.\n\n\nConclusions\n\nA similar efficacy was found between endoscopic DCR with lacrimal sac marsupialization and the other endoscopic techniques in this study. Studies with larger patient samples are needed. Appropriate follow-up and postoperative care are recommended for all cases.\n\n\nData availability\n\nFigshare: Dacryocystorhinostomy dataset 2019, celis-aguilar et al.https://doi.org/10.6084/m9.figshare.7716500.v47.\n\nThis dataset includes the following files:\n\nDCRSPSSFEB2016f10002.csv (dataset containing surgical information on all patients)\n\ndata coding dacryocystorhinostomy article celis et al.docx (data dictionary)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe results of this research were presented at the AAO-HNSF Annual Meeting & OTO Experience, Chicago 2017.\n\nWe acknowledge the support of Hospital Civil de Culiacán and Universidad Autonoma de Sinaloa.\n\n\nReferences\n\nJin HR, Yeon JY, Choi MY: Endoscopic dacryocystorhinostomy: creation of a large marsupialized lacrimal sac. J Korean Med Sci. 2006; 21(4): 719–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarle M, Cabrera N, Naser A, et al.: Dacriocistorrinostomía endoscópica: Experiencia de 4 años del Hospital Clínico de la Universidad de Chile. Rev Otorrinolaringol Cir Cabeza Cuello. 2015; 75(3): 220–226. Publisher Full Text\n\nMassegur H, Trias E, Ademà JM: Endoscopic dacryocystorhinostomy: modified technique. Otolaryngol Head Neck Surg. 2004; 130(1): 39–46. PubMed Abstract | Publisher Full Text\n\nHernández-Ortiz A, Campos-Navarro LA: Dacriocistorrinostomía endoscópica. An Orl Mex. 2008; 53(2): 91–99. Reference Source\n\nTsirbas A, Wormald PJ: Endonasal dacryocystorhinostomy with mucosal flaps. Am J Ophthalmol. 2003; 135(1): 76–83. PubMed Abstract | Publisher Full Text\n\nWormald PJ: Powered endoscopic dacryocystorhinostomy. Laryngoscope. 2002; 112(1): 69–72. PubMed Abstract | Publisher Full Text\n\nCelis-Aguilar E: Dacryocystorhinostomy dataset 2019. figshare. Dataset. http://www.doi.org/10.6084/m9.figshare.7716500.v3\n\nDave TV, Mohammed FA, Ali MJ, et al.: Etiologic analysis of 100 anatomically failed dacryocystorhinostomies. Clin Ophthalmol. 2016; 10: 1419–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOnerci M, Orhan M, Ogretmenoğlu O, et al.: Long-term results and reasons for failure of intranasal endoscopic dacryocystorhinostomy. Acta Otolaryngol. 2000; 120(2): 319–322. PubMed Abstract | Publisher Full Text\n\nAli MJ, Psaltis AJ, Bassiouni A, et al.: Long-term outcomes in primary powered endoscopic dacryocystorhinostomy. Br J Ophthalmol. 2014; 98(12): 1678–80. PubMed Abstract | Publisher Full Text\n\nYigit O, Samancioglu M, Taskin U, et al.: External and endoscopic dacryocystorhinostomy in chronic dacryocystitis: comparison of results. Eur Arch Otorhinolaryngol. 2007; 264(8): 879–885. PubMed Abstract | Publisher Full Text\n\nSmirnov G, Tuomilehto H, Teräsvirta M, et al.: Silicone tubing is not necessary after primary endoscopic dacryocystorhinostomy: a prospective randomized study. Am J of Rhinol. 2008; 22(2): 214–217. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "45383",
"date": "22 Mar 2019",
"name": "Basil Mohammednather Saeed",
"expertise": [
"Reviewer Expertise Rhinology",
"endoscopic sinus surgery",
"rhinoplasty"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paragraph of 'Standard lacrimal sac surgery':\n\nHow much is the dilution of adrenaline?\nIn the results section:\nIn Table 1, the number of females and males in each group has to be mentioned fully, and what was the other ocular surgery in one patient? In the 'Efficacy of the procedures' paragraph, the last word needs correction. In the 'Follow up' paragraph, the first sentence was repeated on the next page.\nThe 'Discussion' was not that concise as the author concentrated on the comparison with external DCR, whilst your work was a comparison between different endoscopic procedures that was not satisfactorily covered.\n\nIs the work 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? Yes",
"responses": []
},
{
"id": "49429",
"date": "11 Jun 2019",
"name": "Michael K. Yoon",
"expertise": [
"Reviewer Expertise Ophthalmic plastic surgery",
"lacrimal surgery"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe their experience of endoscopic DCR with marsupialization versus endoscopic DCR with \"other techniques\". They compared their first 7 cases of endoDCR with marsupialization to the others. They found similar results (non-statistically significant) in comparing the surgical techniques.\nMajor comments: Patients included in this study are not the same. Of the 24 patients, 2 had congenital disease and two had previous DCR (unclear endo or external). Thus, 16% of their patients had atypical disease.\n\nIt is unclear how various patients were assigned to have marsupialization versus other types of surgery.\n\nThe authors stated they collected subjective symptomatology outcomes, however this is not reported.\n\nThree patients were lost to follow-up (all in the non-marsupialization group). These should be excluded from the analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/8-259
|
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