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https://openalex.org/W4223457172	https://harmreductionjournal.biomedcentral.com/track/pdf/10.1186/s12954-022-00622-8	English	null	Integration of a community-based harm reduction program into a safety net hospital: a qualitative study	Harm reduction journal	2,022	cc-by	9,491	Khan et al. Harm Reduction Journal (2022) 19:35 https://doi.org/10.1186/s12954-022-00622-8 Khan et al. Harm Reduction Journal (2022) 19:35 https://doi.org/10.1186/s12954-022-00622-8 Open Access Abstract Background: Community-based harm reduction programs reduce morbidity and mortality associated with drug use. While hospital-based inpatient addiction consult services can also improve outcomes for patients using drugs, inpatient clinical care is often focused on acute withdrawal and the medical management of substance use disorders. There has been limited exploration of the integration of community-based harm reduction programs into the hospital setting. We conducted a qualitative study to describe provider perspectives on the implementation of a harm reduc‑ tion in-reach program. Methods: We conducted 24 semi-structured interviews with providers from three different primary work sites within a safety net hospital in Boston, MA, in 2021. Interviews explored perceived facilitators and barriers to the implemen‑ tation of the harm reduction in-reach program in the hospital setting and solicited recommendations for potential improvements to the harm reduction in-reach program. Interviews were analyzed using an inductive approach that incorporated principles of grounded theory methodology to identify prevailing themes. Results: Twenty-four participants were interviewed from the harm reduction in-reach program, inpatient addic‑ tion consult service, and the hospital observation unit. Thematic analysis revealed seven major themes and multiple facilitators and barriers to the implementation of the harm reduction in-reach program. Participants highlighted the impact of power differences within the medical hierarchy on inter-team communication and clinical care, the persis‑ tence of addiction-related stigma, the importance of coordination and role delineation between care team members, and the benefits of a streamlined referral process. Conclusions: Harm reduction programs offer accessible, patient-centered, low-barrier care to patients using drugs. The integration of community-based harm reduction programs into the inpatient setting is a unique opportunity to bridge inpatient and outpatient care and expand the provision of harm reduction services. Trial registration: Not applicable. Keywords: Harm reduction, Substance use, Patient-centered care, Addiction consult service Integration of a community‑based harm reduction program into a safety net hospital: a qualitative study Ghulam Karim Khan1, Leah Harvey1,2, Samantha Johnson2,3, Paul Long1, Simeon Kimmel1,2,3, Cassandra Pierre2,4 and Mari‑Lynn Drainoni2,5,6 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Harm reduction is a collection of practical strategies and ideas designed to decrease the health, social, and/ or economic consequences of drug use and serve as an alternative to abstinence-based policies and program- ming. It acknowledges that people who use drugs have Correspondence: ghulam.khan@bmc.org p g g 1 Section of General Internal Medicine, Department of Medicine, Boston University School of Medicine, 801 Massachusetts Ave, 2nd floor, Boston, MA 02118, USA Full list of author information is available at the end of the article Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Khan et al. Harm Reduction Journal (2022) 19:35 a right to use drugs and to use them in a safe manner. Examples of harm reduction programs include syringe service programs (SSPs), opioid overdose education and naloxone distribution (OEND), and low-barrier super- vised consumption sites [1, 2]. Harm reduction is a social movement which seeks to affirm the dignity and rights of people who use drugs. The concept of harm reduc- tion grew from informal, grassroots advocacy efforts led by people who use drugs themselves in response to the widespread moralistic view of drug use during the early HIV/AIDS epidemic [3, 4]. In the USA, the government- sponsored War on Drugs exemplified the antagonistic and punitive response to drug use and addiction. Typi- cally, harm reduction programs function in the commu- nity outside of medical centers due to legal and logistical barriers as well as debates about the role of harm reduc- tion in clinical care [5, 6]. Until recently, addiction care and harm reduction specifically had been marginalized or excluded from general medical care, creating further barriers to the integration of harm reduction services and approaches into medical centers [2]. the first month after discharge, as well as a lower 30-day readmission rate [12, 13]. Inpatient addiction consult services are also able to start medications for opioid use disorder as well as link patients to outpatient care [14]. Study design We conducted qualitative interviews with 24 individu- als working in harm reduction or who have contact with the harm reduction program from the observation unit or ACS. The semi-structured interviews were conducted in order to gain a better understanding of the implemen- tation of a community-based harm reduction in-reach program. Though harm reduction programs have been present in the community for decades, addiction consult ser- vices have recently been implemented in inpatient medi- cal settings. Inpatient addiction consult services typically offer clinical care, pharmacologic treatment, behavioral interventions, and patient-centered education for indi- viduals who are hospitalized and use drugs. In contrast to community-based harm reduction programs, inpa- tient addiction consult services are often focused on the medical management of substance use disorders and acute withdrawal [10]. Limited qualitative data suggest that inpatient addiction consult services can decrease substance use while improving emotional well-being and socioeconomic circumstances [11]. Patients who are seen by multi-disciplinary addiction consult teams while hos- pitalized are more likely to reduce use of substances in Modeling studies suggest that addiction consult services are cost-effective while also saving lives [15]. f Despite the proven benefit of community-based harm reduction programs and high hospitalization rates of people who use drugs (PWUD), there has been lim- ited exploration of the integration of community-based harm reduction programs into the hospital setting. PWUD often experience addiction-related stigma (e.g., poorly controlled withdrawal or pain, loss of autonomy, room searches) during hospitalization and, as a result, delay necessary hospital care and often elect to leave the hospital before they are clinically optimized as a patient-directed discharge [10, 16, 17]. Though inpa- tient addiction consult services have helped to improve medical management of addiction and withdrawal from substances, integrating harm reduction specialists into the hospital setting could offer a more patient-centered approach to care and could facilitate improved clinical outcomes and safer discharges [10, 18]. Community harm reduction programs reduce morbid- ity and mortality associated with drug use and address racial and health-related inequities in a cost-effective manner. SSPs, OEND programs, and supervised con- sumption, also called overdose prevention sites, have all been shown to lessen the harmful consequences of injec- tion drug use. Harm reduction programs provide educa- tion on how to safely inject as well as how to reduce risk of infection [1, 2]. Increased utilization of SSPs and access to naloxone can play a significant role in mitigating the overdose crisis and related outbreaks of infectious dis- eases [7]. OEND programs have been found to increase readiness for substance use treatment and decrease self- reported opioid use [8]. Likewise, SSPs are effective at reducing HIV transmission when implemented as part of a multifaceted addiction program [9].h In 2019, Project TRUST, a harm reduction program at Boston Medical Center (BMC), initiated an in-reach program in the BMC short-stay observation unit. We conducted a qualitative study with staff from the harm reduction program, observation unit, and addiction con- sult service (ACS). Our aims were to describe barriers and facilitators to the implementation of a community- based harm reduction in-reach program within the hos- pital and to solicit suggestions for improvement from the perspectives of both community-based harm reduction specialists and hospital-based healthcare providers. Data collection Th i The semi-structured interview guide (see “Appendix A”) included nine open-ended questions. The initial por- tion of the guide explored the participants’ understand- ing of housing insecurity and available support services for patients experiencing housing insecurity admitted to the observation unit. Participants were then asked to describe their perception of the function and value of the harm reduction in-reach program. Subsequent questions explored perceived facilitators and barriers to the use of the harm reduction in-reach program. Participants were specifically asked to describe their department’s support of the harm reduction in-reach program, their impres- sion of the consult referral processes and logistics, edu- cational resources, staff availability, and patient desire for harm reduction services. The interview guide closed with a question regarding the impact of the COVID-19 pandemic on the availability of support services in the hospital and an exploration of any changes to the harm reduction in-reach program that participants would like to see pursued. BMC also operates a harm reduction program called Project TRUST (PT). PT is a community-based drop- in site located across the street from BMC with a street outreach team. Through PT, BMC employs harm reduc- tion specialists, a nurse practitioner, and an infectious diseases and addiction medicine physician who provide harm reduction education and safer consumption sup- plies (e.g., sterile injection and smoking equipment), infectious disease testing and prevention, addiction treatment resources (e.g., detoxification referrals, bridge buprenorphine prescriptions), and offer patient naviga- tion assistance. PT leverages BMC’s expertise in addic- tion medicine, existing infrastructure, and extensive resources through an integrated prevention and support services navigation model that addresses the barriers to care and services faced by PWUD. Since 2019, PT has also conducted a novel harm reduction hospital in-reach program in the observation unit. PT harm reduction spe- cialists are consulted to come into the inpatient setting to provide harm reduction counseling and facilitate post- discharge linkage to care for PWUD who are admitted to Participants completed a written demographic form at the time of data collection. Between January and March 2021, an interviewer (GKK) conducted confi- dential semi-structured interviews with 24 participants. The institutional review board of Boston Medical Center and the Boston University Medical Campus approved all study protocols and allowed a waiver of documenta- tion of consent by the participants. Interviews were con- ducted virtually over Zoom Video Communications Inc. (Zoom, Version 5, 2021). Study siteh The study was conducted at Boston Medical Center (BMC), the largest safety net hospital in New England [19]. BMC is a 514-bed academic medical center with a commitment to providing accessible care to the com- munity. Approximately 72% of BMC patients come from underserved populations, including the low-income and elderly, and the majority are publicly insured. Thirty-two Khan et al. Harm Reduction Journal (2022) 19:35 Khan et al. Harm Reduction Journal (2022) 19:35 Page 3 of 11 BMC. PT harm reduction specialists advocate to other care hospital-based care teams on behalf of their patients. Both ACS and PT are frequently consulted in the care of PWUD admitted to the BMC observation unit. percent of patients do not speak English as their pri- mary language [20]. Thirty-five percent of BMC patients were identified as African-American, 24% as Hispanic or Latino/a, and 25% as white [21]. BMC also serves as a national hub for substance use disorder (SUD) resources. BMC’s comprehensive substance use disorder manage- ment includes an office-based addiction treatment pro- gram, a low-barrier SUD urgent care clinic, integrated addiction and prenatal care programs, a multifaceted program for adolescents and young adults who use sub- stances, a community-based harm reduction and street outreach program, and an inpatient addiction consult service [22]. Recruitment Study participants were individuals who worked in the hospital observation unit, on ACS, or as part of the harm reduction in-reach program. We employed a convenience sampling strategy. An initial recruitment email asking if individuals were willing to participate in a semi-struc- tured, confidential interview was sent to potential partic- ipants in each work site via departmental contact listings. Individuals who replied to the recruitment email indicat- ing interest in participating were then contacted by study personnel, and eligibility was assessed according to inclu- sion criteria: age > 18 years old, job description (nurse, physician, midlevel provider, public health navigator, or social worker), and English speaking. BMC’s observation unit is a 28-bed inpatient unit where patients with lower clinical acuity and anticipated length of stay of less than 24 h can be admitted. Patients with skin and soft tissue infections, chest pain and other acute clinical conditions with concomitant substance use disorder are frequently admitted to this unit and may experience acute withdrawal from opioids or other sub- stances. For patients who would not otherwise be admit- ted to the hospital, the observation unit setting facilitates care coordination beyond what is typically possible in the emergency room and enables access to addiction-related resources that are less readily available in the ambulatory setting. One such resource is ACS, a physician-led inpa- tient medical consultation team that provides guidance on the intensive medical management of substance use disorders and acute withdrawal, including medication initiation and linkage to outpatient substance use disor- der treatment with methadone and buprenorphine, and peer recovery coaching and social work support. Data collection Th i Each of the 24 interviews lasted between 20 and 45 min and were audio-recorded with participants’ permission. Khan et al. Harm Reduction Journal (2022) 19:35 Khan et al. Harm Reduction Journal (2022) 19:35 Page 4 of 11 Table 1 Participant characteristics Results In total, 24 participants were interviewed. A majority of participants were recruited from ACS [16]. Four partici- pants each were recruited from the community-based harm reduction in-reach program and the observation unit. Most participants self-identified as clinical provid- ers [20], including nurse practitioners, physician assis- tants, social workers, and physicians. Other participants were identified as harm reduction specialists [4]. Twenty participants were identified as White, two as Black or African-American, and two as Asian. Thirteen partici- pants were identified as female, and 11 participants were identified as male (Table 1). Interviewees from all three primary work sites identi- fied conflicts between the structure of the US healthcare system and the ethos of harm reduction as barriers to the implementation of a community-based harm reduction in-reach program. A harm reduction worker explained: I think I have issues with our medical system over- all and how – and the for-profit system that we have with insurance and all of that, but you see it really impact those who are the most disadvantaged and need the most resources because there is no flex- ibility within a lot of these institutions to meet the needs of the patients that have it, have the highest needs. They set systems for those who have the lowest needs and it’s the oftentimes that they’re just making – they’re trying to get these patients that don’t fit the mold to fit the mold instead of changing the mold to fit the patients. – Participant 1 Data analysis Audio recordings were professionally transcribed verba- tim for qualitative analysis. Transcripts were reviewed by the interviewer (GKK) for accuracy. Analysis took an inductive approach that incorporated principles of grounded theory methodology [23]. To create the code- book, two members of the research team (GKK and LH) coded the first transcript independently and met to dis- cuss their findings. Thereafter, the two researchers coded sets of three transcripts independently and met regularly to update the codebook in an iterative fashion. This pro- cess continued until all of the interviews had been coded, and consensus had been achieved across all transcripts. All transcripts were coded and analyzed using NVivo 12.0 (QSR International Pty Ltd, version 12, 2018). N Gender Male 11 Female 13 Race White 20 Black or African-American 2 Asian 2 Primary work site Observation unit 4 Harm reduction in-reach program 4 Addiction consult service 16 Role Clinical provider 20 Harm reduction specialist 4 N Following the completion of the coding process, mem- bers of the study team (GKK and LH) engaged in the- matic analysis. Emerging themes were identified and discussed by the two coders and then shared with the larger research team for feedback and further discussion using a grounded theory approach [23]. The research team focused on developing themes related to the aims of describing facilitators and barriers to the implementation of a community-based harm reduction in-reach program as well as identifying desired changes to an existing com- munity-based harm reduction in-reach program. addiction care. Ultimately, participants expressed the desire to develop accessible, low-barrier harm reduction programs with staffing that reflect the patient popula- tions they serve. 1. The US healthcare system neglects social determi- nants of health and incentivizes expediency, which often conflicts with harm reduction work. Themesh – Participant 1 People who use drugs are often wary of medical staff and so they often will ask for us, or when they see us, they’ll tell us stuff that they wouldn’t normally tell, tell the medical staff, whether it be nurses, doctors, social workers, this often may mean, you know, I’m in a lot of pain, but I don’t want to say this, because they’re gonna think I’m drug seeking, that’s some- thing that we get constantly and I hate to say this, but often, I think our clients, and I’ve done it myself like, they feel overwhelmed or pressured a little bit to say what they think people want to hear. – Partici- pant 15 2. Addiction-related stigma toward patients negatively impacts both patients and harm reduction special- ists, contributing to provider burnout and patient mistrust of the medical system. While the inherent conflict between harm reduction work and system-level financial priorities was frequently implicated as contributing to staff burnout and cited as a key barrier to care, addiction-related stigma that patients face was also described as an important barrier by both ACS providers and harm reduction specialists. This stigma is thought to be perpetuated by a misperception of substance use disorders as a choice or moral failing rather than as a disease process. One addiction consult team member stated: Addiction-related stigma acts as a barrier to the provi- sion of care to patients by creating a cycle of mistrust that often results in patient-directed discharges. Alternatively, harm reduction programs offer an opportunity to foster trust between patients and the medical system. 3. The inpatient setting and inherent power hierar- chies in the clinical environment feel unfamiliar and unwelcoming to community-based harm reduction specialists. Yeah, like the way they treat and judge. You know, like the revolving door conversations that they’re not going to get no better, you’re going to see them again next week. While if you are more educated about the disease of addiction, and what it can do to a person, it’s not like that person wanted to grow up to be a person who shoots drugs and overdose. That’s not their dreams when they were a little kid, like things happened to them before they even pick up the drug. Themesh Thematic analysis revealed seven major themes. Partici- pants identified themes ranging from attitudes around the practice of harm reduction in the USA to specific reflections on the community-based harm reduction in- reach program and referral process. An area that was repeatedly discussed was the impact of innate power dif- ferences within the medical hierarchy on workplace com- munication and clinical care. Participants also described the barrier of addiction-related stigma and empha- sized the need for the clear delineation of work roles in Harm reduction philosophies in healthcare attempt to be patient-centered and focus on the patient’s experi- ence and values. In doing so, the patient’s needs can be prioritized over efficiency and expediency, which could potentially incur a higher financial cost for the health system. For example, a patient might be kept in the Page 5 of 11 Page 5 of 11 Khan et al. Harm Reduction Journal (2022) 19:35 Khan et al. Harm Reduction Journal (2022) 19:35 like taking off a Band-Aid and you’re cured. – Par- ticipant 6 hospital beyond when they are “medically” ready for dis- charge, in order to arrange for an appropriate discharge that addresses housing and safety. The harm reduction specialists, in particular, voiced frustrations and per- ceived differences in priorities for their patients between the greater health system and their services. One harm reduction worker described: Participants explained that addiction-related stigma is often exemplified by interactions between clinical staff and patients. Negative interactions and micro- aggressions can create an unwelcoming environment for patients, which can disrupt clinical care and potentially prompt patients to leave the hospital. Participants from all three work sites described situations in which stigma has disrupted a therapeutic relationship between patients and providers. An ACS member describes: When I was an outreach worker, and I was doing outreach, I was just pissed all the time because you’re literally just looking at people dying. You’re going why is this just down to money, but a lot of our – the systems in the hospital, a lot of them are established around keeping everything profitable and keeping everything going. That causes a lot of tension. Themesh But they just haven’t dealt with and don’t know how to deal with and found something to take them out- side that pain, and they’re running with it. And it’s something that became their best friend, their love, their mother, their father, that disease of addiction is there for them to take them out of the pain that they don’t want to feel. And the longer they stay out there, the more baggage that comes on top that they have to work to get out of that recover piece. So, if you understand that, okay educated about that, then you will understand that it’s not easy. It’s not Participants from the harm reduction in-reach pro- gram highlighted the difficulties arising from working in a new environment. The harm reduction in-reach pro- gram intentionally employs people with lived experience of addiction. One harm reduction worker explained how this intended facilitator of a successful harm reduction program can actually become a barrier: I think one of the harder things for the outreach workers specifically is they still feel that too, that hierarchy. We really try to hire from the community. Most all of our outreach workers are peers in some sort, so most of them have used substances them- selves and had their own struggles with substance use disorder or what have you. What ends up being difficult for the outreach workers is that they don’t get much more respect initially from the provid- ers and nurses on the floor or within the medical – Khan et al. Harm Reduction Journal (2022) 19:35 Page 6 of 11 or within medical clinics that we’re going into. The patients can also sense that. – Participant 1 or within medical clinics that we’re going into. The patients can also sense that. – Participant 1 or within medical clinics that we’re going into. The patients can also sense that. – Participant 1 therapeutic alliance between patients and their care teams. The above participant explains that harm reduction specialists with lived experience are often not treated as professionals with expertise and a valuable skill set. As a result, the harm reduction specialists have found the hierarchical world of inpatient medicine unwelcoming and difficult to navigate. 5. Clearly delineated roles reduce redundancy and streamline harm reduction services. The potential for miscommunication is compounded when roles are not clearly delineated. The services pro- vided by community-based harm reduction specialists can potentially overlap with hospital-based addiction specialists. One ACS clinician identified the importance of routine and clear task delegation: fi In addition to the power discrepancies between harm reduction specialists and hospital staff, harm reduction specialists also face an unfamiliar communication style in the inpatient setting. I think that it would be helpful to have a daily rou- tine of who to expect at what time, so that those point people will be the point people to talk to if we need to delegate screening for treatment programs or whatever, so that we’re not all doing the same thing at the same time. – Participant 11 They understand the hidden curriculum in the OBS unit, which might be for example; if someone is oth- erwise ready for discharge, an advocate might not be as well received by storming in and saying there’s no way you can let this patient out the door right [now]. There’s a hidden curriculum that we learn in medical school and residency in terms of how teams interact and make suggestions and disagree with one another. – Participant 4 Clearly defined roles can facilitate the provision of effi- cient, patient-centered harm reduction services. Many participants also cited a need for a centralized, stream- lined process to consult the harm reduction specialists. The hospital environment was viewed as frequently inhospitable to harm reduction specialists who have less experience in the inpatient setting and are less familiar with inpatient workflows and clinical communication. 6. Harm reduction programs should reflect the patient populations they serve. 6. Harm reduction programs should reflect the patient populations they serve. In considering the role that a harm reduction team should play in the hospital environment, participants across all three work sites emphasized the specific need for harm reduction specialists to identify with the patient populations they serve. A harm reduction specialist described the in-reach team: 4. or within medical clinics that we’re going into. The patients can also sense that. – Participant 1 Miscommunication negatively impacts patient care and hinders the provision of harm reduction services. 4. Miscommunication negatively impacts patient care and hinders the provision of harm reduction services. Participants cited difficulties in communication as a common barrier to the provision of harm reduction services to patients. Harm reduction specialists do not document their conversations and services in the patient chat. One ACS clinician explained that although this issue is not specific to harm reduction, it is an important barrier nonetheless. Forming a care team pushes it – pushes them to col- laborate and get rid of that hierarchy, so that’s really what we’ve tried to be at Project TRUST is make the outreach workers empowered enough to feel like they can talk up, they can advocate because they are an example for our patients, and if our patients feel like okay, I see somebody looks like me, has the same life experience as me, they are advocating for me. They’re getting what they want. They are able to talk with the doctors like a normal person. It’s not this person that’s just over them that’s smarter than them or doesn’t understand them. – Participant 1 So I think that’s not a - it’s not a problem that’s exclusive in any way to these teams. I think it just, whenever you have more - the more people that are involved there’s always more risk of kind of poor communication because there are more people who need to communicate, and then there’s more risk of harming patient trust or therapeutic alliance by them perceiving kind of lack of team cohesion or lack of communication. – Participant 23 Staffing the harm reduction in-reach program with peers with lived experience facilitates the development of a therapeutic relationship between clinical staff and patient. The addition of harm reduction specialists to an already extensive care team necessarily increases and can complicate inter-team communication as well as communication between providers and patients. Any miscommunication can fracture trust and threaten the While many participants explained that harm reduc- tion specialists with lived experience are best able to provide accessible harm reduction services, several Khan et al. Harm Reduction Journal (2022) 19:35 Page 7 of 11 participants further expounded on the ideal for harm reduction specialists to racially and ethnically reflect the populations they serve. or within medical clinics that we’re going into. The patients can also sense that. – Participant 1 As one harm reduction specialist explained, each patient has different goals and priorities: We’ve made our interventions purposely very flexible as far as time periods because we know that, espe- cially with this population that we deal with, they – there is no set formula to getting somebody clean or getting somebody to invest in even caring about themselves enough to use clean needles every day. – Participant 1 We’ve made our interventions purposely very flexible as far as time periods because we know that, espe- cially with this population that we deal with, they – there is no set formula to getting somebody clean or getting somebody to invest in even caring about themselves enough to use clean needles every day. – Participant 1 or within medical clinics that we’re going into. The patients can also sense that. – Participant 1 of a scheduled “addiction care check-in” for all provid- ers involved in the care of patients with substance use disorders. Participants from all three primary work sites highlighted the need for an automated referral pathway that could identify high-risk patients early in their clini- cal course in order to involve the harm reduction in- reach program as early as possible and potentially reduce patient-directed discharges. I think having a really diverse group of people in there, I think is helpful, especially patients coming from different backgrounds, people of color, et cetera, I think connecting with people is important in that respect. – Participant 12 That could look a whole bunch of different ways. At BMC, we have a lot of the IT infrastructure in terms of the basics in place, but I don’t think we’ve lever- aged it as much as we could to do things like have algorithms that might generate an automatic refer- ral. And in a case like this, that might look like some- one with a substance use disorder diagnosis on their list who’s had two or more OBS presentations or you could hash out what the algorithm would look like, but I think that would be really facilitating so that the Project TRUST team gets pulled in earlier in the admission rather than when if a patient might be feeling like their withdrawal is uncontrolled and not having a good experience and considering leaving in a patient-directed manner. – Participant 4 7. The referral process should be low barrier and facili- tate the identification of patients who would benefit from harm reduction services. 7. The referral process should be low barrier and facili- tate the identification of patients who would benefit from harm reduction services. 7. The referral process should be low barrier and facili- tate the identification of patients who would benefit from harm reduction services. Harm reduction programs work to meet patients where they are, with respect to their substance use. Success- ful harm reduction programs are flexible in the provi- sion of their services. Discussion Harm Reduction Journal (2022) 19:35 Page 8 of 11 Community-based harm reduction specialists typically have limited experience with the structure and workflow of the inpatient setting [31]. Consequently, they struggle to gain acceptance from primarily hospital-based work- ers and often experience similar stigma as the PWUD whom they are consulted to see [32]. One of the ACS cli- nicians aptly named the “hidden curriculum” that exists in the hospital. Hospital-based providers undergo years of training that facilitate an effective and efficient form of communication that is medical treatment-focused, rather than harm reduction-focused [33]. Community-based harm reduction specialists are not always privy to this communication style and therefore can be seen as inter- rupting the efficiency of clinical work flow when they are providing their consultation services to PWUD.fi complications of substance use [24]. At the same time, costs and overcrowding are often cited as pressures to decrease hospital length of stay and to discharge patients as early as medically feasible [25]. While the financial feasibility of harm reduction in-reach repre- sents an area for additional research, we do know that inpatient addiction medicine consultation is effective at reducing repeat hospitalizations and morbidity asso- ciated with substance use disorders and these results suggest that harm reduction in-reach may be a valuable mechanism to support inpatient addiction medicine consultation [12, 26]. Alongside a lack of institutional and systemic sup- port for harm reduction philosophies, participants cited addiction-related stigma as a major barrier to the success of a harm reduction in-reach program. The ACS clini- cians and harm reduction specialists interviewed were intimately familiar with the spectrum of addiction care, including harm reduction and non-abstinence-focused strategies. Observation unit staff, however, had less experience with harm reduction. As a result, there were significant discrepancies in participants’ comfort treat- ing patients who use drugs, knowledge of substance use disorder treatment and harm reduction strategies, and consideration of addiction as a disease process. Addic- tion and harm reduction specialists both emphasized the negative impact of ongoing addiction-related stigma in the hospital setting, which has been demonstrated to be a significant barrier to the implementation of compre- hensive harm reduction programs [27]. Multiple partici- pants suggested that negative interactions and perception of stigma could increase patient-directed discharges and disrupt the provision of care, leading to negative health outcomes and hospital re-admissions. Discussion Patient-directed discharges represent gaps in our addiction care that could be partially addressed by harm reduction programs and inpatient addiction consult services [17].hl Difficulties in communication highlight the impor- tance of clear role delineation when implementing a harm reduction in-reach program. As many participants described, unclear roles between addiction medicine consultants and harm reduction specialists can be frus- trating for both patients and other hospital staff. [34]. However, it is important to note that patients often feel more comfortable communicating their needs and val- ues to the community-based harm reduction specialist, with whom they can more closely identify due to shared experience. [35]. As such, the harm reduction in-reach program may arrive at different clinical conclusions from the addiction consult service. For example, patients may feel pressured to commit to abstinence and pursue phar- macologic treatment for their SUD rather than acknowl- edging that their current priority is to continue to use. This represents a missed opportunity for harm reduc- tion services, such as safer injection education, and often contributes to a discrepancy between the care plan and patient behaviors. Furthermore, community-based harm reduction specialists who are stigmatized upon entering the hospital environment feel frustrated that they are not being heard or treated as professional colleagues by the hospital-based workers [32]. The conflict between the ethos of harm reduction and the US healthcare system and ongoing addiction-related stigma contributes to burnout among harm reduction and addiction specialists and negatively impacts PWUD [28]. Harm reduction specialists can potentially bridge these gaps in care and offer an accessible and patient-cen- tered model of care delivery. Harm reduction specialists, who often have lived experience with drug use, continue to refine their expertise by working with clients in the community setting [29]. When these harm reduction specialists enter the hospital setting, they bring those skills and relationships with them. Our results suggest that patients feel much more comfortable when they are able to speak with peers who look like them, come from their communities, and, in some cases, know them from before their hospitalization [30]. With respect to the harm reduction in-reach program itself, participants identified the need for the program to reflect the patient population it serves. Our findings are consistent with prior studies proposing that harm reduc- tion in-reach programs should be staffed by peers with lived experience and who are diverse in race, ethnicity, and gender identity [36]. Discussion In this study, a community-based harm reduction in- reach program was identified as a valued program by ACS staff, hospital observation unit staff, and harm reduction specialists. While the need for additional harm reduction services was consistently described, there was a wide variation in perspectives on how to better imple- ment harm reduction services in the hospital. Partici- pants also described multiple barriers and facilitators to the success of a community-based harm reduction in- reach program. These included power hierarchies across hospital systems, addiction-related stigma, and miscom- munication within the clinical setting.i Bearing this in mind, one ACS staff member theorized the role that community-based harm reduction special- ists could play in the hospital setting: But I think that integrating community low barrier organizations in our healthcare system could make hospitals much more welcoming places for people experiencing homelessness or with substance use disorders, provide better continuity of care and to places like our bridge clinics that could have ongoing communication with these sort of community-based low-barrier organizations, and help sort of follow people who right now are completely falling through the cracks who get discharged, and we never hear or see from them again, until they’re re admitted. – Participant 14 Harm reduction specialists specifically described the inherent contradiction between the philosophy and practice of harm reduction and the US healthcare sys- tem, which incentivizes patient volume and clinical expediency. They explained that the provision of harm reduction services can include counseling, motivational interviewing, and patient advocacy, which are thought- ful, time-consuming, and often at odds with hospital prioritization of the rapid turnover of beds. However, other addiction clinicians and harm reduction spe- cialists suggested that the overall cost-effectiveness of harm reduction in-reach may outweigh the immedi- ate cost of extending a hospital admission. The exist- ing literature supports this idea and demonstrates that community programs decrease downstream medical By being more patient-centered than traditional clinical care, community-based harm reduction specialists could help make the hospital a more welcoming environment and interrupt the cycle of stigma and disengagement from care. When asked about how to design and integrate a harm reduction program into the traditional hospital workflow, participants suggested a variety of models. Suggestions ranged from modeling the service off of a traditional medical consult (e.g., cardiology consult) to the creation Khan et al. Harm Reduction Journal (2022) 19:35 Khan et al. Discussion Participants reported that the most positive interactions involving the harm reduc- tion in-reach program occurred when PWUD knew that harm reduction specialists were coming from their own communities.i Participants suggested a wide variety of specific logis- tical adaptations to better facilitate the integration of community-based harm reduction in-reach programs Khan et al. Harm Reduction Journal (2022) 19:35 Page 9 of 11 into the inpatient setting. For example, some participants suggested modeling the harm reduction in-reach referral system after a traditional inpatient specialty service con- sultation. Many participants cited the benefit of having a separate referral process for the harm reduction in-reach program that is distinct from other addiction services, while maintaining close working relationships between the two teams. While addiction consult services in hos- pitals often offer counseling and education around safer injection practices, they generally approach patients from a medical perspective rather than with a harm reduction lens [37]. To facilitate the consultation process, partici- pants requested a clear and automated process for involv- ing harm reduction specialists, particularly in the care of high-risk patients who may wish to leave prior to medical optimization. While this need has been described with respect to the identification of patients who would ben- efit from addiction medicine consults, it was emphasized by participants as a priority for any harm reduction in- reach program [37].h using a convenience sample, which may have inadvert- ently introduced a selection bias of participants who were more supportive of harm reduction hospital in-reach programs. The morbidity and mortality associated with the ongo- ing overdose crisis have continued to increase over the past decade. The COVID-19 pandemic further com- pounded the impact of the overdose crisis by reducing access to SSPs and other harm reduction services and exacerbating underlying mental health issues [38–40]. Harm reduction programs offer a powerful and patient- centered tool to mitigate the negative consequences of substance use [41]. As safe consumption sites and other harm reduction approaches in use in other countries come to the USA, there will be an opportunity and need to integrate these into inpatient settings. The lessons learned from the integration of harm reduction workers into the observation unit may promote effective imple- mentation and mitigate challenges. The existing literature focuses on the successes of harm reduction programs in the outpatient setting or on the implementation of inpatient addiction medicine services. Conclusionh The integration of community-based harm reduction programs into the inpatient setting is a unique opportu- nity to bridge inpatient and outpatient care and expand the provision of harm reduction services. Future areas for investigation include explorations of patient perspectives on harm reduction in-reach programs as well as the clini- cal effectiveness and cost-effectiveness of harm reduction in-reach programs. Discussion This study is unique in that it focuses on the integration of community-based harm reduction programs into the inpatient environment. Because of its qualitative nature, the study is able to explore different perspectives around how to best implement harm reduction in-reach pro- grams, as well as describe some of the potential impli- cations on clinical care. Our results describe important facilitators and barriers to the integration of a commu- nity-based harm reduction in-reach program into the inpatient setting. The results augment the existing lit- erature on potential conflicts between harm reduction philosophies and privatized medical systems. Our quali- tative analysis reaffirms the continued negative impact of addiction-related stigma, contributing to both pro- vider burnout and patient mistrust of the medical sys- tem. This study also highlights the importance of clear communication between harm reduction specialists and inpatient addiction specialists. It adds the voices of both harm reduction and addiction specialists to the literature as they identify ways to improve the provision of harm reduction services to PWUD who are hospitalized. References 1. Olding M, Ivsins A, Mayer S, Betsos A, Boyd J, Sutherland C, et al. A low- barrier and comprehensive community-based harm-reduction site in Vancouver. Canada Am J Public Health. 2020;110(6):833–5. 1. Olding M, Ivsins A, Mayer S, Betsos A, Boyd J, Sutherland C, et al. A low- barrier and comprehensive community-based harm-reduction site in Vancouver. Canada Am J Public Health. 2020;110(6):833–5. 2. Laura Vearrier MDDB. The value of harm reduction for injection drug use: a clinical and public health ethics analysis. Dis Mon. 2019;65(5):119–41. 2. Laura Vearrier MDDB. The value of harm reduction for injection drug use: a clinical and public health ethics analysis. Dis Mon. 2019;65(5):119–41. p y 3. O’Hare P. Merseyside, the first harm reduction conferences, and the early history of harm reduction. Int J Drug Policy. 2007;18(2):141–4. 3. O’Hare P. Merseyside, the first harm reduction conferences, and the early history of harm reduction. Int J Drug Policy. 2007;18(2):141–4. 8. The COVID-19 pandemic has drastically affected our healthcare system. In what ways has it affected the referral of unstably housed patients in the BMC Observation Unit to the Project Trust medical out- reach team? 5. Heller D, McCoy K, Cunningham C. An invisible barrier to integrating HIV primary care with harm reduction services: philosophical clashes between the harm reduction and medical models. Public Health Rep. 2004;119(1):32–9. 6. Hawk KF, Vaca FE, D’Onofrio G. Reducing fatal opioid overdose: prevention, treatment and harm reduction strategies. Yale J Biol Med. 2015;88(3):235–45. 9. How would you like to see the Project Trust medical outreach team evolve and what changes to the refer- ral process for unstably housed patients in the BMC Observation Unit would you like to see? 7. Saloner B, McGinty EE, Beletsky L, Bluthenthal R, Beyrer C, Botticelli M, et al. A public health strategy for the opioid crisis. Public Health Rep. 2018;133(1_suppl):24S-34S. 7. Saloner B, McGinty EE, Beletsky L, Bluthenthal R, Beyrer C, Botticelli M, et al. A public health strategy for the opioid crisis. Public Health Rep. 2018;133(1_suppl):24S-34S. 8. Winhusen T, Wilder C, Lyons MS, Theobald J, Kropp F, Lewis D. Evalua‑ tion of a personally-tailored opioid overdose prevention education and naloxone distribution intervention to promote harm reduction and treat‑ ment readiness in individuals actively using illicit opioids. Drug Alcohol Depend. 2020;216:108265. 9. Fernandes RM, Cary M, Duarte G, Jesus G, Alarcao J, Torre C, et al. Abbreviations ACS: Addiction consult service; BMC: Boston Medical Center; OEND: Opioid overdose education and naloxone distribution; PWUD: People who use drugs; PT: Project TRUST; SUD: Substance use disorder; SSPs: Syringe service programs. 10. Sharma M, Lamba W, Cauderella A, Guimond TH, Bayoumi AM. Harm reduction in hospitals. Harm Reduct J. 2017;14(1):32. https://doi.org/10. 1186/s12954-017-0163-0 Funding g GKK is supported by the Health Resources & Services Administration (Grant Number T32HP1000028). LH is supported by the Boston University Clinical HIV/AIDS Research Training program (Award Number T32AI052074). 15. Barocas JA, Savinkina A, Adams J, Jawa R, Weinstein ZM, Samet JH, et al. Clinical impact, costs, and cost-effectiveness of hospital-based strategies for addressing the US opioid epidemic: a modelling study. Lancet Public Health. 2022;7(1):e56–64. References Effectiveness of needle and syringe Programmes in people who inject drugs—an overview of systematic reviews. BMC Public Health. 2017;17(1):309. Declarations 16. Biancarelli DL, Biello KB, Childs E, Drainoni M, Salhaney P, Edeza A, et al. Strategies used by people who inject drugs to avoid stigma in healthcare settings. Drug Alcohol Depend. 2019;198:80–6. Acknowledgements 11. Hyshka E, Morris H, Anderson-Baron J, Nixon L, Dong K, Salvalaggio G. Patient perspectives on a harm reduction-oriented addiction medicine consultation team implemented in a large acute care hospital. Drug Alcohol Depend. 2019;204:107523. The authors would like to thank all of the participants from the Project TRUST harm reduction in-reach program, hospital observation unit, and inpatient addiction consult service. 12. Wakeman SE, Kane M, Powell E, Howard S, Shaw C, Regan S. Impact of inpatient addiction consultation on hospital readmission. J Gen Intern Med. 2021;36(7):2161–3. Competing interests h h d l h p g The authors declare that they have no competing interests. 17. Simon R, Snow R, Wakeman S. Understanding why patients with sub‑ stance use disorders leave the hospital against medical advice: a qualita‑ tive study. Subst Abus. 2020;41(4):519–25. Appendix A: Semi‑structured interview guide Interview questions: factors may include but are not limited to depart- mental buy-in, referral processes and logistics, avail- able educational resources, staff availability, staff knowledge of the referral program, Project Trust medical outreach team services, and patient desire for referral. Received: 4 March 2022 Accepted: 2 April 2022 Received: 4 March 2022 Accepted: 2 April 2022 7. What factors do you feel act as barriers to the refer- ral of unstably housed patients in the BMC Obser- vation Unit to the Project Trust medical outreach team? Barriers may include but are not limited to departmental buy-in, referral processes and logis- tics, available educational resources, staff availability, staff knowledge of the referral program, Project Trust medical outreach team services, and patient desire for referral. Author contributions GKK and LH analyzed the data and drafted the manuscript. GKK finalized the manuscript. MLD and CP provided extensive guidance, feedback, and editing during the paper development process. GKK conducted the interviews. PL and SJ assisted in recruitment and drafting the manuscript. SK assisted in drafting the manuscript. All authors read and approved the final transcript. 13. Wakeman SE, Metlay JP, Chang Y, Herman GE, Rigotti NA. Inpatient addiction consultation for hospitalized patients increases post-dis‑ charge abstinence and reduces addiction severity. J Gen Intern Med. 2017;32(8):909–16. 14. Trowbridge P, Weinstein ZM, Kerensky T, Roy P, Regan D, Samet JH, et al. Addiction consultation services—linking hospitalized patients to outpa‑ tient addiction treatment. J Subst Abuse Treat. 2017;79:1–5. Appendix A: Semi‑structured interview guide Interview questions: 1. What clinical problems are unstably housed patients presenting to the BMC Observation Unit with? 1. What clinical problems are unstably housed patients presenting to the BMC Observation Unit with? 2. What is your understanding of support services available for unstably housed patients in the BMC Observation Unit? 3. What is your familiarity with the Project Trust medi- cal outreach team referral program for unstably housed patients in the BMC Observation Unit? 4. What do you think the value of the Project Trust medical outreach team referral program for unstably housed patients in the BMC Observation Unit is, and why do you use this referral program? This study has several limitations. First, our sample size was limited. We recruited from a single site hospital with extensive addiction resources and services that may not be generalizable. Likewise, the majority of participants were clinical providers and the non-clinical perspective is likely underrepresented. However, the heterogene- ity of the sample reflects the diverse roles and perspec- tives of individuals who provide care for PWUD in this inpatient setting. Second, participants were selected 5. What is your understanding of how referrals of unstably housed patients in the BMC Observation Unit to the Project Trust medical outreach team pro- gram works? 6. What factors do you feel facilitate referrals of unsta- bly housed patients in the BMC Observation Unit to the Project Trust medical outreach team? Facilitating Khan et al. Harm Reduction Journal (2022) 19:35 Page 10 of 11 Page 10 of 11 Khan et al. Harm Reduction Journal (2022) 19:35 factors may include but are not limited to depart- mental buy-in, referral processes and logistics, avail- able educational resources, staff availability, staff knowledge of the referral program, Project Trust medical outreach team services, and patient desire for referral. factors may include but are not limited to depart- mental buy-in, referral processes and logistics, avail- able educational resources, staff availability, staff knowledge of the referral program, Project Trust medical outreach team services, and patient desire for referral. Boston Medical Center, Boston, MA, USA. 4 Department of Infection Control, Boston Medical Center, Boston, MA, USA. 5 Evans Center for Implementation and Improvement Sciences, Boston University School of Medicine, Boston, MA, USA. 6 Department of Health Law, Policy and Management, Boston University School of Public Health, Boston, MA, USA. Author details 1 1 Section of General Internal Medicine, Department of Medicine, Boston University School of Medicine, 801 Massachusetts Ave, 2nd floor, Boston, MA 02118, USA. 2 Section of Infectious Diseases, Department of Medicine, Boston University School of Medicine, Boston, MA, USA. 3 Project TRUST, y 18. Naeger S, Mutter R, Ali MM, Mark T, Hughey L. Post-discharge treatment engagement among patients with an opioid-use disorder. J Subst Abuse Treat. 2016;69:64–71. Page 11 of 11 Page 11 of 11 Khan et al. Harm Reduction Journal (2022) 19:35 Khan et al. Harm Reduction Journal (2022) 19:35 19. Medicine Io. America Health Care Safety Net: Intact but Endangered. Lewin ME, Altman S, editors. Washington, DC: The National Academies Press; 2000. 302 p. Publisher’s Note 2012;307(11):1143. 33. Emanuel EJ, Fuchs VR. Shortening medical training by 30%. JAMA. 2012;307(11):1143. 34. Brothers TD, Fraser J, Macadam E, Morgan B, Francheville J, Nidumolu A, et al. Implementation and evaluation of a novel, unofficial, trainee-organ‑ ized hospital addiction medicine consultation service. Substance Abuse. 2020:1–8. 35. Basset T, Faulkner A, Repper J, Stamou E. Lived experience leading the way: peer support in mental health. London: Together UK; 2010. 36. Greer AM, Luchenski SA, Amlani AA, Lacroix K, Burmeister C, Buxton JA. Peer engagement in harm reduction strategies and services: a critical case study and evaluation framework from British Columbia, Canada. BMC Public Health. 2016;16(1):452. https://doi.org/10.1186/ s12889-016-3136-4. 37. Weinstein ZM, Wakeman SE, Nolan S. Inpatient addiction consult service. 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Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 38. Prevention CfDCa. Drug Overdose Deaths 2021. Available from: https:// www.cdc.gov/drugoverdose/deaths/index.html. 39. COVID-19 & Substance Use 2021. Available from: https://www.drugabuse. gov/drug-topics/comorbidity/covid-19-substance-use. 40. Aponte-Melendez Y, Mateu-Gelabert P, Fong C, Eckhardt B, Kapadia S, Marks K. The impact of COVID-19 on people who inject drugs in New York City: increased risk and decreased access to services. Harm Reduct J. 2021;18(1):10. https://doi.org/10.1186/s12954-021-00568-3. 41. Des Jarlais DC. Harm reduction in the USA: the research perspective and an archive to David Purchase. Harm Reduct J. 2017;14(1):51. https://doi. org/10.1186/s12954-017-0178-6. 41. Des Jarlais DC. Harm reduction in the USA: the research perspective and an archive to David Purchase. Harm Reduct J. 2017;14(1):51. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 20. BMC Facts 2017. 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https://openalex.org/W2905879714	https://www.sibscript.ru/jour/article/download/2586/2255	Russian	null	THE IMAGES OF AMERICA AND EUROPE IN THE LINGUISTIC CONSCIOUSNESS OF NATIVE ARABIC SPEAKERS*	Vestnik Kemerovskogo gosudarstvennogo universiteta	2,018	cc-by	5,522	Вестник Кемеровского государственного университета • 2018 • № 4 Вестник Кемеровского государственного университета • 2018 • № 4 Поступила в редакцию 28.07.2018. Принята к печати 31.10.2018. Поступила в редакцию 28.07.2018. Принята к печати 31.10.2018. Аннотация: В статье рассматриваются способы репрезентации обра- зов Америка и Европа в языковом сознании носителей арабской культуры. Актуальность выбранной темы обусловлена современными геополитически- ми факторами и экономическими предпосылками современного общества. Объектом исследования является языковое сознание носителей арабской культуры. Предмет изучения – образы стран, сложившиеся в нем. Результаты исследования основываются на материале, полученном в ходе проведенного свободного ассоциативного эксперимента среди носителей арабской культуры. Анализ построенных ассоциативных полей позволил разделить полученные вербальные реакции на ассоциации смежности и сходства, выделить наиболее частотные ответы на предъявляемые стимулы, оценить ценностно-эмоцио- нальную составляющую исследуемых образов и увидеть сходства и различия в восприятии исследуемых стран арабскими студентами, показать иерархию семантических связей в оценках стимулов. Предложенные выводы позволя- ют судить о том, что языковое сознание является динамическим явлением, на формирование которого большое влияние оказывают современные полити- ческие, экономические и социокультурные изменения и преобразования. Ключевые слова: психолингвистика, язы- ковое сознание, образ, ассоциативное поле, реакции Для цитирования: Баландина Е. С. Образы стран в языковом сознании носителей арабской культуры (на примере образов Америка и Европа) // Вестник Кемеровского государственного университета. 2018. № 4. С. 190–197. DOI: https://doi.org/10.21603/2078-8975-2018-4-190-197 УДК 821.111(73) ОБРАЗЫ СТРАН В ЯЗЫКОВОМ СОЗНАНИИ НОСИТЕЛЕЙ АРАБСКОЙ КУЛЬТУРЫ (НА ПРИМЕРЕ ОБРАЗОВ АМЕРИКА И ЕВРОПА)* Екатерина С. Баландина a, @, ID a Южно-Уральский государственный университет (национальный исследовательский университет), 454080, Россия, г. Челябинск, пр-т Ленина, 76 @ nenash_ek@mail.ru ID https://orcid.org/0000-0002-1360-7413 * Статья выполнена при поддержке Правительства РФ (Постановление № 211 от 16.03.2013), соглашение № 02.A03.21.00 Введение социальные мифы, формируемые в так называемом рефлексивном слое сознания. Образы рефлексивного слоя сознания, в том числе и социальные мифы, кон- струируются в относительном отрыве от реальной действительности, с которой они связаны через дру- гие – чувственные – образы, прямо, непосредственно отображающие реальное бытие людей [2]. Однако, с нашей точки зрения, глобализация мирового про- странства, развитие экономических связей привели к тому, что представления, мировоззрения людей и общественное сознание в значительной мере стали формироваться средствами массовой информации. Практически все сферы человеческой жизни нахо- дятся под их влиянием. Правильный выбор адресатов и механизмов во многом определяет успех имиджевых технологий [3]. Следовательно, на восприятие образов стран в настоящее время политических игр и эконо- мических разногласий также оказывают существен- ное воздействие современные средства массовой ин- формации, которые воздействуют на формирование Межкультурная коммуникация является важной частью информационного поля общества. От её успешного использования зависит эффективность реализации разного рода социальных, коммерче- ских и политических проектов. При этом становит- ся необходимым изучение исторических, социоло- гических, культурологических, лингвистических аспектов межкультурной коммуникации с целью углубления знаний о народе, который является парт нером в общении [1]. В отечественной науке психо- лингвистический подход к межкультурному обще- нию ведётся на основе изучения взаимоотношения языка, культуры и сознания. Образы стран – это фрагменты языкового созна- ния, существующие как социальные автостереотипы (у представителей анализируемой страны) и как на- циональные предрассудки (у иностранцев). Подоб- ные ментальные образования трактуются в совре- менной философии и социальной психологии как 190 Вестник Кемеровского государственного университета • 2018 • № 4 конфликты, грамотно построить государственную политику в сфере межкультурных отношений [8]. Среди научных работ, посвященных сформу- лированной проблеме, можно выделить различ- ные направления исследования: изучение образов стран представителями российской культуры, ана- лиз образов стран в политическом дискурсе, меж- культурной коммуникации, метафорическое моде- лирование образов-стран и т. д. Научная новизна представленных результатов обусловлена тем, что впервые была предпринята попытка целенаправ- ленного исследования образов стран, сложившихся в языковом сознании носителей арабской культу- ры определенной возрастной группы (19–23 года), выстроена схема анализа полученного материала, позволяющая описать всестороннее содержание анализируемых образов. образа той или иной страны за счет различных мани- пуляционных стратегий. конфликты, грамотно построить государственную политику в сфере межкультурных отношений [8]. конфликты, грамотно построить государственную политику в сфере межкультурных отношений [8]. у ф р у ур [ ] Среди научных работ, посвященных сформу- лированной проблеме, можно выделить различ- ные направления исследования: изучение образов стран представителями российской культуры, ана- лиз образов стран в политическом дискурсе, меж- культурной коммуникации, метафорическое моде- лирование образов-стран и т. д. Введение Научная новизна представленных результатов обусловлена тем, что впервые была предпринята попытка целенаправ- ленного исследования образов стран, сложившихся в языковом сознании носителей арабской культу- ры определенной возрастной группы (19–23 года), выстроена схема анализа полученного материала, позволяющая описать всестороннее содержание анализируемых образов. Помимо этого, каждый этнос характеризуется своими особыми системами восприятия мира. Они формируются в процессе практической деятельно- сти людей на основе их собственного опыта и тра- диций, унаследованных от предыдущих поколений [4]. Этнокультурные стереотипы и этнические преду- беждения, формируемые при этом, играют значимую роль в моделировании определенного образа той или иной страны [5]. Под этнокультурным стереотипом понимается обобщенное представление о типичных чертах, характеризующих какой-либо народ. Под эт- ническим предубеждением понимается предвзятое мнение о национальных свойствах какого-либо эт- носа, возведенное во враждебную психологическую установку по отношению к данному этносу [6]. Стереотипное представление об определенной стране складывается из образа самой страны и образа населяющего ее народа. Образ страны складывается из представлений о внешней и внутренней полити- ке государства, экономике и культуре. Образ нации складывается из представления о национальном характере и национальных пристрастиях, внешнем виде представителя того или иного народа, особен- ностей образа жизни [7]. Выявление таких особенно- стях восприятия представляет огромный интерес. Методология исследования Изучение обра- зов сознания позволит оптимизировать межкуль- турное общение, предотвратить межэтнические 191 Вестник Кемеровского государственного университета • 2018 • № 4 Вестник Кемеровского государственного университета • 2018 • № 4 В качестве испытуемых для эксперимента было привлечено 100 арабских студентов в возрасте 19– 23 лет, обучающихся на различных факультетах Баг- дадского университета. Полученный синхронный срез языка, представляющий собой построенные ассоциативные поля, дает возможность описывать образы сознания определенной возрастной группы носителей выбранной культуры, поскольку он ов- нешняет знания, ассоциированные со словом-сти- мулом в данной конкретной национальной культу- ре. Достоверность результатов, полученных в ходе эксперимента, подтверждается положением о том, что в практике психолингвистики для проведения анализа образа и включения данных в словарь до- статочным является ассоциативное поле, состоя- щее из 100 ассоциатов [13]. В нашем эксперименте на предъявляемые стимульные слова от 100 респон- дентов мы получили 100 реакций, тем самым ни один студент не пропустил интересующий нас стимул. Важно отметить, что анкеты, предлагаемые студентам в ходе эксперимента, были составлены на их родном языке, однако в данной статье для удобства восприятия анализируемые реакции были переведены на русский язык при сохранении фор- мально грамматических характеристик ассоциаций. с элементами различного рода лексических объеди- нений парадигматического характера. В арабском ассоциативном поле могут быть выделены следую- щие ассоциаты, относящиеся к данной группе и со- ставляющие 8 % от общего количества полученных ответов: – دلبстрана (3), – ةينيتاللا اكيرمأЛатин- ская Америка, – نابايلاЯпония, – ايقيرفأАфрика, – ناكمместо, – ةيرحلا لاثمتстатуя свободы. Детерминационные – к ним относятся ассоциа- тивные пары, в которых содержание ответа-реак- ции входит в содержание слова-стимула в качестве одного из признаков этого содержания. В построен- ном нами ассоциативном поле арабских студентов можно выделить следующий ряд ассоциатов, от- носящихся к данной подгруппе (25 %): словофор- мы и словосочетания, выявляющие качественные характеристики образа: – يوقсильная (6), – ميظع великая (5), – ةئيسплохая (4), – يكذумная (3), – مخضогромная, – ديعبдалекая, – ميظع دلبве- ликая страна, – ريقفбедная; несогласованные со словом-стимулом глаголы: – رفسلاпутешество- вать (2), – ةبقارمконтролировать. Следует также отметить, что на данный стимул было получено 2 отказа на ассоциирование. На стимульное слово Европа мы получили спи- сок реакций, которые представлены следующим списком. Методология исследования К ассоциациям по предметно-смысло- вой смежности относятся реакции, составляющие 66 % от общего числа ответов: – يبنجأиностранец (14), – رفسпутешествие (11), – ريوطتразвитие (7), – ةحايسтуризм (5), – ةفاقثкультура (4), – ملح мечта (4), – ملعнаука (3), – مارتحاуважение (2), – تامظنملاорганизации, – ةريشأتвиза, – يملحмоя мечта, – ةلادعсправедливость, – يملح سيلне моя мечта, – عونتразнообразие, – مدقتпрогресс, – ةرجه иммиграция, – ةينغأпесня, – ةصقистория, – رحبلا море, – ةيملاعلاмир, – داصتقاэкономика, – ةطيرخ карта, – ةطيرخкрасивая жизнь. Методология исследования Теоретической основой исследования послу- жило обоснованное в психолингвистике представ- ление о том, что явления действительности, вос- принимаемые человеком в процессе деятельности и общения, отображаются в его языковом созна- нии. Это отображение не только фиксирует при- чинные и пространственные связи явлений и эмо- ций, вызываемые в ходе восприятия, но и обладает определенной этнокультурной обусловленностью, динамичностью и вариативностью [9]. Цель данной статьи состоит в выявлении, фик- сации и анализе фрагмента языкового сознания арабских студентов, содержащего осознаваемые и неосознаваемые знания об Америке и Европе. Одним из средств доступа к сознанию, а также базой для исследования национально-культурной специфики образов языкового сознания является ассоциативный эксперимент. Ассоциативный экс- перимент – ценный источник информации, способ- ствующий вскрытию объективно существующих в психике носителей языка семантических связей слов, он является одним из способов доступа к со- знанию с целью изучения содержания того или ино- го фрагмента языкового сознания [10]. Методоло- гическим основанием применения ассоциативного эксперимента стало положение о том, что ассоциа- ции на каждое конкретное слово-стимул формиру- ют естественно-языковое поле, имеющее не только структурно-лексикографический, но и онтологиче- ский статус [11]. По мнению В. Ф. Петренко, кате- гории сознания задают определенную рамку вос- приятия мира, в контексте которой строятся более частные, так называемые имплицитные теории, или имплицитные модели, т. е. слабо упорядочен- ные и плохо рефлексируемые формы организации знаний субъекта в различных содержательных областях его обыденного сознания и житейского опыта [12]. Проанализированный и классифициро- ванный определенным образом материал позволя- ет вскрыть внутреннее содержание и смыслы рас- сматриваемых образов. Актуальность представленной темы во многом объясняется современной геополитической обста- новкой, когда на первый план выходят вопросы, связанные с построением дружественных отно- шений между государствами и нормализацией по- литической обстановки в целом. Состояние мира в условиях глобализации обусловлено не только позитивными результатами интеграционных про- цессов, но и обострением противоречий и конфлик- тов. Это приводит к тому, что в настоящий момент становится особенно важным выстроить межкуль- турное общение между представителями различ- ных стран, основанное на доверии и взаимоува- жении. В подобном эффективном взаимодействии значимую роль играют образы стран, сложившие- ся в языковом сознании, на формирование которых влияют экономическая ситуация, социокультурная среда, образование, религия и политическая об- становка. Таким образом, значимость подобных исследований обусловлена необходимостью фун- даментальных разработок лингвистической науки, связанных с проблемами восприятия и отражения действительности и способами ее фиксации и ре- презентации в языковом сознании. Результаты исследования Следовательно, несмотря на некоторую напряжен- ность в отношениях, существующую между Аме- рикой и арабскими странами, опрошенные студен- ты воспринимают анализируемый образ в большей степени с положительной точки зрения. Данную мысль подтверждает и анализ полного ассоциатив- ного поля, где распределение ценностно-эмоцио- нальных положительных и отрицательных ассоци- атов соотносится как 74 % к 26 %. Образ Европа вызвал следующие наиболее час тотные реакции: – يبنجأиностранец (14), – رفس путешествие (11), – ريوطتразвитие (7), – ارتلجنا Англия (6), – اسنرفФранция (5), – ةحايسтуризм (5). Европа в языковом сознании арабских студентов ассоциируется, в первую очередь, с путешествиями, поскольку общая частотность родственных реак- ций – رفسпутешествие (11) и – ةحايسтуризм (5) опережает частотность ассоциата – يبنجأиностра- нец (14). Европа представляется развитой – ضرأ землей (4), – داحتالاСоюзом (2) и связана, прежде всего, с такими странами, как – ارتلجناАнглия (6) и – اسنرفФранция (5). Ценностно-эмоциональ- ный спектр всего ассоциативного поля составляют исключительно положительно окрашенные слова- ответы, отражающие многоаспектное восприятие исследуемого образа. Для арабских студентов наиболее частотные ре- акции на стимульное слово Америка представле- ны следующим списком ответов: – ةيرحلاсвобода (11), – ةوقلاсила (9), – ملحмечта (8), – برحلاвойна (7), – يوقсильная (6), – ميظعвеликая (5), – شيجلا армия (5). Выделенные реакции демонстрируют две противоположные точки зрения респондентов на исследуемый стимул. С одной стороны, Амери- ка представляется сильной, великой страной-меч- той, где свобода как наивысшая ценность стала своеобразным символом, олицетворяющим данное государство. С другой стороны, внешнеполити- ческие конфликты с арабским миром несомненно оказали влияние на восприятие образа Америки, что объясняет значительную частотность реакций – برحلاвойна (7) и – شيجلاармия (5). Таким об- разом, ценностно-эмоциональный спектр ядра ис- следуемого стимульного слова представлен 75 % положительными и 24 % отрицательных реакций. Далее нам представляется интересным класси- фицировать полученные ответы по семантическим полям. Тем самым мы получим возможность более глубокого изучения содержания исследуемых об- разов (таблицы 1 и 2). Таблица 1. Семантические поля образа Америка Table 1. Semantic fields of the image of America Таблица 1. Семантические поля образа Америка Table 1. Результаты исследования Рассмотрим более подробно характеристику ответов-реакций на глубинном уровне отношений, су- ществующих между содержаниями слов-ассоциатов. Стимульное слово Америка вызвало определен- ные ассоциации у опрошенных нами студентов, которые мы можем классифицировать следующим образом. К ассоциациям по предметно-смысло- вой смежности относятся ассоциативные пары слов, не имеющие общих существенных признаков в своем содержании, в основных значениях членов которых отражаются реальные отношения смежно- сти между предметами и явлениями действитель- ности или содержаниями абстрактных понятий [14]. Среди ответов, данных арабскими студента- ми, мы выделили 64 % реакций, которые отнесли к данной группе: – ةيرحلاсвобода (11), – ةوقلاсила (9), – ملحмечта (8), – برحلاвойна (7), – شيجلاар- мия (5), – مالفأкино (4), – ريمدتразрушения (2), – ةيطارقميدдемократия, – ةينغأпесня, – لكاشمпро- блемы, – لبقتسملا تاينقتтехнологии будущего, – بورحلا عيبرарабская весна, – يملحмоя мечта, – دجمслава, – ةنجلاрай, – مدقتпрогресс, – يبحмоя любовь, – ةبآكдепрессия, – ةيلامسأرкапитализм, – ةبالصтвердость, – داصتقاэкономика, – رقف бедность, – لمعработа, – ةيملاعلاмир, – انلнаша. Классификационные ассоциации составили 27 %: – ارتلجناАнглия (6), – اسنرفФранция (5), – ضرأ земля (4), – داحتالاСоюз (2), – ايسآАзия, – ايسور Россия, – نطولاРодина, – يناثلا تيبلاвторой дом, – دلبلاстрана, – ةراقконтинент, – ناكمместо, ضرأ – مالحألاместо мечты, – ةراضحцивилизация. Детерминационные – 7 % ассоциативно- го поля: – ربكأбольшая (4), – ميظعвеликая (2), – ليمجкрасивая. Таким образом, наиболее частотными ассоциа- циями на выбранные стимульные слова являются реакции предметно-смысловой смежности, харак- теризующие широкий спектр предметов и явлений, связанных в языковом сознании носителей араб- ской культуры с исследуемыми образами. Клас- сификационные реакции в полученных ассоциа- тивных полях составляют примерно 25 % от всех ответов, тогда как наименьший процент имеют группы детерминационных ассоциаций. В свою очередь, ассоциации по сходству можно разделить на две подгруппы. Классификационные – ассоциативные пары, члены которых имеют в своих содержаниях хотя бы один общий существенный признак. К ним относятся реакции, соотносимые 192 Вестник Кемеровского государственного университета • 2018 • № 4 На следующем этапе анализа было рассмотрено ядро исследуемых ассоциативных полей. Относи- тельно объема ядра и принципов его выделения ученые придерживаются различных точек зрения, поскольку эти довольно неоднозначные определе- ния не дают возможности выделить основные ха- рактеристики ядерных ассоциаций [15]. В статье мы будем придерживаться мнения, что ядро ассо- циативного поля составляет около 47 % от общего числа ответов, т. е. одиночные реакции и реакции, имеющие небольшой индекс частотности, относят- ся к периферии ассоциативного поля [16]. Результаты исследования Semantic fields of the image of America Семантическое поле % Реакции Характеристики: 19 % – положительные 10 % – ميظعвеликая (5), – يكذумная (3), – مخضогромная, – ميظع دلب великая страна – отрицательные 8 % – ةئيسплохая (4), – ريقفбедная, отказ ассоциирования (3) – нейтральные 1 % – ديعبдалекая (Военный) конфликт 16 % – برحلاвойна (7), – شيجلاармия (5), – ريمدتразрушения (2), – لكاشمпроблемы, – بورحلا عيبرарабская весна Мощь и величие страны 16 % – ةوقلاсила (9), – يوقсильная (6), – ةبالصтвердость Личные ассоциации 11 % – ملحмечта (8), – يملحмоя мечта, – يبحмоя любовь, – انلнаша Ценности 11 % – ةيرحلاсвобода (11) Место 9 % – دلبстрана (3), – ةينيتاللا اكيرمأЛатинская Америка, – نابايلا Япония, – ايقيرفأАфрика, – ناكمместо, – ةنجلاрай, – ةيملاعلاмир Социальная сфера 6 % – مالفأкино (4), – ةينغأпесня, – دجمслава Экономическая сфера 4 % – ةبآكдепрессия, – داصتقاэкономика, – رقفбедность, – لمعработа Действия 3 % رفسلاпутешествовать (2), ةبقارمконтролировать Политическая сфера 2 % – ةيطارقميدдемократия, – ةيلامسأرкапитализм Научная сфера 2 % – لبقتسملا تاينقتтехнологии будущего, – مدقتпрогресс Таблица 1. Семантические поля образа Америка Table 1. Semantic fields of the image of America Семантическое поле % Реакции Характеристики: 19 % – положительные 10 % – ميظعвеликая (5), – يكذумная (3), – مخضогромная, – ميظع دلب великая страна – отрицательные 8 % – ةئيسплохая (4), – ريقفбедная, отказ ассоциирования (3) – нейтральные 1 % – ديعبдалекая (Военный) конфликт 16 % – برحلاвойна (7), – شيجلاармия (5), – ريمدتразрушения (2), – لكاشمпроблемы, – بورحلا عيبرарабская весна Мощь и величие страны 16 % – ةوقلاсила (9), – يوقсильная (6), – ةبالصтвердость Личные ассоциации 11 % – ملحмечта (8), – يملحмоя мечта, – يبحмоя любовь, – انلнаша Ценности 11 % – ةيرحلاсвобода (11) Место 9 % – دلبстрана (3), – ةينيتاللا اكيرمأЛатинская Америка, – نابايلا Япония, – ايقيرفأАфрика, – ناكمместо, – ةنجلاрай, – ةيملاعلاмир Социальная сфера 6 % – مالفأкино (4), – ةينغأпесня, – دجمслава Экономическая сфера 4 % – ةبآكдепрессия, – داصتقاэкономика, – رقفбедность, – لمعработа Действия 3 % رفسلاпутешествовать (2), ةبقارمконтролировать Политическая сфера 2 % – ةيطارقميدдемократия, – ةيلامسأرкапитализм Научная сфера 2 % – لبقتسملا تاينقتтехнологии будущего, – مدقتпрогресс 193 Вестник Кемеровского государственного университета • 2018 • № 4 Таблица 2. Семантические поля образа Европа Table 2. Результаты исследования Semantic fields of the image of Europe Семантическое поле % Реакции Путешествия 32 % – يبنجأиностранец (14), – رفسпутешествие (11), – ةحايسтуризм (5), – ةريشأتвиза, – ةطيرخкарта Место 23 % – ارتلجناАнглия (6), – اسنرفФранция (5), – ضرأземля (4), – داحتالا Союз (2), – ايسآАзия, – ايسورРоссия, – دلبلاстрана, – ةراقконтинент, – ناكمместо, – ةيملاعلاмир Научная сфера 11 % – ريوطتразвитие (7), – ملعнаука (3), – مدقتпрогресс Личные ассоциации 7 % – ملحмечта (4), – يملحмоя мечта, – يملح سيلне моя мечта, – مالحألا ضرأ место мечты Положительные характеристики 7 % – ربكأбольшая (4), – ميظعвеликая (2), – ليمجкрасивая Традиции и история 6 %– ةفاقثкультура (4), – ةصقистория, – ةراضحцивилизация Социальная сфера 3 % – عونتразнообразие, – ةينغأпесня, – ةطيرخкрасивая жизнь Ценности 3 % – مارتحاуважение (2), – ةلادعсправедливость Экономическая сфера 3 % – تامظنملاорганизации, – ةرجهиммиграция, – داصتقاэкономика Дом 2 % – نطولاРодина, – يناثلا تيبلاвторой дом Таблица 2. Семантические поля образа Европа Table 2. Semantic fields of the image of Europe Полученные данные позволяют сделать вывод, что в содержательном плане доминируют два се- мантических поля, демонстрирующие глубинное восприятие образа: семантическое поле мощи и силы как признания могущества Америки и ее обширного влияния и семантическое поле (воен- ного) конфликта как отражение современного по- литического и военного противостояния между го- сударствами. Семантическое поле характеристик составляет 19 % и представлено относительно ровным распределением положительно и отрица- тельно окрашенных ассоциатов, свидетельствую- щих о двух противоположных точках зрения на ис- следуемый образ. Далее по наполнению следуют семантическое поле ценностей, представленное единичным, но наиболее частотным ответом, груп- па личностно-ориентированных реакций и семан- тическое поле места, отражающее географические ассоциативные связи образа. Оставшиеся выделен- ные семантические поля фиксируют наиболее важ- ные, яркие аспекты и сферы влияния, которыми характеризуется анализируемый образ в языковом сознании арабских студентов: политика, экономи- ка, наука, социальная жизнь (кино, шоу-бизнес). культуры и традиций исследуемого образа. Тре- тьим по значимости и процентному соотношению выступает семантическое поле ответов, связанных с развитием и прогрессом, что указывает на при- знание научной базы Европейского Союза. Инте- ресным, с нашей точки зрения, является не только выделение в отдельную группу личных ассоциаций – ملحмечта (4), – يملحмоя мечта и т. д., но и появ- ление двух реакций в семантическом поле Дом, от- ражающих восприятие исследуемого образа в ка- честве второй Родины. Заключение Представленные семантические поля позво- ляют увидеть сходства и различия между иссле- дуемыми образами. В образах Америка и Европа опрошенные респонденты в одинаковой степени акцентировали внимание на группе ассоциаций, связанных с личными мечтами и желаниями жить, учиться, работать или просто посмотреть эти места. Однако целостное восприятие значительно отличает- ся. Америка представляется, с одной стороны, стра- ной-мечтой, с провозглашенной ценностью свобода, а с другой – страной, породившей военный конфликт. Европа, наоборот, в языковом сознании арабских сту- дентов связана с отдыхом и научным прогрессом, по- этому не вызывает отрицательных реакций. Приведенные данные показывают, что основны- ми смысловыми ассоциациями на анализируемый образ являются ответы, входящие в семантические поля путешествия и места. Тем самым мы можем судить о том, что образ Европа вызывает у моло- дых людей ассоциации, связанные с отдыхом, что приводит к отсутствию в семантическом поле ха- рактеристик отрицательных ассоциатов и выделе- нию группы реакций, связанных с определением Следовательно, на основе анализа образов Аме- рика и Европа мы можем сделать вывод, что язы- ковое сознание является динамическим и подвиж- ным явлением, изменяющимся под воздействием внешних факторов – экономических, политических и социально-культурных условий. Содержание 194 Вестник Кемеровского государственного университета • 2018 • № 4 выми, порой кратковременными ассоциативными связями, отражающими современное состояние об- щественного мнения и языкового сознания в целом. существующих в нем образов репрезентирует не только устойчивые отношения в долговремен- ной перспективе, но и оперативно дополняется но- Литература р ур 1. Кечина Э. А., Гришенина Ю. А. 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The relevance of the chosen topic is due to the current geopolitical factors and the economic prerequisites governing the modern society. The object of the present study is the linguistic consciousness of the native Arab speakers. The subject is the country images that are formed in the mind of Arab culture members. The results of the research are based on the material obtained in the course of the free associative experiment. An analysis of the constructed associative fields made it possible to divide the verbal reactions into contiguity and similarity associations; to select the most frequent responses to the presented stimuli; to evaluate the value- emotional component of the images and to see the similarities and differences in the perception of the countries by Arab students; to show the hierarchy of semantic links and fields in estimates of incentives. The results of the study show that linguistic consciousness is a dynamic phenomenon, the formation of which is influenced by the modern political, economic, and sociocultural changes and transformations. Keywords: psycholinguistics, linguistic consciousness, image, associative field, associations For citation: Balandina E. S. Obrazy stran v iazykovom soznanii nositelei arabskoi kulʹtury (na primere obra zov Amerika i Evropa) [The Images of America and Europe in the Linguistic Consciousness of Native Arabic Speakers]. Bulletin of Kemerovo State University, no. 4 (2018): 190–197. DOI: https://doi.org/10.21603/2078- 8975-2018-4-190-197 Литература И., Сахарный Л. В., Штерн А. С. Психолингвистика // Прикладное языкознание / отв. ред. А. С. Герд. СПб.: СПбГУ, 1996. С. 245–267. 195 Bulletin of Kemerovo State University • 2018 • № 4 THE IMAGES OF AMERICA AND EUROPE IN THE LINGUISTIC CONSCIOUSNESS OF NATIVE ARABIC SPEAKERS* Ekaterina S. Balandina a, @, ID a South Ural State University (national research university),76, Lenin Ave., Chelyabinsk, Russia, 454080 @ nenash_ek@mail.ru ID https://orcid.org/0000-0002-1360-7413 * The article was supported by the Government of the Russian Federation (Decree No. 211 of 03.16.2013), Agreement No. 02.A0 References Semantika = RUDN Journal of Language Studies, Semiotics and Semantics, no. 4 (2017): 1161–1170. g g , ( ) 9. Ufimtseva N. V. Dinamika i variativnost' iazykovogo soznaniia [Dynamics and variability of linguistic consciousness]. Moscow: Institut iazykoznaniia RAN, 2011, 252. g g ( ) 9. Ufimtseva N. V. Dinamika i variativnost' iazykovogo soznaniia [Dynamics and variability of linguistic consciousness]. Moscow: Institut iazykoznaniia RAN, 2011, 252. y 10. Polyakova N. V. Fitonimicheskii obraz Rossii (po dannym assotsiativnogo eksperimenta) [Fitonimic image of Russia (according to the associative experiment)]. 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Opyt kompleksnogo issledovaniia dannykh assotsiativnogo eksperimenta [Experience of complex research of associative experiment data]. Voprosy psikhologii = Questions of psychology, no. 2 (1993): 93–99. i 14. Martinovich G. A. Opyt kompleksnogo issledovaniia dannykh assotsiativnogo eksperimenta [Experience of complex research of associative experiment data]. Voprosy psikhologii = Questions of psychology, no. 2 (1993): 93–99. 15. Sergieva N. S. g ] g 16. Zubkova T. I., Saharnyi L. V., Shtern A. S. Psikholingvistika [Psycholinguistics]. Prikladnoe iazy [Applied linguistics]. Saint-Petersburg: SPbGU, 1996, 245–267. References 1. Kechina E. A., Grischenina Y. A. Obraz Rossii kak avto- i geterostereotip v iazykovom soznanii russkikh i arabskikh studentov [The image of Russia as an autostereotype and a heterostereotype in the linguistic consciousness of Russian and Arab students]. Mezhdunarodnyi nauchno-issledovatelʹskii zhurnal = International Research Journal, no. 8-5 (2016): 112–114. 1. Kechina E. A., Grischenina Y. A. Obraz Rossii kak avto- i geterostereotip v iazykovom soznanii russkikh i arabskikh studentov [The image of Russia as an autostereotype and a heterostereotype in the linguistic consciousness of Russian and Arab students]. Mezhdunarodnyi nauchno-issledovatelʹskii zhurnal = International Research Journal, no. 8-5 (2016): 112–114. , ( ) 2. Tarasov E. F. Obraz Rossii: metodologiia issledovaniia [The image of Russia: research methodology]. Voprosy psikholingvistiki = Questions of psycholinguistics, no. 4 (2006): 69–73. 3. Savel'eva N. Kh., Pikhtovnikova Yu. V. Formirovanie obraza rossiiskogo gosudarstva v britanskikh SMI [Formation of the image of the Russian state in the British mass media]. Filologicheskie nauki. Voprosy teorii i praktiki = Philological Sciences. Theory and practice, no. 3-1 (2018): 56–58.i 4. Ufimtseva N. V. 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Voprosy teorii i praktiki zhurnalistiki = Questions of journalism theory and practice, no. 1 (2018): 87–99.i 8. Balyasnikova O. V., Cherkasova G. A., Stepanova A. A., Ufimtseva N. V. Etnolingvisticheskii aspekt regional'nogo iazykovogo soznaniia [Ethnopsycholinguistic aspect of regional linguistic consciousness]. Vestnik * The article was supported by the Government of the Russian Federation (Decree No. 211 of 03.16.2013), Agreement No. 02.A03.21.0011. 196 Bulletin of Kemerovo State University • 2018 • № 4 Rossiiskogo universiteta druzhby narodov. Seriia: Teoriia Iazyka. Semiotika. Semantika = RUDN Journal of Language Studies, Semiotics and Semantics, no. 4 (2017): 1161–1170.i Rossiiskogo universiteta druzhby narodov. Seriia: Teoriia Iazyka. Semiotika. 15. Sergieva N. S. Prostranstvo i vremia zhiznennogo puti v russkom iazykovom soznanii [Space and in the Russian linguistic consciousness]. Saint-Petersburg: Nauka, 2009, 316. References Prostranstvo i vremia zhiznennogo puti v russkom iazykovom soznanii [Space and lifetime 14. Martinovich G. A. Opyt kompleksnogo issledovaniia dannykh assotsiativnogo eksperimenta [Experience of complex research of associative experiment data]. Voprosy psikhologii = Questions of psychology, no. 2 (1993): 93–99. 15. Sergieva N. S. Prostranstvo i vremia zhiznennogo puti v russkom iazykovom soznanii [Space and lifetime in the Russian linguistic consciousness]. Saint-Petersburg: Nauka, 2009, 316. 14. Martinovich G. A. Opyt kompleksnogo issledovaniia dannykh assotsiativnogo eksperimenta [Experience of complex research of associative experiment data]. Voprosy psikhologii = Questions of psychology, no. 2 (1993): 93–99. 15. Sergieva N. S. Prostranstvo i vremia zhiznennogo puti v russkom iazykovom soznanii [Space and lifetime in the Russian linguistic consciousness]. Saint-Petersburg: Nauka, 2009, 316. p p p y p g f p y gy ( ) 15. Sergieva N. S. 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https://openalex.org/W2947190934	https://europepmc.org/articles/pmc6489344?pdf=render	English	null	Prevalence of and risk factors for hepatitis C virus antibody among people who inject drugs in Cambodia: a national biological and behavioral survey	Harm reduction journal	2,019	cc-by	9,722	RESEARCH Open Access Yi et al. Harm Reduction Journal (2019) 16:29 https://doi.org/10.1186/s12954-019-0299-1 Yi et al. Harm Reduction Journal (2019) 16:29 https://doi.org/10.1186/s12954-019-0299-1 Prevalence of and risk factors for hepatitis C virus antibody among people who inject drugs in Cambodia: a national biological and behavioral survey yan Yi1,2,3* , Phalkun Mun4, Pheak Chhoun2, Navy Chann4, Sovannary Tuot2 and Gitau Mburu5 Abstract Background: Hepatitis C virus (HCV) is a significant global health concern. Despite evidence of the relationship between injecting drug use and HCV, studies on HCV among people who inject drugs in developing countries remain scarce. To address this need, we conducted this study to explore the prevalence of and factors associated with HCV antibody positivity among people who inject drugs in Cambodia. Methods: Data used for this study were collected as part of the National Integrated Biological and Behavioral Survey among people who use and inject drugs conducted in 2017. We used the respondent-driven sampling method to recruit participants in 12 provinces for face-to-face interviews and HIV and HCV antibody testing. Weighted multivariable logistic regression analysis was conducted to identify risk factors associated with HCV antibody positivity. Results: This study included 286 people who inject drugs with a mean age of 31.6 (SD = 7.5) years. The prevalence of HCV antibody among participants in this study was 30.4%, of whom 31.0% were co-infected with HIV. After adjustment for other covariates, the odds of HCV antibody positivity was significantly higher among participants who were in the older age group of 25 to 34 (AOR = 1.85, 95% CI = 1.06–7.92) and ≥35 (AOR = 2.67, 95% CI = 1.24–5.71), were in Vietnamese ethnic group (AOR = 5.44, 95% CI = 2.25–13.14), were living on the streets (AOR = 3.01, 95% CI = 1.29–704), had been sent to a drug rehabilitation center in the past 12 months (AOR = 2.67, 95% CI = 1.21–5.90), had received methadone maintenance therapy in the past 12 months (AOR = 3.02, 95% CI = 1.32–6.92), and were tested positive for HIV (AOR = 3.80, 95% CI = 1.58–9.12) compared to their respective reference group. Conclusion: The prevalence of HCV antibody among people who inject drugs in Cambodia is high, particularly in older and more vulnerable subgroups. Tailor-made interventions are required to increase access to culturally sensitive harm reduction interventions to prevent primary HCV infection and reinfection. In addition, there is an opportunity to expand screening, diagnosis, and treatment with new directly acting antiviral agents. Abstract Keywords: Co-infection, Harm reduction, Injecting drug use, Key population, Resource-limited setting, Viral hepatitis * Correspondence: siyan@doctor.com 1Saw Swee Hock School of Public Health, National University of Singapore and National University Health System, Tahir Foundation Building, 12 Science Drive 2, #10-01, Singapore 117549, Singapore 2KHANA Center for Population Health Research, Phnom Penh, Cambodia Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yi et al. Harm Reduction Journal (2019) 16:29 Yi et al. Harm Reduction Journal (2019) 16:29 Page 2 of 11 Page 2 of 11 Background PWID in resource-limited settings, we conducted a study aiming to determine the prevalence and correlates of HCV antibody positivity in this population in Cambodia so as to inform services, policy, and future re- search programs. g Hepatitis C is a significant global health problem [1]. The global prevalence of hepatitis C virus (HCV) is esti- mated at 3%, translating to at least 185 million people living with the infection worldwide [2]. Persistent HCV infection is a leading cause of hepatic cirrhosis, hepato- cellular cancer, and liver failure [3]. In 2015, an esti- mated 495,000 deaths occurred worldwide attributable to HCV [4]. Besides high mortality rates, HCV infection is associated with significant healthcare costs [5]. Methods Study design, sites, and participants Data used for this study were obtained from the Na- tional Biological and Behavioral Survey (IBBS) con- ducted among people who use and inject drugs from June to December 2017 in the capital city of Phnom Penh and other 11 major provinces. The selection of the study sites was based on a feasibility assessment con- ducted by the study team prior to the main study. These 12 study sites contained 21 operational districts with high-burden of HIV and large population size of people who use and inject drugs. PWID were defined as people who have injected any types of illicit drugs, defined by the Cambodian Law on Control of Drugs, in the past 12 months [17]. To be included in the study, an individ- ual must (1) be at least 18 years old, (2) have a valid study coupon, (3) have not participated in this survey, (4) meet the definition of PWID, and (5) be able and willing to provide a written informed consent to partici- pate in the study. g While the global incidence patterns of HCV have shifted significantly in recent years [2, 6], it has remained a common health problem among people who inject drugs (PWID) [6, 7]. In Cambodia, PWID are a priority population for human immunodeficiency virus (HIV) and harm reduction programming. However, much of the current efforts relate to HIV prevention and treatment among this population [8]. The current focus on prevention and treatment of HIV among PWID is reasonable given the very high HIV prevalence among this group nationally, at 24.8% [9]. In this context, the Cambodian Ministry of Health has set a goal to elimin- ate new HIV infection by 2025 through the implementa- tion of the Cambodia 3.0 Strategy that focuses on key populations such as PWID, female entertainment workers, men who have sex with men, and transgender women [10]. Consequently, a large part of the existing strategic information related to PWID in Cambodia is concerned with HIV, with relatively limited research having been conducted on HCV epidemiology in this population. While a limited number of studies on HCV epidemiology have been conducted in Cambodia, those studies have mostly focused on the general population [11–13]. Where PWID have been included in these stud- ies, their proportion was small [11]. Data collection training A data collection training was conducted for three days for all data research team members. The contents of the training included informed consent process, interview techniques, confidentiality and privacy, and practices of the questionnaire administration. The teams also reviewed the study protocol and tools, so that all members would be thoroughly familiar with them. A laboratory refresher training was also provided to data collection teams re- sponsible for HCV and HIV testing. Team leaders were also encouraged to perform daily reviews with the team members to review progress and communicate any issues that may occur during the data collection. We collected socio-demographic information includ- ing age (continuous), gender, type of the community (urban or rural), years of formal education attained (continuous), average income in the past 6 months (continuous), living situation, and employment status. Information on drug use included types of illicit drugs, frequency of use, and needle and syringe exchange prac- tices in the past 3 months. Sexual behaviors included number of partners and condom use with non-commer- cial and commercial partners (defined as a partner with whom the participant had sex in exchange for money or goods) in the past 3 months. Participants were also asked about community-based HIV and harm reduction services they had received in the past 6 months. Other information included HIV testing, symptoms of sexually transmitted infections (STIs) and care seeking behaviors for the most recent symptoms, use of other substances (tobacco and alcohol) in the past 3 months, as well as experiences with drug rehabilitation and methadone maintenance therapy in the past 12 months. Data collection procedures and tool HCV and HIV test Rapid serological tests for HCV antibody was performed by a laboratory technician from Médecins Sans Frontières (MSF) in Cambodia with capillary blood using OraQuick© (OraSure Technologies, Inc., Bethlehem). OraQuick© is a World Health Organization (WHO) pre-qualified HCV rapid qualitative immunoassay screening test based on detection of immunoglobulin G HCV antibody [20]. A reactive test establishes that a person had been exposed to HCV resulting in the generation of HCV antibodies. The newly detected HCV antibody positive cases were followed up by MSF for further management. Rapid HIV testing was performed by a laboratory tech- nician from the provincial health department using SD Bioline HIV/Syphilis Duo test (Standard Diagnostic, Inc., Korea). The SD Bioline HIV/Syphilis Duo test is a WHO pre-qualified solid-phase immunoassay for the qualita- tive detection of antibodies to all isotypes (IgG, IgM, and IgA) specific to HIV-1/2 and/or Treponema pallidum simultaneously [21]. A non HIV-reactive result estab- lishes that an individual is not HIV-infected. A con- firmatory test was conducted on site using HIV 1/2 Questionnaire development and measures p A structured questionnaire was developed using stan- dardized tools adapted from previous studies among HIV key populations in Cambodia [9, 23–25]. The ques- tionnaire was initially developed in English and then translated into Khmer, the national language of Cambodia. Back-translation was performed to ensure that the “content and spirit” of every original item were maintained. A validated workshop was conducted with representatives of beneficiaries, communities, and key stakeholders working on HIV and harm reduction in Cambodia. The questionnaire was pretested with ten PWID in Phnom Penh, who were later excluded from the main study. Sample size and sampling procedures Harm Reduction Journal (2019) 16:29 Page 3 of 11 Yi et al. Harm Reduction Journal (2019) 16:29 STAT-PAK® Assay (Chembio Diagnostic Systems, Inc., New York) for cases with an HIV reactive result from the SD Bioline HIV/Syphilis Duo test, in keeping with the national HIV testing guidelines. The HIV 1/2 STAT-PAK™Assay is a rapid immunochromatographic screening test for the detection of HIV 1/2 antibodies [22]. Participants received their results verbally in a post-test counseling session after the interview. New HIV-positive cases were linked to a local NGO working in the area. After completion of the data collection, par- ticipants received a gift for their time and transport compensation, costing approximately US$5. [19]. The RDS was implemented in five stages. First, eli- gible four seeds who were well connected to other PWID or PWUD in each location were selected with support from non-governmental organizations (NGOs) working in the area. Second, after obtaining a written in- formed consent, a personal identification number (PIN) was assigned to each seed to be enrolled as a participant. Third, three coupons were given to each seed to refer three additional PWID. For a successful referral, the seed would receive US$2, and the seed was expected to ex- tend to 3–6 “recruitment waves” in each site. Additional seeds would be selected in case the enrolment has been halted because all recruitment chains have “dried up” (i.e., stopped recruiting) or the initial seeds did not re- cruit participants. Finally, recruited individuals were in- vited to become seeds and recruit other peers. Sample size and sampling procedures p p g p The sample size was calculated for the parent study based on an assumption that HIV prevalence would have decreased by 20% between the national survey in 2012 and the current IBBS and the estimated PWID popula- tion size of 1300 [9]. An estimated HIV prevalence of 20% was used, with a margin error of 1.5%, a confidence interval of 95%, a response rate of 90%, and a design ef- fect of 1.5. The minimum required sample size for this study was approximately 280. For the overall IBBS, the sample size was stratified by study sites based on the es- timated population size of people who use non-injecting drugs (PWUD), and roughly 15% of the estimated PWUD in each site were recruited. Data collection was conducted in 21 locations (six locations in Phnom Penh and 15 locations in the remaining provinces), deter- mined based on the required sample size of PWUD in each study site. Approximately 25.0% of the estimated 1068 PWID in Phnom Penh in 2016 [18] were recruited, assuming that there would be no PWID in provinces outside the capital city according to HIV programs re- ports. However, all PWID found in provinces were in- cluded in the study. Injecting drug use is a key driving factor for both HCV and HIV, particularly when injecting is conducted through shared equipment [14]. This situation calls for increased focus on PWID. PWID infected with HCV represent a high-risk group in terms of unsafe injecting practices. Identifying those at high risk for HCV infec- tion could inform education efforts for safer injection among PWID who have not been reached by such ser- vices. Such efforts could also contribute to prevention of co-infection with HIV among those already infected with HCV. Early detection of HCV could lead to earlier treat- ment, given the increasing availability of better-tolerated oral antiviral drugs [15, 16]. Currently, access and treat- ment to pegylated interferon and ribavirin is very limited in Cambodia [11]. Given the health impacts of HCV infection, the poten- tial for HCV/HIV co-infection, the national goal of bet- ter responding to the needs of key and vulnerable populations, and the limited data related to HCV among The respondent-driven sampling (RDS) method was used to recruit study participants, and the Strengthening the Reporting of Observational Studies in Epidemiology for RDS Studies (STROBE-RDS) statement was followed Page 3 of 11 Yi et al. Hepatitis C-related characteristics As shown in Table 1, 21.3% of the study participants re- ported having been tested for HCV and 15.8% receiving HCV education through drop-in centers or peer out- reach workers in the past 12 months. Only 20.8% perceived that their HCV risk was higher than that of the general population. Data analyses All analyses were estimated with sampling weights that corrected for nonresponse and sampling design [26]. The prevalence of HCV antibody was calculated by div- iding the total number of HCV antibody positive partici- pants with the total number of participants tested. Page 4 of 11 Page 4 of 11 Yi et al. Harm Reduction Journal (2019) 16:29 Yi et al. Harm Reduction Journal (2019) 16:29 Yi et al. Harm Reduction Journal (2019) 16:29 Socio-demographic characteristics and behavioral vari- ables of HCV antibody positive participants were com- pared to those of HCV antibody negative participants using Chi-square test (or Fisher’s exact test for an expected cell value of < 5) for categorical variables and Student’s t test or Mann-Whitney U test for continuous variables. Age, level of education, and income were transformed into categorical variables. To identify inde- pendent risk factors for HCV antibody positivity, vari- ables associated with HCV antibody positivity at a significance level of p < 0.05 in bivariate analyses were simultaneously included in a multivariable logistic regression model. Because of their epidemiological im- portance, we included age, gender, level of education, and income in the model regardless of the significance level in bivariate analyses. STATA Version 12.0 for Windows (Stata Corp, TX, USA) was used for the analyses. Prevalence of HCV This study included 286 PWID with a mean age of 31.6 (SD = 7.5). The majority of PWID in the survey (72.0%) were residing in Phnom Penh. Of the total, 30.4% were tested positive for HCV antibody (95% CI = 25.3–36.0%). Almost one-third (31.0%) of the HCV antibody positive individuals were co-infected with HIV. All HCV anti- body positive cases were found in Phnom Penh. Only 11.5% of the HCV antibody positive cases were aware of their HCV infection status prior to the study, and five had received HCV treatment. Ethical considerations This study was approved by the National Ethics Com- mittee for Health Research (NECHR) of the Ministry of Health, Cambodia (No. 193 NECHR). Privacy of the re- spondents was protected by having the data collected in a private place and confidentiality ensured by remov- ing all personal identifiers from the study documents. Participants were fully informed about the risks and benefits they may expect from their participation and the voluntary nature of the study. A written informed consent was obtained from each participant prior to the data collection. HCV hepatitis C virus, PWID people who inject drugs j Chi-square (or Fisher’s exact test when a cell count was smaller than 5) was used Socio-demographic characteristics Table 2 shows that 92.0% of the participants were re- cruited from urban communities; 72.4% were male; and 20.4% were in Vietnamese ethnic group. Less than half (40.2%) were married, and 57.3% had attained primary education or lower. While 39.9% reported living with their family or relatives, 26.9% were living on the streets. The most common job was a laborer or farmer (37.1%), and 12.9% were unemployed. The majority of the partici- pants (78.7%) reported an average monthly income in Table 1 Hepatitis C-related characteristics among HCV antibody positive and negative PWID Hepatitis C-related characteristics Total (n = 286) HCV antibody test result Positive (n = 87) Negative (n = 199) n (%) n (%) n (%) Tested for HCV in lifetime 61 (21.3) 28 (32.2) 33 (16.6) Tested for HCV in the past 12 months (n = 61) 47 (77.0) 22 (78.6) 25 (75.8) Diagnosed or told about having hepatitis C (n = 61) 16 (26.2) 10 (35.7) 6 (18.2) Currently on hepatitis C treatment (n = 16) 8 (50.0) 5 (50.0) 3 (50.0) Received education on hepatitis C in the past 12 months 45 (15.8) 17 (19.5) 28 (14.1) Source of information on hepatitis C received last time Peer educator/outreach worker 23 (50.0) 7 (41.2) 16 (55.2) Drop-in center 27 (58.7) 13 (76.5) 14 (43.3) Perceived HCV risk compared to the general population Higher 59 (20.8) 24 (27.6) 35 (17.8) About the same 75 (26.4) 21 (24.1) 64 (27.4) Lower 32 (11.3) 6 (6.9) 26 (13.2) Do not know 118 (41.5) 36 (41.4) 82 (41.6) HCV hepatitis C virus, PWID people who inject drugs Chi-square (or Fisher’s exact test when a cell count was smaller than 5) was used Table 1 Hepatitis C-related characteristics among HCV antibody positive and negative PWID Yi et al. Socio-demographic characteristics Harm Reduction Journal (2019) 16:29 Page 5 of 11 Table 2 Socio-demographic characteristics of HCV antibody positive and negative PWID Socio-demographic characteristics Total (n = 286) HCV antibody test result Positive (n = 87) Negative (n = 199) n (%) n (%) n (%) P valuea Living in an urban community 263 (92.0) 87 (100.0) 176 (88.4) < 0.001 Male gender 207 (72.4) 66 (75.9) 141 (70.9) 0.38 Age group < 0.001 18–24 54 (18.9) 1 (1.1) 53 (26.6) 25–34 122 (42.7) 43 (49.4) 79 (39.7) ≥35 110 (38.5) 43 (49.4) 67 (33.7) Ethnic group < 0.001 Khmer 227 (79.6) 47 (54.0) 180 (90.9) Vietnamese 58 (20.4) 40 (46.0) 18 (9.1) Current marital status 0.44 Never married 114 (39.9) 30 (34.5) 84 (42.2) Married 115 (40.2) 37 (42.5) 78 (39.2) Widowed/divorced/separated 57 (19.9) 20 (23.0) 37 (18.6) Level of formal education completed 0.009 Primary (0–6 years) 164 (57.3) 61 (70.1) 103 (51.8) Secondary school (7–9 years) 68 (23.8) 17 (19.5) 51 (25.6) High school or higher (≥10 years) 54 (18.9) 9 (10.3) 45 (22.6) Living arrangement < 0.001 On the street (homeless) 77 (26.9) 47 (54.0) 30 (15.1) With family or relatives at home 114 (39.9) 21 (24.1) 93 (46.7) In own dwelling 52 (15.0) 8 (9.2) 44 (22.1) With friends 15 (5.2) 6 (6.9) 9 (4.5) Other 28 (9.8) 5 (5.7) 23 (11.6) Main occupation 0.41 Unemployed 37 (12.9) 11 (12.6) 26 (13.1) Entertainment worker 30 (10.5) 7 (8.0) 23 (11.6) Office worker 75 (26.2) 29 (33.3) 46 (23.1) Laborer/farmer 106 (37.1) 28 (32.2) 78 (39.2) Other 38 (13.3) 12 (13.8) 26 (13.1) Average monthly income in the past 6 months (US$) 0.53 < 100 105 (36.7) 33 (37.9) 72 (36.2) 100–199 120 (42.0) 39 (44.8) 81 (40.7) ≥200 61 (21.3) 15 (17.2) 46 (23.1) HCV hepatitis C virus, PWID people who inject drugs aChi-square (or Fisher’s exact test when a cell count was smaller than 5) was used Table 2 Socio-demographic characteristics of HCV antibody positive and negative PWID Characteristics of substance use Characteristics of substance use the past 6 months of < US$200. A significantly higher proportion of HCV antibody positive participants were living in an urban community (100% vs. 88.4%), were in the age group of ≥35 (49.4% vs. 33.7%), were in Vietnamese ethnic group (46.0% vs. 9.1%), had attained primary education or lower (70.1% vs. 51.8%), and lived on the streets (54.0% vs. 15.1%) compared to that in HCV antibody negative group. the past 6 months of < US$200. A significantly higher proportion of HCV antibody positive participants were living in an urban community (100% vs. 88.4%), were in the age group of ≥35 (49.4% vs. 33.7%), were in Vietnamese ethnic group (46.0% vs. 9.1%), had attained primary education or lower (70.1% vs. 51.8%), and lived on the streets (54.0% vs. 15.1%) compared to that in HCV antibody negative group. As shown in Table 3, the median time since they started injecting drugs was 49 months (IQR = 16–120). About half (50.3%) reported “injection” as the method of their first drug use and that they were firstly introduced to drugs by their friends or colleagues (80.4%). Heroin was the most commonly used drugs in the past 3 months (61.9%), followed by methamphetamine (23.8%). About Yi et al. Characteristics of substance use Harm Reduction Journal (2019) 16:29 Page 6 of 11 Table 3 Characteristics of substance use among HCV antibody positive and negative PWID Substance use characteristics Total (n = 286) HCV antibody test result Positive (n = 87) Negative (n = 199) n (%) n (%) n (%) P valuea Median months using drugs (IQR) 49 (16–120) 96 (36–132) 36 (12–120) < 0.001 Mode of first drug use—injecting 104 (50.3) 51 (58.6) 93 (46.7) 0.06 Injecting drugs in the past 3 months 250 (88.0) 84 (96.6) 166 (84.3) 0.003 Type of drugs most commonly used in the past 3 months Heroin 156 (61.9) 67 (79.8) 89 (53.0) < 0.001 Yama/ice (methamphetamine) 60 (23.8) 21 (25.0) 39 (23.2) 0.75 Ecstasy 9 (3.6) 0 (0.0) 9 (5.4) 0.27 Always used new syringes/needles in the past 3 months 112 (66.7) 27 (61.4) 85 (68.5) 0.54 Used needles/syringes used by someone else in the past 3 months 37 (21.6) 8 (18.2) 29 (22.8) 0.31 Having been sent to a drug rehabilitation center in the past 12 months 119 (41.6) 55 (63.2) 64 (32.2) < 0.001 Having received methadone maintenance therapy in the past 12 months 125 (43.7) 68 (78.2) 57 (28.6) < 0.001 Alcohol drinking ≥3 times per week 84 (29.4) 13 (14.9) 71 (35.7) < 0.001 Binge drinking ≥3times per week (n = 171) 83 (48.5) 13 (36.1) 70 (51.9) 0.09 HCV hepatitis C virus, IQR interquartile range, PWID people who inject drugs aChi-square (or Fisher’s exact test when a cell count was smaller than 5) was used for categorical variables and Mann-Whitney U test for continuous variables Table 3 Characteristics of substance use among HCV antibody positive and negative PWID 45.4% reported having sex when a partner was intoxi- cated in the past 3 months. Of those who reported having sex with partners not in exchange for money or gifts (n = 108), 8.3% reported always using con- doms with the non-commercial partners in the past 3 months. Of the total respondents, 23.3% reported having sex in exchange for money or goods in the past 3 months, of whom 26.8% reported always using condoms with the commercial partners in the past 3 months. Despite their substantial HIV risk, only 27.3% perceived that their HIV risk was higher com- pared to that of the general population. Characteristics of substance use No significant difference was found in comparison of sexual behav- iors in HCV antibody positive and negative group (Table 4). two-thirds (66.7%) reported always using new syringes/ needles for drug injection, and 21.6% using needles or syringes that had been used by someone else in the past 3 months. Less than half (41.6%) had been sent to a drug rehabilitation center, and 43.7% had received methadone maintenance therapy in the past 12 months. The use of other substances was also common, with 29.4% reported alcohol drinking at least three times per week; of them, 48.5% reported binge drinking (drinking at least five units of alcohol on a typical day) on at least 3 days per week in the past 3 months. The median time of using drugs was significantly longer in HCV antibody positive group compared to that in HCV antibody negative group (96 months vs. 36 months). The proportion of participants who reported injecting any kind of drugs (96.6% vs. 84.3%) and injecting heroin (79.8% vs. 53.0%) in the past 3 months was higher in HCV antibody positive group than in HCV antibody negative group. The proportion of participants who reported having been sent to a drug rehabilitation (63.3% vs. 33.2%) and having re- ceived methadone maintenance therapy (78.2% vs. 28.6%) in the past 12 months was significantly higher in HCV antibody positive group. A significantly lower proportion of HCV antibody positive participants reported alcohol drinking ≥3 times per week in the past 3 months (14.9% vs. 35.7%). Access to community-based HIV services Table 5 shows that 68.9% of the study participants reported having received some form of community- based HIV services in the past 6 months. The services included condom and lubricant distribution (71.5%), HIV/syphilis testing (63.1%), HIV education (48.8%), needle and syringe distribution (50.8%), methadone maintenance therapy (43.7%), drop-in services (22.3%), and legal support (5.4%). The proportion of participants who reported having received needles and syringes from community-based harm reduction programs (68.2% vs. 41.9%) and methadone maintenance therapy (78.2% vs. 28.6%) in the past 6 months was significantly higher in HCV antibody positive group than in HCV antibody negative group. Sexual behaviors Table 4 shows that 95.1% of participants reported be- ing sexually active in the past 3 months, with a me- dian number of sex partners of 1.0 (IQR = 0.0–2.0). Only 16.6% reported always using condoms, and Yi et al. Harm Reduction Journal (2019) 16:29 Page 7 of 11 Table 4 Sexual behaviors among HCV antibody positive and negative PWID Sexual behaviors in the past 3 months Total (n = 286) HCV antibody test result Positive (n = 87) Negative (n = 199) n (%) n (%) n (%) P valuea Had sexual intercourse 272 (95.1) 84 (96.6) 188 (94.5) 0.56 Median number of sex partners (IQR) 1.0 (0.0–2.0) 1.0 (0.0–1.0) 1.0 (0.0–2.0) 0.48 Always used condom with any partner 29 (16.6) 6 (13.3) 23 (17.7) 0.50 Had sex when a partner was intoxicated 79 (45.4) 23 (51.1) 56 (43.4) 0.37 Had sex with partners not in exchange for money or gift 108 (61.7) 26 (57.8) 82 (63.1) 0.53 Always used condom with partners not in exchange for money or gift 9 (8.3) 2 (7.7) 7 (8.5) 0.89 Had sex in exchange for money or goods 41 (23.3) 6 (13.3) 35 (26.7) 0.07 Always used condom with partners in exchange for money or goods 11 (26.8) 1 (16.7) 10 (28.6) 0.48 Perceived HIV risk compared to the general population 0.48 Higher 78 (27.3) 28 (32.2) 50 (25.1) About the same 96 (33.6) 28 (32.2) 68 (34.2) Lower 33 (11.5) 7 (8.0) 26 (13.1) Do not know 79 (27.6) 24 (27.6) 55 (27.6) Perceived HIV risk compared to the general population Higher 59 (20.8) 24 (27.6) 35 (17.8) About the same 75 (26.4) 21 (24.1) 64 (27.4) Lower 32 (11.3) 6 (6.9) 26 (13.2) Do not know 118 (41.5) 36 (41.4) 82 (41.6) HCV hepatitis C virus, HIV human immunodeficiency virus, IQR interquartile range aChi-square (or Fisher’s exact test when a cell count was smaller than 5) was used for categorical variables and Mann-Whitney U test for continuous variables Table 4 Sexual behaviors among HCV antibody positive and negative PWID Table 4 Sexual behaviors among HCV antibody positive and negative PWID HCV hepatitis C virus, HIV human immunodeficiency virus, IQR interquartile range aChi-square (or Fisher’s exact test when a cell count was smaller than 5) was used f Factors associated with HCV antibody drug rehabilitation center in the past 12 months (AOR = 2.67, 95% CI = 1.21–5.90), had received methadone maintenance therapy in the past 12 months (AOR = 3.02, 95% CI = 1.32–6.92), and were tested positive for HIV (AOR = 3.80, 95% CI = 1.58–9.12) compared to their respective reference group. Factors associated with HCV antibody positivity among PWID in this study are shown in Table 6. After adjust- ment for other covariates, the odds of HCV antibody positivity was significantly higher among participants who were in the older age group of 25 to 34 (AOR = 1.85, 95% CI = 1.06–7.92) and ≥35 (AOR = 2.67, 95% CI = 1.24–5.71), were in Vietnamese ethnic group (AOR = 5.44, 95% CI = 2.25–13.14), were living on the streets (AOR = 3.01, 95% CI = 1.29–704), had been sent to a irus, HIV human immunodeficiency virus, PWID people who inject drugs, STI sexually transmitted infections sher’s exact test when a cell count was smaller than 5) was used HCV hepatitis C virus, HIV human immunodeficiency virus, PWID people who inject drugs, STI sexually transmitted infections aChi-square (or Fisher’s exact test when a cell count was smaller than 5) was used HCV hepatitis C virus, HIV human immunodeficiency virus, PWID people who inject drugs, STI sexually transmitted infections aChi square (or Fisher’s exact test when a cell count was smaller than 5) was used Discussion Findings from this national survey show that the overall prevalence of HCV antibody among PWID in Cambodia Table 5 Access to community-based HIV and harm reduction services among HCV antibody positive and ne Table 5 Access to community-based HIV and harm reduction services among HCV antibody positive and negative PWID Access to community-based ser- vices in the past 6 months Total (n = 286) HCV antibody test result Positive (n = 87) Negative (n = 199) n (%) n (%) n (%) P valuea Received community-based services 197 (68.9) 66 (75.9) 131 (65.8) 0.09 HIV education 63 (48.8) 18 (40.9) 45 (52.9) 0.20 Condom and lubricant distribution 93 (71.5) 33 (75.0) 60 (69.8) 0.53 Needle and syringe distribution 66 (50.8) 30 (68.2) 36 (41.9) 0.005 HIV/syphilis testing 82 (63.1) 30 (68.2) 52 (60.5) 0.39 Legal support 7 (5.4) 2 (4.5) 5 (5.8) 0.76 Drop-in services 29 (22.3) 6 (13.6) 23 (26.7) 0.09 Methadone maintenance therapy 125 (43.7) 68 (78.2) 57 (28.6) < 0.001 HCV hepatitis C virus, HIV human immunodeficiency virus, PWID people who inject drugs, STI sexually transmitted infections aChi-square (or Fisher’s exact test when a cell count was smaller than 5) was used Page 8 of 11 Yi et al. Harm Reduction Journal (2019) 16:29 Table 6 Factors associated with HCV antibody positivity among PWID in multivariate logistic regression model Variables in the final model AOR (95% CI) P value Age group < 25 Reference 25–34 1.85 (1.06–7.92) 0.009 ≥35 2.67 (1.24–5.71) 0.001 Ethnic group Khmer Reference Vietnamese 5.44 (2.25–13.14) < 0.001 Living arrangement With family or relatives Reference In the street (homeless) 3.01 (1.29–7.04) 0.01 In own dwelling 0.64 (0.20–2.03) 0.45 With friends 0.15 (0.46–10.07) 0.33 Having been sent to a drug rehabilitation center in the past 12 months No Reference Yes 2.67 (1.21–5.90) 0.02 Having received methadone maintenance therapy in the past 12 months No Reference Yes 3.02 (1.32–6.92) 0.009 HIV testing result Negative Reference Positive 3.80 (1.58–9.12) < 0.001 AOR adjusted odds ratio, CI confidence interval, HCV hepatitis C virus, HIV human immunodeficiency virus, PWID people who inject drugs Age, gender, marital status, education level, income, and variables associated with HIV infection in the bivariate analyses at a level of p < 0.05 were simultaneously included in the model HCV infection and has implications for education, ser- vices, and policy in Cambodia. Discussion For instance, of the 87 participants who tested positive for HCV, only ten (11.5%) were aware of their status prior to the study, of which only five had received HCV treatment. Multivariate results showed several factors associ- ated with positive HCV antibody screening results. After adjustment for potential confounding factors, the odds of testing positive for HCV antibody was significantly higher among PWID who were in the older age group of 25 to 34 and ≥35, compared with those aged ≤25. The association of older age with higher odds of testing positive for HCV antibody is similar to findings in previous studies ([31–34]. It is plausible that, as PWID continue to inject, they have more opportunities to acquire HCV, and indeed, dur- ation of injection has been reported as independently predictive of HCV antibody positivity [31, 32, 35] or seroconversion [36]. Our study suggests that HCV antibody positivity was three times more likely to be detected among participants who were living on the streets compared to those living with their family or relatives at home. Other studies have shown that homelessness is predictive of both the likeli- hood to inject [37] and to using shared equipment [38]. On a practical level, this finding provides a useful way in which to categorize profiles of HCV risk among PWID, who were also identified as being at higher risk of HIV in this national survey (Mburu M, Chhoun P, Navy Chann, Tuot S, Mun P, and Yi S: Prevalence and risk factors for HIV infection among people who injct drugs in Cambodia: findings from a national survey, submitted). As such, outreach services to homeless PWID will be essen- tial for prevention of both HCV and HIV. This is particu- larly relevant given that 26.9% of the participants were living on the streets. In addition, the prevalence of HCV antibody was five times higher among participants of Viet- namese ethnic origin. Although ethnicity has been re- ported to be associated with HCV in other studies [39], we suggest that this is likely to be a proxy indicator of other structural risk behavioral or other risk factors, which would need to be further investigated in our context. Discussion AOR adjusted odds ratio, CI confidence interval, HCV hepatitis C virus, HIV human immunodeficiency virus, PWID people who inject drugs Age, gender, marital status, education level, income, and variables associated with HIV infection in the bivariate analyses at a level of p < 0.05 were simultaneously included in the model AOR adjusted odds ratio, CI confidence interval, HCV hepatitis C virus, HIV human immunodeficiency virus, PWID people who inject drugs Age, gender, marital status, education level, income, and variables associated with HIV infection in the bivariate analyses at a level of p < 0.05 were simultaneously included in the model was 30.4%. Prior to this study, a few smaller studies in the country had reported varying HCV antibody preva- lence in the Cambodian general adult populations, ran- ging from 2.3 to 14.7% [27–30]. Among Cambodian people living with HIV, previous studies reported a prevalence between 5.3 and 10.5% [11–13]. Although one of these studies included some PWID, the number was small [11]. In Cambodia, the national response toward people who use and inject drugs has previously included send- ing users to a compulsory drug rehabilitation center, which have generally been associated with poor health outcomes [40, 41]. In China, a recent study also found that having been sent to a compulsory drug rehabilita- tion center increased the odds of positive HCV antibody [33]. While these findings do not necessarily point to causation, they are generally consistent with assertions that structural factors related to arrests and incarcer- ation could promote risky injection and may lead to HCV infection [31]. Indeed, studies from other countries Our study is the first exclusively conducted among PWID and shows very high prevalence of HCV antibody in this population. However, in this study, confirmation of active infection and genotyping data were not feasible due to cost of HCV ribonucleic acid (RNA) testing, and it was not part of the national IBBS protocol. The preva- lence of active hepatitis C would be expected to be lower than the proportion of people with HCV antibody [11]. However, the prevalence of HCV antibody still provides important data related to the potential magnitude of Yi et al. Harm Reduction Journal (2019) 16:29 Page 9 of 11 Page 9 of 11 such as Iran, the United Kingdom, and Australia have shown that the odds of positive HCV infection are higher among PWID with history of incarceration [35, 42, 43]. Limitations of the study y In interpreting our findings, it should be borne in mind that the design, recruitment, methods of data collection, and sample profile could affect what are reported. However, the study aimed to be representative as far as possible by recruiting participants nationally. The cross- sectional nature of this study only captures a snapshot and may not provide valuable information regarding spontaneous clearance of HCV, which is part of natural history of the infection [53]. Personal sensitive data were collected from the participants through a self-reported questionnaire, which can be affected by social desirabil- ity bias as is the case in other studies of PWID [54]. In addition, the HCV antibody prevalence data presented in this paper were based on a rapid test, and did not repre- sent current active HCV infection [55]. A recent review of rapid diagnostics reported that OraQuick ADVANCE® had higher sensitivity (98%, 95% CI 97–98%) compared to the other oral brands (pooled sensitivity: 88%, 95% CI 84–92%) [56]. Nevertheless, other diagnostic methods such as polymerase chain reaction (PCR)-based positiv- ity are required to identify active hepatitis C, but was not feasible in this large national survey. Despite this limitation, the oral rapid screening could be a useful strategy for screening PWID who are unaware of their HCV status [57], in keeping with the global testing and elimination targets [55]. This is particularly relevant in Cambodia, given the high prevalence of HCV antibody found in this study. Our observation that being infected with HIV tripled the odds of being positive for HCV antibody is not ne- cessarily surprising, given the most common shared risk factor for both infections—unsafe injection. In our sample, 21.6% reported using needles or syringes that had been used by someone else in the past 3 months. Indeed, the endemic level of HCV among the general population in Cambodia could be related to the historical popularity—and to some extent, un- safe injecting practices in the country [46, 47]. Other studies, such as from Canada and India, have also re- ported associations between HIV and HCV [48, 49]. Discussion 87 PWID living with HCV; 88.5% of them who were not aware of their HCV antibody status prior to the study would benefit from the close follow-up and link- age to care and treatment services provided by MSF. The association between positive HCV antibody and methadone maintenance therapy is not consistent across the literature. Although it was associated with the increased odds of testing positive for HCV antibody in our own and other studies [33, 35], this association may not necessarily be causal. Indeed, several studies indicate that opioid substitution treatment (OST) could be pro- tective of HCV [44, 45]. However, OST could be an indi- cation of longer-term drug use and dependency that may lead to increased HCV risk. Policy implications These results provide critical information for program- ming and policy. First, there is a need to raise aware- ness of hepatitis C, so as to ensure that detection is increased. Increasing awareness of HCV can also bene- fit HIV testing, prevention, and treatment [49]. Second, there is a need for increased governmental focus on hepatitis C, with intent of increasing screen- ing, diagnosis, and treatment. Currently, access to pegylated interferon and ribavirin is limited in Cambodia [11]. Poor availability of treatment is known to be associated with poor health outcomes among those with hepatitis C in many low- and middle-in- come countries [50]. As opposed to treatment with pegylated interferon and ribavirin, however, the advent of new generation direct-acting antiviral (DAA) that are effective across different genotypes [51, 52] could provide impetus for enhancing screening, diagnosis, and treatment. Although, currently, there is no na- tional program to target hepatitis C in Cambodia, MSF has provided hepatitis C screening and treatment using DAAs free of charge since 2016. Given the high preva- lence of HCV in PWID, this high-risk population should be the target for both prevention and treatment programs in Cambodia. In this study, using the respondent-driven sampling method, we could reach PWID who are vulnerable to or infected with HCV in resource-limited settings. Received: 7 January 2019 Accepted: 9 April 2019 Conclusions Hepatitis C is becoming an important health promotion focus for PWID globally [58]. Understanding the epi- demiology of HCV among this population, who are already heavily burdened by HIV, will inform future pre- vention, care, and treatment of HCV or HCV/HIV co-infection. In Cambodia, HCV/HIV co-epidemic is poorly characterized [58], yet this has relevance for PWID. This article presents findings from a national survey in Cambodian PWID, showing the HCV antibody prevalence of 30.4%. In addition, several important factors including older age, homelessness, Vietnamese ethnicity, having been sent to a drug rehabilitation cen- ter, having received methadone maintenance therapy, and being infected with HIV were independently pre- dictive of HCV infection. Given the health impact of hepatitis C, these findings suggest a number of policy and programmatic implications to promote health of Page 10 of 11 Yi et al. Harm Reduction Journal (2019) 16:29 Yi et al. Harm Reduction Journal (2019) 16:29 Yi et al. Harm Reduction Journal Competing interests Competing interests The authors declare that they have no competing interests. Competing interests Competing interests The authors declare that they have no competing interests. 16. Poordad F, McCone J Jr, Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med. 2011;364(13):1195–206. https://doi.org/10.1056/NEJMoa1010494. 16. Poordad F, McCone J Jr, Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med. 2011;364(13):1195–206. https://doi.org/10.1056/NEJMoa1010494. References CI: Confidence interval; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; IBBS: Biological and behavioral survey; IQR: Interquartile range; MSF: Médecins Sans Frontières; NECHR: National Ethics Committee for Health Research; NGO: Non-governmental organization; OST: Opioid substitution treatment; PCR: Polymerase chain reaction; PIN: Personal identification number; PWID: People who inject drugs; PWUD: People who use drugs; RDS: Respondent-driven sampling; RNA: Ribonucleic acid; SD: Standard deviation 1. Cooke GS, Lemoine M, Thursz M, Gore C, Swan T, Kamarulzaman A, et al. Viral hepatitis and the global burden of disease: a need to regroup. J Viral Hepat. 2013;20(9):600–1. https://doi.org/10.1111/jvh.12123. 1. Cooke GS, Lemoine M, Thursz M, Gore C, Swan T, Kamarulzaman A, et al. Viral hepatitis and the global burden of disease: a need to regroup. J Viral Hepat. 2013;20(9):600–1. https://doi.org/10.1111/jvh.12123. 2. Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology. 2013;57(4):1333–42. https://doi.org/10.1002/hep.26141 2. Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology. 2013;57(4):1333–42. https://doi.org/10.1002/hep.26141. 3. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med. 2001;345(1): 41–52. https://doi.org/10.1056/NEJM200107053450107. 3. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med. 2001;345(1): 41–52. https://doi.org/10.1056/NEJM200107053450107. 4. Stanaway JD, Flaxman AD, Naghavi M, Fitzmaurice C, Vos T, Abubakar I, et al. The global burden of viral hepatitis from 1990 to 2013: findings from the global burden of disease study 2013. Lancet. 2016;388(10049):1081–8. https://doi.org/10.1016/S0140-6736(16)30579-7. Availability of data and materials Data used for this study can be accessed upon request from the Principal Investigator (Dr. Siyan Yi) at siyan@doctor.com. 8. National AIDS Authority. Cambodia country progress report: monitoring progress towards the 2011 UN political declaration on HIV and AIDS. Phnom Penh: National AIDS Authority; 2015. Authors’ information SY (PhD) is Senior Research Fellow at Saw Swee Hock School of Public Health, National University of Singapore, Singapore and Director of KHANA Center for Population Health Research, Cambodia. ST (MA) is Manager and PC (MPH) is Research Fellow at KHANA Center for Population Health Research, Cambodia. PM (MPH) is Chief of Surveillance Unit and NC (MPH) is Deputy Chief of Surveillance Unit, National Center for HIV/AIDS, Dermatology and STD. GM (MPH) is a Researcher at Division of Health Research, Lancaster University, the United Kingdom. 12. Lerolle N, Limsreng S, Fournier-Nicolle I, Ly S, Nouhin J, Guillard B, et al. High frequency of advanced hepatic disease among HIV/HCV co-infected patients in Cambodia: the HEPACAM study (ANRS 12267). J AIDS Clinic Res. 2012;3:161. https://doi.org/10.4172/2155-6113.1000161. 13. van Griensven J, Phirum L, Choun K, Thai S, De Weggheleire A, Lynen L. Hepatitis B and C co-infection among HIV-infected adults while on antiretroviral treatment: long-term survival, CD4 cell count recovery and antiretroviral toxicity in Cambodia. PLoS One. 2014;9(2):e88552. https://doi. org/10.1371/journal.pone.0088552. 13. van Griensven J, Phirum L, Choun K, Thai S, De Weggheleire A, Lynen L. 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J Clin Epidemiol. 2015;68(12):1463–71. https://doi.org/10.1016/j.jclinepi.2015.04.002. Authors’ contributions 9. Chhea C, Heng S, Tuot S. National Popualtion size estimation, HIV related risk behaviors, HIV prevalence among people who use drugs in Cambodia in 2012. Phnom Penh: National Authority for Combating Drugs and KHANA; 2014. SY, ST, and PM designed the study and developed the study protocol and tools. NC, PC, TS, and PM were responsible for trainings and data collection. SY and GM analyzed data and wrote the manuscript. All authors contributed to the conceptualization of the research questions, interpretation of the results, and manuscript writing. All authors read and approved the final manuscript. 10. National Center for HIV/AIDS, Dermatology and STD. Standard operating procedures for HIV testing and counseling (HTC). Phnom Penh: National Center for HIV/AIDS, Dermatology and STD; 2012. 10. National Center for HIV/AIDS, Dermatology and STD. 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https://openalex.org/W2897105212	https://www.scielo.br/j/lajss/a/j8qqVGJ7S3sZVXkKFsk5FFx/?format=pdf&lang=en	English	null	Calibration and evaluation of the Lemaitre damage model using axial- torsion fatigue tests on five engineering alloys	Latin American Journal of Solids and Structures	2,018	cc-by	7,893	1 INTRODUCTION Fatigue crack initiation is a primary type of failure in structural members under cyclic loading. Common examples of fatigue critical members include axles, bearings, crankshafts, pressure vessels, and turbine blades. Such members often experience multiaxial stress-strain conditions originated from multiple applied loads and/or notch effects (Socie and Marquis, 2000; Sharifi et al., 2016; Googarchin et al., 2017). In addition, depending on the nature of the applied loads, the cyclic stresses and strains can be proportional or nonproportional, and of constant or varying amplitude. Many of the multiaxial fatigue life prediction methods that have been developed over the years are based on four components: a constitutive model for the calculation of the cyclic stress-strain response, a fatigue damage parameter, a cycle counting method, and a damage summation rule. A comprehensive overview of the different modeling approaches for each of these four components, including their implications for fatigue life prediction, is presented in the book by Socie and Marquis (2000). A further discussion on these four fundamental issues in multiaxial fatigue is given by Fatemi and Shamsaei (2011). In parallel to the advances in the traditional methods for fatigue life prediction, progress has also been made in the development of fatigue damage evolution rules for metals (see, e.g., Chow and Wei, 1991; Bonora and Newaz, 1998; Jiang, 2000; Lemaitre and Desmorat, 2005; Jiang et al., 2009; Shen et al., 2015; Lopes and Malcher, 2017). As cycle counting under multiaxial loading conditions is still a difficult task, its elimination in many of the damage evolution rules is an attractive feature. For simplicity, a scalar damage quantity is often used to represent fatigue damage, and, hence, no information on the crack orientation is given. A remarkable exception is the incremental fatigue damage model developed by Jiang (2000), which incorporates the critical plane concept in multiaxial fatigue. Despite the progress in the formulation of fatigue damage evolution rules, the experimental evaluation of many of such rules has been limited to simple uniaxial loading conditions. However, efforts to evaluate damage models by using multiaxial tests under more complicated loading conditions can be found in some studies (Jiang, 2000; Jiang et al., 2007; Jiang et al., 2009; Castro and Jiang, 2016; Castro and Jiang, 2017). Also, in a recent work by Lopes and Malcher (2017), the Lemaitre damage model has been evaluated with respect to proportional and nonproportional axial-torsion fatigue data. Keywords Multiaxial fatigue, Life prediction, Damage mechanics, Carbon steel, Aluminum alloy, Magnesium alloy. Received: July 21, 2017 In Revised Form: January 17, 2018 Accepted: January 19, 2018 Available Online: May 02, 2018 Received: July 21, 2017 In Revised Form: January 17, 2018 Accepted: January 19, 2018 Available Online: May 02, 2018 Thematic Section: Solid Mechanics in Brazil 2017 Thematic Section: Solid Mechanics in Brazil 2017 Calibration and evaluation of the Lemaitre damage model using axial- torsion fatigue tests on five engineering alloys Fábio Castroa* Cainã Bemficaa Fábio Castroa* Cainã Bemficaa The Lemaitre damage model is evaluated using fatigue test data from five engineering alloys: 1045 steel, 16MnR steel, 7075-T651 Al alloy, extruded AZ61A Mg alloy, and extruded AZ31B Mg alloy. Tension–compression, torsion, proportional axial-torsion, and 90° out-of-phase axial-torsion loadings were investigated. The results show that the overall accuracy of the fatigue life estimates made by using the Lemaitre model is comparable to those obtained by fatigue models that require the definition of a loading cycle. A simple and effective method is described for determining the material constants of the Lemaitre model. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Abstract Fábio Castroa* Cainã Bemficaa a Departamento de Engenharia Mecânica, Universidade de Brasília – UnB, Brasília, DF, Brasil. E-mail: fabiocastro@unb.br, cainabemfica@gmail.com *Corresponding author. http://dx.doi.org/10.1590/1679-78254340 Received: July 21, 2017 In Revised Form: January 17, 2018 Accepted: January 19, 2018 Available Online: May 02, 2018 2 LEMAITRE DAMAGE MODEL Lemaitre developed a fatigue model for general multiaxial loading based on the framework of continuum thermodynamics (Lemaitre, 1985; Lemaitre and Desmorat, 2005). A scalar quantity, D , is used in this model to describe the fatigue damage at a material point, and a macroscopic crack is predicted to occur when the fatigue damage reaches a critical value, c D . The damage evolution rule takes the following form: s Y D p S         (1) where S and s are material constants and the superposed dot denotes differentiation with respect to time. The quantity p is the equivalent plastic strain rate defined as 2 / 3 p p    ε , where p ε is the plastic strain tensor and the symbol stands for the Euclidean norm of a second-order tensor. Y is expressed as where S and s are material constants and the superposed dot denotes differentiation with respect to time. The quantity p is the equivalent plastic strain rate defined as 2 / 3 p p    ε , where p ε is the plastic strain tensor and the symbol stands for the Euclidean norm of a second-order tensor. Y is expressed as   2 eq 2 2 1 R Y E D     (2) (2) In Eq. (2), E is the Young’s modulus, eq  is the von Mises equivalent stress, and R is a scalar function defined as as     2 2 1 3 1 2 3 R       (3)     2 2 1 3 1 2 3 R           2 2 1 3 1 2 3 R       (3) where  is the Poisson’s ratio. The quantity  is called stress triaxiality and is defined as h eq /    , where h  is the hydrostatic stress. where  is the Poisson’s ratio. The quantity  is called stress triaxiality and is defined as h eq /    , where h  is the hydrostatic stress. 1 INTRODUCTION The damage model developed by Lemaitre (1985) can describe various damage behaviors. Although widely used to estimate ductile fracture, fatigue life prediction based on the Lemaitre model has received much less attention. The experimental evaluation carried out by Lopes and Malcher (2017) indicated that the Lemaitre model Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. ation and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys can provide reasonably accurate fatigue lives for three engineering materials (304 stainless steel, S460N steel, and 6061-T6 Al alloy) under proportional and nonproportional, axial-torsion loading conditions. However, very few torsion tests were examined by Lopes and Malcher (3 torsion tests on the S460N steel), and the fatigue tests on the 6061-T6 Al alloy were limited to the low-cycle fatigue regime. It is desirable to evaluate the Lemaitre model using different materials and testing conditions. In the present work, the Lemaitre damage model is further evaluated based on experimentally observed fatigue lives of five engineering metals: 1045 steel, 16MnR steel, 7075-T651 Al alloy, extruded AZ61A Mg alloy, and extruded AZ31B Mg alloy. A simple and effective method for determining the material constants of the Lemaitre model is also presented. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 2.2 Determination of material constants 2.2 Determination of material constants There are three fatigue-related constants ( S , s, c D ) in the Lemaitre model. To determine these constants, the following objective function based on the squared difference between estimated and observed fatigue lives is minimized:   2 est 1 obs Φ log i n i i N N            (10)   2 est 1 obs Φ log i n i i N N            (10) where n is the number of test data used to determine the material constants, the superscript (i ) denotes the i th test,  obs i N is the observed fatigue life, and  est i N is the fatigue life estimated by using Eq. (9). To solve the minimization problem, an exhaustive search over a pre-defined domain of candidate constants is carried out. The method can be readily implemented in a computer and its computational cost is very low. where n is the number of test data used to determine the material constants, the superscript (i ) denotes the i th test,  obs i N is the observed fatigue life, and  est i N is the fatigue life estimated by using Eq. (9). To solve the minimization problem, an exhaustive search over a pre-defined domain of candidate constants is carried out. The method can be readily implemented in a computer and its computational cost is very low. 2.1 Constant amplitude loading Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engi libration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering allo Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys The above relation implies that The above relation implies that The above relation implies that   2 1 s D D I N     (6) where   2 1 s D D I N       2 1 s D D I N     (6) where 2 eq cycle d 2 s R I p ES             2 eq cycle d 2 s R I p ES             (7) (7) (7) The estimated number of cycles to failure, est N , is determined by integrating Eq. (6) over the whole loading history. If the stress‒strain response becomes stabilized well before the total number of cycles to failure, the value of I can be regarded as a constant. Thus,   c est 2 est 0 0 1 D N s D D I N IN        (8)   c est 2 est 0 0 1 D N s D D I N IN        (8) In view of (8), the following formula for fatigue life prediction can be obtained:   1 2 1 1 s c D N    (9) In view of (8), the following formula for fatigue life prediction can be obtained:     1 2 est 1 1 1 2 s c D N s I      (9)     1 2 est 1 1 1 2 s c D N s I          1 2 est 1 1 1 2 s c D N s I      (9) (9) Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 2.1 Constant amplitude loading 2.1 Constant amplitude loading The Lemaitre model is applicable to general multiaxial loading. For any loading history, the occurrence of fatigue failure can be predicted by numerical integration of Eq. (1) until a critical value. For constant amplitude loading, if a stabilized stress‒strain response can be identified well before the total number of cycles to failure, a convenient expression for fatigue life prediction can be derived as follows. The fatigue damage increment per loading cycle is given by cycle d D D t N    (4) cycle d D D t N    (4) cycle d D D t N    (4) where, as usual in fatigue damage analysis, the variation of damage with the number of loading cycles is regarded as continuous, and the prefix  denotes an infinitesimal. Inserting (1) and (2) in (4), and neglecting the variation of D over a cycle, it follows that   2 eq 2 cycle 1 d 2 1 s s R D p N ES D                (5)   2 eq 2 cycle 1 d 2 1 s s R D p N ES D                (5)   2 eq 2 cycle 1 d 2 1 s s R D p N ES D                (5) Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 2/13 Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial torsion fatigue tests on five engineering alloys bio Castro et al. libration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering allo Fábio Castro et al. Castro et al. ation and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Fábio Castro et al. 3 CYCLIC PLASTICITY MODEL Uncoupling between the elastic-plastic constitutive behavior and fatigue damage is assumed in this paper. Within this framework, the cyclic stress and strain histories at the critical location of the engineering component are first determined by using, for example, a finite element model. Fatigue crack initiation life is then predicted by post-processing the stress-strain data using a fatigue damage model. The stress-strain analysis was performed in this work by using the cyclic plasticity model developed by Jiang and Sehitoglu (1996a). The basic constitutive relations of the model are summarized in Table 1. The model was chosen due to its reasonable predictions of the stress and strain responses for general multiaxial loading, as demonstrated in previous studies (Jiang and Kurath, 1996, 1997; Socie, 1998). Strain-controlled axial fatigue testing with solid cylindrical specimens and strain-controlled axial-torsion fatigue testing with thin-walled tubular specimens were evaluated in this work. For such experimental configurations, the axial and/or shear stress distributions can be assumed uniformly distributed over the cross section of the specimen. By taking advantage of this feature, the numerical implementation of the cyclic plasticity model was achieved by developing a MATLAB script that simulates the stress response of a material point under prescribed axial and shear strains. The stress update algorithm was based on an explicit integration scheme. 3/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. C lib ti d l ti f th L it d d l i i l t i f ti t t fi i i ll Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Fábio Castro et al. Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys bio Castro et al. libration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering allo Table 1: Summary of the Jiang‒Sehitoglu cyclic plasticity model. 3 CYCLIC PLASTICITY MODEL 1) Yield function 2 2 2 0 f k     S   α 2) Flow rule 1 p h    S n n  ε 3) Hardening rule  1 M i i  α α          1 1, 2, ..., i i i i i i i i c r p i M r                      n       α α α α S = deviatoric stress, α = backstress, k = yield stress in shear, n = normal on the yield surface, h = plastic modulus function, p ε = plastic strain, i α = i th backstress part, M = number of backstress parts, p = equivalent plastic strain rate, and i c ,  i r , i  = material constants. S = deviatoric stress, α = backstress, k = yield stress in shear, n = normal on the yield surface, h = plastic modulus function, p ε = plastic strain, i α = i th backstress part, M = number of backstress parts, p = equivalent plastic strain rate, and i c ,  i r , i  = material constants. S = deviatoric stress, α = backstress, k = yield stress in shear, n = normal on the yield surface, h = plastic modulus function, p ε = plastic strain, i α = i th backstress part, M = number of backstress parts, p = equivalent plastic strain rate, and i c ,  i r , i  = material constants. 4 RESULTS AND DISCUSSION ation and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Fatigue life prediction by the Lemaitre model requires as input the stress–strain hysteresis loops at the critical material point. For the AZ61A and AZ31B Mg alloys, the stress–strain loops obtained directly from the fatigue tests were available to the authors. On the other hand, for the 1045 steel, 16MnR steel, and 7075-T651 Al alloy, the stresses and strains were simulated by using the Jiang–Sehitoglu plasticity model. The material constants of the model were determined from the uniaxial cyclic stress-strain curve by using the method described by Jiang and Sehitoglu (1996b). The format of the cyclic stress-strain curve was described by the Ramberg‒Osgood relationship. Table 2 lists the material constants used in this study for the Jiang‒Sehitoglu model. Table 2: Material constants used in the Jiang‒Sehitoglu plasticity model. 4 RESULTS AND DISCUSSION 1045 steel E = 204 GPa,  = 0.27, k = 50 MPa 1 c = 12583.2,  2 c = 1859.5, 3 c = 475.2,  4 c = 164.4,  5 c = 68.9 1 r = 55.8,  2 r = 56.0, 3 r = 55.0,  4 r = 54.5, 5 r = 116.3 1  = 2  =  = 5  = 5 16MnR steel E = 212.5 GPa,  = 0.31, k = 50 MPa 1 c = 18373.8,  2 c = 1721.7, 3 c = 325.3,  4 c = 89.8,  5 c = 31.5 1 r = 67.8,  2 r = 65.9, 3 r = 64.4,  4 r = 63.7, 5 r = 116.5 1  = 2  =  = 5  = 5 7075-T651 Al alloy E = 71.7 GPa,  = 0.3, k = 100 MPa 1 c = 651313.5,  2 c = 121390.2, 3 c = 29596.0,  4 c = 8764.4,  5 c = 3007.4  6 c = 1158.2,  7 c = 489.5,  8 c = 223.3,  9 c = 108.6,   10 c = 55.8 1 r = 28.8,  2 r = 33.9, 3 r = 33.7,  4 r = 33.6, 5 r = 33.5 6 r = 33.4,  7 r = 33.4, 8 r = 33.3, 9 r = 33.3,   10 r = 76.8 1  = 2  =  =   10  = 10 Table 2: Material constants used in the Jiang‒Sehitoglu plasticity model. For all the fatigue experiments, the number of cycles to failure was estimated by using Eq. (9). To obtain the stabilized stresses and strains for the 1045 steel, 16MnR steel, and 7075-T651 Al alloy, elastic-plastic simulations were carried out for 5 loading cycles, and the results at the 5th loading cycle were used to determine the fatigue life. For the AZ61A and AZ31B Mg alloys, the stress-strain hysteresis loops taken from the tests at approximately half the observed fatigue lives were used in Eq. (9). The constants of the Lemaitre model were determined by a least-squares fitting of the tension‒compression and torsion data, as described in Section 2.2, and are listed in Table 3. Table 3: Material constants used in the Lemaitre damage model. 4 RESULTS AND DISCUSSION The Lemaitre model was evaluated using axial-torsion fatigue test data available in the literature (Fatemi and Stephens, 1989a; Zhao and Jiang, 2008; Gao et al., 2009; Yu et al., 2011; Xiong et al., 2012). Five engineering materials were investigated: normalized 1045 steel, 16MnR steel, 7075-T651 Al alloy, extruded AZ61A Mg alloy, and extruded AZ31B Mg alloy. 1045 steel is a medium carbon steel that is widely used in the ground vehicle industry. 16MnR steel (also designated Q345R steel and similar to ASME SA302B) is usually applied to manufacture pressure vessels. 7075 Al alloys are commonly used in aircraft structures. Wrought magnesium alloys are potential candidates for structural applications in transportation and aerospace industries due to their high strength-to- weight ratio (Bettles and Barnett, 2012). The loading paths used in the tests are shown in Fig. 1, where  is the axial strain and  is the shear strain. Fully reversed tension‒compression, fully reversed torsion, proportional (in-phase) loading, and nonproportional (90º out-of-phase) loading were investigated. The test data for 1045 steel were obtained by testing thin-walled tubular specimens (Fatemi and Stephens, 1989a). The experiments on 16MnR steel and 7075-T651 Al alloy were performed by Gao et al. (2009) and Zhao and Jiang (2008), respectively. For these materials, two types of specimens were tested: solid cylindrical specimens for uniaxial loading and thin-walled tubular specimens for torsion and axial-torsion. For the AZ61A and AZ31B Mg alloys, thin-walled tubular specimens were used for all tests (Yu et al., 2011; Xiong et al., 2012). Further details of the fatigue tests used in this work, including the test setup, specimen dimensions, and cracking orientations, can be found in the corresponding references. g p g Failure was defined by a load drop criterion in most of the experiments (5% load drop for the 16MnR steel, AZ61A Mg alloy, and AZ31B Mg alloy, and 10% load drop for the 1045 steel and 7075-T651 Al alloy), the exception being the uniaxial solid specimens of 7075-T651 Al alloy, which were tested to complete fracture. For the materials, loading conditions, and specimen geometries studied, the crack propagation phase was expected to be a small part of the total life. Figure 1: Loading paths used in the fatigue tests. Figure 1: Loading paths used in the fatigue tests. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 4/13 Fábio Castro et al. 4 RESULTS AND DISCUSSION Material S (MPa) s c D 1045 steel 2.1 2.8 0.4 16MnR steel 2.0 2.7 0.8 AZ61A Mg alloy 2.2 1.8 0.1 AZ31B Mg alloy 1.7 2.1 0.1 7075-T651 Al alloy 14.0 0.8 1.0 Table 3: Material constants used in the Lemaitre damage model. Observed fatigue lives and estimates made with the Lemaitre model are compared in Figs. 2-4 for the five materials investigated. A data point with a horizontal arrow denotes a run-out test. The solid diagonal lines in these figures represent a perfect correlation, the two dashed lines mark the factor-of-two boundaries in Figs. 2 and 3, and the two dot lines in Fig. 4 are the factor-of-five boundaries. Observed fatigue lives and estimates made with the Lemaitre model are compared in Figs. 2-4 for the five materials investigated. A data point with a horizontal arrow denotes a run-out test. The solid diagonal lines in these figures represent a perfect correlation, the two dashed lines mark the factor-of-two boundaries in Figs. 2 and 3, and the two dot lines in Fig. 4 are the factor-of-five boundaries. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 5/13 Fábio Castro et al. Fábio Castro et al. Castro et al. ation and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Most of the fatigue life estimates by the Lemaitre model for the 1045 steel and the 16MnR steel were within the factor-of-two boundaries, indicating that the overall accuracy of the model is excellent. Such accuracy is similar to those obtained by fatigue models based on the critical plane approach (Fatemi and Socie, 1988; Gao et al., 2009). One factor that contributed to such successful fatigue life predictions is the capability of the Jiang‒Sehitoglu plasticity model to provide reasonable cyclic stresses and strains. Tables 4 and 5 summarize the accuracy of the elastic-plastic stress-strain analysis carried out for the proportional and nonproportional (90º out-of-phase) axial- torsion tests. Figure 2: Observed live versus estimated live for 1045 steel (a) and 16MnR steel (b). Figure 2: Observed live versus estimated live for 1045 steel (a) and 16MnR steel (b). Figure 3: Observed live versus estimated live for AZ61 Mg alloy (a) and AZ31B Mg alloy(b). Figure 3: Observed live versus estimated live for AZ61 Mg alloy (a) and AZ31B Mg alloy(b). Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 4 RESULTS AND DISCUSSION 6/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Fábio Castro et al. o et al. n and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Figure 4: Observed live versus estimated live for 7075-T651 Al alloy. Figure 4: Observed live versus estimated live for 7075-T651 Al alloy. In these tables, the symbols exp Δ and exp Δ refer to the axial and shear stress ranges of the half-life (or near half-life) stress-strain hysteresis loop obtained from the fatigue experiments, while the symbols num Δ and num Δ are the corresponding quantities calculated from numerical simulations using the Jiang-Sehitoglu plasticity model. The results shown in Tables 4 and 5 indicate that the correlation between the experimentally obtained and the simulated stress ranges are better for the proportional than for the nonproportional tests. This is because the adopted plasticity model does not consider the nonproportional hardening effect. However, this effect is not very significant for the two steels studied (Fatemi and Stephens, 1989b; Jiang and Kurath, 1996; Gao et al., 2009), and therefore the overall accuracy of the estimated fatigue lives is good. For the nonproportional tests with fatigue lives less than 104 cycles, it can also be noticed that the life estimates for the 1045 steel are better than those obtained for the 16MnR steel (4 data points above the upper factor-of-two boundary). This is due to a better agreement between the experimentally obtained and the simulated stress ranges for the 1045 steel. 7/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 bio Castro et al. libration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering allo Fábio Castro et al. Castro et al. ation and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Table 4: Difference between stabilized stress ranges obtained from fatigue experiments on 1045 steel and from numerical simulations using the Jiang-Sehitoglu plasticity model. 4 RESULTS AND DISCUSSION Loading type Specimen ID exp num exp Δ Δ Δ     (%) exp num exp Δ Δ Δ     (%) In-phase axial-torsion 4524 0 5 4523 0 6 4528 -2 3 4516 0 8 4519 0 7 4525 0 -3 4520 1 -4 4515 2 -1 4550 0 4 4514 2 2 4554 1 0 4517 2 3 4526 3 -3 4503 7 -3 4501 6 -3 4522 3 -2 4548 3 -2 4521 0 -1 4509 9 -5 4530 4 -3 90º out-of- phase axial- torsion 45A3 8 18 4583 5 4 4580 18 18 45B2 14 18 4586 13 16 45A5 8 6 45D4 21 14 45D1 13 14 45B5 20 13 4588 8 5 Table 4: Difference between stabilized stress ranges obtained from fatigue experiments on 1045 steel and from numerical simulations using the Jiang-Sehitoglu plasticity model. ence between stabilized stress ranges obtained from fatigue experiments on 1045 steel and from numerical simulations using the Jiang-Sehitoglu plasticity model. Although the Lemaitre model can provide reasonable fatigue life predictions for the AZ61A and AZ31B Mg alloy, its performance is not as good as that for the two steels studied. It is found that the model yields somewhat non-conservative life estimates for the proportional and nonproportional tests. Also, the model has a difficult to correlate the torsion tests. It is noted that for the tension‒compression and torsion tests, the triaxiality function is a constant and, therefore, the fatigue damage in the Lemaitre model depends only on the von Mises equivalent measures of stress and plastic strain. Thus, the significant difference between the cyclic stress-plastic strain curves of extruded Mg alloys under tension‒compression and torsion (Zhang et al., 2011) may have contributed to the less satisfying life predictions for the torsion tests. 8/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. Castro et al. ation and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Table 5: Difference between stabilized stress ranges obtained from fatigue experiments on 16MnR numerical simulations using the Jiang-Sehitoglu plasticity model. 5 FURTHER DISCUSSION The Lemaitre model was used in a previous work by Lopes and Malcher (2017) to estimate fatigue life under axial-torsion loading conditions. The golden-section search was adopted by these authors to determine the damage strength material parameter, S , while maintaining fixed the values for c D and s ( c D = 1, s = 1). In the current work, the three constants ( S , s, c D ) were determined by simply performing an exhaustive search over a range of candidate constants. No pre-defined values for c D and s were assumed. Based on the experience gained from the five engineering metals studied, the fatigue constants in the Lemaitre model are generally within the ranges 0 ≤ S ≤ 14, 0 ≤ s ≤ 3, and 0 ≤ c D ≤ 1. It is also worth mentioning that the evaluation of the fatigue life by Eq. (9) eliminates the need to numerically integrate the damage evolution rule until a critical value, and, hence, the determination of the material constants by an exhaustive search can be performed quickly on a computer. Another advantage of Eq. (9) is that it can be applied to stress‒strain hysteresis loops of any shape (9) is that it can be applied to stress‒strain hysteresis loops of any shape. ( ) In the fatigue damage analysis performed in this work, no coupling between the stress-strain behavior and the damage was considered. This study and many others (see, e.g., Socie, 1998; Gao et al., 2010) indicate that reasonable fatigue life predictions of conventional metallic materials can be made using an uncoupled procedure if an accurate plasticity model is employed. On the other hand, for materials such as porous sintered metals (Ma and Yuan, 2017) and concrete or for metallic materials under large plastic strains, a coupled analysis is required because of the strong coupling between the stress-strain behavior and the damage in the material. The material constants of a multiaxial fatigue model are often determined by using test data from tension‒ compression and torsion tests. Alternatively, the material constants in the Lemaitre model were also determined in the present work by fitting only the tension‒compression data. It was found that the fatigue life predictions for the five materials investigated were not significantly altered as compared to the fatigue life estimates shown in Figs. 2-4. 4 RESULTS AND DISCUSSION Loading type Specimen ID exp num exp Δ Δ Δ     (%) exp num exp Δ Δ Δ     (%) In-phase axial-torsion TU20N 6 6 TU21N 4 2 TU09N 13 19 TU15N 17 21 TU06S 22 23 TU09S 23 23 90º out-of- phase axial- torsion TU07S 32 32 TU08N 31 29 TU06N 28 26 TU03N 30 28 TU14N 29 32 between stabilized stress ranges obtained from fatigue experiments on 16MnR steel and from numerical simulations using the Jiang-Sehitoglu plasticity model. Observed life versus predicted life for the 7075-T651 Al alloy is shown in Fig. 4. Most of the fatigue life estimates are within the factor-of-five boundaries. The overall accuracy of the Lemaitre model for the 7075-T651 Al alloy is comparable to the scatter of the baseline fatigue data obtained from tension‒compression tests (Zhao and Jiang, 2008). Plastic strains in Al alloys are typically very small for fatigue lives higher than 104 cycles and, therefore, high-cycle fatigue life prediction is a difficulty for any fatigue model that incorporates plastic strains. To enable the Lemaitre model to be applicable to high-cycle fatigue life prediction of Al alloy, a small yield stress close to the endurance limit under uniaxial loading (Jiang and Kurath, 1996) was used to simulate the small cyclic plastic strains in the material. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 5 FURTHER DISCUSSION Such feature of the Lemaitre model may be useful when only uniaxial fatigue data are available to the design engineer. A glance at Figs. 2 and 4 shows that the Lemaitre model can correlate well the torsion data of the two steels and of the Al alloy, while the accuracy of the life estimates for the torsion tests on Mg alloys is not so good. An Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 9/13 Fábio Castro et al. o et al. n and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys attempt to improve the life estimates for such torsion data was carried out by relating the damage strength material parameter, S , to the stress triaxiality using a linear relationship,    0.33 0 0 3 S S S S      (11)    0.33 0 0 3 S S S S      (11) where 0 S and 0.33 S are material constants. For cyclic torsion  = 0 and S = 0 S , whereas for tension‒compression  = 0.33 and S = 0.33 S . To determine the four material constants ( 0 S , 0.33 S , s, c D ) in the modified Lemaitre model, the method described in Section 2.2 was applied to a dataset obtained from tension‒compression and torsion tests. Note that in the numerical computation of the integral that defines the value of I, Eq. (7), the expression for  S  in Eq. (11) must be adopted. Figure 5 shows the fatigue lives predicted by the modified where 0 S and 0.33 S are material constants. For cyclic torsion  = 0 and S = 0 S , whereas for tension‒compression  = 0.33 and S = 0.33 S . To determine the four material constants ( 0 S , 0.33 S , s, c D ) in the modified Lemaitre model, the method described in Section 2.2 was applied to a dataset obtained from tension‒compression and torsion tests. Note that in the numerical computation of the integral that defines the value of I, Eq. (7), the expression for  S  in Eq. (11) must be adopted. Figure 5 shows the fatigue lives predicted by the modified Lemaitre model versus the observed fatigue lives for the AZ31B Mg alloy. 5 FURTHER DISCUSSION A significant improvement in the correlation of the torsion data was obtained as compared to the estimates by the original model (refer to Fig. 3b). The overall accuracy of the life predictions for the proportional and nonproportional tests was maintained, except for the nonproportional test with observed life f N = 35 cycles. For AZ61A Mg alloy, the fatigue life estimates based on the original Lemaitre model and its modified version were identical. The use of a damage strength material parameter as function of the stress triaxiality, Eq. (11), to improve the correlation of tests with different levels of stress triaxiality was inspired by the work of Malcher and Mamiya (2014), in which a modification of the damage strength material parameter yielded better results for ductile fracture prediction. Such a modification of the original Lemaitre model requires an appropriate generalization of the damage potential function. Following the standard formulation of continuum elastic-plastic damage mechanics (Lemaitre and Desmorat, 2005), the damage potential function can be obtained by integrating the damage evolution law with respect to the thermodynamic force associated to the damage, Y . This gives the following expression for the damage potential function:      1 D 1 1 s S Y D s S                (12)      1 D 1 1 s S Y D s S                     1 D 1 1 s S Y D s S                (12) where the function  S  is defined by Eq. (11). where the function  S  is defined by Eq. (11). Indeed, for any axial-torsion stress state, the stress triaxiality lies in the range 0 1/3    because whenever 0  the denominator of the expression for the stress triaxiality, 2 2 1/ 3 / 3       , is always equal or greater than . Since the constants of the modified Lemaitre model were determined by using fatigue data with 0  (torsion tests) and 1/ 3  (tension‒compression tests), this may have contributed to the good life estimates. Fatigue experiments covering a wide range of stress triaxiality are needed to better evaluate the modified Lemaitre model. where the function  S  is defined by Eq. (11). For constant amplitude loading and when the stabilized stress–strain response is reached well before the total number of cycles to failure, the fatigue life estimated by the Lemaitre model can be calculated by using Eq. (9). There are cases, however, where the stress response of the material never becomes stabilized. For example, the 1045 steel under high strain amplitude experiences hardening followed by a mild softening for a short period and then a second hardening stage until failure (Fatemi and Stephens, 1989b). Fatigue life prediction based on Eq. (9) can still be used in such cases by taking the stress–strain hysteresis loop at a reference number of cycles (for example, at half of the fatigue life). The results in the present study indicate that such an engineering solution can provide fatigue life estimates with acceptable accuracy. For more general loadings, the variation of the fatigue damage with time (or loading cycle) can be calculated by numerically integrating Eq. (1) or Eq. (5). 10/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. Fábio Castro et al. C lib ti d l ti f th L it d d l i i l t i f ti t t fi i i ll Fábio Castro et al. Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys o et al. n and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Figure 5: Observed live versus estimated live based on the modified Lemaitre model for AZ31B Mg alloy. Figure 5: Observed live versus estimated live based on the modified Lemaitre model for AZ31B M The limited stress states that were employed to evaluate the Lemaitre model may have contributed to its goo correlation of the fatigue lives. Thin-walled tubular specimens under axial-torsion loading inherently experien limited values of stress triaxiality. Indeed, for any axial-torsion stress state, the stress triaxiality lies in the ran The limited stress states that were employed to evaluate the Lemaitre model may have contributed to its good correlation of the fatigue lives. Thin-walled tubular specimens under axial-torsion loading inherently experience limited values of stress triaxiality. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 6 CONCLUSIONS The Lemaitre damage model was experimentally evaluated using axial-torsion fatigue data of five engineering metals. For 1045 steel, 16MnR steel, and 7075-T651 Al alloy, the Lemaitre model provided life predictions with an accuracy comparable to the scatter of the baseline fatigue tests conducted under tension‒compression and torsion (within a factor of 2 for the two steels and within a factor of 5 for the Al alloy). Reasonably accurate fatigue life estimates were also obtained for the AZ61A Mg alloy and the AZ31B Mg alloy, but the model was not able to correlate well some of the torsion tests. A significant improvement in the correlation of the torsion data was 11/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys o et al. n and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys achieved by a modified Lemaitre model in which the damage strength material parameter depends on the stress triaxiality. achieved by a modified Lemaitre model in which the damage strength material parameter depends on the stress triaxiality. Acknowledgments g The authors would like to thank the support provided by CNPq (contracts 308126/2016-5 and 131847/2017- 1). References Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Jiang, Y., Kurath, P., (1996). Characteristics of the Armstrong-Frederick type plasticity models. International Journal of Plasticity, 12(3): 387–415. Jiang, Y., Kurath, P., (1997). Nonproportional cyclic deformation: critical experiments and analytical modeling. International Journal of Plasticity, 13: 743–763. Jiang, Y., Ott, W., Baum, C., Vormwald, M., Nowack, H., (2009). Fatigue life predictions by integrating EVICD fatigue damage model and an advanced cyclic plasticity theory. International Journal of Plasticity, 25: 780–801. Jiang, Y., Sehitoglu, H., (1996a). Modeling of cyclic ratchetting plasticity, Part I: development of constitutive equations. ASME Journal of Applied Mechanics, 63: 720–725. Jiang, Y., Sehitoglu, H., (1996b). Modeling of cyclic ratchetting plasticity, Part II: comparison of model simulations with experiments. ASME Journal of Applied Mechanics, 63: 726–733. Lemaitre, J., (1985). Coupled elasto-plasticity and damage constitutive equations. Computer Methods in Applied Mechanics and Engineering 51:31–49. Lemaitre, J., Desmorat, R., (2005). Engineering Damage Mechanics: Ductile, Creep, Fatigue and Brittle Failures. Springer Science & Business Media. Lopes, J. P., Malcher, L., (2017). Fatigue life estimates under non-proportional loading through continuum damage evolution law. Theoretical and Applied Fracture Mechanics, 88: 64–73. Ma, S., Yuan H., (2017). A continuum damage model for multi-axial low cycle fatigue of porous sintered metals based on the critical plane concept. Mechanics of Materials, 104: 13–25. Malcher, L., Mamiya E. N., (2014). An improved damage evolution law based on continuum damage mechanics and its dependence on both stress triaxiality and the third invariant. International Journal of Plasticity, 56: 232–261. Sharifi, S. M. H., Googarchin, H. S., Forouzesh, F., (2016). Three dimensional analysis of low cycle fatigue failure in engine part subjected to multi-axial variable amplitude thermomechanical load. Engineering Failure Analysis, 62: 128–141. Shen, F., Voyiadjis, G. Z., Hu, W., Meng, Q., (2015). Analysis on the fatigue damage evolution of notched specimens with consideration of cyclic plasticity. Fatigue & Fracture of Engineering Materials and Structures, 38: 1194–1208. Socie, D. F., (1998). An evaluation of methods for estimating fatigue damage under multiaxial nonproportional variable amplitude loading. In: Low Cycle Fatigue and Elasto-Plastic Behavior of Materials, Eds. K.T. Rie and P.D. Portella, 205–210. Socie, D. F., Marquis, G. B., (2000). Multiaxial Fatigue, SAE International, Warrendale. Xiong, Y., Yu, Q., Jiang, Y., (2012). Multiaxial fatigue of extruded AZ31B magnesium alloy. Materials Science and Engineering: A, 546: 119–128. Yu, Q., Zhang, J., Jiang, Y., Li, Q., (2011). References Bettles, C., Barnett, M., (Eds.) (2012). Advances in wrought magnesium alloys: Fundamentals of processing, properties and applications. Publisher: Woodhead Publishing. Bonora, N., Newaz, G. M., (1998). Low cycle fatigue life estimation for ductile metals using a nonlinear continuum damage mechanics model. International Journal of Solids and Structures, 35(6): 1881–1894. Castro, F., Jiang, Y., (2016). Fatigue life and early cracking predictions of extruded AZ31B magnesium alloy using critical plane approaches. International Journal of Fatigue, 88: 236–246. Castro, F., Jiang, Y., (2017). Fatigue of extruded AZ31B magnesium alloy under stress- and strain-controlled conditions including step loading. Mechanics of Materials, 108: 77–86. Chow, C. L., Wei, Y., (1991). A model of continuum damage mechanics for fatigue failure. International Journal of Fracture, 50: 301–316. Fatemi, A., Shamsaei, N., (2011). Multiaxial fatigue: An overview and some approximation models for life estimation. International Journal of Fatigue, 33: 948–958. Fatemi, A., Socie, D., (1988). A critical plane approach to multiaxial fatigue damage including out-of-phase loading. Fatigue & Fracture of Engineering Materials and Structures, 11(3): 149–165. Fatemi, A., Stephens, R. I., (1989a). Biaxial fatigue of 1045 steel under in-phase and 90 deg out-of-phase loading conditions, Multiaxial Fatigue: Analysis and Experiments, Chapter 9, SAE AE-14, 121–137. Fatemi, A., Stephens, R. I., (1989b). Cyclic deformation of 1045 steel under in-phase and 90 deg out-of-phase axial- torsional loading conditions, Multiaxial Fatigue: Analysis and Experiments, Chapter 10, SAE AE-14, 139–147. Gao, Z., Zhao, T., Wang, X., Jiang, Y., (2009). Multiaxial fatigue of 16MnR steel. Journal of Pressure Vessel Technology, 131(2): 021403-1–021403-9. Gao, Z., Qiu, B., Wang, X., Jiang, Y., (2010). An investigation of fatigue of a notched member. International Journal of Fatigue, 32: 1960–1969. Googarchin, H. S., Sharifi, S. M. H., Forouzesh, F., Hosseinpourd, G. H. R., Etesamia, S. M., Malek Zadea, S. (2017). Comparative study on the fatigue criteria for the prediction of failure in engine structure. Engineering Failure Analysis, 79: 714–725. Jiang, Y., (2000). A fatigue criterion for general multiaxial loading. Fatigue & Fracture of Engineering Materials and Structures, 23: 19–32. Jiang, Y., Hertel, O., Vormwald, M., (2007). An experimental evaluation of three critical plane multiaxial fatigue criteria. International Journal of Fatigue, 29: 1490–1502. 12/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Fábio Castro et al. Calibration and evaluation of the Lemaitre damage model using axial-torsion fatigue tests on five engineering alloys Fábio Castro et al. C l b d Fábio Castro et al. References Multiaxial fatigue of extruded AZ61A magnesium alloy. International Journal of Fatigue, 33(3): 437–447. Zhang, J., Yu, Q., Jiang, Y., Li, Q., (2011). An experimental study of cyclic deformation of extruded AZ61A magnesium alloy. International Journal of Plasticity, 27: 768–447. Zhao, T., Jiang, Y., (2008). Fatigue of 7075-T651 aluminum alloy. International Journal of Fatigue, 30: 834–849. Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 13/13 13/13 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62 Latin American Journal of Solids and Structures, 2018, 15(10 Thematic Section), e62
https://openalex.org/W4210733435	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/77A6DE2E66BF22E04317E433EB6D963C/S0955603600012101a.pdf/div-class-title-nhs-health-advisory-service-div.pdf	English	null	NHS Health Advisory Service	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	1,989	cc-by	586	NHS Health Advisory Service The Legal Practice Directorate of the Law Society is currently up-dating its register of expert witnesses who can offer assistance to solicitors in litigation. The NHS Health Advisory Service will shortly be appointing team members for its 1989-1990 pro gramme of visits to services for elderly people and for mentally ill people. g Any members who wish to assist should contact the Information Officer, Edwina Dunne, at the Law Society, 12Chancery Lane, London WC2A 1PL and should enclose the following details: Any members who wish to assist should contact the Information Officer, Edwina Dunne, at the Law Society, 12Chancery Lane, London WC2A 1PL and should enclose the following details: y p p The Director, Professor Philip Seager, would wel come applications from consultant psychiatrists, who arc not already on the Health Advisory Service's panel, to be seconded to teams. Assignments are of three to five weeks' duration but applicants should also state if they would be interested in a continuous three or six months' secondment. Employing Auth orities are reimbursed by the Department of Health for the salaries and expenses of those undertaking assignments. (1) name g (1) name ( ) (2) background and experience ( ) g p (3) services offered (4) illi t d t k di ( ) g (3) services offered (4) illi t (4) willingness to undertake medico-legal work (including negligence) as appropriate. (4) willingness to undertake medico-legal work (including negligence) as appropriate. g The ad hoc multidisciplinary teams (comprising a consultant psychiatrist or geriatrician, a general manager, and managers of nursing, occupational therapy or physiotherapy, and social services) will be visiting Health Districts throughout England and Wales to help the relevant Authorities to assess and improve their servicesfor the client groups concerned, and in particular to advise on the better management of resources and the ways in which the services might be changed or developed in the future. Teams bring their own experience of similar working situations to bear in a form of peer review; visits have proved themselves helpful educational experiences for team members. The final week of each assignment is spent at the Health Advisory Service's headquarters at Sutton writing the report, which is subsequently sub mitted to the Secretary of State and becomes a public document. Diploma in Psychotherapy (University of Liverpool) Applications are invited for the above multidisciplin ary course in psychotherapy which starts its ninth annual intake in October 1989. The course is part- time, extending over two years, and leads to the University qualification of Diploma in Psycho therapy. Application forms and further details are available from: The Secretary, Sub Department of Clinical Psychology, New Medical School, Ashton Street, PO Box 147, Liverpool L69 3BX. Completed application forms should be returned by 28 April Â¡989. https://doi.org/10.1192/pb.13.2.110 Published online by Cambridge University Press 110 110 Miscellany Register of expert witnesses NHS Health Advisory Service Back issues of the Journal Dr John Pippard, Elizabeth House, 9 Princes Avenue, Woodford Green, Essex IG8 OLL, oners runs of back issues of the Journal of Mental Science from 1955-1962 and TheBritish Journal of Psychiatry from 1963-1984. Dr John Pippard, Elizabeth House, 9 Princes Avenue, Woodford Green, Essex IG8 OLL, oners runs of back issues of the Journal of Mental Science from 1955-1962 and TheBritish Journal of Psychiatry from 1963-1984. Applications, accompanied by a curriculum vitae, should be sent as soon as possible to: The Director, NHS Health Advisory Service, Sutherland House, 29-37 Brighton Road, Sutton, Surrey SM2 5AN. https://doi.org/10.1192/pb.13.2.110 Published online by Cambridge University Press
https://openalex.org/W2802638752	https://repositoriosaludmadrid.es//bitstream/20.500.12530/37144/1/PMC5892014.pdf	English	null	Endoscopic submucosal injection of adipose-derived mesenchymal stem cells ameliorates TNBS-induced colitis in rats and prevents stenosis	Stem cell research & therapy	2,018	cc-by	10,763	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: emarranz@salud.madrid.org Conference presentation: this work was partially presented at UEGW 2013 in Berlin and IFATS 2013 11th conference in New York. †Equal contributors 1Gastroenterology Department, La Paz University Hospital, Paseo de la Castellana 261 4th floor, 28046 Madrid, Spain Full list of author information is available at the end of the article Abstract Background: Mesenchymal stem cells have potential applications in inflammatory bowel disease due to their immunomodulatory properties. Our aim was to evaluate the feasibility, safety and efficacy of endoscopic administration of adipose-derived mesenchymal stem cells (ASCs) in a colitis model in rats. Methods: Colitis was induced in rats by rectal trinitrobenzenesulfonic acid (TNBS). After 24 h ASCs (107 cells) or saline vehicle were endoscopically injected into the distal colon. Rats were followed for 11 days. Daily weight, endoscopic score at days 1 and 11, macroscopic appearance at necropsy, colon length and mRNA expression of Foxp3 and IL-10 in mesenteric lymph nodes (MLN) were analyzed. Results: Endoscopic injection was successful in all the animals. No significant adverse events or mortality due to the procedure occurred. Weight evolution was significantly better in the ASC group, recovering initial weight by day 11 (−0.8% ± 10.1%, mean ± SD), whereas the vehicle group remained in weight loss (−6.7% ± 9.2%, p = 0.024). The endoscopic score improved in the ASC group by 47.1% ± 5.3% vs. 21.8% ± 6.6% in the vehicle group (p < 0.01). Stenosis was less frequent in the ASC group (4.8% vs. 41.2%, p < 0.01). Colon length significantly recovered in the ASC group versus the vehicle group (222.6 ± 17.3 mm vs. 193.6 ± 17.9 mm, p < 0.001). The endoscopic score significantly correlated with weight change, macroscopic necropsy score and colon length. Foxp3 and IL-10 mRNA levels in MLN recovered with ASC treatment. Conclusions: ASC submucosal endoscopic injection is feasible, safe and ameliorates TNBS-induced colitis in rats, especially stenosis. Keywords: Inflammatory bowel disease, Cell therapy, Mesenchymal stem cells, Endoscopic treatment Endoscopic submucosal injection of adipose-derived mesenchymal stem cells ameliorates TNBS-induced colitis in rats and prevents stenosis Eduardo Martín Arranz1, María Dolores Martín Arranz1, Tomás Robredo2, Pablo Mancheño-Corvo3, Ramón Menta3, Francisco Javier Alves4, Jose Manuel Suárez de Parga1, Pedro Mora Sanz1, Olga de la Rosa3, Dirk Büscher5, Eleuterio Lombardo3† and Fernando de Miguel2† Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 https://doi.org/10.1186/s13287-018-0837-x Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 https://doi.org/10.1186/s13287-018-0837-x Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 https://doi.org/10.1186/s13287-018-0837-x Open Access Background dysregulation of the innate and adaptive immune system, genetic influences and environmental factors are suggested to intervene in the development of the disease. With current treatments, there is still a substantial proportion of patients in whom remission cannot be achieved, leaving unmet needs in the treatment of IBD and leading to the emergence of new treatments [1]. Inflammatory bowel disease (IBD) includes Crohn’s disease (CD) and ulcerative colitis (UC); it is characterized by chronic and relapsing inflammation of the intestinal tract. Its pathogenesis is not completely understood, but a Mesenchymal stem cells (MSCs) are cells that can be isolated from several adult tissues, including bone mar- row (BM-MSCs) and adipose tissue (ASCs) [2]. Adult MSCs have been proposed as a potential treatment for Page 2 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Although promising, MSC therapy for IBD still raises some questions, such as the best route of administration to optimize bioavailability in the damaged areas. Con- flicting results have been reported to date in terms of intravenous efficacy, and local approaches used in ani- mal experiments, such as intraperitoneal inoculation or colon injection through laparotomy, are impractical for human use [36]. In this study, we propose a new admin- istration route for IBD cell therapy: injecting cells into the colon submucosa using endoscopy, allowing direct cell delivery to the damaged area, while enabling the evaluation of disease severity and evolution. several diseases, including immune-based treatments, due to their multilineage differentiation capabilities that could allow MSCs to repair damaged tissues [1, 3] and their capacity to modulate the function of the majority of immune cells [2, 4]. This treatment could promote the regulation of the inflammatory cascade by inducing tol- erogenic phenotypes in antigen-presenting cells (APCs) [5] while inhibiting the proliferation of cytotoxic T-cells and promoting differentiation toward regulatory phenotypes in lymphocytes (regulatory T cells) and macrophages (M2 macrophages) [6]. The immunomodulatory properties of MSCs rely not only on cell-to-cell interactions but also on effects mediated by a variety of soluble factors [7–9]. The aim of this study was to evaluate the feasibility, safety and efficacy of submucosal endoscopic injection of human ASCs in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model in rats, and the utility of endoscopy to evaluate and follow the course and severity of the disease over time. Background MSCs are considered to have low immunogenicity due to a low expression of major histocompatibility complex (MHC)-I and an absence of MHC-II and clas- sic costimulatory molecules, allowing allogeneic use in the clinical setting or even xenogeneic use for research purposes [10, 11]. Animals Immunocompetent SD-OFA male rats (Charles River, Barcelona, Spain), weighing 375–400 g were used, and were kept in standard stabulation conditions (12 h light/ dark daily cycle) in our facilities with pellet chow and water ad libitum throughout the experiment. Methods Ethics Regarding IBD cell therapy, MSCs from different sources have reported efficacy through various routes of administration (systemic and local) in animal studies on experimental colitis [12]. Thus, MSC treatment reduced the clinical and histopathological severity in experimen- tal animal models of colitis by downregulating the Th1 and Th17-driven inflammatory response (i.e. reduction of pro-inflammatory cytokines such as tumor necrosis factor (TNF), interferon gamma (IFNγ), interleukin (IL)-6, IL-12 or IL-17), increasing the levels of anti-inflammatory cytokines such as IL-10, and promoting the generation of immune cells with immunomodulatory properties such as FoxP3 regulatory T cells, regulatory B cells, and M2 macrophages [13–18]. The engraftment of MSCs on the inflamed gut might play an important role for their thera- peutic effect on IBD models [13, 14, 19, 20] Interestingly, preactivation of MSCs with inflammatory mediators such as IFNγ, IL-1β, IL-25 or poly (I:C) increase their thera- peutic capacity in vivo by increasing their immunomodu- latory properties and capacity to home to the site of inflammation [21–24]. Noteworthy, efficacy on the treat- ment of experimental colitis has been also obtained using components of MSCs, such as conditioned medium and extracellular vesicles, showing that MSC secreted factors can ameliorate colitis [25, 26]. Another aspect of interest is that exposure of MSCs to drugs commonly used in the treatment of IBD do not appear to affect MSC function in vitro [27, 28]. Furthermore, MSC-based therapies have been already used in human studies on fistulizing [29–31] and luminal CD [32–34]. Importantly, Panés et al., re- cently reported for the first time the significant efficacy of the intralesional treatment with allogeneic ASCs (Cx601) of complex perianal fistulas in Crohn’s pa- tients, in a randomized, placebo-controlled phase III clinical trial [35]. All the animal experiments were performed following approval from the Animal Experimental and Welfare Ethics Committee of La Paz University Hospital (CEBA 24-2010), and in accordance with the guidelines of the directive 2010/63/EU from the European Parliament and of the Council on the protection of animals used for scientific purposes and the corresponding Spanish regulations (RD53/2013). Endoscopy Approximately 24 h after colitis induction, the animals were weighed and anesthetized with inhaled isofluorane. Prior to the endoscopy, colon cleansing was performed with a 20-ml room temperature (RT) saline solution enema. ASC isolation For ASC isolation, lipoaspirates were obtained from hu- man adipose tissue after informed consent from other- wise healthy adult male and female donors undergoing cosmetic surgery. The ASC isolation was performed as previously re- ported [37]. Briefly, the lipoaspirates were washed twice with phosphate-buffered saline (PBS) to remove contam- inating debris and red blood cells and digested at 37 °C for 30 min with 0.075% collagenase (Type I, Invitrogen, Carlsbad, CA, USA) in the PBS. The digested sample was washed with 10% fetal bovine serum (FBS), treated with 160 mM ammonium chloride to eliminate the remaining red blood cells, suspended in culture medium (Dulbecco’s modified Eagle medium), containing 10% FBS, and filtered. The cells were seeded onto tissue cul- ture flasks and expanded at 37 °C and 5% carbon Page 3 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Page 3 of 13 (Sigma-Aldrich, Tres Cantos, Spain), 30 mg/ml diluted in 50% ethanol, freshly prepared, was delivered slowly. The rats were kept in a head-down position for 1 min to pre- vent immediate expulsion of TNBS, and were then returned to their cages where they recovered conscious- ness shortly thereafter [40, 41]. dioxide. The culture medium was changed every 3 to 4 days. The cells were passed to a new culture flask when the cultures reached of confluence, and were phenotyp- ically characterized according to their capacity to differ- entiate into chondro-, osteo-, and adipogenic lineages [38, 39]. In addition, ASCs were verified by flow cytome- try staining for specific surface markers: positive for HLA-I, CD73, CD90 and CD105; and negative for HLA- II, CD14, CD19, CD34 and CD45. A pool of six various ASC samples from male and female donors (population doubling 12–14) were used in the study. Experimental design The endoscopy was performed with a videoendoscope GIF-XP-160 (Olympus Optical Co Ltd, Tokyo, Japan), with an outer diameter of 5.9 mm, 180°/90° up/down bending, 100°/100° right/left bending, 103 cm working length, 120° view field, 2 mm working channel and a CV-145 processor (Olympus Optical Co Ltd). On day 0, the rats were weighed and colitis was induced. On day 1, the animals that presented weight loss under- went endoscopic evaluation under anesthesia. After col- onoscopy, treatment was subsequently applied according to the assigned experimental group: ASC group (n = 25), or vehicle (PBS, n = 21). Two additional groups were used: one without colitis induction was used as a healthy control (n = 25); and another with induced colitis but without en- doscopy or treatment, the TNBS group (n = 13), was used as a safety control for the endoscopy and injection. While in the supine position, the endoscope was inserted into the rectum, advancing until the splenic flexure (8–10 cm). All the endoscopies were digitally re- corded for posterior analysis by two different observers. All the animals were weighed daily. On day 11, a sec- ond colonoscopy was performed under anesthesia for the assessment of colonic damage; blood was then ob- tained by cardiac puncture, and the animals were eutha- nized with saturated potassium chloride through intracardiac injection. A medial abdominal incision was then performed for macroscopic evaluation. To assess colitis severity, we developed an endoscopic index, adapted from published animal endoscopic exper- iments [19, 42–45] and human IBD scales [46–48]. The degree of inflammation, ulceration, stenosis, thickening, bleeding and extent of disease were scored individually and a final score was obtained by adding all the vari- ables, ranging from 0 to 25 (Table 1). Cell injection E d Endoscopic needles (23G × 5 mm, 160 cm length, Olym- pus Optical Co Ltd) were used for submucosal injection. Following endoscopic evaluation, a needle was passed through the endoscope’s working channel, then intro- duced tangentially into the submucosa using the dy- namic endoscopic submucosal injection method [49], injecting 0.2 ml PBS containing ASCs or not (total dose On day 0, the animals were weighed and anesthetized with inhaled isofluorane (5% induction and 2% mainten- ance), and feces were removed by gentle manual pressure of the abdomen. While in a supine position, a flexible plastic intravenous catheter (BD Insyte™Autoguard™18G, Becton Dickinson, Madrid, Spain) was inserted 5 cm from the anal verge, and a single bolus of 0.5 ml of TNBS Table 1 Endoscopic score. The score is the result of the sum of each item Inflammation Ulceration type Stenosis Colon thickening Spontaneous bleeding Inflammation longitudinal extent (cm) 0 Normal aspect of the mucosa 0 No ulcers 0 No stenosis 0 Transparent 0 Absent 1 Hyperemia 1 Cicatricial ulcer 1 Mild stenosis 1 Mild 1 Present 2 Ulceration, covering < 1/4 of the circumference 2 Superficial ulcer 2 Stenosis preventing endoscope pass 2 Pronounced 3 Ulceration, covering 1/4 to 1/2 of the circumference 3 Medium depth ulcer 3 Totally opaque 4 Ulceration, covering > 1/2 of the circumference 4 Deep ulcer 5 Circumferential ulceration 5 Necrotic ulcer 6 Circumferential ulceration+ longitudinal extension Table 1 Endoscopic score. The score is the result of the sum of each item Table 1 Endoscopic score. The score is the result of the sum of each item Inflammation Ulceration type Stenosis Colon thickening Spontaneous bleeding Inflammation longitudinal extent (cm) 0 Normal aspect of the mucosa 0 No ulcers 0 No stenosis 0 Transparent 0 Absent 1 Hyperemia 1 Cicatricial ulcer 1 Mild stenosis 1 Mild 1 Present 2 Ulceration, covering < 1/4 of the circumference 2 Superficial ulcer 2 Stenosis preventing endoscope pass 2 Pronounced 3 Ulceration, covering 1/4 to 1/2 of the circumference 3 Medium depth ulcer 3 Totally opaque Page 4 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 1:250 in blocking solution for 1 h at RT, followed by the ABC kit (HRP-based, Vector Laboratories, Burlingame, CA, USA), for 1 h at RT, and DAB as the chromogen. Sections were counterstained with hematoxylin, and mounted with DPX. 107 ASCs) in four different spots surrounding the lesions. Immunohistochemistry Distal colonic tissue was fixed in 10% buffered formalin, embedded in paraffin, cut into 5-μm sections and stained with hematoxylin-eosin. To detect ASCs by immunohistochemistry, tissue sec- tions were dewaxed and rehydrated, and then micro- waved for 20 min in 0.01 M trisodium citrate buffer pH 6.0. After cooling to RT, endogenous peroxidase was blocked by incubating the tissues for 10 min in 1% H2O2 in methanol. After rinsing with TBST buffer, the samples were blocked for 1 h at RT with 5% normal goat serum and 1% bovine serum albumin in TBST, then incubated overnight at 4 °C with anti-human mitochondria anti- body (113-1, Abcam, Cambridge, UK), 1:500 in blocking solution. Detection was performed with goat anti-mouse biotinylated IgG (Life Technologies, Carlsbad, CA, USA) g For the quantitative PCR, we used a CFX96 Touch system (Bio-Rad, Hercules, CA, USA) and Quantimix Easy kit (Biotools, Madrid, Spain) containing 0.3 μM each oligonucleotide and 0.5 × SYBR Green (Life Tech- nologies), for 40 cycles of 20 s at 95 °C, 20 s at 60 °C, 30 s at 72 °C and 2 s at 80 °C (when fluorescence was ac- quired). The oligonucleotide sequences were as follows: Foxp3, forward, 5′-TGG CAG GGA AGG AGT GTC AG-3′, reverse, 5’-GGC TGA CTT CCA AGT CTC GT- 3′; IL-10, forward, 5’-GGC TCA GCA CTG CTA TGT TGC C-3′, reverse, 5’-AGC ATG TGG GTC TGG CTG ACT G-3′; GAPDH, forward, 5′- CGT GGA GTC TAC TGG TGT CTT CAC C-3′, reverse, 5′- GAT GGC ATG GAC TGT GGT CAT GAG C-3′. We used the standard 2-ΔΔCt method to quantitate expression levels. Table 2 Macroscopic damage score. The score is the result of the sum of each item Colon wall vascularization Colon wall thickening Adherences to the colon wall 0 Normal 0 Normal 0 Absence 1 Mild vascular pattern distortion 1 Mild 1 Mild adherences 2 Pronounced vascular pattern distortion 2 Pronounced 2 Moderate adherences 3 Complete absence of vascular pattern 3 Very intense 3 Pronounced adherences Table 2 Macroscopic damage score. The score is the result of the sum of each item Macroscopic evaluation After euthanasia, the abdominal cavity was exposed through medial abdominal incision, allowing visualization of the colon and the presence of adherences. Photographs were taken for evaluation by two different observers. Macroscopic damage was assessed by the degree of colon wall vascularization, wall thickening and presence of ad- herences, for a final score ranging from 0 to 9 (Table 2). The colon was removed to measure its entire length from the colocecal junction to the anal verge, and the distal part excised and fixed in buffered formalin. Mesenteric lymph nodes (MLN) were collected, snap- frozen in liquid nitrogen and stored at −80 °C for further use. RNA isolation, reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR The complete procedure, including submucosal injec- tions, usually took no longer than 10 min. reaction (RT PCR), quantitative PCR Total RNA was isolated from frozen MLN with TRIzol reagent (Ambion, Life Technologies), according to the manufacturer’s recommendations. The average yield was 1.91 ± 0.06 μg RNA per mg MLN. Complementary DNA synthesis was performed with MultiScribe Reverse Transcriptase and oligo (dT)16 (Applied Biosystems, Fos- ter City, CA, USA), according to the manufacturer’s rec- ommendations. Standard PCR was performed with 1u DNA polymerase (Biotools, Madrid, Spain) and 0.5 μM each oligonucleotide for 35 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The sequences of the oligonu- cleotides were as follows: Foxp3, forward, 5’-CAG CTG CCT ACA GTG CCC CTA G-3′, reverse, 5’-CGT TTG CCA GCA GTG GGT AG-3′; IL-10, forward, 5′-GGA TCC AAC GCA GCC TTG CAG AAA C-3′, reverse, 5’-ACG CGT ATT TTT CAT TTT GAG TGT CAC GTA GGC -3′; ß-Actin, forward, 5′-AGA GGG AAA TCG TGC GTG-3′, reverse, 5’-CTG GGT ACA TGG TGG TGC-3′. The PCR products were analyzed on 1. 6% agarose gels in 0.5 × TBE buffer (Tris-Borate-EDTA) containing 1 × REALSafe (Durviz, Valencia, Spain) and with a digital photodocumentation system (Alliance 2.7, UVitec, Cambridge, UK). Cell injection E d This dose and number of injections were chosen taking into account the maximum concentration in which ASC viability was guaranteed and covering the extension of the damaged colon while keeping a low volume of fluid, and thus avoiding potential mucosal damage caused by over injection. Statistics The statistical analyses were performed with SPSS 20.0 (SPSS Inc., Chicago, IL, USA) and Prism 5.01 (GraphPad Software Inc., San Diego, CA, USA). The statistical level of significance was p < 0.05, corrected for multiple com- parisons where appropriate. The quantitative data are expressed as mean ± SD. Page 5 of 13 Page 5 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Feasibility and safety The differences between continuous and qualitative variables were calculated with non-parametric tests: the Kruskal-Wallis or the Mann-Whitney U test. The Wilcoxon signed-rank test was used for paired analysis. The endoscopic injection was successful in all the rats, with formation of a visible submucosal bleb (Fig. 1a-c). Neither significant adverse events nor mortality due to the procedure occurred. For the frequency analysis between qualitative vari- ables we used the chi-squared test or Fisher’s exact test, when necessary (if n < 20 or any value in the expected value table was < 5). We assessed the correct location of the cell injection using immunohistochemistry with anti-human mito- chondria antibody, which allows detection of human ASCs. In a healthy animal in which ASCs were injected following the same method of the experiment, a dense accumulation of positive cells was observed in the colon submucosa 24 h after the injection, with no signs of im- mune reaction (Fig. 1d). Correlations were analyzed by Pearson’s correlation coefficient. Slopes of linear regression were compared with the beta coefficient of the simple linear regression and its standard error. Survival curves were made using the Kaplan-Meier method and the log-rank test. In the colitic rats, we also detected ASCs in the colon submucosa 24 h after cell injection, with intense inflam- matory infiltrate and edema (Fig. 1e). At the end of the experiment, however, we could not detect ASCs by im- munohistochemistry within the submucosa, even in the animals that showed signs of improvement: the extensive areas that were seen in the submucosa of these animals showed cells with a fibroblastic phenotype, less inflam- matory infiltrate and almost absent edema (Fig. 1f). Basal characteristics Twenty-four hours after a TNBS enema, the rats suf- fered significant weight loss (−3.9% ± 2.4% vs. 1.0% ± 1. 4% in the control group, p < 0.001); diarrhea and other signs of discomfort were also evident in the colitic ani- mals. The endoscopic score increased significantly in these animals compared with the control group (16.1 ± 4.9 vs. 1.3 ± 1.9, p < 0.001). The selection of rats to ei- ther treatment group, PBS or ASC, was not biased, given neither weight loss (−3.6% ± 2.8% vs. -3.0% ± 1.9%, re- spectively, p = 0.21) nor endoscopic score (17.6 ± 4.3 vs. 15.0 ± 5.0, respectively, p = 0.08) was statistically different. There was no difference in overall survival rate with either treatment, (85.7% PBS vs. 83.3% ASCs vs. 84.6% TNBS, p is not significant). As expected, the healthy control group suffered no deaths (data not shown). Endoscopic analysis After colitis induction, endoscopic signs of damage were evident, with disappearance of vascular pattern, edema and ulceration (Fig. 3a, b), which improved over time, in particular in the ASC group (Fig. 3d). The ASC-treated rats also suffered an initial weight loss that reached its peak on day 3 (−8.4% ± 4.2%), after which, unlike the other colitic groups, weight recovery was evident, reaching, on average, initial values by the end of the experiment (−0.8% ± 10.1% on day 11, p = 0. 741) (Fig. 2a). In fact, the ASC-treated animals gained weight with a slope comparable to the one observed in the control group (0.93 ± 0.08 vs. 0.89 ± 0.04, respectively, p = 0.601), whereas the slopes for the TNBS and the PBS- treated groups were significantly flatter (0.52 ± 0.09, p = 0. 001, and 0.41 ± 0.09, p < 0.001, respectively) (Fig. 2a). We evaluated the endoscopic mucosal damage following the score described in Table 1, with higher scores for more severe inflammation. There is no validated score for endoscopic evaluation of experimental colitis severity. The most commonly used variable for follow-up is daily weight; therefore, we first analyzed the overall correlation between our colon- oscopy score and weight change, finding a strong correl- ation at both time points evaluated: at day 1, the Pearson coefficient was r = −0.75 (p < 0.001) and at the end of the experiment at day 11 r = −0.78 (p < 0.001) (Additional file 1: Figure S1). Weight recovery was statistically significant in the ASCs group over the PBS group on day 5 (−6.5% ± 5. 86% vs. −11.32% ± 4.18%, respectively, p = 0.009), day 9 (−3.7% ± 9.1% vs. −8.5% ± 6.8%, respectively, p = 0.034), At day 11, the final endoscopic score was 9.1 ± 5.6 in the ASC group compared with 13.3 ± 6.2 in the PBS group (p = 0.052) (Fig. 3e). As expected in an acute col- itis model, both groups improved; however, this im- provement was significantly greater in the ASC group compared with the PBS group (−6.6 ± 2.9 vs. −3.5 ± 5 points, respectively, p = 0.011). This represents a mean improvement of 47.1% ± 5.3% in the ASCs compared with 21.8% ± 6.6% in the PBS group (p = 0.005). Fig. 2 hASC improves weight evolution. Colitis induces weight loss. a Body weight was measured daily. Weight evolution All the control rats gained weight steadily throughout the course of the experiment, reaching a significant a d e f b c Fig. 1 Injection feasibility and cell location. Cells were injected into the submucosa using a 23G endoscopic needle. a, b and c injection procedure, bleb formation and endoscopic appearance after injection. Cells are injected into the submucosa, this is demonstrated by presence of hASC staining (brown) using anti-human mitochondria antibodies in colon submucosa 24 h after injection in a healthy animal (d) or in a colitic animal (e). Eleven days after injection in a colitic animal that recovered weight cells no staining was seen (f) a b a d e f f d Fig. 1 Injection feasibility and cell location. Cells were injected into the submucosa using a 23G endoscopic needle. a, b and c injection procedure, bleb formation and endoscopic appearance after injection. Cells are injected into the submucosa, this is demonstrated by presence of hASC staining (brown) using anti-human mitochondria antibodies in colon submucosa 24 h after injection in a healthy animal (d) or in a colitic animal (e). Eleven days after injection in a colitic animal that recovered weight cells no staining was seen (f) Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Page 6 of 13 day 10 (−1.9% ± 9.4% vs. −8.4% ± 8.6%, respectively, p = 0.013) and day 11 (−0.8% ± 10.1% vs. −6.7% ± 9.2%, respectively, p = 0.037). increase of 9.9% ± 3.4% (p < 0.001) compared with their initial weight (Fig. 2a, b). Regarding the colitic rats, the TNBS group suffered rapid weight loss, reach- ing −11.9% ± 2.1% at day 3; they then began a slight recovery, with mean weight loss at the end of the ex- periment of −8.7% ± 8.2% (p = 0.021) (Fig. 2a). The PBS-treated rats also exhibited a similar pattern, with significant weight loss that reached a maximum on the fifth day after induction with the hapten (−11.3% ± 4.2%). After this day, the rats gained weight slightly, and, on aver- age, did not reach initial values (−6.7% ± 9.2% on day 11, p = 0.024) (Fig. 2a). Weight evolution Individually, some animals from every colitic group re- covered initial weight by the end of the experiment, but the frequency of weight recovery was higher in the ASC- treated animals compared with the TNBS and PBS groups: 60.0% (12/20), 27.3% (3/11) and 38.9% (7/18), re- spectively (p is not significant) (Fig. 2b). Endoscopic analysis Faster improvement was seen in ASC group, being significantly different on days 5, 9, 10 and 11. p < 0.05. b Frequency of weight recovery by treatment group. 60% of animals treated with ASC recovered weight vs. 38.9% in PBS group and 27.3% in TNBS group (p = n.s.). ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid ASC treatment dramatically decreased the develop- ment of stenosis (defined as the presence of a narrowing of the lumen that hinders or prevents passage of the endoscope) when evaluated by endoscopy on day 11 (Fig. 4a, b). While in the PBS group, 7 of 17 evaluable rats (41.2%) developed stenosis, whereas only 1 of 21 rats (4.8%) did so in the ASC group (p = 0.001) (Fig. 4c). Macroscopic evaluation 4 Stenosis development is decreased with ASC treatment. a Endoscopic image from a PBS-treated rat with stenosis. b Macroscopic aspect of the colon of the same rat. The stenosis is marked with a white arrow. c Frequency of stenosis in the PBS and ASC groups. p = 0.01. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid a b c d a b a b b c c Fig. 4 Stenosis development is decreased with ASC treatment. a Endoscopic image from a PBS-treated rat with stenosis. b Macroscopic aspect of the colon of the same rat. The stenosis is marked with a white arrow. c Frequency of stenosis in the PBS and ASC groups. p = 0.01. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid e e Fig. 3 Endoscopic evaluation of the colitis. TNBS induced distal colitis characterized by erythema, edema, loss of vascular pattern and ulceration. This is still evident after 11 days of the onset of the disease. a-b Endoscopic evolution of a PBS-treated rat at day 1 and 11. c-d Endoscopic evolution of an ASC-treated rat at day 1 and 11. e Evolution of the endoscopic score over time per treatment group. The endoscopic score at baseline was not different between groups. As expected this score improved between day 1 and 11 in both groups. The change in ASC group was greater than in the PBS group (−6.6 ± 2.9 vs. −3.5 ± 5 points, p = 0.011). Score at day 11 did not achieve significant difference between groups p = 0.052. p < 0.01, p < 0.001. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid Colitis is known to induce a marked shortening of the colon. In the TNBS group and the PBS group, colon length was shorter than in the control group (204.5 ± 24.6 mm, p < 0.001; 193.6 ± 17.9 mm, p < 0.001, vs. 237.2 ± 16.3 mm, respectively). This shortening was significantly improved in the ASC group, with a mean length of 222.6 ± 17.3 mm, showing no difference from the control group (p = 0.2) and statistically longer than the PBS group (p < 0.001) (Fig. 5f). Colon length also cor- related significantly with the endoscopic score at the end of the experiment, with a Pearson correlation r = −0.33, p = 0.041 (Additional file 3: Figure S3). Macroscopic evaluation Distal colonic samples stained with hematoxylin-eosin were evaluated for ulceration and histologic recovery (Fig. 6). TNBS groups, although there was no statistical signifi- cance among them. Nevertheless, the macroscopic dam- age score in the ASC group (4.7 ± 2.5) decreased to values not statistically different from the control group (Fig. 5e). Macroscopic evaluation Macroscopic evaluation of the abdominal cavity was per- formed after euthanasia at day 11. Vascular pattern dis- tortion, colon wall thickening and fat tissue or visceral adhesions to the colon were assessed (Fig. 5a-d). Both the TNBS and PBS groups showed a significantly higher macroscopic damage score compared with the control group (6.5 ± 2.5, p < 0.001; 6.1 ± 2.7, p < 0.01; 2.2 ± 1.0, respectively). This total score and all the evaluated subi- tems were lower in the ASC group than in the PBS and Fig. 2 hASC improves weight evolution. Colitis induces weight loss. a Body weight was measured daily. Faster improvement was seen in ASC group, being significantly different on days 5, 9, 10 and 11. p < 0.05. b Frequency of weight recovery by treatment group. 60% of animals treated with ASC recovered weight vs. 38.9% in PBS group and 27.3% in TNBS group (p = n.s.). ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid Page 7 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 a b c d e Fig. 3 Endoscopic evaluation of the colitis. TNBS induced distal colitis characterized by erythema, edema, loss of vascular pattern and ulceration. This is still evident after 11 days of the onset of the disease. a-b Endoscopic evolution of a PBS-treated rat at day 1 and 11. c-d Endoscopic evolution of an ASC-treated rat at day 1 and 11. e Evolution of the endoscopic score over time per treatment group. The endoscopic score at baseline was not different between groups. As expected this score improved between day 1 and 11 in both groups. The change in ASC group was greater than in the PBS group (−6.6 ± 2.9 vs. −3.5 ± 5 points, p = 0.011). Score at day 11 did not achieve significant difference between groups p = 0.052. p < 0.01, p < 0.001. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid a b c Fig. 4 Stenosis development is decreased with ASC treatment. a Endoscopic image from a PBS-treated rat with stenosis. b Macroscopic aspect of the colon of the same rat. The stenosis is marked with a white arrow. c Frequency of stenosis in the PBS and ASC groups. p = 0.01. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid a b c Fig. Immunomodulatory effects mediated by ASCs Immunomodulatory effects mediated by ASCs The possible immunomodulatory effect of the local in- jection of ASCs into the colon submucosa was evaluated at day 11 in mesenteric lymph nodes by measuring the mRNA expression of Foxp3, a transcription factor expressed by regulatory T lymphocytes, and of IL-10, an anti-inflammatory cytokine produced by various cell types. The macroscopic damage score correlated significantly with weight change on the day of euthanasia, with a Pearson correlation r = 0.78 (p < 0.001), and with the endoscopic index on the same day (Pearson correlation r = 0.80, p < 0.001) (Additional file 2: Figure S2). Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Page 8 of 13 c b a Fig. 6 Histologic evaluation. a Hematoxylin-eosin staining of a control animal. b-c Sections made at the injection point of TNBS colitis at 11 days (b). Animal treated with PBS injection, showing c b a Fi 6 Hi l i l i H li i i i f a b c d Healthy TNBS +ASC TNBS +PBS a b c d Healthy TNBS +ASC TNBS +PBS e f Fig. 5 Macroscopic evaluation. Colitis is characterized by edema, loss of vascular pattern and presence of adherences. a-d Macroscopic aspect of the colon in a control rat (a), ASC-treated rat (b) and PBS- treated rats with diverse severity grades (c and d). e Macroscopic damage score after sacrifice per group. Higher scores represent more severe damage. The score did not achieve statistical difference between the ASC and PBS group. p < 0.01 p < 0.001. f Colon length in mm per group. Colon length decreases with inflammation, the TNBS and PBS groups had significant shorter colons than the healthy colon group and the ASC-treated group. The ASC group shows recovery and is not different from the healthy control group. p < 0.001. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid a b e e f f Fig. 5 Macroscopic evaluation. Colitis is characterized by edema, loss of vascular pattern and presence of adherences. a-d Macroscopic aspect of the colon in a control rat (a), ASC-treated rat (b) and PBS- treated rats with diverse severity grades (c and d). e Macroscopic damage score after sacrifice per group. Higher scores represent more severe damage. The score did not achieve statistical difference between the ASC and PBS group. p < 0.01 *p < 0.001. Immunomodulatory effects mediated by ASCs Although promising, MSC therapy has yet to address several questions, such as optimal doses and administra- tion routes, selection of patients and elucidation of mechanisms of action, making animal models essential. g Regarding administration route, it has been demon- strated that ASCs have homing capabilities toward dam- aged areas [51, 55]. While convenient, intravenous use remains controversial due to concerns about lung en- trapment that could lead to a reduced number of avail- able cells and therefore reduced efficacy and potential adverse events [19, 52, 54, 56]. Delivering the cells dir- ectly into the damaged area would overcome this issue, and intraperitoneal use has been used in animals, but this approach is impractical in humans. Injecting cells through endoscopy could offer advantages in experimental studies and translational potential for future human use. The TNBS chemical-induced model of colitis is inex- pensive, reproducible, easy to handle and widely used [40, 41, 57]. Although human IBD is better represented by knock-out mouse models, we chose the TNBS model because it leads to distal colitis and can be used in rat strains that can grow enough to make endoscopy a feas- ible tool for evaluation and treatment. Therefore, even if the use of a chemical model is a limitation of our study, it is useful for the evaluation of the endoscopic adminis- tration route and that efficacy data obtained with this model is meaningful. Fig. 7 mRNA expression in MLN.ASC treatment has immunomodulatory effects. Mesenteric lymphatic nodes were isolated and frozen in liquid nitrogen. RNA was isolated and quantitative PCR was made for FoxP3 expression and IL-10. Standard 2-ΔΔCt method to quantitate expression levels was used n = 4. a Foxp3 is significantly increased in ASC treated rats (n = 4). b IL-10 expression did not reach significant difference. p < 0.05. ASCs adipose-derived mesenchymal stem cells, IL-10, interleukin-10, TNBS trinitrobenzenesulfonic acid Our data demonstrate that endoscopy in trained hands is a simple and very effective tool for follow-up and evaluation of colitic rats, with an excellent safety profile, given we did not observe any case of perforation or sig- nificant bleeding due to the injection, and no deaths were attributed to the procedure. Moreover, there were no significant differences in the evolution of the rats with colitis without endoscopy (TNBS group) and the group with endoscopy and PBS injection. Immunomodulatory effects mediated by ASCs f Colon length in mm per group. Colon length decreases with inflammation, the TNBS and PBS groups had significant shorter colons than the healthy colon group and the ASC-treated group. The ASC group shows recovery and is not different from the healthy control group. **p < 0.001. ASCs adipose-derived mesenchymal stem cells, PBS phosphate-buffered saline, TNBS trinitrobenzenesulfonic acid Fig. 6 Histologic evaluation. a Hematoxylin-eosin staining of a control animal. b-c Sections made at the injection point of TNBS colitis at 11 days (b). Animal treated with PBS injection, showing mucosal ulceration (c) TNBS colitis and ASC injection, showing, slight crypt distortion persists, but no ulcers or active inflammation. Scale bar 500 µm which was not statistically different from the control group (p = 0.46) (Fig. 7a). Regarding IL-10 mRNA, the expression of IL-10 was reduced in the TNBS colitic group (0.56 ± 0.2 times) com- pared with the healthy control group (1.07 ± 0.4, p = 0.05), whereas in the ASC-treated animals, IL-10 expression was similar to the control group (1.05 ± 0.1, p = 0.71) (Fig. 7b). As shown in Fig. 7a, Foxp3 expression decreased dra- matically in the untreated colitic rats, being three times smaller in the TNBS group than in the control group (0. 33 ± 0.1 vs. 1.05 ± 0.3, respectively, p = 0.014). The ASC group showed a recovered expression (1.27 ± 0.5 times), Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 Page 9 of 13 Page 9 of 13 Fig. 7 mRNA expression in MLN.ASC treatment has immunomodulatory effects. Mesenteric lymphatic nodes were isolated and frozen in liquid nitrogen. RNA was isolated and quantitative PCR was made for FoxP3 expression and IL-10. Standard 2-ΔΔCt method to quantitate expression levels was used n = 4. a Foxp3 is significantly increased in ASC treated rats (n = 4). b IL-10 expression did not reach significant difference. *p < 0.05. ASCs adipose-derived mesenchymal stem cells, IL-10, interleukin-10, TNBS trinitrobenzenesulfonic acid against commensals. In this setting, MSC treatment could help restore this balance due to its diverse immunomodula- tory properties, promoting both differentiation of lympho- cytes and mononuclear cells toward tolerant phenotypes and suppression of activated lymphocytes [6]. Several studies have tried stem cell therapy in colitic animals with both BM-MSCs and ASCs [13–15, 19, 36, 50–54]. Immunomodulatory effects mediated by ASCs This safety profile is one of the main advantages of the technique, allowing in vivo repeated assessment of the severity and evolution of the disease, and also reducing the number of animals needed. Discussion In the present study, we hypothesized that local adminis- tration by endoscopy of ASCs in the submucosa of coli- tic rats could be an optimal route of administration to increase the bioavailability of ASCs in the damaged areas of the colon and reduce the severity of the disease. Therefore, we evaluated the feasibility, safety and effi- cacy of endoscopic ASC administration in an experimen- tal colitis model, as well as the utility of endoscopy to follow the course of the disease in rats. Our results support endoscopy as a useful administra- tion route for ASC treatment for colitis and its utility for assessing treatment efficacy and severity of the disease in an animal model. We also demonstrate that submuco- sal injection of human ASCs ameliorates the course of TNBS colitis in immunocompetent rats. While it has been used for carcinogenesis [43, 58–63] and colitis [19, 42, 44, 64] evaluation, these studies are heterogeneous in aims, endoscopes and scoring systems. Although lacking a formal validation, the endoscopic score developed for this study showed a significant cor- relation with body weight changes, macroscopic damage and colon length, which are commonly used variables in IBD pathogenesis is complex and involves multiple mechanisms. One of the most significant is the disba- lance between pathogen recognition and tolerance Page 10 of 13 Page 10 of 13 Page 10 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 experimental colitis. Further studies will determine which is the best score for colitis evaluation in murine models. human ASCs are sufficiently well tolerated in this proof- of-concept model. This immunoprivileged status is prob- ably due to a lack of MHC molecules, as has been shown in other recent studies [13–15]. ASC administration demonstrated improvement in weight recovery, in colon length and in the endoscopic damage score. In addition, the macroscopic damage ob- served after euthanasia was numerically smaller in the treated group, although it did not reach statistical signifi- cance. These are among the main variables commonly used in colitic animals, thus supporting the ameliorating effect of the cells, and confirming the results of previous studies [13–15, 19, 36]. Although these studies differ in protocol, severity of the colitis, animal strains, cell type, dose and administration routes, all suggest a beneficial effect of ASC treatment in murine chemical-induced colitis models. Conclusions In conclusion, our study provides evidence that endos- copy is a safe and reliable method to administer cell therapy into the colon and to follow up colitis murine models. ASC treatment ameliorates the course of TNBS colitis and prevents stenosis development. Funding h k This work was partially supported by a grant of the Instituto de Salud Carlos III to FdM (PI10/0317). Availability of data and materials All data generated or analyzed during this study are included in this published article (and its supplementary information file). Acknowledgements The authors would like to thank Jesus Diez for his statistical support, Dr. Carlota Largo Aramburu for her veterinary support and the Hospital Universitario La Paz Inflamatory Bowel Disease Unit and Cell Therapy Laborathory members for their help and support for the study. Discussion If our results are confirmed there would be transla- tional potential, in humans, endoscopic injection could be a simple, well-tolerated route of delivering cells directly into the damaged area, through a technique routinely used in patients with IBD for both diagnostic and therapeutic purposes [66], including pharmacological local injection treatment [67–69]. Cell-based therapies are currently be- ing tested in different phases in humans, reporting efficacy of intravenous infusion with various doses and regimens of BM-MSCs, both autologous and allogeneic [32–34]. Other studies have focused on local treatment for fistu- lizing disease, showing improved healing with BM- MSCs [70] or ASCs [29–31, 35, 70]. One of the main results of our study was the decrease in stenosis development; even though the experiment was not designed for this purpose, the difference be- tween the ASC and the vehicle group is remarkable. Whereas in the PBS group stenosis developed in 41% of the animals, in the ASC group this occurred in 4.8%. This is an observation that, in our opinion, deserves fur- ther studies to elucidate whether ASCs are able to pre- vent stenosis development in other models and if they can also help to reverse established stenosis. Additional files A limitation of our study is the lack of histological scoring; in our hands, microscopic changes in inflamma- tion and healing were inconsistent. Additional file 1: Figure S1. Endoscopic score correlation with weight change at day 1 (A) and 11 (B). (PPTX 2289 kb) Additional file 1: Figure S1. Endoscopic score correlation with weight change at day 1 (A) and 11 (B). (PPTX 2289 kb) Additional file 2: Figure S2. The macroscopic damage score correlates with weight change (A) and with endoscopic score (B). (PPTX 2254 kb) We were able to detect the ASCs in colon submucosa using anti-human mitochondrial antibodies 24 h after injection, proving the correct location of the cells after the endoscopic administration. In agreement with previ- ous studies reporting a short persistence of the cells in vivo, ASCs were not detected after 11 days. The fate of the ASCs in vivo remains largely unknown to date [65]. Additional file 3: Figure S3. The endoscopic score correlation with the colon length 2. (PPTX 37 kb) Abbreviations APCs: Antigen-presenting cells; ASCs: Adipose-derived mesenchymal stem cells; BM-MSCs: Bone marrow mesenchymal stem cells; CD: Crohn’s disease; FBS: Fetal bovine serum; IBD: Inflammatory bowel disease; IL: Interleukin; INF: Interferon; MHC: Major histocompatibility complex; MLN: Mesenteric lymph nodes; MSCs: Mesenchymal stem cells; PBS: Phosphate-buffered saline; RT: Room temperature; TBST: Tris-buffered saline with Tween 20; TNBS: Trinitrobenzenesulfonic acid; UC: Ulcerative colitis g y In order to determine whether the endoscopic admin- istration of ASCs resulted in immunomodulatory effects in the colitic rats, we determined the levels of the anti- inflammatory cytokine IL-10 and the transcription factor Foxp3, characteristic of regulatory T cells. 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Gastroenterology. 2009;136(3): 978–89. https://doi.org/10.1053/j.gastro.2008.11.041. Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. Chao K, Zhang S, Qiu Y, Chen X, Zhang X, Cai C, Peng Y, Mao R, Pevsner- Fischer M, Ben-Horin S, Elinav E, Zeng Z, Chen B, He Y, Xiang AP, Chen M. Human umbilical cord-derived mesenchymal stem cells protect against experimental colitis via CD5(+) B regulatory cells. Stem Cell Res Ther. 2016;7: 109. https://doi.org/10.1186/s13287-016-0376-2. Authors’ contributions EM, MDM, FdM and EL designed the study, analyzed and interpreted the data. EM wrote the draft, MDM, FdM, OR, DB, FdM and EL reviewed the draft and made important contributions to the final manuscript: All authors We cannot completely rule out a potential immuno- genicity using xenogeneic cells; however, it appears that Page 11 of 13 Page 11 of 13 Page 11 of 13 Martín Arranz et al. Stem Cell Research & Therapy (2018) 9:95 collected data and reviewed contributed and approved the final version of the manuscript. 11. Griffin MD, Ritter T, Mahon BP. Immunological aspects of allogeneic mesenchymal stem cell therapies. Hum Gene Ther. 2010;21(12):1641–55. https://doi.org/10.1089/hum.2010.156. 12. Lopez-Santalla M, Mancheño-Corvo P, Escolano A, Menta R, DelaRosa O, Abad JL, Büscher D, Redondo JM, Bueren JA, Dalemans W, Lombardo E, Garin MI. Biodistribution and efficacy of human adipose-derived mesenchymal stem cells following intranodal administration in experimental colitis. Front Immunol. 2017;8:638. https://doi.org/10.3389/fimmu.2017.00638. Ethics approval and consent to participate All the animal experiments were performed following approval from the Animal Experimental and Welfare Ethics Committee of La Paz University Hospital (CEBA 24-2010), and in accordance with the guidelines of the directive 2010/63/EU from the European Parliament and of the Council on the protection of animals used for scientific purposes and the corresponding Spanish regulations (RD53/2013. 13. Gonzalez Rey E, Anderson P, González MA, Rico L, Büscher D, Delgado M. Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis. Gut. 2009;58(7):929–39. https://doi.org/ 10.1136/gut.2008.168534. 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References • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2159646784	https://figshare.com/articles/journal_contribution/Supplementary_Table_1_from_Radiation_Survivors_Understanding_and_Exploiting_the_Phenotype_following_Fractionated_Radiation_Therapy/22517167/1/files/39979759.pdf	English	null	Radiation Survivors: Understanding and Exploiting the Phenotype following Fractionated Radiation Therapy	Molecular cancer research	2,013	cc-by	616	Gy x1 SD) G Sym 0.05) 8.83 IFI27 5.20 BST 4.86 IFIT 3.89 IFIT 3.53 OAS 3.46 CXC 3.13 RSA 3.07 IFIT2 3.02 HSH 2.93 IFIT3 0.05) 0.04 FUT 0.04 DHO 0.04 CD3 0.04 DFF 0.04 CLU 0.04 C19 0.04 DUS 0.03 MCM 0.03 CAM 0.03 CDC regulated an diation (eith ble. .* No ge PC3 DU145* 0Gy x1 (SD) Gene Symbol 1Gy x 10 (MF) Gene Symbol 10Gy x1 (SD) Gene Symbol 1Gy x 10 (MF) Gene Symbol 10Gy x1 (SD) <0.05) 8.83 IFI27 34.33 FCGBP 6.96 MMP1 5.45 CFB 3.91 5.20 BST2 22.15 BMP3 3.76 C12orf39 4.90 OASL 3.64 4.86 IFITM1 17.81 TTN 3.60 SUNC1 3.26 SLPI 3.36 3.89 IFIT1 12.37 KRT20 3.58 ATF3 3.24 APOBEC3G 3.19 3.53 OASL 11.73 OASL 3.09 MAMDC2 2.67 TXNIP 2.98 3.46 CXCL11 11.36 KCNK15 2.88 KLRG2 2.57 LCN2 2.94 3.13 RSAD2 10.28 MUC2 2.83 SNAI2 2.57 AZU1 2.79 3.07 IFIT2 8.93 IFITM1 2.75 IFI44 2.53 ATF3 2.75 3.02 HSH2D 8.32 ABCA1 2.64 FST 2.51 IFIT3 2.56 2.93 IFIT3 7.96 GINS2 0.59 HRK 2.51 IFIT1 2.54 <0.05) 0.04 FUT10 0.50 CHN1 0.57 NA NA MCM3 0.61 0.04 DHODH 0.49 HIST1H1E 0.52 NA NA WDR76 0.58 0.04 CD302 0.49 HIST1H3B 0.51 NA NA MCM6 0.54 0.04 DFFB 0.48 E2F2 0.49 NA NA HIST1H4L 0.51 0.04 CLUL1 0.46 HIST1H1D 0.49 NA NA HIST1H4D 0.51 0.04 C19orf51 0.46 CD274 0.48 NA NA GINS2 0.49 0.04 DUSP2 0.46 MCM6 0.46 NA NA UNG 0.48 0.03 MCM3 0.45 WDR76 0.46 NA NA HIST1H1E 0.36 0.03 CAMKV 0.40 HIST1H4D 0.32 NA NA 0.03 CDCA7 0.32 HIST1H4L 0.31 NA NA regulated and downregulated genes 24h after radiation. Figures highlights top microarray changes in adiation (either fractionated or single dose). Upregulated genes are in bold and downregulated genes are able. .* No genes were significantly downregulated in DU145 after fractionated radiation. PC3 DU145* 0Gy x1 (SD) Gene Symbol 1Gy x 10 (MF) Gene Symbol 10Gy x1 (SD) Gene Symbol 1Gy x 10 (MF) Gene Symbol 10Gy x1 (SD) <0.05) 8.83 IFI27 34.33 FCGBP 6.96 MMP1 5.45 CFB 3.91 5.20 BST2 22.15 BMP3 3.76 C12orf39 4.90 OASL 3.64 4.86 IFITM1 17.81 TTN 3.60 SUNC1 3.26 SLPI 3.36 3.89 IFIT1 12.37 KRT20 3.58 ATF3 3.24 APOBEC3G 3.19 3.53 OASL 11.73 OASL 3.09 MAMDC2 2.67 TXNIP 2.98 3.46 CXCL11 11.36 KCNK15 2.88 KLRG2 2.57 LCN2 2.94 3.13 RSAD2 10.28 MUC2 2.83 SNAI2 2.57 AZU1 2.79 3.07 IFIT2 8.93 IFITM1 2.75 IFI44 2.53 ATF3 2.75 3.02 HSH2D 8.32 ABCA1 2.64 FST 2.51 IFIT3 2.56 2.93 IFIT3 7.96 GINS2 0.59 HRK 2.51 IFIT1 2.54 <0.05) 0.04 FUT10 0.50 CHN1 0.57 NA NA MCM3 0.61 0.04 DHODH 0.49 HIST1H1E 0.52 NA NA WDR76 0.58 0.04 CD302 0.49 HIST1H3B 0.51 NA NA MCM6 0.54 0.04 DFFB 0.48 E2F2 0.49 NA NA HIST1H4L 0.51 0.04 CLUL1 0.46 HIST1H1D 0.49 NA NA HIST1H4D 0.51 0.04 C19orf51 0.46 CD274 0.48 NA NA GINS2 0.49 0.04 DUSP2 0.46 MCM6 0.46 NA NA UNG 0.48 0.03 MCM3 0.45 WDR76 0.46 NA NA HIST1H1E 0.36 0.03 CAMKV 0.40 HIST1H4D 0.32 NA NA 0.03 CDCA7 0.32 HIST1H4L 0.31 NA NA regulated and downregulated genes 24h after radiation. Figures highlights top microarray changes in adiation (either fractionated or single dose). Upregulated genes are in bold and downregulated genes are able. .* No genes were significantly downregulated in DU145 after fractionated radiation. 51 49 91 64 * Gen Symb B ASL PI OBE NIP N2 U1 F3 T3 0Gy x1 (SD) Ge Sym <0.05) 8.83 IFI27 5.20 BST2 4.86 IFITM 3.89 IFIT1 3.53 OAS 3.46 CXC 3.13 RSA 3.07 IFIT2 3.02 HSH 2.93 IFIT3 <0.05) 0.04 FUT 0.04 DHO 0.04 CD3 0.04 DFFB 0.04 CLU 0.04 C19o 0.04 DUS 0.03 MCM 0.03 CAM 0.03 CDC regulated an diation (eith ble. .* No ge
https://openalex.org/W4304099177	https://www.researchsquare.com/article/rs-2114246/latest.pdf	English	null	Increasing Impacts of Summer Extreme Precipitation and Heatwaves in Eastern China	Research Square (Research Square)	2,022	cc-by	10,372	Increasing Impacts of Summer Extreme Precipitation and Heatwaves in Eastern China Yulong Yao State Key Laboratory of Tropical Oceanography South China Sea Institute of Oceanology Chinese Academy of Sciences Wei Zhang (  wz19@princeton.edu ) Princeton University https://orcid.org/0000-0001-7179-5697 Ben Kirtman University of Miami Rosenstiel School of Marine and Atmospheric Science Research Article Keywords: Extreme Precipitation, Heatwaves, Eastern China, CMIP6, Climate Projection, SSP Scenarios Posted Date: October 10th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2114246/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Climatic Change on September 19th, 202 See the published version at https://doi.org/10.1007/s10584-023-03610-4. Increasing Impacts of Summer Extreme Precipitation and Heatwaves in Eastern China Yulong Yao State Key Laboratory of Tropical Oceanography South China Sea Institute of Oceanology Chinese Academy of Sciences Wei Zhang (  wz19@princeton.edu ) Princeton University https://orcid.org/0000-0001-7179-5697 Ben Kirtman University of Miami Rosenstiel School of Marine and Atmospheric Science Research Article Keywords: Extreme Precipitation, Heatwaves, Eastern China, CMIP6, Climate Projection, SSP Scenarios Posted Date: October 10th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2114246/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Climatic Change on September 19th, 202 See the published version at https://doi.org/10.1007/s10584-023-03610-4. Version of Record: A version of this preprint was published at Climatic Change on September 19th, 2023. See the published version at https://doi.org/10.1007/s10584-023-03610-4. Abstract Observational and modeling analysis suggests an increased frequency of heatwaves and extreme precipitation in an anthropogenically warmed climate. However, the accurate link between extreme precipitation events (EPEs) and heatwaves (HWs), and changes in these extremes and associated socio- economic impacts in eastern China have not been fully resolved. This study examines historical and future changes of summer EPEs and HWs in eastern China based on observations, reanalysis, and model outputs from the Coupled Model Intercomparison Project Phase 6. The results show that EPEs and HWs in eastern China have increased in the past four decades and are projected to rise in the future. The Yangtze River Basin and its southern regions will be confronted with the compound disaster of HWs and EPEs in the future projections. High values of the annual mean total person-times (estimated as population density multiplied by event frequency) affected by EPEs and HWs are observed in the North China Plain, Yangtze River Delta, Sichuan Basin, and southeast coast. The total person-times affected by EPEs show a slightly decreasing trend under both scenarios. However, the total person-times affected by HWs under Shared Socioeconomic Pathway (SSP) 245 scenario maximize at around 4.0 billion, lower than the peak person-times (about 5.0 billion) under SSP585 scenario. We further investigate the linkage of such extreme events with sea surface temperature and western North Pacific subtropical high anomalies. The correlations between the mean-state and extreme precipitation and maximum temperature anomalies both shifted from negative in the historical period to positive under future projections. Research Article Version of Record: A version of this preprint was published at Climatic Change on September 19th, 2023. See the published version at https://doi.org/10.1007/s10584-023-03610-4. Page 1/25 Page 1/25 Page 1/25 1. Introduction Previous studies have highlighted the significant impacts of large-scale atmospheric circulation (i.e., high-pressure anticyclones) and sea surface temperature (SST) on HWs (Wu and Wang, 2015, Wang et al. 2017, Rodrigues et al. 2019, Zheng and Wang, 2019, Xu et al. 2020). Moreover, the physical mechanisms dominating the variations and changes of EPEs and HWs in China have not been fully resolved. Previous studies have highlighted the significant impacts of large-scale atmospheric circulation (i.e., high-pressure anticyclones) and sea surface temperature (SST) on HWs (Wu and Wang, 2015, Wang et al. 2017, Rodrigues et al. 2019, Zheng and Wang, 2019, Xu et al. 2020). Summer EPEs in eastern China is primarily driven by the East Asian summer monsoon and western Pacific subtropical high (Zhou and Yu 2005, Zhu et al. 2011). Tropical cyclones (TCs) have also been reported to significantly influence summer precipitation in both eastern and southern China (Wu et al. 2007, Y. Ma et al. 2017). Nevertheless, till today, the knowledge of physics behind the EPEs and HWs remains limited. For example, the dominated SST mode affects the EPEs and HWs at a given study area remains ambiguous. Therefore, it is of great scientific interest to investigate the different influential factors of EPEs and HWs in eastern China. China as the most populated country in the world has experienced an increase in the frequency of HWs and EPEs during the past several decades (Sun and Ao 2013, Ma et al. 2015, Luo and Lau 2017, S. Ma et al. 2017, Ning et al. 2022). In 2022, China issued the first national red alert for extreme drought and heatwaves, with the maximum 2-m air temperature above 40 °C (104 °F) over a period of 48 hours or more in eastern China. Eastern China, in particular, features several megacities and accounts for over 70% of the national population, thus, its role in the socioeconomic development of China is unshakable (Zhu et al. 2011, Zhang et al. 2015). However, the economy and society of Eastern China are remarkably vulnerable to summer HWs and EPEs (Sun et al. 2014, Chen et al. 2017, Zhang et al. 2017, Zhang and Zhou 2020). The recent frequent occurrence of extreme temperature and precipitation inevitably raises questions regarding the effects of anthropogenic climate change on the intensity and frequency of HWs and EPEs in Eastern China under different scenarios of greenhouse gases emission. 1. Introduction Global-scale warming has been unequivocal over the past century and the global mean surface temperature is projected to increase by 1.1 to 5.4 ℃ in 2100 depending on different scenarios of greenhouse gases emissions (IPCC, 2014). Within an anthropogenically warming climate, extreme weather and climate events have been shown to occur more frequently and intensely (Coumou and Rahmstorf 2012, Dosio et al. 2018). Many previous studies suggest an increasing trend in extreme temperature, flood, and tropical cyclones over the past several decades, profoundly impacting the global ecosystem and socio-economic sectors (Emanuel 2005, Hansen et al. 2012, Perkins et al. 2012, Westra et al. 2013, Fischer and Knutti 2015, Donat et al. 2016). Heatwaves (HWs) and extreme precipitation events (EPEs) are among the most threatening acute meteorological events, which have received considerable attention from policymakers, the scientific community, and the general public (Stott et al. 2004, Lau and Nath 2012, Campbell et al. 2018, Wei Li et al. 2018, Ning et al. 2022). Climate model simulation and projections have shown an increasing frequency of EPEs and HWs as the Earth’s climate becomes warmer (O'Gorman and Schneider 2009, Fischer et al. 2013, Bao et al. 2017, Z. Li et al. 2019, Raghavendra et al. 2019). Extreme precipitation is more likely to occur in warmer seasons, as the saturation water vapor pressure increases by roughly 7%/ ℃ based on the Clausius–Clapeyron relation (Boer 1993, Trenberth et al. 2003, Held and Soden 2006). Allan and Soden (2008) addressed the changes of extreme precipitation in a warmed climate and Page 2/25 Page 2/25 concluded that wet regions become wetter and dry regions become drier. To the best of our knowledge, however, little research has been focused on understanding the relationship between HWs and EPEs within the context of global warming. Will HWs and EPEs occur simultaneously and exert a double threat on social and economic development? concluded that wet regions become wetter and dry regions become drier. To the best of our knowledge, however, little research has been focused on understanding the relationship between HWs and EPEs within the context of global warming. Will HWs and EPEs occur simultaneously and exert a double threat on social and economic development? Moreover, the physical mechanisms dominating the variations and changes of EPEs and HWs in China have not been fully resolved. 2.2.1 Observational and reanalysis data The observational and reanalysis data used in this study are summarized in Table S1. The daily 2-m maximum surface air temperature (Tasmax) and precipitation (Pr) were obtained from the China Daily Surface Temperature/Precipitation Dataset (V2.0) at China Meteorological Data Service Center (CMDC) based on 2,472 meteorological stations in China (Chen et al. 2017). The daily satellite SST data were obtained from the National Oceanic and Atmospheric Administration (NOAA) Optimum Interpolation Sea Surface Temperature (OISST) High Resolution Dataset Version 2.1 (Huang et al. 2021). The monthly 500- hPa and 850-hPa geopotential height (hereafter referred to as Z500 and Z850, respectively) were extracted from the fifth-generation atmospheric analysis (ERA5) at the European Centre for Medium- Range Weather Forecasts (ECMWF) (Hersbach et al. 2018). To evaluate the potential impacts of HWs (EPEs), we estimated the total person-times in eastern China computed as the population density multiplied by the corresponding frequency of HWs (EPEs). The historical population density data in China were obtained from the Data Center for Resources and Environmental Sciences, Chinese Academy of Sciences (RESDC). This dataset includes historical population density every five years from 1990 to 2015 on 1-km grids (Xu 2017). The downscaled annual population data in China under different Shared Socioeconomic Pathways (SSPs) scenarios (1 km from 2010 to 2100) were applied following Y. Chen et al. (2020). 2. Materials and methods 2.2 Data sources 2.2 Data sources 1. Introduction Global climate models are primary tools for investigating possible future changes in climate extremes (Jiang et al. 2015, Y. Li et al. 2019, Wang et al. 2019, Zhang et al. 2022). The Coupled Model Intercomparison Project Phase 6 (CMIP6) incorporates the most complete scientific experiments and the greatest amount of simulation data of the past 20 years (Eyring et al. 2016, Simpkins 2017). However, the commonality and difference between summer HWs and EPEs in the current and future have not yet been fully addressed by CMIP6. In this study, we focus on examining the spatiotemporal variation characteristics and projections of HWs and EPEs in eastern China during extended summer (June–September) based on historical data and CMIP6 models. The potential impacts of HWs and EPEs are evaluated on the population in eastern China from the present to the future. Finally, we investigate the potential drives of summer HWs and EPEs and establish the relationship between such events. Scientific answers to these questions can provide a comprehensive picture of the changes in and impact of HWs and EPEs in eastern China, allowing for the development of future response strategies and early-warning systems for extreme climate hazards. Page 3/25 Page 3/25 2.3.1 Definition of heatwave Either absolute or relative thresholds can be used to define HWs. A fixed t temperature of 35 °C is commonly used to define an HW event in China (T quantify the differences in regional climatology, a relative threshold (i.e., d adopted in this study. For each calendar day, an HW event is identified if T percentile threshold for at least three consecutive days based on the 1979 (Hobday et al. 2016, Chen et al. 2017, Freychet et al. 2017). The climatolo from data within an 11-day window centered on each calendar day and th moving average. This seasonally varying threshold allows heatwave even year (Hobday et al. 2016). Following several previous studies (Wang et al apply four metrics to represent HW characteristics, including the number number of heatwave days (HWT), average heatwave duration (HWDU), an (HWI). The details of their definition are listed in Table S4. Either absolute or relative thresholds can be used to define HWs. A fixed threshold of a daily maximum temperature of 35 °C is commonly used to define an HW event in China (Tan et al. 2010). To better quantify the differences in regional climatology, a relative threshold (i.e., defined by percentile) for HWs is adopted in this study. For each calendar day, an HW event is identified if Tasmax exceeded the 95th percentile threshold for at least three consecutive days based on the 1979–2019 climatology period (Hobday et al. 2016, Chen et al. 2017, Freychet et al. 2017). The climatological threshold is calculated from data within an 11-day window centered on each calendar day and then applied with a 31-day moving average. This seasonally varying threshold allows heatwave events to occur at any time of the year (Hobday et al. 2016). Following several previous studies (Wang et al. 2017, Yao and Wang, 2021), we apply four metrics to represent HW characteristics, including the number of heatwaves (HWN), the total number of heatwave days (HWT), average heatwave duration (HWDU), and heatwave mean intensity (HWI). The details of their definition are listed in Table S4. 2.2.2 Future data preparation and model evaluation The daily Tasmax and Pr outputs obtained from the CMIP6 models are used to assess the projected HWs and EPEs during 2021–2100 (Eyring et al. 2016). We use the SSP scenarios with different radiative forcing conditions (i.e., representative concentration pathway, hereafter RCP) to quantify future projections up to 2100 based on CMIP6 models (O'Neill et al. 2016). To compare the simulation against the observations and to analyze both a medium and a high emission pathway, we use only the models that have outputs for the three simulations: historical, SSP245, and SSP585. SSP245 is considered a “middle of the road” pathway, which combines intermediate population growth, emission, and challenges, for both mitigation and adaptation, stabilizing at 4.5 W/m2 (RCP4.5). On the other hand, SSP585 describes a “fossil-fueled development” pathway, incorporating rapid population growth and high emission, stabilizing at 8.5 W/m2 (RCP8.5) and resulting in high challenges for mitigation (Costa and Rodrigues 2021). The simulations are bilinearly interpolated into a 0.5° × 0.5° common grid. All climate model data are derived from the first realization (r1i1p1f1) to equally estimate each model. The multi- model ensemble mean method with equal weight across models is used to reduce model uncertainty. To evaluate the agreement of the daily historical Tasmax and Pr from CMIP6 with observations during the historical period, we use the Taylor diagrams to present the goodness of fit to observations by their Page 4/25 Page 4/25 correlation, centered root-mean-square difference (RMSD), and standard deviation (Taylor 2001). Fig. S1 displays the relative skills of Tasmax for 28 climate models (Table S2) and Pr for 32 climate models (Table S3) for the extended summer (June–September). The performance in terms of Tasmax is typically superior to that of Pr when comparing climate models based on historical data (1979–2014) during the same observational period. To ensure the mutual exclusion of HWs and EPEs during the extended summer, we did not select individual models with sufficiently high prediction performances of Tasmax and Pr. Instead, based on the Tasmax variable, six climate models with high performance (RMSD and standard deviation below 0.5, correlation exceeding 0.5) were selected, namely, EC-Earth3-Veg, GFDL- ESM4, INM-CM4-8, MPI-ESM1-2-HR, MPI-ESM1-2-LR, and MRI-ESM2-0. Finally, INM-CM4-8 was excluded after comparing the spatial patterns of extreme precipitation with the observation data set (Fig. S2 and S3). The remaining five models were then used to detect future variability of HWs and EPEs in eastern China. 3.1 Spatiotemporal characteristics of summer EPEs during 1979–2019 Large spatial variations exist in the mean frequency of EPEs and amount of extreme precipitation (AEP), but as shown in Fig. 1, different spatial patterns of EPEs and AEP are identified. The regions with the largest EPEs are apparent in the south of the Yangtze River Basin and its western part, with an average of 3–5 EPEs yr-1 in the past 41 summers (Fig. 1a). Meanwhile, the regions with large mean AEP appear in the southeast coast, Yangtze River Basin, North China Plain, and the southern coast of Northeast Plain, ranging from 60 to 110 mm (Fig. 1b). Notably, we find a correspondence of spatial patterns between mean EPEs and precipitation, and the consistent structure between mean AEP and EPE thresholds (Fig. S4). High annual mean summer precipitation values (over 200 mm) are apparent in the South of the Yangtze River Basin, the southeast coast, and the central and western parts of the Yangtze River Basin, exhibiting a decreasing trend from southeast to northwest (Fig. S4a). The spatial distribution of the EPE threshold is similar to that of AEP, with high values reaching 30–70 mm (Fig. S4b). Furthermore, the spatial pattern of ratios between AEP and total precipitation shows consistency with that of the EPE threshold, with high values (over 25%) apparent in the North China Plain, eastern of the Yangtze River Basin, and southeast coast (Fig. S4c). In general, regions with more summer rainfall are prone to have more EPEs, while regions with high values of AEP are apparent in the eastern coastal areas and the Yangtze River Basin, and the percentage of AEP in total precipitation in these regions is also higher than other regions. Moreover, the spatial pattern of the 1-day EPEs (total number) suggest consistency with mean frequency of EPEs, with high values located in the south of the Yangtze River Basin and its western part, ranging from 120 to 190 counts (Fig. 2c). Meanwhile, the trend in total 1-day EPEs is increased by 149.9±87.1 counts per decade over 1979–2019, indicating the impact of warming climate on regional precipitation. As shown in Fig. 2d, there is a remarkable interannual variation in time series of total 1-day EPEs. 2.3.2 Extreme precipitation event A threshold of 1 mm is typically used to distinguish between wet and dry days because extremely light precipitation (< 1 mm) may be occasionally recorded as no precipitation (Guo et al. 2018, Wu et al. 2018). Similarly, to represent the regional characteristics of precipitation changes, the relative threshold definition (the percentile-based method) is used to identify EPEs in this study (Huang et al. 2018, Wu et al. 2018). Following Gao et al. (2017) and Huang et al. (2018), we applied the 95th percentile to identify extremely unusual precipitation events in this study. Therefore, for each grid point in eastern China, an EPE was defined as the daily precipitation exceeding the 41-year average of the 95th percentile for the summers from 1979 to 2019. The method for calculating the 95th percentile precipitation is described in Bonsal et al. (2001). If the precipitation at each point exceeds this threshold, an EPE is considered to have occurred. Further, if the precipitation for 1 day exceeds the threshold of a certain day, then namely a 1-day Page 5/25 EPE event. Besides, we utilize the Pearson’s correlation analysis and linear trend test to further analyze the observations and model data. The significance level was calculated using the Mann-Kendall test. 3.3 HWs and EPEs in CMIP6 projection Based on CMIP6 model projection, we evaluate the spatiotemporal distribution of HWs and EPEs by 2100 under two SSP/RCP-based scenarios. The result shows high HWT and EPE frequencies in the Yangtze River Basin and southern coastal regions. Under SSP245 scenario, HWT and frequency of EPEs reached 20–30 days and 3–6 counts, respectively, under SSP585 scenario, they reached 50–70 days and 5–6 counts, respectively (Fig. 4a, b). The southeastern coast shows relatively larger values of HWT and AEP than other regions in eastern China. HWT (AEP) reached 20–30 days (40–45 mm) under SSP245 scenario and 50–70 days (40–50 mm) under SSP585 scenario (Fig. 4c, d), indicating that a higher rate of population growth and radiative forcing conditions triggers more severe impacts on temperature and precipitation extremes. In general, the spatial patterns of HWN are consistent with those of HWT under SSP245 and SSP585 scenarios (Fig. 4). The regions with high HWN and EPE frequencies are identified in the Yangtze River Basin and southern coastal regions under SSP245 scenario, reaching 3.5–4.5 counts and 4–6 counts, respectively (Fig. 4e). Under SSP585 scenario, the regions with high value of HWN expanded, but the frequency of EPEs shows negligible changes (Fig. 4f). In the southeast coast of China, both HWN and AEP show relatively high values, reaching 3.5–4.5 counts and 40–45 mm under SSP245 scenario and 3.5–5.0 counts and 40–50 mm under SSP585 scenario (Fig. 4g, h). Yet, the differences between SSP245 and SSP585 scenarios are trivial in terms of HWDU and HWI (Fig. S5). The enhanced number of future HWs is accompanied with a rapid increase of HWT, and the high-value areas are extended to the northwestern and northeastern parts of the study area from SSP245 to SSP585 scenarios. Specifically, the high values of multi-year mean HWT range from 8 to 13 days over 1979–2019 (Fig. 3), which is projected to increase to 20–30 days and 50–70 days under SSP245 and SSP585 scenarios (Fig. 4). This indicates that the HWT increases from the semi-moon scale in the historical period to the near-month scale under SSP245 scenario and the bimonthly scale under SSP585 scenario. It is interesting to find that compared to the changes in the HW characteristics, there are limited changes in the frequency of EPEs and AEP from SSP245 to SSP585 scenarios. 3.2 Spatiotemporal characteristics of summer HWs during 1979–2019 The high-value HWT areas are detected over the Yangtze River Basin and its northern regions, especially in the Yangtze River Delta and Sichuan Basin, where the corresponding HW duration ranges from 8 to 13 days (Fig. 3a). The high-value HWDU areas are relatively scattered, but mainly appear around the Yangtze River Basin, and the corresponding HW duration is 5–7 days/count (Fig. 3b). The HWN spatial pattern is similar to that of HWT, but the high values also appear in the Northeast Plain (Fig. 3c). The HWI spatial pattern shows an increasing trend from south to north, reaching a maximum of 7–10 ℃/count in Page 6/25 northeastern China (Fig. 3d). Notably, high-value HWT and HWDU regions are both found in the Yangtze River Dealt, where is one of the most developed economies and densest population areas in China. northeastern China (Fig. 3d). Notably, high-value HWT and HWDU regions are both found in the Yangtze River Dealt, where is one of the most developed economies and densest population areas in China. Additionally, increasing trends are observed in the regional averaged HW metrics (Fig. 3). Both HWT and HWN metrics exhibit rapid increases over 1979–2019, reaching 0.69±0.22 days per decade and 0.12±0.03 counts per decade, respectively (Fig. 3e, g, p < 0.01). The trends in HWDU and HWI are 0.19±0.08 days/count per decade and 0.18±0.08 ℃/count per decade over 1979–2019, respectively (Fig. 3f, h, p < 0.1). We note that the spatial patterns and temporal variations of both HWT and HWN are very similar. Since HWT is equal to HWN multiplied by HWDU, HWT is mainly affected by HWN (Fig. 3). Furthermore, summer HWs in eastern China has strong interannual variations and severe HWs that usually occur during major El Niño years (e.g., 1982/83, 1997/98, 2009/10). 3.4 Potential socioeconomic impact estimates The person-times describe the potential socioeconomic impacts from extreme event exposure with respect to population. Fig. 5 shows the spatial distributions of the annual mean person-times affected by EPEs and HWs. Consistent spatial patterns are detected for EPEs and HWs in eastern China, with high person-times in high-density population areas, such as the North China Plain, Yangtze River Delta, Sichuan Basin, and southeast coast. Time series of the total person-times affected by EPEs and HWs exhibit a significantly rising trends, showing a growth rate of 11.3 million per decade for EPEs and 21.6 million per decade for HWs (p < 0.01). The person-times affected by EPEs is generally higher than that affected by HWs in eastern China, while the increase rate of person-times of HWs is nearly 2-fold than EPEs. Figure S6 shows the future changes in total person-times affected by EPEs and HWs in Eastern China under SSP245 and SSP585 scenarios from 2021 to 2100. The total person-times affected by EPEs range between 3.5 and 4.5 billion in eastern China, showing a slightly decreasing trend under both scenarios (Figs. S6a and S6c). The total person-times affected by HWs under SSP585 scenario show a linear increasing trend, peak at around 2065, and then decline by the end of 21st century (Fig. S6d). Compared with the SSP585 scenario, we detect a lagged peak year at around 2080 for person-times with the SSP245 scenarios (Fig. S6b). We note that the total person-times affected by HWs under SSP245 scenario maximize at around 4.0 billion, lower than the peak person-times (about 5.0 billion) under SSP585 scenario. Considering the total population projection curves will diverge after 2040 between two future projections (green and blue lines in Fig. S6e), the potential socioeconomic impacts (as suggested by total person-times) in eastern China from EPEs and HWs under the SSP585 scenario is more severe than that under the SSP245 scenario. 3.3 HWs and EPEs in CMIP6 projection It is worth noting that summer HWs and EPEs pose a dual threat to the Yangtze River Basin and its southern region, which has the highest risk of natural disasters in the future. Page 7/25 Page 7/25 4.1 Increased HWs and EPEs in a warming climate Our results in HWs are consistent with several previous studies that eastern China has experienced frequent HWs in recent decades, and such HWs are expected to increase in frequency, severity, and duration (Guo et al. 2017, Wang et al. 2017, Dosio et al. 2018, Z. Li et al. 2019, Ning et al. 2022). The number of EPEs significantly increases in eastern China and is projected to continue to rise by 2100 (Wang and Zhou, 2005, Feng et al. 2011, Liu et al. 2015, Zhang et al. 2017, Dong et al. 2020). Specifically we find that the frequency of EPEs shows upward trends with 0.09±0.01 counts per decade and 0.21±0.01 counts per decade under SSP245 and SSP585 scenarios, the trends of HWN also increased from 0.31±0.01 counts per decade under SSP245 to 0.50±0.02 counts per decade under SSP585 (p < 0.01, Fig. 6a, c). The increase in the atmospheric water-holding capacity associated with a temperature increase (the Clausius–Clapeyron relation) considerably influences the changes in extreme precipitation intensity at a Page 8/25 rate of ~7%/℃ under warmer climates (Pall et al. 2007, Allan and Soden 2008, Utsumi et al. 2011). The increase in AEP and HWT reaches 0.14±0.03 mm per decade and 2.61±0.10 days per decade under the SSP245 scenario, under SSP585 scenario, the frequency of AEP and HWT rises at a rate of 0.37±0.03 mm per decade and 7.77±0.21 days per decade (p < 0.01, Fig. 6b, d). Notably, the trends of HWT and AEP under the SSP585 are 3.0-fold and 2.6-fold higher than those of SSP245, respectively. While the trends of HWN and EPE under the SSP585 are 1.6-fold and 2.3-fold higher than those of SSP245, respectively. This suggests that the intensity of HWs and EPEs changes faster than the frequency. The HWs are becoming more frequent, longer lasting, and more intense in eastern China under global warming background (Fig. S7). On the other hand, global warming will alter atmospheric circulation and evaporation in some regions, providing a richer source of water for precipitation (IPCC 2018). The synergy of these two factors increases the probability of EPEs in humid regions (Allan and Soden 2008) (Fig. S8). Summer Tasmax anomalies in eastern China are shown to increase by to 1 ℃ in 2020 and are projected to be 2.5 ℃ and 4.8 ℃ warmer under SSP245 and SSP585 scenarios than the period of 1850-1900 (Fig. S9). 4.1 Increased HWs and EPEs in a warming climate Consequently, the Yangtze River Basin and its southern regions will face the combined disasters of HWs and EPEs in summer in the future projections, becoming a regional climate change hotspot. 4.2 Underlying mechanisms and connections between HWs and EPEs Anthropogenic factors contributes to the rising occurrence of heavy precipitation and high-temperature extremes globally (Fischer and Knutti 2015, Dosio et al. 2018). The rapid increase in the risk of summer HWs and EPEs in easter China can also be partially attributed to global warming (Sun et al. 2014, Liu et al. 2015, Freychet et al. 2017). Furthermore, SST anomaly (SSTA) plays an important role in modulating HWs and EPEs in China (Zhu et al. 2011, Wang et al. 2017, Wei et al. 2020). When the SSTAs in the Indian Ocean and Tropical North Atlantic are positive, they contribute to the positive rainfall anomalies in South China and the southeast coast (Fig. 7c, d). The long-term climate variations in China in summer may be related to the warming trend of SST in the Indian Ocean (Hu et al. 2003) and teleconnections from the North Atlantic (Shang et al. 2020). Previous studies have shown that summer precipitation over central eastern and southern China are attributed to the warming trend of the ENSO-like SSTAs in the tropical Pacific, Indian Ocean, and North Atlantic, which will trigger anomalous anticyclonic circulation over Philippine Sea (Yang and Lau, 2004, Weijing Li et al. 2018, J. Liu et al. 2019). When the SSTAs in the west Pacific, Tropical North Atlantic, and the Indian Ocean are positive, they can cause widespread warming in eastern China (Fig. 7e, g, h). The summer three-ocean SSTAs have a greater influence on Tasmax than on precipitation in eastern China, but the influences have significant variations from region to region. For example, the influence of Indian Ocean SST on precipitation and maximum temperature in eastern China is opposite (Fig. 7c, g). Notably, the west Pacific and Niño 3.4 SST anomalies alone have little influence on the summer precipitation in eastern China (Fig. 7a, b, f), which confirms that tropical Indian Ocean SST warming acts like a capacitor affecting summer climate ndian Ocean SST warming acts like a capacitor affecting summer climate Page 9/25 Page 9/25 Page 9/25 anomalies over the Indo-western Pacific and East Asia, and the three-ocean interactions through ocean- atmosphere coupling can modulate climate variability (Xie et al. 2009, Cai et al. 2019, Wang 2019). Moreover, summer EPEs and HWs in eastern China are closely related to the strength and location of the western North Pacific subtropical high (WNPSH), and their influences vary across space (Zhu et al. 4.2 Underlying mechanisms and connections between HWs and EPEs mean anomalous precipitation and Tasmax is negative in eastern China, while the regions with the positive correlation area appear in the eastern coast of China under SSP245 scenario and expand rapidly under SSP585 scenario (Fig. 8a, c, e). Climate models suggest that EPEs and HWs will become more common in an anthropogenically warmed climate (IPCC, 2014). The correlations between the frequencies of EPEs and HWs shifted from negative in the historical period to positive under future projections (Fig. 8b, d, f). These results reveal a distinct link between future rainfall and temperature, with increased EPEs significantly associated with HWs during summer (Raghavendra et al. 2019). In addition, summer precipitation and maximum temperature were negatively correlated, consistent with the argument that warmer summers tend to be dryer, but this negative correlation is largely reduced in extreme cases. HWs are strongly linked to global warming, and previous studies have shown a significant increase in global HW activity from present climate to future projections (Hu et al. 2003, Freychet et al. 2017, Dosio et al. 2018, Perkins-Kirkpatrick and Lewis 2020). However, the compound disasters of HWs and EPEs in a warming climate are rarely studied. In eastern China, as the correlations between the frequencies of EPEs and HWs shifts from negative to positive in summer, this will lead to future loss of life and property and enormous socioeconomic consequences. It further supports the urgent need for policymakers to take action to curb greenhouse emissions. 4.2 Underlying mechanisms and connections between HWs and EPEs 2011, Freychet et al. 2017, Zhang et al. 2017). Summer Tasmax and frequency of HWs have a higher correlation with WNPSH than precipitation and frequency of EPEs (Fig. S10). The high value area of the correlation coefficient between anomalous Tasmax and 500-hPa geopotential height (GPH) appeared in the northern and eastern coasts of China, reaching above 0.6 (p < 0.05), the correlation coefficient between the anomalous frequency of HWs and GPH was mainly range from 0.4 to 0.6 (p < 0.05), and the spatial distribution of high value areas is scattered (Fig. S10 c, d). Notably, the intensification of the WNPSH is favorable for more summer HWs in eastern China under present climate, and more monsoon rainfall and HWs in future projections (Q. Liu et al. 2019, X. Chen et al. 2020, Li et al. 2021). In addition to WNPSH, other factors such as the tropical cyclones, Pacific Decadal Oscillation/Interdecadal Pacific Oscillation, and Atlantic Multidecadal Oscillation may contribute to the interdecadal or multidecadal variations in the EPEs and HWs in eastern China (Ding et al. 2009, Zhang et al. 2017). Detailed analysis of the impacts of these longer timescale factors on EPEs and HWs is beyond the scope of this manuscript. Intuitively, we expect cooler summers when it rains, while heatwaves often accompany droughts (Trenberth and Shea, 2005). In the historical period, the correlation between (Trenberth and Shea, 2005). In the historical period, the correlation between mean anomalous precipitation and Tasmax is negative in eastern China, while the regions with the positive correlation area appear in the eastern coast of China under SSP245 scenario and expand rapidly under SSP585 scenario (Fig. 8a, c, e). Climate models suggest that EPEs and HWs will become more common in an anthropogenically warmed climate (IPCC, 2014). The correlations between the frequencies of EPEs and HWs shifted from negative in the historical period to positive under future projections (Fig. 8b, d, f). These results reveal a distinct link between future rainfall and temperature, with increased EPEs significantly associated with HWs during summer (Raghavendra et al. 2019). In addition, summer precipitation and maximum temperature were negatively correlated, consistent with the argument that warmer summers tend to be dryer, but this negative correlation is largely reduced in extreme cases. 5. Conclusion Page 10/25 EPEs and HWs can significantly affect the socioeconomic losses and human health risks related to extreme climate events during summer, especially in the densely populated and economically active areas of the East Asian summer monsoon region. This study attempted to investigate the current and future spatiotemporal characteristics of EPEs and HWs in eastern China and the impacts of HWs on EPEs changes in a warming world. The main conclusions based on the above analysis are as follows: EPEs and HWs can significantly affect the socioeconomic losses and human health risks related to extreme climate events during summer, especially in the densely populated and economically active areas of the East Asian summer monsoon region. This study attempted to investigate the current and future spatiotemporal characteristics of EPEs and HWs in eastern China and the impacts of HWs on EPEs changes in a warming world. The main conclusions based on the above analysis are as follows: 1. The regions with the largest EPEs are apparent in the south of the Yangtze River Basin and its western part, with an average of 3–5 EPEs yr-1 during 1979–2019, the regions with large mean AEP appear in the southeast coast, Yangtze River Basin, North China Plain, and the southern coast of Northeast Plain, ranging from 60 to 110 mm. The metrics of HWs all show increase trends over 1979–2019, and the increased HWN resulted in a rapid increase of HWT, with rates of 0.69±0.22 days per decade and 0.12±0.03 counts per decade, respectively. 2. In terms of spatial variation, the high values of multi-year mean HWT are 20–30 days and 50–70 days under SSP245 and SSP585 scenarios, respectively. The increased HWDU and HWN lead to a rapid increase in HWT. However, there is little change in the frequency of EPEs and AEP from SSP245 to SSP585 scenarios. In terms of temporal variation, HWs and EPEs are both increased in future projections. The Yangtze River Basin and its southern regions will face the compound disaster of HWs and EPEs in summer in the future projections. 3. High values of the annual mean total person-times affected by EPEs and HWs are observed in the North China Plain, Yangtze River Delta, Sichuan Basin, and southeast coast, with over four million person- times. The total person-times affected by EPEs range between 3.5 billion to 4.5 billion in eastern China shows a slightly decrease trend under both scenarios. 5. Conclusion The total person-times affected by HWs under SSP245 scenario maximize at around 4.0 billion, lower than the peak person-times (about 5.0 billion) under SSP585 scenario. 4. The increased EPEs and HWs are primarily caused by anthropogenic warming. Positive summer SSTs (West Pacific, Tropical North Atlantic, and the Indian Ocean) can cause widespread warming in eastern China and a regional increase in rainfall in the Yangtze River Basin. Moreover, summer Tasmax and frequency of HWs have a higher correlation with WNPSH than precipitation and frequency of EPEs. We detect strong positive correlation between mean-state (Tasmax or Precipitation) and extreme climate (HWs or EPEs), this implies that improved prediction of mean-state climate can provide insights into the extreme prediction. In eastern China, summer precipitation and temperature were negatively correlated, consistent with the argument that warmer summers tend to be dryer, but this negative correlation is largely reduced in extreme cases. As the correlations between the frequencies of EPEs and HWs shifts from negative to positive in summer, this will lead to future loss of life and property and enormous socioeconomic consequences. Data Availability All the datasets used in this study are publicly available. The NOAA High Resolution SST V2.1 data is provided by the NOAA/OAR/ESRL PSD, Boulder, Colorado, USA, from their Web site at https://psl.noaa.gov/data/gridded/data.noaa.oisst.v2.highres.html. ERA5 dataset is available at https://www.ecmwf.int/en/forecasts/datasets/reanalysis-datasets/era5. The daily 2-m maximum surface air temperature and precipitation datasets are available at http://data.cma.cn/en. The daily climate model outputs are obtained from https://esgf-node.llnl.gov/projects/cmip6/. Declarations Page 11/25 References 1. Allan RP, Soden BJ (2008) Atmospheric warming and the amplification of precipitation extremes. Science 321: 1481-1484. 2. Bao J, Sherwood SC, Alexander LV et al (2017) Future increases in extreme precipitation exceed observed scaling rates. Nat Clim Chang 7: 128-132. 2. Bao J, Sherwood SC, Alexander LV et al (2017) Future increases in extreme precipitation exceed observed scaling rates. Nat Clim Chang 7: 128-132. 3. 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Trenberth KE, Shea DJ (2005) Relationships between precipitation and surface temperature. Geophys Res Lett 32: L14703. 67. Trenberth KE, Shea DJ (2005) Relationships between precipitation and surface temperature. Geophys Res Lett 32: L14703. 68. Utsumi N, Seto S, Kanae S (2011) Does higher surface temperature intensify extreme precipitation? Geophys Res Lett 38: L16708. 68. Utsumi N, Seto S, Kanae S (2011) Does higher surface temperature intensify extreme precipitation? Geophys Res Lett 38: L16708. 69. Wang P, Tang J, Sun X et al (2017). Heat waves in China: Definitions, leading patterns, and connections to large‐scale atmospheric circulation and SSTs. J Geophys Res: Atmos 112: 10679- 10699. Page 15/25 Page 15/25 70. Wang C (2019) Three-ocean interactions and climate variability: A review and perspective. Clim Dyn 53: 5119-5136. 71. Wang P, Hui P, Xue D et al (2019) Future projection of heat waves over China under global warming within the CORDEX-EA-II project. Clim Dyn 53: 957-973. 72. Wang Y, Zhou L (2005) Observed trends in extreme precipitation events in China during 1961–2001 and the associated changes in large‐scale circulation. Geophys Res Lett 32: L09707. 73. Wei J, Wang W, Shao Q et al (2020) Heat wave variations across China tied to global SST modes. J Geophys Res: Atmos 125: e2019JD031612. 74. Westra S, Alexander LV, Zwiers FW (2013) Global increasing trends in annual maximum daily precipitation. J Clim 26: 3904-3918. 75. Wu, L, Wang C (2015) Has the western Pacific subtropical high extended westward since the late 1970s? J Clim 28: 5406-5413. 76. Wu X, Guo S, Yin J et al (2018) On the event-based extreme precipitation across China: Time distribution patterns, trends, and return levels. J Hydrol 562: 305-317. 77. Wu Y, Wu S, Zhai P (2007) The impact of tropical cyclones on Hainan Island's extreme and total precipitation. Int J Climatol 27: 1059-1064. 78. Xie S, Hu K, Hafner J et al (2009) Indian Ocean capacitor effect on Indo–western Pacific climate during the summer following El Niño. J Clim 22: 730-747. 79. References Recent changes in the summer precipitation pattern in East China and the background circulation. Clim Dyn 36: 1463-1473. References Xu P, Wang L, Liu Y et al (2020) The record‐breaking heat wave of June 2019 in Central Europe. Atmos Sci Lett 21: e964. 80. Xu X (2017) The 1 km grid dataset of China's population spatial distribution. htt // d /DOI/DOI ?DOIid 32 80. Xu X (2017) The 1 km grid dataset of China's population spatial distribution. https://www.resdc.cn/DOI/DOI.aspx?DOIid=32. 81. Yang F, Lau KM (2004) Trend and variability of China precipitation in spring and summer: linkage to sea‐surface temperatures. Int J Climatol 24: 1625-1644. 82. Yao Y, Wang C (2021) Variations in summer marine heatwaves in the south China sea. J Geophys Res: Oceans, 126: e2021JC017792. 82. Yao Y, Wang C (2021) Variations in summer marine heatwaves in the south China sea. J Geophys Res: Oceans, 126: e2021JC017792. 83. Zhang Q, Zheng Y, Singh VP et al (2017) Summer extreme precipitation in eastern China: Mechanisms and impacts. J Geophys Res: Atmos 122: 2766-2778. 84. Zhang W, Kirtman B, Siqueira L et al (2022). Decadal variability of southeast US rainfall in an eddying global coupled model. Geophys Res Lett 49: e2021GL096709. 85. Zhang W, Pan S, Cao L et al (2015) Changes in extreme climate events in eastern China during 1960–2013: A case study of the Huaihe River Basin. Quatern Int 380: 22-34. 86. Zhang W, Zhou T (2020) Increasing impacts from extreme precipitation on population over China with global warming. Sci Bull 65: 243-252. 86. Zhang W, Zhou T (2020) Increasing impacts from extreme precipitation on population over China with global warming. Sci Bull 65: 243-252. 87. Zheng J, Wang C (2019) Hot summers in the Northern Hemisphere. Geophys Res Lett 46: 10891- 10900. Page 16/25 88. Zhou T, Yu R (2005) Atmospheric water vapor transport associated with typical anomalous summer rainfall patterns in China. J Geophys Res: Atmos 110: D08104. 89. Zhou Y, Liu Y, Wu W et al (2015) Integrated risk assessment of multi-hazards in China. Nat Hazards 78: 257-280. 90. Zhu Y, Wang H, Zhou W et al (2011). Recent changes in the summer precipitation pattern in East China and the background circulation. Clim Dyn 36: 1463-1473. 90. Zhu Y, Wang H, Zhou W et al (2011). Recent changes in the summer precipitation pattern in East China and the background circulation. Clim Dyn 36: 1463-1473. 90. Zhu Y, Wang H, Zhou W et al (2011). 88. Zhou T, Yu R (2005) Atmospheric water vapor transport associated with typical anomalous summer rainfall patterns in China. J Geophys Res: Atmos 110: D08104. Figures Figure 1 Page 17/25 Page 17/25 Page 17/25 Topography and the Kuroshio path in the study area. Eastern China is in the east of the cyan line (100 °E). Figure 2 Spatial distributions of the multi-year average (a) frequency of EPEs and (b) AEP in summer over 1979– 2019. Spatial distribution of total (c) 1-day EPEs and (d) its corresponding variation in summer over Figure 2 Figure 2 Spatial distributions of the multi-year average (a) frequency of EPEs and (b) AEP in summer over 1979– 2019. Spatial distribution of total (c) 1-day EPEs and (d) its corresponding variation in summer over Page 18/25 Page 18/25 Page 18/25 1979–2019. Purple lines represent the Yangtze River Basin and the dashed line represents the linear trend. Page 19/25 Figure 3 Spatial distributions of the multi-year average of summer heatwa corresponding temporal variations (right column) in eastern Chin Figure 3 Spatial distributions of the multi-year average of summer heatwave metrics (left column) and their corresponding temporal variations (right column) in eastern China from 1982 to 2019. (a, e) mean HWT, Figure 3 Figure 3 Spatial distributions of the multi-year average of summer heatwave metrics (left column) and their corresponding temporal variations (right column) in eastern China from 1982 to 2019. (a, e) mean HWT, Spatial distributions of the multi-year average of summer heatwave metrics (left column) and their corresponding temporal variations (right column) in eastern China from 1982 to 2019. (a, e) mean HWT, Page 19/25 (b, f) mean HWDU, (c, g) mean HWN, and (d, h) mean HWI. The purple lines delimit the Yangtze River Basin and the dashed lines represent the linear trend. Figure 4 Spatial distributions of multi year averaged (left column) HWT (shading area Unit: Figure 4 Figure 4 Spatial distributions of multi-year averaged (left column) HWT (shading area, Unit: Days) with the frequency of EPEs (blue contour line, Unit: Counts) and AEP (green contour line, Unit: mm), (right column) Page 20/25 Page 20/25 HWN (shaded area, Unit: Counts) with the frequency of EPEs and AEP under SSP245 (a, c, e, and g) and SSP585 (b, d, f, and h) scenarios in eastern China over 2021–2100. The purple lines present the Yangtze River Basin. Figure 5 Spatial patterns of multi-year average person-times affected by frequencies of (a) EPEs and (b) HWs in eastern China over 1979–2019. The inset charts represent the annual average of person-times affected by EPEs (green line) and HWs (pink line). Dashed lines represent the linear trend and the purple lines present the Yangtze River Basin. Figure 5 Figure 5 Spatial patterns of multi-year average person-times affected by frequencies of (a) EPEs and (b) HWs in eastern China over 1979–2019. The inset charts represent the annual average of person-times affected by EPEs (green line) and HWs (pink line). Dashed lines represent the linear trend and the purple lines present the Yangtze River Basin. Page 21/25 Page 21/25 Figure 6 Temporal variations of the regionally averaged summer frequency of EPEs and AEP compared with HWN under SSP245 and SSP585 scenarios in eastern China over 2021–2100. (a) Frequency of EPEs and HWN under SSP245, (b) AEP and HWT under SSP245, (c) EPEs and HWN under SSP585, and (d) AEP and HWT under SSP585. Dashed blue and red lines represent the linear trend. Figure 6 Figure 6 Temporal variations of the regionally averaged summer frequency of EPEs and AEP compared with HWN under SSP245 and SSP585 scenarios in eastern China over 2021–2100. (a) Frequency of EPEs and HWN under SSP245, (b) AEP and HWT under SSP245, (c) EPEs and HWN under SSP585, and (d) AEP and HWT under SSP585. Dashed blue and red lines represent the linear trend. Page 22/25 Page 22/25 Figure 7 Anomalous summer rainfall (left column, unit: mm) and Tasmax fie onto the (a, e) West Pacific SSTA (0–10°N, 130°E–150°E), (b, f) Niñ (c, g) Indian Ocean SSTA (10°S–20°N, 50°E–90°E), and (d, h) Tropic Figure 7 Anomalous summer rainfall (left column, unit: mm) and Tasmax fields (right column, unit: ℃) regressed onto the (a, e) West Pacific SSTA (0–10°N, 130°E–150°E), (b, f) Niño 3.4 SSTA (5°S–5°N, 170°W–120°W), (c, g) Indian Ocean SSTA (10°S–20°N, 50°E–90°E), and (d, h) Tropical North Atlantic SSTA (0°–25°N, 15°W–90°W). The areas significant at the 95% confidence level are dotted. The purple lines delimit the Yangtze River Basin. Figure 7 Anomalous summer rainfall (left column, unit: mm) and Tasmax fields (right column, unit: ℃) regressed onto the (a, e) West Pacific SSTA (0–10°N, 130°E–150°E), (b, f) Niño 3.4 SSTA (5°S–5°N, 170°W–120°W), (c, g) Indian Ocean SSTA (10°S–20°N, 50°E–90°E), and (d, h) Tropical North Atlantic SSTA (0°–25°N, 15°W–90°W). The areas significant at the 95% confidence level are dotted. The purple lines delimit the Yangtze River Basin. Page 23/25 Page 23/25 Figure 8 The correlation maps between summer (a, c, and e) precipitation and Tasmax anomalies, (b, d, and f) frequencies of EPEs and HWs anomalies from the historical period (1979–2019) to future projections (2021–2100). The areas significant at the 95% confidence level are dotted. The purple lines delimit the Yangtze River Basin. Figure 8 Figure 8 The correlation maps between summer (a, c, and e) precipitation and Tasmax anomalies, (b, d, and f) frequencies of EPEs and HWs anomalies from the historical period (1979–2019) to future projections (2021–2100). The areas significant at the 95% confidence level are dotted. The purple lines delimit the Yangtze River Basin. Page 24/25 Page 24/25 SupplementaryInformation.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryInformation.docx Page 25/25
https://openalex.org/W2898518492	https://europepmc.org/articles/pmc6343082?pdf=render	English	null	The evolving role of cardiac magnetic resonance in primary mitral regurgitation: ready for prime time?	European heart journal. Cardiovascular imaging	2,018	cc-by	7,452	Boyang Liu1, Nicola C. Edwards1, Dudley Pennell2, and Richard P. S 1Department of Cardiology, University Hospital Birmingham and Institute of Cardiovascular Science, University of Birmingham, Edgbaston, Birmingham B15 2TH, UK; and 2CMR Unit, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK Received 19 January 2018; editorial decision 6 September 2018; accepted 16 September 2018; online publish-ahead-of-print 25 October 2018 A ﬁfth of patients with primary degenerative mitral regurgitation continue to present with de novo ventricular dysfunction following sur- gery and higher rates of heart failure, morbidity, and mortality. This raises questions as to why the left ventricle (LV) might fail to recover and has led to support for better LV characterization; cardiac magnetic resonance (CMR) may play a role in this regard, pending further research and outcome data. CMR has widely acknowledged advantages, particularly in repeatability of measurements of volume and ejec- tion fraction, yet recent guidelines relegate its use to cases where there is discordant information or poor-quality imaging from echocardi- ography because of the lack of data regarding the CMR-based ejection fraction threshold for surgery and CMR-based outcome data. This article reviews the current evidence regarding the role of CMR in an integrated surveillance and surgical timing programme. Keywords Keywords cardiac magnetic resonance • primary mitral regurgitation • echocardiography • ventricular remodelli Introduction Despite clear American Heart Association/American College of Cardiology/European Society of Cardiology (AHA/ACC/ESC) guide- lines on the timing of surgery, a fifth of patients with severe primary mitral regurgitation (MR) continue to present post-operatively with reduced ejection fraction and an increased risk of congestive cardiac failure.1,2 These data raise questions regarding the sole use of 2D echocardiography-based dimensions and ejection fraction as Class 1 indications for surgery. Recent guidelines by the American Society of Echocardiography in collaboration with the Society for Cardiovascular Magnetic Resonance acknowledged the role of car- diac magnetic resonance (CMR) but relegated its use to cases where there is discordant information or poor-quality imaging on echo.3 CMR has widely acknowledged advantages in monitoring size and function of the LV, and there are preliminary data highlighting dis- crepancies in quantification of primary MR with echo. This article reviews the current evidence on the role of CMR in an integrated surveillance and surgical timing programme. Complete analysis of the components of the mitral valve (MV) and subvalvar apparatus are required to identify the lesion, aetiology, and type of dysfunction in MR. Such an analysis requires assessment of the leaflets, annulus, chordae tendonnae, and papillary muscles. .................................. REVIEW REVIEW European Heart Journal - Cardiovascular Imaging (2019) 20, 123–130 doi:10.1093/ehjci/jey147 Corresponding author. Tel: þ44 (121) 371 4035; Fax: þ44 (121) 4629. E-mail: boyang.liu@nhs.net V C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Cardiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. * Corresponding author. Tel: þ44 (121) 371 4035; Fax: þ44 (121) 4629. E-mail: boyang.liu@nhs.net V C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Cardiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Identification of prolapse and assessment of leaflets Assessment and measurements on 2D and 3D echo offer high accur- acy and reproducibility due to high-temporal resolution and improved frame rates. While 2D transthoracic echo (TTE) is recom- mended as the first-line examination, 3D TTE acquisitions provide in- cremental value in establishing the lesion, leaflet, and location of the segments involved, with an accuracy that is equivalent to 2D multi- plane transoesophageal echo (TOE).4 3D TOE has the greatest over- all accuracy with a higher specificity in the segmental localization of 124 B. Liu et al. defects (92.8%) compared with surgical findings and is particularly ef- fective in the localization of flail defects (99%). Moreover, 3D TOE has the advantage of producing both static and dynamic measure- ments of MV anatomy and pathology that can predict both the com- plexity of repair and likely outcome at surgery, including prolapse height, prolapse volume, and anterior leaflet surface area and leaflet length (Supplementary data online, Table S1).4,5 superior displacement of PM tips during systole as one mechanism contributing to MV prolapse.12 3D TOE has demonstrated PM dis- placement secondary to progressive LV dilatation with resultant leaf- let tethering, as well as the relationship between PMs and LV wall which may assist surgical planning of MV repair.13 Although CMR can accurately locate, measure size, and track PM displacement, as well as identify PM infarction/fibrosis,14 the clinical implications of these find- ings in primary degenerative MR remain unclear. ................................................................................................ Data comparing echo with CMR for the detection of leaflet abnor- malities are limited. Annulus Two difficulties have been encountered in establishing an optimal imaging strategy for the MV annulus: firstly, the surgical reference for sizing is performed in a de-aired heart, and therefore, these measure- ments do not correspond to ‘live’ imaging; secondly, 3D TOE has highlighted that the annulus is not fixed but dynamic throughout the cardiac cycle, with change in size (early systolic area contraction) and shape (deepening of the saddle-shape). There are no data on the ac- curacy of CMR-based assessment of MV annular dimensions com- pared with echo or surgical findings in primary MR, meaning that 3D echo is currently gold standard.10 Severity of MR: a potential role for CMR? Echo is the principal imaging modality to diagnose and establish sever- ity of MR but requires integration of multiple qualitative, semi-quanti- tative, and quantitative parameters.3 The echocardiographer must also weigh up the different specificity of each parameter and is encouraged to use quantitative measures to grade MR severity, including pulsed Doppler, volumetric and flow convergence [prox- imal isovelocity surface area (PISA)] methods (Supplementary data online, Table S1). Despite the recommendation to quantify MR, a re- cent communication from the ACC recognized that inadequate qual- ity of echo may be a barrier to optimal care of almost a quarter of patients with MR and that many decisions regarding severity of MR are still made on echo by visually estimating severity based on colour, with a minority of reports providing regurgitant volumes and frac- tion.15 Even when recommendations are followed, quantification by echo is subject to several errors, including practical issues such as over-estimation of non-holosystolic Doppler profiles and failure to account for regurgitant blood volume on the atrial side of the MV annulus in patients with extensive bileaflet prolapse which is not ac- countable when using a Simpson’s method for quantifying LV end- systolic volume.16 Pulsed Doppler methods to estimate differential LV and aortic stroke volumes are valuable, particularly in the pres- ence of multiple jets, constrained and non-holosystolic jets when PISA and vena contracta (VC) are less reliable. However, limitations include the combination of several parameters that carry intrinsic error and increase as the root sum square so that, even under re- search conditions, the volume bias and limits of agreement between observers for aortic stroke volume (4.7± 29.1 mL) and mitral stroke volume (2.9± 19.6 mL) are poor.17 Likewise, questions have been raised about the reliability of flow convergence even amongst experts from university institutions.18 When grading MR as severe or non- severe, the overall interobserver agreement for PISA measurements was poor: 0.37 [95% confidence interval (CI): 0.16–0.58]. A significant source for this variation is the frame-to-frame and beat-to-beat vari- ation in PISA distance which have been quantified as 15–25% even in expert hands, highlighting the importance of multi-frame averaging [estimated at approximately 35 beats if clinicians require an effective regurgitant orifice area (EROA) with CI of ±10%] if precision is to be ................................................................................................. Identification of prolapse and assessment of leaflets CMR requires a dedicated set of sequences that requires time and experience and those most commonly used have lower temporal and spatial resolution than echo.6 However, using a set protocol with dedicated valve imaging planes on CMR, one group diagnosed the presence of posterior mitral valve leaflet prolapse with 100% sensitivity and anterior mitral valve leaflet prolapse with 78% sensitivity when compared with 2D TTE, but the study did not com- pare accuracy to surgical findings.7 In another study of 27 patients, CMR correctly identified the presence or absence of a segmental de- fect in 82% comparing to 2D TTE.8 In 43 patients using surgical find- ings as the reference standard, results on CMR and 2D TOE were concordant in terms of diagnosing prolapse and identification of the leaflet at fault but data on identification of the scallop at fault was not given.9 Correct identification not only of the leaflet at fault but of the specific scallop is now the minimum standard in preoperative assess- ment of primary MR, and the degree of accuracy and amount of infor- mation provided by 3D echo far surpasses that of CMR in this respect. Moreover, CMR cannot yet deliver the same high-quality en- face view that is standard in 2D and 3D echo as part of pre-operative planning. In summary, echo remains the current gold standard for anatomic and functional assessment of the MV apparatus in primary MR, ena- bling precise localization of the abnormality and determination of aetiology, as well as being key for planning and guiding intervention. There are a few small studies on the role of CMR in defining mitral apparatus anatomy but restricted spatial and temporal resolution remains a limiting factor. Assessment of chordae and papillary muscles The location and attachment of the primary, secondary, and tertiary chordae are critical to current surgical strategies for repair. The loca- tion, length, and thickness of the mitral chordae are best measured on 2D echo due to higher spatial resolution and are best visualized on 2D TOE from the transgastric view.11 Although the subvalvular apparatus, including chordae, leaflets, and papillary muscle attach- ments, can be seen on CMR, detailed assessment is much more lim- ited compared with 2D echo which, remains the modality of choice. Assessment of papillary muscle (PM) location, displacement, and function (whether ruptured, fibrotic, infarcted, or displaced) is im- portant for establishing the mechanism of the MR yet there are no comparative data on the utility of echo and CMR in this regard. Data with echo are more compelling; 2D TTE has demonstrated abnormal 125 Cardiac magnetic resonance imaging in primary MR achieved.19 While this may be impractical in routine clinical practice, any degree of averaging will reduce variation. Additionally, the future validation and adoption of automated averaging EROA20 and auto- mated 3D colour flow Doppler quantification systems21 may en- hance the accuracy and consistency of echocardiographic MR quantification. (see Table 2). In recent small prospective studies, CMR quantification of regurgitant volume better predicted LV remodelling after surgery [change in left ventricular end-diastolic volume (LVEDV), r = 0.85; P < 0.0001]; whereas no correlation was present for echo (r = 0.32; P = 0.1).31 In addition, a post-operative decrease in LV internal dimen- sions most closely correlated (r = 0.69) with the regurgitant volume by CMR.26 These studies of post-operative outcome both suggested that a lower threshold for severe MR grading may be needed for CMR and is supported by two recent prospective observational stud- ies. In 109 asymptomatic MR patients, severe MR was defined as a regurgitant volume >55 mL or regurgitant fraction >40% using the SVol-AVf method on baseline CMR.32 Using these cut-offs, over a mean follow-up of 2.5years, subjects with a regurgitant volume of >55 mL had a surgery-free survival rate of 21% at 5years compared with 91% in those with regurgitant volume <_55 mL. Assessment of chordae and papillary muscles Similarly a se- cond study on 258 asymptomatic patients found a regurgitant volume of >_50 mL to possess 77% sensitivity and 78% specificity for identify- ing mortality or indication for MV surgery over a median follow-up of 5years.27 In contrast to current guideline-based echocardiographic approaches, estimation of MR severity by CMR (Supplementary data online, Table S1) is reliant on a small number of quantitative methods that have been validated against other reference standards, including cardiac catheteriza- tion,22 quantitative Doppler TTE,23 and current single-frame 2D PISA- based methods.24 Studies that compare the reproducibility of TTE and CMR in the same patients however are limited. In a prospective study of 26 patients with a range of severity in MR, regurgitant volume was similar between quantitative Doppler, flow convergence TTE, and CMR, but variability in measurements by all methods was poor between observers [Doppler -10 mL (95% CI -76 to 56mL); PISA: -4mL (-21 to 13mL); and CMR 0.7mL (-30 to 32mL)].24 Interobserver variability of regurgi- tant fraction was significantly lower by CMR than TTE in a retrospective study of 70 patients (0.1± 7.3% vs. -5.5± 15%) although this finding only applied to re-analysis of acquired datasets.25 Another retrospective study comparing quantitative assessment of MR highlighted that quantifi- cation by echo was often not achievable in practice, being feasible only in 44 out of 72 patients.26 Where an integrative approach to echocardiographic-based MR severity grading has been applied as per American Society of Echocardiography guidance, intra- and interob- server disagreement is quoted at 20% and 40% on echo.27 On CMR based categorization (severe MR defined as regurgitant volume >_60mL), discordance is reported at 5% and 10%, respectively. Assessment of chordae and papillary muscles Furthermore, in this prospective observational study of 258 asymptom- atic patients, a quarter of patients had a discrepant degree of severity diagnosed on CMR and echo, with the CMR classification offering super- ior prognostic value for predicting the composite endpoint of mortality or MV surgery during the median follow-up of 5years.27 However, there are also limitations to quantifying MR severity by CMR, including the presence of non-compensated Eddy current-induced fields, variability in volumetric analysis according to placement of the basal slice and in phase velocity mapping according to the plane used for measuring aortic flow.28 4D flow phase contrast CMR may be able to overcome some of these limitations but this currently remains a research tool.29 Additionally, echo can easily be combined with exercise testing for cases where exertional symptoms are disproportionate to resting haemo- dynamics,30 whereas this is understandably more challenging for CMR. In summary (Figure 1), CMR quantification of MR is simple, feasible in a greater proportion of patients and from the limited comparative studies, may be more reproducible. However, outcome data remain limited and present guidelines are derived from large echo-based outcome studies such that more data are needed which compare the primary analysis of newly acquired CMR datasets. In summary, single measures of MR severity by echo have wide lim- its of agreement therefore a multi-parametric approach must be used. While CMR has limitations, it uses fewer parameters and observation- al data suggest it is better at predicting severity, need for surgery and LV outcomes, although standardized CMR cut-off points for severe MR need to be agreed upon. Importantly, it remains to be seen whether surgical intervention based on CMR parameters alone can lead to an improvement in patient outcome compared with echo. Chamber size and function: Achilles heel of echo? Serial TTE is the recommended method for measurement of cham- ber size and function in primary MR, with CMR only indicated when echo is ‘unsatisfactory’.3,33 Using a left ventricular end-systolic linear dimension (LVESd) >40 mm on echo is however inherently ‘unsatis- factory’ as a guide to intervention, as it is a poor marker of end- systolic volume in primary MR due to the preferential spherical remodelling process that takes place at the apex and mid-ventricular level.34 On the other hand, accurate CMR volumes may be sufficient to monitor patients, since an indexed LVEDV of <100 mL/m2 from a single baseline CMR conveyed a 90% surgery-free survival rate at 5years.32 Recognising that delaying surgery until the onset of ven- tricular dysfunction with left ventricular ejection fraction (LVEF) <60% or LVESd >40 mm, may be associated with an outcome pen- alty,35,36 the recent AHA/ACC focused update have introduced an additional Class IIa indication as progressive deterioration in LVEF or LVESd on serial imaging.37 Finally, mitral regurgitant volume:LVEDV ratio, which is best measured on CMR, has been proposed as an use- ful indicator that can detect the presence of excessive ventricular dilatation for the degree of MR, thereby highlighting the presence of additional myopathic processes such as ischaemia.38 Furthermore, reminiscent of animal models of volume overload where cardiac de- compensation contributed to further increases in LVEDV in the pres- ence of static volume overload (Supplementary data online, Table S2),39 it is reasonable to hypothesize that longitudinal regurgitant vol- ume: LVEDV ratio measurements may be able to identify Are severity cut-offs equivalent on echo and CMR? Several studies have found a discrepancy in the grading of severity be- tween CMR and TTE, with a tendency for echo to over-estimate pri- mary MR (Table 1).26,27,31 These data support a role for CMR in such cases where echo grading is qualitative, indeterminate, or incomplete 126 B. Liu et al. gure 1 The relative benefits of echocardiography and cardiac magnetic resonance in the management of primary degenerative mit Figure 1 The relative benefits of echocardiography and cardiac magnetic resonance in the management of primary degenerative mitral regurgitation. Figure 1 The relative benefits of echocardiography and cardiac magnetic resonance in the management of primary degenerative mitral regurgitation. asymptomatic primary MR patients who are in the early transitional stages of decompensation. For now, although it is not known whether serial imaging by CMR would result in improved patient out- comes compared with echo, the acknowledged greater reliability of CMR volumetric measurements argues for more routine use in surveillance. LVEF is problematic in MR, whether measured by echo or CMR, as it is a measure of chamber function that is affected by changes in loading conditions. In recognition of this inherent weakness in serial LVEF, recent efforts have focused on identifying more sensitive ‘sub-acute’ markers of LV impairment. These include echo based tissue Doppler (TDI) for measurement of myocardial systolic and diastolic velocities (TDI),40 2D asymptomatic primary MR patients who are in the early transitional stages of decompensation. For now, although it is not known whether serial imaging by CMR would result in improved patient out- comes compared with echo, the acknowledged greater reliability of CMR volumetric measurements argues for more routine use in surveillance. 127 Cardiac magnetic resonance imaging in primary MR ...................................................................................................................................................................................................................... Table 1 Example cases highlighting the differing beneﬁts of echo vs. CMR Case 1: 20-year-old $ Case 2: 69-year-old $ Case 3: 45-year-old # Case 4: 45-year-old # Moderate on TOE Severe on TOE Severe on TOE MV anatomy Severe on CMR Moderate on CMR Severe on CMR Echo vs. CMR Bileaﬂet myxomatous degeneration resulting in holosystolic CW Doppler, systolic ﬂow reversal in RUPV. MR jet is posteriorly directed, wrapping around LA. VC 6 mm. Bileaﬂet myxomatous degeneration resulting in multiple jets. Are severity cut-offs equivalent on echo and CMR? MR is mainly anteriorly directed (VC area 79 mm2, systolic ﬂow reversal in RUPV, PISA radius 10 mm, LV SVol - LVOT SVol = 60 mL) Complex valve lesion with ﬂail A3/P3 scallops from chordal rupture, involv- ing the posteromedial commissure. EROA 70 mm2, LV SVol - LVOT SVol = 104 mL, systolic ﬂow reversal in LUPV. Severe MR due to ﬂail P2/P3 with ruptured secondary chord (arrows) from posteromedial papillary muscle. PISA 14 mm, LV SVol - LVOT SVol = 68 mL, systolic ﬂow reversal in LUPV. Colour Doppler Colour Doppler Colour Doppler CMR en-face valve view CMR - end systole CMR - end diastole CMR - aortic ﬂow A2P2 and A3P3 scallops BSA 1.87; LVEDVi 98 mL/m2; LVESV 26 mL/m2; LVSV 133 mL; LVEF 73%; AVﬂow 80 mL; LA volume 43 mL/ m2; MRvol 55 mL; MRfraction 41%. BSA 1.77; LVEDVi 76 mL/m2 LVESVi 18 mL/m2; LV SV 102 mL; LVEF 76%; AVﬂow 72 mL; MRvol 30 mL; MRfraction 30%. BSA 2.5; LVEDVi 88 mL/m2; LVESVi 21 mL/m2; LVSV 169 mL; LVEF 76%; MRvol 77 mL; MRfraction 46%. ...................................................................................................................................................................................................................... Table 1 Example cases highlighting the differing beneﬁts of echo vs. CMR Case 1: 20-year-old $ Case 2: 69-year-old $ Case 3: 45-year-old # Case 4: 45-year-old # Moderate on TOE Severe on TOE Severe on TOE MV anatomy Severe on CMR Moderate on CMR Severe on CMR Echo vs. CMR Table 1 Example cases highlighting the differing beneﬁts of echo vs. CMR CMR en-face valve view Colour Doppler Colour Doppler Colour Doppler Colour Doppler CMR en-face valve view CMR - end systole A2P2 and A3P3 scallops CMR - aortic ﬂow CMR - end diastole A2P2 and A3P3 scallops CMR - end systole CMR - end diastole CMR - aortic ﬂow BSA 1.77; LVEDVi 76 mL/m2 LVESVi 18 mL/m2; LV SV 102 mL; LVEF 76% AVﬂow 72 mL; MRvol 30 mL MRfraction 30%. BSA 2.5; LVEDVi 88 mL/m2; LVESVi 21 mL/m2; LVSV 169 mL; LVEF 76%; MRvol 77 mL; MRfraction 46%. BSA 1.87; LVEDVi 98 mL/m2; LVESV 26 mL/m2; LVSV 133 mL; LVEF 73%; AVﬂow 80 mL; LA volume 43 mL/ m2; MRvol 55 mL; MRfraction 41%. BSA 1.77; LVEDVi 76 mL/m2 LVESVi 18 mL/m2; LV SV 102 mL; LVEF 76%; AVﬂow 72 mL; MRvol 30 mL; MRfraction 30%. BSA 2.5; LVEDVi 88 mL/m2; LVESVi 21 mL/m2; LVSV 169 mL; LVEF 76%; MRvol 77 mL; MRfraction 46%. Discussion points: Case 1: VC of 6 mm suggests moderate MR with CMR quantiﬁcation demonstrating borderline severe MR. Note that the additional end-systolic LV volume below leaﬂets and above MV annulus (shaded area) in extensive prolapses may not be accounted on Simpson’s biplane, but can be taken into account on a CMR short-axis stack. Case 2: Multiple jets of severe MR visualized on TOE, but repeated longitudinal CMR measuring only moderate MR. Case 3: Agreement between echo and CMR despite the presence of complex MR with commissure involvement. Case 4: Echo remains gold standard for MV anatomy assessment. Scallops can be visualized on CMR using dedi- cated valve planes, but there is neither 3D visualization nor sufﬁcient spatial resolution for chordal characterization. AVﬂow, aortic valve ﬂow; BSA, body surface area; CW, continuous wave; LUPV, left upper pulmonary vein; LVEDVi, left ventricular end-diastolic volume index; LVESVi, left ventricular end-systolic volume index; MRfraction, mitral regurgitant fraction; MRvol, mitral regurgitation volume; RUPV, right upper pulmonary vein; SVol, stroke volume. Are severity cut-offs equivalent on echo and CMR? (PASP) >50 mmHg on echo is a Class IIA indication to repair.1,2 Although questions have been raised about the accuracy of PASP by echo45 and the general estimation of the probability of pulmonary hypertension has not been tested in primary MR,46 CMR methods of measuring pressure are not in routine use.47,48 The main benefit of CMR at present lies in assessment of RV chamber size, function, and hypertrophy.49 Doppler measurement of TR Vmax remains central to the management of primary MR and the role of CMR in quantification of RV size and function is supportive only. speckle tracking echo for measurement of strain, strain rate, and tor- sion,41 and the CMR equivalents that include grid-tagging and feature/tis- sue tracking. The Holy Grail of all techniques is to measure detect sub- clinical LV dysfunction in primary MR42 although all are affected by load- ing conditions and none are yet included in current valve guidelines.43 Therefore, at present, accurate and reliable measurement of volumes and ejection fraction remain at the centre of management of patients with asymptomatic primary MR and highlights an important area where CMR can be complementary to existing management pathways. (PASP) >50 mmHg on echo is a Class IIA indication to repair.1,2 Although questions have been raised about the accuracy of PASP by echo45 and the general estimation of the probability of pulmonary hypertension has not been tested in primary MR,46 CMR methods of measuring pressure are not in routine use.47,48 The main benefit of CMR at present lies in assessment of RV chamber size, function, and hypertrophy.49 Doppler measurement of TR Vmax remains central to the management of primary MR and the role of CMR in quantification of RV size and function is supportive only. Are severity cut-offs equivalent on echo and CMR? Discussion points: Case 1: VC of 6 mm suggests moderate MR with CMR quantiﬁcation demonstrating borderline severe MR. Note that the additional end-systolic LV volume below leaﬂets and above MV annulus (shaded area) in extensive prolapses may not be accounted on Simpson’s biplane, but can be taken into account on a CMR short-axis stack. Case 2: Multiple jets of severe MR visualized on TOE, but repeated longitudinal CMR measuring only moderate MR. Case 3: Agreement between echo and CMR despite the presence of complex MR with commissure involvement. Case 4: Echo remains gold standard for MV anatomy assessment. Scallops can be visualized on CMR using dedi- cated valve planes, but there is neither 3D visualization nor sufﬁcient spatial resolution for chordal characterization. AVﬂow, aortic valve ﬂow; BSA, body surface area; CW, continuous wave; LUPV, left upper pulmonary vein; LVEDVi, left ventricular end-diastolic volume index; LVESVi, left ventricular end-systolic volume index; MRfraction, mitral regurgitant fraction; MRvol, mitral regurgitation volume; RUPV, right upper pulmonary vein; SVol, stroke volume. Discussion points: Case 1: VC of 6 mm suggests moderate MR with CMR quantiﬁcation demonstrating borderline severe MR. Note that the additional end-systolic LV volume below leaﬂets and above MV annulus (shaded area) in extensive prolapses may not be accounted on Simpson’s biplane, but can be taken into account on a CMR short-axis stack. Case 2: Multiple jets of severe MR visualized on TOE, but repeated longitudinal CMR measuring only moderate MR. Case 3: Agreement between echo and CMR despite the presence of complex MR with commissure involvement. Case 4: Echo remains gold standard for MV anatomy assessment. Scallops can be visualized on CMR using dedi- cated valve planes, but there is neither 3D visualization nor sufﬁcient spatial resolution for chordal characterization. AVﬂow, aortic valve ﬂow; BSA, body surface area; CW, continuous wave; LUPV, left upper pulmonary vein; LVEDVi, left ventricular end-diastolic volume index; LVESVi, left ventricular end-systolic volume index; MRfraction, mitral regurgitant fraction; MRvol, mitral regurgitation volume; RUPV, right upper pulmonary vein; SVol, stroke volume. 128 B. Liu et al. Are severity cut-offs equivalent on echo and CMR? Table 2 Current guideline indications for CMR and TTE and proposed additive value of CMR to these recommendations Current recommendations for CMR (1) Assessment by echo is unsatisfactory (2) Discrepancy between MR severity and clinical ﬁndings • ASE/SCMR 2017 • ESC/EACTS 2012 • AHA/ACC 2014 Current recommendations for TTE (1) Moderate MR and preserved LV function requires echocardiographic assessment every 1–2 years (2) Severe MR and preserved LV function requires echocardiographic assessment every 6–12 months • ESC/EACTS 2012 • AHA/ACC 2014 Revised recommendations for CMR (1) Unsatisfactory assessment by echo, speciﬁcally: a. Indeterminant ‘moderate-severe’ MR. b. Inability to perform quantitative measurement of MR by echo when severe MR cannot be deﬁnitively included or excluded. c. Borderline ventricular function (LVEF 55–65%) in moderate–severe MR. d. Fall in LVEF below 60% in asymptomatic patients, where a change of <10% has occurred compared to the previous echocardiographic study. (2) Discrepancy between clinical, exercise, and echo ﬁndings (including stress): a. Symptoms with moderate MR and preserved LV function. b. Moderate MR with impaired or dilated LV. (3) Routine use of CMR: a. Moderate MR patients as baseline reference for LV size and function. b. Every 12–24 months for severe MR with preserved LV function (interdigitating with TTE to give imaging assessment every 6–12 months by one modality). Future of CMR imaging in MR? (1) Markers of adverse LV remodelling: a. Strain imaging with tagging or tissue tracking. b. Fibrosis imaging with LGE and T1/ECV mapping. (2) 4D ﬂow phase-contrast CMR Table 2 Current guideline indications for CMR and TTE and proposed additive value of CMR to these recommendations (1) Assessment by echo is unsatisfactory (2) Discrepancy between MR severity and clinical ﬁndings b. Moderate MR with impaired or dilated LV. a. Moderate MR patients as baseline reference for LV size and function. a. Strain imaging with tagging or tissue tracking. b. Fibrosis imaging with LGE and T1/ECV mapping. ECV, extra-cellular volume; LGE, late gadolinium enhancement. ...................................................................... Current limitations and future directions Left atrium (LA) volume above 60mL/m2 on echo has become a Class IIA indication for MV surgery in the European guidelines, al- though 2D TTE appears to under-estimate size compared with a 3D CMR approach.44 A multi-slice short-axis volumetric approach on CMR is considered the gold standard for structural assessment of the LA (Supplementary data online, Table S3) with more accurate and re- liable measurement again arguing for a greater role. CMR remains a modality which is less readily available than echo in many centres and which demands expertise. The most commonly used sequences require acquisition over several cardiac cycles and employ breath-holding that some patients cannot manage, although alternatives are now available that cover the entire LV in a fraction of the time and without need for breath-holding.50 There is no current direct method to quantify MR severity by CMR and much of the data that compare accuracy of techniques in determining outcomes are derived from small, often single centre studies that do not all Summary: when should you do a CMR for MR? 7. Han YC, Peters DC, Salton CJ, Bzymek D, Nezafat R, Goddu B et al. Cardiovascular magnetic resonance characterization of mitral valve prolapse. JACC Cardiovasc Imaging 2008;1:294–303. 8. Gabriel RS, Kerr AJ, Raffel OC, Stewart RA, Cowan BR, Occleshaw CJ. Mapping of mitral regurgitant defects by cardiovascular magnetic resonance in moderate or severe mitral regurgitation secondary to mitral valve prolapse. J Cardiovasc Magn Reson 2008;10:16. Any assessment of a patient attending with primary MR must include a detailed echocardiogram using high-quality 2D and 3D imaging to de- fine the aetio-pathogenesis, involving careful assessment of the leaflets, annulus, chords, and papillary muscles. CMR plays a secondary role in this regard. In the assessment of severity of MR, echo may be sufficient in many cases, but CMR can quantify regurgitation in more cases than 2D or 3D and does so with greater reliability. There appears to be a tendency for echo to over-estimate severity of MR and there are data to suggest that CMR measures may deliver better long-term out- comes, although prospective CMR-based interventional studies are lacking. At present, there are sufficient data not only to support the use of CMR where 2D or 3D echo is incomplete, or results are inde- terminate but also to promote a greater role in an integrated surveil- lance and surgical timing programme for patients with moderate- severe MR. CMR has a recognized primacy compared to 2D echo in measurement of LV volumes and ejection fraction, not least due to the limitations of linear measurements on 2D or M-mode in primary MR. This accuracy suggests that CMR should have a bigger part in monitoring, independent of its advantages in measuring atrial and right-sided volumes. In the future, more comparative research is needed to define the roles of each modality against outcomes in pro- spective studies, and this should also seek to confirm or refute the util- ity of advanced echo techniques and tissue characterization by CMR. 9. Stork A, Franzen O, Ruschewski H, Detter C, Mullerleile K, Bansmann PM et al. Assessment of functional anatomy of the mitral valve in patients with mitral re- gurgitation with cine magnetic resonance imaging: comparison with transesopha- geal echocardiography and surgical results. Eur Radiol 2007;17:3189–98. 10. Grewal J, Suri R, Mankad S, Tanaka A, Mahoney DW, Schaff HV et al. 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https://openalex.org/W4394579942	https://rr.peercommunityin.org/download/t_recommendations.reply_pdf.a3f711b3a0698645.5043495f5f52525f534d495f4573706f7274735f5374616765325f526573705f6c65747465722e706466.pdf	English	null	Synergistic Mindset Intervention in Competitive Situations	Peer community in registered reports	2,024	cc-by	3,476	1 1 1 March 27th, 2024 March 27th, 2024 Dear Dr. Veli-Matti Karhulahti: Dear Dr. Veli-Matti Karhulahti: Please find enclosed the revised version of our manuscript, Applying a Synergistic Mindsets Intervention to an Esports Context (PCI Registered Reports #364) (word count = 24262). The revised manuscript is 87 pages long and includes one table and four figures. We are grateful for the thoughtful comments received during Stage 2 of the review process. Your suggestions helped us to improve the overall quality and robustness of our project. We are pleased to submit the revised Stage 2 version of our manuscript for your consideration. In this revised version, all amended sections have been highlighted in green for easy identification. Additionally, we have provided a detailed summary of deviations from the original registration, ensuring transparency and clarity. We thank you again for considering our work. We look forward to hearing from you and hope you will find this revision appropriate for acceptance in PCI Registered Reports. Sincerely yours, Gratefully, Maciej Behnke Daniël Lakens Kate Petrova Patrycja Chwiłkowska Szymon Jęśko Białek Maciej Kłoskowski Wadim Krzyżaniak Patryk Maciejewski Lukasz D. Kaczmarek Kacper Szymański Jeremy P. Jamieson James J. Gross Maciej Behnke Veli-Matti Karhulahti (Recommender) #1. As noted by LM, the results section could use some clarifications especially in reporting the hypothesis tests. I personally prefer a structure that clearly reports “H1 = outcome, H2 = outcome etc”, as it helps readers to easily locate and assess key findings. That's an option to consider. I also highly support IR’s request to add descriptives. Reply: We aligned the hypothesis with the results across the manuscript, including the Presents Study, Analysis, Results and Design Table. We hope this clarification will help readers find and evaluate critical findings. As we stated in the response to Reviewer 2 #2, we showed the descriptives (M, SD) for the tested variables and all self-reports in Tables S4 and S5. Furthermore, we plan to make all raw psychophysiological and video data from our study publicly available as an open database, accompanied by an article that offers a detailed description of the dataset. Recognizing the dataset's considerable value and its potential for broad reuse in research, we 2 believe that a separate paper is warranted to describe and provide an overview of the collected data fully. Changes in the manuscript: See Analysis section, line numbers 881 - 883. Results section, line numbers 1005:1010, 1026:1037, and Table 1. Design Table. #2. Regarding the optimality of the created conditions and the limits they set to the intervention, I agree with LM and IR that some further engagement with previous related knowledge would make the discussion even more informative. E.g., to my memory, already the first-ever doctoral dissertation on videogames (Elizabeth Ellen Moulds, 1978) investigated arousal in the competitive gaming situation. It could be useful to review the experimental literature of the past decades in detail to further assess whether the stress response in the present study is unique or echo those in previous studies involving similar conditions. Reply: In the updated version of the manuscript, we reviewed the findings from the relevant literature in the discussion. hanges in the manuscript: See Discussion section, line numbers 1139 – 1168, 1245-12 #3. JK and IR point out some wordings in the discussion that should be rephrased. I made similar notes and would also suggest removing the first three words of the phrase “For this reason, we observed no effects..” (p. 47). Many other reasons could be too. Veli-Matti Karhulahti (Recommender) I would also rethink some wordings in the last sentences: "However, we found the limited impact of SMI on the performance outcomes and challenge/threat affective responses... In sum, we recommend the SMI as a prevention tool." Please reconsider the conclusion of ‘limited impact’ for inferred no effects. As to the framing “we recommend the SMI”, would it carry similar meaning if phrased “the findings lead us to recommend the SMI”? I think it's a small but important difference. Reply: Thank you for these suggestions; we incorporated them into the manuscript. Changes in the manuscript: See Conclusion section, line numbers: 1303. #4. Please add the date July 5 2023 as the time of updating the randomisation plan. Reply: Added. ly: In the revised version of the paper, we explicitly clarified which findings support, do Reply: Added. Changes in the manuscript: See Supplementary Materials, line number 4874. #5. Last, I recommend stating all theoretical implications in the discussion, even if briefly, as per the last column of the design table (Theory that could be shown wrong). They're already implicitly in the text, but being explicit would be preferred. They re already implicitly in the text, but being explicit would be preferred. In my experience, readers tend to draw their own theoretical inferences if they’re not spelled out. You may also add a new results column to the design table; some Stage 2 RRs have it and it's often informative. If you wish to discuss any of the revisions before the next (perhaps final) version, I can be contacted as usual. Reply: In the revised version of the paper, we explicitly clarified which findings support, do 3 3 not support, or are in contrast to the theoretical models. We also added a column with the results. Changes in the manuscript: See Discussion section, line numbers 1105-1108,1182-1185, 1206-1207, and Table 1. Design Table. Lee Moore (Reviewer 1) I enjoyed reading this Stage 2 PCI registered report entitled 'Applying a synergistic mindsets intervention to an esports context'. When reflecting on the criteria and key issues to consider at Stage 2, I felt the authors did a great job with the manuscript. Indeed: • A direct url to the approved protocol was included. • The introduction was largely the same as the stage 1 manuscript with only mi changes made and these were appropriate and transparently flagged. • The authors stayed true to their approved Stage 1 protocol and where they deviated this was appropriate, clearly articulated, and well-justified (e.g., additional exploratory analyses). • Additional exploratory analyses were informative, justified, performed appropriately, methodological sound, and clearly distinguished from the pre- registered analyses. • The conclusions aligned to the preregistered aims/hypotheses and were appropriate, evidence-based, and well-communicated. Reply: Thank you! Beyond these points, I just have a series of relatively minor suggestions that I believe could help the authors improve the manuscript further: Beyond these points, I just have a series of relatively minor suggestions that I believe could help the authors improve the manuscript further: #1. Remove (including stress) from the abstract, stress is a process, not an emotion linked to negative affect (note: if the authors are referring to feeling stressed, the wording should better reflect this). #1. Remove (including stress) from the abstract, stress is a process, not an emotion linked to negative affect (note: if the authors are referring to feeling stressed, the wording should better reflect this). Reply: Clarified. Reply: Clarified. Changes in the manuscript: See Abstract section, line number: 37-38. Changes in the manuscript: See Abstract section, line number: 37-38. #2. In the methods, it would be good to see a breakdown of gender/sex (i.e., proportion of the sample that were male, female, etc.). I also felt more information could be added beyond stage 1 details relating to missing data and outliers (e.g., stating the precise number linked to physiological variables). #2. In the methods, it would be good to see a breakdown of gender/sex (i.e., proportion of the sample that were male, female, etc.). I also felt more information could be added beyond stage 1 details relating to missing data and outliers (e.g., stating the precise number linked to physiological variables). Reply: As we only invited male gamers, we decided not to include a breakdown of gender/sex. We provided additional details on how many participants completed each phase of the study. Given the extensive volume of variables collected in this study (n=1752), providing detailed information on missing data and outliers for each would be impractical. Therefore, we opted for a more streamlined approach by aggregating these details at the apparatus level. Additionally, we provided the missing data for variables used in the analysis (Table S6). 4 Changes in the manuscript: See Participants section, line numbers 270-272; Analysis Plan section, line numbers 803 – 810; Supplementary Materials section, line number 4895. Changes in the manuscript: See Participants section, line numbers 270-272; Analysis Plan section, line numbers 803 – 810; Supplementary Materials section, line number 4895. #3. Regarding the results, some proof-reading of the inferential statistics reported is needed (e.g., page 44, paragraph 2), units should be included for relevant variables (e.g., bpm for HR), and the formatting of tables/figures should match the rest of the text (e.g., font style). I also found some of the wording tricky to interpret (e.g., a lack of increased challenge cardiovascular reactivity [TPT reactivity), and the results were generally relatively difficult to follow and link to the hypotheses noted on pages 12 and 13. I would therefore recommend the authors reflect and consider how best to restructure and rewrite the results section so it is as clear to the reader as possible. Reply: We have incorporated the suggestions from both the Reviewer and the Recommender. We aligned the hypothesis with the results across the manuscript, including the Presents Study section, Analysis, Results, and Design Table. Changes in the manuscript: See Results section, line numbers 995 – 1010, 1027-1037. Changes in the manuscript: See Results section, line numbers 995 – 1010, 1027-1037. #4. The discussion would benefit from being more evidence-based and via the inclusion of more relevant citations/references (e.g., page 48, paragraph 3; page 52, paragraph 2).In particular, I would like to see the authors expand on their discussion regarding the post-intervention test not being high-pressured or stressful enough; what other factors could they have manipulated beyond performance-contingent rewards (see Baumeister and Showers [1986] for some suggestions like performance-contingent punishments, social comparison and evaluation, ego relevance, etc.)? Relatedly, other key discussion points could be elaborated upon (e.g., page 53, paragraph 2, lines 1206-1207). Finally, the writing could be checked to ensure it remains formal and scientific throughout (e.g., avoid colluqualisms like 'don't'). Reply: We have incorporated the suggestions from both the Reviewer and the Recommender. In the revised version of the manuscript, we reviewed the findings from the relevant literature in the discussion and included additional citations/references. Changes in the manuscript: See Discussion section, line numbers 1139 – 1168, 1245-1252. hanges in the manuscript: See Discussion section, line numbers 1139 – 1168, 1245-12 Reply: Clarified. We hope this clarification will help readers evaluate the results. Furthermore, we did not adjust the fonts, as we do not know the journal in which our work will be published. We will adjust the font according to the guidelines of the chosen journal once it is confirmed. Ivan Ropovik (Reviewer 2) I understand that fitting it into the manuscript may be difficult, but there is no reason not to include it in the supplementary materials. I also cannot see the generated file with the analysis outputs – a knitted html, pdf or something of that sort. Reply: We regret any confusion caused previously. The analysis outputs were initially placed in the folder named "Mplus_code." To improve clarity and intuitiveness, we have renamed this folder to "Mplus_analysis_code_and_outputs" in the updated version of our repository. We included descriptives for the study variables in the supplementary materials, which, unfortunately, due to our shortcomings, the Reviewer did not find. We also apologize for this inconvenience. The descriptives (M, SD) for the study variable are presented in Tables S4 and S5. Additionally, we are in the process of making all raw psychophysiological and video data from our study publicly available through an open database. This initiative will be complemented by a detailed article dedicated to describing the dataset. Given the dataset's substantial value and its potential applicability across various research questions, we are committed to detailing it in a standalone paper rather than as supplementary material. This forthcoming publication will offer an in-depth overview of the entire dataset collected during our project, ensuring its maximum utility for future research. p j g y Finally, we reformatted our R script file into R markdown and generated a knitted HTML file with the analysis outputs from R. #2. The introduction, rationale and stated hypotheses (where applicable) are the same as the approved Stage 1 submission. The track changes document is not very clear but I believe it is the case. As a sidenote, I think it okay not to track some low-level edits (like changing to past tense) as it clutters the track changes document a lot. Reply: Thank you! Due to the lack of clarity of the tracked version, we also included the marked version, which was much clearer. 3. The authors adhered precisely to the registered study procedures. Any unregistered exploratory analyses are justified, methodologically sound, and informative. Yes, they are, IMO. Of course there were several acknowledged deviations. To me, several of the deviations are not really deviations _from the research plan_ at all. But okay, it is great to see that the authors were really very diligent about disclosing any change. Ivan Ropovik (Reviewer 2) My review of the Stage II report is going to be way shorter than my review of the Stage I report. That is because have done a great job making their research workflow very open, accountably owning the minor mistakes, and being frank about the lack of clear signal from the data in several respects. Due to the fact that the data do not support a more straightforward stylized narrative, I think that the paper would have much harder time in many mainstream journals that are rather “novelty-oriented”. In my view, it is always very refreshing to see a study that reads like a report from a real study. Now to the point of what should be the target of my comments: 5 #1. The data are able to test the authors’ proposed hypotheses (or answer the proposed research question) by passing the approved outcome-neutral criteria. In my view, the study is still informative, even though the situational context created to observe and test the target effect did not turn out to be optimal. This should probably be assessed by a substantive reviewer knowledgeable about gaming and affective responses, as it is potentially a limiting factor. Yes, the fact that esports competition may not represent a threat-type situation may be seen as logical, but only now, ex post. Although this aspect has a foul taste of failure, I think it is equally worthy of being published. #1. The data are able to test the authors’ proposed hypotheses (or answer the proposed research question) by passing the approved outcome-neutral criteria. In my view, the study is still informative, even though the situational context created to observe and test the target effect did not turn out to be optimal. This should probably be assessed by a substantive reviewer knowledgeable about gaming and affective responses, as it is potentially a limiting factor. Yes, the fact that esports competition may not represent a threat-type situation may be seen as logical, but only now, ex post. Although this aspect has a foul taste of failure, I think it is equally worthy of being published. In that respect, I think the reader of the study would benefit from seeing the descriptives for the study variables to see how much limiting factor are these floor effects. Please, in the revision, include a table with basic descriptives. Ivan Ropovik (Reviewer 2) I think that the deviations had little chance to jeopardize the integrity of the RR. Reply: Thank you! Reply: Thank you! 6 4. The authors’ conclusions are justified given the evidence. The authors offer a measured interpretation of the results, the narrative is not overblown. The discussion would, however, benefit from taking more into account the constrains on generality posed by the present design and tightening several too-much-generalizing claims accordingly. 4. The authors’ conclusions are justified given the evidence. The authors offer a measured interpretation of the results, the narrative is not overblown. The discussion would, however, benefit from taking more into account the constrains on generality posed by the present design and tightening several too-much-generalizing claims accordingly. Reply: We toned down some of our claims in the discussion. We also supported our claims with additional relevant citations/references. We hope that the revised version is a more balanced and cautious interpretation of our findings. Changes in the manuscript: See Discussion section, line numbers: 1111, 1115, 1139-1168, 1303. #5. I now see that the authors refer to supplementary materials, I just cannot see them on the osf repo. #5. I now see that the authors refer to supplementary materials, I just cannot see them on the osf repo. Reply: We apologize for the misunderstanding. The supplementary materials were included in the folder PCI_RR_Stage2, named PCI_RR_SMI_Esports_SuppMat_Stage2.docx. In the updated version of the manuscript, we included a direct link to the supplementary materials to avoid this inconvenience. Changes in the manuscript: See line number: 1315. Changes in the manuscript: See line number: 1315. #6. I tend to agree how the authors resolved unexpected situations with the pre-reg deviations, except for the randomization schedule. Unequal groups would have induced no causal bias whatsoever – only some drop in power if H0 is indeed false. But I agree that the fix is an okay solution, albeit a one to a non-problem. Reply: Thank you for your understanding. #7. I do not see how the present data support the claim that SMI is a universal prevention tool. #7. I do not see how the present data support the claim that SMI is a universal prevention tool. Reply: We no longer refer to the SMI as the “universal” prevention tool. However, we believe that our findings support recommending the SMI as a prevention tool (but not as a performance-enhancing tool). We found some evidence that it can have important benefits (e.g., changing one’s mindsets) and found no evidence that the SMI causes harm. Even small changes resulting from SMI that harness these psychological adaptations may have implications for broader applications to promote long-term mental health. Changes in the manuscript: See Discussion section, line numbers: 1115. #8. The model did not fit the data well. Even with very modest N and low magnitudes of interrelationships, the formal test of the model, the chi-square test, had enough teeth to reject the exact fit hypothesis. As you can see, the value of the chi^2 is almost 4 times as large, which is not terrible, but it points to model-data deviations inconsistent with purely random sampling variability. Also, the CFI is not reaching the (rather benevolent) threshold preached by Hu & Bentler (1999). I would say that even the approximate fit is mediocre at best. 7 Reply: Corrected. We included the Reviewer’s suggestions as the additional limitation of our study. Changes in the manuscript: See Limitations section, line numbers 1282-1292. Jacob Keech (Reviewer 4) Well done to the authors for completing this study and submitting a well-written Stage 2 Registered Report. In my view, all necessary information has been adequately described, the analyses have been competently conducted, and the manuscript is well written. Reply: Thank you! I have three minor comments which I recommend addressing: #1. Double check the sentence structure for accuracy where the SMI abbreviation has been used throughout. For example, a “the” is missing in this sentence: “we adapted and validated SMI in real-world performance”. Reply: Corrected. Reply: Corrected. #2. For the measure of Situational Affect Regulation, is there any indication of validity when using half of the items from the original scale? Reply: The validity of using half of the items from the original scale had not been tested prior to our study. We reduced the number of items to minimize participants' burden and maintain engagement, as it was only a manipulation check in our study. Leveraging the extensive experience of our team members in measuring affect regulation, we (MB, KP, JG) carefully selected the items to ensure they were representative of each factor. In the specific context of our study, we determined that reducing the scale from 2 to 1 item per factor was a pragmatic decision despite recognizing it as a less-than-ideal solution that may have compromised the scale's reliability and validity to some extent. We clarified this issue. Changes in the manuscript: See Measures section, line number 653-654. #3. Line 2068: This phrase seems to overstep the results “that the SMI is the universal prevention tool that is recommended.” The data suggest that the intervention may be promising, but there is no evidence yet that it is effective for universal prevention. Reply: We toned down some narratives in the discussion, and we no longer refer to the SMI as the “universal” prevention tool. Changes in the manuscript: See Discussion section, line number 1115.
https://openalex.org/W2782841819	https://eprints.gla.ac.uk/271412/1/271412.pdf	English	null	Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival	PLOS genetics	2,018	cc-by	15,576	RESEARCH ARTICLE Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival Arie Zackay1¤, James A. Cotton2, Mandy Sanders2, Asrat Hailu3, Abedelmajeed Nasereddin1, Alon Warburg1, Charles L. Jaffe1* Arie Zackay1¤, James A. Cotton2, Mandy Sanders2, Asrat Hailu3, Abedelmajeed Nasereddin1, Alon Warburg1, Charles L. Jaffe1* 1 Dept Microbiology & Molecular Genetics, The Kuvin Center for the Study of Infectious & Tropical Diseases, IMRIC, Hebrew University–Hadassah Medical School, Jerusalem, Israel, 2 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom, 3 Dept Microbiology, Immunology & Parasitology, Addis Ababa University, Addis Ababa, Ethiopia 1 Dept Microbiology & Molecular Genetics, The Kuvin Center for the Study of Infectious & Tropical Diseases, IMRIC, Hebrew University–Hadassah Medical School, Jerusalem, Israel, 2 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom, 3 Dept Microbiology, Immunology & Parasitology, Addis Ababa University, Addis Ababa, Ethiopia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Equinom Ltd., Givat Brenner, Israel * cjaffe@cc.huji.ac.il ¤ Current address: Equinom Ltd., Givat Brenner, Israel * cjaffe@cc.huji.ac.il ¤ Current address: Equinom Ltd., Givat Brenner, Israel * cjaffe@cc.huji.ac.il OPEN ACCESS Leishmania donovani is the main cause of visceral leishmaniasis (VL) in East Africa. Differ- ences between northern Ethiopia/Sudan (NE) and southern Ethiopia (SE) in ecology, vectors, and patient sensitivity to drug treatment have been described, however the relation- ship between differences in parasite genotype between these two foci and phenotype is unknown. Whole genomic sequencing (WGS) was carried out for 41 L. donovani strains and clones from VL and VL/HIV co-infected patients in NE (n = 28) and SE (n = 13). Chromo- some aneuploidy was observed in all parasites examined with each isolate exhibiting a unique karyotype. Differences in chromosome ploidy or karyotype were not correlated with the geographic origin of the parasites. However, correlation between single nucleotide poly- morphism (SNP) and geographic origin was seen for 38/41 isolates, separating the NE and SE parasites into two large groups. SNP restricted to NE and SE groups were associated with genes involved in viability and parasite resistance to drugs. Unique copy number varia- tion (CNV) were also associated with NE and SE parasites, respectively. One striking exam- ple is the folate transporter (FT) family genes (LdBPK_100390, LdBPK_100400 and LdBPK_100410) on chromosome 10 that are single copy in all 13 SE isolates, but either double copy or higher in 39/41 NE isolates (copy number 2–4). High copy number (= 4) was also found for one Sudanese strain examined. This was confirmed by quantitative polymer- ase chain reaction for LdBPK_100400, the L. donovani FT1 transporter homolog. Good cor- relation (p = 0.005) between FT copy number and resistance to methotrexate (0.5 mg/ml MTX) was also observed with the haploid SE strains examined showing higher viability than the NE strains at this concentration. Our results emphasize the advantages of whole genome analysis to shed light on vital parasite processes in Leishmania. Citation: Zackay A, Cotton JA, Sanders M, Hailu A, Nasereddin A, Warburg A, et al. (2018) Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival. PLoS Genet 14(1): e1007133. https://doi.org/10.1371/journal. pgen.1007133 Introduction Leishmania donovani, together with L. infantum, are the main causative agents of visceral leish- maniasis (VL). The World Health Organization (WHO) estimates that this disease causes an estimated 200,000 to 400,000 new VL cases worldwide, and >40,000 deaths yearly. The major- ity of VL cases occur on the Indian subcontinent, Brazil, and East Africa with most cases in the latter region found in Sudan, South Sudan and Ethiopia [1]. While treatment regimens for VL, including combination therapy based on existing drugs, have improved safety and prognosis, they are still suboptimal, and new drugs are urgently needed. High parasite resistance on the Indian subcontinent to the pentavalent antimonial sodium stibogluconate (SSG) has led to its discontinuation, however SSG is still part of the primary treatment regimen for VL in Ethiopia. The situation in Ethiopia is further complicated by the presence of AIDS, a leading cause of adult illness and death in this country [2, 3]. Between 20–40% of VL patients are co-infected with HIV and relapses, up to 50% at a year post-treatment, are frequently observed [2, 4]. Treatment following relapse normally utilizes alternative drugs like liposomal amphotericin B, pentamidine and paromomycin. Paromomycin is an antibiotic recently approved to treat VL in India and is in clinical trials in Africa [5]. Differences in the dose of paromomycin required to treat VL patients from northern Ethiopia (NE) and Sudan, as compared to southern Ethio- pia (SE) and Kenya have been reported [5, 6]. Interestingly, other differences in the ecology, sand fly vectors and parasites have been described between NE and SE. Endemic regions of NE are typically semi-arid, with commercial monoculture fields and scattered Acacia–Balanite for- ests [7–9]. Phlebotomus orientalis is the primary vector in this region. On the other hand, trans- mission in SE occurs in areas of savannah and forest where termite mounds abound; and Phlebotomus martini and Phlebotomus celiae have been implicated as vectors [7, 8, 10, 11]. Molecular characterization including multilocus enzyme electrophoresis (MLEE), multilocus microsatellite typing (MLMT), protein and DNA sequence analysis of individual genes, and k26—PCR targeting the hydrophilic acylated surface protein B (HASPB) repeat region have been used to characterize the L. donovani complex in East Africa [7, 12–15]. MLMT analysis indicated that different genetic populations and subpopulations are present in NE and SE [13]. Whole genome sequence analysis of Ethiopian Leishmania donovani Competing interests: The authors have declared that no competing interests exist. Author summary Approximately 200,000–400,000 new cases of visceral leishmaniasis (VL) occur annually resulting in an estimated 40,000 deaths. Almost 90% of the reported cases are caused by the Leishmania donovani occurring primarily in East Africa and the Indian subcontinent. Parasites in East Africa are more polymorphic than those isolated in other regions, and differences in vectors, biotopes and patient response to drugs are found in Ethiopia. Large-scale whole genome sequence (WGS) analysis of L. donovani strains and clones iso- lated from VL and HIV+-VL co-infected patients in Ethiopia was carried out. Single nucleotide polymorphism (SNP) and gene copy number variation (CNV) analysis shows genetic differences correlated with geographic regions in Ethiopia. These differences are associated with distinct biological processes and molecular functions, and may be associ- ated with genes involved in drug resistance and parasite survival. Editor: Joshua M. Akey, Princeton University, UNITED STATES Editor: Joshua M. Akey, Princeton University, UNITED STATES Received: October 28, 2016 Accepted: November 28, 2017 Published: January 9, 2018 Received: October 28, 2016 Accepted: November 28, 2017 Published: January 9, 2018 Copyright: © 2018 Zackay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All read data files are available from the EMBL Nucleotide Sequence Database (ENA) database (accession number ERP016010). Funding: This study was funded by grant OPPGH5336 from the Bill and Melinda Gates Foundation (to CLJ, AH and AW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Aneuploidy Chromosome copy number was predicted based on whole chromosome median read coverage as described in Material and Methods. The predicted values for each chromosome in the patient isolates and their clones are given in Fig 1 and S2 Table. Normalized read depth, showed that 68.5% of the chromosomes had a predicted chromosome (PCHR) copy number of 2 ± 0.5, i.e. disomic, with smaller percentages showing higher copy numbers; i.e., trisomic (3 ± 0.5) in 23.4%, tetrasomic (4 ± 0.5) in 6.7%, pentasomic (5 ± 0.5) in 1% and hexasomic (6 ± 0.5) in 0.4%, similar to what was previously reported for Nepalese L. donovani strains [16]. Several strains, mostly patient isolates and clones belonging to the SE population; show intermediate ploidy (mixoploidy) for specific chromosomes, e.g. chromosomes 1, 6 and 23 (S2 Table), suggestive of parasite populations with mixed polysomic diversity, perhaps due to vari- ation in chromosomal amplification between individual cells in culture. Differences in chro- mosome aneuploidy between SE patient strain and its clones was not significant (unpaired two samples student’s t-test, p = 0.2), suggesting that aneuploidy in the clonal population is not due to selection of clones exhibiting different ploidy for identical chromosomes. On the other hand, there is a significant difference in chromosome aneuploidy (unpaired two samples stu- dent’s t-test, p = 0.028) when SE parasites, patient and clones, were compared to NE parasites, patient and clones. Cluster analysis based on chromosome copy number provides insight on three levels: first, that similarities and differences exist between all parasites examined; second, validation that clones derived from the same patient strain are highly similar; and third, that differences in chromosome copy are not correlated with the parasite geographic origin. Similar to earlier studies [16, 19, 20, 25] where aneuploidy was examined in clinical isolates of Leishmania from India and Nepal (L. donovani), in sand flies from Turkey (L. infantum), and in laboratory strains (L. major, L. braziliensis, L. donovani, L. infantum, L. mexicana and L. panamensis); all the Ethiopian L. donovani lines examined showed aneuploidy, and each has a different karyo- type. Seven chromosomes (17, 25, 27, 30, 32, 34 and 36) were disomic in all lines examined, as was chromosome 3 with the exception of one strain AM554. Four of these chromosomes (30, 32, 34 and 36) were used for normalization of average chromosome read coverage as described by Rodgers et al [18]. Results WGS of 41 L. donovani parasites, both clones and isolates, from 15 Ethiopian patients was car- ried out and analyzed (S1 Table). All 18 patient strains were isolated in the years 2009 and 2010 in NE (n = 9) or SE (n = 10). The parasites analyzed were isolated from spleens, bone marrows and skin lesions. Five of the patients had AIDS at the time the parasites were isolated; and in three cases, two strains obtained from different organs (either spleen and skin, or spleen and bone marrow), were analyzed. None of the patients received treatment for VL prior to par- asite isolation. A Sudanese strain, isolated in 1998, was also included in some analysis (S1 Table). Whole genome sequence analysis of Ethiopian Leishmania donovani analyzed by WGS. In this report 41 patient strains and clones from NE and SE are analyzed and compared by WGS. This study provides insights into the population structure, and genetic differences of parasites circulating in the distinct ecologies of Ethiopia. The evidence of geno- mic variation between the two L. donovani populations (NE and SE) may provide an additional insight about parasite virulence, development of drug resistance and give new directions for new treatment strategies. Introduction Recent developments in whole genome sequencing (WGS) and computational analysis allow the in-depth exploration and comparison of leishmanial genomes at a high level of reso- Recent developments in whole genome sequencing (WGS) and computational analysis allow the in-depth exploration and comparison of leishmanial genomes at a high level of reso- lution and accuracy [16–18]. Genomes of individual Leishmania species [18]; of L. infantum or L. donovani strains in limited geographic regions of Turkey or India/Nepal respectively [16, 19, 20], and strains showing differences in drug resistance and tropism [16, 20–24] have been PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 2 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Whole genome sequence analysis of Ethiopian Leishmania donovani Chromosome copy number was compared using clones from several strains (AM560, n = 4; GR356, n = 9; GR363sp, n = 3; GR363sk, n = 3; GR364sp, n = 3; GR383, n = 5). In one case (GR364sk, n = 3) two clones and the original patient strain were examined. Validation of aneu- ploidy similarities was carried out using clustering based on ploidy patterns taking into account all 36 chromosomes. What is readily apparent, from both the heat map and dendro- gram (Fig 1), that in most cases karyotypes of clones isolated from the same strain are highly similar. For instance, the karyotypes of the five GR383 clones (I, II, X, XII and XIII) are almost identical (unpaired student’s t-test, p > 0.73), and fall in a tight cluster. This is also seen for all the clones of GR363sp, GR363sk, GR364sk and AM560, and 8/9 clones of GR356. Only two clones, GR356/cl.I and GR364sk/cl.II, show karyotypes significantly different from their sister lines or the patient strain from which they were derived, and fail to cluster with the former (Fig 1). Interestingly, clone GR356/cl.1 groups with its sister lines based on SNP analysis (Figs 2 and 3), and exhibits a k26-PCR amplicon identical to the other NE strains (290 bp). Of note, clone GR364sk/cl.II had a k26-PCR amplicon (450 bp) similar in size to SE strains, even though it was derived from GR364sk, a NE strain with a 290 bp amplicon typical of the NE region [7]. This strain was isolated from a HIV-VL patient, and the sister clone, GR364sk/cl.I which is very similar to the patient strain also has a k26—PCR product of 290 bp. Clone GR364sk/cl.II also groups with the SE strains by SNP analysis (Figs 2 and 3), but is distinct from them suggesting that this patient might have had a mixed infection. Cluster analysis of the karyotype data doesn’t separate the SE and NE strains by geographic region instead they are mixed together, interspersed among each other. Despite this, some dif- ferences in chromosome copy number between the two populations are apparent. Chromo- somes 13, 15, 16, 23, 28, and 31 show significantly higher average ploidy for SE strains compared to NE strains, while chromosome 4 is the only chromosome where the NE strains show a significantly higher average ploidy than the SE population (Table 1 and S2 Table). Aneuploidy Interestingly, disomy was also observed for five of these chromosomes, 17, 25, 30, 34 and 36, in all the 17 Indian and Nepalese L. donovani lines originally studied PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 3 / 26 Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 1. Comparison of aneuploidy profiles of Leishmania donovani from northern and southern Ethiopia. Chromosome numbers are listed down the right side of the heat map. Across the bottom the L. donovani isolates, clones and parental patient strains, analyzed are indicated. The dendrogram on the top shows clusters of the strains and clones based on similarity of aneuploidy profiles and was generated by comparison of the Euclidean distances between aneuploidy profiles among strains (R packages; stats, gplots). Bar under dendrogram indicates the parasite source: Red–southern Ethiopia and Blue–northern Ethiopia. Insert: color key indicating ploidy of chromosomes in heat map. The minimum number of chromosomes, 2, is diploid. Green indicates chromosomal copy number > 4 and pink 4. https://doi org/10 1371/journal pgen 1007133 g001 Fig 1. Comparison of aneuploidy profiles of Leishmania donovani from northern and southern Ethiopia. Chromosome numbers are listed down the right side of the heat map. Across the bottom the L. donovani isolates, clones and parental patient strains, analyzed are indicated. The dendrogram on the top shows clusters of the strains and clones based on similarity of aneuploidy profiles and was generated by comparison of the Euclidean distances between aneuploidy profiles among strains (R packages; stats, gplots). Bar under dendrogram indicates the parasite source: Red–southern Ethiopia and Blue–northern Ethiopia. Insert: color key indicating ploidy of chromosomes in heat map. The minimum number of chromosomes, 2, is diploid. Green indicates chromosomal copy number > 4 and pink 4. https://doi.org/10.1371/journal.pgen.1007133.g001 [16], and in almost all 206 strains from the Indian subcontinent recently examined [20]. Sev- eral other chromosomes (18, 19, 21 and 28) that were disomic in all the Indian and Nepalese lines showed somewhat more diverse ploidy (2 to 3 copies) in the Ethiopian lines. Chromo- some 31 was polysomic in all the lines used in this study. Even GR363sk/cl.I and GR363sk/cl. II, which showed the least aneuploidy of all the lines examined, were still trisomic for chromo- some 31. Chromosome 31 is polysomic in every Old World Leishmania strain examined to date [16, 19, 25]. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 4 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 In addition, we found that SE strains show a significantly higher average total chromosome somy (2.52 ± 0.09) than the NE strains, (2.35 ± 0.06; paired t-test, p = 0.0016), perhaps indicating that the SE strains tend towards higher polyploidy than the NE strains (S2 Table). Finally, parasites from different organs of three HIV-VL co-infected patients were exam- ined. Parasites isolated from the skin or spleen of the same patient form separate groups, and the karyotypes of parasites isolated from the respective sites show greater differences, in most cases, than clones generated from the same site, either skin or spleen. GR364 spleen and skin strains, with the exception of GR364sk/cl.II, form separate clusters (Fig 1). Overall these five lines (skin–original patient strain and clone I, versus spleen—all three clones) show no signifi- cant difference in chromosome ploidy distribution, i.e. karyotype. However, when each GR364 spleen and skin chromosome was compared on an individual basis, 6 out of 36 chro- mosomes (Ld4, 5, 8, 14, 20 and 31) show significant differences in ploidy (p = 0.0004, 0.04, 0.01, 0.05, 0.01 and 0.001, respectively). This probably accounts for the fact that the skin and spleen strains form separate subgroups (Fig 1). Likewise, GR363sk and GR363sp taken from the skin and spleen, respectively, of the same patient cluster in separate branches of the den- drogram (Fig 1). Significant overall differences in ploidy between the GR363 skin and the spleen clones (two tailed paired t-test, p = 0.0067) was observed. Interestingly, the spleen clones from this patient demonstrate on average higher ploidy than the skin clones. Finally, strains isolated from the spleen and bone marrow of one patient, LDS373sp and LDS373bm respectively, also show different karyotypes (7/36 chromosomes differ), even though they clus- ter together on the dendrogram. These parasites also show different k26—PCR fragment sizes, gene CNV and SNP profiles. An overall difference in ploidy was found in chromosomes 1, 4, 6, 20 and 35 by the comparison of all spleen against skin clones. The differences in PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 5 / 26 Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 2. Phylogentic tree and similarity analysis based on SNP. Panel A. Cluster analysis based on Euclidian dis between SNP similarity profiles among all 41 NE (black bar under name) and SE (red bar under name) lines. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Th resulting phylogenetic tree describes the genetic diversity among Ethiopia isolates within the two main Leishma populations. B. Comparison between SNP profiles of Leishmania donovani strains and clones from northern an southern Ethiopia. Heat map allows visualization of similarities between all SNPs from SE and NE strains and c Each colored square in the matrix indicates the percent SNP similarity for a strain/clone listed on the right com to strain/clone listed along the bottom of the matrix. The heat map was generated with R packages; stats and gpl green range indicates on identity level 70% and the pink < 70%. Darker green/pink point to higher or lower identity, respectively. https://doi.org/10.1371/journal.pgen.1007133.g002 Fig 2. Phylogentic tree and similarity analysis based on SNP. Panel A. Cluster analysis based on Euclidian distance between SNP similarity profiles among all 41 NE (black bar under name) and SE (red bar under name) lines. The resulting phylogenetic tree describes the genetic diversity among Ethiopia isolates within the two main Leishmania populations. B. Comparison between SNP profiles of Leishmania donovani strains and clones from northern and southern Ethiopia. Heat map allows visualization of similarities between all SNPs from SE and NE strains and clones. Each colored square in the matrix indicates the percent SNP similarity for a strain/clone listed on the right compared to strain/clone listed along the bottom of the matrix. The heat map was generated with R packages; stats and gplots. The green range indicates on identity level 70% and the pink < 70%. Darker green/pink point to higher or lower identity, respectively. https://doi.org/10.1371/journal.pgen.1007133.g002 https://doi.org/10.1371/journal.pgen.1007133.g002 6 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 3. Principal component analysis (PCA) based on SNP of Leishmania donovani NE and SE lines. PCA analysis was performed after pruning SNPs showing high linkage disequilibrium (LD). SNPs with LD>0.9 are not included. Panel A. PCA of all 41 NE and SE lines. The two atypical SE parasites, AM422 and AM553, fall in eigenvectors 1 and 2 relatively far from both populations (EV1 = 0.229 EV2 = -0.954 and EV1 = 0.945 EV2 = 0.265, respectively). Two NE parasites, GR364sk/cl.II and the atypical LDS373bm, fall with the SE population. Despite the short distance between typical SE (red circles) and NE (black circles) population, these two populations are physically well defined. Panel B. PCA of 39 NE and SE lines without atypical SE strains. The typical SE population (red circles) is very homogenous and falls in a tight cluster unlike the NE population, which is very heterogeneous but still distinct from the SE population. LDS373bm falls equidistance between the SE population and LDS b h i l d f h i Li GR k/ l II ill l h i h h SE l i (bl k i l Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 3. Principal component analysis (PCA) based on SNP of Leishmania donovani NE and SE lines. PCA analysis was performed after pruning SNPs showing high linkage disequilibrium (LD). SNPs with LD>0.9 are not included. Panel A. PCA of all 41 NE and SE lines. The two atypical SE parasites, AM422 and AM553, fall in eigenvectors 1 and 2 relatively far from both populations (EV1 = 0.229 EV2 = -0.954 and EV1 = 0.945 EV2 = 0.265, respectively). Two NE parasites, GR364sk/cl.II and the atypical LDS373bm, fall with the SE population. Despite the short distance between typical SE (red circles) and NE (black circles) population, these two populations are physically well defined. Panel B. PCA of 39 NE and SE lines without atypical SE strains. The typical SE population (red circles) is very homogenous and falls in a tight cluster unlike the NE population, which is very heterogeneous but still distinct from the SE population. LDS373bm falls equidistance between the SE population and LDS373sp both isolated from the same patient. Line GR364sk/cl.II still clusters together with the SE population (black circle on the middle of the SE cluster). Interestingly lines from spleen and skin taken from the same patients (GR363sp and sk; and GR364 sp and sk) are clustered together and create a well-defined population. https://doi.org/10.1371/journal.pgen.1007133.g003 Fig 3. Principal component analysis (PCA) based on SNP of Leishmania donovani NE and SE lines. PCA analysis was performed after pruning SNPs showing high linkage disequilibrium (LD). SNPs with LD>0.9 are not included. Panel A. PCA of all 41 NE and SE lines. The two atypical SE parasites, AM422 and AM553, fall in eigenvectors 1 and 2 relatively far from both populations (EV1 = 0.229 EV2 = -0.954 and EV1 = 0.945 EV2 = 0.265, respectively). Two NE parasites, GR364sk/cl.II and the atypical LDS373bm, fall with the SE population. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Single nucleotide polymorphism analysis SNP calling, compared to the L. donovani reference strain (BPK282A1), for each of the strains and clones was carried out as described in material and methods. The two parasite popula- tions, SE and NE, show significantly different number of SNPs on average, ~153K and ~168K (p < 0.043) respectively, compared to the Indian reference strain (R). In addition, the percent- age of homozygous and heterozygous SNPs within each geographic population (S3 Table), rep- resented by a single alternate allele (A), show significant differences i.e., for the SE (mean homozygous AA = 87.2% and heterozygous RA = 12.7%, respectively, p<0.05) and the NE populations (mean homozygous AA = 83.3% and heterozygous RA = 16.6%, respectively, p<0.05). The relationship between SE and NE strains and clones based on whole genome SNP pair-wise analysis is shown in Fig 2. Each colored square in the matrix indicates the percent SNP similarity for a strain/clone listed on the left compared to strain/clone listed along the bot- tom of the matrix. Unlike the chromosomal aneuploidy profiles (Fig 1), SNP population analy- sis divides the L. donovani population into large clades or groups based on geographical distribution (Fig 2). This is easily seen both in the hierarchical cluster tree where SE and NE parasites each form separate groups with clones from each strain showing highest similarity to each other (Fig 2A) and the heat map (Fig 2B). The only exceptions are three atypical strains/ clones (AM422, AM553, and LDS373bm) which fall outside the main clades, and clone GR364sk/cl.II, as mentioned above, which groups with the SE rather than the NE parasites. The two atypical SE strains; AM422 and AM553 fall closest to the NE clade, yet are distinct from the NE strains. Interestingly, LDS373bm does not cluster with the spleen strain, LDS373sp, isolated from the same patient, even though the karyotypes are similar. AM422, AM553 and LDS373bm seem to have SNP profiles falling between the NE and SE populations. p g p p Principal component analysis on the SNPs was also used to examine the population struc- ture. SNPs showing high linkage disequilibrium were removed prior to analysis by SNP prun- ing [26] (S4 Table). As can be seen in Fig 3A, two clusters or near-clades containing most the NE (black circles) or SE (red circles) strains and clones are observed. Whole genome sequence analysis of Ethiopian Leishmania donovani Table 1. Average chromosome ploidy as a separator of the two Ethiopian parasite populations. Chromosome SEmean NEmean SEmean/ NEmean p-value 4 2.13 2.63 0.81 0.041 13 3.19 2.44 1.31 0.002 15 2.68 2.08 1.29 0.038 16 2.62 2.14 1.22 0.02 23 3.94 2.82 1.40 0.018 28 2.32 2.01 1.15 0.034 31 4.36 3.66 1.19 0.002 Where multiple clones exist for an individual isolate, the value used in calculating average chromosome ploidy is the median ploidy of the clones for that strain. Table 1. Average chromosome ploidy as a separator of the two Ethiopian parasite populations. vidual isolate, the value used in calculating average chromosome ploidy is the median ploidy of the clones for that strain. chromosome ploidy of strains isolated from different organs may result from clonal selection of the parasites in the host due to specific selective pressures at the infection site. Despite the short distance between typical SE (red circles) and NE (black circles) population, these two populations are physically well defined. Panel B. PCA of 39 NE and SE lines without atypical SE strains. The typical SE population (red circles) is very homogenous and falls in a tight cluster unlike the NE population, which is very heterogeneous but still distinct from the SE population. LDS373bm falls equidistance between the SE population and LDS373sp both isolated from the same patient. Line GR364sk/cl.II still clusters together with the SE population (black circle on the middle of the SE cluster). Interestingly lines from spleen and skin taken from the same patients (GR363sp and sk; and GR364 sp and sk) are clustered together and create a well-defined population. Fig 3. Principal component analysis (PCA) based on SNP of Leishmania donovani NE and SE lines. PCA analysis was performed after pruning SNPs showing high linkage disequilibrium (LD). SNPs with LD>0.9 are not included. Panel A. PCA of all 41 NE and SE lines. The two atypical SE parasites, AM422 and AM553, fall in eigenvectors 1 and 2 relatively far from both populations (EV1 = 0.229 EV2 = -0.954 and EV1 = 0.945 EV2 = 0.265, respectively). Two NE parasites, GR364sk/cl.II and the atypical LDS373bm, fall with the SE population. Despite the short distance between typical SE (red circles) and NE (black circles) population, these two populations are physically well defined. Panel B. PCA of 39 NE and SE lines without atypical SE strains. The typical SE population (red circles) is very homogenous and falls in a tight cluster unlike the NE population, which is very heterogeneous but still distinct from the SE population. LDS373bm falls equidistance between the SE population and LDS373sp both isolated from the same patient. Line GR364sk/cl.II still clusters together with the SE population (black circle on the middle of the SE cluster). Interestingly lines from spleen and skin taken from the same patients (GR363sp and sk; and GR364 sp and sk) are clustered together and create a well-defined population. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 7 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 https://doi.org/10.1371/journal.pgen.1007133.t001 Whole genome sequence analysis of Ethiopian Leishmania donovani however the NE strains show more diverse distribution with each strain and its clones forming a separate cluster. LDS373bm no longer groups with the SE strains. SNPs in protein coding regions were examined, and SNPs unique to the SE and/or NE pop- ulations identified. No significant difference in the percentage of synonymous, nonsynon- ymous or nonsense mutations was found between SE and NE parasite populations: 47%, 52.6% and 0.4% versus 50.0%, 49.8% and 0.2%, respectively. Altogether 683 common genes containing at least one SNP causing either a nonsynonymous or a nonsense mutation are pres- ent in both NE and SE parasites. The remaining SNPs resulting in nonsynonymous or non- sense mutations are only found in either SE (412 genes) or NE (595 genes) parasite populations (S5 Table). As such, SNPs in these genes are unique markers for parasites in each geographic region. Gene Ontology enrichment analysis of the “unique” SNP containing genes found in each population indicates that the proteins are involved in different biological processes (S6 Table). A web based semantic cluster algorithm, REVIGO, was used to remove redundant GO terms [27]. After removal of redundant GO terms the remaining terms were graphed as scatterplots in two-dimensional space according to semantic similarity. Semantically similar GO terms should remain close together in the plot, and size of the circle indicates the frequency of GO term. Unique SNPs in NE parasites (Fig 4A and S7 Table) are associated with biological pro- cesses such as tRNA aminoacylation for protein translation, glutamine family metabolism, reg- ulation of transferase activity e.g. protein kinases, and phosphate ion transport, while those in SE parasites are primarily associated with cation transmembrane transport, purine nucleoside triphosphate and nucleobase metabolism and DNA conformation (Fig 4B). Similar differences are also noted when the molecular functions of the genes with unique SNPs are analyzed. Unique SNPs in the NE population are concentrated mainly in genes involved in glutamine family biosynthesis and metabolism, tRNA aminoacetylation, pyrimidine metabolism, and cyclins involved in protein kinase regulation during cell division. On the other hand, unique SNPs associated with the SE population are found in genes such as glutathione metabolism, protein translation initiation and elongation factors, transport and oxidoreductase activity (S5–S7 Tables). Several genes associated with the development of leishmanial drug resistance also contain nonsynonymous SNPs and/or nonsense mutations. A unique SNP, only present in the NE population, was identified in the aquaglyceroporin (LdBPK_310030)gene, a protein that plays a role in trivalent antimony (SbIII) uptake, located on chromosome 31 [20]. This unique het- erozygote nonsynonymous mutation g.7444A>T causes an amino acid exchange (Ser251Thr) in TML-5 of aquaglyceroporin, and is only found in the NE population (S5 Table). The MRPA gene (PGPA) encodes an ABC-thiol transporter (LdBPK_230290.1)that sequesters thiol-Sb conjugates and is also involved in antimony resistance [28]. This gene contains several unique nonsynonymous SNPs unique to the NE (four homozygote and one heterozygote), and SE (two homozygote) populations (S5 Table). It is not clear how these unique SNPs affect trans- porter function, as no difference in response to antimonial chemotherapy between L. donovani isolates from NE and SE has been reported. Single nucleotide polymorphism analysis The two atypical SE strains, AM422 and AM553, are outliers falling far outside both clusters (EV1 = 0.229, EV2 = -0.954 and EV1 = 0.945 EV2 = 0.265 respectively), while the two atypical NE strains, GR364sk/ cl.II and LDS373bm, group together with the rest of the SE population confirming the results shown in Fig 2. PCA was repeated excluding the two atypical SE strains to allow better separa- tion of the remaining 39 isolates (Fig 3B). All the SE strains still group together in one dense cluster that includes GR364sk/cl.II suggesting that these strains are more homogenous, PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 8 / 26 Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 4. Scatterplots after reduction of semantic redundancy in enriched Gene Ontology (GO) terms for unique SNP containing genes of northern and southern Leishmania donovani populations. Removal of redundant GO terms was carried out using the web program REVIGO with C set at 0.7 [27]. Enriched GO terms are graphed in two- dimensional semantic space with terms that semantically similar closer together. The semantic space units have no intrinsic meaning. Enrichment p-values are shown by circle color as indicated in the key to the right of each panel, while circle diameter indicates the frequency of the GO term i.e. general terms are larger. Numbers in each circle refer to the GO terms listed in S7 Table. A. Northern Ethiopia (NE). Panel B. Southern Ethiopia (SE). https://doi org/10 1371/journal pgen 1007133 g004 https://doi.org/10.1371/journal.pgen.1007133.g004 https://doi.org/10.1371/journal.pgen.1007133.g004 in average CNV between the NE and SE populations was noted (Table 2). Sixty-two different genes showed significant differences in CN between the SE and NE populations (S9 Table and S1 Fig). In the SE strains, genes with an average CN < 1.5 are primarily found on chromo- somes 10, 11, 12, 22, 27, 31, 34, and 36; and include the folate-biopterin transporters, ABC transporters, ATP-binding cassette protein, D-lactate dehydrogenase, branched-chain amino acid aminotransferase, amastin-like proteins, phosphoglycerate mutase, tartrate-sensitive acid phosphatase, mitogen activated protein kinase homolog, as well as numerous uncharacterized proteins. Genomic CNV of the atypical NE strains was very similar to that observed for the SE strains. On the other hand, only one gene, an amastin-like protein (LdBPK_341700) on chro- mosome 34, shows an average CN <1.5 in a majority of NE strains. Interestingly, low copy number genes were more prevalent in the SE population with 84% of all strains and clones exhibiting an average CN < 1.5 over all genes. Table 2. Summary of copy number variation (CNV) in each L. donovani strain. WHO Code No. complete gene deletions (CNV = 0) No. haploid genes (CNV = 1) No. A summary of the absolute number of different genes showing CN6¼2 over all isolates examined in this study e.g., complete gene deletion (CN = 0), deletion of one copy (CN = 1) or greater than two copies of a gene (CN>2). Isolates from southern Ethiopia (SE) have codes beginning with AMxxx, where xxx represents the strain number. Isolates from northern Ethiopia (NE) have codes beginning with either GRxxx or LDSxxx, where xxx represents the strain number. #Multiple clones exist for the same patient strain, and the value given represents the average number of genes showing gene copy number different from CN = 2. https://doi.org/10.1371/journal.pgen.1007133.t002 Gene copy number variation Comparative read coverage was examined for 41 SE and NE isolates using a sliding window (5000 bp) in order to detect genomic copy number variation (CNV) as described in Material and Methods. Chromosome somy was not taken into account at this stage. While both increases, and decreases in CN were observed (S8 Table), increases in CN (>2) were more prevalent occurring 83% of the time. In addition, a significant overall difference (p < 0.00001) PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 9 / 26 Whole genome sequence analysis of Ethiopian Leishmania donovani PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 10 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 10 / 26 nt strain, and the value given represents the average number of genes showing gene copy number different from CN = 2. Whole genome sequence analysis of Ethiopian Leishmania donovani One striking difference between the NE and SE strains and clones is in the number of folate/biopterin transporter (FBT) gene(s) on chromosome 10 (Table 3, S10 Table and S2 Fig). Leishmania are auxotrophs for folic acid, and 14 different members of FBT family have been identified in L. infantum [29]. Several of these genes are known to play roles in parasite drug resistance and viability. Eight of the 13 L. donovani FBT homologs are located on chromosome 10, of which 7/8 are present in a tandem array. This chromosome is disomic in most leishman- ial strains examined to date [16, 18–20, 25]. Interestingly, three of the FBT genes present in this tandem array on chromosome 10 (LdBPK_100390, LdBPK_100400 and LdBPK_100410) show gene amplification in several NE (GR364sk, GR364sp and GR356) and Sudanese para- sites (S10 Table). In addition, LdBPK_355160 on chromosome 35, an ortholog of L. infantum biopterin transporter 1, also a member of the FBT family, is amplified in the NE strain GR383 (CN = 5), but not the other NE isolates. SE parasites exhibit the opposite trend for these three genes (LdBPK_100390, LdBPK_100400 and LdBPK_100410)on chromosome 10 showing loss of heterozygosity in eight, ten, and ten out of eleven SE strains, respectively (S10 Table). Loss of heterozygosity was also observed for one additional FBT gene on chromosome 10 (LdBPK_100380) in the SE strain AM553. Interestingly, two atypical NE strains, LDS373bm and GR364sk/cl.II, which group with the SE strains by SNP analysis also show loss of heterozygosity for the same three FBT genes on chromosome 10. The average haploid CN taking into account chromosome somy for these three genes is 2.75 in the NE strains versus 1.05, 1.25 and 1.25 respectively in the SE strains (Table 3). Genomic CN for LdBPK_100400 (L. infantum FT1 homologue) in the NE and SE leishman- ial strains was also determined by qPCR (Fig 5) in 20 strains and clones using a novel dual priming oligonucleotide system [30]. qPCR tended to give higher CNs for this gene than found by computational analysis (cn.mops), however there was a good correlation overall between FT1 CN based on computational estimation with cn.mops [31] and qPCR (ρ = 0.91). Strain LDS373bm also showed loss of heterozygosity by qPCR, similar to what was found above by computational analysis. polyploidy genes (CNV>2) Average polyploidy Average CNV MHOM/ET/2009/AM421 0 19 19 3.31 2.16 MHOM/ET/2009/AM422 0 7 19 3.42 2.77 MHOM/ET/2010/AM546 0 18 20 3.15 2.13 MHOM/ET/2010/AM548 0 18 18 3.16 2.08 MHOM/ET/2010/AM551 0 17 25 3.12 2.26 MHOM/ET/2010/AM552 0 16 21 3.14 2.21 MHOM/ET/2010/AM553 0 4 24 3 2.71 MHOM/ET/2010/AM554 0 16 17 3.17 2.12 MHOM/ET/2010/AM560# 0 14 44 3.16 2.65 MHOM/ET/2010/AM563 0 19 53 3.3 2.71 MHOM/ET/2009/GR356# 0 2 31 3.1 3.01 MHOM/ET/2010/GR363sp# 0 4 5 3.8 2.62 MHOM/ET/2010/GR363sk# 0 3 5 3.8 2.74 MHOM/ET/2010/GR364sp# 0 4 22 3.32 2.96 MHOM/ET/2010/GR364sk# 0 7 34 3.31 2.95 MHOM/ET2010/GR379 0 3 16 3.25 2.90 MHOM/ET/2010/GR383# 0 5 74 4.51 4.23 MHOM/ET/2009/LDS373bm 2 10 158 3.04 2.88 MHOM/ET/2009/LDS373sp 0 2 18 3.38 3.15 MHOM/SD/1998/S3570 0 4 27 3.77 3.42 Table 2. Summary of copy number variation (CNV) in each L. donovani strain. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 11 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Whole genome sequence analysis of Ethiopian Leishmania donovani Table 3. Haploid gene copy number variation in leishmania donovani from Northern and Southern Ethiopia. Gene Id Annotation Southern Ethiopia Northern Ethiopia CN§ Chr ploidy CN CN§ Chr ploidy CN LdBPK _080780; LdBPK _080790 Amastin-like protein 2.3 2.9 3.3 1.7 2.5 2.2 LdBPK _100390 Folate/biopterin transporter 1.2 2.1 1.3 2.7 2.0 2.8 LdBPK _100400; LdBPK _100410 Folate/biopterin transporter 1.0 2.1 1.0 2.7 2.0 2.8 LdBPK _191340; LdBPK _191350 Glycerol uptake protein 2.9 2.0 3.0 2 2.3 2.3 LdBPK _231050 Quinone oxidoreductase 3 4.5 6.7 2.0 3.0 3.0 LdBPK _231240 Hydrophilic acylated surface protein 2.8 4.5 6.2 2.0 3.0 3.0 LdBPK_271940 D-lactate dehydrogenase 1.1 2.0 1.1 2.0 2.0 2.0 LdBPK_271950 Branched-chain amino acid aminotransferase 1.1 2.0 1.1 2.0 2.0 2.0 LdBPK_282040 PUC 2.9 2.2 3.2 1.8 2.0 1.8 LdBPK _282050 Zinc transporter 2.9 2.2 3.2 1.8 2.0 1.8 LdBPK _291620; LdBPK _291630 Major facilitator superfamily 1.7 2.5 2.1 3.0 2.1 3.2 LdBPK _301630 Ferric reductase 2.3 2.0 2.3 3.2 2.0 3.2 LdBPK _303210 Mitosatin 3.5 2.0 3.5 2.0 2.0 2.0 LdBPK _303220 RNA binding protein 3.5 2.0 3.5 2.0 2.0 2.0 LdBPK _303230 3-hydroxy-3-methylglutaryl-CoA reductase 3.5 2.0 3.5 2.0 2.0 2.0 LdBPK _311470 PUC 1.1 4.4 2.5 2.0 3.8 3.8 LdBPK _311630 PUC 2.9 4.3 6.4 2.0 3.8 3.8 LdBPK _311640 Diphthine synthase-like protein 2.9 4.4 6.4 2.0 3.8 3.8 LdBPK _311650 PUC, DUF84 2.9 4.4 6.4 2.0 3.8 3.8 LdBPK _311660 3-ketoacyl-coa thiolase 2.9 4.4 6.4 2.0 3.8 3.8 LdBPK _311920 Mannosyltransferase 2.5 4.4 5.6 2.0 3.8 3.8 LdBPK _311930 Ubiquitin-fusion protein 2.5 4.4 5.6 2.0 3.8 3.8 LdBPK _341700 Amastin-like protein 2.5 2.0 2.5 1.4 2.0 1.4 LdBPK _342650; LdBPK_342660 Amastin-like protein 1.2 2.0 1.2 2.5 2.0 2.5 LdBPK _366740 Tartrate-sensitive acid phosphatase 1.2 2.0 1.2 2.0 2.0 2.0 LdBPK _366750 PUC 1.2 2.0 1.2 2.0 2.0 2.0 LdBPK _366760 Mitogen activated protein kinase 1.1 2.0 1.2 2.0 2.1 2.0 https://doi.org/10.1371/journal.pgen.1007133.t003 gene dosage may be correlated with differences in protein expression [18]. For instance, the region on chromosome 10 containing LdBPK_100480, LdBPK_100510, LdBPK_100520 and LdBPK_100521 encodes a Zn-binding protein whose function is unknown, two tandem copies of gp63 and an uncharacterized protein, respectively (S8 and S9 Tables). This region is ampli- fied (CN = 3) in 3/10 SE strains and one SE-like NE clone, GR364sk/cl.II that clusters by SNP analysis with the SE strains. In addition, both methods show significant difference in FT1 CN between the SE and NE populations. The mean FT1 CN for the two different methods and populations is as follows: qPCR; SEmean = 0.79, NEmean = 2.44, p = 0.00034; cn.mops; SEmean = 1, NEmean = 2.6, p = 0.00009 confirming the trend toward loss of heterozygosity in the SE and amplification in the NE strains / clones examined. FT1 is the main transporter for folate. Resistance to methotrexate (MTX) is correlated with reduced folate uptake [32, 33], and CN for this gene was reduced in some resistant parasites [29, 34]. Therefore, the effect of MTX on the viability of eight SE and NE strains/clones that vary in FT1 CN was examined (Fig 6). Several of the SE and NE strains examined also show CNV for other FBT genes on chromosome 10 flanking FT1 (S10 Table). All of the SE strains/ clones tested are single copy for FT1 and are significantly less sensitive (6–30% growth inhibi- tion) to MTX (0.5 mg/ml) than the NE strains/clones (42–78% growth inhibition), p = 0.005; and a linear correlation (r2 = 0.937) between CN, for the genes demonstrating CNV on chro- mosomes 10 (LdBPK_100380, LdBPK_100390, LdBPK_100400 and LdBPK_100410)and 35 (LdBPK_355160), and sensitivity to MTX was observed (Fig 6 and S11 Table). NE parasites are significantly more sensitive (p = 0.02 to 0.0009) to inhibition by MTX over a wide range of concentrations (33 to 900 μg/ml) when grown at limiting folate concentrations (S3 Fig). A high correlation between FT1 CN and sensitivity to MTX was found (Pearson’s correlation coefficient ρ = 0.85, p = 0.007). Plasticity in gene organization has been reported for several Leishmania species with the number of gene copies varying between isolates from the same species [35–37] and changes in PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 12 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 5. Comparison of folate transporter 1 (LdBPK_100400.1) genomic DNA copy number in northern and southern Ethiopian Leishmania donovani strains by quantitative polymerase chain reaction (qPCR) and cn.mops (GCNV). L. donovani strains and clones are shown on the x-axis, while estimated FT1 copy number is given on the y-axis. SE strains (n = 9, line under x-axis), and NE (n = 9) and Sudanese (S3570) strains (dashed line under x-axis). https://doi org/10 1371/journal pgen 1007133 g005 Fig 5. Comparison of folate transporter 1 (LdBPK_100400.1) genomic DNA copy number in northern and southern Ethiopian Leishmania donovani strains by quantitative polymerase chain reaction (qPCR) and cn.mops (GCNV). L. donovani strains and clones are shown on the x-axis, while estimated FT1 copy number is given on the y-axis. SE strains (n = 9, line under x-axis), and NE (n = 9) and Sudanese (S3570) strains (dashed line under x-axis). https://doi.org/10.1371/journal.pgen.1007133.g005 Fig 5. Comparison of folate transporter 1 (LdBPK_100400.1) genomic DNA copy number in northern and southern Ethiopian Leishmania donovani strains by quantitative polymerase chain reaction (qPCR) and cn.mops (GCNV). L. donovani strains and clones are shown on the x-axis, while estimated FT1 copy number is given on the y-axis. SE strains (n = 9, line under x-axis), and NE (n = 9) and Sudanese (S3570) strains (dashed line under x-axis). https://doi.org/10.1371/journal.pgen.1007133.g005 on chromosome 23 has been associated with drug resistance in vitro [40, 41]. Part of this region is also amplified in some wild-type strains [40, 41]. Deletions (CN = 1) or duplications (CN = 4) of part of the H-region (9 kb) were seen in several SE and NE parasites, respectively. The deleted region was found in 3/10 SE strains and contains the genes coding for the ABC- thiol transporter (MRPA) (LdBPK_230290), involved in resistance to antimony [42], and argi- ninosuccinate synthase (LdBPK_230300), involved in arginine synthesis [43]. Interestingly a similar region is amplified in 8 clones from 2 different NE patient strains, and contains the genes coding for argininosuccinate synthase (LdBPK_230300), a putative uncharacterized pro- tein (LdBPK_230270), the Terbinafine resistance locus protein (Yip1) (LdBPK_230280), and the PTR1 gene (LdBPK_230310). These genes are present in the H-region and frequently amplified in some drug resistant cell lines [44], however they were unchanged, diploid, in all the other parasites belonging to NE and SE populations. All other strains are diploid for this region. Gp63 is a protease involved in parasite virulence and survival [38, 39], and is frequently present on chromosome 10 in other species as a multicopy gene family e.g., L. infantum (LinJ.10.0490, 10.0500, 10.0510, 10.0520 and 10.0530) or L. major (LmjF. 10.0460, 10.0465, 10.0470 and 10.0480). Likewise, on chromosome 19 there are two glycerol uptake proteins (LdBPK_191340 and LdBPK_191350) that have an additional gene copy (CN = 3) in 9/10 SE strains and the SE-like clone (GR364sk/ cl.II). Interestingly, in other species these genes are part of a tandem multicopy gene family (L. infantum 7 genes, L. major 6 genes, L. braziliensis 8 genes) that may be involved in the remod- eling of lipids on glycerol phosphoinositol lipid anchors. Amplification of the 48 kb H-region PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 13 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 This is similar to CN analysis of anti- mony resistant and sensitive L. donovani strains from Nepal where amplification was not observed for the H-region genes [16], even though MRPA, and in some cases the PTR1 gene, were shown to be amplified in naturally resistant parasites examined by other techniques [45, 46]. Finally, CNV was also found in part of a 15.8 kb region located on chromosome 36 known as the MAPK locus (LdBPK_366740, LdBPK_366750, LdBPK_366760 and LdBPK_366770). Amplification of this region was found in antimony resistant L. donovani from Nepal and on chromosome 23 has been associated with drug resistance in vitro [40, 41]. Part of this region is also amplified in some wild-type strains [40, 41]. Deletions (CN = 1) or duplications (CN = 4) of part of the H-region (9 kb) were seen in several SE and NE parasites, respectively. The deleted region was found in 3/10 SE strains and contains the genes coding for the ABC- thiol transporter (MRPA) (LdBPK_230290), involved in resistance to antimony [42], and argi- ninosuccinate synthase (LdBPK_230300), involved in arginine synthesis [43]. Interestingly a similar region is amplified in 8 clones from 2 different NE patient strains, and contains the genes coding for argininosuccinate synthase (LdBPK_230300), a putative uncharacterized pro- tein (LdBPK_230270), the Terbinafine resistance locus protein (Yip1) (LdBPK_230280), and the PTR1 gene (LdBPK_230310). These genes are present in the H-region and frequently amplified in some drug resistant cell lines [44], however they were unchanged, diploid, in all the other parasites belonging to NE and SE populations. This is similar to CN analysis of anti- mony resistant and sensitive L. donovani strains from Nepal where amplification was not observed for the H-region genes [16], even though MRPA, and in some cases the PTR1 gene, were shown to be amplified in naturally resistant parasites examined by other techniques [45, 46]. Finally, CNV was also found in part of a 15.8 kb region located on chromosome 36 known as the MAPK locus (LdBPK_366740, LdBPK_366750, LdBPK_366760 and LdBPK_366770). Amplification of this region was found in antimony resistant L. donovani from Nepal and PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 14 / 26 Discussion Genome wide sequencing (WGS) and analysis of pathogens has proven widely useful for inves- tigations on molecular epidemiology and evolution; genotype—phenotype associations; identi- fication of genes involved in various biological processes such as drug resistance and virulence; as well as new targets for drug and vaccine development [16–18, 21, 48, 49]. Developments in next generation sequencing over the last two decades have provided a relatively low cost, fast pipeline for the exploration and comparison of Leishmania genomes. This study focused on comparison and analysis of WGS data from a large number of L. donovani strains and clones (n = 41) originating from fifteen VL patients in southern and northern Ethiopia. Previous studies on population genetics using multilocus microsatellite typing (MLMT) or individual gene sequences suggest that L. donovani is comprised of distinct populations associated with specific geographic regions in East Africa [7, 15, 50], and that African parasites are in large part distinct from those found on the Indian subcontinent [13, 17]. Differences have also been documented in the parasites, sand fly vectors and host responses between these geographic regions e.g., sensitivity of antigen based serodiagnostic assays [51], clinical response to paro- momycin [6], incidence of PKDL [52, 53]. Our whole-genome sequence data confirms the presence of two very different populations of L. donovani in the region, exemplified by an absolute difference of ~15,000 SNPs between the NE and SE populations. These parasite popu- lations likely arose due to unique evolutionary pressures associated with local sand fly species, hosts, reservoirs, ecology, and other factors. A unique advantage of whole-genome data is that it gives us a comprehensive catalog of genetic variation that could underpin these adaptations. When chromosome aneuploidy is analyzed, a picture appears suggesting great diversity among the Ethiopian strains within each population. This picture is unlike that shown for the Ethiopian reference strain LV9 where all 35 chromosomes, except for chromosome 31, were disomic [18]. This reference strain was extensively passaged in numerous laboratories since first isolated from a VL patient in 1967. Unlike the reference strain, all the isolates used in this study were rapidly cryopreserved and only briefly cultured under identical conditions prior to DNA purification for WGS, yet they still show unique, highly variable karyotypes compared to strains from the same geographic population or strains isolated from different organs of an identical patient. Whole genome sequence analysis of Ethiopian Leishmania donovani Whole genome sequence analysis of Ethiopian Leishmania donovani Fig 6. Folate transporter gene copy number is correlated with greater sensitivity to methotrexate. Effect of Methotrexate on parasite growth (%Dead) was plotted as a function of the total gene copy number of folate (LdBPK_100380; LdBPK_100390; LdBPK_100400 and LdBPK_100410) and biopterin transporters (LdBPK_355160) showing variation on chromosomes 10 and 35, respectively (FT+BT). CN was predicted by cn.mops. %Dead— percentage parasites killed following incubation with 0.5mg/ml methotrexate as described in Material and Methods. Average of n = 3 independent experiments ± s.e. Isolates from southern Ethiopia (SE) have codes beginning with AM. Isolates from northern Ethiopia (NE) have codes beginning with GR. https://doi org/10 1371/journal pgen 1007133 g006 Fig 6. Folate transporter gene copy number is correlated with greater sensitivity to methotrexate. Effect of Methotrexate on parasite growth (%Dead) was plotted as a function of the total gene copy number of folate (LdBPK_100380; LdBPK_100390; LdBPK_100400 and LdBPK_100410) and biopterin transporters (LdBPK_355160) showing variation on chromosomes 10 and 35, respectively (FT+BT). CN was predicted by cn.mops. %Dead— percentage parasites killed following incubation with 0.5mg/ml methotrexate as described in Material and Methods. Average of n = 3 independent experiments ± s.e. Isolates from southern Ethiopia (SE) have codes beginning with AM. Isolates from northern Ethiopia (NE) have codes beginning with GR. https://doi.org/10.1371/journal.pgen.1007133.g006 associated with higher gene dosage in drug resistant lines [16] [47]. Interestingly, CN of 3/4 genes found in this locus were significantly lower (p 0.5 x 10−7) in the SE and SE-like NE strains/clones than the NE strains/clones (S8 and S12 Tables). No significant different in CNV between the NE and SE parasites, CN = 2 in 40/41 strains and clones, was observed in the case of the histidine secretory acid phosphatase (LDBPK_366770) which is considered part of the MAPK-locus and amplified in antimony resistant parasites [16]. The only exception was seen with the SE-like NE strain (LDS373bm) that show a complete deletion of LDBPK_366770, as well as LDBPK_366780 (CN = 0). When haploid gene CN is calculated, taking into account both gene CNV and chromosome ploidy, most of the differences between the NE and SE parasite populations (Table 3 and S13 Table) still remain even though chromosome polyploidy is statistically more common in SE parasites. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 15 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Whole genome sequence analysis of Ethiopian Leishmania donovani Similar to previous reports, chromosome 31 was supernumerary in all 41 Ethiopian strains and clones studied. This appears to be a defining characteristic in all Leishmania species exam- ined so far [16, 18, 19, 25, 55]. As genes involved in iron metabolism and related functions are highly enriched on chromosome 31, it has been suggested the chromosome polyploidy arose to expedite iron uptake, and that expression of Iron–Sulfur proteins that are important in oxi- dation-reduction reactions, and synthesis of metabolites essential for parasite survival and growth [55]. One interesting finding is that strains concurrently isolated from different organs of identi- cal patients, in the two cases examined, have significantly different karyotypes. Thus, while clones and/or parental strain from spleens of each patient clearly grouped together, they clus- tered separately from clones originating from the skin of the same patient. While changes in specific chromosome ploidy associated with parasite tropism were not identified, these results suggest that the aneuploidy patterns observed are a result of parasite origin (spleen or skin), and the differing conditions, perhaps temperature or host immune responses, to which the parasite is exposed. SNP analysis, similar to MLMT [15], clearly shows that the Ethiopian L. donovani strains group, in large part, into two main populations, NE and SE, delineated by geography rather than clinical history (VL or HIV-VL co-infection; spleen or skin). Interestingly, the NE popu- lation appears to be more polymorphic than the SE population (Figs 2 and 3), reflecting the finding from MLMT data that inbreeding is higher for SE strains than NE strains [15]. In most cases, clones generated from an individual strain (GR363sp/sk, GR364sp/sk, GR383, GR356, AM560, etc), show more limited genetic polymorphism than that observed between strains, generally clustering together regardless of the method used for analysis (chromosome aneu- ploidy, SNPs or gene CNV). This was the case even when the patient strain(s) were isolated from different organs, such as skin and spleen, of the same HIV-VL co-infected patient, show- ing that the genotypes present in visceral organs can spread systemically in immunosuppressed patients to the skin where they get transmitted to sand flies. Only in one case, GR364sk/cl.II, did a cloned line fail to cluster with other clones generated from the patient strain. Instead, this clone grouped with SE strains both by SNP and CNV analysis. Discussion This indicates that chromosome aneuploidy, unlike SNPs, cannot be used to map leishmanial population genetics. Interestingly, MLMT analysis of L. donovani strains from Libo Kemkem, a previously non-endemic region in NE where an outbreak of VL occurred in 2004–2005, also demonstrated high genetic diversity among parasites isolated from patients, including a unique genetic group that shared several alleles with strains from SE [50]. Leishmania chromosome ploidy in individual cells can change rapidly in response to envi- ronmental conditions, even routine culture, resulting in mosaic aneuploidy [54], however in this study individual clones generated from patient strains generally showed similar karyotypes clustering together, and when examined, were highly similar to the parental patient isolate. These results suggest that the average karyotype for each strain is relatively stable i.e., mini- mally affected by culturing and/or cloning prior to DNA extraction. Downing et al [16] also reported that chromosome aneuploidy was stable in culture for 17 Nepalese L. donovani strains even though they also showed diverse karyotypes. This suggests that the karyotypes observed for the parasites in this study are probably very similar to the original patient isolate, assuming changes don’t take place upon differentiation from intracellular amastigote to extracellular promastigote. However, this can only be confirmed by direct measurement of aneuploidy in parasites taken directly from patients without prior culturing, something not currently possible. 16 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 Ethics statement This study was conducted according to the Helsinki declaration, and was reviewed and approved by the Institutional Review Board (IRB), Medical Faculty, Addis Ababa University. Written informed consent was obtained from each adult study participant. Cloning Eight patient strains were cloned prior to DNA extraction for WGS. The cloning procedure was carried out essentially as described [61]. Parasites origin and patient pathology For this work 18 L. donovani strains isolated from 15 patients with VL in Ethiopia during the years 2009–2010 were selected for WGS (S1 Table). The selection was based on three criteria: 1) geographical origin (northern or southern Ethiopia); 2) Patient’s pathology such as HIV/VL co-infection versus VL; and 3) source of parasites, skin versus spleen. Parasites were cultivated in M199 medium with supplements and rapidly frozen [7]. Additional Ethiopian (GR373 [7]) and Sudanese (LEMS3570, kindly provided by Prof. Patrick Bastein, National Reference Cen- ter of Leishmania, University Hospital Centre of Montpellier, France) strains were included in analysis of FT1 copy number, and are also listed in S1 Table. All parasites used in this study were characterized by ITS1 -, cpb—and k26—PCR ([7] and S4 Fig). Whole genome sequence analysis of Ethiopian Leishmania donovani these parasites and see if they form a separate genetic group. AM422 and AM553, both isolated in SE, fell outside the main NE and SE populations when SNPs were analyzed by two methods (Figs 2 and 3). Neither of these isolates was from patients co-infected with HIV. AM422 showed a k26 PCR amplicon typical of NE strains (290 bp) and originated in the Omo valley near the border with South Sudan, while AM553 had a unique k26 amplicon (360 bp). The unique SNP profiles for these two isolates suggest that additional genotypes are circulating in SE, perhaps a result of the more varied ecology in this region. Gene CNV analysis also identifies differences that are typical of each geographic parasite population, NE and SE. Candidate genes that can be attributed to essential biological processes like drug resistance, virulence and parasite viability demonstrate differential CN among SE and NE strains and clones. While it is not clear which environmental and host factors resulted in the selective amplification of different genes in the NE and SE populations, many of them are essential genes important for parasite survival. Amplification or deletion of specific genes may give the parasites a growth advantage in the sand fly vector or human host. In the NE par- asites, there are three times more copies of folate/biopterin transporter (FBT) genes on chro- mosome 10. Leishmania are folic acid auxotrophs, and LDBPK_100400 is a homologue to the Leishmania infantum FT1 transporter that was defined as the main folate transporter in this Leishmania species. It is also known that FT1 transporter expression is upregulated in log phase of promastigote stage [34, 44], the stage found in sand fly midgut. Therefore, increased folate concentration in sand fly midgut may result in better parasite growth in the vector and provide the parasite with a better chance for survival and infection. While contamination dur- ing cloning can’t be ruled out, parasites isolated from HIV co-infected patients have been shown to be more polymorphic than those isolated from patients with VL, and differences fol- lowing patient treatment have been noted [56–58]. The chromosome karyotype and SNPs for this clone are distinct from all the SE strains suggesting that contamination, if it occurred, did not take place during generation of the cloned line. SNP analysis, similar to MLMT [15], clearly shows that the Ethiopian L. donovani strains group, in large part, into two main populations, NE and SE, delineated by geography rather than clinical history (VL or HIV-VL co-infection; spleen or skin). Interestingly, the NE popu- lation appears to be more polymorphic than the SE population (Figs 2 and 3), reflecting the finding from MLMT data that inbreeding is higher for SE strains than NE strains [15]. In most cases, clones generated from an individual strain (GR363sp/sk, GR364sp/sk, GR383, GR356, AM560 ) h li i d i l hi h h b d b i SNP analysis also identified three strains, AM422, AM553 and LDS373bm, that didn’t fall, as expected, together with their respective geographic genotypes, SE and NE. LDS373bm and LDS373sp were isolated in parallel from different organs, bone marrow and spleen respec- tively, of the same HIV co-infected patient. The latter parasite (sp) is genetically similar to other parasite strains belonging to the NE population, while the former (bm) belongs to another genotype. These parasites are also different by k26 PCR typing of the HASP B repeat region [7]. This patient was apparently infected by at least two genotypes, with the genotype present in the bone marrow perhaps less virulent and only surviving in immune suppressed hosts. While several reports using MLMT, multilocus sequence typing and kDNA RFLP show that HIV-VL patients can be sequentially infected with genetically different parasites [58–60], to our knowledge this is the first time that an HIV-VL patient was shown to be simultaneously infected with two genetically different parasites. The amplicon (290 bp) seen for LDS373sp was typical of most NE strains examined (37/41), while for LDS373bm it was larger (410 bp), and observed in 4/41 NE strains, all HIV-VL co-infected patients. WGS of other parasites exhibiting the 410 bp amplicon was not carried out. It would be interesting to analyze more of PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 17 / 26 Whole genome sequence analysis of Ethiopian Leishmania donovani Genomic DNA was sheared into 400–600-base pair fragments by focused ultrasonication (Covaris Adaptive Focused Acoustics technology (AFA Inc., Woburn, USA)) and standard Illumina libraries were prepared. 100 base pair paired end reads were generated on the HiSeq 2000 v3 according to the manufacturer’s standard sequencing protocol [62]. Raw sequence data was deposited in the European Nucleotide Archive with the accession number ERP016010. Sequence reads were mapped against the reference genome Leishmania donova- ni_21Apr2011 [16] using SMALT (version 0.7.4 https://sourceforge.net/projects/smalt/) to produce bam files. SMALT was used to index the reference using a kmer size of 13 and a step size of 2 (-k 13 -s 2) and the reads aligned. Reads were mapped if they had an identity of at least 90% to the reference genome and mapped uniquely to the genome. Reads in pairs were mapped independently, and marked as properly paired if they mapped in the correct orienta- tion no more than 1.5 kb apart. PCR duplicate reads were identified using Picard v1.92(1464) and flagged as duplicates in the bam files. Prediction of chromosome aneuploidy Aneuploidy was predicted based on whole chromosome median read coverage. For the nor- malization of median read coverage over all 36 chromosomes for a given strain, the average median coverage of four stable diploid chromosomes (chromosome 30, 32, 34 and 36) was cal- culated and taken as the mean read coverage for a diploid chromosome (DCmean). These four chromosomes have been previously shown to be diploid in almost all L. donovani isolates examined, and have been used for prediction of chromosome copy number [18, 20]. The pre- dicted chromosome copy number is calculated as the fold change compared to DCmean. This prediction was applied to all 41 L. donovani sequences over 36 chromosomes, and saved in a 4136 chromosome copy number matrix. We used R (version 3.1.3) [63] to evaluate of similari- ties and differences between strains and clones, and their chromosome aneuploidy patterns and computed a heat map over the chromosome copy number matrix with R heatmap.2() function from the gplots (version 2.17.0) R package. The numerical data for the creation of the heat map (Fig 1) is given in the supporting information (S2 Table). Median chromosome ploidy was used to compare average ploidy between NE and SE strains in Table 1. For isolates with multiple clones, average chromosome ploidy was calculated by determining the median somy of all the clones from an individual strain separately for each of the 36 chromosomes (S2 Table). Next generation sequencing and sequence analysis DNA was purified from Leishmania promastigotes that were harvested in their stationary growth stage in ~20ml M199 medium. DNA extraction was carried out as described by [7] PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 18 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 SNP analysis For the identification of population typical SNPs a procedure was implemented based on SNP and indel calling with the Genome Analysis Toolkit (GATK version 3.1) [64], VCFtools (ver- sion 0.1.12b) [65] and a custom R script based on Bioconductor packages (www.bioconductor. org) (Release (3.2)). The parameters for the first filtering procedure with GATK were set as fol- lows: Emission confidence threshold > 10, Calling confidence threshold >50, read- depth > 500. A calculation of level of similarity between all 41 samples based on SNP profiles was computed with VCFtools, and processed with R based procedures into a similarity matrix. Finally, this matrix was visualized with heatmap.2() function from the gplots R package. Princi- pal component analysis (PCA) following pruning of SNPs with high linkage disequilibrium was run using the Bioconductor package SNPRelate [26]. For the creation of unique SE or NE SNP profiles, only SNPs that were identified in >4 or >7 of the SE or NE clones and strains, respectively, were taken for final analysis. This protocol created a consensus SNP profile for each parasite population, as repetition of SNPs in more than 1/3 of the strains and clones for For the identification of population typical SNPs a procedure was implemented based on SNP and indel calling with the Genome Analysis Toolkit (GATK version 3.1) [64], VCFtools (ver- sion 0.1.12b) [65] and a custom R script based on Bioconductor packages (www.bioconductor. org) (Release (3.2)). The parameters for the first filtering procedure with GATK were set as fol- lows: Emission confidence threshold > 10, Calling confidence threshold >50, read- depth > 500. A calculation of level of similarity between all 41 samples based on SNP profiles was computed with VCFtools, and processed with R based procedures into a similarity matrix. Finally, this matrix was visualized with heatmap.2() function from the gplots R package. Princi- pal component analysis (PCA) following pruning of SNPs with high linkage disequilibrium For the identification of population typical SNPs a procedure was implemented based on SNP and indel calling with the Genome Analysis Toolkit (GATK version 3.1) [64], VCFtools (ver- sion 0.1.12b) [65] and a custom R script based on Bioconductor packages (www.bioconductor. org) (Release (3.2)). The parameters for the first filtering procedure with GATK were set as fol- lows: Emission confidence threshold > 10, Calling confidence threshold >50, read- depth > 500. Whole genome sequence analysis of Ethiopian Leishmania donovani each population supported the accuracy of SNP calling and served as an additional quality con- trol step. As a final step, unique SNP profiles were detected with VCFtool for each population. Unique SNPs present in coding regions that affect protein translation, namely non-synonymous or nonsense mutation(s) that change the amino acid or cause a stop codon were identified by a self-implemented R procedure based on Bioconductor packages. The mean absolute number of SNPs was compared between the SE and NE parasite populations, as well as to the Indian refer- ence strain. Mean absolute SNPs for an individual patient isolate was calculated by averaging the SNPs from all clones of the respective strain. Variation in the number of SNPs between clones of an individual isolate was small, and % standard error varied from = 0.34–6.0 x 10−3, except for GR364sk (% s.e. = 0.064) when the atypical clone GR364sk/cl.II was included. In the absence of the atypical clone the % s.e. = 0.001. Drug sensitivity Parasites 2 x 106 / ml were added in triplicate to 96-well plates and incubated with 0.5 mg/ml MTX (Sigma catalog number M8407) and in non-drug treated medium in final volume of 200 μl for 66 hours at 26˚C. AlamarBlue (25 μl/well; AbD Serotec) was added and the viability was measured after four hours (λex = 544; λem = 590; Fluroscan Ascent FL, Thermo) [67]. The percentage of killing was calculated as the fraction of fluorescence level of MTX treated wells compared to the non-drug treated wells. Prediction of gene copy number variation The detection of copy number variations (CNV) and aberrations was done using the R- bio- conductor [66] (www.bioconductor.org) package cn.mops (version 1.16.1) (Copy Number estimation by a Mixture Of PoissonS). cn.mops detected copy number variation as the normal- ized read depth variation at a certain genomic position over all 41 strains and clones. For that cn.mops calculated the read count matrices across all BAM files. For this analysis, the genomic window length was set to 5 Kbp and used as a sliding window for the prediction of genomic CNV. Therefore, a genomic position of certain strain or clone is considered as one with "CNV" if it shows a significant change in normalized read depth (> or < two i.e., diploidy) in a window length of 5 Kbp compared to other strains or clones at the same given genomic posi- tion. Further analysis genomic CNV that is localized in coding regions was carried out. SNP analysis A calculation of level of similarity between all 41 samples based on SNP profiles was computed with VCFtools, and processed with R based procedures into a similarity matrix. Finally, this matrix was visualized with heatmap.2() function from the gplots R package. Princi- pal component analysis (PCA) following pruning of SNPs with high linkage disequilibrium was run using the Bioconductor package SNPRelate [26]. For the creation of unique SE or NE SNP profiles, only SNPs that were identified in >4 or >7 of the SE or NE clones and strains, respectively, were taken for final analysis. This protocol created a consensus SNP profile for each parasite population, as repetition of SNPs in more than 1/3 of the strains and clones for PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 19 / 26 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 S4 Fig. Characterization of Leishmania donovani strain LEMS3570 from Sudan by ITS1 -, cpb—and k26 –PCR. Don–L. donovani reference strain MHOM/SD/1962/1S Sudan cl2; Inf–L. infantum reference strain MHOM/TN/1980/IPT1; 3570 –LEMS3570. (TIF) S4 Fig. Characterization of Leishmania donovani strain LEMS3570 from Sudan by ITS1 -, cpb—and k26 –PCR. Don–L. donovani reference strain MHOM/SD/1962/1S Sudan cl2; Inf–L. infantum reference strain MHOM/TN/1980/IPT1; 3570 –LEMS3570. (TIF) Whole genome sequence analysis of Ethiopian Leishmania donovani 95˚C. HRM PCR and analysis were performed using a Rotor-Gene 6000 real-time PCR analyzer (Corbett Life Science, Australia). All reactions were carried out in duplicate and a negative-control reaction without parasite DNA was included in each experiment. For the cal- culation of the FT1 relative copy number (CNrel) the threshold (Ct) for all FT1 amplified sam- ples were compared with their corresponding alpha-tubulin amplified samples as followed: CNrel = 2Ct(alphatubulin)-Ct(FT1). The CNrel was further normalized based on the mean of the six lowest predicted relative CN. The mean value was considered as GCN = 1. S2 Fig. Folate/biopterin transporter genes on chromosomes 10 and 35 showing regional differences in Copy Number Variation (CNV) associated with southern and northern Ethi- opian L. donovani parasites. (T ) S2 Fig. Folate/biopterin transporter genes on chromosomes 10 and 35 showing regional differences in Copy Number Variation (CNV) associated with southern and northern Ethi- opian L. donovani parasites. (TIF) S2 Fig. Folate/biopterin transporter genes on chromosomes 10 and 35 showing regional differences in Copy Number Variation (CNV) associated with southern and northern Ethi- opian L. donovani parasites. (TIF) S3 Fig. Growth inhibition of Leishmania donovani promastigotes from northern and southern Ethiopia by methotrexate as a function of folate concentration in the culture medium. A crosshatch titration on two parasite clones (SE—AM560/cl.IV and NE–GR383/cl. XIII) is shown below. Parasites were cultured in medium containing increasing concentrations of folic acid (FA) from 0 to 28.5 μg/ml (x-axis). To each of the culture conditions, increasing concentrations of methotrexate (MTX) from 0 to 1800 μg/ml (y-axis) was added, and parasite growth measured after 70 hours. The measurement of live promastigotes was carried out with alamarBlue1 assay as described by Shimony and Jaffe [67]. (TIF) Supporting information S1 Fig. Coding regions on chromosomes showing copy number variation for individual northern and southern Ethiopian Leishmania donovani strains. Genes that are not diploid are indicated by symbols. (TIF) Quantitation of folate transporter 1 (FT1) gene copies by qPCR The polycistronic folate/biopterin transporters (FT) on chromosome 10 have high DNA sequence similarity [29]. FT1 (LDBPK_100400) dual priming oligonucleotides (FT1-DPO) were used to specifically evaluate genomic CN for this gene by qPCR. FT1-DPO primers, for- ward FT1-DPO 5’-CGCCAGAACCCGAAGCCTGIIIIIGCACTGG-3’ and reverse FT1-DPO 5’-GTTCATCACAGTCGCGATGAGTIIIIIAATCATTATG-3’, were designed to include a polydeoxyinosine linker (IIIII) [30] that allows the specific primer annealing to the LDBPK_10 0400 (FT1 ortholog) gene, and not to the other LD FT homologues. Specificity of the FT1- DPO PCR product was confirmed by cloning and sequencing of the amplicon. The L. dono- vani housekeeping gene alpha-tubulin was used for normalization. The qPCR conditions were the same for both FT1-DPO and housekeeping genes, and was carried out as follows: DNA (~10–20 ng) or no DNA control was added to HRM PCR Kit reaction mix (10 μl, QIAGEN GmbH, Germany) containing the FT1—DPO primers (1 μM each final concentration), and ultra-pure PCR-grade water (final volume 25 μl/PCR). Amplification conditions were as fol- lows: 3 min denaturation at 95˚C, followed by 40 cycles of denaturation 1 s at 95˚C; annealing 20 s at 55˚C; and extension 1 s at 65˚C. HRM Ramping was carried out at 0.2˚C/s from 65 to PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007133 January 9, 2018 20 / 26 S1 Table. Strains used in this study. (DOCX) S2 Table. A. Chromosome ploidy of the Leishmania donovani parasites analyzed by whole genome sequencing and used to compare strains from northern (NE) and southern (SE) Ethio- pia. B. Chromosome ploidy of each strain and clone analyzed by whole genome sequencing. (XLSX) S3 Table. A. Absolute number of SNPs in Ethiopian Leishmania donovani strains analyzed by whole genome sequencing. B. Absolute number of SNPs in each Ethiopian Leishmania dono- vani strain and clone analyzed by whole genome sequencing. (XLSX) S3 Table. A. Absolute number of SNPs in Ethiopian Leishmania donovani strains analyzed by whole genome sequencing. B. Absolute number of SNPs in each Ethiopian Leishmania dono- vani strain and clone analyzed by whole genome sequencing. (XLSX) S3 Table. A. Absolute number of SNPs in Ethiopian Leishmania donovani strains analyzed by whole genome sequencing. B. Absolute number of SNPs in each Ethiopian Leishmania dono- vani strain and clone analyzed by whole genome sequencing. (XLSX) S4 Table. Data from PCA analysis of SNPs in 41 Ethiopian Leishmania donovani isolates. (DOCX) Whole genome sequence analysis of Ethiopian Leishmania donovani S6 Table. Gene ontology enrichment in Leishmania donovani from Ethiopia based on SNPs unique to northern and southern parasite populations. (XLSX) S7 Table. REVIGO analysis of GO ontology terms for unique SNPs in northern and south- ern Ethiopian Leishmania donovani populations. (XLSX) S8 Table. Copy number variation of genes in northern and southern Ethiopian Leishmania donovani. (XLSX) S9 Table. Summary of genes showing differences in copy number variation between north- ern or southern Ethiopian Leishmania donovani populations. (XLSX) S10 Table. Predicted copy number variation of genes belonging to the folate/biopterin transporter family in Ethiopian Leishmania donovani strains. (DOCX) S11 Table. Folate transporter gene copy number is correlated with greater sensitivity to Methotrexate. (DOCX) S12 Table. Copy number variation (CNV) in the MAPK locus among SE and NE strain and clones. (DOCX) S13 Table. Haploid gene copy number variation in southern and northern Ethiopian Leish- mania donovani populations. (XLS) S11 Table. Folate transporter gene copy number is correlated with greater sensitivity to Methotrexate. (DOCX) S12 Table. Copy number variation (CNV) in the MAPK locus among SE and NE strain and clones. (DOCX) S13 Table. Haploid gene copy number variation in southern and northern Ethiopian Leish- mania donovani populations. (XLS) S4 Table. Data from PCA analysis of SNPs in 41 Ethiopian Leishmania donovani isolates (DOCX) S4 Table. Data from PCA analysis of SNPs in 41 Ethiopian Leishmania donovani isolates. (DOCX) S5 Table. List of SNPs causing nonsynonymous and nonsense mutations that are unique for northern or southern Ethiopian Leishmania donovani populations. (XLS) S5 Table. 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https://openalex.org/W4391537199	https://hal.univ-lorraine.fr/hal-04438059/document	English	null	Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues	Mycology	2,024	cc-by	11,818	To cite this version: Chloé Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, et al.. Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues. Experimental Mycology, 2024, 15 (2), pp.255-271. 10.1080/21501203.2023.2301003. hal-04438059v2 Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues clamps affect fungal diversity assessment from plant root tissues Chloé Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, Damien Blaudez, Coralie Bertheau tissues Chloé Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, Damien Blaudez, Coralie Bertheau Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, Damien Blaudez, Coralie Bertheau An International Journal on Fungal Biology ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/tmyc20 ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/tmyc20 HAL Id: hal-04438059 https://hal.univ-lorraine.fr/hal-04438059v2 Submitted on 20 Sep 2024 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License Distributed under a Creative Commons Attribution - ShareAlike 4.0 International Licen Mycology An International Journal on Fungal Biology Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues Chloé Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, Damien Blaudez & Coralie Bertheau ABSTRACT ARTICLE HISTORY Received 6 October 2023 Accepted 27 December 2023 KEYWORDS ITS2; fungi; annealing temperature; metabarcoding; high-throughput sequencing; blocking primers; Urtica dioica ARTICLE HISTORY Received 6 October 2023 Accepted 27 December 2023 High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity. CONTACT Coralie Bertheau coralie.bertheau-rossel@univ-fcomte.fr Université de Franche-Comté, CNRS, Chrono-environnement, Montbéliard F-25200, France Supplemental data for this article can be accessed online at https://doi.org/10.1080/21501203.2023.2301003. © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Chloé Viottia, Michel Chalota,b, Peter G. Kennedyc, François Maillardc, Sylvain Santonid, Damien Blaudeze and Coralie Bertheaua Peter G. Kennedyc, François Maillardc, Sylvain Santonid, Damien Blaudeze aCNRS, Chrono-environnement, Université de Franche-Comté, Montbéliard, France; bFaculté des Sciences et Technologies, Université de Lorraine, Nancy, France; cDepartment of Plant & Microbiology, University of Minnesota, St. Paul, MN, USA; dAGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France; eUniversité de Lorraine, CNRS, LIEC, Nancy, France Université de Franche-Comté, CNRS, Chrono-environnement, Montbéliard Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues Chloé Viottia, Michel Chalota,b, Peter G. Kennedyc, François Maillardc, Sylvain Santonid, Damien Blaudeze and Coralie Bertheaua Chloé Viottia, Michel Chalota,b, Peter G. Kennedyc, François Maillardc, Sylvain Santonid, Dam and Coralie Bertheaua Chloé Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, Damien Blaudez & Coralie Bertheau To cite this article: Chloé Viotti, Michel Chalot, Peter G. Kennedy, François Maillard, Sylvain Santoni, Damien Blaudez & Coralie Bertheau (2024) Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues, Mycology, 15:2, 255-271, DOI: 10.1080/21501203.2023.2301003 To link to this article: https://doi.org/10.1080/21501203.2023.2301003 © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Published online: 04 Feb 2024. Submit your article to this journal Article views: 1493 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tmyc20 MYCOLOGY 2024, VOL. 15, NO. 2, 255–271 https://doi.org/10.1080/21501203.2023.2301003 1. Introduction Several primers have been designed to amplify ITS2 subregions exclusively belonging to the fungal kingdom (i.e. fungal-specific primers). However, most widely used fungal-specific primers also amplified DNA from other eukaryotes such as plants, for exam ple, fITS7/ITS4, gITS7/ITS4, or 5.8Fun/ITS4-Fun (Ihrmark et al. 2012; Lindahl et al. 2013; Taylor et al. 2016; Koyama et al. 2019; Ricks et al. 2019; Botnen et al. 2020; Li et al. 2020; Chen et al. 2022). and have subsequently been used for fungi (Cregger et al. 2018; Taerum et al. 2020). Most notably, Taerum et al. (2020) showed that PNA clamps reduced plant reads while increasing the number of fungal reads and associated diversity. Although some studies conducted on plant tissues have combined fungal-specific primers with ITS PNA clamps (i.e. ITS3/ITS4; 5.8S-Fun/ITS4-Fun; fITS7/ITS4; ITS1/ITS2) (Hamonts et al. 2018; Sun et al. 2020; Do et al. 2021; Lee and Hawkes 2021), to our knowledge, no studies have addressed which fungal taxa are affected when the ITS2 region is amplified. Moreover, it has been shown that blocking oligos can change fungal relative abundances and co-inhibit cer tain species with universal primers when sequencing arthropods (Piñol et al. 2015). Consequently, there is currently no consensus on the most appropriate PCR protocol for preferentially amplifying fungi over plants. Yet, in the study of fungal diversity within plant roots, the selection of primers, Ta, and PNA clamps is pivotal for achieving this aim, and there is a notable absence of a systematic comparison regarding the impact and interplay of these technical elements. When working on environmental samples, it is recommended to combine primers with degenerate positions (Ihrmark et al. 2012) and a lower annealing temperature (Ta) than typically recommended (Bellemain et al. 2010) to improve the taxonomic coverage and facilitate amplification (Toju et al. 2012; Brown et al. 2018). Since amplification efficiency and specificity depend on the Ta (Yu and Wu 2011), Ta can be adjusted to limit the plant-fungal DNA co- amplification. Moreover, some rare ITS variants may be amplified only at a high Ta (Schmidt et al. 2013). However, increasing Ta can significantly reduce the yield of products (Rychlik et al. 1990) and the diversity recovered (Schmidt et al. 2013). Since the optimal Ta is often only determined based on electrophoresis agar ose gel analysis (Terashima et al. 2002; Green et al. 2004; Banos et al. 1. Introduction from environmental samples (Nilsson et al. 2019; Baldrian et al. 2021). Yet, HTS applied to fungal com munities associated with plants is challenging, given that plant DNA can contaminate the sequencing results. For example, amplicons deriving from plant DNA have been found to be dominant in many meta barcoding studies exploring the plant mycobiota (Almario et al. 2017; Fabiańska et al. 2019). As plant- fungal DNA co-amplification can significantly reduce the sequencing depth, this might lead to an under estimation of the plant-associated fungal diversity (Mayer et al. 2021). New techniques such as host- associated microbiome PCR, aim to co-amplify micro bial markers and host genes to determine microbial load and community composition by adjusting the host: microbe ratio (Lundberg et al. 2021). Fungi are ubiquitous microorganisms that play a number of critical ecological roles in ecosystems (Peay et al. 2016; Dighton 2018). Notably, many fungal species across diverse lineages interact with and grow in close association with cultivated and wild plants (Nguyen et al. 2016; Zeilinger et al. 2016). Fungi are frequently found both around and within plant tissues, particularly in roots, as part of the root microbiome (Trivedi et al. 2020), and are involved in both plant nutrient acquisition and resistance to pathogens (Smith and Read 2010; Waqas et al. 2015; Diagne et al. 2020). Thus, there has been a growing interest in characterising the diversity and composition of plant-associated fungal communities over recent dec ades (Trivedi et al. 2020; Bollmann-Giolai et al. 2022). High-throughput amplicon sequencing (HTS), also called metabarcoding, has emerged as a powerful tool for analysing complex microbial communities The ribosomal internal transcribed spacer (ITS) region is considered the primary fungal DNA barcode (500–700 bases) (Schoch et al. 2012). The ITS region Published online 04 Feb 2024 C. VIOTTI ET AL. 56 256 comprises ITS1 and ITS2, two highly variable spacers linked by the 5.8S gene. The ITS1 and ITS2 subregions are more commonly targeted (250–400 bases) com pared with the entire ITS region being too long for metabarcoding with Illumina Miseq sequencing, which remains the most widely used HTS sequencing platform (Schoch et al. 2012; Tedersoo et al. 2015). Nevertheless, the ITS2 region generates less taxo nomic bias than the ITS1 due to lower length variation with universal primer sites and has been proposed as a better alternative to ITS1 in the characterisation of fungal communities (Yang et al. 2018; Nilsson et al. 2019). 2.1. Sample collection and DNA extraction In 2021, root samples from six adjacent stinging nettle (U. dioica) individuals were collected in Etobon (Bourgogne-Franche-Comté, France). Fine roots were first washed with tap water, followed by three baths of sterile distilled water, and an ultrasonic bath for 20 min to remove the soil attached to the roots. Samples were stored at −20 °C until analysis. Roots from an axenic in vitro stinging nettle from Rovaniemi (Luke, Finland) were also collected and processed identically. Total DNA was extracted from 50 mg of roots using the DNeasy PowerSoil kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s instructions. Three extractions were performed on the same pool of roots and then com bined for both environmental and in vitro roots. DNA from all samples was quantified with a Qubit® dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). Additionally, a synthetic fungal mock community SynMock, (Palmer et al. 2018) comprising 12 ITS constructs was used as control, either alone or in combination with different ratios of in vitro stinging nettle to fungal mock DNA (i.e. 50:50 and 80:20). 1. Introduction 2018), but were also found to align with the ITS sequence of the herbaceous plant Urtica dioica, as extracted from the NCBI database. A two-step PCR was performed to prepare sequencing libraries. PCR1 was designed to achieve amplification of the target regions and to add Illumina Nextera trans posase sequence to the amplicons. Both forward and reverse primers were amended with frameshift sequences (FS), after the Illumina Nextera transposase sequence, to improve sequence diversity and overall read quality (Caporaso et al. 2011). PCR1 was per formed in triplicates for each sample. The reaction mixture (20 µL final volume) consisted of 4 µL of 5× Phusion Green High-Fidelity reaction buffer, 0.4 µL of 10 mmol dNTPs, 0.8 µL of each 10 μmol primers, 0.4 µL of Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific, Illkirch, France) and 10 ng of DNA template. The following cycling parameters were then used for amplification: An initial denaturation stage at 98 °C for 30 s; 35 cycles of denaturation at 98 °C for 15 s, annealing at 57 °C and 68 °C (fITS7 and gITS7) or 63 °C (5.8S-Fun) for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. For the PCR mix with PNA, 0.8 μL of 10 μmol PNA was added. PCR conditions were the same as before, except an additional step at 78 °C for 30 s was included before the primer annealing step. The quality of the PCR1 products was verified through electrophoresis on 1% agarose gels. Purification of PCR1 amplicons was achieved using Agencourt AMPure XP beads (Beckman Coulter, Chaska, MN, USA) at a bead-to- DNA ratio of 1:1. In PCR2 (Illumina dual indexing PCR), each cleaned PCR1 product within the same sample received a unique combination of forward and reverse primers (respectively, N7 and S5 Illumina dual index et al. 2012) (abbreviated thereafter fITS7 and gITS7, respectively) and 5.8S-Fun/ITS4-Fun (Taylor et al. 2016) (abbreviated thereafter 5.8S-Fun) (Table 1). Although, the clamps used in this study were designed to target the plant 5.8S nuclear RNA gene of Populus species (Cregger et al. 2018), but were also found to align with the ITS sequence of the herbaceous plant Urtica dioica, as extracted from the NCBI database. A two-step PCR was performed to prepare sequencing libraries. 1. Introduction PCR1 was designed to achieve amplification of the target regions and to add Illumina Nextera trans posase sequence to the amplicons. Both forward and reverse primers were amended with frameshift sequences (FS), after the Illumina Nextera transposase sequence, to improve sequence diversity and overall read quality (Caporaso et al. 2011). PCR1 was per formed in triplicates for each sample. The reaction mixture (20 µL final volume) consisted of 4 µL of 5× Phusion Green High-Fidelity reaction buffer, 0.4 µL of 10 mmol dNTPs, 0.8 µL of each 10 μmol primers, 0.4 µL of Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific, Illkirch, France) and 10 ng of DNA template. The following cycling parameters were then used for amplification: An initial denaturation stage at 98 °C for 30 s; 35 cycles of denaturation at 98 °C for 15 s, annealing at 57 °C and 68 °C (fITS7 and gITS7) or 63 °C (5.8S-Fun) for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. For the PCR mix with PNA, 0.8 μL of 10 μmol PNA was added. PCR conditions were the same as before, except an additional step at 78 °C for 30 s was included before the primer annealing step. The quality of the PCR1 products was verified through electrophoresis on 1% agarose gels. Purification of PCR1 amplicons was achieved using Agencourt AMPure XP beads (Beckman Coulter, Chaska, MN, USA) at a bead-to- DNA ratio of 1:1. In PCR2 (Illumina dual indexing PCR), each cleaned PCR1 product within the same sample received a unique combination of forward and reverse primers (respectively, N7 and S5 Illumina dual index mock community was made from ITS-like fragments of different lengths and used as a control (Castaño et al. 2020) to assess both the influence of primers and PNA clamps, and to test the efficiency of plant DNA exclusion (Gohl et al. 2016; Pauvert et al. 2019). 1. Introduction 2018), the specific taxa impacted by varying Ta are therefore not described. This study aims to evaluate the combined effects of primer pairs, associated Ta, and PNA clamps in deter mining the fungal community diversity and composi tion associated with plant roots. The plant used in this study is stinging nettle, Urtica dioica (L.). Although this plant grows in a wide range of habitats (Taylor 2009) and has raised scientific and commercial interests (Viotti et al. 2022), limited information is available on its associated microorganisms. Here, three primer pairs used in the literature were studied i) fITS7/ITS4, which is fungi-specific, ii) gITS7/ITS4, a universal pri mer pair amplifying fungi, plants, and protists, and iii) 5.8S-Fun/ITS4-Fun which is designed to be fungi- specific and to generate longer amplicons than the two other primer pairs (Taylor et al. 2016). The influ ence of a higher Ta (i.e. equal to the melting tempera ture of the primers) compared to the commonly used one was investigated for each primer pair, as well as the addition of PNA. Variations in relative abundance, OTU richness, and diversity indices of nettle root fun gal communities were evaluated under various con ditions of Ta, PNA clamp use, and primer pair choice. The fungal taxa impacted by changes in PCR condi tions were also identified. Finally, primer pairs and PNA clamps were evaluated using a synthetic mock community SynMock (Palmer et al. 2018) either alone or with varying ratios of plant to fungal DNA. The Regardless of fungal primer pairs and PCR conditions, peptide nucleic acid (PNA) clamps have also been described as an efficient approach to discriminate fungal DNA from plant DNA (Giangacomo et al. 2021). These clamps are synthetic oligomers that bind to host- derived DNA and block its amplification during PCR (Lundberg et al. 2013; Fitzpatrick et al. 2018). PNA clamps were initially designed for and used in bacterial microbiome studies (Sakai and Ikenaga 2013; Fitzpatrick et al. 2018; Reigel et al. 2020; Kawasaki and Ryan 2021), MYCOLOGY 257 MYCOLOGY 257 et al. 2012) (abbreviated thereafter fITS7 and gITS7, respectively) and 5.8S-Fun/ITS4-Fun (Taylor et al. 2016) (abbreviated thereafter 5.8S-Fun) (Table 1). Although, the clamps used in this study were designed to target the plant 5.8S nuclear RNA gene of Populus species (Cregger et al. 2.2. DNA amplification and amplicon sequence library preparation The fungal 5.8S and ITS2 region from nettle root sam ples, the SynMock community, and three negative con trols were amplified with three Illumina-compatible primer pairs both with and without PNA clamps. The three primer pairs were fITS7/ITS4, gITS7/ITS4 (Ihrmark Table 1. Amplicon size, fungal specificity, and the sequences of primers used. Primers Specificity for fungi Sequences (5’–3’) References fIT7 98% GTGARTCATCGAATCTTTG Ihrmark et al. (2012) gITS7 99% GTGARTCATCGARTCTTTG Ihrmark et al. (2012) ITS4 98% TCCTCCGCTTATTGATATGC White et al. (1990)) Amplicons size (bp): 292 5.8S-Fun 95% AACTTTYRRCAAYGGATCWCT Taylor et al. (2016) ITS4-Fun 90% AGCCTCCGCTTATTGATATGCTTAART Taylor et al. (2016) Amplicons size (bp): 440 PNA - CGAGGGCACGTCTGCCTGG Cregger et al. (2018) C. VIOTTI ET AL. 8 258 from the mock community. OTU richness and Shannon’s index were calculated with the vegan package (Oksanen et al. 2019) and compared between treatments using Kruskal-Wallis test fol lowed by multiple comparisons. The effect of pri mer pair, PNA clamps, and a higher Ta was measured using linear mixed-effects models from the nlme package (Pinheiro et al. 2019). Fungal OTUs composition differences within the three primer pairs with or without PNA or higher Ta were plotted with a non-metric multi- dimensional scaling (NMDS) using the “meta MDS” function in the vegan package, based on the Bray-Curtis dissimilarity matrix. To determine the effects of the different primer pairs, PNA clamps, and a higher Ta potential on OTUs com position, we performed a permutational multivari ate analysis of variance (PERMANOVA) based on Bray-Curtis dissimilarity using the “adonis” func tion from the vegan package. Bray-Curtis dissim ilarity was calculated with the “vegdist” function from the vegan package between primer pairs with or without PNA or with a higher Ta for envir onmental samples and the mock community to measure the dissimilarity of fungal community composition. Statistical analyses and data visuali sation were performed using R (R Core Team 2020) and were considered significant at P < 0.05. oligos). Samples were again cleaned afterwards, using AMPure XP magnetic beads, pooled in equimolar con centrations, and sequenced using 2 × 300 bp MiSeq v3 sequencing (Illumina Inc., San Diego, CA, USA). 3. Results 3. Results 3. Results 2.3. Data processing and bioinformatic analysis Three PCR controls were performed for each pri mer pair. Read counts obtained in PCR controls were subtracted from those of the samples. The “amptk” pipeline described in Palmer et al. (2018) was used to process the raw demultiplexed.fastq files. Following the removal of primers, sequences were trimmed to 250 bp, denoised using UNOISE3 (Edgar 2016) and clustered into opera tional taxonomic units (OTUs) at 97% similarity. Read counts in the OTU x sample matrix were adjusted to account for the tag-switch between samples with the amptk filter function “−t” that assesses the number of reads of mock community taxa present in environmental samples and removes all reads under this threshold from each sample. A hybrid algorithm that integrates results from USEARCH global alignment against the UNITE database v8 (Nilsson et al. 2018) and both UTAX and SINTAX Bayesian classifiers were used to assign taxonomy. The environmental dataset was rarefied to 49,007 sequences per sample, and the mock community dataset was rarefied to 138,195 sequences per sample. 3. Results 2.4. Statistical analyses fITS7/ITS4 246,913 ± 11,955a 218,033 ± 17,439ab 236,290 ± 6,130ab 229,954 ± 14,493ab 204,413 ± 7,241ab 228,534 ± 42,884ab gITS7/ITS4 205,999 ± 19,578ab 231,118 ± 6,776ab 242,530 ± 3,204ab 189,718 ± 3,601ab 217,261 ± 9,906ab 223,649 ± 14,853ab 5.8S-Fun/ITS4-Fun 169,416 ± 10,723ab 176,245 ± 13,371ab 204,221 ± 2,730ab 164,275 ± 16,281ab 200,470 ± 7,536ab 185,277 ± 10,225b Different letters indicate significant differences between primer pairs and the tested conditions using a Kruskal-Wallis analysis followed by a post-hoc test using Fisher’s least significant difference, P < 0.05. the recovery of the 12 members of the mock compared with the other primer pairs (Figure 1b). gITS7/ITS4 205,999 ± 19,578 231,118 ± 6,776 242,530 ± 3,204 189,718 ± 3,601 217,261 ± 9,906 223,649 ± 14,853 5.8S-Fun/ITS4-Fun 169,416 ± 10,723ab 176,245 ± 13,371ab 204,221 ± 2,730ab 164,275 ± 16,281ab 200,470 ± 7,536ab 185,277 ± 10,225b Different letters indicate significant differences between primer pairs and the tested conditions using a Kruskal-Wallis analysis followed by a post-hoc test using Fisher’s least significant difference, P < 0.05. Figure 1. Variation in the SynMock community composition depending on the primer pair used (fITS7/ITS4 in red; gITS7/ITS4 in green and 5.8S-Fun/ITS4-fun in blue), the addition of PNA clamps (+PNA) and the ratio of plant-mock DNA (50:50 or 80:20). (a) OTU Bray- Curtis dissimilarity between classic amplification (Ta = 57 °C) and the various conditions tested. (b) Relative abundance of the 12 OTUs composing the SynMock. 5.8S-Fun/ITS4-Fun 169,416 ± 10,723ab 176,245 ± 13,371ab 204,221 ± 2,730ab 164,275 ± 16,281ab 200,470 ± 7,536ab 185,277 ± 10,225b Different letters indicate significant differences between primer pairs and the tested conditions using a Kruskal-Wallis analysis followed by a post-hoc test using Fisher’s least significant difference, P < 0.05. Different letters indicate significant differences between primer pairs and the tested conditions using a Kruskal-Wallis analysis followed by a post-hoc test using Fisher’s least significant difference, P < 0.05. Figure 1. Variation in the SynMock community composition depending on the primer pair used (fITS7/ITS4 in red; gITS7/ITS4 in green and 5.8S-Fun/ITS4-fun in blue), the addition of PNA clamps (+PNA) and the ratio of plant-mock DNA (50:50 or 80:20). (a) OTU Bray- Curtis dissimilarity between classic amplification (Ta = 57 °C) and the various conditions tested. (b) Relative abundance of the 12 OTUs composing the SynMock. compared with the other primer pairs (Figure 1b). 2.4. Statistical analyses We used a synthetic mock community to evaluate the potential bias of three primer pairs alone or combined with PNA clamps and to assess the impact of plant-fungal DNA co-amplification at different ratios. Sequencing of the mock commu nity generated 11,322,947 fungal sequences before subsampling. The number of fungal sequences did not differ significantly between the three primer pairs and associated conditions (i.e. PNA clamps, different ratios of plant-fungal DNA) (Table 2). gITS7 primers added with plant DNA was the only treatment that generated plant reads (i.e. 464 and 2,052 Viridiplantae reads for 50:50 plant-mock DNA and 80:20 plant-mock DNA, respectively). The three primer pairs allowed All variables were checked for their homoscedas ticity (Bartlett test) and normal distribution (Shapiro-Wilk test). Data that did not fit a normal distribution after transformation were analysed with non-parametrical tests. Differences in the number of fungal sequences from environmental samples and relative abundance of Viridiplantae kingdom among primer pairs and PCR conditions were assessed using a Kruskal-Wallis test followed by multiple comparisons from the “kruskal” func tion of the agricolae package (de Mendiburu 2019). Tukey’s HSD multiple compari son post-hoc analyses were used to assess the differences in the number of fungal sequences MYCOLOGY 259 259 Table 2. Mean number of fungal sequences (±SE) obtained using the SynMock community for the three primer pairs (Ta = 57 °C) and the various combined treatments tested. Primer pairs Ta = 57 °C +PNA 50:50 plant-mock 50:50 +PNA 80:20 plant-mock 80:20 +PNA fITS7/ITS4 246,913 ± 11,955a 218,033 ± 17,439ab 236,290 ± 6,130ab 229,954 ± 14,493ab 204,413 ± 7,241ab 228,534 ± 42,884ab gITS7/ITS4 205,999 ± 19,578ab 231,118 ± 6,776ab 242,530 ± 3,204ab 189,718 ± 3,601ab 217,261 ± 9,906ab 223,649 ± 14,853ab 5.8S-Fun/ITS4-Fun 169,416 ± 10,723ab 176,245 ± 13,371ab 204,221 ± 2,730ab 164,275 ± 16,281ab 200,470 ± 7,536ab 185,277 ± 10,225b Different letters indicate significant differences between primer pairs and the tested conditions using a Kruskal-Wallis analysis followed by a post-hoc test using Fisher’s least significant difference, P < 0.05. Table 2. Mean number of fungal sequences (±SE) obtained using the SynMock community for the three primer pairs (Ta = 57 °C) and the various combined treatments tested. Table 2. Mean number of fungal sequences (±SE) obtained using the SynMock community for the three primer pairs (Ta = 57 °C) and the various combined treatments tested. 2.4. Statistical analyses Moreover, 5.8S-Fun exhibited a higher number of additional OTUs (H17,36 = 39.7, P = 0.001), which exhibited a higher relative abundance (H17,36 = 44.3, P < 0.001). Experimentally reducing the plant-fungal DNA ratio did not impact the relative the recovery of the 12 members of the mock community, and the OTU Bray-Curtis dissimilarity between the different treatments for each primer pair was low (<0.1) (Figure 1a). However, 5.8S-Fun exhibited differences in individual OTU relative abundances, ranging from less than 1% to 9.6% C. VIOTTI ET AL. 0 260 removing primers, short (<200 bp), low-quality sequences, and chimeras. The mean number of fungal sequences significantly differed between the three primer pairs and the associated treatments before subsampling (H8,18 = 20.2; P < 0.01). fITS7 generated a significant higher average number of fungal sequences than 5.8S-Fun (165,377 ± 7,786 and 99,339 ± 9,131 sequences respectively; Table S1) but did not differ significantly from gITS7 (114,656 ± 12,595 sequences). PNA clamps significantly increased the average number of fungal sequences for gITS7 (210,501 ± 14,611) and 5.8S-Fun (156,901 ± 3,197), while the higher Ta did not result in significant differences (Table S1). abundance of OTUs. On the contrary, adding PNA resulted in slight but significant changes in OTU relative abundance with the fITS7 and gITS7 pri mers (<2.5%; Figure 1b). 3.2. Plant-fungal DNA co-amplification in root samples We amplified the same environmental samples with different PCR conditions (primer pairs, PNA clamps, a higher Ta) to assess the impact on the level of plant- fungal DNA co-amplification. Illumina sequencing of environmental samples generated 4,167,783 fungal sequences and 346,249 Viridiplantae sequences after gure 2. Abundance, richness and diversity of fungi in Urtica dioica roots for the three primer pairs tested fITS7/ITS4, gITS7/ITS4, an 8S-Fun/ITS4-fun (Ta = 57 °C), the addition of PNA clamps (+PNA) and the increase of Ta (Ta = 68 °C or 63 °C). (a–c) Relative abundanc f reads of Viridiplantae, fungi, and other phyla (i.e. Amoebozoa, Choanoflagellozoa, Heterolobosa, Ichthyosporia, Metazoa, Protist hizaria, rhodoplantae, Stramenopila, and NA); (d–f) Richness and (g–i) Shannon’s index. Boxes with the same letters did not dif gnificantly from each other using a Tukey-adjusted comparison and Kruskal-Wallis analysis followed by a post-hoc test using Fishe ast significant difference, respectively, P < 0.05. Figure 2. Abundance, richness and diversity of fungi in Urtica dioica roots for the three primer pairs tested fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun (Ta = 57 °C), the addition of PNA clamps (+PNA) and the increase of Ta (Ta = 68 °C or 63 °C). (a–c) Relative abundance of reads of Viridiplantae, fungi, and other phyla (i.e. Amoebozoa, Choanoflagellozoa, Heterolobosa, Ichthyosporia, Metazoa, Protista, Rhizaria, rhodoplantae, Stramenopila, and NA); (d–f) Richness and (g–i) Shannon’s index. Boxes with the same letters did not differ significantly from each other using a Tukey-adjusted comparison and Kruskal-Wallis analysis followed by a post-hoc test using Fisher’s least significant difference, respectively, P < 0.05. MYCOLOGY GY MYCOLOGY 261 elevated Ta significantly lowered the OTU richness with 5.8S-Fun and mostly with gITS7 for which the richness dropped by 40% (F8,18 = 22.2; P < 0.001). At Ta = 57 °C, 5.8S-Fun resulted in significantly more OTUs than fITS7 and gITS7 (Figure 2f). On the contrary, the primer pair had a greater impact on the Shannon index than Ta (F8,18 = 36.8; P < 0.001 and F8,18 = 12.7; P < 0.01, respectively). 5.8S-Fun resulted in a significantly higher Shannon index than the two other primer pairs (Figure 2g–i), and a higher Ta sig nificantly increased it with fITS7 and gITS7 (H8,18 = 21.2; P < 0.01), while Hill’s index resulted in the exact opposite trend (Figure S1, H8,18 = 21.2; P < 0.001). 3.2. Plant-fungal DNA co-amplification in root samples Primer pairs and Ta significantly impacted the relative abundance of Viridiplantae reads (H8,18 = 48.8; P < 0.001). gITS7 exhibited a four times higher relative abundance of Viridiplantae reads than fITS7 (Figure 2a,b), and it accounted for 19.2% of the relative abundance with 5.8S-Fun at both Ta = 57 °C and 63 °C (Figure 2c). Finally, PNA clamps completely inhibited plant DNA co- amplification with 5.8S-Fun, while fITS7 and gITS7 still recovered 0.1% and 0.5% of Viridiplantae sequences, respectively (Figure 2a–c). A higher Ta with fITS7 significantly divided it by seven (Figure 2a). We observed a similar trend but with a lower decrease for gITS7 with higher Ta (Figure 2b). The percentage of diminution in the number of OTUs of the different treatments compared to 5.8S- Fun at Ta = 57 °C, which gave the highest OTU rich ness, was calculated to assess the quantification of fungal richness at different read depths. All the treat ments exhibited a similar percentage of diminution compared to 5.8S-Fun between 8,000 and 32,000 read depths, with gITS7 at Ta = 68 °C that showed the high est decrease (Figure 3). Below a read depth of 2,000, fITS7 and gITS7 at Ta = 68 °C exhibited the lowest percentage of diminution, followed by fITS7 at Ta = 57 °C and gITS7 added with PNA clamps. PNA clamps significantly mitigated the decrease in OTUs by low ering the sequencing depth between 2,000 and 32,000 read depth with gITS7 (Figure 3). 3.3. Fungal richness and diversity depending on PCR conditions and read depth We compared the impact of different PCR conditions using environmental samples on the fungal richness and diversity to further assess the accuracy of primers. After subsampling, Ta and, at a lesser extent, the primer pairs had a significant impact on OTU richness (Figure 2d–f, F8,18 = 75.7; P < 0.001 and F8,18 = 21.0; P < 0.05, respectively), and Hill’s index (Figure S1, F8,18 = 51.0; P < 0.001 and F8,18 = 21.8; P < 0.001 respec tively) compared to PNA clamps (P > 0.05). An Figure 3. Percentage reduction in the number of OTUs for each primer pairs (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) compared to the number of OTUs obtained with 5.8S-Fun, subsampled at various sequencing depths. Figure 3. Percentage reduction in the number of OTUs for each primer pairs (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) compared to the number of OTUs obtained with 5.8S-Fun, subsampled at various sequencing depths. 262 C. VIOTTI ET AL. C. VIOTTI ET AL. 262 Figure 4. Fungal community composition in Urtica dioica roots depending on the primer pair used fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun /ITS4-fun (Ta = 57 °C), the addition of PNA clamps (+PNA) and a higher Ta (Ta = 68 °C or 63 °C). (a) Non-metric multidimensional scaling (NMDS) analysis of the fungal communities, based on OTUs composition; (b) Relative abundance and F-value from linear mixed-effect models measuring the effect of primers, Ta, PNA, the number of OTUs and the number of reads on the relative abundances of fungal classes and (c) Genera. Figure 4. Fungal community composition in Urtica dioica roots depending on the primer pair used fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun /ITS4-fun (Ta = 57 °C), the addition of PNA clamps (+PNA) and a higher Ta (Ta = 68 °C or 63 °C). (a) Non-metric multidimensional scaling (NMDS) analysis of the fungal communities, based on OTUs composition; (b) Relative abundance and F-value from linear mixed-effect models measuring the effect of primers, Ta, PNA, the number of OTUs and the number of reads on the relative abundances of fungal classes and (c) Genera. 3.4. Fungal taxonomic diversity and abundance relative abundances of the main fungal classes (Figure 4b). 5.8S-Fun, on the other hand, showed a significantly higher abundance of Dothideomycetes (+10%, F8,18 = 13.5; P < 0.001) and Sordariomycetes (+13%, H8,18 = 23.9; P < 0.01), a lower abundance of Olpidiomycetes (-18% H8,18 = 23.4; P < 0.01) (Figure 4b) and more OTUs assigned to Dothideomycetes (+20 OTUs, F8,18 = 32.9; P < 0.001), Eurotiomycetes (+5 OTUs, H8,18 = 19.3; P = 0.01) and Pezizomycetes (+4 OTUs, H8,18 = 20.8; P < 0.01) compared to the two other primers We studied the fungal diversity and abundance from the same environmental samples to identify the classes and genera impacted by different PCR conditions. PCR con ditions notably affected fungal community structure (Figure 4a). PERMANOVA analysis showed that the pri mer pair was the most impacting factor (R2 = 0.75; P = 0.001), followed by Ta (R2 = 0.14; P = 0.001), while PNA clamps had no significant effect (R2 = 3.25e−3; P > 0.05). fITS7 and gITS7 were not significantly different in the MYCOLOGY 26 MYCOLOGY 263 (Figure S2). The elevated Ta did not significantly impact the relative abundances of the main classes with 5.8S- Fun. On the contrary, a Ta = 68 °C with fITS7 and gITS7 significantly increased the relative abundances of Eurotiomycetes, Pezizomycetes, Sordariomycetes, and Tremellomycetes (H8,18 = 18.4; P < 0.05). The number of reads impacted these relative abundances more than the number of OTUs (Figure 4b). A higher Ta resulted in fewer OTUs assigned to the main classes but with a higher number of reads (Figure S2). PNA clamps only significantly increased by 2.5 times the relative abun dance of Mortierellomycetes with gITS7 (F8,18 = 2.8; P < 0.05) but did not impact the number of reads. Also, four OTUs assigned to the Sordariomycetes were not recov ered with fITS7 when PNA was added (Figures 4b, S2). 55% of the relative abundance (Figures 5c,6a). On the contrary, fITS7 and gITS7 exhibited less specific OTUs that were poorly abundant (<4%). OTUs specific to 5.8S- Fun mainly belonged to the phylum Ascomycota while those specific to the two other primer pairs were mainly associated with the phylum Basidiomycota (Figure 5b,c). A higher Ta resulted in a few specific OTUs that accounted for less than 4% of the relative abundance (Figures 5b,c,6a). 4. Discussion 4.1. Primer pairs and plant-fungal DNA ratio affect plant DNA co-amplification Since co-amplification of plant and fungal DNA can occur in studies focusing on the fungal microbiome associated with plant tissues, several methods have been proposed to alleviate this limitation (Harrison et al. 2021), notably by using fungal-specific primer pairs. In the present study focusing on plant roots, the fITS7 primer pair resulted in fewer plant reads than 5.8S- Fun, which has been described to exclude Viridiplantae sequences (Taylor et al. 2016). The non-specific fungal primer gITS7 yielded the highest number of plant reads (i.e. more than 20% of the relative abundance) in agree ment with previously published results (Ihrmark et al. 2012). These results confirmed that different primer pairs led to varying levels of plant-fungal DNA co- amplification. This can be attributed to varying degrees of mismatch between the primer and the plant sequence, resulting in plant/fungal DNA co- amplification that varies depending on the plant species studied. Additionally, in our study, artificial modification of the plant-fungal DNA ratio to 50:50 and 80:20 before 3.4. Fungal taxonomic diversity and abundance PNA clamps gave few, low abundant, and specific OTUs that covered a wide range of fungal classes from phyla Basidiomycota and Ascomycota (Figures 5c,6a). OTU Bray-Curtis dissimilarity confirmed that the main differences were between fITS7–5.8S-Fun, gITS7–5.8S-Fun, fITS7, and gITS7 at Ta = 57 °C and Ta = 68 °C (Figure 6b). Although the dissimilarity was low between Ta = 57 °C and PNA clamps, few OTUs were recovered only when they were added or not (Figure 7). These OTUs mainly represented by the phylum Ascomycota accounted for less than 1% of the relative abundance. Most OTUs could not be classified to either the genus (40%– 75%) or the species (50%–83%) level. At the genus level, 5.8S-Fun had significant differ ences in relative abundances compared to fITS7 and gITS7, and allowed the recovery of the genus Plenodomus that accounted for 10.5% ± 0.4 of the rela tive abundance (Figure 4c, S2). Unclassified genera exhibited a higher relative abundance (+20% F8,18 = 38.1, P < 0.001), with 1.5 times more unassigned OTUs (F8,18 = 21.5, P < 0.001) and associated reads (F8,18 = 38.1, p < 0.001) (Figure 4c, S2). 5.8S-Fun also resulted in sig nificant lower relative abundances of Didymella (H8,18 = 18.0, P < 0.05), Titaea (H8,18 = 18.6, P < 0.05), Pyrenochaeta (H8,18 = 23.4, P < 0.01) and Olpidium (H8,18 = 23.4; P < 0.01) (Figure 4c). A higher Ta mostly impacted the genus Olpidium, with a strong decrease in relative abundance (H8,18 = 24.0; P < 0.01) and a two- and eight-fold decrease in Olpidium-assigned OTUs with fITS7 and gITS7, respectively (H8,18 = 24.0; P < 0.01) (Figure 4, S2). On the contrary, a higher Ta significantly increased the relative abundances of unclassified gen era and Exophiala with fITS7 and gITS7 (Figure 4c). 3.5. Primer pair specificity We identified the OTUs recovered only in specific PCR conditions and their relative abundance to assess the taxonomic coverage of primers depending on the Ta and the use of PNA clamps. Sixteen percent of OTUs were shared to all PCR conditions tested (with the three primer pairs, with or without PNA clamps, with higher Ta = 68–63 °C, Figure 5a), and accounted for 95% of the relative abundance with the fITS7 and gITS7 primers but only for 43% with 5.8S-Fun (Figure 6a). 5.8S-Fun gave the highest number of specific OTUs, accounting for 264 C. VIOTTI ET AL. C. VIOTTI ET AL. Figure 5. Representative classes and phyla of (a) non-specific or (b–c) specific OTUs of fungal community in Urtica dioica roots accordin to primer pairs (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) and conditions used, as well as the number of OTUs corresponding “Common” corresponds to the OTUs that were found in all PCR conditions with the three primer pairs. “Non-specific” corresponds to th OTUs that were found in at least one condition (Ta = 57 °C; +PNA or Ta = 68–63 °C) of PCR with the three primer pairs. Figure 5. Representative classes and phyla of (a) non-specific or (b–c) specific OTUs of fungal community in Urtica dioica roots according to primer pairs (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) and conditions used, as well as the number of OTUs corresponding. “Common” corresponds to the OTUs that were found in all PCR conditions with the three primer pairs. “Non-specific” corresponds to the OTUs that were found in at least one condition (Ta = 57 °C; +PNA or Ta = 68–63 °C) of PCR with the three primer pairs. DNA amplification of the mock community resulted in no plant reads with the fungal-specific primers fITS7 and 5.8S-Fun with only a few plant reads detected using the more sensitive primer gITS7. Thus, the plant-fungal DNA ratio appears to substantially affect fungal sequence recovery from plant tissues under our experimental con ditions. This result is in agreement with Lundberg et al. (2021) which reported a recovery of 100% of bacterial sequences from 24% of bacterial DNA. Variations in the plant-fungal DNA ratio within plant tissues might explain why Bodenhausen et al. (2019) observed up to 75% of plant sequences in Petunia roots, despite using a fungal-specific primer pair. 3.5. Primer pair specificity Consequently, we suggest that plant roots with sparse fungal endophyte colonisa tion might be more susceptible to plant DNA contam ination limitation. DNA amplification of the mock community resulted in no plant reads with the fungal-specific primers fITS7 and 5.8S-Fun with only a few plant reads detected using the more sensitive primer gITS7. Thus, the plant-fungal DNA ratio appears to substantially affect fungal sequence recovery from plant tissues under our experimental con ditions. This result is in agreement with Lundberg et al. (2021) which reported a recovery of 100% of bacterial MYCOLOGY 265 265 Figure 6. (a) Relative abundance of specific and non-specific OTUs for each primer pair used (fITS7/ITS4: red; gITS7/ITS4: green; 5.8S- Fun/ITS4-fun: blue), with the addition of PNA clamps (+PNA) and a higher annealing temperature (Ta = 68 °C or 63 °C); (b) OTU Bray- Curtis dissimilarity between the different primer pairs (Ta = 57 °C), with or without PNA clamps (+PNA), or with an annealing temperature of 68 °C or 63 °C. Figure 6. (a) Relative abundance of specific and non-specific OTUs for each primer pair used (fITS7/ITS4: red; gITS7/ITS4: green; 5.8S- Fun/ITS4-fun: blue), with the addition of PNA clamps (+PNA) and a higher annealing temperature (Ta = 68 °C or 63 °C); (b) OTU Bray- Curtis dissimilarity between the different primer pairs (Ta = 57 °C), with or without PNA clamps (+PNA), or with an annealing temperature of 68 °C or 63 °C. 4.2. Primer pairs affect fungal diversity and composition estimations independently of plant DNA co-amplification reads, the fITS7 primer pair yielded the highest number of fungal reads and 5.8S-Fun the lowest. Therefore, 5.8S-Fun does not appear to be the most suitable primer pair when the samples are plant tissues with a high plant:fungal DNA ratio (e.g. endophytes or pathogens with low colonisa tion levels), as it may result in low fungal In this study, among the three primer pairs tested on a plant mainly colonised by endophytic fungi and saprotrophs (Yung et al. 2021), the majority of sequences were fungal. After the removal of plant Figure 7. Number of OTUs found in at least two replicates, either without or with the addition of PNA clamps (+PNA), mean relative abundance in percentage ±SE for each primer pair (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) and the percentage of represented fungal classes. Figure 7. Number of OTUs found in at least two replicates, either without or with the addition of PNA clamps (+PNA), mean relative abundance in percentage ±SE for each primer pair (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) and the percentage of represented fungal classes. Figure 7. Number of OTUs found in at least two replicates, either without or with the addition of PNA clamps (+PNA), mean relative abundance in percentage ±SE for each primer pair (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) and the percentage of represented fungal classes. Figure 7. Number of OTUs found in at least two replicates, either without or with the addition of PNA clamps (+PNA), mean relative abundance in percentage ±SE for each primer pair (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun) and the percentage of represented fungal classes. C. VIOTTI ET AL. 266 assessment than plant-DNA contamination in our experimental conditions (i.e. high read depths). sequencing depth. Additionally, we observed a higher number of additional OTUs, although they represented a low proportion in terms of relative abundance, that were not expected to be recovered from the mock community with 5.8S- Fun. This may suggest a higher index bleed with this primer pair or, more likely, potential chimeras, following the results of Li et al. (2020) that high lighted a higher non-specific amplification (i.e. chi mera and mismatch) with 5.8S-Fun. However, 5.8S- Fun exhibited significantly higher OTU richness and Shannon’s index in environmental samples com pared to fITS7 and gITS7. This finding is inconsis tent with results based on soil samples (Li et al. 2020) and those from Ihrmark et al. (2012) that found more diverse amplicon communities with gITS7. 4.2. Primer pairs affect fungal diversity and composition estimations independently of plant DNA co-amplification Among the three primer pairs, members of Ascomycota were the most prevalent, following the trend of other studies (Li et al. 2020; Yung et al. 2021). The 5.8S-Fun primer pair yielded sig nificantly more OTUs assigned to Ascomycota and Chytridiomycota (e.g. Plenodomus) than fITS7 and gITS7. This is consistent with the fact that fITS7 excludes some Ascomycota (Ihrmark et al. 2012). Furthermore, differentiating taxa within the phy lum Chytridiomycota is challenging with variable markers such as the ITS region due to high genetic divergence and limited reference sequences avail able (Ishii et al. 2015; Tedersoo et al. 2015). Moreover, in a previous study, 5.8S-Fun yielded the lowest Basidiomycota abundance (Li et al. 2020). As the taxonomic specificity and coverage vary depending on the primer used (Baldrian et al. 2021), the impact of primer choice on the recov ered diversity and richness of plant root-associated communities will depend on the fungal taxa har boured by the plant (e.g., dominance of Basidiomycota phylum over Ascomycota). Despite varying levels of plant DNA co-amplification, fITS7 sequencing depth. Additionally, we observed a higher number of additional OTUs, although they represented a low proportion in terms of relative abundance, that were not expected to be recovered from the mock community with 5.8S- Fun. This may suggest a higher index bleed with this primer pair or, more likely, potential chimeras, following the results of Li et al. (2020) that high lighted a higher non-specific amplification (i.e. chi mera and mismatch) with 5.8S-Fun. However, 5.8S- Fun exhibited significantly higher OTU richness and Shannon’s index in environmental samples com pared to fITS7 and gITS7. This finding is inconsis tent with results based on soil samples (Li et al. 2020) and those from Ihrmark et al. (2012) that found more diverse amplicon communities with gITS7. Among the three primer pairs, members of Ascomycota were the most prevalent, following the trend of other studies (Li et al. 2020; Yung et al. 2021). The 5.8S-Fun primer pair yielded sig nificantly more OTUs assigned to Ascomycota and Chytridiomycota (e.g. Plenodomus) than fITS7 and gITS7. This is consistent with the fact that fITS7 excludes some Ascomycota (Ihrmark et al. 2012). Furthermore, differentiating taxa within the phy lum Chytridiomycota is challenging with variable markers such as the ITS region due to high genetic divergence and limited reference sequences avail able (Ishii et al. 2015; Tedersoo et al. 2015). 4.3. Increased Ta reduces plant co-DNA amplification but alters the assessment of fungal diversity 4.3. Increased Ta reduces plant co-DNA amplification but alters the assessment of fungal diversity Modifying Ta can contribute to limiting plant DNA co-amplification. In this study, increasing Ta signifi cantly decreased plant-fungal DNA co-amplification with the fITS7 and gITS7 primers, but not with 5.8S- Fun. Although Ta can limit plant-fungal DNA co- amplification with some primers, it decreased the number of OTUs recovered, but not the sequencing depth. Schmidt et al. (2013) also described a decrease in the number of OTUs with a higher Ta when targeting the ITS1 region. Despite a lower OTU richness, raising the Ta allowed the recovery of specific OTUs. Shannon’s index, which was signif icantly higher with the fITS7 and gITS7 primer pairs, confirmed this observation. These specific OTUs, mainly assigned to Basidiomycota were found with the fITS7 and 5.8S-Fun primers. In addition, the relative abundance of Ascomycota increased despite a lower number of OTUs, suggesting that higher Ta decreases the recovery of Ascomycota-related fungi. That is contradictory with Larena et al. (1999), who used a high Ta (i.e. 62–64 °C) for the selective asco mycetous primer ITS4A. Additionally, increasing Ta highly impacted the relative abundance of the genus Olpidium with fITS7 and gITS7. Nevertheless, higher Ta with 5.8S-Fun had no impact on the diver sity of rare OTUs nor the relative abundances of fungal classes or genera, suggesting that this primer pair is less sensitive to changes in Ta. Our results indicate that while increasing Ta might help limit plant DNA contamination, it simultaneously reduces fungal diversity and alters fungal community composition. 4.2. Primer pairs affect fungal diversity and composition estimations independently of plant DNA co-amplification Moreover, in a previous study, 5.8S-Fun yielded the lowest Basidiomycota abundance (Li et al. 2020). As the taxonomic specificity and coverage vary depending on the primer used (Baldrian et al. 2021), the impact of primer choice on the recov ered diversity and richness of plant root-associated communities will depend on the fungal taxa har boured by the plant (e.g., dominance of Basidiomycota phylum over Ascomycota). Despite varying levels of plant DNA co-amplification, fITS7 and gITS7 resulted in comparable taxa coverage with only a few differences in the number of OTUs of some fungal classes that exhibited the same relative abundances. Overall, our results con firm that the primer pairs have a much more sub stantial impact on fungal diversity and composition 4.4. PNA clamps efficiently exclude plant sequences without modifications in fungal diversity and composition PNA clamps have been described in previous stu dies and were first used to inhibit the host DNA amplification in studies that aimed to characterise bacterial communities associated with plant MYCOLOGY 267 tissues. They were later used for fungal studies (Lundberg et al. 2013). It was shown that PNA clamps are not universal and need to be designed based on the plant species (Fitzpatrick et al. 2018). Although the clamps used in this study were designed for Populus species, they efficiently excluded U. dioica sequences. In Populus roots, a primer mixture tagging the fungal ITS2 region, combined with 5.8S PNA, reduced plant reads from 98.4% to 1.2% (Cregger et al. 2018). In our study, the same concentration of ITS PNA clamp fully inhibited plant reads with 5.8S-Fun while few reads (<1%) were still recovered with the two other primer pairs. The efficiency of PNA clamps has been described as concentration-dependent (Belda et al. 2017), but our result suggests that the concentration of PNA should be adjusted depending on the primer pair used. Clamping in plant roots did lead to a greater sequencing recovery of other eukaryotes besides fungi, which still accounted for less than 1% in relative abundance, even with the non-fungal specific gITS7 primers. These results suggest that blocking plant DNA amplification did not result in an over- amplification of non-plant and non-fungal sequences. PNA clamps are useful for excluding plant sequences without introducing bias into the determination of fungal community composition. 4.5. PNA clamps improve sequencing depth and fungal richness determination for non-fungal specific primers at a low read depth 4.5. PNA clamps improve sequencing depth and fungal richness determination for non-fungal specific primers at a low read depth Although we demonstrated that PNA clamps signifi cantly excluded plant contamination in our experi ments, they did not enhance the observed fungal diversity. This might largely be explained by the fact that fungal reads accounted for at least 75% of total reads, even with gITS7, which is not fungal-specific. Consequently, when the plant sequence contamina tion is relatively low, and the sequencing depth is high, as it was the case in our experimental condi tions, using PNA clamps seems unnecessary for esti mating plant-associated fungal diversity and composition. 4.4. PNA clamps efficiently exclude plant sequences without modifications in fungal diversity and composition Still, at artificially reduced sequencing depths, and particularly below 2,000 sequences per sample, we observed a substantial decrease in fungal richness for the non-fungal specific primers gITS7, which was alleviated by the use of PNA clamps, align ing it with the fungal-specific fITS7. When using non- specific primers, for instance, to ensure high taxo nomic coverage (Op De Beeck et al. 2014), consider ing that rarefying OTU datasets at relatively low read depth as a normalisation approach is commonly employed (McKnight et al. 2019; Tedersoo et al. 2022), using PNA clamps appears as a suitable method to reduce plant DNA contamination and minimise the loss of fungal richness. PNA clamps increased the proportion of fungal reads, contrary to Borodušķe et al. (2023), but did not result in an over-amplification of fungal classes and genera. Also, PNA clamps did not impact OTU richness, Shannon index, or Hill index, and did not increase the recovery of rare microbial taxa, as described in Moccia et al. (2020). Instead, our results align with those of Fitzpatrick et al. (2018) on plant bacterial communities but differ from those of Steven et al. (2018) based on bee bacterial communities (Muñoz-Colmenero et al. 2020). PNA clamps slightly impacted the fungal com position during the amplification of the mock commu nity. Clamping impacted several OTU relative abundances, with variations ranging from 2.5% to less than 1%, using fITS7 and gITS7 primers. This phe nomenon was not observed in environmental samples except for Mortierellomycetes and gITS7 primers. PNA had little impact on the fungal composition com pared with the unclamped samples. These results, in agreement with those of Reigel et al. (2020) on coral- associated bacteria, indicate a proportional DNA ampli fication of microbial communities with the increase in fungal sequences. Collectively, our results suggest that Acknowledgments Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H. 2010. ITS as an environmental DNA barcode for fungi: An in silico approach reveals potential PCR biases. BMC Microbiol. 10(1):189. doi:10.1186/1471-2180-10-189 . The work was funded by the European Union’s Horizon 2020 research and innovation programme under Grant Agreement ERA-NET Cofund SusCrop (N°771134), being part of the Joint Programming Initiative on Agriculture, Food Security and Climate Change (FACCE-JPI). CV has received a Ph-D grant from the Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation (MESRI). DB has received funding from the French PIA project “Lorraine Université d’Excellence” Deepsurf, reference ANR-15-IDEX-04-LUE. We thank Marie- Laure Toussaint for the sampling of the nettles and Stephane Pfendler for performing the fungal DNA extractions. Bodenhausen N, Somerville V, Desiró A, Walser JC, Borghi L, van der Heijden MGA, Schlaeppi K. 2019. Petunia- and Arabidopsis-specific root microbiota responses to phos phate supplementation. Phytobiomes J. 3(2):112–124. doi:10.1094/PBIOMES-12-18-0057-R . Bollmann-Giolai A, Malone JG, Arora S. 2022. Diversity, detec tion and exploitation: Linking soil fungi and plant disease. Curr Opin Microbiol. 70:102199. doi: 10.1016/j.mib.2022. 102199 . Borodušķe A, Ķibilds J, Fridmanis D, Gudrā D, Ustinova M, Seņkovs M, Nikolajeva V. 2023. Does peptide-nucleic acid (PNA) clamping of host plant DNA benefit ITS1 amplicon-based characterization of the fungal endophyte community? Fungal Ecol. 61:101181. doi: 10.1016/j.funeco. 2022.101181 . 5. Conclusions In this study, we tested the efficiency of primer pairs, Ta, and PNA clamps to limit plant DNA contamination while assessing fungal diversity and composition in plant tissues. We highlight that using fungal-specific primer pairs to exclude plant sequences yielded mixed results, specifically that fungal-specific primer (5.8S-Fun) pairs led to similar levels of plant DNA contamination as the non-fungal specific primer (gITS7). Furthermore, increasing Ta reduced plant DNA contamination and improved the recovery of rare fungal taxa but had a substantial negative impact on the overall fungal richness. Finally, we C. VIOTTI ET AL. 268 demonstrated that PNA clamps efficiently suppressed plant amplification without introducing bias in fungal diversity and composition. The positive effects of PNA clamps on the fungal richness quantification were particularly significant at low read depth, and appear to be suitably combined with universal primers. 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https://openalex.org/W3205822131	https://www.nature.com/articles/s43246-021-00209-z.pdf	English	null	Artificial intelligence for search and discovery of quantum materials	Communications materials	2,021	cc-by	11,885	Artiﬁcial intelligence for search and d of quantum materials Valentin Stanev 1,2✉, Kamal Choudhary3, Aaron Gilad Kusne1,3, Johnpierre Paglione 2,4,5 & Ichiro Takeuchi1,2 Artiﬁcial intelligence and machine learning are becoming indispensable tools in many areas of physics, including astrophysics, particle physics, and climate science. In the arena of quantum materials, the rise of new experimental and computational techniques has increased the volume and the speed with which data are collected, and artiﬁcial intelligence is poised to impact the exploration of new materials such as superconductors, spin liquids, and topolo- gical insulators. This review outlines how the use of data-driven approaches is changing the landscape of quantum materials research. From rapid construction and analysis of compu- tational and experimental databases to implementing physical models as pathﬁnding guidelines for autonomous experiments, we show that artiﬁcial intelligence is already well on its way to becoming the lynchpin in the search and discovery of quantum materials. I I n the last decade, a broad new topic has emerged at the forefront of condensed matter physics. It includes recently discovered exotic systems such as two-dimensional (2D) materials1, topological insulators2, topological superconductors3, and Weyl semimetals4 alongside several materials that have been bafﬂing researchers for many years—unconventional superconductors5–7 and quantum spin liquids8—in the loosely-deﬁned family of “quantum materials”9. The unifying theme connecting these seemingly disparate systems is the idea of emergence—they cannot be understood as simple ensembles of noninteracting fermions or bosons in “free” space; rather, their unique properties spring out of the collective behavior of a macroscopic number of interacting quantum particles. These materials host various exotic excitations, e.g., “relativistic” fermions in Dirac materials, Majorana bound states in topological superconductors, and skyrmions in chiral magnets10, which not only enrich our fundamental understanding of matter but also hold the promise of “quantum” leaps in a multitude of technological arenas. Because of its high carrier mobility, graphene stands to change the future of high-speed electronics and communication devices11. Topological insulators, which exhibit quantized anomalous quantum Hall effect12–14, possess spin-momentum-locked conduction channels that are immune to backscattering and can be used to make perfect spin-ﬁltering devices15. p p g But arguably the most exciting prospect is the use of topological superconductors in fault- tolerant quantum computing16. Quantum information processing based on manipulating topologically protected Majorana states provides an inherent cover against the threat of deco- herence—the Achilles heel of most embodiments of qubits. 1 Department of Materials Science and Engineering, University of Maryland, College Park, MD 20742, USA. 2 Maryland Quantum Materials Center, University of Maryland, College Park, MD 20742, USA. 3 National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. 4 Department of Physics, University of Maryland, College Park, MD 20742, USA. 5 The Canadian Institute for Advanced Research, Toronto, ON M5G 1Z, Canada. ✉email: vstanev@umd.edu REVIEW ARTICLE COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsm Artiﬁcial intelligence for search and d of quantum materials The complexities and rich phy- sics of these materials make them simultaneously one of the most promising targets and a particularly challenging subject for the emerging AI-enabled methods. Only exhaustive high- dimensional data encompassing a diverse range of physical properties can help scientists develop a comprehensive under- standing of materials such as unconventional superconductors, with complex physics often rooted in strong electron–electron interactions, or graphene superlattices, hosting a multitude of competing phases29. Thus, data need to be fused from disparate sources with vastly different modalities and veracities: theoretical models and ﬁrst-principles computational tools, various experi- mental measurements, and results from previous studies. This review provides a vision for the future where ML algorithms are commonly proliferated for the creation, analysis, and visualiza- tion of such high-dimensional heterogeneous data collections. The focus of the article is superconductivity, which is one of the most intensely pursued topics in all of condensed matter physics: the continued research effort, combined with its relatively long history, has led to signiﬁcant accumulation of computational and experimental data—a prerequisite for the use of ML tools. However, the tools being developed to study superconductors have the potential to dramatically increase the efﬁciency of the experimental and computational studies of all quantum materials, representing a veritable breakthrough in the exploration of these complex physical systems. (For some common AI terminology and recent examples of ML applied to quantum materials pro- blems see Fig. 1 and Box 1.) Still, this vision is not without its share of potential pitfalls. In the sections below, we discuss the beneﬁts and challenges associated with AI-based approaches to novel quantum materials. These exciting developments notwithstanding, for now, con- ventional DFT remains the workhorse of computational materials science. It has been shown that the calculations can be combined with ML algorithms to efﬁciently and accurately predict materials properties such as melting temperature, bandgap, shear modulus, and heat capacity49–51. DFT methods were also at the center of some of the early data-driven approaches to quantum materials, especially in the search for new superconductors. The discovery of superconducting materials has always been driven by serendipity and individual researchers’ intuition. However, the complex physics of cuprates and other strongly correlated materials (for example organic charge-transfer salts, exhibiting a variety of exotic phases: unconventional superconductivity, Mott insulator, spin liquid, etc.52) has forced scientists to explore more advanced data- centric approaches. In ref. Artiﬁcial intelligence for search and d of quantum materials Great strides are being made toward realizing topological superconducting qubits using existing materials (e.g., by inducing super- conductivity in topological insulators through the proximity effect)17,18; however, there is very little progress in systematically identifying many true “intrinsic” topological superconductors. p g y y y g y p g p In general, the search for new quantum materials presents an immense scientiﬁc challenge. The rich tapestries of quantum physics, which make them so exciting are also the very reasons 1 MMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat REVIEW ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z which render them so difﬁcult to decipher. The notorious case in point is the superconductivity in cuprate materials, whose fun- damental mechanism still eludes us after three decades of con- certed research efforts5,6,19. For some strongly correlated quantum materials such as spin liquids even the theoretical lan- guage necessary to adequately describe them is yet to be fully developed. When there is no guidance from rigorous theory, we are often left with serendipity to work with while exploring new compounds. There is, however, a glimmer of hope in an unusual form: research tools using Artiﬁcial Intelligence (AI) are coming to the rescue. high-quality data. Not surprisingly, in physical sciences, these algorithms were ﬁrst adopted in ﬁelds with abundant and highly standardized measurements. In fact, the vast amounts of data generated in particle physics have for decades required the use of automated algorithms for data analysis21,33. The importance of ML in this area is exempliﬁed by the central role it played in the discovery of the Higgs boson at the Large Hadron Collider at CERN33. which render them so difﬁcult to decipher. The notorious case in point is the superconductivity in cuprate materials, whose fun- damental mechanism still eludes us after three decades of con- certed research efforts5,6,19. For some strongly correlated quantum materials such as spin liquids even the theoretical lan- guage necessary to adequately describe them is yet to be fully developed. When there is no guidance from rigorous theory, we are often left with serendipity to work with while exploring new compounds. There is, however, a glimmer of hope in an unusual form: research tools using Artiﬁcial Intelligence (AI) are coming to the rescue. Artiﬁcial intelligence for search and d of quantum materials When it comes to materials data, sophisticated computational ﬁrst-principles tools are the order of the day, and enormous databases with millions of computed property entries are now readily available at everyone’s ﬁngertips34–38. In the realm of quantum materials, however, the situation is less auspicious. The most widely used computational method for simulating materials properties is Density Functional Theory (DFT), which has served as the backbone of materials by design in areas such as energy and magnetic materials (see, for example, refs. 39–41). Yet, DFT is built on several commonly used but essentially uncontrolled approximations. While the trends of the physical properties (e.g., as a function of composition) calculated by conventional DFT are generally correct, the veracity of the calculated parameters depends signiﬁcantly on computational details. For quantum materials, calculated properties can become even more tenuous and inaccurate, due for example, to self-interaction error origi- nating in electron–electron interactions, spin-orbit coupling, magnetism, etc. This severely limits the utility of conventional DFT for entire classes of quantum materials. A very promising emerging research area addresses this challenge by using ML methods to systematically improve the approximations made in DFT calculations42–45. AI methods are also being used to accel- erate high-level methods such as Quantum Monte Carlo (QMC)46,47 and Dynamical Mean Field Theory (DMFT)48, which are more accurate than DFT but can be orders-of-magnitude slower and higher in computational cost. The explosive increase in computational power and volume of accessible data is fueling the growth of AI as it transforms many aspects of our everyday lives with emerging technologies such as self-driving vehicles and Internet of Things devices. AI, especially its subﬁeld of Machine Learning (ML), is also becoming a bread- and-butter analysis tool, with important applications in virtually all sciences, from cancer research20 to astrophysics21. ML has already made its mark on condensed matter and materials physics by providing a robust platform for extracting a parsimonious description of a materials system from experimental and com- putational data22–28. When the task at hand is the discovery of new quantum materials, then one obvious strategy is to unleash ML on information obtained from existing compounds to make predictions on compositions and collective behaviors of their atomic constituents. In this review, we discuss the ongoing research spearheading the effort to turn AI into a ubiquitous and indispensable tool for the study of quantum materials. Artiﬁcial intelligence for search and d of quantum materials 53, a series of DFT calculations of the band structure of hole-doped cuprates were compared, in order to identify physical parameters governing superconductivity in these materials. The authors of ref. 54 went further and proposed several distinct characteristics of the band structure of cuprates as key precursors of their superconductivity. These characteristics were then used as criteria to ﬁlter through the calculated electronic structures of all materials in Inorganic Crystal Structure Database (ICSD), containing hundreds of thousands of existing crystal structures, and over 100 materials were identiﬁed as potential high-temperature superconductors. A similar approach was used recently in ref. 55, where p-terphenyl—an organic material that shows hints of a possible superconducting transition at ≈123 K when doped with potassium—was used as a prototype. A database containing the electronic structure of more than 10,000 organic crystals was surveyed for compounds with a similar density of states (DOS)56, and as a result, 15 compounds were proposed as candidate superconductors. COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat Box 1 \| Key terms Query strategies are typically some mix of exploration (aiming to maximize new information) and exploitation (aiming to optimize a property). g, g, erimental design): Subﬁeld of ML, designing algorithms able to interactively query an information source to obtain ne typically some mix of exploration (aiming to maximize new information) and exploitation (aiming to optimize a property The studies mentioned above combine DFT calculations with heuristic criteria to search for novel superconductors. Such cri- teria unavoidably suffer from various biases and can be difﬁcult to generalize. A much more rigorous approach is offered by ML methods, which provide a systematic way to extract important predictors of materials properties from complex high- dimensional data. As an example, an investigation in 2015 used structural and electronic properties data from the AFLOW Online Repositories to deﬁne and calculate various ﬁngerprints that encode materials’ electronic band structures, as well as crystallographic and constitutional information57. These ﬁnger- prints were then used to create both regression and classiﬁcation ML models for the critical temperature of several hundred superconductors. The classiﬁcation model separating the mate- rials into two groups based on their critical temperature showed good predictive performance. Such models can be used to screen hundreds of thousands of existing and potential materials stored in computational databases. lattice vibrations creates an effective attractive electron–electron interaction leading to Cooper pair formation. However, an ab initio calculation of electron-phonon coupling strength requires knowledge of properties such as the phonon DOS, which are computationally expensive, especially for complex materials. In a recent publication, it was shown that a neural network model can be trained to create high-ﬁdelity phonon DOS predictions. The authors utilized phonon DOS calculations of ~1500 crystalline solids as a training dataset58. The predictions of the model were able to capture the main features of phonon DOS, even for crystalline solids with elements not present in the training set. Thus, by reducing the computational cost of ab initio methods, the use of ML can signiﬁcantly enhance the throughput of computational materials screening. In yet another example of incorporating AI into DFT for superconductivity, ML feature selection methods have been uti- lized to derive a simple analytical expression improving the Allen- Dynes approximation59. The Allen-Dynes analytical formula is based on the Eliashberg-Migdal theory and is commonly used to predict the critical temperature of electron-phonon paired superconductors, reducing the number of required DFT calcula- tions. AI for computational quantum materials In application after application, it has been demonstrated that ML can turn complex multidimensional data into actionable knowl- edge (see, for example, refs. 30–32). The success of ML methods, however, depends crucially on access to prodigious amounts of COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat 2 REVIEW ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z Fig. 1 Commonly used machine-learning tasks and algorithms. The ﬁgure shows the most common types of machine-learning tasks and algorithms, as well as recent examples of these algorithms applied to quantum materials problems. See Box 1 for the deﬁnitions of some key machine-learning terms. Fig. 1 Commonly used machine-learning tasks and algorithms. The ﬁgure shows the most common types of machine-learning tasks and algorithms, a well as recent examples of these algorithms applied to quantum materials problems. See Box 1 for the deﬁnitions of some key machine-learning terms Fig. 1 Commonly used machine-learning tasks and algorithms. The ﬁgure shows the most common types of machine-learning tasks and algorithms, as well as recent examples of these algorithms applied to quantum materials problems. See Box 1 for the deﬁnitions of some key machine-learning terms. Fig. 1 Commonly used machine-learning tasks and algorithms. The ﬁgure shows the most common types of machin well as recent examples of these algorithms applied to quantum materials problems. See Box 1 for the deﬁnitions of Box 1 \| Key terms Box 1 \| Key terms Artiﬁcial intelligence: “Intelligence” demonstrated by machines able to perceive their environment and take autonomous actions to achieve a goa Machine learning: Branch of AI, a method for automated model building. Design of systems that can learn from data and make decisions with minim human intervention. Supervised learning: Subﬁeld of ML, aimed at creating predictive models based on both input and output (target) data. Subdivides into: Classiﬁcation: Modeling target data with discrete categories. Regression: Modeling continuous target data. Commonly used algorithms include Linear Regression, Support Vector Machines, Decision Trees, Gaussian Processes, Neural Networks. Unsupervised learning: Subﬁeld of ML, aimed at discovering internal representations from input data only. The two most widely used subﬁelds a Dimensionality reduction: Creating low-dimensional projections of high-dimensional data. Clustering: Finding groups of related data points. Commonly used algorithms include K-means Clustering, Hierarchical Clustering, Hidden Markov Models. Active learning (optimal experimental design): Subﬁeld of ML, designing algorithms able to interactively query an information source to obtain ne outputs. Query strategies are typically some mix of exploration (aiming to maximize new information) and exploitation (aiming to optimize a propert Artiﬁcial intelligence: “Intelligence” demonstrated by machines able to perceive their environment and take autonomous actions to achieve a goal. Machine learning: Branch of AI, a method for automated model building. Design of systems that can learn from data and make decisions with minimal human intervention. Subﬁeld of ML, aimed at creating predictive models based on both input and output (target) data. Subdivides into: ing target data with discrete categories. vised learning: Subﬁeld of ML, aimed at creating predictive models based on both input and output (target) data. Su ﬁcation: Modeling target data with discrete categories. Regression: Modeling continuous target data. Commonly used algorithms include Linear Regression, Support Vector Machines, Decision Trees, Gaussian Processes, Neural Networks. Unsupervised learning: Subﬁeld of ML, aimed at discovering internal representations from input data only. The two most widely used subﬁelds are: Dimensionality reduction: Creating low-dimensional projections of high-dimensional data. Cl i Fi di f l d d i g g g p p ommonly used algorithms include K-means Clustering, Hierarchical Clustering, Hidden Markov Models. Commonly used algorithms include K-means Clustering, Hierarchical Clustering, Hidden Markov Models. Active learning (optimal experimental design): Subﬁeld of ML, designing algorithms able to interactively query an information source to obtain new outputs. COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat Box 1 \| Key terms The increase of the accuracy achieved in the new formula p In addition to directly predicting a property of interest (such as Tc) from existing DFT data, ML can be used to accelerate the intermediate computational steps. For example, in conventional superconductors, the coupling between the electrons and the 3 REVIEW ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z can improve the computational pipeline, which in turn can lead to the discovery of novel superconductors faster. explore the interplay between particle–particle interactions, band structure, and constrained dimensionality (leading to effects such as the long-sought Wigner crystal75). The search for new 2D materials and a systematic comparison of their properties is still in its infancy. High-throughput DFT has been used in the last several years to compile publicly available databases of potential 2D materials76–79. In ref. 80, a JARVIS-DFT dataset containing results of 2D and 3D DFT calculations was used to develop hand- crafted structural descriptors as inputs into gradient boosting models to predict properties including exfoliation energies, for- mation energies, and bandgaps. From the synthesis point of view, the exfoliation energy is particularly crucial for 2D materials design, yet its DFT is prohibitively expensive. These models were then used to discover exfoliable materials satisfying speciﬁc property requirements. In ref. 81, the Computational 2D Materials Database (C2DB) was used to create an ML model (again based on gradient boosting) utilizing composition and structural pre- dictors to classify potential 2D materials as having low, medium, or high stability. The model was used to discover potential 2D materials suitable for photoelectrocatalytic water splitting. In contrast to superconductors, the concept of materials with intrinsic topologically nontrivial states is relatively new2,3. The current ﬂurry of activities was initially spearheaded by a theore- tical prediction60 and the subsequent experimental conﬁrmation of the existence of topological insulators61,62—materials with insulating bulk but protected metallic surface states. Despite the signiﬁcant efforts in the last decade, however, only a handful of distinct topological insulators, as well as other topological mate- rials such as Dirac and Weyl semimetals4,63–65 are known, and given that many of them are difﬁcult to synthesize and/or suffer from deleterious properties—such as the presence of bulk trivial states undermining the surface states, the community is always on the lookout for new topological materials. Box 1 \| Key terms p g To address this issue, several groups have developed algorithms such as symmetry indicators and spin-orbit spillage that use materials chemistry and symmetry in combination with electro- nic structures to calculate its topological properties, opening the door to automated screening for new topological insulators and semimetals66–68. Based on these ideas, several searches through ICSD generated extensive lists of candidates corresponding to various types of topological materials69–73. Such developments make the ﬁeld a fertile ground for the application of ML methods (for an example see Fig. 2). A very recent work constructed a gradient boosting model (a particular type of powerful supervised learning algorithm) that can predict the topology of a given known material based only on “coarse-grained” chemical com- position and crystal symmetry predictors with an accuracy of 90%74. Similar to superconductivity, such models can be used to accelerate the search for novel materials by providing fast and efﬁcient means to predict the possible topological nature of a given candidate. 1 Beyond monolayers, there is a vast conﬁguration space of possible van der Waals heterostructures, which can be formed by stacking different 2D layers. Such hybrid structures add addi- tional degrees of freedom to their functionalities, creating 2D and 3D materials with untold tunability. Because DFT is too time- consuming to explore more than a small fraction of all possible combinations, incorporating ML in the process provides a prac- tical alternative. Recent work has trained several ML models for predicting the interlayer distance and bandgap of bilayer heterostructures82. The models showed good accuracy on both tasks and were used to predict the properties of nearly 1500 hypothetical bilayer structures based on less than 300 DFT cal- culations. This underscores the promise of combining DFT and ML for rapid computational screening to identify new hybrid heterostructures with interesting and desirable properties. g Another class of quantum materials systems is 2D materials1. Since the breakthrough discovery of graphene in 2004, many other 2D materials families (such as monolayers of hexagonal boron nitride, silicene, germanene, stanene, phosphorene, and transition metal dichalcogenides) have been synthesized1. They have been the focus of a concerted investigation, both due to their rich potential in technologies such as electronics, sensing, and energy storage, and because they offer an entirely new avenue to COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z In an early pioneering work, Villars and Phillips demonstrated that using just three stoichiometric descriptors (associated with ele- mental makeups), the 60 known superconductors at the time (in the 80 s) with Tc > 10 K can all be clustered in three islands83. Based on this observation the authors made predictions for potential high-temperature superconductors83,84. In another early work, Hirsch used statistical methods to look for correlations between normal state properties and Tc of the metallic elements in the ﬁrst six rows of the periodic table85. g y This increase of the available experimental data, together with the surge of popularity of AI topics and the appearance of general-purpose ML libraries, led to a recent groundswell of activities introducing sophisticated ML methods in the study of superconductivity91–96. In one notable example91, Stanev et al. considered more than 16,000 different compositions extracted from the MatNavi SuperCon database, which contains an exhaustive list of known superconductors as well as many “clo- sely-related” materials varying only by small changes in stoi- chiometry. Compared to the early exercise by Villars and Philips in the 80 s, the orders-of-magnitude increase in the number of data points has led to the possibility to create a robust ML pipeline. The regression models developed to predict the values of Tc for different superconducting families used over one hundred stoichiometric descriptors, demonstrated strong predictive power and high accuracy, and offered valuable insights into the origins of superconductivity mechanisms in different materials groups (see Fig. 3b). The models also demonstrated an important lim- itation of ML in failing to extrapolate to materials families not included in the training set. A pipeline was then created to search for potential new superconductors among the roughly 110,000 different compositions contained in ICSD, which resulted in predictions of possible Tc’s above 20 K in 35 known compounds that had previously not been tested for superconductivity. With time, more and more of the experimental data collected by generations of scientists are becoming easily accessible to researchers. Thanks to tedious curation efforts over decades by researchers at several institutes around the world, there are large databases with compiled experimental report entries such as the Phase Equilibria Diagram and the ICSD, arguably one of the largest compendiums of experimental materials data86. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z Fig. 3 Examples of machine-learning methods applied to experimental data. a Three clusters (shown with blue, green, and red), representing groups of related materials, containing topologically nontrivial materials. The materials representations are extracted from a neural network model predicting the Bravais lattice from composition—for details, see ref. 87. The clusters can be used to search for new topological materials. b The measured vs predicted ln(Tc) of various superconductors based on a random forest model presented in ref. 91. The same model can predict Tc of several distinct superconducting classes (blue markers: low-Tc materials; green markers: iron-based superconductors; red markers: cuprate superconductors). Fig. 3 Examples of machine-learning methods applied to experimental data. a Three clusters (shown with blue, green, and red), representing groups of related materials, containing topologically nontrivial materials. The materials representations are extracted from a neural network model predicting the Bravais lattice from composition—for details, see ref. 87. The clusters can be used to search for new topological materials. b The measured vs predicted ln(Tc) of various superconductors based on a random forest model presented in ref. 91. The same model can predict Tc of several distinct superconducting classes (blue markers: low-Tc materials; green markers: iron-based superconductors; red markers: cuprate superconductors). the datasets. In fact, one is often hard-pressed to ﬁnd extensive databases of functional materials in general. A rare exception is the MatNavi database, managed by the National Institute of Materials Science (NIMS) in Japan. It is an experimental database encompassing well-curated information on materials with a variety of functional properties. Transcribing published journal results into formatted databases can be a massive undertaking, which NIMS materials data scientists have managed to do for decades. Some of the collected information is already being used for data-driven materials exploration88,89. The Pauling File also contains up to tens of thousands of individual composition entries (and corresponding speciﬁc physical property quantities) diligently entered over decades90. experimental materials data are becoming just as prevalent. One natural task would be to apply them to databases amassing experimentally known compounds and to build models for making predictions. In the study of superconductors—a ﬁeld with more than a century of extensive research history—there are large volumes of accumulated information. Sometimes even a small fraction of this data can be used to discover important trends. COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat Applying ML to experimental databases: making predictions from known materials With the ability to quickly recognize patterns in large collections of quantitative information, applications of AI approaches in Fig. 2 Example of Density Functional Theory used to predict topological properties of materials. Periodic table trend showing the percentage chance Fig. 2 Example of Density Functional Theory used to predict topological properties of materials. Periodic table trend showing the percentage chance (color coded) that a compound containing any given element can be topologically nontrivial based on spin-orbit spillage approach, based on spillage data for 4835 nonmagnetic materials (reproduced from ref. 68 under the terms of the Creative Commons CC BY license). The spillage is a machine-learnable quantity, and an accurate classiﬁcation model predicting it (utilizing gradient boosting decision trees) was developed in ref. 73. 4 COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat Fig. 2 Example of Density Functional Theory used to predict topological properties of materials. Periodic table trend showing the percentage chance (color coded) that a compound containing any given element can be topologically nontrivial based on spin-orbit spillage approach, based on spillage data for 4835 nonmagnetic materials (reproduced from ref. 68 under the terms of the Creative Commons CC BY license). The spillage is a machine-learnable quantity, and an accurate classiﬁcation model predicting it (utilizing gradient boosting decision trees) was developed in ref. 73. Fig. 2 Example of Density Functional Theory used to predict topological properties of materials. Periodic table trend showing the percentage chance (color coded) that a compound containing any given element can be topologically nontrivial based on spin-orbit spillage approach, based on spillage data for 4835 nonmagnetic materials (reproduced from ref. 68 under the terms of the Creative Commons CC BY license). The spillage is a machine-learnable quantity, and an accurate classiﬁcation model predicting it (utilizing gradient boosting decision trees) was developed in ref. 73. COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat 4 4 COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z REVIEW ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z Because of their comprehensive and exhaustive nature, such databases have become the de-facto standard-bearers for materials exploration and development, and they often serve as a starting point for building ML models (as already discussed above for use in con- junction with purely computational approaches). While these databases rarely contain functional properties, phase formation and stability as well as phase diagrams can be used as blueprints for navigating the materials exploration process. For example, a recent work used ICSD data to train a neural network to predict crystal structure information87. The network’s activations maps were then used to group materials according to their composi- tional and structural similarity, providing lists of materials potentially sharing properties with known superconductors or topological insulators (see Fig. 3a) Unfortunately, when it comes to experimental databases of known quantum materials, usually there are very few entries in One interesting ﬁnding from this pipeline is that most of the newly identiﬁed possible superconductors possess a ﬂat/nearly- 5 5 REVIEW ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z ﬂat band just below the Fermi energy, leading to an increase in the electronic DOS. Such enhanced DOS has long been con- sidered a promising way to boost Tc. However, a very recent study using experimental Tc and DFT-based DOS calculations failed to discover a strong and consistent correlation between super- conductivity and peaks in the electronic DOS97. Yet, the strongest conclusion from the latter study was on “…the restrictions that the current availability and organization of materials data place on reliable machine-learning and data-based experimentation,” underscoring the need for a more systematic approach to col- lecting and organizing quantum materials data. order parameter104,105. Pump-probe techniques permit the study of highly excited and out-of-equilibrium states and phases, sometimes revealing hidden tendencies106. With these techniques, even a single measurement of one material can generate large volumes of high-dimensional data necessitating the use of sophisticated statistical methods. In these instances, ML algorithms are implemented for uncovering phy- sics working behind the scene, which affects the collective beha- vior of materials at an atomic scale. In one recent example, a neural network model was used to analyze Spectroscopic Imaging Scanning Tunneling Microscopy (SISTM) images of the CuO2 planes, where transport takes place in high-temperature super- conducting cuprates107. The model was designed to recognize different symmetry-breaking ordered electronic states. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z It was trained on a set of artiﬁcial images, each generated to represent one of four distinct states, differing by their fundamental wave- vector. Various forms of heterogeneity, intrinsic disorder, and topological defects were added to these images to mimic the experimental data. The model was then applied to experimental images from carrier-doped cuprates. Analyzing the noisy and complex data, the model was able to discover the existence of a translational-symmetry-breaking ordered state. The presence of this particular ordered state has important implications for the- ories aiming to explain the mysterious pseudogap phase of these materials. g g g q This, however, is far easier said than done: the experimental exploration of quantum materials is a remarkably diverse and distributed endeavor, and it is not uncommon for tens or even hundreds of research groups to study the same material, with each group applying its unique toolbox and interrogation tech- niques. Also, many experiments are extremely resource-intensive and can only produce limited datasets. Combining numerous small datasets covering the same material can lead to issues with data consistency and incomplete metadata. Furthermore, the quantum materials community has yet to fully embrace the open data paradigm, and a signiﬁcant fraction of the collected data are only published in unstructured form (i.e., text and images) and are not made easily available to other researchers. (Signiﬁcant fraction of the collected information—especially “negative” results—is never published. For a rare example of work reporting both negative and positive results see ref. 98). In another notable example, researchers applied an ML method to the Angle-Resolved Photoemission Spectroscopy (ARPES) data of optimally doped and under-doped cuprates108. The use of a restricted Boltzmann machine model allowed the recovery of a “hidden” feature in the spectra: prominent peak structures pre- sent both in the normal and anomalous self-energies of the single- particle spectral function. These peaks structures cancel each other in the total self-energy and were only discovered by a model respecting physical constraints such as causality (encoded in the Kramers-Kronig relation). The use of ML allowed researchers to solve a non-linear underdetermined problem, and to obtain important information directly from experimental data, without the use of any theoretical model. The results clariﬁed the role of the energy dissipation and quantum entanglement in the super- conducting phase and provide a way to ﬁnally identify the boson responsible for the pairing in cuprates. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z g p ) Thus, if one wants to construct an experimental database of quantum materials, the researcher may have to resort to the laborious manual extraction of data points from published articles. The signiﬁcant effort required by this process at least partially explains the scarcity of databases in the ﬁeld. However, emerging AI-driven automatic generation of databases can provide an alter- native to the slow and tedious process. One example is the recently created database of almost 40,000 Curie and Néel phase-transition temperatures of magnetic materials99. It is fully auto-generated and completely open-source. The database was produced using Natural Language Processing (NLP) and related ML methods, applied to the texts of published chemistry and physics articles. The method is quite precise, with the accuracy of the extracted transition tem- peratures reaching 82%. In a very recent extension of this work, the same authors were able to reconstruct the phase diagrams of well- known magnetic and superconducting compounds, again using text data100. They also demonstrated that it is possible to predict the phase-transition temperatures of compounds not present in the database. (A yet another recent effort to organize various experi- mental results into a curated database focuses on digitization of plots extracted from published papers101.) Beyond superconductivity, ML was recently applied to neutron scattering data of a frustrated magnet, exhibiting a complex phase diagram, and used to extract model Hamiltonians and to identify different magnetic regimes109. An autoencoder—dimensionality reduction architecture based on a neural network—was trained to create a compressed representation of three-dimensional diffuse scattering, over a wide range of spin Hamiltonians. The auto- encoder was able to ﬁnd optimal Hamiltonian parameters matching observed scattering and heat capacity signatures. The autoencoder was also able to categorize different magnetic behaviors and eliminate background noise and artifacts in raw data. Thus, ML can augment many traditional diffraction and inelastic neutron scattering data analysis tools, which are often time-consuming and error-prone. COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat Autonomous materials laboratories (cluster) measurements from different points of a combinatorial library, and ML algorithms have been routinely applied to a large number of X-ray diffraction patterns to delineate structural phases and rapidly construct a composition-structure relationship120,124,129,130. The authors of ref. 124 took this idea a step further and used a comprehensive ML algorithm for on- the-ﬂy analysis of synchrotron diffraction data from combina- torial libraries to facilitate a search for rare-earth-free permanent magnets (see Fig. 4b). Unsupervised ML for rapid data reduction is now commonly being applied to high-throughput character- ization data from a variety of spectroscopic techniques, including Raman spectroscopy131, Time-of-Flight Secondary Ion Mass Spectrometry132, and X-ray photoelectron spectroscopy133. Armed with predictions of potential quantum materials generated by theory or the ML techniques discussed above, experiments can be designed for systematic synthesis and characterization of novel compounds. However, even with the predictions providing blueprints, one often has to negotiate potentially large multi- dimensional parameter space as in any materials exploration and optimization task. One proven platform to effectively host such experiments is the high-throughput approach, which permits the interrogation of libraries that contain hundreds or even thou- sands of different compounds111. Over the years combinatorial approaches have been effectively used to explore new superconductors112 and map their compositional phase diagrams (for an example demonstrating the ability of this method to discover superconducting compositions see Fig. 4a). It has also been used to optimize the synthesis of a topological Kondo insulator SmB6 in a thin-ﬁlm form113. p y y p p py This close integration of experimental tools and ML can be considered as the ﬁrst step towards an emerging AI-driven paradigm for materials exploration. It relies on active learning—a branch of AI dedicated to providing a systematic and rigorous approach for identifying the best experiment to perform to achieve an objective. This can be either ﬁnding the shortest path toward a material that optimizes some desired properties or identifying a series of experiments that maximizes knowledge of the explored space. Recently, materials physicists have begun to capitalize on active learning to accelerate experimental research134. For example, active learning has been used to advise experimentalists on the next best experiment to perform in the search for various functional materials135–137. COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z Fig. 4 Accelerating experimental discovery processes. a High-throughput methods can be used to perform validation of predicted materials. Visualization of resistivity vs temperature curves measured at different parts of a Fe-B composition spread. The horizontal range of each curve covers 2–300 K. The resistivity range for each curve is color coded (reproduced from ref. 141 under the terms of the Creative Commons CC BY license); b On-the-ﬂy analysis of synchrotron diffraction data124. A snapshot photo from the experiment. The upper screen shows the scanning stage with a thin-ﬁlm library inside a beamline hutch. The bottom screen is a laptop, where unsupervised ML of diffraction patterns is carried out after each measurement. The same setup has also been used to carry out active-learning-based autonomous experiments. Fig. 4 Accelerating experimental discovery processes. a High-throughput methods can be used to perform validation of predicted materials. Visualization of resistivity vs temperature curves measured at different parts of a Fe-B composition spread. The horizontal range of each curve covers 2–300 K. The resistivity range for each curve is color coded (reproduced from ref. 141 under the terms of the Creative Commons CC BY license); b On-the-ﬂy analysis of synchrotron diffraction data124. A snapshot photo from the experiment. The upper screen shows the scanning stage with a thin-ﬁlm library inside a beamline hutch. The bottom screen is a laptop, where unsupervised ML of diffraction patterns is carried out after each measurement. The same setup has also been used to carry out active-learning-based autonomous experiments. Extracting latent knowledge from materials characterization data data In an ironic twist, in some areas of experimental materials phy- sics, the issue is not the scarcity but the overabundance of data. In fact, negotiating the large quantities of data churned out in real- time by modern experimental labs is a signiﬁcant emerging challenge. Continual improvements in materials characterization instrumentation coupled with the ever-increasing might of readily available computers for data-acquisition and memory storage have created the capacity to collect data on an unprecedented scale and with high speed. Through advances in scanning transmission electron microscopy, it is now possible to determine atomic column positions with picometer level precision (see, for example, refs. 102,103). Spectroscopy and scanning probe mea- surements can provide detailed information about materials properties including electronic structure and symmetry of an Another very recent work reported a convolutional-neural- network-based classiﬁer designed to distinguish topological from trivial materials and trained on X-ray Absorption Spectroscopy (XAS) data110. The model showed high accuracy in distinguishing the different classes of materials, demonstrating the potential of ML methods in recognizing topological character embedded in complex spectral features. XAS is a widely used materials char- acterization technique, and the ability to decipher the topological character of material from XAS signatures can substantially simplify and expedite the experimental identiﬁcation of topolo- gically nontrivial materials. OMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat 6 REVIEW ARTICLE COMMUNICATIONS MATERIALS \| (2021) 2:105 \| https://doi.org/10.1038/s43246-021-00209-z \| www.nature.com/commsmat Outlook As physicists focus more and more on materials with unusual properties grounded in many-body quantum mechanics, their research methods have to evolve. The ongoing revolution in AI is a great opportunity for the condensed matter physics community, and ML tools are starting to play an important role in the study of quantum materials. From accelerating the ﬁrst-principles com- putational tools to helping analyze high-dimensional experi- mental data, these tools have already left their mark on the ﬁeld. But even more exciting developments are gradually turning into reality. Researchers are working on automated research systems controlled by AI-guided robots. These systems will allow scien- tists to build penetrating multidimensional pictures of these complex materials, and at the same time accelerate the search and discovery process. Autonomous materials exploration can be particularly effective when coupled with high-throughput experimentation. In a recent study, a real-time closed-loop, autonomous system for materials exploration and optimization (CAMEO) was demonstrated on a combinatorial library.140 The goal of CAMEO is to ﬁrst map the structural phase diagram across the compositional landscape and then use the phase map as a blueprint to optimize a physical property of interest. It was used to quickly ﬁnd the optimum composition of phase change memory materials with the largest bandgap difference between amorphous and crystalline states within a Ge–Sb–Te thin-ﬁlm composition spread. To achieve this, analysis of ellipsometry measurements from the library and remotely-controlled synchrotron diffraction were carried out simultaneously. Combining these two sources of information, CAMEO discovered a novel composition with phase change performance superior to known materials in the ﬁeld, for only about one-tenth of the time required to measure every point in the library. Apart from reshaping the experimental process, the use of such autonomous systems will reduce the many superﬂuous hurdles and dramatically lower the effort and time needed for running an experiment. This will allow scientists to focus on the most chal- lenging and important parts of the research process, while also making science more “democratic” and equitable. Received: 5 July 2021; Accepted: 12 September 2021; Received: 5 July 2021; Accepted: 12 September 2021; Even more revolutionary AI-enabled systems, designed to help human scientists in optimizing every step of the research process, are already being considered and developed. These systems will coordinate a host of computational and experimental probes to help efﬁciently search a vast and mostly unexplored composi- tional space. Autonomous materials laboratories Another work already demonstrated the potential of these methods in the ﬁeld of quantum materials: an active-learning framework designed to discover the material with the highest Tc was evaluated on about 600 known superconductors138. The framework did signiﬁcantly better than pure random guessing, highlighting the impact it can have. A signiﬁcant hurdle in the more widespread use of high- throughput methods is the need for special characterization tools, often required for the exploration of many classes of quantum materials. For instance, for detecting topological states, one typically turns to angle-resolved photoemission spectroscopy (ARPES) to look for their signature in electronic structure114. Antiferromagnetism—whose role in the superconductivity of several classes of materials is hotly debated—is best probed using neutron scattering115. X-ray magnetic circular dichroism has been used to investigate the electronic structure of quantum spin liquid α-RuCl3116 These advanced techniques require unique synchrotron or neutron facilities, and their implementation in the screening of combinatorial libraries has been limited to date117,118 The high-throughput approach generates large high- dimensional datasets, requiring analysis techniques that can rapidly turn raw data into knowledge with limited or no human supervision119. This community adopted dimensionality reduc- tion and data mining techniques early on120–123, gradually creating a diverse ML toolbox for the rapid digestion of combi- natorial data124–130. One very common task is to quickly group The active-learning algorithms guiding the exploration can indeed decisively outperform exhaustive experimental approa- ches, leading to a similar amount of actionable knowledge for only a fraction of the time and resources. This has profound implications: with a signiﬁcant reduction in the number of iterative experimental runs, it may now be possible to incorporate 7 REVIEW ARTICLE COMMUNICATIONS MATERIALS \| https://doi.org/10.1038/s43246-021-00209-z Fig. 5 Schematic for the autonomous experimental systems of the future. The AI in charge of the autonomous lab will have full control over the synthesis, characterization, and modeling tools. It will extract prior knowledge from structured databases, publications, and human guidance, do experiments and computations and be able to distill its ﬁndings in a succinct mathematical form. Such autonomous laboratories are already being implemented in other areas of materials science142. (Icons used in the ﬁgure are from INCORS). Fig. 5 Schematic for the autonomous experimental systems of the future. The AI in charge of the autonomous lab will have full control over the synthesis, characterization, and modeling tools. Autonomous materials laboratories It will extract prior knowledge from structured databases, publications, and human guidance, do experiments and computations and be able to distill its ﬁndings in a succinct mathematical form. Such autonomous laboratories are already being implemented in other areas of materials science142. (Icons used in the ﬁgure are from INCORS). to maximize the knowledge gain and drastically accelerate pro- gress. In its ultimate form, fully autonomous laboratories con- trolled by AI in command of various synthesis and characterization will orchestrate entire experimental campaigns, update their knowledge, and continue to explore until the desired goal is reached (see Fig. 5 for a schematic). These AI “scientists”, dedicated to the exploration of quantum materials, can help us ﬁnally solve some of the most enduring mysteries of physics. active-learning-guided tools at synchrotron and neutron facilities for quick screening of quantum materials libraries in closed-loop cycles. However, the examples of active learning discussed above only provided support for an important but limited set of questions, leaving the majority of the experimental design, execution, and analysis to human experts. This situation is rapidly changing, and truly autonomous experimental systems are beginning to arrive on the scene. 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https://openalex.org/W2151031617	https://europepmc.org/articles/pmc3963981?pdf=render	English	null	Intensity of Territorial Marking Predicts Wolf Reproduction: Implications for Wolf Monitoring	PloS one	2,014	cc-by	9,078	Luis Llaneza1., Emilio J. Garcı´a1, Jose´ Vicente Lo´ pez-Bao2,3. 1 A.RE.NA. Asesores en Recursos Naturales, S.L., Lugo, Spain, 2 Research Unit of Biodiversity (UO/CSIC/PA), Oviedo University, Mieres, Spain, 3 Grimso¨ Wildlife Research Station, Swedish University of Agricultural Sciences (SLU), Riddarhyttan, Sweden Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Regional wolf surveys were funded by the Regional Government of Asturias, the Spanish Environmental Ministry and the Regional Government of Galicia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. E-mail: jv.lopezbao@gmail.com . These authors contributed equally to this work. . These authors contributed equally to this work. tools [10], camera trapping [12], and statistical procedures [13,14], the implementation of complex approaches over large spatial scales (i.e. regions or countries) or large populations of these species is resource demanding. Consequently, these methods are still commonly restricted to threatened species, small populations or small distribution ranges [14–16]. Because resources for wildlife management are limited [17] (especially deteriorated under the current global financial crisis) and budgets are generally dependent on the conservation status of species [18], the availability of handsome funds to survey large carnivores over large spatial scales is not always guaranteed. This uncertainty is especially important if species or populations are not threatened or are delisted from any protective conservation status [19]. But, the management of contentious species such as large canids, bears or felids must require regular estimations of their abundance regardless of their conservation status in order to guide decision-makers, especially for those populations living in human-dominated landscapes [20]. Abstract Background: The implementation of intensive and complex approaches to monitor large carnivores is resource demanding, restricted to endangered species, small populations, or small distribution ranges. Wolf monitoring over large spatial scales is difficult, but the management of such contentious species requires regular estimations of abundance to guide decision- makers. The integration of wolf marking behaviour with simple sign counts may offer a cost-effective alternative to monitor the status of wolf populations over large spatial scales. Methodology/Principal Findings: We used a multi-sampling approach, based on the collection of visual and scent wolf marks (faeces and ground scratching) and the assessment of wolf reproduction using howling and observation points, to test whether the intensity of marking behaviour around the pup-rearing period (summer-autumn) could reflect wolf reproduction. Between 1994 and 2007 we collected 1,964 wolf marks in a total of 1,877 km surveyed and we searched for the pups’ presence (1,497 howling and 307 observations points) in 42 sampling sites with a regular presence of wolves (120 sampling sites/year). The number of wolf marks was ca. 3 times higher in sites with a confirmed presence of pups (20.3 vs. 7.2 marks). We found a significant relationship between the number of wolf marks (mean and maximum relative abundance index) and the probability of wolf reproduction. Conclusions/Significance: This research establishes a real-time relationship between the intensity of wolf marking behaviour and wolf reproduction. We suggest a conservative cutting point of 0.60 for the probability of wolf reproduction to monitor wolves on a regional scale combined with the use of the mean relative abundance index of wolf marks in a given area. We show how the integration of wolf behaviour with simple sampling procedures permit rapid, real-time, and cost- effective assessments of the breeding status of wolf packs with substantial implications to monitor wolves at large spatial scales. Citation: Llaneza L, Garcı´a EJ, Lo´pez-Bao JV (2014) Intensity of Territorial Marking Predicts Wolf Reproduction: Implications for Wolf Monitoring. PLoS ONE 9(3): e93015. doi:10.1371/journal.pone.0093015 Editor: Ce´dric Sueur, Institut Pluridisciplinaire Hubert Curien, France Editor: Ce´dric Sueur, Institut Pluridisciplinaire Hubert Curien, France Received June 13, 2013; Accepted February 28, 2014; Published March 24, 2014 Received June 13, 2013; Accepted February 28, 2014; Published March 24, 2014 Copyright: 2014 Llaneza et al. Collection of wolf marks We used 42 sampling sites with a regular presence of wolves during the last two decades (39 in Asturias and 3 in Lugo). Each sampling site comprised 2–5 valleys and the surrounding mountains covering approximately 150–200 km2 (see Fig. 1b). This area was of a similar size than the average home range size reported in wolves in N Iberia [41]. All sites were regularly sampled between 1994 and 2007 around the pup-rearing period (summer-autumn) within different regional wolf surveys (from 1994 to 2004 in Asturias and between 2006 and 2007 in Lugo). Information from these surveys was comparable since the same field procedures (see details below) were implemented in all surveys throughout the study period [42]. Each sampling site was surveyed on average 3 years (range 1–6), with a total of 120 sampling sites/year (hereafter sites, note that this is the sample size that will be used for subsequent analyses). Within each site, wolf marks were searched in transects along existing paths and trails, on foot or using a vehicle (,10 km h21) covering as much selected area as possible (see Fig. 1b). Wolf monitoring over large spatial scales is difficult. However, the integration of wolf marking behaviour with simple indexes resulting from sign counts (e.g. visual and scent marks such as faeces) may offer a good alternative to monitor wolves over large areas. Sign counts have been widely used as indexes of relative abundance of large carnivores. Government agencies have implemented them as a relative low-cost approach to gathering data at large scales using rangers, technical staff or volunteers [10,21]. Although there have been attempts to correlate these indexes with densities [31], few efforts have been carried out in order to link sign counts with demographic parameters such as breeding status. The most popular attempt is the use of snow- tracking to infer reproduction [28,32]; but it is a time-delayed method dependent on long periods of good snow conditions the next winter after the breeding period, absent, for instance, in most of the southern wolf’s European range. Faeces and ground scratching marks serve as visual and scent marks in wolves [34–36]. Based on their visual function facilitating their detection by field observers with different levels of expertise compared with urine –which is also dependent on snow conditions-, we considered both visual wolf marks for this study (hereafter wolf marks). Shape, size, content, smell and spatial position (i.e. Integrating Wolf Marking Behaviour into Monitoring As a consequence of multiple conflicts surrounding wolves (Canis lupus), a development of simple and efficient tools to regularly assess the status of populations is a pressing need. Methods to survey wolf populations involve sign surveys, howling, radio tracking, questionnaires, or statistics of livestock damages and hunting bags [21–23]. More recently, the use of non-invasive samples became popular [24–26]. In Europe, wolf management relies on an estimated number of individuals or packs, with the former estimation being less accurate as population size or range increases, and the quality of both wolf estimates varies widely across countries and within populations (see http://www.kora.ch/ sp-ois/wolf-ois) [27]. Thus, non-invasive samples or intensive radio-tracking are usually implemented over small spatial scales or small populations (,500 individuals) [16,24,26,28], whereas questionnaires or hunting statistics are commonly used over vast areas and large populations, increasing uncertainty (see http:// www.kora.ch/sp-ois/wolf-ois) [27]. On the other hand, small populations are annually surveyed (e.g. Scandinavian or the Western Alps wolf populations) [16,28], whereas a lack of temporal continuity or partial assessment covering small popula- tion fractions (usually corresponding to different administrative limits such as counties or national parks) is normal in large populations. This is the case for the NW Iberian wolf population – the main wolf population in Western Europe [29] - where the last comprehensive view of the status of the whole population (as a result of several Spanish regional surveys and a Portuguese national census) was ten years ago (estimated population size between 1999 and 2003 of ca. 320 packs or ca. 2000–3000 individuals) [30], and only some regional surveys are regularly implemented (e.g. county of Asturias). date, no studies have attempted to establish a real-time link between wolf marking behaviour and breeding status in a pack [39]. We discuss the application of the method proposed here for wolf monitoring over large spatial scales (i.e. countries or entire wolf populations). Study area The study was carried out in the Cantabrian Mountains, N Iberia (ca. 9,000 km2; Fig. 1a), covering the rugged region of Asturias and a small mountain area within Galicia (east of Lugo province, hereafter Lugo; Fig. 1a). Vegetation is mainly comprised by scrublands, woodlands, and grasslands (pastures). Scrublands are mainly composed by Calluna vulgaris, Genista spp., Erica spp. and Vaccinium spp..Woodlands are dominated by beech (Fagus sylvatica), oaks (mainly Quercus petraea and Quercus pyrenaica), birch (Betula pubescens) and anthropogenic chesnut trees (Castanea sativa). Isolated trees or small groups of holly (Ilex aquifolium) and rowan (Sorbus aucuparia) often occur scattered through mature or secondary forests. Scrublands are the predominant habitat type and the level of fragmentation of forests here is high [40]. Snow is present irregularly (both seasonally and annually) between December and March. Introduction Large carnivores are actively managed in an ebb and flow between human interests and conservation goals. The relative weight of social, economic, conservation or ecological factors [1–6] tip the balance towards either side continuously, turning the management of large carnivores into one of the most complex tasks within wildlife management. To achieve an adaptive management approach [7], decision-makers require reliable and updated estimates of their status to establish, for instance, trends, conservation actions, sustainable hunting quotas or population controls [3,8,9]. Independent of the target, primary management step is the ability to obtain objective, reliable and economically affordable abundance estimates that are easily reproducible over time. However, despite the fact that the wide range of methods available to survey these elusive species has [10,11], significantly increased due to promising advances in non-invasive molecular March 2014 \| Volume 9 \| Issue 3 \| e93015 March 2014 \| Volume 9 \| Issue 3 \| e93015 1 PLOS ONE \| www.plosone.org Integrating Wolf Marking Behaviour into Monitoring Integrating Wolf Marking Behaviour into Monitoring March 2014 \| Volume 9 \| Issue 3 \| e93015 Collection of wolf marks (a) Map showing the study area located in the Cantabrian Mountains (N Spain). (b) Scheme showing the spatial distribution of transects within a typical sampling site. doi:10.1371/journal.pone.0093015.g001 placed on the abovementioned landscape features represented some kind of marking behaviour [39]. This assumption also relies on the fact that wolf marks were deposited on conspicuous places on a landscape level that enhance them as marks [32,35,36,39,43]. This procedure decrease the probability to include faeces from young and lone wolves since they located more often their faeces off-trail [25,26,39]. Potential differences in marking behaviour among pack members [38] were not considered in this study. Furthermore, considering the posterior spatial distribution of rendezvous sites -based on the results from the howling and observation points - transects did not cross rendezvous sites, decreasing the probability to count pup faeces as wolf marks as well as avoiding the inclusion of notable aggregations of faeces in these sites as a consequence of its regular use by pack members (e.g. resting sites) [32]. elements often used by wolves as marking places (paths and trails, particularly focusing on junctions and mountain passes; see Fig. 1b) [34,42,43] where the probability of the detection of wolf marks by other intra and inter-pack individuals is maximized. Mountain passes may favour wolf movements between valleys in mountain- ous areas turning these places into important landmarks on a landscape level. One to several wildlife trails/paths usually cross mountain passes being an important place for territorial marking. For example, considering 10 transects crossing such passes in our study area, out of the 164 wolf marks found in a total of 83.5 km surveyed (an average of 8.5 km per transect), 82% of wolf marks were located in these particular conspicuous sites (ranging from 40% to 100% of wolf marks detected per transect). The ability of wolves to deposit their marks in conspicuous sites (both on a large and fine spatial scale) [34,35,39,43] indicates a degree of control about where to locate their marks. For example, wolf faeces, whether deposited as scent marks or not, are recognized as important visual marks and a powerful sources of odour [34,35,39]. Although it is difficult to distinguish between faeces with and without (i.e. Collection of wolf marks distance to villages) were, in combination, diagnostic attributes to determine wolf faeces. In those cases that there were clear doubts on the identity of the species, faeces were not collected. We considered the criteria used to identify wolf faeces and the experience of field-observers reliable since a posterior trial determining wolf faeces using these diagnostic attributes and a parallel DNA analyses confirmed that 90% of faeces were correctly assigned to wolves (n = 108, unpublished data). On the other hand, ground scratching marks were identified on the basis of size, length, intensity (i.e. excluding those marks compatible with dogs) [36]. The presence of wolf faeces accompanying this sign was an important factor determining wolf scratching. Territoriality in wolves is indicated by the means of visual and scent marks such as faeces, urine, ground scratching or anal gland segregations [32–36]. Pack members accumulate these marks, for instance, in the edges of their territories or the surroundings of the rendezvous sites [32–36]. As a general rule, only mature, dominant wolves, breeds every year within the pack [32], and they show an intense territorial marking behaviour compared to other members of the pack [27,37–39]. Thus, we hypothesised that as pack members, particularly the breeding pair, use visual and scent marks to indicate possession and defence of the pack’s territory, high levels of marking behaviour around the breeding season in an area could reflect the existence of an organised pack and a successful breeding pair. Since a biological meaningful units to monitor wolf populations are the pack and the number of wolf reproductions, then high abundance of wolf marks around this period could be used as an indicator of successful reproduction (i.e. the presence of pups) [39] and may offer a cost-effective tool to monitor wolves (i.e. number of wolf reproductions). However, to As random sampling is not effective to locate wolf marks [42], surveys in this mountainous region were focused on landscape March 2014 \| Volume 9 \| Issue 3 \| e93015 March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 2 Integrating Wolf Marking Behaviour into Monitoring Figure 1. Study area and sampling design. (a) Map showing the study area located in the Cantabrian Mountains (N Spain). (b) Scheme showing the spatial distribution of transects within a typical sampling site. doi:10.1371/journal.pone.0093015.g001 Figure 1. Study area and sampling design. March 2014 \| Volume 9 \| Issue 3 \| e93015 Reproductive success Two complementary methods, simulated howling and direct observation points, were used to assess wolf reproduction (i.e. presence of pups in the rendezvous sites). First, human-simulated howling elicited the response of adults and pups in the rendezvous site [44]. In each howling point, the observer emitted between 3 to 5 howls, each separated between 5 and 8 s long breaks [44]. If wolves did not reply within 2 minutes, then the observer repeated this process 2–3 times. The selection of the howling points was based on the landscape configuration (e.g. distribution of potential rendezvous sites based on the availability of refuge and areas with low human activity) [45], the environmental conditions (selection of the best places ensuring optimal conditions to simulate howling and to hear replies), and information gathered previously in the collection of wolf marks [42]. Howling sessions started at sunset and spanned the early night-time hours, avoiding rainy or windy nights, and were carried out between August and October when reply rate is remarkable [44] (occasionally early November). Second, observation points were used to contact pups in the rendezvous sites. The observer used 86 or 106 binoculars and telescopes with 20–606 zoom lenses to scan potential rendezvous sites and the surrounding areas during at least one hour. Observation points were carried out at sunrise and sunset in the same period of howling sessions. p p We then evaluated the relationship between wolf reproduction (i.e. the presence of pups) and the mean and the maximum relative indexes of abundance of wolf marks (predictors). Since the same site was sampled several years, we built generalised linear mixed models (GLMMs) with binomial error distribution and logit link. We included site and year as random factors in the models. Although sampling effort per site (total transect length) did not differ between sites with and without a confirmed presence of pups or between months, its variance was considerable (72 km), which could potentially affect the studied relationships. Therefore, we included in the models a covariate, the total transect length, in order to statistically control their potential effect. Additionally, we included a second covariate in the models (month) to account for temporal differences in wolf mark detectability and abundance. Results A total of 1,877 km were surveyed in 463 transects with an average length (6 s.d.) of transects of 15.668.5 km per site. Overall, 1,964 wolf marks were found and 1,497 howling (mean number per site = 12.5, range 1–43) and 307 observations points (mean number per site = 2.5, range 1–18) were used to confirm the presence of pups. Out of the 120 sites surveyed, wolf reproduction was confirmed in 84 cases (70%). Ethics statement In Spain, wolves north of river Duero are in Annex V of the European Habitats Directive (92/43/EEC); being a game species in Galicia and a species with a special regime (no game species) in Asturias. Fieldwork procedures (collection of wolf marks and the methods used to confirm the presence of pups) were specifically approved by the Regional Governments of Asturias and Galicia as well as the Spanish Ministry of Environment (Picos de Europa National Park) as a part of regional and local wolf monitoring activities. We did not handle any individual. Integrating Wolf Marking Behaviour into Monitoring October and early November. The number of wolf marks by transect was noted. of the presence of pups, the two other comparisons inform how invested the effort in detecting pups was. We assumed higher values of howling points in areas without confirmed wolf reproduction would reduce the probability of false absences of pups. Reproductive success Finally, as total transect length per site positively correlated with the number of transects (Spearman coefficient correlation, rs = 0.845, P,0.0001) and the number of transects per site could influence the maximum relative index of abundance (the probability to find higher indexes increase with the number of transects), we included this factor as another covariate in the model with the maximum relative index of abundance. For each model, we estimated the marginal and the conditional R2 following Nakagawa and Schielzeth (2013) [46]. Marginal R2 represents the variance explained by fixed factors (relative indexes of abundance of wolf marks, total transect length, month, number of transects per site) whereas Conditional R2 is interpreted as variance explained by both fixed and random factors. We predicted the probability of wolf reproduction based on the intensity of wolf marking behaviour and determined the cutting points for both relative abundance indexes when the probability of wolf reproduction was higher than 0.60, 0.80 and 0.99 (these values were selected according to their informative and conserva- tive value from a management point of view). All GLMMs were fitted with SAS 9.2 (procedure GLIMMIX) [47]. We only considered reproduction when pups responded positively to howling or when they were observed at the rendezvous site or its surroundings. Sampling effort was nearly constant among sites with four days invested per site; which overall, accounted for 480 days along the study period. Collection of wolf marks excretion) an intention of commu- nication [35,38], pack members seems to use substrates and conspicuous places differently according to their position in the social hierarchy [38], and some faeces perhaps had other functions than territorial marking during the period of study (e.g. for marking empty food caches) [35,39], we considered that all faeces Taking into account the landscape configuration and the availability of paths and trails, 1 to 9 transects were sampled within each site with an average number of 4 transects per site (median = 4), varying in length from 1 to 18 km (average length per transect = 4.1 km, ntransects = 463). All transects were con- ducted once around the pup rearing period. Seventy-seven percent of transects were surveyed between August and September, when all individuals of the pack are relatively stable around rendezvous sites [32]. The remaining surveys were carried out between March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 3 Integrating Wolf Marking Behaviour into Monitoring March 2014 \| Volume 9 \| Issue 3 \| e93015 Integrating Wolf Marking Behaviour into Monitoring Integrating Wolf Marking Behaviour into Monitoring The mean number of wolf marks was ca. 3 times higher in sites with a confirmed presence of pups (20.3 vs. 7.2 marks in sites with and without confirmed reproduction, respectively) with the mean relative index of abundance lower in sites without confirmed reproduction (0.43; n = 36) compared to sites with confirmed reproduction (1.61; n = 84; Fig. 2). The same pattern was observed for the maximum relative index of abundance (1.19 vs. 3.16 respectively; Fig. 2). We found a significant relationship between the mean and the maximum relative abundance index and the probability of wolf reproduction (x22 = 17.52, d.f. = 1, P,0.0001 and x2 = 14.55, d.f. = 1, P,0.001, respectively; Table 1; Fig. 3) when month, total transect length and number of transects were controlled for (all covariates with a P.0.285; Table 1). The model using the mean relative abundance index of wolf marks showed a marginal R2 of 0.79 and a conditional R2 of 0.84, whereas in the case of the model considering the maximum relative abundance of wolf marks, marginal R2 was 0.63 and conditional R2 was 0.77. These results indicate that the explained variance by the random factors (pack and year) was small. Our models predicted a probability of wolf reproduction higher than 0.60, 0.80 and 0.99 for a mean relative abundance index of 0.65, 0.93 and 1.84 wolf marks per km respectively; and a maximum relative abundance index of 0.71, 1.45 and 3.88 marks per km, respectively (Fig. 3). Predicted probabilities of pup presence by both indexes were positively correlated (Spearman coefficient correlation, rs = 0.843, P,0.0001). In fact, considering the three established cutting points and focusing on the mean relative abundance index, we observed 100% of agreement with the maximum relative abundance index in determining wolf reproduction for a probability of 0.60, 97% for a probability of 0.80, and 96% for a probability of 0.99. The proportion of false positives increased towards lower values of cutting points for the probability of pup presence (Table 2). Within each selected cutting point, this proportion was always higher for the maximum relative abundance index (Table 2), although differences were not significant (Z-test, all P-values.0.100; Table 2). Wolf marking behaviour and sign counts Individual asymmetries in territorial marking behaviour can influence indices of abundance based on marks [25,26,48,49]. Thus, understanding the link between different marking behav- iours and the abundance of marks is the only way to efficiently interpret monitoring procedures based on marks (i.e. sign counts). Territoriality in carnivorous mammals is commonly indicated by using scent or visual marking, such as urine, faeces or ground scratching, playing these marks an important role in territory maintenance and defence, indication of breeding status, demar- cation of valuable places such as rich-food patches or home sites and intra- or inter-specific communication [34–36,50]. Repro- ductively active individuals - dominants in social species - assume a high cost in territorial marking behaviour compared to subordi- nates [38,51]. In the case of wolves, an assumption that only the mature pair breeds [32], translates into a higher territorial marking intensity by the breeding individuals [26,38,45], especially in conspicuous sites, to increase the probability of the mark’s detection by conspecifics. On the contrary, lone wolves or young individuals tend to deposit less marks in these conspicuous sites compared to breeding wolves and other pack members [25,32,39]. In the case of lone wolves this behaviour may be particular important to decrease the probability of an encounter with territory holders. Higher levels of sex hormones detected in the wolf faeces located in particular conspicuous sites such as junctions supports these behavioural patterns [38]. As predicted, high levels of wolf marking behaviour around breeding season (pup rearing period) will reflect the existence of a successful breeding pair and the presence of pups. The observation that the number of wolf marks was three times higher in sites with a confirmed presence of pups compared with sites where wolves were present but pups were not confirmed is worth mentioning. Because pups are mainly stationary around rendezvous sites in the first months of life, they can not be responsible for a higher number of faeces in these areas. On the other hand, an absence of temporal differences in the intensity of territorial marking (we only detected a non-significant decrease in November, see Table 1) suggests constancy in this behaviour during this period. Figure 2. Box-plot showing mean and maximum relative abundance indexes of wolf marks in sites with and without confirmed wolf reproduction (presence of pups). Grey box indicates sites with confirmed wolf reproduction, whereas white box refers to sites without confirmed wolf reproduction. Wolf marking behaviour and sign counts The continuous line within the box indicate the median; whereas the discontinuous line shows the mean value. The lower end of the box indicate the 25th percentile, the upper end of the box indicate the 75th percentile, and the error bars shows the 10th and 90th percentiles. Points denote outliers. Figure 2. Box-plot showing mean and maximum relative abundance indexes of wolf marks in sites with and without confirmed wolf reproduction (presence of pups). Grey box indicates sites with confirmed wolf reproduction, whereas white box refers to sites without confirmed wolf reproduction. The continuous line within the box indicate the median; whereas the discontinuous line shows the mean value. The lower end of the box indicate the 25th percentile, the upper end of the box indicate the 75th percentile, and the error bars shows the 10th and 90th percentiles. Points denote outliers. Discussion Wolf marking behaviour and sign counts Data analyses For each site, we considered two measures of relative abundance of wolf marks. First, we calculated a mean relative index of abundance of wolf marks as the ratio between the total number of marks found and the sum of the length of all surveyed transects (in kilometres). Second, we identified the transect with the highest value of relative index of abundance (i.e. the ratio between the marks found and the length for each transect) and we considered this value as the maximum relative index of abundance per site. Moreover, we categorised all sites in a binary variable, according to the confirmed presence of pups. p ( ) Comparing sites with and without reproduction, average total transect length was similar, 14.8 km and 17.7 km respectively, and we did not detect significant differences between groups (Mann- Whitney U test, P = 0.151, n = 120), indicating homogeneity in the sampling effort invested among sites in relation with the number of kilometers surveyed. However, the number of howling and observation points was different between groups. Whereas the mean number of howling points was significantly higher in sites without a confirmed reproduction (16 vs. 11; Mann-Whitney U test, P = 0.005, n = 120); the number of observation points showed the opposite pattern (2 vs. 3; Mann-Whitney U test, P = 0.029, n = 120). Although observation points were made in all sites regardless of the confirmation of reproduction, they particularly confirmed wolf reproduction in those sites where howling sessions were positive. Differences in sampling effort among sites could affect either the value of the relative indexes of abundance used or the probability to detect wolf reproduction, which could ultimately affect our results. Therefore, we first tested whether the sampling effort was different between sites with and without confirmed wolf repro- duction by comparing transect length as well as the number of howling and observation points using Mann-Whitney U tests. While the former comparison gives us an idea about the homogeneity in the sampling effort to find wolf marks irrespective March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 4 Determining wolf reproduction Predicted probability of presence of pups against the mean and the maximum relative abundance index of wo Continuous black and grey line refers to the mean and the maximum relative abundance index of wolf marks, respectively (6 s.e., dot Striped line show the cutting point of 0.60; whereas the vertical black and grey bars show the cutting points of 0.80 and 0.99 for the mea maximum relative abundance index, respectively. Numbers for these cutting points refers to the number of wolf marks km21 for the mean Figure 3. Predicted probability of presence of pups against the mean and the maximum Continuous black and grey line refers to the mean and the maximum relative abundance index o Striped line show the cutting point of 0.60; whereas the vertical black and grey bars show the cuttin maximum relative abundance index, respectively. Numbers for these cutting points refers to the num the maximum (right) relative abundance index. For clarity, we also show a zoom (bottom) on these correspondence between the established cutting points and the values of the relative abundance ind of wolf marks km21. Figure 3. Predicted probability of presence of pups against the mean and the maximum relative abundance index of wolf marks. Continuous black and grey line refers to the mean and the maximum relative abundance index of wolf marks, respectively (6 s.e., dotted lines). Striped line show the cutting point of 0.60; whereas the vertical black and grey bars show the cutting points of 0.80 and 0.99 for the mean and the maximum relative abundance index, respectively. Numbers for these cutting points refers to the number of wolf marks km21 for the mean (left) and the maximum (right) relative abundance index. For clarity, we also show a zoom (bottom) on these predicted probabilities, with lines denoting the correspondence between the established cutting points and the values of the relative abundance indexes, for the first range of values of the number of wolf marks km21. doi:10.1371/journal.pone.0093015.g003 Figure 3. Predicted probability of presence of pups against the mean and the maximum relative abundance index of wolf marks. Continuous black and grey line refers to the mean and the maximum relative abundance index of wolf marks, respectively (6 s.e., dotted lines). Determining wolf reproduction Simple mark/sign counts provide valuable information about presence, distribution or relative abundance of large carnivores [10]. However, gathering data on demographic parameters doi:10.1371/journal.pone.0093015.g002 March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 5 Integrating Wolf Marking Behaviour into Monitoring Table 1. Parameter estimates (6 s.e.) in the models testing the relationship between the mean and the m abundance of wolf marks (marks km21) and wolf reproduction. Mean relative abundance index Maximum relative abundan Model-effect Parameter estimate (± s.e.) Parameter estimate (± s.e. Intercept 21.8861.14 20.5161.17 Relative abundance of wolf marks 3.5360.84* 1.3260.34* Sampling effort (km) 20.0160.04 0.0560.07 Number of transects - 20.2160.30 Month September 0.5160.84 0.9960.87 October 0.3961.07 0.7961.05 November 21.4861.27 20.7761.21 The level ‘‘month (August)’’ is included in the intercept. * Significant at P,0.001. Note that the covariate ‘‘number of transects’’ was not included in the model with the mean relative abundance index (see text for details). doi:10.1371/journal.pone.0093015.t001 Table 1. Parameter estimates (6 s.e.) in the models testing the relationship between the mean and the maximum relative abundance of wolf marks (marks km21) and wolf reproduction. Mean relative abundance index Maximum relative abundance index Model-effect Parameter estimate (± s.e.) Parameter estimate (± s.e.) Intercept 21.8861.14 20.5161.17 Relative abundance of wolf marks 3.5360.84* 1.3260.34* Sampling effort (km) 20.0160.04 0.0560.07 Number of transects - 20.2160.30 Month September 0.5160.84 0.9960.87 October 0.3961.07 0.7961.05 November 21.4861.27 20.7761.21 The level ‘‘month (August)’’ is included in the intercept. * Significant at P,0.001. Note that the covariate ‘‘number of transects’’ was not included in the model with the mean relative abundance index (see text for details). doi:10.1371/journal.pone.0093015.t001 Table 1. Parameter estimates (6 s.e.) in the models testing the relationship between the mean and the maximum relative abundance of wolf marks (marks km21) and wolf reproduction. Table 1. Parameter estimates (6 s.e.) in the models testing the relationship between the mean and the maximum relative abundance of wolf marks (marks km21) and wolf reproduction. Table 1. Parameter estimates (6 s.e.) in the models testing the relationship between the mean and the maximum relative abundance of wolf marks (marks km21) and wolf reproduction. Significant at P,0.001. Note that the covariate ‘‘number of transects’’ was not included in the model with the mean relative abundance index (see text for details). doi:10 1371/journal pone 0093015 t001 Figure 3. Determining wolf reproduction Moreover, because pack members seems to use substrates and conspicuous places differently for marking behaviour [38], the influence of different levels of territorial marking, according to individual attributes and the conspicuous sites considered, on the studied relationships deserves further investigation. The level of confidence determining the probability of wolf reproduction in a given area could be adapted according to different management goals and population scenarios. For example, we recommend a reliable level in small populations (cutting points of 0.80 or 0.99) decreasing the probability of false positives (type I error, see Table 2), whereas this probability could be conservative (0.60) at large populations. The mean relative abundance index showed a low probability of false positive (below 0.10 for all cutting points); being zero for the cutting point of 0.99. Thus, as a general rule, we suggest a cutting point of 0.60 in the probability of wolf reproduction to monitor wolves on a regional scale and the use of the mean instead of the maximum relative abundance index of wolf marks. This option will reduce the influence of short transects with a high number of wolf marks or a small number of transects per site on the probability of wolf reproduction. By combining this method with different spatial criteria, such as the distance among packs in different scenarios (saturated, non-saturated or recovering areas) or simulated wolf territories, it would be possible to improve the estimates on the number of wolf packs. In addition, we highlight that a similar approach based on wolf marking behaviour could determine the probability of the occurrence of non-breeding packs. This application would require the understanding of differences in the intensity of territorial marking in areas with lonely or floaters individuals and areas with established non-breeding pairs or family groups. p g Our findings would objectively reduce the level of uncertainty in the estimation of the number of packs, particularly when they are not confirmed with other procedures [53]. For example, many monitoring programmes simulate howls to locate pups [22,23], but this method also has drawbacks in relation with the subjectivity of the observer determining the participation of pups in a chorus howl and the fact that wolves occasionally do not respond, as well as its logistical constraints when it is applied over large scales [54]. Determining wolf reproduction Mean relative abundance index Maximum relative abundance index Cutting point Proportion of false positives (number of total cases) Proportion of false positives (number of total cases) 0.60 0.08 (75) 0.18 (101) 0.80 0.07 (61) 0.14 (80) 0.99 0.00 (26) 0.04 (22) doi:10.1371/journal.pone.0093015.t002 Table 2. Proportion of false positives (Type I error) using the 0.60, 0.80 and 0.99 cutting points for the predicted probability of wolf reproduction based on the mean and the maximum relative abundance index. Table 2. Proportion of false positives (Type I error) using the 0.60, 0.80 and 0.99 cutting points for the predicted probability of wolf reproduction based on the mean and the maximum relative abundance index. generally required other intensive, invasive, and expensive approaches (e.g. radiotracking or DNA analyses), often feasible for research-oriented efforts applied over small areas or small populations. Excluding snow-tracking – a time-delayed approach used to determine the occurrence of reproduction events based on the number of individuals detected (tracks) in the following winter - which shows several logistical constraints associated to snow conditions, spatial scale (high cost and significant field effort needed) and, sometimes, interpretation [28], to our knowledge, this is the first time that it is established a real-time relationship between intensity of territorial marking and the probability of wolf reproduction. training of observers, resulting in high correct assignment rates [52]. On the other hand, even although we used a multiple sampling approach to determine reproduction events, it is well-known that both methods are not completely efficient [53,54], and the existence of an important number of false absences could affect our results. However, in our case, sampling effort was higher in those sites where pups were not detected, decreasing the probability of false absences of wolf reproduction. In fact, for both relative abundance indexes, the 25th percentiles of sites with confirmed wolf reproduction did not overlap or showed a low overlapping with the 75th and 90th percentiles, respectively, of sites with unconfirmed wolf reproduction (Fig. 2), suggesting that the number of false absences was small. Finally, since the probability of wolf reproduction was independent of a sampling effort, indicating the importance of a stratified sampling to monitor wolves based on wolf marking behaviour, more studies are needed to optimize sampling design (e.g. minimum transect length per site). Determining wolf reproduction For wolf surveys covering large areas, it has been suggested that the effectiveness of simulated howling could be improved by selecting howling sites in those places with the largest concentra- tions of wolf marks during the breeding period [42]. In other words, selecting sites where the probability of wolf reproduction is high based on the intensity of territorial marking as this study shows. In wolf surveys based on howling, this method could add an objective approach to determine the probability of wolf reproduc- tion when high relative abundance of wolf marks are found in a given area, but the presence of pups is not verified using howling. Although our analyses were controlled for mark detectability, we acknowledge that prior to generalising the use of this method, it is necessary to develop similar trials in other landscape configu- rations and wolf populations evaluating quantitative changes in the values of the relative abundance indexes in established cutting points for the probability of wolf reproduction (0.60, 0.80 and 0.99). For example, several studies suggest that wolves use linear infrastructures to place a significant proportion of their territorial marks [31,32,34,42,43], but how landscape configuration influ- ences this marking behaviour is unknown. Another important concern in the use of this method would be the feasibility in the identification of wolf marks. However, features described above may be efficiently used as diagnostic attributes, along with the Determining wolf reproduction Striped line show the cutting point of 0.60; whereas the vertical black and grey bars show the cutting points of 0.80 and 0.99 for the mean and the maximum relative abundance index, respectively. Numbers for these cutting points refers to the number of wolf marks km21 for the mean (left) and the maximum (right) relative abundance index. For clarity, we also show a zoom (bottom) on these predicted probabilities, with lines denoting the correspondence between the established cutting points and the values of the relative abundance indexes, for the first range of values of the number of wolf marks km21. doi:10.1371/journal.pone.0093015.g003 Figure 3. Predicted probability of presence of pups against the mean and the maximum relative abundance index of wolf marks. Continuous black and grey line refers to the mean and the maximum relative abundance index of wolf marks, respectively (6 s.e., dotted lines). Striped line show the cutting point of 0.60; whereas the vertical black and grey bars show the cutting points of 0.80 and 0.99 for the mean and the maximum relative abundance index, respectively. Numbers for these cutting points refers to the number of wolf marks km21 for the mean (left) and the maximum (right) relative abundance index. For clarity, we also show a zoom (bottom) on these predicted probabilities, with lines denoting the correspondence between the established cutting points and the values of the relative abundance indexes, for the first range of values of the number of wolf marks km21. doi:10.1371/journal.pone.0093015.g003 March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 6 Integrating Wolf Marking Behaviour into Monitoring Table 2. Proportion of false positives (Type I error) using the 0.60, 0.80 and 0.99 cutting points for the predicted probability of wolf reproduction based on the mean and the maximum relative abundance index. Mean relative abundance index Maximum relative abundance index Cutting point Proportion of false positives (number of total cases) Proportion of false positives (number of total cases) 0.60 0.08 (75) 0.18 (101) 0.80 0.07 (61) 0.14 (80) 0.99 0.00 (26) 0.04 (22) doi:10.1371/journal.pone.0093015.t002 Table 2. Proportion of false positives (Type I error) using the 0.60, 0.80 and 0.99 cutting points for the predicted probability of wolf reproduction based on the mean and the maximum relative abundance index. Acknowledgments We are in debt to wolf biologists Victor Sazatornil, Vicente Palacios, Alberto Ferna´ndez, Javier Naves, Andre´s Ordiz, Antonio Uzal and Miguel Rico who participated in the fieldwork. We thank to the staff, ranges and technicians of these institutions for their help. JVLB was supported by the Spanish Ministry of Economy and Competitiveness. This is scientific paper no. 1 from the Iberian Wolf Research Team (IWRT). We propose that this procedure could be easily exported to other wolf populations and countries. Across Europe, transbound- ary cooperation in wolf management is a pressing need [29]. This fact requires coordination among all authorities and actions including compatibility and/or convergence in monitoring meth- ods and target information. Since this method does not rely upon complex procedures or equipment, it can be relatively straight- forward and quick to conduct by rangers, technical staff or volunteers, and easily implemented in routine samplings. The use of the number of packs may turn estimates more homogeneous in a transboundary context. References 20. Llaneza L, Lo´pez-Bao JV, Sazatornil V (2012) Insights into wolf presence in human-dominated landscapes: the relative role of food availability, humans and landscape attributes. 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John Wiley & Sons, New York. 8. Jhala Y, Qureshi Q, Gopal R (2011) Can the abundance of tigers be assessed from their signs? J Appl Ecol 48: 14–24. 26. Galaverni M, Palumbo D, Fabbri E, Caniglia R, Greco C, et al. (2011) Monitoring wolves (Canis lupus) by non-invasive genetics and camera trapping: a small-scale pilot study. Eur J Wildlife Res 58: 47–58. 9. Peek J, Dale B, Hristienko H, Kantar L, Loyd KA, et al. References (2012) Management of large mammalian carnivores in North America. The Wildlife Society Technical Review 12–1. The Wildlife Society, Bethesda, Maryland, USA. 9. Peek J, Dale B, Hristienko H, Kantar L, Loyd KA, et al. (2012) Management of large mammalian carnivores in North America. The Wildlife Society Technical 27. Kaczensky P, Chapron G, von Arx M, Huber D, Andre´n H, et al. (2013) Status, management and distribution of large carnivores - bear, lynx, wolf and wolverine - in Europe. Report to the EU Commission, 272 p. ˚ Review 12–1. The Wildlife Society, Bethesda, Maryland, USA. 10. Long RA, MacKay P, Zielinski WJ, Ray JC (2008) Noninvasive survey methods for carnivores. Island Press, Washington D.C. 28. Liberg O, Aronson A˚ , Sand H, Wabakken P, Maartmann E, et al. (2012) Monitoring of wolves in Scandinavia. Hystrix 23: 29–34. 11. Boitani L, Powell RA (2012) Carnivore ecology and conservation: a handbook of techniques. Oxford University Press, London, UK. 29. Linnell JDC, Boitani L (2012) Building biological realism into wolf management policy: the development of the population approach in Europe. Hystrix 23: 80– 91. 12. O’Connell AF, Nichols JD, Karanth KU (2011) Camera traps in animal ecology. Methods and analyses. Springer, Tokyo. y p g y 13. Royle JA, Nichols JD, Karanth KU, Gopalaswamy AM (2009) A hierarchical model for estimating density in camera-trap studies. J Appl Ecol 46: 118–127. 30. Alvares F, Barroso I, Blanco JC, Correia J, Cortes Y, et al. (2005) Wolf status and conservation in theIberianPeninsula. Frontiers of wolf recovery: Southwest- ern U.S. and the world. Colorado Springs, Colorado. 14. Karanth KU, Gopalaswamy AM, Kumar NS, Vaidyanathan S, Nichols JD, et al. (2011) Monitoring carnivore populations at the landscape scale: occupancy modelling of tigers from sign surveys. J Appl Ecol 48: 1048–1056. 31. Creˆte M, Messier F (1986) Evaluation of indices of gray wolf (Canis lupus), density in hardwood-conifer forests of Southwestern Quebec. Can Field Nat 101: 147– 152. 15. Kendall KC, States U, Survey G, Rocky N, Science M, et al. (2009) Demography and genetic structure of a recovering grizzly bear population. J Wildl Manage 73: 3–17. 32. Mech LD, Boitani L (2003) Wolves: behavior, ecology, conservation. University of Chicago Press. J g 16. Marucco F, Boitani L (2012) Wolf population monitoring and livestock depredation preventive methods in Europe. Hystrix 23: 1–4. 33. Kleiman DG (1966) Scent marking in the Canidae. Symp Zool Soc Lond 18: 167– 177. 17. Integrating Wolf Marking Behaviour into Monitoring determine the level of accuracy required in wolf estimates (individuals or packs). For example, at small populations such as the critically endangered population located in Sierra Morena (S Spain; ,50 individuals) [27,29] the appropriate level of informa- tion would estimate both the number of individuals, using intensive approaches and the number of packs. But, the former level of accuracy may be unnecessary at large populations, such as the NW Iberian wolf population, assuming higher levels of uncertainty until the population would not be below a critical value, such as a given annual number of packs, number of reproductions or effective population size (minimum number of breeding pairs). In this regard, adopting a cost effective approach, the number of packs could serve as sentinel information to shift between intensive (individuals and packs) and simple and extensive (packs) monitoring procedures and vice versa. Concluding Remarks We show how the integration of wolf marking behaviour with simple sampling procedures (sign counts) permit rapid, real-time, and cost effective assessments of the breeding status of wolf packs with substantial implications to monitor wolves over large spatial scales. The assessment of the number of packs using this approach would provide a simple way to regularly assess the status of wolf populations providing acceptable estimates of demographic parameters such as reproduction success or annual effective population sizes. This information is essential for an efficient, adaptive management framework in wolf populations, even when populations are not threatened. Author Contributions Conceived and designed the experiments: JVLB LLL. Performed the experiments: LLL EJG JVLB. Analyzed the data: JVLB. Contributed reagents/materials/analysis tools: LLL JVLB. Wrote the paper: JVLB LLL. Conceived and designed the experiments: JVLB LLL. Performed the experiments: LLL EJG JVLB. Analyzed the data: JVLB. Contributed reagents/materials/analysis tools: LLL JVLB. Wrote the paper: JVLB LLL. Surveying wolves at large spatial scales In the last years, the convenience of estimating the number of wolves or packs (or the number of reproductions) has been the focus of an intense and controversial debate. Finally, wolf biologists and managers seem to agree about the robust and biological meaning of packs as a unit to monitor wolves [16], which could be easily estimated with the method proposed here. However, monitoring and management are interactive processes, and therefore, management goals for each wolf population will March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 7 Integrating Wolf Marking Behaviour into Monitoring Integrating Wolf Marking Behaviour into Monitoring Integrating Wolf Marking Behaviour into Monitoring Integrating Wolf Marking Behaviour into Monitoring 46. Nakagawa S, Schielzeth H (2013) A general and simple method for obtaining R2 from Generalized Linear Mixed-effects Models. Methods Ecol Evol 4: 133–142. 37. Scandura M, Iacolina L, Capitani C, Gazzola A, Mattioli L, et al. (2011) Fine- scale genetic structure suggests low levels of short-range gene flow in a wolf population of the Italian Apennines. Eur J Wildlife Res 57: 949–958. g gg g g population of the Italian Apennines. Eur J Wildlife Res 57: 949–95 47. Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O (2006) SAS for Mixed Models. 2nd ed. SAS Institute Inc., Cary, NC. 38. Barja I, Silva´n G, Rosellini S, Pin˜eiro A, Illera MJ, et al. (2008) Quantification of sexual steroid hormones in faeces of Iberian wolf (Canis lupus signatus): a non- invasive sex typing method. Repro Domest Anim 43: 701–707. y 48. Palomares F, Roques S, Cha´vez C, Silveira L, Keller C, et al. (2012) High proportion of male faeces in Jaguar populations. PLOS ONE 7: e52923. 48. Palomares F, Roques S, Cha´vez C, Silveira L, Keller C, et al ( p invasive sex typing method. Repro Domest Anim 43: 701–707. proportion of male faeces in Jaguar populations. PLOS ONE 7: e52 39. Rothman RJ, Mech LD (1979) Scent-marking in lone wolves and newly formed pairs. Anim Behav 27: 750–760. 49. Ralls K, Sharma S, Smith DA, Bremner-Harrison S, Cypher BL, et al. (2010) Changes in Kit Fox Defecation Patterns During the Reproductive Season: Implications for Noninvasive Surveys. J Wildl Manage 74: 1457–1462. 40. Garcı´a D, Quevedo M, Obeso JR, Abajo A (2005) Fragmentation patterns and protection of montane forest in the Cantabrian range (NW Spain). Forest Ecol Manag 208: 29–43. 50. Gorman ML (1990) Scent-marking strategies in mammals. Rev Suisse Zool 97: 3- 29. 41. Garcı´a E, Llaneza L, Palacios V, Lo´pez-Bao JV, Sazatornil V, et al. (2012) Primeros datos sobre la ecologı´a espacial del lobo en Galicia. Abstract-book of the III Iberian Wolf Congress, pp. 44. 51. Sands J, Creel S (2004) Social dominance, aggression and faecal glucocorticoid levels in a wild population of wolves (Canis lupus). Anim Behav 67: 387–396. 52. Godinho R, Castro D, Lo´pez-Bao JV, Garcı´a E, Rio-Maior H, et al. (2011) Genetic assessment of error rates in identifying wolf (Canis lupus) faeces in four areas of the Iberian Peninsula. References Leader-Williams N, Albon SD (1988) Allocation of resources for conservation. Nature 336: 533–535. 34. Barja I, Miguel FJD, Ba´rcena F (2005) Faecal marking behaviour of Iberian wolf in different zones of their territory. Folia Zool 54: 21–29. 18. Palomares F, Rodrı´guez A, Revilla E, Lo´pez-Bao JV, Calzada J (2011) Assessment of the conservation efforts to prevent extinction of the Iberian lynx. Conserv Biol 25: 4–8. 35. Peters RP, Mech LD (1975) Scent-marking in Wolves. Am Sci 63: 628–637. 36. Zub K, Theuerkauf J, Jedrzejewski W, Jedrzejewska B, Schmidt K, et al. (2003) Wolf pack territory marking in the Bialowieza Primeval Forest (Poland). Behaviour 140: 635–648. 19. Gude JA, Mitchell MS, Russell RE, Sime CE, Bangs EE, et al. (2012) Wolf population dynamics in the U.S. Northern Rocky Mountains are affected by recruitment and human-caused mortality. J Wildl Manage 76: 108–118. March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org 8 46. Nakagawa S, Schielzeth H (2013) A general and simple method for obtaining R2 from Generalized Linear Mixed-effects Models. Methods Ecol Evol 4: 133–142. Integrating Wolf Marking Behaviour into Monitoring Abstract-book of the X Jornadas Espan˜olas de Conservacio´n y Estudio de Mamı´feros: p. 64. Fuengirola, Spain. g pp 42. Llaneza L, Ordiz A, Palacios V, Uzal A (2005) Monitoring wolf populations using howling points combined with sign survey transects. W B P 1: 108–117. 43. Vila` C, Urios V, Castroviejo J (1994) Use of faeces for scent marking in Iberian wolves (Canis lupus). Can J Zool 72: 374–377. Conservacio´n y Estudio de Mamı´feros: p. 64. Fuengirola, Spain. 53. Blanco JC, Corte´s Y (2012) Surveying wolves without snow: a critical review of the methods used in Spain. Hystrix 23: 35–48. ( p ) J Z 44. Harrington FH, Mech LD (1982) An analysis of howling response parameters useful for wolf pack censusing. J Wildl Manage 46: 686–693. the methods used in Spain. Hystrix 23: 35–48. 54. Fuller TK, Sarnpson BA (1988) Evaluation of a simulated howling survey for wolves. J Wildl Manage 52: 60–63. p g J g 45. Ausband DE, Mitchell MS, Doherty K, Zager P, Mack CM, et al. (2010) Surveying predicted rendezvous sites to monitor gray wolf populations. J Wildl Manage 74: 1043–1049. March 2014 \| Volume 9 \| Issue 3 \| e93015 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9
https://openalex.org/W2015133380	https://xn--archivoespaoldearte-53b.revistas.csic.es/index.php/aea/article/download/591/588/586	es		Un tímpano románico inédito procedente de Labastida (Álava)	Archivo español de arte/Archivo español de arte	1,996	cc-by	1,426	AEA, 274, 1996 VARIA 205 UN TÍMPANO ROMÁNICO INÉDITO PROCEDENTE DE LABASTIDA (ÁLAVA) En el claustro del Museo de San Telmo de San Sebastián se conservan, empotrados en una pared, restos de un tímpano que ha pasado desapercibido hasta la fecha. Únicamente conozco una nota explicativa a una foto que aparece en el catálogo del Museo de San Telmo de Gonzalo Manso de Zúñiga, donde señala su procedencia de la localidad alavesa de Labastida, lo fecha en el siglo X y añade que son restos de un sepulcro i. Sin embargo la iconografía nos indica que se trata de un tímpano y que su cronología se sitúa hacia finales del siglo xii o principios del xiii. De Labastida se conocen hasta once ermitas que han desaparecido —Nuestra Señora de los Angeles de Toloño, Nuestra Señora de Remélluri, San Andrés de Muga, San Clemente, San Cristóbal, Santa Eulalia, El Humilladero, San Julián, San Martín, San Román y Santiago— sin que se sepa en la mayoría de ellas a qué época pertenecieron y de cuál proceden los restos del tímpano 2. El tímpano (Fig. 1) presenta a dos ángeles que sostienen un crismón trinitario con las letras habituales, P, S, X, Alfa y Omega. Como es conocido es a partir del tímpano de Jaca cuando se difunde por todo el arte románico este tipo de Crismón que incluye la S referida al Espíritu Santo. La parte superior de la P está parcialmente fracturada, por lo que no sabemos si en esta parte tendría cruz o si carecería de ella, pero en cualquier caso parece difícil que, a parte de la duda de la cruz, pudiese existir otro elemento iconográfico en esa parte del tímpano. Los dos ángeles que sostienen el crismón están también deteriorados, pero se conservan las cabezas nimbadas, se aprecia parte de las vestimentas y las alas, habiéndose perdido en ambos casos la parte inferior, lo que nos impide saber sobre qué superficie se sostendrían o si por el contrario estarían elevados. El motivo iconográfico de los dos ángeles que sostienen el crismón procede de los soldados que guardaban el Lábaro imperial romano, que se convertirán en victorias aladas que custodiaban dicho estandarte. Este motivo debió servir para desarrollar la conocida fórmula de la imago clipeata en los sarcófagos paganos, esquema que pasará íntegramente a los sarcófagos cristianos que ya hacia el año 400 incorpora el monograma de Cristo ^. La presencia de los dos ángeles sosteniendo o elevando el crismón trinitario cobra un especial significado eucarístico al encontrarse una invocación a las tres personas en algunos pasajes del Ordo Missae, como ha recordado Serafín Moralejo a propósito del baldaquino de Compostela 4. En cualquier caso no es una novedad este sentido eucarístico, pues ya desde el siglo 11 aparece el Cordero junto al crismón, lo que refuerza la idea de salvación de la segunda persona e incide por tanto en el sacramento eucarístico. No olvidemos que la fórmula iconográfica del crismón junto al agnus Dei, fórmula eucarística por excelencia, tuvo una amplia repercusión en el entorno de Álava, y así aparece en Armentia o Aguilar de Codés. Al ámbito aragonés se incorporó el motivo de los dos ángeles a partir de las fórmulas creadas por el taller del maestro Gilduin en Saint-Sernin de Toulouse, que ahí lo hacían sosteniendo el busto de Cristo. Luego, los dos ángeles incorporaron el crismón trinitario de origen jaqués hasta completar la fórmula iconográfica que vemos en el tímpano de Labastida y que tanto desarrollo tuvo en el área navarro-aragonés. Así aparecen ángeles sosteniendo el crismón en la por1 MANSO DE ZÚÑIGA, Gonzalo: Museo San Telmo, Bilbao, 1976, p. 74. 2 ENCISO VIANA, Emilio y CANTERA ORIVE, Julián: Catálogo monumental de la diócesis de Vitoria, vol. 1, Vitoria, 1967, pp. 199-206, señalan la existencia documental de estas once ermitas desaparecidas, en algún caso con descripciones o citas hechas hace un siglo. 3 Para este tipo de iconografía vid. OCON ALONSO, D.: Tímpanos románticos en españoles: reinos de Navarra y Aragón, Madrid, Universidad Complutense, 1985, vol. 2, pp. 100-108. 4 MORALEJO, Serafín: «Ars sacra et sculpture romane: le trésor et le chantier de Compostelle», en Ler cahiers de SaintMichel de Cuixà, Il (1980), p. 215. (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://archivoespañoldearte.revistas.csic.es AEA, 274, 1996 VARIA 206 Fig. 7. Restos del tímpano procedentes de Labastida (Álava). San Sebastian, Museo de San Telmo. Fig. 8. Carrillo, Bodegón. Madrid, Comercio de Arte. (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://archivoespañoldearte.revistas.csic.es AEA 274, 1996 VARIA 207 tada oeste de San Salvador de Ejea de los Caballeros (Zaragoza), San Pedro el Viejo de Huesca, San Felices de Uncastillo (Zaragoza), Tamarite de Litera (Huesca), Sainte Engrace (Pyrénées). De todas éstas la primera receptora de la iconografía del crismón sostenido por dos ángeles se encuentra en San Pedro el Viejo de Huesca hacia la mitad del siglo xii; luego estaría la iglesia de Sainte Engrance y Tamarite que se fechan hacia el último tercio del siglo xii; Uncastillo todavía se sitúa en el siglo xii, hacia su final, y Ejea de los Caballeros sobrepasa las primeras décadas del XIII. Para Labastida no contamos con fechas precisas, pero teniendo en cuenta las anteriores y su factura popular, todo nos induce a creer que estaría más cerca de la última que de la primera. Estilísticamente el tímpano de Labastida presenta unas características sumamente populares. Se trata, en efecto, de un escultor rural que interpreta una iconografía oficial, y como suele ser frecuente en este tipo de obras populares, no se encuentran relaciones estilísticas claras. De hecho, con los otros cuatro tímpanos existentes en Álava —Añes, Zambrana y dos en Armenia— no guarda ninguna relación estilística, aspecto que es extensivo a los dos únicos crismones alaveses existentes, el de Armentia y el de Zambrana. Agustín GÓMEZ GÓMEZ Doctor por la Universidad del País Vasco UN BODEGÓN FIRMADO POR «CARRILLO» El número de cuadros firmados, dentro de la pintura española es tan relativamente escaso que siempre es de celebrar la aparición de uno de ellos con estas características. Es el caso del bodegón que nos ocupa (1,25 x 2,25 m), en el comercio, y del cual sabemos el nombre del autor, Carrillo, cuya firma se encuentra en el papel del envoltorio situado en el centro de la composición (Fig. 8 y 9.) Desafortunadamente, nada mas se sabe de este artista. Tan sólo el «Lexikon» i menciona a un tal Juan Carrillo, ubicándolo en Sevilla hacia 1513. Esta fecha no coincide con la del cuadro estudiado ya que la aparición del bodegón en España se sitúa a finales del xvi y, dados los elementos presentes en esta obra, no se puede aceptar una datación anterior. La obra es interesante al tratarse de un «Bodegón» en el más estricto sentido de la palabra 2, en el cual las figuras humanas aparecen en un interior mezcladas con aumentos. Esta definición de bodegón procede del «Tesoro de la Lengua española» de Covarrubias (1611) donde aparece el término como: «El sótano o portal baxo, dentro del cual está la bodega a donde el que no tiene quien le guise la comida la halla allí aderezada y juntamente con las bebidas». Dos años antes, en 1599, se recogen, en el testamento de Pantoja de la Cruz y como pintados por él, unos «bodegones de Italia, pinturas grandes». No se sabe con precisión el tipo de pintura a la que alude pero trae a la memoria las obras del norte de Italia las cuales, fechadas en época similar e imbuidas de un aire flamenco, serían llamadas «bodegones» en España. Estos cuadros recogen escenas con vendedores de aves, de pescados o de frutas ^. El cuadro que nos ocupa encaja a la perfección dentro 1 «AUgemeines Lexikon der Bildenden Künstler», Thieme-Becker, 1932, T. VI: «Carrillo, Juan: spanische Maler in Sevilla um 1513». 2 Véase PÉREZ SÁNCHEZ: «La Nature Morte espagnole de XVIIe ème siècle a Goya», 1971, pp. 13-16. 3 Ver: PÉREZ SÁNCHEZ: «Pintura española de bodegones y floreros. De 1600 a Goya», Catálogo Exposición Museo del Prado, 1983, p. 24. Por su parte, en el Diccionario de la Real Academia Española se define «bodegón» como «Tienda donde se guisa y se sirven comidas ordinarias». De estas dos últimas acepciones, que identifican el bodegón con la venta de algún producto alimenticio, puede proceder la idea, sin confirmar, de que estos cuadros colgaban de las paredes a la entrada de tiendas o mesones, anunciando la mercancía. (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://archivoespañoldearte.revistas.csic.es
https://openalex.org/W3119418050	http://www.bioinformation.net/016/973206300161060.pdf	English	null	Association of occlusal wear facets in patients with temporomandibular disorders	Bioinformation	2,020	cc-by	4,594	Declaration on Publication Ethics: The authors state that they adhere with COPE guidelines on publishing ethics as described elsewhere at https://publicationethics.org/. The authors also undertake that they are not associated with any other third party (governmental or non-governmental agencies) linking with any form of unethical issues connecting to this publication. The authors also declare that they are not withholding any information that is misleading to the publisher in regard to this article. Faazila Fathima1, Sreedevi Dharman2,* & P Senthil Murugan3 1Saveetha Dental college and hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 77, India; 2Department of Oral Medicine &Radiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 77, India; 3Department of Oral Surgery, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 77,India; Sreedevi Dharman – Email.id: sreedevi@saveetha.com The authors are responsible for the content of this article. The Editorial and the publisher has taken reason in accordance to publishing ethics with adequate peer reviews deposited at PUBLONS. this article. The Editorial and the publisher has taken reasonable steps to check the content of the article ate peer reviews deposited at PUBLONS. Declaration on official E-mail: Declaration on official E mail: The corresponding author declares that official e-mail from their institution is not available for all authors The corresponding author declares that official e-mail from their institution is not available for all authors www.bioinformation.net Volume 16(12) Research Arti Association of occlusal wear facets in patients with temporomandibular disorders Research Article Author contacts: Faazila Fathima - 151501018.sdc@saveetha.com; Sreedevi Dharman - sreedevi@saveetha.com; Senthil Murugan - senthilmuruganp.sdc@saveetha.com Received October 7, 2020; Revised October 27, 2020; Accepted October 27, 2020; Published December 31, 2020 DOI: 10.6026/973206300161060 The authors are responsible for the content of this article. The Editorial and the publisher has taken reasonable steps to check the content of the article in accordance to publishing ethics with adequate peer reviews deposited at PUBLONS. Background: The present study was conducted in a university setting (Saveetha dental college and hospitals, Chennai, India). Thus the data available is of patients from the same geographic location and have similar ethnicity. The retrospective study was carried out with the help of digital case records of 98 patients who reported to the hospital. Ethical clearance to conduct this study was obtained from the Scientific Review Board of the hospital. The ethical approval number for the present study is SDC/SIHEC/2020/DIASDATA/0619-0320. The present study was conducted in a university setting (Saveetha dental college and hospitals, Chennai, India). Thus the data available is of patients from the same geographic location and have similar ethnicity. The retrospective study was carried out with the help of digital case records of 98 patients who reported to the hospital. Ethical clearance to conduct this study was obtained from the Scientific Review Board of the hospital. The ethical approval number for the present study is SDC/SIHEC/2020/DIASDATA/0619-0320. Background: TMD is a functional and pathological condition affecting temporomandibular joint, the masticatory musculature, or both in the maxillofacial region [1]. Approximately 33% of the population has at least one TMD symptom and 3.6% to 7% of the population has TMD with more severity to cause them to seek treatment [2]. Etiology of TMD is multifactorial, and is related to a number of dental and medical conditions, such as occlusion, posture, parafunctional habits, restorative procedures, orthodontic treatment, emotional stress, trauma, anatomy of the disc, pathophysiology of the muscles, genetic and psychosocial factors such as age, and gender [3]. Typical signs and symptoms of TMD are pain, limited mouth opening, joint sounds, mandibular deviation, and chewing disability. These symptoms may exist alone or in combination. However, it cannot be stated that these factors are predisposing TMD or are only coincidental [4]. Due to the etiological complexity and variety of signs and symptoms that may represent other pathologies, recognition and differentiation of TMDs is not very clear to the professional. Therefore, routine screening should be combined with the anamnesis and selective clinical examination, for the professional to perform an accurate diagnosis and therefore develop the proper treatment plan [5]. Teeth wear is considered as a visible sign of physiological functional wear and a rather rapid onset in bruxism. It is probably seen more as a result of functional disturbance in the masticatory system [6]. Abstract: Abstract: Occlusal changes were important factors in temporomandibular disorder (TMD). It is of interest to evaluate the association of occlusal wear facets in TMD patients. We used a dataset of 49 patients with and without TMD for this study. Occlusal wear facets were evaluated using Smith and Knight tooth wear index. Data shows that teeth wear was present more in patients with TMD (55%). The age group 26-40 years showed high prevalence of teeth wear (grade1) in TMD patients (P value = 0.034). TMD was present more in females than males. Female (54%) patients with TMD showed more teeth wear compared to males. Most patients with TMD showed posterior teeth wear (61%) than generalized teeth wear. Thus, association was present between occlusal teeth wear and TMD patients especially in the age group of 26-40 years. Hence, proper evaluation of occlusal factors will aid in early diagnosis of TMDs. Keywords: Bruxism; clenching; occlusal wear; temporomandibular disorder. ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 1060 ©Biomedical Informatics (2020) Sampling: 98 patients were reviewed from patient’s records and data were extracted. Only relevant data was included to minimize sampling bias. Simple random sampling method was carried out. Cross verification of data for error was done by presence of additional reviewer and by photographic evaluation. Background: Mechanical tooth surface wear is caused by harmful or parafunctional mandibular movements. Parafunctional habits such as bruxism, tooth clenching, gum chewing, biting foreign objects, and prolonged nail biting might increase the risk of developing TMD [7]. Parafunctional activities are usually harmless, until the forces exerted exceed the structural tolerance [8]. Bruxism and clenching reportedly lead to joint space reduction, followed by disc compression, and resulting pain in the masticatory muscles. Occlusion is treated not only as the ratio of contact between teeth, but also as a dynamic, morphological and functional relation, presenting a great influence on chewing, swallowing and speech [9]. Tooth wear alters the existing occlusal plane introducing deflective occlusal interferences [2]. Occlusal/skeletal factors, such as crossbite, overjet and centric relation (CR) to maximum intercuspation (MI) discrepancy greater than 2 mm could be considered occlusal risk factors for TMD [6]. These are of serious concern to the patient and the dental professionals because of its relation to the temporo mandibular joint disorders. Therefore, it is of interest to evaluate the association of occlusal wear facets in TMD patients. is Table 1:Table shows the frequency of different age groups among patients with TMD. It is evident from the table that TMD was present more in the age group of 26- 40years(46.9%). Table 1:Table shows the frequency of different age groups among patients with TMD. It is evident from the table that TMD was present more in the age group of 26- 40years(46.9%). 40years(46.9%). Age group Frequency Percentage Below 25years 15 30.6 26-40years 23 46.9 41-55years 8 16.3 Above 55 years 3 6.2 Total 49 100 40years(46.9%). Age group Frequency Percentage Below 25years 15 30.6 26-40years 23 46.9 41-55years 8 16.3 Above 55 years 3 6.2 Total 49 100 Table 2: Table shows the frequency of gender among patients with TMD. It is evident from the table that TMD was present more in females (53.1%) compared to males. Table 2: Table shows the frequency of gender among patients with TMD. It is evident from the table that TMD was present more in females (53.1%) compared to males. Gender Frequency Percentage Gender Frequency Percentage Male 23 46.9 Female 26 53.1 Total 49 100 Results: Out of 49 patients with TMD, 46.9% of them belonged to the age group of 26-40 years, 30.6% belonged to the age group below 25 years, 16.3% belonged to the age group of 41-55 years and 6.2% belonged to the age group above 55 years (Figure 1). Majority of the patients with TMD were in the age group of 26-40 years (Table 1). 26 (53.1%) out of 49 TMD patients were females and 23 (46.9%) were males (Figure 2). It is thus evident that TMD was present more in females compared with males (Table 2). On comparison of Figure 3 and Figure 4, it was observed that in the age group below 25 years in TMD patients, 33.4% had grade1 and 6.6% had grade2 (p value-0.034 (p<0.05), hence statistically significant) while in the same age group, patients without TMD had no teeth wear and grade0 was only observed (pvalue-0.00 (p<0.05), hence statistically significant). In the age group of 26-40years, patients with TMD showed 30.4% grade 1, 13.1% grade2 and 4.4% grade4 while in the same age group of patients without TMD majority did not show teeth wear and 21.1% had grade1. Grade 2 (62.5%) was the most prevalent teeth wear grade in 41-55 years of age in TMD patients while the same age group patients without TMD also showed higher prevalence of grade 2(50%). However, in the age group of above 55 years, the groups showed high prevalence of teeth wear of which, 66.7% of the patients with TMD had grade2 and in patients without TMD, 75% had grade 2. On comparison of Figure 5 and Figure 6, it was observed that in patients with TMD, 43.5% of males and 65.4% of females had teeth wear (p value- 0.34 (p>0.05), hence statistically not significant), while in patients without TMD, 40.8% of the males and only 36.4% of the females showed presence of teeth wear (p value- 0.823 (p>0.05), hence statistically not significant). It was observed from Figure 7 that higher teeth wear grades were prevalent in TMD patients compared to patients without TMD (p value- 0.340 (p>0.05), hence statistically not significant). Teeth wear grade1 was present in 51.8% of TMD patients and 48.2% in patients without TMD; grade2 was present in 68.7% of TMD patients and 31.3% in patients without TMD. Grade3 was present equally in both the groups and grade 4 was observed only in TMD patients. Grade 2: Teeth wear of less than 1/3rd of the tooth Grade 2: Teeth wear of less than 1/3rd of the tooth Data Collection: A single calibrated examiner evaluated the digital case records of patients who reported to Saveetha Dental College from June 2019 to March 2020. For the present study, inclusion criteria were data of patients with TMD and patients without TMD having no systemic diseases and records of these patients with incomplete data were excluded from the study. Clinical examination, dental status and photographs of these patients’ records were reviewed. Grading was done using, Smith and Knight tooth wear index (TWI) [10] for evaluation of occlusal wear facets based on extent to which teeth wear was present. The gradings are as follows: Grade 0: No teeth wear Grade 1: Loss of enamel surface characteristics Materials and methods: Study designs and Study setting: Materials and methods: Study designs and Study setting: ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 1061 ©Biomedical Informatics (2020) Grade 4: Pulpal exposure due to teeth wears Grade 4: Pulpal exposure due to teeth wears Results: Teeth wear grade2 showed the highest prevalence among patients with TMD. Posterior teeth wear was observed more in TMD patients (61.%) while generalised teeth wear was more in patients without TMD (57.2%)(p value-0.178 (p>0.05), hence statistically not significant) (Figure 8). Overall it was observed that 55.1% of the patients with TMD had teeth wear and 38.8% of patients without TMD had teeth wear. Chi square test was done and association between age group and teeth wear grade was found to be statistically significant (P<0.05). While association between gender, TMD and teeth wear was statistically not significant (P>0.05). Grade 3: Teeth wear causing dentin exposure for more than 1/3rd teeth wear Figure 3: Bar graph depicts association between age and teeth wear grade in TMD patients. X-axis denotes age and Y-axis denotes number of TMD patients. Teeth wear grades (grade1-green) were more in the age group of 26-40 years and grade2 (yellow) in 41- 55years when compared to other age groups in patients with TMD. Chi square test was done and association was statistically significant. (Pearson’s chi square value=23.300, p value=0.034 (>0.05)). Figure 2: Bar graph showing frequency distribution of gender among patients with TMD. X-axis denotes gender and Y-axis denotes number of patients with TMD. It is observed that 53.1% of the TMD patients were females (light green). Figure 2: Bar graph showing frequency distribution of gender among patients with TMD. X-axis denotes gender and Y-axis denotes number of patients with TMD. It is observed that 53.1% of the TMD patients were females (light green). Statistical analysis: y The collected data was tabulated and analysed with Statistical Package for Social Sciences for Windows, version20.0 (SPSS Inc., Chicago, IL, USA) and results were obtained. Categorical variables were expressed in frequency and percentage. Chi square test was used to test association between categorical variables. Chi square tests were carried out using age, gender and occlusal wear facets as independent variables and temporo mandibular disorder as dependent variable. The statistical analysis was done by pearson chi square test. P value < 0.05 was considered statistically significant. Figure 1: Bar graph showing frequency distribution of age group of TMD patients. The X-axis denotes age and Y-axis denotes number of patients with TMD. It is observed that 46.9% of the TMD patients belonged to the age group of 26-40years(blue). Figure 1: Bar graph showing frequency distribution of age group of TMD patients. The X-axis denotes age and Y-axis denotes number of patients with TMD. It is observed that 46.9% of the TMD patients belonged to the age group of 26-40years(blue). ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 1062 ©Biomedical Informatics (2020) The early rehabilitation treatment in asymptomatic patients may be indicated in order to prevent the occlusal collapse and, consequently, to reduce the risk of future joint problems. In the past, studies have suggested that occlusal interferences were main factors in TMD development, thus, validating irreversible occlusal therapies as the definitive treatment of the disorder [18]. Based on that, occlusal adjustments, full mouth rehabilitation and orthodontic treatment became very popular as the treatment of choice for TMD. This study could pave way for future research to be done for better understanding of the signs and symptoms of TMD and aid in early intervention and better treatment modalities for improved results in clinical practice. The limitations of the study include limited sample size and limited demographics. Figure 5: Bar graph depicts the association between gender and teeth wear grade in patients with TMD. X-axis denotes gender and Y-axis denotes the number of patients with TMD. Teeth wear grades (grade1-green, grade 2-yellow and grade 3-purple) were more in females compared to males in patients with TMD. Chi square test was done and association was statistically not significant. (Pearson’s chi square value = 4.522, p value = 0.340 (>0.05)). ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 10 p g p Figure 5: Bar graph depicts the association between gender and teeth wear grade in patients with TMD. X-axis denotes gender and Y-axis denotes the number of patients with TMD. Teeth wear grades (grade1-green, grade 2-yellow and grade 3-purple) were more in females compared to males in patients with TMD. Chi square test was done and association was statistically not significant. (Pearson’s chi square value = 4.522, p value = 0.340 (>0.05)). Discussion: [20] reported that males showed increased teeth wear compared to females and ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 1063 ©Biomedical Informatics (2020) Figure 4: Bar graph depicts association between age and teeth wear grade in patients without TMD. X-axis denotes age and Y-axis denotes the number of patients without TMD. Teeth wear grade1 (green) was more in the age group of 41-55 years and grades (grade2-yellow and grade3-purple) was more in above 55years in patients without TMD. Chi square test was done. (Pearson’s chi square value=44.468, p value=0.000(>0.05), statistically significant). Figure 4: Bar graph depicts association between age and teeth wear grade in patients without TMD. X-axis denotes age and Y-axis denotes the number of patients without TMD. Teeth wear grade1 (green) was more in the age group of 41-55 years and grades (grade2-yellow and grade3-purple) was more in above 55years in patients without TMD. Chi square test was done. (Pearson’s chi square value=44.468, p value=0.000(>0.05), statistically significant). attributed this to strong masseter function and greater muscle fiber mass. As observed in the present study that posterior teeth wear was more in TMD patients, several other studies [15] also reported that the posterior teeth wear was more in TMD patients. They also stated that these changes are associated with joint changes, particularly increasing the risk of cracking, and disc displacement. The occlusal instability caused by the changes in posterior teeth may cause TMD since the occlusal changes; muscle changes and joint changes exceed the adaptive threshold of the stomatognathic system. The early rehabilitation treatment in asymptomatic patients may be indicated in order to prevent the occlusal collapse and, consequently, to reduce the risk of future joint problems. In the past, studies have suggested that occlusal interferences were main factors in TMD development, thus, validating irreversible occlusal therapies as the definitive treatment of the disorder [18]. Based on that, occlusal adjustments, full mouth rehabilitation and orthodontic treatment became very popular as the treatment of choice for TMD. This study could pave way for future research to be done for better understanding of the signs and symptoms of TMD and aid in early intervention and better treatment modalities for improved results in clinical practice. The limitations of the study include limited sample size and limited demographics. attributed this to strong masseter function and greater muscle fiber mass. As observed in the present study that posterior teeth wear was more in TMD patients, several other studies [15] also reported that the posterior teeth wear was more in TMD patients. They also stated that these changes are associated with joint changes, particularly increasing the risk of cracking, and disc displacement. The occlusal instability caused by the changes in posterior teeth may cause TMD since the occlusal changes; muscle changes and joint changes exceed the adaptive threshold of the stomatognathic system. Discussion: Figure 3: Bar graph depicts association between age and teeth wear grade in TMD patients. X-axis denotes age and Y-axis denotes number of TMD patients. Teeth wear grades (grade1-green) were more in the age group of 26-40 years and grade2 (yellow) in 41- 55years when compared to other age groups in patients with TMD. Chi square test was done and association was statistically significant. (Pearson’s chi square value=23.300, p value=0.034 (>0.05)). Thus, from this study it was observed that teeth wear was present more in patients with TMD (55%). Study population showed teeth wear was more prevalent in the age group of 26-40 years. Also, females showed more prevalence of teeth wear than males. Most patients with TMD showed posterior teeth wear than generalised teeth wear. Similar to the present study, Costa et al. [11] reported a high incidence of teeth with wear facets, observed in patients with TMD dysfunction. Few authors as well observed similar results in their study [4,12]. Increased prevalence of teeth wears in TMD patients were attributed to high incidence of grinding and clenching habits in these patients and due to the obvious connection between the structures of temporo mandibular joint and the dental occlusion [13]. In accordance to the present study, Luther et al. [14] also reported that increased teeth wear in TMD patients was reported in younger age group, while few authors stated no association between age and teeth wear was present [15,16]. As in this study 26-40 years showed higher incidence of teeth wear in TMD patients, Renecker et al. [17] as well reported similar findings. They suggested that severe teeth wear is mostly the result of bruxing activity and to a lesser extent as a result of functional wear. Increased teeth wear was observed in females than males in the present as well as in studies done by other authors [11,14,18]. According to Luther [14] the high incidence in women may be related to hormonal changes that occur at this stage of life.In addition, psychological factors and increased levels of somatization, that is, depression or anxiety have been closely linked to TMD which may also be the reason why females are more affected [18]. In contrast, Yadav [19] and Solberg et al. more in females than males. Female (54%) patients with TMD showed more teeth wear compared to males. Most patients with TMD showed posterior teeth wear (61%) than generalized teeth wear. Thus, association was present between occlusal teeth wear and TMD patients especially in the age group of 26-40 years. Hence, proper evaluation of occlusal factors will aid in early diagnosis of TMDs. more in females than males. Female (54%) patients with TMD showed more teeth wear compared to males. Most patients with TMD showed posterior teeth wear (61%) than generalized teeth wear. Thus, association was present between occlusal teeth wear and TMD patients especially in the age group of 26-40 years. Hence, proper evaluation of occlusal factors will aid in early diagnosis of TMDs. Figure 7: Bar graph depicts association between TMD and teeth wear grade. X-axis denotes teeth wear grade and Y-axis denotes number of patients with and without TMD. Teeth wear grades (grade1, 2,3,4) were more commonly observed in patients with TMD (dark green). Chi square test was done and association between TMD and teeth wear grade among patients was statistically not significant. (Pearson’s chi square value=4.518, p value=0.340 (>0.05)). Conclusion: Data shows that teeth wear was present more in patients with TMD (55%). The age group 26-40 years showed high prevalence of teeth wear (grade 1) in TMD patients (P value = 0.034). TMD was present ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 1064 ©Biomedical Informatics (2020) ©Biomedical Informatics (2020) Clinical significance: g The study helps us to understand the manifestation of occlusal teeth wear in patients with temporo mandibular disorder. Figure 6: Bar graph depicts the association between gender and teeth wear grade in patients without TMD. X-axis denotes gender and Y-axis denotes the number of patients without TMD. Teeth wear grades (grade1-green, grade2-yellow and grade3-purple) were more in males compared to females in patients without TMD. Chi square test was done and association was statistically not significant. (Pearson’s chi square value=0.910, p value=0.823 (>0.05)). Figure 7: Bar graph depicts association between TMD and teeth wear grade. X-axis denotes teeth wear grade and Y-axis denotes number of patients with and without TMD. Teeth wear grades (grade1, 2,3,4) were more commonly observed in patients with TMD (dark green). Chi square test was done and association between TMD and teeth wear grade among patients was statistically not significant. (Pearson’s chi square value=4.518, p value=0.340 (>0.05)). Acknowledgment: g This research was supported by Saveetha Dental College and Hospital. We thank the department of Oral Medicine and Radiology, Saveetha Dental College for providing insight and expertise that greatly assisted this research. Figure 6: Bar graph depicts the association between gender and teeth wear grade in patients without TMD. X-axis denotes gender and Y-axis denotes the number of patients without TMD. Teeth wear grades (grade1-green, grade2-yellow and grade3-purple) were more in males compared to females in patients without TMD. Chi square test was done and association was statistically not significant. (Pearson’s chi square value=0.910, p value=0.823 (>0.05)). ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 16(12): 1060-1068 (2020) 1065 ©Biomedical Informatics (2020) References: [1] Huang GJ et al. J Dent Res 2002 81:284. [PMID: 12097315] [2] https://www.elsevier.com/books/management-of temporomandibular-disorders-and- occlusion/okeson/978-0-323-08220-4 Figure 8: Bar graph depicts the association between TMD and teeth wear. X-axis denotes Teeth wear and Y-axis denotes the number of patients with and without TMD. Posterior teeth wear was observed more in TMD patients (dark green) and generalised teeth wear was observed more in patients without TMD (brown). Chi square test was done and association was statistically not significant. (Pearson’s chi square value=3.451, p value=0.178(>0.05)). occlusion/okeson/978-0-323-08220-4 [3] Magnusson T et al. Acta Odontologica Scandinavica 2005 63:99. [PMID: 16134549] [4] Fujita Y et al. Bull Tokyo Dent Coll 2003 44:201. [PMID: 15103917] ] [5] Ishigaki S et al. Cranio 1992 10:289. [PMID: 1291102] [6] https://www.academia.edu/23447841/Functional_Occlus ion_From_TMJ_to_Smile_Design_2007_ g [7] Poveda-Roda R et al. Medicina Oral Patología Oral y Cirugia Bucal 2013 18:e392. [PMID: 23385515] [ ] [8] Cairns BE Journal of Oral Rehabilitation 2010 37:391. [PMID: 20337865] [9] Gesch D et al. Angle Orthod 2004 74:512. [PMID: 15387030] [10] Lopez-Frias FJ et al. Journal of Clinical and Experimental Dentistry 2012 4:e48. [PMID: 24558525] y [11] Costa MD et al. Dental Press Journal of Orthodontics 2012 17:261. [12] Pergamalian A et al. J Prosthet Dent. 2003 90:194. [PMID: 12886214] [13] John MT et al. Pain 2005 118:61. [PMID: 16154702] [14] Luther F British Dental Journal 2007 202:E3. [PMID: 17220828] [15] Seligman DA et al. Journal of Dental Research 1988 67:1323. [PMID: 3049715] Figure 8: Bar graph depicts the association between TMD and teeth wear. X-axis denotes Teeth wear and Y-axis denotes the number of patients with and without TMD. Posterior teeth wear was observed more in TMD patients (dark green) and generalised teeth wear was observed more in patients without TMD (brown). Chi square test was done and association was statistically not significant. (Pearson’s chi square value=3.451, p value=0.178(>0.05)). [16] Clarke NG et al. Journal of Oral Rehabilitation 1984 11:123. [17] Reneker J et al. J Orthop Sports Phys Ther 2011 41: 408. [PMID: 21335932] [18] Henrikson T & Nilner M Journal of Orthodontics 2003 30:129. [PMID: 12835429] [19] Yadav S The Journal of Indian Prosthodontic Society 2011 11:98. [PMID: 22654349] [20] Solberg WK et al. The Journal of Prosthetic Dentistry 1972 28:412. [PMID: 4403492] References: Edited by P Kangueane y g Citation: Fathima et al. 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https://openalex.org/W2150610973	https://europepmc.org/articles/pmc3335589?pdf=render	English	null	The Soluble PlasminogenActivator Receptoras a Biomarker on Monitoring the Therapy Progress of Pulmonary TB-AFB(+) Patients	Tuberculosis research and treatment	2,010	cc-by	4,230	Hindawi Publishing Corporation Tuberculosis Research and Treatment Volume 2010, Article ID 406346, 6 pages doi:10.1155/2010/406346 Hindawi Publishing Corporation Tuberculosis Research and Treatment Volume 2010, Article ID 406346, 6 pages doi:10.1155/2010/406346 Hindawi Publishing Corporation Tuberculosis Research and Treatment Volume 2010, Article ID 406346, 6 pages doi:10.1155/2010/406346 Tri Yudani Mardining Raras1 and Iin Noor Chozin2 Tri Yudani Mardining Raras1 and Iin Noor Chozin2 1Laboratory of Biochemistry-Molecular Biology, Faculty of Medicine, Brawijaya University, Malang 65145, Indonesia 2Department of Pulmonology, Faculty of Medicine, Brawijaya University Malang-Dr. Saiful Anwar Hospital Malang, Malang 65145, Indonesia 1Laboratory of Biochemistry-Molecular Biology, Faculty of Medicine, Brawijaya University, Malang 65145, Indonesia 2Department of Pulmonology, Faculty of Medicine, Brawijaya University Malang-Dr. Saiful Anwar Hospital Malang, Malang 65145, Indonesia Correspondence should be addressed to Tri Yudani Mardining Raras, daniraras@yahoo.com Received 29 June 2010; Accepted 8 September 2010 Academic Editor: William N. Rom Copyright © 2010 T. Y. Mardining Raras and I. Noor Chozin. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The role of soluble soluble urokinase-type plasminogen activator receptor (suPAR) as a biological marker for TB treatment eﬃcacy on active pulmonary TB-AFB(+) patients was investigated. Twenty pulmonary TB-AFB(+) patients participated in a cohort study for six months. The plasma suPAR level was measured using ELISA method before treatment, two months, four months and six months after treatment. At the same time clinical parameters were also measured. Results indicated that all patients (n = 20) showed highest plasma suPAR levels before treatment (median 12.775 ng/mL) and signiﬁcantly decreased ( P = .0001 <.05, R2 = .890) after 2 months (median 8.019 ng/mL) and 4 months (median 5.771 ng/mL) of treatment, respectively. However, only slightly declined after 6 months therapy (median 5.009 ng/mL), near control group level (median 4.772 ng/mL). Interestingly, the signiﬁcant reduced of suPAR level was parallel to treatment eﬃcacy and correlated with other clinical and laboratory parameters, that is, decreasing of patients’ complaints, increasing of BMI (r = −0.281), thoracic imaging improvement, sputum conversion, decreasing of ESR (r = 0.577) and monocytes count (r = 0.536) with exception the width of lesion in thoracic imaging. In conclusion, the suPAR level in could reﬂect the progress of TB therapy. 2. Material and Methods 2.1. Subjects. This study was conducted in Malang, where the prevalence of new smears or cultures positive for TB was 107/100.000 population per year between 2008 and 2009. For the study, 5 tuberculin skin test positive healthy community controls and 20 positive, newly diagnosed pulmonary TB with sputum AFB-(Acid-fast bacillu) positive patients were enrolled. The inclusive criteria were patients with category I of pulmonary TB who regularly took medication, were male and female aged between 15–50 years, had a BMI (Body Mass Index) >16, agreed to be the subject of the research, and signed the informed consent. Exclusion criteria were TB patients with other diseases (severe bacterial pneumonia, HIV-AIDS, and heart disease, diabetes mellitus, heart and kidney problems), extra pulmonary TB, pregnant patients, and patients with psychiatric problems. 2.7. Statistical Analysis. All statistical analysis on suPAR was carried out using SPSS software version and the statistical programming language. Analyzed statistically, using longitu- dinal model analysis, bivariate analysis, multivariate analysis and linear regression, these analysis were carried out with the SAS 9.13 (SAS Institute, Cary, NC, USA) and SPSS 16 (SPSS Inc, Chicago, IL) program. The diﬀerence between the patients and the controls was analyzed using one-way ANOVA. The association between the immune parameters and the extent of disease was assessed using the Mann Whitney U-test. A P-value of P ≤.05 was judged signiﬁcant. The patients received a six-month (26 weeks) directly observed short course antituberculosis therapy as recom- mended by the Indonesian National Tuberculosis Program based on WHO TB guidelines. 2.8. Ethics. Written consent was obtained from all partici- pants. The study was approved by the Ethics Committees, Saiful Anwar Public Hospital, Brawijaya University. 2.2. TB Treatment. The drug regimen consisted of a ﬁxed weight-dependent combination of INH (320–400 mg/day), rifampicin (480–600 mg/day), ethambutol (800–1200 mg/ day), and pyrazinamide (1000–1250 mg/day) for the two- month intensive phase, followed by rifampicin and INH for the four-month continuation phase. 1. Background Concentrations of the respective analyses were determined using SPSS version 3.4 software. 1. Background receptor for serine protease urokinase plasminogen activator (uPA). UPAR can be cleaved from the cell surface by a number of protease, such as chymotrypsin, phospholipases C, and uPA, to yield a soluble form of the receptor (suPAR) that has intrinsic chemotactic properties [1, 2]. One study has demonstrated elevated levels of suPAR in pulmonary TB patient with higher suPAR levels in smear microscopy- positive patients compared to smear-negative patients [3]. SuPAR is an inﬂammatory biomarker and thus not speciﬁc for TB, but it is elevated by several infectious diseases and s to reﬂect the severity of disease and may hence be used as a prognostic tool. However, little is known about suPAR as a prognostic marker in extrapulmonary TB [4]. The success in controlling Tuberculosis (TB) depends to a great extent on the appropriate diagnosis, the right therapy, treatment monitoring, and evaluation. Particularly concern- ing the treatment, the long duration of therapy may adversely aﬀect patient adherence to treatment and lead to failure of the recommended treatment program. This urges a stringent monitoring program that could show the progress of the therapy earlier than normal therapy (min. 6 months). In the last decade, several biological markers have been intensively studied. Such markers could contribute, the improvement of the quality of clinical trials; and therefore enable develop- ment and validation of new therapeutic strategies. Assuming that serum levels of suPAR reﬂect the progress of the therapy, we investigated to access in prospective man- ner whether serum levels of suPAR may be used to monitor One of the most intensively investigated biomarkers is urokinase plasminogen activator receptor (uPAR), a cellular Tuberculosis Research and Treatment 2 of anti-TB drugs treatment based on WHO guideline (2HRHZE/4H3R3) [5, 6]. Serum was separated by centrifu- gation (6000 x g) at 4◦C for 7 min and aliquots of 500 mL stored at −80◦C. treatment of pulmonary TB-AFB(+) patients. In the present study, the suPAR levels of TB pulmonary patients were measured during-six-month therapy and compared with other routine laboratory evaluation including Body Mass Index (BMI), monocyte count, Erytrocyte Sedimentation Rate, (ESR) and thorax imaging. 2.6. Enzyme-Linked Immunoassaysorbant Assays. Serum suPAR measurement was done in duplicate using com- mercially available ELISA kits according to the manufacture’s protocol (suPARnostic, ViroGates A/S, Copenhagen, Den- mark) [7]. Plate reading was conducted using a Biotech micro plate reader set to 450 nm, with the wavelength correction set to 650 nm. 3. Results Study materials were 3 mL blood specimens obtained from new patients through aspiration using injective needle before they started on antituberculosis diagnosis at two, four, and six months after the initiation Tuberculosis Research and Treatment 3 Table 1: Clinical data of patients included in the study. No Patients’ characteristics Frequency (n) Percentage (%) 1 Number of patients 20 100% Male 10 50% Female 10 50% 2 Age (years); age median = 30 years 19–30 12 60% 31–40 4 20% 41–50 4 20% 3 Width of lesion classiﬁcation Minimal lesion 1 5% Moderate advance lesion 7 35% Far advance lesion 12 60% 4 Patients’ complaints Chronic cough >3 weeks 20 100% Decrease of appetite 15 75% Low grade fever 10 50% Shortness of breath 9 45% Hemoptoe 9 45% Chest pain 2 10% Table 1: Clinical data of patients included in the study. Table 2: Comparison between medical records used to monitor TB theraphy progress in Indonesia, that is, patients complaints, BMI (kg/m2), width of lesion, sputum AFB, erythrocyte sedimentation rate (mm/hour), monocyte count (cell/mm2), and soluble urokinase plasminogen activator receptor during the 6-month therapy. Table 2: Comparison between medical records used to monitor TB theraphy progress in Indonesia, that is, patients complaints, BMI (kg/m2), width of lesion, sputum AFB, erythrocyte sedimentation rate (mm/hour), monocyte count (cell/mm2), and soluble urokinase plasminogen activator receptor during the 6-month therapy. (kg/m ), width of lesion, sputum AFB, erythrocyte sedimentation rate (mm/hour), monocyte count (cell/mm ), and soluble urokinase plasminogen activator receptor during the 6-month therapy. During anti-TB-drugs treatment No. 3. Results Twenty patients with active pulmonary TB were observed during the six-month treatment. Characteristics of the twenty patients are presented in Table 1. 2.3. Routine Examination. Patient’s complaint was con- ducted prior to the clinical and laboratory examination, body mass index, monocyte count, and Erythrocyte Sedimenta- tion Rate (ESR). All were measured according to the standard procedure conducted in Indonesian Hospital [5]. The number of female pulmonary TB patients who fulﬁlled the inclusion and exclusion criteria were (50%) equal to the number of male patients (50%). Distribution based on age in this study showed that TB patients were mainly dominated by those aged between 19–30 years. Measurement of suPAR concentration indicated that there was a high level of suPAR in infected patients before treatment (median 12.775 ng/mL), but suPAR levels dropped signiﬁcantly (P = .0001 < .05) after two months (median 8.019 ng/mL) and four months (median 5.771 ng/mL) of therapy. However, at the sixth month of treatment, there was only slightly decrease although signiﬁcant reached (median 5.009 ng/mL) (Figure 1(a)). These were almost similar to the healthy control group (median 4.772 ng/mL). 2.4. ChestX-Ray Grading of Disease. Standard postero-an- terior and lateral chest radiographs were taken prior to therapy and were read by a pulmonologist. The grading included the following categories: (i) minimal lesion, that is, lesion width is less than area restricted to median line, apex and front costae, solitaire lesion can be everywhere, and there is no cavity found, (ii) moderate advanced, that is, width of cavity is less than one lobe, and if there is cavity, should be more than one lobe. (iii) far advance is width of lesion that more than minimal and moderate lesion, if with cavity, should not be more than 4 cm. With regard to clinical symptoms (Table 2), chronic cough is the most common complaint, however, decreasing of appetite, low grade fever, shortness of breath, haemoptoe, and chest pain were also found. After the second and the sixth month of treatment, some patients still complained about coughing, shortness of breath, and chest pain. Con- cerning sputum conversion, before treatment, all patients (n = 20) had sputum-AFB(+) but after the two-month 2.5. Sampling Handling. 3. Results Medical records and level of suPAR Before Treatment 2 months 4 months 6 months P-value <.05 1 Patients’ Complaints Chronic cough >3 weeks 20 (100%) 17 (85%) 5 (25%) Decrease of appetite 15 (75%) 0 (0%) 0 (0%) Low grade fever 10 (50%) 0 (0%) 0 (0%) Shortness of breath 9 (45%) 5 (25%) 3 (15%) Hemoptoe 9 (45%) 0 (0 %) 0(0 %) Chest pain 2 (10 %) 1 (5 %) 1 (5 %) 2 BMI 18.28 ± 2.12 19.18 ± 2.31 20.36 ± 2.35 .0001 3 Width of lesion classiﬁcation Thoracic Imaging n = 20 Thoracic Imaging n = 20 Minimal Lesion Mod Advance Lesion Far Advance Lesion 1 (5%) 7 (35%) 12 (60%) ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ Worsen : 0 (0%) Unchange : 3 (15%) Getting better : 17 (85%) ⎫ ⎪⎪⎪⎬ ⎪⎪⎪⎭ ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ Worsen : 1 (5%) Unchange : 0 (0%) Getting better : 19 (95%) ⎫ ⎪⎪⎪⎬ ⎪⎪⎪⎭ 4 Sputum AFB(+) 20 (100%) 5 (25%) 0(0%) 5 ESR 84.15 ± 32.47 44.10 ± 29.97 19.30 ± 13.29 .0001 6 Monocytes Count 847.00 ± 244.52 662.00 ± 241.72 496.50 ± 129.67 0.0001 7 Level of suPAR 12.775 ± 3.940 8.019 ± 1.458 5.771 ± 0.796 5.009 ± 0.842 .0001 Tuberculosis Research and Treatment 4 Proﬁle of suPAR level during treatment y = −1.277x + 11.725 R2 = 0.890 0 5 10 15 20 25 30 0 2 4 6 During treatment (months) suPAR level (ng/mL) P-value .0001<.05 (a) Proﬁle of ESR during treatment y = −10.15x + 76.25 R2 = 0.9 0 20 40 60 80 100 120 140 0 2 4 6 During treatment (months) ESR (mm/hour) P-value .0001<.05 (b) Proﬁle of monocyte count during treatment y = −55.98x + 817.79 R2 = 0.95 0 200 400 600 800 1000 1200 1400 1600 0 2 4 6 Monocyte count (cell/mm3) P-value .0001<.05 During treatment (months) (c) Proﬁle of BMI during treatment y = 0.34x + 18.37 R2 = 0.99 0 5 10 15 20 25 30 0 2 4 6 During treatment (months) BMI (kg/m2) P-value .0001<.05 (d) Figure 1: (a) Serum levels of soluble urokinase receptor (suPAR), (b) sedimentation rate of erythrocytes (ESR), (c) monocyte count (d) Body Mass Index (BMI) in 20 TB patient before (0), during (2 and 4 months), and after (6 months) completion of anti-TB treatment. Individual proﬁle (dotted line) and median (thick line) levels are shown. 3. Results Proﬁle of ESR during treatment y = −10.15x + 76.25 R2 = 0.9 0 20 40 60 80 100 120 140 0 2 4 6 During treatment (months) ESR (mm/hour) P-value .0001<.05 (b) Proﬁle of BMI during treatment y = 0.34x + 18.37 R2 = 0.99 0 5 10 15 20 25 30 0 2 4 6 During treatment (months) BMI (kg/m2) P-value .0001<.05 (d) Proﬁle of suPAR level during treatment y = −1.277x + 11.725 R2 = 0.890 0 5 10 15 20 25 30 0 2 4 6 During treatment (months) suPAR level (ng/mL) P-value .0001<.05 (a) Proﬁle of ESR during treatment y = −10.15x + 76.25 R2 = 0.9 0 20 40 60 80 100 120 140 0 2 4 6 During treatment (months) ESR (mm/hour) P-value .0001<.05 (b) Proﬁle of BMI during treatment Proﬁle of monocyte count during treatment y = −55.98x + 817.79 R2 = 0.95 0 200 400 600 800 1000 1200 1400 1600 0 2 4 6 Monocyte count (cell/mm3) P-value .0001<.05 During treatment (months) (c) (d) (c) Figure 1: (a) Serum levels of soluble urokinase receptor (suPAR), (b) sedimentation rate of erythrocytes (ESR), (c) monocyte count (d) Body Mass Index (BMI) in 20 TB patient before (0), during (2 and 4 months), and after (6 months) completion of anti-TB treatment. Individual proﬁle (dotted line) and median (thick line) levels are shown. treatment, ﬁfteen patients turned to be AFB(−), and by the end of the six-month treatment, all patients showed sputum conversion. During observation, most patients (19) showed high level ESR, and it decreased signiﬁcantly (P- value .0001 < .05) from time to time during the six-month treatment. ESR usually increased in active processes, but normal ESR did not eliminate the diagnosis of TB [5]. In this papaer, the highest median of ESR before treatment was 84.2 mm/hour, and then it dropped to 44.1 mm/hour after-two-month treatment. The lowest of the ESR level was reached after six-month treatment (19.3 mm/hour). (Figure 1(b)) A signiﬁcant correlation between suPAR and ESR was found (P-value .0001 < .05), with a quite strong correlation coeﬃcient (r = 0.577). weight reached the highest BMI mean (median 20.36 kg/m2). (Figure 1(d)). There was a signiﬁcant correlation (P-value .012 < .05) albeit weak (r = −0.281) between the decrease in suPAR with the increasing of BMI. 3. Results Finally, thoracic imaging of the patients who were diagnosed to have active pulmonary TB showed far advanced lesions (n = 12), moderate advanced lesions (n = 7), and minimal lesions (n = 1). However, there was no signiﬁcant correlation between suPAR level with the width of lesions on thoracic imaging of the TB patients before and after treatment (Table 3). 4. Discussion Increased suPAR levels pretreatment may be a result of mobilization of macrophage into the bronchi and the increased immune activation and inﬂammation caused by the active infection. Adherence and migration of monocytes involves a functional interaction between uPAR and integrins [8]. The reduction trend of suPAR during six-month therapy was signiﬁcantly in accordance with the decline in ESR, however, only signiﬁcantly correlated with monocyte count. We only found light monocytosis before the treatment which decreased in count from time to time during anti-TB drugs treatment, the same is found in previous ﬁnding [9] that showed that mild absolute or relative monocytosis was found in 29% to 60% of TB patients. Considering that suPAR attach on monocytes and macrophage and play a role in cell migration [10], however, seems that in this study, the level of suPAR did not reﬂect the number of monocytosis. It is possible that release of suPAR in the blood not necessary statistically comparable with the process of monocytosis. forced to come out through cough [12]. It is shown that in thoracic imaging can occur in one to three ﬁrst month of anti-TB drugs treatment, and this can be better or stable in six months in 90% cases [9]. However, after 6 months of treatments there was one patient who still underwent destroyed pulmonary with wider atelectasis. In this study, suPAR level among patients with far advanced lesion, moderate advanced lesion, and minimal lesion did not diﬀer signiﬁcantly. This is in contrast with ﬁnding previous ﬁnding [13] in which the suPAR level correlated signiﬁcantly with width of lesion. To our knowledge, these could be due to the use of diﬀerent method in measuring the lesion. An interesting phenomenon was found in which the suPAR level was much higher (28.000 ng/mL) in one patient with pulmonary TB miliary type. Pulmonary TB miliary type is known as a severe type of pulmonary TB due to hematogen spread process that aﬀect all parts of the lung [10]. Whether this one case reﬂects severity of the disease due to hematogen spread still needs further proof in further study. This was the ﬁrst research done in Indonesia studying the suPAR level during anti-TB drugs treatment. This research was aimed at obtaining the applicable use of suPAR as a complement to monitor treatment eﬃcacy. All 20 patients in the current study responded clinically to the TB treatment. 4. Discussion TB has remained in the third rank of infectious disease in Indonesia for years. Lack of accurate method in monitoring progress in TB therapy could be one of the causes of this problem. The present study aimed to investigate the possible use of suPAR as a biomarker in pulmonary TB-AFB(+) patients, hence allow stratiﬁcation of treatment regimens with possibly shortening of treatment in a majority of patients that may not require a six-month treatment period and intensiﬁed regimens in those with an increased risk for poor response and relapse. The same trend was found on monocytes count. Before treatment, monocytes count showed the highest concen- tration (median 847 cell/mm3) However, it dropped signif- icantly (P = .0001 < .05) after two (median 662 cell/mm3) and-six-month treatment (median 497 cell/mm3) (Figure 1(c)). The decreasing suPAR level correlated signiﬁcantly (r = 0.536) with the decreasing of monocytes count (P- value .000 < .05). We observed light monocytosis before treatment, which went down in count during the treatment period. In the normal condition, monocytes count in blood was in the range of 0.3–0.8 × 103 cell/mm3. It is surprising to see in general that the range of suPAR level in patients with active pulmonary TB (8.539 ng/mL– 28.000 ng/mL) was rather diﬀerent from African people which ranged between 0.9 ng/mL to 45 ng/mL [2]. Whether In contrast, BMI of the TB patients rose gradually but sig- niﬁcant (P = .012) after two (median 19.18 kg/m2) and the six-month of treatment. By the end of therapy, all patients’ Tuberculosis Research and Treatment Tuberculosis Research and Treatment 5 Table 3: Result of ANOVA Test. No Width of lesion classiﬁcation Test Sig Sig < 0.05 Conclusion 1 Min lession, mod advance, far advance Anova test Sig F 0.901 Not Signiﬁcant 2 Min lession, mod advance T-test Sig t 0.61 Not Signiﬁcant 3 Min lession, far advance T-test Sig t 0.792 Not Signiﬁcant 4 Mod advance, far advance T-test Sig t 0.716 Not Signiﬁcant Table 3: Result of ANOVA Test. Table 3: Result of ANOVA Test. these are genetically inﬂuenced, it needs further study. It was found that suPAR level was elevated before treatment and drastically dropped after two- and four- month therapy. A decrease in suPAR after 8-month of Tb-treatment among treatment responders was also found in the study of Eugen- Olsen et al. [3]. 4. Discussion The positive eﬀect of treatment was also reﬂected in decrease in the inﬂammatory status as measured by suPAR levels. Thus, we conclude that decreasing suPAR level may reﬂect treatment eﬃcacy, but further studies involving TB patients that do not respond to TB treatment is necessary to determine if suPAR remains high for example, in TB-MDR patients. The drastic decrease of suPAR level in the ﬁrst two months was weakly associated with raise of BMI. The raise of appetite of TB patient might be due to decreasing of TNF-alpha release [11]. These acts aﬀect the immune status condition which sifted to Th2, resulting in the pressure on Th1 or the existence of Th1 signal transduction resistance on macrophage by tuberculosis bacilli and infection severity [8]. By the end of the initial phase treatment, there were ﬁve patients who did not undergo sputum conversion yet, but at the end of continuation phase, all the patients underwent sputum conversion. Patient’s sputum conversion occurred in 75%–85% cases after 2-month treatment and 96% cases after six-month treatment [9]. Although the trend of the drop of suPAR level and sputum conversion look similar, nevertheless it is hard to correlate the sputum conversion with the level of suPAR in the blood, since suPAR is not a direct product of Mycobacterium. References [1] M. Jo, K. S. Thomas, L. Wu, and S. L. Gonias, “Soluble Urokinase-type plasminogen activator receptor inhibits can- cer cell growth and invasion by direct Urokinase-independent eﬀects on cell signaling,” The Journal of Biological Chemistry, vol. 278, no. 47, pp. 46692–46698, 2003. [2] P. Rabna, A. Andersen, C. Wejse et al., “High mortality risk among individuals assumed to be TB-negative can be pre- dicted using a simple test,” Tropical Medicine and International Health, vol. 14, no. 9, pp. 986–994, 2009. [3] J. Eugen-Olsen, P. Gustafson, N. Sidenius et al., “The serum level of soluble urokinase receptor is elevated in tuberculosis patients and predicts mortality during treatment: a com- munity study from Guinea-Bissau,” International Journal of Tuberculosis and Lung Disease, vol. 6, no. 8, pp. 686–692, 2002. [4] J. F. D. Siawaya, M. Ruhwald, J. Eugen-Olsen, and G. Walzl, “Correlates for disease progression and prognosis during con- current HIV/TB infection,” International Journal of Infectious Diseases, vol. 11, no. 4, pp. 289–299, 2007. [5] PDPI. Perhimpunan Dokter Paru Indonesia, Tuberkulosis : Pedoman Diagnosis dan Penatalaksanaan di Indonesia, Indah Oﬀset Citra Graﬁka, Jakarta, Indonesia, 2006. [6] WHO, TB/HIV A Clinical Manual. Stop TB Dept. Dept of HIV/AIDS. Dept Child and Adolescent Health and Develop- ment, World Health Organization, Geneva, Switzerland, 2004. [7] ViroGates. suPARnostic ELISA Kit: Enzyme-linked immunosorbent assay for quantitative determination of soluble urokinase plasminogen activator receptor in human plasma. Copenhagen, Denmark, 2007. [8] P. F. Barnes and B. Wizel, “Type 1 cytokines and the pathogenesis of tuberculosis,” American Journal of Respiratory and Critical Care Medicine, vol. 161, no. 6, pp. 1773–1774, 2000. [9] W. N. Rom and S. M. Garay, Tuberculosis, Lippincott Williams & Wilkins, Philadelphia, Pa, USA, 2nd edition, 2004. [10] O. G. Wilhelm, S. Wilhelm, G. M. Escott et al., “Cellular glycosylphosphatidylinositol-speciﬁc phospholipase D regu- lates urokinase receptor shedding and cell surface expression,” Journal of Cellular Physiology, vol. 180, no. 2, pp. 225–235, 1999. [11] R. van Crevel, T. H. M. Ottenhoﬀ, and J. W. M. van der Meer, “Innate immunity to Mycobacterium tuberculosis,” Clinical Microbiology Reviews, vol. 15, no. 2, pp. 294–309, 2002. [12] H. Alsagaﬀand A. dan Mukty, Dasar-Dasar Ilmu Penyakit Paru, Airlangga University Press, Surabaya, Indonesia , 2002. [13] J. F. D. Siawaya, N. B. Bapela, K. Ronacher et al., “Immune parameters as markers of tuberculosis extent of disease and early prediction of anti-tuberculosis chemotherapy response,” Journal of Infection, vol. 56, no. 5, pp. 5. Conclusion The decreasing of suPAR level in pulmonary TB patients with active AFB(+) was a manifestation of treatment eﬃcacy. suPAR level elevated almost three times in TB patients before treatment and drastically dropped after the ﬁrst two-month treatment, followed by the gradual decrease until the end of sixth month treatment that was comparable to suPAR level of the healthy control. The reduction trend of suPAR level was correlated to other clinical and laboratory parameters, that is, disappearance of patients’ complaints, increasing of BMI, declining of ESR, and fewer monocytes number. However, suPAR concentration did not reﬂect the pulmonary lesion width on thoracic imaging; hence, suPAR could not be used as a sole biological marker to monitor the eﬃcacy of the treatment. Patients who have active pulmonary TB with AFB spu- tum(+) were found mostly in pulmonary TB patients with far advanced lesion, followed by moderate advanced lesion then minimal lesion. Pulmonary lesion is considered to be a key parameter used to diagnose and monitor the outcome of the treatment. As the disease progresses its resulted in the softening of caseosa necrosis, forming cavity containing a lot of M. tuberculosis and bronchial wall erosion which then are Tuberculosis Research and Treatment 6 Acknowledgments This study was supported by Faculty of Medicine, Brawijaya University. The authors thank to suPARnostic, ViroGates A/S, Copenhagen, Denmark for giving special price for the kit. Special thanks for Dr. Jesper Eugene-Olsen, Danemark for editing the paper. References 340–347, 2008.
W4385202581.txt	https://www.mdpi.com/1996-1944/16/14/5154/pdf?version=1690031395	en		Modification of Polycaprolactone with Plant Extracts to Improve the Aging Resistance	Materials	2,023	cc-by	11,878	materials Article Modification of Polycaprolactone with Plant Extracts to Improve the Aging Resistance Krzysztof Moraczewski 1, * , Magdalena Stepczyńska 1 , Rafał Malinowski 2 , Tomasz Karasiewicz 1 , Bartłomiej Jagodziński 1 and Piotr Rytlewski 1 1 2 * Faculty of Materials Engineering, Kazimierz Wielki University, Chodkiewicza 30, 85-064 Bydgoszcz, Poland; m.stepczynska@ukw.edu.pl (M.S.); tomakara@ukw.edu.pl (T.K.); bar.jag@ukw.edu.pl (B.J.); prytlewski@ukw.edu.pl (P.R.) Łukasiewicz Research Network—Institute for Engineering of Polymer Materials and Dyes, Marii Skłodowskiej-Curie 55, 87-100 Toruń, Poland; malinowskirafal@gmail.com Correspondence: kmm@ukw.edu.pl Abstract: Natural extracts of plant origin are used as anti-aging compounds of biodegradable polymers. Coffee, cocoa, or cinnamon extracts in amounts from 0.5 to 10 wt.% were added to the polycaprolactone matrix. The manufactured materials were aged at elevated temperatures with increased relative humidity and continuous exposure to UV radiation for 720, 1440, or 2160 h. The performance of the proposed extracts was compared with the retail anti-aging compound, butylated hydroxytoluene. Visual assessment, FTIR analysis, melt flow rate, tensile strength, impact tensile strength, thermogravimetry, and differential scanning calorimetry tests were conducted. Results showed that the use of lower contents of the tested extracts is particularly advantageous. When the content of the extract did not exceed 1 wt.%, no unfavorable influence on the properties of the materials was observed. The stabilizing performance during accelerated aging was mostly similar to or greater than that of the reference compound used. Keywords: polycaprolactone; polyphenols; plant extracts; accelerated aging Citation: Moraczewski, K.; Stepczyńska, M.; Malinowski, R.; Karasiewicz, T.; Jagodziński, B.; Rytlewski, P. Modification of Polycaprolactone with Plant Extracts to Improve the Aging Resistance. Materials 2023, 16, 5154. https:// doi.org/10.3390/ma16145154 Academic Editors: Andrea Sorrentino and Alfonso Maffezzoli Received: 20 June 2023 Revised: 11 July 2023 Accepted: 20 July 2023 Published: 21 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 1. Introduction As materials and products are utilized, they undergo aging which can lead to a decline in their visual and functional characteristics [1]. Due to the prevalence of this phenomenon, multiple research centers worldwide are dedicated to conducting studies aimed at mitigating its negative impact [2–4]. Currently, the demand for polymeric materials is on the rise, leading to extensive research on the aging mechanisms of these materials. The research focuses on understanding the complex aging processes and their effects on the physical and chemical properties of polymeric materials, as well as the influence of processing on said aging phenomena [5–8]. Extensive research has been conducted over the years to enhance the resistance of polymers to atmospheric, chemical, and thermal factors. Anti-aging compounds have proven to be the most effective approach to improving the stability of polymer materials. These compounds, whether natural or synthetic, are designed to increase the resilience of polymer materials against external factors such as high temperatures, UV radiation, and chemicals [9–12]. As the push for more durable polymers continues, a growing global focus on ecological sustainability has also continued. The primary objective is to find effective solutions to protect the environment. One promising approach is promoting and implementing biodegradable polymers across various industries and applications. The biodegradable polymer is completely transformed by microorganisms (bacteria and fungi) into carbon dioxide, water, and humus [13] and can be successfully processed 4.0/). Materials 2023, 16, 5154. https://doi.org/10.3390/ma16145154 https://www.mdpi.com/journal/materials Materials 2023, 16, 5154 2 of 22 using most standard polymer processing technologies, including the most popular: thermoforming, extrusion, injection, or blow molding [14–18]. As of late, there has been an increasing demand for polymer materials that can withstand external factors while being entirely biodegradable at the end of their lifespan. To achieve this, one must select a suitable biodegradable polymer/anti-aging compound combination. One of the widely used biodegradable polymers is polycaprolactone (PCL) [19–25]. PCL is an aliphatic polyester, obtained from caprolactone by ring-opening polymerization. It is easily mixed with many other polymers, therefore it is very often used as a plasticizer increasing the flexibility of polymers, as well as an additive increasing biodegradability. In combination with starch, it is used to make rigid plastic from which disposable plates or cups are made, which can be disposed of by composting. This polymer has found many biomedical applications. It is commonly used in the production of implants and absorbable surgical threads due to the gradual and slow decomposition caused by the hydrolysis of ester bonds in the human body, which takes approximately two years [26–29]. When it comes to using anti-aging compounds for biodegradable polymers, certain limitations must be considered. These compounds must not disrupt the composting process and should be non-toxic, non-volatile, and safe for contact with food, as the packaging industry is a major user of biodegradable polymers, particularly for food packaging. It would be highly beneficial to opt for plant-based products, as they decompose easily and do not impede the composting process. Additionally, plant-based substances are environmentally friendly. Substances containing natural polyphenols derived from plants show enormous potential as effective anti-aging compounds due to their composition and chemical structure. Polyphenols are readily available and do not require special chemical synthesis processes [30,31]. Natural antioxidants are classified into various groups, with polyphenols being the largest among them. They exhibit a wide range of molecular weights, structures, and biological and physicochemical properties. Due to the diverse nature of polyphenols in terms of structures and properties, it can be challenging to systematize them. Phenolic compounds can be very generally divided into four general groups: phenolic acids, phenolic diterpene, flavonoids, and volatile oils [32]. They are secondary metabolites widespread in the plant world (usually in the form of glycosides or esters) but not found in animals. They are found in fruits, vegetables, plant-based beverages, spices, and herbs. Plant polyphenols can act in several of the following ways: reducing agents, free radical blockers, metal ion chelating agents that catalyze oxidative reactions, and agents preventing reactions caused by a single active oxygen atom [33–37]. Natural polyphenols are becoming a popular ecological substitute for synthetic antiaging compounds due to their versatility. The literature offers insights into how different polyphenols can stabilize various polymer types. One can find the use of such polyphenols as gallic, vanillic, caffeic, cinnamic, sinapic, syringic, ferulic, coumaric, and ascorbic acids, tocopherols, quercetin to stabilize polyethylene, polypropylenes, starch, cellulose, polylactide, polyhydroxyalkanoates, and poly (ethylene oxide) [38–43]. The main objective of the conducted study was to determine whether natural plantderived substances containing polyphenols could potentially serve as anti-aging compounds for polycaprolactone. The proposed approach will improve the resistance of biodegradable polycaprolactone to external factors without negatively affecting biodegradation during composting. The results of selected properties of unaged and aged PCL containing from 0.5 to 10 wt.% of coffee, cocoa, or cinnamon extract are presented. Detailed studies of the influence of the proposed extracts on selected properties of polycaprolactone have already been carried out and published [44]. In this paper, selected properties of PCL containing natural anti-aging compounds subjected to the accelerated aging process were determined and compared to properties of materials containing a commercially used anti-aging compound. Materials 2023, 16, 5154 3 of 22 2. Materials and Methods The materials matrix used was polycaprolactone (PCL) type Capa 6800 from Perstorp, Sweden. It had a mass melt flow rate (MFR) of 2–4 g/10 min (160 ◦ C, 5 kg), density (ρ) of 1.10 g/dm3 , mean molecular weight of 80,000, melting point 58–60 ◦ C, and glass transition temperature of −60 ◦ C. To enhance its properties, 3 natural compounds of plant origin were added: coffee (Coffea arabica L.) extract, cocoa (Theobroma cacao L.) extract, and cinnamon (Cinnamomum cassia Presl) extract, sourced from Agrema Sp. z o.o., Wrocław, Poland. The coffee extract contained 45 wt.% of polyphenols, while cocoa and cinnamon extracts had 5 wt.% of polyphenols. Chlorogenic acid, flavonoids, and phenolic acids were the main polyphenols in coffee, cocoa, and cinnamon extracts, respectively. To assess the quality of the acquired materials, they were compared to a commonly used synthetic anti-aging compound. Butylated hydroxytoluene (BHT) from Sigma-Aldrich, St. Louis, MO, USA, was used as the reference compound, with a density of 1.04 g/dm3 . BHT is a popular anti-aging additive for multiple polymers [45–47]. Initially, the test materials were created through an extrusion process. The granules of the PCL composites under examination were extruded with the aid of a co-rotating twin screw extruder BTSK 20/40D (Bühler, Germany). The parameters of each extruder zones were 115, 115, 120, and 120 with a head temperature of 120 ◦ C. 0.5; 1; 3; 5 or 10 wt.% of the proposed natural anti-aging extract was added to the polymer matrix, giving samples marked as P0.5, P1, P3, P5, and P10, respectively. A sample of pure PCL was designated as P. Usually, the content of traditional anti-aging compounds typically used in the processing and use of polymeric materials does not exceed a few percent. In the conducted research, in addition to the small contents of the extracts, larger amounts of these materials were used. Unlike synthetic anti-aging compounds, the active substance is only a part of the weight of the extract added to the polymer; therefore, it seems advisable to use higher concentrations of extracts. As a reference sample, a material containing 2 wt.% BHT was also extruded. The reference material was designated as the R sample. During the second phase of processing, the test samples were created. Using a PlastiCorder Lab Station (from Brabender, Germany), composite films were extruded with a single-screw extruder that had a flat-faced head with a mouthpiece width of 170 mm. The cylinder zones were heated to temperatures of 75, 85, and 95 ◦ C, while the head temperature was at 100 ◦ C. Afterwards, we placed the melted polymer onto cold thermostatic rolls that were at around 20 ◦ C. The resulting film had a thickness of roughly 0.5 mm. The accelerated aging process was carried out using a CCK 40/300NG (Dycometal, Barcelona, Spain) climatic chamber. The aging process was performed in a closed chamber at a temperature of 40 ◦ C, 70% relative humidity, and UV irradiation for 720, 1440, or 2160 h. The apparatus was equipped with 8 UV lamps (PHILIPS SUPER ACTINIC TL 60 W/10-R ISL, Philips, Amsterdam, The Netherlands) with a wavelength range of 350–400 nm with a peak intensity of 370 nm. The sample/lamp distance was kept as small as possible by placing the samples directly under the UV lamps. The distance between the lamps and the surface of the samples was approx. 10 cm. The distance used was selected based on the experience of the operator of the climatic chamber. A shorter distance would cause the surface temperature of the samples to be too high, which could melt them. If the lamps were too far apart, the intensity of UV radiation could be too low to effectively age the material. Color tests were carried out on films with a thickness of 500 µm using a BYK colorimeter (BYK-Gardner GmbH, Geretsried, Germany) designed for measuring the color and gloss of materials. The color measurement was quantified using a color space standardized by the CIELab system based on 3 components: L, a, and b. The component “L” describes the lightness of the material, the component “a” describes the color in the range from green to red, and the component “b” describes the color in the range from blue to yellow. The color difference ∆E and browning index (BI) [48] were calculated according to the equations: ∆E = q ∆L2 + ∆a2 + ∆b2 (1) Materials 2023, 16, 5154 4 of 22 100 BI = h a+1.75L 5.645L+a−0.3012b − 0.31 i (2) 0.17 where ∆L is the difference in L between 2 samples (virgin PLA and PLA with specific extract content) while, similarly, ∆a and ∆b are the differences in the a and b coordinates, respectively. For samples without the extracts, instead of the browning index, the yellowness index (YI) was calculated according to the YID1925 standard from Equation (3) by converting the L, a, and b color scale into the XYZ color scale. 0.847Z YI = 100 1 − (3) Y An FTIR Nicolet iS10 spectrophotometer (ThermoScientific, Waltham, MA, USA) was used to record sample spectra. The attenuated total reflection method (ATR-FTIR) was used to obtain Fourier transform infrared spectra (FTIR). The analyzed spectrum was an average of 16 measurements taken between the wave number range of 4000 to 650 cm−1 . An MP 600 plastometer (Tinius Olsen, Horsham, PA, USA) was used in the melt flow rate (MFR) tests. The MFR (100 ◦ C, 5 kg) was determined using 12 individual samples. The final values of MFR were calculated as the arithmetic mean of 10 results (2 extreme values were neglected). An Instron 3367 (Instron, Norwood, MA, USA) tensile machine was used in mechanical properties tests. The extension rate for testing each sample was 200 mm/min. Tensile tests were carried out on standardized samples in the formation of paddles with measuring section of 100 × 10 × 4 mm. Mechanical parameters were determined using 12 individual samples. The final values were calculated as the arithmetic means of 10 results, with 2 extreme ones being neglected. An IMPASS-15 (ATS FAAR, Cassina dè Pecchi, Italy) impact hammer was used in impact tests. A 4J hammer was utilized with a speed of 2.9 m/s to break samples in the form of 80 × 10 × 4 mm bars. The impact strength was determined through 12 individual samples, and the final values were calculated as the arithmetic means of 10 results after disregarding 2 extreme values. A Q500 differential scanning calorimeter (TA Instruments, New Castle, DE, USA) was used in thermal properties tests. The experiments were conducted under a nitrogen atmosphere, utilizing samples weighing approximately 7 mg and a temperature range from −70 to 80 ◦ C. The data were captured in 3 separate cycles, which included first heating, cooling, and a second heating process with a heating rate of 10 ◦ C/min. A Q200 thermogravimetric analyzer (TA Instruments, USA) was used in the thermal stability tests. The experiments were performed under a nitrogen atmosphere. We utilized samples that weighed around 22.5 mg and conducted the tests within a temperature range from 20 to 600 ◦ C while heating them. 3. Results and Discussion 3.1. Visual Evaluation For the polymeric components and parts used in indoor and outdoor applications, changes in color because of aging phenomena could be an important problem and challenge. The color differences of the samples before and after aging were assessed by comparing their photographic images. Pictures of selected un-aged samples and samples aged for 720 or 2160 h are shown in Figure 1. To support the visual assessment with experimental data, the color tests in the CIElab color space were carried out and some of the parameters are presented in Table 1. Materials 2023,2023, 16, 5154 Materials 16, x FOR PEER REVIEW 5 of 5 22of 22 Figure 1. Photos of selected samples before and after the aging process. Approximate sample size Figure 1. mm. Photos of selected samples before and after the aging process. Approximate sample size 10 × 10 10 × 10 mm. Materials 2023, 16, 5154 6 of 22 Table 1. Color test results. Aging Time [h] Extract Content [%wt] 720 2160 L ∆E BI/YI * L ∆E BI/YI * L ∆E BI/YI * - 91.67 - 3.37 * 91.52 3.37 13.07 * 92.65 14.35 27.79 * 0.5 85.88 13.04 0.78 82.51 29.86 4.89 91.94 3.47 −0.34 1 69.87 35.10 12.81 60.14 47.62 22.48 91.12 4.41 −0.39 3 71.66 33.32 11.63 46.39 56.79 31.79 45.05 53.53 28.01 5 62.45 37.98 12.11 42.76 56.28 29.44 49.63 47.99 24.16 10 55.54 40.30 12.02 38.17 57.71 26.76 38.4 57.06 24.75 0.5 79.23 22.80 6.40 88.35 18.89 0.73 91.77 4.16 −0.37 1 71.75 33.32 11.65 82.44 29.16 5.41 91.25 5.45 −0.17 3 54.84 48.99 23.37 59.84 49.02 25.41 85.78 15.94 2.40 5 44.65 54.35 29.49 53.16 51.71 29.45 67.58 40.92 17.10 10 35.04 59.74 30.34 41.28 53.72 23.31 55.35 40.69 15.28 83.18 17.73 3.33 89.34 18.14 0.26 90.92 6.66 0.06 1 82.66 20.41 4.31 86.84 20.59 2.14 88.79 11.22 1.28 3 57.61 48.48 22.27 79.09 31.45 8.37 81.59 24.90 6.67 Cinnamon extract Cocoa extract Coffee extract P 0 0.5 5 57.76 47.96 21.80 68.54 43.56 18.68 68.01 42.70 19.21 10 46.8 53.46 28.32 47.9 53.49 31.68 61.58 45.74 24.13 R - 92.31 0.64 8.59 * 90.91 6.31 17.13 * 92.64 18.13 32.61 * * YI. The color of samples P and R were identical. The ∆E value of sample R was 0.64. According to the CIELab color space rules, when ∆E < 1, the standard observer will not notice the difference between the two colors [43,49]. The introduction of the tested extracts to the PCL matrix resulted in clear changes in the color of the material. The samples turn from non-transparent white into brown, the shade of which depends on the type of extract and the intensity of the extract content. The value of the L component, which indicates the brightness of the samples, decreased with the increase in the extract content, with the simultaneous increase in the ∆E parameter, which for all samples had values above five, i.e., the color change was easily noticeable with a distinct impression of two different colors. The more extract in the material, the more intense the brown was, which was confirmed by the growing BI values. Due to the accelerated aging process, as an effect of UV radiation, the color of the samples changed. The first easily noticeable effect of polymer aging is the yellowing of the material. It is caused by several factors, including UV radiation, heat, or liquids. The effect of UV radiation on matter is related to the processes of photolysis, i.e., the cleavage (breaking) of chemical bonds due to radiation absorption. This process breaks chemical bonds and creates two compounds each with one unpaired electron, which stimulates the formation of very reactive free radicals. They react with other compounds, creating a new compound and another free radical. The reason for the color change (yellowing) is precisely the formation of these radicals (because these new molecules reflect some light, in many cases yellow). Such a yellowing effect was observed in the case of the pure PCL sample (sample P), where after 2160 h of aging the material had a yellow shade. A numerical representation of the yellowness effect is a substantial change in the calculated YI value from 3.37 for unaged PCL to 27.79 for PCL aged for 2160 h. Interestingly, the unfavorable color change was not stopped by the addition of the reference anti-aging compound (BHT) Materials 2023, 16, 5154 7 of 22 because sample R also turned yellow. The calculations showed that the yellowing effect was even more intense than pure PCL, as the YI value increased from 0.64 for the unaged R sample to 32.61 for the R sample aged 2160 h. In the case of samples containing the tested extracts, accelerated aging caused a decrease in color intensity, which was caused by the influence of UV radiation on the chromophore groups. This effect was most visible at low extract contents, where after 2160 h of aging the color practically disappeared completely. The visual observations were confirmed by the values of the L parameters obtained with the use of a colorimeter and the calculated values of ∆E and BI. The L component in most cases increased with the aging time, confirming the increase in brightness of the samples. The decrease in the intensity of the brown color visible in the photos was confirmed by the lower BI value of individual samples with increasing aging time, as well as a decrease in the ∆E parameter. Due to the high energy, ultraviolet radiation can cause photochemical reactions in the illuminated object. Chemical decomposition is also subject to the chemical decomposition of compounds giving color to the tested materials. This process is stronger, resulting in the darker colors of the samples, which is confirmed by the presented results. The exceptions were samples containing coffee extract, in the case of which the shortest aging period was followed by a remarkably interesting effect of “enhancing” the color, visible in the photos and supported by numerical results. Color change from colorless to brown should not be a problem in the possible use of tested material films. They can be successfully used in applications where color is not of great importance, e.g., disposable dishes, straws, general medical applications, or garbage bags. The biggest advantage of these products will be their biodegradability and lower environmental burden, which may be more important for the potential consumer. 3.2. Spectroscopic Studies The analysis of the spectrum of pure PCL and the materials containing the tested extracts (not shown here) showed the occurrence of absorption bands typical of this polymer. The introduction of extracts into the matrix did not cause any significant changes in the spectrum. Only the appearance of a small band in the range of 3600–3200 cm−1 was observed, the intensity of which increased with the amount of extract in the material. This band originates from the -OH groups of polyphenols contained in the extracts. The lack of significant changes in the image of the spectra suggests that the added extracts tend to accumulate in the core of the material and not in its surface layer. In the FTIR analysis, detailed attention was focused on the 1800–1650 and 1500–1000 cm−1 regions allowing the assessment of the degree of oxidation of the surface layer. The 1720 cm−1 peak is from stretching vibrations of carbonyl group C=O, the 1420 cm−1 peak is from deformation vibrations of CH2 –C=O, the 1294 cm−1 peak is from C–O stretch in the crystalline phase, the 1242 cm−1 peak is from asymmetric COC stretching, and the 1189 cm−1 peak is from OC-O stretching. Two groups of spectra are visible in Figure 2. Spectra of unaged samples (solid line) with lower peak intensities and spectra of aged samples (dashed line) with higher peak intensities. Thus, it can be seen that because of accelerated aging (mainly because of UV radiation), there was a significant oxidation of the surface layer of the tested samples. The lack of differences between the samples without and with the extracts indicates that unfortunately, the addition of extracts did not reduce the oxidation of the polymer surface. The applied extracts, however, were slightly more effective than the reference anti-aging compound, which, interestingly, also did not limit oxidation. Materials 2023, FOR PEER REVIEW Materials 2023, 16, 16,x5154 (a) 8 of 22 8 of 22 0,9 P 0h P 2160h 0,8 P0.5 coffee extract 0h P0.5 coffee extract 2160h 0,7 P10 coffee extract 0h P10 coffee extract 2160h Absorbance 0,6 R 0h R2160h 0,5 0,4 0,3 0,2 0,1 0 1800 1775 1750 1725 1700 1675 1650 Wavenumber [cm-1] (b) 0,4 P 0h P 2160h 0,35 P0.5 coffee extract 0h P0.5 coffee extract 2160h P10 coffee extract 0h Absorbance 0,3 P10 coffee extract 2160h R 0h 0,25 R2160h 0,2 0,15 0,1 0,05 0 1500 1400 1300 1200 1100 1000 Wavenumber [cm-1] −1 −1 Figure FTIR spectra of 1800–1650 (a) 1800–1650 and (b) 1500–1000 cm of regions of samples. selected samples. Figure 2. 2. FTIR spectra of (a) cm−1cm and (b) 1500–1000 cm−1 regions selected 3.3. Melt Flow Rate 3.3. Melt Flow Rate The MFR value for sample P, which was pure PCL, was 1.65 g/10 min. Upon adding The MFR value for sample P, which was pure PCL, was 1.65 g/10 min. Upon adding the reference anti-aging compound (sample R), the MFR increased slightly to 1.89 g/10 min. the reference anti-aging compound (sample R), the MFR increased slightly to 1.89 g/10 TheThe addition of of tested extracts alsoresulted resultedininchanges changes MFR. The min. addition tested extractstotothe thepolymer polymer matrix matrix also inin MFR. MFR result for all samples is presented in Table 2. The MFR result for all samples is presented in Table 2. Comparing the MFR of pure PCL to the MFR of PCL mixed with coffee extract, the latter showed an increase from 0.11 g/10 min for 0.5 wt.% to 0.31 g/10 min for 10 wt.%. Similarly, adding 0.5 wt.% of cocoa extract resulted in a significant increase in MFR by 0.43 g/10 min compared to the value of sample P. However, unlike the coffee extract, increasing the amount of cocoa extract decreased the MFR, with a difference of only 0.06 g/10 min for the sample with 10 wt.%. As for cinnamon extract, after an initial increase of 0.14 g/10 min at 0.5 wt.%, the MFR value decreased. The lowest MFR value was obtained for the sample containing 3 wt.% of cinnamon extract, with a subsequent increase in extract content leading to an increase in MFR. The highest MFR value was obtained for Materials 2023, 16, 5154 9 of 22 the sample containing 10 wt.% of the extract, which was higher by 0.29 g/10 min than the pure PCL value. Table 2. The results of the melt flow rate (MFR) tests. Aging Time [h] Extract Content [%wt] 720 1440 2160 MFR [g/10 min] MFR [g/10 min] MFR [g/10 min] MFR [g/10 min] - 1.65 ± 0.07 1.83 ± 0.05 1.85 ± 0.06 1.89 ± 0.09 0.5 1.76 ± 0.08 1.9 ± 0.08 1.98 ± 0.11 2.12 ± 0.15 1 1.85 ± 0.03 1.92 ± 0.04 2.04 ± 0.04 2.27 ± 0.16 3 1.84 ± 0.03 1.96 ± 0.03 2.14 ± 0.09 2.43 ± 0.18 5 1.90 ± 0.03 1.96 ± 0.04 2.16 ± 0.15 2.46 ± 0.16 10 1.96 ± 0.04 2.19 ± 0.084 2.84 ± 0.14 2.77 ± 0.15 0.5 2.08 ± 0.08 2.19 ± 0.08 2.59 ± 0.09 2.95 ± 0.05 1 1.99 ± 0.03 2.33 ± 0.08 2.63 ± 0.11 2.99 ± 0.11 3 2.05 ± 0.04 2.25 ± 0.12 2.50 ± 0.11 3.16 ± 0.08 5 1.95 ± 0.05 2.03 ± 0.05 2.48 ± 0.11 2.87 ± 0.12 10 1.71 ± 0.04 1.71 ± 0.03 1.96 ± 0.11 2.42 ± 0.15 Cinnamon extract Cocoa extract Coffee extract P 0 0.5 1.79 ± 0.02 1.8 ± 0.08 1.88 ± 0.06 2.03 ± 0.10 1 1.68 ± 0.02 1.81 ± 0.05 1.85 ± 0.11 2.09 ± 0.06 3 1.60 ± 0.03 1.77 ± 0.07 1.93 ± 0.09 2.22 ± 0.07 5 1.76 ± 0.06 1.99 ± 0.04 2.15 ± 0.09 2.72 ± 0.07 10 1.94 ± 0.04 2.07 ± 0.10 2.47 ± 0.12 3.39 ± 0.16 R - 1.89 ± 0.03 1.95 ± 0.03 1.91 ± 0.05 2.06 ± 0.17 The increase in MFR after the addition of the extracts was caused by the migration of short polyphenol molecules between the PCL polymer chains. This caused an increase in the distance between the macromolecules, weakening their mutual forces, thus facilitating a chain slip under the pressure increase in the flow of the polymer mass [50]. Changes in the MFR value as a function of aging time for selected samples are shown in Figure 3. Aging the P sample increased the MFR of pure PCL by 0.2 g/10 min compared to the unaged sample. The use of the reference anti-aging compound slightly limited this increase, as the MFR value of the R sample aged for 2160 h increased by 0.17 g/10 min. Changes at a similar level were observed in the samples with coffee extract and cinnamon extract. As a result of accelerated aging, the MFR values of these materials increased by 0.36 g/10 min for coffee extract and 0.24 g/10 min for cinnamon extract. Unfortunately, it only applied to the lowest extract contents, i.e., 0.5 wt.%. Increasing the content of the extracts was disadvantageous considering the stability of the flow parameter. With the increase in the amount of coffee and cinnamon extracts in the material, the difference between the MFR values of the unaged sample and the aged sample increased significantly, reaching the maximum at 10 wt.% of the extracts. Therefore, for 10 wt.% of coffee extract, the difference between the unaged sample and the sample aged for 2160 h was 0.81 g/10 min and for 10 wt.% of the cinnamon extract was 1.45 g/10 min. Thus, coffee and cinnamon extracts with higher contents intensify the adverse effects occurring during aging, negatively affecting the rheological properties of the material. An interesting effect was observed for cocoa extract. In this case, the MFR value was most similar to unaged PCL, and the smallest changes in this value due to accelerated aging were obtained for the sample with the highest extract content, i.e., 10 wt.%. Materials 2023, 16, x FOR PEER REVIEW aging, negatively aﬀecting the rheological properties of the material. An interesting eﬀect was observed for cocoa extract. In this case, the MFR value was most similar to unaged PCL, and the smallest changes in this value due to accelerated aging were obtained for the 10 of 22 sample with the highest extract content, i.e., 10 wt.%. P P0.5 coffee extract P10 cocoa extract P0.5 cinnamon extract R 2,7 2,5 2,3 MFR [g/10 min] Materials 2023, 16, 5154 10 of 22 2,1 1,9 1,7 1,5 0 720 1440 2160 Aging time [h] Figure Melt flow (MFR) values of selected samples as a function of aging Figure 3. 3. Melt flow raterate (MFR) values of selected samples as a function of aging time. time. Thesignificant significantincrease increaseininMFR MFR due due to to the number The the aging aging of ofsamples samplescontaining containinga alarge large of extracts can be explained by the fact that in the case of polyphenols, there is a number of extracts can be explained by the fact that in the case of polyphenols, there is acertain limit amount above which theythey loselose their anti-aging amounts, these certain limit amount above which their anti-agingproperties. properties.In In larger larger amounts, compounds, instead of stopping the aging process, accelerate it significantly. these compounds, instead of stopping the aging process, accelerate it significantly. As As aa result, we observe a significant increase in flow because of intensified degradation processes. result, we observe a significant increase in flow because of intensified degradation processes. 3.4. Tensile Tests 3.4. Tensile Tests Based on the static tensile strength tests, the tensile strength (σM ) and tensile strain at break (εB )on were determined. The aging (K) of polymeric materials calculated Based the static tensile strength tests,coefficients the tensile strength (σM) and tensile strain at from(εEquation (4) were also The 2160 h polymeric aging timematerials values were used as the break B) were determined. Thedetermined. aging coeﬃcients (K) of calculated values for “after aging”. from Equation (4) were also determined. The 2160 h aging time values were used as the (σM ·εB )after aging values for “after aging”. K= K (σ∙M ·εB )befor aging ∙ (4) (4) The results of static tensile strength tests on unaged and aged samples together with K The are results of staticintensile tests on unaged and aged samples together with values presented Tablestrength 3. K values are presented in Table 3. The value of σM of pure, unaged PCL was 45.8 MPa. When the reference anti-aging compound was added, the tensile strength of the unaged sample R increased by 3.6 MPa. As PCL is highly elastic at ambient temperature, the obtained deformations are large. The εB values of the unaged P and R samples were 1531% and 1463%, respectively. Strengthening of the materials was also observed after adding the tested extracts to the matrix. Even a small content of all tested extracts caused a significant increase in the σM value. The σM values of samples containing 0.5 wt.% of coffee, cocoa, or cinnamon extract increased by 3.7; 4.5, and 5.2 MPa. Unfortunately, with higher contents of cocoa and cinnamon extracts, the tensile strength deteriorated. In the case of cocoa extract already at a content of 1 wt.%, the obtained values of σM were lower than the values of pure PCL and they decreased further along with the increase in the extract content. For cinnamon extract, a similar decrease in tensile strength was observed from 5 wt.% of extract. The exception was the coffee extract, for which the tensile strength was better than for pure PCL even at higher extract contents. The parameter εB of samples containing coffee extract was also at a similar level, where the obtained values ranged from 1473 to 1537; therefore, they were close to the value of the sample P. In the case of cocoa and cinnamon extracts, there was a Materials 2023, 16, 5154 11 of 22 decrease in the εB value from 1519 to 1266% for cocoa extract and from 1472 to 1182% for cinnamon extract with an increase in the extract content. The initial increase in strength is due to a slight increase in the crystallinity of the materials (which is confirmed by further DSC studies). The deterioration of mechanical properties at elevated levels of extracts may be related to the high content of the carrier introduced into the matrix together with the active substances of the extracts and the solubility of extracts. A detailed description of changes in mechanical properties is provided in the previous work [44]. Table 3. Tensile test results. Aging Time [h] Extract Content [%wt] 720 1440 2160 K σM [MPa] εB [%] σM [MPa] εB [%] σM [MPa] εB [%] σM [MPa] εB [%] - 45.8 ± 4.9 1531 ± 29 54.8 ± 3.7 1494 ± 62 51.9 ± 4.7 1393 ± 87 22.0 ± 2.0 745 ± 65 0.23 0.5 49.5 ± 5.7 1473 ± 132 53.5 ± 8.2 1445 ± 157 53.0 ± 5.1 1464 ± 127 42.2 ± 8.1 1299 ± 17 0.75 1 52.9 ± 3.5 1537 ± 24 56.6 ± 6.2 1486 ± 121 57.7 ± 5.9 1527 ± 114 47.8 ± 1.4 1310 ± 6 0.77 3 47.9 ± 4.6 1484 ± 109 50.5 ± 4.8 1372 ± 93 54.0 ± 3.7 1454 ± 90 46.6 ± 3.0 1279 ± 52 0.84 5 51.5 ± 2.6 1522 ± 76 54.2 ± 4.4 1423 ± 102 51.2 ± 9.0 1368 ± 196 41.1 ± 10.3 1287 ± 39 0.67 10 48.3 ± 4.3 1480 ± 47 41.5 ± 5.0 1292 ± 161 28.0 ± 2.5 892 ± 280 29.8 ± 3.6 911 ± 197 0.38 0.5 50.3 ± 4.0 1519 ± 35 53.2 ± 5.5 1466 ± 146 49.9 ± 5.4 1405 ± 114 21.7 ± 2.5 739 ± 38 0.21 1 43.5 ± 4.7 1465 ± 125 49.1 ± 2.8 1411 ± 75 48.1 ± 3.5 1404 ± 78 22.4 ± 3.5 729 ± 38 0.26 3 44.5 ± 2.7 1445 ± 67 42.7 ± 1.6 1299 ± 56 34.0 ± 8.3 1046 ± 309 21.3 ± 2.0 724 ± 63 0.24 5 42.4 ± 3.8 1323 ± 68 38.9 ± 1.1 1226 ± 49 34.7 ± 1.9 1181 ± 41 20.8 ± 3.0 715 ± 20 0.27 10 33.8 ± 2.1 1266 ± 15 35.9 ± 2.4 1181 ± 56 33.0 ± 2.1 1112 ± 41 20.8 ± 5.3 717 ± 26 0.35 51.0 ± 2.2 1472 ± 83 48.7 ± 5.6 1351 ± 126 48.4 ± 4.9 1384 ± 118 20.1 ± 1.0 614 ± 89 0.16 1 50.6 ± 7.0 1494 ± 47 48.3 ± 1.8 1405 ± 81 44.5 ± 2.9 1362 ± 84 19.3 ± 0.5 602 ± 86 0.15 3 59.8 ± 7.4 1349 ± 75 41.3 ± 2.2 1254 ± 35 35.7 ± 6.4 1205 ± 108 22.0 ± 5.5 584 ± 211 0.16 Cinnamon extract Cocoa extract Coffee extract P 0 0.5 5 43.5 ± 5.4 1251 ± 58 39.5 ± 2.0 1210 ± 42 35.0 ± 3.1 1194 ± 49 22.3 ± 6.2 906 ± 179 0.37 10 28.3 ± 3.0 1182 ± 92 29.8 ± 2.2 1080 ± 46 26.0 ± 5.1 1018 ± 135 16.6 ± 0.9 29 ± 9 0.01 R - 49.4 ± 3.9 1463 ± 104 55.7 ± 5.0 1510 ± 91 51.2 ± 4.1 1467 ± 76 20.7 ± 1.3 562 ± 110 0.16 As the best tensile properties were obtained for low extract contents, a more detailed analysis of the influence of accelerated aging on the mechanical parameters was carried out on these materials (Figure 4). The tensile strength of sample P decreased significantly after 2160 h of accelerated aging. Initially, however, material strengthening was observed as the recorded value of σM after 720 h aging increased by 9.0 MPa. After this time, the tensile strength of the material deteriorated, with a large decrease in the value of σM by 23.8 MPa after 2160 h of aging. The initial strengthening of the material with a further decrease in tensile strength with increasing aging time was also observed for the R sample. The total decrease in the value of σM after 2160 h of aging for this material was 28.7 MPa. Thus, the reference anti-aging compound did not inhibit the adverse effect of accelerated aging on PCL tensile strength. Materials2023, 2023, 16, 16, 5154 x FOR PEER REVIEW Materials 12 of 22 12 of 22 (a) P P1 coffee extract P0.5 cocoa extract P0.5 cinnamon extract R 70 60 σM [MPa] 50 40 30 20 10 0 720 1440 2160 Aging time [h] (b) P P1 coffee extract P0.5 cocoa extract P0.5 cinnamon extract R 2000 1600 εB [%] 1200 800 400 0 0 720 1440 2160 Aging time [h] Figure 4. Values of (a) tensile strength (σM ) and (b) strain at break (εB ) of selected samples as a Figure 4. Values of (a) tensile strength (σM) and (b) strain at break (εB) of selected samples as a functionofofaging aging time. function time. Cocoa andstrength cinnamon characterized by a similar ineffectiveness. As in the The tensile of extracts sample Pwere decreased significantly after 2160 h of accelerated case of samples P and R, for samples containing andas cinnamon extracts, aging. Initially, however, material strengthening wascocoa observed the recorded value after of σM2160 h of accelerated aging there was a sharp drop in the value of σ . For materials containing M after 720 h aging increased by 9.0 MPa. After this time, the tensile strength of the material 0.5 wt.%. cocoa and cinnamon extracts, the σ values decreased by 28.6 MPa and 30.9 MPa, M deteriorated, with a large decrease in the value of σM by 23.8 MPa after 2160 h of aging. The respectively, compared to the values of unaged samples. On the other hand, the initial strengthening of the material with a further decrease in tensile strength with coffee extract showed the ability toobserved inhibit the in tensile strength. This increasing aging time was also fordecrease the R sample. The total decrease in substance the value oflimited the decrease in tensile strength after 2160 h of accelerated aging observed in the case of σM after 2160 h of aging for this material was 28.7 MPa. Thus, the reference anti-aging other materials. The most stable parameters as a function of aging time were found in the compound did not inhibit the adverse effect of accelerated aging on PCL tensile strength. material 1 wt.% of coffee whereby the difference between the Cocoacontaining and cinnamon extracts were extract characterized a similar ineffectiveness. Asvalues in the of the case of samples P aged and R,samples for samples containing cocoa and cinnamon extracts, aftermuch 2160 hsmaller unaged and the was only 5.1 MPa. The difference was therefore ofthan accelerated aging there was a sharp drop in the value of It σMis . For materials containing the differences observed in the other samples. worth noting that the0.5 positive wt.%. cinnamon the σM values decreased by 28.6 MPa and 30.9 MPa,extract effectcocoa of theand coffee extract extracts, was maintained even at higher concentrations. The coffee positively influenced the stability of the tensile strength up to 5 wt.%. The coffee extract also had a positive effect on the recorded deformations of aged samples. Unlike other materials, the decrease in εB of a sample containing 0.5 wt.%. coffee extract after 2160 h of Materials 2023, 16, 5154 13 of 22 accelerated aging was much smaller. The εB value decreased from 1473 to 1299%, i.e., by 174 units, so the material was still very elastic. This decrease was much smaller than in the other samples, where the εB values dropped by 780–1455 units, and most of the samples became very brittle. The initial increase in tensile strength observed after shorter aging times was associated with an increase in the degree of crystallinity of the materials. As demonstrated by the DSC tests presented below, the degree of crystallinity of the materials remained at a similar level despite the longer aging time; however, a decrease in tensile strength and strain at the break was observed. The decrease in mechanical properties is probably related to the degradation of the amorphous phase, which is more susceptible to UV radiation [48]. The aging coefficient characterizes the degree of material degradation as an effect of aging processes. A value of K close to zero indicates a high susceptibility to aging while values closer to one indicate high aging resistance [43]. Calculations of the K coefficient confirm that the coffee extract has the best anti-aging properties, as the obtained K values for most samples are exceedingly high. In the case of the remaining extracts, the calculated K values did not differ significantly from the values obtained for the P and R samples. 3.5. Impact Tensile Tests The results of the impact strength (atU ) tests are presented in Table 4. Table 4. Impact tensile strength results (atU ); NB—did not break. Extract Content [%wt] 0 720 1440 2160 atU [kJ/m2 ] atU [kJ/m2 ] atU [kJ/m2 ] atU [kJ/m2 ] - NB NB NB 353.2 ± 34.2 0.5 NB NB NB NB 1 NB NB NB NB 3 NB NB NB NB 5 NB NB NB NB 10 268.2 ± 28.4 282.2 ± 21.4 172.9 ± 26.1 167.2 ± 26.6 0.5 NB NB NB 272.3 ± 23.0 1 NB NB NB 249.6 ± 13.7 3 187.7 ± 26.1 290.6 ± 55.2 221.3 ± 19.1 224.4 ± 18.0 5 187.6 ± 32.6 202.9 ± 18.6 202.5 ± 23.4 192.6 ± 17.2 10 149.5 ± 27.6 192.1 ± 19.3 180.3 ± 24.9 184.6 ± 12.2 NB NB NB 231.5 ± 31.0 Cinnamon extract Cocoa extract Coffee extract P Aging Time [h] 0.5 5 173.5 ± 20.7 191.5 ± 28.8 199.5 ± 26.6 189.6 ± 13.3 10 134.6 ± 16.1 138.6 ± 13.7 163.0 ± 9.3 160.4 ± 8.1 R - NB NB NB 325.3 ± 55.5 1 NB NB 316.8 ± 83.0 237.6 ± 15.3 3 182.8 ± 28.0 223.1 ± 39.4 226.9 ± 32.7 195.3 ± 19.4 During the tensile impact test, only a few samples broke, while P and R, along with most materials containing coffee extract and samples with up to 3 wt.% of cocoa and cinnamon extracts, showed no signs of fracture. However, an observed decrease in the value of atU was noticed in samples with cocoa and cinnamon extract when the extract content exceeded 3 wt.%. The samples containing 10 wt.% mass of extracts exhibited the lowest values with a decrease of 38.2 or 48.2 kJ/m2 , respectively, compared to samples Materials 2023, 16, 5154 14 of 22 containing 3 wt.% of extracts. The material containing 10 wt.% of coffee extract had the highest atU value among the samples that broke. The atU value was about twice that of samples with the same concentration of cocoa or cinnamon extracts. The negative effect on impact tensile strength during the tensile test may be due to the active substance carrier contained in the added extracts and the difference in the solubility of contained polyphenols. According to [36], the type and amount of polyphenols present in the polymer matrix determines how much of them can dissolve. Most of the time, polyphenols lose some of their solubility as their concentration rises. The matrix develops precipitation, which has a detrimental effect on the created materials’ mechanical properties. The impact tensile strength of pure PCL did not change under the influence of accelerated aging up to 1440 h. Up to that aging time, the R samples did not break during the impact test. Only after 2160 h of accelerated aging did the material break, and the recorded value of atU was 353.2 kJ/m2 . Furthermore, the material with the reference anti-aging compound did not break until 1440 h of accelerated aging. The rupture of the R sample took place after 2160 h of aging, and the obtained atU value was 325.3 kJ/m2 , i.e., 27.9 kJ/m2 lower than the value of the aged P sample. Again, the most favorable anti-aging effects were observed with lower extract contents. Materials containing 0.5 wt.% cocoa and cinnamon extracts under the influence of accelerated aging behaved similarly to pure PCL and the material containing the reference anti-aging compound. Up to 1440 h of aging the samples did not break. The atU values obtained after 2160 h of aging were 272.3 kJ/m2 for cocoa extract and 231.5 kJ/m2 for cinnamon extract. However, they were lower than the value obtained for the aged R sample. Therefore, it can be assumed that cocoa and cinnamon extracts showed lower anti-aging properties than the reference compound. The coffee extract had better properties than the other extracts and the reference antiaging compound. Even with 0.5 wt.% of coffee extract and after 2160 h of accelerated aging, the samples did not break during the tensile impact test. The positive effect of the coffee extract was maintained also at higher contents, as even samples containing 5 wt.% did not break after the longest accelerated aging time. 3.6. Thermogravimetric Analysis Figure 5 shows the thermogravimetric (TG) curves of selected samples before and after the aging process. In the TG analysis the temperature of the loss of 5% of the mass of defining Materials 2023, 16, x FOR PEER REVIEWthe tested sample T5% was determined. The T5% value was taken as a parameter 15 of 22 the beginning of thermal degradation of the material. The T5% results of all samples are shown in Table 5. 120 ––––––– ––– ––– ––––––– ––– ––– ––––––– ––– ––– ––––––– ––– ––– 100 Weight (%) 80 P 0h P 2160h P0.5 coffee extract 0h P0.5 coffee extract 2160h P10 coffee extract 0h P10 coffee extract 2160h R 0h R 2160h 60 40 20 0 200 300 400 Temperature (°C) 500 600 Universal V4.5A TA Instruments Figure 5. TG curves of selected samples beforebefore and after aging Figure 5. TG curves of selected samples andthe after the process. aging process. Table 5. Results of thermogravimetric tests. Extract Content [%wt] 0 T5% [°C] Aging Time [h] 720 1440 T5% [°C] T5% [°C] 2160 T5% [°C] Materials 2023, 16, 5154 15 of 22 Table 5. Results of thermogravimetric tests. Aging Time [h] Extract Content [%wt] - 720 1440 2160 T5% [◦ C] T5% [◦ C] T5% [◦ C] T5% [◦ C] 371.7 342.1 324.5 312.4 0.5 357.7 349.1 357.4 340.6 1 346.6 353.1 349.1 353.7 3 339.0 330.9 342.4 342.8 5 326.1 299.7 312.9 321.8 10 261.6 305.3 303.4 267.4 0.5 362.4 366.6 334.0 336.2 1 366.9 352.4 353.2 330.7 3 357.0 357.4 333.1 344.2 5 346.3 338.9 338.6 331.4 10 313.3 317.1 297.8 307.8 Cinnamon extract Cocoa extract Coffee extract P 0 0.5 369.4 363.5 350.1 338.1 1 368.3 353.1 339.2 341.1 3 362.2 346.1 346.6 334.0 5 353.8 346.2 359.2 326.5 10 350.3 315.8 310.7 297.4 R - 362.8 350.3 330.5 323.8 When PCL is subjected to thermal degradation in an inert atmosphere, the polyester chains break down due to an ester pyrolysis reaction, which results in the release of CO2 , H2 O, and carboxylic acids. Through pyrolysis, the chains of macromolecules experience random cleavages [51]. The P sample has a T5% value of 371.7 ◦ C. Adding the reference antiaging agent reduced the T5% by 8.9 ◦ C. The tested extracts also reduced the T5% compared to the value obtained for the P and R samples. When 0.5% of coffee, cocoa, or cinnamon extracts were added to PCL, T5% decreased by 14.0, 9.4, and 2.3 ◦ C, respectively, compared to pure PCL. However, the most significant decrease was observed in samples containing 10% extracts, with T5% values being lower by 110.1 ◦ C for coffee extract, 58.4 ◦ C for cocoa extract, and 74.3 ◦ C for cinnamon extract compared to sample P. The observed decrease in thermal resistance after adding the extracts may be caused by two factors. The first is the introduction to the matrix of compounds that are characterized by lower thermal resistance than the matrix material [52]. Secondly, the plasticizing effect of the extracts was visible in the MFR studies. According to reports [53], incorporating plasticizers into biopolyesters leads to a reduction in the thermal stability of the polymer. The thermal resistance of pure PCL decreased significantly with the progress of the accelerated aging time. The T5% value of the P sample decreased approximately linearly with increasing aging time (Figure 6). The difference in T5% between the sample aged for 2160 h and the unaged sample was 59.3 ◦ C. By adding a reference anti-aging compound to the matrix, the material’s thermal resistance was enhanced. The T5% value of the R sample aged for 2160 h was lower by 39.0 ◦ C than the unaged sample, so the decrease was smaller than that observed for the P sample. P P1 coffee extract P1 cocoa extract P0.5 cinnamon extract R 380 370 360 350 T5% [°C] Materials 2023, 16, 5154 accelerated aging time. The T5% value of the P sample decreased approximately linearly with increasing aging time (Figure 6). The diﬀerence in T5% between the sample aged for 2160 h and the unaged sample was 59.3 °C. By adding a reference anti-aging compound to the matrix, the material’s thermal resistance was enhanced. The T5% value of the R sample aged for 2160 h was lower by 39.0 °C than the unaged sample, so the decrease 16 of 22was smaller than that observed for the P sample. 340 330 320 310 300 0 720 1440 2160 Aging time [h] Figure 6. The temperature of 5% weight loss (T aging time. ) values of selected samples as a function of 5% Figure 6. The temperature of 5% weight loss (T5%) values of selected samples as a function of aging time. With lowcontents contents of extracts to weight), 1% by the weight), thesubstances proposedpositively substances With low of extracts (up to(up 1% by proposed positively in thermal asaging a function of agingcausing time without influencedinfluenced the changesthe in changes thermal resistance as resistance a function of time without too causing too large of an initial decrease in thermal resistance. In the case of cocoa large of an initial decrease in thermal resistance. In the case of cocoa and cinnamon extracts,and cinnamon extracts, the behavior ofthese samples containing these extracts under the influence the behavior of samples containing extracts under the influence of accelerated aging similar toaging that ofwas sample R. As time progressed, decrease the value a ofwas accelerated similar to the thataging of sample R. As the aaging timeinprogressed, of T5% was observed but changes werebut smaller than inwere the case of sample P, the which was observed the changes smaller than in case of decrease in the value of T5%the indicates better anti-aging properties of the extracts. The differences in T between the sample P, which indicates better anti-aging properties of the extracts. 5% The diﬀerences in aged for 2160 h and the unaged samples were 26.2 ◦ C for 1 wt.% of cocoa extract Tsamples 5% between the samples aged for 2160 h and the unaged samples were 26.2 °C for 1 wt.% ◦ C for 0.5 wt% of cinnamon extract. In the case of coffee extract, the course of 31.3extract ofand cocoa and 31.3 °C for 0.5 wt% of cinnamon extract. In the case of coﬀee extract, changes in thermal resistance as a function of aging time was different. Despite the initial the course of changes in thermal resistance as a function of aging time was diﬀerent. decrease in T5% after adding 1 wt.% of coffee extract, the value of this parameter did not Despite the initial decrease in T5% after adding 1 wt.% of coﬀee extract, the value of this change significantly due to accelerated aging. The T5% after 2160 h of accelerated aging parameter did not change due to accelerated aging. Thesample. T5% after 2160ith of was practically at the samesignificantly level as the value obtained for the unaged Thus, accelerated aging was practically at the same level as the value obtained for the was the coffee extract that showed the best anti-aging properties, taking into accountunaged the sample. Thus,init limiting was thethe coﬀee extract that showed the best anti-aging properties, taking effectiveness decrease in thermal resistance. into account the eﬀectiveness in limiting the decrease in thermal resistance. 3.7. Differential Scanning Calorimetry 3.7. Diﬀerential Scanning Calorimetry The analysis of the DSC results focused on the first and second heating runs. Through the The first analysis heating, we determined howfocused the selected thermal properties the samples were of the DSC results on the first and second of heating runs. Through affected by accelerated aging. The second heating, undertaken after removing the thermal the first heating, we determined how the selected thermal properties of the samples were history, helped to determine how accelerated aging affected the structure of the tested aﬀected by accelerated aging. The second heating, undertaken after removing the thermal materials. Using the DSC test, the melting point (Tm1 and Tm2 ) and degree of crystallinity (Xc1 and Xc2 ) for each heating cycle were determined. Unfortunately, the DSC curves did not show the glass transition of PCL despite the suitable temperature range. The values of Xc were calculated based on Formula (1), assuming that the value of the enthalpy change of 100% crystalline PCL (∆Hm100% ) is 139.5 J/g [54]. ∆Hm Xc = ·100% (5) ∆Hm100% Examples of the curves of the first and second heating of selected unaged and aged samples are shown in Figure 7. Xc were calculated based on Formula (1), assuming that the value of the enthalpy change of 100% crystalline PCL (ΔHm100%) is 139.5 J/g [54]. ∆ X % ∙ 100% (5) 17 of 22 Examples of the curves of the first and second heating of selected unaged and aged samples are shown in Figure 7. (a) 1.0 ––––––– ––– ––– ––––––– ––– ––– ––––––– ––– ––– ––––––– ––– ––– Heat Flow (W/g) 0.5 0.0 P 0h P 2160h P0.5 coffee extract 0h P0.5 coffee extract 2160h P10 coffee extract 0h P10 coffee extract 2160h R 0h R 2160h -0.5 -1.0 -1.5 -2.0 30 Exo Up (b) 40 50 60 Temperature (°C) 1.0 ––––––– ––– ––– ––––––– ––– ––– ––––––– ––– ––– ––––––– ––– ––– 0.5 Heat Flow (W/g) Materials 2023, 16, 5154 ∆ 0.0 70 80 Universal V4.5A TA Instruments P 0h P 2160h P0.5 coffee extract 0h P0.5 coffee extract 2160h P10 coffee extract 0h P10 coffee extract 2160h R 0h R 2160h -0.5 -1.0 -1.5 -2.0 Exo Up 30 50 Temperature (°C) 70 Universal V4.5A TA Instruments Figure7.7.DSC DSCcurves curves unaged and aged samples containing coffee extract; (a) first (b) second Figure ofof unaged and aged samples containing coﬀee extract; (a) first heatheat (b) second heat. heat. Based the first first heating heating (Table (Table6), 6),ititcan canbebe concluded that Basedon on the the results results of the concluded that thethe accelaccelerated aging process didsignificantly not significantly the phase changes the materials. tested erated aging process did not affectaﬀect the phase changes of the of tested The melting point (Tm1 ) of sample P was 61.2 ◦ C with a degree of crystallinity (Xc1 ) of 46.7%. The addition of the reference compound or the tested extracts to the matrix did not significantly affect the thermal parameters of unaged samples. Only a slight increase in the degree of crystallinity was observed for the samples containing the tested extracts, probably caused by the appearance of additional nucleation centers (active substance carrier particles). As a result of the accelerated aging process, the Tm1 value slightly increased, which was related to the increasing degree of crystallinity Xc1 of the samples, which in practically all cases reached its maximum after 2160 h of aging. The observed increase in the degree of crystallinity with aging is a typical phenomenon accompanying the conditioning of polymeric materials for a long time at elevated temperatures, which is related to the ordering of the polymer chains. Materials 2023, 16, 5154 18 of 22 Table 6. Results of the first heating scan. Aging Time [h] 0 Extract Content [%wt] 1440 2160 Tm1 [◦ C] Xc1 [%] Tm1 [◦ C] Xc1 [%] Tm1 [◦ C] Xc1 [%] Tm1 [◦ C] Xc1 [%] - 61.2 46.7 62.8 54.2 62.5 58.5 64.0 56.8 0.5 61.2 48.2 62.9 55.2 62.6 55.7 63.9 57.2 1 61.0 49.0 61.8 53.8 62.7 55.3 64.6 55.5 3 60.5 47.8 63.8 51.3 63.1 54.7 63.3 54.1 62.5 48.3 63.1 51.4 62.1 53.9 63.5 53.7 58.2 43.3 63.1 49.7 62.9 52.4 62.8 50.9 0.5 60.3 49.0 62.5 55.5 62.9 56.1 64.4 58.6 1 61.3 49.1 62.8 53.8 64.4 57.0 64.0 57.7 3 59.5 48.2 65.1 52.2 63.8 53.8 63.4 55.8 5 59.1 48.7 62.7 53.4 63.0 52.4 63.3 54.9 10 59.6 44.4 62.7 50.8 63.5 51.2 62.7 53.0 0.5 59.2 47.7 63.6 52.8 62.9 58.0 63.9 58.5 1 59.5 47.7 63.2 53.4 64.4 56.7 64.2 58.7 3 62.6 49.3 63.4 51.9 63.8 55.5 64.2 58.1 5 58.5 46.1 62.2 51.4 63.0 52.7 63.8 57.3 10 60.5 46.6 63.2 51.3 63.5 55.7 62.7 57.5 R - 60.1 46.1 62.0 55.3 62.6 58.5 64.3 65.2 Cocoa extract 5 10 Cinnamon extract Coffee extract P 720 By analyzing the results obtained based on the second heating (Table 7), i.e., when the influence of the accelerated aging process itself was eliminated, it was found that the melting point (Tm2 ) of the unaged P sample decreased to 58.6 ◦ C with a simultaneous decrease in the degree of crystallinity (Xc2 ) to 42.8%. A similar dependence was observed for the unaged samples containing the tested anti-aging compounds, where a decrease in these two parameters was also observed. However, no major influence of the accelerated aging process on the melting point (Tm2 ) and the degree of crystallinity (Xc2 ) of the tested materials was noticed. The obtained differences between the samples aged for 2160 h and the aged samples were insignificant. This was applied to both pure PCL and materials containing the reference anti-aging compound as well as the tested extracts. Contrary to the studies presented above, in the case of DSC studies, there was no clear relationship between the contents of individual extracts and the values of the recorded parameters of aged materials. Regardless of whether the material contained 0.5 or 10 wt.% of the extract, the values of the tested samples at individual stages of aging were similar. Materials 2023, 16, 5154 19 of 22 Table 7. Results of the second heating scan. Aging Time [h] 0 Extract Content [%wt] - 1440 2160 Tm2 [◦ C] Xc2 [%] Tm2 [◦ C] Xc2 [%] Tm2 [◦ C] Xc2 [%] Tm2 [◦ C] Xc2 [%] 58.6 42.8 56.5 44.5 55.8 47.3 56.2 50.5 0.5 57.9 44.3 56.1 45.0 56.2 45.4 56.4 44.1 1 58.4 45.4 56.3 44.0 55.9 47.0 56.9 44.3 3 57.0 47.1 57.4 42.6 56.5 46.1 56.1 43.5 57.3 45.7 57.3 42.9 56.3 46.3 56.4 44.8 55.2 42.7 57.2 41.6 56.2 44.5 55.6 41.0 0.5 56.6 44.6 56.1 44.1 57.0 47.4 56.5 45.6 1 58.3 47.3 56.3 45.5 56.3 45.3 56.5 43.4 3 55.8 49.6 58.3 43.9 55.9 45.4 56.2 46.2 5 56.1 46.0 56.3 43.8 57.4 42.6 56.3 43.7 10 56.8 43.0 56.7 43.3 55.9 42.2 56.0 39.4 0.5 56.5 46.1 56.7 46.8 56.0 46.3 56.9 43.8 1 56.3 47.5 56.4 43.5 56.8 44.9 56.3 47.2 3 58.2 48.7 56.7 43.8 53.3 42.9 56.2 46.4 5 56.7 45.6 56.2 40.7 56.2 44.4 55.9 44.1 10 55.5 42.5 56.9 41.7 56.5 45.4 55.6 46.7 R - 56.7 48.2 56.1 46.0 56.3 46.4 56.4 52.6 Cocoa extract 5 10 Cinnamon extract Coffee extract P 720 4. Conclusions The purpose of the study was to investigate the potential of utilizing coffee, cocoa, and cinnamon extracts as natural anti-aging agents in a biodegradable polymer known as polycaprolactone. The effectiveness of these extracts was evaluated through FTIR analysis and by observing changes in selected properties of polycaprolactone in relation to extract concentration and aging duration. The results were compared against those of a control sample containing a synthetic anti-aging compound, BHT. The research suggests that the plant proposed extracts possess anti-aging properties. However, it is important to note that higher concentrations of the extracts can alter the properties of polycaprolactone, making it beneficial to use amounts below 1 wt.%. At this concentration, the extracts can effectively prevent the harmful effects of accelerated aging while maintaining the original properties of polycaprolactone-based materials. Additionally, using lower concentrations of the extracts can prevent the decrease in mechanical strength, thermal resistance, and increase in melt flow index during the aging process. Coffee extract in an amount up to 1 wt.% turned out to be the best anti-aging compound, in most cases giving results better than the results obtained for the reference anti-aging compound. The material with coffee extract after 2160 h of aging was characterized by an acceptable increase in the melt rate by approx. 0.4 g/10 min (0.17 g/10 min in the case of the reference anti-aging compound), a decrease in tensile strength by approx. 6.0 MPa (28.6 MPa for the reference anti-aging compound), a decrease in the strain at fracture of approximately 200 units (901 units for the reference anti-aging compound), no breakage of the samples in the tensile impact test (the sample with the reference anti-aging compound cracked), and no loss of thermal stability (32.3 ◦ C reduction in resistance with the reference anti-aging compound). Materials 2023, 16, 5154 20 of 22 Based on the results obtained, plant extracts could be utilized in an industrial setting to produce eco-friendly packaging materials as an alternative to traditional polymer products. Author Contributions: Conceptualization, K.M.; methodology, K.M., M.S., R.M., T.K. and B.J.; validation, K.M.; formal analysis, K.M. and B.J.; investigation, K.M.; data curation, K.M., M.S., R.M., P.R., T.K. and B.J.; writing—original draft preparation, K.M.; visualization, K.M.; supervision, P.R. All authors have read and agreed to the published version of the manuscript. Funding: The studies were financed by the National Science Centre, project 2015/17/D/ST8/02618. Data Availability Statement: Data are contained within the article. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Vohlidal, J. Polymer Degradation: A Short Review. Chem. Teach. Int. 2021, 3, 213–220. [CrossRef] Kutz, M. Handbook of Environmental Degradation of Materials, 3rd ed.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 1–684. [CrossRef] Mierzwa-Hersztek, M.; Gondek, K.; Kopeć, M. 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https://openalex.org/W2461498912	https://europepmc.org/articles/pmc4924100?pdf=render	English	null	(-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model	Scientific reports	2,016	cc-by	8,737	(-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model XiaoyinZhu1,2 JingweiTian2 Songmei Sun2 Qiuju Dong2 Fangxi Zhang2 & XiumeiZhang1 received: 02 July 2015 accepted: 13 June 2016 Published: 28 June 2016 Chronic cerebral hypoperfusion (CCH) is one of the most common causes of vascular dementia (VaD) and is recognised as an etiological factor in the development of Alzheimer’s disease (AD). CCH can induce severe cognitive deficits, as assessed by the water maze task, along with neuronal loss in the hippocampus. However, there are currently no effective, approved pharmacological treatments available for VaD. In the present study, we created a rat model of CCH using bilateral common carotid artery occlusion and found that (-)-SCR1693, a novel compound, prevented rats from developing memory deficits and neuronal damage in the hippocampus by rectifying cholinergic dysfunction and decreasing the accumulation of the phospho-tau protein. These results strongly suggest that (-)- SCR1693 has therapeutic potential for the treatment of CCH-induced VaD. Vascular dementia (VaD) is the most common cause of dementia after Alzheimer’s disease (AD)1. VaD is defined as a loss of cognitive function resulting from ischaemic, ischaemic-hypoxic or haemorrhagic brain tissue lesions due to cardiovascular disease and cardiovascular pathological changes2. Chronic cerebral hypoperfusion (CCH) is a major cause of VaD and can result from disorders that affect the cerebral vascular system, including hyperten- sion, diabetes, generalised atherosclerosis, and cigarette smoking3. A study focusing on the pathogenetic mech- anism of VaD has revealed that, similar to AD, cholinergic abnormalities are associated with a disturbance in cognitive function in patients with VaD4. Cholinergic neurons that project into the hippocampus play a critical role in learning and memory function, and the cholinergic terminals in the presynaptic membrane are sensitive to ischaemic insults5. These findings suggest the possibility of using cholinergic substances as therapeutic interven- tions in patients with VaD. The inhibition of brain acetylcholinesterase (AChE) can increase synaptic concentra- tions of acetylcholine, which may improve cognitive dysfunction and neuropathology in patients suffering from cerebral ischaemic dementia6. However, many clinical studies have revealed that memantine and the AChE inhib- itors donepezil, galantamine and rivastigmine only have modest beneficial effects on the cognitive symptoms of VaD and provide no concomitant global or clinical benefits in most cases7. The use of AChE inhibitors is limited because of adverse drug reactions, which include increased patient mortality7. This limitation indicates that the inhibition of cholinergic abnormalities may not completely prevent the development of VaD. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 Results ( ) SCR1 (-)-SCR1693 prevents CCH-induced learning and memory deficits. Clinical observations and experimental research have indicated that CCH could lead to learning/memory deficits in subjects who are mid- dle-aged and older18. As shown in Fig. 1A, the ischaemic rats in the model group exhibited significantly longer escape latencies than the rats in the sham group from day 1 to day 5 (day 1, p = 0.012; day 2, p = 0.011; day 3, p = 0.019; day 4, p = 0.024; day 5, p = 0.016; n = 6) in the water maze task, which was initiated 4 weeks after BCCAo. On days 2–5, there were no significant differences in escape latencies between the rats in the donepezil and (-)-SCR1693 groups vs. the sham group, except in the 0.3 mg/kg (-)-SCR1693 group on day 3 (p = 0.036). We found that on days 3–5, 3 mg/kg of (-)-SCR1693 significantly shortened the escape latencies prolonged by permanent BCCAo (day 3, p = 0.036; day 4, p = 0.006; day 5, p = 0.026) compared with the model group. On day 4, the escape latencies of the rats in the group that received 1 mg/kg of (-)-SCR1693 were significantly shorter compared with the model group (p = 0.005). Although the mean escape latency of the rats that received 1 mg/ kg of donepezil was lower on days 4 and 5, no statistically significant difference was observed compared with the model group during the trial. g p g In the probe trial, Fig. 1B shows that the number of times the rats crossed the platform area for the experimen- tal and sham groups was 4.83 ± 1.07 and 2.67 ± 1.37 (p = 0.015, n = 6), respectively. Administering 1 mg/kg and 3 mg/kg of (-)-SCR1693 significantly increased the number of times the rats crossed compared with the model group (5.33 ± 1.25 and 5 ± 0.82, p = 0.038 and p = 0.028, respectively), which was similar to the sham group. The number of times the rats crossed the platform area in the group that received 1 mg/kg of donepezil was not different from that in the model group. In Fig. 1C, representative swim paths during the probe test are shown, and they reveal shorter swimming distances and less time spent in the target quadrant for the ischaemic rats, while (-)-SCR1693 ameliorated these changes. (-)-SCR1693 alleviates neuronal damage caused by CCH. www.nature.com/scientificreports/ tau can be cleaved, which increases the propensity of tau to oligomerise and eventually form filamentous aggre- gates13. The exact function that tau oligomers and filaments serve in the cell dysfunction/death process has not yet been clearly defined. The enzyme GSK-3β is one of a group of proline-directed kinases that can phosphorylate tau. Gene-knockout studies indicate that both tau and GSK-3β bind to the same region of presenilin 1 (PS1), residues 250–298, whereas the binding domain on tau is the microtubule-binding repeat region14. The ability of PS1 to bring tau and GSK-3β into close proximity suggests that PS1 may regulate the interaction between tau and GSK-3β14. Protein kinase B, also known as Akt, is activated by phosphorylation at serine 473 (Ser473) and threonine 308 (Thr308). Activated Akt phosphorylates a wide range of substrates, resulting in the activation of anti-apoptotic (survival) factors and the inactivation of pro-apoptotic factors15. Akt down-regulates the activities of GSK-3β by phosphorylating serine residue 9 (Ser9)16. β y p p y g The novel compound (-)-SCR1693, whose name is gem-dimethyl-tacripyrine hydrochloride and whose chemical name is (-)-ethyl 5-amino-4-(2-chlorophenyl) -2,7,7-trimethyl-1,4,6,7,8,9-hexahydrobenzo[b][1,8] naphthyridine-3-carboxylate hydrochloride, shows multiple activities at the enzymatic and cellular levels, includ- ing the inhibition of both tau hyperphosphorylation and AChE activity. (-)-SCR1693 is being developed as a new drug for the treatment of dementia associated with AD, and its efficacy in AD has been demonstrated in animal models. Two major pathological pathways leading to the development of AD have been hypothesised: the amyloid cascade and vascular injury. The vascular hypothesis suggests that ischaemic changes and hypoperfusion asso- ciated with aging, as well as other vascular risk factors, disturb the blood supply and metabolism. This distur- bance leads to neuronal and/or neuroglial energy failure that not only causes injury but also accelerates amyloid over-production and reduces clearance17, eventually leading to AD pathology. This vascular hypothesis has also been suggested to contribute to VaD pathology. Thus, medications administered for AD are probably effective in the treatment of VaD. (-)-SCR1693 has been proposed to improve cognitive dysfunction and prevent neuro- degeneration in VaD patients by inhibiting AChE and increasing synaptic concentrations of acetylcholine, thus inhibiting tau hyperphosphorylation. Based on these properties, a new trial was initiated using BCCAo rats to test the effects of long-term (-)-SCR1693 administration on behaviour and on neuronal survival and tau pathology. Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 (-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model XiaoyinZhu1,2 JingweiTian2 Songmei Sun2 Qiuju Dong2 Fangxi Zhang2 & XiumeiZhang1 g y p y p p The bilateral common carotid artery occlusion (BCCAo) rat model is a commonly used model of VaD. Surgical ligation of both the common carotid arteries in rats produces a CCH condition8. Previous studies have revealed that CCH induces severe cognitive deficits, as assessed by the water maze task, along with neuronal loss in the hippocampus9. In addition to cholinergic abnormalities, CCH treatment induced learning/memory alterations, increased microtubule-associated protein tau hyperphosphorylation, and caused imbalances in the phosphorylation system by activating glycogen synthase kinase 3β (GSK-3β) and Akt10. In humans, tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth, and all of these func- tions of tau are modulated by site-specific phosphorylation11. Hyperphosphorylation of tau can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the pathogenesis of AD and other tauopathies, such as VaD12. After phosphorylation at additional sites, including Ser396/404, 1Department of Pharmacology, Shandong Univeristy School of Medicine 44#, Wenhua Xi Road, Jinan, Shandong, 250012 P.R. China. 2School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, P.R. China. Correspondence and requests for materials should be addressed to X.Z. (email: zxy_sptenic@163.com) Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 1 www.nature.com/scientificreports/ Results ( ) SCR1 Global ischaemia is known to often lead to delayed neuronal death in the CA1 region of the hippocampus. The cognitive deficits caused by ischaemia have been shown to be closely correlated with neuronal cell damage in the hippocampal CA1 region19. In addition, granule neurons of the dentate gyrus receive inputs from the entorhinal cortex through the perforant pathway and send signals to the CA3 pyramidal neurons through the mossy fibre pathway, which are critical pathways for memory processing and recording. Therefore, neuronal degeneration in these areas, together with synaptic deficits, may underlie the basis of the mild memory impairments observed in CCH mice20. Previous studies have shown neuronal damage, such as pyknosis of nuclei and vacuolation of neuropil, in pyramidal neurons of the CA1–CA4 region of the hippocampus and granule neurons of the dentate gyrus (DG) in post-ischaemic rats19.hf The neuroprotective effects of donepezil and (-)-SCR1693 were evaluated by measuring neuronal cell density in the hippocampus after 34 days of global cerebral ischaemia. Representative photomicrographs of the Nissl staining results for each group are shown in Fig. 2. In the sham group, the neurons in the DG, CA1, CA3, and CA4 regions were normal and did not show any cell damage. In ischaemic rats, the numbers of cells in these regions were markedly decreased and the neurons exhibited a shrunken morphology (solid arrows) and vacuolation (hollow arrows). These results demonstrate that ischaemia leads to obvious neuron loss, disordered arrangement, vacuolation of the cell body of neurons, pyknosis of nuclei, and coagulation necrosis in the DG, CA1, CA3, and CA4 regions of the model group. Similar changes were observed in the donepezil group, but these changes were ameliorated in the group that received 3 mg/kg of (-)-SCR1693. In 2 out of 6 rats in the 0.3 mg/kg group and 3 out of 6 rats in the 1 mg/kg group, the histological lesions in the hippocampus were attenuated, but there were no Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 2 www.nature.com/scientificreports/ Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, mean daily escape latencies during the training phase are shown. Results ( ) SCR1 When the ANOVA indicated significant treatment effects, the means were separated using Tukey’s multiple comparison test. Bars represent mean ± SEM for replicate samples (n = 6). p < 0.05, p < 0.01 vs. Sham group; #p < 0.05, ##p < 0.01 vs. Model group. Representative swim paths during the probe test showed that spatial memory deficits improved in ischaemic rats treated with (-)-SCR1693. statistically significant differences in the number of surviving neurons in the groups that received 0.3 or 1 mg/kg of (-)-SCR1693 compared with the model group. The histograms show that the number of surviving neurons in the model group was markedly decreased compared with that in the sham rats. Obvious recovery of neuron loss was observed in rats treated with 3 mg/kg of (-)-SCR1693, while the neuron loss could not be suppressed follow- ing treatment with 1 mg/kg of donepezil. (-)-SCR1693 inhibits AChE activity in the hippocampus and blood plasma. Inhibition of AChE activity could decrease the hydrolysation of Ach and increase the concentration of ACh. In Fig. 3a, we show that CCH significantly increased the activity of AChE in the rat hippocampus compared with the sham group (p = 0.002, n = 6). Hippocampal AChE activity in the donepezil group was significantly lower than in the model group (p = 0.018). All three doses of (-)-SCR1693 significantly decreased the activity of AChE compared with the model group (0.3 mg/kg, p = 0.025; 1 mg/kg, p = 0.001; 3 mg/kg, p = 0.000). g p g g p g g p g g p As shown in Fig. 3b, blood plasma AChE activity was slightly lower in the model group than in the sham group, but the difference was not statistically significant (p = 0.636). Donepezil (p = 0.001 vs. sham, p = 0.003 vs. model), 1 mg/kg of (-)-SCR1693 (p = 0.013 vs. sham, p = 0.038 vs. model), and 3 mg/kg of (-)-SCR1693 (p = 0.000 vs. sham, p = 0.001 vs. model) significantly decreased blood plasma AChE activity compared with the sham and model groups. (-)-SCR1693 (1 mg/kg) led to a similar decrease in hippocampal AChE activity and higher plasma AChE activity compared with donepezil, while 3 mg/kg of (-)-SCR1693 led to a similar change in plasma AChE activity and lower hippocampal AChE activity compared with donepezil. Results ( ) SCR1 (B) The number of times the rats crossed the place where the platform had been hidden during the training phase of the probe trial (swimming 120 s without platform) is shown. (C) Representative swim paths during the probe test are shown. Statistical analysis was performed using one-way ANOVA. When the ANOVA indicated significant treatment effects, the means were separated using Tukey’s multiple comparison test. Bars represent mean ± SEM for replicate samples (n = 6). p < 0.05, *p < 0.01 vs. Sham group; #p < 0.05, ##p < 0.01 vs. Model group. Representative swim paths during the probe test showed that spatial memory deficits improved in ischaemic rats treated with (-)-SCR1693. Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, mean daily escape latencies during the training phase are shown. (B) The number of times the rats crossed the place where the platform had been hidden during the training phase of the probe trial (swimming 120 s without platform) is shown. (C) Representative swim paths during the probe test are shown. Statistical analysis was performed using one-way ANOVA. When the ANOVA indicated significant treatment effects, the means were separated using Tukey’s multiple comparison test. Bars represent mean ± SEM for replicate samples (n = 6). p < 0.05, *p < 0.01 vs. Sham group; #p < 0.05, ##p < 0.01 vs. Model group. Representative swim paths during the probe test showed that spatial memory deficits improved in ischaemic rats treated with (-)-SCR1693. Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, mean daily escape latencies during the training phase are shown. (B) The number of times the rats crossed the place where the platform had been hidden during the training phase of the probe trial (swimming 120 s without platform) is shown. (C) Representative swim paths during the probe test are shown. Statistical analysis was performed using one-way ANOVA. Results ( ) SCR1 According to the data, (-)-SCR1693 may produce fewer side effects and may be more efficient in inhibiting AChE activity than donepezil. (-)-SCR1693 up-regulates the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. The enzyme-linked immunosorbent assay (ELISA) (Fig. 4) revealed that hippocampal BDNF levels in the model group were significantly higher than in the sham group (p = 0.049, n = 6). These results indi- cate that ischaemia leads to an increase in BDNF gene expression, which is in accordance with a previous study21. Exposure to 3 mg/kg of (-)-SCR1693 significantly elevated hippocampal BDNF expression (p = 0.013, vs. sham group; p = 0.045, vs. model group). Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (h neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the mo Long term administration of ( ) SCR1693 and donepezil attenuated chronic hypoperfusion Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Results ( ) SCR1 Model group. Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 4 www.nature.com/scientificreports/ Figure 3. The effects of (-)-SCR1693 on acetylcholinesterase (AChE) activity in the hippocampus and blood plasma (mean ± SEM, n = 6). (A) Hippocampal AChE activity. (B) Blood plasma AChE activity. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 3. The effects of (-)-SCR1693 on acetylcholinesterase (AChE) activity in the hippocampus and blood plasma (mean ± SEM, n = 6). (A) Hippocampal AChE activity. (B) Blood plasma AChE activity. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 4. The effects of (-)-SCR1693 on hippocampal brain-derived neurotrophic factor (BDNF) ( ± ) h # d l ** Figure 4. The effects of (-)-SCR1693 on hippocampal brain-derived neurotrophic factor (BDNF) expression (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 4. The effects of (-)-SCR1693 on hippocampal brain-derived neurotrophic factor (BDNF) expression (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. (-)-SCR1693 modulates the phosphorylation of AKT, GSK-3β and tau in the rat hippocamp T l th h i d l i lt d t h h l ti i d th lt ti f t t l (-)-SCR1693 modulates the phosphorylation of AKT, GSK-3β and tau in the rat hippocampus. To explore the mechanisms underlying altered tau phosphorylation, we examined the alterations of total Akt, p-Akt, total GSK-3β, and p-GSK-3β. Phosphorylation of Akt at Ser473 reflects an increased activity of Akt22. Akt phosphorylation could lead to a decreased activity of GSK-3β, thus reducing tau phosphorylation and activating the cell survival-supporting functions of the Akt pathway22. Western blotting (Fig. 5) showed that the levels of total Akt did not change dramatically, while p-Akt immunoreactivity in the hippocampus after CCH treatment decreased by 16%. (-)-SCR1693 (3 mg/kg) reversed this change. The ratio of p-GSK-3β to GSK-3β decreased by 12% following CCH and increased by approximately 10% in rats that were treated with (-)-SCR1693 com- pared with control rats. Results ( ) SCR1 Total GSK-3β and p-GSK-3β levels did not change after CCH or after treatment with (-)-SCR1693 and donepezil. (-)-SCR1693 modulates the phosphorylation of AKT, GSK-3β and tau in the rat hippocampus. To explore the mechanisms underlying altered tau phosphorylation, we examined the alterations of total Akt, p-Akt, total GSK-3β, and p-GSK-3β. Phosphorylation of Akt at Ser473 reflects an increased activity of Akt22. Akt phosphorylation could lead to a decreased activity of GSK-3β, thus reducing tau phosphorylation and activating the cell survival-supporting functions of the Akt pathway22. Western blotting (Fig. 5) showed that the levels of total Akt did not change dramatically, while p-Akt immunoreactivity in the hippocampus after CCH treatment decreased by 16%. (-)-SCR1693 (3 mg/kg) reversed this change. The ratio of p-GSK-3β to GSK-3β decreased by 12% following CCH and increased by approximately 10% in rats that were treated with (-)-SCR1693 com- pared with control rats. Total GSK-3β and p-GSK-3β levels did not change after CCH or after treatment with (-)-SCR1693 and donepezil. Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 5 www.nature.com/scientificreports/ Figure 5. The effects of (-)-SCR1693 on the ratios of p-Akt/Akt, p-GSK-3β/GSK-3β and p-tau/tau (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. The gels were run under the same experimental conditions. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 5. The effects of (-)-SCR1693 on the ratios of p-Akt/Akt, p-GSK-3β/GSK-3β and p-tau/tau (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. The gels were run under the same experimental conditions. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Assay Test Concentration (M) % Inhibition of Control Values % of Control Values Reference Compound IC50 Ref (M) nH Ref 1st 2nd Mean CDK5 /p35 3.0E-05 −5 105.2 104.1 104.6 staurosporine 2.1E-08 1.2 GSK3beta 3.0E-05 −3 106.4 98.8 102.6 staurosporine 9.9E-08 1.6 MARK1 3.0E-05 −4 100.7 107.8 104.2 staurosporine 1.5E-08 2.9 MARK2 3.0E-05 7 93.5 91.5 92.5 H-89 8.3E-06 2.9 MARK3 3.0E-05 4 95.8 96.9 96.3 staurosporine 1.7E-08 1.6 MARK4 3.0E-05 2 98.5 97.1 97.8 hymenialdisine 3.6E-08 1.4i Table 1. Enzyme and cell-based assays profile of (-)-SCR1693. Discussion h In the present study, we investigated the mechanisms underlying CCH-induced spatial learning and memory deficits, and we examined whether the novel compound (-)-SCR1693 could ameliorate these deficits in a CCH rat model. We also compared the effects of (-)-SCR1693 with the effects of donepezil, which has been shown to improve hippocampal neuronal damage and reduce learning and memory deficits in the hippocampus of a global ischaemic gerbil model23. Our findings suggest that (-)-SCR1693 exhibits beneficial effects on CCH-induced cognitive and memory deficits and that its effects on these cognitive deficits are mediated by attenuating CCH-induced dysfunction of the central cholinergic system and the tau protein in the brain. y g y p Surgical ligation of both of the common carotid arteries in rats produces a chronic, global hypoperfusion state, which is less severe than the 4-vessel occlusion (4-VO) animal model24. Impaired learning and memory have been confirmed using the Morris water maze task at ~7 days post-surgery25. In our present behavioural experiment, learning performance in the water maze task was severely impaired in the BCCAo rats, as indicated by an increase in the time required to find the hidden platform and a decrease in the number of times the rats crossed the place where the hidden platform was previously located. These results are in agreement with previous studies showing that chronic cerebral hypoperfusion results in impaired learning performance26. Our behavioural experiment shows that (-)-SCR1693 prevents learning and memory deficits and is more effective than donepezil.h p g yif p Neurons have a high demand for oxygen and a limited endogenous reserve of glycogen. Thus, adult neurons are vulnerable to ischaemic conditions27. Hippocampal neurons are particularly sensitive and undergo selective and delayed degeneration in response to global ischaemia28. The pyramidal neurons in the hippocampus are crit- ically involved in spatial learning and memory, and degeneration of these neurons results in cognitive deficits29. We found that CCH caused hippocampal neural damage, including neuronal loss, disordered arrangement, pyk- nosis of nuclei, vacuolation of the neuronal cell body, and coagulation necrosis in the model group. (-)-SCR1693 significantly attenuated the bilateral ligation-induced histological lesions in the hippocampus, while vacuola- tion and pyknosis of neurons were still observed in the donepezil group. Results ( ) SCR1 Tau can be phosphorylated at multiple sites, such as Ser396/404, which increases the propensity of tau to oli- gomerise and eventually form filamentous aggregates13. While the protein levels of total tau in all of the groups were similar, a dramatic increase in the levels of p-tau (phosphorylated at Ser396) was observed in the hip- pocampus of the BCCAo rats. (-)-SCR1693 rectified this CCH-induced change in p-tau, and this effect was dose dependent (Fig. 5). However, there were no obvious changes in the levels of p-Akt and p-GSK-3β between the ischaemic group and either the control group or the (-)-SCR1693 group. We consider the small variations in p-Akt and p-GSK-3β to be unrelated to the dramatic (-)-SCR1693-induced decrease in the levels of p-tau. The enzymatic activity of GSK-3β in vitro. To determine the effects of (-)-SCR1693 on the activity of the upstream enzymes of tau, an additional cellular functional assay was conducted by Cerep (Le Bois l’Eveque, France). The effects of the compounds on the activity of human CDK5/p35, GSK-3β, MARK1, MARK2, MARK3 and MARK4 were evaluated by measuring the phosphorylation of the corresponding substrates using human recombinant enzymes and the LANCE® detection method. The results shown in Table 1 demonstrate that (-)-SCR1693 did not affect the activities of human CDK5/p35, GSK-3β, MARK1, MARK2, MARK3 and MARK4 in vitro. The inhibitory effect of (-)-SCR1693 on tau hyperphosphorylation depends on the interaction of PS1 with tau. The ability of PS1 to bring tau and GSK-3β into close proximity suggests that PS1 may play an important role in regulating the phosphorylation of tau via GSK-3β14. In the present study, (-)-SCR1693 did not alter either the phosphorylation or enzymatic activity of GSK-3β. Thus, we speculated that the interaction between PS1 and tau may explain the effect of (-)-SCR1693 on tau. The anti-tau antibody was incubated with pro- tein extracts from the hippocampus. The immunocomplexes were then precipitated by incubation with Protein A+G Agarose and detected using the anti-PS1 antibody. As shown in Fig. 6, more PS1 was precipitated in the Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 6 www.nature.com/scientificreports/ Figure 6. Binding of PS1 to tau using co-immunoprecipitation. (A) Protein bands represent the levels of PS1 binding to tau. (B) The effects of (-)-SCR1693 on the interaction between tau and PS1 (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. p < 0.05 vs. Results ( ) SCR1 Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 6. Binding of PS1 to tau using co-immunoprecipitation. (A) Protein bands represent the levels of PS1 binding to tau. (B) The effects of (-)-SCR1693 on the interaction between tau and PS1 (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. **p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. ischaemia model group than in the control group, which indicates that CCH results in an increase in PS1 binding to tau. (-)-SCR1693 at doses of 1 mg/kg and 3 mg/kg prevented the binding of tau to PS1, but donepezil did not have this effect. Discussion h Thus, the neuroprotective effects of (-)-SCR1693 contribute to cognitive rehabilitation, and its efficacy is greater than that of donepezil.i gfi y g p Cholinergic deficits in VaD have been observed in preclinical and clinical studies30,31, and evidence of cho- linergic changes in animal models of VaD has recently been reviewed32. The VaD-induced increase in AChE activity in the hippocampal areas could lead to reductions in the efficiency of cholinergic neurotransmission, such as a decrease in ACh levels in the synaptic cleft. This effect would contribute to the progression of cognitive impairment and other types of neurological dysfunction seen in patients and rats with VaD33. Our data showed that long-term administration of (-)-SCR1693, a potent AChE inhibitor, significantly inhibited AChE activity in a dose-dependent manner. The inhibition of serum AChE activity by donepezil has been linked to its adverse side effects, which include nausea, vomiting, diarrhoea, abdominal pain, weight loss, anorexia34 and hepatotox- icity associated with serum alanine aminotransferase elevation in up to 50% of patients35. We could reasonably conclude that (-)-SCR1693 might have similar AChE-inhibiting effects in the hippocampus, but with less adverse side effects, as higher doses of (-)-SCR1693 did not cause more adverse side effects than donepezil. A previous study reported that BDNF is involved in adult hippocampal neurogenesis and memory recovery under ischaemic conditions36 and that BDNF expression may be involved in the CREB-dependent neuroprotective mechanisms of donepezil in ischaemic injuries23. Neurodegenerative disorders may indeed affect neurotrophic factor function by reducing the adaptation of neurons to disease-related alterations37. Our results indicate that CCH induces an increase in BDNF expression, which was previously considered to be induced by neurogenesis after ischae- mia38. (-)-SCR1693 also increased the levels of BDNF, which may contribute to the protection of neurons in the hippocampus. pp p To investigate the possible molecular mechanisms underlying CCH-induced cognitive impairment and the effects of (-)-SCR1693, we further investigated tau-related molecules and pathways in the CCH rat brain. Under pathological conditions in which there is an imbalance in the phosphorylation/dephosphorylation of tau, aber- rant tau phosphorylation at Ser396 can increase the propensity of tau to oligomerise and eventually form filamen- tous aggregates13. The present study found that the levels of p-tau increased significantly in the hippocampi of the BCCAo rats, while (-)-SCR1693 inhibited the CCH-induced phosphorylation of tau at Ser396. Treatment and control materials Treatment and control materials. (-)-SCR1693 (light yellow solid, purity = 98%, provided by Jiangsu Simovay Pharmaceutical Co., Ltd) was suspended in 1% (w/v) carboxymethyl cellulose sodium (SCMC) solution. Donepezil (Donepezil Hydrochloride Tablets, Eisai, China), which was used as a positive control, was pulverised and suspended in 1% (w/v) SCMC solution. Rats in the sham group were given 1% (w/v) SCMC solution. Rat surgery and treatment. Male Sprague–Dawley (SD) rats weighing between 200 and 250 g were pur- chased from Shandong Luye Pharmaceutical Co. Ltd, China. Animals were housed at 50–70% humidity, a temper- ature of 22–24 °C, and under a 12:12 h light/dark cycle. All experiments were conducted in accordance with the guidelines of the Ministry of Health of PR China and the Animal Care Committee of China Medical University. The study protocol was approved by the Experimental Animal Research Committee of Yantai University. Food and water were freely available during all phases of the experiment. Rats were acclimated to the facility for 5 days prior to surgery. The rats were anaesthetised with 3 mL/kg of 10% (w/v) chloral hydrate administered by intra- peritoneal injection. Through a midline cervical incision, the bilateral common carotid arteries were exposed and gently separated from the carotid sheath and vagus nerve. Each artery of the rats assigned to the ischaemic group was ligated with a 5–0 silk suture, while rats in the sham group underwent the same operation, including the neck incision and isolation of arteries, but without ligation. During recovery, the rats were kept in the animal quarters with free access to food and water. After the operation, the ischaemic animals were randomly divided into 6 groups (n = 12): a sham group, a model group, a 1 mg/kg/day donepezil group, and (-)-SCR1693 groups consisting of 0.3, 1, or 3 mg/kg/day. The SCMC or drugs were administered once daily for 4 weeks by gavage at a dosing volume of 1 mL/kg. Four weeks later, the spatial memory retention of the rats was tested, after which the rats were sacrificed to conduct histopathologic and biochemical examinations. During the behavioural test, the drug was administered 60 min before the trials. Morris water maze. Spatial memory performance was evaluated 4 weeks after BCCAo or sham surgery using the Morris water maze29. The Morris water maze device is composed of a circular pool placed in a room with conspicuous symbols on the pool wall. Discussion h Active GSK-3β can Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 7 www.nature.com/scientificreports/ cause hyperphosphorylation of the tau protein at Ser39611, and its upstream enzyme, activated Akt, increases Aβ protease levels. This increase inhibits GSK-3β and activates multiple survival signals to promote neuron growth and survival39,40. The present study found that CCH led to a minor decrease in p-Akt (active form) and p-GSK-3β (inactive form), while (-)-SCR1693 increased p-Akt and p-GSK-3β expression to almost normal levels. The effects of (-)-SCR1693 on the phosphorylation of Akt and GSK-3β in the rat hippocampus are clearly not the main cause of the inhibition of tau hyperphosphorylation. In addition, cellular functional assays conducted by CEREP confirmed that (-)-SCR1693 did not affect the enzymatic activity of GSK-3β. Therefore, (-)-SCR1693 inhibited the hyperphosphorylation of tau at Ser396 without affecting the enzymatic activity of GSK-3β. PS1 has been proven to bring tau and GSK-3β into close proximity to regulate the phosphorylation of tau by GSK-3β14. In our co-immunoprecipitation study, CCH resulted in more interactions between tau and PS1, which may be the reason why the levels of p-tau increased without changes in p-GSK-3β levels. (-)-SCR1693 decreased the precipitation of PS1, which suggests that (-)-SCR1693 may inhibit the interactions between tau and PS1. Therefore, the phos- phorylation of tau by GSK-3β was inhibited. Donepezil did not show similar effects on PS1 and the tau protein. However, further research is needed to investigate the precise mechanism of action of (-)-SCR1693. g p In summary, the present study shows that (-)-SCR1693 attenuates the learning/memory impairment that is due to CCH-induced neuron damage. The proposed mechanisms of the neuroprotective effects of (-)-SCR1693 include the rectification of cholinergic deficits, an increase in survival signals such as BDNF, and a decrease in the accumulation of the phospho-tau protein. Our results strongly suggest that (-)-SCR1693 has therapeutic potential for the treatment of VaD-induced cholinergic dysfunction and CCH and shows more therapeutic potential than donepezil. The pathological changes that occur after permanent bilateral ligation of the common carotid arteries are quite similar to those observed in patients suffering from multi-infarct dementia, Binswanger’s and AD41. Therefore, the present findings suggest that (-)-SCR1693 may be a promising therapeutic agent for the treatment of other diseases involving vascular dementia. However, further studies are needed to explore its precise mecha- nism of action. Materials and Methods Treatment and control materials. Treatment and control materials The pool was 150 cm in diameter, 50 cm deep, and filled to a height of 30 cm with water to cover a platform (10 cm in diameter). The platform and the interior of the device were painted black to prevent visibility of the platform, which was submerged approximately 1.5 cm below the surface of the water and remained in the middle of quadrant II. The swimming activity of each rat was monitored by a video camera linked to a computer with an image analyser. On days 1–5, the rats were placed in the water (24 ± 1 °C) with their backs to the platform and allowed to swim until they found the platform (maximum swim time 60 s). The latency of each rat climbing onto the hidden platform was recorded. If the animal failed to locate the platform in 60 s, it was placed on the platform and left there for 20 s and the result was recorded as 60 s. Each rat was placed into the water at the same point in one quadrant and then the three other quadrants. On day 6, the platform was removed from the pool and the number of times rats crossed the place where the hidden platform had been pre- viously hidden in 120 s was recorded. www.nature.com/scientificreports/ www.nature.com/scientificreports/ solution. Neuronal damage was examined and evaluated by counting the number of surviving neurons per field of view (×400) in the DG, CA1, CA3, and CA4 regions using light microscopy (Leica, DMIRB). solution. Neuronal damage was examined and evaluated by counting the number of surviving neurons per field of view (×400) in the DG, CA1, CA3, and CA4 regions using light microscopy (Leica, DMIRB). solution. Neuronal damage was examined and evaluated by counting the number of surviving neurons per field of view (×400) in the DG, CA1, CA3, and CA4 regions using light microscopy (Leica, DMIRB). Measurement of AChE activity. The remaining rats were anaesthetised with 3 mL/kg of 10% (w/v) chloral hydrate administered by intraperitoneal injection, and blood samples were obtained from the abdominal aorta. After myocardial perfusion with 0.9% (w/v) sodium chloride solution, the skull was dissected and the hippocam- pus was separated on ice. The tissue was homogenised in ice-cold saline to determine the relative biochemical index. AChE activity in blood plasma and the hippocampus was assayed using an AChE assay kit (#A024, Nanjing Jiancheng Bioengineering Institute, China) via a spectrometric method42. Measurement of BDNF. BDNF expression in the hippocampus was measured using the BDNF Emax® ImmunoAssay System (#G7610, Promega Corporation, USA) as per the manufacturer’s instructions. Western blot analysis. Protein samples were homogenised in lysis buffer (20 mM Tris, 150 mM NaCl, 1% (v/v) Triton X-100) containing 1 mM PMSF, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and leupeptin. The samples were then incubated for 30 min at 4 °C and centrifuged for 10 min at 13,000 × g. The supernatant (lysate) was collected, and 15 μg protein was loaded into each lane. Samples were separated on 8–12% SDS-polyacrylamide gels and electro-blotted onto nitrocellulose membranes (Millipore). After blocking with 5% (w/v) non-fat milk, the blots were incubated with the following primary antibodies: rabbit anti-phospho-Akt (p-Akt-Ser473, 1:400, Santa Cruz), rabbit anti-Akt (1:400, Santa Cruz), rabbit anti-phospho-GSK-3β (p-GSK-3β-Ser9, 1:1000, Cell Signaling Technology), rabbit anti-GSK-3β (1:1000, Cell Signaling Technology), mouse anti-phospho-tau (p-tau-Ser396, 1:1000, Cell Signaling Technology), mouse anti-tau (1:1000, Cell Signaling Technology), rabbit anti-presenilin 1 (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz). Conjugated goat anti-rabbit or goat anti-mouse IgG was detected with enhanced chemilumines- cence (ECL) (Pierce® ECL Western Blotting Substrate). β-actin was used as an internal reference for relative quantification. Cellular functional assays in vivo. www.nature.com/scientificreports/ The effects of (-)-SCR1693 on the activities of human CDK5/p35, GSK3β, MARK1, MARK2, MARK3 and MARK4 were evaluated by measuring the phosphorylation of the respec- tive corresponding substrates using a human recombinant enzyme and the LANCE® detection method (Le Bois l’Evêque - B.P. 1 - 86600 Celle-L’Evescault - FRANCE). Co-immunoprecipitation. Protein extracts from the rat hippocampi of each group were preincubated with 1 μg of anti-tau antibody at 4 °C overnight and then incubated with 40 μL Protein A+G Agarose at 4 °C for 3 h. The mixtures were centrifuged at 2500 rpm for 5 min, and then the supernatant was discarded. After being washed 5 times in phosphate buffered saline, the immunoprecipitates were eluted by incubation with loading buffer at 100 °C for 5 min. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis for detection of tau and PS1. Statistical analysis. Statistical analysis of the data was conducted according to the Curran-Everett and Benos methods. All data were expressed as the mean ± SEM, n = 6. The behavioural data were analysed using one-way repeated-measures ANOVA or two-way repeated-measures ANOVA followed by the Tukey test for mul- tiple comparisons among different groups. Neurochemical data were compared using one-way ANOVA followed by the Tukey test for multiple comparisons. P < 0.05 was considered statistically significant. Histopathological examination. Histopathological examination. After behavioural examination, six rats from each group were anaesthe- tised with 3 mL/kg of 10% (w/v) chloral hydrate administered by intraperitoneal injection. After myocardial per- fusion with 4% (w/v) paraformaldehyde solution, the brains of the 6 rats in each group were separated and fixed in paraformaldehyde solution overnight. 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https://openalex.org/W2245035074	https://europepmc.org/articles/pmc4734427?pdf=render	English	null	Chemiresistive/SERS dual sensor based on densely packed gold nanoparticles	Beilstein journal of nanotechnology	2,015	cc-by	3,899	Abstract Chemiresistors are a class of sensitive electrical devices capable of detecting (bio)chemicals by simply monitoring electrical resis- tance. Sensing based on surface enhanced Raman scattering (SERS) represents a radically different approach, in which molecules are optically detected according to their vibrational spectroscopic fingerprint. Despite different concepts are involved, one can find in the literature examples from both categories reporting sensors made of gold nanoparticles. The same building blocks appear because both sensor classes share a common principle: nanometric interparticle gaps are needed, for electron tunneling in chemi- resistors, and for enhancing electromagnetic fields by plasmon coupling in SERS-based sensors. By exploiting such nano-gaps in self-assembled films of gold nanoparticles, we demonstrate the proof of concept of a dual electrical/optical sensor, with both chemiresistive and SERS capabilities. The proposed device is realized by self-assembling 15 nm gold nanoparticles into few micrometers-wide strips across commercially available interdigitated electrodes. The dual-mode operation of the device is demon- strated by the detection of a biologically relevant model analyte, 4-mercaptophenyl boronic acid. Chemiresistive/SERS dual sensor based on densely packed gold nanoparticles Sanda Boca1, Cosmin Leordean1, Simion Astilean1,2 and Cosmin Farcau1 Full Research Paper Open Access Address: 1Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on Bio-Nano-Sciences, Babes-Bolyai University, 42 T. Laurian, 400271 Cluj-Napoca, Romania and 2Faculty of Physics, Babes-Bolyai University, 1 M Kogalniceanu, 400084 Cluj-Napoca, Romania Email: Cosmin Farcau - cosmin.farcau@phys.ubbcluj.ro * Corresponding author Keywords: colloidal nanoparticles; convective self-assembly; interparticle gaps; surface enhanced Raman scattering; chemiresistor Beilstein J. Nanotechnol. 2015, 6, 2498–2503. doi:10.3762/bjnano.6.259 Received: 03 September 2015 Accepted: 10 December 2015 Published: 29 December 2015 Associate Editor: N. Motta © 2015 Boca et al; licensee Beilstein-Institut. License and terms: see end of document. Introduction sensor must be able to identify and quantify the investigated analyte, ideally by the use of a low-cost chip made by simple manufacturing procedures. Noble metal nanoparticles (NPs) demonstrated repeatedly their effectiveness as transducing elements in optical sensors based on surface plasmon reso- nance, surface-enhanced fluorescence or surface-enhanced The development of optical sensors is still following an ascen- dant slope nowadays and ongoing efforts are made to get them more accurate, rapid, portable, and inexpensive. This is mostly due to the promising potential they present in application fields such as health care, food contamination, environmental safety and security, among others [1]. Today, a reliable type of optical 2498 2498 Beilstein J. Nanotechnol. 2015, 6, 2498–2503. inter-particle distance. Plasmon coupling and SERS enhance- ment are also known to strongly depend on inter-particle nanoscale gaps. Thus, a dense particle organization and the molecular capping layer (preventing metal particles from touching each other) control both SERS enhancement and the electrical resistance of the particle assembly. External molec- ular species adsorbed on the nanoparticles can modify the elec- trical response of the film, and can also be identified by their SERS spectrum (graphs and spectra are just for illustration). The advantages offered by such a dual sensor would be: an electrical readout, which is always preferred since it is the most simple and inexpensive method to implement in a final product/ device; complementary, by optics (vibrational spectroscopy, SERS), specific information can be retrieved on the identity of the analyte. Such a DEOS would thus combine unique advan- tages of both electric and optical sensors. Raman spectroscopy (SERS) [2,3]. Among these analytical techniques, SERS is particularly interesting because it can specifically identify the analyte by the unique vibrational signa- ture of chemical groups. Another class of promising sensors are chemiresistors, which are sensitive electrical devices capable of detecting (bio)chemi- cals by simply monitoring electrical resistance. They require simple DC circuitry, and are therefore ideal for developing wearable/portable devices for protection from chemical expo- sure, or for environmental monitoring. Assemblies of gold NPs were recently proposed as chemiresistor sensors, exhibiting reli- able responses to the analyzed compounds [4]. In most cases very small particles were used (3–7 nm), functionalized with specific ligands that facilitate their assembly into thin nanopar- ticle films through cross-linking reactions, and connected to (micro)electrodes [5,6]. Introduction Figure 1: Schematic representation of the concept of a dual electric/ optical sensor made of a nanoparticle assembly connected by metal electrodes on a substrate. In this work we propose a dual electrical/optical sensor (DEOS) capable of exploiting both chemiresistive and SERS-based sensing. The device consists of a self-assembled film of spher- ical gold nanoparticles that is electrically connected by interdig- itated electrodes (IDE) on a commercially available chip. We tested the electrical response of the sensor to external stimuli and validated its capability to detect the model analyte mercap- tophenyl boronic acid. In contrast with previous nanoparticle- based chemiresistors, which use small nanoparticles (3–7 nm), we used larger gold nanoparticles (15 nm) in the sensor design, which allow one to obtain useful SERS measurements at the same time. Thus, we were able to optically detect and identify the analyte by its vibrational SERS signature. To our knowl- edge this is the first demonstration of a dual electrical/optical sensing concept based on gold nanoparticles, combining unique advantages of both chemiresistor and SERS sensors. Dual sensing concept Figure 1: Schematic representation of the concept of a dual electric/ optical sensor made of a nanoparticle assembly connected by metal electrodes on a substrate. The main idea of this work stems from the fact that the same physical systems (noble metal colloids) and the same fabrica- tion tools (colloidal self-assembly) could in principle yield novel nano-enabled devices with both optical and electrical functionalities. Colloidal gold nanoparticles as building blocks A request for a good control of the NP assembling process is represented by the chemical stability of the used colloid. We observed that by increasing the concentration of the colloid, this stability decreases proportionally. Hence, to increase the stability of the concentrated collidal solution, the originally citrate-capped AuNPs were functionalized with folic acid mole- cules by electrostatic interaction between the negative surface of the particles and the positive amino groups of folic acid molecules. Folic acid is a low molecular weight vitamin com- A graphical summary describing the envisaged DEOS concept is given in Figure 1. An array of noble metal colloids assem- bled between electrodes on a solid substrate can lead to elec- trical charge flow through the nanoparticle assembly by applying a voltage on the electrodes. This system can work as a resistive sensor based on the dependence of tunneling currents on the inter-NP tunnel barrier, which can be affected by the molecular species adsorbed on the NPs, or by changes of the 2499 Beilstein J. Nanotechnol. 2015, 6, 2498–2503. spaced by 100 µm. The optical microscopy image presented in Figure 2b displays the IDE gold electrodes that are connected by gold nanoparticle strips. The zoom in AFM image (Figure 2c) shows that the gold nanoparticle strips adopted an elongated shape near the gold electrodes, in agreement with the fact that the meniscus is deformed by topographical features during CSA. Finally, the SEM image in Figure 2d indicates that the gold nanoparticles in the strips are assembled with a high density packing. This is due to the good stability of the colloid during assembly, achieved by capping NPs with FA. The current–voltage (I–V) behavior of the assembled films was linear in the measured range, indicating an ohmic behavior (Figure S3, Supporting Information File 1). pound, which has been shown to be an effective targeting vector of various cancer cell lines which over-express folate receptors [7]. It also proved to be an effective capping ligand for linking onto various polymer backbones or biological molecules. Optical absorption spectra of the colloidal suspension recorded before and after incubation with folic acid confirmed the capping of AuNPs (redshift of the dipolar plasmon band) (Figure S1, Supporting Information File 1). DLS measurements correlate with the optical absorption results: for FA-capped nanoparticles (21 nm) the obtained hydrodynamic diameter was larger than for bare colloidal nanoparticles (18 nm) (Figure S2, Supporting Information File 1). Dual-mode sensing Figure 3 presents results on the dual-sensing capabilities of the developed nanoparticle-based DEOS. The experimental setup for the resistive sensing is depicted in Figure 3a. The electri- cally connected IDE with assembled Au NPs is immersed in a methanol (MeOH) bath, by taking care to leave the two elec- trode tracks outside of the liquid. Since the Au NPs are dispersable in water, the exposure to alcohol does not damage markedly the assembled film. Assembly of gold nanoparticles on IDE In the conventional way, convective self-assembly (CSA) was used for growing thin colloidal films on planar substrates, by particle deposition from a colloidal suspension along the sub- strate–liquid–air triple contact line (called also meniscus) [9,10]. Further, the technique was refined in order to allow for the fabrication of parallel strips of nanoparticle films on various substrates [11]. One such variant was named stop&go convec- tive self-assembly (SG-CSA) [12]. If up to now SG-CSA was employed only on planar solid substrates like glass slides [13] or PET films [12], in the present work, we used as substrate commercial IDE. We chose here SG-CSA and not a simpler variant because it is neccessary for the assembly proccess. It is known that topography on a substrate strongly influences the assembly of nanoparticles, especially when the NPs are smaller than those topographical features. SG-CSA allows to restrict the meniscus movement at will, by imposing (through computer- controlled software) a periodic movement and stopping of the substrate. During the 'stop' phase the particles are accumulated at the meniscus and deposited on the substrate. During the 'go' phase, the meniscus is rapidly translated to the next stop pos- ition. Our setup also imposes a straight triple contact line, which ensures deposition of strips of desired geometry. Furthermore, the gold electrodes are positioned perpendicular to the meniscus, and parallel to the translation direction. This ensures that during the 'go' phase no undesired/uncontrolled pinning by the electrodes occurs. Figure 2: (a) Optical image of the IDE (Dropsens) electrically connected by conductor wires; (b) Optical image of a region containing Au NPs assembled as a strip between a pair of Au IDE. (c) AFM image of the region shown in b); (d) SEM image showing the dense NP assembly. Colloidal gold nanoparticles as building blocks Zeta potential measurements of the concentrated suspension show an average value of ζ = −32 mV, which prove a good electrostatic stability of the folic acid-capped AuNPs in the aqueous system [8]. Figure 2: (a) Optical image of the IDE (Dropsens) electrically connected by conductor wires; (b) Optical image of a region containing Au NPs assembled as a strip between a pair of Au IDE. (c) AFM image of the region shown in b); (d) SEM image showing the dense NP assembly. Sensor characterization An optical image of the commercial IDE chip is presented in Figure 2a. As better seen in Figure 2b it consists of 10 µm wide gold electrodes spaced by 10 µm. Their height is 150 nm. Strips made of gold nanoparticles are deposited through SG-CSA, perpendicular to the IDE with the purpose of electrically connecting them. Five gold nanoparticle strips were fabricated, Upon immersion of the sensor from air into MeOH a large and instant drop of the resistance was observed, from about 93 MΩ to about 7 MΩ for the sample discussed here. The resistance slowly increases, and stabilizes around 20 MΩ after 8–10 min. 2500 Beilstein J. Nanotechnol. 2015, 6, 2498–2503. Figure 3: (a) Scheme of the experimental setup for chemiresistive detection; (b) Chemiresistor response: electrical resistance as function of time, as the sensor is exposed to different media; (c) SERS spectra on the chemiresistor surface before and after exposure to MBA. Figure 3: (a) Scheme of the experimental setup for chemiresistive detection; (b) Chemiresistor response: electrical resistance as function the sensor is exposed to different media; (c) SERS spectra on the chemiresistor surface before and after exposure to MBA. This behavior could be interpreted as an accommodation of the NP assembly into the solvent environment; at the same time it could indicate small disturbances in the microscopic NP arrangement, induced by the solvent. Subsequently, the analyte (here MBA in methanol solution) is pipetted (one drop) into the methanol bath (the final MBA solution concentration is 10−5 M). Again, the resistance of the NP assembly suddenly drops, to 1.3 MΩ and stabilizes at 1.7 MΩ within 1–2 min. This change in resistance represents a drop of more than 90% from the value it had before adding the MBA analyte. Qualitatively, we observed the same behavior across several samples, although the resistance values and change of resistance are not exactly the same. Clearly, sample resistance depends on the precise geometry of the NP strips that connect the IDEs. For the resistance change, values between 65 and 90% were observed. The resistance drop upon adsorption of the analyte can be understood by considering that charge transport through the NP assembly occurs mainly by electron tunneling. The tunneling resistance depends exponentially on the interparticle distance l (surface to surface): RT ≈ eβl, where β is a tunneling decay constant that describes the tunneling of electrons along the organic capping molecules. Sensor characterization By changing the molecular content of the interparticle gap both l and β can be expected to suffer modifications, which translate into changes of the macroscopic resistance. A decrease of the resistance upon analyte sorption has been already observed in chemiresistors based on rigidly cross-linked nanoparticle films [6,14]. The effective permit- tivity in the environment of the nanoparticles can increase due to the adsorption of analytes, which can replace ligands or fill up inter-molecular voids on the surface of the nanoparticles. This can decrease the activation energy for electron transfer, which is in turn observed as a resistance decrease. MBA molecules can replace folic acid ligands from the surface of the gold particles. MBA is smaller than folic acid, and this observation is another argument sustaining the above discus- sion. Afterwards, the sample was removed from the liquid environ- ment, dried, and SERS spectra were measured. Figure 3c presents the SERS spectra recorded on the NP wires at the beginning of the experiment, i.e., before adsorbing the analyte, and after MBA adsorption, after the chemiresistor experiment. After MBA exposure, two intense vibrational bands are observed at 1078 and 1587 cm−1. These bands are assigned to the benzene ring, specifically being attributed to βCCC + νCS and νCC with β and ν denoting bending and stretching modes, respectively [15,16]. This is an indication for the adsorption of MBA molecules. These bands were observed also in control experiments performed on another kind of gold SERS substrate without capping ligands such as folic acid on the NPs (see Supporting Information File 1). Some less intense bands, e.g., at 1182 and 1487 cm−1, can also be observed, which were present in the spectrum measured before MBA exposure, due to folic acid molecules. Note that a flat gold film deposited by thermal evaporation did not exhibit any visible Raman bands, under the same experimental conditions (exposure to methanol, MBA solution, drying). The SERS enhancement factor, EF, for this kind of self-assembled Au nanoparticle films is on the order 106 to 107, as we previously demonstrated in similar systems. Conclusion This work demonstrated the proof-of-concept of a nanoparticle- based dual-mode (bio)chemo-sensor capable of working as both chemiresistor and SERS substrate. At the heart of the proposed DEOS lie strips of gold nanoparticles formed by convective self-assembly, without implying lithographic procedures. The NP strips are fabricated directly across interdigitated electrodes on commercially available chips, making the DEOS easy to handle, and compatible with existing electrochemistry equip- ment. The adsorption of MBA model molecular analyte on the nanoparticles caused a change of electrical resistance, proving the chemiresistor ‘half’ of the DEOS. By subsequent SERS analyses information on the identity of the species affecting the resistance was obtained through its specific vibrational Raman bands. Further development is necessary, e.g., by introducing molecular recognition elements, in order to improve the selec- tivity of the sensor. Knowledge and technology existing on chemiresistive biosensors on one side and SERS detection on the other, can be commonly exploited for developing advanced dual sensors benefiting from the advantages of the two separat- ed, up to now, research fields. For example, by integration with microfluidics technology, the simultaneous operation in both chemiresistor and SERS-based modes should readily become possible. We expect that this innovative type of dual sensor will combine and multiply the benefits offered by its two facets, electric and optic, which were previously only separately exploited for (bio)sensing. Sample characterization: Optical extinction spectra were measured with a Jasco V-670 spectrophotometer. Particle morphology was determined by transmission electron microscopy (TEM) using a JEOL JEM1010 microscope. Zeta potential and hydrodynamic diameter of the particles were determined by using a Zetasizer Nano-ZS90 (Malvern Instru- ments). SERS spectra were recorded with a confocal Raman microscope (Witec alpha300 R) using a 100× objective (NA = 0.9) and 785 nm laser. Topography was studied by inter- mittent contact mode AFM measurements using the same Witec system. Electrical measurements were performed with a Keithley electrometer; a voltage of 1 V was applied in the chemiresistor experiments. Sensor characterization It was calculated using the equation: where ISERS is the SERS intensity, IRaman is the Raman inten- sity, NSERS is the number of probed molecules in SERS, and NRaman is the number of probed molecules in Raman measure- where ISERS is the SERS intensity, IRaman is the Raman inten- sity, NSERS is the number of probed molecules in SERS, and NRaman is the number of probed molecules in Raman measure- Moreover, in our system MBA is a thiol-terminated molecule while folic acid is not, and therefore one can expect that the 2501 Beilstein J. Nanotechnol. 2015, 6, 2498–2503. changed from yellow to burgundy-red, the heat was stopped, and stirring continued until the colloid reached room tempera- ture. Folic acid was mixed with the colloidal nanoparticles and stirred overnight at RT. The capped AuNPs were purified and concentrated by centrifugation. ments [17]. Further studies about the sensitivity and selectivity of the DEOS are required too fully ascribe these on the quanti- tative level, especially as a comparative assessment of the elec- tric and the optical sensing approaches. Finally, although the measurements reported above are done sequentially, and not simultaneous, one can easily accept the fact that the progress to a simultaneous experiment is only a technical matter; it requires technologies such as PDMS molding of a fluidic chamber, and online analyte injection, which we might tackle in the near future. Convective self-assembly of gold nanoparticles on IDE: Commercial IDE were used as platforms for NP assembly. These were washed in ethanol, water and dried. An UV–ozone treatment (NovaScan PSDP-UVT) was performed to increase the hydrophilicity of the IDE surface. The convective self- assembly (CSA) deposition setup consists in a glass deposition plate placed at an angle of 30° over the horizontal IDE fixed substrate. A drop (5 µL) of gold nanoparticles was placed in the wedge between the plate and IDE. Strip-like assemblies of gold NPs are formed by computer-controlled periodic movement and stopping of the substrate. The process is performed under ambient conditions at 22–25 °C and a humidity of 32–35%. Supporting Information File 1 Supporting Information File 1 Additional experimental data. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-259-S1.pdf] Materials and reagents: Hydrogen tetrachloroaurate(III) trihy- drate (HAuCl4·3H2O), trisodium citrate (C6H5Na3O7), 4-mercaptophenylboronic acid 90% (HSC6H4B(OH)2), folic acid (C19H19N7O6) were purchased from Sigma-Aldrich. Gold interdigitated electrodes (IDE) were purchased from Dropsens. [http://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-6-259-S1.pdf] Supporting Information Supporting Information provides optical extinction and DLS characterization of the gold colloid, current–voltage characteristics of the gold nanoparticle strips, and SERS of MBA on AuFoN substrate. Acknowledgements Gold nanoparticle synthesis and stabilization: Colloidal gold nanoparticles (AuNPs) were synthesized by the aqueous reduc- tion of HAuCl4 with trisodium citrate. An amount of 100 mL of 10−3 M HAuCl4·3H2O was boiled. A solution of 38.8·10−3 M sodium citrate (10 mL) was added under stirring. After the color This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-II-RU-TE-2011-3-0134. S. Boca acknowledges COST Action Raman4Clinics. 2502 Beilstein J. Nanotechnol. 2015, 6, 2498–2503. 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https://openalex.org/W2980936999	https://e-docs.geo-leo.de/bitstream/11858/8976/1/JGRC_JGRC23671.pdf	English	null	Inferring Air‐Sea Carbon Dioxide Transfer Velocities From Sea Surface Scatterometer Measurements	Journal of geophysical research. Oceans	2,019	cc-by	10,355	Key Points: Key Points: Key Points: • Derivation of new air-sea flux parameterization for CO2 fluxes 1Centrum für Erdsystemforschung und Nachhaltigkeit, Universität Hamburg, Hamburg, Germany 1Centrum für Erdsystemforschung und Nachhaltigkeit, Universität Hamburg, Hamburg, Germany • Application of new parameterization to satellite scatterometer data Abstract C-band microwave sea surface radar backscatter observations from the FINO-2 tower in the western Baltic are analyzed with respect to their relevance for air-sea CO2 transfer velocity parameterizations. The scatterometer measurements observed from a height of 25 m above the sea surface using a multifrequency scatterometer instrument of the University of Hamburg were obtained quasi-simultaneously with eddy covariance CO2 flux measurements. Both data sets are merged here to derive a gas transfer velocity parameterization based on radar backscatter measurements. At the location of the FINO-2 tower, the resulting time-averaged gas transfer velocity amounts to 26.95 cm/hr. In combination with ΔPCO2 measurements available from the vicinity of the FINO-2 platform, a time-mean CO2 flux of 0.23 μmol·m−2·s−1 into the Baltic was estimated. Applied to monthly mean satellite-based C-band ASCAT scatterometer data, the newly derived gas transfer velocity parameterization provides estimates of seasonal and annual mean global maps of air-sea transfer velocities. The new results agree in their general pattern with previous estimates using wind speed parameterization. However, the backscatter-based transfer velocities appear smaller at higher latitudes. Globally averaged air-sea CO2 fluxes would thereby be reduced by 20%. To what extent this is a robust result, or if it depends on the fact that the training data set did not represent conditions, has to be investigated in the future. • Provision of seasonal and annual mean CO2 estimates Correspondence to: D. Stammer, detlef.stammer@uni-hamburg.de Citation: Ghobadian, M., & Stammer, D. (2019). Inferring air-sea carbon dioxide transfer velocities from sea surface scatterometer measurements. Journal of Geophysical Research: Oceans, 124, 7974–7988. https://doi.org/10.1029/ 2019JC014982 Received 30 JAN 2019 Accepted 30 SEP 2019 Accepted article online 17 OCT 2019 Published online 19 NOV 2019 Citation: Ghobadian, M., & Stammer, D. (2019). Inferring air-sea carbon dioxide transfer velocities from sea surface scatterometer measurements. Journal of Geophysical Research: Oceans, 124, 7974–7988. https://doi.org/10.1029/ 2019JC014982 ©2019. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Received 30 JAN 2019 Accepted 30 SEP 2019 Accepted article online 17 OCT 2019 Published online 19 NOV 2019 1. Introduction Air-sea gas exchange processes play an important role in climate change since they are the controlling mechanism for the uptake of anthropogenic CO2 by the ocean. Without this uptake, about 25% more anthro- pogenic CO2 concentration would exist in the atmosphere with severe consequences for global temperature rise (Pachauri et al., 2014). Quantifying the ongoing air-sea gas flux and monitoring the ocean sink of anthro- pogenic carbon, therefore, is key for better projections of global warming. But although air-sea fluxes of CO2 have been studied for many decades, considerable uncertainties remain in existing time-mean estimates (Jonsson et al., 2008; Lansø et al., 2015; Takahashi et al., 2002; Yu et al., 2014). It is conceivable that uncer- tainties are even larger for the time-varying flux component. For a better quantification of the oceanic CO2 uptake, both uncertainties have to be reduced. n general terms, the air-sea flux of CO2 can be expressed as In general terms, the air-sea flux of CO2 can be expressed as In general terms, the air-sea flux of CO2 can be expressed as F = 𝛼𝜅 ( PCO2,water −PCO2,air ) , (1) (1) where 𝜅represents the gas transfer velocity (in cm/hr), 𝛼the solubility of gas in seawater (in mol·m−3·atm−1), and (PCO2,water −PCO2,air) stands for the partial pressure difference between seawater and the air above (in μatm), henceforth referred to as ΔPCO2. According to equation (1), the CO2 flux depends linearly on 𝜅and ΔPCO2. To reduce uncertainties in the time-varying air-sea fluxes of CO2, we therefore need to improve estimates of these two parameters as a function of space and time. This holds especially for parameterizations of the transfer velocity 𝜅, which typically is described as a function of large-scale observable parameters. Among the most common gas transfer velocity parameterizations are empirical relations with wind speed at 10 m height (e.g., Ho et al., 2006; Liss & Merlivat, 1986; Nightingale et al., 2000; Wanninkhof, 1992; Wanninkhof & McGillis, 1999; Wanninkhof et al., 2009) because wind controls air-sea gas exchange through its influence on the surface waves and especially turbulence and wind speed near the ocean surface can be used as a proxy to estimate gas transfer velocity. The resulting traditional wind-based parameterization of gas transfer velocity is of the form ©2019. The Authors. Inferring Air-Sea Carbon Dioxide Transfer Velocities From Sea Surface Scatterometer Measurements M. Ghobadian1 and D. Stammer1 M. Ghobadian1 and D. Stammer1 1. Introduction This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 𝜅= a (U∕U0)b (Sc)n, (2) (2) 𝜅= a (U∕U0)b (Sc)n, 𝜅= a (U∕U0)b (Sc)n, 7974 GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 1. Experimental site in the Western Baltic Sea. Shown is the bottom iowtopo2 topography (in m). The red star mark the location of the FINO-2 tower. Figure 1. Experimental site in the Western Baltic Sea. Shown is the bottom iowtopo2 topography (in m). The red sta mark the location of the FINO-2 tower. where U is the wind speed at 10 m height above sea level, U0 = 1 m/s, a (in cm/hr) and b are coefficients determined by experimental data, Sc is the Schmidt number, and the exponent n is an empirical value which varies between one half and two thirds depending on the friction velocity and mean square slope of the surface waves (Jähne et al., 1989). However, several processes other than just wind speed mediate air-sea fluxes as well, such as surface films or wave breaking which are difficult to account for in wind speed parameterization (Broecker et al., 1978; Merlivat & Memery, 1983). Equation (2) is therefore not a sufficient descriptor of all factors or physical processes involved (Banner & Phillips, 1974; Bock et al., 1999; Melville, 1996; Weiss et al., 2007). Takahashi et al. (2002) quantified this uncertainty as variation in the global oceanic CO2 uptake of 50% or more as a consequence of the different wind exponents in the regional gas transfer velocity parameterizations that account for these difficulties. In this paper we attempt to improve estimates of the time-varying gas transfer velocity parameterizations, 𝜅. Improvements will be sought by using surface roughness as the primary controlling observable; because of the strong dependence of transfer velocity on small-scale turbulence and wave breaking, surface roughness or surface wind stress should be a better proxy for estimating surface CO2 transfer as compared to one depen- dent only on wind speed. An effective way to estimate small-scale surface roughness can be accomplished using radar backscatter measurements from the sea surface as measured by radar instruments from space. 1. Introduction It therefore appears to be an important step to test the usefulness of such globally recorded radar backscatter data for estimates of surface CO2 fluxes over the global ocean as a function of time and geographic position. First respective approaches were investigated by Glover et al. (2002) based on altimetric data sets. Building on these earlier results, here we analyze the usefulness of radar cross-section measurements as input for a new 𝜅parameterization which intrinsically takes into account various environmental forcing mechanisms. The study is based on multipolarization scatterometer observations obtained in the C-band (5.3 GHz) at incidence angles of 35◦, 45◦, and 55◦in the western Baltic. The resulting new 𝜅parameterization will then be transferred and applied to global satellite-based C-band scatterometer sets to obtain new global estimates of time-varying air-sea CO2 gas fluxes. The remaining paper is structured as follows: Section 2 describes the methodology and the experimental data from the Western Baltic Sea. Section 3 presents a new backscatter-based CO2 flux parameterization derived from scatterometer data. Its pilot application to the satellite scatterometer data to obtain new estimates of global gas CO2 fluxes will be discussed in section 4, followed by concluding remarks in section 5. GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER 7975 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 2. (a) The research platform FINO-2 on which the scatterometer antenna was mounted at a height of 25 m above the sea surface. (b) The Multi3Scat antenna. Pictures taken by Mayk Fischer. Figure 2. (a) The research platform FINO-2 on which the scatterometer antenna was mounted at a height of 25 m above the sea surface. (b) The Multi3Scat antenna. Pictures taken by Mayk Fischer. 2. Methodology Our study is based on C-band surface roughness measurements obtained on the German research platform FINO-2 using a microwave scatterometer. The research platform is located in the western Baltic, 35 km north of the Island Rügen at a water depth of 54 m (Figure 1). The experimental setup is illustrated in Figure 2, showing in its left panel the FINO-2 tower on which the scatterometer antenna was mounted pointing in a westward direction (Figure 2b). Several other sensors also present on the FINO-2 platform measured wind speed and direction, air temperature, air pressure, solar radiation, atmospheric humidity, and precipitation, at different heights from 30 to 100 m, every 10 m. Also collected near the platform were observations of water surface temperatures, wind, and wave parameters. The sea surface temperature and significant wave height (SWH) were measured by a buoy about 150 m away from the platform and provided data points every 30 min. 2.1. Scatterometer Observation of Sea Surface The radar backscatter measurements were carried out as part of the German SOPARN project during a 27-month period from 2011 to 2013 using the multifrequency and multipolarization scatterometer Table 1 Technical Specification of the Multi3Scat Instrument Type Doppler scatterometer Antenna Dual polarized parabolic antenna Microwave bands L S C X Ku Frequency (GHz) 1.0 2.4 5.3 10.0 15.0 Wavelength (cm) 30.0 12.5 5.7 3.0 2.0 Gain (dBi) 14 22 28 33 35 3 dB beamwidth (◦) 22 10 4.5 2.5 1.5 35◦ 83.3 13.8 2.7 0.79 0.33 Footprint (m2) 45◦ 132.6 21.6 4.3 1.2 0.51 55◦ 260.8 40.8 8.15 2.29 0.96 Table 1 Technical Specification of the Multi3Scat Instrument Type Doppler scatterometer Antenna Dual polarized parabolic antenna Microwave bands L S C X Ku Frequency (GHz) 1.0 2.4 5.3 10.0 15.0 Wavelength (cm) 30.0 12.5 5.7 3.0 2.0 Gain (dBi) 14 22 28 33 35 3 dB beamwidth (◦) 22 10 4.5 2.5 1.5 35◦ 83.3 13.8 2.7 0.79 0.33 Footprint (m2) 45◦ 132.6 21.6 4.3 1.2 0.51 55◦ 260.8 40.8 8.15 2.29 0.96 GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER Journal of Geophysical Research: Oceans Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 3. Time series of the data measured at the FINO-2 platform. Each point indicates a bin average representing 30 min of observations. (a) Radar backscatter at 5.3 GHz, VV polarization, and 55◦incidence angle. The black dots correspond to backscattering at wind speed above 3 m/s; the red dots are the backscattering at wind speed below 3 m/s. (b) Wind speed over the entire measurement period with same color coding as above. (c) Water temperature measured in blue and air temperature extrapolated to the temperature at 10 m height in red. (d) Significant wave height. Figure 3. Time series of the data measured at the FINO-2 platform. Each point indicates a bin average representing 30 min of observations. (a) Radar backscatter at 5.3 GHz, VV polarization, and 55◦incidence angle. The black dots Figure 3. Time series of the data measured at the FINO-2 platform. Each point indicates a bin average representing 30 min of observations. (a) Radar backscatter at 5.3 GHz, VV polarization, and 55◦incidence angle. The black dots correspond to backscattering at wind speed above 3 m/s; the red dots are the backscattering at wind speed below 3 m/s. (b) Wind speed over the entire measurement period with same color coding as above. (c) Water temperature measured in blue and air temperature extrapolated to the temperature at 10 m height in red. (d) Significant wave height. Multi3Scat of the University of Hamburg. The scatterometer instrument consists of an antenna, a microwave unit, a control unit, a direct digital synthesizer module, and a data storage unit. The antenna was mounted with a fixed westward looking orientation because this is the prevailing wind direction at the platform loca- tion. The technical overview of the Multi3Scat is summarized in Table 1 (see also Kern et al., 2009, for a helicopter-based application of the instrument). Multi3Scat of the University of Hamburg. The scatterometer instrument consists of an antenna, a microwave unit, a control unit, a direct digital synthesizer module, and a data storage unit. The antenna was mounted with a fixed westward looking orientation because this is the prevailing wind direction at the platform loca- tion. The technical overview of the Multi3Scat is summarized in Table 1 (see also Kern et al., 2009, for a helicopter-based application of the instrument). GHOBADIAN AND STAMMER 7976 Journal of Geophysical Research: Oceans Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 4. Time series of (a) hourly averaged CO2 flux from eddy covariance system (gray) and corrected flux (black), and (b) partial pressure in the air and water surface, collected at the FINO-2 platform from November 2011 until August 2012. Figure 4. Time series of (a) hourly averaged CO2 flux from eddy covariance system (gray) and corrected flux (black), and (b) partial pressure in the air and water surface, collected at the FINO-2 platform from November 2011 until August 2012. Figure 4. Time series of (a) hourly averaged CO2 flux from eddy covariance system (gray) and corrected flux (black), and (b) partial pressure in the air and water surface, collected at the FINO-2 platform from November 2011 until August 2012. becomes smoother (red dots in Figures 3a and 3b). These measurements were therefore eliminated from further analysis. Shown in the remaining panels of the figure are time series of wind speed, air temperature, sea surface temperature, and SWH. Clearly seen from the figure is a seasonal signal showing higher backscatter values during winter time conditions and lower values during the summer, in agreement with respective modula- tions of the wind stress. Stronger winds are present from October to February, and weaker wind are observed from April to June. The bulk of the measurements was obtained for moderate wind speeds between 5 to 10 m/s; only occasionally were values above 20 m/s reached (cf. the top two panels). Horizontal currents (not shown) are weak near the FINO-2 platform (about 10 cm/s), and the sea surface temperature typically varies between 10◦and 25◦(Figure 3c). Amplitudes of SWH were typically found between 0.5 to 1.5 m (Figure 3d); associated wave periods of about 7 to 8 s are relatively short except during strong storms. Journal of Geophysical Research: Oceans Measurements of radar backscatter were obtained at about 5 min intervals at five microwave bands L, S, C, X, and Ku (corresponding to 1.0, 2.3, 5.3, 10.0, and 15.0 GHz, respectively), in four polarization combinations (HH, HV, VV, and VH; the first and second letter denoting the polarization in horizontal [H] or vertical [V] directions of the transmitted and the received signal, respectively) and with three incidence angles (35◦, 45◦, and 55◦). In this study the analysis is limited to the multipolarization C-band data because this corresponds in frequency to available satellite data. Because the two cross-polarized data sets are identical, only the VH polarized signal will be analyzed. And because the platform itself has a negative influence on the wind and thus on the surface roughness pattern, we will limit the analysis here to westward wind situations, that is, to upwind situations. An example of the resulting C-band backscatter signal from the sea surface is shown in Figure 3a in the form of a time series of the radar backscatter observations received with VV polarization and an incidence angle 55◦at different sea surface and weather conditions. The shown backscatter data are in agreement with pre- vious findings (e.g., Jones & Schroeder, 1978; Keller et al., 1989; Plant et al., 1985); however, measurements become very noisy as soon as the wind speed declines below 3 m/s for which conditions the sea surface GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER 7977 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 13.8 m above the sea surface, respectively. The open-path infrared gas analyzer provided measurements of the densities of carbon dioxide and water vapor in turbulent air structures with a resolution of 10 Hz. Based on the eddy covariance technique, these data were used in conjunction with sonic anemometer air turbulence data to determine the fluxes of CO2 and H2O integrated over 30 min intervals. The eddy covari- ance measurements were performed from November 2011 until August 2012, as described by Lammert and Ament (2015). Additional water PCO2 measurements were obtained from cargo ships moving along a transect between Helsinki in the Gulf of Finland and Lübeck/Gdynia in the southwest of the Baltic Sea (Schneider et al., 2014). These PCO2measurements were obtained via equilibration of surface water with air and subsequent detection of the equilibrium CO2 concentration using infrared spectroscopy as described by Schneider et al. (2014). Combined with the eddy covariance data, the ship-based measurements provided estimates of partial pressure differences of CO2 in the air and seawater. Figure 4 shows the resulting daily averaged CO2 flux time series (top panel), together with time series of the partial pressure differences of CO2 in the air and seawater during 2011 and 2012 (bottom panel). During March, the surface water PCO2drops, presumably as a result of intensive primary production. The increase from there toward summer PCO2values can be rationalized by the temperature solubility effect on PCO2in the water. CO2 fluxes are maximum during winter time when they reach values of up to 0.15 mg·m−2·s−1 from the atmosphere into the ocean. Because these enhanced CO2 fluxes occur in the presence of weak air-sea PCO2difference (see bottom panel), they need to be associated with enhanced gas transfer velocities, 𝜅, result- ing from enhanced surface wind speeds and an associated sea surface roughness. A detailed investigation of the PCO2in the Baltic Sea can be found in Schneider et al. (2009). As can be seen from the figure, the estimates of winter CO2 fluxes are extremely noisy due to random errors in atmospheric PCO2measurements (Lammert & Ament, 2015). To reduce this noise, we exclude all flux data exceeding the range of one monthly standard deviation of the individual 30 min data points. Mean and standard deviations were computed for each month individually over the observational period, thereby accounting for the seasonal variations in the mean. 2.2. CO2 Transfer Velocity Derived From Eddy Covariance Observations Mounted on the FINO-2 platform was also an eddy covariance setup producing CO2 flux estimates quasi-simultaneous to the Multi3Scat measurements. The setup consisted of a three-component sonic anemometers (USA1) and an open-path infrared gas analyzer (LICOR 7500) for CO2 and H2O. These instru- ments were operated on a 9 m long boom pointing southward from the platform at heights of 6.8 and GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER 7978 7978 Journal of Geophysical Research: Oceans Journal of Geophysical Research: Oceans The gas analyzer counts the number of CO2 molecules in the sample air volume, it therefore determines the density of CO2 but not the mixing ratio. Moreover, the turbulent CO2 flux determined by the eddy covariance system from the CO2 density fluctuations is affected by fluctuations in air temperature and air pressure, as well as H2O density fluctuations. Eddy covariance flux measurements therefore need to be corrected for density fluctuations not related to air-sea gas exchange. For this purpose, a Webb-Pearman-Leuning density correction (Webb et al., 1980) was applied to the FINO-2 eddy covariance measurements. While usually small, in some cases, the correction can be of the same order as the turbulent flux itself; then the density correction of Webb would not be a sufficient correction for all CO2 flux values. To test this, we compare the flux velocity of CO2 to that of heat and water vapor, as suggested by Weiss et al. (2007). Upon this test, any CO2 flux data with flux velocities higher than 10 times the flux velocities of heat flux measured simultaneous to the CO2 flux where eliminated. For further details, see Lammert and Ament (2015). Based on the corrected eddy covariance flux time series also shown in Figure 4a, we can infer the underlying gas transfer velocity, 𝜅, from the eddy covariance data using equation (1), as 𝜅= FCO2 𝛼ΔPCO2 . (3) (3) To compare our resulting transfer velocity 𝜅with published methods, it needs to be converted into the normalized transfer velocity, 𝜅660, as To compare our resulting transfer velocity 𝜅with published methods, it needs to be converted into the normalized transfer velocity, 𝜅660, as 𝜅660 = 𝜅( Sc 660)−0.5. (4) (4) Here, Sc is the Schmidt number that depends on the kinematic viscosity and molecular diffusivity as pro- posed by Wanninkhof (1992) and 660 corresponds to the CO2 Schmidt number for seawater at a temperature of 20 ◦C. We calculated the Schmidt number for this normalization using simultaneous salinity and surface temper- ature measurements from the Western Baltic Sea following Wanninkhof (1992) and using the coefficients GHOBADIAN AND STAMMER Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 5. CO2 transfer velocity cm/hr normalized with Sc number versus wind speed, from direct eddy covariance measurements (red dots). The averaged values of transfer velocity over 0.5 m/s classes of wind speed are shown in black bullets. The error bars correspond to standard deviations of each window. The curves with gray patterns describe model results of Wanninkhof (1992) (star), Wanninkhof and McGillis (1999) (plus), Nightingale et al. (2000) (dot), Weiss et al. (2007) (circle), and Takahashi et al. (2009) (diamond). Figure 5. CO2 transfer velocity cm/hr normalized with Sc number versus wind speed, from direct eddy covariance measurements (red dots). The averaged values of transfer velocity over 0.5 m/s classes of wind speed are shown in black bullets. The error bars correspond to standard deviations of each window. The curves with gray patterns describe model results of Wanninkhof (1992) (star), Wanninkhof and McGillis (1999) (plus), Nightingale et al. (2000) (dot), Weiss et al. (2007) (circle), and Takahashi et al. (2009) (diamond). provided there. Figure 5 shows the resulting CO2 transfer velocity 𝜅660 for the western Baltic Sea, as a func- tion of wind speed, representing the entire 10-month period from November 2011 to August 2012. Values reach up to 120 cm/hr for wind speed of 20 m/s. We recall, however, that 80% of these transfer velocity val- ues were obtained at wind speeds below 10 m/s and that the interval between 100 and 300 cm/hr contains less than 5% of the total number of data points. Nevertheless, we can compare results to those using tradi- tional parameterizations. To this end, the data were fit to a quadratic function of wind speed, leading to a coefficient of 0.28. (5) 𝜅660 = 0.28 · (U∕U0)2 cm∕hr. (5) Here U is the wind at 10 m elevation above sea level and U0 = 1 m/s. The correlation coefficient between the fitted line and the original data points is 0.63; using any other exponent (e.g., linear or cubic) would lead to a smaller correlation. The coefficient of the gas transfer parameterization of 0.28 shows close agreement with findings from Takahashi et al. (2009) (0.26) and Wanninkhof (1992) (0.31). Overall, our fitted curve lies between those from Takahashi et al. (2009) and Wanninkhof (1992). Here U is the wind at 10 m elevation above sea level and U0 = 1 m/s. GHOBADIAN AND STAMMER 7979 Journal of Geophysical Research: Oceans Journal of Geophysical Research: Oceans The correlation coefficient between the fitted line and the original data points is 0.63; using any other exponent (e.g., linear or cubic) would lead to a smaller correlation. The coefficient of the gas transfer parameterization of 0.28 shows close agreement with findings from Takahashi et al. (2009) (0.26) and Wanninkhof (1992) (0.31). Overall, our fitted curve lies between those from Takahashi et al. (2009) and Wanninkhof (1992). Figure 6. Time series of the CO2 transfer velocity cm/hr normalized by the Sc number from direct eddy covariance measurements (black dots). The red curve describes the 12 hr moving mean. Figure 6. Time series of the CO2 transfer velocity cm/hr normalized by the Sc number from direct eddy covariance measurements (black dots). The red curve describes the 12 hr moving mean. Figure 6. Time series of the CO2 transfer velocity cm/hr normalized by the Sc number from direct eddy covariance measurements (black dots). The red curve describes the 12 hr moving mean. GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER 7980 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 7. Upwind C-band radar backscatter from Multi3Scat observations versus gas transfer velocity computed from the direct eddy covariance observations, both in the Western Baltic Sea. The data are averaged over the period from November 2011 to August 2012, providing one data point every 1 m/s wind speed. Red, green, and blue correspond VV, HH, and VH polarization radar backscatter, respectively. The error bars represent one standard deviation of the uncertainty. In comparison to traditional transfer velocity parameterizations (also included in the figure), all results appear to agree mostly for wind speeds below 10 m/s. However, differences become prominent for larger wind speeds, especially with respect to Wanninkhof and McGillis (1999) and Weiss et al. (2007). In an root-mean-square difference sense, our find- ings are closest to the gas transfer velocity of Nightingale et al. (2000) with a root-mean-square difference of 10 cm/hr and a correlation coef- ficient of 0.89, followed by the parameterization of Wanninkhof (1992) with nearly the same root-mean-square difference and correlation coef- ficient. For Weiss et al. (2007) and Wanninkhof and McGillis (1999), the root-mean-square differences reach values of about 14 and 19 cm/hr, respectively. A time series of the transfer velocity 𝜅660 reveals its strong seasonal variation likely results from changes in the wind-induced turbulence (Figure 6). Journal of Geophysical Research: Oceans Enhanced 𝜅660 values occur during winter months and sub- stantially reduced transfer velocities during summer time. In contrast, the stress at the surface potentially is enhanced during spring due to reduced atmospheric stability. Figure 7. Upwind C-band radar backscatter from Multi3Scat observations versus gas transfer velocity computed from the direct eddy covariance observations, both in the Western Baltic Sea. The data are averaged over the period from November 2011 to August 2012, providing one data point every 1 m/s wind speed. Red, green, and blue correspond VV, HH, and VH polarization radar backscatter, respectively. The error bars represent one standard deviation of the uncertainty. from the direct eddy covariance ic Sea. The data are averaged over the 2012, providing one data point every e correspond VV, HH, and VH vely. The error bars represent one The seasonality in 𝜅660 suggests that a relation like equation (5) should be derived as a function of season. In our case, such a seasonally depen- dent relation between the gas transfer velocity and wind speed varies the coefficient of equation (5) from 0.11 for winter and autumn and 0.42 for summer and spring, that is, by about a factor of 4; a fact that could have a strong impact on global estimates of CO2 fluxes. We do note that a backscatter-based parameterization would already account for such a seasonality as the backscatter is affected by all relevant processes. 3. Backscatter-Based CO2 Flux Parameterization The goal of this paper is to derive a 𝜅660 parameterization in the form of surface radar backscatter dependence. Shown in Figure 7 is an empirical relation between the upwind C-band radar backscatter mea- surements and the simultaneous eddy covariance based 𝜅660 gas transfer velocity shown in Figure 6. Figure 7 shows the relations separately for VV (red), HH (green), and VH (blue) polarizations. For each polarization, valid data pairs were binned in 1 dB intervals, and the means are plotted for each bin connected by a line with the bin-STD superimposed as error bars. Only scatterometer data that overlap in time with the eddy covariance observation from November 2011 to August 2012 are used in this analysis. The figure reveals the differences in 𝜅660 dependencies on the VV, HH, and VH radar backscatter data, which can be rationalized by the different impacts of surface processes such as Bragg scattering, wave breaking, or steep waveforms on backscatter in each polarization. Fitting a power law to each resulting mean value leads to an empirical relationship between 𝜅660 and the upwind backscatter for each microwave frequency band and polarization pq (i.e., VV, HH, and VH), respectively, in the form of 𝜅= A · 𝜎B pq. (6) (6) Table 2 Empirically Coefficients of the Scatterometer Retrieved Gas Transfer Velocity From the Experiments by the Multi3Scat at C-Band Radar Frequency and Different Polarizations and Their Related Statistics 𝜅= A · 𝜎B pp Setting A (cm/hr) B RMSE (cm/hr) r VV 6, 516 1.1 10.05 0.90 C-band HH 9, 332 0.9 9.70 0.91 VH 50, 234 0.9 9.29 0.92 Table 2 Empirically Coefficients of the Scatterometer Retrieved Gas Transfer Velocity From the Experiments by the Multi3Scat at C-Band Radar Frequency and Different Polarizations and Their Related Statistics 𝜅= A · 𝜎B pp Setting A (cm/hr) B RMSE (cm/hr) r VV 6, 516 1.1 10.05 0.90 C-band HH 9, 332 0.9 9.70 0.91 VH 50, 234 0.9 9.29 0.92 Table 2 Empirically Coefficients of the Scatterometer Retrieved Gas Transfer Velocity From the Experiments by the Multi3Scat at C-Band Radar Frequency and Different Polarizations and Their Related Statistics GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 8. Seasonal variation of the averaged gas transfer velocity in the Western Baltic Sea. The panel shows in red observational gas transfer velocity, in blue and turquoise the wind-based gas transfer velocities of Nightingale et al. (2000) and Weiss et al. (2007), respectively, and in green colors the scatterometer-based gas transfer velocity. Figure 8. Seasonal variation of the averaged gas transfer velocity in the Western Baltic Sea. The panel shows in red observational gas transfer velocity, in blue and turquoise the wind-based gas transfer velocities of Nightingale et al. (2000) and Weiss et al. (2007), respectively, and in green colors the scatterometer-based gas transfer velocity. Here 𝜎pq has logarithmic values with units of dB. The coefficient A has units of cm/hr. All respective empir- ically determined coefficients A and B of the scatterometer-based gas transfer velocities obtained from the Multi3Scat observations are shown in Table 2 for C-band at all polarizations. Also summarized are the root-mean-square differences and correlation coefficients. It can be seen from the table that HH polarization leads to better agreement as compared to VV, with smaller root-mean-square error and better correlation coefficient r. Here 𝜎pq has logarithmic values with units of dB. The coefficient A has units of cm/hr. All respective empir- ically determined coefficients A and B of the scatterometer-based gas transfer velocities obtained from the Multi3Scat observations are shown in Table 2 for C-band at all polarizations. Also summarized are the root-mean-square differences and correlation coefficients. It can be seen from the table that HH polarization leads to better agreement as compared to VV, with smaller root-mean-square error and better correlation coefficient r. With the results at hand, we can now summarize the seasonal cycle present in transfer velocity 𝜅660 at the FINO-2 location. To this end, we computed the climatological monthly means from the scatterometer-based gas transfer velocities and compared them with the monthly mean values of the transfer velocity observed through the eddy covariance method. Results are plotted in Figure 8 and confirm that the largest gas transfer velocity values exist during the winter season from November to January and the smallest in April and August. We note that, except in May, the transfer velocities computed from radar backscatter data are smaller in value than those observed. In contrast, the wind-based model of Nightingale et al. GHOBADIAN AND STAMMER (2000) (labeled N2000 in the figure) produces smaller values than observations in all cold months and larger values than obser- vations in all warm months, except March. Finally, the model of Weiss et al. (2007) (labeled W2007 in the figure) produces particularly large overestimation at high wind speed (the light blue bars). We also note that during the cold season, HH backscatter retrieved gas transfer veloc- ity is better correlated with observations, while during stable atmospheric conditions and small-scale wave breaking and surface roughness, VV backscatter correlates better with the eddy covariance observations. Figure 9. Scatter diagram of the CO2 flux from observation in the Western Baltic Sea against the computed flux using microwave backscatter in the same area. The correlation between the two data sets is r = 0.83. Only a small offset about 0.027 μmol·m−2·s−1 can be detected. Using ΔPCO2 observations shown in Figure 4 and invoking equation (1), we computed the bulk surface CO2 fluxes associated with the scatterometer-based 𝜅660 time series (Figure 9). A comparison of the resulting fluxes with those obtained directly from the eddy covariance system (also shown in the figure) reveals a tight relation between both estimates in the range above −0.5 μmol·m−2·s−1. The spread below results from a cutoff in eddy covariance observations due to instrumental drifts or biases due to thermal expansion. Despite those differences for high negative magnitudes, the correlation between the two data sets is high (r ∼0.83). Overall, only a small offset of about 0.027 μmol·m−2·s−1 can be detected across the entire range. Figure 9. Scatter diagram of the CO2 flux from observation in the Western Baltic Sea against the computed flux using microwave backscatter in the same area. The correlation between the two data sets is r = 0.83. Only a small offset about 0.027 μmol·m−2·s−1 can be detected. GHOBADIAN AND STAMMER 7982 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 10. Seasonal variation of monthly averaged CO2 flux in the Western Baltic Sea from the direct eddy covariance measurements (in dark gray) and the estimated flux using scatterometer data (in light gray). Figure 10. Seasonal variation of monthly averaged CO2 flux in the Western Baltic Sea from the direct eddy covariance measurements (in dark gray) and the estimated flux using scatterometer data (in light gray). GHOBADIAN AND STAMMER As before for the transfer velocity, we can isolate the seasonal cycle also in the surface fluxes of CO2 at the FINO-2 location (Figure 10). The figure compares the CO2 fluxes obtained from the eddy covariance system with those computed from the scatterometer observations at C-band, VV polarization. A good agreement between both estimates is obvious, especially during the cold season. However, during summer months, the scatterometer-based estimates seem to overestimate the fluxes into the ocean during April and May due to the enhanced scatterometer-based 𝜅660 during those months. During winter months, we can identify a CO2 flux at the FINO-2 location from the ocean into the atmosphere when no primary productivity takes place in the water. In contrast, between May and August, the flux reverses when the primary productivity in the Baltic is large. It remains unclear, however, whether the negative values during January present in both estimates are an artifact and, if true, what could lead to such flux reversal during this month relative to the rest of the season. 4. Global Satellite Scatterometer-Based CO2 Flux Estimates Platform-based radar backscatter measured by Multi3Scat compared to ASCAT radar backscatter from the same time (temporal difference of less than 1 hr) and location (spatial difference of less than 0.05◦), in C-band, VV polarization, and upwind direction. A systematic bias about 13.5 dB is obvious. angles with respect to the satellite track, which allow computing the antenna look direction. Combining this information with wind direction from ASCAT Level 2 products, we can extract the radar backscatter relevant for a particular azimuth angle. Azimuth angle is the horizontal angle between the wind and the antenna beam of the scatterometer. We collected upwind and downwind direction radar backscatter for gas transfer computation. Figure 11 shows the comparison of the backscatter magnitudes of both data sets at the FINO-2 location, revealing similar temporal changes. To make the backscatter-based algorithm inferred from the Multi3Scat data applicable to the ASCAT data, the algorithm was adjusted for the apparent miscalibration of the Multi3Scat data by 13.5 dB before feeding the ASCAT data into the new gas transfer parameterization equation (6) with the A and B parameters as specified in Table 2 for the C-band and VV polarization. Shown in Figure 12 is a comparison of the respective gas transfer velocity estimates, revealing only a relatively small root-mean-square difference between both of 7.3 cm/hr and correlation coefficient of 0.81. Figure 12. The retrieved transfer velocity from platform-based scatterometer data (Multi3Scat) versus ASCAT radar backscatter using our new parameterization. The RMS difference between both is 7.3 cm/hr; the correlation coefficient is r = 0.81. Encouraged by this finding, we took the next step of applying the global ASCAT radar backscatter data to our new algorithm to produce maps of the global gas transfer velocity. To this end, we resampled ASCAT Global Data Service (GDS) Level 1 𝜎0 fields on a 25 km Swath Grid. The data are available every 5 days along a swath of close to 1,000 km width. The ASCAT incidence angle ranges from 25◦to 65◦. Detailed information about the instrument, performance, calibration, processing, and generation of Levels 1 and 2 products of ASCAT are presented by EUMETSAT (2004, 2005, 2009a). The satellite track azimuth angle, beam azimuth angle, and wind direction provide an azimuth direction. There- fore, we collected upwind and downwind direction radar backscatter for gas transfer computation. 4. Global Satellite Scatterometer-Based CO2 Flux Estimates Ultimately, a scatterometer-based 𝜅660 parameterization should be applicable to satellite-based data sets to infer time-varying CO2 fluxes over the global ocean. However, to be fully applicable, our parameterization inferred at the FINO-2 location would need to be tested at various other locations of the global ocean against in situ measurements. In the absence of such required data sets, we nevertheless test if such a step toward global time-varying estimates from satellite scatterometer is possible at all. To this end, we use, in a pilot setting, global space-borne vertically polarized advanced scatterometer (ASCAT) C-Band radar backscatter data, available during 2012 from the MetOp-B satellite (see www.eumetsat.int), to provide the first global maps of radar backscatter-based CO2 flux fields. We extracted the ASCAT data with spatial differences to the FINO-2 position of less than 0.05◦and tempo- ral differences relative to our scatterometer measurements of less than 60 min. For comparison we use the C-band, VV polarization, and incidence angles of 35◦, 45◦, and 55◦from both data sources. Prior to the com- parison, the backscatter data were grouped into specific ranges of wind direction for which a FINO-2-based algorithm exists and the Multi3Scat observations were averaged over 60 min for each incidence angle. From ASCAT, only data in upwind directions were used. In order to extract those satellite data relevant to the upwind direction, we combined the satellite track azimuth angle, beam azimuth angle, and wind direction. The wind direction for this purpose is computed from the Geophysical Model Function inversion which converts the sigma naught values to wind speed and direction. The ASCAT instrument consists of two sets of three antennas. The radar beams are directed 45◦forward, orthogonally, and 45◦backward relative to the satellite's flight direction, on both sides of the satellite ground track. From the satellite track, we have the moving direction of the satellite. From the beam azimuth angle, we get the antenna side (left or right). Three values of sigma naught correspond to three antennas look GHOBADIAN AND STAMMER GHOBADIAN AND STAMMER 7983 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 11. Platform-based radar backscatter measured by Multi3Scat compared to ASCAT radar backscatter from the same time (temporal difference of less than 1 hr) and location (spatial difference of less than 0.05◦), in C-band, VV polarization, and upwind direction. A systematic bias about 13.5 dB is obvious. Figure 11. 4. Global Satellite Scatterometer-Based CO2 Flux Estimates The air-sea gas transfer velocity with respect to the azimuth direction and incidence angle was then computed for each satellite track using our new algorithm. Global maps of resulting satellite-based air-sea CO2 transfer velocity 𝜅660 estimates are shown in Figure 13 on a 4◦latitude and 5◦longitude grid, separately for each season. The highest transfer velocities are found in high latitudes of both hemispheres during respective winter and autumn conditions there, while the lowest value resides on the equator because Figure 12. The retrieved transfer velocity from platform-based scatterometer data (Multi3Scat) versus ASCAT radar backscatter using our new parameterization. The RMS difference between both is 7.3 cm/hr; the correlation coefficient is r = 0.81. ure 12. The retrieved transfer velocity from platform-based Figure 12. The retrieved transfer velocity from platform-based scatterometer data (Multi3Scat) versus ASCAT radar backscatter using our new parameterization. The RMS difference between both is 7.3 cm/hr; the correlation coefficient is r = 0.81. GHOBADIAN AND STAMMER 7984 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 13. Estimates of global seasonal air-sea CO2 transfer velocity derived from ASCAT satellite scatterometer using the new FINO-2-based algorithm. Shown are fields for (a) winter, (b) spring, (c) summer, and (d) fall. Figure 13. Estimates of global seasonal air-sea CO2 transfer velocity derived from ASCAT satellite scatterometer using the new FINO-2-based algorithm. Shown are fields for (a) winter, (b) spring, (c) summer, and (d) fall. of low wind speeds in this region. In autumn and winter, Northern Hemisphere midlatitudes show higher values than the other seasons. Similar patterns were reported previously by Wanninkhof (1992) from wind speed dependencies. of low wind speeds in this region. In autumn and winter, Northern Hemisphere midlatitudes show higher values than the other seasons. Similar patterns were reported previously by Wanninkhof (1992) from wind speed dependencies. In a final step, air-sea CO2 flux are calculated from equation (1) on the basis of the new transfer veloc- ity parameterization using ASCAT radar backscatter data and monthly averaged ΔPCO2 available from Takahashi et al. (2014). Climatological monthly mean ΔPCO2 fields compiled on a 4◦latitude and 5◦longi- tude grid were retrieved from the LDEO database (NDP-094) at http://cdiac.ornl.gov/oceans/doc.html for Figure 14. 4. Global Satellite Scatterometer-Based CO2 Flux Estimates Estimates of global seasonal air-sea CO2 fluxes using the ASCAT scatterometer-based transfer velocity estimates shown in Figure 13 together with monthly averaged ΔPCO2 from the LDEO database NDP-094 (http://cdiac.ornl.gov/oceans/doc.html) (Takahashi et al., 2014), after rearrangement on a 4◦ latitude and 5◦longitude grid for the Reference Year 2005. Shown are fields for (a) winter, (b) spring, (c) summer, and (d) fall. Figure 14. Estimates of global seasonal air-sea CO2 fluxes using the ASCAT scatterometer-based transfer velocity estimates shown in Figure 13 together with monthly averaged ΔPCO2 from the LDEO database NDP-094 (http://cdiac.ornl.gov/oceans/doc.html) (Takahashi et al., 2014), after rearrangement on a 4◦ latitude and 5◦longitude grid for the Reference Year 2005. Shown are fields for (a) winter, (b) spring, (c) summer, and (d) fall. GHOBADIAN AND STAMMER 7985 Journal of Geophysical Research: Oceans 10.1029/2019JC014982 Figure 15. (left column) Time-mean of (top) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux from this study, averaged over Year 2012. The right column shows respective difference between our results and Wanninkhof (1992). p) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux from this study, averaged over Year 2012. The right en our results and Wanninkhof (1992). Figure 15. (left column) Time-mean of (top) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux from this study, averaged over Year 2012. The right column shows respective difference between our results and Wanninkhof (1992). the Reference Year 2005. This is an updated version of Takahashi et al. (2009) representing non-El Niño years using a database of about 6.5 million PCO2 data (see Takahashi et al., 2014, for details). Figure 14 shows the resulting global seasonal scatterometer derived air-sea CO2 flux fields. Positive values indicate the direction of the flux from the ocean into atmosphere, and negative values correspond to flux into the ocean. Compared with the wind-only parameterization, the distribution of the flux from our parameteri- zation agrees well with the previous study of Wanninkhof (1992) for each season. The equatorial areas show Figure 16. (left column) Zonal averages of (top) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux, plotted as function of latitude for individual seasons. In the right column, respective annual mean values of (top) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux are compared to results from Wanninkhof (1992). The transfer velocity estimates are Schmidt number corrected. Figure 16. 5. Concluding Remarks We also thank Stefan Kern for his support and advice regarding the use of satellite data. The CO2 flux data is provided by the Institute of Meteorology, The newly derived parameterization derived in the Western Baltic was applied subsequently to global monthly mean satellite-based ASCAT scatterometer fields to estimate seasonal and annual global maps of air-sea transfer velocities and CO2 fluxes over the global ocean. We show that the new results agree in their general pattern with the previous studies using wind speed parameterization. Differences between the traditional wind-based gas transfer estimation and the new radar backscatter-gas transfer estimation were illustrated by calculating monthly averaged gas transfer velocities. In all months, the estimated gas transfer velocity using radar backscatter results in a very close value to observations with smaller root-mean-square error of 9.29 cm/hr compared to the traditional wind-based gas transfer parameterizations. A comparison reveals lower values in the backscatter-based results, especially at higher latitudes. Globally radar-based air-sea CO2 fluxes would thereby lower by 20%. University of Hamburg, notable F. Ament and A. Lammert. B. Schneider from the Institut für Ostseeforschung in Warnemünde provided the partial pressure of CO2. The meteorological data from the FINO-2 platform and buoy data are provided by Bundesamt für Seeschifffahrt und Hydrographie (BSH). Scatterometer data can be made available on request through the Integrated Climate Data Center (ICDC, http://icdc.cen.uni-hamburg.de/1. html) of the Center for Earthsystem Science and Sustainability (CEN) of the Universität Hamburg authors are expected to curate the above data for at least 5 years after publication and provide a transparent process to make the data available to anyone upon request. Contribution to the DFG funded excellence cluster CliSAP of the Universität Hamburg. Funded in part through the BMBF-supported SOPRAN project. The results presented are promising, but leave room for further improvements. Most of all, a dedicated experiment is required to extend our algorithm derived for only upwind conditions to the full spectrum of available wind directions. In that way, all satellite-based scatterometer data can be used to estimate surface CO2 fluxes, enabling an unprecedented temporal resolution in flux data. Moreover, it needs to be investigated to what extent the algorithm derived here is biased low by using a training data set that does not represent high-wind conditions. Journal of Geophysical Research: Oceans 10.1029/2019JC014982 positive and higher flux than the other areas. Midlatitudes (north and south) show negative flux. Flux val- ues are larger in high latitudes during winter and autumn as compared to the remaining seasons. However, a detailed comparison shows that, on annual averages, our estimates are smaller than that of wind-based parameterizations (see Figure 15). positive and higher flux than the other areas. Midlatitudes (north and south) show negative flux. Flux val- ues are larger in high latitudes during winter and autumn as compared to the remaining seasons. However, a detailed comparison shows that, on annual averages, our estimates are smaller than that of wind-based parameterizations (see Figure 15). Comparing results from our study with those available from Wanninkhof (1992), we find similar spatial variation but smaller values of air-sea CO2 transfer velocity and very close values for air-sea CO2 fluxes. The largest differences in the transfer velocity are noticed in the North Atlantic and Pacific Oceans which becomes clear in plots of zonally averaged gas transfer velocity and fluxes shown in Figure 16. In the equa- torial region, the seasonal distribution of both transfer velocity and flux is similar. In the North Hemisphere, summer shows lower transfer velocity, and winter and autumn shows higher transfer velocity. Seasonal changes of gas transfer velocity in the Northern Hemisphere are larger than in the Southern Hemisphere. In 60◦north, winter and autumn show strong transfer velocity near the north Pacific Currents. In the South- ern Hemisphere, high seasonal variation is more around 60◦. Large values of gas flux are noticed at high latitudes, over 60◦north and south because of the high transfer velocity in these areas. The strong air-sea CO2 flux in the middle east of Pacific Ocean in the presence of small gas transfer velocity is the effect of large partial pressure differences in this area. In the tropical area, the ocean acts as a source of CO2 because of upwelling. The North Pacific and Atlantic act as a sink of the gas flux. In the belt of 60◦ south, partial pressure differences control the gas flux, thereby producing high flux even though the transfer velocity is low. This area acts as a strong source of air-sea CO2 flux. 5. Concluding Remarks The aim of this paper was to develop an algorithm by which local scatterometer data can be used to estimate a time-varying air-sea transfer velocity 𝜅parameterization and to apply the algorithm to actual satellite data over the global ocean. The study is based on local multifrequency and multipolarization scatterometer obser- vations obtained in the Western Baltic. In combination with ΔPCO2 measurements obtained simultaneously to the scatterometer data, we were able to develop such an algorithm and to use it to estimate surface CO2 fluxes. The results agree with previous results using a wind speed parameterization. However, they reveal a seasonally varying coefficient to wind speed as would result from the effect of different environmental con- ditions on the surface roughness and air sea transfer velocity, such as wind, short waves, wave breaking, atmospheric stability, and near-surface turbulence. Acknowledgments We thank an anonymous referee for a detailed and very constructive review. We thank M. Gade, M. Fischern, K.-W. Gurgel, and T. Schlick who all were involved in the FINO-2 measurement campaign from which data are evaluated here. Their technical support during the scatterometer measurements and data processing is gratefully acknowledged. We also thank Stefan Kern for his support and advice regarding the use of satellite data. The CO2 flux data is provided by the Institute of Meteorology, University of Hamburg, notable F. Ament and A. Lammert. B. Schneider from the Institut für Ostseeforschung in Warnemünde provided the partial pressure of CO2. The meteorological data from the FINO-2 platform and buoy data are provided by Bundesamt für Seeschifffahrt und Hydrographie (BSH). Scatterometer data can be made available on request through the Integrated Climate Data Center (ICDC, http://icdc.cen.uni-hamburg.de/1. html) of the Center for Earthsystem Science and Sustainability (CEN) of the Universität Hamburg authors are expected to curate the above data for at least 5 years after publication and provide a transparent process to make the data available to anyone upon request. Contribution to the DFG funded excellence cluster CliSAP of the Universität Hamburg. Funded in part through the BMBF-supported SOPRAN project. Acknowledgments We thank an anonymous referee for a detailed and very constructive review. We thank M. Gade, M. Fischern, K.-W. Gurgel, and T. Schlick who all were involved in the FINO-2 measurement campaign from which data are evaluated here. Their technical support during the scatterometer measurements and data processing is gratefully acknowledged. Banner, M. L., & Phillips, O. (1974). On the incipient breaking of small scale waves. Journal of Fluid Mechanics, 65(04), 647–656. Bock, E. J., Hara, T., Frew, N. M., & McGillis, W. R. (1999). Relationship between air-sea gas transfer and short wind waves. Journal of Geophysical Research, 104(C11), 25,821–25,831. 4. Global Satellite Scatterometer-Based CO2 Flux Estimates (left column) Zonal averages of (top) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux, plotted as function of latitude for individual seasons. In the right column, respective annual mean values of (top) air-sea CO2 transfer velocity and (bottom) air-sea CO2 flux are compared to results from Wanninkhof (1992). The transfer velocity estimates are Schmidt number corrected. GHOBADIAN AND STAMMER 7986 Journal of Geophysical Research: Oceans Acknowledgments We thank an anonymous referee for a detailed and very constructive review. We thank M. Gade, M. Fischern, K.-W. Gurgel, and T. Schlick who all were involved in the FINO-2 measurement campaign from which data are evaluated here. Their technical support during the scatterometer measurements and data processing is gratefully acknowledged. We also thank Stefan Kern for his support and advice regarding the use of satellite data. The CO2 flux data is provided by the Institute of Meteorology, 5. Concluding Remarks All together, we conclude that the use of surface backscatter data to derive surface CO2 fluxes should receive further attention than devoted to it in the past. References Banner, M. L., & Phillips, O. (1974). On the incipient breaking of small scale waves. Journal of Fluid Mechanics, 65(04), 647–656. Bock, E. J., Hara, T., Frew, N. M., & McGillis, W. R. (1999). Relationship between air-sea gas transfer and short wind waves. Journal of Geophysical Research, 104(C11), 25,821–25,831. 7987 GHOBADIAN AND STAMMER Journal of Geophysical Research: Oceans f ghtingale, P. D., Liss, P. S., & Schlosser, P. (2000). Measurements of air-sea gas transfer during an open ocean algal bloom. 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https://openalex.org/W2156025330	https://hal.univ-lorraine.fr/hal-02496101/file/Dong2011.pdf	English	null	Control strategies of DC-bus voltage in islanded operation of microgrid	null	2,011	cc-by	2,625	To cite this version: Bo Dong, Yongdong Li, Zhixue Zheng. Control strategies of DC-bus voltage in islanded operation of microgrid. 2011 4th International Conference on Electric Utility Deregulation and Restructuring and Power Technologies (DRPT), Jul 2011, Weihai, China. pp.1671-1674, 10.1109/DRPT.2011.5994166. hal-02496101 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02496101 https://hal.univ-lorraine.fr/hal-02496101v1 Submitted on 7 Apr 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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Distributed under a Creative Commons Attribution 4.0 International License Control Strategies of DC-Bus Voltage in Islanded Operation of Microgrid - the association of the local generators and local loads in order to minimize the energy losses in the electricity transport; - the possibility of electricity and heat cogeneration in order to increase the energy generation efficiency; - the ease of using communication means in order to optimize the total electricity generation and to increase the quality and the reliability of the power system[1, 2]. - the ease of using communication means in order to optimize the total electricity generation and to increase the quality and the reliability of the power system[1, 2]. One unit of the microgrid is established in the lab as shown in Fig 1. It consists of a wind turbine (2KW), photovoltaic panels (1KW), batteries (one cell is 12V, 65AH and six cells series connected) and super-capacitor (72V, 70F). The stability of DC- and AC-bus voltage is very important. When microgrid is connected to the utility grid, the voltage of DC-bus voltage (200V) is regulated by the inverter. However when the microgrid works in islanded operation, DC-bus voltage must be regulated by renewable energy generations and storage system. And the magnitude and frequency of AC bus are controlled by droop character of parallel inverters[3-5]. Keywords-microgrid; grid-connected operation; islanded operation; storage system Control Strategies of DC-Bus Voltage in Islanded Operation of Microgrid Bo Dong Department of Electrical Engineering Tsinghua University Beijing, China dongb07@mails.tsinghua.edu.cn Zhixue Zheng Department of Electrical Engineering Tsinghua University Beijing, China zheng-zx09@mails.tsinghua.edu.cn Yongdong Li Department of Electrical Engineering Tsinghua University Beijing, China liyd@mail.tsinghua.edu.cn Yongdong Li Department of Electrical Engineering Tsinghua University Beijing, China liyd@mail.tsinghua.edu.cn Microgrid is one of the solutions. The advantages of the microgrid consist of Microgrid is one of the solutions. The advantages of the microgrid consist of Abstract—This paper focuses on the energy management system and stability of DC bus in both grid-connected and islanded operations in microgrid system. The microgrid system consists of wind turbines, photovoltaic panels, batteries and super- capacitors, also includes both AC and DC zones. Voltage of DC bus must be kept stable especially in islanded operation. In grid- connected operation voltage of DC bus is controlled by inverter. Real power from renewable energy generations and storage system can be transferred to AC zone through DC bus. In islanded operation, inverter must be controlled to keep magnitude and frequency of AC bus stable, so storage system is used to regulate voltage of DC bus. Simulation results in the paper show that voltage of DC bus can be kept steady and power can be kept balance with the strategy in microgrid system. Abstract—This paper focuses on the energy management system and stability of DC bus in both grid-connected and islanded operations in microgrid system. The microgrid system consists of wind turbines, photovoltaic panels, batteries and super- capacitors, also includes both AC and DC zones. Voltage of DC bus must be kept stable especially in islanded operation. In grid- connected operation voltage of DC bus is controlled by inverter. Real power from renewable energy generations and storage system can be transferred to AC zone through DC bus. In islanded operation, inverter must be controlled to keep magnitude and frequency of AC bus stable, so storage system is used to regulate voltage of DC bus. Simulation results in the paper show that voltage of DC bus can be kept steady and power can be kept balance with the strategy in microgrid system. A. In Grid-Connected Operation Voltage of DC bus can be controlled by inverter in grid- connected operation. Control strategy of inverter is shown in Fig. 2. Real power from photovoltaic panels, wind turbine and storage system is all delivered to DC load and AC bus through DC bus when voltage of DC bus is stable. If the power is not enough for the loads, microgrid will absorb power from utility grid. If power from microgrid is more than needed, it will transfer power to utility grid. Fig 3.Power flow in the system Fig 2.Block diagram of the controller for inverter Power difference includes high frequency and low frequency power. Super-capacitor as a fast-dynamic storage unit is used to absorb high frequency power, and battery as a long-term storage unit is controlled to absorb low frequency power for a long time. We can get power reference with the control strategy as shown in Fig. 4 where K is a gain determined by rating and state of charge of super-capacitor and batteries[8]. storage P batt P sc P batt ref I − sc ref I − Fig 4.Control strategy of storage system storage P storage P Fig 2.Block diagram of the controller for inverter I. INTRODUCTION (HEADING 1) More and more renewable energy generations such as wind turbines and photovoltaic panels are integrated into grid, which will lead grid instability because their fluctuate power. p p Fig 1.Hybrid DC and AC linked microgrid Fig 1.Hybrid DC and AC linked microgrid Fig 3.Power flow in the system m U is the modulated voltage of chopper. Fig 7.Voltage of DC bus when microgrid works from grid-connected to island Fig 7.Voltage of DC bus when microgrid works from grid-connected to island When DC loads change in islanded operation, the voltage of DC bus should be kept constant by storage system, and different K will lead different response speed. From Fig. 8 and Fig. 9, we can see when DC loads changed or power from wind turbine and photovoltaic panels was fluctuant rapidly, the super capacitors must be release or absorb more power. According to equation (4) and linear relationship between m ref I − and L ref I − , m ref U − is obtained by equation (5): ( ) m ref in L ref L U U PI I I − − = + − (5) (5) The average modulation function of converter is obtained by equation (6): m ref dc U m U − = (6) (6) Fig 7.Voltage of DC bus when microgrid works from grid-connected to island So we can get control strategy of Bi-DC/DC converter for voltage of DC bus from upper equations as shown in Fig. 6. In islanded operation storage system is used to not only keep voltage of DC bus stable but also absorb or release the power difference between renewable energy generations and loads with the control strategy[6, 7]. When DC loads change in islanded operation, the voltage of DC bus should be kept constant by storage system, and different K will lead different response speed. From Fig. 8 and Fig. 9, we can see when DC loads changed or power from wind turbine and photovoltaic panels was fluctuant rapidly, the super capacitors must be release or absorb more power. When DC loads change in islanded operation, the voltage of DC bus should be kept constant by storage system, and different K will lead different response speed. From Fig. 8 and Fig. 9, we can see when DC loads changed or power from wind turbine and photovoltaic panels was fluctuant rapidly, the super capacitors must be release or absorb more power. 1/Uout 1/Uout Fig 8.Voltage of DC bus (up) and current of load (down) when K=0.3 Fig 6. B. In Islanded Operation Inverter must supply real and reactive power to AC zone in order to keep magnitude and frequency of AC bus stable in islanded operation when there some faults happened in utility grid. So voltage of DC bus must be regulated by Bi-DC/DC converters which connect batteries and super-capacitor to DC bus. Also because power from wind turbine and photovoltaic panels is fluctuant, storage system must absorb or release the power difference between renewable energy generations and loads which can be calculated by equation (1). Fig 4.Control strategy of storage system Bi-DC/DC converter is a bidirectional buck-boost converter as shown in Fig. 5. We should control PWM of T1 and T2 to make voltage of DC bus stable and absorb the power calculated by control strategy in Fig. 4. Fig 5.Bi-DC/DC converter storage batt sc wind solar load P P P P P P = + = + − （1） where sto ra g e P is the total power of storage system including batteries and super-capacitor. b a tt P and sc P are power released from battery and super-capacitor respectively. And w in d P is generated by wind turbine. s o la r P is generated by photovoltaic panels. lo a d P is power absorbed by DC and AC loads. Power flow in the microgrid system is shown in Fig. 3. Very little power is absorbed or released by DC bus. When voltage of DC bus is stable and power absorbed by DC bus is zero in a whole period and real power from renewable energy generations and storage system is all delivered to inverter through DC bus. Fig 5.Bi-DC/DC converter Fig 5.Bi-DC/DC converter Control strategy of Bi-DC/DC converter as shown in Fig. 5 is very important to keep voltage of DC bus stable in islanded operation. The storages exchange power with others by the DC-capacitor: 2 dc dc dU d t C I = (2) storage m ref batt m ref sc m ref I I I − − − − − − = + where (8) (2) In Fig. B. In Islanded Operation 4 we can get equation (9) and (10): batt m ref storage m ref I K I − − − − = (9) (1 ) I K I batt m ref storage m ref I K I − − − − = (9) (1 ) sc m ref sto ra ge m ref I K I − − − − = − (10) batt m ref storage m ref I K I − − − − = (9) where C is the equivalent capacitor, dc U is the voltage of DC bus, dc U is the voltage of DC bus, (1 ) sc m ref sto ra ge m ref I K I − − − − = − (10) dc I is the current applied to the DC-bus capacitor. dc I is the current applied to the DC-bus capacitor. K is determined by rating and SOC of super-capacitor and batteries. Also the gain will affect voltage of DC bus in transient operation when DC loads change or power from wind turbine and photovoltaic panels is fluctuant rapidly. The super capacitors as fast-dynamic storage system must absorb or release power quickly. We can make K=0.1～0.3 in transient operation.. After transition the strategy controls K=0.7～0.9, because the batteries store more energy. According to equation (2), L ref I − is obtained by equation (3): (3): (3): ( ) m re f d c re f d c o u t I P I U U I − − = − + (3) For inductor L, equation (4) describes the relationship between voltage and current: in m L L dI d t U U = − (4) III. SIMULATION AND EXPERIMENTAL RESULTS (4) When there are some faults happened in utility grid, mircogrid will work in islanded operation. At the time, voltage of DC bus is not kept by inverter. Instead, the storage system and renewable energy generations will keep DC-bus voltage constant with control strategies. Fig. 7 shows that when t=0.2s microgrid system began to work in islanded operation, voltage of DC bus can be kept stable. where L is the equivalent inductance, where L is the equivalent inductance, in U is the voltage of battery or super capacitor, in U is the voltage of battery or super capacitor, ACKNOWLEDGMENT This paper and its related research are supported by grants from the Power Electronics Science and Education Development Program of Delta Environmental & Educational Foundation. Fig 9.Voltage of DC bus (up) and current of load (down) when K=0.7 In order to make the voltage of DC bus stable more quickly, the equivalent inductance L can be changed smaller. In Fig 10 L ref I − is set to 10A first, when t=1s L ref I − is changed to 20A. And the equivalent inductance L is set 5mH in pink line and 1mH in yellow line. m U is the modulated voltage of chopper. Block diagram of the controller for charging In equation (3) m ref I − is the sum current of renewable energy generations and storage system: m ref wind m ref sc m ref storage m ref I I I I − − − − − − − = + + (7) Fig 8.Voltage of DC bus (up) and current of load (down) when K=0.3 3 3 Fig 9.Voltage of DC bus (up) and current of load (down) when K=0.7 IV. CONCLUSION This paper introduces the strategies of different converters in the hybrid DC and AC bus linked microgrid. Also strategies for grid-connected, islanded and transition operations are described in the paper. With these strategies, the power between microsources, storage systems, loads and utility grid could be kept balance. In island operation, the microsources and storage systems are used to keep the voltage of DC bus steady and the simulation results are described. REFERENCES [1] Z. Tao, L. Peng, and B. Francois, "Power management strategies of a DC-coupled hybrid power system in a microgrid for decentralized generation," in Power Electronics and Applications, 2009. EPE '09. 13th European Conference on, 2009, pp. 1-10. [1] Z. Tao, L. Peng, and B. Francois, "Power management strategies of a DC-coupled hybrid power system in a microgrid for decentralized generation," in Power Electronics and Applications, 2009. EPE '09. 13th European Conference on, 2009, pp. 1-10. Fig 10a.Currents of inductance L Fig 10b.Transient operation Fig 10a.Currents of inductance L [2] J. Zhenhua and Y. Xunwei, "Hybrid DC- and AC-Linked Microgrids: Towards Integration of Distributed Energy Resources," in Energy 2030 Conference, 2008. IEEE, 2008, pp. 1-8. [3] K. De Brabandere, B. Bolsens, J. Van den Keybus, A. Woyte, J. Driesen, R. Belmans, and K. U. Leuven, "A voltage and frequency droop control method for parallel inverters," in Power Electronics Specialists Conference, 2004. PESC 04. 2004 IEEE 35th Annual, 2004, pp. 2501-2507 Vol.4 [4] R. H. Lasseter, “MicroGrids,” in Proc. IEEE Power Eng. Winter Meeting, New York, Jan. 2002. [5] P. Piagi and R. H. Lasseter, “Autonomous control of microgrids,” in Proc. IEEE Power Eng. Soc. Meeting, Montreal, Canada, Jun. 2006. [Online]. Available: http://certs.lbl.gov/CERTS_P_DER.html. Fig 10a.Currents of inductance L [6] H. Nikkhajoei and R. H. Lasseter, "Distributed Generation Interface to the CERTS Microgrid," Power Delivery, IEEE Transactions on, vol. 24, pp. 1598-1608, 2009. [7] G. Fang and M. R. Iravani, "A Control Strategy for a Distributed Generation Unit in Grid-Connected and Autonomous Modes of Operation," Power Delivery, IEEE Transactions on, vol. 23, pp. 850- 859, 2008. [8] Haihua Zhou, Bhattacharya, “Composite energy storage system with flexible energy management capability for micro-grid applications”, Energy Conversion Congress and Exposition, 2010, pp. 2558-2563. Fig 10b.Transient operation Fig 10b.Transient operation 4
https://openalex.org/W3000709529	https://zenodo.org/record/5519425/files/fpls-10-01651.pdf	English	null	Mediterranean Long Shelf-Life Landraces: An Untapped Genetic Resource for Tomato Improvement	Frontiers in plant science	2,020	cc-by	19,656	HYPOTHESIS AND THEORY published: 10 January 2020 doi: 10.3389/fpls.2019.01651 Mediterranean Long Shelf-Life Landraces: An Untapped Genetic Resource for Tomato Improvement Miquel À. Conesa 1, Mateu Fullana-Pericàs 1, Antonio Granell 2 and Jeroni Galmés 1 1 Department Biologia-INAGEA, Universitat de les Illes Balears, Balearic Islands, Spain, 2 Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain The Mediterranean long shelf-life (LSL) tomatoes are a group of landraces with a fruit remaining sound up to 6–12 months after harvest. Most have been selected under semi- arid Mediterranean summer conditions with poor irrigation or rain-fed and thus, are drought tolerant. Besides the convergence in the latter traits, local selection criteria have been very variable, leading to a wide variation in fruit morphology and quality traits. The different soil characteristics and agricultural management techniques across the Mediterranean denote also a wide range of plant adaptive traits to different conditions. Despite the notorious traits for fruit quality and environment adaptation, the LSL landraces have been poorly exploited in tomato breeding programs, which rely basically on wild tomato species. In this review, we describe most of the information currently available for Mediterranean LSL landraces in order to highlight the importance of this genetic resource. We focus on the origin and diversity, the main selective traits, and the determinants of the extended fruit shelf-life and the drought tolerance. Altogether, the Mediterranean LSL landraces are a very valuable heritage to be revalued, since constitutes an alternative source to improve fruit quality and shelf-life in tomato, and to breed for more resilient cultivars under the predicted climate change conditions. Edited by: Edited by: Conxita Royo, Institute of Agrifood Research and Technology (IRTA), Spain Reviewed by: Brian Farneti, Fondazione Edmund Mach, Italy Antonio Ferrante, University of Milan, Italy Correspondence: Miquel À. Conesa ma.conesa@uib.es Edited by: Conxita Royo, Institute of Agrifood Research and Technology (IRTA), Spain Reviewed by: Brian Farneti, Fondazione Edmund Mach, Italy Antonio Ferrante, University of Milan, Italy Correspondence: Miquel À. Conesa ma.conesa@uib.es Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Received: 20 July 2019 Accepted: 22 November 2019 Published: 10 January 2020 Keywords: drought tolerance, extended fruit shelf-life, fruit quality traits, gas exchange, Mediterranean landraces, tomato, yield INTRODUCTION The Mediterranean “long shelf-life” (LSL) or “long storage” tomatoes (Solanum lycopersicum L.) are a group of landraces that converge in three characteristic traits. First, the LSL fruit phenotype, allowing tomato fruits to remain sound, without symptoms of wrinkling or deterioration, for up to 6-12 months after harvest (e.g., Casals et al., 2011; Bota et al., 2014; Manzo et al., 2018; Tranchida- Lombardo et al., 2018a). In contrast with the commercial practice for most tomato cultivars, LSL fruits are harvested fully ripen on the vine, needing neither postharvest ripening nor cold temperature storage, which maximizes fruit quality. Second, landraces are drought tolerant as a result of typical open ﬁeld cultivation under semi-arid Mediterranean summer conditions, meaning that plants are irrigated only during ﬁrst stages after transplantation and rely on occasional rain-fed afterwards. This has been an unconscious selection (Zohary, 2004; Meyer et al., 2012) imposed by local conditions and the efforts of local farmers to cultivate tomato during last centuries in the Received: 20 July 2019 Accepted: 22 November 2019 Published: 10 January 2020 Diversity and Distribution of the Mediterranean LSL Landraces Noteworthy, the largest array of LSL landraces exists in the Eastern Iberian Peninsula, the Balearic Islands, southern Italy and Sicily; all these regions forming part of the Crown of Aragón from the end of 13th to early 18th centuries (Bisson, 1986). This could have been a factor promoting the expansion of particular landraces, cultural practices and tomato uses across the kingdom, and was suggested as a reason explaining the lack of country- speciﬁc population structure when comparing non-LSL traditional accessions from Spain and Italy (García-Martínez et al., 2013). In the following, we summarize the occurrence of Mediterranean LSL landraces for which information exists in literature. Those landraces have been compiled in Table 1. In this study, we review most of the available information characterizing the Mediterranean LSL landraces, to focus on the origin, distribution and diversity, the underlying causes of the extended shelf-life, drought tolerance and variation in fruit morphology and quality traits, and the potentiality of this important genetic resource for future tomato improvement. Apart of a reservoir of useful alleles, Mediterranean LSL landraces also represent a fascinating treasure of culture and heritage, stories and possibilities of ancient tomato cultivation in the Mediterranean area. The ‘de Ramellet’ tomato from the Balearic Islands is a landrace, or population of landraces (Zeven, 1998; Camacho- Villa et al., 2005) consisting of diverse inbreeding lines maintained in family orchards, small farms and cooperatives, used for self-consumption and sold in small markets, cultivated following traditional management practices, and with seed being self-stored and maintained within families over decades. This selection and seed storage system, and the lack of a single morphological type preferred by the Balearic people, resulted in an enormous heterogeneity in fruit morphology and size due to variable preferences among growers (Figure 1), as documented through a prospection for variation across the Balearic Islands (Conesa et al., 2010; Ochogavía et al., 2011; Bota et al., 2014). Results denoted that some growers maintain different landraces based on the particular fruit properties and uses they consider interesting, e.g., a ﬂat-fruit landrace for fresh consumption (rubbed on bread), and an elongated-fruit landrace for cooking and canning after pre-cooking, which is another factor boosting variation within this landrace. Balearics recognize ‘de Ramellet’ as a particularly acid tomato, unsuitable to consume in salad. It is used for cooking, but the most generalized use is to rub on bread. Diversity and Distribution of the Mediterranean LSL Landraces In this regard, consumers appreciate that the skin, which is thick and hard, remains completely free of pericarp after spreading, which is uncommon in diverse Iberian Peninsula LSL landraces, and especially in commercialized ‘de Ramellet’-like F1 hybrids. Some landraces have proven to be tolerant to pests (Ximénez- Embún et al., 2018). Citation: Conesa MÀ, Fullana-Pericàs M, Granell A and Galmés J (2020) Mediterranean Long Shelf-Life Landraces: An Untapped Genetic Resource for Tomato Improvement. Front. Plant Sci. 10:1651. doi: 10.3389/fpls.2019.01651 January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org LSL Landraces for Tomato Improvement Conesa et al. Mediterranean basin (e.g., Pernice et al., 2010; Galmés et al., 2011; Galmés et al., 2013; Mercati et al., 2015; Patanè et al., 2016; Siracusa et al., 2018). Besides the convergence in extended shelf- life and drought tolerance, the third deﬁning trait is the huge variation in fruit morphology and quality traits, plant habit and agronomic performance within and among landraces (Ochogavía et al., 2011; Bota et al., 2014). This trait responds to different selection criteria across regions, including tomato cultivation practices, soil characteristics, cultural practices, culinary uses and preferences in local populations (Zeven, 1998; Camacho-Villa et al., 2005). shelf-life of fruits has been a determining selection factor, together with plant drought tolerance. Consequently, traits characterizing most non-LSL landraces, like fruit shape, size and color, are usually very variable in the Mediterranean LSL landraces. Frontiers in Plant Science \| www.frontiersin.org ORIGIN, DIVERSITY AND DISTRIBUTION OF THE LSL LANDRACES The published names are indicated, together with some general details for common shelf-life (d. are days and m. are months) and fruit size and shape as described in the literature. TABLE 1 \| Mediterranean long shelf-life (LSL) landraces ordinated by regions of origin. The published names are indicated, together with some general details for common shelf-life (d. are days and m. are months) and fruit size and shape as described in the literature. Landrace Region Shelf-life Fruit size Fruit shape References de Ramellet Balearic Islands 6–12 m.; 49% fruits after 6 m. 47–57 g (see Tables 3 and 4) round-slightly ﬂat (but see Figure 1) 1–6 de Penjar Catalonia/Valencian Comm. 127 d. (78–139 d.) 38–86 g (25–121 g) round (but variable) 7,8 Vesuviano (Piennolo Vesuviano; Pomodorino del Piennolo del Vesuvio) Campania–Vesuvius area 50% fruits after 6 m. 20–35 g oval; apex 9–16 ecotype Fiaschella Campania–Vesuvius area 11,13,14 ecotype Lampadina Campania–Vesuvius area 11,13,14 ecotype Patanara Campania–Vesuvius area 11,13,14 ecotype Re Umberto Campania–Vesuvius area 11,13,14 Acampora Campania–Vesuvius area 16 Lucariello Campania–Vesuvius area 5–10 m.; (56% fruits after 60 d.) heart-shape; apex 17,16,18 Pomodoro di Ercolano Campania–Vesuvius area ca. 26 g 15,16 Piennolo Giallo Campania–Napoli– Visciano 16 Giallo Beneventano Campania–Benevento (very long) 17,16 Castel di Sasso Campania–Caserta 16 Seccagno 1 Campania–Avellino 16 Corbarino (Pomodorino di Corbara) Campania–Salerno 37% fruits after 60 d. 15–21 g; > 21 g round/oval-pear/elongate; variable apex 19,13,20 Nocerino Campania–Salerno– Nocera 12–21 g round/oval-pear/elongate; variable apex 19,9,16 Casarbore Campania 16 Crovarese (Corbarino type) Campania (commercial) (long) 17,21,16,18 Principe Borghese Campania (commercial) 15–18 g round/oval/elongate; apex 19,22,23,9,11, 24,13,14,15,16 Regina Apulia 22 g 9,10,25,16 ecotype di Fasano Apulia –Fasano 22 g 25 ecotype di Monopoli Apulia –Monopoli 22 g 25 ecotype di Ostuni Apulia –Ostuni 22 g 9,10,25,16 Locale di Altamura Apulia –Bari ca. 22 g 15,16 Pummidora Scimona (Pomodoro da Serbo Giallo) Apulia–Grecìa 26 Locale di Arnesano Apulia –Lecce ca. 39 g 15,16 Giallo di Pitigliano Tuscany (several months) 27 Perina a Punta della Valtiberina Tuscany (up to next spring) (link in 27) Rosso di Pitigliano (da Serbo Rosso) Tuscany 27 Tondino Liscio da Serbo Toscano (Liscio da Serbo; Pomodorino da Appendere; Tondino Maremmano) Tuscany (for winter) 27 Buttigghieddu d'appenniri Sicily–Agrigento ca. 17 g 9,10,15,16 Kachi di Sciacca Sicily–Agrigento ca. 31 g 15,16 Linosa Sicily–Agrigento 9,10,16 Mezzocachi di Montallegro Sicily–Agrigento ca. 37 g 15,16 Piriddu (Piruddu) Sicily–Agrigento– Siculana 9,10,16 Pizzottello di Montallegro Sicily–Agrigento 18–21 g (ca. ORIGIN, DIVERSITY AND DISTRIBUTION OF THE LSL LANDRACES After its introduction in Europe by the 16th century, the tomato suffered a rapid diversiﬁcation (Sims, 1980; Ruiz et al., 2005; Rodriguez et al., 2011). Tomato was initially used as ornamental, what probably promoted increased variation in traits like fruit colour and shape. This is evidenced in pictograms from the second half of the 16th century, frequently representing chimeric plants mirroring the existing diversity (e.g., Fuchs, ca. 1550; Matthiolus, 1586; see Daunay et al., 2007, and Peralta et al., 2008). Diversiﬁcation was particularly important in the Mediterranean basin, which is considered a secondary centre of diversity of the species (Esquinas-Alcázar and Nuez, 1995; Cebolla-Cornejo et al., 2007; Mazzucato et al., 2008; Terzopoulos and Bebeli, 2010; García-Martínez et al., 2013). Despite the exact introduction of tomato in the Mediterranean basin is unknown, in the Balearic Islands several paintings dated ca. 1630–1650 show leaves and fruits similar to the ‘de Ramellet’ landrace (Sa Nostra, 1994), denoting that selection for this typical landrace could extend back for four centuries in this region. Genetic studies in tomato suggest notoriously different selection related to geographic differences and regional preferences (Sim et al., 2011; Sim et al., 2012; Blanca et al., 2015). Accordingly, selection for fruit colour, size and shape varied depending on the region which, together with low levels of out-crossing typical in tomato crop, led to a large number of family- or local-inherited varieties (Watson, 1996; Zeven, 1998), and the maintenance of very local landraces associated to regional uses and culture in the Mediterranean basin. The ‘de Penjar’ tomato from Catalonia and Valencia has similar fruit morphology and cultivation practices than ‘de Ramellet’ (Casals et al., 2011). The close vicinity and historical and cultural links with the Balearic Islands presupposed a close relationship between both landraces, although morphological and physiological variation in ‘de Ramellet’ seems to be wider (Bota et al., 2014; Fullana-Pericàs et al., 2017; Fullana-Pericàs et al., 2019) and genetic variation denotes particular identity for the Balearic accessions (A. Granell, unpubl.). Yield and fruit size have been in general the main drivers for tomato selection (Bai and Lindhout, 2007; Rodriguez et al., 2011; Lin et al., 2014). Instead, in the Mediterranean basin, the extended January 2020 \| Volume 10 \| Article 1651 2 LSL Landraces for Tomato Improvement Conesa et al. TABLE 1 \| Mediterranean long shelf-life (LSL) landraces ordinated by regions of origin. ORIGIN, DIVERSITY AND DISTRIBUTION OF THE LSL LANDRACES 27 g) round; apex 22,23,24,30,15,16 Pizzutello di Licata Sicily–Agrigento ca. 21 g 15,16 Pizzutello di Montallegro Sicily–Agrigento ca. 27 g 15,16 Pizzutello di Sciacca Sicily–Agrigento < 12 g elongate; apex 22,24,28,15,16 San Andrea (di Lampedusa) Sicily–Agrigento 9,16 Albicocca di Lipari Sicily–Messina ca. 33 g 29 Locale di Basicò Giallo Sicily Messina 18 21 g (ca 27 g) round; apex 22 24 15 16 January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 3 LSL Landraces for Tomato Improvement Conesa et al. TABLE 1 \| Continued Landrace Region Shelf-life Fruit size Fruit shape References Locale di Filicudi Sicily–Messina 12–15 g (ca. 20 g) round 22,23,24,30,15,16 Locale di Pollara (Locale di Salina 9) Sicily–Messina < 12 g (11–21 g) round 22,24,15,16 Locale di Salina 1 Sicily–Messina ca. 21 g 15,16 Locale di Salina 2 Sicily–Messina 15–18 g round; apex 22,24,15,16 Locale di Salina 3 Sicily–Messina ca. 42 g 15,16 Locale di Salina 4 Sicily–Messina ca. 24 g 15,16 Locale di Salina 5 Sicily–Messina ca. 48 g 15,16 Locale di Salina 6 Sicily–Messina 12–15 g round 22,24,12,28,15,16 Locale di Salina 10 Sicily–Messina ca. 26 g 15,16 Locale di Vulcano Sicily–Messina ca. 17 g 15,16 Mazzarrà San Andrea Sicily–Messina ca. 23 g 15,16 Ruccaloru Sicily–Messina – S.Pierniceto 12–15 g (ca. 25 g) elongate 22,24,15,16 Poma Sicily–Palermo–Cefalù 9,16 Albicocca di Favignana Sicily–Trapani–Egadi Is. ca. 25 g 29 Locale di Custonaci Sicily–Trapani 18–21 g (ca. 28 g) round; apex 22,24,15,16 Paceco Sicily–Trapani 9 Patataro Sicily–Trapani–Marsala 9,10,16 Pizzutello d'Inverno Sicily–Trapani–Marsala 9,10,16 Pizzutello di Nubia Sicily–Trapani 9,10,16 Pizzutello di Paceco Sicily–Trapani 10,16 Sinacori Sicily–Trapani–Paceco 9,10,16 Giallo Piccolo a Punta Sicily ca. 31 g 29 Landrace Siciliana Sicily (long) 17 Percopara Sicily ca. 41 g 29 Pizzutello (di Stagnone)? Sicily–Trapani ca. 25 g 29 Rosso Sicily ca. ORIGIN, DIVERSITY AND DISTRIBUTION OF THE LSL LANDRACES 24 g 29 Stella Sicily(Palermo) 9,10 The references for the information are indicated: 1-Galmés et al., 2011; 2-Ochogavía et al., 2011; 3-Galmés et al., 2013; 4-Bota et al., 2014; 5-Fullana-Pericàs et al., 2017; 6-Fullana- Pericàs et al., 2019; 7-Casals et al., 2012; 8-Figàs et al., 2015; 9-Mercati et al., 2015; 10-Abenavoli et al., 2016; 11-Fattore et al., 2016; 12-Guida et al., 2017; 13-Sacco et al., 2017; 14- Manzo et al., 2018; 15-Siracusa et al., 2018; 16-Tranchida-Lombardo et al., 2018b; 17-Landi et al., 2017; 18-Tranchida-Lombardo et al., 2018a; 19-Andreakis et al., 2004; 20-Pernice et al., 2010; 21-Tamburino et al., 2017; 22-Siracusa et al., 2012; 23-Siracusa et al., 2013; 24-Patanè et al., 2016; 25-Renna et al., 2018; 26-Laghetti et al., 2008; 27-Berni et al., 2018; 28- Giorio et al., 2018; 29-Barbagallo et al., 2008; 30-Patanè et al., 2017. The references for the information are indicated: 1-Galmés et al., 2011; 2-Ochogavía et al., 2011; 3-Galmés et al., 2013; 4-Bota et al., 2014; 5-Fullana-Pericàs et al., 2017; 6-Fullana- Pericàs et al., 2019; 7-Casals et al., 2012; 8-Figàs et al., 2015; 9-Mercati et al., 2015; 10-Abenavoli et al., 2016; 11-Fattore et al., 2016; 12-Guida et al., 2017; 13-Sacco et al., 2017; 14- Manzo et al., 2018; 15-Siracusa et al., 2018; 16-Tranchida-Lombardo et al., 2018b; 17-Landi et al., 2017; 18-Tranchida-Lombardo et al., 2018a; 19-Andreakis et al., 2004; 20-Pernice et al., 2010; 21-Tamburino et al., 2017; 22-Siracusa et al., 2012; 23-Siracusa et al., 2013; 24-Patanè et al., 2016; 25-Renna et al., 2018; 26-Laghetti et al., 2008; 27-Berni et al., 2018; 28- Giorio et al., 2018; 29-Barbagallo et al., 2008; 30-Patanè et al., 2017. under names like ‘Pizzutello’ and ‘Locale’ followed by the name of the region of provenance (Table 1). Those are in general cultivated in very small family orchards, mostly for self- consumption (Sacco et al., 2017). This results in relatively low productive landraces as compared to commercial cultivars (Siracusa et al., 2013). Fruits are small, in many cases below 25 g. Fruit shape is variable, from round to elongated or pear- shaped, frequently with a prominent stylar end tip, which is variable or absent across landraces (Table 1). Despite the extended shelf-life might have been the primary selection criteria, most have high to very high sugar content (Table 2), denoting that this has also been an important selective trait. The references for the information are indicated: 1-Galmés et al., 2011; 2-Ochogavía et al., 2011; 3-Galmés et al., 2013; 4-Bota et al., 2014; 5-Fullana-Pericàs et al., 2017; 6-Fullana- Pericàs et al., 2019; 7-Casals et al., 2012; 8-Figàs et al., 2015; 9-Mercati et al., 2015; 10-Abenavoli et al., 2016; 11-Fattore et al., 2016; 12-Guida et al., 2017; 13-Sacco et al., 2017; 14- Manzo et al., 2018; 15-Siracusa et al., 2018; 16-Tranchida-Lombardo et al., 2018b; 17-Landi et al., 2017; 18-Tranchida-Lombardo et al., 2018a; 19-Andreakis et al., 2004; 20-Pernice et al., 2010; 21-Tamburino et al., 2017; 22-Siracusa et al., 2012; 23-Siracusa et al., 2013; 24-Patanè et al., 2016; 25-Renna et al., 2018; 26-Laghetti et al., 2008; 27-Berni et al., 2018; 28- Giorio et al., 2018; 29-Barbagallo et al., 2008; 30-Patanè et al., 2017. Selection for the LSL Fruit Phenotype A clear indication that extended shelf-life was the main selective trait is indeed the local name of these landraces. Thus, names for ‘de Ramellet,’ ‘de Penjar,’ ‘Alcobaça’ in the Balearic Islands and Iberian Peninsula, and ‘da Serbo,’ ‘da Appendere,’ ‘del Piennolo,’ ‘d'Inverno,’ etc. in Italian landraces, all refer to the use of tomatoes for long-term-over-winter-storage, disposed in bunches, or to be hung (e.g., Casals et al., 2011; Bota et al., 2014; Mercati et al., 2015; Manzo et al., 2018). The ‘Regina’ tomato includes diverse drought-adapted LSL landraces in Puglia, with average shelf-life of ca. 6 months (Renna et al., 2018). Also, some LSL landraces in Tuscany are ‘Tondino Liscio da Serbo Toscano,’ ‘Giallo di Pitigliano,’ ‘Rosso di Pitigliano,’ and ‘Perina a Punta della Valtiberina’ (Berni et al., 2018), from which little has been published. This might be extensible to further regions in Italy. In the Balearic Islands, the ‘de Ramellet’ name (‘small-bunch’ in Catalan) may refer to the fruit bunch in the plant, or perhaps to the strings anciently produced to store fruits. Thus, an ancient practice still performed to date consists in sewing the tomato pedicels to a main rope, threading fruit strings of variable length, anciently hung on the roof beams in ventilated sheds (Figure 2). This was industrialized ca. 1930 in Banyalbufar, a village of Mallorca that build up an industry and exported ‘de Ramellet’ mainly to Catalonia (Fairchild, 1927). This could be an important route for genetic material exchange between Mallorca and Catalonia. In fact, the ‘de Penjar’ tomatoes from Catalonia and Valencia are stored in very similar bunches, also sewing the pedicels to a main rope (Figure 2). It is worth mentioning that other traditional ways to preserve ‘de Ramellet’ exist in the Balearic Islands, like hanging tomatoes in very densely branched olive tree woods through the fruit bunch stems (Figure 2), or disposing individual fruits onto hurdle to ensure ventilation. LSL landraces might also be found in other Mediterranean regions, but little information has been reported to our knowledge. For example, landraces in Greece have been tested for genetic, morphological and agronomic traits (Terzopoulos and Bebeli, 2008; Terzopoulos et al., 2009; Terzopoulos and Bebeli, 2010; Koutsika-Sotiriou et al., 2016), showing that local selection favoured plant morphology and fruit shape and ﬂavour over shelf-life (Terzopoulos and Bebeli, 2010), which would hinder for the existence of LSL landraces. ORIGIN, DIVERSITY AND DISTRIBUTION OF THE LSL LANDRACES Drought tolerance is common in Sicilian landraces because anciently, but still today, have been cultivated under rain-fed conditions. To some extent, this also responds to the increased fruit quality obtained under such conditions (e.g., Siracusa et al., 2012; Siracusa et al., 2013; Sacco et al., 2017). Various ‘de Ramellet’-like and ‘de Penjar’-like F1 hybrids are widely produced by professional growers, and some seed companies produce different ‘de Ramellet’-like and ‘de Penjar’- like varieties improved with biotic tolerances and uniform color. The original landrace material for such breeding is in many cases uncertain and product traceability suggests that in some cases the same F1 hybrid is sold either as ‘de Penjar’ or ‘de Ramellet’ depending on the market (i.e., Catalonia-Valencia or Balearic Islands, respectively). LSL accessions similar to ‘de Ramellet’ and ‘de Penjar,’ with poorly deﬁned and little extended use, and mainly of unknown origin, are found in most part of the Mediterranean coast of the Iberian Peninsula, and also inland regions like Andalusia and Cáceres (R. Fernández-Muñoz, pers. comm.). The latter location is only ca. 250 km away from Alcobaça in Portugal, location from which the ‘Alcobaça’ landrace was described (Almeida, 1961; Kopeliovitch et al., 1980). This could indicate that LSL landraces anciently existed in the Iberian Peninsula aside the Mediterranean coast. Most LSL accessions occurring outside Catalan-speaking regions are recognized as ‘Alcobaça,’ textually meaning ‘de Penjar’ (“for hanging”) in Spanish. In fact, some commercial tomatoes are recognized as ‘Alcobaça’ also in Algeria (Maamar et al., 2015). Diverse landraces from mount Vesuvio region in Campania are grouped into the ‘Vesuviano’ landrace, which is cultivated since the end of the 19th century (Ercolano et al., 2014). Nevertheless, different ecotypes are also recognized, like ‘Fiaschella,’ ‘Lampadina,’ ‘Patanara,’ ‘Re Umberto’ and ‘Principe Borghese’ (Table 1), all included in the Protected Designation of Origin regulation (PDO) ‘Pomodorino del Piennolo del Vesuvio.’ Tomatoes under this PDO may have a Attending to literature, Sicily is by far the richest Italian region for LSL landraces, with outstanding groups of landraces January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 4 LSL Landraces for Tomato Improvement Conesa et al. ORIGIN, DIVERSITY AND DISTRIBUTION OF THE LSL LANDRACES FIGURE 1 \| Morphological variation in fruit size and shape in the Balearic LSL landrace ‘de Ramellet.’ Left: ‘de Ramellet’ fruit (blue dots) as compared to diverse tomatoes with variable size and shape, based on the ﬁrst two Principle Components (73% of total variation explained) resulting from morphological data collected from transverse sections with Tomato Analyzer (Brewer et al., 2006). Each accession is represented by an average of all scanned fruits. Representative fruits are shown along the plot. PC1 and PC2 mainly explain fruit size and fruit elongation, respectively. Modiﬁed from Conesa et al. (2010). Right: Variation found in a prospection across the Balearic Islands to create the UIB-collection. Modiﬁed from Ochogavía et al. (2011). FIGURE 1 \| Morphological variation in fruit size and shape in the Balearic LSL landrace ‘de Ramellet.’ Left: ‘de Ramellet’ fruit (blue dots) as compared to diverse tomatoes with variable size and shape, based on the ﬁrst two Principle Components (73% of total variation explained) resulting from morphological data collected from transverse sections with Tomato Analyzer (Brewer et al., 2006). Each accession is represented by an average of all scanned fruits. Representative fruits are shown along the plot. PC1 and PC2 mainly explain fruit size and fruit elongation, respectively. Modiﬁed from Conesa et al. (2010). Right: Variation found in a prospection across the Balearic Islands to create the UIB-collection. Modiﬁed from Ochogavía et al. (2011). THE EXTENDED SHELF-LIFE IN MEDITERRANEAN LSL LANDRACES minimum of 6.5 °Brix, which denotes that sugar content has been an important selective trait (Manzo et al., 2018). Some other renowned LSL landraces in Campania are ‘Lucariello,’ which is a ‘Vesuviano’-like tomato with shelf-life of 5–10 months (Tranchida-Lombardo et al., 2018a), and ‘Corbarino.’ The latter is used either fresh, canned or to store hung, with notorious diversity in fruit shape among landraces (Table 1). Most ‘Corbarino’ landraces are very vigorous, outperforming commercial F1 hybrids in yield and quality traits (Andreakis et al., 2004; Sinesio et al., 2007; Lisanti et al., 2008), and are particularly drought tolerant (Pernice et al., 2010). Frontiers in Plant Science \| www.frontiersin.org Selection for the LSL Fruit Phenotype Fruit quality traits measured at harvest time unless months (m.) or days (d) indicated in brackets. is shown after the average in brackets. Fruit quality traits measured at harvest time unless months (m.) or days (d) indicated in brackets. Landrace Region Cultivation Yield (kg pl−1) Fruit num. Fruit weight (g) °Brix Titratable ac. (g citric ac. 100g−1) pH Firmness Refs. Ramellet (n = 6) Balearic Islands OF–WW; WD (20%PET) 4.1; 1.3 76; 60* 57; 23* 1,2 Ramellet (n = 158) Balearic Islands OF–WW 1.3 (0.1– 2.8) 29 (4– 75) 47 (9– 164) 5.9(0d)– (170d)5.1* 1.48(0d)–(170d) 0.71* 3.85(0d)– (170d)4.49* 1.14(0d)– (170d)1.56* 3,4 Ramellet (n = 48) Balearic Islands OF–WW; WD (40%PET) 2.6; 2.1* 89; 74* 30; 28 4.8; 5.7 1.09; 1.15* 4.25; 4.31* 5 Ramellet (n = 4) Balearic Islands OF; GH 4.2; 2.8 105; 76 6.1; 5.9 0.58; 0.53 4.15; 4.14 53.1; 50.2 6 Penjar (n = 4) Catalonia/Val. Comm. OF–WW 3.0 (1.7– 3.1) 67 (31– 116) 5.3 (4.8– 6.6) 7 Penjar (n = 27) Cat./Val Comm./Bal. Is. OF–WW 2.9 (1.6– 4.3) 64 (25– 121) 6.9 (5.0– 9.5) 8 Penjar (n = 2agro; n = 5qualit) Valencian Comm. OF–WW 2.5 (0.3– 6.5) 200 (183– 217) 4.7 (4.1– 5.4) 0.56 (0.39–0.78) 4.3 (4.1– 4.5) 9 Penjar (n = 3) Valencian Comm. WW–OF; GH 3.7; 2.4 70; 51 6.6; 7.2 0.42; 0.45 4.37; 4.33 60.9; 45.3 6 Penjar (n = 12) Valencian Comm. OF–WW (36–86) 6.6 (4.1– 8.7) 0.54 (0.35–0.81) 4.25 (3.98– 4.45) 10 PenjarVAL (n = 6) Valencian Comm. Selection for the LSL Fruit Phenotype Landraces of supposed Greek origin have been documented in Greek-speaking regions in Southern Italy (Grecìa), like ‘pummidora scimona,’ (‘pomodoro da serbo giallo’ in Italian), a yellow-colored LSL landrace (Laghetti et al., 2008). January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 5 LSL Landraces for Tomato Improvement Conesa et al. TABLE 2 \| Agronomic and fruit quality traits in Mediterranean long shelf-life (LSL) landraces, ordinated by regions of origin. For landrace details, see Table 1. The number of accessions in the average (n) is indicated unless is one. Cultivation conditions and different water treatments indicated when available: OF, open ﬁeld in Mediterranean summer; GH, greenhouse; WW, full irrigation replacing potential evapotranspiration (PET); WD, water deﬁcit (number in brackets indicate the % of covered PET); and RF, rain-fed. Yield and fruit number are per plant unless units are indicated. Total soluble solids, titratable acidity and ﬁrmness are in °Brix, g citric ac. 100 g−1 and shore units, respectively, unless units are indicated. When different water treatments were applied, data is indicated in the same order as treatments and separated by semicolon. Asterisk () indicates signiﬁcant differences between treatments, when available. For any trait, if a range was indicated for different accessions, it is shown after the average in brackets. Fruit quality traits measured at harvest time unless months (m.) or days (d) indicated in brackets. TABLE 2 \| Agronomic and fruit quality traits in Mediterranean long shelf-life (LSL) landraces, ordinated by regions of origin. For landrace details, see Table 1. The number of accessions in the average (n) is indicated unless is one. Cultivation conditions and different water treatments indicated when available: OF, open ﬁeld in Mediterranean summer; GH, greenhouse; WW, full irrigation replacing potential evapotranspiration (PET); WD, water deﬁcit (number in brackets indicate the % of covered PET); and RF, rain-fed. Yield and fruit number are per plant unless units are indicated. Total soluble solids, titratable acidity and ﬁrmness are in °Brix, g citric ac. 100 g−1 and shore units, respectively, unless units are indicated. When different water treatments were applied, data is indicated in the same order as treatments and separated by semicolon. Asterisk () indicates signiﬁcant differences between treatments, when available. For any trait, if a range was indicated for different accessions, it is shown after the average in brackets. Selection for the LSL Fruit Phenotype OF–WW; WD (40%PET) 2.3; 1.5* 89; 79 35; 23 4.4; 5.6 0.84; 1.08* 4.30; 4.25 5 PenjarCAT (n = 6) Catalonia OF–WW; WD (40%PET) 1.7; 1.7 96; 86 15; 18* 5.7; 5.8 1.30; 0.92 4.24; 4.31 5 DFD Catalonia GH–WW 5.5 11 Ramellet/Penjar improved (n = 3) Commercial improved WW–OF; GH 3.7; 3.5 97; 64 6.1; 6.5 0.45; 0.51 4.31; 4.3 53.9; 47.9 6 Ramellet/Penjar hybrids F1 (n = 2) Commercial F1 WW–OF; GH 4.4; 3.4 100; 76 6.5; 6.6 0.49; 0.54 4.25; 4.16 59.8; 58.3 6 Piennolo del Vesuvio Campania OF–WW; RF (wet year) 5.3; 5.1 160; 183 32.8; 27.5* 4.7; 6.2* 4.45; 4.56 12 Vesuvio Campania– Ercolano OF–RF 25 7.5 0.34 13 Piennolo Vesuviano Campania– Ercolano N/A (ﬁeld collected) 6.9(0m.)– (6m.)7.9* 0.53(0m.)–(6m.)0.50 4.36(0m.)– (6m.)4.45 14 Lucariello Campania OF–RF 8.0 15 Ercolano Campania– Ercolano OF–RF 26 7.7 0.35 13 Corbarino Campania OF–RF 7.2 15 Corbarino (n = 4) Campania– Sarno Valley OF–4m. irrig.(April–Sept) 73.9 t/ha 18 6.3 0.4 4.5 16 Corbarino PC01 Campania OF–WW; WD (50%PET); RF 114; 98; 62 t/ha 17; 17; 13 5.5; 5.6; 7.3 17 Corbarino PC05 Campania OF–WW; WD (50%PET); RF 122; 112; 72 t/ha 17; 17; 13 6.3; 6.8; 9.1 17 Principe Borghese Campania (commercial) OF–RF 17 7.4 0.32 13 Principe Borghese Campania (commercial) OF–RF 13.7 t/ha 18 Principe Borghese Campania (commercial) OF–RF 16.8 t/ha 15 7.6 0.31 19 Regina–Fasano ecotype Puglia–Fasano OF– RF 20 6.4c 20 Regina–Monopoli ecotype Puglia– Monopoli OF– RF 22 7.0b 20 Regina–Ostuni ecotype Puglia–Ostuni OF–RF 25 7.6 20 (Contin January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 6 LSL Landraces for Tomato Improvement Conesa et al. TABLE 2 \| Continued Landrace Region Cultivation Yield (kg pl−1) Fruit num. Fruit weight (g) °Brix Titratable ac. (g citric ac. 100g−1) pH Firmness Refs. Selection for the LSL Fruit Phenotype Altamura Puglia–Bari OF– RF 22 6.9 0.34 13 Arnesano Puglia–Lecce OF– RF 39 7.7 0.33 13 Buttigghieddu Sicily–Agrigento OF– RF 17 7.4 0.31 13 Kachi di Sciacca Sicily–Agrigento OF– RF 31 6.8 0.32 13 Mezzocachi di Montallegro Sicily–Agrigento OF– RF 37 6.2 0.28 13 Pizzottello di Montallegro Sicily–Agrigento OF–RF (18.5– 19.8 t/ha) (19–27) (7.0–8.0) 0.29 4.07 22.7 21,19,18,22,13 Pizzutello di Licata Sicily–Agrigento OF–RF (21–22) (6.2–6.7) 0.34 13 Pizzutello di Montallegro Sicily–Agrigento OF– RF 22 6.2 0.32 13 Pizzutello di Sciacca Sicily–Agrigento OF– RF 13.6 t/ha 12 8.1 0.38 18,13 Albicocca di Lipari Sicily–Messina GH–WW; WD (50%PET) 33; 16* 5.5; 6.6* 2.09; 2.41 4.25; 4.19 23 Locale di Basicò Giallo Sicily–Messina OF– RF 11.7 t/ha 27 6.5 0.26 18,13 Locale di Basicò Rosso Sicily–Messina OF– RF 24 6.7 0.3 13 Locale di Filicudi Sicily–Messina OF– RF (20.8– 23.4 t/ha) 14 8.2 0.34 4.08 20.4 19,18,22 Locale di Filicudi Sicily–Messina OF– RF 20 7 0.36 13 Locale di Pollara Sicily–Messina OF– RF 18.2 t/ha 12 7.6 0.29 18,13 Locale di Salina 1 Sicily–Messina OF– RF 21 6.8 0.34 13 Locale di Salina 2 Sicily–Messina OF– RF 28.9 t/ha 22 7.5 0.34 18,13 Locale di Salina 3 Sicily–Messina OF– RF 42 6.6 0.34 13 Locale di Salina 4 Sicily–Messina OF– RF 24 7.4 0.32 13 Locale di Salina 5 Sicily–Messina OF– RF 48 5.6 0.27 13 Locale di Salina 6 Sicily–Messina OF– RF 16.1 t/ha 15 7.4 0.33 18,13 Locale di Salina 6 Sicily–Messina OF–WW; RF (dryY) 3.2; 3.1 306; 283 11; 11 4.9; 6.0* 12 Locale di Salina 6 Sicily–Messina OF–WW; rainfed(wetY) 3.4; 3.2 251; 238 14; 14 5.5; 7.5* 4.39; 4.57 12 Locale di Salina 10 Sicily–Messina OF– RF 26 7.3 0.31 13 Locale di Vulcano Sicily–Messina OF– RF 17 7.5 0.26 13 Mazzarrà S. Selection for the LSL Fruit Phenotype Andrea Sicily–Messina OF– RF 23 7.3 0.28 13 Ruccaloru Sicily–Messina –S.Pierniceto OF– RF 13.2 t/ha 25 7.2 0.28 18,13 Albicocca di Favignana Sicily–Trapani (Egadi Is.) GH–WW; WD (50%PET) 25; 24 6.0; 7.8* 1.86; 2.75* 4.02; 4.22 23 Locale di Custonaci Sicily–Trapani OF– RF 22 t/ha 28 7.3 0.29 18,13 Giallo Piccolo a Punta Sicily GH– WW; WD (50%PET) 31; 26* 5.5; 7.3* 1.63; 2.75* 4.21; 4.11 23 Percopara Sicily GH– WW; WD (50%PET) 41; 23* 6.0; 6.8* 1.69; 2.28* 4.30; 4.27 23 Pizzutello di Stagnone Sicily–Trapani GH– WW; WD (50%PET) 27; 24 5.8; 6.8* 1.93; 2.24 4.27; 4.23 23 Rosso Sicily GH– WW; WD (50%PET) 24; 16* 5.9; 6.3 1.80; 2.80* 4.10; 3.94 23 The references for the information are indicated: 1-Galmés et al., 2011; 2-Galmés et al., 2013; 3-Ochogavía et al., 2011; 4-Bota et al., 2014; 5-Fullana-Pericàs et al., 2019; 6-Figàs et al., 2018; 7-Casals et al., 2011; 8-Casals et al., 2012; 9-Cebolla-Cornejo et al., 2013; 10-Figàs et al., 2015; 11-Saladie et al., 2007; 12-Guida et al., 2017; 13-Siracusa et al., 2018; 14-Manzo t l 2018 15 T hid L b d t l 2018 16 A d ki t l 2004 17 P i t l 2010 18 P t è t l 2016 19 Si t l 2013 20 R t l 2018 21 Si The references for the information are indicated: 1-Galmés et al., 2011; 2-Galmés et al., 2013; 3-Ochogavía et al., 2011; 4-Bota et al., 2014; 5-Fullana-Pericàs et al., 2019; 6-Figàs et al., 2018; 7-Casals et al., 2011; 8-Casals et al., 2012; 9-Cebolla-Cornejo et al., 2013; 10-Figàs et al., 2015; 11-Saladie et al., 2007; 12-Guida et al., 2017; 13-Siracusa et al., 2018; 14-Manzo et al., 2018; 15-Tranchida-Lombardo et al., 2018a; 16- Andreakis et al., 2004; 17-Pernice et al., 2010; 18-Patanè et al., 2016; 19-Siracusa et al., 2013; 20-Renna et al., 2018; 21-Siracusa et al., 2012; 22-Patanè et al., 2017; 23-Barbagallo et al., 2008. in most Italian “da Serbo” landraces is performed without detaching fruits from the truss, and tying together different trusses twisted arround a twine, sometimes conforming a circle (Figure 2), (Mercati et al., 2015; Sacco et al., 2017; Manzo et al., 2018). This is also common in the ‘Vesuviano’ landraces from Among Italian landraces, the individual fruit threading performed in ‘de Ramellet’ and ‘de Penjar’ is not common. Frontiers in Plant Science \| www.frontiersin.org Selection for the LSL Fruit Phenotype Conesa et al., 2014). Actually, the dfd mutant seems to bear the alc mutation (Vrebalov et al., 2004; Bota et al., 2014). The alc mutation has been suggested as responsible for the LSL phenotype also in ‘da Serbo’ Italian ‘Alcobaça’ landrace (Almeida, 1961; Leal and Tabim, 1974). First reports for shelf-life in ‘Alcobaça’ were up to 316 days (Leal and Tabim, 1974), although further reports are considerably shorter (up to 33 days; Kopeliovitch et al., 1980; Mutschler 1984b; Mutschler et al., 1988; Mutschler et al., 1992; Dias et al., 2003). The alc mutation is present in ‘de Penjar’ from Catalonia and Valencia and ‘de Ramellet’ from the Balearic Islands (Casals et al., 2012; Bota et al., 2014). The ‘delayed fruit deterioration’ mutant (dfd) was described as a regionalized cultivar grown in speciﬁc areas around the Mediterranean, with dramatically delayed softening and unknown genetic background (Saladie et al., 2007). Such description ﬁts with LSL landraces, while fruit size, shape and shelf-life are similar to ‘de Penjar’ (Rose, unpubl., cf. Conesa et al., 2014). Actually, the dfd mutant seems to bear the alc mutation (Vrebalov et al., 2004; Bota et al., 2014). The alc mutation has been suggested as responsible for the LSL phenotype also in ‘da Serbo’ Italian Selection for the LSL Fruit Phenotype It seems to be performed in the ‘Regina’ landrace from Puglia, by tying fruit pedicels with a cotton thread in bunches called ‘ramasole’ (Sacco et al., 2017; Renna et al., 2018). Fruit hanging January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 7 Conesa et al. LSL Landraces for Tomato Improvement FIGURE 2 \| Different ways to store hung long shelf-life tomatoes across the Mediterranean. (A) Typical ‘de Ramellet’ strings with fruit pedicels needle-sewn to a main rope (Banyalbufar, Mallorca; courtesy: Aina Socies - Associació de Varietats Locals de Mallorca). (B) ‘de Ramellet’ hung by pedicels on wild olive tree branches (Artà, Mallorca; courtesy: Toni Muñoz). (C) Needle-sewing of ‘de Penjar’ strings (Alcalà de Xivert, Castelló; courtesy: Associació de Productors i Comercialitzadors de Tomata de Penjar d'Alcalà de Xivert). (D) Typical Sicilian ‘da Serbo’ trusses (Sicily). (E) Typical ‘piennoli’ of ‘Pomodorino del Piennolo del Vesuvio’ (Ercolano, Napoli; courtesy: Rosario Custro). FIGURE 2 \| Different ways to store hung long shelf-life tomatoes across the Mediterranean. (A) Typical ‘de Ramellet’ strings with fruit pedicels needle-sewn to a main rope (Banyalbufar, Mallorca; courtesy: Aina Socies - Associació de Varietats Locals de Mallorca). (B) ‘de Ramellet’ hung by pedicels on wild olive tree branches (Artà, Mallorca; courtesy: Toni Muñoz). (C) Needle-sewing of ‘de Penjar’ strings (Alcalà de Xivert, Castelló; courtesy: Associació de Productors i Comercialitzadors de Tomata de Penjar d'Alcalà de Xivert). (D) Typical Sicilian ‘da Serbo’ trusses (Sicily). (E) Typical ‘piennoli’ of ‘Pomodorino del Piennolo del Vesuvio’ (Ercolano, Napoli; courtesy: Rosario Custro). Campania, whose bunches are called ‘piennolo’ or ‘spunzilli’ (Figure 2), (Sacco et al., 2017). ‘Alcobaça’ landrace (Almeida, 1961; Leal and Tabim, 1974). First reports for shelf-life in ‘Alcobaça’ were up to 316 days (Leal and Tabim, 1974), although further reports are considerably shorter (up to 33 days; Kopeliovitch et al., 1980; Mutschler 1984b; Mutschler et al., 1988; Mutschler et al., 1992; Dias et al., 2003). The alc mutation is present in ‘de Penjar’ from Catalonia and Valencia and ‘de Ramellet’ from the Balearic Islands (Casals et al., 2012; Bota et al., 2014). The ‘delayed fruit deterioration’ mutant (dfd) was described as a regionalized cultivar grown in speciﬁc areas around the Mediterranean, with dramatically delayed softening and unknown genetic background (Saladie et al., 2007). Such description ﬁts with LSL landraces, while fruit size, shape and shelf-life are similar to ‘de Penjar’ (Rose, unpubl., cf. Frontiers in Plant Science \| www.frontiersin.org Genetic Basis of the LSL Phenotype Fernández-Muñoz). All bear the alc mutation except the latter, from which the two discordant nor products were described (Kumar et al., 2018). In this regard, Kumar et al. (2018) compared ethylene emission in ‘Ailsa Craig’ with four different ‘de Penjar’ accessions, and found reduced ethylene production in three of the ‘de Penjar,’ but increased in the Penjar-2 accession, emitting slightly higher ethylene at breaker stage than ‘Ailsa Craig’. This suggests that the above indicated opposite responses of alc- bearing accessions towards ethylene emission may not be only related to this mutation, but could respond to intrinsic variation within LSL landraces. In fact, Kumar et al. (2018) described novel NOR mutations within the studied ‘de Penjar’ accessions, all in the Penjar-1 accession and thus, not related to the differences reported for ethylene production. Further, they found good correlation of ethylene emission with other ripening-related metabolites like jasmonic acid in all the ‘de Penjar’ accessions except Penjar-4. Consequently, results suggest that the LSL fruit phenotype may be also related to non-ethylene mediated ripening regulation, as described from non-ripening mutants (e.g., Moore et al., 2002; Vrebalov et al., 2002). This variability in ethylene production within LSL landraces, uncertainties on the different role of ethylene in LSL vs. non-LSL landraces, and the impact of ethylene in fruit quality traits and aroma highlights the importance of deepening in the knowledge of ethylene production pathways within LSL landraces to better understand extended shelf-life and ripening process in tomato. The impact of the alc mutation on the extended fruit shelf-life was assessed by introducing the mutation in ‘M82’ tomato by CRISPR/Cas9 transformation (Yu et al., 2017). The shelf-life reported was only up to 40 days, which is similar to early reports for ‘Alcobaça,’ and much shorter than that in most LSL landraces. This agrees with past indications that alc may be necessary but not sufﬁcient to explain the LSL phenotype in the Mediterranean landraces (Conesa et al., 2014; Casals et al., 2015). Altogether, some controversy exists among the possible existence of different mutations in the MADS-box, SBP-box and NAC- family genes (Seymour et al., 2013; Kumar et al., 2018), which may be responsible for part of the variation reported in shelf-life across LSL landraces. Genetic Basis of the LSL Phenotype yp The tomato ripening mutants like rin, nor, Nr, Gr, and Cnr, among others, result in a strong ripening impairment frequently related to ethylene insensitivity (Barry and Giovannoni, 2007; Giovannoni, 2007), and with pleiotropic effects on colour and ﬂavour. Because of that, rin and nor are used only in heterozygous in breeding programs, resulting in delayed and altered ripening and extended shelf-life (Garg et al., 2008; Kosma et al., 2010; Klee and Giovannoni, 2011). On the contrary, tomatoes with the LSL phenotype differ from the latter mutants in that fruit ripening is not badly impaired, but rather exhibit normal climateric ripening. The LSL phenotype has been related to the alc mutation (e.g., Kopeliovitch et al., 1981; Lobo et al., 1984; Mutschler, 1984a; Casals et al., 2012; Conesa et al., 2014), described in the January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 8 LSL Landraces for Tomato Improvement Conesa et al. described an effect of the alc mutation related to reduced ethylene production (i.e., 25% less than ‘Rutgers’ cultivar; Mutschler, 1984b). However, postharvest storage attributed to this landrace is commonly lower than 60 days, which differs from the 6–8 months reported for most LSL landraces. This also endorses previous indications that the alc mutation may be needed but not sufﬁcient to explain extended fruit shelf-life in LSL landraces (Conesa et al., 2014; Casals et al., 2015). Moreover, this denotes opposite responses attributable to the alc mutation on the ethylene production, either reducing it (Mutschler, 1984b) and increasing it (Saladie et al., 2007). landraces (Mercati et al., 2015). However, its presence has not been reported in Italian landraces, to our knowledge; whereas it is absent in ‘Corbarino’ and ‘Lucariello’ (Tranchida-Lombardo et al., 2018a). This suggests that mutations other than alc may also result in the LSL phenotype, particularly in Italian landraces. Several studies support that alc and nor mutations are allelic (Tigchelaar et al., 1976; Lobo et al., 1984; Benites et al., 2010; Casals et al., 2012; Kumar et al., 2018). Recently, studies of NAC- family genes described diverse nor alleles and nor-like genes involved in the tomato ripening process (Wang et al., 2018). Also, Kumar et al. (2018) described two different cDNA products for nor added up to alc in ‘de Penjar’/‘de Colgar.’ The accessions considered included two from Catalonia (i.e, ‘de Penjar’), one from Málaga and one from Cáceres (i.e., ‘Alcobaça’), (pers. comm. R. Genetic Basis of the LSL Phenotype Factors Determining the LSL Phenotype Contrary to non-ripening mutants, most of which have an impairment related to ethylene insensitivity causing deﬁcient ripening and pleiotropic effects on fruit quality (Barry and Giovannoni, 2007; Giovannoni, 2007), ripening in LSL landraces is fully achieved on the vine, and fruit quality is one of the most appreciated traits (Saladie et al., 2007; Siracusa et al., 2013; Bota et al., 2014; Casals et al., 2015; Patanè et al., 2017). Ethylene production and respiration rates in LSL fruits has been poorly studied to date. Saladie et al. (2007) found a delay of seven days to each red-ripe stage in dfd as compared to ‘Ailsa Craig’, although the peak in ethylene production was similar, and the climacteric respiratory burst was more pronounced in dfd (i.e., ca. 16 vs. 12 nl ethylene g−1 h−1, and 55 vs. 25 ml CO2 g−1 h−1). Nevertheless, authors attributed such differences to already known variation occurring within the tomato crop (Saladie et al., 2007). Apart from ethylene, the maintenance of fully-ripen fruits sound over months of storage at ambient temperature suggests a dramatic slow-down of the respiratory metabolism and, particularly, an effective prevention of transpirational water loss in LSL fruits (Bargel and Neinhuis, 2004; Saladie et al., 2007; Conesa et al., 2014; Casals et al., 2015). A main effect of fruit cuticle properties on non-LSL tomato shelf-life has already been suggested (Bargel and Neinhuis, 2004; Bargel and Neinhuis, 2005; Domínguez et al., 2011), and related to increased waxes during ripening in nor, rin and ‘Alcobaça’ (Kosma et al., 2010). Increased cutin accumulation, but not cuticle thickness, seems important in dfd (Saladie et al., 2007). Therefore, it seems that cuticle properties –composition and cutin matrix architecture— in fruits with the LSL phenotype may be key in maintaining fruit impermeability. In a recent screening for genetic diversity across a collection of Italian LSL landraces, Tranchida-Lombardo et al. (2018b) found high variation in ethylene-responsive genes, relating it to the extended fruit shelf-life. However, they found no polymorphism in nor and alc mutations, suggesting that genetic determinants of the LSL fruit phenotype may be different between Italian and ‘de Ramellet’–‘de Penjar’ landraces. In the ‘Alcobaça’ landrace, it was Studies in tomato ripening mutants suggest a role for fruit water loss in triggering ethylene synthesis leading to ripening ﬁnalization and over-ripening. This agrees with initiation of fruit ripening in the tomato wild relatives Solanum chilense and S. Frontiers in Plant Science \| www.frontiersin.org Genetic Basis of the LSL Phenotype peruvianum, only once fruit is detached from the plant (reviewed in Barry and Giovannoni, 2007). Consequently, the ability to January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 9 LSL Landraces for Tomato Improvement Conesa et al. maintain fruit water for longer time can also be a factor preventing over-ripening in LSL fruits. morphological, physiological and molecular levels, as evidenced by a huge amount of studies in diverse crops (reviewed in Tuberosa, 2012; Nuccio et al., 2018; Sreeman et al., 2018 and Tardieu et al., 2018). Particularly in Mediterranean LSL tomato landraces, studies of drought tolerance have focused mainly on leaf gas exchange traits, water use efﬁciency, solute accumulation and hormonal signalling, which biases this review. Hence, this also highlights the need to explore further mechanisms improving drought tolerance in this tomato landrace group. Diverse authors suggested the thick skin and high antioxidant content as responsible for the extended fruit shelf-life in Italian landraces (Barbagallo et al., 2008; Siracusa et al., 2012; Mercati et al., 2015; Patanè et al., 2016; Manzo et al., 2018; Renna et al., 2018). Moreover, fruit size has been inversely correlated with shelf-life in ‘Alcobaça’ (Leal and Tabim, 1974; Mutschler et al., 1992) and ‘de Penjar’ (Casals et al., 2012). This correlation was non-signiﬁcant in ‘de Ramellet,’ probably because of variation in shelf-life sensitivity to water availability (Conesa et al., 2014) and high variation in fruit size and shape (Bota et al., 2014). In fact, many Italian LSL landraces have smaller fruits than ‘de Ramellet’ and ‘de Penjar’ (< 21 g; Siracusa et al., 2012; Patanè et al., 2016) but the shelf-life is not longer than that reported for the latter landraces, being two- to ten-fold bigger (Casals et al., 2012; Bota et al., 2014). Carbon isotopic composition (d13C) in leaf tissue has proven to be an efﬁcient and reliable method to assess water use efﬁciency in plants (e.g., Farquhar et al., 1982; Condon et al., 1990; Donovan and Ehleringer, 1994; Bacon, 2004; Galmés et al., 2011). It was measured in a diverse array of traditional tomato accessions including cherry, fresh market, processing and LSL (Fullana-Pericàs et al., 2019), grown under full irrigation (WW) and water deﬁcit (WD). The LSL group included accessions from Eastern Iberian Peninsula and Balearic Islands (LSL-big) and Italian accessions (LSL-cherry). Genetic Basis of the LSL Phenotype d13C increased in all groups under WD as compared to WW (Figure 3) showing that, irrespective of the tomato type, traditional accessions have mechanisms allowing adaptation to water shortage through increased water use efﬁciency. Despite the huge variation in shelf-life reported for LSL tomatoes (e.g., Leal and Tabim, 1974; Mutschler et al., 1988; Saladie et al., 2007; Casals et al., 2011; Conesa et al., 2014; Manzo et al., 2018; Tranchida-Lombardo et al., 2018a) could result from different mutations and even epigenetics (Mirouze and Paszkowski, 2011; Osorio et al., 2013; Schmitz et al., 2013; Zhong et al., 2013; Bressan et al., 2014; Van Oosten et al., 2014; Giovannoni et al., 2017), factors affecing shelf-life in LSL fruits like fruit size (Leal and Tabim, 1974; Mutschler et al., 1992; Casals et al., 2011; Bota et al., 2014), titratable acidity (Bota et al., 2014), sugar content (Kumar et al., 2018), antioxidant content (Barbagallo et al., 2008; Siracusa et al., 2012; Mercati et al., 2015; Patanè et al., 2016; Renna et al., 2018) are to a high extent inﬂuenced by cultivation conditions. Therefore, a part of the reported variation may be environmentally-driven. When comparing among groups, LSL-big had highest (less negative) d13C under both water treatments (Figures 3B, C), which would indicate lower water use efﬁciency. Higher d13C frequently occurs under drought and is related to stomatal closure to prevent water loss. However, low d13C values found also under WW denote that this is an intrinsic trait in LSL-big, which could relate to constitutive tighter stomatal control as compared to the remaining groups, and result in higher improvement of water use efﬁciency. In fact, fruit shelf-life in ‘de Ramellet’ showed some interaction with water availability during cultivation, denoting that traits conferring the LSL phenotype were impaired by too high irrigation in some landraces, but not others (Conesa et al., 2014). Since ‘de Ramellet’ is homozygous for alc, this mutation is not sufﬁcient to explain the variation in shelf-life within this landrace. Hence, too high irrigation could be impairing cuticle properties in the sensitive genotypes, either composition, component proportions or biomechanic properties, resulting in impaired LSL phenotype (Conesa et al., 2014; Conesa et al., 2015). It is noteworthy that in both treatments the largest difference in d13C among groups was between LSL-big and LSL-cherry, highlighting strikingly different strategies to adapt to harsh conditions between both LSL groups. Genetic Basis of the LSL Phenotype Contrary to LSL-big, the lowest d13C values in LSL-cherry, especially under WD (Figures 3B, C), pointed to an adaptive strategy minimizing operation with partially closed stomata, either through deeper roots providing higher water availability, or with a water- conservative stomatal behaviour (i.e., fast closure under drought). The big plant size and high vigor and yield in the LSL-cherry under drought (Andreakis et al., 2004; Sinesio et al., 2007; Lisanti et al., 2008) better points to the former strategy. The lowest difference between treatments in d13C occurring in LSL- cherry (Figure 3) also endorsed a drought-tolerance strategy involving mechanisms to increase access to water. In this regard, LSL-big had the highest difference between treatments in d13C (Figure 3), denoting the existence of further mechanisms increasing drought tolerance in this group. DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES In agricultural systems, drought refers to a scenario where water transpired by the plant cannot be fully replaced by soil water availability, forcing plants to reduce transpiration by stomatal closure, which limits growth and yield (Tardieu et al., 2018). Drought tolerant crops have mechanisms allowing plants to withstand eventual or permanent water shortage with lower yield reduction as compared to non-tolerant crops (Tuberosa, 2012). These mechanisms may be found at very different As compared to WW, stomatal closure under WD to prevent water loss is evidenced in a reduction in stomatal conductance (gs), meaning either lower water loss and lower CO2 intake through the stomata. Consequently, net photosynthesis (AN) is also reduced. Adaptive mechanisms at leaf level allow plants to minimize the reduction in AN as compared to gs, that is, increase the intrinsic January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 10 LSL Landraces for Tomato Improvement Conesa et al. 3 \| Water use efﬁciency as inferred from d13C (‰) isotopic composition of leaves from tomato plants grown under full irrigation (WW) and water deﬁcit (WD) in Fullana-Pericàs et al. (2019). Tomato accessions (n = 171) were separated in ﬁve groups depending on the accession type, including non-long shelf-life erry; n = 29), fresh market (Fresh; n = 50), long shelf-life from eastern Iberian Peninsula and Balearic Islands (LSL-big; n = 63) and from Italy (LSL-cherry; nd processing accessions (Processing; n = 13). (A) Percent of d13C in WD as compared to WW. ANOVA differences between WD and WW were signiﬁcant ps (P < 0.001). (B) Values of d13C in WW and (C) in WD. For each accession group, boxplots represent the average and the median (red and black lines pectively), the 75% interval (box), the 90% interval (error bars) and the outliers (isolated points). In each plot, letters on top indicate ANOVA-Tukey among groups (P < 0.05). FIGURE 3 \| Water use efﬁciency as inferred from d13C (‰) isotopic composition of leaves from tomato plants grown under full irrigation (WW) and water deﬁcit (WD) treatments in Fullana-Pericàs et al. (2019). DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES Despite stomatal closure under WD resulted in reduction of both gs and gm, the differential degree of reduction was explained by leaf anatomical adaptations occurring under WD. A close inspection of mesophyll anatomy showed a notorious increase in intercellular airspaces (Figures 4A, B), resulting in increased surface of cells having chloroplasts exposed to airspaces (Sc) under WD, thus windows for chloroplasts to access CO2. Further, chloroplasts were tightly positioned against the cell membrane to minimize the CO2 pathway (Figures 4C, D; Galmés et al., 2013). The positive correlation between gm and Sc under WD demonstrated that WUEi improvement was a consequence of increased gm (as compared to gs) achieved through increased Sc. Since CO2 is ﬁnally ﬁxed by Rubisco, the Sc/[Rubisco] ratio may be a key trait explaining the efﬁciency in CO2 ﬁxation (Onoda et al., 2017). Accordingly, this ratio positively correlated with WUEi in both water treatments, although for the same Sc/[Rubisco] the WUEi was scaled-down under WW as compared to WD (Figure 5). In this regard, the non-‘de Ramellet’ accession was the single one reducing the Sc/ [Rubisco] ratio under WD (Figure 5) and thus, the described leaf anatomical adaptation in ‘de Ramellet’ is not occurring in all tomato varieties. Further knowledge on stomatal and mesophyll characteristics in Mediterranean landraces, and their response to drought conditions, may unravel important patterns suitable to breed for novel tomato cultivars more tolerant to water scarcity. Di d h i i l di l d f Si il Leaf veins are a necessary connection for water supply to the stomata and thus, a key link between leaf function and stomatal and mesophyll anatomy (Brodribb and Holbrook, 2003; Brodribb et al., 2010; Scoffoni et al., 2016). This link has been described across diverse species and growth forms (e.g., Brodribb et al., 2007; Flexas et al., 2013b; Sack et al., 2015), but has been much less explored within crop species. In a comparison of domesticated tomato accessions including ‘de Ramellet’ with the tomato wild relative species (Solanum sect. Lycopersicon, sect. Lycopersicoides and sect. Juglandifolia), the domesticated had consistently larger leaf size and higher AN. This was achieved with the lowest vein density and more elastic cell walls (i.e., lowest bulk modulus of elasticity, ϵmax), (Conesa et al., unpubl.). DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES (2016) found that accessions with highest yield had also among the highest WUEi, standing out very tolerant and productive landraces like ‘Locale di Custonacci’. In such LSL landraces, drought tolerance was linked to leaf proline accumulation. Since proline accumulation is related to osmoregulation capacity, this could agree with d13C results above (Figure 3) suggesting that the drought tolerance strategy in LSL- cherry accessions is better related to maintenance of water intake than to water conservation. Nevertheless, important differences in the response to drought have been described in Italian landraces. In ‘Locale di Salina 6’ and ‘Pizzutello di Sciacca,’ Giorio et al. (2018) found a trade-off between gs and ABA accumulation. In ‘Crovarese’ under short-term water stress, Tamburino et al. (2017) also found high reduction of gs and increase of ABA and proline. In several LSL landraces from Campania including ‘Lucariello,’ ‘Crovarese’ and ‘Giallo Benaventano,’ Landi et al. (2017) described the constitutive activation of ROS detoxiﬁcation machinery as a fast response under stress. This involved enzymes that reduced the accumulation of ROS resulting from drought-induced photorespiration, with a parallel proline accumulation, thus also triggering osmotic adjustments as a response to drought. Deepening in the role of proline and ROS detoxiﬁcation in Mediterranean LSL landraces, and the comparison of the drought response in LSL landraces from different regions may unmask alternative strategies to the described above for ‘de Ramellet’ to adapt to water scarcity. At leaf mesophyll level, mechanisms to minimize growth and yield impairment under drought due to lower CO2 intake through stomata involve improvement of the CO2 delivery pathways from the substomatal cavity to the Rubisco, leading to improved mesophyll conductance (gm). This is a frequently described mechanism across plant groups and growth forms (Flexas et al., 2013a). Gas exchange and leaf anatomy measurements in ‘de Ramellet’ unravelled the mechanistic basis of improved gm under water deﬁcit conditions in this LSL landrace (Galmés et al., 2011; Galmés et al., 2013). In ‘de Ramellet,’ yield correlated positively with AN and negatively with WUEi, indicating that yield increases were at expenses of proportionally higher increases in gs (i.e, water consumption), (Galmés et al., 2011). As compared to WW, there was an increase of WUEi under WD, achieved through increased gm per unit water transpired, i.e., increased gm/gs ratio (Galmés et al., 2013). DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES (2018) found a trade-off between gs and ABA accumulation. In ‘Crovarese’ under short-term water stress, Tamburino et al. (2017) also found high reduction of gs and increase of ABA and proline. In several LSL landraces from Campania including ‘Lucariello,’ ‘Crovarese’ and ‘Giallo Benaventano,’ Landi et al. (2017) described the constitutive activation of ROS detoxiﬁcation machinery as a fast response under stress. This involved enzymes that reduced the accumulation of ROS resulting from drought-induced photorespiration, with a parallel proline accumulation, thus also triggering osmotic adjustments as a response to drought. Deepening in the role of proline and ROS detoxiﬁcation in Mediterranean LSL landraces, and the comparison of the drought response in LSL landraces from different regions may unmask alternative strategies to the described above for ‘de Ramellet’ to adapt to water scarcity. Leaf veins are a necessary connection for water supply to the accessions, irrespective of the severity of the stress (Patanè et al., 2016; Guida et al., 2017; Giorio et al., 2018). Moreover, AN, gs and WUEi values under drought were close to those reported for ‘de Ramellet’ (Galmés et al., 2011) and, similarly, the AN to gs relationship indicated a saturation of AN and thus, a disproportionate, suboptimal water consumption under non- stressing conditions (Guida et al., 2017; Giorio et al., 2018). Overall, despite maximum AN and gs varied considerably among Mediterranean LSL landraces and studies, WUEi under stress was grossly similar across landraces, with values at low gs (i.e., 0.15–0.30 mol m−2 s−1) close to 90–110 µmol mol−1 in “de Ramellet” (Galmés et al., 2011), Sicilian landraces (Guida et al., 2017; Giorio et al., 2018) and “Vesuviano” (Guida et al., 2017). water use efﬁciency (WUEi, as AN/gs). Leaf adaptations to increase WUEi in ‘de Ramellet’ occur at leaf surface and leaf mesophyll levels. At leaf surface level, stomatal anatomy modiﬁcations expected to provide higher WUEi are related to smaller stomata, higher stomatal density, and higher abaxial/adaxial ratio (Franks and Beerling, 2009). In agreement with that, ‘de Ramellet’ showed reduced stomatal size and increased stomatal density under WD as compared to WW. However, it is remarkable that WD adaptation resulted in increased stomatal density because of an increased adaxial density (Galmés et al., 2013). The lack of similar studies in other LSL, and even non-LSL landraces limits understanding if this is a common strategy for drought adaptation in Mediterranean landraces. In diverse Sicilian LSL landraces, Patanè et al. Frontiers in Plant Science \| www.frontiersin.org DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES Tomato accessions (n = 171) were separated in ﬁve groups depending on the accession type, including non-long shelf-life cherry (Cherry; n = 29), fresh market (Fresh; n = 50), long shelf-life from eastern Iberian Peninsula and Balearic Islands (LSL-big; n = 63) and from Italy (LSL-cherry; n = 16), and processing accessions (Processing; n = 13). (A) Percent of d13C in WD as compared to WW. ANOVA differences between WD and WW were signiﬁcant in all groups (P < 0.001). (B) Values of d13C in WW and (C) in WD. For each accession group, boxplots represent the average and the median (red and black lines inbox, respectively), the 75% interval (box), the 90% interval (error bars) and the outliers (isolated points). In each plot, letters on top indicate ANOVA-Tukey differences among groups (P < 0.05). January 2020 \| Volume 10 \| Article 1651 11 Frontiers in Plant Science \| www.frontiersin.org LSL Landraces for Tomato Improvement Conesa et al. accessions, irrespective of the severity of the stress (Patanè et al., 2016; Guida et al., 2017; Giorio et al., 2018). Moreover, AN, gs and WUEi values under drought were close to those reported for ‘de Ramellet’ (Galmés et al., 2011) and, similarly, the AN to gs relationship indicated a saturation of AN and thus, a disproportionate, suboptimal water consumption under non- stressing conditions (Guida et al., 2017; Giorio et al., 2018). Overall, despite maximum AN and gs varied considerably among Mediterranean LSL landraces and studies, WUEi under stress was grossly similar across landraces, with values at low gs (i.e., 0.15–0.30 mol m−2 s−1) close to 90–110 µmol mol−1 in “de Ramellet” (Galmés et al., 2011), Sicilian landraces (Guida et al., 2017; Giorio et al., 2018) and “Vesuviano” (Guida et al., 2017). In diverse Sicilian LSL landraces, Patanè et al. (2016) found that accessions with highest yield had also among the highest WUEi, standing out very tolerant and productive landraces like ‘Locale di Custonacci’. In such LSL landraces, drought tolerance was linked to leaf proline accumulation. Since proline accumulation is related to osmoregulation capacity, this could agree with d13C results above (Figure 3) suggesting that the drought tolerance strategy in LSL- cherry accessions is better related to maintenance of water intake than to water conservation. Nevertheless, important differences in the response to drought have been described in Italian landraces. In ‘Locale di Salina 6’ and ‘Pizzutello di Sciacca,’ Giorio et al. DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES Thus, as compared to wild tomato species, the domesticated accessions minimized the space occupied by the vein system, which maximizes the space occupied by mesophyll cells, maximizing AN. This contravenes general patterns in plants linking high AN Diverse drought experiments including landraces from Sicily and Campania denoted the lack of AN and gs differences among January 2020 \| Volume 10 \| Article 1651 12 LSL Landraces for Tomato Improvement Conesa et al. FIGURE 4 \| Cross sections of a ‘de Ramellet’ leaﬂet formed under (A) well-watered and (B) water stress (WS) conditions. (C) Detail of the mesophyll cells and airspaces under WS. Notice the chloroplasts (bright) tightly positioned against the cell membrane and cell wall. (D) Magniﬁcation of a few cells in C to highlight intercellular airspaces, that have been whitened to ease visualization. Black bar represents 200 µm in A and B, 40 µm in C and 10 µm in D. All images from the experiment described in Galmés et al. (2013). FIGURE 4 \| Cross sections of a ‘de Ramellet’ leaﬂet formed under (A) well-watered and (B) water stress (WS) conditions. (C) Detail of the mesophyll cells and airspaces under WS. Notice the chloroplasts (bright) tightly positioned against the cell membrane and cell wall. (D) Magniﬁcation of a few cells in C to highlight intercellular airspaces, that have been whitened to ease visualization. Black bar represents 200 µm in A and B, 40 µm in C and 10 µm in D. All images from the experiment described in Galmés et al. (2013). landraces and local population heritage (Zeven, 1998; Camacho- Villa et al., 2005). This is particularly true for in the Mediterranean LSL landraces (Bota et al., 2014; Patanè et al., 2016; Siracusa et al., 2018), making the high trait variability a characteristic trait deﬁning many of such landraces. with high vein density and high hydraulic conductance (e.g., Brodribb et al., 2007; Sack et al., 2015), highlighting that crop domestication might have led to alternative trait relationships in particular species like tomato. Nevertheless, within ‘de Ramellet’ higher vein density was related to higher hydraulic conductance (Galmés et al., 2013). Variation in Fruit Morphology There is a notorious difference in fruit shape between LSL landraces from Italy and from the Iberian Peninsula and Balearic Islands. The latter are in general round to ﬂat fruits, while Italian landraces are usually elongated, oval, pear-shaped and frequently bear a stylar end tip. Differences exist also in fruit size, with most Italian LSL landraces having small fruits, up to 30–40 g, while most Iberian and Balearic LSL landraces have bigger fruits (Table 2). To our knowledge, variation in fruit size and shape has been widely characterized only in the Balearic ‘de Ramellet’ (Figure 1), which is one of the most variable Mediterranean LSL landraces. Most fruits are ﬂat or round, existing also accessions with ellipsoid, heart, obovoid, oxheart, and rectangular fruit shapes (Table 3; Figure 1). DROUGHT TOLERANCE IN MEDITERRANEAN LSL LANDRACES Knowledge on the impact of leaf venation and hydraulics modiﬁcations under drought across diverse tomato LSL landraces may highlight important traits and trait- relationships at leaf level endowing superior capacity to overcome drought, suitable to be considered in breeding programs aimed to adapt tomato crop to future climate change conditions. TRAIT VARIABILITY WITHIN MEDITERRANEAN LSL LANDRACES A common characteristic in landraces is the high heterogeneity in most plant and fruit traits, resulting from variable selection criteria across local populations, due to the close link between January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 13 LSL Landraces for Tomato Improvement Conesa et al. FIGURE 5 \| Relationship between intrinsic water use efﬁciency (net photosynthesis and stomatal conductance ratio, AN/gs) and the ratio between the mesophyll cell surface exposed to airspaces (Sc) and the Rubisco concentration. Dots are different “de Ramellet” accessions grown under well- watered (black, soild regression line) and water-stress (grey, dashed regression line) conditions. The triangles represent a processing accession used as control. Plotted from data in Galmés et al. (2013). Ramellet’ from derived F1 hybrids, with consistently uniform coloration. Variation in Plant Morphology, Agronomic Traits and Yield Similar to fruit morphology variation, plant traits are also diverse within ‘de Ramellet’ (Ochogavía et al., 2011; Bota et al., 2014). When grown with no tutoring and no pruning, most accessions had plant volumes of 200–400 dm3 (Table 3). Regarding plant habit, most plants were climbing-erect, with 5–8 principal branches, and 8% of the accessions had potato leaf instead of the typical 5–9 leaﬂets leaves. There was also variation in phenology, with most accessions having relatively late production as compared to non-‘de Ramellet,’ and on average most productive period ranged between 85–120 days after transplantation (Ochogavía et al., 2011). Although root traits have not been assessed in detail in ‘de Ramellet,’ the variability in aerial plant parts' morphology suggest also differences in root, either extension and growth patterns. FIGURE 5 \| Relationship between intrinsic water use efﬁciency (net photosynthesis and stomatal conductance ratio, AN/gs) and the ratio between the mesophyll cell surface exposed to airspaces (Sc) and the Rubisco concentration. Dots are different “de Ramellet” accessions grown under well- watered (black, soild regression line) and water-stress (grey, dashed regression line) conditions. The triangles represent a processing accession used as control. Plotted from data in Galmés et al. (2013). FIGURE 5 \| Relationship between intrinsic water use efﬁciency (net photosynthesis and stomatal conductance ratio, AN/gs) and the ratio between the mesophyll cell surface exposed to airspaces (Sc) and the Rubisco concentration. Dots are different “de Ramellet” accessions grown under well- watered (black, soild regression line) and water-stress (grey, dashed regression line) conditions. The triangles represent a processing accession used as control. Plotted from data in Galmés et al. (2013). Agronomic evaluation of 48 ‘de Ramellet’ accessions grown outdoors under well-watered (WW) and water deﬁcit (WD) treatments showed a low effect of irrigation and ﬁeld conditions on fruit size (Table 4; Fullana-Pericàs et al., 2019). Most accessions produced 1.7–4.0 kg plant−1, and very variable fruit number per plant, from 25–150. In ca. 30% of the accessions, yield was higher under WD than under WW or invariable between treatments, denoting a low impact of water shortage, or a negative effect of higher water availability on yield. Results are similar in ‘de Penjar,’ being also variable among accessions and experiments (Table 2). Despite rare, up to 4% of the accessions had some degree of stylar end tip, all being elongated and pear-shaped fruits (Figure 1). Variation in Plant Morphology, Agronomic Traits and Yield Since the frequency of this trait in Italian tomatoes, this could represent some degree of introgression between Mallorcan and Italian landraces (Conesa et al., 2010). Added to the variation in fruit shape and size, fruit colour is also variable within the Balearic ‘de Ramellet.’ While most are kind of ‘pink tomatoes’ (with a colourless cuticle lacking ﬂavonoids), red fruited landraces (with yellow cuticle) also occur in ‘de Ramellet’ (Conesa et al., 2015). Most landraces have yellow to orange coloration in the half part closest to the pedicel (Figures 2A, B), which is attributable to pleiotropic effects of the alc mutation. This coloration-deﬁcient trait is appreciated by local consumers and helps recognizing ‘de Literature reports for yield in Italian LSL landraces also denote large differences across landraces and experiments, with values in open-ﬁeld and rain-fed conditions ranging from 12–16 t/Ha in e.g. ‘Locale di Salina 6,’ ‘Locale Giallo di Basilicò,’ ‘Pizzutello di Sciacca’ and the commercial ‘Principe Borghese,’ to ca. 100 t/Ha in ‘Corbarino’ (Table 2). Yield values per plant reported for ‘Piennolo Vesuviano’ (ca. 5.2 kg plant−1) and ‘Locale TABLE 3 \| Descriptive plant and fruit traits in “de Ramellet” (158 accessions) cultivated outdoors in Mediterranean summer under low irrigation (WW; see Table 2 for details). Fruit shape % acc. Fruit weight % acc. 6–month shelf–life % acc. Heart 4.9 <25 g 7.6 100% 2.1 Ellipsoid 6.3 25–50 g 50 99–75% 17.6 Flat 56.3 50–70 g 38 74–50% 38 Round 30.3 >70 g 4.4 49–25% 24.6 Obovoid 1.4 24–1% 13.4 Oxheart 0.7 0% 4.2 Rectangular 0.7 Plant volume % acc. Plant habit % acc. Num. branches % acc. <100 dm3 7 Climbing/erect 47 3–4 17 100–200 dm3 25 Typical indet. 43 5–6 32 200–400 dm3 50 Creeping 8 7–8 32 400–500 dm3 10 Small bush 2 9–10 6 >500 dm3 8 11 3 Data shown represents the % of accessions in each category or interval. Fruit traits include shape categories based on Tomato Analyzer (Brewer et al., 2006), average fruit weight, and accessions with >50% of fruits intact after 6–month shelf–life (170 days). Plant morphological traits are plant volume (based on two horizontal axes and maximum height), plant habit, and the number of principal branches. Data from, or recalculated from, Ochogavía et al. (2011) and Bota et al. (2014). Data shown represents the % of accessions in each category or interval. Variation in Fruit Quality Attributes Despite selection for the LSL phenotype converged in the need for fresh vegetables over-winter, organoleptic and physical properties of the fruit is contrastingly different across regions (Causse et al., 2010). In the Balearic Islands, ‘de Ramellet’ is recognized by higher acidity than most tomatoes (> 1 g citric acid 100 ml−1 at open- ﬁeld; Table 2), whereas most Italian LSL landraces have lower values (ca. 0.2–0.4 g citric acid 100 ml−1 at open-ﬁeld; Table 2). On the contrary, sugar content is lower in ‘de Ramellet’ (ca. 6.0 °Brix) than in most Italian landraces (ca. 7-8 °Brix), (Table 2), indicating that sugar content has been an important selective trait in Italian LSL landraces. Reports of acidity and sugar content for ‘de Penjar’ are similar to ‘de Ramellet’ (Table 2). On the one hand, the variation in fruit quality traits among LSL landraces denotes that the LSL phenotype is mostly unlinked to fruit quality attributes, agreeing with the different taste and The variation in fruit quality traits among LSL landraces, and among reports for the same landrace, may be in part the result of TABLE 4 \| Agronomic traits in a selection of 48 ‘de Ramellet’ accessions cultivated outdoors in Mediterranean summer under low irrigation (WW) and water deﬁcit (WD; see Table 2 for details), at a commercial production ﬁeld. Yield per plant (total) WW (% accessions) WD (% accessions) <600g 8.3 8.3 600–1,150g 4.2 10.4 1,150–1,700g 8.3 22.9 1,700–2,250g 16.7 16.7 2,250–2,850g 27.1 22.9 2,850–4,000g 22.9 16.7 4,000–6,000g 12.5 2.1 Average yield (g plant–1) 2603.4 ± 178.7 2017.1 ± 145.4 Average % WD vs WW 82.4 ± 4.3 (10–157%) % acc. WD > WW 29.2% (539–3648 g) Num. fruits per plant (total) WW (% accessions) WD (% accessions) <25 2.1 10.4 25–50 18.8 18.8 50–75 10.4 22.9 75–100 29.2 22.9 100–150 29.2 22.9 150–200 8.3 0.0 >200 2.1 2.1 Average num. fruits (plant−1) 90.2 ± 6.1 73.6 ± 5.7 Average % WD vs WW 85.5 ± 4.6 (15–168%) % acc. WD > WW 33.3% (48–235) Fruit weight (marketable) WW (% accessions) WD (% accessions) <25g 4.2 6.3 25–50g 22.9 20.8 50–70g 56.3 56.3 >70g 16.7 16.7 Average fruit weight (g) 57.4 ± 2.0 54.6 ± 2.4 Average % WD vs WW 101.1 ± 6.0 (31–340%) % acc. WD > WW 35.4% (43–89 g) The accession selection represents the highest genetic diversity in the UIB–collection. Variation in Plant Morphology, Agronomic Traits and Yield Fruit traits include shape categories based on Tomato Analyzer (Brewer et al., 2006), average fruit weight, and accessions with >50% of fruits intact after 6–month shelf–life (170 days). Plant morphological traits are plant volume (based on two horizontal axes and maximum height), plant habit, and the number of principal branches. Data from, or recalculated from, Ochogavía et al. (2011) and Bota et al. (2014). January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 14 LSL Landraces for Tomato Improvement Conesa et al. plants grown under different cultivation practices and environmental conditions, having important effects on fruit quality (e.g., Kirda et al., 2004; Patanè and Cosentino, 2010; Casa and Rouphael, 2014; D'Esposito et al., 2017). Cultivation conditions rendering smaller fruits, like drought stress, are in general resulting in a ‘concentration’ effect and ‘tastier’ fruits (Zanor et al., 2009; Panthee et al., 2013; Figàs et al., 2015; Tieman et al., 2017). Fullana-Pericàs et al. (2019) compared fruit quality attributes in diverse ‘de Ramellet’ grown under WW and WD treatments. WD signiﬁcantly increased sugar content, although acidity differences between WW and WD were non-signiﬁcant. Nevertheless, acidity reports for ‘de Ramellet’ in further experiments have been contrastingly different (Table 2), which indicates an effect of the environment and agronomical practices irrespective of, or in combination with, water availability. As compared to rain-fed conditions, the lower sugar content under irrigation has also been reported in Italian landraces like ‘Locale di Salina 6,’ ‘Piennolo Vesuviano’ and ‘Corbarino,’ (Table 2). di Salina 6’ (ca. 3.2 kg plant−1) are not very different to those in ‘de Ramellet’ and ‘de Penjar,’ although fruit number per plant is dramatically higher in Italian landraces, and often associated to the smaller fruit size (Table 2). MEDITERRANEAN LSL LANDRACES AS A RESOURCE FOR IMPROVING TOMATO CROP IN A CLIMATE CHANGE SCENARIO Most breeding efforts in tomato focused earlier in increasing yield and biotic stress resistances (Bai and Lindhout, 2007; Foolad, 2007; Labate and Robertson, 2012; Foolad and Panthee, 2012; Lin et al., 2014). In the 1980-90 decades there was also interest in extending fruit shelf-life, seeking for fruits supporting days-to-weeks export and storage, and having low shipping and transportation damage (Bai and Lindhout, 2007; Foolad, 2007; Díez and Nuez, 2008; Arah et al., 2015). More recently, there has been increasing interest in breeding for drought tolerance, aiming to adapt the tomato crop to climate change conditions, since water scarcity will be a main limitation to yield in important productive areas for tomato like the Mediterranean basin (Lane and Jarvis, 2007; Rockstrom et al., 2007; Lobell and Gourdji, 2012; Dwivedi et al., 2016). However, most of the improvement programs related to extended shelf-life and drought tolerance involved mutants (e.g., rin, nor; Garg et al., 2008; Klee and Giovannoni, 2011) and wild tomato species (e.g., Solanum pennellii; Bolger et al., 2014), respectively. Agricultural biodiversity is a prime resource for novel breeds adaptation (Lane and Jarvis, 2007; Frison et al., 2011). The vast majority of LSL landraces are maintained in home-gardens and orchards for self-consumption, through own selection and seed storage, with a tight and ancient link to the local gastronomy and culture (e.g., Andreakis et al., 2004; Siracusa et al., 2012; Bota et al., 2014; Mercati et al., 2015; Patanè et al., 2016). Changes in cultural practices and lower interest in agriculture in upcoming generations leads to a dramatic genetic erosion in landraces across the Mediterranean (Cebolla-Cornejo et al., 2007; Terzopoulos and Bebeli, 2008; Mazzucato et al., 2008; Bota et al., 2014; Cortés-Olmos et al., 2015; Mercati et al., 2015). Consequently, there is urgent need for researchers, breeders, growers, public managers and policy makers to work collaboratively towards the characterisation, conservation, develop protection regulations and revalorisation of Mediterranean LSL landraces, to preserve this rich local g p g y Paradoxically, the Mediterranean LSL landraces, with dramatically extended shelf-life, and selected for centuries under severe drought conditions, are an almost neglected TABLE 5 \| Fruit quality parameters corresponding to ‘de Ramellet’ accessions in Table 3. Variation in Fruit Quality Attributes On the other hand, this describes a diverse array of quality attributes and fruit morphologies in the LSL landraces, making those a very rich resource to improve ﬂavour linked to extended shelf-life. There is also variation in fruit quality traits in the LSL landraces during postharvest conservation, with decreased sugar content and titratable acidity, and increased pH and ﬁrmness, over six-month shelf-life in ‘de Ramellet’ (Table 5). Very similar results have been reported for ‘de Penjar,’ with a general decrease of different sugars and volatile compounds during conservation, although the proﬁle for acids was variable (Casals et al., 2015). In both ‘de Ramellet’ and ‘de Penjar,’ most changes occurred during the ﬁrst two months, with lower changes afterwards. Trends for ﬁrmness showed variable patterns in ‘de Ramellet,’ with a dramatically increase during the ﬁrst two months, followed by a slow decrease afterwards, globally leading to increased ﬁrmness after six months (Ochogavía et al., 2011; Bota et al., 2014). Flavour has been largely neglected during tomato improvement and currently many consumers demand tomatoes tasting “like before.” Notorious efforts are being made to decipher determinants of tomato ﬂavour and aroma in order to breed for it in the near future (Brugarolas et al., 2009; Zanor et al., 2009; Causse et al., 2010; Klee and Tieman, 2013; Tieman et al., 2017). Different from biotic and abiotic resistances, for which wild relatives have been a key resource (Foolad, 2007), yearned tomato ﬂavour relies on extant heirloom and landraces (Brugarolas et al., 2009; Figàs et al., 2015; Baldina et al., 2016; Tieman et al., 2017). In this regard, preferences for taste are very dependent on the uses and regions considered and thus, a diverse array of fruit quality traits must be considered (Causse et al., 2010). Particularly in the Mediterranean, the large variation observed for LSL landraces in fruit size, shape and quality attributes, constitute a very suitable resource to seek for ancient tomato ﬂavour, and as a source to breed for improved tomato taste. Variation in Fruit Quality Attributes Yield and fruit number are per plant and correspond to total production (i.e., marketable and damaged), while average fruit weight corresponds only to marketable fruits. In each interval, values correspond to the % of accessions in the WW and WD treatments. The global effect of WD on each trait is indicated as the average and SE in each treatment (asterisk in WD indicates signiﬁcant differences between treatments; P < 0.05), the average of the % in WS as compared to WW (the variation range shown in brackets), and the % of accessions with higher value in WS than in WW (the variation range of such accessions under WD shown in brackets). Data recalculated from Fullana–Pericàs et al. (2019). 48 ‘de Ramellet’ accessions cultivated outdoors in Mediterranean summer under low irrigation (WW) and water deﬁcit (WD; duction ﬁeld The accession selection represents the highest genetic diversity in the UIB–collection. Yield and fruit number are per plant and correspond to total production (i.e., marketable and damaged), while average fruit weight corresponds only to marketable fruits. In each interval, values correspond to the % of accessions in the WW and WD treatments. The global effect of WD on each trait is indicated as the average and SE in each treatment (asterisk in WD indicates signiﬁcant differences between treatments; P < 0.05), the average of the % in WS as compared to WW (the variation range shown in brackets), and the % of accessions with higher value in WS than in WW (the variation range of such accessions under WD shown in brackets). Data recalculated from Fullana–Pericàs et al. (2019). January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 15 LSL Landraces for Tomato Improvement Conesa et al. genetic resource for tomato improvement. Unlike many non- LSL varieties, LSL landraces do not need to be harvested at breaker stage and ﬁnish ripening in chamber-storage to increase shelf-life. Furthermore, due to the ability to maintain fruits sound, LSL tomatoes have higher resistance to microbial attack than most tomatoes, and high cicatrization capacity after damage on the vine like cracking, which probably enhances microbial attack prevention (pers. obs.; Figure 6). Thus, the LSL phenotype is a valuable trait to reduce costs of harvesting and storage previous to commercialization (Arah et al., 2015). uses across Mediterranean landraces. MEDITERRANEAN LSL LANDRACES AS A RESOURCE FOR IMPROVING TOMATO CROP IN A CLIMATE CHANGE SCENARIO Harvest Max Min 2 months 6 months Firmness (kg cm−2) 1.14 ± 0.04a 1.82–2.86 0.19–0.60 1.80 ± 0.03c 1.57 ± 0.04b Sugar content (°Brix) 5.90 ± 0.06c 7.13–8.75 4.00–5.00 5.48 ± 0.07b 5.10 ± 0.08a Titratable acidity (g citric ac. 100 ml−1) 1.48 ± 0.03c 1.95–2.75 0.80–1.00 1.09 ± 0.02b 0.71 ± 0.02a pH 3.85 ± 0.01a 4.11−4.28 3.27−3.57 4.19 ± 0.01b 4.49 ± 0.02c Fruit ﬁrmness (with penetrometer), sugar content (total soluble solids), titratable acidity and pH are shown at harvest and after two and six months (55 and 170 days, respectively) of postharvest storage in a ventilated shed at ambient temperature, as frequently performed by self-consumption growers. Values correspond to averages and standard error for all the ‘de Ramellet’ accessions tested. Letters denote statistically signiﬁcant differences within each parameter by ANOVA-Tukey (P < 0.001). At harvest time, variation ranges of the 10 accessions with maximum (Max) and with minimum (Min) values are shown to demonstrate variability in the extremes for each trait. Data from Ochogavía et al. (2011) and Bota et al. (2014). Fruit ﬁrmness (with penetrometer), sugar content (total soluble solids), titratable acidity and pH are shown at harvest and after two and six months (55 and 170 days, respectively) of postharvest storage in a ventilated shed at ambient temperature, as frequently performed by self-consumption growers. Values correspond to averages and standard error for all the ‘de Ramellet’ accessions tested. Letters denote statistically signiﬁcant differences within each parameter by ANOVA-Tukey (P < 0.001). At harvest time, variation ranges of the 10 accessions with maximum (Max) and with minimum (Min) values are shown to demonstrate variability in the extremes for each trait. Data from Ochogavía et al. (2011) and Bota et al. (2014). January 2020 \| Volume 10 \| Article 1651 Frontiers in Plant Science \| www.frontiersin.org 16 LSL Landraces for Tomato Improvement Conesa et al. FIGURE 6 \| Resistance of ‘de Ramallet’ fruits during post-harvest storage. (A) Fruit with slight wrinkling due to water loss six months after harvest. (B) Fruit with important scariﬁcation a month after a microbial attack, showing the zone of attack (left) and the rest of the fruit (right) remaining intact, without wrinkling. (C) Fruit with an important microbial attack emptying half part of the fruit (left) but not the other part (right) which remains intact. Fruits as in B and C can remain as in the picture up to 6 months. MEDITERRANEAN LSL LANDRACES AS A RESOURCE FOR IMPROVING TOMATO CROP IN A CLIMATE CHANGE SCENARIO (D) Fruit completely dry after suffering a microbial attack during postharvest storage. Notice that the dark part was attacked and that the clear half of the fruit maintained integrity until complete water loss. The fruit remains as in the picture for decades with no further deterioration. FIGURE 6 \| Resistance of ‘de Ramallet’ fruits during post-harvest storage. (A) Fruit with slight wrinkling due to water loss six months after harvest. (B) Fruit with important scariﬁcation a month after a microbial attack, showing the zone of attack (left) and the rest of the fruit (right) remaining intact, without wrinkling. (C) Fruit with an important microbial attack emptying half part of the fruit (left) but not the other part (right) which remains intact. Fruits as in B and C can remain as in the picture up to 6 months. (D) Fruit completely dry after suffering a microbial attack during postharvest storage. Notice that the dark part was attacked and that the clear half of the fruit maintained integrity until complete water loss. The fruit remains as in the picture for decades with no further deterioration. heritage (Zeven, 1998; Camacho-Villa et al., 2005; Negri et al., 2009; Frison et al., 2011; Elia and Santamaria, 2013; Corrado et al., 2014; Garcia-Mier et al., 2014; Dwivedi et al., 2016; Casañas et al., 2017). tomato for fruit quality, shelf-life and cultivation under the predicted climate change conditions. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article. AUTHOR CONTRIBUTIONS The Mediterranean LSL tomatoes are a group of landraces with dramatically extended fruit shelf-life after harvest, mostly occurring in Eastern Iberian Peninsula, the Balearic Islands, and Southern Italy. In these areas, LSL landraces are commercialized in local markets and are an important part of the local culture and heritage. Besides extended shelf-life, most LSL landraces are drought tolerant, as a consequence of ancient selection under Mediterranean summer conditions with poor irrigation or rain-fed. Therefore, LSL landraces constitute an alternative to wild species as a source of genes to improve drought stress tolerance. Moreover, the large variation in fruit morphology, fruit quality traits and ﬂavor within and among landraces makes the LSL landraces an attractive source to breed for future tomato cultivars with a genetic background conferring extended shelf-life and drought tolerance. Currently, genetic erosion is a prime in most Mediterranean LSL landraces and there is an urgent need to preserve and revalue this important genetic resource, which has notorious traits suitable to improve MC and JG conceived the idea and the review scheme. MC drafted the manuscript. All authors did signiﬁcant contributions improving the ﬁnal version of the manuscript. 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https://openalex.org/W3015388576	https://europepmc.org/articles/pmc7154125?pdf=render	English	null	Embryology and Morphological (Mal)Development of UPJ	Frontiers in pediatrics	2,020	cc-by	2,384	Embryology and Morphological (Mal)Development of UPJ 1 Division of Pediatric Urology, Department of Pediatric Surgery, Ege University, Izmir, Turkey, 2 Gaziantep Maternity and Children’s Hospital, Pediatric Urology, Gaziantep, Turkey Kidney parenchyma and collecting system arise from two different embryologic units as a result of a close interaction between them. Therefore, their congenital abnormalities are classiﬁed together under the same heading named CAKUT (congenital abnormalities of the kidney and urinary tract). The pathogenesis of CAKUT is thought to be multifactorial. Ureteropelvic junction obstruction (UPJO) is the most common and most investigated form of CAKUT. Despite years of experimental and clinical research, and the information gained on the embryogenesis of the kidney; its etiopathogenesis is still unclear. It involves both genetic and environmental factors. Failure in development of the renal pelvis, failure in the recanalization of ureteropelvic junction, abnormal pyeloureteral innervation, and impaired smooth muscle differentiation are the main proposed mechanisms for the occurrence of UPJO. There are also single gene mutations like AGTR2, BMP4, Id2 proposed in the etiopathogenesis of UPJO. MINI REVIEW MINI REVIEW published: 07 April 2020 doi: 10.3389/fped.2020.00137 published: 07 April 2020 doi: 10.3389/fped.2020.00137 Edited by: Edited by: Alberto Parente, Consultant, Córdoba, Spain Alberto Parente, Consultant, Córdoba, Spain Reviewed by: Huixia Zhou, Bayi Children’s Hospital, China Andres Gomez Fraile, University Hospital October 12, Spain Correspondence: Ali Avanoglu ali.avanoglu@gmail.com INTRODUCTION Reviewed by: Huixia Zhou, Bayi Children’s Hospital, China Andres Gomez Fraile, University Hospital October 12, Spain The role of embryology in medical education is often underrated. Even clinicians dealing with congenital abnormalities consider in-depth knowledge on embryology unnecessary. Studies about urinary tract obstruction date back to forties (1), but there is still scarce information on how ureteropelvic junction obstruction (UPJO) develops. We believe, clinicians and embryologists shall work together to obtain further progress. The aim of this review is to demystify the current knowledge on the embryo-pathogenesis of UPJO to clinicians to promote future research. Correspondence: Ali Avanoglu ali.avanoglu@gmail.com THE CONCEPT OF CAKUT Specialty section: This article was submitted to Pediatric Urology, a section of the journal Frontiers in Pediatrics Specialty section: This article was submitted to Pediatric Urology, a section of the journal Frontiers in Pediatrics Congenital anomalies of the kidney and urinary tract (CAKUT) refer to all the developmental abnormalities of kidney and ureter (2). The concept of CAKUT is based on the close interaction of the ureteric bud and metanephric mesenchyme in the development of kidney and ureter. Received: 27 January 2020 Accepted: 11 March 2020 Published: 07 April 2020 The main steps in the formation of the metanephric kidney and ureter are; formation of the ureteric bud from the Wolﬃan duct, its dorsal growth into the caudal portion of the nephric cord and branching of the ureter when it invades the mesenchyme. This appositional growth continues until the formation of the terminal nephrons in 32nd week in human embryo (3). Keywords: ureteropelvic junction obstruction, embryology, genetics, congenital anomalies of the kidney and urinary tract, BMP4 THE GENETICS CAKUT is thought to be multifactorial. There are familial cases with diﬀerent occurrence, so genetic penetrance is regarded to be incomplete or variable. Also, there are several single gene mutations like Id2, PAX2, EYA, AGTR2, BMP4, SOX17, CHD1L, DSTYK proposed by the experimental and clinical studies about the etiopathogenesis of UPJO (16–19). However, mutations in these mostly results in more than one form of CAKUT. For example, mutant mice has a 3% chance of developing CAKUT when AGTR2 is inactivated, but it can be any type and happens randomly within the same pedigree (2). Miyazaki et al. showed angiotension type 1 lacking mice failed to develop a renal pelvis (11). They also showed hypoplastic smooth muscle and lacking peristalsis in the ureters of mutant mice. Reminding the results of Miyazaki’s experimental study, Kajbafzadeh et al. showed increased smooth muscle cell apoptosis and collagen ﬁbers while a decreased number of nerve terminals in the UPJO specimens compared to normal ureteropelvic junctions from autopsies (12). These studies strongly suggest defective muscle and nerve structure in the site of obstruction, but it is still unknown if these are the causative changes or the results of the obstruction. Later, Yiee et al. compared intrinsic, and extrinsic cases focusing on the muscle distribution. Their ﬁndings support a causative role by revealing a diﬀerent muscle density between them (13). Among these, Adamts1 and Id2 are reported to lead to a more restricted phenotype resembling human UPJO (17). Interestingly, the macroscopic morphology of the kidney of the Id2 knock-out mice even shows the high-insertion of the ureter into the pelvis (17). BMP4 also has noteworthy features. It has an essential role in embryonic development shown by the fatality of the homozygous null mutations. Heterozygous mutation results, on the other hand, in multiple abnormalities including all types of CAKUT. It is also shown to cause ectopic budding of the ureter (like Mackie and Stephens described) (20). BMP4’s role may seem too wide to explain UPJO alone; however, two screening studies showed its association with UPJO (21, 22). The study from China revealed BMP4 mutation in three cases with UPJO which were not apparent in the controls (21). Same study failed to show any speciﬁc mutation in Id2 gene. The other one from Brazil showed Chang et al. generated an animal model of UPJO with a mutation in a calcineurin protein subunit (14). Citation: Avanoglu A and Tiryaki S (2020) Embryology and Morphological (Mal)Development of UPJ. Front. Pediatr. 8:137. doi: 10.3389/fped.2020.00137 Experimental studies with knock-out mice support this interactive development delivering the renal parenchymal and ureteric abnormalities together. In fact, most popular theory about this close interaction by Mackie and Stephens was even earlier than these. They hypothesized that the association of renal parenchymal abnormalities with vesicoureteral reﬂux and other ureteric April 2020 \| Volume 8 \| Article 137 Frontiers in Pediatrics \| www.frontiersin.org Embryology of UPJO Avanoglu and Tiryaki abnormalities were the result of initial ectopic budding of the ureter (4). shape of the pelvis and faulty mesenchyme with abnormal pyeloureteral peristalsis which they concluded as the cause of UPJO. CAKUT accounts for one of the most frequent congenital abnormalities detected by routine fetal sonography (5), but the spectrum is wide. Ureteropelvic junction obstruction is the most common form of CAKUT with an estimated incidence of 1/1,000–1,500 (6). Based on the studies about peristalsis, Lye et al. speculated that peristalsis in the urinary tract becomes more important in late gestation when the fetus stays upside down and urine travels against gravity. They concluded that failure of peristalsis results in a functional obstruction manifested by hydronephrosis (15). THEORIES ON UPJO PATHOGENESIS In fact, none of the above studies describe the macroscopic ﬁndings of the surgeon which are as follows: mostly there is narrow but patent lumen, ureter inserts the pelvis in a level higher than ureter and pelvis ﬁrst meet, they are attached to each other between these two levels and there is ﬁbrotic tissue around. Stephen Koﬀhas an interesting idea about this that he never published. He believes UPJO is a consequence of temporary vesicoureteral reﬂux during the fetal life. He says reﬂux disrupts the position of the ureter and UPJ, and then pelvic drainage. When this lasts long enough, it results in inﬂammation and the ﬁbrotic attachments around and UPJO becomes permanent (Koﬀ, personal communication). The ﬁrst theory was obliteration-recanalization by Ruano- Gil and Tejedo-Mateu which they raised on their ﬁndings on 45 normal human embryos of 5–55 mm (7). They said the ureter becomes obstructed beginning when the fetus is 14 mm, this process starts in the middle zone and progresses to the entire lumen, and then recanalization occurs after the fetus is 22 mm (7). Later, Alcaraz et al. also supported the existence of an obstructive phenomena of the ureter with their study on human and rat embryos (8); however, showed that this obstruction site didn’t reach the ureteropelvic junction. After that, obstruction-recanalization theory to explain UPJO was abandoned by the majority. Also others think this obstruction phenomenon can only be the collapse of the ureter before the passage of the urine (2). Despite the above theories and two very interesting speculations, further studies are still required to reveal etiopathogenesis of UPJO. Other early studies about the subject were pathological analyses of the specimens with UPJO. They all noted the changes in the ureteropelvic junction (UPJ) without attribution to the etiology (9). Zhang et al. were also researchers who analyzed UPJO specimens. They showed that UPJs were thicker with enlarged muscularis propria, increased perifascicular ﬁbrosis and inﬂammation in cases with intrinsic UPJO (10). They also couldn’t make a statement whether these changes were causative but showed that they were not apparent in the extrinsic cases. Frontiers in Pediatrics \| www.frontiersin.org REFERENCES 14. Chang CP, McDill BW, Neilson JR, Joist HE, Epstein JA, Crabtree GR, et al. Calcineurin is required in urinary tract mesenchyme for the development of the pyeloureteral peristaltic machinery. J Clin Invest. (2004) 113:1051–8. doi: 10.1172/JCI20049 1. Peters CA. Obstruction of the fetal urinary tract. J Am Soc Nephrol. (1997) 8:653–63. 2. Ichikawa I, Kuwayama F, Pope JC, Stephens FD, Miyazaki Y. Paradigm shift from classic anatomic theories to contemporary cell biological views of CAKUT. Kidney Int. (2002) 61:889–98. doi: 10.1046/j.1523-1755.2002.00188.x 15. Lye CM, Fasano L, Woolf AS. Ureter myogenesis: putting teashirt into context. J Am Soc Nephrol. (2010) 21:24–30. doi: 10.1681/ASN.2008111206 16. dos Santos Junior ACS, de Miranda DM, Simões e Silva AC. Congenital anomalies of the kidney and urinary tract: an embryogenetic review. Birth Defects Res C Embryo Today Rev. (2014) 102:374–81. doi: 10.1002/bdrc.21084 3. Saxen L. Ontogenesis of the vertebrate excretory system. In: Barlow P, Green P, Wylie C, editors. Ontogenesis of the Kidney. Cambridge: Cambridge University Press (1987). p. 13–8. 17. Aoki Y, Mori S, Kitajima K, Yokoyama O, Kanamura H, Okada K, et al. Id2 haploinsuﬃciency in mice leads to congenital hydronephrosis resembling that in humans. Genes Cells. (2004) 9:1287–96. doi: 10.1111/j.1365-2443.2004.00805.x 4. Mackie GG, Stephens FD. Duplex kidneys: a correlation of renal dysplasia with position of the ureteral oriﬁce. J Urol. (1975) 114:274–80. doi: 10.1016/S0022-5347(17)67007-1 5. Caiulo VA, Caiulo S, Gargasole C, Chiriacò G, Latini G, Cataldi L, et al. Ultrasound mass screening for congenital anomalies of the kidney and urinary tract. Pediatr Nephrol. (2012) 27:949–53. doi: 10.1007/s00467-011-2098-0 18. Yerkes E, Nishimura H, Miyazaki Y, Tsuchida S, Brock JW, Ichikawa I. Role of angiotensin in the congenital anomalies of the kidney and urinary tract in the mouse and the human. Kidney Int Suppl. (1998) 54:75–7. doi: 10.1046/j.1523-1755.1998.06715.x 5. Caiulo VA, Caiulo S, Gargasole C, Chiriacò G, Latini G, Cataldi L, et al. Ultrasound mass screening for congenital anomalies of the kidney and urinary tract. Pediatr Nephrol. (2012) 27:949–53. doi: 10.1007/s00467-011-2098-0 6. Klein J, Gonzalez J, Miravete M, Caubet C, Chaaya R, Decramer S, et al. Congenital ureteropelvic junction obstruction: human disease and animal models. Int J Exp Pathol. (2011) 92:168–92. doi: 10.1111/j.1365-2613.2010.00727.x 19. Vivante A, Kohl S, Hwang DY, Dworschak GC, Hildebrandt F. Single-gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in humans. Pediatr Nephrol. (2014) 29:695–704. doi: 10.1007/s00467-013-2684-4 7. Ruano Gil D, Coca Payeras A, Tejedo Mateu A. THE GENETICS The mutant mice had abnormal renal mesenchyme and lack of a funnel-shaped ureteropelvic junction. They showed no abnormality in the nerve distribution. They correlated abnormal April 2020 \| Volume 8 \| Article 137 2 Avanoglu and Tiryaki Embryology of UPJO the association of BMP4 mutation with UPJO and multicystic dysplastic kidney (22). Despite years of clinical and experimental research, there is no solid theory or genetic mutation to explain this frequent abnormality yet. Despite promising results of these papers, data to acknowledge a causative role of any gene is still lacking. CONCLUSION AA and ST contributed to the literature search and drafting the manuscript of this review. The etiopathogenesis and impacts of ureteropelvic junction obstruction has long been an interesting area for researchers. REFERENCES Obstruction and normal recanalization of the ureter in the human embryo. its relation to congenital ureteric obstruction. Eur Urol. (1975) 1:287–93. 20. Miyazaki Y, Ichikawa I. Ontogeny of congenital anomalies of the kidney and urinary tract, CAKUT. Pediatr Int. (2003) 45:598–604. doi: 10.1046/j.1442-200X.2003.01777.x 21. He JL, Liu JH, Liu F, Tan P, Lin T, Le XL. Mutation screening of BMP4 and Id2 genes in Chinese patients with congenital ureteropelvic junction obstruction. Eur J Pediatr. (2012) 171:451–456. doi: 10.1007/s00431-011- 1561-z 8. Alcaraz A, Vinaixa F, Tejedo-Mateu A, Fores M, Gotzens V, Mestres C, et al. Obstruction and recanalization of the Ureter during embryonic development. J Urol. (1991) 145:410–6. doi: 10.1016/S0022-5347(17)38354-4 9. Starr NT, Maizels M, Chou P, Brannigan R, Shapiro E. Microanatomy and morphometry of the hydronephrotic obstructed renal pelvis in asyptomatic infants. J Urol. (1992) 148:519–24. doi: 10.1016/S0022-5347(17)36643-0 22. Reis GS Dos, Simões E Silva AC, Freitas IS, Heilbuth TR, Marco LA De, Oliveira EA, et al. Study of the association between the BMP4 gene and congenital anomalies of the kidney and urinary tract. J Pediatr. (2014) 90:58– 64. doi: 10.1016/j.jped.2013.06.004 10. Zhang PL, Peters CA, Rosen S. Ureteropelvic junction obstruction: morphological and clinical studies. Pediatr Nephrol. (2000) 14:820–6. doi: 10.1007/s004679900240 11. Miyazaki Y, Tsuchida S, Nishimura H, Pope JC IV, Harris RC, McKanna JM, et al. Angiotensin induces the urinary peristaltic machinery during the perinatal period. J Clin Invest. (1998) 102:1489–97. doi: 10.1172/JCI4401 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 12. Kajbafzadeh AM, Payabvash S, Salmasi AH, Monajemzadeh M, Tavangar SM. Smooth muscle cell apoptosis and defective neural development in congenital ureteropelvic junction obstruction. J Urol. (2006) 176:718–23. doi: 10.1016/j.juro.2006.03.041 Copyright © 2020 Avanoglu and Tiryaki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 13. Yiee JH, Johnson-Welch S, Baker LA, Wilcox DT. Histologic diﬀerences between extrinsic and intrinsic ureteropelvic junction obstruction. Urology. (2010) 76:181–4. Frontiers in Pediatrics \| www.frontiersin.org REFERENCES doi: 10.1016/j.urology.2010.02.007 April 2020 \| Volume 8 \| Article 137 Frontiers in Pediatrics \| www.frontiersin.org 3
https://openalex.org/W2904473079	https://europepmc.org/articles/pmc6290385?pdf=render	English	null	Retroperitoneal hematoma from lumbar artery as an unusual complication after rectal perforation with massive retroperitoneal emphysema	Journal of surgical case reports	2,018	cc-by	2,068	Takashi Miyata1,, Yuta Fujiwara1, Koji Nishijima1, Fumio Futagami1, Takashi Nakamura1, Takahisa Yamaguchi2, Koichi Okamoto2, Isamu Makino2, Tomoharu Miyashita2, and Tetsuo Ohta2 Takashi Miyata1,, Yuta Fujiwara1, Koji Nishijima1, Fumio Futagami1, Takashi Nakamura1, Takahisa Yamaguchi2, Koichi Okamoto2, Isamu Makino2, Tomoharu Miyashita2, and Tetsuo Ohta2 1Department of Surgery, Japanese Red Cross Kanazawa Hospital, Kanazawa, Ishikawa 9218162, Japan and 2Department of Gastroenterological Surgery, Division of Cancer Medicine Graduate School of Medicine, Kanazawa University, Kanazawa, Ishikawa 9208641, Japan *Correspondence address. Department of General and Digestive Surgery, Kanazawa Medical University Hospital, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 9200293, Japan. Tel: +81-76-286-2211; Fax: +81-76-286-4626; E-mail: ryutami5383917@gmail.com INTRODUCTION the case of spontaneous retroperitoneal hematoma occurring in a woman with adult Still’s disease undergoing high-dose steroid and cyclosporine therapy who was precisely diagnosed and successfully treated. the case of spontaneous retroperitoneal hematoma occurring in a woman with adult Still’s disease undergoing high-dose steroid and cyclosporine therapy who was precisely diagnosed and successfully treated. Retroperitoneal hematoma is a rare clinical entity that can be fatal and requires rapid treatment for patient survival [1]. It is frequently seen with a complication such as femoral artery catheterization, pelvic or lumbar trauma, rupture of aneur- ysms, or anticoagulation therapy [2, 3]. However, retroperiton- eal hematoma caused by postoperative peritonitis with retroperitoneal emphysema is extremely rare. We herein report Abstract The patient was a 54-year-old woman diagnosed with adult Still’s disease, undergoing high-dose steroid and immunosup- pressant therapy for 5 years, who was admitted to our hospital with abdominal pain. Computed tomography (CT) revealed pneumoperitoneum around the rectum and a large quantity of retroperitonal emphysema around the inferior vena cava, aorta and left kidney. An emergency laparotomy was performed. Intraoperative observation revealed a perforation on the mesenteric side of the rectum due to diverticulum, and Hartmann’s operation was performed. Deep tenderness and anemia were observed 4 days postoperatively. CT revealed extravasation in the left retroperitoneal space and a retroperitoneal hematoma, and emergency embolization of lumbar arteries was performed. Retroperitoneal bleeding associated with peri- tonitis after surgery is very rare. We surmised that higher-dose immunosuppressive therapy and gastrointestinal perfor- ation with emphysema in the retroperitoneum induced lumbar artery bleeding. Clinicians should consider these factors as a potential cause of retroperitoneal hematoma. Journal of Surgical Case Reports, 2018;12, 1–4 Journal of Surgical Case Reports, 2018;12, 1–4 doi: 10.1093/jscr/rjy332 Case Report doi: 10.1093/jscr/rjy332 Case Report doi: 10.1093/jscr/rjy332 Case Report p Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: October 22, 2018. Accepted: November 14, 2018 CASE REPORT A second emergency laparotomy was performed, which revealed cecal perforation with no obvious masses (Fig. 5b). Perforation resulting from diverticulum was suspected, and an ileostomy without intraperitoneal anastomosis was performed because of concern about anastomotic leakage. Postoperatively the patient developed an intra-abdominal abscess, surgical site infection (Clavien-Dindo IIIa) and pneumonia (Clavien-Dindo II), which were treated conservatively. Although it took time to rehabili- tate the patient and control the adult Still’s disease, she was discharged on Day 212 after the ﬁrst operation. The patient is now doing well with comfortable activity of daily life. Figure 2: Laparotomy revealed the peritoneal ﬂuid collection. seen as a complication of interventions such as femoral artery catheterizations and pelvic or lumbar trauma [2]. In this report, we present an extremely rare case of retroperitoneal hema- toma after surgery of rectal perforation with peritonitis. Two clinical circumstances, pneumoretroperitoneum and immuno- suppressive therapy, were potential etiologic factors leading to retroperitoneal hematoma. Pneumoretroperitoneum caused by colonic retroperitoneal perforation is a rare clinical condition, the most common cause of which is diverticular disease [4]. Colonic retroperitoneal per- foration leads to the spread of abscess, gas and necrosis along the retroperitoneum, occasionally extending to the mediasti- num. The proposed surgical procedure for this condition is colonic resection and radical debridement [4]. To the best of our knowledge, however, there have been no reports in the English literature of the postoperative complication of retro- peritoneal hematoma associated with pneumoretroperitoneum caused by colonic retroperitoneal perforation. CASE REPORT A 54-year-old woman was referred to our hospital because of abdominal pain. She had a history of adult Still’s disease at age Received: October 22, 2018. Accepted: November 14, 2018 Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2 \| T. Miyata et al. 2 Figure 1: (a) Enhanced abdominal CT showed free air around the rectum (yellow arrow), and strongly suggested perforation of the rectum. (b) Massive continuous pneumoretroperitoneum from rectum was revealed (yellow arrows). Figure 1: (a) Enhanced abdominal CT showed free air around the rectum (yellow arrow), and strongly suggested perforation of the rectum. (b) Massive continuous pneumoretroperitoneum from rectum was revealed (yellow arrows). Figure 2: Laparotomy revealed the peritoneal ﬂuid collection. 49 and underwent treatment with oral prednisolone, 90 mg/day and cyclosporine, 175 mg/day. A physical examination revealed deep tenderness in the abdomen. Laboratory data showed slight leukocytosis (white blood cell count 9100/μL) with a mod- erately elevated C-reactive protein level (9.3 mg/dL), while other data, including blood coagulation factor, were within normal ranges. Computed tomography (CT) revealed a small amount of extra-intestinal free air around the rectum and massive retro- peritoneal emphysema between the rectum and the left kidney (Fig. 1a and b). Arterial aneurysm was not conﬁrmed. Based on a preoperative diagnosis of rectal perforation, emergency lapar- otomy was performed, which conﬁrmed peritoneal ﬂuid collec- tion (Fig. 2) and rectum perforation on the retroperitoneal side. After aspiration of the pus and irrigation of the area with saline, Hartmann’s operation was performed (Fig. 3a). Although she was undergoing immunosuppressive treatment, patho- logical study disclosed no association between diverticulum perforation and cytomegalovirus enteritis (Fig. 3b). On post- operative Day 4, she suffered a sudden intolerable left ﬂank pain; her hemoglobin level was 7.5 g/dL, and slight prolongation of prothrombin time was recognized. CT revealed a left retro- peritoneal hematoma and extravasation from the left ﬁrst lum- bar arteries (Fig. 4a). Emergency transarterial angiography and lumbar artery embolization was performed (Fig. 4b). On Day 20 after the ﬁrst operation the patient felt a sudden right ﬂank pain, and CT conﬁrmed intra-abdominal free air (Fig. 5a). DISCUSSION The association between steroid use and gastrointestinal ulceration or perforation is now well established. Chronic high- dose steroid treatment has been related to decline of some cell- Retroperitoneal hematoma is a well-known, but rare, clinical condition that can be potentially lethal [1]. It is most frequently Retroperitoneal hematoma after surgery for rectal perforation \| 3 Retroperitoneal hematoma after surgery for rectal perforation \| 3 gure 3: (a) The resected specimen showed perforation at the rectum (red arrow), and diverticulitis was apparent (yellow arrows). (b) A photomicrograph showed verticulum perforation over the posterior wall of the rectum (red arrow). Figure 3: (a) The resected specimen showed perforation at the rectum (red arrow), and diverticulitis was apparent (yellow arrows). (b) A photomicrograph showed diverticulum perforation over the posterior wall of the rectum (red arrow). Figure 3: (a) The resected specimen showed perforation at the rectum (red arrow), and diverticulitis was apparent (yellow arrows). (b) A photomicrograph showed diverticulum perforation over the posterior wall of the rectum (red arrow). Figure 3: (a) The resected specimen showed perforation at the rectum (red arrow), and diverticulitis was apparent (yellow arrows). (b) A photomicrograph showed diverticulum perforation over the posterior wall of the rectum (red arrow). Figure 4: (a) Contrast extravasation from lumbar artery and hematoma expansion lifting the left kidney was identiﬁed (yellow arrow). (b) Angiography revealed extravasation of contrast medium (red arrow) from the left ﬁrst lumbar artery (yellow arrow). Figure 4: (a) Contrast extravasation from lumbar artery and hematoma expansion lifting the left kidney was identiﬁed (yellow arrow). (b) Angiography revealed extravasation of contrast medium (red arrow) from the left ﬁrst lumbar artery (yellow arrow). a short period. Although the immunosuppressive therapy must be considered as potentially inducing the event, thus far there have been no reports of retroperitoneal hematoma associated with immunosuppressive therapy for adult Still’s disease. mediated lymphocyte functions, suppression of bactericidal activ- ities of neutrophils and fragility of tissue [5]. The present patient, who had adult Still’s disease, had been taking high-dose steroids for a long time, and gastrointestinal perforation occurred twice in mediated lymphocyte functions, suppression of bactericidal activ- ities of neutrophils and fragility of tissue [5]. The present patient, who had adult Still’s disease, had been taking high-dose steroids for a long time, and gastrointestinal perforation occurred twice in mediated lymphocyte functions, suppression of bactericidal activ- ities of neutrophils and fragility of tissue [5]. CONSENT The patient discussed in this case report provided her informed consent for publishing the information in this report. The patient discussed in this case report provided her informed consent for publishing the information in this report. REFERENCES In conclusion, despite not ﬁnding any obvious cause of bleeding, we experienced an extremely rare case of retroperi- toneal hematoma after surgery for colonic perforation of the rectum with retroperitoneal emphysema in a woman undergo- ing chronic high-dose immunosuppressive therapy for adult Still’s disease. Retroperitoneal bleeding should be suspected, and early treatment may be essential for improved prognosis when patients receiving a higher dose of immunosuppressants present with abdominal pain. 1. Estivill Palleja X, Domingo P, Fontcuberta J, Felez J. Spontaneous retroperitoneal hemorrhage during oral anti- coagulant therapy. Arch Intern Med 1985;145:1531–4. 1. Estivill Palleja X, Domingo P, Fontcuberta J, Felez J. Spontaneous retroperitoneal hemorrhage during oral anti- coagulant therapy. Arch Intern Med 1985;145:1531–4. 2. Pode D, Caine M. Spontaneous retroperitoneal hemorrhage. J Urol 1992;147:311–8. 3. Chan YC, Morales JP, Reidy JF, Taylor PR. Management of spontaneous and iatrogenic retroperitoneal haemorrhage: conservative management, endovascular intervention or open surgery? Int J Clin Pract 2008;62:1604–13. 4. Montori G, Di Giovanni G, Mzoughi Z, Angot C, Al Samman S, Solaini L, et al. Pneumoretroperitoneum and pneumomediasti- num revealing a left colon perforation. Int Surg 2015;100:984–8. 4. Montori G, Di Giovanni G, Mzoughi Z, Angot C, Al Samman S, Solaini L, et al. Pneumoretroperitoneum and pneumomediasti- num revealing a left colon perforation. Int Surg 2015;100:984–8. FUNDING In general, the pathophysiology of spontaneous retroperi- toneal bleeding is yet to be determined. It has been hypothe- sized that diffused occult vasculopathy and minor trauma in the microcirculation in the retroperitoneum may give rise to fragility and proneness to rupture [6]. Others have suggested that anticoagulation-induced immune microangiopathy may be responsible [7]. In this case, drainage of the abscess in the retroperitoneal space, vascular tissue weakness caused by chronic high-dose immunosuppressants, and anticoagulation resulting from peritonitis were considered the inciting factors leading to spontaneous retroperitoneal bleeds. No authors have direct or indirect commercial and ﬁnancial incentives associated with publication of the article. No authors have direct or indirect commercial and ﬁnancial incentives associated with publication of the article. DISCUSSION The present patient, who had adult Still’s disease, had been taking high-dose steroids for a long time, and gastrointestinal perforation occurred twice in 4 \| T. Miyata et al. Figure 5: (a) CT showed free air around the right lateral region (yellow arrows). (b) Intraoperative ﬁndings showed perforation of the cecum with no obvious masses (yellow arrow). Figure 5: (a) CT showed free air around the right lateral region (yellow arrows). (b) Intraoperative ﬁndings showed perforation of the cecum with no obvious masses (yellow arrow). ACKNOWLEDGEMENTS We thank Hugh McGonigle, from Edanz Group (www. edanzediting.com/ac), for editing a draft of the article. 5. ReMine SG, McIlrath DC. Bowel perforation in steroid treated patients. Ann Surg 1980;192:581–6. 5. ReMine SG, McIlrath DC. Bowel perforation in steroid treated patients. Ann Surg 1980;192:581–6. 6. Torres GM, Cernigliaro JG, Abbitt PL, Mergo PJ, Hellein VF, Fernandez S, et al. Iliopsoas compartment: normal anatomy and pathologic processes. Radiographics 1995;15:1285–97. 6. Torres GM, Cernigliaro JG, Abbitt PL, Mergo PJ, Hellein VF, Fernandez S, et al. Iliopsoas compartment: normal anatomy and pathologic processes. Radiographics 1995;15:1285–97. CONFLICT OF INTEREST STATEMENT 7. McCort JJ. Intraperitoneal and retroperitoneal hemorrhage. Radiol Clin North Am 1976;14:391–405. 7. McCort JJ. Intraperitoneal and retroperitoneal hemorrhage. Radiol Clin North Am 1976;14:391–405. The authors declare no conﬂicts of interest.
https://openalex.org/W2942598356	http://revista.abrasd.com.br/index.php/rbsd/article/download/327/188	English	null	For a general theoretical unified axiomatic reduction of the natural spaces	Revista Brasileira de Sociologia do Direito	2,019	cc-by	4,596	Revista Brasileira de Sociologia do Direito Revista Brasileira de Sociologia do Direito 1 Emeritus Full Professor Dr. of Sociology of Law at the Federal University of Pernambuco, Recife. Founder Full Professor Dr. of Sociology of Law at the Catholic University of Pernambuco, Recife, initiating the teaching and research of Sociology of Law in Brazil. Ex-Professor of the discipline “Man and Ethos” in the Master Course in Philosophy of the Federal University of Pernambuco. Associated with Prof. Dr. Solange Souto, he was Professor of Scientific Methodology in the Integrated Teaching of the Faculty of Medicine, Federal University of Pernambuco, Recife. Dr. in Social Sciences (Dr. rer. soc.) by the Faculty of Sociology of the University of Bielefeld, Germany. Doctoral thesis on General Sociological Theory entitled Most General Scientific Foundations of the Social. Ex-Chief-Researcher of the Federal University of Pernambuco. 1-A Researcher of the National Council of Scientific and Technological Development (CNPQ) and consultant of this organ. Fulbright Researcher in the United States, where he was Visiting Research Fellow in the Southwestern Legal Center (Dallas) and Visiting Research Scholar of the Graduate Faculties of the Columbia University (New York). Visiting Professor of the University of Cologne (Köln, Germany) in a program of the Alexander von Humboldt Foundation. Visiting Professor of the Center for Interdisciplinary Research of the University of Bielefeld.  This is an expanded English version of the article originally published in volume 5, issue 3 of this journal. Palavras-chave: Campos. Teoria axiomática unificada. Postulados. Teoremas. Keywords: Fields. Axiomatic unified theory. Postulates. Theorems. 1 INTRODUCTION At the end of his career, Albert Einstein was pursuing a dream far more ambitious than the theory of relativity. He was trying to find an equation that explained all physical reality - a theory of everything. He failed, but others have taken up the challenge in a remarkable quest that is shedding light on unsuspected secrets of the cosmos. It is the dream of a theory of everything through Newton, Maxwell, Einstein, Bohr, Schrödinger, Feynman, Gell-Mann, Weinberg, and other great physicists, charting their progress toward an all-embracing, unifying theory. (…) toward an ultimate, as-yet-unfinished masterpiece.” The Theory of Everything: The Quest to Explain All Reality [Audiobook] By Dr. Don Lincoln (Author, Narrator), Experimental physicist, Fermi National Accelerator Laboratory The Theory of Everything: The Quest to Explain All Reality [Audiobook] By Dr. Don Lincoln (Author, Narrator), Experimental physicist, Fermi National Accelerator Laboratory The Theory of Everything: The Quest to Explain All Reality [Audiobook] By Dr. Don Lincoln (Author, Narrator), Experimental physicist, Fermi National Accelerator Laboratory However -- the present author must say -- “everything” or “all” (HAWKING, 2010) is not restricted to the domain of physics, or to any other specific domain of human knowledge. The present study is a modest attempt to include, by theoretical reduction, all scientifically cognizable reality: the physical, mental and social fields (spaces). It is here initially suggested that there is no real separation of the physical, mental and social fields, a factor that should facilitate a unified theory of these fields. The usual separation is thus a mere abstraction, not a reality. And the idea of a continuous interaction between nature and the human being approximates the obvious. Moreover, nature is the ambiental space where the human mental and social interactive with nature occurs. It is necessary to be attentive that nature – be it terrestrial or already primordially cosmic – is in the human and in his or her exterior, forming his or her environment. Therefore, the physical space (nature) will be a constant along the postulates and theorems of the present study. The spatial reach is magnificent because it is already initially cosmic. The physical, mental and social spaces present a progressive complexity from the physical space on, and there is a maximum of complexity in the mental and social spaces, above all in the social space, which is a derivation of the mental one. ■■■ ■■■ FOR A GENERAL THEORETICAL UNIFIED AXIOMATIC REDUCTION OF THE NATURAL SPACES PARA UMA REDUÇÃO AXIOMÁTICA UNIFICADA TEÓRICA GERAL DOS ESPAÇOS NATURAIS Cláudio Fernando da Silva Souto1 RESUMO: Este estudo introduz uma teoria unificada de máxima amplitude. Seu desenvolvimento repousa na pressuposição factual de que os espaços (campos) físicos, mentais e sociais são inseparáveis e são apenas separáveis pela abstração. Essa suposição facilita a formulação dessa teoria dos espaços unificada aparentemente sem precedentes. Cinco postulados (ou axiomas) são induzidos da realidade e 23 teoremas são deduzidos dos postulados. Todas as proposições devem ser confirmadas ou confir veis má por pesquisa empírica. É importante ressaltar que há um máximo de complexidade nos espaços mental e social, sobretudo no espaço social, que é uma derivação do mental. A teoria apresentada é determinista e, como tal, não abandona o terreno da probabilidade, no máximo é apenas altamente provável. ABSTRACT: This study introduces a unified theory of utmost amplitude. Its development rests on the factual presupposition that the physical, mental and social spaces (fields) are inseparable and are only separable by abstraction. This assumption facilitates the formulation of this apparently without precedent unified theory of spaces. Five postulates (or axioms) are induced from reality and 23 theorems are deduced from the postulates. All propositions must be confirmed or may be confirmed by empirical research. Importantly, there is a maximum of complexity in the mental and social spaces, above all in the social space, which is a derivation from the mental one. The presented theory is deterministic and, as such, does not abandon the terrain of probability, at the utmost is only highly probable. ABSTRACT: This study introduces a unified theory of utmost amplitude. Its development rests on the factual presupposition that the physical, mental and social spaces (fields) are inseparable and are only separable by abstraction. This assumption facilitates the formulation of this apparently without precedent unified theory of spaces. Five postulates (or axioms) are induced from reality and 23 theorems are deduced from the postulates. All propositions must be confirmed or may be confirmed by empirical research. Importantly, there is a maximum of complexity in the mental and social spaces, above all in the social space, which is a derivation from the mental one. The presented theory is deterministic and, as such, does not abandon the terrain of probability, at the utmost is only highly probable. Palavras-chave: Campos. Teoria axiomática unificada. Postulados. Teoremas. Keywords: Fields. Axiomatic unified theory. Postulates. Theorems. 2 Cláudio Fernando da Silva Souto 1 INTRODUCTION Thence the general theoretical unified reduction of these spaces do not exceed an only tentative stage. Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 It is worthy of note that not even in Physics, whose object, although complex, is less complex, it was achieved a unified theory of its fields (SOUTO, 2006). Already Schenberg observed that “generally speaking our intuition (…) is the intuition of the world that more or less corresponds to that one of Newtonian physics, not to the relativistic physics and still less to the quantic mechanics” (SCHENBERG, 1985, p. 143). Perhaps this is so because our intuition has the presentiment of a fundamental unity subjacent to the specificities. 2 POSTULATES 1. In the physical, mental and social spaces (fields), the more energy and the less distance, the more attraction. 1. In the physical, mental and social spaces (fields), the more energy and the less distance, the more attraction. 2. In the physical, mental and social spaces (fields), attraction varies in the direct reason of affinity and in the inverse reason of distance. 3. In the physical, mental and social spaces (fields), the less distance the more attraction and repulsion. 4. In the physical, mental and social spaces, since these fields are separable only by abstraction, the equilibrium in each one of these fields (spaces) always causes equilibrium in the other two fields, and the disequilibrium in each one of these fields (spaces) always causes disequilibrium in the other two fields. 5. In the physical, mental and social spaces (fields), the larger the idea of similarity, the larger the agreeableness, and the feeling of agreeableness, or of preponderant agreeableness in face of something, always causes approximation or a resultant of approximation towards this something, with emission of energy of equilibrating (cohesive, integrative) mental waves. Inversely, the larger the idea of dissimilarity, the larger the disagreeableness, and the feeling of disagreeableness, or of preponderant disagreeableness in face of something, always causes, respectively, separation or a resultant of separation towards this something, with emission of energy of disequilibrating (discohesive, desintegrative) mental waves. Cláudio Fernando da Silva Souto 3 CLEARING UP 3 CLEARING UP Concerning the second postulate, we as yet do not have scientific data about the velocity of the human thought, presumably greater than the velocity of light – data that would make possible a satisfactory quantification of the unified proposition. It is still for notice that affinity is object of study of both Chemistry and Physics. The quantic physicist David Bohm warns that “(…) if we were more rapid than light (…) our atoms would be dispersed and we would fall into pieces” (BOHM, 1988, p. 122). However here it is not the case that our bodies move quicker than the light, but only it is referred the possibility that a human mental emission be more rapid than the light. It seems clear that by the reference to velocity of thought the obvious mention is to a possible radiant energy not confined to the space of cerebral neurons. Regarding quantification, it is noteworthy Einstein’s classical thinking, according to which “in so far as mathematical propositions refer to reality, they are not exact (“sicher”), and in so far as they are exact, they do not refer to reality.” (EINSTEIN apud ALDRUP, 1977, p. 174). In any branch of knowledge, the difficult mission of the theoretician is to proceed beyond the academically usual. In the mere repetition, or in the merely formal, be it logical or mathematical, is not created a content of knowledge. In a similar way, Lakshmikantham et al., in their study “The Hybrid Grand Unified Theory”, state: “to solve long standing unsolved problems, to resolve unanswered questions and unsettled issues of physics, mathematical modeling is inadequate. We need qualitative modeling, which is the complement of mathematical modeling”. Moreover “mathematical models they can only describe the behavior of a physical system but cannot explain their behavior” (2009, p. 44; 90). It is also noteworthy that Newton asks tolerance for his work, since he was exploring a so difficult subject (Newton, final of the Preface, first edition of the Principia). In the present theoretical construction, the postulate 5 represents the maximum of complexity. And in this postulate, the mental is represented by idea and feeling, being implicit the will, the motor of action. Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 3 CLEARING UP 2019 Present science is orientated by humility. Physics, the more developed human science, affirms itself indeterminate, inexact, uncertain, even in its deterministic propositions, that are only highly probable (HEISENBERG, 1958, 1986). Even so Einstein states: “I still believe in the possibility of a model of reality – that is to say, of a theory which represents things themselves and not merely the probability of their occurrence.” (EINSTEIN, 1954, p. 276). Nevertheless, the real and the only probable are inseparable in the present stage of substantive science. It must be observed that the high probability in science (even in the deterministic construction of Physics) pays the price of approximation. of evidence. As in the Newton’s classical law of the universal gravitation. At bottom there is a tendency to the obvious: the more the mass and the less the distance, the more the attraction. Or in the important equation of energy by Einstein, in which remains the tendency to the obvious: the energy would vary in the direct reason of mass and velocity. The far-famed Einstein’s equation of energy, if refers to light, does not present the greater dematerialization of referring to the human thought, energetic modality presumably more subtle and more rapid. It seems to exist a growing “dematerialization” of nature that goes from the attraction of masses passing by the attraction and repulsion of electricity, until the sophistication of the mental and social in refined movements of approximation and separation -- for here is the human mental and social in a greater complexity and with greater wealth of nuances. The human mind has as infra-structures the physical- chemical and the organic but is a new and more subtle reality. However, in spite of this subtlety, the mental and the social are explicable, as all seems to indicate, by a theoretical deterministic simplicity. It seems also undeniable that thought is an energetic modality that is part of nature and, as such, would deserve a rigorous scientific consideration. However, alluding to Psychology itself, Hofstätter affirms that “our science still waits its Newton” (HOFSTÄTTER, 1981, p. 5). Cláudio Fernando da Silva Souto Cláudio Fernando da Silva Souto It seems even possible a more general unified theoretical explanation, because referred to the behavior of superior animals (SOUTO, 1990; SOUTO, 1991, p. 303-309; SOUTO, 2000, p. 138-146). 3 CLEARING UP All this is not astonishing: for Newton, nature is satisfied with simplicity and dislikes the pomp of superfluous causes (NEWTON, 1955, p. 270). In the present article 23 theorems are deduced from the postulates. Other theorems are deducible. It is known that the theoretical scientific activity reaches its maximum of rigor in the axiomatic construction. But the word “always”, implicit or explicit, in the construction of deterministic postulates, means only a high degree of probability. Reichenbach affirmed classically that, rigorously speaking, we cannot state nothing about reality, whose validity may be affirmed with more than probability. (REICHENBACH, 1949, p. 10). The axioms or postulates are obtained by induction of the concrete particular and from them less general propositions, the theorems, are deducible, theorems that would be explained by the postulates. According to the scientific protocol of the rigorous theoretical axiomatic construction, no postulate is deduced from other postulates and the postulates are necessarily in small number. All propositions must be confirmed or may be confirmed by empirical research. For preparatory antecedents of the present construction, Cláudio Souto, 1984, 1986, 1987, 1988, 2002, 2006, 2015, 2018 (all these bibliographical references). 4 THEOREMS 1. (Ceteris paribus), in the physical, mental and social spaces, the less space, the less time, and the more space, the more time. 2. In the physical, mental and social spaces, the larger the idea of similarity in the interactive system (and consequently the larger the agreeableness of feeling), the more rapidly the passage of time will be experimented. 2. In the physical, mental and social spaces, the larger the idea of similarity in the interactive system (and consequently the larger the agreeableness of feeling), the more rapidly the passage of time will be experimented. Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 3. If there is force of repulsion between interactive poles in the physical, mental and social spaces, the greater the distance between these poles, the smaller the repulsive force between them. 4. If there is force of attraction between interactive poles in the physical, mental and social spaces, the smaller the distance between these poles, the greater the attractive force between them. 5. In the physical, mental and social spaces, (ceteris paribus) if there is agreeableness of feeling (corresponding to approximation) the eventual emotional intensity is smaller, with less dissipation of energy, than if there is disagreeableness of feeling (corresponding to disapproximation, separation). 6. In the physical, mental and social spaces, if there is permanent equilibrium of the interactive system, the resulting process is associative. 6. In the physical, mental and social spaces, if there is permanent equilibrium of the interactive system, the resulting process is associative. 7. In the physical, mental and social spaces, the more the similarity among the interactive poles (such as defined in function of what they accept), the greater the equilibrium of the interactive system. The decisive is the idea of similarity with what is accepted. 7. In the physical, mental and social spaces, the more the similarity among the interactive poles (such as defined in function of what they accept), the greater the equilibrium of the interactive system. The decisive is the idea of similarity with what is accepted. 8. 4 THEOREMS In the physical, mental and social spaces, the more the equilibrium of the interactive system, the more promptly this equilibrium controls any event that may disturb the integrity of the system. 15. In the physical, mental and social spaces, the smaller the distance among interactive poles, the lesser energy will be necessary for communication and control. 15. In the physical, mental and social spaces, the smaller the distance among interactive poles, the lesser energy will be necessary for communication and control. 16. In the physical, mental and social spaces, in the interaction of peace, there is the minimum of energy necessary for communication and control. 16. In the physical, mental and social spaces, in the interaction of peace, there is the minimum of energy necessary for communication and control. 17. In the physical, mental and social spaces, change is a constant, and the greater the idea of similarity among interactive poles, the greater the equilibrium of the respective system of interacts, and the fewer material language (language condensed in material signs) is necessary for communication and control – be this communication dialogistic internal (mental dialogue of the individual with himself) or exteriorized. 17. In the physical, mental and social spaces, change is a constant, and the greater the idea of similarity among interactive poles, the greater the equilibrium of the respective system of interacts, and the fewer material language (language condensed in material signs) is necessary for communication and control – be this communication dialogistic internal (mental dialogue of the individual with himself) or exteriorized. 18. In the physical, mental and social spaces, the more the socialization (exposition to social patterns) is made in the direction of similarity among interactive poles, the more the equilibrium of the system of interacts corresponding to these poles. 18. In the physical, mental and social spaces, the more the socialization (exposition to social patterns) is made in the direction of similarity among interactive poles, the more the equilibrium of the system of interacts corresponding to these poles. 19. In the physical, mental and social spaces, the more the socialization is made in the direction of the similarity among interactive poles, the more peace. 19. In the physical, mental and social spaces, the more the socialization is made in the direction of the similarity among interactive poles, the more peace. 20. 4 THEOREMS In the physical, mental and social spaces, the smaller the distance among interactive poles, the greater the relative index of interaction among them (the relativity of the index of interaction has reference to the number of interacting poles, and this index refers to the frequency and duration of the interactive process). 9. In the physical, mental and social spaces, if the idea of similarity is greater than the one necessary for the permanent equilibrium of the interactive system, the resultant process is of the greatest agreeableness (= greatest affective suaveness = peace). 10. In the physical, mental and social spaces, the more the peace of the interactive system, the greater the equilibrium of this system. 10. In the physical, mental and social spaces, the more the peace of the interactive system, the greater the equilibrium of this system. 11. In the physical, mental and social spaces, the greater the standardization (uniformization) of the element idea of interactive poles, the grater the similarity among these poles and consequently the greater the equilibrium of the corresponding system of interaction. 11. In the physical, mental and social spaces, the greater the standardization (uniformization) of the element idea of interactive poles, the grater the similarity among these poles and consequently the greater the equilibrium of the corresponding system of interaction. Cláudio Fernando da Silva Souto 12. In the physical, mental and social spaces, if interactive poles have their element idea scientifically standardized, to this extent there is the maximum possibility of equilibrium of the corresponding system of interaction (maximum possibility of peace). 13. In the physical, mental and social spaces, if interactive poles have their element idea standardized, scientific-empirically inclusive, to the extent of this standardization, the element “feeling” in the general direction of the feeling of agreeableness, is the main factor of change (smaller or larger alteration) of the corresponding interactive system (and not the element “idea”, which, in the mentioned extension, may be considered a constant). 13. In the physical, mental and social spaces, if interactive poles have their element idea standardized, scientific-empirically inclusive, to the extent of this standardization, the element “feeling” in the general direction of the feeling of agreeableness, is the main factor of change (smaller or larger alteration) of the corresponding interactive system (and not the element “idea”, which, in the mentioned extension, may be considered a constant). 14. 4 THEOREMS In the physical, mental and social spaces, in so far as the socialization is made in terms of dissimilarity among interactive poles, in this extent the poles thus socialized will tend towards disequilibrating behavior of the interactive system. 20. In the physical, mental and social spaces, in so far as the socialization is made in terms of dissimilarity among interactive poles, in this extent the poles thus socialized will tend towards disequilibrating behavior of the interactive system. Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 Revista Brasileira de Sociologia do Direito, v. 6, n. 2, maio/ago. 2019 21. In the physical, mental and social spaces, the larger the separation by the idea of dissimilarity, the larger the disagreeableness. And the larger the disagreeableness, the larger the tendency towards conflict (mental conflict: internal conflict, or external conflict: fight). 22. In the physical, mental and social spaces, in the approximation by the idea of similarity, the larger the approximation, the larger the agreeableness, and the larger the agreeableness, the larger the tendency towards cooperation and peace, with larger stability of the integration. And the larger the competition (“pacific fight”, process of separation in the mental and social spaces), the larger the instability of the mental and social integration. 23. In the physical, mental and social spaces (justice scientifically defined as the feeling of agreeableness in face of what is thought as ought to be), if a system of interacts is considered essentially (mainly) just by one or more interactive poles (therefore essentially similar to what they accept), such poles experiment a situation of relative and healthful affective suaveness. Contrarily, the oscillating situations of agreeableness and disagreeableness, above all the situations of disagreeableness -- since they are not relatively mild --, favor disequilibrating pathological conditions in the individual and consequently in the concerning interactive system. 5 FINAL REMARKS Newton (1955, passim) and Einstein (1954, passim) still represent the most important steps in the way of science. Both are characterized by science and modesty. are characterized by science and modesty. Einstein do not consider Newton a figure of the past, but has him in highest consideration. Let us transcribe Einstein’s own words about Newton: “(…) brilliant genius, who determined the course of western thought, research, and practice like no one else before or since.(…) The whole evolution of our ideas about the processes of nature, with which we have been concerned so far, might be regarded as an organic development of Newton’s ideas. (…) Who would presume today to decide the question whether the law of causation and the differential law, these ultimate premises of the Newtonian view of Cláudio Fernando da Silva Souto Cláudio Fernando da Silva Souto nature, must definitely be abandoned? (…) Certain is that a conviction, akin to religious feeling, of the rationality or intelligibility of the world lies behind all scientific work of a higher order” (EINSTEIN, 1955, p. 253; 261-262). The present author is happy of humbly trying to continue the tradition of theoretical reduction. ■■■ REFERENCES ALDRUP, Dieter. 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Recebido em: 10/02/2019 Autor convidado
https://openalex.org/W4282918900	https://hal.inrae.fr/hal-03699002/file/Paes-MS-2022.pdf	English	null	Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions	MSystems	2,022	cc-by	12,794	To cite this version: Charlotte Paës, Thierry Gidenne, Karine Bébin, Joël Duperray, Charly Gohier, et al.. Early In- troduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions. mSystems, 2022, 18 p. 10.1128/msystems.00243-22. hal-03699002 Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions Charlotte Paës, Thierry Gidenne, Karine Bébin, Joël Duperray, Charly Gohier, Emeline Guené-Grand, Gwénaël Rebours, Céline Barilly, Béatrice Gabinaud, Laurent Cauquil, et al. Distributed under a Creative Commons Attribution 4.0 International License Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions Charlotte Paës,a,b Thierry Gidenne,a Karine Bébin,b Joël Duperray,c Charly Gohier,d Emeline Guené-Grand,e Gwénaël Rebours,f Céline Barilly,a Béatrice Gabinaud,a Laurent Cauquil,a Adrien Castinel,g Géraldine Pascal,a Vincent Darbot,a Patrick Aymard,a Anne-Marie Debrusse,a Martin Beaumont,a Sylvie Combesa aGenPhySE, Université de Toulouse, INRAE, ENVT, Castanet-Tolosan, France bCCPA, Janzé, France cEVIALIS, Lieu dit Talhouët, Saint-Nolff, France dMiXscience, Bruz, France eWisium, Chierry, France fTECHNA, Couëron, France gGeT-PlaGe, Genotoul, INRAE, Castanet-Tolosan, France ABSTRACT In mammals, the introduction of solid food is pivotal for the establishment of the gut microbiota. However, the effects of the ﬁrst food consumed on long-term micro- biota trajectory and host response are still largely unknown. This study aimed to investigate the inﬂuences of (i) the timing of ﬁrst solid food ingestion and (ii) the consumption of plant polysaccharides on bacterial community dynamics and host physiology using a rabbit model. To modulate the ﬁrst exposure to solid nutrients, solid food was provided to suckling rabbits from two different time points (3 or 15 days of age). In parallel, food type was modulated with the provision of diets differing in carbohydrate content throughout life: the food either was formulated with a high proportion of rapidly fermentable ﬁbers (RFF) or was starch-enriched. We found that access to solid food as of 3 days of age accelerated the gut microbiota maturation. Our data revealed differential effects according to the digestive segment: precocious solid food ingestion inﬂuenced to a greater extent the development of bacterial communities of the appendix vermiformis, whereas life course polysaccharides inges- tion had marked effects on the cecal microbiota. Greater ingestion of RFF was assumed to promote pectin degradation as revealed by metabolomics analysis. However, transcriptomic and phenotypic host responses remained moderately affected by experimental treatments, suggesting little outcomes of the observed microbiome modulations on healthy subjects. In conclusion, our work highlighted the timing of solid food introduction and plant polysaccha- rides ingestion as two different tools to modulate microbiota implantation and functionality. Downloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. IMPORTANCE Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe–host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota proﬁles in older individuals. This study also provided in-depth analysis of dietary polysaccharide effects on intestinal microbiota. HAL Id: hal-03699002 https://hal.inrae.fr/hal-03699002v1 Submitted on 20 Jun 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License RESEARCH ARTICLE Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions The type of plant poly- saccharides reaching the gut through the lifetime was described as an important modu- lator of the cecal microbiome and its activity. These ﬁndings will contribute to better deﬁne the interventions that can be employed for modulating the ecological succession of young mammal gut microbiota. KEYWORDS ﬁrst food, gut health, gut microbiota, intestinal development, metabolomics, microbiota development, microbiota maturation, polysaccharides, starch, young mammal Month YYYY Volume XX Issue XX Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession T he mammalian gut is colonized by a variety of microorganisms, leading to the con- cept that the host and its inhabiting microbiota constitute a “superorganism,” also called holobiont (1). The symbiotic microbiota is essential for the nutrition and health of the host (2). The microbial ecosystem evolves concomitantly with the host chronological devel- opment (3). Microbiota development is ﬁrst under the inﬂuence of the maternal milk, a substrate containing various microbiota-shaping compounds (4, 5). The introduction of solid food later in life represents a new step in the dynamic construction of the gut microbiota with the ingestion of components resistant to host digestion, such as plant cell walls and speciﬁc starches (6). This dietary shift modiﬁes the substrates present in the luminal milieu, leading to dramatic changes in the bacterial population in terms of both diversity and composition (7, 8). As a result of this new gut environment, the microbiota deploys metabolic adaptations with increased capacities to degrade com- plex plant carbohydrates (3, 9). Later in life, bacterial communities continue their adap- tation to the host diet (10). For these reasons, the introduction of solid food and diet composition are considered to be major drivers of the microbial succession in the di- gestive tract (7, 11). Recent studies have shown that precocious supply of solid food before weaning can modulate the establishment of intestinal bacterial communities and enzymatic activities in several mammalian species (12–16). As a result, the host–microbiota dialogue at the muco- sal interface may be affected, such as in lambs, for which inﬂammation system (12) and digestive tract development (13) modulations were observed following early solid food supplementation. Although these studies demonstrated the inﬂuence of the timing of solid food introduction on the host-microbiota codevelopment, the understanding of how early-life solid food ingestion affects the gut microbiota trajectory and the long-term host response remains insufﬁcient. 10.1128/msystems.00243-22 2 Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions Among the substances that inﬂuence the gut microbiome of mammals, dietary ﬁbers are known to affect the microbiota in late childhood or adulthood, as they are important substrates for bacterial fermentations (17, 18). The term “dietary ﬁber” encompasses a variety of complex carbohydrates with different physicochemical prop- erties such as resistant starch, nonstarch polysaccharides from plant walls, or nondiges- tible oligosaccharides (19). The time required by the developing microbial ecosystems to adapt to speciﬁc ﬁbrous carbohydrate substrates remains largely unexplored. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Our approach was to provide a dynamic follow-up of the holobiont, evaluated with various measurements, to provide a comprehensive understanding of the impact of different feeding patterns on mammals. In these species, modeling the effects of early- life exposure to solid food is particularly challenging, as it is difﬁcult to capture juvenile feeding patterns (food and milk) without disrupting mother–offspring interactions. To bypass this issue, we propose herein to use a neonatal rabbit model, characterized by a short contact time with the nursing doe for suckling (less than 5 min every 24h) (20). This experimental design allowed us to track the early ingestion of solid food together with a control of milk ingestion. Moreover, 1-week-old rabbits are capable of consuming solid food simultaneously with large amounts of milk (21), making the newborn rabbit a good model to study the timing of solid food introduction in early life. Finally, rabbits are hindgut fermenters that rely heavily on their gut microbiota for digestion and health, allowing for a relevant study of symbiosis in mammals. As commonly observed in mammals, the rabbit cecal microbiota is dominated by Bacteroidetes and Firmicutes phyla (22) and followed by others including Proteobacteria (23). Ruminococcaceae and Lachnospiraceae are abundant families of the cecal microbiota, with a distinctive feature being the poor colonization of the gut by Lactobacilli (24). The present study aimed to further investigate the mechanisms by which preco- cious solid food ingestion affects microbiota establishment, with attention given to the dietary polysaccharides. We investigated how the introduction of solid food affects in- testinal maturation by providing solid nutrients to suckling rabbits as of 3 or 15 days of age. We examined the impact of two types of plant polysaccharides (rapidly Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession FIG 1 (A) Suckling rabbits’ ingestion pattern (milk and pellets) from d3 to weaning. Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions The effects of early solid feeding and plant polysaccharides ingestion on bacterial communities were assessed at ﬁve developmental stages in two intestine sections with distinct physiological functions: the cecum, which contributes greatly to host nutrition (25), and the appendix vermiformis, a specialized lymphoid organ at the apex of the cecum (26). Extensive investigation of the cecal ecosystem was then per- formed with the assessment of predictive functional proﬁling and quantitative meta- bolic signatures. Gene expression in this tissue was analyzed as a proxy of gut health. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. g p y p y g Our ﬁndings highlighted that the bacterial communities of rabbit pups quickly responded to the precocious ingestion of solids with both taxonomic and metabolic changes. Although starter feeding resulted in an acceleration of the gut microbiota to- ward a steady state and increased acetate and butyrate levels early in life, impacts on microbiota activities and host remained moderate after weaning. Functional analysis revealed bacterial specialization depending on the type of polysaccharides ingested throughout the weaning transition. Taken together, those results underlined key fac- tors to modulate the gut microbiota trajectories (either maturation rate or endpoints). Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions (B and C) Focus on early consumption of gels in the nest (kinetics and total amounts consumed). Error bars stand for standard error of the mean (n = 15–16 litters of 10 rabbits/group). Green stars emphasize signiﬁcant differences in gel ingestion between the groups with access to early feeding (STA1/RFF1: , P , 0.05; , P , 0.01). Post-hoc test with correction was used to compare the mean values of each group at different dates. FIG 1 (A) Suckling rabbits’ ingestion pattern (milk and pellets) from d3 to weaning. (B and C) Focus on early consumption of gels in the nest (kinetics and total amounts consumed). Error bars stand for standard error of the mean (n = 15–16 litters of 10 rabbits/group). Green stars emphasize signiﬁcant differences in gel ingestion between the groups with access to early feeding (STA1/RFF1: , P , 0.05; *, P , 0.01). Post-hoc test with correction was used to compare the mean values of each group at different dates. fermentable ﬁbers [RFF] and starch) consumed during suckling and thereafter, on the gut microbiota. The effects of early solid feeding and plant polysaccharides ingestion on bacterial communities were assessed at ﬁve developmental stages in two intestine sections with distinct physiological functions: the cecum, which contributes greatly to host nutrition (25), and the appendix vermiformis, a specialized lymphoid organ at the apex of the cecum (26). Extensive investigation of the cecal ecosystem was then per- formed with the assessment of predictive functional proﬁling and quantitative meta- bolic signatures. Gene expression in this tissue was analyzed as a proxy of gut health. Our ﬁndings highlighted that the bacterial communities of rabbit pups quickly responded to the precocious ingestion of solids with both taxonomic and metabolic changes. Although starter feeding resulted in an acceleration of the gut microbiota to- ward a steady state and increased acetate and butyrate levels early in life, impacts on microbiota activities and host remained moderate after weaning. Functional analysis revealed bacterial specialization depending on the type of polysaccharides ingested throughout the weaning transition. Taken together, those results underlined key fac- tors to modulate the gut microbiota trajectories (either maturation rate or endpoints). fermentable ﬁbers [RFF] and starch) consumed during suckling and thereafter, on the gut microbiota. RESULTS At d30, se- rum hydroperoxyde levels tended to be lower in the STA1 group than in the STA– group (P = 0.082). These results therefore suggest moderate impact on the animal response of early feeding practice or dietary polysaccharide modulations. The timing of solid food introduction and lifelong dietary polysaccharides ingestion slightly affect the host development and immune response. Afterwards, we analyzed the consequences of our nutritional interventions on the rabbit’s growth and health. Overall mortality rate was low with 1.3% of deaths from d3 to weaning and one dead rabbit found afterwards. Live weights were equal between the groups during the suckling period (403 6 5 g/rabbit at d21), at weaning (984 6 6 g/rabbit), and dur- ing the exclusive solid-feeding period (1652 6 11 g/rabbit at d50) (Fig. S3). The cecum of the rabbits from the RFF1 group was 32% heavier compared to the STA1 group at d30 (P , 0.05) (Table S2). Systemic and local immune response were assessed by measuring the levels of plasma IgG and cecal IgA and the concentration of hydroperoxydes in serum (Fig. S4). IgA concentrations varied greatly with age, with highest levels at d18 due to passive immunity (i.e., milk IgA), but did not vary with the treatments. Plasma IgG concentra- tions were lower in the STA1 group compared to the STA group at d30 (–1.2 mg/mL, P , 0.05) and compared to the RFF1 rabbits at d58 (–1.2 mg/mL, P , 001). At d30, se- rum hydroperoxyde levels tended to be lower in the STA1 group than in the STA– group (P = 0.082). These results therefore suggest moderate impact on the animal response of early feeding practice or dietary polysaccharide modulations. Characteristics of the rabbit hindgut microbiota. As expected, a shift in microbial composition of the hindgut was observed over time, with marked effects observed when the solid food ingestion sharply increased together with a gradual decrease in milk intake (between d18 and d32). The microbial community structure and composi- tion were in consonance with the sequential microbiota colonization previously observed in the cecum of healthy rabbits (23, 27). All the phyla and families were affected by age. One substantial pattern observed over time was the gradual and sig- niﬁcant increase in Firmicutes relative abundance (150% between d18 and d58, P , 0.001) together with a decrease in Bacteroidetes abundance (–45% between d18 and d58, P , 0.001; Fig. 2). 10.1128/msystems.00243-22 4 RESULTS Voluntary precocious solid food ingestion is modulated by the polysaccharide content of the diet. We ﬁrst examined suckling rabbits’ feeding behavior to validate the stimulation of early-life solid food ingestion (Fig. 1A). Gel food was consumed from d7 with a 35% higher ingestion of RFF-enriched gels compared to starch-enriched gels (Fig. 1B and C: 5.8 6 0.4 g of RFF gels/rabbit, i.e., 1.5 g/rabbit in dry matter versus 4.3 6 0.3 g of STA gels/rabbit, i.e., 1.1 g/rabbit in dry matter). The number of visits to the gel cups counted within two litters indicated that all the kits were responding to the gel stimuli (Fig. S2 in the supplemental material). At d17, gel consumption accounted for 3% of the raw ingestion and 10% of the total dry matter (major supply Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession from the milk with limited pellet consumption). Altogether, our results conﬁrmed that providing starter food gels reduces the age of ﬁrst solid food consumption and that early-life solids ingestion is dependent on the polysaccharide content of the meal. early-life solids ingestion is dependent on the polysaccharide content of the meal. The timing of solid food introduction and lifelong dietary polysaccharides ingestion slightly affect the host development and immune response. Afterwards, we analyzed the consequences of our nutritional interventions on the rabbit’s growth and health. Overall mortality rate was low with 1.3% of deaths from d3 to weaning and one dead rabbit found afterwards. Live weights were equal between the groups during the suckling period (403 6 5 g/rabbit at d21), at weaning (984 6 6 g/rabbit), and dur- ing the exclusive solid-feeding period (1652 6 11 g/rabbit at d50) (Fig. S3). The cecum of the rabbits from the RFF1 group was 32% heavier compared to the STA1 group at d30 (P , 0.05) (Table S2). Systemic and local immune response were assessed by measuring the levels of plasma IgG and cecal IgA and the concentration of hydroperoxydes in serum (Fig. S4). IgA concentrations varied greatly with age, with highest levels at d18 due to passive immunity (i.e., milk IgA), but did not vary with the treatments. Plasma IgG concentra- tions were lower in the STA1 group compared to the STA group at d30 (–1.2 mg/mL, P , 0.05) and compared to the RFF1 rabbits at d58 (–1.2 mg/mL, P , 001). RESULTS FIG 2 Distribution of bacterial taxa in the cecum (A, B) and the appendix (C, D) of rabbits regardless of the experimen age). Relative abundances of phyla (A, C) and the 10 most abundant families (B, D) are presented. FIG 2 Distribution of bacterial taxa in the cecum (A, B) and the appendix (C, D) of rabbits regardless of the experimental treatments (n = 27–30 rabbits/ age). Relative abundances of phyla (A, C) and the 10 most abundant families (B, D) are presented. 10.1128/msystems.00243-22 5 Downloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. ingestion on the cecal microbiota throughout life. Lower bacterial richness was observed in the RFF1 group rather than in STA1 rabbits at d30, d38, and d58, to- gether with a lower InvSimpson index at d30 and d38 (P , 0.05; Fig. 3A). The cecal microbiota structure of RFF1 rabbits was further from the corresponding 58-day-old state compared to the STA1 based on wUniFrac distances calculation (P , 0.01 when signiﬁcant; Fig. 3B). At the phylum level, the ratio Firmicutes/Bacteroidota, which is known to increase during the transition from young to adult (28), was lower in the RFF1 group at d38 compared to STA1 (4 versus 7; P , 0.05) as a consequence of more abundant Bacteroidota (16% in RFF1 at d38, P , 0.05; Fig. 3C). Differences in main family relative abundances were observed, such as lower proportions of Ruminococcaceae in the RFF1 group before weaning together with higher relative abundances of Bacteroidaceae (Fig. 3D and Table S3C). Twenty abundant bacterial OTUs were highly differentially abundant over time according to the dietary levels of polysaccharides (Fig. 3F). Bacteria from Ruminococcaceae and Lachnospiraceae families accounted for the majority of the differentially abundant OTUs (Table S3B). Downloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Our results support the idea that precocious solid food introduction and dietary plant polysaccharide content both modulate the establishment of the microbial com- munity in a main site of fermentation. Knowing that the development of lymphoid organs after birth is markedly inﬂuenced by microbiota exposure (29), the effects of our treatments on the bacterial colonization of the adjacent appendix were then assessed. To this end, the same methodology presented previously was used. RESULTS In both gut segments, the dominance of the Firmicutes was driven by an early increase of Lachnospiraceae and Ruminococcaceae between d18 and d25 (P , 0.001) followed by a stabilization. The proportion of Bacteroidaceae sharply decreased between d18 and d25 (P , 0.001 in cecum and appendix) and showed a regular depletion afterwards. The differentiation between the cecum and the appendix ecosystem structure was initiated from d58 based on wUniFrac distances (Fig. S5). We will focus below on the effects of early food introduction (comparison STA–/STA1) and diet composition (comparison STA1/RFF1). nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. p p Precocious solid food ingestion and dietary polysaccharides differently affect the developing cecal microbiota. Effects of precocious ingestion of solid food on the cecal microbiota. To assess effects of postnatal ingestion of solid food on the cecal microbiota, we compared bacterial communities of the groups STA– and STA1 (early access to solid food). The InvSimpson index, which reﬂects species richness and evenness, was higher in 30-day-old STA1 rabbits (P , 0.05; Fig. 3A). Throughout the experiment, the cecal microbiota structure of STA1 rabbits was found closer to the corresponding 58-day-old state compared to the STA– group (P , 0.01; Fig. 3B), emphasizing a more mature gut ecosystem when precocious access to solid food was given. The relative abundances of the two main phyla were affected at d30 with 10% less Bacteroidota and 10% more Firmicutes in the cecum of STA1 rabbits compared to the STA– group (P , 0.05; Fig. 3C). At the family level, we observed lower abundances of Bacteroidaceae before weaning in STA1 rabbits (Fig. 3D and Table S3A). Six bacterial operational taxonomic units (OTUs) with relative abundance superior to the quantita- tive threshold of 0.5% were highly differentially abundant (jlog2 fold changej . 2) between STA– and STA1 during the preweaning period (Fig. 3E and Table S3B). Effects of dietary polysaccharides on the cecal microbiota. The comparison between STA1 and RFF1 groups was made to assess the effect of the polysaccharide Month YYYY Volume XX Issue XX Early-Life Nutrition and Gut Microbiota Succession mSystems FIG 2 Distribution of bacterial taxa in the cecum (A, B) and the appendix (C, D) of rabbits regardless of the experimental treatments (n = 27–30 rabbits/ age). Relative abundances of phyla (A, C) and the 10 most abundant families (B, D) are presented. 10.1128/msystems.00243-22 5 Month YYYY Volume XX Issue XX 10.1128/msystems.00243-22 RESULTS Alpha-diversity metrics were not modiﬁed by the type of plant polysaccharides consumed, and main taxa abundances only slightly differed between STA1 and RFF1 appendix commun- ities (Fig. 4A, C, and D). Appendix microbiota of 30-day-old and 38-day-old RFF1 rab- bits were closer to their corresponding 58-day-old state compared to the STA1 group (Fig. 4B). Ten OTUs from a wide variety of taxa were highly differentially abundant according to diet composition, some of them being affected in the cecum as well (Fig. 4F and Table S3D). Taken together, those results highlight different responses of the appendix and cecal luminal communities to our two nutritional strategies. Precocious supply of solids affects microbiota composition in both sites, whereas the nature of the dietary polysac- charides has more pronounced effects on the cecal communities. Precocious solid food ingestion and dietary polysaccharide type shift the gut metabolomic proﬁle and predicted functionality across time frames. As a next step, we used NMR-based metabolomics to investigate if the observed modulations of the microbiota composition were associated with adjustments of its metabolic activity. Twenty- nine metabolites were detected in cecal content metabolome from d18. In addition, we inferred the functional proﬁle of cecal bacterial communities. As expected, cecal metabo- lome was under the inﬂuence of age (Fig. S6). We will focus herein on the dietary speciﬁc effects. Effects of precocious ingestion of solid food on the cecal metabolomic proﬁle and predicted functionality. The relative concentrations of the luminal cecal acetate and butyrate, as well as the cecal content in amino acids glycine, leucine, lysine, valine, threonine, phenylalanine, and glutamate, increased at d18 when rabbits had precocious access to solid food (Fig. 5A and B; Table S4A). From d25, similar metabolome proﬁles, pH and NH3 concentrations were observed between the STA– and STA1 groups (Table S2). Based on the metagenomic functional pathways that could be inferred (72% of the ecosys- tem relative abundances), no differential metabolic potentials were highlighted between STA– and STA1 groups. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Downloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Effects of dietary polysaccharides on the cecal metabolomic proﬁle and pre- dicted functionality. Ingestion of the RFF-rich diet was associated with decreased rel- ative concentrations of cecal amino acids phenylalanine, valine, as well as glycine at d18 and d58 when compared to the diet enriched in starch (Fig. 5B; Table S4B). FIG 3 Legend (Continued) Bacteroidaceae and Ruminococcaceae family abundances. (E and F) Log2 fold change superior to j2j of the OTUs signiﬁcantly affected by the timing of solid food introduction (STA–/STA1), and dietary polysaccharide ratio (RFF1/STA1) based on differential abundance analysis (DESeq2). “unk. genus” stands for unknown genera. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA–/STA1), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1) (, P , 0.05; , P , 0.01; , P , 0.001). RESULTS The vermiformis appendix microbiota is affected by early solid food ingestion and to a lesser extent by the nature of the polysaccharides ingested. Effects of precocious ingestion of solid food on the vermiformis appendix microbiota. The effect of early solid food ingestion on the appendix microbiota alpha diversity was Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession FIG 3 Analysis of cecal microbiota implantation and colonization according to the experimental groups before and after weaning (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Comparison of alpha–diversity indices. (B) Dissimilarity between the bacterial communities and the 58 days of age state, (C) Distribution of main phyla. (D) (Continued on next page) Month YYYY Volume XX Issue XX nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. FIG 3 Analysis of cecal microbiota implantation and colonization according to the experimental groups before and after weaning (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Comparison of alpha–diversity indices. (B) Dissimilarity between the bacterial communities and the 58 days of age state, (C) Distribution of main phyla. (D) (Continued on next page) FIG 3 Analysis of cecal microbiota implantation and colonization according to the experimental groups before and after weaning (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Comparison of alpha–diversity indices. (B) Dissimilarity between the bacterial communities and the 58 days of age state, (C) Distribution of main phyla. (D) (Continued on next page) Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession similar to the one observed in the cecum (Fig. 4A). At d18 and d38, the appendix microbiota structure of STA1 rabbits was found closer to the corresponding 58-day- old state compared to the STA– group (P , 0.01; Fig. 4B). The changes of the appendix communities at the phyla level (Fig. 4C) resulted in a greater maturity indicator Firmicutes/Bacteroidota ratio in the STA1 group at d38 compared to STA– treatment (7 versus 4; P , 0.01). Around weaning, Bacteroidaceae abundances were reduced in the appendix of STA1 rabbits (at d30 STA–: 19% 6 9%, STA1: 8% 6 3%; at d38 STA–: 7% 6 4%, STA1: 3% 6 1%, P , 0.01) (Fig. 4D). Seven OTUs were highly differentially abundant according to the timing of solid food introduction (Fig. 4E). Effects of dietary polysaccharides on the appendix microbiota. 10.1128/msystems.00243-22 7 Month YYYY Volume XX Issue XX RESULTS Cecal levels of glucose and glutamate were lower in the RFF1 group after weaning. Ingestion of RFF led to the highest cecal relative concentrations of methanol at d18, d25, d30, and d58 (from 1.5- to 2.0-fold increase, P , 0.001) and galactose from d25 onwards (from 1.7- to 2.5-fold increase, P , 0.001). Cecal acetate content was higher in the cecum of 30-day-old (135%, P , 0.05) rabbits from the RFF1 group compared to the STA1 group (Table S4B). The number of functional pathways inferred from well- covered OTUs was affected by the nature of the dietary polysaccharides from d30. 10.1128/msystems.00243-22 8 FIG 3 Legend (Continued) 10.1128/msystems.00243-22 7 Month YYYY Volume XX Issue XX Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession Among the 48 metabolic pathways inﬂuenced by the type of plant polysaccharides ingested (Table S4C) 3 were highly differentially abundant between STA1 and RFF1 FIG 4 Analysis of appendix microbiota implantation and colonization according to the experimental groups before and after weaning (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Comparison of alpha-diversity indices. (B) Dissimilarity between the bacterial communities and the 58 days of age state. (C) Distribution of main phyla. (D) Bacteroidaceae and Ruminococcaceae family abundances. (E and F) Log2 fold change superior to j2j of the OTUs signiﬁcantly affected by the timing of solid food introduction (STA–/STA1) and dietary polysaccharide ratio (RFF1/STA1) based on differential abundance analysis (DESeq2). “unk. genus” stands for unknown genera. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA–/STA), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1) (, P , 0.05; , P , 0.01; , P , 0.001). Early-Life Nutrition and Gut Microbiota Succession nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Downloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. FIG 4 Analysis of appendix microbiota implantation and colonization according to the experimental groups before and after weaning (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Comparison of alpha-diversity indices. (B) Dissimilarity between the bacterial communities and the 58 days of age state. (C) Distribution of main phyla. (D) Bacteroidaceae and Ruminococcaceae family abundances. (E and F) Log2 fold change superior to j2j of the OTUs signiﬁcantly affected by the timing of solid food introduction (STA–/STA1) and dietary polysaccharide ratio (RFF1/STA1) based on differential abundance analysis (DESeq2). “unk. genus” stands for unknown genera. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA–/STA), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1) (, P , 0.05; , P , 0.01; , P , 0.001). Among the 48 metabolic pathways inﬂuenced by the type of plant polysaccharides ingested (Table S4C), 3 were highly differentially abundant between STA1 and RFF1 (jlog2 fold changej . 1 and P , 0.01). The latter were predicted to be associated with hexitol fermentation and aerobic respiration, and they were downregulated with the Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession RFF diet. FIG 3 Legend (Continued) Interestingly, pathways related to plant polysaccharides degradation (L-rham- d ) l t d i th RFF1 t d38 (P , 0 05) FIG 5 NMR measurements in cecal luminal content (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Relative concentrations of the main cecal short-chain fatty acids throughout the time (B) Relative concentrations of the amino acids affected by one of the nutritional interventions in 18-day-old rabbits. False discovery rate procedure was used to identify differential metabolite levels. Tukey’s post hoc test was then performed on the selected metabolites. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA– /STA1), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1) (, P , 0.05; , P , 0.01; , P , 0.001). nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. FIG 5 NMR measurements in cecal luminal content (Mean 6 SEM, n = 9–10 rabbits/age/group). (A) Relative concentrations of the main cecal short-chain fatty acids throughout the time (B) Relative concentrations of the amino acids affected by one of the nutritional interventions in 18-day-old rabbits. False discovery rate procedure was used to identify differential metabolite levels. Tukey’s post hoc test was then performed on the selected metabolites. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA– /STA1), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1) (, P , 0.05; , P , 0.01; , P , 0.001). RFF diet. Interestingly, pathways related to plant polysaccharides degradation (L-rham- nose and mannan) were upregulated in the RFF1 group at d38 (P , 0.05). Our results indicate that precocious supply of solid food mainly modiﬁed cecal met- abolic activities in early life, whereas long-lasting change of diet through different polysaccharide ingestion modulated microbial activities over the time including a reshaping of glycan digestion. Precocious solid food ingestion and dietary polysaccharide type had moderate effects on the cecal mucosal transcriptome. To evaluate the host response to the observed changes in luminal environment, the expression of selected genes in the cecal tissue was assessed. As expected, we highlighted an age-related cecal mucosa maturation with high production of transcripts associated with barrier function in early life (genes encoding for antimicrobials peptides, mucins, and tight junctions) followed by upregulation of genes involved in both innate and adaptative immunity at d58 (re- dox signaling, cytokines, and IgA transport) (Fig. 6). 10.1128/msystems.00243-22 DISCUSSION Postnatal ingestion of solid food is known to be one major inﬂuential factor of microbiota implantation in mammals (11, 30). Recent research suggests that early solids ingestion (i.e., early food diversiﬁcation) could be beneﬁcial for health (31), presumably through a reshaping of gut microbial colonization (32, 33). Our study aimed to analyze further how early-life inges- tion of solid foods affects hindgut microbiota implantation. Two dietary approaches were used to broaden our understanding of microbiota engineering strategies: on one hand, a short and early nutritional intervention was performed to investigate the effect of the timing of solid food introduction, and on the other hand, a long-term dietary modulation enabled us to study dietary polysaccharide effects on the holobiont development. Overall, precocious ingestion of starch-rich solid food accelerated microbiota matura- tion in both cecum and appendix. Indeed, when rabbits had early access to solid food enriched in starch, we observed more diverse bacterial communities the week preceding the weaning. Faster stabilization of the microbiota with early feeding practice was also evi- denced in the STA1 group compared to the STA–. This is in agreement with previous stud- ies (15, 34, 35, 36) indicating the potential for an earlier introduction of solid feed to reduce the time required for the stabilization of the hindgut bacterial community. Such an enhancement may be advantageous later in life by improving the digestive capacity to cope with dietary shifts (37, 38), by providing the level of microbiota diversity required to inhibit allergic and autoimmune disorders (32), and by stimulating normal components of the immune system (39). But because ﬁrst gut colonizers play a pivotal role in fermenting the numerous substances present in the milk (40), a precipitated replacement of these bac- terial communities may also be detrimental for the host’s health, as suggested by prospec- tive human studies (15, 41). When looking at the cecal tissue, decreased expression of the B lymphocytes activator gene TNFSF13B was observed at the end of gel supplementation, possibly in response to bacterial signaling (42). However, the downregulation of this gene involved in the adaptive immunity did not modify the cecal IgA content of suckling rabbits. Moreover, it was proposed that a transient reduction of the mucosal IgA content before weaning might be beneﬁcial for the colonization by segmented ﬁlamentous bacteria (43, 44). FIG 3 Legend (Continued) The expression of the IgA-secretion Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession Early Life Nutrition and Gut Microbiota Succession FIG 6 Screening of the expression of 48 genes of the rabbit cecal mucosa related to health and quantiﬁed with microﬂuidic chip assay. Data were normalized to the average expression in the 18-day-old STA– group. Focus was put on the genes signiﬁcantly affected by one of the nutritional treatments. False discovery rate procedure was used to identify those genes. Tukey’s post hoc test was then performed on the selected genes. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA–/STA1), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1). The horizontal black lines stand for fold change mean values (, P , 0.05; , P , 0.01; , P , 0.001). Month YYYY Volume XX Issue XX 10.1128/msystem ded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Downloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. FIG 6 Screening of the expression of 48 genes of the rabbit cecal mucosa related to health and quantiﬁed with microﬂuidic chip assay. Data were normalized to the average expression in the 18-day-old STA– group. Focus was put on the genes signiﬁcantly affected by one of the nutritional treatments. False discovery rate procedure was used to identify those genes. Tukey’s post hoc test was then performed on the selected genes. Orange stars represent signiﬁcant differences induced by precocious solid food introduction (STA–/STA1), whereas green stars emphasize signiﬁcant differences induced by the food composition (STA1/RFF1). The horizontal black lines stand for fold change mean values (, P , 0.05; , P , 0.01; *, P , 0.001). Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession stimulating cytokine TNFSF13B (TNF superfamily member 13b) was reduced in STA1 compared to STA– and RFF1 rabbits at d18 (P , 0.01, 0.5-fold decrease). At d30, the gene expression of the epithelial differentiation marker ALPI (intestinal alkaline phos- phatase) was markedly upregulated in cecal mucosa of the RFF1 group compared to STA1 (P , 0.01). Month YYYY Volume XX Issue XX DISCUSSION Decreased plasma IgG content after precocious ingestion of starch-rich solid food also pointed out immune modulations due to early solid food introduction. Whether these changes in the humoral immunity can be beneﬁcial for the host needs to be further eluci- dated. Histological examination of intestinal epithelium, at the interface of the microbiota and the gut mucosal immunity, would help us to investigate further this question. Still, these ﬁndings conﬁrmed the importance of early-life solid food ingestion on the host– bacterial symbiosis. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Early ingestion of starch-rich diet promptly stimulated the growth of species belonging to the Ruminococcaceae family in the appendix, despite concomitant ingestion of large quantities of milk that might create a niche constraint on the gut microbiome (5, 23, 45). This compositional change is in line with the pattern observed after later introduction of solid food in young mammals (46, 47). The cecal luminal metabolome was also modiﬁed after solid food introduction, highlighting a quick adaptation of the developing microbiota activity to new dietary substrates and environmental transitions. Notably, the fermentative activities, assessed by cecal acetate and butyrate concentrations, transiently increased after the ingestion of small quantities of solid food. The levels of several amino acids also increased when plant-based food was introduced in the diet at an early stage. Although those amino acids can be of different origins (48), we hypothesize that poorly digestible plant protein from solid food partly reached the cecum, thus precociously modifying the amino acids content of the hindgut environment, whereas milk proteins are highly Month YYYY Volume XX Issue XX 10.1128/msystems.00243-22 11 mSystems Early-Life Nutrition and Gut Microbiota Succession digested in the upper gastrointestinal tract (49). These modulations of the substrates avail- able in the cecal luminal content likely explains some long-term effect of early life solid food ingestion on taxonomic modiﬁcations, such as a drop in the Bacteroidaceae family, a “milk-oriented microbiota” characteristic taxon (50). After the establishment of the pioneer species, early supply of solid food was thus found to drive the second colonization of gut communities. Dietary ﬁber fractions represent major modulators of digestive physiology due to their inﬂuence on the nutrient rate of passage, mucosal functionality, and gut micro- biota (51). The particularity of this study was to modulate starch and digestible ﬁber fractions only while maintaining a balanced diet. DISCUSSION Our results showed that the type of dietary complex plant carbohydrates shaped both composition and functions of the hindgut microbiota, with major effects observed when the ingestion of solid food became predominant over milk. Not surprisingly, the type of polysaccharides ingested modulated the distribution of species from Firmicutes and Bacteroidota phyla, which encompass plant-degrading bacteria (52). At the family level, we found that the distinct carbohydrates contents of STA and RFF diets drove a differential establishment of Lachnospiraceae and Ruminococcacae, presumably because they encompass two plant degrader groups characterized by different carbohydrate- active enzymes within their genomes (53). When rabbits ingested high amounts of starch throughout their lives, the cecal communities reached a mature state faster (27). We hypothesized that resistant starch (54), which has the ability to be quickly fermented and to modulate the gut microbiota (10), may have contributed to this stabilization. Indeed, glucose is readily absorbed in the rabbit small intestine (55), and the higher levels of free glucose observed in the cecum of rabbits that received the enriched-starch diet could be related to the bacterial degradation of starch re- sistant to host digestion. Diet enrichment with RFF was associated with decreased diversity in the cecum and appendix, with a lower InvSimpson index outlining the dominance of some abundant species in those ecosystems. This can be attributed to the speciﬁc dietary content of RFF diet but also can be due to an improved ﬁber fraction digestibility as reported when increasing digestible ﬁber to starch ratio (56). Interestingly, higher levels of cecal methanol were found when rabbits received higher quantities of RFF. This must be explained by high dietary concentrations of pectins, a substrate that can be fermented into methanol by speciﬁc intestinal bac- teria such as members of the Bacteroides genus (57). Consistently, the RFF diet was associated with increased cecal acetate content before weaning, in line with the pectin in vitro fermentation process assessed in human stools (58). PICRUSt predic- tions were sketchy and should be treated with caution. However, these predictions seem in line with our hypothesis since increased expression of glycans-degradation pathways was observed in the cecal microbiome of the rabbits that received the RFF diet. In terms of host response, lifelong ingestion of a diet rich in RFF was associated with increased gene expression of ALPI, considered as a marker of epithelial differentiation and a regulator of intestinal inﬂammation (59). DISCUSSION Moreover, animals that ingested more RFF had heavier cecum, which may highlight faster development of this organ in response to fer- mentable nonstarch polysaccharides. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Interestingly, we observed different distribution drivers of microbial popula- tions in the two contiguous digestive segments investigated, even though these sections do not differentiate from each other during the neonatal phase (60). Cecal communities were mostly affected by the type of polysaccharides substrates provided, with pronounced effects on alpha diversity and OTU composition once solid food ingestion overtook milk consumption. In contrast, the composition of the appendix microbiota was substantially inﬂuenced by food ingestion stimula- tion in early life. These differential effects can be linked to the dualism of func- tions between the cecum and appendix, respectively related to nutrition and immunity (26). Differences in peristaltic movements may also explain these dis- tinct microbiota (61): the narrow lumen of the appendix limits the nutrient ﬂow, Month YYYY Volume XX Issue XX 10.1128/msystems.00243-22 12 mSystems Early-Life Nutrition and Gut Microbiota Succession which could induce an inertia of the microbiota. The appendix has long been considered as a degenerating organ, but it could represent a reservoir for beneﬁcial bacteria that can reinoculate the bowel (62). Moreover, the microbial communities of the gut-associated lymphoid tissue are essential for the immune system development (63). We thus believe that the effects of early-life stimuli on the colonization of lymphoid organs deserve increased attention. We demonstrated that very early ingestion of solid food in infant rabbits, although in small quantities, induced changes in gut microbiota colonization and activity, with an acceleration of the ecological species succession and increased production of short-chain fatty acids. Postnatal solid ingestion showed more impact on the appendix microbiota pattern, whereas the type of dietary plant polysaccharides mainly modiﬁed microbiota composition and functions in the cecum. In sum, we showed that the gut microbiota de- velopment trajectory was partly modiﬁed by the type of plant polysaccharides ingested, whereas its maturation speed was more dependent on the timing of solid food introduc- tion. However, no strong impact on the endpoint studied was achieved, suggesting a dilution of the effects observed at maturity. Those results evidence the possibility of shap- ing the developing microbiota with nutrition leverage. MATERIALS AND METHODS Animals and experimental design. Animals were raised and handled at the INRAE experimental unit (PECTOUL, Castanet-Tolosan, France) according to the European Union’s recommendations con- cerning the protection of animals used for scientiﬁc purposes (2010/63/EU) and in agreement with the French legislation (NOR: AGRG1238753A 2013). The experiment received the approval of the local ethics committee (SSA_2019_001). Forty-eight crossbred litters (Hyplus PS19 x Hyplus PS59) were raised until weaning with their doe using a mother–litter separate feeding system (64). Two days after parturition (d2), litter size was stand- ardized to 10 kits. At d3, litters were allocated to three experimental groups (n = 16 litters/group), differ- ing by feeding practices After 36 days, kits were weighed, weaned, and moved to cages of 5 rabbits according to their group and litter of origin. A follow-up of individual rabbit weight was then performed at d50. Controlled suckling was applied once a day in the morning between 0800 h and 0900 h from d2 to d21. Afterwards, free access for nursing was given to the doe rabbits. Milk ingestion was determined as the difference between doe weight before and after nursing at d3, d7, d10, d14, d17, and d21 (65). Litter weight after suckling was recorded at d3, d14, d21, and d28. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Plastic cups were set up within the litter’s nest from d3 to d18. They were used as feeders dedicated to the kits and were ﬁlled with food in a hydrated gel in two experimental groups. Pelleted food was offered ad libitum to all the litters from d15 to weaning, in metal feeders. During the exclusive solid- feeding period (d36–d58), a restricted feeding at 75% of ad libitum ingestion was applied, following common breeding practices. Rabbits received water ad libitum through water nipples. No antibiotic treatment was administered during the experiment. Experimental diets. Throughout the experiment, rabbits had access to a diet either rich in starch (STA diet) or enriched with rapidly fermentable ﬁbers (RFF diet). In the early feeding groups (recorded as STA1 and RFF1), the litters were fed from d3 to d17 nutritional substrates in a hydrated gel form (to optimize the ingestion [66]) in addition to maternal milk, whereas no gels were distributed to the group STA– (Fig. 7). Gel food ingestion was measured daily from d7 on a dry matter basis. DISCUSSION An important matter to resolve in future research will be to understand the implication of early modiﬁcations of the micro- biota on the young mammal immune response in a challenging environment. Month YYYY Volume XX Issue XX MATERIALS AND METHODS In addition, webcams were used to record within two litters the number of visits to the gels for each kit, as an attempt to assess individuals’ feeding behavior. Pelleted food was offered to all the litters from d15 to the end of the experiment. Animals were maintained on the same diet as per their experimental groups (STA or RFF pellets). Two diets were formulated that differed only in the ratio of RFF to starch (0.6 for the STA diet and 2.0 for the RFF diet; Table S1). The RFF component includes pectins, b-glucans, fructans and gums. It excludes the Neutral Detergent Fibers (NDF) fraction, i.e., lignin, hemicellulose, and cellulose. From an analytical point of view, RFF fraction represents the Total Dietary Fibers minus the NDF fraction (51). To achieve balanced diet formulation, the same ingredients were used between the two experimental diets with higher levels of cereals (barley, wheat) and sunﬂower meal in the STA diet and higher concentra- tions of wheat bran fraction, beet, and grape pulp in the RFF diet. The food gels were processed with the corresponding kits’ pellets (either STA or RFF pellets) trans- formed into mash supplemented with attractive vanilla aroma (66) (0.06%, Phodé, Terssac, France) and mixed with hot water (75%) and agar (0.6%). In accordance with the preferences of rabbits in the suck- ling period (64), 2.5-mm-diameter pellets were provided ad libitum from d15 to weaning and switched to pellets with a diameter of 4 mm from weaning to d58. Sample collection. At d18, d25, d30, d38, and d58, 10 kits per group were weighed and then eutha- nized by electronarcosis followed by exsanguination. Those dates correspond to key developmental Month YYYY Volume XX Issue XX 10.1128/msystems.00243-22 13 mSystems Early-Life Nutrition and Gut Microbiota Succession FIG 7 Experimental design. Before weaning, food was offered ad libitum. Afterwards, access to food was restricted (hatched area). Diamonds stand for the sampling dates (n = 30 rabbits/date). STA-, access to starch- enriched food in a pellet form from d15 to d58; STA1, access to starch-enriched food from d3 to d18 in a gel form and from d15 to d58 in a pellet form; RFF1, access to food rich in rapidly fermentable ﬁbres from d3 to d18 in a gel form and from d15 to d58 in a pellet form. FIG 7 Experimental design. Before weaning, food was offered ad libitum. MATERIALS AND METHODS Cecal content metabolome was analyzed by using 1H nuclear magnetic res- onance (NMR)-based metabolomics, following the experimental procedure and the data processing pre- viously described (46). y DNA extraction and 16S rRNA gene sequencing. Total microbial genomic DNA was extracted from 50 mg of cecal or appendix luminal content using the Quick-DNA Fecal/Soil Microbe 96 Kit (ZymoResearch, Irvine, CA, USA) after mechanical lyses by bead beating, according to manufacturer’s instructions. 16S rRNA gene V3-V4 region was ampliﬁed using the primer set 343 F/784 R (343 F: 59– CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG-39 and 784 R: 59-GGAGTTCAGACGTGTGCTCTT CCGATCTTACCAGGGTATCTAATCCT-39) (68, 69). Thirty PCR ampliﬁcation cycles were carried out with an annealing temperature of 65°C. The PCR products were controlled by gel electrophoresis. Single multi- plexing was then performed using 6bp indexes, this indexes were added to R784 during a second PCR with 12 cycles using forward primer (59-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC- 39) and reverse primer (59-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGT-39). The resulting PCR products were puriﬁed and sequenced by MiSeq Illumina Sequencing at the Genomic and Transcriptomic Platform (INRA, Toulouse, France) as previously described (23). PhiX was used as a quality and calibration control for the sequencing run. Negative (ultra-pure water) and positive control (four standard bacterial strains) were sequenced within samples to assess sequencing performance. DNA extraction and 16S rRNA gene sequencing. Total microbial genomic DNA was extracted from 50 mg of cecal or appendix luminal content using the Quick-DNA Fecal/Soil Microbe 96 Kit (ZymoResearch, Irvine, CA, USA) after mechanical lyses by bead beating, according to manufacturer’s instructions. 16S rRNA gene V3-V4 region was ampliﬁed using the primer set 343 F/784 R (343 F: 59– CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG-39 and 784 R: 59-GGAGTTCAGACGTGTGCTCTT CCGATCTTACCAGGGTATCTAATCCT-39) (68, 69). Thirty PCR ampliﬁcation cycles were carried out with an annealing temperature of 65°C. The PCR products were controlled by gel electrophoresis. Single multi- plexing was then performed using 6bp indexes, this indexes were added to R784 during a second PCR with 12 cycles using forward primer (59-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC- 39) and reverse primer (59-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGT-39). The resulting PCR products were puriﬁed and sequenced by MiSeq Illumina Sequencing at the Genomic and Transcriptomic Platform (INRA, Toulouse, France) as previously described (23). PhiX was used as a quality and calibration control for the sequencing run. Negative (ultra-pure water) and positive control (four standard bacterial strains) were sequenced within samples to assess sequencing performance. Sequence analysis. We obtained 12,298,433 16S ribosomal DNA sequences from 296 samples (41,548 6 11,734 reads per sample). MATERIALS AND METHODS Afterwards, access to food was restricted (hatched area). Diamonds stand for the sampling dates (n = 30 rabbits/date). STA-, access to starch- enriched food in a pellet form from d15 to d58; STA1, access to starch-enriched food from d3 to d18 in a gel form and from d15 to d58 in a pellet form; RFF1, access to food rich in rapidly fermentable ﬁbres from d3 to d18 in a gel form and from d15 to d58 in a pellet form. stages of the rabbit’s digestive bacterial ecosystem (23). At d18, rabbits that visually exhibited an interest in gel food were selected for euthanasia, whereas a random selection was performed at the subsequent dates. Blood samples were collected during slaughter in EDTA tubes and dry tubes for plasma and se- rum preparation, respectively. Immediately after euthanasia, the cecum, the appendix vermiformis, the stomach, and the spleen were isolated and weighed full. Afterwards, cecal and appendix contents were collected in sterile tubes and stored at 280°C until further analysis. Proximal cecum tissue was ﬂash fro- zen and preserved at 280°C. pH was measured at the junction of the ileum and cecum quickly after slaughter (VWR Collection SP225, Radnor, PA, USA), and additional cecal luminal content was diluted in H2SO4 (at 2% wt/vol) to quantify ammonia (NH3) concentrations. ELISA measurements and evaluation of oxidative stress. Total plasma IgG and cecal IgA levels were determined in duplicates by sandwich ELISA in 96-well plates coated with speciﬁc polyclonal goat anti-rabbit IgG or IgA antibodies (Bethyl Laboratories, Montgomery, TX, USA) with plate reading at 450 nm as previously described (45). IgG was quantiﬁed by using a reference IgG serum (Bethyl Laboratories). For IgA analysis, 12 samples were pooled to build a reference for the standard curve con- struction. Oxidative stress status of the rabbits was assessed on serums by quantiﬁcation of the deriva- tives of reactive oxygen metabolites (hydroperoxydes primarily) following the procedure of the d-ROM test (Diacron International, Grosseto, Italy). nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Evaluation of the cecal metabolome. Ammonia was measured quantitatively with a colorimetric method using a continuous ﬂow analyzer (SAN11, Skalar, Norcross, GA, USA) as previously described (67). Results were expressed according to the liquid phase of the cecal content after determination of its dry matter at 103°C for 24h. MATERIALS AND METHODS Eventually, 232 OTU out of the 1,197 identiﬁed were used for the prediction. The MetaCyc database was used to classify and inter- pret the relevant metabolic pathways. Cecal tissue gene expressions. Cecal tissue total RNA extraction was performed in TRI reagent using the Direct-zol kit (ZymoResearch) following the manufacturer’s instructions (n = 149 sam- ples extracted). cDNA was prepared from 1 mg RNA with Superscript II (ThermoFisher Scientiﬁc, Waltham, MA, USA) following the manufacturer’s instructions. RNA and cDNA quantity and qual- ity were determined using NanoDrop-2000 (Thermo Fisher Scientiﬁc). High throughput real-time qPCR was performed using the Biomark microﬂuidic system with three 96.96 Dynamic Array IFC for gene expression (Fluidigm, San Francisco, CA, USA). The sequences of the primers used are presented in a supplemental table (see table_primer.txt available at https://doi.org/10.15454/ QSTXWF). The relative fold changes of target genes expression were calculated with the 2-DDCt method with GAPDH as the housekeeping gene and STA– group at d18 as the reference samples. Calculations and statistical analysis. All statistical analyses were performed using R software (ver- sion 4.0), with the corresponding code available at https://github.com/paescharlotte/early_life_nutrition _rabbit/. All the analyses were done with linear mixed models including age, experimental group, and their interaction as ﬁxed effects, litter as random effect, and a correction for age heteroscedasticity when necessary (to satisfy the homogeneity of variances assumption). False discovery rate adjustments (BH procedure) were used for multiple testing. Microbiome, metabolomics, predicted pathways, and host transcriptome data were analyzed with separate investigations of the effect of the timing of solid food introduction (comparisons STA–/STA1 groups) and dietary polysaccharides (comparisons STA1/RFF1 groups) to support eased interpretation. Within-community diversity was evaluated with the R phyloseq package af- ter rarefaction of the OTU table at 8,802 sequences (74). To examine differences in community structure, weighted UniFrac distances (wUniFrac) were calculated on the rareﬁed count matrix. A PERMANOVA was used to perform pairwise comparison between groups using the “adonis” func- tion of the R phyloseq package. To evaluate community evolution dynamics to reach stability, i.e., maturity, the wUniFrac distances to reach 58-day-old microbiota structures were evaluated at the different sampling points. Bacterial relative abundances at the phylum and family levels were fourth root transformed, whereas metabolites relative concentrations and gene expression data were log10 transformed. MATERIALS AND METHODS Sequence processing was performed using the Galaxy-sup- ported pipeline FROGS (70). After discarding sequences with primer mismatch and after deleting sequences with erroneous sequencing length (,400 or .500 nucleotides) and ambiguous base, Month YYYY Volume XX Issue XX 10.1128/msystems.00243-22 14 mSystems Early-Life Nutrition and Gut Microbiota Succession 10,141,290 sequences were left (82% kept, 34,261 6 10,458 reads per sample). One low-sequence sample was discarded so that the number of reads per sample ranged between 8,802 and 56,641. Chimeric DNA sequences were detected using VSEARCH (71) and were removed. Clustering was performed using SWARM 21 (aggregation distance of 3) (72). After that, we assigned the opera- tional taxonomic units (OTUs) to the obtained paired sequences using the BLAST algorithm against the SILVA SSU Ref NR 138 data set 22 (73). A total of 1,197 OTUs were kept (min: 224 – max: 927 OTUs per sample). An OTU table with taxonomic afﬁliations as well as metadata was then built for subsequent analysis with the phyloseq package (74). 10,141,290 sequences were left (82% kept, 34,261 6 10,458 reads per sample). One low-sequence sample was discarded so that the number of reads per sample ranged between 8,802 and 56,641. Chimeric DNA sequences were detected using VSEARCH (71) and were removed. Clustering was performed using SWARM 21 (aggregation distance of 3) (72). After that, we assigned the opera- tional taxonomic units (OTUs) to the obtained paired sequences using the BLAST algorithm against the SILVA SSU Ref NR 138 data set 22 (73). A total of 1,197 OTUs were kept (min: 224 – max: 927 OTUs per sample). An OTU table with taxonomic afﬁliations as well as metadata was then built for subsequent analysis with the phyloseq package (74). Prediction of microbial functional proﬁles. PICRUSt2 analysis was used to predict the cecal micro- bial community functions with the unrareﬁed OTU abundance table as input (75). Nearest sequenced taxon index (NSTI) values are an indicator of PICRUSt2 prediction accuracy, with lower scores indicating availability of closely related genomes (75). NSTI scores across all bacterial samples were relatively high (0.42 6 0.35). Therefore, OTUs with insufﬁcient reference genome coverage were removed from the analysis, i.e., predictions with NSTI scores superior to 0.5. This threshold was chosen as a compromise between accuracy (removal of samples with low NSTI scores) and reliability (72% of the total relative abundances kept within our microbial communities), as presented in Fig. S1. MATERIALS AND METHODS Based on the threshold of 0.5% of relative abundances for OTUs quantitative repeatability (14), OTU differential abundance testing was performed using the DESeq2 package with multiple comparison corrections (76). Likewise, the number of MetaCyc metabolic pathways per treatment was analyzed through DeSeq procedure. nloaded from https://journals.asm.org/journal/msystems on 20 June 2022 by 147.100.179.233. Data Availability. Amplicon sequences are available online (NCBI accession PRJNA615661). Analysis scripts are available via https://github.com/paescharlotte/early_life_nutrition_rabbit/. Other data used can be accessed at https://doi.org/10.15454/QSTXWF. REFERENCES 16. Choudhury R, Middelkoop A, de Souza JG, van Veen LA, Gerrits WJJ, Kemp B, Bolhuis JE, Kleerebezem M. 2021. Impact of early-life feeding on local intestinal microbiota and digestive system development in piglets. Sci Rep 11:4213. https://doi.org/10.1038/s41598-021-83756-2. 1. Thursby E, Juge N. 2017. Introduction to the human gut microbiota. Bio- chem J 474:1823–1836. https://doi.org/10.1042/BCJ20160510. 1. Thursby E, Juge N. 2017. Introduction to the human gut microbiota. 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J Evolutionary Biology 22:1984–1999. https://doi.org/10 .1111/j.1420-9101.2009.01809.x. 13. Wang W, Li C, Li F, Wang X, Zhang X, Liu T, Nian F, Yue X, Li F, Pan X, La Y, Mo F, Wang F, Li B. 2016. Effects of early feeding on the host rumen tran- scriptome and bacterial diversity in lambs. Sci Rep 6:32479. https://doi .org/10.1038/srep32479. 27. Combes S, Michelland RJ, Monteils V, Cauquil L, Soulié V, Tran NU, Gidenne T, Fortun-Lamothe L. 2011. SUPPLEMENTAL MATERIAL Supplemental material is available online only. Supplemental material is avai FIG S1, DOCX ﬁle, 0.1 MB. FIG S2, DOCX ﬁle, 0.04 MB. FIG S3, DOCX ﬁle, 0.03 MB. FIG S4, DOCX ﬁle, 0.1 MB. FIG S5, DOCX ﬁle, 0.1 MB. FIG S6, DOCX ﬁle, 0.1 MB. TABLE S1, XLSX ﬁle, 0.01 MB. TABLE S2, XLSX ﬁle, 0.01 MB. TABLE S3, XLSX ﬁle, 0.02 MB. TABLE S4, XLSX ﬁle, 0.02 MB. FIG S1, DOCX ﬁle, 0.1 MB. FIG S2, DOCX ﬁle, 0.04 MB. FIG S3, DOCX ﬁle, 0.03 MB. FIG S4, DOCX ﬁle, 0.1 MB. FIG S5, DOCX ﬁle, 0.1 MB. FIG S6, DOCX ﬁle, 0.1 MB. TABLE S1, XLSX ﬁle, 0.01 MB. TABLE S2, XLSX ﬁle, 0.01 MB. TABLE S3, XLSX ﬁle, 0.02 MB. TABLE S4, XLSX ﬁle, 0.02 MB. 10.1128/msystems.00243-22 15 Month YYYY Volume XX Issue XX Month YYYY Volume XX Issue XX Month YYYY Volume XX Issue XX mSystems Early-Life Nutrition and Gut Microbiota Succession ACKNOWLEDGMENTS We gratefully acknowledge M. Moulis and J.-M. Bonnemere for their assistance at the rabbit experimental unit, as well as M. Segura, who contributed to sample preparation, and C. Bannelier, who performed the food analysis. We thank the people of the research teams NED (https://genphyse.toulouse.inra.fr/groups/ned) and SYSED (https://genphyse .toulouse.inra.fr/groups/sysed) for their assistance during data collection. We especially thank C. Knudsen for her careful proofreading. We acknowledge L. Gress and S. Fourre, who performed the quantitative Fluidigm assay. The authors are grateful to the Genotoul bioinformatics platform Toulouse Midi-Pyrenees and the Sigenae group for providing computing and storage resources thanks to Galaxy instance http://sigenae-workbench .toulouse.inra.fr. We thank the metabolomics platform Metatoul-AXIOM in Toulouse. We declare that we have no conﬂicts of interest. REFERENCES Br J Nutr 18:385–392. https://doi.org/10.1079/bjn19640035. 7. Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, Angenent LT, Ley RE. 2011. Succession of microbial consortia in the devel- oping infant gut microbiome. Proc Natl Acad Sci U S A 108(Suppl 1): 4578–4585. https://doi.org/10.1073/pnas.1000081107. 21. Paës C, Aymard P, Debrusse A-M, Laperruque F, Bannelier C, Bébin K, Duperray J, Gohier C, Guené-Grand E, Rebours G, Gidenne T, Fortun-Lamothe L, Combes S. 2020. 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Catatan: Pernyataan hak cipta dan lisensi ini berlaku untuk buku ini secara keseluruhan, termasuk semua konten yang terkandung di dalamnya, kecuali disebutkan sebaliknya. Hak cipta dari website, aplikasi, atau halaman eksternal yang dijadikan contoh, dipegang dan dimiliki oleh sumber aslinya ii KATA PENGANTAR Puji syukur kepada Tuhan Yang Maha Esa, sehingga Buku Ajar Pengolahan Citra Digital ini dapat disusun dengan baik meskipun perlu penyempurnaan lebih lanjut. Pengolahan Citra Digital merupakan mata kuliah Konsentrasi Kecerdasan Buatan di Program Studi Informatika Universitas Muhammadiyah Sidoarjo. Buku ajar Pengolahan Citra Digital ini dikhususkan untuk pegangan Mahasiswa Informatika Fakultas Sains dan Teknologi Universitas Muhammadiyah Sidoarjo. Mata kuliah prasarat yang harus diselesaikan sebelum mengambil mata kuliah ini adalah: 1. Algoritma Pemograman 2. Pengantar Kecerdasan Buatan 3. Statistik Komputasi Di era komputerisasi ini perkembangan terkonologi komputasi cerdas komputer kemajuannya sangat pesat karena dituntut untuk mampu membuat teknologi terapan dengan kecepatan komputasi yang tinggi dengan ketelitian dan kualitas yang maksimal. Penulisan buku ajar Pengolahan Citra Digital ini ditulis dalam 16 BAB yang mengacu pada capaikan pembelajaran mata kuliah pengolahan citra digital. Dengan selesainya penulisan buku ajar ini penulis mengucapkan terima kasih kepada semua pihak yang telah iii memberikan bahan-bahan tulisan baik langsung maupun tidak langsung. Penulis juga mengucapkan terima kasih khususnya kepada: 1. Dr. Hidayatullah, M.Si pemangku pimpinan tertinggi yaitu Rektor Universitas Muhammadiyah Sidoarjo yang telah memberikan dan memfasilitasi dalam penulisan buku ajar ini. 1. Dr. Hidayatullah, M.Si pemangku pimpinan tertinggi yaitu Rektor Universitas Muhammadiyah Sidoarjo yang telah memberikan dan memfasilitasi dalam penulisan buku ajar ini. 2. Perpustakaan Universitas Muhammadiyah Sidoarjo yang telah memfasilitasi dan mengkoordinasi dalam penulisan buku ajar ini. 2. Perpustakaan Universitas Muhammadiyah Sidoarjo yang telah memfasilitasi dan mengkoordinasi dalam penulisan buku ajar ini. Akhir kata, kritik dan saran sangat diharapkan untuk penyempurnaan buku ajar ini. Harapan kami semoga buku ajar ini dapat digunakan sebagai tambahan informasi dan bermanfaat bagi aktivitas pembelajaran mata kuliah Pengolahan Citra Digital di Program Studi Informatika, Fakultas Sains dan Teknologi, Universitas Muhammadiyah Sidoarjo. Penulis iv Bab I. Pengantar Pengolahan Citra Digital 1.1 Definisi Citra Digital Bab I. Pengantar Pengolahan Citra Digital 1.1 Definisi Citra Digital Citra digital adalah salah satu bentuk representasi visual dari dunia nyata dalam bentuk digital yang dapat dipahami dan diolah oleh komputer. Citra ini terdiri dari elemen-elemen titik yang disebut piksel, yang tersusun dalam baris dan kolom. Setiap piksel memiliki nilai numerik yang menggambarkan tingkat kecerahan atau warna pada posisi tertentu dalam citra. Citra digital dapat diperoleh dari berbagai sumber, seperti kamera digital, scanner, atau hasil simulasi komputer.(E Woods & C Gonzalez, 2008). Dalam definisi ini, kami menjelaskan bahwa citra digital adalah representasi visual dalam bentuk digital yang terdiri dari piksel-piksel dengan nilai numerik. Daftar Isi KATA PENGANTAR ........................................................... iii Daftar Isi ................................................................................. v Bab I. Pengantar Pengolahan Citra Digital ..............................1 Bab 2. Dasar Matematika untuk Pengolahan Citra ..................6 Bab 3. Pengambilan Citra ......................................................11 Bab 4. Pengambilan dan Praproses Citra ...............................18 BAB 5. Pengolahan Intensitas dan Transformasi ...................23 Bab 6. Filtrasi dalam Domain Spasial ...................................29 Bab 7. Transformasi Citra dan Pengolahan Frekuensi ...........33 Bab 8. Transformasi Wavelet ................................................37 Bab 9. Analisis dan Segmentasi Citra ....................................40 Bab 10. Segmentasi Citra ......................................................44 Bab 11. Ekstraksi Fitur dan Pengenalan Pola ........................49 Bab 12. Pengenalan Pola .......................................................55 Bab 13. Aplikasi Lanjutan .....................................................62 Bab 14. Pengolahan Citra dalam Realitas Virtual dan Augmented ............................................................................67 Bab 15. Kasus Studi ..............................................................71 Bab 16. Kesimpulan ..............................................................76 Biodata Penulis ....................................................................81 v e) Pengenalan Pola Pengenalan Objek: Mengidentifikasi objek dalam citra. Pengenalan Objek: Mengidentifikasi objek dalam citra. Pengenalan Karakter: Mentranskripsikan teks dari citra tulisan tangan atau cetakan. Pengenalan Karakter: Mentranskripsikan teks dari citra tulisan tangan atau cetakan. Pengenalan Wajah: Mengidentifikasi wajah dalam citra.. Pengenalan Wajah: Mengidentifikasi wajah dalam citra.. 1.2 Tujuan Pengolahan Citra Tujuan utama dari pengolahan citra digital adalah memproses dan memanipulasi citra digital untuk berbagai keperluan. Pengolahan citra ini melibatkan serangkaian operasi yang bertujuan untuk meningkatkan kualitas citra, mengekstraksi informasi penting, dan membuat citra lebih mudah dipahami atau digunakan dalam berbagai aplikasi(Svoboda et al., 2007). Berikut adalah beberapa tujuan utama dari pengolahan citra digital: a) Peningkatan Kualitas Citra a) Peningkatan Kualitas Citra Penajaman Kontras: Memperjelas perbedaan antara area gelap dan terang dalam citra. Peningkatan Kejelasan: Memperbaiki ketajaman dan rincian dalam citra. Reduksi Noise: Mengurangi gangguan acak yang dapat mengaburkan citra. Reduksi Noise: Mengurangi gangguan acak yang dapat mengaburkan citra. b) Ekstraksi Fitur Tepi Deteksi: Menemukan lokasi tepi atau perubahan tajam dalam citra. 1 Ekstraksi Tekstur: Mengidentifikasi pola tekstur dalam citra. Ekstraksi Fitur Bentuk: Mendeteksi objek dan bentuk khusus dalam citra. c) Segmentasi Citra Segmentasi Warna: Membagi citra menjadi kelompok berdasarkan warna. Segmentasi Objek: Memisahkan objek dari latar belakang dalam citra. Pemisahan Wilayah: Membagi citra menjadi wilayah-wilayah homogen. d) Kompresi Citra Reduksi Ukuran: Mengurangi ukuran citra untuk penyimpanan atau transmisi yang efisien. Kompresi Tanpa Kehilangan: Mengurangi ukuran citra tanpa kehilangan banyak informasi. e) Pengenalan Pola Pengenalan Objek: Mengidentifikasi objek dalam citra. Pengenalan Karakter: Mentranskripsikan teks dari citra tulisan tangan atau cetakan. Pengenalan Wajah: Mengidentifikasi wajah dalam citra.. 1.3 Aplikasi Pengolahan Citra Pengolahan citra digital memiliki beragam aplikasi yang sangat relevan di berbagai bidang. Kemampuan untuk memproses dan menganalisis citra digital membuka pintu bagi banyak inovasi dan perbaikan dalam berbagai industri dan disiplin ilmu. Berikut adalah beberapa aplikasi utama dari pengolahan citra digital: Ekstraksi Tekstur: Mengidentifikasi pola tekstur dalam citra. Ekstraksi Fitur Bentuk: Mendeteksi objek dan bentuk khusus dalam citra. Ekstraksi Tekstur: Mengidentifikasi pola tekstur dalam citra. Ekstraksi Fitur Bentuk: Mendeteksi objek dan bentuk khusus dalam citra. d) Kompresi Citra Reduksi Ukuran: Mengurangi ukuran citra untuk penyimpanan atau transmisi yang efisien. Kompresi Tanpa Kehilangan: Mengurangi ukuran citra tanpa kehilangan banyak informasi. 1.3 Aplikasi Pengolahan Citra Pengolahan citra digital memiliki beragam aplikasi yang sangat relevan di berbagai bidang. Kemampuan untuk memproses dan menganalisis citra digital membuka pintu bagi banyak inovasi dan perbaikan dalam berbagai industri dan disiplin ilmu. Berikut adalah beberapa aplikasi utama dari pengolahan citra digital: 2 a) Kedokteran Diagnosa Medis: Pengolahan citra digunakan untuk mendiagnosis penyakit dan mengidentifikasi gangguan dalam citra medis seperti MRI (Magnetic Resonance Imaging) dan CT scan (Computed Tomography). Pemantauan Kesehatan: Pengawasan perkembangan penyakit dan pemantauan kondisi pasien. Pencitraan Bedah: Bantuan dalam prosedur bedah dan pencitraan endoskopi. a) Kedokteran Diagnosa Medis: Pengolahan citra digunakan untuk mendiagnosis penyakit dan mengidentifikasi gangguan dalam citra medis seperti MRI (Magnetic Resonance Imaging) dan CT scan (Computed Tomography). Pemantauan Kesehatan: Pengawasan perkembangan penyakit dan pemantauan kondisi pasien. Pencitraan Bedah: Bantuan dalam prosedur bedah dan pencitraan endoskopi. b) Industri Manufaktur b) Industri Manufaktur Kontrol Kualitas: Memeriksa produk-produk untuk kecacatan atau ketidaksesuaian. Kontrol Kualitas: Memeriksa produk-produk untuk kecacatan atau ketidaksesuaian. Pemantauan Proses: Mengamati dan mengontrol proses produksi untuk meningkatkan efisiensi. Pemantauan Proses: Mengamati dan mengontrol proses produksi untuk meningkatkan efisiensi. Desain Produk: Mengembangkan desain produk dan prototipe dengan bantuan pemodelan citra. c) Kehutanan Pengawasan Hutan: Memantau keadaan hutan dan deteksi perubahan, termasuk pemantauan kebakaran hutan. c) Kehutanan Pengawasan Hutan: Memantau keadaan hutan dan deteksi perubahan, termasuk pemantauan kebakaran hutan. Penelitian Ekologi: Studi lingkungan dan perkembangan tumbuhan dan satwa liar. Penelitian Ekologi: Studi lingkungan dan perkembangan tumbuhan dan satwa liar. d) Robotika Penglihatan Komputer: Menggunakan pengolahan citra untuk navigasi dan pengenalan dalam robotika. Kendaraan Otonom: Dalam kendaraan otonom seperti mobil tanpa pengemudi. Kendaraan Otonom: Dalam kendaraan otonom seperti mobil tanpa pengemudi. e) Keamanan Pengawasan Video: Memonitor area keamanan melalui kamera pengawas dan mendeteksi perilaku mencurigakan. Pengenalan Wajah: Mengenali individu melalui analisis citra wajah. ) Pengawasan Video: Memonitor area keamanan melalui kamera pengawas dan mendeteksi perilaku mencurigakan. pengawas dan mendeteksi perilaku mencurigakan. Pengenalan Wajah: Mengenali individu melalui analisis citra wajah. Pengenalan Wajah: Mengenali individu melalui analisis citra wajah. 3 f) Hiburan Efek Visual: Pencitraan digital digunakan untuk menciptakan efek visual dalam film dan permainan video. Pengolahan Gambar: Mengedit dan memperbaiki citra untuk keperluan hiburan. g) Astronomi Pencitraan Astronomi: Analisis citra astronomi untuk mendapatkan pemahaman tentang alam semesta dan objek- objek langit. 1.4 Sejarah Pengolahan Citra Digital Pengolahan citra digital memiliki sejarah yang menarik yang dimulai pada awal abad ke-20 dan berkembang pesat seiring dengan perkembangan teknologi komputer. Sejarah ini mencakup beberapa tonggak penting dalam perkembangan pengolahan citra digital. 1.3 Aplikasi Pengolahan Citra a) Awal Pengolahan Citra Analog Sebelum era digital, pengolahan citra dilakukan secara analog menggunakan teknik fotografi dan teknik pencitraan kimia. Teknik ini terbatas dalam hal manipulasi citra dan pengambilan tindakan lebih lanjut berdasarkan hasil pencitraan. b) Era Awal Pengolahan Citra Digital (1940-an - 1950-an) b) Era Awal Pengolahan Citra Digital (1940-an - 1950-an) Pada tahun 1940-an dan 1950-an, komputer pertama digunakan untuk pemrosesan citra. Salah satu contohnya adalah komputer ENIAC yang digunakan untuk mengolah citra radar selama Perang Dunia II. Ini menjadi titik awal pengolahan citra digital. b) Era Awal Pengolahan Citra Digital (1940-an - 1950-an) Pada tahun 1940-an dan 1950-an, komputer pertama digunakan untuk pemrosesan citra. Salah satu contohnya adalah komputer ENIAC yang digunakan untuk mengolah citra radar selama Perang Dunia II. Ini menjadi titik awal pengolahan citra digital. c) Pengembangan Teknik Pengolahan Citra Digital (1960-an - 1970-an) c) Pengembangan Teknik Pengolahan Citra Digital (1960-an - 1970-an) Pada tahun 1960-an dan 1970-an, pengolahan citra digital mulai berkembang sebagai disiplin ilmu tersendiri. Teknik- 4 teknik seperti pemrosesan histogram dan deteksi tepi mulai dikembangkan. Pada saat ini, penggunaan komputer menjadi lebih umum dalam pengolahan citra. d) Munculnya Standar Pengolahan Citra (1980-an - 1990-an) Pada tahun 1980-an dan 1990-an, industri dan organisasi internasional mulai mengembangkan standar untuk pengolahan citra digital. Hal ini membantu memfasilitasi pertukaran dan pemrosesan citra antar platform dan aplikasi. e) Era Modern Pengolahan Citra Digital (2000-an - Sekarang) Dengan perkembangan pesat teknologi komputer dan perangkat keras yang kuat, pengolahan citra digital telah mencapai tingkat yang belum pernah terjadi sebelumnya. Algoritma-algoritma canggih seperti pengenalan pola dan pembelajaran mesin digunakan dalam berbagai aplikasi, termasuk pengenalan wajah, kendaraan otonom, dan banyak lagi. 5 Bab 2. Dasar Matematika untuk Pengolahan Citra 2.1. Konsep Dasar Citra Bab 2. Dasar Matematika untuk Pengolahan Citra 2.1. Konsep Dasar Citra Citra adalah representasi visual dari dunia nyata atau objek dalam bentuk yang dapat dipahami oleh komputer. Untuk memahami pengolahan citra digital dengan baik, penting untuk memahami konsep dasar berikut: Piksel adalah unit dasar dari citra Setiap piksel memiliki koordinat spasial (baris dan kolom) yang menentukan posisinya dalam citra. Setiap piksel memiliki nilai numerik yang menggambarkan tingkat kecerahan (grayscale) atau kombinasi warna (citra berwarna). Resolusi mengacu pada jumlah piksel dalam citra. Citra dengan resolusi tinggi memiliki lebih banyak detail karena memiliki lebih banyak piksel. Citra dengan resolusi tinggi memiliki lebih banyak detail karena memiliki lebih banyak piksel. Citra dengan resolusi rendah cenderung lebih kasar. c. Mode Citra Mode citra mengacu pada jenis informasi yang diwakili oleh setiap piksel. Citra grayscale hanya memiliki informasi kecerahan. Citra berwarna memiliki informasi kecerahan dan warna, biasanya dalam mode RGB (Merah, Hijau, Biru). d. Citra Biner Citra biner hanya memiliki dua nilai piksel yang mungkin (hitam dan putih). Biasanya digunakan untuk segmentasi citra. e. Format Citra Format citra mengacu pada cara data citra disimpan dalam memori komputer, seperti JPEG, PNG, atau BMP. c. Citra Biner c. Citra Biner Citra biner hanya memiliki dua nilai piksel yang mungkin: hitam dan putih. Ini adalah hasil dari proses segmentasi, di mana piksel-piksel dikelompokkan menjadi dua kelas berdasarkan ambang tertentu. Representasi citra biner menggunakan nilai biner, sering kali 0 untuk hitam dan 1 untuk putih. 2.2. Representasi Citra Digital Representasi citra digital adalah cara untuk menggambarkan citra dalam bentuk yang dapat dipahami oleh komputer. Ini melibatkan penggunaan matriks atau array angka untuk mewakili tingkat kecerahan atau warna setiap piksel dalam citra. Dalam pengolahan citra digital, ada beberapa jenis representasi citra yang penting untuk dipahami: a. Citra Grayscale Citra grayscale adalah citra yang hanya memiliki tingkat kecerahan sebagai informasi. Setiap piksel dalam citra grayscale direpresentasikan oleh satu angka, biasanya dalam rentang 0 hingga 255. Nilai 0 mewakili warna hitam, sementara nilai 255 mewakili warna putih. a. Citra Grayscale Citra grayscale adalah citra yang hanya memiliki tingkat kecerahan sebagai informasi. Setiap piksel dalam citra grayscale direpresentasikan oleh satu angka, biasanya dalam rentang 0 hingga 255. Nilai 0 mewakili warna hitam, sementara nilai 255 mewakili warna putih. b. Citra Berwarna (Mode RGB) d. Citra Biner Citra biner hanya memiliki dua nilai piksel yang mungkin (hitam dan putih). Biasanya digunakan untuk segmentasi citra. e. Format Citra Format citra mengacu pada cara data citra disimpan dalam memori komputer, seperti JPEG, PNG, atau BMP. e. Format Citra Format citra mengacu pada cara data citra disimpan dalam memori komputer, seperti JPEG, PNG, atau BMP. e. Format Citra Format citra mengacu pada cara data citra disimpan dalam memori komputer, seperti JPEG, PNG, atau BMP. 6 b. Citra Berwarna (Mode RGB) b. Citra Berwarna (Mode RGB) Citra berwarna mengandung informasi kecerahan dan warna. Representasi paling umum untuk citra berwarna adalah mode RGB (Merah, Hijau, Biru). Setiap piksel dalam citra RGB direpresentasikan oleh tiga nilai, masing-masing mewakili tingkat merah, hijau, dan biru dalam rentang 0 hingga 255. d. Matriks Citra Citra digital dapat direpresentasikan sebagai matriks dua dimensi di mana setiap elemen matriks mewakili nilai piksel di lokasi yang sesuai dalam citra. Matriks ini dapat diolah 7 menggunakan operasi matematika untuk menghasilkan efek tertentu pada citra. 2.4. Transformasi Citra Transformasi citra adalah proses mengubah citra digital dari domain spasial (pada piksel individu) ke domain frekuensi atau domain lain untuk tujuan tertentu. Ini adalah salah satu konsep dasar dalam pengolahan citra digital dan sering digunakan untuk meningkatkan citra atau mengekstraksi informasi penting. Berikut adalah beberapa transformasi citra yang umum digunakan: Transformasi citra adalah proses mengubah citra digital dari domain spasial (pada piksel individu) ke domain frekuensi atau domain lain untuk tujuan tertentu. Ini adalah salah satu konsep dasar dalam pengolahan citra digital dan sering digunakan untuk meningkatkan citra atau mengekstraksi informasi penting. Berikut adalah beberapa transformasi citra yang umum digunakan: a) Transformasi Fourier a) Transformasi Fourier Transformasi Fourier digunakan untuk mengubah citra dari domain spasial ke domain frekuensi. Ini berguna untuk menganalisis spektrum frekuensi citra dan dapat digunakan dalam proses seperti penyaringan frekuensi dan pemulihan citra. c) Transformasi Geometrik Transformasi geometrik digunakan untuk mengubah bentuk atau posisi objek dalam citra. Ini termasuk rotasi, pemantulan, dan skalasi. Transformasi geometrik digunakan untuk mengubah bentuk atau posisi objek dalam citra. Ini termasuk rotasi, pemantulan, dan skalasi. b) Transformasi Wavelet b) Transformasi Wavelet Transformasi wavelet membagi citra menjadi komponen frekuensi berbeda pada berbagai tingkat resolusi. Ini memungkinkan deteksi dan representasi fitur citra pada tingkat resolusi yang berbeda. Transformasi wavelet membagi citra menjadi komponen frekuensi berbeda pada berbagai tingkat resolusi. Ini memungkinkan deteksi dan representasi fitur citra pada tingkat resolusi yang berbeda. c) Transformasi Geometrik 2.3. Operasi Dasar Citra Digital Operasi dasar pada citra adalah serangkaian operasi matematika dan transformasi yang diterapkan pada citra untuk mengubahnya atau menghasilkan citra baru. Ini adalah langkah awal dalam pengolahan citra digital. Beberapa operasi dasar pada citra melibatkan manipulasi tingkat kecerahan, kontras, dan distribusi nilai piksel. Berikut adalah beberapa operasi dasar yang umumnya digunakan: a. Peningkatan Kecerahan dan kontras Peningkatan Kecerahan: Operasi ini meningkatkan tingkat kecerahan seluruh citra dengan menambahkan nilai tetap ke setiap piksel. Ini membuat citra terlihat lebih terang. Peningkatan Kontras: Operasi ini meningkatkan kontras citra dengan menggandakan seluruh rentang nilai piksel sehingga perbedaan antara piksel-piksel yang berdekatan lebih jelas. p p g Peningkatan Kontras: Operasi ini meningkatkan kontras citra dengan menggandakan seluruh rentang nilai piksel sehingga perbedaan antara piksel-piksel yang berdekatan lebih jelas. b. Transformasi Logaritmik Operasi ini mengubah tingkat kecerahan piksel dengan menggunakan logaritma. Hal ini berguna untuk meningkatkan detail di area dengan tingkat kecerahan rendah. b. Transformasi Logaritmik Operasi ini mengubah tingkat kecerahan piksel dengan menggunakan logaritma. Hal ini berguna untuk meningkatkan detail di area dengan tingkat kecerahan rendah. c. Transformasi Gamma c. Transformasi Gamma Transformasi gamma adalah operasi non-linear yang digunakan untuk memodifikasi kontras citra. Ini berguna untuk menyesuaikan tingkat kecerahan dalam kondisi tertentu. d. Operasi Konvolusi Operasi konvolusi melibatkan penggunaan kernel atau matriks konvolusi untuk mengubah nilai piksel dalam citra. Ini dapat digunakan untuk penghalusan, deteksi tepi, atau filter citra. e. Transformasi Citra Transformasi citra melibatkan perubahan koordinat piksel dalam citra. Ini bisa termasuk rotasi, skalasi, atau pemantulan. e. Transformasi Citra Transformasi citra melibatkan perubahan koordinat piksel dalam citra. Ini bisa termasuk rotasi, skalasi, atau pemantulan. e. Transformasi Citra Transformasi citra melibatkan perubahan koordinat piksel dalam citra. Ini bisa termasuk rotasi, skalasi, atau pemantulan. 8 2.4. Transformasi Citra 2.4. Transformasi Citra Bab 3. Pengambilan Citra Pengambilan citra adalah tahap pertama dalam pengolahan citra digital di mana citra direkam atau diperoleh dari sumber aslinya. Proses pengambilan citra dapat bervariasi tergantung pada jenis sumber citra dan tujuannya. Dalam subbab ini, kita akan membahas beberapa aspek penting terkait pengambilan citra: Hardware Pengambilan Citra Kamera Digital: Kamera digital adalah salah satu sumber citra paling umum. Mereka dapat digunakan untuk mengambil gambar diam (foto) atau merekam video. Kamera digital modern dapat menghasilkan citra beresolusi tinggi dengan berbagai fitur pengaturan. Scanner: Scanner digunakan untuk mengambil citra dari dokumen cetak atau gambar yang telah ada. Mereka menghasilkan citra dengan tingkat resolusi yang tinggi dan kualitas yang baik. Sensor Citra: Sensor citra digunakan dalam berbagai aplikasi seperti pengawasan, penglihatan mesin, dan kendaraan otonom. Mereka dapat berupa kamera termal, sensor radar, atau lidar. d) Transformasi Histogram d) Transformasi Histogram d) Transformasi Histogram Transformasi histogram adalah teknik yang digunakan untuk mengubah distribusi tingkat kecerahan dalam citra.Ini berguna untuk meningkatkan kontras dan penyesuaian tingkat kecerahan. ) g Transformasi histogram adalah teknik yang digunakan untuk mengubah distribusi tingkat kecerahan dalam citra.Ini berguna untuk meningkatkan kontras dan penyesuaian tingkat kecerahan. e) Transformasi Logaritmik dan Eksponensial e) Transformasi Logaritmik dan Eksponensial Transformasi logaritmik dan eksponensial digunakan untuk mengubah nilai piksel dalam citra secara non-linear. Ini sering Transformasi logaritmik dan eksponensial digunakan untuk mengubah nilai piksel dalam citra secara non-linear. Ini sering 9 digunakan untuk meningkatkan kontras di area dengan kecerahan rendah atau tinggi. 10 10 Bab 3. Pengambilan Citra Prinsip Pengambilan Citra yang Baik Parameter Pengambilan Citra Resolusi: Resolusi citra mengacu pada jumlah piksel dalam citra. Resolusi tinggi menghasilkan citra dengan detail yang tinggi, sementara resolusi rendah menghasilkan citra dengan detail yang lebih rendah. Kedalaman Warna: Kedalaman warna mengacu pada jumlah warna yang dapat direpresentasikan dalam citra. Citra berwarna memiliki kedalaman warna yang lebih tinggi dibandingkan citra grayscale. Kecepatan Pengambilan Citra: Kecepatan pengambilan citra penting dalam aplikasi bergerak seperti kendaraan otonom atau pengawasan real-time. Prinsip Pengambilan Citra yang Baik 11 Pencahayaan: Pencahayaan yang baik sangat penting untuk pengambilan citra yang berkualitas. Pastikan kondisi pencahayaan sesuai dengan kebutuhan.Fokus: Pastikan citra fokus dengan baik untuk menghindari citra yang buram atau kabur.Kompensasi Guncangan: Dalam situasi bergerak, seperti pengambilan citra dari kendaraan bergerak, penggunaan stabilisasi atau teknologi anti-guncangan dapat membantu menjaga kestabilan citra. a) Kamera Digital Kamera digital adalah perangkat keras pengambilan citra yang paling umum digunakan oleh pengguna umum. Mereka terdiri dari lensa optik untuk mengumpulkan cahaya dan sensor gambar untuk merekam citra. Sensor gambar dalam kamera digital dapat berupa CCD (Charge-Coupled Device) atau CMOS (Complementary Metal-Oxide-Semiconductor). 3.1. Hardware Pengambilan Citra Hardware pengambilan citra adalah perangkat keras yang digunakan untuk mendapatkan citra dari berbagai sumber. Perangkat keras ini berperan penting dalam pengambilan citra karena menentukan resolusi, kualitas, dan kemampuan untuk beradaptasi dengan berbagai kondisi pengambilan citra. Dalam subbab ini, kita akan membahas beberapa jenis hardware pengambilan citra yang umum digunakan: b) Scanner Scanner adalah perangkat keras yang digunakan untuk mengambil citra dari dokumen cetak, foto, atau gambar lainnya. Mereka bekerja dengan menggulung atau meletakkan dokumen di bawah pemindai yang memiliki sensor untuk mengukur tingkat reflektansi pada setiap titik. 12 Kamera Spesialis c) Ada kamera yang dirancang khusus untuk aplikasi tertentu seperti kamera termal untuk pengukuran suhu, kamera satelit untuk pengambilan citra bumi, dan kamera endoskopi untuk pemeriksaan dalam tubuh manusia. e) Perangkat Penangkap Video Perangkat penangkap video adalah perangkat keras yang digunakan untuk merekam citra bergerak dalam bentuk video. Mereka sering digunakan dalam pengawasan video dan rekaman film. f) Kedalaman Warna dan Resolusi Keputusan dalam memilih hardware pengambilan citra termasuk kedalaman warna (bit per piksel) dan resolusi (jumlah piksel). Pemilihan kedalaman warna dan resolusi tergantung pada aplikasi dan kebutuhan spesifik. d) Sensor Citra Sensor citra adalah perangkat keras yang digunakan untuk mengambil citra di berbagai aplikasi seperti pengawasan keamanan, kendaraan otonom, dan fotografi udara. Jenis sensor mencakup sensor radar, lidar, dan sensor termal. e) Perangkat Penangkap Video e) Perangkat Penangkap Video e) Penempatan Perspektif b) Pengaturan Fokus b) Pengaturan Fokus b) Fokus yang baik sangat penting untuk mendapatkan citra yang tajam dan jelas. Pengaturan fokus melibatkan penyesuaian lensa kamera atau sensor untuk memfokuskan cahaya pada sensor gambar. Fokus yang baik sangat penting untuk mendapatkan citra yang tajam dan jelas. Pengaturan fokus melibatkan penyesuaian lensa kamera atau sensor untuk memfokuskan cahaya pada sensor gambar. 3.2. Teknik Pengambilan Citra Teknik pengambilan citra merujuk pada berbagai metode yang digunakan untuk mengoptimalkan proses pengambilan citra dan memastikan bahwa citra yang dihasilkan sesuai dengan 13 tujuan aplikasi tertentu. Berikut adalah beberapa teknik pengambilan citra yang umum digunakan: Pencahayaan yang Dikontrol Pencahayaan yang Dikontrol a) Pencahayaan yang baik adalah kunci untuk pengambilan citra yang berkualitas. Teknik pengaturan pencahayaan termasuk penggunaan cahaya alami, cahaya buatan, dan penggunaan reflektor atau difusor untuk mengendalikan intensitas cahaya. Pencahayaan yang baik adalah kunci untuk pengambilan citra yang berkualitas. Teknik pengaturan pencahayaan termasuk penggunaan cahaya alami, cahaya buatan, dan penggunaan reflektor atau difusor untuk mengendalikan intensitas cahaya. Penggunaan Filter Optik Penggunaan Filter Optik Filter optik digunakan untuk mengatur karakteristik cahaya yang mencapai sensor gambar. Filter polarisasi, filter ND (Neutral Density), dan filter inframerah adalah contoh filter optik yang dapat digunakan untuk efek tertentu. g) Penggunaan Teknik Khusus Dalam aplikasi tertentu, seperti fotografi makro, fotografi inframerah, atau fotografi malam, teknik pengambilan citra khusus mungkin diperlukan. Dalam aplikasi tertentu, seperti fotografi makro, fotografi inframerah, atau fotografi malam, teknik pengambilan citra khusus mungkin diperlukan. d) Penggunaan Tripod dan Stabilisasi d) Penggunaan Tripod dan Stabilisasi Penggunaan tripod atau perangkat stabilisasi dapat membantu menjaga kestabilan kamera selama pengambilan citra. Ini adalah penting dalam kondisi pengambilan citra yang memerlukan eksposur lama atau ketika kamera berada dalam gerakan. e) 14 Penempatan perspektif yang baik adalah penting dalam pengambilan citra objek atau lanskap. Teknik seperti pengaturan sudut pandang, tinggi kamera, dan penempatan objek dapat memengaruhi tampilan akhir citra. 3.3. Kualitas Citra Kualitas citra merujuk pada sejauh mana citra yang diambil mencerminkan objek atau scene asli dengan benar dan detail yang baik. Kualitas citra adalah faktor kunci dalam pengolahan citra digital karena hasil akhirnya sangat bergantung pada kualitas citra yang digunakan. Berikut adalah beberapa aspek penting yang memengaruhi kualitas citra: f) Pengaturan Kamera f) Pengaturan Kamera Pengaturan kamera yang tepat termasuk penyesuaian kecepatan rana, bukaan lensa, ISO, dan mode eksposur. Pengaturan ini dapat disesuaikan untuk mencapai hasil yang diinginkan dalam pengambilan citra. Penggunaan Teknik Khusus d) Distorsi Distorsi adalah penyimpangan dari bentuk atau penampilan objek atau scene asli dalam citra. Distorsi dapat disebabkan oleh lensa kamera, pergeseran perspektif, atau faktor teknis lainnya. e) Kebisingan (Noise) Kebisingan adalah gangguan acak dalam citra yang dapat mengurangi ketajaman dan kualitas citra. Sumber kebisingan meliputi sensor kamera, pencahayaan yang rendah, atau kompresi citra. c) Kecerahan dan Kontras c) Kecerahan dan Kontras c) Kecerahan mengacu pada tingkat pencahayaan dalam citra, sementara kontras mengacu pada perbedaan antara tingkat kecerahan yang berbeda dalam citra. Citra yang terlalu terang atau terlalu gelap dapat mengurangi kualitasnya. Kecerahan mengacu pada tingkat pencahayaan dalam citra, sementara kontras mengacu pada perbedaan antara tingkat kecerahan yang berbeda dalam citra. Citra yang terlalu terang atau terlalu gelap dapat mengurangi kualitasnya. Resolusi a) Resolusi citra mengacu pada jumlah piksel dalam citra.Citra dengan resolusi tinggi memiliki lebih banyak detail dan lebih baik dalam merepresentasikan objek atau scene yang kompleks. 15 b) Kedalaman Warna Kedalaman warna mengacu pada jumlah warna yang dapat direpresentasikan dalam citra. Citra dengan kedalaman warna yang tinggi dapat menangkap detail warna yang halus. c) Kecerahan dan Kontras f) Artefak(Artifact) Artefak adalah elemen yang tidak ada dalam objek atau scene asli, tetapi muncul dalam citra karena proses pengambilan citra atau kompresi. Artefak dapat mengurangi kualitas citra secara signifikan. 16 g) Gangguan (Interference) Gangguan adalah gangguan elektromagnetik atau gangguan lainnya yang dapat memengaruhi transmisi atau pengambilan citra. Ini dapat terjadi dalam situasi di mana banyak perangkat elektronik beroperasi secara bersamaan. 17 17 17 4.2. Segmentasi Citra Segmentasi citra adalah proses penting dalam pengolahan citra digital di mana citra dibagi menjadi beberapa bagian atau segmen, dengan tujuan mengidentifikasi dan memisahkan objek atau area yang menarik dalam citra. Segmentasi digunakan dalam berbagai aplikasi, termasuk pengenalan objek, pengukuran area, pemrosesan citra medis, dan analisis citra satelit. Berikut adalah beberapa teknik dan konsep utama dalam segmentasi citra: Bab 4. Pengambilan dan Praproses Citra Bab 4. Pengambilan dan Praproses Citra Bab 4. Pengambilan dan Praproses Citra Bab 4. Pengambilan dan Praproses Citra 4.1. Perbaikan Kualitas Citra Praproses citra adalah tahap yang sangat penting dalam pengolahan citra digital di mana citra mentah yang diambil dari sumber asli diperbaiki dan ditingkatkan sebelum tahap pengolahan selanjutnya. Salah satu aspek utama dari praproses citra adalah perbaikan kualitas citra. Ini melibatkan serangkaian teknik untuk menghilangkan gangguan dan meningkatkan kualitas citra. Berikut adalah beberapa teknik yang umum digunakan dalam perbaikan kualitas citra: 4.1. Perbaikan Kualitas Citra Praproses citra adalah tahap yang sangat penting dalam pengolahan citra digital di mana citra mentah yang diambil dari sumber asli diperbaiki dan ditingkatkan sebelum tahap pengolahan selanjutnya. Salah satu aspek utama dari praproses citra adalah perbaikan kualitas citra. Ini melibatkan serangkaian teknik untuk menghilangkan gangguan dan meningkatkan kualitas citra. Berikut adalah beberapa teknik yang umum digunakan dalam perbaikan kualitas citra: a) Peningkatan Kecerahan dan Kontras Peningkatan kecerahan dan kontras adalah salah satu langkah pertama dalam perbaikan kualitas citra. Ini dapat dilakukan dengan teknik seperti penyesuaian tingkat kecerahan, kontras, dan histogram citra. b) Reduksi Kebisingan (Noise Reduction) Kebisingan adalah gangguan acak dalam citra yang dapat mengurangi kualitasnya. Teknik seperti filter median atau filter Gaussian dapat digunakan untuk mengurangi kebisingan. c) Peningkatan Tepi (Edge Enhancement) Peningkatan tepi adalah teknik untuk meningkatkan kontur dan detail dalam citra. Filter tepi seperti filter Sobel atau filter Canny dapat digunakan untuk tujuan ini. d) Peningkatan Ketajaman (Sharpening) Peningkatan ketajaman digunakan untuk memperjelas detail dalam citra. Filter peningkatan ketajaman, seperti filter Laplacian, dapat digunakan. e) Penghapusan Artefak (Artifact Removal) 18 Artefak adalah elemen yang tidak ada dalam objek asli, tetapi muncul dalam citra. Teknik penghapusan artefak digunakan untuk menghilangkan artefak yang disebabkan oleh kompresi atau proses pengambilan citra. f) Koreksi Distorsi (Distortion Correction) Jika citra mengalami distorsi seperti pergeseran perspektif, teknik koreksi distorsi digunakan untuk mengembalikannya ke bentuk asli. f) Koreksi Distorsi (Distortion Correction) Jika citra mengalami distorsi seperti pergeseran perspektif, teknik koreksi distorsi digunakan untuk mengembalikannya ke bentuk asli. g) Pemulihan Citra yang Rusak (Image Restoration) Jika citra mengalami kerusakan, misalnya karena kerusakan akibat kompresi atau transmisi, teknik pemulihan citra digunakan untuk memulihkannya sebisa mungkin. g) Pemulihan Citra yang Rusak (Image Restoration) Jika citra mengalami kerusakan, misalnya karena kerusakan akibat kompresi atau transmisi, teknik pemulihan citra digunakan untuk memulihkannya sebisa mungkin. a) Teknik Segmentasi Citra • Thresholding: Teknik ini melibatkan pemilihan ambang tertentu sehingga piksel dengan intensitas di atas ambang menjadi satu segmen, sementara piksel dengan intensitas di bawah ambang menjadi segmen lain. • Segmentasi Berdasarkan Warna: Pada citra berwarna, segmentasi dapat dilakukan berdasarkan komponen warna (misalnya, merah, hijau, biru) atau dalam ruang warna tertentu seperti HSV (Hue, Saturation, Value). • Metode Region Growing: Metode ini memulai dengan satu atau beberapa titik benih (seed) dan memperluas 19 segmen dengan mempertimbangkan piksel tetangga yang memiliki kriteria kesamaan tertentu. segmen dengan mempertimbangkan piksel tetangga yang memiliki kriteria kesamaan tertentu. • Metode Clustering: Metode ini mengelompokkan piksel ke dalam kelompok berdasarkan kemiripan mereka dalam ruang fitur, seperti ruang warna atau domain spasial. • Segmentasi Berbasis Tepi: Metode ini mengidentifikasi tepi objek dengan menemukan perubahan tajam dalam tingkat kecerahan atau warna citra. Tepi ini digunakan sebagai batas objek. b) Pemilihan Metode Segmentasi yang Tepat Pemilihan metode segmentasi yang tepat sangat tergantung pada jenis citra dan karakteristik objek yang akan di-segmentasi. Beberapa metode mungkin lebih cocok untuk citra medis, sementara yang lain lebih cocok untuk citra alam atau citra satelit. c) Evaluasi Segmentasi Evaluasi hasil segmentasi adalah langkah penting untuk mengukur sejauh mana segmen mencerminkan objek yang sebenarnya. Metrik evaluasi seperti IoU (Intersection over Union) digunakan untuk mengukur overlap antara hasil segmentasi dengan referensi yang benar. 4.3. Ekstraksi Fitur a) Fitur Citra Fitur citra adalah karakteristik, informasi, atau atribut tertentu yang dapat diukur atau diekstraksi dari citra. Contoh fitur citra termasuk tekstur, warna, bentuk, tepi, dan intensitas piksel. 4.3. Ekstraksi Fitur a) Fitur Citra Fitur citra adalah karakteristik, informasi, atau atribut tertentu yang dapat diukur atau diekstraksi dari citra. Contoh fitur citra termasuk tekstur, warna, bentuk, tepi, dan intensitas piksel. b) Teknik Ekstraksi Fitur Ekstraksi Histogram: Histogram citra mengukur distribusi intensitas piksel dalam citra. Ini digunakan 20 untuk mengidentifikasi tingkat kecerahan, kontras, dan distribusi warna dalam citra. Ekstraksi Tepi (Edge Detection): Teknik ini digunakan untuk menemukan garis tepi atau kontur dalam citra. Algoritma seperti operator Sobel atau Canny dapat digunakan. Ekstraksi Tepi (Edge Detection): Teknik ini digunakan untuk menemukan garis tepi atau kontur dalam citra. Algoritma seperti operator Sobel atau Canny dapat digunakan. Ekstraksi Tekstur: Tekstur menggambarkan pola ulang yang terlihat dalam citra. Teknik ekstraksi tekstur seperti GLCM (Gray-Level Co-Occurrence Matrix) dapat digunakan. Ekstraksi Warna: Warna adalah fitur penting dalam citra berwarna. Ruang warna seperti RGB, HSV, atau LAB digunakan untuk ekstraksi fitur warna. a) Teknik Segmentasi Citra Ekstraksi Bentuk: Ekstraksi bentuk mencakup pengukuran karakteristik geometris dari objek dalam citra, seperti area, perimeter, atau bentuk. Ekstraksi Fitur Transformasi: Transformasi seperti transformasi Fourier atau transformasi wavelet digunakan untuk ekstraksi fitur dalam domain frekuensi. c) Seleksi Fitur c) Seleksi Fitur Seleksi fitur adalah proses memilih subset fitur yang paling relevan dan penting untuk tugas tertentu. Hal ini dapat membantu mengurangi dimensi data dan meningkatkan efisiensi dalam analisis citra. d) Penggunaan Fitur dalam Aplikasi 21 Fitur-fitur yang diekstraksi dapat digunakan dalam berbagai aplikasi, termasuk pengenalan pola, klasifikasi objek, deteksi objek, atau analisis citra medis. 22 22 BAB 5. Pengolahan Intensitas dan Transformasi Peningkatan kontras dan kejelasan adalah salah satu tahap penting dalam pengolahan citra digital yang bertujuan untuk meningkatkan perbedaan antara tingkat kecerahan piksel dalam citra. Hal ini membantu memperjelas detail dan objek dalam citra, sehingga hasilnya lebih informatif dan dapat digunakan dalam berbagai aplikasi. Berikut adalah beberapa teknik dan konsep utama dalam peningkatan kontras dan kejelasan citra: Peningkatan Kontras • Histogram Equalization: Teknik ini meratakan distribusi intensitas piksel dalam citra sehingga seluruh rentang intensitas digunakan secara optimal. Ini efektif untuk meningkatkan kontras secara umum dalam citra. • Histogram Equalization: Teknik ini meratakan distribusi intensitas piksel dalam citra sehingga seluruh rentang intensitas digunakan secara optimal. Ini efektif untuk meningkatkan kontras secara umum dalam citra. • Kontrast Stretching: Metode ini memperluas rentang intensitas dalam citra sehingga citra memiliki rentang kecerahan yang lebih besar. Ini berguna dalam situasi di mana citra memiliki kontras rendah. • Enhancement dengan Operasi Titik: Operasi titik digunakan untuk mengubah intensitas piksel secara khusus dalam citra, seperti meningkatkan kecerahan objek tertentu. Peningkatan Kejelasan • Filtrasi: Filter spatialis digunakan untuk memperbaiki kejelasan citra dengan meningkatkan kontras pada tingkat spasial yang berbeda. Contohnya termasuk filter peningkatan ketajaman (sharpening) dan filter anisotropic diffusion. • Transformasi Histogram: Transformasi histogram seperti transformasi gamma digunakan untuk mengontrol kontras dan kecerahan dalam citra. 23 • Metode Deteksi Tepi: Tepi dalam citra adalah area di mana terjadi perubahan tajam dalam intensitas. Mendeteksi dan meningkatkan tepi dapat meningkatkan kejelasan objek dalam citra. • Metode Deteksi Tepi: Tepi dalam citra adalah area di mana terjadi perubahan tajam dalam intensitas. Mendeteksi dan meningkatkan tepi dapat meningkatkan kejelasan objek dalam citra. Penggunaan dalam Aplikasi Peningkatan kontras dan kejelasan adalah tahap yang penting dalam berbagai aplikasi pengolahan citra, termasuk pemrosesan citra medis, pemrosesan citra satelit, pengenalan pola, dan analisis citra forensik. Peningkatan kontras dan kejelasan adalah tahap yang penting dalam berbagai aplikasi pengolahan citra, termasuk pemrosesan citra medis, pemrosesan citra satelit, pengenalan pola, dan analisis citra forensik. 5.1. Histogram Equalization Histogram equalization adalah salah satu teknik yang digunakan dalam pengolahan citra untuk meningkatkan kontras citra dengan meratakan distribusi intensitas pikselnya. Teknik ini berguna ketika citra memiliki kontras yang rendah atau distribusi intensitas yang tidak merata. Berikut adalah beberapa konsep dan langkah-langkah dalam histogram equalization: a. Histogram Citra 5.1. Histogram Equalization Histogram equalization adalah salah satu teknik yang digunakan dalam pengolahan citra untuk meningkatkan kontras citra dengan meratakan distribusi intensitas pikselnya. Teknik ini berguna ketika citra memiliki kontras yang rendah atau distribusi intensitas yang tidak merata. Berikut adalah beberapa konsep dan langkah-langkah dalam histogram equalization: a. Histogram Citra a. Histogram Citra Histogram citra adalah plot yang menunjukkan sebaran intensitas piksel dalam citra. Histogram ini menggambarkan berapa banyak piksel dalam citra yang memiliki intensitas tertentu. b. Prinsip Histogram Equalization Prinsip dasar histogram equalization adalah meratakan histogram citra sehingga intensitas piksel terdistribusi secara merata di seluruh rentang intensitas yang mungkin.Ini dilakukan dengan mengubah intensitas piksel dalam citra asli sehingga histogram yang diperoleh menjadi histogram yang merata. 24 c. Langkah-langkah Histogram Equalization Langkah-langkah dalam melakukan histogram equalization adalah sebagai berikut: 1. Hitung Histogram: Hitung histogram dari citra asli untuk mengetahui sebaran intensitas piksel. 1. Hitung Histogram: Hitung histogram dari citra asli untuk mengetahui sebaran intensitas piksel. 2. Hitung Distribusi Kumulatif: Hitung distribusi kumulatif dari histogram. Distribusi kumulatif adalah jumlah kumulatif dari frekuensi piksel dalam histogram. 2. Hitung Distribusi Kumulatif: Hitung distribusi kumulatif dari histogram. Distribusi kumulatif adalah jumlah kumulatif dari frekuensi piksel dalam histogram. 3. Normalisasi Distribusi Kumulatif: Normalisasikan distribusi kumulatif sehingga nilainya berada dalam rentang [0, 255] (untuk citra dengan 8-bit per channel). 3. Normalisasi Distribusi Kumulatif: Normalisasikan distribusi kumulatif sehingga nilainya berada dalam rentang [0, 255] (untuk citra dengan 8-bit per channel). 4. Transformasi Citra: Gunakan distribusi kumulatif yang dinormalisasi untuk mengubah intensitas piksel dalam citra asli. 4. Transformasi Citra: Gunakan distribusi kumulatif yang dinormalisasi untuk mengubah intensitas piksel dalam citra asli. 5. Hasil Equalization: Hasil dari langkah ini adalah citra yang telah ditingkatkan kontrasnya dengan intensitas piksel yang merata. 5. Penggunaan dalam Aplikasi Hasil Equalization: Hasil dari langkah ini adalah citra yang telah ditingkatkan kontrasnya dengan intensitas piksel yang merata. d. Keuntungan Histogram Equalization Histogram equalization efektif dalam meningkatkan kontras citra, terutama dalam kasus citra dengan kontras yang rendah atau distribusi intensitas yang tidak merata. e. Batasan Histogram Equalization Histogram equalization tidak selalu cocok untuk semua jenis citra. Pada beberapa kasus, dapat menyebabkan peningkatan noise atau menghasilkan citra dengan pencahayaan yang tidak alami. 5.2. Operasi Logaritmik Operasi logaritmik adalah salah satu teknik pengolahan citra yang digunakan untuk meningkatkan citra dengan tingkat kecerahan yang rendah atau citra yang memiliki distribusi intensitas yang sangat luas. Teknik ini berdasarkan prinsip 25 penggunaan logaritma untuk mengkompresi atau memperluas rentang intensitas piksel dalam citra. Berikut adalah beberapa konsep dan langkah-langkah dalam operasi logaritmik: penggunaan logaritma untuk mengkompresi atau memperluas rentang intensitas piksel dalam citra. Berikut adalah beberapa konsep dan langkah-langkah dalam operasi logaritmik: a. Prinsip Dasar Operasi Logaritmik Prinsip dasar operasi logaritmik adalah menggunakan fungsi logaritma untuk merubah intensitas piksel dalam citra. Operasi ini cenderung meningkatkan detail pada piksel dengan intensitas rendah sambil menjaga detail pada piksel dengan intensitas tinggi. b. Rumus Operasi Logaritmik Rumus dasar operasi logaritmik adalah sebagai berikut: s=c∗log⁡(1+r)s=c∗log(1+r) s=c∗log⁡(1+r)s=c∗log(1+r) Di mana: c. $s$ adalah intensitas piksel dalam citra hasil. d. $c$ adalah konstanta yang digunakan untuk mengontrol penguatan. e. $r$ adalah intensitas piksel dalam citra asli. f. Keuntungan Operasi Logaritmik Bab 6. Filtrasi dalam Domain Spasial 6.1. Operasi Konvolusi Bab 6. Filtrasi dalam Domain Spasial 6.1. Operasi Konvolusi 6.1. Operasi Konvolusi Operasi konvolusi adalah salah satu teknik yang paling umum digunakan dalam pengolahan citra dalam domain spasial. Teknik ini digunakan untuk menerapkan filter atau kernel pada citra asli dengan tujuan menghasilkan citra hasil yang telah ditingkatkan atau memiliki fitur tertentu yang diinginkan. Berikut adalah beberapa konsep dan langkah-langkah dalam operasi konvolusi: a) Prinsip Dasar Operasi Konvolusi a) Prinsip Dasar Operasi Konvolusi ) p p Prinsip dasar operasi konvolusi adalah menggeser filter atau kernel melintasi citra asli dan menghitung jumlah produk titik antara elemen-elemen filter dan piksel yang tumpang tindih. Hasil dari setiap perhitungan tersebut ditempatkan di lokasi yang sesuai dalam citra hasil. Hasil operasi konvolusi adalah citra baru yang telah difilter dengan kernel yang digunakan. b) Kernel Konvolusi Kernel konvolusi adalah matriks berukuran kecil yang digunakan dalam operasi konvolusi. Kernel ini dapat memiliki berbagai bentuk dan nilai-nilai yang berbeda tergantung pada tujuan pengolahan citra. Sebagai contoh, kernel Gaussian digunakan untuk penyaringan rata-rata, sementara kernel Sobel digunakan untuk deteksi tepi. f. Keuntungan Operasi Logaritmik f. Keuntungan Operasi Logaritmik Operasi logaritmik efektif untuk meningkatkan detail pada citra yang memiliki intensitas rendah. Ini juga berguna untuk mengkompresi rentang intensitas dalam citra yang sangat luas, menjadikannya lebih sesuai untuk tampilan atau analisis. g. Batasan Operasi Logaritmik Operasi logaritmik tidak selalu cocok untuk semua jenis citra. Pada beberapa kasus, penggunaan yang tidak tepat dapat menghasilkan citra yang tidak diinginkan dengan tingkat kecerahan yang tidak alami. 26 5.3. Transformasi Gamma Transformasi gamma adalah salah satu teknik pengolahan citra yang digunakan untuk mengubah kecerahan dan kontras citra dengan mengontrol nilai gamma. Teknik ini memungkinkan pengguna untuk menyesuaikan intensitas piksel dalam citra sesuai dengan preferensi atau kebutuhan tertentu. Berikut adalah beberapa konsep dan langkah- langkah dalam transformasi gamma: a. Prinsip Dasar Transformasi Gamma Transformasi gamma didasarkan pada prinsip menggunakan fungsi matematika berbentuk pangkat untuk mengubah intensitas piksel dalam citra. Fungsi transformasi gamma memiliki rumus umum berikut: s=c∗rγs=c∗rγ Di mana: s=c∗rγs=c∗rγ Di mana: b. $s$ adalah intensitas piksel dalam citra hasil. c. $c$ adalah konstanta yang digunakan untuk mengontrol penguatan. d. $r$ adalah intensitas piksel dalam citra asli. e. $\gamma$ adalah parameter gamma yang mengontrol bentuk transformasi. f. Parameter Gamma Parameter gamma ($\gamma$) dapat digunakan untuk mengontrol bentuk transformasi gamma. Nilai $\gamma < 1$ akan menghasilkan peningkatan kontras dalam citra dan meningkatkan detail pada piksel dengan intensitas rendah. Nilai $\gamma > 1$ akan menghasilkan pengurangan kontras dalam citra dan meningkatkan detail pada piksel dengan intensitas tinggi. Nilai $\gamma = 1$ tidak mengubah citra, sehingga hasilnya sama dengan citra asli. 27 g. Keuntungan Transformasi Gamma Transformasi gamma memungkinkan kontrol yang fleksibel atas tingkat kecerahan dan kontras dalam citra. Ini berguna untuk mengoreksi citra yang memiliki tingkat kecerahan atau kontras yang tidak sesuai. g. Keuntungan Transformasi Gamma Transformasi gamma memungkinkan kontrol yang fleksibel atas tingkat kecerahan dan kontras dalam citra. Ini berguna untuk mengoreksi citra yang memiliki tingkat kecerahan atau kontras yang tidak sesuai. h. Batasan Transformasi Gamma Penggunaan yang tidak tepat dari transformasi gamma dapat menghasilkan citra yang terlihat tidak alami atau distorsi yang tidak diinginkan. 28 28 e) Batasan Operasi Konvolusi Kernel konvolusi yang salah atau penggunaan yang tidak tepat dapat menghasilkan citra yang tidak diinginkan atau merusak informasi dalam citra asli. c) Langkah-langkah Operasi Konvolusi c) Langkah-langkah Operasi Konvolusi Langkah-langkah dalam melakukan operasi konvolusi adalah sebagai berikut: Langkah-langkah dalam melakukan operasi konvolusi adalah sebagai berikut: 1. Tempatkan kernel konvolusi di atas citra asli sehingga pusat kernel berada pada piksel yang sedang diproses. 2. Hitung jumlah produk titik antara elemen-elemen kernel dan piksel yang tumpang tindih. 3. Hasil perhitungan tersebut ditempatkan pada lokasi yang sesuai dalam citra hasil. 29 4. Geser kernel ke piksel berikutnya dan ulangi langkah- langkah 2-3 hingga seluruh citra telah diproses. 4. Geser kernel ke piksel berikutnya dan ulangi langkah- langkah 2-3 hingga seluruh citra telah diproses. 4. Geser kernel ke piksel berikutnya dan ulangi langkah- langkah 2-3 hingga seluruh citra telah diproses. d) Keuntungan Operasi Konvolusi Operasi konvolusi memungkinkan kita untuk melakukan berbagai jenis penyaringan dan pemrosesan citra dalam domain spasial. Ini berguna untuk menghilangkan noise, meningkatkan kontras, dan mendeteksi fitur dalam citra. ) Keuntungan Operasi Konvolusi Operasi konvolusi memungkinkan kita untuk melakukan berbagai jenis penyaringan dan pemrosesan citra dalam domain spasial. Ini berguna untuk menghilangkan noise, meningkatkan kontras, dan mendeteksi fitur dalam citra. e) Batasan Operasi Konvolusi 6.2. Deteksi Tepi Deteksi tepi adalah salah satu aplikasi utama dari teknik filtrasi dalam pengolahan citra. Tujuan dari deteksi tepi adalah untuk menemukan garis tepi atau perubahan tajam dalam tingkat kecerahan citra. Tepi ini sering kali mewakili batas objek dalam citra. Berikut adalah beberapa konsep dan teknik yang digunakan dalam deteksi tepi: Deteksi tepi adalah salah satu aplikasi utama dari teknik filtrasi dalam pengolahan citra. Tujuan dari deteksi tepi adalah untuk menemukan garis tepi atau perubahan tajam dalam tingkat kecerahan citra. Tepi ini sering kali mewakili batas objek dalam citra. Berikut adalah beberapa konsep dan teknik yang digunakan dalam deteksi tepi: a) Prinsip Dasar Deteksi Tepi Prinsip dasar dalam deteksi tepi adalah mengidentifikasi perubahan tajam dalam intensitas piksel dalam citra. Tepi dapat muncul sebagai perbedaan tingkat kecerahan, warna, atau tekstur. b) Operator Deteksi Tepi Operator deteksi tepi adalah filter khusus yang digunakan untuk menemukan tepi dalam citra.Operator Sobel dan operator Prewitt adalah beberapa contoh operator yang sering digunakan untuk deteksi tepi. c) Jenis Tepi Tepi yang dapat dideteksi mencakup tepi tajam dan tepi tumpul. Tepi tajam adalah perubahan tajam dalam tingkat kecerahan, sementara tepi tumpul adalah perubahan yang lebih lembut.Deteksi tepi juga dapat menghasilkan tepi 30 positif (meningkatnya kecerahan) dan tepi negatif (penurunan kecerahan). positif (meningkatnya kecerahan) dan tepi negatif (penurunan kecerahan). positif (meningkatnya kecerahan) dan tepi negatif (penurunan kecerahan). d) Metrik Evaluasi Deteksi Tepi Untuk mengevaluasi kualitas deteksi tepi, metrik seperti sensitivitas, spesifisitas, dan kesalahan lokal dapat digunakan. e) Keuntungan Deteksi Tepi Deteksi tepi digunakan dalam berbagai aplikasi, termasuk segmentasi citra, pengenalan objek, dan analisis citra medis.Ini membantu dalam pemisahan objek dari latar belakang dalam citra. g f) Batasan Deteksi Tepi Deteksi tepi dapat dipengaruhi oleh noise dalam citra, sehingga prefiltering sering diperlukan sebelumnya. 6.3. Filtrasi Non-Linear Filtrasi non-linear adalah teknik pengolahan citra yang melibatkan operasi terhadap piksel dalam citra dengan menggunakan fungsi non-linear. Berbeda dengan operasi konvolusi linear yang menggunakan kernel, operasi non-linear memproses piksel berdasarkan karakteristik tertentu tanpa mempertimbangkan hubungan dengan piksel tetangga. Berikut adalah beberapa konsep dan teknik dalam filtrasi non-linear: f) Batasan Deteksi Tepi Deteksi tepi dapat dipengaruhi oleh noise dalam citra, sehingga prefiltering sering diperlukan sebelumnya. 6.3. Filtrasi Non-Linear Filtrasi non-linear adalah teknik pengolahan citra yang melibatkan operasi terhadap piksel dalam citra dengan menggunakan fungsi non-linear. Berbeda dengan operasi konvolusi linear yang menggunakan kernel, operasi non-linear memproses piksel berdasarkan karakteristik tertentu tanpa mempertimbangkan hubungan dengan piksel tetangga. 6.2. Deteksi Tepi Berikut adalah beberapa konsep dan teknik dalam filtrasi non-linear: a) Prinsip Dasar Filtrasi Non-Linear Prinsip dasar dalam filtrasi non-linear adalah menggunakan fungsi non-linear yang tidak bergantung pada konvolusi atau hubungan dengan piksel tetangga. Filtrasi non-linear sering digunakan untuk mengatasi situasi di mana ada perubahan mendadak dalam intensitas piksel, seperti dalam citra dengan noise yang kuat. a) Prinsip Dasar Filtrasi Non-Linear Prinsip dasar dalam filtrasi non-linear adalah menggunakan fungsi non-linear yang tidak bergantung pada konvolusi atau hubungan dengan piksel tetangga. Filtrasi non-linear sering digunakan untuk mengatasi situasi di mana ada perubahan mendadak dalam intensitas piksel, seperti dalam citra dengan noise yang kuat. b) Median Filter Median filter adalah salah satu teknik filtrasi non-linear yang paling umum digunakan. Pada median filter, nilai piksel tengah dari himpunan piksel tetangga diurutkan, dan nilai median digunakan sebagai nilai piksel yang baru. 31 Median filter efektif dalam mengurangi noise berbentuk titik (salt-and-pepper noise) dalam citra. c) Filter Bilateral Filter bilateral adalah teknik filtrasi non-linear yang lebih kompleks yang mempertimbangkan kedua jarak spasial dan perbedaan intensitas piksel.Filter ini memungkinkan pemrosesan citra yang lebih halus, mengurangi noise, dan mempertahankan tepi dengan baik. d) Keuntungan Filtrasi Non-Linear Filtrasi non-linear efektif dalam menghilangkan noise non- Gaussian dan menjaga detail dalam citra. Filter seperti median filter sangat berguna dalam mengatasi jenis noise seperti salt-and-pepper noise. e) Batasan Filtrasi Non-Linear Filtrasi non-linear dapat mempengaruhi detail halus dalam citra, sehingga penggunaan yang berlebihan dapat menghasilkan citra yang tampak "halus" atau "kabur". Beberapa teknik filtrasi non-linear juga lebih komputasi- intensif dibandingkan dengan operasi konvolusi linear. 32 32 Bab 7. Transformasi Citra dan Pengolahan Frekuensi Bab 7. Transformasi Citra dan Pengolahan Frekuensi 6. Transformasi Fourier Transformasi Fourier adalah teknik penting dalam pengolahan citra yang digunakan untuk menganalisis citra dalam domain frekuensi. Ini memungkinkan kita untuk mengonversi citra dari domain spasial ke domain frekuensi, di mana kita dapat menganalisis komponen-komponen frekuensi yang membentuk citra. Berikut adalah beberapa konsep dan langkah-langkah dalam Transformasi Fourier: Konsep Dasar Transformasi Fourier 6. Transformasi Fourier Transformasi Fourier adalah teknik penting dalam pengolahan citra yang digunakan untuk menganalisis citra dalam domain frekuensi. Ini memungkinkan kita untuk mengonversi citra dari domain spasial ke domain frekuensi, di mana kita dapat menganalisis komponen-komponen frekuensi yang membentuk citra. 6.2. Deteksi Tepi Berikut adalah beberapa konsep dan langkah-langkah dalam Transformasi Fourier: Konsep Dasar Transformasi Fourier Transformasi Fourier adalah teknik matematis yang digunakan untuk menganalisis dan merepresentasikan sinyal atau citra dalam domain frekuensi.Dalam konteks pengolahan citra, kita dapat mengonversi citra dari domain spasial (di mana citra direpresentasikan sebagai matriks piksel) ke domain frekuensi (di mana citra direpresentasikan sebagai spektrum frekuensi). Transformasi Fourier 1D dan 2D Transformasi Fourier 1D dan 2D Transformasi Fourier 1D digunakan untuk menganalisis sinyal 1D, seperti sinyal audio.Transformasi Fourier 2D digunakan untuk menganalisis citra 2D. Citra 2D dipecah menjadi komponen-komponen frekuensi yang berbeda, seperti frekuensi rendah (komponen global) dan frekuensi tinggi (detail lokal). Spektrum Citra Spektrum citra adalah representasi citra dalam domain frekuensi. Ini menunjukkan berapa banyak energi terkandung dalam berbagai frekuensi.Pada citra, komponen frekuensi rendah (spektrum rendah) mengandung informasi tentang bagian dasar atau bentuk objek, sedangkan komponen frekuensi tinggi (spektrum tinggi) mengandung detail atau tepi. 33 Keuntungan Transformasi Fourier Keuntungan Transformasi Fourier Transformasi Fourier membantu dalam analisis dan pengolahan citra dalam domain frekuensi, yang dapat bermanfaat dalam berbagai aplikasi seperti kompresi citra, restorasi citra, dan pengenalan pola. Ini memungkinkan pemisahan informasi citra ke dalam berbagai komponen frekuensi, yang dapat dianalisis dan diproses secara terpisah. Batasan Transformasi Fourier Transformasi Fourier adalah teknik linier, yang berarti bahwa operasi yang kompleks mungkin diperlukan untuk mengatasi beberapa aspek dalam citra, seperti penekanan detail atau penghilangan noise. 7.1 Pengenalan Transformasi Fourier Transformasi Fourier adalah teknik penting dalam pengolahan citra yang digunakan untuk menganalisis dan merepresentasikan citra dalam domain frekuensi. Pemahaman konsep dasar Transformasi Fourier sangat relevan dalam pemrosesan citra digital. Berikut adalah beberapa konsep dasar dalam pengenalan Transformasi Fourier: a) Konsep Dasar Transformasi Fourier Transformasi Fourier adalah teknik matematis yang digunakan untuk menganalisis sinyal atau citra dalam domain frekuensi. Konsep utama adalah bahwa citra dalam domain spasial (waktu atau ruang) dapat dipecah menjadi komponen-komponen sinusoidal dalam domain frekuensi. b) Transformasi Fourier 1D dan 2D ) Transformasi Fourier 1D digunakan untuk menganalisis sinyal 1D seperti sinyal audio atau sinyal citra baris. Transformasi Fourier 2D digunakan untuk menganalisis 34 citra 2D, di mana citra dipecah menjadi berbagai komponen frekuensi. c) Komponen Spektral c) Komponen Spektral Spektrum citra adalah representasi citra dalam domain frekuensi. Ini menunjukkan sebaran energi atau amplitudo dalam berbagai frekuensi. Komponen frekuensi rendah dalam spektrum citra mengandung informasi tentang struktur dasar atau objek besar dalam citra. Komponen frekuensi tinggi dalam spektrum citra mengandung detail atau tepi dalam citra. d) Keuntungan Transformasi Fourier d) Keuntungan Transformasi Fourier Transformasi Fourier membantu dalam analisis dan pengolahan citra dalam domain frekuensi, yang berguna dalam berbagai aplikasi seperti kompresi citra, penghilangan noise, dan deteksi pola. Ini memungkinkan kita untuk memahami komponen-komponen frekuensi yang membentuk citra, yang dapat membantu dalam perbaikan citra atau ekstraksi fitur. e) Batasan Transformasi Fourier Transformasi Fourier hanya merupakan alat analisis, sehingga penggunaan yang efektif memerlukan strategi pengolahan selanjutnya berdasarkan analisis frekuensi. e) Batasan Transformasi Fourier Transformasi Fourier hanya merupakan alat analisis, sehingga penggunaan yang efektif memerlukan strategi pengolahan selanjutnya berdasarkan analisis frekuensi. 7.2 Penggunaan Transformasi Fourier dalam Pengolahan Citra 7.2 Penggunaan Transformasi Fourier dalam Pengolahan Citra 35 35 7. Transformasi Wavelet Transformasi Wavelet adalah teknik yang sangat berguna dalam pengolahan citra digital. Ini memungkinkan kita untuk menganalisis citra dalam berbagai tingkat resolusi dan mengidentifikasi detail serta fitur dengan lebih baik daripada Transformasi Fourier. Transformasi Wavelet bekerja dengan memecah citra menjadi komponen- komponen frekuensi pada berbagai skala, memungkinkan analisis yang lebih adaptif(Mallat, 2008). Berikut adalah beberapa konsep dan aplikasi dalam Transformasi Wavelet: 7. Transformasi Wavelet Transformasi Wavelet adalah teknik yang sangat berguna dalam pengolahan citra digital. Ini memungkinkan kita untuk menganalisis citra dalam berbagai tingkat resolusi dan mengidentifikasi detail serta fitur dengan lebih baik daripada Transformasi Fourier. Transformasi Wavelet bekerja dengan memecah citra menjadi komponen- komponen frekuensi pada berbagai skala, memungkinkan analisis yang lebih adaptif(Mallat, 2008). Berikut adalah beberapa konsep dan aplikasi dalam Transformasi Wavelet: Konsep Dasar Transformasi Wavelet Konsep Dasar Transformasi Wavelet Transformasi Wavelet adalah teknik yang digunakan untuk merepresentasikan citra (atau sinyal) sebagai kombinasi dari fungsi-fungsi wavelet. Fungsi-fungsi wavelet adalah fungsi matematis yang berbentuk gelombang kecil, mirip dengan gelombang air yang bergerak. Wavelet memiliki dua parameter penting: skala (scale) dan posisi (position). Skala mengontrol berapa kali gelombang tersebut mengulang dalam rentang tertentu, sementara posisi mengontrol lokasinya dalam citra. Transformasi Wavelet 1D dan 2D Transformasi Wavelet 1D dan 2D Transformasi Wavelet 1D dan 2D Transformasi Wavelet 1D digunakan untuk menganalisis sinyal 1D seperti sinyal audio atau baris citra. Transformasi Wavelet 2D digunakan untuk menganalisis citra 2D. Citra dipecah menjadi komponen-komponen frekuensi pada berbagai skala dan orientasi. Fungsi-Fungsi Wavelet Populer Beberapa fungsi wavelet populer yang digunakan dalam praktik adalah Haar wavelet, Daubechies wavelet, dan Morlet wavelet. Masing-masing dari ini memiliki karakteristik dan aplikasi yang berbeda. 36 Bab 8. Transformasi Wavelet Transformasi Wavelet memungkinkan analisis citra dalam berbagai tingkat resolusi, sehingga kita dapat mengidentifikasi fitur dan detail pada berbagai skala. Ini berguna dalam berbagai aplikasi seperti kompresi citra, penghilangan noise, deteksi tepi, dan pengenalan objek. Bab 8. Transformasi Wavelet Aplikasi Transformasi Wavelet Transformasi Wavelet digunakan dalam berbagai aplikasi seperti kompresi citra (JPEG2000), analisis citra medis (deteksi tumor), pengenalan pola (pencocokan sidik jari), dan pemulihan citra (penghilangan noise). 8.1 Pengenalan Transformasi Wavelet 8.1 Pengenalan Transformasi Wavelet Transformasi Wavelet adalah teknik penting dalam pengolahan citra yang digunakan untuk menganalisis citra dalam domain frekuensi yang lebih adaptif dibandingkan dengan Transformasi Fourier. Ini memungkinkan kita untuk memahami detail dan fitur citra dalam berbagai tingkat resolusi. Berikut adalah beberapa konsep dasar dalam pengenalan Transformasi Wavelet: 8.1 Pengenalan Transformasi Wavelet Transformasi Wavelet adalah teknik penting dalam pengolahan citra yang digunakan untuk menganalisis citra dalam domain frekuensi yang lebih adaptif dibandingkan dengan Transformasi Fourier. Ini memungkinkan kita untuk memahami detail dan fitur citra dalam berbagai tingkat resolusi. Berikut adalah beberapa konsep dasar dalam pengenalan Transformasi Wavelet: a) Konsep Dasar Transformasi Wavelet Transformasi Wavelet adalah teknik yang digunakan untuk merepresentasikan sinyal atau citra sebagai gabungan dari fungsi-fungsi wavelet. Fungsi-fungsi wavelet adalah fungsi matematis yang berbentuk gelombang kecil dan dapat digunakan untuk menganalisis sinyal atau citra dalam berbagai tingkat resolusi. b) Transformasi Wavelet 1D dan 2D Transformasi Wavelet 1D digunakan untuk menganalisis sinyal 1D seperti sinyal audio atau baris citra. Transformasi Wavelet 2D digunakan untuk menganalisis citra 2D. Citra dipecah menjadi berbagai 37 skala dan orientasi, memungkinkan analisis detail dalam berbagai resolusi. skala dan orientasi, memungkinkan analisis detail dalam berbagai resolusi. skala dan orientasi, memungkinkan analisis detail dalam berbagai resolusi. c) c) Skala dan Resolusi Salah satu konsep penting dalam Transformasi Wavelet adalah penggunaan skala berbeda untuk menganalisis berbagai tingkat resolusi dalam citra. Dengan menggunakan skala yang berbeda, kita dapat mengidentifikasi fitur dan detail dalam citra dengan baik, baik pada tingkat resolusi tinggi maupun rendah. Transformasi Wavelet 1D dan 2D d) Keuntungan Transformasi Wavelet Transformasi Wavelet lebih adaptif daripada Transformasi Fourier karena dapat mengidentifikasi detail dalam berbagai tingkat resolusi. Ini berguna dalam berbagai aplikasi seperti kompresi citra, deteksi tepi, pengenalan objek, dan pemulihan citra. e) Batasan Transformasi Wavelet Transformasi Wavelet memerlukan pemahaman yang mendalam tentang teori wavelet, yang bisa menjadi kompleks. e) Batasan Transformasi Wavelet Transformasi Wavelet memerlukan pemahaman yang mendalam tentang teori wavelet, yang bisa menjadi kompleks. 8.2 Aplikasi Transformasi Wavelet dalam Pengolahan Citra 8.2 Aplikasi Transformasi Wavelet dalam Pengolahan Citra Transformasi Wavelet adalah alat yang kuat dalam pengolahan citra digital yang memiliki berbagai aplikasi yang luas. Ini memungkinkan analisis citra dalam berbagai tingkat resolusi dan dapat digunakan untuk berbagai tujuan. Berikut adalah beberapa aplikasi utama Transformasi Wavelet dalam pengolahan citra: Transformasi Wavelet adalah alat yang kuat dalam pengolahan citra digital yang memiliki berbagai aplikasi yang luas. Ini memungkinkan analisis citra dalam berbagai tingkat resolusi dan dapat digunakan untuk berbagai tujuan. Berikut adalah beberapa aplikasi utama Transformasi Wavelet dalam pengolahan citra: Transformasi Wavelet adalah alat yang kuat dalam pengolahan citra digital yang memiliki berbagai aplikasi yang luas. Ini memungkinkan analisis citra dalam berbagai tingkat resolusi dan dapat digunakan untuk berbagai tujuan. Berikut adalah beberapa aplikasi utama Transformasi Wavelet dalam pengolahan citra: a) Kompresi Citra Salah satu aplikasi paling terkenal dari Transformasi Wavelet adalah dalam kompresi citra. Teknik ini digunakan dalam format kompresi seperti JPEG2000.Transformasi Wavelet memungkinkan representasi citra yang efisien dengan menghapus 38 informasi yang kurang penting pada resolusi yang lebih rendah. b) b) Penghilangan Noise Transformasi Wavelet digunakan untuk menghilangkan noise dari citra. Dengan menganalisis citra pada berbagai skala, noise dapat diidentifikasi dan dihilangkan pada tingkat resolusi yang sesuai. Teknik penghilangan noise berbasis wavelet sering digunakan dalam citra medis dan fotografi. b) Penghilangan Noise Transformasi Wavelet digunakan untuk menghilangkan noise dari citra. Dengan menganalisis citra pada berbagai skala, noise dapat diidentifikasi dan dihilangkan pada tingkat resolusi yang sesuai. Teknik penghilangan noise berbasis wavelet sering digunakan dalam citra medis dan fotografi. c) Deteksi Tepi Transformasi Wavelet membantu dalam deteksi tepi dalam citra. Dengan mengidentifikasi perubahan tajam dalam intensitas pada berbagai skala, tepi dapat ditemukan dengan lebih baik. Ini memiliki aplikasi dalam visi komputer dan analisis citra medis. d) Pemulihan Citra Dalam pemulihan citra, Transformasi Wavelet digunakan untuk mengembalikan atau memperbaiki citra yang rusak atau kabur. Contohnya termasuk penghilangan goresan pada foto dan pemulihan citra satelit. Analisis dan Segmentasi Citra Analisis dan segmentasi citra adalah langkah penting dalam pengolahan citra yang bertujuan untuk memahami konten citra, mengidentifikasi objek, dan memisahkan objek dari latar belakang. Dalam subbab ini, kita akan menjelaskan konsep dasar dan teknik dalam analisis serta segmentasi citra. Metode Analisis Citra Beberapa metode analisis citra meliputi ekstraksi fitur, deskripsi citra, dan pengenalan pola. Ekstraksi fitur melibatkan identifikasi dan pengukuran fitur-fitur seperti tepi, sudut, atau tekstur dalam citra. Deskripsi citra adalah representasi abstrak dari citra yang membantu dalam memahami karakteristiknya.Pengenalan pola mencakup pengenalan objek atau pola tertentu dalam citra berdasarkan fitur yang telah diekstraksi. Konsep Dasar Analisis Citra Analisis citra adalah proses ekstraksi informasi yang berguna dari citra, seperti fitur objek, warna, tekstur, dan bentuk. Ini melibatkan metode matematis dan komputasi untuk menganalisis setiap piksel dalam citra. Transformasi Wavelet 1D dan 2D d) Pemulihan Citra Dalam pemulihan citra, Transformasi Wavelet digunakan untuk mengembalikan atau memperbaiki citra yang rusak atau kabur. Contohnya termasuk penghilangan goresan pada foto dan pemulihan citra satelit. e) Pengenalan Pola Transformasi Wavelet juga digunakan dalam pengenalan pola. Dengan menganalisis fitur-fitur berdasarkan skala dan orientasi, fitur-fitur yang penting dapat diidentifikasi untuk pengenalan objek. Ini memiliki aplikasi dalam pengenalan wajah dan sidik jari. 39 Bab 9. Analisis dan Segmentasi Citra Konsep Dasar Segmentasi Citra Segmentasi citra adalah proses memisahkan citra menjadi bagian-bagian yang homogen atau objek-objek yang berbeda. Tujuannya adalah untuk mengidentifikasi area atau objek yang 40 memiliki karakteristik serupa, seperti warna, tekstur, atau intensitas. Metode Segmentasi Citra Metode segmentasi citra meliputi pemisahan objek dari latar belakang, deteksi tepi, segmentasi berbasis warna, dan pengelompokan berbasis fitur. Algoritma tersedia untuk segmentasi citra seperti metode pemambangan berkas, pemambangan level ambang, dan pemambangan berbasis grafik. Aplikasi Analisis dan Segmentasi Citra Analisis dan segmentasi citra digunakan dalam berbagai aplikasi, termasuk visi komputer, pengenalan karakter, analisis medis, dan pemrosesan citra satelit. Contoh aplikasi termasuk deteksi objek dalam lalu lintas, pemantauan citra medis, dan pengenalan wajah dalam pengamanan. Analisis Tekstur Citra 9.1. Pengenalan Analisis Tekstur Analisis tekstur adalah salah satu aspek penting dalam pengolahan citra yang berkaitan dengan karakterisasi pola dan struktur dalam citra. Pengenalan analisis tekstur penting dalam berbagai aplikasi, termasuk visi komputer, pengolahan citra medis, dan pemrosesan citra geologi. Dalam subbab ini, kita akan menjelaskan konsep dasar dan tujuan dari analisis tekstur citra. a. Konsep Dasar Analisis Tekstur Tekstur dalam citra merujuk pada pola-pola visual yang menggambarkan bagaimana piksel-piksel berinteraksi satu sama lain dalam citra. Contoh tekstur termasuk pola berulang seperti garis-garis, titik-titik, atau variasi kecil dalam intensitas warna 41 b. Tujuan Analisis Tekstur Citra Tujuan utama dari analisis tekstur adalah untuk mengukur, menganalisis, dan menggambarkan karakteristik tekstur dalam citra. Ini membantu dalam mengklasifikasikan citra berdasarkan tekstur, deteksi anomali, dan ekstraksi fitur. b. Tujuan Analisis Tekstur Citra Tujuan utama dari analisis tekstur adalah untuk mengukur, menganalisis, dan menggambarkan karakteristik tekstur dalam citra. Ini membantu dalam mengklasifikasikan citra berdasarkan tekstur, deteksi anomali, dan ekstraksi fitur. c. Metode Analisis Tekstur Citra Metode analisis tekstur mencakup statistik, metode struktural, dan transformasi. Metode statistik melibatkan pengukuran statistik seperti mean, variansi, dan matriks korelasi dari piksel-piksel dalam citra. Metode struktural berfokus pada pengenalan pola seperti arah, orientasi, dan perbedaan intensitas dalam citra. Metode transformasi menggunakan transformasi matematis seperti Transformasi Fourier atau Transformasi Wavelet untuk menganalisis tekstur dalam domain frekuensi atau skala. d. Aplikasi Analisis Tekstur Citra Analisis tekstur citra memiliki berbagai aplikasi, termasuk dalam pengenalan pola (seperti pengenalan karakter), pengawasan kualitas produk, dan diagnostik medis (seperti deteksi tumor pada citra medis). 9.2. Metode Analisis Tekstur Metode analisis tekstur adalah teknik-teknik yang digunakan untuk mengukur dan menggambarkan tekstur dalam citra. Dalam subbab ini, kita akan membahas beberapa metode penting dalam analisis tekstur citra. a. Metode Statistik untuk Analisis Tekstur Metode statistik adalah salah satu pendekatan utama dalam analisis tekstur. Metode ini melibatkan pengukuran statistik dari distribusi intensitas piksel 42 dalam citra. Beberapa parameter statistik yang umum digunakan termasuk mean, variansi, dan matriks korelasi. Matriks korelasi mengukur hubungan antara intensitas piksel dalam citra, yang dapat memberikan informasi tentang tekstur. dalam citra. Beberapa parameter statistik yang umum digunakan termasuk mean, variansi, dan matriks korelasi. Matriks korelasi mengukur hubungan antara intensitas piksel dalam citra, yang dapat memberikan informasi tentang tekstur. dalam citra. Beberapa parameter statistik yang umum digunakan termasuk mean, variansi, dan matriks korelasi. Matriks korelasi mengukur hubungan antara intensitas piksel dalam citra, yang dapat memberikan informasi tentang tekstur. b. Analisis Tekstur Citra Metode Spasial untuk Analisis Tekstur Metode spasial berfokus pada deteksi pola dan struktur dalam citra. Metode ini dapat mencakup pengenalan pola berulang, seperti garis-garis atau titik-titik, serta analisis kontur dan orientasi. b. Metode Spasial untuk Analisis Tekstur Metode spasial berfokus pada deteksi pola dan struktur dalam citra. Metode ini dapat mencakup pengenalan pola berulang, seperti garis-garis atau titik-titik, serta analisis kontur dan orientasi. 9.3. Teknik seperti operator Laplacian dari Gauss (LoG) sering digunakan untuk deteksi tekstur. 9.3. Teknik seperti operator Laplacian dari Gauss (LoG) sering digunakan untuk deteksi tekstur. a. Metode Transformasi untuk Analisis Tekstur Metode transformasi melibatkan penggunaan transformasi matematis untuk menganalisis tekstur dalam domain frekuensi atau skala.Transformasi Fourier dan Transformasi Wavelet adalah contoh transformasi yang digunakan untuk menganalisis tekstur. Transformasi Wavelet, khususnya, dapat memberikan pemahaman yang mendalam tentang struktur tekstur pada berbagai skala. b. Aplikasi Metode Analisis Tekstur Metode analisis tekstur digunakan dalam berbagai aplikasi, termasuk pemrosesan citra medis, pengawasan kualitas produk, dan identifikasi bahan dalam citra satelit. Misalnya, dalam citra medis, analisis tekstur dapat membantu dalam deteksi dan klasifikasi tumor berdasarkan karakteristik tekstur mereka. 43 Bab 10. Segmentasi Citra Bab 10. Segmentasi Citra Segmentasi citra adalah proses penting dalam pengolahan citra yang bertujuan untuk memisahkan citra menjadi bagian-bagian yang homogen atau objek-objek yang berbeda. Segmentasi membantu dalam mengidentifikasi objek, batas, dan fitur dalam citra, yang merupakan langkah awal dalam berbagai aplikasi pengolahan citra. Dalam subbab ini, kita akan membahas konsep dasar dan teknik segmentasi citra. Konsep Dasar Segmentasi Citra Segmentasi citra adalah proses memisahkan citra menjadi wilayah-wilayah yang memiliki karakteristik serupa. Wilayah ini disebut segmen. Tujuan dari segmentasi adalah untuk mengidentifikasi objek atau fitur dalam citra, memisahkan objek dari latar belakang, atau membagi citra menjadi wilayah yang homogen untuk analisis lebih lanjut. Algoritma Segmentasi Terkenal Beberapa algoritma segmentasi terkenal termasuk metode ambang (thresholding), pemambangan wilayah (region growing), pemambangan berkas (watershed), dan pemrosesan berbasis grafik. Aplikasi Segmentasi Citra Segmentasi citra memiliki banyak aplikasi, termasuk dalam pengenalan objek, visi komputer, analisis medis, pengolahan citra satelit, dan pengenalan wajah. 10.1. Thresholding Thresholding adalah salah satu teknik segmentasi citra paling sederhana dan efektif yang digunakan untuk memisahkan objek dari latar belakang berdasarkan nilai intensitas piksel. Dalam subbab ini, kita akan mendiskusikan konsep dasar dan metode thresholding serta penerapannya dalam pengolahan citra. Metode Segmentasi Citra Metode Segmentasi Citra Metode segmentasi citra dapat dibagi menjadi beberapa kategori, termasuk segmentasi berbasis warna, segmentasi berbasis tepi, segmentasi berbasis tekstur, dan segmentasi berbasis kontur. Segmentasi berbasis warna menggunakan informasi warna untuk memisahkan objek dalam citra. Segmentasi berbasis tepi mengidentifikasi tepi atau batas antara objek dengan latar belakang. Segmentasi berbasis tekstur memisahkan objek berdasarkan perbedaan tekstur atau pola dalam citra. Segmentasi berbasis kontur berfokus pada identifikasi fitur kontur dalam citra. 44 Algoritma Segmentasi Terkenal Konsep Dasar Thresholding Konsep Dasar Thresholding Thresholding adalah proses yang melibatkan pemilihan nilai ambang (threshold) tertentu. Semua piksel dalam citra dengan intensitas di atas nilai ambang ini dianggap sebagai bagian dari objek, sedangkan piksel dengan intensitas di bawah nilai ambang dianggap sebagai latar belakang. Tujuan utama dari thresholding adalah untuk memisahkan objek dari latar belakang dan menciptakan citra biner di mana objek direpresentasikan sebagai piksel bernilai satu (putih) dan latar belakang sebagai piksel bernilai nol (hitam). Metode Thresholding Sederhana Metode thresholding sederhana termasuk thresholding global dan thresholding lokal. Metode Thresholding Sederhana Metode thresholding sederhana termasuk thresholding global dan thresholding lokal. 45 Dalam thresholding global, satu nilai ambang digunakan untuk seluruh citra. Ini cocok untuk citra dengan latar belakang homogen. Dalam thresholding global, satu nilai ambang digunakan untuk seluruh citra. Ini cocok untuk citra dengan latar belakang homogen. Dalam thresholding lokal, nilai ambang dihitung secara adaptif untuk setiap piksel berdasarkan lingkungan sekitarnya. Ini berguna untuk citra dengan perubahan intensitas yang signifikan. Algoritma Thresholding Terkenal Beberapa algoritma thresholding terkenal termasuk algoritma Otsu, algoritma Kapur, dan algoritma adaptif Sauvola. Algoritma Otsu mencoba untuk menentukan nilai ambang yang memaksimalkan varian antara kelas objek dan latar belakang. Algoritma Kapur menggunakan pendekatan entropi untuk menentukan nilai ambang yang optimal. Aplikasi Thresholding Thresholding memiliki banyak aplikasi, termasuk dalam pengenalan karakter, deteksi tepi, pemrosesan citra medis, dan analisis objek dalam visi komputer. Contoh aplikasi termasuk pengenalan karakter dalam OCR (Optical Character Recognition) dan deteksi tepi dalam pemrosesan citra. 10.2. Metode Berbasis Kontur Metode berbasis kontur dalam segmentasi citra adalah teknik yang berfokus pada identifikasi dan pemisahan objek dalam citra berdasarkan kontur atau batas yang membatasi objek. Dalam subbab ini, kita akan menjelaskan konsep dasar dan beberapa metode berbasis kontur yang umum digunakan dalam pengolahan citra. Konsep Dasar Metode Berbasis Kontur Konsep Dasar Metode Berbasis Kontur Metode berbasis kontur bertujuan untuk mengidentifikasi dan mengekstraksi batas atau kontur dari objek dalam citra. 46 Kontur adalah garis yang menggambarkan perbatasan antara objek dengan latar belakang dalam citra. Teknik ini memungkinkan segmentasi citra berdasarkan karakteristik bentuk dan kontur objek. Metode Berbasis Kontur Terkenal Beberapa metode berbasis kontur yang umum digunakan meliputi: Deteksi Tepi: Teknik ini mengidentifikasi tepi atau perubahan tajam dalam intensitas citra, yang sering mengindikasikan batas objek. Transformasi Hough: Digunakan untuk menentukan garis atau bentuk tertentu dalam citra. Aktif Kontur (Snakes): Merupakan model deformable yang dapat digunakan untuk menentukan kontur objek dengan mengikuti batas objek yang sebenarnya dalam citra. Kontur Aktif (Active Contours): Merupakan perluasan dari snakes yang digunakan untuk menentukan kontur objek dengan meminimalkan energi dalam citra. Aplikasi Metode Berbasis Kontur p Metode berbasis kontur memiliki banyak aplikasi, termasuk dalam pengenalan objek, deteksi pola, dan analisis bentuk. Contoh aplikasi termasuk pengenalan wajah dalam visi komputer, pemrosesan citra medis (misalnya, deteksi tumor berdasarkan bentuknya), dan pengawasan industri (misalnya, Metode berbasis kontur memiliki banyak aplikasi, termasuk dalam pengenalan objek, deteksi pola, dan analisis bentuk. Contoh aplikasi termasuk pengenalan wajah dalam visi komputer, pemrosesan citra medis (misalnya, deteksi tumor berdasarkan bentuknya), dan pengawasan industri (misalnya, pengenalan komponen dalam lini produksi). 10.3. Segmentasi Berdasarkan Warna Segmentasi Berdasarkan Warna Segmentasi berdasarkan warna adalah salah satu teknik penting dalam pengolahan citra yang digunakan untuk memisahkan objek dalam citra berdasarkan perbedaan warna atau spektral. 47 Dalam subbab ini, kita akan menjelaskan konsep dasar dan metode yang digunakan dalam segmentasi berdasarkan warna serta penerapannya dalam pengolahan citra. Konsep Dasar Segmentasi Berdasarkan Warna Segmentasi berdasarkan warna melibatkan pemisahan objek dalam citra berdasarkan perbedaan warna piksel-pikselnya. Warna adalah atribut visual yang penting dalam citra, dan informasi warna dapat digunakan untuk memisahkan objek dari latar belakang atau mengelompokkan objek berdasarkan karakteristik warna mereka. Konsep Dasar Segmentasi Berdasarkan Warna Segmentasi berdasarkan warna melibatkan pemisahan objek dalam citra berdasarkan perbedaan warna piksel-pikselnya. Warna adalah atribut visual yang penting dalam citra, dan informasi warna dapat digunakan untuk memisahkan objek dari latar belakang atau mengelompokkan objek berdasarkan karakteristik warna mereka. Metode Segmentasi Berdasarkan Warna Metode Segmentasi Berdasarkan Warna Metode segmentasi berdasarkan warna dapat mencakup pemisahan warna berdasarkan skala warna tunggal, skala warna ganda (misalnya, RGB atau HSV), atau pemrosesan warna dalam domain spektral. Algoritma segmentasi berdasarkan warna sering mengggunakan teknik seperti pemambangan berkas berbasis warna, ambang warna, dan analisis spektral. Metode segmentasi berdasarkan warna sering digunakan dalam aplikasi seperti pengenalan objek berwarna, analisis citra medis berwarna, dan pemrosesan citra berwarna alami. Aplikasi Segmentasi Berdasarkan Warna Segmentasi berdasarkan warna memiliki berbagai aplikasi, termasuk dalam visi komputer, pemrosesan citra medis, pengenalan objek dalam citra alami, dan pemantauan warna dalam industri. Contoh aplikasi termasuk pengenalan objek berwarna dalam berbagai industri seperti otomotif dan makanan, serta identifikasi struktur anatomi berwarna dalam citra medis. 48 Bab 11. Ekstraksi Fitur dan Pengenalan Pola Bab 11. Ekstraksi Fitur dan Pengenalan Pola 8. Ekstraksi Fitur Ekstraksi Fitur dan Pengenalan Pola Ekstraksi fitur dan pengenalan pola adalah tahap penting dalam pengolahan citra yang bertujuan untuk mengidentifikasi dan menggambarkan karakteristik objek dalam citra, serta mengklasifikasikan objek berdasarkan fitur-fitur ini. Dalam subbab ini, kita akan membahas konsep dasar ekstraksi fitur, metode ekstraksi fitur yang umum digunakan, dan aplikasi dalam pengenalan pola. Bab 11. Ekstraksi Fitur dan Pengenalan Pola 8. Ekstraksi Fitur Ekstraksi Fitur dan Pengenalan Pola Ekstraksi fitur dan pengenalan pola adalah tahap penting dalam pengolahan citra yang bertujuan untuk mengidentifikasi dan menggambarkan karakteristik objek dalam citra, serta mengklasifikasikan objek berdasarkan fitur-fitur ini. Dalam subbab ini, kita akan membahas konsep dasar ekstraksi fitur, metode ekstraksi fitur yang umum digunakan, dan aplikasi dalam pengenalan pola. Konsep Dasar Ekstraksi Fitur Konsep Dasar Ekstraksi Fitur p Ekstraksi fitur adalah proses mengkonversi citra ke dalam bentuk representasi yang lebih sederhana, kompak, dan informatif yang disebut fitur. Fitur-fitur ini mencerminkan karakteristik objek dalam citra seperti bentuk, tekstur, warna, atau pola. Pengenalan pola adalah tugas untuk mengklasifikasikan objek atau pola dalam citra berdasarkan fitur-fitur yang diekstraksi. Metode Ekstraksi Fitur Beberapa metode umum dalam ekstraksi fitur citra termasuk: Ekstraksi Fitur Geometrik: Ini melibatkan pengukuran fitur geometrik seperti panjang, lebar, area, dan bentuk objek. Ekstraksi Fitur Statistik: Ini melibatkan pengukuran statistik seperti mean, variansi, skewness, dan kurtosis dari distribusi intensitas piksel dalam citra. Ekstraksi Fitur Geometrik: Ini melibatkan pengukuran fitur geometrik seperti panjang, lebar, area, dan bentuk objek. Ekstraksi Fitur Statistik: Ini melibatkan pengukuran statistik seperti mean, variansi, skewness, dan kurtosis dari distribusi intensitas piksel dalam citra. Ekstraksi Fitur Tekstur: Ini melibatkan ekstraksi fitur yang mencerminkan pola tekstur dalam citra, seperti energi, kontras, dan homogenitas tekstur. Ekstraksi Fitur Frekuensi: Ini melibatkan analisis dalam domain frekuensi, seperti ekstraksi fitur spektral atau penggunaan transformasi Fourier. 49 Pengenalan Pola Pengenalan pola adalah tugas untuk mengklasifikasikan objek atau pola berdasarkan fitur-fitur yang diekstraksi. Metode yang umum digunakan dalam pengenalan pola mencakup pengenalan pola berbasis aturan, pengenalan pola berbasis pembelajaran mesin, dan pengenalan pola berbasis jaringan saraf tiruan. Pengenalan Pola Pengenalan pola adalah tugas untuk mengklasifikasikan objek atau pola berdasarkan fitur-fitur yang diekstraksi. Metode yang umum digunakan dalam pengenalan pola mencakup pengenalan pola berbasis aturan, pengenalan pola berbasis pembelajaran mesin, dan pengenalan pola berbasis jaringan saraf tiruan. Aplikasi Ekstraksi Fitur dan Pengenalan Pola Aplikasi Ekstraksi Fitur dan Pengenalan Pola Ekstraksi fitur dan pengenalan pola memiliki berbagai aplikasi, termasuk dalam pengenalan wajah, pengenalan karakter optik (OCR), analisis citra medis, dan pengawasan keamanan. Contoh aplikasi termasuk pengenalan wajah dalam sistem keamanan, pengenalan karakter dalam dokumen tercetak, dan identifikasi anomali dalam citra medis. Pilihan fitur adalah salah satu langkah penting dalam ekstraksi fitur yang melibatkan seleksi fitur-fitur yang paling relevan atau informatif dari sekumpulan fitur yang tersedia dalam citra atau data(Haralick et al., 1973). Dalam subbab ini, kita akan membahas konsep dasar pilihan fitur, pentingnya seleksi fitur, serta metode-metode yang umum digunakan dalam proses ini. Konsep Dasar Pilihan Fitur Konsep Dasar Pilihan Fitur Pada dasarnya, setiap citra atau data dapat memiliki banyak fitur yang digunakan untuk menggambarkan karakteristiknya. Namun, tidak semua fitur tersebut relevan atau informatif dalam tugas pengenalan atau klasifikasi. Pilihan fitur adalah proses memilih subset fitur yang paling relevan atau memiliki kontribusi signifikan dalam menjelaskan variabilitas data. 50 Pilihan fitur membantu dalam mengurangi dimensi data, meningkatkan akurasi model, mengurangi overfitting, dan meningkatkan efisiensi komputasi. Metode Pilihan Fitur Metode Pilihan Fitur Metode Pilihan Fitur Beberapa metode umum dalam pilihan fitur meliputi: Metode Filter: Metode ini memilih fitur berdasarkan metrik statistik atau informasi seperti korelasi, mutual information, atau uji chi-squared. Metode Wrapper: Metode ini memilih fitur dengan menggambarkan performa model pembelajaran mesin tertentu, seperti algoritma klasifikasi, ketika berbagai subset fitur digunakan. Metode Embedded: Metode ini menggabungkan pilihan fitur ke dalam proses pelatihan model pembelajaran mesin, seperti algoritma pengklasifikasi decision tree yang memungkinkan pemilihan fitur selama pelatihan. Pentingnya Pilihan Fitur Pilihan fitur memiliki dampak besar dalam efisiensi dan efektivitas proses pengenalan dan klasifikasi. Dengan mengurangi dimensi data, pilihan fitur dapat mengurangi kompleksitas permasalahan dan mempercepat waktu komputasi. Selain itu, dengan memilih fitur yang paling relevan, kita dapat meningkatkan akurasi dan generalisasi model. 11.2 Ekstraksi Fitur Tekstur 51 Ekstraksi fitur tekstur adalah salah satu teknik penting dalam pengolahan citra yang bertujuan untuk menggambarkan pola tekstur dalam citra. Pola tekstur mencerminkan distribusi spasial intensitas piksel dalam citra. Dalam subbab ini, kita akan membahas konsep dasar ekstraksi fitur tekstur, metode- metode yang umum digunakan, serta penerapannya dalam pengenalan pola(Haralick et al., 1973). Konsep Dasar Ekstraksi Fitur Tekstur Konsep Dasar Ekstraksi Fitur Tekstur Pola tekstur dalam citra mencakup pola-pola seperti garis- garis, titik-titik, atau struktur yang mengulang. Ekstraksi fitur tekstur bertujuan untuk mengukur dan menggambarkan pola tekstur dalam citra dengan cara yang bermakna dan informatif. Fitur tekstur dapat digunakan dalam berbagai aplikasi, termasuk klasifikasi citra berdasarkan tekstur, deteksi cacat dalam bahan, dan analisis citra medis. Metode Ekstraksi Fitur Tekstur Beberapa metode umum dalam ekstraksi fitur tekstur meliputi: Histogram Warna: Metode ini mengukur distribusi warna piksel dalam citra dan menggambarkan pola warna. Matrix Korespondensi: Metode ini menggunakan matriks korespondensi seperti matriks korelasi atau matriks ko- occurrence untuk menggambarkan hubungan antara intensitas piksel. 52 Transformasi Wavelet: Transformasi wavelet digunakan untuk menggambarkan tekstur pada berbagai skala. Transformasi Wavelet: Transformasi wavelet digunakan untuk menggambarkan tekstur pada berbagai skala. Metode Statistik: Metode ini menggunakan statistik seperti energi, kontras, dan homogenitas untuk mengukur tekstur. Aplikasi Ekstraksi Fitur Tekstur Aplikasi Ekstraksi Fitur Tekstur Ekstraksi fitur tekstur memiliki berbagai aplikasi, termasuk dalam pengenalan objek berdasarkan tekstur, deteksi anomali, dan analisis citra medis. Contoh aplikasi termasuk deteksi retakan pada permukaan bahan, pengenalan pola pada kain tekstil, dan identifikasi jaringan atau tumor dalam citra medis. 11.3 Ekstraksi Fitur Bentuk Ekstraksi Fitur Bentuk Ekstraksi fitur bentuk adalah salah satu aspek penting dalam pengolahan citra yang bertujuan untuk menggambarkan karakteristik geometris atau morfologi objek dalam citra. Dalam subbab ini, kita akan membahas konsep dasar ekstraksi fitur bentuk, metode-metode yang umum digunakan, serta penerapannya dalam pengenalan pola. Konsep Dasar Ekstraksi Fitur Bentuk Ekstraksi fitur bentuk melibatkan pengukuran dan deskripsi karakteristik geometris objek dalam citra, seperti panjang, lebar, area, perimeter, dan bentuk keseluruhan. Fitur-fitur bentuk ini membantu dalam pemisahan objek berdasarkan bentuk atau morfologi mereka. Ekstraksi fitur bentuk penting dalam berbagai aplikasi, termasuk pengenalan objek, pengawasan industri, dan pemrosesan citra medis. Metode Ekstraksi Fitur Bentuk Metode Ekstraksi Fitur Bentuk 53 Beberapa metode umum dalam ekstraksi fitur bentuk meliputi: Ekstraksi Fitur Geometrik: Metode ini melibatkan pengukuran fitur-fitur geometrik seperti panjang, lebar, area, dan perimeter objek. Transformasi Hough: Metode ini digunakan untuk mendeteksi garis atau bentuk tertentu dalam citra. Deskriptor Bentuk: Metode ini menggambarkan bentuk objek dengan menggunakan deskriptor seperti momen bentuk, convex hull, atau fitur-fitur Fourier. Pentingnya Ekstraksi Fitur Bentuk Pentingnya Ekstraksi Fitur Bentuk Ekstraksi fitur bentuk memiliki peran penting dalam pengenalan objek berdasarkan bentuknya. Fitur-fitur bentuk membantu dalam mengklasifikasikan objek berdasarkan morfologi mereka, yang sering kali lebih deskriptif daripada fitur-fitur seperti tekstur atau warna. Konsep Dasar Ekstraksi Fitur Tekstur Ekstraksi fitur bentuk penting dalam aplikasi seperti pengenalan karakter optik (OCR), pemantauan geometri objek dalam proses produksi, dan identifikasi bentuk objek dalam analisis citra medis. Ekstraksi fitur bentuk memiliki peran penting dalam pengenalan objek berdasarkan bentuknya. p g j y Fitur-fitur bentuk membantu dalam mengklasifikasikan objek berdasarkan morfologi mereka, yang sering kali lebih deskriptif daripada fitur-fitur seperti tekstur atau warna. Ekstraksi fitur bentuk penting dalam aplikasi seperti l k k ik (OC ) i bj k Fitur-fitur bentuk membantu dalam mengklasifikasikan objek berdasarkan morfologi mereka, yang sering kali lebih deskriptif daripada fitur-fitur seperti tekstur atau warna. Ekstraksi fitur bentuk penting dalam aplikasi seperti pengenalan karakter optik (OCR), pemantauan geometri objek dalam proses produksi, dan identifikasi bentuk objek dalam analisis citra medis. 54 54 Bab 12. Pengenalan Pola Pengenalan pola adalah salah satu cabang penting dalam bidang pengolahan citra dan pemrosesan sinyal yang bertujuan untuk mengidentifikasi pola atau objek dalam data, termasuk citra, suara, atau data lainnya. Dalam subbab ini, kita akan membahas konsep dasar pengenalan pola, metode-metode yang umum digunakan, serta aplikasi dalam berbagai bidang.(Duda et al., 2006) Konsep Dasar Pengenalan Pola Konsep Dasar Pengenalan Pola Pengenalan pola mencakup proses pembelajaran dari data atau citra untuk mengklasifikasikan atau mengidentifikasi pola yang telah dipelajari sebelumnya. Tujuan pengenalan pola adalah untuk menggambarkan pola dengan fitur atau karakteristik yang dapat digunakan dalam klasifikasi atau identifikasi. Pengenalan pola adalah bidang multidisiplin yang mencakup statistik, pembelajaran mesin, pengolahan citra, dan banyak lagi. Metode Pengenalan Pola Beberapa metode umum dalam pengenalan pola meliputi: K-Nearest Neighbors (K-NN): Metode ini membandingkan objek yang akan dikenali dengan objek-objek tetangganya dalam ruang fitur dan mengklasifikasikannya berdasarkan mayoritas tetangga terdekatnya. 55 Support Vector Machines (SVM): SVM adalah algoritma pembelajaran mesin yang digunakan untuk mengklasifikasikan data dalam dua atau lebih kelas. Jaringan Saraf Tiruan (Neural Networks): Neural networks adalah model komputasi yang terinspirasi oleh struktur jaringan saraf manusia yang digunakan untuk tugas pengenalan pola kompleks. Aplikasi Pengenalan Pola Pengenalan pola memiliki berbagai aplikasi, termasuk dalam pengenalan wajah, pemrosesan citra medis, analisis sentimen dalam teks, dan pengenalan karakter optik (OCR). Contoh aplikasi termasuk penggunaan pengenalan wajah dalam sistem keamanan, pengenalan karakter dalam dokumen cetak, atau pengenalan suara dalam asisten virtual. 12.1 Pengenalan Pola Citra 12.1 Pengenalan Pola Citra Pengenalan pola citra adalah salah satu aspek penting dalam pengolahan citra yang bertujuan untuk mengidentifikasi objek, pola, atau karakteristik dalam citra atau rangkaian citra. Dalam subbab ini, kita akan membahas konsep dasar pengenalan pola citra, metode-metode yang umum digunakan, serta aplikasi dalam berbagai bidang. Konsep Dasar Pengenalan Pola Citra Pengenalan pola citra melibatkan proses pengklasifikasian objek atau pola dalam citra berdasarkan fitur-fitur yang diekstraksi dari citra tersebut. Fitur-fitur ini mencakup informasi seperti bentuk, warna, tekstur, atau konteks spasial objek dalam citra. 56 Pengenalan pola citra memiliki berbagai aplikasi, termasuk pengenalan karakter optik (OCR), pengenalan objek dalam visi komputer, dan analisis citra medis. Metode Pengenalan Pola Citra Beberapa metode umum dalam pengenalan pola citra meliputi: Pengenalan Pola Berbasis Fitur: Metode ini menggambarkan objek dalam citra dengan menggunakan fitur-fitur seperti ekstraksi bentuk, tekstur, atau warna, dan kemudian mengklasifikasikan objek berdasarkan fitur-fitur ini. Pengenalan Pola Berbasis Model: Metode ini memodelkan objek dalam citra dengan menggunakan model geometris seperti model aktor, model Markov tersembunyi, atau jaringan saraf tiruan. Pengenalan Pola Berbasis Statistik: Metode ini menggunakan pendekatan statistik untuk mengklasifikasikan objek dalam citra berdasarkan distribusi fitur-fitur yang diekstraksi. Aplikasi Pengenalan Pola Citra Pengenalan pola citra memiliki berbagai aplikasi, seperti: Pengenalan pola citra memiliki berbagai aplikasi, seperti: Pengenalan Karakter Optik (OCR): Digunakan untuk mengkonversi teks yang tercetak dalam dokumen ke dalam teks digital. Pengenalan Wajah: Digunakan dalam sistem keamanan, pengenalan wajah dalam foto, atau pengenalan emosi wajah. Analisis Citra Medis: Digunakan untuk mendeteksi dan mengklasifikasikan patologi dalam citra medis, seperti citra MRI atau CT. 57 12.2 Klasifikasi Citra Klasifikasi citra adalah salah satu aspek penting dalam pengenalan pola yang bertujuan untuk mengklasifikasikan citra atau objek dalam citra ke dalam kategori atau kelas yang telah ditentukan. Dalam subbab ini, kita akan membahas konsep dasar klasifikasi citra, metode-metode yang umum digunakan, serta aplikasinya dalam berbagai bidang(Sonka et al., 2014). 12.2 Klasifikasi Citra Konsep Dasar Klasifikasi Citra Klasifikasi citra melibatkan proses mengatributkan label kategori atau kelas pada citra berdasarkan karakteristik visual atau fitur-fitur yang diekstraksi dari citra tersebut. Fitur-fitur yang digunakan dalam klasifikasi dapat mencakup bentuk, tekstur, warna, atau fitur-fitur lain yang relevan. Tujuan klasifikasi citra adalah untuk mengenali objek atau pola dalam citra dan membedakannya ke dalam kategori yang telah ditentukan. Metode Klasifikasi Citra Metode Klasifikasi Citra Beberapa metode umum dalam klasifikasi citra meliputi: K-Nearest Neighbors (K-NN): Metode ini mengklasifikasikan citra berdasarkan mayoritas label dari K citra terdekat dalam ruang fitur. K-Nearest Neighbors (K-NN): Metode ini mengklasifikasikan citra berdasarkan mayoritas label dari K citra terdekat dalam ruang fitur. Support Vector Machines (SVM): SVM adalah algoritma pembelajaran mesin yang digunakan untuk mengklasifikasikan citra dalam dua atau lebih kelas dengan mencari hyperplane terbaik yang memisahkan kelas-kelas tersebut. 58 Jaringan Saraf Tiruan (Neural Networks): Neural networks adalah model komputasi yang digunakan untuk tugas-tugas klasifikasi citra yang kompleks. Aplikasi Klasifikasi Citra Klasifikasi citra memiliki berbagai aplikasi yang luas, seperti: Klasifikasi citra memiliki berbagai aplikasi yang luas, seperti: Klasifikasi citra memiliki berbagai aplikasi yang luas, seperti: Pengenalan Wajah: Dalam sistem keamanan atau pengenalan wajah dalam foto dan video. Pengenalan Objek: Untuk mengklasifikasikan objek dalam citra atau video, seperti pengenalan plat nomor kendaraan. Pengenalan Objek: Untuk mengklasifikasikan objek dalam citra atau video, seperti pengenalan plat nomor kendaraan. Pemrosesan Citra Medis: Untuk mengklasifikasikan struktur anatomi dalam citra medis atau untuk mendeteksi penyakit. Pemrosesan Citra Medis: Untuk mengklasifikasikan struktur anatomi dalam citra medis atau untuk mendeteksi penyakit. 12.3 Aplikasi Pengenalan Pola Citra Aplikasi Pengenalan Pola Citra 12.3 Aplikasi Pengenalan Pola Citra Aplikasi Pengenalan Pola Citra Aplikasi pengenalan pola citra adalah salah satu aspek paling menarik dalam pengolahan citra dan pengenalan pola. Dalam subbab ini, kita akan membahas berbagai aplikasi praktis dari teknik pengenalan pola citra yang telah kita pelajari sebelumnya. Aplikasi di Bidang Medis Aplikasi di Bidang Medis Pengenalan Tumor: Teknik pengenalan pola citra digunakan dalam analisis citra medis untuk mendeteksi dan mengklasifikasikan tumor dalam citra MRI atau CT scan. Pendeteksian Retina: Dalam oftalmologi, pengenalan pola digunakan untuk mendeteksi masalah retina seperti retinopati diabetik. 59 Aplikasi di Bidang Keamanan Aplikasi di Bidang Keamanan Pengenalan Wajah: Pengenalan pola citra digunakan dalam sistem keamanan untuk mengidentifikasi individu berdasarkan wajah mereka. Pengenalan Plat Nomor: Dalam sistem pemantauan lalu lintas, teknik pengenalan pola digunakan untuk mengenali plat nomor kendaraan. Aplikasi dalam Teknologi Otomotif Aplikasi dalam Teknologi Otomotif Panduan Parkir Otomatis: Dalam mobil modern, teknologi pengenalan pola digunakan untuk panduan parkir otomatis. Kendaraan Otonom: Teknik pengenalan pola citra adalah komponen kunci dalam kendaraan otonom untuk mengidentifikasi objek di jalan. 4. Analisis Sentimen dalam Media Sosial Bab 13. Aplikasi Lanjutan Subbab ini akan membahas beberapa aplikasi lanjutan dari pengolahan citra digital dan pengenalan pola yang mencakup berbagai bidang(Sonka et al., 2014). Aplikasi dalam Pengenalan Karakter dan Tulisan Tangan Aplikasi dalam Pengenalan Karakter dan Tulisan Tangan Aplikasi dalam Pengenalan Karakter dan Tulisan Tangan OCR (Optical Character Recognition): Teknik pengenalan pola citra digunakan dalam OCR untuk mengubah teks yang tercetak dalam dokumen ke dalam teks digital yang dapat diedit. OCR (Optical Character Recognition): Teknik pengenalan pola citra digunakan dalam OCR untuk mengubah teks yang tercetak dalam dokumen ke dalam teks digital yang dapat diedit. Pengenalan Tulisan Tangan: Dalam aplikasi seperti pengenalan cek atau dokumen tangan, teknik pengenalan pola tulisan tangan digunakan. Aplikasi dalam Industri 60 Pengawasan Kualitas Produk: Dalam industri manufaktur, pengenalan pola citra digunakan untuk pengawasan kualitas produk, seperti mendeteksi cacat pada produk yang diproduksi. Robotic Vision: Teknik pengenalan pola citra digunakan dalam robotika untuk membantu robot mengidentifikasi objek dan berinteraksi dengan lingkungan mereka. 61 61 Bab 13. Aplikasi Lanjutan Metode-Metode Pengolahan Citra 3D Beberapa metode umum dalam pengolahan citra 3D meliputi: Segmentasi 3D: Metode ini digunakan untuk memisahkan objek dalam citra 3D berdasarkan fitur-fitur seperti intensitas, tekstur, atau morfologi. Rekonstruksi 3D: Proses rekonstruksi 3D digunakan untuk membuat model 3D dari serangkaian citra 2D, seperti yang sering terjadi dalam pemindaian CT atau MRI. Visualisasi 3D: Teknik visualisasi digunakan untuk membuat representasi visual dari data 3D, memungkinkan pemahaman yang lebih baik tentang struktur objek. Konsep Dasar Citra 3D Citra 3D adalah representasi data spasial yang menggambarkan objek atau lingkungan dalam tiga dimensi, mencakup panjang, lebar, dan kedalaman. Citra 3D dapat diperoleh dari berbagai sumber, termasuk pemindaian laser, pemindaian CT (Computed Tomography), pemindaian MRI (Magnetic Resonance Imaging), kamera stereo, atau bahkan pemodelan 3D. 1. Citra Medis dan Diagnostik 1. Citra Medis dan Diagnostik Segmentasi Organ dan Jaringan: Dalam radiologi, pengolahan citra digunakan untuk segmentasi organ tubuh atau jaringan dalam citra medis, seperti MRI atau CT scan, untuk analisis lebih lanjut. Pengenalan Pola pada Citra Histologi: Dalam patologi, analisis citra digital digunakan untuk mengenali pola histologi dalam citra mikroskopis untuk diagnosis penyakit. 2. Pengenalan Objek dalam Robotika 2. Pengenalan Objek dalam Robotika Navigasi Kendaraan Otonom: Pengolahan citra dan pengenalan pola digunakan dalam kendaraan otonom untuk mengidentifikasi rambu lalu lintas, pejalan kaki, atau kendaraan lainnya di jalan. Navigasi Kendaraan Otonom: Pengolahan citra dan pengenalan pola digunakan dalam kendaraan otonom untuk mengidentifikasi rambu lalu lintas, pejalan kaki, atau kendaraan lainnya di jalan. Manipulasi Objek: Dalam robotika industri, robot digunakan untuk mengidentifikasi, mengambil, dan memanipulasi objek berdasarkan citra. 3. Pengenalan Tulisan Tangan dan Optik Karakter (OCR) 3. Pengenalan Tulisan Tangan dan Optik Karakter (OCR) Pengenalan Tulisan Tangan: OCR diperluas untuk mengenali tulisan tangan manusia dalam dokumen sehari-hari, seperti cek atau nota. Pengenalan Optik Karakter dalam Bahasa yang Berbeda: OCR ditingkatkan untuk mengenali karakter dalam bahasa yang berbeda, termasuk bahasa yang tidak menggunakan alfabet Latin. 62 Pengenalan Emosi dalam Gambar atau Video: Teknik pengenalan pola digunakan untuk mengidentifikasi emosi wajah dalam gambar atau video yang diunggah ke media sosial. Analisis Sentimen Teks: Dalam pengolahan bahasa alami, analisis sentimen digunakan untuk menentukan perasaan atau pandangan dalam teks yang diposting di media sosial. Pengenalan Emosi dalam Gambar atau Video: Teknik pengenalan pola digunakan untuk mengidentifikasi emosi wajah dalam gambar atau video yang diunggah ke media sosial. Analisis Sentimen Teks: Dalam pengolahan bahasa alami, analisis sentimen digunakan untuk menentukan perasaan atau pandangan dalam teks yang diposting di media sosial. 13. Pengolahan Citra 3D 13. Pengolahan Citra 3D Konsep Dasar Citra 3D Aplikasi dalam Kedokteran Aplikasi dalam Kedokteran Pemodelan Organ dan Jaringan: Citra 3D digunakan untuk memodelkan organ tubuh manusia untuk perencanaan bedah, diagnosis penyakit, atau pemantauan perkembangan penyakit. 63 Navigasi Bedah: Dalam bedah, pengolahan citra 3D digunakan untuk navigasi dan panduan bedah yang presisi. Aplikasi dalam Robotika Aplikasi dalam Robotika Panduan Navigasi Robot: Citra 3D digunakan dalam robotika untuk membantu robot mengenali dan berinteraksi dengan lingkungan mereka, seperti robot otonom yang beroperasi di bawah air. Aplikasi dalam Pemodelan Pemodelan Arsitektur: Citra 3D digunakan dalam industri arsitektur untuk membuat model bangunan dan lingkungan yang lebih realistis. Pemodelan untuk Desain Produk: Dalam industri manufaktur, pengolahan citra 3D digunakan untuk membuat model produk yang akan diproduksi, memungkinkan perancang untuk melakukan pengujian virtual sebelum produksi. 13.1 Pengenalan Citra 3D Konsep Dasar Citra 3D Citra 3D adalah representasi visual dari objek atau lingkungan dalam tiga dimensi, yang mencakup panjang, lebar, dan kedalaman. Untuk menghasilkan citra 3D, berbagai teknologi pemindaian digunakan, seperti pemindaian laser, pemindaian CT (Computed Tomography), pemindaian MRI (Magnetic Resonance Imaging), atau kamera stereo. Citra 3D sering direpresentasikan dalam format voxel (volume pixel) atau titik 3D. Metode Pengenalan Citra 3D Pengenalan citra 3D melibatkan berbagai metode, termasuk: Segmentasi 3D: Metode ini digunakan untuk memisahkan objek atau struktur tertentu dalam citra 3D, seringkali berdasarkan ambang batas intensitas atau ciri-ciri morfologi. 64 Rekonstruksi 3D: Proses ini menghasilkan model 3D dari serangkaian citra 2D, seperti yang sering terjadi dalam pemindaian CT atau MRI. Rekonstruksi 3D: Proses ini menghasilkan model 3D dari serangkaian citra 2D, seperti yang sering terjadi dalam pemindaian CT atau MRI. p Visualisasi 3D: Teknik visualisasi digunakan untuk menghasilkan representasi grafis yang memungkinkan pengguna untuk memahami dan berinteraksi dengan citra 3D. Aplikasi Pengenalan Citra 3D Pengenalan citra 3D memiliki berbagai aplikasi, termasuk: Pengenalan citra 3D memiliki berbagai aplikasi, termasuk: Kedokteran: Digunakan dalam pemodelan organ tubuh manusia untuk perencanaan bedah, diagnosis penyakit, atau pemantauan perkembangan penyakit(Ma et al., 2004). Robotika: Membantu robot mengenali dan berinteraksi dengan lingkungan mereka, seperti robot otonom dan robot bedah. Industri Manufaktur: Digunakan untuk pemodelan produk dan pengawasan kualitas produk. 13.2 Pencitraan Medis Subbab ini akan membahas aplikasi pengolahan citra dalam dunia medis yang sering disebut "pencitraan medis." Pencitraan medis adalah salah satu bidang utama dalam pengolahan citra 3D yang digunakan untuk diagnosis penyakit, perencanaan bedah, dan pemantauan perkembangan penyakit. Konsep Dasar Pencitraan Medis Pencitraan medis melibatkan pengambilan, pemrosesan, dan analisis citra untuk membantu dalam diagnosis, pemantauan, dan perawatan penyakit(Bankman, 2008). Sumber citra medis meliputi CT scan, MRI, ultrasonografi, sinar-X, dan banyak lagi. Metode-Metode Pencitraan Medis Metode-Metode Pencitraan Medis Beberapa metode umum dalam pencitraan medis meliputi: Pencitraan Tomografi Komputer (CT Scan): Metode ini menggunakan sinar-X untuk menghasilkan serangkaian gambar potongan tubuh dalam tiga dimensi. 65 Pencitraan Resonansi Magnetik (MRI): MRI menggunakan medan magnet dan gelombang radio untuk menghasilkan gambaran detail dari dalam tubuh. Ultrasonografi: Pencitraan ultrasonik menggunakan gelombang suara tinggi untuk menghasilkan gambar organ dalam tubuh. Aplikasi Pencitraan Medis Pencitraan medis memiliki berbagai aplikasi yang krusial dalam dunia medis, termasuk: Diagnosis Penyakit: Digunakan untuk mendiagnosis penyakit seperti kanker, stroke, atau cedera jaringan lunak. Perencanaan Bedah: Pencitraan medis membantu dokter merencanakan operasi dengan presisi, meminimalkan risiko, dan memaksimalkan efisiensi. Perencanaan Bedah: Pencitraan medis membantu dokter merencanakan operasi dengan presisi, meminimalkan risiko, dan memaksimalkan efisiensi. Pemantauan Perkembangan Penyakit: Digunakan untuk memantau respons pasien terhadap perawatan dan perkembangan penyakit. Pemantauan Perkembangan Penyakit: Digunakan untuk memantau respons pasien terhadap perawatan dan perkembangan penyakit. 66 66 Bab 14. Pengolahan Citra dalam Realitas Virtual dan Augmented Konsep Dasar Realitas Virtual (VR) dan Augmented Reality (AR) Realitas Virtual (VR): VR adalah teknologi yang menciptakan lingkungan simulasi yang sepenuhnya imersif. Pengguna dapat sepenuhnya terlibat dalam dunia maya yang diciptakan oleh komputer, seringkali menggunakan headset VR. Augmented Reality (AR): AR adalah teknologi yang memadukan elemen dunia nyata dengan elemen digital, seperti grafis, suara, atau video. Ini biasanya dicapai melalui aplikasi di perangkat mobile atau headset AR. Metode-Metode Pengolahan Citra dalam VR dan AR Beberapa metode yang digunakan dalam pengolahan citra Metode-Metode Pengolahan Citra dalam VR dan AR Beberapa Metode-Metode Pengolahan Citra dalam VR dan AR Beberapa metode yang digunakan dalam pengolahan citra dalam VR dan AR meliputi: metode yang digunakan dalam pengolahan citra dalam VR dan AR meliputi: Pengenalan Objek: Teknik pengenalan citra digunakan untuk mengenali objek dalam lingkungan nyata dan mengintegrasikan objek digital dalam AR. Pemodelan 3D: Pemodelan objek dan lingkungan 3D sangat penting dalam VR untuk menciptakan dunia maya yang imersif. 14.1 Realitas Virtual (VR) Subbab ini akan memperkenalkan konsep dasar Realitas Virtual (VR) dalam konteks pengolahan citra. VR adalah teknologi yang menciptakan lingkungan simulasi yang sepenuhnya imersif, di mana pengguna dapat sepenuhnya terlibat dalam dunia maya yang diciptakan oleh komputer. 13.2 Pencitraan Medis Pelacakan Gerakan: Pelacakan gerakan adalah komponen kunci dalam VR dan AR untuk memastikan bahwa objek digital berinteraksi secara realistis dengan gerakan pengguna. Aplikasi dalam VR dan AR 67 VR dan AR memiliki berbagai aplikasi, termasuk: VR dan AR memiliki berbagai aplikasi, termasuk: Gaming: VR digunakan dalam game yang sepenuhnya imersif, sementara AR digunakan dalam game yang memadukan elemen digital dengan dunia nyata. Pendidikan: VR digunakan dalam pembelajaran virtual yang mendalam, sementara AR digunakan untuk memperkaya pengalaman pembelajaran dalam lingkungan nyata. Pariwisata: Aplikasi AR digunakan dalam panduan wisata yang memungkinkan pengguna melihat informasi tambahan tentang tempat wisata yang mereka kunjungi. Perawatan Kesehatan: VR digunakan dalam terapi dan pelatihan medis, sedangkan AR digunakan dalam visualisasi dan panduan bedah. Konsep Dasar VR VR menciptakan lingkungan maya yang mensimulasikan dunia nyata atau menciptakan dunia fiksi sepenuhnya. Pengguna biasanya menggunakan headset VR yang dilengkapi dengan sensor gerak untuk mengalami VR. Lingkungan VR sering kali mencakup visualisasi 3D yang sangat realistis, audio 3D, serta pelacakan gerakan pengguna untuk memberikan pengalaman imersif(Sherman & Craig, 2018). Pengolahan Citra dalam VR Pengolahan citra adalah komponen kunci dalam pengalaman VR. Beberapa aspek pengolahan citra dalam VR meliputi: 68 Visualisasi 3D: VR memerlukan representasi visual yang realistis dari dunia maya. Ini melibatkan pemodelan objek dan lingkungan 3D dengan tekstur dan pencahayaan yang memadai. Pelacakan Gerakan: Sensor dan kamera pada headset VR digunakan untuk melacak gerakan kepala dan tangan pengguna, sehingga citra VR dapat berubah sesuai dengan perubahan orientasi dan lokasi pengguna. Rendering Stereo: VR seringkali menggunakan rendering stereo untuk menghasilkan citra 3D terpisah untuk mata kiri dan kanan pengguna, menciptakan efek tiga dimensi. Konsep Dasar AR AR memadukan informasi digital dengan lingkungan fisik, menghasilkan pengalaman di mana objek atau informasi digital muncul dalam dunia nyata(Azuma, 1997). Aplikasi AR sering dijalankan pada perangkat mobile, tablet, atau headset AR khusus. Pengolahan Citra dalam AR Pengolahan citra adalah aspek penting dalam AR. Beberapa aspek pengolahan citra dalam AR meliputi: Pengolahan Citra dalam AR Aplikasi VR VR memiliki berbagai aplikasi, termasuk: Gaming: VR digunakan dalam game yang sepenuhnya imersif, memungkinkan pemain untuk merasakan lingkungan dan interaksi dengan objek dalam dunia maya. Pendidikan: VR digunakan dalam pembelajaran virtual yang mendalam, memungkinkan siswa untuk mengalami konsep- konsep abstrak dalam lingkungan 3D. Pelatihan: VR digunakan dalam pelatihan simulasi, seperti pelatihan pilot pesawat atau latihan bedah. 14.2 Realitas Augmented (AR) Pengolahan Citra dalam AR g Pengolahan citra adalah aspek penting dalam AR. Beberapa aspek pengolahan citra dalam AR meliputi: 69 Pengenalan Objek: Teknik pengenalan citra digunakan untuk mengenali objek dalam lingkungan nyata dan menambahkan objek digital yang sesuai. Pelacakan Posisi: AR memerlukan pelacakan posisi perangkat dan objek dalam lingkungan nyata agar objek digital dapat berinteraksi secara realistis dengan dunia nyata. Visualisasi Augmented: Proses ini melibatkan pemrosesan citra dan tampilan objek digital sehingga mereka tampak seperti bagian integral dari lingkungan nyata. Aplikasi AR AR memiliki berbagai aplikasi, termasuk: AR memiliki berbagai aplikasi, termasuk: Gaming: AR digunakan dalam game yang memadukan elemen digital dengan dunia nyata, seperti Pokémon GO. Gaming: AR digunakan dalam game yang memadukan elemen digital dengan dunia nyata, seperti Pokémon GO. Pendidikan: AR digunakan dalam pembelajaran interaktif di mana siswa dapat melihat model 3D, info tambahan, atau animasi dalam buku teks. Penggunaan Harian: Aplikasi AR digunakan dalam navigasi, memandu pengguna menuju tujuan dengan menampilkan rute dan info penting dalam pandangan kamera perangkat. 70 70 Bab 15. Kasus Studi Bab 15. Kasus Studi 15. Studi Kasus Pengolahan Citra Subbab ini akan memperkenalkan beberapa studi kasus dalam pengolahan citra untuk memberikan pemahaman yang lebih dalam tentang bagaimana teknik-teknik pengolahan citra digunakan dalam berbagai aplikasi dunia nyata. Kasus Studi 1: Pengolahan Citra Medis untuk Diagnosis Penyakit Deskripsi Kasus Studi: Dalam dunia medis, pengolahan citra digunakan untuk mendiagnosis penyakit seperti kanker. Sebuah kasus studi dapat berfokus pada bagaimana citra medis seperti CT scan atau MRI digunakan dalam deteksi dan diagnosis kanker. Teknik Pengolahan Citra: Segmentasi citra, ekstraksi fitur, dan klasifikasi citra adalah teknik utama yang digunakan dalam pengolahan citra medis. Sebagai contoh, penggunaan pengolahan citra untuk memisahkan area yang mencurigakan pada gambar CT scan. Sumber Referensi: Anda dapat merujuk ke penelitian medis dan artikel yang membahas penggunaan pengolahan citra dalam diagnosis kanker. Kasus Studi 2: Pengolahan Citra dalam Keamanan Kasus Studi 2: Pengolahan Citra dalam Keamanan Deskripsi Kasus Studi: Pengolahan citra digunakan dalam sistem keamanan, seperti pemantauan CCTV. Studi kasus dapat membahas bagaimana teknik pengolahan citra digunakan dalam mendeteksi intrusi atau perilaku mencurigakan. Teknik Pengolahan Citra: Teknik deteksi tepi, pelacakan objek, dan analisis pola adalah teknik yang relevan dalam aplikasi keamanan. Penggunaannya untuk mendeteksi gerakan mencurigakan dalam rekaman video. Kasus Studi 2: Pengolahan Citra dalam Keamanan Deskripsi Kasus Studi: Pengolahan citra digunakan dalam sistem keamanan, seperti pemantauan CCTV. Studi kasus dapat membahas bagaimana teknik pengolahan citra digunakan dalam mendeteksi intrusi atau perilaku mencurigakan. g Teknik Pengolahan Citra: Teknik deteksi tepi, pelacakan objek, dan analisis pola adalah teknik yang relevan dalam aplikasi keamanan. Penggunaannya untuk mendeteksi gerakan mencurigakan dalam rekaman video. Sumber Referensi: Jurnal dan konferensi tentang keamanan dan pengolahan citra dalam sistem keamanan. 71 Kasus Studi 3: Pengolahan Citra dalam Augmented Reality Deskripsi Kasus Studi: Augmented Reality (AR) menggunakan pengolahan citra untuk menampilkan objek digital dalam dunia nyata. Studi kasus dapat mencakup penggunaan AR dalam industri periklanan atau pendidikan. Aplikasi AR Teknik Pengolahan Citra: Pengenalan objek, pelacakan posisi, dan visualisasi augmented adalah teknik utama dalam aplikasi AR. Sumber Referensi: Buku dan artikel tentang AR serta penggunaan pengolahan citra dalam AR. Kasus Studi 4: Pengolahan Citra dalam Identifikasi Wajah Deskripsi Kasus Studi: Identifikasi wajah adalah aplikasi yang semakin umum dalam keamanan, pengelolaan identitas, dan pengawasan. Studi kasus dapat fokus pada penggunaan teknologi pengolahan citra untuk mengenali individu berdasarkan wajah mereka. Teknik Pengolahan Citra: Deteksi wajah, ekstraksi fitur wajah, dan pengenalan pola adalah teknik utama dalam identifikasi wajah. Penggunaan algoritma pengenalan wajah dalam aplikasi keamanan. 15.1 Aplikasi dalam Bidang Kesehatan Dalam dunia kesehatan, pengolahan citra memiliki peran penting dalam diagnosis penyakit, pemantauan perkembangan penyakit, dan perencanaan bedah. Salah satu aplikasi yang akan kita telusuri adalah penggunaan pengolahan citra dalam mendiagnosis penyakit kulit seperti melanoma. Implementasi dengan Python: Dalam studi kasus ini, kita akan melihat bagaimana teknik pengolahan citra dapat diterapkan dengan menggunakan 72 Python. Kita akan memanfaatkan library Python seperti OpenCV dan scikit-image. Langkah-langkah Implementasi: Pengambilan Citra: Pertama, kita akan mempelajari bagaimana citra kulit pasien diambil, baik melalui kamera medis atau smartphone. Praproses Citra: Citra kulit yang diambil akan diproses untuk meningkatkan kualitasnya. Praproses ini mungkin melibatkan perbaikan kualitas citra dan perbaikan kontras. Segmentasi Citra: Teknik segmentasi akan digunakan untuk mengidentifikasi area yang mencurigakan pada kulit yang perlu dianalisis lebih lanjut. Ekstraksi Fitur: Fitur-fitur penting dari kulit, seperti ukuran, bentuk, atau warna, akan diekstraksi untuk analisis lebih lanjut. Klasifikasi Citra: Citra kulit akan diklasifikasikan menggunakan model machine learning atau deep learning. M d l i i k dil tih t k b d k t l i k lit Ekstraksi Fitur: Fitur-fitur penting dari kulit, seperti ukuran, bentuk, atau warna, akan diekstraksi untuk analisis lebih lanjut. Ekstraksi Fitur: Fitur-fitur penting dari kulit, seperti ukuran, bentuk, atau warna, akan diekstraksi untuk analisis lebih lanjut. Klasifikasi Citra: Citra kulit akan diklasifikasikan menggunakan model machine learning atau deep learning. Model ini akan dilatih untuk membedakan antara lesi kulit normal dan lesi yang mencurigakan(Qasim Gilani et al., 2023). 15.2 Aplikasi dalam Bidang Teknik Klasifikasi Citra: Citra kulit akan diklasifikasikan menggunakan model machine learning atau deep learning. Model ini akan dilatih untuk membedakan antara lesi kulit normal dan lesi yang mencurigakan(Qasim Gilani et al., 2023). Aplikasi AR 15 2 Aplikasi dalam Bidang Teknik 15.2 Aplikasi dalam Bidang Teknik Deskripsi Kasus Studi: Deskripsi Kasus Studi: Dalam dunia teknik, pengolahan citra memiliki banyak aplikasi yang penting, termasuk inspeksi kualitas produk, pemantauan infrastruktur, dan analisis data sensor. Sebagai contoh, kita akan menjelajahi penggunaan pengolahan citra dalam inspeksi kualitas produk. p Implementasi dengan Python: Implementasi dengan Python: Dalam studi kasus ini, kita akan memahami bagaimana teknik pengolahan citra dapat diimplementasikan menggunakan bahasa pemrograman Python. Kita akan menggunakan library Python seperti OpenCV untuk menjalankan proses pengolahan citra. Langkah-langkah Implementasi: 73 Pengambilan Citra: Pertama, kita akan mempelajari bagaimana citra produk yang akan diinspeksi diambil menggunakan kamera atau perangkat pemrosesan citra. Pengambilan Citra: Pertama, kita akan mempelajari bagaimana citra produk yang akan diinspeksi diambil menggunakan kamera atau perangkat pemrosesan citra. Praproses Citra: Citra produk akan diproses untuk menghilangkan noise, meningkatkan kontras, dan mempersiapkannya untuk analisis lebih lanjut. Segmentasi Citra: Teknik segmentasi akan digunakan untuk memisahkan area produk dari latar belakang dan memungkinkan analisis fokus pada produk itu sendiri. Ekstraksi Fitur: Fitur-fitur produk seperti bentuk, warna, atau tekstur akan diekstraksi untuk melakukan pemantauan kualitas. Ekstraksi Fitur: Fitur-fitur produk seperti bentuk, warna, atau tekstur akan diekstraksi untuk melakukan pemantauan kualitas. Klasifikasi Citra: Citra produk akan diklasifikasikan sebagai produk yang memenuhi standar kualitas atau memiliki cacat. Ini dapat dilakukan menggunakan metode machine learning atau deep learning. 15.3 Aplikasi dalam Bidang Hiburan Bidang hiburan memiliki banyak aplikasi pengolahan citra, seperti pengembangan efek visual untuk film dan televisi, pemrosesan citra dalam game, dan pengenalan gerakan dalam tari atau seni pertunjukan. Dalam studi kasus ini, kita akan menjelajahi penggunaan pengolahan citra dalam pengembangan efek visual untuk film. Implementasi dengan Python: Kita akan melihat bagaimana teknik pengolahan citra dapat diimplementasikan dengan menggunakan bahasa pemrograman Python. Kita akan memanfaatkan berbagai library Python yang relevan untuk tugas-tugas dalam bidang hiburan. Langkah-langkah Implementasi: Pengambilan Citra atau Frame: Pertama, kita akan memahami bagaimana citra atau frame dari adegan film diambil menggunakan perangkat perekaman atau kamera. Langkah-langkah Implementasi: g g p Pengambilan Citra atau Frame: Pertama, kita akan memahami bagaimana citra atau frame dari adegan film diambil menggunakan perangkat perekaman atau kamera. 74 Praproses Citra atau Frame: Citra atau frame akan diproses untuk meningkatkan kualitasnya, menghilangkan noise, dan mempersiapkannya untuk manipulasi lebih lanjut. Efek Visual: Teknik pengolahan citra akan digunakan untuk menghasilkan efek visual yang sesuai dengan kebutuhan film, seperti efek ledakan, penghapusan latar belakang, atau penciptaan karakter digital. Aplikasi AR Integrasi dengan Footage Asli: Citra hasil efek visual akan diintegrasikan dengan footage asli untuk menciptakan tampilan yang mulus dalam film(Foley et al., 1994). 75 75 Bab 16. Kesimpulan Bab 16. Kesimpulan 16. Tantangan dan Arah Masa Depan dalam Pengolahan Citra Digital 16. Tantangan dan Arah Masa Depan dalam Pengolahan Citra Digital Tantangan dalam Pengolahan Citra Digital: Kompleksitas Data: Citra digital semakin kompleks dan berukuran besar, yang menghasilkan tantangan dalam pemrosesan dan analisis. Variabilitas dan Noise: Variabilitas dalam citra dan adanya noise dapat mempengaruhi akurasi algoritma pengolahan citra. Interpretasi dan Subyektivitas: Interpretasi citra seringkali bersifat subyektif dan dapat bervariasi antara individu. Kehandalan dan Keamanan: Dalam aplikasi medis atau keamanan, kehandalan dan keamanan sistem pengolahan citra sangat penting. Kecepatan dan Efisiensi: Beberapa aplikasi memerlukan pengolahan citra secara real-time, sehingga kecepatan dan efisiensi algoritma sangat penting. Arah Masa Depan dalam Pengolahan Citra Digital: Pengolahan Citra 3D: Pengolahan citra 3D akan terus berkembang dalam berbagai aplikasi, termasuk pencitraan medis dan realitas virtual. Deep Learning dan AI: Penggunaan metode deep learning dan kecerdasan buatan akan terus diperluas dalam analisis citra, termasuk segmentasi dan pengenalan pola. Pengolahan Citra dalam AR dan VR: Pengolahan citra akan menjadi inti dalam pengalaman realitas virtual dan augmented, menghadirkan elemen-elemen digital yang realistis. Pengolahan Citra Medis yang Lebih Lanjut: Teknologi pengolahan citra akan terus digunakan dalam mendiagnosis penyakit secara lebih akurat dan tepat waktu. Kombinasi Data Multi-modal: Penggabungan data citra dengan data lain seperti teks dan sensorik akan memberikan informasi yang lebih kaya dan komprehensif. 76 Big Data dan Skalabilitas: Pengolahan citra pada data yang sangat besar (big data) memerlukan teknik yang efisien untuk menyimpan, mengakses, dan menganalisis citra dalam skala besar(Gonzalez et al., 2009). Pengolahan Citra 4D: Pengolahan citra dalam dimensi waktu (4D) semakin penting dalam aplikasi seperti pemantauan cuaca, pergerakan lalu lintas, dan pencitraan medis dinamis. Privasi dan Keamanan Citra: Dalam konteks pengenalan wajah dan identifikasi citra pribadi, tantangan privasi dan keamanan citra menjadi semakin signifikan. Interpretasi Konteks: Tantangan untuk mengembangkan algoritma yang dapat menginterpretasikan konteks citra, seperti lingkungan sekitarnya, untuk pengambilan keputusan yang lebih baik. Pengolahan Citra pada Perangkat Bergerak: Pengolahan citra pada perangkat mobile yang memiliki sumber daya terbatas memerlukan algoritma yang hemat daya dan berkinerja tinggi. Interaksi Manusia-Mesin: Pengembangan antarmuka manusia- mesin yang lebih intuitif untuk mengintegrasikan interaksi manusia dengan citra digital(Szeliski, 2022). Aplikasi AR 16.2 Arah Penelitian Masa Depan Arah Penelitian Masa Depan dalam Pengolahan Citra Digital: Deep Learning dan Neural Networks: Pengembangan arsitektur neural networks yang lebih kompleks dan metode deep learning yang lebih canggih untuk pengenalan pola dan segmentasi citra(Prince, 2012). Pengolahan Citra 3D dan 4D: Perkembangan teknik pengolahan citra untuk data citra dalam tiga dimensi (3D) dan dalam dimensi waktu (4D), terutama dalam bidang kedokteran, ilmu material, dan realitas virtual. Penggabungan Data Multi-modal: Penelitian untuk mengintegrasikan data citra dengan data dari berbagai sumber lain seperti teks, sensorik, dan data geospasial untuk analisis yang lebih holistik. 77 Privasi dan Keamanan Citra: Pengembangan teknik untuk melindungi privasi individu dalam citra serta mencegah manipulasi dan kebocoran citra. Privasi dan Keamanan Citra: Pengembangan teknik untuk melindungi privasi individu dalam citra serta mencegah manipulasi dan kebocoran citra. Realitas Virtual dan Augmented Reality (VR/AR): Penelitian dalam pengolahan citra yang mendukung pengalaman realitas virtual dan augmented yang lebih imersif dan realistis. Kecerdasan Buatan dalam Pengolahan Citra Medis: Penerapan kecerdasan buatan dalam diagnosis dan perawatan medis melalui analisis citra yang lebih akurat dan cepat. Pengolahan Citra pada Edge Devices: Penelitian tentang pengolahan citra pada perangkat edge seperti kamera pintar dan kendaraan otonom untuk aplikasi real-time. Interaksi Manusia-Mesin yang Lebih Lanjut: Pengembangan antarmuka manusia-mesin yang lebih canggih dan intuitif untuk berinteraksi dengan citra digital(Nixon & Aguado, 2019). 78 78 Daftar Pustaka Azuma, R. T. (1997). A survey of augmented reality. Presence: Teleoperators & Virtual Environments, 6(4), 355–385. Bankman, I. (2008). Handbook of medical image processing and analysis. Elsevier. Duda, R. O., Hart, P. E., & others. (2006). Pattern classification. John Wiley & Sons. 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Cambridge University Press. Qasim Gilani, S., Syed, T., Umair, M., & Marques, O. (2023). Skin Cancer Classification Using Deep Spiking Neural Network. Journal of Digital Imaging, 1–11. Sherman, W. R., & Craig, A. B. (2018). Understanding virtual reality: Interface, application, and design. Morgan Kaufmann. Sonka, M., Hlavac, V., & Boyle, R. (2014). Image processing, analysis, and machine vision: Cengage Learning. Inc,. Svoboda, T., Kybic, J., & Hlavac, V. (2007). Image processing, analysis & and machine vision-a MATLAB companion. Thomson Learning. Szeliski, R. (2022). Computer vision: algorithms and applications. Springer Nature. 80 80 Biodata Penulis Biodata Penulis Rohman Dijaya merupakan dosen tetap Fakultas Sains dan Teknologi, Universitas Muhammadiyah Sidoarjo yang mengampu beberapa mata kuliah diantaranya: Pengolahan Citra Digital, Grafika Komputer dan Kecerdasan Buatan. Putra pertama dari pasangan bapak Ambyah dan Ibu Rokhmah ini lahir di Gresik, 23 April 1990 yang mengawali kariernya sebagai praktisi Teknologi Informasi tahun 2010 – 2017 dan menjadi trainer dan dosen Teknik Informatika sejak 2016. Latar belakang pendidikan Penulis antara lain: S-1 Teknik Informatika, Universitas Muhammadiyah Gresik di Gresik (lulus tahun 2013). S-2 Teknik Informatika, Institut Teknologi Sepuluh Nopember Surabaya (lulus tahun 2016), dan S-3. Penulis terlibat dalam penelitian dan pengabdian kepada masyarakat baik didanai oleh Ristekdikti maupun dana mandiri tentang kecerdasan buatan dalam pengembilan keputusan atau otomatisasi serta teknologi informasi sebagai teknologi tepat guna dalam peningkatan masyarakat desa mandiri. Selain terlibat dalam penelitian dan pengabdian yang didanai oleh ristekDikti maupun Mandiri penulis juga mengabdikan diri dalam pendampingan kelompok masyarakat dilingkungan desa (mitra) Universitas Muhammadiyah Sidoarjo melalui kegiatan pengabdian masyarakat dosen ataupun mahasiswa melalui kegiatan Kuliah Kerja Nyata. 81
https://openalex.org/W4388125385	https://www.ijonmes.net/index.php/ijonmes/article/download/241/114	English	null	The Effect of The Art Education Status of Gifted Students on Their Professional Preferences	International journal of modern education studies	2,023	cc-by	9,317	\| Volume 7 - No 1 - June 2023 - ISSN 2618-6209 \| \| Volume 7 - No 1 - June 2023 - ISSN 2618-6209 \| Research Article Citation: Bağrıaçık, B., Emir, S.(2023).The Effect of The Art Education Status of Gifted Students on Their Professional Preferences.International Journal of Modern Education Studies, 7(1), 128-152. h //d i /10 51383/ij 2023 241 The Effect of The Art Education Status of Gifted Students on Their Professional Preferences Belgin BAĞRIAÇIK 1- Serap EMİR 2 Abstract: This study aims to investigate the effect of gifted students' art education on their professional preferences. The survey model, one of the quantitative research models, was used. A total of 320 students from the Çukurova Science and Art Center art field and general talent field, which were determined by the convenience sampling method, constitute the research sample group. The occupational preference inventory prepared by Atli and Kendal (2017) was used in the research. The inventory comprises six sub-dimensions: investigative, entrepreneurial, artistic, social, realistic, and traditional. Independent Sample t-Test and Anova Test were applied for the analyses, and Cohen's d and Eta Square tests were applied to calculate the effect level in meaningful data. As a result of the research on general talent and art students, it has been determined that there are significant differences in gender, school type, school level, and age. Special Talent, Vocational Choice, Art Education, Professional İnterest. Citation: Bağrıaçık, B., Emir, S.(2023).The Effect of The Art Education Status of Gifted Students on Their Professiona Preferences.International Journal of Modern Education Studies, 7(1), 128-152. 1 PhD, Adana Cukurova Science and Art Center, Adana Turkey. belginyuzgec@hotmail.com, Orcid ID: 0000-0001-7335-1432 1 PhD, Adana Cukurova Science and Art Center, Adana Turkey. belginyuzgec@hotmail.com, 2 Prof. Dr. Istanbul University- Cerrahpaşa, Education Faculty, İstanbul, Turkey. emirserap@gmail.com, Orcid ID: 0000-0001-7577-6012 Orcid ID: 0000-0001-7577-6012 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original authors and source are credited. International Journal of Modern Education Studies INTRODUCTION It is important both personally and socially that a person can express himself/herself, that they will not get bored of dealing with it throughout their life, and that he prefers a profession suitable for their personality. It is essential for the individual to choose a profession according to their existing abilities to benefit themselves and the society in which they live because it affects their economic level, social environment, living standard, job satisfaction, and job efficiency. For this reason, choosing a profession is seen as an important turning point in one's life. Selecting a profession that conflicts with the personality traits and interests of the individual negatively affects both their private life and professional life (Aydemir, 2018). Abstract: The reflection of the individual on their characteristics, interests, training, life experiences, and professional preferences will benefit both themselves and the society they live in (Miller & Cummings, 2009). Many methods are used to measure the interests of individuals. However, while one of the most preferred methods is inventory application, another is to ask a person directly about their interests. Although it is seen as a conventional method to determine the areas of interest in line with the answers received, it is also possible to reveal the interests by considering the social appreciation (dignity, prestige) against the attitudes related to the job and profession (Kuzgun, 2000). For this reason, inventory and scales should be used in determining interest, where both validity and reliability factors are considered (Herr, Cromer, & Niles, 2004). Holland's (1997) RIASEC professional interest model on identifying professional interests, cognitive abilities, and academic achievement is among the leading models (Vock, Köller, & Nagy, 2013). Dutch Model of Professional Interest The vocational Preference Inventory (VPI), initially developed as a short personality test, was primarily used to evaluate professional interests (Gottfredson, 1996). Holland collects the occupational choice inventory in six sub-dimensions. These dimensions were determined as "Realist," "Investigative," "Artistic," "Social," "Entrepreneurial," and "Conventional" (RIASEC). Each type is characterized by a constellation of interests, preferred activities, beliefs, abilities, values, and characteristics. A Holland code (typically the first letters of the RIASEC type that the person most closely resembles) is generated based on evaluations (Nauta, 2010). Realistic: The realistic dimension is associated with technical and mechanical skills, a dogmatic and practical approach to work, and an interest in working outdoors, with machines or hands. People in this group are more prone to take action than mental activities; they look for logical and concrete solutions while solving problems. They like nature, plants, and animals. Among the professions specific to this type, there are professions such as carpentry, agricultural technicians, and engineering. Researcher: The researcher includes scientific skills and interests, an intellectual and curious personality, and mathematics and research skills. People in this group enjoy experimenting and observing, researching abstract concepts, and solving problems using analysis synthesis steps. Profession groups that include physics, chemistry, biology, mathematics, and social sciences are among the professions specific to this type. Artistic: The artistic dimension is associated with innovative and creative features, interests, and skill sets in the arts, including visual and performing arts and creative writing. In addition to the fact that the people in this group have high imagination, developed creative aspects, and can produce original ideas, they have personality traits that work individually rather than in group work and do not like systematic and regular work. Among the professions specific to this type are departments that include fine arts, literature, theater, architecture, and cinema. Social: This area is characterized by a social and harmonious attitude, interest in helping others in areas such as teaching or counseling, and interpersonal skills. It can be said that the people in this group like to communicate with people and avoid being involved in mechanical work. As characteristics specific to this area, it is stated that individuals have more humane, sociable, benevolent, and human aspects. Professionals specific to this type include psychologists, teachers, and tourism guides. Dutch Model of Professional Interest Entrepreneur: The entrepreneurial dimension is characterized by an ambitious and dominant personality and leadership skills related to sales, law, and trade; extroverts affect those around them and attach importance to power and prestige. The people in this group have improved leadership characteristics, strong rhetoric, high persuasion skills, and energetic, ambitious, and extroverted characteristics. These individuals enjoy competition and taking risks very much. Occupations such as policy, operator, lawyer, and finance departments can be examples of this type of occupation group. Traditional: This area is characterized by a systematic and practical approach to work in general, strong office and organizational ability, and conservative values. Individuals have a more traditionally planned structure with certain rules, and enjoy routine work. At the same time, they are individuals with limited imagination, regular, meticulous, and enjoy dealing with numerical data. Among the professions specific to this type, jobs such as accountants, bankers, and office clerks can be given as examples (Holland, 1997). The closer the types of people are to each other, the higher the harmony between their profession and personality is. While adjacent types of features are called adaptive (e.g., realistic versus traditional; entrepreneur versus social), opposing areas are also referred to as maladaptive (e.g., traditional versus artistic; researcher versus entrepreneur). In addition, according to this theory, people's success in their careers depends on the harmony between their professions and their personalities (Kamaşak and Bulutlar, 2010: 122). 130 130 Figure 1. Holland's Occupational Choice Hexagon Figure 1. Holland's Occupational Choice Hexagon Source: (Kamaşık and Bulutlar, 2010: 122) Source: (Kamaşık and Bulutlar, 2010: 122) Source: (Kamaşık and Bulutlar, 2010: 122) Source: (Kamaşık and Bulutlar, 2010: 122) Even if the individual's professional preferences vary at every age and in every period, their interests or areas of talent play an active role in the profession they want to choose. Many studies have been conducted on occupational preferences from the primary school to university (Flexer, 2008; Bezanson, 2003; Can&Taylı, 2014). Early identification of individuals' interests and orientations is important regarding professional preferences, national added value, social benefit, and individual satisfaction. In particular, it will be beneficial for society to determine the interests of individuals whose particular talent areas are more advanced than their peers and to receive training in this direction. These students are considered more successful than their peers (Gagne, 2003; Sternberg, Jarvin, & Grigerenko, 2010), and the education they need may also differ (Kaya, Ogurlu, & Hizli, 2017). Special, talented individuals differ from their peers with their metacognitive characteristics (Kail, 2000), intellectual development (Achter, Lubinski, Benbow & Eftekhari-Sanjani, 1999), their ability to comprehend and combine academic and emotional knowledge to solve problems (Gottfredson, 2003). For this reason, they begin to think about their careers earlier than their peers (Kerr and Sodano, 2003; Silverman, 1993). In terms of education and career development, the gifted individual is confronted at a younger age than their peers with the issue that their preferences (i.e., interests and values) are sufficient to produce mature, valid information and can evaluate this information and can help clarify the current complexity (Schmidt, Lubinski, & Benbow, 1998). Some researchers state that intellectually gifted and highly successful students differ from their peers in terms of their intellectual abilities and professional preferences (Stapf, 2003). Specially talented individuals will carry their current potential to the highest level with the training they need in areas or areas for their outstanding talents. When determining the fields of verbal, mathematical, visual, or auditory ability, it is crucial that they have a tendency and interest in the areas (Chen and Wong, 2013). Of course, talent alone is insufficient for positive and successful vocational training development. However, it is crucial to choose a profession that matches their interests, needs, abilities, and personal life. (Lofquist & Dawis, 1991; Lubinski & Benbow, 2000). Figure 1. Holland's Occupational Choice Hexagon In particular, the process is central to success, professional interests, and decision-making for the selected job and post-professional satisfaction. (Gottfredson, 1996). While making a professional choice, the individual is expected to consider their ability in the areas they are interested in and enjoys working in (Chen and Wong, 2013). Previous studies have determined factors such as gender differences (Ferriman, Lubinski, and Benbow, 2009; Kerr and Sodano, 2003) and high potentiality (Achter vd., 1999; Silverman, 1993) affect the occupational preferences of exceptionally talented individuals. Her education, culture, environmental expectations and interests, and abilities are active in her career choices. In addition to determining the individual's area of interest, receiving an education in that area of interest will enable them to develop their interest and ability. With the art education of gifted individuals who are prone to the field of art, it was observed that they developed self-confidence (Bayav, 2007; Koca, 2007), self- esteem (Barış, 2002; Toy, 2006), social skills (Barış, 2008), creative thinking skills (Zimmerman, 2009; Köse, 2006; Keser, 2019), visual perception skills (Carroll, 1987), environmental awareness (Durmuş, 2009) and entrepreneurship characteristics (Mohamed Helmy Elfiel, 2019). Science and Art Centers (BİLSEM) have been established in our country to provide specially talented individuals with the education they need for their field of interest. Individuals with high artistic skills are educated in the areas of "Visual Arts" and "Music," while individuals with interest and skills in mathematics, science, and social sciences are educated in the field of "General Talent" (MEB, 2016). Although, there are studies indicating that the education they receive in BİLSEM affects children's personality development and their skill and talent development in the field they are related to (Sözel, 2019). In the literature review, there are many studies to determine the professional preferences of individuals with unique talents (Kher-Durlabhji, Lacina-Gifford, Carter, & Lalande, 1997; Kara 2019), the problems they encounter when determining their professional preferences (Kaya, Ogurlu, & Hizli, 2017; Bostan, Bostan, Öztürk &Öztürk, 2020;.), the guidance needed when determining a career (Yusof, Mokhtar, Sulaiman & Mohtar, 2020; Chen & Wong, 2013), the change of professional preferences over the years (Schmidt, Lubinski, & Benbow, 1998), or to investigate the differences between individuals with unique talents and their peers with normal development (Miller & Cummings, 2009; Vock, Köller & Nagy, 2013).However, no study has been found on the effect of the art education status of specially talented individuals on their professional preferences. The Aim of the Study The individual's basic skills, characteristics, abilities, and equipment should be considered in occupational preferences. To determine these characteristics of the individual, it was aimed to give an idea to exceptionally talented individuals about their professional 132 132 preferences by using the Holland Professional Choice inventory and to determine whether their status of receiving art education from these individuals affects their preferences. For this purpose, answers to the following questions will be sought; preferences by using the Holland Professional Choice inventory and to determine whether their status of receiving art education from these individuals affects their preferences. For this purpose, answers to the following questions will be sought; 1. Is there a significant difference in the occupational preferences of gifted students according to the field they are diagnosed with? 2. Is there a significant difference in the professional preferences of gifted general and art talent students according to gender? 3. Is there a significant difference in the professional preferences of gifted general and art talent students according to their school levels? 4. Is there a significant difference in the professional preferences of gifted general and art talent students according to age? 5. Is there a significant difference in the professional preferences of gifted general talent students and gifted art talent students according to the type of school? 5. Is there a significant difference in the professional preferences of gifted general talent students and gifted art talent students according to the type of school? METHOD Research Method: The survey model, one of the quantitative research models, was used. According to Karasar (2004), screening models aim to describe a past or present situation as it is. The event, person, or object subject to the research is tried to be defined as it is under its conditions, and no effort is made to change or affect them in any way. Participants The convenience sampling method was used in the study. The appropriate sampling method is explained as the selection of the sample from the unit that can be easily applied due to time and labor limitations (Büyüköztürk et al., 2009). A total of 320 students ranging from fifth to twelfth-grade students studying general talent, music, and visual arts at the Adana Science and Art Center make up the study's sample group.35% (112) of the students participating in the study were female in the field of art (SA), 23.1% (74) were female in the field of general talent (GYA), 15% (48) were male in the area of art (SA), and 26.9% (86) were male in the field of general talent (GYA). Table.1 Traditional: A systematic and practical approach to work is characterized by solid bureau and organizational capability and conservative values. The scale consists of 30 items, and a 9-point Likert rating was used. The Cronbach Alpha value of the scale varies between .65 and .85. In the studies using this scale, Cronbach's alpha value was found to be .72 (Bostan, Bostan, Öztürk &Öztürk, 2020). In this study, .78 for the realistic sub-dimension, .81 for the researcher sub-dimension, .74 for the artist sub- dimension, .86 for the social sub-dimension, .84 for the entrepreneur sub-dimension, and .84 for the traditional sub-dimension. In this study, Cronbach's alpha value was found to be 82. Table.1 133 Demographic Characteristics of the Sample Group Area of Diagnosis Female Male Total f % f % f % Artspace 112 35,0 48 15.0 160 50.0 Demographic Characteristics of the Sample Group Area of Diagnosis Female Male Total f % f % f % Artspace 112 35,0 48 15.0 160 50.0 Demographic Characteristics of the Sample Group General Capability Area 74 23.1 86 26,9 160 50.0 Total 186 58.1 134 41.9 320 100 General Capability Area 74 23.1 86 26,9 160 50.0 Total 186 58.1 134 41.9 320 100 In the first part, the researcher creates a personal information form. This form consists of gender, school type, school level, and age variables. The occupational preference inventory prepared by Atli and Keldal (2017) was used. In the inventory, there are sub-dimensions that determine the 6-person type of realistic, researcher, artistic, social, entrepreneurial, and traditional personality. Realistic: Technical and mechanical skills are characterized by a dogmatic and practical approach to work and an interest in working outdoors, with machines or hands. Realistic: Technical and mechanical skills are characterized by a dogmatic and practical approach to work and an interest in working outdoors, with machines or hands. Researcher: Scientific skills and interests are characterized by an intellectual and curious personality and mathematical and research abilities. Researcher: Scientific skills and interests are characterized by an intellectual and curious personality and mathematical and research abilities. Artistic: It is characterized by creative and creative features and a set of interests and skills in the arts, including visual and performing arts and creative writing. Artistic: It is characterized by creative and creative features and a set of interests and skills in the arts, including visual and performing arts and creative writing. Social: A social and harmonious attitude is characterized by an interest in helping others in areas such as teaching or counseling and interpersonal skills. Social: A social and harmonious attitude is characterized by an interest in helping others in areas such as teaching or counseling and interpersonal skills. Entrepreneur: Characterized by an extroverted, ambitious, and dominant personality and leadership skills in areas of interest in sales, law, and commerce. Entrepreneur: Characterized by an extroverted, ambitious, and dominant personality and leadership skills in areas of interest in sales, law, and commerce. Traditional: A systematic and practical approach to work is characterized by solid bureau and organizational capability and conservative values. Data Collection and Analysis Personal information forms and scales were prepared on the form and applied to students, and the data used in the study were obtained. Before the inventory was applied, the researcher created the informative text containing the necessary explanations, and volunteerism was taken as a basis for the study participation. Students were asked to fill in International Journal of Modern Education Studies the items in the inventory according to the most appropriate option. It took a student approximately 20-30 minutes to complete the inventory. Before the study, the number of participants was determined by applying the G Power power analysis test. The statistical analyses to be used in light of the data collected from the participants were determined. A normality test was applied to examine the distribution of the data to determine the analysis of the relationship between the art education status of gifted students and their professional preferences. Correlation analysis was performed with Frequency Distribution Test, Independent Sample t-test, and ANOVA test in normally distributed data. Cohen d and Eta Square values were calculated to determine the effect level. Ethical consideration In this study, all rules stated to be followed within the "Higher Education Institutions Scientific Research, and Publication Ethics Directive" scope was observed. None of the actions stated under the title "Actions Against Scientific Research and Publication Ethics," which is the second part of the directive, was not taken. Ethical review board name: Istanbul University Cerrahpaşa Scientific Publication Ethics Board Date of ethics review decision: 27.05.2022 Ethics assessment document issue number: 2022/131 Ethics assessment document issue number: 2022/131 FINDINGS This section includes the findings and comments obtained by analyzing the research questions determined. Table.2 Analysis Table Regarding the Field of Diagnosis and Professional Preferences of Especially Talented Students Art Field Realistic Investigator Artistic Social Enterprising Conventional R I A S E C N 160 160 160 160 160 160 𝜒 7.33 8.12 9.50 8.54 4.93 3.38 ss 3.62 3.81 3.29 3.53 3.24 3.02 General Talented Field N 160 160 160 160 160 160 𝜒 7.42 8.84 7.66 8.36 4.97 3.99 ss 3.63 3.88 3.81 3.65 3.30 3.42 s Table Regarding the Field of Diagnosis and Professional Preferences of Especially Talented Analysis Table Regarding the Field of Diagnosis and Professional Preferences of Especially Students 135 According to the results of the analysis made for the first research question, when the field of special talented students' diagnosed and their occupational preferences were examined, it was found that the scoring average of the students in the area of art was high in the artistic, social and research sub-dimensions, and the scoring average of the students in the field of general talent was high in the researcher, social and artistic sub-dimensions (Table.2). Table. 3 e. 3 va Test Results Regarding Gender and Occupational Preferences of Especially Talented Student Anova Test Results Regarding Gender and Occupational Preferences of Especially Talented Students Anova Test Results Regarding Gender and Occupational Preferences of Especially Talented Students Variable Source of Variance Sum of Squares sd Mean of Squares F p \|Ƞ2 Difference Realistic Between Groups 18.948 4 6,316 0.480 .696 In-group Total 4155.799 4174.747 316 320 13,151 Investigator Between Groups 120.234 4 40.078 2,740 0.043 .024 GTF Girl>AF Girl>GTF Boy>AF Boy In-group Total 4621.654 4741.888 316 320 14.625 Artistic Between Groups 674.327 4 .776 19.613 .000 .157 AF Girl>GTF Girl>AF Boy>GTF Boy In-group Total 3521.560 4295.888 316 320 11.461 Social Between Groups 75.481 4 25.160 1.976 .117 In-group Total 4023.719 4099.200 316 320 12.733 Enterprising Between Groups 2.001 4 0.667 0.062 .980 In-group Total 3393.199 3395.200 316 320 10.738 Conventional Between Groups 95.370 4 31.790 3.097 ,027 .029 GTF Boys>AF Boys>GTF Test Results Regarding Gender and Occupational Preferences of Especially Talented Students 136 In-group Total 3244.117 3339.488 316 320 10,266 Girls>AF Girls Table. 4 Descriptive Statistical Table on Gender and Occupational Preferences of Special Ability Students Gender N 𝜒 ss Art Field Girl 112 8.46 3.827 Researcher General Talented Field Girl 74 9.34 3.833 Art Field Male 48 7.31 3.685 General Talented FieldMale 86 8.42 3.888 TOTAL 320 8.48 3.855 Art Field Girl 112 10.11 3.085 Artistic General Talented Field Girl 74 9.05 3.789 Art Field Male 48 8.08 3.338 General Talented Field Male 86 6.47 3.419 TOTAL 320 8.58 3.670 Art Field Girl 112 3.21 2.749 ConventionalGeneral Talented Field Girl 74 3.35 3.345 Art Field Male 48 3.75 3.570 General Talented Field Male 86 4.53 3.412 TOTAL 320 3.68 3.236 Table. 4 e Statistical Table on Gender and Occupational Preferences of Special Ability Students There was no significant difference in the professional preferences of gifted General Talent Field (GTF) students and gifted Art Field (AF) students according to gender in realistic, social and entrepreneurial sub-dimensions (p>.05). In the researcher sub- dimension according to gender in the professional preferences of gifted general talent students and gifted artfield students,024) (p<.05, Table.3). According to the Post Hoc test, this significance was found to be high between the researcher score average of the female students with GTF (© χ= 9.34)and the research score average of the female students with AF (8.46); high between the research score average of the female students with AF (8.46)and the research score average of the male students with GTF (8.42); high between the research score average of the male students with GTF (8.42) and the research score average of the male students with AF (7.31) (Table.4). Again, when the artistic sub-dimension is examined, it is seen that there iAF moderate ( .157). This difference was found to be high between the artistic score average of AF female students (© χ= 10.11) and the artistic score average of AF female students (9.05); high between the artistic score average of AF female students (9.05) and the artistic score average of AF male students (8.08); high between the artistic score average of AF male students (8.08) and the artistic score average of AF male students (6.47) (Table.4). In addition, in the traditional sub-dimension, there is a weak level (.02=029). According to this difference; AF female students' artistic score average (© χ= 10.11) and GTF female students' artistic score average (149.05) were found to be high; AF female students' artistic score average (159.0516) and GTF male students' artistic score average (178.08) were found to be high;19 AF male student' artistic score average (8.08) GTF male students' artistic score average (186.47) were found to be high (Table 4). Table. 5 Anova Test Results Regarding School Level and Professional Preferences of Special Talented Students Variable Source of Variance Sum of Squares sd Mean of Squares F p Ƞ2 Difference Realistic Between Groups 28.739 4 9.580 0.730 .535 In-group Total 4146.008 4174.747 316 320 13,120 Investigator Between Groups 164,504 4 54.835 3,786 .011 0,072 GTF Secondary School>AF Secondary School>GTF High School >AF High School In-group Total 4577.887 4741.887 316 320 14.485 Artistic Between Groups 356.201 4 118,734 9.524 .000 .074 AF High School >AF Secondary School>GTF Secondary School>GTF High School In-group Total 3939.687 4295.888 316 320 12.467 Social Between Groups 21.465 4 7.155 0.554 645 In-group Total 4077.735 4099.200 316 320 12.904 Anova Test Results Regarding School Level and Professional Preferences of Special Talented St 138 Enterprising Between Groups 142,194 4 47.389 4604 .004 .033 AF High School >GTF Secondary School>AF Secondary School>GTF High School In-group Total 3253.006 3395.200 316 320 10.294 Conventional Between Groups 61.602 4 20.534 1.980 .117 In-group Total 3277.885 3339.487 316 320 10.373 Table. 6 Descriptive Statistical Table on School Level and Professional Preferences of Special Talented Students Score School Level N 𝜒 sd Investigator AF Secondary School 131 8.40 3.745 GTF Secondary School 124 9.15 3.742 AF High School 26 6.58 3.818 GTF High School 39 7.90 4.191 TOTAL 320 8.48 3.855 Artistic AF Secondary School 131 9.26 3297 GTF Secondary School 124 8.20 3.788 AF High School 26 10.35 3.013 GTF High School 39 6.33 4.038 TOTAL 320 8.58 (3,670) Enterprising AF Secondary School 131 4.59 3.108 GTF Secondary School 124 5.40 3.368 AF High School 26 6.35 3.286 GTF High School 39 3.82 2.955 TOTAL 320 4.95 3.262 There was no significant difference in the professional preferences of gifted General Talent Field (GTF) students and gifted Art Field (AF) students in realistic, social and traditional sub-dimensions according to the school level (p>.05). \|According to the school tive Statistical Table on School Level and Professional Preferences of Special Talented ts There was no significant difference in the professional preferences of gifted General Talent Field (GTF) students and gifted Art Field (AF) students in realistic, social and traditional sub-dimensions according to the school level (p>.05). \|According to the school level, there is a weak level in the researcher sub-dimension in the professional preferences of gifted general talent students and gifted art field students (.02=072) (p<.05, Table.5). Table. 5 According to the Post Hoc test, this significance was found to be high between the researcher score average of the secondary school students with GTF (χ= 9.15) and the research score average of the secondary school students with AF (8.40); high between the research score average of the secondary school students with AF (8.40)and the research score average of the high school students with GTF (7.90); high between the research score average of the high school students with GTF (7.90) and the research score average of the high school students with AF () (Table.6). Again, when the artistic sub-dimension is examined, it is seen that there iAF low level of 074). This difference was found to be high between the artistic score average of AF high school students (χ= 10.35) and the artistic score average of AF secondary school students (9.26); high between the artistic score average of AF secondary school students (9.26) and the artistic score average of GTA secondary school students (8.20); high between the artistic score average of GTF secondary school students (8.20) and the artistic score average of GTF high school students (6.33) (Table.6). In addition, a low level of significance was found in the entrepreneur sub-dimension (p<.05, Table.6). Accordingly, it was determined that the mean entrepreneurial score of AF high school students was high (χ= 6.35) and the meanentrepreneurial score of GTF secondary school students was high (5.40); the mean entrepreneurial score of GTF secondary school students was high (5.40) and the mean entrepreneurial score of AF secondary school students was high (4.59); the mean entrepreneurial score of AF secondary school students was high (4.59) and the mean entrepreneurial score of GTF high school students was high (3.82) (Table.6). Table. 7 Anova Test Results Regarding the Age Levels and Professional Preferences of Especially Talented Students Variable Source of Variance Sum of Squares sd Mean of Squares F p Ƞ2 Difference Realistic Between Groups 77.511 6 37.483 1.188 .315 .045 In-group Total 4097.236 4174.747 314 320 12.699 Investigator Between Groups 188.834 6 37,767 2.605 ,025 .062 GTF 12-14 Years>GTF 10-11 Years>AF 10-11 Years>AF 12-14 Years>GTF 15-17 Years>AF In-group Total 4553.053 4741.888 314 320 14,500 va Test Results Regarding the Age Levels and Professional Preferences of Especially Talented dents Anova Test Results Regarding the Age Levels and Professional Preferences of Especially Students 140 15-17 Years Artistic Between Groups 384.194 6 76.839 6.168 .000 .079 AF 15-17 Years old>AF 12-14 Years old>AF 10-11 Years old>GTF 12-14 Years old>GTF 10-11 Years old>GTF 15-17 Years old In-group Total 3911.694 4295.888 314 320 12,458 Social Between Groups 18.618 6 3.724 0.287 .920 In-group Total 4080.582 4099.200 314 320 12.995 Enterprising Between Groups 112.160 6 22.432 2.145 .060 In-group Total 3283.040 3395.200 314 320 10.456 Conventional Between Groups 83.411 6 16.682 1.609 .157 In-group Total 3256.076 3339.487 314 320 10,370 Table. 8 Descriptive Statistical Table on Age Levels and Occupational Preferences of Special Ability Students Score Age Level N 𝜒 sd AF 10-11 Years 68 8.71 3.856 GTF 10-11 Years 86 8.90 3,505 AF 12-14 Years 56 8.02 3.430 Years old>GTF 12-14 Years old>GTF 10-11 Years old>GTF 15-17 Years old Social Between Groups 18.618 6 3.724 0.287 .920 In-group Total 4080.582 4099.200 314 320 12.995 Enterprising Between Groups 112.160 6 22.432 2.145 .060 In-group Total 3283.040 3395.200 314 320 10.456 Conventional Between Groups 83.411 6 16.682 1.609 .157 In-group Total 3256.076 3339.487 314 320 10,370 Table. 8 Descriptive Statistical Table on Age Levels and Occupational Preferences of Special Ability Students Score Age Level N 𝜒 sd Investigator AF 10-11 Years 68 8.71 3.856 GTF 10-11 Years 86 8.90 3,505 AF 12-14 Years 56 8.02 3.430 GTF 12-14 Years 36 9.86 4.223 Social Between Groups 18.618 6 3.724 0.287 .920 In-group Total 4080.582 4099.200 314 320 12.995 Enterprising Between Groups 112.160 6 22.432 2.145 .060 In-group Total 3283.040 3395.200 314 320 10.456 Conventional Between Groups 83.411 6 16.682 1.609 .157 In-group Total 3256.076 3339.487 314 320 10,370 Table. Table. 7 8 Descriptive Statistical Table on Age Levels and Occupational Preferences of Special Ability Students Score Age Level N 𝜒 sd Investigator AF 10-11 Years 68 8.71 3.856 GTF 10-11 Years 86 8.90 3,505 AF 12-14 Years 56 8.02 3.430 GTF 12-14 Years 36 9.86 4.223 Table. 8 Descriptive Statistical Table on Age Levels and Occupational Preferences of Special Ability Students Score Age Level N 𝜒 sd Investigator AF 10-11 Years 68 8.71 3.856 GTF 10-11 Years 86 8.90 3,505 AF 12-14 Years 56 8.02 3.430 GTF 12-14 Years 36 9.86 4.223 tical Table on Age Levels and Occupational Preferences of Special Ability Students AF 15-17 Years 35 7.00 4.109 GTF 15-17 Years 39 7.90 4.191 TOTAL 320 8.48 3.855 Artistic AF 10-11 Years 68 9.21 3.258 GTF 10-11 Years 86 7.97 3.546 AF 12-14 Years 56 : 9.45 3.379 GTF 12-14 Years 36 8.44 4.212 AF 15-17 Years 35 10.14. (3.246) GTF 15-17 Years 39 6.33 3.716 TOTAL 320 8.58 (3,670) As an answer to another research question, no significant difference was found in the professional preferences of gifted General Talent Field (GTF) students and gifted Art Field (AF) students according to their age level in realistic, social, entrepreneurial and traditional sub-dimensions (p>.05). According to the school level, there is a weak level in the researcher sub-dimension in the professional preferences of gifted general talent students and gifted art field students (.02=072) (p<.05, Table.7). According to the Post Hoc test, this significance was found to be high between the researcher score average of the students aged 12-14 years (© χ= 9.86) and the research score average of the students aged 10-11 years (); high between the research score average of the students aged 10-11years (8.90) and the research score average of the students aged 10-11 years (8.71); high between the research score average of the students aged 10-11 years (8.71) and the research score average of the students aged 12-14 years (); high between the research score average of the students aged 12-14 years (8.02) and the research score average of the students aged 15-17 years (); high between the research score average of the students aged 15-17 years (7.90) and the research score average of the students aged 15-17 years (Table).8). Again, when the artistic sub-dimension is examined, it is seen that there is a low level of. Table. 8 This difference was found to be high between the artistic score average of AF 15-17 years old students (© χ= 10.14) and the artistic score average of AF 12-14 years old students (9.45); high between the artistic score average of AF 12-14 years old students (9.45) and the artistic score average of AF 10-11 years old students (9.21); high between the artistic score average of AF 10-11 years old students (9.21) and the artistic score average of AF 10-11 years old students (8.44); high between the artistic score average of AF 12-14 years old students () and the artistic score average of GTF 10-11 years old students (7.97); high between the artistic score average of GTA 10-11 years old students (7.97) and the artistic score average of GTF 15-17 years old students (6.33) (Table).8). 142 Table.9 Table.9 Anova Test Results Regarding the Type of School and Professional Preferences of Special Talented Students Variable Source of Variance Sum of Squares sd Mean of Squares F p \|Ƞ2 Difference Realistic Between Groups 39.063 4 13.021 (0.995 ) 395 In-group Total 4135.684 4174.747 316 320 13088 Investigator Between Groups 89.894 4 29.965 2.035 .109 In-group Total 4651.993 4741.888 316 320 14.721 Artistic Between Groups 277.176 4 92.392 7.265 .000 .065 AF Private School>AF Public School>GT F Private School>GT F Public School In-group Total 4018.711 4295.888 316 320 12.717 Social Between Groups 22.617 4 7.539 0.584 .626 In-group Total 4076.583 4099.200 316 320 12.901 Enterprising Between Groups 29.275 4 9.758 0.916 0.433 In-group Total 3365.925 3395.200 316 320 10.652 Conventional Between Groups 49.640 4 16.547 1.589 192 In-group Total 3289.847 3339.487 316 320 10.411 t Results Regarding the Type of School and Professional Preferences of Special Talented Table. 10 Table. 10 Descriptive Statistical Table on School Type and Professional Preferences of Special Talented Students Score School Type N 𝜒 sd Artistic state school 75 9.41 3.133 state school 70 7.44 3.933 AF Private School 85 9.58 3.434 GTF Private School 90 7.83 3.724 TOTAL 320 8.58 (3,670) Descriptive Statistical Table on School Type and Professional Preferences of Special Talented Students As an answer to the last research question; no significant difference was found in the professional preferences of gifted General Talent Field (GTF) students and gifted Art Field (AF) students according to school type; realistic, researcher, social, entrepreneurial, and traditional sub-dimensions (p>.05, Table.9). However, when we look at the artistic sub- dimension, it is seen that there is a low level of 074). This difference was found to be high between the artistic score average of the AF private school students (©χ= 9.58) and the artistic score average of the AF public school students (9.41); high between the artistic score average of the AF public school students (9.41)and the artistic score average of the GTF private school students (7.83); high between the artistic score average of the GTF private school students (7.83) and the artistic score average of the GTF public school students (7.44) (Table 10). Conclusions Regarding the Research Question Science and Art Centers are the leading institutions that provide education to gifted students in our country. In these institutions, the student receives training only in the field they are diagnosed in. While students diagnosed in the area of General Talent are studying in the fields of social sciences, science, and mathematics, students diagnosed in art receive only music or visual arts education. Considering the field of diagnosis and professional interests of these students, it was seen that the mean score of the students in the field of art was high in the artistic, social, and research sub-dimensions, and the mean score of the students in the area of general talent was high in the researcher, social and artistic sub- dimensions. When the results are examined, it is seen that there are common sub- dimensions in both areas. The fact that gifted students are curious, research-loving, questioning, and sensitive to their environment and the society they live in is compatible with the researcher and social sub-dimension, and the fact that they are sensitive, idealistic, aesthetic, emotional, introverted, and creative is compatible with the artistic sub-dimension (Yusof, Mokhtar, Sulaiman and Mohtar, 2020). On the other hand, the study showed that the entrepreneurship sub-dimension had the lowest mean score in both areas. Similar results in the literature. CONCLUSION AND DISCUSSION In studies investigating the effect of art education on the individual, there are three different opinions cognitive approach, psychological approach, and self-developmental approach. While the cognitive approach explains the use of art in the evaluation of children's knowledge about the changing world, the psychological approach explains art as the reflection of the inner worlds of individuals. In the third approach, art education is where individuals establish a relationship with the society they are in, understand their self- development, and become a tool for communicating with society (Zimmerman and Zimmerman, 2000). Art aims to reveal the meaning in the content of the work, not to define it by the appearance of the resulting products. Art stimulates entrepreneurship and independence feelings in the individual. The work of art is expected to be original. Art education aims to educate tastes and emotions, to create a perspective towards a beautiful work, and to give an aesthetic view to every stage of daily life. Art should be included in the educational process of each individual starting from preschool, not only for being a profession but also International Journal of Modern Education Studies with or without special abilities (Aral, 1999). Interest and skill areas and life experiences play a significant role in the professional preferences of individuals. If the individual realizes these areas early, it will also make it easier for him/her to direct their life. The gifted individual begins to research the profession they want to do before their peers (Schmidt, Lubinski, & Benbow, 1998). They need differentiated or enriched education to develop their areas of interest. Providing these children with the education they need from an early age is seen as a national gain (Madeja, 1983). Even if the field in which the child is diagnosed is different, their education about any of the branches of art during their education will provide them with an aesthetic perspective, and socialization, explore the entrepreneurial spirit, and develop their artistic ability, if any (Hurwitz, 1983). Conclusions Regarding the Research Question When examined according to genders, it was seen that in the researcher sub- dimension, female students in the field of general talent had the highest mean score, followed by female students in the field of art, male students in the field of general talent and male students in the field of art, respectively. In their study, Webb ve diğ(2002) found that gifted female students had more investigative characteristics than male students. In another dimension of art, female students in the art field have the highest mean score. In contrast, the mean scores of female students in the general talent field, male students in the art field and male students in the general talent field are observed, respectively. In the traditional sub-dimension, the highest average score of male students in the general talent area is seen. Then, the art field is listed for male students, the general talent field as female students, and the art field as female students. Conclusions Regarding the Research Question According to the school level, the mean scores of secondary school students in the field of general talent, secondary school students in the field of art, high school students in the field of art, and high school students in the field of general talent are ranked in the research sub-dimension. Similarly, when the age levels were examined, it was determined that the students between the ages of 12-14 had the highest general ability, and the students between the ages of 15-17 had the lowest average score in the research sub-dimension. It is seen that as the students' school level or age level increases, there is a decrease in the direction of the researcher. Future anxiety is gradually moving away from the questioning student model due to the anxiety of being able to settle in the university (Kumandaş and Kutlu, 2014). In the artistic sub-dimension, the average score of high school students in art is the highest. Afterward, secondary school scores in the field of art, a secondary school in the field of general talent, and high school scores in general talent are listed. The artistic score of the student receiving art education increases, and the artistic score of the student who does not receive art education decreases as the school level increases. The student's art education changes their creativity, imagination, and perspective towards events as well as the development of their artistic ability (Zimmerman, 2009). The highest average score in the entrepreneurship sub-dimension is seen in high school students in the art field. Then, the mean scores of secondary school students in general talent, secondary school students in the art field, and high school students in the general talent field are respectively followed. In his study, Shavinina (2008) defined the characteristics of the gifted entrepreneurial individual as; innovative, creative, capable of working independently, not afraid of difficulties, perfectionist, and not like to be bound by rules. On the other hand, Lena and Lindemann (2014) defined the artist as a creative, perfectionist person who enjoys working independently and producing original works. It can be interpreted that the state of the gifted student's art education affects the entrepreneurship aspect. Conclusions Regarding the Research Question In the artistic sub-dimension, it was determined that the student between the ages of 15-17 had the highest mean score, and the student between the ages of 15-17 had the lowest mean score. According to the "maturation theory" developed by Arnold Gesell et al., children are born with some innate abilities, and their abilities begin to emerge as they mature. (Ulutaş, Ersoy, 2004). In this process, education is of great importance. If a suitable environment is created for the child and the right people can guide them, they can develop their talent. These are intense feelings of aesthetics and creativity. Supporting it in the early period will be productive, creative individuals who understand and perceive the beauties in the environment (Feeney and Moravcik, 1987). Lowenfield (1947) and Gardner (1980) reported that when adults are not given the necessary support and equipment, their artistic 146 International Journal of Modern Education Studies abilities will be blunted, and at the same time, their inner skills will be lost if they intervene too much about the product offered by an adult (Ulutaş, Ersoy, 2004). RECOMMENDATIONS Based on the survey results; Based on the survey results; • Investigation of the factors underlying the vocational choices of gifted students, the overlap between the professions they want to choose and their interests, and the interests and professional preferences of students graduating from Science and Art Centers, • Organizing information seminars for gifted students on professional promotion days and what they should pay attention to in their professional preferences, • Organizing training on the professional preferences of exceptionally talented students for their teachers and parents, • It is recommended that art education courses be held by art teachers in public schools affiliated with the Ministry of National Education. Conclusions Regarding the Research Question Significant differences were observed only in the artistic sub-dimension when the school types of gifted students were examined. Accordingly, private school students diagnosed from the art field with the highest average score, then the average score of public school students diagnosed from the art field, the average score of private school students diagnosed from the general talent field, and the average score of students going to the diagnosed public school from the general talent field, respectively. It is thought that one of the reasons for this difference is that art lessons are taught by classroom teachers at the primary school level in public schools affiliated with the Ministry of National Education, and art lessons are taught by art teachers in private schools. Students attending the Science and Art Center support the elimination of deficiencies by receiving the art education they need in these centers, whether their formal education is private or public school. 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Zimmerman, E. and Zimmerman, L. (2000). Art education and early childhood education: the young child as creator and meaning maker within a comminity context. Young Children, November; 87-92. Zimmerman, E. and Zimmerman, L. (2000). Art education and early childhood education: the young child as creator and meaning maker within a comminity context. Young Children, November; 87-92. Zimmerman, E. and Zimmerman, L. (2000). Author(s)’ statements on ethics and conflict of interest Ethics statement: We hereby declare that research/publication ethics and citing principles have been considered in all the stages of the study. We take full responsibility for the content of the paper in case of dispute. Ethics statement: We hereby declare that research/publication ethics and citing principles have been considered in all the stages of the study. We take full responsibility for the content of the paper in case of dispute. p p p Statement of interest: We have no conflict of interest to declare. p p p Statement of interest: We have no conflict of interest to declare. Funding: None Acknowledgements: None
https://openalex.org/W4365516842	https://nguhist.elpub.ru/jour/article/download/1440/947	Russian	null	Guizhou Ethnic Groups in the Qing Dynasty Miao Albums: Miao, Yizu, Gelao	Vestnik Novosibirskogo gosudarstvennogo universiteta. Seriâ: istoriâ, filologiâ	2,023	cc-by	7,153	Аннотация ц Исследование ставит своей целью оценить значимость сведений о народах Гуйчжоу из цинских «альбомов об инородцах» в собрании научной библиотеки Санкт-Петербугского университета (НБ СПбГУ) для исследова- ния истории крупных этнических групп провинции – мяо, ицзу и гэлао. В одном альбоме из собрания НБ СПбГУ есть 72 описания с иллюстрациями, в другом описаний с иллюстрациями сохранилось 28. В них дано описание нескольких десятков групп мяо, нескольких групп ицзу, буи и гэлао, но отрывочные данные об истории можно найти только в разделах об ицзу и в отдельных случаях о мяо. Исследование показывает, из каких источников, начиная с эпохи Хань, заимствовались сведения в «альбомы», а также расширяет и до- полняет представленную в них информацию. Альбомы ценны своими изображениями обычаев народов Гуй- чжоу, а также заметками этнографического характера, а не информацией об истории различных этнических групп. К ру Ключевые слова «альбомы об инородцах», эпоха Цин, этнография, Гуйчжоу, источниковедение, ицзу, гэлао, хмонги Благодарности Ключевые слова «альбомы об инородцах», эпоха Цин, этнография, Гуйчжоу, источниковедение, ицзу, гэлао, хмонги Благодарности Благодарности Исследование выполнено за счет гранта Российского научного фонда № 22-28-00119, https://rscf.ru/project/22- 28-00119 р Исследование выполнено за счет гранта Российского научного фонда № 22-28-00119, https://rscf.ru/project/22- 28-00119 Для цитирования Для цитирования Завидовская Е. А. Народы Гуйчжоу в цинских «альбомах об инородцах»: мяо, ицзу, гэлао // Вестник НГУ. Се- рия: История, филология. 2023. Т. 22, № 4: Востоковедение. С. 47–60. DOI 10.25205/1818-7919-2023-22-4- 47-60 Для цитирования Завидовская Е. А. Народы Гуйчжоу в цинских «альбомах об инородцах»: мяо, ицзу, гэлао // Вестник НГУ. Се- рия: История, филология. 2023. Т. 22, № 4: Востоковедение. С. 47–60. DOI 10.25205/1818-7919-2023-22-4- 47-60 Ekaterina A. Zavidovskaya Institute of China and Modern Asia of the Russian Academy of Sciences Moscow, Russian Federation Bryansk State University Bryansk, Russian Federation katushaza@yahoo.com, https://orcid.org/0000-0002-8369-7776 Institute of China and Modern Asia of the Russian Academy of Sciences Moscow, Russian Federation Bryansk State University Bryansk, Russian Federation katushaza@yahoo.com, https://orcid.org/0000-0002-8369-7776 Institute of China and Modern Asia of the Russian Academy of Sciences katushaza@yahoo.com, https://orcid.org/0000-0002-8369-7776 katushaza@yahoo.com, https://orcid.org/0000-0002-8369-7776 Научная статья УДК 025.171 DOI 10.25205/1818-7919-2023-22-4-47-60 Народы Гуйчжоу в цинских «альбомах об инородцах»:мяо, ицзу, гэлао Екатерина Александровна Завидовская Институт Китая и Современной Азии Российской академии наук Москва, Россия Брянский государственный университет Брянск, Россия k t h @ h htt // id /0000 0002 8369 7776 Институт Китая и Современной Азии Российской академии наук Москва, Россия Брянский государственный университет Брянск, Россия k h @ h h // id /0000 0002 8369 7776 Abstract Abstract This paper aims to evaluate textual descriptions of Guizhou ethnic groups found in the Qing dynasty Miao albums as a source for the study of history of large ethnic groups such as Miao, Yizu and Gelao. Two Miao albums about ethnic groups of Guizhou are housed at the library of Saint Petersburg State University, one of them has 72 descriptions with hand-drawn plates, only 28 out of 40 descriptions with plates remain in the other one. Among them are dozens of de- scriptions of Miao groups, several groups of Yizu, Buyi and Gelao, mainly with information on their costume and cus- toms. Scarce information on history is found only in the descriptions of Yizu, since Yizu had been holding positions of local tusi chieftains and had established their own polities which the Chinese empire was forced to interact with. Rare descriptions of Miao have mentions of historical events. This study traces the historical sources, starting from the Han dynasty, where information was borrowed from, for the Miao albums. The paper restores these pieces of infor- mation and provides a commentary. Apparently, the value of Miao albums is presented by their visual images of Guizhou people together with accompanying ethnographic notes rather than the historical accounts. K d y Miao albums, Qing dynasty, ethnography, Guizhou, source studies, Miao, Yizu, Gelao Acknowledgements g This study was funded by the Russian Scientific Fund, project no. 22-28-00119, https://rscf.ru/project/22-28-00119 For citation g This study was funded by the Russian Scientific Fund, project no. 22-28-00119, https://rscf.ru/project/22-28-00119 i i Zavidovskaya E. A. Guizhou Ethnic Groups in the Qing Dynasty Miao Albums: Miao, Yizu, Gelao. Vestnik NSU. Se- ries: History and Philology, 2023, vol. 22, no. 4: Oriental Studies, pp. 47–60. (in Russ.) DOI 10.25205/1818-7919- 2023-22-4-47-60 Введение Термин «альбомы об инородцах», или «альбомы о сотне / обо всех мяо» (百苗圖 баймяо- ту), в данном исследовании используется для обозначения рукописных книг с иллюстрация- ми, изображающими различные народы Гуйчжоу, создававшихся в основном в середине – конце цинского периода (середина XVIII – конец XIX в.). Существовали также альбомы о народах провинций Юньнань, Хайнань, Хунань и Тайвань, но их количество, дошедшее до настоящего времени, значительно уступает гуйчжоуским. Помимо картин, отражающих основные особенности той или иной этнической группы, эти источники интересны сопрово- ждающими их рукописными комментариями. Последние представляют собой документ, зна- комящий с восприятием этих некитайских этносов представителями конфуциански образо- ванного чиновничества. Изначально альбомы создавались в Гуйчжоу для чиновников с целью помочь им в ознакомлении с отличительными чертами «подведомственных» народов, особенностями их внешности или обычаев. В Восточном отделе Научной библиотеки СПбГУ хранятся два «альбома об инородцах» с шифрами Xyl F-25a, Xyl F-27, описывающих народы провинции Гуйчжоу. Альбом Xyl F-25a (без оригинальной обложки, названия, даты) включает 72 иллюстрации на листах размером 32,4 × 27,6 см. В верхнем правом углу картины наклеены небольшие листочки ри- совой бумаги с краткими описаниями каждого народа, причем эти описания выполнены в жанре поэзии «цы бамбуковых ветвей» (竹枝詞 чжучжицы). Их авторство неизвестно, а жанр чжучжицы использовался для поэтической характеристики нравов народов юго- запада империи и, реже, для описания других экзотических народов. Таким образом, описа- ния в альбоме Xyl F-25a весьма кратки. Второй альбом с шифром Xyl F-27 имеет название «Полное собрание изображений мяо провинции Гуйчжоу» (全黔苗圖 Цюаньцянь мяоту, без предисл., б. д.) и представляет собой гармонику из 56 листов. Первый и последний листы на- клеены на оригинальную обтянутую узорной тканью деревянную обложку. Из сорока описа- ний и иллюстраций к ним сохранились только 28. Они выполнены в прозаической форме и гораздо более пространны и информативны, чем сведения из альбома Xyl F-25a. Обзор источников сведений о народах Гуйчжоу Источниками, откуда в более или менее полной форме прозаические описания заимство- вались в «альбомы об инородцах», были следующие сочинения: 1) труд Тянь Жучэна (田汝成, 1503–1557) «Записи об услышанном в жарком пограничье» (炎徼紀聞 Яньцзяо цзи- вэнь, 1558, 4 цз. 1), четвертый цзюань которого посвящен истокам, облику, обычаям 29 на- родностей Гуйчжоу; 2) «Общие описания Гуйчжоу» (貴州通志 Гуйчжоу тунчжи) 1673 и 1692 гг. издания, содержащие иллюстрации с изображением 31 народности Гуйчжоу; 3) «Общее описание Гуйчжоу» (貴州通志 Гуйчжоу тунчжи, 46 цз.) 1741 г. издания, главным составителем которого выступил маньчжурский сановник Ортай (鄂爾泰, 1677–1745), вошло в состав «Полного собрания книг по четырем разделам» (四庫全書 Сыкуцюаньшу, 1772– 1782). Исследование о народах Гуйчжоу Линь Юэхуа охватывает гораздо больший спектр источников о провинции [Lin Yueh-hwa, 1941, p. 268]. Важно отметить, что часть описанных в исследуемых альбомах этнических групп не упомянута в вышеперечисленных трудах, источник сведений о них, сформировавшийся во второй половине XVIII в., установить сложнее. В альбоме Xyl F-25a представлено 72 отдельных описания, которые можно разделить на группы: 1) описания отдельных народов (мулао, яожэнь, божэнь, маньжэнь и др.); 2) описа- ния различных групп мяо (более распространенное название – хмонги, всего 35 групп мяо); 3) описания различных групп лунцзя (5 групп); 4) описания различных групп гэлао (6 групп); 5) описания различных групп чжунцзя (современное название буи, 4 группы); 6) описания групп, связанных с ханьцами, или их потомков (цайцзя-цзы, сунцзя-цзы, лиминь-цзы и проч.), 7) описание двух групп ицзу (черные лоло, белые лоло) и «женщины-чиновника». В альбоме Xyl F-27 сохранилось лишь 28 описаний из 40, перечень которых совпадает с частью описа- ний народов из альбома Xyl F-25a. Для получения развернутых сведений об истории и обычаях каждой из упомянутых в аль- бомах народностей имеет смысл проанализировать данные о них в доступных источниках. В большинстве альбомных описаний сведения об истории той или иной группы отсутствуют. Лишь небольшая часть описаний содержит краткие упоминания о прошлом данной народно- сти, эти краткие сведения необходимо восстанавливать в развернутом виде по другим источ- никам. Задача данного исследования состоит в аккумуляции и аналитическом обзоре сведе- ний по истории нескольких крупных этнических групп. 1 URL: https://ctext.org/wiki.pl?if=gb&res=909592&remap=gb (дата обращения 20.01.2023). Мяо (хмонги) Народ мяо(цзу) 苗(族) характеризуется этнографами следующим образом: «Мяо, хмонг (самоназвание) – народ на юге Китая, севере Вьетнама, в Лаосе, Таиланде, Бирме. Относятся к южноазиатской переходной расе, близки к яо. Говорят на языке мяо группы мяо-яо авст- роазиатской семьи. <…> Различаются несколько групп мяо – белые (хмонг дау), красные (хмонг пе), черные (хмонг ду), синие, пестрые, имеющие свои диалекты» [Крюков, 1988а, с. 317]. ] В «Книге о Цянь [Гуйчжоу]» (黔書 Цяньшу, 1690, 2 цз.) цинского чиновника и поэта Тянь Вэня (田雯, 1635–1704) упомянуты 28 разных видов мяо. «Общее описание Гуйчжоу» 1741 г. включает 13 видов мяо, в официальном каталоге «Изображения данников правящей дина- стии Цин» (皇清職貢圖 Хуанцин чжигунту – далее «Чжигунту», ред. Фу Хэн 傅恒, предисл. 1751) имеется 23 описания разных видов мяо (не только из Гуйчжоу). В исследуемом альбо- ме Xyl F-25a описаны 35 видов мяо. Сочинение «Воззрения о юге Гуйчжоу» (黔南識略 Цянь- нань шилюэ, 1749, 18 цз.) служившего в Гуйчжоу Ниугулу Айбида (愛必達, 1711–1766) гово- рит о том, что «есть сто видов мяо». Изданная в 1847 г. книга Ло Жаодяня (羅繞典, 1793– 1854) «Очерки чиновника, ведающего югом Цянь [Гуйчжоу]» (黔南職方紀略 Цяньнань чжифан цзилюэ) упоминает 52 вида мяо. Таким образом, количество выделяемых в отдель- ные виды мяо росло на протяжении XVII–XIX вв. В «альбомах об инородцах» пусть и описано много разных видов мяо, но не приведено сведений об их ранней истории, чуть больше сведений сообщается об эпохах Мин и Цин. Выделяется описание № 33 цзюгу-мяо 九股苗 («мяо из Цзюгу», илл. 1) из альбома Xyl F-27. Оно начинается c древней истории и упоминания деяний полководца Чжугэ Ляна (諸葛亮, 181–234) во время его южного похода 225 г., ниже приведем отрывок из него: 與偏橋黑苗同類武侯南征戮之殆盡僅存九人遂名九股㪚…前明播州之乳為楊應龍羽翼雖調兵十数萬誅滅楊應龍而九股未剿伏莽刦掠時出為害由地曠而險猝難制伏雍正十年勾連蠢動合楚粤黔三省兵剿撫兼施搜繳兵甲建城安汎焉 [Цзюгу-мяо] одного племени с хэй-мяо («черными мяо») из Пяньцяо. У-хоу [Чжугэ Лян] во время по- хода на юг почти всех их истребил. Остались только девять человек. Оттого и прозвали их «цзюгу» («де- вять бёдер»)… Во время бочжоуского мятежа при прежней [династии] Мин [цзюгу-мяо] помогали Ян Инлуну 2. Несмотря на то что переброшенные войска численностью более ста тысяч [солдат] уничтожили Ян Инлуна, «цзюгу» не были полностью истреблены, прятались в чащах, постоянно чинили грабежи и всякое зло. Поскольку [их] земли обширны и труднодоступны, [их] было трудно обуздать. В 10-м году Юнчжэн [1732] сговорились и учинили смуту. Объединенные войска трех провинций Хунань, Гуандун и Гуйчжоу провели тотальную карательную операцию, нашли и изъяли всё оружие, построили крепость для поддержания спокойствия и несения охраны там. 2 Ян Инлун (楊應龍, 1551–1600) – местный старейшина (тусы) в Бочжоу (播州) провинции Гуйчжоу, в 1600 г. войска Мин вели против него военную кампанию. 3 Данный принцип управления некитайскими народами путем использования их правящей верхушки возник еще в конце II – начале I в. до н. э., в XIV в. был воплощен завоевавшими страну монголами в систему тусы [Ла- рин, 1994, с. 61]. 4 Тин (廳) – административный район, вместо уезда, подчиненный области фу (府), в местностях, заселенных нацменьшинствами. Мяо (хмонги) Основной массив сведений о восстании Ян Инлуна содержится в «Правдивых записях императора Сянь Шэнь-цзуна [девиз правления Ваньли (萬曆), 1563–1620]» (神宗顯皇帝實錄 Шэньцзун сяньхуанди шилу). Упоминание цзюгу-мяо впервые встречается в историческом труде Ли Хуалуна (李化龍, 1554–1611) «Полная книга об [областях] Пин и Бо» (平播全書 Пинбо цюаньшу) эпохи Мин, где «Цзюгу» 九股 – это название местности. Цзюгу-мяо в эпоху Цин относились к группе «нецивилизованных» мяо (生苗 шэнмяо), не находившихся под властью тусы (土司) 3. В эпоху Мин этноним цзюгу-мяо указывал на группы мяо, проживав- ших на южном берегу реки Циншуй-цзян, они нередко пересекали реку и нападали на армию Мин, потому у китайцев рано сложилось о них представление. В «Общем описании Гуй- чжоу» 1673, 1692 гг. они названы «черными мяо из Цзюгу» (九股黑苗 цзюгу хэймяо), при этом очевидно, что приведенные выше сведения из альбома Xyl F-27 заимствованы из «Об- щего описания Гуйчжоу» 1741 г., где были значительно дополнены в сравнении с более ран- ними источниками (рис. 1). В монографии В. Л. Ларина приводятся данные из «Собрания основных материалов по Гуйчжоу “Правдивых записей о правлении [династии] Цин”», подтверждающие связь этой группы мяо с местностью Цзюгу: «Наибольшее беспокойство цинским властям доставляли “нецивилизованные мяо” тинов 4 Гучжоу, Тайгун, Цинцзян и Даньцзян, расположенных по берегам Циншуйцзяна и его верховьев – Цзюгухэ и Даньцзяна. Издревле мяо этих террито- рий, обобщенно именовавшиеся мяо Цзюгу (Цзюгу-мяо) или мяо Циншуйцзяна, “не подчи- няются монаршей власти, по натуре безрассудны, не уважают закон. Затаившись в [своих] укреплениях, часто покидают [их], вредят народу, грабят путников, и если не ликвидировать этого мятежа, территории не будут спокойны”» [Ларин, 1994, с. 111] (цит. по: [Циншилу Гуйчжоу…, 1964, с. 321]). Таким образом, «альбомы об инородцах» нельзя считать информа- тивным источником по истории мяо, в основном они содержат данные этнографического ха- рактера. Рис. 1. Описание и иллюстрация, изображающая цзюгу-мяо. Альбом Xyl F-27. НБ СПбГУ Fig. 1. Description and the image of jiugu-miao. Album Xyl F-27. Academic Library of Saint-Petersburg State University Рис. 1. Описание и иллюстрация, изображающая цзюгу-мяо. Альбом Xyl F-27. НБ СПбГУ Рис. 1. Описание и иллюстрация, изображающая цзюгу-мяо. Альбом Xyl F-27. НБ СПбГУ Fig. 1. Description and the image of jiugu-miao. Album Xyl F-27. Academic Library of Saint-Petersburg State University y ig. 1. Description and the image of jiugu-miao. Album Xyl F-27. Academic Library of Saint-Petersburg State University Наименование мяо менялось на протяжении исторического периода, зафиксированного в китайских источниках. По данным сочинения «Планы сражающихся царств» (戰國策 Чжаньгоцэ, V–III вв. до н. Мяо (хмонги) э.) можно представить место расселения мяо – «где-то в бассейне среднего течения Янцзы на севере провинций Цзянси и Хунань, в области озер Дунтин- Поянху». В «Книге истории» (尚書 Шаншу, не позже VII в. до н. э.) встречается термин «мяоминь» 苗民 [Итс, 1972, с. 139]. Географическое сопоставление позволяет считать «сань- мяо» 三苗 (или мяоминь) создателями неолитических культур Дунтин-Поянху и основным субстратом некитайского населения царства Чу, где «мань» (蠻) – это предки туцзя (土家) и мяо-яо (苗瑤). В самом названии коренного населения царства Чу «мань-и» (蠻夷) содер- жится указание на два компонента: «мань» – предки мяо, «и» – предки туцзя [Там же, с. 144, 172]. «Поздняя книга Хань» (後漢書 Хоуханьшу, цз. 116) содержит легенду о том, что предки мяо происходят из района Елан [Lin Yueh-hwa, 1941, p. 273]. В источниках эпох Тан – Сун народы района Уси упоминались как «мяожэнь» (苗人), это и были предки современных мяо. Лишь в династийной истории Юань (元史 Юаньши, 1370) появились письменные упомина- ния о разных группах варваров-мяо (苗蠻 мяомань), которые делятся на «гуйчжоу мяомань» (貴州苗蠻), «пинфа мяомань» (平伐苗蠻), «цзыцзян мяомань» (紫薑苗蠻), упомянуты также «мяолао» (苗獠), «шэнмяо» (生苗) и др. [Yang Zhiqiang, 2010, p. 2]. Термин «мань» как обо- значение всей совокупности местных народов был заменен на «мяо» в эпоху Мин. Тогда же мяо начали делить на «нецивилизованных» (生 шэн), не желающих принимать китайские традиции и считавшихся «варварами», и «цивилизованных» (熟 шу), более склонных к асси- миляции, кроме того шэнмяо не находились во власти тусы, а шумяо были включены в чис- ло подчинявшихся тусы [Lee, Tapp, 2010, p. xxi]. 5 Гуй-фан (鬼方), «край духов» – старое название Гуйчжоу. 6 Исследование Дж. Хермана показывает, что история о том, что в период Шу-Хань Хо Цзи оказал помощь У-хоу (Чжугэ Ляну) в поимке Мэн Ху, за что был пожалован титулом вана Лодянь, а потомки Хо Цзи, проживав- шие в Шуйси, носят титул «да гуйчжу» 大鬼主, фигурирует в «Правдивых записях Тай-цзу (Хунъу)» (太祖洪武實錄, 71 цз.), а также в раннем минском описании Гуйчжоу «Новое описание Гуйчжоу с картами» (貴州圖經新志 Гуйчжоу туцзин синьчжи, 17 цз., периода Хунчжи, 1488–1505) [Herman, 2007, p. 270]. 7 Шу-Хань (蜀漢, 221–263), или Шу (蜀) – одно из государств периода Троецарствия (220–280). 8 Цзи Хо (濟火, 175–238), другие имена Цзицзи Хо, Хо Цзи, вождь ицзу, житель области Цзанкэ эпохи Хань. Дальний предок тусы рода Ань. 5 Гуй-фан (鬼方), «край духов» – старое название Гуйчжоу. 6 Исследование Дж. Хермана показывает, что история о том, что в период Шу-Хань Хо Цзи оказал помощь У-хоу (Чжугэ Ляну) в поимке Мэн Ху, за что был пожалован титулом вана Лодянь, а потомки Хо Цзи, проживав- шие в Шуйси, носят титул «да гуйчжу» 大鬼主, фигурирует в «Правдивых записях Тай-цзу (Хунъу)» (太祖洪武實錄, 71 цз.), а также в раннем минском описании Гуйчжоу «Новое описание Гуйчжоу с картами» (貴州圖經新志 Гуйчжоу туцзин синьчжи, 17 цз., периода Хунчжи, 1488–1505) [Herman, 2007, p. 270]. 7 漢 у у уц , ц , р д у , ) [ , , p ] 7 Шу-Хань (蜀漢, 221–263), или Шу (蜀) – одно из государств периода Троецарствия (220–280). 8 濟火 8 Цзи Хо (濟火, 175–238), другие имена Цзицзи Хо, Хо Цзи, вождь ицзу, житель области Цзанкэ эпохи Х Дальний предок тусы рода Ань. 5 Гуй-фан (鬼方), «край духов» – старое название Гуйчжоу. 6 Ицзу (устар. лоло, голо, носу-и) На восток и на запад от Лодяня такие земли, как Цзыци 10, Елан 11 и Цзангэ 12 были цар- ствами; Тэтан, Байи и Цзюдао – были округами-дао 13. Все они суть племена лоло. с министром [Чжугэ] Ляном разбил Мэн Ху 9, отличился, потом получил титул вана Лодяня – это дальний предок рода Ань. На восток и на запад от Лодяня такие земли, как Цзыци 10, Елан 11 и Цзангэ 12 были цар- ствами; Тэтан, Байи и Цзюдао – были округами-дао 13. Все они суть племена лоло. Линь Юэхуа приводит данные о происхождении термина «лоло» из сочинения Ло Жаодя- ня «Очерки чиновника, ведающего югом Цянь [Гуйчжоу]» (1847): «Когда У-хоу [Чжугэ Лян] усмирил государства юга, он приказал главам больших родов вести своих подчиненных. Ло Цзихо (羅濟火) большого рода Цзяньнин (建甯) имел своих подчиненных в регионе между Цзангэ и Елан, они были названы именем Лодянь (羅甸)… В конце Суй [581–618] – начале Тан [618–907] способные из числа мань были подняты до статуса «гуйчжу» (鬼主) 14. Люди Лодянь были тогда названы «гуйчжу семьи Ло», что сокращенно звучит как «ло гуй» (羅鬼). Этот термин потом был ошибочно изменен на «лулу» (盧鹿), а затем опять на «лоло» (羅羅)» [Lin Yueh-hwa, 1941, p. 272–274]. p В сочинении Тянь Вэня «Книга о Цянь [Гуйчжоу]» (1690) также есть разъяснение проис- хождения этнонима «лоло»: «Цзи Хо был черным лулу (黑盧鹿), был дальним предком семьи Ань (安) из Шуйси (水西 – ныне уезд Цяньси 黔西). У него были глубоко посаженные глаза, звериное лицо, белые зубы. Синей тканью он оборачивал волосы на голове в форме рога 15… Мань (蠻) его поддерживали» [Ibid., p. 273]. Почему в «альбомах об инородцах» фигурируют отрывочные сведения об ицзу времен глубокой древности? Это связано с тем, что они являлись основателями нескольких государ- ственных образований, включая Муэгэ (慕俄格). Деяния правивших там родов были зафик- сированы в исторических хрониках с эпохи Хань до эпохи Цин. Исследование Дж. Хермана, посвященное роли государства ицзу в истории освоения провинций Гуйчжоу и Юньнань, со- общает, что в 225 г. вождь ицзу То-ачжэ (妥阿哲, др. имена – Цзи Хо, Хо Цзи) захватил Ло- гэнь (в районе совр. г. Бицзе), в том же году Чжугэ Лян встретил его там и заручился его поддержкой кампании царства Шу-Хань против восставшей на юге армии Мэн Ху. То-ачжэ обеспечил армию Чжугэ-ляна провизией, людьми и лошадьми, за что Чжугэ Лян удостоил То-ачжэ наследственного титула правителя-вана Лодянь (羅甸王). 9 Речь идет о Мэн Хо (孟獲), который также являлся вождем ицзу и родился в уезде Цюцзин. После смерти правителя царства Шу Лю Бэя (劉備, 162–223) Мэн Хо восстал против царства Шу-Хань. По легендарным дан- ным китайских источников, Чжунгэ Лян захватывал и освобождал Мэн Хо семь раз, в результате тот присягнул на верность царству Шу. 11 Елан (夜郎) – государство, существовавшее на западе Гуйчжоу в эпоху Хань, в тот период находилось в 20 ли на восток от уезда Тунцзы (桐梓) на севере Гуйчжоу. Одно из самых крупных царств юго-западных варва- ров (синань-и), центр Елан располагался в районе современного Цзуньи (尊義) в Гуйчжоу [Итс, 1972, с. 213]. 12 Цзангэ (牂牁) – царство, упоминаемое уже в хрониках об эпохе Чжоу. В эпоху Хань при императоре У-ди на территории бывшего царства Целань (且蘭) была учреждена управа Цзангэ-цзюнь (牂牁郡), в эпоху Тан семь крупных семей Цзангэ управляли частью Гуйчжоу наряду с царствами Муэгэ, Юйши, Бадэдянь, далее ставшими территорией с юга округа Цзуньи-фу до округов Сынань (司南), Шицянь (石阡). Территория совр. Автономной области юго-востока Гуйчжоу (黔東南自治州). 10 Цзыци (自杞) – государственное образование, существовавшее на востоке Юньнани и западе Гуйчж риод Южной Сун (1127–1260), основное население составляли «темные варвары» (умань), т. е. ицзу. Ицзу (устар. лоло, голо, носу-и) Народность ицзу (彝族) характеризуется этнографами следующим образом: «Ицзу (само- назв. ий) – народ на Юго-Западе Китая: провинции Сычуань (Авт. район Ляншань народно- сти ицзу), Юньнань (Авт.округ Хунхэ и Авт.уезд Цзянчэн народностей хани и ицзу, Авт. Ок- руг Эшань и Авт. уезды Милэ, Нинлан и Лунань народности ицзу), Гуйчжоу (Авт. уезд Вэйнин народностей ицзу, хуэй, мяо), Хунань и Гуанси-Чжуанский автономный район. Отно- сятся к восточноазиатской расе большой монголоидной расы, говорят на языке ицзу лоло- бирманской подгруппы центральной группы сино-тибетской семьи. Существует пиктогра- фическая письменность. Этническая общность ицзу сформировалась после падения государ- ства Наньчжао (VII–XIII вв.), когда часть была оттеснена в горы Ляншань, дав начало ицзу. Ляншаньские ицзу сохраняли независимость от Китая до середины ХХ в.» [Крюков, 1988б, с. 189]. В исследуемых «альбомах об инородцах», как правило, присутствуют два отдельных опи- сания для «черных лоло» (黑猓玀 хэй ло / голо, носу-и) – высшей социальной группы и «бе- лых лоло» (白猓玀 бай ло / голо) – низшей социальной группы. Стихотворное описание (№ 16) альбоме Xyl F-25a содержит такие строки о черных голо (лоло), в которых упомянут истори- ческий персонаж Цзи Хо: 黑猓玀在大定府第一。昔年濟火長南夷。蔓衍猓玀徧水西。地屬鬼方人信鬼。蠻文引結縂無稽。黑猓玀在大定府第一。昔年濟火南夷昔年濟火長南夷。蔓衍猓玀徧水西蔓衍猓玀徧水西。地屬鬼方人信鬼。蠻文引結縂無稽。 Черные голо в Дадин-фу. Часть один. р Д фу В былые годы Цзи Хо возглавлял южных инородцев нань-и. В былые годы Цзи Хо возглавлял южных инородцев Расселились голо повсюду в Сишуй. 5 Земля относилась к Гуй-фан 5, люди верят в духов. Письменность маней из узелков не имеет оснований. Предыдущее исследование автора уже было посвящено источникам сведений о черных и белых лоло в исследуемых альбомах [Завидовская, 2022]. В нем мы показали, что наиболее ранним подробным материалом о двух группах ицзу было сочинение Тянь Жуйчэна «Записи об услышанном в жарком пограничье» (炎徼紀聞 Яньцзяо цзивэнь 6), откуда почти без изме- нений данные поступили в «Общее описание Гуйчжоу» 1673 г. Приведем оттуда выдержку: 羅羅本盧鹿，而訛為今稱。有黑白二種。居平逺、大定、黔西、威寧者，為黑羅羅，亦曰烏蠻。黑者為大姓。羅俗尚鬼，故又曰羅鬼。蜀漢時有濟火者。從丞相亮破孟護有功，後封羅甸王，即安氏遠祖。自羅甸東西，若自杞、夜郎、牂牁則以國名；若特唐、白衣、九道則以道名，皆羅羅之種也。羅羅本盧鹿，而訛為今稱。有黑白二種。居平逺、大定、黔西、威寧者，為黑羅羅，亦曰烏蠻。黑者為大姓。羅俗尚鬼，故又曰羅鬼。蜀漢時有濟火者。從丞相亮破孟護有功，後封羅甸王，即安氏遠祖。自羅甸東西，若自杞、夜郎、牂牁則以國名；若特唐、白衣、九道則以道名，皆羅羅之種也。 Лоло изначально [обозначались как] «лулу», в результате ошибки получили нынешнее наименование. Есть два племени – черные и белые. Проживающие в Пинъюане, Дадине, Цяньси и Вэйнине – это черные лоло, называемые также «умань» («темные варвары»). Кланы черных – великие. В обычаях Ло [принято] почитание демонов, поэтому называются еще «логуй» («демоны-ло»). В эпоху Шу-Хань 7 жил Цзи Хо 8, с министром [Чжугэ] Ляном разбил Мэн Ху 9, отличился, потом получил титул вана Лодяня – это дальний предок рода Ань. 13 Дао (道) – административная единица типа округа в юго-западных регионах. 14 Гуйчжу (鬼主) – название лица, возглавлявшего ритуалы поклонения духам. В «Книге Тан» сказано: «Вар- вары-и (夷) почитают демонов-гуй. Так называют главу жертвоприношения. Когда духов призывают и провожают, необходимо, чтобы их сопровождали солдаты. Когда глава возглавлял только сто семей, то назывался «малым гуйчжу» [Lin Yueh-hwa, 1941, p. 274] (цит. по «Книге Тан»). 15 9 Речь идет о Мэн Хо (孟獲), который также являлся вождем ицзу и родился в уезде Цюцзин. После смерти правителя царства Шу Лю Бэя (劉備, 162–223) Мэн Хо восстал против царства Шу-Хань. По легендарным дан- ным китайских источников, Чжунгэ Лян захватывал и освобождал Мэн Хо семь раз, в результате тот присягнул на верность царству Шу. 10 Цзыци (自杞) – государственное образование, существовавшее на востоке Юньнани и западе Гуйчжоу в пе- риод Южной Сун (1127–1260), основное население составляли «темные варвары» (умань), т. е. ицзу. 11 Елан (夜郎) – государство, существовавшее на западе Гуйчжоу в эпоху Хань, в тот период находилось в 20 ли на восток от уезда Тунцзы (桐梓) на севере Гуйчжоу. Одно из самых крупных царств юго-западных варва- ров (синань-и), центр Елан располагался в районе современного Цзуньи (尊義) в Гуйчжоу [Итс, 1972, с. 213]. 12 Цзангэ (牂牁) – царство, упоминаемое уже в хрониках об эпохе Чжоу. В эпоху Хань при императоре У-ди на территории бывшего царства Целань (且蘭) была учреждена управа Цзангэ-цзюнь (牂牁郡), в эпоху Тан семь крупных семей Цзангэ управляли частью Гуйчжоу наряду с царствами Муэгэ, Юйши, Бадэдянь, далее ставшими территорией с юга округа Цзуньи-фу до округов Сынань (司南), Шицянь (石阡). Территория совр. Автономной области юго-востока Гуйчжоу (黔東南自治州). 13 Дао (道) – административная единица типа округа в юго-западных регионах. 14 Гуйчжу (鬼主) – название лица, возглавлявшего ритуалы поклонения духам. В «Книге Тан» сказано: «Вар- вары-и (夷) почитают демонов-гуй. Так называют главу жертвоприношения. Когда духов призывают и провожают, необходимо, чтобы их сопровождали солдаты. Когда глава возглавлял только сто семей, то назывался «малым гуйчжу» [Lin Yueh-hwa, 1941, p. 274] (цит. по «Книге Тан»). 15 Прическа в форме рога у высшего сословия ицзу – носу называлась «вместилище небесного духа» [Итс, 1972, с. 139]. 13 Дао (道) – административная единица типа округа в юго-западных регионах. 14 Ицзу (устар. лоло, голо, носу-и) Потомки То-ачжэ расшили область своего влияния, установив контроль над автохтонным народом пу (濮), проживав- шим в горах района Шуйси, внук То-ачжэ покинул Логэнь ради города Мугэбай-чжагэнь на западе Гуйчжоу, где было объявлено об основании царства Муэгэ [Herman, 2007, p. 26–27]. 12 Цзангэ (牂牁) – царство, упоминаемое уже в хрониках об эпохе Чжоу. В эпоху Хань при императоре У-ди на территории бывшего царства Целань (且蘭) была учреждена управа Цзангэ-цзюнь (牂牁郡), в эпоху Тан семь крупных семей Цзангэ управляли частью Гуйчжоу наряду с царствами Муэгэ, Юйши, Бадэдянь, далее ставшими территорией с юга округа Цзуньи-фу до округов Сынань (司南), Шицянь (石阡). Территория совр. Автономной области юго-востока Гуйчжоу (黔東南自治州). 14 Гуйчжу (鬼主) – название лица, возглавлявшего ритуалы поклонения духам. В «Книге Тан» сказано: «Вар- вары-и (夷) почитают демонов-гуй. Так называют главу жертвоприношения. Когда духов призывают и провожают, необходимо, чтобы их сопровождали солдаты. Когда глава возглавлял только сто семей, то назывался «малым гуйчжу» [Lin Yueh-hwa, 1941, p. 274] (цит. по «Книге Тан»). 15 15 Прическа в форме рога у высшего сословия ицзу – носу называлась «вместилище небесного духа» [Итс, 1972, с. 139]. Рис. 2. Иллюстрация, изображающая женщину-чиновника. Альбом Xyl F-25a. НБ СПбГУ Fig. 2. Illustration depicting a female official. Album Xyl F-25a. Academic Library of Saint-Petersburg State University Рис. 2. Иллюстрация, изображающая женщину-чиновника. Альбом Xyl F-25a. НБ СПбГУ Fig. 2. Illustration depicting a female official. Album Xyl F-25a. Academic Library of Saint-Petersburg State University Говоря о наличии государственности у ицзу, Р. Ф. Итс сообщает, что «в китайских пись- менных памятниках южносунской эпохи мяо были под прямым воздействием государства ицзу» [Итс, 1972, с. 266]. Во время войн Тан и Наньчжао ицзу расширили свои территории, охватив всю современную Гуйчжоу. В VIII в. государства ицзу отправляли посольства с да- рами ко двору Наньчжао. В свою очередь, Наньчжао организовало 37 племенных представи- тельств на территориях ицзу [Herman, 2007, p. 29]. В танских источниках клан Аванжэнь, расширявший свои владения в областях Пусянь и Пуань, назван царством Юйши (于矢), та- ким образом, территория Гуйчжоу в VIII в. была под властью двух царств носу-и – Муэгэ и Юйши [Ibid., p. 30–31]. С целью усиления буферной зоны с экспансионистски настроенным Наньчжао в 846 г. танское правительство наделило главу правящего клана ицзу титулом вана государства Лодянь (羅甸國王 Лодянь гован). Первое описание носу-и оставил танский по- сланник Фань Чо (樊綽) в труде «Книга о варварах-мань» (蠻書 Маньшу). Ицзу (устар. лоло, голо, носу-и) С основанием Се- верной Сун знатные роды с территории Муэгэ, заявлявшие о своих ханьских корнях, вклю- чая род Ян (楊) из Бочжоу (播州), получили почетные титулы в обмен на признание своего вассалитета. Монгольское завоевание Дали 1253 г. пошатнуло изолированное положение царства Муэгэ, оно было покорено монголами в 1283 г., но контроль монголов над террито- рией не был длительным [Ibid., p. 39]. Не меньший интерес для исследования устройства общества черных лоло (носу-и) пред- ставляют описания «женщины-чиновника» (女官 нюйгуань). «Женщина-чиновник из демо- нов ло[ло]» (羅鬼女官 логуй нюйгуань) представляет собой отражение системы наследования в обществе черных лоло (носу-и). Если находившийся у власти тусы или туму рано умирал, а его наследник был слишком мал, то главная жена тусы вела дела. Такого рода система управления существовала в таких тусы, как Шуйси (水西), Уса (烏撒), Пуань (普安), Юннин (永寧). Старшая жена (耐徳 найдэ) могла быть только из семьи тусы, т. е. из той же социаль- ной прослойки [Баймяоту…, 2004, с. 9]. В альбоме Xyl F-25а приведено такое описание «женщины-чиновника» (рис. 2): 女官在大定府代理土務第二。銀梅點額髻盤螺。細襇長裙委地拖。馬上頳衣輝磊落。蠻兒爭看女官过。 Женщина-чиновник в Дадин-фу исполняет обязанности ответственного за земляные работы. Часть два. Серебряные цветы на лбу, волосы собраны в улитку. Серебряные цветы на лбу, волосы собраны в улитку. Серебряные цветы на лбу, волосы собраны в улитку. Длинная юбка в тонкую складку, ползет по земле. С лошади багряная одежда ниспадает слоями. р Сыновья варваров борются, чтобы увидеть, как проходит женщина-чиновник. Сыновья варваров борются, чтобы увидеть, как проходит женщина-чиновник. Ранние сведения о женщинах-чиновниках и системе передачи власти у ицзу встречаем в юаньском сочинении Ли Цзина (李京) «Краткое описание Юньнань» (雲南志略 Юньнань чжилюэ), сочинении минского автора Се Чжаочжэ (謝肇淛) «Кратко о Юньнань» (滇略 Дяньлюэ) [Чжань Юэхай, 2015, с. 25]. «Общее описание Гуйчжоу» 1692 г. содержит ксилографическую иллюстрацию с назва- нием «Официальный выход женщины-чиновника [из демонов]-ло» (羅鬼女官出行之圖 Ло- нюйгуань чусинчжи ту). Изображена процессия из пяти женщин, две служанки несут шлейф длинной юбки женщины-чиновника, за ними следуют еще две служанки со сложенным зон- тиком. Текст описания следующий: «У лоло управляет делами женщина-чиновник, всеми местными управами-фу и по-прежнему как военный правитель. В стихотворении Тянь Вэня “Женщина-чиновник из Дунчуань 16” (東川女官 Дунчуань нюйгуань) сказано: “Я взираю на женщину-чиновника как взираю на картину. 16 Префектура Дунчуань-фу была основана при династии Мин на территории Юньнани, сейчас это провинция в Сычуань, но Тянь Вэнь описал женщину-чиновника из рода Ань из Шуйси, Гуйчжоу. 17 Янь Либэнь (閻立本, ок. 600 – 673 г.) китайский художник. 18 Чжаньлу (湛盧) – чудесный меч, выкованный древним оружейником Оу Е (歐冶). ( ) у 18 Чжаньлу (湛盧) – чудесный меч, выкованный древним оружейником Оу Е (歐冶). 17 Янь Либэнь (閻立本, ок. 600 – 673 г.) китайский художник. Ицзу (устар. лоло, голо, носу-и) Янь Либэнь 17 нарисовал картины о поднесении дани, я взираю на женщину-чиновника как на диковинный сон, диковинный странный облик и походка, я взираю как женщина-чиновник входит в разрушенный буддистский храм, при северном ветре в сумраке кричат злые горные духи, древние духи при тусклом светильнике открывают патру, старейший Будда превратился в стремительного льва, черепахи-оборотни и лисы вымазаны пудрой и краской для бровей, демоны-асуры в латах и шлемах дуют в губ- ной органчик и свирель, у женских особей темной тканью обмотана лохматая голова, у муж- ских особей глаза цвета лазури, в кулаке [держат] усы. Манерой держаться женщина-чи- новник как мужчина, выполощенная в огне холстина оборачивает ее тело, на девяти раттано- вых тростях красные кораллы, в ушах золотые кольца со свисающими жемчужинами, на та- лии висит пара [мечей] чжаньлу 18, вышитая юбка волочится по земле, короткая куртка в ло- тосах. Женщина-чиновник из Шуйси”». Итак, мы видим, что в официальной хронике провинции отдельное описание с иллюстрацией посвящено одному из аспектов управления подвластными ицзу народами – институту регентства у тусы. Добавим, что стихотворение цинского сановника и поэта Тянь Вэня из «Сборника Зала тяги к старине» (古歡堂集 Гухуань танцзи, цз. 7) положило начало традиции стихотворений о «нюй-гуань». цинского сановника и поэта Тянь Вэня из «Сборника Зала тяги к старине» (古歡堂集 Гухуань танцзи, цз. 7) положило начало традиции стихотворений о «нюй-гуань». Гэлао Гэлао (犵狫) проживают в основном в автономном уезде Лунлинь Гуанси-Чжуанского ав- тономного района [Народы Восточной Азии, 1965, с. 32]. По данным И. В. Самариной, пред- ками народа гэлао, по мнению большинства исследователей, являются автохтонные племена юга Китая. Языки гэлао принадлежат к тай-кадайской, или крадайской, языковой семье и входят в состав кадайской ветви (ветви кра) [Самарина и др., 2011, с. 9]. В альбоме Xyl F-25a содержится описание шести групп гэлао: 1) «гэлао свиного навоза» (豬屎犵狫 чжуши-гэлао); 2) «беззубые гэлао» (打牙犵狫 дая-гэлао) (рис. 3); 3) «водные гэ- лао» (水犵狫 шуй-гэлао); 4) «гэлао с круглыми крышками под котлы» (鍋圈犵狫 гоцзюань- гэлао); 5) «местные гэлао» (土犵狫 ту-гэлао); 6) «гэлао в халате-накидке» (披袍犵狫 пипао- гэлао). В труде Ло Жаодяня (1847) к гэлао отнесены также «кующие железо гэлао» (打鐵犵狫 тате гэлао), «красные гэлао» (紅犵狫 хун гэлао), лаодан, гэдоу (犵兜) [Lin Yueh-hwa, 1941, p. 279]. Рис. 3. Иллюстрация «беззубые гэлао» с описанием. Альбом Xyl F-25a. НБ СПбГУ Fig. 3. Illustration of the “toothless gelao” with a description. Album Xyl F-25a. Academic Library of Saint-Petersburg State University Рис. 3. Иллюстрация «беззубые гэлао» с описанием. Альбом Xyl F-25a. НБ СПбГУ Fig. 3. Illustration of the “toothless gelao” with a description. Fig. 3. Illustration of the “toothless gelao” with a description. Alb X l F 25 A d i Lib f S i P b S U i Album Xyl F-25a. Academic Library of Saint-Petersburg State University Все описания содержат только этнографические сведения, не сообщая ничего об истории народа. В качестве примера приведем стихотворное описание «беззубых гэлао» из альбома Xyl F-25a: 打牙犵狫在黔西州。桶群無摺織青羊。最愛夫家不忍傷。新婦一齊齲齒笑。打牙風俗大荒唐。打牙犵狫在黔西州。桶群無摺織青羊。最愛夫家不忍傷。新婦一齊齲齒笑。打牙風俗大荒唐。打牙犵狫在黔西州。桶群無摺織青羊。最愛夫家不忍傷。新婦一齊齲齒笑。打牙風俗大荒唐。打牙犵狫在黔西州。桶群無摺織青羊。新婦一齊齲齒笑。打牙風俗大荒唐。 Дая-гэлао (беззубые гэлао) в Цяньси-чжоу. «Юбки-ведра» без складок ткут из шерсти горала. Больше всего любят семью мужа – не смеют вредить ей Когда молодые жены покажут улыбки с больными зубами. [Поймете, что] обычай вырывать зубы совершенно абсурден. Исследование И. В. Самариной опирается на данные современных китайских ученых и сообщает, что «предками гэлао были пу (濮) и лао (獠). Пу не представляли собой единого этноса. Это был многоэтнический союз племен, бытовавший на территории современной провинции Юньнань к западу от реки Юаньцзян. Впоследствии они расселились на террито- рии современных провинций Сычуань, Юньнань, Гуйчжоу и в Северном Вьетнаме. Первые сведения о пу, или «ста / всех пу» (百濮 байпу), встречаются в китайских исторических хро- никах начиная с ханьской эпохи при упоминании неханьских народов Гуйчжоу, Сычуани, Хунани и Гуанси. Гэлао к эпохе Мин возникло деление гэлао на группы [Hostetler, 2001, p. 109]. В эпохи Юань, Мин, Цин в хрониках упоминаются тулао (土狫), потомки переселившихся в Юньнань лао. При Мин велись войны с неханьским населением Гуйчжоу, включая гэлао и тулао [Самарина и др., 2011, с. 17–19]. Обобщая всё богатство этнографических сведений о народах Гуйчжоу, приведенных в альбомах Xyl F-25a, Xyl F-27, можно выделить следующие превалирующие аспекты: 1) особенности семейно-брачных отношений, куда относятся обычаи, связанные со знаком- ством молодых людей, свадебным ритуалом, функционированием семьи до и после рожде- ния детей; 2) похоронная обрядность, включая ритуалы, связанные с захоронением умершего, способы захоронения, ритуалы после похорон; 3) культы различных божеств, шаманизм и ритуалы излечения (магическая медицина). Китайские источники, как правило, не дают интерпретаций данных явлений. Последняя стала возможной уже на современном этапе ис- следования этих обществ через призму этнографии и антропологии. Заключение Представленный в статье анализ текстов описаний ряда неханьских народов провинции Гуйчжоу из «альбомов об инородцах» эпохи Цин, хранящихся в Восточном отделе Научной библиотеки СПбГУ, позволяет представить место данного жанра книжной продукции в сис- теме источников о юго-западных окраинах Китая. Было показано, что описания разных групп мяо (хмонгов), ицзу, гэлао содержат только сведения этнографического характера об особен- ностях костюма, прически и наиболее характерных обычаях. Мы показали, по каким источ- никам можно восстановить более полные данные об истории этих народностей. При этом описание черных лоло (носу-и) содержит краткие упоминания событий древней истории, что связано с главенствующей ролью ицзу в сложившейся системе управления провинцией через посредство местных старейшин тусы. Ицзу сформировали на территории Гуйчжоу свои го- сударства, с которыми приходилось стоить отношения Китайской империи, отсюда большее внимание, уделенное обычаям и системе передачи власти у ицзу в альбомах («женщина- чиновник»). Тем не менее, очевидно, что «альбомы об инородцах» ценны больше своей ху- дожественной составляющей, содержащаяся в них описательная информация представляет собой краткие выдержки из более подробных источников, выявленных автором статьи. Гэлао В «Истории поздней Хань» есть упоминание о том, что «прочие “лао” (獠) получили название “главных лао” (主獠)», подробнее лао описаны в «Истории Вэй» (魏書 Вэйшу) [Lin Yueh-hwa, 1941, p. 275]. В эпоху Западная Хань на территории современных пров. Гуйчжоу и Юньнань было создано государство Елан. Оно упоминается в гл. 116 «Исторических записок» Сыма Цяня. В 130 г. до н. э. государство Елан входит в состав Ханьской империи, на его месте учрежда- ется военный округ. В «Истории Поздней Хань» при описании жителей государства Елан встречается этноним «варвары-лао» (夷獠 и-лао). Вторая часть этого этнонима – лао, запи- санная тем же иероглифом (獠), используется и во вьетнамском историческом памятнике «Полное собрание исторических записок Дайвьета» при описании «сильных» и «своенрав- ных» варваров, населявших земли Вубинь (武平), Кыудык (九德) и Тансыонг (新昌). Как ука- зывается в комментарии к этому памятнику, этноним лао употреблялся в узком и широком смысле. Употребляя его в узком смысле, китайские авторы имели в виду тайские народы, жившие в пределах современных пров. Гуйчжоу, Гуандун и Гуанси-Чжуанского автономного района. Начиная с эпохи Восточная Хань (25–220) в исторических хрониках вместо пу начи- нают упоминаться лао, проживавшие на территории современных пров. Хунань, Гуйчжоу и Гуанси-Чжуанского автономного района… Древние лао считаются предками не только гэлао, но и тулао, мулао и, возможно, ли, а также других тайских народов, включая чжуан, буи. В этнографической науке Китая ляо считаются потомками лоюэ, т. е. той части народов юэ (越), которую вьетнамские исследователи связывают с предками вьетнамцев. Название «гэлао» (仡僚) впервые появляется в эпоху Суй. В исторических записях тан- ского и сунского периодов встречаются упоминания о народе «гэлао» в различном иеро- глифическом написании (葛老，革老). До эпохи Сун в источниках использован только этно- ним «лао» ( 獠), при Сун использовали термины «лао» и «цилао». Этноним гэлао в современном китайском написании впервые встречается в книге «Собрание шуток о юж- ных манях» (溪蠻叢笑 Циманьцунсяо, ок. 1195) Чжу Фу (朱輔). Начиная с эпохи Мин (1368– 1644) во многих исторических хрониках отмечается, что в древности гэлао назывались «лао» (獠) [Самарина и др., 2011, с. 22]. Л. Хостетлер, опираясь на работы Ломбард-Салмон [Lom- bard-Salmon, 1972, p. 147], утверждает, что этноним «гэлао» начал употребляться в эпоху Сун, к эпохе Мин возникло деление гэлао на группы [Hostetler, 2001, p. 109]. В эпохи Юань, Мин, Цин в хрониках упоминаются тулао (土狫), потомки переселившихся в Юньнань лао. При Мин велись войны с неханьским населением Гуйчжоу, включая гэлао и тулао [Самарина и др., 2011, с. 17–19]. Список литературы Итс Р. Ф. Этническая история юга Восточной Азии. Л.: Наука, 1972. 305 с. Завидовская Е. А. Анализ источников описаний народностей провинции Гуйчжоу из цин- ских «альбомов об инородцах» // Человек и культура Востока. Исследования и переводы. М.: ИКСА РАН, 2022. С. 154–181. , Крюков М. В. Мяо // Народы мира: Историко-этнографический справочник. М.: Сов. эн- цикл., 1988а. С. 317–318. Крюков М. В. Ицзу // Народы мира: Историко-этнографический справочник. М.: Сов. эн- цикл., 1988б. С. 189. Ларин В. Л. Юго-западный Китай во второй половине XVII–70-х годах XIX в.: проблемы региональной истории. М.: Наука, 1994. 335 с. Народы Восточной Азии. М.: Наука, 1965. 1027 с. Самарина И. В., Мазо О. М., Нгуен Ван Лой. 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https://openalex.org/W2158565065	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-13-S17-S21	English	null	Mathematical modeling of the immune system recognition to mammary carcinoma antigen	BMC bioinformatics	2,012	cc-by	11,561	© 2012 Bianca et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mathematical modeling of the immune system recognition to mammary carcinoma antigen Carlo Bianca1 Ferdinando Chiacchio2 Francesco Pappalardo3 Marzio Pennisi4* From Asia Pacific Bioinformatics Network (APBioNet) Eleventh International Conference on Bioinformatics (InCoB2012) Bangkok, Thailand. 3-5 October 2012 Bangkok, Thailand. 3-5 October 2012 Abstract The definition of artificial immunity, realized through vaccinations, is nowadays a practice widely developed in order to eliminate cancer disease. The present paper deals with an improved version of a mathematical model recently analyzed and related to the competition between immune system cells and mammary carcinoma cells under the action of a vaccine (Triplex). The model describes in detail both the humoral and cellular response of the immune system to the tumor associate antigen and the recognition process between B cells, T cells and antigen presenting cells. The control of the tumor cells growth occurs through the definition of different vaccine protocols. The performed numerical simulations of the model are in agreement with in vivo experiments on transgenic mice. PROCEEDINGS Open Access * Correspondence: mpennisi@dmi.unict.it 4Dipartimento di Matematica e Informatica, Università di Catania, Catania, Italy Full list of author information is available at the end of the article Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Background developing cannot be easily predicted and controlled. Among human cancers, the mammary carcinoma repre- sents a major cause of concerns in women, since it belongs to the class of endogenous cancers which escape immuno- surveillance of the IS. The immune system (IS) is a complex system of organs, cells and molecules whose main task is to protect living organisms from external pathogens such as viruses and bacteria. Nevertheless the effectiveness of the IS in tumors disease is nowadays under discussion among biologists and physicians. As stated by the immunosurveillance the- ory [1,2], biotechnology engineered naked mice (mice without immune system) show the developing of multiple variants of malignant tumors that are not usually visible in wild mice, thus suggesting that the immune system plays an important role also against tumors. Indeed most mutated malignant cells are recognized and eliminated by immune system mechanisms right after their birth, and tumors that usually arise are indeed poorly immunogenic tumors, originating from malignant cells which escape from immune surveillance. Some tumors are caused by exogenous factors (e.g., smoke for lung cancer), and the elimination of the exogenous cause would in theory pre- vent the risk of developing the tumor. However many other tumors are caused by endogenous factors and their The risk of appearance of mammary carcinoma is usually estimated by analyzing the family history of can- cer, and breast cancer screening in young women is highly recommended since the achievement of earlier diagnosis could greatly improve the outcomes of the treatment. Strong family history of cancer usually entitles higher risks of developing the tumor, thus suggesting that tumor hereditary is encoded into the DNA. Some gene tests such as the genetic screening for the BRCA genes [3] are nowadays possible and may determine the risk of cancer. Indeed the analysis of the genome of indi- viduals will be useful to better estimate the risk of cancer. Biologists and physicians are exploring novel immuno- preventive treatments that can avoid the development of breast cancer in patients with high risks of malignant cell mutations. Among others, Lollini et al. [4] have devel- oped a cellular vaccine, called Triplex, which is able to elicit complete prevention of mammary carcinogenesis in HER-2/neu transgenic mice. Background A simplified version of the mathema- tical model proposed in the present paper has been analytically investigated in [15,16]. The simplified model does not include the role of the associated antigens, plasma B cells, interleukins 2 and 12 and the antigen pre- senting cells. Therefore the mathematical model of the present paper is a robust extension, from the biological viewpoint, of the model analyzed in [16]. In the present paper we restrict our attention to the comparison of numerical solutions of the model with in vivo experi- ments and sensitivity analysis of the model parameters. The model described in this paper can be also developed in order to take into account many biological phenom- ena, like chemotaxis, spatial cell dynamics or cluster for- mation. If spatial cells dynamics needs to be included, one can use the mathematical framework of the kinetic theory for active particles, see [17] and the reference therein. According to the latter framework, cells are grouped in functional subsystems which express a speci- fic strategy (called activity) and the time evolution of the subsystem is represented by a distribution function over the cells microscopic state (position, velocity and activ- ity). In this framework the mathematical modeling of the chemotaxis phenomenon and the formation of tumor at tissue scale can be included as shown in [18] and [19,20]. Th d f ll S “Th The repeated administration of the vaccine, realized with the aim to increase the antigenicity of the tumor associated antigens, maintains the immune system response ready against newborn cancer cells. However, even if vaccines are usually less toxic than standard drugs, uncontrolled administration of the vaccine can induce undesirable effects such as autoimmune diseases. Therefore the optimization of the vaccination protocol constitutes a fundamental and open problem. In the in vivo experiments it is not usually possible to reach an optimum vaccination protocol that maximizes the efficacy of the tumor depletion on the one hand and minimizes the risk of side effects on the other hand, because of the large variability cases. Indeed vaccination protocols are usually determined heuristically basing on best practice and previous experience. Moreover the cost of in vivo experiments can be prohibitive. In order to understand whether it was possible to gain complete prevention of mammary carcinogenesis with fewer injections, a (multi) agent-based model named Sim- Triplex [5] has been developed. Background Triplex combines three dif- ferent elements (the tumor antigen with two adjuvants) * Correspondence: mpennisi@dmi.unict.it 4Dipartimento di Matematica e Informatica, Università di Catania, Catania, Italy Full list of author information is available at the end of the article Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 2 of 15 that stimulate the immune system response with different actions [4]. Vaccine cells have been engineered to present and release the tumor associated antigen p-185 (that is also the oncogene of the tumor) with the addition of Allogeneic MHC-class I molecules to easier recognizing by cytotoxic T cells. Moreover, thanks to transduction of interleukin-12 genes, they release interleukin-12 mole- cules that have a broad range of costimulatory functions in boosting the immune response against tumors. consist in partial integro-differential equations and allow both the modeling of proliferative/destructive events and the modeling of mutations occurring in the onset of tumor, [13]. Further modeling approaches for the immune system-cancer competition include cellular automata, agent-based models, see the recent expository paper [14]. Most of the mathematical models of the IS summarize the response of the immune system in a single popula- tion of cells, named effector cells, which perform the task of destroying cancer cells. This simplifying assump- tion allows to reduce the complexity of the dynamics of immune system but it neglects the recognition process that occurs among the different cells that constitutes the response of the IS to the tumor antigen. It is worth stressing that differently from the vaccine for virus or bacteria, cancer vaccines require repeated admin- istration for the the entire life of the host. This is due to the low antigenicity of the cancer cells, the capability to escape the immune system surveillance. Moreover present cancer immunoprevention research is unable to find better vaccines able to assure complete, long-term protection. g The ODE-based model proposed in this paper has been derived from a biological conceptual model that is a good representation of the biological scenario (see Figure 1). The model takes into account both the humoral and cel- lular response of the immune system and the recognition process that involves the following entities: vaccine cells (VC), cancer cells (CC), tumor associated antigens (TAA), Plasma B cells (B), thymus cytotoxic lymphocytes (TC), thymus helper (TH) lymphocytes, antibodies (AB), interleukins 2 and 12 (IL2 and IL12), and antigen pre- senting cells (APC). Background It is worth noticing that SimTriplex has been also employed for other pathologies [6-10]. However agent-based models do not allow the development of asymptotic analysis of the competition and an easy investigation of parameters’ space. Different mathematical tools have been developed in order to model complex biological systems and among others, immune system-cancer competition. The most famous approach is the ODE-based model where the over- all system is decomposed in different cell populations whose time evolution is depicted by solutions of a non- linear ODE system (nonlinear terms take care of the inter- actions among two or more cell populations), see paper [11] for a review of ODE models available in the literature and [12] for a comparison between ODE models with and without delay. The present paper is organized as follows: Section “The Triplex vaccine in vivo experiments” briefly deals with the phenomenological analysis of the biological system. Sec- tion “The ODE-based model” is devoted to the description Kinetic theory models have been also proposed for the immune system-cancer competition. These models Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 3 of 15 Figure 1 Conceptual model of the in vivo experiment. On top vaccine cells (VC) are administered through intravenous injections, and then recognized by Cytotoxic T cells (TC) and Antibodies (AB) that kill them. Killed VC release both Interleukin-12 (IL12) and Tumor associated antigens (TAA). TAA are captured by antigen presenting cells (APC) and then presented to T helper cells (TH). IL12 stimulates both TH and TC actions. TH release interleukin-2 (IL2) which boosts TH, TC, and B actions and stimulates B cells to differentiate into plasma B cells (B). B release AB, and both AB and stimulated TC kill cancer cells (CC), which further release TAA. Figure 1 Conceptual model of the in vivo experiment. On top vaccine cells (VC) are administered through intravenous injections, and then recognized by Cytotoxic T cells (TC) and Antibodies (AB) that kill them. Killed VC release both Interleukin-12 (IL12) and Tumor associated antigens (TAA). TAA are captured by antigen presenting cells (APC) and then presented to T helper cells (TH). IL12 stimulates both TH and TC actions. TH release interleukin-2 (IL2) which boosts TH, TC, and B actions and stimulates B cells to differentiate into plasma B cells (B). B release AB, and both AB and stimulated TC kill cancer cells (CC), which further release TAA. Background of the ODE-based model. Section “Sensitivity analysis” introduces the sensitivity analysis technique. Section “Results and discussion” compares, by means of numerical simulations, the mathematical model with the computa- tional model SimTriplex. Finally Section “Conclusions” concludes the paper with a critical analysis and research perspective of the model. For interested readers Additional File 1 presents a simplified version of the model by cou- pling the differential model with an algebraic model. over-express the activated rat HER-2/neu oncogene. The description does not pretend to be exhaustive from the biological point of view but highlights the essentials of the experiments in order to motivate our study. The Triplex vaccine has been obtained from a mam- mary carcinoma of a FVBneuN #202 (H-2q) mouse, transgenic for the rat protooncogene c-neu, and com- bines different stimuli: • The p185neu oncoantigen; Materials and methods • The H-2q MHC molecules (allogeneic for H-2d BALBneuT mice); The Triplex vaccine in vivo experiments The Triplex vaccine in vivo experiments • The interleukin-12 (vaccine cells are engineered with the genes coding for murine IL-12). This section briefly deals with the in vivo experiment car- ried on BALB-neuT neu virgin female mice groups which Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 4 of 15 subclasses for successful cancer prevention indicates that humoral immune responses is more important than cellu- lar responses driven by cytotoxic T cells [4]. The experiment starts at the sixth week of age, where BALB-neuT mice start the vaccination protocol. Mice are divided in different groups, one for control untreated group, and one for each protocol tested. All vaccine protocols that have been tested are built upon the same 4-week cycle which consists in twice-weekly intra peritoneum vaccinations (Tuesday and Friday) for the first 2 weeks followed by 2 weeks of rest. Recent investigations [21] show that the Triplex vaccine progressively looses its efficacy with the advancement of tumor progression, both in terms of tumor incidence and multiplicity (see Figure 2). In particular, tumor develop- ment is remarkably delayed in mice receiving the early protocol with respect to untreated mice, whereas protocols started later have produced only a negligible delay. Furthermore in vivo tests show that the Triplex vaccine is ineffective against larger tumor targets. Thus, the triplex vaccine demonstrates very effective at preventing mam- mary carcinoma onset in tumor-free mice but is ineffective against established local tumors. The Prophylactic, lifelong Chronic vaccination proto- col of cancer-prone HER-2/neu transgenic mice with cells expressing HER-2, allogeneic MHC antigens and IL-12 demonstrated able to completely prevent the onset of mammary carcinoma. The Early vaccination protocol (which counts only three 4-week cycles at the beginning of the experiment) produces a significant delay in the onset of tumors, but all mice eventually succumbe to mammary carcinoma. Other tested proto- cols demonstrated much less effective, with little or no gain in efficacy when compared to untreated control mice. It should be therefore clear that any vaccination proto- col should be started early enough to avoid carcinoma in situ formation. On the other hand it should be advisable to minimize the number of administrations in order to both maintain complete efficacy and reduce the risk of any undesirable effect. In order to help biologists in find- ing better vaccination protocols, a (multi) agent model named SimTriplex has been developed in [5]. The Triplex vaccine in vivo experiments The model has been inspired by the work of Celada and Seiden [22] and uses an approach that models ab initio the interac- tion and diffusion kinetics of each relevant biological entity. SimTriplex has been tuned with the in vivo It is worth stressing that maximal prevention against mammary carcinoma required all the three vaccine com- ponents (HER-2/neu, allogeneic MHC antigens, and IL-12) and was due to the induction of both cellular and humoral immune responses. Although cellular and humoral immune responses are taken into account in the vaccine administration, the relative importance of antibody Figure 2 Triplex vaccine efficacy measured in in vivo experiments with respect to the advancement of the tumor. The abscissa represents the main temporal stages of tumor progression: from atypical hyperplasia up to mammary carcinoma. The ordinate shows the rate of inhibition of tumor burden entitled with the use of the vaccine. The red line represents the achievable efficacy of the Triplex vaccine in preventing the tumor burden whether the first protocol administration is delayed at successive stages of tumor progression. Figure 2 Triplex vaccine efficacy measured in in vivo experiments with respect to the advancement of the tumor. The abscissa represents the main temporal stages of tumor progression: from atypical hyperplasia up to mammary carcinoma. The ordinate shows the rate of inhibition of tumor burden entitled with the use of the vaccine. The red line represents the achievable efficacy of the Triplex vaccine in preventing the tumor burden whether the first protocol administration is delayed at successive stages of tumor progression. Figure 2 Triplex vaccine efficacy measured in in vivo experiments with respect to the advancement of the tumor. The abscissa represents the main temporal stages of tumor progression: from atypical hyperplasia up to mammary carcinoma. The ordinate shows the rate of inhibition of tumor burden entitled with the use of the vaccine. The red line represents the achievable efficacy of the Triplex vaccine in preventing the tumor burden whether the first protocol administration is delayed at successive stages of tumor progression. Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 5 of 15 variables are summarized in table 1; In Figure 1 we show the conceptual model for the biological problem. experiments and demonstrated able to coherently repro- duce the behaviors of the entities involved in the in vivo immunoprevention experiment. g y Vaccine cells The vaccine cells dynamics is described by equation (1). Vaccine cells (VC) are injected into the host through intraperitoneal vaccination with a predefined dosage. The inoculation of the vaccine cells is modeled by a function kin(t, q) which adds q vaccine cells to the cells in the host at time t whether a that time an injection was scheduled. Since vaccine cells come from the exter- nal, this term represents the only source element in the equation. Vaccine cells die for multiple causes, such as natural death (term -μ1VC), are inhibited by cytotoxic T cells that recognize vaccine cells thanks to their allo- genic-MHC class II molecules (term -a19TCVC), or by specific antibodies that are able to directly kill vaccine cells by complement mechanism (term -a17ABVC). If cancer cells are able to escape immunosurveillance, the cancer takes over, tends to compete with the healthy cells for nutrients, and could be able to kill the host. How- ever the immune system response can be helped in recog- nizing harmful cells by an external agent represented in our case by a vaccine. The induced immune response is the result of a complex network of interactions between IS cells which mainly depends from cells receptors. IS cells which present, through their receptors, specific tumor antigens can trigger a complex process whose final result is the eradication of the tumor. Specific interactions, which involve cell receptors, cannot be described with an ODE population model, see [5], however if we assume that the vaccine cells activate IS cells at given ratios we can model the subsequent immune response of activated cells. dVC dt = kin(t, q) −μ1VC −(α19TC + α17AB)VC (1) (1) The Triplex vaccine in vivo experiments In addition the use of SimTriplex as a predictive tool yielded to encouraging results [23]. variables are summarized in table 1; In Figure 1 we show the conceptual model for the biological problem. Model parameters The model contains 44 parameters which have a specific biological meaning. These parameters, assumed as con- stants, modify the rate of variations of the populations due to natural death, interactions with other population and release of new quantities. Accordingly: parameters referring to natural death of entities are denoted by μi, where i identifies the population under consideration; parameters referring to the interaction between popula- tions i and j are identified by ai j; finally parameters referring to releasing processes are identified by gi j, where i refers to the released entity and j to the releas- ing entity. The ODE-based model The competition between immune system cells and can- cer cells reminds the well known Predator-Prey (PP) model described by Lotka-Volterra equations. There is a population of prey, represented by the cancer cells, with an infinite set of food resources (nutrients coming from the host blood) and, differently from the classical PP, multiple populations of predators (the effectors cells) cooperate through cell-cell and cell-molecule interactions to neutralize the prey. Differently from the classical pre- dator-prey models, predator survival does not depends on the number of prey, since predator populations exist normally and, in absence of the prey, their number oscil- late around given equilibrium levels. Interleukin 2 Interleukin 2 is mainly released by T helper cells (term g64TH). As previously stated, interleukin 2 stimulates T helper priming, and primed T helper cells produce further interleukin 2. It is subjected to normal degrada- tion (-μ6IL2) and it is partially absorbed for mitotic and stimulation signals in cytotoxic T cells priming (term -a69TCIL2) and B cells duplication (term -a63BIL2). dTH dt = γ40APC + α46 IL2 IL2 + s1 TH + α45 IL12 IL12 + s2 TH −μ4TH (3) dIL2 dt = γ64TH −(α63B + α69TC)IL2 −μ6IL2 (6) (3) (6) Interleukin 12 (2) Interleukin 12 (IL12) is mainly introduced through vaccine administrations, so it depends on the vaccine dosage. In previous in vivo experiments [24] interleukin 12 was intro- duced separately, but after transduction of IL2 genes inside vaccine cells [4], it is released by killed vaccine cells, so it is proportional to the number of killed vaccine cells (term g51(a19TC + a17AB + μ1)VC). IL2 is subjected to normal degradation (-μ6IL12) and it is partially absorbed for mitotic and stimulation signals by cytotoxic and helper T cells priming (terms -a59TCIL12 and -a54THIL12). P-185 tumor associated antigens Antigens are subjected to nat- ural degradation (term μ2TAA) and phagocytosis by antigen presenting cells (-a20APCTAA), such as dendri- tic cells, macrophages and B cells. Moreover antibodies can bind to free antigens producing immune complexes (term -a27ABTAA). number of cancer cells (VC and TAA respectively) that are inhibited (terms g21(a19TC+ a17AB+μ1)VC and g28 (a88+a89TC)VC). These two terms represent the source elements of the equation. Antigens are subjected to nat- ural degradation (term μ2TAA) and phagocytosis by antigen presenting cells (-a20APCTAA), such as dendri- tic cells, macrophages and B cells. Moreover antibodies can bind to free antigens producing immune complexes (term -a27ABTAA). dB dt = γ34TH + α36 IL2 IL2 + s3 B −μ3B (4) (4) dTAA dt = γ21(α19TC + α17AB + μ1)VC + γ28(α88 + α89TC + α87AB)CC+ −(μ2 + α20APC + α27AB)TAA (2) dTAA dt = γ21(α19TC + α17AB + μ1)VC + γ28(α88 + α89TC + α87AB)CC+ −(μ2 + α20APC + α27AB)TAA (2) dTAA dt = γ21(α19TC + α17AB + μ1)VC + γ28(α88 + α89TC + α87AB)CC+ −(μ2 + α20APC + α27AB)TAA (2) T helper cells The key role of T helper cells is to stimulate both the humoral and cellular branches of the immune response by direct receptor binding or the releasing of specific cytokines that boost the immune response, such as interleukin 2. T helper cells are activated by specialized APC such as dendritic cells, macrophages or presenting B cells which present major histocompatibility class II/ peptide complexes. Since we are dealing with a monoclonal model, pre- sentation is not directly modeled, so the percentage of activated T helper cells is estimated from the number of antigen presenting cells (APC) existing in the system (term g40APC). Interleukins 12 and 2 (IL12 and IL2) contribute to stimulate T helper cells priming and dupli- cation (terms α46 ( IL2 IL2+s1 )TH and α45( IL12 IL12+s2 )TH). It is worth noting that, since interleukin 2 is released by T helper cells, these cells are able to self-stimulate their activities. The death factor is modeled by -μ4TH term. dIL12 dt = γ51(α19TC + α17AB + μ1)VC −(α54TH + α59TC + μ5)IL12 (5) (5) Antibodies Plasma B lymphocytes may absolve to multiple functions in building the immune response chain against patho- gens. In a first stage they can act as specialized antigen presenting cells, by recognizing pathogens through their specialized “Y-shaped” receptors, and can then present peptidic sequences to T helper cells. As a consequence of a successful interaction with T helper cells, they can be stimulated to differentiate into plasma B cells, which release antibodies with the same receptors shape, or B memory cells, which readily act against new appearance of previously encountered pathogens. Since there is no in vivo experimental evidence of B memory cells, as also suggested by the need of a chronic vaccination to achieve complete protection against tumor onset, we decided to do not include for now memory B cells into the model [4]. Antibodies represent the main result of the humoral immune response. Antibodies (AB) are released by plasma B (B) cells (term g73B) and are subjected to normal degra- dation (modeled by -μ7AB term). More-over they disap- pear in absolving their functions: binding to specific targets, i.e. antigens (term a72TAAAB), cancer and vaccine cells (terms a78CCAB and a71VCAB, respectively). dAB dt = γ73B −[α78CC + α71VC + α72TAA]AB −μ7AB (7) P-185 tumor associated antigens Equation(2) models tumor associated antigens dynamics. Tumor associated antigens (TAA) can be released by dead or killed vaccine or cancer cells. Accordingly we suppose that the number of released antigens is propor- tional to both the number of vaccine cells and the The network of organs, cells and molecules involved in the immune system is very large. In the model of the present paper we include only the entities recognized as fundamental for the biological process. We also assume that the IS-cancer competition occurs only in one hybrid organ which includes all the physical involved compartments (peritoneum, mammary gland, lymph nodes and so on). Table 1 Model variables Variable Description Short name x1 Number of injected vaccine cells VC x2 Number of P-185 tumor associated antigens TAA x3 Number of activated B cells B x4 Number of activated T helper cells TH x5 Number of interleukin 12 molecules IL12 x6 Number of interleukin 2 molecules IL2 x7 Number of released antibodies AB x8 Number of cancer cells CC x9 Number of activated cytotoxic cells TC x0 Number of activated antigen presenting cells APC Both the humoral and cellular responses of the immune system are taken into account with their main entities, plasma B cells (B), cytotoxic and helper T lymphocytes (TC and TH), antibodies (AB), antigen presenting cells (APC) and interleukins 2 and 12 (IL2 and IL12), while the pathogens are represented by the cancer cells (CC), the vaccine cells (VC) and the P-185 tumor associated anti- gens (TAA). The interactions among the various entities of the immune system network, the external stimulus of the vaccine and the cancer cells are modelled by a system of ten nonlinear ordinary differential equations whose Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 6 of 15 Page 6 of 15 cells (TH) positive feedback (term g34TH) and is there- fore able to release specific antibodies against cancer cells. We include the B as APC function in equation (10). Interleukin 2 (IL2) released by T helper cells plays an adjuvant role in stimulating B cells duplication (term α36( IL2 IL2+s3 )B). Death is modeled by -μ3B term. number of cancer cells (VC and TAA respectively) that are inhibited (terms g21(a19TC+ a17AB+μ1)VC and g28 (a88+a89TC)VC). These two terms represent the source elements of the equation. Cancer cells Cancer cells growth (CC) is modeled through the term 1 −CC cmax k −α88 CC . The term -a88CC is used to Cancer cells growth (CC) is modeled through the term 1 −CC cmax k −α88 CC . The term -a88CC is used to k k ll b h ll Cancer cells growth (CC) is modeled through the term 1 −CC cmax k −α88 CC . The term -a88CC is used to take into account CC killing by other immune system cells that are considered of minor importance for the process and are consequently not explicitly modeled, such as take into account CC killing by other immune system cells that are considered of minor importance for the process and are consequently not explicitly modeled, such as In equation(4) we only consider the behavior of the B cells population (B) that has been activated by T helper Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 7 of 15 Natural Killer cells which can kill cancer cells that under- express the major histocompatibility class I complex. The term p models the continuous production of newborn cancer cells. Due to transgenic nature of HER-2/neu mice new cancer cells are in fact continuously introduced into the host. The other terms describe cancer cells death mainly due to antibodies (term -a87ABCC) and cytotoxic T cells (term -a89TCCC) actions. As matter of a fact acti- vated cytotoxic T cells can kill cancer cells by direct cyto- toxicity and specific immunoglobulins can kill cancer cells by complement and other mechanisms. Since we consider populations that are activated by vaccine administrations, we set the following initial con- ditions: VC (0) = TAA (0) = B (0) = TH (0) = IL12 (0) = IL2 (0) = AB (0) = CC (0) = TC (0) = APC (0) = 0 The parameters in the model have been derived from literature, from measurements made during the in vivo experiment and from the SimTriplex model. Some para- meters which belong to the class of free parameters that any model has, were chosen into reasonable rages in such a way that the model was able to reproduce in vivo mean survivals for the untreated, early, and chronic vaccina- tions using a trial and error technique with mean-square evaluation, see table 2. Cancer cells dCC dt = 1 −CC cmax k −α88 CC −(α89TC + α87AB)CC + p (8) (8) Sensitivity analysis d d In order to understand which parameter may be consid- ered fundamental in this process, it is significant to inves- tigate the sensitivity of the model to the alteration of the parameters. Choosing a parameter in a suitable range while retaining fixed the others, represents the classical way to do sensitivity analysis. This methodology clearly owns limitations i.e., results are strongly bounded to the values of fixed parameters, and different sets of values for the fixed parameters may entitle completely different results. Cytotoxic T cells During the in vivo experiment, biological dynamics is observed in time slices that are not smaller than eight hours. For this reason, we set the simulation time step equal to (Δ(t) = 8 hrs). This biological motivation also determined the SimTriplex time-step. The choice of the physical time-step allows to compare the results of the two models. Both models are supposed to simulate the dynamics of entities inside a volume of 1μl, which corre- sponds to a small portion of mammary gland of mice. Cytotoxic T cells priming (TC) depends mainly on vac- cine cells (VC). Vaccine cells are engineered with allo- geneic major histocompatibility class I complex in order to easier presentation (term g91VC). Duplication is instead indirectly stimulated by T helper cells through the release of interleukin 2 (term α96( IL2 IL2+s96 )TC)). Nat- ural death is modeled with the term -μ9TC. dTC dt = γ91VC + α96 IL2 IL2 + s96 TC −μ9TC (9) (9) Antigen presenting cells With the term antigen presenting cells we indicate a class of different types of cells, such as dendritic cells, macro- phages, but also B cells, whose focal mission is to recognize, capture, and process antigens in order to present small antigenic sequences named peptides in conjunction with MHC class molecules to both cytotoxic and helper T cells. Antigen Presenting cells (APC) are then depending on the quantity of the antigens that have been released (term g02TAA), and can die (term -μ0APC). Partial rank correlated coefficients (PRCC) [25] is a sta- tistical approach used to bypass the above mentioned limitations. It works by calculating the partial correlation on rank-transformed data between input (model para- meters) and output (entities behaviors). Such a technique does not depend on the values of fixed parameters and permits to vary all the parameters at the same time, allowing to study the influence of input parameters on the model outcomes. Nevertheless the methodology can be in principle easily applied and used with any kind of continuous or discrete model. Antigen presenting cells dAPC dt = γ02TAA −μ0APC (10) (10) We thus designed the following set of ten non linear ODEs that is able to model the considered system of cell populations and interactions: ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ dVC dt = kin(t, q) −μ1VC −(α19TC + α17AB) VC dTAA dt = γ21(α19TC + α17AB + μ1)VC + γ28(α88 + α89TC + α87AB)CC+ −(μ2 + α20APC + α27AB)TAA dB dt = γ34TH + α36 IL2 IL2+s3 B −μ3B dTH dt = γ40APC + α46 IL2 IL2+s1 TH + α45 IL2 IL2+s2 TH −μ4TH dIL12 dt = γ51(α19TC + α17AB + μ1)VC −(α54TH + α59TC + μ5)IL12 dIL2 dt = γ64TH −(α63B + α69TC)IL2 −μ6IL2 dAB dt = γ73B −[α78CC + α71VC + α72TAA]AB −μ7AB dCC dt = 1 − cc cmax k −α88 CC −(α89TC + α87AB) CC + p dTC dt = γ91VC + α96 IL2 IL2+s96 TC −μ9TC dAPC dt = γ02TAA −μ0APC ⎧ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩ dVC dt = kin(t, q) −μ1VC −(α19TC + α17AB) VC dTAA dt = γ21(α19TC + α17AB + μ1)VC + γ28(α88 + α89TC + α87AB)CC+ −(μ2 + α20APC + α27AB)TAA dB dt = γ34TH + α36 IL2 IL2+s3 B −μ3B dTH dt = γ40APC + α46 IL2 IL2+s1 TH + α45 IL2 IL2+s2 TH −μ4TH dIL12 dt = γ51(α19TC + α17AB + μ1)VC −(α54TH + α59TC + μ5)IL12 dIL2 dt = γ64TH −(α63B + α69TC)IL2 −μ6IL2 dAB dt = γ73B −[α78CC + α71VC + α72TAA]AB −μ7AB dCC dt = 1 − cc cmax k −α88 CC −(α89TC + α87AB) CC + p dTC dt = γ91VC + α96 IL2 IL2+s96 TC −μ9TC dAPC dt = γ02TAA −μ0APC The methodology we used to perform sensitivity analysis (LHS-PRCC) is briefly described as follows. The interested reader can found more information about the methodol- ogy in [26]. Parameters space is initially sampled using a Monte-Carlo technique. In this case we use a technique named Latin-Hypercube-Sampling (LHS) [27]. The techni- que divides the random parameter distributions into N Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 8 of 15 (where N represents the chosen sample size) equal prob- ability intervals that are then sampled. Antigen presenting cells The choice for N h ld b t l t k 1 h k i th b f After sampling an LHS matrix X of sampled parameters is built. Each row represents an unique set of variables f th d l l d ith t l t Table 2 Model parameters Param Description Value(estimate) Ref μ1 VC (VC) death rate ln(2) = 9 In vivo a19 VC killing rate by TC cells (TC) (killing) 0.001 Estimated a17 VC killing rate by AB (AB) molecules (killing) 0.001 Estimated q No. Antigen presenting cells of cancer cells to inject at every vaccine administration 50 SimTriplex g21 released TAA (TAA) rate by killed VC 3 Estimated g28 released TAA rate by killed CC (CC) 3 Estimated μ2 TAA natural degradation rate ln(2) = 9 In vivo a20 Binding rate between TAA and APC cells 0.0005 Estimated a27 Binding rate between TAA and AB (IC formation) 0.00001 Estimated g34 plasma B cells (B) activation rate by TH cells (TH) 0.05 Estimated a36 B stimulation rate by IL2 (IL2) 0.0035 Estimated s3 B duplication stimulation threshold due to IL2 400 Estimated μ3 B cells natural death rate (half life) ln(2) = 15 [32] g40 TH cells (TH) activation rate by APC (APC) cells 0.15 Estimated a46 TH cells stimulation rate by IL2 (IL2) (duplication) 0.009 Estimated s1 duplication stimulation threshold due to IL2 1000 Estimated a45 TH cells cells stimulation rate by (IL12) IL12 (duplication) 0.009 Estimated s2 duplication stimulation threshold due to IL12 1000 Estimated μ4 TH cells natural death rate (half life) ln(2) = 15 Estimated g51 IL12 molecules release rate by VC 10 SimTriplex a54 absorbed IL12 rate by TH cells for mitotic signals 0.00009 Estimated a59 absorbed IL12 rate by TC cells for mitotic signals 0.001 Estimated μ5 IL12 molecules natural degradation rate ln(2) = 9 [33] g64 IL2 release rate by TH 5 Estimated a63 absorbed IL2 rate by B cells for mitotic signals 0.0001 Estimated a69 absorbed IL2 rate by TC cells for mitotic signals 0.0001 Estimated μ6 IL2 molecules natural degradation rate ln(2) = 3 Estimated g73 Released AB molecules rate by B cells 3 SimTriplex a78 AB - CC binding rate 0.0001 Estimated a71 AB - VC binding rate 0.001 Estimated a72 AB - TAA binding rate (IC formation) = a72 - μ7 AB natural degradation rate ln(2) = 7 [34] cmax CC (CC) growth saturation threshold 107 Estimated k CC duplication rate 0.0226 SimTriplex p No. Antigen presenting cells of newborn CC due to transgenic nature of mice 3 SimTriplex a88 CC death rate due to other IS entities 0.0000001176 Estimated a89 CC killing rate by TC cells 0.00004 Estimated a87 CC killing rate by AB 0.00004 Estimated g91 TC cells activation rate by VC 0.2 Estimated a96 TC cells duplication rate due to IL2 0.05 Estimated s96 duplication stimulation threshold thanks to IL2 400 Estimated μ9 TC cells natural death rate ln(2) = 21 [35] g02 APC (APC) activation rate due to TAA (TAA) 0.07 Estimated μ0 APC natural death rate ln(2)/15 [36] Table 2 Model parameters (where N represents the chosen sample size) equal prob- ability intervals that are then sampled. The choice for N should be at least k + 1, where k is the number of para- meters varied, but usually much larger to ensure accuracy. In our trials we set N = 1000. (where N represents the chosen sample size) equal prob- ability intervals that are then sampled. The choice for N should be at least k + 1, where k is the number of para- meters varied, but usually much larger to ensure accuracy. In our trials we set N = 1000. After sampling an LHS matrix X of sampled parameters is built. Each row represents an unique set of variables for the model sampled without replacement. The model is then solved for each row of X, and the model output values are stored into an output matrix Y. Page 9 of 15 Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Each matrix is then rank-transformed (XR and YR). X and Y can be used to calculate the Pearson correlation coefficient. XR and YR can be used to calculate the Spearman or rank correlation coefficient (RCC) and the partial rank correlation coefficient (PRCC). stop the simulation and, therefore, to consider a mouse as dead if the total number of cancer cells reached 105 cells. Over such a threshold the formation of carcinoma in situ can be considered an inevitable circumstance. Since in vivo experiments demonstrated that the vaccine progressively loses its efficacy when such an event occurs [21], this threshold represents a point of no return that halves between survival and death. Antigen presenting cells PRCC between an input parameter xj Î XR, j ≤k and output y Î YR is then computed by considering the resi- duals xj −ˆxj and y −ˆy where ˆxj and ˆy are given by the following regression models: Carcinoma in situ formation entitles a lot of different processes such as formation of physical barriers around the tumor mass and vascularization processes that are not described at this stage, even because this goes beyond the scope of the model. This means that both the ODE-based model and the SimTriplex models can- not be considered accurate in describing the in vivo experiment if the cancer cells threshold is overcome. Therefore the numerical simulations presented here refer to interactions where the number of cells do not go beyond this threshold. ˆxj = c0 + k p=1,p̸=j cpxp and ˆy = b0 + k p=1,p̸=j bpxp Results and discussion The outcome of the in vivo experiment has been mainly represented by the mice tumor-free survivals, and the Kaplan Meier survival curves [4] for each mice group treated with a given vaccination protocol have been built accordingly (see Figure 3). As previously stated, the success of failure of a treat- ment has been determined mainly by the survival rates of the mice involved in the experiment. Even if some measurements were made during the in vivo experi- ment, it was not possible to keep track of the time One of the first problems in modeling the process was to determine how to translate the biological concept of death in mathematical/computational terms. When developing the SimTriplex model, it has been decided to Figure 3 Kaplan-Meier survival curves. Kaplan-Meier survival curves given by the in vivo experiment for the Untreated (red circles), Early (purple triangles) and Chronic (blue squares) vaccination protocols. Figure 3 Kaplan-Meier survival curves. Kaplan-Meier survival curves given by the in vivo experiment for the Untreated (red circles), Early (purple triangles) and Chronic (blue squares) vaccination protocols. Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 10 of 15 Page 10 of 15 silico mice survivals for all vaccine protocols tested were in good agreement with mean survivals showed in in vivo experiment. evolution of the involved entities. Such measurements are not possible in vivo experiments, or can be achieved just partially in vitro for multiple reasons, i.e. it is not possible to do the measure too frequently due to wet- lab requirements, it is not possible to take the measure at present time with current technology, or simply because the measure entitles the need to kill the host. Since the biological behavior of the involved entities may change in a consistent manner even from mouse to mouse, we mainly focused in qualitatively analyzing can- cer cells behaviors and the response times of the princi- pal outcomes of immune response, i.e. antibodies and cytotoxic T cells behaviors for the Chronic, Early and Untreated protocols. One of SimTriplex main features is represented by the possibility of simulating different individuals. Tuning of free parameters has been executed in order to reproduce the same population survival curves for the vaccination protocols tested in vivo [5]. Results and discussion Moreover, during its tuning phase, SimTriplex entities behaviors have been accu- rately checked by biologists in order to verify that they were qualitatively in line with both biologists assump- tions and last immunological knowledge. The use of SimTriplex as a predictive tool, in conjunction with var- ious optimization techniques [28-30], to find better vac- cination protocols showed indeed that it represented a good approximation of the in vivo experiment [23], and therefore can be used to substitute missing in vivo data. In Figure 4 we compare the number of cancer cells (CC) behavior for the three protocols. As the Figure 4 shows, there is a slight delay between SimTriplex and the ODE model curves for both Chronic and Early protocols, whereas the Untreated protocol exhibits negligible difference, since both models use the same parameters for the growth law. Such a delay remains in line with in vivo experiment expectations. Indeed the behaviors are qualitatively in agreement, suggesting that the cancer cells dynamics is well described by the ODE model. The Chronic protocol (see Figure 4, left panel) plot suggests that after an initial growth phase, cancer cells are kept under control from the immune system thanks to the repeated administration of the vaccine. Bearing all the above in mind, we initially checked that the mathematical model mice survivals for all the tested vaccine protocols were in tune with mean survi- vals showed in the in vivo experiment, obtaining a good agreement between the two experiments. For the miss- ing in vivo data, mainly represented by entities time- behaviors, we compared ODE behaviors obtained numerically with the ones obtained by SimTriplex, high- lighting similarities and differences. The above behavior does not happen in the Early case (see Figure 4, center panel), where the vaccination pro- tocol is only able to delay the development of the can- cer, and the threshold on the number of cancer cells that entitles high risks of carcinoma in situ is reached at around at 44 weeks of age in SimTriplex, and at 47 weeks in the ODE model. In in vivo experiments the middle of the Kaplan Meyer survival curve for the early protocol is reached approximately at 52 weeks of age [4], with carcinoma in situ formation between 5 to 9 weeks earlier. We would note here that, in order to compare the results, we looked in SimTriplex for “mean virtual mouse”, i.e. Results and discussion Moreover the cytotoxic T cells peaks observed in Sim- Triplex for both the Chronic and Early protocols (see Figure 5, left and center panels) are a lot higher than those showed by the ODE model. In in vivo experiments it was observed that antibodies covered a major role in eradicating the tumor, whereas cytotoxic activity was estimated to be of secondary importance [23]. So from this point of view the ODE model may indeed be more accurate in describing this aspect of the immune response. From the LHS-PRCC analysis we found that 15 para- meters that correlated significantly with the number of cancer cells. For some parameters a negative or positive correlation was somewhat expected, for example it is tri- vial to observe that the dead rate and the activation rate of APC (μ0 and g02) positively and negatively correlate with cancer cells behavior, respectively (see Figure 7). The time correlation of some parameters indeed brings out some interesting findings we show as follows. p Finally we analyze the number of antibodies (AB) behavior in Figure 6. The untreated plots (see Figure 6, right panel) are practically flat for both the simulations and show negligible difference. Only at the end of the experiment SimTriplex plots show the appearing of anti- bodies. This may be due to the presence in SimTriplex of Natural killer cells that are able to kill cancer cells which under-express the MHC molecules, giving rise to a week response at late stages. For the Chronic and Early protocols (Figure 6, left and center), the antibodies time-behavior is very similar, but with higher AB peaks in the ODE model than those observed in SimTriplex simulations. This can be seen as a consequence of the weaker cytotoxic immune response observed in the ODE model which requires, in accord with in vivo The first interesting finding is related to the mechanisms driving the immune response against the tumor. The cyto- toxic immune response against the tumor is influenced by parameters such as g91 which represents the vaccine cells killing rate and a89 which represents the cancer cells kill- ing rate by cytotoxic T cells. By taking a look at g91 and a89 (Figure 8) PRCC time plots it is possible to observe that the two parameters show a strong negative correlation just at the beginning of the experiment. Results and discussion a mouse whose death occurs near the middle of the Kaplan Meier curves for the tested protocols. The ODE model demonstrated able to reproduce the available in vivo experimental data, in particular the in Figure 4 Number of cancer cells (CC) behaviors for the Chronic, Early and Untreated protocols. Blue solid lines identify SimTriplex simulations, red dashed lines the ODE model numerical results. Plots are presented on a log-scale to improve comparison. Figure 4 Number of cancer cells (CC) behaviors for the Chronic, Early and Untreated protocols. Blue solid lines identify SimTriplex simulations, red dashed lines the ODE model numerical results. Plots are presented on a log-scale to improve comparison. Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 11 of 15 Page 11 of 15 observations, a stronger humoral immune response in order to deplete the tumor. The number of cytotoxic T cells (TC) behavior is shown in Figure 5. The untreated plot (see Figure 5, right panel) is flat for both the models since in absence of vaccination there is no cytotoxic T activation. Even in this case we observe that the ODE model plots for the Chronic and Early vaccine protocols are a little bit delayed with respect to the SimTriplex plots, even if they remain in the expected range of the in vivo experi- ment. This could partially justify the delays observed in the cancer cells plots for the ODE plots (see Figure 4, left and center panels). We used PRCC to analize the effects of the most important input parameters which influence more the behavior of Cancer Cells. We plotted for these entities the PRCCs over the entire time course of the experiment to how the parameters sensitivity varies as the process behavior advances. The analysis has been executed by supposing that the administration of the vaccine follows the Chronic protocol. In this way it is possible to study which mechanisms mainly drive the immune response against cancer cells and which parameters should be tweaked in vivo in order to obtain a strong immune response with the minimal effort. To this end we kept constant the parameter related to the quantity of injected vaccine cells (q) and the parameters related to the tumor growth (k, p, cmax). Results and discussion The correlation becomes weaker and weaker as time goes on, becoming Figure 5 Number of cytotoxic T cells (TC) behaviors for the Chronic, Early and Untreated protocols. Blue solid lines identify SimTriplex simulations, red dashed lines the ODE model numerical results. Figure 5 Number of cytotoxic T cells (TC) behaviors for the Chronic, Early and Untreated protocols. Blue solid lines identify SimTriplex simulations, red dashed lines the ODE model numerical results. Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 12 of 15 Figure 6 Number of antibodies (AB) behaviors for the Chronic, Early and Untreated protocols. Blue solid lines identify SimTriplex simulations, red dashed lines the ODE model numerical results. Figure 6 Number of antibodies (AB) behaviors for the Chronic, Early and Untreated protocols. Blue solid lines identify SimTriplex simulations, red dashed lines the ODE model numerical results. totally not significative starting from (around) day 160. The humoral immune response instead is driven by para- meters such as g73 which represents the antibodies release rate and a87 which represents the cancer cells killing rate by antibodies. These parameters show a strong negative correlation which grows fast and lasts up to the end of the experiment (see Figure 9). This means that after an initial stage needed to start the immune response, variations of cytotoxic T cells related parameters do not influence can- cer cells behavior, thus suggesting how cytotoxic T cells are not fundamental for the complete eradication of the Tumor, which is instead strongly correlated with humoral immune response related parameters for all the time length of the experiment. This fact confirms the observa- tion made by Palladini et. Al. [23], where in vivo observations showed that the immune response against the tumor was mainly driven by antibodies. totally not significative starting from (around) day 160. The humoral immune response instead is driven by para- meters such as g73 which represents the antibodies release rate and a87 which represents the cancer cells killing rate by antibodies. These parameters show a strong negative correlation which grows fast and lasts up to the end of the experiment (see Figure 9). Results and discussion This means that after an initial stage needed to start the immune response, variations of cytotoxic T cells related parameters do not influence can- cer cells behavior, thus suggesting how cytotoxic T cells are not fundamental for the complete eradication of the Tumor, which is instead strongly correlated with humoral immune response related parameters for all the time length of the experiment. This fact confirms the observa- tion made by Palladini et. Al. [23], where in vivo Another interesting finding can be derived by taking a look at g21 and a72 PRCC time plots (see Figure 10). The g21 parameter represents the antigens release rate by vac- cine cells whereas the a72 parameter represents the rate of interaction between antibodies and antigens which brings to the formation of immune-complexes. The g21 plot clearly shows a negative correlation with the number of cancer cells. However it is interesting to observe that this correlation becomes weaker at the end of the experi- ment when the vaccine protocol is used to maintain under control the number of cancer cells, thus suggesting that the role of antigens becomes less important at the end of the experiment. In addition the a72 PRCC plot Figure 7 μ0 and g02 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Figure 7 μ0 and g02 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. CC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted CC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes hown in gray Figure 7 μ0 and g02 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Bianca et al. Results and discussion BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 13 of 15 Figure 8 g91 and a89 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Figure 8 g91 and a89 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. shows a positive correlation with the number of cancer cells, suggesting that a higher interaction rate between antibodies and antigens negatively influences the effects of the treatment. This becomes more evident at the end of the experiment (just around day 300) when the last 4 vaccination cycles are administered. Every vaccination cycle seems to reinforce the correlation between the input and the output parameters, affecting negatively the immune response. This can be explained by the fact an higher interaction rate between antibodies and antigens would entitle that more antibodies are recruited in bind- ing antigens, and then fewer antibodies (which as discussed earlier play a major role in the immune system response against the tumor) are employed in killing can- cer cells. When the number of cancer cells is kept under control, antigens may enter in competition with cancer cells and may negatively influencing the immune response. From the sensitivity analysis we can conclude that the antigenic administration should be then stronger during the first phases of the immune response, and then reduced once the humoral immune response is well established in order to reduce the risk that too many antibodies are involved in binding antigens instead can- cer cells. This model speculation is in line with the Figure 9 g73 and a87 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Figure 9 g73 and a87 PRCC time plots. Conclusions The mathematical model proposed in this paper is based on nonlinear ordinary differential equations. The model simulates the competition between the immune system and the mammary carcinoma under the action of an external force field (the vaccine). Three different proto- cols of the vaccine have been taken into account: Untreated, Early, and Chronic. The biological role of vac- cine cells, cancer cells, tumor associated antigens, plasma B cells, thymus cytotoxic lymphocytes, thymus helper lymphocytes, antibodies, interleukins 2 and 12, and anti- gen presenting cells has been taken into account. These results are certainly useful to research activity in immunology addressed to improve the efficacy of the treat- ment and to modulate the activation of the immune system in order to prevent side effects such as autoimmune dis- eases. Of course, different choices of initial conditions and of the parameters may modify the competition dynamics. We plan to investigate the optimal protocol using mathematical tecniques which are currently under inves- tigation. Results will be pubblished in due course. Numerical simulations of the model have been per- formed for different vaccination protocols and results were compared with a previously developed multi-agent model, called SimTriplex. For the tested vaccination pro- tocols, the ODE-based model is able to qualitatively repro- duce the time evolution not only for the number of cancer cells, but also for antibodies and cytotoxic T cells, main outcomes of humoral and cell mediated immune responses. From a quantitative point of view the mathema- tical model showed, respectively, a weaker and a stronger immune response of cytotoxic T cells and antibodies with respect to the SimTripex model, showing indeed better agreement with the in vivo observations and speculations. Results and discussion Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Figure 9 g73 and a87 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Page 14 of 15 Figure 10 g21 and a72 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. Figure 10 g21 and a72 PRCC time plots. Partial Rank Correlated Coefficients are computed on the number of cancer cells (CC), and are plotted over time (blue lines). PRCC plot of Dummy parameter (red lines) is presented for comparison. The plot portions where the correlation becomes significant (p <0.01) are shown in gray. biological effect of high antigen stimulation that usually suppresses the immune response [31]. some cases may negatively influence the immune response. It is then possible to conclude that a reduction of the intensity of vaccine administrations in later stages, when the immune response is already set, is advisable. This has been also highlighted in [8], where an ABM model developed to illustrate the effects of the same vaccine in cancer immunotherapy, suggested to apply the golden standard vaccination procedure (initial boost followed by sparse recalls) also to cancer vaccines. g The first author was partially supported by the FIRB project-RBID08PP3J- Metodi matematici e relativi strumenti per la modellizzazione e la simulazione della formazione di tumori, competizione con il sistema immunitario, e conseguenti suggerimenti terapeutici. The research was also partially supported by the INDAM-GNFM-Young Researchers Project-prot. n.43-Mathematical Modelling for the Cancer-Immune System Competition Elicited by a Vaccine and by University of Catania under PRA grant. The authors would like to thank Prof. P.L. Lollini and coworkers for supplying data and Prof. S. Motta for useful discussions. Additional material Additional File 1: Additional File 1 – On the coupling of differential and algebraic models. References 1. Burnet M: Cancer: a biological approach. III. Viruses associated with neoplastic conditions. IV. Practical applications. Br Med J 1957, 1:841-847. 2 Th L R ti t h l ti ti i l ti t 1. Burnet M: Cancer: a biological approach. III. Viruses associated with neoplastic conditions. IV. Practical applications. Br Med J 1957, 1:841-847. 22. Seiden PE, Celada F: A Model for Simulating Cognate Recognition and Response in the Immune System. J Ther Biol 1992, 158(3):329-357. 2. Thomas L: Reactions to homologous tissue antigens in relation to hypersensitivity. In Cellular and Humoral Aspects of the Hypersensitive States. Hoebers-Harper. New York;Lawrence HS 1959:529-532. 2. Thomas L: Reactions to homologous tissue antigens in relation to hypersensitivity. 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J Exp Med 2001, 194(9):1195-1205. 25. Saltelli A: Sensitivity Analysis in Practice: A Guide to Assessing Scientific Models. Hoboken, NJ: Wiley; 2008. 26. Competing interests 20. Bianca C: An existence and uniqueness theorem for the Cauchy problem for thermostatted-KTAP models. Int Journal of Math Analysis 2012, 6:813-824. The authors declare that they have no competing interests. Authors’ contributions 17. Bianca C: On the modelling of space dynamics in the kinetic theory for active particles. Math Comput Modelling 2010, 51:72-83. CB: Provided mathematical expertise and analytical solution, wrote the paper. FC: Performed sensitivity analysis, wrote the paper. CB: Provided mathematical expertise and analytical solution, wrote the paper. FC: Performed sensitivity analysis, wrote the paper. 18. Bellouquid A, Bianca C: Modelling aggregation-fragmentation phenomena from kinetic to macroscopic scales. Math Comput Modelling 2010, 52:802-813. FP: Designed the model, analyzed data, supervised the project, wrote the paper. MP: Designed the model, analyzed data, performed numerical simulations and sensitivity analysis, wrote the paper. 19. Bianca C: Kinetic theory for active particles modelling coupled to Gaussian thermostats. Applied Mathematical Sciences 2012, 6:651-660. Published: 13 December 2012 21. Nanni P, Nicoletti G, Palladini A, Croci S, Murgo A, Antognoli A, Landuzzi L, Fabbi M, Ferrini S, Musiani P, Iezzi M, De Giovanni C, Lollini PL: Antimetastatic activity of a preventive cancer vaccine. Cancer Res 2007, 67(22):11037-11044, Erratum in: Cancer Res. 2007 Dec 15;67(24).12034. Author details 1 1Dipartimento di Scienze Matematiche, Politecnico di Torino, Torino, Italy. 2Dipartimento di Ingegneria Informatica e delle Telecomunicazioni, Università di Catania, Catania, Italy. 3Dipartimento di Scienze del Farmaco, Università di Catania, Catania, Italy. 4Dipartimento di Matematica e Informatica, Università di Catania, Catania, Italy. 1Dipartimento di Scienze Matematiche, Politecnico di Torino, Torino, Italy. 2Dipartimento di Ingegneria Informatica e delle Telecomunicazioni, 15. Bianca C, Pennisi M: The triplex vaccine effects in mammary carcinoma: A nonlinear model in tune with SimTriplex. Nonlinear Analysis: Real World Applications 2012, 13:1913-1940. 16. Bianca C, Pennisi M, Motta S, Ragusa MA: Immune System Network and Cancer Vaccine. AIP Conference Proceedings 2011, 1389:945-948. Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 Bianca et al. BMC Bioinformatics 2012, 13(Suppl 17):S21 http://www.biomedcentral.com/1471-2105/13/S17/S21 This article has been published as part of BMC Bioinformatics Volume 13 Supplement 17, 2012: Eleventh International Conference on Bioinformatics (InCoB2012): Bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/ supplements/13/S17. ordinary and delay differential equations. Journal of Theoretical Medicine 1997, 2:117-128. ordinary and delay differential equations. Journal of Theoretical Medicine 1997, 2:117-128. 13. Bianca C: Mathematical modeling for keloid formation triggered by virus: Malignant effects and immune system competition. Math Models Methods Appl Sci 2011, 21:389-419. 14. Bianca C, Pennisi M: Immune system modeling by top-down and bottom-up approaches. International Mathematical Forum 2012, 7(3):109-128. References Marino S, Hogue IB, Ray CJ, Kirschner DE: A methodology for performing global uncertainty and sensitivity analysis in systems biology. Journal of Theoretical Biology 2008, 254:178-196. , 5. Pappalardo F, Castiglione F, Lollini PL, Motta S: Modelling and Simulation of C I ti i Bi i f ti 2005 21(12) 2891 2897 5. Pappalardo F, Castiglione F, Lollini PL, Motta S: Modelling and Simulation of Cancer Immunoprevention vaccine. Bioinformatics 2005, 21(12):2891-2897. 5. Pappalardo F, Castiglione F, Lollini PL, Motta S: Modelling and Simulation of Cancer Immunoprevention vaccine. Bioinformatics 2005, 21(12):2891-2897. 27. Mckay M, Beckman R, Conover W: Comparison of 3 methods for selecting values of input variables in the analysis of output from a computer code. Technometrics 1979, 21:239-245. 6. Halling-Brown M, Pappalardo F, Rapin N, Zhang P, Alemani D, Emerson A, Castiglione F, Doroux P, Pennisi M, Miotto O, Churchill D, Rossi E, Moss DS, Sansom CE, Bernaschi M, Lefranc MP, Brunak S, Motta S, Lollini PL, Basford KE, Brusic V, Shepherd AJ: ImmunoGrid: Towards Agent-based Simulations of the Human Immune System at a Natural Scale. Philosophical Transactions A 2010, 368(1920):2799-2815. 28. Pappalardo F, Mastriani E, Lollini PL, Motta S: Genetic Algorithm against Cancer. Lecture Notes in Computer Science 2006, 3849:223-228. 29. Pappalardo F, Pennisi M, Castiglione F, Motta S: Vaccine protocols optimization: in silico experiences. Biotechnology Advances 2010, 28:82-93. 7. Pappalardo F, Halling-Brown MD, Rapin N, Zhang P, Alemani D, Emerson A, Paci P, Duroux P, Pennisi M, Palladini A, Miotto O, Churchill D, Rossi E, Shepherd AJ, Moss DS, Castiglione F, Bernaschi M, Lefranc MP, Brunak S, Motta S, Lollini PL, Basford KE, Brusic V: ImmunoGrid, an integrative environment for large-scale simulation of the immune system for vaccine discovery, design, and optimization. Briefings in Bioinformatics 2009, 10(3):330-340. 30. Pennisi M, Catanuto R, Pappalardo F, Motta S: Optimal vaccination schedules using Simulated Annealing. Bioinformatics 2008, 24(15):1740-1742. 30. Pennisi M, Catanuto R, Pappalardo F, Motta S: Optimal vaccination schedules using Simulated Annealing. Bioinformatics 2008, 24(15):1740-1742. 31. Abbas AK, Litchman AH, Pilllai S: Cellular and molecular immunology. Elsevier;, 7 2011. 32. Mattioli CA, Tomasi TB: The life-span of IgA plasma Cells From the Mouse intestine. J Exp Med 1973, 138(2):452-460. 8. Pennisi M, Pappalardo F, Palladini A, Nicoletti G, Nanni P, Lollini PL, Motta S: Modeling the competition between lung metastases and the immune system using agents. BMC Bioinformatics 2010, 11(Suppl 7):S13. 33. Acknowledgements h f h The first author was partially supported by the FIRB project-RBID08PP3J- Metodi matematici e relativi strumenti per la modellizzazione e la simulazione della formazione di tumori, competizione con il sistema immunitario, e conseguenti suggerimenti terapeutici. The research was also partially supported by the INDAM-GNFM-Young Researchers Project-prot. n.43-Mathematical Modelling for the Cancer-Immune System Competition Elicited by a Vaccine and by University of Catania under PRA grant. The authors would like to thank Prof. P.L. Lollini and coworkers for supplying data and Prof. S. Motta for useful discussions. The sensitivity analysis gave two major results. First it confirmed the major role of humoral immune response also observed in in vivo experiments [23], then showed that during later stages of the experiment antigens loose their role of activating the immune response and in Page 15 of 15 Page 15 of 15 References Remick DG, Friedland JS: Cytokines in Health and Disease. Marcel Dekker, New York;, 2nd Review and Expand 1997. 34. Peppard JV, Orlans E: The biological half-lives of four rat immunoglobulin isotypes. Immunology 1980, 40:683-686. 34. Peppard JV, Orlans E: The biological half-lives of four rat immunoglobulin isotypes. Immunology 1980, 40:683-686. 9. Pennisi M, Pappalardo F, Motta S: Agent based modeling of lung metastasis-immune system competition. Lecture Notes in Computer Science 2009, 5666:1-3. y gy 35. Pardoll D: T cells take aim at cancer. PNAS 2002, 99(25):15840-15842. y y 35. Pardoll D: T cells take aim at cancer. PNAS 2002, 99(25):15840-15842. 36. Zehn D, Cohen CJ, Reiter Y, Walden P: Extended presentation of specific MHC-peptide complexes by mature dendritic cells compared to other types of antigen-presenting cells. European Journal of Immunology 2004, 34(6):1551-1560. 10. Pappalardo F, Forero IM, Pennisi M, Palazon A, Melero I, Motta S: Simb16: Modeling induced immune system response against B16-melanoma. PLoS ONE 2011, 6(10), art. no. e26523. 11. Eftimie R, Bramson JL, Earn DJD: Interactions between the immune system and cancer: A brief review of non-spatial mathematical models. Bull Math Biol 2011, 73:2-32. doi:10.1186/1471-2105-13-S17-S21 Cite this article as: Bianca et al.: Mathematical modeling of the immune system recognition to mammary carcinoma antigen. BMC Bioinformatics 2012 13(Suppl 17):S21. 12. Baker CTH, Bocharov GA, Paul CAH: Mathematical modelling of the interleukin-2 T-cell system:A comparative study of approaches based on 12. Baker CTH, Bocharov GA, Paul CAH: Mathematical modelling of the interleukin-2 T-cell system:A comparative study of approaches based on
https://openalex.org/W2109745988	https://www.scielo.br/j/ibju/a/tjNS77tmJnPKYJQ9r4GYM7Q/?lang=en&format=pdf	English	null	Resiniferatoxin for detrusor instability refractory to anticholinergics	International Braz J Urol	2,004	cc-by	2,826	RESINIFERATOX Urological Neurology International Braz J Urol Official Journal of the Brazilian Society of Urology RESINIFERATOX Urological Neurology International Braz J Urol Official Journal of the Brazilian Society of Urology RESINIFERATOX Urological Neurology International Braz J Urol Official Journal of the Brazilian Society of Urology Vol. 30 (1): 53-58, January - February, 2004 Division of Urology, State University of Campinas, UNICAMP, Campinas, São Paulo, Brazil Division of Urology, State University of Campinas, UNICAMP, Campinas, São Paulo, Bra ABSTRACT Purpose: We have evaluated the clinical and urodynamic effects of intravesical instillation of resiniferatoxin in patients with idiopathic detrusor instability refractory to anticholinergics. Materials and Methods: There were 30 women, median age 56 years old with detrusor insta- bility for over 6 months and a history of anticholinergic use with no response or intolerable collateral effects. A 50 nM solution of resiniferatoxin was prepared for intravesical instillation. All patients were evaluated for urinary symptoms, as well as for urodynamic assessments before and 30 days after instillation. Tolerability was analyzed during the instillation. Results: A clinical improvement was observed in 30% of the patients with urinary urgency and in 33% of the patients with urge-incontinence. The mean maximum cystometric capacity before application was 303.9 ± 78.9 and after application 341 ± 84.6. No significant difference was observed (p = 0.585). The mean maximum amplitude of the contractions diminished from 47.86 ± 29.64 to 38.72 ± 30.77 (p = 0.002). Conclusions: Resiniferatoxin, in this concentration, proved to be useful in a small percent- age of patients regarding clinical detrusor instability. Maximum amplitude of the involuntary contrac- tions was significantly reduced and in 33% patients the involuntary contractions disappeared. Further studies with different concentrations are recommended. Key words: bladder; urodynamics; urination disorders; bladder instillations; toxins Int Braz J Urol. 2004; 30: 53-8 RESINIFERATOXIN FOR DETRUSOR INSTABILITY RESINIFERATOXIN FOR DETRUSOR INSTABILITY required volumes in the following manner: 9.5 mL of pure ethanol, 90 mL of 0.9% saline solution and 0.5 mL of RTX, producing a 50 nM solution in 10% etha- nol using the saline solution as a vehicle. This solu- tion was prepared just before each instillation. A 14F Foley catheter was used for intravesical instillation of the medication and left in the bladder for 30 min- utes. Patients were asked about pain and the intensity of these symptoms was analyzed using a visual ana- logical scale. Zero meaning no discomfort and 10 in- dicating unbearable sensation. mediated micturition reflex, since lidocaine instilla- tion reduces these contractions (9). Lidocaine is a potent C fiber inhibitor and less potent in the case of A delta fibers. As this anesthetic improves involun- tary contractions in patients with detrusor instability, it has been suggested that this type of nerve fiber con- tributes to the etiology of this disease (9). RTX was effective in reducing the frequency, urgency and incontinence episodes in patients with detrusor hyperreflexia. As it did not produce anatomic dysreflexia episodes in spinal cord trauma patients, it proved to be a good alternative in such patients (10,11). Since RTX has been successfully used in patients with detrusor hyperreflexia, it could also be effective in the treatment of detrusor instability. All patients were asked about urinary symp- toms on the 30th day following the instillation. Cystometry was performed before and 30 days after RTX instillation. The same researcher per- formed all the urodynamic tests. A double lumen 8F catheter (one lumen for saline infusion rate of 50 mL/ min and other to measure the intravesical pressure) and a 4F rectal catheter-balloon to measure abdomi- nal pressure were used. MATERIALS AND METHODS A prospective cohort study was conducted comprising 30 women, median age 56 years-old (age range 24 to 88) with urodynamic proven detrusor in- stability for more than 6 months and unsuccessful use of anticholinergics for at least 40 days or severe col- lateral effects. All the patients underwent urine test to rule out urinary tract infection. This study received the approval of the Hospital Ethics Committee for research in humans. The cystometric parameters evaluated were maximum cystometric capacity, maximum ampli- tude of the involuntary contractions and the pres- ence of urgency or urinary leakage during these contractions. The influence of intravesical instillation of RTX was accessed by comparing the results of the various parameters utilizing McNemar’s test and the Wilcoxon rank sum test for non-parametric samples. P < 0.05 was considered statistically significant. All patients underwent a work-up for DI that included history and physical examination, so that patients with neurological, cardiovascular, renal, he- patic and psychiatric disorders as well as those pa- tients with malignant diseases or pregnant patients could be excluded from the study. INTRODUCTION tus found in Morocco, presents pharmacological ac- tivity in detrusor instability. It is known as resiniferatoxin (RTX), an analogue of capsaicin but a thousand times more potent (6). RTX seems to in- terfere in the non-myelinized C fibers responsible for the micturition reflex in patients with medullar le- sions. It has very little effect on the myelinized delta A fibers present in the pelvis and responsible for trans- mitting sensorial information to the encephalic cen- ter in normal individuals (7). RTX has a homovanylic ring in its structure its biological activity is able to treat some lower urinary tract functional disorders (8). Lastly, evidence exists indicating that involun- tary detrusor contractions also depend on C fiber Detrusor instability (DI) is responsible for lower urinary tract symptoms and it is characterized by involuntary contractions of the detrusor during the filling phase of the bladder, during urodynamic as- sessment (1). These contractions have been attributed to neurogenic (2,3) or myogenic (4) alterations and, recently, the focus has been on C sensory fibers (2) and atropine (3) resistant parasympathetic transmit- ters. Anticholinergics have been used as first line treat- ment, despite the side effects (5). Recently, experi- mental studies have demonstrated that a substance isolated from the euphorbia species, a common cac- 53 RESINIFERATOXIN FOR DETRUSOR INSTABILITY RESINIFERATOXIN FOR DETRUSOR INSTABILITY Table 2 – Resiniferatoxin effect on urgency. Urgency Before % After % No 3 10 12 40 Yes 27 90 18 60 Total 30 100 30 100 p = 0.0077 Table 2 – Resiniferatoxin effect on urgency. tions before and after treatment. The mean pre-instil- lation was 47.86 ± 29.64 cm H2O and post instilla- tion was 28.72 ± 30.77 cm H2O (p = 0.0002). The mean reduction was 40%. In 33.33% the involuntary contractions disappeared. The histograms of Figure-2 demonstrate the distribution of contraction amplitude before and af- ter RTX. DISCUSSION Urgency was present in 90% of the patients, and decrease to 60% after RTX instillation (Table- 2). Resiniferatoxin is a potent agonist of type 1 vanilloid receptors in rats and humans (12). These receptors are localized on the dorsal ganglionar neu- rons (12). A study has demonstrated that the increase in bladder volume triggered the first contraction due to the attachment of RTX to the type 1 receptors in the C fibers (7). However, it is not known if desensi- tization or degeneration of the nerve endings of the bladder wall occurs. It was suggested that the C fi- bers were more responsible than the A delta fibers for the involuntary contractions. In normal individu- als, desensitization of these fibers does not provoke any reaction (10), but finding out why sensorial in- formation becomes preponderant in the C fibers may explain the physiopathology of idiopathic detrusor instability. The increased sensorial information in the C fibers could be provoked by the liberation of ex- cessive bladder NGF (nerve growth factor) (13) mus- culature, also observed in infravesical obstruction. Although this study suggests the involvement of C fibers in the etiology of bladder instability, the exist- ence of other abnormalities cannot be ignored, espe- cially because the same improvement was not ob- served in the present study. The fact that the patients were extremely refractory to any type of treatment or presented bad results with any kind of proposed medi- Urge-incontinence was present in 83.33% of the patients, and decrease to 50% after RTX instilla- tion (Table-3). Resiniferatoxin instillation produced no sig- nificant changes in nocturia and enuresis. Supra-pubic pain also did not improve sig- nificantly because 60% reported pain before treatment and 46.67% after treatment (p = 0.134). The urodynamic examination before and after RTX treat- ment did not demonstrate significant alterations of urinary leakage during involuntary contractions (p = 0.077). Despite the fact, no statistically significant difference regarding maximum cystometric capacity was noted (p = 0.585), this finding may be related to the fact that 6 patients presented reduction in bladder capacity post RTX instillation. The histograms in Figure-1 demonstrate the distribution of the patients before and after RTX in- stillation for maximum cystometric capacity. The mean maximum cystometric capacity pre-instillation was 303.9 ± 78.9 and post instillation 341 ± 84.6. There were significant differences between the maximum amplitude of the involuntary contrac- Table 3– Resiniferatoxin effect on urge-incontinence. RESULTS No significant difference was observed in urinary frequency after treatment. Nevertheless, in 3 cases, the condition worsened and an improvement was observed in 5 cases (Table-1). RTX was supplied in 1 mg packages by Sigma Company. They were diluted into 10 µM stock solu- tions in pure ethanol and conserved in dark flasks at 4°C. This solution was then utilized to prepare the Table 1 – Resiniferatoxin effect on urinary frequency. Frequency Before % After % Once / 3 to 5 hours 8 26.66 12 40 Once / 2 hours 11 36.67 8 26.67 More than once/ 2 hours 11 36.67 10 33.33 Total 30 100 30 100 p = 0.207 Table 1 – Resiniferatoxin effect on urinary frequency. 54 REFERENCES 1. Abrams P, Blaivas JG, Stanton SL, Andersen JT: The standardisation of terminology of lower urinary tract function. The International Continence Society Com- mittee on Standardisation of Terminology. Scand J Urol Nephrol Suppl. 1988; 114: 5-19. 2. De Groat WC: A neurological basis for the overactive bladder. Urology. 1997; 50 (suppl. 6A): 36-52. 3. O’Reilly B, Kosaka AH, Knight GF, Chang TK, Ford AP, Rymer JM, et al.: P2X receptors and their role in female idiopathic detrusor instability. J Urol. 2002; 167: 157-64. 4. Brading AF: A myogenic basis for the overactive blad- der. Urology. 1997; 50 (suppl. 6A): 57-67. 5. Chapple CR: Muscarinic receptor antagonists in the treatment of overactive bladder. Urology. 2000; 55 (suppl. 5A): 33-46. 6. Szallasi A, Blumberg PM: Resiniferatoxin and ana- logs provide novel insights into the pharmacology of the vanilloid receptor. Life Sci. 1990; 47: 1399- 408. 7. Silva C, Ribeiro MJ, Cruz F: The effect of intravesical resiniferatoxin in patients with idiopathic detrusor in- stability suggests that involuntary detrusor contractions are triggered by C-fiber input. J Urol. 2002; 168: 575- 9. These authors reported that there were no strong complaints during instillation and that the pain score was 3 on the same analogical scale, similar to the score in this study. Silva and collaborators (7) performed the RTX instillation with urodynamic con- trol to verify the alterations caused and the innumer- ous phasic contractions of the detrusor that began slowly and became more spaced out at the end of the infusion. They reported that the RTX suppressed or diminished the involuntary contractions. 8. Szallasi A, Conte B, Goso C, Blumber PM, Manzini S: Vanilloid receptors in the urinary bladder: regional distribution, localization on sensory nerves, and spe- cies-related differences. Naunyn schmiedebergs arch pharmacol [Multi]. 1993; 347: 624. 9. Yokoyama O, Komatsu K, Kodama K, Yotsuyanagi S, Niikura S, Namiki M: Diagnostic value of intravesical lidocaine for overactive bladder. J Urol. 2000; 164: 340-3. 10. Lazzeri M, Beneforti P, Turini D: Urodynamic effects of intravesical resiniferatoxin in humans: preliminary results in stable and unstable detrusor. J Urol. 1997; 158: 2093-6. RESINIFERATOXIN FOR DETRUSOR INSTABILITY RESINIFERATOXIN FOR DETRUSOR INSTABILITY 440 ± 130 mL during the first 30 days and 90 days. This increase was observed in 92% of a total of 11 women. The maximum cystometric capacity of the 11 patients (472 ± 139 mL) increased 30 days after instil- lation, returning to a volume above the initial volume (413 ± 153 mL) after 90 days. Our findings differ from that data because there was no difference in maximum cystometric capacity pre and post RTX. This may be partly because 6 patients presented reduced capacity after RTX. Silva’s et al. (7) results showed an improve- ment in 91% of the incontinent patients, while in our study improvement was observed in only 33% of the patients, 30 days after instillation. The reason for these different findings is probably that our patients had more severe symptoms and were refractory to other treat- ments. Silva et al (7) used RTX as first line treatment in no refractory DI patients and this could be the rea- son of such difference. The urinary frequency also dif- fered, it was 9.7 ± 3.2 times a day after 30 days de- creasing in relation to the previous frequency, while in our study 73% of the patients reported that after instil- lation they urinated at least once every 2 hours. The urodynamic parameters in this present study did not demonstrate the significant change observed in the Silva’s et al (7) study, with the exception of the ampli- tude of the involuntary contractions that decreased from 40 ± 28.86 to 28.72 ± 30.77 mL. This reduction was noticed in 25 patients. Instillations were well tolerated with no interruption due to pain or discomfort. Further studies are recom- mended to access the role of RTX in this subset of patients. DISCUSSION Urge-Incontinence Before % After % No 5 16.67 15 50 Yes 25 83.33 15 50 Total 30 100 30 100 p = 0.0044 Table 3– Resiniferatoxin effect on urge-incontinence. 55 RESINIFERATOXIN FOR DETRUSOR INSTABILITY B cation should obviously be taken into consideration. N th l ti l i t b d ith There is only one study in literature using RTX f DI (7) I th t t d 50 M l ti f RTX A B Figure 1 – Maximum cystometric capacity before (A) and after (B) resiniferatoxin instillation. A Figure 2 – Maximum involuntary contractions before (A) and after (B) resiniferatoxin instillation. A B Figure 1 – Maximum cystometric capacity before (A) and after (B) resiniferatoxin instillation. B A B A A Figure 1 – Maximum cystometric capacity before (A) and after (B) resiniferatoxin instillation. B A Figure 2 – Maximum involuntary contractions before (A) and after (B) resiniferatoxin instillation. A B B B A Figure 2 – Maximum involuntary contractions before (A) and after (B) resiniferatoxin instillation. There is only one study in literature using RTX for DI (7). In that study a 50 nM solution of RTX was applied in 13 patients with DI and urodynamic were performed at 30 and 90 days after instillation. The maximum cystometric capacity and urinary volume at the first involuntary contraction were measured after instillation. The volume that triggered off the first con- traction increased from a mean of 170 ± 109 mL to cation should obviously be taken into consideration. Nevertheless, partial improvement was observed with RTX, which reduced symptoms of urgency in up to 30% of the patients. The reason for the long-term ef- fect of RTX is still unknown, but may involve the down-regulation mechanism of the C fiber receptors and of neuropeptides such as substance P and CGRP (14). cation should obviously be taken into consideration. Nevertheless, partial improvement was observed with RTX, which reduced symptoms of urgency in up to 30% of the patients. The reason for the long-term ef- fect of RTX is still unknown, but may involve the down-regulation mechanism of the C fiber receptors and of neuropeptides such as substance P and CGRP (14). 56 Correspondence address: Dr. Marcelo Thiel Rua Barão de Jaguara, 601 / 122 Campinas, SP, 13015-001, Brazil Fax: + 55 19 3233-6016 E-mail: thiel7@uol.com.br CONCLUSIONS Intravesical instillation of 50 nM RTX solu- tion clinically improved a small percentage of pa- tients. The maximum amplitude of the involuntary contractions diminished significantly, and in 33% patients the involuntary contractions disappeared. 11. Cruz F, Guimarães M, Silva C, Reis M: Supression of bladder hyperreflexia by intravesical resiniferatoxin. Lancet. 1997; 350: 640-1. 12. Hayes P, Meadows HJ, Gunthorpe MJ, Harries MH, Duckworth DM, Cairns W, et al.: Cloning and func- 57 RESINIFERATOXIN FOR DETRUSOR INSTABILITY tional expressions of a human orthologue of rat vanilloid receptor-1. Pain. 2000; 88: 205-215. tional expressions of a human orthologue of rat vanilloid receptor-1. Pain. 2000; 88: 205-215. 14. Avelino A, Cruz C, Nagy I, Cruz F: Vanilloid receptor 1 expression in the rat urinary tract. Neuroscience. 2002; 109: 787-98. 13. Steers WD: Rat: overview and innervation. Neurourol Urodyn. 1994; 13: 97-118. 13. Steers WD: Rat: overview and innervation. Neurourol Urodyn. 1994; 13: 97-118. Received: September 24, 2003 Accepted: October 20, 2003 Received: September 24, 2003 Accepted: October 20, 2003 Correspondence address: Dr. Marcelo Thiel Rua Barão de Jaguara, 601 / 122 Campinas, SP, 13015-001, Brazil Fax: + 55 19 3233-6016 E-mail: thiel7@uol.com.br 58 58
https://openalex.org/W4387764880	https://www.jmir.org/2023/1/e39995/PDF	English	null	Detecting Prolonged Stress in Real Life Using Wearable Biosensors and Ecological Momentary Assessments: Naturalistic Experimental Study	JMIR. Journal of medical internet research/Journal of medical internet research	2,023	cc-by	11,627	Corresponding Author: p g Rayyan Tutunji, BA, MSc Donders Institute for Brain, Cognition, and Behaviour Radboud University Medical Center Kapittelweg Nijmegen, 6525EN Netherlands Phone: 31 36168494 Email: rayyan.tutunji@donders.ru.nl p g Rayyan Tutunji, BA, MSc Donders Institute for Brain, Cognition, and Behaviour Radboud University Medical Center Kapittelweg Nijmegen, 6525EN Netherlands Phone: 31 36168494 Email: rayyan.tutunji@donders.ru.nl Detecting Prolonged Stress in Real Life Using Wearable Biosensors and Ecological Momentary Assessments: Naturalistic Experimental Study Rayyan Tutunji1, BA, MSc; Nikos Kogias1, MSc; Bob Kapteijns1, MSc; Martin Krentz1, MSc; Florian Krause1, PhD; Eliana Vassena1,2, PhD; Erno J Hermans1, PhD 1Donders Institute for Brain, Cognition, and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands 2Behavioural Science Institute, Radboud University, Nijmegen, Netherlands JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al Original Paper J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 1 (page number not for citation purposes) Abstract (J Med Internet Res 2023;25:e39995) doi: 10.2196/39995 (J Med Internet Res 2023;25:e39995) doi: 10.2196/39995 Abstract Background: Increasing efforts toward the prevention of stress-related mental disorders have created a need for unobtrusive real-life monitoring of stress-related symptoms. Wearable devices have emerged as a possible solution to aid in this process, but their use in real-life stress detection has not been systematically investigated. Objective: We aimed to determine the utility of ecological momentary assessments (EMA) and physiological arousal measured through wearable devices in detecting ecologically relevant stress states. Methods: Using EMA combined with wearable biosensors for ecological physiological assessments (EPA), we investigated the impact of an ecological stressor (ie, a high-stakes examination week) on physiological arousal and affect compared to a control week without examinations in first-year medical and biomedical science students (51/83, 61.4% female). We first used generalized linear mixed-effects models with maximal fitting approaches to investigate the impact of examination periods on subjective stress exposure, mood, and physiological arousal. We then used machine learning models to investigate whether we could use EMA, wearable biosensors, or the combination of both to classify momentary data (ie, beeps) as belonging to examination or control weeks. We tested both individualized models using a leave-one-beep-out approach and group-based models using a leave-one-subject-out approach. Results: During stressful high-stakes examination (versus control) weeks, participants reported increased negative affect and decreased positive affect. Intriguingly, physiological arousal decreased on average during the examination week. Time-resolved analyses revealed peaks in physiological arousal associated with both momentary self-reported stress exposure and self-reported positive affect. Mediation models revealed that the decreased physiological arousal in the examination week was mediated by lower positive affect during the same period. We then used machine learning to show that while individualized EMA outperformed EPA in its ability to classify beeps as originating from examinations or from control weeks (1603/4793, 33.45% and 1648/4565, 36.11% error rates, respectively), a combination of EMA and EPA yields optimal classification (1363/4565, 29.87% error rate). Finally, when comparing individualized models to group-based models, we found that the individualized models significantly outperformed the group-based models across all 3 inputs (EMA, EPA, and the combination). Conclusions: This study underscores the potential of wearable biosensors for stress-related mental health monitoring. However, it emphasizes the necessity of psychological context in interpreting physiological arousal captured by these devices, as arousal can be related to both positive and negative contexts. Moreover, our findings support a personalized approach in which momentary stress is optimally detected when referenced against an individual’s own data. Introduction Stress-related mental disorders such as major depression and anxiety disorders have gained increased recognition in the public eye. While a vast body of research exists regarding these disorders, studies have mostly focused on retrospective assessments of afflicted individuals. More recently, an increased interest has emerged in determining what makes some individuals more resilient to developing these disorders than others [1-4]. Investigating resilience, however, first requires an investigation of individual variation in stress reactivity before the development of psychological illness [1]. Following contemporary transactional frameworks of stress reactivity [5-7], such an approach would require the ability to assess how environmental or psychological stressors trigger biological and psychological responses depending on subjective appraisals of the degree to which an individual’s well-being is threatened. A strong motivation for this effort is the need to establish early warning signals for the onset of stress-related disorders. The ability to unobtrusively detect states of stress in daily life would enable early ecological interventions in those at risk by either flagging risk states to health-care providers or by delivering in-the-moment personalized interventions during these periods [8], thereby preventing or improving negative outcomes in patients [9], and ultimately reducing the societal and economic burdens of psychiatric illness on society [10]. Previous studies have used ecological momentary assessments (EMA) [11] to investigate stress reactivity in real life. These studies use repeated questionnaires (beeps) in daily life to investigate stress-related psychological processes [12-14]. Such methods used in stress-related disorders have identified real-life behavioral patterns that may explain or predict the onset of psychiatric illness [15,16]. They have also given insight into the effects of stress exposure on mood and its links to depression [17]. Despite providing substantial insights, these methods are often intrusive (ie, require active participation of patients), can lack feasibility in psychiatric populations, and may be influenced by careless responses or lower subjective insight into symptoms and associated states [18]. Furthermore, the sparse sampling of subjective states may miss time windows in which stressors occur. More recently, the reliability of such measures has also come into question, showing the importance of accounting for measurement errors [19]. Additionally, within the transactional model, the reliance on subjective assessments of stressors may also be conflated with the outcomes of interest that are being measured, such as mood measures that are often seen as indicators of mental health [20]. KEYWORDS biosensor; devices; ecological momentary assessments; experience sampling; machine learning; mental disorder; mental health; monitoring; physiological; prevention; psychological; smartwatches; stress; wearables biosensor; devices; ecological momentary assessments; experience sampling; machine learning; mental disorder; mental health; monitoring; physiological; prevention; psychological; smartwatches; stress; wearables physiological markers such as skin conductance (SC) and heart rate (HR). These measures have been extensively validated in laboratory-based studies using controlled stress-induction protocols [21], showing increased HR and SC and decreased HR variability in response to stressors [22,23]. However, these autonomic physiological parameters are also associated with general arousal [24], including high-arousal states for positive affect [23]. Thus, using EPA may be more complicated in daily life than in the lab. While acute stress may trigger arousal, arousal itself may not necessarily signal the presence of stress. Both positive and negative affective states may thus be related to arousal measures [25]. The relationship of autonomic physiological responses to stressors in real life is not well understood. Some studies have attempted to investigate the physiology of daily life stress using scenarios or methods that are restrictive or burdensome [26,27]. For instance, a study using wearable biosensors could replicate lab findings to some extent [28]. However, this study lacked an environmental stressor and relied on the assumption that subjective stress measures can be taken as the “ground truth.” Overall reports of stressed states in this study were also relatively low when compared to the nonstress states. Finally, it did not allow probing the consequences of the accumulation of stress over a prolonged period, a key aspect when considering mental health. A recent review of the associations between subjective stress and HR measures also reflects these limitations, showing mixed results and inconsistency in findings [29]. (J Med Internet Res 2023;25:e39995) doi: 10.2196/39995 (J Med Internet Res 2023;25:e39995) doi: 10.2196/39995 J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 1 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 https://www.jmir.org/2023/1/e39995 XSL•FO RenderX XSL•FO RenderX Tutunji et al JOURNAL OF MEDICAL INTERNET RESEARCH J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 2 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 Introduction Participants completed 2 weeks of EMA one during Participants completed 2 weeks of EMA, one during an examination period (ie, stress week) and the other occurring on average 16 days (minimum=10; maximum=33) outside of these periods (ie, control week, demographics in Tables 1 and 2). We maintained at least one week between the end of one week and the start of the other to ensure sufficient recovery time from the stressor. Compliance rates were overall high, with 84% (70.56/84) of surveys completed within the allocated 1-hour window during both weeks. When accounting for missing and poor-quality physiology (EPA) data, completion rates dropped to between 76 and 77% (within ranges for other EMA studies) [32]. Compliance rates did not differ significantly between the weeks for either measure. Gender distribution was similar to that of students enrolled at the university (13739/24104, 57% female; according to the Radboud University website). We were unable to fully counterbalance the order of weeks due to the early termination of recruitment but instead controlled for it in all statistical analyses. Participants also filled out questionnaires and participated in magnetic resonance imaging sessions, which are outside the scope of this study and will be reported elsewhere. Participants completed 2 weeks of EMA, one during an examination period (ie, stress week) and the other occurring on average 16 days (minimum=10; maximum=33) outside of these periods (ie, control week, demographics in Tables 1 and 2). We maintained at least one week between the end of one week and the start of the other to ensure sufficient recovery time from the stressor. Compliance rates were overall high, with 84% (70.56/84) of surveys completed within the allocated 1-hour window during both weeks. When accounting for missing and poor-quality physiology (EPA) data, completion rates dropped to between 76 and 77% (within ranges for other EMA studies) [32]. Compliance rates did not differ significantly between the weeks for either measure. Gender distribution was similar to that of students enrolled at the university (13739/24104, 57% female; according to the Radboud University website). We were unable to fully counterbalance the order of weeks due to the early termination of recruitment but instead controlled for it in all statistical analyses. Participants also filled out questionnaires and participated in magnetic resonance imaging sessions, which are outside the scope of this study and will be reported elsewhere. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 3 (page number not for citation purposes) Introduction These issues indicate a growing need for novel and more reliable methods for passive and ambulatory mental-health monitoring. To this end, we aimed to investigate the ability of active EMA measures, passive EPA monitoring, and the combination of these 2 methods to detect stress in real life. We investigated a population of first-year medical and biomedical students known to experience increased psychological distress [30]. Participants collected EMA and EPA data once during a week culminating in a high-stakes examination (ie, stress week) and another without (ie, control week). In line with the transactional framework of the stress response, this naturalistic experimental design allowed us to objectively manipulate prolonged stressor exposure while allowing EMA-based assessments of stress appraisals (through measures of event, activity, and social stress; see below) and separating these from EMA-based measures of mood reactivity as well as EPA-based measures of physiological reactivity to stressors. We first validated our protocol by testing between-week differences in EMA-based subjective appraisals of stress. We then assessed the impact of examinations and stress weeks on mood and physiology outcomes. Finally, we used individualized machine learning models to classify per time point (beep) which week participants were in using either mood, physiological, or a combination of both measures. This was done to investigate the utility of wearables as passive monitors of stress in ecologically relevant scenarios above mood-related EMA measures. We predicted increased autonomic physiological responses and negative affect and decreased positive affect in stress weeks. We expected that both EPA and EMA measures would successfully identify prolonged stress The emergence of widely accessible wearable biosensors has raised the question of whether these devices can be used for ecological physiological assessments (EPA), either as an add-on or an alternative to EMA, in mental health monitoring. Wearable biosensors offer continuous recording of autonomic https://www.jmir.org/2023/1/e39995 XSL•FO RenderX XSL•FO RenderX Tutunji et al JOURNAL OF MEDICAL INTERNET RESEARCH states and predicted that models combining EPA and EMA would outperform the single models. The study time line is shown in Figure 1. Figure 1. Study time line. Diagram portraying the sequence of participation in the study with counterbalanced weeks. Ecological momentary assessment (EMA) surveys included stress and affect assessments 6 times a day, in addition to sleep and evening questionnaires once a day. The wristband continuously measured physiological arousal, temperature, and movement. Assessing Daily-Life Stress Through EMA and EPA g y g The comparison of the stress week (examination week) versus the control week allowed us to determine individualized patterns of stress reactivity. During these weeks, participants received 6 surveys a day at fixed intervals through SMS text message links. Participants were given a 1-hour window to fill out the surveys (like previous studies [33]). Individual surveys are referred to as beeps in the EMA literature. Surveys assessed different psychological aspects related to stress, including event, activity, social, and physical stress, as well as positive affect (PA) and negative affect (NA) outcomes. The first questionnaire of the day contained a sleep quality assessment, and the last included a self-reflection questionnaire. Participants were instructed to wear an Empatica E4 wristband (Empatica) recording ambulatory EPA data throughout both weeks (collected passively, continuously, and in the background). Participants were instructed to charge and synchronize the watch to researcher-specific accounts once a day for 1 hour. A detailed explanation was given to participants on the E4 operation with a practice session during the intake interview. The E4 devices collected blood pulse volume, electrodermal activity, 3-axis movement, and body temperature. EMA surveys consisted of questions regarding subjective stress used for validating our experimental paradigm and mood questions (PA and NA) relating to our subjective outcome measures filled in on a 7-point Likert scale. Questions in the validation set probed four types of stress, as follows: (1) event-related stress assessed the most prominent event that occurred in between EMA beeps; (2) activity-related stress questions probed the activity participants were engaged in upon receiving the beep; (3) social-related stress addressed stress that may arise from the social context participants were present in (either being alone or with someone); and (4) physical-related stress was used as a control measure to account for environmental and physical demands. Mood outcome questions consisted of 4 items assessing positive mood and 5 items assessing negative mood based on the positive and negative affect schedule (PANAS), as validated in previous work [34]. EMA items on a reversed scale were first inverted. Items for each scale were summed to create a single score for each of the scales (ie, a single measure for event, activity, social, and physical stress). Total item scores were then rescaled, and a participant-centered measure was derived. Surveys that were not filled out within the assigned time window were excluded from further analyses. Experimental Design rst and third quantiles indicating 50% of participants had completion rates in the given range. EPA data cleaning was performed using Python (version 3.6.1; Python Software Foundation) [35]. Additional packages used for preprocessing included NumPy (version 1.18.1; Travis Oliphant) [36] and pandas (version 1.0.3; Wes McKinney) [37]. Time stamps for each survey instance were used to classify surveys as belonging to a stress or control week. Ten-minute time windows before each survey were selected for the extraction of physiology features acquired from the E4. Preprocessed interbeat interval (IBI) data were deemed too sparse to offer meaningful temporal domain analysis, with an average of 27% of IBIs successfully detected in our selected time window. This is within the margins of the manufacturer’s signal loss estimates for daily use. We instead selected average HR features from the resulting processed files from Empatica. The devices use a strict proprietary detection algorithm for the detection of IBIs, so these files can be used with minimal processing to derive global HR features. These features included the mean, minimum, and maximum HR. Raw SC was processed for offline use with the PyPhysio package (version 2.1; Andrea Bizzego) [38]. A minimum threshold of 0.01 µsiemens was set for the SC levels deemed of acceptable quality based on previous recommendations of a threshold between 0.01 and 0.05 µsiemens [39]. Data were first despiked to remove artifacts due to sudden hand motions using standard settings in the library. Data were then denoised to remove remaining artifacts through windowed filtering of changes in the signal greater than 0.02 µsiemens between subsequent samples. Additionally, an elliptic filter with a cut-off frequency set between 0.8 and 1.1 was applied to the data. SC data were subsequently deconvolved using a Bateman impulse response function into phasic and tonic components from which specific features were extracted (mean tonic activity, magnitude, area under the curve, and the number of phasic responses). The raw temperature measures were used to calculate the mean skin temperature as well as the slope as a function of change in skin temperature within the acquired time window. A total of 2 participants had a watch with faulty temperature sensors. These measures were substituted from the population mean and SD to avoid the loss of participants’ data due to missing data points in statistical models. The other sensors on this device were tested, and no errors were detected in other recordings. Assessing Daily-Life Stress Through EMA and EPA The same was done for outcome measure items relating to PA and NA. Experimental Design Finally, the root mean squared displacement in each time window was calculated from the accelerometer data. The extracted features were collected into a single data frame used for statistical analysis. https://www.jmir.org/2023/1/e39995 Experimental Design We recruited 84 right-handed, first-year bachelor’s students in the medical or biomedical science majors from Radboud Health Academy spanning 3 academic years (2017, 2018, and 2019). Participants were recruited through flyer distribution, presentations in common lecture halls, and web-based advertisements. A participant withdrew during testing, resulting in a total sample size of 83 participants used in the analysis. The programs were selected due to their structured examination weeks that occur every 5th and 10th week of a semester, allowing us to examine a period with higher stress levels during examination weeks as an ecological prolonged stressor. Right-handed participants were selected as participants were instructed to wear watches on their nondominant hand to reduce motion-related noise, with studies showing better signal from the left wrist than the right [31]. Only participants with no history of psychiatric illness were included in the study. Recruitment was stopped following the COVID-19 outbreak (March 2020). Table 1. Descriptive statistics showing sex and program distributions, and week ordering. Table 1. Descriptive statistics showing sex and program distributions, and week ordering. Table 1. Descriptive statistics showing sex and program distributions, and week ordering. Table 1. Descriptive statistics showing sex and program distributions, and week ordering. Students, n (%) Demographic items Sex, n (%) 51 (61.4) Female 32 (38.6) Male Course program, n (%) 61 (73.5) Medicine 22 (26.5) Biomedical sciences First week, n (%) 27 (32.5) Examination week 56 (67.5) Control week Students, n (%) Demographic items Sex, n (%) Female Male Course program, n (%) Medicine Biomedical sciences First week, n (%) Examination week Control week Control week J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 3 https://www.jmir.org/2023/1/e39995 (page number not for citation purposes) O X https://www.jmir.org/2023/1/e39995 XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al Table 2. Completion rates for each of the examination and control weeks for both ecological momentary assessment (EMA) and ecological physiological assessment (EPA) measures. Control week Examination week Compliance Rates Third quantile, n (%)a Mean (%) First quantile, n (%)a Third quantile, n (%)a Mean (%) First quantile, n (%)a 40 (95) 35.89 (85) 34 (81) 39 (93) 35.51 (85) 34 (81) EMA 37 (88) 32.36 (77.05) 29 (69) 37 (88) 32.15 (76.55) 29 (69) EMA with EPA aFirst and third quantiles indicating 50% of participants had completion rates in the given range. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 4 (page number not for citation purposes) Examination Periods are Associated With Increased Self-Reported Stress We found a significant increase in prominent stressful events (ie, event-related stress, β=.30; 95% CI 0.18-0.42; P<.001) and current reports of stress (ie, activity-related stress, β=.51; 95% CI 0.30-0.71; P<.001) in the examination stress versus control week. Social stress was not significantly different between the 2 weeks. The control items measuring physical stress also did not differ significantly between the weeks, showing that increases in subjective stress were likely due to our experimental manipulation instead of environmental or physical changes (Figure 2A, Table S1 in Multimedia Appendix 1). As anticipated, not all beeps in stress weeks were subjectively reported as stressful, while some beeps during the control week were subjectively rated as stressful. To quantify this, subjective stress variables for event, social, and activity stress were aggregated across both weeks. A median split was then used to estimate the percentage of incongruent self-report beeps (ie, false positives in stress weeks and false negatives in control weeks). On average, across participants, 45% (2157/4794) of the beeps yielded self-reported stress incongruent with the week type. Machine learning models using self-reported stress assessments in a LOBO approach to classify week types achieved similar error rates, with 43% (2016/4794) of beeps being classified as the wrong week type. Ethical Considerations randomforest packages) [40,41]. Initial analyses examined overall differences in subjective stress between the 2 weeks to establish the validity of the experimental manipulation. We then tested for the effect of an examination week on affect and physiology. We additionally tried replicating previous findings associating momentary stress with physiology and mood. Mediation analysis was then used to explain the apparent differences in the relationships between the week type and momentary analyses. Additional covariates were added to all models. Covariates were selected to control for potential population differences and behavioral differences that may arise from being in an examination period. These covariates can be divided into subject-level, day-level, and beep-level covariates. Subject-level covariates modeled as fixed effects included sex, study program, and order of the weeks (ie, stress or control week first). Day-level covariates included the days relative to start (ie, day 1, day 2, day 3, etc), beep number, self-reported sleep duration, and the previous night’s alcohol consumption. Beep-level covariates modeled included hunger, caffeine intake, exercise, and sexual activity. Additionally, ambient temperature and accelerometer-derived movement were modeled for the EPA models. For further details regarding the surveys, code, and statistical modelling approaches see Multimedia Appendix 1 [42] All procedures carried out were approved by the regional medical ethical review board (METC Oost-Nederland, protocol ID 2014-288). Written informed consent was obtained from all participants in Dutch following an intake interview where a detailed explanation of the procedures was carried out. Participants were given unique identifiers to maintain anonymity for all data acquired, with encrypted key files maintained by selected study personnel. In order to ensure the anonymity of the wearable data, participants were also provided with a study-specific account for data synchronization instead of personal accounts. Participants who completed all parts of the study were awarded 150 euros (US $158). The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Tutunji et al Tutunji et al J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 5 (page number not for citation purposes) Statistical Analysis All statistical analyses were conducted in R (version 3.6.1, Ross Ihaka and Robert Gentleman) using generalized linear mixed effects models and random forest models (lmer and https://www.jmir.org/2023/1/e39995 https://www.jmir.org/2023/1/e39995 J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 4 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Machine Learning Models One of our goals was to assess the usability of ambulatory, nonintrusive measures to determine whether someone is currently in a stressed state. To this end, random forest models were used to determine the ability to classify whether participants’ beeps were in the stress or control week using the collected EMA mood and ambulatory EPA outcome data. Due to the subjective nature of mood items, participant-centered mood was used in all models. We conceptualized mood and physiology as outcomes of stressed states based on previous findings [17,43]. Individualized models were estimated using a Leave-One-Beep-Out (LOBO) approach at a single-participant level, where models were trained on individuals’ n-1 beep data and tested on the removed beep, repeating until all beeps had been removed. This is similar to the Leave-One-Trial-Out method used in other fields [44]. A total of 3 models were tested, as follows: Model 1 tested the ability to classify week type from (momentary) PA and NA, Model 2 from EPA data, and Model 3 from the combination of both. Models were tested against a bootstrap error distribution (n=10,000), with group effects tested using 2-tailed paired sample t tests against the mean subject-level bootstrap error. We tested the generalizability of the random forest models to a population level using a Leave-One-Subject-Out (LOSO) analysis in which models were trained on N-1 participants data set and tested on the removed participant, repeating until each participant had been removed once from the data set. Model predictions using the LOBO were then compared to those of the LOSO method to estimate the generalizability of machine learning models based on the data. In accordance with our expectations, we also saw an increase in NA (β=.12; 95% CI 0.08-0.17; Pfdr<.001), and a decrease in PA (β=–.08; 95% CI –0.11 to –0.05; Pfdr<.001) during the stress week (Figure 2B). Unexpectedly, we found a decrease in physiology arousal-related measures during the examination week, including the number of SC responses (log-mean –0.27; 95% CI –0.42 to –0.12; Pfdr<.001), and maximum HR (β=–.10, 95% CI –0.16 to –0.03; Pfdr=.003). Figure 2B, Table S2 in Multimedia Appendix 1. https://www.jmir.org/2023/1/e39995 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al Figure 2. Fixed effects estimates of the between-week difference. (A) Event-related stress (pertaining to the most prominent event since the last survey) and activity-related stress (relating to the current activity participants are engaged in) are significantly higher in the stress week compared to the control week. (B) This is accompanied by increased negative affect, decreased positive affect, and decreases in averages of multiple arousal-related physiological measures. Error bars represent 95% CI. AUC: area under the curve; HR: heart rate; SC: skin conductance. measures. Error bars represent 95% CI. AUC: area under the curve; HR: he Momentary Subjective Stress is Associated With Mood and Physiology p 9 ; ; Momentary Subjective Stress is Associated With Mood and Physiology To explore the dynamics underlying the unexpected average decrease in measures of physiological arousal during the stress week, we investigated the link between in-the-moment fluctuations in subjective stress and outcome measures (ie, within the same beep mood and physiological arousal). We found a positive association between NA and activity-related (β=.06; 95% CI 0.01-0.12; Pfdr=.05), social (β=.22; 95% CI 0.18-0.27; Pfdr<.001), and physical stress (β=.15; 95% CI 0.12-0.18; Pfdr<.001). The opposite was true for PA for event-related (β=–.12; 95% CI –0.19 to –0.06; Pfdr<.001), activity-related (β=–.17; 95% CI –0.25 to –0.09; Pfdr<.001), social (β=–.28; 95% CI –0.34 to –0.22; Pfdr<.001), and physical stress (β=–.23; 95% CI –0.27 to –0.18; Pfdr <.001). The magnitude of SC responses was associated with activity (β=.08; 95% CI 0.02-0.15; Pfdr=.02), event (β=.07; 95% CI 0.02-0.13; Pfdr=.02) and physical stress (β=.03; 95% CI 0.00-0.06; Pfdr=.04). For HR measures, mean (β=–.04; 95% CI 0.08 to –0.01; Pfdr=.04) and minimum (β=–.02; 95% CI –0.03 to –0.00; Pfdr=.01) HR were negatively associated with social stress. Thus, within-beep fluctuations in subjective stress are associated with expected mood changes and increases in physiological arousal (Figure 3, Table S3 in Multimedia Appendix 1). ; activity-related (β=–.17; 95% CI –0.25 to –0.09; Pfdr<.001), social (β=–.28; 95% CI –0.34 to –0.22; Pfdr<.001), and physical stress (β=–.23; 95% CI –0.27 to –0.18; Pfdr <.001). The magnitude of SC responses was associated with activity (β=.08; 95% CI 0.02-0.15; Pfdr=.02), event (β=.07; 95% CI 0.02-0.13; Pfdr=.02) and physical stress (β=.03; 95% CI 0.00-0.06; Pfdr=.04). For HR measures, mean (β=–.04; 95% CI 0.08 to –0.01; Pfdr=.04) and minimum (β=–.02; 95% CI –0.03 to –0.00; Pfdr=.01) HR were negatively associated with social stress. JOURNAL OF MEDICAL INTERNET RESEARCH Thus, within-beep fluctuations in subjective stress are associated with expected mood changes and increases in physiological arousal (Figure 3, Table S3 in Multimedia Appendix 1). Momentary Subjective Stress is Associated With Mood and Physiology J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 6 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Momentary Subjective Stress is Associated With Mood and Physiology To explore the dynamics underlying the unexpected average decrease in measures of physiological arousal during the stress week, we investigated the link between in-the-moment fluctuations in subjective stress and outcome measures (ie, within the same beep mood and physiological arousal). We found a positive association between NA and activity-related (β=.06; 95% CI 0.01-0.12; Pfdr=.05), social (β=.22; 95% CI 0.18-0.27; Pfdr<.001), and physical stress (β=.15; 95% CI 0.12-0.18; Pfdr<.001). The opposite was true for PA for event-related (β=–.12; 95% CI –0.19 to –0.06; Pfdr<.001), J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 6 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 XSL•FO RenderX Tutunji et al Figure 3. Effect estimates for the associations between within-beep fluctuations in subjective stress and measures of mood and physiology. (A) Event-related, (B) activity-related, (C) social-related, and (D) physical-related stress are generally associated with a decrease in positive affect, an increase in negative affect, and increases in some of the measures of physiological arousal. P values are corrected for multiple comparisons using false discovery rate corrections. Error bars represent 95% CIs. AUC: area under the curve; HR: heart rate; SC: skin conductance. ery rate corrections. Error bars represent 95% CIs. AUC: area under the curve; HR: heart rate; SC: skin conductance. tive Mood is Related to Increased Arousal and iates Week Changes the number of SC responses (β=.08; 95% CI Pfdr=.04), and mean (β=.01; 95% CI 0.001-0.02; i i (β 01 95% CI 0 001 0 02 P 03) d the number of SC responses (β=.08; 95% CI 0.02-0.06; Pfdr=.04), and mean (β=.01; 95% CI 0.001-0.02; Pfdr =.01), minimum (β=.01; 95% CI 0.001-0.02; Pfdr=.03), and maximum HR (β=.01; 95% CI 0.001-0.02; Pfdr=.03; Figure 4, Table S4 in Multimedia Appendix 1). Thus, in addition to subjective stress, PA is also positively associated with momentary physiological arousal. the number of SC responses (β=.08; 95% CI 0.02-0.06; Pfdr=.04), and mean (β=.01; 95% CI 0.001-0.02; Pfdr =.01), minimum (β=.01; 95% CI 0.001-0.02; Pfdr=.03), and maximum HR (β=.01; 95% CI 0.001-0.02; Pfdr=.03; Figure 4, Table S4 in Multimedia Appendix 1). Thus, in addition to subjective stress, PA is also positively associated with momentary physiological arousal. the number of SC responses (β=.08; 95% CI 0.02-0.06; Pfdr=.04), and mean (β=.01; 95% CI 0.001-0.02; Pfdr =.01), minimum (β=.01; 95% CI 0.001-0.02; Pfdr=.03), and maximum HR (β=.01; 95% CI 0.001-0.02; Pfdr=.03; Figure 4, Table S4 in Positive Mood is Related to Increased Arousal and Mediates Week Changes To investigate whether the observed decreases in physiological arousal during stress weeks could instead be linked to reduced PA, we tested the within-beep association between affect and physiological arousal. Increased PA was related to increase in To investigate whether the observed decreases in physiological arousal during stress weeks could instead be linked to reduced PA, we tested the within-beep association between affect and physiological arousal. Increased PA was related to increase in Multimedia Appendix 1). Thus, in addition to subjective stress, PA is also positively associated with momentary physiological arousal. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 7 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al Figure 4. Relationship between momentary affect and physiology. Arousal-related physiological measures (magnitude of skin conductance responses and mean and minimum heart rate) were linked to positive affect but not to negative affect. P values are corrected for multiple comparisons using false discovery rate correction. Error bars represent 95% CI. AUC: area under the curve; HR: heart rate; SC: skin conductance. (based on affect), 36.11% (SD 2.72%) for Model 2 (based on physiology), and 29.87% (SD 3.45%) for Model 3 (based on affect and physiology combined). Hence, the combined model outperformed the single-variable models. All models performed significantly above chance on an individual level for all but one subject (Figure 5, Multimedia Appendix 1). Group-level effects were further tested with 2-tailed paired-samples t tests with FDR correction comparing the LOBO models to the mean bootstrapped error. Model 1 (affect, meandiff –16.29; t80=–64.06; Pfdr<.001), Model 2 (physiology, meandiff –13.87; t78=–50.38; Pfdr<.001), and Model 3 (combination, meandiff –19.45; t78=–48.94; Pfdr<.001) all performed above chance. Paired-samples, 2-tailed t tests comparing the within-participant error rates between the LOBO models showed that Model 3 (ie, combined EMA and EPA) outperformed Model 1 using mood alone (meandiff 3.64; t78=19.20; P<.001), which in turn outperformed Model 2 using EPA alone (meandiff 2.60; t78=14.65; P<.001). While overall the EMA mood models performed better, for some participants, models 1 and 2 had almost equivalent performance. Next, to confirm that the observed average decrease in physiological arousal observed during the stress weeks is due to the decrease in PA, we assessed whether PA statistically mediated the effects of week type on reductions in physiological arousal. We specifically focused on the arousal measures linked to subjective stress and PA: the number of SC responses and their magnitudes. Results indicated that PA significantly mediated the relationship between SC magnitude and week type (7.3%, mediating estimate=–0.013; 95% CI –0.03 to 0.00; P=.03) but not fully (direct estimate=–0.166; 95% CI –0.23 to –0.10; P<.001), indicating potential additional mechanisms are at play. The effect of week type on the number of SC responses was not mediated by PA. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 8 (page number not for citation purposes) Tutunji et al 7 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 https://www.jmir.org/2023/1/e39995 XSL•FO RenderX XSL•FO RenderX Machine Learning Classification of Beeps Using Mood and Physiology We next examined to what extent prolonged stress (ie, stress vs control week) can be classified from individual beeps using machine learning based on affect, physiological arousal, or a combination of both using individualized LOBO models. The mean subject-level error was 33.45% (SD 2.21%) for Model 1 https://www.jmir.org/2023/1/e39995 XSL•FO RenderX XSL•FO RenderX Figure 5. Random-forest classification error estimates. Average error estimates and error bars (representing the SE of the mean) for each of the random forest models. Combinations of mood and physiology yield superior classification, and individually trained and tested models (Leave-One-Beep-Out [LOBO]) perform better than models trained on group-level data (Leave-One-Subject-Out [LOSO]). Chance levels estimated from the permutation test and 95% CI are shown in blue. Significance levels between bars indicate between model comparisons, and above bars indicate model comparisons to chance levels. P values are corrected for multiple comparisons using false discovery rate. Individualized Models Offer Better Predictions Than Group-Based Models We next investigated the generalizability of these models from an individualized approach to a population-level one (using group-level classification) through LOSO cross-validation. M1 showed that only model 1 (affect, meandiff –3.53; t80=–3.59; Pfdr=.005), and model 3 (combination, meandiff –6.55; t78=–6.81; Pfdr<.001) performed better than chance. Model 2 did not perform above chance (physiology, meandiff –1.34; t78=–1.54; Pfdr>.99). Tutunji et al JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al Figure 5. Random-forest classification error estimates. Average error estimates and error bars (representing the SE of the mean) for each of the random forest models. Combinations of mood and physiology yield superior classification, and individually trained and tested models (Leave-One-Beep-Out [LOBO]) perform better than models trained on group-level data (Leave-One-Subject-Out [LOSO]). Chance levels estimated from the permutation test and 95% CI are shown in blue. Significance levels between bars indicate between model comparisons, and above bars indicate model comparisons to chance levels. P values are corrected for multiple comparisons using false discovery rate. [ ]) p g p ( j [ ]) p and 95% CI are shown in blue. Significance levels between bars indicate between model comparisons, and above bars indicate model comparisons to chance levels. P values are corrected for multiple comparisons using false discovery rate. showed that only model 1 (affect, meandiff –3.53; t80=–3.59; Pfdr=.005), and model 3 (combination, meandiff –6.55; t78=–6.81; Pfdr<.001) performed better than chance. Model 2 did not perform above chance (physiology, meandiff –1.34; t78=–1.54; Pfdr>.99). Individualized Models Offer Better Predictions Than Group-Based Models We next investigated the generalizability of these models from an individualized approach to a population-level one (using group-level classification) through LOSO cross-validation. M1 using affect (45.85%, SD 9.50%), M2 using physiology (48.42%, SD 8.05%), and M3 using the combination (42.44%, SD 9.00%) were again tested against their bootstrapped counterparts. We next investigated the generalizability of these models from an individualized approach to a population-level one (using group-level classification) through LOSO cross-validation. M1 using affect (45.85%, SD 9.50%), M2 using physiology (48.42%, SD 8.05%), and M3 using the combination (42.44%, SD 9.00%) were again tested against their bootstrapped counterparts. We additionally directly compared the classification errors between the individualized and group models for each participant using a paired-sample t test with FDR correction. All individualized LOBO models performed better than the group-level LOSO models. LOSO Model 1 (mood) performed significantly worse than the equivalent LOBO model (meandiff 11.43; t80=12.17; Pfdr<.001). LOSO Model 2 (physiology) was also significantly worse than the LOBO counterpart (meandiff For some individual participants, LOSO models performed significantly above chance level (Model 1-affect n=45, 54.1%; Model 2-physiology n=30, 37.9%; and Model 3-combination n=55, 69.6%) in classifying week type (Multimedia Appendix 1). Group-level analysis using a 2-tailed paired sample t test J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. Discussion This study investigated physiological and psychological responses to ecological stressors in daily life (ie, examination weeks in students) to determine the usability of passive monitoring technologies for detecting prolonged stress. We used EMA and EPA to track subjective stress, mood, and arousal-related physiology. Our findings confirmed an overall increase in subjective stress during examination weeks. As hypothesized during the stress week, NA increased and PA decreased. Contrary to what was expected, lower SC and HR arousal measures were recorded during the stress week. At a beep-to-beep time scale, increased subjective stress was associated with increased NA, decreased PA, and increased SC responses. Interestingly, PA was also associated with increased SC responses and partially mediated the between-week differences in SC we found. Thus, the observed decreases in physiological arousal measures were (at least partially) due to a reduction in PA. Using a machine learning approach, we showed that the combination of individual mood and physiology was best able to detect whether individual beeps stemmed from stress or control weeks. We conclude that passive monitoring with wearable biosensors can detect prolonged stress, highlighting the importance of mood measures to dissociate positive and negative arousal. In line with previous work, the stress week resulted in increases in self-reported stress, validating our paradigm [45]. We observed expected changes in mood, with increased NA and decreased PA. However, arousal measures were surprisingly reduced during the stress week. The observed overall decrease in physiological arousal during stress weeks appears at odds with the positive association between within-beep subjective stress and increased arousal in our analysis and previous works [22,26,28]. This finding reveals a dissociation between prolonged and acute stress. While prolonged stress leads to increased within-beep peaks in self-reported acute stress, it also results more generally in decreased PA and decreased overall average arousal. Our results suggest that reduced arousal may be linked to reduced PA seen in the stress weeks (irrespective of peaks in subjective stress). Our mediation analysis corroborates this mechanistic link, confirming that PA partially mediates the effect of week type on reduced arousal. While this may seem counterintuitive, SC and HR measures are known to respond to both positive and negative events, showing that physiological arousal is not valence specific [23,46]. This fits with a recent review observing the most consistent link between high-arousal subjective states and increased HR measures [29]. Tutunji et al 9 (page number not for citation purposes) J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 9 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al in positive mood that persist outside of peak moments of acute stress. 8.11; t78=8.61; Pfdr<.001). LOSO Model 3 (combination) similarly performed worse than the LOBO model counterpart (meandiff 12.61; t78=11.74; Pfdr<.001). In sum, individual models vastly outperformed group-based models. We subsequently tested the ability of machine learning models to classify individual beeps as stemming from stress or control weeks with physiology, mood, or a combination of both. Physiology models could classify beeps almost as well as mood models (3.85% difference on average). However, and more importantly, combination models showed the highest accuracy. Hence, the addition of mood questions to physiological arousal provides valuable information for prolonged stress detection. This converges with the mixed models and mediation results: accounting for valence through mood is necessary to distinguish stress-induced from PA-induced arousal. Our findings provide a mechanistic explanation for why previous studies using SC trigger-based EMA to detect stress captured positive arousal instead [46]. In addition to demonstrating that affect and arousal offer better than chance classification levels, we also show that they achieve higher accuracy in classifying week type than classification based on a median split across explicit subjective stress measures. Using a combination of EPA and mood EMA may also reduce issues related to measurement errors seen within EMA. Additionally, assessing mood and physiological arousal may offer a more nuanced measure of stress states that is not dependent on activities or events that occurred since the previous beep. This approach is also common in laboratory research on stress, where mood questionnaires and physiological arousal measures are often used to quantify stress [48]. In sum, combining a wearable biosensor with minimally invasive mood assessment might offer the best approach to detecting stress in both healthy and clinical populations, offering a more feasible approach than full EMA batteries. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 10 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 Discussion This was an intentional design choice: the goal was to test the ability of physiology and mood measures combined to detect momentary stress states during heightened periods of stress, which may be required, for example, for detecting warning signals. Furthermore, the accuracy achieved with our real-life models is also on par with more recent laboratory studies classifying affect from wearables and infrared cameras [50]. Hence, the classification accuracy found in this study represents what might occur in the general population during real-life stress periods (including stressful moments in regular weeks and regular moments in stressful weeks) and supersedes directly asking about stress. It may also be argued that the uncontrolled nature of the study is a detriment to the findings and has an impact on the reliability of the proposed measure. However, the ecological validity of this study is rather a strength in providing a necessary translation of laboratory measures to a real-life setting [56]. Additionally, we controlled for several potential confounds that may impact the reliability of our measures, such as those differences in behavior across weeks (ie, alcohol intake, sleep, caffeine, and exercise). Finally, and worth noting, this study focused on a relatively smaller sample of students, which may limit generalizability to other contexts. However, we also note that an examination stressor may resemble many real-life stressors and daily hassles, such as work deadlines. Yet, these results may not generalize to more severe, traumatic, and stressful life events. Acute stressful events may lead to very different arousal responses, and future research is needed to address this topic. However, having to rely on the occurrence of such events in a study may prove difficult and would require longer periods of assessment in the hopes of capturing these types of acutely stressful moments. Indeed, this is an issue that has already been addressed by previous attempts at classifying stress from such devices [28]. This study provides early evidence for the successful detection of prolonged periods of stress in individuals. However, some limitations warrant discussion. This study was purely cross-sectional, meaning that we are unable to make connections in how our findings can be used for predicting mental health outcomes and resilience in the long term. Prospective and longitudinal designs with explicit measures of resilience are needed for this. Data Availability The data sets generated during and/or analyzed during this study are not publicly available due to the sensitive and potentially identifiable nature of the data but are available from the corresponding author on reasonable request. Data repository also includes all scripts used in preprocessing and statistical analysis, which are also included on the GitHub repository linked in Multimedia Appendix 1. Discussion Within a theoretical framework of affect dynamics, these findings also align with the circumplex theory of emotion and valence, linking the 2 on a grid-like schema of valence and arousal [47]. Thus, the net effect of prolonged stress exposure stems from a reduction in overall arousal driven by reductions Besides demonstrating the utility of physiological monitoring, our results highlight the importance of individualized approaches in stress detection. Classification models trained and tested on individuals’ own data (LOBO) performed significantly better than those trained on group data (LOSO). Our individualized approach offers drastic improvements in the classification of stress states in comparison with group approaches [28]. This supports findings in previous work, where large between-participant differences in dynamic ranges of responses limited applications of machine learning at the population level, pointing toward the need for individualization [28]. Intuitively, the same experience can generate different physiological and psychological responses in different individuals based on a multitude of factors, such as sex, appraisal, or clinical traits. For example, patients with anxiety may display a very different physiological response to stress than those with depression (hyper- vs hypoactivation) [49]. This is a key strength of the current approach, fully aligned with recent developments in personalized psychiatry: individualized models allow for greater prediction accuracy than a one-size-fits-all approach. Worth noting is that classification accuracy of our machine learning models was relatively low in this study compared to many other ML studies. Previous studies found limited applicability of such algorithms due to variance in ranges of physiological responses between participants [28]. However, by using a within-participant design, we circumvent such issues. Importantly, lower accuracy stems from the ecological design: J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 10 (page number not for citation purposes) XSL•FO RenderX Tutunji et al JOURNAL OF MEDICAL INTERNET RESEARCH models can also partially explain variance related to wrist displacement. Furthermore, we refrain from including HR variability metrics in our analysis for this specific reason, and such measures, while more specific to sympathetic nervous system activity, are also more susceptible to noise. our models did not classify weeks but rather individual beeps within the weeks (approximately 70% of beeps were correctly assigned to the weeks in the best models). Through the median split of our data based on self-reported stress, we clearly demonstrate that even during a stress week, participants are not stressed 100% of the time. Discussion This is also important within the context of promoting resilience, as prospective detection of vulnerability is the next step in this line of research, with the aim of identifying early warning signals [51]. More research (some of which is currently underway [52]) will be needed to extend these results into prospective stress detection algorithms [49]. Additionally, it is important to consider the reliability and validity of the devices used in this study. Previous research has shown that these devices offer reliable measures of SC for stress detection [53,54], but this may not extend to daily life scenarios, which are inherently noisier [55]. While we cannot eliminate noise from our data, preprocessing steps such as despiking and filtering allowed us to derive a cleaner signal. Additionally, the inclusion of an accelerometer-derived motion component in our In conclusion, this study shows that EPA may be used for monitoring stress-related mental health but highlights the importance of affect ratings to dissociate changes in arousal due to stress versus PA. A combination of physiology and mood measures is optimal for detecting prolonged stress, and personalized approaches to modeling these variables are necessary. If successfully implemented at a wider scale, our findings may have implications for disease prevention, potentially helping to reduce the overall disease burden of stress-related disorders through personalized early-warning systems and treatment strategies. Acknowledgments The authors would like to acknowledge Mike van Engelenburg from the technical group for his assistance with setting up the EMA platform and Annemieke Smeets for her assistance with recruitment. This work was supported by the European Research Council (ERC2015-CoG 682591). J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 11 (page number not for citation purposes) References 1. Kalisch R, Baker DG, Basten U, Boks MP, Bonanno GA, Brummelman E, et al. The resilience framework as a strategy to combat stress-related disorders. Nat Hum Behav 2017;1(11):784-790 [doi: 10.1038/s41562-017-0200-8] [Medline: 31024125] 1. Kalisch R, Baker DG, Basten U, Boks MP, Bonanno GA, Brummelman E, et al. 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Stuyck H, Dalla Costa L, Cleeremans A, Van den Bussche E. Validity of the Empatica E4 wristband to estimate resting-state heart rate variability in a lab-based context. Int J Psychophysiol 2022;182:105-118 [doi: 10.1016/j.ijpsycho.2022.10.003] [Medline: 36252721] 54. J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 15 (page number not for citation purposes) Abbreviations EMA: ecological momentary assessment EPA: ecological physiological assessment HR: heart rate IBI: interbeat interval LOBO: Leave-One-Beep-Out LOSO: Leave-One-Subject-Out NA: negative affect PA: positive affect PANAS: positive and negative affect schedule SC: skin conductance EMA: ecological momentary assessment J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 14 (page number not for citation purposes) J Med Internet Res 2023 \| vol. 25 \| e39995 \| p. 14 (page number not for citation purposes) https://www.jmir.org/2023/1/e39995 https://www.jmir.org/2023/1/e39995 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Tutunji et al Edited by A Mavragani; submitted 01.06.22; peer-reviewed by D Goyal, B Breil; comments to author 20.10.22; revised version received 18.01.23; accepted 14.09.23; published 19.10.23 Please cite as: Tutunji R, Kogias N, Kapteijns B, Krentz M, Krause F, Vassena E, Hermans EJ Detecting Prolonged Stress in Real Life Using Wearable Biosensors and Ecological Momentary Assessments: Naturalistic Experimental Study J Med Internet Res 2023;25:e39995 URL: https://www.jmir.org/2023/1/e39995 doi: 10.2196/39995 PMID: 37856180 Edited by A Mavragani; submitted 01.06.22; peer-reviewed by D Goyal, B Breil; comments to author 20.10.2 received 18.01.23; accepted 14.09.23; published 19.10.23 ©Rayyan Tutunji, Nikos Kogias, Bob Kapteijns, Martin Krentz, Florian Krause, Eliana Vassena, Erno J Hermans. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 19.10.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. ©Rayyan Tutunji, Nikos Kogias, Bob Kapteijns, Martin Krentz, Florian Krause, Eliana Vassena, Erno J Hermans. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 19.10.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. ©Rayyan Tutunji, Nikos Kogias, Bob Kapteijns, Martin Krentz, Florian Krause, Eliana Vassena, Erno J Hermans. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 19.10.2023. Edited by A Mavragani; submitted 01.06.22; peer-reviewed by D Goyal, B Breil; comments to author 20.10.22; revised version received 18.01.23; accepted 14.09.23; published 19.10.23 Please cite as: Tutunji R, Kogias N, Kapteijns B, Krentz M, Krause F, Vassena E, Hermans EJ Detecting Prolonged Stress in Real Life Using Wearable Biosensors and Ecological Momentary Assessments: Naturalistic Experimental Study J Med Internet Res 2023;25:e39995 URL: https://www.jmir.org/2023/1/e39995 doi: 10.2196/39995 PMID: 37856180 Abbreviations This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. https://www.jmir.org/2023/1/e39995 https://www.jmir.org/2023/1/e39995 XSL•FO RenderX
W3203671382.txt	https://www.frontiersin.org/articles/10.3389/fcimb.2021.736204/pdf	en		Fecal Microbiota Transplantation Protects the Intestinal Mucosal Barrier by Reconstructing the Gut Microbiota in a Murine Model of Sepsis	Frontiers in cellular and infection microbiology	2,021	cc-by	8,721	ORIGINAL RESEARCH published: 22 September 2021 doi: 10.3389/fcimb.2021.736204 Fecal Microbiota Transplantation Protects the Intestinal Mucosal Barrier by Reconstructing the Gut Microbiota in a Murine Model of Sepsis Xiaowei Gai 1,2,3, Huawei Wang 1, Yaqing Li 4, Haotian Zhao 5, Cong He 1, Zihui Wang 1 and Heling Zhao 1* Edited by: Gislane Lelis Vilela de Oliveira, São Paulo State University, Brazil Reviewed by: Helioswilton Sales-Campos, Universidade Federal de Goiás, Brazil Parag Kundu, Institut Pasteur of Shanghai (CAS), China Correspondence: Heling Zhao zhheling@sina.com Specialty section: This article was submitted to Microbiome in Health and Disease, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 04 July 2021 Accepted: 07 September 2021 Published: 22 September 2021 Citation: Gai X, Wang H, Li Y, Zhao H, He C, Wang Z and Zhao H (2021) Fecal Microbiota Transplantation Protects the Intestinal Mucosal Barrier by Reconstructing the Gut Microbiota in a Murine Model of Sepsis. Front. Cell. Infect. Microbiol. 11:736204. doi: 10.3389/fcimb.2021.736204 1 Department of Intensive Care Unit, Hebei General Hospital, Shijiazhuang, China, 2 Graduate School of Hebei Medical University, Hebei Medical University, Shijiazhuang, China, 3 Department of Intensive Care Unit, Qinhuangdao Jungong Hospital, Qinhuangdao, China, 4 Department of Infection, Hebei General Hospital, Shijiazhuang, China, 5 Department of Ultrasound, Hebei General Hospital, Shijiazhuang, China The gastrointestinal (GI) tract has long been hypothesized to play an integral role in the pathophysiology of sepsis, and gut microbiota (GM) dysbiosis may be the key factor. Previous studies have shown that the gut ﬂora was signiﬁcantly altered in critically ill patients. This study aimed to observe what kind of GM dysbiosis is in the early stage of sepsis and whether the application of fecal microbiota transplantation (FMT) can reconstruct the GM of septic mice and restore its protective function on the intestinal mucosal barrier. The study investigated the effect of FMT on gut microbiota, mucosal barrier function, inﬂammatory response, and survival in a murine model of sepsis established by cecal ligation and puncture (CLP). It is found that FMT can not only reduce morbidity and mortality and restore the abundance and diversity of the gut ﬂora in septic mice, but can also improve the intestinal barrier function by reducing epithelial cell apoptosis, improving the composition of the mucus layer, upregulating the expression of tight junction proteins, and reducing intestinal permeability and the inﬂammatory response. After FMT, Lachnospiraceae contributed the most to intestinal protection through enhancement of the L-lysine fermentation pathway. FMT offers a microbemediated survival advantage in a murine model of sepsis. Therefore, an improved understanding of the connection between microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis. Keywords: sepsis, fecal microbiota transplantation, intestinal mucosal barrier, gut microbiota, critical care Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 1 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice INTRODUCTION MATERIALS AND METHODS Sepsis continues to be the leading cause of mortality in the intensive care unit (Fay et al., 2017; Haak and Wiersinga, 2017). The World Health Organization has recognized sepsis as a global health priority (Fay et al., 2019). Despite signiﬁcant advancement in our understanding of the pathophysiology of sepsis, treatment of sepsis is still limited to antibiotics, aggressive ﬂuid resuscitation, vasopressor administration, and supportive care, and no targeted therapeutics for sepsis are approved for usage in patients (Ames et al., 2018). Sepsis is deﬁned as a life-threatening organ dysfunction caused by a dysregulated host response to infection (Ames et al., 2018; Fay et al., 2019; Gong et al., 2019). The syndrome can be induced by a wide variety of microbes by deﬁnition and the gastrointestinal tract is the largest pool of bacteria (Fay et al., 2019). It is known that the human gastrointestinal tract contains trillions of bacteria that comprise a complex ecosystem known as the intestinal microbiota that has relevant implications in human health and disease (Bassetti et al., 2020). The symbiotic relationship between microbiota and the host is mutually beneﬁcial (Ekmekciu et al., 2017). The host provides an important habitat and nutrients for the microbiome, and the gut microbiota supports the development of the metabolic system and the maturation of the intestinal immune system by providing beneﬁcial nutrients, for example, by the synthesis of vitamins and short-chain fatty acids (SCFAs) (Shi et al., 2017). Resident microbiota can out-compete pathogens for space, metabolites, and nutrients, and inhibit pathogens by calibrating the host immune response (Bassetti et al., 2020). However, the microbiome is markedly altered in critical illness. Studies have shown that the microbial diversity is diminished within 6 hours of admission to the intensive care unit, and this lack of diversity has been associated with poor outcomes in critically ill patients (Fay et al., 2019). Considering GM dysbiosis is one of the most important factors that can lead to pathological bacterial translocation and systemic infection, it may be feasible to develop novel therapeutic strategies against gut-derived sepsis by modulating the microbiota (Wang et al., 2019). More than 90% of the commensal organisms may be lost during the early stage of critical illness, making it nearly impossible that a single or several probiotic species would be able to completely replenish the diversity of the GM without intervention. Transfer of healthy donor feces containing thousands of microbial species, termed FMT, facilitates the replenishment of diminished commensal bacteria and may guide the patient’s microbiota toward a healthy state (Wang et al., 2019). Fecal microbiota transplantation has been successfully applied to a series of diseases (Otani and Coopersmith, 2019; Zeng et al., 2019). Even though the evidence is limited to some case reports on the treatment of septic patients, the improved clinical outcomes following FMT are promising (Schmidt et al., 2018; Limketkai et al., 2019; Wang et al., 2019). However, during sepsis, the exact mechanism of action for the use of FMT on the intestine is still unknown (Haussner et al., 2019). Considering the important role of microbiota in sepsis, we wonder whether the use of fecal microbiota transplantation in the early stage of sepsis can inhibit or even reverse the clinical outcome. Animals Experiments Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org All experimental procedures were performed by the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health) and were approved by the Animal Ethics Committee of Hebei Medical University (identiﬁcation number: 202152). Male C57BL/6 mice, approximately 6–8-weeks old and weighing 20 –25 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number: SCXK(京)20160006). The animals were housed in a temperature-controlled environment (20°C-23°C and 45%-55% humidity) with a 12 h light-dark cycle. The mice were allowed to acclimate to the housing conditions for one week before the experiments started. The experiment was divided into two parts: an acute experiment and a 7-day mortality observation experiment. All experimental animals were randomly divided into four groups: the sham operation group, the sepsis model group, the fecal microbiota transplantation group, and the normal group (healthy donor mice). The ten normal mice were only used to collect feces and make fecal bacteria liquid. The mice were euthanized at 12, 24, and 48 hours following cecal ligation and puncture for acute studies, respectively (Sham: n=6 per time point; CLP: n= 10/11 per time point; FMT: n=10/11 per time point). Ten mice in each group of the sham group, the CLP group, and the FMT group were used for a 7-day mortality observation experiment. All animals had free access to food and water. All surgery was performed under anesthesia, and every effort was made to minimize suffering. Sepsis Model Sepsis was induced by cecal ligation and puncture as previously described (Rittirsch et al., 2009; Khailova et al., 2013). Mice were anesthetized using an intraperitoneal injection at 50 mg/kg of 2% sodium pentobarbital. To perform the surgery, the cecum was exposed by a 1.5 cm midline incision in the abdomen. The cecum was ligated at 1cm from the cecal tip using a single suture, punctured with a squeeze to extrude a small amount (droplet) of feces from the perforation sites, and returned to the peritoneal cavity. The location of the cecal ligation and the size of the puncture or hole was determined in each mouse. The amount of extruded cecal content was kept the same to ensure the consistency of the model. The laparotomy was closed with silk sutures. Sham controls were subjected to the same procedures, except that there was no ligation or puncture of the cecum. Animals were resuscitated by subcutaneously injecting prewarmed normal saline (37°C; 5 mL per 100 g body weight). Recovery of these mice was assessed and recorded 2 hours after surgery, and their survival was recorded daily, including diet, fur, bloating, defecation, mobility, abnormal behavior, and response to stimuli. Tissue, blood, and feces samples of all mice were collected when they were euthanized. Fecal Microbiota Transplantation Fresh feces were collected from ten healthy C57BL/6 mice, homogenized in 10 mL of sterile phosphate-buffered saline (PBS), and centrifuged for 30 sec at 800 ×g, 4°C, to pellet the 2 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice particulate matter. The optical density (OD) value of the supernatant slurry was checked to calculate the concentration of total bacteria (OD = 0.5 represents 108 cells). For each mouse, 1×109.8 bacterial cells (sum of the total bacterial population within 2 g cecal contents) were centrifuged for 5 min at 13,000 ×g, 4°C, and then bacterial pellets were resuspended in 0.2 mL PBS (Li et al., 2015; Li et al., 2017). Mice in the FMT group received a single dose of fecal microbiota just prior to cecal ligation and puncture and were treated for three consecutive days (Khailova et al., 2013). The mice in the CLP group and the Sham group were gavaged with 0.2 mL PBS once a day as a control. and rinsed the specimen three times in PBS for 5 min each and covered the specimen with a DAPI coverslip. Transmission Electron Microscopy (TEM) For TEM, two 0.5×0.5 cm mini-segments of intestinal tissue from each group were excised and placed in a ﬁxative for TEM at 4°C for 2-4 h. The segments were washed in 0.1 M PBS three times for 15 min each time, postﬁxed in 1% osmium tetroxide in PBS, dehydrated in a graduated series of ethanol solution, and embedded by baking in an oven at 60°C for 48 h. Samples were cut into 60-80 nm sections and stained with uranyl acetate and lead citrate. The sections were analyzed by electronic microscopy (HT7700 TEM; Hitachi Inc., Tokyo, Japan). Serum IL-6, IL-10, and TNF-a Analysis Enzyme-linked immunosorbent assay was used to determine the concentrations of TNF-a (Lot: RXQJYSXLD4, Elabscience, China), IL-6 (Lot: 449268JEI6, Elabscience, China), and IL-10 (Lot: C8QEY2KAVE, Elabscience, China) in serum according to the manufacturer’s instructions. Serum was collected after centrifuging blood for 10 min at 4°C and 800 ×g and stored at -80°C until the assay was performed. Western Blot The snap-frozen tissues were subjected to homogenization in 250mL of lysis buffer as previously described. Samples (30 mg of protein for each condition) were transferred onto PVDF membranes and then incubated with antibodies (Li et al., 2017). The following were used as primary antibodies: caspase 3 (ab323519, Abcam), myeloid differentiation factor 88 (MyD88) (SC74532, Santa Cruz), toll-like receptor 4 (TLR4) (AF7017, Afﬁnity), ZO-1 (AF 5145, Afﬁnity), occludin (DF7504, Afﬁnity), and NF-kB(ab16502, Abcam). Immunoreactive bands were revealed using a 1:10,000 dilution of secondary antibody conjugated to horseradish peroxidase (goat antirabbit IgG, BE0101, Bioeasy; goat anti-mice IgG, BE0102, Bioeasy). The blots were re-probed with antibodies against b-actin (EASYBIO) and GAPDH (Bioworld) to ensure equal loading and transfer of proteins. All critical blots and immunoprecipitation experiments were repeated at least three times. Sample Processing for Animal Experiments After the mice were euthanized, their GI tracts were quickly removed. The colons were gently separated, by cutting at the cecum-colon junction and rectum and divided into two parts. One-half of each colon was immediately frozen in liquid nitrogen and then stored at -80°C until further use. The other part ones were preserved in Carnoy’s ﬁxative (dry methanol: chloroform: glacial acetic acid in the ratio 60:30:10) (Johansson et al., 2011; Desai et al., 2016). The Carnoy’s ﬁxative was made fresh with anhydrous methanol, chloroform, and glacial acetic acid. The colons were ﬁxed in Carnoy’s solution for 3 h followed by transfer to fresh Carnoy’s solution for 2-3 h. The colons were then washed in dry methanol for 2 h, placed in cassettes, and stored in fresh dry methanol at 4°C. Samples were then embedded in parafﬁn, and cut into sections (5mm thick). Cecal contents from each animal were divided into replicates, and they were all instantly ﬂash-frozen in liquid nitrogen and then stored at -80°C until the use for microbiome evaluation. Real-Time PCR Total RNA was isolated from colonic tissue using the RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China) as described in the manufacturer’s protocol. The RNA concentrations were quantiﬁed at 260 nm, and their purity and integrity were determined using a NanoDrop. Reverse transcription and real-time PCR assays were performed to quantify steady-state messenger RNA levels of TLR4, MyD88, and NF-kB at 48 h following CLP. Complementary DNA was synthesized from 0.1 mg of total RNA. The following cycling protocol was used: denaturation at 95°C (15 min) and 40 cycles of 95°C (10 s), 60°C (32 s). The reporter dye emission (SYBR green) was detected by an automated sequence detector combined with ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Immunohistochemistry and Immunoﬂuorescence The colonic sections were mounted onto polylysine-coated slides, deparafﬁnized, rehydrated, and placed in a 3% citrate buffer to repair antigens. After being pretreated with 3% H2O2 for 30 min, the sections were blocked with goat serum for 20 min. Sections were incubated overnight at 4°C with a rabbit polyclonal to MUC2 blocking antibody (MUC2, ab90007, Abcam Ltd.; Occludin, ab168986, Abcam Ltd; caspase 3, ab44976, Abcam Ltd.). The sections were washed with PBS and incubated with a secondary antibody for 30 min, rewashed, and incubated with peroxidase-conjugated streptavidin for 30 min. DAB developed, hematoxylin counterstained, dehydrated, and mounted. The secondary antibody of MUC 2 was prepared in 0.5% Triton X100 PBS buffer, and the Alexa Fluor™ 488 donkey anti-rabbit antibody was diluted 1:1000. The specimen was incubated in this solution at 37°C for 1 h. We aspirated the secondary antibody Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Microbiome Evaluation Fecal samples were collected at the time mice were euthanized and frozen until DNA extraction samples were sent to the Shanghai Personal Biotechnology Co., Ltd., where bacterial DNA was extracted. PCR ampliﬁcation of the bacterial 16S rRNA genes V3V4 region was performed using the forward primer 338F (5’ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The Illumina MiSeq sequencing platform was ampliﬁed (Illumina, San Diego, CA, USA) using the NovaSeq-PE250 sequencing strategy. Sequence denoising or OTU clustering was performed according to the 3 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice QIIME2 dada 2 analysis process or the Vsearch software analysis process. The resulting sequences were clustered with 100% similarity against the GreenGenes to the database (v. 13.8; https:// greengenes.secondgenome.com/) (Fay et al., 2019) to assign bacterial operational taxonomic units. Alpha and beta diversity comparisons were performed using QIIME2 and taxonomic summaries were generated with QIIME2. Comparison of sample compositions and identiﬁcation of statistically signiﬁcant differences were performed with LEfSe using the correction for independent comparisons. Microbial functions were predicted by using PICRUSt (Phylogenetic investigation of communities by reconstruction of unobserved states) based on highquality sequences. group had a mortality rate of 30% at 24 h and a 50% survival rate at 7 days. There were no deaths at 24 h in the FMT group, and a 90% survival at 7 days, and no deaths in the Sham group. Compared with the Sham group and the FMT group, the mortality of the CLP group was signiﬁcantly higher, P < 0.05 (Figure 1A). Apoptosis The expression of caspase 3 in the CLP group was signiﬁcantly higher than that of the other two groups at 12 and 24 h, but the average integral optical density (IOD) of caspase 3 in the FMT group was close to that of the CLP group at 48 h (Figure 1B). Therefore, we performed a quantitative analysis of caspase 3 protein. The results of the western blot showed that the expression of caspase 3 in the CLP group at 24 and 48 h was higher than that in the FMT group, P < 0.001 (Figure 1C). Statistical Analysis The Kaplan-Meier estimator was used to draw the survival curve of the mice, and the log-rank method was used to compare the survival rates between different groups. The measurement data had a normal distribution, the variance was uniform, and the oneway ANOVA and LSD tests were used for comparison between multiple groups. The Student’s t-test was used to compare the two independent groups, and the measurement result was expressed as the mean ± standard deviation (mean ± sd). The Kruskal-Wallis H test was used for data with non-normal distribution and/or uneven variance. The results were expressed in medians (interquartile range). SPSS 21.0, GraphPad Prism 8.0, Photoshop CS5, Image-Pro Plus, and ImageJ were used for data analysis, and P < 0.05 was considered statistically signiﬁcant. Mouse Serum Inﬂammatory Factors TNF-a, IL-6, and IL-10 The expression of IL-6 in the CLP group was the highest at 24 h after modeling, and then decreased, but it was still higher than that in the Sham group at 48 h. Expression of IL-6 in the FMT group was slightly higher than that in the CLP group at 12 h, but there was no signiﬁcant difference, to the contrary, IL-6 levels were markedly lower than the CLP group at 24 and 48 h. The TNF-a level in the CLP group continued to increase, and it was the highest among the three groups at 48 h, however, the IL-10 level was lowest at 24 h after modeling (Figure 2). The Thickness of the Mucus Layer (nm) and MUC2 Expression The AB-PAS method was used to detect the colonic mucus layer thickness at 12, 24, and 48 h after sepsis modeling in the three groups. The mucus layer thickness of mice in the CLP group was signiﬁcantly lower than that of the Sham group during the same timepoint (P < 0.0001). The thickness of the mucus layer in the RESULTS Mortality Among Three Groups The survival rates of the Sham group, CLP group, and FMT group were compared 7 days following sepsis modeling. The CLP A B C FIGURE 1 \| Survival analysis, and caspase 3 expressions among the Sham, CLP, and FMT groups. (A) Seven-day mortality observations in the Sham (red), CLP (black), and FMT groups (blue). There were signiﬁcant differences between the CLP group and the other two groups, P < 0.05, but there was no signiﬁcant difference between the FMT group and the Sham group; (B) Immunohistochemistry of the colon [under a digital microscope (20 × 10)]. The average integral optical density of caspase 3 between the Sham, CLP, and FMT groups at 12, 24, and 48 h are shown on the right side. (C) Relative expression of caspase 3 compared with GAPDH between the CLP and FMT groups at 24 and 48 h. P < 0.05, *P < 0.001, *P < 0.0001, ns, no statistical difference, respectively. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 4 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice FMT group was signiﬁcantly greater than that in the CLP group (P < 0.01). Compared with the Sham group, the thickness of the mucus layer in the FMT group had no difference at 24 h (P = 0.4473) but had a signiﬁcant difference at 12 and 48 h (P < 0.0001, and P < 0.05, respectively) (Figure 3A). The ﬂuorescence expressions of MUC2 at 12, 24, and 48 h in the three groups were observed by digital microscope and recorded, and the gray value of the green channel was calculated and statistically analyzed. Except that the expression levels of MUC2 in the Sham group and the FMT group were not statistically different at 12 h, there was a signiﬁcant difference between each pair at 24 and 48 h (P < 0.0001). The expression of MUC2 in the CLP group at 12, 24, and 48 h was lower than that of the other two groups (Figure 3B). expression of these two proteins in the CLP group was signiﬁcantly lower than that in the other two groups at 24 or 48 h, (P < 0.001). The relative expression of the two proteins was highest in the Sham group, intermediate in the FMT group and lowest in the CLP group (Figure 5B). TLR4, MyD88, and NF-kB Protein Levels and mRNA Levels We analyzed and compared the expression of TLR4, MyD88, and NF-kB relative to GAPDH at 24 and 48 h in the three groups. Except that there was no difference in the expression of TLR4 protein between the Sham and FMT groups, expression levels of the other two proteins in the three groups were signiﬁcantly different compared with each other. Expression of the three proteins in the CLP group at 24 and 48 h was signiﬁcantly higher than those in both the Sham and the FMT groups at the same time points (P < 0.05) (Figure 6A). Coincidently, mRNA expression trends in the three groups were similar to the trends in protein expression. The relative expressions of TLR4, MyD88, and NF-kB, in the CLP group, were signiﬁcantly higher than those in the Sham and FMT groups. Expression of MyD88, and NF-kB had a signiﬁcant difference in the CLP group compared with the Sham and FMT groups(P < 0.05). There was no difference in the expression of the three indicators between the Sham group and the FMT group (Figure 6B). Transmission Electron Microscope Intestinal epithelial cells and intracytoplasmic organelles in the CLP group were signiﬁcantly swollen, and the microvilli were arranged neatly, with uniform thickness and partial shedding. The tight junctions and the structure of the intermediate junctions were blurred, and the gap between junctions was slightly widened in the CLP group. Portions of the desmosomes and tension wires had disappeared. The intercellular space was widened and the mitochondria had swelled. Intestinal epithelial cells and intracytoplasmic organelles in the FMT group were slightly swollen. The microvilli were arranged neatly and uniformly in thickness, and the local area was slightly detached. The tight junctions between epithelial cells and the structure of the intermediate junctions were fuzzy, and the gap was slightly widened in the FMT group. The number of desmosomes was slightly reduced, the surrounding tension ﬁlaments were abundant, and the mitochondria were slightly swollen (Figure 4). 16SrRNA Sequence Analysis Krona Species Composition Firmicutes and Bacteroidetes were the main bacteria, accounting for 55% and 33% of the abundance found in the normal mice, respectively. The Sham group was dominated by Firmicutes, at 66%. Proteobacteria was the dominant bacteria in the CLP group. The relative abundance of Proteobacteria observed in the mortality observation of the CLP groups following sepsis modeling at 12 h, 24 h, and 7-day were 48%, 66%, and 42%, respectively. The relative abundance of unclassiﬁed bacteria in the CLP group at 48 h and the 7-day mortality observation in the FMT group was 70% and 41%. The FMT group at 24 and 48 h were dominated by Firmicutes and Bacteroidetes, accounting for Tight Junction Proteins We compared the average integral OD of occludin between the CLP and the FMT groups at 12, 24, and 48 h. The results showed that occludin expression in the CLP group at 12, 24, and 48 h was signiﬁcantly lower than expression in the FMT group (Figure 5A). We further veriﬁed occludin and ZO-1 protein expression by western blot test. The results showed the relative FIGURE 2 \| The serum IL-6, TNF-a, and IL-10 among the Sham, CLP, and FMT groups at 12, 24, and 48 h. The concentration of serum IL-6 (pg/mL) and IL-10 (pg/ mL) in the FMT group at 12 h after sepsis modeling were signiﬁcantly higher than in the Sham group (P < 0.05), and there was no signiﬁcant difference in TNF-a levels (pg/mL) among the three groups. The serum IL-6 level in the CLP group was signiﬁcantly higher than in the Sham group and the FMT group (P < 0.001) at 24 h. The TNF-a levels in both the CLP and FMT groups were higher than in the Sham group (P < 0.001 and P < 0.01, respectively). The IL-10 level in the CLP group was lower than in the Sham group and the FMT group (P < 0.001). The serum IL-6 level in the CLP group was higher than in the Sham group at 48 h (P < 0.01). The TNF-a level in the CLP group was higher than in the Sham group and FMT group (P < 0.001). The TNF-a level in the FMT group was higher than in the Sham group (P < 0.05). There was no signiﬁcant difference among the three groups in the IL-10 level at 48 h. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 5 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice A B FIGURE 3 \| The mucus layer thickness and MUC2 expression in the Sham, CLP, and FMT groups at 12, 24, and 48 h. (A) The AB-PAS method was used to measure the thickness of the mucus layer in the Sham, CLP, and FMT groups at 12, 24, and 48 h under a digital microscope (20 × 10). The blue color indicates goblet cell secretion and a mucus layer. The histogram on the right shows the statistical results of the comparison of the three groups. There was no signiﬁcant difference between the Sham group and the FMT group in mucus layer thickness at 24 h. (B) MUC2 expression (green ﬂuorescence) was observed in the three groups at 12, 24, and 48 h. The histogram shows the statistical results of all three groups. There was no signiﬁcant difference between the Sham group and the FMT group at 12 h. P < 0.05, P < 0.01, P < 0.0001, ns, no signiﬁcant difference. there was a slight recovery in the 7-day still alive mice in the CLP group, a signiﬁcant difference compared with the normal mice. The richness and diversity of the ﬂora in the FMT group were higher than that observed in the CLP group at all time points, and there was more fecal diversity than the normal mice at 24 h in the FMT group. There was no signiﬁcant difference in fecal richness and diversity in the 7-day mortality FMT mice compared with the CLP group at the same timepoint (Figure 7B). 48%, 27%, and 37%, 26%, and the composition closely resembled the composition found in mice. The main bacteria in the FMT group at 12 h were Proteobacteria and Verrucomicrobiae, with a relative abundance of 33% and 39%, respectively. Verrucomicrobiae accounted for 22% in the CLP group at 24 h (Figure 7A). Alpha Diversity Analysis The 12 h Sham group had a similar fecal richness and diversity compared with normal mice. The fecal richness and diversity were signiﬁcantly lower in the CLP group at 12, 24, and 48 h and 7-day mortality than those of normal mice. We observed that the fecal richness and diversity were lowest at 48 h in the CLP group, but Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Beta Diversity Analysis The dimensionality reduction of multi-dimensional microbial data was performed through NMDS analysis, and the main 6 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice FIGURE 4 \| Intestinal epithelial cell junctions. Intestinal epithelial cell junctions in the CLP group and FMT group at 48 h. The images on the left were enlarged 4K, and those on the right were enlarged 10K. ▲ Tight junction and the intermediate junction between intestinal epithelial cells. ■ The space between intestinal epithelial cells. trends of data changes were displayed through the distribution of samples on a continuous sorting axis. The data were also classiﬁed by cluster analysis. In the NMDS analysis, the clusters within the group were well and the difference between the groups was large (Figure 7C). that infectious diseases had the highest relative abundance among human diseases in the KEGG secondary functional pathway (Figure 7E). Metabolite Analysis We further analyzed the species composition of the differential pathways, and found that the Lachnospiraceae contributed the most to L-lysine fermentation to acetate and butanoate (Figure 7F). LefSe (LDA Effect Size) Analysis LEfSe analysis can directly perform simultaneous difference analysis on all classiﬁcation levels, and at the same time, it emphasizes ﬁnding robust differences between groups, that is, marker species. The main species found in normal mice were Firmicutes and Bacteroides. The species with high levels in the Sham group were Firmicutes and Lactobacillus, while the differential species in the CLP group were Bacillales and Staphylococcaceae at 12 h, Enterobacteriales and Proteobacteria at 24 h, Planococcaceae at 48 h, and Deltaproteobacteria, Desulfovibrionales, and Erysipelatoclostridium at 7-day mortality. The FMT group was modeled with high levels of bacteria, including Verrucomicrobiae, Akkermansia, and Ruminococcus at 12 h, Lachnospiraceae group, Biﬁdobacteriales, Actinobacteria at 24 h, and Burkholderiaceae, Bacteroides, and Butyricimonas at 48 h (Figure 7D). DISCUSSION The commensal microbiome has been shown to play a key role in intestinal immunity because microbes regulate the maturation of the mucosal immune system, support local mucosal immunity, regulate cell growth, and maintain the epithelial barrier function (Haussner et al., 2019). Sepsis alters the composition of the ﬂora and disrupts the balance between the host and the gut (Schmidt et al., 2018; Liu et al., 2019). Striking abnormalities have been reported in the intestinal microbiota of critically ill patients with sepsis, with a wide inter-individual variation and a low bacterial diversity (Avila et al., 2020; Bassetti et al., 2020). In this context, FMT is an effective strategy for adjusting the dysbiosis and restoring the normal gut microﬂora in patients with sepsis (Avila et al., 2020). Fecal microbiota transplantation refers to the transplantation of functional bacteria from the feces of healthy donors into the patient’s GI tract to restore the intestinal micro ecological Functional Analysis The core of the KEGG database is a biological metabolic pathway analysis database, in which metabolic pathways are classiﬁed into six categories, including metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. We found Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 7 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice A B FIGURE 5 \| Comparison of tight junction protein expression. (A) Comparison of occluding expression in the CLP and FMT groups at 12, 24 add 48 h, respectively, and observed under a digital microscope (20 × 10). Signiﬁcant results are shown in the box plot on the right, *P < 0.0001; (B) Relative expression of occludin and ZO-1 compared with b-actin expression in the Sham, CLP, and FMT groups at 24 or 48 h. SB, CB, TB, SD, CD, and TD represent the 24 h Sham, CLP, and FMT groups and 48 h Sham, CLP, and FMT groups, respectively. the intestinal ﬂora of the 7-day mortality CLP group still accounted for 42%, which was signiﬁcantly higher than that of normal mice. The analysis of the bacterial ﬂora in the FMT group revealed that Firmicutes and Bacteroidetes were the most prevalent and the relative abundance of Proteobacteria was low, meaning that the bacterial composition is similar to that of normal mice. Besides, the Alphaproteobacterial load increased and the Betaproteobacterial load decreased in the FMT group. All of these observations indicate that the intestinal ﬂora of septic mice had been replenished after FMT. It is believed that FMT can balance and subsequently to treat diseases related to microbial imbalances (Wang et al., 2019; Zeng et al., 2019). This study was an attempt to explore whether FMT can maintain the integrity of the intestinal ﬂora and protect the intestinal barrier function in mice with sepsis. We found that there was a ﬂora imbalance 12-h after the modeling of sepsis. Proteobacteria had an absolute advantage, but Firmicutes and Bacteroidetes decreased, which was consistent with previous studies (Otani and Coopersmith, 2019). The relative abundance of Firmicutes and Bacteroidetes recovered slightly over time, but the amount of Proteobacteria in Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 8 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice A B FIGURE 6 \| TLR4, MyD88, and NF-kB expression and mRNA levels. (A) Expression of TLR4, MyD88, and NF-kB compared to GAPDH protein among Sham, CLP, and FMT groups at 24 and 48 h. SB, CB, TB, SD, CD, and TD represent 24 h Sham, CLP, and FMT groups and 48 h Sham, CLP, and FMT groups, respectively. (B) Relative TLR4, MyD88, and NF-kB levels in the colon. P < 0.05, *P < 0.01. This indicated that Lachnospiraceae may be the key bacteria for the effectiveness of FMT. Tight junctions play an important role in maintaining the integrity of the mucosal epithelium (Zihni et al., 2016; Xu et al., 2018), and it is generally deﬁned as a life-threatening organ failure in the setting of critical illness (Guttman and Finlay, 2009; Vermette et al., 2018). Critical illness induces hyper-permeability of the gut barrier which begins as early as 1 hour after the onset of sepsis and lasts at least 48 hours (Yoseph et al., 2016; Otani and Coopersmith, 2019). We compared transmission electron microscopy results in 48-h between the CLP and the FMT groups. The TJs of the septic mice were blurred more than those of the FMT mice. The cell gap was signiﬁcantly wider, and both cells and organelles were swollen. We suppose that the mice had not only intestinal barrier dysfunction but also cellular dysfunction. The main functions of occludin are regulating and sealing the TJs (Shawki and McCole, 2017; Wang et al., 2017). That ZO-1 outperformed other TJ markers may reﬂect the concomitant organ epithelial injury which occurs in MODS (Vermette et al., 2018). Therefore, we observed the expression of the above two proteins in 24-h and 48-h in the three groups, and the results showed that occludin and ZO-1 in the CLP group were signiﬁcantly lower than those in the Sham group and the FMT group. Levels of the two indicators in the FMT group were higher than those in the CLP group, or they were similar to those in the Sham group. Research has shown that the thickness of the mucus layer is dependent on commensal bacteria (Otani and Coopersmith, 2019). We performed blinded thickness measurements of the colonic mucus layer in each mouse using Alcian blue-stained sections. We further validated the thickness of the mucus layer by immunoﬂuorescence staining of the MUC2 mucins using a-MUC2 antibody (Desai et al., 2016) and observed that the thickness of the mucus layer in septic mice signiﬁcantly reduced, and the thickness in the FMT group was signiﬁcantly increased. This change was consistent with the changes in the ﬂora of the two groups. TLR4 is the best-characterized pathogen-recognition receptor. Its downstream effects are varied, and the TLR/ MyD88/p38 MAPK/NF-kB pathway is popularly believed to maintain the intestinal bacterial balance by increasing the diversity of the ﬂora and restoring and protecting intestinal ﬂora from external interference (Jeon et al., 2018). However, in the observation of the 7-day mortality CLP group, the richness and the diversity of bacterial ﬂora were both lower than those of the FMT group at 24 -h and 48 -h. As aforementioned, the dose and the number of infusions of FMT may have contributed to this result (Jeon et al., 2018). This experiment conﬁrmed that the 7-day mortality rate of septic mice was signiﬁcantly higher than that of the FMT group. Early application of FMT can effectively reduce the mortality of septic mice, most likely due to the reconstruction of intestinal ﬂora. The GM supports the development of the metabolic system and the maturation of the intestinal immune system by providing beneﬁcial nutrients, such as synthesizing vitamins and short-chain fatty acids (SCFAs) (Shi et al., 2017). Butyrate, especially, can promote the release of the mucoprotein so as to maintain the mucus barrier (Johansson and Hansson, 2016; Wang et al., 2019). Studies have shown that SCFAs have anti-inﬂammatory and immunoregulatory activities and may reduce butyrate-producing bacteria such as Ruminococcaceae, Faecalibacterium, and Roseburia (Cammarota et al., 2015). Therefore, we compared the relative abundance of Ruminococcaceae in each group, and the results suggest that fecal bacteria signiﬁcantly reduced in the sepsis model group, while in the FMT group, the bacterial counts were similar to or even slightly higher than those found in the normal mice. (In our study, the relative abundance of the normal mice was 0.07%, 0.0025% in the CLP group, and 0.067% in the FMT group in 48-h). It appears that Ruminococcaceae plays a role in the inﬂammatory response, and fecal microbiota transplantation can effectively improve the intestinal bacteria composition, thereby improving the inﬂammatory state. We found through the functional prediction that the relative abundance of intestinal ﬂora was signiﬁcantly different in infectious diseases. Further analysis of the species composition of the different pathways revealed that Lachnospiraceae contributed the most to L-lysine fermentation to acetate and butanoate, consistent with previous studies (Shen et al., 2018). Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 9 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice A B C D E F FIGURE 7 \| Microbiota analysis. (A) Krona species at phylum among the Normal, Sham, CLP, and FMT groups. (B) Alpha diversity. P < 0.05, P < 0.01 **P < 0.001. (C) NMDS two-dimensional sorting diagram. The closer the distance between the two points in the ﬁgure, the smaller the difference between the microbial communities in the two samples. (D) LEfSe (LAD Effect Size). A longer bar denotes a more signiﬁcant difference in the taxon. The color of the bar graph indicates the most abundant sample group corresponding to the taxon. (E) Predicted abundance map of KEGG secondary functional pathways. (F) Species composition map of differential MetaCys metabolic pathways. The abscissa shows different groups. The order of the samples in the groups was sorted according to the similarity of the data; the ordinate was the relative abundance of the metabolic pathways, and the species different levels of contribution to the metabolic pathways were displayed in different colors at different levels. (In order to show the results better, we used abbreviations to represent each group: N = normal mice, 7C = CLP group mortality observation, 7T = FMT group mortality observation, SA = Sham group 12 h; CA, CB, and CD = CLP group at 12, 24, and 48 h time points, respectively, and TA, TB, and TD = FMT group at 12, 24, and 48 h time points, respectively). the three groups. It was observed that the amount of apoptosis protein caspase 3 was different between the CLP group and the FMT group, and the amount of apoptosis in the CLP group was signiﬁcantly higher than that of the FMT group. This study has certain limitations: First, we selected the C57BL/ 6 mouse as our animal model. It is of common knowledge that the human gut microbiota varies greatly, and it is affected by many factors, including host-intrinsic, host-extrinsic, and play a critical role in the inﬂammatory response (Piton and Capellier, 2016; Tian et al., 2016). In this study, the TLR4/ MyD88/NF-kB pathway in septic mice increased signiﬁcantly at both the protein level and the gene level, and the inﬂammatory factors TNF-a and IL-6 increased at different levels in 24-h and 48-h after modeling, while IL-10 decreased in 24-h and 48-h after modeling. FMT treatment decreased the inﬂammatory response. This is consistent with the pathological score of bowel injury in Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 September 2021 \| Volume 11 \| Article 736204 Gai et al. FMT in Septic Mice environmental. Precise microbiome modulation, therefore, is still in its infancy (Schmidt et al., 2018). Second, FMT has been shown to be highly efﬁcient in the treatment of recurrent C. difﬁcile infection (Jeon et al., 2018; Schmidt et al., 2018), but its application in sepsis has only been rarely reported. FMT is often preceded by preparatory antibiotic treatment in clinical practice, which makes it difﬁcult to disentangle its effects (Schmidt et al., 2018). While antimicrobials are one of the fundamental and often life-saving modalities in septic patients, they can also pave the way for subsequent harm because of the resulting damage to the gut microbiome (Bhalodi et al., 2019). It has been reported that the association between antibiotic exposure and subsequent sepsis is related to microbiome depletion, rather than the severity of illness (Haak et al., 2018). Therefore, our initial experiment did not involve the use of antibiotics, but its combined application with FMT was included in subsequent studies. Third, it has been reported that the biological activity of the fresh fecal microbiota liquid is not affected by two hours of storage on ice (Hamilton et al., 2012). In our experiment, the fresh fecal bacteria liquid was kept on ice and was transplanted within 1 hour. It is speculated that its biological activity is not affected, but this needs to be conﬁrmed in further research. Fourth, We did not attempt to discern the complete reconstruction time of intestinal ﬂora after initiating FMT, nor did we compare the frequency and the number of transplantation, but it appears that early application of FMT has a protective effect on the intestinal function of septic mice. Finally, in view of the limited predictive function of PICRUSt, we will further use the full RNA transcriptome from both stool and mucosa-adherent microbiota for veriﬁcation. Lachnospiraceae contributes the most to intestinal protection through enhancement of the L-lysine fermentation pathway, resulting in the production of acetate and butanoate, and maybe the key bacteria in short-chain fatty acid metabolism that promotes the success of fecal microbiota transplantation. CONCLUSION ACKNOWLEDGMENTS GM imbalance exists early in sepsis. Fecal microbiota transplantation can not only improve morbidity and effectively reduce mortality in septic mice, but can also effectively reduce epithelial cell apoptosis, improve the composition of the mucus layer, upregulate the expression of TJ proteins, and reduce intestinal permeability and the inﬂammatory response, thus protecting the intestinal barrier function. In our study, after FMT, the abundance and diversity of the gut ﬂora were restored, and the microbial characteristics of the donors changed. This article was submitted as a preprint to BioRxiv as Gai et al. 2020. 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Intensive Care 7, 17. doi: 10.1186/s40560-019-0372-6 Piton, G., and Capellier, G. (2016). Biomarkers of Gut Barrier Failure in the ICU. Curr. Opin. Crit. Care 22 (2), 152–160. doi: 10.1097/MCC.000000 0000000283 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Gai, Wang, Li, Zhao, He, Wang and Zhao. 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https://openalex.org/W3124008525	https://link.springer.com/content/pdf/10.1007/s12403-020-00381-6.pdf	English	null	Low-Level Environmental Heavy Metals are Associated with Obesity Among Postmenopausal Women in a Southern State	Exposure and health	2,021	cc-by	10,092	Shelbie Stahr1 · Tung‑chin Chiang2 · Michael A. Bauer3 · Gail A. Runnells4 · Lora J. Rogers5 · Huyen Vi Do4 Susan A. Kadlubar2 · L. Joseph Su4 Shelbie Stahr1 · Tung‑chin Chiang2 · Michael A. Bauer3 · Gail A. Runnells4 · Lora J. Rogers5 Susan A. Kadlubar2 · L. Joseph Su4 Received: 21 August 2020 / Revised: 23 November 2020 / Accepted: 15 December 2020 / Published online: 17 January 2021 © The Author(s) 2021 Exposure and Health (2021) 13:269–280 https://doi.org/10.1007/s12403-020-00381-6 Exposure and Health (2021) 13:269–280 https://doi.org/10.1007/s12403-020-00381-6 ORIGINAL PAPER Abstract Both arsenic and cadmium are reported to be toxic to humans. The use of saliva as a biomarker of low-level exposures to these elements has not been adequately explored, and the putative relationship between exposure and obesity is unclear. This cross-sectional study aims to investigate the relationship between salivary arsenic and cadmium concentrations and their association with obesity. Arsenic and cadmium concentrations were analyzed in human saliva samples by Inductively Coupled Plasma-Mass Spectrometry on 270 randomly selected women who participated in the Arkansas Rural Community Health Study. Multivariable logistic regression was performed to evaluate the association between heavy metal concentra- tions and obesity. Stratified logistic regression was performed based on menopausal status. Generalized linear models were used to evaluate weight gain velocity. Significant positive associations were observed in postmenopausal women for both arsenic (OR = 4.43, 95% CI 1.91–10.28) and cadmium (OR = 2.72, 95% CI 1.23–5.99) concentrations, as well as significant trends among tertiles (p < 0.01 and p = 0.01, respectively). No relationship with obesity was evident among premenopausal women for either metal. A dose–response relationship was observed between increasing weight gain velocity and increas- ing metal concentrations. At concentrations well below governmental and industrial standards for acute toxicity, significant associations between obesity and concentration of these heavy metals are evident. The rate at which individuals gain weight is affected by metal concentrations and may play a role in the rapid increase in weight in postmenopausal women. These results might explain, in part, the missing variability in the increasing obesity pandemic in certain population exposed to these environmental toxicants. Keywords Arsenic · Cadmium · Obesity · Saliva · Menopause Vol.:(012345678 1 3 Keywords Arsenic · Cadmium · Obesity · Saliva · Menopause * Shelbie Stahr SStahr@uams.edu Tung‑chin Chiang TChiang@uams.edu Michael A. Bauer MBAUER2@uams.edu Gail A. Runnells RunnellsGailAnn@uams.edu Lora J. Rogers LJRogers@uams.edu Huyen Vi Do dohuyenv@gmail.com Susan A. Kadlubar sakadlubar@gmail.com L. Joseph Su ljsu@uams.edu 1 Interdisciplinary Biomedical Sciences, Department of Clinical and Translational Sciences, University of Arkansas Medical Sciences, 4301 W. Markham St. Slot #601, Little Rock, AR 72205, USA 2 Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas Medical Sciences, 4301 West Markham, # 820, Little Rock, AR 72205, USA 3 Department of Biomedical Informatics, College of Medicine, University of Arkansas Medical Sciences, 4018 W Capitol Ave, Little Rock, AR 72205, USA 4 Department of Epidemiology, Fay W. Introduction Hundreds of millions of dollars are spent to prevent obe- sity in the United States annually (Lanza et al. 2010; Kruger et al. 2004); however, obesity rates over the past 50 years continue to climb (Wang et al. 2020). It is esti- mated that approximately 38% of US adults are defined as obese (NCHS 2016). Factors contributing to obesity include lack of physical activity and exercise, imbalance of energy intake and expenditure, a high sedentary lifestyle, intake of high-caloric foods, stress, genetics, and health/ medical conditions (Eunice Kennedy Shriver National Institute of Child Health and Human Development 2016; Lopomo et al. 2016). The National Institute of Health has concluded that the greatest contributor to weight gain is the imbalance between caloric intake and energy expendi- ture (Eunice Kennedy Shriver National Institute of Child Health and Human Development 2016), but recent stud- ies demonstrate that these factors do not fully explain the obesity problem (Park et al. 2017; Lopomo et al. 2016). Lesser known consequences of arsenic exposure have been observed in both experimental and epidemiologi- cal studies regarding the relationship between arsenic and obesity. An in vitro study published in 2011 concluded that prolonged inorganic arsenite exposure resulted in decreased expression in glucose transporter type 4 (GLUT4) and sev- eral adipogenic genes (Xue et al. 2011). This study indicates that prolonged exposure to arsenic causes significant effects on adipocytes (Xue et al. 2011). Similarly, an in utero study published in 2016 found that mice with early life exposure to arsenite resulted in abnormal metabolism, increased body weight, as well as other cardiometabolic risk factors (Ditzel et al. 2016). Unlike these experimental studies, epidemio- logical studies are less consistent. High BMI was associated with a low percentage of urinary arsenic excreted as mono- methylarsonic acid in women (Gomez-Rubio et al. 2011). Contrarily, in 2010 a study evaluating if arsenic was asso- ciated with body composition in reproductive-age women found that there was no association between the two after adjusting for food consumption and lifestyle factors (Ronco et al. 2010). y p p As of 2019, arsenic ranks number one on the Agency for Toxic Substances and Disease Registries (ATSDR) list of hazardous substance Priority List (ATSDR 2020). This list is created by utilizing an algorithm that takes into account the toxicity, potential for human exposure, as well as the frequency of human exposure (ATSDR 2020). Abstract Boozman College of Public Health, University of Arkansas Medical Sciences, 4301 W. Markham St. # 820, Little Rock, AR 72205, USA 5 COPH Department of Epidemiology Slot 721‑21, Winthrop P. Rockefeller Cancer Institute, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, 4104 Outpatient Circle, Little Rock, AR 72205‑7101, USA 1 Interdisciplinary Biomedical Sciences, Department of Clinical and Translational Sciences, University of Arkansas Medical Sciences, 4301 W. Markham St. Slot #601, Little Rock, AR 72205, USA 1 Interdisciplinary Biomedical Sciences, Department of Clinical and Translational Sciences, University of Arkansas Medical Sciences, 4301 W. Markham St. Slot #601, Little Rock, AR 72205, USA 2 Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas Medical Sciences, 4301 West Markham, # 820, Little Rock, AR 72205, USA 3 Department of Biomedical Informatics, College of Medicine, University of Arkansas Medical Sciences, 4018 W Capitol Ave, Little Rock, AR 72205, USA 4 Department of Epidemiology, Fay W. Boozman College of Public Health, University of Arkansas Medical Sciences, 4301 W. Markham St. # 820, Little Rock, AR 72205, USA 5 COPH Department of Epidemiology Slot 721‑21, Winthrop P. Rockefeller Cancer Institute, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, 4104 Outpatient Circle, Little Rock, AR 72205‑7101, USA (0121 3456789) 3 270 S. Stahr et al. 2006; Munoz et al. 2018; Surdu et al. 2015). Arsenic has also been identified as an endocrine disrupter in various in vivo, in utero, and cell culture models (Chatterjee and Chatterji 2010; Davey et al. 2007; Waalkes et al. 2004). These disruptions can cause adverse developmental, repro- ductive, neurological, and immune effects (Alvarado-Cruz et al. 2018; Ashrap et al. 2019; Chen et al. 2009; Iavicoli et al. 2009; Liu et al. 2015; White et al. 2019). One study reported that an increase in arsenic resulted in decreased levels of estradiol that presented in a dose–response man- ner within a rat model (Chatterjee and Chatterji 2010). The study further describes that arsenic may mimic the estrogen mechanisms that disrupt the endocrine signaling pathway and reproductive failures. Study Population Arsenic and cadmium, among other elements, are in the multi-element ICP-MS stock standard and quality control standard solutions. The internal standard used for ICP-MS analysis was Yttrium (10 µg/L). Saliva samples were homog- enized by inverting several times prior to analysis. All sam- ples were analyzed in kinetic energy discrimination (KED) mode. Blank Oragene DNA stabilizing solution without saliva was tested and confirmed no measurable trace ele- ment of interest. Data obtained from the Arkansas Rural Community Health (ARCH) Study cohort, formerly known as Spit for the Cure (Bondurant et al. 2011; Lee et al. 2014; McElfish et al. 2019) was utilized in the present study. All 75 counties in Arkansas are represented in this cohort, including an overrepresenta- tion of African Americans. The ARCH population was not intended to be representative of the entire female population of Arkansas. In addition to being a racially diverse cohort, it is also comprised of individuals with a higher education attainment compared to the overall state average. Data were collected at baseline that contained information regarding, family and medical history, specific information regarding breast cancer history and treatment, reproductive health, physical activity, and socioeconomic status. For each par- ticipant, a baseline 2 mL self-collection saliva sample was collected at the time of study enrollment. Baseline informa- tion was collected through various community events from a total of 26,347 women between the ages of 18 and 100. To verify that all samples utilized produced valid num- bers, samples were compared against the Background Equiv- alent Concentration (BEC) (Wilschefski and Baxter 2019; Thomas 2004; CASRN E. S. C. E. S. 2014). The BEC is used to assure that the signals used in sample quantifica- tion are not due to “signal noise” resulting from background interference (CASRN E. S. C. E. S. 2014). The literature states that utilization of the BEC is a more reliable assess- ment in real-world sample matrices in regards to instrument performance (Thomas 2004), due to the quantification of the background level. In the present study, three arsenic concentrations were below its respective BEC value, and one cadmium fell below its respective BEC. For purposes of analyses, these values were recorded as “0.00” due to the variable being analyzed in tertiles. When reporting the measures of central tendency, these values were excluded from the analysis. Study Population Less than 1.0% of cadmium concentra- tions were recorded below BEC and 1.1% of arsenic concen- trations fell below their respective BEC values. A random sample of 270 subjects (~ 1% of the ARCH cohort) was selected and concentrations for arsenic and cadmium were quantified for the present study. This study is pilot in nature to explore our hypothesis. Future studies utilizing a larger sample size from the cohort with prospec- tive follow-up will be conducted as well as the addition of more underlying conditions. Baseline questionnaire data were used for analysis, as well as 400 µL of the baseline saliva sample. Introduction 1 3 3 271 Low-Level Environmental Heavy Metals are Associated with Obesity Among Postmenopausal Women… The obesity epidemic has dramatically increased, par- ticularly in the southern region of the United States (CDC 2019), including Arkansas. Among women, Arkansas has the highest prevalence of obesity compared to all other states (America’s Health Rankings 2020). This study conducted in an Arkansas population aimed to identify other putative contributors to the growing obesity epidemic. We sought to evaluate the potential association of arsenic and cadmium with obesity among women in Arkansas, where obesity and exposure to these elements are prevalent. solution and stored in the dark at room temperature until analysis. To obtain arsenic and cadmium concentrations, samples were analyzed using a Thermo Fisher Scientific iCAP RQ ICP-MS (Thermo Fisher Scientific, Waltham, MA). Multi-element ICP-MS stock standard and internal stand- ards of 10 µg/mL was purchased from Inorganic Ventures, Christiansburg, VA. A second source quality control stand- ard (100 µg/mL) was purchased from SPEX CertiPrep, Metuchen, NJ. Nitric acid trace metal grade and ethyl alco- hol was purchased from Fisher Chemical, Fair Lawn, NJ. The sample diluent used to prepare the standards, quality controls, and samples consisted of 2% (v/v) nitric acid and ethanol. Introduction Exposure to high levels of arsenic can cause skin lesions, cardiovascu- lar disease, neurological effects, diabetogenic effects, and various forms of cancer (ATSDR 2011a; IARC 2012a). The effect of arsenic exposure in low doses remains con- troversial in the literature (Schmidt 2014). General popula- tion exposures to arsenic are largely attributed to contami- nated food, water, and air ingestion averaging between 20 and 300 µg/day (IARC 2012a). Arsenic (As) is a naturally occurring metalloid that is ubiquitous in the environment. Arkansas is among several states along the Mississippi Delta region, which reports the highest levels of arsenic in the U.S. (Smith et al. 2017). Individuals residing in the state experience chronic low-dose exposures to these met- als, and the health effects are not yet clear.f Cadmium (Cd) has long been recognized as an environ- mental risk factor for multi-organ dysfunction and has been determined to play a role in the pathogenesis of obesity, diabetes, and the metabolic syndrome (Tinkov et al. 2017). Like arsenic, cadmium is ubiquitous in the environment and is in the top ten environmental chemicals of concern to envi- ronmental health agencies (Park et al. 2017). There is an abundance of literature focusing on cadmium exposure via in utero, perinatal, and infant exposure, but limited data on the relationship between adult exposure and obesity exist (Park et al. 2017). Human studies have produced conflicting results (Tinkov et al. 2017). Data from the National Health & Nutrition Examination Survey (NHANES), reported urine cadmium concentrations were negatively associated with BMI, waist circumference, and obesity (Padilla et al. 2010). However, a study utilizing blood cadmium levels reported a positive association between cadmium and BMI, as well as cadmium and dyslipidemia (Zhou et al. 2016). f While arsenic is known to have harmful health effects regardless of gender, there are some consequences that are unique to women. Numerous studies have shown that preg- nant women, fetuses, and neonates all suffer adverse preg- nancy outcomes when exposed to arsenic (Milton et al. 2005, 2017; Rahman et al. 2009; Farzan et al. 2013; Ronco et al. 2010). Particularly for exposed mothers, hypertensive disorders have been reported at higher rates than in the general population. Studies of pregnant women in Chile and Romania have identified associations between adverse health outcomes among mothers consumed drinking water with arsenic concentrations of 40 μg/L (Hopenhayn et al. Other Covariates Sociodemographic information provided in the baseline survey such as race, hormone use, region of residence, education, breastfeeding/parity, alcohol use, age, physical activity, and age at last menstrual cycle were included to identify confounding effects and effect modification. The variables race, hormone use, region, education, breastfeed- ing/ parity, and alcohol use were analyzed as categorical variables, while the variables age, physical activity, and age at last menstrual cycle were analyzed as continuous vari- ables. Physical activity was a composite variable that varied from walking to vigorous activity, resulting in total hours per week. Race was analyzed as a categorical variable, white/ European American (EA), black/African American (AA), and other. In the final stratified models, other racial groups were excluded from the analysis due to insufficient sample size (n = 12) and race was analyzed as a dichotomous vari- able, EA and AA. Hormone use was analyzed as a dichoto- mous variable (yes/no), relating to the participants’ men- strual cycle. Region was analyzed as a dichotomous variable as either Rural or Urban, classified by the subjects’ zip code at baseline using the rural–urban commuting area (RUCA) codes. Participants’ parity and breastfeeding practices were combined to create a composite variable with the following three possible outcomes: Nulliparous and never breastfed, given birth to at least one child and never breastfed, or given birth to at least one child and breastfed. This composite was created to avoid multicollinearity being introduced into the statistical models. Linear regression models were attempted to observe the relationship between arsenic and cadmium and their associa- tion to obesity, while using BMI as the continuous outcome. Due to majority of participants had very low concentrations of these heavy metals, the data were heavily skewed such that normalization could not be achieved (data not shown). However, after producing stratified models, a log10 normal- ization was attempted and successful for postmenopausal women and two regression models examining arsenic and cadmium as continuous exposures with the outcome of obe- sity was performed producing odds ratios (ORs) and 95% confidence intervals (95% CI). Heavy metal data of premen- opausal women were unable to be adequately normalized, and therefore linear regression was not possible at the time, this is likely due to the small sample size. Unconditional logistic regression was used to estimate unadjusted and multivariable-adjusted ORs and 95% CIs to estimate associations between increased likelihood for obesity and each metal concentration category. Statistical Analyses Weight Gain Velocity = (Weight at enrollment −Weight at age 18) (Age at enrollment −8) Differences in patient characteristics between non-obese and obese individuals were evaluated using X2 test for cat- egorical variables and t-tests for continuous variables. Both arsenic and cadmium concentrations were categorized into three levels based on the overall cohort distribution; the first level belonging to individuals with the lowest third of the specific element concentration, the second level comprised of the middle 33% of the concentrations, and the highest tertile contains the highest third of the specific element con- centrations. Distributions of both arsenic and cadmium were determined by evaluating the measures of central tendency. Obesity Participants were asked to refrain from eating or drinking at least 30 min prior to providing a saliva sample. Participants were also discouraged from providing a sputum sample, as mucus was not desired. Roughly 200 µL of a spit sample was mixed with equal parts Oragene DNA (OG-250) stabilizing solution (DNA Genotek, Ottawa, Ontario, Canada) (Barai et al. 2017), inverted 3–4 times to ensure distribution of At baseline, participants self-reported their height (in.) and weight (lb.), to which the participants’ body mass index (BMI) was calculated with the following formula: BMI = [weight (kg)/height (m2)]. If a participant had a BMI of greater than or equal to 30, they were defined as obese. If the BMI was between 18.5 and less than 30, they were defined as non-obese. Individuals with unreliable data, 1 3 272 S. Stahr et al. the analysis if a covariate utilized in the specific model was recorded as missing (n = 14). As previously described, data obtained for the current study were obtained from the exist- ing ARCH study cohort. The ARCH cohort was established to study the risk and effects of breast cancer. In the current study, breast cancer cases were excluded (n = 8), to omit any potential bias that were associated with any treatment, life- style, or genetic factors. such as outliers or data entry errors, were removed from the analysis (n = 4). The weight gain velocity (WGV) was calculated with the formula below to observe potential associations in the rate of weight gain with different concentrations of arsenic and cadmium. Individuals were asked to self-report their best approximation of their weight at age 18 during the base- line survey. If there were missing records for an individual’s weight, age, or weight at age 18, their WGV was reported as missing (n = 1): Other Covariates Tests for trend were performed by assessing median of arsenic and cadmium concentration tertiles as ordinal variables in the logistic regression model to observe their associations with obesity. These models were created due to the low concen- trations of both arsenic and cadmium, to better observe a difference between relative “low”, “medium”, and “high” values. Exclusions Participants were excluded from the study to eliminate potential bias. Individuals that recorded non-reliable or miss- ing value for height or weight, or a record of a BMI < 18.5 or > 60 were excluded from the analysis (n = 11). When ana- lyzing the adjusted models, individuals were excluded in Potential confounding variables for the association between obesity and arsenic and cadmium concentrations were included in the multivariable logistic regression mod- els. Confounding variables were selected based on a priori knowledge, as well as a 10% change in the beta coefficient. 1 3 3 273 Low-Level Environmental Heavy Metals are Associated with Obesity Among Postmenopausal Women… The most parsimonious model was used for each analysis to estimate associations with obesity and arsenic and cad- mium concentrations. The following covariates were used in at least one of the models presented: age, ethnicity, meno- pausal status, parity/breastfeeding history, age at last men- strual cycle, and alcohol use. Stratified models were used to analyze obesity with arsenic and cadmium concentrations for both premenopausal and postmenopausal women, using two different models. Generalized linear models were used to examine the regression between WGV and various metal quartiles, and comparisons were made using the lowest quartile as a reference. WGV was analyzed utilizing arsenic and cadmium quartiles rather than tertiles due to the larger sample size allowing for further categorizations to better investigate the relationship. form of education obtained. The distribution of age since menopause among postmenopausal women is rather uni- form, regardless of obesity status, with roughly 30% in each category. When the variable of parity and breastfeed- ing were combined into one composite variable, premeno- pausal non-obese women mainly gave birth to children and did breastfeed (52.8%) compared to the other categories, whereas premenopausal obese women, as well as all post- menopausal women primarily gave birth to children and did not participate in breastfeeding (50.0%, 54.8%, and 48.9% respectively). The majority of women in the study, both non-obese and obese, premenopausal and postmeno- pausal women reported their alcohol consumption as never to once a year. The distribution of BMI was similar among premeno- pausal and postmenopausal women. Among premenopausal women, the non-obese women had a mean BMI of 24 kg/ m2, whereas obese women had a mean BMI of 38 kg/m2. Similarly, postmenopausal non-obese women had a BMI of 27 kg/m2, and obese women recorded a higher BMI of 37 kg/ m2. Exclusions Premenopausal women displayed an almost 23-lb differ- ence between non-obese and obese self-reported weight at age 18, whereas postmenopausal women reported a smaller gap of 7.4 lb. Regardless of the menopausal group, the non- obese women reported lower weights at age 18 compared to obese women. The mean ages for each group of women ranged from 44 years of age to 59 years of age. Premenopau- sal women recorded more hours per week participating in physical activity compared to postmenopausal women. The age at which women underwent menarche was consistent across all groups. Interaction was assessed between arsenic and cadmium using a multiplicative model utilizing 75% cutoff points to determine the effects of “high” and “low” concentrations of each heavy metal. The test for interaction did not produce significant results; however, high arsenic values appear to enhance the associations. Therefore, we produced separate final models for arsenic and cadmium. i Each analysis performed was two-sided with p values of α less than 0.05. All analyses were performed using SAS version 9.3. Results Demographic information stratified by both obesity and menopausal status is shown in Table 1. The cohort over- all was split evenly between non-obese and obese women (51.9% vs. 48.1%, respectively). The study population consists of 78 women who identified as being premeno- pausal and 192 postmenopausal women. Among non-obese premenopausal women, the majority self-identified as European American (EA) (77.8%), whereas the major- ity of the obese premenopausal women self-identified as African American (AA) (59.5%). In the postmenopausal group, a large percentage of both non-obese and obese individuals identified as EA (69.2% and 60.2%, respec- tively). Regardless of obesity status, the majority of study participants live in rural regions of Arkansas. No premeno- pausal women reported using hormone therapy. Among postmenopausal women, similar percentages reported using hormones for both non-obese and obese individuals (11.5% and 11.4%, respectively). Premenopausal women differed in education status when separated by obesity. The majority of non-obese individuals attended some college or technical school, whereas obese individuals primarily completed a high school degree or GED. The majority of postmenopausal women, regardless of obesity status, completed a high school degree or GED as their highest Distributions of both arsenic and cadmium are detailed in Table 2. All concentrations, regardless of element, are below the safety limits set by the Centers for Disease Control and Prevention (CDC), World Health Organization (WHO), Food and Drug Administration (FDA), and the Agency for Toxic Substances and Disease Registry (ATSDR) (ATSDR 2011a, b). The mean arsenic values for both premenopausal and postmenopausal women are higher for obese women compared to non-obese women (0.028 µg/L, 0.023 µg/L vs. 0.022 µg/L, 0.020 µg/L, respectively). When cadmium con- centrations were considered, mean distributions differed by menopausal status. Premenopausal women had higher mean cadmium concentrations among non-obese women com- pared to obese (0.026 µg/L and 0.019 µg/L, respectively). Among postmenopausal women, those that were obese had a higher mean cadmium concentration compared to those who identified as non-obese (0.110 µg/L and 0.020 µg/L, respectively). It should be noted that one individual in the study population had a significantly higher cadmium con- centration that the rest of the participants; however, when excluding this participant, the relationships between obese and non-obese individuals remain (results not shown). 1 3 S. Stahr et al. 274 Table 1 Sociodemographic characteristics of the population by obese status and menopausal status SD standard deviation, BMI Body mass index, lbs. Pounds, hrs. Hours, yrs. Results Years † Chi-square test for categorical variables Characteristic Premenopausal women Postmenopausal women Non-obese† Obese† Non-obese† Obese† N = 36 (%) N = 42 (%) N = 104 (%) N = 88 (%) Race AA/African American 6 (16.7) 25 (59.5) 26 (25.0) 33 (37.5) EA/European American 28 (77.8) 17 (40.5) 72 (69.2) 53 (60.2) Missing 2 (5.5) 0 (0.0) 6 (5.8) 2 (2.3) Region Rural 36 (100.0) 37 (88.1) 99 (95.2) 84 (95.5) Urban 0 (0.0) 5 (11.9) 4 (3.8) 3 (3.4) Missing 0 (0.0) 0 (0.0) 1 (1.0) 1 (1.1) Hormone use Yes 0 (0.0) 0 (0.0) 12 (11.5) 10 (11.4) No 36 (100.0) 42 (100.0) 92 (88.5) 78 (88.6) Missing 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Education Less than high school 2 (5.6) 3 (7.1) 16 (15.4) 14 (15.9) High school graduate or GED 11 (30.5) 16 (38.1) 42 (40.4) 35 (39.8) Some college or technical school 14 (38.9) 15 (35.7) 26 (25.0) 30 (34.1) College or post graduate 9 (25.0) 8 (19.1) 20 (19.2) 9 (10.2) Missing 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Menopausal status Premenopausal 36 (100.0) 42 (100.0) 0 (0.0) 0 (0.0) Postmenopausal for ≤ 10 years 0 (0.0) 0 (0.0) 34 (32.7) 29 (33.0) Postmenopausal for 10–21 years 0 (0.0) 0 (0.0) 33 (31.7) 34 (38.6) Postmenopausal for > 21 years 0 (0.0) 0 (0.0) 37 (35.6) 25 (28.4) Missing 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Children and breastfeeding Not given birth and not breastfeed 2 (5.5) 5 (11.9) 8 (7.7) 12 (13.6) Given birth and not breastfed 15 (41.7) 21 (50.0) 57 (54.8) 43 (48.9) Given birth and breastfed 19 (52.8) 16 (38.1) 38 (36.5) 30 (34.1) Missing 0 (0.0) 0 (0.0) 1 (1.0) 3 (3.4) Alcohol use Never to once a year 26 (72.2) 26 (61.9) 78 (75.0) 77 (87.5) Once a month 5 (13.9) 11 (26.2) 10 (9.6) 3 (3.4) Once a week to several times a week 4 (11.1) 4 (9.5) 14 (13.5) 5 (5.7) Every day 1 (2.8) 1 (2.4) 2 (1.9) 2 (2.3) Missing 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.1) Mean‡ (SD) Mean‡ (SD) Mean‡ (SD) Mean‡ (SD) BMI 24 (3.0) 38 (6.0) 27 (2.7) 37 (4.8) Missing 0 0 0 0 Weight at 18 years of age (lbs.) 117.4 (16.5) 140.3 (38.8) 122.2 (26.1) 129.6 (29.3) Missing 0 0 2 0 Baseline Age (yrs.) 44 (6.7) 46 (5.2) 59 (8.8) 58 (8.1) Missing 0 0 0 0 Physical Activity (hrs. SD standard deviation, BMI Body mass index, lbs. Pounds, hrs. Hours, yrs. Years Results A significant increase in risk for obesity can be seen with increasing cadmium concentrations, as well as a significant trend (P for trend = 0.01). These trends suggest a dose–response for both arsenic and cadmium, and their relationship with an increased risk for obesity. Observing arsenic as a continuous variable for postmenopausal women was also produced and yielded statistically significant results similar to results of the logistic regression models. For every 10 unit increase in arsenic, the odds of being obese increase by a factor of 19.01. Similarly, for every 10 unit increase in cadmium, the odds of being obese increase by a factor of 2.64. Models were only ran among postmenopausal women, as pre- menopausal women did not fit a normal distribution among heavy metals. Weight gain velocity was calculated to identify potential associations between metal concentrations and the rate of weight gain since 18 years of age. Figure 1 displays the comparison of mean metal concentrations displayed in quartiles compared to their respective weight gain velocities. While only the first and fourth arsenic quartile show a statisti- cally significant difference (p < 0.05), the overall positive trend can be seen for increasing metal concentration and increasing weight gain velocity for both cadmium and arsenic that suggest dose–response relationships. While these results are promising, the mechanisms of these relationships have yet to be ascertained. Furthermore, there are several biologically plausible hypotheses of the associations between arsenic and obesity seen in this study. Arsenic is typically measured in urine, blood, hair, and fingernails/toenails as appropriate biospecimens for expo- sure, while saliva is not as commonly used. Cadmium is more commonly measured in blood, urine, and kidney sam- ples. A study published in West Bengal, India, analyzed saliva and concluded that salivary arsenic could be used as a potential biomarker of arsenic exposure, compared to urine arsenic concentrations (r = 0.60) (Bhowmick et al. 2013). A similar study published in Environmental Health and Pre- ventative Medicine concluded that saliva might be utilized as a biological monitoring tool for arsenic, due to their cor- relation analysis finding significant positive correlations between total arsenic in urine and saliva (r = 0.794, p < 0.01) (Wang et al. 2017). Results per week) 17.5 (1207.3) 20.5 (1590.7) 15.3 (1290.6) 12.1 (874.3) Missing 0 0 0 0 Age at first menstrual cycle 13 (2.0) 13 (2.0) 13 (1.8) 12 (1.8) Missing 0 1 0 0 Sociodemographic characteristics of the population by obese status and menopausal status 1 3 Low-Level Environmental Heavy Metals are Associated with Obesity Among Postmenopausal Women… 275 Table 2 Descriptive statistics by element concentration, obese status, and menopausal status Excluding concentrations that were below BEC Mean, Median, Min. and Max. are shown in µg/L Arsenic Premenopausal women Postmenopausal women N Mean Median SD Min Max N Mean Median SD Min Max Obese 41 0.028 0.017 0.044 0.008 0.261 88 0.023 0.022 0.012 0.008 0.086 Non-obese 35 0.022 0.019 0.011 0.007 0.048 103 0.020 0.017 0.012 0.007 0.090 Cadmium Obese 42 0.019 0.016 0.019 0.003 0.092 88 0.110 0.012 0.655 0.002 6.144 Non-obese 36 0.026 0.017 0.030 0.002 0.169 104 0.020 0.011 0.029 0.001 0.209 Table 2 Descriptive statistics by element concentration, obese status, and menopausal status g *Mean, Median, Min. and Max. are shown in µg/L associations found in this study relating to obesity were evi- dent only among postmenopausal women. While it is known that weight gain is a common result after menopause (Al- Safi and Polotsky 2015; Lovejoy 2003), a change in fat dis- tribution also occurs (Lovejoy 2003; Al-Safi and Polotsky 2015). While the mechanism is unclear, there are several factors that influence this distribution, such as hormone replacement therapy, age, and body composition (Lovejoy 2003). Another factor that should be recognized are estrogen levels. While there is sparse epidemiologic data regarding the relationship between estrogen and arsenic, the results produced from the current study support the existing evi- dence. The relationship seen between arsenic and obesity among postmenopausal women in the current study sup- ports the relationship of a decrease in estrogens as a result of increased arsenic concentrations seen in other publications (Chatterjee and Chatterji 2010). Table 3 displays the associations between obesity and metal concentrations. No models comprised of premenopausal women produced significant relationships or trends regard- ing metal concentration and obesity. Among postmenopausal women, relationships between obese and heavy metals were evident. A significant association was observed for increas- ing concentrations of arsenic and risk for obesity, including a significant trend (P for trend < 0.01). Although the relation- ship with obesity was less robust, cadmium concentrations followed a similar direction. Results We also found a significantly correla- tion in our pilot study of 100 volunteers for the concentra- tion of arsenic between saliva and urine (r = 0.60, p < 0.01) after adjusting for age, race, sex, BMI, and smoking status Discussion 1 Regression between the weight gain velocity LS-means and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simple linear regression 1 3 Table 3 Odds Ratios and 95% Confidence Intervals for association between element concentrations and obese status a Adjusted for: age, ethnicity, and parity/breastfeeding b Adjusted for: age, ethnicity, alcohol use, parity/Breastfeeding, and age at first menstrual cycle Premenopausal women Postmenopausal women Element concentrations Non-obese Obese OR (95% CI) Element concentrations Non-obese Obese OR (95% CI) Arsenica (N = 34) (N = 42) Arsenicb (N = 97) (N = 83) ≤ 0.014 (µg/L) 11 15 1.00 (ref.) ≤ 0.015 (µg/L) 42 18 1.00 (ref.) > 0.014—≤ 0.024 (µg/L) 12 15 0.72 (0.22–2.29) > 0.015—≤ 0.023 (µg/L) 31 29 2.74 (1.20–6.27) > 0.024 (µg/L) 11 12 0.63 (0.18–2.15) > 0.023 (µg/L) 24 36 4.43 (1.91–10.28) Trend 0.44 Trend < 0.01 Arsenic (continuous)b 19.01 (3.39–106.48) Cadmiuma (N = 34) (N = 42) Cadmiumb (N = 97) (N = 83) ≤ 0.009 (µg/L) 11 15 1.00 (ref.) ≤ 0.008 (µg/L) 39 20 1.00 (ref.) > 0.009—≤ 0.021 (µg/L) 11 15 0.98 (0.31–3.13) > 0.008—≤ 0.019 (µg/L) 32 27 1.57 (0.70–3.48) > 0.021 (µg/L) 12 12 0.70 (0.21–2.34) > 0.019 (µg/L) 26 36 2.72 (1.23–5.99) Trend 0.56 Trend 0.01 Cadmium (continuous)b 2.64 (1.33–5.21) Fig. Discussion Associations between both salivary arsenic and cadmium concentrations and obesity were examined in a pilot study using data from a cohort of Arkansan women. The 1 3 276 S. Stahr et al. Fig. 1 Regression between the weight gain velocity LS-means and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simple linear regression Table 3 Odds Ratios and 95% Confidence Intervals for association between element concentrations and obese status a Adjusted for: age, ethnicity, and parity/breastfeeding b Adjusted for: age, ethnicity, alcohol use, parity/Breastfeeding, and age at first menstrual cycle Premenopausal women Postmenopausal women Element concentrations Non-obese Obese OR (95% CI) Element concentrations Non-obese Obese OR (95% CI) Arsenica (N = 34) (N = 42) Arsenicb (N = 97) (N = 83) ≤ 0.014 (µg/L) 11 15 1.00 (ref.) ≤ 0.015 (µg/L) 42 18 1.00 (ref.) > 0.014—≤ 0.024 (µg/L) 12 15 0.72 (0.22–2.29) > 0.015—≤ 0.023 (µg/L) 31 29 2.74 (1.20–6.27) > 0.024 (µg/L) 11 12 0.63 (0.18–2.15) > 0.023 (µg/L) 24 36 4.43 (1.91–10.28) Trend 0.44 Trend < 0.01 Arsenic (continuous)b 19.01 (3.39–106.48) Cadmiuma (N = 34) (N = 42) Cadmiumb (N = 97) (N = 83) ≤ 0.009 (µg/L) 11 15 1.00 (ref.) ≤ 0.008 (µg/L) 39 20 1.00 (ref.) > 0.009—≤ 0.021 (µg/L) 11 15 0.98 (0.31–3.13) > 0.008—≤ 0.019 (µg/L) 32 27 1.57 (0.70–3.48) > 0.021 (µg/L) 12 12 0.70 (0.21–2.34) > 0.019 (µg/L) 26 36 2.72 (1.23–5.99) Trend 0.56 Trend 0.01 Cadmium (continuous)b 2.64 (1.33–5.21) Fig. Discussion One hypothesized explanation for this relationship is that arsenic is stored and accumulates in adi- pose tissue. In the current study, postmenopausal women had a significant correlation with increasing arsenic concentra- tions and obesity. Fat distribution in women changes with menopausal status, where central/abdominal obesity is more prevalent among postmenopausal women, as are various hormone stores within this white adipose tissue (Garaulet et al. 2002; Ferrara et al. 2002; Hodson et al. 2015; Lovejoy, 2003; Ceja-Galicia et al. 2017). The decreased urinary arse- nic concentration associated with an increase in BMI could possibly be explained by the urinary quantification. With the arsenic being stored in adipose tissues and only a marginal amount being excreted in the urine, it is possible that a low amount of arsenic is associated with a high BMI. In real- ity, these results are due to a large concentration of arsenic present, but it is stored in the adipose tissue, thus causing a bias moving away from the null value, and could explain a number of study findings in the literature. It should also be noted that the literature identifies that arsenic concentrations in saliva are considerably lower when compared to blood and urine (Wang et al. 2017; Bhowmick et al. 2013; Yuan et al. 2008), which was a major technical issue a few decades ago because the lower detection limit of Atomic Absorption Spectroscopy or earlier version of ICP- MS was at part per million (ppm) or part per billion (ppb) level. The newer version of ICP-MS, as used in this study, is capable of detecting concentrations as low as parts per trillion (ppt). This allows us to reach extremely low detec- tion limits within our population who were exposed to trace amounts of arsenic and cadmium that previous generation of instruments could not achieve. While arsenic and cadmium produce concentrations lower in saliva compared to blood and urine, their exposures remain the same, and with this study, we wish to identify the potential use of saliva as a biomarker for various metal exposures. Additionally, another primary advantage of this study is the ability to quantify both arsenic and cadmium concentrations simultaneously, allowing the concurrent observation of multiple elements and their relationship with obesity, with independent analy- ses per each element of interest. Discussion Positive aspects of utilizing saliva as a bio- specimen are that it is a relatively stable matrix compared to blood and urine, the processing and storage of the sample is less tedious compared to other biofluids, as well as the non- invasive procurement (Bhowmick et al. 2013; Wang et al. 2017). Limitations of the use of saliva are related to the fact that the matrix has a different chemical composition com- pared to blood or urine, the concentrations are considerably lower in saliva compared to traditional biological samples used (often 1 to 2 orders of magnitude lower than blood), and a lack of Standard Reference Material set by the US National Institute of Standards and Technology (Bhowmick et al. 2013; Wang et al. 2017). different results, depending upon the urine dilution adjust- ment method used (Bulka et al. 2017). In the present study, we report a more accurate representation of the arsenic-BMI relationship by the use of saliva as a biospecimen with less variation due to physiological factors. Figure 1 depicts the results from the regression analy- sis of weight gain velocity. The study identified a positive association between the rate of weight gain since 18 years of age and elevated concentrations of salivary arsenic and cadmium. As shown in Table 3, a clear relationship between these metals and obesity is evident. Further analysis of this relative weight gain suggests that low amounts of metal con- centrations affect the rate at which weight gain occurs in the lifespan. While the current study reports concentrations of As and Cd well below the governmental guidelines, a more diverse sample size with a larger range of concentrations could identify a more robust relationship between WGV and metal concentration. It is known that rapid weight gain causes nutritional deficiencies, excess adipose tissue accu- mulation, and increased pressure on various organ systems. With the concentrations of metals in the current study all below 1 ppb, these low concentrations could contribute to meaningful differences in WGV, thus further investigations into these relationships are needed. The relationship between urinary arsenic concentra- tion and BMI remains inconsistent in the exiting published reports. A study published in Toxicology and Applied Phar- macology in 2011 found that a decrease in urinary arsenic concentration is associated with an increase in BMI (Gomez- Rubio et al. 2011). Discussion 1 Regression between the weight gain velocity LS-means and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simple linear regression on between the weight gain velocity LS-means and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simple linear 1 3 Table 3 Odds Ratios and 95% Confidence Intervals for association between element concentrations and obese status Premenopausal women Postmenopausal women Element concentrations Non-obese Obese OR (95% CI) Element concentrations Non-obese Obese OR (95% CI) Arsenica (N = 34) (N = 42) Arsenicb (N = 97) (N = 83) ≤ 0.014 (µg/L) 11 15 1.00 (ref.) ≤ 0.015 (µg/L) 42 18 1.00 (ref.) > 0.014—≤ 0.024 (µg/L) 12 15 0.72 (0.22–2.29) > 0.015—≤ 0.023 (µg/L) 31 29 2.74 (1.20–6.27) > 0.024 (µg/L) 11 12 0.63 (0.18–2.15) > 0.023 (µg/L) 24 36 4.43 (1.91–10.28) Trend 0.44 Trend < 0.01 Arsenic (continuous)b 19.01 (3.39–106.48) Cadmiuma (N = 34) (N = 42) Cadmiumb (N = 97) (N = 83) ≤ 0.009 (µg/L) 11 15 1.00 (ref.) ≤ 0.008 (µg/L) 39 20 1.00 (ref.) > 0.009—≤ 0.021 (µg/L) 11 15 0.98 (0.31–3.13) > 0.008—≤ 0.019 (µg/L) 32 27 1.57 (0.70–3.48) > 0.021 (µg/L) 12 12 0.70 (0.21–2.34) > 0.019 (µg/L) 26 36 2.72 (1.23–5.99) Trend 0.56 Trend 0.01 Cadmium (continuous)b 2.64 (1.33–5.21) Fig. 1 Regression between the weight gain velocity LS-means and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simple linea regression Fig. 1 Regression between the weight gain velocity LS-means and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simple lin regression s and arsenic quartiles and between the weight gain velocity LS-mean and cadmium quartiles determined using simp a Adjusted for: age, ethnicity, and parity/breastfeeding b Adjusted for: age, ethnicity, alcohol use, parity/Breastfeeding, and age at first menstrual cycle enic quartiles and between the weight gain velocity LS-mean and a Adjusted for: age, ethnicity, and parity/breastfeeding b Adjusted for: age, ethnicity, alcohol use, parity/Breastfeeding, and age at first menstrual cycle Fig. 1 Regression between the weight gain velocity LS-means and regression 1 3 Low-Level Environmental Heavy Metals are Associated with Obesity Among Postmenopausal Women… 277 (not published). Discussion With the sam- ple size of 77 participants, we are unable to produce pre- cise results, compared to that of the postmenopausal results where the sample sizes are more than doubled. However, a study published in Biological Trace Element Research pro- duced similar results. Women aged 18–35 of childbearing- age were analyzed for relationships between urinary arsenic concentrations and BMI as well as the fat mass percentage (Ronco et al. 2010). The results concluded that no significant relationships could be obtained. Therefore, while the cur- rent study has a smaller sample size for the stratification of premenopausal women, the results are consistent with the current literature. arsenic and cadmium concentration with smoking status (results not shown). Different occupational exposures can be related to elevated levels of arsenic and cadmium (IARC 2012a; b), as well as the risk for obesity (Church et al. 2011). Smoking is also an established cause of increased arsenic and cadmium concentrations in the body (Pappas 2011) and is associated with various occupational exposures (Sterling and Weinkam 1990). Another concern at baseline, several essential variables, such as height, weight, and weight at age 18 were collected as self-report data. These data were collected at the same interview as the saliva sample, which is a limitation of cross-sectional studies. We are unable to confirm the temporal relationship between the arsenic and cadmium concentrations with obesity. Another restriction is that the number of subjects included in subset analyses, par- ticularly among premenopausal subjects, was relatively low. In the current study, the significant findings were among the postmenopausal women, while the premenopausal category failed to establish any significant correlations. With the sam- ple size of 77 participants, we are unable to produce pre- cise results, compared to that of the postmenopausal results where the sample sizes are more than doubled. However, a study published in Biological Trace Element Research pro- duced similar results. Women aged 18–35 of childbearing- age were analyzed for relationships between urinary arsenic concentrations and BMI as well as the fat mass percentage (Ronco et al. 2010). The results concluded that no significant relationships could be obtained. Therefore, while the cur- rent study has a smaller sample size for the stratification of premenopausal women, the results are consistent with the current literature. Acknowledgments This study is indebted to volunteers and partici- pants contributed to the ARCH. Discussion Funding This research was supported by a grant from the Arkansas Breast Cancer Research Program and the University of Arkansas for Medical Sciences. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest. Consent to Participate Informed consent was obtained from all indi- vidual participants included in the study. Consent for Publication The authors affirm that human research par- ticipants provided informed consent for publication of the aggregate data produced in the study. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Discussion i In contrast, fewer results published have concluded that an increase in urinary arsenic concentration is associated with a decrease in BMI (Ronco et al. 2010); however, the inverse relationship can still be explained by the same mech- anism as described above. With a low BMI, there is not an abundance of adipose tissue to be utilized for arsenic storage, and therefore the majority is excreted in the urine. These results can inaccurately depict that high urinary arse- nic is associated with a low BMI, where, in reality, there is no arsenic being stored in the body and is all excreted, thus biasing the results. This study has several limitations. First, there are covari- ates that were not recorded at baseline, such as smoking status and occupation, which would preferably be included in the analyses. Smoking and the effects on heavy metal con- centrations are controversial in the literature (Garhammer et al. 2004). A separate pilot study consisting of 100 indi- viduals did not provide any significant association between By utilizing saliva, these potential biases can be avoided, as saliva does not undergo the same biological processes as urine. A paper published in Environmental Health Per- spectives in 2017 suggested that the relationship between arsenic and obesity measured in urine can have drastically 1 278 S. Stahr et al. arsenic and cadmium concentration with smoking status (results not shown). Different occupational exposures can be related to elevated levels of arsenic and cadmium (IARC 2012a; b), as well as the risk for obesity (Church et al. 2011). Smoking is also an established cause of increased arsenic and cadmium concentrations in the body (Pappas 2011) and is associated with various occupational exposures (Sterling and Weinkam 1990). Another concern at baseline, several essential variables, such as height, weight, and weight at age 18 were collected as self-report data. These data were collected at the same interview as the saliva sample, which is a limitation of cross-sectional studies. We are unable to confirm the temporal relationship between the arsenic and cadmium concentrations with obesity. Another restriction is that the number of subjects included in subset analyses, par- ticularly among premenopausal subjects, was relatively low. In the current study, the significant findings were among the postmenopausal women, while the premenopausal category failed to establish any significant correlations. 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Best Pract Res ClinObstetGynaecol 29:548–553 In conclusion, the relationship between arsenic and obe- sity, as well as cadmium and obesity were observed in post- menopausal women, both with an evident dose–response relationship. The relationship is not evident among premeno- pausal women for either element. Measurement in saliva, rather than in blood or urine, produced meaningful results. While these concentrations are below the regulatory and governmental limits, they still show a significant associa- tion with obesity. The data presented in this study regarding low levels of arsenic and cadmium can inform governmental limits and guidelines regarding appropriate levels of human exposure. Information presented in the current study has revealed other possible explanations to the growing obesity epidemic. Additional research is needed to further exam- ine the magnitude of effect arsenic and cadmium have on obesity. This study has demonstrated that saliva can be an appropriate biospecimen for various elements and should be further investigated. Future research including smoking habits to more specifically analyze metal concentrations as well as any potential confounders is necessary; ambient air pollution measurements to assess the sources of exposure to these metals is also needed. Alvarado-Cruz I, Alegria-Torres JA, Montes-Castro N, Jimenez-Garza O, Quintanilla-Vega B (2018) Environmental epigenetic changes, as risk factors for the development of diseases in children: a sys- tematic review. Ann Glob Health 84:212–224 America’s Health Rankings (2020) ’Health of Women and Children: Public Health Impact: Obesity - Women’, United Health Founda- tion, America’s Health Rankings. https://www.americashealthr ankings.org/explore/health-of-women-and-children/measure/ Obesity_women/state/ALL. 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https://openalex.org/W3034975298	https://figshare.com/articles/journal_contribution/Optimal_allocation_of_subjects_in_a_matched_pair_cluster-randomized_trial_with_fixed_number_of_heterogeneous_clusters/12848110/1/files/24405934.pdf	English	null	Optimal allocation of subjects in a matched pair cluster-randomized trial with fixed number of heterogeneous clusters	Journal of applied statistics	2,020	cc-by	760	Supplementary material for: Optimal allocation of subjects in a matched pair cluster randomized trial with ﬁxed number of heterogeneous clusters Supplementary material for: Optimal allocation of subjects in a matched pair cluster randomized trial with ﬁxed number of heterogeneous clusters Satya Prakash Singh, Pradeep Yadav Satya Prakash Singh, Pradeep Yadav Equations whose label does not include numbers appear in the main texts. Equations whose label does not include numbers appear in the main texts. Equations whose label does not include numbers appear in the main texts. ion of equations (14) and (15): (1/N)(1 −⇢i) {(1/N)(1 −⇢i) + ⇠i⇢i}2 = (1/N)(1 −⇢j) {(1/N)(1 −⇢j) + ⇠j⇢j}2 p (1 −⇢i) {(1/N)(1 −⇢i) + ⇠i⇢i} = ± p (1 −⇢j) {(1/N)(1 −⇢j) + ⇠j⇢j}, for i 6= j = 1, . . . , m (A) (A) e solutions from (A), the solution based on the following system of equations Out of two possible solutions from (A), the solution based on the following system of equations p (1 −⇢i) {(1/N)(1 −⇢i) + ⇠i⇢i} = − p (1 −⇢j) {(1/N)(1 −⇢j) + ⇠j⇢j}, for i 6= j = 1, . . . , m results in an infeasible solution. This can be easily veriﬁed for a particular case when m = 2, and ⇢i = ⇢for i = 1, 2. Therefore, we solve the system based on the following system of equations results in an infeasible solution. This can be easily veriﬁed for a particular case when m = 2, and ⇢i = ⇢for i = 1, 2. Therefore, we solve the system based on the following system of equations i = 1, 2. Therefore, we solve the system based on the following system of equations p (1 −⇢i) {(1/N)(1 −⇢i) + ⇠i⇢i} = p (1 −⇢j) {(1/N)(1 −⇢j) + ⇠j⇢j}, for i 6= j = 1, . . . , m (B) (B) After some algebra, (B) can be written as After some algebra, (B) can be written as (1/N)(1 −⇢j) p (1 −⇢i) + ⇠j⇢j p (1 −⇢i) = (1/N)(1 −⇢i) q (1 −⇢j) + ⇠i⇢i q (1 −⇢j) ⇠i⇢i q (1 −⇢j) −⇠j⇢j p (1 −⇢i) = (1/N)(1 −⇢j) p (1 −⇢i) −(1/N)(1 −⇢i) q (1 −⇢j) ⇠i⇢i −⇠j⇢j s (1 −⇢i) (1 −⇢j) = (1/N) p (1 −⇢i){ q (1 −⇢j) − p (1 −⇢i)} ⇣ ⇢i −⇢j q (1−⇢i) (1−⇢j) ⌘ 0 @⇠i ⇠j 1 A = (1/N) p (1 −⇢i){ q (1 −⇢j) − p (1 −⇢i)}; i 6= j = 1, . . . Satya Prakash Singh, Pradeep Yadav Supplementary material for: Optimal allocation of subjects in a matched pair cluster randomized trial with ﬁxed number of heterogeneous clusters , ⇣ ⇢i −⇢i+k q (1−⇢i) (1−⇢i+k) ⌘ 0 @ ⇠i ⇠i+k 1 A = (1/N) p (1 −⇢i){ p (1 −⇢i+k) − p (1 −⇢i)}; k = 1, . . . , m The last system of equations is consist of m−1 equations for (i, i + 1) 2 {(1, 2), (2, 3), . . . , (m−1, m)}, which together with (13) can be written in matrix form as given in (14) and (15). Derivation of equation (20): Derivation of equation (20): Suppose ⇢j’s are independently distributed as Uniform(aj, bj) for j = 1, . . . , m. For this prior, (19) reduces to Suppose ⇢j’s are independently distributed as Uniform(aj, bj) for j = 1, . . . , m. For this prior, (19) reduces to B(⇠, N) = 1 2σ2 m X j=1 h 1 (bj −aj) Z bj aj ⇠j (1/N)(1 −⇢j) + ⇠j⇢j d⇢j i = 1 2σ2 m X j=1 h 1 (bj −aj) Z bj aj ⇠j 1/N + ⇢j(⇠j −1/N)d⇢j i . (C) (C) Let us assume that yj = 1/N + ⇢j(⇠j −1/N), then (C) can be written as Let us assume that yj = 1/N + ⇢j(⇠j −1/N), then (C) can be written as Let us assume that yj = 1/N + ⇢j(⇠j −1/N), then (C) can be written as Let us assume that yj = 1/N + ⇢j(⇠j −1/N), then (C) can be written as B(⇠, N) = 1 2σ2 m X j=1 h 1 (bj −aj) Z 1/N+bj(⇠j−1/N) 1/N+aj(⇠j−1/N) ⇠j yj dyj (⇠j −1/N) i = 1 2σ2 m X j=1 h ⇠j (bj −aj)(⇠j −1/N) Z 1/N+bj(⇠j−1/N) 1/N+aj(⇠j−1/N) dyj yj i = 1 2σ2 m X j=1 h ⇠j (bj −aj)(⇠j −1/N) Z (1/N)(1−bj)+bj⇠j (1/N)(1−aj)+aj⇠j dyj yj i = 1 2σ2 m X j=1 h. ⇠j (bj −aj)(⇠j −1/N) log . (1/N)(1 −bj) + bj⇠j (1/N)(1 −aj) + aj⇠j i . 2 2
https://openalex.org/W2045256860	https://curationis.org.za/index.php/curationis/article/download/454/394	English	null	An example of a self-instructional teaching unit for nursing students	Curationis	1,982	cc-by	3,877	TITLE Human motivation and ego-defence mechanisms, and their application to nursing care. Inability to handle his situation adequately affects the patient’s self-image making him anxious and caus ing him to adopt one or other ego-defence mechanism. This is a normal reaction, but one needs to know when the patient is using defences excessively because this makes him rigid and unopen to the care planned for him. INTRODUCTION Designing effective teaching units is a very important part of the work of the tutor. As part of a course in curriculum development at the University of the Orange Free State, tutors are given practice in this skill. Much attention is given to varying the teaching methods and adapting them according to the unit objectives, which in turn arise from a task analysis. The emphasis of the teaching methods is also on developing higher levels of thinking in the student. An example of such a teaching unit is presented here SPECIFIC OBJECTIVES • Define motivation and identify the biological and the psycho-social forms of motives. • Define ego-defence mechanism and describe the rela tionship between this and motivation. • Identify, from a given description of a patient’s be haviour, his felt needs, and plan for appropriate nursing care. TARGET GROUP • Identify situations in which the patient demonstrates excessive use of one or two defences and plan for the appropriate nursing care. Second year students: Diploma in General Nursing Second year students: Diploma in General Nursing COURSE OBJECTIVE pp p g • Identify your own habitual defence mechanisms. Students will be able to understand human behaviour in health and in illness, and be able to use this knowledge in the care of patients as individuals and in groups. OPSOMMING Die ontwerp van onderrigeenhede waarin die onderrigmetodes met die doelstellings ooreenkom en waarin hoër vlakke van denke by die student gestimuleer word, word aan die hand van ’n voorbeeld, oor motivering en verdedigingsmega- nismes, geillustreer. COURSE identification sublimation. Introductory psychology • Identify in yourself (or in others) a lack of motiva tion and analyse the possible causes thereof. RATIONALE All behaviour is directed towards the satisfaction of a person’s needs and the fulfilment of his aspirations. Nursing involves mainly helping the patient to meet his needs. The nurse must have insight into the processes involved in motivation such as why a lonely person will have difficulty in respecting himself. Concept psychology and how it is applied to nursing practice Diagram 1. The effects of hypothalamic stimulation on the body and its influence on behaviour. Psychology Scientific study of behaviour and experi ence. dge about reality is gained 1 EQ UILIBRIUM Science A method of study by which knowledge about reality is gained largely under controlled conditions rather than by casual observation. Behaviour All observable (overt) and non-observable (covert) reactions to stimulation. Experience Mental processes occuring within the indi vidual as a result of exposure to a situation. Application The extent to which the nurse-practitioner is able to use the knowledge gained from psychology in her relationships with others and in patient interven tion. ENTERING BEHAVIOUR g • Describe, in your own words in writing, without the help of your prescribed sources, the defensive be havior characteristics of each of the following Mechanisms: Before the student proceeds with the learning, she must have acquired knowledge concerning Before the student proceeds with the learning, she must have acquired knowledge concerning — the concept psychology and how it is applied to nursing practice; regression — the organic structure of the body in relation to emo tion; rationalisation projection — the effect of hypothalamic stimulation on the body, and its influence on behaviour. displacement CURATIONIS VOL. 5 NO. 4 VOL. 5 NO. 4 46 CURATIONIS Final test A final test will be given at the end of the week, on a given date. The student will be tested on all the work covered in the unit. The questions will, by and large, be on application of the concepts motivation and ego de fence mechanisms to the planning of care for a (hypo thetical) patient. Consult: 1. Fisher, E.E. Psychology for Nurses and the Health Team. Juta & Company, Cape Town, 1981 4th Ed (pages 2-6.) Maslow’s hierarchy of human needs must be seen in re lation to patient care. See flow-diagram 1. Relationship between motivation, anxiety and ego-defence mechanisms 2. McNaught, A.B. Callander, R Nurses’ illustrated physiology E. & S. Livingstone London 1971 2nd Ed. (pages: 94 and 150) See flow-diagram 2 The estimated study time for this unit is one week The estimated study time for this unit is one week The endocrine system Secretes special chemicals (hormones) which influence the behavioural reactions' of the individual, such as energy levels, and reaction-time. Hierarchy of human needs (Maslow) Hierarchy of human needs (Maslow) The hypothalamus Is the main seat of emotional expression. Is closely associated with the autonomic nervous system. unit. The student must obtain a 100 % score and if not, must study the unit again and re-test herself until at least 100 % accurate. The student must first answer the questions and then give herself a score against the cor rect answers provided. Is sensitive to conditions of stress and operates through the sympathetic stimulation to restore body equilibrium. The autonomic nervous system The parasympathetic system generally has a soothing effect on body tissues. The sympathetic system has a stimulating effect, its output being directly related to the energy levels needed by the body. Effects of hypothalamic stimulation on the body and its influence on behaviour See diagram 1. The student who has not mastered the above con cepts must do so before proceeding with the rest of the unit. The organic structure of the body in relation to emotion The autonomic nervous system Progress-test Possible motives underlying the patient’s behaviour must be identified and the necessary care planned. See flow-diagram 3. A progress test is included by which the student must test herself. The answers are provided at the end of this 47 CURATIONIS (2) Safety needs protection; familiar environment; non-fear provoking situations. Become dominant in newly admitted patients; in disasters etc. (1) Biological needs air, food, water, sleep exercise, elimina tion etc. -Aroused first and become dominant if not satisfied -If adequately met needs of the higher levels are felt -Most urgent in states of shock due to dehydration or blood loss, and in respiratory distress. Higher level needs arise when lower ones are satisfied. Regression to lower needs occurs when higher needs are frustrated. anxiety and ego-defence mechanisms. CURATIONIS CURATIONIS Flow-diagram 2: Relationship between motivation, anxiety and ego-defence mechanisms. Flow-diagram 2: Relationship between motivation, anxiety and ego-defence mechanisms. Flow-diagram 1: Hierarchy of Human Needs (Maslow) Flow-diagram 1: Hierarchy of Human Needs (Maslow) Flow-diagram 1: Hierarchy of Human Needs (Maslow) (5) Self-actualisation achievement; attain ment of goals; competence Dominant in the con valescing patient. (4) Self-esteem respect; recognition, honour, prestige, independence. Dominant in the loss of status for the hospital ised patient; exposure to medical treatments etc. (3) Belonging needs affection; friendship acceptance; interperso nal relationships. Strongly felt by children in separating states, by the neglected patient and the homeless patient. (2) Safety needs protection; familiar environment; non-fear provoking situations. Become dominant in newly admitted patients; in disasters etc. (1) Biological needs air, food, water, sleep exercise, elimina tion etc. -Aroused first and become dominant if not satisfied -If adequately met needs of the higher levels are felt -Most urgent in states of shock due to dehydration or blood loss, and in respiratory distress. Higher level needs arise when lower ones are satisfied. Regression to lower needs occurs when higher needs are frustrated. (Maslow) (5) Self-actualisation achievement; attain ment of goals; competence Dominant in the con valescing patient. (4) Self-esteem respect; recognition, honour, prestige, independence. Dominant in the loss of status for the hospital ised patient; exposure to medical treatments etc. (3) Belonging needs affection; friendship acceptance; interperso nal relationships. Strongly felt by children in separating states, by the neglected patient and the homeless patient. (2) Safety needs protection; familiar environment; non-fear provoking situations. Become dominant in newly admitted patients; in disasters etc. (1) Biological needs air, food, water, sleep exercise, elimina tion etc. -Aroused first and become dominant if not satisfied -If adequately met needs of the higher levels are felt -Most urgent in states of shock due to dehydration or blood loss, and in respiratory distress. Higher level needs arise when lower ones are satisfied. Regression to lower needs occurs when higher needs are frustrated. anxiety and ego-defence mechanisms. (Maslow) (5) Self-actualisation achievement; attain ment of goals; competence Dominant in the con valescing patient. (4) Self-esteem respect; recognition, honour, prestige, independence. Dominant in the loss of status for the hospital ised patient; exposure to medical treatments etc. (3) Belonging needs affection; friendship acceptance; interperso nal relationships. Strongly felt by children in separating states, by the neglected patient and the homeless patient. Flow-diagram 3: Identifying motives underlying behaviour and planning care ow-diagram 3: Identifying motives underlying behaviour and planning care Possible motives underlying the behaviour of an adult patient who refuses hospital treatment and wishes to sign the Refusal of Hospital Treatment form, and the care which can be provided 48 C A O S O 5 O 4 Possible motives underlying the behaviour of an adult patient who refuses hospital treatment a the Refusal of Hospital Treatment form, and the care which can be provided 48 On motivation create situations that encourage a hopeless patient to identify with, and therefore gain support from, other patients who are facing illness bravely; * All behaviour is motivated, that is based on need- arousal. * Much of our behaviour has unconscious motivation. * Much of our behaviour has unconscious motivation. p g y create a ward atmosphere which provides tension- outlets for the patients, for example suitable indoor- games and cheerful conversation; * Need-arousal puts the individual in a state of tension and readiness to meet the felt need. * Needs that cannot be met puts the individual in a state of disequilibrium or anxiety. recognise, in the patients’ behaviour, excessive use of defences, or inability to formulate defences, and plan for the necessary support systems. q y * Motivation is inferred from the individual’s behav Skills The nurse is able to identify the patient’s needs and compile a compre hensive care plan to meet those needs; Concepts to be learned iour and, in the case of the patient, assists in the plan ning of nursing care. iour and, in the case of the patient, assists in the plan ning of nursing care. On motivation biological drives emotional drives psycho-social needs hierarchy of human needs (Maslow’s theory) unconscious motivation. ANSWER: The student is given the specific objectives, the entering behaviour and the task description with the following instructions. a) Teach him how to use hospital equipment (eli minate anxiety) 1. Study the diagram depicting Maslow’s hierrarchy of needs (flow diagram 1) and test yourself with the few questions below. y b) Show skill when carrying out procedures on him. y b) Show skill when carrying out procedures on him. 1. Define the concept hierarchy of needs. Confine your answer to about 20 words. 1. Define the concept hierarchy of needs. Confine your answer to about 20 words. c) Keep patient and ward environment very clean. c) Keep patient and ward environment very clean. d) Assist him with domestic problems. On ego-defence mechanisms * In most people anxiety is dealt with by the adoption of appropriate ego-defence mechanisms. * Inability to cope imposed by illness renders the patient more predisposed to anxiety, and makes him adopt defensive behaviour. On ego-defence mechanisms the concept ego-defence and the processes involved displacement identification projection rationalisation regression sublimation On ego-defence mechanisms the concept ego-defence and the processes involved displacement identification projection rationalisation regression sublimation p * Overuse of one or two ego-defence mechanisms makes the individual rigid and it is difficult for him to enter into meaningful relationships with others. CURATIONIS CURATIONIS VOL.5 NO. 4 CURATIONIS ANSWER: All other higher level needs recede. For example, if there is no water, nothing else matters. All other higher level needs recede. For example, if there is no water, nothing else matters. Attend the film Angry Boy which illustrates how un conscious motivation affects the behaviour of both chil dren and adults. (Running time = 31 minutes) 3. Name any three ways to help the patient fulfil the needs in level 2. Study ego-defence mechanisms in Vlok (1973: 327- 341); or any other relevant literature. Study ego-defence mechanisms in Vlok (1973: 327- 341); or any other relevant literature. 2. Study the flow-diagrams 2 and 3. 2. Study the flow-diagrams 2 and 3. Attend the film Anger at work which demonstrates a case of displacement of anger. (Running time = 19V2 minutes.) OR The film Neurotic Behaviour which includes illustra tions of some classical ego-defence mechanisms. (Running time = 19 minutes) Attend the film Anger at work which demonstrates a case of displacement of anger. Attend the film Anger at work which demonstrates a case of displacement of anger. 2. When the needs in level 1 are unsatisfied, how can you explain the patient’s condition in a few words with regard to the higher level needs? The film Neurotic Behaviour which includes illustra tions of some classical ego-defence mechanisms. (Running time = 19 minutes) The film Neurotic Behaviour which includes illustra tions of some classical ego-defence mechanisms. (Running time = 19 minutes) ANSWER: a) Teach him how to use hospital equipment (eli minate anxiety) ANSWER: If not satisfied with your own answers, study flow- diagram 1 again. The lower (biological)needs dominate and the higher needs emerge only when the lower ones have been met. The lower (biological)needs dominate and the higher needs emerge only when the lower ones have been met. 2. Study the flow-diagrams 2 and 3. 2. Study the flow-diagrams 2 and 3. ANSWER: 1. Assessment of patient — general condition — prevailing needs. 2. Devise a plan to provide the patients’ needs. 3. Use the plan and observe patients’ response to care. 4. Evaluate patients’ condition and decide whether further intervention is necessary. 1. Assessment of patient — general condition — prevailing needs. p g 2. Devise a plan to provide the patients’ needs. 3. Use the plan and observe patients’ response to care. These mechanisms are ego-defensive in that they prevent the lowering of the person’s self-esteem and prevent disintegration of his person. 4. Evaluate patients’ condition and decide whether further intervention is necessary. DECEMBER 1982 CURATIONIS 49 3. For each of the behaviours described here, identify the ego-defence mechanism employed. (The college time-table on FILMS shows students when and where the above films will be showing.) 3.1. The patient states that she could not seek medi cal advice early because she had nobody to remain with her children when she was, in fact, afraid of the possible outcome of the consulta tion. 3.1. The patient states that she could not seek medi cal advice early because she had nobody to remain with her children when she was, in fact, afraid of the possible outcome of the consulta tion. PROGRESS-TEST The student must obtain full marks in this test, if not she must follow the instructions at the end of this test. It is in the student’s own interest to do so. Item 1 Indicate (/) whether the following statements are true oi false :- Indicate (/) whether the following statements are true oi false :- 1. The nurse can help to reduce the patient’s anxiety by leaving the patient to sort out things for himself. PROCEED TO TEST YOURSELF WITH THE FOL LOWING QUESTIONS: (First write down your own answer before consulting the one provided. Study again the areas in which you gave a wrong answer.) 3.2. Nurse Judy is concerned about a patient in her ward who appears lonely and never seems to get visitors. So, whenever nurse Judy gets the chance, she spends a few minutes engaging the patient in conversation. 1. Define the concept motivation and support your definition with a suitable example. ANSWER: A person meets a situation which he cannot handle, experiences threats in his environment or encounters any other impediment which makes him uncomfortable. His energy levels rise to help him overcome the handicap by direct means (e.g. fight or flight) and this makes him tense. If no suitable means are available, he becomes anxi ous. Because anxiety cannot be relieved by physi cal fear-reducing measures, the person uncon sciously develops psychological mechanisms which bury his anxiety in unconsciousness, and make him comfortable with himself again. 4. Name the steps that must be covered in the process of providing nursing care for the patients. TRUE/FALSE 2. The so-called difficult patient may be motivated to escape from pain. ANSWER: 3.3 Peter is a sixteen-year-old boy. His parents often scold him for his poor performance at school. Peter spends most of his time playing football. His club-members call him Pele after the Brazilian football star whose style and mannerisms Peter has adopted. Motivation is the arousal of an internal stimulus, which may be conscious or unconscious, which energises the individual and makes him behave in a certain way. p 3.4 A student constantly accuses her group-mates of untidiness and says they are always throwing tis sues about. But looking at her own behaviour one can say that hers is worse than theirs. Example: A thirsty person is motivated to drink water. 2. Describe, in a paragraph of not more than 120 words, the process by which ego-defence mecha nisms occur. ANSWER: 3.1. — Rationalisation 3.2. — Identification 3.3. — Identification 3.4. — Projection Instructions: — Write down in your exercise book the answer(s) to each item without looking at the answers which are provided at the back of the unit. TRUE/FALSE 1. Ego-defence mechanisms are behaviour patterns used to reduce or remove . . . which cannot be handled by physical means. 1. Ego-defence mechanisms are behaviour patterns used to reduce or remove . . . which cannot be handled by physical means. 5. The patient who is constantly seeking the attention of the nurse for no apparent reason can be said to be using the mechanism of regression. y p y 2. The ego-defence mechanisms make the individual comfortable with himself and better able to adapt to the requirements of society. Therefore, they play an important part in interpersonal. . . . g TRUE/FALSE Item 3 4. To make the patient develop the urge to get well the nurse should use threats to force him to co-operate in his own treatment. Fill in the missing words. Do not rewrite the whole sen tence; simply write down the word(s): Item 2 Indicate with a ring around the alternative which best describes the behaviour: - p p p 3. To motivate a person is to . . . needs in him, and we can see whether or not a person is motivated by his . . . 1. If the patient behaves rudely towards the nursing staff as a result of his anxiety about his illness, which of the following ego-defences would suit this behav iour? 4. When a patient is dyspnoeic, he becomes restless and fidgety, refuses to co-operate with the nurse who comes to change her bed-linen because at this moment the . . . . is dominant and the patient cannot be motivated to do anything else. 4. When a patient is dyspnoeic, he becomes restless and fidgety, refuses to co-operate with the nurse who comes to change her bed-linen because at this moment the . . . . is dominant and the patient cannot be motivated to do anything else. 1.1. Sublimation 1.2. Projection 1.3. Displacement FINAL INSTRUCTIONS: ANSWERS TO PROGRESS-TEST 3.1. Displacement Item 1 item 2 Number Answer Number Answer 1. False 1. 1.3 2. True 2. 2.1 3. False 3. 3.4 4. False 4. 4.6 5. True 5. 5.7 Item 3 Number Answer 1. anxiety 2. relations 3. arouse; behaviour 4. biological (physical) need. 3.2. Sublimation 3.3. Projection 3.4. Rationalisation 4. When an elderly patient shows regressive behaviour by soiling her bed-linen and refusing to use the bedpan, the nursing staff should: 4.1. Tell the patient that the nurses have a big work load, and unless she behaves they will ask the doctor to discharge her. 4.2. Not scold her at all. 4.2. Not scold her at all. 4.3. Give her love and attention 4.4. Wash the patient with cold water so that she will not soil her bed again. g 4.5. Let the patient do as much for herself as poss ible to prevent total dependence. 4.6. (4.2; 4.3; and 4.5) 4.6. (4.2; 4.3; and 4.5) 4.7. (4.3; 4.5) 4.7. (4.3; 4.5) 4.8. All of the above. FINAL INSTRUCTIONS: 1.4. None of the above 2. When a person is very critical of others and always finding fault, it can be assumed that he is using the mechanism of:- 1. Determine your performance: 1. Determine your performance: . . . . out of 15 1. Determine your performance: . . . . out of 15 . . . . out of 15 2. Full marks? You have mastered the task! Very good. 2.1. Projection 2.2. Aggression g 3. Any mistakes made require you to refer back to the Presentation technique and study the areas in which your answers were wrong. Then re-answer the ques tions which were answered incorrectly until you have all the answers correct. 3. Any mistakes made require you to refer back to the Presentation technique and study the areas in which your answers were wrong. Then re-answer the ques tions which were answered incorrectly until you have all the answers correct. 2.3. Sublimation 2.4. Displacement 3. The mother who punishes her child and justifies her action by saying that it is for the child’s own good whereas she may in fact be relieving her own anger is using the mechanism of: ANSWERS TO PROGRESS-TEST TRUE/FALSE — Do not consult your references while answering the questions. 3. The patient who complains that nobody loves him 50 S VOL. 5 NO. 4 CURATIONIS VOL. 5 NO. 4 and that he feels alone in the world can be helped to overcome this feeling by assuring him that the doctor attending him is very experienced in his job. TRUE/FALSE and that he feels alone in the world can be helped to overcome this feeling by assuring him that the doctor attending him is very experienced in his job. TRUE/FALSE 5.6. All of the above. SOURCES 5. To plan for nursing care, the nurse must assess the needs of the patient. This can be done by: 1. Altschul, A. Psychology for nurses Bailliere Tindall. London 1975 4th Ed. 5.1. Asking the patient how he feels. 2. Fisher, E.E. Psychology for nurses and the health team Juta & Company Cape Town 1981 4th Ed. 5.2. Telling the patient that you will not help him unless he tells you the truth. 3. McNaught, A.B.; Callander, R. Nurses illustrated physiology E. & S. LIVINGSTONE London 1971 2nd Ed. 5.3. Observing the behaviour of the patient. 5.4. Examining the patient physically to determine his nutritional state. 4. Vlok, Manual o f Advanced Nursing Juta & company Johannes burg. DESEMBER 1982 51 CURATIONIS
https://openalex.org/W2944700777	https://europepmc.org/articles/pmc6566074?pdf=render	English	null	Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius	Frontiers in genetics	2,019	cc-by	17,418	Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius Maria Favia1, Robert Fitak2,3, Lorenzo Guerra1, Ciro Leonardo Pierri1, Bernard Faye4, Ahmad Oulmouden5, Pamela Anna Burger2 and Elena Ciani1 1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”, Bari, Italy, 2 Research Institute of Wildlife Ecology, Vetmeduni, Vienna, Austria, 3 Department of Biology, Duke University, Durham, NC, United States, 4 CIRAD, UMR SELMET, Montpellier, France, 5 Département Sciences du Vivant, Université de Limoges, Limoges, France Myostatin, a negative regulator of skeletal muscle mass in animals, has been shown to play a role in determining muscular hypertrophy in several livestock species, and a high degree of polymorphism has been previously reported for this gene in humans and cattle. In this study, we provide a characterization of the myostatin gene in the dromedary (Camelus dromedarius) at the genomic, transcript and protein level. The gene was found to share high structural and sequence similarity with other mammals, notably Old World camelids. 3D modeling highlighted several non-conservative SNP variants compared to the bovine, as well as putative functional variants involved in the stability of the myostatin dimer. NGS data for nine dromedaries from various countries revealed 66 novel SNPs, all of them falling either upstream or downstream the coding region. The analysis also conﬁrmed the presence of three previously described SNPs in intron 1, predicted here to alter both splicing and transcription factor binding sites (TFBS), thus possibly impacting myostatin processing and/or regulation. Several putative TFBS were identiﬁed in the myostatin upstream region, some of them belonging to the myogenic regulatory factor family. Patterns of SNP distribution across countries, as suggested by Bayesian clustering of the nine dromedaries using the 69 SNPs, pointed to weak geographic differentiation, in line with known recurrent gene ﬂow at ancient trading centers along caravan routes. Myostatin expression was investigated in a set of 8 skeletal muscles, both at transcript and protein level, via Digital Droplet PCR and Western Blotting, respectively. No signiﬁcant differences were observed at the transcript level, while, at the protein level, the only signiﬁcant differences concerned the promyostatin dimer (75 kDa), in four pair-wise comparisons, all involving the tensor fasciae latae muscle. Beside the mentioned band at 75 kDa, additional bands were observed at around 40 and 25 kDa, corresponding to the promyostatin monomer and the active C-terminal myostatin dimer, respectively. Their weaker intensity suggests that the unprocessed myostatin dimers could act as important reservoirs of slowly available myostatin forms. Keywords: Camelus dromedarius, myostatin, skeletal muscle, Single Nucleotide Polymorphisms, Next Generation Sequencing, Digital Droplet PCR, Western Blot, 3D protein comparative modeling Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius Under this assumption, the sequential cleavage steps may contribute additional layers of control within an already complex regulatory framework. Correspondence: Lorenzo Guerra lorenzo.guerra1@uniba.it Specialty section: This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Genetics Received: 30 January 2019 Accepted: 07 May 2019 Published: 07 June 2019 ORIGINAL RESEARCH published: 07 June 2019 doi: 10.3389/fgene.2019.00502 Edited by: Edited by: Edward Hollox, University of Leicester, United Kingdom Reviewed by: Kieran G. Meade, Teagasc, The Irish Agriculture and Food Development Authority, Ireland Reviewed by: Kieran G. Meade, Teagasc, The Irish Agriculture and Food Development Authority, Ireland René Massimiliano Marsano, University of Bari Aldo Moro, Italy René Massimiliano Marsano, University of Bari Aldo Moro, Italy *Correspondence: Lorenzo Guerra lorenzo.guerra1@uniba.it INTRODUCTION responsive type II receptors (ActIIRA or, preferentially, ActIIRB) and two type I receptors, either activin (ALK4) or TGF-β (ALK5) (Lee and McPherron, 2001; Rebbapragada et al., 2003). Signaling is hence initiated by phosphorylation of SMAD2 or SMAD3, operated by the type I receptors, followed by translocation of SMADs to the nucleus for modulation of gene expression (Huang et al., 2011). In particular, in skeletal muscle, myostatin is known to block the transcription of genes responsible for the myogenesis, among which MyoD, a transcriptional factor that is involved in skeletal muscle development and repair (Megeney et al., 1996; Cornelison et al., 2000; Guttridge et al., 2000; Montarras et al., 2000). Beside the above mentioned canonical pathway, two other pathways have been highlighted, involving MAPK activation or inhibition of Akt signaling (Elkina et al., 2011). Myostatin (alias growth and diﬀerentiation factor-8, GDF8), a member of the transforming growth factor-β (TGF-β) super- family, is a negative regulator of skeletal muscle mass in animals during development and growth. It is exclusively expressed in skeletal muscle during embryogenesis, while in adults is also detected, at a much lower level, in other tissues (e.g., heart, adipose tissue, mammary gland) (McPherron et al., 1997; Ji et al., 1998; Sharma et al., 1999; Morissette et al., 2006; Shyu et al., 2006; Allen et al., 2008). Expression in these tissues can be upregulated under pathological conditions, such as myocardial infarction (Sharma et al., 1999), obesity (Allen et al., 2008) or experimentally induced skeletal muscle atrophy (Rodriguez et al., 2014), while it can be down regulated during chronic exercise (Carlson et al., 1999; Reardon et al., 2001; Kim et al., 2005; Matsakas et al., 2006; Allen et al., 2009; Gustafsson et al., 2010). Myostatin genomic organization was ﬁrst provided for the murine species by McPherron et al. (1997) who also reported on the highly conserved nature of the myostatin transcript across several species. The myostatin gene (MSTN) comprises three exons and two introns. The nucleotide sequence coding for the active form of myostatin (109 a.a) is located in the 3′ terminal of the third exon (Gonzalez-Cadavid et al., 1998). Eﬀects of abolishing myostatin function were ﬁrst explored by McPherron et al. (1997) in mutant mice where the entire mature C-terminal region was deleted, showing a two- to three-fold increase in skeletal muscle mass in mutant compared to wild-type animals. INTRODUCTION Mutations at the myostatin gene, responsible for a signiﬁcantly increased skeletal muscle mass, were also shown to naturally occur in several livestock species, like cattle, sheep, pigs, dogs, horses, rabbit, poultry (for a review, see Aiello et al., 2018), and human (Schuelke et al., 2004). In particular, the high level of polymorphism previously described for the myostatin gene in humans (Ferrell et al., 1999) was conﬁrmed in cattle in a survey of 678 animals from 28 European breeds by using Single Strand Conformation Polymorphism (SSCP) analysis, followed by Sanger sequencing of the PCR re-ampliﬁed SSCP bands (Dunner et al., 2003). A total of 10 silent, 3 missense and 6 disruptive mutations were detected in the above study, giving origin to 20 distinct haplotypes whose sequence variation and breed distribution patterns supported the hypothesis that origin of muscular hypertrophy (also known in cattle as “double muscle” phenotype) was the result of both (i) European dispersal of the common variant nt821(del11) and (ii) arising and maintaining of various (mostly disruptive) mutations in single breeds. , ; , ) Like other TGF-β super-family members, myostatin is synthesized as a precursor protein (375 amino acids), referred to as pre-promyostatin. After translocation to the endoplasmic reticulum, it goes through a ﬁrst cleavage to remove a 24-amino acid signal peptide and it forms a disulﬁde-linked homodimer (promyostatin dimer). Within the Golgi, the promyostatin dimer may be further cleaved by the furin family of protein convertases to generate two NH2-terminal (27.7 kDa, each) and two disulﬁde- linked COOH-terminal fragments (12.4 kDa, each) (Lee and McPherron, 2001; Thies et al., 2001). The two NH2-terminal fragments (also referred to as pro-domains) may complex with the COOH-terminal dimer (also referred to as active myostatin) via a non-covalent bound that maintains myostatin in a latent state by rendering it unable to engage its receptors (Wolfman et al., 2003; Jiang et al., 2004). The “latent myostatin complex” (Lee and McPherron, 2001; Thies et al., 2001) may be secreted in the extracellular space, though it has been shown that, in skeletal myocytes, myostatin is mainly secreted as uncleaved promyostatin (Anderson et al., 2008; Pirruccello-Straub et al., 2018). In the extracellular space, the action of furin protein convertase and metalloproteinases (like BMP-1, TLL-1, and TLL- 2) may ﬁnally convert the uncleaved promyostatin and the latent complex, respectively, into the active form of myostatin (Lakshman et al., 2009). Citation: Favia M, Fitak R, Guerra L, Pierri CL, Faye B, Oulmouden A, Burger PA and Ciani E (2019) Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius. Front. Genet. 10:502. doi: 10.3389/fgene.2019.00502 June 2019 \| Volume 10 \| Article 502 1 Frontiers in Genetics \| www.frontiersin.org Dromedary Myostatin Characterization Favia et al. Frontiers in Genetics \| www.frontiersin.org • Reverse 5′CCTAAGTTTTCGAGCTAGGAGATC3′ • Reverse 5′CCTAAGTTTTCGAGCTAGGAGATC3′ Myostatin Transcription Initiation and Termination Sit Transcription initiation and termination sites were identiﬁed using the RACE (Rapid ampliﬁcation of cDNA ends) PCR approach implemented in the SMARTer RACE cDNA Ampliﬁcation Kit (CloneTech) following manufacturer’s instructions. Precursor Prediction The SignalP 4.11, the Combined Signal Peptide Predictor (CoSiDe)2 and the Signal-3L 2.03 online tools were interrogated for predicting the most probable location of the signal peptide cleavage site. • 2nd reaction: 5′GGCTGTGTAATGCATGTATGTGGAGA CAAA3′ For the 3′RACE-PCR, the following primers were used: • 1st reaction: 5′TGTGCACCAAGCAAACCCCAGAGGTT CGGC3′ • Reverse 5′TGTGCACCAAGCAAACCCCAGAGGTT CGGC3′ The PCR conditions were: initial step at 95◦C for 2 min; 35 cycles of a three-step thermal proﬁle of 95◦C for 30 s (denaturation), 61◦C for 30 s (annealing), 72◦C for 60 s (elongation); a ﬁnal elongation step at 72◦C for 5 min. For both reactions, 3 µl of cDNA were added to a solution of 12.5 µl Master Mix (Multiplex PCR Kit, QIAGEN), 1 µl of each primer (Forward and Reverse), 7.5 µl water. After separation on a 2% agarose gel, PCR products were excised from the gel, puriﬁed using the QIAquick Gel Extraction Kit (QIAGEN) and sequenced on both directions with internal primers, using the Sanger method. 1http://www.cbs.dtu.dk/services/SignalP/ 2http://sigpep.services.came.sbg.ac.at/coside.html 3http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/ Ampliﬁcation and Sequencing of the Myostatin cDNA A nested PCR was developed, with external forward and reverse primers falling in the 5′UTR and 3′UTR, respectively. The primer sequences were: • Forward 5′CCTTGGCATTACTCAAAAGCAA3′ • Forward 5′CAGTACGATGTCCAGAGAGATGACA GCAGT3′ 5′CAGTACGATGTCCAGAGAGATGACA • Reverse 5′TGTGCACCAAGCAAACCCCAGAGGTT CGGC3′ Sequences Alignment Sequences obtained via Sanger method (RACE PCR and cDNA amplicons, see above) were aligned using ClustalOmega (Sievers et al., 2011). For the 5′RACE-PCR, the following primers were used: r the 5′RACE-PCR, the following primers were used • 1st reaction: 5′ATCCTCAGTAAACTTCGCCTGGAAAC AGCT3′ INTRODUCTION Notwithstanding, in serum, myostatin has been shown to exist mainly as a latent complex (Hill et al., 2002; Zimmers et al., 2002; Lee, 2010). The active myostatin present in plasma circulates bound to several proteins (Miura et al., 2006; Cash et al., 2009; Walker et al., 2015), including follistatin, FSTL3, GASP1, GASP2 and decorin, that prevent it binding to the receptor and activating signaling (Hill et al., 2002, 2003; Lee, 2008; Cotton et al., 2018). Composite pools of myostatin are hence available at the various compartments, suggesting that extracellular processing of the protein may be a key regulatory step for its signaling (Anderson et al., 2008). The presence, at various extents, of the myostatin active form in the extracellular space and in the serum is consistent with the postulated autocrine, paracrine (Gao et al., 2013), and/or endocrine manner of function (Zimmers et al., 2002). y p g Old World camels include both wild (Camelus ferus) and domestic (Camelus dromedarius and Camelus bactrianus) species. Despite diﬀerences in muscularity can be observed among distinct populations and/or individuals, these are not dramatic as those observed in other livestock species and no evident “double muscle” phenotype has been described so far (B. Faye, personal communication). The myostatin gene has been previously characterized in various dromedary populations from Pakistan and India, although only 256 bp of exon 1 and 375 bp of exon 2, respectively, were considered in the analyses (Shah et al., 2006; Agrawal et al., 2017). A more comprehensive sequence polymorphism analysis of the myostatin gene was performed in our laboratories, where more than 3.6 kb of genomic sequence, Upon binding to the target cell, myostatin induces the formation of a heterotetrameric complex made of two activin June 2019 \| Volume 10 \| Article 502 June 2019 \| Volume 10 \| Article 502 2 Dromedary Myostatin Characterization Favia et al. RNA Retro-Transcription including the three exons, small part of the introns and part of the 3′ and 5′ ends, was sequenced in a total of 22 dromedaries from three diﬀerent Northern African geographic regions (Muzzachi et al., 2015). In this study, to further expand the knowledge base about myostatin, we followed up by (i) characterizing the gene structure (transcriptional initiation/termination sites; exon/intron boundaries), (ii) analyzing polymorphism of the complete genomic sequence and of the partial cDNA in a set of animals from various sampling sites, (iii) investigating expression patterns at both the transcript and the protein level in diﬀerent skeletal muscles. RNA Retro Transcription After quality control using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientiﬁc), 50 µl of total RNA was retro-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientiﬁc), which is based on a combination of oligo(dT) and random primers, following manufacturer’s instruction. Characterization of the Full-Length Myostatin cDNA The PCR conditions were: initial step at 95◦C for 2 min; 35 cycles of a three-step thermal proﬁle of 95◦C for 30 s (denaturation), 57◦C for 30 s (annealing), 72◦C for 60 s (elongation); a ﬁnal elongation step at 72◦C for 5 min. RNA Isolation From Skeletal Muscles Skeletal muscles were sampled at slaughterhouses from seven diﬀerent animals (Sudan, 3; Egypt, 2; Mauritania, 2). For each animal, a small sample of frozen muscle tissue (100 mg), previously stored in tubes containing RNAlater (QIAGEN), was ﬁnely chopped by using a sharp scalpel in 2 ml RLT buﬀer (RNeasy Midi Kit, QIAGEN) supplemented with β-mercaptoethanol following the manufacturer’s instructions. The sample was then homogenized using the T 10 basic Ultra- Turrax homogenizer (IKA). After homogenization, the sample was added with 4 ml of RNase-/DNase-free water plus 65 µl of Proteinase K (SIGMA, ≥0.6 Units/µl). After an incubation step at 55◦C for 20 min, samples were processed following manufacturer’s instruction. Internal primers were: • Forward 5′CAGTACGATGTCCAGAGAGATGACA GCAGT3′ Whole-Genome Variant Identiﬁcation Whole Genome Variant Identiﬁcation Another set of SNVs from the realigned and recalibrated alignment ﬁles was generated using the GATK HAPLOTYPECALLER and ﬁltering criteria as described above. SNVs on scaﬀolds putatively assigned to the X and Y chromosome, with a minimum allele count < 2, missing a genotype in more than ﬁve individuals, with 4 > DP > 30 per genotype, and deviating from Hardy–Weinberg equilibrium (p < 0.0001) in VCFTOOLS v0.1.12b (Danecek et al., 2011) were further excluded. This set of SNVs was used as a training set to perform variant quality score recalibration in GATK, assigning a probability of error to the training set of 0.1. This recalibration develops a Gaussian mixture model across the various annotations in the high-quality training dataset then applies the model to all variants in the initial dataset. The process has been shown to outperform the ‘hard’ ﬁltering of variants (e.g., Pirooznia et al., 2014). After variant recalibration, all SNVs with LOD score < −5.0 and 4 > DP > 30 per genotype were excluded. Polymorphism Analysis Sample Collection and Whole-Genome Sequencing Whole blood from 25 Old World camels was collected during routine veterinary procedures or as part of a monitoring program of the wild camel population in Mongolia. These samples included nine dromedaries (C. dromedarius), seven domestic Bactrian camels (C. bactrianus), and nine wild camels (C. ferus). Dromedary camels were selected to represent a variety of geographic locations: Pakistan (1), Kenya (1), Kingdom of Saudi Arabia (3), Sudan (1), United Arab Emirates (1), Qatar (1), Canary Islands – Spain (1). Domestic Bactrian camel originated from Mongolia or Kazakhstan, while all the wild camels originated from Mongolia. DNA was extracted using the Master PureTM DNA puriﬁcation kit for blood (Epicentre version III) and generated a 500 bp paired-end library for each sample. Each library was sequenced with a single lane of an Illumina HiSeq (Illumina, United States) according to standard protocols. Whole-Genome Read Processing and Alignments (5ntu.pdb), were thus aligned by using ClustalW (see Pierri et al., 2010, and references therein) for investigating chemical-physical properties of the amino acid regions showing variants between the human and the dromedary sequences. Accession numbers of the sequences considered in this study, together with additional details concerning the databases and the online tools used in this study, are summarized in Supplementary Table S1. (5ntu.pdb), were thus aligned by using ClustalW (see Pierri et al., 2010, and references therein) for investigating chemical-physical properties of the amino acid regions showing variants between the human and the dromedary sequences. Accession numbers of the sequences considered in this study, together with additional details concerning the databases and the online tools used in this study, are summarized in Supplementary Table S1. Then, SPDBV was used for generating a 3D model of the dromedary myostatin protein according to protocols described in Pierri et al. (2010). The obtained 3D comparative model was energetically minimized. A total of 100 steps of energy minimization were performed for relaxing the obtained 3D model by using the energy minimization tools implemented in Chimera. WhatIF and Chimera biochemical tools were used for checking the correct 3D model packing. PyMOL was used for manual inspection of the investigated 3D models and for generating ﬁgures (see Pierri et al., 2010, and references therein). Comparative Protein Modeling g Myostatin orthologs were searched through and sampled from Mammalia. The crystallized structure of the myostatin was available under the PDB_ID 5ntu (Cotton et al., 2018). The retrieved sequences, including the proposed crystallized structure • 2nd reaction: 5′CCTGCTGTACTCCCACAAAGATGTCT CCAA3′ After separation on a 2% agarose gel, PCR products were excised from the gel, puriﬁed using the QIAquick Gel Extraction Kit (QIAGEN) and sequenced using the following primers: 1http://www.cbs.dtu.dk/services/SignalP/ 2http://sigpep.services.came.sbg.ac.at/coside.html 3http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/ 5′RACE: 5′TTTGTCTCCACATACATGCATTACACAGCC3′ 3′RACE: 5′CCTGCTGTACTCCCACAAAGATGTCTCCAA3′ 5′RACE: 5′TTTGTCTCCACATACATGCATTACACAGCC3′ 5 RACE: 5 TTTGTCTCCACATACATGCATTACACAGCC3 3′RACE: 5′CCTGCTGTACTCCCACAAAGATGTCTCCAA3′ 3′RACE: 5′CCTGCTGTACTCCCACAAAGATGTCTCCAA3′ Frontiers in Genetics \| www.frontiersin.org June 2019 \| Volume 10 \| Article 502 3 Dromedary Myostatin Characterization Favia et al. Whole-Genome Read Processing and Alignments The 3′ end of sequence reads were trimmed to a minimum phred- scaled base quality score of 20 (probability of error < 1.0%) and trimmed reads < 50 bp in length were excluded using POPOOLATION v1.2.2 (Koﬂer et al., 2011). All processed reads were aligned to the C. ferus CB1 reference genome (Genbank accession: AGVR01040332.1) using BWA v0.6.2 (Li and Durbin, 2009) with parameters ‘−n 0.01 −o 1 −e 12 −d 12 −l 32.’ Duplicate reads were removed and alignments were ﬁltered to only include reads that were properly paired and unambiguously mapped with a mapping quality score > 20. Reads around insertions/deletions were realigned and a base quality score recalibration was performed using the Genome Analysis Toolkit (GATK) v3.1-1 following guidelines presented by Van der Auwera et al. (2013). As input into the base quality score recalibration step, a stringently ﬁltered set of single nucleotide variants (SNVs) was generated using the overlap of three diﬀerent variant-calling algorithms [SAMTOOLS v1.1] (Li et al., 2009); [GATK HAPLOTYPECALLER v3.1-1] (Van der Auwera et al., 2013); [ANGSD v0.563] (Korneliussen et al., 2014). The overlapping SNVs were ﬁltered to exclude those with a quality score (Q) < 20, depth of coverage (DP) > 750 (∼30X/individual), quality by depth (QD) < 2.0, strand bias (FS) > 60.0, mapping quality (MQ) < 40.0, inbreeding coeﬃcient < −0.8, mapping quality rank sum test (MQRankSum) < −12.5, and read position bias (ReadPosRankSum) < −8.0. Furthermore, SNVs were excluded if three or more were found within a 20 bp window, were within 10 bp of an insertion/deletion, or were found in an annotated repetitive region. Phylogenetic Analysis The analysis of the evolutionary relationships among orthologous myostatin sequences was conducted using MEGA5 (Tamura et al., 2011). Orthologous sequences of myostatin/growth diﬀerentiation factor 8 with E-value lower than 10ˆ-55, query coverage higher than 70% and % of identical amino acids ranging between 40 and 100% were aligned by using ClustalW implemented in Jalview. For Arthropoda, Aves, and Mammalia, due to the existence of more than one hundred of sequences complying with the above criteria, we imposed a ﬁlter on the ﬁrst 30 sequences for each taxonomic group. Redundant sequences with 100% identical amino acids were removed from the multiple sequence alignment. A ﬁnal set of 83 protein sequences (see Supplementary Data Sheet S1) were retained for tree building. In detail, the tree was built from the ungapped multiple sequence alignment applying the maximum likelihood method with the JTT model for the amino acid substitutions and a gamma distribution (ﬁve discrete gamma categories) for the rates among sites. A total of 100 bootstrap samplings were applied to test the robustness of the tree. In silico Functional Prediction The web-based analysis tool by the Human Splicing Finder Version 3.0.2 (Desmet et al., 2009), available at http:// www.umd.be/HSF3/index.html, was used to predict putative functional eﬀect of SNP variants in terms of potential alteration of splicing patterns. The in silico tool TFBIND (Tsunoda and Takagi, 1999), available at http://tfbind.hgc.jp/, was used to identify transcription factor binding sites (TFBS) and their possible disruption due to the presence of Single Nucleotide Polymorphisms. • Assay 1: Probe 1 5′-/56-FAM/CTACAGAGT/ZEN/CTGATCTTCT AATGC/3IABkFQ/-3′ Primer 1 5′-GACGGAAACAATCATTACC-3′ Primer 2 5′-GAGCTAAACTTAAAGAAGCAA-3′ • Assay 1: Probe 1 5′-/56-FAM/CTACAGAGT/ZEN/CTGATCTTCT AATGC/3IABkFQ/-3′ Primer 1 5′-GACGGAAACAATCATTACC-3′ Primer 2 5′-GAGCTAAACTTAAAGAAGCAA-3′ • Assay 1: Probe 1 5′-/56-FAM/CTACAGAGT/ZEN/CTGATCTTCT AATGC/3IABkFQ/-3′ Primer 1 5′-GACGGAAACAATCATTACC-3′ Primer 2 5′-GAGCTAAACTTAAAGAAGCAA-3′ • Assay 2: Probe 1 5′-/5HEX/AAGGGATTC/ZEN/AAACCATCTT CTC/3IABkFQ/-3′ ′ ′ Digital Droplet PCR Conditions g p A 20-µl reaction mixture was prepared comprising of 10 µl ddPCR SupermixTM for probes (no dUTP) (Bio-Rad), 1 µl primers and probe mix for Assay 1, 1 µl primers and probe mix for Assay 2, 2 µl cDNA, 6 µl RNase-/DNase-free water. The ﬁnal concentration of primers and probe was 900 and 250 nM, respectively. The ampliﬁcation conditions were 10 min DNA polymerase activation at 95◦C, followed by 40 cycles of a two-step thermal proﬁle of 30 s at 94◦C for denaturation, and 60 s at 60◦C for annealing and extension, followed by a ﬁnal hold of 10 min at 98◦C for droplet stabilization, and cooling to 4◦C. A thermal cycler (T100TM; Bio-Rad) was used, and the temperature ramp rate was set to 2◦C/s, with the lid heated to 105◦C, according to the Bio-Rad recommendations. A negative (no template) and a positive control were included. The latter consisted, for both assays, of a synthetic oligonucleotide (gBlocks Gene Fragment, by IDT), with a size of 467 bp, including junctions between exons 1 and 2 and between exons 2 and 3, designed based on the predicted sequence for the myostatin transcript in Camelus dromedarius (XM_010991955). In the reaction preparation, for the positive control, 2 µl of the above synthetic oligonucleotide were added, at a ﬁnal concentration of 1 ng/ml. For all the considered muscles, two biological and two technical replicates were included in the experiment. RNA Isolation and cDNA Synthesis Skeletal muscles were sampled at slaughterhouses (Kingdom of Saudi Arabia) from two adult animals. For each animal, eight muscles, representative of the diﬀerent anatomical regions of the body were taken: brachiocephalicus (head/neck), deltoid, extensor carpi radialis, and tensor fasciae latae (forelimbs), semitendinosus and coccygeus (trunk), biceps femoris, and peroneus longus (hindlimbs). For all muscles, sampling occurred within 30 min post-mortem. Immediately after collection, samples were stored in tubes containing RNAlater (QIAGEN). For RNA isolation and cDNA synthesis, the procedures described above were adopted. Data Analysis After the thermal cycling, the plates were transferred to a droplet reader (QX200TM; Bio-Rad). The software package provided with the ddPCR system was used for data acquisition (QuantaSoftTM 1.6.6.0320; Bio-Rad). The rejection criterium for the exclusion of a reaction from subsequent analysis was a low number of droplets measured (<10,000 per 20 µl PCR). The data from the ddPCR are given in target copies/µl reaction. The signiﬁcance of diﬀerences among muscles was tested using ANOVA (Analysis of Variance). Bayesian Clustering The identiﬁed SNPs were used for clustering the nine dromedary samples by adopting the Bayesian algorithm implemented in the STRUCTURE software v. 2.2 (Falush et al., 2007). The analysis was performed without providing a priori information on population membership, adopting the “admixture model” option and a burn-in period of 10,000 generations, followed by 100,000 iterations. Five independent runs were performed for each K value (number of clusters to be tested), and the results were visually inspected for reproducibility. K values ranging from 1 to 9 were tested, and the K value showing the highest probability was discussed. By partitioning the reaction volume into thousands of droplets, this technique allows absolute quantiﬁcation of nucleic acids without the need of a standard curve, with improved precision over classical quantitative PCR (qPCR). • Assay 1: Probe 1 5′-/56-FAM/CTACAGAGT/ZEN/CTGATCTTCT AATGC/3IABkFQ/-3′ Primer 1 5′-GACGGAAACAATCATTACC-3′ Primer 2 5′-GAGCTAAACTTAAAGAAGCAA-3′ • Assay 2: Probe 1 5′-/5HEX/AAGGGATTC/ZEN/AAACCATCTT CTC/3IABkFQ/-3′ Primer 1 5′-GGTCATGATCTTGCTGTA-3′ Primer 2 5′-GTCTGTTACCTTGACTTCTA-3′ • Assay 1: Probe 1 5′-/56-FAM/CTACAGAGT/ZEN/CTGATCTTCT AATGC/3IABkFQ/-3′ Primer 1 5′-GACGGAAACAATCATTACC-3′ Primer 2 5′-GAGCTAAACTTAAAGAAGCAA-3′ • Assay 2: Probe 1 5′-/5HEX/AAGGGATTC/ZEN/AAACCATCTT CTC/3IABkFQ/-3′ Primer 1 5′-GGTCATGATCTTGCTGTA-3′ Primer 2 5′-GTCTGTTACCTTGACTTCTA-3′ Digital Droplet PCR Assay Design The Digital Droplet PCR method is based on end-point ﬂuorescence signal detection, and the intensity of signal observed for positive droplets, varying with primer/template combination, is not considered for target quantiﬁcation. However, in this system, droplets are interpreted as either “positive” or “negative,” depending whether target ampliﬁcation occurred or not, based on a settled ﬂuorescence cut-oﬀ. Two diﬀerent assays, with probes targeting the two possible exon junctions in the myostatin gene (between exon 1 and 2, and between exon 2 and 3), were used. FAM- (6-carboxy-ﬂuorescein) and HEX- (hexa-chloro- ﬂuorescein) labeled probes were used, respectively, in Assay 1 and Assay 2. Probes and primers sequences were as follows: Identiﬁcation and Characterization of Variants at the Myostatin Locus The publicly available Camelus ferus myostatin sequence (GenBank Accession No AGVR01040332) was BLASTed against our Old World camel genomes and the contig-8645394 (Camelus dromedarius), contig-8938518 (Camelus bactrianus) and contig-7907533 (Camelus ferus) were retrieved and used in the comparative analysis of the myostatin locus at the June 2019 \| Volume 10 \| Article 502 Frontiers in Genetics \| www.frontiersin.org 4 Dromedary Myostatin Characterization Favia et al. nucleotide level. Moreover, from the ﬁnal set of SNVs described in the sub-section above, the Camelus dromedarius Single Nucleotide Polymorphisms (SNPs) falling in the contig- 8645394 (Supplementary Data Sheet S2) were selected for further inspection. Frontiers in Genetics \| www.frontiersin.org Myostatin Gene Organization Myostatin Gene Organization The RACE-PCR approach allowed to map the start transcription site (Supplementary Figure S1A) at 109 bp upstream the start-codon, in a position that is 24 and 25 bp downstream compared to the usual human and mouse transcription initiation sites, respectively4. The transcriptional termination site (Supplementary Figure S1B) was mapped 215 bp downstream the stop-codon, much earlier than in human and mouse where a 1561 and 1448 bp 3′UTR is usually reported4. No evidence was found, by combined RT-PCR and RACE approaches, for alternative splicing events or alternative 5′ or 3′ ends (Supplementary Figure S2). Based on the above, a 5292 bp genomic locus was identiﬁed for myostatin in C. dromedarius. The locus was highly conserved among the three Old World camelids species (Supplementary Figure S3). Comparative analysis of the C. dromedarius genomic sequence (contig- 8645394) with the obtained cDNA sequences conﬁrmed, as in other species, the presence of three exons and two introns, with a predicted C. dromedarius myostatin full length cDNA of 1452 bp (Supplementary Figure S4A) and a protein of 375 amino acids (Figure 1). The latter is consistent with the predicted protein 4http://genome-euro.ucsc.edu/index.html Comparative 3D Protein Modeling Comparative 3D Protein Modeling Sequences from nine Artiodactyla species, including the three phylogenetically close Old World camelids (C. dromedarius, C. bactrianus, and C. ferus), one New World camelid (Vicugna pacos), the two Bos taurus subspecies, i.e., B. taurus taurus (a non “double muscle” Hereford subject) and B. taurus indicus, the wild yak (Bos mutus), the buﬀalo (Bubalus bubalis) and the bison (Bison bison) were aligned with the human myostatin sequence (Accession no. ABI48419.1), and the human myostatin C-terminal domain solved by X-ray diﬀraction (residues 46-375 out of 375) (Figure 2A). C. dromedarius, C. ferus, C. bactrianus, and V. pacos share 100% of identical amino acids. Six, or fourteen, variants are observed among the above cited Camelidae sequences and the human myostatin sequence (Accession No. ABI48419.1), or the corresponding taurine myostatin sequence, respectively (Figure 2A). Out of them, 5 in the contrast with the human sequence and 13 in the contrast with the taurine sequence are variants occurring at diﬀerent sites, while one, at position 164, presented diﬀerent variants when contrasted with the human and the cattle sequence, respectively. In addition, it is possible to observe that the 6 variants detected in the contrast with the human sequence are conservative (Figure 2A), while 9, out of the 14 variants detected in the contrast with the cattle sequence are not conservative (Figure 2A). No variants were observed when contrasting among them the sequences from the ﬁve species belonging to the Bovidae family. A notable exception was B. bubalis, for which variants where observed at positions 101, 117 and 141. In all the above cases, the nucleotides observed in B. bubalis were diﬀerent from those observed in all the other eight sequences. Figure 2B presents the 3D comparative model of the C. dromedarius myostatin dimer, and highlights the variants observed between the Camelidae myostatin sequences and the human/bovine myostatin. 5https://www.uniprot.org/uniprot/ Protein Extraction and Western Blotting Protein Extraction and Western Blotting A small sample of frozen muscle tissue (100 mg), previously stored in tubes containing RNAlater (QIAGEN), was ﬁnely chopped and homogenized by using a sharp scalpel in 300 µl Ripa buﬀer [10 mM Tris-HCl pH 7.4, 140 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) Na-deoxycholate, 0.1% (v/v) SDS, 1 mM June 2019 \| Volume 10 \| Article 502 Frontiers in Genetics \| www.frontiersin.org 5 Dromedary Myostatin Characterization Favia et al. NaF, 1 mM EDTA, 1 mM Na3VO4] supplemented with 1x protease inhibitor cocktail (Sigma), and then by using the T 10 basic Ultra-Turrax homogenizer (IKA). After homogenization, the sample was kept on ice for 30 min, and then vortexed for 5 min. At the end, sample was centrifuged for 20 min at 4◦C at 13,000 × g to remove unbroken cells, nuclei and cell debris. The supernatant, containing solubilized proteins, was recovered and protein concentration was measured by the method of Bradford (Bradford, 1976). An aliquot of 20 µg of protein for each sample was diluted in Laemmli buﬀer not containing DTT or β-mercaptoethanol, heated at 95◦C for 5 min, and separated by 12 % (v/v) Tris/HCl SDS/PAGE. The separated proteins were transferred to Immobilon P (Millipore) in Trans-Blot semidry electrophoretic transfer cell (Amersham Biosciences) for immunoblotting. The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls. The densitometric quantiﬁcation and image processing of the considered bands were carried out using Adobe Photoshop and the Image software package (version 1.61, National Institutes of Health, Bethesda, MD, United States). The total lane density of transferred proteins on the membrane stained with Coomassie Blue dye was used for the normalization of the proteins of our interest. The signiﬁcance of diﬀerences among muscles, for each considered band, was tested using ANOVA (Analysis of Variance). Post hoc t-tests were performed to determine where the groups diﬀered. All p-levels for post hoc t-tests were adjusted using Bonferroni correction. NaF, 1 mM EDTA, 1 mM Na3VO4] supplemented with 1x protease inhibitor cocktail (Sigma), and then by using the T 10 basic Ultra-Turrax homogenizer (IKA). After homogenization, the sample was kept on ice for 30 min, and then vortexed for 5 min. Protein Extraction and Western Blotting At the end, sample was centrifuged for 20 min at 4◦C at 13,000 × g to remove unbroken cells, nuclei and cell debris. The supernatant, containing solubilized proteins, was recovered and protein concentration was measured by the method of Bradford (Bradford, 1976). An aliquot of 20 µg of protein for each sample was diluted in Laemmli buﬀer not containing DTT or β-mercaptoethanol, heated at 95◦C for 5 min, and separated by 12 % (v/v) Tris/HCl SDS/PAGE. The separated proteins were transferred to Immobilon P (Millipore) in Trans-Blot semidry electrophoretic transfer cell (Amersham Biosciences) for immunoblotting. The used primary antibody was a rabbit polyclonal anti-MSTN antibody against the C-terminal region (300–349 aa) of mouse myostatin (TA343358, OriGene; dilution 1:1000) that presented broad species reactivity, including artiodactyls. The densitometric quantiﬁcation and image processing of the considered bands were carried out using Adobe Photoshop and the Image software package (version 1.61, National Institutes of Health, Bethesda, MD, United States). The total lane density of transferred proteins on the membrane stained with Coomassie Blue dye was used for the normalization of the proteins of our interest. The signiﬁcance of diﬀerences among muscles, for each considered band, was tested using ANOVA (Analysis of Variance). Post hoc t-tests were performed to determine where the groups diﬀered. All p-levels for post hoc t-tests were adjusted using Bonferroni correction. size for most of the species5. The dromedary myostatin protein also showed all the hallmarks present in other TGF-ß family members, including an N-terminal signal sequence for secretion, a pro-region followed by the proteolytic processing RSRR site, and a C-terminal domain containing nine cysteine residues. In particular, the signal peptide was consistently predicted to have an 18 amino acid length by SignalP 4.1 and Signal-3L 2.0, while a length of 23 amino acids was predicted by CoSiDe. Evolutionary Relationships Among Myostatin Proteins The inferred maximum likelihood tree of myostatin protein sequences (Figure 3) highlighted the presence of three supported clusters (bootstrap value higher than 60%), corresponding to Arthropoda, Reptilia and Amphibia, that may reﬂect a diﬀerent attitude in the regulation of skeletal muscle growth in the diﬀerent taxonomic groups. Interestingly, within Mammalian sequences, the highest bootstrap value (99%) was observed for the cluster grouping myostatin sequences belonging to the Bovidae family. June 2019 \| Volume 10 \| Article 502 Frontiers in Genetics \| www.frontiersin.org 6 Dromedary Myostatin Characterization Favia et al. FIGURE 1 \| Amino acidic sequence of the C. dromedarius myostatin as inferred from the cDNA sequence. (A) Schematic outline. The three protein domains (signal peptide, pro-region, and active peptide) are highlighted in different colors (yellow, blue, and green, respectively). The two most likely residues involved in the signal peptide cleavage (see main text) are indicated by black arrows. Similarly, the residue (D, for aspartic acid) shown to be essential for BMP/tolloid protease cleavage, and the motif (RSRR) needed for recognition by furin convertase, are highlighted. (B) Amino acidic sequence of the C. dromedarius myostatin, with the three protein domains highlighted in different colors, as in (A). The above mentioned hallmarks are also depicted here (signal peptide cleavage, black arrows; BMP/tolloid protease cleavage residue and furin convertase recognition motif, bold). In addition, the nine conserved cysteine residues in the active peptide are indicated (bold and white). FIGURE 1 \| Amino acidic sequence of the C. dromedarius myostatin as inferred from the cDNA sequence. (A) Schematic outline. The three protein domains (signal peptide, pro-region, and active peptide) are highlighted in different colors (yellow, blue, and green, respectively). The two most likely residues involved in the signal peptide cleavage (see main text) are indicated by black arrows. Similarly, the residue (D, for aspartic acid) shown to be essential for BMP/tolloid protease cleavage, and the motif (RSRR) needed for recognition by furin convertase, are highlighted. (B) Amino acidic sequence of the C. dromedarius myostatin, with the three protein domains highlighted in different colors, as in (A). The above mentioned hallmarks are also depicted here (signal peptide cleavage, black arrows; BMP/tolloid protease cleavage residue and furin convertase recognition motif, bold). In addition, the nine conserved cysteine residues in the active peptide are indicated (bold and white). Polymorphism Analysis and in silico Functional Prediction myostatin sequences with sequences generated in this study, are presented, for completeness, in Supplementary Figures S4A,B. myostatin sequences with sequences generated in this study, are presented, for completeness, in Supplementary Figures S4A,B. Analysis by Human Splicing Finder highlighted, for the intronic polymorphisms, a potential role in alteration of splicing for G66148C, predicted to break an ESE (exonic splicing enhancer) site (Supplementary Figure S6A), A66460G, predicted to generate a new donor site and a new ESS (exonic splicing silencer) site (Supplementary Figure S6B), while no signiﬁcant splicing motif alteration was detected for T66461C (Supplementary Figure S6C). TFBS analysis, performed for each SNP using the two input sequences harboring the alternative alleles, highlighted the presence of disrupted TFBSs for all the three loci (Table 2). Moreover, we analyzed the potential transcriptional factor binding sites in the DNA sequence of 8 kb of the C. dromedarius myostatin gene upstream region (included in the contig-8645394). A total of 10677 putative binding sites were identiﬁed (Supplementary Data Sheet S3). A graphical outline of the most signiﬁcant predicted regulatory motifs in the 1.5 kb proximal to the transcription initiation site of the C. dromedarius myostatin gene is presented in Supplementary Figure S7. In addition, in this region two SNPs were present (T63437C and A64026G) (Table 1), out of which presented, for completeness, in Supplementary Figures S4A,B. Analysis by Human Splicing Finder highlighted, for the intronic polymorphisms, a potential role in alteration of splicing for G66148C, predicted to break an ESE (exonic splicing enhancer) site (Supplementary Figure S6A), A66460G, predicted to generate a new donor site and a new ESS (exonic splicing silencer) site (Supplementary Figure S6B), while no signiﬁcant splicing motif alteration was detected for T66461C (Supplementary Figure S6C). TFBS analysis, performed for each SNP using the two input sequences harboring the alternative alleles, highlighted the presence of disrupted TFBSs for all the three loci (Table 2). Moreover, we analyzed the potential transcriptional factor binding sites in the DNA sequence of 8 kb of the C. dromedarius myostatin gene upstream region (included in the contig-8645394). A total of 10677 putative binding sites were identiﬁed (Supplementary Data Sheet S3). A graphical outline of the most signiﬁcant predicted regulatory motifs in the 1.5 kb proximal to the transcription initiation site of the C. dromedarius myostatin gene is presented in Supplementary Figure S7. Frontiers in Genetics \| www.frontiersin.org Polymorphism Analysis and in silico Functional Prediction In addition, in this region two SNPs were present (T63437C and A64026G) (Table 1), out of which The results of the sequence polymorphism analysis for the myostatin locus are presented in Table 1. As can be observed, only three polymorphisms, two transitions (A66460G and T66461C) and one transversion (G66148C), were identiﬁed inside the myostatin gene, all located deep in the intron 1. On the other side, a total of 45 and 21 variant sites were identiﬁed in our study in the upstream and downstream regions, respectively, with an overall average density of one SNP every about 1.5 kb. In order to interpret the observed patterns of SNP distribution across samples from diﬀerent countries, we performed a Bayesian clustering analysis of the nine dromedary samples using the 69 identiﬁed SNPs. At K = 7 (Supplementary Figure S5), the analysis highlighted that the sample from Pakistan was well diﬀerentiated, and the same occurred for a pair of samples, one from Kingdom of Saudi Arabia and one from United Arab Emirates, respectively. The rest of the samples were clearly “admixed,” suggesting that most of the considered SNPs do not follow a geographic pattern. Putative variants in the myostatin coding region, inferred from aligning previously published June 2019 \| Volume 10 \| Article 502 7 Dromedary Myostatin Characterization Favia et al. Dromedary Myostatin Characterization Comparative analysis of myostatin protein sequences. (A) The alignment of myostatin orthologous sequences sampled from various mammalian species . Blue arrowheads indicate the variants detected between the four considered Camelidae sequences and the ﬁve Bovidae sequences at 13 speciﬁc arrowheads indicate the variants detected between the four considered Camelidae sequences and the Homo sapiens sequence at 5 speciﬁc sites, m the sites previously cited. The gray arrowhead indicates the position of two different variants detected in H. sapiens and in Bovidae in correspondence m C. dromedarius. Orange “boxes” indicate variations between the H. sapiens sequence retrieved from refseq_database and the sequence of the human myostatin. Amino acid codes and numbering refers to the C. dromedarius myostatin. (B) Lateral view of the 3D comparative model of the C. dromedarius mer. The protein is reported in green/magenta cartoon representation. Variants observed between Camelidae myostatin sequences and human/Bovidae re reported in black (5)/blue (13) spheres in chain A, and dark-yellow (5)/cyan (13) spheres in chain B, respectively. The only site of C. dromedarius howing a variation both in H. Polymorphism Analysis and in silico Functional Prediction sapiens and in Bovidae locates at site 164 (Q164 for C. dromedarius, E164 in H. sapiens, K164 in B. taurus) and is gray spheres. Notably, variants observed between C. dromedarius and Bovidae occur at different sites with respect to those detected between rius and H. sapiens, with the exclusion of residues at site 164. Residues C339/C340 of chain A and chain B, forming inter-monomer disulphide bridges, in red spheres. R65 of chain A and chain B, involved in interactions with T114, is indicated by red sticks. was also observed as being polymorphic by aligning -8645394 (Supplementary Data Sheet S2) with the and contig_13989_126 (Accession No. LSZX01094446.1). TFBS analysis, repeated for each SNP using the two input sequences \| Comparative analysis of myostatin protein sequences. (A) The alignment of myostatin orthologous sequences sampled from various mammalian species d. Blue arrowheads indicate the variants detected between the four considered Camelidae sequences and the ﬁve Bovidae sequences at 13 speciﬁc arrowheads indicate the variants detected between the four considered Camelidae sequences and the Homo sapiens sequence at 5 speciﬁc sites, m the sites previously cited. The gray arrowhead indicates the position of two different variants detected in H. sapiens and in Bovidae in correspondence m C. dromedarius. Orange “boxes” indicate variations between the H. sapiens sequence retrieved from refseq_database and the sequence of the human myostatin. Amino acid codes and numbering refers to the C. dromedarius myostatin. (B) Lateral view of the 3D comparative model of the C. dromedarius imer. The protein is reported in green/magenta cartoon representation. Variants observed between Camelidae myostatin sequences and human/Bovidae re reported in black (5)/blue (13) spheres in chain A, and dark-yellow (5)/cyan (13) spheres in chain B, respectively. The only site of C. dromedarius howing a variation both in H. sapiens and in Bovidae locates at site 164 (Q164 for C. dromedarius, E164 in H. sapiens, K164 in B. taurus) and is y gray spheres. Notably, variants observed between C. dromedarius and Bovidae occur at different sites with respect to those detected between arius and H. sapiens, with the exclusion of residues at site 164. Residues C339/C340 of chain A and chain B, forming inter-monomer disulphide bridges, FIGURE 2 \| Comparative analysis of myostatin protein sequences. (A) The alignment of myostatin orthologous sequences sampled from various mammalian species is presented. Polymorphism Analysis and in silico Functional Prediction Blue arrowheads indicate the variants detected between the four considered Camelidae sequences and the ﬁve Bovidae sequences at 13 speciﬁc sites. Black arrowheads indicate the variants detected between the four considered Camelidae sequences and the Homo sapiens sequence at 5 speciﬁc sites, different from the sites previously cited. The gray arrowhead indicates the position of two different variants detected in H. sapiens and in Bovidae in correspondence of Q164 from C. dromedarius. Orange “boxes” indicate variations between the H. sapiens sequence retrieved from refseq_database and the sequence of the human crystallized myostatin. Amino acid codes and numbering refers to the C. dromedarius myostatin. (B) Lateral view of the 3D comparative model of the C. dromedarius myostatin dimer. The protein is reported in green/magenta cartoon representation. Variants observed between Camelidae myostatin sequences and human/Bovidae myostatin are reported in black (5)/blue (13) spheres in chain A, and dark-yellow (5)/cyan (13) spheres in chain B, respectively. The only site of C. dromedarius myostatin showing a variation both in H. sapiens and in Bovidae locates at site 164 (Q164 for C. dromedarius, E164 in H. sapiens, K164 in B. taurus) and is indicated by gray spheres. Notably, variants observed between C. dromedarius and Bovidae occur at different sites with respect to those detected between C. dromedarius and H. sapiens, with the exclusion of residues at site 164. Residues C339/C340 of chain A and chain B, forming inter-monomer disulphide bridges, are reported in red spheres. R65 of chain A and chain B, involved in interactions with T114, is indicated by red sticks. FIGURE 2 \| Comparative analysis of myostatin protein sequences. (A) The alignment of myostatin orthologous sequences sampled from various mammalian species is presented. Blue arrowheads indicate the variants detected between the four considered Camelidae sequences and the ﬁve Bovidae sequences at 13 speciﬁc sites. Black arrowheads indicate the variants detected between the four considered Camelidae sequences and the Homo sapiens sequence at 5 speciﬁc sites, different from the sites previously cited. The gray arrowhead indicates the position of two different variants detected in H. sapiens and in Bovidae in correspondence of Q164 from C. dromedarius. Orange “boxes” indicate variations between the H. sapiens sequence retrieved from refseq_database and the sequence of the human crystallized myostatin. Amino acid codes and numbering refers to the C. dromedarius myostatin. (B) Lateral view of the 3D comparative model of the C. dromedarius myostatin dimer. Quantitative Myostatin Transcript Analysis in Dromedary Skeletal Muscles We investigated the quantitative expression of myostatin transcripts in eight dromedary skeletal muscles (deltoid, extensor carpi radialis, coccygeus, biceps femoris, peroneus longus, semitendinosus, tensor fasciae latae, brachiocephalicus) by Digital Droplet PCR. Two diﬀerent assays, with probes targeting the two possible exon junctions in the myostatin gene, were used. Number of droplets generated in each experiment replicate, together with plot of raw data, are shown in Supplementary Figure S8. All the replicates passed the cut-oﬀ value (>10,000 droplets). Figure 4 presents the results of the Polymorphism Analysis and in silico Functional Prediction The protein is reported in green/magenta cartoon representation. Variants observed between Camelidae myostatin sequences and human/Bovidae myostatin are reported in black (5)/blue (13) spheres in chain A, and dark-yellow (5)/cyan (13) spheres in chain B, respectively. The only site of C. dromedarius myostatin showing a variation both in H. sapiens and in Bovidae locates at site 164 (Q164 for C. dromedarius, E164 in H. sapiens, K164 in B. taurus) and is indicated by gray spheres. Notably, variants observed between C. dromedarius and Bovidae occur at different sites with respect to those detected between C. dromedarius and H. sapiens, with the exclusion of residues at site 164. Residues C339/C340 of chain A and chain B, forming inter-monomer disulphide bridges, are reported in red spheres. R65 of chain A and chain B, involved in interactions with T114, is indicated by red sticks. the latter was also observed as being polymorphic by aligning the contig-8645394 (Supplementary Data Sheet S2) with the publicly available contig4726 (Accession No. JDVD01004726.1) and contig_13989_126 (Accession No. LSZX01094446.1). TFBS analysis, repeated for each SNP using the two input sequences harboring the alternative alleles, suggested the disruption of June 2019 \| Volume 10 \| Article 502 Frontiers in Genetics \| www.frontiersin.org 8 Dromedary Myostatin Characterization Favia et al. FIGURE 3 \| Phylogenetic analysis of myostatin protein sequences. Numbers indicate bootstrap values higher than 60/100. one (NFKB_Q6) and three (COUP_01, MYB_Q6, and T3R_01) transcription factors binding sites for loci T63437C and A64026G, respectively (data not shown). absolute quantiﬁcation experiments for the eight muscles, and for the two probes, expressed as target copies/µl. The FAM- labeled probe (probe 1, targeting the junction between exon 1 and 2) produced systematically higher values compared to the HEX-labeled probe (probe 2, targeting the junction between exon 2 and 3). However, trends among diﬀerent muscles were similar for both probes, as also supported by a correlation coeﬃcient higher than 0.78 (data not shown). Analysis of variance among means of diﬀerent muscles did not ﬁnd signiﬁcant diﬀerence for any of the two assays. Myostatin Protein Expression in Dromedary Skeletal Muscles The # (hash) symbol indicates the only SNP, located in the myostatin gene (intron 2), that was not observed in our study but was evident when aligning the contig-8645394 with both contig4726 and contig_13989_126. Variant sites are presented based on the region where they are located (either in the exons or introns of the myostatin locus, in the 5′ or in the 3′ UTR, or in the upstream/downstream regions). Site positions refer to the C. dromedarius contig-8645394. Circles (◦) indicates those SNPs that could also be observed when aligning the contig-8645394 with the publicly available contig4726 (Accession No. JDVD01004726.1). Asterisks (∗) indicates those SNPs that could also be observed when aligning the contig-8645394 with the publicly available contig_13989_126 (Accession No. LSZX01094446.1). The # (hash) symbol indicates the only SNP, located in the myostatin gene (intron 2), that was not observed in our study but was evident when aligning the contig-8645394 with both contig4726 and contig_13989_126. the active C-terminal myostatin dimer, we performed Western Blot analyses using a polyclonal antibody raised against the C-terminus. Moreover, protein electrophoretic separation was run under non-reducing conditions in order to preserve the integrity of the disulphide bonds in the C-terminal domain. As shown in Figure 5A, a major band is present at an apparent molecular mass of 75 kDa, corresponding to the expected mass for the promyostatin dimer. Additional, weaker bands were observed at around 40 and 25 kDa, corresponding to the promyostatin monomer and the active C-terminal myostatin dimer, respectively. Densitometric analysis of Western Blots, performed on ﬁve diﬀerent protein extracts for each muscle, highlighted signiﬁcant diﬀerences (ANOVA, p = 0.0001) among muscle types for the promyostatin dimer (Figure 5B), while no signiﬁcant diﬀerence was observed for both the promyostatin monomer and the active C-terminal myostatin dimer, respectively (Figure 5B). Post hoc tests highlighted four signiﬁcant (p < 0.0017) pair-wise comparisons, all of them involving the tensor fasciae latae muscle (vs. deltoid, extensor carpi radialis, coccygeus, and brachiocephalicus). in other species for the myostatin gene. As expected, a high sequence similarity was observed among our experimentally obtained transcript sequence for C. dromedarius myostatin and the publicly available predicted sequences for the other two species within the Camelus genus, in line with the relatively recent divergence times between them, estimated in 5– 8 mya between one-humped and two-humped domestic camels (Wu et al., 2014), and about 0.7 million years ago between C. bactrianus and C. Myostatin Protein Expression in Dromedary Skeletal Muscles We investigated the expression of myostatin at the protein level on the same set of dromedary skeletal muscles previously described for quantitative transcript analysis. In order to detect June 2019 \| Volume 10 \| Article 502 Frontiers in Genetics \| www.frontiersin.org 9 Dromedary Myostatin Characterization Favia et al. TABLE 1 \| Single Nucleotide Polymorphisms identiﬁed in the considered population sample (nine C. dromedarius animals from seven countries). Target region Size (bp) Polymorphism Upstream 64736 G13066T; T13901A◦; C14568A◦; T14745G; C16352T; G16640A◦; G17867A; T25076C◦; T29288A; G31182A; G31464A; C32869T◦∗; C33308G; G33597C; G34295T; T34901C◦∗; G35625A◦∗; C35782T◦; G38778A; T40104C◦∗; T41149C∗; T42365A; C43439A◦; C47473T; C48225T; G48931T◦∗; C49083T∗; G49630C◦∗; T50414C; T50514G∗; A50637C◦; T50897C∗; T52618C◦; G52669T; T53222C◦∗; G53398A◦∗; C55949T◦∗; A56391G; G57208A◦∗; T58686C◦∗; G59091T◦∗; A59187T∗; A60587G; T63437C; A64026G◦∗ 5′ UTR 109 None Exon 1 373 None Intron 1 1801 G66148C◦; A66460G∗; T66461C∗ Exon 2 374 None Intron 2 2039 None# Exon 3 381 None 3′ UTR 215 None Downstream 21956 A72141T; A72526G; A73136C; G75845A◦∗; T75862C; G76270A◦∗; T77354C◦∗; T78572A; G78774A◦; G78993A∗; T80531G; C81364T◦; C81499T◦; G83533C; T86423C◦; T87060A◦; G89595A◦; A89886T; C90627A◦; A90686T; T90834C◦ Variant sites are presented based on the region where they are located (either in the exons or introns of the myostatin locus, in the 5′ or in the 3′ UTR, or in th upstream/downstream regions). Site positions refer to the C. dromedarius contig-8645394. Circles (◦) indicates those SNPs that could also be observed when alignin the contig-8645394 with the publicly available contig4726 (Accession No. JDVD01004726.1). Asterisks (∗) indicates those SNPs that could also be observed whe aligning the contig-8645394 with the publicly available contig_13989_126 (Accession No. LSZX01094446.1). The # (hash) symbol indicates the only SNP, located in th myostatin gene (intron 2), that was not observed in our study but was evident when aligning the contig-8645394 with both contig4726 and contig_13989_126. Nucleotide Polymorphisms identiﬁed in the considered population sample (nine C. dromedarius animals from seven countries). Variant sites are presented based on the region where they are located (either in the exons or introns of the myostatin locus, in the 5′ or in the 3′ UTR, or in the upstream/downstream regions). Site positions refer to the C. dromedarius contig-8645394. Circles (◦) indicates those SNPs that could also be observed when aligning the contig-8645394 with the publicly available contig4726 (Accession No. JDVD01004726.1). Asterisks (∗) indicates those SNPs that could also be observed when aligning the contig-8645394 with the publicly available contig_13989_126 (Accession No. LSZX01094446.1). Myostatin Protein Expression in Dromedary Skeletal Muscles ferus (Ji et al., 2009). Also, myostatin orthologs showed a high percentage of identical amino acids through Mammalia, which may explain the low bootstrap values observed in the maximum likelihood tree. A notable exception was represented by the Bovidae sequences, that clustered with high bootstrap values. Peculiar selection constraints may have played a role in shaping the evolutionary history of myostatin in Bovidae. Indeed, besides the well documented human- mediated positive selection experienced in recent times by the Bos taurus myostatin gene, particularly in specialized beef breeds, evidence for a more remote action of positive selection on this gene, operating during the time of divergence of Bovinae and Antilopinae, has been produced by Tellgren et al. (2004) in a systematic analysis of myostatin sequence evolution in ruminants. These periods of positive selective pressure on myostatin may correlate with changes in skeletal muscle mass. In fact, the early bovid fossil record, dating back to around 17 million years ago, had a body mass estimate of only around 20 kg. Hence, the hypothesis is that selective pressures on myostatin drove this increase in body mass coupled to an increase in skeletal muscle mass, in turn driven by ecological changes in the environments of the various species. In a recent paper, the phylogenetic relationships between camelids and other mammalian species were investigated using whole-genome sequence data (Wu et al., 2014). The authors report estimated June 2019 \| Volume 10 \| Article 502 Myostatin Gene Organization and Protein Comparative Modeling dromedarius, Ala in B. taurus), 243 (Gly in C. dromedarius; Glu in B. taurus), 356 (Lys in C. dromedarius; Glu in B. taurus), 357 (Glu in C. dromedarius, Gly in B. taurus) that may produce a diﬀerent charge network in myostatin dimer, favoring diﬀerent monomer/monomer interactions. In particular, among the described variants, it is worth noting that the residue at site 114 forms intra-chain binding interactions with Y111 and H112 and inter-chain binding interactions with R65. R65, Y111 and H112 together with K153 (R153 in H. sapiens) were already described as “fastener” residues associated with muscle- and obesity-related divergence time between camelids and cattle lower than those between other mammals. This seeming discrepancy with our results may arise from the fact that single gene phylogenies could not reﬂect the complex evolutionary history of a whole genome and they could be less reliable in inferring genome- scale events. Myostatin protein consists of a non-covalently held complex of the N-terminal propeptide and a disulﬁde-linked dimer of C-terminal fragments (Lee and McPherron, 2001). Variants detected respectively in B. taurus and H. sapiens, in correspondence of the C. dromedarius K49 and L53 may be involved in the stability of the myostatin dimer due to their location close to the disulphide bonds occurring among C339 and C340 residues at the myostatin dimer interface (Jiang et al., 2004). Notably, the myostatin N-terminal domain contains a region (residues 49–67) highly similar to the key latency-determining regions of the TGF-β superfamily (Walton et al., 2010). Thus, it is expected that variations observed in our comparative analysis at this region, above all in correspondence of the C. dromedarius K49 (Thr in B. taurus myostatin, Lys TABLE 2 \| Results of the TFBS analysis for the three intronic SNPs detected in this study. SNP AC ID Score Strand Consensus Signal A66460G Allele A M00103 V$CLOX_01 0.778624 (+) NNTATCGATTANYNW GGTATTAATTAGCTG M00104 V$CDPCR1_01 0.790948 (−) NATCGATCGS GGTATTAATT M00134 V$HNF4_01 0.769283 (−) NNNRGGNCAAAGKTCANNN ATTTAAATTTTGGTATTAA Allele G M00211 V$PADS_C 0.825857 (+) NGTGGTCTC TTTGGTGTT M00212 V$POLY_C 0.787056 (+) CAATAAAACCYYYYKCTN CATTTAAATTTTGGTGTT M00279 V$MIF1_01 0.741931 (−) NNGTTGCWWGGYAACNGS GGTGTTAATTAGCTGCTA M00280 V$RFX1_01 0.776836 (−) NNGTNRCNWRGYAACNN GTGTTAATTAGCTGCTA G66148C Allele G M00143 V$PAX5_01 0.787091 (+) NCNNNRNKCANNGNWGNRKRGCSRSNNN GAGACAGGCACCTTAACAGAGAAGGCAT Allele C M00262 V$STAF_01 0.767919 (+) NTTWCCCANMATGCAYYRCGNY TTAACACAGAAGGCATGACAAG T66461C Allele T M00103 V$CLOX_01 0.778624 (+) NNTATCGATTANYNW GGTATTAATTAGCTG M00104 V$CDPCR1_01 0.790948 (−) NATCGATCGS GGTATTAATT M00252 V$TATA_01 0.829231 (+) STATAAAWRNNNNNN GTATTAATTAGCTGC Allele C M00185 V$NFY_Q6 0.779548 (−) TRRCCAATSRN CTAATTAGCTG AC, transcription factor matrix code. Myostatin Gene Organization and Protein Comparative Modeling Myostatin Gene Organization and Protein Comparative Modeling The myostatin gene has been largely studied in several livestock and model species. Very recently, the gene has been mapped to chromosome 5 in Camelus dromedarius (Elbers et al., 2019). By combining experimental and in silico approaches, we here describe, for the ﬁrst time, the gene organization and the protein structure in the one-humped Old World camelid species. We conﬁrmed the major features observed June 2019 \| Volume 10 \| Article 502 Frontiers in Genetics \| www.frontiersin.org 10 Dromedary Myostatin Characterization Favia et al. in TGF-β1) and L53 (Ile in H. sapiens myostatin, Leu in TGF- β1) may contribute toward its latency, according to Walton et al. (2010). More in general, all the cited variants locate at the monomer/monomer interface. Notably, we observed, in Camelidae myostatin sequences, not conservative substitutions compared to the B. taurus taurus myostatin sequence at position 49 (Lys in C. dromedarius; Thr in B. taurus), 88 (Arg in C. dromedarius; Leu in B. taurus), 100 (Asp in C. dromedarius; Ala in B. taurus),114 (Thr in C. dromedarius; Arg in B. taurus), 129 (Met in C. dromedarius, Thr in B. taurus), 167 (Thr in C. dromedarius, Ala in B. taurus), 243 (Gly in C. dromedarius; Glu in B. taurus), 356 (Lys in C. dromedarius; Glu in B. taurus), 357 (Glu in C. dromedarius, Gly in B. taurus) that may produce a diﬀerent charge network in myostatin dimer, favoring diﬀerent monomer/monomer interactions. In particular, among the described variants, it is worth noting that the residue at site 114 forms intra-chain binding interactions with Y111 and H112 and inter-chain binding interactions with R65. R65, Y111 and H112 together with K153 (R153 in H. sapiens) were already described as “fastener” residues associated with muscle- and obesity-related in TGF-β1) and L53 (Ile in H. sapiens myostatin, Leu in TGF- β1) may contribute toward its latency, according to Walton et al. (2010). More in general, all the cited variants locate at the monomer/monomer interface. Notably, we observed, in Camelidae myostatin sequences, not conservative substitutions compared to the B. taurus taurus myostatin sequence at position 49 (Lys in C. dromedarius; Thr in B. taurus), 88 (Arg in C. dromedarius; Leu in B. taurus), 100 (Asp in C. dromedarius; Ala in B. taurus),114 (Thr in C. dromedarius; Arg in B. taurus), 129 (Met in C. dromedarius, Thr in B. taurus), 167 (Thr in C. Frontiers in Genetics \| www.frontiersin.org June 2019 \| Volume 10 \| Article 502 Myostatin Gene Organization and Protein Comparative Modeling TFBS analysis in the myostatin upstream region allowed to predict several potential transcriptional factor binding sites, mainly belonging to the large family of dimerizing transcription factors harboring a basic helix-loop-helix (bHLH) structural motif, such as MYOD (myogenic diﬀerentiation), MYOG (myogenin), MYC (myelocytomatosis viral oncogene), MAX (MYC Associated Factor X), TAL1 (T-Cell Acute Lymphocytic Leukemia), SREBP (Sterol Regulatory Element-Binding Protein), AHR (Aryl Hydrocarbon Receptor), ARNT (Aryl hydrocarbon receptor nuclear translocator), HEN (Nescient Helix-Loop- Helix 1), HLF (Hepatic Leukemia Factor), USF (Upstream Transcription Factor) (Jones, 2004; Sailsbery and Dean, 2012). Out of them, MYOD and MYOG belong to the myogenic regulatory factor (MRF) family known to play key roles in the determination and diﬀerentiation of skeletal muscle (Botzenhart et al., 2018). Besides them, a role in regulating myostatin expression has been largely demonstrated for CREB (Xie et al., 2018), MEF (Bo Li et al., 2012; Estrella et al., 2015) and C/EBP (Allen et al., 2010; Deng et al., 2012), for which potential binding sites were also detected in our in silico analysis of the dromedary myostatin gene upstream sequence. Moreover, in the about 400 bp region upstream to the transcriptional start site, three TATA boxes and one CCAAT box were observed, consistently with previous reports Myostatin Gene Organization and Protein Comparative Modeling ID, transcription factor label, with V meaning “vertebrate.” SCORE, similarity (0.0–1.0) between a registered sequence for the transcription factor binding sites and the input sequence. STRAND, strandness. + and −means forward and reverse strands that the transcription factor binds, respectively. CONSENSUS, consensus sequence (ﬁxed) of the transcription factor binding sites. S = C or G, W = A or T, R = A or G, Y = C or T, K = G or T, M = A or C, N = any base pair. SIGNAL, sub-sequence from the input sequence at the position corresponding to the consensus sequence. Default cut-offs by Tsunoda and Takagi, 1999 were adopted. The analysis was performed, for each SNP, using the two input sequences harboring the alternative alleles. June 2019 \| Volume 10 \| Article 502 11 Dromedary Myostatin Characterization Favia et al. FIGURE 4 \| Absolute quantiﬁcation of the myostatin transcript in skeletal muscles. Results of the Digital Droplet PCR analysis for the eight considered dromedary muscles (1, deltoid; 2, extensor carpi radialis; 3, coccygeus; 4, biceps femoris; 5, peroneus longus; 6, semitendinosus; 7, tensor fasciae latae; 8, braciocephalicus) and for the two used probes (u, Probe 1; N, 2 Probe 2) are presented as mean ± SD of the four replicates. FIGURE 4 \| Absolute quantiﬁcation of the myostatin transcript in skeletal muscles. Results of the Digital Droplet PCR analysis for the eight considered dromedary muscles (1, deltoid; 2, extensor carpi radialis; 3, coccygeus; 4, biceps femoris; 5, peroneus longus; 6, semitendinosus; 7, tensor fasciae latae; 8, braciocephalicus) and for the two used probes (u, Probe 1; N, 2 Probe 2) are presented as mean ± SD of the four replicates. phenotypes (Gonzalez-Freire et al., 2010; Santiago et al., 2011; Bhatt et al., 2012; Garatachea et al., 2013; Szlama et al., 2015). Furthermore, variations of the residue at site 100 may inﬂuence the release of the active form from the myostatin propeptide complex due to its location close to the myostatin propeptide TLD cleavage target site (consisting of the dipeptide 98-RD-99) (Szlama et al., 2013). deep intronic regions (Mendes de Almeida et al., 2017; Vaz- Drago et al., 2017). The three intronic SNPs detected in this study were in silico predicted to have the potential of altering both splicing and TFBS, thus suggesting they may play a role in myostatin processing and/or regulation. Frontiers in Genetics \| www.frontiersin.org Genetic Sequence Polymorphism and Functional Prediction Unexpectedly, the Next Generation Sequencing (NGS) of the whole myostatin locus in nine dromedaries from a variety of geographic locations in Asia, Africa and Europe did not allow the identiﬁcation of novel intra-genic variants and only conﬁrmed the presence of the three SNPs in intron 1 previously identiﬁed by Muzzachi et al. (2015) via Sanger-sequencing of a reduced portion of the gene on a diﬀerent set of Northern African dromedaries. This result seems to support the hypothesis, formulated by Muzzachi et al. (2015) that the low diversity observed at the myostatin locus in Camelus dromedarius may reﬂect the peculiar evolutionary history of this species, which likely developed as domesticates from a low variable wild ancestor population. Evidence about the existence of functional variants located in introns is growingly accumulating (Lee et al., 2015; Mou et al., 2015; Hong et al., 2018; Ostrovsky et al., 2018), not only restricted to exon–intron boundaries but also in June 2019 \| Volume 10 \| Article 502 12 Dromedary Myostatin Characterization Favia et al. FIGURE 5 \| Myostatin protein expression. (A) Representative Western Blot of the eight considered dromedary muscles (1, deltoid; 2, extensor carpi radialis; 3, coccygeus; 4, biceps femoris; 5, peroneus longus; 6, semitendinosus; 7, tensor fasciae latae; 8, brachiocephalicus) performed using a rabbit polyclonal antibody (1:1000 dilution) that speciﬁcally binds the carboxy-terminal domain (Origene, TA343358). (B) Western Blot densitometric analysis of the promyostatin dimer (75 kDa), the promyostatin monomer (40 kDa) and the active C-terminal dimer (25 kDa), respectively. For each lane, optical density (OD) of the considered band is presented as a ratio over the total density of all the proteins transferred on the membrane and stained with Coomassie blue. FIGURE 5 \| Myostatin protein expression. (A) Representative Western Blot of the eight considered dromedary muscles (1, deltoid; 2, extensor carpi radialis; 3, coccygeus; 4, biceps femoris; 5, peroneus longus; 6, semitendinosus; 7, tensor fasciae latae; 8, brachiocephalicus) performed using a rabbit polyclonal antibody FIGURE 5 \| Myostatin protein expression. (A) Representative Western Blot of the eight considered dromedary muscles (1, deltoid; 2, extensor carpi radialis; 3, coccygeus; 4, biceps femoris; 5, peroneus longus; 6, semitendinosus; 7, tensor fasciae latae; 8, brachiocephalicus) performed using a rabbit polyclonal antibody (1:1000 dilution) that speciﬁcally binds the carboxy-terminal domain (Origene, TA343358). Genetic Sequence Polymorphism and Functional Prediction (B) Western Blot densitometric analysis of the promyostatin dimer (75 kDa), the promyostatin monomer (40 kDa) and the active C-terminal dimer (25 kDa), respectively. For each lane, optical density (OD) of the considered band is presented as a ratio over the total density of all the proteins transferred on the membrane and stained with Coomassie blue. by Spiller et al. (2002) in the bovine species and Du et al. (2011) in the ovine species. On the contrary, in the above region, we detected four E-boxes, unlike (Spiller et al., 2002) and (Du et al., 2005), who detected three and ﬁve E-boxes, respectively. considered as providing a good representation of the species genetic diversity, and (ii) a limited diﬀerentiation may exist also among geographically distant samples, as pointed out by the preliminary results of the ﬁrst world-wide Camelus dromedarius genetic diversity survey performed using genome-wide RAD- sequencing (Ciani et al., 2017)6, and in line with the known recurrent gene ﬂow at ancient trading centers along the caravan routes (Almathen et al., 2016). Most of the variants detected in our population sample, representative of seven diﬀerent countries across the African and the Euro/Asiatic continent, could be also detected by aligning our Camelus dromedarius contig-8645394 with contigs available in public databases, representative of animals of African and Asiatic descent (Accession No. LSZX01094446.1, from a Targui animal sampled in Algeria, and Accession No. JDVD01004726, from an animal sampled in the Kingdom of Saudi Arabia, respectively). This result suggests that (i) our population sample, despite being limited in size, could be 6https://pag.confex.com/pag/xxv/meetingapp.cgi/Paper/23709 Frontiers in Genetics \| www.frontiersin.org AUTHOR CONTRIBUTIONS MF performed the experiments, analyzed the data, prepared the ﬁgures and contributed toward writing the manuscript. RF performed the experiments and analyzed the data. LG and CP performed the experiments, analyzed the data, and contributed to the discussion and toward writing the manuscript. BF helped in sample collection and contributed to the discussion. AO performed the RACE-PCR experiments and contributed to the discussion. PB performed the experiments, analyzed the data, and contributed to the discussion. EC designed the research, performed the experiments, analyzed the data, and wrote the manuscript. All authors have read and approved the ﬁnal manuscript. ETHICS STATEMENT In our experimental conditions, no signiﬁcant diﬀerences were observed for the eight considered muscles by any of the two assays. These results are in line with those previously published by Morrison et al. (2014) who did not ﬁnd signiﬁcant variation in myostatin expression levels, assessed via Quantitative Real-time PCR, among four skeletal muscles (rectus abdominis, longus colli, adductor, pectoralis transversus) in the horse species. A similar scenario was observed in this study also at the protein level where no signiﬁcant diﬀerence was observed among muscle types for the three myostatin forms (25, 40, and 75 kDa), with the exception of four pair- wise comparisons, all involving the tensor fasciae latae muscle, where a signiﬁcantly lower expression was observed only for the promyostatin dimer. For the other two myostatin forms, tensor fasciae latae displayed weaker bands although they did not reach signiﬁcance when contrasted to other muscles. The generally low expression of the three myostatin forms in the tensor fasciae latae muscle tempted us to speculate about a possible relationship with the ﬁber type composition of this muscle, which has been reported, in various species, to be mainly of the fast glycolytic type (Ariano et al., 1973; Abe et al., 1987; Manabe et al., 2000; Sazili et al., 2005; Bakou et al., 2015). In fact, some authors previously reported about a negative correlation between myostatin and the fast phenotype of skeletal muscles (Bouley et al., 2005; Hennebry et al., 2009; Baan et al., 2013). However, muscle phenotypes may be aﬀected by many endogenous and exogenous factors, such as stage of maturity (Kugelberg, 1976), level of activity (Goldspink, 1983), diﬀerent sampling regions of the same muscle (Torrella et al., 2000), histological method (Karlsson et al., 1999). Hence, the known large variability of muscle ﬁber phenotypes, coupled to the lack of speciﬁc data for the dromedary camels, makes it hazardous to extend the mentioned correlation to the species under study. Tissue sampling was done on slaughterhouses from dead animals. Blood sampling from 25 Old World camels was collected during routine veterinary procedures or as part of a monitoring program of the wild camel population in Mongolia. Frontiers in Genetics \| www.frontiersin.org Myostatin Expression Proﬁling in Skeletal Muscles In order to quantify the level of myostatin transcript in dromedary skeletal muscle, we adopted a Digital Droplet June 2019 \| Volume 10 \| Article 502 13 Dromedary Myostatin Characterization Favia et al. PCR approach. In our experiments, we used two diﬀerent probes designed on the two exon-junctions of the myostatin gene and diﬀerentially dye-labeled. The FAM-labeled probe gave systematically higher values compared to the HEX- labeled one. This result agrees with the known evidence that FAM has a stronger signal compared to other dyes. Indeed, this feature may determine a larger number of droplets, where target ampliﬁcation occurred for both assays, to be designed as “positive” for the FAM-labeled probe compared to the HEX-labeled probe at a given ﬂuorescence threshold. Alternatively, a diﬀerent absolute quantiﬁcation in a two-probe system targeting the same gene may arise from the presence of alternative transcripts that may reduce the ampliﬁcation/detection eﬃciency of one of the two assays, but not necessarily both. However, in our study, given the systematic and consistent diﬀerences between the two assays across eight diﬀerent skeletal muscles, a major role for the alternative transcript phenomenon appears rather unlike. Finally, we cannot exclude a minor role of stochastic factors in aﬀecting the observed results. level of expression. The above results ﬁt well with multiple evidences that, in muscle, myostatin resides primarily as unprocessed promyostatin (Anderson et al., 2008; Pirruccello- Straub et al., 2018) and that the active mature growth factor is signiﬁcantly less abundant in this compartment (Hill et al., 2002, 2003; Zimmers et al., 2002; Anderson et al., 2008; Lakshman et al., 2009). Moreover, it must be pointed out that the observed 25 kDa bands, suggestive of the active myostatin form, could, in our study, reﬂect the amount of myostatin dimers, deriving from the latent complex generated by furin cleavage, and artiﬁcially “activated” by the experimental SDS environment (Wehling et al., 2000), rather than reﬂecting a physiologically activated form. Based on the above, the unprocessed or partially processed myostatin dimers could act as important reservoirs of slowly available myostatin forms, and the sequential cleavage steps contribute an additional layer of control, within an already complex regulatory framework. REFERENCES Anderson, S. B., Goldberg, A. L., and Whitman, M. (2008). Identiﬁcation of a novel pool of extracellular pro-myostatin in skeletal muscle. J. Biol. Chem. 283, 7027–7035. doi: 10.1074/jbc.M706678200 Abe, J., Fujii, Y., Kuwamura, Y., and Hizawa, K. (1987). Fiber type diﬀerentiation and myosin expression in regenerating rat muscles. Acta Pathol. Jpn. 37, 1537–1547. doi: 10.1111/j.1440-1827.1987.tb02465.x Ariano, M. A., Armstrong, R. B., and Edgerton, V. R. (1973). Hindlimb muscle ﬁber populations of ﬁve mammals. J. Histochem. Cytochem. 21, 51–55. doi: 10.1177/21.1.51 Agrawal, V., Gahlot, G. C., Ashraf, M., Khicher, J. P., and Thakur, S. (2017). Sequence analysis and phylogenetic relationship of myostatin gene of bikaneri Camel (Camelus dromedarius). J. Camel Pract. Res. 24:73. doi: 10.5958/2277- 8934.2017.00011.X Baan, J. A., Kocsis, T., Keller-Pinter, A., Muller, G., Zador, E., Dux, L., et al. (2013). The compact mutation of myostatin causes a glycolytic shift in the phenotype of fast skeletal muscles. J. Histochem. Cytochem. 61, 889–900. doi: 10.1369/0022155413503661 Aiello, D., Patel, K., and Lasagna, E. (2018). The myostatin gene: an overview of mechanisms of action and its relevance to livestock animals. Anim. Genet. 49, 505–519. doi: 10.1111/age.12696 Bakou, S. N., Nteme Ella, G. S., Aoussi, S., Guiguand, L., Cherel, Y., and Fantodji, A. (2015). Fiber composition of the grasscutter (Thryonomys swinderianus. Temminck 1827) thigh muscle: an enzyme-histochemical study. J. Cytol. Histol. 6:311. doi: 10.4172/2157-7099.1000311 Allen, D. L., Bandstra, E. R., Harrison, B. C., Thorng, S., Stodieck, L. S., Kostenuik, P. J., et al. (2009). Eﬀects of spaceﬂight on murine skeletal muscle gene expression. J. Appl. Physiol. 106, 582–595. doi: 10.1152/japplphysiol.90780. 2008 Bhatt, S. P., Nigam, P., Misra, A., Guleria, R., Luthra, K., Jain, S. K., et al. (2012). Association of the Myostatin gene with obesity, abdominal obesity and low lean body mass and in non-diabetic Asian Indians in north India. PLoS One 7:e40977. doi: 10.1371/journal.pone. 0040977 Allen, D. L., Cleary, A. S., Hanson, A. M., Lindsay, S. F., and Reed, J. M. (2010). CCAAT/enhancer binding protein-delta expression is increased in fast skeletal muscle by food deprivation and regulates myostatin transcription in vitro. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, R1592–R1601. doi: 10.1152/ajpregu. 00247.2010 Bo Li, Z., Zhang, J., and Wagner, K. R. (2012). Inhibition of myostatin reverses muscle ﬁbrosis through apoptosis. J. Cell Sci. 125(Pt 17), 3957–3965. doi: 10. 1242/jcs.090365 Allen, D. L., Cleary, A. S., Speaker, K. J., Lindsay, S. F., Uyenishi, J., Reed, J. M., et al. (2008). SUPPLEMENTARY MATERIAL S = C or G, W = A or T, R = A or G, Y = C or T, K = G or T, M = A or C, N = any base pair. SIGNAL, sub-sequence from the input sequence at the position corresponding to the consensus sequence. Default cut-offs by Tsunoda and Takagi, 1999 were adopted. The analysis was performed, for each SNP, using the two input sequences harboring the alternative alleles. DATA SHEET S3 \| Results of the TFBIND analysis for the SNPs present in the C. dromedarius myostatin gene upstream region (8 kb). AC, transcription factor matrix code. ID, transcription factor label, with V meaning “vertebrate”. SCORE, similarity (0.0–1.0) between a registered sequence for the transcription factor binding sites and the input sequence. STRAND, strandness. + and −means forward and reverse strands that the transcription factor binds, respectively. CONSENSUS, consensus sequence (ﬁxed) of the transcription factor binding sites. S = C or G, W = A or T, R = A or G, Y = C or T, K = G or T, M = A or C, N = any base pair. SIGNAL, sub-sequence from the input sequence at the position corresponding to the consensus sequence. Default cut-offs by Tsunoda and Takagi, 1999 were adopted. The analysis was performed, for each SNP, using the two input sequences harboring the alternative alleles. FIGURE S7 \| Graphical outline of the major predicted regulatory motifs in the 1.5 kb proximal to the transcription initiation site of the C. dromedarius myostatin gene. TATA boxes (highlighted in yellow), E-boxes (highlighted in gray), CREBP1 (light green), CREB_01 (orange), MYOGNF1 (blue), CEBP_01 (purple), MEF (pink), MYOD (dark green), and the CCAAT box (highlighted in purple) are presented. FIGURE S8 \| Graphical evaluation of the Digital Droplet PCR performance. (A) 2D-dot plot of ﬂuorescent signals detected in the Digital Droplet PCR experiments. 2D-dot plot of ﬂuorescent signals detected in the Digital Droplet PCR experiments. Fluorescence results are plotted as two-dimensional dot plots. Gray dots correspond to empty droplets. Blue dots correspond to droplets containing at SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene. 2019.00502/full#supplementary-material FIGURE S1 \| Electropherograms of 5’ and 3’ RACE PCR products. (A) Partial sequence of the 5’ untranslated region (UTR) of the C. dromedarius myostatin transcript, together with a partial sequence of the universal oligonucleotide provided in the SMARTer RACE cDNA ampliﬁcation kit (Clonetech). (B) Partial sequence of the 3’ untranslated region (UTR) of the C. dromedarius myostatin transcript, together with part of the poly(A) tail. In general, densitometric analysis highlighted that the promyostatin dimer is the most expressed form in all the considered muscles while the active myostatin has the lowest FIGURE S2 \| RACE PCR products separated via 2% agarose gel electrophoresis. The upper arrow corresponds to 366 base pairs, while the lower arrow corresponds to 316 base pairs. June 2019 \| Volume 10 \| Article 502 14 Dromedary Myostatin Characterization Favia et al. FIGURE S3 \| Comparative analysis of the myostatin genomic locus in the three Old World camelid species. Dromedary, C. dromedarius (contig-8645394). Wild camel, C. ferus (contig-7907533). Bactrian camel, C. bactrianus (contig-8938518). Highlighted, in the C. dromedarius sequence, the 5’ and 3’ UTR regions (light blue) and the three exons (yellow). least one copy of the sequence complementary to Probe 1. Green dots correspond to droplets containing at least one copy of the sequence complementary to Probe 2. Orange dots correspond to droplets containing at least one copy of the sequence complementary to Probe 1 and at least one copy of the sequence complementary to Probe 2 (double-positive droplets). The observed pattern is relatively well balanced and dot clouds are well separated, suggesting (i) the absence of signiﬁcant bias in the ampliﬁcation of the two regions targeted by the two considered probes (exon1/exon2, and exon2/exon3) and (ii) the applicability of the BIORAD proprietary auto-analysis tool for target quantiﬁcation. (B) Number of droplets generated in the Digital Droplet PCR experiments. Results for eight dromedary skeletal muscles, each from two different animals (biological replicate), analyzed in duplicate (technical replicate) are shown. FIGURE S3 \| Comparative analysis of the myostatin genomic locus in the three Old World camelid species. Dromedary, C. dromedarius (contig-8645394). Wild camel, C. ferus (contig-7907533). Bactrian camel, C. bactrianus (contig-8938518). Highlighted, in the C. dromedarius sequence, the 5’ and 3’ UTR regions (light blue) and the three exons (yellow). least one copy of the sequence complementary to Probe 1. SUPPLEMENTARY MATERIAL Green dots correspond to droplets containing at least one copy of the sequence complementary to Probe 2. Orange dots correspond to droplets containing at least one copy of the sequence complementary to Probe 1 and at least one copy of the sequence complementary to Probe 2 (double-positive droplets). The observed pattern is relatively well balanced and dot clouds are well separated, suggesting (i) the absence of signiﬁcant bias in the ampliﬁcation of the two regions targeted by the two considered probes (exon1/exon2, and exon2/exon3) and (ii) the applicability of the BIORAD proprietary auto-analysis tool for target quantiﬁcation. (B) Number of droplets generated in the Digital Droplet PCR experiments. Results for eight dromedary skeletal muscles, each from two different animals (biological replicate), analyzed in duplicate (technical replicate) are shown. FIGURE S4 \| Putative SNPs in the myostatin coding region, as inferred from alignment among previously published myostatin sequences and sequences generated in this study. (A) Consensus sequence of the myostatin coding region. Highlighted, in yellow, the exonic region, and, in red, the variant sites. Numbering of polymorphism positions refers to the contig AGVR01040332. (B) Summary table of the inferred polymorphisms showing the nature of the putative variants, together with the reference literature, and the prediction of the variant effects (in case of a missense mutation, the alternative amino acids are reported, otherwise “none” is entered). FIGURE S4 \| Putative SNPs in the myostatin coding region, as inferred from alignment among previously published myostatin sequences and sequences generated in this study. (A) Consensus sequence of the myostatin coding region. Highlighted, in yellow, the exonic region, and, in red, the variant sites. Numbering of polymorphism positions refers to the contig AGVR01040332. (B) Summary table of the inferred polymorphisms showing the nature of the putative variants, together with the reference literature, and the prediction of the variant effects (in case of a missense mutation, the alternative amino acids are reported, otherwise “none” is entered). TABLE S1 \| Sequence accession numbers (left panel) and links to the webpages of databases and softwares (right panel) considered in this study. TABLE S1 \| Sequence accession numbers (left panel) and links to the webpages of databases and softwares (right panel) considered in this study. DATA SHEET S1 \| Amino acidic sequences of the 83 non redundant myostatin proteins used for building the maximum likelihood tree presented in Figure 3. The data are in the “multiple sequence alignment” format. SUPPLEMENTARY MATERIAL FIGURE S5 \| Plot of the Bayesian clustering analysis perfoemed on the nine considered dromedaries using the 69 identiﬁed SNPs. The plot shows the results obtained for K = 7, that was identiﬁed as the most likely output by visual inspection of the probability values associated to each tested K value (from 1 to 9). Numbers indicate different samples (1, United Arab Emirates; 2, Qatar; 3-4-5, Kingdom of Saudi Arabia; 6, Austria; 7, Kenya; 8, Sudan; 9, Pakistan). Colors indicate the seven different clusters. The proportion of each individual sample in each inferred cluster is shown in the y-axis. FIGURE S5 \| Plot of the Bayesian clustering analysis perfoemed on the nine considered dromedaries using the 69 identiﬁed SNPs. The plot shows the results obtained for K = 7, that was identiﬁed as the most likely output by visual inspection of the probability values associated to each tested K value (from 1 to 9). Numbers indicate different samples (1, United Arab Emirates; 2, Qatar; 3-4-5, Kingdom of Saudi Arabia; 6, Austria; 7, Kenya; 8, Sudan; 9, Pakistan). Colors indicate the seven different clusters. The proportion of each individual sample in each inferred cluster is shown in the y-axis. DATA SHEET S1 \| Amino acidic sequences of the 83 non redundant myostatin proteins used for building the maximum likelihood tree presented in Figure 3. The data are in the “multiple sequence alignment” format. DATA SHEET S2 \| Sequence of the Camelus dromedarius contig-8645394. Exons (highlighted in yellow), 5’ and 3’ UTR regions (underlined), SNPs (highlighted in red), the GAT codon for the aspartic acid essential for BMP/tolloid protease cleavage (highlighted in green), and the 12 nucleotides coding for the RSRR motif (highlighted in gray), needed for recognition by furin convertase, are shown. FIGURE S6 \| Results of the Human Splicing Finder analysis for the three intronic SNPs detected in this study. (A) G66148C. (B) A66460G. (C) T66461C. DATA SHEET S3 \| Results of the TFBIND analysis for the SNPs present in the C. dromedarius myostatin gene upstream region (8 kb). AC, transcription factor matrix code. ID, transcription factor label, with V meaning “vertebrate”. SCORE, similarity (0.0–1.0) between a registered sequence for the transcription factor binding sites and the input sequence. STRAND, strandness. + and −means forward and reverse strands that the transcription factor binds, respectively. CONSENSUS, consensus sequence (ﬁxed) of the transcription factor binding sites. REFERENCES MyoD(-/-) satellite cells in single-ﬁber culture are diﬀerentiation defective and MRF4 deﬁcient. Dev. Biol. 224, 122–137. doi: 10.1006/dbio.2000.9682 Guttridge, D. C., Mayo, M. W., Madrid, L. V., Wang, C. Y., and Baldwin, A. S. Jr. (2000). NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia. 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https://openalex.org/W4380081039	https://www.frontiersin.org/articles/10.3389/fimmu.2023.1150754/pdf	English	null	PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma	Frontiers in immunology	2,023	cc-by	16,387	TYPE Original Research PUBLISHED 09 June 2023 DOI 10.3389/fimmu.2023.1150754 OPEN ACCESS OPEN ACCESS EDITED BY Christian Kellner, LMU Munich University Hospital, Germany REVIEWED BY Cornelia Brunner, Ulm University Medical Center, Germany Michalis Koullouros, Flinders Medical Centre, Australia CORRESPONDENCE Noel E. Donlon donlonn@tcd.ie †PRESENT ADDRESSES Maria Davern, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States Jasmine Yun-Tong Kung, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States ‡These authors have contributed equally to this work and share ﬁrst authorship RECEIVED 25 January 2023 ACCEPTED 11 May 2023 PUBLISHED 09 June 2023 CITATION Davern M, Gaughan C, O’ Connell F, Moran B, Mylod E, Sheppard AD, Ramjit S, Yun-Tong Kung J, Phelan JJ, Davey MG, Ryan EJ, Butler C, Quinn L, Howard C, Tone E, Phoenix E, Butt WT, Lynam-Lennon N, Maher SG, Ravi N, Donohoe CL, Reynolds JV, Lysaght J and Donlon NE (2023) PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma. Front. Immunol. 14:1150754. doi: 10.3389/fimmu.2023.1150754 COPYRIGHT © 2023 Davern, Gaughan, O’ Connell, Moran, Mylod, Sheppard, Ramjit, Yun-Tong Kung, Phelan, Davey, Ryan, Butler, Quinn, Howard, Tone, Phoenix, Butt, Lynam-Lennon, Maher, Ravi, Donohoe, Reynolds, Lysaght and Donlon. This is an open-access article distributed under the terms of the Creative Commons Attribution Maria Davern 1,2†‡, Caoimhe Gaughan 1‡, Fiona O’ Connell 3, Brendan Moran 3, Eimear Mylod 3, Andrew D. Sheppard 1, Sinead Ramjit 3, Jasmine Yun-Tong Kung 2†, James J. Phelan 1, Matthew G. Davey 3, Eanna J. Ryan 3, Christine Butler 3, Laura Quinn 3, Claudine Howard 3, Emily Tone 3, Eimear Phoenix 3, Waqas T. Butt 3, Niamh Lynam-Lennon 1, Stephen G. Maher 1, Narayanasamy Ravi 3, Claire L. Donohoe 3, John V. Reynolds 3, Joanne Lysaght 1 and Noel E. Donlon 3 1Cancer Immunology and Immunotherapy Group, Department of Surgery, Trinity St. James’s Cancer Institute, Trinity Translational Medicine Institute, Dublin, Ireland, 2Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States, 3Department of Surgery, Trinity St. James’s Cancer Institute, Trinity Translational Medicine Institute, St. 1Cancer Immunology and Immunotherapy Group, Department of Surgery, Trinity St. James’s Cancer Institute, Trinity Translational Medicine Institute, Dublin, Ireland, 2Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States, 3Department of Surgery, Trinity St. James’s Cancer Institute, Trinity Translational Medicine Institute, St. James’s Hospital, Trinity College Dublin, Dublin, Ireland PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma Donlon donlonn@tcd.ie †PRESENT ADDRESSES Maria Davern, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States Jasmine Yun-Tong Kung, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States ‡These authors have contributed equally to this work and share ﬁrst authorship RECEIVED 25 January 2023 ACCEPTED 11 May 2023 PUBLISHED 09 June 2023 CITATION Davern M, Gaughan C, O’ Connell F, Moran B, Mylod E, Sheppard AD, Ramjit S, Yun-Tong Kung J, Phelan JJ, Davey MG, Ryan EJ, Butler C, Quinn L, Howard C, Tone E, Phoenix E, Butt WT, Lynam-Lennon N, Maher SG, Ravi N, Donohoe CL, Reynolds JV, Lysaght J and Donlon NE (2023) PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma. Front. Immunol. 14:1150754. doi: 10.3389/fimmu.2023.1150754 COPYRIGHT © 2023 Davern, Gaughan, O’ Connell, Moran, Mylod, Sheppard, Ramjit, Yun-Tong Kung, Phelan, Davey, Ryan, Butler, Quinn, Howard, Tone, Phoenix, Butt, Lynam-Lennon, Maher, Ravi, Donohoe, Reynolds, Lysaght and Donlon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma Frontiers in Immunology OPEN ACCESS EDITED BY Christian Kellner, LMU Munich University Hospital, Germany REVIEWED BY Cornelia Brunner, Ulm University Medical Center, Germany Michalis Koullouros, Flinders Medical Centre, Australia CORRESPONDENCE Noel E. Donlon donlonn@tcd.ie †PRESENT ADDRESSES Maria Davern, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States Jasmine Yun-Tong Kung, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States ‡These authors have contributed equally to this work and share ﬁrst authorship RECEIVED 25 January 2023 ACCEPTED 11 May 2023 PUBLISHED 09 June 2023 CITATION Davern M, Gaughan C, O’ Connell F, Moran B, Mylod E, Sheppard AD, Ramjit S, Yun-Tong Kung J, Phelan JJ, Davey MG, Ryan EJ, Butler C, Quinn L, Howard C, Tone E, Phoenix E, Butt WT, Lynam-Lennon N, Maher SG, Ravi N, Donohoe CL, Reynolds JV, Lysaght J and Donlon NE (2023) PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma. Front. Immunol. 14:1150754. doi: 10.3389/fimmu.2023.1150754 COPYRIGHT © 2023 Davern, Gaughan, O’ Connell, Moran, Mylod, Sheppard, Ramjit, Yun-Tong Kung, Phelan, Davey, Ryan, Butler, Quinn, Howard, Tone, Phoenix, Butt, Lynam-Lennon, Maher, Ravi, Donohoe, Reynolds, Lysaght and Donlon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS EDITED BY Christian Kellner, LMU Munich University Hospital, Germany REVIEWED BY Cornelia Brunner, Ulm University Medical Center, Germany Michalis Koullouros, Flinders Medical Centre, Australia CORRESPONDENCE Noel E. Introduction immunophenotyping immune checkpoint expression proﬁles of circulating T cells and circulating soluble Th1, Th2, Treg, pro- inﬂammatory and pro-angiogenic factors in peripheral circulation (5). These ﬁndings highlighted the immunosuppressive effects of major oncologic surgery in OGJ cancer patients whereby surgery dampened systemic Th1 immune responses and promoted an immunosuppressive Th2 systemic immune response and increased circulating pro-angiogenic factors (5). These interesting ﬁndings suggest that the wound healing response induced by surgical tumour resection (Th2-like and regulator T cell-like) may be promoting metastatic dissemination and tumour recurrence and inhibiting cancer immune surveillance via dampening of the Th1-like immune response (5). However, it remains unknown if surgery dampens the cytotoxic immune response suppressing killing ability of lymphocytes. In this study we hypothesized that based on the ﬁndings from our previous study that surgery inhibits the cytotoxic ability of patient-derived lymphocytes and perhaps the use of immune checkpoint blockade (ICB) may abrogate these surgery-induced immunosuppressive effects. Recent clinical data from the Checkmate 577 trial, which led to the FDA approval of nivolumab for treating OGJ cancer patients in the adjuvant setting with residual disease, demonstrated that nivolumab signiﬁcantly increased progression-free survival compared with the placebo arm (saline arm) (22.4 months versus 11 months) (6). Collectively, a rationale is highlighted to utilize immunotherapy in the perioperative surgical window to harness the immunomodulatory effects of surgery and promote anti-tumour Th1 responses. Therefore, our timely study aims to assess the therapeutic potential for incorporating ICB in the perioperative setting to boost Th1 cytotoxic immunity. Oesophagogsatric junctional adenocarcinoma (OGJ) is an aggressive malignancy with a high propensity to metastasise (1). Clinical presentation of OGJ is often at the more advanced stages due to its indolent nature (1). The current treatment remains surgical en bloc resection, with multimodal perioperative therapy namely perioperative FLOT chemotherapy or preoperative CROSS chemoradiotherapy (2). FLOT treatment comprises 5-FU (anti- metabolite), oxaliplatin (platinum agent) and docetaxel (taxane) consisting of 4 neoadjuvant (pre-surgery) cycles and 4 adjuvant (post-surgery) cycles (2). The CROSS chemoradiotherapy regimen includes neoadjuvant carboplatin (platinum agent) and paclitaxel (taxane) with concomitant daily 1.8 Gy doses of radiotherapy with a cumulative dose of 41.4 Gy (2). The 5-year survival rate for OGJ cancer patients is between 20-30%, with approximately 70% of patients failing to achieve a complete pathologic response to neoadjuvant treatment (no viable tumour cells at time of surgical tumour resection) (3). OPEN ACCESS James’s Hospital, Trinity College Dublin, Dublin, Ireland CITATION Davern M, Gaughan C, O’ Connell F, Moran B, Mylod E, Sheppard AD, Ramjit S, Yun-Tong Kung J, Phelan JJ, Davey MG, Ryan EJ, Butler C, Quinn L, Howard C, Tone E, Phoenix E, Butt WT, Lynam-Lennon N, Maher SG, Ravi N, Donohoe CL, Reynolds JV, Lysaght J and Donlon NE (2023) PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma. Introduction: This timely study assesses the immunosuppressive effects of surgery on cytotoxic Th1-like immunity and investigates if immune checkpoint blockade (ICB) can boost Th1-like immunity in the perioperative window in upper gastrointestinal cancer (UGI) patients. Methods: PBMCs were isolated from 11 UGI patients undergoing tumour resection on post-operative days (POD) 0, 1, 7 and 42 and expanded ex vivo using anti-CD3/28 and IL-2 for 5 days in the absence/presence of nivolumab or ipilimumab. T cells were subsequently immunophenotyped via ﬂow cytometry to determine the frequency of T helper (Th)1-like, Th1/17-like, Th17-like and regulatory T cell (Tregs) subsets and their immune checkpoint expression proﬁle. Lymphocyte secretions were also assessed via multiplex ELISA (IFN-g, granzyme B, IL-17 and IL-10). The 48h cytotoxic ability of vehicle-, nivolumab- and ipilimumab-expanded PBMCs isolated on POD 0, 1, 7 and 42 against radiosensitive and radioresistant oesophageal adenocarcinoma tumour cells (OE33 P and OE33 R) was also examined using a cell counting kit-8 (CCK-8) assay to determine if surgery affected the killing ability of lymphocytes and whether the use of ICB could enhance cytotoxicity. Front. Immunol. 14:1150754. doi: 10.3389/fimmu.2023.1150754 COPYRIGHT © 2023 Davern, Gaughan, O’ Connell, Moran, Mylod, Sheppard, Ramjit, Yun-Tong Kung, Phelan, Davey, Ryan, Butler, Quinn, Howard, Tone, Phoenix, Butt, Lynam-Lennon, Maher, Ravi, Donohoe, Reynolds, Lysaght and Donlon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: Th1-like immunity was suppressed in expanded PBMCs in the immediate post-operative setting. The frequency of expanded circulating Th1-like cells was signiﬁcantly decreased post-operatively accompanied by a decrease in IFN-g Frontiers in Immunology 01 frontiersin.org Davern et al. OPEN ACCESS 10.3389/ﬁmmu.2023.1150754 production and a concomitant increase in the frequency of expanded regulatory T cells with an increase in circulating levels of IL-10. Interestingly, PD-L1 and CTLA-4 immune checkpoint proteins were also upregulated on expanded Th1- like cells post-operatively. Additionally, the cytotoxic ability of expanded lymphocytes against oesophageal adenocarcinoma tumour cells was abrogated post-surgery. Of note, the addition of nivolumab or ipilimumab attenuated the surgery-mediated suppression of lymphocyte cytotoxicity, demonstrated by a signiﬁcant increase in tumour cell killing and an increase in the frequency of Th1-like cells and Th1 cytokine production. Conclusion: These ﬁndings support the hypothesis of a surgery-mediated suppression in Th1-like cytotoxic immunity and highlights a rationale for the use of ICB within the perioperative setting to abrogate tumour-promoting effects of surgery and ameliorate the risk of recurrence. nivolumab and ipilimumab, PD-1, TIGIT, perioperative, Th1 immunity, immunosuppression, surgery, oesophageal adenocarcinoma KEYWORDS Introduction And of the 30% that experience a complete pathologic response to treatment, up to 60% of those patients experience tumour recurrence within 5 years. In addition, of the patients who are prescribed FLOT perioperative treatment, only 33% of patients actually receive the complete 8 cycles of FLOT chemotherapy due to therapy-associated toxicities meaning that the majority of patients do not complete their anti-cancer treatment (4). The high tumour recurrence rate post-surgery in conjunction with the substantial dose limiting toxicities of current ﬁrst-line adjuvant therapies highlights the clinical need to identify novel adjuvant therapies for OGJ cancer patients (4). A recent study by Donlon et al, proﬁled systemic immunity in OGJ cancer patients perioperatively via ﬂow cytometry Frontiers in Immunology frontiersin.org 02 Davern et al. 10.3389/ﬁmmu.2023.1150754 Peripheral blood mononuclear cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque (GE Healthcare, USA). Whole blood was diluted 1 in 2 with phosphate-buffered saline (PBS, Gibco, USA) and layered onto 15 ml of Ficoll-Paque™(GE Healthcare, USA) and centrifuged at 2000 rpm for 25 minutes with the brake turned off. The buffy coat layer containing PBMCs was removed, washed with PBS (Gibco, USA) and resuspended at a density of 1x 10(6) cells/ml in complete RPMI with recombinant human IL-2 (10 units/ml) (ImmunoTools, Germany). TABLE 1 Patient demographics and clinical data. Age (years) 65.8 Sex ratio (M:F) 7:5 Diagnosis (no. patients) OSCC 4 Adenosquamous carcinoma 1 OGJ 5 Gastric adenocarcinoma 2 Pathological tumour stage (no. patients) T0 0 T1 6 T2 0 T3 6 T4 0 Pathological nodal status (no. patients) Positive 5 Negative 7 TABLE 1 Patient demographics and clinical data. Ethical approval Ethical approval was granted from the St. James’s Hospital Ethics Committee. All samples were collected with prior informed written consent for sample and data acquisition from patients attending St. James’s Hospital and the Beacon Hospital. This study was carried out in accordance with the World Medical Cell lines Isogenic radiosensitive (OE33 P) and radioresistant (OE33 R) OGJ tumour cells were cultured in Roswell Park Memorial Institute 1640 Medium with 2 mM L-glutamine (ThermoFisher Scientiﬁc, Ireland) and supplemented with 1% (v/v) penicillin-streptomycin (50 U/ml penicillin 100mg/ml streptomycin) and 10% (v/v) foetal bovine serum (FBS) (ThermoFisher Scientiﬁc, Ireland) (complete RPMI). Cells were detached using trypsin-EDTA (Gibco, USA). Mycoplasma testing was routinely carried out by PCR every three months. Serum and plasma isolation Serum and plasma samples were obtained from patients on POD 0, 1, 7, 42, respectively. Whole blood samples were collected in BD Bioscience EDTA vacutainer blood tubes and were centrifuged at 3,000 RPM at room temperature. The plasma and serum were removed and stored at -80°C for future experimentation. Patient cohort 12 patients undergoing surgical resection of OGJ tumours in St James’s Hospital Dublin and the Beacon Hospital, Dublin from April 2021 to July 2021 were recruited for this study. Inclusion criteria included: newly diagnosed patients with cancers of the upper gastrointestinal tract who may or may not have received neoadjuvant chemoradiotherapy prior to their surgery. Exclusion criteria included patients with metastasis. Based on power calculations we estimated a cohort size of 10 patients would be required to detect statistical signiﬁcance therefore, we recruited a total of 12 patients. Whole blood was obtained from patients on the day of surgical tumour resection prior to surgery (before anaesthesia): post-operative day (POD) 0 (12 patients), 1 day post-surgery (POD 1) (12 patients), 7 days post-surgery (POD 7) (10 patients) and 6 weeks post-surgery (POD 42) (4 patients). Some patients were unavailable to follow up with for non-medical reasons. The cohort consisted of 7 males and 5 females, with an average age of 65.8 years. The patient demographics are detailed in Table 1. Methods Association’s Declaration of Helsinki guidelines on medical research involving human subjects. Patient samples were pseudonymised to protect the privacy rights of the patients. Cell counting kit-8 assay Following a 48h co-culture of lymphocytes with OE33 P and OE33 R tumour cells, the viability of tumour cells was assessed using a cell counting kit (CCK-8, Sigma, USA) viability assay. 5 µl of CCK-8 solution was added to each well for 1h at 37°CC, 5% CO2. The optical density was measured using the Versa Max microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm and 650 nm (reference wavelength). Formula: (viability OE33 cell- lymphocyte co-culture)-(viability PBMCs alone)/(viability untreated OE33 cells alone) × 100 = % live cells. Overall, systemic Th1 immune responses appeared to be dampened in the immediate post-operative setting in OGJ cancer patients paralleled by an increase in regulatory T cell immunity. The frequency of expanded circulating CD4+CCR6-CXCR3+ Th1-like cells was signiﬁcantly decreased on POD 42 compared with POD 0 (Figures 1B, P, 25.1 ± 4.8 vs. 39.5 ± 5.6%, p=0.05). Similarly, the frequency of expanded circulating CD8+CCR6-CXCR3+ Th1-like cells was signiﬁcantly decreased on POD 7 compared to POD 0 (Figures 1B, P, 51.7 ± 8.1 vs. 66.7 ± 3.9%, p=0.02). This was accompanied by a decrease in IFN-g production from expanded PBMCs on POD 7 compared with POD 0, that trended toward statistical signiﬁcance, indicative of a Th1 suppressed immune response in the post-operative setting (Figure 1C, 1,081 ± 661.6 vs. 10,682 ± 4,156%, p=0.07). We did not observe any signiﬁcant Co-culture with cancer cells OE33 P cells and OE33 R cells were seeded at density of 1 x 104 in 100 ml/in a ﬂat 96 well plate in complete RPMI and were allowed adhere overnight at 37 °C, 5% CO2. The next day the media was replaced with 50 ml of complete RPMI and 50 µl of media only (untreated wells) or 50 µl of vehicle-, nivolumab- or ipilimumab- expanded lymphocytes were added at a density of 1 x 105 cells in 50 µl of complete RPMI to achieve an effector to target ratio (E:T) of 10:1. Expanded lymphocytes were also cultured alone to account for the absorbance of lymphocytes using the CCK-8 assay. Cells were left to incubate for 48h at 37°CC, 5% CO2. Concomitant T cell activation and immune checkpoint blockade For the purpose of this study these immune proteins were compared between patients who had lower than median IFN-g levels versus higher than median IFN-g levels to determine if there were particular proteins that were differentially expressed based on circulating levels of IFN-g. We chose IFN-g in this instance, as patients with higher circulating levels of IFN-g in their serum on POD 0 also had a greater nivolumab-mediated increase in IFN-g by expanded lymphocytes ex vivo. Based on this observation we sought to investigate what might be different in the circulating factors between patients with high versus low IFN-g in their serum. Th1 immunity is dampened in expanded PBMCs in the immediate post-operative setting The balance between Th1 and regulatory T cell (Treg) immune responses has a profound impact on anti-cancer immunity. Additionally, Th17 immune responses can have either a tumour- promoting or tumour-suppressive effect, which is often context dependent. Therefore, this study assessed the frequency of expanded circulating Th1-, Th17- and Th1/17-like CD3+CD4+ and CD3+CD8+ cells from OGJ cancer patients who underwent surgical tumour resection to determine how major oncologic surgery affects systemic Th1, Treg and Th17 immunity (Figure 1A). Overall, systemic Th1 immune responses appeared to be dampened in the immediate post-operative setting in OGJ cancer patients paralleled by an increase in regulatory T cell immunity. The frequency of expanded circulating CD4+CCR6-CXCR3+ Th1-like cells was signiﬁcantly decreased on POD 42 compared with POD 0 (Figures 1B, P, 25.1 ± 4.8 vs. 39.5 ± 5.6%, p=0.05). Similarly, the frequency of expanded circulating CD8+CCR6-CXCR3+ Th1-like cells was signiﬁcantly decreased on POD 7 compared to POD 0 (Figures 1B, P, 51.7 ± 8.1 vs. 66.7 ± 3.9%, p=0.02). This was accompanied by a decrease in IFN-g production from expanded PBMCs on POD 7 compared with POD 0, that trended toward statistical signiﬁcance, indicative of a Th1 suppressed immune response in the post-operative setting (Figure 1C, 1,081 ± 661.6 vs. 10,682 ± 4,156%, p=0.07). We did not observe any signiﬁcant The balance between Th1 and regulatory T cell (Treg) immune responses has a profound impact on anti-cancer immunity. Additionally, Th17 immune responses can have either a tumour- promoting or tumour-suppressive effect, which is often context dependent. Therefore, this study assessed the frequency of expanded circulating Th1-, Th17- and Th1/17-like CD3+CD4+ and CD3+CD8+ cells from OGJ cancer patients who underwent surgical tumour resection to determine how major oncologic surgery affects systemic Th1, Treg and Th17 immunity (Figure 1A). Statistical analysis Statistical analysis was performed using GraphPad Prism 9.1.1 software (GraphPad Prism, San Diego, California). Signiﬁcance level of p ≤0.05 was used for all analyses. CCK-8 data and ﬂow cytometry data were analysed using a two-way ANOVA statistical analysis. Spearman correlative analysis was used to assess correlations. Flow cytometry staining Vehicle-, nivolumab- or ipilimumab-expanded lymphocytes were stained with zombie NIR viability dye, CD8-BV421, CD4- BV510, CD3-FITC, CXCR3-PE, CCR6-APC, CTLA-4-PerCPCy5.5, TIGIT-PE/Cy7, PD-L1-PerCPCy5.5, PD-1-PECy/7, CD25-FITC and FOXP3-BV421 (Biolegend, USA). Intracellular FoxP3 permeabilization and ﬁxation buffer was used for FoxP3 staining (Biolegend, USA). Cells were acquired using the BD FACs Canto II (BD Biosciences) using Diva software and analysed using FlowJoTM v10.7. Concomitant T cell activation and immune checkpoint blockade A sterile 6-well plate was coated with 10 µg/ml of goat anti- mouse IgG (Sigma, USA) and 10 µg/ml of anti-CD3/anti-CD28 diluted in PBS (BioLegend, USA). PBMCs were seeded at a density of 1x 10(6) cells/ml in 2 ml of media with 10 IU of recombinant IL-2 cytokine (Biolegend, USA) in anti-CD3/28 pre-coated wells. Nivolumab and ipilimumab were each added to one well each at a concentration of 10 µg/ml. Nivolumab and Ipilimumab were kindly donated by the Beacon Hospital. Vehicle-treated PBMCs were treated with an isotype control antibody (IgG4,10 µg/ml, Biolegend, USA). Plates were incubated at 37°CC, 5% CO2 for 5 days. The media was topped up with an additional 1 ml of media every 2 days containing fresh IL-2 (10 IU) and 10 ug/ml of isotype frontiersin.org 03 Davern et al. 10.3389/ﬁmmu.2023.1150754 supernatants were run undiluted. All assays were run as per manufacturer’s recommendation, an overnight supernatant incubation protocol was used for all assays. Analyte concentrations were calculated using Discovery Workbench software (version 4.0). Values outside the kits limit of detection were not reported. For the purpose of this study these immune proteins were compared between patients who had lower than median IFN-g levels versus higher than median IFN-g levels to determine if there were particular proteins that were differentially expressed based on circulating levels of IFN-g. We chose IFN-g in this instance, as patients with higher circulating levels of IFN-g in their serum on POD 0 also had a greater nivolumab-mediated increase in IFN-g by expanded lymphocytes ex vivo. Based on this observation we sought to investigate what might be different in the circulating factors between patients with high versus low IFN-g in their serum. control, nivolumab or ipilimumab. On day 5 of the T cell expansion a total of 6 ml of media was present in each well which contained the lymphocytes which were activated in the presence of a vehicle, nivolumab or ipilimumab. 1 ml of supernatant was harvested from each well and stored at -80°C for future experimentation on day 5 of the T cell expansion protocol. supernatants were run undiluted. All assays were run as per manufacturer’s recommendation, an overnight supernatant incubation protocol was used for all assays. Analyte concentrations were calculated using Discovery Workbench software (version 4.0). Values outside the kits limit of detection were not reported. Frontiers in Immunology Quantiﬁcation of serum and lymphocyte supernatant immune proteins Serum samples were processed according to MSD (Meso Scale Discovery) multiplex protocol. To assess interferon-gamma (IFN- g), granzyme B (GrB), interleukin-10 (IL-10) and IL-17 cytokines, a custom 4 spot U-PLEX ELISA kit was used (Meso Scale Diagnostics, USA). Serum samples were diluted 1:2 for analysis and lymphocyte 04 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. Quantiﬁcation of serum and lymphocyte supernatant immune proteins CD4 Th1 CD4 Th1/17 CD4 Th17 CD4 Tregs CD8 Th1 CD8 Th1/17 CD8 Th17 GrB IFNγ IL-10 IL-17A GrB IFN-γ IL-10 IL-17A CD4 Th1 CD4 Th1/17 CD4 Th17 CD4 Tregs CD8 Th1 CD8 Th1/17 CD8 Th17 GrB IFNγ IL-10 IL-17A GrB IFN-γ IL-10 IL-17A serum PBMC serum PBMC * * * * * * * * * * * * * -1.0 -0.5 0 0.5 1.0 Treg POD 0 POD 1 POD 7 POD 42 0 10 20 30 40 50 % CD4+FoxP3+cells ** Th1 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 50 100 % CCR6-CXCR3+cells CD4+ cells CD8+ cells * * 0 10000 20000 30000 40000 IFNy levels of IFNy (pg/ml) POD 42 POD 0 POD 1 POD 7 p=0.07 0 100 200 300 IL-10 levels of IL-10 (pg/ml) POD 42 POD 0 POD 1 POD 7 Day 0 Day 1 Day 7 Day 42 0 5 10 15 IL-10 levels of IL-10 (pg/ml) ** * Experimental Schematic Day 0 Day 1 Day 7 Day 42 0 20 40 60 IFN-y levels of IFNy (pg/ml) 0 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 42 POD 0 POD 1 POD 7 Day 0 Day 1 Day 7 Day 42 0 5 10 15 GrB levels of GrB (pg/ml) 0 5000 10000 15000 20000 25000 IL-17 levels of IL-17 (pg/ml) POD 42 POD 0 POD 1 POD 7 Th17 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 20 40 60 % CCR6+CXCR3-cells CD4+ cells CD8+ cells Th1/17 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 20 40 60 % CCR6+CXCR3+cells CD4+ cells CD8+ cells Day 0 Day 1 Day 7 Day 42 0 10 20 30 IL-17a levels of IL-17a (pg/ml) ** * 1.0 0.7 -0.9 -0.4 0.7 1.0 -0.9 -0.7 -0.9 -0.9 1.0 0.9 -0.4 -0.7 0.9 1.0 Th1CD4 cells Th1CD8 cells Tumour regression grade Nodal stage Th1CD4 cells Th1CD8 cells Tumour regression grade Nodal stage * * * * * -1.0 -0.5 0 0.5 1.0 CCR6 CXCR3 POD 0 POD 1 POD 7 POD 42 CD4 cells CD8 cells A B D E F G I H J K L M N C O P URE 1 e Th1 immune response was dampened in expanded PBMCs in the immediate postoperative setting. Quantiﬁcation of serum and lymphocyte supernatant immune proteins A A Experimental Schematic Day 0 Day 1 Day 7 Day 42 0 20 40 60 IFN-y levels of IFNy (pg/ml) Day 0 5 10 15 levels of GrB (pg/ml) E F Day 0 Day 1 Day 7 Day 42 0 20 40 60 IFN-y levels of IFNy (pg/ml) E 0 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 42 POD 0 POD 1 POD 7 D 0 10000 20000 30000 40000 IFNy levels of IFNy (pg/ml) POD 42 POD 0 POD 1 POD 7 p=0.07 C Th1 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 50 100 % CCR6-CXCR3+cells CD4+ cells CD8+ cells * * B D B E C Th17 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 20 40 60 % CCR6+CXCR3-cells CD4+ cells CD8+ cells H 1 2 3 4 5 % CD4+FoxP3+cells 0 5000 10000 15000 20000 25000 IL-17 levels of IL-17 (pg/ml) POD 42 POD 0 POD 1 POD 7 Day 0 Day 1 Day 7 Day 42 0 10 20 30 IL-17a levels of IL-17a (pg/ml) ** * I J K 7 POD 42 OD 7 Day 0 Day 1 Day 7 Day 42 0 10 20 30 IL-17a levels of IL-17a (pg/ml) ** * J Th1/17 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 20 40 60 % CCR6+CXCR3+cells CD4+ cells CD8+ cells G L Treg POD 0 POD 1 POD 7 POD 42 0 10 20 30 40 50 % CD4+FoxP3+cells ** K N I G H K J L L N CD4 Th1 CD4 Th1/17 CD4 Th17 CD4 Tregs CD8 Th1 CD8 Th1/17 CD8 Th17 GrB IFNγ IL-10 IL-17A GrB IFN-γ IL-10 IL-17A CD4 Th1 CD4 Th1/17 CD4 Th17 CD4 Tregs CD8 Th1 CD8 Th1/17 CD8 Th17 GrB IFNγ IL-10 IL-17A GrB IFN-γ IL-10 IL-17A serum PBMC serum PBMC * * * * * * * * * * * * * -1.0 -0.5 0 0.5 1.0 0 100 200 300 IL-10 levels of IL-10 (pg/ml) POD 42 POD 0 POD 1 POD 7 Day 0 Day 1 Day 7 Day 42 0 5 10 15 IL-10 levels of IL-10 (pg/ml) ** * 1.0 0.7 -0.9 -0.4 0.7 1.0 -0.9 -0.7 -0.9 -0.9 1.0 0.9 -0.4 -0.7 0.9 1.0 Th1CD4 cells Th1CD8 cells Tumour regression grade Nodal stage Th1CD4 cells Th1CD8 cells Tumour regression grade Nodal stage * * * * * -1.0 -0.5 0 0.5 1.0 CCR6 CXCR3 POD 0 POD 1 POD 7 POD 42 CD4 cells CD8 cells L M N O P M 0 100 200 300 IL-10 levels of IL-10 (pg/ml) POD 42 POD 0 POD 1 POD 7 N O FIGURE 1 The Th1 immune response was dampened in expanded PBMCs in the immediate postoperative setting. Quantiﬁcation of serum and lymphocyte supernatant immune proteins PBMCs were isolated from peripheral blood cancer patients on POD 0 (n=12), POD 1 (n=12), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 d recombinant IL-2 (A). The frequency of expanded Th1 (CXCR3+CCR6-) (B), Th1/17 (CXCR3-CCR6-) (G) and Th17 (CXCR3+CCR6+) (H) CD3+CD4+ d CD3+CD8+ cells and Treg cells (CD4+FOXP3+) (K) was assessed by ﬂow cytometry. The soluble levels of IFN-g (C, E), GrB (D, F), IL-17 (I, J) and 10 (M, N) were measured in the serum from whole blood and in the supernatant from expanded PBMCs on POD 0, 1, 7 and 42. (L) shows the rrelation values between cytokine production and the frequency of expanded lymphocytes following a 5-day expansion. (O) shows the signiﬁcant rrelation values between T cell subset frequencies post-5-day expansion with clinical characteristics. (P) shows representative dot plots for the quency of Th1, Th17 and Th1/17 cells. Data presented as mean ± SEM. Two-way ANOVA for (B–K) and (M, N). Spearman correlation used for n-parametric correlative analysis for (L, O). The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A ue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. Quantiﬁcation of serum and lymphocyte supernatant immune proteins patients who had higher levels of CD8+ Th1-like cells had a decreased nodal stage or In summary, our ﬁndings suggest that an IL-10-driven immunosuppressive window is propagated by resection of OGJ tumours in the post-operative setting and this is accompanied by a dampening of a Th1 immune response, which is maintained ex vivo in expanded PBMCs. Overall, this data highlights the need to introduce immunotherapies in the post-operative setting to exploit this surge in immune activation and skew it towards an anti-cancer Th1 immune response to enhance cancer immune surveillance and reduce the high tumour recurrence rate in the adjuvant setting. Quantiﬁcation of serum and lymphocyte supernatant immune proteins 0.1 ± 0.6%, p<0.001, p,0.001 and p=0.009). Induction of immunosuppression post-surgery was further underlined by a signiﬁcant increase in the frequency of expanded regulatory T cells (Tregs) by POD 7 (Figure 1K, 14.7 ± 4.0 vs. 5.1 ± 1.2%, p=0.04). We did not detect any differences in the levels of IL-10 in the supernatant of expanded PBMCs (Figure 1N). Furthermore, we observed that the frequency of expanded CD4+ and CD8+ Th1-like cells following 5-day T cell activation was signiﬁcantly correlated with a worse pathologic response to neoadjuvant treatment (r= -0.9, p=0.03). The presence of nodal metastasis also signiﬁcantly correlated with the frequency of expanded CD8+ Th1-like cells following 5-day T cell activation (r= -0.6, p=0.03, respectively). This might indicate that patients who responded poorly to treatment and had more advanced disease may have a dampened Th1 immune response as the expansion of Th1- like cells ex vivo was attenuated. The frequency of expanded T helper subsets was also correlated with the levels of T helper- associated cytokines in the supernatant from expanded PBMC cultures and in peripheral circulation of cancer patients (Figure 1L). Not surprisingly, the levels of Th1-associated cytokines such as GrB and IFN-g correlated with the frequency of expanded Th1-like CD4 cells ex vivo (Figure 1L). In addition, the levels of pro-inﬂammatory cytokine 1L-17 in the supernatant of expanded PBMCs also positively correlated with the levels of expanded Th1-like CD4 cells ex vivo (Figure 1L). It was observed that the frequency of expanded Th1 like CD8+ important it is on the efﬁcacy of cancer treatments and responses. Therefore, we carried out statistical analysis between the ﬁve female patients and seven male patients to determine if there were any statistically signiﬁcant differences in any of the immunological parameters between the two groups which included the frequency of circulating immune cells, levels of 5-day expanded lymphocyte- secreted cytokines and immune checkpoint expression of proﬁles of the different T cell subsets (Figure S1). The two groups were comparable however, recognizing the heterogeneity of patient samples and the low number of numbers in both groups, there may be a statistically signiﬁcant difference if more patient samples were included. changes in the production of GrB in the secretome of expanded lymphocytes post-operatively compared with pre-operative levels (Figure 1D). The levels of IFN-g and GrB in the serum of whole blood was not signiﬁcantly altered post-operatively compared with pre-operative levels (Figures 1E, F). Quantiﬁcation of serum and lymphocyte supernatant immune proteins Although we observed a signiﬁcant increase in the circulating levels of IL-17 in the serum of whole blood post-operatively on POD 7 and POD 42 compared with pre-operative levels (Figure 1J), the frequency of expanded Th1/17 and Th17 cells remained unchanged as did the levels of IL- 17 secretions from expanded lymphocytes (Figures 1G–I). Interestingly, we identiﬁed a signiﬁcant increase in the levels of circulating IL-10 in the serum of whole blood in OGJ cancer patients post-operatively on POD 1, 7 and 42 highlighting that the immunosuppressive arm of the immune system was engaged post-oesophagectomy, which may be detrimental in the elaboration of anti-cancer Th1 immune responses and cancer immune surveillance, conversely promoting tumour recurrence and dissemination in the post-operative adjuvant setting (Figure 1M, 3.1 ± 0.5, 1.7 ± 0.1, 2.6 ± 1.2 vs. 0.1 ± 0.6%, p<0.001, p,0.001 and p=0.009). Induction of immunosuppression post-surgery was further underlined by a signiﬁcant increase in the frequency of expanded regulatory T cells (Tregs) by POD 7 (Figure 1K, 14.7 ± 4.0 vs. 5.1 ± 1.2%, p=0.04). We did not detect any differences in the levels of IL-10 in the supernatant of expanded PBMCs (Figure 1N). Furthermore, we observed that the frequency of expanded CD4+ and CD8+ Th1-like cells following 5-day T cell activation was signiﬁcantly correlated with a worse pathologic response to neoadjuvant treatment (r= -0.9, p=0.03). The presence of nodal metastasis also signiﬁcantly correlated with the frequency of expanded CD8+ Th1-like cells following 5-day T cell activation (r= -0.6, p=0.03, respectively). This might indicate that patients who responded poorly to treatment and had more advanced disease may have a dampened Th1 immune response as the expansion of Th1- like cells ex vivo was attenuated. The frequency of expanded T helper subsets was also correlated with the levels of T helper- associated cytokines in the supernatant from expanded PBMC cultures and in peripheral circulation of cancer patients (Figure 1L). Not surprisingly, the levels of Th1-associated cytokines such as GrB and IFN-g correlated with the frequency of expanded Th1-like CD4 cells ex vivo (Figure 1L). In addition, the levels of pro-inﬂammatory cytokine 1L-17 in the supernatant of expanded PBMCs also positively correlated with the levels of expanded Th1-like CD4 cells ex vivo (Figure 1L). It was observed that the frequency of expanded Th1-like CD8+ cells negatively correlated with nodal stage i.e. Quantiﬁcation of serum and lymphocyte supernatant immune proteins PBMCs were isolated from peripheral blood of cancer patients on POD 0 (n=12), POD 1 (n=12), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 (A). The frequency of expanded Th1 (CXCR3+CCR6-) (B), Th1/17 (CXCR3-CCR6-) (G) and Th17 (CXCR3+CCR6+) (H) CD3+CD4+ and CD3+CD8+ cells and Treg cells (CD4+FOXP3+) (K) was assessed by ﬂow cytometry. The soluble levels of IFN-g (C, E), GrB (D, F), IL-17 (I, J) and IL-10 (M, N) were measured in the serum from whole blood and in the supernatant from expanded PBMCs on POD 0, 1, 7 and 42. (L) shows the correlation values between cytokine production and the frequency of expanded lymphocytes following a 5-day expansion. (O) shows the signiﬁcant correlation values between T cell subset frequencies post-5-day expansion with clinical characteristics. (P) shows representative dot plots for the frequency of Th1, Th17 and Th1/17 cells. Data presented as mean ± SEM. Two-way ANOVA for (B–K) and (M, N). Spearman correlation used for non-parametric correlative analysis for (L, O). The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. 05 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. changes in the production of GrB in the secretome of expanded lymphocytes post-operatively compared with pre-operative levels (Figure 1D). The levels of IFN-g and GrB in the serum of whole blood was not signiﬁcantly altered post-operatively compared with pre-operative levels (Figures 1E, F). Although we observed a signiﬁcant increase in the circulating levels of IL-17 in the serum of whole blood post-operatively on POD 7 and POD 42 compared with pre-operative levels (Figure 1J), the frequency of expanded Th1/17 and Th17 cells remained unchanged as did the levels of IL- 17 secretions from expanded lymphocytes (Figures 1G–I). Interestingly, we identiﬁed a signiﬁcant increase in the levels of circulating IL-10 in the serum of whole blood in OGJ cancer patients post-operatively on POD 1, 7 and 42 highlighting that the immunosuppressive arm of the immune system was engaged post-oesophagectomy, which may be detrimental in the elaboration of anti-cancer Th1 immune responses and cancer immune surveillance, conversely promoting tumour recurrence and dissemination in the post-operative adjuvant setting (Figure 1M, 3.1 ± 0.5, 1.7 ± 0.1, 2.6 ± 1.2 vs. PD-L1 expression was signiﬁcantly increased on the surface of expanded Th1-like cells in the immediate postoperative setting Due to the reduction in expanded circulating Th1-like cells in the immediate post-operative period, we investigated whether there was a corresponding increase in the expression of inhibitory immune checkpoint proteins on the surface of Th1-like cells (Figure 2B), Th1/17-like (Figure 2C), Th17-like cells (Figure 2D) and Treg cells (Figure 2E) as outlined in the experimental schematic in Figure 2A. Expression of TIGIT on expanded circulating T cells did not signiﬁcantly change (Figure 2). Interestingly, the frequency of PD-L1 expression on expanded circulating CD4+CCR6-CXCR3+ Th1-like cells was signiﬁcantly increased on POD 7 compared to POD 1 (Figure 2F, 18.55 ± 5.8 vs. 7.4 ± 1.2%, p=0.04). The frequency of CTLA-4 expression on expanded circulating CD8+CCR6-CXCR3+ Th1/17-like cells was signiﬁcantly increased on POD 7 compared to POD 1 (Figure 2G, 42.4 ± 10.7 vs. 17.8 ± 5.7%, p=0.04). The frequency of PD-1 expression on expanded circulating Treg cells was signiﬁcantly increased on POD 7 compared to POD 1 (Figure 2I, 28.8 ± 6.5 vs. 19.4 ± 6.5%, p=0.04). Finally, the frequency of PD-1 expression on expanded circulating CD8+CCR6-CXCR3+ Th1/17-like cells was signiﬁcantly increased on POD 7 compared to POD 1 (Figure 2H, 26.2 ± 5.2 vs. 14.7 ± 4.2%, p=0.04). Moreover, the frequency of PD-L1+ Th1/17- like cells following a 5-day activation correlated with worse overall survival (Figure 2J). The correlation between the frequencies of expanded Th subsets and the expression of immune checkpoints on surface of Th1, Th1/17, Th17 and Treg cells was also carried out and depicted in Figures 2K–N. It was observed that the frequency of expanded Th1-like CD8+ cells negatively correlated with nodal stage i.e. patients who had higher levels of CD8+ Th1-like cells had a decreased nodal stage or fewer number of lymph nodes with metastasis. This suggests that in patients with more advanced stage disease CD8 anti-tumour immunity may be blunted (Figure 1O). Similarly, patients who had higher levels of expanded CD4+ or CD8+ Th1-like immune cells had an improved pathologic response to treatment with lower tumour regression scores (Figure 1O). Again underpinning the importance of Th1 immune responses in disease progression and treatment response.Several studies have highlighted major differences between female versus male immunity and how Frontiers in Immunology 06 frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. A B D E F G I H J K L M N C FIGURE 2 Expression of PD-L1 on the surface of expanded Th1-like cells was signiﬁcantly increased post-operatively. PD-L1 expression was signiﬁcantly increased on the surface of expanded Th1-like cells in the immediate postoperative setting PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti- CD3/28 and recombinant IL-2 (A). The immune checkpoint expression proﬁle of expanded Th1 (CXCR3+CCR6-) (B), Th1/17 (CXCR3-CCR6-) (C) and Th17 (CXCR3+CCR6+) (D) CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) (E) is displayed as a fold change in expression that is normalised to the levels of immune checkpoints on POD 0. The percentage of speciﬁc T cell subsets within the entire T cell population are shown and was determined by ﬂow cytometry. (F) Shows the percentage of Th1-like cells expressing PD-L1and, (G, H) highlight percentage of expanded Th1/17-like cells expressing CTLA-4 and PD-1, respectively. (I) Highlights the percentage of expanded Treg cells expressing PD-1. (K–N) Presents the correlation values between the frequency of expanded circulating CD4+ and CD8+ T helper subsets and their respective IC expression proﬁles for Th1-like cells, Th1/17-like cells, Th17-like cells and Treg cells. (J) Showcases the signiﬁcant correlations between the frequency of IC expression on T helper subsets with clinical parameters in cancer patients. Data presented as mean ± SEM. Two-way ANOVA analysis for F-I. Spearman correlation used for non-parametric correlative analysis for (J–N). The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. A B D E F G H K C A B A D E K D E G H K C D E B C K L M N H F K L I J I J N FIGURE 2 Expression of PD-L1 on the surface of expanded Th1-like cells was signiﬁcantly increased post-operatively. PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti- CD3/28 and recombinant IL-2 (A). The immune checkpoint expression proﬁle of expanded Th1 (CXCR3+CCR6-) (B), Th1/17 (CXCR3-CCR6-) (C) and Th17 (CXCR3+CCR6+) (D) CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) (E) is displayed as a fold change in expression that is normalised to the levels of immune checkpoints on POD 0. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients Our ﬁndings have identiﬁed an immunosuppressive window created by cancer surgery in the post-operative setting, which may likely contribute to the suppression of cancer immune surveillance and tumour dissemination and recurrence. Therefore, these results highlight the urgent clinical need to identify therapeutics that can counteract the propagation of immunosuppression mediated by surgery in the adjuvant setting. Subsequently, we explored the use of ICB namely, single agent anti-PD-1 and anti-CTLA-4 therapy to determine if these agents might enhance anti-tumour Th1 immunity post-operatively using our ex vivo PBMC expansion protocol (Figure 3A). As the levels of IFN-g in patients on POD 0 was the only mediator that signiﬁcantly correlated with a favourable response to ICB, we next set out to understand what is different between the soluble immune factors in systemic circulation between patients who have higher circulating levels of IFN-g versus patients with lower levels. This might contribute to an explanation as to why patients with higher IFN-g levels possess PBMCs that are predisposed to experiencing a nivolumab-induced increase in Th1 immunity versus those that don’t respond in this way. We examined the difference in the levels of 35 soluble immune proteins in 10 patients with high versus low IFN-g levels using the median level as a cut-off for high versus low. Of note only 4 soluble immune proteins were differentially expressed between patients with high versus low IFN-g levels. In the sub-cohort of patients with higher levels of IFN-g, there was a signiﬁcant difference in the levels of circulating soluble immune checkpoint proteins, speciﬁcally these patients had signiﬁcantly higher levels of soluble PD-L2 (Figure 3K, 2882 ± 170.6 vs. 2176 ± 121.4 pg/ml, p=0.008) and signiﬁcantly lower levels of soluble CTLA-4 (Figure 3L, 5.11 ± 1.1 vs. 8.4 ± 1.2 pg/ml, p=0.04), which might explain why these patients responded better to nivolumab over ipilimumab. Moreover, in the patients with higher circulating levels of IFN-g there was a signiﬁcantly higher level of soluble IL-12p40, an important Th1-like cytokine indicating that these patients may have an already primed and ongoing anti-tumour immune response (Figure 3M, 252.6 ± 32.2 vs. 163.9 ± 15.43 pg/ml, p=0.04). Lastly, patients with higher circulating levels of IFN-g also had a signiﬁcantly lower circulating level of MIP-3a (Figure 3N, 12.9 ± 4.8 vs. 23.1 ± 5.6 pg/ml, p=0.04). Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients Ipilimumab signiﬁcantly increased the frequency of expanded CD4+CCR6-CXCR3+ Th1-like cells compared with untreated cells on POD 7 (Figure 3B, 37.9 ± 7.6 vs. 30.4 ± 6.1%, p=0.03). In addition, nivolumab signiﬁcantly increased the frequency of expanded CD8+CCR6-CXCR3+ Th1-like cells compared to untreated cells on POD 7 (Figure 3C, 58.8 ± 7.3 vs. 51.2 ± 8.1%, p=0.04). Both single agent nivolumab and ipilimumab decreased the frequency of expanded Th17-like cells on POD 7 (Figures 3D, E). Furthermore, nivolumab and ipilimumab signiﬁcantly reduced the frequency of expanded Treg cells on POD 1 (Figure 3F, 3.3 ± 0.6, 3.2 ± 0.7% vs. 6.7 ± 1.5, p=0.04 and p=0.04). Additionally, we observed in a sub-cohort of 7/10 patients that single agent nivolumab signiﬁcantly increased the production of IFN-g by expanded PBMCs on POD 0, 1 and 7 (Figure 3G). Complementary ﬁndings demonstrated that GrB production was signiﬁcantly enhanced by single agent nivolumab on POD 0 in the same sub-cohort of 7/10 patients (Figure 3G). Furthermore, expanded PBMCs from 3 patients did not exhibit an increase in IFN-g or GrB levels (Figure 3H). Interestingly, in the sub-cohort of patients that experienced an enhancement in Th1 immunity in their nivolumab-expanded PBMCs a concomitant increase in IL-10 production was also noted on POD 0 and 1 in parallel, which may represent an immunosuppressive mechanism of resistance to nivolumab treatment (Figure 3I). In summary, only a sub-cohort of patients possessed PBMCs that experienced a nivolumab-mediated increase in Th1 immunity measured by an increase in IFN-g secretion by expanded PBMCs. These patients were enriched with higher levels of circulating IFN-g, which might indicate that these patients had an ongoing anti- tumour immune response that was amenable to nivolumab- mediated enhancement in Th1 immunity. In our study we also conducted correlative analysis between the magnitude of response to ICB (magnitude of response characterised by alteration in PBMC secretion of IFN-g in PBMC culture following 5 days of T cell activation) in expanded PBMCs with circulating serum proteins in cancer patients on POD 0, 1 and 7 to determine if there was a circulating factor that might indicate if the use of ICB could enhance Th1 immune responses in a patients PBMCs. PD-L1 expression was signiﬁcantly increased on the surface of expanded Th1-like cells in the immediate postoperative setting The percentage of speciﬁc T cell subsets within the entire T cell population are shown and was determined by ﬂow cytometry. (F) Shows the percentage of Th1-like cells expressing PD-L1and, (G, H) highlight percentage of expanded Th1/17-like cells expressing CTLA-4 and PD-1, respectively. (I) Highlights the percentage of expanded Treg cells expressing PD-1. (K–N) Presents the correlation values between the frequency of expanded circulating CD4+ and CD8+ T helper subsets and their respective IC expression proﬁles for Th1-like cells, Th1/17-like cells, Th17-like cells and Treg cells. (J) Showcases the signiﬁcant correlations between the frequency of IC expression on T helper subsets with clinical parameters in cancer patients. Data presented as mean ± SEM. Two-way ANOVA analysis for F-I. Spearman correlation used for non-parametric correlative analysis for (J–N). The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. 07 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. did not. We performed this analysis by correlating the fold-change in nivolumab- and ipilimumab-induced alterations in IFN-g, GrB and IL-10 produced by PBMCs with the levels of circulating IFN-g, GrB and IL-10 in the serum of whole blood in matched patients on POD 0, 1 and 7 (Figure 3J). On POD 0 there was a positive correlation between the nivolumab-induced increase in IFN-g production by PBMCs ex vivo with the circulating levels of IFN-g in the serum of whole blood in cancer patients on POD 0. This correlation suggests that patients who have higher levels of circulating IFN-g in their peripheral blood may possess a particular immune state that makes their PBMCs more amenable to nivolumab-mediated enhancement in Th1 immunity. Frontiers in Immunology Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients (A) Experimental schematic. PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ml). The frequency of expanded Th1 (CXCR3+CCR6-), Th17 (CXCR3-CCR6-) and Th1/17 (CXCR3+CCR6+) CD4+ and CD8+ ce and Treg cells (CD4+FOXP3+) was assessed by ﬂow cytometry. The levels of IFN-g, GrB and IL-10 were measured in the supernatant from expande PBMCs on POD 0, 1, 7 and 42. (B–F) Displays the effect of nivolumab and ipilimumab treatment on the frequency of expanded CD4+ and CD8+ CXCR3+CCR6- Th1-like cells on POD 7, the frequency of expanded CD4+ and CD8+ CXCR3+CCR6+ Th17-like cells on POD 7 and the frequency of Treg cells on POD 1. (G) Presents the sub-cohort of patients (7/10) whose PBMCs increased their IFN-g and GrB production following nivolumab treatment. (H) Presents the sub-cohort of patients whose PBMCs had no increase or showed a decrease in their IFN-g and GrB production followin nivolumab treatment. (I) Shows two sub-cohorts of patients whose PBMCs had an increase (7/10) or a decrease (3/10) in their IL-10 production following nivolumab treatment. (J) Correlation plot showing a signiﬁcant correlation between the effect of nivolumab on lymphocyte secretion of IFN-g following a 5-day T cell activation with the circulating levels of IFN-g in the serum of matched patients on POD 0. (K–N) Shows the soluble circulating factors that were signiﬁcantly altered in the serum of patients who had higher than median levels of circulating IFN-g compared with patients who had lower than median levels. All data presented as mean ± SEM. Two-way ANOVA. Correlative analysis: Spearman. The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients In consideration of the observed increase in IC expression and subsequent reduction in Th1-like cells following surgery, we postulated that the use of ICB might attenuate the global increased expression of IC proteins contributing to the enhancement in Th1 immune responses (Figure 4A). Overall, nivolumab treatment signiﬁcantly decreased PD-1 expression on the surface of T helper subsets as expected (Figures 4B–G). Surprisingly, nivolumab also decreased PD-L1 expression on the surface of Th1-like, Th1/17-like and Th17 like cells (Figures 4B–E). Given that ICB increased IFN-g, GrB and IL-10 in only a sub- cohort of patients we wondered if there might be circulating factors in patients’ serum that might correlate with this favourable response to ICB (favourable response to ICB: nivolumab/ipilimumab- mediated increase in GrB or IFN-g). Identifying any correlations between a favourable response to ICB ex vivo and circulating factors in patients’ serum might reveal a potential explanation behind why some patients’ PBMCs responded favourably to ICB whilst others 08 frontiersin.org Davern et al. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients 10.3389/ﬁmmu.2023.1150754 veh niv ipi 0 10 20 30 CD4+Foxp3+ cells POD 1 % CD4+Foxp3+cells * * veh niv ipi 0 20 40 60 80 CD4+Th1 cells POD 7 % CD4+CXCR3+CCR6- * veh niv ipi 0 20 40 60 80 100 CD8+Th1 cells POD 7 % CD8+CXCR3+CCR6- * veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 10000 20000 30000 40000 IFNy levels of IFNy (pg/ml) POD 0 POD 1 POD 7 POD 42 * * * veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 400 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 0 POD 1 POD 7 POD 42 * p=0.06 veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 10000 20000 30000 40000 IFNy levels of IFNy (pg/ml) POD 0 POD 1 POD 7 POD 42 veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 400 500 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 0 POD 1 POD 7 POD 42 Sub-cohort - ICB induced increase in Th1 immunity Sub-cohort - ICB had no effect/decreased Th1 immunity veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 50 100 150 200 250 IL-10 levels of IFNy POD 0 POD 1 POD 7 POD 42 * * veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 IL-10 levels of IFNy POD 0 POD 1 POD 7 POD 42 Dichotomous effect of ICB on IL-10 productoin veh niv ipi 0 2 4 6 8 10 CD8+Th17 cells POD 7 % CD8+CXCR3-CCR6+ * veh niv ipi 0 10 20 30 CD4+Th17 cells POD 7 % CD4+CXCR3-CCR6+ * * IFNy low IFNy high 0 20 40 60 MIP3α pg/ml ✱ IFNy low IFNy high 0 100 200 300 400 IL-12p40 pg/ml ✱ IFNy low IFNy high 0 5 10 15 CTLA-4 pg/ml ✱ IFNy low IFNy high 0 1000 2000 3000 4000 PD-L2 pg/ml ✱✱ 0 2 4 6 8 0 10 20 30 40 lymphocyte production of IFN-γ post-nivolumab (pg/ml) circulating serum levels of IFN-γ (pg/ml) r=0.9 p=0.05 A B D E F G I H J K L C Schematic of experimental setup: M N FIGURE 3 Single agent nivolumab abrogated the surgery-induced dampening of anti-tumour Th1 immune responses in a subset of patients. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients veh niv ipi 0 10 20 30 CD4+Foxp3+ cells POD 1 % CD4+Foxp3+cells * * veh niv ipi 0 20 40 60 80 CD4+Th1 cells POD 7 % CD4+CXCR3+CCR6- * veh niv ipi 0 20 40 60 80 100 CD8+Th1 cells POD 7 % CD8+CXCR3+CCR6- * veh niv ipi 0 2 4 6 8 10 CD8+Th17 cells POD 7 % CD8+CXCR3-CCR6+ * veh niv ipi 0 10 20 30 CD4+Th17 cells POD 7 % CD4+CXCR3-CCR6+ * * A B D E F C Schematic of experimental setup: Schematic of experimental setup: A B veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 10000 20000 30000 40000 IFNy levels of IFNy (pg/ml) POD 0 POD 1 POD 7 POD 42 * * * veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 400 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 0 POD 1 POD 7 POD 42 * p=0.06 Sub-cohort - ICB induced increase in Th1 immunity G 0 2 4 6 8 0 10 20 30 40 lymphocyte production of IFN-γ post-nivolumab (pg/ml) circulating serum levels of IFN-γ (pg/ml) r=0.9 p=0.05 J Sub-cohort - ICB induced increase in G veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 400 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 0 POD 1 POD 7 POD 42 * p=0.06 duced increase in munity J Th1 immunity IFNy low IFNy high 0 5 10 15 CTLA-4 pg/ml ✱ L IFNy low IFNy high 0 5 10 15 CTLA-4 pg/ml ✱ IFNy low IFNy high 0 1000 2000 3000 4000 PD-L2 pg/ml ✱✱ K L IFNy low IFNy high 0 1000 2000 3000 4000 PD-L2 pg/ml ✱✱ K L K veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 10000 20000 30000 40000 IFNy levels of IFNy (pg/ml) POD 0 POD 1 POD 7 POD 42 veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 400 500 2000 4000 6000 8000 GrB levels of GrB (pg/ml) POD 0 POD 1 POD 7 POD 42 Sub-cohort - ICB had no effect/decreased Th1 immunity H Sub-cohort - ICB had no effect/decreased Sub-cohort - ICB had no effect/decreased H Th1 immunity veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 50 100 150 200 250 IL-10 levels of IFNy POD 0 POD 1 POD 7 POD 42 * * veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 IL-10 levels of IFNy POD 0 POD 1 POD 7 POD 42 Dichotomous effect of ICB on IL-10 productoin I I Dichotomous effect of ICB on IL-10 productoin Dichotomous effect of ICB on IL-10 productoin IFNy low IFNy high 0 20 40 60 MIP3α pg/ml ✱ N IFNy low IFNy high 0 100 200 300 400 IL-12p40 pg/ml ✱ M veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 IL-10 levels of IFNy POD 0 POD 1 POD 7 POD 42 t of ICB on IL-10 toin iv ipi veh niv ipi veh niv ipi IL-10 D 1 POD 7 POD 42 veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 100 200 300 IL-10 levels of IFNy POD 0 POD 1 POD 7 POD 42 chotomous effect of ICB on IL-10 productoin N M MIP3α FIGURE 3 Single agent nivolumab abrogated the surgery-induced dampening of anti-tumour Th1 immune responses in a subset of patients. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients (A) Experimental schematic. PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ml). The frequency of expanded Th1 (CXCR3+CCR6-), Th17 (CXCR3-CCR6-) and Th1/17 (CXCR3+CCR6+) CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) was assessed by ﬂow cytometry. The levels of IFN-g, GrB and IL-10 were measured in the supernatant from expanded PBMCs on POD 0, 1, 7 and 42. (B–F) Displays the effect of nivolumab and ipilimumab treatment on the frequency of expanded CD4+ and CD8+ CXCR3+CCR6- Th1-like cells on POD 7, the frequency of expanded CD4+ and CD8+ CXCR3+CCR6+ Th17-like cells on POD 7 and the frequency of Treg cells on POD 1. (G) Presents the sub-cohort of patients (7/10) whose PBMCs increased their IFN-g and GrB production following nivolumab treatment. (H) Presents the sub-cohort of patients whose PBMCs had no increase or showed a decrease in their IFN-g and GrB production following nivolumab treatment. (I) Shows two sub-cohorts of patients whose PBMCs had an increase (7/10) or a decrease (3/10) in their IL-10 production following nivolumab treatment. (J) Correlation plot showing a signiﬁcant correlation between the effect of nivolumab on lymphocyte secretion of IFN-g following a 5-day T cell activation with the circulating levels of IFN-g in the serum of matched patients on POD 0. (K–N) Shows the soluble circulating factors that were signiﬁcantly altered in the serum of patients who had higher than median levels of circulating IFN-g compared with patients who had lower than median levels. All data presented as mean ± SEM. Two-way ANOVA. Correlative analysis: Spearman. The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. FIGURE 3 Single agent nivolumab abrogated the surgery-induced dampening of anti-tumour Th1 immune responses in a subset of patients. (A) Experimental schematic. FIGURE 3 Single agent nivolumab abrogated the surgery-induced dampening of anti-tumour Th1 immune responses in a subset of patients. (A) Experimental schematic. PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ml). The frequency of expanded Th1 (CXCR3+CCR6-), Th17 (CXCR3-CCR6-) and Th1/17 (CXCR3+CCR6+) CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) was assessed by ﬂow cytometry. The levels of IFN-g, GrB and IL-10 were measured in the supernatant from expanded PBMCs on POD 0, 1, 7 and 42. (B–F) Displays the effect of nivolumab and ipilimumab treatment on the frequency of expanded CD4+ and CD8+ CXCR3+CCR6- Th1-like cells on POD 7, the frequency of expanded CD4+ and CD8+ CXCR3+CCR6+ Th17-like cells on POD 7 and the frequency of Treg cells on POD 1. (G) Presents the sub-cohort of patients (7/10) whose PBMCs increased their IFN-g and GrB production following nivolumab treatment. (H) Presents the sub-cohort of patients whose PBMCs had no increase or showed a decrease in their IFN-g and GrB production following nivolumab treatment. (I) Shows two sub-cohorts of patients whose PBMCs had an increase (7/10) or a decrease (3/10) in their IL-10 production following nivolumab treatment. (J) Correlation plot showing a signiﬁcant correlation between the effect of nivolumab on lymphocyte secretion of IFN-g following a 5-day T cell activation with the circulating levels of IFN-g in the serum of matched patients on POD 0. (K–N) Shows the soluble circulating factors that were signiﬁcantly altered in the serum of patients who had higher than median levels of circulating IFN-g compared with patients who had lower than median levels. All data presented as mean ± SEM. Two-way ANOVA. Correlative analysis: Spearman. The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ml). The frequency of expanded Th1 (CXCR3+CCR6-), Th17 (CXCR3-CCR6-) and Th1/17 (CXCR3+CCR6+) CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) was assessed by ﬂow cytometry. The levels of IFN-g, GrB and IL-10 were measured in the supernatant from expanded PBMCs on POD 0, 1, 7 and 42. (B–F) Displays the effect of nivolumab and ipilimumab treatment on the frequency of expanded CD4+ and CD8+ CXCR3+CCR6- Th1-like cells on POD 7, the frequency of expanded CD4+ and CD8+ CXCR3+CCR6+ Th17-like cells on POD 7 and the frequency of Treg cells on POD 1. (G) Presents the sub-cohort of patients (7/10) whose PBMCs increased their IFN-g and GrB production following nivolumab treatment. (H) Presents the sub-cohort of patients whose PBMCs had no increase or showed a decrease in their IFN-g and GrB production following nivolumab treatment. (I) Shows two sub-cohorts of patients whose PBMCs had an increase (7/10) or a decrease (3/10) in their IL-10 production following nivolumab treatment. (J) Correlation plot showing a signiﬁcant correlation between the effect of nivolumab on lymphocyte secretion of IFN-g following a 5-day T cell activation with the circulating levels of IFN-g in the serum of matched patients on POD 0. (K–N) Shows the soluble circulating factors that were signiﬁcantly altered in the serum of patients who had higher than median levels of circulating IFN-g compared with patients who had lower than median levels. All data presented as mean ± SEM. Two-way ANOVA. Correlative analysis: Spearman. The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. 09 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients TH1 veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 20 40 60 80 0 1 7 42 CD4+ 0 1 7 42 CD8+ % CCR6-CXCR3+PD-1+cells * * veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT PD-1 PD-L1 CTLA-4 TIGIT veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT PD-1 PD-L1 CTLA-4 TIGIT CD4+ T cells CD8+ T cells POD 0 POD 1 POD 7 POD 42 1 2 3 4 veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT PD-1 PD-L1 CTLA-4 TIGIT CD4+ T cells CD8+ T cells POD 0 POD 1 POD 7 POD 42 1 2 3 4 Th1 cells fold change Th1/17 cells fold change Th17 cells fold change Treg cells fold change TH1 veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 20 40 60 80 % CCR6-CXCR3+CTLA-4+cells * * 0 1 7 42 CD4+ 0 1 7 42 CD8+ veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT CD4+ T cells POD 0 POD 1 POD 7 POD 42 1 2 3 4 A B D E F G C Schematic of experimental setup FIGURE 4 Single agent nivolumab and ipilimumab treatment signiﬁcantly decreased the expression of PD-1 and CTLA-4 on the surface of expanded Th1-like cells ex vivo. PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ ml) (A). The IC expression proﬁle of expanded Th1 (CXCR3+CCR6-) (B), Th1/17 (CXCR3+CCR6+) (C), Th17 (CXCR3-CCR6-) (D) and CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) (E) was assessed by ﬂow cytometry. The percentage of T cell subsets positive for an immune checkpoint is summarized in a heat map format. (F, G) Display the effects of ICB on the expression of PD-1 and CTLA-4 on the surface of expanded PBMCs ex vivo. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients (B–E) In each heat map a value greater than 1 demonstrates ICB increased the percentage of cells positive for an immune checkpoint compared with the effect of the vehicle on that T cell subset in a particular POD timepoint sample be it POD 0, 1, 7 or 42. Similarly, a value less than 1 indicates a decrease and a value equal to 1 indicates no effect was observed. All data presented as mean ± SEM. Two-way ANOVA analysis for (F, G). p<0.05, p<0.01, p<0.001. A Schematic of experimental setup Schematic of experimental setup A A veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT PD-1 PD-L1 CTLA-4 TIGIT CD4+ T cells CD8+ T cells POD 0 POD 1 POD 7 POD 42 1 2 3 4 Th1/17 cells fold change C TH1 veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT PD-1 PD-L1 CTLA-4 TIGIT CD4+ T cells CD8+ T cells POD 0 POD 1 POD 7 POD 42 1 2 3 4 Th17 cells fold change D veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT PD-1 PD-L1 CTLA-4 TIGIT Th1 cells fold change B D B C B Treg cells fold change veh niv ipi veh niv ipi veh niv ipi veh niv ipi PD-1 PD-L1 CTLA-4 TIGIT CD4+ T cells POD 0 POD 1 POD 7 POD 42 1 2 3 4 E TH1 veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 20 40 60 80 0 1 7 42 CD4+ 0 1 7 42 CD8+ % CCR6-CXCR3+PD-1+cells * * ** H1 F F F E fold change CD4 CD8 TH1 veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi veh niv ipi 0 20 40 60 80 % CCR6-CXCR3+CTLA-4+cells * * 0 1 7 42 CD4+ 0 1 7 42 CD8+ G G FIGURE 4 Single agent nivolumab and ipilimumab treatment signiﬁcantly decreased the expression of PD-1 and CTLA-4 on the surface of expanded Th1-like cells ex vivo. Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients PBMCs were isolated from peripheral blood of Cancer patients on POD 0 (n=11), POD 1 (n=11), POD 7 (n=11) and POD 42 (n=4) and activated ex vivo for 5 days using plate bound anti-CD3/28 and recombinant IL-2 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ ml) (A). The IC expression proﬁle of expanded Th1 (CXCR3+CCR6-) (B), Th1/17 (CXCR3+CCR6+) (C), Th17 (CXCR3-CCR6-) (D) and CD4+ and CD8+ cells and Treg cells (CD4+FOXP3+) (E) was assessed by ﬂow cytometry. The percentage of T cell subsets positive for an immune checkpoint is summarized in a heat map format. (F, G) Display the effects of ICB on the expression of PD-1 and CTLA-4 on the surface of expanded PBMCs ex vivo. (B–E) In each heat map a value greater than 1 demonstrates ICB increased the percentage of cells positive for an immune checkpoint compared with the effect of the vehicle on that T cell subset in a particular POD timepoint sample be it POD 0, 1, 7 or 42. Similarly, a value less than 1 indicates a decrease and a value equal to 1 indicates no effect was observed. All data presented as mean ± SEM. Two-way ANOVA analysis for (F, G). p<0.05, p<0.01, *p<0.001. 10 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. cytotoxicity of OE33 P cells was signiﬁcantly reduced on POD 1 compared to POD 0 (Figure 5B, 80.44 ± 15.8 vs. 58.25 ± 11.4% p=0.04). Similarly, lymphocyte-mediated cytotoxicity of OE33 R cells was signiﬁcantly reduced on POD 7 compared to POD 0 (Figure 5B, 105.7 ± 14.8 vs. 67.27 ± 9.5%, p=0.03). Overall, cancer surgery signiﬁcantly impaired the cytotoxic ability of expanded circulating lymphocytes by POD 7. Moreover, CTLA-4 blockade signiﬁcantly decreased CTLA-4 on the surface of T helper subsets but also surprisingly decreased the expression of PD-1 on the surface of Th-1-like, Th1/17-like and Th17-like cells (Figures 4B–G). Overall, treatment with ICB reduced the expression of IC proteins on Th1-like cells, which may be a contributing factor in the unleashing of anti-tumour Th1 immunity following ICB. Interestingly, lymphocyte-mediated killing of OE33 P cells was negatively correlated with the frequency of PD-1+CD8+ T cells suggesting that the PD-1 axis may be hindering the ability of expanded lymphocytes to kill tumour cells (Figure 5C). Single agent nivolumab abrogated the surgery-induced dampening of Th1 anti-tumour immune responses in a subset of patients Additionally, lymphocyte-mediated killing of OE33 R cells was negatively correlated with the frequency of CTLA-4+CD8+ T cells suggesting that the CTLA-4 axis may be hindering the ability of expanded lymphocytes to kill tumour cells as well (Figure 5C). This prompted us to next investigate if the addition of anti-PD-1 ICB or anti-CTLA-4 ICB might attenuate the surgery-mediated suppression in lymphocyte killing of OE33 P and OE33 R tumour cells (Figure 6A). The cytotoxic ability of expanded lymphocytes is impaired in the immediate postoperative setting, an effect which is attenuated by ICB The cytotoxic ability of expanded lymphocytes is impaired in the immediate postoperative setting, an effect which is attenuated by ICB Given the immunosuppressive milieu in the immediate postoperative setting, we investigated whether this would reduce the cytotoxic ability of expanded circulating lymphocytes to kill radiosensitive and radioresistant OGJ tumour cells (OE33 P and OE33 R isogenic cell line) (Figure 5A). Lymphocyte-mediated POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 100 200 300 400 * OE33 P (radiosensitive tumour cells) % viability (control) OE33 R (radioresistant tumour cells) A B Research Question: Does oncologic surgery affect the cytotoxic ability of lymphocytes? Clinical Significance: If surgery abrogates the cytotoxic ability of lymphocytes this might contribute to immune evasion, tumour recurrence and metastatic disemination in the post-operative period. C Experimental Schematic OE33 P killing OE33 R killing Th1 CD8+CTLA-4+ Th1 CD8+PD-1+ OE33 P killing OE33 R killing * * -1.0 -0.5 0 0.5 1.0 FIGURE 5 Lymphocyte-mediated killing of OE33 P and OE33 R cells is impaired in the immediate post-operative setting. (A) Experimental schematic. (B) OE33 P and OE33 R cells were co-cultured with PBMCs isolated from Cancer patients undergoing surgical tumour resection on POD 0 (n=7), 1 (n=10), 7 (n=10) and week 6 (n = 4) in an effector:target ratio (E:T) of 10:1 1 x 105:1 x 104for 48h. PBMCs were pre-activated using plate bound anti-CD3/28 and IL-2 for 5 days. A CCK8 assay was used to determine the viability of OE33 P and OE33 R cells. (C) Presents a correlation matrix between lymphocyte-mediated killing of OE33 P and OE33 R cells and the expression of PD-1 and CTLA-4 on the surface of expanded circulating CD8+ T cells that were present in the tumour cell:PBMC co-culture. A value greater than 0 (red) is a positive correlation and a value below zero (blue) is a negative correlation. Spearman correlation used for non-parametric correlative analysis. The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. Data were analysed using a two-way ANOVA and presented as percentages ± SEM. A Research Question: Does oncologic surgery affect the cytotoxic ability of lymphocytes? Clinical Significance: If surgery abrogates the cytotoxic ability of lymphocytes this might contribute to immune evasion, tumour recurrence and metastatic disemination in the post-operative period. Experimental Schematic Experimental Schematic Experimental Schematic POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 100 200 300 400 * * OE33 P (radiosensitive tumour cells) % viability (control) OE33 R (radioresistant tumour cells) B C OE33 P killing OE33 R killing Th1 CD8+CTLA-4+ Th1 CD8+PD-1+ OE33 P killing OE33 R killing * * -1.0 -0.5 0 0.5 1.0 FIGURE 5 POD 0 POD 1 POD 7 POD 42 POD 0 POD 1 POD 7 POD 42 0 100 200 300 400 * * OE33 P (radiosensitive tumour cells) % viability (control) OE33 R (radioresistant tumour cells) B C FIGURE 5 C OE33 P killing OE33 R killing Th1 CD8+CTLA-4+ Th1 CD8+PD-1+ OE33 P killing OE33 R killing * * -1.0 -0.5 0 0.5 1.0 C B OE33 P (radiosensitive tumour cells) OE33 R (radioresistant tumour cells) OE33 R (radioresistant tumour cells) FIGURE 5 Lymphocyte-mediated killing of OE33 P and OE33 R cells is impaired in the immediate post-operative setting. (A) Experimental schematic. (B) OE33 P and OE33 R cells were co-cultured with PBMCs isolated from Cancer patients undergoing surgical tumour resection on POD 0 (n=7), 1 (n=10), 7 (n=10) and week 6 (n = 4) in an effector:target ratio (E:T) of 10:1 1 x 105:1 x 104for 48h. PBMCs were pre-activated using plate bound anti-CD3/28 and IL-2 for 5 days. A CCK8 assay was used to determine the viability of OE33 P and OE33 R cells. (C) Presents a correlation matrix between lymphocyte-mediated killing of OE33 P and OE33 R cells and the expression of PD-1 and CTLA-4 on the surface of expanded circulating CD8+ T cells that were present in the tumour cell:PBMC co-culture. A value greater than 0 (red) is a positive correlation and a value below zero (blue) is a negative correlation. Spearman correlation used for non-parametric correlative analysis. The r2 value indicates the correlation value. A red square indicates a positive correlation i.e. R2>0. A blue square indicates a negative correlation i.e. R2<0. An asterisk depicts the signiﬁcant correlations i.e. r2 value has a p=0.05. Data were analysed using a two-way ANOVA and presented as percentages ± SEM. Frontiers in Immunology 11 frontiersin.org Davern et al. Davern et al. A Research Question: Does oncologic surgery affect the cytotoxic ability of lymphocytes? 10.3389/ﬁmmu.2023.1150754 Nivolumab signiﬁcantly enhanced the killing of OE33 R cells compared with untreated cells on POD 0 (Figure 6B, 57.9 ± 15.9 vs. 71.43 ± 15.7%, p=0.04) and POD 1 (Figure 6C, 63.5 12.9 vs. 73.52 8.9%, p=0.03. Nivolumab also signiﬁcantly enhanced the killing of OE33 P cells on POD 1 (Figure 6C, 63.5 ± 12.9 vs. 73.5 ± 8.9%, p=0.04) and POD 7 (Figure 6D, 71.1 ± 9.1 vs. 79.6 ± 10.6%, p=0.04). Ipilimumab signiﬁcantly enhanced the killing of OE33 P cells compared with untreated cells on POD 0 (Figure 6B, 41.4 ± 9.6 vs. 71.6 ± 9.7%, p=0.02), POD 1 (Figure 6C, 60.7 ± 9.2 vs. 73.5 ± 8.9%, p=0.04) and POD 7 (Figure 6D, 56.1 ± 12.1 vs. 79.7 ± 10.6%, p=0.04). Likewise, Ipilimumab signiﬁcantly enhanced the killing of OE33 R cells compared with untreated cells on POD 0 (Figure 6B, 51.4 ± 14.3 vs. 71.43 ± 15.7%, p=0.03) and POD 1 (Figure 6C, 60.7 ± 9.2 vs. 73.5 ± 8.9%, p=0.04). ICB did not signiﬁcantly improve the ability of lymphocytes isolated from patients 6 weeks post-surgery in killing OE33 P or OE33 R tumour cells (Figure 6E). Overall, the use of ICB abrogated the surgery-induced reduction in the cytotoxic ability of expanded PBMCs against radiosensitive and radioresistant OGJ tumour cells ex vivo. Discussion Considering the systemic tumour-promoting environment in the body created during the post-operative period previously The ﬁndings from our study supported the previously reported immunosuppressive effects of tumour resection (5).Firstly, we identiﬁed that an immunosuppressive phenotype prevailed in the immediate postoperative setting. This was characterised by a reduction in the frequency of expanded circulating Th1-like cells post-operatively with a subsequent decrease in IFN-g production. Donlon et al, recently showed that systemic Th1 immune responses were dampened in the post-operative setting illustrated by a decrease in the circulating levels of Th1 cytokines such as IFN-g and IL-12p40 (5). g y g p ( ) Furthermore, our results suggested a potential role for ICs in propagating these observed immunosuppressive effects, as we noted an increase in the expression of PD-L1 on the surface of expanded circulating Th1-like immune cells ex vivo one-week post-surgery. Previously, the immune checkpoint expression proﬁle was evaluated on circulating T cells immediately post-surgery and identiﬁed that TIM-3 was upregulated on T cells immediately post-surgery before returning to baseline by week 6 (5). In contrast, PD-L1 immune checkpoint expression was not upregulated on circulating T cells immediately post-surgery and was indeed found to be downregulated on circulating T cells by week 6 post-surgery (5). However, this study did not proﬁle immune checkpoint expression on T helper subsets, which may explain the discrepancies (5). In summary, these results together might indicate that the activation of Th1 immunity post-surgery might be inhibited by upregulation of PD-L1 immune checkpoint suggesting that blockade of the PD-1-PD-L1 axis may enhance Th1 immune responses post-surgery for this patient cohort. Moreover, the results from this study revealed that the cytotoxic ability of PBMCs was abrogated in the immediate post-operative setting by day 7. The reduced killing ability of PBMCs against radiosensitive and radioresistant tumour cells was observed, strengthening the hypothesis that tumour resection dampens Th1 immunity and anti- cancer cytotoxic responses. This provided us with further rationale to test our hypothesis that targeting the PD-1/PD-L1 pathway might be useful in abrogating the observed surgery- induced immunosuppression. In our study, we also found that several immune checkpoints including PD-1, PD-L1 and CTLA-4 were expressed at signiﬁcantly higher levels on the surface of 5-day expanded post-operative Th1 and Th1/17 cells compared with 5-day expanded pre-operative T cells. Discussion This might suggest that post-operative T cells may be more prone to T cell exhaustion, creating a rationale for administering ICB in the post-operative setting as the target of ICB would be more highly expressed. Additionally, this potentially implicates the PD-1/ PD-L1 pathway as a mediator of the decrease in anti-tumour Th1 cells and reduced cytotoxic ability of expanded circulating lymphocytes. Complementary ﬁndings were identiﬁed in a lung cancer study, where PD-1 expression was upregulated on T cells following surgical resection (9). Th1/17 immune cells have potent anti-tumour properties which combine the cancer-ﬁghting properties of Th1 cells and the ability of Th17 cells to self-renew and regenerate (15). In our study, single agent ipilimumab signiﬁcantly decreased PD-1 expression on the surface of Th1/17 cells. The reduction in PD-1 expression may prevent inhibition or exhaustion of these potent anti-tumour cells. The landmark CheckMate 577 phase III trial found that adjuvant nivolumab signiﬁcantly improved patient outcomes, doubling disease-free survival following surgical resection of oesophageal or gastroesophageal cancers compared to placebo in patients who had received neoadjuvant chemoradiotherapy (6). Additionally, the adverse effect proﬁle was favourable (6). Our ﬁndings complement those of that trial, providing a mechanistic rationale for the beneﬁcial effects reported in CheckMate 577, namely by identifying postoperative immunosuppression and reduced anti-tumour cytotoxic ability of lymphocytes, which is abrogated by the addition of ICB. We found that nivolumab had signiﬁcantly attenuated the depletion of Th1 cells and enhanced the cytotoxic ability of expanded circulating PBMCs in the immediate postoperative setting. A reduction in the cytotoxic ability of lymphocytes in the immediate post-operative setting may likely facilitate immune evasion of tumour cells, formation of micrometastatic deposits and tumour recurrence. Our study shows that use of ICB can enhance lymphocyte-mediated killing of tumour cells both radiosensitive and radioresistant tumour cells, which is critical given the high level of radiation resistance in oesophageal (16) and gastric cancer patients (17). Considering the systemic tumour-promoting environment in the body created during the post-operative period previously characterised by an increase in pro-survival tumour factors, pro- angiogenic and pro-inﬂammatory mediators, the ability to prevent or abrogate the suppression of anti-tumour immunity would be a useful tool in promoting cancer immune surveillance, preventing the growth of micrometastatic disease and reduce the risk of cancer recurrence (5, 7–9). Discussion Surgery creates a unique immune environment with immunosuppressive, pro-inﬂammatory, pro-angiogenic and pro- proliferative effects (7–9). Combining immunotherapy with surgery A B D E C FIGURE 6 Nivolumab and ipilimumab enhanced lymphocyte-mediated killing of OE33 P and OE33 R cells in the immediate post-operative setting. (A) Experimental schematic. OE33 P and OE33 R cells were co-cultured with PBMCs isolated from cancer patients on POD 0 (n = 11) (B), 1 (n = 11) (C), 7 (n = 11) (D) and week 6 (n = 4) (E) in an E:T ratio of 10:1 (100,000:10,000) for 48h. PBMCs were pre-activated using plate bound anti-CD3/28 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ml) for 5 days. A CCK8 assay was used to determine the viability of OE33P and OE33R cells. Two-way ANOVA statistical analysis for (B–E). p<0.05, p<0.01. B C C B D E E D E D FIGURE 6 Nivolumab and ipilimumab enhanced lymphocyte-mediated killing of OE33 P and OE33 R cells in the immediate post-operative setting. (A) Experimental schematic. OE33 P and OE33 R cells were co-cultured with PBMCs isolated from cancer patients on POD 0 (n = 11) (B), 1 (n = 11) (C), 7 (n = 11) (D) and week 6 (n = 4) (E) in an E:T ratio of 10:1 (100,000:10,000) for 48h. PBMCs were pre-activated using plate bound anti-CD3/28 in the presence or absence of nivolumab (niv) (10 µg/ml) or ipilimumab (ipi) (10 µg/ml) for 5 days. A CCK8 assay was used to determine the viability of OE33P and OE33R cells. Two-way ANOVA statistical analysis for (B–E). p<0.05, **p<0.01. 12 12 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1150754 Davern et al. continued to increase 6 weeks post-surgery. Prior studies have also identiﬁed an increase in immunosuppressive cytokines post- operatively in other cancer types (5). Treg cells are a key producer of this immunosuppressive cytokine which function to inhibit Th1 immune responses (12). Activation of the PD-1/PD-L1 pathway in Th1 cells mediates their conversion into Treg cells via reduced STAT activation (13). Our ﬁndings, identifying an increase in PD-L1 expression on Th1 cells in parallel with an increase in the circulating levels of IL-10 in the immediate postoperative may suggest that the PD-1 axis is a mechanism for decreasing Th1 cells and increasing Treg cells post-operatively. Shigematsu et al (14), demonstrated that Treg cells suppressed cytotoxic lymphocyte mediated-killing of tumour cells. Frontiers in Immunology Discussion The authors observed that co-culturing Treg cells with cytotoxic lymphocytes and tumor cells decreased tumor cell killing and depletion of Treg cells in the co-culture system enhanced cytotoxic lymphocyte mediated-tumor cell killing (14). to turn an immunosuppressive effect into a therapeutic opportunity is an attractive and rational strategy. In the case of cancer resection, this creates an ideal environment for the promotion of micrometastatic residual disease, pro-tumorigenesis and tumour recurrence (7, 8). recurrence (7, 8). The ﬁndings from our study supported the previously reported immunosuppressive effects of tumour resection (5).Firstly, we identiﬁed that an immunosuppressive phenotype prevailed in the immediate postoperative setting. This was characterised by a reduction in the frequency of expanded circulating Th1-like cells post-operatively with a subsequent decrease in IFN-g production. Donlon et al, recently showed that systemic Th1 immune responses were dampened in the post-operative setting illustrated by a decrease in the circulating levels of Th1 cytokines such as IFN-g and IL-12p40 (5). Furthermore, our results suggested a potential role for ICs in propagating these observed immunosuppressive effects, as we noted an increase in the expression of PD-L1 on the surface of expanded circulating Th1-like immune cells ex vivo one-week post-surgery. Previously, the immune checkpoint expression proﬁle was evaluated on circulating T cells immediately post-surgery and identiﬁed that TIM-3 was upregulated on T cells immediately post-surgery before returning to baseline by week 6 (5). In contrast, PD-L1 immune checkpoint expression was not upregulated on circulating T cells immediately post-surgery and was indeed found to be downregulated on circulating T cells by week 6 post-surgery (5). However, this study did not proﬁle immune checkpoint expression on T helper subsets, which may explain the discrepancies (5). In summary, these results together might indicate that the activation of Th1 immunity post-surgery might be inhibited by upregulation of PD-L1 immune checkpoint suggesting that blockade of the PD-1-PD-L1 axis may enhance Th1 immune responses post-surgery for this patient cohort. Moreover, the results from this study revealed that the cytotoxic ability of PBMCs was abrogated in the immediate post-operative setting by day 7. The reduced killing ability of PBMCs against radiosensitive and radioresistant tumour cells was observed, strengthening the hypothesis that tumour resection dampens Th1 immunity and anti- cancer cytotoxic responses. This provided us with further rationale to test our hypothesis that targeting the PD-1/PD-L1 pathway might be useful in abrogating the observed surgery- induced immunosuppression. Discussion Th1 cells play a pivotal role in performing cancer immune surveillance and mediating anti-tumour immunity to ﬁrstly prevent tumour formation and secondly eradicate tumours (10). Key Th1 mediators include IFN-g and GrB which are essential components in the direct killing of tumour cells and inducing tumour cell apoptosis (11). Furthermore, our ﬁndings identiﬁed a concomitant increase in circulating levels of IL-10 in cancer patients post-operatively, which Frontiers in Immunology frontiersin.org 13 Davern et al. Davern et al. 10.3389/ﬁmmu.2023.1150754 Of note we also observed that anti-PD-1 therapy enhanced anti- tumour Th1-like immunity prior to surgery. The beneﬁcial effect of anti-PD-1 therapy in the neoadjuvant setting complements and supports the favourable ﬁndings from clinical trials that led to the FDA approval of nivolumab in combination with chemotherapy in the neoadjuvant setting in oesophagogastric junctional carcinomas providing supporting data for its efﬁcacy. Our goal was to ask the question if ICB might also be advantageous in the adjuvant setting for promoting anti-tumour Th1-like immunity and abrogate the immunosuppressive effects surgery has on anti-tumour Th1 responses which we found it to be the case. immunosuppressive window created by surgery in the post- operative setting and the potential for ICB to harness the surgery- induced wound healing response that tends to be tumour-promoting and skew it towards a Th1-mediated immune response to prevent cancer recurrence and improve survival outcomes. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Our ﬁndings support existing literature in the creation of a tumour-promoting, immunosuppressive environment within the body following major OGJ oncologic surgery, including a depletion of circulating Th1 cells, reduced anti-cancer cytotoxic ability of lymphocytes and upregulation of immune checkpoint molecules (19). Additionally, we found that the use of ICB within the post- operative setting attenuated the surgery-mediated reduction in Th1 cytotoxic immunity. Given the poor prognosis associated with OGJ cancers, and the recent beneﬁcial effects identiﬁed using ICB, the immediate post-operative period represents a viable therapeutic window of opportunity. This study underlines the Author contributions Acknowledging that the numbers are too low to draw any conclusions, we noted an interesting observation in the three patient-derived PBMC samples that did not respond to nivolumab, characterised by the absence of a nivolumab-mediated increase in IFN-g production by PBMCs ex vivo. Responders were characterised by an increase in IFN-g production by PBMCs ex vivo. Non- responders had an overall higher expression of CTLA-4 on CD4+Th1-like cells compared with responders to nivolumab on POD 0 (14.0 vs 4.0%) (data not shown). Additionally, in all three patients that did not respond to nivolumab the expression of CTLA-4 on CD4+ Th1 cells was increased after nivolumab treatment on POD 0 (14.0 and 23.0%, n=3), an effect that was not observed in patients who responded to nivolumab (4.0 vs. 3.0%, n=7) (data not shown). This may suggest that nivolumab-mediated upregulation of CTLA-4 may be one mechanism inhibiting the nivolumab-mediated increase in IFN-g production by PBMCs ex vivo. This supports a rationale for dual blockade with nivolumab and ipilimumab to potentially help overcome these inhibitory effects on IFN-g production by PBMCs. In a previous study published by our lab, we reported that dual nivolumab-ipilimumab treatment increased production of IFN-g more substantially than single agent alone in oesophageal adenocarcinoma patient-derived T cells ex vivo (18). MD and CG contributed equally to this work and share ﬁrst authorship. MD, CG, and ND wrote the manuscript. MD and ND conceptualized the study. MD, CG, FO’C, BM, EM, AS, ND, JP, JL, SGM, NLL, MGD and JY-T carried out experiments and data analysis. FO’C, EM, SR, ER, MGD, NLL, SGM, CB, LQ, CH, ET, EP, WB, JL, NR, CD, JR contributed to sample acquisition. All authors contributed to the article and approved the submitted version. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Data availability statement The original contributions presented in the study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author. An interesting caveat we observed in this study was that ICB only increased IFN-g and granzyme B production in expanded PBMCs in only a subset of patients. This ﬁnding is reminiscent of clinical response rates in that only a subset of patients will respond to ICB. Correlative analysis between ICB-mediated IFN-g and GrB production by expanded PBMCs with immune factors in patients’ serum uncovered that patient’s with a higher level of circulating IFN-g on POD 0 correlated positively with increased production of IFN-g and GrB by PBMCs on POD 0. This observation suggests that these patients may have been pre-poised to have a better response to ICB perhaps because they had a higher level of immune activation against the tumour indicated by increased IFN-g levels in the blood. Ethics statement The studies involving human participants were reviewed and approved by Tallaght University Hospital/St James Hospital. The patients/participants provided their written informed consent to participate in this study. Frontiers in Immunology Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2023.1150754/ full#supplementary-material Frontiers in Immunology frontiersin.org 14 Davern et al. 10.3389/ﬁmmu.2023.1150754 10.3389/ﬁmmu.2023.1150754 Davern et al. References 10. Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T. Regulatory T cells: how do they suppress immune responses? Int Immunol (2009) 21:1105–11. doi: 10.1093/intimm/dxp095 1. Donlon NE, Power R, Hayes C, Reynolds J. v., Lysaght J. Radiotherapy, immunotherapy, and the tumour microenvironment: turning an immunosuppressive milieu into a therapeutic opportunity. Cancer Lett (2021) 502:84–96. doi: 10.1016/ j.canlet.2020.12.045 1. Donlon NE, Power R, Hayes C, Reynolds J. v., Lysaght J. Radiotherapy, immunotherapy, and the tumour microenvironment: turning an immunosuppressive milieu into a therapeutic opportunity. Cancer Lett (2021) 502:84–96. doi: 10.1016/ j.canlet.2020.12.045 10. Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T. Regulatory T cells: how do they suppress immune responses? Int Immunol (2009) 21:1105–11. doi: 10.1093/intimm/dxp095 11. Cai J, Wang D, Zhang G, Guo X. The role of PD-1/PD-L1 axis in treg development and function: implications for cancer immunotherapy. Onco Targets Ther (2019) 12:8437–45. doi: 10.2147/OTT.S221340 2. Bakos O, Lawson C, Rouleau S, Tai LH. Combining surgery and immunotherapy: turning an immunosuppressive effect into a therapeutic opportunity. J Immunother Cancer (2018) 6. doi: 10.1186/s40425-018-0398-7 2. Bakos O, Lawson C, Rouleau S, Tai LH. Combining surgery and immunotherapy: turning an immunosuppressive effect into a therapeutic opportunity. J Immunother Cancer (2018) 6. doi: 10.1186/s40425-018-0398-7 12. 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https://openalex.org/W2173736706	https://repository.ubn.ru.nl//bitstream/handle/2066/149113/149113.pdf	English	null	Psychometric properties of the Dutch version of the Evidence-Based Practice Attitude Scale (EBPAS)	Health research policy and systems	2,015	cc-by	9,760	Psychometric properties of the Dutch version of the Evidence-Based Practice Attitude Scale (EBPAS) Sonsbeek, A.M.S. van; Hutschemaekers, G.J.M.; Veerman, J.W.; Kleinjan, M.; Aarons, G.A.; Tiemens, B.G. 2015, Article / Letter to editor (Health Research Policy and Systems, 13, (2015), article 69) Doi link to publisher: https://doi.org/10.1186/s12961-015-0058-z 2015, Article / Letter to editor (Health Research Policy and Systems, 13, (2015), article 69) Doi link to publisher: https://doi.org/10.1186/s12961-015-0058-z 2015, Article / Letter to editor (Health Research Policy and Systems, 13, (2015), article 69) Doi link to publisher: https://doi.org/10.1186/s12961-015-0058-z Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/149113 Download date: 2024-10-24 Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 DOI 10.1186/s12961-015-0058-z Open Access Psychometric properties of the Dutch version of the Evidence-Based Practice Attitude Scale (EBPAS) Maartje A. M. S. van Sonsbeek1,2, Giel J. M. Hutschemaekers1,2, Jan W. Veerman1, Marloes Kleinjan3, Gregory A. Aarons4,5 and Bea G. Tiemens1,2 Abstract Background: The Evidence-Based Practice Attitude Scale (EBPAS) was developed in the United States to assess attitudes of mental health and welfare professionals toward evidence-based interventions. Although the EBPAS has been translated in different languages and is being used in several countries, all research on the psychometric properties of the EBPAS within youth care has been carried out in the United States. The purpose of this study was to investigate the psychometric properties of the Dutch version of the EBPAS. Methods: After translation into Dutch, the Dutch version of the EBPAS was examined in a diverse sample of 270 youth care professionals working in five institutions in the Netherlands. We examined the factor structure with both exploratory and confirmatory factor analyses and the internal consistency reliability. We also conducted multiple linear regression analyses to examine the association of EBPAS scores with professionals’ characteristics. It was hypothesized that responses to the EBPAS items could be explained by one general factor plus four specific factors, good to excellent internal consistency reliability would be found, and EBPAS scores would vary by age, sex, and educational level. Results: The exploratory factor analysis suggested a four-factor solution according to the hypothesized dimensions: Requirements, Appeal, Openness, and Divergence. Cronbach’s alphas ranged from 0.67 to 0.89, and the overall scale alpha was 0.72. The confirmatory factor analyses confirmed the factor structure and suggested that the lower order EBPAS factors are indicators of a higher order construct. However, Divergence was not significantly correlated with any of the subscales or the total score. The confirmatory bifactor analysis endorsed that variance was explained both by a general attitude towards evidence-based interventions and by four specific factors. The regression analyses showed an association between EBPAS scores and youth care professionals’ age, sex, and educational level. Conclusions: The present study provides strong support for a structure with a general factor plus four specific factors and internal consistency reliability of the Dutch version of the EBPAS in a diverse sample of youth care professionals. Hence, the factor structure and reliability of the original version of the EBPAS seem generalizable to the Dutch version of the EBPAS. © 2015 van Sonsbeek et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: m.van.sonsbeek@propersona.nl 1Behavioural Science Institute, Radboud University, Montessorilaan 3, 6525 HR Nijmegen, The Netherlands 2Pro Persona Centre for Education & Science (ProCES), Postbus 27, 6870 AA Renkum, The Netherlands Full list of author information is available at the end of the article Background There is increasing evidence that the values and beliefs of professionals play an important role in the de- gree to which innovations are initiated and incorpo- rated into common practice [11,12]. On the one hand, attitudes of professionals toward EBP can be a precur- sor to the decision of whether or not to try a new prac- tice [13,14], and if professionals do decide to try a new practice, the affective or emotional component of atti- tudes can impact decision processes regarding the ac- tual implementation and use of the innovation [12]. On the other hand, behaviour can influence attitudes [15]. Engaging in a behaviour, such as continued use of an evidence-based intervention until familiarity is devel- oped, using a data monitoring system to track specific indicators of change, or attending collaborative meet- ings with one’s peers, may change attitudes and beliefs about EBP [5]. Previous studies [16-18] confirmed the four-factor structure of the EBPAS in samples from the United States. These studies also suggested adequate internal consistency reliability for the EBPAS total score (Cron- bach’s alpha ranging from 0.79 to 0.77) and good in- ternal consistency reliability for the subscale scores (Cronbach’s alpha ranging from 0.93 to 0.74) in three samples. Only the Divergence subscale had a somewhat lower reliability estimate (Cronbach’s alpha ranging from 0.66 to 0.59 across studies). Construct validity was sup- ported, in part, by finding acceptable model-data fit for confirmatory factor analysis (CFA) models in both United States samples and a Greek sample [16-18,21,22]. Accept- able fit indices were found for both a first-order structure (in which the individual items loaded on four factors) and a higher order structure (in which the four first-order fac- tors were indicators of a more global higher order con- struct). Construct validity was also supported by the association of EBPAS scores with mental health clinic structures and policies [18], culture and climate [23], and leadership [24]. Evidence of content validity was obtained by asking an expert panel to rate the relevance of each item of the EBPAS for each proposed construct [16]. Content validity was supported because every item In order to tailor implementation efforts to meet the needs and characteristics of professionals in youth care institutions, we have to consider professionals’ attitudes toward adopting EBP [16,17]. Background steps of the EBP process [20] as defined by Sackett et al. [3]. Because most of our professionals are not trained in these five steps, we chose to use an instrument that as- sesses a general attitude toward using evidence-based interventions. The dissemination and implementation of evidence-based practice (EBP) to improve the quality of care and out- comes for clients and their families is a critical concern worldwide [1]. EBP is the integration of the best available research with clinical expertise in the context of client characteristics, culture, and preferences [2,3]. The purpose of EBP is to promote effective psychological practice and enhance public health by applying empirically supported principles of psychological assessment, case formula- tion, therapeutic relationship, and intervention. EBP also holds the promise to increase cost-effectiveness [4]. EBP may not only comprise the use of efficacious interventions, but may also include innovations such as data monitoring systems, alerts to target prescribing practices, and routine outcome monitoring (ROM) with feedback to clinicians [5-7]. The EBPAS consists of 15 items measured on a 5- point Likert scale ranging from 0 (Not at all) to 4 (To a very great extent). The items of the EBPAS are organized into four dimensions. The Appeal subscale (four items) assesses the extent to which the professional would adopt an evidence-based intervention if it was intuitively appealing, could be used correctly, or was being used by colleagues who were happy with it. The Requirements subscale (three items) assesses the extent to which the professional would adopt an evidence-based intervention if it was required by the supervisor, agency, or state. The Openness subscale (four items) assesses the extent to which the professional is generally open to trying new interventions and would be willing to try or use more structured or manualized interventions. The Divergence subscale (four items) assesses the extent to which the professional perceives evidence-based interventions as not clinically useful and less important than clinical ex- perience. The EBPAS total score is computed by first re- verse scoring the Divergence scale item scores and then computing the overall mean [17]. The EBPAS total score represents one’s global attitude toward adoption of evidence-based interventions. The higher the score, the more positive the attitude toward evidence-based interventions. Multiple factors at community, organizational, and in- dividual levels influence the dissemination and implemen- tation of EBP in real-world mental healthcare settings [8-10]. Abstract Keywords: Attitude, EBPAS, Evidence-based practice, Professionals, Youth care * Correspondence: m.van.sonsbeek@propersona.nl 1Behavioural Science Institute, Radboud University, Montessorilaan 3, 6525 HR Nijmegen, The Netherlands 2Pro Persona Centre for Education & Science (ProCES), Postbus 27, 6870 AA Renkum, The Netherlands Full list of author information is available at the end of the article © 2015 van Sonsbeek et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Background Aarons [18] developed the Evidence-Based Practice Attitude Scale (EBPAS) to as- sess professionals’ attitudes toward adopting EBP in mental health and welfare settings. The EBPAS asks for professionals’ feelings about using evidence-based inter- ventions, which are defined as new types of therapy or treatments and interventions with specific guidelines or components that are outlined in a manual or that are to be followed in a predetermined way. There are two other instruments to assess the attitudes of professionals to- ward adopting EBP. However, one is a non-validated sur- vey questionnaire that was not designed to assess change over time [19]. The other was developed to measure pro- fessionals’ views about and implementation of the five Page 3 of 12 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 imperative to test the EBPAS in other countries to facili- tate cross-cultural comparisons. Even though there are similarities between youth care in the Netherlands and the United States, such as types of disorders treated, substan- tial differences exist regarding organizational structures, financial barriers, types of services, training and back- ground of professionals, and client attitudes [33]. was on average rated as at least moderately relevant, important, and representative of the factor it was pur- ported to assess. Criterion validity was supported by studies showing that EBPAS scores predict adoption and use of evidence-based interventions [14,25,26]. To date, two studies have examined changes over time of EBPAS scores, with both reporting little variation over time [27,28]. The purpose of this study was to investigate the psy- chometric properties of the Dutch version of the EBPAS. We first translated the EBPAS into Dutch and evaluated the forward and back-translation. Second, we examined the factor structure and internal consistency reliability of the Dutch version of the EBPAS in a diverse sample of youth care professionals. We hereby replicated the ex- ploratory factor analysis and confirmatory factor ana- lyses by Aarons et al. [16] and Aarons [18]. In addition, we conducted a confirmatory bifactor analysis to evalu- ate the plausibility of subscales, to determine the extent to which the EBPAS total score reflects a single factor, and to evaluate the feasibility of applying a unidimen- sional model structure to a measure with heterogeneous indicators. Third, we examined the association of EBPAS scores with age, sex, and educational level of the profes- sionals. Background Based on the literature [32], it was hypothesized that (1) responses to the EBPAS items could be explained by one general factor (attitude toward evidence-based interventions) plus four specific factors (Requirements, Appeal, Openness, and Divergence), (2) good to excel- lent internal consistency reliability would be found, (3) EBPAS scores would vary by age (with older profes- sionals scoring higher on the Requirements, Openness, and Divergence subscales and lower on the EBPAS total score), (4) EBPAS scores would vary by sex (with women scoring higher on the Appeal and Requirements subscales and EBPAS total score), and (5) EBPAS scores would vary by educational level (with professionals with university education scoring higher on the Appeal and Openness subscales and lower on the Requirements subscale). Furthermore, several studies have examined EBPAS scores in relation to individual differences between profes- sionals (e.g. education, level of experience, age, discipline, and sex) and organizational characteristics (e.g. structures and policies, climate and culture, and leadership). Higher educational attainment was associated with a lower likeli- hood of adopting evidence-based interventions if re- quired, greater willingness to adopt given the appeal of evidence-based interventions, and more general open- ness to evidence-based interventions [16,24,29,30]. As with educational attainment, years of experience was associated with lower Requirements scores but also as- sociated with lower Openness and EBPAS total scores [16,29]. Years of experience was related to higher Di- vergence scores for autism early intervention profes- sionals [29] and lower Divergence scores for mental health professionals [16]. Contrasting educational at- tainment with years of experience demonstrated differ- ent patterns suggesting a more restrained openness to adopting an evidence-based intervention for those with higher educational attainment and lower enthusiasm for an evidence-based intervention given more on-the- job experience [16]. Results concerning differences by age, discipline, and sex were inconsistent. Some studies found that older professionals had higher Requirements [16] and Openness scores [23], while other studies found that younger professionals had higher EBPAS total scores [21,31]. However, older professionals also had higher Divergence scores [23]. The relationship be- tween age and attitude toward evidence-based interven- tions is possibly affected by job tenure. Concerning discipline, one study did not find any difference in EBPAS scores [18], but another study in a United States nationally representative sample found that profes- sionals trained in social work had higher Openness and EBPAS total scores than professionals trained in psych- ology [16]. Background Sex differences were absent in two studies [18,21], but in two other studies women had higher Ap- peal, Requirements, and EBPAS total scores [16,24]. Participants l f A total of 276 youth care professionals completed the survey. The results of six professionals were excluded; one endorsed two answers for most items, one com- pleted the survey twice, one was not a clinician, and three filled out 0 (Not at all) for all items. Filling out Not at all for every item (straight lining), produced in- consistent results such as “I do not know better than academic researchers how to care for my clients” con- trary to “I am not willing to use new and different types of therapy/interventions developed by researchers”. Thus, the final sample size consisted of 270 professionals. Of the respondents, 140 (51.9%) worked in a mental healthcare institution and 130 (48.1%) worked in a child welfare institution. Data on age, sex, and educational level were missing for 85 (31.5%), 30 (11.1%), and 44 (16.3%) respondents, respectively. The mean age of the remaining respondents was 43.15 years (SD = 11.03; range, 23–63) and 71.3% (n = 171) were female. The re- spondents’ primary discipline was education (n = 78; 34.5%), psychology (n = 66; 29.2%), nursing (n = 33; 14.6%), social work (n = 12; 5.3%), medicine (psychia- trists and physicians; n = 12; 5.3%), non-verbal therapies (e.g. psychomotor therapy; n = 10; 4.4%), teaching (n = 8; 3.5%), and other (n = 7; 3.1%). Because of the relatively few professionals within each discipline, we created groups based on level of education. This resulted in two groups: higher vocational education (education, nursing, social work, non-verbal therapies, and teaching; n = 125; 55.3%) and university education (education, psychology, and medicine; n = 94; 41.6%). The ‘other’ category was excluded from the analyses (n = 7; 3.1%). For each participating institution an appointment was made to discuss how and when the EBPAS was assessed. The first two institutions and one department of the third institution preferred a pen and paper survey of the EBPAS. The coordinators of these institutions and this department scheduled the survey sessions within regular team meetings. The surveys were administered to the whole group of professionals at these meetings. After the meetings, the professionals returned the survey in an en- velope which was sent to the principal investigator. The other department of the third institution and the fourth and fifth institution preferred a web-based survey of the EBPAS. Methods Setting Th The present study took place within the Academic Center Youth Nijmegen (ACYN), a multidisciplinary col- laboration between the Radboud University and multiple youth care institutions in the South-East of the Netherlands. One of the main aims of ACYN is improv- ing the care for youth by making this care more evi- dence based. Among other things, ACYN stimulates the youth care institutions to use more evidence-based inter- ventions and innovations such as ROM with feedback to clinicians. The coordinators of seven youth care institu- tions were contacted and the study was described to them in detail. Permission was sought to survey the profes- sionals of the collaborating departments. Five institutions Although the EBPAS has been translated to different languages and is currently being used in several coun- tries (e.g. in Iran, Israel, Japan, Korea, Norway, Romania, and Sweden), a Dutch version of the EBPAS has not yet become available [32]. Further, all research on the psychometric properties of the EBPAS within youth care has been carried out in the United States. It is Page 4 of 12 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 agreed to participate: one large mental healthcare institu- tion and four institutions for child welfare. retrieving information from the electronic personal files and in reaching professionals of specific locations, as well as absence of professionals after filling out the EBPAS, it was not possible to obtain demographic in- formation of all professionals. Participants l f The professionals of this department and these institutions received a personalized survey invita- tion email with a web-link to electronic questionnaire system NetQuestionnaires version 6.5 [34]. Profes- sionals who did not respond within a week received a reminder email. After the surveys were completed, the answers were automatically saved in the electronic ques- tionnaire system. The data were exported to an SPSS file. Data collection was conducted from November 2011 through November 2012. Procedure With permission of the original author (GAA), a Dutch version of the EBPAS was constructed. First, a forward translation was conducted. The EBPAS was translated into Dutch by the first and last author of this article. The authors emphasized conceptual equivalence of the questionnaire rather than literal translation. Then, a back-translation was made by a bilingual, native English- speaking translator. The first and last author of this art- icle compared the back-translation with the original version and discussed the differences. Subsequently, an- other bilingual, native English-speaking translator made a literal Dutch translation of the original version, com- pared the first Dutch translation with the second Dutch translation, and compared the differences with the ori- ginal version of the EBPAS. It was concluded that no significant differences appeared during the translation process. Only small adjustments were suggested, which were incorporated by the first and last author of this article. The final Dutch version of the EBPAS can be obtained from the first author. Exploratory factor analysis (EFA) Exploratory factor analysis (EFA) We examined descriptive statistics (means and standard deviations) at the item level, for the EBPAS subscales, and the EBPAS total score. We also examined item-total cor- relations. Internal consistency reliability was examined using Cronbach’s alpha. An EFA was conducted using data from the randomly selected half of the sample (n = 127). The EFA suggested a four-factor solution in accordance with simple structure criteria, scale reliabilities, and parallel analysis. The EFA model accounted for 61% of the variance in the data. Table 1 shows overall means and standard deviations, item-total correlations, eigenvalues, internal consistency reliabilities, and item loadings for each of the scales. Cron- bach’s alphas ranged from 0.67 to 0.89, showing acceptable to good values for the different subscales. The overall scale alpha was 0.72. The factors represented four subscales of attitudes toward adoption of evidence-based interventions in keeping with the hypothesized dimensions: Require- ments (three items, α = 0.89), Appeal (four items; α = 0.75), Openness (four items; α = 0.77), and Divergence (four items; α = 0.67). Item analyses showed that the reli- ability coefficient for all the subscales would not signifi- cantly improve by removing items from the subscale. To assess the factor structure of the Dutch version of the EBPAS, we first replicated the analyses by Aarons et al. [16] and Aarons [18]. Two separate fac- tor analytic procedures were conducted. The sample was divided by randomly selecting approximately 50% of cases and assigning cases to either an exploratory (n = 127) or confirmatory (n = 143) analysis group. An exploratory factor analysis (EFA) was conducted on the one half of the sample using Principal Axis Factor- ing in order to partition systematic and error variance in the solution [37,38]. Promax oblique rotation was used allowing for factor intercorrelations [37]. Items were retained on a factor if they loaded at least 0.32 on the primary factor and less than 0.32 on all other factors [39]. Item-total correlations and scale reliabilities were also used to assess scale structure. Confirmatory factor analyses CFA were conducted on the other half of the sample to test the factor structure derived in the EFA. Be- cause professionals were nested within institutions, models were adjusted for the nested data structure using the TYPE = COMPLEX procedure within MPlus (cf. [40]). Analyses SPSS SPSS statistical software version 20 [35] was used for the assessment of internal consistency reliability and the ex- ploratory factor analysis. Mplus software version 6 [36] was used for the confirmatory factor analyses, confirma- tory bifactor analysis, and multiple linear regression ana- lyses. For one respondent (0.4%), data of seven questions were missing. These missing data were handled in SPSS by imputing the missing values through expectation maximization, which makes use of all available data in estimating parameters. We used the imputed dataset for our analyses. Because we used two different data collec- tion methods, we also tested for differences in mean total score between the pen and paper group and the web-based group in SPSS. Since we found no differences After the data collection was finished, the coordinators of the institutions received an overview of participating professionals. Subsequently, the coordinators collected information about the age, sex, and education of the professionals through the electronic personal files of the institutions. If demographic information was miss- ing, the coordinators directly asked the professionals to provide the information. Due to technical issues with Page 5 of 12 Page 5 of 12 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 we have merged the results of both groups for the analyses. Exploratory factor analysis (EFA) First-order confirmatory factor analysis (CFA) A first-order CFA was conducted using data from the other randomly selected half of the sample (n = 143), specifying the factor structure identified in the EFA. CFA items were constrained to load only on the primary factor indicated in the EFA, thus providing a highly stringent test of the factor structure. As in the EFA, fac- tor intercorrelations were allowed. CFA factor loadings confirmed the EFA-based a priori factor structure and the model demonstrated acceptable fit (χ2(84) = 179.17, CFI = 0.90, TLI = 0.87, RMSEA = 0.09, SRMR = 0.08) fur- ther supporting the original EBPAS factor structure. Fac- tor loadings ranged from 0.40 to 0.99 and all factor loadings were statistically significant. Factor intercorrela- tions ranged from 0.04 to 0.50. The Openness subscale had a weak positive correlation with the Requirements subscale (r = 0.33, P <0.01) and a moderate positive cor- relation with the Appeal subscale (r = 0.51, P <0.01). The Divergence subscale had no significant correlations with the Requirements (r = 0.04, P >0.05), Appeal (r = −0.05, P >0.05), or Openness (r = −0.23, P >0.05) subscales. The results of the first-order CFA are shown in Figure 1. In addition, we conducted a confirmatory bifactor ana- lysis with the following features [41,42]: (1) each item had a non-zero loading on both the general factor and the specific factor that it was designed to measure, but zero loadings on the other specific factors, (2) the spe- cific factors were uncorrelated with each other and with the general factor, and (3) all error terms associated with the items were uncorrelated. The model was identified by fixing the variance of each latent factor to 1. Because the observed variables are measured on a 5-point Likert scale Robust Maximum Likelihood estimation was used. Commonly accepted rules of thumb for fit indices in confirmatory factor analyses include a comparative fit index (CFI) and Tucker–Lewis Index (TLI) value near 0.95 or greater, a root mean square error of approximation (RMSEA) value near 0.06 or less, and a standardized root mean square residual (SRMR) near 0.08 or less [43]. Second-order confirmatory factor analysis (CFA) Table 1 EBPAS subscale and item means, standard deviations, item-total correlations, eigenvalues, Cronbach’s alpha, and exploratory factor analysis loadings Table 1 EBPAS subscale and item means, standard deviations, item-total correlations, eigenvalues, Cronbach’s alpha, and e and item means, standard deviations, item-total correlations, eigenvalues, Cronbach’s alpha, and n = 270 for means, standard deviations, item-total correlations, and Cronbach’s alpha; n = 127 for exploratory factor analysis and eige SD, Standard deviation; EV, Eigenvalue; α, Cronbach’s alpha; factor loadings <0.30 are not shown. CFA among the total sample of professionals (n = 270). In the total sample, the model could be identified, but the la- tent variable covariance matrix was not positive definite. out of 15) were weaker for the general factor than for the grouping factors and some loadings were negative. According to Reise et al. 44], if items primarily reflect the general factor and have low loadings on the grouping factors, subscales make little sense. However, when items have substantial loadings on both the general factor and the grouping factors, subscales will make sense. There- fore, the findings indicate that variance is explained both by a general factor (Attitude toward evidence-based in- terventions) and by specific factors above and beyond the general factor (Requirements, Appeal, Openness, and Divergence). This further supports the original EBPAS fac- tor structure. The results of the confirmatory bifactor ana- lysis are shown in Figure 3. This indicated a correlation greater or equal to one be- tween two variables: item 9 (intuitively appealing) and item 10 (makes sense). By correcting the model for this correlation, we found a good fit (χ2(85) = 146.48, CFI = 0.96, TLI = 0.95, RMSEA = 0.05, SRMR = 0.05) and our re- sults exactly matched the results of Aarons et al. [16]. Fac- tor loadings ranged from 0.45 to 0.99. All factor loadings were statistically significant, except for Divergence on At- titude (P = 0.21). The results of the second-order CFA are shown in Figure 2. Second-order confirmatory factor analysis (CFA) Second order confirmatory factor analysis (CFA) In replication of Aarons et al. [16], a second-order CFA was conducted to test if all lower order EBPAS factors are indicators of a higher order construct that might be regarded as general attitude toward adoption of evidence- based interventions. With data from the randomly se- lected half of the sample that was used for the first-order CFA (n = 143), the model could not be identified. Because we assumed that our subgroup was too small for a second-order CFA, we also conducted the second-order After identifying a measurement model with accept- able fit, we examined the association of the Dutch EBPAS subscale and total scores with characteristics of the youth care professionals (i.e. age, sex, and educa- tional level). Rather than replicating Aarons [18] mul- tiple stage analytic approach, we replicated Aarons et al. [16] regression analyses because we already adjusted for nested data with the TYPE = COMPLEX procedure within MPlus. We used two-tailed tests. van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Page 6 of 12 Table 1 EBPAS subscale and item means, standard deviations, item-total correlations, eigenvalues, Cronbach’s alpha, and exploratory factor analysis loadings EBPAS subscales and total M SD Item-total correlation EV α Scale 1 Scale 2 Scale 3 Scale 4 Requirements 2.38 0.78 4.19 0.89 Agency required 2.36 0.86 0.67 0.97 Supervisor required 2.41 0.84 0.60 0.85 State required 2.37 0.87 0.63 0.76 Appeal 3.13 0.54 1.94 0.75 Makes sense 3.22 0.71 0.44 0.84 Intuitively appealing 3.09 0.78 0.46 0.74 Get enough training to use 3.18 0.68 0.50 0.37 Colleagues happy with intervention 3.03 0.71 0.53 0.60 Openness 2.63 0.62 1.56 0.77 Will follow a treatment manual 2.74 0.77 0.59 0.66 Like new therapy types 2.57 0.78 0.43 0.58 Therapy developed by researchers 2.70 0.78 0.57 0.74 Therapy different than usual 2.51 0.87 0.60 0.52 Divergence 1.46 0.65 1.41 0.67 Research-based treatments not useful 1.15 0.86 0.41 0.48 Will not use manualized therapy 0.81 0.89 0.43 0.44 Clinical experience more important 2.07 0.91 0.25 0.66 Know better than researchers 1.81 0.98 0.32 0.56 EBPAS total 2.67 0.41 0.72 n = 270 for means, standard deviations, item-total correlations, and Cronbach’s alpha; n = 127 for exploratory factor analysis and eigenvalues. SD, Standard deviation; EV, Eigenvalue; α, Cronbach’s alpha; factor loadings <0.30 are not shown. Regression analyses d l The bifactor model simultaneously assessed the specific factors Requirements, Appeal, Openness, and Diver- gence as well as the general factor Attitude toward evidence-based interventions shared by those specific factors. The bifactor model demonstrated a good fit (χ2(75) = 107.37, CFI = 0.97, TLI = 0.96, RMSEA = 0.04, SRMR = 0.04). The majority of the factor loadings were statistically significant, except for items 3 and 6 on Attitude (P = 0.38 and P = 0.83), and items 14 and 15 on Appeal (P = 0.16 and P = 0.07). Most factor loadings (10 Regression models using the maximum likelihood estima- tion with robust standard errors were used to assess the as- sociation of EBPAS scores with characteristics of youth care professionals. For these analyses, in order to enable a good comparison with the study of Aarons et al. [16], the whole sample was used in a first-order factor model. A second- order factor model could not be identified because the number of iterations was exceeded. The results are shown in Table 2. For the Requirements subscale, willingness to van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Page 7 of 12 Page 7 of 12 Figure 1 Confirmatory factor analysis model of the EBPAS. n = 143, χ2 (84) = 179.17, CFI = 0.90, TLI = 0.87, RMSEA = 0.09, SRMR = 0.08, P < 0.05, * P < 0.01; all factor loadings are significant at P < 0.01. Figure 1 Confirmatory factor analysis model of the EBPAS. n = 143, χ2 (84) = 179.17, CFI = 0.90, TLI = 0.87, RMSEA = 0.0 P < 0.01; all factor loadings are significant at P < 0.01. adopt evidence-based interventions given the requirements to do so decreased with higher levels of education. Control- ling for age and sex, professionals with university education scored significantly lower than professionals with higher vo- cational education. For the Appeal subscale, no associations with characteristics of youth care professionals were found. For the Openness subscale, greater openness to new prac- tices was associated with higher levels of education. Con- trolling for age and sex, professionals with university education scored significantly higher than professionals with higher vocational education. Finally, for the Diver- gence subscale, greater divergence between evidence-based interventions and current practice was associated with being older, being male, and lower levels of education. Regression analyses d l While holding constant other variables in the model, older professionals scored significantly higher than younger pro- fessionals, males scored significantly higher than females, and professionals with higher vocational education scored significantly higher than professionals with university education. Discussion The current study contributes to the development and cross-validation of the EBPAS by examining the van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Page 8 of 12 Figure 2 Second-order confirmatory factor analysis model of the EBPAS. n = 270, χ2 (85) = 146.48, CFI = 0.96, TLI = 0.95, RMSEA = 0.05, SRMR = 0.05; All factor loadings are significant at P < 0.01, except for Divergence on Attitude (in italics; P = 0.21). Estimation of correlated residuals between two Appeal subscale items is indicated by a double-headed arrow. Figure 2 Second-order confirmatory factor analysis model of the EBPAS. n = 270, χ2 (85) = 146.48, CFI = 0.96, TLI = 0.95, RMSEA = 0.05, SRMR = 0.05; All factor loadings are significant at P < 0.01, except for Divergence on Attitude (in italics; P = 0.21). Estimation of correlated residuals between two Appeal subscale items is indicated by a double-headed arrow. consistency reliability and the mean loading of items on their predicted factors was substantial to good. These find- ings are highly congruent with studies of the original ver- sion of the EBPAS conducted in the United States [32]. psychometric properties of the Dutch version of the EBPAS. Besides that, the present research is the first to assess a bifactorial solution of attitude toward evidence- based interventions. The results provide strong support for a structure with a general factor plus four specific factors of the Dutch version of the EBPAS in a diverse sample of youth care professionals of both a mental healthcare institution and institutions for child welfare. The general factor is attitude toward evidence-based in- terventions and the specific factors involve willingness to adopt evidence-based interventions given the intuitive appeal, willingness to adopt evidence-based interventions if required, general openness toward evidence-based inter- ventions, and perceived divergence of usual practice with evidence-based interventions. The EBPAS total score and subscale scores demonstrated acceptable to good internal Generally consistent with previous findings, Openness subscale scores were positively correlated with Appeal and Requirements subscale scores. Professionals who are more open toward adoption of evidence-based interven- tions may also endorse positive attitudes toward the intui- tive appeal of these interventions and the requirements to adopt these interventions. Discussion Contrary to previous findings, we did not find a statistically significant correlation be- tween the extent to which professionals would adopt evidence-based interventions if they were intuitively ap- pealing and if they were required. The Divergence subscale was not significantly correlated with any of the other van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Page 9 of 12 Figure 3 Confirmatory bifactor analysis model of the EBPAS. n = 270, χ2 (75) = 107.37, CFI = 0.97, TLI = 0.96, RMSEA = 0.04, SRMR = 0.04; All factor loadings are significant at P < 0.05, except for items ‘Clinical experience important’ and ‘Know better than researchers’ on the general factor (in italics; P = 0.83 and P = 0.38), and items ‘Colleagues like it’ and ‘Enough training’ on Appeal (in italics; P = 0.16 and P = 0.07). Figure 3 Confirmatory bifactor analysis model of the EBPAS. n = 270, χ2 (75) = 107.37, CFI = 0.97, TLI = 0.96, RMSEA = 0.04, SRMR = 0.04; All factor loadings are significant at P < 0.05, except for items ‘Clinical experience important’ and ‘Know better than researchers’ on the general factor (in italics; P = 0.83 and P = 0.38), and items ‘Colleagues like it’ and ‘Enough training’ on Appeal (in italics; P = 0.16 and P = 0.07). required. Moreover, we did not find a statistically signifi- cant correlation between the Divergence subscale and the general Attitude toward adoption of evidence-based inter- ventions. While the three distinct dimensions Require- ments, Appeal and Openness possibly address a common subscales. Therefore, the extent to which professionals per- ceive evidence-based interventions as not clinically useful does not appear to be meaningfully associated with open- ness to evidence-based interventions, appeal of these inter- ventions, and willingness to adopt these interventions if Table 2 Association of characteristics of youth care professionals with EBPAS scores Requirements (R2 = 0.01) Appeal (R2 = 0.02) Openness (R2 = 0.06) Divergence (R2 = 0.15) Variable ß b z ß b z ß b z ß b z Age 0.025 0.001 0.32 –0.031 −0.001 −0.54 –0.153 −0.003 −1.11 0.262 0.005 4.18 Sex 0.039 0.047 1.18 –0.137 −0.141 −1.70 –0.153 −0.112 −1.62 0.223 0.170 1.97* Educational level –0.101 −0.100 −2.99 0.060 0.051 1.31 0.137 0.082 3.93 –0.232 −0.145 −3.05** Reference groups for dummy-coded variables are as follows: sex reference is female and discipline reference is higher vocational education. Discussion social work, psychology, marriage and family therapy, nursing) in the United States, and this has been developing for a longer period of time than in the Netherlands. Besides this, males and older professionals reported more perceived divergence than females and younger professionals. The sex difference is in contrast to studies with the original version of the EBPAS, in which this ef- fect was not found [32]. This suggests that additional re- search is needed to explore when, where, and why sex differences in attitudes toward evidence-based interven- tions might operate. The age difference is in accordance with one earlier study [23]. It is conceivable that younger professionals, like professionals with a university degree, are more familiar with evidence-based interventions be- cause they have had more prior exposure to these inter- ventions during their education than older professionals [48]. Being older may result in giving more importance to own clinical experience and less appreciation of research-based interventions. Some limitations should be noted. First, the sample was not large and thus statistical power was limited. Fur- ther, data on age was missing for almost one-third of the respondents and data about level of experience was not available, limiting the direct comparisons that could be made with previous studies. Additionally, only one men- tal healthcare organization was included; although this was a large one, caution should be exercised when inter- preting the generalizability to other mental healthcare institutions. Furthermore, while our sample consisted of professionals of a mental healthcare institution and insti- tutions for child welfare, our findings may not generalize to professionals in individual practice or to other sectors. Finally, because only information about respondents was available, it was not possible to compare respondents with non-respondents to examine a potential non- response bias. The results also demonstrated associations between EBPAS scores and characteristics of youth care profes- sionals. Professionals with university education scored higher on Openness and lower on both Requirements and Divergence than professionals with higher voca- tional education. Professionals with university education may have received more education related to evidence- based interventions. In addition, it is likely that the drawbacks of overreliance upon unsystematic clinical observations and experience alone, were raised. It may also be that professionals with university education have had more training and applied practice in evidence-based interventions. Discussion P <0.05, * P <0.01. Table 2 Association of characteristics of youth care professionals with EBPAS scores Page 10 of 12 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Page 10 of 12 attitude toward evidence-based interventions, Divergence seems to be a separate construct in the Dutch sample. report more openness and less divergence. However, professionals with university education also seem less willing to adopt evidence-based interventions if re- quired. It may be that professionals with additional education and practice are more autonomous and as- sertive in making independent decisions about utilizing evidence-based interventions [16] and therefore less willing to accept external pressure than their colleagues with lower education. Possible explanations for the finding that the Diver- gence factor loading was not statistically significant in the higher order model might lie in cultural, political and/or time differences between youth care institutions in the United States and the Netherlands. A possible cultural difference is that Dutch professionals may view research as separate from using evidence-based inter- ventions [45,46]. There are three items in the EBPAS that explicitly use the term “research/researchers”: two items of the Divergence subscale (“Research-based treat- ments/interventions are not clinically useful” and “I know better than academic researchers how to care for my clients”) and one item of the Openness subscale (“I am willing to use new and different types of therapy/inter- ventions developed by researchers”). The item scores of the Dutch sample appear to be somewhat higher on the first two items and somewhat lower on the last item compared to scores found in the United States samples. Dutch professionals may feel more discrepancy between their clinical views and scientific results or perceive re- search as confining their ability to make independent decisions about therapy, while they still can be open to evidence-based interventions, can find these interven- tions appealing, and are willing to adopt these interven- tions if required. A possible time difference is that evidence-based interventions may be more widely dis- seminated and implemented in the United States than in the Netherlands. The main body of research about attitudes toward evidence-based interventions (begin- ning in 2004) is from the United States, exposure to the concept and meaning of evidence-based interventions may be more common across different professional dis- ciplines (e.g. Acknowledgements This study was funded in part by a grant from ZonMw, the Dutch organization for Health Research and Development (Grant Number 157031002). The authors would like to thank the youth care institutions and professionals that participated in the study and made this work possible. Abbreviations ACYN A d i 11. Damanpour F. Organizational innovation: a meta analysis of effects of determinants and moderators. Acad Manage J. 1991;34(3):555–90. 12. Frambach RT, Schillewaert N. Organizational innovation adoption: a multi- level framework of determinants and opportunities for future research. J Bus Res. 2002;55(2):163–76. ACYN: Academic Center Youth Nijmegen; CFA: Confirmatory factor analysis; CFI: Comparative fit index; EBP: Evidence-based practice; EBPAS: Evidence- Based Practice Attitude Scale; EFA: Exploratory factor analysis; RMSEA: Root mean square error of approximation; ROM: Routine Outcome Monitoring; SRMR: Standardized root mean square residual; TLI: Tucker–Lewis Index. 12. Frambach RT, Schillewaert N. Organizational innovation adoption: a multi- level framework of determinants and opportunities for future research. J Bus Res. 2002;55(2):163–76. 13. Fishbein M, Hennessy M, Yzer M, Douglas J. Can we explain why some people do and some people do not act on their intentions? Psychol Health Med. 2003;8(1):3–18. doi:10.1080/1354850021000059223. 13. Fishbein M, Hennessy M, Yzer M, Douglas J. Can we explain why some people do and some people do not act on their intentions? Psychol Health Med. 2003;8(1):3–18. doi:10.1080/1354850021000059223. References 1. Aarons GA, Cafri G, Lugo L, Sawitzky A. Expanding the domains of attitudes towards evidence-based practice: the evidence based practice attitude scale- 50. Admin Pol Ment Health. 2012;39(5):331–40. doi:10.1007/s10488-010-0302-3. 1. Aarons GA, Cafri G, Lugo L, Sawitzky A. Expanding the domains of attitudes towards evidence-based practice: the evidence based practice attitude scale- 50. Admin Pol Ment Health. 2012;39(5):331–40. doi:10.1007/s10488-010-0302-3. 2. APA Presidential Task Force on Evidence-Based Practice. Evidence-based practice in psychology. Am Psychol. 2006;61(4):271–85. doi:10.1037/0003-066X.61.4.2712. 2. APA Presidential Task Force on Evidence-Based Practice. Evidence-based practice in psychology. Am Psychol. 2006;61(4):271–85. doi:10.1037/0003-066X.61.4.2712. 3. Sackett DL, Rosenberg WMC, Gray JAM, Haynes RB, Richardson WS. Evidence based medicine: what it is and what it isn’t. Br Med J. 1996; 312(7023):71–2. 3. Sackett DL, Rosenberg WMC, Gray JAM, Haynes RB, Richardson WS. Evidence based medicine: what it is and what it isn’t. Br Med J. 1996; 312(7023):71–2. ACYN: Academic Center Youth Nijmegen; CFA: Confirmatory factor analysis; CFI: Comparative fit index; EBP: Evidence-based practice; EBPAS: Evidence- Based Practice Attitude Scale; EFA: Exploratory factor analysis; RMSEA: Root mean square error of approximation; ROM: Routine Outcome Monitoring; SRMR: Standardized root mean square residual; TLI: Tucker–Lewis Index. Discussion More research is needed to confirm associations between EBPAS scores and characteristics of youth care professionals, structures and policies of youth care institutions, culture and climate, and leadership. Furthermore, the sensitivity to change of the Dutch version of the EBPAS should be investigated to be able to examine how attitudes towards evidence-based interventions might influence behaviour, how behaviour might influence change in attitudes, and whether attitudes predict adoption, implementation, and sustainment of evidence-based interventions. Finally, for implementation of evidence-based interventions within youth care, it will be useful to examine factors that might moderate the rela- tionship between attitudes and behaviour. Such behav- iours are not limited to the use of efficacious treatment models, but can also include use of innovations such as data monitoring systems, alerts to target prescribing prac- tices, and ROM. Although the referent for the EBPAS are interventions that have research support and may be man- ualized and structured, attitudes towards these interven- tions can also reflect attitudes toward other clinical innovations. Using information obtained by the Dutch version of the EBPAS may ultimately enable a better tai- loring of implementation efforts to the readiness of youth care professionals to adopt evidence-based initiatives. Authors’ contributions MAMSS, GJMH, JWV and BGT conceptualized the specific research questions and analytic approach for this manuscript. MAMSS and BGT were responsible for the translation of the EBPAS into Dutch. Analyses were conducted by MAMSS and MK. MAMSS wrote the first draft of the manuscript, and all authors edited and revised portions of the manuscript. All authors read and approved the final manuscript. The Dutch version of the EBPAS is available from the first author. Conclusions Glisson C, Schoenwald SK. The ARC organizational and community intervention strategy for implementing evidence-based children’s mental health treatments. Ment Health Serv Res. 2005;7(4):243–59. doi:10.1007/s11020-005-7456-1. 9. Glisson C, Schoenwald SK. The ARC organizational and community intervention strategy for implementing evidence-based children’s mental health treatments. Ment Health Serv Res. 2005;7(4):243–59. doi:10.1007/s11020-005-7456-1. 10. Raghavan R, Bright CL, Shadoin AL. Toward a policy ecology of implementation of evidence-based practices in public mental health settings. Implement Sci. 2008;3:26. doi:10.1186/1748-5908-3-26. 10. Raghavan R, Bright CL, Shadoin AL. Toward a policy ecology of implementation of evidence-based practices in public mental health settings. Implement Sci. 2008;3:26. doi:10.1186/1748-5908-3-26. 11. Damanpour F. Organizational innovation: a meta-analysis of effects of determinants and moderators. Acad Manage J. 1991;34(3):555–90. Author details 1 1Behavioural Science Institute, Radboud University, Montessorilaan 3, 6525 HR Nijmegen, The Netherlands. 2Pro Persona Centre for Education & Science (ProCES), Postbus 27, 6870 AA Renkum, The Netherlands. 3Department of Epidemiology, Trimbos Institute, Da Costakade 45, 3521 VS Utrecht, The Netherlands. 4Department of Psychiatry, University of California, 9500 Gilman Drive, San Diego, La Jolla, CA 92093-0812, USA. 5Child and Adolescent Services Research Center, 3020 Children’s Way, San Diego, CA 92123, USA. Received: 20 February 2015 Accepted: 1 November 2015 Received: 20 February 2015 Accepted: 1 November 2015 Conclusions The present study provides strong support for the ori- ginal four-factor structure and internal consistency reli- ability of the Dutch version of the EBPAS in a diverse sample of youth care professionals. This supports the utility of the EBPAS in varied countries and settings. Be- cause we focused on real-world professionals, the study also provides support for ecological validity. 4. Barnoski R. Outcome evaluation of Washington State’s research-based programs for juvenile offenders. Olympia, WA: Washington State Institute for Public Policy; 2004. y 5. Aarons GA, Glisson C, Green PD, Hoagwood K, Kelleher KJ, Landsverk JA, et al. The organizational social context of mental health services and clinician attitudes toward evidence-based practice: a United States national study. Implement Sci. 2012;7:56. doi:10.1186/1748-5908-7-56. 5. Aarons GA, Glisson C, Green PD, Hoagwood K, Kelleher KJ, Landsverk JA, et al. The organizational social context of mental health services and clinician attitudes toward evidence-based practice: a United States national study. Implement Sci. 2012;7:56. doi:10.1186/1748-5908-7-56. y 6. Bickman L. A measurement feedback system (MFS) is necessary to improve mental health outcomes. J Am Acad Child Adolesc Psychiatry. 2008;47(10): 1114–9. 6. Bickman L. A measurement feedback system (MFS) is necessary to improve mental health outcomes. J Am Acad Child Adolesc Psychiatry. 2008;47(10): 1114–9. The study suggests four directions for future research. First, the factor structure of the Dutch version of the EBPAS needs to be further established. Second, additional research is needed to further establish the construct and criterion validity of the Dutch version of the EBPAS. Third, the sensitivity to change of the Dutch version of the EBPAS should be investigated. Finally, it is recom- mended to examine factors that might moderate the rela- tionship between attitudes and behaviour of youth care professionals. 7. Bickman L, Kelley SD, Breda C, de Andrade AR, Riemer M. Effects of routine feedback to clinicians on mental health outcomes of youths: results of a randomized trial. Psychiat Serv. 2011;62(12):1423–9. 7. Bickman L, Kelley SD, Breda C, de Andrade AR, Riemer M. Effects of routine feedback to clinicians on mental health outcomes of youths: results of a randomized trial. Psychiat Serv. 2011;62(12):1423–9. 8. Greenhalgh T, Robert G, Macfarlane F, Bate P, Kyriakidou O. Diffusion of innovations in service organizations: systematic review and recommendations. Milbank Q. 2004;82(4):581–629. 8. Greenhalgh T, Robert G, Macfarlane F, Bate P, Kyriakidou O. Diffusion of innovations in service organizations: systematic review and recommendations. Milbank Q. 2004;82(4):581–629. 9. Discussion Professionals whose edu- cation includes and emphasizes exposure to and train- ing in research may be more likely to understand, value, and utilize research [47]. Such professionals may This study has added to the knowledge about the EBPAS in general and for the Netherlands specifically. To our knowledge, this is the first study that used the EBPAS in the Netherlands. Additional research is needed to further establish the factor structure of the Dutch ver- sion of the EBPAS. Both the first-order structure and higher order structure require more consideration and investigation. Additionally, it is not clear if the factor structure would vary for child mental health versus child welfare professionals. Future research should examine factor structure differences by professional primary dis- cipline, level of experience, and sector. In addition, the van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 van Sonsbeek et al. Health Research Policy and Systems (2015) 13:69 Page 11 of 12 Page 11 of 12 construct and criterion validity of the Dutch version of the EBPAS have to be further established. More research is needed to confirm associations between EBPAS scores and characteristics of youth care professionals, structures and policies of youth care institutions, culture and climate, and leadership. Furthermore, the sensitivity to change of the Dutch version of the EBPAS should be investigated to be able to examine how attitudes towards evidence-based interventions might influence behaviour, how behaviour might influence change in attitudes, and whether attitudes predict adoption, implementation, and sustainment of evidence-based interventions. Finally, for implementation of evidence-based interventions within youth care, it will be useful to examine factors that might moderate the rela- tionship between attitudes and behaviour. Such behav- iours are not limited to the use of efficacious treatment models, but can also include use of innovations such as data monitoring systems, alerts to target prescribing prac- tices, and ROM. Although the referent for the EBPAS are interventions that have research support and may be man- ualized and structured, attitudes towards these interven- tions can also reflect attitudes toward other clinical innovations. Using information obtained by the Dutch version of the EBPAS may ultimately enable a better tai- loring of implementation efforts to the readiness of youth care professionals to adopt evidence-based initiatives. construct and criterion validity of the Dutch version of the EBPAS have to be further established. Competing interests Bifactor models and rotations: exploring the extent to which multidimensional data yield univocal scale scores. J Pers Assess. 2010;92:544–59. doi:10.1080/00223891.2010.496477. 20. Rubin A, Parrish DE. Validation of the Evidence-Based Practice Process Assessment Scale. Res Soc Work Pract. 2011;21(1):106–18. doi:10.1177/1049731509347851. 45. Lauteslager M. Wetenschap, akkoord, maar welke wetenschap? 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https://openalex.org/W2529695194	http://www.vliz.be/imisdocs/publications/295851.pdf	English	null	Landscape mapping at sub-Antarctic South Georgia provides a protocol for underpinning large-scale marine protected areas	Scientific reports	2,016	cc-by	13,121	Oliver T. Hogg1,2,3, Veerle A. I. Huvenne2, Huw J. Griffiths1, Boris Dorschel4 & Katrin Linse1 Global biodiversity is in decline, with the marine environment experiencing significant and increasing anthropogenic pressures. In response marine protected areas (MPAs) have increasingly been adopted as the flagship approach to marine conservation, many covering enormous areas. At present, however, the lack of biological sampling makes prioritising which regions of the ocean to protect, especially over large spatial scales, particularly problematic. Here we present an interdisciplinary approach to marine landscape mapping at the sub-Antarctic island of South Georgia as an effective protocol for underpinning large-scale (105–106 km2) MPA designations. We have developed a new high-resolution (100 m) digital elevation model (DEM) of the region and integrated this DEM with bathymetry- derived parameters, modelled oceanographic data, and satellite primary productivity data. These interdisciplinary datasets were used to apply an objective statistical approach to hierarchically partition and map the benthic environment into physical habitats types. We assess the potential application of physical habitat classifications as proxies for biological structuring and the application of the landscape mapping for informing on marine spatial planning. Rates of species extinction currently exceed those of speciation, leading to a decline in global biodiversity1–3. Anthropogenic activities are significant drivers of this decline4, with the marine environment experiencing sig- nificant and increasing pressure from extractive industries such as fishing, marine deposit mining, and oil and gas extraction, activities, which are increasingly proliferating into deep-sea environments5. Furthermore, the world’s oceans are becoming warmer and more acidic6, driving changes in the distributions of marine species7, and impinging on ecosystem services8,9.i p g g y Under financial, political and social constraints, two key questions that precede any conservation strategy are how and where to prioritise limited resources and effort to maximise the scientific robustness of conser- vation strategies in an increasingly exploited, yet still poorly understood marine environment. In response to international frameworks such as the Convention on Biological Diversity10, conservation efforts have increas- ingly prioritised the protection of regions and ecosystems as a holistic and geopolitically achievable approach to conservation. In recent years this has led to the establishment of several large-scale (105–106 km2) marine protected areas (MPAs) at sites including Ascension Island (2016), Pitcairn Island (2015), South Georgia and South Sandwich Islands (2012) and the Chagos Archipelago (2010). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 23 March 2016 accepted: 18 August 2016 Published: 03 October 2016 Landscape mapping at sub- Antarctic South Georgia provides a protocol for underpinning large- scale marine protected areas received: 23 March 2016 accepted: 18 August 2016 Published: 03 October 2016 Oliver T. Hogg1,2,3, Veerle A. I. Huvenne2, Huw J. Griffiths1, Boris Dorschel4 & Katrin Linse1 Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 www.nature.com/scientificreports/ www.nature.com/scientificreports/ biogeographical knowledge of the marine environment through in situ sampling is typically spatially very patchy and in some instances, notably the deep sea, virtually non-existent.hi biogeographical knowledge of the marine environment through in situ sampling is typically spatially very patchy and in some instances, notably the deep sea, virtually non-existent.hi y p y This paucity in knowledge is particularly problematic for the scientific underpinning of large-scale MPA desig- nations. Frequently, ambitious political and social demand for high-impact, low-cost and large-scale management solutions conflict with scientific data that only support designation on localised scale, or in a generalised way (i.e. a region is known to have high faunal diversity but little is known about its distribution). As a consequence, MPA designation tends to be scientifically underpinned by the data that are available, but not scientifically driven in their design. By default the spatial configuration of many MPAs therefore tends to be delineated by geopolitical boundaries (e.g. extent of Exclusive Economic Zones) and located in often remote overseas territories that elicit minimal stakeholder opposition. pp In response to the challenge of limited biological data, habitat mapping can be used to greatly increase the value of limited in situ data for marine conservation, by inferring associations between ground-truthed sam- ple data, remotely sensed geophysical data (e.g. topography; surficial geology), and physical environmental data (e.g. oceanography). In this way, in the absence of sufficient biological data, geophysical and biophysical attrib- utes such as depth, substrate type, and geomorphology can act as useful surrogates for biological communities or assemblages13. Analysis of the interaction between physical and biological variables helps create a more compre- hensive spatial and temporal understanding of how biodiversity is structured across a region, and can help con- strain the bio-physical processes driving this structuring. Furthermore remotely sensed data are typically orders of magnitude faster and cheaper to collect per unit area than ground-truthed sample data14. g p p g p Marine landscape mapping is a form of top-down habitat mapping that delineates and describes the marine environment on the basis of physical characteristics. Biological data are then used to ground-truth the map to test how effective proxy habitat classification is for modelling biological distributions. Marine landscape mapping is often adopted as a broad-scale approach to habitat classification15,16, but is a technique with application across spatial scales17–19. www.nature.com/scientificreports/ Through this approach, different geomorphic features such as canyons, pinnacles, rocky ridges and muddy basins can be mapped, either for their own intrinsic conservation value20, or for use as proxies of par- ticular types of benthic habitats21. This makes it a technique with potential application for assessing risk to benthic communities and in the proposing of MPAs designations.h p p g g The waters surrounding the sub-Antarctic island of South Georgia are one of the largest recently designated MPAs, and include one of the largest and most isolated continental shelf areas in the Southern Ocean. A combi- nation of early separation from a continental land mass, a large shelf area, a high degree of geographic isolation, and the proximity of nutrient-rich currents have led to the evolution of a biologically rich and distinct island22. In 2012 the region (including the South Sandwich Islands archipelago to the south-east) was designated an IUCN category IV marine protected area, covering a total area of over one million km2,23.h g y p g The regional waters around South Georgia represent a particularly good model system in which to develop, test, and implement a landscape mapping protocol. The region is one of a number of recent additions to the list of extremely large-scale MPAs. There has been no concerted effort (expeditions to the region) with the specific purpose of mapping physical habitats or biotopes. As such, all data from the region are (from a habitat mapping perspective) based on opportunistic historical data collection. This is important in developing a protocol that has transferability to other marine systems, as the suite of data available at South Georgia is comparable with most other potential large-scale MPA sites. The existence of a number of comprehensive datasets including seabed geophysical data; modelled oceanographic data, sediment data, and an extensive biological dataset provides an ideal opportunity for interdisciplinary habitat mapping. In addition to being a good location to test landscape mapping models, it is also an internationally important site to understand and conserve because of its speciose and unique benthic fauna, commercially important fisheries and the presence of a globally important population of higher predators12,22.h g p The aim of this study is to develop a fully automated and objective marine landscape mapping protocol that adopts a hierarchically nested clustering approach to classify physical habitat types across spatial scales. www.nature.com/scientificreports/ This pro- tocol should be robust, repeatable and have universal application for other proposed or established marine pro- tected areas. It should also enable assessment of the ecological relevance of physical habitat classifications, their application as proxies for benthic biological structuring, and as such the application of the landscape mapping protocol for informing marine spatial planning. Oliver T. Hogg1,2,3, Veerle A. I. Huvenne2, Huw J. Griffiths1, Boris Dorschel4 & Katrin Linse1 The basis of many of these MPAs centre on the presence of particularly vulnerable, keystone, or charismatic species; the presence of large numbers (or proportions) of endemic species; and the presence of high biodiversity across taxonomic levels (i.e. biodiversity hotspots)11,12. The scientific underpinning for the creation of such vast reserves is based predominantly on in situ data such as biological sampling (e.g. by trawling, dredging and diving) and direct observation (e.g. by divers or camera systems). In situ sampling and observation, however, is intrinsically expensive, time-consuming, and logistically difficult, especially when attempting to cover large areas, inaccessible or remote regions. As such, 1British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 OET, U.K. 2National Oceanography Centre, University of Southampton Waterfront Campus, European Way, Southampton SO14 3ZH, Southampton, U.K. 3University of Southampton, Waterfront Campus, European Way, Southampton SO14 3ZH, U.K. 4Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Mitte, Am Alten Hafen 26, 27568 Bremerhaven, Germany. Correspondence and requests for materials should be addressed to O.T.H. (email: olgg@bas.ac.uk) Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 1 Methodology S d T Study area. The greater South Georgia region, which forms the focus of this paper, is defined here by a bounding box of ~900 km (45°E to 19°E) by ~580 km (63°S to 50°S), covering an area of 530,000 km2 (Fig. 1). The region includes the South Georgia shelf, the Shag Rock shelf (to the west of South Georgia), the surrounding continental slopes and adjacent deep sea. It does not, however, extend to include the South Sandwich Islands region of the SGSSI MPA. The rationale for this geographical delimitation at the expense of covering the entire > 1 million km2 of the SGSSI MPA was based on the limited spatial extent of input datasets (notably the modelled oceanographic dataset). Data compilation. Bathymetry gridding process. We constructed a new high resolution (100 m) bathy- metric grid of the South Georgia continental shelf, slope and surrounding open-ocean. The grid was derived from multibeam and single-beam echo-sounding data compiled from numerous research institutes, universities and commercial fisheries (see Supplementary materials Table 1). Given the diverse range of data sources, the echo-sounding data varied greatly in terms of collection method, data quality, and file format. As such the process of incorporating different data streams into the compilation varied depending on their provenance. p gf p p g p Data curated at the BAS Polar Data Centre and collected during 36 research cruises between 1994 and 2 were gridded at a 100m resolution using MB-System23. Unprocessed data from recent BAS cruises to the reg Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 2 www.nature.com/scientificreports/ Figure 1. The South Georgia and South Sandwich Island marine protected area. Dark green areas demark the no-take zones around South Georgia, South Sandwich Islands, Shag Rocks and Clerke Rocks. Light green indicates depths less than 700 m in which bottom fishing is prohibited. Hashed boxes with red border are additional benthic closed areas established in 2013 in which bottom fishing is prohibited. The purple borders around SSI are a 12nm pelagic no-take zone. The large black hashed area south of 60° S falls within the SGSSI Maritime Zone in a region for which no fishing licenses are issued. In all other regions of the SGMZ bottom fishing is prohibited with the sole exception of the narrow pale blue region which includes the depths between 700m and 2250m. Within this region bottom fishing is permitted by license. Methodology S d T Single-beam bathymetry data were obtained from the UK Hydrographic Office (UKHO), BAS and commercial fisheries vessels. The spatial coverage of respective datasets is summarised in Fig. 2. g As with a number of previous bathymetric compilations24–26 we used TOPOGRID to grid the digital ele- vation model. TOPOGRID is an interpolation function in ArcGIS (version 10.1) built around the ANUDEM algorithm27,28, which has been shown as an effective approach to integrating spatially discontinuous data with different sampling densities whilst minimising standard error in the model26. The ANUDEM algorithm runs over multiple iterative cycles. It starts by gridding data at a coarse spatial resolution before interpolating at successively finer resolutions, until the pre-defined resolution (in this case 100m) is reached. TOPOGRID is particularly well-suited to constraining fine-scale topographic features whilst imposing constraints to prevent erroneous sinks being formed in the output digital elevation model (DEM)29.h g p g The quality of the data varied between surveys as a result of a number of factors including the echo-sounder equipment used, the calibration of this equipment (i.e. the use of different sound-velocity profiles), the sea state conditions at the time of data collection, and the degree and quality of data post-processing and cleaning. This variability was manifest in two key regards: the effective spatial resolution of the data (i.e. at what spatial scale the bathymetry could be rendered at), and the presence of acquisition artefacts (artificial ‘sinks’ or ‘peaks’) in the data. i TOPOGRID reads data as point ‘xyz’ (longitude, latitude, depth) records for each data source. Creating a bathymetric compilation from a point cloud is inherently problematic given the variability in the quality of the data from different sources. Specifically, this is a problem when regions of data points from different sources (which may vary in spatial resolution by 10 s or 100 s of meters) overlap. The algorithm will treat each point as equally valid, and draw a spline between them, potentially creating steps in the data as it moves between points from different sources. To remove this artefact, we ranked our datasets hierarchically based on data quality. Using spatial coverage masks of each dataset, regions of overlapping data were cut from all masks except for the mask representing the best-quality data available from that particular region. Methodology S d T The region of interest for this study is delineated by the grey shaded box. The inset shows the position of the South Georgia and South Sandwich Islands MPA relative to South America, the Antarctic continent and the Polar Front (dashed line). Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Background bathymetry is The GEBCO_2014 Grid, version 20141103 (http://www.gebco.net). Figure 1. The South Georgia and South Sandwich Island marine protected area. Dark green areas demark the no-take zones around South Georgia, South Sandwich Islands, Shag Rocks and Clerke Rocks. Light green indicates depths less than 700 m in which bottom fishing is prohibited. Hashed boxes with red border are additional benthic closed areas established in 2013 in which bottom fishing is prohibited. The purple borders around SSI are a 12nm pelagic no-take zone. The large black hashed area south of 60° S falls within the SGSSI Maritime Zone in a region for which no fishing licenses are issued. In all other regions of the SGMZ bottom fishing is prohibited with the sole exception of the narrow pale blue region which includes the depths between 700m and 2250m. Within this region bottom fishing is permitted by license. The region of interest for this study is delineated by the grey shaded box. The inset shows the position of the South Georgia and South Sandwich Islands MPA relative to South America, the Antarctic continent and the Polar Front (dashed line). Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Background bathymetry is The GEBCO_2014 Grid, version 20141103 (http://www.gebco.net). were cleaned using CARIS HIBS & SIPS (version 8.1) to remove erroneous points. Data collected by the Alfred Wegener Institute, Bremerhaven (AWI) over the course of 22 cruises to the region between 1985 and 2013 as well as data collected on a recent R/V Nathaniel B. Palmer cruise were processed and gridded to 100m resolution using Fledermaus (version 7.0). Single-beam bathymetry data were obtained from the UK Hydrographic Office (UKHO), BAS and commercial fisheries vessels. The spatial coverage of respective datasets is summarised in Fig. 2. were cleaned using CARIS HIBS & SIPS (version 8.1) to remove erroneous points. Data collected by the Alfred Wegener Institute, Bremerhaven (AWI) over the course of 22 cruises to the region between 1985 and 2013 as well as data collected on a recent R/V Nathaniel B. Palmer cruise were processed and gridded to 100m resolution using Fledermaus (version 7.0). Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 Methodology S d T Points that were deemed to be creating false peaks and sinks were manually removed using the ArcGIS Editor toolbox. After this iterative process, the final DEM was considered to be as accurate as possible, with a minimum amount of sampling artefact but without excessive data smoothing. Ideally, only cleaned data would be input into the interpolation. Given the number of data points involved in an analysis of this scale ( > 1.0 × 108), however, this was impractical. After gridding the data with TOPOGRID, the new interpolated 100m resolution raster data was therefore overlaid with the point cloud of original input data. Points that were deemed to be creating false peaks and sinks were manually removed using the ArcGIS Editor toolbox. After this iterative process, the final DEM was considered to be as accurate as possible, with a minimum amount of sampling artefact but without excessive data smoothing. Bathymetry Derivatives. The new 100 m resolution DEM was used to calculate five bathymetry derivative data- sets, which alongside oceanographic and net primary productivity data (summarised in Table 1) formed the basis for the landscape mapping analysis. These bathymetry derivatives included measures of slope angle, seabed rugosity, topography, aspect and curvature. Slope was calculated using LandSerf (version 2.3) multi-scale analysis. To ‘smooth out’ sampling artefacts and noise in the data, the effective resolution of the slope was reduced by introducing a window scale of 10 grid cells (i.e. 1000 m) with an inverse linear distance decay, whereby the analysis takes into account the slope value of surrounding cells (in this case with a diameter of 10) to give greater importance to cells closer to the target cell. The effect of this is to remove finer-scale variation in slope morphology but retain larger topographic features. hfi Topographic position index (TPI) was calculated using Land Facet Corridor Tools extension for ArcGIS. TPI provides a measure of whether a cell is positioned on a peak, in a depression, or in a region of constant gra- dient (flat or constant slope) relative to the surrounding cells. It can account for local scale topography versus broader-scale features by changing the size of the window of reference. For this analysis a window size of 10 was used. Terrain ruggedness index (TRI) was calculated using SAGA GIS Terrain Analysis Morphometry tools as a measure of rugosity. Methodology S d T In this way we created a continuous mosaic of masks for the entire South Georgia region with no overlapping parts and only the best available data representing each region. To avoid artificial ‘steps’ in the data, a 500m buffer was used to create a region of no-data on the boundaries between different data layers. This had the effect of ‘smoothing out’ any abrupt changes at the boundaries of adjoining data layers during interpolation. These masks were then used to extract the respective point data to input into TOPOGRID. Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 3 www.nature.com/scientificreports/ tificreports/ Figure 2. The spatial coverage of data sources used in the bathymetry compilation. Multi-beam data derived from BAS cruises are shown in green; AWI and other multi-beam (see Supplementary materials Table 1 for sources) are shown in grey; single-beam data is shown in blue and UK Hydrographic Office and coastline data is shown in red. For the remaining white areas The GEBCO_2014 Grid, version 20141103 (http://www.gebco. netdata) was used. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Figure 2. The spatial coverage of data sources used in the bathymetry compilation. Multi-beam data derived from BAS cruises are shown in green; AWI and other multi-beam (see Supplementary materials Table 1 for sources) are shown in grey; single-beam data is shown in blue and UK Hydrographic Office and coastline data is shown in red. For the remaining white areas The GEBCO_2014 Grid, version 20141103 (http://www.gebco. netdata) was used. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Figure 2. The spatial coverage of data sources used in the bathymetry compilation. Multi-beam data derived from BAS cruises are shown in green; AWI and other multi-beam (see Supplementary materials Table 1 for sources) are shown in grey; single-beam data is shown in blue and UK Hydrographic Office and coastline data is shown in red. For the remaining white areas The GEBCO_2014 Grid, version 20141103 (http://www.gebco. netdata) was used. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Ideally, only cleaned data would be input into the interpolation. Given the number of data points involved in an analysis of this scale ( > 1.0 × 108), however, this was impractical. After gridding the data with TOPOGRID, the new interpolated 100m resolution raster data was therefore overlaid with the point cloud of original input data. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Abiotic Variables Description Unit Scale Topography Digital elevation model (DEM) of bathymetry Bathymetric compilation of multibeam, singlebeam and soundings data (see Table 1 for sources) interpolated using TOPOGRID algorithm. m 100 m Slope A first derivative of DEM representing the rate of change in depth from one cell to its neighbours. ° 100 m Terrain Ruggedness Index A measure of rugosity calculated as the ratio of the three-dimensional DEM surface area to the two-dimensional planar area of a cell. — 100 m Eastness = sin(aspect/57.296) A first derivative of DEM providing a measure of the easterly orientation of the slope on a continuous scale (−1 to + 1). — 100 m Northness = cos(aspect/57.296) A first derivative of DEM providing a measure of the northerly orientation of the slope on a continuous scale (−1 to + 1). — 100 m Profile curvature A second derivative of DEM measuring the rate of change in the slope gradient. — 100 m Topographic Position Index (TPI) A measure of whether a cell is positioned on a topographic peak, in a depression on in a region of constant gradient. — 100 m Satellite derived variables Net primary productivity 5-year mean net primary productivity calculated using VGPM, a global “chlorophyll-based” model that estimate net primary production from chlorophyll using a temperature-dependent description of chlorophyll-specific photosynthetic efficiency31. Mg C/m2/day 1/12° Oceanography Three year means derived from the Proudman Oceanographic Laboratory Coastal Ocean Modelling System (POLCOMS) South Georgia high-resolution dataset29. Summer Seabed Temperature Three-year austral summer (Dec–Feb) bottom temperature mean. °C 2800 m Summer Seabed Salinity Three-year austral summer (Dec–Feb) bottom salinity mean. PSU 2800 m Summer Seabed Current U Three-year austral summer (Dec–Feb) mean measure of the easterly orientation of the current on a continuous scale (−1 to + 1). — 2800 m Summer Seabed Current V Three-year austral summer (Dec–Feb) mean measure of the northerly orientation of the current on a continuous scale (−1 to + 1). — 2800 m Summer Seabed Current Magnitude Three-year austral summer (Dec–Feb) mean measure of current magnitude m/s 2800 m Winter Seabed Temperature Three-year austral winter (Jun–Aug) bottom temperature mean. °C 2800 m Winter Seabed Salinity Three-year austral winter (Jun-Aug) bottom salinity mean. PSU 2800 m Winter Seabed Current U Three-year austral winter (Jun–Aug) mean measure of the easterly orientation of the current on a continuous scale (−1 to + 1). www.nature.com/scientificreports/ — 2800 m Winter Seabed Current V Three-year austral winter (Jun–Aug) mean measure of the northerly orientation of the current on a continuous scale (−1 to + 1). — 2800 m Winter Seabed Current Magnitude Three-year austral winter (Jun–Aug) mean measure of current magnitude m/s 2800 m Seabed Temperature Range Temperature differential between three-year summer and winter means. °C 2800 m Table 1. Abiotic variables included in the landscape mapping analysis. Table 1. Abiotic variables included in the landscape mapping analysis. to be particularly good at resolving tidal processes, topographically steered currents, and freshwater fluxes from island runoff29 to be particularly good at resolving tidal processes, topographically steered currents, and freshwater fluxes from island runoff29. f Satellite derived net primary productivity data (NPP) were accessed through Oregon State University (http:// www.science.oregonstate.edu/ocean.productivity/). Here NPP is defined as a function of chlorophyll, available light, and photosynthetic efficiency. The data are derived from the Vertically Generalized Production Model (VGPM)30, MODIS surface chlorophyll concentrations (Chlsat), MODIS 4-micron sea surface temperature data (SST4), and MODIS cloud-corrected incident daily photosynthetically active radiation (PAR). Euphotic depths are calculated from Chlsat. The data were extracted as monthly means over a five year period (2010–2014) with a grid cell resolution of 1/12 degree of latitude (~9275 m) by longitude (~5465 m). R (version 3.1.2) was used to define the geographic region of interest, create a data matrix of each month’s mean NPP, and transpose this into a raster dataset with the correct geographic projection and each grid cell pixel representing a mean NPP for five-year of monthly data. i y y To standardise the spatial extent and resolution of each input variable, each dataset was converted to the same file format (.img), spatial units (meters) and projection (South Georgia Lambert Conic Conformal, WGS1984), with each raster resampled using nearest neighbour analysis to the same spatial resolution. For the oceanographic and satellite primary productivity data which had coarser spatial resolutions (see Table 2), data were resampled to 100m using ArcGIS spatial analyst spline (with barrier) interpolation. Spline with barrier interpolation was selected on the basis of its suitability for environmental variables that change over gradients. The spline barrier used was a polygon of the coastline of South Georgia to prevent values being interpolated across the physical boundary of the island. Landscape Mapping. Methodology S d T TRI is calculated as the square root of the sum of squared difference between the bathymetric value of a cell and its 8 surrounding cells. g Aspect was calculated using the spatial analyst toolbox in ArcGIS. It provides a measure of the geographical ori- entation of a region as a circular data variable, from which we created two variables: northness = cos (aspectπ/180), as a measure of orientation on the north-south axis, and eastness = sin (aspectπ/180), as a measure of orientation on the east-west axis.i Profile curvature was calculated using the spatial analyst toolbox in ArcGIS. It is a second derivative index of bathymetry that measures the surface shape of the seabed in the steepest downhill direction quantifying the rate at which slope gradient changes. Regions with constant gradient return a value at or approaching zero, concave and convex slopes return large negative and positive values respectively. Other abiotic variables. Oceanographic data were derived from a high-resolution 3D hydrodynamic model of the South Georgia shelf and adjacent open-ocean29. The model is based on the Proudman Oceanographic Laboratory Coastal Ocean Modelling System (POLCOMS) providing salinity, temperature, current magnitude and current direction data at a spatial resolution of 2.8km. Surface and seabed data, taken as a summer (Dec–Feb) and winter (Jun–Aug) mean were averaged over three years (1999–2001). The model is validated against an exten- sive CTD dataset at South Georgia, shore-based tide gauges, and satellite temperature data. It has been shown Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Abiotic Variables PC1 PC2 PC3 PC4 PC5 PC6 Depth −0.652 — — — 0.673 — Slope — — — 0.958 — — Terrain Ruggedness Index — — — 0.959 — — Curvature — — — — — 0.807 Topographic Position Index — — — — — 0.812 Primary Productivity — — — — — — Summer Seabed Temperature — — — — 0.926 — Summer Seabed Salinity 0.951 — — — — — Summer Seabed Current U — 0.875 — — — — Summer Seabed Current V — — 0.968 — — — Summer Seabed Current Magnitude — 0.861 — — — — Winter Seabed Temperature — — — — 0.970 — Winter Seabed Salinity 0.933 — — — — — Winter Seabed Current U — 0.882 — — — — Winter Seabed Current V — — 0.974 — — — Winter Seabed Current Magnitude — 0.861 — — — — Seabed Temperature Range −0.763 — — — — — Variance Explained (%) 16.60% 18.40% 11.70% 11.80% 14.80% 7.70% Cumulative Variance (%) 16.60% 35.00% 46.70% 58.50% 73.30% 81.00% Eigenvalues 3.7053 3.3623 2.0678 1.8257 1.5012 1.3138 Table 2. Component matrix showing correlation between the Varimax rotated PCs and the original input variables. High factor loads (r < −0.6 or r > 0.6) are highlighted in bold; Low factor loads (r < −0.3 or r > 0.3) are omitted. Table 2. Component matrix showing correlation between the Varimax rotated PCs and the original input ariables. High factor loads (r < −0.6 or r > 0.6) are highlighted in bold; Low factor loads (r < −0.3 or r > 0.3) re omitted. cluster; (5) assessment of the stability of the clustering solution based on calculation of membership values and corresponding confusion indices31.ht cluster; (5) assessment of the stability of the clustering solution based on calculation of membership values and corresponding confusion indices31.ht The software used for PCA, K-means clustering, cluster validation, and raster map creation was R version 3.0.0. Given the large size of the South Georgia datasets, this was run remotely on the University of Southampton Iridis4 high performance cluster computing system high memory nodes. Principal component analysis (PCA). Principal component analysis was conducted on 19 abiotic environmental variables (Table 1). The input variables were all standardised to have zero-means and unit variance, thus giving them equal weight in the PCA. www.nature.com/scientificreports/ PCA reduces the input variables down to a new set of linearly independent vari- ables called principal components (PCs) that account for most of the variance observed in the original data32. It acts as an objective means of data reduction when dealing with multiple input variables without the need for an a priori assessment of which variables should form the basis of the analysis33, whilst removing collinearity in the data. We followed the Kaiser-Guttman criterion32 retaining only PCs with Eigen values greater than one. In order to maximise the independence of the PCs and simplify the interpretation of the factor loading pattern, a varimax rotation of the PCs was computed. These rotated PCs were the input for the K-means analysis. K-means clustering and defining optimal number of clusters. The K-means clustering algorithm is a widely used method for partitioning marine environmental data18,19,34,35. K-means works by partitioning n observations into a pre-defined k number of clusters whereby each observation is assigned to the cluster that minimises the distance of that point to the cluster centroid. The most subjective element in K-means clustering is the requirement for the input of a predefined number of clusters into the algorithm. There are several indices that can be used to define the optimal number of clusters36, with many available in R through the NbClust package37. We used two separate indi- ces that have been shown to be offer effective solutions. The C-H criterion38 evaluates the validity of different clus- ter solutions based on the proportion of the total variance explained by variance between clusters (between group sum of squares - SSB)36. The second approach was an elbow method which plots variance within clusters (within group sum of squares – SSW) against an increasing number of K-means clusters (here ranging from 2 to 15). As the number of clusters increases the SSW decreases. The optimal cluster solution is defined by a change in gradient or elbow in the graph representing the point at which increasing the number of clusters further will not greatly reduce SSW. Marine landscape and confidence map. Once the optimal cluster solution was decided, the results of the subse- quent K-means analysis (predefined for number of clusters) were converted to a raster grid in R and exported to ArcGIS to create a marine landscape map of the region at the same spatial resolution and projection as the origi- nal abiotic input variables. www.nature.com/scientificreports/ The statistical approach to mapping marine landscapes presented in this study is based on an unsupervised mapping protocol developed for shallow water shelf environments18 and subsequently adapted for use in the high–resolution analysis of submarine canyon systems19.hi Landscape Mapping. The statistical approach to mapping marine landscapes presented in this study is based on an unsupervised mapping protocol developed for shallow water shelf environments18 and subsequently adapted for use in the high–resolution analysis of submarine canyon systems19.hi The protocol for landscape mapping can be summarised in five steps. (1) principal component analysis (PCA) of the gridded environmental variables; (2) determination of the optimal cluster solution; (3) K-means clustering of the principal components; (4) plotting the optimal cluster solution as a landscape map and assigning environ- mental meaning to each cluster based on the relationship between the original environmental variables and each Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 5 Results h Bathymetric Compilation. The new 100 m resolution South Georgia Bathymetric Dataset (SGBD) (Fig. 3) covers an area of 530,000 km2, of which 54.4% (288,470 km2) is derived from multibeam data, 10.2% (54,022 km2) from singlebeam data and the remaining 35.4% (187,508 km2) is derived from The GEBCO_2014 Grid, version 20141103 (http://www.gebco.netdata). The SGBD offers significant improvements on the pre-existing bathyme- tric knowledge of the region24, specifically in terms of the inclusion of new high resolution datasets in the inter- polation, the resulting increased spatial resolution of the grid (100m), and its wider geographical extent. The grid is projected in Lambert Conformal Conic (LCC) projection optimised for South Georgia to give a more realistic representation of the shape of topographical features.hih p p p g p The high resolution of the SGBD enables us to better constrain fine-scale topographic features. The shelf is dominated by deeply incised glacial cross-shelf troughs (Fig. 3), extending from coastal fjords, in most cases to terminal moraine fields at or near the shelf break. The troughs form deep (100–160 m) and narrow depressions (~2 km) that converge further offshore to form large (up to 20 km) canyons cut into the shelf. The higher reso- lution grid enables us to delineate the extent of the shelf itself far more accurately, and pick out in much sharper detail the shelf break, including the presence of complex gully systems on the steep continental slope. The SGBD covers a bathymetric range of 0–5500 m. The inclusion of the deep sea in the compilation reveals that these regions are not homogenous but exhibit complex and sudden changes in topography (Supplementary materials Fig. 1). This includes geological features with some of the steepest slopes in the region (drop-offs from 2500 m to > 4500 m with slope angle > 45°). Landscape Mapping. PCA. Principal component analysis was conducted using 19 abiotic input variables (Table 1). After running the entire landscape mapping protocol and conducting a qualitative assessment of the output map however, aspect was excluded from the analysis as it was creating ecologically meaningless clusters in the deep sea (i.e. regions > 3000m depth with very low slope gradients but different orientation). As such the principal component analysis was re-run with the remaining 17 abiotic variables from Table 1. Retaining only PCs with Eigen values greater than 1, PCA resulted in six PCs, explaining 81% of the total variance. www.nature.com/scientificreports/ Using the highest and second highest membership value for each data point, the confusion index can be calculated for all points to offer a quantitative measure of the uncer- tainty associated with the classification of each point into clusters by K-means. The confusion index is calculated by formula 2: µ µ = − CI (2) max i i ( 1) max (2) where CI is the confusion index, μmax i is the membership value of the cluster with maximum μik at location i, and μ(max−1)i is the second largest membership value at the same i location. The CI of any given point can range between 0 and 1, with uncertainty in the K-means clustering solution between two or more clusters at that loca- tion increasing as CI approaches 1. where CI is the confusion index, μmax i is the membership value of the cluster with maximum μik at location i, and μ(max−1)i is the second largest membership value at the same i location. The CI of any given point can range between 0 and 1, with uncertainty in the K-means clustering solution between two or more clusters at that loca- tion increasing as CI approaches 1. Nested-hierarchical clustering. Once a stable landscape map is produced which minimises within-cluster vari- ance, and is qualitatively assessed to offer a good representation of the physical marine landscapes of the region, the landscape mapping protocol can begin again. This time the analysis is delineated by, and done in isolation for each of the cluster solutions derived from this first iteration. In this way a nested hierarchical clustering structure for the region is created, whereby each successive level of the hierarchy provides progressively more detail on the physical environment characterising each region. This method of hierarchically nested clustering provides a phys- ical habitat classification system that is specific to a given region of interest. This is in marked contrast to more generalised models of habitat classification40, which offer a one-size-fits-all solution that is not always ecological meaningful across all marine environments (e.g. deep-sea environments41). This hierarchical approach also offers the opportunity to assess environmental conditions across spatial scales and in turn the interaction between fau- nal communities and environment over these different scales. www.nature.com/scientificreports/ Boxplots of the abiotic input variables against the K-means cluster solutions were used to characterise and offer interpretation of the environmental conditions driving the classification of each cluster. Interpretation of these boxplots was used to assess the performance of the automated landscape classification Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 6 www.nature.com/scientificreports/ in terms of the perceived ecological meaningfulness of the classifications. Given that all input variables were standardised at the start to provide them equal weight in the PCA analysis, qualitative assessment was made as to whether any of the variables were disproportionately influencing cluster classification and if so whether these needed to be removed and the analysis re-run.i y Once the final clustering solution was achieved, the stability of the clusters was assessed by creating a separate set of cluster membership maps. Cluster membership is a methodology adapted from fuzzy K-means classifi- cation19,31,39, and is defined by calculating the relative inverse distance squared in attribute space between each individual data point and the centroids of all the K-means clusters. The sum membership value to all centroids of any given point equals 1. As such, a data point with a high membership value (approaching 1) would indicate that point is dominated by membership to one cluster. A data point with lower membership values, spread over a number of clusters, is less well characterised by membership to its K-means designated cluster. Membership value is expressed by formula 1: µ = × ∑= d 1 ( ) 1 (1) ik ik k n d 2 1 1 ( ) ik 2 (1) where μik is the membership value of the ith data point to cluster k, n is the number of clusters, and dik is the dis- tance between data point i and cluster centroid k. Using the highest and second highest membership value for each data point, the confusion index can be calculated for all points to offer a quantitative measure of the uncer- tainty associated with the classification of each point into clusters by K-means. The confusion index is calculated by formula 2: where μik is the membership value of the ith data point to cluster k, n is the number of clusters, and dik is the dis- tance between data point i and cluster centroid k. Results h Using the ‘elbow’ method, where a change in the gradient of the graph identifies the optimal cluster solution, seven clusters were assessed to be a good fit for the data. To validate this result, the C-H criterion was applied to the data with the same cascade in cluster solutions (Fig. 4b). A clear optimum of seven clusters was found, hence a final K-means clustering using seven clusters was carried out. K-Means. A total of 52,996,140 grid cells with six rotated PC variables were clustered using K-means, in a cas- cade from two to fifteen cluster solutions. The resulting within group sum of squares were plotted against number of clusters (Fig. 4a). Using the ‘elbow’ method, where a change in the gradient of the graph identifies the optimal cluster solution, seven clusters were assessed to be a good fit for the data. To validate this result, the C-H criterion was applied to the data with the same cascade in cluster solutions (Fig. 4b). A clear optimum of seven clusters was found, hence a final K-means clustering using seven clusters was carried out. Marine landscape. The results of the seven cluster K-means is presented in Fig. 5a. A description of the physical characteristics of each cluster was obtained based on interpretation of boxplots showing the correlation between the original 17 abiotic input variables and each of the seven clusters (Fig. 6). Four of the seven clusters (1, 4, 5, and 6) demonstrated a clear, distinct suite of physical conditions. Clusters 1 and 6 were characterised by the prevalence of relatively strong bottom currents in a northerly (x = 0.05 m/s) and south-easterly (x = 0.1 m/s) direction respectively. These currents appeared to be associated with large-scale, deep-sea topographic features (especially cluster 6). Cluster 4 was regionally widespread but locally spatially constrained. It demonstrated no dependence on depth or oceanography but was dominated by locally complex seabed topography. Cluster 4 exhibited steep slopes ( > 10°), high terrain ruggedness (rugosity) and a large range in curvature and topographic position values suggesting a region of topographic peaks and depressions. Cluster 5 was the most spatially discrete cluster and was characterised by shallow water; flat terrain; large annual temperature range; low salinity; and high primary productivity. We interpret this cluster as the con- tinental shelf of South Georgia and Shag Rock.hfi The three remaining clusters (clusters 2, 3 and 7) are more difficult to constrain. Results h The rotated components matrix (Table 2) shows the factor loads which explain the correlation between Varimax rotated PCs Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 7 www.nature.com/scientificreports/ Figure 3. New bathymetric compilation for South Georgia gridded to a spatial resolution of 100 m. Transects (A–F) denote depth-profile plots shown in Supplementary materials Fig. 1. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]) TOPOGRID (Spatial Analyst Tools) to grid datasets listed in Supplementary materials Table 1. www.nature.com/scientificreports/ Figure 3. New bathymetric compilation for South Georgia gridded to a spatial resolution of 100 m. Transects (A–F) denote depth-profile plots shown in Supplementary materials Fig. 1. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]) TOPOGRID (Spatial Analyst Tools) to grid datasets listed in Supplementary materials Table 1. Figure 3. New bathymetric compilation for South Georgia gridded to a spatial resolution of 100 m. Transects (A–F) denote depth-profile plots shown in Supplementary materials Fig. 1. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]) TOPOGRID (Spatial Analyst Tools) to grid datasets listed in Supplementary materials Table 1. Figure 3. New bathymetric compilation for South Georgia gridded to a spatial resolution of 100 m. Transects (A–F) denote depth-profile plots shown in Supplementary materials Fig. 1. Figure was created using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]) TOPOGRID (Spatial Analyst Tools) to grid datasets listed in Supplementary materials Table 1. (with Eigen values > 1) and the original abiotic input variables and excludes any factor loads < 0.3. Assessment of the rotated components matrix showed only one variable (depth) had a high factor load for multiple PCs, all other variables had an exclusive relationship with specific PCs. PC 1 had high loads (r < −0.6 or r > 0.6) for the varia- bles depth, seabed temperature range and seabed salinity; PC 2 for current magnitude (specifically on an easterly axis); PC 3 for current on a northerly axis; PC 4 for slope gradient and terrain ruggedness; PC 5 for depth and seabed temperature; and PC 6 for curvature and topographic positioning. Primary productivity was not related to a specific PC, but did play a role in the distinction between classes (see below). K-Means. A total of 52,996,140 grid cells with six rotated PC variables were clustered using K-means, in a cas- cade from two to fifteen cluster solutions. The resulting within group sum of squares were plotted against number of clusters (Fig. 4a). Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Optimal K-means cluster solution. Calculated as, (a) the number of clusters versus the Calinski- Harabasz (C–H) criterion, whereby the optimal cluster solution corresponds to the first local maximum of the C-H value; and (b) the number of clusters versus the within group sum of squares based on Varimax rotated PCs, whereby the optimal cluster solution is identified by an ‘elbow’ or change in the gradient of the slope. For both indices the best solution is identified as 7 clusters (marked in red). Figures created using R (version 3.0 [www.r-project.org]). Figure 4. Optimal K-means cluster solution. Calculated as, (a) the number of clusters versus the Calinski- Harabasz (C–H) criterion, whereby the optimal cluster solution corresponds to the first local maximum of the C-H value; and (b) the number of clusters versus the within group sum of squares based on Varimax rotated PCs, whereby the optimal cluster solution is identified by an ‘elbow’ or change in the gradient of the slope. For both indices the best solution is identified as 7 clusters (marked in red). Figures created using R (version 3.0 [www.r-project.org]). extremities of the South Georgia shelf and the lower gradient regions of the slope represented by cluster 7. The only bathymetric exclusion from cluster 7 were shelf environments with depths < 250 m. Confidence map. The confusion index map (Fig. 7) provided an assessment of the confidence values associated with the membership of each grid square to its given cluster defined through K-means partitioning. In general under a good cluster solution, high confusion values (denoted by white in Fig. 7) would be expected to only occur in the ‘transition zone’ on the boundaries between clusters. Confusion within clusters however would imply the clustering solution is not particularly stable and that those particular grid-cells do not have a strong affinity with a single K-means cluster.i g Mapping the confusion indices revealed high confidence in the clustering of the South Georgia shelf (and to a lesser extent Shag Rocks). There was very little within-cluster confusion, which was restricted to the edges of cross-shelf troughs (presumably as a result of seabed topography), inshore shelf (presumably resulting from a distinct salinity and temperature gradients) and at a single anomaly on the south-western edge of the shelf break. With these exceptions the cluster is delineated by a well-defined uncertainty ‘halo’. Results h All three clusters have a deep-sea element (depths > 2500 m). Cluster 3 had a large but northerly-restricted distribution. It exclusively represented deep-sea environments with low sea-bed temperature (x = 0.5 °C), but was primarily defined by its significantly higher sea-surface primary productivity (x = 420 Mg C/m2/day). Cluster 2 also represented exclu- sively deep-sea (2500–4500 m) environments with low sea-bed temperature (x = 0.75 °C). It had a largely, but not exclusively southern spatial distribution. However it was the drivers of other clusters (primary productivity for cluster 3 and temperature for cluster 7) that distinguished them from it rather than its own characterisation that sets it apart. As such depending on the interpretation of the importance of surface primary productivity on deep-sea benthic environments (cluster 3) it is entirely plausible to group clusters 2 and 3 together as a generic deep-sea, flat abyssal plain with stable low temperature.h l Cluster 7 was characterised by annually-stable, warmer sea-bed temperatures (x = 1.4 °C). Though mostly occurring in depths of 2000–3000 m the overall bathymetric range of the cluster was wide with the deeper 8 Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 www.nature.com/scientificreports/ Generally there was an increased level of background confusion (most notably in deep water) as a result of sampling artefact caused by high-resolution multibeam swath lines transecting regions of lower data quality. This was notable across clusters but was particularly apparent in the bottom-left quadrant of the plot that had been subject to a large-scale AWI multibeam survey (Fig. 2).ii Both clusters defined by prevailing currents (clusters 1 and 6) were characterised by regions of well-defined zones of lower uncertainty. Cluster 3 was subdivided by a well-defined line of uncertainty forming an arc in the top-centre of the map. Inspection of the bathymetry dataset revealed the arc demarked the contour of a large topographic dome 100 km in diameter and 1000 m in height, with the uncertainty reflecting this change in topography (potentially conflicting with cluster 4). In contrast the boundary of clusters 2 and 3 in the top-right quadrant were not delineated by any uncertainty. The major driver of the separation of these two clusters was Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 9 www.nature.com/scientificreports/ Figure 5. Hierarchically nested marine landscape maps. Showing (a) distribution of 7 cluster classes across the whole study region as defined by k-means cluster analysis; (b) re-clustering of cluster 5 taken from first k-mean partition (Fig. 5a) whereby the shelf (previously a single cluster) is now split into 6 sub-clusters; and (c) re-clustering of cluster 5 - sub-cluster 5 (Fig. 5b) whereby sub-cluster 5 is partitioned into 7 further third-tier clusters. Data for figures gridded in R (version 3.0) and visualised using ArcGIS (version 10.1 [www.esri.com/ software/Arcgis]). Figure 5. Hierarchically nested marine landscape maps. Showing (a) distribution of 7 cluster classes across the whole study region as defined by k-means cluster analysis; (b) re-clustering of cluster 5 taken from first k-mean partition (Fig. 5a) whereby the shelf (previously a single cluster) is now split into 6 sub-clusters; and (c) re-clustering of cluster 5 - sub-cluster 5 (Fig. 5b) whereby sub-cluster 5 is partitioned into 7 further third-tier clusters. Data for figures gridded in R (version 3.0) and visualised using ArcGIS (version 10.1 [www.esri.com/ software/Arcgis]). higher net primary productivity occurring in cluster 3. Inspection of the primary productivity data revealed an abrupt change in primary productivity at this boundary. This lack of a gradual change would explain the subse- quent confidence in the cluster designation. www.nature.com/scientificreports/ The certainty of cluster 4 was hard to quantify given it consisted of predominantly spatially small topographic features. In general however the well constrained clusters of lower uncertainty delineated by narrow transition zones between clusters supports the K-means partition as robust physical landscape classification of the region. Nested Hierarchical Clustering. Given the stability and spatially discrete nature of the shelf cluster, cluster 5 was selected to test the functionality of a nested hierarchical clustering solution. The original abiotic input datasets were clipped to the spatial extent of cluster 5. The landscape mapping protocol was then re-run to create a new set of shelf sub-clusters (Fig. 5b,c). Assessment of the shelf sub-clusters (Supplementary materials Fig. 2) revealed that annual temperature range, salinity, and slope dominated near-shore locations (cluster 2). A combination of depth (cluster 1), stronger current regimes (cluster 3 and 4), and complex topography (cluster 6) dominated on the outer shelf locations. A body of annually-stable cold bottom water drove designation of the large spatial coverage of sub-cluster 5. A third nested clustering of this sub-cluster 5 partitioned the shelf environment further still, delineating seven distinct clusters (Fig. 5c, Supplementary materials Fig. 3). Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 10 www.nature.com/scientificreports/ Figure 6. Box plots of K-means derived clusters versus 17 original abiotic variables. The x-axis seven k-means clusters, and the y-axis the respective units of each original abiotic variable. Descrip variable including their units are summarised in Table 1. In each box plot the middle line represent the upper and lower extent of the box represent the first and third quartiles. The whiskers are the m and minimum observed values (excluding statistical outliers - values > 1.5 x the interquartile range colours denote the corresponding landscape map cluster colours from Fig. 5a. Figures created in R [www.esri.com/software/Arcgis]). Figure 6. Box plots of K-means derived clusters versus 17 original abiotic variables. The x-axis denote the seven k-means clusters, and the y-axis the respective units of each original abiotic variable. Descriptions of each variable including their units are summarised in Table 1. In each box plot the middle line represents the median, the upper and lower extent of the box represent the first and third quartiles. The whiskers are the maximum and minimum observed values (excluding statistical outliers - values > 1.5 x the interquartile range). Box plot colours denote the corresponding landscape map cluster colours from Fig. 5a. Discussion Th T The TOPOGRID algorithm, as in previous studies24–26 proved a robust methodology for gridding a digital ele- vation model from a compilation of disparate datasets, integrating spatially discontinuous data with different sampling densities. The input bathymetry data varied considerably in terms of the state of its post-processing. Approximately ten iterative cycles were required to obtain a final DEM, cleaning most erroneous soundings. TOPOGRID was notably sensitive when interpolating joins between bathymetry datasets. Hence a large buffer of 500m was created to smooth the boundary zone.f The TOPOGRID algorithm, as in previous studies24–26 proved a robust methodology for gridding a digital ele- vation model from a compilation of disparate datasets, integrating spatially discontinuous data with different sampling densities. The input bathymetry data varied considerably in terms of the state of its post-processing. Approximately ten iterative cycles were required to obtain a final DEM, cleaning most erroneous soundings. TOPOGRID was notably sensitive when interpolating joins between bathymetry datasets. Hence a large buffer of 500m was created to smooth the boundary zone.f y When compiling and interpolating multiple disparate bathymetry datasets into a DEM, the effective resolu- tion of each grid is limited to, but not necessarily equal to, the pre-defined resolution (in this case 100m). As such when the interpolated bathymetric grid is used to create derivative datasets for landscape mapping, areas where the interpolation is underpinned by many data points will contain far more detail (albeit limited to 100m resolu- tion) than regions of sparse data which appear comparatively smooth. The effect of this is that these high-density data regions appear to have higher topographic complexity (e.g. high rugosity) when in fact the model is simply recording an artefact of sampling provenance. Rather than gridding the DEM at the resolution of the coarsest resolution data (in this case ~900 m), and in doing so removing fine-scale topographic details, we advocate the high-resolution analysis undertaken here with the caveats discussed taken into account when qualitatively assess- ing partitioning of environmental parameters. The statistical approach to marine landscape mapping adopted in this study was proposed by Verfaillie et al.18. The utilisation of a statistical approach is intended to remove highly subjective decisions associated with classical landscape mapping protocol42,43, namely the selection of ecologically relevant abiotic input variables and the classifying of those variables into relevant classes. www.nature.com/scientificreports/ Figures created in R (version 3.0 [www.esri.com/software/Arcgis]). Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 11 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Confusion Index map quantifying clustering uncertainty across the study region. The confusion index is overlaid on the seven-cluster landscape map showing where zones of higher uncertainty (white) correspond with the boundaries between clusters and where there are instances of intra-cluster uncertainty. Data for figures gridded in R (version 3.0 [www.esri.com/software/Arcgis]) and visualised using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Figure 7. Confusion Index map quantifying clustering uncertainty across the study region. The confusion index is overlaid on the seven-cluster landscape map showing where zones of higher uncertainty (white) correspond with the boundaries between clusters and where there are instances of intra-cluster uncertainty. Data for figures gridded in R (version 3.0 [www.esri.com/software/Arcgis]) and visualised using ArcGIS (version 10.1 [www.esri.com/software/Arcgis]). Discussion Th T So whilst the process starts as an unsupervised classification based on statistical clustering, the resultant landscape map is qual- itatively assessed on the basis of perceived biological, oceanographic and geomorphological meaningfulness. Any input variable deemed to be confounding the results is removed from the analysis and the protocol is re-run. For example, after the first iteration of this analysis, slope aspect was excluded from the analysis as deep-sea environ- ments with relatively low slope gradients (<3°) were being partitioned based on minor variations in orientation of the slope. The aim of this approach is to strip down the analysis to retain only partitions with potential ecological relevance at a broad spatial scale. The process can then be re-run over iteratively smaller spatial subsets forming a nested hierarchical landscape classification (Fig. 5). As the importance of input variables as ecological drivers is scale-dependent, all input variables were included at the start of each new clustering level. This approach retains broad-scale gradients driving clusters across the entire region whilst identifying finer-scale geomorphic features nested within these broad-scale clusters.i A significant omission from this analysis is substrate type, which has been shown as a major driver of species composition45. It was not included in this analysis given the limited spatial extent of the sedimentology dataset in the region, and as such the difficulty in interpolating across such a wide area. Backscatter data were available for the regions with multibeam coverage (see Fig. 2), but interpretation was particularly sensitive to ship-specific calibrations which made a backscatter compilation problematic. It was our assessment, therefore, that the inclu- sion of geomorphology data layers in the PCA (slope angle, topography, rugosity) should effectively represent different sediment regimes in the analysis. For example homogenous flat regions are likely to be mud at South Georgia and steep escapements hard substrate. The downside of this approach is the spatial scale of the analysis will tend to pick out large-scale features (e.g. large escarpments, canyons, and gullies) but not sedimentary fea- tures with lower topographic profiles such as moraine fields, which have been suggested as important driver of marine biodiversity46.f y Our results demonstrate that the iterative unsupervised landscape mapping protocol is effective at creating meaningful (Fig. 5) statistically stable (Fig. 7) partitions. Depth, current, and seabed topography are all shown to drive broad-scale clustering. Discussion Th T At a finer scale on the South Georgia shelf, current regimes were shown to be less important with clustering driven by depth, sea-bed temperatures, coastal salinity, and topography (Fig. 5b,c, Supplementary materials 1 and 2). Taken in isolation this analysis provides valuable information pertaining to the nature of different physical habitats, their spatial distribution, and degree of heterogeneity20. In itself, this provides useful information for policy makers in terms of physical proxies for faunal richness with application in predictive modelling of taxa, functional traits, assemblages, and diversity13,21,47.h g g y The next step will now be to quantify the biological meaningfulness of our physical landscape partitions. In the first instance, this will involve the inclusion of a regional biological dataset into the analysis22, to test for cor- relations between our proposed hierarchical clusters and biogeographical trends. The advantage of a landscape mapping approach for underpinning large-scale MPAs such as at South Georgia and South Sandwich Islands (SGSSI), as opposed to more holistic bottom-up approaches that integrate biological data in the analysis from the outset48–50, is largely on account of the spatial scale of the analysis. For example, although at South Georgia more than 25,000 biological point samples have been recorded22, spread over an area in excess of 1 million km2, the data exist at a spatial scale that is of limited application to draw statistically meaningful conclusions. Furthermore knowledge of benthic communities rapidly declines as a function of distance from the South Georgia continental shelf, with significant paucity in sampling of the region’s deep-sea environments. The biological data will be inval- uable, however, in testing the output of the model to see how well it acts as a proxy for biological distributions. In doing this it will be important to better constrain other confounding influences on regional biogeography such as, paleo-environment and the presence of glacial refugia during the last glacial maximum51; the impact of iceberg scouring on biological communities52,53; and the effect of bathymetric divides (such as between South Georgia and Shag Rocks) as barriers to genetic transfer54,55.f g g Given sampling effort for many groups is uneven and most species in the region are rare, inferring distribution patterns at high taxonomic resolution (i.e. genus or species) over a large spatial extent will be problematic. Discussion Th T An objective means of defining partitions in the physical environment should be considered particularly useful for regions such as South Georgia where proposed and reviewed hierarchical definitions40 have not yet been established. This study represents the first application of this methodology over such a large regional scale (107 km2). The results demonstrate that a statistical protocol is highly effective at reducing large numbers of sometimes collinear environmental input variables to a smaller number of relevant principal components. It is also effective at defining the optimal number of clusters and the spatial delineation of those clusters. p Adopting a ‘blind’ statistical approach to the data partitioning is problematic, however, as it does not remove all need for subjective supervision of the abiotic input variables and a careful evaluation of the process and its results is necessary. Firstly, PCA will remove collinearity in the data but if the model input includes for example many oceanographic variables and few topographic derived variables, then the output will be oceanographic-centric placing greater emphasis on the importance of these variables than anything else. Hence a balanced choice of input variables is needed. Secondly, K-means finds similar sized clusters based on spherical partitions in the multi-dimensional PC space. This is not always a correct representation of the reality, as meaningful clusters can vary in spatial extent and shape: for example, large numbers of data points might be clustered as deep-sea envi- ronments with fairly wide-ranging characteristics, whilst smaller rocky outcrops that punctuate that homogenous region would have much tighter environmental constraints. An alternative approach could be density-based spa- tial clustering (e.g. DBSCAN44), in which clusters are not restricted to a spherical designation. Such an approach may also be advantageous to large-scale datasets due to the ‘noise’ of data outliers that exists in datasets of this scale. The disadvantage of such an approach, however, is the requirement for a definition of high point-density. Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 12 www.nature.com/scientificreports/ Finally, the protocol ensures that input variables are standardised and given equal weighing in the PCA. Not all input variables will drive biological distribution patterns to the same degree, but for areas that have not been studied much before, it is impossible to estimate these varying degrees of importance a priori.i p y g g p p To resolve these problems, our protocol is based on iterative refinement of each cluster solution. Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 Discussion Th T More achievable (and perhaps with greater application in spatial planning) would be the mapping of a standardised (for sampling effort) measure of species level biodiversity to identify the presence of ‘biodiversity hotspots’ and correlate these with our physical landscape clusters. This approach would be helpful in understanding the role of habitat heterogeneity as a potential proxy for biodiversity (i.e. beta diversity). In addition to this, it would also be feasible to categorise certain taxa into functional groups (based on traits such as feeding, locomotive and repro- ductive strategies) and assess the relationship between these faunal aggregations and the clusters solutions. This could be used to assess the distributions of vulnerable marine ecosystems or habitat forming taxonomic groups. y g g p It is also important to consider what the priorities are in terms of marine management. For example is it the presence of rare or endemic species ? If so then the methodology will have to reflect the fact that most species are rare22, so distribution is not well constrained. Is it the presence of species richness zones ? Or the presence of habitat forming fauna including vulnerable marine ecosystems (VMEs) such as coral gardens and sponge assemblage ? Alternatively benthic communities that offer an ecosystem service such as fisheries or carbon seques- tration46 may be considered of importance to identify. The marine landscape map produced here provides a first-level, baseline picture of the spatial pattern in the marine regions around South Georgia, and can be used as a tool to start developing a method to answer each of the questions above. Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 13 www.nature.com/scientificreports/ References e e e ces 1. Butchart S. H. M. et al. Global biodiversity: Indicators of recent declines. Science 328, 1164–1168, doi: 10.1126/science.1187512.2 (2010). 2. Hooper, D. U. et al. A global synthesis reveals biodiversity loss as a major driver of ecosystem change. Nature 486, 105–108, doi 10.1038/nature11118 (2012). 3. Ceballos, G. et al. Accelerated modern human–induced species losses: Entering the sixth mass extinction. Science Advances 1(5) doi: 10.1126/sciadv.1400253 (2015). 4. Halpern, B. 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Conclusionsh The methodology presented here provides an objective assessment of the physical attributes of the benthic environment over nested spatial scales, providing analysis from broad-scale drivers of biogeography such as large-scale (10–100 s km) abiotic gradients (e.g. depth and temperature) to smaller-scale features (100 m–1 kms) such as local topography. Currently this level of analysis on the factors driving biogeography is lacking from most marine spatial planning frameworks. We argue that it provides the potential for a holistic overview of the marine environment, and meaningful information to aid policy-makers to manage the region’s marine environment. Furthermore the approach is adaptable to different input variables and as such transferable globally to different proposed or established MPAs. References et al. Why is the South Orkney Island shelf (the world’s first high seas marine protected area) a carbon immobilization hotspot ? 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Griffiths, H. J., Linse, K. & Barnes, D. K. A. Distribution of macrobenthic taxa across the Scotia Arc, Southern Ocean. Antarctic Science 20(3), 213–226 (2008). Acknowledgementsh g This work was supported by the National Environmental Research Council [grant number NE/L002531/1] and builds on results from the CODEMAP project (ERC Starting Grant 258482), through which V. Huvenne is funded. The authors acknowledge additional bathymetry data provided with courtesy of the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research and the National Science Foundation Grant PLR 1246111 to I.W.D. Dalziel and L.A. Lawver at the Institute for Geophysics, The University of Texas at Austin. The authors acknowledge the use of the IRIDIS High Performance Computing Facility, and associated support services at the University of Southampton, in the completion of this work. 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Marine Biodiversity and Ecosystem Functioning: Frameworks, methodologies, and integration. (Oxford, 2012). g 6. Barnes, D. K. A. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Hogg, O. T. et al. Landscape mapping at sub-Antarctic South Georgia provides a protocol for underpinning large-scale marine protected areas. Sci. Rep. 6, 33163; doi: 10.1038/srep33163 (2016) How to cite this article: Hogg, O. T. et al. Landscape mapping at sub-Antarctic South Georgia provides a protocol for underpinning large-scale marine protected areas. Sci. Rep. 6, 33163; doi: 10.1038/srep33163 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports \| 6:33163 \| DOI: 10.1038/srep33163 15
https://openalex.org/W3162067739	http://kau.diva-portal.org/smash/get/diva2:1554166/FULLTEXT01	English	null	Reevaluating the Influence of Leaders Under Proportional Representation: Quantitative Analysis of Text in an Electoral Experiment	Frontiers in psychology	2,021	cc-by	8,547	http://www.diva-portal.org http://www.diva-portal.org This is the published version of a paper published in Frontiers in Psychology. Citation for the original published paper (version of record): Fredén, A., Sikström, S. (2021) Reevaluating the Influence of Leaders Under Proportional R Quantitative Analysis of Text in an Electoral Experiment Frontiers in Psychology, 12: 1-8 https://doi.org/10.3389/fpsyg.2021.604135 Access to the published version may require subscription. Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. N.B. When citing this work, cite the original published paper. Copyright © 2021 Fredén and Sikström. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-83997 ORIGINAL RESEARCH published: 12 May 2021 doi: 10.3389/fpsyg.2021.604135 Edited by: Kim F. Nimon, University of Texas at Tyler, United States Reviewed by: Kathrin J. Hanek, University of Dayton, United States Jan Ketil Arnulf, BI Norwegian Business School, Norway Oyvind Lund Martinsen, BI Norwegian Business School, Norway Correspondence: Sverker Sikström sverker.sikstrom@psy.lu.se Keywords: leaders, parties, voting, primacy, proportional representation, statistical semantics Annika Fredén1 and Sverker Sikström2 Annika Fredén1 and Sverker Sikström2* 1 Department of Political, Historical, Religious and Cultural Studies, Karlstad University, Karlstad, Sweden, 2 Department of Psychology, Lund University, Lund, Sweden We propose that leaders play a more important role in voters’ party sympathy in proportional representation systems (PR) than previous research has suggested. Voters, from the 2018 Swedish General Election, were in an experiment asked to describe leaders and parties with three indicative keywords. Statistical models were conducted on these text data to predict their vote choice. The results show that despite that the voters vote for a party, the descriptions of leaders predicted vote choice to a similar extent as descriptions of parties. However, the order of the questions mattered, so that the ﬁrst questions were more predictive than the second question. These analyses indicate that voters tend to conﬂate characteristics of leaders with their parties during election campaigns, and that leaders are a more important aspect of voting under PR than previous literature has suggested. Overall, this suggests that statistical analysis of words sheds new light of underlying sympathies related to voting. INTRODUCTION Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Most current election studies measure political sympathy through approval rating scales (see for example, Mueller, 1970; Van der Eijk and Franklin, 2009; Oscarsson and Holmberg, 2013). However, a measure like a score on a scale tells little about the contents of the voter’s evaluation. What role does the leader play? How much relates to policy? This study approaches party preference from a new angle, asking voters directly what they think about when they think about parties, and to what extent leaders intertwine with descriptions of the party. The focus of the study is proportional representation systems (PR), where electoral studies tend to center on ideology, parties and party identiﬁcation rather than leaders (Granberg and Holmberg, 1988; McCall Rosenbluth and Shapiro, 2018; Oscarsson and Holmberg, 2020). Nevertheless, the party leaders should be important as spokespersons and concrete representations of policy orientation, especially in a political landscape where many voters switch parties from one election to the next (Fieldhouse et al., 2020). This study argues for the inclusion of leader perceptions in studying voters’ behavior, also under proportional representation. Received: 09 September 2020 Accepted: 14 April 2021 Published: 12 May 2021 Citation: We collected voters’ free text descriptions in a real-life election campaign—the 2018 Swedish General Election. The party system contains a large number of smaller parties, which makes it possible to examine the inﬂuence of leaders for those too. In order to emphasize the party vs. the leader in the experiment, half of the sample was randomly assigned to describe the leaders ﬁrst, whereas the other half started by describing the parties. Drawing on ﬁndings from the psychology literature (Murdock, 1962; Sullivan, 2019), the belief was that a primacy eﬀect should make a statement that comes ﬁrst matter more for the voting decision than a statement that comes after, independently of whether it concerns the party, or the leader. From these perspectives, the overarching expectation is that voters’ descriptions of leaders during election times are equally important for their choices as their descriptions of the parties. Their respective predictive powers will also depend on the order of the descriptive task, since more important, concrete and consistent descriptions should be remembered earlier. These claims are supported by the following hypotheses. In current media, the party leader is the concrete representation of the abstract concept of a party. Because concrete and simple representations are usually easier to understand and remember (see e.g., Kahneman, 2011), the hypothesis is that the leader representation will be essential for shaping the voter’s associations to a party. At the same time, party policies are important shortcuts for orienting oneself in a party system with a clear left-right ideological spectrum. The argument is that voters under proportional representation can have diﬃculties separating leaders’ policy messages from their parties, and parties from their leaders. Citizens thus need both representations: the concrete of the leader, and the more stable ideological reference to the party, to form an association of a political unit. This leads to the ﬁrst hypothesis: The text descriptions were analyzed using latent semantics, which is a natural language processing (NLP) approach to quantitative text (Landauer and Dumais, 1997) which we combine with machine learning (ML) to predict voting behavior. This method allows examination of how respondents’ descriptions of parties and leaders co-occurred, and how these descriptions can be related to vote choice. In line with the argument, the descriptive words of leaders and parties predicted vote choice to the same extent, whereas the order of questions mattered. Citation: Fredén A and Sikström S (2021) Reevaluating the Inﬂuence of Leaders Under Proportional Representation: Quantitative Analysis of Text in an Electoral Experiment. Front. Psychol. 12:604135. doi: 10.3389/fpsyg.2021.604135 May 2021 \| Volume 12 \| Article 604135 1 Frontiers in Psychology \| www.frontiersin.org Quantitative Analysis of Text Fredén and Sikström The focus on voters’ own responses in the present study is rather unique: so far, the materials that are the focus in related studies are usually party manifestos, press releases or related materials (Klüver and Sagarzazu, 2016; Crabtree et al., 2018). When leaders are the focus, the current trend is survey experiments where leader qualities are experimentally manipulated (see for example Tavares et al., 2018). Fewer studies refer to “real” political leaders, which is the starting-point in this study. Media scholars have been somewhat more tempted to follow this path where, for example, Aaldering et al. (2018) start from the perspective that the tone of the media coverage of leaders has a mediating impact on the propensity to vote for a party. Still, current research tends to look at leader impact during election campaigns more generally, without asking the voters themselves. Sullivan, 2019). More important for the present study, is that text written early tends to carries more important content. In particular, Kjell et al. (2019) showed a semantic primacy eﬀect, where words generated early in the description of a mental state were more predictive of rating scale scores, than words generated later. This ﬁnding matches the current experiment well, in the sense that the descriptions that voters give ﬁrst should be more strongly associated with vote intention than the descriptions that they give later. The words that the voter comes up with ﬁrst are the words that are most easily accessible, and represent the voter’s primary view of a political unit (i.e., the mental representation of the party and/or the leader), whereas words that generated later are less informative the voter’s representation of the political unit. Following this line of argument we propose that: Primacy hypothesis (H2). In the condition where voters are asked to ﬁrst describe leaders and then describe parties, the description of leaders will be a more important indicator of vote choice than the description of parties. The opposite pattern will be found in the conditions were voters are asked to describe parties ﬁrst. Citation: The words that the respondent gave ﬁrst predicted the vote intention better that the words that came second. Frontiers in Psychology \| www.frontiersin.org 1The Social Democrats, the largest party in Sweden, was not included in the survey. However, their main competitor, the Moderates, is included. Since the previous dominance of Social Democrats in Sweden is an exception rather than rule in similar PR contexts (see, for example, McCall Rosenbluth and Shapiro, 2018), this sample should still be suﬃciently representative for established proportional representation party systems today. MATERIALS AND METHODS The case for the study is the proportional representation system of Sweden, which was long dominated by the single party Social Democrats governments. More recently it has oriented toward coalitions of parties (Bäck and Bergman, 2016; Fredén, 2021). The party system of 2018 consisted of three bigger parties (the Social Democrats, the Moderates, and the Sweden Democrats) and ﬁve smaller parties (Greens, Liberals, Left party, Centre party, and Christian Democrats). The focus of the present study is the parties that characterize these types of PR systems, namely, these smaller parties. One circumstance that could direct voters more toward leaders over parties in general is if the parties coordinate before the election, or if the parties run more independently. If the negotiations between the parties after the election are supposed to matter more, that is, if the blocs are more loosely organized, then, candidate evaluations potentially matter more Leader conﬂation hypothesis (H1). The words a voter uses to describe a party leader tend to be similar and are at least as indicative for his or her vote choice as the words used to describe the party. The second hypothesis concerns how the order of the descriptive task potentially aﬀects the predictive powers of free text descriptions. A well-studied eﬀect in the memory literature is the primacy eﬀect (e.g., Murdock, 1962). This eﬀect shows that items that are presented ﬁrst are usually better remembered than items presented later. The theoretical basis for the primacy eﬀect is not fully understood, however, a view typically taken in the literature relates to the ﬁrst items receives more attention or are rehearsed more than the later items (Anderson and Hubert, 1963; May 2021 \| Volume 12 \| Article 604135 Frontiers in Psychology \| www.frontiersin.org 2 Quantitative Analysis of Text Fredén and Sikström since the leaders will then have a crucial role in the post-election negotiations. In the 2018 general election, the parties competed more independently than in the previous elections (Aylott and Bolin, 2019). Three of the parties had new party leaders since the previous election (the Greens, the Christian Democrats, and the Moderates), and three of the parties were at risk of not reaching the four percent electoral threshold (the Greens, the Christian Democrats, and the Liberals). Selection of Political Parties For pragmatic reasons, we had to select a smaller number of parties to include for the descriptive task in the experiment. Including too many parties in the survey experiment would also have made the task more cumbersome and risk increasing participant fatigue. Previous political science research mainly focuses on leader eﬀects of bigger parties (compare research from the US context as well as previous research on the Swedish context such as for example Oscarsson and Holmberg, 2016). Here, the focus is on party characteristics that are typical for proportional representation, that is, smaller parties whose fortune is more insecure during elections times, and where the leader may play a less salient role. The survey includes the three smallest parties that were at risk of not reaching electoral representation—the Liberals, the Greens, and the Christian Democrats—and the major right-wing party, the Moderates, which was a potential leader of government. This implies a mix of parties in terms of size, their positions on the left-right-scale, as well as the gender of the leader (two male leaders, and two female leaders). In order to draw conclusions about potential leader eﬀects under proportional representation, this sample of four parties should thus serve as a relevant reference1. Sample The sample consists of self-recruited participants, who participated in the survey voluntary (with no extra reward). Eleven thousand six hundred twenty-one were invited to take the survey experiment, and 58% (6,776) responded. Mullinix et al. (2015) show that convenience samples, in general, generate eﬀects that are very similar to population-based samples. Since the main interest here is the global relationship between party and leader perceptions, rather than contents, levels of support or word counts concerning speciﬁc parties, sample characteristics should matter relatively little (compare Mutz et al., 2019). The number of unique words is high: 10,010 related to parties and 8,165 related to leaders. Most important, standard socio-economic characteristics are evenly spread between the randomized treatment groups. Respondents come from all age groups, education levels and gender (for more detailed information of sample characteristics, see the Supplementary Table A1). Also party support is evenly spread between the two treatment groups. Supporters of the main parties Social Democrats and Moderates are underrepresented compared with election results, whereas supporters of smaller parties Study Design g The aim of the study was to collect evaluations of political parties and their leaders in a real-life campaign using a survey experimental design, where we would (1) examine voters’ leader descriptions in relation to their party descriptions (2) examine the impact of priming the respondent with the leader descriptive task vs. the party descriptive task. The experiment was part of a methods-oriented survey at the Swedish National Election Studies Program/LORe Internet Campaign panel, managed by the SOM-Institute, University of Gothenburg. It was released 2 weeks before the general election on 9 September 2018 (respondents continued to submit their responses up to the Election Day, but most of the respondents submitted their answers in the period 25–31 August). Before entering the study, participants agreed to participate by accepting the data and investigation procedures in the LORe Internet campaign panel, in accordance with current ethics and GDPR standards. MATERIALS AND METHODS The presence of a strengthened populist party, the Sweden Democrats, oriented the campaign toward issues as well as the four percent electoral threshold, since the established blocs needed the smaller parties to reach the threshold to survive as government alternatives. One of the main opinion polls indicated a tight race between the three bigger parties Social Democrats, Moderates, and Sweden Democrats (Bergman, 2018) and most polls suggested a close race between the traditional left-socialist bloc and the center-of-right bloc (see for example Sifo, 2018). are overrepresented (compare Valmyndigheten, 2019, and Supplementary Table A2 for distributions of vote intentions over treatments in this experiment). Seventy-nine percent of the respondents indicated that they were very certain about their party choice when they took the survey experiment (corresponding to 6 or 7 on a scale from 1 to 7, where 1 stands for not certain at all, and 7 for absolutely certain). The study sample is thus a group of relatively convinced voters. Since the impact of leaders on choice may be stronger among volatile and unknowledgeable voters (Oscarsson and Holmberg, 2016), the potential leader inﬂuence on the perception of a political party should not be particularly great here. Instead, the experiment should rather underestimate than overestimate primacy eﬀects and leader conﬂation. Frontiers in Psychology \| www.frontiersin.org Method: Latent Semantic Analysis Predicting Voting From Text Data The novelty of this study is to collect free text descriptions of political units (leaders and parties), as a complement to the standard approval rating scales. The primary interest was to study how well these three keywords generated by the participants predict their voting behavior, and to what extent priming respondents with one descriptive task over the other would inﬂuence the results. To our knowledge, the best methods for doing this builds on a combination of NLP and ML. NLP methods allow quantiﬁcation of texts (e.g., keywords) to a high dimensional representation to which an individual’s word descriptions are compared. ML allows us to investigate whether this representation predicts an outcome variable, which in our case is voting behavior. To do this, we used Latent Semantic Analysis (LSA), a quantitative text analytical approach that quantiﬁes and systematize voters’ responses. This data-driven (unsupervised) method is suitable for measuring meaning in word expressions by quantifying how similar the words are to each other. The method resembles factor analysis, since words that are similar in meaning receives similar semantic representations. In this study, we ﬁrst created a high dimensional (N = 300) semantic representation based on the 135,806 words in the dataset. A semantic space was created based on the words generated by the participants. The method is described in detail in Kjell et al. (2019) (see also Landauer and Dumais, 1997). First a word-by-word co-occurrence matrix is created where each cell The semantic similarity score (SS) between two sets of words is calculated by taking the cosine of the angle between two associated semantic vectors, which in this case is mathematically equivalent with multiplying each dimension with each other and summing them. This score, bounded between -1 and +1, which is high when the word sets are similar in meanings and small when they are unrelated. For example, descriptions such as “right” get a high score relative to a “conservative” dimension, since these are close in meanings, whereas descriptions such as for example “solidarity” gets a lower score relative to an “authority” dimension, since these word representations are less similar to each other. The semantic representations of the three words that the participants generated can be used to predict vote choice. This is done by using the semantic representation as predictors in logistic regression, where 1 represent choosing the speciﬁc party, 0 choosing some other party. Experimental Procedure The online survey experiment proceeded as follows. Participants were randomly assigned to starting with either the task of describing parties (n = 3,428), or the task describing the leaders of the same four parties (n = 3,348). The party item was formulated as follows “What does the following party represent for you?” “Please enter up to three descriptive keywords, or leave blank if you do not know about the party.” The party leader item, in turn, was formulated as follows: “What does the following party leaders/spokespersons represent for you?” “Please enter up to three descriptive keywords, or leave blank if you do not know about the party leader/spokesperson.” Respondents were provided with party abbreviations in brackets when they were May 2021 \| Volume 12 \| Article 604135 Frontiers in Psychology \| www.frontiersin.org 3 Quantitative Analysis of Text Fredén and Sikström represents the number of times two words have been generated in the same answer by a participant. Then each cell is normalized by logarithm plus one. Finally, a data compression algorithm (singular value decomposition) is applied to this matrix, where the ﬁrst 300 resulting dimensions are maintained (i.e., the dimensions are ordered by how much information they maintain from the original matrix, so the ﬁrst dimensions are the most important)4. This results in a representation where each word is associated with a vector (normalized to the length of one) that represent how semantically similar the words are in the dataset. Since the data material concerns keywords on parties and politicians (and little irrelevant text information) this method is suitable for categorizing responses. The three words from the individual are summarized to one semantic representation, by adding the vector associated to each word and normalize the length of the resulting vector to one. This representation allows to measure the semantic similarities scores between two texts, as well as make predictions to a numerical variable, for example vote intention, as described below. to describe the leaders. Since this is how leaders are usually presented in the media, we believe that is a valid way of collecting words on leaders. See the Supplementary Material for the original formulations in Swedish. On the next page, the descriptive task was shifted—those who had not described leaders described parties, and vice versa2. All respondents described all four parties and their four party leaders. The survey institute decided the order in which the parties appeared3. Experimental Procedure On the following screen, the respondent indicated three important issues. After these items, the respondent declared his or her vote intention. The experiment ﬁnished by responding to a question about certainty of vote decision on a scale from1 (not certain at all) to 7 (very certain). For screenshots of the experiment’s online format, see the Supplementary Material. 2The average number of words that the respondent used for describing parties and leaders decreased for parties and leaders depending on whether it was the ﬁrst or last descriptive task. It decreases somewhat more for parties (from on average 2.3 to 1.9 words) than for leaders (from 2.0 to 1.8 words). 4Another option would have been to compare the voters’ text descriptions with general text materials such as for example google n-grams, however, in this case, there was reason to create a semantic space of words related to political parties and leader descriptions. 5See for example Medium (2019) for a discussion on correlation measures for binary outcomes. 2The average number of words that the respondent used for describing parties and leaders decreased for parties and leaders depending on whether it was the ﬁrst or last descriptive task. It decreases somewhat more for parties (from on average 2.3 to 1.9 words) than for leaders (from 2.0 to 1.8 words). 3The descriptive statistics show that the average number of words is very similar describing the four leaders, independent of their internal order in the survey experiment (ranging from 1.96 to 2.03 when the leader descriptive task comes ﬁrst, to 1.80–1.87 when it comes last). When it comes to the party descriptive task, the number of keywords is associated with size rather than order: the greatest number of words (2.46) is for the Moderate party and the lowest (2.17) for the Christian Democrats when party descriptions came ﬁrst, and 2.05 for Moderates (highest) vs. 1.80 for Christian Democrats (lowest) when party descriptions came last. Since the general patterns are similar and the diﬀerences relatively small, we do not believe that an internal order eﬀect is driving the results and main conclusions. Moreover, previous research indicates that the ﬁrst and second words are most important for measuring the respondent’s semantic representation (Kjell et al., 2019). 4Another option would have been to compare the voters’ text descriptions with general text materials such as for example google n-grams, however, in this case, there was reason to create a semantic space of words related to political parties and leader descriptions. Frontiers in Psychology \| www.frontiersin.org 5See for example Medium (2019) for a discussion on correlation measures for binary outcomes. Method: Latent Semantic Analysis Predicting Voting From Text Data The resulting predicted vote choice were then correlated with the empirical value of the vote intention. Here we used point-biserial correlation, which is a suitable method for dichotomous dependent variables5. Based on the text data from a speciﬁc word question (e.g., about the party “Moderates”), we can predict to what extent voters are likely to choose a party (e.g., “Moderates,” “Liberals,” etc.). For example, if Liberal party voters tended to enter the words “liberal” and “school” together and other voters used these word combinations to a less extent (or used words with very diﬀerent meanings), such systematic co-occurrence patterns will translate into r-scores that are higher for the Liberal party relative to other parties. The predictions are evaluated with a 10-fold cross-validation procedure, which means that the text data from the experiment was randomly divided into a training set consisting of 90% of the data, were the empirical values of vote intentions were 3The descriptive statistics show that the average number of words is very similar describing the four leaders, independent of their internal order in the survey experiment (ranging from 1.96 to 2.03 when the leader descriptive task comes ﬁrst, to 1.80–1.87 when it comes last). When it comes to the party descriptive task, the number of keywords is associated with size rather than order: the greatest number of words (2.46) is for the Moderate party and the lowest (2.17) for the Christian Democrats when party descriptions came ﬁrst, and 2.05 for Moderates (highest) vs. 1.80 for Christian Democrats (lowest) when party descriptions came last. Since the general patterns are similar and the diﬀerences relatively small, we do not believe that an internal order eﬀect is driving the results and main conclusions. Moreover, previous research indicates that the ﬁrst and second words are most important for measuring the respondent’s semantic representation (Kjell et al., 2019). May 2021 \| Volume 12 \| Article 604135 Frontiers in Psychology \| www.frontiersin.org 4 Quantitative Analysis of Text Fredén and Sikström that the most central word is identical to the most central leader descriptions that come ﬁrst: “school” [skola]. In addition, more abstract concepts such as “freedom” and the “EU” are signiﬁcant in the party descriptions that precede leader descriptions. The interpretation of the diﬀerence between words coming ﬁrst or last is less straight-forward for parties than for leaders. 6The sample sizes in the sixteen diﬀerent correlation models (the word descriptions of four parties and their leaders described ﬁrst or last, correlated with respondents’ vote intentions) varied between 2,475 and 2,844. 7The only exception is the Moderate party descriptions, where the second party descriptive task predicted vote choice to a greater extent that the ﬁrst party descriptive task. One potential explanation is that the preceding leader descriptions ampliﬁed voters’ associations to the party more in this case because the leader is a potential Prime Minister. Correlations The analyses were performed in the Matlab version of the online statistical software semanticexcel.com (Sikström et al., 2020). Below we test the hypotheses more directly, i.e., how well the written descriptions of leaders and parties predicted voting intention. Table 1 and Figure 3 show the point biserial correlation (r) between the empirical value of vote intention and the predicted value of vote choice. Table 1 shows how well descriptions of the party’s leader or party predicted vote choice for the four parties that were included in the survey items (the Moderates, the Liberals, the Christian Democrats, and the Greens). These analyses support the ﬁrst hypothesis that voters’ descriptions of leaders are associated with vote choice to the same extent as their description of parties. Overall, leader descriptions (r = 0.125, s = 0.0093) mattered as much as party descriptions (r = 0.127, s = 0.0093) concerning these four focal parties. Method: Latent Semantic Analysis Predicting Voting From Text Data The size of the cloud, i.e., the number of central words following the LSA, is the same size in the two treatments. One observation is that the words on the right, i.e., where the party descriptions come last, are more inﬂuenced by policy-laden words (for example, “right” [höger]), which are features that may detach voters from a party. It is possible that the leader descriptions that preceded these descriptions inﬂuenced the party words in that direction. used in the predictions, and then evaluated on the remaining 10% of the data. This procedure is repeated 10 times, with diﬀerent training and test data sets, so all data points receive a predicted value. The Supplementary Material provides a general overview of this method. We thus predicted vote choice based on the survey items that contained up to six words per political unit (three related to the party, three related to the party leader) and the vote intention item, which were collected during the experiment. This allowed direct comparison of predictive powers of words related to leaders, vs. words related to parties (H1). We separated the sample into test order, where one condition consisted of respondents answering the party leader questions ﬁrst, and the other condition answered the party items ﬁrst. This allowed us to investigate whether test order inﬂuenced the results (H2).6 The descriptions suggest that participants describe leaders and parties with rather similar concepts if it is their ﬁrst associative task. Nevertheless, personal characteristics such as “boring” [tråkig] and “clear”/“unclear” [tydlig/otydlig] are signiﬁcant words following the ﬁrst descriptive leader task. This suggests that primacy of leaders can inﬂuence voters to think about issues and personal characteristics simultaneously, and that evaluations of leaders and party contents in conjunction predict vote choice to the greatest extent. To get a qualitative overview of the data, the words in the dataset were also visualized in word clouds, following the methods speciﬁed in Kjell et al. (2019). The words that were representative for voters’ descriptions of leaders and parties were grouped together, where the words in the center of the clouds are the most representative (i.e., words with the highest semantic similarity with other words in the same condition), and font size represents frequency. Then, these descriptions were divided by order, i.e., coming ﬁrst or last as descriptive tasks. Descriptives First, we evaluate leader and party descriptions depending on the order of the question. Figure 1 summarizes descriptions of all four leaders, where the left side of the ﬁgure shows words that are indicative results of the leader question being second (i.e., after the party question), whereas the right- hand side presents the result when the leader question was presented ﬁrst. When the party leaders were described ﬁrst, the descriptions relate to politics and party characteristics, for example “school” [skola], “conservative” [konservativ], as well as personal characteristics, such as for example “boring” [tråkig]. On the other hand, when leaders were described after the parties, the word clouds contain less ideological and issue-related words, and more characteristics related to personal qualities: “trustworthy” [trovärdig], “competent” [duktig]. These ﬁndings give some ﬁrst support to the hypothesis that the leader and party descriptions tend to conﬂate, especially if the leader item precedes the party item. Second, we ﬁnd support of the primacy eﬀect stated in the second hypothesis. The descriptions that the voters gave ﬁrst, in general, predicted vote choice better independently of the descriptive task. Thus, for example, if leaders were described ﬁrst, then the descriptions of these predicted vote choice better than the descriptions of the parties that came afterward. The ﬁrst question had a higher correlation for parties (r = 0.145, s = 0.013 vs. r = 0.110, s = 0.013) as well as for party leaders (r = 0.149, s = 0.013 vs. r = 0.101, s = 0.013). The correlation for the ﬁrst questions were signiﬁcantly higher than the correlation for second questions (p = 0.0026, N = 2,607 (participant) ∗8 (questions), z = 3.0 (see Meng et al., 1992)7. Figure 3 illustrates the general pattern that we found. The graph compares the predictive powers of vote choice at t1 (when the party or leader is described ﬁrst) and at t2 (when party or Figure 3 illustrates the general pattern that we found. The graph compares the predictive powers of vote choice at t1 (when the party or leader is described ﬁrst) and at t2 (when party or For comparison, Figure 2, in turn, shows word clouds for the party descriptions, where the left-hand side shows words indicative of party descriptions given ﬁrst, and the right side party descriptions after leader descriptions. Descriptives The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of party descriptive texts was signiﬁcant t(59,515) = 23.61, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 2 \| Word descriptions of parties under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the party descriptions is 59,515, and the number of unique words is 10,010. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of party descriptive texts was signiﬁcant t(59,515) = 23.61, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. leader is described last). This supports the conclusion that the order of the descriptive task matters (Hypothesis 2). conceptualization of the party. In our experiment, we ﬁnd that associations that are prior to others predict vote choice best, which demonstrates that a primacy eﬀect occurs in the vote decision-making process. To summarize, the latent semantic analyses support the claim that voters’ descriptions of leaders and parties are of similar importance for predicting their vote choice. In line with our ﬁrst hypothesis, the leader descriptions from the three keywords predicted vote intention to the same extent as party descriptions did. Leader descriptions given before party descriptions were more inﬂuential and explicitly related to policy. This suggests that voters often conﬂate representations of leaders and parties, and that these concepts may be exchanged in the voter’s mental representation within the context of voting behavior. In addition, the generally clearer descriptions that voters entered in the ﬁrst party association task appear to matter more for choice than the more diverging words that summarized the last descriptive task. Thus, the more solid picture of the party and its leader predicted vote choice better than the less coherent ﬁgure. Nevertheless, the analysis shows that the leader descriptions, which are more oriented toward evaluation of personal qualities, can be part of this solid Frontiers in Psychology \| www.frontiersin.org Descriptives The number of plotted words has been limited to 100. The total number of words for the party descriptions is 59,515, and the number of unique words is 10,010. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of party descriptive texts was signiﬁcant t(59,515) = 23.61, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 1 \| Word descriptions of leaders under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the leader descriptions is 53,372, and the number of unique words is 8,165. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of leader descriptive texts was signiﬁcant t(53,372) = 23.69, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 2 \| Word descriptions of parties under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the party descriptions is 59,515, and the number of unique words is 10,010. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of party descriptive texts was signiﬁcant t(59,515) = 23.61, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 2 \| Word descriptions of parties under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the party descriptions is 59,515, and the number of unique words is 10,010. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. Descriptives Interestingly, we ﬁnd May 2021 \| Volume 12 \| Article 604135 Frontiers in Psychology \| www.frontiersin.org 5 Quantitative Analysis of Text Fredén and Sikström FIGURE 1 \| Word descriptions of leaders under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the leader descriptions is 53,372, and the number of unique words is 8,165. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of leader descriptive texts was signiﬁcant t(53,372) = 23.69, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 2 \| Word descriptions of parties under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the party descriptions is 59,515, and the number of unique words is 10,010. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of party descriptive texts was signiﬁcant t(59,515) = 23.61, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 1 \| Word descriptions of leaders under different conditions. The ﬁgures show words arranged in word clouds. The number of plotted words has been limited to 100. The total number of words for the leader descriptions is 53,372, and the number of unique words is 8,165. Words in color were signiﬁcant following Bonferroni correction for multiple comparisons. The ﬁgure shows color-coded data-points that signiﬁcantly discriminate between the high and the low value of the scale parties ﬁrst (left) or leaders ﬁrst (right) using semantic tests. The semantic t-test comparing the two sets of leader descriptive texts was signiﬁcant t(53,372) = 23.69, p = 0.0000. For a detailed description of the method, see the Supplementary Figure notes. FIGURE 2 \| Word descriptions of parties under different conditions. The ﬁgures show words arranged in word clouds. DISCUSSION The models include respondents with valid key word responses. The actual sample sizes of the prediction models vary between 2,844 (Moderate party described ﬁrst) and 2,475 (Green’s spokesperson I. Lövin described second). Forthcoming studies should also elaborate more upon how important leaders are for party success, and how important leaders are as spokespersons for certain policy proﬁles. For example, the choice of leader has an impact on how voters view the party’s ideological leaning, which in turn aﬀects voting behavior. When voters tend to be more volatile, and rely upon various media sources for their decisions, these kinds of mechanisms become even more important to scrutinize. One avenue for future research is the duration of such leader eﬀects, and the potential variation over diﬀerent contexts. This study examined a proportional context with a less predictable outcome than usual as a populist party had grown stronger relative to the established parties. Potentially, this made the 2018 Swedish election more similar to other countries where we have seen similar patterns, such as Denmark, Norway, and the United Kingdom. It would be fruitful to replicate the study in these other contexts in order to test the generalizability of the relatively strong leader inﬂuence we found in this experiment. FIGURE 3 \| Prediction of voting intention based on order of the descriptive task. The ﬁgure is based on Table 1 and show the party average Pearson correlation coefﬁcient between predicted and empirical voting intention (r) at t1 (party or leader described ﬁrst) and t2 (party or leader described last). The difference between the correlations at t1 and t2 are signiﬁcant with p < 0.001. FIGURE 3 \| Prediction of voting intention based on order of the descriptive task. The ﬁgure is based on Table 1 and show the party average Pearson correlation coefﬁcient between predicted and empirical voting intention (r) at t1 (party or leader described ﬁrst) and t2 (party or leader described last). The difference between the correlations at t1 and t2 are signiﬁcant with p < 0.001. FIGURE 3 \| Prediction of voting intention based on order of the descriptive task. The ﬁgure is based on Table 1 and show the party average Pearson correlation coefﬁcient between predicted and empirical voting intention (r) at t1 (party or leader described ﬁrst) and t2 (party or leader described last). The difference between the correlations at t1 and t2 are signiﬁcant with p < 0.001. ETHICS STATEMENT Using this kind of text analytical approach advances knowledge about how voters think when they think about parties and leaders, and how these associations guide the vote choice process. This knowledge may have practical implications, as it suggests that creating positive associations to the leader and make them stand in the front of the party’s policy message is a potentially successful party strategy. Leader and party descriptions are not separate from policy positions, and the leader’s role as spokespersons should not be underestimated. Clarity and uniqueness in the policy message, as well as repetition of it, would make such associative patterns even more salient. The inﬂuence of leaders can be a problem if this has consequences for party survival that are not rooted in policy responsiveness between voters and parties, but rather in personal characteristics of the leader that can be less relevant. Nevertheless, if the parties’ The studies involving human participants were reviewed and approved by the University of Gothenburg. The participants provided their written informed consent to participate in this study. DATA AVAILABILITY STATEMENT The datasets presented in this article are not readily available because the dataﬁles may still contain personal identiﬁable information. Parts of the dataset may be available on upon request, after some additional screening by data managers at the Laboratory of Opinion Research at the University of Gothenburg. Requests to access the datasets should be directed to AF, annika.freden@kau.se. showed that a combination of policy and personal characteristics had greater predictive power than personal characteristics that are less associated with the party. Studying voters’ own free text responses thus revealed that leader inﬂuence on political sympathy is salient also in PR. Frontiers in Psychology \| www.frontiersin.org DISCUSSION The results from an electoral experiment and a LSA lent support to the hypothesis that descriptions of leaders had about equally as strong predictive power as descriptions of parties in the 2018 Swedish general election campaign. We also found clear evidence that the order of the questions matter: descriptions of leaders or parties that were given ﬁrst mattered more for the decision and were qualitatively diﬀerent from descriptions given second. We thus revealed a primacy eﬀect in an electoral context, where voters were asked to describe party leaders and parties in free text. One potential implication is that the piece of information that the campaign currently emphasizes, be it the leader or the party, is inﬂuencing the voter’s mindset. The analysis also May 2021 \| Volume 12 \| Article 604135 6 Quantitative Analysis of Text Fredén and Sikström TABLE 1 \| Prediction of voting intention based on participants written descriptions. Describe parties Describe leaders Party First Second Leader First Second Average Green 0.104 0.070 I. Lövin 0.144 0.131 0.112 Liberals 0.186 0.059 J. Björklund 0.153 0.084 0.121 Christian Democrats 0.073 0.060 E. Busch Thor 0.087 0.024 0.061 Moderates 0.215 0.252 U. Kristersson 0.211 0.164 0.211 Average 0.145 0.110 0.149 0.101 0.126 The sample sizes in the sixteen different correlation are based on the samples of the respective treatments group (n = 3,428 when parties are described ﬁrst, n = 3,348 when leaders are described ﬁrst). The models include respondents with valid key word responses. The actual sample sizes of the prediction models vary between 2,844 (Moderate party described ﬁrst) and 2,475 (Green’s spokesperson I. Lövin described second). paint a coherent picture of party policies and leader, it will facilitate voters’ possibility to predict the leaders’ forthcoming abilities to negotiate with other parties. In the studied election, previous policy orientations had to be reconsidered since the election resulted in unclear majorities. 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Methods 24, 92–115. doi: 10.1037/ met0000191 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Klüver, H., and Sagarzazu, I. (2016). Setting the agenda or responding to voters? political parties, voters and issue attention. West Eur. Politics 39, 380–398. doi: 10.1080/01402382.2015.1101295 Copyright © 2021 Fredén and Sikström. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). ACKNOWLEDGEMENTS experimental data, developed a strategy for the more complex analysis in collaboration with SS, and drafted the manuscript. SS performed the statistical analysis and provided important revisions. Both authors contributed to the article and approved the submitted version. The authors would like to thank the reviewers and research assistant Alexander Rangfält. FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.604135/full#supplementary-material This work was supported by Vetenskapsrådet Grant 2017-02941. Publication fees were supported by Lund University. This work was supported by Vetenskapsrådet Grant 2017-02941. Publication fees were supported by Lund University. AUTHOR CONTRIBUTIONS AF and SS developed the study concept and raised the funds that were necessary to conduct the experiment (with AF as main applicant). AF was responsible for the ﬁnal survey experimental design, the contact with the Lore opinion lab at the University of Gothenburg, performed the statistical overview analysis of the May 2021 \| Volume 12 \| Article 604135 7 Quantitative Analysis of Text Fredén and Sikström REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Landauer, T. K., and Dumais, S. T. (1997). A solution to Plato’s problem: the latent semantic analysis theory of acquisition, induction, and representation of knowledge. Psychol. Rev. 104, 211–240. doi: 10.1037/0033-295X.104.2.211 knowledge. Psychol. Rev. 104, 211–240. doi: 10.1037/0033-295X.1 McCall Rosenbluth, F., and Shapiro, I. (2018). 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https://openalex.org/W3013466680	https://europepmc.org/articles/pmc7177876?pdf=render	English	null	Neighborhood Walkability and Active Transportation: A Correlation Study in Leisure and Shopping Purposes	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	10,215	Received: 28 February 2020; Accepted: 24 March 2020; Published: 25 March 2020 Abstract: A walkable environment is a crucial factor for promoting active transportation. The purpose of this study is to examine the association between neighborhood walkability and active transportation for noncommuting purposes (leisure and shopping) in Seoul, Korea. The Walkability Score is used as a measure of walkability, and a multilevel logistic regression model is employed to measure the odds of active transportation (i.e., walking and cycling; nonmotorized trips) at two levels: individual (level 1) and neighborhood (level 2). The results of the study showed that the Walkability Score was signiﬁcantly correlated with higher odds of active transportation in shopping models. Speciﬁcally, every one-point increase in the Walkability Score was associated with 1.5%–1.8% higher odds of active transportation in shopping models. However, there was no signiﬁcant correlation between the two in leisure models. Meanwhile, individual characteristics associated with the odds of active transportation diﬀered in the leisure and shopping models. Older age was positively correlated with the odds of active transportation in the leisure model, while females showed a positive correlation in the shopping model. Based on the study, urban and transportation planners can recommend urban policies to promote active transportation in an urban setting. Keywords: active transportation; walking; cycling; leisure trip; shopping trip; Walk Score; Walkability Score; multilevel logistic regression model; Seoul Article Neighborhood Walkability and Active Transportation: A Correlation Study in Leisure and Shopping Purposes Eun Jung Kim 1 , Jiyeong Kim 1 and Hyunjung Kim 2,* Eun Jung Kim 1 , Jiyeong Kim 1 and Hyunjung Kim 2,* 1 Department of Urban Planning, Keimyung University, 1095 Dalgubeol-daero, Dalseo-gu, Daegu 42601, Korea; kimej@kmu.ac.kr (E.J.K.); th154@naver.com (J.K.) 2 Department of Civil and Environmental Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Korea J g , J y g y j g 1 Department of Urban Planning, Keimyung University, 1095 Dalgubeol-daero, Dalseo-gu, Daegu 42601, Korea; kimej@kmu.ac.kr (E.J.K.); th154@naver.com (J.K.) 2 Department of Civil and Environmental Engineering, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 08826, Korea * Correspondence: urbanistar@snu.ac.kr; Tel.: +82-2-880-8903 International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2020, 17, 2178; doi:10.3390/ijerph17072178 1. Introduction An increase in sedentary behavior and a proportionate growth in chronic diseases have been considered the most critical public health issues in the modern world, prompting several researchers in the public health and urban planning ﬁelds to investigate the environmental impact of promoting higher levels of physical activity [1,2]. As a means to promote physical activity, walking and cycling can be considered feasible daily activities for most people [3–5]. Among the diﬀerent types of physical activities, walking and cycling are considered suitable for all age groups, given that they do not require special skills or facilities, and allow people to manage the intensity of their own movements [5]. Therefore, eﬀorts to enhance walking and cycling within the community have been gaining momentum lately [5]. Promoting physical activity can help prevent a rise in the overweight and obese population and reduce the risk of potential chronic ailments such as respiratory diseases and Type 2 diabetes, as well as mortality risk from cardiovascular diseases and cancer [3,6–10]. Enhanced physical activity can also beneﬁt mental health as it can improve emotions and the sense of recognition [11], reduce Int. J. Environ. Res. Public Health 2020, 17, 2178; doi:10.3390/ijerph17072178 www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 2178 2 of 16 pressure [12,13], and depression [14]. From the urban and transportation planning perspective, walking and cycling, usually termed as “nonmotorized transport” or “active transportation,” can be considered an important means for promoting sustainable cities and for providing social, environmental, and economic beneﬁts [4,15]. Therefore, there is a growing emphasis on the importance of active transportation in light of urban problems such as traﬃc congestion, environmental pollution, energy shortage, and an increase in the obese population. It is imperative to promote environmental and policy approaches to encourage physical activity considering that it can beneﬁt all citizens in the neighborhood [16–18]. Accordingly, eﬀorts to ﬁnd an adequate built environment for physical activity, especially a walkable environment, has attracted the attention of policymakers, urban and transportation researchers, and public health scientists. Several studies have identiﬁed built environmental factors that most signiﬁcantly inﬂuence walking behavior in urban and suburban areas [4,7,19–21], and eﬀorts were made to develop a methodology to objectively measure the level of walkability. Consequently, several indices such as the Walk Score, Walkability Index, and Pedestrian Index of the Environment were developed combining various built environmental variables that inﬂuence walking behavior [7,19–32]. 1. Introduction The Walk Score is one of the popular indices that objectively measures neighborhood walkability, taking into account the accessibility of amenities in the vicinity (e.g., grocery stores, restaurants, shopping centers, coﬀee shops, parks, schools) and pedestrian friendliness (e.g., intersection density and average block length) [25], and is currently used in various ﬁelds, including public health, real estate, and urban planning [33–37]. Studies have veriﬁed whether the Walk Score is appropriate to describe the level of walkability, and correspondingly, several works of the literature showed that a higher level of Walk Score is positively correlated with walking behavior [7,26,29,30,38–42]. A number of studies have investigated the association between walkability and health indicators using the Walk Score [42–44]. Xu and Wang examined the impact of the neighborhood environment on physical inactivity and obesity in Washington, D.C. in the United States. Using a multilevel regression model, they found that street connectivity was negatively associated with obesity, while Walk Score was negatively associated with physical inactivity. They also found that the obesity risk varied depending on urbanicity levels and gender, with a higher Walk Score linked to a lower risk of obesity in urban areas for females [43]. Wasﬁet al. examined the inﬂuence of neighborhood walkability on the Body Mass Index (BMI) of urban Canadians using the Walk Score and the National Population Health Survey of Canada, and found that neighborhood walkability inﬂuences BMI trajectories for males [42]. McCormack et al. explored the relationship between walkability and waist circumference, waist-to-hip ratio, and BMI in Calgary, Alberta, Canada. Correspondingly, they found that a higher Walk Score was associated with lower odds of having a large waist circumference; neighborhoods with a lower Walk Score had higher odds of a high waist circumference, BMI, and waist circumference–BMI risk [44]. In short, previous studies have shown that developing a walkable environment can inﬂuence physical activity, which can lead to a signiﬁcant population health impact. 2. Research Background Manaugh and El-Geneidy examined the relationship between the trip purpose and walkability in nonmotorized mode of transportation; however, they considered noncommuting trip purposes mainly for shopping [30]. Lefebvre-Ropars et al. examined the association between walking time and the built environment using the Pedestrian Index of the Environment (PIE). They considered both shopping and leisure as noncommuting trips, and found that the PIE was more strongly correlated to the choice of walking for work, leisure, or shopping in very short trips [21]. However, they did not consider cycling as active transportation. In sum, previous studies on noncommuting active trips mainly focused on leisure walking/cycling, or investigated either leisure or shopping trips, or mainly focused on walking. Korea is facing a severe health problem related to physical inactivity [45]. According to the Community Health Survey of Korea, the walking rate (the percentage of people who walked more than ﬁve days a week for more than 30 min a day in a week) in the country decreased overall during the past decade, from 50.6% in 2008 to 42.9% in 2018, while the proportion of individuals who are obese increased from 21.6% in 2008 to 31.8% in 2018 [46]. This increase in physical inactivity and obesity can be considered a social problem in Korea, which underscores the importance of developing pedestrian-friendly urban environments [47–50]. Accordingly, the city of Seoul is promoting “Walkable City, Seoul” as a major urban policy to enhance citizens’ health and reduce traﬃc congestion [51]. A pedestrian-oriented traﬃc environment is being modeled with increasing safe walking zones and connecting touristic spots. Furthermore, bicycling is being promoted by expanding bike-related infrastructure, such as a sharing bike system. g y A recent study by Kim et al. examined the association between walkability and active commuting (i.e., walking and cycling to work or school) in Seoul using the Walkability Score, and empirically found a positive correlation between walkability and active commuting [52]. This raises another question as to whether walkability will also have a signiﬁcant positive association in the case of noncommuting trips (e.g., leisure and shopping trips) with active transportation (i.e., walking and cycling). In addition, based on trip purposes, would a walkable environment associate similarly or diﬀerently within leisure and shopping trips? Especially, noncommuting trips are an intentional trip that may more likely depend on the neighborhood environment than a commuting trip [30]. 2. Research Background Over the past several decades, rapid industrialization and urbanization have led to signiﬁcant lifestyle changes resulting in an increasing overweight and obese population, creating a signiﬁcant public health burden in many countries [5,45]. A practical way to encourage physical activity on a daily basis is to promote active transportation. Therefore, several studies identiﬁed that a walkable environment has a positive association with active transportation [7,21,26–32]. For example, Reyer et al. used the Walkability Index and the Walk Score to explore the link to active transportation and found a tendency toward more active travel in more walkable neighborhoods [7]. Likewise, Knuiman et al. examined the relationship of neighborhood walkability and accessibility to a destination using walking as transportation in Perth, Australia. They found that accessibility to local destination, land use mix, and street connectivity are important determinants for promoting walking as a means of transportation [32]. 3 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 Studies regarding the relationship between walkability and noncommuting active trips mainly considered the leisure purpose of walking or cycling. Investigating the neighborhood walkability in Canada, de Sa and Arden found that respondents living in highly walkable 500 m buﬀer zones (upper quartiles of the walkability index) were more likely to walk or cycle for leisure than those living in low-walkable buﬀer zones. When a 1000 m buﬀer zone was applied, respondents in more walkable neighborhoods were more likely to walk or cycle for both leisure and transport-related purposes [27]. Dyck et al. examined the association between leisure-time physical activity and perceived neighborhood environmental walkability in Belgium, Australia, and the United States. Except for the city of Ghent, Belgium, there was a positive linear association with recreational walking and leisure time physical activity [28]. Thielman et al. estimated the association between walkability and physical activity by transport walking and leisure time physical activity in Canada at the national level. They found that walkability was associated with transport walking in all age groups and city sizes. However, it had an inverse association with leisure-time physical activity among young adults and in large cities [29]. Some studies examined walkability for shopping purposes [21,30,31], but there are few studies that examined both leisure and shopping trips depending on the neighborhood walkability. For example, Habibian and Hosseinzadeh examined walkability across trip purposes including commuting, educational, and shopping, although they did not consider trips for leisure [31]. 3.1. Study Area 1. Study Area The study focuses on the city of Seoul, and uses travel mode data from the Household Travel Diary Survey adapted from the Korea Transport Database [54]. The total number of home-based trips for leisure and shopping purposes in the survey were 9998 and 8578, respectively. The city of Seoul consists of 424 neighborhoods with their own administrative oﬃces. The neighborhood is referred to as “dong” in Korea, which is the most disaggregated administrative unit. Of the 424 neighborhoods, some had very few sample numbers in the survey. For example, in the case of Jamwon-dong, only four individuals responded to the survey. Neighborhoods that had notably few samples such as Jamwon-dong were excluded from the multilevel modeling, given that ensuring a suﬃcient number of samples is one of the most important issues in multilevel analyses [55–57]. For unbiased results, some studies suggested ”30/30” and “20/50” rules that indicated the minimum number of observations per group/minimum number of groups. Kreft recommended the “30/30” [58], while Hox recommended the “‘20/50” rule [59]. To prevent biased estimate of parameters, we employed a minimum of 30 respondents per neighborhood for the study. Out of the 424 neighborhoods, the number of corresponding neighborhoods was 129 and 91 in the leisure and shopping models, respectively. Overall, the study used 5742 individuals nested within 129 neighborhoods and 3722 individuals nested within 91 neighborhoods in the leisure and shopping trip models, respectively, as shown in Figure 1. The study focuses on the city of Seoul, and uses travel mode data from the Household Travel iary Survey adapted from the Korea Transport Database [54]. The total number of home-based trips r leisure and shopping purposes in the survey were 9998 and 8578, respectively. The city of Seoul onsists of 424 neighborhoods with their own administrative offices. The neighborhood is referred to s “dong” in Korea, which is the most disaggregated administrative unit. Of the 424 neighborhoods, ome had very few sample numbers in the survey. For example, in the case of Jamwon-dong, only four dividuals responded to the survey. Neighborhoods that had notably few samples such as Jamwon- ong were excluded from the multilevel modeling, given that ensuring a sufficient number of samples one of the most important issues in multilevel analyses [55–57]. For unbiased results, some studies uggested ”30/30” and “20/50” rules that indicated the minimum number of observations per roup/minimum number of groups. 3.1. Study Area 1. Study Area Kreft recommended the “30/30” [58], while Hox recommended the 20/50” rule [59]. To prevent biased estimate of parameters, we employed a minimum of 30 spondents per neighborhood for the study. Out of the 424 neighborhoods, the number of orresponding neighborhoods was 129 and 91 in the leisure and shopping models, respectively. Overall, he study used 5742 individuals nested within 129 neighborhoods and 3722 individuals nested within 1 neighborhoods in the leisure and shopping trip models, respectively, as shown in Figure 1. (a) (b) Figure 1. Study area: (a) Neighborhoods with at least 30 individual respondents for leisure trips (N = 129); (b) Neighborhoods with at least 30 individual respondents for shopping trips (N = 91). Figure 1. Study area: (a) Neighborhoods with at least 30 individual respondents for leisure trips (N = 129); (b) Neighborhoods with at least 30 individual respondents for shopping trips (N = 91). (a) (b) (b) (a) igure 1. Study area: (a) Neighborhoods with at least 30 individual respondents for leisure trips (N = 29); (b) Neighborhoods with at least 30 individual respondents for shopping trips (N = 91). Figure 1. Study area: (a) Neighborhoods with at least 30 individual respondents for leisure trips (N = 129); (b) Neighborhoods with at least 30 individual respondents for shopping trips (N = 91). 2. Research Background Some studies found that the distance and duration of noncommuting walking were substantially longer than they were for commuting purpose, as well as the importance of urban forms on noncommuting trips [21,53]. In particular, compared to commuting, a noncommuting trip can induce more walking and cycling if an adequate walkable and bikeable built environment is provided in the neighborhood. This study expands the scope of Kim et al.’s [52] research by considering the Walkability Score and active transportation diﬀerentiating the trip purposes to noncommuting. The purpose of this study is 4 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 to examine the association between the level of walkability measured by the Walkability Score and active transportation for noncommuting trips in Seoul, Korea. Speciﬁcally, the relationship between the walkability level and active transportation (i.e., walking and cycling) in noncommuting trips (i.e., shopping and leisure trip) will be examined considering both individual characteristics and the built environment of the neighborhood by conducting a multilevel analysis in Seoul. t. J. Environ. Res. Public Health 2020, 17, x FOR PEER REVIEW 4 of 17 2 Measures 3.2. Measures As mentioned earlier, this research expands on a study by Kim et al. [52]. In this study, an nvestigation of the correlation between noncommuting active trips (i.e., leisure and shopping) and he Walkability Score was conducted. Accordingly, independent variables were basically derived om related data sources and methodology from Kim et al. [52], differentiating the trip purposes sed as the dependent variables. The measurements and data sources of variables used in this study re shown in Table 1. As mentioned earlier, this research expands on a study by Kim et al. [52]. In this study, an investigation of the correlation between noncommuting active trips (i.e., leisure and shopping) and the Walkability Score was conducted. Accordingly, independent variables were basically derived from related data sources and methodology from Kim et al. [52], diﬀerentiating the trip purposes used as the dependent variables. The measurements and data sources of variables used in this study are shown in Table 1. Int. J. Environ. Res. Public Health 2020, 17, 2178 5 of 16 3.2.2. Neighborhood-level Variables (Level 2) 3.2.2. Neighborhood-level Variables (Level 2) This study employed the neighborhood as a spatial unit for multilevel modeling at level 2. All neighborhood-level variables in this study used the mean values of each neighborhood. • Walkability Score • Individual socioeconomic status variable • Individual socioeconomic status variable At the individual level, several socioeconomic status variables were considered as confounding factors, including age, gender, household income, and car ownership. These individual variables were also acquired from the 2016 Household Travel Diary Survey from the Korea Transport Database [54]. • Dependent variables: Travel mode (motorized vs. nonmotorized modes) As dependent variables, this study used active transportation (e.g., walking and cycling) separately for leisure and shopping purposes. From the Household Travel Diary Survey, there are four travel modes—walking, bicycling, public transport, and private automobile. For the analysis, they were coded as binaries—1 for nonmotorized modes (walking and cycling) and 0 for motorized modes (public transport and private automobile). • Walkability Score As a key independent variable, this study employed the Walkability Score, which was assessed in Seoul by Kim et al. [61]. Moreover, a recent study found that the Walkability Score is a reliable index to measure environmental walkability in the city [62]. The value of the Walkability Score is calculated by the Walk Score algorithm, which basically calculates the accessibility of utilitarian destinations with a distance decay function. They include nine amenities essential to everyday life—grocery stores, restaurants, shopping centers, coﬀee shops, banks, parks, schools, books, and entertainment. The Walkability Score calculates the closest network distance from each amenity and then awards 100% of the maximum points to amenities located within a network distance of 400 m, 75% within 800 m, 40% within 1.2 km, and 12.5% within 1.6 km of a given location [61]. By combining the accessibility of nine types of utilitarian destinations, the scores are normalized on a scale of 0 to 100. Additionally, poor pedestrian friendliness is considered as a penalty element. It considers the intersection density and average block length as factors of pedestrian friendliness. Areas with lower intersection density (no penalty: intersections per square mile > 200) and longer average block length (no penalty: average block length < 120 m) receive penalties up to 10% of the total score. The Walk Score ranges from 0 (car-dependent) to 100 (walkers’ paradise) [25]. Geospatial data used for assessing the Walkability Score in Seoul were collected from both governmental websites and private companies. More detailed information on measures, data sources, and calculation methods is found in Kim et al. [61]. • Neighborhood environmental variables 3.2.1. Individual-level Variables (Level 1) All individual-level variables of this study were acquired from the 2016 Household Travel Diary Survey from the Korea Transport Database [54], which is a traﬃc-related survey conducted every ﬁve years across the country and considered as a nationwide passenger survey in Korea. This survey examines the travel diary of household members aged ﬁve and older on weekdays, and is conducted through home visits and online using a self-reported questionnaire [60]. • Dependent variables: Travel mode (motorized vs. nonmotorized modes) • Dependent variables: Travel mode (motorized vs. nonmotorized modes) • Neighborhood environmental variables The Walkability Score is adapted from the Walk Score, which is an index that combines various variables that represent urban form such as density, diversity, and destination accessibility [21,62]. Since the Walk Score itself is a composite index of neighborhood walkability, there are some studies that include only itself as a neighborhood environmental variable [7,63]. However, one study found that there are some urban form elements such as land use diversity and walking route supply that the Walk Score does not include [21]. Therefore, this study considered land use mix and sidewalk length as additional neighborhood environmental variables. Land use mix is an important urban environmental Int. J. Environ. Res. Public Health 2020, 17, 2178 6 of 16 variable for promoting walking and cycling. Some studies have shown that the higher the land use mix, the more people walk and/or are physically active [31,32,64,65]. The entropy index was used to measure a land use mixture of residential, commercial, industrial, and greenspaces. It ranges from 0 (single use) to 1 (perfect mixing). Moreover, sidewalk length (i.e., total length of sidewalks per square kilometer) was considered in this study, given that it was positively associated with the minutes of neighborhood-based walking for transportation [66]. The data used in the analysis were obtained from the public source of the National Spatial Data Infrastructure Portal [67], while the neighborhood environmental variables were captured by ArcGIS. Table 1. Measurement, data source, and descriptive statistics of variables. Variable Measurement Data Source Dependent Variable Travel mode Binary: 0 = Motorized mode, 1 = Nonmotorized mode Household Travel Diary Survey from the Korea Transport Database [54] Individual Variables (Level 1) Age Continuous: Age Household Travel Diary Survey from the Korea Transport Database [54] Gender Binary: 0 = male, 1 = female Income Ordinal: 1 = less than 1 million won, 2 = 1–2 million won, 3 = 2–3 million won, 4 = 3–5 million won, 5 = 5–10 million won, 6 = more than 10 million won Car ownership Binary: 0 = no, 1 = yes Neighborhood Variables (Level 2) Walkability Score Continuous: Walkability Score Kim et al. [61] Land use mix 1 Continuous: 0 (single use)–1 (perfect mixing) National Spatial Data Infrastructure Portal [67] Sidewalk length Continuous: Length of sidewalk per square kilometer 1 Land Use Mix = −1 nP i=1 pi × ln(pi) ! • Neighborhood environmental variables / ln(n), where pi is the proportion of the land use type of i, i = residential, commercial, industrial, and greenspaces, n = total number of land uses in the mix (=4). Table 1. Measurement, data source, and descriptive statistics of variables. 4.1. Descriptive Statistics of Variables As shown in Table 2, the proportions of individuals using nonmotorized modes of transportation for leisure and shopping purposes were 81.8% and 76.8%, respectively. In both the samples, the ratio of the motorized mode to nonmotorized mode was approximately 1 to 4. From the individual variables, the participants for the leisure and shopping trips reported a mean age of 61.1 and 53.5 years, respectively. The median income level of households per month was 4 and 3, respectively, for each subsample; the value of 3 corresponds to 2–3 million won while 4 corresponds to 3–5 million won. The variable of household income ranged from 1 (less than 1 million won) to 6 (more than 10 million won). In the leisure and shopping trip group, the proportion of female participants was 61.4% and 90.9%, and the proportion of car ownership was 54.8% and 63.3%, respectively. Table 2. Descriptive statistics of variables. Variable Measurement Leisure Purpose Shopping Purpose % Mean (SD) % Mean (SD) Dependent Variable Travel mode Binary: 0 = Motorized mode 18.2% 23.2% 1 = Nonmotorized mode 81.8% 76.8% Individual Variables (Level 1) Age Continuous: Age 61.1 (16.7) 53.5 (15.1) Gender Binary: 0 = male 38.7% 9.1% 1 = female 61.4% 90.9% Income Ordinal: Household income level 4 1 3 2 Car ownership Binary: 0 = no 45.2% 36.7% 1 = yes 54.8% 63.3% Neighborhood Variables (Level 2) Walkability Score Continuous: Walkability Score 67.55(9.0) 67.71 (9.8) Land use mix 3 Continuous: 0 (single use)–1 (perfect mixing) 0.52(0.3) 0.53 (0.3) Sidewalk length 3 Continuous: Length of sidewalk per square kilometer 1.99(0.6) 1.95 (0.6) 1 This is a median value and it corresponds to 3–5 million won, 2 this is a median value and it corresponds to 2–3 million won, 3 square root-transformed, SD: standard deviation. Table 2. Descriptive statistics of variables. Table 2. Descriptive statistics of variables. 1 This is a median value and it corresponds to 3–5 million won, 2 this is a median value and it corresponds to 2–3 million won, 3 square root-transformed, SD: standard deviation. The mean values of the neighborhood environmental variables were slightly diﬀerent because the target areas (number of neighborhoods for leisure model = 129, number of neighborhoods for shopping model = 91) varied between leisure and shopping travels, although it was basically similar in the two samples. 3.3. Data Analysis A multilevel logistic regression model was used, with the two odds of nonmotorized trips (walking and cycling) for (1) leisure purposes and (2) shopping purposes. An odds ratio (OR) is a statistic that quantiﬁed the association between an outcome and exposure. When calculating a logistic regression analysis, the regression coeﬃcient is an estimated increase in the log probability of the outcome according to a unit increase in the exposure value [68]. If the OR is exactly 1, there is no correlation between outcome and exposure. When the OR is greater than 1, then there is a positive association, conversely if the OR is less than 1, then there is a negative correlation between outcome and exposure. Applying this concept here, this study examined the correlation between outcome (odds of nonmotorized trips for noncommuting) and exposure (individual and neighborhood variables). The multilevel data included two levels: individual (level 1) and neighborhood (level 2). Individuals living in the same neighborhood shared the same Walkability Score and the neighborhood environmental characteristics including land use mix and sidewalk length at the corresponding level. R software was used for the analysis in this study. 7 of 16 7 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 4.1. Descriptive Statistics of Variables In the samples of leisure and shopping purposes, the mean value of Walkability Score was 67.55 (SD = 9.0) and 67.71 (SD = 9.8), respectively. Meanwhile, the mean value of land use mix was 0.52 (SD = 0.3) and 0.53 (SD = 0.3), and the mean value of sidewalk length was 1.99 (SD = 0.6) and 1.95 (SD = 0.6), respectively. Both variables of land use mix and sidewalk length were square root-transformed for use in the multilevel analysis. Int. J. Environ. Res. Public Health 2020, 17, 2178 8 of 16 4.2. Results of Multilevel Logistic Models: Odds of Active Transportation for Leisure and Shopping Purposes 4.2. Results of Multilevel Logistic Models: Odds of Active Transportation for Leisure and Shopping Purpo 4.2. Results of Multilevel Logistic Models: Odds of Active Transportation for Leisure and Shopping Purposes This study conducted a multilevel logistic model because individual variables were nested within neighborhoods. Generally, there are three phases of procedure for multilevel logistic regression: (step 1) an empty model without predictors to assess variation of log-odds between clusters, (step 2) an intermediate model to assess the variation of the lower-level eﬀects between clusters, and (step 3) a ﬁnal model to test a research hypothesis [69]. Based on the procedure, this study ﬁrst ran a model without predictors and found it was necessary to employ the multilevel analysis. In the second phase, we tested which variables were associated with the odds of outcome (active transportation) by performing a likelihood-ratio test. The ﬁnal model was then presented according to theoretical importance as well as the statistical signiﬁcance of variables. There are two ﬁnal models each in the leisure and shopping models. Model 1 considers individual and neighborhood variables, but only considered the Walkability Score as a neighborhood variable, while Model 2 included additional neighborhood-level variables in Model 1. Speciﬁcally, the dependent variable is binary, for example, an individual using nonmotorized transportation (=1, and 0 otherwise) for leisure and shopping purposes. Model 1 included age, gender, income, and car ownership as individual variables (level 1) and the Walkability Score as a neighborhood variable (level 2). Meanwhile, Model 2 basically included all variables used in Model 1, but added land use mix and sidewalk length as neighborhood variables (level 2). The results of multilevel logistic models are shown in Table 3. 4.1. Descriptive Statistics of Variables Since Models L–2 and S–2 contained theoretically important neighborhood environmental variables such as land use mix and sidewalk length, the results from both Model 1 (L–1 and S–1) and Model 2 (L–2 and S–2) are reported in this section. 4.2.1. Odds of Active Transportation for Leisure Purposes 4.2.1. Odds of Active Transportation for Leisure Purposes The results of the analysis are as follows. First, the Walkability Score was insigniﬁcant in both Models L–1 and L–2. At the 0.1 signiﬁcance level, there was no evidence of statistical interaction between the Walkability Score and the odds of nonmotorized trips in both Model L–1 (p = 0.145) and Model L–2 (p = 0.178). Second, age was positively associated with the odds of nonmotorized trips in this model. Older adults were more likely to use active transportation for leisure purposes. The results are similar to a previous study, which found that young adults had a negative association with leisure physical activity [29]. Third, car ownership was a statistically signiﬁcant predictor of nonmotorized trips. Individuals having a car are much less likely to walk or cycle for leisure purpose. Fourth, gender and household income were not associated with the odds of nonmotorized trips in both the models. Previous studies also showed diﬀerent associations on gender and the Walk Score (e.g., positive association of lower risk of obesity for females in urban areas [43], while showing no signiﬁcant inﬂuence on BMI for females [42]). With respect to income level, a previous study showed that lower income individuals were more likely to walk and cycle for commuting purposes [52]. For economic reason, the lower the income, the more likely it is to commute by walking and cycling. On the other hand, because travel for leisure and shopping purposes are less sensitive to income than commuting travel, it is understandable that there no signiﬁcant correlation between income level and the odds of nonmotorized trips in this study. Fifth, unlike previous studies [31,32,64–66], additional neighborhood environmental variables, including land use mix and sidewalk length, had no statistical relationships with the odds of nonmotorized trips in Model L–2. 9 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 Table 3. Results of the multilevel logit regression analyses for estimating environmental correlates of active transportation in leisure and shopping purposes. Variable Odds of Nonmotorized Trip for Leisure Purpose Odds of Nonmotorized Trip for Shopping Purpose Model L–1 Model L–2 Model S–1 Model S–2 OR p-Value 95% CI OR p-Value 95% CI OR p-Value 95% CI OR p-Value 95% CI Lower Upper Lower Upper Lower Upper Lower Upper Intercept. * p < 0.001, p < 0.01, * p < 0.05; 1 square root-transformed; OR = Odds Ratio; CI = Conﬁdence Interval; ICC = Intraclass Correlation Coeﬃcient. 4.2.2. Odds of Active Transportation for Shopping Purposes 4.2.2. Odds of Active Transportation for Shopping Purposes The results for the shopping model can be summarized as follows. First, similar to previous studies [30,31], the Walkability Score was signiﬁcantly associated with the odds of nonmotorized trips in both Model S–1 and Model S–2. Every one-point increase in the Walkability Score was associated with 1.5% and 1.8% higher odds of nonmotorized trips in Model S–1 (OR: 1.015, 95% CI = 1.003–1.026) and Model S–2 (OR: 1.018, 95% CI = 1.005–1.031), respectively. Second, the female gender was positively correlated with the odds of nonmotorized trips in both models, as they tended to walk and cycle more for shopping purposes. Third, as with leisure models, car ownership was signiﬁcantly associated with nonmotorized trips. Individuals with cars were associated with lower odds of nonmotorized trips for shopping. Fourth, there were contradictory results depending on individual variables. For example, age was a signiﬁcant predictor in leisure models similar to the previous study [29]; however, it was insigniﬁcant in shopping models. Income level had no signiﬁcance in both leisure and shopping models. Fifth, similar to leisure models, there were no signiﬁcant correlations between the odds of nonmotorized trips and neighborhood variables such as land use mix and sidewalk length. Finally, according to the intraclass correlation coeﬃcient (ICC) value, about 4.5% of the total variance in the odds of nonmotorized trips for shopping purposes was accounted for by diﬀerences of the Walkability Scores between neighborhoods in Model S–1. The ICC represents the proportion of the between-group variance compared to the total variance [70]. The interpretation of the ICC is the expected correlation between randomly selected observations from the same group [71]. In this study, the ICC is the proportion of variance in the odds of nonmotorized trips (outcome variable) that is explained by the neighborhood (level 2). It indicated that neighborhood walkability was one of the most important factors aﬀecting individuals’ behavior of active transportation for shopping purposes. Model S–2 showed about 4.7% explanatory power of neighborhood variables (level 2), but the signiﬁcance of additional neighborhood environmental variables, such as land use mix and sidewalk length, were not guaranteed. There are some arguments that if ICC is close to zero (typically less than 4%–5%), there is lesser need to use a multilevel analysis [72,73]. However, it is still necessary to use multilevel analysis in nested data [73]. 4.2.1. Odds of Active Transportation for Leisure Purposes 1.662 0.266 0.766 2.557 1.540 0.375 0.587 2.493 0.879 0.785 −0.051 1.809 0.871 0.789 −0.138 1.881 Individual Variables (Level 1) Age 1.012 * <0.001 1.008 1.017 1.013 * 0.000 1.008 1.017 1.004 0.227 0.997 1.011 1.003 0.374 0.996 1.010 Gender (reference: male) 1.043 0.600 0.885 1.202 1.050 0.547 0.892 1.208 1.755 * <0.001 1.473 2.036 1.735 * 0.000 1.444 2.026 Income 1.044 0.248 0.971 1.117 1.043 0.263 0.969 1.116 0.996 0.925 0.904 1.087 0.983 0.731 0.886 1.080 Car Ownership (reference: no) 0.519 * <0.001 0.301 0.736 0.523 * 0.000 0.306 0.739 0.646 * <0.001 0.407 0.885 0.652 * 0.001 0.410 0.894 Neighborhood Variables (Level 2) Walkability Score 1.009 0.145 0.997 1.020 1.009 0.178 0.996 1.022 1.015 * 0.013 1.003 1.026 1.018 0.008 1.005 1.031 Land use mix 1 1.082 0.699 0.681 1.483 0.944 0.789 0.518 1.369 Sidewalk length 1 1.000 1.000 0.797 1.203 0.949 0.646 0.727 1.172 ICC 9.3% 9.2% 4.5% 4.7% N 5742 3722 * p < 0.001, ** p < 0.01, * p < 0.05; 1 square root-transformed; OR = Odds Ratio; CI = Conﬁdence Interval; ICC = Intraclass Correlation Coeﬃcient. nalyses for estimating environmental correlates of active transportation in leisure and shopping purposes. Int. J. Environ. Res. Public Health 2020, 17, 2178 10 of 16 10 of 16 5. Discussion This study examined the correlation between the level of walkability and noncommuting trips by conducting multilevel logistic regression analysis with the two odds of active transportation (i.e., walking and cycling) for leisure and shopping purposes. It is an expansion of a prior study that revealed a positive correlation between neighborhood walkability and active commuting in Seoul [52], since it is likely that noncommuting trips are more inﬂuenced by the neighborhood environment than commuting trips when people walk or cycle [30]. As a result, we empirically discovered that the walkability level of Seoul’s neighborhoods was positively correlated with the probability of active transportation for shopping purposes while showing no statistical correlation in leisure purposes. Discussions based on the ﬁndings are presented as follows. First, the correlation between the Walkability Score and the nonmotorized trips varied in the leisure and shopping models. In the shopping models, the Walkability Score was positively correlated with the odds of nonmotorized trips (Model S–1: OR = 1.015, 95% CI = 1.003–1.026; Model S–2: OR = 1.018, 95% CI = 1.005–1.031). However, there was no signiﬁcant correlation between the Walkability Score and the odds of nonmotorized trips for leisure purposes. This diﬀers from the ﬁndings of previous literature that showed that the odds ratios of walking for the Pedestrian Index of the Environment (PIE) were 1.05 for leisure and 1.04 for shopping purposes [21]. This diﬀerence is interpreted based on the indicators included in the walkability index. While the PIE considers the 5Ds of urban form, including density, diversity, design, destination accessibility, and distance to transit [21], the Walkability Score mainly considers destination accessibility [25]. As mentioned earlier, the Walkability Score was assessed based on the accessibility of amenities including grocery stores, restaurants, shopping centers [61]. Therefore, it can be demonstrated that the Walkability Score is an index based on the accessibility of amenities that 11 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 are more suitable for shopping than for leisure purposes. Second, other neighborhood-level variables had no signiﬁcant correlation with the odds of nonmotorized trips both in the leisure and shopping models. From Models L–2 and S–2, no variable was signiﬁcant among land use mix and sidewalk length. This result is diﬀerent from those of previous studies in which active transport (e.g., walking) and/or physical activity was positively correlated with land use mix [31,64,65]. 5. Discussion Meanwhile, some studies showed mixed-use development may not provide a positive eﬀect on walking and cycling in some high-density cities because these cities would reduce the chance for people walking or cycling as they can move easily to another area by public transportation [5,74,75]. This might be applied to Seoul because it is one of the high-density cities [76] and has good public transportation in its neighborhoods. Regarding sidewalk length, this analysis showed a similar result to the previous study that estimated the association between sidewalk length and walking for diﬀerent trip purposes [66]. It found that neighborhood-based walking for transportation had a positive association with sidewalk length but had no association in recreation walking, which was in line with our research on active transportation for leisure and shopping purposes. Third, the types of individual variables that were signiﬁcantly associated with the odds of nonmotorized trips diﬀer in the leisure and shopping models. Speciﬁcally, older age was positively correlated with the odds of nonmotorized trips for leisure purposes, while female gender had a positive correlation with the odds of nonmotorized trips for shopping purposes. These results can be used to develop various urban policies. For example, older people were more likely to have higher odds of nonmotorized trips for leisure purposes in this study. This ﬁnding presents an important issue for further research on age-friendly community design. Environmental factors that promote leisure walking (e.g., greener landscape, well-designed street furniture, wide pedestrian roads, safer pathway) in the development of age-friendly communities may be considered. In addition, a higher Walk Score was correlated to a lower risk of obesity for females in urban areas [43]. As noted from the result, the female gender tends to walk and cycle for shopping purposes; therefore, planning a wider choice of commercial facilities in a walkable and bikeable distance in urban areas or creating a more walkable environment for shopping could induce more physical activity for females. Finally, according to ICC values, the level of walkability of the neighborhood was an important factor in inﬂuencing individuals’ odds of nonmotorized trips for shopping purposes. From the results of the multilevel analyses, the proportion accounted for by the Walkability Score in the odds of nonmotorized trips for shopping purposes was about 4.5%. Although the ICC value was high at 9.3% in the leisure model (Model L–1), the Walkability Score was insigniﬁcant; therefore, it was not discussed. 5. Discussion Additionally, compared to the results of the previous study by Kim et al. [52] (ICC was 2.1% for active commuting model), active trips for shopping had a higher ICC value (4.5%) in this study. This indicates that the Walkability Score is a walkability index that responds more sensitively and eﬀectively to the odds of nonmotorized trips for shopping purposes when compared to commuting purposes. This study has several limitations; directions for future studies to address them follow. At the outset, even though this study found a signiﬁcant correlation between the walkability and odds of active transportation for shopping purposes, it showed no correlation between them for leisure models. This may be due to the Walkability Score’s sensitive nature toward walking and cycling for shopping purposes. For further direction of the study, the Walkability Score can be developed and customized based on trip purposes. For example, it will be possible to develop a walkability index, “Walkability Score for Leisure,” where people can search for environmental conditions for leisure purposes in their daily lives by considering the characteristics and contents of each facility that can attract and aﬀect leisure trips. Second, this study examined the correlation of neighborhood walkability with active transportation in Seoul. The city of Seoul is promoting policies regarding walking and cycling, and the neighborhood environment is being developed toward a more walkable environment [51]. Future research will be able to experiment with other cities in Korea, which could help identify practical policies in urban design and transportation planning that can be used across the country. Third, since we focused on active transportation for noncommuting trips, this study combined walking and cycling into nonmotorized transportation. However, walkers and cyclists can have diﬀerent characteristics of 12 of 16 12 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 shopping and leisure trips. In a further study, an analysis comparing walking and cycling behavior on each trip’s purposes could be discussed. Fourth, the mean ages of the participants for leisure and shopping purposes were relatively high, corresponding to middle-aged and older adults. This can reﬂect that retired people may have more time for recreational and shopping activities, and thus they are likely to spend their time on the active mode of the trip. Nevertheless, the older age of respondents remains a limitation. Author Contributions: Conceptualization, E.J.K. and H.K.; Methodology, E.J.K. and H.K.; Software, E.J.K. and J.K.; Validation, E.J.K., J.K., and H.K.; Formal Analysis, E.J.K. and J.K.; Investigation, E.J.K.; Resources, E.J.K.; Data Curation, E.J.K. and J.K.; Writing—Original Draft Preparation, E.J.K., J.K., and H.K.; Writing—Review and Editing, E.J.K. and H.K.; Visualization, J.K.; Supervision, E.J.K.; Project Administration, E.J.K.; Funding Acquisition, E.J.K. All authors have read and agreed to the published version of the manuscript. 5. Discussion Future studies need to properly extract samples and use them for analysis so that respondents are not biased at a speciﬁc age group. Finally, this study is a cross-sectional design that cannot identify causal relationship over time. Cross-sectional analysis cannot identify whether individuals walk frequently because of its walkable environmental conditions or whether the individuals who walk a lot more choose to live in walkable neighborhoods. For further study, a longitudinal analysis can be performed to examine the causality between walkable environment and active transportation. Despite these limitations, this study is signiﬁcant in several aspects. First, since the correlation of environmental walkability and active trips was analyzed using a multilevel logistic model, factors from both individual and neighborhood levels were taken into account. Although the ICC values were generally low at 4.5% in Model S–1 and 4.7% in Model S–2, it is suggested that multilevel modeling is required when dealing with nested data [73]. That is, because individual-level variables were nested within the neighborhood, the multilevel analysis allowed an examination of the impact of multilevel factors on the dependent variable (active transportation). Second, there are few studies that comprehensively examined active transportation for both leisure and shopping purposes. In this study, we compared the probability of active transportation (i.e., walking and cycling) for each travel purpose (i.e., leisure and shopping purposes) depending on the Walkability Score. Based on our ﬁndings, a tailored policy guideline can be provided based on trip purposes. Finally, we found that the Walkability Score, measured in Seoul, had a signiﬁcant validity for examining the odds of active transportation for shopping purposes. However, this study demonstrated that more variables should be considered when assessing active transportation, especially for leisure purposes, as they aﬀect not just accessibility to local destinations but also other urban form factors such as density, diversity, and design, and the quality of urban environments. From the ﬁndings of this study, policymakers and researchers from the urban and transportation planning and public health ﬁelds can obtain a comprehensive understanding of enhancing active travel in leisure and shopping, based on the primary concerns. References 1. Carr, L.J.; Dunsiger, S.I.; Marcus, B.H. Validation of walk score for estimating access to walkable amenities. Br. J. Sports Med. 2010, 45, 1144–1148. [CrossRef] [PubMed] 2. Shashank, A.; Schuurman, N. Unpacking walkability indices and their inherent assumptions. Health Place 2019, 55, 145–154. [CrossRef] [PubMed] 3. Nykiforuk, C.I.J.; McGetrick, J.A.; Crick, K.; Johnson, J.A. 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The results of this study showed that the association between walkability and active transportation varies depending on trip purposes. The results remind urban policymakers of the need to diﬀerentiate policy remedies while promoting active transportation. Based on this study, various policy suggestions for promoting active transportation were established, and it is expected that citizens will be encouraged to walk and cycle and increase physical activity to enjoy a better quality of life. 13 of 16 13 of 16 Int. J. Environ. Res. Public Health 2020, 17, 2178 Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2017R1A2B4005440). Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2017R1A2B4005440). Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4287321948	https://zenodo.org/records/4607474/files/GSCARR-2021-0019.pdf	English	null	Traumatic brain injury: Future application of nanomedicine	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	4,748	Abstract Traumatic brain injury (TBI) is currently a rising player in the cause of disability and neurological dysfunction worldwide. TBI is a common occurrence in the military and extreme activities, sports arena and accidents. Severe TBI can be fatal but mild TBI persists and progressively deteriorates brain homeostasis and physiology. Apart from the physical disabilities, psychological complexities arise in people with mild TBI. Despite the seriousness of this hazard, treatments for TBI are not adequate, mostly due to the brain being involved. Nanoparticle (NP) therapy seems to be an effective alternative to combat TBI. This review outlines the state of TBI and describes the probable medical support that nanomedicine can provide. Keywords: Traumatic brain injury; Blast TBI; Pathophysiology; Blood brain barrier; Nanoparticle therapy Nasima Khanam and Debjani Nath * Department of Zoology, University Of Kalyani, Kalyani Nadia, West Bengal, India Pin 741235. Department of Zoology, University Of Kalyani, Kalyani Nadia, West Bengal, India Pin 741235. GSC Advanced Research and Reviews, 2021, 06(02), 020–027 Department of Zoology, University Of Kalyani, Kalyani Nadia, West Bengal, India Pin 741235. GSC Advanced Research and Reviews, 2021, 06(02), 020–027 Publication history: Received on 08 January 2021; revised on 04 February 2021; accepted on 06 February 2021 Article DOI: https://doi.org/10.30574/gscarr.2021.6.2.0019  Corresponding author: Debjani Nath Department of Zoology, University Of Kalyani, Kalyani Nadia, West Bengal, PIN 741235. Copyright © 2021 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. or(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.  Corresponding author: Debjani Nath Department of Zoology, University Of Kalyani, Kalyani Nadia, West Bengal, PIN 741235. 1.1. TBI due to blast explosion A bTBI resulting from a bomb or improvised explosive devices (IEDs) explosion is a major concern. bTBI is caused by blast shock pressure waves which last over short periods, milliseconds. The diffuse interaction of the brain with the pressure waves triggers a complex cascade of events that disturb the brain structure and function. bTBI occurs as mentioned by Marklund (2020) (1) the primary blast injury is caused by sudden rise in atmospheric pressure from the explosion itself; (2) the secondary blast injury results from released fragments which may cause a penetrating TBI; (3) the tertiary blast injury occurs when an individual is thrown against or into a solid object by the blast forces, and (4) the quaternary blast injury results from, e.g., blood loss from associated injuries. The more controversial fifth mechanisms may result from markedly delayed injuries due to the initial components released at time of explosion.[7] Most of the head injuries in warfare are bTBI. 1.2. TBI due to penetrative ballistic injury Another common form of brain injury is penetration by a foreign body. Physical disruption of neurons, glial cells and fibre tracts occur when a foreign object pierces the bony skull and passes into the or through the brain substance. Brain damage may be further complicated by ischemia and haemorrhage [6]. 1.3. TBI in military air force To understand the military air force TBI, firstly it is essential to have a brief idea of the underlying causative factors. When it comes to brain injury, it is convenient to understand forces and the impact of forces on the intra-cerebral pressure and homeostasis of the brain [8]. The repercussions of pushing the body against the natural forces to fly high and battle can be dire in extreme cases. The gravitational force or G force acts on blood and blood vessels. When pushed to the limit, blood collects in the lower extremities of the body blocking the adequate blood supply in the brain. Fighter pilots are trained to manage up to 8 or 9 G for longer periods, where as untrained people may pass out between 3 or 4 G. Fighter pilots are equipped with specialized suits, taught specialized breathing and muscle tensing techniques and trained in centrifuges with artificial Gs. Nevertheless, there is a limit to what people can tolerate. High acceleration of the body in the longitudinal direction (Gz) may lead to loss of vision or even loss of consciousness of the pilot leading to fatal accidents. The unpredictable circumstances of warfare steer accidents where an impact on the head, in addition to the already stressful conditions lead to TBI. mTBI or concussions usually occur between 80-100G. It is natural for mTBI to go unnoticed, hence the lack of documentation. But the cytoarchitectural and functional damage in the brain due to trauma persists and creates complications. Mostly neurodegenerative diseases follow, along with psychiatric symptoms or disability. 1. Introduction Traumatic Brain Injury is caused when an external force hits the head, strong enough to modify brain functioning and cause pathological changes. Traumatic brain injury or TBI can result from an accidental impact on the head during a car accident or an impact from a fall or sports activity, also from non-impact events like blast waves, rapid acceleration or deceleration that generates brain movement within the cranium [1, 2]. TBI has been the stealth factor disrupting the ability to speak, see, hear, walk, think, also control emotions. Symptoms of TBI range from mild to severe. Mild TBI often goes undetected initially but ‘silently’ makes an appearance only to hinder an individual’s normal flow of life that can be discussed with special reference to the military forces. Worldwide incidence of TBI is about 10 million with many deaths. Military cases alone account for 4 lakh cases. The gravity of TBI requires special attention for military personnel since they are in the higher-risk zone for combat and non-combat exposures [3, 4] and often face multiple TBIs. The risk of incessant neurological degradation and pain in case of recurrent injuries pose a serious threat to military personnel. Mild TBI (mTBI) or concussions are a common injury in warfare. mTBI is often listed as one of the prime rationales behind post-traumatic stress disorder PSTD and repetitive mTBIs also pose the threat of depression leading to suicide after deployment [5]. Military TBI is essentially different from civilian TBI. The phases of neural injury from TBI are traditionally divided into primary mechanical injury and delayed secondary injury. The primary process refers to the injury inflicted during the impact while the secondary process points out to the cascade of molecular events after the insult of the primary injury, generally occurring minutes or days later, determined partly by the host response. The military TBI includes two additional modes of mechanical injury - penetrating ballistic injury and blast injury [6]. But TBI due to explosive blast (bTBI) seems to be the prevalent type in the battlefield. GSC Advanced Research and Reviews, 2021, 06(02), 020–027 2. Pathophysiology of TBI 2. Pathophysiology of TBI 2. Pathophysiology of TBI Figure 1 Pathophysiology of TBI Figure 1 Pathophysiology of TBI 21 GSC Advanced Research and Reviews, 2021, 06(02), 020–027 The pathophysiology of TBI represents an intricate system of damage which throws the brain cells in disarray, crippling the natural state. The pathophysiology of TBI represents an intricate system of damage which throws the brain cells in disarray, crippling the natural state. Severe TBI patients who suffer heavy injury are mainly comatose or with haemorrhage. When hit, the manifestation of TBI triggers mechanical disturbance in cellular integrity. There is a redistribution of ions and neurotransmitters resulting from transient cell membrane disruptions, mainly rapid, unregulated influx of sodium(Na+), efflux of potassium(K+) and rise in levels of intra-axonal calcium(Ca²+) [9,10,11] ( Figure 1). There may be changes in glutamate stimulated Ca²+ signalling. The increasing Ca²+ in the axons activates calpain , a protease, initiating proteolysis of cytoskeletal proteins creating irreversible axonal pathology. Ca²+ rise also induces N- methyl-D-aspartate (NMDA) receptor activation, further depolarizing the neurons. It has been observed that Ca²+ build-up in mitochondria leads to oxidative stress and impairment of mitochondrial function and acidosis and edema due to lactate production. Cognitive deficits in TBI correspond to Ca²+ accumulation. On the other side, axons mainly in the white matter tracts of the brain, also experience rotational and shear forces during rapid acceleration and deceleration which cause diffuse axonal injury. Pressure waves from blast which pass through the brain, stretch the axons and vasculature and compress the glial cells and neurons [12]. A hallmark after TBI is the alteration in cerebral glucose metabolism (CMRglc). After head injury there is a period of hyperglycolysis, followed by a state of glucose metabolic depression, yet the cause of these phenomena is only speculative [13]. Over the time upregulation of aquaporins (AQP1 and AQP4) on endothelial cells disrupts the blood brain barrier (BBB) by allowing passive diffusion of water [14]. Consequently, circulating macrophages, neutrophils and lymphocytes gather promoting inflammation. The cascade of neuroinflammation follows generating reactive oxygen species (ROS), proteolytic enzymes, cytokines and chemokines [15]. Matrix metalloproteinases are also implicated in the destruction of BBB [16]. Continual stimulation of the resident microglial cells, astrocytes accompanied by the infiltration of peripheral immune cells aggravates inflammation. Microglial alteration, with retracted processes, is a distinctive feature of bTBI [17]. TBI also alters the cerebral blood flow (CBF) in the brain. 2. Pathophysiology of TBI In the case of mTBI, after an initial period of decreased CBF, nitric oxide release leads to unresponsive vasodilation [18]. Activation of Wallerian degeneration along with hypoxia and inflammation is a common scenario of the brain after a traumatic injury [19]. 3. Treatment Primary injury is due to sudden direct impact and cannot be treated. Hence treatment strategies are directed towards alleviating the secondary responses. Currently, the treatment provided is intensive care management and palliative. Primary injury is due to sudden direct impact and cannot be treated. Hence treatment strategies are directed towards alleviating the secondary responses. Currently, the treatment provided is intensive care management and palliative. No two TBIs are the same. The extent of injury varies in each event and also depends on the host resilience. Thus the symptoms and effects experienced by each person differ and require attention in an almost personalized manner. The severity of TBI is currently determined using the Glasgow Coma Scale (GCS) based on the level of consciousness after the injury (Table 1). Table 1 Simplified GCS data for classification of TBI BRAIN INJURY GCS Severe <8-9 Moderate 8 or 9-12 Minor 13 or > 13 Table 1 Simplified GCS data for classification of TBI Table 1 Simplified GCS data for classification of TBI Over the past two decades, TBI has emerged as one of the leading concerns for the mental health of military personnel. Patients with mTBI suffer concussions that recoup but they are trailed by difficulty in cognitive and motor abilities. Severe TBI patients may be comatose [19]. In spite of the grim situation, the only treatments currently available are pressure dressings and dealing with external wounds. Regardless of the alarming issue, the clinical and pre-clinical trials conducted could not produce successful therapies [20]. Along these lines, the use of nanoparticles in the treatment of TBI is now being actively considered. As mentioned above, the clinical trials failed at procuring definitive treatments. The primary protagonist of this stalemate is the mighty blood brain barrier (BBB). Although therapeutic drugs have been developed, none are sufficiently effective since they do not reach their destination. The BBB is the anatomical barrier that fortifies the brain 22 GSC Advanced Research and Reviews, 2021, 06(02), 020–027 cells against the surrounding vasculature in the brain parenchyma. The BBB is the protective selective filter that coordinates the trading of molecules with the blood vessels due to the presence of brain microvascular endothelial cells (BMECs). BBB also includes astrocytes and pericytes. The BMECs present a single layer of compact, almost non- fenestrated endothelial cells, sealed together by tight junctions. 4. Nanomedicine for TBI The discipline of nanoscale materials, namely particles ranging from 1-100 nm, is rapidly on the rise. The nanostructural elements have found their way in myriad aspects of neuroscience because of their unique properties which have the potential to uncover previously implausible areas. Nanomedicine, in these regards, seems to be the pragmatic alternative for drug delivery to the brain. Biocompatible tunable nanoparticle (NP) carriers have the capacity to cross the BBB. NPs that are biocompatible, biodegradable [22], have higher stability in blood, low immunogenicity and non- toxic for the brain are considered for drug delivery. Therefore mainly organic NPs are used in nanomedicine, viz, liposomes, solid lipid NP, polymeric NP etc. Perhaps the most advantageous characteristics of NPs is their small size and large surface to volume ratio. This feature accounts for the faster rate of drug release and greater bioavailability. For infiltrating the BBB, the NP carriers are coated with biospecific molecules according to requirements in a process called functionalization. This facilitates targeted delivery in the brain [23]. To ensure entry into the brain NPs are tuned to follow mainly three broad pathways. Receptor-mediated transcytosis (RMT) is the process through which NPs are conjugated with ligands whose receptors are present in the apical surface of BBB. Upon ligand binding, the cell membrane invaginates into intracellular coated vesicles and the macromolecule is uptaken, while the receptor is recycled. Another mechanism of transport is the adsorptive-mediated transcytosis (AMT) where NPs conferred with positive charges electrostatically interact with the negatively charged endothelial cells. However, this pathway remains ambiguous due to non-specific drug accumulation tendencies. Lastly, the cell-mediated transcytosis involves immune cell dependent uptake, via neutrophils, monocytes and macrophages. This method is exploited to “hitchhike” NPs into the brain [23]. For greater access to the CNS of NPs, the nasal route of drug delivery has become popular as of late. The roof of the nasal palate is perhaps the sole region of the body where direct uptake of NP into the CNS, bypassing the BBB, is a possibility [21]. It is also advantageous that TBI often results in leaky BBB making NP drug delivery feasible. In the case of TBI, the brain injury is not concentrated in just any single facet, it is varied and a complex network operates. This represents broader prospects of therapeutic targets for NPs. 3. Treatment The efflux transporters of BMECs prevent the entry of arbitrary substances, ions and enzymes, in the brain parenchyma while the influx transporters allow the inward movement of essential nutrients and necessary factors to the brain cells [21]. The BBB, thus, competently sustains the brain homeostasis and functional and architectural integrity. Granted, this impediment limits the traditional therapies, it is still a possibility that non-conventional therapies can carry forward the research. The emergence of nanoparticles (NPs) for neurological research is squarely due to this obstacle. The nanoparticles have been shown to traverse the influx transporters into the brain parenchyma. Thus NPs shed a light in this dark corner of brain studies and suggests an optimistic commencement of a new era of neurological studies. 4. Nanomedicine for TBI The secondary injury, post-TBI, initiates a cascade of chain reactions that serve as potential targets for NP therapeutics. To date, there has been quite a good amount of work in targeting NPs within the brain. Perhaps neuroinflammation of the brain post-TBI has been the subject of much research. Microglia and astrocyte damage is one of the major concerns in bTBI. Hubbard et al [24] designed hemostatic nanoparticles delivering dexamethasone (hDNPs) functionalized with GRGDS ( glycine-arginine-glycine-aspartic acid-serine) peptide targeting platelets, aimed at reducing bleeding and neuroinflammation. hDNPs were shown to reduce microglia and astrocyte degeneration and also reduced apoptosis levels (Figure 2). hDNPs further diminished anxiety, a pressing problem of bTBI among war veterans. Assisting the military personnel and civilians to lead a normal life without the traumatic experience of the injuries is imperative. The administration of hDNPs in blast-exposed rodents increased their survivability after the traumatic event. 23 23 GSC Advanced Research and Reviews, 2021, 06(02), 020–027 Figure 2 Probable NP therapeutic targets of TBI Figure 2 Probable NP therapeutic targets of TBI Post primary injury, the inflammation of the brain cells is responsible for a large amount of degeneration of neurons and damage in the hippocampus, resulting in behavioural and memory deficits. Thioester core NPs were used to mitigate the ROS generated in the secondary biochemical pathways of TBI. Though further research is warranted, this approach has a potential for future therapies. CeONPs (cerium oxide nanoparticles) have also demonstrated similar ROS scavenging functions. Additionally, CeONPs reduced the glutamate and calcium induced excitotoxicity, consequently reducing neuronal damage and oxidative stress. CeONPs have inherent superoxide dismutase (SOD)- like activity owing to its redox property combined with the increased surface area and nanoscale quantum lattice alterations [25]. Oxygen Reactive Polymer (ORP) NPs lowered the secondary complications due to ROS and abated H + O+ levels by threefold [26]. ROS levels were also cut down by cross-linked NPs, which were synthesized from polysorbate-80. This approach also eases the secondary neuroinflammation and also prevents cognitive and sensorimotor disabilities to some extent [27]. Yet another method was devised where non-steroidal anti-inflammatory drugs, here Ibuprofen, were armed with tocopherol conjugated with tetra ethylene glycol. This nanoprodrug was able to cross the BBB in the injured regions and conferred neuroprotection [28]. Redox-active nitroxide radical-containing nanoparticles, RNPs, administered to scavenge ROS, displayed an effect in enhancing cognitive behaviour, lowering the contusion volume and providing neuroprotection [29]. 5. Discussion More than 2.6 million victims of TBI are around and the number of people with TBI related physical and mental disabilities is ever increasing. This is especially true when the life of war veterans is looked into. Yet, felicitous facilities are lacking for proper medical support. Preventive measures for TBI are a far cry from a realistic viewpoint. Since the primary injury is unfixable the alternative is to preserve that which remains. The foremost hurdle, in this case, is the missing timely detection of mTBI. The absence of prompt action exacerbates the condition. Soldiers need periodic screening for mTBI. Since each traumatic event is different from the other, the extent of injury varies. This requires special attention to follow the treatment methods with individuality in an almost personalized manner. Even after TBI has been identified the existing surgical and management procedures do not guarantee recovery. The greatest hindrance in treating brain injury as well as other neurological problems is the BBB. Although NPs have successfully overcome this initial barrier, there is still a long way for translational work. Promising in vitro studies are all at preclinical levels. Since NP therapy revolves around particles of nanoscale dimensions and their interactions with the brain which itself is an enigma, profound research concerning every aspect of the brain and subsequent effects on the patients has to be carried out meticulously. Moreover, nanomedicine is now at the outset stage. For advancing clinical trials NP production and cost-effectiveness also need improvement. An important factor to be considered is that a neutral zeta potential is critical for successful delivery across the BBB, however, very few reports are currently available [36]. Despite these challenges, the preclinical trials have provided conclusive results which paved the way to clinical trials. Thorough and ample research in this field is the need of the hour where numerous soldiers and civilians suffer from TBI which gradually leads to physical impairments, depression and even suicide. 4. Nanomedicine for TBI Breakdown of BBB in TBI fuels the destruction of brain homeostasis. Rouzi et al [30] developed PLGA NPs (poly (lactide- co-glycolide) nanoparticles) loaded with cerebrolysin which interfered with BBB breakdown and edema formation in rats with concussive head injury(CHI). The brain-derived neurotrophic factor, BDNF of the neurotrophin family, when transported to the brain via PLGA NPs which were coated with surfactant poloxamer 188, improved neurological state and cognitive abilities of the trauma induced mice [31]. A measure that attempts to minimize secondary neuron degradation is the application of neuroprotective agents. Targeting neurons with siRNA containing NPs, while taking advantage of the leaky BBB after primary impact, may serve to reduce the downstream after effects of TBI [32]. In the case of TBI, timely diagnosis is an important factor for a patient's recovery. But again, mTBI tends to go unnoticed most of the time. NPs have been exploited for diagnostic purposes. Gold NPs coated with silica serve as detection tools for neuron-specific enolase (NSE) and S100-ß proteins which are typical TBI markers [33]. For detection and monitoring of the necrotic brain cells in TBI, PEG-PLGA NPs (polyethylene glycol-PLGA NPs) encapsulating both near- infrared (NIR) fluorophores and perfluorocarbons (PFCs) in conjugation with cyanide dye 800CW is employed [34]. Further, KO et al [35] have used nanofluidics for better identification of TBI markers. Track-Etched magnetic NanoPOre (TENPO) has enabled efficient biomarker profiling, relevant characterization of plasma and serum using biomarkers from healthy and injured patients and prediction of history and intensity of TBI. Nonetheless, there still remains many relatively unexplored areas of TBI for NP therapy like excitotoxicity, ionic imbalance and lipid peroxidation. 24 24 GSC Advanced Research and Reviews, 2021, 06(02), 020–027 6. Conclusion Malaise of TBI has plagued human lives in a rather complicated manner. The paucity of treatment and relief measures have added to the misery. Nanoparticle therapy for TBI is now an active field of research with novel designs cropping up every day. In the future NP therapeutics, in all probability, may emerge as the front in the treatment of traumatic brain injury, raising the quality of life of TBI patients. Acknowledgments Acknowledgments All authors gratefully acknowledge the University of Kalyani, Kalyani Nadia, WB, India for financial support. Disclosure of conflict of interest No competing financial or academic interests exist. No competing financial or academic interests exist. References [1] Prins M, Greco T, Alexander D, Giza CC. The pathophysiology of traumatic brain injury at a glance. Disease models & mechanisms. 2013; 6(6): 1307-15. [2] Sulhan S, Lyon KA, Shapiro LA, Huang JH. Neuroinflammation and blood–brain barrier disruption following traumatic brain injury: Pathophysiology and potential therapeutic targets. Journal of neuroscience research. 2020; 98(1): 19-28. [2] Sulhan S, Lyon KA, Shapiro LA, Huang JH. 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JAMA psychiatry. 2013; 70(7): 686-91. [6] Risdall JE, Menon DK. Traumatic brain injury. Philosophical Transactions of the Royal Society B: Biological Sciences. 2011; 366(1562): 241-50. 7] Marklund N. Blast-Induced Brain Injury. InManagement of Severe Traumatic Brain Injury. 202 25 GSC Advanced Research and Reviews, 2021, 06(02), 020–027 [8] Evans V. Newton's laws, G-forces and the impact on the brain. Australasian Journal of Neuroscience. 2020; 30(1): 24. [9] Pearn ML, Niesman IR, Egawa J, Sawada A, Almenar-Queralt A, Shah SB, Duckworth JL, Head BP. Pathophysiology associated with traumatic brain injury: current treatments and potential novel therapeutics. Cellular and molecular neurobiology. 2017; 37(4): 571-85. [10] Zetterberg H, Smith DH, Blennow K. Biomarkers of mild traumatic brain injury in cerebrospinal fluid and blood. Nature Reviews Neurology. 2013; 9(4): 201. [11] Wolf JA, Stys PK, Lusardi T, Meaney D, Smith DH. Traumatic axonal injury induces calcium influx modulated by tetrodotoxin-sensitive sodium channels. Journal of Neuroscience. 2001; 21(6): 1923-30. [12] Duckworth JL, Grimes J, Ling GS. Pathophysiology of battlefield associated traumatic brain injury. Pathophysiology. 2013; 20(1): 23-30. [13] Yoshino A, Hovda DA, Kawamata T, Katayama Y, Becker DP. References Dynamic changes in local cerebral glucose utilization following cerebral concussion in rats: evidence of a hyper-and subsequent hypometabolic state. Brain research. 1991; 561(1): 106-19. [14] Winkler EA, Minter D, Yue JK, Manley GT. Cerebral edema in traumatic brain injury: pathophysiology and prospective therapeutic targets. Neurosurgery Clinics. 2016; 27(4): 473-88. [15] Hall ED, Vaishnav RA, Mustafa AG. Antioxidant therapies for traumatic brain injury. Neurotherapeutics. 2010; 7(1): 51-61. [16] Abdul-Muneer PM, Pfister BJ, Haorah J, Chandra N. Role of matrix metalloproteinases in the pathogenesis of traumatic brain injury. Molecular neurobiology. 2016; 53(9): 6106-23. [17] Xu L, Schaefer ML, Linville RM, Aggarwal A, Mbuguiro W, Wester BA, Koliatsos VE. Neuroinflammation in primary blast neurotrauma: Time course and prevention by torso shielding. Experimental neurology. 2016; 277: 268-74. [18] McGinn MJ, Povlishock JT. Pathophysiology of traumatic brain injury. Neurosurgery Clinics. 2016; 27(4): 397- 407. [19] Moore J, Alzate‐Correa D, Dasgupta D, Lawrence W, Dodd D, Mathews C, Valerio I, Rink C, Higuita‐Castro N, Gallego‐Perez D. Micro‐and Nanoscale Biointerrogation and Modulation of Neural Tissue–From Fundamental to Clinical and Military Applications. Nanotechnology and Microfluidics. 2020: 383-417. [20] Sharma S, Ifergan I, Kurz JE, Linsenmeier RA, Xu D, Cooper JG, Miller SD, Kessler JA. Intravenous immunomodulatory nanoparticle treatment for traumatic brain injury. Annals of neurology. 2020; 87(3): 442- 55. A. Nanoparticle brain delivery: a guide to verification methods. Nanomedicine. 2020; 15(04): 409-32 [21] Yokel RA. Nanoparticle brain delivery: a guide to verification methods. Nanomedicine. 2020 [22] Tosi G, Duskey JT, Kreuter J. Nanoparticles as carriers for drug delivery of macromolecules across the blood-brain barrier. Expert Opinion on Drug Delivery. 2020; 17(1): 23-32. [23] Sim TM, Tarini D, Dheen ST, Bay BH, Srinivasan DK. Nanoparticle-based technology approaches to the management of neurological disorders. International Journal of Molecular Sciences. 2020; 21(17): 6070. [24] Hubbard WB, Lashof-Sullivan M, Greenberg S, Norris C, Eck J, Lavik E, VandeVord P. Hemostatic nanoparticles increase survival, mitigate neuropathology and alleviate anxiety in a rodent blast trauma model. Scientific reports. 2018; 8(1): 1-2. [25] Bailey ZS, Nilson E, Bates JA, Oyalowo A, Hockey KS, Sajja VS, Thorpe C, Rogers H, Dunn B, Frey AS, Billings MJ. Cerium oxide nanoparticles improve outcome after in vitro and in vivo mild traumatic brain injury. Journal of Neurotrauma. 2020; 37(12): 1452-62. [26] Xu J, Ypma M, Chiarelli PA, Park J, Ellenbogen RG, Stayton PS, Mourad PD, Lee D, Convertine AJ, Kievit FM. GSC Advanced Research and Reviews, 2021, 06(02), 020–027 References Theranostic oxygen reactive polymers for treatment of traumatic brain injury. Advanced Functional Materials. 2016; 26(23): 4124-33. [27] Yoo D, Magsam AW, Kelly AM, Stayton PS, Kievit FM, Convertine AJ. Core-cross-Linked nanoparticles reduce neuroinflammation and improve outcome in a mouse model of traumatic brain injury. ACS nano. 2017; 11(9): 8600-11. 26 GSC Advanced Research and Reviews, 2021, 06(02), 020–027 [28] Clond MA, Lee BS, Jeffrey JY, Singer MB, Amano T, Lamb AW, Drazin D, Kateb B, Ley EJ, John SY. Reactive oxygen species-activated nanoprodrug of Ibuprofen for targeting traumatic brain injury in mice. PLoS One. 2013; 8(4): e61819. [29] Takahashi T, Marushima A, Nagasaki Y, Hirayama A, Muroi A, Puentes S, Mujagic A, Ishikawa E, Matsumura A. Novel neuroprotection using antioxidant nanoparticles in a mouse model of head trauma. Journal of Trauma and Acute Care Surgery. 2020; 88(5): 677-85. [30] Ruozi B, Belletti D, Sharma HS, Sharma A, Muresanu DF, Mössler H, Forni F, Vandelli MA, Tosi G. PLGA nanoparticles loaded cerebrolysin: studies on their preparation and investigation of the effect of storage and serum stability with reference to traumatic brain injury. Molecular neurobiology. 2015; 52(2): 899-912. [31] Khalin I, Alyautdin R, Wong TW, Gnanou J, Kocherga G, Kreuter J. Brain-derived neurotrophic factor delivered to the brain using poly (lactide-co-glycolide) nanoparticles improves neurological and cognitive outcome in mice with traumatic brain injury. Drug delivery. 2016; 23(9): 3520-8. [32] Kwon EJ, Skalak M, Lo Bu R, Bhatia SN. Neuron-targeted nanoparticle for siRNA delivery to traumatic brain injuries. ACS nano. 2016; 10(8): 7926-33. [33] Wang Y, Zhao P, Mao L, Hou Y, Li D. Determination of brain injury biomarkers by surface-enhanced Raman scattering using hollow gold nanospheres. RSC advances. 2018; 8(6): 3143-50. [34] Cruz LJ, Que I, Aswendt M, Chan A, Hoehn M, Löwik C. Targeted nanoparticles for the non-invasive detection of traumatic brain injury by optical imaging and fluorine magnetic resonance imaging. Nano Research. 2016; 9(5): 1276-89. [35] Ko J, Hemphill M, Yang Z, Sewell E, Na YJ, Sandsmark DK, Haber M, Fisher SA, Torre EA, Svane KC, Omelchenko A. Diagnosis of traumatic brain injury using miRNA signatures in nanomagnetically isolated brain-derived extracellular vesicles. Lab on a Chip. 2018; 18(23): 3617-30. [36] Bony BA, Kievit FM. A Role for Nanoparticles in Treating Traumatic Brain Injury. Pharmaceutics. 2019; 11(9): 473. 27
https://openalex.org/W2513928403	https://www.repo.uni-hannover.de/bitstream/123456789/822/1/c6ra06774c.pdf	English	null	Facile fabrication of highly efficient modified ZnO photocatalyst with enhanced photocatalytic, antibacterial and anticancer activity	RSC advances	2,016	cc-by	12,215	Waseem Raza,a Syed Mohammad Faisal,b Mohammad Owais,b D. Bahnemanncd and M. Muneera Waseem Raza,a Syed Mohammad Faisal,b Mohammad Owais,b D. Bahnemanncd and M M nee a The degradation of organic pollutants in the aqueous medium using semiconductor photocatalysts has become an attractive method for detoxiﬁcation of water. ZnO is an eﬃcient photocatalyst with few disadvantages such as: (i) the wide band gap (3.37 eV); and (ii) the fast recombination rate of photogenerated electron–hole pairs which limit the photodegradation eﬃciency of bare ZnO photocatalyst. Herein, we report a strategy for the suppression of electron–hole pair recombination rate, extention the absorption edge in the visible region and enhancement of photocatalytic eﬃciency by introducing rare earth metal as a dopant. We present the fabrication of pure and Er/Nd doped ZnO semiconductor photocatalysts with hexagonal wurtzite structure using sol gel method. The prepared photocatalysts were characterized by standard analytical techniques, such as XRD, SEM-EDS, TEM, FTIR, XPS, BET, TGA, DTA, DSC, PL, DRS and UV-vis spectroscopy. The photocatalytic activity of pure and doped ZnO nanoparticles (NPs) was investigated by studying the degradation of two diﬀerent organic dyes as a function of irradiation time. The results indicate that the photocatalytic activity of doped ZnO was found to be higher than bare ZnO for degradation of dyes. This may be attributed predominantly due to decrease in the recombination rate by the eﬃcient charge separation of photoinduced electron– hole pair as inferred from PL studies. The results also indicate that parameters such as amount of photocatalyst dose, initial pH and diﬀerent quenchers play a signiﬁcant role for degradation of model dyes. The synthesized photocatalyst was recycled four times for degradation of dye with very little decrease in eﬃciency. Interestingly, comparative in vitro antibacterial and anticancer potential of the pure and Er/Nd doped ZnO NPs were also investigated against human pathogenic bacterial strains and various human cancer cell lines. The result of our study clearly revealed that Nd doped ZnO NPs showed better antibacterial as well as anticancer eﬃcacy as compared to pure and Er doped ZnO NPs. Received 14th March 2016 Accepted 5th July 2016 DOI: 10.1039/c6ra06774c www.rsc.org/advances DOI: 10.1039/c6ra06774c www.rsc.org/advances new methods for dyeing textile, paper and pulp, a large amount of harmful waste containing dyes are continuously released into the water system. Most dyes contain a high degree of aroma- ticity, substituted with azo (–N]N) and hydroxyl (–OH) groups which are non-biodegradable.2 These dyes are polluting water and hazardous to health. RSC Advances RSC Advances Cite this: RSC Adv., 2016, 6, 78335 Received 14th March 2016 Accepted 5th July 2016 DOI: 10.1039/c6ra06774c www.rsc.org/advances Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Waseem Raza,a Syed Mohammad Faisal,b Mohammad Owais,b D. Bahnemanncd and M. Muneera Therfore, it is important to develop techniques which are helpful for their remediation. The tech- niques such as occulation, precipitation, adsorption, physi- cochemical, biological and sedimentation for the degradation of dyes/organic pollutants from water bodies are not fully eﬀective because they do not completely degrade the pollut- ants.3 Hence, there is a vital need to develop a new clean and ecofriendly technique for treating such environmental pollut- ants.4 The degradation of organic dyes in aqueous phase using advanced oxidation processes (AOPs) is an eﬃcient approach. Purication of waste water using photocatalysis is one of the most widely used AOPs.5 aDepartment of Chemistry, Aligarh Muslim University, Aligarh-202002, India. E-mail: m.muneer.ch@amu.ac.in; readermuneer@gmail.com; Tel: +91-571-2700920 ext. 3365 bInterdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India cPhotocatalysis and Nanotechnology, Institut fuer Technische Chemie, Gottfried Wilhelm Leibniz Universitaet Hannover, Callinstrasse 3, D-30167 Hannover, Germany dPhotoactive Nanocomposite Materials, Saint-Petersburg State University, Ulyanovskaya str. Peterhof, Saint-Petersburg, 198504 Russia † Electronic supplementary information (ESI) available: The supporting information contains the following results: X-ray photoelectron spectroscopy, Brunauer–Emmett–Teller, thermal analysis measurement (TGA and DSC), the plot of ln C/C0 vs. irradiation time, eﬀect of catalyst loading, eﬀect of pH, apparent rate constant and R2 value for degradation of dyes in the presence of doped ZnO NPs. See DOI: 10.1039/c6ra06774c This journal is © The Royal Society of Chemistry 2016 RSC Advances PAPER View Article Online View Journal \| View Issue RSC Advances PAPER View Article Online View Journal \| View Issue Facile fabrication of highly eﬃcient modiﬁed ZnO photocatalyst with enhanced photocatalytic, antibacterial and anticancer activity† Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1. 1. Water pollution from diﬀerent sources such as agricultural run- oﬀ, eﬄuent from dyes & pesticides industries and chemical spill are major concern all over the world.1 Due to the development of aDepartment of Chemistry, Aligarh Muslim University, Aligarh-202002, India. E-mail: m.muneer.ch@amu.ac.in; readermuneer@gmail.com; Tel: +91-571-2700920 ext. 3365 bInterdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India Recently, semiconductor based heterogeneous photo- catalysis has attracted strong attention for the degradation of This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 78335–78350 \| 78335 View Article Online View Article Online RSC Advances pollutants via photo-splitting of water using renewable solar energy.1,6,7 The reactive species generated can also kill micro- organisms. Many semiconductor based heterogeneous photo- catalysts, such as SnO2, Bi2O3, WO3, TiO2 and ZnO have been used.8–10 Among the various semiconductor photocatalysts ZnO and TiO2 are known as excellent photocatalysts for waste water treatment.11,12 TiO2 is universally recognized, extensively inves- tigated and widely employed as the most active photocatalyst. ZnO is a suitable alternative to TiO2 due to its high reactivity, higher volume to area ratio, large excitation binding energy (60 meV), environmentally friendly nature, low cost and long life span.4,13,14 ZnO is an n-type II–VI semiconductor and has a wurtzite structure.15 However, the practical application of ZnO is restricted to UV-light due to its wide band gap and fast recombination rate of photogenerated charge carrier.16 There- fore, extensive researches are being carried out for the improvement of electrical and optical properties of bare ZnO. Development of solar light driven ZnO can eﬀectively be carried out by doping with nonmetals, metals, metal oxides, and carbon materials.17–20 During doping process the higher valency metal ions can replace the Zn2+ ions in the ZnO lattice.12,15,21,22 Recently, doping of ZnO with rare earth metals has attracted much attention due to the f orbital that allows adsorption of more pollutants on the catalyst surface reducing the band gap energy which may in turn improve the photocatalytic activity. Rare earth metals have good ability to trap the photogenerated charge carrier and reduce the recombination rate, which may intern increase the photocatalytic activity of doped ZnO.23 Among them Er and Nd doped ZnO have attracted strong attention due to the optical intra-4f transition which can cause an emission at 1.54 mm with minimum loss in optical communication.24 prepared photocatalyst was tested by studying the degradation of two diﬀerent organic dyes under visible light illumination. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Zinc acetate dihydrate was purchased from Sigma-Aldrich. Reactive Red 241 (RR 241) was supplied by Atul India Ltd. Val- sad, Gujrat, India. Triton-X, nitric acid (HNO3, 98wt%), sodium hydroxide (NaOH), isopropanol (IPA), potassium iodide (KI) and ammonium solution were obtained from Merck, while erbium nitrate hexahydrate, neodymium nitrate hexahydrate Methylene Blue (MB) were purchased from Central Drug House, India. Benzoquinone (BQ) was obtained from Alfa Aesar. 2.2 Preparation of pure and doped ZnO NPs The photocatalytic activity of the Zn(CH3COO)2$2H2O + 4NH4OH / Zn(NH3)4 2+ + 2CH3COOH + 6H2O (1) Zn(NH3)4 2+ + 2OH / ZnO + 4NH3 + H2O (2) (2) 2.2 Preparation of pure and doped ZnO NPs All reagents were of analytical grade and used without further purication. Doped ZnO NPs were prepared by sol gel method using zinc acetate dihydrate as a precursor and erbium nitrate hexahydrate/neodymium nitrate hexahydrate as dopants. In a typical procedure 6 g, 0.023 M of zinc acetate dihydrate and 5 mL Triton-X were dissolved in 50 mL double distilled water in a round bottom ask under vigorous stirring for 60 min at room temperature to form a transparent solution. To the above solution, the desired mole percent of dopants in ammonium solution was added drop wise. The solution was stirred continuously at 40 C until the formation of a white gel. The obtained gel was lefor 24 h, which was ltered and washed thoroughly with water and ethanol. The obtained material was dried at 120 C for 2 h in an oven and then manually ground into a ne powder and calcinated at 600 C for 4 h. The pure ZnO NPs were fabricated using the same method without the addition of dopant. The formation of ZnO NPs can be explained by the chemical reaction given below. A wide range of methods have been employed for the prep- aration of doped ZnO photocatalysts, such as autocombustion, co-precipitation, solvothermal processes, precipitation and ball- milling.25–29 However, these methods are expensive as they require complex equipment. Currently, synthesis of metal doped ZnO photocatalysts using sol–gel process has attracted considerable attention because of its acceptable costs, simplicity, simple experimental conditions, good crystalline quality and easy to handle.30 This method is also helpful for synthesis of ZnO with diﬀerent morphologies, various sizes and higher surface area. The photocatalytic activity also depends upon the size and shape of fabricated ZnO. Controlling the morphology of ZnO can increase the photodegradation rate of pollutants.31 Herein, we report the synthesis of pure and Er/Nd doped ZnO NPs using sol gel method. The characterization of the synthesized materials was carried out using standard analytical techniques, such as X-ray diﬀraction analysis (XRD), scanning electron microscopy (SEM), electron dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), UV-vis spectroscopy, UV-vis diﬀuse-reection spectroscopy (DRS), Fourier transformed infrared spectroscopy (FTIR), diﬀerential thermal analysis (DTA), thermo gravimetric analysis (TGA), diﬀerential scanning calorimetry (DSC), Brunauer– Emmett–Teller (BET), X-ray photoelectron spectroscopy (XPS) and photoluminescence (PL). 1. In addition, we have also studied the antibacterial and anticancer activity of the prepared materials against human pathogenic bacterial strains and various human cancer cell lines. 2.4 In vitro antibacterial studies The photocatalytic activity of the prepared samples was evalu- ated by studying the degradation of an aqueous solution of two diﬀerent organic dyes (RR-241 and MB) under visible light illumination using (500 W) tungsten halogen linear lamp as a light source. In a typical procedure, required amount of photocatalyst was added to 200 mL of an aqueous solution of dyes such as RR-241 (14 ppm) or MB (10 ppm) in the immersion well photochemical reactor. Prior to illumination, the solution was rst ultrasonicated for 10 min and then stirred in the presence of photocatalyst in the dark for 15 min to ensure the establishment an adsorption–desorption equilibrium. In order to maintain the temperature (20 0.5 C) of dye solution to minimize the eﬀects of heating, the photoreactor was sur- rounded by refrigerated owing water. The zero time reading was given to a blank solution kept in the dark, but otherwise treated similarly to the irradiated solution. At a given time interval 5 mL suspension was withdrawn and centrifuged at 4000 rpm for 30 min to remove the photocatalyst powder completely. The photocatalytic degradation of RR-241 and MB dyes were monitored using a Perkin Elmer (Lamda 35) spectrophotometer. 2.4.1 Bacterial strain. The bacterial strains used for screening were Gram negative Escherichia coli (American Type Culture Collection (ATCC)® 25922™; ATCC, Manassas, VA, USA), Gram positive Listeria monocytogene (Microbial Type Culture Collection Gene Bank (MTCC)® 657™; MTCC, IMTECH, Chandigarh, India) and Staphylococcus aureus (ATCC 25923). The E. coli strain was cultured in Luria Bertani broth while L. monocytogene and S. aureus were cultured in a brain heart infusion broth medium for 12–18 hours at 37 C. 2.4.2 Agar well diﬀusion assay. The antibacterial potential of the pure and doped ZnO NPs were evaluated using agar well diﬀusion method.32 The inoculum was prepared by diluting the overnight cultures with sterile normal saline to a 0.5 McFarland standard. The agar petri plates were prepared by spreading the 1 107 CFU per 50 mL of mature broth culture of specic bacterial strains with a sterile L-shaped glass rod. The well of 8 mm was created in each petri-plate with the help of sterile yellow tips. Various formulations of ZnO suspended in sterile phosphate buﬀer saline was used to evaluate the antibacterial activity. 2.4 In vitro antibacterial studies The experimental procedure was performed under sterile conditions using bio-safety level 2 (BSL-2) hoods and petri plates incubated at 37 C for 12–18 h. The susceptibility of our formulation was determined on the basis of diameter of the zone of inhibition against human pathogenic strain E. coli, S. aureus and L. mon- ocytogene. The inhibition zone was measured in triplicate with various forms of ZnO NPs, the average of the three was calcu- lated for the antibacterial activity and compared with the reference drug gentamicin as a control. 3.1 XRD analysis The crystal structure of fabricated NPs was investigated by powder X-ray diﬀraction analysis. Fig. 1 displays the XRD patterns of pure and Er/Nd doped ZnO calcinated at 600 C for 4 h. The peaks at 31.80, 35.2, 36.9, 47.50, 56.50, 62.80, 66.20, 67.80, 69.10, 78.20 could be indexed to (100), (002), (101), (102), (110), (103), (200), (112), (201) and (202) crystal planes of hexagonal wurtzite ZnO (P63mc, a ¼ 3.1495, c ¼ 5.106), respec- tively.34,35 The XRD patterns of doped ZnO exhibit the same pattern as pure ZnO indicating that the products are in a pure hexagonal phase. The XRD analysis also indicate that there is no change in the crystal structure upon doping.36 Furthermore, all diﬀraction peaks are intense and sharp, indicating that the prepared NPs are in crystalline nature without any amorphous phase.4,37 The average crystallite size of pure and Er/Nd doped ZnO was estimated by Debye Scherer formula: This journal is © The Royal Society of Chemistry 2016 2.3 Characterization XRD analysis of the fabricated samples were carried out with a Shimadzu diﬀractometer (model 6100) with Cu Ka radiations (l ¼ 1.5418 ˚A) operated at a voltage of 30 kV and current of 15 MA in the 2q range of 20–80. A JEOL JSM-6510 scanning elec- tron microscope (SEM) was used to study the surface morphology and elemental analysis of pure and doped ZnO NPs. The optical properties of the prepared NPs were charac- terized using UV-vis absorption spectroscopy and UV-vis diﬀuse-reection spectroscopy with a Perkin Elmer spectro- photometer (l ¼ 35). FTIR spectra of the fabricated NPs were This journal is © The Royal Society of Chemistry 2016 78336 \| RSC Adv., 2016, 6, 78335–78350 RSC Advances View Article Online RSC Advances View Article Online Paper recorded in KBr powder as a reference at room temperature using a Perkin-Elmer Spectrum 2 in the range 400–4000 cm1. PL spectra of the prepared materials were recorded using Hitachi (F-2700). A Xe lamp was used as the excitation source during PL study and PL spectra was performed at an excitation wavelength of 290 nm. Thermal analysis (TGA/DTA) measure- ment of the prepared NPs was done using Shimadzu (model 60H) and DSC using Shimadzu (model 60). X-ray photoelectron spectroscopy (XPS) was carried out by a PHI VersaProbe II with AES photoelectron spectrometer. The Brunauer–Emmett–Teller (BET) analysis was done using Quantachrome Autosorb Auto- mated Gas Sorption System Instruments. dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT is a colorimetric assay, which is based on the ability to cleave the tetrazolium ring of the MTT using the mitochon- drial dehydrogenase enzyme in viable cells to form insoluble formazan crystals, which deposits in living cells. This insoluble formazan is then dissolved by addition of a suitable solvent into a purple colored solution. The absorbance of this colored solution can be measured at 570 nm.33 The amount of formazan crystal product is directly proportional to the number of living cells. 3.2 FTIR analysis To conrm the nature of the reaction intermediate and identi- cation of various functional groups which participate in the formation of ZnO NPs, FTIR analysis was carried out in the wavenumber range 400 to 4000 cm1 using KBr powder at room temperature as shown in Fig. 2. The peaks below 500 cm1 was assigned to the stretching vibration of Zn–O.39 The stretching vibration corresponds to the bond between dopant and oxygen (Nd/Er–O) may appear between 948 and 1113 cm1 (ref. 15, 40 and 41). A sharp absorption band at 1623 cm1 in the higher energy region was ascribed to O–H bending vibration.39 The ZnO surface contains hydroxyl groups and water molecules either chemisorbed or physisorbed as conrmed by the broad absorption band at around 3440 cm1.42 The peaks at 2920 and 2842 cm1 correspond to the –CH– stretching band which belongs to Triton-X.39,43 The sharp peak at 2332 cm1 shows atmospheric CO2 absorbed on the metal cation.15,44 nm in the visible region. The band gap energy of pure and Er/Nd doped ZnO NPs was calculated by eqn (4),49 nm in the visible region. The band gap energy of pure and Er/Nd doped ZnO NPs was calculated by eqn (4),49 nm in the visible region. The band gap energy of pure and Er/Nd doped ZnO NPs was calculated by eqn (4),49 ahn ¼ A(hn Eg)n/2 (4) (4) where a, n, Eg, and A are the absorption coeﬃcient, light frequency, band gap, and a constant, respectively. Moreover, n depends on the characteristics of the transition in a semi- conductor for direct transition n ¼ 1 and for indirect transition n ¼ 4. The value of n for ZnO was 1 due to direct transition.50 The band gap energy of pure and doped ZnO NPs is calculated from the plot between (ahn)2 vs. photon energy (hn). As seen from the inset in Fig. 3 the band gap of pure ZnO was found to be 3.3, while the band gap of Er and Nd doped ZnO was 2.74 and 2.66, respectively. 3.2 FTIR analysis These results are consistent with previous results.51–55 The band gap energy was found to decrease from 3.37 to 2.66 aer doping, which may be due to increased defects introduced by Er and Nd doping or substitutional impurity located in the lattice position of ZnO.56 A higher defect makes the electronic transitions from the lled valence band to the energy level of the defect more probable than the transitions to the conduction band.57 3.3 Optical properties and band gap The optical characterization of the synthesized material was done using UV-vis diﬀuse reectance spectroscopy (DRS). DRS is a good technique for examining the light harvesting ability of synthe- sized hexagonal semiconductor photocatalysts.45,46 Fig. 3 presents the DRS spectra of fabricated pure and Er/Nd doped ZnO NPs. A strong absorption at 372 nm in the UV region corresponding to pure ZnO may be due to band to band transitions.47,48 The Er/Nd doped ZnO NPs showed the broad absorption near 452 and 465 2.5 In vitro cell viability analysis by MTT assay 2.5.1 Cell culture. Human breast cancer cell line (MDA-MB- 231), human liver cancer cell line (Hep3B) and human cervical cancer cell line (HeLa) were used in the present study and cell lines were procured from the Cell Repository – National Centre for Cell Science, Pune (India). Cell lines were cultured in the Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 10% heat inactivated fetal bovine serum (FBS) and 0.5% antibiotic antimycotic solution (Sigma-Aldrich Co, St Louis, MO, USA) at 37 C in a humidied incubator maintaining 5% CO2. D ¼ 0:9l b cos q (3) (3) 2.5.2 MTT assay. The anticancer potential of the pure and Nd/Er doped ZnO NPs were determined using 3-(4,5- This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 78335–78350 \| 78337 View Article Online RSC Advances Table 1 Crystallite size of pure and doped ZnO NPs with diﬀerent dopant concentrations Table 1 Crystallite size of pure and doped ZnO NPs with diﬀerent dopant concentrations where D is the crystallite size, l is the X-ray wavelength (0.154 nm), q is the Bragg diﬀraction angle and b is the full width at half maximum (FWHM). The crystallite size of pure and doped ZnO NPs were calculated using the most intense peak (101). The crystallite size of doped ZnO was found to be smaller than that of the pure ZnO as given in Table 1. The decrease in crystallite size of Er/Nd doped ZnO is mainly due to the formation of Er–O–Zn or Nd–O–Zn linkages on the surface of the doped samples, which may hinder the growth of ZnO particles to some degree.15,38 3.4 Material morphology analysis (SEM-EDS and TEM) This indicate the formation of a Zn–O bond which reveals the oxidation of Zn2+ in bound ZnO.58–61 The energy diﬀerence between two peaks is 22 eV, which agreed well with the standard value, i.e., 22.97 eV.60 The absorptions at 530.6 and 532.4 eV are due to lattice oxygen and chemisorbed oxygen (OH) caused by the surface hydroxyl in wurtzite ZnO as shown in Fig. 1S(B) ESI†.61,62 Fig. 2S(A) ESI†displays peaks at 978.2 and 984.3 eV corresponding to Nd 3d5/2 and Nd3/2, respectively, which reveals that Nd exists as +3 oxidation state in Nd doped ZnO.62 The peaks position of Nd 3d are closely related to the value of Nd2O3 in literature data, which suggests that Nd substitutes the Zn.63,64 Fig. 2S(B) ESI†shows the two binding energy peaks at 184.3 and 188.1 eV corresponding to the 4d5/2 and 4d3/2, respectively, indicate the presence of Er3+ ions in Er doped ZnO.65–67 Based on both the XRD and XPS results, we conclude that the Nd and Er dopants are successfully incorporated into the ZnO lattice. C. We could see that pure and Er/Nd doped ZnO form hexagonal rod-like structures with the aggregation of prepared ZnO NPs possessing non-uniform morphology lying on top of one another. Aer doping with diﬀerent concentrations of Er and Nd the surface becomes rough, implying the deposition of Er and Nd on the surface of ZnO. Dopants also decrease the aggregation of NPs. The grain size of doped NPs was gradually decreased as the dopant concentration increased. As the crys- tallite size decreases upon doping, the aggregation of NPs also decreases which lead to an increase in surface area. The percentage of doping in ZnO NPs was conrmed by EDS analysis, the results are illustrated in Fig. 5A and B which indi- cate the existence of Nd and Er metal ions in the ZnO lattice. The atomic ratio of Nd and Er in ZnO was measured from EDX and found to be 1.75% and 4.55%, respectively. The gures also show the presence of Zn, O, Er and Nd demonstrating that erbium and neodymium ions are successfully doped into the ZnO NPs. The surface morphology and particle size of pure and doped ZnO NPs were further analyzed by TEM as shown in Fig. 6A and B. TEM images of prepared ZnO NPs clearly indicate the hexagonal rod like structure. 3.4 Material morphology analysis (SEM-EDS and TEM) The morphology of pure and Er/Nd doped ZnO NPs calcinated at 600 C for 4 h were investigated by SEM as shown in Fig. 4A– Fig. 1 XRD pattern of pure and Er/Nd doped ZnO NPs. Fig. 2 FTIR Spectra of pure and Er/Nd doped ZnO NPs. Fig. 2 FTIR Spectra of pure and Er/Nd doped ZnO NPs. Fig. 2 FTIR Spectra of pure and Er/Nd doped ZnO NPs. Fig. 2 FTIR Spectra of pure and Er/Nd doped ZnO NPs. Fig. 2 FTIR Spectra of pure and Er/Nd doped ZnO NPs. Fig. 1 XRD pattern of pure and Er/Nd doped ZnO NPs. Fig. 1 XRD pattern of pure and Er/Nd doped ZnO NPs. This journal is © The Royal Society of Chemistry 2016 78338 \| RSC Adv., 2016, 6, 78335–78350 RSC Advances View Article Online RSC Advances View Article Online Paper Paper Fig. 3 UV-vis diﬀuse reﬂectance (DRS) spectra and the inset of the ﬁgure shows the plots of (ahn)2 vs. photon energy (hn) for pure and Er/Nd doped ZnO NPs. d on 17 August 2016. Downloaded on 02/12/2016 07:42:52. ensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 UV-vis diﬀuse reﬂectance (DRS) spectra and the inset of the ﬁgure shows the plots of (ahn)2 vs. photon energy (hn) for pure and Er/Nd doped ZnO NPs. Fig. 3 UV-vis diﬀuse reﬂectance (DRS) spectra and the inset of the ﬁgure shows the plots of (ahn)2 vs. photon energy (hn) for pure and Er/Nd doped ZnO NPs. Fig. 3 UV-vis diﬀuse reﬂectance (DRS) spectra and the inset of the ﬁgure shows the plots of (ahn)2 vs. photon energy (hn) for pure and Er/Nd doped ZnO NPs. synthesized ZnO NPs. The wide survey scan spectra of Nd and Er doped ZnO NPs are presented in Fig. 7A and B, respectively, which indicate the presence of only Zn, O, C, Nd and Er elements. The obtained binding energies were corrected by using C 1s 282.8 eV as the reference. It could be seen from Fig. 1S(A) ESI†, that the peaks appearing at 1020 and 1042 eV, are in good agreement with the binding energy value of Zn 2p and ascribed to the core level of Zn 2p3/2 and 2p1/2, respectively. 3.4 Material morphology analysis (SEM-EDS and TEM) The average particle size of doped NPs was found to be in the range between 9 to 30 nm. Small particles on the surface may be due to the presence of dopants. This journal is © The Royal Society of Chemistry 2016 This journal is © The Royal Society of Chemistry 2016 3.6 BET analysis Table 2 BET surface area, pore volume and pore diameter of pure and Er/Nd doped ZnO NPs Parameter Pure ZnO 5% Er-doped ZnO 2% Nd-doped ZnO BET surface area (m2 g1) 38.23 64.65 73.22 Pore volume (cm3 g1) 0.312 0.332 0.381 Pore diameter D (nm) 12.67 15.78 19.34 Fig. 8 The PL spectra of pure and Er/Nd doped ZnO NPs. Table 2 BET surface area, pore volume and pore diameter of pure and Er/Nd doped ZnO NPs Fig. 5 (A and B) EDS image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 8 The PL spectra of pure and Er/Nd doped ZnO NPs. Fig. 5 (A and B) EDS image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. Fig. 6 (A and B) TEM image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. BET surface area of 2% Nd was found to be higher than that of pure ZnO and 5.0% Er doped ZnO. Fig. 6 (A and B) TEM image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. Fig. 8 The PL spectra of pure and Er/Nd doped ZnO NPs. Fig. 6 (A and B) TEM image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. photocatalytic activity decreases when an electron and hole recombine to emit a photon.68–70 Therefore, the photocatalytic activity of lower PL intensity would be higher. The PL intensity of ZnO decreases upon doping with Er and Nd as shown in Fig. 8. Hence, the recombination rate decreases by the eﬃcient charge separation of photoinduced electron–hole pairs. The PL spectra of Nd doped ZnO NPs shows the lowest intensity peak among all. Therefore, photocatalytic performance of Nd-doped ZnO would be higher than pure and Er doped ZnO. It could be seen from the gure that pure and doped ZnO NPs reveal a UV emission band at approximately 329, 345, and a yellow emission band around 585 nm. Generally the UV emission band is due to the near band-edge emission (NBE), and the yellow emission in the visible region may be attributed by deep level emission (DLE). The UV emission was ascribed to the free BET surface area of 2% Nd was found to be higher than that of pure ZnO and 5.0% Er doped ZnO. 3.6 BET analysis To further obtain more accurate information regarding doped ZnO, we employed X-ray photoenergy spectroscopy (XPS) anal- ysis to examine the chemical composition and oxidation state of The BET surface area, pore diameter and pore volume of synthesized pure and Er/Nd doped ZnO NPs were examined This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 78335–78350 \| 78339 View Article Online Paper View Article Online View Article Online RSC Advances n adsorption–desorption and Barrett–Joyner– ) analysis in liquid nitrogen. The nitrogen sorption isotherms (Fig. 3S, ESI†) showed a Type III hysteresis loop shape according to the IU The BET surface, pore diameter and pore v 2.0% Nd and 5.0% Er doped ZnO NPs are gi M image of (A) pure ZnO, (B) Er-doped ZnO, and (C) Nd-doped ZnO NPs. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 (A–C) SEM image of (A) pure ZnO, (B) Er-doped ZnO, and (C) Nd-doped ZnO NPs. Fig. 4 (A–C) SEM image of (A) pure ZnO, (B) Er-doped ZnO, and (C) Nd-doped ZnO NPs. III hysteresis loop shape according to the IUPAC classication. The BET surface, pore diameter and pore volume of pure and 2.0% Nd and 5.0% Er doped ZnO NPs are given in Table 2. The using nitrogen adsorption–desorption and Barrett–Joyner– Halenda (BJH) analysis in liquid nitrogen. The nitrogen adsorption–desorption isotherms (Fig. 3S, ESI†) showed a Type This journal is © The Royal Society of Chemistry 2016 This journal is © The Royal Society of Chemistry 2016 78340 \| RSC Adv., 2016, 6, 78335–78350 Paper Paper BET surface area of 2% Nd was found to be higher than that of pure ZnO and 5.0% Er doped ZnO. photocatalytic activity decreases when an electron and hole recombine to emit a photon.68–70 Therefore, the photocatalytic activity of lower PL intensity would be higher. The PL intensity of ZnO decreases upon doping with Er and Nd as shown in Fig. 8. Hence, the recombination rate decreases by the eﬃcient charge separation of photoinduced electron hole pairs The PL Fig. 5 (A and B) EDS image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. Fig. 6 (A and B) TEM image of (A) Er-doped ZnO, and (B) Nd-doped ZnO NPs. This journal is © The Royal Society of Chemistry 2016 3.9 Cytotoxicity and antibacterial activity Fig. 9 DTA plot of pure and Er/Nd-doped ZnO NPs. In vitro results of the agar well diﬀusion susceptibility test reveal that Nd-doped ZnO NPs show better antibacterial activity as compared to pure and Er-doped NPs. Moreover, it was observed that Nd-doped ZnO NPs show nearly equivalent bactericidal potential as compared to the standard drug gentamicin, while pure and Er-doped NPs have shown admirable bactericidal potential against Gram negative as well as Gram positive path- ogenic strains. The presence of zone of inhibition by the various ZnO NPs against bacterial strains clearly indicates the bacteri- cidal potential of the NPs. A bar graph and zone of inhibition are presented in Fig. 10. recombination of photogenerated electrons in the conduction band and hole in the valence band.71 The DLE emission in the visible regions would arise due to the intrinsic or extrinsic defects in ZnO NPs. 3.7 Photoluminescence spectroscopy PL is a well-known powerful technique for investigating the eﬀects of impurity, eﬃciency of charge carrier trapping and predicting the photocatalytic eﬃciency of semiconductor pho- tocatalyst. The PL spectrum of ZnO NPs originates from irra- diative recombination of excited or photoinduced charge carriers. The electron and hole play a great role in deciding the photocatalytic activity of a semiconductor photocatalyst. The Fig. 7 XPS spectra of (A) Nd, and (B) Er-doped ZnO NPs. This journal is © The Royal Society of Chemistry 2016 RSC Adv 2016 6 78335–78350 \| 78341 Fig. 7 XPS spectra of (A) Nd, and (B) Er-doped ZnO NPs. This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 78335–78350 \| 78341 View Article Online View Article Online RSC Advances Paper Fig. 9 DTA plot of pure and Er/Nd-doped ZnO NPs. The rst step from 70 to 300 C was ascribed to the removal of free water, and weight loss in the second step from 300 to 500 C may be due to decomposition of bound water and a hydroxyl group. Finally, the weight loss above 500 C was due to the crystallization of ZnO.73 The rst step from 70 to 300 C was ascribed to the removal of free water, and weight loss in the second step from 300 to 500 C may be due to decomposition of bound water and a hydroxyl group. Finally, the weight loss above 500 C was due to the crystallization of ZnO.73 The DSC analysis of pure and doped ZnO NPs heated at 20 C per minute from 30 to 550 C is given in Fig. 7S–9S, ESI†. From the DSC curve, 4 endothermic peaks were found at 50, 428, 367 and 393 C. The peak at 50 C for all NPs indicates the evapo- ration of adsorbed water molecules. The peaks at 428, 367 and 393 C are ascribed to crystal transformation from cubic to the wurtzite ZnO.74 3.8 Thermal analysis (TGA, DTA and DSC) To investigate the thermal stability of pure and Nd/Er doped ZnO, TGA, DTA and DSC were carried out under a nitrogen atmosphere. The total weight loss, i.e., 1.653% was observed for pure ZnO NPs in the temperature range from 100 to 800 C (Fig. 4S, ESI†) and shows good thermal stability up to 100 C. In contrast to this Er-doped ZnO NPs display the loss of 4.453% weight (Fig. 5S, ESI†) in the range of 100 to 800 C. The Nd- doped ZnO NPs exhibit a total weight loss of approximately 1.476% in the range of 100 to 800 C (Fig. 6S, ESI†). The initial weight loss for all NPs between 100 and 500 C was caused by the evaporation of physically or chemically adsorbed water and hydroxide molecules.72 The weight loss above 500 C might be due to conversion of ZnO into crystalized wurtzite ZnO.15 Cytotoxicity eﬀects of pure and Nd/Er-doped ZnO NPs were examined in HeLa, hep3B and MDA-MB-231 cells using 3-(4,5- dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were placed in a at bottom 96-well plate at a density of 5 103 cells per well and allowed to grow for 24 h at 37 C. Aer incubation the culture medium was then replaced with the 100 mL fresh medium containing increasing concen- trations of pure and Nd/Er doped ZnO NPs. The cells were incubated for 48 h and cell viability was measured by adding 20 mL of MTT dye (5 mg mL1 in phosphate-buﬀered saline) per well. The plates were incubated for a further 4 h and thus for- mazan crystals formed due to the reduction of dye by viable cells in each well which were dissolved in 200 mL dimethyl sulfoxide The DTA curve of pure and Nd/Er doped ZnO NPs are shown in Fig. 9. The DTA plot reveals three main steps of weight loss. Fig. 10 Bar graph showing the diameter of the zone of inhibition (in mm) produced by pure and Nd/Er-doped ZnO NPs against E. coli, S. aureus and L. monocytogene. Fig. 10 Bar graph showing the diameter of the zone of inhibition (in mm) produced by pure and Nd/Er-doped ZnO NPs against E. coli, S. aureus and L. monocytogene. This journal is © The Royal Society of Chemistry 2016 78342 \| RSC Adv., 2016, 6, 78335–78350 Paper Fig. 3.8 Thermal analysis (TGA, DTA and DSC) 11 Dose dependent anticancer potential of pure and Nd/Er-doped ZnO Nps against HeLa, Hep3B and MDA-MB-231 human cancer cell lines. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 11 Dose dependent anticancer potential of pure and Nd/Er-doped ZnO Nps against HeLa, Hep3B and MDA-MB-231 human cancer cell lines cer potential of pure and Nd/Er-doped ZnO Nps against HeLa, Hep3B and MDA-MB-231 human cancer cell Fig. 11 Dose dependent anticancer potential of pure and Nd/Er-doped ZnO Nps against HeLa, Hep3B and MDA-MB-231 human cancer cell lines. Fig. 11 Dose dependent anticancer potential of pure and Nd/Er-doped ZnO Nps against HeLa, Hep3B and M ines oxygen species (ROS) such as hydroxyl radicals, hydrogen peroxide and super oxides which react with cell content such as protein lipid DNA to cause cell death.75–77 (DMSO). The absorbance was recorded in an ELISA plate reader (iMARK Microplate Absorbance Reader, Bio Rad, California, USA) at 570 nm. The absorption values were expressed as percent cell viability compared with untreated control group considered as 100% viable. The results indicate that there is a decrease in cell metabolic activity of all targeted cells with increasing concentrations of ZnO NPs. It could be seen from Fig. 11 that MDA-MB-231 cells show higher damage compared to the other two cells. This showed that the MDA-MB-231 cell lines were more sensitive to the cytotoxic eﬀects of ZnO NPs. At 512 mM concentration the toxicity of Nd-doped ZnO NPs was found to be higher than pure and Er-doped ZnO NPs. This journal is © The Royal Society of Chemistry 2016 3.10 Photocatalytic activity The photocatalytic degradation of two diﬀerent chromophoric dyes such as MB and RR-241, two typical organic pollutants discharged by textile industries, were chosen to evaluate the photocatalytic eﬃciency of pure and doped ZnO NPs under visible light illumination. The characteristic absorption values of MB and RR-241 observed at 663 and 542 nm were used to monitor the photocatalytic performance of pure and doped ZnO NPs. The control experiments indicate that MB and RR-241 were resistant to degradation during irradiation without photo- catalyst. However, little degradation of both dyes was observed in the presence of photocatalyst in the dark due to adsorption of dyes on the catalyst surface. Therefore, both catalyst and light are needed for eﬀective decomposition. All prepared samples Several studies have been reported in the literature which suggest that an increase in in vitro cytotoxicity and antibacterial activity with increasing pure and doped hexagonal ZnO rods may be due to: (i) NPs gathering in the cytoplasm or accumu- lating on the bacterial surface which disturbed the cellular function and membrane function; (ii) generation of reactive Fig. 12 (A) Change in the absorbance of an aqueous solution of MB (lmax ¼ 663) in the presence of 2.0% Nd-doped ZnO NPs. (B) Change in the absorbance of an aqueous solution of RR-241 (lmax ¼ 542) in the presence of 5.0% Er-doped ZnO NPs at diﬀerent time intervals during irradiation under visible light. Fig. 12 (A) Change in the absorbance of an aqueous solution of MB (lmax ¼ 663) in the presence of 2.0% Nd-doped ZnO NPs. (B) Change in the absorbance of an aqueous solution of RR-241 (lmax ¼ 542) in the presence of 5.0% Er-doped ZnO NPs at diﬀerent time intervals during irradiation under visible light. RSC Adv., 2016, 6, 78335–78350 \| 78343 This journal is © The Royal Society of Chemistry 2016 View Article Online RSC Advances Fig. 13 (A and B) Change in concentration of MB and RR-241 in the presence of pure and diﬀerent % of Nd and Er doped ZnO NPs, respectively, under visible light irradiation. (C and D) Kinetic study of degradation of Mb and RR-241 in the presence of pure and diﬀerent % of Nd and Er doped ZnO NPs, respectively, under visible light irradiation. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. cle is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 17 August 2016. 3.10 Photocatalytic activity 16 Photostability of 2% Nd-doped ZnO NPs within four consec- utive cycles for degradation of MB under visible light illumination. Paper Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper Fig. 15 Decolorization eﬃciency of MB in the presence of diﬀerent radical scavengers over 2% Nd-doped ZnO under visible light illumination. Paper ons Attribution 3.0 Unported Licence. signicantly increases with increasing dopant percentage up to 2.0% (Nd) and 5.0% (Er). Further, an increase in dopant percentage lead to a decrease in degradation of both dyes. However, 30.7% and 19.4% degradation of MB RR-241 dyes takes place over pure ZnO aer 10 min due to a dye-sensitized mechanism. To understand the photodegradation of dyes more accu- rately and quantitatively, we calculated the kinetics in the presence of pure and doped ZnO NPs. The plot of ln(C/C0) vs. irradiation time (t) (as shown in Fig. 10SA and B†), for MB (2.0% Nd) and RR-241 (5.0% Er doped ZnO NPs) indicate a good liner t. All R2 (correlation coeﬃcients) values were found to be higher than 0.9, which demonstrate that the photodegradation of MB and RR-241 by the NPs follow pseudo-rst-order kinetics, and the slope of the linear curve could be considered as the rate constant kapparent. The ln C0/C vs. irradiation time using all prepared ZnO NPs for degrada- tion of MB and RR-241 under visible light illumination is given in Fig. 13C and D. The results indicate that the kinetic rate of MB and RR-241 catalyzed by Er and Nd doped ZnO enhanced with increasing Er and Nd concentration, and the Nd (2%) and Er (5%) doped ZnO display the highest photo- degradation rate. Further, an increase in dopant percentage lead to a decrease in degradation of both dyes. The calculated kapparent values of the synthesized NPs are listed in Tables 1S and 2S†, respectively. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 15 Decolorization eﬃciency of MB in the presence of diﬀerent radical scavengers over 2% Nd-doped ZnO under visible light illumination. Fig. 15 Decolorization eﬃciency of MB in the presence of diﬀerent radical scavengers over 2% Nd-doped ZnO under visible light illumination. 3.10.1 Eﬀect of diﬀerent operational parameters on the degradation of dyes. 3.10 Photocatalytic activity Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 13 (A and B) Change in concentration of MB and RR-241 in the presence of pure and diﬀerent % of Nd and Er doped ZnO NPs, respectively, under visible light irradiation. (C and D) Kinetic study of degradation of Mb and RR-241 in the presence of pure and diﬀerent % of Nd and Er doped ZnO NPs, respectively, under visible light irradiation. presence of pure or desired amount of Er/Nd doped ZnO NPs under constant stirring and bubbling of atmospheric oxygen. Fig. 12A and B display that 100% and 78.6% decomposition of MB and RR-241 dyes take place aer 10 min in the presence of 2.0% Nd and 5.0% Er doped ZnO NPs, respectively. were able to degrade both dyes under investigation, as observed from the decrease in the characteristic absorbance and decol- orization indicating the loss of chromophoric groups of dyes and conversion into small aromatic intermediates. Nd and Er doped ZnO NPs exhibit higher activity than pure ZnO under visible light illumination. The results indicate that photo- catalytic activity of doped ZnO was improved by introducing Er and Nd metal ions into the lattice of ZnO. To compare the photocatalytic performance of pure and diﬀerent mole% doped ZnO more precisely, we plotted (C)/(C0) vs. irradiation time for degradation of MB and RR-241, the results are depicted in Fig. 13A and B. The results indicate that the doped NPs provided enhanced photodecomposition of dyes as compared to bare ZnO, and the photocatalytic activity In a typical experiment an aqueous solution of MB (10 ppm, 200 mL) or RR-241 (14 ppm, 200 mL) was illuminated in an immersion well photoreactor with a visible lamp in the Fig. 14 (A and B) Comparative study for the degradation of MB and RR-241 in the presence of Nd and Er (with varying dopant%) under visible light irradiation. Fig. 14 (A and B) Comparative study for the degradation of MB and RR-241 in the presence of Nd and Er (with varying dopant%) under visible light irradiation. This journal is © The Royal Society of Chemistry 2016 78344 \| RSC Adv., 2016, 6, 78335–78350 RSC Advances View Article Online RSC Advances View Article Online Fig. 15 Decolorization eﬃciency of MB in the presence of diﬀerent radical scavengers over 2% Nd-doped ZnO under visible light illumination. Fig. This journal is © The Royal Society of Chemistry 2016 Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The degradation eﬃ- ciency was found to increase as the dopant percentage increases up to 2% Nd and 5% Er doped ZnO NPs. It then decreases, suggesting that 2% Nd and 5% Er doped ZnO NPs could be more eﬃcient for achieving high degradation eﬃciency due to an increased charge separation ability. Therefore, a precise amount of dopant could be critical for getting high photo- catalytic activity.78 The presence of Nd3+ and Er3+ ions on the surface of ZnO may promote the following reactions: Nd/Er3+ + e / Nd/Er2+ (6) Nd/Er2+ + O2 / Nd/Er3+ + O2c (7) Nd/Er3+ + e / Nd/Er2+ (6) Nd/Er2+ + O2 / Nd/Er3+ + O2c (7) (6) (7) Further increase in dopant percentage lead to a decrease in the eﬃciency of photocatalyst. This may be due the fact that at higher dopant concentrations, 7.0% (Er) and 2.5% (Nd), multiple trapping of charge carriers occurs, hence the recom- bination of photogenerated electron–hole pairs becomes easier and fewer charge carriers reach the surface to initiate the degradation of the dye. In addition, at high dopant percentages the blocking of light rays or shadowing eﬀect was observed. The turbidity of powder takes place due to aggregation of the cata- lyst particles, which decreases the penetration depth of light. The aggregation of ZnO powder covers part of the photosensi- tive surface thereby decreasing the number of surface active sites, which leads to a decrease in the photocatalytic activity of the photocatalyst due to an increase in the number of electron– hole recombination centers.79 Similar results have also been reported in the literature.15,80–84 ZnO–OH + H+ / ZnO–OH2 + (+ve at pH < pHzpc) (8) ZnO–OH + OH / ZnO–O + H2O (ve at pH > pHzpc) (9) (8) The results indicate that the degradation of both dyes increases with increasing pH from 4 to 7.8 in the case of MB, and from 4 to 8.6 in the case of RR-241. A further increase in pH leads to decreased degradation of both dyes. The highest eﬃ- ciency is observed at pH 7.8 (in the case of MB), and 8.6 (in the case of RR-241), which may be due to the fact that the dye molecules have a good interaction with the surface of the catalyst at these pH values. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The pH of the reaction mixture plays an important role in the degradation of dyes because it determines the surface charge properties of the catalyst, adsorption/desorption of dyes on the surface of the catalyst, and charges on the dye molecules. Therefore, it is important to study the degradation of dyes at varying pH values of the reaction mixture. In order to see the role of pH on the decolorization of MB and RR-241, we per- formed experiments at diﬀerent pH values varying from 4 to 10 using 5.0% Er and 2.0% Nd-doped ZnO NPs. The pH of the reaction mixture was maintained before irradiation by adding an aqueous solution of either HNO3 or NaOH. The eﬀect of pH on degradation of MB and RR-241 was studied in analogous experimental conditions. Fig. 12S(A)† shows the change in % degradation vs. pH (4.0 to 10) for the degradation of MB in the presence of 2.0% Nd doped ZnO, whereas, Fig. 12S(B)† shows the change in % degradation vs. pH value (4.0 to 10) for the degradation of RR-241 in the presence of 5.0% Er doped ZnO. It could be seen from Fig. 12S(A)† that the decomposition of MB dye increases upon increases in pH from 4 to 7.8 and a further increase in pH leads to a decrease in MB degradation. The degradation of RR-241 increases with increasing pH from 4 to 8.6, further increases lead to decreased degradation of RR-241 dye as shown in Fig. 12S(B).† The rate of decomposition of dyes depends on the adsorption of dyes on the surface of ZnO NPs. However, adsorption of dyes depends upon the ionic structure of dyes and point charge value of ZnO. Since the zero point charge for doped ZnO (pHzpc) is about 8.3.23 The surface of the catalyst remains positively charged below pHzpc whereas the catalyst surface is negatively charged above pHzpc, according to eqn (8) and (9), respectively. The photocatalytic activity of the prepared materials was found to increase as the molar ratio of dopants increased from 0 to 2.0% in the case of Nd, and 0–5% in the case of Er doped ZnO NPs. This may be because under visible light illumination dopants act as scavengers to trap the electron and prevent the electron hole recombination rate. The 4f electron transitions of dopants leads to the optical adsorption of ZnO and also supports the charge carrier separation. This journal is © The Royal Society of Chemistry 2016 78346 \| RSC Adv., 2016, 6, 78335–78350 3.10 Photocatalytic activity The eﬃciency of pure and doped catalysts were investigated by degradation of two diﬀerent chromophoric dyes such as MB (cationic dye) and RR-241 (anionic dye) at other optimum conditions under visible light illumination with continuous purging of air. The percentage degradation (h) of dyes was calculated using the eqn (5), Fig. 16 Photostability of 2% Nd-doped ZnO NPs within four consec- utive cycles for degradation of MB under visible light illumination. h ¼ C0 C C0 100% (5) (5) Scheme 1 A plausible mechanism for the degradation of dyes using hexagonal nanorod ZnO under visible light irradiation. Scheme 1 A plausible mechanism for the degradation of dyes using hexagonal nanorod ZnO under visible light irradiation. This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 78335–78350 \| 78345 View Article Online RSC Advances Paper where C0 is the initial concentration and C is the concentration at a particular time (t). g L1 (Er) doped ZnO NPs, and then the % degradation of dyes decreases. The initial increase in the % degradation may be due to an increase in total active surface area, the availability of active sites on the catalytic surface and the penetration of visible light. However, at a higher catalyst dose the decrease in % degradation may be due to less visible light penetration and a decrease in surface area because of agglomeration. Therefore, fewer photons may reach the active surface sites.85,86 Hence, all the experiments were carried out at optimal catalyst loading. Fig. 14A and B shows the % degradation of MB and RR-241 over pure and diﬀerent % of Nd and Er doped ZnO NPs, respectively, under 10 min illuminations. It was estimated from the results that the degradation of MB and RR-241 over Nd doped ZnO NPs follows the degradation order as Nd(2%)ZnO > Nd(2.5%)ZnO > Nd(1.5%)ZnO > Nd(1.0)ZnO > Nd(0.5)ZnO > ZnO. The degradation of MB and RR-241 over Er doped ZnO NPs follows the degradation order as Er(5%)ZnO > Er(7%)ZnO > Er(3%)ZnO > Er(1.0)ZnO > ZnO. The degradation eﬃciency of MB and RR-241 reaches 100% and 89.5% over (2%) Nd, and 87.2% and 78.6% over (5%) Er doped ZnO NPs aer 10 min, respectively. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The low degradation rate under highly acidic and highly alkaline pH may due to the fact that ZnO NPs may undergo photocorrosion through self-oxidation at pH lower than 4. Moreover, in a strongly alkaline environment, ZnO may also undergo dissolution as given in eqn (10)–(12), respectively.87 In order to optimize the photocatalyst dose, we performed the experiment at diﬀerent loading levels of dopant (0.5 to 2.0 g L1) to ensure the use of the minimum required amount of catalyst for highest degradation of dyes. As shown in Fig. 11SA and B,† the % degradation of RR-241 and MB increases with an increase in catalyst dose from 0.5 to 1.5 g L1 (Nd), and 0.5 to 1.0 This journal is © The Royal Society of Chemistry 2016 78346 \| RSC Adv., 2016, 6, 78335–78350 RSC Advances View Article Online RSC Advances View Article Online Paper Paper ZnO + 2h+ / Zn2+ + 1/2O2 ZnO + 2H+ / Zn2+ + H2O ZnO + H2O + 2OH / Zn(OH)4 2 a possible mechanism for degradation of dyes over doped ZnO NPs is shown in Scheme 1. The mechanism involving degra- dation of dyes using irradiation of ZnO could be visualized as follows. On absorption of photons of energy equal to or greater than the band gap of the prepared ZnO NPs, an electron may be promoted from the valence band to the conduction band leaving behind an electron vacancy or “hole”. Generally, the photocatalytic degradation of dyes in the presence of bare ZnO was found to be lower than doped materials due to the recom- bination of electron and hole pairs. The enhanced photo- catalytic activity of doped ZnO NPs is not only due to the fact that dopants can improve the absorption eﬃciency of the NPs to solar light, but they also suppress the recombination rate of e/h+ pairs by trapping the electron, which improved the separation of the charge carrier. Furthermore, the higher separation of charge carried by doped ZnO NPs was conrmed by PL spectra. The trapped electron is picked up by oxygen to generate superoxide radical anions. At the same time the hole generated at the valence band (hvb +) may react with surface bound H2O to produce the highly reactive hydroxyl radical. The superoxide radical anions (cO2 ) and hydroxyl radicals (cOH) produced under visible light irradiation are responsible for the degrada- tion of dyes.89,90 3.10.2 Trapping experiment. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To nd out the role of dopants on the degradation of dyes, we performed experiments to examine the main reactive species – hydroxyl radical, super- oxide radical anion and hole – using diﬀerent scavengers. Therefore, diﬀerent scavengers were added to the aqueous solution of dyes to investigate the inhibitory eﬀects of scaven- gers. It is clearly seen from Fig. 15 that the maximum decom- position of MB dye takes place without any scavenger. However, potassium iodide (KI) caused the highest negative eﬀect on degradation eﬃciency of dye, indicating that hydroxyl radicals and holes are the major reactive species for the decolorization of MB in the presence of Nd-doped ZnO NPs. In addition, the role of hydroxyl radicals was investigated by the addition of isopropyl alcohol (IPA) and the results indicate that the degra- dation of MB was found to be inhibited signicantly. Further- more, a very small change in degradation of MB is observed aer the addition of benzoquinone (quencher for superoxide radical anion). The mechanism for trapping reactive species is given below. In addition, doped ZnO NPs have a small crystallite size and therefore possess a large surface area as inferred from BET, TEM and XRD. Photocatalytic activity also depends upon surface area; a larger surface area provides higher activity due to increased adsorption area. Hence, the hexagonal rod like doped ZnO NPs provide eﬃcient photocatalytic activity.91 I + cOH / Ic + OH (13) I / Ic + e (14) e + hvb + / heat (15) Ic + I / I2c (16) I2c / I2 + e (17) IPOH + cOH / IPOc + H2O (18) BQ + O2c / BQc + O2 (19) I + cOH / Ic + OH (13) I / Ic + e (14) e + hvb + / heat (15) Ic + I / I2c (16) I2c / I2 + e (17) IPOH + cOH / IPOc + H2O (18) BQ + O2c / BQc + O2 (19) (13) (14) On the other hand a dye-sensitized mechanism may also be possible as indicated in experiments carried out for degradation of both dyes over pure ZnO NPs under a visible light source. Dyes such as MB and RR-241 are also visible light-absorbing and get excited in visible light. Open Access Article. Published on 17 August 2016. Downloaded on 02/12/2016 07:42:52. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The excited dye can transfer the electron to the conduction band of ZnO NPs.92–94 The elec- tron can be trapped by the dopant at the surface or by the impurity band preventing the recombination rate of the charge carrier. The trapped electrons may subsequently be transferred to adsorbed oxygen to generate the superoxide radical anion (O2c) which may act as a strong reducing agent and may form hydrogen peroxide as indicated in the following equations. The hydroxyl radical (cOH) formed in the reaction mixture may act as a strong oxidizing agent. These ROS are responsible for the degradation of dyes and killing of bacterial and cancer cells. 3.10.3 Photostability and reusability of doped ZnO NPs. In addition to photocatalytic performance, the reusability of the fabricated photocatalyst is also very important. To investigate the stability of a hexagonal nanorod, we conduct four successive photocatalytic experiments for degradation of MB under visible light irradiation in the presence of recycled 2% Nd-doped ZnO NPs, by keeping all other parameters unchanged. Fig. 16 shows that 2% Nd-doped ZnO NPs exhibited eﬀective stability under visible light illumination, however, a slight reduction in eﬃ- ciency from 100% to 95.6% was observed aer the third cycle. Therefore, synthesized doped ZnO NPs could be an eﬃcient photocatalyst for photoxidation of pollutants with higher reus- ability potential. Hence, doping semiconductors with suitable metal ions enhances the stability of photocatalysts.78,83 Dye + visible light / dye* (20) Doped ZnO + visible light / ecb + hvb + (21) Dye* + doped ZnO / dyec+ + ecb (22) Nd/Er3+ + e / Nd/Er2+ (23) Nd/Er2+ + O2 / Nd/Er3+ + O2c (24) ecb + O2 / O2c (25) hvb + + H2O / cOH + H+ (26) Dye + visible light / dye* (20) Doped ZnO + visible light / ecb + hvb + (21) Dye* + doped ZnO / dyec+ + ecb (22) Nd/Er3+ + e / Nd/Er2+ (23) Nd/Er2+ + O2 / Nd/Er3+ + O2c (24) ecb + O2 / O2c (25) hvb + + H2O / cOH + H+ (26) (20) 3.10.4 Mechanism of degradation of dyes. On the basis of the above experimental results and previous studies,88 3.10.4 Mechanism of degradation of dyes. 4. Conclusion 12 P. V. Korake, R. S. Dhabbe, A. N. Kadam, Y. B. Gaikwad and 12 P. V. Korake, R. S. Dhabbe, A. N. Kadam, Y. B. Gaikwad and K. M. Garadkar, J. Photochem. Photobiol., B, 2014, 130, 11–19. K. M. Garadkar, J. Photochem. Photobiol., B, 2014, 130, 11–19. In summary, we have successfully synthesized the hexagonal pure and Er/Nd doped ZnO nanorods by a simple and facile sol gel method using zinc acetate dihydrate as a precursor. All doped ZnO NPs exhibit smaller crystallite size, red shiin absorbance spectra, which allow utilization of visible light, greater separation of e/h+ pairs and more adsorption of dyes on the surface of doped ZnO as compared to bare ZnO NPs. The fabricated doped ZnO NPs show better photocatalytic activity for the degradation of MB and RR-241 dyes under visible light. The enhanced photocatalytic activity of doped ZnO NPs may be due to the smaller crystalline size and higher surface area. The dopants on the surface may act as electron wells to enhance the charge carrier inferred from EDX, PL, XPS and FTIR. In addition to superb photocatalytic activity and higher photostability, doped ZnO also exhibits better anticancer and antibacterial activity than pure ZnO NPs. 13 P. Gomathisankar, K. Hachisuka, H. 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This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. On the basis of the above experimental results and previous studies,88 (26) RSC Adv., 2016, 6, 78335–78350 \| 78347 This journal is © The Royal Society of Chemistry 2016 View Article Online RSC Advances RSC Advances Paper Paper O2c + H2O + 2H+ / H2O2 + O2 (27) H2O2 + e / OH + cOH (28) O2c + H2O + 2H+ / H2O2 + O2 (27) H2O2 + e / OH + cOH (28) (27) 8 J. Zhang, K. Yu, Y. Yu, L.-L. Lou, Z. Yang, J. Yang and S. Liu, J. Mol. Catal. A: Chem., 2014, 391, 12–18. 9 X. Chen and S. Mao, Chem. Rev., 2007, 107, 2891–2959. 10 R. Kozhummal, Y. Yang, F. G¨uder, A. Hartel, X. Lu, U. M. K¨uç¨ukbayrak, A. Mateo-Alonso, M. Elwenspoek and M. Zacharias, ACS Nano, 2012, 6, 7133–7141. cOH/O2c + absorbed dye/dyec+ / degradation products (29) cOH/O2c + absorbed dye/dyec+ / degradation products (29) 11 J. Mu, C. Shao, Z. Guo, Z. Zhang, M. Zhang, P. Zhang, B. Chen and Y. Liu, ACS Appl. Mater. Interfaces, 2011, 3, 590–596. 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https://openalex.org/W4282579199	https://www.frontiersin.org/articles/10.3389/fimmu.2022.906650/pdf	English	null	B10 Cells Are Associated With Clinical Prognosis During Adult Symptomatic Acute HBV Infection	Frontiers in immunology	2,022	cc-by	4,943	Edited by: Yan-Mei Jiao, Fifth Medical Center of the PLA General Hospital, China Reviewed by: Tao Dong, University of Oxford, United Kingdom Ping Ma, Tianjin Second People’s Hospital, China Correspondence: Xinyue Chen chenxydoc@ccmu.edu.cn Jing Zhang zjyouan@ccmu.edu.cn Edited by: Yan-Mei Jiao, Fifth Medical Center of the PLA General Hospital, China Reviewed by: Tao Dong, University of Oxford, United Kingdom Ping Ma, Tianjin Second People’s Hospital, China Tianjin Second People’s Hospital, China Keywords: hepatitis B, acute, interleukin-10, regulatory B cell, cytokine, prognosis, B10 Correspondence: Xinyue Chen chenxydoc@ccmu.edu.cn Jing Zhang zjyouan@ccmu.edu.cn Correspondence: Xinyue Chen chenxydoc@ccmu.edu.cn Jing Zhang zjyouan@ccmu.edu.cn B10 Cells Are Associated With Clinical Prognosis During Adult Symptomatic Acute HBV Infection 1 The Third Unit, Department of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China, 2 The First Unit, Department of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China, 3 Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China There are few reports about the role of B10 cells in acute hepatitis B (AHB) infection. In this study, based on 48 acute hepatitis B infected patients, we analysis the correlation of B10 cells with HBV clinical prognosis. The results showed that B10 was positively correlated with HBsAg and HBeAg and inversely correlated with anti-HBs. The level of B10 in one week before HBsAg clearance was signiﬁcantly lower than 2 weeks prior to HBsAg clearance and after 1-2 weeks of HBsAg clearance. B10 cell frequency displayed no correlation with HBV DNA; however, it showed signiﬁcant temporal synchronization with hepatic inﬂammatory markers such as ALT. B10 level also associated with hospitalization time. These results indicated that B10 is closely related to the clinical prognosis of acute HBV infection. INTRODUCTION Although adult acute hepatitis B is mostly self-limited, more than 100,000 people still die from the disease each year, especially in areas with restricted health resources, according to Global Burden of Disease Study (1). There are signiﬁcant differences in immunological pathogenesis and clinical outcome between acute and chronic HBV infection. For spontaneous clearance of the HBV in the acute setting within several months, both the innate and the adaptive immune system have been proven to be critical (2–4). Otherwise, inﬂammation will aggravate or turn into chronic persistent infection. The main function of regulatory B cell is to secrete IL-10, so it is also known as B10 cell (5). B10 cells act as an negative regulator and inhibit excessive immune response and maintain homeostasis especially in autoimmune diseases (6). In last two decades, the immunomodulatory effect of B10 on inﬂammation, autoimmune diseases, and cancer have attracted much attention (7–9). Previous studies were mostly limited to chronic hepatitis B infection (10, 11). However, it is clear that acute hepatitis B virus infection has a more intense dynamics of virological and immunological process. Here, a cohort of patients with acute hepatitis B who made eventually favorable recovery was analyzed and we attempted to elucidate the relationship between B10 cells and the clinical course. Specialty section: This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology Received: 28 March 2022 Accepted: 17 May 2022 Published: 13 June 2022 Specialty section: This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology Received: 28 March 2022 Accepted: 17 May 2022 Published: 13 June 2022 Keywords: hepatitis B, acute, interleukin-10, regulatory B cell, cytokine, prognosis, B10 ORIGINAL RESEARCH published: 13 June 2022 doi: 10.3389/fimmu.2022.906650 Citation: Liu Y, Du X, Lu J, Ma L, Jing Y, Ben H, Chen X and Zhang J (2022) B10 Cells Are Associated With Clinical Prognosis During Adult Symptomatic Acute HBV Infection. Front. Immunol. 13:906650. doi: 10.3389/fimmu.2022.906650 June 2022 \| Volume 13 \| Article 906650 Frontiers in Immunology \| www.frontiersin.org B10 Cells in AHB Liu et al. MATERIALS AND METHODS Inc., Ashland, OR, USA) was used as the ﬂow analysis software. Patient Demography and Clinical Outcomes Patient Demography and Clinical Outcomes In total, 48 adult hospitalized patients with AHB were enrolled, which ages ranging from 20 to 71 years (y) The most common chief complaints were dark urine (47/48, 97.9%) scleral or skin jaundice (45/48, 93.8%) and fatigue (42/48, 87.5%). All of the patients displayed signiﬁcant abnormalities in liver biochemistry enzyme level. The clinical characteristics of all enrolled patients are listed in Table 1 and the details in Supplementary Table 1 (Supplementary 1). After routine symptomatic treatment and liver protection treatment (but antiviral therapy-free), all 48 patients recovered smoothly. That is, abnormal liver function returned to normal, accompanied by undetectable HBV DNA, and clearance of both HBsAg and HBeAg. At the end of the 48- week follow-up, only 2 patients (4.17%) still did not produce effective Anti-HBs. None of the 48 cases turned chronic. This study was guided by the Declaration of Helsinki and the ethical principles developed by the American Human Research Protection Program (AHRPP), and approved by the Ethical Committee of Beijing You An Hospital, Capital Medical University. All patients provided written informed consent. Statistical Analysis We conducted a retrospective observational single-center study. The acute hepatitis B (AHB) patients were initially screened according to the Chinese Management Scheme of Diagnostic and Therapy Criteria of Viral Hepatitis criteria (12): Patients needed to meet the following criteria: no history of hepatitis B virus infection (HBsAg negative), presence of clinical symptoms and signs of AHB, signiﬁcant increase in serum transaminase with or without elevated bilirubin, positive for HBsAg and/or HBeAg and/or HBV DNA, and immunoglobulin M antibody to hepatitis B core antigen positive (IgM anti-HBc). Exclusion criteria were HCV, HDV, HEV, or HIV co-infection. This case series has been described in our previous study (13). Among these cases, we chose the patients with antiviral treatment intervention-free. Overall, there were 48 patients in this study. The blood samples collection was performed between -2 and 24 weeks after the acute hepatitis B diagnosis was conﬁrmed. 24 patients with chronic hepatitis B (CHB) and 10 healthy people (normal controls, NC) as a comparison group. All fresh blood samples were tested immediately or gathering tested within 2 weeks. All data were analyzed using SPSS 20.0 (IBM Inc, ArMonk, NY, USA) and the GraphPad Prism 7 software (San Diego, CA, USA). Continuous variables were expressed in mean ± SD or median (interquartile range) as appropriate. Qualitative and quantitative differences between subgroups were analyzed by chi-squared or Fisher’s exact tests for categorical parameters and Student’s t-test or Mann–Whitney test for continuous parameters, paired or unpaired, as appropriate. The Pearson correlation was used to analyze the linear correlation of normally distributed data, otherwise the Spearman rank correction test was used. P < 0.05 was considered statistically signiﬁcant. HBV Markers Serologic markers for HBV infection were analyzed by Roche e601 automatic electrochemical luminescence instrument Roche kits (lower limit of detection of HBsAg was 0.05 IU/mL, range from 0.05 to 52,000 IU/mL), according to the manufacturer’s instructions. Quantitation of HBV DNA was performed by polymerase chain reaction using the Roche Cobas/Taqman Real-Time PCR 2.0 System(Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions (lower limit of detection was 20 IU/mL). TABLE 1 \| Baseline Clinical and Virological Characteristics. Characteristic n = 48 Sex (M/F) 30/18 Age (y), mean±SD 39.6 ± 13.8 ALT (U/L), median (range) 1043 (64.1-6367) AST (U/L), median (range) 367 (35.5-3129) TBil (mmol/L), median (range) 83.50 (14.1-379.4) HBV DNA (log10 IU/mL) mean±SD 3.59±1.77 HBsAg (IU/mL), median (range) 934.7 (0.05-52000) HBeAg (S/CO), median (range) 6.21 (0.08-1193) Transmission Route sexual 15 (31.3%) tattooing/dental/body piercing 6 (12.5%) healthcare exposure 2 (4.17%) uncertain 25 (52.1%) Incubation period (days), median (range) 37.0 (21-102) Average onset day†(days), mean±SD 11.27±6.45 M, male; F, female; y, year; SD, standard deviation; ALT, alanine transaminase; AST, aspartate transaminase; TBIL, total bilirubin. *Incubation period, depends on data or transmission route available. †Onset day also means the ﬁrst day of collecting blood sample. B10 Conﬁrming Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (Ficoll-Hypaque density gradient, Axis-shield, Germany) from 10 ml fresh heparinized blood sample. And then PBMCs were incubated with 1 mM CpG-B ODN2006 (In vivoGen, San Diego, CA, USA) for 96 h at 37°C. PMA (3 ng/ml) and ionomycin (100 ng/ml) were added during the last 4h in the presence of 10 mg/ ml brefeldin A (all from Sigma, St Louis, MO, USA). Cells were then surface stained for the markers CD19 Paciﬁc Blue- A, and stained intracellularly with anti-IL-10 APC. So we could assess the ability and the frequency of IL-10 production from puriﬁed B cells (14). All antibodies including CD19-PE and IL-10-APC used in ﬂow cytometry were ordered from BD Pharmingen (BD Pharmingen, San Diego, CA, USA). PBMCs were acquired on a FACS Canton ﬂow cytometer (Becton- Dickinson, Franklin Lakes, NJ, USA). FlowJo 7.6.1 (Tree Star Frontiers in Immunology \| www.frontiersin.org June 2022 \| Volume 13 \| Article 906650 2 B10 Cells in AHB Liu et al. Correlation Analysis Between Initial B10 Level and Length of Hospital Stay g p y We also analyzed the relationship between B10 level at the admission day (i.e., before the HBsAg clearance) and length of hospital stay. The B10 level of patients with hospitalization time more than 4 weeks was higher than that of patients within 4 weeks (Figure 3A). Baseline TBIL levels also show signiﬁcance between Analysis of the Correlation of B10 and HBV Markers B10 within one and two weeks before HBsAg turned negative and one to two weeks after HBsAg negative were estimated. The level of B10 in one week before HBsAg turning negative was signiﬁcantly lower than before and after one to two weeks of that (Figure 2A). Compared with the chronic hepatitis B (CHB) group and the healthy normal control (NC) group, the CHB group showed the highest B10 level, while the NC group showed the lowest expression level. There were statistical differences among the three groups (Figure 2B). In this study, we used CD19 as the marker of B cells and the percentage of IL-10 in Breg or regulatory B cells as the level of B10 (Figure 1A). In order to analyze the relationship between B10 and prognosis of acute hepatitis B, we ﬁrst analyzed the relationship between B10 and HBV DNA, HBsAg, Anti-HBs and HBeAg. We found that B10 was positively correlated with HBsAg (r = 0.245, P = 0.015; Figure 1B) and HBeAg (r = 0.181, P = 0.045; Figure 1D), and negatively correlated with anti- HBs (r = -0.408, P = 0.007; Figure 1C). There was no correlation between B10 and HBV DNA (r = -0.153, P = 0.298; Figure 1E). Analysis of B10 Level Before and After HBsAg Seroconversion In order to further analyze the temporal relationship between B10 and the clearance of acute hepatitis B surface antigen. The level of A B C FIGURE 1 \| B10 positively associated with HBsAg and HBeAg and negatively associated with anti-HBs. We deﬁned B10 as the level of IL-10 in CD19+ cells (A) and analyzed the relationship of B10 with HBsAg (B), anti-HBs (C), HBeAg (D) and HBV DNA (E). A B B C FIGURE 1 \| B10 positively associated with HBsAg and HBeAg and negatively associated with anti-HBs. We deﬁned B10 as the level of IL-10 in CD19+ cells (A) and analyzed the relationship of B10 with HBsAg (B), anti-HBs (C), HBeAg (D) and HBV DNA (E). June 2022 \| Volume 13 \| Article 906650 Frontiers in Immunology \| www.frontiersin.org 3 B10 Cells in AHB Liu et al. Liu et al. A B FIGURE 2 \| B10 signiﬁcantly decreased before HBsAg serum clearance in AHB, show signiﬁcance among the 3 groups. AHB, acute hepatitis (B) CHB, chronic hepatitis (B) NC, normal control. (A). The dynamics of B10 in each patient with two weeks before HBsAg clearance, one week and within 1-2 weeks after HBsAg clearance. (B). The average of B10 show signiﬁcance among the 3 groups. the CHB group (n=24) was the highest, followed by the AHB group (n=48), and the lowest was the NC group (n=10). B A B FIGURE 2 \| B10 signiﬁcantly decreased before HBsAg serum clearance in AHB, show signiﬁcance among the 3 groups. AHB, acute hepatitis (B) CHB, chronic hepatitis (B) NC, normal control. (A). The dynamics of B10 in each patient with two weeks before HBsAg clearance, one week and within 1-2 weeks after HBsAg clearance. (B). The average of B10 show signiﬁcance among the 3 groups. the CHB group (n=24) was the highest, followed by the AHB group (n=48), and the lowest was the NC group (n=10). The role of B lymphocytes in the pathogenesis of hepatitis B may have been underestimated (15). It is thought that the robust speciﬁc T lymphocyte response largely determines the clearance or persistence of HBV infection. Related research also focuses on the depletion mechanisms of HBV-speciﬁc T lymphocyte responses (16, 17). Analysis of B10 Level Before and After HBsAg Seroconversion B lymphocytes comprise a major part of adaptive immunity, and their main functions in resisting viral infection include a series of events, including complex antigen identiﬁcation and antigen presentation, as well as the ultimate production of viral antigen-speciﬁc antibodies (18). Mizoguchi et al. ﬁrst coined the term regulatory B cell to describe B cells that suppress disease in a mouse inﬂammatory bowel disease model (19, 20). B lymphocytes can perform duplex immunomodulation. A prominent example is that B lymphocytes are highly capable of antigen processing and presentation and are necessary for activating CD4+ T lymphocytes. In recent years, attention has been paid to the potential of B lymphocytes in negative immunomodulation, manifested by a number of studies on autoimmune diseases and inﬂammatory diseases (21–26). the 2 groups (Figure 3D), but not ALT, AST, HBsAg, HBeAg, and HBV DNA (Figures 3B, C, E–G). The hospitalization time of acute hepatitis B patients are not only related to the time of HBsAg clearance, but also related to the time of liver function recovery. We also analyzed the relationship between B10 and ALT. The levels of ALT (Figure 4A) and B10 (Figure 4B) gradually decreased with a smooth recovery from the disease. B10 was positively correlated with ALT (r = 0.293, P = 0.001; Figure 4C). DISCUSSION Indeed, it signiﬁcantly increased at the peak of inﬂammatory activities and decreased when inﬂammation was eased, suggesting that it can inhibit inﬂammation. The inﬂammation levels of acute hepatitis B can be self-limiting, synchronically related to the levels of B10 frequency, indicating the latter’s function in inhibiting overdevelopment of inﬂammation to prevent spread. In the AHB recovery phase, B10 drop to a low level and prevent the disease from turning into a chronic persistent infection. Even if the P values have statistical signiﬁcance, the absolute values of Pearson correlation coefﬁcient (r) between B10 cell and HBsAg, HBeAg and ALT are not strong enough. A larger sample size to verify the correlation is expected. It is no doubt that regulatory T cells can perform negative regulation by means of IL-10, and this has been conﬁrmed for chronic hepatitis B. B10 cells exerted dysregulation in T cell function through IL-35 dependent mechanism and depend on cell-to-cell contact style (27, 28). B B C mechanism and depend on cell to cell contact style (27, 28). With AHB standard management procedure and no antiviral intervention, we could conclude that the length of hospital stay is directly related to severity of illness. In this study, the frequency of B10 cell was higher in patients hospitalized for longer than 4 weeks than in patients hospitalized for less than 4 weeks. These results further suggest that B10 is positively correlated with the course and prognosis of acute liver inﬂammatory disease. Before and after HBsAg clearance, we also clearly observed the critical time point which the expression of B10 frequency is the lowest. Accompanied by HBsAg clearance and anti-HBs appearance gradually, B10 return to the previous normal level. These regulatory cells are also regulated by regulators and may be related to IL-35 or IL-21 (8), which needs to be conﬁrmed by further studies. FIGURE 4 \| B10 temporal positively associated with ALT during acute HBV infection. Dynamic of ALT (A) and B10 (B) during hospitalization stay time. The positive correlation between B10 and ALT during hospitalization stay time (C). Previous study revealed that the depletion of Treg cells in the PBMCs of patients with chronic hepatitis B did not alter the frequency of B10 cells or their ability to produce IL-10 (11), indicating regulatory T or B lymphocyte have their own unique pathway to inﬂuence immune response. DISCUSSION Regulatory B cells are a subgroup of B cells. Regulatory B cells are determined by their secretion of negative cytokine IL-10, so the B cells secreting IL-10 are also known as B10. Very little was found in the literature on the question of the role of B10 cells in acute HBV infection. In this study, we found that B10 was closely related to the prognosis of acute HBV by series of samples from 48 cases of acute HBV patients. A B D E F G C FIGURE 3 \| Compared B10 levels and other liver function index between different hospitalization stay time patients. The admitting day’s B10 levels was signiﬁcant lower in patients within 4 weeks of hospitalization stay than those of more than 4 weeks (A). TBIL levels also show signiﬁcance between the 2 groups (D), but not ALT (B), AST (C), HBsAg (E), HBeAg (F), and HBV DNA (G). D C C D B E F F E G G FIGURE 3 \| Compared B10 levels and other liver function index between different hospitalization stay time patients. The admitting day’s B10 levels was signiﬁcant lower in patients within 4 weeks of hospitalization stay than those of more than 4 weeks (A). TBIL levels also show signiﬁcance between the 2 groups (D), but not ALT (B), AST (C), HBsAg (E), HBeAg (F), and HBV DNA (G). 4 June 2022 \| Volume 13 \| Article 906650 Frontiers in Immunology \| www.frontiersin.org B10 Cells in AHB Liu et al. Liu et al. A B C FIGURE 4 \| B10 temporal positively associated with ALT during acute HBV infection. Dynamic of ALT (A) and B10 (B) during hospitalization stay time. The positive correlation between B10 and ALT during hospitalization stay time (C). inﬂammation changes rapidly but can eventually recover in a relatively short time. This study revealed the obvious correlation between the B10 frequency and the changes in liver inﬂammation: the B10 frequency increased signiﬁcantly when liver inﬂammation intensiﬁed and decreased rapidly to normal during disease recovery. This also fully demonstrates the potential leading role B10 cell plays in regard to inﬂammation and immunity. A As stated above, because the secretion of IL-10 is not limited to a particular cell or cell subset, B10 cells may also comprise different cell subsets and may have certain heterogeneities. Frontiers in Immunology \| www.frontiersin.org REFERENCES 12. Chinese Society of Infectious Diseases and Parasitology, CSoH. Management Scheme of Diagnostic and Therapy Criteria of Viral Hepatitis. Chin J Hepatol (2000) 6:324–9. doi: 10.3760/j.issn:1007- 3418.2000.06.001 1. 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Ferrari C, Penna A, Bertoletti A, Valli A, Antoni AD, Giuberti T, et al. Cellular Immune Response to Hepatitis B Virus-Encoded Antigens in Acute and Chronic Hepatitis B Virus Infection. J Immunol (1990) 145:3442–9. 15. Zhang S, Zhao J, Zhang Z. Humoral Immunity, the Underestimated Player in Hepatitis B. Cell Mol Immunol (2018) 15:645–8. doi: 10.1038/cmi.2017.132 4. Dunn C, Peppa D, Khanna P, Nebbia G, Jones M, Brendish N, et al. Temporal Analysis of Early Immune Responses in Patients With Acute Hepatitis B Virus Infection. Gastroenterology (2009) 137:1289–300. doi: 10.1053/ j.gastro.2009.06.054 16. Rehermann B, Lau D, Hoofnagle JH, Chisari FV. Cytotoxic T Lymphocyte Responsiveness After Resolution of Chronic Hepatitis B Virus Infection. J Clin Invest (1996) 97:1655–65. doi: 10.1172/JCI118592 5. Tedder TF. B10 Cells: A Functionally Deﬁned Regulatory B Cell Subset. J Immunol (2015) 194:1395–401. doi: 10.4049/jimmunol.1401329 17. Seto WK, Lo YR, Pawlotsky JM, Yuen MF. Chronic Hepatitis B Virus Infection. Lancet (2018) 392:2313–24. doi: 10.1016/S0140-6736(18)31865-8 6. Matsushita T. Regulatory and Effector B Cells: Friends or Foes? J Dermatol Sci (2019) 93:2–7. doi: 10.1016/j.jdermsci.2018.11.008 18. Oliviero B, Cerino A, Varchetta S, Paudice E, Pai S, Ludovisi S, et al. Enhanced B-Cell Differentiation and Reduced Proliferative Capacity in Chronic Hepatitis C and Chronic Hepatitis B Virus Infections. J Hepatol (2011) 55:53–60. doi: 10.1016/j.jhep.2010.10.016 7. Cai X, Zhang L, Wei W. Regulatory B Cells in Inﬂammatory Diseases and Tumor. SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and approved by Ethical Committee of Beijing YouAn Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2022.906650/ full#supplementary-material DISCUSSION Also, there are other IL- 10-producing immune cells such as T cell, monocytes and NK cells, not involved in this assay with regret (29–31). We speculate that these immune cells focus on secreting IL-10, forming a complex and interacting regulatory network, and exert inhibitory effects organically. Furthermore, the lack of acute fulminant hepatitis and liver specimens is the deﬁciency of this study. Additionally, the development about HBsAg-speciﬁc and/or HBcAg-speciﬁc B cells maybe novel targets for antiviral strategies toward functional cure of chronic HBV infection attracted more attentions (32–34). The failure to explore the IL-10 is an important inhibitory cytokine in the human body, with a low absolute concentration in peripheral blood. In various microenvironments, it inhibits pro-inﬂammatory cytokines, Th1 cell responses, and T lymphocyte proliferation, performing multiple negative regulations. In a certain sense, the number of IL-10-producing Breg cells and the IL-10 production level reﬂect the changes in the body’s inﬂammation level and immune status. According to a pioneer study by Dr. Maini and her colleagues in 2012, IL-10 levels in plasma and liver inﬂammation levels of chronic hepatitis B patients were positively correlated, suggesting that the elevation of IL-10 levels is a protective mechanism (10). In terms of the disease course, compared to chronic hepatitis B, acute hepatitis B is characterized by the clinical features of acute onset and self-limiting viral clearance. In acute hepatitis B, June 2022 \| Volume 13 \| Article 906650 Frontiers in Immunology \| www.frontiersin.org 5 B10 Cells in AHB Liu et al. DATA AVAILABILITY STATEMENT Thirteenth Five-Year Major Science and Technology Projects (2017ZX10202202-005-010), the Capital Health Research and Development Projects (2020-1-3011). 2022 Research project on education and teaching reform of Capital Medical University (2022JYY260). Thirteenth Five-Year Major Science and Technology Projects (2017ZX10202202-005-010), the Capital Health Research and Development Projects (2020-1-3011). 2022 Research project on education and teaching reform of Capital Medical University (2022JYY260). The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. AUTHOR CONTRIBUTIONS phenotype features of those HBV-speciﬁc B cells in the environment of acute hepatitis B is also one of the short- comings of this study. YL, XC, and JZ conceived and designed the experiments; YL and XD performed the experiments; YL and HB analyzed the data; XD, LM, JL, and YJ contributed patients recruiting and management. YL and JZ wrote the paper. All authors contributed to the article and approved the submitted version. In conclusion, we found that B10 is closely related to the clinical prognosis of acute HBV infection. Whether B10 is one of the pathogenic mechanisms of acute fulminant hepatitis and whether intervention of B10 cells is helpful to the elimination of chronic HBV deserves further study. REFERENCES Le Bert N, Salimzadeh L, Gill US, Dutertre CA, Facchetti F, Tan A, et al. Comparative Characterization of B Cells Speciﬁc for HBV Nucleocapsid and Envelope Proteins in Patients With Chronic Hepatitis B. J Hepatol (2020) 72:34–44. doi: 10.1016/j.jhep.2019.07.015 26. Mielle J, Audo R, Hahne M, Macia L, Combe B, Morel J, et al. 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Clin Sci (Lond) (2016) 130:907–19. doi: 10.1042/CS20160069 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 28. Liu Y, Luo Y, Zhu T, Jiang M, Tian Z, Tang G, et al. Regulatory B Cells Dysregulated T Cell Function in an IL-35-Dependent Way in Patients With Chronic Hepatitis B. Front Immunol (2021) 12:653198. doi: 10.3389/ ﬁmmu.2021.653198 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 29. Bedke T, Muscate F, Soukou S, Gagliani N, Huber S. IL-10-Producing T Cells and Their Dual Functions. 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Mucosal Immunol (2022) 15:268–78. doi: 10.1038/s41385-021-00481-9 11. Gong Y, Zhao C, Zhao P, Wang M, Zhou G, Han F, et al. Role of IL-10- Producing Regulatory B Cells in Chronic Hepatitis B Virus Infection. Dig Dis Sci (2015) 60:1308–14. doi: 10.1007/s10620-014-3358-1 June 2022 \| Volume 13 \| Article 906650 Frontiers in Immunology \| www.frontiersin.org 6 B10 Cells in AHB Liu et al. 24. Daïen CI, Tan J, Audo R, Mielle J, Quek LE, Krycer JR, et al. Gut-Derived Acetate Promotes B10 Cells With Antiinﬂammatory Effects. JCI Insight (2021) 6:e144156. doi: 10.1172/jci.insight.144156 32. Salimzadeh L, Le Bert N, Dutertre CA, Gill US, Newell EW, Frey C, et al. PD-1 Blockade Partially Recovers Dysfunctional Virus-Speciﬁc B Cells in Chronic Hepatitis B Infection. J Clin Invest (2018) 128:4573–87. doi: 10.1172/ JCI121957 25. Chodisetti SB, Fike AJ, Domeier PP, Choi NM, Soni C, Rahman Z. TLR7 Negatively Regulates B10 Cells Predominantly in an Ifng Signaling Dependent Manner. Front Immunol (2020) 11:1632. doi: 10.3389/ ﬁmmu.2020.01632 33. June 2022 \| Volume 13 \| Article 906650 REFERENCES doi: 10.1002/hep.23977 Copyright © 2022 Liu, Du, Lu, Ma, Jing, Ben, Chen and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 31. Gori S, Soczewski E, Fernandez L, Grasso E, Gallino L, Merech F, et al. Decidualization Process Induces Maternal Monocytes to Tolerogenic IL-10- Producing Dendritic Cells (DC-10). Front Immunol (2020) 11:1571. doi: 10.3389/ﬁmmu.2020.01571 June 2022 \| Volume 13 \| Article 906650 Frontiers in Immunology \| www.frontiersin.org
https://openalex.org/W4379659628	https://ejournal.itn.ac.id/index.php/jati/article/download/6361/3714	Indonesian	null	MENENTUKAN STATUS KEGIATAN PEMBANGUNAN DAN PEMELIHARAAN POS KEAMANAN WILAYAH JAWA BARAT MENGGUNAKAN ALGORITMA C.45	JATI	2,023	cc-by-sa	2,730	Vol. 7 No. 1, Februari 2023 Vol. 7 No. 1, Februari 2023 JATI (Jurnal Mahasiswa Teknik Informatika) MENENTUKAN STATUS KEGIATAN PEMBANGUNAN DAN PEMELIHARAAN POS KEAMANAN WILAYAH JAWA BARAT MENGGUNAKAN ALGORITMA C.45 Faisal Nurdiana, Arif Rinaldi Dikananda Program Studi Manajemen Informatika, STMIK IKMI Cirebon Jl. Perjuangan No. 10B, Karyamulya Cirebon, Indonesia Faisalnurdiana18@gmail.com ABSTRAK Tujuan dari proyek akhir ini adalah untuk mengetahui bagaimana perkembangan kebersihan dan lingkungan keamanan di kabupaten/kota jawa barat dan memahami keinginan seluruh warga local serta pendataan yang dari luar daerah dalam menjaga dan menciptakan lingkungan keamanan dalam kebersihan dan agar segera terwujud: siskamling-klaster system, serta adanya kesadaran individu dan semua warga negara sangat penting dalam melaksanakan perubahan lingkungan keamanan dan masalah kebersihan, baik ke alam lingkungan serta lingkunan social, Hasil dari proyek akhir ini adalah menghasilkan akurasi sebesar 97.58% dari total dataset sebanyak 330 data, dengan klasifikasi daaerah yang ada kegiatan pembangunan dan pemeliharaan pos keamanan di jawa barat sebanyak 322 daerah daan 8 daerah yang belum melaksankan kegiatan pembangunan dan pemeliharaan pos keamanan. Kata Kunci: Environment, Security Post, Classification C4.5 2.1. Keamanan Dan Ketertiban Lingkungan 2.1. Keamanan Dan Ketertiban Lingkungan , , Kegiatan Siskamling merupakan wadah bagi masyarakat untuk berpartisipasi dalam meningkatkan kesadaran hukum dan menegakkan supremasi hukum. Perlindungan lingkungan dapat dilakukan dengan sebaik-baiknya ketika masyarakat berpartisipasi secara aktif. Pentingnya peran serta masyarakat dalam penyelenggaraan sistem keamanan lingkungan didasari oleh adanya fenomena kriminalitas yang semakin kompleks dalam kehidupan masyarakat, termasuk maraknya perampokan.[2] , = − \| \| \| \| ∗ ( 1 ) Keterangan : S = himpunan kasus A = atribut n = jumlah partisi atribut A \|Si\| = jumlah kasus pada partisi ke-i \|S\| = jumlah kasus dalam S Sementara itu, perhitungan nilai entropy dapat dilihat pada persamaan 2 (2) Keterangan: S = himpunan kasus A = fitur n = jumlah partisi S pi = proporsi dari Si terhadap S [12] − \| \| \| \| ∗ ( 1 ) = − ∗ log ( 1 ) Keterangan : S = himpunan kasus A = atribut n = jumlah partisi atribut A \|Si\| = jumlah kasus pada partisi ke-i \|S\| = jumlah kasus dalam S S = himpunan kasus A = atribut n = jumlah partisi atribut A \|Si\| = jumlah kasus pada partisi ke-i Gambar 1. Tahapan Knowledge Discovery in Database Gambar 1. Tahapan Knowledge Discovery in Database \|S\| = jumlah kasus dalam S Sementara itu, perhitungan nilai entropy dapat dilihat pada persamaan 2 p p (2) Keterangan: S = himpunan kasus A = fitur n = jumlah partisi S pi = proporsi dari Si terhadap S [12] = − ∗ log (2) Keterangan: Gambar 1. Tahapan Knowledge Discovery in Database 1. PENDAHULUAN 7 No. 1, Februari 2023 JATI (Jurnal Mahasiswa Teknik Informatika) Dengan adanya penelitian ini diharapkan dapat memaksimalkan system siskamling di setiap daerah yang ada di provinsi jawa barat agar di setiap daerah yang ada di jawa barat menjadi daerah yang aman serta nyaman bagi pendatang maupun warga asli. a) pilih atribut sebagai akar c) bagi kasus dalam cabang c) bagi kasus dalam cabang d) ulangi proses untuk setiap cabang sampai semua kasus pada cabang memiliki kelas yang sama Untuk memilih atribut sebagai akar, didasarkan pada nilai gain tertinggi dari atribut-atribut yang ada. Untuk menghitung gain digunakan persamaan 1. 1. PENDAHULUAN Pada beberapa dekade lalu, sistem penjagaan seperti ini dikenal dengan Siskamling, dan tempatnya bernama Poskamling. Hal ini pernah booming di ganggang, di RT-RT, dan di RW-RW seluruh Indonesia meski timbul tenggelam. Namun semangat Siskamling, yang semestinya bisa untuk pengelolaan Kamtibmas lingkungan bersama Poskamlingnya, naik turun bak ombak dilautan, Slamet Pribadi[4] Pada saat yang sama populasi tumbuh secara bertahap dan proporsional. Upaya masyarakat dalam mendorong, urbanisasi dan migrasi untuk meningkatk an pendapatan, pendidikan, kesejahteraan, lingkungan yang aman dan kehidupan yang lebih layak dilakukan dengan memanfaatkan dan mengembangkan potensi daerah [1], Mencitakan keamanan dan ketertiban masyarakat tidak hanya pelaksanaan peraturan perundangundangan Oleh karena itu Dinas Pemberdayaan Masyrakat dan Desa melakukan suatu program dimana di setiap desa/kelurahan yang ada di jawa barat khususnya di wilayah kab/kota cirebon diadakan system siskamling, dengan tujuan untuk meminalmilisir tindak kejahatan yang ada di masyarkat, baik itu tindak pidana pencurin atau sebagainnya serta menciptakan lingkungan yang nyaman dan aman. dan penegakan hukum, tetapi masyaraka juga dapat ik ut serta dalam menciptakan keamanan dan ketertiba paling tidak di lingkungannya sendiri. [2], maka dari itu dibuatlah system keamanan lingkungan atau biasa disebut siskamling, Siskamling (Sistem Keamanan Lingkungan) merupakan salah satu upaya pencegahan bahaya, keamanan dan gangguan untuk menciptakan rasa aman dan nyaman di masyarakat. Untuk mendukung kegiatan Siskamling yang direncanakan dapat dilaksanakan dengan baik, diperlukan sistem pemantauan pelaksanaan kegiatan.[3] Data diatas merupakan data dari www.opendata.jabar.com di, ada beberapa yang berjumlah 15.936 data yang berisikan atribut kode_pr ovinsi,nama_provinsi, bps_kode_kabupaten_kota, bp s_nama_kabupaten_kota, bps_kode_kecamatanbps_n ama_kecamatan,bps_kode_desa_kelurahan, bps_nam a_desa_kelurahan, kemendagri_kode_kecamata, kem endagri_nama_kecamatan, kemendagri_kode_desa_k elurahan, kemendagri_nama_desa_kelurahan, status_ kegiatan, tahun, dan id Salah satu metode dalam SISKAMLING adalah e-siskamling yang telah di terapkan di wilayah jember, E-Siskamling ini dilaunching pada 6 April 2017, diresmikan oleh Kepala Kepolisian Daerah Jawa Timur Irjen.Pol.Drs. Macfud Arifin, SH pada saat digelarnya rangkaian kegiatan kunjungan kerja Kapolda Jatim di Polres Jember. Kapolda Jatim sangat mengapresiasi program e-siskamling ini, sebab aplikasi ini merupakan yang pertama di Indonesia. Selain itu, Kapolri juga mengapresiasi inovasi-inovasi dari Polres Jember dengan memberikan penghargaan WBBM, penghargaan ini merupakan penghargaan tertinggi di bidang inovasi dan pelayanan (https://nusantaranews/ diakses pada 10 Juli 2018). Kemanan serta kenyamanan bagi masryarakat merupakan hal yang utama, Dengan hasil akurasi pemberdayaan masyarakat terhadap pembangunan dan pemeliharaan pos keamanaan lingkungan dengan tujuan menciptakan lingkungan yang nyaman dan aman serta sehat, yang memungkinan warga nya hidup dengan damai dan bisa menjadi pelopor bagi daerah lain untuk menjaga serta melestarikan lingkungan alam sekitar kita [5] 505 Vol. 2.2. Data Mining Dalam penelitian ini, digunakan metode Knowledge Discovery in Database (KDD) yang memiliki beberapa tahapan dan proses yang harus dilakukan. Tahapan-tahapan tersebut mencakup Selection, Preprocessing, Transformation, Data Mining, serta Evaluation. Dalam proses dilakukan identifikasi dan ekstraksi informasi penting yang terdapat dalam data yang diolah. Setiap tahapan dalam proses ini memiliki peran penting dalam menghasilkan hasil yang akurat dan bermakna dari data yang diolah. Data mining menurut David Hand, Heikki Mannila, dan Padhraic Smyth dari MIT adalah analisa terhadap data (biasanya data yang berukuran besar) untuk menemukan hubungan yang jelas serta menyimpulkannya yang belum diketahui sebelumnya dengan cara terkini dipahami dan berguna bagi pemilik data tersebut.[6], Data mining, di sisi lain, adalah kegiatan yang melibatkan pengumpulan dan penggunaan data historis untuk menemukan keteraturan, pola, atau hubungan dalam kumpulan data besar.[7], Klasifikasi dalam informasi mining ialah tata cara pendidikan informasi buat memprediksi nilai dari sekelompok atribut[8], data mining merupakan proses menemukan pola yang dalam, menarik, dan baru, serta pola deskriptif, dapat dipahami, dan prediktif dalam data skala besar.[9] 3.1. Selection Tahap selection memasukkan operator Read Excel dalam membaca data Excel yang akan memakai Rapidminer versi 10.0. Selanjutnya menggunakan operator Select Attributes, yang berfungsi untuk memilih atribut mana yang ingin digunakan. Operator select atribut berfungsih untuk mengatur peran dari 1 atau lebih atribut yang akan digunakan 2.3. Algoritma C4.5 Salah satu algoritma yang dapat digunakan untuk membuat pohon keputusan (decission tree) adalah algoritma C4.5. Algoritma C4.5 merupakan algoritma yang sangat populer yang digunakan oleh banyak peneliti di dunia,[10], Algoritma C4.5 memiliki akurasi yang tinggi ketika sebagian besar dari parameter data yang digunakan adalah nominal.[11]. Secara umum algoritma C4.5 untuk membangun pohon keputusan adalah sebagai berikut: 1. Seleksi Data (Data Selection) Pada proses seleksi data dilakukan menggunakan operator Select Attributes dan menghasilkan dataset dengan atribut terpilih untuk digunakan pada proses selanjutnya yaitu pemilihan data. Berikut adalah hasil dari seleksi data menggunakan Select Attributes: g) Performance. Dimana setiap operator memiliki tugas serta fungsihnya masing-masing Sesuai dengan metode serta algoritma yang kita gunakan dalam memproses suatu data set pada data mining, Gambar 3. Hasil Proses Select Attributes Pada Gambar 3 terlihat bahwa atribut pada dataset status kegiatan pembangunandan pos keamanan di wilayah Jawa Barat telah diseleksi sesuai kebutuhan proyek, dimana hanya 4 atribut dari total 15 atribut yang digunakan 4. HASIL DAN PEMBAHASAN 4. HASIL DAN PEMBAHASAN 4.1. Penerapan algoritma C4.5 untuk menentukan status kegiatan pembangunan dan pemeliharaan pos keamanan yang ada di wilayah Jawa Barat 4.1. Penerapan algoritma C4.5 untuk menentukan status kegiatan pembangunan dan pemeliharaan pos keamanan yang ada di wilayah Jawa Barat 3.2. Preprocessing T h Tahapan Preprocessing dilakukan melalui Rapidminer dengan menggunakan operator select atribut. Operator select atribut digunakan untuk memilih data yang digunakan pada setiap atributnya Pada operator select atribut ini terdapat atribut yang a kan di gunakan dalam tahapan selanjutnya, yaitu atrib 506 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 7 No. 1, Februari 2023 ut: bps_nama_desa_kelurahan, bps_nama_kabupaten _kota, bps_nama_kecamatan, status_kegiataan, berada pada tahapan yang berbed, berikut merupakan tahapn klasifikasi: berada pada tahapan yang berbed, berikut merupakan tahapn klasifikasi: karena dalam atribut tersebut akan berpengaruh terhadap hasil akhir. 3.3. Data Mining Dalam proses data minin akan terlihat operator apa saja yang digunakan dari awal proses hingga mendapatkan hasil yang sesuai, dimana kali ini operator yang digunakan yaitu: Gambar 3. Hasil Proses Select Attributes a) Read Excel b) Select Atribut c) Set Role d) Split Data e) Decision Tree f) Apply Mode g) Performance. 3.4. Evaluasi Tahapan terakhir dalam proses suatau data mining ialah proses evaluasi atau hasil akhir dengan menggunakan metode yang di pilih, pada proyek tugas akhir kali ini menggunakan operator Performance Clasification, dimana dalam operator ini akan muncul hasil akhir dari setiap proses yang dilakukan. Berdasarkan informasi tersebut, telah diketahui bahwa terdapat perbedaan jumlah data yang signifikan antara jumlah data awal dan jumlah data setelah dilakukan proses pemilihan data. Jumlah data awal pada dataset sebelum dilakukan pemilihan data.  Hasil Operator Split Data  Hasil Operator Split Data  Hasil Operator Split Data  Hasil Operator Split Data Gambar 6. Hasil Performance (Classification) Gambar 6. Hasil Performance (Classification) Berdasarkan pada gambar 4.6, diketahui hasil Performance Classification, di dapatkan nilai akurasi sebesar 97.58% dari total dataset sebanyak 330 data, dengan klasifikasi daerah yang ada kegiatan pembangunan dan pemeliharaan pos keamanan di Jawa Barat sebanyak 322 daerah dan 8 daerah yang belum melaksanakan kegiatan pembangunan dan pemeliharaan pos keamanan Gambar 5. Proses penentuan rasio pada operator Split Data Bedasarkan gambar diatas, terdapat perbandingan rasio dari data utuh dibagi menjadi dua bagian, perbandingan ini bernilai 0.8 dan 0.2, data dengan rasio 0.8 merupakan data training dan data dengan rasio 0.2 merupakan data testing. Hasil Dari Status kegiatan pembangunan dan pemeliharaan pos keamanan yang ada di wilayah Jawa Barat dengan menggunakan algotirma C4.5 3. Data Mining Untuk melakukan klasifikasi, pada proyek tugas akhir ini digunakan algoritma C4.5 Classifikasi. Dataset yang telah dilakukan proses seleksi data, pemilihan data dan transformasi data sudah dapat diolah pada proses data mining dengan menggunakan algoritma C4.5 Classifikasi. Tabel 1. Hasil Proses C4.5 WALED KOTA ADA 0.9 0.1 ADA MEKARSARI TIDAK ADA 0.9 0.1 ADA AMBIT ADA 0.9 0.1 ADA CIUYAH ADA 0.9 0.1 ADA KARANGSARI ADA 0.9 0.1 ADA CIKULAK KIDUL ADA 0.9 0.1 ADA CIKULAK ADA 0.9 0.1 ADA CISAAT ADA 0.9 0.1 ADA LEUWEUNGGAJAH ADA 1.0 0.0 ADA TENJOMAYA ADA 1.0 0.0 ADA DAMARGUNA ADA 1.0 0.0 ADA JATISEENG ADA 1.0 0.0 ADA JATISEENG KIDUL ADA 1.0 0.0 ADA CILEDUG KULON ADA 1.0 0.0 ADA CILEDUG WETAN ADA 1.0 0.0 ADA CILEDUG LOR ADA 1.0 0.0 ADA CILEDUG TENGAH ADA 1.0 0.0 ADA BARISAN ADA 0.9 0.1 ADA LOSARI KIDUL ADA 0.9 0.1 ADA PANGGANGSARI ADA 0.9 0.1 ADA LOSARI LOR TIDAK ADA 0.9 0.1 ADA MULYASARI ADA 0.9 0.1 ADA KALIRAHAYU ADA 0.9 0.1 ADA KALISARI ADA 0.9 0.1 ADA AMBULU ADA 0.9 0.1 ADA TAWANGSARI ADA 0.9 0.1 ADA Gambar 6. Hasil operator Decision Tree Gambar 6. Hasil operator Decision Tree Bedasarkan gambar diatas, pada proses DecisionTree ini didapatkan klasifikasi jumlah data status kegiatan pembangunan dan pemeliharaan pos kemanan yang ada di Jawa Barat sebagai berikut: Dari data diatas dapat di ketahui daerah mana saja yang sudah menerapkan kegiatan pos kemanan/siskamling serta daerah mana yang belum optimal dalam penerapan pos kemanan/siskamling. 2. Pemilihan Data (Data Pre-Processing) Tahap kedua adalah tahap pemilihan data, tahap ini dilakukan dengan menggunakan operator Set Role dan operator Split Data pada aplikasi Rapidminer. Pemilihan data dilakukan untuk memilih serta menghapus data yang tidak relevan, Berikut merupakan hasil dari proses PreProcessing: Penerapan Algoritma C4,5 untuk menentukan status kegiatan pembangunan dan pemeliharaan pos keamanan yang ada di wilayah Jawa Barat dilakukan menggunakan software Rapidminer.  Hasil Operator Set Role Gambar 2. Proses Klasifikasi Menggunakan Rapidminer  Hasil Operator Set Role Gambar 4. Proses operator Set Role Gambar 2. Proses Klasifikasi Menggunakan Rapidminer Gambar 4. Proses operator Set Role Pada Operator Set Role di dapatkan atribut dan target role apa yang akan di proses pada tahapan selanjutnya, yaitu Atribut status_kegiatan dengan target role berbentuk label. Berdasarkan Gambar 4.1, proses klasifikasi menggunakan beberapa operator yang terdapat pada Rapidminer. Operator yang digunakan tersebut memiliki fungsi serta tujuan masing-masing dan 507 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 7 No. 1, Februari 2023  Hasil Operator Split Data Gambar 5. Proses penentuan rasio pada operator Split Data yang telah dibuat. Tahap evaluasi menggunakan operator Performance (Classification). yang telah dibuat. Tahap evaluasi menggunakan operator Performance (Classification). 4. Evaluasi Bedasarkan proses yang telah di lalui, didapatkan sebuah pohon keputusan atau data yang telah di olah sesui dengan metedo yang diterapkan, Adapun data set Setelah mengolah data dengan menggunakan algoritma C4.5 Classification pada aplikasi Rapidminer, tahap selanjutnya adalah evaluasi hasil 508 Vol. 7 No. 1, Februari 2023 JATI (Jurnal Mahasiswa Teknik Informatika) yang ada tidak semunya di masukan dalam laporan tugas akhir no. 2, p. 293, 2019, doi: 10.31544/jtera.v4.i2.2019.293-302. no. 2, p. 293, 2019, doi: 10.31544/jtera.v4.i2.2019.293-302. [4] W. Guan et al., “Clinical Characteristics of Coronavirus Disease 2019 in China,” N. Engl. J. Med., vol. 382, no. 18, pp. 1708–1720, 2020, doi: 10.1056/nejmoa2002032. 5. KESIMPULAN DAN SARAN Dari hasil proyek akhir ini, dapat disimpulkan bahwa untuk di wilayah Jawa Barat khususnuya wilayah Kab/Kota Cirebon masi banyak yang belum menerapkan program system keamanan lingkungan, sebagai mana yang telah di anjurkan oleh Dinas Pemberdayaan Masyrakat Dan Desa Jawa Barat, [5] S. Rahayu, “1914-Article Text-3776-1-10- 20210924,” vol. 5, pp. 6077–6084, 2021. [6] Muhammad Robith Adani, “Mengenal Konsep Data Mining Beserta Contoh Implementasinya,” Sekawanmedia.Co.Id, 2021. Hasil dari proyek akhir ini adalah menghasilkan akurasi sebesar 97.58% dari total dataset sebanyak 330 data, dengan klasifikasi daaerah yang ada kegiata n pembangunan dan pemeliharaan pos keamanan di jawa barat sebanyak 322 daerah daan 8 daerah yang belum melaksankan kegiatan pembangun an dan pemeliharaan pos keamanan, [7] R. Avrizal and S. Dwiasnati, “Implementasi Data Mining dengan Algoritma C4.5 pada Penjualan Obat,” Format J. Ilm. Tek. Inform., vol. 8, no. 1, p. 77, 2019, doi: 10.22441/format.2019.v8.i1/010. [8] H. Hozairi, A. Anwari, and S. Alim, “Implementasi Orange Data Mining Untuk Klasifikasi Kelulusan Mahasiswa Dengan Model K-Nearest Neighbor, Decision Tree Serta Naive Bayes,” Netw. Eng. Res. Oper., vol. 6, no. 2, p. 133, 2021, doi: 10.21107/nero.v6i2.237. Dengan adanya proyek akhir ini dihapakan Dinas PemberdayaanMasyarakat Dan Desa dapat me ngevaluasi daerah - daerah yang belum menerapkan s ystem pemeliharaan dan pembangunan pos keamanan agar dapat terwujudnya lingkungan yang aman dan nyaman bagi seluruh warga masyrkat [9] Normah, B. Rifai, S. Vambudi, and R. Maulana, “Analisa Sentimen Perkembangan Vtuber Dengan Metode Support Vector Machine Berbasis SMOTE,” J. Tek. Komput. AMIK BSI, vol. 8, no. 2, pp. 174–180, 2022, doi: 10.31294/jtk.v4i2. Bagi dinas - dinas terkait yang menangani perm asalahan yang berkaitan dengan proyek akhir ini agar dapat memaksimalkan dan melakukan sosialisasi serta evaluasi untuk daerah-daerah yang sudah dan belum menerapkan program yang telah di susun sedemikian rupa [10] S. Febriani and H. Sulistiani, “Analisis Data Hasil Diagnosa Untuk Klasifikasi Gangguan Kepribadian Menggunakan Algoritma C4.5,” 89Jurnal Teknol. dan Sist. Inf., vol. 2, no. 4, pp. 89–95, 2021. DAFTAR PUSTAKA [1] D. Mirwansyah, R. Riyayatsyah, and D. Martadinata, “Pemetaan Pemukiman dan Potensi Wilayah Desa Berbasis WebGis,” METIK J., vol. 4, no. 2, 2020, doi: 10.47002/metik.v4i2.187. [11] M. F. Hasani and Sutikno, “Implementasi Algoritma C4.5 Untuk Klasifikasi Penyakit Infeksi Saluran Kemih Berbasis Web,” J. Ilm. Inform. Komput., vol. 26, no. 3, pp. 233–246, 2021, doi: 10.35760/ik.2021.v26i3.4710. [2] N. Amallia, “Partisipasi Masyarakat Dalam Sistem Keamanan Lingkungan Untuk Meningkatkan Keamanan Dan Ketertiban Masyarakat,” Jisip, vol. 2, no. 1, pp. 1–9, 2020, doi: 10.24967/fisip.v2i1.653. [12] Yuli Mardi, “Data Mining : Klasifikasi Menggunakan Algoritma C4 . 5 Data mining merupakan bagian dari tahapan proses Knowledge Discovery in Database ( KDD ) . Jurnal Edik Informatika,” J. Edik Inform., vol. 2, no. 2, 2019. [3] E. Andika, L. Nurlani, and R. R. Kusmana, “Desain Pemanfaatan RFID dan SMS Gateway untuk Monitoring Sistem Keamanan Lingkungan Desa,” JTERA (Jurnal Teknol. Rekayasa), vol. 4, 509
https://openalex.org/W2785954573	https://centaur.reading.ac.uk/87015/8/1-s2.0-S0079661117303154-main.pdf	English	null	A window on the deep ocean: The special value of ocean bottom pressure for monitoring the large-scale, deep-ocean circulation	Progress in oceanography/Progress in Oceanography	2,018	cc-by	26,341	Article Published Version Creative Commons: Attribution 4.0 (CC-BY) Published Version Hughes, C. W., Williams, J., Blaker, A., Coward, A. and Stepanov, V. (2018) A window on the deep ocean: the special value of ocean bottom pressure for monitoring the large-scale, deep-ocean circulation. Progress in Oceanography, 161. pp. 19-46. ISSN 0079-6611 doi: https://doi.org/10.1016/j.pocean.2018.01.011 Available at https://centaur.reading.ac.uk/87015/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1016/j.pocean.2018.01.011 To link to this article DOI: http://dx.doi.org/10.1016/j.pocean.2018.01.011 It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1016/j.pocean.2018.01.011 To link to this article DOI: http://dx.doi.org/10.1016/j.pocean.2018.01.011 Publisher: Elsevier All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . A B S T R A C T We show how, by focusing on bottom pressure measurements particularly on the global continental slope, it is possible to avoid the “fog” of mesoscale variability which dominates most observables in the deep ocean. This makes it possible to monitor those aspects of the ocean circulation which are most important for global scale ocean variability and climate. We therefore argue that such measurements should be considered an important future component of the Global Ocean Observing System, to complement the present open-ocean and coastal elements. Our conclusions are founded on both theoretical arguments, and diagnostics from a ﬁne-resolution ocean model that has realistic amplitudes and spectra of mesoscale variability. These show that boundary pressure variations are coherent over along-slope distances of tens of thousands of kilometres, for several vertical modes. We illustrate the value of this in the model Atlantic, by determining the time for boundary and equatorial waves to complete a circuit of the northern basin (115 and 205 days for the ﬁrst and second vertical modes), showing how the boundary features compare with basin-scale theoretical models, and demonstrating the ability to monitor the meridional overturning circulation using these boundary measurements. Finally, we discuss applicability to the real ocean and make recommendations on how to make such measurements without con- tamination from instrumental drift. A window on the deep ocean: The special value of ocean bottom pressure for monitoring the large-scale, deep-ocean circulation Chris W. Hughesa,b,⁎, Joanne Williamsb, Adam Blakerc, Andrew Cowardc, Vladimir a University of Liverpool, UK b National Oceanography Centre, Liverpool, UK c National Oceanography Centre, Southampton, UK a University of Liverpool, UK b National Oceanography Centre, Liverpool, UK c National Oceanography Centre, Southampton, UK Contents lists available at ScienceDirect Contents lists available at ScienceDirect ⁎ Corresponding author at: University of Liverpool, UK. 1 Now at Hydrometeorological Center of Russia, and University of Reading, UK. E-mail address: cwh@liv.ac.uk (C.W. Hughes). Central Archive at the University of Reading Reading’s research outputs online Progress in Oceanography 161 (2018) 19–46 E-mail address: cwh@liv.ac.uk (C.W. Hughes). https://doi.org/10.1016/j.pocean.2018.01.011 Received 18 October 2017; Received in revised form 16 January 2018; Accepted 17 January 2018 Available online 30 January 2018 0079-6611/ © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ⁎ Corresponding author at: University of Liverpool, UK. 1 Now at Hydrometeorological Center of Russia, and University of Re 2. Model descriptions The model diagnostics are mainly from the National Oceanography Centre run N006 of the 1/12° global NEMO model. This is a single integration of NEMO v3.6 encompassing years 1958–2012 (inclusive), though it has more recently been extended to 2015. The model is forced by the Drakkar Surface Forcing data set version 5.2, which supplies surface air temperature, winds, humidity, surface radiative heat ﬂuxes and precipitation (Dussin et al., 2014; Brodeau et al., 2010). To prevent excessive drifts in global salinity due to deﬁciencies in the fresh water forcing, sea surface salinity is relaxed toward climatology with a piston velocity of 33.33 mm day−1 psu−1. Sea ice is represented by the Lou- vain-la-Neuve Ice Model version 2 (LIM2) sea-ice model (Timmerman et al., 2005). Bottom topography is represented as partial steps and bathymetry is derived from ETOPO2 (U.S. Department of Commerce, 2006). Climatological initial conditions for temperature and salinity were taken in January from PHC2.1 (Steele et al., 2001) at high lati- tudes, MEDATLAS (Jourdan et al., 1998) in the Mediterranean, and Levitus et al. (1998) elsewhere. More details of the model and valida- tion of its representation of the AMOC can be found in Moat et al. (2016). To put these numbers into context with the mesoscale variability, Fig. 1 (top) shows the standard deviation of sea level from 20 years of satellite altimetry (trend, annual and semiannual cycle removed). This is deliberately plotted using a saturated colour scale, in order to show how few regions approach variability of only a few centimetres. It is not just the amount of variability that matters, but also its spectrum in both space and time. For the frequency spectrum, given a certain standard deviation, it is helpful for climate monitoring if the variability is dominated by the highest frequencies, since high fre- quencies can be averaged out more eﬀectively if sampling frequency is high enough. Fig. 1 (bottom), updated from Hughes and Williams (2010), illustrates the variability in the shape of the spectrum in a re- latively intuitive way: it simply shows the colours which would be perceived if the spectrum of sea level variability was translated to a light spectrum, with periods 2–24 weeks mapped on to the visible range, corresponding to wavelengths of 380–760 nm. 1. Introduction Progress in Oceanography 161 (2018) 19–46 to a typical requirement for local sea level which is measured in dec- ades (Hughes and Williams, 2010). presence of complex topography. Our purpose in this paper is to illustrate the value of ocean bottom pressure measurements, and to make the case that such measurements, in particular regions, should be a major part of a global ocean observing system. In the following sections, we will see that bottom pressure is quieter than sea level, and has a “whiter” characteristic spectrum (meaning that it will appear more blue in the spectral colour plots). We will also ﬁnd that mesoscale variability is strongly damped by steep topography, and give a theoretical reason why that should be expected. Focusing on the steep topography of the continental slope, we will show how this allows us to see global scale ocean processes and to access diagnostics which test simple theoretical representations of the global ocean circulation, particularly the AMOC. The Argo ﬂoat program sampling is now suﬃciently dense that a similar noise reduction is apparent in estimates of upper ocean heat content (Wunsch, 2016), although the sampling is signiﬁcantly poorer than altimetry, and even altimetry leaves signiﬁcant room for im- provement with the present nadir-sampling systems only measuring thin lines along the ocean surface. These systems are providing very important inventory information; how much water there is in the ocean and in diﬀerent density classes. What they cannot generally do is pro- vide useful transport estimates. To the extent that the ocean is in geostrophic balance, pressure and sea level represent naturally integrating variables, pressure diﬀerence at a particular latitude and depth being proportional to the integrated horizontal current perpendicular to the section. Unfortunately, to ob- tain a useful integral it must be from boundary to boundary, otherwise the end points are likely to be in regions of strong mesoscale variability and the integral will still be dominated by the mesoscale (Wunsch, 2008). For sea level this is a problem because the boundaries are in shallow water where locally-driven dynamics can dominate, as the di- rect eﬀect of wind stress on sea level is inversely proportional to the depth. Viscous processes also become important in shallow water, so geostrophic balance does not hold. Furthermore, the boundaries are the most troublesome region for satellite altimetry. 1. Introduction Here, special measures must be taken to apply the standard path-length corrections to alti- metry, tidal variability is typically larger and more complicated than in the open ocean, and temporal aliasing is more important (Vignudelli et al., 2011). We will make these arguments based on diagnostics from a ﬁne resolution global ocean model. While we will only illustrate these ar- guments with one model, we have investigated a number of diﬀerent models with a range of resolutions and architectures, and the general ﬁndings we present are robust. Section 2 describes the model runs, and general aspects of the data analysis, Section 3 discusses the variability and spectra of model sea level and bottom pressure, demonstrating how diﬀerent bottom pres- sure is and describing some general features. Section 4 presents a the- oretical argument explaining why the mesoscale signal is so strongly suppressed in bottom pressure, particularly over steep topography. Section 5 focuses on the Atlantic continental slope, illustrating the striking coherence of dynamical signals over large distances, and making some links to theoretical ideas and simple models, particularly in the context of the AMOC. Finally, in Section 6 we discuss how this can be applied in the real ocean, highlighting the capabilities and de- ﬁciencies of present technology and some possibilities for the future. To give an idea of the size of the signals we are interested in, a good rule of thumb is that, at mid-latitudes where the Coriolis parameter f is about − 10 4 s−1, a sea level diﬀerence of 1 cm (or a pressure diﬀerence of 1 mbar = 1 hPa) reﬂects a transport of 1 Sv (Sv stands for sverdrup, a unit of 106 m3 s−1), on the assumption that the associated geostrophic ﬂow penetrates to 1000 m depth. This is the transport associated with about a 5% change in the Atlantic meridional overturning circulation (AMOC), for example, and is the size of change we might aspire to monitor if changes in global ocean circulation are considered. 1. Introduction measurements are one such variable, but can be diﬃcult to interpret because the integral involves the entire Earth system, not just the ocean. Somewhat more focused is the Earth’s gravity ﬁeld as measured by the GRACE satellite mission. This has provided extremely valuable information about variations in total ocean mass and the sources of water responsible for these changes (Dieng et al., 2015) and is a crucial element of the ocean and Earth observation system, although it does suﬀer from some of the same ambiguities as Earth rotation, the inﬂu- ence on long time scales of long term plastic deformation of the earth, particularly with respect to the pole tide, remains contentious (Wahr et al., 2015), and it is limited to providing relatively coarse resolution information on ocean bottom pressure variations. In monitoring the global ocean circulation we are faced with a major challenge in the form of the wide disparity in length scales in- volved. A recent review (Wunsch, 2016) highlighted how this challenge limits what can be said about large-scale, integral properties of the ocean. In essence, the issue is that ocean currents are dominated by mesoscale variability (Ferrari and Wunsch, 2009), with natural length scales of order 10–100 km, so that any one in situ measurement is only representative of a very small region of the ocean. Quantiﬁcation of mapping accuracy requires a knowledge of the frequency-wavenumber spectrum of ocean variability. To this end, Wortham and Wunsch (2014) have made an eﬀort to characterise this spectrum as seen in the primary physical variables of pressure (and sea level), velocity and density (or temperature and salinity). Their spectrum varies regionally, and most of this variation is designed to reﬂect the varying character- istics of mesoscale eddies around the world. A second way to obtain large-scale information is to have good sampling over the entire ocean. In this respect, satellite altimetry is a particularly powerful system, with suﬃcient sampling to average out most of the mesoscale variability. Once the trend and seasonal cycle has been removed, the measured variability in global mean sea level has a standard deviation of only 2.5 mm, a level of noise which allows for detection of a trend of 1 mm yr−1 from only 2 years of data, compared One method of obtaining large-scale information is to use a variable which intrinsically integrates some property. Earth rotation C.W. Hughes et al. 2. Model descriptions There is no atmospheric pressure forcing, so the sea level can be considered to be equivalent to the inverse barometer-corrected dynamic topography provided in the satellite data. The output data are averaged over 5-day periods which start at the beginning of each year, giving × 73 5-day means per year (the last day of leap years is thus not saved). The model is volume conserving (Boussinesq), so we calculate bottom pressure from sea level (multiplied by acceleration due to gravity and surface density) plus an integral of gravity times density using hydro- static balance exactly as implemented in the model, then subtract oﬀ the global area-averaged pressure at each time to enforce mass con- servation. The corresponding adjustment to global area-averaged sea level was also made, as described by Greatbatch (1994). More detailed explanation of these colour plots and their scale bars is given in the appendix, but they should not be interpreted in a very quantitative way. For present purposes, the value of these colour spectrum plots is as a qualitative condensation of a combination of information about amplitude of variability (brightness) and spectral shape (colour), which we can also exploit when looking at model di- agnostics. Blue colours tend to represent relatively higher variability at high frequencies, and similar colours are often representative of similar processes, but more detailed analysis is needed to conﬁrm this. We will not attempt similar diagnostics for the spatial spectrum because, as we will ﬁnd, bottom pressure is strongly inﬂuenced by topography, so the along-slope and across-slope variations can be very diﬀerent, something which is diﬃcult to account for with wavenumber spectra in the The nominal 1/12° resolution is on the tripolar ORCA12 grid, which is regular in longitude south of 20°N, with Mercator latitude spacing 20 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. k id ll I h h h i h h id i d 1959 2012 i l i f b i f . 1. Sea level variability from satellite altimetry, after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation over 22 years, 1993–2014 inclusiv a saturated scale, and (bottom) the spectrum of variability (from 1024 weeks from 1993 to mid-2012) as described in the text, the appendix, and in Hughes and Williams (2010 Fig. 1. 3. Spectral analyses In the case of the Arctic, the colour is the blue which we will usually come to associate with wind stress forcing, and the entire deep Arctic basin has extremely well correlated variability (not shown), consistent with the barotropic mode ﬁrst noted by Hughes and Stepanov (2004) and nicely elucidated by Fukumori et al. (2015). The Mediterranean is similar, though a diﬀer- ence in character between the eastern and western basins is apparent. The well-known regions of high barotropic variability in the subpolar North Paciﬁc, the south east Paciﬁc and Indian Ocean sectors of the Southern Ocean (Chao and Fu, 1995; Fu and Davidson, 1995) show up clearly. In the Argentine Basin, the range of colours is indicative of the complex interplay of mesoscale eddies and barotropic basin modes which is known to occur here (Fu et al., 2001; Weijer et al., 2007; Hughes et al., 2007; Fu, 2007). Perhaps the most obvious feature, though, is in the Caribbean Sea, which shows up as bright red in the spectral colour plot. It was this feature of the spectrum, repeated across a wide range of ocean models, which led to the discovery of the Rossby whistle: a 120-day baroclinic basin mode in the region, excited by baroclinic instability of the Caribbean Current (Hughes et al., 2016). This clear, coherent mode dominates the regional bottom pressure, despite having a standard deviation of less than 2 mbar. The spectrum plots in Figs. 1 and 2 focus on timescales associated with mesoscale variability, which is appropriate as that is the main source of “noise” with respect to our aim of measuring large-scale variability. As noted by Hughes and Williams (2010), we see that in many regions the coastal and shelf sea variability has a blue tinge in the spectrum, and is separated by a region of lower variability near the top of the continental slope from a diﬀerently-coloured spectrum of high variability oﬀshore. This is particularly apparent at mid to high lati- tudes near the western boundary of the North and South Atlantic, but also occurs elsewhere (as many of the features described in this section are at quite small scales, we recommend that the reader uses the elec- tronic version of this paper to zoom in on the regions being discussed). As a reminder, blue represents spectral slopes signiﬁcantly shallower than − f 2, so “white noise” would appear intensely blue. 2. Model descriptions for example the plume of high variability stretching to the west and steadily north from the tip of South Africa in the model which suggests a too-regular path of Agulhas rings penetrating into the South Atlantic. However, overall, the similarity in both amplitude and spectral shape (colour) is suﬃcient to give us conﬁdence that the model can be used to investigate the inﬂuence of mesoscale variability on observation of large-scale processes. factor = β 0.12 as described in that paper (this is of importance mainly for the variability at periods of a few days or less, including the tides). It is forced using 6-hourly atmospheric pressure and wind stress from the ERA-40 reanalysis (Uppala et al., 2005) as well as 17 components of diurnal and semidiurnal tidal forcing. For present purposes, tides are removed by ﬁltering before forming 5-day means. We use a time series of 1080 5-day means, which covers approximately 14.8 years. Ideally we would have a single-layer model with exactly the same conﬁguration and forcing as the NEMO run, but this is not available. However, there are some advantages to using AGBOM, as it ﬁlls in some missing physics such as tides and the response to varying air pressure, which are absent from the NEMO run. As we will see, the AGBOM re- sults do appear consistent with NEMO in the relevant frequency ranges, which testiﬁes to the robustness of the barotropic modelling, since the model conﬁgurations and discretisations are quite diﬀerent. We expect the main inﬂuence of atmospheric pressure variability on bottom pressure to be seen on time scales shorter than a month (Stepanov and Hughes, 2006), and this expectation is consistent with the spectra we present in the next section. For comparison, Fig. 3 repeats these sea level diagnostics for ocean bottom pressure. We report bottom pressure in mbar, equivalent to hPa. Applying hydrostatic balance as a scaling factor, 1 mbar pressure is equivalent to approximately 1 cm of sea level (the scaling can vary by about 2–3% depending on the local water density and gravity used). Note that, for standard deviation, the colour scale amplitude has been halved compared to the sea level plots, but still the area occupied by saturated scale values has markedly reduced. Similarly, for the spectral colour, the brightness of the plot is exaggerated compared to Fig. 3. Spectral analyses In this section we start by examining and discussing global maps related to the amplitude and spectra of sea level variability, showing how it is partitioned into signals related to steric and mass (bottom pressure) variability. We discuss the known forms of variability, and draw attention to the relative quietness of bottom pressure, especially along the continental slope. We then present more detailed spectral analyses, averaged over diﬀerent regions chosen based on what we have learned from the maps. These highlight the fact that the part of the spectrum which represents mesoscale variability is much weaker in bottom pressure than in sea level, especially on the steep continental slopes. Here, we focus particularly on the spatial distribution of the varia- bility and its spectrum. In addition to the tropics, we see a very quiet region along the eastern boundary continental slope. Compared to sea level (Figs. 1 and 2) we see an even more marked and, especially in the Atlantic, a broader minimum in bottom pressure variability along the western boundary continental slope. The spectrum is generally less “red” than sea level (hence the overall blue colour), with the exception of regions of intense eddy activity where the eddy variability also dominates bottom pressure. Fig. 2 shows the same diagnostics, standard deviation and spectrum of sea level, from the NEMO model as were shown from altimetry in Fig. 1. It is immediately clear that the model is performing well in re- producing much of the variability of the real ocean, not just in ampli- tude but also in spectral characteristics. The two ﬁgures are not pre- cisely comparable, being based on diﬀerent lengths of time series, with diﬀerent temporal sampling (5-day means for the model, nominal 20- day low-pass ﬁlter for altimetry) and diﬀerent spatial ﬁltering (de- termined by resolution and parameterised friction in the model, and by a compromise between satellite track spacing, observed scales of variability, instrumental noise and high frequency ocean processes for the altimetry). Nonetheless, many features of the observations are re- produced in the model at very similar amplitudes. p Several features stand out in the bottom pressure variability. There is often evidence of strong topographic inﬂuence, even in regions where the colour of the spectrum suggests a mesoscale inﬂuence, with entire sub-basins having a rather constant colour. 2. Model descriptions Sea level variability from satellite altimetry, after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation over 22 years, 1993–2014 inclusiv on a saturated scale, and (bottom) the spectrum of variability (from 1024 weeks from 1993 to mid-2012) as described in the text, the appendix, and in Hughes and Williams (2010 Fig. 1. Sea level variability from satellite altimetry, after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation over 22 years, 1993–2014 inclusive, on a saturated scale, and (bottom) the spectrum of variability (from 1024 weeks from 1993 to mid-2012) as described in the text, the appendix, and in Hughes and Williams (2010). Fig. 1. Sea level variability from satellite altimetry, after subtraction of a linear trend, annual and semiannual cycle, showing (top) standar on a saturated scale, and (bottom) the spectrum of variability (from 1024 weeks from 1993 to mid-2012) as described in the text, the a chosen to make square grid cells. In the northern hemisphere, the grid distorts to avoid producing a pole in the Arctic Ocean, instead having two poles over land. Over ocean points, the linear resolution (square root of grid cell area) thus varies between 9.27 km at the equator, 2.09 km at 77°S (the southernmost point), and 1.75 km in the Canadian Arctic. period 1959 to 2012 inclusive, after subtraction of a mean, annual cycle, semiannual cycle and linear trend, determined by simultaneous least squares ﬁtting. The exception is the spectral colour plots, which use a period from 1980 to January 2008 (this gives a time series length of 2048 values, representing 28 years and 20 days). In addition to the NEMO data, we also show some diagnostics from the Advanced Global Barotropic Ocean Model (AGBOM). This is a global ocean model with constant water density, at 0.25° resolution, based on that described in Stepanov and Hughes (2004). It uses a simple parameterisation of self-attraction and loading with a proportionality Preliminary analyses showed that annual and semiannual signals frequently show clearly above the background spectrum, but higher harmonics generally do not, though they can with suﬃcient averaging of spectra. Most of the diagnostics presented here are from the 54-year 21 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. 2. Model descriptions 2; the eﬀect is equivalent to multiplying the spectral power by a factor of 10 (i.e. the time series are multiplied by 10 before calculating the col- ours), which is why the Arctic and Southern Ocean, among other re- gions, are so much brighter than in the sea level spectrum shown in Fig. 2. The quieter nature of bottom pressure has been noted before, in both coarse resolution (Vinogradova et al., 2007) and eddying models (Bingham and Hughes, 2008a), and the remarkably quiet nature of much of the tropics has led to ocean bottom pressure measurements being used to determine the annual cycle of mass exchange between ocean and land (Hughes et al., 2012; Williams et al., 2014; Hsu and Velicogna, 2017). 3. Spectral analyses We interpret this blue colour on the shelf as the result of the strong inﬂuence of wind stress forcing in this region. The minimum of variability near the top of the continental slope is also visible in the standard deviation. This drop- oﬀin eddy amplitude near the western boundary is referred to by Zhai et al. (2010) as the “eddy graveyard”. We have interpreted much of the bottom pressure variability in terms of barotropic modes, but it is not clear that bottom pressure has to be dominated by barotropic dynamics (by which we mean dynamics associated with depth-independent pressure variations). Baroclinic modes also have a bottom pressure signature, especially over weakly sloping topography and close to the equator. In addition, topography There are some clear diﬀerences between model and observations, 22 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. uses mode coupling between barotropic and baroclinic modes, so that arotropic signals in bottom pressure can be the result of baroclinic ocesses, as in the Caribbean Sea mode. Such mode coupling can also region thus represent a combination of baroclinic signals and barotropi signals induced by the baroclinic variability. A simple example of such mode coupling is a baroclinic equatoria g. 2. Sea level variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on urated scale, and (bottom) the spectrum of variability from a 28-year subset starting in 1980, as described in the text, the appendix, and in Hughes and Williams (2010). Fig. 2. Sea level variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on saturated scale, and (bottom) the spectrum of variability from a 28-year subset starting in 1980, as described in the text, the appendix, and in Hughes and Williams (2010). Fig. 2. Sea level variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on a saturated scale, and (bottom) the spectrum of variability from a 28-year subset starting in 1980, as described in the text, the appendix, and in Hughes and Williams (2010). Fig. 2. 3. Spectral analyses 4, integral, must tend to zero, meaning density changes do not sig niﬁcantly disrupt the relationship between sea level and bottom pres g. 3. Bottom pressure variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation a saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980, shown brighter than the equivalent sea level spectrum (power increased by a factor o ). W. Hughes et al. Progress in Oceanography 161 (2018) 19–4 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 3. Bottom pressure variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviat on a saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980, shown brighter than the equivalent sea level spectrum (power increased by a facto 10). Fig. 3. Bottom pressure variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on a saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980, shown brighter than the equivalent sea level spectrum (power increased by a factor of 10). Fig. 3. Bottom pressure variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semi on a saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980, shown brighter than the equivalent se 10). integral, must tend to zero, meaning density changes do not sig- niﬁcantly disrupt the relationship between sea level and bottom pres- sure. component, cause a coastal signal which is seen in bottom pressure but not in steric sea level variability. The latter point is conﬁrmed in Fig. 4, which shows the variability and spectral colour of the steric component of sea level variability (i.e. the part that is attributable to water column density changes rather than bottom pressure changes). The low varia- bility around coasts and most shelf sea regions conﬁrms that the variability here is predominantly barotropic, with sea level and bottom pressure varying in step as they would in a homogeneous ocean. 3. Spectral analyses Sea level variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannu saturated scale, and (bottom) the spectrum of variability from a 28-year subset starting in 1980, as described in the text, the appendix region thus represent a combination of baroclinic signals and barotropic signals induced by the baroclinic variability. causes mode coupling between barotropic and baroclinic modes, so that barotropic signals in bottom pressure can be the result of baroclinic processes, as in the Caribbean Sea mode. Such mode coupling can also result from nonlinear dynamics. The intense blue bands in the spectrum of sea level either side of the Paciﬁc equator (Figs. 1 and 2) are asso- ciated with Tropical Instability Waves (Legeckis, 1977). Farrar (2011) has recently shown how these waves, which have periods of about 30 days, can radiate beyond the permitted latitude range for baroclinic Rossby waves of that period by partially converting to barotropic waves. The interesting colours in the bottom pressure spectrum in this causes mode coupling between barotropic and baroclinic modes, so that barotropic signals in bottom pressure can be the result of baroclinic processes, as in the Caribbean Sea mode. Such mode coupling can also result from nonlinear dynamics. The intense blue bands in the spectrum of sea level either side of the Paciﬁc equator (Figs. 1 and 2) are asso- ciated with Tropical Instability Waves (Legeckis, 1977). Farrar (2011) has recently shown how these waves, which have periods of about 30 days, can radiate beyond the permitted latitude range for baroclinic Rossby waves of that period by partially converting to barotropic waves. The interesting colours in the bottom pressure spectrum in this A simple example of such mode coupling is a baroclinic equatorial Kelvin wave which, when it reaches the eastern boundary, will still cause a coastal sea level change despite the water depth becoming too shallow for this to be a baroclinic mode (i.e. shallower than the ther- mocline depth). This may be what we are seeing on the Atlantic coast of Africa, where the coastal bottom pressure variability has a pink hue in the spectral colour plot. Baroclinic processes, such as the Kelvin wave and associated coastal-trapped waves which may have a baroclinic 23 mponent, cause a coastal signal which is seen in bottom pressure but t in steric sea level variability. The latter point is conﬁrmed in Fig. 3. Spectral analyses In the limit of zero depth this is inevitable, as the steric signal, being a depth In comparison with Fig. 2, Fig. 4 shows that most of the blue-purple “haze” visible in the sea level spectral colour originates from the bottom pressure variability, and steric variability produces a sharper-looking plot. We can also see how, for example over many small islands and seamounts in the Indian and Paciﬁc ocean, the sea level spectrum is continuous from deep to shallow water, but the partitioning of 24 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. ariability shifts sharply from being dominated by steric variations in e deep water to bottom pressure variations in shallow water. It seems at such small topographic features oﬀer little dynamical obstacle to e propagation of open-ocean sea level signals to the coast, despite the witch between steric and bottom pressure-related sea level change. In ct, Williams and Hughes (2013) showed (in a diﬀerent high-resolution odel) that the relationship between small-island sea level and sur- unding open ocean sea level is practically unaﬀected by the existence the island topography, for 5-day mean values. From a single model run mode coupling and nonlinear processes mean that it is impossible to determine which variability is the result o purely barotropic processes. In Fig. 5, we show the variability of invers barometer-corrected sea level (or bottom pressure, which is equivalen in this model) from the AGBOM model. Since this model has constan density, all processes in the model are due to barotropic dynamics. Th spectral colour is almost everywhere blue to purple, supporting the ide that much of the bottom pressure variability, particularly at high lat tudes and in broad shelf seas, is the result of purely barotropic pro cesses. In contrast most of the more colourful features in Fig 3 are absen g. 4. Steric variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on urated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980. Fig. 4. Steric variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980. Fig. 4. 3. Spectral analyses Steric variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on a saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980. Fig. 4. Steric variability from 54 years of ocean model data (5-day means), after subtraction of a linear trend, annual and semiannua saturated scale, and (bottom) the spectrum of variability from a 28 year subset starting in 1980. variability shifts sharply from being dominated by steric variations in the deep water to bottom pressure variations in shallow water. It seems that such small topographic features oﬀer little dynamical obstacle to the propagation of open-ocean sea level signals to the coast, despite the switch between steric and bottom pressure-related sea level change. In fact, Williams and Hughes (2013) showed (in a diﬀerent high-resolution model) that the relationship between small-island sea level and sur- rounding open ocean sea level is practically unaﬀected by the existence of the island topography, for 5-day mean values. mean that it is impossible to determine which variability is the result of purely barotropic processes. In Fig. 5, we show the variability of inverse barometer-corrected sea level (or bottom pressure, which is equivalent in this model) from the AGBOM model. Since this model has constant density, all processes in the model are due to barotropic dynamics. The spectral colour is almost everywhere blue to purple, supporting the idea that much of the bottom pressure variability, particularly at high lati- tudes and in broad shelf seas, is the result of purely barotropic pro- cesses. In contrast, most of the more colourful features in Fig. 3 are absent From a single model run, mode coupling and nonlinear processes 25 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. om Fig. 5, strongly suggesting that these features are the result, either rectly or indirectly, of baroclinic processes. This includes the deep- ater tropics, and most of the eastern boundary regions. It is striking ow the low variability region in the barotropic model extends right ound the continental slope of the North Paciﬁc. This is not so dramatic the North Atlantic where even barotropic variability is signiﬁcant in he western basins. Though this variability is generally smaller than ther the steric variability seen in Fig. 3. Spectral analyses 4, or the total bottom pressure i bilit i Fi 3 it i bl i it d t th t t l b tt suggesting that purely barotropic processes are a signiﬁcant fraction o the total in this region. Putting these diagnostics together we see that pure barotropi variability accounts for much of the blue-white “wash” seen in the se level variability in Figs. 1 and 2, and especially on broad shelf seas though much of the shelf sea variability on narrower shelves and clos to the shelf break is, though locally barotropic, induced by baroclini processes. The blue-white “wash” is removed when focusing on steri l l h i Fi 4 R b i l f th b g. 5. Sea level or bottom pressure variability from 15 years of barotropic ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showin op) standard deviation on a saturated scale, and (bottom) the spectrum of variability, shown brighter than the equivalent sea level spectrum (power increased by a factor of 10). W. Hughes et al. Progress in Oceanography 161 (2018) 194 ig. 5. Sea level or bottom pressure variability from 15 years of barotropic ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showi top) standard deviation on a saturated scale, and (bottom) the spectrum of variability, shown brighter than the equivalent sea level spectrum (power increased by a factor of 10). Fig. 5. Sea level or bottom pressure variability from 15 years of barotropic ocean model data (5-day means), after subtraction of a linear trend, annual and semiannual cycle, showing (top) standard deviation on a saturated scale, and (bottom) the spectrum of variability, shown brighter than the equivalent sea level spectrum (power increased by a factor of 10). Fig. 5. Sea level or bottom pressure variability from 15 years of barotropic ocean model data (5-day means), after subtraction of a linea (top) standard deviation on a saturated scale, and (bottom) the spectrum of variability, shown brighter than the equivalent sea level sp suggesting that purely barotropic processes are a signiﬁcant fraction of the total in this region. from Fig. 5, strongly suggesting that these features are the result, either directly or indirectly, of baroclinic processes. This includes the deep- water tropics, and most of the eastern boundary regions. 3. Spectral analyses While the spectral colour maps are a useful qualitative indicator of the dynamics, they do not provide good quantitative information, and are limited to representing a particular subset of the frequencies which can be resolved. To address this issue, we use the maps to guide us in choice of regions to average over, in order to investigate the spectral shapes and amplitudes in more detail. The most obvious geographical division, in the deep ocean at least, is latitude, so in Fig. 6 we show power spectra averaged over the deep ocean in latitude bands, with a secondary partitioning based on how energetic the steric sea level variability is (as an attempt to isolate the inﬂuence of nonlinear me- soscale eddy variability). We deﬁne “quiet” as regions where the standard deviation of steric sea level variability is less than 6 cm, and “energetic” as where it is greater than 9 cm. It is also worth remarking that, at periods longer than 10 years, the NEMO bottom pressure spectra start to slope up again. Given that this is not the case for wind stresses (not shown), and that barotropic pro- cesses have no memory on timescales longer than a few months, we would not expect this to be the case in AGBOM, though the time series is too short to conﬁrm this. This decadal-to-multidecadal bottom pres- sure variability may be related to climate-scale ocean dynamics, or to baroclinic ocean model adjustment processes. Since we have identiﬁed the continental slope as an important re- gion, Fig. 7 shows the spectra averaged over diﬀerent regions deﬁned by topography. Here, shallow means all regions shallower than 200 m, and deep means regions deeper than 3200 m as before. For the range of depths in between, however, we only include points which are on the global continental slope, excluding seamounts and isolated islands. This region is deﬁned by starting at a latitude on the Atlantic coast of the USA where the continental slope happens to be monotonic, and fol- lowing depth contours from this section until they close after passing round Europe, Africa, Asia and the Americas. Some deep contours also pass round Australia and New Zealand, and some shallow contours enter and pass round the Arctic and Mediterranean (more detail in the Atlantic is given later in this paper, including a map of the Atlantic part of the continental slope, Fig. 8). 3. Spectral analyses The deﬁnition of the continental slope is thus limited to very long contours in the depth range 200–3200 m. Fig. 6 contains a lot of information. Focusing ﬁrst on sea level (red) at latitudes equatorward of 30°, we see that the spectrum is divided into two regions with a very clear breakpoint. The higher frequency part of the spectrum is often a very straight line, indicating a power law. However, the gradient of the line varies, between almost −4 at the lowest latitudes to about −3 at 15–30°, and becomes shallower still at higher latitudes. As noted above, the annual cycle and its harmonics are very clear with such averaging, with harmonics as high as 5 cycles per year being clearly visible in some cases. As we move to higher latitudes, a diﬀerence between the quiet and energetic regions becomes more apparent. In the quiet regions, the breakpoint in the spectrum is still visible out to latitudes of 45–60°, but in the energetic regions it becomes more of a smooth, rounded transi- tion rather than a breakpoint. This suggests that the rounded spectrum is indicative of the more nonlinear processes in the higher latitude energetic regions, whereas the breakpoint is related to more linear processes. Although not especially energetic in terms of sea level, the shallow region is the most energetic in bottom pressure. Because the deﬁnition of “energetic” is based on steric sea level, and steric signals are small in shallow water, there are no points which are both energetic and shallow. As expected, the shallow signals are dominated by bottom pressure, but at the lower frequencies a large fraction of this represents a locally barotropic signal which is induced by baroclinic variability, as it is absent in the purely barotropic AGBOM. Finally, at the highest latitudes, the breakpoint is lost and, parti- cularly in the Arctic, the spectrum becomes much more noisy. This is informative in itself. The spectra are formed by area-weighted aver- aging of the modulus-squared fourier transforms of the time series from each grid point in each region. For a single gridpoint, this would pro- duce a very noisy spectrum, but for multiple gridpoints the diﬀerent realisations of time series with the same (or similar) underlying spec- trum, but diﬀerent phasing of the components, averages out that noise. 3. Spectral analyses amplitude of this mesoscale variability and barotropic shelf sea varia- bility means that, without spatial averaging, sea level is a poor monitor of large-scale ocean circulation except perhaps in a few very quiet re- gions near eastern boundaries and near the poles. g Bottom pressure can be signiﬁcantly quieter than sea level. Even in bottom pressure the mesoscale variability is too large for climatological monitoring in many regions, especially in the western basins. We have seen that steep topography alone is not suﬃcient to suppress such variability, as the sea level variability over many small tropical islands and shallow seamounts is almost the same as in nearby deep water. The continental slope, however, is both steep and long. These are the characteristics needed to suppress mesoscale variability, as we will discuss in the next section. The result is illustrated in Fig. 3, which shows strong suppression of variability over the continental slope. Combined with Fig. 5, which suggests that a signiﬁcant fraction of even the small variability seen over these regions results from (presumably large-scale) barotropic processes, this suggests that bottom pressure on the continental slope is the most promising variable to monitor in order to measure large-scale, climatological changes in the ocean circulation. Turning now to the other curves in Fig. 6, blue shows NEMO bottom pressure, and black shows the AGBOM sea level or bottom pressure. We see that bottom pressure is much quieter than sea level at low fre- quencies, but approaches sea level at high frequencies where barotropic processes become dominant. In fact, the barotropic model spectra are often more energetic than the NEMO bottom pressure spectra at the very highest frequencies (periods shorter than about two weeks), a fact which may be attributable to the eﬀect of atmospheric pressure forcing (compare Stepanov and Hughes (2006), who ﬁnd that the inverse barometer correction ceases to be a good approximation at these timescales on a basin scale). Otherwise, the AGBOM and NEMO bottom pressure spectra tend to be very similar in quiet regions. In the more energetic regions, however, the diﬀerence between NEMO and AGBOM bottom pressure spectra tends to have a shape reﬂecting the sea level spectra, suggesting that bottom pressure is being inﬂuenced by the energetic mesoscale variability as we deduced from the spectral colour maps. 3. Spectral analyses It is striking how the low variability region in the barotropic model extends right round the continental slope of the North Paciﬁc. This is not so dramatic in the North Atlantic where even barotropic variability is signiﬁcant in the western basins. Though this variability is generally smaller than either the steric variability seen in Fig. 4, or the total bottom pressure variability in Fig. 3, it is comparable in magnitude to the total bottom pressure variability on much of the western continental slope, from Fig. 5, strongly suggesting that these features are the result, either directly or indirectly, of baroclinic processes. This includes the deep- water tropics, and most of the eastern boundary regions. It is striking how the low variability region in the barotropic model extends right round the continental slope of the North Paciﬁc. This is not so dramatic in the North Atlantic where even barotropic variability is signiﬁcant in the western basins. Though this variability is generally smaller than either the steric variability seen in Fig. 4, or the total bottom pressure variability in Fig. 3, it is comparable in magnitude to the total bottom pressure variability on much of the western continental slope, Putting these diagnostics together we see that pure barotropic variability accounts for much of the blue-white “wash” seen in the sea level variability in Figs. 1 and 2, and especially on broad shelf seas, though much of the shelf sea variability on narrower shelves and close to the shelf break is, though locally barotropic, induced by baroclinic processes. The blue-white “wash” is removed when focusing on steric sea level, as shown in Fig. 4. Remembering our rule of thumb sug- gesting we want to measure large-scale signals of order 1 cm, the large 26 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. waves are limited to frequencies below a strongly latitude dependent cutoﬀfrequency. This frequency is given by = f π βR 2 / 1, where R1 is the baroclinic Rossby radius and β is the meridional gradient of the Coriolis parameter. The green boxes in Fig. 6 show the range of this cutoﬀ frequency for each latitude range, based on zonally averaged values of the Rossby radius taken from Chelton et al. (1998). Where a clear breakpoint is visible, it does indeed lie in this band. 3. Spectral analyses However, in the Arctic, the time series are all strongly correlated, so there are no diﬀerent realisations, and no averaging eﬀect is found. The noisiness of the spectra is an indication of the number of spatial degrees of freedom in the variability, so large-scale processes (or small regions) will result in noisier spectra. We see this eﬀect at the high frequency end of many of the curves, where large-scale barotropic processes be- come dominant. In the deep ocean we see a pattern consistent with Fig. 6. The pure barotropic AGBOM spectrum is indiﬀerent to whether or not there is energetic steric variability, but some fraction of the steric variability is seen in the NEMO bottom pressure, lifting the spectrum above the barotropic spectrum in energetic regions, but much less so in quiet regions. Over the continental slope, a more interesting result arises. Here, the NEMO bottom pressure is comparable to that in quiet deep ocean regions, whatever the local steric variability. Particularly at frequencies above about 0.3 cycles per year (periods shorter than about 3 years) bottom pressure on the slope does not seem to be signiﬁcantly Lin et al. (2008) and Hughes and Williams (2010) attributed the breakpoints to Rossby wave processes. In linear dynamics, Rossby 27 6. Spectra of NEMO sea level (red), NEMO bottom pressure (blue) and barotropic model sea level (black) averaged over the deep ocean (depth > 3200 m) in latitude ban ctra are further divided according to the amplitude of steric sea level variability in NEMO, with “quiet’ deﬁned as less than 6 cm standard deviation, and “energetic” as more tha dard deviation. Green bars mark the range of linear baroclinic cut-oﬀfrequencies for each latitude range. Some representative power laws are shown in the bottom left plo rpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) W. Hughes et al. Progress in Oceanography 161 (2018) Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 6. Spectra of NEMO sea level (red), NEMO bottom pressure (blue) and barotropic model sea level (black) averaged over the deep ocean (depth > 3200 m) in latitude bands. The spectra are further divided according to the amplitude of steric sea level variability in NEMO, with “quiet’ deﬁned as less than 6 cm standard deviation, and “energetic” as more than 9 cm standard deviation. 3. Spectral analyses Green bars mark the range of linear baroclinic cut-oﬀfrequencies for each latitude range. Some representative power laws are shown in the bottom left plot. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) 28 g. 7. Spectra of NEMO sea level (red), NEMO bottom pressure (blue) and barotropic model sea level (black) averaged over the ocean in regions deﬁned by topography. Shallow is ions shallower than 200 m, deep is all regions deeper than 3200 m, and in between is the continental slope (see text for a more detailed deﬁnition). The top and bottom rows sho ctra further divided according to the amplitude of steric sea level variability in NEMO, with deﬁnitions as in Fig. 6. The green box simply provides a constant reference level f mparison. Some representative power laws are shown in the top left plot. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version s article.) W. Hughes et al. Progress in Oceanography 161 (2018) 19– Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 7. Spectra of NEMO sea level (red), NEMO bottom pressure (blue) and barotropic model sea level (black) averaged over the ocean in regions deﬁned by topography. Shallow is all regions shallower than 200 m, deep is all regions deeper than 3200 m, and in between is the continental slope (see text for a more detailed deﬁnition). The top and bottom rows show spectra further divided according to the amplitude of steric sea level variability in NEMO, with deﬁnitions as in Fig. 6. The green box simply provides a constant reference level for comparison. Some representative power laws are shown in the top left plot. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) wb is vertical velocity at the bottom, and H is ocean depth, with the sea ﬂoor at = − z H. If f is the Coriolis parameter ( = f ϕ 2Ωsin where Ω is the Earth’s angular rotation rate and ϕ is latitude), p is pressure, and ̂k is the upward unit vector, we can use geostrophic balance ̂ = × ∇ v k ρf p to substitute for vb in terms of pressure. 3. Spectral analyses Writing this in a coordinate system in which x is measured along the direction of the depth gradient (positive towards deep water) and y is along the depth contour, we obtain contaminated by eﬀects associated with local steric variability, al- though there is signiﬁcant variability above the purely barotropic dy- namics in AGBOM. This suggests that, on the continental slope, we have a window into processes beyond the purely barotropic or purely me- soscale. It is worth noting that even the purely barotropic processes (black) are less energetic over the slope than either the deep or shallow ocean. We will investigate this in more detail following a discussion of the theory, but we conclude this section by noting the clear message of these diagnostics. Although mesoscale variability may dominate pres- sures, currents and sea level in the ocean interior, a quite diﬀerent spectrum is apparent in bottom pressure in many regions, and espe- cially on the continental slope. Bottom pressure is a special variable which gives access to diﬀerent processes in diﬀerent frequency ranges than those accessible by other physical ocean variables. This opens up particular opportunities for ocean monitoring. = − ∂ ∂ = ∂ ∂ ∂ ∂ w u H x ρf p y H x 1 , b b b (1) (1) which leads to the scaling which leads to the scaling ∼ w u S, b b (2) (2) ∼ w u S, b b where S is the bottom slope, and ub is the bottom horizontal ﬂow to- ward deeper water, related to the along-slope gradient of bottom pressure. A second scaling for wb can be obtained from the vorticity equation. Consider the inviscid equation of motion, with Boussinesq and “con- ventional” approximations: 4. Theory of mesoscale suppression on the continental slope If we take ̂ ∇× k· of (3) we obtain the vertical vorticity equation: (6) ̂ ∇ + = ∇ + v k ω f ζ w f · ( ) ·[ ( )], (4) ̂ + ∇ + = ∇ + v k ω ζ f ζ w f · ( ) ·[ ( )], t (4) (4) where the terms on the right hand side derive from the time depen- dence, ∇ v f · , and ∇ v ζ · terms respectively in (4). Here, R is the Earth’s radius (arising from = f β R ϕ / tan , approximated as R for mid-latitude regions). where ζ is the vertical component of vorticity ω. Taking representative horizontal velocity to be U, horizontal eddy length scale L, vertical length scale H and time scale T, this allows us to derive a scaling for wb. First, we note that ω f/ scales as the Rossby number = U fL Ro / , so that, dimensionally, ̂ ̂ + ∼ ± k ω k f f (1 Ro). After an integral from top to bottom, the right hand side of (4) becomes − ± f w w ( )(1 Ro) a b , where wa is vertical velocity at the surface, which can be taken as zero for the mesoscale (the Ekman pumping velocity is much smaller than other vertical velocities in this scaling). Dimensionally, introducing a factor H on the left hand side to account for the vertical integral, (4) can then be written Setting these two scalings for bottom velocity, (2) and (6), to be equal, gives ⎜ ⎟ ⎜ ⎟ ∼⎛ ⎝ ± ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ u U H LS Tf L R (1 Ro) 1 , , Ro . b (7) (7) The three bracketed terms are precisely the terms which are assumed to be small in the quasigeostrophic approximation, so they are small for the (large) proportion of the mesoscale eddy ﬁeld which can be de- scribed by quasigeostrophic scaling. In more detail, for the terms to be small, timescales must be long compared to the inertial period and length scales short compared to the Earth’s radius, both of which are clearly true of mesoscale eddies. The third requirement, that the Rossby + ∇ + ∼ ± v H ζ f ζ fw [ · ( )] (1 Ro). 4. Theory of mesoscale suppression on the continental slope The argument for suppression of mesoscale energy in bottom pres- sure on the continental slope centres on the relationship between bottom pressure pb and vertical velocity w. The kinematic boundary condition on near-bottom velocity (neglecting a viscous boundary layer) is ∇ = − v H w · b b, where vb is the horizontal velocity at the bottom, ̂ ⎜ ⎟ + + × = −∇⎛ ⎝ + + ⎞ ⎠ v k ω v v f p ρ ( ) \| \| 2 Φ , t 0 2 (3) (3) where v is the horizontal velocity, = ∇× ω u is the relative vorticity where v is the horizontal velocity, = ∇× ω u is the relative vorticity 29 tic continental slope deﬁned as described in the text for the NEMO model. Dots indicate reference nodes every 500 km along the 2000 m contour, with circl ng-slope distance in units of 10,000 km. Colours indicate how points at other depths in the range 100–3200 m are mapped to the distance variable. (For inter colour in this ﬁgure legend, the reader is referred to the web version of this article.) l. Progress in Oceanography 161 (2 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. The Atlantic continental slope deﬁned as described in the text for the NEMO model. Dots indicate reference nodes every 500 km along the 2000 m contour, with circled n nting along-slope distance in units of 10 000 km Colours indicate how points at other depths in the range 100–3200 m are mapped to the distance variable (For interpre Fig. 8. The Atlantic continental slope deﬁned as described in the text for the NEMO model. Dots indicate reference nodes every 500 km along the 2000 m contour, with circled numbers representing along-slope distance in units of 10,000 km. Colours indicate how points at other depths in the range 100–3200 m are mapped to the distance variable. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) ⎜ ⎟ ⎜ ⎟ ∼⎛ ⎝ ± ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ w HU L Tf L R (1 Ro) 1 , , Ro , b (6) (curl of the 3D velocity), and Φ is the gravity potential. 4. Theory of mesoscale suppression on the continental slope t b (5) (5) caling the remaining terms and rearranging, this become Scaling the remaining terms and rearranging, this becomes 30 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. number be small, is eﬀectively the deﬁnition of mesoscale (or larger) rather than submesoscale. distance along the continental slope. The ability of the mesoscale to excite only ﬂows with much longer scales along than across depth contours therefore means that the mesoscale-induced along-topography ﬂows tend to be an integral of mesoscale inﬂuences over length scales typically much larger than the mesoscale itself. As this integral will tend to include forcing of both signs, the integration will usually have the eﬀect of reducing even this component in comparison with typical mesoscale pressure gradients. The exception to this is when the depth contours are closed over distances which are not large compared to the mesoscale length scale, so no averaging occurs. This explains why small islands do not act as a signiﬁcant barrier to bottom pressure signals, and why the suppression is limited in the case of the Caribbean Sea Rossby whistle mode (Hughes et al., 2016). In fact, closed depth contours (more strictly, closed contours of H f/ ) lead to a coupling of stratiﬁca- tion and bottom pressure which excites a barotropic circulation around the closed contours, enabling baroclinic disturbances to rapidly skip across such closed contour regions (Marshall, 2011). The averaging eﬀect of long contours is clearly an important consideration. If the terms in the ﬁnal brackets are small, then ub can only become comparable to U if the ﬁrst term H LS / is large. This term can be in- terpreted as the aspect ratio of the eddies divided by the slope, and clearly shows how steeper slopes result in smaller u U / b ratios. Another interpretation is that H LS / is the width of the “extended slope” divided by the eddy length scale, where “extended slope” means an imaginary slope with constant gradient S, and its width is the horizontal distance over which it extends from top to bottom of the water column. Note that the eddies can be smaller than the extended slope, and the total scaling can still be small if the Rossby number is small. 4. Theory of mesoscale suppression on the continental slope Thus, we see that, for mesoscale eddies, the relative suppression of bottom velocities in comparison with mid-water velocities is de- termined mainly by the Rossby number, the bottom slope, and the eddy scale. For example, a slope of 0.1 in water 2 km deep leads to = H LS / 1 at an eddy length scale of 20 km, meaning that bottom velocity sup- pression occurs for all eddies larger than 20 km at = Ro 1, or 2 km at = Ro 0.1. The continental slope can be about ﬁve times steeper or gentler than a slope of 0.1 in extreme cases, showing that a large fraction of the mesoscale energy cannot penetrate to the bottom over typical continental slopes. There are clearly processes with small length scales or large Rossby numbers which can penetrate to a steeply sloping bottom without attenuation (and even processes which are bottom- trapped), but the bulk of the mesoscale variability cannot do so. Near the foot of the continental slope, the topographic constraint weakens, and bottom-trapped Rossby waves are often observed (e.g. Hogg (2000)), particularly at periods shorter than about 10 days such that Tf 1/ is only moderately small. Over the abyssal plain, slopes may not be strong enough to constrain the bottom velocity so strongly. In these regions, the small bottom pressure variability indicated by NEMO within most subtropical gyres (Fig. 3) must be attributed to the fact that the energy input is at the surface, and the surface-intensiﬁed stratiﬁ- cation (especially at lower latitudes) results in weakened ﬂow below the thermocline. When the Rossby number is the main constraint we can write the mesoscale suppression factor as To summarise this scaling analysis in simple terms, the vorticity balance places a constraint on the vertical stretching of water columns, which limits the possible size of the vertical velocity at the bottom. The vertical velocity at the bottom is coupled to the horizontal velocity via the fact that ﬂow cannot pass through the seaﬂoor, so horizontal bottom velocities lead to larger vertical velocities where the slope is steep. For steep continental slopes and typical mesoscale conditions, these two scalings turn out to be incompatible if we use the same horizontal velocity scaling in each case. 5. Atlantic variability and continental slope bottom pressure For the sake of providing a concrete example, we will focus on the Atlantic Ocean, for which there is an established interest in the long- term climate variability particularly related to the AMOC. Studies of the Paciﬁc and Indian oceans would be of interest in their own right, but only one basin is necessary in order to establish the general principles. A coordinate system for the Atlantic continental slope is devised as shown in Fig. 8. This allows us to plot bottom pressure values in two dimensions, distance along the slope (colours), and depth (time is the third dimension). As this coordinate system is used in many subsequent plots, it is worth describing in some detail. For the sake of providing a concrete example, we will focus on the Atlantic Ocean, for which there is an established interest in the long- term climate variability particularly related to the AMOC. Studies of the Paciﬁc and Indian oceans would be of interest in their own right, but only one basin is necessary in order to establish the general principles. Furthermore, the inﬂuence of this mesoscale suppression is cumu- lative. Since most of the mesoscale variability is generated in the open ocean and propagates toward the western boundary (e.g. Zhai et al., 2010), once it encounters the continental slope and the mesoscale suppression scaling becomes of order 1 or less, this interaction will inﬂuence the eddy propagation so that it does not enter the shallower slope region at all. The raw scaling only applies to eddies generated over the slope. A coordinate system for the Atlantic continental slope is devised as shown in Fig. 8. This allows us to plot bottom pressure values in two dimensions, distance along the slope (colours), and depth (time is the third dimension). As this coordinate system is used in many subsequent plots, it is worth describing in some detail. The argument so far has been in terms of velocities and, in parti- cular, the horizontal velocity component that is constrained at the bottom, ub, is that perpendicular to depth contours; there is no con- straint on the velocity along depth contours as these have no associated vertical velocity. Translating this into pressures, the constraint on u U / b should be interpreted as a constraint tending to reduce the bottom pressure gradient along depth contours in relation to a typical mid- depth pressure gradient. 4. Theory of mesoscale suppression on the continental slope As a result, the horizontal bottom velocity (and hence the along-slope pressure gradient) at the bottom must be much smaller than the typical near-surface velocities (pressure gradients), and mesoscale variability is suppressed in bottom pressures on the continental slope. We have neglected the viscous boundary layer in this scaling on the grounds that it is not generally found to produce a large local perturbation in pressure. That is not to say that it is unimportant in indirectly shaping the larger-scale pressure ﬁeld. On the contrary, we suspect that it is important for models to represent the slopes and the frictional processes on those slopes as well as possible, and that this may be one of the main limitations of the present generation of ocean models. ± = = H LS HU fL S gHη f L S Ro (1 Ro) , 2 2 3 (8) (8) So far we have presented diagnostics concerning the size and the spectrum of variability, and made a scaling analysis. These suggest that we should see large-scale, coherent variability in bottom pressure on the continental slope, and hence that this would be a good place to monitor large-scale ocean circulation variability while minimising the mesoscale noise. In the next section, we will test this suggestion in more detail. where the last equality invokes geostrophic balance to express the ve- locity scale in terms of a sea level scale η (hence = gη f L Ro / 2 2). The factor ± (1 Ro) has been dropped from all except the ﬁrst form because, as this makes clear, there is no constraint on the scaling when the Rossby number approaches 1. From the ﬁnal scaling we see that, for a given amplitude of sea level variability, the suppression is sharply de- pendent on length scale. For example, for = η 0.1 m, we obtain = Ro 1/4 at mid-latitudes, and hence a mesoscale suppression factor of 1/4 for length scale 20 km. However, for a 40 km length scale, this suppression factor becomes 1/32. 5. Atlantic variability and continental slope bottom pressure Locally, this means that bottom pressure will be close to being a function of H, with that function varying slowly with Initially, the 2000 m depth contour is followed around the basin as a reference, starting deliberately on the Paciﬁc side of South America so any link between Paciﬁc and Atlantic values can be seen. The contour consists of a set of straight line (actually very short great-circle) seg- ments between positions where the linear interpolation of depths be- tween neighbouring grid points is 2000 m, thus avoiding any rectan- gular zigzagging around grid boxes. The along-slope distance is then calculated as the sum of the lengths of these short line segments. The 31 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. black dots in Fig. 8 are every 500 km distance along this contour, and are referred to as “nodes”. The ringed numbers are every 10,000 km along, and thus represent distance in units of 10,000 km. This is used as the distance axis in later plots. Colours are an additional indicator of distance, with a colour change every 1000 km. Note that the distance is deﬁned following the 2000 m contour which, though not as convoluted as a coastline, can be quite convoluted in places. Thus, although the nodes are separated by 500 km along the 2000 m contour, the great- circle distance between nodes is typically (median value) about 390 km. black dots in Fig. 8 are every 500 km distance along this contour, and are referred to as “nodes”. The ringed numbers are every 10,000 km along, and thus represent distance in units of 10,000 km. This is used as the distance axis in later plots. Colours are an additional indicator of distance, with a colour change every 1000 km. Note that the distance is deﬁned following the 2000 m contour which, though not as convoluted as a coastline, can be quite convoluted in places. Thus, although the nodes are separated by 500 km along the 2000 m contour, the great- circle distance between nodes is typically (median value) about 390 km. away from the coast, but has no equivalent in shallow regions. In ad- dition to these gaps, the density of points is strongly inﬂuenced by the steepness of the slope, with greater density along the northern boundary where the slope is gentle, and much more sparse coverage in regions of steep slopes. 5.1. Variability and vertical (cross-slope) structure It is striking how uniform the structure is in these EOFs. EOF1 has a single node at almost all distances, that node being between about 800 and 1500 m depth except where it is forced deeper by the absence of shallow data (together with the subtraction of the average over the available depth range). The increased deep variability in some locations has an inﬂuence on the EOF structure, but this does not seem to dom- inate. Fig. 9 (top) shows the standard deviation of bottom pressure as a function of distance and depth. Note that depth here is the depth of the bottom of the ocean. In the diagnostics presented here, horizontal po- sition is a function of depth, and any signal that is described as ‘in- dependent of depth’ could equally well be described as ‘independent of cross-slope distance’, for a given along-slope distance as deﬁned in Fig. 8. There may or may not be variation in the vertical above a given point. Thus, the usual ideas of barotropic and baroclinic modes are not appropriate interpretations when looking at bottom pressure on the continental slope, unless it is vertical. To emphasize this, the depth- averaged bottom pressure at a given distance will be represented by 〈〉 p H, where the subscript H emphasizes that the averaging variable is depth of the ocean ﬂoor rather than the vertical coordinate z. In the case of EOF2, there are generally two nodes, though there is more variability in the depths of these except along the eastern boundary, where the structure is very uniform. The deep variability has more inﬂuence on EOF2 than on EOF1. The depth average, 〈〉 p H, plus the ﬁrst two EOFs typically explain about 90% of the total variance, though this can be as little as 50% in small regions. This is illustrated in the bottom panel of Fig. 9, which shows the standard deviation of the residual after subtracting the depth average and the ﬁrst 2 EOFs. This is below 0.5 mbar in most of the basin, though larger in northern regions, near Cape Hatteras, and along parts of the South American coast, particularly those inﬂuenced by the Antarctic Circumpolar Current. In terms of the residual after subtracting the depth average, the ﬁrst two EOFs typically explain over 80% of the variance. 5. Atlantic variability and continental slope bottom pressure From this we can see that, even at 1/ 12° resolution, parts of the continental slope are barely resolved (not resolved, if we take the criterion of no more than one vertical grid step per horizontal step as the deﬁnition of resolved). Typical climate models will have much lower resolution than this, which calls into question how realistic their representation of boundary processes can be. Contours are then followed at other depths, to a maximum of 3200 m, at an interval of 1 m, to identify all the gridpoints associated with each continuous contour (excluding seamounts and other closed contour regions on the slope). Below 3200 m, contours do not pass through Drake Passage and also start to spread down the mid-Atlantic ridge. For each contour, the point nearest to each node of the reference contour is labelled as having the same distance as that node, with distance linearly interpolated between points matched to nodes. If the distance to a node is greater than 1000 km, the point is ignored, thus preventing the use of shallow contours which pass round the Arctic, Mediterranean, or parts of the Gulf of Mexico and Caribbean Sea. This procedure provides a usable distance value for points oﬀthe reference contour. Returning to the bottom pressure standard deviation in Fig. 9, we see that a large fraction of the slope has a standard deviation below 2 mbar, as we suspected based on the map (Fig. 3). Variability is lower near the equator and on the eastern boundary (distances above about 4). There are also deep maxima in variability in the northern Labrador Sea (3.0–3.2) and south of Iceland (3.6–3.8), locations where deep water formation and mixing processes are likely to be important (these are large Rossby number processes, so not subject to damping according to the scaling argument above). Another deep maximum occurs near Cape Hatteras (2.4) where the deep western boundary current passes beneath the Gulf Stream. We suspected that a signiﬁcant portion of this variability would be due to large-scale barotropic processes. To remove these, we calculated a depth-averaged bottom pressure 〈〉 p H at each distance and time, and subtracted this oﬀ. The residual standard deviation is shown in the middle panel of Fig. 9. It is clear that this mode accounts for a sig- niﬁcant part of the variability (typically about half the variance). 5. Atlantic variability and continental slope bottom pressure For each point on the continental slope, we now have a depth and a distance. Bottom pressures were extracted from these points, and a mean, trend, annual and semiannual cycle were ﬁtted and removed. The two dimensional ﬁelds at each time were then regridded onto a regular grid every 50 km in distance and 10 m in depth, from 100 m to 3200 m, using a Delauney triangulation, which treats depths in metres and distances in kilometres as equivalent. After subtracting the depth average, 〈〉 p H to look at higher vertical mode structures, we performed an Empirical Orthogonal Function (EOF) analysis of the time series at each distance, returning a set of basis functions in depth and time, for each distance. The middle and lower panels of Fig. 10 shows composites of the ﬁrst two depth basis functions. These functions are calculated independently at each dis- tance. The only processing to improve the appearance of the plot is the choice of arbitrary sign at each point to maintain continuity in distance. This interpolation gives values at all depths and distances, which is unrealistic given the gaps in shallow contours. To account for this, we calculate a mapping error estimate which is the geometric mean of the horizontal distance of each point on the regular grid from the points used in the triangulation. Points for which this distance is greater than 200 km are left blank. 5.1. Variability and vertical (cross-slope) structure The depth average, 〈〉 p H, and ﬁrst two EOFs together are therefore suﬃcient to describe most of what is seen along the con- tinental slope. Fig. 9 can thus be imagined as looking into the Atlantic from the south, and unwrapping the continental slope into a straight strip. Ver- tical lines mark the equator, and grey masks regions of missing data. Reading from the left, the ﬁrst grey patch at distance about 1.9–2.2 represents the Caribbean Sea and the Gulf of Mexico, with the tip of Florida at about 2.2, and Cape Hatteras at 2.4. Around 3.0–3.1 is the top of the Labrador Sea, where some shallow contours are lost into Baﬃn Bay or to circumnavigate the Arctic. Between about 3.7 and 4.0, a deeper range connects to the Arctic between Iceland and Scotland. The Strait of Gibraltar is at about 4.57. 5.2. Coherence along the slope The position of the actual gridpoints used to map this standard deviation is shown in the top panel of Fig. 10. In addition to the gaps discussed above, a sparse region in the top kilometre at distances of 1.1–1.2 reﬂects the Vitoria-Trindade ridge, a feature oﬀthe Brazilian coast which diverts deep contours (including the 2000 m reference) far Each of the three modes 〈〉 p ( H, EOF1, EOF2) has an associated time series at each distance. In Fig. 11, we show the cross-correlations be- tween those time series at each distance with each other distance. For the depth-independent mode, 〈〉 p H, this conﬁrms that the 32 ability is highly coherent over the whole basin, with positive cor- tions almost everywhere The correlations are for Fourier ﬁltered eastern Iceland), and 4–6 (Scotland to South Africa, the entire eas boundary) The scales of these regions imply strong correlations o 9. Standard deviation of bottom pressure on the Atlantic continental slope as a function of distance and depth of the ocean ﬂoor (compare with Fig. 8). The top panel shows the r removing mean, trend, annual and semiannual cycles). The middle panel shows the residual after removing the depth average (cross-slope average, 〈〉 p H) at each distance and bottom panel shows the residual after further removing the signal explained by the ﬁrst two EOFs at each distance. Hughes et al. Progress in Oceanography 161 (2018) 1 W. Hughes et al. Progress in Oceanography 161 (2018 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. g. 9. Standard deviation of bottom pressure on the Atlantic continental slope as a function of distance and depth of the ocean ﬂoor (compare with Fig. 8). The top panel shows the to fter removing mean, trend, annual and semiannual cycles). The middle panel shows the residual after removing the depth average (cross-slope average, 〈〉 p H) at each distance and ti h b l h h id l f f h i h i l l i d b h ﬁ O h di Fig. 9. Standard deviation of bottom pressure on the Atlantic continental slope as a function of distance and depth of the ocean ﬂoor (compare with Fig. 8). The top panel shows the total (after removing mean, trend, annual and semiannual cycles). 5.2. Coherence along the slope Position of the points used in this analysis (top), and the structures of the ﬁrst two EOFs of bottom pressure at each distance, after subtracting the depth average (cross-slope average, 〈〉 p H) (middle and bottom). EOFs are dimensionless and have a variance of 1. Fig. 10. Position of the points used in this analysis (top), and the structures of the ﬁrst two EOFs of bottom pressure at each distance, after subtracting the depth average (cross-slope average, 〈〉 p H) (middle and bottom). EOFs are dimensionless and have a variance of 1. Fig. 10. Position of the points used in this analysis (top), and the structures of the ﬁrst two EOFs of bottom pressure at each distance, a average, 〈〉 p H) (middle and bottom). EOFs are dimensionless and have a variance of 1. The northern block (Cape Hatteras to Iceland) is split into two subregions separated at around the northern limit of the Labrador Sea (3.1). Similarly, the eastern block (Scotland to South Africa) is split into subregions at the Strait of Gibraltar (4.57), although in this case it is more like the start of a reduction in correlation to the north rather than the separation of two clear regions. Within the subregions, the corre- lations are especially strong, usually above 0.8 or 0.9. It is noticeable that the boundaries between blocks and sub-blocks often lie in the re- gions of large deep variability identiﬁed before. indicates that points on the eastern boundary correlate with one an- other if they are at equal distances from the equator. This suggests a signal propagating away from the equator suﬃciently slowly that lags in signal propagation become important enough to reduce the corre- lation in the higher frequency band. Points at equal distances from the equator can then still correlate because they are at equal lags. Assessment of statistical signiﬁcance depends on the spectral con- tent at each point, but the correlations within blocks are so strong as to be clearly signiﬁcant with any reasonable estimate of degrees of freedom (only 14 degrees of freedom are needed for a correlation of 0.5 to be signiﬁcant at the 95% level; we have 54 years of data and, as we shall see, the data are not dominated by the longest periods). We will, however, consider this in more detail when looking at lagged correla- tions later on. 5.2. Coherence along the slope The middle panel shows the residual after removing the depth average (cross-slope average, 〈〉 p H) at each distance and time. The bottom panel shows the residual after further removing the signal explained by the ﬁrst two EOFs at each distance. variability is highly coherent over the whole basin, with positive cor- relations almost everywhere. The correlations are for Fourier-ﬁltered time series, showing periods shorter than 1.5 years below the diagonal, and periods between 1.5 and 10 years above. The correlations are stronger at the lower frequencies, except for those involving the strip at distances 0.0–0.29 which is on the Paciﬁc side of South America, where long-period correlations drop oﬀsharply, even becoming slightly ne- gative, showing that there is a clear distinction between basins for this large-scale mode. eastern Iceland), and 4–6 (Scotland to South Africa, the entire eastern boundary). The scales of these regions imply strong correlations over distances of measured in tens of thousands of kilometres. In contrast to the depth-independent mode, 〈〉 p H, there is clear communication be- tween the Paciﬁc and Atlantic (distances 0–0.29 in the Paciﬁc are correlated with the western South Atlantic distances 0.29–1.4), al- though the tip of South America provokes a drop in correlation. The smaller block is from about 1.4 to 1.93, with a weaker extension to 2.35 (the north coast of South America and the eastern Caribbean, with extension to north Florida). In addition, there is an oﬀ-diagonal block showing correlation between the eastern and western boundary equator positions (marked with black lines), which shows strong cor- relations between the equator-spanning small block and the entire eastern boundary. Perhaps more striking is the result for EOF1. At low frequencies (above the diagonal), this shows three large blocks of strong correla- tion, and one smaller one. The large blocks are at distances 0–1.4 (the east Paciﬁc and southern hemisphere western boundary of the Atlantic to about the easternmost tip of Brazil), 2.4–3.8 (Cape Hatteras to 33 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. 10. Position of the points used in this analysis (top), and the structures of the ﬁrst two EOFs of bottom pressure at each distance, after subtracting the depth average (cross age, 〈〉 p H) (middle and bottom). EOFs are dimensionless and have a variance of 1. . Hughes et al. Progress in Oceanography 161 (2018) 1 Fig. 10. 5.2. Coherence along the slope Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass periods between 1.5 and 10 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. g g g py ( ) 9 Fig. 13. Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the co periods between 1.5 and 10 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Fig. 13. Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass periods between 1.5 and 10 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. highlight the importance of the continental slope still further. They are roughly consistent with baroclinic Kelvin wave speeds at the equator: Brandt et al. (2016) calculate Kelvin wave speeds of 2.47 and 1.32 m s−1 for the ﬁrst and second modes respectively. However, the Kelvin wave speed for a vertical boundary will become steadily slower as the waves propagate polewards, because the stratiﬁcation weakens. The faster propagation speeds seen here are consistent with the fact that the true boundary waves on a slope are not Kelvin waves, but a hybrid mixture between baroclinic Kelvin waves and topographic Rossby waves, which become more barotropic and faster as the slope scale comes to exceed the Kelvin wave trapping scale, which is the relevant Rossby radius (Huthnance, 1978). For example, Cartwright et al. (1980) calculate a barotropic shelf wave speed of about 7.9 m s−1 oﬀthe Scottish coast (translating from the quoted wavenumber of 53 degrees per 100 km at the K1 tidal period). with more interruption by local noise. The same broad blocks are visible, though more weakly, and the same oﬀ-diagonal equatorial correlation and eastern boundary lagged correlation structure are also apparent. Figs. 12–14 show Hovmoeller plots of the diﬀerent modes in dif- ferent frequency bands. The ﬁrst, Fig. 12 shows only a representative 5.5-year period so that the structure can be clearly seen. 5.2. Coherence along the slope At higher frequencies (below the diagonal), the northern block is similar if a bit weaker, but the other blocks are signiﬁcantly weaker, especially the eastern boundary block, and the oﬀ-diagonal correlation between eastern and western equators is missing. However, the curious high-correlation feature extending down and to the right of the diag- onal from the eastern equator gives a clue to the reason for this. This For EOF2, we see a picture broadly similar to that for EOF1, but 34 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 11. Cross correlations between the time series for each vertical (cross-slope) mode at each distance along the slope, with that at each other distance. Values above the diagonal are for periods between 1.5 and 10 years, and values below the diagonal are for periods shorter than 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Black lines indicate the positions of the equator on the western and eastern boundaries. C.W. Hughes et al. Progress in Oceanography 161 (2018) 19–46 Fig 11 Cross correlations between the time series for each vertical (cross slope) mode at each distance along the slope with that at each other distance Values above the diagonal are fo Fig. 11. Cross correlations between the time series for each vertical (cross-slope) mode at each distance along the slope, with that at each other distance. Values above the diagonal are for periods between 1.5 and 10 years, and values below the diagonal are for periods shorter than 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Black lines indicate the positions of the equator on the western and eastern boundaries. Fig. 12. Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass periods shorter than 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Fig. 12. Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass periods shorter than 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. 35 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 13. 5.2. Coherence along the slope Again, this highlights the basin-scale nature of the depth-independent mode, 〈〉 p H, with perhaps additional variability in the northern region (note that the boundary values are coherent over the entire basin, but this does not preclude anticorrelation with values in the interior). No lags can be discerned, which is to be expected as the largest-scale signals should propagate with a barotropic Kelvin wave speed of around 200 m s−1, as was found in the western North Atlantic from bottom pressure mea- surements on the continental slope (Elipot et al., 2013). More interesting are the plots for EOF1 and EOF2. Here, we can clearly see the eﬀect of ﬁnite propagation speeds. Propagation is toward the western equator (equators are again marked as vertical lines), and away from the eastern equator. Wave speeds are slowest near the equator, and faster further away, but it must be remembered that the concept of along-slope distance depends on length scales and on how convoluted the 2000 m contour is. Various techniques have been ap- plied in an attempt to estimate wave speeds, but we do not generally ﬁnd robust numbers. We can see that speeds near the equator are be- tween about 2 and 3 m s−1 for EOF1, and between about 1 and 1.5 m s−1 for EOF2. Speeds clearly get faster away from the equator, but meaningful quantiﬁcation of these speeds is diﬃcult. These speeds At longer periods (now showing the entire range of time), Fig. 13 shows that these wave propagation lags become insigniﬁcant, as lags along the eastern boundary can no longer be seen. For EOF1 there are hints of a slower equatorward propagation along the western boundary (particularly 2.5–3.1), but this is irregular in nature and too slow to be related to the boundary waves, it may be an advective phenomenon, if it is signiﬁcant. Another feature which becomes apparent at longer periods is the contrast in amplitudes between eastern and western boundaries. Signals are much weaker, and rather constant amplitude on the east, with a hint of ampliﬁcation north of about the Strait of Gibraltar (4.57). This is consistent with idealised theories, e.g. Kawase (1987) and Johnson and 36 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 14. 5.2. Coherence along the slope Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass periods longer than 10 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. C.W. Hughes et al. Progress in Oceanography 161 (2018) 19–46 Fig. 14. Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along t periods longer than 10 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Fig. 14. Hovmoeller plots showing the temporal variations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass periods longer than 10 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. iations in the three vertical (cross-slope) modes as a function of distance along the continental slope and time, after ﬁltering to pass and semiannual cycles were removed at an earlier stage. roughly 50-year period, which is the longest possible within this dataset after detrending. While the model physics should still be consistent, the probability of being dominated by long-term thermohaline adjustment is high, and the eﬀective number of degrees of freedom is so low that it is dangerous to conclude anything from apparent correlations. The large, quadratic-in-time variability in the region 2.1–3.0 seems to ex- plain the unusual oscillation in the low frequency spectrum of bottom pressure in energetic continental slope regions, which can be seen in Fig. 7. Marshall (2002a,b) which assume that low frequency signals propagate eﬀectively instantly along eastern boundaries without attenuation or ampliﬁcation. Ampliﬁcation is expected poleward of the highest lati- tude at which Rossby waves can propagate for a given frequency. Marshall and Johnson (2013) give a nice overview of how theoretical models treat boundary waves (usually with a vertical sidewall), and of the importance of this process for a wide range of issues. On western boundaries, they are found (in theory) to decay in amplitude toward the equator, unlike our model results (except perhaps close to the equator), where we see an ampliﬁcation in many places. There is clearly rapid transmission of information along the western boundary, but a more complex response than the simple, linear, vertical sidewall theory would predict. It should be noted though, that the theoretical results are for buoyancy forcing only. 5.2. Coherence along the slope With wind stress variability too, angular momentum balance at each latitude requires that there be changing sidewall pressures as seen in the AMOC response to winds (Elipot et al., 2017). Here we have a rare case where diagnostics from a realistic model can address a problem of basin scale dynamics rather than being overwhelmed by mesoscale variability. Returning to the high frequency data, we look in more detail at the long distance coherence of the signal when accounting for lags. We calculated lags relative to four diﬀerent regions distinguished by col- ours in Fig. 15. Roughly, these regions are the eastern boundary (red), a northern region (orange), a north tropical western boundary region south of the Gulf of Mexico and north of the equator (green), and a southern hemisphere western boundary region (blue). The results of this analysis are shown in Fig. 15, which shows both correlations (Fig. 15a and c) and calculated lags (Fig. 15b and d. The precise extent of the reference regions is marked by the paired dots of matching colour at the top of Fig. 15a and c. For completeness, Fig. 14 shows periods longer than 10 years. The contrast between east and west is now even more evident for EOF1, partial barriers to communication of western boundary signals appear at about Cape Hatteras (2.4) and the Gulf of Mexico (2.1). Some signal does appear to eventually propagate to the equator, from where it ra- pidly appears all along the eastern boundary. However, we caution against interpreting this plot in too much detail. Although periods as short as 10 years should be present, the variability is dominated by a Absolute values of the lags are chosen to be consistent between the diﬀerent curves as described below, so they can be interpreted as the time taken for a signal to propagate to each point from the eastern boundary equator (or, for negative values, minus the time taken to propagate from that point to the eastern boundary equator). The cor- relations are plotted as thin lines, with large dots on top only for points that are statistically signiﬁcant. Lags are plotted only for these 37 Hughes et al. Progress in Oceanography 161 (2018) 1 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. ﬁcant points. he calculation was performed as follows. 5.2. Coherence along the slope We started by selecting expected coherent regions by eye, from the Hovmoeller plot, 12 Th d k d ti i ﬁlt d t i d reference time series T0. Then, all time series in the region were related with T0, with a range of diﬀerent lags, to identify the la which the correlation was best. Each time series was then shifted by b t l d th i l ( th ti i ) 5. Best lagged correlations, and the lag at which the best correlation is found, for EOF1 and EOF2 time series compared with reference time series based on data from betwee ing-coloured dots. Lags are shown only where the correlations are signiﬁcant at the 99% level, and correlations are plotted with a thin line where they are not signiﬁcant a The time series are ﬁltered to pass periods between 20 days and 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. See main text for . (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Hughes et al. Progress in Oceanography 161 (2018) 1 15. Best lagged correlations, and the lag at which the best correlation is found, for EOF1 and EOF2 time series compared with reference time series based on data from between hing-coloured dots. Lags are shown only where the correlations are signiﬁcant at the 99% level, and correlations are plotted with a thin line where they are not signiﬁcant at The time series are ﬁltered to pass periods between 20 days and 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. See main text for m Fig. 15. Best lagged correlations, and the lag at which the best correlation is found, for EOF1 and EOF2 time series compared with reference time series based on data from between the matching-coloured dots. Lags are shown only where the correlations are signiﬁcant at the 99% level, and correlations are plotted with a thin line where they are not signiﬁcant at this level. The time series are ﬁltered to pass periods between 20 days and 1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. See main text for more details. 5.2. Coherence along the slope (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) signiﬁcant points. This same growth is seen in lags with respect to the northern region (orange), so the con- stant for that region is chosen to align it with the eastern region lags. Similarly, where both are signiﬁcant, the lags for the north tropical western boundary region (green) have very similar variations to the eastern region (red), so the constant lag for this region is found by aligning the two (tracing the signals back in time to the western boundary at the equator). For the northern boundary region (orange) we have two options, as the lags show it preceding the north tropical western boundary region (green), and lagging behind the eastern boundary region (red), so two diﬀerent constant lags can be calculated, with a diﬀerence which is the time taken for a signal to perform a complete circuit. This region is therefore plotted with both lags (the eastern region is also repeated using the same oﬀset). Finally, the constant lag for the south western boundary region (blue) is chosen to align it with the other lags at the western boundary equator. The whole graph can be considered to be periodic in y, with a period of 115 days for EOF1 and 205 days for EOF2. Translating these delays to mean propagation speeds is not straightforward. The along-slope distance from eastern equator to western equator is 39,000 km, but the sum of great-circle distances between nodes over the same stretch of continental slope is only 28,133 km. Using these two extremes for the distance, we obtain a mean along-slope propagation speed for EOF1 of 3.7–5.2 m s−1, and for EOF2 we obtain 2.0–2.7 m s−1. Although the range is wide, the mean values are clearly faster than the corresponding equatorial Kelvin wave speeds. These are important parameters for understanding timescales of basin adjustment, which could not realistically be computed otherwise. Again, the clean nature of the continental slope bottom pressure signal has made it possible to distinguish basin scale dynamics which could not be seen in other, mesoscale-contaminated signals. Note, though, that these numbers may depend on the model representation of topo- graphy and friction, so they may be diﬀerent in the real world, and diﬀerent again in coarser resolution models. It is worth noting that, for EOF1, signiﬁcant correlations for the northern region (orange) overlap the signiﬁcant lags from the north tropical western region (green) on both the eastern and western boundaries. signiﬁcant points. reference time series T0. Then, all time series in the region were cor- related with T0, with a range of diﬀerent lags, to identify the lag at which the correlation was best. Each time series was then shifted by this best lag, and the regional average (now over the entire region) was recalculated to obtain a new reference time series T1. This process was iterated to convergence (four iterations was suﬃcient). The best lag, and the associated best correlation, were then calculated relative to the converged regional average time series T4, for all points, not just those in the selected region. The calculation was performed as follows. We started by selecting the expected coherent regions by eye, from the Hovmoeller plot, Fig. 12. The procedure worked on time series ﬁltered to pass periods between 20 days and 1.5 years and normalised to a standard deviation of 1 (periods shorter than 20 days were removed in order to permit shifts of the time series of less than 5 days, using a Fourier method; normalisation avoids skewing the results to focus on regions of highest variability). For each region, an iterative method was then applied to determine the best lagged correlations. Initially, all the time series in the central 2000 km of that region were averaged together to provide a Finally, a constant was added to the lags derived for each region. For the eastern region (red), the constant was chosen so that the lag is 38 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 16. Cross correlations for sea level variability in the NEMO model, along a mer- idional section of the Atlantic at 23°W from 55°S to 64°N. Values above the diagonal use time series ﬁltered to pass periods between 1.5 and 10 years, those below use 0–1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Black lines mark the equator. reference time series at all possible lags, and the correlation at the best lag chosen. These 1000 correlations were then sorted to determine the 99% conﬁdence level (this ranged between 0.10 and 0.25 for the dif- ferent time series). What we ﬁnd is that, when accounting for lags, the correlations are indeed coherent over very large distances, as they are at lower fre- quencies without accounting for the lags. signiﬁcant points. Furthermore, the lagged correlations remain signiﬁcant to some distance either side of the re- ference regions, with consistently varying lags where the curves overlap. For example, in EOF1, the eastern boundary signal (red) re- mains detectable as far as Greenland (3.3) and, though the correlation drops oﬀrapidly, the north tropical western mode (green) also obtains a small part of its variance from the Greenland coast, as well as com- municating with much of the eastern boundary. The south western boundary signal (blue) involves signals propagating from the Paciﬁc, and extends to the Atlantic equator. For EOF2 we ﬁnd a similar pattern, though correlations tend to drop oﬀmore rapidly, reducing the overlap of regions with signiﬁcant correlations, and the corresponding lags are larger, corresponding to the slower propagation speeds. These lags are all consistent with expectations for the propagation of continental shelf waves and equatorial Kelvin waves. The consistency of the lags allows us to estimate various propaga- tion times. We ﬁnd times to cross from the western boundary to the east at the equator of 28 and 40 days respectively for EOF1 and EOF2 (using slightly diﬀerent regions or weightings varies these numbers by ± 1 day). Given a distance of approximately 5900 km, these times cor- respond to speeds of 2.44 and 1.71 m s−1, comparable to the ﬁrst and second baroclinic mode Kelvin wave speeds calculated by Brandt et al. (2016) which are 2.47 and 1.32 m s−1. We can also determine the time to complete a full circuit of the North Atlantic, which is 115 days for EOF1 and 205 days for EOF2 (these estimates have larger uncertainties of about 5 days for EOF1 and 20 days for EOF2). The travel time along the continental slope, from eastern equator to western equator, is therefore 87 days for EOF1 and 165 days for EOF2. Fig. 16. Cross correlations for sea level variability in the NEMO model, along a mer- idional section of the Atlantic at 23°W from 55°S to 64°N. Values above the diagonal use time series ﬁltered to pass periods between 1.5 and 10 years, those below use 0–1.5 years. Linear trend, annual, and semiannual cycles were removed at an earlier stage. Black lines mark the equator. zero at the eastern equator. As signals propagate along the eastern boundary away from the equator, the lag grows. signiﬁcant points. On the east, the lags show signals propagating into the northern region. On the west, they show signals propagating away from the northern region. This means an estimate for the basin circuit time can be made purely from these two region, without needing the eastern region. The fact that all three lags agree in the eastern region is therefore an independent test of this circuit time. The circuit time es- timate for EOF2 is more fragile as it relies purely on the small region of overlap between signiﬁcant lags for the northern (orange) and north tropical western (green) regions, which also involve rather low corre- lations. To conclude this subsection, it is worth emphasizing the exceptional nature of these long correlation scales. For comparison with Fig. 11, Fig. 16 shows cross-correlations for NEMO model sea level along a mid- Atlantic meridional section at 23°W. The distance is again measured in units of 10,000 km, so the scale is comparable to Fig. 11 (the whole distance scale is less than a quarter of the total scale for the continental slope). These correlations are typical of the open ocean, with some large-scale correlation apparent near the equator (black lines), but nothing approaching the clarity and large-scale nature of the boundary signals. 5.3. The meridional overturning circulation The exception, at mid- depths for the longest periods, is because there is little variability to capture in this depth range. At these longest periods, the AMOC anomalies truly do seem to consist of a northward shallow ﬂow and a southward return ﬂow, with a node at intermediate depths, across the entire Atlantic basin. Here, we have focused on the boundary signals themselves rather than their relevance to the AMOC, but it is still worth demonstrating their link to the AMOC explicitly. The theoretical argument is straightforward. Integration of geostrophic balance from west to east across the ocean basin at constant latitude and depth, leads to the fol- lowing balance: In contrast, at the shortest periods, there is variability at all depths, and the amplitude rises toward the equator. This suggests a scaling proportional to f 1/ , and that the boundary signals may be best thought of as pressure signals at the boundary, with the AMOC as an incidental result of their presence. That is consistent with the independent pro- pagation we see along eastern and western boundaries, with similar amplitudes. (9) = − fT p p , E W where T is the zonally integrated northward mass transport (in kilo- grammes per metre of depth per second), and pE and pW are bottom pressures at the eastern and western end of the section respectively. Assuming a midlatitude value = − f 10 4 s−1, and a density of about 1000 kg m−3, this leads to a 1 mbar pressure diﬀerence producing a net northward volume transport of 1 Sv km−1. It is therefore straightfor- ward to see from Figs. 12–14 how the eastern and western boundary pressures contribute to net meridional transport. At intermediate periods, the deep AMOC variability is small, but there is no systematic latitude dependence outside the tropics. This is the regime in which the eastern boundary signals are much smaller than those on the west but also extremely coherent in space (also true at longer periods). The reconstruction has diﬃculties in the shallow North Atlantic, and particularly the Gulf of Mexico latitudes (about 20–30°N), but this is to be expected given the complicated geometry here, and even these regions improve at the longest timescales. 6. Application to the real ocean We have shown above that, in the NEMO model, continental slope bottom pressure measurements provide a means of extracting large- scale information about the ocean circulation, and the AMOC in par- ticular. If this carries over to the real ocean, and if such measurements can be made with the necessary accuracy, then this would make a strong case for such measurements to be considered an important component of the Global Ocean Observing System. Similarly, a positive value of EOF2 on the west would lead to a southward transport above about 500 m and at depth (typically below about 1.8 km but somewhat variable), and a northward transport at intermediate depths. Again, the same on the east would produce the opposite AMOC change. We test that these relationships between boundary pressure and AMOC hold in the model by diagnosing the AMOC (in the sense of zonally-integrated meridional transport per unit depth) and comparing with that predicted based on the boundary pressures. This involves some complications because the pressure values at a given distance along the slope are not all at the same latitude. Equally, the model grid is not perfectly aligned with latitude lines north of about 20°N, though it remains within 1 degree of a constant in the Atlantic to about 55°N. Accordingly, we use the latitude associated with the reference 2000 m depth contour to deﬁne the latitudes of the pressure measurements, and average the AMOC in 1-degree bins for comparison, relying on the spatial coherence of the signals in order for the two datasets to match. The sign associated with the boundary pressure’s contribution to the AMOC is determined by the direction of the contour at that point; it is negative where increasing distance moves north along the contour, and positive where it moves south. In this case we also use monthly means The continental slope represents a very small fraction of the global ocean area and, as a result, there are rather few measurements made in this region. It is poorly monitored using Argo ﬂoats which rarely enter these regions and tend to spend very little time there when they do enter. The diﬃculty seems to be, in some ways, one of perception. Because the slope region is so small, it seems disproportionate to spend resources here which could be used to improve open-ocean sampling. 5.3. The meridional overturning circulation Signiﬁcance of the correlations was assessed by a Monte Carlo method: 1000 time series with the same spectrum as each reference time series were generated. These were each correlated with the actual The most obvious quantity to attempt to recover in the Atlantic is the meridional overturning circulation (AMOC), which has received a 39 C.W. Hughes et al. Progress in Oceanography 161 (2018) 19–46 rather than 5-day means as an extra ﬁlter on ageostrophic high fre- quency variability. great deal of attention as a signiﬁcant climatic mode of the Earth system in recent decades. It is not our purpose here to revisit all this work. Bingham and Hughes (2008b) showed that much of the AMOC at 42°N could be recovered simply from boundary pressure measurements in a 1/4° resolution ocean model (and almost all of that from the western boundary only, at interannual periods), and Bingham and Hughes (2009a) looked in more detail at 50°N in the same model, and in 100 years of a coarser-resolution climate model run. These ideas have been used to measure the AMOC using the WAVE array at 43°N (Hughes et al., 2013; Elipot et al., 2013, 2014), and it has been shown how the measurements of the RAPID array at 26°N (McCarthy et al., 2012) can be interpreted in the same way (Elipot et al., 2017). The argument that eddies do not dominate the AMOC signal has also been made based on observations, and theory in a vertical sidewall context (Kanzow et al., 2009) (though note that this argument relates to the integrated transports, not to the pathways of ﬂow and tracer transport, which are very strongly inﬂuenced by eddy variability). Fig. 17 shows the resulting AMOC variability (left) in three diﬀerent frequency bands, and the percentage of AMOC variance explained (right) by using only boundary pressure measurements. It should be recalled that these do not represent all the contributions to the zonal integral in this depth range. There are also contributions from the Mediterranean, Gulf of Mexico and Caribbean Sea which we are missing out, as well as any portions of the Mid-Atlantic Ridge which rise above 3200 m depth. Nonetheless, with the exception of a region around the equator where the geostrophic argument breaks down, a large part of the AMOC variability is captured by these boundary measurements, and this pro- portion generally increases at longer timescales. 5.3. The meridional overturning circulation Overall, at intermediate and long timescales, the unexplained residual standard deviation (not shown) is less than 0.3 Sv km−1 over most of this depth range, for la- titudes more than 10 degrees from the equator. The east-west symmetry of the depth-independent mode, 〈〉 p H, is testament to the fact that there can be rather little net northward transport across each latitude section (a net transport must be balanced by either an accumulation of mass, a ﬂow through the Bering Strait, or a net gain or loss of mass from evaporation and precipitation, so it is tightly constrained by these integral properties). The AMOC represents a ﬂow which is to the north at some depths and south at others, and is therefore reﬂected in the other EOF modes. From the vertical structures of the EOFs (Fig. 10), a positive value of EOF1 on the western boundary would correspond to a southward ﬂow above about 1300 m, and a northward return ﬂow below that depth; a negative AMOC anomaly. The same on the east would produce a positive AMOC anomaly. In summary, the highly-correlated, basin-scale boundary pressure signals are indeed a good diagnostic of the AMOC. 6.1. Real ocean illustrations First we need to show that this mesoscale suppression occurs in the 6. Application to the real ocean 3 and 5), which suggests that the dominant signal seen was the depth-independent mode, consistent with the fact that no propagation lag could be seen over this distance. real ocean and is not a model artefact. We showed in Section 3 that the model represents the mesoscale energy and its spectrum well. In fact, a number of experiments have demonstrated this at the Atlantic western boundary. In Fig. 18 we give an example from the continental slope near Halifax, Nova Scotia (distance 2.8 in the model slope diagnostics), from the RAPID-Scotian array (Hughes et al., 2013). We see that, after 5-day averaging (blue), the variability over almost 4 years drops below 2 mbar except at the deepest point. Furthermore, subtracting an estimate of the depth average based on the four shal- lowest instruments, this drops below 1 mbar (below 0.6 mbar for the two central depths). Comparing with Fig. 9 at distance 2.8, this matches expectations both for amplitude and structure, with the amplitudes rising both in deeper and in shallower water. Our model continental slope diagnostics do not extend below 3200 m depth, but it can be seen from Fig. 3 that this amplitude increase continues at greater depths as a result of weakly-damped mesoscale variability at the foot of the con- tinental slope. The altimetry result highlights further the need for bottom pressure measurements: without them it is impossible to distinguish the diﬀerent vertical modes, and the depth-independent mode is liable to dominate. Fig. 18 shows the importance of the depth-independent mode. Once this is subtracted, the vertical structures of the remaining observed varia- bility are quite robust, with the ﬁrst EOF being almost linear with depth (Elipot et al., 2013) apart from instruments on the gently-sloping tail of the continental slope, and having a quite diﬀerent spectral structure from the depth-independent mode, with less variability at the shortest periods. Other sites show similar amplitudes, and a similar reduction on subtracting the depth average, making it possible to discern large-scale correlations and basin-scale responses to forcing (Elipot et al., 2013, 2014, 2017). To the extent that it has been possible to test it, the real ocean does indeed seem to behave in a manner similar to the model. We have focused here on the continental slope, for monitoring ocean dynamics, but an even quieter region is the deep tropical ocean. 6. Application to the real ocean However, if we view the ocean in the latitude-depth plane, rather than latitude-longitude, the continental slope spans most of the active ocean depth range. This is the appropriate viewpoint for the meridional overturning circulation. Just by looking at the ocean “sideways”, we obtain a very diﬀerent viewpoint on where to expend resources. First we need to show that this mesoscale suppression occurs in the First we need to show that this mesoscale suppression occurs in the 40 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 17. Diagnostics of zonally-integrated northward water volume transport across the Atlantic (MOC) in the NEMO model as a function of depth and latitude, using monthly values after subtracting mean, linear trend, and annual and semiannual cycles. Left hand panels show the standard deviation in Sv km−1 (or, equivalently, 103 m2 s−1). Right hand panels show the percentage of variance in the MOC which is explained by the boundary pressures using Eq. (9). Negative values are plotted as grey. The time series are divided into long periods (longer than 10 years, top), medium (1.5–10 years, middle), and short periods (2 months to 1.5 years, bottom). g g g py Fig. 17. Diagnostics of zonally-integrated northward water volume transport across the Atlantic (MOC) in the NEMO model as a function of depth and latitude, using monthly values after subtracting mean, linear trend, and annual and semiannual cycles. Left hand panels show the standard deviation in Sv km−1 (or, equivalently, 103 m2 s−1). Right hand panels show the percentage of variance in the MOC which is explained by the boundary pressures using Eq. (9). Negative values are plotted as grey. The time series are divided into long periods (longer than 10 years, top), medium (1.5–10 years, middle), and short periods (2 months to 1.5 years, bottom). To some extent, the continental slope signals can be discerned in sea level measurements, again conﬁrming the relevance of these arguments to the real ocean. Hughes and Meredith (2006) used satellite altimetry to show that signals with periods shorter than 1 year were correlated over very long distances around the global continental slope. One block of correlation they found stretched from Cape Hatteras right round the north of the Atlantic to tropical Africa (their Figs. 6. Application to the real ocean Here, the dynamical signals are so small that bottom pressure can, in principle, be used to monitor changes in global ocean mass. In practice, this has so far been limited to the annual cycle (Hughes et al., 2012; Williams et al., 2014), which has been determined to an accuracy of ± 0.3 mbar, or about ± 1100 Gt of water, but the principle works well at long timescales too (Hughes et al., 2012). Thus, tropical ocean bottom pressure monitoring would provide a valuable contribution to the global sea level budget. 6.2. Achieving the accuracy required Red is after subtracting the depth average based on the 4 shallowest points (those above 3200 m depth, as used in the model analysis), with pale red for 20-min sampling and dark for 5-day means. The dashed blue line shows the expected reduction resulting from this subtraction if the time series at each depth were uncorrelated. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Kontoyiannis, 1990; Polster et al., 2009). We must ﬁnd an independent way to remove this drift. Several possibilities are available with present technology. The most obvious technique is to use bottom pressures determined from satellite gravimetry. Indeed, some success has been claimed for this method by Landerer et al. (2015), who construct a time series of the AMOC from GRACE data. This is an interesting development, but should be treated with some caution. The patterns of bottom pressure seen by GRACE are limited to large horizontal scales, and the illustra- tions in Landerer et al. (2015) show a mode in which contrasting pressure anomalies appear over the entire shelf versus a wide region of the western abyssal plain. This is in contrast to what we see in ocean models where the relevant region is just the thin strip of the continental slope. Furthermore, at 26°N the inference is made based on broad pressure anomalies to east and west being representative of the deep branch of the AMOC, where they could equally well have been inter- preted as representing the shallow branch, producing a time series with the opposite sign. It is possible that the broad-scale patterns seen by GRACE are indirectly reﬂecting the AMOC, especially if the variations are dominated by a large-scale response to wind stress variations over the time period considered, as suggested by Elipot et al. (2017). Or perhaps GRACE is seeing transient changes in broad water mass prop- erties associated with AMOC transport anomalies. What is clear is that satellite gravimetry does not have the spatial resolution to distinguish between the shallow and deeper parts of the continental slope, espe- cially in the presence of neighbouring larger signals both on the con- tinental shelf and at the foot of the slope, so any such measurement relies on larger-scale correlations. 6.2. Achieving the accuracy required We are left with the question of how to make these measurements to the required accuracy. While bottom pressure recorders can be ex- cellent for high-frequency measurements, they are prone to a long- period drift which, although it is well characterised as an exponential plus linear drift, can be very diﬀerent from instrument to instrument and even between deployments of the same instrument (Watts and 41 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 18. Standard deviations of bottom pressures as a function of depth, from 4 years of measurements at the RAPID-Scotian array near Halifax, Nova Scotia. Black shows the result from the raw 20-min sampled data after removal of tides and instrumental drift. Blue shows the variability based on 5-day means of the same data. Red is after subtracting the depth average based on the 4 shallowest points (those above 3200 m depth, as used in the model analysis), with pale red for 20-min sampling and dark for 5-day means. The dashed blue line shows the expected reduction resulting from this subtraction if the time series at each depth were uncorrelated. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) signals. Similar arguments limit the use of upper-ocean and sea level measurements to derive the deep, large scale circulation. Although the inﬂuence of the AMOC has been noted (Bingham and Hughes, 2009b; Duchez et al., 2014; Goddard et al., 2015; McCarthy et al., 2015), it is an indirect link partially masked by other eﬀects such as the inﬂuence of coastal winds (Woodworth et al., 2014, 2017). Following satellite gravimetry, the most straightforward method to obtain bottom pressure at a point is to combine satellite altimetry with hydrographic measurements which allow the calculation of the steric sea level. The diﬀerence between the two is then a measure of ocean bottom pressure. Williams et al. (2015) investigated this approach using collocated tall moorings and bottom pressure recorders as part of the RAPID array at 26°N. 6.2. Achieving the accuracy required They found that it was crucial to have informa- tion right to the surface, that very careful calibration of the moored instruments was required at the start and end of each deployment and that, even with ideal sampling and calibration, accuracy is limited to about 1–2 mbar in 5000 m depths (somewhat less in shallower water, but errors tend to be dominated by surface waters). This approach is therefore marginally feasible, but requires great care in its im- plementation. It may be the best approach for tropical measurements related to global ocean mass. Such a system should incorporate a good quality bottom pressure recorder as a check on system integrity, and to dramatically reduce errors at periods much shorter than the instrument deployment length. The method pursued by the WAVE group (Hughes et al., 2013) fo- cuses not on the pressures themselves, but on diﬀerences between pressures at diﬀerent depths on the slope. Since the most interesting information is not in the depth-independent mode, 〈〉 p H, but in the depth-varying modes, this is suﬃcient to capture these modes. For a vertical sidewall, this would simply be a matter of measuring density at the boundary and using hydrostatic balance to compute the pressure diﬀerences. For a sloping wall, the horizontal pressure diﬀerences are also important (in fact dominant, on all but the steepest slopes, at intra- annual periods). The horizontal diﬀerences can be measured by mea- suring near-bottom currents and invoking geostrophic balance. The detailed theory, known as the Stepping Method, is given by Hughes et al. (2013), who applied this method to the ﬁrst deployment of the RAPID-Scotia array, which was designed with this methodology in mind. Elipot et al. (2013) also applied the method at the predecessors to this array, for which the relevant data was available, but less well sampled. This method requires only bottom and near-bottom mea- surements, thus making the array more robust and simple to maintain than one which also includes moorings reaching to the surface. Fig. 18. Standard deviations of bottom pressures as a function of depth, from 4 years of measurements at the RAPID-Scotian array near Halifax, Nova Scotia. Black shows the result from the raw 20-min sampled data after removal of tides and instrumental drift. Blue shows the variability based on 5-day means of the same data. 6.2. Achieving the accuracy required While the GRACE results are very interesting, they cannot be used as a means to monitor the AMOC on longer timescales without detailed validation and understanding of how the measured signal relates to the AMOC and further understanding of the inﬂuence of plastic deformation of the earth on the observed We reproduce here (Fig. 19), Fig. 11 from Hughes et al. (2013), which shows how accurately the pressure diﬀerence budget can be closed with this measurement system. The residual errors are 0.16 mbar for the depth distance of 600 m between RS1 and RS2, 0.37 mbar for the 1675 m distance between RS4 and RS1, and 0.48 mbar for the 2300 m distance between RS5 and RS1. This method therefore meets the sub- millibar requirement for resolving the signals of interest. Note, though, that RS3 was excluded from this analysis because it showed multiple adjustment periods, and there may be other kinds of deviation from exponential plus linear drift in other cases. Fig. 19 also demonstrates the eﬀectiveness of removal of the high frequency tidal signals when data are sampled in such a way as to re- solve the tides. This can become more of an issue with satellite data, either altimetry or gravimetry, for which a tidal model must be used and aliasing to long periods remains an issue. Long period tides remain in the data but their departure from equilibrium (the equilibrium tide can be calculated accurately from ﬁrst principles) is expected to be limited to periods shorter than a few months, and to be predominantly geostrophic. In this sense, the non-equilibrium tides are simply part of the signal to be monitored. The largest long period tides have ampli- tudes of up to about 3 mbar (Egbert and Ray, 2003). It is, therefore, important at the 1 mbar level to calculate and subtract oﬀthe long period equilibrium tides, including the pole tide. 42 Despite the success of this method, it would be nice to have a more Another proposed system is the use of a reference pressure g. 19. Diﬀerences between directly-measured bottom pressure diﬀerences and those inferred from density and current measurement. Diﬀerences are between (RS2, RS4 RS5) an e Fig. 18). The top panel shows the raw values, together with the exponential-plus-linear instrument drift that is ﬁtted. 6.2. Achieving the accuracy required The bottom panel shows the remainder after removing gether with its standard deviation in Pa (100 Pa = 1 hPa = 1 mbar). Reproduced from Hughes et al. (2013). W. Hughes et al. Progress in Oceanography 161 (2018) Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Fig. 19. Diﬀerences between directly-measured bottom pressure diﬀerences and those inferred from density and current measurement. Diﬀerences are between (RS2, RS4 RS5) and RS1 (see Fig. 18). The top panel shows the raw values, together with the exponential-plus-linear instrument drift that is ﬁtted. The bottom panel shows the remainder after removing drift, together with its standard deviation in Pa (100 Pa = 1 hPa = 1 mbar). Reproduced from Hughes et al. (2013). Despite the success of this method, it would be nice to have a more straightforward means of monitoring bottom pressures without the need for drift corrections. As Fig. 19 shows, the present technology works well apart from the exponential-plus-linear drift, so all that is needed is a small number of measurements without drift to correct this. Another proposed system is the use of a reference pressure in a closed chamber, together with a diﬀerential pressure sensor (Gennerich and Villinger, 2015). The targeted trend accuracy is 0.1 mbar yr−1, which would be a major step forward, but the instrument currently exists in concept only. We would like to encourage these developments, and other in- itiatives to measure ocean bottom pressures. The required precision and stability is of order − 10 7– − 10 8 over a year or longer, if we are aiming at 0.1 mbar (1 mm) in depths of 1000–5000 m. Perhaps a method using ﬂuids rather than crystals would avoid the unknown source of the drift in the quartz crystal sensors presently used? Perhaps some optical technique could provide a better solution? Although the technology is improving, we are still using essentially the system that was developed in the 1970s, it may be time to explore new options. One possibility has been put forward by Sasagawa and Zumberge (2013), who use a known weight acting over a known area to produce a reference pressure at intermittent times. They claim a precision of 1.16 mbar per calibration point, which can reach our required precision if suﬃcient (order 10 or more) calibrations can be made per deploy- ment. 6.3. Final thoughts To summarise, continental slope bottom pressure measurements can make an extremely valuable contribution to a Global Ocean Observing System, monitoring basin-scale dynamics uncontaminated by the me- soscale variability which dominates most other measurements, and al- lowing for measurement of the AMOC. This is possible with current technology if we combine bottom pressure recorders with near-bottom density and current measurements, and are willing to sacriﬁce the less dynamically-interesting depth-independent mode (though a combina- tion of altimetry and a full-depth mooring, plus bottom pressure re- corders, could also provide this mode to about 1–2 mbar accuracy, permitting monitoring of variations in the Arctic throughﬂow plus precipitation minus evaporation, though only with an accuracy of a few sverdrups). Future developments may make direct measurements of bottom pressure more accessible, and these should be encouraged, but an important start can be made with the present technology. Though the accuracies required may seem daunting, these are simply a reﬂec- tion of assuming a 1 sverdrup target accuracy, together with an equivalence of 1 mbar to 1 sverdrup per kilometre at mid latitudes (the pressure signals reduce, and hence the required accuracy increases, at low latitudes). Any system which aims for 1 Sv transport accuracy is implicitly determining bottom pressure at the 1 mbar level. It would seem sensible to make that accuracy an explicit aim, to be sure we are not fooling ourselves when we consider how well the ocean state is monitored. The main thing to note is that, just as with light, there is no way to infer the spectrum from the colour, since many diﬀerent spectra are consistent with the three weighted averages which determine a given colour. Only the forward calculation can be done, to ﬁnd the colour given a particular spectrum. This is how we produce the diﬀerent colour bars on the plots in Figs. 1–5. A particular shape of spectrum is chosen, and various parameters changed, with the resulting colour plotted as a function of those varying parameters. In the ﬁrst three colour bars, the chosen spectrum is a Gaussian as a function of period (i.e. a Gaussian Spectral Power Distribution, as de- scribed in the appendix of Hughes and Williams (2010)). The two varied parameters are the period of the peak (x-axis), and the overall brightness (y-axis). 6.3. Final thoughts Thus, the ﬁrst colour bar shows the intense colours which result from a sharply-peaked spectrum peaking at diﬀerent per- iods, the second shows the same for a broader Gaussian, and the third for a still broader Gaussian (the white lines show the actual Gaussian for a particular period). The fourth colour bar shows the colours for particular power laws, i.e. spectra in which power is proportional to frequency raised to powers in the range −4 to 0. Again, the y-axis represents varying overall brightness. In this case the colours range smoothly from orange- red, through grey-white near a power of −2, to blue at power zero. Note that we do not generally expect steep power laws (−2 or below) to persist to the lowest measured frequencies (there are great diﬃcul- ties with calculating spectra if they do). However, the mapping to colour is only sensitive to our chosen range of periods from 2 to 24 weeks, so the power law only need extend over this range to dom- inate the colour. In an unfortunate clash of terminology, it turns out that broadband white light has what is usually referred to as a “red” spectrum, with power spectral density proportional to frequency raised to the power −2. As a result, such “red” spectra appear in these spectral colour plots as a grey scale (the Wortham and Wunsch (2014) spectrum is “red” in the 2–24 week period range, and hence appears as grey). Similarly, a “white” spectrum (power spectral density independent of 6.2. Achieving the accuracy required However, although a recent deployment of two instruments de- monstrated a clear improvement of the drift, almost eliminating the exponential part and signiﬁcantly reducing the linear trend, a re- maining, mostly linear trend diﬀerence between the instruments re- mained (Sasagawa et al., 2016), amounting to about 5 mbar after 1.5 years. This is very good progress, but not quite at the required ac- curacy yet. Finally, if we did have a very stable pressure sensor, this would then bring into focus the fact that the seaﬂoor itself is not perfectly stable. Secular vertical land motions of order 1 mm yr−1 are a very familiar 43 Progress in Oceanography 161 (2018) 19–46 C.W. Hughes et al. Supercomputing Service (http://www.archer.ac.uk). ATB and JW would like to acknowledge the support of the European Union’s Horizon 2020 research and innovation program under grant agreement No. 633211 (AtlantOS). We would also like to thank the two reviewers for their careful reading and constructive comments. The research materials supporting this publication can be accessed by contacting the corresponding author. feature to those working with tide gauges, and much larger values can be found in tectonically-active areas (indeed, the aim of several bottom pressure measurement experiments has been to measure motion of the seaﬂoor, not ocean dynamics). There is no equivalent of Global Navigation Satellite Systems like GPS to monitor the seaﬂoor, so for the present we would have to rely on placement of instruments in stable positions, and/or accurate models of vertical land movement. In fact, it is not the geometrical motion that is relevant for ocean dynamics, but the motion relative to geopotential surfaces: what we need to know is what level the instrument is at, which involves knowing the Earth’s gravity ﬁeld. Satellite data can help with this, but an intriguing possi- bility for the future may be the use of highly accurate optical clocks. These are now approaching an accuracy of − 10 18 (Margolis, 2014). General relativity predicts that a change in geopotential height causes a clock to run faster if it is raised through a height δh by a factor of approximately gδh c / 2, where c is the speed of light. The factor g c / 2 is about − 10 16, meaning that a clock accuracy of − 10 18 would make it possible to measure level diﬀerences of 1 cm. Appendix A. Interpreting the colour spectrum plots The colour spectrum plots in Figs. 1–5 are designed to give a qua- litative representation of the diﬀerent spectra at each point in the ocean, in a manner that can be naturally interpreted. We do this by exploiting the way the eye interprets spectra of light. In a person with normal vision, three numbers are suﬃcient to describe any colour including its brightness, because the eye has three diﬀerent colour-sensitive receptors with diﬀerent ranges of sensitivity. Thus, the eye is only sensitive to these three diﬀerent weighted averages of the visible spectrum. A particular perceived colour can be produced from many diﬀerent spectra. For example, the “sodium or- ange” of many old street lamps is produced by a spectrum dominated by a very narrow band at about 590 nm wavelength, but the same per- ceived colour can be produced by a combination of red and yellow light. The vertical land movement problem is only an issue for direct measurement of bottom pressure (or satellite gravity measurements, where it becomes a rather diﬀerent issue). For the indirect methods, either altimetry plus a tall mooring or the Stepping Method, precise positioning of the instruments in the ocean is not a major issue and it suﬃces to know depths to a few metres. The details of how we calculate colours from a spectrum are given in the appendix to Hughes and Williams (2010), and we use exactly the same parameters here except that we divide by a ten times smaller normalizing factor of × 1.6 105 in the bottom pressure plots, in order to make them brighter (note however that there is an error in that ap- pendix: the matrices given as A4 and A5 are both transposed). In this appendix we describe in more detail how to interpret the colours and the scales attached to the ﬁgures in this paper. 6.2. Achieving the accuracy required The technological chal- lenges may be formidable, but there is nothing which cannot be over- come in principle. feature to those working with tide gauges, and much larger values can be found in tectonically-active areas (indeed, the aim of several bottom pressure measurement experiments has been to measure motion of the seaﬂoor, not ocean dynamics). There is no equivalent of Global Navigation Satellite Systems like GPS to monitor the seaﬂoor, so for the present we would have to rely on placement of instruments in stable positions, and/or accurate models of vertical land movement. In fact, it is not the geometrical motion that is relevant for ocean dynamics, but the motion relative to geopotential surfaces: what we need to know is what level the instrument is at, which involves knowing the Earth’s gravity ﬁeld. Satellite data can help with this, but an intriguing possi- bility for the future may be the use of highly accurate optical clocks. These are now approaching an accuracy of − 10 18 (Margolis, 2014). General relativity predicts that a change in geopotential height causes a clock to run faster if it is raised through a height δh by a factor of approximately gδh c / 2, where c is the speed of light. The factor g c / 2 is about − 10 16, meaning that a clock accuracy of − 10 18 would make it possible to measure level diﬀerences of 1 cm. The technological chal- lenges may be formidable, but there is nothing which cannot be over- come in principle. Progress in Oceanography 161 (2018) 19–46 Progress in Oceanography 161 (2018) 19–46 frequency, index 0) appears as an intense blue. Clim. 27, 6439–6455. http://dx.doi.org/10.1175/JCLI-D-13-00052.1. Dieng, H.B., Palanisamy, H., Cazenave, A., Meyssignac, B., von Schuckmann, K., 2015. The sea level budget since 2003: inference on the deep ocean heat content. Surv. G h 36 209 229 h d d i 10 100 10 12 01 9314 6 A third spectral shape was used in the generation of Fig. 1b of Hughes and Williams (2010). In that case the spectra were modelled as two power laws: zero for low frequencies, and −4 for high frequencies, with the varying parameter being the period at which the switch be- tween power laws occurs. This was designed to represent an extreme version of sea level spectra like those in Fig. 6, many of which show very steep power laws at high frequency and much gentler power laws at low frequency, with a sharp transition at a period related to the shortest baroclinic Rossby wave period. The range of colours produced with that spectral shape was very similar to that in our second colour bar, for periods shorter than 100 days (i.e. the more intense reds were absent, as they are in the power law colour bar). Geophys. 36, 209–229. http://dx.doi.org/10.1007/s10712-015-9314-6. y g Egbert, G.D., Ray, R.D., 2003. Deviation of long-period tides from equilibrium: kinematics and geostrophy. J. Phys. Oceanogr. 33, 822–839. http://dx.doi.org/10.1175/1520- Egbert, G.D., Ray, R.D., 2003. Deviation of long-period tides from equilibrium: kinematics and geostrophy. J. Phys. Oceanogr. 33, 822–839. http://dx.doi.org/10.1175/1520- 0485(2003)33<822:DOLTFE>2.0.CO;2. Dussin, R., Barnier, B., Brodeau, L., 2014. The Making of Drakkar Forcing Set DFS5. DRAKKAR/MyOcean Rep. 05-10-14, LGGE, Grenoble, France. Elipot, S., Hughes, C., Olhede, S., Toole, J., 2013. Coherence of western boundary pres- sure at the RAPID WAVE array: boundary wave adjustments or deep western boundary current advection? J. Phys. Oceanogr. 43, 744–765. http://dx.doi.org/10. 1175/JPO-D-12-067.1. Elipot, S., Frajka-Williams, E., Hughes, C.W., Willis, J.K., 2014. The observed North Atlantic meridional overturning circulation: its meridional coherence and ocean bottom pressure. J. Phys. Oceanogr. 44, 517–537. http://dx.doi.org/10.1175/JPO-D- 13-026.1. Looking at the relationship between spectrum and colour, we can draw some general conclusions. Pale–mid-blue and orange-red are quite common in smooth spectra, and generally represent more or less power in the high frequencies than would be found with a −2 power law (again, over the 2–24 week period range). 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W3199946842.txt	https://zenodo.org/records/8146972/files/2006-Metais%20isolantes%20e%20transicao%20de%20fase.pdf	fr		Metais, isolantes e transições de fase metal-isolante	Zenodo (CERN European Organization for Nuclear Research)	2,006	cc-by	7,779	V AGNER B ERNAL B ARBETA ; R ENATO F. J ARDIM Artigo METAIS, ISOLANTES E TRANSIÇÕES DE FASE METAL-ISOLANTE Embora muito progresso tenha ocorrido no entendimento Although much progress has been observed in dos fenômenos relacionados ao processo de condução understanding the electronic conduction eletrônica, particularmente após o desenvolvimento da mechanisms in crystalline solids, specially after the mecânica quântica, vários aspectos básicos ainda não são development of quantum mechanics, many basic plenamente compreendidos. A principal razão para isso é o issues are, at present, not fully clear. The main fato de os modelos necessários para se descrever estas reason is that the models needed to describe this situações físicas envolverem um problema de muitos corpos phenomenon involve an interacting many-body interagentes. Um dos assuntos que tem se mostrado problem. One of the research fields that has bastante complexo para o seu estudo é o de sistemas que shown an intense activity involves compounds apresentam uma transição entre uma fase condutora that undergo an electronic phase transition from (metálica) e uma fase isolante ou semicondutora. Estas a metallic to an insulating or semiconducting transições de fase, genericamente chamadas de transições state. The so-called metal-insulator transitions, do tipo metal-isolante, têm sido estudadas de forma observed in a large family of oxides, attracted the extensiva, particularmente em óxidos e em sulfetos. Além attention of many researchers specially after the do interesse científico no estudo desses materiais, existe um observation of superconducting properties in grande interesse tecnológico, motivado especialmente pela some of these compounds. Besides the possibility observação de propriedades supercondutoras em altas of finding superconductivity, many other features temperaturas em algumas famílias de óxidos. Além disso, a make these systems technologically interesting. possibilidade de se modificar as propriedades de condução Modification of the electrical conduction elétrica através de variação de parâmetros termodinâmicos properties with thermodynamic parameters like como temperatura, pressão e campo magnético, abre temperature, pressure and magnetic field, makes grandes perspectivas para o uso destes compostos na these compounds also suitable for the construção de sensores. Pretende-se com este artigo realizar development of many types of sensors. This article uma breve descrição a respeito dos modelos de condução is intended to briefly review the main electronic elétrica, discutindo-se ainda em que situações um portador conduction models and some of the situations de carga itinerante pode se tornar localizado, ou seja, where an itinerant charge carrier become levando um composto a apresentar uma transição de fase localized, i.e., show a metal-insulator transition. do tipo metal-isolante. Será analisado particularmente o The specific situation of transition metal oxides caso de óxidos de metais de transição. will be detailed. VAGNER BERNAL BARBETA - Professor do Departamento de Física do Centro Universitário da FEI RENATO F. JARDIM - Professor do Departamento de Física dos Materiais e Mecânica da Universidade de São Paulo 50 P E S Q U I S A & T E C N O LO G I A F E I - Nº 28 Palavras-chave: teoria de bandas, transição metal-isolante, óxidos de metais de transição. Keywords: band theory, metal insulator transition, transition metal oxides. METAIS, ISOLANTES E TRANSIÇÕES DE FASE METAL-ISOLANTE Introdução Uma das formas de se agrupar os materiais pela observação de sua condutividade elétrica, separandoos em dois grandes grupos: os metais e os isolantes, incluindo-se neste último os semicondutores. Os metais, ou condutores, têm atraído desde há muito tempo o interesse dos cientistas e diversos modelos físicos foram criados para explicar os processos de condução elétrica nesses materiais. Os compostos metálicos são aqueles que apresentam uma baixa resistividade elétrica, ou seja, alta condutividade elétrica. Tipicamente um metal possui uma resistividade que varia entre 10-2 e 10-6 !cm. Além de possuírem uma boa condutividade elétrica são também bons condutores de calor, já que os portadores de carga (em geral elétrons livres) que promovem a condução elétrica também auxiliam o processo de condução térmica. Por esse motivo, os termos metal e condutor (de eletricidade e de calor) são utilizados como sinônimos em diversos textos, inclusive neste. Além de elementos químicos como alumínio, cobre, estanho e ouro, entre outros, diversos óxidos e sulfetos também podem ser classificados como materiais metálicos. Entre eles pode-se citar o TiO, CrO2, NbO, RuO2, ReO3, TiS, CoS2 e CuS2. Os isolantes e semicondutores são materiais que apresentam uma grande resistividade elétrica, com valores da ordem de 103 a 1017 !cm. A resistividade elétrica desta classe de materiais normalmente decresce com o aumento da temperatura. Além dos elementos como o silício e o germânio, encontram-se neste grupo materiais como o NiO, CoO, MnO, Fe2O3, Cr2O3, MnS, FeF2, MnS2 e o CuO. Uma outra categoria de compostos apresenta um comportamento metálico em uma faixa de tempera- tura e um comportamento isolante em outra, ou seja, apresenta uma transição de fase metal-isolante, podendo-se citar como exemplos o V2O3, VO2, VO, V6O13, Ti2O3, NbO2, Fe2O4, V3O5, V4O7, Ti3O5, NiS, CrS e o FeS. O primeiro modelo a explicar com relativo sucesso o processo de condução elétrica em metais foi proposto por P. Drude [1], no início do século XX. No modelo de Drude os elétrons de condução são imaginados como constituindo um “gás” de partículas livres e independentes, e a estas partículas foram aplicados conceitos semelhantes àqueles utilizados na teoria cinética dos gases. Um dos grandes sucessos do modelo de Drude foi prever com enorme concordância a lei de Wiedemann-Franz, que estabelece a relação entre as condutividades térmica e elétrica, o que se verificou mais tarde ter sido resultado de um erro de cálculo cometido por Drude. Porém, este modelo é falho, não sendo capaz de explicar diversas observações experimentais [2]. O modelo de Sommerfeld [3], o qual incorporou elementos da mecânica quântica, resolveu parcialmente as falhas do modelo de Drude. A grande diferença entre estes modelos é que o modelo de Sommerfeld considera que os elétrons formam um “gás” de férmions, ou seja, partículas com spin semiinteiro e que obedecem, portanto, ao princípio de exclusão de Pauli. Com essa modificação Sommerfeld pôde explicar, por exemplo, a contribuição dos elétrons livres ao calor específico, que era erroneamente prevista pelo modelo de Drude, embora outras falhas continuassem a existir, como a não previsão do efeito Hall positivo. É interessante notar que o modelo de Sommerfeld, assim como o de Drude, é considerado como um modelo clássico de condução, já que para a descrição do comportamento dinâmico dos elétrons não se consi- deram os princípios da mecânica quântica. A incorporação, de forma definitiva, da mecânica quântica ao estudo dos metais ocorreu com o desenvolvimento de modelos de elétrons quase livres, como no chamado modelo de bandas, introduzido por Wilson em 1931 [4]. No modelo de bandas existem faixas de valores de energia (bandas), dentro das quais a energia do elétron deve se encontrar, existindo regiões de energia proibida (gaps) separando essas regiões de energias permitidas. As propriedades de transporte dos materiais estão ligadas às energias dessas bandas e à maneira como elas são preenchidas. A introdução do conceito de bandas de energia, além de melhor explicar o porquê da existência de sistemas metálicos e isolantes, permitiu entender o comportamento de outra classe de materiais, os chamados semicondutores, cuja resistividade diminui muito rapidamente com o aumento da temperatura [2, 5]. Embora tenha apresentado grande sucesso para a explicação das propriedades de transporte eletrônico e seja a base para o entendimento do comportamento de diversos sistemas metálicos, isolantes e semicondutores, a teoria de bandas também apresenta falhas quando aplicada a certos compostos. É interessante notar, por exemplo, que materiais aparentemente semelhantes como o MnO (isolante) e TiO (condutor), ambos como a mesma estrutura cristalina e portanto devendo apresentar o mesmo esquema de bandas de energia, possuem comportamentos bastante distintos, variando a resistividade entre eles por um fator de cerca de 1020. A explicação para esse tipo de comportamento foi feita, em 1949, por Mott [6]. Segundo Mott, o estado isolante pode ocorrer em um sistema que a priori seria metálico, se for P E S Q U I S A & T E C N O L O G I A F E I - Nº 28 51 Artigo levado em conta o efeito do potencial coulombiano resultante da transposição de um elétron “livre” para um sítio já ocupado por outro elétron “livre”. Nesse caso, existirá uma força de atração coulombiana U entre a carga removida e o respectivo “buraco” criado na região onde este se encontrava, formando uma quasepartícula de nome éxciton. Esta quase-partícula não é livre e o estado metálico pode ser obtido se esse potencial coulombiano for mascarado, através do aumento do número destas quase-partículas acima de um certo valor crítico. Ou, pensando de uma forma distinta, abaixo de um certo valor crítico de elétrons portadores de carga, o efeito do potencial coulombiano deixa de ser mascarado e os elétrons, que seriam livres, ficam ligados a seus buracos, formando um éxciton, que não pode participar do processo de condução elétrica. O que se verifica, na visão proposta por Mott, é que em alguns sistemas não se pode considerar os elétrons como independentes, ou seja, não se pode negligenciar a correlação elétronelétron, já que nesses casos é ela quem irá determinar as propriedades de transporte eletrônico do sistema. Neste caso, a descrição deixa de ser a de um gás de férmions (partículas independentes) para ser dada por um líquido de férmions (partículas interagentes). Na formulação de Mott encontra-se embutida a possibilidade de um sistema isolante tornar-se metálico se houver algum parâmetro externo que venha a ser variado e que faça com que o número de éxcitons seja maior do que o valor crítico de mascaramento do potencial. Acima deste valor crítico os portadores de carga tornam-se livres e a sua quantidade deve crescer muito rapidamente, o que deve ocasionar uma transição de fase de primeira ordem. Ao longo dos anos foram descobertos diversos tipos de sistema que apresentam transições de fase 52 P E S Q U I S A & T E C N O LO G I A F E I - Nº 28 metal-isolante. Em certos casos o estado isolante ocorre devido a outros efeitos que não aquele proposto originalmente por Mott, como, por exemplo, no caso de isolantes do tipo “transferência de carga”. O estudo das transições de fase metal-isolante é, portanto, bastante profícuo, e as razões pelas quais tais transições ocorrem em alguns tipos de compostos ainda não são plenamente compreendidas. Para o estudo de tais sistemas costuma-se fazer alterações em um ou mais parâmetros termodinâmicos (como pressão e temperatura), realizar a introdução de desordem, ou alterar o grau de interação elétron-elétron ou elétron-rede, como forma de se verificar como estes parâmetros influenciam as propriedades de transporte e a transição entre as duas fases eletrônicas. Modelos clássicos de condução elétrica Pouco tempo após a descoberta do elétron por J. J. Thomson em 1887, P. Drude propôs um modelo para o entendimento do comportamento metálico. Em seu modelo, um condutor pode ser entendido como sendo composto de uma série de íons positivos, imóveis, em torno dos quais se encontra uma nuvem de elétrons móveis [1]. Estes elétrons móveis, chamados também de elétrons de condução, são aqueles que se encontram menos ligados ao núcleo dos átomos. Eles formam um gás de partículas onde são negligenciadas as interações elétron-íon e elétron-elétron, ou seja, os elétrons são livres e independentes. O único mecanismo de interação que se considera é o eventual espalhamento dos elétrons de condução pelos núcleos (íons), através de choques semelhantes ao de esferas rígidas. Com isso é possível utilizar as idéias da teoria cinética dos gases, com a diferença de que num gás o choque ocorre entre as partículas do próprio gás, enquanto neste modelo de condução eletrônica o choque é entre as partículas do gás de elétrons com os íons fixos. O processo de equilíbrio térmico é estabelecido, portanto, através do choque dos elétrons de condução com os íons da redondeza, fazendo com que após a colisão o elétron tenha uma velocidade que seja compatível com a temperatura na região. A distribuição estatística de velocidades assim obtida deve seguir a mesma de um gás de partículas, isto é, deve seguir a distribuição de Maxwell-Boltzmann. No modelo de Drude, a r densidade de corrente j , que se estabelece r quando um campo elétrico E é aplicado, pode ser escrita como: r ne 2l r j = E mv0 sendo n a densidade de elétrons (quantidade de elétrons por unidade de volume), e a carga do elétron, l o livre caminho médio (distância média percorrida entre colisões sucessivas), m a massa do elétron e v0 a velocidade média estimada por Drude a partir da distribuição de Maxwell-Boltzmann (dependente da temperatura T) e que é dada por: 1 " 8k T % 2 v0 = $$ B '' # m( & Ou seja, a resistividade ) pode ser escrita como: )= mv0 ne 2l Uma das previsões mais impressionantes da teoria de Drude foi a explicação da lei empírica de Wiedemann-Franz, a qual estabelece que a razão entre a condutividade térmica k e a condutividade elétrica * e = 1 / ) é diretamente proporcional à temperatura, sendo a constante de proporcionalidade aproximada- METAIS, ISOLANTES E TRANSIÇÕES DE FASE METAL-ISOLANTE mente a mesma para uma ampla variedade de metais (cerca de 2,45x10"8 W!/K2). Posteriormente, observou-se que os cálculos realizados por Drude continham um fator dois errado, o que fazia com que seu modelo não apresentasse a concordância que se acreditava possuir [2]. O modelo prevê ainda uma contribuição eletrônica ao calor específico de (3/2)kB por elétron, o que não é observado experimentalmente. Nas medidas de efeito Hall, realizadas em alguns materiais, é obtido um sinal diferente daquele esperado teoricamente (o chamado efeito Hall positivo). Além disso, a variação da resistividade elétrica com a temperatura, para altas temperaturas, também é erroneamente prevista, pois segundo o modelo ) deve variar com T 1/2 , enquanto experimentalmente observava-se uma variação linear. Com o desenvolvimento da mecânica quântica, o modelo de Drude foi superado. Sommerfeld [3] foi o primeiro a propor um outro modelo, que em essência era muito semelhante àquele proposto por Drude. Considerou, porém, que os elétrons livres devem obedecer ao princípio de exclusão de Pauli, ou seja, que duas partículas do gás de elétrons não podem ocupar o mesmo estado quântico. Assim, a distribuição de Maxwell-Boltzmann (MB) para as velocidades após o choque deve ser substituída pela distribuição de Fermi-Dirac (FD) fFD (+ ) , mostrada abaixo, a qual nos fornece a probabilidade de um estado com energia + se encontrar ocupado. fFD (+ ) = 1 e (+ , µ ) / kBT +1 onde µ é o potencial químico do material, que é função da temperatura T. Na temperatura de zero absoluto µ = + F , sendo + F (energia de Fermi) o valor da máxima energia que um elétron livre de um metal pode ter em T = 0 K. É interessante notar que na distribuição de Maxwel-Boltzmann fMB (+ ) = Ae ,+ / kBT , em T = 0 K, a energia dos elétrons é estritamente nula, aumentando a quantidade de partículas com energia diferente de zero à medida que a temperatura aumenta. No caso da distribuição de FD, a T = 0 K, todos os valores de energia estarão ocupados até o limite de + = + F . Assim, no caso da distribuição de FD, a velocidade média dos elétrons a uma dada temperatura é muito maior do que aquela obtida por Drude usando a distribuição de MB. Deste modo, enquanto que para Drude o livre caminho médio l esperado era da ordem da distância interatômica a, para o modelo de Sommerfeld obtinha-se l ~ 100 a. Deve-se observar que a troca da distribuição de MB pela distribuição de FD faz com que o modelo continue sendo essencialmente clássico, sendo os elétrons livres ainda considerados como partículas que são espalhadas pela rede cristalina e que não interagem entre si. O modelo de Sommerfeld, no entanto, permite a solução de alguns problemas que a teoria de Drude apresenta, como o fato de prever erroneamente um alto valor para a contribuição eletrônica do calor específico. Embora os chamados modelos clássicos de elétrons livres (Drude e Sommerfeld) tenham sucesso na explicação dos efeitos de condução elétrica e condução térmica de vários sistemas, apresentam também uma série de falhas. Entre os problemas mais notáveis, pode-se destacar: ! não explicam como a resistividade elétrica varia para diferentes tipos de materiais; ! não prevêem corretamente como é a variação das propriedades de transporte elétrico com a temperatura e nem a variação destas com a pressão; não explicam porque alguns materiais são isolantes e outros condutores. Por exemplo, o diamante e o grafite são formados de carbono e, no entanto, um deles é isolante e o outro condutor; ! não são capazes de explicar o efeito Hall positivo. Muitas das dificuldades teóricas impostas pelo modelo de Drude, e posteriormente pelo modelo de Sommerfeld, só foram efetivamente resolvidas com a incorporação, de forma definitiva, das idéias da mecânica quântica. O chamado modelo de bandas [4, 5], baseado na existência de níveis de energia quantizados nos átomos e no fato de os elétrons terem um comportamento ondulatório, levou ao abandono dos modelos clássicos de condução elétrica, embora em muitas situações eles continuem a ser usados, por fornecerem uma boa descrição qualitativa de diversos sistemas metálicos. ! O modelo de bandas O modelo de bandas foi proposto a partir dos desenvolvimentos da mecânica quântica. Neste caso, o estado de uma partícula qualquer deve ser descrito em termos de um conjunto de números quânticos, a partir dos quais as propriedades do estado dessa partícula podem ser obtidas. Considere-se um sólido cristalino qualquer, formado por uma série de átomos dispostos de forma regular. Para se entender como podem ser dispostos os elétrons que compõem os átomos deste sólido é preciso resolver a equação de Schroedinger para cada um deles. A partir da solução desta equação, é possível obter as funções de onda que descrevem as propriedades dessas partículas, assim como as energias associadas a elas. Este tipo de problema é bastante complexo, até P E S Q U I S A & T E C N O L O G I A F E I - Nº 28 53 Artigo mesmo para um átomo isolado, tendo sido desenvolvidos diversos métodos para a realização destes cálculos de forma aproximada. Uma abordagem possível é considerar um conjunto muito grande de átomos do mesmo tipo muito afastados entre si, de modo que cada um não interaja com os outros. Neste tipo de situação os elétrons de cada um dos átomos poderão ser descritos por um mesmo conjunto de números quânticos e cada um deles terá o mesmo diagrama de níveis de energia, que pode ser obtido por métodos numéricos. Ao serem aproximados dois átomos, estes passam a interagir entre si e os níveis de energia do sistema composto por esses dois átomos modificam-se, de forma a atender ao princípio de exclusão de Pauli. Assim, estes átomos isolados, ao serem aproximados uns dos outros para se formar uma rede cristalina, apresentam níveis de energia distintos, fazendo com que um nível de energia do átomo isolado seja dividido em N níveis de energia muito próximos, sendo N o número de átomos da rede. O que era um conjunto de átomos isolados, cada um descrito pelo mesmo diagrama de níveis de energia, passa a ser um sistema de átomos que interagem, que não são mais descritos por níveis e sim por bandas de energia, com valores de energia dentro de uma banda muito próximos uns dos outros, de modo que este diagrama parece ser contínuo. Quanto menor for a distância interatômica, maior será a largura que se observa nas bandas de energia. A figura 1 mostra uma representação da abertura dos níveis e energia quando ocorre a diminuição da distância interatômica, onde se observa a formação de bandas de energia e de um gap de energia proibida. Este tipo de abordagem é utilizado em um método de cálculo de estruturas eletrônicas chamado de Tight Binding [5]. 54 P E S Q U I S A & T E C N O LO G I A F E I - Nº 28 Fig. 1 – Bandas de energia em um cristal com a variação da distância interatômica A altura da barreira de potencial a ser vencida pelos elétrons mais internos para que estes saiam de um átomo e se desloquem para outro é tão grande quando comparada com a sua energia, que a probalidade destes elétrons passarem (tunelarem) para um átomo vizinho é extremamente pequena. Por este motivo, os elétrons mais internos podem ser considerados como sendo localizados e suas funções de onda são, basicamente, as mesmas que aquelas obtidas considerando-se os elétrons pertencentes a um átomo isolado. Por outro lado, os elétrons mais externos podem ter energia maior que a barreira de potencial a que estão sujeitos e por isso podem movimentar-se pela rede cristalina. São estes elétrons mais externos os responsáveis pela maioria das propriedades coletivas dos sólidos cristalinos, tais como condutividade térmica, condutividade elétrica, etc. Quando a banda mais externa está completamente cheia, ela é chamada de “banda de valência”; este é o caso de materiais isolantes (que são também chamados de isolantes de banda). Por outro lado, se a banda mais externa está semipreenchida, ela é chamada de “banda de condução”; este é o caso de materiais condutores. Note que, se por alguma razão, a distância interatômica de um cristal for diminuída, a última banda completamente preenchida de um isolante pode se superpor à banda seguinte, que se encontrava completamente vazia, formando assim uma nova banda semipreenchida e o material deixará de apresentar um comportamento isolante, passando a apresentar um comportamento condutor. Uma abordagem alternativa para a descrição quântica de um metal cristalino é resolver a equação de Schroedinger para um determinado elétron da camada mais externa. Se este for considerado livre, a existência de bandas de energia não é observada e os resultados são aqueles obtidos por Sommerfeld. Assim, em um modelo de elétrons livres, a relação de dispersão (relação entre a energia + e o vetor de onda k) é parabólica e é dada por: += p2 2m sendo h = = h 2k 2 h 2( 2m (h é a constante de Planck), m a massa do elétron e k= p h O modelo apresentado anteriormente pode ser melhorado quando considerado o efeito do potencial periódico da rede cristalina ao qual o elétron está sujeito, resultando assim em um elétron quase livre. Um resultado importante que se obtém neste caso é o surgimento natural de regiões de r energia proibida, quando o vetor k (definido a seguir) estiver próximo a certos valores, chamados de limites das zonas de Brillouin. Deste modo, bandas de energia são formadas e, conforme proposto por Wilson [4], o esquema de preenchimento destas bandas é quem irá determinar as propriedades de condução do material cristalino. A função de onda do elétron quase-livre será dada, conforme o teorema de Bloch [7], por uma função de onda de uma partícula livre multiplicada por uma função moduladora u(x,y,z), que possui a mesma periodicidade da rede METAIS, ISOLANTES E TRANSIÇÕES DE FASE METAL-ISOLANTE embora classicamente o elétron devesse continuar sendo acelerado à medida que k aumenta. Isso é semelhante a imaginar que a partícula tem massa positiva, porém a carga do elétron tenha um sinal contrário, ou seja, positivo. Neste caso, o sistema se comporta como se o portador de carga fosse uma carga positiva. Isto explica a possibilidade dos portadores de carga serem elétrons ou buracos, dependendo de como a banda se encontra preenchida (pouco ocupada ou muito ocupada). Fig. 2 – Relação de dispersão obtida para um elétron quase livre, sujeito a um potencial periódico. cristalina. Isto é, a função de onda - k ( x, y, z) do elétron será dada por: r r - k ( x, y, z) = u( x, y, z).e ik .r r O vetor k , embora semelhante ao vetor que define o momento de um elétron no modelo de Sommerfeld (modelo de elétron livre), não possui o mesmo significado. Neste caso k é um número quântico que descreve o estado em que um elétron de Bloch pode se encontrar e não representa o momento do elétron. A energia total do elétron quaselivre não é apenas cinética, mas possui também um termo potencial, devido à rede, fazendo assim com r que a energia de um elétron no + ( k ) r estado k dependa do potencial na região onde o elétron se encontra. A expressão da relação de dispersão será, portanto, mais complicada que a obtida para o caso de um elétron livre (relação de dispersão parabólica) e possuirá descontinuidades quando k = n(/a. No entanto, exceto quando as energias sejam tais que k se aproxime dos valores k = n(/a, o elétron poderá movimentar-se praticamente livre, sem perceber o efeito da rede cristalina, comportando-se de forma semelhante a um elétron realmente livre (relação de dispersão parabólica). A figura 2 apresenta a relação de dispersão de um elétron quase-livre, para o caso unidimensional, onde se observam os gaps de energia quando k = n(/a. Considerando-se apenas uma dimensão, como, por exemplo, a direção x, a velocidade de grupo vx da onda associada ao portador de carga pode ser dada por: vx = df d (1 / . ) Sendo f = + / h e . = 2 ( / kx , é possível escrever vx como sendo dado por: vx = 1 /+ h /kx Pode-se, portanto, escrever que a aceleração ax, à qual está sujeito o elétron em uma dada direção, que será dada por: ax = dv x dt = 1 d 2+ dkx h dkx2 dt = 1 d 2+ h 2 dkx2 Fx É possível definir uma massa efetiva m* para o sistema pela relação: 1 m* Fig. 3 – Curva de dispersão com as diferentes regiões de comportamento eletrônico. = ax Fx = 1 d 2+ h 2 dkx2 ou seja, pela mudança da concavidade da curva de dispersão (+ versus k), pode-se ter uma massa efetiva m, que será positiva ou negativa. Assim, ao ser analisada a curva de dispersão, mostrada na figura 3, observam-se duas regiões de comportamento distinto: na região I tem-se que v cresce à medida que k aumenta; na região II, v decresce quando k aumenta. Portanto, a região II se comporta como se o sistema tivesse uma massa negativa, pois a velocidade diminui A densidade de corrente devido a um elétron pode ser obtida a partir da velocidade de grupo da onda associada a ele. Portanto, a densidade de corrente será dada por: j x = , ev x = , e /+ h /kx No caso tridimensional, a expressão acima pode ser escrita como: r e r j = , 0 k+ h A densidade de corrente total será então dada pela soma sobre todos os estados ocupados da contribuição de cada elétron. Quando todos os estados estão completamente preenchidos (banda cheia), para cada contribuição ao longo de uma certa direção, existe uma contribuição igual com sinal oposto, o que leva a uma densidade de corrente líquida nula. Ou seja, se uma banda estiver completamente preenchida, a corrente total será P E S Q U I S A & T E C N O L O G I A F E I - Nº 28 55 Artigo nula, pois para cada elétron viajando em uma dada direção (kx positivo), existe outro com mesma velocidade viajando na direção oposta (k x negativo). Por outro lado, se uma banda se encontra parcialmente ocupada, mais elétrons podem estar viajando em uma direção do que na direção oposta, o que irá resultar em uma corrente líquida no sistema. Considerando-se a curva de dispersão, haverá uma assimetria nos valores de k assumidos pelos elétrons, ou seja, mais elétrons terão o valor de kx > 0 do que o contrário (ou vice-versa). É por este motivo que bandas completamente preenchidas não podem contribuir para o processo de condução elétrica, mas apenas aquelas que se encontram parcialmente preenchidas. Deve-se notar que a condição fundamental para que ocorra condução de corrente para uma banda semipreenchida é que haja um desequilíbrio na população dos estados (para kx > 0 e kx < 0), o que é feito, por exemplo, aplicando-se um campo elétrico externo ao material. O mesmo tipo de desequilíbrio pode ser obtido se for aplicado um gradiente de temperatura no material, resultando, nesse caso, no aparecimento do efeito termoelétrico. Cada banda que surge ao se constituir o material cristalino pode acomodar até 2N estados eletrônicos, sendo N o número de celas primitivas do cristal e o fator 2 é devido às duas possíveis orientações do spin. É possível concluir, portanto, que um cristal pode ser um isolante somente se o número de elétrons de valência em uma cela primitiva do cristal é um número par. Caso este número seja ímpar, estes deveriam, a princípio, levar a um comportamento condutor, já que não é possível preencher completamente uma banda com um número ímpar de elétrons. 56 P E S Q U I S A & T E C N O LO G I A F E I - Nº 28 Outra observação interessante é que, devido ao fato de as funções de onda de Bloch descreverem estados que são estacionários quando o potencial é perfeitamente periódico, não ocorre atenuação dessas ondas. Ou seja, em um cristal perfeito não haverá espalhamento dos elétrons e a resistência elétrica deve ser nula em T = 0 K. A resistência elétrica irá aparecer devido ao efeito de agitação térmica, defeitos e impurezas, que causam irregularidades no potencial periódico a que os elétrons estão sujeitos. Embora o entendimento conceitual obtido com o modelo de bandas seja bastante grande, é importante salientar aqui que ele não consegue explicar a contento o comportamento observado para compostos como o MnO, o FeO, o NiO e o CrO. Nestes óxidos, a quantidade de elétrons de valência na cela unitária é impar e seria esperado que esses sistemas tivessem um comportamento metálico. No entanto, observa-se experimentalmente que eles possuem um comportamento isolante. A grande falha do modelo de bandas está no fato de não levar em consideração os efeitos de correlação eletrônica, isto é, os elétrons continuam sendo considerados não interagentes entre si (elétrons independentes). A proposição de que os efeitos de correlação eletrônica poderiam ser os responsáveis pela observação de um comportamento isolante em sistemas que aparentemente seriam metálicos foi levantada por Mott em 1949 [6]. A descrição do estado metálico, visando incorporar os efeitos de correlações eletrônicas, avançou muito com a aplicação da teoria de líquido de Fermi [8], que foi inicialmente desenvolvida por Landau para explicar o comportamento do 3He superfluído. Óxidos de metais de transição com elétrons do tipo d A forma com que as bandas de energia de um material se configuram, em especial a de valência e a de condução, determina fortemente as suas propriedades. De modo geral, a banda s dos metais se superpõe à banda d. Porém, nos óxidos de metais de transição, a banda s se encontra deslocada alguns elétron-volts acima da energia de Fermi +F. Por outro lado, a banda 2p, proveniente do oxigênio que liga dois metais de transição adjacentes, encontra-se completamente preenchida e, no caso geral, ela se encontra alguns elétron-volts deslocada para baixo de +F. Assim, as únicas bandas na região próxima à energia de Fermi são aquelas provenientes de orbitais do tipo d, associadas ao metal de transição que forma o composto, sendo estas bandas, portanto, as responsáveis pelas propriedades de condução elétrica desses materiais. Os orbitais d que se encontram na região de +F são do tipo 3d, que apresentam uma degenerescência de 10, incluídos aqui os dois possíveis valores de spin, ou seja, existem 5 tipos distintos de orbitais que resultam na mesma energia. Neste caso, obtém-se 3 orbitais do tipo t2g (dxy, dxz e dyz) e 2 orbitais do tipo eg (dx2-y2 e d3z2-r2). A figura 4 apresenta uma representação dos orbitais t2g e eg. Fig. 4 – Representação dos orbitais possíveis para o nível 3d. METAIS, ISOLANTES E TRANSIÇÕES DE FASE METAL-ISOLANTE No caso dos óxidos de metais de transição, esta degenerescência é resolvida pela anisotropia do campo cristalino, que surge devido à presença do átomo ligante, no caso o oxigênio. Quando o sistema apresenta a menor simetria (ortorrômbica), todos os cinco orbitais encontramse resolvidos (apresentam diferentes energias), conforme pode ser visto na figura 5. bandas semipreenchidas por apresentarem um número ímpar de elétrons. No entanto, é verificado experimentalmente que todos os quatro compostos são isolantes, isto é, existe um gap de energia entre uma banda que se encontra completamente cheia e a banda seguinte que deve se encontrar completamente vazia. Esse gap de energia aparece devido a efeitos de correlação Fig. 5 – Abertura dos orbitais 3d pelo campo cristalino, para diferentes tipos de simetrias do cristal. Os números abaixo dos níveis representam a degenerescência. Assim, ao serem considerados materiais como o MnO (3d5), FeO (3d6), CoO (3d7) e NiO (3d8), verificase que todos possuem a mesma estrutura sal gema (cúbica, com cátions nos sítios octaétricos, semelhante à do NaCl). Nestes compostos a banda 3d, sob ação do campo cristalino, se separa em duas sub-bandas, uma inferior (t2g) com degenerescência 6, e uma superior (eg) com degenerescência 4, conforme mostrado na figura 5. Existe, neste caso, a possibilidade de abertura de um gap de energia entre estas duas sub-bandas. Isso acontecerá caso a largura da banda seja pequena, ou da ordem da abertura das duas subcamadas, o que é esperado acontecer nestes sistemas. Assim, utilizando-se argumentos de teoria de bandas, espera-se que o único composto isolante deste grupo seja o FeO, que apresenta uma banda completamente cheia (t 2g, com 6 elétrons). Os outros deveriam ser condutores, já que possuem as eletrônica, que não são levados em conta em um modelo de elétrons independentes. Compostos como o MnO e o CoO, que por uma análise de teoria de bandas pura deveriam ser condutores mas que são na realidade isolantes, são conhecidos como isolantes de Mott. Nos isolantes convencionais (isolantes de banda) os elétrons são considerados como fracamente correlacionados, ao contrário dos isolantes de Mott, onde existe forte correlação eletrônica. Este efeito de correlação eletrônica é responsável pelas propriedades isolantes, pois o potencial coulombiano de repulsão entre os elétrons faz com que a movimentação dos mesmos seja energeticamente desfavorável, como discutido a seguir. Isolantes de Mott Embora a teoria de bandas tenha sucesso em explicar o comportamento de diversos sistemas, observa- se que ela falha para o caso de muitos óxidos de metais de transição que possuem uma banda d parcialmente preenchida. Um exemplo típico de um sistema desse tipo é o NiO, discutido anteriormente, que deveria ser um condutor por uma análise de teoria de bandas, mas que é na realidade um isolante. Peierls foi um dos primeiros a observar que a correlação elétronelétron não poderia ser desprezada em alguns sistemas, já que poderia existir uma forte repulsão coulombiana entre os elétrons e que esta energia de interação U poderia ser a razão pela qual alguns sistemas aparentemente metálicos fossem na realidade isolantes [9]. Em 1949, Mott [6] propôs uma forma para entender de que modo essas correlações eletrônicas podem levar materiais como o NiO a serem isolantes. Tais compostos são chamados genericamente de isolantes de Mott. Distingue-se, entre dois tipos de isolantes de Mott (termo genérico para isolantes que não podem ser entendidos por teoria de bandas), os isolantes de Mott-Hubbard e os isolantes de transferência de carga. Essa distinção ficou clara com o trabalho de Zaanen et al. [10], em 1985, e pode ser entendida pelo esquema de níveis de energia mostrado na figura 6. Nos isolantes de Mott-Hubbard, o gap de energia depende de U, isto é, está relacionado com a energia necessária para promover um elétron da banda 3d para a banda 3d, já que a banda 3d é separada em duas devido ao efeito da energia potencial U, formando um gap d-d. Por outro lado, nos isolantes do tipo transferência de carga, o gap depende de 1, isto é, está relacionado com a energia necessária para promover um elétron da banda 2p (do oxigênio) para a banda 3d (do metal de transição). Zaanen et al. mostraram que se U > 1, o gap é do tipo transferência de carga, enquanto que P E S Q U I S A & T E C N O L O G I A F E I - Nº 28 57 Artigo Fig. 6– Diagrama mostrando as energias em isolantes do tipo (a) Mott-Hubbard e (b) transferência de carga, sem a energia de interação U (esquerda) e com a energia U (direita). se U < 1, o gap é do tipo MottHubbard (do tipo d-d). Transição de fase metal-isolante Existem diferentes mecanismos físicos responsáveis pelo estabelecimento do estado isolante em um sistema metálico, que podem ser agrupados em duas grandes categorias: aqueles em que ocorre a diminuição da mobilidade dos portadores de carga, como, por exemplo, quando ocorre o aumento da massa efetiva; e aqueles em que ocorre a redução a zero do número de portadores, como, por exemplo, através da abertura de um gap de energia em torno da energia de Fermi [11]. Na realidade, em transições de fase metal-isolante os dois mecanismos podem estar agindo de forma simultânea, o que às vezes torna o estudo deste tipo de transição bastante complexo. A teoria de líquido de Fermi, utilizada para explicar o estado metálico quando não há quebra de simetrias, mostra que ao ser introduzida a interação elétronelétron, não existe alteração do número de portadores de carga, o que significa que a única forma deste modelo ser compatível com uma transição metal-isolante contínua é que ocorra uma divergência na 58 P E S Q U I S A & T E C N O LO G I A F E I - Nº 28 massa efetiva m da quase-partícula formada, ou seja, o estado isolante pode ocorrer neste modelo através da diminuição da mobilidade do portador de carga [12]. Em outras situações como, por exemplo, quando ocorre ordenamento magnético e o surgimento, portanto, de uma superestrutura que diminui o grau de simetria do sistema, pode haver uma abertura dos níveis de energia e conseqüentemente o surgimento de um gap de energia no nível de Fermi, ou seja, o estado isolante é alcançado nesta situação pela diminuição do número de portadores de carga. Note que, neste caso, a teoria de líquido de Fermi não mais se aplica (já que existe quebra de simetria no sistema). No caso de óxidos de metais de transição, devido às interações de supertroca, o estado isolante mais comum é aquele que se estabelece devido à quebra de simetria, como no caso de ordenamento antiferromagnético do sistema de spins. Outra possível situação de quebra de simetria é o ordenamento orbital. Como o orbital t 2g é triplamente degenerado em uma simetria cúbica, o isolante de Mott pode se estabelecer devido a um ordenamento orbital alternado (similar ao ordenamento antiferromagnético de spins), ou uniforme (similar ao ordenamento ferromagnético de spins), ou outros mais complicados. O ordenamento de carga também pode levar ao surgimento de gaps de energia, o mesmo acontecendo no caso das distorções Jahn-Teller. Diversas outras situações podem levar a uma transição M-I. No caso da chamada transição de Anderson, por exemplo, a introdução de desordem acima de uma concentração crítica faz com que o portador de carga fique localizado, isto é, mesmo a densidade de estados não apresentando um gap em torno da energia de Fermi o estado isolante se estabelece, pois os portadores possuem uma mobilidade extremamente reduzida. Segundo Ioff e Regel [13], quando um sistema metálico passa a ter um grau de desordem cada vez maior, existirá um limite de desordem a partir do qual este comportamento metálico não mais será possível. Isto é, quando se aumenta o grau de desordem, diminui-se o livre caminho médio, até que em um certo limite este será comparável à distância interatômica, ou seja, l ~ a. Nesse caso, o livre caminho médio será dado aproximadamente por: l ~ 1/2(kF. Baseado neste critério, Mott propôs que uma transição M-I deveria ocorrer quando esse limite fosse ultrapassado [6]. Assim, para pequenos graus de desordem, o elétron pode difundir de um sítio para outro, mas não para grandes graus de desordem, pois estão localizados e formam, neste caso, o que se chama de um “vidro de Fermi” (Fermi glass). Nesta descrição, na qual não se leva em conta a interação elétron-elétron, pode modificar significativamente o comportamento do sistema (no caso em que a interação coulombiana entre os elétrons localizados apresenta um papel dominante, tem-se os chamados “vidros de Coulomb”). Deve haver, portanto, um ponto de desordem crítica, onde um sistema condutor METAIS, ISOLANTES E TRANSIÇÕES DE FASE METAL-ISOLANTE passa a ter um comportamento isolante; a esse tipo de transição é dado o nome de “transição de Anderson” [14]. Diversas questões sobre o estado metálico e a transição para o estado isolante necessitam ainda ser esclarecidas, já que em alguns sistemas observa-se um aumento da massa efetiva, mas não obrigatoriamente uma transição para um estado isolante. Em um cristal iônico, por exemplo, a alteração de parâmetros termodinâmicos externos pode fazer com que o portador de carga altere seu estado de fracamente acoplado aos fônons para fortemente acoplado. Esta alteração pode ocorrer quando o portador de carga polariza uma pequena região ao seu redor, ficando preso ao poço de potencial resultante da deformação criada, formando uma quase-partícula chamada de polaron pequeno. Embora haja um grande aumento da massa efetiva do portador e um autoaprisionamento do mesmo, isso não significa necessariamente que este ficará localizado, já que o tunelamento entre diferentes sítios pode ainda ser expressivo, embora seja sugerido que em materiais reais estes polarons (pequenos) sejam sempre localizados devido à presença de impurezas e discordâncias [15]. No caso de polarons pequenos, quando a temperatura é alta, ou seja, T > 2D/2 (2D é a temperatura de Debye, característica do material), um elétron pode saltar para um íon vizinho através de hopping ativado por efeito térmico, ou seja, através da interação do polaron com as vibrações da rede (fônons). Quando a temperatura é baixa (T < 2D/2), a passagem do elétron associado ao polaron de um íon para outro pode se dar também através de hopping, porém com a energia de ponto zero fazendo o papel da agitação térmica. Assim, além das situações em que existe uma abertura previsível das bandas de energia devido à ocorrência de quebras de simetria (como no caso de uma transição de fase estrutural do cristal), o efeito de localização eletrônica em um metal pode ser iniciado por três diferentes mecanismos [16]: desordem estática (transição de Anderson), forte interação elétron-elétron (transição de Mott-Hubbard) e forte acoplamento elétron-rede (polarons pequenos localizados). Conclusões Um grande progresso tem sido verificado no entendimento das propriedades de transporte elétrico nos materiais, desde as proposições originais de Drude. A incorporação da mecânica quântica, com o abandono da idéia de elétrons livres e independentes, tem permitido uma melhor compreensão dos fenômenos de transporte de diversos sistemas, sejam eles elementos simples como o cobre ou silício, sejam eles complexos como os sulfetos e os óxidos. Em particular, os óxidos de metais de transição, por apresentarem propriedades como, por exemplo, ordenamento antiferromagnético, ordenamento de cargas e ordenamento orbital, entre outras, têm criado um grande desafio para o seu entendimento, e por isso mesmo um campo muito fértil para a busca e o teste de novos modelos. O melhor entendimento do comportamento destes sistemas poderá no futuro auxiliar o desenvolvimento de novos materiais com propriedades de interesse tecnológico, como isolantes com constantes dielétricas extremamente altas, ou condutores com propriedades de transporte térmico e elétrico ajustadas para o atendimento de uma determinada necessidade. Além disso, um melhor entendimento do estado metálico e isolante poderá auxiliar muito no entendimento de fenômenos como a supercondutividade, que possui um inegável interesse tecnológico. " Referências [1] DRUDE, P. Zur Elektronentheorie der Metalle. Annalen der Physik, v. 1, p. 566, 1900. [2] ASCHROFT, N. W. e MERMIN, N. D. Solid State Physics. New York: Thomson Learning, 1976. [3] SOMMERFELD, A. The Statistical theory of thermoelectric, galvano- and thermomagnetic phenomena in metals. Rev. Mod. Phys., v. 3, p. 1, 1931. [4] WILSON, A. H. The Theory of Electronic Semiconductors I. Proceedings of the Physical Society of London A, v. 133, p. 458-491, 1931; WILSON, A. H. The Theory of Electronic Semiconductors II. Proceedings of the Physical Society of London A, v. 134, p. 277-287, 1931. [5] SINGLETON, J. Band theory and electronic properties of solids. Oxford: Oxford University Press, p. 32, 2001. [6] MOTT, N. F. The Basis Of the electron theory of metals, with special reference to the transition metals. Proceedings of the Physical Society of London A, v. 62, p. 416, 1949. [7] BLOCH, F. Über die quantenmechanik der electronen in kristallgittern. Z. Phys., v. 52, p. 555-600, 1928. [8] LANDAU, L. D. Sov. Phys. JETP, v. 3, p. 920, 1957; LANDAU, L. D. Sov. Phys. JETP, v. 5, p. 101, 1957; LANDAU, L. D. Sov. Phys. JETP, v. 8, p. 70, 1957. [9] PEIERLS, R. Zur Theorie der galvanomagnetischen Effekte. Z. Phys.,v. 53, p. 255-266, 1929. [10] ZAANEN, J.; SAWATZKY, G. A; ALLEN, J. W. Band gaps and electronic structure of transitionmetal compounds. Physical Review Letters, v. 55, p. 418-421, 1985. [11] RAYCHAUDHURI, A. K. Metal-insulator transition in perovskite oxides: a lowtemperature perspective. Advances in Physics, n. 1, p. 21-46, 1995. [12] IMADA, M.; FUJIMORI, A. e TOKURA, Y. Metal Insulator Transitions. Rev. of Mod. Physics, v. 70, p. 1039, 1998. [13] IOFFE, A. F e REGEL, A. R. Non-crystalline, amorphous and liquid electronic semiconductors. Proc. Semiconduct., v. 4, p. 239-291, 1960. [14] ANDERSON, P. W. Absence of diffusion in certain random lattices. Physical Review, v. 109, p. 1492-1505, 1958. [15] BRONOLD, F. X. e HOLGER, F. Anderson localization of polaron states, Physical Review B, v. 66, p. 073102(1)-07310(4), 2002; DERAEDT, H. e LAGENDIJK, A. Phys. Rev. B, v. 27, p. 6097, 1983; LÖWEN, H. Phys. Rev. B, v. 37, p. 8661, 1988; RANNINGER, J. e THIBBLIN, U. Phys. Rev. B, v. 45, p. 7730, 1992; ALEXANDROV, A. S. et al. Phys. Rev. B, v. 49, p. 9915, 1994. [16] MOTT, N. F. Metal-insulator transition. Rev. of Mod. Physics, v. 40, p. 677-683, 1968; EDWARDS, P. P. et al. The metal insulator transition: a perspective. Phil. Trans. R. Soc. Lond. A, v. 356, p. 5-22, 1998. P E S Q U I S A & T E C N O L O G I A F E I - Nº 28 59
https://openalex.org/W4242467259	https://ora.ox.ac.uk/objects/uuid:290ee603-db6e-4d71-bc4e-ff3de4f3815a/files/mbabaa37265f3e9d64fa4ec44f9dd3427	English	null	Drama as a community engagement strategy for malaria in rural Cambodia	Wellcome open research	2,017	cc-by	11,962	RESEARCH ARTICLE Drama as a community engagement strategy for malaria in rural Cambodia [version 1; referees: 1 approved, 2 approved with reservations] 1 2 2 3 2 4 2 2 Quality Use of Medicines and Pharmacy Research Centre, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Faculty of Medicine, University of Amsterdam, Amsterdam, Netherlands Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK Provincial Health Department, Battambang, Cambodia National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia The Ethox Centre, University of Oxford, Oxford, UK 1 2 3 4 5 6 7 Open Peer Review Discuss this article (0) Comments Referee Status: Invited Referees version 1 published 29 Sep 2017 1 2 3 report report report , Sustainable Gillian F. Black Livelihoods Foundation, South Africa 1 , Oxford University Mary Chambers Clinical Research Unit (OUCRU), Vietnam 2 , Kenya Medical Alun Davies Research Institute-Wellcome Trust Research Programme, Kenya University of Oxford, UK 3 Open Peer Review Discuss this article (0) Comments Referee Status: Invited Referees version 1 published 29 Sep 2017 1 2 3 report report report , Sustainable Gillian F. Black Livelihoods Foundation, South Africa 1 , Oxford University Mary Chambers Clinical Research Unit (OUCRU), Vietnam 2 , Kenya Medical Alun Davies Research Institute-Wellcome Trust Research Programme, Kenya University of Oxford, UK 3 29 Sep 2017, :95 (doi: ) First published: 2 10.12688/wellcomeopenres.12594.1 29 Sep 2017, :95 (doi: ) Latest published: 2 10.12688/wellcomeopenres.12594.1 v1 Open Peer Review Referee Status: Invited Referees version 1 published 29 Sep 2017 1 2 3 report report report Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Abstract This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This work was supported by the Wellcome Trust [096527 and 106698]. Grant information: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 29 Sep 2017, :95 (doi: ) First published: 2 10.12688/wellcomeopenres.12594.1 Renly Lim ( ) Corresponding author: renly.lim@outlook.com : Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation, Writing – Review Author roles: Lim R & Editing; : Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Tripura R J Peto T Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Investigation, Methodology, Project Sareth M Administration; : Investigation, Methodology, Project Administration; : Project Administration, Supervision; : Sanann N Davoeung C Nguon C Project Administration, Supervision; : Conceptualization, Data Curation, Funding Acquisition, Project Administration, Supervision, Cheah PY Writing – Review & Editing Competing interests: The authors declare that they have no competing interests. Lim R, Tripura R, J Peto T How to cite this article: et al. Drama as a community engagement strategy for malaria in rural Cambodia Wellcome Open Research 2017, :95 (doi: [version 1; referees: 1 approved, 2 approved with reservations] 2 ) 10.12688/wellcomeopenres.12594.1 © 2017 Lim R . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This work was supported by the Wellcome Trust [096527 and 106698]. Grant information: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 29 Sep 2017, :95 (doi: ) First published: 2 10.12688/wellcomeopenres.12594.1 Abstract Renly Lim ( ) Corresponding author: renly.lim@outlook.com : Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation, Writing – Review Author roles: Lim R & Editing; : Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Tripura R J Peto T Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Investigation, Methodology, Project Sareth M Administration; : Investigation, Methodology, Project Administration; : Project Administration, Supervision; : Sanann N Davoeung C Nguon C Project Administration, Supervision; : Conceptualization, Data Curation, Funding Acquisition, Project Administration, Supervision, Cheah PY Writing – Review & Editing Competing interests: The authors declare that they have no competing interests. Lim R, Tripura R, J Peto T How to cite this article: et al. Drama as a community engagement strategy for malaria in rural Cambodia Wellcome Open Research 2017, :95 (doi: [version 1; referees: 1 approved, 2 approved with reservations] 2 ) 10.12688/wellcomeopenres.12594.1 © 2017 Lim R . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This work was supported by the Wellcome Trust [096527 and 106698]. Grant information: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 29 Sep 2017, :95 (doi: ) First published: 2 10.12688/wellcomeopenres.12594.1 Renly Lim ( ) Corresponding author: renly.lim@outlook.com : Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation, Writing – Review Author roles: Lim R & Editing; : Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Tripura R J Peto T Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Investigation, Methodology, Project Sareth M Administration; : Investigation, Methodology, Project Administration; : Project Administration, Supervision; : Sanann N Davoeung C Nguon C Project Administration, Supervision; : Conceptualization, Data Curation, Funding Acquisition, Project Administration, Supervision, Cheah PY Writing – Review & Editing Competing interests: The authors declare that they have no competing interests. Lim R, Tripura R, J Peto T How to cite this article: et al. Drama as a community engagement strategy for malaria in rural Cambodia Wellcome Open Research 2017, :95 (doi: [version 1; referees: 1 approved, 2 approved with reservations] 2 ) 10.12688/wellcomeopenres.12594.1 © 2017 Lim R . Abstract : Countries in Southeast Asia are working to eliminate Background multidrug-resistant falciparum malaria, a major cause of mortality in tropical regions. Malaria is declining but transmission persists in many rural areas and among forest workers and isolated populations. In these remote communities, conventional health services and education are limited. Mobilising and educating these populations require new approaches as many people are illiterate and do not attend village meetings. This article describes a qualitative study to assess the feasibility of a drama project as a community engagement strategy. , Sustainable Gillian F. Black Livelihoods Foundation, South Africa 1 : A drama project was conducted in twenty villages in Cambodia with Methods three key messages: to use insecticide-treated bednets and repellents, to get early diagnosis and treatment, and to learn about risks of forest-acquired malaria. Qualitative interviews were conducted with the drama team members, village malaria workers, local health staffs and villagers, to explore the feasibility of using drama to engage the community and the associated challenges. : 29 people were interviewed, which included 18 semi-structured Results interviews and one focus group discussion. Analysis of the interviews resulted in development of the following seven themes: i) exposure to malaria and engagement activities, ii) readiness and barriers to participation, iii) understanding and learning about malaria using drama, iv) entertainment value and engagement method preferences, v) challenges to community engagement, vi) future participation and vii) sustainability. The event saw a very positive response, with an encouraging average participation rate of 66%. The project faced several challenges including logistic problems, rescheduling due to raining season, and time- and budget-constraints. (0) Comments : Our evaluation demonstrated that the drama project was Conclusions feasible in promoting awareness and understanding of malaria prevention and Page 1 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 control. Audience members perceived drama as entertaining and as the preferred choice of engagement activity. Participatory drama could be considered as part of the community engagement for malaria elimination. control. Audience members perceived drama as entertaining and as the preferred choice of engagement activity. Participatory drama could be considered as part of the community engagement for malaria elimination. control. Audience members perceived drama as entertaining and as the preferred choice of engagement activity. Participatory drama could be considered as part of the community engagement for malaria elimination. Renly Lim ( ) Corresponding author: renly.lim@outlook.com : Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation, Writing – Review Author roles: Lim R & Editing; : Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Tripura R J Peto T Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Supervision; : Investigation, Methodology, Project Sareth M Administration; : Investigation, Methodology, Project Administration; : Project Administration, Supervision; : Sanann N Davoeung C Nguon C Project Administration, Supervision; : Conceptualization, Data Curation, Funding Acquisition, Project Administration, Supervision, Cheah PY Writing – Review & Editing Competing interests: The authors declare that they have no competing interests. sts: The authors declare that they have no competing interests. Lim R, Tripura R, J Peto T How to cite this article: et al. Drama as a community engagement strategy for malaria in rural Cambodia Wellcome Open Research 2017, :95 (doi: [version 1; referees: 1 approved, 2 approved with reservations] 2 ) 10.12688/wellcomeopenres.12594.1 © 2017 Lim R . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This work was supported by the Wellcome Trust [096527 and 106698]. Grant information: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 29 Sep 2017, :95 (doi: ) First published: 2 10.12688/wellcomeopenres.12594.1 Page 2 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Introduction and language that resonates with villagers are popular in rural communities. In light of the need to convey messages about malaria and the VMW programme in a comprehensible and appropri- ate fashion, traditional Cambodian drama was used as a means to supplement existing text-based messaging on malaria and other efforts rolled out by the local authorities and education system. The Village Drama Against Malaria (VDAM) was a pilot project that sought to encourage people to prevent mosquito bites by using insecticide-treated nets and repellents, get early diagnosis and treatment from the VMWs, and to raise awareness about the risk of malaria in local forests. Countries in South East Asia are working towards the elimina- tion of Plasmodium falciparum, a major cause of mortality in tropical regions1,2. In Cambodia, malaria prevention and control programmes have made significant progress and malaria cases have declined substantially3. Unfortunately, emergent artemisinin- resistant P. falciparum threatens the recent gains4–7. In January 2016, the Cambodian National Centre for Parasitology, Entomology and Malaria Control (CNM) announced the Malaria Elimination Action Framework (MEAF) 2016–2020, which aims to eliminate P. falciparum by 2020 and P.vivax by 20258. The elimination efforts are underpinned by CNM’s village malaria worker (VMW) programme, where the VMWs provide early, free diagnosis and treatment in settlements with the highest prevalence9. Achieving elimination is complicated by several factors. For example, in areas of low and falling transmis- sion, those at highest risk of infection, forest goers and migrant groups, are the hardest to reach with health services and health education10,11. Moreover, the success of the VMW programme is predicated on the sustained motivation of isolated VMWs and local communities making use of the services they offer. This paper describes a qualitative study to: This paper describes a qualitative study to: i) assess the feasibility of the VDAM project as a community engagement strategy, ii) use drama and related workshops to convey the three key messages of the project (to use insecticide-treated nets and repellents, to get early diagnosis and treatment, and to raise awareness about the risk of malaria in local forests); and iii) describe the challenges faced by the VDAM team, local team members and villagers. Methods Setting In rural communities affected by malaria, awareness and under- standing of the disease and the VMW programme is indispensable to support elimination efforts. Such communities however often record lower literacy rates compared to urban areas: 77 percent com- pared to 93 percent in the 2014 census12. Using printed media such as leaflets and posters to raise malaria awareness, may therefore be less successful in these areas. In developing countries, folk media such as folk songs, dramas, puppet shows and dance have been used as community engagement strategies for health educa- tion and to encourage research participation13–15. Khmer dramas which use comedy and music to tell stories with local references Setting The Village Drama Against Malaria (VDAM) project16 was a joint initiative of the Battambang Provincial Health Department and the Mahidol-Oxford Tropical Medicine Research Unit (MORU), funded by the Wellcome Trust. MORU has a long-standing collabo- ration with the Battambang Provincial Health Department. The project was conducted in 20 of the 49 remote villages in the Samlout District of Battambang Province, which is located in the western Cambodia near the Thailand border (Figure 1). These 20 Figure 1. Location of the 20 drama villages in the Samlout district of Battambang province. Figure 1. Location of the 20 drama villages in the Samlout district of Battambang province. Page 3 of 17 Page 3 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 productions did so to weave together stories and health education. The project mixed art and drama with modern technologies, such as using drones to film the villages, and social media (Facebook and YouTube) to share photos and videos across the province. The ‘Village Drama Against Malaria’ Facebook page was updated regularly after every drama performance depicting highlights of the 3-day events. villages were selected by the provincial health department based on their high P. falciparum incidence from VMW records in 2014, and a proven prevalence of subclinical malaria infections from cross- sectional surveys in early 2015. Village populations ranged from approximately 150 to 3000 residents with an average of 800 (Table 1). Villagers are generally poor and most families are involved in small-scale agriculture. The villages typically have only basic infrastructure: poor access to potable water, sanitation and irrigation systems, limited electricity supplies, minimal post-primary education, and poor availability of health services. The villagers consist of majority ethnic Khmer with some ethnic minorities, chiefly the Por group, living in some villages close to the forests along the Thai border. Planning The VDAM team first introduced the project to and sought approval and support from the national authorities. Meetings were held in the target communities with village leaders, key opinion leaders identified by staff of the provincial health department and other prominent members to deliberate on the proposed project. The VDAM community engagement project The VDAM project combined Cambodian drama, art, music workshops and village concerts and competitions to provide health education in a fun and engaging way. Cambodian drama uses caricatures in a funny and irreverent manner and the VDAM Professional drama performers were recruited in Battambang province. A series of workshops were organised to train the team on the malaria messages to incorporate into the drama perform- ance. The final main script, storyline and key messages were approved by the provincial health authorities. Table 1. Audience numbers, total population and percentage attendance in each of the twenty villages. Table 1. Audience numbers, total population and percentage attendance in each of the twenty villages. attendance in each of the twenty villages. Number Village Audience numbers Population Percentage attendance 1 Outreng + Peamta 350 522 67.0 2 Vealroleum 250 291 85.9 3 Ting Moung 70 153 45.8 4 Phnom Rei 500 614 81.4 5 Ouda 650 1012 64.2 6 Srea Chipov 550 1537 35.8# 7 ChamlongRemeangLue 450 1450 31.0# 8 Tasanh Chueng 350 949 36.9 9 Boeung Run 600 814 73.7 10 Prey Thum/MaiKo 350 313 111.8* 11 Ou Choam Leu 350 456 76.8 12 KampongLpov 450 298 151.0* 13 Chhak Rokar 500 1781 28.1# 14 Srae Reach 450 562 80.1 15 Outating 150 164 91.5 16 Kanhchang 450 649 69.3 17 Prey Rumchek 600 3361 17.9# 18 Ou Samriel 550 793 69.4 19 Tanoun 450 822 54.7 20 Kantuot 550 1020 53.9 Key: #: These villages are very large in size, making it difficult for villagers to attend the drama event. The majority of villagers commute on foot. * : Percentage attendance exceeded 100% because participants came from nearby villages to attend the drama event. Page 4 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Ethical statement The study was approved by the Oxford Tropical Research Ethics Committee (OXTREC; 1015-13), National Ethics Committee for Health Research Cambodia (18th Feb 2016 NECHR 0051), and registered on clinicaltrials.gov (NCT01872702). Verbal informed consent was obtained from all participants prior to commencing the interviews and questionnaires. Drama workshops and performances The project, which took place from June until September 2016, comprised a series of three-day workshops with the drama group travelling from one village to another every third day. During the workshops, villagers shared their talents and local stories which were integrated as part of the drama performance. School children were invited to participate to sing songs about malaria in Khmer and create drawings with the malaria theme. Teenagers and young people were invited to participate in the drama. On the third evening, the activities included singing competitions, songs and a quiz on malaria, followed by a 45-minute drama performance by both locals and professional drama team members. These events took place in the village square, usually near a school or temple, starting about 6pm and ending at 10pm or 11pm. Results Analysis of the data resulted in the following seven themes: i) exposure to malaria and engagement activities, ii) readiness and barriers to participation, iii) understanding and learning about malaria using drama, iv) entertainment value and engagement method preferences, v) challenges to community engagement, vi) future participation; and vii) sustainability. i) exposure to malaria and engagement activities, Planning Verbal instead of written consent was obtained due to cultural reasons, and due to the low literacy level in the remote villages. The con- sent process was recorded using an audio recorder. Verbal consent for this study was approved by OXTREC in accordance with their guidelines on consent (https://www.admin.ox.ac.uk/curec/ resources/informed-consent/), as well as the local ethics commit- tee. Approvals for the VDAM project, use of social media as well as filming and photography for all public events were obtained from the national and provincial authorities prior to the start of the project. In each village, the team and village leaders met with the local community members and VMWs to explain the purpose of the project and to introduce the activities involved. Key opinion leaders in each village played an active role throughout the project development and progress. Data collection li ii) readiness and barriers to participation, Our sampling method was purposive and limited by practicalities. In July 2016, 18 semi-structured interviews and one focus group discussion were conducted. The semi-structured interviews included six villagers who attended the drama performance, five professional actors, two VDAM organisers, two provincial health department staff, one VMW, one village leader and one local health staff. One focus group discussion took place, which involved eleven youths aged between 14 and 16 who participated in the performance. iv) entertainment value and engagement method preferences, v) challenges to community engagement, vi) future participation; and vi) future participation; and vii) sustainability. Interviews were conducted by two interviewers (RL and PYC) in English and Khmer with the help of a Khmer social scientist (NS). All interviews, including the focus group discussion, were recorded with permission of the interviewees. In addition, data were obtained from field notes based on observations and informal conversations. Challenges to community engagement Reaching the forest goers and migrant population was a particular challenge. On each visit, forest workers typically spend one week to ten days in the forest; they make two trips per month, whereas migrants move in and out of the villages depending on planting and harvest seasons. For many forest workers who were unable to attend the event, their partners said that they would get their husbands to use repellents when they go into the forest in the future. The village leader and local health staff raised the villagers’ con- cerns regarding rumours that healthcare personnel came to the villages to collect their blood and sell it. Some respondents also described how other villagers believe in spiritual and psychic mediums to cure their illnesses. Some villagers reported limited understanding of the project aim, and this led to reservations about participation. Questions raised included, ‘who are [the drama team]?’, ‘where do they come from?’ and ‘what are they doing here?’. Understanding and learning about malaria using drama To assess if the project can be used to convey the three key messages of the project (to use insecticide-treated nets and repel- lents, to get early diagnosis and treatment, and to raise awareness about the risk of malaria in local forests), interviewers asked the villagers about the lessons that they had learnt from the drama. When asked about the drama, villagers faced no difficulty recapitulating the storyline, which comprised two main characters, one of whom slept under a bednet while the other slept without protection. The latter contracted malaria and went to meet the VMW, who performed a blood test to confirm the malaria diag- nosis. He was given a three-day malaria medication and later recovered. The respondents felt that the team gave simple and straightforward key messages which were easy to understand to the local community, including people who were illiterate. “…….because they believe in the superstitions like they believe in burning incense for the offering. They said they don’t want to do blood test or to have vaccinations.” “And some people they said that oh...maybe take their blood for selling. Because they don't know. Because in Cambodia they sell blood.” (local health staff) (local health staff) Some villagers said that the drama would bring about behavioural changes in their daily lives. Compared with before the drama, solely relying on mosquito nets, they would now use mosquito repellents during the day. Exposure to malaria and engagement activities Exposure to malaria and engagement activities Interviews with local villagers revealed that either they themselves had contracted malaria, or know people who had contracted malaria. Villagers who had contracted malaria said that they suffered from its symptoms and economic complications, includ- ing inability to work and loss of income. Villagers who had personal experiences with malaria recognized its seriousness, and would seek diagnosis and treatment from VMWs. Some villagers shared stories of immigrants and outsiders who died after contracting malaria in the villages. Interview guides explored the following areas: i) personal history and experiences with malaria and community engagement, ii) perceptions of malaria prevention and treatment, iii) understanding of malaria messages conveyed via drama; “Since one of the actors from this group died, then never has any other group which does the drama performance come to perform at this village any more.” iv) opinions on the community engagement method, v) challenges to organizing the community engagement or attending the event and (Village leader) vi) feedback and suggestions on community engagement. Community engagement activities were few and far between, and involved only showing videos and films, without active participation from the community. Staff from the local health authority felt that the existing health education methods, through healthcare centres, hospitals, advertisement (e.g. banners), media (e.g. television) and local service providers were limited and not comprehensive. Others commended the unique approach of the current participatory drama project. Audio recordings of interviews were transcribed and translated where necessary into English by professional translators. Two authors (RL and PYC) performed manual line-by-line coding of all interview transcripts in Microsoft Word. Using thematic analysis, the content of the codes were used to develop the themes that are presented below and to explore patterns of these themes across the transcripts/data sources. Page 5 of 17 Page 5 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Readiness and barriers to participation as mosquitoes while singing songs about malaria. They were seen dancing away on the open ground in front of the stage. The catchy tunes meant villagers could easily join in the singing, and often were able to remember the messages in the songs. as mosquitoes while singing songs about malaria. They were seen dancing away on the open ground in front of the stage. Exposure to malaria and engagement activities The catchy tunes meant villagers could easily join in the singing, and often were able to remember the messages in the songs. Many villagers expressed their readiness to participate in the drama activities, as reflected by an encouraging average participa- tion rate of 66% (range 18% to 100%) (Table 1). The number of attendees was between 70 and 650, with an average of 430 people. Many young people and children volunteered to participate during the workshops to showcase their performance talents. For many, this was the first time they received drama training and had the opportunity to perform in public. Amongst the activities held during the evening (singing competi- tions, songs and a quiz on malaria, drama performance), drama was reported to be the favourite, followed by songs on malaria. Audience members used Facebook, which is very popular with Cambodians, to upload pictures and videos of the performances. Villagers said that their motivation to join the workshops stemmed from a desire to learn and understand more about malaria, and they hoped to get more information to eliminate malaria in their villages. Village leaders were interested in the project because they wanted to know more about the on-going projects conducted by MORU and the provincial health department. The provincial health department and local healthcare staff commented that health education without entertainment may not be appealing to the villagers. Combining arts and science led people to remember key messages better. Using drama to attract people in the village, and incorporate key messages about malaria achieved both entertainment and education purposes. The lack of transportation meant villagers often commuted on foot, making accessibility to the performance location potentially difficult. A few villages such as Srea Chipov and Prey Rumchek are very large in size. Villagers may not be able to attend the drama due to the distance, which potentially explains the lower coverage in these villages (Table 1). Interviews with participants in other villages, however, showed that they had no problem walking to the pagodas or schools where the drama performances were conducted. The villagers who were interviewed said that they went with their entire families. They were happy to participate in the workshops, which they felt were ‘real performances’ instead of video shows and films. Challenges to community engagement Men who go to the forest would bring mosquito nets with them and wear long-sleeved clothing. Social relationships in Cambodia embody a hierarchical pattern. The villagers' attitudes and participation appear to be heavily influenced by the village leaders and monks, who are respected members of the community. Establishing trust and good relation- ships with local village and spiritual leaders, and gaining their support implied subsequent participation from the villagers. Sustainability h O The MORU team worked closely with the provincial health department and village leaders. Interviews with these key members suggested that villagers gained a better understanding of the purpose of malaria elimination projects after they conveyed these messages to the villagers. Some villagers said they were now more willing to do blood testing, which was conducted as part of malaria research conducted by MORU. Challenges to drama as community engagement Due to the timing of approvals, the project was conducted during the rainy season. Travelling from one village to another was considerably slower and everything took longer. Perform- ance dates were often rescheduled due to the degraded road conditions. Frequent heavy downpours also made it more difficult for villagers to participate in the drama workshops and performance. Malaria incidence occurs throughout the year but most malaria cases are confined to the months during or directly after the rainy season; it would have been more appropriate to deliver the malaria messages before the rainy season begins. In a few villages with large population of over 1000 people, the villages consisted of several hamlets which were scattered. It was too far for villagers to walk to the event location, which resulted in lower participation rates in those villages. Interviewee: “For me, I really think this is very valuable for the community.” Interviewer: “Really, uncle? So it provides benefits for the community?” Interviewer: “Really, uncle? So it provides benefits for the community?” Interviewee: “Yes, it really does. And recently people have involved in activities, for example: blood testing and so on.” (audience) During the focus group discussion, youths felt that they learned how to perform in public and to express their thoughts. They further requested for materials related to ways of preventing malaria so that they could spread such awareness in schools. At the end of the project, video recordings and photos of the workshops and performances in the form of DVDs were distrib- uted to all village leaders as souvenirs. All photos and videos were taken at public places and with formal written permission from the national and provincial authorities. Another challenge faced by the VDAM team was the time pressure. It was crucial to allocate enough time in each village without compromising the ability to convey the key malaria messages. However, with twenty villages to perform in, the length of time spent in each village was limited to only three days. Sustainability h O As a result, we were unable to reach a large number of young men who were mainly forest workers who spend a significant amount of time in the forests, and the mobile and migrant population who were not in the villages. Entertainment value and engagement method preferences Th d f d ib d f d i i Entertainment value and engagement method preferences The drama performance was described as funny and entertaining, and made learning and understanding about malaria much more interesting compared with the conventional health education methods. Villagers especially enjoyed watching children dressed The VDAM organisers commented that the project which was conducted from June to September 2016 coincided with the Page 6 of 17 Page 6 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 rainy season in Cambodia. Roads that were already a bumpy ride for motorists were further battered due to the heavy downpour, making them inaccessible via small trucks used by the local drama team. The performance schedule often needed rearrangements because bridges connecting villages collapsed due to the flood. project. Taking ‘top-down’ approach, support was garnered from the provincial health department, village leaders and other respected members of the community which would then con- vince the local communities to participate. Taking a ‘bottom-up’ approach, local communities were encouraged to actively participate in the planning and execution of the 3-day event and to contribute stories and ideas to the drama performance, an approach the villagers claimed they have never experienced before. Future participation Many interviewees requested that the VDAM team organise the event again and said that they would participate in the future because very few events are organised in their villages. Some wanted their family members to participate in the event so that they would remember the key messages more effectively. The local health staff commented health education without properly engaging the community results in minimal impact. Our community engagement project used traditional Cambodian drama to incorporate key messages about malaria prevention and elimination. A similar method was used in a study in Laos which utilized traditional folk songs known as ‘lam’ as an educa- tional medium for preventing HIV/AIDS13. Based on interviews in the focus group discussions, the authors concluded that tradi- tional folk songs could be used to motivate individual behavioural changes and to encourage community action for disease preven- tion. However, traditional songs were less enticing to the younger generations who preferred pop songs. Our engagement project used drama and various music genres which appealed to both the younger and older generations. Discussion Our results showed that the VDAM project was a feasible method to engage the rural communities and to convey the key malaria messages. Villagers said they learned more about malaria through the drama, enjoyed watching and participating in the performances and were keen to participate in future events. To our knowledge, the current paper is the first assessment of using drama to engage the community for malaria control and elimination. Limitations Since this engagement project had time and budget limitations, a longitudinal evaluation was not feasible. Furthermore, it was not possible to differentiate the VDAM project from other engage- ment and educational projects provided by the provincial health department, schools and VMWs. Since there was no before and after comparison made, it was not possible to determine the extent of changes in knowledge of malaria amongst villagers. The ultimate indicator of success is the elimination of malaria in these villages, which would be attributable to many factors. Drama within a community engagement strategy Drama within a community engagement strategy Community engagement is increasing recognized as integral to successful and ethical health-related research17. To maximize the efficiency of the community engagement programme, a combi- nation of ‘top-down’ and ‘bottom-up’ approaches are needed18. Such a combination of approaches was taken to promote VDAM Page 7 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Acknowledgements This project was conducted by the Mahidol-Oxford Tropical Medi- cine Research Unit (MORU) in collaboration with the Battambang Provincial Health Department. MORU works in Cambodia in partnership with the National Center for Parasitology Entomology and Malaria Control. Data availability Our sample size was small as we were limited by practicalities: chiefly the interviewers could not attend all of the remote villages where the drama took place. The performances tended to end quite late at night and villagers had to go home immediately after the event ended to wake up early the next day. Many people also appeared to be shy and somewhat reluctant to be interviewed. Due to ethical and security considerations, the data that supports the findings in this study can be accessed only through the Data Access Committee at Mahidol Oxford Tropical Medicine Research Unit (MORU). The data sharing policy can be found here: http:// www.tropmedres.ac/data-sharing. The application form for datasets under the custodianship of MORU Tropical Network can be found as a Supplementary File. Abbreviations The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CNM: Cambodian National Centre for Parasitology, Entomology and Malaria Control; MEAF: Malaria Elimination Action Frame- work; MORU: Mahidol-Oxford Tropical Medicine Research Unit; VDAM: Village Drama Against Malaria; VMW: village malaria worker. Conclusions We conclude that although there were many challenges, vil- lage drama and its associated activities such as competitions and workshops is feasible as a community engagement strategy. Audience members recalled the plot of the performance and the malaria- related messages, indicating that the key messages were clear and concrete. Villagers perceived drama as entertaining and as the pre- ferred choice of community engagement activity. Using drama is a promising way to engage communities and could be considered as part of the community engagement for malaria elimination. Competing interests The authors declare that they have no competing interests. Grant information This work was supported by the Wellcome Trust [096527 and 106698]. 1. Lwin KM, Imwong M, Suangkanarat P, et al.: Elimination of Plasmodium falciparum in an area of multi-drug resistance. Malar J. 2015; 14: 319. PubMed Abstract \| Publisher Full Text \| Free Full Text 2. Tripura R, Peto TJ, Chalk J, et al.: Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies. Malar J. 2016; 15(1): 181. PubMed Abstract \| Publisher Full Text \| Free Full Text Ethical statement The study was approved by the Oxford Tropical Research Ethics Committee (OXTREC; 1015-13), National Ethics Committee for Health Research Cambodia (18th Feb 2016 NECHR 0051), and registered on clinicaltrials.gov (NCT01872702). Verbal informed consent was obtained from all participants prior to commencing the interviews and questionnaires. Verbal consent for this study was approved by OXTREC in accordance with their guidelines on consent (https://www.admin.ox.ac.uk/curec/resources/informed- consent/) as well as the local ethics committee. Approvals for the VDAM project, use of social media as well as filming and pho- tography for all public events were obtained from the national and provincial authorities prior to the start of the project. We thank the communities, village leaders, school directors, health centres, and local authorities in Battambang that participated in this project. We also thank Nicholas Day (MORU), Kayna Kol (MORU), Chhouen Heng (MORU), Kem Sovann (MORU), Coco Snethlage (MORU), Mark Droogleever (MORU), Pachararit Wis- mol (MORU), and Abby Taylor (Wellcome Trust, UK). Photography and videography by Nicky Almasy at N/A Productions. Permission has been obtained by the named individuals to be acknowledged in the manuscript. 3. Maude RJ, Nguon C, Ly P, et al.: Spatial and temporal epidemiology of clinical malaria in Cambodia 2004–2013. Malar J. 2014; 13: 385. PubMed Abstract \| Publisher Full Text \| Free Full Text 4. Noedl H, Se Y, Schaecher K, et al.: Evidence of Artemisinin-Resistant Malaria in Western Cambodia. N Engl J Med. 2008; 359(24): 2619–2620. PubMed Abstract \| Publisher Full Text 5. Phyo AP, Nkhoma S, Stepniewska K, et al.: Emergence of artemisinin-resistant 2. Tripura R, Peto TJ, Chalk J, et al.: Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies. Malar J. 2016; 15(1): 181. PubMed Abstract \| Publisher Full Text \| Free Full Text Supplementary materialh Supplementary materialh Supplementary material Supplementary File 1: The application form for datasets supporting this research, under the custodianship of MORU Tropical Network. pp y Supplementary File 1: The application form for datasets supporting this research, under the custodianship of MORU Tropical Network. Cl k h h d Supplementary File 1: The application form for datasets supporting this research, under the custodianship of Click here to access the data. References 1. Lwin KM, Imwong M, Suangkanarat P, et al.: Elimination of Plasmodium falciparum in an area of multi-drug resistance. Malar J. 2015; 14: 319. PubMed Abstract \| Publisher Full Text \| Free Full Text 2. Tripura R, Peto TJ, Chalk J, et al.: Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies. Malar J. 2016; 15(1): 181. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Maude RJ, Nguon C, Ly P, et al.: Spatial and temporal epidemi malaria in Cambodia 2004–2013. Malar J. 2014; 13: 385. PubMed Abstract \| Publisher Full Text \| Free Full Text 4. Noedl H, Se Y, Schaecher K, et al.: Evidence of Artemisinin-Re Western Cambodia. N Engl J Med. 2008; 359(24): 2619–2620. PubMed Abstract \| Publisher Full Text 5. Phyo AP, Nkhoma S, Stepniewska K, et al.: Emergence of artem 1. Lwin KM, Imwong M, Suangkanarat P, et al.: Elimination of Plasmodium falciparum in an area of multi-drug resistance. Malar J. 2015; 14: 319. PubMed Abstract \| Publisher Full Text \| Free Full Text References 1. Lwin KM, Imwong M, Suangkanarat P, et al.: Elimination of Plasmodium falciparum in an area of multi-drug resistance. Malar J. 2015; 14: 319. PubMed Abstract \| Publisher Full Text \| Free Full Text 2. Tripura R, Peto TJ, Chalk J, et al.: Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies. Malar J. 2016; 15(1): 181. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Maude RJ, Nguon C, Ly P, et al.: Spatial and temporal epidemiology of clinical malaria in Cambodia 2004–2013. Malar J. 2014; 13: 385. PubMed Abstract \| Publisher Full Text \| Free Full Text 4. Noedl H, Se Y, Schaecher K, et al.: Evidence of Artemisinin-Resistant Malaria in Western Cambodia. N Engl J Med. 2008; 359(24): 2619–2620. PubMed Abstract \| Publisher Full Text 5. Phyo AP, Nkhoma S, Stepniewska K, et al.: Emergence of artemisinin-resistant Page 8 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 malaria on the western border of Thailand: a longitudinal study. Lancet. 2012; 379(9830): 1960–6. PubMed Abstract \| Publisher Full Text \| Free Full Text 6. Ashley EA, Dhorda M, Fairhurst RM, et al.: Spread of Artemisinin Resistance in Plasmodium falciparum Malaria. N Engl J Med. 2014; 371(5): 411–423. PubMed Abstract \| Publisher Full Text \| Free Full Text 7. Tun KM, Imwong M, Lwin KM, et al.: Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis. 2015; 15(4): 415–21. PubMed Abstract \| Publisher Full Text \| Free Full Text 8. Cambodia Malaria Elimination Action Framework (2016–2020). 2016. Reference Source 9. Canavati SE, Lawpoolsri S, Quintero CE, et al.: Village malaria worker performance key to the elimination of artemisinin-resistant malaria: a Western Cambodia health system assessment. Malar J. 2016; 15(1): 282. PubMed Abstract \| Publisher Full Text \| Free Full Text 10. Guyant P, Canavati SE, Chea N, et al.: Malaria and the mobile and migrant population in Cambodia: a population movement framework to inform strategies for malaria control and elimination. Malar J. 2015; 14(1): 252. PubMed Abstract \| Publisher Full Text \| Free Full Text 11. Wangroongsarb P, Satimai W, Khamsiriwatchara A, et al.: Respondent-driven sampling on the Thailand-Cambodia border. II. Knowledge, perception, practice and treatment-seeking behaviour of migrants in malaria endemic zones. Malar J. References 2011; 10(1): 117. PubMed Abstract \| Publisher Full Text \| Free Full Text 12. National Institute of Statistics, Directorate General for Health, ICF International: Cambodia Demographic and Health Survey 2014. National Institute of Statistics, Directorate General for Health, and ICF International: Phnom Penh, Cambodia, and Rockville, Maryland, USA. 2015. Reference Source Cambodia Demographic and Health Survey 2014. National Institute of Statistics, Directorate General for Health, and ICF International: Phnom Penh, Cambodia, and Rockville, Maryland, USA. 2015. Reference Source malaria on the western border of Thailand: a longitudinal study. Lancet. 2012; 379(9830): 1960–6. malaria on the western border of Thailand: a longitudinal study. Lancet. 2012; 379(9830): 1960–6. 6. Ashley EA, Dhorda M, Fairhurst RM, et al.: Spread of Artemisinin Resistance in Plasmodium falciparum Malaria. N Engl J Med. 2014; 371(5): 411–423. P bM d Ab t t \| P bli h F ll T t \| F F ll T t 6. Ashley EA, Dhorda M, Fairhurst RM, et al.: Spread of Artemisinin Resistance Plasmodium falciparum Malaria. N Engl J Med. 2014; 371(5): 411–423. PubMed Abstract \| Publisher Full Text \| Free Full Text 13. Yoshida I, Kobayashi T, Sapkota S, et al.: Evaluating educational media using traditional folk songs (‘lam’) in Laos: a health message combined with oral tradition. Health Promot Int. 2012; 27(1): 52–62. PubMed Abstract \| Publisher Full Text 7. Tun KM, Imwong M, Lwin KM, et al.: Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis. 2015; 15(4): 415–21. PubMed Abstract \| Publisher Full Text \| Free Full Text 14. Kroeger A, Meyer R, Mancheno M, et al.: Health education for community-based malaria control: an intervention study in Ecuador, Colombia and Nicaragua. Trop Med Int Health. 1996; 1(6): 836–46. PubMed Abstract \| Publisher Full Text 15. Mahasarinand P: Stop popping pills. 2016. Reference Source 16. Lim R, Peto TJ, Tripura R, et al.: Village Drama Against Malaria. Lancet. 2016; 388(10063): 2990. PubMed Abstract \| Publisher Full Text 17. Tindana PO, Singh JA, Tracy CS, et al.: Grand challenges in global health: community engagement in research in developing countries. PLoS Med. 2007; 4(9): e273. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Atkinson JA, Vallely A, Fitzgerald L, et al.: The architecture and effect of participation: a systematic review of community participation for communicable disease control and elimination. Implications for malaria elimination. Malar J. References 2011; 10: 225. PubMed Abstract \| Publisher Full Text \| Free Full Text 14. Kroeger A, Meyer R, Mancheno M, et al.: Health education for community-based malaria control: an intervention study in Ecuador, Colombia and Nicaragua. Trop Med Int Health. 1996; 1(6): 836–46. PubMed Abstract \| Publisher Full Text 14. Kroeger A, Meyer R, Mancheno M, et al.: Health education for community-based malaria control: an intervention study in Ecuador, Colombia and Nicaragua. Trop Med Int Health. 1996; 1(6): 836–46. PubMed Abstract \| Publisher Full Text 8. Cambodia Malaria Elimination Action Framework (2016–2020). 2016. Reference Source 15. Mahasarinand P: Stop popping pills. 2016. Reference Source 15. Mahasarinand P: Stop popping pills. 2016. Reference Source 9. Canavati SE, Lawpoolsri S, Quintero CE, et al.: Village malaria worker performance key to the elimination of artemisinin-resistant malaria: a Western Cambodia health system assessment. Malar J. 2016; 15(1): 282. PubMed Abstract \| Publisher Full Text \| Free Full Text 16. Lim R, Peto TJ, Tripura R, et al.: Village Drama Against Malaria. Lancet. 2016; 388(10063): 2990. PubMed Abstract \| Publisher Full Text 10. Guyant P, Canavati SE, Chea N, et al.: Malaria and the mobile and migrant population in Cambodia: a population movement framework to inform strategies for malaria control and elimination. Malar J. 2015; 14(1): 252. PubMed Abstract \| Publisher Full Text \| Free Full Text 17. Tindana PO, Singh JA, Tracy CS, et al.: Grand challenges in global health: community engagement in research in developing countries. PLoS Med. 2007; 4(9): e273. PubMed Abstract \| Publisher Full Text \| Free Full Text 17. Tindana PO, Singh JA, Tracy CS, et al.: Grand challenges in global health: community engagement in research in developing countries. PLoS Med. 2007; 4(9): e273. PubMed Abstract \| Publisher Full Text \| Free Full Text 11. Wangroongsarb P, Satimai W, Khamsiriwatchara A, et al.: Respondent-driven sampling on the Thailand-Cambodia border. II. Knowledge, perception, practice and treatment-seeking behaviour of migrants in malaria endemic zones. Malar J. 2011; 10(1): 117. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Atkinson JA, Vallely A, Fitzgerald L, et al.: The architecture and effect of participation: a systematic review of community participation for communicable disease control and elimination. Implications for malaria elimination. Malar J. 2011; 10: 225. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Alun Davies 1,2 Kenya Medical Research Institute-Wellcome Trust Research Programme, Kilifi, Kenya 1 The Centre for Tropical Medicine, University of Oxford, Oxford, UK 2 The Centre for Tropical Medicine, University of Oxford, Oxford, UK 2 Overall, I feel that the novel approach described in this project is worthy of disseminating to wider engagement audiences. The study is described in a fairly clear way; however, aspects could be improved on to strengthen the article. Firstly, I would encourage the authors to clearly reference the Facebook and YouTube links explicitly with hyperlinks within the document if this is possible. The footage and images within these links clearly convey: the nature of the engagement; the participants and audiences involved in the activities; some of the challenges the authors and implementers encountered; and the sheer enjoyment of the participants in taking part in the activities. These are incredibly beautiful and striking images which bring the article alive and so are worth making the most of (assuming of course that the participants are happy for their images to be shared). There is a risk that readers may interpret the project as using a ‘deficit model’ of engagement, where educated researchers enter ‘illiterate’ communities to fill the knowledge gaps. Though the main aim of the activities described is explicitly stated as addressing health knowledge gaps related to Malaria control, I feel that more of an emphasis could be made of the close collaboration between researchers and community members required in implementing this project, drawing on existing local talent and knowledge, towards addressing goals of engagement/health promotion. This collaboration was especially evident in the videos. In relation to this, if the data-set included evidence of a strengthening relationship, mutual-understanding or trust between researchers and participants, then this would be worth including. There is some ambiguity about the overall aim of the engagement/research. The abstract states that the purpose of the project is to assess the feasibility of a drama project as a community engagement strategy. The description however, lacks an ethical argument as to why the authors selected health-promotion activities as a vehicle for engaging communities. Playing devil’s advocate, it could be argued that health promoting activities such as “using insecticide-treated bednets and repellents, etc.” rather than being the role of health researchers, is the work of the the Cambodian National Centre for Parasitology, Entomology and Malaria Control (CNM), through their Malaria Elimination Action Framework (MEAF) 2016–2020. References Atkinson JA, Vallely A, Fitzgerald L, et al.: The architecture and effect of participation: a systematic review of community participation for communicable disease control and elimination. Implications for malaria elimination. Malar J. 2011; 10: 225. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Atkinson JA, Vallely A, Fitzgerald L, et al.: The architecture and effect of participation: a systematic review of community participation for communicable disease control and elimination. Implications for malaria elimination. Malar J. 2011; 10: 225. PubMed Abstract \| Publisher Full Text \| Free Full Text 12. National Institute of Statistics, Directorate General for Health, ICF International: 12. National Institute of Statistics, Directorate General for Health, ICF International: Page 9 of 17 Page 9 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Version 1 13 October 2017 Referee Report Open Peer Review Current Referee Status: Current Referee Status: Current Referee Status: Alun Davies 1,2 Since the discussion presents a case that this is an engagement project ‘integral to successful and ethical health-related research,’ more clarity is needed in arguing why conducting health-promotion activities addresses ethical goals of community engagement. For example, did the implementers feel that they were well placed to conduct these activities, given their knowledge of malaria, to conduct health Page 10 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 promotion? Was it a form of addressing ‘justice’ and ‘beneficence’ in research through offering community members means of improving their health? Was the purpose of the engagement to strengthen community members understanding of Malaria control, in order to complement informed consent for a specific MORU study (addressing ‘respect for persons’)? Or was it a means of strengthening relationships between research host communities and researchers (there is some evidence of this – leaders felt that participation offered an opportunity to learn about the work of MORU)? … or was it a combination of all of these? I feel that using health-promotion as a means of engagement is entirely justifiable (see for example Participants in the CE and Consent Workshop (2013) or (Nakibinge et al., 2009 ), but I think the ambiguity could be cleared with a short description/justification of why they are using it. 1 2 In relation to the above paragraph it would be helpful to provide a clearer description of the Malaria Elimination Action Framework (MEAF) 2016–2020. Is this a guideline document, or a group of government interventions, or both? Is the Battambang Provincial Health Department responsible for implementing this framework? If so, what are they already doing on the ground? Does MORU have a “Malaria elimination’, or related study which this engagement project is part of, or is the project a stand-alone? Alun Davies 1,2 I agree with other referees with regards to the following points agree with other referees with regards to the following points Requiring reference to the value of working with participatory visual methods as a commu engagement approach in public health and health research Requiring reference to the value of working with participatory v engagement approach in public health and health research engagement approach in public health and health research The relatively small sample size, but that this is acknowledged as a limitation The overlaps with regards to the data presented within the findings themes and how these could be streamlined In addition to the above points, I would have liked to have seen more participant quotes to highlight some of the qualitative findings, for example, in the following areas: a) the drama bringing about reported behaviour change; b) a quote from the children’s song; c) a quote highlighting that drama was the preferred activity; d) evidence of establishing trust by community leaders translating to subsequent participation; and e) evidence of a desire for further participation. This article provides a description of the creative use of a culturally appropriate method to engage communities with health and health research. As such it is worthy of being indexed. References 1. Participants in the Community Engagement and Consent Workshop , Kilifi, Kenya , March 2011: Consent and community engagement in diverse research contexts. . 2013; J Empir Res Hum Res Ethics 8 (4): 1-18 \| PubMed Abstract Publisher Full Text 2. Nakibinge S, Maher D, Katende J, Kamali A, Grosskurth H, Seeley J: Community engagement in health research: two decades of experience from a research project on HIV in rural Uganda. . Trop Med Int Health 2009; (2): 190-5 \| 14 PubMed Abstract Publisher Full Text Is the work clearly and accurately presented and does it cite the current literature? Partly Are all the source data underlying the results available to ensure full reproducibilit No source data required I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 06 October 2017 Referee Report doi:10.21956/wellcomeopenres.13638.r26534 Are sufficient details of methods and analysis provided to allow replication by others? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly Page 11 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: Referee Expertise: Public/community engagement with health research I have read this submission. I believe that I have an appropriate level of expertise to confirm th it is of an acceptable scientific standard, however I have significant reservations, as outlined above. doi:10.21956/wellcomeopenres.13638.r26534 Mary Chambers Oxford University Clinical Research Unit (OUCRU), Ho Chi Minh City, Vietnam Oxford University Clinical Research Unit (OUCRU), Ho Chi Minh City, Vietnam It is timely to have a publication describing the strengths and weaknesses of community engagement activities. Pressure from funders and increasing researcher interest in engagement means that more biomedical trials and research studies have a community engagement component. However, publications evaluating their effectiveness are scarce. The authors clearly state the reasons for and the methods of engagement. The genre of engagement seems appropriate for the population served, as backed by the evaluation. The data collection is described clearly, although I would like the authors to clarify the timeframe between the activities and the interviews/focus group. This is relevant if they are assessing recall of the theatre script content and attributing the engaging methods of health promotion for people's ability to remember the key health messages. The number of participants is small and ideally they would have interviewed more villagers, and at different time points. The authors acknowledge this as a limitation to the study. The discussion is clear and conclusions appropriate. However, I would question the overall conclusion that village drama could be considered as part of CE for malaria elimination without some discussion as to whether these activities are prohibitively costly for local health systems to conduct. The question of sustainability as an imbedded CE/public health activity is not addressed. Perhaps the conclusion that they may be effective as part of an externally funded research engagement strategy would be more appropriate. These minor limitations do not prevent this from being a well written and useful contribution to the literature about community engagement in a biomedical research setting. Page 12 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: I have read this submission. Gillian F. Black Sustainable Livelihoods Foundation, Cape Town, South Africa Gillian F. Black Sustainable Livelihoods Foundation, Cape Town, South Africa Gillian F. Black Sustainable Livelihoods Foundation, Cape Town, South Africa Sustainable Livelihoods Foundation, Cape Town, South Africa Sustainable Livelihoods Foundation, Cape Town, South Africa doi:10.21956/wellcomeopenres.13638.r26534 I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 05 October 2017 Referee Report doi:10.21956/wellcomeopenres.13638.r26538 doi:10.21956/wellcomeopenres.13638.r26538 Is the work clearly and accurately presented? For the most part, the work is clearly and accurately presented, but important clarifications are required at certain points. It is emphasized throughout that drama is the key engagement strategy being evaluated. However, the VDAM community engagement approach (at each site) comprised of a 3-day workshop including art, music, village concerts and competitions, with a (participatory) drama performance taking place on the final evening. As far as I can understand, drama was not used in isolation in any of the participant villages. So using the term 'drama project' is questionable. The authors could perhaps consider another term that better reflects the multi-pronged nature of the community engagement approach taken. The authors explain that the project took place between June and September 2016, and that the interviews and focus group discussions were conducted in July of that year. Details are provided about the groups who were included in these processes, and the number of respondents. Were all of the respondents from the same village? (i.e. are all responses based on a single 3-day workshop event?). It is important to know this as it will indicate which of the study areas are represented in the results and conclusions. (At several points in the paper, the authors refer to ‘the’ event or ‘the’ performance, which indicates that the results are indeed based on the implementation of a single workshop activity. Please do clarify if this was the case). Page 13 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 It is explained that the project also included modern technologies such as drones to film the villages and social media to share photos and videos across the province. These elements of the engagement process do not inform the research findings described in the article. I was left wondering why this information was included in the paper, what the main purpose of this province wide dissemination was, and what the ethical implications of these disseminations approaches were. The average rate of event participation is calculated to be 66% across all the villages, and this statistic is highlighted in the paper abstract. Is the work clearly and accurately presented? The authors explain that a single 3-day workshop was held for each village, that there is appreciable variation in the size of the villages, and that the majority of villagers commute on foot, making it potentially difficult for residents of the largest villages to attend the drama events (due to walking distance). The suggestion that walking distance may explain lower participation rates in larger villages could be substantiated by a formal but simple analysis of population size versus percent attendance. A comment on the feasibility/practicality/value of implementing more than one workshop in the larger villages would be helpful, too. I felt that certain parts of the results section (e.g. page 6 ‘Combining arts and science…’ and ‘Social relationships in Cambodia…’) would be more appropriately placed in the discussion. Current literature In the discussion, the authors mention that - to their knowledge - ‘the current paper is the first assessment of using drama to engage ‘ the community’ for malaria control and elimination’. M Frishkopf and his colleagues have recently published research showing that a performing arts public health intervention in northern Ghana was effective to convey key messages about malaria transmission to local audiences (Frishkopf M. et al, 2016 ) 1 Additional reference to growing international recognition of both the value and ethics of community engagement in health-related research could be included. (King KF et al., 2014 . Wallerstein and Duran, 2010 ). 2 3 The key community engagement intervention strategy that is discussed in this paper is participatory drama. Drama is a visual method of engagement. Some reference to the value of working with participatory visual methods as a community engagement approach in public health and health research would add an important element to the current article (Mitchell and Sommer, 2016 ) 4 sufficient details of methods and analysis provided to allow replication by others? Are sufficient details of methods and analysis provided to allow replication by others? On page 5, the authors explain that their sampling method was purposive and limited by practicalities, but. To enable replicability, it would have been helpful to be given a brief description of the process through which interview respondents and focus group members were selected. The authors also explain that semi-structured interviews were done with various groups, including five professional actors one village malaria worker. The results section describes thematic key responses from different participant groups – especially the villagers - but there is no mention of responses from the actors or the VMWs. A complete understanding of the feasibility of the engagement strategy will only be gained through including the perspectives of all research respondents in the analysis. The description of the drama workshops and performances indicates that the drama was partly developed before the village engagement activities began, and then further developed during the workshop. This further development included participation from teenagers and young people, at each site, by the incorporation of their personal stories and experiences. This process of drama development is a very i t t t f th th d l d h t it t I ld t th t bit Page 14 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 important part of the methodology and approach to community engagement. I would suggest that a bit more detail is provided here as to the steps taken, for the purposes of replicability by others in the field. In the interest of the word count, this could perhaps be written in detail as supplementary material. important part of the methodology and approach to community engagement. I would suggest that a bit more detail is provided here as to the steps taken, for the purposes of replicability by others in the field. In the interest of the word count, this could perhaps be written in detail as supplementary material. The section describing the main storyline for the play is currently included in the results section (page 6). It does not describe the results of the data analysis, and would be more appropriately placed in the methods section. However, the storyline description is not necessary to the understanding of the project and could be left out entirely. Are the conclusions drawn adequately and supported by the results? Seven themes were surfaced through analysis of the data generated by the interviews and focus group discussions. There is a large overlap between the areas explored in the interview guides and the resulting themes. This is not entirely surprising, given the small sample size in each respondent group, but it is worth noting as a study limitation. The groups that participated in the interviews/focus group discussions, and the number of respondents in each of these groups, is described on page 5. For example, we know that there was 1 local health staff respondent and 1 village leader respondents. However, at several points in the results sections, the writing indicates that there were multiple respondents in these groups. (‘Staff from the local health authority felt…’, and ‘ Village leaders were interested because…’. The text needs to more accurately reflect the singular nature of these responses, and of the other interviewed groups that contained a single respondent. I wonder if sustainability is a relevant theme to be discussed this paper, based on the results of the data analysis that are described. The actual responses cited in this section refer to better understanding about malaria elimination projects that were gained through the engagement approach, which would seem to fit under theme 3 (understanding and learning about malaria using drama). Also, increased willingness of villagers to do blood testing does not speak to the sustainability of the engagement approach that is being evaluated, but rather to its impact. I would suggest revisiting this section and thinking about which parts are results that could be included under other themes, and which parts could be moved to the methods or discussion sections. The conclusions that are drawn are based on both the rates of participation in each village (averaging out at 66%) and the results from interviews and focus group sessions. In the section on data collection, the authors do acknowledge that their sampling was limited by practicalities, but the small sample size of each respondent group is a key limitation and this needs to be noted in the conclusion section. Additional Comments In the results section of the abstract, it is indicated that 'The event saw a very positive response...' Again this indicates that – even although the project included community engagement activities in 20 villages - the article is based on a single engagement event. If this is the case, readers should be made aware of this in the abstract. In the conclusion of the abstract, it is stated that 'the drama project was feasible in promoting awareness and understanding of malaria prevention and control'. This statement could be qualified by adding 'in this context' (i.e. rural settings in S.E. Asia). I would make the same suggestion for the final concluding statement in the paper. In the discussion, it would have been interesting to hear some of the authors reflections on the ethical In the discussion, it would have been interesting to hear some of the authors reflections on the ethical Page 15 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 considerations of using this specific approach to community engagement, having gained this important experience. For example, as far as I can see, approvals for the use of social media, filming and photography were only gained from the provincial authorities. Is this level of approval sufficient? If so, why is this considered to be the case? Did the project process foster expectations about the elimination of malaria in the district? If so, how were such expectations managed by the MORU team? considerations of using this specific approach to community engagement, having gained this important experience. For example, as far as I can see, approvals for the use of social media, filming and photography were only gained from the provincial authorities. Is this level of approval sufficient? If so, why is this considered to be the case? Did the project process foster expectations about the elimination of malaria in the district? If so, how were such expectations managed by the MORU team? In the discussion, a sentence or two indicating the authors learning from using a professional, touring drama team as the lead performers in a participatory process would also have valuable. Page 3. The explanation of literacy rates needs to be clarified For example : The 2014 census indicated literacy rates to be 93% and 77% in urban and rural areas, respectively. The ethical statement on page 5 is duplicated on page 8. This interesting research article provides important insights into the possibilities and challenges for multi-pronged, creative community engagement strategies to contribute to malaria elimination in rural South East Asia. With a little more reference to directly resonant publications, greater attention to grammar in some places, the repositioning of certain content, and the inclusion of ethical reflection, it will make an excellent and needed contribution to the community engagement in global health literature. Additional Comments On page 5, the authors explain that approval for the main script, storyline and key messages was sought from provincial health authorities. Was approval for these things also sought, at any stage, from villagers, VMW or local health staff? The authors explain that the average number of attendees at each village performance was 430. This is a large audience size for a community drama event. Were there any implications of this, for example background noise levels, or children running around? Were microphones/speakers used in this process? I wonder if this element of the drama engagement was explored through the interviews or focus groups. These are also important methodological details for the purposes of replicability. Minor comments In five cases, the spelling/formatting of the village names differs between the map (Figure 1) and Table 1. The ethical statement on page 5 is duplicated on page 8. References 1. Frishkopf M, Hamze H, Alhassan M, Zukpeni I, Abu S, Zakus D: Performing arts as a social technology for community health promotion in northern Ghana. . 2016; (1): Family Medicine and Community Health 4 22-36 Publisher Full Text 2. King KF, Kolopack P, Merritt MW, Lavery JV: Community engagement and the human infrastructure of global health research. . 2014; : 84 \| BMC Med Ethics 15 PubMed Abstract Publisher Full Text 3. Wallerstein N, Duran B: Community-based participatory research contributions to intervention research: the intersection of science and practice to improve health equity. . 2010; Am J Public Health 100 : S40-6 \| Suppl 1 PubMed Abstract Publisher Full Text 4. Mitchell CM, Sommer M: Participatory visual methodologies in global public health. . Glob Public Health (5-6): 521-7 \| 11 PubMed Abstract Publisher Full Text Is the work clearly and accurately presented and does it cite the current literature? Partly Is the work clearly and accurately presented and does it cite the current literature? Partly Page 16 of 17 Wellcome Open Research 2017, 2:95 Last updated: 13 OCT 2017 Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Partly No competing interests were disclosed. Competing Interests: Referee Expertise: Participatory visual methods for community engagement in public health and health science research I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Are the conclusions drawn adequately supported by the results? Partly I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Page 17 of 17
https://openalex.org/W2030284965	https://europepmc.org/articles/pmc4413869?pdf=render	English	null	Creaming and Parking in Quasi-Marketised Welfare-to-Work Schemes: Designed Out Of or Designed In to the UK Work Programme?	Journal of social policy	2,014	cc-by	8,779	Jnl Soc. Pol. (2015), 44, 2, 277–296 © Cambridge University Press 2014. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Jnl Soc. Pol. (2015), 44, 2, 277–296 © Cambridge University Press 2014. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S0047279414000841 Abstract ‘Creaming’ and ‘parking’ are endemic concerns within quasi-marketised welfare-to-work (WTW) systems internationally, and the UK’s ﬂagship Work Programme for the long-term unemployed is something of an international pioneer of WTW delivery, based on outsourcing, paymentbyresultsandproviderﬂexibility.IntheWorkProgrammedesign,providers’incentives to ‘cream’ and ‘park’ differently positioned claimants are intended to be mitigated through the existence of nine payment groups (based on claimants’ prior beneﬁt type) into which different claimants are allocated and across which job outcome payments for providers differ. Evaluation evidence suggests however that ‘creaming’ and ‘parking’ practices remain common. This paper offers original quantitative insights into the extent of claimant variation within these payment groups, which, contrary to the government’s intention, seem more likely to design in rather than design out ‘creaming’ and ‘parking’. In response, a statistical approach to differential payment setting is explored and is shown to be a viable and more effective way to design a set of alternative and empirically grounded payment groups, offering greater predictive power and value-for-money than is the case in the current Work Programme design. 10.1017/S0047279414000841 Creaming and Parking in Quasi-Marketised Welfare-to-Work Schemes: Designed Out Of or Designed In to the UK Work Programme? ELEANOR CARTER∗and ADAM WHITWORTH∗∗ ∗Department of Geography, University of Shefﬁeld, UK email: ecarter1@shefﬁeld.ac.uk ∗∗Department of Geography, University of Shefﬁeld, UK email: adam.whitworth@shefﬁeld.ac.uk Quasi-marketised WTW governance: convergence in logics and tensions The past ﬁfteen years have seen considerable changes in how welfare-to-work (WTW) is organised and delivered across the advanced economies with a consistent trend – though with inevitable variation within it (van Berkel et al., 2012) – towards ‘work ﬁrst’ activation delivered by a mixture of contractualism, managerialism and marketisation (van Berkel, 2010; van Berkel et al., 2012; de Graaf and Sirovatka, 2012). In the UK context, this is evident in the progression through New Deals from 1997, Employment Zones from 2001, Pathways to Work from 2007, Flexible New Deals from 2009 and, most recently, in the 278 eleanor carter and adam whitworth 278 form of the Work Programme since 2011, each more fully and deeply embracing these principles than its predecessor. The UK has seen remarkable cross-party agreement to this approach, aided by the transfer of key policy actors such as Lord Freud from Labour to the Conservatives in 2009. Hence, the arrival of a Conservative-Liberal Democrat Coalition government in the 2010 general election after thirteen years of Labour government, and the roll-out of its ﬂagship Work Programme WTW ‘does nothing to break out of the policy paradigm established by Labour; it simply takes it further and faster’ (Lister and Bennett, 2010: 102). Delivery within the Work Programme takes place through contracts between the Department for Work and Pensions (DWP) and large-scale, mainly private sector, ‘Prime providers’ (Primes) who can both deliver services themselves and/or sub-contract to organisations within large and complex supply chains which sit underneath each Prime. The programme is structured geographically into eighteen large ‘regional’1 Contract Package Areas (CPAs) with two or three Primes in each CPA to whom claimants are randomly allocated from the public sector Jobcentre Plus if they have not found work within an initial period of Jobcentre Plus provision, usually nine to twelve months. Whilst Flexible New Deal prescribed mandatory service components, the Work Programme operates a ‘black box’ delivery model, with providers having signiﬁcant ﬂexibility over interventions to, in principle, support experimentation and harness providers’ specialisation and innovation. Quasi-marketised WTW governance: convergence in logics and tensions At the heart of the Work Programme is payment- by-results for job outcomes: providers receive a small initial ‘attachment fee’ for taking a client on (though this ended in April 2014); receive a main ‘job outcome payment’ if a participant has been supported into ‘sustained paid work’ (of three months or six months duration depending on the payment group); and with further ‘sustainment payments’ available every four weeks for keeping a participant in employment (up to a maximum of twenty-four months). In these ways, Work Programme both draws from and contributes to the shared international trend towards WTW models built around principles and practices of quasi-marketisation and new public management. However, awareness of the international evidence around such WTW systems highlights a range of by now well-known problems, tensions and risks concerning their ability to achieve what are often conﬂicting policy aims around efﬁciency, performance, innovation, personalisation and equity. Despite extensive work analysing cross- national trends towards different forms of ‘new’ WTW governance regimes since the late 1990s, there remains a distinct lack of clear evaluation evidence as to their effects (Bredgaard and Larsen, 2008; de Graaf and Sirovatka, 2012). The piecemeal evidence that does exist suggests that effects are uncertain, variable and contingent on context and implementation (van Berkel and van der Aa, 2005; Bredgaard and Larsen, 2008; de Graaf and Sirovatka, 2012). In part, this reﬂects the difﬁculty in robustly evaluating what have tended to be rapidly evolving creaming and parking in the uk work programme 279 reforms, because new ‘implementation hazards’ are thrown up as policies are implemented and as policy makers respond to problems, disappointments and shifting priorities as they emerge (Bredgaard and Larsen, 2008; van Berkel, 2010; Finn, 2011a; Finn, 2011b). The patchwork of evidence that does exist suggests that outsourced provision is not more effective than public services in achieving job outcomes, but that claimants do tend to be more satisﬁed with outsourced provision. The evidence also suggests that quasi-marketised WTW can – but might not (e.g., as in the Netherlands) – generate efﬁciency savings and that cost-cutting makes the ‘hardest to help’ claimants vulnerable, is often hampered by signiﬁcant (and often signiﬁcantly underestimated) ongoing pressures to re- regulate and is often achieved by providers retreating to a relatively generic and basic offer (Struyven and Steurs, 2005; Davies, 2008; Bredgaard and Larsen, 2008; Considine et al., 2011; de Graaf and Sirovatka, 2012). ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? Among these issues, of particular focus in this paper are internationally shared concerns and experiences relating to the inequitable treatment of ‘harder to help’ claimants within the context of quasi-marketised WTW programmes that operate on a payment-by-results basis – i.e., what the WTW literature refers to as problems of ‘creaming’ and ‘parking’. This is particularly true when such schemes also operate a light touch minimum standards and monitoring framework, face tight performance targets and costs pressures and/or are dominated by for- proﬁt providers, all of which apply in the case of the UK Work Programme. In the context of WTW interventions, ‘creaming’ refers to provider behaviour that prioritises attention for unemployed claimants with fewer barriers to work and who are therefore felt to be easier, cheaper and also more likely to move into paid work and release outcome payments. In contrast, ‘parking’ refers to provider behaviour that deliberately neglects giving time, energy or resources to unemployed claimants with more substantial barriers to work, given that such claimants are considered to be relatively unlikely to move into paid work and/or to require considerable, and usually expensive, employment support to make a move into paid work likely (and hence an outcome payment). The practice of creaming and parking clearly cuts against the explicit policy intention to ‘narrow the performance gap between easier and harder to help claimants’ (NAO, 2012: 5), but the ﬁnancial and psychological implications of the practice are also of concern. Creaming and parking effectively translate to overpayment by government for any given employment outcome secured, and for individual programme participants who are denied meaningful support there are serious ramiﬁcations since unemployment, when coupled with lack of progression and ability to control the future life course, is associated with reduced mental 280 eleanor carter and adam whitworth well-being (Fryer, 1986). Given that providers receive payments based on job outcomes, there is a core tension around how best to deal with the inevitable variation in the likelihood of different claimants’ successfully moving into paid work (and hence releasing outcome payments). It is important therefore for programme design to ensure that the incentives of the economically rational provider to cream or park claimants, depending on type, is mitigated such that meaningful and equitable employment support is provided for all, irrespective of the level, complexity and cost of their support needs. ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? creaming and parking in the uk work programme 281 • Different programmes or streams for different claimant groups so that differently tailored support, performance criteria and outcome payments can be more easily and more explicitly operated for different types of claimants. • Different programmes or streams for different claimant groups so that differently tailored support, performance criteria and outcome payments can be more easily and more explicitly operated for different types of claimants. • Different programmes or streams for different claimant groups so that differently tailored support, performance criteria and outcome payments can be more easily and more explicitly operated for different types of claimants. • Differential pricing for individual participants within a holistic programme so that providers are incentivised to work meaningfully with all unemployed claimants, irrelevant of the level of their support needs/costs because the outcome payments attached to claimants increase in line with these support needs/costs. • Milestone payments can be used to incentivise providers to focus on all claimants despite their different places and trajectories on their employment journey (e.g., for moving claimants closer to work even if they do not move into work, for transitions from unemployment into work, or to reward job retention or progression for those in work). • Claimant leverage can be used systemically to seek to drive up provider performance by creating effective channels for unemployed participants to exercise voice (e.g., effective complaints processes and/or customer surveys) and exit (e.g., participant agency around choosing or switching providers or interventions). • ‘Star ratings’, as used in the Australian WTW model, where provider performance is rated based on their outcome performance after having controlled for the characteristics of claimants and their local areas, hence seeking to create a fairer comparison between providers with more and less difﬁcult caseloads. Of these potential policy responses, the Work Programme seeks to mitigate creaming and parking through minimum service guarantees from Prime providers and, particularly, via the use of nine separate payment groups with differential payments across these groups. Of these two mechanisms, weaknesses inthesubstance,consistencyand,insomecases,eventhepossibleenforceabilityof providers’ minimum service guarantees render these a far less useful and reliable protectionthantheycouldbe(Finn,2012).Moreover,theinherentvulnerabilityof Work Programme providers’ minimum service guarantees combines with highly imperfect participant awareness both of these guarantees and of the complaints procedures – particularly the existence of the Independent Case Examiner – through which claimants might complain and seek improved service. ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? Mitigating such risks is a perennial design challenge within quasi-marketised WTW schemes using payment-by-results, and one that international experiences show to be difﬁcult to resolve. This might be expected to be particularly true during periods of sustained economic difﬁculty, when jobs are scarce, when the economic context is more challenging than it was at the point at which contracts and performance targets were drawn up, when funding is largely or wholly based on successful job outcomes such that up-front funding for more costly interventions is harder to ﬁnd and when providers are explicitly for-proﬁt. These are all situations applicable to the Work Programme since its inception, though Rees et al. (2013) ﬁnd that sector makes less difference than might perhaps be expected given the sharp ﬁnancial pressures experienced by all providers within the scheme to date. More broadly, it should be noted that failings in meeting the support needs of harder-to-help claimants are not exclusive to outsourced provision, and, in part, reﬂect the reality that providers – of whatever sector – simply may not have the scope or resources to affect certain characteristics of claimants (e.g., mental or physical health conditions) or their local areas (e.g., a chronic lack of suitable jobs locally) that are relevant to their (un)employment. Despite inevitable variation in results, the international evidence is, on balance, clear about the risk and existence of creaming and parking in outsourced, payment-by-results WTW models (Struyven and Steurs, 2005; van Berkel and van der Aa, 2005; Bredgaard and Larsen, 2008; van Berkel et al., 2012; de Graaf and Sirovatka, 2012). The international evidence is equally clear about the central, dynamic, enormously challenging and largely trial-and-error process of programme design attempts to effectively guard against practices of creaming and parking (Struyven and Steurs, 2005; Finn, 2009; Finn, 2011a; Finn, 2012). A range of different design approaches have been proposed and pursued internationally to mitigate the risks around creaming and parking, and these can be grouped into six main strands that can be employed either in isolation or in combination: • Accelerator/progressive pricing systems in which payments for job outcomes increase in size as providers reach further into their caseloads, in principle incentivising them to support claimants with more complex needs who are assumed not to be amongst the early movers into paid work. ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? This payment model is signiﬁcantly creaming and parking in the uk work programme 283 predicated on realising successful job outcomes such that up-front funding to providers for interventions is tight while pressures to deliver job outcomes are intense, due both to the reliance on outcome payments but also to the permanent threat of contract termination for providers deemed by the DWP to be underperforming.2 On ﬁrst impressions, the complex system of payment groups, differential pricing and milestone payments appears a reasonably robust primary defence against provider incentives to cream and park claimants. Such a view would certainly tally with DWP’s own conﬁdence that it will ‘ensure that providers have strong incentives to help all of their customers’, and will close the performance gap between the easiest- and hardest-to-help (DWP, 2012: 6). However, the Work Programme payment groups are deﬁned relatively crudely on the basis simply of thetypeofbeneﬁtbeingclaimedatthepointofreferral.Thisisincontrasttoother systems internationally – most notably the Australian Jobseeker Classiﬁcation Instrument (JSCI) – that combine complex differential pricing systems with signiﬁcantly more subtle proﬁling instruments. One parking risk is that payments simply may not be high enough to realistically cover the intervention costs of the hardest to help, and recent evidence suggests that this may indeed be an important ongoing problem in the scheme (WPSC, 2013; Riley et al., 2014). Of central concern to the present paper, however, is the widely discussed but as yet unquantiﬁed risk that the Work Programme’s broad payment groups may actually offer up incentives for providers to cream and park given that, within such broad and heterogeneous payment groups, all claimants attract the same job outcome payment, but there is known to be substantial variation in support needs/costs, employment likelihood and risk for providers across those claimants (WPSC, 2011, 2013). Given that these issues have been widely experienced and discussed internationally, one may ask how Work Programme’s rather simplistic payment groups came to be, with the DWP offering differential pricing across payment groups as the primary systemic defence against creaming and parking incentives within the programme. Chris Grayling, then Minister for Employment within the DWP explained, ‘we needed to ﬁnd something that was simple to administer that was likely to be reasonably reﬂective’ and that ‘was simple, easy to understand, where there was no scope for debate and discussion . . . ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? In practice, therefore, there is a reliance within the Work Programme on the effectiveness of the payment groups and the differential pricing structure to defend against creaming and parking (Finn, 2011b: 2). As shown in Table 1, Work Programme participants are grouped into nine payment groups across which maximum possible payment levels for providers vary. This segmentation operates, in the DWP’s view, according to some notion of the average difﬁculty of securing transitions to employment for different 282 eleanor carter and adam whitworth TABLE 1. Work Programme payment groups and payment levels Claimant Group number Customer Group Time of referral Basis for referral Max Payment Year 1 starters (£) % of Work Programme population 1 JSA 18–24 From 9 months JSA Mandatory 3,810 20.09 2 JSA 25+ From 12 months JSA Mandatory 4,395 44.94 3 JSA Early Access From 3 months JSA Mandatory or voluntary depending 6,600 24.65 4 JSA ex-IB From 3 months JSA Mandatory 6,600 0.71 5 ESA vol- unteers At any time from WCA Voluntary 3,700 1.76 6 New ESA claimants Mandatory when expected to be ﬁt for work in 3–6 months; voluntary from WCA Mandatory or voluntary depending 6,500 5.92 7 ESA ex-IB Mandatory when expected to be ﬁt for work in 3–6 months; voluntary from WCA Mandatory or voluntary depending 13,720 1.03 8 IB/IS (Eng- land only) At any time Voluntary 3,285 0.23 9 JSA prison leavers On release Mandatory 6,600 0.67 claimants. As noted above, providers can receive three types of payments – an initial attachment fee (this disappeared in April 2014), a job outcome payment when a claimant moves into ‘sustained employment’ (work of three or six months TABLE 1. Work Programme payment groups and payment levels claimants. As noted above, providers can receive three types of payments – an initial attachment fee (this disappeared in April 2014), a job outcome payment when a claimant moves into ‘sustained employment’ (work of three or six months depending on the payment group) and further monthly sustainment payments when a claimant remains in paid work. ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? There will be variations within each group, that is inevitable but we think as a broad average it gives the providers a sensible basis to work with’ (Grayling in WPSC, 2011: Ev40; 28). Robert Devereux, Permanent Secretary of the DWP, mirrors this sentiment that the Work Programme payment groups and differential payments system ‘begins to move us towards trying to reﬂect some of the average difﬁculty [of moving claimants into sustained employment]. Everything we have done here takes us really a long way forward compared with where we were’ (PAC, 2012: 26). 284 eleanor carter and adam whitworth 284 It is undoubtedly true that the Work Programme’s differential payment design is administratively simple, easy to explain and understand, and more nuanced then previous schemes. Yet, it has also been repeatedly pointed out that the current segmentation based on beneﬁt type is an overly – and, in our view, unduly – crude basis on which to proxy distance to labour market and, as a consequence, on which to calibrate groups and differential payment levels (Lane et al., 2013; WPSC, 2011; WPSC, 2013). Rather, the more appropriate question is not, as Robert Devereux suggests, whether the current Work Programme design is more complex than previous designs in a relative sense – it is (WPSC, 2013: 33) – but, rather, whether or not it is adequate and effective in an absolute sense in mitigating provider incentives to cream and park and, in so doing, to deliver effective support for all claimants as well as value-for-money for the taxpayer. Here, unfortunately, there is growing and consistent evidence from within DWP’s ofﬁcial evaluation (Newton et al., 2012; Lane et al., 2013), from select committees (PAC, 2012; PAC, 2013; WPSC, 2011; WPSC, 2013) and from academic research (Rees et al., 2013; Rees et al., 2014) that this is not the case. Indeed, the ﬁrst phase of the DWP commissioned qualitative evaluation of the Work Programme is as clear as any government commissioned evaluation is ever likely to be: ‘the available evidence to date suggests that providers are engaging in creaming and parking, despite the differential payments regime’ (Newton et al., 2012: 124). ‘Creaming’ and ‘parking’ in the Work Programme: designed out or designed in? One further telling piece of evidence is that Prime providers are using in-house proﬁling to develop their own claimant streams rather than adopting DWP’s claimant groups as the structure for their activities, suggesting that the current payment- group-based differentiated payments do not correspond to providers’ views of claimants’ support needs and distance to labour market (Newton et al., 2012; Lane et al., 2013). Indeed, one provider suggested that the RAG rating proﬁling system33 used by some Primes and their end-to-end providers to ‘triage’ their caseloads was in effect a mechanism for creaming and parking: So from day one you’re categorised and if you’re a green customer you’ve got an anticipated job start date, you’ve got an action plan to work towards that, and you have to be seen so that is once or twice a week. So you’re pushed. If you’re amber your job start date is obviously further away, and it’s the expectation that you’ll have activity at least once a fortnight. If you’re red it could be a phone call once a month. So people are not using the word parking because it’s politically incorrect, but it’s happening [Tier 2 provider]. (Rees et al., 2014) The focus of this paper is to offer an original, programme-wide quantitative assessment of the extent of claimant variation within payment groups and, as a result, the extent to which the Work Programme’s current payment-group based differential payment system may be designing in rather than, as intended, designing out provider incentives to cream and park claimants. Following on from this, the paper explores the potential for greater use of statistical proﬁling techniques to better calibrate Work Programme’s differential payment levels with creaming and parking in the uk work programme 285 claimants’ distance from the labour market as one element towards creating an improved future Work Programme; a timely empirical contribution given recent requestsfromtheDWPSelectCommitteeforfullerconsiderationofthisapproach (WPSC, 2013). Data and methods Hence, our JSA Early Access cohort omits voluntary ‘vulnerable’ participants and under represents JSA repeaters. New ESA claimants can be either mandatory8 or voluntary9 participants depending upon their circumstances. It is not possible to estimate voluntary participation, and our New ESA payment group is comprised therefore only of mandatory ESA WRAG participants. These individuals were identiﬁed according to the severity of their health condition where we mimicked as closely as possible the questions within the Work Capability Assessment (WCA) from the various health-related survey questions within Understanding Society. DWP (2013:10–11) reports that 56 per cent of the ESA decisions resulting from the WCA, place individuals within the WRAG group rather than the Support Group and the least severely disabled 56 per cent of these ESA claimants in the survey are therefore classiﬁed by us as ESA WRAG and are mandated into this payment group. Having identiﬁed the pseudo-payment groups, employment outcomes over approximately the following twelve months were tracked. Of the 933 individuals originally identiﬁed in Wave 1, 528 individuals remain across our four pseudo- payment groups once survey attrition, missing employment data and residency beyond Great Britain10 are accounted for. Of these 528 individuals, 59 per cent fall in the JSA twenty-ﬁve plus payment group and roughly 14 per cent in the remaining three payment groups. The key outcome variable is transition into ‘sustained employment’ as it is this that triggers the Work Programme job outcome payment. Transitions to ‘sustained employment’ are deﬁned as six months of cumulative employment for the JSA eighteen to twenty-four and JSA twenty-ﬁve plus groups and as three months of cumulative employment for the JSA Early Access and New ESA groups. Whilst providers can also receive sustainment payments for helping claimants to remain in paid employment, the job outcome payment is a key outcome measure of the programme and makes for asensiblebasisonwhichtoevaluatetheadequacyofthepaymentgroupstructure. Around one-third of our cases in each payment group experience a transition into employment over this period, although only 18 per cent of cases show a transition into the ‘sustained employment’ that is the focus in the modelling below. Binary logistic regression models are used to estimate these claimants’ predictedprobabilityoftransitionintosustainedwork,giventheircharacteristics, and Understanding Society offers a rich set of explanatory variables for potential inclusion. Data and methods To explore these issues, the analyses below make use of the ﬁrst two waves of the special licensed geocoded version of Understanding Society, the UK’s largest household survey, to look at whether the current policy design links in to evidence of differing likelihoods of sustained employment transitions amongst a cohort of individuals identiﬁed to ﬁt within matched pseudo-payment groups. Some form of proﬁling within WTW systems is almost inevitable and is, in different countries and for different schemes, usually based on simple administrative eligibility criteria, such as in the Work Programme (e.g., beneﬁt type), advisor discretion or statistical modelling. Statistical approaches within WTW are considerably more developed in the US, but improvements in data quality and availability in the UK over the past decade have been shown to enable statistical proﬁling to an acceptable degree of accuracy, even if there will always be error, and evaluations of ‘acceptable accuracy’ will always to some extent be subjective (Hasluck, 2004; Bryson and Kasparova, 2003; Matty, 2013). Work Programme pseudo-payment groups were identiﬁed from Wave 1 of the Understanding Society survey (University of Essex. 2009–2011) in accordance withDWP’sserviceproviderguidance4 (summarisedinTable 1above).Duetothe small cohort size of some Work Programme payment groups, the Understanding Society data, despite their size, do not allow all nine claimant groups, or all parts of claimant groups, to be identiﬁed. However, four key payment groups – Jobseeker’s Allowance (JSA)5 claimants aged eighteen to twenty-four, JSA claimantsagedtwenty-ﬁveplus,JSAEarlyAccess,andESA6 WorkRelatedActivity Group (WRAG) – were included and together these payment groups account for around 90 per cent of the Work Programme referrals to date. In total, 933 such pseudo Work Programme individuals were identiﬁed in Wave 1, with 57 per cent of these cases in the JSA twenty-ﬁve plus payment group. The identiﬁcation of individuals in the JSA eighteen to twenty-four and the JSA twenty-ﬁve plus payment groups is relatively straightforward. The JSA Early Access group contains three separate types of claimants: mandatory entry of young adults not in education, employment or training (NEETs); mandatory entry of ‘JSA repeaters’;7 and voluntary early entry of a series of pre-identiﬁed ‘vulnerable’ JSA claimants (e.g., homeless, carers). It is possible in the Understanding Society survey to identify eligible NEET participants, but not voluntary ‘vulnerable group’ participants. JSA repeaters can be identiﬁed through the employment history module, but only for the one-quarter of the eleanor carter and adam whitworth 286 main sample of whom this is asked. Data and methods Model testing identiﬁed the set of variables shown in Figure 1 as the most parsimonious11and variables are retained either when they are statistically signiﬁcant or when they show reasonable effect sizes and their lack of statistical signiﬁcance could plausibly be due to small sub-group sample sizes. This is in recognition of the fact that our sample size is limited and that our desire is to focus on messages and effect sizes at least as much as simple statistical signiﬁcance (Ziliak and McCloskey, 2008). It also reﬂects our desire to present creaming and parking in the uk work programme 287 Figure 1. Explaining transitions into sustained employment Figure 1. Explaining transitions into sustained employment exploratory analyses and ideas around the potential role of statistical proﬁling as one potential element within a reformed Work Programme. Follow-on research with larger and richer administrative and/or Prime provider data that are not currently available in the public domain could be incorporated as a next step. Identifying and mitigating provider incentives to cream and park through enhanced proﬁling Figure 1 summarises the odds ratios from the ﬁnal regression model with asterisks used to highlight effects that are statistically signiﬁcant at the 5 per cent level. The substantive direction and size of these effects is as expected, though the restricted sample sizes for some sub-groups limits our ability to detect statistical signiﬁcance for some ﬁndings even when these effects are relatively large in size (e.g., experiencing health problems, English not ﬁrst language). Our focus is less on individual effects within the model and instead offers model ﬁt as a way of evaluating the current Work Programme design, as well as highlighting the potential of statistical approaches to drive subtler proﬁling and payment setting to better protect against creaming and parking. There is no agreement in the literature as to how best to evaluate the model ﬁt of binary logit models such as these and a box to the lower-right of Figure 1 shows two commonly used model ﬁt statistics – pseudo-R2 and predicted groups (i.e., whether an individual’s transition to employment is successfully predicted). As discussed below, although these measures are problematic, when 288 eleanor carter and adam whitworth 288 modelling rare events such as these they are presented here in order to get a feel for how our statistical model from Understanding Society performs compared with the current DWP Work Programme design. The lower section relates to the Understanding Society regression and shows a pseudo-R2 of 11 per cent, with 6.3 per cent of the actual transitions into sustained employment being correctly predicted. By way of comparison, the upper section presents equivalent model ﬁt statistics for a separate regression (not shown) relating to the current DWP Work Programme ‘model’ in which only payment groups are used as the predictor of transitions into sustained work. This DWP model offers virtually no explanatory power on these measures and this raises serious questions about the evidence for, and logic of, the current Work Programme payments structure. More importantly, in terms of this paper’s argument, this comparison highlights the relative superiority of a statistical approach to proﬁling and policy structuring. However, one technical challenge is that neither model ﬁt is well suited to assess the power and potential of a statistical approach, as a viable proﬁling strategy in this case, given that the sustained employment transition outcome variable being modelled is relatively rare. Identifying and mitigating provider incentives to cream and park through enhanced proﬁling Pseudo-R2 statistics from logit modelslackthetransparent,agreedupon,andconceptuallysoundfoundationsof their better-known OLS counterparts and, consequently, are often not reported. As a result, a variety of such statistics exist, all with health warnings for users and all tending to produce relatively low values when seeking to model rare events. The principles of the predicted groups measure is more helpful given that it is this, after all, that Prime providers will be paid for – can we use statistical models to differentiate between different types of unemployed claimants in terms of their likelihoods of moving into sustained work? The measure in its current form, however, is not well suited to this task, given that the threshold value to allocate cases to groups is predicted probabilities above 0.5 (predicted a sustained work transition) or below 0.5 (not predicted a sustained work transition). Although arbitrary,thisdefaultisusuallyunproblematic.Whenoutcomesarerare,however, a threshold value of 0.5 for the predicted probabilities is unrealistic as a basis to allocate cases given that, almost by deﬁnition, these long-term unemployed individuals are not (in a probabilistic sense) highly likely to enter sustained employment. Hence, and as shown in the left pane of Figure 3, few cases gain predicted probabilities above 0.5, leaving an inevitably low percentage of the cases that do actually move into sustained work with correct predictions. The default predicted group measure is not a helpful one in these circumstances. Moreover, the policy imperative for the Work Programme in terms of proﬁling and differential payments is the particular need to differentiate between the relativelikelihoodofunemployedindividualsmovingintosustainedemployment far more than it is to fully account for those employment transitions; the two are related, but they are not identical. creaming and parking in the uk work programme 289 Figure 2. Claimant segmentation and predictive accuracy Figure 2. Claimant segmentation and predictive accuracy Recent work by the DWP to estimate the likelihood that the unemployed will become long-term unemployed (Matty, 2013), the inverse in many ways to our focus, faces a similar problem and a similar technical challenge. It suggests segmentation analysis as a way forward as both a more conceptually and technically meaningful approach to the evaluation of model power in these circumstances. Identifying and mitigating provider incentives to cream and park through enhanced proﬁling In this segmentation approach shown in Figure 2, claimants’ predicted likelihoods of moving into sustained employment are broken into ten equally sized deciles with the 10 per cent of cases with the highest probabilities in the far left bar of Figure 2 through to the 10 per cent of cases with the lowest probabilities in the far right bar of Figure 2. For each segmented group the vertical axis shows the percentage of each group that did actually move successfully into sustained work in these survey data. For example, the far left bar shows that, of the 10 per cent of cases predicted most likely to move into sustained employment, around 37 per cent actually did so. In general, and crucially, the model correctly orders claimants’ relative likelihoods of moving into sustained work as shown by the gradual decline from left to right, such that at the far right of Figure 2 less than 2 per cent of the least likely claimants to move into sustained work actually do so. Due to the relatively small sample size of just over ﬁfty cases in each decile in Figure 2, there is some inevitable crudeness in these data, shown most clearly by the slight jump in decile 6, and greater subtlety and smoothness would be expected with larger datasets. Even with these data, however, Figure 2 highlights that it is possible to use the relative positioning of the predicted probabilities to 290 eleanor carter and adam whitworth Figure 3. From predicted probabilities to an improved differential payments system Figure 3. From predicted probabilities to an improved differential payments system fulﬁl the key policy task required for meaningfully differentiating between the long-term unemployed in terms of their likelihood of moving into paid work. The statistical model is not perfectly predictive, naturally, but it is superior to the current DWP model in an absolute sense (see Figure 1) and, most importantly in terms of its policy function, it is able to accurately and sensibly differentiate between different types of unemployed claimants (see Figure 2). In our view, this offers positive proof of concept for the potential of statistical proﬁling as a viable route towards subtler statistically based approaches to differential payment structures. The future availability of larger, richer administrative and/or Prime provider data could offer real traction for improving such approaches further. Identifying and mitigating provider incentives to cream and park through enhanced proﬁling At the same time, statistical proﬁling approaches should not be seen as a ‘magic bullet’ for solving the challenge of creaming and parking within the Work Programme, but, instead, as one necessary element in a broader evolution towards a redesigned Work Programme offering better calibrated incentives for providers and improved value-for-money for the taxpayer. How, though, might a statistical approach to proﬁling and payment structures potentially be operationalised in a redesigned future Work Programme? Ignoring for a moment the horizontal lines and labelling in Figure 3, the grey dots in the left pane of Figure 3 plot the individual predicted probabilities of moving into sustained work for our Understanding Society sample. Although the logic of our approach steps beyond the existing payment groups structure, quantifying and visualising this variation within the existing payment groups is creaming and parking in the uk work programme 291 a powerful visual insight into the extent to which highly cost-pressured, mainly for-proﬁt Prime providers are currently facing considerable incentives to proﬁle, cream and park claimants in the Work Programme. Speciﬁcally, if one assumes economic rationality on the part of providers then, given that all claimants within each payment group share the same payment level, those individuals with the lowest predicted probabilities of moving into sustained work seem to be at considerable risk of being parked, while those individuals with the highest predicted probabilities seem liable to be creamed. Indeed, given the likelihood of increased intervention costs for harder-to-place claimants, the incentives to park and cream are stronger than these probabilities suggest. Equally, individuals in different payment groups, but with equivalent predicted probabilities of moving into sustained work, might be expected to be treated differently by Primes where payment levels differ substantially between those payment groups. The black triangles in this left pane of Figure 3 reﬂect the payment group’s average probability of moving into sustained work – in other words DWP’s basis for the current payment groups and pricing structure. Figure 3 makes clear that this, in Chris Grayling’s words, ‘broad average’ (Grayling in WPSC, 2011: 28) is precisely that, and is a poor basis for proﬁling and price setting given that virtually all of the variation shown is within rather than between payment groups. Identifying and mitigating provider incentives to cream and park through enhanced proﬁling One obvious way to seek to better protect against creaming and parking given these two conﬂicting features of the current Work Programme system – large individual variation within payment groups combined with ﬁxed payment levels for those payment groups – is to calibrate payment levels more closely with modelled likelihoods of moving into sustained employment. Although visualised here within payment groups for ease of explanation, there is no need to retain the existing payment groups within any such redesign, and, indeed, it may well be more logical to move beyond them as the Work Programme evolves. The right pane of Figure 3 shows two potential redesigns. Firstly, one might in principle think about individualised payment levels, though this does not represent the preferred way forward in our view. The black triangles in this right pane of Figure 3 reﬂect the payment levels that providers currently receive once an individual in each payment group moves into sustained employment (i.e., the attachment fee plus job outcomes payment). The vertical spread of grey markers show one potential payment redesign: individualised payments based on each individual’s predicted probability of moving into sustained paid work and calculated as an inverse share of the current budgetary pot (i.e., this proposal is cost-neutral). As a result, providers are given strengthened ﬁnancial incentives to work with individuals with lower predicted probabilities who are farther from the labour market (markers lower down in the left pane) and who are most at risk of being parked in the current framework. The ﬂip-side of this analysis, of course, is the recognition that the DWP must currently be systematically overpaying providers for many job outcomes if it is accepted that parking is taking place 292 eleanor carter and adam whitworth and that payment levels reﬂect average payment group likelihoods of moving into sustained work. Hence, the need for an improved payment structure relates importantly also to efﬁciency (taxpayer value-for-money) as well as to equity (creaming and parking). In our view, however, despite the improvement to claimant segmentation that statistical proﬁling can offer, it may well be sensible to remain realistic about the error that will always occur at the individual level within such approaches. Hence, a sensible route forward is, in our view, to think instead more about creating alternative, empirically based and probabilistically grounded payment groups rather than seeking fully individualised payment levels. Identifying and mitigating provider incentives to cream and park through enhanced proﬁling The horizontal lines and plot labelling in Figure 3 show how such an approach might work. In this example, Figure 3 identiﬁes three new groups of claimants with low probabilities/high payments, medium probabilities/medium payments and high probabilities/low payments, respectively. Relating this back to Figure 2 above, it may be for example that the top three deciles with the highest predicted probabilities form one group (with lower payments), the central three deciles form a second group (with medium payments) and the bottom four deciles with the lowest predicted probabilities form a third group (with higher payments), or some such equivalent. This is the essence of the Job Services Australia model that has been in place since 2009 in Australia and in which claimants are placed into one of four differently paying streams according to their distance from the labour market as based on a detailed statistical proﬁling exercise known as the Job Seeker Classiﬁcation Instrument. Discussion OutsourceddeliveryofWTWsupport,basedonpayment-by-results,isbecoming anincreasinglywidespreadpolicyframeworkacrossadvancedeconomies,andthe UK Work Programme is in many ways at the vanguard of this trend. A perennial problem within such frameworks is the difﬁculty in incentivising what are often proﬁt-seeking providers to support all claimants effectively, despite their differing support costs and their differing likelihoods of realising outcome payments. Within the Work Programme, it is differential pricing across administratively deﬁned payment groups that seeks to calibrate provider incentives across the whole range of claimants and, in so doing, to mitigate risks of creaming and parking for claimants and to realise value-for-money for taxpayers. The evaluation literature to date, however, suggests that the current Work Programme design is not achieving these aims and that creaming and parking do seem to be taking place. At the heart of this problem lie the overly simplistic current payment groups, and a pricing mechanism that hides signiﬁcant within-group variation and that, as a consequence, is a poor counter to providers’ economically rational incentives to cream and park differently positioned claimants. creaming and parking in the uk work programme 293 The modelling work presented above offers original and timely programme- wide quantitative evidence on this debate for the ﬁrst time. It highlights that the current Work Programme payment groups offer virtually no explanatory power given that the vast majority of the variation in predicted probabilities is within rather than between payment groups. The incentives for creaming and parking within this context are considerable and there is a real risk that the current payment groups unintentionally design such practices in rather than out. In response, the analyses highlight that there is viable potential in the UK context for a statistical approach to proﬁling and improved payment calibration in order to create alternative, empirically grounded and probabilistically rooted payment groups as in the Australian model. The result of doing so would be to better calibrate Work Programme price setting, more effectively design out creaming and parking and better protect taxpayer value-for-money than is the case currently. Given that a switch towards some form of statistical proﬁling approach is relatively simple, and inexpensive to implement and explain, it is unclear why it would not be seriously considered given its empirical advantages to the current Work Programme design. Discussion These analyses in our view offer positive proof of concept, and further work with larger and richer administrative and/or Prime provider data is now needed to progress this work towards use in the ﬁeld. p p g At the same time, statistical approaches to improved proﬁling, segmentation and payment calibration clearly should not be expected to offer a magic bullet to these problems and, indeed, they come with problems of their own (Struyven and Steurs, 2005: 221; OECD, 2012: 110–112). The Australian system, for example, towards which these analyses point, has undergone successive waves of policy reform as it has sought to respond variously to problems experienced around parking, employer engagement, participant group misallocation and ‘gaming’ by providers (Finn, 2011b). As outlined above, a range of approaches can be taken to achieve the multiple aims of quasi-marketised WTW schemes, such as the Work Programme, and many of these approaches should be considered as necessary complements to one another rather than as alternative options. While statistical approaches offer clear advantages to the current Work Programme payment structure, other complementary design changes are also likely to be necessary if both the hardest-to-help claimants and value-for-money for the taxpayer are to be supported effectively. Obvious areas to enhance within the Work Programme include tightening up on the existing variable and often unduly vague minimum service guarantees, greater attention to, and usage of, claimant experiences and claimant choice. Key questions also remain around the adequacy of current resources to cover the intervention costs of those with complex support needs, whether the current balance between up-front and outcome-based payments is the right one and whether intermediate outcome payments might also be used more creatively. One example of the latter might be to incentivise providers to reducethedistancetolabourmarketforthoseclaimantswiththemostsubstantial 294 eleanor carter and adam whitworth 294 barriers where transitions to work itself simply may not be realistic in the short- term. As outlined above, the trends towards these new governance regimes within WTW delivery run considerably ahead of the evidence of their positive impacts yet there seems little likelihood of anything but a general continuation of these shifts. If that is the case, and we are to remain within the logic and practice of that paradigm, then the least that we should do is to remain ﬂexible to reshaping those policies as evidence emerges regarding their weaknesses and limitations. Discussion The present analyses offer a starting point for one aspect of these discussions. It is hopedthattheywillstimulateseriousacademicandpolicydebateintheUKabout theevidentneedforWorkProgrammeevolutionarounditscurrentlyproblematic approach to proﬁling and differential pricing that seems to be designing in, rather than designing out, provider incentives to cream and park. Notes 1 The North East and Scotland are examples of the scale of CPAs. 1 The North East and Scotland are examples of the scale of CPAs. 2 In March 2014, Newcastle College Group became the ﬁrst Prime provider to have one of its contracts ended, despite the ﬂawed design of the minimum performance levels and despite NCG technically not having breached those minimum performance levels (NAO, 2014). 2 In March 2014, Newcastle College Group became the ﬁrst Prime provider to have one of its contracts ended, despite the ﬂawed design of the minimum performance levels and despite NCG technically not having breached those minimum performance levels (NAO, 2014). 3 RAG rating uses a trafﬁc light system to group individuals either as red (hard to move into work), amber (moderately difﬁcult) or green (easy to move into work). 3 RAG rating uses a trafﬁc light system to group individuals either as red (hard to move into work), amber (moderately difﬁcult) or green (easy to move into work). ) ( y ) g ( y ) 4 Department for Work and Pensions (2014). y g 4 Department for Work and Pensions (2014). 5 JSAreferstoJobseeker’sAllowance,themainout-of-workbeneﬁtforunemployedindividuals without a certiﬁed health condition in the UK. 6 ESA refers to Employment and Support Allowance, the main out-of-work beneﬁt for the unemployed with a certiﬁed health condition in the UK (although ESA can also be claimed by those in work). 7 Those who have received JSA in twenty-two of the past twenty-four months. 8 ESA Work Related Activity Group (WRAG). 8 ESA Work Related Activity Group (WRAG). 9 ESA claimants with child under ﬁve and either three to six month prognosis or a full-time carer. 10 Some survey cases relate to Northern Ireland but Work Programme operates only in Great Britain. 10 Some survey cases relate to Northern Ireland but Work Programme operates only in Great Britain. 11 Also tested but not retained in ﬁnal model were: age, ethnicity, household type, vocational qualiﬁcations, number of children, work experience, born in UK, urban/rural, uses public transport rather than car. 11 Also tested but not retained in ﬁnal model were: age, ethnicity, household type, vocational qualiﬁcations, number of children, work experience, born in UK, urban/rural, uses public transport rather than car. 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https://openalex.org/W2900918048	https://www.frontiersin.org/articles/10.3389/fncel.2018.00442/pdf	English	null	Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface	Frontiers in cellular neuroscience	2,018	cc-by	9,415	Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPA-type glutamate receptor; AZLM, active-zone-like membrane; BSA, Bovine serum albumin; Fc, Fragment crystallizable; LTP, Long-term potentiation; LTD, Long-term depression; LRRTM, Leucine rich repeat transmembrane; NLG, Neuroligin; NMDA, N-methyl-D-aspartate; NRX, Neurexin; PALM, Photo-activated localization microscopy; PSLM, Postsynaptic-like membrane; SEP, Super-ecliptic pHluorin; SN, Signal to noise; STORM, Stochastic optical reconstruction microscopy; SynCAM, Synaptic cell adhesion molecule; TIRFM, Total internal reflection fluorescence microscopy. REVIEW published: 21 November 2018 doi: 10.3389/fncel.2018.00442 Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface Tomoo Hirano* Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan Regulation of exo- and endocytosis of α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) plays a critical role in the expression of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) at excitatory central synapses. Enhanced AMPAR exocytosis or endocytosis has been suggested to contribute to LTP or LTD, respectively. However, several unsettled fundamental questions have remained about AMPAR exo- and endocytosis in the basal condition and during synaptic plasticity: (1) Does the size of each exo- or endocytosis event, and/or do the frequencies of these events change during LTP or LTD? If they change, what are the time courses of the respective changes? (2) Where does the exo- or endocytosis preferentially occur in each condition: inside or in the vicinity of postsynaptic membrane, or in the extrasynaptic membrane? (3) Do different types of AMPAR, such as GluA1 homo-tetramer, GluA1/2 hetero-tetramer and GluA2/3 hetero-tetramer, show distinct exo- and endocytosis changes? To address these questions, we developed new methods to observe individual events of AMPAR exo- or endocytosis with a high signal to noise (SN) ratio in a culture preparation using total internal reﬂection ﬂuorescence microscopy (TIRFM). In these studies, hippocampal neurons were cultured on a neurexin (NRX)-coated glass coverslip, which induced formation of postsynaptic-like membrane (PSLM) directly on the glass surface. Then, a super-ecliptic pHluorin (SEP)-tagged AMPAR subunit such as GluA1 (GluA1-SEP) was expressed in neurons and its ﬂuorescence changes during LTP induced by high frequency electrical ﬁeld stimulation were observed with TIRFM, which showed different time courses of exocytosis changes of GluA1-, GluA2-, or GluA3-SEP in and around PSLM. In addition, a new method to detect individual endocytosis events of AMPAR was developed by combining TIFRM observation of GluA-SEP around PSLM with a rapid extracellular pH exchange method using a U-tube. Recent results on exo- and endocytosis changes of GluA-SEP during N-methyl-D-aspartate (NMDA)-induced Edited by: Enrica Maria Petrini, Fondazione Istituto Italiano di Technologia, Italy Reviewed by: Yoichi Araki, Johns Hopkins University, United States Takashi Hayashi, National Center of Neurology and Psychiatry (Japan), Japan Reviewed by: Yoichi Araki, Johns Hopkins University, United States Takashi Hayashi, National Center of Neurology and Psychiatry (Japan), Japan *Correspondence: Tomoo Hirano thirano@neurosci.biophys.kyoto- u.ac.jp Received: 28 July 2018 Accepted: 05 November 2018 Published: 21 November 2018 Received: 28 July 2018 Accepted: 05 November 2018 Published: 21 November 2018 Received: 28 July 2018 Accepted: 05 November 2018 Published: 21 November 2018 INTRODUCTION Fernández-Monreal et al., 2012). Both clathrin-dependent and - independent AMPAR endocytosis occur, and the contribution of the former to LTD induction has been reported (Glebov et al., 2015; Zheng et al., 2015). AMPAR endocytosis might occur not only in the extrasynaptic membrane but also in the vicinity of postsynaptic membrane (Blanpied et al., 2002; Kennedy and Ehlers, 2006; Lu J. et al., 2007; Tao-Cheng et al., 2011; Fujii et al., 2017, 2018). The involvement of GluA1 homo-tetramer in LTD induction has been suggested (Sanderson et al., 2016), although involvement of GluA2-lacking AMPAR in LTP or LTD has been debated (Passafaro et al., 2001; Plant et al., 2006; Adesnik and Nicoll, 2007; Gray et al., 2007; Lu Y. et al., 2007). AMPAR subtype changes might also take place during LTD. Long-term potentiation (LTP) and long-term depression (LTD) at hippocampal glutamatergic synapses have been regarded as basic cellular mechanisms of learning and memory, and intensively studied (Malinow and Malenka, 2002; Kauer and Malenka, 2007; Collingridge et al., 2010; Huganir and Nicoll, 2013). Originally changes in functional properties of α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) were considered as molecular mechanisms of LTP or LTD. More recently, changes in the number of AMPAR on the postsynaptic membrane attracted much attention of synaptic physiologists as primary mechanisms of LTP or LTD expression (Malinow and Malenka, 2002; Kennedy and Ehlers, 2006; Derkach et al., 2007; Shepherd and Huganir, 2007; Huganir and Nicoll, 2013). AMPAR exocytosis, endocytosis, lateral movement on the plasma membrane and trapping in the postsynaptic membrane regulate the number of postsynaptic AMPARs (Malinow and Malenka, 2002; Shepherd and Huganir, 2007; Makino and Malinow, 2009; Opazo and Choquet, 2011; Huganir and Nicoll, 2013; Wu et al., 2017). g LTP and LTD have been studied mainly by electrophysiological recording, immuno-cytological staining and biochemical assays combined with pharmacological or molecular biological manipulations and/or use of transgenic mice (Malinow and Malenka, 2002; Kauer and Malenka, 2007; Collingridge et al., 2010; Huganir and Nicoll, 2013). The use of live-cell imaging techniques in the analyses of LTP and LTD mechanisms has increased. Technical advancements have made it possible to detect individual events of exo- or endocytosis of AMPAR. Development of a pH-sensitive variant of green fluorescent protein called super-ecliptic pHluorin (SEP) enabled selective monitoring of proteins in neutral pH conditions, such as on the cell-surface, but not proteins inside intracellular organelles with acidic luminal solution (Miesenböck et al., 1998). INTRODUCTION SEP has been widely used in studies on AMPAR trafficking during LTP or LTD (Ashby et al., 2004; Lin and Huganir, 2007; Yudowski et al., 2007; Lin et al., 2009; Araki et al., 2010; Kennedy et al., 2010; Tanaka and Hirano, 2012; Rathje et al., 2013; Jullié et al., 2014; Tanaka et al., 2014; Fujii et al., 2017, 2018; Rosendale et al., 2017; Temkin et al., 2017; Wu et al., 2017). Total internal reflection fluorescence microscopy (TIRFM) provides very high signal/noise (SN) ratio images by limiting the depth of the visualization zone (Axelrod, 2001), and has also been used in live-cell imaging studies of AMPAR dynamics (Yudowski et al., 2007; Wang et al., 2008; Lin et al., 2009; Araki et al., 2010; Tanaka and Hirano, 2012; Jullié et al., 2014; Tanaka et al., 2014; Fujii et al., 2017, 2018; Rosendale et al., 2017). A rapid extracellular pH exchange method combined with the use of SEP made it possible to record individual endocytosed vesicles (Merrifield et al., 2005; Jullié et al., 2014; Rosendale et al., 2017). Enhancement of AMPAR exocytosis was suggested to contribute to LTP expression (Shi et al., 1999; Hayashi et al., 2000; Passafaro et al., 2001; Kopec et al., 2006; Park et al., 2006; Plant et al., 2006; Lin et al., 2009; Kennedy et al., 2010; Patterson et al., 2010; Huganir and Nicoll, 2013). Exocytosis was reported to occur around the postsynaptic membrane and in extra-synaptic membrane away from synapses (Kennedy et al., 2010). In the latter case, lateral movement of AMPAR and trapping of it on the postsynaptic membrane are necessary to accumulate AMPAR in the postsynaptic membrane (Opazo et al., 2010, 2012; Opazo and Choquet, 2011; Chen et al., 2015). However, the extent to which each pathway contributes to LTP expression remains an open question. There are four types of AMPAR subunits GluA1–4 (Hollmann and Heinemann, 1994; Dingledine et al., 1999). In hippocampal glutamatergic synapses GluA1/GluA2 hetero- tetramer and GluA2/GluA3 hetero-tetramer are the main postsynaptic receptors (Dingledine et al., 1999). On the other hand, GluA1 homo-tetramer is present in some types of hippocampal neurons, and its involvement in synaptic plasticity has also been reported (Iino et al., 1990; Plant et al., 2006; Lu Y. et al., 2007; Sanderson et al., 2016). Citation: Hirano T (2018) Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface. Front. Cell. Neurosci. 12:442. doi: 10.3389/fncel.2018.00442 November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 1 AMPA Receptor Exo- and Endocytosis Hirano Hirano LTD suggested that suppression of AMPAR exocytosis rather than enhancement of AMPAR endocytosis primarily contributes to LTD expression, although the NMDA application transiently enhances clathrin-dependent endocytosis of GluA1-containing AMPAR. Keywords: exocytosis, endocytosis, LTP, LTD, hippocampus, AMPA receptor, total internal reﬂection ﬂuorescence microscopy, live-cell imaging Frontiers in Cellular Neuroscience \| www.frontiersin.org FORMATION OF PSLM ON NEUREXIN-COATED GLASS Several types of cell-adhesion molecules are found at synapses such as Neuroligin (NLG), Neurexin (NRX), Synaptic cell adhesion molecule (SynCAM), EphrinB, leucine rich repeat transmembrane (LRRTM) and N-Cadherin. Among them presynaptic membrane protein NRX and postsynaptic membrane protein NLG have been studied extensively (Levinson and El-Husseini, 2005; Dean and Dresbach, 2006; Craig and Kang, 2007; Südhof, 2008; Bukalo and Dityatev, 2012). Both of them have different subtypes and various splice variants. There are five NLG genes NLG 1–4 and NLG 4Y, and there are six NRX genes NRX 1α, 1β, 2α, 2β, 3α, 3β. NRXs undergo extensive alternative splicing, which could potentially generate >2,000 variants. Among these variants, splicing insertion of site 4 in β-NRX promotes GABAergic synapse formation, whereas β-NRX without site 4 insertion promotes glutamatergic synapse formation. It is also known that NLG 1 with splice insertion at site B promotes glutamatergic synapse formation, and that NLG 2 is primarily found at GABAergic synapses. FIGURE 1 \| Formation of postsynaptic-like membrane (PSLM). NRX, Neurexin; NLG, Neuroligin; BSA, Bovine serum albumin; Fc, fragment crystallizable of immunoglobulin. This ﬁgure is newly drawn based on our previous publications (Tanaka and Hirano, 2012; Tanaka et al., 2014), and copyright permission is not required. FIGURE 2 \| PSLM and normal synapses observed with total internal reﬂection ﬂuorescence microscopy (TIRFM) or with conventional epi-ﬂuorescence. (A) Scheme of PSLM and a normal synapse on NRX-coated glass. Excitation light (yellow) reaches only PSLM and the lower parts of dendrites in TIRFM (left), whereas it covers the whole area under epi-ﬂuorescence (right). At a normal synapse, postsynaptic PSD95 signal is apposed to presynaptic vglut1 signal. (B) PSD95-EGFP signal (green) and vglut1 signal (magenta) recorded with TIRFM (left) or with epi-ﬂuorescence (right), respectively. Arrows indicate PSLMs that are clearly observed with TIRFM and are not accompanied by vglut1 signals, and arrowheads indicate normal synapses which are not clearly observed with TIRFM. These ﬁgure panels were ﬁrst published in Tanaka et al. (2014), and copyright permission was obtained. NLG expressed in non-neuronal cells co-cultured with neurons induces formation of presynaptic structures in axons, while NRX when similarly expressed induces formation of postsynaptic structures in dendrites (Scheiffele et al., 2000; Graf et al., 2004). Furthermore, NRX attached to beads induces clustering of postsynaptic proteins (Graf et al., 2004). INTRODUCTION Thus, there may be specific regulatory mechanisms for each of these subtypes of AMPAR composed of different combinations of subunits, but this has not yet been precisely clarified. Recently, we developed a new method to further improve the SN ratio and spatiotemporal resolution of live-cell imaging data of SEP-tagged AMPAR. We induced formation of postsynaptic-like membrane (PSLM) directly on the surface of a glass coverslip, and then studied the dynamics of GluA-SEP Enhancement of AMPAR endocytosis has been suggested as a primary mechanism for LTD expression (Beattie et al., 2000; Lee et al., 2002; Ashby et al., 2004; Lin and Huganir, 2007; November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 2 AMPA Receptor Exo- and Endocytosis Hirano around PSLM during LTP or LTD expression (Tanaka and Hirano, 2012; Tanaka et al., 2014; Fujii et al., 2017). In this review, I will briefly summarize recent results on AMPAR dynamics during synaptic plasticity obtained using GluA-SEP, PSLM and TIRFM. FORMATION OF PSLM ON NEUREXIN-COATED GLASS These findings prompted us to test whether a glass coverslip coated with NRX could induce formation of postsynaptic structures on the glass surface. We considered that such postsynaptic structures formed directly on and parallel to the glass surface would be an ideal model of postsynaptic structure which could be used in live-cell fluorescence imaging experiments using TIRFM (Figures 1, 2), because application of TIRFM to such structures would be efficient and effective. TIRFM can provide very high contrast fluorescence images by decreasing background signals. Excitation light reaches only about 100 nm above the glass surface in an inverted microscope equipped for TIRFM (Figure 2). FIGURE 2 \| PSLM and normal synapses observed with total internal reﬂection ﬂuorescence microscopy (TIRFM) or with conventional epi-ﬂuorescence. (A) Scheme of PSLM and a normal synapse on NRX-coated glass. Excitation light (yellow) reaches only PSLM and the lower parts of dendrites in TIRFM (left), whereas it covers the whole area under epi-ﬂuorescence (right). At a normal synapse, postsynaptic PSD95 signal is apposed to presynaptic vglut1 signal. (B) PSD95-EGFP signal (green) and vglut1 signal (magenta) recorded with TIRFM (left) or with epi-ﬂuorescence (right), respectively. Arrows indicate PSLMs that are clearly observed with TIRFM and are not accompanied by vglut1 signals, and arrowheads indicate normal synapses which are not clearly observed with TIRFM. These ﬁgure panels were ﬁrst published in Tanaka et al. (2014), and copyright permission was obtained. FIGURE 2 \| PSLM and normal synapses observed with total internal reﬂection ﬂuorescence microscopy (TIRFM) or with conventional epi-ﬂuorescence. (A) Scheme of PSLM and a normal synapse on NRX-coated glass. Excitation light (yellow) reaches only PSLM and the lower parts of dendrites in TIRFM (left), whereas it covers the whole area under epi-ﬂuorescence (right). At a normal synapse, postsynaptic PSD95 signal is apposed to presynaptic vglut1 signal. (B) PSD95-EGFP signal (green) and vglut1 signal (magenta) recorded with TIRFM (left) or with epi-ﬂuorescence (right), respectively. Arrows indicate PSLMs that are clearly observed with TIRFM and are not accompanied by vglut1 signals, and arrowheads indicate normal synapses which are not clearly observed with TIRFM. These ﬁgure panels were ﬁrst published in Tanaka et al. (2014), and copyright permission was obtained. FIGURE 2 \| PSLM and normal synapses observed with total internal reﬂection ﬂuorescence microscopy (TIRFM) or with conventional epi-ﬂuorescence. (A) Scheme of PSLM and a normal synapse on NRX-coated glass. Frontiers in Cellular Neuroscience \| www.frontiersin.org FORMATION OF PSLM ON NEUREXIN-COATED GLASS (A–C) Averaged time courses of GluA1–3 ﬂuorescence intensity in PSLM (red) and in non-PSLM (black) measured every 4 min before and after the ﬁeld stimulation (arrows). Data in the presence of APV (+APV) are also shown (dotted lines). Error bars indicate SEM. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. (D–F) GluA-super-ecliptic phluorin (SEP) signals (green) and PSD95-RFP signal (magenta) are shown. PSD95-RFP was recorded before the stimulation, and images of the two signals were overlaid. GluA-SEP signals in PSLM and non-PSLM are indicated by arrows and arrowheads, respectively. Scale bar, 2 µm. These ﬁgure panels were ﬁrst published in Tanaka and Hirano (2012), and copyright permission is not necessary. FIGURE 3 \| Changes of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) subunit number by long-term potentiation (LTP)-inducing stimulation. (A–C) Averaged time courses of GluA1–3 ﬂuorescence intensity in PSLM (red) and in non-PSLM (black) measured every 4 min before and after the ﬁeld stimulation (arrows). Data in the presence of APV (+APV) are also shown (dotted lines). Error bars indicate SEM. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. (D–F) GluA-super-ecliptic phluorin (SEP) signals (green) and PSD95-RFP signal (magenta) are shown. PSD95-RFP was recorded before the stimulation, and images of the two signals were overlaid. GluA-SEP signals in PSLM and non-PSLM are indicated by arrows and arrowheads, respectively. Scale bar, 2 µm. These ﬁgure panels were ﬁrst published in Tanaka and Hirano (2012), and copyright permission is not necessary. not apposed to a presynaptic terminal releasing glutamate. We considered that glutamate released from nearby presynaptic terminals activates N-methyl-D-aspartate (NMDA) receptors and induces the changes in the amount of GluA-SEP in PSLM, because an antagonist of NMDA receptor APV suppresses the changes. but that were not apposed to a presynaptic marker were observed with TIRFM (Figure 2). Such postsynaptic-like membrane (PSLM) exhibited accumulation of homer, another marker protein of postsynaptic density and AMPAR. We chose relatively large neurons possessing spines which presumably corresponded to pyramidal neurons for the experiments. Conditioning stimulation inducing either LTP or LTD increased or decreased the amount of AMPAR in PSLM, respectively (Tanaka and Hirano, 2012; Fujii et al., 2018). g One possible factor contributing to the increase in the amount of GluA-SEP on the surface is enhancement of GluA-SEP exocytosis. Individual events of GluA-SEP exocytosis can be observed around PSLM by high frequency live-cell TIRFM imaging (Figure 4). FORMATION OF PSLM ON NEUREXIN-COATED GLASS LTP-inducing electrical stimulation increases the frequency of GluA-SEP exocytosis. We reported a transient (about 1 min) increase of GluA1-SEP exocytosis frequency around PSLM and a subsequent increase for several minutes outside PSLM. We also found that GluA1-SEP exocytosis does not occur in the center of PSLM, but rather it occurs in the periphery of PSLM or outside of PSLM. Exocytic domain adjacent to the postsynaptic membrane was previously reported (Kennedy et al., 2010). FORMATION OF PSLM ON NEUREXIN-COATED GLASS Excitation light (yellow) reaches only PSLM and the lower parts of dendrites in TIRFM (left), whereas it covers the whole area under epi-ﬂuorescence (right). At a normal synapse, postsynaptic PSD95 signal is apposed to presynaptic vglut1 signal. (B) PSD95-EGFP signal (green) and vglut1 signal (magenta) recorded with TIRFM (left) or with epi-ﬂuorescence (right), respectively. Arrows indicate PSLMs that are clearly observed with TIRFM and are not accompanied by vglut1 signals, and arrowheads indicate normal synapses which are not clearly observed with TIRFM. These ﬁgure panels were ﬁrst published in Tanaka et al. (2014), and copyright permission was obtained. Glass coating with NRX was performed utilizing biotin- avidin interaction and an antibody which was described in detail elsewhere (Tanaka and Hirano, 2012; Tanaka et al., 2014; Figure 1). Briefly, glass coverslips were coated with bovine serum albumin (BSA) conjugated with biotin. Then, streptavidin, which binds to biotin, was overlaid. Next, the anti-fragment crystallizable (Fc) region of human immunoglobulin conjugated to biotin, which binds to streptavidin, was applied. Finally, NRX 1β without splicing insertion at site 4 fused to the FC region of human immunoglobulin was applied so that it was captured by the anti-Fc antibody. When hippocampal neurons overexpressing NLG 1 with splice insertion at site B were cultured on the NRX-coated coverslip, many punctate structures that contained PSD95, a marker protein of postsynaptic density, November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 3 AMPA Receptor Exo- and Endocytosis Hirano Hirano FIGURE 3 \| Changes of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) subunit number by long-term potentiation (LTP)-inducing stimulation. (A–C) Averaged time courses of GluA1–3 ﬂuorescence intensity in PSLM (red) and in non-PSLM (black) measured every 4 min before and after the ﬁeld stimulation (arrows). Data in the presence of APV (+APV) are also shown (dotted lines). Error bars indicate SEM. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. (D–F) GluA-super-ecliptic phluorin (SEP) signals (green) and PSD95-RFP signal (magenta) are shown. PSD95-RFP was recorded before the stimulation, and images of the two signals were overlaid. GluA-SEP signals in PSLM and non-PSLM are indicated by arrows and arrowheads, respectively. Scale bar, 2 µm. These ﬁgure panels were ﬁrst published in Tanaka and Hirano (2012), and copyright permission is not necessary. FIGURE 3 \| Changes of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) subunit number by long-term potentiation (LTP)-inducing stimulation. ENDO-AND EXOCYTOSIS REGULATION DURING LTD AROUND PSLM NMDA application induces LTD in hippocampal culture preparations, which is accompanied by a decrease in amount of cell-surface AMPAR (Lee et al., 1998; Beattie et al., 2000; Collingridge et al., 2010; Fernández-Monreal et al., 2012). Enhancement of clathrin-dependent endocytosis has been considered to contribute to LTD expression (Glebov et al., 2015; Zheng et al., 2015). In order to obtain better understanding of the process of LTD expression, Fujii et al. (2017, 2018) used PSLM for analyses of AMPAR endocytosis during LTD. Individual endocytic events of cell surface molecules, including AMPAR, have been detected by the combination of a rapid extracellular pH change method with the use of SEP (Jullié et al., 2014; Rosendale et al., 2017). The extracellular pH change was performed with a θ tube in these studies. We used a U-tube instead of a θ tube for the extracellular pH exchange (Fujii et al., 2017, 2018; Figure 6). The use of a U-tube enables us to remove the applied solution from the experimental chamber, although the speed of solution exchange is somewhat slower than that of a method using a θ tube. Using the combination of rapid extracellular pH change and GluA1-SEP, individual events of GluA1-SEP endocytosis were observed around PSLM. FIGURE 4 \| Two examples of GluA1-SEP (green) exocytosis (arrows) shown together with PSD95-RFP (magenta). The numbers indicate time (seconds) after the ﬁeld stimulation. These ﬁgure panels were ﬁrst published in Tanaka and Hirano (2012), and copyright permission is not necessary. Changes in the cell surface amounts and in the exocytosis frequencies of GluA2-SEP or GluA3-SEP also occur during LTP expression (Tanaka and Hirano, 2012). GluA2-SEP and GluA3-SEP show different time courses of these changes (Figure 3). Co-expression experiments of GluA1-SEP/GluA2, GluA1/GluA2-SEP or GluA2/GluA3-SEP were also performed. Based on the experimental results, we proposed the following scheme as a mechanism of the expression of LTP (Figure 5). (1) Exocytosis of GluA1 homo-tetramer occurs particularly in the periphery of PSLM immediately after the conditioning stimulation. (2) A few minutes after the conditioning stimulation, exocytosis of GluA1/GluA2 hetero-tetramer increases for several minutes outside PSLM. Some of the exocytosed GluA1/GluA2 hetero-tetramers may move into PSLM by diffusion on the plasma membrane. (3) From about 20 min after the conditioning stimulation exocytosis of GluA2/GluA3 gradually increases outside PSLM. The above scheme suggests that changes in the distribution of AMPAR subtypes are likely to occur during LTP. EXOCYTOSIS REGULATION IN LTP AROUND PSLM We reported that high frequency electrical field stimulation used to induce LTP increases the amount of SEP-tagged GluA subunit of AMPAR in and outside PSLM in a hippocampal culture preparation (Figure 3, Tanaka and Hirano, 2012). The increase is somewhat larger in PSLM. By the way, PSLM is November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 4 AMPA Receptor Exo- and Endocytosis Hirano normal cellular processes. These points will be discussed later. normal cellular processes. These points will be discussed later. FIGURE 4 \| Two examples of GluA1-SEP (green) exocytosis (arrows) shown together with PSD95-RFP (magenta). The numbers indicate time (seconds) after the ﬁeld stimulation. These ﬁgure panels were ﬁrst published in Tanaka and Hirano (2012), and copyright permission is not necessary. ENDO-AND EXOCYTOSIS REGULATION DURING LTD AROUND PSLM I presume that exo- and endocytosis of GluA1/GluA2 and GluA2/GluA3 hetero- tetramers are in equilibrium in a basal condition. This combination method is also useful to precisely determine the amount of cell surface AMPAR, because some SEP fluorescence arises from molecules located in endoplasmic reticulum with relatively neutral luminal pH (Paroutis et al., 2004; Rathje et al., 2013). By subtracting fluorescence signals at pH 5.5 from those at pH 7.3, cell-surface signals can be isolated. This method allowed us to precisely analyze cell-surface amounts of GluA1-SEP and GluA2-SEP during LTD expression (Fujii et al., 2018). The thus estimated cell-surface amounts of both GluA1-SEP and GluA2-SEP gradually decrease after the NMDA application. The decrease of GluA1-SEP is sustained for more than 30 min, while that of GluA2-SEP tends to recover (Fujii et al., 2018). Regarding changes of AMPAR exocytosis during LTP expression, some unclear or conflicting observations have been reported about sites of exocytosis, AMPAR subtype specificity and precise time courses. Some previous studies reported different molecular regulation mechanisms between constitutive AMPAR exocytosis and regulated exocytosis during LTP induction (Ahmad et al., 2012; Temkin et al., 2017; Wu et al., 2017). Yudowski et al. (2007), Lin et al. (2009) and Makino and Malinow (2009) reported that the majority of GluA1 exocytosis occurred in extrasynaptic membrane, whereas Kennedy et al. (2010) found exocytic domains adjacent to postsynaptic density. The involvement of GluA2-lacking AMPAR such as GluA1 homo-tetramer in LTP has also been controversial (Passafaro et al., 2001; Plant et al., 2006; Adesnik and Nicoll, 2007; Gray et al., 2007; Lu Y. et al., 2007). Furthermore, there has been little precise information about how exocytosis of each subtype of AMPAR, such as GluA1/2 or GluA2/3 hetero- tetramer changes during LTP expression. Tanaka and Hirano (2012) provided some answers or information regarding these questions. However, I would like to note the following. First, PSLM is an artificial structure and may not necessarily express all normal functions of hippocampal glutamatergic postsynaptic membrane. Second, over-expressed GluA1-SEP might affect The LTD-inducing NMDA application transiently increases the size of individual GluA1-SEP endocytic events and also the frequency of GluA1-SEP endocytosis for about 1 min. Interestingly this transiently enhanced large endocytosis is clathrin-dependent, whereas constitutive endocytosis of GluA1-SEP does not depend on clathrin (Fujii et al., 2017, 2018). ENDO-AND EXOCYTOSIS REGULATION DURING LTD AROUND PSLM Considering that the cell-surface amounts of molecules are regulated by the balance of endo- and exocytosis, we examined changes of GluA1-SEP exocytosis after the LTD-inducing NMDA application (Fujii et al., 2018). We found that after the NMDA application, GluA1-SEP exocytosis is transiently enhanced and then decreased. Taking endocytosis, plays a predominant role in LTD expression (Figure 7). In addition, the cell-surface amount, and endo- and exocytosis of GluA2-SEP after the NMDA application were examined. We showed that GluA2-SEP exhibits different temporal profiles from those of GluA1-SEP, suggesting FIGURE 5 \| Scheme of exocytosis changes of each AMPAR subtype during LTP expression. In a basal condition (Basal), exo- and endocytosis of GluA1/GluA2 and GluA2/GluA3 hetero-tetramer are in equilibrium. Soon after the high frequency electrical stimulation (Transient, around 1 min after the stimulation), exocytosis of GluA1 homo-tetramer occurs in the periphery of PSLM (red arrow), and exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). In the following period (Intermediate, about 3–10 min after the stimulation), exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). Finally (Late, about 20 min after the stimulation) exocytosis of GluA2/GluA3 increases outside PSLM (blue arrow). Some AMPAR exocytosed outside PSLM are likely to move into PSLM by lateral diffusion on the plasma membrane. This ﬁgure is newly drawn based on our previous publication (Tanaka and Hirano, 2012), and copyright permission is not required. FIGURE 5 \| Scheme of exocytosis changes of each AMPAR subtype during LTP expression. In a basal condition (Basal), exo- and endocytosis of GluA1/GluA2 and GluA2/GluA3 hetero-tetramer are in equilibrium. Soon after the high frequency electrical stimulation (Transient, around 1 min after the stimulation), exocytosis of GluA1 homo-tetramer occurs in the periphery of PSLM (red arrow), and exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). In the following period (Intermediate, about 3–10 min after the stimulation), exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). Finally (Late, about 20 min after the stimulation) exocytosis of GluA2/GluA3 increases outside PSLM (blue arrow). Some AMPAR exocytosed outside PSLM are likely to move into PSLM by lateral diffusion on the plasma membrane. This ﬁgure is newly drawn based on our previous publication (Tanaka and Hirano, 2012), and copyright permission is not required. FIGURE 6 \| Rapid extracellular pH exchange method using a U-tube and detection of individual AMPAR endocytosis. ENDO-AND EXOCYTOSIS REGULATION DURING LTD AROUND PSLM This result is consistent with a previous study reporting that basal endocytosis of AMPAR does not depend on clathrin, but that the NMDA-induced endocytosis depends on clathrin (Glebov et al., 2015). Thus, there are at least two independent endocytosis pathways for AMPAR. We also reported that clathrin-dependent GluA1-SEP endocytosis induced by the NMDA application preferentially takes place in the periphery of PSLM, which is likely to correspond to the endocytic zone adjacent to the postsynaptic membrane (Blanpied et al., 2002; Lu J. et al., 2007). T i t h t f Gl A1 SEP d t i Transient enhancement of GluA1-SEP endocytosis seems to be insufficient to explain slowly developing LTD November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 5 AMPA Receptor Exo- and Endocytosis Hirano Hirano FIGURE 5 \| Scheme of exocytosis changes of each AMPAR subtype during LTP expression. In a basal condition (Basal), exo- and endocytosis of GluA1/GluA2 and GluA2/GluA3 hetero-tetramer are in equilibrium. Soon after the high frequency electrical stimulation (Transient, around 1 min after the stimulation), exocytosis of GluA1 homo-tetramer occurs in the periphery of PSLM (red arrow), and exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). In the following period (Intermediate, about 3–10 min after the stimulation), exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). Finally (Late, about 20 min after the stimulation) exocytosis of GluA2/GluA3 increases outside PSLM (blue arrow). Some AMPAR exocytosed outside PSLM are likely to move into PSLM by lateral diffusion on the plasma membrane. This ﬁgure is newly drawn based on our previous publication (Tanaka and Hirano, 2012), and copyright permission is not required. FIGURE 6 \| Rapid extracellular pH exchange method using a U-tube and detection of individual AMPAR endocytosis. When a bulb on a U-tube is open (Bulb open), the pH 6.0 solution ﬂows inside U-tube and the extracellular pH 7.3 solution is also soaked into the U-tube. When the bulb is closed (Bulb closed), the intra U-tube pH 6.0 solution leaks out to the extracellular solution. In this pH 6.0 condition, only SEP signals from intracellular vesicles with near-neutral luminal pH such as those immediately after endocytosis can be detected. This ﬁgure is newly drawn based on our previous publication (Fujii et al., 2017), and copyright permission is not required. expression. ENDO-AND EXOCYTOSIS REGULATION DURING LTD AROUND PSLM When a bulb on a U-tube is open (Bulb open), the pH 6.0 solution ﬂows inside U-tube and the extracellular pH 7.3 solution is also soaked into the U-tube. When the bulb is closed (Bulb closed), the intra U-tube pH 6.0 solution leaks out to the extracellular solution. In this pH 6.0 condition, only SEP signals from intracellular vesicles with near-neutral luminal pH such as those immediately after endocytosis can be detected. This ﬁgure is newly drawn based on our previous publication (Fujii et al., 2017), and copyright permission is not required. FIGURE 6 \| Rapid extracellular pH exchange method using a U-tube and detection of individual AMPAR endocytosis. When a bulb on a U-tube is open (Bulb open), the pH 6.0 solution ﬂows inside U-tube and the extracellular pH 7.3 solution is also soaked into the U-tube. When the bulb is closed (Bulb closed), the intra U-tube pH 6.0 solution leaks out to the extracellular solution. In this pH 6.0 condition, only SEP signals from intracellular vesicles with near-neutral luminal pH such as those immediately after endocytosis can be detected. This ﬁgure is newly drawn based on our previous publication (Fujii et al., 2017), and copyright permission is not required. expression. Considering that the cell-surface amounts of molecules are regulated by the balance of endo- and exocytosis, we examined changes of GluA1-SEP exocytosis after the LTD-inducing NMDA application (Fujii et al., 2018). We found that after the NMDA application, GluA1-SEP exocytosis is transiently enhanced and then decreased. Taking all these results together, it was suggested that sustained suppression of AMPAR exocytosis, rather than enhanced endocytosis, plays a predominant role in LTD expression (Figure 7). In addition, the cell-surface amount, and endo- and exocytosis of GluA2-SEP after the NMDA application were examined. We showed that GluA2-SEP exhibits different temporal profiles from those of GluA1-SEP, suggesting differential regulation of GluA2-SEP. However, how each type of AMPAR consisting of GluA1–3, such as GluA1/2 or November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 6 AMPA Receptor Exo- and Endocytosis Hirano Hirano FIGURE 7 \| Scheme of exo- and endocytosis changes of GluA1-containing AMPAR during long-term depression (LTD) expression. In a basal condition (Basal) exo- and endocytosis of GluA1-containing AMPAR are in equilibrium. MERITS AND DEMERITS OF PSLM High SN ratio images of fluorescent molecules can be obtained around PSLM with TIRFM through the reduction of background signals (Figure 2). Parallel formation of PSLM on the glass surface enables a simple interpretation of imaging data about how synaptic proteins are localized in and around PSLM. PSLM is also stable and does not move, whereas dendritic spines in which postsynaptic membrane is located occasionally move in culture and in vivo (Deng and Dunaevsky, 2005). In addition, PSLM can be found much more easily under TIRFM than conventional postsynaptic membranes (Tanaka et al., 2014). These points are significant merits of using PSLM for analyses of postsynaptic processes in a basal condition and during synaptic plasticity. Importantly, similar changes of GluA1-SEP dynamics during LTD expression were observed in conventional synapses. Using oblique illumination, we studied the exo- and endocytosis changes of GluA1-SEP around conventional synapses in hippocampal neurons cultured on glass that was not coated with NRX (Fujii et al., 2018). Although, the SN ratio of fluorescence images of GluA1-SEP around synapses observed with oblique illumination was inferior to that obtained using PSLM and TIRFM, qualitatively similar results were obtained. ENDO-AND EXOCYTOSIS REGULATION DURING LTD AROUND PSLM Immediately after the N-methyl-D-aspartate (NMDA) application (Transient, around 1 min after the onset of NMDA application), clathrin-dependent endocytosis and exocytosis of AMPAR increase in the periphery of PSLM. In the following period (Intermediate, about 3–10 min), AMPAR exocytosis is suppressed. Finally (Late) exo- and endocytosis of AMPAR go into equilibrium in a low level. How each type of AMPAR changes during LTD remains to be clariﬁed. This ﬁgure is newly drawn based on our previous publications (Fujii et al., 2017, 2018), and copyright permission is not required. FIGURE 7 \| Scheme of exo- and endocytosis changes of GluA1-containing AMPAR during long-term depression (LTD) expression. In a basal condition (Basal) exo- and endocytosis of GluA1-containing AMPAR are in equilibrium. Immediately after the N-methyl-D-aspartate (NMDA) application (Transient, around 1 min after the onset of NMDA application), clathrin-dependent endocytosis and exocytosis of AMPAR increase in the periphery of PSLM. In the following period (Intermediate, about 3–10 min), AMPAR exocytosis is suppressed. Finally (Late) exo- and endocytosis of AMPAR go into equilibrium in a low level. How each type of AMPAR changes during LTD remains to be clariﬁed. This ﬁgure is newly drawn based on our previous publications (Fujii et al., 2017, 2018), and copyright permission is not required. FIGURE 7 \| Scheme of exo- and endocytosis changes of GluA1-containing AMPAR during long-term depression (LTD) expression. In a basal condition (Basal) exo- and endocytosis of GluA1-containing AMPAR are in equilibrium. Immediately after the N-methyl-D-aspartate (NMDA) application (Transient, around 1 min after the onset of NMDA application), clathrin-dependent endocytosis and exocytosis of AMPAR increase in the periphery of PSLM. In the following period (Intermediate, about 3–10 min), AMPAR exocytosis is suppressed. Finally (Late) exo- and endocytosis of AMPAR go into equilibrium in a low level. How each type of AMPAR changes during LTD remains to be clariﬁed. This ﬁgure is newly drawn based on our previous publications (Fujii et al., 2017, 2018), and copyright permission is not required. GluA2/3 hetero-tetramer, changes during LTD has not been reported. Transient and simultaneous enhancement of both exo- and endocytosis of GluA1 after the onset of NMDA application might contribute to substitutions of AMPAR subtypes. GluA2-containing AMPAR might be replaced by Ca2+ permeable AMPAR lacking GluA2 (Sanderson et al., 2016). should influence the amount of AMPAR at a postsynaptic membrane (Opazo et al., 2010, 2012; Opazo and Choquet, 2011; Chen et al., 2015). should influence the amount of AMPAR at a postsynaptic membrane (Opazo et al., 2010, 2012; Opazo and Choquet, 2011; Chen et al., 2015). FUTURE DIRECTIONS There are many different types of synapses. Some are excitatory, and the others are inhibitory. There are also large differences in presynaptic transmitter release probability among synapses, which is likely to affect postsynaptic properties (Konnerth et al., 1990; Miyawaki and Hirano, 2011; Biederer et al., 2017). Synapses between a pair of neurons often change their characteristics during development (Pouzat and Hestrin, 1997; Yu and Goodrich, 2014). There are many types of synaptic adhesion molecules such as NLG, NRX, SynCAM, EphrinB, LRRTM and N-cadherin. In addition, there are different subtypes and different splice variants of NRX and NLG as explained above. They are differently distributed among synapses, and some of them are co-localized at a synapse. Combination of pre- and postsynaptic adhesion molecules are likely to play critical roles in determination of synaptic properties (Levinson and El-Husseini, 2005; Dean and Dresbach, 2006; Craig and Kang, 2007; Südhof, 2008; Bukalo and Dityatev, 2012). Changing a glass-coating presynaptic adhesion molecule, adding another coating molecule, or changing cultured neuronal type might reveal synapse-type specific postsynaptic properties. Such experiments might also provide useful information about determinant molecules for functional properties of a particular type of synapse. Another interesting extension of the culture method on the coated-glass surface is application to studies on presynaptic mechanisms. By coating glass surface with postsynaptic adhesion molecule, formation of presynaptic structure has been achieved (Funahashi et al., 2018). By this method, presynaptic active- zone-like membrane (AZLM) was formed on the glass surface coated with NLG, and single exocytosis event of a synaptic vesicle was visualized. Using an original experimental system, we also demonstrated fast diffusion of a synaptic vesicle protein synaptophysin tagged with SEP on the plasma membrane after membrane fusion of synaptic vesicle membrane, and also distinct distribution patterns of synchronous and asynchronous synaptic-vesicle release locations (Südhof, 2012; Kaeser and Regehr, 2014; Kavalali and Jorgensen, 2014; Maschi and Klyachko, 2017). Thus, the novel imaging preparations enabled by pre- and postsynaptic structure formation directly on the glass surface coated with a synaptic adhesion molecule combined with TIRFM, are expected to shed light on detailed molecular dynamics underlying synaptic transmission and plasticity. Over-expression of AMPAR-SEP is also artificial manipulation which could affect normal cellular processes. Overexpression of a subunit of AMPAR such as GluA1 inevitably increases its relative amount, potentially affecting normal cellular processes. FUTURE DIRECTIONS Indeed, we found that GluA2-SEP behaves somewhat differently depending on whether it is co-expressed with GluA1 (Tanaka and Hirano, 2012). I also note that SEP is not small in size and could affect AMPAR functions and regulations. One way to overcome these problems is to label endogenous AMPAR with a small fluorescent molecular probe (Wakayama et al., 2017). LATERAL MOVEMENT OF AMPAR ON THE PLASMA MEMBRANE However, PSLM is certainly an artificial structure deficient in interaction with presynaptic structures, which could potentially affect some functions of postsynaptic membrane. Thus, certain care should be taken in interpretation of results obtained using PSLM. Nevertheless, PSLM retains essential properties of postsynaptic membrane, as evidenced by the accumulation of postsynaptic proteins such as PSD95 and homer, and dynamic changes of the amount of AMPAR relevant to the expression of LTP and LTD. Furthermore, it was demonstrated that exo- and endocytic changes of GluA1-SEP during LTD at conventional postsynaptic membranes were qualitatively similar to those observed at PSLM as explained above Fujii et al. (2018). Thus, AMPAR moves around on the plasma membrane by lateral diffusion, and this movement has been studied by live-cell imaging of a fluorescent quantum dot attached to AMPAR (Bats et al., 2007; Groc et al., 2008). Diffusion is much faster in extrasynaptic membrane than in postsynaptic membrane. Notably, there is little movement of AMPAR in the postsynaptic membrane for a long time. Thus, AMPAR can be trapped at a postsynaptic membrane. Together with the balance of exo- and endocytosis, the efficiency of trapping of AMPAR at a postsynaptic membrane or the balance of coming-in and going-out of AMPAR to and from a postsynaptic membrane November 2018 \| Volume 12 \| Article 442 Frontiers in Cellular Neuroscience \| www.frontiersin.org 7 AMPA Receptor Exo- and Endocytosis Hirano PSLM can be regarded as a useful experimental model and can provide guiding results and/or ideas that would be worth rigorously examining at conventional synapses. 2017). I would also like to note that application of super- resolution fluorescence imaging techniques, Stochastic optical reconstruction microscopy (STORM) and Photo-activated localization microscopy (PALM) in particular, were likely to match very well with analyses on PSLM, which is formed parallelly in a single focal plane (Dani et al., 2010; Maglione and Sigrist, 2013; Baddeley and Bewersdorf, 2018). STORM and PALM are used with TIRFM in most cases, and parallel formation of PSLM on the glass surface should facilitate image capture processes of STORM or PALM. FUNDING This work was supported by a grant 18H02526 to TH from Japan Society for the Promotion of Science, and a grant from Takeda Science Foundation in Japan. ACKNOWLEDGMENTS Development of new fluorescent proteins such as pH-sensitive red fluorescent proteins will enable us to simultaneously monitor multiple proteins, and will certainly promote the analyses (Shen et al., 2014; Martineau et al., I thank Drs. S. Kawaguchi, H. 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https://openalex.org/W2903635579	https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=2260&context=theses	English	null	UTILIZING A LIPID BILAYER-BASED SOLVENT TO ENHANCE BUTANOL PRODUCTION FROM A CONTINUOUS FERMENTATION OF CLOSTRIDIA PASTEURIANUM	null	2,020	cc-by	20,729	UTILIZING A LIPID BILAYER-BASED SOLVENT TO ENHANCE UTILIZING A LIPID BILAYER-BASED SOLVENT TO ENHANCE BUTANOL PRODUCTION FROM A CONTINUOUS FERMENTATION BUTANOL PRODUCTION FROM A CONTINUOUS FERMENTATION OF CLOSTRIDIA PASTEURIANUM OF CLOSTRIDIA PASTEURIANUM John Nunes University of Rhode Island, Jjnunes8@gmail.com Follow this and additional works at: https://digitalcommons.uri.edu/theses Terms of Use All rights reserved under copyright. Recommended Citation Recommended Citation Nunes, John, "UTILIZING A LIPID BILAYER-BASED SOLVENT TO ENHANCE BUTANOL PRODUCTION FROM A CONTINUOUS FERMENTATION OF CLOSTRIDIA PASTEURIANUM" (2018). Open Access Master's Theses. Paper 1279. https://digitalcommons.uri.edu/theses/1279 This Thesis is brought to you by the University of Rhode Island. It has been accepted for inclusion in Open Access Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons-group@uri.edu. For permission to reuse copyrighted content, contact the author directly. UTILIZING A LIPID BILAYER-BASED SOLVENT TO ENHANCE BUTANOL PRODUCTION FROM A CONTINUOUS FERMENTATION OF CLOSTRIDIA PASTEURIANUM BY JOHN NUNES A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMICAL ENGINEERING CHEMICAL ENGINEERING UNIVERSITY OF RHODE ISLAND 2018 BY JOHN NUNES Abstract This work highlights the capabilities of a lipid bilayer-based solvent for butanol extraction. Previous work in our group has shown that lipid bilayers have a high affinity for butanol extraction. Here, we show higher butanol partition coefficients than previously seen in other solvents. Our partition coefficients were found using a quantitative NMR method to allow for in situ measurements to be taken which resulted in higher partition coefficients than previously found by HPLC methods. Two lipids were used for these experiments in order to examine the effect that lipid bilayer phase has on the butanol partition coefficient and we see that a mixed phase bilayer (DPPC/DOPC) resulted in the highest butanol partition coefficient. Additionally, butanol’s effect on bilayer size was examined by dynamic light scattering. DPPC vesicles showed the largest change in size when butanol was added due to the smaller spaces in between lipid head groups in a DPPC bilayer. Working in collaboration with Dr. Carmen Scholz’s group at the University of Alabama Huntsville, a continuous fermentation was developed that used glycerol as a feedstock for Clostridium pasteurianum. We also utilized our lipid bilayer solvent in an extractive fermentation of these continuous cultures. Our results showed an increase in butanol production and yield at lower dilution rates, which can also be attributed to the extraction of butanol from the system. Acknowledgments First, I’d like to thank my parents for their support and love. I’d like to thank Dr. First, I’d like to thank my parents for their support and love. I’d like to thank Dr. Bothun for his help and guidance throughout this process and also for giving me the great opportunity to work with him. I’d like to thank my family and friends for being so supportive of me with a special thank you to my lab mates at URI who have helped me and provided constructive feedback countless times. I’d like to thank the URI Chemical Engineering department for welcoming me and their assistance. Finally, I’d like to thank Becky for always being there when I needed her and pushing me to succeed. iii Table of Contents Abstract ...........................................................................................................................ii Acknowledgments ......................................................................................................... iii Table of Contents .......................................................................................................... iv List of Tables ................................................................................................................. vii Table of Figures............................................................................................................ viii Chapter 1 ........................................................................................................................ 1 Chapter 2 ........................................................................................................................ 5 2.1 Butanol Toxicity and Cell Inhibition .................................................................... 5 2.2 Vesicle Characterization ...................................................................................... 7 2.3 Extractive Fermentation ...................................................................................... 9 References ................................................................................................................... 11 CHAPTER 3 ................................................................................................................... 13 3.1 Introduction ....................................................................................................... 13 3.2 Methods/Materials............................................................................................ 19 3.2.1 Vesicle Preparation ...................................................................................... 19 3.2.2 Nuclear Magnetic Resonance (NMR) ........................................................... 20 3.2.3 Dynamic Light Scattering ............................................................................. 22 3.3 Results and Discussion ....................................................................................... 23 iv iv 3.3.1 Partition coefficient with increasing lipid concentrations ........................... 23 3.3.2 Liposome Swelling ....................................................................................... 28 3.4 Conclusions ........................................................................................................ 31 References ................................................................................................................... 32 Chapter 4 ...................................................................................................................... 36 4.1 Introduction ....................................................................................................... 36 4.2 Materials and Methods ..................................................................................... 38 4.2.1 Vesicle Preparation ...................................................................................... 38 4.2.2 Fermentation Media .................................................................................... 39 4.2.3 Bioreactor Schematic and Operation .......................................................... 39 4.2.4 Quantitative NMR ........................................................................................ 42 4.2.5 HPLC Analysis ............................................................................................... 44 4.3.1 Validating qNMR for determining butanol concentration .......................... 44 4.3.2 Butanol extracted by vesicle solvent ........................................................... 45 4.3.3 Total butanol production ............................................................................. 46 4.4 Conclusions ........................................................................................................ 49 References ................................................................................................................... 50 Chapter 5 ...................................................................................................................... 52 5.1 Conclusions ........................................................................................................ 52 v 5.2 Future Work ....................................................................................................... 54 Appendix 1 ................................................................................................................... 56 Bibliography ……………………………………………………………………………………………………………61 vi List of Tables List of Tables Table 2.1: Lipids used in this study with molecular weight and structure. Soy lecithin is a mixture and the structure shown is representative and the molecular weight is an average (Avanti Polar Lipids, Inc, Alabaster, AL). ........................................................... 8 Table 3.1: Table of NHOC, AKI fuel properties of ethanol, butanol, gasoline. NHOC – Net heat of combustion, AKI – Anti-knock index (Harvey, Meylemans 2011). ......... 145 Table 3.2: DPPC and DOPC were the lipids used in this study with molecular weight and structure shown. ................................................................................................. 189 Table 3.3: Butanol/Lipid mass and mole fraction partition coefficients at a lipid concentration of 4 g/L and a butanol concentration of 5 g/L. Our results show that the equimolar mixture of DPPC and DOPC provide the highest butanol partition coefficient................................................................................................................... 267 Table 4.1: Compositions of the glycerol media used during the fermentation. ...... 3940 vii vii Table of Figures Table of Figures Table of Figures Figure 2.1: [Left] Additional butanol in presence of fermentation inhibits cell growth. [Right] Butanol production decreases when additional butanol is present in the system (Venkataramanan et al. 2014). .......................................................................... 6 Figure 2.2: Pathway of glycerol fermentation by Clostridium pasteurianum (Kubiak et al. 2012). ......................................................................................................................... 7 Figure 2.3: The four phases that can be present in a liposome. The interdigitated gel occurs once butanol is added to the system and returns to a fluid phase after the melting temperature (Tm) (Kurniawan et al. 2012) ………………………………………………… 9 Figure 3.1: NMR spectra of butanol without vesicles present (red) and in the presence of vesicles (green). This difference is measurable and can then be used to find butanol/lipid partition coefficients. ........................................................................... 245 Figure 3.2: Partition coefficients of butanol in DPPC (A), DOPC (B) and 50:50 DPPC:DOPC (C). All partition coefficient experiments were conducted at 25 oC and at a butanol concentration of 5 g/L. A “” represents p < 0.05, while “n.s.” represents p > 0.05. This basis is used throughout the results presented. (D) The number of butanol molecules per lipid molecule based on KP results at a butanol concentration of 5 g/L and lipid concentrations of 4 g/L. ................................................................. 256 Figure 3.3: Change in vesicle size with (A) increasing butanol concentration and (B) increasing acetic acid, 1,3 propanediol and ethanol concentration (DPPC only). A table containing the slopes of linear fits through the intercept. ............................. 2930 Figure 4.1: Schematic of the continuous fermentation with vesicle extraction process. P1-P4 are the pumps. (A) Fresh media inlet being fed into (B) the bioreactor with the C. Pasteurianum fermentation. (C) was the waste collection. (D) housed the vesicle solution being sent through (E) the hollow fiber membrane used to extract butanol. ...................................................................................................................................... 42 Figure 4.2: NMR spectrum of a sample from the continuous fermentation. The maleic acid peak (6.25 ppm) is compared to the visible butanol peaks (1.4, 1.25, 0.8 ppm) in order to find how much butanol is in solution. ......................................................... 434 viii Figure 4.3: A comparison between HPLC and qNMR with an internal calibrant. Random samples were chosen from the experiments and tested with both HPLC and NMR. On average there was an 8% difference between them. ................................ 456 Figure 4.3: A comparison between HPLC and qNMR with an internal calibrant. Random samples were chosen from the experiments and tested with both HPLC and NMR. Chapter 1 Chapter 1 Table of Figures On average there was an 8% difference between them. ................................ 456 Figure 4.3: A comparison between HPLC and qNMR with an internal calibrant. Random samples were chosen from the experiments and tested with both HPLC and NMR. On average there was an 8% difference between them. ................................ 456 Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution rates (DR). The blue bar represents the total butanol measured by qNMR therefore outside of the vesicles. The orange bar represents the butanol found by calculation using the partition coefficients from previous work. 60 min is highlighted here as that was the final time point both trials had in common. ......... 467 Figure 4.5: Total butanol produced (g) over time in the reactor, in the solvent and in total for two dilution rates (A - 0.18 hr-1, B - 0.09 hr-1). The DR of 0.09 hr-1 showed almost triple the total butanol production of the DR 0.18 hr-1. A table showing the production of butanol per hour, the percent increase of butanol totals and the butanol yields. ............................................................................................................ 489 Figure 4.6: Ratio of moles of butanol to moles of lipid present for two dilution rates. ...................................................................................................................................... 50 Figure 5.1: Mole fraction-based butanol/lipid partition coefficients using an equimolar mixture of DPPC and DOPC. ..................................................................... 534 Figure 5.2: A dilution rate 0.09 provided an increase of 94% in butanol produced. We also see that the amount of butanol in the reactor rises once the extraction begins indicating an increase in production by the C. pasteurianum. .................................. 545 ix INTRODUCTION Growing energy demands for the world will require innovative options for energy production. Alternative fuel can be an important and viable option to expand on current energy choices (Dresselhaus and Thomas 2001). Ethanol is a common fuel additive used today in fuel to enhance the fuel’s properties. However, when present in large amounts, ethanol can be corrosive to car engines. Due to the corrosive nature of ethanol, car engines would need to undergo modifications to combat this corrosion (Surisetty et al. 2011). In contrast, butanol offers fuel properties superior to those of ethanol and thus has the potential to be a more innovative fuel source (Nanda et al. 2017; Lee et al. 2008; Dürre 2007; MacLean and Lave 2003). The fuel properties of butanol are so similar to gasoline that butanol is considered a “drop-in” biofuel meaning it is capable of being used in car engines with limited or no modifications (Dürre 2007). In addition to its use as a biofuel, butanol has a number of other uses in the chemical industry such as in the production of paints, lacquers, and resins (Harvey and Meylemans 2011; Nanda et al. 2017). Batch fermentation utilizing the Clostridia strain has also shown promise when it comes to butanol production (Zheng et al. 2009; Jones and Woods 1986; Tomas et al. 2003; Lee et al. 2008) 1 Butanol production via fermentation does come with certain limitations, namely, that butanol is toxic to cells (Sardessai and Bhosle 2002). This toxicity can severely impact the production of butanol and life of the cells (Venkataramanan et al. 2014). In an effort to reduce the impact butanol can have on a fermentation, extractive fermentation has been utilized to remove butanol as it is formed in a fermentation setting. By removing the butanol, not only does this limit the toxicity that butanol exhibits which increases cell growth but it also drives the reaction to produce more butanol due to Le Chatelier’s principle. The effectiveness of an extractive fermentation is based on the solvent choice. While many solvents have been used to extract butanol, this work focuses on vesicles as the solvent of choice. Vesicles show excellent potential as a solvent for butanol extraction due to their biocompatibility and high preliminary partition coefficient results for butanol into vesicles (Kurniawan et al. 2012). INTRODUCTION 2 The overall goal of this work was to design and operate a continuous fermentation process coupled with extractive fermentation utilizing a vesicle solvent. In order to establish an extractive fermentation, details about the vesicle solvent needed to be found. Partition coefficients were found using a quantitative nuclear magnetic resonance (qNMR) approach. When butanol partitions into a bilayer, the bilayer expands to include the butanol (Löbbecke and Cevc 1995). This phenomenon was observed using dynamic light scattering during this work. A continuous fermentation of Clostridia pasteurianum was then established in collaboration with the University 2 of Alabama chemistry department. Following this, an extraction system was set up using vesicles as the solvent and a hollow fiber membrane to bring the solvent into contact with the fermentation broth. Chapter 2 will provide a detailed background on relevant information in regards to this research. Chapter 3 contains a manuscript that is in preparation to be published which covers butanol partition coefficient results as well as butanol’s effect on vesicle size. Chapter 4 contains another manuscript format that is in its preliminary stages of editing. This chapter contains information regarding continuous fermentation and extractive fermentation along with our current results. Chapter 5 contains conclusions and potential ideas for future work on this project. The appendix will show NMR background and procedures used. References Dresselhaus, M. S. and Thomas, I. L. (2001): Alternative energy technologies. In Nature 414 (6861), pp. 332–337. DOI: 10.1038/35104599. Dresselhaus, M. S. and Thomas, I. L. (2001): Alternative energy technologies. In Nature 414 (6861), pp. 332–337. DOI: 10.1038/35104599. Dresselhaus, M. S. and Thomas, I. L. (2001): Alternative energy technologies. In Nature 414 (6861), pp. 332–337. DOI: 10.1038/35104599. Dürre, P. (2007): Biobutanol. An attractive biofuel. In Biotechnology Journal 2 (12), pp. 1525–1534. DOI: 10.1002/biot.200700168. Harvey, B.G. and Meylemans, H.A. (2011): The role of butanol in the development of sustainable fuel technologies. In Journal of Chemical Technology and Biotechnology 86 (1), pp. 2–9. DOI: 10.1002/jctb.2540. Harvey, B.G. and Meylemans, H.A. (2011): The role of butanol in the development of sustainable fuel technologies. In Journal of Chemical Technology and Biotechnology 86 (1), pp. 2–9. DOI: 10.1002/jctb.2540. Jones, D.T. and Woods, D.R. (1986): Acetone Butanol Fermentation Revisited. In Microbiological Reviews 50 (4), pp. 484–524. Jones, D.T. and Woods, D.R. (1986): Acetone Butanol Fermentation Revisited. In Microbiological Reviews 50 (4), pp. 484–524. Jones, D.T. and Woods, D.R. (1986): Acetone Butanol Fermentation Revisited. In Microbiological Reviews 50 (4), pp. 484–524. 3 Kurniawan, Y.; Venkataramanan, K.P.; Scholz, C.; Bothun, G.D. (2012): n-Butanol partitioning and phase behavior in DPPC/DOPC membranes. In The Journal of Physical Chemistry. B 116 (20), pp. 5919–5924. DOI: 10.1021/jp301340k. Kurniawan, Y.; Venkataramanan, K.P.; Scholz, C.; Bothun, G.D. (2012): n-Butanol partitioning and phase behavior in DPPC/DOPC membranes. In The Journal of Physical Chemistry. B 116 (20), pp. 5919–5924. DOI: 10.1021/jp301340k. Lee, S.Y.; Park, J.H.; Jang, S.H.; Nielsen, L.K.; Kim, J.; Jung, K.S. (2008): Fermentative butanol production by Clostridia. In Biotechnology and Bioengineering 101 (2), pp. 209–228. DOI: 10.1002/bit.22003. Löbbecke, L. and Cevc, G. (1995): Effects of short-chain alcohols on the phase behavior and interdigitation of phophatidylcholine bilayer membranes. In Biochimica et biophysica acta 1237 (1), pp. 59–69. MacLean, H.L and Lave L.B. (2003): Evaluating automobile fuel/propulsion system technologies. In Progress in Energy and Combustion Science 29, pp. 1–69. Nanda, S.; Golemi-Kotra, D.; McDermott, J.C.; Dalai, A.K.; Gökalp, I.; Kozinski, Janusz A. (2017): Fermentative production of butanol. Perspectives on synthetic biology. In New Biotechnology 37 (Pt B), pp. 210–221. DOI: 10.1016/j.nbt.2017.02.006. Sardessai, Y.; Bhosle, S. (2002): Tolerance of bacteria to organic solvents. In Research in Microbiology 153 (5), pp. 263–268. DOI: 10.1016/S0923-2508(02)01319-0. Surisetty, V.R.; Dalai, A.K.; Kozinski, J. (2011): Alcohols as alternative fuels. An overview. In Applied Catalysis A: General. DOI: 10.1016/j.apcata.2011.07.021. Tomas, C. References A.; Welker, N. E.; Papoutsakis, E. T. (2003): Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program. In Applied and Environmental Microbiology 69 (8), pp. 4951–4965. DOI: 10.1128/AEM.69.8.4951-4965.2003. Venkataramanan, K.P.; Kurniawan, Y.; Boatman, J.J.; Haynes, C.H.; Taconi, K.A.; Martin, L. (2014): Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation. In Journal of Biotechnology 179, pp. 8–14. DOI: 10.1016/j.jbiotec.2014.03.017. Venkataramanan, K.P.; Kurniawan, Y.; Boatman, J.J.; Haynes, C.H.; Taconi, K.A.; Martin, L. (2014): Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation. In Journal of Biotechnology 179, pp. 8–14. DOI: 10.1016/j.jbiotec.2014.03.017. Zheng, Y.; Li, L.; Xian, M.; Ma, Y.; Yang, J.; Xu, X.; He, D. (2009): Problems with the microbial production of butanol. In Journal of Industrial Microbiology & Biotechnology 36 (9), pp. 1127–1138. DOI: 10.1007/s10295-009-0609-9. 4 BACKGROUND This chapter covers a variety of topics and provides an up-to-date review of each of them. First, butanol’s inhibitory effect on cell growth is discussed. Then, information about butanol’s effect on vesicle phase is presented. Finally, extractive fermentation of butanol is shown with a variety of solvents shown. 2.1 Butanol Toxicity and Cell Inhibition 2.1 Butanol Toxicity and Cell Inhibition The inherent toxicity of butanol becomes an issue in a fermentation setting as butanol will kill cells (Sardessai and Bhosle 2002). Seen in Figure 2.1 below where butanol is added after two days and a decreased optical density is measured, this implies that the cell growth was stunted after the addition of butanol (Venkataramanan et al. 2014). Butanol is known to partition between the headgroups of a lipid bilayer causing a cell membrane to fluidize, killing the cell (Bowles and Ellefson 1985; Vollherbst-Schneck et al., 1984). The toxicity of a solvent is related to the solvent’s log P. The log P is a value representing how well a solvent will partition in an equimolar octanol/water mixture (Sardessai and Bhosle 2002). A The inherent toxicity of butanol becomes an issue in a fermentation setting as butanol will kill cells (Sardessai and Bhosle 2002). Seen in Figure 2.1 below where butanol is added after two days and a decreased optical density is measured, this implies that the cell growth was stunted after the addition of butanol headgroups of a lipid bilayer causing a cell membrane to fluidize, killing the cell (Bowles and Ellefson 1985; Vollherbst-Schneck et al., 1984). The toxicity of a solvent is related to the solvent’s log P. The log P is a value representing how well a solvent will partition in an equimolar octanol/water mixture (Sardessai and Bhosle 2002). A 5 log P lower than 4 means that the solvent will be highly toxic to cells; butanol shows a log P around 0.8 (Sardessai and Bhosle 2002). Figure 2.1: [Left] Additional butanol in presence of fermentation inhibits cell growth. [Right] Butanol production decreases when additional butanol is present in the system (Venkataramanan et al. 2014). Figure 2.1: [Left] Additional butanol in presence of fermentation inhibits cell growth. [Right] Butanol production decreases when additional butanol is present in the system (Venkataramanan et al. 2014). The Clostridia species of bacteria has shown an increased ability to produce butanol. Clostridium acetobutylicum has been shown to produce butanol around 10 g/L (Monot et al., 1982). Additionally, Clostridium pasteurianum can produce butanol well and also shows superior resistance to butanol’s toxicity. Clostridium pasteurianum has been shown to grow even when using crude glycerol, which is a byproduct of conventional biodiesel production (Venkataramanan et al. 2012). The glycerol pathway for butanol production is seen in Figure 2.2. 2.1 Butanol Toxicity and Cell Inhibition 6 Figure 2.2: Pathway of glycerol fermentation by Clostridium pasteurianum (Kubiak et al. 2012). Figure 2.2: Pathway of glycerol fermentation by Clostridium pasteurianum (Kubiak et al. 2012). 2.2 Vesicle Characterization Phospholipids were used in this study because they are normally present in a cell membrane. This is important because butanol has shown that in the presence of cell membranes, it gathers in the space between the lipid head groups along the lipid/water interface (Bowles and Ellefson 1985; Vollherbst-Schneck et al., 1984). In this study the phospholipids used were zwitterionic with varying degrees of unsaturation and can be seen in Table 2.1 below. 7 7 Table 2.1: Lipids used in this study with molecular weight and structure. Soy lecithin is a mixture; the structure shown is representative and the molecular weight is an average (Avanti Polar Lipids, Inc, Alabaster, AL). Alabaster, AL). Alabaster, AL). Lipid Name Structure Molecular Weight [g/mol] Dipalmitoylhosphatidylcholine (DPPC) 734.05 Dioleoylphosphatidylcholine (DOPC) 786.13 L-a-phosphatidylcholine (Soy Lecithin) 775.04 ) Lipid Name Structure Molecular Weight [g/mol] Dipalmitoylhosphatidylcholine (DPPC) 734.05 Dioleoylphosphatidylcholine (DOPC) 786.13 L-a-phosphatidylcholine (Soy Lecithin) 775.04 Once amphiphilic, both hydrophobic and hydrophilic, lipids enter a water environment the hydrophilic head groups point towards the aqueous environment while the hydrophobic tails of the lipids interact with each other. This forms a lipid bilayer with an aqueous core. The lipid bilayer is normally represented by two phases and an intermediate phase. When below the melting temperature of the bilayer the phase is a gel like system. This changes to a fluid phase when above the melting temperature. The intermediate phase occurs when the bilayer approaches its melting temperature. When butanol is in the presence of a lipid bilayer a fourth phase has been shown to form known as the interdigitated phase which causes the lipid head 8 8 groups to space out and the tails to lie side by side instead of being end-to-end. Each of these phases can be viewed in Figure 2.3. e phases can be viewed in Figure 2.3. Figure 2.3: The four phases that can be present in a vesicle. The interdigitated gel occurs once butanol is added to the system and returns to a fluid phase after the melting temperature (Tm) is reached (Kurniawan et al. 2012). 2.3 Extractive Fermentation 2.3 Extractive Fermentation Extractive fermentation is the process of removing an end product from a fermentation using a solvent to extract it. Removing a product then lowers the end products potential inhibition on the bacteria’s growth (Dhamole et al. 2012). When a product is removed, the fermentation should be pushed to produce more of the missing product, in this case butanol. Extractive fermentation can take two different forms: direct addition of a solvent to the fermentation (Dhamole et al. 2012) or passing the fermentation broth through an extraction unit to bring the media in contact with a solvent (Zhang et al. 2017; Roffler et al. 1988). Extractive fermentation’s effectiveness at increasing butanol yields has been demonstrated a 9 number of times. A liquid-liquid extraction using oleyl alcohol running counter currently across a plate column showed a 70% increase over a batch fermentation (Roffler et al. 1988). Long chain alcohols also provided high butanol partitioning with 2-Ethyl-1,3-hexanediol reported as having a mass partition coefficient of 8.1 (Barton and Daugulis 1992). While organic solvents have been shown to increase butanol production, these solvents tend to also be toxic to the bacteria. Thus, extractants with a biocompatibility towards butanol are becoming more relevant. number of times. A liquid-liquid extraction using oleyl alcohol running counter currently across a plate column showed a 70% increase over a batch fermentation (Roffler et al. 1988). Long chain alcohols also provided high butanol partitioning with 2-Ethyl-1,3-hexanediol reported as having a mass partition coefficient of 8.1 (Barton and Daugulis 1992). While organic solvents have been shown to increase butanol production, these solvents tend to also be toxic to the bacteria. Thus, extractants with a biocompatibility towards butanol are becoming more relevant. 10 Zhang et al. uses mixtures of aliphatic fatty acids and oleyl alcohol to extract butanol from a fermentation and shows an improvement of 11% over using oleyl alcohol alone (Zhang et al. 2017; Dhamole et al. 2012). Surfactant micelles have been shown to increase butanol productivity by over 200% with a mass partition coefficient of 3.5. Ionic liquid solvents have also shown to be effective solvents for butanol (Cascon et al. 2011; Davis and Morton 2008). Tetrahexyammonium dihexylsulfosuccinate ([THA][DHSS]) showed a mass partition coefficient with a value of 7.99 presented (Cascon et al. 2011). 1-Butyl-3-methylimidazolium bis(triflouromethylsulfonyl)imide ([bmim][Tf2N]) was shown to have a distribution coefficient of 14 (Davis and Morton 2008). 2.3 Extractive Fermentation Vesicles provide a highly biocompatible solvent with a high affinity towards butanol. In addition to the toxicity inferred from the low log P of butanol, the log P also highlights the ability for butanol to partition. It has already been stated that butanol has a fluidizing effect on a cell membrane due to its partitioning between the headgroups of the bilayer. Combining both of these concepts leads to the idea that 10 cell membranes could be used as a solvent to extract butanol. Some preliminary studies that use model cell membranes to extract butanol have shown high partition coefficients (Kurniawan et al. 2013; Kurniawan et al. 2012). The partition coefficient is the ratio of the concentration of a solute between two solvents. As of this writing vesicles have not been used to extract butanol from a fermentation. References Barton, E.W. and Daugulis, A.J. (1992): Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200. In Applied Microbiology Biotechnology 36 (5). DOI: 10.1007/BF00183241. Barton, E.W. and Daugulis, A.J. (1992): Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200. In Applied Microbiology Biotechnology 36 (5). DOI: 10.1007/BF00183241. Bharti, S.K. and Roy, R. (2012): Quantitative 1H NMR spectroscopy. In TrAC Trends in Analytical Chemistry 35, pp. 5–26. DOI: 10.1016/j.trac.2012.02.007. Bharti, S.K. and Roy, R. (2012): Quantitative 1H NMR spectroscopy. In TrAC Trends in Analytical Chemistry 35, pp. 5–26. DOI: 10.1016/j.trac.2012.02.007. Bowles L.K., and Ellefson W.L. (1985): Effects of butanol on Clostridium acetobutylicum. In Applied and Environmental Microbiology 50 (Nov. 1985), pp. 1165–1170. Bowles L.K., and Ellefson W.L. (1985): Effects of butanol on Clostridium acetobutylicum. In Applied and Environmental Microbiology 50 (Nov. 1985), pp. 1165–1170. Bowles L.K., and Ellefson W.L. (1985): Effects of butanol on Clostridium acetobutylicum. In Applied and Environmental Microbiology 50 (Nov. 1985), pp. 1165–1170. Cascon, H.R.; Choudhari, S.K.; Nisola, G.M.; Vivas, E.L.; Lee, D.; Chung, W. (2011): Partitioning of butanol and other fermentation broth components in phosphonium and ammonium-based ionic liquids and their toxicity to solventogenic clostridia. In Separation and Purification Technology 78 (2), pp. 164–174. DOI: 10.1016/j.seppur.2011.01.041. Darbeau, R.W. (2006): Nuclear Magnetic Resonance (NMR) Spectroscopy. A Review and a Look at Its Use as a Probative Tool in Deamination Chemistry. In Applied Spectroscopy Reviews 41 (4), pp. 401–425. DOI: 10.1080/05704920600726175. Davis, S. E. and Morton, S. A. (2008): Investigation of Ionic Liquids for the Separation of Butanol and Water. In Separation Science and Technology 43 (9-10), pp. 2460– 2472. DOI: 10.1080/01496390802122089. Dhamole, P.B.; Wang, Z.; Liu, Y.; Wang, B.; Feng, H. (2012): Extractive fermentation with non-ionic surfactants to enhance butanol production. In Biomass and Bioenergy 40, pp. 112–119. DOI: 10.1016/j.biombioe.2012.02.007. Kubiak, P.; Leja, K.; Myszka, K.; Celińska, E.; Spychała, M.; Szymanowska-Powałowska, D. (2012): Physiological predisposition of various Clostridium species to synthetize 1,3-propanediol from glycerol. In Process Biochemistry 47 (9), pp. 1308–1319. DOI: 10.1016/j.procbio.2012.05.012. 11 Kurniawan, Y.; Venkataramanan, K.P.; Piernavieja, M.; Scholz, C.; Bothun, G.D. (2013): Role of ionic strength on n-butanol partitioning into anionic dipalmitoyl phosphatidylcholine/phosphatidylglycerol vesicles. In The Journal of Physical Chemistry. B 117 (28), pp. 8484–8489. DOI: 10.1021/jp403735h. Kurniawan, Y.; Venkataramanan, K.P.; Piernavieja, M.; Scholz, C.; Bothun, G.D. (2013): Role of ionic strength on n-butanol partitioning into anionic dipalmitoyl phosphatidylcholine/phosphatidylglycerol vesicles. In The Journal of Physical Chemistry. B 117 (28), pp. 8484–8489. DOI: 10.1021/jp403735h. p p y p p y g y f y Chemistry. B 117 (28), pp. 8484–8489. DOI: 10.1021/jp403735h. Kurniawan, Y.; Venkataramanan, K.P.; Scholz, C.; Bothun, G.D. (2012): n-Butanol partitioning and phase behavior in DPPC/DOPC membranes. In The Journal of Physical Chemistry. B 116 (20), pp. 5919–5924. DOI: 10.1021/jp301340k. Malz, F. and Jancke, H. (2005): Validation of quantitative NMR. In Journal of Pharmaceutical and Biomedical Analysis 38 (5), pp. 813–823. DOI: 10.1016/j.jpba.2005.01.043. Kurniawan, Y.; Venkataramanan, K.P.; Scholz, C.; Bothun, G.D. (2012): n-Butanol partitioning and phase behavior in DPPC/DOPC membranes. In The Journal of Physical Chemistry. B 116 (20), pp. 5919–5924. DOI: 10.1021/jp301340k. Kurniawan, Y.; Venkataramanan, K.P.; Scholz, C.; Bothun, G.D. Bowles L.K., and Ellefson W.L. (1985): Effects of butanol on Clostridium acetobutylicum. In Applied and Environmental Microbiology 50 (Nov. 1985), pp. 1165–1170. (2012): n-Butanol partitioning and phase behavior in DPPC/DOPC membranes. In The Journal of Physical Chemistry. B 116 (20), pp. 5919–5924. DOI: 10.1021/jp301340k. Malz, F. and Jancke, H. (2005): Validation of quantitative NMR. In Journal of Pharmaceutical and Biomedical Analysis 38 (5), pp. 813–823. DOI: 10.1016/j.jpba.2005.01.043. Monot, F., Martin, J., Petitdemange, H., and Gay, R. (1982): Acetone and butanol production by clostridium acetobutylicum in a synthetic medium. In Applied and Environmental Microbiology 44 (6), pp. 1318–1324. Pauli, G.F.; Jaki, B.U.; Lankin, D.C. (2007): A routine experimental protocol for qHNMR illustrated with Taxol. In Journal of Natural Products 70 (4), pp. 589–595. DOI: 10.1021/np060535r. Roffler, S. R.; Blanch, H. W.; Wilke, C. R. (1988): In situ extractive fermentation of acetone and butanol. In Biotechnology and Bioengineering 31 (2), pp. 135–143. DOI: 10.1002/bit.260310207. Sardessai, Y. and Bhosle, S. (2002): Tolerance of bacteria to organic solvents. In Research in Microbiology 153 (5), pp. 263–268. DOI: 10.1016/S0923-2508(02)01319- 0. 0. Venkataramanan, K.P.; Boatman, J.J.; Kurniawan, Y.; Taconi, K.A.; Bothun, G.D.; Scholz, C. (2012): Impact of impurities in biodiesel-derived crude glycerol on the fermentation by Clostridium pasteurianum ATCC 6013. In Applied microbiology and biotechnology 93 (3), pp. 1325–1335. DOI: 10.1007/s00253-011-3766-5. Venkataramanan, K.P.; Kurniawan, Y.; Boatman, J.J.; Haynes, C.H.; Taconi, K.A.; Martin, L. (2014): Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation. In Journal of biotechnology 179, pp. 8–14. DOI: 10.1016/j.jbiotec.2014.03.017. Venkataramanan, K.P.; Kurniawan, Y.; Boatman, J.J.; Haynes, C.H.; Taconi, K.A.; Martin, L. (2014): Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation. In Journal of biotechnology 179, pp. 8–14. DOI: 10.1016/j.jbiotec.2014.03.017. Vollherbst-Schneck K., Sands J., and Montenecourt B. (1984): Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum. In Applied and Environmental Microbiology 47 (Jan 1984), pp. 193–194. Vollherbst-Schneck K., Sands J., and Montenecourt B. (1984): Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum. In Applied and Environmental Microbiology 47 (Jan 1984), pp. 193–194. Zhang, S.; Huang, X.; Qu, C.; Suo, Y.; Liao, Z.; Wang, J. (2017): Extractive fermentation for enhanced isopropanol and n-butanol production with mixtures of water insoluble aliphatic acids and oleyl alcohol. In Biochemical Engineering Journal 117, pp. 112– 120. DOI: 10.1016/j.bej.2016.10.006. 12 BUTANOL PARTITIONING INTO A LIPID BILAYER AS EXAMINED BY NMR NHOC – Net heat of combustion, AKI – Anti-knock index (Harvey and Meylemans 2011). BUTANOL PARTITIONING INTO A LIPID BILAYER AS EXAMINED BY NMR This chapter has been prepared in manuscript format with the intent to publish in the area of colloidal science or bioenergy. The work done here represents a collaborative effort with Dr. Scholz’s group at the University of Alabama Huntsville. 3.1 Introduction Growing energy demands world-wide require innovative options for sustainable energy production. Alternative energies that are naturally derived are an important and viable option to expand on current energy choices beyond fossil fuels (Dresselhaus and Thomas 2001). Biofuels such as ethanol are commonly used as a fuel additive (typically 10 to 15% by volume) to lower greenhouse gas emissions, however, ethanol is corrosive to car engines and has a lower energy density than gasoline (MacLean and Lave 2003; Surisetty et al. 2011). Due to the corrosive nature of ethanol, car engines require modification in order to use gasoline with higher ethanol content such as E85 or pure ethanol (Surisetty et al. 2011). Therefore, alternatives to ethanol are sought that are compatible with conventional engines and have higher energy densities similar to gasoline (Table 3.1). Butanol can be produced by fermentation as a biofuel and the properties of butanol are similar to those of gasoline, allowing it to be used as a fuel without any further modifications to most engines (Surisetty et al. 2011; Dürre 2007; Harvey and 13 Meylemans 2011). Butanol can also be produced from glycerol, which is the primary by-product of biodiesel production – 10 kg of glycerol are produced for every 100 kg of biodiesel (Yazdani and Gonzalez 2007). Unless further refined, this crude glycerol by-product has limited uses and is often discarded as a waste product. Hence, Meylemans 2011). Butanol can also be produced from glycerol, which is the primary by-product of biodiesel production – 10 kg of glycerol are produced for every 100 kg of biodiesel (Yazdani and Gonzalez 2007). Unless further refined, this crude glycerol by-product has limited uses and is often discarded as a waste product. Hence, producing butanol from glycerol achieves two goals of biorefining; creating a value- added alternative fuel from a bio-derived feedstock that is a by-product of biodiesel production. producing butanol from glycerol achieves two goals of biorefining; creating a value- added alternative fuel from a bio-derived feedstock that is a by-product of biodiesel production. Table 3.1: Table of NHOC, AKI fuel properties of ethanol, butanol, gasoline. Table 3.1: Table of NHOC, AKI fuel properties of ethanol, butanol, gasoline. NHOC – Net heat of combustion, AKI – Anti-knock index (Harvey and Meylemans 2011). Fuel NHOC Density (g/mL) AKI Gasoline 32.3 0.74 87 Ethanol 21.1 0.79 113 n-Butanol 26.8 0.81 87 Fuel NHOC Density (g/mL) AKI Gasoline 32.3 0.74 87 Ethanol 21.1 0.79 113 n-Butanol 26.8 0.81 87 One promising method to produce butanol is by fermentation using the Clostridia strain of bacteria (Nanda et al. 2017; Jones and Woods 1986; Monot et al., 1982). Fermentation by Clostridia species produces butanol, acetic acid, butyric acid, acetone and ethanol (Nanda et al. 2017). Various Clostridia strains have shown promising results for butanol production with yields of 10 g/L (C. beijerinckii )(Zhang One promising method to produce butanol is by fermentation using the Clostridia strain of bacteria (Nanda et al. 2017; Jones and Woods 1986; Monot et al., 1982). Fermentation by Clostridia species produces butanol, acetic acid, butyric acid, acetone and ethanol (Nanda et al. 2017). Various Clostridia strains have shown promising results for butanol production with yields of 10 g/L (C. beijerinckii )(Zhang One promising method to produce butanol is by fermentation using the Clostridia strain of bacteria (Nanda et al. 2017; Jones and Woods 1986; Monot et al., 1982). 14 et al. 2017), 9 g/L (C. pasteurianum) (Jensen et al. 2012) and 17 g/L after being metabolically engineered (C. acetobutylicum) (Lee et al. 2008). Furthermore, crude glycerol, despite containing impurities such as salts, methanol, and free fatty acids from biodesiel production, has been shown to be a capable feedstock for the fermentation of C. pasteurianum to produce butanol (Venkataramanan et al. 2012). Efforts are currently underway to increase butanol production by Clostridia via metabolic engineering, fermentation optimization, and/or extractive fermentation. A limitation to any fermentation process is product inhibition of cellular activity as high product concentrations are toxic to bacteria. Butanol toxicity is responsible for the low butanol concentrations achieved by fermentation. Butanol is a small amphiphilic molecule that partitions into the lipid bilayer of bacterial membrane leading to cell growth inhibition due to membrane fluidization (Sardessai and Bhosle 2002; Bowles and Ellefson 1985; Kurniawan et al. 2012; Vollherbst-Schneck et al., 1984). Membrane fluidization reduces lipid ordering within the membranes, which in turn can make the membrane more permeable and reduce the function of membrane-bound proteins. In order to minimize butanol toxicity during fermentation, butanol can be removed as it is being produced via extractive fermentation. Table 3.1: Table of NHOC, AKI fuel properties of ethanol, butanol, gasoline. NHOC – Net heat of combustion, AKI – Anti-knock index (Harvey and Meylemans 2011). In addition to reducing membrane fluidization, removing butanol from the fermentation broth drives the metabolic reaction to produce more butanol. Several solvents have been shown to be effective 15 at removing butanol from cell cultures by extractive fermentation. For example, oleyl alcohol is a commonly used solvent that has been shown to increase butanol productivity by 20% with C. beijerinckii (Zhang et al. 2017) and by 24% with C. acetobutylicum (Roffler et al. 1988). However, organic solvents themselves can be toxic to cells and limit butanol production (Lemos et al. 2017). A variety of non- organic solvents have been studied with the intent of finding a solvent capable of extracting butanol without inhibiting cellular activity. at removing butanol from cell cultures by extractive fermentation. For example, oleyl alcohol is a commonly used solvent that has been shown to increase butanol productivity by 20% with C. beijerinckii (Zhang et al. 2017) and by 24% with C. acetobutylicum (Roffler et al. 1988). However, organic solvents themselves can be toxic to cells and limit butanol production (Lemos et al. 2017). A variety of non- organic solvents have been studied with the intent of finding a solvent capable of extracting butanol without inhibiting cellular activity. One novel approach to extractive fermentation is to use molecular self-assemblies in aqueous phases, such as surfactant micelles, which exhibit a high affinity for butanol partitioning. For example, a 225% increase in butanol and acetone production was observed when non-ionic Pluronic surfactant L62 was added as micelles directly to a fermentation (Dhamole et al. 2012). Dhamole et al. investigated a wide range of surfactants in addition to L62 and found that the micelles assisted in the ‘capture’ of butanol (Dhamole et al. 2015), presumably into the amphiphilic and/or hydrophobic regions of the self-assembly. This capability has also been reported for lipid bilayer vesicles, which would provide a sustainable alternative to synthetic surfactants given that lipids can be isolated from natural or biological resources. Though scarcely investigated, lipid bilayer-based solvents show promise as they provide high butanol partition coefficients (KP) and are biocompatible (Kurniawan et al. 2012; Kurniawan et al. 2013). 16 This study examines the solvent characteristics of lipid bilayer vesicles; specifically, vesicle structure (swelling) and partitioning as a function of butanol concentration and lipid composition (ratio of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)). Table 3.1: Table of NHOC, AKI fuel properties of ethanol, butanol, gasoline. NHOC – Net heat of combustion, AKI – Anti-knock index (Harvey and Meylemans 2011). DPPC and DOPC were chosen because they have been previously investigated by our group and they offer different chemical structures, which have been shown to influence butanol partitioning (Kurniawan et al. 2012). DPPC and DOPC are similar molecules with a phosphocholine headgroup, but DPPC has saturated C16 tails and DOPC contains a double bond on each of its C18 tails (Table 3.2). 17 17 17 able 3.2: DPPC and DOPC were the lipids used in this study with molecular weight and structure shown. Lipid Name Structure M.W. (g/Mol) 1,2-dipalmitoyl-sn- glycero-3- phosphocholine (DPPC) 734.05 1,2-dioleoyl-sn- glycero-3- phosphocholine (DOPC) 786.13 shown. When DOPC forms a vesicle, the presence of the double bond creates more space between the lipid head groups than DPPC, which has been shown to increase butanol partitioning (Kurniawan et al. 2012; Kurniawan et al. 2013). Mixtures of DOPC and DPPC have been shown to have high butanol partition coefficients due to the inclusion of DOPC, which forms fluid phases in the bilayer along with the gel-like DPPC phases (Kurniawan et al. 2012). As butanol is extracted by a vesicle, the bilayers expand and the vesicle size becomes larger (Tierney et al. 2005; Löbbeckeand Cevc 1995). The degree of expansion and its relation to butanol partitioning, which are critical to evaluating the solvent capacity of lipid vesicles, have not been examined. In 18 this work, vesicle size was examined by dynamic light scattering (DLS) and an in situ quantitative nuclear magnetic resonance (qNMR) approach was developed to determine the amount of butanol in the lipid phase. By using this approach, the butanol partition coefficients could be found without requiring the separation of the lipids as high performance liquid chromatography (HPLC) would require (Zhang et al. 2004; Kitamura 1999). 3.2 Methods/Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, >99% purity) and 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC, >99% purity) were purchased from Avanti Polar Lipids (Alabaster, AL) dissolved in chloroform and were used without further purification Deuterium oxide (99 9 atom % D) was purchased from Sigma-Aldrich 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, >99% purity) and 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC, >99% purity) were purchased from Avanti Polar Lipids (Alabaster, AL) dissolved in chloroform and were used without further purification. Deuterium oxide (99.9 atom % D) was purchased from Sigma-Aldrich. Deionized (DI) ultrafiltered water was obtained from a Millipore Direct Q-3 purifier. Butanol, ethanol, 1,3 propanediol, butyric acid and acetic acid were purchased from Sigma Aldrich. 3.2.1 Vesicle Preparation 3.2.1 Vesicle Preparation Vesicles were prepared using the Bangham method (Bangham et al. 1965). In short, lipids previously dissolved in chloroform were placed under a flow of nitrogen gas until the chloroform evaporated and a thin-film of lipids remained. The lipids were dried further by placing them in a vacuum chamber for 30 min. DI water or deuterium oxide was added before the lipid/water solution was placed in a heat bath deuterium oxide was added before the lipid/water solution was placed in a heat bath 19 to form vesicles. This allowed for nuclear magnetic resonance experiments. Following this, they were sonicated for 10 min at 42 OC. to form vesicles. This allowed for nuclear magnetic resonance experiments. Following this, they were sonicated for 10 min at 42 OC. 3.2.2 Nuclear Magnetic Resonance (NMR) NMR was used to determine the lipid/water butanol partitioning coefficient. During all quantitative NMR (qNMR) experiments the protocol described in Pauli et al. 2007 was followed using a 400 MHz NMR (Bruker Biospin AG, Magnet System 400’54 Ascend). NMR experiments were carried out with a non-spinning sample and 13C decoupling to prevent side satellites from forming in the spectra, which can interfere with primary peaks when analyzing spectra. Sixteen scans and two mock scans were used to capture the NMR spectra. The MestReNova software was used to analyze data collected via NMR. After ensuring the spectrum baseline was properly phased, the integral of each peak associated with an analyte of interest was taken. To ensure consistency when measuring, the integral lengths and starting points were recorded and duplicated for each sample. A calibration curve was constructed relating the concentration of butanol to the area under the peaks of butanol. A concentration range of 0.9 to 15 g/L of butanol was prepared for NMR analysis. Each sample prepared was 500 µL in volume. A spectrum was obtained for each sample and analysis was done by integrating each butanol signal with reference to the water peak. The area under the peaks were recorded for each of the concentrations via integration in the MestReNova program. A plot of 20 concentration versus area under the peak allowed for a relationship to be found between the NMR data and quantitative results. A quadratic polynomial fit was used to match this data and fit through concentration = 0 with an R2 of 0.996. concentration versus area under the peak allowed for a relationship to be found between the NMR data and quantitative results. A quadratic polynomial fit was used to match this data and fit through concentration = 0 with an R2 of 0.996. Partition coefficients were found using an in-situ method where butanol and vesicles were present during testing. When butanol molecules are captured by a vesicle, the NMR signal associated with the captured butanol is hidden. On this basis, the number of moles of butanol in the water phase (nb,w) were measured. 3.2.2 Nuclear Magnetic Resonance (NMR) The partition coefficient of butanol in lipid was measured for each lipid composition and calculated using equation (1): 𝐾𝑝= 𝑛𝑏,𝐿 𝑛𝑏,𝐿+𝑛𝐿 𝑛𝑏,𝑤 𝑛𝑏,𝑤+𝑛𝑤 ⁄ (1) (1) Where KP is the mole fraction lipid-water partition coefficient of butanol in lipid, nb,L is the moles of butanol in the lipid phase, nL is the total moles of lipid, nb,w is the moles of butanol in the water phase, and nw is the total moles of water (Kurniawan et al. 2012). The number of moles of butanol in the lipid phase (nb,L) was calculated as nb,L = nb - nb,w. Partition coefficients were measured at lipid concentrations ranging from 0.4 g/L to 4 g/L (2.5 – 5 mM) and a butanol concentration of 5 g/L was used following preliminary fermentation results in our group. It should be noted that 5 g/L butanol is well below the water solubility limit (73 g/L at 25 oC), therefore, the experiment results were not influenced by butanol phase separation. Each NMR sample was 500 µL in volume and control experiments were conducted with only 21 butanol and D2O present, and then additional samples that contained a mixture of butanol, lipid and D2O. One butanol solution was prepared and used for the control and lipid samples. The original butanol concentration was measured in the butanol/D2O sample and then used to find the moles of butanol in the lipid phase (nb,L). 3.2.3 Dynamic Light Scattering As butanol molecules partition into a lipid bilayer the space between the head groups expands to accommodate the butanol (Tierney et al. 2005; Löbbecke and Cevc 1995). Bilayer expansion can be examined by measuring the hydrodynamic diameter of the vesicles via light scattering while adding butanol. A Malvern Zetasiver Nano ZA was used for DLS experiments. First, the swelling effect of butanol alone was studied. Different liposome solutions (10mM DPPC, 10mM DOPC, 10mM 50/50 DOPC/DPPC) were prepared and modified by vesicle extrusion using a polycarbonate track-etched 100 nm membrane First, the swelling effect of butanol alone was studied. Different liposome solutions (10mM DPPC, 10mM DOPC, 10mM 50/50 DOPC/DPPC) were prepared and modified by vesicle extrusion using a polycarbonate track-etched 100 nm membrane (Whatman). An initial DLS test was completed to identify the size of the liposomes with no butanol present. Butanol was then added in small amounts to the vesicles and the hydrodynamic diameter of the liposomes was measured after each butanol addition until a butanol concentration of 20 g/L was reached. This was chosen to match and exceed the higher butanol yields that have been reported (Roffler et al. 1988). Ethanol, 1,3 propanediol and acetic acid were also studied following the same 1988). Ethanol, 1,3 propanediol and acetic acid were also studied following the same 1988). Ethanol, 1,3 propanediol and acetic acid were also studied following the same 22 procedure. In these studies, only DPPC was examined because DPPC showed the largest change in size during previous experiments. procedure. In these studies, only DPPC was examined because DPPC showed the largest change in size during previous experiments. 3.3 Results and Discussion 3.3.1 Partition coefficient with increasing lipid concentrations 3.3.1 Partition coefficient with increasing lipid concentrations Figure 3.1 shows two NMR spectra of butanol overlaid on each other; a spectrum of butanol without vesicles (red) and a spectrum of butanol in the presence of vesicles (green). When vesicles are present, the NMR shows butanol peaks at lower intensity compared to when vesicles are not present. Comparing the spectra provides direct evidence that the butanol extracted into the vesicles is shielded from NMR and that in situ measurements can be used to determine partitioning. This was further validated by centrifuging a butanol+vesicle sample, removing the vesicles with captured butanol, and analyzing the residual butanol in the supernatant by qNMR. These results were comparable to direct calculations in situ (results not shown). 23 23 Figure 3.1: NMR spectra of butanol without vesicles present (red) and in the presence of vesicles (green). The y-axis shows a relative intensity and the x-axis shows chemical peak shift. This difference is measurable and can then be used to find butanol/lipid partition coefficients. Figure 3.1: NMR spectra of butanol without vesicles present (red) and in the presence of vesicles (green). The y-axis shows a relative intensity and the x-axis shows chemical peak shift. This difference is measurable and can then be used to find butanol/lipid partition coefficients. Butanol partition coefficients were determined using qNMR at a butanol concentration of 5 g/L and three different lipid compositions: DPPC, DOPC and an equimolar mixture. For all lipid compositions KP increased with lipid concentration. The equimolar mixture showed the highest butanol partition coefficient, consistent with previous work using Langmuir monolayers (Kurniawan et al. 2013),while DOPC had the second highest and DPPC showed the lowest (Figure 3.2 A-C). The number of butanol molecules per each lipid composition was calculated based on the KP results 24 (Figure 3.2 D). The results showed that the equimolar composition provided the highest butanol to lipid ratio with a value of 7.1 moles butanol per mole of lipid while DPPC had the lowest with 4.5 moles of butanol per mole of lipid. Fi 3 2 P titi ffi i t f b t l i DPPC (A) DOPC (B) d 50 50 DPPC DOPC (C) All Figure 3.2: Partition coefficients of butanol in DPPC (A), DOPC (B) and 50:50 DPPC:DOPC (C). All partition coefficient experiments were conducted at 25 oC and at a butanol concentration of 5 g/L. 3.3 Results and Discussion A “” represents p < 0.05, while “n.s.” represents p > 0.05. This basis is used throughout the results presented. (D) The number of butanol molecules per lipid molecule based on KP results at a butanol concentration of 5 g/L and lipid concentrations of 4 g/L. The partition coefficients measured here follow a similar trend to those found previously, where DPPC showed the lowest partition coefficient and a mixture of 25 DPPC:DOPC showed a higher KP value (Kurniawan et al. 2012). To our knowledge, butanol partition coefficients for DOPC alone have not yet been shown but the trend presented in Kurniawan et al. 2012 showed that with increasing DOPC present, the partition coefficient rose. We found while that DOPC alone provided an increased KP compared to DPPC, the equimolar mixture still resulted in the highest KP. This suggests that the multiphase bilayer (gel and fluid) would be a more suitable solvent for butanol extraction. Butanol partition coefficients have been reported for other solvents. In most instances the partition coefficient reported is a mass-based partition coefficient and in order to effectively compare these to our own partition coefficients we needed to convert our mole fraction partition coefficients (Table 3.3). These converted values can be seen in the table below. DPPC:DOPC showed a higher KP value (Kurniawan et al. 2012). To our knowledge, butanol partition coefficients for DOPC alone have not yet been shown but the trend presented in Kurniawan et al. 2012 showed that with increasing DOPC present, the partition coefficient rose. We found while that DOPC alone provided an increased KP compared to DPPC, the equimolar mixture still resulted in the highest KP. This suggests that the multiphase bilayer (gel and fluid) would be a more suitable solvent for butanol extraction. Butanol partition coefficients have been reported for other solvents. In most instances the partition coefficient reported is a mass-based partition coefficient and in order to effectively compare these to our own partition coefficients we needed to convert our mole fraction partition coefficients (Table 3.3). These converted values can be seen in the table below. Table 3.3: Butanol/Lipid mass and mole fraction partition coefficients at a lipid concentration of 4 g/L and a butanol concentration of 5 g/L. Our results show that the equimolar mixture of DPPC and DOPC provide the highest butanol partition coefficient. 3.3 Results and Discussion Butanol Partition coefficient Lipid Mass basis Mole fraction basis DPPC 22 ± 5 900 ± 190 DPPC:DOPC (50:50) 33 ± 10 1370 ± 365 DOPC 28 ± 8 1220 ± 480 Butanol Partition coefficient Lipid Mass basis Mole fraction basis DPPC 22 ± 5 900 ± 190 DPPC:DOPC (50:50) 33 ± 10 1370 ± 365 DOPC 28 ± 8 1220 ± 480 26 26 The surfactant L62 used by Dhamole et al. yielded a mass partition coefficient of 3.5 and showed an increase in butanol productivity of over 200%. For organic solvents, a number of long chain alcohols were examined with the highest partition coefficient amongst them obtained with 2-ethyl-1,3-hexanediol with a mass partition coefficient of 8.1 reported (Barton, Daugulis 1992). Ionic liquid solvents have also shown to be effective solvents for butanol (Cascon et al. 2011; Davis and Morton 2008). Tetrahexyammonium dihexylsulfosuccinate ([THA][DHSS]) yielded a mass partition coefficient of 7.99 (Cascon et al. 2011) and 1-butyl-3-methylimidazolium bis(triflouromethylsulfonyl)imide ([bmim][Tf2N]) a mass partition coefficient of 14 (Davis, Morton 2008). Each lipid composition provided a higher mass partition coefficient than partition coefficients examined. While the trend reported here for butanol partitioning and lipid composition is a similar trend to previous results reported by our group, the values we found using NMR showed higher butanol lipid partition coefficients and higher butanol-lipid molecular ratios. One significant difference between this study and our previous work was this study analyzed each sample using NMR which allows for in situ measurements. While HPLC was used previously which requires the sample to be separated before being analyzed. This separation could be the cause for such a distinct difference between both the partition coefficients and the butanol/lipid ratios. 27 3.3.2 Liposome Swelling DPPC, DOPC and an equimolar mixture of DPPC and DOPC had an increase in size as butanol was added as seen in Figure 3.3A. A linear fit was applied to each data series plotted through change in size = 0. The slope of this line with units of (diameter)/ (g/L butanol) allowed for a quantitative comparison between lipid compositions (tabulated in Figure 3.3). DPPC showed the largest change in size per butanol added with a slope of 1.4. DOPC and an equimolar mixture of DPPC and DOPC showed smaller slopes of 0.8 and 1.1, respectively. 3.3 Results and Discussion The polydispersity index of the samples, which is a measure of size homogeneity and used to identify aggregation or sample destabilization, was less than 0.3 for these experiments. This indicates that the changes in size were not due to vesicle aggregation or butanol phase separation (into lipid-stabilized butanol droplets in water – i.e. an emulsion). Acetic acid, 1,3 propanediol and ethanol were tested in the same way as butanol and the changes in vesicle size can be seen in Figure 3.3B. Acetic acid, ethanol and 1,3 propanediol showed a change in vesicle size (slope) of 0.3, 0.2 and 0.5, respectively. The vesicles used were 10 mM DPPC and, when compared with the butanol results, the changes in size for acetic acid, 1,3 propanediol, and ethanol were two to five times lower than when butanol was present. 28 Change in vesicle size ((diameter)/ (g/L butanol) Lipid Butanol Acetic acid 1,3 Propanediol Ethanol DPPC 1.42 0.33 0.58 0.25 DPPC/DOPC (50:50) 1.06 DOPC 0.81 Figure 3.3: Change in vesicle size with (A) increasing butanol concentration and (B) increasing acetic cid, 1,3 propanediol and ethanol concentration (DPPC only). A table containing the slopes of linear fits through the intercept. Change in vesicle size ((diameter)/ (g/L butanol) Lipid Butanol Acetic acid 1,3 Propanediol Ethanol DPPC 1.42 0.33 0.58 0.25 DPPC/DOPC (50:50) 1.06 DOPC 0.81 Figure 3.3: Change in vesicle size with (A) increasing butanol concentration and (B) increasing acetic acid, 1,3 propanediol and ethanol concentration (DPPC only). A table containing the slopes of linear fits through the intercept. Figure 3.3: Change in vesicle size with (A) increasing butanol concentration and (B) increasing acetic acid, 1,3 propanediol and ethanol concentration (DPPC only). A table containing the slopes of linear fits through the intercept. ure 3.3: Change in vesicle size with (A) increasing butanol concentration and (B) increasing acetic 29 To our knowledge, the use of vesicles for in situ extractive butanol fermentation and the effect of butanol and lipid composition on vesicle solvent characteristics have not been previously studied. Comparing butanol partitioning and swelling results provides insight into the interdependence of these phenomena. qNMR was conducted at lipid:butanol mass ratios > 0.73 (Figure 3.2), which corresponds to swelling conditions > 10 g/L butanol where the lipid concentration was 10 mM or 7.34 g/L (Figure 3.3A). This provides direct evidence that butanol partitioning led to swelling. 3.3 Results and Discussion As butanol swells the vesicles, more space is created in the vesicles to accommodate more butanol. This was observed for qNMR measurements at low lipid concentrations (0.4 g/L) where the molar ratio of captured butanol to lipid was extremely high with ratios of 14, 24 and 24 for DPPC, DOPC and the equimolar mixture respectively. While butanol has not been studied extensively, ethanol is known to increase vesicle size at higher concentrations due to bilayer expansion and lipid interdigitation (Löbbecke, L. and Cevc, G. 1995). Butanol also leads to bilayer expansion and our previous results showed that DPPC and DPPC:DOPC vesicles entered an interdigitated phase at butanol concentrations above 10 g/L (Kurniawan et al. 2012). According to this work, vesicle size increased linearly before and after butanol caused lipid interdigitation. This indicates that size of the vesicle does not reflect the effects of interdigitation, which is known to expand lipid bilayers (Löbbecke and Cevc 1995), at the conditions examined. 30 With respect to lipid composition, all vesicles swelled in the presence of butanol, but swelling was not correlated to the butanol partition coefficient, meaning a greater increase in size does not reflect a higher partition coefficient. This can be seen for DPPC, which showed the greatest change in size and the lowest partition coefficient. In turn, DOPC showed the smallest change in size with a high partition coefficient. This observation is consistent with the concept of free-space provided by unsaturated lipids with double bonds (DOPC). The free-space allows the bilayers to accommodate butanol with less expansion compared to saturated lipids (DPPC), which are tightly packed and must “unpack” to accommodate butanol. With respect to lipid composition, all vesicles swelled in the presence of butanol, but swelling was not correlated to the butanol partition coefficient, meaning a greater increase in size does not reflect a higher partition coefficient. This can be seen for DPPC, which showed the greatest change in size and the lowest partition coefficient. In turn, DOPC showed the smallest change in size with a high partition coefficient. This observation is consistent with the concept of free-space provided by unsaturated lipids with double bonds (DOPC). The free-space allows the bilayers to accommodate butanol with less expansion compared to saturated lipids (DPPC), which are tightly packed and must “unpack” to accommodate butanol. 3.4 Conclusions The vesicle swelling tests revealed two specific traits about the effect of butanol on lipid vesicle solvents. The first is that butanol creates a larger swelling effect on vesicles when compared to other fermentation products. This can be attributed to the vesicles ability to preferentially extract butanol over other products. This is due to the greater hydrophobicity of butanol compared to acetic acid, 1,3-propanediol, and ethanol. Secondly, the butanol partition coefficient of vesicles does not correlate with the change in vesicle size with added butanol. After examining the partition coefficient results it is clear that the equimolar mixture of DOPC and DPPC is the most effective at extracting butanol. 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In Chemistry and Physics of Lipids 127 (1), pp. 113–120. DOI: 10.1016/j.chemphyslip.2003.09.013. Zhang, X.; Patel, A.B.; Graaf, R.A.; Behar, K.L. (2004): Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. In Chemistry and Physics of Lipids 127 (1), pp. 113–120. DOI: 10.1016/j.chemphyslip.2003.09.013. Zhang, X.; Patel, A.B.; Graaf, R.A.; Behar, K.L. (2004): Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. In Chemistry and Physics of Lipids 127 (1), pp. 113–120. DOI: 10.1016/j.chemphyslip.2003.09.013. 35 35 CHAPTER 4 LIPID VESICLE-BASED IN-SITU EXTRACTION FERMENTATION OF BIOBUTANOL PRODUCED BY CLOSTRIDIUM PASTEURIANUM This chapter is written in manuscript format with the intent to submit once additional results are obtained. The work presented here represents a collaborative effort with Dr. Carmen Scholz’s group at the University of Alabama in Huntsville. 4.1 Introduction 4.1 Introduction Butanol is becoming a highly sought after fuel source as it has similar fuel properties to that of gasoline and could even be used in a mixture of diesel (Atabani et al. 2012; Surisetty et al. 2011). While current butanol synthesis is usually undertaken by the conversion of fossil fuels (Surisetty et al. 2011), butanol can also be produced via fermentation. Fermentation can offer a biofriendly, renewable process that could potentially replace fossil fuels as the industrial source of butanol (Nanda et al. 2017). Even crude glycerol, an unrefined by-product of biodiesel, can be effectively utilized for butanol production (Venkataramanan et al. 2012). Batch fermentations using Clostridia strain of bacteria have shown promising results of butanol yields (Kubiak et al. 2012; Lee et al. 2008; Groot et al. 1989), however, in order for fermentation to become a viable butanol production option, a more 36 industrial approach is needed. Continuous fermentation eliminates several negatives that batch production would have in an industrial setting, such as long down-times required for cleaning and sterilizing the equipment in between batches (Lee et al. 2008). Continuous cultures of Clostridium acetobutylicum and C. beijerinckii achieved butanol yields of 0.42 g/g (Huang et al. 2004) and 0.38 g/g (Qureshi and Blaschek 2000). Groot, et al. compared batch and continuous fermentations with a product recovery process integrated and found the yields of both to be comparable to each other (Groot et al. 1989). industrial approach is needed. Continuous fermentation eliminates several negatives that batch production would have in an industrial setting, such as long down-times required for cleaning and sterilizing the equipment in between batches (Lee et al. 2008). Continuous cultures of Clostridium acetobutylicum and C. beijerinckii achieved 2008). Continuous cultures of Clostridium acetobutylicum and C. beijerinckii achieved butanol yields of 0.42 g/g (Huang et al. 2004) and 0.38 g/g (Qureshi and Blaschek 2000). Groot, et al. compared batch and continuous fermentations with a product recovery process integrated and found the yields of both to be comparable to each other (Groot et al. 1989). Product removal is an important process that increases butanol production. Extractive fermentation removes product as it is formed to both limit the toxic effects of butanol (Sardessai and Bhosle 2002) and drive the fermentation to produce more butanol. Recent work showed butanol yields of 0.36 g/g using a mixture of oleyl alcohol and dodecanoic acid as a solvent (Zhang et al. 2017). 4.1 Introduction Another study combined continuous fermentation using metabolically engineered C. acetobutylicum with ex situ recovery and found butanol yields of 0.18 g/g and 0.3 g/g (Lee et al. 2016). While organic solvents have been shown to be effective, biocompatible self- assembly solvents have recently been shown to be effective solvents (Dhamole et al. 2012; Vian et al. 2017). 37 This work showcases the potential of the C. pasteurianum strain of bacteria for butanol production. Additionally, a continuous fermentation process was used with ex situ recovery and to our knowledge this is the first time C. pasteurianum has been used in a continuous fermentation. Finally, a novel lipid bilayer self-assembled solvent was used due to the high affinity that lipid bilayers show towards butanol extraction (Kurniawan et al. 2012; Kurniawan et al. 2013). 4.2 Materials and Methods L-α-phosphatidylcholine (95%) (Soy-Lecithin) was purchased from Avanti Polar Lipids (Alabaster, AL) in granule form and was used without further purification. Deuterium oxide (99.9 atom % D) and maleic acid (traceCERT, qNMR standard) were purchased from Sigma-Aldrich. Clostridia pasteurianum ATCC 6013 strain was used for these experiments. Deionized (DI) ultrafiltered water was obtained from a Millipore Direct Q-3 purifier. A 400 MHz NMR (Bruker Biospin AG, Magnet System 400’54 Ascend) was used for the NMR analysis. 4.2.1 Vesicle Preparation Soy-Lecithin was added to preheated DI water at a temperature over 40oC. The lipids were heated and stirred until dissolved. Then the vesicle solution was autoclaved at a temperature of 120 oC before being placed in the glove box. The experiments here used 2 g/L of lipid. 38 4.2.2 Fermentation Media The media used for this fermentation was a glycerol-based media. The compositions used for each volume can be seen in Table 4.1. The CaCO3 solution was added immediately before inoculation of the bacteria after everything was mixed and autoclaved. Table 4.1: Compositions of the glycerol media used during the fermentation. Table 4.1: Compositions of the glycerol media used during the fermentation. Volume 500 ml 1000 ml K2HPO4 1.87 g 3.74 g KH2PO4 0.715 g 1.43 g (NH4)2SO4 1.1 g 2.2 g Yeast extract 0.55 g 1.1 g MgSO4/FeSO4 solution 5 ml 10 ml Trace metal solution 1 ml 2 ml Glycerol 12.5 g 25 g CaCO3 solution 2.5 ml 5.0 ml CaCO3 solution is prepared by slowly adding 22g of CaCO3 to 36.3 ml of HCl, then slowly adding HCl until the solution turns clear. Trace metal solution SL7 is composed of 10mL of 25% HCl solution per liter, 1.5 g/L FeCl2·4H2O, 190 mg/L CoCl2·6H2O, 100 mg/L MnCl2·4H2O, 70 mg/L ZnCl2, 62 mg/L H3BO3, 36 mg/L Na2MoO4·2H2O, 24 mg/L NiCl2·6H2O, 17 mg/L CuCl2·2H2O. 4.2.3 Bioreactor Schematic and Operation 4.2.3 Bioreactor Schematic and Operation A schematic of the bioreactor system is shown in Figure 4.1. Prior to butanol extraction with vesicles, bacteria were first grown in batch culture until an optical 39 density (O.D.) of 0.7 and a pH of 5 was obtained, signifying that the conditions were appropriate for butanol production. A dilution rate (DR) was chosen, more on that below, and the continuous fermentation was then started. Fresh media was pumped into the bioreactor while simultaneously removing media from the bioreactor to prevent accumulation. The fermentation continued in this way until 98% turnover of the bioreactor volume had been achieved which was considered reaching steady state. However, to ensure we did reach steady state an additional hour was spent and the O.D., pH and composition were checked at the start and end of that hour and compared to each other. Following this the extraction process was started. A hollow fiber membrane (SpectrumLabs, 300kD, 20 cm EL, modified polyether sulfone or mPES) was used to contact the vesicle solvent with the fermentation broth. Analysis was completed on samples taken from the bioreactor (B) and vesicle solvent container (D). density (O.D.) of 0.7 and a pH of 5 was obtained, signifying that the conditions were appropriate for butanol production. A dilution rate (DR) was chosen, more on that below, and the continuous fermentation was then started. Fresh media was pumped into the bioreactor while simultaneously removing media from the bioreactor to prevent accumulation. The fermentation continued in this way until 98% turnover of the bioreactor volume had been achieved which was considered reaching steady state. Table 4.1: Compositions of the glycerol media used during the fermentation. However, to ensure we did reach steady state an additional hour was spent and the O.D., pH and composition were checked at the start and end of that hour and compared to each other. Following this the extraction process was started. A hollow fiber membrane (SpectrumLabs, 300kD, 20 cm EL, modified polyether sulfone or mPES) was used to contact the vesicle solvent with the fermentation broth. Analysis was completed on samples taken from the bioreactor (B) and vesicle solvent container (D). 40 Figure 4 1 Schematic of the continuous fermentation with vesicle extraction process P1 P4 are the Figure 4.1: Schematic of the continuous fermentation with vesicle extraction process. P1-P4 are the pumps. (A) Fresh media inlet being fed into (B) the bioreactor with the C. Pasteurianum fermentation. (C) was the waste collection. (D) housed the vesicle solution being sent through (E) the hollow fiber membrane used to extract butanol. 𝐷𝐹= 𝑄𝑀 𝑉𝑅 ( 4.1 ) ( 4.1 ) ( 4 Dilution rate was found by dividing the flow rate of the media in to the reactor (𝑄𝑀) by the initial volume of the reactor (𝑉𝑅). Two dilution rates were used in these experiments, 0.18 hr-1 and 0.09 hr-1. The flow rates remained consistent throughout the experiments. The reactor’s initial volume was changed in order to change the dilution rate. 41 4.2.4 Quantitative NMR qNMR was performed to analyze butanol concentration and determine extraction performance. Maleic acid (Sigma, standard for quantitative NMR, TraceCERT) was used as an internal calibrant as its NMR shift is unique compared to butanol. Samples were prepared with a 90/10 split of H2O/D2O in pre-weighed NMR tubes. The volume of each sample was the same with 450 µL of a sample and 50 µL of internal calibrant solution. The NMR spectrum was collected for each sample using the same acquisition parameters as previously used in chapter 3, with the exception of the delay time. The delay time was extended from 20 s to 30 s to accommodate the longer relaxation time of maleic acid. The following equation was used to analyze this data and convert the intensity of a butanol peak to purity or mass fraction of butanol. Figure 4.2 shows an NMR spectrum of a sample from the vesicle solution after extraction. The maleic acid peak located at 6.25 ppm is compared to the available butanol peaks using Equation 4.2 (Malz and Jancke 2005). Table 4.1: Compositions of the glycerol media used during the fermentation. 42 Figure 4.2: NMR spectrum of a sample from the continuous fermentation. The maleic acid peak (6.25 ppm) is compared to the visible butanol peaks (1.4, 1.25, 0.8 ppm) in order to find how much butanol is in solution. Figure 4.2: NMR spectrum of a sample from the continuous fermentation. The maleic acid peak (6.25 ppm) is compared to the visible butanol peaks (1.4, 1.25, 0.8 ppm) in order to find how much butanol is in solution. ppm) is compared to the visible butanol peaks (1.4, 1.25, 0.8 ppm) in order to find how much butanol is in solution. 𝑃𝑥= 𝐼𝑥 𝐼𝑠𝑡𝑑 𝑁𝑠𝑡𝑑 𝑁𝑥 𝑀𝑥 𝑀𝑠𝑡𝑑 𝑚𝑠𝑡𝑑 𝑚𝑡𝑜𝑡𝑎𝑙 𝑃𝑠𝑡𝑑 ( 4.2 ) ( 4.2 ) ( 4.2 ) ( 4 Once the purity is known, it can be converted to a concentration of butanol by using the partition coefficient and the molecular weights. This tells us the concentration of butanol outside of the vesicles. The butanol partition coefficient of soy-lecithin was then found and used to calculate the number of moles of butanol captured by the vesicles. 43 4.2.5 HPLC Analysis Samples taken from the bioreactor were centrifuged immediately to separate any bacteria. The supernatant was analyzed for butanol concentration using an HPLC as described previously in Kurniawan et al. 2012. Each sample was measured at least in duplicate. The HPLC used was a Varian ProStar pump system with a Varian Star 800 Module Interface. Module Interface. 4.3 Results and Discussion 4.3.1 Validating qNMR for determining butanol concentration Samples were taken at random time points from the bioreactor and butanol concentration was measured by both HPLC and NMR (Figure 4.3). This comparison was done to validate the qNMR approach, which is the basis for determining butanol partitioning into, and extraction by, lipid vesicles in situ. While HPLC is commonly used to determine fermentation product concentrations, it is not suitable for these in situ measurements – the vesicles would have needed to have been separated from the vesicle solvent phase prior to analysis. Good agreement was observed between qNMR and HPLC. 44 Figure 4.3: A comparison between HPLC and qNMR with an internal calibrant. Random samples were chosen from the experiments and tested with both HPLC and qNMR. On average there was an 8% difference between them. 0 1 2 3 4 5 1 2 3 4 5 6 Butanol [g/L] Sample Number HPLC NMR 0 1 2 3 4 5 1 2 3 4 5 6 Butanol [g/L] Sample Number HPLC NMR Figure 4.3: A comparison between HPLC and qNMR with an internal calibrant. Random samples were chosen from the experiments and tested with both HPLC and qNMR. On average there was an 8% difference between them. Figure 4.3: A comparison between HPLC and qNMR with an internal calibrant. Random samples were chosen from the experiments and tested with both HPLC and qNMR. On average there was an 8% difference between them. 4.3.2 Butanol extracted by vesicle solvent The mass of butanol extracted by the vesicle solvent was found by measuring the amount of butanol outside of the vesicles by qNMR, and then calculating the amount of butanol within the vesicle bilayers using previously measured partition coefficients for soy-lecithin. At butanol concentrations of 1 g/L, around the concentration found in our dilution rate 0.18 hr-1, the lecithin/water mole fraction partition coefficient was approximately 200. As seen in figure 4.4, the amount of butanol extracted increased over time, and similar results were observed at 60 min for the two dilution rates. This was expected given that the solvent flow was unchanged between dilution 45 45 rates. The difference in dilution rate results can instead be seen in the total butanol produced as discussed below. Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution rates (A 0.18 h-1 and B 0.09 h-1). 4.3 Results and Discussion The blue bar represents the total butanol measured by qNMR therefore outside of the vesicles. The orange bar represents the butanol found by calculation using the partition coefficients from previous work. 60 min is highlighted here as that was the final time point both trials had in common 0 0.5 1 1.5 0 10 20 30 40 50 60 Butanol [g] Time [min] A) Inside Outside 0 0.5 1 1.5 0 40 80 120 Butanol [g] Time [min] B) 0 0.5 1 1.5 0 10 20 30 40 50 60 Butanol [g] Time [min] A) Inside Outside 0 0.5 1 1.5 0 40 80 120 Butanol [g] Time [min] B) 0 0.5 1 1.5 0 10 20 30 40 50 60 Butanol [g] Time [min] A) Inside Outside Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution rates (A 0.18 h-1 and B 0.09 h-1). The blue bar represents the total butanol measured by qNMR therefore outside of the vesicles. The orange bar represents the butanol found by calculation using the partition coefficients from previous work. 60 min is highlighted here as that was the final time point both trials had in common. Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilut Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution rates (A 0.18 h-1 and B 0.09 h-1). The blue bar represents the total butanol measured by qNMR therefore outside of the vesicles. The orange bar represents the butanol found by calculation using the partition coefficients from previous work. 60 min is highlighted here as that was the final time point both trials had in common. Figure 4.4: Amount of butanol (g) extracted by the vesicle solvent as a function of time at two dilution rates (A 0.18 h-1 and B 0.09 h-1). The blue bar represents the total butanol measured by qNMR therefore outside of the vesicles. The orange bar represents the butanol found by calculation using the partition coefficients from previous work. 4.3 Results and Discussion 60 min is highlighted here as that was the final time point both trials had in common. 4.3.3 Total butanol production 4.3.3 Total butanol production The total butanol produced during continuous culture with vesicle-based solvent extraction is shown in figure 4.5. The lower dilution rate, DR = 0.09 h-1, produced more butanol compared to the higher dilution rate with a productivity of 1.11 g/h and a yield of 0.12 g butanol/g glycerol. For comparison, Roffler et al. conducted a 46 fed-batch extractive fermentation of butanol with oleyl alcohol and produced over 500 g of butanol after 50 hours with a yield of 0.18 g butanol/g glucose. More recently, a batch extractive fermentation using oleyl alcohol exhibited a yield of 0.28 g/g (Zhang et al. 2017). Continuous cultures of Clostridium acetobutylicum and C. beijerinckii achieved butanol yields of 0.42 g/g (Huang et al. 2004) and 0.38 g/g (Qureshi and Blaschek 2000) without extraction present. The total time of these experiments was longer than our time in each case. While our time did not exceed 2 hours, in all of these studies, butanol production increases significantly after 10 hours of extractive fermentation. fed-batch extractive fermentation of butanol with oleyl alcohol and produced over 500 g of butanol after 50 hours with a yield of 0.18 g butanol/g glucose. More recently, a batch extractive fermentation using oleyl alcohol exhibited a yield of 0.28 g/g (Zhang et al. 2017). Continuous cultures of Clostridium acetobutylicum and C. beijerinckii achieved butanol yields of 0.42 g/g (Huang et al. 2004) and 0.38 g/g (Qureshi and Blaschek 2000) without extraction present. The total time of these experiments was longer than our time in each case. While our time did not exceed 2 hours, in all of these studies, butanol production increases significantly after 10 hours of extractive fermentation. 47 Both dilution rates showed that over time the total butanol produced increased with time. This is attributed to the vesicle extraction system which drove the reaction to form more butanol by removing the product. The butanol production was increased by 32% and 94% for DR of 0.18 h-1 and 0.09 h-1 respectively, higher than previously reported values for oleyl alcohol solvents. Oleyl alcohol has shown butanol productivity increases of 20% (Zhang et al. 2017) and 24% (Roffler et al. 1988). 4.3 Results and Discussion When the percent increase of butanol is calculated at 60 minutes for the DR of 0.09 h-1, a value of 61% is found which is almost double the value for DR 0.18 h-1 at the same timepoint. At the lower dilution rate, we see that the amount of butanol in the reactor rises even after the extraction process is underway. This is in agreement with another continuous fermentation result that showed lower dilution rates produced more butanol (Huang et al. 2004). This highlights the importance of lower dilution 47 rates as the bacteria are given more time to grow and produce butanol. Initial results also show the vesicle solvent extracts more butanol when in the presence of higher amounts of butanol. so show the vesicle solvent extracts more butanol when in the presence of highe mounts of butanol. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 10 20 30 40 50 60 70 Butanol [g] Time [min] A) Vesicle Reactor Total 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 20 40 60 80 100 120 140 Butanol [g] Time [min] B) Vesicle Reactor Total amounts of butanol. Dilution Rate (hr-1) Butanol Production Rate (g/hr) Increased Butanol Production Butanol Yield (g butanol / g glycerol) 0.09 1.11 94% 0.12 0.18 0.29 32% 0.07 Figure 4.5: Total butanol produced (g) over time in the reactor, in the solvent and in total for two dilution rates (A - 0.18 hr-1, B - 0.09 hr-1). The DR of 0.09 hr-1 showed almost triple the total butanol production of the DR 0.18 hr-1. A table showing the production of butanol per hour, the percent increase of butanol totals and the butanol yields. Figure 4.5: Total butanol produced (g) over time in the reactor, in the solvent and in total for two Figure 4.5: Total butanol produced (g) over time in the reactor, in the solvent and in total for two dilution rates (A - 0.18 hr-1, B - 0.09 hr-1). The DR of 0.09 hr-1 showed almost triple the total butanol production of the DR 0.18 hr-1. A table showing the production of butanol per hour, the percent increase of butanol totals and the butanol yields. increase of butanol totals and the butanol yields. 4.3 Results and Discussion 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 10 20 30 40 50 60 70 Butanol [g] Time [min] A) Vesicle Reactor Total 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 20 40 60 80 100 120 140 Butanol [g] Time [min] B) Vesicle Reactor Total Dilution Rate (hr-1) Butanol Production Rate (g/hr) Increased Butanol Production Butanol Yield (g butanol / g glycerol 0.09 1.11 94% 0.12 0.18 0.29 32% 0.07 Figure 4.5: Total butanol produced (g) over time in the reactor, in the solvent and in total for two 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 10 20 30 40 50 60 70 Butanol [g] Time [min] A) Vesicle Reactor Total 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 20 40 60 80 100 120 140 Butanol [g] Time [min] B) Vesicle Reactor Total 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 10 20 30 40 50 60 70 Butanol [g] Time [min] A) Vesicle Reactor Total 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 20 40 60 80 100 120 140 Butanol [g] Time [min] B) Vesicle Reactor Total 48 Molar ratios of butanol to lipid for each experiment were calculated and are presented in Figure 4.6. The ratios here are consistent with our previous results, which showed butanol/lipid ratios from 4 to 7 as seen in Chapter 3. Molar ratios of butanol to lipid for each experiment were calculated and are presented in Figure 4.6. The ratios here are consistent with our previous results, which showed butanol/lipid ratios from 4 to 7 as seen in Chapter 3. Figure 4.6: Ratio of moles of butanol to moles of lipid for two dilution rates. 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 140 mol butanol / mol lipid Time [min] 0.09 hr-1 0.18 hr-1 Figure 4.6: Ratio of moles of butanol to moles of lipid for two dilution rates. 4.4 Conclusions Initially, we are seeing that the addition of our lipid bilayer solvent increases the butanol production by 94% with a yield of 0.12 g/g. Of the dilution rates tested, the lower value provided higher butanol production due to the additional time that this allowed the bacteria to grow. More results are needed in order to determine how much of this increased butanol production is due to the extraction or due to the dilution rate’s influence. To our knowledge this is the first continuous fermentation 49 of C. pasteurianum completed and the first time a vesicle solvent has been used to extract butanol. of C. pasteurianum completed and the first time a vesicle solvent has been used to extract butanol. extract butanol. References Atabani, A.E.; Silitonga, A.S.; Badruddin, I.A.; Mahlia, T.M.I.; Masjuki, H.H.; Mekhilef, S. (2012): A comprehensive review on biodiesel as an alternative energy resource and its characteristics. In Renewable and Sustainable Energy Reviews 16 (4), pp. 2070– 2093. DOI: 10.1016/j.rser.2012.01.003. Atabani, A.E.; Silitonga, A.S.; Badruddin, I.A.; Mahlia, T.M.I.; Masjuki, H.H.; Mekhilef, S. (2012): A comprehensive review on biodiesel as an alternative energy resource and its characteristics. 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This is due to the greater hydrophobicity of butanol compared to the others. The butanol partition coefficients showed that the equimolar mixture of DOPC and DPPC are the most effective at extracting butanol (Figure 5.1). This is supported by previous work done in our group showing two phase bilayers create favorable partitioning conditions. We also found our butanol/lipid partition coefficients using NMR which resulted in higher partition coefficients being found compared to HPLC methods. 52 52 Figure 5.1: Mole fraction-based butanol/lipid partition coefficients using an equimolar mixture of DPPC and DOPC. Figure 5.1: Mole fraction-based butanol/lipid partition coefficients using an equimolar mixture of DPPC and DOPC. Our initial fermentation and extraction results show promising increases in butanol production. We also showed that a lower dilution rate produced more butanol (Figure 5.2), which is consistent with previous continuous fermentations in the literature. The inclusion of our lipid bilayer solvent resulted in an increase of 90% in butanol production from the start of the extraction. 53 53 Figure 5.2: A dilution rate of 0.09 provided an increase of 94% in butanol produced. We also see that the amount of butanol in the reactor rises once the extraction begins indicating an increase in production by the C. pasteurianum. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 20 40 60 80 100 120 140 Butanol [g] Time [min] Vesicle Reactor Total Figure 5.2: A dilution rate of 0.09 provided an increase of 94% in butanol produced. We also see that the amount of butanol in the reactor rises once the extraction begins indicating an increase in production by the C pasteurianum Figure 5.2: A dilution rate of 0.09 provided an increase of 94% in butanol produced. We also see that Figure 5.2: A dilution rate of 0.09 provided an increase of 94% in butanol produced. We also see that the amount of butanol in the reactor rises once the extraction begins indicating an increase in production by the C. pasteurianum. 5.2 Future Work The next step that this work should take would be to finish the continuous fermentation tests. This would then reveal how much of the increased butanol production is due to the continuous fermentation process or effectiveness of our vesicle solvent. Also, our future experiments in this area should take place over a longer time frame as some literature showed a rapid increase in butanol production after longer times. 54 Our current extraction method involves bringing the fermentation broth into contact with the vesicle solvent using a hollow fiber membrane. Another method could instead add the vesicles directly to the fermentation media. Finally, a method should be established for the reclamation of butanol from the vesicles after the extraction takes place. A couple of different options exist for breaking apart vesicles. One option would be to dissolve the vesicles by using a surfactant like Triton x-100 or a detergent to release the butanol. 55 55 NMR Background, Procedures and Calibration Curve Nuclear magnetic resonance (NMR) is the phenomenon when nuclei absorb and then emit electromagnetic energy. A magnetic field is applied to the sample which causes the individual spins of the nuclei to align before the magnetic field is turned off and the molecule relaxes. NMR spectroscopy utilizes this phenomenon to collect data. The electromagnetic energy is released upon the molecule relaxing and this resonance is collected as a frequency which is then converted into chemical shift (Darbeau 2006). Nuclear magnetic resonance (NMR) is the phenomenon when nuclei absorb and then emit electromagnetic energy. A magnetic field is applied to the sample which causes the individual spins of the nuclei to align before the magnetic field is turned off and the molecule relaxes. NMR spectroscopy utilizes this phenomenon to collect data. The electromagnetic energy is released upon the molecule relaxing and this resonance is collected as a frequency which is then converted into chemical shift (Darbeau 2006). NMR’s quantitative capabilities are based on the direct proportionality between the intensity generated, Ix, and the number of nuclei, Nx, that correspond to the resonance. There are multiple ways that this relationship can be utilized. One method, involves adding a known amount of a specific substance to a sample and comparing that known amount and its NMR signal to the unknown amount and the corresponding signal. This proportion is related by a spectrometer constant, Ks (Malz, Jancke 2005). The spectrometer constant is dependent on the equipment and the acquisition parameters used. Therefore, to keep Ks constant the acquisition parameters used must be the same for each sample (Bharti, Roy 2012; Malz, Jancke 2005). 𝐼𝑥= 𝐾𝑠𝑁𝑥 𝐼𝑥= 𝐾𝑠𝑁𝑥 56 56 56 A molar ratio can be calculated based on the ratio of two different nmr signals. The spectrometer constant can be canceled out due to each signal appearing within the same spectrum so the parameters must be the same. This leads to the following: A molar ratio can be calculated based on the ratio of two different nmr signals. The spectrometer constant can be canceled out due to each signal appearing within the same spectrum so the parameters must be the same. This leads to the following: 𝑛𝑥 𝑛𝑦 = 𝐼𝑥 𝐼𝑦 𝑁𝑦 𝑁𝑥 𝑛𝑥 𝑛𝑦 = 𝐼𝑥 𝐼𝑦 𝑁𝑦 𝑁𝑥 Molar fractions can be calculated by adding a total summation term for n, I and N. NMR Background, Procedures and Calibration Curve Finally, if the purity of one substance is known, this can be used to calculate the purity associated with the resonance peak of a different substance which is shown here: 𝑃𝑥= 𝐼𝑥 𝐼𝑠𝑡𝑑 𝑁𝑠𝑡𝑑 𝑁𝑥 𝑀𝑥 𝑀𝑠𝑡𝑑 𝑚𝑠𝑡𝑑 𝑚𝑡𝑜𝑡𝑎𝑙 𝑃𝑠𝑡𝑑 𝑃𝑥= 𝐼𝑥 𝐼𝑠𝑡𝑑 𝑁𝑠𝑡𝑑 𝑁𝑥 𝑀𝑥 𝑀𝑠𝑡𝑑 𝑚𝑠𝑡𝑑 𝑚𝑡𝑜𝑡𝑎𝑙 𝑃𝑠𝑡𝑑 Where I is the intensity of the resonance peak, N is the number of nuclei associated with the resonance peak, M is the molecular weight, m is the mass of the sample, and P is the purity. Subscript x is related to the substance of unknown purity while std refers to the qNMR standard used (Malz, Jancke 2005; Bharti, Roy 2012). An example of a spectrum that has undergone this analysis can be seen in Figure A.1. Adding in a standard proved useful for obtaining quantitative information in a setting where multiple chemicals were present in a solution however another approach was taken to quantitatively measure butanol alone in solution. When butanol was the only measurable solute in solution, the concentration of butanol was found by using a calibration curve. The calibration curve was based on the same principle as the previous technique where the intensity of the signal is related to the corresponding 57 protons. The curve was constructed by relating the integral of butanol (as related to the water peak) to a known concentration and fitted using a 3rd degree polynomial. Then, when an unknown butanol signal is measured the concentration could be found in this manner. A sample of a butanol spectrum used to make the calibration curve is included in Figure A.2 and the calibration curve can be seen in Figure A.3. protons. The curve was constructed by relating the integral of butanol (as related to the water peak) to a known concentration and fitted using a 3rd degree polynomial. Then, when an unknown butanol signal is measured the concentration could be found in this manner. A sample of a butanol spectrum used to make the calibration curve is included in Figure A.2 and the calibration curve can be seen in Figure A.3. Quantitative NMR acquisition parameters were set according to this procedure (Pauli et al. 2007). In short, the parameters that needed to be adjusted were as followed: sample spin, 13C satellite removal, relaxation delay time, spectral window, transmitter position, pulse width, acquisition time, number of scans, receiver gain, dummy pulses, 13C decoupling. NMR Background, Procedures and Calibration Curve The goal of changing these parameters was to provide a clearer NMR signal and eliminate potential side peaks from appearing. Quantitative NMR acquisition parameters were set according to this procedure (Pauli et al. 2007). In short, the parameters that needed to be adjusted were as followed: sample spin, 13C satellite removal, relaxation delay time, spectral window, transmitter position, pulse width, acquisition time, number of scans, receiver gain, dummy pulses, 13C decoupling. The goal of changing these parameters was to provide a clearer NMR signal and eliminate potential side peaks from appearing. transmitter position, pulse width, acquisition time, number of scans, receiver gain, dummy pulses, 13C decoupling. The goal of changing these parameters was to provide a clearer NMR signal and eliminate potential side peaks from appearing. 58 58 Figure A.1: Raw NMR data from an extractive fermentation test (Results seen in Chapter 4). This shows where the integrals were taken. Once taken, the absolute values were found in the MestReNova program “Table of Integrals”. This was using an internal calibrant maleic acid (seen at 6.3 ppm). Figure A.1: Raw NMR data from an extractive fermentation test (Results seen in Chapter 4). This shows where the integrals were taken. Once taken, the absolute values were found in the MestReNova program “Table of Integrals”. This was using an internal calibrant maleic acid (seen at 6.3 ppm). 59 59 Figure A.2: One NMR spectrum used in the process of creating a calibration curve. The D2O peak (4.7 ppm) is used to reference to the other butanol peaks (3.5, 1.5-1.2, 0.8 ppm). Figure A.2: One NMR spectrum used in the process of creating a calibration curve. The D2O peak (4 Figure A.2: One NMR spectrum used in the process of creating a calibration curve. The D2O peak (4.7 ppm) is used to reference to the other butanol peaks (3.5, 1.5-1.2, 0.8 ppm). Figure A.2: One NMR spectrum used in the process of creating a calibration curve. The D2O peak (4.7 ppm) is used to reference to the other butanol peaks (3.5, 1.5-1.2, 0.8 ppm). Figure A.1: The calibration curve of butanol from NMR. Each butanol shift was recorded as a peak. Peak 1 – 3.5 ppm, peak 2 – 1.5-1.2 ppm, peak 3 – 0.8 ppm. NMR Background, Procedures and Calibration Curve y = 0.0463x3 - 0.0105x2 + 0.0558x R² = 1 y = 0.006x3 - 0.0034x2 + 0.0284x R² = 0.9999 y = 0.0143x3 - 0.0066x2 + 0.0395x R² = 1 0 0.05 0.1 0.15 0.2 0.25 0 0.5 1 1.5 2 2.5 3 3.5 Butanol Concentration Area Under Peak BtOH Calibration Peak 1 Peak 2 Peak 3 Poly. (Peak 1) Poly. (Peak 2) Poly. (Peak 3) Figure A.1: The calibration curve of butanol from NMR. Each butanol shift was recorded as a peak. Peak 1 – 3.5 ppm, peak 2 – 1.5-1.2 ppm, peak 3 – 0.8 ppm. y = 0.0463x3 - 0.0105x2 + 0.0558x R² = 1 y = 0.006x3 - 0.0034x2 + 0.0284x R² = 0.9999 y = 0.0143x3 - 0.0066x2 + 0.0395x R² = 1 0 0.05 0.1 0.15 0.2 0.25 0 0.5 1 1.5 2 2.5 3 3.5 Butanol Concentration Area Under Peak BtOH Calibration Peak 1 Peak 2 Peak 3 Poly. (Peak 1) Poly. (Peak 2) Poly. (Peak 3) ibration curve of butanol from NMR. Each butanol shift was recorded as a peak. 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https://openalex.org/W3005726478	http://europepmc.org/articles/pmc6781420?pdf=render	English	null	A Enfermagem: diálogo com o passado no compromisso com o presente	null	2,019	cc-by	1,848	Rev. Latino-Am. Enfermagem 2019;27:e3220 DOI: 10.1590/1518-8345.0000-3220 www.eerp.usp.br/rlae Nursing: dialogue with the past in the commitment to the present By the end of 2020, the campaign aims to increase the influence of nurses and maximize their contributions to ensure that everyone has access to health and health care. More information at: http://www.nursingnow.org/ How to cite this article Angerami ELS. Nursing: dialogue with the past in the commitment to the present. Rev. Latino-Am. Enfermagem. 2019;27:e3220. [Access ___ __ ____]; Available in: ___________________ . DOI: http://dx.doi.org/10.1590/1518-8345.0000-3220. year day month URL 1 Universidade de São Paulo, Escola de Enfermagem de Ribeirão Preto, PAHO/WHO Collaborating Centre for Nursing Research Development, Ribeirão Preto, SP, Brazil. * The Nursing Now campaign is an initiative of PAHO/WHO and ICN and aims to train nurses to assume the central role in the confrontation of the health challenges of the 21st century. By the end of 2020, the campaign aims to increase the influence of nurses and maximize their contributions to ensure that everyone has access to health and health care. More information at: http://www.nursingnow.org/ Nursing: dialogue with the past in the commitment to the present Emilia Luigia Saparoti Angerami1 https://orcid.org/0000-0001-9733-4952 Emilia Luigia Saparoti Angerami1 https://orcid.org/0000-0001-9733-4952 In the 1960s, the world went through profound political-social transformations; nursing, in this context, expanded spaces and assumed new tasks. The reduced number of nurses was asked to treat patients, administer wards and train human resources. In the 1960s, the world went through profound political-social transformations; nursing, in this context, expanded spaces and assumed new tasks. The reduced number of nurses was asked to treat patients, administer wards and train human resources. Nursing entered the university, assuming research as a promising element for professional development. The first few studies, of the descriptive type, were important for showing the status quo of the profession, the human capital available and to be trained in relation to the gaps in the services and in education, as well as to be researchers. The problems investigated answered the questions made, contributing to the advancement of knowledge and, above all, to awaken interest in the continuity of research and in the multiplication of the themes under study. In this century, in 2019, the global Nursing Now campaign* of the World Health Organization (WHO), assumed by the international leaders of nursing, puts us in the central position to “reach the goal of universal coverage” of the populations, among other actions. Topics that have long been concerns in my professional life associated with teaching, practice and research in human resources remain present in the face of problems related to the low number of professionals in the services, unjust salaries, bad working conditions, lack of recognition of the work of nurses, difficulty in recruiting nurses and students, staff retention and brain drain. Additionally, gender issues, low wages, violence in the services, overload of the work of women, precariousness of human and material resources and poor working conditions have also been subjects of concern. Nursing entered the university, assuming research as a promising element for professional development. The first few studies, of the descriptive type, were important for showing the status quo of the profession, the human capital available and to be trained in relation to the gaps in the services and in education, as well as to be researchers. The problems investigated answered the questions made, contributing to the advancement of knowledge and, above all, to awaken interest in the continuity of research and in the multiplication of the themes under study. Nursing: dialogue with the past in the commitment to the present Nursing entered the university, assuming research as a promising element for professional development. The first few studies, of the descriptive type, were important for showing the status quo of the profession, the human capital available and to be trained in relation to the gaps in the services and in education, as well as to be researchers. The problems investigated answered the questions made, contributing to the advancement of knowledge and, above all, to awaken interest in the continuity of research and in the multiplication of the themes under study. In this century, in 2019, the global Nursing Now campaign* of the World Health Organization (WHO), assumed by the international leaders of nursing, puts us in the central position to “reach the goal of universal coverage” of the populations, among other actions. Topics that have long been concerns in my professional life associated with teaching, practice and research in human resources remain present in the face of problems In this century, in 2019, the global Nursing Now campaign* of the World Health Organization (WHO), assumed by the international leaders of nursing, puts us in the central position to “reach the goal of universal coverage” of the populations, among other actions. Topics that have long been concerns in my professional life associated with teaching, practice and research in human resources remain present in the face of problems related to the low number of professionals in the services, unjust salaries, bad working conditions, lack of recognition of the work of nurses, difficulty in recruiting nurses and students, staff retention and brain drain. Additionally, gender issues, low wages, violence in the services, overload of the work of women, precariousness of human and material resources and poor working conditions have also been subjects of concern. Two theses of professors from the University of São Paulo’s School of Nursing in Ribeirão Preto were milestones of research in nursing in Brazil and Latin America, and show the concern over the social recognition of the profession. The conclusions of the first thesis(1) show the need for the community to become aware 1 Universidade de São Paulo, Escola de Enfermagem de Ribeirão Preto, PAHO/WHO Collaborating Centre for Nursing Research Development, Ribeirão Preto, SP, Brazil. * The Nursing Now campaign is an initiative of PAHO/WHO and ICN and aims to train nurses to assume the central role in the confrontation of the health challenges of the 21st century. How to cite this article Angerami ELS. Nursing: dialogue with the past in the commitment to the present. Rev. Latino-Am. Enfermagem. 2019;27:e3220. [Access ___ __ ____]; Available in: ___________________ . DOI: http://dx.doi.org/10.1590/1518-8345.0000-3220. year day month URL 2 Rev. Latino-Am. Enfermagem 2019;27:e3220. of the role of nursing; according to the authors, nurses, through their professional performance, should contribute so that this degree of perception is achieved quickly, suggesting that: “nurses are responsible for the construction of their own image”(1). In the second thesis, the researcher, a sociologist, when analyzing nursing as a profession in a teaching hospital, reports the inconsistency and lack of definition of roles among the nursing categories, with the profession being poorly integrated into the social system of hospitals and undervalued in global society(2). The topicality of these results reveals the value of replicating studies from time to time, as while the temporal comparison is valuable, it is possible to go beyond it, reviewing strategies, advances and setbacks, motivations and even making new questions involving persistent themes. After forty years of Alma Ata(3) and considering the profound transformation that took place in health care services with the assumption of the prevention of diseases as main goal, nursing was considered key to achieving the goals proposed by WHO, “Health for all by the year 2000”. The commitment of nurses to achieve this goal was intense, in the organization of services, in government programs and in the training of human resources. In Brazil and throughout Latin America, several programs have been created and their contribution to health remains vigorous. The recent call to nurse leaders to once again assume a central position for the success of the new WHO proposal induces essential reflections, as the current working conditions are considered unsatisfactory and the human and material resources available are insufficient to meet the population that knocks on the doors of health care services and is denied shelter, which in turn creates problems of legal and ethical proportions. Transforming services or changing paradigms presupposes the group’s adherence to the project and acceptance of the empowerment of leadership, not as an attribute imposed on the individual, although its development is feasible. Being a leader implies changes in power relations because it is a process grounded in human relations. How to cite this article In a world in constant transformation, where technology extinguishes and creates professions as if by magic, paradigms are built and deconstructed at the speed of light, making people tense and competitive in the pursuit of purposes, and sometimes incapable of dealing with small slips. In a constantly moving environment, nurses must be well informed and ready to present innovative proposals, maintaining an ethical stance in view of the responsibility assumed by them in the exercise of their profession. In the position of leaders, they are those in charge of the management of situations and with the competence to reduce frictions that can cause uncontrollable crises; the ability of the leader manifests itself especially in conflicts. The respect for the work performed begins at the base of the pyramid, as it is in the you/me relationship, with the recognition of the other as an equal, that the first movement of leadership is revealed. If patients do not know the nurses treating them, they will not know when and how to count on these nurses and their responsibility for the quality of care. Being a leader is a compromise, one that nurses are being asked to assume once more at this time Corresponding author: Emilia Luigia Saparoti Angerami E-mail: elsanger@eerp.usp.br https://orcid.org/0000-0001-9733-4952 Copyright © 2019 Revista Latino-Americana de Enfermagem This is an Open Access article distributed under the terms of the Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon your work, even commercially, as long as they credit you for the original creation. This is the most accommodating of licenses offered. Recommended for maximum dissemination and use of licensed materials. References 1. Alcantara G de. Enfermagem moderna como categoria profissional: obstáculos a sua expansão na sociedade brasileira. Tese [Cátedra]. Ribeirão Preto: Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo; 1963. a-Santos C. A enfermeira como categoria ocupacional num moderno hospital-escola brasileiro. Te do]. Ribeirão Preto: Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo; 1968. 2. Ferreira-Santos C. A enfermeira como categoria ocupacional num moderno hospital-escola brasileiro. Tese [Doutorado]. Ribeirão Preto: Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo; 1968. 3. Organização Pan-americana de Saúde. Organização Mundial de Saúde. Declaração de Alma-Ata. Conferência Internacional sobre Cuidados Primários em Saúde. URSS, 6-12 set 1978. Disponível em: https://apps.who.int/iris/ bitstream/handle/10665/39228/9241800011_por.pdf;jsessionid=75CCB6D051D08CE03D0BB3152D09872E?sequen 2. Ferreira-Santos C. A enfermeira como categoria ocupacional num moderno hospital-escola brasileiro. Tese [Doutorado]. Ribeirão Preto: Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo; 1968. 3 Organização Pan americana de Saúde Organização Mundial de Saúde Declaração de Alma Ata Conferência [Doutorado]. Ribeirão Preto: Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo; 1968. 3. Organização Pan-americana de Saúde. Organização Mundial de Saúde. Declaração de Alma-Ata. Conferência Internacional sobre Cuidados Primários em Saúde. URSS, 6-12 set 1978. Disponível em: https://apps.who.int/iris/ bitstream/handle/10665/39228/9241800011_por.pdf;jsessionid=75CCB6D051D08CE03D0BB3152D09872E?sequen ce=5. Acesso: 27 maio 2019. www.eerp.usp.br/rlae
https://openalex.org/W1577045840	https://www.repository.cam.ac.uk/bitstreams/2be82b7e-4d3c-4e28-9c10-32697df174f5/download	English	null	Fine-scale precipitation in the high-entropy alloy Al0.5CrFeCoNiCu	Materials science and engineering. A, Structural materials: proporties, microstructures and processing/Materials science & engineering. A, Structural materials: properties, microstructure and processing	2,015	cc-by	5,604	1. Introduction site results in reduced rates of substitutional diffusion [4]. The phase equilibria and properties of the AlxCrFeCoNiCu sys- tem have been widely investigated [1,5–11], with Al0.5CrFeCoNiCu receiving particular attention [12–18]. The original investigation of Al0.5CrFeCoNiCu reported the presence of two face-centred cubic (fcc, A1 in Strukturbericht notation) phases below the solidus temperature in the as-cast condition: one constituting the den- dritic material and the other the interdendritic material. At lower temperatures, the dendritic fcc phase was reported to order fully to an L12 structure [12]. The majority of subsequent studies have conﬁrmed the presence of a miscibility gap in the fcc solid solution below the solidus temperature [13–15], but the nature of the L12 formation is disputed. Some studies have suggested that no fcc ordering is involved and that the L12 phase forms directly below the solidus at the same time as the two fcc phases [14]. In contrast, other studies have demonstrated that the L12 phase only pre- cipitates in the dendritic fcc phase after cooling to lower tem- peratures, i.e., that the fcc ordering is partial [15,16]. One of the most recent Al0.5CrFeCoNiCu studies identiﬁed only one fcc phase alongside an L12 phase in melt-spun material [18], in agreement with the full fcc L12 → ordering proposed originally. Reports have also identiﬁed a Ni–Al-based B2 phase and a Cr–Co-Fe-based s phase in the alloy, following prolonged exposures at temperatures in the range 700–1000 °C [14,16]. High-entropy alloys (HEAs) are commonly deﬁned as materials that contain ﬁve or more elements in near-equiatomic ratios [1]. Such alloys have been referred to as ‘high-entropy’ because, in contrast to conventional alloys, their multi-principal component compositions theoretically deliver signiﬁcantly higher entropies of mixing. It was initially proposed that this high mixing entropy may be sufﬁcient to stabilise solid solutions in preference to in- termetallic compounds, which might otherwise be expected to form [1]. However, a recent study has suggested that the like- lihood of formation of strongly bonded intermetallic compounds increases with the number of alloying elements [2]. Judicious al- loying is therefore required if intermetallic phase formation is to be avoided. Importantly, it has been suggested that HEAs comprising compositionally complex solid solutions may possess superior mechanical properties and enhanced phase stability, compared to their conventional counterparts. This has been attributed to the local distortions of the crystal caused by the presence of elements with different atomic radii. a r t i c l e i n f o Article history: Received 27 April 2015 Received in revised form 24 July 2015 Accepted 2 August 2015 Available online 4 August 2015 Keywords: High-entropy alloys Precipitation Kinetics Phase transformations Electron microscopy Article history: Received 27 April 2015 Received in revised form 24 July 2015 Accepted 2 August 2015 Available online 4 August 2015 Keywords: High-entropy alloys Precipitation Kinetics Phase transformations Electron microscopy The high-entropy alloy Al0.5CrFeCoNiCu has been shown to consist of two stable, face-centred cubic solid solutions at temperatures approaching its solidus: one rich in Cr, Fe, Co & Ni (dendritic) and the other rich in Cu (interdendritic). While some studies have suggested that the high-temperature microstructure may be metastably retained to room temperature through rapid cooling, evidence of phase decom- position has also been reported. In this study, ﬁne-scale precipitation has been observed in samples of Al0.5CrFeCoNiCu that have been rapidly cooled after casting, and water quenched following ageing for 1000 h at 1000 °C. Contrary to previous reports, in the as-cast state, the two face-centred cubic phases, as well as an L12 phase, were found in both dendritic and interdendritic areas, with the dendritic areas having undergone a spinodal decomposition. After ageing and quenching, L12 precipitates were found in both dendritic and interdendritic areas, and precipitates of the Cr-, Fe-, Co- and Ni-enriched face-centred cubic phase were found in the Cu-rich interdendritic regions. Given the nature of the heat treatments applied, the results suggest that precipitation in the alloy is rapid and cannot be avoided, even when the material is cooled quickly to room temperature. q y p & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). site results in reduced rates of substitutional diffusion [4]. Fine-scale precipitation in the high-entropy alloy Al0.5CrFeCoNiCu E.J. Pickering n, H.J. Stone, N.G. Jones Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, UK Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, UK Contents lists available at ScienceDirect Contents lists available at ScienceDirect n Corresponding author. Fax: þ44 1223 334567. E-mail address: ejp57@cam.ac.uk (E.J. Pickering). Materials Science & Engineering A 645 (2015) 65–71 Materials Science & Engineering A 645 (2015) 65–71 http://dx.doi.org/10.1016/j.msea.2015.08.010 0921-5093/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ea.2015.08.010 ors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). http://dx.doi.org/10.1016/j.msea.2015.08.010 0921-5093/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://crea herent mixture of A2 and B2 phases, accompanied by precip http://dx.doi.org/10.1016/j.msea.2015.08.010 0921-5093/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E-mail address: ejp57@cam.ac.uk (E.J. Pickering). 2. Experimental An ingot of Al0.5CrFeCoNiCu weighing 40 g was prepared via arc melting in an inert atmosphere from elemental metals with 99.95% purity. To increase compositional homogeneity, the ingot was inverted and remelted ﬁve times. A short section, approxi- mately 10 mm long, was cut from the as-cast ingot, encapsulated in an evacuated argon-backﬁlled quartz ampoule, and heat treated at 1000 °C for 1000 h, before quenching in water [15]. Specimens for transmission-electron microscopy (TEM) were prepared from both as-cast and heat-treated material by mechanically thinning slices to below 100 m μ , spark eroding 3 mm discs, and ion-milling. The interdendritic constituent ion milled more readily than the dendritic material, and many interfaces between the two con- stituents were located in electron-transparent regions. Bright- and dark-ﬁeld TEM images were taken using an FEI Tecnai Osiris mi- croscope, using an accelerating voltage of 200 kV. The same mi- croscope was also used for scanning-TEM (STEM) imaging, using a high-angle annular dark-ﬁeld (HAADF) detector, as well as energy- dispersive X-ray mapping in STEM mode (STEM-EDX). X-ray dif- fraction (XRD) was carried out on a Bruker D8 ADVANCE instru- ment in Bragg–Brentano geometry using Cu Kα radiation. Data was collected between angles of 20 and 120° 2θ using a step size of 0.03° and a counting time of 3 s per step. Analyses of the resulting spectra were carried out in Wavemetrics Igor Pro and the peaks ﬁtted with Gaussian functions that accounted for both K 1 α and K 2 α radiation. The interface between the dendritic and the interdendritic re- gion shown in Fig. 1a was studied using HAADF STEM imaging, with the material oriented along a 100 〈 〉zone axis, Fig. 2a. The selected-area diffraction patterns (SADPs) from this orientation, Fig. 2b and c, showed that the intensities of the L12 reﬂections were lower in the interdendritic material. This difference was more pronounced than in the diffraction patterns taken from the 114 〈 〉orientation, Fig. 1b and c. STEM-EDX mapping was carried out on the area marked with a box in Fig. 2d, with the resulting elemental maps shown at the bottom of Fig. 2. The STEM-EDX mapping revealed a nano-scale spinodal mi- crostructure in the dendritic material, which is shown at higher magniﬁcation in Fig. 3. 2. Experimental The EDX data indicated that the two components of the spinodal were rich in Cr, Co, Fe & Ni, and rich in Cu & Al, respectively. Cuboidal particles were also observed in the Cu-rich component, which were consistent in scale and mor- phology with the L12 precipitates seen in Fig. 1d. These particles were enriched in Ni & Al in comparison to the matrix and ex- hibited some solubility for Cu. The precipitates present in the interdendritic material with rectangular cross-sections are located at the bottom of the EDX maps in Fig. 2. They were found to be rich in Cr, Co, Fe & Ni. This evidence, and their absence in superlattice dark-ﬁeld imaging, suggested that they possessed an fcc crystal structure similar to that of the matrix. Also observed were the ﬁne L12 precipitates identiﬁed in the dark-ﬁeld image in Fig. 1d. These precipitates were enriched in Ni & Al, and showed some solubility for Cu, Co & Fe, but did not exhibit any enrichment in Cr. 1. Introduction These distortions have been hypothe- sised to impede dislocation motion and atomic diffusion [1,3,4]. In addition, it has also been suggested that the variation in bonding potential energy experienced by an atom in moving from site to Studies of AlxCrFeCoNiCu alloys with higher aluminium con- tents ( x 1 ≥ ) have identiﬁed the occurrence of spinodal decom- position and ordering of the dendritic constituent to form a co- herent mixture of A2 and B2 phases, accompanied by precipitation E.J. Pickering et al. / Materials Science & Engineering A 645 (2015) 65–71 66 of Cu-rich particles [12,8,11]. In this study, high-resolution elec- tron microscopy has been used to show, for the ﬁrst time, that a spinodal decomposition also occurs in the dendritic constituent of Al0.5CrFeCoNiCu, leading to the formation of two fcc phases, fol- lowing rapid cooling from the molten state. In addition, nano-scale fcc precipitates were observed in the interdendritic regions in the as-cast state, as well as after ageing and quenching. L12 pre- cipitates were also observed, consistent with previous reports [15,16]. Notably, the L12 precipitates were not uniquely conﬁned to either the dendritic or interdendritic areas, but were identiﬁed in both regions in both the as-cast state and after ageing and quenching. interdendritic materials is shown in the bright-ﬁeld image in Fig. 1a, in which the dendritic and interdendritic regions are la- belled 1 and 2, respectively. The interdendritic material did not appear to be homogeneous and contained precipitates that had rectangular cross sections, which seemed to have similar crystal- lographic orientations. These precipitates are indicated with ar- rows in Fig. 1a. Electron diffraction patterns from both the den- dritic and interdendritic regions are shown in Fig. 1b and c, re- spectively. Both patterns contained superlattice reﬂections con- sistent with the presence of an L12 phase, and showed that the dendritic and interdendritic regions had the same crystallographic orientation. Dark-ﬁeld imaging, using the reﬂection indicated in Fig. 1b and c, enabled the L12 phase present in both regions to be examined using the same objective aperture location. The result- ing image is shown in Fig. 1d, and conﬁrmed the existence of L12 precipitates and an fcc matrix in both the dendritic and inter- dendritic regions, with the L12 precipitates appearing larger in the dendritic material. In the interdendritic material the larger rec- tangular-cross-sectioned precipitates did not appear bright in dark-ﬁeld images using the superlattice reﬂections. 3.2. 1000 °C 1000 h Interestingly, these precipitates were observed in the dendritic material in the as-cast state, not in the interdendritic material. It was noted that the intensity of the L12 reﬂections was diminished in the dendritic region (1), Fig. 4b. This may be indicative of a lower volume fraction of the L12 phase, but it could also have resulted from changes in the scattering contrast of the constituent elements, or have been a consequence of size-induced peak broadening. The large precipitates indicated in Fig. 4a appeared dark in dark-ﬁeld imaging. A similar interface between dendritic and interdendritic re- gions was also analysed from material heat treated for 1000 h at 1000 °C. Fig. 4 shows the results of TEM, STEM-HAADF and STEM- EDX investigations, with the dendritic and interdendritic regions labelled 1 and 2, respectively. Bright-ﬁeld TEM revealed relatively large precipitates in both the dendritic and the interdendritic material, labelled (i) and (ii) in Fig. 4a. The edges of the pre- cipitates in the dendritic material appeared diffuse, while the precipitates present in the interdendritic material were more nu- merous and generally had a more deﬁned shape. SADPs from both dendritic and interdendritic regions comprised predominantly fcc reﬂections, and demonstrated that both regions were very closely oriented, Fig. 4b and c. L12 superlattice reﬂections were again observed in the SADPs, and dark-ﬁeld images were formed using the reﬂections marked by the arrows in Fig. 4b and c. The resulting dark-ﬁeld image is shown in Fig. 4d and revealed extensive L12 precipitation in both dendritic and interdendritic areas, with the L12 precipitates in the interdendritic material appearing coarser. These coarse L12 precipitates were of a similar size to those ob- served in the Cu-rich spinodal material in the as-cast specimen. A STEM-HAADF image of the same interface is shown in Fig. 4e. EDX mapping was performed on the sub-region indicated by the box, and the elemental concentration maps obtained are pre- sented at the end of Fig. 4. These revealed that the dendritic ma- terial was enriched in Cr, Co, Fe & Ni, and the interdendritic ma- terial was Cu-rich. Consistent with the results obtained from the as-cast material, the L12 precipitates in the dendritic material were enriched in Ni, Al & Cu relative to the matrix, while the L12 pre- cipitates in the Cu-rich interdendritic material were enriched in Ni & Al. 3.1. As-cast material A region containing an interface between dendritic and 200 nm 200 nm 1 2 2 1 Fig. 1. TEM images and electron diffraction patterns from the as-cast material. (a) Bright-ﬁeld image showing dendritic material (top, labelled 1) and interdendritic material (bottom, labelled 2). Precipitates with rectangular cross-sections are arrowed. (b) and (c) show 114 〈 〉selected-area diffraction patterns (SADPs) from dendritic (1) and interdendritic (2) materials, respectively. (d) Dark-ﬁeld image formed using the superlattice reﬂection indicated in the SADPs. 200 nm 200 nm 1 2 1 2 2 200 nm Fig. 1. TEM images and electron diffraction patterns from the as-cast material. (a) Bright-ﬁeld image showing dendritic material (top, labelled 1) and interdendritic material (bottom, labelled 2). Precipitates with rectangular cross-sections are arrowed. (b) and (c) show 114 〈 〉selected-area diffraction patterns (SADPs) from dendritic (1) and interdendritic (2) materials, respectively. (d) Dark-ﬁeld image formed using the superlattice reﬂection indicated in the SADPs. 67 E.J. Pickering et al. / Materials Science & Engineering A 645 (2015) 65–71 200 nm 1 2 Fe Al Ni Cr Co Cu 2 1 Fig. 2. The results of STEM imaging and STEM-EDX mapping of as-cast Al0.5CrFeCoNiCu. (a) HAADF image of the region examined, with dendritic and interdendritic regions identiﬁed by 1 and 2, respectively. (b) and (c) 100 〈 〉SADPs from dendritic (1) and interdendritic (2) materials, respectively. STEM-EDX maps for Cr, Co, Fe, Al, Ni & Cu, using the region indicated by the box in (a), are shown at the bottom of the ﬁgure. 200 nm 1 2 200 nm Co Cr Fe Cr Cu Ni Al Ni Cu Al Fig. 2. The results of STEM imaging and STEM-EDX mapping of as-cast Al0.5CrFeCoNiCu. (a) HAADF image of the region examined, with dendritic and interdendritic regions identiﬁed by 1 and 2, respectively. (b) and (c) 100 〈 〉SADPs from dendritic (1) and interdendritic (2) materials, respectively. STEM-EDX maps for Cr, Co, Fe, Al, Ni & Cu, using the region indicated by the box in (a), are shown at the bottom of the ﬁgure. 4. Discussion The precipitation sequence for the aged material in Fig. 5 does not include the large Cu- and Ni-rich precipitates that were ob- served. These have been omitted as it is unclear at what point in the sequence they formed, and because their crystal structures have not been determined. Given their enrichment in fcc-forming elements, it is likely that the precipitates are also fcc, but this was not conﬁrmed. The results of this study conﬁrm that two fcc phases are the predominant constituents in Al0.5CrFeCoNiCu in both the as-cast state, and after ageing at 1000 °C for 1000 h followed by quenching. One is enriched in Cr, Co, Fe & Ni, which will subse- quently be referred to as fcc1, while the other is enriched in Cu, with some solubility for Ni & Al, fcc2. Similar fcc phases have been identiﬁed in many of the previous Al0.5CrFeCoNiCu studies [7,13– 15] and the results obtained in this study are consistent with there being two stable fcc phases at temperatures immediately below the solidus, fcc1 and fcc2 [15,16]. On cooling from the solidus in the as-cast state, our ﬁndings suggest that fcc1, which is likely to have some solubility for Cu at high temperatures, spinodally de- composes into fcc1 and fcc2 at lower temperatures, relieving any Cu supersaturation. At lower temperatures still, the solubility of Ni & Al in fcc2 decreases and, as a result, a Ni- & Al-rich L12 phase precipitates. This occurs in the fcc2 present in both the spinodally decomposed dendritic material and in the interdendritic regions. The solubility of Cr, Co & Fe is also reduced in the interdendritic fcc2 at low temperatures, which leads to the precipitation of fcc1 as precipitates with rectangular cross-sections. This sequence is A previous study reported that the L12 precipitates found in aged Al0.5CrFeCoNiCu were conﬁned to the dendritic material [15]. Here, the use of high-resolution electron microscopy has demon- strated that the L12 precipitates are, in fact, formed in both den- dritic and interdendritic materials, in the as-cast state as well as the aged condition. The same study also observed small deviations around 1000 °C in differential-scanning calorimetry thermograms for both as-cast and aged material, which could not be explained. In light of the results of this study, it is possible that these de- viations were due to the dissolution of a second population of L12 precipitates in the interdendritic regions. 3.2. 1000 °C 1000 h These interdendritic L12 precipitates showed some solubility for Co & Fe, but no detectable Cr. Another phase was also observed to be present in the interdendritic material, in the form of Cr-, Co- & Fe-rich precipitates that were smaller than the L12 particles. One 50 nm Fe Al Ni Cr Co Cu Fig. 3. STEM-EDX elemental composition maps of the dendritic material in the as-cast state, showing a spinodally decomposed structure, with L12 phase precipitation in the Cu-rich component. E.J. Pickering et al. / Materials Science & Engineering A 645 (2015) 65–71 68 68 E.J. Pickering et al. / Materials Science & Engineering A 645 (2015) 65–71 Cr Co Fe Cr Al Al Ni Cu maps of the dendritic material in the as-cast state, showing a spinodally decomposed structure, with L12 phase precipitation in the Fig. 3. STEM-EDX elemental composition maps of the dendritic material in the as-cast state, showing a spinodally decomposed str Cu-rich component. Fig. 3. STEM-EDX elemental composition maps of the dendritic material in the as-cast state, showing a spinodally decomposed structure, with L12 phase precipitation in the Cu-rich component. outlined in Fig. 5a. The size of the precipitates observed was consistent with the high cooling rate that would have been ex- perienced by the as-cast material as a result of rapid cooling on a water-chilled copper hearth. of these precipitates is circled in the Cr, Co & Fe elemental maps. These small precipitates were not visible in bright-ﬁeld imaging, and they did not appear bright in the dark-ﬁeld images formed using the L12 superlattice reﬂections. These observations were consistent with the precipitates having an fcc structure. In the material aged at 1000 °C for 1000 h, experimental evi- dence suggests that the same two fcc phases are likely to have been stable at high temperature. However, the composition of both phases was altered subtly on ageing [12,13,15,16]. This ap- pears to have eliminated the driving force for the spinodal de- composition of the dendritic material on cooling. However, this resulted in fcc1 retaining signiﬁcant levels of Cu, Ni & Al, and these elements subsequently formed L12 precipitates at lower tem- peratures. In the interdendritic Cu-rich fcc2, a reduction in tem- perature again led to decreased solubility for the other alloying elements, resulting in the formation of L12 precipitates and fcc1. This sequence is shown in Fig. 5b. 3.2. 1000 °C 1000 h Importantly, the size of the precipitates was found to be consistent with the rapid cooling rates provided by water quenching. STEM-EDX mapping revealed that the large precipitates ob- served in the bright-ﬁeld images, labelled (i) and (ii) in Fig. 4a and e, were not the same in the dendritic and the interdendritic re- gions. The precipitates in the dendritic area, labelled (i), appeared to be Ni-rich, while the more well-deﬁned precipitates in the in- terdendritic material, labelled (ii), were enriched in Cu. SADPs from regions containing these precipitates showed extra diffrac- tion spots, indicating the presence of other phases besides the fcc and L12 phases described above. However, no information was gathered that could further elucidate their crystal structures. 4. Discussion An alternative possibility is that these deviations were caused by the dissolution of fcc precipitates that had formed in their counterpart fcc phase, for instance the circled precipitates in Fig. 4. A separate study of 69 E.J. Pickering et al. / Materials Science & Engineering A 645 (2015) 65–71 200 nm 200 nm 200 nm 1 (ii) (i) 2 2 1 Fe Al Ni Cr Co Cu (ii) (i) (ii) (i) ld TEM image of a region containing an interface between dendritic (1) and interdendritic (2) materials following exposure at 1 e large precipitates in the dendritic and the interdendritic material are labelled (i) and (ii), respectively. (b) and (c) show 110 〈 ( ) i l (d) k ﬁld i f h h i ( ) f d i h l i ﬂ i d i (b) 200 nm 1 (ii) (i) 2 1 1 2 2 200 nm 200 nm 200 nm 200 nm Fe Al Ni Cr Co Cu (ii) (i) (ii) (i) Fig. 4. (a) Bright-ﬁeld TEM image of a region containing an interface between dendritic (1) and interdendritic (2) materials following exposure at 1000 °C for 1000 h and water quenching. The large precipitates in the dendritic and the interdendritic material are labelled (i) and (ii), respectively. (b) and (c) show 110 〈 〉SADPs from dendritic (1) and interdendritic (2) areas, respectively. (d) Dark-ﬁeld image of the area shown in (a) formed using the superlattice reﬂection arrowed in (b) and (c). (e) HAADF-STEM image of the area shown in (a). EDX maps, from the sub-region indicated by the box in (e), are shown at the bottom of the ﬁgure. The large precipitates labelled (i) and (ii) are indicated. An example of a small precipitate enriched in Cr, Co & Fe in the interdendritic area is circled. Cr Co Cr Fe Ni (i) Al Ni Cu Fig. 4. (a) Bright-ﬁeld TEM image of a region containing an interface between dendritic (1) and interdendritic (2) materials following exposure at 1000 °C for 1000 h and water quenching. The large precipitates in the dendritic and the interdendritic material are labelled (i) and (ii), respectively. (b) and (c) show 110 〈 〉SADPs from dendritic (1) and interdendritic (2) areas, respectively. (d) Dark-ﬁeld image of the area shown in (a) formed using the superlattice reﬂection arrowed in (b) and (c). (e) HAADF-STEM image of the area shown in (a). 4. Discussion EDX maps, from the sub-region indicated by the box in (e), are shown at the bottom of the ﬁgure. The large precipitates labelled (i) and (ii) are indicated. An example of a small precipitate enriched in Cr, Co & Fe in the interdendritic area is circled. Al0.5CrFeCoNiCu has reported the presence of ﬁne Cu-rich acicular precipitates in dendritic material following cooling from the liquid state at rates between 10 and 50 °C min1 [17]. The results of the present study suggest that these precipitates were in fact part of the fcc fcc fcc 1 1 2 → + spinodal decomposition, and that the scale of this spinodal decomposition increases with decreasing cooling rate. Critically, the results of the present study are in contradiction with the ﬁndings of the original Al0.5CrFeCoNiCu investigation [12], and also with one of the most recent studies of the alloy [18]. E.J. Pickering et al. / Materials Science & Engineering A 645 (2015) 65–71 70 As-Cast Dendritic FCC1 FCC1 FCC1 FCC2 FCC2 + L12 FCC2 + L12 + FCC1 FCC2 + L12 + FCC1 FCC1 + L12 FCC2 + L12 + FCC1 FCC1 + L12 FCC2 + L12 + FCC1 FCC2 + L12 FCC2 FCC1 FCC2 Aged Interdendritic T T Decreasing temperature Dendritic Interdendritic Fig. 5. A schematic representation of the precipitation sequence in Al0.5CrFeCoNiCu on cooling from casting, and during water quenching after exposure for 1000 h at 1000 °C. strongly resemble the results of spinodal decomposition seen in as-cast material here. As-Cast Dendritic FCC1 FCC1 FCC1 FCC2 FCC2 + L12 FCC2 + L12 + FCC1 FCC2 + L12 + FCC1 FCC2 + L12 FCC2 Interdendritic FCC2 + L12 + FCC1 FCC1 + L12 FCC2 + L12 + FCC1 FCC1 + L12 FCC1 FCC2 Aged T T Decreasing temperature Dendritic Interdendritic Dendritic In explaining the results of Al0.5CrFeCoNiCu investigations, authors have often referred to the precipitation kinetics as being slow [12,14], following the belief that diffusion in HEAs is ‘slug- gish’ [1,3,4]. However, the same studies present results that sug- gest quite the opposite; precipitation and spinodal structures are observed in as-cast AlxCrFeCoNiCu alloys, which are not consistent with slow kinetics [12,15,17,18]. The precipitation, and spinodal decomposition, observed in the alloy during rapid cooling in this study provides evidence that kinetics are not slow or ‘sluggish’. 4. Discussion Indeed, the L12 precipitates observed here are of the same scale that would be expected to form in Ni-based superalloys quenched from above their γ’ solvus temperatures, suggesting that the pre- cipitation characteristics are similar to existing alloys. Fig. 5. A schematic representation of the precipitation sequence in Al0.5CrFeCoNiCu on cooling from casting, and during water quenching after exposure for 1000 h at 1000 °C. Fig. 6. XRD data showing the 311 { } reﬂection of Al0.5CrFeCoNiCu after heat treatment at 1000 °C for 1000 h and water quenching. The peak was ﬁtted (bold line) using three Gaussian functions (thin lines below data) corresponding to the two fcc and the L12 phases. The K 1 α and K 2 α contributions were accounted for in all cases. 5. Summary and conclusions The microstructure of Al0.5CrFeCoNiCu has been characterised using a combination of high-resolution electron microscopy techniques. Samples were studied in the as-cast state, and after ageing for 1000 h at 1000 °C followed by water quenching. In the as-cast state, Al0.5CrFeCoNiCu was found to comprise two fcc phases and an L12 phase. One fcc phase was rich in Cr, Co, Fe & Ni, while the other was Cu rich. The L12 phase was rich in Ni & Al, with some solubility for Cu. The two fcc phases and the L12 phase were all present in both the dendritic and the interdendritic ma- terial. In the dendritic regions, the two fcc phases appeared to have formed through a spinodal decomposition. After ageing for 1000 h at 1000 °C followed by water quench- ing, L12 precipitates were found in both the dendritic and inter- dendritic areas, with precipitates of the fcc phase rich in Cr, Co, Fe & Ni also appearing in the Cu-rich interdendritic material. Fine- scale precipitation was prevalent in both as-cast and aged material after rapid cooling, demonstrating that the precipitation kinetics were not slow, as is often claimed for HEAs. Fig. 6. XRD data showing the 311 { } reﬂection of Al0.5CrFeCoNiCu after heat treatment at 1000 °C for 1000 h and water quenching. The peak was ﬁtted (bold line) using three Gaussian functions (thin lines below data) corresponding to the two fcc and the L12 phases. The K 1 α and K 2 α contributions were accounted for in all cases. Acknowledgements Both studies reported the existence of only one Cu-rich fcc phase, consistent with fcc2, and an L12 phase rich in Cr, Co, Fe & Ni in the material at low temperatures. These ﬁndings are surprising for two reasons. First, it is unusual to ﬁnd an L12 phase rich in Cr, Co, Fe & Ni, as the elements are known to form stable fcc solid solu- tions in their respective ternary systems [19,20]. Second, the XRD patterns that are presented by the more recent study show pro- nounced asymmetry on a number of fcc peaks [18], which could be consistent with the presence of another fcc phase. XRD spectra from the two material conditions examined in the present manuscript were reported in a previous study, and showed evi- dence of at least two contributions to each fcc reﬂection [16]. Here, using a similar analysis approach with data obtained from a higher-resolution instrument, contributions from at least three phases were identiﬁed in the high-angle reﬂections, where peak separation is greatest. By way of example, the measured data and associated ﬁt for the 311 { } reﬂections from a sample in the heat treated condition are shown in Fig. 6. The ﬁt comprises three Gaussian functions, individually shown below the experimental data, which correspond to the two fcc phases and the L12 phases identiﬁed in Fig. 4. Unfortunately, the XRD patterns presented by the original study are not clear enough to discern whether asymmetry is present or not [12], but it is clear that in dark-ﬁeld imaging the dendritic areas did not appear to comprise solely an L12 phase (Fig. 5 in [12]). Indeed, the dark features in the ﬁgure The authors acknowledge funding from Rolls-Royce plc and the EPSRC under the Rolls-Royce/EPSRC Strategic Partnership (EP/ M005607/1). Requests for access to the underlying research data should be directed to the corresponding author, and will be con- sidered against commercial interests and data protection. This paper can also be viewed at https://www.repository.cam. ac.uk/handle/1810/249206. [6] M.-R. Chen, S.-J. Lin, J.-W. Yeh, S.-K. Chen, Y.-S. Huang, M.-H. Chuang, Effect of vanadium addition on the microstructure, hardness, and wear resistance of Al0.5CoCrCuFeNi high-entropy alloy, Metal. Mater. Trans. A 37A (2006) References [1] J.-W. Yeh, S.-K. Chen, S.-J. Lin, J.-Y. Gan, T.-S. Chin, T.-T. Shun, C.-H. Tsau, S.- Y. Chang, Nanostructured high-entropy alloys with multiple principal ele- ments: novel alloy design concepts and outcomes, Adv. Eng. Mater. 6 (2004) 299–303. [2] O. Senkov, J. Miller, D. Miracle, C. 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https://openalex.org/W4309516558	https://www.nature.com/articles/s41598-022-22026-1.pdf	English	null	CA125 as a predictor of endometrial cancer lymphovascular space invasion and lymph node metastasis for risk stratification in the preoperative setting	Scientific reports	2,022	cc-by	5,494	(2022) 12:19783 \| https://doi.org/10.1038/s41598-022-22026-1 CA125 as a predictor of endometrial cancer lymphovascular space inva and lymph node metastasi stratification in the preope setting K. Shawn LyBarger1,2, Hunter A. Miller3 & Hermann B. Frieboes3,4,5, Endometrial cancer (EC) is the most common malignancy of the female repr antigen 125 (CA125) is a serum tumor marker widely reported in EC patients poor prognostic factors such as grade 3 tumors, deep myometrial invasion, (LNM), and extra-uterine disease. This retrospective study stratifies pre-ope evaluate odds ratios (OR) and relative risk (RR) between CA125 levels and th lymphovascular space invasion (LVSI), grade, and stage. Patient charts for w with a diagnosis of EC and pre-operative or within one week CA125 measure to January 2015 at a regional hospital were reviewed. OR and RR were deter maximum likelihood estimation for CA125 levels as the predictor with stagin and +LNM as outcomes. The largest increase in risk for patients having stage greater (1.52-fold risk) while largest increase in risk for patients having stag greater (1.67-fold risk), both at CA125 ≥ 222U/ml. Patients with CA125 ≥ 122 increased risk of +LNM, with maximum increase in risk of 98% (1.98-fold risk with CA125 ≥ 175U/ml had significantly increased risk of +LVSI, with maximu (1.39-fold risk) at 222U/ml. This study shows that elevated CA125 levels corr stage, +LVSI, and +LNM in patients with EC. Abbreviations RR Relative risk CA125 Cancer antigen 125 EC Endometrial cancer FIGO International Federation of Gynecology and Obstetrics MI Myometrial invasion DVT Deep vein thrombosis LVSI Lymphovascular space invasion +LVSI Positive LVSI diagnosis LNM Lymph node metastasis +LNM Positive LNM diagnosis PEN 1Sarah Cannon Cancer Institute, HCA MidAmerica, HCA Midwest Health, USA. 2Department of Obstetrics and Gynecology, University of Missouri, Kansas of Bioengineering, University of Louisville, Louisville, KY, USA. 4Department of P University of Louisville, Louisville, KY, USA. 5UofL Health – Brown Cancer Cen Louisville, KY, USA. 6Center for Predictive Medicine, University of Louisville, kristopher.lybarger@hcahealthcare.com www.nature.com/scientificreports www.nature.com/scientificreports CA125 as a predictor of endometrial cancer lymphovascular space invasion and lymph node metastasis for risk stratification in the preoperative setting OPEN K. Shawn LyBarger1,2, Hunter A. Miller3 & Hermann B. Frieboes3,4,5,6 Endometrial cancer (EC) is the most common malignancy of the female reproductive system. Cancer antigen 125 (CA125) is a serum tumor marker widely reported in EC patients, particularly those with poor prognostic factors such as grade 3 tumors, deep myometrial invasion, lymph node metastasis (LNM), and extra-uterine disease. This retrospective study stratifies pre-operative CA125 levels to evaluate odds ratios (OR) and relative risk (RR) between CA125 levels and the likelihood of +LNM, lymphovascular space invasion (LVSI), grade, and stage. Patient charts for women 18 years or older with a diagnosis of EC and pre-operative or within one week CA125 measurement from January 2000 to January 2015 at a regional hospital were reviewed. OR and RR were determined by unconditional maximum likelihood estimation for CA125 levels as the predictor with staging, grade, +LVSI and +LNM as outcomes. The largest increase in risk for patients having stage I/II/III disease was 52% greater (1.52-fold risk) while largest increase in risk for patients having stage III/IV disease was 67% greater (1.67-fold risk), both at CA125 ≥ 222U/ml. Patients with CA125 ≥ 122U/ml had significantly increased risk of +LNM, with maximum increase in risk of 98% (1.98-fold risk) at 222U/ml. Patients with CA125 ≥ 175U/ml had significantly increased risk of +LVSI, with maximum increase in risk of 39% (1.39-fold risk) at 222U/ml. This study shows that elevated CA125 levels correspond to increased stage, +LVSI, and +LNM in patients with EC. Abbreviations RR Relative risk CA125 Cancer antigen 125 EC Endometrial cancer FIGO International Federation of Gynecology and Obstetrics MI Myometrial invasion DVT Deep vein thrombosis LVSI Lymphovascular space invasion +LVSI Positive LVSI diagnosis LNM Lymph node metastasis +LNM Positive LNM diagnosis 1Sarah Cannon Cancer Institute, HCA MidAmerica, HCA Midwest Health, Kansas City, MO 64132, USA. 2Department of Obstetrics and Gynecology, University of Missouri, Kansas City, MO, USA. 3Department of Bioengineering, University of Louisville, Louisville, KY, USA. 4Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA. 5UofL Health – Brown Cancer Center, University of Louisville, Louisville, KY, USA. 6Center for Predictive Medicine, University of Louisville, Louisville, KY, USA. email: kristopher.lybarger@hcahealthcare.com www.nature.com/scientificreports/ Endometrial cancer (EC) is the most common malignancy of the female reproductive system in developed countries. According to the American Cancer Society, approximately 66,570 new cases of cancer of the body of the uterus were diagnosed in 2021, and almost 12,940 women will die from the disease1. A rising incidence of EC has been reported partly due to the obesity epidemic in the United States2. Consequently, the number of women diagnosed with EC is expected to significantly increase in coming years.hi Endometrial cancer (EC) is the most common malignancy of the female reproductive system in developed countries. According to the American Cancer Society, approximately 66,570 new cases of cancer of the body of the uterus were diagnosed in 2021, and almost 12,940 women will die from the disease1. A rising incidence of EC has been reported partly due to the obesity epidemic in the United States2. Consequently, the number of women diagnosed with EC is expected to significantly increase in coming years.hi g gi y g y The identification of those at highest risk of recurrence is vital in treatment planning. Since 1998 the Inter- national Federation of Gynecology and Obstetrics (FIGO)3 has undergone several iterations of the surgical staging of EC. In an effort to achieve postoperative pathological staging, the primary surgical procedure for patients with all stages is a total hysterectomy with bilateral salpingo-oophorectomy and some form of lymph node sampling, whether sentinel lymph nodes or systematic lymphadenectomy. The addition of pelvic and or para-aortic lymph node dissection depending on various factors, such as tumor size, grade, subtype, myometrial invasion (MI), lymphovascular space invasion (LVSI), cervical involvement, and extrauterine metastasis is also performed4,5. Despite this, extensive lymphadenectomy has been linked to an increased incidence of surgical related complication such as lymphedema, deep vein thrombosis (DVT), intraoperative hemorrhage, and lym- phocyst formation5. Further, lymph node sampling does not necessarily improve survival6. Several studies have argued whether a lymphadenectomy should be performed in patients with tumor size < 2 cm, tumor grade 1 or 2, endometrioid subtype, and < 50% myometrial invasion7, since the chance of lymph node metastasis (LNM) is < 5%8. While routine lymphadenectomy does not improve disease-free or overall survival, positive lymph nodes do aid in the prognosis and management of the disease. www.nature.com/scientificreports/ Knowledge of patients with a higher risk of lymph node involvement preoperatively would be beneficial when planning for surgery and discussing prognosis and surgical procedure with patients.h g p p The use of cancer antigen 125 (CA125) as a predictor of endometrial cancer lymphovascular space invasion and lymph node metastasis remains understudied. CA125 is a serum tumor marker widely used in epithelial ovarian cancers9. Ever since it was found that serum CA125 levels increased with the clinical course of some malignancies10, its use as a tumor marker has been reported in EC patients, particularly those with poor prognos- tic factors such recurrences, grade 3 tumors, deep myometrial invasion, LNM, and extrauterine disease. Previous work has evaluated preoperative serum CA125 levels as a predictor of metastatic disease and survival11,12. While studies have attempted to stratify CA125 utility, its clinical value in the evaluation of EC remains ambiguous since it can be mildly elevated in other non-malignant conditions13,14. Its use either in combination with imaging or alone may be able to help predict or stratify patients in a preoperative setting. Further, the marker is considered partially helpful in evaluating patients with significant comorbidities and perioperative risk who may not be candidates for comprehensive staging15. Additionally, CA125 has been incorporated into several preoperative predictive models with varying degrees of success. Clinically, CA125 can be a sensitive marker when using a cutoff of 35U/ml since only around 1% of healthy women have greater levels16. Furthermore, 6% of women with benign disease and 23% of women with no gynecological cancers have levels ≥ 35U/ml16, with elevated levels in over 80% of women with non-mucinous ovarian cancer. Ideally, to minimize overtreatment and complications following a lymphadenectomy in EC patients, risk factors for + LNM could be used to distinguish patients more likely to benefit from lymphadenectomy versus those who would not. Therefore, the purpose of this study was to identify odds ratios (OR) and relative risks (RR) between pre-operative CA125 levels and the likelihood of + LNM, + LVSI, high grade, and advanced stage. \| https://doi.org/10.1038/s41598-022-22026-1 Scientific Reports \| (2022) 12:19783 www.nature.com/scientificreports/ Results A total of 890 patients with a diagnosis of endometrial cancer were included in the primary analysis, out of which 413 had pre-operative staging information, 414 had pre-operative grade information, 449 had LVSI information, and 411 had LNM information (Fig. 1). Patient statistics are summarized in Table 1.hi g Median and average CA125 levels across all groups is shown in Fig. 2. There is a significant increase in both median and average CA125 for + LNM and + LVSI. Additionally, CA125 generally increased as stage and grade increased.i RR and OR trends between CA125 levels and all patient classifications are shown in Figs. 3 and 4, respectively. Generally, RR and OR at a CA125 threshold below 35U/ml was associated with a better outcome (0 ≤ RR < 1, 0 ≤ OR < 1) and above 35U/ml was associated with a worse outcome (RR > 1, OR > 1). RR and OR tended to increase above a CA125 threshold of 35U/ml for all patient classifications.ii i RR and OR values that were found to be statistically significant are shown for all patient classifications in Fig. 5 The trends between OR and RR were the same. Patients having a CA125 of 9U/ml, 10U/ml, or 12U/ml had a decrease in risk of + LNM by 87, 88, and 72% respectively (0.13 fold, 0.12 fold, and 0.28 fold risk). This was the only decrease in risk that was significant. The largest increase in risk for patients having stage II/III/ IV disease was 52% greater (1.52 fold risk) than for stage I while the largest increase in risk for patients having stage III/IV disease was 66% greater (1.66 fold risk), both at a CA125 level of 222U/ml or greater. The lowest CA125 threshold showing a significantly increased risk for stage II/III/IV (compared to stage I) and stage III/IV (compared to stage I/II) was 117U/ml and 120U/ml, respectively. Patients with a CA125 of 122U/ml or greater had a significantly increased risk of + LNM, with a maximum increase in the risk of 97% (1.97 fold risk) at a CA125 of 222U/ml. Patients with a CA125 of 199 U/ml or greater had a significantly increased risk of + LVSI, with a maximum increase in the risk of 38% (1.38 fold risk) at a CA125 of 222U/ml. No significant differences were found with respect to grade. Scientific Reports \| (2022) 12:19783 \| https://doi.org/10.1038/s41598-022-22026-1 www.nature.com/scientificreports/ Figure 1. Results Classification of patient population based on outcome measures. Patients within each group are not mutually exclusive, with 342 patients having information available for each of the four groups. LVSI = lymphovascular space invasion; LNM = lymph node metastasis. Figure 1. Classification of patient population based on outcome measures. Patients within each group are not mutually exclusive, with 342 patients having information available for each of the four groups. LVSI = lymphovascular space invasion; LNM = lymph node metastasis. Table 1. Summary statistics of patient population. LVSI Lymphovascular space invasion, LNM Lymph node metastasis. 95% CI is shown in parentheses for Average Age at Diagnosis, Median CA125, and Average CA125. Patient population (n = 890) With staging information CA125 ≤ 35 CA125 > 35 Average age at diagnosis Median CA125 (U/ml) Average CA125 (U/ml) Total 413 (46.4% of patients) 340 (82.3%) 73 (17.7%) 62.4 (61.3–63.5) 12 (11–13) 66.0 (39.22–101.79) Stage I 307 (34.5%) 289 (94.1%) 18 (5.9%) 61.5 (60.2–62.8) 10 (9–11) 15.8 (13.1–19.3) Stage II 23 (2.6%) 16 (69.6%) 7 (30.4%) 68.5 (63.9–73.0) 13 (7–33) 24.2 (14.8–33.6) Stage III 56 (6.3%) 27 (48.2%) 29 (51.8%) 63.4 (60.2–66.6) 38.5 (23.0–58.5) 109.6 (67.2–159.5) Stage IV 27 (3.0%) 8 (29.6%) 19 (70.4%) 65.1 (61.6–68.7) 98 (37–175) 582.3 (197.9–1069.2) With LVSI information Total 449 (50.5% of patients) 365 (81.3%) 84 (18.7%) 62.8 (61.7–63.8) 12 (11–13) 66.6 (39.5–99.83) −LVSI 341 (76.0%) 307 (90.0%) 34 (10.0%) 62.3 (61.19–63.5) 11 (9–11) 31.3 (19.6–45.9) +LVSI 108 (24.0%) 58 (53.7%) 50 (46.3%) 64.2 (61.9–66.4) 32.5 (24–40) 178.1 (81.1–316.8) With LNM information Total 411 (46.2% of patients) 341 (83.0%) 70 (17.0%) 62.7 (61.6–63.7) 12 (11–13) 53.3 (34.9–76.9) −LNM 364 (88.6%) 324 (89.0%) 40 (11.0%) 62.6 (61.5–63.8) 11 (10–12) 36.6 (21.4–58.9) +LNM 47 (11.4%) 17 (36.2%) 30 (63.8%) 63.1 (59.8–66.4) 55 (34–83) 184.4 (97.8–297.4) With grade information Total 414 (46.5% of patients) 341 (82.5%) 73 (17.5%) 62.7 (61.7–63.8) 12 (11–13) 62.5 (36.43–99.7) Grade 1 281 (67.8%) 253 (90.0%) 28 (10.0%) 62.1 (60.8–63.3) 11 (10–12) 23.5 (17.3–31.3) Grade 2 62 (15.0%) 52 (83.9%) 10 (16.1%) 63.8 (60.7–66.8) 12 (9.0–13.5) 30.2 (17.8–45.7) Grade 3 71 (17.1%) 36 (50.7%) 35 (49.3%) 64.7 (62.1–67.4) 34 (20–52) 244.9 (104.9–432.6) Table 1. Summary statistics of patient population. LVSI Lymphovascular space invasion, LNM Lymph node metastasis. 95% CI is shown in parentheses for Average Age at Diagnosis, Median CA125, and Average CA125. Discussion i fi di Main findings. It is well known that improved preoperative risk stratification for EC is crucial due to the increased number of cases in the United States as well as the morbidity and mortality in high risk and advanced- stage disease. In the current analysis, we demonstrate that an elevated preoperative CA125 is associated with an increased risk of elevated stage, + LVSI, + LNM, and higher grade, all of which are associated with less favora- ble outcomes. In particular, this study demonstrates that a CA125 level of ≥ 222U/ml is associated with a 1.66 (1.14–2.44 95%CI) fold risk of having stage III/IV disease. Since a proportion of EC patients are first identified and some incidentally treated by general gynecologists, being able to risk-stratify patients preoperatively into low-risk versus high-risk is of utmost importance to ensure not only accurate patient referral to gynecologic oncologists but also to select appropriate surgical procedures. https://doi.org/10.1038/s41598-022-22026-1 Scientific Reports \| (2022) 12:19783 \| www.nature.com/scientificreports/ Figure 2. Median CA125 (U/ml) (left) and average CA125 (U/ml) (right) for all patient classifications. LVSI = lymphovascular space invasion; LNM = lymph node metastasis. Bars represent 95% CI. For medians, a Wilcoxon rank-sum test was used for statistical significance. For averages, a student’s T-test was used. p ≤ 0.05; p ≤ 0.01; p ≤ 0.001. Figure 2. Median CA125 (U/ml) (left) and average CA125 (U/ml) (right) for all patient classifications. LVSI = lymphovascular space invasion; LNM = lymph node metastasis. Bars represent 95% CI. For medians, a Wilcoxon rank-sum test was used for statistical significance. For averages, a student’s T-test was used. p ≤ 0.05; p ≤ 0.01; *p ≤ 0.001. Figure 3. Relative risk (RR) for all CA125 thresholds from 1 to 222. RR for baseline CA125 of 35U/ml is equal to 1.0. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Figure 3. Relative risk (RR) for all CA125 thresholds from 1 to 222. RR for baseline CA125 of 35U/ml is equal to 1.0. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Scientific Reports \| (2022) 12:19783 \| https://doi.org/10.1038/s41598-022-22026-1 www.nature.com/scientificreports/ Figure 4. Odds ratio (OR) for all CA125 thresholds from 1 to 222. Methods d d Study design subjects and settings. This is a large single-center retrospective chart review of 890 patients diagnosed and surgically staged for EC between January 2000 to January 2015 at WellSpan York Hos- pital. Female patients over the age of 18 years old with a diagnosis of endometrial cancer and a pre-operative or within one week CA125 level were eligible for inclusion. All research was performed in accordance with the relevant institutional guidelines and regulations. Data collection. Data were abstracted using the PowerChart and E-care electronic medical records systems. Collected data included patient age at diagnosis, preoperative CA125 value, the procedure performed (total, subtotal or radical hysterectomy), both preoperative as well as postoperative histology, cancer grade (1/2/3), positive/negative lymph nodes, positive/negative lymphovascular invasion, pathologic stage (I/II/III/IV), pelvic/ abdominal metastasis, race/ethnicity, physical exam findings, radiological imaging, and laboratory findings. The reference range of CA 125 is 0–35 units/mL (0–35 kU/L). The cutoff of 35 kU/L for CA 125 was determined from the distribution of values in healthy individuals to include 99% of the normal population23. CA 125 meas- urements were obtained with electrochemiluminescence Elecsys CA 125 II assay (Roche, Indianapolis, IN) and quantified by Labcorp (Burlington, NC). Currently, the standard method for assessing LVSI is light microscopic examination of haematoxylin and eosin (H&E) stained sections as the presence of tumor emboli within vascular channels lined by endothelial cells. The majority of patients had either endometroid adenocarcinoma (75.2%), serous (5.5%), mucinous (1.3%), clear cell (1.3%), mixed cell (4.2%) or carcinosarcoma (3.0%) histology, while the remainder had other histology subtypes (9.5%). Patients were subjected to either systematic lymphadenec- tomy or sentinel node sampling as determined by the operating physician in accordance with standard clinical protocol. Both pre-operative and post-operative pathology were analyzed by the same clinical oncology/pathol- ogy team. Exclusion criteria. Patients with a historical diagnosis of endometrial cancer, those who did not have a documented CA1-25 level, pregnant women, or those who had been previously staged surgically were excluded from the study. Outcome variables. The primary outcome was to determine OR and RR between a preoperative CA125 level and the likelihood of lymph node metastases, lymphovascular invasion, advanced stage, and high grade. Based on CA125 levels, a secondary outcome was to identify CA125 ranges that correspond to these variables. Statistical analysis. www.nature.com/scientificreports/ elevated in the preoperative workup, it may be able to predict the need for further imaging, tests or biopsies prior to surgery, or better prepare patients for potential procedures to be incurred intraoperatively in the event of advanced malignancies.i elevated in the preoperative workup, it may be able to predict the need for further imaging, tests or biopsies prior to surgery, or better prepare patients for potential procedures to be incurred intraoperatively in the event of advanced malignancies.i g Limitations of this study include a single-center retrospective study and lack of evaluation of a specific age associated with CA125. In addition, since elevated CA125 levels can occur in various benign gynecologic condi- tions and other disease states, such as leiomyoma and endometriosis, elevated CA125 may not be an ideal serum biomarker in premenopausal patients. Interpretation. Endometrial cancer is staged according to well defined and evidence-based surgical stag- ing protocols, as clinical staging is less accurate and cannot assess LVSI, grade, or LNM. Proper surgical staging including lymph node assessment is crucial, especially in those with high-risk preoperative features to provide the best prognostic information. Previous studies have examined the predictive accuracy of high-risk features in endometrial cancer and serum markers such as CA125. Levels of CA125 ≥ 21.2 U/mL predicted + LVSI in women with EC in a study by Zhou et al.17. Logistic regression models were used in Yang et al.18 and Anton et al.19 to predict + LNM in EC patients. CA125 was found to be useful for predicting high-risk patients, includ- ing + LNM, by Zamani et al.20. Preoperative CA125 levels were significantly associated with deep myometrial invasion, advanced stage, and extrauterine and node metastasis in a study by Chen et al.21. Our study confirmed similar findings with a CA125 level of ≥ 222U/ml having up to a 38% (1.38 fold risk, 1.03–1.84 95%CI) increase in the risk of LVSI. Also, using the same level gives a significantly increased risk of + LNM with the maximum increase in the risk of 98% (1.98 fold risk, 1.14–3.43 95% CI) for a CA125 of ≥ 222U/ml. These data are further supported by the study by Antonsen et al.22, showing CA125 as a strong predictor for the presence of + LNM. Conclusion Our study was able to stratify CA125 levels with respect to increases in stage, + LVSI, + LNM, and grade in patients with EC. Further, we were able to quantify specific values at which the risk for the presence of high-risk features in the preoperative patient with EC could be utilized to better risk-stratify patients before surgery. These data could also be used to minimize potential postoperative risks in patients who are poor surgical candidates. Evaluation of CA125 in relation to other potential clinical variables to enable a multivariable analysis will be considered in follow-on work. Discussion i fi di OR for baseline CA125 of 35U/ml is equal to 1.0. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Figure 4. Odds ratio (OR) for all CA125 thresholds from 1 to 222. OR for baseline CA125 of 35U/ml is equal to 1.0. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Figure 5. Relative risk (RR) (A) and odds ratio (OR) (B) of significant (p ≤ 0.05) CA125 thresholds. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Figure 5. Relative risk (RR) (A) and odds ratio (OR) (B) of significant (p ≤ 0.05) CA125 thresholds. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Figure 5. Relative risk (RR) (A) and odds ratio (OR) (B) of significant (p ≤ 0.05) CA125 thresholds. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Figure 5. Relative risk (RR) (A) and odds ratio (OR) (B) of significant (p ≤ 0.05) CA125 thresholds. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Bars represent 95% CI. Strengths and limitations. In contrast to previous work, this study stratifies specific CA125 ranges at which there is an increase in relative risk of having a more advanced stage, higher grade, + LNM, and + LVSI. In this way, patients can be better risk-stratified in the preoperative period, allowing for more informed discussions regarding treatment options and having the improved knowledge of these risks during surgery. Previous studies have found that patients with deep myometrial invasion, + LVSI, or lymph node metastasis can have significantly elevated CA125. As such, CA125 levels are a marker in both preoperative and recurrent settings. If CA125 is https://doi.org/10.1038/s41598-022-22026-1 Scientific Reports \| (2022) 12:19783 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ tionship between patient groups and outcomes26. For CA125 averages and medians, 95% confidence intervals were produced with a non-parametric bootstrap method using boot function from the boot package where the number of resampling iterations was 1000 and the statistic was the average or median, respectively. P values for comparing medians and means were calculated using Wilcoxon rank-sum test and unpaired student’s T-test, respectively. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. tionship between patient groups and outcomes26. For CA125 averages and medians, 95% confidence intervals were produced with a non-parametric bootstrap method using boot function from the boot package where the number of resampling iterations was 1000 and the statistic was the average or median, respectively. P values for comparing medians and means were calculated using Wilcoxon rank-sum test and unpaired student’s T-test, respectively. P values for OR and RR were calculated with Fisher’s exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation. Data classification. OR and RR were determined for CA125 levels as the predictor with staging, grade, + LVSI, and + LNM as outcomes. All RR and OR values were calculated relative to a CA125 level of 35U/ ml or below being considered normal. Staging was subclassified as follows: stage I versus stage II/III/IV; stage I/II versus stage III/IV; stage I/II/III versus stage IV. Grade was subclassified as follows: grade 1 versus grade 2/3; grade 1/2 versus grade 3. CA125 thresholds were incremented by 1U/ml from 1 to 222 with OR and RR re- calculated at every threshold. A CA125 level of 35 U/ml was used as a baseline, as values at or below this level are considered normal. Ethics approval. The study was approved by Institutional Review Board (IRB) at York Hospital Human Subjects Research Protection Program Office (IRB number 731831-1). The study was exempt from informed consent. References 1. ACS. Key Statistics for Endometrial Cancer. Available from: https://www.cancer.org/cancer/endometrial-cancer/about/key-stati stics.html (2021). 2. Panyavaranant, P. & Manchana, T. Preoperative markers for the prediction of high-risk features in endometrial cancer. World J Clin. Oncol. 11(6), 378–388 (2020).h 3. Mutch, D. G. The new FIGO staging system for cancers of the vulva, cervix, endometrium and sarcomas. Gynecol. Oncol. 115(2) 325–328 (2009). 4. Denny, L. & Quinn, M. FIGO cancer report 2015. Int. J. Gynaecol. Obstet. 131(Suppl 2), S75 (2015). y p y pp 5. Volpi, L. et al. Long term complications following pelvic and para-aortic lymphadenectomy for endometrial cancer, incidenc potential risk factors: A single institution experience. Int. J. Gynecol. Cancer 29(2), 312–319 (2019). b Th l f l h d l f d l l potential risk factors: A single institution experience. Int. J. Gynecol. Cancer 29(2), 312–319 (2019). 6. Neubauer, N. 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CA 125: A useful marker in endometrial carc 10. Duk, J. M. et al. CA 125: A useful marker in endometrial carcinoma. Am. J. Obstet. Gynecol. 155(5), 1097–1102 (1986). 11. Ross, M. S. et al. Cancer antigen 125 is associated with disease status in uterine carcinosarcoma. Rare Tumors 11, 203636131 ( ) y 11. Ross, M. S. et al. Cancer antigen 125 is associated with disease status in uterine carcinosarcoma. Rare Tumors 11, 20363613198 (2019). 12. Goksedef, B. P. C. et al. Data availabilityh y The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. Received: 1 June 2022; Accepted: 7 October 2022 References Preoperative serum CA 125 level as a predictor for metastasis and survival in endometrioid endometrial cancer. J. Obstet. Gynaecol. Can. 33(8), 844–850 (2011). 13. Dong, C., Liu, P. & Li, C. Value of HE4 combined with cancer antigen 125 in the diagnosis of endometrial cancer. Pak. J. Med. Sci. 33(4), 1013–1017 (2017).i 4. 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A novel model to estimate lymph node metastasis in endometrial cancer patients. Clinics 72(1), 30–35 (2017 20. Zamani, N. et al. The utility of CA125 and HE4 in patients suffering from endometrial cancer. Int. J. Women’s Health Reprod 19. Anton, C. et al. A novel model to estimate lymph node metast 19. Anton, C. et al. A novel model to estimate lymph node metastasis in endometrial cancer patients. Clinics 72(1), 30 35 (2017). 20. Zamani, N. et al. The utility of CA125 and HE4 in patients suffering from endometrial cancer. Int. J. Women’s Health Reprod. Sci. 8(1), 95–100 (2020). 21. Chen, Y. L. et al. Value of pre-operative serum CA125 level for prediction of prognosis in patients with endometrial cancer. Aust. N. Z. J. Obstet. Gynaecol. 51(5), 397–402 (2011). 2. Antonsen, S. L. et al. HE4 and CA125 levels in the preoperative assessment of endometrial cancer patients: A prospective multi center study (ENDOMET). Acta Obstet. Gynecol. Scand. 92(11), 1313–1322 (2013).i y ( ) y ( ) ( ) 23. Davelaar, E. M. et al. Comparison of seven immunoassays for the quantification of CA 125 antigen in serum. Clin. Chem. 44(7), 1417–1422 (1998).h n, T. R. The R Project for Statistical Computing. Methods d d Data analyses were conducted in the R programming language (version 4.0.2)24, with scripts that utilized functions from the following R packages: Odds ratios (OR) and relative risk (RR) were calculated by unconditional maximum likelihood estimation (Wald) using oddsratio and riskratio functions from epitools package25. In this retrospective study, both RR and OR were calculated to improve data interpreta- tion and avoid exaggerated estimates, as OR has the possibility to overestimate event odds when there is a rela- https://doi.org/10.1038/s41598-022-22026-1 Scientific Reports \| (2022) 12:19783 \| www.nature.com/scientificreports/ Acknowledgements h f ll k l g Authors gratefully acknowledge Dr. Timothy McGuiness (formerly at Gynecologic Oncology, WellSpan Health, York, PA) for assistance with the clinical program coordination. Competing interests h The authors declare no competing interests. Author contributions Clinical data collection and initial assessment: K.S.L. Clinical program coordination: K.S.L. Odds ratios and relative risk analysis: H.A.M. Development of manuscript structure and data presentation: K.S.L., H.A.M., H.B.F. Writing and revision of the manuscript: K.S.L., H.A.M., H.B.F. Additional information Correspondence and requests for materials should be addressed to K.S.L. Correspondence and requests for materials should be addressed to K.S.L. References Available from: ww h j f p g p j g 5. Aragon, T. J. CRAN—Package epitools. Available from: https://cran.r-project.org/web/packages/epitools/index.html (2021). h 25. Aragon, T. J. CRAN—Package epitools. Available from: https://cran.r-project.org/web/packages/ 25. Aragon, T. J. CRAN—Package epitools. Available from: https://cran.r-project.org/web/packages/epitools/index.html (2021). 26. Ranganathan, P., Aggarwal, R. & Pramesh, C. S. Common pitfalls in statistical analysis: Odds versus risk. Perspect. Clin. Res. 6(4), 25. Aragon, T. J. CRAN Package epitools. Available from: https://cran.r project.org/web/packages/epitools/index.html (2021). 26. Ranganathan, P., Aggarwal, R. & Pramesh, C. S. Common pitfalls in statistical analysis: Odds versus risk. Perspect. Clin. Res. 6(4), 222–224 (2015). 6. Ranganathan, P., Aggarwal, R. & Pramesh, C. S. Common pitfalls in statistical analysis: Odds versus risk. Perspect. Clin. Res. 6(4) 222–224 (2015). Scientific Reports \| (2022) 12:19783 \| https://doi.org/10.1038/s41598-022-22026-1 www.nature.com/scientificreports/ © The Author(s) 2022 Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 https://doi.org/10.1038/s41598-022-22026-1 Scientific Reports \| (2022) 12:19783 \|
https://openalex.org/W4310114260	https://dmsjournal.biomedcentral.com/counter/pdf/10.1186/s13098-022-00959-x	English	null	Hypoglycemia unawareness identified by continuous glucose monitoring system is frequent in outpatients with type 2 diabetes without receiving intensive therapeutic interventions	Diabetology & metabolic syndrome	2,022	cc-by	9,149	Abstract Background: Patients with diabetes are prone to asymptomatic hypoglycemia (AH) due to diminished ability to perceive the onset of hypoglycemia. However, the actual prevalence and influencing factors of AH in outpatients with type 2 diabetes (T2DM) have not been well investigated. Methods: A total of 351 outpatients with T2DM underwent glucose monitoring by continuous glucose monitor- ing system (CGMS) for consecutive 72 h without changing their lifestyle and treatment regimens. Hypoglycemia is defined as a blood glucose level less than 3.9 mmol/L, which was further divided into Level 1 hypoglycemia (blood glucose 3.0–3.9 mmol/L) and Level 2 hypoglycemia (blood glucose < 3.0 mmol/L). Univariate and multivariate logistic regression analyses were used to determine the possible risk factors of AH. Results: In all 351 subjects studied, 137 outpatients (39.0%) were captured AH events, in which Level 1 AH and Level 2 AH accounted for 61.3% and 38.7%, respectively. 85 (62.0%) of the AH patients experienced nocturnal asympto- matic hypoglycemia (NAH) and 25 (18.2%) exclusively NAH. Multivariate logistic regression analysis demonstrated that patients with younger age, lower hemoglobin A1c (HbA1c), and higher systolic blood pressure (SBP) levels were associated with increased risk of AH. While after further grading of AH, male sex and Dipeptidylpeptidase-4 inhibitors (DPP4i) regime were shown to be associated with lower risk of Level 2 AH. †Bingkun Huang and Qiuhui Jiang contributed equally to this work and share first authorship †Bingkun Huang and Qiuhui Jiang contributed equally to this work and share first authorship Correspondence: shixiulin2002@163.com; xmlixuejun@163.com 1 Department of Endocrinology and Diabetes, Xiamen Diabetes Institute, Xiamen Clinical Medical Center for Endocrine and Metabolic Diseases, Xiamen Diabetes Prevention and Treatment Center, Fujian Key Laboratory of Diabetes Translational Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China Full list of author information is available at the end of the article Hypoglycemia unawareness identified by continuous glucose monitoring system is frequent in outpatients with type 2 diabetes without receiving intensive therapeutic interventions Bingkun Huang1†, Qiuhui Jiang1,2†, Ting Wu1,2, Qingbao Shen1, Wengui Wang1, Shoubi Wang1,3, Yinxiang Huang1, Shunhua Wang1, Peiying Huang1, Mingzhu Lin1, Xiulin Shi1 and Xuejun Li1* Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 https://doi.org/10.1186/s13098-022-00959-x Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 https://doi.org/10.1186/s13098-022-00959-x Diabetology & Metabolic Syndrome Open Access Introduction Hypoglycemia is well-recognized as one of the most severe complications and therapy-limited factors of patients with diabetes (reviewed by Amiel SA et al.) [1]. It has been reported that hypoglycemia could not only cause unpleasant symptoms, impaired cognitive func- tion, diminished productivity, but also result in long- term chronic vascular complications of diabetes and affect the lifespan [2]. Theoretically, when hypoglyce- mia occurs, counter-regulatory systems are normally activated and patients with diabetes tend to develop the characteristic neuroglycopenic and adrenergic symptoms of hypoglycemia, which is regarded as a protective mech- anism [3, 4]. Whereas, in clinical practice, there are quite a few patients experiencing hypoglycemia episodes with- out any sensed symptoms, which is defined as asympto- matic hypoglycemia (AH). AH accounted for 36% of the hypoglycemia events that occurred while subjects were awake, according to the Diabetes Control and Compli- cations Trial (DCCT) [4]. Studies by Gold et al. [5] and Akram et al. [6] have revealed that being the most criti- cal risk factor for severe hypoglycemia, impaired percep- tion of hypoglycemia could lead to a six-fold increase in the frequency of severe hypoglycemia, thus significantly increasing the potential risks of severe adverse events [7]. Additionally, antecedent hypoglycemia attacks could shift glycemic thresholds for autonomic and sympto- matic responses to lower plasma glucose concentrations and further aggravate impaired awareness of hypogly- cemia, with evidence that avoiding such exposure could restore consciousness and terminate the vicious cycle of recurrent hypoglycemia [8–10]. Nocturnal asymptomatic hypoglycemia (NAH), a significant problem noted in the AH population, is more likely to be ignored because patients do not monitor their blood glucose (BG) while sleeping, which accounted for 55% of severe hypoglyce- mic events in the DCCT [4]. It has been reported that NAH could cause life-threatening events, such as “dead in bed” syndrome [10, 11]. g g Rates and influencing factors of AH have been widely reported in type 1 diabetes (T1DM) [17–19] and T2DM [20, 21] patients with intensive treatment who are prone to hypoglycemia. It is generally assumed that the risk of developing hypoglycemia in daily diabetes outpatients is relatively lower because their medical conditions are relatively stable. A small sample size research (31 partici- pants) reported that 83.1% (69 out of 83) hypoglycemia episodes were unrecognized in outpatients with T2DM [16]. Another study in 222 outpatients with T2DM dem- onstrated that the AH was frequently seen [22]. Introduction Given the high prevalence, potential and prolonged harms, and insidious onset of AH, estimating the actual rate and promptly identifying episodes of AH have important implications for diabetes care. In view of the insufficient attention to T2DM outpatients without receiving inten- sive therapeutic interventions, our study enrolled such an entity and aimed to determine the influencing factors of AH in T2DM outpatients via CGMS. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 10 Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 Conclusions: Hypoglycemia unawareness could be frequently observed at either daytime or nighttime, although NAH was more common, in outpatients with T2DM. Relative relax HbA1c targets should be considered for patients who are prone to AH. Keywords: Hypoglycemia unawareness, Asymptomatic hypoglycemia, Continuous glucose monitoring system, Type 2 diabetes high proportion of AH, including 62.5% of T1DM patients and 46.6% of T2DM patients. Data recorded via 72-h CGMS [14] revealed that the incidence of NAH in T2DM patients was twofold higher than that of daytime hypoglycemia. Similarly, a 1-year prospective study of 350 insulin-dependent children with diabetes revealed that 56% of severe hypoglycemic episodes occurred at night [15]. McNally et al. [14, 16] noted the unrecognized hypoglycemia in patients with T2DM and took neces- sary treatment adjustments, significantly reducing the frequency of hypoglycemia. Thereby, concerning the reli- ability and accuracy, it is considerable for CGMS to be brought into the routine management of T2DM. Research design and methods Study designh This study included 351 outpatients with T2DM in the First Affiliated Hospital of Xiamen University from Jan- uary 2018 to March 2022. Exclusions included children (< 18 years), pregnant women, and patients who had the following: perioperative, bleeding disorders, cog- nitive dysfunction, alcohol abuse, taking beta-block- ers or selective serotonin reuptake inhibitors (SSRIs), and incomplete data due to dropped probe. The stud- ies involving human participants were reviewed and approved by The First Affiliated Hospital of Xiamen The continuous glucose monitoring system (CGMS) is rapidly improving diabetes management and has been proved to be superior to daily self-monitoring blood glu- cose (SMBG) in the detection of hypoglycemia and in improving glycemic control in T2DM individuals [12]. By using CGMS, Chico et al. [13] detected a substantially Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 Page 3 of 10 Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 University. Enrolled patients completed the informed consent and wore the CGMS device at the outpatient clinic. In order to reflect the effectiveness and safety of the current treatment regimen of the subjects, we did not ask them to change their previous diet and exercise habits during the CGM test. Thus, they went home to lead a normal life for three consecutive days. Clinical data of baseline characteristics, such as Glycated hemo- globin A1c (HbA1c), BMI (calculated as weight in kilograms divided by the square of height in meters), diabetes duration, age, diabetes medications, complica- tions, and diabetic complications, were collected. CGM (continuous glucose monitoring) test ran for three days on average, and data were obtained through iPro™ ver- sion 2 (Medtronic Minimed, MMT-7745WW). The glu- cose value is recorded every 5 min, for a total of 288 readings a day. Before the CGM test, we provided uni- fied blood glucose meters (Contour Plus Blood Glucose Meter, 7600P) to each patient and instructed them to provide at least four self-monitoring blood glucose (SMBG) tests per day. Besides, notebooks were pro- vided to each patient to record the frequency and time of suspicious hypoglycemia if they had symptoms such as hunger, palpitations, tremulousness, sweats, and dizziness. After three days of monitoring, patients returned to the outpatient clinic, where their SMBG metrics were imputed into the system and the CGM data was downloaded. Definition of hypoglycemiai i yp g y According to the definition of the International Hypoglycemia Study Group [1], hypoglycemia is defined as a blood glucose level less than 3.9 mmol/L, which was further divided into Level 1 hypoglyce- mia (BG 3.0–3.9 mmol/L) and Level 2 hypoglycemia (BG < 3.0 mmol/L) according to the minimum blood glu- cose value. AH was defined as BG < 3.9 mmol/L without any sensed symptoms. Daytime asymptomatic hypogly- cemia (DAH) was defined as BG < 3.9 mmol/L during 6:00–24:00. NAH was defined as BG < 3.9 mmol/L during 0:00–6:00. A single hypoglycemic episode was defined as BG below 3.9 mmol/L for at least 15 min [23]. Two sepa- rate hypoglycemic events were defined when the time interval between two hypoglycemic events was equal to or greater than 30 min. Research design and methods Study designh The CGM data recorded in this article included the glucose standard deviation (SD), mean blood glucose (MBG), the mean amplitude of glycemic excursions (MAGE), time in range (TIR, 3.9– 10.0 mmol/L), time below range (TBR, < 3.9 mmol/L), time above range (TAR, > 10.0 mmol/L), the number of hypoglycemic events, and the corresponding duration of hypoglycemic episodes. Statistical analysis Preliminary statistical analyses included descriptive sta- tistics and assessment of distributions. Variables are pre- sented as the mean ± standard deviation, median with interquartile range or percentages where appropriate. The statistical significance of differences between different groups was assessed by t test or Kruskal–Wallis test for continuous variables and by Chi-square test for categorical variables. The univariate and multivariate logistic regres- sion analyses were used to determine clinical predictors of hypoglycemia. Those found to be significant in univari- ate models and some characteristics selected a priori were adjusted for in multivariate logistic regression models. Statistical analyses were performed with SAS version 9.3 statistical software. All the statistical tests were two-sided. P-value < 0.05 was considered statistically significant. Results Characteristics and CGM data of the enrolled outpatients The characteristics of the total 351 outpatients with T2DM were shown in Table 1. There were 195 (55.6%) men and 156 (44.4%) women with an average age of 55.4 ± 14.4 years. The average systolic blood pressure (SBP) was 125.5 ± 15.9 mmHg, and the average diastolic blood pressure (DBP) was 75.9 ± 10.0 mmHg. The mean BMI was 22.9 ± 3.1 kg/m2. The median duration of dia- betes was 6.0 years, and the mean HbA1c was 7.1 ± 1.5% (54 ± 16 mmol/mol). According to 3-day CGM data and self-reports of the subjects, none experienced sympto- matic hypoglycemia. Overall, AH events were captured in 137 outpatients (39.0%), in whom Level 1 and Level 2 AH accounted for 61.3% and 38.7%, respectively. 112 (31.9%) of the AH population occurred during the daytime, and 85 (24.2%) experienced NAH. Hypoglycemia episodes that occurred exclusively at night accounted for 18.2% of the AH population and 29.4% of the NAH population. The number of hypoglycemic events among those with daytime asymptomatic hypoglycemia (DAH), nocturnal asympto- matic hypoglycemia (NAH), asymptomatic hypoglycemia occurred exclusively in the daytime (DAH), asymptomatic hypoglycemia occurred exclusively at night (NAH), and asymptomatic hypoglycemia occurred in both daytime and nighttime were 3.0 (1.0, 10.0), 6.0 (3.0, 12.0), 1.0 (1.0, 4.0), 1.0 (0.0,4.0), and 8.0 (3.0, 14.5), respectively. The Total TIR was 84.9 (66.7, 96.0). The MAGE, SD, and MBG were 4.2 ± 2.2 mmol/L, 1.9 ± 0.9 mmol/L, and 8.2 ± 2.1 mmol/L, respectively. Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH Diabetology & Metabolic Syndrome (2022) 14:180 Page 4 of 10 Table 1 Clinical characteristics and CGM data of the enrolled outpatients Characteristics n = 351 Sex Male 195 (55.6) Female 156 (44.4) Age, years 55.4 ± 14.4 SBP, mmHg 125.5 ± 15.9 DBP, mmHg 75.9 ± 10.0 BMI, kg/m2 22.9 ± 3.1 Duration, years 6.0 (2.0, 11.0) HbA1c, % 7.1 ± 1.5 HbA1c, mmol/mol 54 ± 16 C-P, ng/mL 1.4 (0.6,2.0) Comorbidity Hypertension 45 (12.8) Hyperlipidemia 59 (16.8) Fatty liver 19 (5.4) CCVD 37 (10.5) Medication Metformin 97 (27.6) DPP-4i 57 (16.2) SGLT-2i 15 (4.3) α-GI 65 (18.5) GLP-1ra 6 (1.7) TZD 9 (2.6) SU 78 (22.2) Insulin therapy 137 (39.0) Long-acting insulin 78 (22.2) Premixed insulin 36 (10.3) Short-acting insulin 58 (16.5) Complication DR 54 (15.4) DPN 45 (12.8) DPVD 17 (4.8) DN 17 (4.8) DF 2 (0.6) CGM data AH (%) 137 (39.0) Total TIR, % 84.9 (66.7,96.0) Nighttime TIR, % 96.8 (76.0, 100.0) Daytime TIR, % 81.9 (61.6,95.6) Total TBR, % 2.1 (0.8,5.8) Nighttime TBR, % 1.8 (0.0, 10.2) Daytime TBR, % 1.1 (0.2,3.7) Total TAR, % 12.7 (2.2,32.1) Nighttime TAR, % 0.0 (0.0, 17.1) Daytime TAR, % 16.2 (2.9,37.6) Level 1 AH (%) 84 (23.9) Total TIR, % 89.4 (77.6,97.2) Nighttime TIR, % 96.3 (80.4, 100.0) Daytime TIR % 88 9 (72 6 97 0) ( ld ld Table 1 Clinical characteristics and CGM data of the enrolled outpatients Characteristics n = 351 Sex Male 195 (55.6) Female 156 (44.4) Age, years 55.4 ± 14.4 SBP, mmHg 125.5 ± 15.9 DBP, mmHg 75.9 ± 10.0 BMI, kg/m2 22.9 ± 3.1 Duration, years 6.0 (2.0, 11.0) HbA1c, % 7.1 ± 1.5 HbA1c, mmol/mol 54 ± 16 C-P, ng/mL 1.4 (0.6,2.0) Comorbidity Hypertension 45 (12.8) Hyperlipidemia 59 (16.8) Fatty liver 19 (5.4) CCVD 37 (10.5) Medication Metformin 97 (27.6) DPP-4i 57 (16.2) SGLT-2i 15 (4.3) α-GI 65 (18.5) GLP-1ra 6 (1.7) TZD 9 (2.6) SU 78 (22.2) Insulin therapy 137 (39.0) Long-acting insulin 78 (22.2) Premixed insulin 36 (10.3) Short-acting insulin 58 (16.5) Complication DR 54 (15.4) DPN 45 (12.8) DPVD 17 (4.8) DN 17 (4.8) DF 2 (0.6) CGM data AH (%) 137 (39.0) Total TIR, % 84.9 (66.7,96.0) Nighttime TIR, % 96.8 (76.0, 100.0) Daytime TIR, % 81.9 (61.6,95.6) Total TBR, % 2.1 (0.8,5.8) Nighttime TBR, % 1.8 (0.0, 10.2) Daytime TBR, % 1.1 (0.2,3.7) Total TAR, % 12.7 (2.2,32.1) Nighttime TAR, % 0.0 (0.0, 17.1) Daytime TAR, % 16.2 (2.9,37.6) Level 1 AH (%) 84 (23.9) Total TIR, % 89.4 (77.6,97.2) Nighttime TIR, % 96.3 (80.4, 100.0) Daytime TIR, % 88.9 (72.6,97.0) Table 1 (continued) Characteristics n = 351 Total TAR, % 6.2 (1.0,23.2) Nighttime TAR, % 0.0 (0.0,2.2) Daytime TAR, % 8.2 (1.3,26.8) Level 2 AH (%) 53 (15.1) Total TIR, % 74.8 (67.0,85.7) Nighttime TIR, % 81.0 (65.5,95.5) Daytime TIR, % 74.3 (63.3–85.3) Total TBR, % 4.8 (2.5,8.6) Nighttime TBR, % 4.6 (1.3, 18.2) Daytime TBR, % 3.4 (1.0,7.2) Total TAR, % 18.3 (3.9,28.5) Nighttime TAR, % 0.7 (0.0, 19.0) Daytime TAR, % 20.6 (5.3,33.8) DAH (%) 112 (31.9) Hypoglycemic events 3.0 (1.0, 10.0) Hypoglycemic duration, min 27.0 (11.5,94.5) NAH (%) 85 (24.2) Hypoglycemic events 6.0 (3.0, 12.0) Hypoglycemic duration, min 51.0 (23.0, 103.0) DAH(%) 52 (14.8) Hypoglycemic events 1.0 (1.0, 3.0) Hypoglycemic duration, min 12.0 (4.5, 25.0) NAH(%) 25 (7.1) Hypoglycemic events 1.0 (0.0, 4.0) Hypoglycemic duration, min 6.0 (0.0, 37.0) Both DAH and NAH (%) 60 (17.1) Hypoglycemic events 8.0 (3.0, 14.5) Hypoglycemic duration, min 68.0 (27.0,135.0) MAGE, mmol/L 4.2 ± 2.2 SD, mmol/L 1.9 ± 0.9 MBG, mmol/L 8.2 ± 2.1 Data were present as Mean ± SD or Median (25th percentile, 75th percentile); SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, HbA1c glycated hemoglobin A1c, C-P C-Peptide, CCVD cardio-cerebral vascular disease, DPP-4i Dipeptidylpeptidase-4 inhibitors, SGLT-2i sodium–glucose cotransporter 2 inhibitors, α-GI alpha-glucosidase inhibitors, GLP-1RA glucagon- like peptide-1 receptor agonists, TZD thiazolidinedione, SU sulfonylurea, DR diabetic retinopathy, DPN diabetic peripheral neuropathy, DPVD diabetic peripheral vascular disease, DN diabetic nephropathy, DF diabetic foot, CGM continuous glucose monitoring, Level 1 AH BG 3.0–3.9 mmol/L, Level 2 AH BG < 3.0 mmol/L, Total TIR total time in range (3.9–10.0 mmol/L), Nighttime TIR time in range (3.9–10.0 mmol/L) during 0:00–6:00, Daytime TIR time in range (3.9–10.0 mmol/L) during 6:00–24:00, Total TBR total time below range (< 3.9 mmol/L), Nighttime TBR time below range (< 3.9 mmol/L) during 0:00–6:00, Daytime TBR time below range (< 3.9 mmol/L) during 6:00–24:00, Total TAR total time above range (> 10.0 mmol/L), Nighttime TAR time above range (> 10.0 mmol/L) during 0:00–6:00, Daytime TAR time above range (> 10.0 mmol/L) during 6:00–24:00, DAH daytime asymptomatic hypoglycemia (defined as 6:00–24:00 blood glucose < 3.9 mmol/L), DAH* asymptomatic hypoglycemia occurred exclusively in the daytime, NAH nocturnal asymptomatic hypoglycemia (defined as 0:00–6:00 blood glucose < 3.9 mmol/L), NAH* asymptomatic hypoglycemia occurred exclusively at night, both DAH and NAH asymptomatic hypoglycemia episodes occurred in both daytime and nighttime, MAGE mean amplitude of plasma glucose excursion, SD glucose standard deviation, MBG mean blood glucose Table 1 (continued) (52.1 ± 14.6 years old vs. Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH 55.9 ± 14.1 years old, P < 0.05) and lower HbA1c (6.8 ± 1.3% vs. Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH In Table 2, compared to outpatients without AH, outpatients in the AH group showed younger age Huang et al. Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH 7.2 ± 1.6%, a of the enrolled n = 351 195 (55.6) 156 (44.4) 55.4 ± 14.4 125.5 ± 15.9 75.9 ± 10.0 22.9 ± 3.1 6.0 (2.0, 11.0) 7.1 ± 1.5 54 ± 16 1.4 (0.6,2.0) 45 (12.8) 59 (16.8) 19 (5.4) 37 (10.5) 97 (27.6) 57 (16.2) 15 (4.3) 65 (18.5) 6 (1.7) 9 (2.6) 78 (22.2) 137 (39.0) 78 (22.2) 36 (10.3) 58 (16.5) 54 (15.4) 45 (12.8) 17 (4.8) 17 (4.8) 2 (0.6) 137 (39.0) 84.9 (66.7,96.0) 96.8 (76.0, 100.0) 81.9 (61.6,95.6) 2.1 (0.8,5.8) 1.8 (0.0, 10.2) 1.1 (0.2,3.7) 12.7 (2.2,32.1) 0.0 (0.0, 17.1) 16.2 (2.9,37.6) 84 (23.9) 89.4 (77.6,97.2) 96.3 (80.4, 100.0) 88.9 (72.6,97.0) 1.0 (0.6,2.6) 0.1 (0.0,6.3) 0.8 (0.2, 1.5) Table 1 (continued) Characteristics n = 351 Total TAR, % 6.2 (1.0,23.2) Nighttime TAR, % 0.0 (0.0,2.2) Daytime TAR, % 8.2 (1.3,26.8) Level 2 AH (%) 53 (15.1) Total TIR, % 74.8 (67.0,85.7) Nighttime TIR, % 81.0 (65.5,95.5) Daytime TIR, % 74.3 (63.3–85.3) Total TBR, % 4.8 (2.5,8.6) Nighttime TBR, % 4.6 (1.3, 18.2) Daytime TBR, % 3.4 (1.0,7.2) Total TAR, % 18.3 (3.9,28.5) Nighttime TAR, % 0.7 (0.0, 19.0) Daytime TAR, % 20.6 (5.3,33.8) DAH (%) 112 (31.9) Hypoglycemic events 3.0 (1.0, 10.0) Hypoglycemic duration, min 27.0 (11.5,94.5) NAH (%) 85 (24.2) Hypoglycemic events 6.0 (3.0, 12.0) Hypoglycemic duration, min 51.0 (23.0, 103.0) DAH(%) 52 (14.8) Hypoglycemic events 1.0 (1.0, 3.0) Hypoglycemic duration, min 12.0 (4.5, 25.0) NAH(%) 25 (7.1) Hypoglycemic events 1.0 (0.0, 4.0) Hypoglycemic duration, min 6.0 (0.0, 37.0) Both DAH and NAH (%) 60 (17.1) Hypoglycemic events 8.0 (3.0, 14.5) Hypoglycemic duration, min 68.0 (27.0,135.0) MAGE, mmol/L 4.2 ± 2.2 SD, mmol/L 1.9 ± 0.9 MBG, mmol/L 8.2 ± 2.1 Data were present as Mean ± SD or Median (25th percentile, 75th percentile); SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, HbA1c glycated hemoglobin A1c, C-P C-Peptide, CCVD cardio-cerebral vascular disease, DPP-4i Dipeptidylpeptidase-4 inhibitors, SGLT-2i sodium–glucose cotransporter 2 inhibitors, α-GI alpha-glucosidase inhibitors, GLP-1RA glucagon- like peptide-1 receptor agonists, TZD thiazolidinedione, SU sulfonylurea, DR diabetic retinopathy, DPN diabetic peripheral neuropathy, DPVD diabetic peripheral vascular disease, DN diabetic nephropathy, DF diabetic foot, CGM continuous glucose monitoring, Level 1 AH BG 3.0–3.9 mmol/L, Level 2 AH BG < 3.0 mmol/L, Total TIR total time in range (3.9–10.0 mmol/L), Nighttime TIR time in range (3.9–10.0 mmol/L) during 0:00–6:00, Daytime TIR time in range (3.9–10.0 mmol/L) during 6:00–24:00, Total TBR total time below range (< 3.9 mmol/L), Nighttime TBR time below range (< 3.9 mmol/L) during 0:00–6:00, Daytime TBR time below range (< 3.9 mmol/L) during 6:00–24:00, Total TAR total time above range (> 10.0 mmol/L), Nighttime TAR time above range (> 10.0 mmol/L) during 0:00–6:00, Daytime TAR time above range (> 10.0 mmol/L) during 6:00–24:00, DAH daytime asymptomatic hypoglycemia (defined as 6:00–24:00 blood glucose < 3.9 mmol/L), DAH* asymptomatic hypoglycemia occurred exclusively in the daytime, NAH nocturnal asymptomatic hypoglycemia (defined as 0:00–6:00 blood glucose < 3.9 mmol/L), NAH* asymptomatic hypoglycemia occurred exclusively at night, both DAH and NAH asymptomatic hypoglycemia episodes occurred in both daytime and nighttime, MAGE mean amplitude of plasma glucose excursion, SD glucose standard deviation, MBG mean blood glucose Data were present as Mean ± SD or Median (25th percentile, 75th percentile); SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, HbA1c glycated hemoglobin A1c, C-P C-Peptide, CCVD cardio-cerebral vascular disease, DPP-4i Dipeptidylpeptidase-4 inhibitors, SGLT-2i sodium–glucose otransporter 2 inhibitors, α-GI alpha-glucosidase inhibitors, GLP-1RA glucagon- ke peptide-1 receptor agonists, TZD thiazolidinedione, SU sulfonylurea, DR diabetic retinopathy, DPN diabetic peripheral neuropathy, DPVD diabetic peripheral vascular disease, DN diabetic nephropathy, DF diabetic foot, CGM ontinuous glucose monitoring, Level 1 AH BG 3.0–3.9 mmol/L, Level 2 AH BG < 3.0 mmol/L, Total TIR total time in range (3.9–10.0 mmol/L), Nighttime TIR time in range (3.9–10.0 mmol/L) during 0:00–6:00, Daytime TIR time n range (3.9–10.0 mmol/L) during 6:00–24:00, Total TBR total time below ange (< 3.9 mmol/L), Nighttime TBR time below range (< 3.9 mmol/L) during 0:00–6:00, Daytime TBR time below range (< 3.9 mmol/L) during 6:00–24:00, Total TAR total time above range (> 10.0 mmol/L), Nighttime TAR time above ange (> 10.0 mmol/L) during 0:00–6:00, Daytime TAR time above range > 10.0 mmol/L) during 6:00–24:00, DAH daytime asymptomatic hypoglycemia defined as 6:00–24:00 blood glucose < 3.9 mmol/L), DAH* asymptomatic hypoglycemia occurred exclusively in the daytime, NAH nocturnal asymptomatic hypoglycemia (defined as 0:00–6:00 blood glucose < 3.9 mmol/L), NAH* symptomatic hypoglycemia occurred exclusively at night, both DAH and NAH symptomatic hypoglycemia episodes occurred in both daytime and nighttime, MAGE mean amplitude of plasma glucose excursion, SD glucose standard deviation, MBG mean blood glucose (52.1 ± 14.6 years old vs. Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH 55.9 ± 14.1 years old, P < 0.05) and lower HbA1c (6.8 ± 1.3% vs. 7.2 ± 1.6%, Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 Page 5 of 10 Table2 Clinical characteristics and CGM data of non-AH and AH patients Non-AH(n=214) AH (n=137) Table2 Clinical characteristics and CGM data of non-AH and AH patients Table2 Clinical characteristics and CGM data of non-AH and AH patients Data were present as Mean ± SD or Median (25th percentile, 75th percentile). Data were present as Mean ± SD or Median (25th percentile, 75th percentile). SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, HbA1c glycated hemoglobin A1c, C-P C-Peptide, DPP-4i Dipeptidylpeptidase-4 inhibitors, α-GI alpha-glucosidase inhibitors, SU sulfonylurea, DR diabetic retinopathy, DPN diabetic peripheral neuropathy, DPVD diabetic peripheral vascular disease, DN diabetic nephropathy, DF diabetic foot, CGM continuous glucose monitoring, Level 1 AH BG 3.0–3.9 mmol/L, Level 2 AH BG < 3.0 mmol/L, DAH daytime asymptomatic hypoglycemia (defined as 6:00–24:00 blood glucose < 3.9 mmol/L), DAH* asymptomatic hypoglycemia occurred exclusively in the daytime, NAH nocturnal asymptomatic hypoglycemia (defined as 0:00–6:00 blood glucose < 3.9 mmol/L), NAH* asymptomatic hypoglycemia occurred exclusively at night, both DAH and NAH asymptomatic hypoglycemia episodes occurred in both daytime and nighttime, MAGE mean amplitude of plasma glucose excursion, SD glucose standard deviation, MBG mean blood glucose, TBR time below range (< 3.9 mmol/L), Total TIR time in range (3.9–10.0 mmol/L), Nighttime TIR time in range (3.9–10.0 mmol/L) during 0:00–6:00, Daytime TIR time in range (3.9–10.0 mmol/L) during 6:00–24:00, Total TAR time above range (> 10.0 mmol/L), Nighttime TAR time above range (> 10.0 mmol/L) during 0:00–6:00, Daytime TAR time above range (> 10.0 mmol/L) during 6:00–24:00 * P < 0.05 for the specified comparison of AH group and non-AH group; P < 0.01 for the specified comparison of AH group and non-AH group; * P < 0.001 for the specified comparison of AH group and non-AH group Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH Diabetology & Metabolic Syndrome (2022) 14:180 Page 6 of 10 P < 0.05). According to the data of CGM, glucose SD (2.2 ± 1.0 mmol/L vs. 1.7 ± 0.9 mmol/L, P < 0.001) and MAGE (4.8 ± 2.3 mmol/L vs. 3.8 ± 2.0 mmol/L, P < 0.001) in the AH group were significantly higher than those without AH, while MBG (7.5 ± 1.4 mmol/L vs. 8.7 ± 2.2 mmol/L, P < 0.001) was significantly lower. Nighttime TIR in the AH group was significantly lower than that in the non-AH group [92.0 (75.8 ,100.0) vs. 100.0 (76.4, 100.0), P = 0.002]. Patients had AH epi- sodes exhibited lower Total TAR [92.0 (1.4,25.0) vs. 14.7 (3.0,38.7), P < 0.01], lower Daytime TAR [12.3 (2.0,31.6) vs. 18.5 (4.1,44.6), P < 0.01], and lower Nighttime TAR [0.0 (0.0, 10.0) vs. 0.0 (0.0,23.6), P < 0.05]. In order to deter- mine the influencing factors of AH, we conducted a series of analyses. After controlling for all factors identified through univariate analyses (Additional file 1: Table S1), multivariate analysis (Table 3) demonstrated that patients with younger age [0.975 (0.955–0.997), P = 0.023], lower HbA1c levels [0.759 (0.620–0.929), P = 0.008], and higher SBP [1.024 (1.002–1.047), P = 0.033] had a significantly higher risk of experiencing overall AH episodes. TAR [6.2 (1.2,20.1) vs. 14.7 (3.0,38.7), P < 0.01], lower Nighttime TAR [0.0 (0.0,2.5) vs. 0.0 (0.0,23.6), P < 0.01], and lower Daytime TAR [8.2 (1.6,26.3) vs. 18.5 (4.1,44.6), P < 0.01] were more vulnerable to Level 1 AH. Multivari- ate analyses (Table 3) controlled for factors associated with high regimen distress in univariate analyses (Addi- tional file 1: Table S1) and found that lower HbA1c levels [0.683 (0.527–0.887), P = 0.004] and higher SBP [1.033 (1.008–1.059), P = 0.010] were also associated with Level 1 AH. Comparisons between the Level 2 AH group and the non‑AH group and assessment of influencing factors for Level 2 AHh There was greater percentage of female in the Level 2 AH group (60.4% vs. 39.6%, P < 0.05). The age (47.0 ± 15.8 vs. 55.9 ± 14.1, P < 0.001) of the Level 2 AH group was sig- nificantly lower and the C-Peptide (C-P) [0.5 (0.1,1.6) vs. 1.5 (0.7, 2.2), P < 0.01] level was lower. Significantly statistical difference was observed in the use of Dipep- tidylpeptidase-4 inhibitors (DPP4i) (1.9% vs. 16.8%, P < 0.01) and insulin regime (52.8% vs. 36.9%, P < 0.05) between Level 2 AH group and non-AH group. The MAGE (5.2 ± 2.5 mmol/L vs. 3.8 ± 2.0 mmol/L, P < 0.001) and glucose SD (2.5 ± 1.0 mmol/L vs. 1.7 ± 0.9 mmol/L, P < 0.001) in the AH group were significantly higher than those without AH, while MBG (7.6 ± 1.6 mmol/L vs. 8.7 ± 2.2 mmol/L, P < 0.001) was significantly lower. There were significant statistical differences in Total TIR [78.1 (67.0,86.0) vs. 85.3 (61.3,96.9), P < 0.05] and Night- time TIR [81.5 (65.5,95.8) vs. 100.0 (76.4,100.0), P < 0.001] between two groups. After controlling for all factors Comparisons between the Overall AH group and the non‑AH group and assessment of influencing factors for Overall AH SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, HbA1c glycated hemoglobin A1c, C-P C-Peptide, DPP-4i Dipeptidylpeptidase-4 inhibitors, α-GI alpha-glucosidase inhibitors, SU sulfonylurea, DR diabetic retinopathy, DPN diabetic peripheral neuropathy, DPVD diabetic peripheral vascular disease, DN diabetic nephropathy, DF diabetic foot, CGM continuous glucose monitoring, Level 1 AH BG 3.0–3.9 mmol/L, Level 2 AH BG < 3.0 mmol/L, DAH daytime asymptomatic hypoglycemia (defined as 6:00–24:00 blood glucose < 3.9 mmol/L), DAH* asymptomatic hypoglycemia occurred exclusively in the daytime, NAH nocturnal asymptomatic hypoglycemia (defined as 0:00–6:00 blood glucose < 3.9 mmol/L), NAH* asymptomatic hypoglycemia occurred exclusively at night, both DAH and NAH asymptomatic hypoglycemia episodes occurred in both daytime and nighttime, MAGE mean amplitude of plasma glucose excursion, SD glucose standard deviation, MBG mean blood glucose, TBR time below range (< 3.9 mmol/L), Total TIR time in range (3.9–10.0 mmol/L), Nighttime TIR time in range (3.9–10.0 mmol/L) during 0:00–6:00, Daytime TIR time in range (3.9–10.0 mmol/L) during 6:00–24:00, Total TAR time Non-AH(n = 214) AH (n = 137) Overall AH (n = 137) Level 1 AH (n = 84) Level 2 AH (n = 53) Sex Male 124 (57.9) 71 (51.8) 50 (59.5) 21 (39.6) * Female 90 (42.1) 66 (48.2) 34 (40.5) 32 (60.4) Age, years 55.9 ± 14.1 52.1 ± 14.6* 55.3 ± 12.9 47.0 ± 15.8*** SBP, mmHg 125.2 ± 15.9 126.0 ± 16.0 128.1 ± 16.0 122.5 ± 15.7 DBP, mmHg 76.3 ± 10.6 75.3 ± 9.0 76.0 ± 8.3 74.2 ± 10.0 BMI, kg/m2 23.1 ± 3.0 22.5 ± 3.1 22.4 ± 2.9 22.8 ± 3.4 Duration, years 6.0 (2.0, 11.0) 6.0 (2.3, 11.0) 6.0 (3.0,11.0) 6.0 (2.0,14.0) HbA1c, % 7.2 ± 1.6 6.8 ± 1.3* 6.7 ± 1.2** 7.0 ± 1.6 HbA1c, mmol/mol 56 ± 17 51 ± 15* 50 ± 13 53 ± 17 C-P, ng/mL 1.5 (0.7, 2.2) 1.2 (0.4, 1.9) 1.5 (1.1,2.6) 0.5 (0.1,1.6) Comorbidity Hypertension 27 (12.6) 18 (13.1) 14 (16.7) 4 (7.5) Hyperlipidemia 36 (16.8) 23 (16.8) 16 (19.0) 7 (13.2) Medication Metformin 63 (29.4) 34 (24.8) 25 (29.8) 9 (17.0) DPP-4i 36 (16.8) 21 (15.3) 20 (23.8) 1 (1.9) ** α-GI 42 (19.6) 23 (16.8) 18 (21.4) 5 (9.4) SU 46 (21.5) 32 (23.4) 23 (27.4) 9 (17.0) Insulin 79 (36.9) 58 (42.3) 30 (35.7) 28 (52.8) * Complication DR 32 (15.0) 22 (16.1) 11 (13.1) 11 (20.8) DPN 31 (14.5) 14 (10.2) 8 (9.5) 6 (11.3) DPVD 11 (5.1) 6 (4.4) 3 (3.6) 3 (5.7) DN 11 (5.1) 6 (4.4) 4 (4.8) 2 (3.8) DF 2 (0.9) 0 (0.0) 0 (0.0) 0 (0.0) CGM data MAGE, mmol/L 3.8 ± 2.0 4.8 ± 2.3*** 4.4 ± 2.0* 5.2 ± 2.5* SD, mmol/L 1.7 ± 0.9 2.2 ± 1.0* 1.9 ± 0.9 2.5 ± 1.0* MBG, mmol/L 8.7 ± 2.2 7.5 ± 1.4* 7.4 ± 1.3* 7.6 ± 1.6* Total TIR, % 85.3 (61.3, 96.9) 84.8 (70.8, 95.4) 89.4 (79.7, 96.8) 78.1 (67.0, 86.0) * Nighttime TIR, % 100.0(76.4, 100.0) 92.0 (75.8, 100.0) 96.8 (80.6, 100.0) 81.5 (65.5, 95.8) * Daytime TIR, % 81.1 (55.4.95.9) 84.7 (67.8, 95.3) 89.3 (73.7, 96.8) * 76.0 (63.3, 85.9) Total TAR, % 14.7 (3.0, 38.7) 9.2 (1.4, 25.0) 6.2 (1.2, 20.1) 18.2 (3.9, 28.6) Nighttime TAR, % 0.0 (0.0, 23.6) 0.0 (0.0, 10.0) * 0.0 (0.0, 2.5) 0.0 (0.0, 19.9) Daytime TAR, % 18.5 (4.1, 44.6) 12.3 (2.0, 31.6) 8.2 (1.6, 26.3) ** 19.0 (5.3, 34.8) Huang et al. OR odds ratio, CI confidence interval, DPP-4i Dipeptidylpeptidase-4 inhibitors, α-GI alpha-glucosidase inhibitors, SU sulfonylurea, HbA1c glycated hemoglobin A1c, SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index Discussion Previous studies [24, 28] had demonstrated that inten- sive insulin treatment or sulfonylurea (SU) could predict the episodes of hypoglycemia in T2DM. Our study also showed a higher rate of insulin therapy (52.8% vs. 36.9%) in the Level 2 AH group than in the non-AH group. The univariate analysis (Additional file 1: Table S1) indicated that patients with the use of insulin were associated with higher risk of Level 2 AH (P = 0.018). However, this asso- ciation did not exist when accounting for other influenc- ing factors in the multiple logistic regression models. This may be due to the substantial effect of the adjusted confounders. Additionally, neither univariate nor multi- variate analysis demonstrated the association between SU and the risk of AH. Overall, our study observed that outpatients treated with insulin or SU did not predispose to asymptomatic hypoglycemia, which was consistent with the conclusion of Monnier et al. [22, 29]. This inter- esting finding reminds us to screen all diabetic patients for a history of hypoglycemia, rather than focusing only on those using insulin or insulin secretagogues. In the current study, 137 (39.0%) of the total 351 outpa- tients with T2DM experienced AH episodes, in which Level 1 AH and Level 2 AH accounted for 61.3% and 38.7%, respectively. 85 (62.0%) of the AH patients expe- rienced NAH and 25 (18.2%) exclusively NAH. Multivari- ate logistic regression analysis demonstrated that patients with younger age, lower HbA1c, and higher SBP levels were significantly associated with increased risk of AH. While after further grading of AH, male sex and DPP4i regime were shown to be associated with lower risk of Level 2 AH. Previously, Chico et al. [13] detected unrecognized hypoglycemias in 46.6% (14 out of 30) of the type 2 patients with diabetes by using CGMS and noticed that 73.7% of all events occurred at night, tremendously pro- voking attention from academia. Further, Gehlaut et al. [24] applied CGMS on 108 non-hospitalized patients with T2DM to detect the episodes of hypoglycemia. Dur- ing the five-day study, 53 (49.1%) participants were cap- tured of hypoglycemia by CGMS, but only 24.5% (13 out of 53) had self-reported symptoms, while the majority (75%) of patients were unaware of any hypoglycemia epi- sodes. The prevalence of NAH was 73.6% (37 out of 53), and hypoglycemia occurred only at night was 20.8% (14 out of 53). Comparisons between the Level 1 AH group and the non‑AH group and assessment of influencing factors for Level 1 AH As for Level 1 AH, patients with lower HbA1c exhib- ited a higher prevalence of AH (6.7 ± 1.2% vs. 7.2 ± 1.6%, P < 0.01). The MBG of the AH group was substantially lower (7.4 ± 1.3 vs. 8.7 ± 2.2, P < 0.001) while the MAGE was higher (4.4 ± 2.0 vs. 3.8 ± 2.0, P < 0.001). Daytime TIR [89.3 (73.7,96.8) vs. 81.1 (55.4,95.9), P < 0.05] was core- lated with Level 1 AH episodes. Patients with lower Total Table3 Predictors of AH, Level 1 AH, and Level 2 AH on the basis of logistic regression models OR odds ratio, CI confidence interval, DPP-4i Dipeptidylpeptidase-4 inhibitors, α-GI alpha-glucosidase inhibitors, SU sulfonylurea, HbA1c glycated hemoglobin A1c, SBP Multivariate analysis Predictor AH Level 1 AH Level 2 AH OR (95%Cl) P OR (95%Cl) P OR (95%Cl) P Sex 0.822 (0.494–1.368) 0.450 1.610 (0.886–2.923) 0.118 0.391 (0.191–0.799) 0.010 Age 0.975 (0.955–0.997) 0.023 0.996 (0.971–1.021) 0.746 0.962 (0.936–0.990) 0.008 Duration 1.016 (0.969–1.066) 0.506 0.986 (0.931–1.044) 0.626 1.041 (0.978–1.108) 0.211 Metformin 0.816 (0.440–1.513) 0.519 0.943 (0.492–1.883) 0.867 0.715 (0.274–1.862) 0.492 DPP-4i 0.937 (0.460–1.908) 0.858 2.011 (0.927–4.361) 0.077 0.110 (0.014–0.855) 0.035 α-GI 0.993 (0.517–1.906) 0.982 1.272 (0.627–2.577) 0.505 0.647 (0.225–1.859) 0.419 SU 1.218 (0.635–2.334) 0.553 1.112 (0.545–2.272) 0.770 1.361 (0.511–3.624) 0.538 HbA1c 0.759 (0.620–0.929) 0.008 0.683 (0.527–0.887) 0.004 0.940 (0.727–1.216) 0.639 insulin 1.261 (0.682–2.333) 0.460 1.197 (0.593–2.414) 0.616 1.198 (0.510–2.813) 0.679 SBP 1.024 (1.002–1.047) 0.033 1.033 (1.008–1.059) 0.010 1.000 (0.969–1.032) 0.999 DBP 0.981 (0.950–1.013) 0.238 0.980 (0.946–1.016) 0.272 0.991 (0.946–1.038) 0.692 BMI 0.961 (0.882–1.047) 0.365 0.920 (0.831–1.018) 0.108 1.063 (0.944–1.198) 0.310 Table3 Predictors of AH, Level 1 AH, and Level 2 AH on the basis of logistic regression models Page 7 of 10 Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 glucose control to investigate the incidence of hypogly- cemia. However, the target population of our study was T2DM outpatients whose status was relatively stable with "well-controlled" glucose levels. Differences in samples may contribute to differences of results. Secondly, we used the CGMS to detect the hypoglycemia episodes, which is expected to discover additional unrecognized hypoglycemia events than traditional SMBG. identified through univariate analyses (Additional file 1: Table S1), multivariate analyses found that female sex [0.391 (0.191–0.799), P = 0.010] and younger age [0.962 (0.936–0.990), P = 0.008] were independent predictors of Level 2 AH episodes. Comparisons between the Level 1 AH group and the non‑AH group and assessment of influencing factors for Level 1 AH In addition, patients treated with DPP4i tended to have lower risk of Level 2 AH [0.110 (0.014–0.855), P = 0.035]. (Table 3). Discussion Diabetology & Metabolic Syndrome (2022) 14:180 Page 8 of 10 Page 8 of 10 association between gender and AH, but when further dividing AH episodes into Level 1 and Level 2 AH, we noticed that women are prone to Level 2 AH, which was in accordance with a prospective study by Zhang et al. [27]. The exact reason was unclear. As such, a larger sam- ple size is needed in the future to verify the relationship between gender and AH. consumption and physical activities, it is impossible to determine whether these factors play a role in the occurrence of AH in T2DM. Finally, our study is a ret- rospective study, which has recalling bias. Therefore, further large samples of prospective studies are needed to consolidate the conclusions of this study, including long-term follow-up of the hypoglycemic outpatients after treatment and lifestyle adjustments. Prior research found that lower HbA1c was an inde- pendent predictor of AH [28, 30, 31]; this was also highly salient in the current cohort. Currently, HbA1c is known to be the most used parameter to assess glycemic control and important index in the treatment of hyperglycemia, which has been used as the primary endpoint for many CGM studies [23]. However, new data support the need to devote attention to TIR and TAR, for a comprehensive evaluation of glycemic control among the diabetes popu- lation [12, 23, 32]. Indeed, some clinicians may choose to target the reduction of the TAR and minimize hypogly- cemia, thereby arriving at more time in the target range. Whereas, recent recommendations from an international consensus on TIR emphasized that targets should be individualized and each 5% increase in TIR correlated with clinically meaningful benefits [23]. For the present study, in terms of lower HbA1c levels and higher percent- ages of TIR, the AH group seemed to had better blood glucose control than the non-AH group, but when inte- grated with MAGE, SD, and TAR, the higher glucose variability (GV) of the AH group is noteworthy. Accord- ingly, given the substantially high risk of AH in T2DM outpatients, clinicians should be vigilant in preventing hypoglycemia in such populations and avoid aggressively attempting to achieve near-normal glucose or HbA1c lev- els in whom the risks of lower glycemic targets may far outweigh the potential benefits on diabetic complica- tions. Abbreviations AH A AH: Asymptomatic hypoglycemia; T2DM: Type 2 diabetes; CGMS: Continuous glucose monitoring system; BG: Blood glucose; NAH: Nocturnal asymptomatic hypoglycemia; DAH: Daytime asymptomatic hypoglycemia; NAH: Asymp- tomatic hypoglycemia occurred exclusively at night; DAH: Asymptomatic hypoglycemia occurred exclusively in the daytime; HbA1c: Glycated hemo- globin A1c; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; DPP4i: Dipeptidylpeptidase-4 inhibitors; DCCT: Diabetes Control and Complications Trial; SMBG: Self-monitoring blood glucose; T1DM: Type 1 diabetes; SD: Glu- cose standard deviation; MBG: Mean blood glucose; MAGE: Mean amplitude of glycemic excursions; TIR: Time in range; TBR: Time below range; TAR: Time above range; CP: C-Peptide; GV: Glucose variability. Funding This work was funded by the Natural Science Foundation of Fujian Province, China (No.2021J011363). Discussion So far, SD, MAGE, and MBG measured by CGMS, as key metrics for GV, has been confirmed to be closely correlated with hypoglycemia events [22, 33–35]. Provid- ing unprecedented access to a range of new indicators of glucose control, CGMS, which would help clinicians and patients with diabetes to overcome the limitations of HbA1c and SMBG, should be brought into the rou- tine management of diabetes to facilitate prompt therapy adjustment. Acknowledgements We would like to give sincere thanks to the research staff who participated in this work and the patients for supporting the program. Author contributions QJ and BH wrote the draft of the manuscript. XS performed the statistical analysis. All authors were involved in the study design, interpretation of data, and review of the manuscript. We would like to give sincere thanks to the research staff who participated in this work and the patients for supporting the program. All authors read and approved the final manuscript. Our study had several strengths. Firstly, compared with similar domestic studies, the sample size of our study is larger. Secondly, after installing the CGMS device, all the participants returned home without any changes of routine life, making the risks of AH highly reliable. Besides, several weaknesses should be noted. First, our CGM data only covered three days of glu- cose metrics, which might underestimate the inci- dence of AH. Second, since we did not collect data of other possible causes of hypoglycemia, such as alcohol Conclusions In summary, a high incidence of AH, of which NAH accounted for the main part, was observed in T2DM out- patients who did not receive intensive therapy. Relative relax HbA1c targets should be considered for patients who are prone to AH. We suggest CGMS should be inter- mittently used in the routine management of T2DM out- patients to timely capture hypoglycemia and minimize hypoglycemia exposure to benefit from better overall glu- cose control. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13098-022-00959-x. Additional file 1: Table S1. Analysis of influencing factors of AH, Level 1 AH, and Level 2 AH by univariate logistic regression model. Discussion In our study, none of the participants reported symptomatic hypoglycemia, and 39.0% of the target patients experienced AH events, which was in parallel with previous studies. Notably, all patients experienced hypoglycemic episodes claimed they had no symptoms of hypoglycemia. We speculate that the heterogeneity of symptom response in diabetic individuals could took into consideration and selective bias might play a role. It is also possible that the patients were asymptomatic due to recurrent episodes of hypoglycemia. In our study, the univariate logistic analysis indicated that DPP4i regime was associated with lower risks of Level 1 and Level 2 AH, yet the association between DPP4i and Level 1 AH was substantially attenuated after further adjusting for other confounders. This may stem from the fact that, in our present study, elder age was a protective factor for AH, and the average age of the Level 1 AH group was substantially higher than that of the Level 2 AH group (55.3 ± 12.9 vs. 47.0 ± 15.8). Thus, after controlling for age and other affecting factors, the protective effect of DPP4i in the Level 1 group disap- peared. Overall, DPP4i have been proved to be substan- tially associated with lower risks of hypoglycemia [26]. Further studies are needed to confirm the causal relation- ship between DPP-4i regime and the risk of AH.h Although plenty of studies [12, 17, 22, 23, 25] had indi- cated that older age, longer diabetes duration, and micro- vascular complications were independently correlated with increased prevalence of hypoglycemia, in our pre- sent study, multivariate logistic regression analysis found no significance of these variables in predicting AH events except for younger age in the Level 2 AH group. Vari- ous explanations could be proposed. Firstly, most prior studies were conducted during hospitalization, and they focused merely on the insulin-used population with poor The link between the diagnosis of hypertension and a higher risk of hypoglycemia had been identified in pre- vious studies [27, 28]. In the present study, 12.8% of the subjects had combined hypertension. 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Page 9 of 10 Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 Competing interests The authors have declared that no competing interests exist. 16. Weber KK, Lohmann T, Busch K, Donati-Hirsch I, Riel R. High frequency of unrecognized hypoglycaemias in patients with Type 2 diabetes is discov- ered by continuous glucose monitoring. Exp Clin Endocrinol Diabetes. 2007;115:491–4. https://doi.org/10.1055/s-2007-984452. Consent for publication Consent for publication All authors have agreed to submit this manuscript for publication. 15. Bergada I, Suissa S, Dufresne J, Schiffrin A. Severe hypoglycemia in IDDM children. Diabetes Care. 1989;12:239–44. https://doi.org/10.2337/diacare. 12.4.239. Ethics approval and consent to participate 14. McNally PG, Dean JD, Morris AD, Wilkinson PD, Compion G, Heller SR. Using continuous glucose monitoring to measure the frequency of low glucose values when using biphasic insulin aspart 30 compared with biphasic human insulin 30: a double-blind crossover study in individuals with type 2 diabetes. Diabetes Care. 2007;30:1044–8. https://doi.org/10. 2337/dc06-1328. pp p p This study has been approved by the Ethical Review Authority in The First Affiliated Hospital of Xiamen University (Number: 2020-055). Participants gave informed consent to participate in the study before taking part. Competing interests Competing interests The authors have declared that no competing interests exist. Competing interests The authors have declared that no competing interests exist. glycemic variability in patients with type 2 diabetes mellitus. Exp Clin Endocrinol Diabetes. 2011;119:59–61. https://doi.org/10.1055/s-0030- 1262874. 32. Beck RW, Bergenstal RM, Riddlesworth TD, Kollman C, Li Z, Brown AS, et al. Validation of time in range as an outcome measure for diabetes clinical trials. Diabetes Care. 2019;42:400–5. https://doi.org/10.2337/dc18-1444. 33. Kilpatrick ES, Rigby AS, Goode K, Atkin SL. Relating mean blood glucose and glucose variability to the risk of multiple episodes of hypoglycaemia in type 1 diabetes. Diabetologia. 2007;50:2553–61. https://doi.org/10. 1007/s00125-007-0820-z. 34. Monnier L, Colette C, Boegner C, Pham TC, Lapinski H, Boniface H. Con- tinuous glucose monitoring in patients with type 2 diabetes: why? when? whom? Diabetes Metab. 2007;33:247–52. https://doi.org/10.1016/j.diabet. 2006.11.007. 35. Marling CR, Shubrook JH, Vernier SJ, Wiley MT, Schwartz FL. Characteriz- ing blood glucose variability using new metrics with continuous glucose monitoring data. J Diabetes Sci Technol. 2011;5:871–8. https://doi.org/10. 1177/193229681100500408. References The continuous glucose moni- toring system is useful for detecting unrecognized hypoglycemias in patients with type 1 and type 2 diabetes but is not better than frequent 31. Engler B, Koehler C, Hoffmann C, Landgraf W, Bilz S, Schoner C, et al. Relationship between HbA1c on target, risk of silent hypoglycemia and Page 10 of 10 Huang et al. Diabetology & Metabolic Syndrome (2022) 14:180 glycemic variability in patients with type 2 diabetes mellitus. Exp Clin Endocrinol Diabetes. 2011;119:59–61. https://doi.org/10.1055/s-0030- 1262874. 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https://openalex.org/W2748476405	https://earth-planets-space.springeropen.com/track/pdf/10.1186/s40623-017-0696-1	English	null	Geomagnetic and ionospheric response to the interplanetary shock on January 24, 2012	Earth, planets and space	2,017	cc-by	13,298	Abstract We have examined multi-instrument observations of the magnetospheric and ionospheric response to the interplan- etary shock on January 24, 2012. Apart from various instruments, such as ground and space magnetometers, photom- eters, and riometers used earlier for a study of possible response to a shock, we have additionally examined variations of the ionospheric total electron content as determined from the global navigation satellite system receivers. World- wide ground magnetometer arrays detected shock-induced sudden commencement (SC) with preliminary and main impulses throughout the dayside sector. A magnetic field compression was found to propagate through the magne- tosphere with velocity less than the local Alfven velocity. Though the preliminary pulse was evident on the ground, its signature was not observed by the THEMIS and GOES satellites in the magnetosphere. The SC was accompanied by a burst of cosmic noise absorption recorded along a latitudinal network of riometers in the morning and evening sectors. The SC also caused an impulsive enhancement of dayside auroral emissions (shock aurora) as observed by the hyperspectral all-sky imager NORUSCA II at Barentsburg and the meridian scanning photometer at Longyearbyen (both at Svalbard). The VHF EISCAT radar (Tromsø, Norway) observed a SC-associated increase in electron density in the lower ionosphere (100–180 km). The system for monitoring geomagnetically induced currents (GICs) in power lines at the Kola Peninsula recorded a burst of GIC during the SC. A ≤10% positive pulse of the ionospheric total electron content caused by the SC in the dusk sector was found. On the basis of the multi-instrument information, a validated theory of the magnetosphere–ionosphere response to IP shock may be constructed. Keywords: Storm commencement, Magnetometers, Riometers, GPS/TEC technique, EISCAT radar, Dayside shock aurora m commencement, Magnetometers, Riometers, GPS/TEC technique, EISCAT radar, Dayside shock simplicity of such an impact, the complexity of impulsive and quasiperiodic phenomena stimulated by an IP shock in the ultra-low-frequency (ULF) band (timescales from few seconds to tens of minutes) turns out to be surpris- ingly large. An observed response to an IP shock or dis- continuity may be rather complicated due to the nearly simultaneous excitation of several transient processes. The IP shock is known to stimulate a global compression of the magnetosphere (Araki 1994), with excitation of transient field-aligned current systems (Fujita et al. 2005), generation of transient ULF waves in a wide frequency band from Pc1 (Parkhomov 1990) to Pc5–6 (Yumoto et al. © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract 1994), triggering of VLF wave activity and enhance- ment of energetic electron precipitation (Gail et al. 1990), brightening of the dayside aurora (“shock aurora”) at auroral (Spann et al. 1998; Vorobiev et al. 2008) and sub-auroral (Liou et al. 2003) latitudes, sudden vertical Geomagnetic and ionospheric response to the interplanetary shock on January 24, 2012 V. B. Belakhovsky1, V. A. Pilipenko2, Ya. A. Sakharov3, D. L. Lorentzen4,5 and S. N. Samsonov6 Belakhovsky et al. Earth, Planets and Space (2017) 69:105 DOI 10.1186/s40623-017-0696-1 Open Access Correspondence: space.soliton@gmail.com 2 Geophysical Center, Moscow, Russia Full list of author information is available at the end of the article Introduction An impact of interplanetary (IP) shock or tangential dis- continuity onto the magnetosphere, observed by ground magnetometers as storm sudden commencement (SC), is a convenient probing signal for the experimental study of near-Earth space. Most SC events are associated with IP shocks (Smith et al. 1986), and, therefore, we will use both terms interchangeably. During a relatively short time period (<10 min), significant energy and momentum is pumped into the magnetosphere from the solar wind. Modelers often use a SC event to validate the numerical models of the solar wind–magnetosphere–ionosphere interaction (Fujita et al. 2003; Sun et al. 2015). Despite the *Correspondence: space.soliton@gmail.com 2 Geophysical Center, Moscow, Russia Full list of author information is available at the end of the article Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 2 of 25 Page 2 of 25 shifting of the ionospheric plasma (Yumoto et al. 1989), prompt injection and energization up to MeV energies of magnetospheric particles (Blake et al. 1992), and trigger- ing of a nightside “compression” magnetic bay or even a substorm (Lyons 1996; Liou et al. 2003). In addition, a SC pulse serves as an indicator of the onset of a more intense solar wind driving and buffeting the magnetosphere. (Motoba et al. 2009). The PI was related to diffuse auro- ral brightening that occur several degrees equatorward of the preexisting discrete aurora, while the MI was associ- ated with the discrete auroral brightening poleward of the diffuse aurora (Nishimura et al. 2016). f Because of multiple aspects of the IP shock impact on the magnetosphere and ionosphere, any comprehen- sive description can be achieved only with the use of multiple instruments, both in space and on the ground. Magnetometers, riometers, optical imagers, satellite particle detectors, and ionospheric radars have become a standard tool for monitoring the solar wind–magne- tosphere–ionosphere coupling. For an examination of the ionospheric response to solar wind pressure pulses, Jayachandran et al. (2011) suggested using the GPS global navigation satellite system. This technique that provides information on total electron content (TEC) along a radio path satellite—ground receiver is sensitive enough to detect the ionospheric response to transient and periodic disturbances in the ULF band (Watson et al. 2015; Belakhovsky et al. 2016). The case study of a TEC response to an IP shock by Jin et al. (2016) suggested that a shock-generated TEC variation is a “duplication” of the shock aurora observed by UV sensors onboard the Defense Meteorological Satellite Program (DMSP) sat- ellites. However, the idea that TEC variation is merely a “duplication” of the shock aurora met with some issues. The observed shock-generated TEC enhancement was a single 2–3-min pulse with a delayed start and peak time indicating the propagating shock compression, while the diffuse shock aurora can last much longer, ~10–20 min. Therefore, a possible mechanism for the modification of the ionosphere by a magnetospheric ULF disturbance has not been firmly established. An examination of the impact on the ionosphere by disturbances of differ- ent physical natures with simultaneous data from mag- netometers, riometers, ionospheric radars, and GPS/TEC receivers may provide an insight into the mechanism of the magnetosphere–ionosphere coupling.h gf g g Though the basic physics of the related phenomena is qualitatively understood, some aspects of the IP shock impact on the magnetosphere–ionosphere system have not been sufficiently established. Based on the analy- sis of ground magnetometer data, Araki (1994) sug- gested a phenomenological model of SC, in which an observed disturbance is interpreted as the superposition of a stepwise compression of the geomagnetic field (DL component) and bipolar impulse, composed of a short preliminary impulse (PI) followed by a main impulse (MI). The nature of a stepwise increase in the geomag- netic field (DL) is now clearly identified as a result of global compression of the magnetosphere. The MI and PI are supposedly associated with transient magneto- spheric field-aligned current (FAC) systems driven by different mechanisms. The sequential occurrence of two transient FAC systems in the magnetosphere produces large-scale ionospheric vortex-like current systems on the morning and evening sides. The MI vortex is clock- wise/anticlockwise driven by the FAC flowing into/out of the ionosphere on the dawn/duskside, while the PI vortex has an opposite polarity. The PI currents are sup- posed to be related to inertial currents of a compressional wave propagating through the magnetosphere, while the MI currents are associated with diamagnetic currents of tailward-propagating pressure structures along the magnetopause.hi The magnetospheric cusp is the first region to directly respond to IP shocks in geospace, so detailed studies focusing on the shock-related phenomena around the dayside cusp are essential in revealing the physics of its early response. Dayside auroras observed right after the impingement of solar wind pressure pulses or IP shocks, referred to as “shock aurora,” exhibit some features that are different from typical auroral activity. Auroral behav- iors were suggested to differ during PI and MI, indicating that these differences can be used potentially as optical counterparts of SC-related FACs (Motoba et al. 2009). Initial brightening of shock aurora usually occurs near the ionospheric footprint of the dayside cusp and propa- gates toward dawnside and duskside along the auroral oval with speeds of several km/s (Holmes et al. 2014). In the afternoon sector, the auroral counterpart of the PI was attributed to mainly proton precipitation, while the MI observed shortly thereafter was assumed to be accompanied by both proton and electron precipitation The IP shock impact has an important practical aspect as a driver of geomagnetically induced currents (GICs). GICs represent a significant challenge for society, given its strong dependence on a stable electricity supply (e.g., Knipp 2015 and references therein). GICs arise from induced geoelectric fields that are caused by geomag- netic field variations. The largest GICs are produced by fast changes in the geomagnetic field (dB/dt). The main phase of a geomagnetic storm or onset of a substorm is a cause of a high dB/dt and, therefore, is a risk factor for power systems at high latitudes. For example, the Hydro Quebec collapse was associated with a storm-related dB/ dt = ~480 nT/min. However, GIC impacts to power sys- tems have been observed at much smaller rates of dB/ Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 3 of 25 Page 3 of 25 encounter with the IP shock. The IP shock arrives at GEOTAIL on ~1500 UT, when the solar wind and IMF parameters increased to N = ~20 cm−3, B = ~20 nT, and Bz = ~15 nT (Fig. 1). The observed step-like jumps in the plasma density, velocity, temperature, and IMF magni- tude evident in Fig. 1 make it possible to associate the recorded parameters from the GEOTAIL disturbance with a fast forward shock. dt = ~100 nT/min (Kappenman 2006). Some power sys- tem failures have been associated with the occurrence of a SC or sudden impulse (SI) prior to the storm main phase (Zhang et al. 2015). Although the ΔB associated with SC/SIs is small compared to ΔB during substorms, the dB/dt can still be large enough to induce dangerous GICs in power systems. As an example, the destruction of the New Zealand power system transformer reported by Béland and Small (2004) coincided with a SC. Distur- bances with dB/dt > 100 nT/min can occur during SC in the auroral region, and the magnitude of the SC-associ- ated dB/dt is not necessarily related to the intensity of the following magnetic storm (Fiori et al. 2014). This shock causes an increase in SYM-H index to ~20 nT and a short impulsive increase in the auroral elec- trojet index (AE) to ~800 nT (Fig. 1). After that, the AE remained at elevated levels. Geomagnetic response on the ground to SC In this study, we consider the magnetospheric and ion- ospheric response to the IP shock on January 24, 2012, as observed with satellite magnetometers and particle detectors, ground magnetometers and riometers, aurora imagers, and the EISCAT radar. In addition, we have used data from the GPS satellite navigation system to reveal an ionospheric response to the IP shock. We attempt to compose a comprehensive pattern of the global magneto- sphere–ionosphere response, because such multi-instru- ment studies can provide modelers with the potential to cross-calibrate different effects. In our case study, we attempted to consider some fine details of the geomag- netic field, ionosphere, and auroral response to IP shocks using local multi-instrument observations. In particular, we examined the correspondence between the fine struc- ture of the SC geomagnetic disturbance with the auroral, ionospheric, and TEC short-time variations. We discuss the features of the SC-related phenomena that are well understood and those that still require further considera- tion and modeling. We analyze data from latitudinal networks of magnetic stations in different local time (LT) sectors. The codes of the selected stations, their geographic coordinates, and geomagnetic latitudes Φ and longitudes Λ are given in Table 1. The locations of magnetic stations, riom- eters, optical images, and GPS receivers are shown in Fig. 2. Movements of the pierce points of the satellite/ GPS receiver radio paths intersecting the ionosphere at 300 km are shown for the period 1500–1530 UT. During the SC, the noon meridian is near the eastern coast of Canada, and the CARISMA magnetometer array (Mann et al. 2008) is in the morning sector. The profile of the CARISMA stations along geomagnetic longitude Λ of ~330° showing SC-associated magnetic disturbances at various latitudes in the morning sector (LT = ~8.5) is shown in Fig. 3. A negative PI (marked by the empty tri- angle) preceding a positive MI is observed at very high geomagnetic latitudes only, Φ > 72°. At lower latitudes (below ~68°), only positive MI (marked by the filled tri- angle) is recorded. Interplanetary shock on January 24, 2012h In this LT sector, the MI triggers quasi-monochromatic slowly decaying pulsations (coined Psc4–5 by Saito and Matsushita (1967)) at various latitudes, with periods from T = ~5 min at Φ ~ 72° (RANK) to T = ~1.5 min at Φ = ~58°–60° (PINA and THRF). An oscillatory response at intermediate latitudes Φ = ~66° (GILL) is lacking.h Interplanetary shock on January 24, 2012 The arrival of an IP shock is revealed according to the OMNI database on January 24, 2012, at ~1500 UT as a rapid jump in the solar wind velocity from ~400 up to ~700 km/s, an interplanetary magnetic field (IMF) mag- nitude B increase from ~3 up to ~30 nT, and the enhance- ment of the solar wind density N up to ~15 cm−3. The shock impact occurs during northward IMF Bz = ~15 nT. The resultant increase in the solar wind flow dynamic pressure is P = ~10 nPa. However, the OMNI database shows a pre-shock spike (a few minutes before the arrival of the IP shock). The examination of the WIND satellite raw magnetic and plasma 3-s data reveals that this pre- shock spike is the result of an error introduced by the algorithm in the OMNI database, predicting propagated solar wind data to the subsolar bow shock.h The stations along the Russian Arctic shore (AMD, DIK, TIX, and PBK) are in the night sector. The signa- ture of the SC-associated PI and MI can be seen even at some of those nighttime stations (e.g., TIX). The SC triggers the development of a magnetic bay at PBK with amplitude up to ~500 nT (Fig. 4), though the IMF Bz remains northward both before and after the IP shock. The auroral indices demonstrate a bursty increase during SC up to AE = ~800 nT (Fig. 1), amplitude upper index (AU) = ~500 nT, and amplitude lower (AL) = ~−300 nT (the latter are not shown). The SC triggers a “compres- sion” bay at PBK, while a further association of this mag- netic bay with a substorm needs more evidence. Therefore, we present the 1-min solar wind and IMF data from the GEOTAIL satellite, located at a point with GSE coordinates (30.25, −0.62, 3.15) RE during the Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 4 of 25 g. Interplanetary shock on January 24, 2012h 1 The interplanetary shock detected on January 24, 2012, during time interval 1400–1900 UT by GEOTAIL: the solar wind velocity [Vx compo- nt (km/s)], solar wind density N (cm−3), plasma temperature (K), IMF magnetic field magnitude B (nT) and component Bz (nT), SYM-H index (nT), d AE index (nT) Fig. 1 The interplanetary shock detected on January 24, 2012, during time interval 1400–1900 UT by GEOTAIL: the solar wind velocity [Vx compo- nent (km/s)], solar wind density N (cm−3), plasma temperature (K), IMF magnetic field magnitude B (nT) and component Bz (nT), SYM-H index (nT), and AE index (nT) We analyze in detail the multi-instrument data from the dusk sector LT = ~17 (Scandinavia) (Fig. 2). The IMAGE magnetometers (Tanskanen 2009) record a “clas- sical” SC, with a positive MI and a preceding negative PI at 1504 UT (Fig. 5). At a latitude of ~67°, the PI changes its polarity from negative (TRO) to positive (NOR), while the MI changes polarity between 68° and 74°. This polar- ity reversal of the geomagnetic disturbance is commonly interpreted as evidence of a vortex-like structure of the ionospheric currents associated with PI and MI. interpreted as evidence of a vortex-like structure of the ionospheric currents associated with PI and MI. At latitudes of ~65°–67° (NOR, IVA, TRO, and SOD), fast-decaying transient Psc5 pulsations with T = ~5–6 min are triggered by the SC (Fig. 5). A period of these pulsations is latitude dependent, so they can be associated with Alfvenic oscillations of different geo- magnetic shells, but not with cavity oscillations. Their At latitudes of ~65°–67° (NOR, IVA, TRO, and SOD), fast-decaying transient Psc5 pulsations with T = ~5–6 min are triggered by the SC (Fig. 5). A period of these pulsations is latitude dependent, so they can be associated with Alfvenic oscillations of different geo- magnetic shells, but not with cavity oscillations. Their At latitudes of ~65°–67° (NOR, IVA, TRO, and SOD), fast-decaying transient Psc5 pulsations with T = ~5–6 min are triggered by the SC (Fig. 5). A period of these pulsations is latitude dependent, so they can be associated with Alfvenic oscillations of different geo- magnetic shells, but not with cavity oscillations. Their Belakhovsky et al. Interplanetary shock on January 24, 2012h Earth, Planets and Space (2017) 69:105 Page 5 of 25 Page 5 of 25 Table 1 Magnetometers, riometers, and GPS receivers Stations Code Geographic latitude (°) Geographic longitude (°) Geomagnetic latitude (°) Geomagnetic longitude (°) Ny Alesund NAL 78.92 11.95 75.2 112.1 Longyearbyen LYR 78.20 15.82 75.1 113.0 Barentsburg BAR 78.07 14.23 75.4 110.4 Hornsund HOR 77.00 15.60 74.1 109.6 Hopen Island HOP 76.51 25.10 73.1 115.1 Bear Island BJN 74.50 19.20 71.5 108.1 Nordkapp NOR 71.09 25.79 67.7 109.4 Tromso TRO 69.70 18.94 66.6 102.9 Abisko ABK 68.35 18.82 65.3 101.8 Ivalo IVA 68.56 27.30 65.1 108.6 Kiruna KIR 67.84 20.42 64.7 102.6 Sodankyla SOD 67.37 26.63 63.9 107.3 Oulu OUJ 64.52 27.23 60.9 106.1 Hankasalmi HAN 62.25 26.60 58.7 104.5 Jyväskylä JYV 62.42 25.28 58.5 105.7 Lovozero LOZ 67.97 35.08 64.2 114.5 Dixon DIK 73.50 80.60 67.9 155.7 Tixie TIX 71.59 128.78 65.7 196.9 Pebek PBK 70.83 170.90 64.8 223.3 Amderma AMD 69.47 61.42 61.7 147.7 Taloyoak TALO 69.54 266.45 72.3 330.9 Rankin Inlet RANK 62.82 267.89 72.2 336.0 Fort Churchill FCHU 58.76 265.92 68.3 333.5 Gillam GILL 56.37 265.36 66.0 333.1 Sanikiluaq SNKQ 56.50 280.80 66.5 357.0 Island Lake ISLL 53.86 265.34 63.6 333.4 Pinawa PINA 50.20 263.96 60.0 331.7 Thief River Falls THRF 48.03 263.64 57.8 331.5 Table 1 Magnetometers, riometers, and GPS receivers propagation, but with an apparent velocity even larger than that of the PI. waveforms differ considerably from the Psc4–5 pulsa- tions on the morning flank (Fig. 3). Therefore, the oscil- latory responses of the magnetosphere on the IP shock at the morning and evening flanks are independent. (See figure on next page.) Fig. 2 A map with selected magnetometers (triangles), riometers (asterisks), and GIC stations (boxes) in Scandinavia used for the study of the SC event on January 24, 2012 (upper panel). Movement of the GPS pierce points (intersections with the ionosphere at 300 km of raypaths between GPS satellite and ground receiver) during the period 1500–1530 UT is shown by a line between two asterisks. Bottom panel the same as in the upper panel, but for Canada. Dark dots denote the geomagnetic projections of the THEMIS and GOES satellites Magnetic and particle response to SC in the near‑Earth space l A detailed examination of the PI at stations IVA–SOD– OUL–HAN, covering latitudes from ~59° to ~65° (Fig. 5), reveals its apparent poleward propagation. This effect may be interpreted as a delay owing to different Alfven pulse propagation times from an excitation region in the equatorial magnetosphere toward the ionosphere along different field lines with latitude-dependent Alfven peri- ods TA(Φ). A peak of the MI also demonstrates poleward During this event, the THEMIS-A, D, and E probes are near the morning side magnetopause (in GSE coordi- nates X = ~5 RE, Y = ~−8 RE) above the magnetospheric equatorial plane (Z ~ 5 RE). With respect to the distance from the Earth, they are located at 1503 UT in the follow- ing order: probe E (11.43 RE), D (11.37 RE), and A (11.21 RE). Their geomagnetic projections are shown in Fig. 2 i g p g Fig. 2 A map with selected magnetometers (triangles), riometers (asterisks), and GIC stations (boxes) in Scandinavia used for the study of the SC event on January 24, 2012 (upper panel). Movement of the GPS pierce points (intersections with the ionosphere at 300 km of raypaths between GPS satellite and ground receiver) during the period 1500–1530 UT is shown by a line between two asterisks. Bottom panel the same as in the upper panel, but for Canada. Dark dots denote the geomagnetic projections of the THEMIS and GOES satellites Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 6 of 25 Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 7 of 25 The magnetograms (X-component in 104 nT) of the latitudinal profile of stations TALO, RANK, FCHU, GILL, ISLL, PINA, and THRF from the MA array during the time period 1440–1540 UT. Geomagnetic latitudes are indicated near the station codes. Gray line shows variations of tric absorption at RANK. The PI and MI impulses are denoted with empty and filled triangles, respectfully Fig. 3 The magnetograms (X-component in 104 nT) of the latitudinal profile of stations TALO, RANK, FCHU, GILL, ISLL, PINA, and THRF from the CARISMA array during the time period 1440–1540 UT. Geomagnetic latitudes are indicated near the station codes. Gray line shows variations of riometric absorption at RANK. The PI and MI impulses are denoted with empty and filled triangles, respectfully ~1503:15 UT with an amplitude ΔB = ~70–80 nT (Fig. 6, upper panel). Magnetic and particle response to SC in the near‑Earth space About 30 s later, a short pulse of the plasma Earthward (anti-sunward) speed is detected with a peak amplitude Vx ~ 1000 km/s (probe E), ~700 km/s (probe D), and ~300 km/s (probe A). The plasma density Np starts to grow gradually ~40 s later. (bottom panel). The satellite data are presented in the solar magnetospheric (GSM) coordinate system, with the X-axis directed toward the Sun, the XZ plane containing the dipole axis, and Y toward the dusk. p The THEMIS onboard magnetometers detect a strong compression of the magnetospheric magnetic field at Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 8 of 25 Fig. 4 Data from the Russian Arctic shore magnetometers (X-component in 104 nT) AMD, DIK, TIK, and PBK and riometer TIX during the period 1400–2000 UT. Geomagnetic latitudes and LT are indicated near the station codes Fig. 4 Data from the Russian Arctic shore magnetometers (X-component in 104 nT) AMD, DIK, TIK, and PBK and riometer TIX during the period 1400–2000 UT. Geomagnetic latitudes and LT are indicated near the station codes Detailed comparison of the compressional front recorded by the THEMIS probes reveals a clear propa- gation effect, indicating the time delay between the compression fronts in component Bt at probes E and D of Δt = 15 s and between probes D and A of Δt = 10 s (Fig. 6, top panel). These delays correspond to the appar- ent velocity of the magnetic compression propagation VED = ~300 km/s and VDA = ~240 km/s. For the simulta- neously recorded local plasma and magnetic field param- eters (Np = 0.8 cm−3, background electron temperature Te = 800 eV, and B = 75 nT), the local Alfven velocity is VA (km/s) = 22 B (nT)/ N (cm−3) = ~1800 km/s, and the sound velocity is Vs = ~280 km/s. Magnetic and particle response to SC in the near‑Earth space The estimated com- pression front propagation velocity, neglecting the wave front orientation, is much less than the local Alfven velocity, so the Alfven Mach number is estimated to be MA = ~0.2.h There is the possibility of estimating the shock veloc- ity Vsh and shock normal to the front n from single point measurements using the coplanarity theorem (i.e., the absence of E parallel to n) from the relationships (Oliveira 2017): Vsh = ρ2 ⇀υ2 −ρ1 ⇀υ1 ρ2 −ρ1 ⇀n n = ± (B1 −B2) × (B1 × B2) (B1 −B2) × (B1 × B2) , Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 9 of 25 ρ and V are the plasma density and velocity and B magnetic field vector The indices 1 and 2 denote According to the Harvard-Smithsonian Center for Astrophysics IP shock database (www cfa harvard edu/ The magnetometer data (X-component in 104 nT) along the latitudinal profile HOR-BJN-NOR-TRO-IVA-SOD-OUL-HAN from the IMAGE array the time period 1500–1530 UT Fig. 5 The magnetometer data (X-component in 104 nT) along the latitudinal profile HOR-BJN-NOR-TRO-IVA-SOD-OUL-HAN from the IMAGE array during the time period 1500–1530 UT where ρ and V are the plasma density and velocity and B is the magnetic field vector. The indices 1 and 2 denote the medium parameter prior to (upstream) and after (downstream) a shock, respectively. According to the Harvard-Smithsonian Center for Astrophysics IP shock database (www.cfa.harvard.edu/ shocks/wi_data), the shock under study is a fast forward shock with the front normal orientation characterized by Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 10 of 25 g. 6 The THEMIS-A (black), E (green), and D (red) FGS magnetometer data (in GSM coordinates): total magnitude Bt, total plasma density Np; sma velocities Vx and Vy; fluxes of electrons with E = 31 keV, and components Bx and By during the time period 1502–1506 UT Fig. 6 The THEMIS-A (black), E (green), and D (red) FGS magnetometer data (in GSM coordinates): total magnitude Bt, total plasma density Np; plasma velocities Vx and Vy; fluxes of electrons with E = 31 keV, and components Bx and By during the time period 1502–1506 UT and Vs = ~54 km/s. The resultant Alfven Mach number is MA = 5.8. Magnetic and particle response to SC in the near‑Earth space The delay between the absorption onset and MI may signify either that the PI causes electron accel- eration in the upper ionosphere or that the MI stimu- lates electron pitch angle diffusion into the loss cone in the equatorial plane. In the latter case, a time delay between the geomagnetic and riometer responses is caused by the difference of energetic (>30 keV) electron velocity and Alfven velocity upon the propagation along a field line.h In an inhomogeneous system, a fast mode wave must be partially converted into an Alfven wave. For an azi- muthally large-scale disturbance such as the SC, the excited toroidal Alfven mode should have a dominating radial component of the wave electric field Ex, an azi- muthal component of the magnetic field By, and plasma velocity Vy. However, the expected Alfven wave signa- tures can hardly be found in the THEMIS data (Fig. 6).h The geosynchronous satellite, GOES-13, is in the morning sector (~10 LT) during the SC. The onboard magnetometer records the Earth’s magnetic field com- ponents, with Bp perpendicular to the satellite’s orbital plane, Be pointing earthward parallel to the satellite- Earth center line, and Bn perpendicular to both Bp and Be and pointing eastward. A strong compression of magne- tospheric magnetic field (as evident from the compres- sional component Bp) with an amplitude ΔB = ~70 nT (Fig. 7) is detected. The azimuthal component Bn shows the excitation of the transient long-period toroidal Psc5 pulsations (T = ~5–6 min) and long-lasting short- period Pc3–4 (T = ~40–50 s) pulsations triggered by the SC-related disturbance of the azimuthal component ΔBn = ~10 nT.l i The auroral response to the SC in the post-noon sec- tor has been examined with data from two optical instru- ments at Svalbard. A keogram and time variations of auroral emission intensity for a fixed scan angle have been constructed from digital images from the hyper- spectral all-sky camera NARUSCA II (Barentsburg) (Sigernes et al. 2012). Figure 9 shows the onset of discrete auroral forms triggered by the SC in several spectral lines. The emission lines at wavelengths 486.1 and 656.3 nm are from the proton impact excitation of hydrogen (Hβ and Hα) emissions selected for a scanning angle of −30°. The forbidden 557.7, 630.0, and 636.4 nm emissions are due to electron impact excitation of atomic oxygen [OI]. Magnetic and particle response to SC in the near‑Earth space An estimate using the above formulas for the time 1503 UT for the magnetic field pulse detected inside the magne- tosphere by THEMIS-E gives Vsh = ~770 km/s. The esti- mated velocity is higher than the velocity determined from the angle between the normal and geocentric solar eclip- tic (GSE) X-axis θxn = ~−30° and φ = ~170°. Indeed, for the jump of the magnetic field and velocity owing to the IP shock recorded by WIND satellite on 1440 UT located in the solar wind at (196, −39, −15) RE, the above rela- tionships give Vsh = ~625 km/s for local VA = ~94 km/s and Vs = ~54 km/s. The resultant Alfven Mach number is MA = 5.8. An estimate using the above formulas for the time 1503 UT for the magnetic field pulse detected inside the magne- tosphere by THEMIS-E gives Vsh = ~770 km/s. The esti- mated velocity is higher than the velocity determined from Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 11 of 25 the time delay between THEMIS-D and A. Nonetheless, for local magnetospheric parameters, the Alfven Mach number is MA = ~0.4. Despite some discrepancies, the values of the effective Mach number obtained with both techniques indicate that the compression disturbance can- not be imagined as a shock inside the magnetosphere. energetic electrons with E = ~30–100 keV into the bot- tom ionosphere. Riometers of the Finland network show an occurrence of isolated SC-associated bursts of pre- cipitation at all latitudes from 59° to 74° (Fig. 8). Notice a reversal of the MI polarity between ABK and HOR. The riometer data show apparent poleward propagation. A detailed comparison of riometer absorption and geomagnetic variations (black and red lines) in the morning sector (Fig. 3) and in the post-noon sector (Fig. 8) at sites where both instruments are available demonstrates that the onset of energetic electron pre- cipitation responsible for riometer bursts nearly coin- cides with the onset of PI and is slightly ahead (~1 min) of the MI. The peaks of the riometer absorption nearly coincide with the MI. In the night sector (TIX), the PI also coincides with the onset of the riometer absorption increase. Magnetic and particle response to SC in the near‑Earth space A comparison of hydrogen and oxygen emissions shows that the main proton precipitation (second panel) starts nearly simultaneously (~1509 UT) with main electron precipitation (third panel). In the SC-induced electron precipitation, the intensity of the green emission is much higher than the intensity of the red emission, in contrast to typical dayside cusp aurora. Therefore, for SC-induced precipitation, electrons with higher energies (≫1 keV) dominate.h An immediate particle flux response to the SC looks different in different energy channels of the electron detectors. The magnetic field compression at ~1504 UT recorded by THEMIS and GOES is accompanied by a positive ~5–6 min duration pulse of flux of ener- getic electrons with E > 10 keV (Figs. 6, 7). At higher E, up to relativistic energies of ~2 MeV, this positive pulse is closely followed by a gradual increase in fluxes (not shown).hl The growth of particle fluxes with E = ~30–40 keV starts nearly simultaneously with the positive PI on the ground in the same morning sector (SNKQ) (Fig. 7). The flux of 40-keV electrons ΔJe rapidly increases from ~4 × 104 e/(cm2 s str) to ~2 × 105 e/(cm2 s str) (Fig. 7). A rapid enhancement, more than four times, of fluxes of electrons with relativistic energies, E > 2 MeV, is also observed (Fig. 7). The detected rates of increase, ΔJe/Je, in energetic and relativistic electron fluxes are larger than the bound imposed by adiabatic betatron acceleration, ~ΔB/B = ~0.7. The main growth of the auroral emission intensities is preceded by a weak intensity increase that starts around 1505 UT (Fig. 9). This auroral intensification starts nearly simultaneously with the onset of the riometer burst at NOR and the positive PI in the magnetic record at BAR. Riometer and auroral optical response to SC Riometer observations of cosmic noise absorption pro- vide the capability of monitoring the precipitation of Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 12 of 25 Fig. 7 The magnetometer [magnetic field Be, Bn, and Bp/Bt components in (nT)] and particle (fluxes of electrons Je with E = 40 keV and >2 MeV [in e/ (cm2 s sr)] data from the geosynchronous satellite GOES-13 and conjugate ground station SNKQ [X-component in (104 nT)] during the time interval 1450–1530 UT Fig. 7 The magnetometer [magnetic field Be, Bn, and Bp/Bt components in (nT)] and particle (fluxes of electrons Je with E = 40 keV and >2 MeV [in e/ (cm2 s sr)] data from the geosynchronous satellite GOES-13 and conjugate ground station SNKQ [X-component in (104 nT)] during the time interval 1450–1530 UT The keogram constructed with data from the meridian scanning photometer at a somewhat higher latitude (LYR) shows a simultaneous activation of the red (630.0 nm) and green (557.7 nm) emissions (Fig. 10). The main increase in auroral intensity is preceded by a weak green line emis- sion enhancement at scanning angle ~160° that started at ~1505 UT, simultaneous with the PI and the onset of the riometer burst (HOR). This weak enhancement has the form of three narrow auroral poleward moving arcs that eventually merge into a wide-latitude auroral increase.h The onset of the main increase in red and green emis- sion intensities above LYR (scan angle 90°) is delayed Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 13 of 25 Fig. 8 Cosmic noise absorption (black line) and X-component magnetic field (red line) variation along the profile of stations HOR-ABK-IVA-SOD-OUL- JYV from the Finland riometer network and IMAGE array during 1450–1550 UT Fig. 8 Cosmic noise absorption (black line) and X-component magnetic field (red line) variation along the profile of stations HOR-ABK-IVA-SOD-OUL- JYV from the Finland riometer network and IMAGE array during 1450–1550 UT ~4 min as compared with the peak of the magnetic dis- turbance at LYR (Fig. 10). α > 15° during the SC event is shown in Fig. 2 (bottom panel). The TEC data derived from signals from different GPS satellites received at station CHUR demonstrate that a gradual vTEC growth starts right after the SC (Fig. 11, bottom panel). The relative increase, ∆(vTEC)/(vTEC), reaches 40%. Riometer and auroral optical response to SC The TEC growth during the SC in the morning sector is observed at latitudes above 60° only. These variations are possibly the result of enhancement of the global electric field of the ionosphere (Nayak et al. 2016). Ionospheric TEC response to SC as observed by the GPS system We have examined the TEC response using 30-s data from the worldwide GPS receivers compiled into the IGS system. The slant TEC data have been converted into ver- tical TEC data, vTEC. The TEC response in the morning hours (Canadian sector) is shown in Fig. 11 (bottom panel). The location of the pierce points for all radio paths with elevation angles In the evening sector (Scandinavia), vTEC variations have been constructed for different radio paths between Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 14 of 25 Fig. 9 Keogram of 630.0 nm emission intensity (scan angle—UT) produced from the digital image of the NORUSCA hyperspectral all-sky imager (Barentsburg); intensity variations (in Rayleigh) of emissions produced by proton precipitation 486.1 nm (multiplied by 5) and 656.3 nm, and by elec- tron precipitation 557.7, 630.0, and 636.4 nm, riometer (in dB), and geomagnetic (X-component) (in nT) variations at stations HOR and BAR during the time interval 1500–1530 UT Fig. 9 Keogram of 630.0 nm emission intensity (scan angle—UT) produced from the digital image of the NORUSCA hyperspectral all-sky imager (Barentsburg); intensity variations (in Rayleigh) of emissions produced by proton precipitation 486.1 nm (multiplied by 5) and 656.3 nm, and by elec- tron precipitation 557.7, 630.0, and 636.4 nm, riometer (in dB), and geomagnetic (X-component) (in nT) variations at stations HOR and BAR during the time interval 1500–1530 UT available GPS satellites and the KIRU receiver. The loca- tion of relevant pierce points with α > 15° during the SC event is shown in Fig. 2 (upper panel). With a back- ground of a gradual increase in vTEC, a superposed posi- tive pulse of vTEC is observed with an amplitude up to Δ(vTEC) = ~1 TECu (Fig. 11, upper panel). The relative increase, ∆(vTEC)/vTEC, produced by the pulse is about 8–9%. The onset of the TEC pulse coincides with the PI at sta- tion KIR, but no PI signatures can be seen in the TEC wave- forms. The peak of the TEC pulse coincides with the peak of the geomagnetic MI at KIR. The observed ionospheric response may be caused by different mechanisms. To iden- tify a responsible mechanism, it is important to determine the vertical structure of an ionospheric disturbance. Such a structure can be measured with VHF incoherent radar. available GPS satellites and the KIRU receiver. Ionospheric TEC response to SC as observed by the GPS system The loca- tion of relevant pierce points with α > 15° during the SC event is shown in Fig. 2 (upper panel). With a back- ground of a gradual increase in vTEC, a superposed posi- tive pulse of vTEC is observed with an amplitude up to Δ(vTEC) = ~1 TECu (Fig. 11, upper panel). The relative increase, ∆(vTEC)/vTEC, produced by the pulse is about 8–9%. Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 15 of 25 ams of 630.0, 557.7, and 6563 nm emissions recorded by meridian scanning photometer at LYR (upper panels), vertical intensities of 557.7 nm emissions (in Rayleigh), riometer variations (in dB), and geomagnetic variations (X-component) (in nT) at station HOR during val 1500–1530 UT Fig. 10 Keograms of 630.0, 557.7, and 6563 nm emissions recorded by meridian scanning photometer at LYR (upper panels), vertical intensities of 630.0 nm and 557.7 nm emissions (in Rayleigh), riometer variations (in dB), and geomagnetic variations (X-component) (in nT) at station HOR during the time interval 1500–1530 UT Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 16 of 25 AT d t th SC l i t i th i h i l l it V a b 1 a The vTEC data (in TECu) from satellites with α > 15° in the Scandinavian sector from the KIRU GPS receiver for various GPS satellites 02, 04, 05, 10, 12, 13, 25, 29, 31) during the time period 1450–1520 UT. Bottom panel shows magnetic variations from station KIR (X-com- nt in [104 nT]); b vTEC data (in TECu) from satellites with α > 15° in the Canadian sector from the CHUR GPS receiver for various GPS satellites 02, 04, 10, 13, 16, 23, 30). Bottom panel shows magnetic variations from station FCHU (X-component in 104 nT). Location of the ionospheric e points during the SC is shown in Fig. 2 b Fig. 11 a The vTEC data (in TECu) from satellites with α > 15° in the Scandinavian sector from the KIRU GPS receiver for various GPS satellites (PRN#02, 04, 05, 10, 12, 13, 25, 29, 31) during the time period 1450–1520 UT. Bottom panel shows magnetic variations from station KIR (X-com- ponent in [104 nT]); b vTEC data (in TECu) from satellites with α > 15° in the Canadian sector from the CHUR GPS receiver for various GPS satellites (PRN#02, 04, 10, 13, 16, 23, 30). Ionospheric TEC response to SC as observed by the GPS system Bottom panel shows magnetic variations from station FCHU (X-component in 104 nT). Location of the ionospheric pierce points during the SC is shown in Fig. 2 EISCAT radar response to the SCh clear signatures in the ionospheric plasma velocity Vi coherent with the magnetic disturbance are observed. EISCAT reveals sharp increases in ion and electron tem- peratures, Ti and Te, at heights h > 140 km. In addition to the Ne enhancement, a short-time (1505–1509 UT) burst of electron temperature Te at h > 150 km and ion EISCAT radar response to the SCh The EISCAT incoherent radar system in Tromsø provides a vertical profile up to 200 km for the main ionospheric parameters with a cadence of 1 min. The EISCAT-meas- ured electron density, Ne, evidently shows an impul- sive response to the SC (Fig. 12). At the same time, no Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 17 of 25 Fig. 12 Altitude variations of ionospheric parameters measured by EISCAT during 1430–1530 UT: electron density Ne, ion temperature Ti, electron temperature Te, and ion velocity Vi. Dashed vertical line denotes the moment of PI recorded in the TRO magnetometer data Fig. 12 Altitude variations of ionospheric parameters measured by EISCAT during 1430–1530 UT: electron density Ne, ion temperature Ti, electron temperature Te, and ion velocity Vi. Dashed vertical line denotes the moment of PI recorded in the TRO magnetometer data temperature Ti at h > 140 km is detected. The Ne burst lasts longer than the Te and Ti bursts (from 1504 to 1511 UT). (~1 min). The onset and duration of the EISCAT-meas- ured Ne pulse and the GPS-detected TEC pulse are nearly the same, 1504–1511 UT, which indicates that both pulses are manifestations of the same disturbance. ) The most evident response in the electron density Ne can be seen at h = 100–180 km (Fig. 13). A growth of Ne in the E-layer (120 km) starts simultaneously with the riometer increase and the PI. The increase in Ne from ~1010 to ~1.7 × 1011 m−3 reaches a maximum simulta- neously with the MI peak, but decays during a period of ~2 min, which is longer than the duration of the MI GIC response to the SCi An interesting and significant manifestation of the SC is the burst of GIC in the ground technological systems. The Polar Geophysical Institute (Apatity, Russia) has deployed and operated a system for recording GIC in Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 18 of 25 Fig. 13 Ionospheric and geomagnetic observations during January 24, 2012, at 1430–1530 UT (from top to bottom): altitude distribution of electron density Ne variations measured by EISCAT; variations of Ne at altitude z = 121 km; and magnetic field variations [X-component in (nT)] at TRO and riometer variations (in dB) at IVA Fig. 13 Ionospheric and geomagnetic observations during January 24, 2012, at 1430–1530 UT (from top to bottom): altitude distribution of electron density Ne variations measured by EISCAT; variations of Ne at altitude z = 121 km; and magnetic field variations [X-component in (nT)] at TRO and riometer variations (in dB) at IVA power lines since 2010 (Sakharov et al. 2009; Viljanen 2011). This system consists of five stations elongated in a latitudinal direction in the Kola Peninsula and in Karelia. In this study, we use data from the stations Vykhodnoj (VKH) with geographic coordinates 68.83°N, 33.08°E and Kondopoga (KND) with coordinates 62.21°N, 34.28°E (see map in Fig. 2, upper panel). caused by the SC is about two times higher than the GIC during the subsequent substorm intensifications, though the SC amplitude (~400 nT) is less than the amplitude of the substorm-associated magnetic bay (~600 nT). This fact matches well with a higher amplitude of dX/dt dur- ing the SC as compared with that during the substorm intensifications at ~1830 UT and ~2130 UT. During the SC, a rapid burst of GIC is recorded at both stations VKH and KND (Fig. 14). The amplitude of the GIC at VKH reaches ~30 A. Variations of the GIC are similar to variations of the magnetic field derivative, dX/ dt, at the nearby magnetic station LOZ. During the SC, the dX∕dt level reaches ~180 nT/min. The GIC intensity impact on the geomagnetic field and ionosphere We have presented a comprehensive picture of the IP shock impact onto the magnetosphere, ionosphere, and geomagnetic field, as detected by various instruments. Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 19 of 25 Fig. 14 GIC recordings (in A) at stations VKH and KND on January 24, 2012, at 1400–2400 UT (two bottom panels). Upper panel shows geomagnetic variations (X-component in [104 nT]) and magnetic field variability dX/dt (in nT/s) at nearby magnetic station LOZ Fig. 14 GIC recordings (in A) at stations VKH and KND on January 24, 2012, at 1400–2400 UT (two bottom panels). Upper panel shows geomagnetic variations (X-component in [104 nT]) and magnetic field variability dX/dt (in nT/s) at nearby magnetic station LOZ We hope that the inter-calibration of the observed effects will help modelers to validate the models of the solar wind–magnetosphere–ionosphere coupling during the SC. Though our observations of the SC event can be basically understood in the framework of existing under- standing, some features still require additional considera- tion and modeling. the magnetosphere, but some reaches the Earth. The ground signal is gradually built up with a time, gov- erned by the propagation time of the MHD disturbance from different parts of the magnetopause and the time of sweeping the dayside magnetosphere by a shock. At the front of a fast compressional wave, an Alfven pulse can be generated. This pulse was suggested to produce a high- latitude PI (Nishida 1964). The transmission of an Alfven pulse through the ionosphere should cause a negative H-component disturbance at the ground, preceding a MI. Therefore, the MI and PI are two nearly overlapping wave A rapid displacement of the magnetopause caused by an IP shock induces a magnetic disturbance propagat- ing as a fast compressional wave inward in the magneto- sphere. Most of this wave energy is scattered throughout Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 20 of 25 Page 20 of 25 The lack of an oscillatory Psc5 response at intermedi- ate latitudes, Φ = ~66°, is probably caused by the plas- mapause, because in its vicinity the radial gradient of the Alfven period, TA(Φ), becomes smooth. Therefore, a mode conversion into Alfven oscillations of a geo- magnetic shell is weak in this region. Drivers with band- limited spectral content, exciting transient Alfven wave response in a limited range of latitudes only, may be dif- ferent. impact on the geomagnetic field and ionosphere Since the IP shock discussed here has very similar properties to the one simulated by Oliveira and Raeder (2014), with exactly the same shock impact angle, their conclusions support the argument of the lack of cavity mode excitation in observed in the event discussed in this paper. i Our estimate of the compressional front velocity, from either timing analysis or the jump in plasma parameters, does not support the idea of the shock wave propagation pattern through the magnetosphere. All obtained val- ues of MA = ~0.2–0.4 in the outer magnetosphere indi- cate that the IP shock cannot survive when propagating through the magnetosphere, so the compression front cannot be imagined as a weak shock inside the magne- tosphere, in contrast to the modeling predictions (Sam- sonov et al. 2007). The fact that an apparent propagation velocity of the magnetic field compression is less than the local VA does not exclude the possibility that this com- pression is caused by the fast mode, because in an inho- mogeneous system, the group velocity of this mode is less than VA (Allan and Wright 1998). The equatorward portion (~65°–79°) of the morning auroral oval is occupied by energetic electron precipita- tion observed from the ground as a region with diffuse green emissions, while in the poleward part of the oval (~75°–83°), there is a region of soft electron precipita- tion corresponding to red emission aurora (Zhou et al. 2003). Diffuse green aurora is primarily caused by the precipitation of energetic electrons (0.1–30 keV) over a broad latitude range (CPS type plasma). The red dis- crete aurora is dominated by soft electron precipitation (0.1–1 keV), which corresponds to injections of magne- tosheath plasma. Two types of responses of the dayside/ morning aurora to an IP shock were observed: (1) a “fast” (in ~1 min) onset of the green diffuse aurora manifested as a relatively uniform luminosity structure in the equa- torward edge of the oval and (2) a “slow” (with a delay of ~4–5 min) substantial enhancement of a discrete red emission band at a higher latitude (Motoba et al. 2009; Liu et al. 2015). This two-step development of the post- noon shock aurora supposes the operation of several mechanisms of auroral intensification. impact on the geomagnetic field and ionosphere The wave energy can be transported to a resonant magnetic shell as an evanescent large-scale magneto- pause surface mode or as a waveguide/cavity mode (e.g., Hartinger et al. 2012). Both the surface mode and wave- guide mode would be inevitably resonantly converted into the Alfven field line oscillations. processes of different physical nature. However, there are still no reliable in situ confirmations of the mechanism of the PI generation at the compressional wave front. In the present event, neither the satellite magnetometers nor the plasma sensors have revealed any clear signature of excited Alfven waves. In contrast, the nature of a short pulse of earthward plasma velocity behind the magnetic compression front (at 1502:20 UT in Fig. 6) is unclear. According to global MHD modeling (Samsonov et al. 2007), the transmitted shock should propagate as a weak shock with MA slightly greater than 1 through the mag- netosphere and then partially (~30%) reflect from the plasmapause/ionosphere. Further, the interaction of the reflected shock with the magnetopause should launch a secondary fast compressional wave moving inward into the magnetosphere, etc. This scenario in effect may sug- gest an effective excitation of the MHD cavity mode trapped between the magnetopause and the plasma- pause/ionosphere. However, these theoretical predic- tions on effective reflection of a fast mode from the inner magnetosphere and subsequent formation of a cavity mode have not been supported by our observations. Upon analysis of the space and ground magnetometer data, no global coherent oscillatory response to the SC has been found. In our opinion, a very rare occurrence of the global cavity mode is a natural consequence of a weak reflection of a large-scale MHD mode with scale size ~10 RE from a small target (Earth) with scale size ~1 RE. The lack of cavity mode excitation in our analysis may be supported by numerical MHD simulation of IP shock impacts on the magnetosphere with different nor- mal orientations by Oliveira and Raeder (2014). These authors concluded that the shock impact angle plays a major role in effectively compressing the magneto- sphere, thereby leading to a more favorable scenario for cavity mode excitation due to symmetric magnetosphere compression. They found that inclined strong shocks did not lead to cavity wave excitation, and only shocks with normal incidence did. impact on the geomagnetic field and ionosphere The fast response may be related to a fast magnetosonic pulse propagat- ing across the magnetosphere from the region of the IP shock impact on the magnetopause, with a velocity ~103 Only latitudinally localized transient Psc5 pulsations at the dawn and dusk flanks have been observed that cor- respond to the independent response of resonant field lines in the morning and evening sectors (Samsonov et al. 2011). Some characteristic features of these pulsations indicate their resonant nature, including a frequency dependence on latitude and an apparent poleward prop- agation along the latitudinal magnetometer array (not shown). Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 21 of 25 Page 21 of 25 km/s, whereas a slow intense response is caused by the disturbance propagating along the magnetospheric flanks with a shock velocity in the magnetosheath. inductive downward electric field on the SC compres- sional front propagating from the dayside magnetopause is a probable candidate for triggering a substorm, but not the disturbances generated by the motion of the flank magnetopause. In the case under examination, the IMF is northward both before and after the SC, which excludes the possibility of substorm triggering associated with a southward IMF. A possible trigger may be transported by a fast compression front from the dayside magneto- pause via the magnetosphere or by a disturbance of the solar wind flow around the magnetopause. According to our observations and timing estimates, the inductive downward electric field transported through the magne- tosphere to the magnetotail by a fast mode front may be a probable candidate to trigger the substorm. However, whether all substorms are triggered is still a controversial issue, because not all magnetic bays are associated with auroral substorms. Although ~50% of the IP shocks do produce negative magnetic bays at nightside, only less than 10% of the SC causes an auroral breakup (Liou et al. 2003). Therefore, it is likely that the observed magnetic bay at PBK is not associated with the auroral breakup but with enhanced convection due to a sudden compression and should be classified as a “compression bay.”h y g In the photometer observations at LYR, a response to the IP shock has been observed in the green and red channels starting at ~1505 UT in the equatorward part of the auroral oval (Fig. 10). impact on the geomagnetic field and ionosphere The response develops into two polar moving arcs with comparable intensities (~1 kR) in both channels until 1509 UT, at which time the intensity of the green channel overcomes the red emis- sion line. After this time, the green channel has three to four times the intensity of the red, and the two arcs stop their poleward movement. In addition, a new green arc is formed in the poleward part of the oval (between 30° and 60° scan angle) starting at ~1511 UT. At this time, a very strong proton arc forms, and at ~1519 UT, it com- pletely subsides in the equatorward portion of the oval (between 130° and 165° scan angles). Therefore, our observations do not coincide completely with the previ- ous observations (Motoba et al. 2009; Liu et al. 2015). We do observe a two-step development with weak poleward moving arcs, which after a few minutes stop their pole- ward movement and increase substantially in intensity. However, we do not observe a difference in the red and green channel in terms of behavior, except for the added poleward arc of the green emission channel. i The propagation of the PI from high to low latitudes is often interpreted as an instantaneous transmission of atmospheric THo mode along the ionosphere–ground waveguide (Kikuchi and Hashimoto 2016), though some researchers (e.g., Yumoto et al. 1997; Chi et al. 2001) consider this mechanism questionable because of the observed finite time delay between the PI detected at separated sites. Engebretson et al. (1999) revealed dis- persive propagation of the PI in early morning hours at speeds decreasing from ~150 to ~50 km/s directly away from a source near the cusp using ground observatories in the Arctic and Antarctic regions. For the event under study, the observed poleward PI propagation pattern contradicts the model of atmospheric THo mode near- instantaneous transmission. A sudden brightening of the aurora in response to the IP shock is commonly observed on the dayside within a few hours of LT. The initial brightening is known to immediately propagate to the midnight sector, occupying most of the oval in ~20 min. In contrast to this auroral transient, a sudden brightening of auroral patches with lifetimes of 5–6 min in the midday sub-auroral zone detached from the oval is observed within 1 min of the SC (Liou et al. 2003). impact on the geomagnetic field and ionosphere We are not aware of any model that would be capable of estimating the energy spectrum of precipitat- ing electrons from TEC disturbances. A TEC response to IP shock may be considered as a good observational test for the study of the impul- sive magnetosphere–ionosphere interaction. To date, a TEC response to the SC has not been well examined and understood. A similar effect was reported by Jay- achandran et al. (2011), who found an isolated ~0.6–1.2 TECu pulse produced by a sudden compression of the magnetosphere using CHAIN receivers, but not by the SC. An application of a GPS triangulation technique revealed that the TEC changes propagated with a speed of 3–6 km/s in the anti-sunward direction near noon and ~8 km/s in the sunward direction in the pre-noon lower latitude sector. Simultaneous with the TEC pulse, a small (0.1–0.2 dB) but detectable pulse in absorption was observed. The authors proposed that these TEC changes were seemingly due to electron density enhance- ment in the F region caused by particle precipitation. In the current examined event, the driver of the SC is dif- ferent from the event considered in (Jayachandran et al. 2011). The case study of TEC response to IP shock by Jin et al. (2016) suggested that a shock-generated TEC variation is a “duplication” of the shock aurora due to the same mechanism. The core of this mechanism is the generation of electromagnetic VLF waves that cause elec- tron pitch angle scattering into the loss cone and diffuse aurora. This mechanism was proposed to be responsible for the fast anti-sunward propagation of the diffuse auro- ral-related TEC signal with velocity of ~13 km/s. At the same time, newly established upward field-aligned cur- rents may increase electron precipitation into the iono- sphere and cause discrete aurora along the oval poleward boundary. These currents were suggested to be responsi- ble for the random intensification of the auroral-related TEC signal near the poleward oval boundary. However, the idea that the TEC variation is a “duplication” of the shock aurora met with some issues. The observed shock- generated TEC enhancement was a single 2–3-min pulse with a delayed start and peak time, indicating a propagat- ing shock compression, while the diffuse shock aurora lasted ~10–20 min. impact on the geomagnetic field and ionosphere This auroral transient is associ- ated with the precipitation of preexisting trapped central plasma sheet particles caused by magnetospheric com- pression. The reason for the midday sub-auroral patch being separated from the oval is not understood. We do not think that our MSP observations could be interpreted as a manifestation of the short-lived midday sub-auroral patch, because the Svalbard MSP is located at a latitude that would be too high. Adiabatic compression of the magnetospheric mag- netic field, ΔB, results in a particle energization and flux enhancement, ΔJ, that follows as ΔJ/J ~ ΔB/B. Accord- ing to GOES-13 observations, the sudden enhancement of fluxes of energetic protons is ΔJ/J = ~3 and relativis- tic electrons is ΔJ/J = ~4, while the magnetic field com- pression is just ΔB/B = ~0.6. Therefore, assuming that GOES-13 recorded the enhancement of the energetic particle and magnetic field in the same flux tube, it may be concluded that the detected particle increase is much larger than the limit imposed by the adiabatic betatron mechanism. However, upon the magnetospheric com- pression, the westward induction electric field produces an Earthward plasma drift. As a result of this additional Solar wind changes are believed to externally trigger a substorm (e.g., McPherron et al. 1986; Liou et al. 2003), though it remains a controversial issue whether the sub- storm triggering is caused by the sudden IMF changes that occur simultaneously with a pressure increase, or if the compression itself has a potential to trigger a sub- storm. In addition, the magnetospheric compression and inward (anti-sunward) displacement of magnetospheric plasma is to be accompanied by an induction electric field, Eλ ~ ∂Bz/∂t. Keika et al. (2009) proposed that the Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 22 of 25 Page 22 of 25 time variation, the betatron acceleration increases by a factor of ~2.5 during the SC (Mal’tsev 1996). In general, the problem of the particle energization by the sudden magnetosphere compression is a separate complicated problem, and a simple interpretation based on the idea of betatron acceleration is insufficient to comply with observations. ionization rate occurs at an altitude of 110–160 km. This altitude range corresponds well to the ionospheric den- sity enhancement and plasma heating detected by EIS- CAT. Therefore, the precipitation of soft electrons caused by the shock compression may be responsible for the TEC pulse. impact on the geomagnetic field and ionosphere Along with an additional ionization, the modulation of the ionospheric plasma density may be caused by the interaction of an incident MHD pulse with the iono- sphere–atmosphere–ground system. The precipitation of energetic electrons and ionization of the lower iono- sphere during the SC, responsible for the disturbance recorded by riometers, can influence the bottom E and D layers only. However, a contribution of these layers into TEC is small as compared with that of the F layer. A shear Alfven wave in the magnetosphere does not produce plasma/magnetic field compression, so any compres- sional effects arise upon interaction of this mode with the anisotropic inhomogeneous ionosphere. The responsible mechanisms may comprise an advection across a steep lateral gradient of the ionospheric plasma, or a plasma compression, ΔN/N ~ ΔB/B. According to these mecha- nisms, the lack of Ne response in the F layer evidenced by the EISCAT observations is hard to comprehend. Another possible mechanism is related to field-aligned plasma transport (Belakhovsky et al. 2016). The FAC and flux transported by an Alfven pulse incident onto the ionosphere from the magnetosphere causes an additional accumulation of plasma in regions with strong vertical gradients of the plasma density vertical profile, Ne(z). A significant part of an Alfvenic pulse FAC can be trans- ported by a background suprathermal electron flow into the ionosphere. These electrons do not ionize the iono- spheric plasma additionally, so they cannot be detected by riometers, but they can pump the ionospheric TEC. As a result, the plasma density in the bottom ionosphere may increase. The EISCAT-observed increase in Ne at altitudes of 110–180 km during the TEC pulse is in accordance with this mechanism. The TEC modulation due to transient pumping into the lower ionosphere of the field-aligned electron flux transported by an Alfven wave should be related to ground magnetic variations as ∂t{vTEC(t)} ∝ B(g)(t). In a realistic ionosphere, several mechanisms may operate simultaneously, so it is hard to discriminate their contribution to the TEC variations. A nearly simultaneous response in riometer, TEC, and EIS- CAT Ne may be interpreted as a SC-stimulated precipi- tation of electrons with a wide energy range, from soft (≤keV) to energetic (tens of keV). Whil h j i f GIC di f During the SC event under examination, the TEC response has been only detected for the MI. Funding Th This research is supported by Grant # 16-17-00121 from the Russian Science Foundation (VP, VB) and Grant # 246725 from the Norwegian Research Council (DL). Author details 1 Institute of Physics of the Earth, Moscow, Russia. 2 Geophysical Center, Moscow, Russia. 3 Polar Geophysical Institute, Apatity, Russia. 4 UNIS, Longyear- byen, Svalbard, Norway. 5 BCSS, Bergen, Norway. 6 Institute of Cosmophysical Research and Aeronomy, Yakutsk, Russia. The conclusions derived from the multi-instrument observations of the magnetosphere–ionosphere effects caused by the IP shock on January 24, 2012, follow. The THEMIS determination of the Mach number of the compressional front from both timing analysis and the jump in the plasma parameters (MA ~ 0.2–0.4) does not support the idea of a shock wave propagation pat- tern through the magnetosphere. Clear resonant tran- sient Psc5 oscillations are independently generated on the morning and evening flanks, but no signatures of theoretically predicted cavity mode excitation have been found. We could not find a reliable in situ confirmation of the mechanism of PI generation owing to mode con- version at the front of fast mode propagation through the magnetosphere. The observed poleward propagation pattern of the dayside PI contradicts the model of instan- taneous atmospheric THo mode transmission. Though the SC amplitude is much less than the amplitude of the subsequent substorm magnetic bays, the GIC intensity at the Kola Peninsula power lines caused by the SC is higher than the GIC during the substorm intensifications.f Availability of data and materials The THEMIS data were downloaded from the site, themis.ssl.berkeley.edu, and GOES data from the site, http://satdat.ngdc.noaa.gov/sem/goes/data/. We thank the national institutes that support INTERMAGNET (www.intermagnet. org). The geomagnetic activity and solar wind data were obtained from the OMNIWeb facility (https://omniweb.gsfc.nasa.gov). The authors acknowledge the open data policies of CARISMA, CANMOS (http://gsc.nrcan.gc.ca/geo- mag), the Technical University of Denmark (www.space.dtu.dk), AARI (http:// geophys.aari.ru), MACCS (http://space.augsburg.edu), the Finland riometer network (www.sgo.fi/Data/Riometer/) maintained by the Sodankyla Geophysi- cal Observatory, and IMAGE magnetometer array (http://space.fmi.fi/image/) maintained by the Finnish Meteorological Institute. GPS 30-s data were down- loaded from the IGS Web site (http://garner.ucsd.edu). Conclusionh VBB processed satellite and ground data, VAP wrote the article, YaAS main- tained the GIC stations, DLL maintained and analyzed photometer data, and SNS processed the riometer data. All authors read and approved the final manuscript. The analysis of a SC event recorded simultaneously by various instruments on the ground and in space has shown a surprisingly large variety of associated effects. Though our observations of these effects basically con- firm the earlier results and can be principally understood in the framework of existing notions, some features still require additional consideration and modeling.h Abbreviations CHAIN C di CHAIN: Canadian High Arctic Ionospheric Network; FAC: field-aligned current; GIC: geomagnetically induced current; GPS: global positioning system; IGS: International GNSS Service; IP: interplanetary; MHD: magnetohydrodynamic; SC: sudden commencement; TEC: total electron content; VLF: very low fre- quency; VHF: very high frequency; ULF: ultra-low frequency. impact on the geomagnetic field and ionosphere The energy of the shock aurora electrons, which are diffuse on closed field lines along the oval and discrete along the oval pole- ward boundary, is mainly 1–10 keV, although the range may vary from case to case. The corresponding maximum While the vast majority of GIC studies focus on severe geomagnetic storms, such space weather phenomenon Belakhovsky et al. Earth, Planets and Space (2017) 69:105 Page 23 of 25 Page 23 of 25 as the SC can also produce very large dB∕dt at various latitudes, from an auroral region to the geomagnetic equator (Fiori et al. 2014). Importantly, the SC events are often the precursors to strong geomagnetic storms. Operators of power grid lines take SC as an occurrence of short circuits in the system. The dB∕dt levels of 65–120 nT/min were reported during severe magnetic storms at mid-latitudes that led to power equipment failures (Kap- penman 2003; Pulkkinen et al. 2012). The dB∕dt levels at the equator can be comparable to those in high-latitude regions (Carter et al. 2015). Given previous equipment failures reported for dB∕dt levels less than 100 nT/min (Kappenman 2003), the recorded dB∕dt of ~180 nT/min (Fig. 14) confirms that IP shock impacts are likely to be a significant factor in power stability problems. For infra- structure not designed to cope with space weather, future studies investigating the direct impact of IP shocks on power grids are strongly encouraged. in detail with the fine temporal structure of the SC using a comprehensive set of geophysical observations, including magnetometers, riometers, auroral imagers, satellite detectors, and ionospheric radars. To date, the TEC modulation by the SC has not been comprehen- sively modeled, but on the basis of this multi-instrument information, a validated theory of the magnetosphere– ionosphere response to an IP shock may be constructed. 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https://openalex.org/W2784462923	https://hal-cyu.archives-ouvertes.fr//hal-01918175/file/Thermal%20desorption%20of%20formamide%20and%20methylamin.pdf	English	null	Thermal desorption of formamide and methylamine from graphite and amorphous water ice surfaces	Astronomy & astrophysics	2,018	public-domain	15,300	To cite this version: Henda Chaabouni, Stephan Diana, T. Nguyen, François Dulieu. Thermal desorption of formamide and methylamine from graphite and amorphous water ice surfaces. Astronomy and Astrophysics - A&A, 2018, 612, pp.A47. 10.1051/0004-6361/201731006. hal-01918175 HAL Id: hal-01918175 https://cyu.hal.science/hal-01918175v1 Submitted on 10 Nov 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Public Domain Astronomy & Astrophysics A&A 612, A47 (2018) https://doi.org/10.1051/0004-6361/201731006 © ESO 2018 ABSTRACT methods: laboratory: molecular – molecular processes – astrochemistry – techniques: spectroscopic – ISM: molecules – dust, extinction H. Chaabouni, S. Diana, T. Nguyen, and F. Dulieu LERMA, Université de Cergy-Pontoise, Observatoire de Paris, PSL Research University, Sorbonne Université, UPMC Univ. Paris 06, UMR 8112, CNRS, 5 mail Gay Lussac, 95000 Cergy-Pontoise, France e-mail: Henda.Chaabouni@u-cergy.fr, Francois.Dulieu@obspm.fr versité de Cergy-Pontoise, Observatoire de Paris, PSL Research University, Sorbonne Université, UPMC Univ. Paris 0 NRS, 5 mail Gay Lussac, 95000 Cergy-Pontoise, France Received 18 April 2017 / Accepted 27 December 2017 ABSTRACT Context. Formamide (NH2CHO) and methylamine (CH3NH2) are known to be the most abundant amine-containing molecules in many astrophysical environments. The presence of these molecules in the gas phase may result from thermal desorption of interstellar ices. ms. The aim of this work is to determine the values of the desorption energies of formamide and methylamine from terstellar dust grain surfaces and to understand their interaction with water ice. Methods. Temperature programmed desorption (TPD) experiments of formamide and methylamine ices were performed in the sub- monolayer and monolayer regimes on graphite (HOPG) and non-porous amorphous solid water (np-ASW) ice surfaces at temperatures 40–240 K. The desorption energy distributions of these two molecules were calculated from TPD measurements using a set of inde- pendent Polanyi–Wigner equations. p y g q Results. The maximum of the desorption of formamide from both graphite and ASW ice surfaces occurs at 176 K after the desorption of H2O molecules, whereas the desorption proﬁle of methylamine depends strongly on the substrate. Solid methylamine starts to desorb below 100 K from the graphite surface. Its desorption from the water ice surface occurs after 120 K and stops during the water ice sublimation around 150 K. It continues to desorb from the graphite surface at temperatures higher than160 K. Conclusions. More than 95% of solid NH2CHO diffuses through the np-ASW ice surface towards the graphitic substrate and is released into the gas phase with a desorption energy distribution Edes = 7460–9380 K, which is measured with the best-ﬁt pre-exponential factor A = 1018 s−1. However, the desorption energy distribution of methylamine from the np-ASW ice surface (Edes = 3850–8420 K) is measured with the best-ﬁt pre-exponential factor A = 1012 s−1. A fraction of solid methylamine monolayer of roughly 0.15 diffuses through the water ice surface towards the HOPG substrate. This small amount of methylamine desorbs later with higher binding energies (5050–8420 K) that exceed that of the crystalline water ice (Edes = 4930 K), which is calculated with the same pre-exponential factor A = 1012 s−1. The best wetting ability of methylamine compared to H2O molecules makes CH3NH2 molecules a refractory species for low coverage. Other binding energies of astrophysical relevant molecules are gathered and compared, but we could not link the chemical functional groups (amino, methyl, hydroxyl, and carbonyl) with the binding energy properties. Implications of these high binding energies are discussed. Key words. Thermal desorption of formamide and methylamine from graphite and amorphous water ice surfaces H. Chaabouni, S. Diana, T. Nguyen, and F. Dulieu 1. Introduction Recent thermal desorption experiments of Souda (2016) have shown that methylamine- water interaction is inﬂuenced by the porosity of the ASW ice. An efﬁcient incorporation of CH3NH2 molecules in the porous water ice ﬁlm, deposited on Nickel Ni(111) substrate at 20 K has been observed, with a narrow desorption peak at 160 K, during water ice crystallization. In contrast, no diffusion of CH3NH2 molecules in the ﬁlm interior of the non-porous water ice, deposited at surface temperature 120 K, has been observed in its TPD spectra, even by hydrogen bonding formation with H2O molecules. Despite this advanced work, no experimental values for the desorption energies of methylamine have been pro- vided on water ice. However, a desorption energy value (6584 K) of CH3NH2 is given in http://kida.obs.u-bordeaux1.fr/ species/154/CH3NH2.html from a previous estimation of the OSU gas-grain code of Eric Herbst’s group. Within molecu- lar clouds, the desorption of molecules from icy mantles back into the gas-phase occurs either by thermal, or non-thermal pro- cesses. Non-thermal desorption of molecules may result from the exothermic reactions occurring on the grain surface (Duley & Williams 1993; Vasyunin & Herbst 2013; Dulieu et al. 2013; Minissale et al. 2016) and the impact of cosmic rays or ultra- violet photons (Leger et al. 1985; Hartquist & Williams 1990). Thermal desorption process occurs during the collapse of the dense clouds and the birth of protostars (Garrod et al. 2007). The gravitational energy is converted into radiation, provoking the warm-up of the grain and the desorption of molecules from the icy mantle to the gas environment. In laboratory, temperature- programmed desorption (TPD) is the most effective method for the measurement of ice sublimations over a signiﬁcantly shorter timescale and for the determination of the energy required for desorption. In this work, we investigate thermal desorption experiments of formamide (NH2CHO) and methylamine (CH3NH2) adsor- bate in the sub-monolayer and monolayer regimes on two sur- f h f h f h hi hl i d l i large-scale molecular outﬂow L1157-B1 through the gas-phase reactions between NH2 and H2CO. For prestellar core condi- tions (T < 10 K), despite the inclusion of this very efﬁcient reaction in their chemical network, Vasyunin et al. (2017) do not really overestimate the abundance of formamide in the prestellar core L1544. In these cold prestellar sources, formamide remains undetected according to Jiménez-Serra et al. (2016). 1. Introduction The sce- nario of the gas-phase formation route of formamide relies on the computational work of Barone et al. (2015) and that of Skouteris et al. (2017), who studied the deuteration aspects and conﬁrmed that the gas-phase route for the formation of formamide in the ﬁrst cold prestellar phase is in agreement with the observa- tions. The theoretical discussion (Song & Kästner 2016) about the presence or the absence of the barrier for this key reac- tion (NH2 + H2CO →NH2CHO + H) in the gas phase will end when experimental work is carried on. On the other hand, in the solid state, many experimental reports about the formation of formamide have been published. Energetic electron bombard- ment of CO–NH3 ice mixtures (Jones et al. 2011), ion irradiation of H2O–HCN ices at 18 K (Gerakines et al. 2004), and during the warm-up of photolyzed ice mixtures of H2O, CH3OH, CO, and NH3 (Bernstein et al. 1995), led to the formation of NH2CHO. Recent laboratory experiments of Fedoseev et al. (2016) have also produced formamide molecules by hydrogenation and UV photolysis of NO in CO-rich interstellar ice analogues. The chemical link in the solid phase of HNCO and NH2CHO has been ruled out by H-bombardment experiments of HNCO at low surface temperature (Noble et al. 2015), which do not lead to detectable amounts of NH2CHO molecules. Our group demon- strates that formamide can be produced with high efﬁciencies even without the help of external energy (Nguyen et al., in prep.). A last aspect of the solid phase chemical route concerns the binding energy of formamide. Dawley et al. (2014) have inves- tigated thermal desorption of thick mixed H2O–NH2CHO ices on a silicate (SiO2) grain analogue from 70 to 400 K. These authors have observed a delayed desorption peak of the water ice at 160–200 K, resulting from the diffusion of H2O molecules during the phase transition of formamide accumulated in the ice, followed by a large desorption proﬁle at higher tempera- tures (200–380 K), corresponding to the diffusion of molecules through the high surface area of silicate. The desorption activa- tion energy of formamide measured experimentally from SiO2 substrate has been found to be 14.7 kcal mol−1 (or 7397 K) with a pre-factor A = 1013 s−1. Recently, Wakelam et al. 1. Introduction Non-thermal desorption of molecules may result from the exothermic reactions occurring on the grain surface (Duley & Williams 1993; Vasyunin & Herbst 2013; Dulieu et al. 2013; Minissale et al. 2016) and the impact of cosmic rays or ultra- violet photons (Leger et al. 1985; Hartquist & Williams 1990). Thermal desorption process occurs during the collapse of the dense clouds and the birth of protostars (Garrod et al. 2007). The gravitational energy is converted into radiation, provoking the warm-up of the grain and the desorption of molecules from the icy mantle to the gas environment. In laboratory, temperature- programmed desorption (TPD) is the most effective method for the measurement of ice sublimations over a signiﬁcantly shorter timescale and for the determination of the energy required for desorption. I thi k i ti t th l d ti i t A&A 612, A47 (2018) into the gas phase is often more puzzling than other complex organic molecules because of its high binding energy and its very low interaction with water. In summary, the main forma- tion route of NH2CHO, by gas-phase or surface reactions, has not yet been settled and more observational and experimental work is needed. Another interesting fundamental organic com- pound in biochemistry studied in this work is the methylamine, CH3NH2, which is known to be the most abundant amine- containing molecule after formamide (NH2CHO). Methylamine has been detected in the gas phase through its 202−> 110Aa− state transition in the direction of Sgr B2 and Ori A (Fourikis et al. 1974) towards the giant molecular cloud Sgr B2(N) with a fractional abundance as high as 3 × 10−7 relative to molecu- lar hydrogen (Nummelin et al. 2000). It has also been detected in the coma of comet 67P/Churyumov-Gerasimenko (Altwegg et al. 2016) by the Rosetta space mission. In the gas phase, the forma- tion of the methylamine molecules is completely dependent on radicals CH• 3 and NH• 2 produced by UV photons (Garrod et al. 2008). In the solid phase, the hydrogenation experiments of HCN molecules by H atoms at low surface temperature (Theule et al. 2011) have shown the formation of the fully saturated species CH3NH2 with methanimine as an intermediate. The desorption of these molecules occurred between 140 and 150 K, at slightly lower temperatures than the desorption of the amorphous water ice in their experimental conditions. 1. Introduction (Raunier et al. 2004), but it is no longer in the list of con- ﬁrmed molecules in interstellar ices (Boogert et al. 2015). As for numerous molecules, especially interstellar complex organic molecules, astrochemists would like to put constraint on the for- mation pathway of formamide. In order to discriminate different scenarios of star formation, solid-state and gas-phase formation routes are usually debated because they imply different physi- cal conditions. For both chemical routes, there are arguments coming from observations and others belonging to the physi- cal or chemical properties of NH2CHO. On the observational side, López-Sepulcre et al. (2015) have noticed that NH2CHO is correlated to isocyanic acid (HNCO) abundances. Further- more, the ﬁrst detection of deuterated formamide towards the low protostar IRAS 16293 by ALMA-PILS, which was a D/H ratio of 2% similar to HNCO (Coutens et al. 2016), has been found to be in agreement with the hypotheses that these species are chemically related through grain-surface formation. On the other hand, Codella et al. (2017) have explained well the obser- vation of formamide in the brightest shocked region of the Formamide, NH2CHO, is a species of great relevance in prebi- otic chemistry (Saladino et al. 2012; Barone et al. 2015). This species contains a (quasi) peptide bond (–NH–(C=O)) that is very active in the synthesis of nucleic acid bases, carboxylic acids, sugars (Saladino et al. 2012), and amino acids responsi- ble for the formation of proteins. It is also well known to be the precursor species for the formation of purine and pyrimi- dine bases during the course of chemical evolution leading to the origin of life (Bhushan et al. 2016). Formamide has been observed in the gas phase in several astronomical environments, such as prestellar and protostellar objects (Kahane et al. 2013), massive hot molecular cores (Bisschop et al. 2007), hot cori- nos (López-Sepulcre et al. 2015), and comets, such as C/20012 F6 (Lemmon), C/2013 R1 (Lovejoy) (Biver et al. 2014), and Hale-Bopp (Bockelée-Morvan et al. 2000). Formamide has been once tentatively identiﬁed in interstel- lar ices of star-forming regions by ISO-SWS infrared spectra Article published by EDP Sciences A47, page 1 of 12 A47, page 1 of 12 A47 (2018) into the gas phase is often more puzzling than other complex organic molecules because of its high binding energy and its very low interaction with water. 1. Introduction In summary, the main forma- tion route of NH2CHO, by gas-phase or surface reactions, has not yet been settled and more observational and experimental work is needed. Another interesting fundamental organic com- pound in biochemistry studied in this work is the methylamine, CH3NH2, which is known to be the most abundant amine- containing molecule after formamide (NH2CHO). Methylamine has been detected in the gas phase through its 202−> 110Aa− state transition in the direction of Sgr B2 and Ori A (Fourikis et al. 1974) towards the giant molecular cloud Sgr B2(N) with a fractional abundance as high as 3 × 10−7 relative to molecu- lar hydrogen (Nummelin et al. 2000). It has also been detected in the coma of comet 67P/Churyumov-Gerasimenko (Altwegg et al. 2016) by the Rosetta space mission. In the gas phase, the forma- tion of the methylamine molecules is completely dependent on radicals CH• 3 and NH• 2 produced by UV photons (Garrod et al. 2008). In the solid phase, the hydrogenation experiments of HCN molecules by H atoms at low surface temperature (Theule et al. 2011) have shown the formation of the fully saturated species CH3NH2 with methanimine as an intermediate. The desorption of these molecules occurred between 140 and 150 K, at slightly lower temperatures than the desorption of the amorphous water ice in their experimental conditions. Recent thermal desorption experiments of Souda (2016) have shown that methylamine- water interaction is inﬂuenced by the porosity of the ASW ice. An efﬁcient incorporation of CH3NH2 molecules in the porous water ice ﬁlm, deposited on Nickel Ni(111) substrate at 20 K has been observed, with a narrow desorption peak at 160 K, during water ice crystallization. In contrast, no diffusion of CH3NH2 molecules in the ﬁlm interior of the non-porous water ice, deposited at surface temperature 120 K, has been observed in its TPD spectra, even by hydrogen bonding formation with H2O molecules. Despite this advanced work, no experimental values for the desorption energies of methylamine have been pro- vided on water ice. However, a desorption energy value (6584 K) of CH3NH2 is given in http://kida.obs.u-bordeaux1.fr/ species/154/CH3NH2.html from a previous estimation of the OSU gas-grain code of Eric Herbst’s group. Within molecu- lar clouds, the desorption of molecules from icy mantles back into the gas-phase occurs either by thermal, or non-thermal pro- cesses. 2. Experimental methods The experiments were performed using the FORMOLISM (FORmation of MOLecules in the InterStellar Medium) appara- tus. The set-up is dedicated to study the interaction of atoms and molecules on surfaces of astrophysical interest. The experimen- tal set-up is brieﬂy described here and more details are given in earlier papers (Amiaud et al. 2007; Chaabouni et al. 2012). The apparatus is composed of an ultra-high vacuum (UHV) stain- less steel chamber with a base pressure 10−10 mbar. The sample holder is located in the centre of the main chamber. It is ther- mally connected to a cold ﬁnger of a closed-cycle He cryostat. The temperature of the sample is measured in the range 40– 350 K by a calibrated platinum (Pt) diode clamped to the sample holder, which is made of 1 cm diameter copper block, covered with a highly orientated pyrolytic graphite (HOPG, ZYA-grade) slab. p p p Since our desorption experiments are performed in the sub- monolayer and monolayer regimes, the dimensionless surface coverage (θ = N/Nmono ≤1) is deﬁned as the number density of molecules exposed on the surface (N) by the maximum surface number density of molecules Nmono prior to multi-layer desorp- tion (Noble et al. 2012). At surface coverage saturation (θ = 1), the ﬁrst monolayer of molecules covering the surface is deﬁned as the maximum number density of molecules that populate 1015 adsorption site per cm2 on a ﬂat surface, such as graphite. This deﬁnition of one monolayer unit (1 ML = 1015 molecules cm−2) is kept over this study; this includes amorphous surfaces, such as non-porous ASW ice, where the saturation of the ﬁrst layer of molecules can thus be observed at a value, which differs from the exact value of the deﬁned 1 ML. In our experiments, the ﬁrst monolayer of solid formamide covering the surface of graphite at 40 K was reached after 90 min of exposure time, and that of solid methylamine after beam deposition during 10 min. The ﬂuxes of formamide and methylamine molecules coming from the gas phase and hitting the surface of the sam- ple holder are deﬁned as the ratio of the amount of molecules in the designed 1 ML divided by the exposure time required for surface saturation. These ﬂuxes derived from TPD data and the method described in Noble et al. 1. Introduction (2017) have estimated the binding energy of formamide on water ice sur- face to be (6300 ± 1890) K using a semi-empirical theoretical approach. The discrepancy can obviously come from the cal- culation method, but could also ﬁnd its origin in the amount of NH2CHO interacting with the water ice, which is high in the ﬁrst case (Dawley et al. 2014) and very low in the second case (Wakelam et al. 2017). Experiments performed with thin layers on the surface of the substrate are required for a bet- ter comparison. Anyhow, formamide has a very high value of binding energy that is much higher than that of H2O, which is around 4930 K for crystalline water ice, estimated from the results of Fraser et al. (2001) using a pre-exponential factor A = 1012 s−1. At the current state of the astrochemistry, it is not mandatory to involve the direct formation of formamide on dust grains to explain its observation (or non-observation) in various media, as long as the gas-phase reaction between NH2 and H2CO is supposed to be efﬁcient. If the correlation between HNCO and NH2CHO is conﬁrmed at lower spacial In this work, we investigate thermal desorption experiments of formamide (NH2CHO) and methylamine (CH3NH2) adsor- bate in the sub-monolayer and monolayer regimes on two sur- faces. These surfaces are that of the highly orientated pyrolytic graphite (HOPG) as a laboratory model for carbonaceous grains and the surface of the H2O non-porous amorphous solid water (np-ASW) ice, covering the HOPG substrate and acting as an extremely relevant astrophysical surface analogue. The aim of A47, page 2 of 12 H. Chaabouni et al.: Thermal desorption of formamide and methylamine QMS of the intact parent molecules NH2CHO (m/z = 45 amu), NH2CH3 (m/z = 31 amu), and H2O (m/z = 18 amu), which are only converted into positive ions by electron impact ionization. The QMS is also moved to the upper position to prepare the ﬁlms of the non-porous amorphous solid water (np-ASW) ice on the graphite surface, using a micro-channel array doser (1 cm in diameter), located 2 cm in front of the surface in the UHV cham- ber. 1. Introduction The np-ASW ice ﬁlm of 10 ML thickness is grown on top of the graphite (HOPG) surface, maintained at the temperature of 110 K, by spraying water vapour under a constant deposi- tion pressure in the vacuum chamber of 2 × 10−9 mbar. Water vapour is obtained from deionized liquid H2O, puriﬁed by sev- eral pumping cycles under cryogenic vacuum conditions (Noble et al. 2012). After deposition, the temperature of the surface is kept constant at 110 K for 30 min, until the background pressure in the vacuum chamber is stabilized at 10−10 mbar. Then the sam- ple was cooled to the base temperature 40 K prior to formamide or methylamine deposition. During the cooling of the water ice from 110 to 40 K, the structure and the morphology of the water ice is not changed. Water ice deposited at 110 K remains in the same compact (non-porous) amorphous state at 40 K. this work is to understand the interaction of these two amino molecules with the water ice and to study the effect of the graphitic substrate on the desorption processes. For this purpose, we determine the desorption energy distributions of formamide and methylamine on both surfaces, using TPD measurements and Polanyi–Wigner equation. We compare these distributions to the binding energy of pure water ice. This paper is orga- nized as follows: Sect. 2 explains the experimental methods, Sect. 3 presents the experimental results and the TPD measure- ments of formamide (NH2CHO) and methylamine (CH3NH2) on graphite (HOPG) and on np-ASW ice surfaces, Sect. 4 describes the model developed to measure the desorption energy distribu- tions of formamide and methylamine molecules from graphite and water ice surfaces, Sect. 5 presents the discussion of the results, and ﬁnally Sect. 6 summarizes the main conclusions of this work. 2. Experimental methods (2012) are estimated to be 2.6 × 1010 molecules cm−2 s−1 for NH2CHO beam and 1.6 × 1012 molecules cm−2 s−1 for CH3NH2 beam. In our laboratory experiments, the viscous, liquid formamide is introduced in a pyrex ﬂask, which is placed in a ceramic bath containing a silicone oil acting as a regulator for the tempera- ture. Because of the low source vapour pressure of formamide in the beam line at room temperature, the liquid is warmed up to 50 ◦C using a heating plate with a motor speed of 280 revolutions per minute. Methylamine compound is also used under its aque- ous solution with a weight percent in water of 40 wt%, meaning that there is 40 g of NH2CH3 solute for every 100 g of water solvent. Methylamine is more volatile than formamide and its source pressure at the inlet of the beam line is set to 1.00 mbar at room temperature. At these experimental conditions, formamide and methylamine beams were aimed at the surface of the HOPG substrate, held at 40 K, for several deposition times, using a triply differentially pumped beam line, orientated 60◦with respect to the surface of the sample holder. 3.1. Formamide p The UHV chamber is also equipped with a movable quadrupole mass spectrometer (Hiden Analytical QMS) operat- ing in the range 1–100 amu (atomic mass unit) with a Channel- tron detector. The QMS is orientated face-on to the beam line to characterize the gas composition of formamide and methy- lamine beams. It is placed 5 mm in front of the surface of the sample holder to apply the TPD technique using a custom Lab- View software that maintains a linear heating rate of 0.2 K s−1. During the warm-up phase of the sample, from 40 to 240 K, the species desorbing from the surface into the gas phase are ion- ized, or fragmented into cracking patterns, by electron impact in the ion source of the QMS. The TPD curves shown in next sections exhibit the most intensive signals monitoring with the 3.1.1. Formamide on graphite surface Figure 1 shows the desorption curves of formamide (NH2COH) given by the QMS signals of m/z = 45, for several exposure doses (0.15, 0.30, 0.61, and 1.00 ML) on the graphite HOPG surface. The ﬁfth TPD spectrum corresponds to the multi-layer regime of formamide given by the higher exposure dose of 1.66 ML. All the TPD spectra exhibit desorption peaks between 160 and 200 K. Although the integrated area under the TPD curves increases with the exposure dose of formamide from 0.15 to 1.66 ML, the desorption peak remains at the same temperature of 176 K, mainly for surface coverage higher than 0.30 ML. For the lowest dose of 0.15 ML, the TPD peak is shifted to a slightly A47, page 3 of 12 A&A 612, A47 (2018) A47 (2018) Fig. 2. TPD curves of formamide (NH2CHO) giving the desorption rates (ML s−1) of the mass (m/z = 45) as a function of the temperature of the surface (K). The red line indicates the TPD signal of NH2CHO deposited on graphite (HOPG) surface at 40 K; the black dashed line shows the TPD signal of NH2CHO deposited on the np-ASW ice sur- face at 40 K; and the blue dashed line indicates the TPD signal of H2O (m/z = 18) for 10 ML thickness of the np-ASW ice ﬁlm prepared at 110 K on the HOPG surface and cooled down to 40 K (scaled by 0.0007). Fig. 1. Desorption rates of formamide (NH2CHO; m/z = 45), expressed (ML s−1) as a function of the temperature of the surface for several expo- sure doses (0.15, 0.30, 0.61, 1.00, and 1.66 ML) of solid formamide on the cold HOPG surface at 40 K. The linear heating rate of the samples is 0.2 K s−1. The linear dependence of the integrated area under the TPD curves as a function of the exposure time is shown in the inset. Fig. 2. TPD curves of formamide (NH2CHO) giving the desorption rates (ML s−1) of the mass (m/z = 45) as a function of the temperature of the surface (K). The red line indicates the TPD signal of NH2CHO deposited on graphite (HOPG) surface at 40 K; the black dashed line shows the TPD signal of NH2CHO deposited on the np-ASW ice sur- face at 40 K; and the blue dashed line indicates the TPD signal of Fig. 1. 3.1.2. Formamide on np-ASW ice Figure 2 shows the desorption curve of solid NH2CHO (m/z = 45) on np-ASW ice, peaking at 176 K. For comparison, the des- orption curve of the non-porous ASW ice ﬁlm, given by the signal (m/z = 18) at 150 K, is also shown. Formamide is clearly desorbing from the surface at a higher temperature than the water ice. The TPD curve of formamide from the water ice surface is similar to that obtained previously from the graphite HOPG surface for the same exposure dose of 1 ML. This means that the desorption of formamide molecules into the gas phase is not affected by the water ice substrate. The calculations of the inte- grated areas below the TPD curves show that more than 0.95 ML of solid formamide is likely to diffuse from the surface of the water ice towards the graphite HOPG substrate. This diffusion probably occurs during the warm-up phase of the sample and the reorganization of the water ice. Since our TPD experiments last few minutes, the diffusion timescale of the molecules from the surface of the water ice to the graphitic substrate is expected to be about few seconds. Because formamide desorbs after water, its binding energy from the HOPG surface is expected to be higher than that of H2O molecules. Fig. 3. Desorption rates, expressed (ML s−1), of methylamine (CH3NH2) as a function of the temperature of the surface (K) for several exposure doses of solid methylamine (0.18, 0.26, 0.46, 0.70, 0.80, 1.0, and 1.40 ML) on the cold HOPG surface at 40 K. The linear heating rate of the samples is 0.2 K s−1. strong interaction of the NH2CH3 molecules with the graphitic surface in the sub-monolayer regime. As the surface coverage increases, molecules are forced to populate progressively less tightly bound sites of the graphitic HOPG surface, provoking earlier desorption at about 113 K. The area of the second desorp- tion peak of CH3NH2 at 113 K increases with the increase of the exposure doses in the sub-monolayer regime, and its maximum shifts towards a lower temperature (106 K) once the surface cov- erage is saturated and the ﬁrst monolayer is formed. The strong desorption peak at 106 K for 1.40 ML corresponds to the onset of the multi-layer desorption of solid methylamine, where CH3NH2 is bound to CH3NH2 ice by hydrogen bonds. 3.1.2. Formamide on np-ASW ice strong interaction of the NH2CH3 molecules with the graphitic surface in the sub-monolayer regime. As the surface coverage increases, molecules are forced to populate progressively less tightly bound sites of the graphitic HOPG surface, provoking earlier desorption at about 113 K. The area of the second desorp- tion peak of CH3NH2 at 113 K increases with the increase of the exposure doses in the sub-monolayer regime, and its maximum shifts towards a lower temperature (106 K) once the surface cov- erage is saturated and the ﬁrst monolayer is formed. The strong desorption peak at 106 K for 1.40 ML corresponds to the onset of the multi-layer desorption of solid methylamine, where CH3NH2 is bound to CH3NH2 ice by hydrogen bonds. 3.1.1. Formamide on graphite surface Desorption rates of formamide (NH2CHO; m/z = 45), expressed (ML s−1) as a function of the temperature of the surface for several expo- sure doses (0.15, 0.30, 0.61, 1.00, and 1.66 ML) of solid formamide on the cold HOPG surface at 40 K. The linear heating rate of the samples is 0.2 K s−1. The linear dependence of the integrated area under the TPD curves as a function of the exposure time is shown in the inset. Fig. 2. TPD curves of formamide (NH2CHO) giving the desorption rates (ML s−1) of the mass (m/z = 45) as a function of the temperature of the surface (K). The red line indicates the TPD signal of NH2CHO deposited on graphite (HOPG) surface at 40 K; the black dashed line shows the TPD signal of NH2CHO deposited on the np-ASW ice sur- face at 40 K; and the blue dashed line indicates the TPD signal of H2O (m/z = 18) for 10 ML thickness of the np-ASW ice ﬁlm prepared at 110 K on the HOPG surface and cooled down to 40 K (scaled by 0.0007). higher temperature compared to the higher doses. The inset in Fig. 1 shows the linear evolution of the integrated areas under the TPD curves (for surface dose up to 1 ML and above) as a function of the exposure times. Fig. 3. Desorption rates, expressed (ML s−1), of methylamine (CH3NH2) as a function of the temperature of the surface (K) for several exposure doses of solid methylamine (0.18, 0.26, 0.46, 0.70, 0.80, 1.0, and 1.40 ML) on the cold HOPG surface at 40 K. The linear heating rate of the samples is 0.2 K s−1. 3.2. Methylamine Similarly to formamide, the adsorption–desorption experiments of methylamine CH3NH2 molecules were investigated both on graphite (HOPG) and on np-ASW ice surfaces. 3.2.1. Methylamine on graphite surface Figure 3 shows the desorption curves of methylamine (m/z = 31) for several exposure doses (ML) on graphite surface held at 40 K. For very small doses (0.18, 0.26 ML), molecules occupy the most energetically favourable adsorption sites and are tightly bound to the surface of the HOPG, desorbing late from the surface, at temperatures up to 160 K. This small TPD peak is assigned to the A47, page 4 of 12 3.2.2. Methylamine on np-ASW ice 2001) r(T) = −dN dt = ANae−Edes/kBT, (1) (1) where r(T) is the desorption rate (molecule cm−2 s−1), N is the number density of molecules adsorbed on the surface, expressed (molecules cm−2), a is the order of the desorption process, A is the pre-exponential factor expressed (molecule−a+1 cm2a−2 s−1), which can be considered to be the attempt frequency of molecules at overcoming the barrier to desorption, kB is the Boltzmann constant (kB = 1.38 × 10−23 J K−1), t is the time (s), T is the absolute temperature of the surface (K), and Edes is the activation energy for desorption (kJ mol−1) or (K), where 1 kJ mol−1 = 120 K. Our results are partially in agreement with those of Souda (2016), who observed a broad TPD curve of methylamine from the np-ASW ice of D2O (8 ML), deposited at 120 K on the nickel Ni(111) substrate. This latter comprises two desorption peaks: one peak at 120 K corresponding to multilayer desorption of methylamine and another at about 150 K corresponding to methylamine mixed with water coming from the aqueous solu- tion. In addition, the TPD spectrum of Souda (2016) shows no desorption peak of methylamine at surface temperature 160 K and above, suggesting that, in contrast to our case, no strong interaction between methylamine and the underlying Ni(111) substrate takes place in their experiments. If our experiments performed on the np-ASW ice of 10 ML thickness allowed the incorporation of methylamine from the water ice surface towards the graphite substrate, one can conclude that the diffusion of methylamine molecules through the non-porous water ice sur- face depends on the substrate below the water ice (HOPG, Ni). The nickel Ni(111) surface is expected to have fewer energetic binding sites than HOPG surface. However, if differences of substrates are seen for methylamine, one could say that similar differences are expected for formamide. The role of the substrate is discussed later. In the particular case of simple molecular adsorption, the pre-exponential factor A may also be equated with the frequency of vibration of the bond between the molecule and substrate; this is because every time this bond is stretched during the course of a vibrational cycle can be considered an attempt to break the bond and hence an attempt at desorption. However, we point out that this is a simplistic view of a complex problem. 3.2.2. Methylamine on np-ASW ice This desorption behaviour of methylamine on the np-ASW ice is observed for two close exposure doses of 0.30 and 0.35 ML. The desorption peaks at 137 K, which occur before the desorption of pure water ice at 150 K, and even the crystallization phase of H2O at about 145 K, correspond to CH3NH2 molecules desorbing from the surface of the water ice. Our experimental results show that, when increasing slightly the exposure dose of methylamine on the np-ASW ice surface from 0.30 to 0.35 ML, the fraction of CH3NH2 ice desorbing from the water ice surface at 137 K increases from 0.16 to 0.21 ML, while those desorbing directly from the HOPG surface at T ≥160 K decreases slightly from 0.16 to 0.14 ML, respectively. This means that by increasing the amount of methylamine on top of the water ice surface, CH3NH2 molecules bind further with the water ice by hydrogen bonds and desorb between 120 and 140 K, rather than diffuse through the water ice surface and desorb later from the HOPG substrate at 160–220 K. The fraction of the refractory CH3NH2 molecules desorbing from the HOPG substrate after water ice sublimation is about 0.15 ML. Fig. 4. TPD curves of methylamine giving the desorption rates (ML s−1) of the mass (m/z = 31) as a function of the temperature of the sur- face (K). The thin red line indicates 1 ML of CH3NH2 deposited on graphite HOPG substrate at 40 K; the thick red line represents 0.30 ML of CH3NH2 deposited on the np-ASW ice surface held at 40 K; the thick magenta line indicates 0.35 ML of CH3NH2 deposited on the np-ASW ice surface at 40 K; and the blue dashed line represents TPD curve of H2O (m/z = 18) for 10 ML of the np-ASW ice ﬁlm prepared at 110 K on the HOPG surface and cooled down to 40 K (scaled by 0.001). of molecules from the surface into the gas phase can be described in terms of an Arrhenius law, given by the Polanyi–Wigner Eq. (1) (Noble et al. 2012; Dohnálek et al. 3.2.2. Methylamine on np-ASW ice The physical observable parameter is the desorption ﬂux; the pre-exponential factor and the binding energy are actually two degenerated parameters linked in the Polanyi–Wigner equation. Therefore, at anytime, a couple of solutions (A, Edes) should be considered. Because the peaks of the TPD curves of formamide appear at a single temperature value for all the exposure doses up to 1.61 ML, the desorption of formamide is considered to be of ﬁrst order, and the desorption energy is expected to be dependent on the coverage (Fraser et al. 2001). 3.2.2. Methylamine on np-ASW ice The desorption peaks at 137 K, which occur before the desorption of pure water ice at 150 K, and even the crystallization phase of H2O at about 145 K, correspond to CH3NH2 molecules desorbing from the surface of the water ice. Our experimental results show that, when increasing slightly the exposure dose of methylamine on the np-ASW ice surface from 0.30 to 0.35 ML, the fraction of CH3NH2 ice desorbing from the water ice surface at 137 K increases from 0.16 to 0.21 ML, while those desorbing directly from the HOPG surface at T ≥160 K decreases slightly from 0.16 to 0.14 ML, respectively. This means that by increasing the amount of methylamine on top of the water ice surface, CH3NH2 molecules bind further with the water ice by hydrogen bonds and desorb between 120 and 140 K, rather than diffuse through the water ice surface and desorb later from the HOPG substrate at 160–220 K. The fraction of the refractory CH3NH2 molecules desorbing from the HOPG substrate after water ice sublimation is about 0.15 ML. 0.35 ML) deposited on the surface during 5 and 6 min, respec- tively (thick red and magenta lines). These curves are compared to the TPD curve of solid methylamine on the graphite (HOPG) surface for a 1 ML exposure dose (thin red line) and to the H2O TPD curve (m/z = 18) of pure np-ASW ice, with 10 ML thick- ness, grown on the HOPG surface at 110 K (blue dashed line). As shown in Fig. 4, the large TPD peak of solid CH3NH2 (1 ML) from the HOPG surface shows a multi-layer desorption peak at lower surface temperatures centred at 106 K, a shoulder at about 140–150 K resulting from the mixture of the methylamine with the water coming from the aqueous phase, and a long desorp- tion tail at higher temperatures, up to 220 K. When CH3NH2 is deposited on top of the np-ASW ice ﬁlm of 10 ML thickness, prepared at 110 K and cooled down to 40 K, the maximum of the desorption peak of methylamine is shifted to the higher tem- perature, 137 K, and a large desorption peak with a maximum centred at 160 K appears as a tail at higher surface tempera- tures up to 220 K. 3.2.2. Methylamine on np-ASW ice Figure 4 shows the TPD curves of solid methylamine (m/z = 31) from the np-ASW ice surface for two small doses (0.30 and A47, page 4 of 12 H. Chaabouni et al.: Thermal desorption of formamide and methylamine Fig. 4. TPD curves of methylamine giving the desorption rates (ML s−1) of the mass (m/z = 31) as a function of the temperature of the sur- face (K). The thin red line indicates 1 ML of CH3NH2 deposited on graphite HOPG substrate at 40 K; the thick red line represents 0.30 ML of CH3NH2 deposited on the np-ASW ice surface held at 40 K; the thick magenta line indicates 0.35 ML of CH3NH2 deposited on the np-ASW ice surface at 40 K; and the blue dashed line represents TPD curve of H2O (m/z = 18) for 10 ML of the np-ASW ice ﬁlm prepared at 110 K on the HOPG surface and cooled down to 40 K (scaled by 0.001). 0.35 ML) deposited on the surface during 5 and 6 min, respec- tively (thick red and magenta lines). These curves are compared to the TPD curve of solid methylamine on the graphite (HOPG) surface for a 1 ML exposure dose (thin red line) and to the H2O TPD curve (m/z = 18) of pure np-ASW ice, with 10 ML thick- ness, grown on the HOPG surface at 110 K (blue dashed line). As shown in Fig. 4, the large TPD peak of solid CH3NH2 (1 ML) from the HOPG surface shows a multi-layer desorption peak at lower surface temperatures centred at 106 K, a shoulder at about 140–150 K resulting from the mixture of the methylamine with the water coming from the aqueous phase, and a long desorp- tion tail at higher temperatures, up to 220 K. When CH3NH2 is deposited on top of the np-ASW ice ﬁlm of 10 ML thickness, prepared at 110 K and cooled down to 40 K, the maximum of the desorption peak of methylamine is shifted to the higher tem- perature, 137 K, and a large desorption peak with a maximum centred at 160 K appears as a tail at higher surface tempera- tures up to 220 K. This desorption behaviour of methylamine on the np-ASW ice is observed for two close exposure doses of 0.30 and 0.35 ML. 4.2. Desorption energies of methylamine where Ni is the population of molecules desorbing from the surface with a desorption energy Ei at a surface temperature T. Similar calculations of the desorption energy distribution of methylamine have been carried out both on graphite HOPG and np-ASW ice surfaces. Assuming a ﬁrst order desorption process of methylamine on graphite surface, the best ﬁts of the TPD curves for various values of the surface exposure coverage, N (0.25, 0.46, 0.71, and 1.0 ML) of methylamine, obtained from the model with the pre-exponential factor A = 1012 s−1, are shown in Fig. 6. p gy p We also assume that the desorption rate r(T) is the sum of the desorption rates ri(T), i.e. r(T) = n X i=1 ri(T) = AN1e−E1/kBT + ANie−Ei/kBT + · · · , (5) where ri(T) = ANie−Ei/kBT (6) r(T) = n X i=1 ri(T) = AN1e−E1/kBT + ANie−Ei/kBT + · · · , (5) (5) where where The top panel of Fig. 7 shows the surface population Ni (ML) of solid methylamine on graphite (HOPG) surface as a function of the desorption energy Ei of methylamine (K), for the ﬁrst monolayer exposure coverage (N = 1 ML). The desorp- tion energy distribution of methylamine from graphite is found to range between 3010 and 8420 K, where a large percentage (∼0.80 ML) of molecules desorbing with low binding energies (3010–4454 K) correspond to the multi-layer and monolayer des- orptions of CH3NH2 species from the graphite surface. The range of the desorption energy (5050–8420 K) is assigned to the surface population (∼0.15 ML) of methylamine that desorbs from the very energetic adsorption sites of the graphite (HOPG) surface. ri(T) = ANie−Ei/kBT (6) is the desorption rate of the molecular population, Ni, desorbing from the surface at a temperature T, with a pre-exponential factor A, and a desorption energy Ei. p gy To ﬁt the TPD data for an exposure dose N of molecules on the surface, we use the desorption rate r(T), given by Eq. (5), then we set two values Emin and Emax for the desorption energy of the molecules from the surface, and we choose a value for the pre-exponential factor A. When we run the program, we obtain the different populations Ni of molecules desorbing from the surface with the desorption energy Ei, at a surface temperature T, varying from Tmin to Tmax. 4.1. Desorption energies of formamide The top panel of Fig. 5 shows the best ﬁts of the TPD curves of formamide for various surface doses (0.14, 0.30, 0.61 and 1.0 ML) on the graphite (HOPG) surface. These best ﬁts are derived from Eq. (5) with a pre-exponential factor A = 1018 s−1. The use of a low pre-exponential factor (A = 1012 s−1) in our modelling simulations does not reproduce the TPD curves on the graphite surface perfectly. The bottom panel of Fig. 5 shows the surface population Ni of formamide molecules (ML) as a func- tion of the desorption energy Ei (K) on the graphite (HOPG) surface for the ﬁrst monolayer exposure coverage (N = 1 ML). As shown in Fig. 5, bottom panel, most of the surface population (Pn i=1 Ni = ∼0.97 ML) of formamide on HOPG surface releases into the gas phase with a desorption energy distribution, ranging from 7460 to 9380 K. 4.2. Desorption energies of methylamine The best ﬁt of the TPD data is obtained when the calculated curve matches well the experimen- tal results, and the three computational parameters (A, Ei, Ni) are well constrained, where the pre-exponential factor A is between 1012 and 1018 s−1, the desorption energy barrier Ei is between Emin and Emax, and all the surface populations Ni satisfy the relation (4). In the case of Fig. 7, bottom panel, where an exposure dose N = 0.30 ML of solid methylamine is deposited on top of the np-ASW ice ﬁlm, the desorption energy distribution of these molecules is reduced to the range (3900–8420 K), with traces of small energies (2770–3610 K) corresponding to methy- lamine multi-layer desorption. The bottom panel of Fig. 7 also shows that a surface population of about 0.15 ML of CH3NH2 leaves the water ice surface with desorption energies (3900– 4500 K), which is lower than the binding energy value (4930 K) of crystalline water ice, calculated with the same pre-exponential factor A = 1012 s−1 from the desorption parameters (Edes, A) of Fraser et al. (2001). Furthermore, the desorption energies (5050–8420 K), which are higher than those of the amorphous water ice, are attributed to the fraction (Pn i=1 Ni = ∼0.15 ML) of methylamine population that diffuses through the water ice surface towards the highest binding sites of the HOPG sub- strate, and then desorbs into the gas phase after water ice sublimation. A&A 612, A47 (2018) The same desorption energy distribution (7460–9380 K) is obtained by ﬁtting the TPD data of formamide deposited on top of the np-ASW ice ﬁlm of 10 ML thickness, covering the graphitic HOPG substrate. This main result is explained by the fast and efﬁcient (>95%) diffusion of NH2CHO molecules from the water ice surface towards the HOPG substrate, at surface temperature, 40 K, or more probably during the warming-up phase of the ices. The bottom panel of Fig. 5 also shows that for a pre-exponential factor A = 1018 s−1, the maximum of the desorption energy distribution of formamide (7700 K) is higher than that of pure amorphous water ice (Edes = 6490 K), calcu- lated with the same pre-exponential value A = 1018 s−1 from the parameters (Edes, A) of the amorphous water ice (Fraser et al. 2001). This means that NH2CHO molecules are more tightly physisorbed to the surface of the HOPG substrate than H2O molecules. where the pre-exponential factor A for desorption (s−1). where the pre-exponential factor A for desorption (s−1). 1 where the pre-exponential factor A for desorption (s−1). The desorption rate r(T) (ML s−1) can be expressed using Eq. (3) p p p The desorption rate r(T) (ML s−1) can be expressed using Eq. (3) r(T)ML s−1 = r(T) β−1 R Tmax Tmin dN dt · dT , (3) (3) where R Tmax Tmin dN dt · dT is the integrated area below the TPD curve of the designed 1 ML exposure dose between Tmin and Tmax. In this model, we consider that each exposure dose N (ML) of molecules on the surface is the sum of the different fraction exposure doses Ni (ML), N = n X i=1 Ni = N1 + N2 + · · · , (4) (4) 4. Analysis For TPD of ﬁrst order, a = 1, the desorption rate r(T) become A simple mathematic model has been developed to ﬁt the TPD curves of formamide and methylamine in the sub-monolayer and monolayer regimes. At any surface coverage, thermal desorption r(T) = −dN dt = ANe−Edes/kBT, (2) r(T) = −dN dt = ANe−Edes/kBT, (2) A47, page 5 of 12 A&A 612, A47 (2018) H. Chaabouni et al.: Thermal desorption of formamide and methylamine Fig. 5. Modelling results of the desorption rates (ML s−1) and the desorption energy distribution (K) of formamide on graphite (HOPG) surface. Top panel: red crosses indicate TPD data of NH2CHO (m/z = 45) on HOPG surface for different exposure doses of 0.14, 0.30, 0.61, and 1.0 ML; blue lines: the best ﬁts of the TPD curves of NH2CHO from the HOPG surface, calculated with the pre-exponential factor A = 1018 s−1 are shown. Bottom panel: the surface population (ML) of NH2CHO on HOPG surface as a function of the desorption energy (K) is shown. The desorption energy distribution (Ei) and the surface population (Ni) of formamide are derived from Eq. (5) with the best pre-exponential factor A = 1018 s−1 for the exposure dose N = Pn i=1 Ni = 1 ML. Fig. 5. Modelling results of the desorption rates (ML s−1) and the desorption energy distribution (K) of formamide on graphite (HOPG) surface. Top panel: red crosses indicate TPD data of NH2CHO (m/z = 45) on HOPG surface for different exposure doses of 0.14, 0.30, 0.61, and 1.0 ML; blue lines: the best ﬁts of the TPD curves of NH2CHO from the HOPG surface, calculated with the pre-exponential factor A = 1018 s−1 are shown. Bottom panel: the surface population (ML) of NH2CHO on HOPG surface as a function of the desorption energy (K) is shown. The desorption energy distribution (Ei) and the surface population (Ni) of formamide are derived from Eq. (5) with the best pre-exponential factor A = 1018 s−1 for the exposure dose N = Pn i=1 Ni = 1 ML. (HOPG) and non-porous ASW ice surfaces in the sub-monolayer and monolayer regimes. The desorption energy distributions, Edes,dist, of formamide and methylamine are obtained with dif- ferent pre-exponential factors: A = 1018 s−1 for formamide and A = 1012 s−1 for methylamine. In the case of formamide (NH2CHO), the decrease in the pre-exponential factor A from 1018 to 1012 s−1, affects the quality of the TPD ﬁts, and reduces the value of the desorption energy of the molecules by 30%. As already mentioned, A and Edes are degenerated parameters that can be derived from a desorption ﬂux at a given temperature T. However, the dynamic of desorption gives more constraints on the couple of parameters. 5. Discussion We developed a model using the Polanyi–Wigner equation to reproduce the TPD proﬁles and determine the desorption energy distributions of NH2CHO and CH3NH2 molecules on graphite A47, page 6 of 12 A47, page 6 of 12 H. Chaabouni et al.: Thermal desorption of formamide and methylamine g. 5. Modelling results of the desorption rates (ML s−1) and the desorption energy distribution (K) of formamide on graphite (HOPG) surfa p panel: red crosses indicate TPD data of NH2CHO (m/z = 45) on HOPG surface for different exposure doses of 0.14, 0.30, 0.61, and 1.0 M ue lines: the best ﬁts of the TPD curves of NH2CHO from the HOPG surface, calculated with the pre-exponential factor A = 1018 s−1 are sho ottom panel: the surface population (ML) of NH2CHO on HOPG surface as a function of the desorption energy (K) is shown. The desorpt ergy distribution (Ei) and the surface population (Ni) of formamide are derived from Eq. (5) with the best pre-exponential factor A = 1018 r the exposure dose N = Pn i=1 Ni = 1 ML. HOPG) and non-porous ASW ice surfaces in the sub-monolayer nd monolayer regimes. The desorption energy distributions, d di of formamide and methylamine are obtained with dif already mentioned, A and Edes are degenerated parameters t can be derived from a desorption ﬂux at a given temperat T However the dynamic of desorption gives more constrai H. Chaabouni et al.: Thermal desorption of formamide and methylamine H. Chaabouni et al.: Thermal desorption of formamide and methylamine H. Chaabouni et al.: Thermal desorption of formamide and methylamine In this work, we want to determine the binding energy distributions of molecules, and once done, we need to be able to compare the different values, originating from different works or analysis methods. where A1 and A2 are the pre-exponential factors (s−1), associ- ated with the desorption energies E1,des and E2,des, respectively, of the adsorbed molecules on the surface, and T is the experi- mental temperature (K) of the surface, at which the desorption is observed. already estimated and discussed previously. Simple molecules (like H2) seems to have a pre-exponential factor for desorption usually close to 1012–13 s−1 (Amiaud et al. 2006). On the con- trary, Doronin et al. (2015) have shown that the same molecule (CH3OH) could have different pre-exponential factors for differ- ent substrates or regimes (multi-layers or sub-monolayer). These authors proposed a method to derive the pre-exponential factor from TPD data. It has been suggested by Tait et al. (2005) that the pre-exponential factor A for the desorption depends on the poten- tial interaction of the adsorbate substrate system, and the number of degrees of freedom of the molecule when it passes from the adsorbate state to the gas-phase transition state. These authors have demonstrated the increase of the pre-factor A for desorp- tion with the chain length of the molecule from the value 1013 to 1019 s−1, and have explained this variation of A by the increase in the rotational entropy available to the molecules in the gas-like transition state for desorption. The difference in the pre- exponential factor values is an experimental fact. In this work, we want to determine the binding energy distributions of molecules, and once done, we need to be able to compare the different values, originating from different works or analysis methods. In Table 1, we can compare the value of the binding energy of methanol (CH3OH) on graphite with those of methylamine and formamide. We can see that even though the pre-exponential factors and analysis methods are different, all the approximated values of binding energies given here are close and can be com- pared to the value of the binding energy of the amorphous water ice derived from the same calculation method. The important result here is that, in any case, the binding energy of CH3OH is lower than that of water. H. Chaabouni et al.: Thermal desorption of formamide and methylamine Experimentalists seeks for getting the best ﬁt from their experiments, and as evidence, the best ﬁt is obtained with extreme differences in the pre-exponential factor values (A = 1018 and A = 1012 s−1) for two apparently similar molecules. The degeneracy of A and Edes has been A47, page 7 of 12 A47, page 7 of 12 A&A 612, A47 (2018) Fig. 6. Modelling results of the desorption rates (ML s−1) of methylamine on graphite (HOPG) surface. Red crosses: TPD data of CH3NH2 (m/z = 31) on HOPG surface for different exposure doses of 0.25, 0.46, 0.71, and 1.0 ML are shown; blue lines: the best ﬁts of the TPD curves of CH3NH2 from the HOPG surface, calculated with the best pre-exponential factor A = 1012 s−1 are represented. Fig. 6. Modelling results of the desorption rates (ML s−1) of methylamine on graphite (HOPG) surface. Red crosses: TPD data of CH3NH2 (m/z = 31) on HOPG surface for different exposure doses of 0.25, 0.46, 0.71, and 1.0 ML are shown; blue lines: the best ﬁts of the TPD curves of CH3NH2 from the HOPG surface, calculated with the best pre-exponential factor A = 1012 s−1 are represented. already estimated and discussed previously. Simple molecules (like H2) seems to have a pre-exponential factor for desorption usually close to 1012–13 s−1 (Amiaud et al. 2006). On the con- trary, Doronin et al. (2015) have shown that the same molecule (CH3OH) could have different pre-exponential factors for differ- ent substrates or regimes (multi-layers or sub-monolayer). These authors proposed a method to derive the pre-exponential factor from TPD data. It has been suggested by Tait et al. (2005) that the pre-exponential factor A for the desorption depends on the poten- tial interaction of the adsorbate substrate system, and the number of degrees of freedom of the molecule when it passes from the adsorbate state to the gas-phase transition state. These authors have demonstrated the increase of the pre-factor A for desorp- tion with the chain length of the molecule from the value 1013 to 1019 s−1, and have explained this variation of A by the increase in the rotational entropy available to the molecules in the gas-like transition state for desorption. The difference in the pre- exponential factor values is an experimental fact. H. Chaabouni et al.: Thermal desorption of formamide and methylamine Table 1 also shows that for the same employed pre-exponential factor A = 1012 s−1, the maxi- mum of the desorption energy distribution (Edes,max = 5265 K) of NH2CHO from graphite (HOPG) substrate is smaller than the value (6871 K) of the mean desorption energy obtained from silicate (SiO2) interstellar grain analogue surface by Dawley et al. (2014). This difference between our result and that of Dawley et al. (2014) is an indication that the desorption energy of formamide depends on the substrate itself. Table 1 shows the maximum of the desorption energies (Edes,max) of formamide and methylamine from different sub- strates, calculated from the model, with the original suitable pre- exponential factors (A), and with a ﬁxed value (A = 1012 s−1) for better comparison with other relevant astrophysical molecules, using the following desorbing ﬂux relationship: More precisely, we ﬁnd that the binding energy of 0.1 ML of formamide adsorbed on graphite substrate is 5600–5900 K, calculated with A = 1012 s−1. These values are smaller than the value given on silicate substrate (Dawley et al. 2014). This differ- ence may not be only related to the composition of the substrate (silicate, graphite), but could also be due to the difference in the morphology of the substrate, which can easily change the r(T) = A1e−E1,des/kBT = A2e−E2,des/kBT, (7) A47, page 8 of 12 A47, page 8 of 12 H. Chaabouni et al.: Thermal desorption of formamide and methylamine H. Chaabouni et al.: Thermal desorption of formamide and methylamine Fig. 7. Modelling results of the desorption energy distributions (K) of methylamine on graphite (HOPG) and np-ASW ice surfaces. Top pan surface population (ML) of CH3NH2 (m/z = 31) as a function of the desorption energy (K) for an exposure dose N = 1 ML of CH3NH2 on t HOPG surface. Bottom panel: surface population (ML) of NH2CH3 as a function of the desorption energy (K) for an exposure dose N = 0.30 M of CH3NH2 on the surface of the np-ASW ice ﬁlm of 10 ML thickness, prepared at 110 K and cooled down to 40 K. The desorption ener distribution (Ei) and the surface population (Ni) of methylamine are derived from Eq. H. Chaabouni et al.: Thermal desorption of formamide and methylamine (5) with the best pre-exponential factor A = 1012 s−1 for the two exposure doses N = Pn i=1 Ni = 1 ML on HOPG and N = Pn i=1 Ni = 0.30 ML on np-ASW ice surfaces. binding energy distribution of the molecules by a few tens of percent (Fillion et al. 2009). In our experiments, the desorption energy of formamide from the surface of the non-porous ASW ice cannot be measured because the water substrate desorbs before formamide, that is deposited on top of it. Actually, the whole binding energy distribution of formamide from graphite (5056–6990 K), calculated with A = 1012 s−1 for 1 ML cover- age, is higher than the binding energy values of water ice in its amorphous (4810 K), and crystalline state (4930 K). Unlike for- mamide, solid methylamine can desorb under different forms, in the multi-layer regime (NH2CH3–NH2CH3) with low desorption energies (3010–3490 K) from the np-ASW ice surface with des- orption energies (3900–4500 K) and even later from the HOPG substrate with higher desorption energies (5050–8420 K). The different desorption energies of methylamine calculated in this work with respect to that of pure amorphous water ice (4810 K) is due to its wetting ability compared to H2O molecules. In fact, CH3NH2 species are able to spread readily and uniformly over the surface of the graphite (HOPG) or the amorphous water ice by hydrogen bonds, thereby forming a thin and continuous ﬁlm in which physisorbed molecules populate a large distribution of surface binding sites. The proportion of molecules desorbing, prior, during, or after the water ice varies with the doses of methylamine, but each time the fraction of methylamine desorb- ing after the water ice is about 0.15 ML. However, the fraction of methylamine co-desorbing with the crystalline phase of the water ice is very small at about 0.03 ML. We can see in Fig. 4 that the desorption of methylamine is very reduced during the desorption of the water ice at around 150 K. The desorption of methylamine at 137 K, before the sublimation of the water ice, is delayed compared with its desorption without water at T = 106 K (Fig. 4), indicating a stronger interaction of methylamine with the water molecule than with itself. In most astrophysical models, the whole desorption of each molecule is described by its (unique) binding energy. H. Chaabouni et al.: Thermal desorption of formamide and methylamine (5) with the best pre-exponential factor A = 1012 s−1 for t two exposure doses N = Pn i=1 Ni = 1 ML on HOPG and N = Pn i=1 Ni = 0.30 ML on np-ASW ice surfaces. binding energy distribution of the molecules by a few tens of percent (Fillion et al. 2009). In our experiments, the desorption energy of formamide from the surface of the non-porous ASW the surface of the graphite (HOPG) or the amorphous water i by hydrogen bonds, thereby forming a thin and continuous ﬁl in which physisorbed molecules populate a large distribution Fig. 7. Modelling results of the desorption energy distributions (K) of methylamine on graphite (HOPG) and np-ASW ice surfaces. Top panel: surface population (ML) of CH3NH2 (m/z = 31) as a function of the desorption energy (K) for an exposure dose N = 1 ML of CH3NH2 on the HOPG surface. Bottom panel: surface population (ML) of NH2CH3 as a function of the desorption energy (K) for an exposure dose N = 0.30 ML of CH3NH2 on the surface of the np-ASW ice ﬁlm of 10 ML thickness, prepared at 110 K and cooled down to 40 K. The desorption energy distribution (Ei) and the surface population (Ni) of methylamine are derived from Eq. (5) with the best pre-exponential factor A = 1012 s−1 for the two exposure doses N = Pn i=1 Ni = 1 ML on HOPG and N = Pn i=1 Ni = 0.30 ML on np-ASW ice surfaces. Fig. 7. Modelling results of the desorption energy distributions (K) of methylamine on graphite (HOPG) and np-ASW ice surfaces. Top panel: surface population (ML) of CH3NH2 (m/z = 31) as a function of the desorption energy (K) for an exposure dose N = 1 ML of CH3NH2 on the HOPG surface. Bottom panel: surface population (ML) of NH2CH3 as a function of the desorption energy (K) for an exposure dose N = 0.30 ML of CH3NH2 on the surface of the np-ASW ice ﬁlm of 10 ML thickness, prepared at 110 K and cooled down to 40 K. The desorption energy distribution (Ei) and the surface population (Ni) of methylamine are derived from Eq. H. Chaabouni et al.: Thermal desorption of formamide and methylamine For some other molecules, such as NH2OH and NH2CHO, the binding energy is higher than that of (H2O) water ice, and we can consider these molecules as refrac- tory species, compared to water molecules. Refractory does not have the same meaning here as sometimes used in shock-regions studies or in geology. Here, it is the remaining part of the molec- ular mantle after the sublimation of water. Before this study, we were wondering if some simple chemical properties could explain the common trends of the binding energies. It is usually discussed in terms of hydrophilic and hydrophobic proprieties of the molecules or in terms of hydrogen bonding formation with water. We can sort molecules in two groups: those with energies are measured for pure species and therefore, the answer would be Edes ≃3000 K with A = 1012 s−1. But in an astro- physical context, the proportion of methylamine in icy mantles is certainly less than a few % as it is not positively detected in ices (Boogert et al. 2015). Therefore, it would be better to take the low concentration limit of our experiments (∼0.15 ML), which would lead to an estimation of the binding energy at around 6000 K, well above the value of those of the water ice. This low coverage value is certainly very dependent on the substrate and represents the wetting properties of the surface for a spe- ciﬁc molecule. This is probably why Souda (2016) did not ﬁnd a late desorption of methylamine in the case of metallic surface Ni(111). The key question is to ﬁnd out whether at low concentra- tion, the binding energy of a speciﬁc molecule is higher than the binding energy of the water ice. Table 1 reveals that the binding energies of methanol (CH3OH) and formaldehyde (H2CO) are lower than that of amorphous (H2O) water ice, and so we can consider these molecules as volatile, or at least they would des- orb with the water ice mantle. For some other molecules, such as NH2OH and NH2CHO, the binding energy is higher than that of (H2O) water ice, and we can consider these molecules as refrac- tory species, compared to water molecules. Refractory does not have the same meaning here as sometimes used in shock-regions studies or in geology. Here, it is the remaining part of the molec- ular mantle after the sublimation of water. H. Chaabouni et al.: Thermal desorption of formamide and methylamine 4. Notes. Col. 4 gives the maximum of the desorption energy (Edes,max) (K) obtained with the best-ﬁt pre-exponential factor A values (s−1) for NH2CHO, NH2CH3, and other astrophysical relevant molecules (NH2OH, CH3OH, H2CO, and H2O) taken from the literature. For a comparison, Col. 5 gives the desorption energies (Edes,max) of all the molecules calculated with the typical pre-exponential factor A = 1012 s−1 for various grain surfaces, using Eq. (7) of ﬂux desorption (A1e−E1,des/kBT = A2e−E2,des/kBT), and the values of A and Edes,max in Col. 4. a binding energy less than that of water, such as CH3OH, NH3, H2CO, and another group with a binding energy, at low cover- age, that are higher than the binding energy of water, among which we can ﬁnd H2O2 (Dulieu et al. 2017), NH2OH (Congiu et al. 2012), and NH2CHO. As a matter of fact, the hydrophobic nature of the methyl (–CH3) group, or the hydrophilic nature of the amino (–NH2) or hydroxyl (–OH) groups, cannot explain our observations. Only detailed calculations could probably give a real understanding of this empirical sorting. energies are measured for pure species and therefore, the answer would be Edes ≃3000 K with A = 1012 s−1. But in an astro- physical context, the proportion of methylamine in icy mantles is certainly less than a few % as it is not positively detected in ices (Boogert et al. 2015). Therefore, it would be better to take the low concentration limit of our experiments (∼0.15 ML), which would lead to an estimation of the binding energy at around 6000 K, well above the value of those of the water ice. This low coverage value is certainly very dependent on the substrate and represents the wetting properties of the surface for a spe- ciﬁc molecule. This is probably why Souda (2016) did not ﬁnd a late desorption of methylamine in the case of metallic surface Ni(111). The key question is to ﬁnd out whether at low concentra- tion, the binding energy of a speciﬁc molecule is higher than the binding energy of the water ice. Table 1 reveals that the binding energies of methanol (CH3OH) and formaldehyde (H2CO) are lower than that of amorphous (H2O) water ice, and so we can consider these molecules as volatile, or at least they would des- orb with the water ice mantle. H. Chaabouni et al.: Thermal desorption of formamide and methylamine Usually, binding A47, page 9 of 12 A47, page 9 of 12 A&A 612, A47 (2018) tributions (Edes,dist) (K), Col. 3, of NH2CHO and CH3NH2 obtained in this work with the best-ﬁt pre-exponential Table 1. Desorption energy distributions (Edes,dist) (K), Col. 3, of NH2CHO and CH3NH2 obtained in this work with the best-ﬁt pre-exponential factor A values, expressed (s−1). Molecule Substrate Edes,dist (A) Edes,max (A) Edes,max References (A = 1012 s−1) K (s−1) K (s−1) K NH2CHO HOPG 7460–9380 (1018) 7700 (1018) 5265 This work Edes,dist = 5056–6990 NH2CHO H2O This work NH2CHO SiO2 7397 (1013) 6871 Dawley et al. (2014) CH3NH2 NH2CH3 3010–3490 (1012) 3307 (1012) 3307 This work CH3NH2 H2O 3900–4500 (1012) 4269 (1012) 4269 This work CH3NH2 HOPG 5050–8420 (1012) 5100 (1012) 5100 This work NH2OH Silicate 6518 (1013) 6080 Congiu et al. (2012) CH3OH CH3OH 4989 (5 × 1014) 4169 Doronin et al. (2015) CH3OH H2O 4366 (2 × 1012) 4310 Martín-Doménech et al. (2014) CH3OH Graphite 5454 (1016) 4257 Doronin et al. (2015) H2CO H2O 3247 (1013) 2899 Noble et al. (2012) H2CO Silicate 3729 (1013) 3040 Noble et al. (2012) H2O Amorphous Au 5600 (1015) 4810 Fraser et al. (2001) Crystalline Au 5773 (1015) 4930 Fraser et al. (2001) Notes. Col. 4 gives the maximum of the desorption energy (Edes,max) (K) obtained with the best-ﬁt pre-exponential factor A values (s−1) for NH2CHO, NH2CH3, and other astrophysical relevant molecules (NH2OH, CH3OH, H2CO, and H2O) taken from the literature. For a comparison, Col. 5 gives the desorption energies (Edes,max) of all the molecules calculated with the typical pre-exponential factor A = 1012 s−1 for various grain surfaces, using Eq. (7) of ﬂux desorption (A1e−E1,des/kBT = A2e−E2,des/kBT), and the values of A and Edes,max in Col. 4. Table 1. Desorption energy distributions (Edes,dist) (K), Col. 3, of NH2CHO and CH3NH2 obtained in this work with the best-ﬁt pre-exponential factor A values, expressed (s−1). Notes. Col. 4 gives the maximum of the desorption energy (Edes,max) (K) obtained with the best-ﬁt pre-exponential factor A values (s−1) for NH2CHO, NH2CH3, and other astrophysical relevant molecules (NH2OH, CH3OH, H2CO, and H2O) taken from the literature. For a comparison, Col. 5 gives the desorption energies (Edes,max) of all the molecules calculated with the typical pre-exponential factor A = 1012 s−1 for various grain surfaces, using Eq. (7) of ﬂux desorption (A1e−E1,des/kBT = A2e−E2,des/kBT), and the values of A and Edes,max in Col. H. Chaabouni et al.: Thermal desorption of formamide and methylamine For methylamine, the main ques- tion is which binding energy shall we propose. Usually, binding binding energy distribution of the molecules by a few tens of percent (Fillion et al. 2009). In our experiments, the desorption energy of formamide from the surface of the non-porous ASW ice cannot be measured because the water substrate desorbs before formamide, that is deposited on top of it. Actually, the whole binding energy distribution of formamide from graphite (5056–6990 K), calculated with A = 1012 s−1 for 1 ML cover- age, is higher than the binding energy values of water ice in its amorphous (4810 K), and crystalline state (4930 K). Unlike for- mamide, solid methylamine can desorb under different forms, in the multi-layer regime (NH2CH3–NH2CH3) with low desorption energies (3010–3490 K) from the np-ASW ice surface with des- orption energies (3900–4500 K) and even later from the HOPG substrate with higher desorption energies (5050–8420 K). The different desorption energies of methylamine calculated in this work with respect to that of pure amorphous water ice (4810 K) is due to its wetting ability compared to H2O molecules. In fact, CH3NH2 species are able to spread readily and uniformly over the surface of the graphite (HOPG) or the amorphous water ice by hydrogen bonds, thereby forming a thin and continuous ﬁlm in which physisorbed molecules populate a large distribution of surface binding sites. The proportion of molecules desorbing, prior, during, or after the water ice varies with the doses of methylamine, but each time the fraction of methylamine desorb- ing after the water ice is about 0.15 ML. However, the fraction of methylamine co-desorbing with the crystalline phase of the water ice is very small at about 0.03 ML. We can see in Fig. 4 that the desorption of methylamine is very reduced during the desorption of the water ice at around 150 K. The desorption of methylamine at 137 K, before the sublimation of the water ice, is delayed compared with its desorption without water at T = 106 K (Fig. 4), indicating a stronger interaction of methylamine with the water molecule than with itself. In most astrophysical models, the whole desorption of each molecule is described by its (unique) binding energy. For methylamine, the main ques- tion is which binding energy shall we propose. H. Chaabouni et al.: Thermal desorption of formamide and methylamine The COMs detected in cold dark prestel- lar cores regions are likely to originate from gas-phase reactions at 10–20 K of precursor molecules, such as formaldehyde and methanol, which are previously formed on icy grain surfaces, then ejected into the gas phase by desorption induced by chem- ical reaction, and ﬁnally followed by gas-phase chemistry via ion molecules (Vasyunin & Herbst 2013), neutral-neutral, and radiative association reactions (Vasyunin et al. 2017). These molecules detectable in the gas phase can in turn be involved in gas-phase reactions to form more complex organic molecules. It is therefore, interesting to explore the interaction of these detected COMs with the ASW ice and with various astrophysical relevant substrates. Formamide and methylamine are probably forming in regions where CO has also started to freeze-out, i.e. in dense molecular clouds. In these regions, the icy mantle might be composed of water ice in the presence of CO molecules or a mixture of CO2 and H2O ices. However, since CO desorbs at 30– 50 K from the amorphous water ice surface (Noble et al. 2012), it is probably that its presence with H2O molecules does not affect the diffusion of formamide and methylamine from the water ice surface towards the substrate, such as graphite HOPG, at the expected temperatures of 110–140 K. Nevertheless, it would be interesting to perform further adsorption–desorption laboratory experiments of formamide and methylamine on mixed or lay- ered CO, CO2, and H2O ices to understand the effect of this relevant astrophysical molecule on the diffusion process of the complex organic molecules through the ASW ice. It would also be interesting to explore other substrates, such as amorphous sil- icon (a-Si) and amorphous carbon (a-C), which better represent the interstellar dust grains composition than HOPG to study the effect of the substrate on the desorption energies and the diffu- sion of these two molecules, and other COMs from the water ice surfaces. If dust grains are heated, as in the case of those located nearby a newly born protostar, the external heat provided by the protostars increases the surface temperature of the ice mantle, provoking desorption from the ice back to the gas phase of the volatile molecules, such as CO, CO2, and H2O or CH3OH. H. Chaabouni et al.: Thermal desorption of formamide and methylamine The fast diffusion of NH2CHO molecules through the water ice may occur during the reorganization of the ices, and the transition phase from the amorphous to the crystalline structure of the ASW ice at about 145 K (Mispelaer et al. 2013; Dawley et al. 2014). In the interstellar medium, dust grains can be composed of rele- vant astrophysical materials, such as amorphous carbon (a-C), amorphous silicon (a-Si), and amorphous silicates (Mg2SiO4 and Fe2SiO4). Amorphous carbon (a-C), such as a diamond, has been shown to consist of graphitic islands, composed of com- pact polycyclic aromatic ring structures, poorly connected with each other Duley et al. (1989). These astrophysical relevant sur- faces, characterized by deepest binding sites and highest binding energies, in comparison to the surface of the amorphous water ices, may affect the diffusion proprieties of the molecules. So methylamine and formamide molecules interact strongly with these amorphous carbon and silicon surfaces after water ice evaporation. Formamide molecules are expected to fully dif- fuse from the water ice surface to these interstellar analogue surfaces. one of the main efﬁcient mechanism included in astrochemical models to feed back the gas phase is the impulsive heating of dust grains by X-rays and cosmic rays, already proposed ear- lier by Leger et al. (1985). Here again, the binding energy of the molecules is still a selective parameter because it explic- itly enters in the calculation of the desorption of each type of molecules. Although this is not exactly the same mechanism than found in thermal desorption, in practice, models sort molecules depending on their binding energies. Therefore, in the context of prestellar cores, a very high binding energy also prevents any return in the gas phase. The second efﬁcient mechanism for pop- ulating the gas phase is the chemical desorption (or reactive desorption) (Takahashi & Williams 2000; Garrod et al. 2007; Dulieu et al. 2013). Here, the energy released during the for- mation of the molecule itself is the promoter of the desorption. But the probability of desorption has been proposed to decrease exponentially with the binding energy, among others factors (Minissale et al. 2016). H. Chaabouni et al.: Thermal desorption of formamide and methylamine Before this study, we were wondering if some simple chemical properties could explain the common trends of the binding energies. It is usually discussed in terms of hydrophilic and hydrophobic proprieties of the molecules or in terms of hydrogen bonding formation with water. We can sort molecules in two groups: those with Formamide and methylamine that diffuse completely or partly through the ASW ice surface towards the graphitic sub- strate and desorb into the gas phase at higher temperatures (≥160 K) are considered as refractory species, which can remain frozen on dust grains even after water ice sublimation. This result leads us to consider different scenarios, in which formamide and methylamine can be formed either in the gas phase and then accreted onto the ice surface or formed by grain-surface chem- istry. In the case of the gas-phase formation, the diffusion of the adsorbed molecules on the colder ice mantle at 10–20 K is an open question, since it depends on the thickness, porosity, and chemical composition of the ice mantle (Mispelaer et al. 2013; Lauck et al. 2015). In the case of grain-surface chem- istry, NH2CHO and CH3NH2 molecules may be formed on icy grain mantles in the interstellar medium, by exothermic reac- tions between mobile radicals, generated by ultraviolet (UV) radiation, or trapped within the bulk of ice mantles of dust grains (Taquet et al. 2012), probably at 30–40 K, once the pro- tostar starts heating its immediate surroundings, as has been suggested by grain surface chemistry models of Garrod & Herbst (2006). The diffusion of the molecules within the water ice A47, page 10 of 12 A47, page 10 of 12 H. Chaabouni et al.: Thermal desorption of formamide and methylamine to the surface of dust grains by hydrogen bonding formation depends on the morphology of the ices (Lauck et al. 2015) and mainly on the surface of the dust grain below the water ice. The diffusion of NH2CHO molecule from the water ice surface to the graphitic substrate is possibly favoured by the formation of larger groups of segregated NH2CHO molecules, which bind together by intermolecular van der Waals interactions, and with H2O molecules by hydrogen bonds through the amino (−NH2) and carbonyl (–C=O) functional groups. H. Chaabouni et al.: Thermal desorption of formamide and methylamine As the thickness of the water ice on the dust grain is reduced, the non-volatile formamide molecules diffuses through the water ice and populates the highest bindings sites of dust grains. Therefore the presence in the gas phase of refractory molecules, if these molecules come from the solid phase, would require a higher ﬁnal temperature of dust grains. These refractory molecules should be observed on a separate snow line. Furthermore, this thermal desorption should be accompanied by a blending of the species and some induced reactivity, as described in for example Theulé et al. (2013), Dulieu et al. (2017), and in Souda (2016) for the H-D exchange in ices. But the presence of molecules in the gas phase coming from the solid phase can arise from the sputtering of the grain mantle, especially in shocks (e.g. Draine 1983; Jones et al. 1994; Tielens et al. 1994). This type of desorption process is not at thermal equi- librium, but is induced by gaseous bombardment with an energy greater than the surface binding energies, provoking the ejection of the material in gas environments. This kind of ballistic kick out mechanism should be sensitive to the binding energy of the molecules. Acknowledgements. This work was supported by the Programme National “Physique et Chimie du Milieu Interstellaire (PCMI)” of CNRS/INSU with INC/INP co-funded by CEA and CNES. H.C would like to thank Prof. Darek Lis, Prof. Jean-Hugues Fillion of the LERMA team for their support and helpful corrections. The authors acknowledge the anonymous referee for con- structive comments that greatly improved the paper. T.N. is granted by the LabEx MICHEM. Formamide is a target molecule of SOLIS, a NOEMA key programme led by C. Ceccarelli and P. Caselli. 6. Conclusions C. A., Gerakines, P. A., & Whittet, D. C. B. 2015, ARA&A, 53, 541 Chaabouni, H., Minissale, M., Manicò, G., et al. 2012, J. Chem. Phys., 137, 234706 Codella, C., Ceccarelli, C., & Caselli, E. A. 2017, A&A, 605, L3 Congiu, E., Chaabouni, H., Laffon, C., et al. 2012, J. Chem. Phys., 137, 054713 , A., Jørgensen, J. K., van der Wiel, M. 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(2012) have suggested that complex organic molecules (COMs) are actively formed in prestellar cores in L1689B before the grav- itational collapsing phase leading to protostar formation and even before the warm-up phase of the grains. Many COMs have been detected in cold dark prestellar cores at low temperature (T ∼10 K). This includes dimethyl ether (CH3OCH3), methyl formate (CH3OCHO), acetaldehyde (CH3CHO), ketene (CH2CO), cyclopropenone (c-C3H2O), propynal (HCCCHO), vinyl cyanide (CH2CHCN), and propyne (CH3CCH) (Jiménez- Serra et al. 2016; Vastel et al. 2014; Bacmann et al. 2012). Here The desorption of formamide NH2CHO and methylamine CH3NH2 molecules has been investigated experimentally on graphite (HOPG) and np-ASW water ice surfaces in the sub- monolayer and monolayer regimes, using the TPD technique. Experimental results show an efﬁcient diffusion of more than 95% of formamide through the np-ASW ice ﬁlm of 10 ML thickness towards the graphite (HOPG) substrate. The large per- centage of solid formamide (>0.95 ML) bound to the graphitic substrate desorbs at higher surface temperature, 176 K, what- ever the surface coverage, after the desorption of the water ice The desorption of formamide NH2CHO and methylamine CH3NH2 molecules has been investigated experimentally on graphite (HOPG) and np-ASW water ice surfaces in the sub- monolayer and monolayer regimes, using the TPD technique. A47, page 11 of 12 A&A 612, A47 (2018) at about 150 K. The diffusion of NH2CHO is likely to occur dur- ing the warming-up phase of the ices at a short timescale of few seconds. Bisschop, S. E., Jørgensen, J. K., van Dishoeck, E. F., & de Wachter, E. B. M. 2007, A&A, 465, 913 Biver, N., Bockelée-Morvan, D., Debout, V., et al. 2014, A&A, 566, L5 ockelée-Morvan, D., Lis, D. C., Wink, J. 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https://openalex.org/W4388764995	https://ijconline.id/index.php/ijc/article/download/1537/627	English	null	Case Reports	Majalah kardiologi Indonesia	2,023	cc-by	137,773	Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology J Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 50-year-old female patient presented to the ER with worsening dyspnea and decrease urine output. There was no prior medical history. Physical exams showed hypotension, tachycardia, tachypnea, and cold extremities. Rales and murmur were found and there was no edema in the lower limbs. The ECG showed atrial fibrillation with RVR. Laboratory evaluation presented with low blood glucose, a slight increase in ureum and creatinine, and BGA revealed metabolic acidosis. Cardiomegaly, pulmonary edema, and bilateral pleural effusion were found in CXR. Echocardiography revealed multiple valve disorders. The patient was treated with dobutamine, norepinephrine, furosemide, sodium bicarbonate, digoxin, spironolactone, and warfarin. Background g Multiple valvular heart diseases (VHD) are still highly prevalent, with rheumatic heart disease (RHD) continuing to be the leading contributor, especially in developing countries. In 2019, 40.5 million people worldwide were predicted to be affected by RHD, with a 2.8 million annual incidence rate. Indonesia ranked fourth in the world in terms of prevalence, with a death rate of 4.8 per 100,000 at-risk individuals. RHD predisposes to atrial fibrillation, which increases the risk for stroke, along with heart failure, pulmonary edema, and cardiorenal syndrome, which all contribute to mortality. Furthermore, in multiple VHD, the manifestation of each valve lesion may be altered by hemodynamic interactions, which poses to diagnostic and therapeutic challenges. This case report aims to analyze and recommend courses of action and key strategies to manage a case with multiple VHD complicated by prolonged cardiogenic shock, acute kidney injury (AKI), and metabolic acidosis. Prolonged cardiogenic shock leading to Acute Kidney Injury and Severe Metabolic Acidosis in Patient with Multiple Valvular Heart Disease: Trouble Never Comes Alone ged cardiogenic shock leading to Acute Kidney Injury and Severe Metabolic Acidosis in Patient with Multiple Valvular Heart Disease: Trouble Never Comes Alone N. Christopher1, P.R.K. Alfera1, W.K. Anggoro2 General Practitioner of Hermina Tangerang Hospital, Banten, Indonesia1 Cardiology department of Hermina Tangerang Hospital, Banten, Indonesia2 Abstract: Case Reports Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Our case highlighted the importance of improving renal perfusion with inotropic and rate control to manage multiple VHD with prolonged cardiogenic shock, AKI, and metabolic acidosis. Further treatment such as a lifelong antibiotic as a secondary prevention and valve surgery are recommended for this case. Keywords: Multiple valvular heart disease, Rheumatic heart disease, Cardiogenic shock Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 ACUTE PERICARDITIS IN MILITARY TRAINEE INDONESIAN AIR FORCE: IMPACT, RECOMMENDATION AND ASSIGNMENT A. Nugraha1, L. Pribadi2, M.G. Suwandi2 dr. M. Munir Airforce Hospital, Malang, Indonesia1 dr. S. Hardjolukito, Airforce Central Hospital, Yogyakarta, Indonesia.2 dr. M. Munir Airforce Hospital, Malang, Indonesia1 dr. S. Hardjolukito, Airforce Central Hospital, Yogyakarta, Indonesia.2 p , g, dr. S. Hardjolukito, Airforce Central Hospital, Yogyakarta, Indonesia.2 Background This case occurs to the military trainee of the Indonesian Air Force (TNI AU) who has only attended the training for 3 weeks. It has an impact on continuing his military training and he would be dismissed of being the prospective soldier candidate of TNI AU. The case becomes the first report of the acute pericarditis in the basic military training institution of the Indonesian Armed Forces (TNI). Case Illustration The male trainee, 19, suffered from severe shortness of breath while doing his activities in the last 3 days before being hospitalized. He complained of cough with thick phlegm and flu, as well as a fever that has subsided. He had a history of smoking without other risk factors of a cardiovascular disease. He was diagnosed with bilateral Pneumonia, PCR Test, Gene expert MTB, blood, and sputum cultures showing the negative results. On the fourth day of the treatment, the patient complained of chest pain when taking a deep breath, a non-contrast CT scan of the thorax was performed and the results was bilateral pneumonia accompanied by pericardial effusion with a volume of 100-250cc. ECG showed (Fig. 1), normal CKMB and CRP. Echocardiography showed mild to moderate posterior pericardial effusion. Background g Cardiac disease has become a major cause of maternal death. Rheumatic Heart Disease (RHD) mostly affect women in reproductive age, linked to mitral valve disease specifically Mitral Stenosis (MS) as complication. Previous cardiac disease with changes in pregnancy including anatomy, hemodynamic, and hormonal changes may predispose pregnant women to arrhythmia. Most common arrhythmia in pregnancy is supraventricular tachycardia (SVT). SVT in 2nd Trimester of Pregnancy – a Case Report A.A. Modjo1, Ahadi2, I.M. Tania3 General Practitioner, ‘Aisyiyah Bojonegoro Hospital1 Department of Cardiology and Vascular Medicine, ‘Aisyiyah Bojonegoro Hospital2 Department of Obstetric and Gynecology, ‘Aisyiyah Bojonegoro Hospital3 p Conclusion The prevention of relapse by optimizing OAINS and the restriction of activities become the main point of successful pericarditis treatment. The impact of pericarditis which generally occurs in a young age with arduous activities has been discussed as a consideration in the assignment of military personnel. Keywords: Pericarditis, Pericardial Effusions, Military. Figure 1. ECG showed Wide Spread ST Elevation Figure 1. ECG showed Wide Spread ST Elevation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 34-year-old multigravida woman with 24-25 weeks of gestation came to ER with complaint of sudden palpitation, dull aching chest pain, dizziness and shortness of breath. She had no relevant history of cardiac disease. Vital sign at the ER with Glassgow Coma Scale (GCS) 456, Blood Pressure (BP) 95/80, Heart Rate (HR) 201, Respiratory Rate (RR) 24x/min, 98% saturation and diastolic murmur grade III-IV/VI. Laboratory examination was in normal limit. ECG showed SVT 201 bpm with narrow QRS complex. The patient was given a carotid sinus massage at first but no respond in ECG. Then patient was admitted with Bisoprolol 5 mg per oral, however there was no change in ECG. Due to the life threatening condition and drugs availability in our hospital, administration of Amiodarone 150 mg intravenous in ICU was initiated, subsequently ECG was convert to sinus rhythm with vital sign GCS 456, BP 110/70, HR 90x/min, RR 20x/min, temperature 36.7oC, 100% saturation with nasal oxygen 4 lpm, and fetal heart rate 136x/min. Echocardiography examination later revealed RHD with severe MS, moderate Mitral Regurgitation (MR), moderate Aorta Stenosis (AS), moderate Aorta Regurgitation (AR), mild Tricuspid Regurgitation (TR) and Left Atrium (LA)-Right Ventricle (RV)-Right Atrium (RA) dilation. Conclusion SVT in pregnancy related with Mitral Stenosis as a complication of RHD required early diagnostic and appropriate management from multidiscipline approach due to concern in maternal and fetal well-being. Keywords: SVT, Mitral Stenosis, RHD, Pregnancy Keywords: SVT, Mitral Stenosis, RHD, Pregnancy Electrocardiography SVT Electrocardiography SVT Keywords: ablation, ECG, PVC, outflow tract (OT). Serial Case of Premature Ventricular Contractions (PVCs) of Outflow Tract Origins P.P. Sakti1, A. Rizal1 Cardiology and Vascular Medicine Department, Dr. Saiful Anwar Teaching Hospital - Medical Faculty of Universitas Brawijaya1 P.P. Sakti1, A. Rizal1 Cardiology and Vascular Medicine Department, Dr. Saiful Anwar Teaching Hospital - Medical Faculty Universitas Brawijaya1 Background: Premature ventricular contractions (PVCs) are defined as myocardium depolarizations that begin in the ventricle. PVCs emerge from a variety of sources, primarily the outflow tracts (OTs). Radiofrequency ablation is the standard treatment for the majority of symptomatic PVCs. Determining the origin of PVCs is critical to achieving better ablation results and lower complications. According to research, ECG features can provide accurate predictions for PVC origin. p g Case Illustration: The first patient was 59-year-old man (weight 70 kg, height 165 cm) with a history palpitations. His Holter examination revealed multifocal PVCs (13%) dominant RVOT origin with episodic bigeminy and couplet multifocal, but no PVCs were found during ECG examination. The patient was diagnosed with frequent PVC septal RVOT epicardial origin. The second patient was a 39-year-old man (weight 80 kg, height 170 cm) suffered from asymptomatic arrythmia during a routine medical checkup. His ECG examination showed sinus rhythm with PVC RBBB and multiphasic pattern in lead V1. He was diagnosed with frequent unifocal PVC posterior LVOT epicardial origin. His Holter monitoring showed episode of PVC unifocal, bigeminy, trigeminy, couplets, and occasional PAC. Both patients are planned for radiofrequency ablation. p p p q y Conclusion: A 12-lead ECG can be used to predict the origin of PVCs and is a non-invasive approach. Identifying the source of PVCs prior to radiofrequency ablation is critical for increasing procedure’s success rate. Conclusion: A 12-lead ECG can be used to predict the origin of PVCs and is a non-invasive approach. Identifying the source of PVCs prior to radiofrequency ablation is critical for increasing procedure’s success Keywords: ablation, ECG, PVC, outflow tract (OT). Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Differing Tumor and Thrombus: When the Heart Explains the Brain J. Koswara1, D. Suwanto2 Cardiologist, Efarina Etaham Hospital, Pematang Siantar, Indonesia2 Background: Primary cardiac tumors are an incredibly rare finding. Cardiac myxomas are the most common type of primary cardiac tumors in adults. 75% of myxomas originate in the left atrium. They are histologically benign but when left untreated they could potentially cause embolism at various site. Nonspecific manifestation may hinder the diagnosis, which can lead to complications. Case Illustration: We present a case of a 57-year-old female with no risk factor for atherosclerosis, admitted at an emergency department an hour after the onset of right sided sensorimotor weakness. Chest X-ray showed an enlarged cardiac silhouette. Brain CT-scan showed normal. A transthoracic echocardiogram revealed a large mobile mass sized 3.7 x 3.7 cm attached to intra-atrial septum and prolapsing into left ventricle through mitral valve from the left atrium. A surgical approach was used to resect the mass in the left atrium and surgery went well. Symptoms were resolved and patient did not report any surgery complications. Conclusion:Cardiac myxomas frequently present with neurological symptoms, especially ischemic events (established stroke or transient ischemic attack). Myxomas and thrombi can be differentiated using echocardiography by assessing the distinguishing features of size, origin, shape, mobility, and prolapse. Surgery should be performed following the diagnosis to prevent dreadful embolic complication of cardiac myxoma. Keywords: Cardiac Mass, Myxoma, Thrombus, Tumor. Figure 1. (a) Left atrial myxoma seen in two-dimensional trans thoracic echocardiography. (b) Left atrial myxoma seen prolapsing through the mitral valve into left ventricle. Figure 1. (a) Left atrial myxoma seen in two-dimensional trans thoracic echocardiography. (b) Left atrial myxoma seen prolapsing through the mitral valve into left ventricle. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Late Treatment of Large Swiss Cheese-like Secundum ASD: Transesophageal and Transthoracic Echocardiography Findings S. Shadiqqa1, R. Nurmeliani2 RSUD Al Ihsan1 RSUD al Ihsan2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Late Treatment of Large Swiss Cheese-like Secundum ASD: Transesophageal and Transthoracic Echocardiography Findings S. Shadiqqa1, R. Nurmeliani2 RSUD Al Ihsan1 RSUD al Ihsan2 Late Treatment of Large Swiss Cheese-like Secundum ASD: Transesophageal and Transthoracic Echocardiography Findings S. Shadiqqa1, R. Nurmeliani2 Late Treatment of Large Swiss Cheese-like Secundum ASD: Transesophageal and Transthoracic Echocardiography Findings S. Shadiqqa1, R. Nurmeliani2 RSUD Al Ihsan1 RSUD al Ihsan2 Background : g Atrial septal defect is one of the most common congenital heart diseases. More than three-quarters of ASD cases are secundum ASDs. [1], [2] In ASD, a left-to-right (L-R) shunt occurs, causing a right-sided volume overload which leads to failure and increased pulmonary atrial pressure. Defect closure can be done surgically or through percutaneous repair, to determine which procedure to perform information about the morphology of the defect is needed. Transthoracic echocardiography (TTE) is the initial examination followed by transesophageal echocardiography (TEE). [3] Objectives assessed from TTE and TEE include the location, size and number of defects, rims deficiency, intraatrial septum and structural changes as a consequence of the hemodynamics L-R shunt.[4] Case : A 64-year-old woman patient complained of shortness of breath which has worsened with daily activities since a year ago. On physical examination there were crackles in both lungs, heart murmur, distention in JVP and swelling in both legs. TTE and TEE examinations revealed multiple defects of secundum ASD, the largest one measured 4-4.4 cm, the appearance of right-sided volume overload, severe tricuspid regurgitation and a high probability of pulmonal hypertension. The patient was then advised to undergo surgical defect closure. Conclusion : Defect closure of ASD can be done surgically or through percutaneous repair. TTE and TEE examinations are necessary to assess the defect's morphology and then consider which method should be performed. L-R shunt will eventually cause a right-sided volume overload, leading to failure and increased pulmonary atrial pressure. [3][4] Keywords: ASD, congenital, failure, secundum Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Report - An Incidental Findings of Lutembacher's Syndrome In Rural Areas P.A. Simanjuntak1, E.J.H. Sinaga1 Department of Cardiology and Vascular Medicine, RSUD Dolok Sanggul, Humbang Hasundutan, Sumatera Utara, Indonesia1 Background : Lutembacher’s syndrome (LS) is a rare cardiovascular anomaly defined as any combination of congenital or iatrogenic atrial septal defect (ASD) with congenital or acquired mitral stenosis (MS). As atrial septal defect acts as a pressure relieving gateway, signs and symptoms of mitral stenosis may be attenuated or delayed in such patients. Patients with LS are often asymptomatic for many years as observed in this case, and this patient may live for up to 50 years before experiencing symptoms of cardiac decompensation. Hemodynamic expression and clinical features are affected by the combined effects of ASD size, mitral stenosis severity, and right ventricle distensibility. Early detection and treatment result in a better prognosis, but in late advanced stage, such patients develop pulmonary hypertension and heart failure with poor prognosis. Case Illustration : Here, we report the case of a 53 years old female with Lutembacher’s syndrome that was incidentally diagnosed when being consulted to cardiologist and treated conservatively for heart failure at Humbang Hasundutan Regional Hospital. Since 2018 this patient has been an internist routine outpatient and treated as congestive heart failure and hypertension. Based on history taking, physical and supporting examination, this patient is on advanced stage with pulmonary hypertension and heart failure are already developed. Conclusion : Delay in identifying and accessing medical center, caused by transportation difficulties, inadequate cardiologists and referral facility, and delay in having prompt treatment particularly in rural areas as Humbang Hasundutan are the cause of delayed in LS diagnosis and treatment as demonstrated in our case. Echocardiography remains the modality of choice for the diagnosis and evaluation of LS. In this case report, transthoracic echocardiography along with color flow imaging, establishes the diagnosis of LS and identifies the type and size of the ASD and the presence and degree of mitral stenosis. p g Keywords: Atrial Septal Defect, Heart Failure, Lutembacher syndrome, Mitral stenosis Trans-Thoracic Echocardiography showed Mitral Valve Area by (a) planimetry and (b) pressure half-time (PHT) (c) left atrial to aortic root ratio (d) the presence of ASD (e) LV size and function Trans-Thoracic Echocardiography showed Mitral Valve Area by (a) planimetry and (b) pressure half-time (PHT) (c) left atrial to aortic root ratio (d) the presence of ASD (e) LV size and function Atypical Atrial Flutter-Fibrillation and Obstructive Sleep Apneu Interconnection D.P. Andryan1, D.Y. Hermanto1 y National Cardiovascular Center Harapan Kita, Faculty of Medicine, Universitas Indonesia Background: Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia in adults, in the world. Obstructive Sleep Apnea (OSA) is one of the overlooked AF comorbid. Untreated OSA is an established risk factor for new-onset atrial AF, progression from paroxysmal AF to persistent AF, and Atrial arrhythmias recurrence after catheter ablation. Case Illustration: Mrs. AT, 42 years old came to the arrhythmia outpatient clinic with complaints of palpitations that had been worse the past 1 year, previously underwent partially successful ablation of Multifocal Atrial Tachycardia. The patient had no history of hypertension, diabetes mellitus, dyslipidemia, or ex-smokers. After re- exploring the comorbid, the patient apparently had complaints of frequent snoring every night. Polysomnography revealed severe OSA. A patient was diagnosed with Atypical Atrial Flutter-Fibrillation and Severe OSA. The patient underwent CPAP therapy for 1 year before undergoing 3D ablation. ENSITE electroanatomic mapping system during tachycardia showed the earliest activation at the right carina. circumferential isolation of the right carina and right PV were delivered. Post ablation, ECG converted to sinus rhythm and resolution of symptom. Conclusion: OSA is an important but overlooked risk factor for AF. Atrial remodeling, diastolic dysfunction, increased autonomic tone, and inflammatory mediators, are potential factors that amplify the risk of AF. Patients with OSA and AF are prone to recurrent AF after catheter ablation, therefore it is necessary to carry out appropriate management for OSA itself. Keywords: Obstructive sleep apneu, Atrial Fibrillation, Atrial Flutter, connection, comorbid Polysomnography & ablation of PV eywords: Obstructive sleep apneu, Atrial Fibrillation, Atrial Flutter, connection, comorbid Keywords: Obstructive sleep apneu, Atrial Fibrillation, Atrial Flutter, connection, comorbid Polysomnography & ablation of PV Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Peripartum Cardiomyopathy with Complete Recovery After 2 Years of Follow Up: A Case Report Y. Pintaningrum1, N.N. Humaera2 Cardiology Department West Nusa Tenggara Central Hospital1 Mataram University2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Peripartum Cardiomyopathy with Complete Recovery After 2 Years of Follow Up: A Case Report Y. Pintaningrum1, N.N. Humaera2 Cardiology Department West Nusa Tenggara Central Hospital1 Mataram University2 Y. Pintaningrum1, N.N. Keywords: peripartum, cardiomyopathy, complete recovery LEFT MAIN CORONARY ARTERY ANEURYSM PRESENTING AS ANTERIOR STEMI: A RARE CLINICAL FINDING H.A.K. Cantika1, D.B. Lululangi1, P.R. Dewi2, U. Bahrudin3 Department of Emergency, KRMT Wongsonegoro State Hospital, Semarang, Indonesia1 Department of Cardiology, KRMT Wongsonegoro State Hospital, Semarang, Indonesia2 tment of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University, Semarang, Indonesia3 Background: Aneurysm of the left main coronary artery (LMCA) in patients with ST-elevation myocardial infarction (STEMI) is unusually reported. LMCA aneurysm is an uncommon coronary disease, found incidentally in 0.1% of patients undergoing routine angiography. Case Illustration: A 40-year-old man presented at our emergency department with a 1-hour onset of an acute typical angina. This patient had no known preexisting risk factors or past coronary artery comorbidities. The electrocardiogram showed ST-segment elevation at V2-V4 lead conforming anterior STEMI. MONACO strategy was initiated promptly upon diagnosis and followed by cardiac catheterization. Coronary angiography revealed 15 – 16 mm diameter aneurysmal dilatation of LMCA with total osteal occlusion in the left anterior descending artery (LAD) and 30% thrombus-type osteal stenosis of the left circumflex. Thrombus formation is favored at the aneurysmal site due to more sluggish flow through a dilated vessel and thus later can present as STEMI. The lack of evidence of any fundamental atherosclerotic risk factors and the presence of stenosis only in the branches of the LMCA led us to also consider possible non-atherosclerotic causes in this patient. Further history-taking acknowledged the patient’s prolonged hospitalization history during childhood, which raised our suspicion of vasculitis causal. A follow-up echocardiography reported reduced left ventricular systolic function with 48% ejection fraction and regional wall motion abnormality. This patient received triple antithrombotic therapy consisting of dual antiplatelet and intravenous anticoagulant and remained stable during intra-hospital care. Antithrombotic management was continued orally in outpatient care. Conclusion: LMCA aneurysms are rare in patients presenting with coronary syndrome. Patient-based approach with comprehensive clinical evaluation remains the management strategy due to the unavailability of a well- established consensus. In this case, the patient obtained optimal medical therapy as the treatment of choice based on clinical considerations. Keywords: left main coronary artery, aneurysm, STEMI Total osteal occlusion in LAD (marked with red pointer) in conjunction with LMCA aneurysm of 15 – 16 mm diameter and 30% thrombus-type osteal stenosis of left circumflex found in coronary angiography. Atypical Atrial Flutter-Fibrillation and Obstructive Sleep Apneu Interconnection Humaera2 Cardiology Department West Nusa Tenggara Central Hospital1 Mataram University2 Background: Peripartum Cardiomyopathy (PPCM) is a systolic disfuction that occurs during late pregnancy or a period of up to 5 months after delivery. PPCM patients may present symptoms earlier, especially in patients with previous cardiac comorbidities. Clinical manifestations of PPCM vary depending on the degree of disease. Case Illustration: A 28-year-old pregnant woman admitted with shortness of breath, accompanied by a dry cough, cold sweating, and fatigue. Patient in 32 weeks of pregnancy with her second time of pregnancy. There are no significant risk factors other than hypertension and no history of other underlying clinical disease. Patient was referred with cardiogenic shock caused by Congestive Heart Failure (NYHA class IV). The results showed valvular abnormalities in the form of mild mitral regurgitation and mild to moderate tricuspid regurgitation, decreased left ventricular systolic function EF 30%, hypokinetic global left ventricle and left ventricular remodeling. Termination of the pregnancy was taken. Comprehensive monitoring was conducted, but due to neonatal complications fetal death has occurred. The patient was treated with furosemide 1x40 mg, spironolactone 1x25 mg, betade 1x2.5 mg, lisinopril 1x5 mg, N acetyl cysteine 3x200 mg, and 1x80 mg of aspirin by controlling of fluid intake 1000 cc/24 hours for the maximum intake. The treatment given was subsequently tappered off according to the patient's condition until 1 year after the onset, treatment was discontinued. After undergoing approximately 2 years of monitoring, an echocardiographic evaluation of the patient was carried out and showed normal results with EF 82%. Background: Peripartum Cardiomyopathy (PPCM) is a systolic disfuction that occurs during late pregnancy or a period of up to 5 months after delivery. PPCM patients may present symptoms earlier, especially in patients with previous cardiac comorbidities. Clinical manifestations of PPCM vary depending on the degree of disease. Conclusion : Comprehensive management can provide a good prognosis in patient with Peripartum Cardiomyopathy. : Comprehensive management can provide a good prognosis in patient with Peripartum athy Keywords: peripartum, cardiomyopathy, complete recovery Acute Coronary Syndromes with Normal Electrocardiogram Finding in Emergency Department : A Case Report S. Raj1, T. Delfian2 1General Practitioner, Hermina Medan General Hospital 2Cardiologist, Hermina Medan General Hospital Background Coronary Artery Diseases (CAD) are number one cause of death worldwide and top three in Indonesia. CAD process results which can be categorized as either Acute Coronary Syndromes (ACS) or Chronic Coronary Syndromes. Diagnostic of ACS including clinical symptoms and vital signs, cardiac marker testing and Electrocardiogram (ECG). ECG in ACS differentiated into two group consist of ST Elevation ACS and Non ST Elevation ACS which may present as transient St-Segment Elevation, persistent or transient ST-segment depression, T-wave inversion, flat T waves, Pseudonormalization of T waves, or the ECG may be normal. Case illustration Female, 52 yo taken by her children to Emergency Department (ED) of Hermina Medan General Hospital with chief of complain dyspnoea triggered by activity with history of chest pain, cold sweat, nausea, vomiting, and syncope. She had nebulized in Clinic Before. Past medical illness was diabetes mellitus uncontrolled. Patient’s Respiratory Rate was 24 x/m with Abnormal blood tests showed leukocytosis 16.800/µL and high blood glucose 314 mg/dL. Chest X Ray was normal. ECG Showed Left Axis Deviation. In ED, patient was treated with Miniaspi 2 tabs, Clopidogrel 4 tabs, and Atorvastatin 40 mg then was consulted to Cardiologist and Internist and was treated for 4 days. y There are four factors independently associated with missed ACS in the ED and failure to hospitalize patients. They are Normal ECG, female sex less than 55 yo, nonwhite race, and chief complain of dyspnoea. Patients with normal ECG will have 22% of a Non-ST Elevation ACS, 19,2% of Serious Cardiac Event Rate, and 5,7% of in-hospital Mortality Rate. LEFT MAIN CORONARY ARTERY ANEURYSM PRESENTING AS ANTERIOR STEMI: A RARE CLINICAL FINDING Keywords: left main coronary artery, aneurysm, STEMI Total osteal occlusion in LAD (marked with red pointer) in conjunction with LMCA aneurysm of 15 – 16 mm diameter and 30% thrombus-type osteal stenosis of left circumflex found in coronary angiography. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Coronary Syndromes with Normal Electrocardiogram Finding in Emergency Department : A Case Report Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Coronary Syndromes with Normal Electrocardiogram Finding in Emergency Department : A Case Report Conclusion Clinician who work in ED should be aware that ACS can present without any objective data of Myocardial ischemic injury. Initial diagnosis depends on patient’s clinical history and clinician’s judgement and treatment should be given immediately. Keywords: ACS, Normal ECG Electrocardiogram of the patient Electrocardiogram of the patient Unprovoked Transformation of Saddle Back to Coved ST-segment Elevation ECG Pattern: A New Paradigm to Recognize the Silent Killer M. Firdaus1, A. Rizal1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Brwaijaya, Dr. Saif Anwar General Hospital, Malang, Indonesia Background g Brugada syndrome (BrS) is a heritable arrhythmia syndrome which usually triggered VF and SCA in young adults by many factors such as psychotropic medications, anesthetic agents, cocaine, excessive alcohol intake and fever. We present a case of young adults with unprovoked transformation of BrS. Case Illustration Case Illustration A 35-year-old man was admitted to ER due to an unexplained syncope. It happened 3 times within 1 hour which preceded by lightheadedness, nausea and vomiting. Before the syncope, he did not feel any palpitation, chest pain nor dyspnea. He had no history of fever, dehydration, alcohol consumption and any medication before. There was no history of sudden cardiac death in his family members. Physical examination, laboratory and echocardiography were within normal limit, but the ECG showed a coved ST-segment elevation with inverted T-wave at V1-V2 with ECG pattern. The ECG then transformed into a saddle back ST-segment elevation 2 weeks later which we assume as the patient’s baseline ECG. The diagnosis of BrS was made. He underwent ICD implantation at Saiful Anwar Hospital Malang as a secondary prevention. Conclusion p Conclusion ECG changes from type 2 to type 1 Brugada pattern is often induced by trigger factors, but even without any provocation, type 1 BrS could still happen. Thorough history taking and ECG investigation are always a mandatory step. Keywords: Brugada, Syncope Keywords: Brugada, Syncope eywo ds g , Sy p Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 A Rare Case of Total AV Block In The Young With Significant Coronary Disease: Complete Revascularization or Pacing First? P.P. Sakti1, A. Rizal1 Department of Cardiology and Vascular Medicine, Saiful Anwar Hospital, Malang1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Rare Case of Total AV Block In The Young With Significant Coronary Disease: Complete Revascularization or Pacing First? P.P. Sakti1, A. Rizal1 Department of Cardiology and Vascular Medicine, Saiful Anwar Hospital, Malang1 Case Illustration A 30 years-old man had chief complaints of syncope occurred for about 20 minutes after playing badminton. The syncope was preceded by chest discomfort and light headedness, after regained consciousness spontaneously, he was fully awake, but he still felt chest discomfort. He was a smoker since the age of 18 years old. His father got ACS at 45 years old and performed CABG at 67 years old. From the physical examination, the findings were normal except for bradycardia of 50 beats per minute. The ECG showed TAVB, atrial rate 100 bpm, ventricular rate 50 bpm. The laboratory test showed elevated cardiac marker. He was then diagnosed with TAVB related to ACS. PCI procedure was performed and revealed CAD 2 vessels disease with culprit lesion in RCA and also significat stenosis in LAD, drug eluting stent was implanted at RCA. The ECG evaluation after procedure was showed that TAVB was converted to 1st degree AV block. He was observed in the following days and the ECG still didn’t turn to sinus rhytm. Background g Total AV block (TAVB) is a highly perilous form of arrhythmia that can manifest in various symptoms such as chest pain, presyncope, and dyspnea, although some patients may not display any symptoms at all. Comprehensive examinations are necessary to exclude the possibility of secondary causes such as myocardial ischemia. This paper will present a case of TAVB in a young patient in setting of acute coronary syndrome with significant lession. Case Illustration Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Single Ventricle with Two Atriums, It is Possible?: A Rare Case from Remote District Hospital at East Nusa Tenggara Conclusion Acute coronary syndrome is one of the complete heart block causes. In a young patient with significant coronary disease that cause TAVB should underwent extensive work-up to find the right decision wether it would be complete revascularization or pacing device treatment to prevent any sequele and obtain the best outcome. Keywords: total AV block, acute coronary syndrome, revascularization, permanent pacemaker ECG at first medical contact showed total AV block ECG at first medical contact showed total AV block Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Single Ventricle with Two Atriums, It is Possible?: A Rare Case from Remote District Hospital at East Nusa Tenggara Single Ventricle with Two Atriums, It is Possible?: A Rare Case from Remote District Hospital at East Nusa Tenggara Eliza1, D.M. Putra2, I.G Sumantra3 Eliza1, D.M. Putra2, I.G Sumantra3 General Practitioner at Waikabubak Hospital, West Sumba, East Nusa Tenggara1 p gg General Practitioner at Reda Bolo Hospital, Southwest Sumba, East Nusa Tenggara2 Cardiology Department at Paramarta Heart and Vascular Hospital, Bandung, West Java3 Cardiology Department at Paramarta Heart and Vascular Hospital, Bandung, West Java3 Background: Hypoplastic right heart syndrome (HRHS) is a rare cyanotic congenital heart disease with under- development of the right ventricle, tricuspid, and pulmonary valves leading to right-to-left shunting of the blood through inter-atrial septal defect. Case Illustration: A 3-month-old male came to the emergency room with a complaint of dyspnea and cyanotic. Vital signs showed heart rate 148 beats per minute, respiratory rate 68 breaths per minute, oxygen saturation 40% with face mask 10 liter per minute. His body weight is 2500 grams. Physical examination showed intercostal retraction and fix splitting murmur. Chest X-ray showed cardiomegaly with CTR 77%, grounded apex, enlarged atrial appendage, and splaying carina. Echocardiography showeda situs solitus, small VSD, moderate ASD sekundum with Right to Left shunt, LV hypertrophy with normal LVEF, pulmonary atresia and stenosis and tricuspid atresia and stenosis with RV hypoplastic. Patient was diagnosed with HRHS based on echocardiography findings. Patient was given diuretics such as furosemide and spironolactone and motivated to be referred for further investigation and management. The patient is temporarily survived because of this CHD condition (ASD and small VSD) help the patient still has an oxygenated blood flow to the body although in the decreased blood oxygen saturation. yg Conclusion: HRHS is a rare cyanotic congenital heart disease that can be deadly if it is diagnosed and treated late. The survival of the newborn depends on the size of the defect in atrial septal and level of development of the right heart structures. In this setting, we can only give conservative therapy to reduce cardiac preload and refer to the cardiovascular center for further diagnosis and management. g g Keywords: hypoplastic right heart syndrome, congenital heart disease, pediatric cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Cardiac tamponade is a life-threatening condition that need urgent pericardiocentesis. Here, the authors report a case of pericardiocentesis with alternative pericardiocentesis kit which is CVC kit. Amiodarone and Oral Verapamil-Induced Hypotension in 34-year-old Woman with Supraventricular Tachycardia and Wolff-Parkinson-White Syndrome E. Rolandika1, V.Y.S. Putri1 RS Baptis Batu1 Amiodarone and Oral Verapamil-Induced Hypotension in 34-year-old Woman with Supraventricular Tachycardia and Wolff-Parkinson-White Syndrome E. Rolandika1, V.Y.S. Putri1 RS Baptis Batu1 Background Supraventricular tachycardia is an arrhythmic condition originating from the sinus node, atrial tissue, accessory pathways and junctional areas. In some cases, patients with WPW have no symptoms until manifesting SVT. Case Illustration a 34-year-old woman came to the ER with palpitations. ECG examination showed a regular and narrow QRS complex tachycardia with pulse was 190 beats/minute, and blood pressure was 100/90 mmHg. The patient was given 150 mg bolus of amiodarone for 15 minutes. The patient was transferred to the ICU and was administered with amiodarone drip 1 mg/minute for 6 hours then 0.5 mg/minute for 18 hours, isoptin SR (verapamil) 240 mg orally, bisoprolol 1x1.25 mg orally, atorvastatin 1x40 mg orally, aspilet 1x80 mg orally, omeprazole injection 1x40 mg intravenously. While in the ICU, the blood pressure dropped to 81/55 mmHg. Amiodarone, verapamil and bisoprolol were stopped, the patient was then given NaCl 0.9% 100 cc in 10 minutes and norepinephrine 0.05 mcg-1 mcg/kg/minute. The patient's blood pressure increased, the pulse rate became 88x/minute. On repeated ECG examination, a shortened PR interval (<0.12 seconds) and a delta wave pattern were found. The patient was observed in the ICU for 2 days, then the patient was discharged without any complications and was given 1x5 mg bisoprolol for home therapy. Conclusion Patients with hemodynamically unstable SVT are recommended to receive synchronized cardioversion, but the patient refused. Amiodarone is a class III antiarrhythmic drug that can interact with β- adrenergic receptors, potassium and sodium channels. Amiodarone can reduce atrial sinus automation and decrease the conduction velocity of the atrioventricular node. In some cases, these effects can lead to bradycardia, hypotension and Torsade de Pointes. Administration of verapamil can also provide negative inotropic effects and peripheral vasodilation that can cause hypotension in patients. p p yp p Keywords: Supraventricular Tachycardia, Wolff-Parkinson-White, Amiodarone, Verapamil Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case illustration A male 46 yo admitted to ER with severe short of breath and chest pain that radiates to back. He was treated with tuberculose and received anti tuberculosis drug by pulmonologist for 2 months. Physical examination showed elevated of JVP, low blood pressure and muffled heart sound. ECG showed low voltage with electrical alternans. From bedside echocardiography, massive pericardial effusion, RV diastolic collapsed and mitral inflow variation > 25% were found. Case Report : Emergency pericardiocentesis with CVC kit at perifer hospital D.J. Simatupang1, Y.S. Situmorang2 Kesrem Hospital Lhokseumawe1 Arun Hospital Lhokseumawe2 Background The association between hyperglycemia (HG) and AHF remains controversial. Several reports have suggested a negative impact of HG on AHF overall survival and mortality yet others have not shown similar findings. Stress-induced Hyperglycemia in Acute Decompensated Heart Failure (AHF): Case Repor D.A.D.L. Sati1, B.A. Prabowo2, A.S. G. Nirwana1 Faculty of Medicine, Jenderal Soedirman University, Purwokerto Central Java, Indonesia1 Cardiologist, Prof. DR. Margono Soekarjo General Hospital, Purwokerto Central Java, Indonesia2 Case Illustration An 87-year-old woman presents to Emergency room complaining of progressive exertional dyspnea over the last two days, she also complains orthopnea, cough, palpitation, leg swollen, and nausea. She has history of uncontrolled chronic heart failure and hypertension but no history of diabetes mellitus. Examination in ER revealed blood pressure 183/90 mmHg, heart rate 88, respiratory rate 26, temperature 36.6, SpO2 99% on 2 lpm O2 nasal cannulae, cardiomegaly, bilateral rales in lungs, and pitting edema. ECG showed sinus rhythm with LVH and 1st-degree AV block. Chest x-ray showed cardiomegaly and pulmonary edema with bilateral pleural effusion. Laboratory shows high random blood glucose (235 mg/dL) with renal insufficiency (ureum 47.2 mg/dL, creatinine 1.53 mg/dL, creatinine clearance 33 ml/min/1.73m2). Patient was diagnosed as acute decompensated heart failure and hyperglycemia. Echocardiography showed concentric LVH with hypokinetic anteroseptal segment, LVEF 30%, TAPSE 18, AR moderate, and MR mild. Patient was treated with IV furosemide, and oral spironolactone, valsartan, ISDN. After 48 hours, Patient condition was improved, and random blood glucose 108 mg/dL. This case is an instance of stress-induced hyperglycemia in AHF. This condition is due to sympathetic nervous system activation and/or excessive production of stress hormones like cortisol. Natural physiological responses to stress result in elevated glucose levels. Keywords: acute decompensated heart failure, stress-induced hyperglycemia Discussion This patient fulfilled criteria for cardiac tamponade and needed to do emergency pericardiocentesis. Pericardiocentesis was done with subxiphoid approach by CVC kit as the absence of pericardiocentesis kit in ER. Two liters of pericardial fluid were withdrawn. Conclusion We describe a case with emergency pericardiocentesis in cardiac tamponade. This case report focused on the importance of clinical judgement and alternative treatment with the limitation of instrument without catheterization laboratory at perifer hospital. Keywords: emergency pericardiocentesis, cardiac tamponade, CVC kit, perifer hospital Keywords: emergency pericardiocentesis, cardiac tamponade, CVC kit, perifer hospital Keywords: emergency pericardiocentesis, cardiac tamponade, CVC kit, perifer hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Persistent Thrombocytopenia in Long-Standing Congestive Hepatopathy Secondary to Severe Mitral Stenosis: A Case Report Persistent Thrombocytopenia in Long-Standing Congestive Hepatopathy Secondary to Severe Mitral Stenosis: A Case Report Persistent Thrombocytopenia in Long-Standing Congestive Hepatopathy Secondary to Severe Mitral Stenosis: A Case Report Z Ramadhanty1 T Rahadiyan2 Conclusion Hyperglycemia can occur in AHF patients. This condition does not imply diabetes mellitus; rather, the stress response caused the hyperglycemia. Once the clinical state is stabilized, the blood glucose level may return to normal. Keywords: acute decompensated heart failure, stress-induced hyperglycemia Persistent Thrombocytopenia in Long-Standing Congestive Hepatopathy Secondary to Severe Mitral Stenosis: A Case Report Z. Ramadhanty1, T. Rahadiyan2 General Practitioner, Cardiology Clinic, dr. Bratanata Military Hospital, Jambi1 Keywords: congestive hepatopathy, mitral stenosis, chronic heart failure, thrombocytopenia, pulmonary hypertension Echocardiography revealed decreased RV function with dilatation of LA, RA, and RV; severe mitral stenosis and severe tricuspid regurgitation; and severe pulmonary hypertension Echocardiography revealed decreased RV function with dilatation of LA, RA, and RV; severe mitral stenosis and severe tricuspid regurgitation; and severe pulmonary hypertension Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Anti-Thrombotic Strategy in Chronic Limb Threatening Ischemia with Deep Vein Thrombosis After Percutaneous Endovascular Intervention: A Case Report T.I. Rahayu1, F. Herfandi2, E.F. Elfi3 General Practitioner, Andalas University, West Sumatera1 Cardiology Resident Mjamil General Hospital West Sumatera2 Anti-Thrombotic Strategy in Chronic Limb Threatening Ischemia with Deep Vein Thrombosis After Percutaneous Endovascular Intervention: A Case Report T.I. Rahayu1, F. Herfandi2, E.F. Elfi3 Anti-Thrombotic Strategy in Chronic Limb Threatening Ischemia with Deep Vein Thrombosis Aft Percutaneous Endovascular Intervention: A Case Report T.I. Rahayu1, F. Herfandi2, E.F. Elfi3 General Practitioner, Andalas University, West Sumatera1 Cardiology Resident, Mjamil General Hospital, West Sumatera2 Cardiologist, M Djamil General Hospital, West Sumatera3 Background : Chronic limb-threatening ischemia (CLTI) represents the end stage of peripheral artery disease where antithrombotic as one of the management strategies. Deep Vein Thrombosis (DVT) is an anticoagulant (OAC)-requiring condition and a compelling indication for antithrombotic therapy in CLTI. Case Illustration : A 65 years old female was admitted with pain in the left foot since 3 weeks before admission. The pain was felt at rest and the tips of the big toe and middle finger of the left foot are blackish. The patient had a history of DVT 3 months ago and treated with rivaroxaban. Localized status of the left foot showed necrosis on the first and third phalange, pain, pallor, pulselessness, paresthesia, poikilothermy, and pretibial edema. There were no arterial pulses in the left dorsalis pedis artery and left anterior tibialis artery. The ABI was 0.14. The vascular doppler showed total occlusion as high as the left anterior tibial artery. We performed Percutaneous Old Balloon Angioplasty (POBA) in the left Anterior Tibial Artery. Subsequently, we started clopidogrel and continued rivaroxaban. The lab's result after POBA showed mild anemia with Hemoglobin 9 mg/dL Therefore, we performed urinalysis and benzidine test with results were microscopic hematuria and negative benzidine test. Persistent Thrombocytopenia in Long-Standing Congestive Hepatopathy Secondary to Severe Mitral Stenosis: A Case Report Z. Ramadhanty1, T. Rahadiyan2 General Practitioner, Cardiology Clinic, dr. Bratanata Military Hospital, Jambi1 Z. Ramadhanty1, T. Rahadiyan2 General Practitioner, Cardiology Clinic, dr. Bratanata Military Hospital, Jambi1 Cardiologist, dr. Bratanata Military Hospital, Jambi, Indonesia2 Background: Congestive hepatopathy is a consequence of a long-term elevated hepatic venous pressures due to chronic right-sided heart failure. This paper aims to present a patient with persistent thrombocytopenia as a rare manifestation of congestive hepatopathy and splenomegaly due to severe mitral stenosis. g p p y p g y Case illustration: A 53-year-old man was admitted to the emergency room with a worsening dyspnea in the last few days, orthopnea, and other signs of right heart failure such as jugular venous distention, hepatomegaly, and pitting oedema. Jaundice was noted and ECG result showed atrial fibrillation. Persistent thrombocytopenia (55- 117x103/uL) in the past few months with normal result of hemoglobin and leucocyte level, slight increase in total bilirubin (2.02mg/dL), SGOT (146u/L), and SGPT (74u/L) was revealed. Coagulation marker (PT and INR) was within therapeutic range. All causes of thrombocytopenia such as viral hepatitis, drug-induced, and immunologic destruction were excluded. Abdominal ultrasonography result showed hepatosplenomegaly and dilated hepatic veins. Echocardiography revealed normal LV function (EF 68%) but decreased RV function (TAPSE 1,25cm); dilatation of LA, RA, and RV; severe mitral stenosis (MV area 0,564cm2 and MV mean PG 10,5mmHg) and severe tricuspid regurgitation (TR Max PG 88,8mmHg); D-shaped LV; and estimated PASP of 108,8mmHg. All these findings conclude that this patient had congestive hepatopathy due to right heart failure and severe pulmonary hypertension secondary to severe mitral stenosis. Prolonged congestive hepatopathy could lead to portal hypertension and redistribution of blood flow and platelets to the splenic pool which further lead to reduced thrombopoietin production and results in persistent thrombocytopenia. p p p y p Conclusion: Persistent thrombocytopenia can be seen in long-standing congestive hepatopathy. If left untreated, the liver can progressively become fibrotic and cirrhotic, leading to liver failure. To prevent that, the underlying cardiac condition should be treated. Persistent Thrombocytopenia in Long-Standing Congestive Hepatopathy Secondary to Severe Mitral Stenosis: A Case Report Z. Ramadhanty1, T. Rahadiyan2 General Practitioner, Cardiology Clinic, dr. Bratanata Military Hospital, Jambi1 As the bleeding occurred, we continued clopidogrel and postponed rivaroxaban as we started in the clinic with consideration to reduce risk of bleeding in this patient. Conclusion : There are several treatment strategies for patients with CLTI. One of the strategies is to treat all patients with CLTI with an antiplatelet and consider clopidogrel as single antiplatelet of choice. Current recommendation in patients with peripheral vascular diseases requiring OAC, OAC should be continued only if a compelling indication exists. After endovascular revascularization, OAC alone should be considered if the bleeding risk are high. Keyword : Anti-Thrombotic, Chronic Limb Threatening Ischemia, Deep Vein Thrombosis Keywords: Anti-Thrombotic, Chronic Limb Threatening Ischemia, Deep Vein Thrombosis Figure 1 Patient’s DSA: Pre-Intervention (A) and Post-Intervention(B) Figure 1 Patient’s DSA: Pre-Intervention (A) and Post-Intervention(B) SPECTRAL CT: STATIC MYOCARDIAL PERFUSION IMAGING IN CHRONIC CORONARY SYNDROME C. Abdillah1, D. Wardana2, Elen1 Faculty of Medicine Universitas Indonesia / National Cardiovascular Center Harapan Kita1 Faculty of Medicine Universitas Diponegoro / Diponegoro National Hospital2 Faculty of Medicine Universitas Indonesia / National Cardiovascular Center Harapan Kita1 Faculty of Medicine Universitas Diponegoro / Diponegoro National Hospital2 Background : The non-invasive technique using multi slice computed tomography (MSCT), especially coronary CT angiography (CCTA), is more preferable option in diagnostic test rather than invasive angiography. Dual Energy CT (DECT) using spectral imaging has been introduced and potentially evaluating the changes in myocardial blood flow as iodine map images. The aim of this study is to present a case of patients with suspected of chronic coronary syndrome using Spectral DECT to identify ischemic lesion Case Illustration : A-64-year old female with dyspnea on effort presented with moderate likelihood of CAD. Coronary CT Angiography with Spectral CT was done in this patient. CCTA found multiple lesions on coronary arteries. Moreover, Spectral CT revealed several segments was already hypoperfused although there was no wall motion abnormality with good ejection fraction and normal myocardial density by conventional monoenergetic CT evaluation. Late iodine enhancement (LIE) did not show any infarcted region. Conclusion : CT has ability to combine evaluation of anatomical detail of coronary anatomy and evaluation of perfusion in single procedure. Spectral imaging CT gives higher diagnostic accuracy regarding ischemia condition which is better than conventional CT. Keywords: Spectral CT, CT angiography, chronic coronary syndrome, myocardial perfusion Spectral CT vs Conventional CT Keywords: Spectral CT, CT angiography, chronic coronary syndrome, myocardial perfusion Spectral CT vs Conventional CT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Inotropes and Vasopressor for Stabilization of Cardiogenic Shock with Extensive Anterior STEMI & Ventricular Extra Systole Patient before Primary PCI; a Case Report S.C. Nugroho1, K.P. Islami1, K. Ruth1 RS Dr. Suyoto General Hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Inotropes and Vasopressor for Stabilization of Cardiogenic Shock with Extensive Anterior STEMI & Ventricular Extra Systole Patient before Primary PCI; a Case Report S.C. Nugroho1, K.P. Islami1, K. Ruth1 RS Dr. SPECTRAL CT: STATIC MYOCARDIAL PERFUSION IMAGING IN CHRONIC CORONARY SYNDROME Suyoto General Hospital1 Inotropes and Vasopressor for Stabilization of Cardiogenic Shock with Extensive Anterior STEMI Ventricular Extra Systole Patient before Primary PCI; a Case Report Inotropes and Vasopressor for Stabilization of Cardiogenic Shock with Extensive Anterior STEMI & Ventricular Extra Systole Patient before Primary PCI; a Case Report S C Nugroho1 K P Islami1 K Ruth1 S.C. Nugroho1, K.P. Islami1, K. Ruth1 RS Dr. Suyoto General Hospital1 S.C. Nugroho1, K.P. Islami1, K. Ruth1 RS Dr. Suyoto General Hospital1 Background g Cardiogenic shock is mentioned to describe as inadequate systemic circulation caused by primary cardiac dysfunction that leads to hypoperfusion and subsequent multi-organ failure. Delayed recognition and treatment of Cardiogenic shock can increase mortality rate. g y The incidence of cardiogenic shock is the most frequent complication in ST- elevation AMI as compared to non- ST-elevation AMI. Norepinephrine (NE) and Dobutamine are Vasopressor and Inotropes that frequently used in clinical for Cardiogenic Shock cases. Case Illustration We hereby present a 55-year old man come to ER with typical angina chest pain 1 hour before arriving. This patient has previous stable angina history but not so clear. There were no type II Diabetic history nor stroke. In first ECG, we found Anterior Extensive STEMI and VES with Cardiogenic Shock status. We do initial oral DAPT, high dose statin, fluid reperfusion and start using Dobutamine 3 µg/Kg/minute as initial Inotropes and NE 0,1 µg/Kg/minute as Vasopressor. We also prepare the patient to send to higher level of Hospital to get Primary PCI. Patient’s vital sign was monitored intensively in ER. After 30 minutes monitoring, the BP was not increasing. We increase NE up to 0,5 µg/Kg/minute and Dobutamine 3 µg/Kg/minute. Targeted MAP and BP was not reached. We substitute the combination, then we increase Dobutamine gradually until 10 µg/Kg/minute and NE to 0,2 µg/Kg/minute, targeted MAP and BP was reached and the VES was not too effected. The patient was stable in this setting. Peripartum Cardiomyopathy: A Case Management Series at Sanjiwani Hospital A.A.G.I. Putra1, G.I. Susanti2, K.E. Bagiari1, A.A.G.R. Budayasa2 1Department of Internal Medicine, Cardiology and Vascular Medicine Division, Sanjiwani Hospital, Gianyar, Bali, Indonesia 2Department of Obstetric and Gynecology, Sanjiwani Hospital, Gianyar, Bali, Indonesia A.A.G.I. Putra1, G.I. Susanti2, K.E. Bagiari1, A.A.G.R. Budayasa2 1Department of Internal Medicine, Cardiology and Vascular Medicine Division, Sanjiwani Hospital, Gianyar, Bali, Indonesia A.A.G.I. Putra1, G.I. Susanti2, K.E. Bagiari1, A.A.G.R. Budayasa2 1Department of Internal Medicine, Cardiology and Vascular Medicine Division, Sanjiwani Hospi Bali, Indonesia 2Department of Obstetric and Gynecology, Sanjiwani Hospital, Gianyar, Bali, Indonesia 2Department of Obstetric and Gynecology, Sanjiwani Hospital, Gianyar, Bali, Indonesia Background: In Peripartum Cardiomyopathy (PPCM), heart function will generally return to normal a few months after delivery, but in some cases poor outcomes might occur, therefore early detection and management are crucial. This case series study aims to get an overview of PPCM cases in a type-B referral hospital in Gianyar, Bali. Case Illustration: This study presents 3 cases of PPCM that showed symptom onset in the antepartum period (the third trimester of pregnancy). 2 out of 3 cases were nulliparous with maternal age <30 years, and went through a Cesarean Section. All three cases underwent treatment in the Intensive Care Unit and showed improvement in their condition. Until now, the heterogeneity of PPCM diagnostic criteria has become an obstacle to its treatment, thus the reported incidence tends to be low. This is likely due to the diagnosis of PPCM which is an exclusion diagnosis from other causes of heart failure. Echocardiography and NT-ProBNP examinations should be done, which, although not specific, may lead to a diagnosis of PPCM when combined with a thorough patient history. The availability of supporting examination modalities in many regions in Indonesia are varied thus referrals are sometimes needed, resulting in the delay of PPCM cases management. Conclusion: All pregnant women who experience dyspnea during the third trimester of pregnancy, along with a family history of heart disease, need to undergo close examination and supervision because of the suspicion of PPCM. Early detection and treatment are the main key to successful management of PPCM cases. Keywords: Early Detection; Echocardiography; Peripartum Cardiomyopathy Keywords: Conclusion: All pregnant women who experience dyspnea during the third trimester of pregnancy, along with a family history of heart disease, need to undergo close examination and supervision because of the suspicion of PPCM. Early detection and treatment are the main key to successful management of PPCM cases. Conclusion In this patient with Cardiogenic Shock with VES and STEMI Anterior Extensive was safely to get 10 µg/Kg/minute of Dobutamine as Inotropes and 0,2 µg/Kg/minute of NE. The Ventricular Extra Systole rhythm was not too effected and not going to lethal rhythm. Keywords: : Cardiogenic Shock Ventricular Extra Systole STEMI Dobutamine Norepinephrine g g y Keywords: : Cardiogenic Shock, Ventricular Extra Systole, STEMI, Dobutamine, Norepinephrine Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Peripartum Cardiomyopathy: A Case Management Series at Sanjiwani Hospital Patient echo study Patient echo study Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 PERICARDIAL EFFUSION IN TB PERICARDITIS AS A SIGN OF WORSE PROGNOSIS : EARLY DETECTION AND PROMPT TREATMENT IS PARAMOUNT. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 PERICARDIAL EFFUSION IN TB PERICARDITIS AS A SIGN OF WORSE PROGNOSIS : EARLY DETECTION AND PROMPT TREATMENT IS PARAMOUNT. Cardiac memory-induced T-wave inversions after temporary ventricular pacing: a case report E.P.B. Mulia1, D.A. Rachmi1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga – Dr. S General Hospital, Surabaya, Indonesia1 E.P.B. Mulia1, D.A. Rachmi1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga – Dr. Soetomo General Hospital, Surabaya, Indonesia1 E.P.B. Mulia1, D.A. Rachmi1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga – Dr. Soetomo General Hospital, Surabaya, Indonesia1 Background: Cardiac memory refers to the T-wave inversions (TWI) on the electrocardiogram (ECG) after a period of abnormal ventricular activation or wide QRS. TWI as cardiac memory manifestations are often confused with other pathological conditions that may manifest as TWI, such as myocardial ischemia or infarction, myopericarditis, takotsubo cardiomyopathy, and cerebrovascular injury. Case illustration: We present a 62-year-old female with symptomatic bradycardia due to sinus pause. She underwent temporary transvenous ventricular pacing immediately. A 12-lead ECG obtained after pacing insertion showed right ventricular paced rhythm (QRS width 150 ms) at 75 bpm (figure 1A). In the following days, intrinsic normal sinus rhythm was resumed at 69 bpm and narrow QRS (97 ms). Additionally, deep TWI were observed in leads II, III, aVF, and V3 to V6 (figure 1B). Echocardiography showed no wall motion abnormality. Serial troponin and electrolytes were within normal range. Coronary angiography performed before permanent pacemaker (PPM) implantation showed no significant stenosis. Due to the distribution of TWI, normal echocardiogram and laboratory results, non-significant coronary angiogram, recent right ventricular pacing, and the direction of the T waves in sinus rhythm followed (remembered) the direction of the QRS complex during the preceding episode of wide QRS or abnormal ventricular activation (ventricular pacing), hence TWI was concluded as a manifestation of cardiac memory after ventricular pacing. Follow-up 12-lead ECG four weeks later showed T-wave morphology returned to normal baseline. This further confirmed the final diagnosis of cardiac memory- induced TWI. lusion: Recognizing cardiac memory phenomenon is essential for physicians to facilitate appropria ation and management, which may help avoid unnecessary hospitalization and further cardiac diagnost Conclusion: Recognizing cardiac memory phenomenon is essential for physicians to facilitate appropriate evaluation and management, which may help avoid unnecessary hospitalization and further cardiac diagnostic test. Keywords: cardiac memory, T-wave inversion, wide QRS, ventricular pacing, bradycardia Transient ST Segment Elevation and Complete Atrioventricular Block as an Uncommon Presentation in Acute Fulminant Myocarditis, How to Deal With it?: A Case Series B. Aji1, I. Prasetya2 Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia1 Department of Cardiology and Vascular Medicine, Dr. Saiful Anwar General Hospital, Malang, East Java, Indonesia2 B. Aji1, I. Prasetya2 B. Aji1, I. Prasetya2 Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia1 Department of Cardiology and Vascular Medicine, Dr. Saiful Anwar General Hospital, Malang, East Java, Indonesia2 B. Aji1, I. Prasetya2 Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia1 Department of Cardiology and Vascular Medicine, Dr. Saiful Anwar General Hospital, Malang, East Java, Indonesia2 PERICARDIAL EFFUSION IN TB PERICARDITIS AS A SIGN OF WORSE PROGNOSIS : EARLY DETECTION AND PROMPT TREATMENT IS PARAMOUNT. L.M.S.I.W. Atmapraja1, D. Elysmasia1 Rumah Sakit Umum Daerah Praya1 Background: Pericarditis is inflammation of the pericardial lining which can be caused by an infection, autoimmune disease, and idiopathy. Tuberculous pericarditis is a rare form of extrapulmonary tuberculosis and can produce a slowly arising pericardial effusion. Tuberculous pericarditis consists of four stages. Beyond third and fourth stages the clinical signs and symptoms of cardiac tamponade or congestion appear may progress to constructive pericarditis. Case Illustration : A 26-year-old woman came to the emergency room at the Praya Regional Public Hospital complaining of shortness of breath for the past 3 days. On physical examination the Heart: ictus cordis is not visible and not palpable, the heart border seems widened, heart sounds away. On chest X-ray examination, a "water bottle-shaped heart" was obtained which indicated a massive pericardial effusion. Electrocardiographic examination revealed sinus tachycardia and low-voltage complexes and electrical alternation. On echocardiographic examination found accumulation of pericardial effusion in the posterior area. The patient was diagnosed with pericarditis with massive pleural effusion and cardiac tamponade. Pericardiocentesis was performed, a rapid molecular TB test was carried out and the results were positive. The patient was diagnosed with tuberculosis pericarditis. Conclusion: The diagnosis of tuberculous pericarditis is challenging. It requires strong clinical suspicion, especially in endemic regions. The findings suggest that pericardial effusion in tuberculous pericarditis in the early clinical stage of TB pericarditis is the most predictive factor of constrictive pericarditis, which indicates a poorer prognosis. Early detection and prompt treatment are needed to reduce progressivity and recurrency as well as to prevent mortality. p y Keywords: Tuberculosis Pericarditis, Cardiac Temponade, early detection, prompt treatment Pericardial Effusion Pericardial Effusion Pericardial Effusion Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Transient ST Segment Elevation and Complete Atrioventricular Block as an Uncommon Presentation in Acute Fulminant Myocarditis, How to Deal With it?: A Case Series Background g Acute myocarditis is a difficult diagnosis to make since it lacks a pathognomonic clinical manifestation. Electrocardiogram (ECG) results in a variety of non-specific abnormalities in myocarditis patients. Nonetheless, ECG is frequently employed as a preliminary myocarditis screening technique. Case illustration A-42-years-old and a-37-years-old male were referred to Emergency Department of Saiful Anwar Hospital with the initial diagnosis of acute coronary syndrome (ACS) and shock condition. The ECG examination for the first patient showed new onset right bundle branch block and complete atrioventricular block, while the second patient showed transient ST segment elevation mimicking spontaneous reperfusion. Both patients had elevated Troponin I 31.8 mcg/dL and 39.2 mcg/dL respectively. Urgent coronary angiography was performed as an initial diagnostic tool to rule out coronary cause in both patients with the result of non-significant stenosis with normal TIMI-3 flow. Transvenous temporary pacemaker and intra-aortic balloon pump were inserted as bridge to recovery. y An accurate working definition of fulminant myocarditis is a sudden and severe myocardial inflammation that causes cardiogenic shock and myocyte necrosis. Fulminant myocarditis must be distinguished from other acute circulatory compromises, the most prevalent of which is an acute coronary syndrome. Serial ECGs, echocardiography, heart catheterization with endomyocardial biopsy, immunohistology, and molecular analysis for the underlying infection and pathogenesis are all required as part of a rapid regular work-up in unstable hemodynamic patients. According to current recommendations, acute heart failure treatment consisting pharmacological and mechanical support is required in such situation. Conclusion ECG abnormalities in myocarditis patients can take many different forms, but none of them are pathognomonic. Although it is typical for fulminant myocarditis to have non-vascular distribution of ECG abnormalities, this shouldn't delay an angiographic examination of the coronary structure. Keywords: fulminant, acute myocarditis, transient STEMI, complete atrioventricular block Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 g y Department of Cardiology, Dr. M. Haulussy General Hospital, Ambon, Indonesia1 L.A. Huningkor1, I. Haryana1 D.F. Soeharto1, M. Dewi2, R. Hermawan1 Majenang Regional General Hospital, Central Java, Indonesia1 Internal Medicine Department, Majenang Regional General Hospital, Central Java, Indonesia2 BACKGROUND: Total ischemic time is an important factor in ischemic heart disease. There are still many patients who are slow to seek medical attention resulting disease's onset of >12 hours during first medical contact. Percutaneous Coronary Intervention (PCI) is the first choice in cases accompanied by persistent symptoms or significant cardiovascular comorbidities. Even so, there are still areas in Indonesia that do not have access to reperfusion therapy. Therefore, an alternative clinical approach is needed in these health facilities. CASE ILLUSTRATION: A 68-year-old male patient complained of left chest pain as if a heavy weight had been placed on him. Complaints have been felt to come and go since 6 days prior and have been aggravating since 1 day before admission. There are also symptoms of shortness of breath since 1-week before admission and swelling in both legs. Tachycardia (122x/minute), elevated JVP, rhonchi on both lungs, and pitting edema were found in physical examination The ECG shows sinus tachycardia, LAD, LVH, ST-segment elevation in V2-V4 accompanied by Q waves in V2-V4 and T wave inversion in V5-V6. Troponin results were 395.2 ng/L and chest X-ray showed pulmonary edema and cardiomegaly. The patient was diagnosed with anteroseptal STEMI Killip II and dilated cardiomyopathy with right-left failure FC III. Primary PCI cannot be performed due to distance and clinical considerations, so conservative treatment is chosen. Intravenous vasodilators, double antiplatelets, anticoagulants, diuretics, and oral digoxin were given during treatment. The patient managed to experience clinical improvement at the end of the treatment period and was allowed to go home without sequelae. p p g q CONCLUSION: Limited facilities are not an obstacle to comprehensive patient management. A complete, rapid, and accurate clinical assessment, as well as careful consideration of benefits and risks, greatly assists the clinical outcome of patients in limited health facilities. used, Heart Failure, Indonesia Patient’s ECG prior and after hospitalization ords: STEMI, Non-reperfused, Heart Failure, Indonesia Patient’s ECG prior and after hospitalization Keywords: STEMI, Non-reperfused, Heart Failure, Indonesia Keywords: STEMI, Non-reperfused, Heart Failure, Indonesia STEMI WITH ONSET >12 HOURS COMPLICATED BY HEART FAILURE IN AREAS FAR FROM ACCESS TO REPERFUSION THERAPY: A CASE REPORT D.F. Soeharto1, M. Dewi2, R. Hermawan1 Majenang Regional General Hospital, Central Java, Indonesia1 Internal Medicine Department, Majenang Regional General Hospital, Central Java, Indonesia2 Atypical de Winter electrocardiography pattern: what is the culprit? D.A. Rachmi1, E.P.B. Mulia2, R.A. Fagi1 RSUD Dr. Soetomo - Universitas Airlangga1 Dr. R. Soetrasno General Hospital, Rembang, Indonesia2 Dr. R. Soetrasno General Hospital, Rembang, Indonesia2 Background. The reported positive predictive value for "de Winter ECG pattern" in predicting acute left anterior descending (LAD) artery occlusion is inconsistent. Case illustration. A 40-year-old man with chest pain for 7 hours came to the emergency room. Troponin I level was elevated. An initial 12-lead ECG demonstrated abnormal Q waves and subtle STE in leads I, aVL, STD in leads II, aVF, and upsloping STD up to 2.0-mm with tall, symmetrical T waves in leads V2 – V4 suggesting de Winter ECG changes. Therefore, extensive anterior MI with acute occlusion of the LAD was suspected (figure 1). However, the presence of prominent R waves in leads V1-V2 should raise suspicion that it might mirror the development of Q waves in posterior leads. With the addition of STE in the lead I and aVL, diagnosis of posterolateral MI is the most likely cause of this atypical de Winter pattern. Coronary angiography revealed total occlusion of the proximal LCx. The primary percutaneous coronary intervention was carried out in the LCx. Postprocedural 15-lead posterior ECG showed complete resolution of the ST segment to baseline in V2 – V4, however, abnormal Q wave and STE were found in posterior leads V7-V9 and more apparent STE in V5-V6, which further supports the diagnosis of posterolateral MI mimicking de Winter ECG pattern. Conclusion. Posterolateral MI may present by mimicking the atypical de Winter ECG pattern. Other ECG findings, such as STE, Q wave, and R/S, and the location of the de Winter pattern in the different ECG leads should be used together to predict the culprit artery and assess the anatomical extent of myocardial ischemia. Recording posterior leads are strongly recommended in patients with atypical de Winter ECG pattern or if the ECG was still non-diagnostic of the anatomical location of MI. culprit artery, de Winter, posterolateral, ST-segment elevation myocardial infarction Figure 1. Initial 12-lead-ECG. Keywords: culprit artery, de Winter, posterolateral, ST-segment elevation myocardial infarction Keywords: culprit artery, de Winter, posterolateral, ST-segment elevation myocardial infarction Figure 1. Initial 12-lead-ECG. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 21-year-old man was referred for cardiac murmur and suspected massive pericardial effusion. He was present with shortness of breath and palpitation. The symptoms began seven days prior, including nausea, vomiting, othopnea, and dyspnea on exertion. Physical examination found tachycardia, tachypnea, elevated jugular vein pressure, bilateral rales, continous thrill, continous murmur along right sternal border, distended abdomen, hepatomegaly, and lower extremity oedema. Blood test showed elevated liver enzymes; chest x-ray showed cardiomegaly, pulmonary oedema, and bilateral pleural effusion. Transthoracic echocardiography (TTE) showed a tunnel-like structure originated from non-coronary sinus, passing anteriorly to the right atrium; pulmonary arterial hypertension; and another cardiac anomaly which is an atrial septal defect. He was treated medically to relieve the heart failure (HF) and the clinical features improved. The patient was then recommended to be referred for further evaluation and surgical treatment. : STEMI, Non-reperfused, Heart Failure, Indonesia Patient’s ECG prior and after hospitalization Aorta-Right Atrial Tunnel with Elevated Liver Enzymes: A Case Report L.A. Huningkor1, I. Haryana1 Department of Cardiology, Dr. M. Haulussy General Hospital, Ambon, Indonesia1 nd Patient’s ECG prior and after hospitalization Patient’s ECG prior and after hospitalization Aorta-Right Atrial Tunnel with Elevated Liver Enzymes: A Case Report Background Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Aorta-right atrial tunnel (ARAT) is an abnormal tubular extracardiac communication between the ascending aorta and the right atrium. It is a very rare genetic anomaly, and sporadic case reports of this disease make it vague and ill understood. In this case, we present a 21-year-old man with symptoms of heart failure and diagnosed as having the first case of ARAT ever reporetd in Ambon, Maluku. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Aorta-right atrial tunnel (ARAT) is an abnormal tubular extracardiac communication between the ascending aorta and the right atrium. It is a very rare genetic anomaly, and sporadic case reports of this disease make it vague and ill understood. In this case, we present a 21-year-old man with symptoms of heart failure and diagnosed as having the first case of ARAT ever reporetd in Ambon, Maluku. Background g Patent foramen ovale (PFO) is a common congenital cardiac lesion, occurring in 25-30% of the population. Although most patients with PFO are asymptomatic, they can be associated with a variety of clinical presentations, both cerebrovascular and non-cerebrovascular related, and might significantly impair patients’ quality of life. This case series aims to highlight key differences across diverse clinical manifestations and how these features play important roles in deciding the management of patients with PFO. Case illustration Six cases of patent foramen ovale from our center were compared and outlined in Table 1. All patients experienced cerebrovascular events in varying degrees, from a transient ischemic attack to stroke, and persisting headache was also a recurring complaint. Holter monitoring and carotid ultrasound were done in an attempt to rule out the cause of cerebrovascular events. Further risk stratification with RoPE scoring and PASCAL classification was done and patients in the probable category were managed with percutaneous PFO closure. Despite case 2 being in the possible PASCAL category, percutaneous closure was performed in consideration of her clinical presentations and quality of life. In all cases, PFO closure seemed to alleviate the chronic headaches and no new cerebrovascular events occurred in these patients. A Case Series of Patent Foramen Ovale: Variation in Clinical Presentations and Management J.A. Setiawan1, S. Ng2, I.M. Pardede2 Pelita Harapan University1 Siloam Hospital Lippo Village2 J.A. Setiawan1, S. Ng2, I.M. Pardede2 Pelita Harapan University1 Siloam Hospital Lippo Village2 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion ARAT is a very rare anomaly that can lead to right sided heart failure which can also result in abnormalities of the liver. TTE is enough to establish clinical diagnosis of ARAT but further evaluation with computed tomographic angiography of the heart, ascending aortography, or cardiac catheterization may be needed. Medical management consists of measures to relieve HF, treat coexistent arrythmias and endocarditis. Treatment of HF may also relieve symptoms of liver abnormalities. Immediate surgery or transcatheter closure after diagnosis is recommended for symptomatic ARAT/ Keywords: Aorta, Right Atrial, Tunnel Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:s pISSN: 2830-3105 / eISSN: 296 doi: 10.30701/ijc.1537 A Case Series of Patent Foramen Ovale: Variation in Clinical Presentations and Management J.A. Setiawan1, S. Ng2, I.M. Pardede2 Pelita Harapan University1 Siloam Hospital Lippo Village2 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Successful Reperfusion of High Thrombus Burden in a Right Coronary Artery Presenting as ST- Elevation Myocardial Infarction Inferior and Symptomatic Bradycardia: A Case Report Successful Reperfusion of High Thrombus Burden in a Right Coronary Artery Presenting as ST- Elevation Myocardial Infarction Inferior and Symptomatic Bradycardia: A Case Report A.A. Budiarto1, Ahadi2, R. Herdyanto3 Medical Intern, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.1 C di l i t D R S d Dj tik G l H it l B j I d i 2 Successful Reperfusion of High Thrombus Burden in a Right Coronary Artery Presenting as ST- Elevation Myocardial Infarction Inferior and Symptomatic Bradycardia: A Case Report A.A. Budiarto1, Ahadi2, R. Herdyanto3 Medical Intern, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.1 Cardiologist, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.2 nterventional Cardiology, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.3 A.A. Budiarto1, Ahadi2, R. Herdyanto3 Medical Intern, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.1 Cardiologist, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.2 Interventional Cardiology, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.3 Background High thrombus burden (HTB) may lead to no-reflow, slow-reflow, and/or distal embolization. HTB in patients with acute myocardial infarction (AMI) with complication bradycardia is a significant cause of morbidity and mortality worldwide and its management is very challenging. y Case Illustration A 60-year-old man with chest pain for one hour at rest with diaphoresis, dizziness, nausea, and abdominal discomfort. He had more than 40 years of smoking history. The patient had no history of hypertension, hepatitis, diabetes mellitus, cerebrovascular disease or other health problems. On examination, he was in pain (VAS 5/10) with a blood pressure of 100/60 mmHg and a heart rate of 45 beats per minute, which was irregular. Killip classification was class I. His electrocardiogram (ECG) showed ST-segment elevation in leads II, III, and aVF with 2nd Degree AV block Mobitz type 2. Aspirin 300 mg, ticagrelor 180 mg, atorvastatin 40 mg and 1 mg sulfa atropine were administered. He was immediately transferred o our hospital for primary percutaneous coronary intervention (PPCI). Coronary angiography (CAG) was performed directly under temporary ventricular pacing. Coronary angiography revealed a right coronary artery (RCA) that was completely occluded in the proximal with a high-burden thrombus (fig 1A). He was managed successfully with triple antiplatelets (aspirin, ticagrelor, and intracoronary GIIb/IIIa inhibitors), anticoagulation, and adjunctive PCI (aspiration thrombectomy and three stents were accurately implanted into the culprit's vessel). arrhythmia was observed during this process and the final angiogram showed flow in the proximal RCA with TIMI flow 3 (fig 1B). Conclusion Conclusion PFO often presents as cerebrovascular events and chronic headaches, hence the presence of these manifestations simultaneously may prompt physicians to include PFO as a differential diagnosis, especially when other causes of cerebrovascular events have been ruled out. Risk calculations such as RoPE score and PASCAL classification remain valuable and may aid in the decision on managing PFO patients, leading to either percutaneous closure or conservative management with antithrombotics. g Keywords: PFO, migraine, stroke, RoPE, PASCAL Table 1: Case details. Table 1: Case details. Indonesian Journal of Cardiology Management of Acute Coronary Syndrome Presenting with Acute Heart Failure in Rural Area: A Case Report g g Conclusion This case report demonstrates successful treatment HTB use of aggressive antiplatelet, anticoagulation, PPCI and temporary pacemakers in STEMI patients with bradycardia but maybe this treatment can't be effective in another case because there is no ideal management strategy. Keywords: High thrombus burden, STEMI, temporary pacemakers, PCI Keywords: High thrombus burden, STEMI, temporary pacemakers, PCI Fig 1. The right coronary artery of a 60-year-old male patient at the (A) beginning and (B) end of the primary percutaneous coronary intervention procedure. Fig 1. The right coronary artery of a 60-year-old male patient at the (A) beginning and (B) end of the primary percutaneous coronary intervention procedure. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Management of Acute Coronary Syndrome Presenting with Acute Heart Failure in Rural Area: A Case Report Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Management of Acute Coronary Syndrome Presenting with Acute Heart Failure in Rural Area: A Case Report Y.D. Tanugerah1, D.N. Sary1 RSUD Muhammad Zein Belitung Timur1 Background: AHF is defined as the sudden emergence of new or worsening heart failure symptoms. ACS is one of the most important causes of AHF. AHF in the setting of acute coronary syndromes is closely associated with reduced survival rates, both during hospitalization and in the long term. Mortality and hospitalization rates are also high in cases of AHF with ACS. The diagnosis of AHF and ACS is made by clinical presentations, ECG, serial cardiac troponin, and emergency echocardiography. In addition, patients with ACS and AHF should immediately undergo coronary angiography and PCI. y g y g g p y Case illustration: We reported a case of a 74-year-old man, without any previous medical history, presenting with shortness of breath and chest pain 2 hours before admission. The physical examination showed high blood pressure, desaturation, and cold extremities. Crackles and wheezing were heard in both lungs. The electrocardiogram (ECG) showed elevation of ST segment in precordial leads. Chest X-Ray showed bilateral pulmonary oedema of both lung fields. The patient was treated conservatively with only oral medications due to limited facilities and resources in the rural area. Conclusion: ACS with AHF is a high-risk group with a mortality rate of 4.2%-5.5% and 1-year mortality rate is 20.6%-26.7%. AHF treatment should be given when required to stabilize the patient and concurrently with the start of ACS therapy. This case report showed adequate conservative therapies based on guidelines had good outcome in patients with ACS and HF. p eywords: Acute Heart Failure, Acute Coronary Syndrome Keywords: Acute Heart Failure, Acute Coronary Syndrome ECG and X-Ray examination shown in Emergency Room ECG and X-Ray examination shown in Emergency Room Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Sildenafil Therapy in a 42-Year-Old Man with Pulmonary Hypertension and Acute Renal Failure: A Case Report SURGICAL APPROACH FOR PATIENT WITH VENTRICULAR SEPTAL DEFECT, MEMBRANOUS SEPTAL ANEURSYM AND LARGE TRICUSPID VALVE VEGETATION F.A. Azmi1, D. Hariyanto1, Kino1 Universitas Andalas1 Background: Ventricular septal defect (VSD) is the most common heart malformation and estimated about 25% of CHDs. One of the long-term risks of VSD is infective endocarditis (IE). Aim of this report is to discuss surgical approach for patient with VSD, membranous septal aneurysm, and large tricuspid valve. pp p p y g p Case Illustration: A 12-year-old girl presented with repeated high fever. There was history of shortness of breath after heavy physical activity. The patient was known for suffering from perimembranous VSD since 3 years old. There was history of repeated cough, cold, and reduced appetite. Body temperature was 38.3oC. Nutritional status was under-weight. Auscultation was grade 3/6 pansystolic murmur on LLSB, which extended laterally, and intensity didn’t increase with inspiration. Laboratory examination showed leukocytosis. ECG results sinus rhythm, LAD and LVH. Chest X-ray showed CTR of 60% and plethora. Echocardiography showed VSD PM 3- 5 mm, left-right shunt and vegetation in septal tricuspid leaflet. Patient was given vancomycin 2x500 mg, gentamicin 1x75 mg, paracetamol 4x300 mg, and captopril 2x6,25 mg. Blood culture was positive for Streptococcus mitis and Streptococcus orafis. Patient was diagnosed with definite infective endocarditis met two major criteria of Duke's modified. Open heart surgery, VSD surgical closure, and evacuation of vegetation were conducted. We didn’t conduct catheterization of the right heart due to vegetation which may complicate as pulmonary embolism if detached. p y Conclusion: We have described a girl diagnosed with VSD PM L-R 3-5 mm shunt with IE. The shunt left-to- right made it easy for the occurrence of IE due to existence of turbulent flow on VSD. IE is natural history of VSD, thus emphasizing the urgency of early diagnosis and management when IE is detected. Keywords: Ventricular septal defect, Membranous Septal Aneurysm, endocarditis infective, vegetation Keywords: Ventricular septal defect, Membranous Septal Aneurysm, endocarditis infective, vegetation Keywords: Ventricular septal defect, Membranous Septal Aneurysm, endocarditis infective, vegetation Transthoracic Echocardiography shows 3-5 mm VSD L-R shunt, MSA and vegetation of tricuspid septal leaflet. sthoracic Echocardiography shows 3-5 mm VSD L-R shunt, MSA and vegetation of tricuspid septal leafle Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Sildenafil Therapy in a 42-Year-Old Man with Pulmonary Hypertension and Acute Renal Failure: A Case Report A Tale of Success: Pericardial Effusion in Pediatric Lupus and Rheumatic Fever I.P. Farissa1, H.H. Satoto1, Y. Herry1, F. Tandri1 Department of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University1 I.P. Farissa1, H.H. Satoto1, Y. Herry1, F. Tandri1 Department of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University1 I.P. Farissa1, H.H. Satoto1, Y. Herry1, F. Tandri1 Department of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University1 Sildenafil Therapy in a 42-Year-Old Man with Pulmonary Hypertension and Acute Renal Failure: A Case Report M.Destiani1, N. Anggita2 1General Practitioner, Kramat Jati Hospital, East Jakarta 2General Practitioner, Hermina Bitung Hospital, Tanngerang Background: Pulmonary hypertension (PH) is one of the world's health issues, its prevalence is estimated at 1% of the world's population and increases to 10% at the age of >65 years. PH is defined as an increase in MAP >20 mmHg. PH is a rare disease, but has high morbidity and mortality rate. g g y y Case Illustration: A 42-year-old man came to emergency room with swollen legs since 2 weeks ago and shortness of breath, he was often sleeping with high pillows. His stomach was getting bigger, accompanied by nausea, vomiting, reduced urine volume. The patient has history of pulmonary tuberculosis treatment. There was increase in respiratory rate (28x/min), oxygen saturation of 74% on room air. Physical examination revealed ronchi in both lung fields, ascites, pitting oedema of inferior extremities. ECG showed RAD axis. Laboratory showed mild thrombocytopenia (141,000), elevated urea (105), blood creatinine (2.4). Chest x-ray resulted cardiomegaly, LAE, RVH. The patient was diagnosed with CHF caused by RHD. The therapies were furosemide 3x40 mg, HCT 1x25 mg, bisoprolol 1x2.5 mg, folic acid 3x1, bicarbonate 3x1, and CaCO3 3x1. On the third day of treatment, the patient experienced worsening shortness of breath, hypotension (70 mmHg/palpation). The patient was transferred to HCU with dobutamine 5mcg/kg/minute. At the maximum dose of dobutamine, the patient was still hypotensive, received norepinephrine 0.2 mcg/kg/minute. Echocardiography resulted LVEF 70%, grade I diastolic dysfunction, TR Severe High Probability for PH. The patient received sildenafil therapy 3x10 mg, on the third day, the dose was increased to 3x20 mg. The patient experienced clinical improvement after five days of using sildenafil. Conclusion: Sildenafil is a class of PDE-5i and is one of therapies for patients with PH. PDE-5i degrades cGMP and causes vasodilation in pulmonary blood vessels where the enzyme is widely excreted. Keywords: Keywords: Pulmonary Hypertension, Sildenafil Keywords: Keywords: Pulmonary Hypertension, Sildenafil Background g Acute rheumatic fever (ARF) is caused by autoimmune response to a group A streptococcal infection. ARF largely affects children between the ages of 5–14 years with risk factors including age, sex, environment, and host susceptibility. Pediatric systemic lupus erythematosus (SLE) is a rare entity, affecting children under 16 years of age. Diagnosis is not always easy. Pericarditis is the most frequent cardiac manifestation of SLE, occurring in 10% to 40% of cases. A-16 years old boy referred with breathlessness 2 weeks prior to admission, not relieved with resting and position changes, accompanied with fever, coughs, odynophagia, and migrating joint pains. He was severely ill, with pressure 113/82 mmHg pulse 113 bpm respiratory rate 30x/min, temperature was 37 ˚C, pansystolic murmur grade IV/6 in apical area, diastolic murmur grade II/4 in Erbs area, gallop. The Laboratory result was leucocytosis, anaemia, elevated ESR, positive ASTO test, positive ANA and ds DNA test. Oropharyngeal swab revealed positive grams diplococcus and blood culture was positive for Klebsiella oxytoca. The echocardiography revealed severe aortic and mitral regurgitation with massive pericardial effusion without tamponade signs. We managed with high dose of steroids, antibiotics, colchicine, and NSAIDs. The evolution was favorable and echocardiography evaluate no longer pericardial effusion. Conclusion Global Longitudinal Strain as predictor of Myocardial Damage in Severe Mitral Regurgitation with Normal Ejection Fraction M.R. Priatama1, A.S. Kuncoro2 Universitas Indonesia1 National Cardiovascular Center Harapan Kita2 Background Myocardial strain imaging using GLS is a novel approach for assessing left ventricular function. From the previous study, GLSalready deteriorated even when EF is still preserved, indicating that GLS may be superior in detecting myocardial damage. It can be suggested that GLS may also have a role in assessment of mitral regurgitation (MR), especially in severe MR with preserved EF where early detection of myocardial damage is needed for making clinical decisions. Case Illustration: A male, 57 years old came to NCCHK, with fatigue and DOE. Patient sleep with 2 pillows stacked and he often wakes up at night due to shortness of breath and cough. There are no chest pain or rapid heart rate. The patient complained a presenting symptoms six months prior to the admission. These symptoms were amplified during mild activity. The echocardiography shows eccentric left ventricular hypertrophy, left ventricular systolic function was normal, dilated left atrium, flail of PML with posteromedial commissure lesion and severe mitral regurgitation splay. GLS score was - 16,3 %. echocardiogr Conclusion The diagnosis of ARF cannot be made using a single test, requiring recognition of a complex of clinical signs divided into major and minor manifestations as well as laboratory investigations aided by application of the Jones criteria. Pediatric-onset SLE diagnosis and management with heavy treatment in a growing being poses some specific problems. Pericardial effusion may happen in pediatric with SLE and ARF requiring prompt management. The use of corticosteroid therapy and treatment with proper adequate antibiotics is crucial in managing pericardial effusion and ARF. Keywords: Massive pericardial effusion, Pediatric-onset SLE, Acute Rheumatic Fever Echo initial Keywords: Massive pericardial effusion, Pediatric-onset SLE, Acute Rheumatic Fever Keywords: Massive pericardial effusion, Pediatric-onset SLE, Acute Rheumatic Fever Echo initial Echo initial Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:su pISSN: 2830-3105 / eISSN: 2964 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: Conclusion: Contrast extravasation with contrast staining can help to differentiate between coronary perforation to pericardial and into the cardiac chamber. Handmade ‘tegaderm’ covered stent may be considered if there is no other option. Contrast extravasation with contrast staining can help to differentiate between coronary perforation to pericardial and into the cardiac chamber. Handmade ‘tegaderm’ covered stent may be considered if there is no other option. Case illustration: A-59 years old man was admitted to emergency department (ED) with chest pain. After careful anamnesis and physical examination, patient was diagnosed with acute ST segment elevation anterior myocardial infarction and sent to the catheterization laboratory. Right radial access was used and coronary angiography at Left Anterior Descending (LAD) artery shows total occlusion. After first Drug Eluting Stent (DES) placed, there is massive contrast extravasation in LAD artery (Ellis type III). Balloon was inflated to compress the perforation, while a second puncture was added through right femoral artery to treat the Right Coronary Artery (RCA) lesion. After delivered one DES stent in RCA, balloon was deflated and there is still massive contrast extravasation. Echocardiography was used to evaluate cardiac tamponade and there is minimal (<10mm) pericardial effusion. Attempt to stop perforation was made with two stacks of DES in Left Main (LM) artery, but failed. Handmade ‘tegaderm’ covered stent was deployed in LM-LAD artery, but contrast extravasation is still present. Echocardiography was used once again and found that stent was protruding into left ventricle base. Procedure was stopped due to maximum contrast was reached. Patient was given additional anticoagulant drug. Consulting with thoracic surgeon was made to retrieve stent surgically, but patient declined. Confirmation with computerized tomography (CT) scan found there is stent from LAD branch to left ventricle with probability of fistula. Background: Background: Coronary artery perforation is one of the most dreadful complications and still convey high mortality risk. Presence of contrast extravasation through frank perforation demand fast and precise decision to prevent further worse outcome. Protruding Stent into Left Ventricle : When Handmade Covered Stent wasn’t Enough V.B.F.P. Untu1, J.A. Pangemanan1, G.E.H. Reppi1, H.J. Sunarto1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R.D.Kandou General Hospital, Manado, Indonesia1 V.B.F.P. Untu1, J.A. Pangemanan1, G.E.H. Reppi1, H.J. Sunarto1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R.D.Kandou General Hospital, Manado, Indonesia1 V.B.F.P. Untu1, J.A. Pangemanan1, G.E.H. Reppi1, H.J. Sunarto1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R.D.Kandou General Hospital, Manado, Indonesia1 Conclusion: GLS is more superior than EF in prediction of myocardial damage in patients with severe MR. Measurements using EF, LVESD and LVEDD are more load dependent, which GLS are less dependent. GLS also have potential role in clinical decision making for timing of intervention in severe MR with preserved LVEF. Keywords: Keywords: Speckle Tracking Echocardiography, Global Longitudinal Strain, Mitral Regurgitation Figure 1. GLS Score -16.3 ds: Keywords: Speckle Tracking Echocardiography, Global Longitudinal Strain, Mitral Regurgitation Keywords: Keywords: Speckle Tracking Echocardiography, Global Longitudinal Strain, Mitr Figure 1. GLS Score -16.3 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Protruding Stent into Left Ventricle : When Handmade Covered Stent wasn’t Enough V.B.F.P. Untu1, J.A. Pangemanan1, G.E.H. Reppi1, H.J. Sunarto1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R.D.Kandou General Hospital, Manado, Indonesia1 Keyword: Coronary Perforation, Protruding Stent, Handmade Covered Stent Keywords: Coronary Perforation, Protruding Stent, Handmade Covered Stent Keywords: Coronary Perforation, Protruding Stent, Handmade Covered Stent Case Illustration A 56 years old woman came to ER with typical chest pain 1.5 hours prior to admission. She has medical history of myasthenia gravis, dyslipidemia, but didn’t take medication routinely. Vital sign and physical examination was normal. The ECG showed ST elevation in anterior extensive lead. Laboratory test revealed elevated troponin. Chest x-ray showed cardiomegaly LA and LV. Echocardiography showed LVEF 40.7% with hypokinetic anterior, anterolateral segment at basal-mid level, apicoanterior. Patient was given loading dose of acetylsalicylic acid, clopidogrel, and statin. The patient underwent fibrinolytic with 1.5 million IU dose of streptokinase, since primary PCI couldn’t be performed. Chest pain was relieved and ECG showed ST elevation resolution indicating successful fibrinolytic. She developed myasthenic crisis that led to respiratory distress at 12-hours admission in ICCU, was managed with ventilator, neostigmine intravenous, corticosteroids and plasmapheresis. Patient condition was improved, extubated, and discharged 1 week later. Background g Myasthenia gravis is an autoimmune disorder affecting neuromuscular transmission leading to muscle weakness. Myasthenic crisis is life-threatening complication, resulting in respiratory failure that requires intubation and mechanical ventilation. Cardiovascular event in patients with myasthenia gravis are rare, but clinicians should bear in mind the potential adverse interaction between myasthenia gravis and cardiovascular disease or its medications. We present a case of acute STEMI patient concomitant with myasthenia gravis which developed into myasthenic crisis. Surviving the Storm: A Case Report of Myasthenic Crisis in Acute STEMI Patient D. Nugraha1, I.P. Farissa1, S. Sungkar1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Diponegoro University- Dr. Kariadi Central General Hospital, Semarang, Indonesia1 D. Nugraha1, I.P. Farissa1, S. Sungkar1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Diponegoro University- Dr. Kariadi Central General Hospital, Semarang, Indonesia1 D. Nugraha1, I.P. Farissa1, S. Sungkar1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Diponegoro University- Dr. Kariadi Central General Hospital, Semarang, Indonesia1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Protruding Stent into Left Ventricle Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Protruding Stent into Left Ventricle Protruding Stent into Left Ventricle Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Protruding Stent into Left Ventricle Protruding Stent into Left Ventricle Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Complete RBBB without the Presence of Cardiovascular Disease: Not Inconsequential as Previously Thought? S. P. Yogita1, S. A. N. Y. Sutarmini2, I G. A. B. K. Jayantika2 S. P. Yogita1, S. A. N. Y. Sutarmini2, I G. A. B. K. Jayantika2 S. P. Yogita , S. A. N. Y. Sutarmini , I G. A. B. K. Jayantika Intern of Cardiology and Vascular Medicine, Bali Mandara Regional Public Hospital, Indonesia1 Departement of Cardiology and Vascular Medicine, Bali Mandara Regional Public Hospital, Indonesia2 Intern of Cardiology and Vascular Medicine, Bali Mandara Regional Public Hospital, Indonesia1 D f C di l d V l M di i B li M d R i l P bli H i l I d i 2 Intern of Cardiology and Vascular Medicine, Bali Mandara Regional Public Hospital, Indonesia1 Departement of Cardiology and Vascular Medicine, Bali Mandara Regional Public Hospital, Indonesia Background: With a prevalence ranging from 0.2% to 2.3%, right bundle branch block (RBBB) is one of the most common electrocardiography (ECG) findings in clinical practice. It is still unknown whether RBBB increases the risk of coronary artery disease (CAD). Bundle branch block is frequently associated with myocardial infarctions, myocarditis, and cardiomyopathy (BBB). Unfortunately, isolated RBBB without structural heart disease remains a possibility. This case study adds to the growing body of evidence that isolated RBBB is not a trivial finding. Case Illustration: A 17-year-old male came for a medical check-up since he will be going to police academy. At the policlinic, the patient was fully conscious with a blood pressure of 120/80 and pulse rate of 58 bpm. He had no symptoms of chest pain or shortness of breath. The patient lives a healthy lifestyle, which includes activities like jogging and running around the park every day. He smokes but only a few cigarettes a month, sometimes even less. There were no other risk factors for coronary artery disease. The patient then proceeded with an ECG, which showed a complete right bundle branch block with normal sinus rhythm, no signs of ischemic and no signs of infarct. Echocardiogram (transthoracic) showed no regional wall motion abnormality with normal left ventricle (LV) ejection fraction. We suggested to proceed with a CT-Angiography but the patient refused, hence the patient went home without any medical treatment. Conclusion Myasthenic crisis is a life-threatening complication of myasthenia gravis. Precipitating factors should be quickly identified and promptly mitigated. It is possible that myocardial infarction or cardiovascular medication could trigger myasthenic crisis in some individuals. However, it is important to note that the relationship between them has not fully understood, and further research is needed to confirm this potential link. Keywords: Acute STEMI, myasthenia gravis, myasthenic crisis Precipitants of myasthenic crisis Precipitants of myasthenic crisis Background g Heart failure caused by low cardiac output state in aortic stenosis (AS) dan mitral stenosis (MS) has poor prognosis with high risk for surgery. The use of loop diuretic to relieve the congestion may induce hyponatremia, worsening cardiac output reduction, hypovolemia, and critical hypotension in this condition. Tolvaptan acts as vasopressin receptor antagonist, helps to maintain hemodynamics and seems to be appropriate in this condition. Therefore, we present the case of tolvaptan's effects and safety in patients with heart failure caused by severe AS and MS. Case Illustration A 35-year-old man scheduled for Double valve replacement surgery complained of exertional dyspnea, altered mental state, ascites, and leg swelling. He has ejection systolic murmur at second right intercostal spaces close to the sternum and mid-diastolic murmur at the apex. Laboratory test showed hyponatremia. Chest X-ray showed cardiomegaly, congestive pulmonum, and pleural effusion. Echocardiography revealed severe AS and MS with preserved LV function (EF 55%). At initial treatment, maintenance dose of furosemide was given but the congestion persisted. After hemodynamic stabilization, Tolvaptan 7,5 mg was added to optimal medical therapy. Dyspnea, ascites and leg swelling were improved. It didn’t cause hypotension significantly (BP 89/68 to 85/69). Urine output increased significantly (750 to 2500cc/day). There was improvement of hyponatremia (127 to 137 mmol/L) after 5 days administration, no worsening of kidney function (creatinine serum 1,13 mg/dL to 1,2 mg/dL). Echocardiography also showed no hypovolemia (IVC 25/19) and no worsening of aortic (VTI 0.4cm2 to 0.4cm2) and mitral valve flow (VTI 0.8cm2- VTI 0.7cm2) Patient maintained stable heart failure and improvement of quality of life until 1-week follow-up. Tolvaptan Use In Low Cardiac Output State Caused By Combination of Aortic And Mitral Stenosis : Hemodynamic Management For Bridging To Surgery F Tandri1, I.P. Farissa1, R. Hidayatulah1, M. F. Ahnaf1 Dr. Kariadi Hospital1 Tolvaptan Use In Low Cardiac Output State Caused By Combination of Aortic And Mitral Stenosis : Hemodynamic Management For Bridging To Surgery F Tandri1, I.P. Farissa1, R. Hidayatulah1, M. F. Ahnaf1 Dr. Kariadi Hospital1 F Tandri1, I.P. Farissa1, R. Hidayatulah1, M. F. Ahnaf1 Dr. Kariadi Hospital1 Complete RBBB without the Presence of Cardiovascular Disease: Not Inconsequential as Previously Thought? Conclusion: Right bundle-branch block has been linked to an increased overall risk of mortality and cardiovascular-related mortality in patients without known cardiovascular disease, as well as a higher frequency of hypertension and more exercise-related limitations, such as decreased aerobic capacity, slower heart rate recovery, and more dyspnoea on exercise testing. However, performing a comprehensive assessment to monitor patients with asymptomatic RBBB imposes a massive healthcare burden. p y p p Keywords: right bundle branch block (RBBB), isolated RBBB, complete RBBB Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Tolvaptan Use In Low Cardiac Output State Caused By Combination of Aortic And Mitral Stenosis : Hemodynamic Management For Bridging To Surgery F Tandri1, I.P. Farissa1, R. Hidayatulah1, M. F. Ahnaf1 Dr. Kariadi Hospital1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 STEMI WITH DIABETIC KETOACIDOSIS, HOW TO MANAGE? F. Yuliani1, Y.R. Ilhami1, W. Erlanda1 RSUP Dr M DJAMIL PADANG1 F. Yuliani1, Y.R. Ilhami1, W. Erlanda1 RSUP Dr M DJAMIL PADANG1 F. Yuliani1, Y.R. Ilhami1, W. Erlanda1 RSUP Dr M DJAMIL PADANG1 Background: Diabetic ketoacidosis (DKA) is an acute life-threatening complication of diabetes mellitus that be signed by trias hyperglycaemia, acidosis, and ketosis. DKA can also be seen concomitantly with a STEMI. Fluid resuscitation to treat DKA could be dilemmatic problem in STEMI patient. Case illustration: A 49 years old woman referred to hospital with sudden chest pain since 7,5 hours before admission. Her physical examination showed she was fully alert, with blood pressure 123/78 mmHg, heart rate 60 beats/min, and minimal rales in both lung fields. Blood gas analyse showed metabolic acidosis, pH 7.32, PCO2 28 mmHg PO2 169 mmHg HCO3- 15 mmol/L BE - 11.2 mmol/L, SO2 99%. Random blood glucose was 526 mg/dL, osmolarity was 316 and fluid deficit 3 litters. Her electrocardiogram showed total AV blocked with ST elevation 1-4 mm at II, III, AVF, V3R-V4R.The patient underwent Temporary Pacemaker and Primary Percutaneous Coronary Intervention in Right Coronary Artery while giving critical ill insulin and fluid therapy with saline 1,5 L. Patient then admitted to CVCU. In CVCU, the patient looked somnolent and Kussmaul breathing with urine ketone (++). We assessed as diabetic ketoacidosis and administrated fluid resuscitation around 3,75 L. Echocardiography revealed EF 43%, TAPSE 1,1, SV 39 ml, CO 4L/i, SVR 1640 dyne.sec/cm5, normal IVC, collapsibility index 22% and central venous catheter (CVC) pressure 12 mmHg. We did strict echocardiography and CVC monitoring. Patient symptom and hemodynamic were improved and discharged at day 10. y Conclusions: When STEMI patient concomitant with DKA, fluid therapy may need to be modified. Volume status and cardiac performance monitoring using central venous catheter and echocardiography should be controlled intensively. Keywords: Diabetic Ketoacidosis, STEMI, Fluid Therapy Keywords: Diabetic Ketoacidosis, STEMI, Fluid Therapy Keywords: Diabetic Ketoacidosis, STEMI, Fluid Therapy c Ketoacidosis, STEMI, Fluid Therapy Conclusion Tolvaptan use in low cardiac output state caused by combination of AS and MS with heart failure is safe before double valve replacement surgery. eywords: Tolvaptan, Heart Failure, Aortic Stenosis, Mitral Stenosis, Double Valve Replacement Surgery Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 57-year-old male with history of hypertension and smoking, was presented to emergency departement with chief complain of intermittent central chest pain, dyspnea, fatigue for two days. To give a brief background, he had a history of a posteriorlateral STEMI 6 weeks earlier without revascularisation therapy. On physical examination, blood pressure was 96/64 mmHg, heart rate 106 bpm, respiratory rate 23 breaths/minute, systolic murmur in apex, no rales. Electrocardiogram was showing sinus rhythm, with ST segment depression in lead V2- V4 and T wave inverted in lead V4-V6, I and aVL. Laboratory study revealed increased WBC: 18750/mL, increased creatinine level: 1,42 mg/dL, CKMB: 15 U/L. Chest X-ray revealed an oval bulge on the left border of the heart and more prominent left cardiac silhouette compared to previous his chest X-ray, cephalisation. He underwent a TTE which accidentally showed LV wall ruptured at basal until mid inferolateral and formed pseudoaneurysm with narrow neck (3 cm), turbulent bidirectional flow showed by colour Doppler, no thrombus, LV dilatation, moderate MR, EF 35%, mild pericardial effusion. He was diagnosed with LV free wall ruptured with pseudoaneurysm and history of late onset posterolateral STEMI. He was given intravenous diuretic, ACE inhibitor, nitrate and beta-blocker. Then, the patient was referred to Harapan Kita National Heart Center for further therapy. He underwent a successfull surgical repair. Background Free wall rupture of the left ventricle (LV) is a mechanical complication following myocardial infarction leading to fatal condition. In rare case, free wall rupture can be forming a pseudoaneurysm. The diagnosis and treatment are challenging. Left Ventricular Free Wall Rupture with Pseudoaneurysm following Inferoposterior Myocardial Infarction: A Case Report F.A. Habibi1, S. Safitri2, R.M. Candrasatria3 Puskesmas Gunung Sari Pesawaran Lampung1 RSUD Menggala2 RSUD Dr.H.Abul Moeloek Provinsi Lampung3 F.A. Habibi1, S. Safitri2, R.M. Candrasatria3 Puskesmas Gunung Sari Pesawaran Lampung1 RSUD Menggala2 RSUD Dr.H.Abul Moeloek Provinsi Lampung3 Keywords: Diabetic Ketoacidosis, STEMI, Fluid Therapy Keywords: Diabetic Ketoacidosis, STEMI, Fluid Therapy Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Conclusion Our patient presented with previous MI and non-specific symtomps but with clinical suspicion finally was leading to find a rare mechanical complication of MI through non-invasive imaging. So the patient could get early appropriate management. pp p g Keywords: Pseudoaneurysm, cardiac rupture, myocardial infarction, case report Figure 1. Transthoracic echocardiography Figure 1. Transthoracic echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 M. Irfan1, M.R. Enoch1, A. Hardin1 Department of Cardiology Abdul Aziz Hospital, Singkawang, West Borneo, Indonesia.1 M. Irfan1, M.R. Enoch1, A. Hardin1 Department of Cardiology Abdul Aziz Hospital, Singkawang, West Borneo, Indonesia.1 M. Irfan1, M.R. Enoch1, A. Hardin1 Indonesian Journal of Cardiology cessful Suppression of Electrical Storm Using A Combination of Medications in The Setting of Acute Coronary Syndrome : A Case Report A. Siagian1, P.S. Prameswari1, S. Laksono1 A. Siagian1, P.S. Prameswari1, S. Laksono1 A. Siagian1, P.S. Prameswari1, S. Laksono1 Department of Cardiology and Vascular Medicine, RS Jantung Diagram Cinere, Depok, Indonesia1 Background Electrical storm is a rare but life threatening condition with high mortality, occuring as complication in 6% of acute coronary syndrome cases. It is defined as a clustering of three or more separated ventricular arrhythmia in 24 hours. The focus of treatment in electrical storm is to stabilize the patient, identifying the etiology, and prevent recurrence. gy p Case Illustration A 49 year-old male was taken to our emergency department complaining of chest pain and shortness of breath since one day. He was previously hospitalized with non-ST elevation myocardial infarction and acute heart failure 10 days before admission. On presentation unstable ventricular tachycardia (VT) was noted. Immediate electrical cardioversion achieved conversion into normal sinus rhythm, but episode of sustained VT returned and the patient was diagnosed with electrical storm. Signs of acute heart failure were found : tachypnea, bilateral rales, distended jugular vein, and pulmonary edema seen on chest x-ray. The patient was diagnoses with NSTEMI according to elevated troponin T. A few hours after admission, cardiac arrest occurred and the patient was placed on mechanical ventilation. Due to acute kidney injury and lack of hemodialysis facility in our hospital, we did not perform percutaneous coronary intervention (PCI) that should be the main focus in treating electrical storm induced by ischemia. Instead, we opted to treat the patient conservatively with a combination of heparinization, dual antiplatelet therapy, treatment of heart failure, and combination of amiodarone and lidocaine as antiarrythmics. Conclusion We opted to treat the patient conservatively without PCI. Our patient probably benefited from administration of lidocaine, an antiarrythmic that is especially useful in ischemic cellular condition. Sedation and treatment of heart failure with diuretics, ACE-inhibitor, and beta blocker also contributes to the suppression of electrical storm. Keywords: acute coronary syndrome, arrhythmia, electrical storm, heart failure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 g Keywords: Transient ST segment depression, High-grade fever, Diffuse ST segment depression, Hyperpyrexia Abstract Background:Intracardiac thrombus are a complication of dilated cardiomyopathy (DCM). Factors implicated in the formation of thrombus in DCM include low-velocity swirling of blood, abnormal endocardial surfaces, atrial fibrillation, and a hypercoagulable state. The most common site for thrombus in DCM is the apex where blood flow is generally slowest. However there is no recommendation for treating thrombus in children. Here we present a case of young children with intracardiac thrombus treated with rivaroxban for 3 months. Case Illustration: A 14 years old female came to Hospital with chief complaint of dyspnea. The vital signs on normal value, with minimal crackles. The Laboratory showed rheumatoid factor were positive with other parameters in normal ranges. The electrocardiogram showed sinus rhythm with left ventricular hypertrophy. Echocardiography showed a thrombus in the left ventricle (LV) with the size is 18x35 mm and decreased of ejection fraction with global hypokinetic. The patient was treated with rivaroxaban for 3 months. The next echocardiography showed the thrombus had disappeared without any complications. Conclusion: LV thrombus can occur due to several factors and one of them is a decrease in ventricular ejection fraction. There is no still guideline recommendation for treating of LV thrombus in children using direct oral anticoagulation. Systemic anticoagulation is indicated when there is evidence of thromboembolic or intracardiac thrombosis. Rivaroxaban has shown to have success as a thrombus dissolution agent among the other agents in adult patient. However we didn’t know the etiology of DCM and thrombus formation for this patient. Rivaroxaban administration in pediatric patients with LV thrombus can reduce or even eliminate the thrombus. Further investigation of rivaroxaban is need to be investigated. eywords: LV thrombus, dilated cardiomyopathy, rivaroxaban Pre and post treated with rivaroxaban for 3 months. Keywords: LV thrombus, dilated cardiomyopathy, rivaroxaban Pre and post treated with rivaroxaban for 3 months. Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Transient Diffuse ST Segment Depression in a Patient with High-Grade Fever: A Diagnostic Pitfall Y.N. Afifah1, E.D.N. Rahmawati1 PKU Muhammadiyah Gamping Hospital, Yogyakarta1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A-37-year-old man admitted to Emergency Department because of dyspnea accompanied with prolonged fever for 3 months with a history of untreated recurrent toothache. From physical examination, there was grade 3/6 holosystolic murmur, high pitched punctum maximum at apex radiating to axilla. Cardiomegaly with congestive pulmonum was noted by chest x-ray. Echocardiography revealed normal Left Ventricle function with vegetation at mitral valve leaflets and severe mitral regurgitation. He was diagnosed as possible IE at presentation. Three sets of blood cultures were taken before the patient was commenced on antibiotic with ampicillin-sulbactam and gentamycin. There was no improvement for 7 days of treatment. Blood culture later revealed Abiotrophia defectiva which resistant to ampicillin-sulbactam, we decided to replace it with Ceftriaxone. On 11th day, patient developed severe kidney injury (Creatine 4,9 mg/dL) and sepsis (Procalcitonin 6,87 ng/ml) which were though as a septic embolism to kidney. A combination of Meropenem and ceftriaxone then administrated but septic shock and respiratory distress syndrome caused the patient death. Background Background Most cases of Infective endocarditis (IE) are caused by Streptococci viridans or Staphylococci species. Abiotrophia defectiva is a rare species which can lead to infective endocarditis. Here we discuss a fatal case of IE due to Abiotrophia defectiva. en a Rare Pathogen Strikes: Abiotrophia defectiva and Infective Endocarditis with Septic Embolism Effendi1, I.P. Farissa1, F. Tandri1, M.F. Ahnaf1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Diponegoro University, Dr. Kariadi Central General Hospital Semarang, Indonesia Transient Diffuse ST Segment Depression in a Patient with High-Grade Fever: A Diagnostic Pitfall Y.N. Afifah1, E.D.N. Rahmawati1 PKU Muhammadiyah Gamping Hospital, Yogyakarta1 Background : High-grade fever is defined as a body temperature above 39°C. Body temperature is known to affect the cardiovascular system. However, it is rare for high-grade fever to induce ST segment depression, which can be a diagnostic pitfall as it mimics coronary occlusion. Case illustration : A 71-year-old female with a history of arrhythmia and hypertension was admitted to the emergency department complaining of fever and chills without any chest pain. She had a body temperature of 41.7°C, elevated blood pressure at 148/66 mmHg, and tachycardia at 110 beats/min. Physical examination revealed cardiomegaly. Echocardiography showed hypertensive heart disease and 64% ejection fraction. Laboratory examination showed normal electrolyte levels, neutrophilia, lymphopenia, and a grey-zone HS Troponin result of 13.80 ng/L. Routine urinary examination suspected cystitis. Electrocardiography displayed sinus tachycardia with diffuse ST segment depression. She was resuscitated with IV hydration and paracetamol infusion. Her temperature returned to normal followed by the ST segment returning to the baseline on serial electrocardiography. Her previous Holter monitoring 1 month earlier confirmed no history of ST segment changes. Three days later, the patient was discharged without any events. The mechanism of ST segment depression triggered by high-grade fever is less understood. It is hypothesized that during high-grade fever, myocardial oxygen demand is elevated due to hyperthermia and tachycardic events. This mechanism may result in transient ischemia shown by the ST segment changes. Conclusion : We documented a patient with transient diffuse ST segment depression during the high-grade fever phase. The electrocardiography resolved after her temperature returned to normal. It is crucial to be cautious in interpreting ST segment changes in patients with high-grade fever, especially without clinical signs and symptoms of acute coronary syndrome. Serial electrocardiography and cardiac biomarker tests can aid in accurate diagnosis and management. Keywords: Transient ST segment depression, High-grade fever, Diffuse ST segment depression, Hyperpyrexia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Th The report underscores the potentially life-threatening nature of IE. It highlights the challenges of diagnosing and treating rare pathogens like Abiotrophia defectiva and also complications of patients with IE. The report underscores the critical role of prompt and aggressive management. Overall, this case report highlights the need for increased awareness and vigilance to infective endocarditis, particularly when rare pathogens are involved. While the outcome in this case was tragic, it provides important lessons for healthcare professionals in diagnosis, treatment, and management of infectious diseases. Keywords: Infective endocarditis, septic, rare pathogen Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 THE IMPORTANCE OF IMMEDIATE REPERFUSION ON STEMI WITH CONDUCTION DISORDER: CASE OF SUCCESSFUL FIBRINOLYTIC ON ANTERIOR STEMI WITH BIFASCICULAR BLOCK A. Almira1, Hendrianus2, Y. Natanael3 Brawijaya Women and Children Hospital Antasari1 Cardiology and Vascular Medicine Resident, Faculty of Medicine Universitas Indonesia, National Cardiovascular Center Harapan Kita2 Ruteng Public Hospital3 Background : Acute myocardial infarction is one of the major leading death worldwide. ST-elevation myocardial infarct (STEMI) is frequently associated with the development of a new bundle branch block or hemiblock result in high mortality rate due to the extensive myocardial necrosis. This case is presented to highlight a case of STEMI with bifascicular block (RBBB and LAFB) and its management in rural area. Case Illustration: A 70 years-old male was referred to rural hospital from primary health centre with typical chest pain 7 hours before admission. At arrival, the patient exhibited mild chest pain with stable vital sign. The electrocardiogram showed sinus rhythm with anterior myocardial infarct followed by RBBB and LAFB, so DAPT and nitrate were given. Before fibrinolytic was administrated, the patient chest pain aggravated and became highly agitated accompanied by sign of shock. Fibrinolytic was administered along with benzodiazepine to sedate the patient. Fibrinolytic therapy was successful as marked by significantly decreased chest pain and agitation. ECG evaluation showed persistent ST elevation in anterior leads. Echocardiography showed decreased left ventricular ejection fraction (EF 20%) and LV aneurysm. After 5 days in the ICU, his clinical condition improved and the patient was discharged in a stable condition. Bifascicular block (RBBB with LAFB or LPFB) is often associated with a poor prognosis and the presence of acute heart failure. Fibrinolytic therapy is crucial in settings where primary PCI cannot be offered in a timely manner, as in rural area. Conclusion: In rural area, immediate reperfusion with fibrinolytic significantly improved the outcome of patients with STEMI complicated by bifascicular block. Keywords: Bifascicular block, fibrinolytic, STEMI, rural area Keywords: Bifascicular block, fibrinolytic, STEMI, rural area Indonesi Car Indonesian J Car pISSN: 2830-310 doi: 10.3 Recurrent Seizures and Arrhythmia : Clinical Entity or Coincidence? R.K. Utami1, A. Rakhmawati1, M.D. Sutadi1 RSUD Dr Tjitrowardojo Purworejo1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 The management of antiplatelet therapy in acute coronary syndrome patients with thrombocytopenia: A Case Report W. Bahagia1 Bhayangkara Palangka Raya Hospital1 Background: Acute myocardial infarction (AMI) is myocardial necrosis resulting from acute obstruction of a coronary artery. Dual antiplatelet therapy (DAPT) consisting of aspirin and a P2Y12 receptor antagonist is a fundamental component of acute coronary syndrome (ACS) management. The management of antiplatelet therapy in ACS patients with thrombocytopenia poses a particular challenge for physicians, as they are at higher risk of both bleeding and, paradoxically, ischaemic events Case Illustration: A 49-year old man came to emergency with a history of hypertension and presented chest pain followed by worsening shortness of breath, nausea, and vomiting 12 hours before admission. Physical examination’s patient was conscious, blood pressure 140/90, a respiration rate of 32 times/minute, and a heart rate 138 times/minute irregular. The electrocardiogram showed irregular narrow QRS complex tachycardia and p wave was absent. Laboratory CBC revealed hemoglobin 9,7 g/dl and platelet count 88 x 109/L. Chest radiograph showed either cardio or lung was normal. Echocardiography showed all chamber dilation, decrease left ventricular systolic function (ejection fraction 46%), segmental hypokinetic, mild mitral regurgitation, and moderate tricuspid regurgitation. Given the patient’s stable hemodynamic, nitroglycerin intravenous drip became started. Aspilet, Clopidogrel, Digoxin, Bisoprolol, Furosemide, Spironolactone, and Atorvastatin were initiated. After six days of adequate treatment, the symptoms resolved and the patient was discharged from hospital. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Recurrent Seizures and Arrhythmia : Clinical Entity or Coincidence? R.K. Utami1, A. Rakhmawati1, M.D. Sutadi1 RSUD Dr Tjitrowardojo Purworejo1 Background : Cardiac arrhythmia is frequently associated with epileptiform seizure. It has been considered as a potential cause of sudden unexpected death in epilepsy. We report a case of a patient with recurrent seizures with high degree atrioventricular block and documented ventricular standstill during seizure which required a cardiac pacemaker. p Case illustration : A 49-year-old woman with a medical history of epilepsy was admitted to the emergency room due to recurrent seizures. She had experienced several episodes of seizure since a week before admission with normal level of consciousness gained between the episodes. The medical history of arrhythmia was unknown. Physical examination showed stable hemodynamics with no remarkable neurologic deficit. Laboratory studies showed elevated white blood cells count and normal metabolic panels. The 12-leads ECG exhibited high degree atrioventricular block. The EEG showed generalized intermittent slowing wave with no epileptiform activity. During hospitalized, she had generalized seizure followed by cardiac arrest with ventricular standstill mode in the monitor. Surface ECG monitoring was high degree atrioventricular block with ventricular standstill (Figure 1). She got return of spontaneous circulation and regained consciousness after the episodes of seizure. The patient was treated with dopamine infusion and referred to higher-level hospital for pacemaker implantation. Conclusion: Recurrent seizures in this patient was most likely caused by atrioventricular node dysfunction, not epilepsy since the EEG showed no epileptiform activity. She needed pacemaker implantation mandatory. Keywords: seizure, arrhythmia, epilepsy, high degree av block Figure 1 ECG showed high degree atrioventricular block with ventricular standstill Figure 1 ECG showed high degree atrioventricular block with ventricular standstill Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964- doi: 10.30701/ijc.1537 The management of antiplatelet therapy in acute coronary syndrome patients with thrombocytopenia: A Background Background Development cardiac tamponade from pericardial effusion is a life-threatening condition requiring prompt diagnostic and therapeutic. In PH, pericardial effusion is related ti higher risk stratification class and an independent mortality risk factor. Dilemma of an Action : Cardiac Tamponade in Severe Pulmonary Hypertension. Should We Drain W. Mubarok1, R.P. Harjoko2, Jefry1 W. Mubarok1, R.P. Harjoko2, Jefry1 y p g y g Departement Cardiology and Vascular Medicine, Dr. Kariadi General Hospital Semarang, Indonesia2 Departement Cardiology and Vascular Medicine, Dr. Kariadi General Hospital Semarang, Indonesia2 Case Illustration 28-year old obess women present with dyspneu, ascites and edema in lower extremity. The complain since 1 years ago. Heart rate was 100 bpm with BP 110/70 mmHg, RR 26 per minutes, SpO2 96%. The ECG was sinus rythm, RAD and RVH. The laboratorium showed hipoalbuminemia, hipokalemia and coagulopathy. Patient already done RHC, there are severe PH with mPAP 70 mmHg with no ASD. In echocardiography, we found dilated RA RV and MPA with LV smallish, decrease RV function, circumferensial pericardial efussion with no sign of cardiac tamponade, TR severe and high probability for PH. Patient got pericardial window and got 500cc over 24 hour. The drain was gradual with improvement in LA filling. So, the presence of of RV failure may significantly alter typical diagnostic findings. Abnormally high intracavitary pressures make RA and RV much more resistant to collapse. Because of ventricular interdependence, right ventricular dilation causes a disproportionate decrease in the size of the left ventricle, which worsens diastolic filling and stroke volume. Rapid drainage has high peri- procedural mortality due to RV decompensation. Gradual drainage, as in our patient, may be safer resulting improvement. Conclusion: The presence of thrombocytopenia in ACS patients predicts significantly worse outcomes. It has been suggested that thrombocytopenia in ACS may reflect a greater burden of atherosclerosis predisposing to heightened platelet consumption, or reflect clinically significant thrombosis, and consequently, its presence should be viewed as a marker of disease severity. DAPT could use in ACS patients by considering the platelet count baseline, state of bleeding, and percutaneous coronary intervention (PCI) planning. Keywords: AMI, NSTE-ACS, Trombocytopenia, Tachyarrhytmia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion The recognition of cardiac tamponade in the presence of severe pulmonary hypertension and RV failure can be even more challenging, because the “classic” findings are often not observed. Deeper understanding in patphysiology and mechanism wolud help the patient workup. Keywords: cardiac tamponade, pericardial effusion, pulmonary hypertension p p y gy p p p Keywords: cardiac tamponade, pericardial effusion, pulmonary hypertension Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 REVASCULARIZATION IN CARDIAC ARREST SURVIVOR: A TRANSIENT ST-ELEVATION MYOCARDIAL INFARCTION POSTEROLATERAL CASE REVASCULARIZATION IN CARDIAC ARREST SURVIVOR: A TRANSIENT ST-ELEVATION MYOCARDIAL INFARCTION POSTEROLATERAL CASE L.B.A. Praha1, M.F. Ahnaf1 Cardiovascular Department, Medical Faculty of Diponegoro University, RSUP Dr. Kariadi L.B.A. Praha1, M.F. Ahnaf1 L.B.A. Praha , M.F. Ahnaf Cardiovascular Department, Medical Faculty of Diponegoro University, RSUP Dr. Kariadi Background: AMI is a frequent cause of cardiac arrests due to VF or pulseless VT. The ESC clearly state in the case of cardiac arrest and ST-segment elevation after ROSC, immediate CAG and revascularization should be performed in order to reduce mortality and improve patient outcomes. p y p p Case Illustration: A 67 years old woman was referred from Magelang hospital complaining of chest pain. Her CAD risk factor was menopause. After an hour, suddenly pulseless VT and cardiac arrest happened, continue CPR and defibrillation 3 times. Presenting on Kariadi, BP 118/75, pulse 84 bpm reguler, RR 20, SpO2 98% NC 3 lpm. There were normal cardiac and pulmonary examinations, ECG in Magelang showed ST elevation in lead I, aVL, ST depression in lead III, V1-V4, and occasional VES, then ECG in Kariadi hospital showed qR lead aVL, V7,8,9 and run of PVC. Chest X Ray revealed Cardiomegaly (LV) and bronchovascular pattern increased. The laboratory test showed leukocytosis (17.4 uL), elevated transaminase enzyme (OT 526, PT 325 U/L) and troponin level >25 ug/L, and metabolic acidosis fully compensated. She was scheduled to early PCI, found CAD3VD+LM Disease and done 1 DES distal LM - proximal LAD, 1 DES mid- distal LAD, 1 DES ostial proximal LCx, and then stabilized in CVCU for further evaluation. Conclusion: Transient STEMI seems to be unique condition. According to ESC guidelines an early invasive approach with a view to revascularization within 24 h of presentation is mandatory. The term “time is muscle” is a basic principle in diagnosis and optimal management of AMI to improve patient outcomes. p p g p g p p Keywords: Revascularization, Cardiac Arrest Survivor, Posterolateral STEMI The early percutaneous coronary intervention had done The early percutaneous coronary intervention had done Indonesian Jou Cardiolog Indonesian J Cardiol 202 pISSN: 2830-3105 / eISS doi: 10.30701/ijc Asymptomatic Obstruction of Bioprosthetic Mitral Valve due to Pannus Formation N.M.A.W. Sari1, K.S. Bhuana1 Universitas Udayana / RSUP IGNG Prof Ngoerah1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background : Pannus is an abnormal fibrovascular tissue, located in the cirmcumference of the bioprosthetic heart valve. The true insidence of pannus is unknown, and thought to be related to incidence of valve thrombosis at 0.1 to 6% per year. Diagnosis of pannus require imaging such as echocardiography. Transthoracic echocardiography provided initial evaluation on transvalvular gradient, and determine the presence of obstruction. Transoesophageal echocardiography provided visualization of the mass and mobility of the leaflet. Management of pannus depend on the severity of the obstruction. Case Illustration : We present a case of a 29-year-old female for routine yearly evaluation after she was performed a mitral valve replacement with BHV around 8 years prior, due to rheumatic severe mitral stenosis. On evaluation, she did not have haeart failure symptoms and only took furosemide when necessary. Physical examination revealed low-grade diastolic murmur at 2nd right intercostal space with vesicular breath sound. Transthoracic echo showed moderate Aortic Regurgitation (AR), suspected pannus on the cirumference of the mitral BHV, with mean transvalvular pressure gradient of 9 mmHg at heart rate (HR) of 69 bpm. Transesophageal echocardiography revealed a restricted motion during diastolic phase on one of the mitral BHV leaflet due to a suspected pannus structure, a moderate calcification on one of the leaflets, and trivial mitral regurgitation. We observed a change of mitral BHV mean transvalvular pressure gradient during procedure into 15mmHg with a HR of 84 bpm, with no change in symptom. The patient was managed conservatively with transthoracic echocardiography reevaluation every 6 months. Conclusion : Pannus formation after mitral bioprosthetic heart valve (BHV) implantation is a rare finding with unclear pathomechanism. An asymptomatic obstruction of the bioprosthetic mitral valve usually require routine symptoms observation and echocardiographic reevaluation. This report hopes to provide an echocardiographic evaluation of BHV mitral valve pannus. Keywords: Transthoracic Echocardiography, Mitral Vave Bioprosthetic, Pannus, Transesophageal Echocardiography, Asymptomatic obstruction. Keywords: Transthoracic Echocardiography, Mitral Vave Bioprosthetic, Pannus, Transesophageal Echocardiography, Asymptomatic obstruction. chocardiography, Asymptomatic obstruction. Top : 2D TEE image at mid-esophageal long axis, showing a mass suspected pannus. Bottom : 3D TEE reconstruction from LV and LA View. Top : 2D TEE image at mid-esophageal long axis, showing a mass suspected pannus. Bottom : 3D TEE reconstruction from LV and LA View. A rare case of intracardiac thymic carcinoma presenting as Superior Vena Cava syndrome Keywords: Thymic Carcinoma, Right Atrial Mass, Superior Vena Cava Syndrome, Anterior mediastinal mass, Intracardiac masses Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Management of NSTEMI Patient with Acute kidney Dysfunction due to Septic Shock at Hasna Medika Cirebon Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Management of NSTEMI Patient with Acute kidney Dysfunction due to Septic Shock at Hasna Medika Cirebon Hospital M.A. Rezza 1, A. Deri2, H.I. Gugun3 Hasna Medika Hospital Cirebon, Jawa Barat, Indonesia Background: The presence of kidney dysfunction in ACS has been associated with worse outcomes, including higher rates mortality and bleeding. Prevalence rates of 42.9% among patients NSTEMI with presenting chronic kidney disease. Management of patients with severe kidney impairment with ACS more complex, consideration of increasing bleeding risk with worsening renal function. Case Illustration: A 46- year-old male present to the hospital with chest discomfort, short of breath, and fatigue, fever and a new cough. Symptoms began approximately 2 weeks before and had progressively worsened with no associated, aggravating, or relieving factors noted. His physical examination showed BP 82/55 mmHg. No JVD present, bilateral rhonchi noted, cold extremities, and physical exam is otherwise normal. The laboratorial presented leukocytosis (19850/mL), troponin T (144 ng/l), aPTT (22.5/s) and a significant elevated urea (289.0 mg/dl) and creatinine blood level (12.01 mg/dl), compared to the historical of last 4-month laboratory result which was urea (31 mg/dl) and creatinine (0.6 mg/dl). ECG showed T wave inversion lead V1-V3, a chest x-ray showed pneumonia and cardiomegaly, and we also performed hemodynamic echocardiography showed ERAP15, SV:35, CO:2.3, SVR:2365 (on norepinephrine support). Due to acute kidney dysfunction we decided to do conservative management. Rehydration treatment with intravenous infusion of lactated ringer’s solution, and followed by intravenous of norepinephrine dose. Loading dose of DAPT, then maintenance dose for the next day, atorvastatin once daily. Bolus dose of intravenous heparin followed by maintenance dose of heparin per hour. The follow up on the day 4th of medication, laboratory evaluation down to 194.9 mg/dl of urea level and 2.0 mg/dl of creatinine level. The laboratory evaluation at the polyclinic visits after a week of discharged, down to 24.6 mg/dl of urea level and 0.77 mg/dl of creatinine level. A rare case of intracardiac thymic carcinoma presenting as Superior Vena Cava syndrome I.N. Wiryawan1, K.S. Bhuana R1 Universitas Udayana / RSUP IGNG Prof Ngoerah1 I.N. Wiryawan1, K.S. Bhuana R1 Universitas Udayana / RSUP IGNG Prof Ngoerah1 Background : Thymic carcinoma is a rare neoplasm that arise in the anterior mediastinum. Thymic carcinoma has an incidence of 0.2 to 1.5% of all malignancies. Thymic carcinoma has varying clinical presentations, from asymptomatic findings to severe tumor mass effect. An intracardiac infiltration of thymoma occur in 4% of all cases. Diagnosis is made from clinical presentation, imaging and histopathologic assessment. Advanced stage of thymic carcinoma carries a worse prognosis. Case Illustration : We present a case of intracardiac thymic carcinoma in a 64-year-old male patient with chief complaint of intermitten swelling of the face with neck lymph node enlargement. Initial chest MRI showed moderate signal intensity on the SVC. He was initially diagnosed as antiphospolipid syndrome due to positive ANA IF. Around 2 years after diagnosis, patient developed severe congestion of the face and neck, and upper extremities. Echocardiography evaluation showed a 5.9 x 5.2 cm mass inside the right atrium. Chest CT Scan showed a Mass from right atrium, extending into the SVC causing type IV SVC obstruction. CT scan also showed varying HU around the mass suggesting a thrombus adjacent to the mass. Patient was planned for sternotomy and tumor evacuation. Intraoperative evaluation showed a mediastinal mass with extensive localized and intracardiac spread. Patient was only performed incisional biopsy with samples taken from both the anterior mediastinum mass and inside the right atrial mass. Hisptopathological diagnosis of Thymic Carcinoma was made. Patient passed away due to lung infection, before receiving definitive treatment. Conclusion : Intracardiac thymic carcinoma is a rare case and often overlooked. Infiltration into the cardiac chamber represent a more advanced disease, such as presented above. This report hopes to provide a comprehensive insight on imaging of intracardiac masses. p g g g Keywords: Thymic Carcinoma, Right Atrial Mass, Superior Vena Cava Syndrome, Anterior mediastinal mass, Intracardiac masses. Background Mitral stenosis (MS) in pregnancy is still one of the most common causes of maternal death. MS is one of the most commonly encountered valvular heart diseases during pregnancy, the majority are rheumatic in origin and poorly tolerated due to cardiocirculatory changes that occur during pregnancy, labor and postpartum. Severe MS is usually poorly tolerated in pregnancy, with 67% of women developing peripartum heart failure even if previously asymptomatic. Successful Balloon Mitral Valvuloplasty in 32-week Pregnant Patient with Severe Mitral Stenosis: A Case Report J.P. Sianipar, W.N. Yuandika, M.F. Ahnaf Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Kariadi General Hospital, Semarang J.P. Sianipar, W.N. Yuandika, M.F. Ahnaf Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Kariadi General Hospital, Semarang Case illustration A 33 years old woman, G3P1A1 31 weeks pregnant came to Dr. Kariadi Hospital with a shortness of breath during daily activity, which was worsened since 26 weeks of pregnancy. The complaint was accompanied by swelling on both legs. She felt more comfortable to sleep with half-sitting position. Chest pain was denied. Physical examinations demonstrated the BMI was 45 kg/m2, diminished S1 and normal S2 sound, irregularly irregular, mid diastolic murmur 2/4 on apex. Echocardiography revealed Severe MS with MVA by planimetry 1.05 cm2, by VTI 0.9 cm2, by PHT 0.9 cm2, mean PG 18 mmHg, Wilkins score 5, moderate tricuspid regurgitation with high probability for pulmonary hypertension. The patient was stabilized with continuous drip furosemide, digoxin, and warfarin. BMV had been done in 32 weeks of pregnancy and managed to reduce the mitral pressure gradient from 17.7 mmHg to 8.6 mmHg with mPAP decrease from 75 mmHg to 62 mmHg. During the procedure patient was under general anesthesia and extubated in recovery room after procedure. Then she was moved to ICU for monitoring in a day, then moved to ward. A rare case of intracardiac thymic carcinoma presenting as Superior Vena Cava syndrome Case Discussion: We describe a case NSTEMI with severe hypoperfusion caused by septic shock induced to kidney parenchymal injury followed by significant decrease of glomerular filtration rate and led to acute kidney dysfunction. In this NSTEMI patient with GFR 5.98 ml/min, we decided conservative therapeutic with unfractionated heparin (UFH) used, DAPT dose and rehydration and norepinephrine support to increase perfusion due to septic shock. We delayed invasive coronary angiography and percutaneous coronary intervention because of concerns about acute kidney injury accelerating their progression to dialysis. Conclusion: UFH remains the default option for anticoagulant NSTEMI patients with kidney impartment can safely and effectively used, despite the complexity of its use and its known toxicity profile. Keywords: acute kidney dysfunction, NSTEMI, septic shock Conclusion: UFH remains the default option for anticoagulant NSTEMI patients with kidney impartment can safely and effectively used, despite the complexity of its use and its known toxicity profile. K d t kid d f ti NSTEMI ti h k Fig. 1 Initial 12-lead ECG showing sinus rhythm, T-wave inversion in lead V1-V3 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 S f l B ll Mit l V l l l t i 32 k P t P ti t ith S Mit l St i A C Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology A 69 years old man with in stent thrombosis after PCI leading to Free Wall Rupture and Massive Pericardial Effusion: a Complex Disastrous Event after PCI A 69 years old man with in stent thrombosis after PCI leading to Free Wall Rupture and Massive Pericardial Effusion: a Complex Disastrous Event after PCI Y. Suryadilaga, M.F. Ahnaf, S. Sungkar1 1 Department of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University, Kariadi General Hospital, Semarang, Indonesia Y. Suryadilaga, M.F. Ahnaf, S. Sungkar1 1 Department of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University, Kariadi General Hospital, Semarang, Indonesia g Case Illustration A 69 years old man was referred with dyspneu and chest. A month ago, he experienced substernal heavy chest pain with diaphoresis. He got treatment and diagnosed posterior STEMI, underwent primary PCI, and a stent was placed in ostial-distal Left Circumflex artery. 3 weeks after, he developed dyspneu and chest discomfort, re- angiography showed in stent thrombosis with LV aneurysm. Echo revealed massive pericardial effusion with free wall rupture. In our ER, he was panting, Blood Pressure was 125/74 (94) HR 94 bpm RR 24 times/minute SpO2 98%, there were no rales, elevated JVP and pitting edema on both ankles. Bedside Echo showed massive localized pericardial effusion without tamponade, LV aneurysm and suspected of free wall rupture. Later he was admitted to ICCU and underwent cardiac surgery. Backgrounds Left ventricular free wall rupture is a rare but severe complication of acute myocardial infarction. It was reported to occur either early after the onset of MI (type I or II, approximately 55%) or during the sub-acute phase accompanied with overt cardiac remodelling (type III, 45%). Factors including old age, female gender, and large infarct size were found to be associated with the risk of free wall rupture. Due to the variable clinical presentations associated with high mortality, it remains a substantial diagnostic and therapeutic challenge for clinicians. Case Illustration Conclusion Balloon mitral valvuloplasty in pregnancy is a relatively safe and effective interventional cardiac procedure, helps the patients with moderate to severe MS which gives optimum results. Keywords: pregnancy, mitral stenosis, balloon mitral valvuloplasty Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 A 69 years old man with in stent thrombosis after PCI leading to Free Wall Rupture and Massive Pericardial Effusion: a Complex Disastrous Event after PCI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Backgrounds Postoperative stroke in cardiac surgery continues to be a major factor that increases morbidity, mortality, and readmission risk, with contemporary reports of a ninefold increase in postoperative death of stroke patients. As cardiac surgical outcomes have improved over the past decades, the demographic of patients being referred for surgery has shifted in favor of patients who are of greater age with more g y p g g comorbidities that may put them at higher risk for postoperative stroke. The incidence of stroke after cardiac surgery is dependent on the procedure itself and the reporting center, but typically ranges from 0.8% to 5.2%. C Ill t ti A 58 years old man felt shortness of breath while taking light activities then underwent mitral valvular replacement. There is no history of atrial fibrillation and neurological sign before cardiac surgery. Vital sign after surgery, blood pressure 106/70 MAP 79 on support dobutamine 10 mcg/kgweight/minutes/ and norepinephrine 0.2mcg/kgweight/minutes, HR 120-130x/minutes irregular, RR 26x/minutes and SpO2 100% on VM Mode SCMV PEEP 5 FiO2 40%, patient showed atrial fibrilation. Sixth day after cardiac surgery patient got seizure tonic clonic with treatment fenitoin injection. Then head MSCT found cytotoxic cerebral edema, showed increased pressure intracranial, and sphenoiditis dextra et sinistra. Patient moved to stroke care unit after got stabilized from ICCU ward. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Y. Suryadilaga, S. Sungkar1 1Department of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University, Kariadi General Hospital, Semarang, Indonesia Y. Suryadilaga, S. Sungkar1 ment of Cardiology and Vascular Medicine, Medical Faculty Diponegoro University, Kariadi General Hospital, Semarang, Indonesia Conclusion Acute myocardial infarction can cause ischemic, mechanical, arrhythmic, embolic, or inflammatory complications. The development of mechanical complications after AMI is associated with significant short-term clinical improvement and long-term survival. Though the incidence of this fatal complication has seen a considerable decline due to primary percutaneous coronary angiography, our observations point to a more prolonged symptom to angiography time as one of the important predictors that leading causes of this complication. Keywords: Free Wall Rupture, STEMI Keywords: Free Wall Rupture, STEMI Keywords: Free Wall Rupture, STEMI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Neurological Complication After Cardiac Surgery: A Case Report Conclusion Patients undergoing cardiac surgery should receive frequent postoperative neurologic checks so that an intervention can be made as quickly as possible when a large stroke occurs. Current interventions can minimize stroke risk,treat perioperative stroke, and manage perioperative hypoxic-ischemic injury. The goals of therapy are to improve oxygenation, maintain an adequate blood pressure for perfusion of vital organs and coagulation status. The underlying cause must be addressed. Keywords: Cardiac surgery, Stroke, Postoperative In Indo pISSN Cardiac Amyloidosis Presented with SND: A Case Report S.A. Alawiyah1, I. Fahri1, S. Hastuti1, J. Endang1 RSUD DR. M YUNUS BENGKULU1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Cardiac Amyloidosis Presented with SND: A Case Report S.A. Alawiyah1, I. Fahri1, S. Hastuti1, J. Endang1 RSUD DR. M YUNUS BENGKULU1 Background: Cardiac Amyloidosis is a restrictive cardiomyophaty characterized by the extracelullular deposition of mis-folded proteins in the heart. It is usually diagnosed late since symptomps are nonspecific and are often disregarded with other conditions. Case Illustration: A 54 years old man presented to emergency departement with sudden onset of syncope. He had medical history of heart failure with preserved ejection fraction and normal coronary artery by coroangiography. On initial physical examination, His blood pressure was 87/62 mmHg with heart rate of 50 bpm. He also had ecchimosys in eyes and had facial droop and difficulty to closing eyes and smiling. He had clear lungs and normal heart sounds without murmurs or gallops. He had mild pitting edema bilaterally. ECG showed 1st degree AV block with episode of junctional rhytm and low voltages in the limb and pre cordial leads. His blood perasure and HR was support with dopamine and hemodynamics was stable, but unable to weaned the inotropic due to hypotension and ECG showed AF SVR. We decided to implant PPM to this patient. Based on the findings above, a diagnosis of amyloidosis complicate with SND was considered and supported by echocardiography illustrated severe concentric ventricular hyperthrophy and longitudinal strain reduction with relative apical sparing showed ‘cherry on top’ on bull’s eye appearance with EF was at 32% by simpsons. He remained asymptomatic after pacing and was discharged three days later. A subsequent follow-up of his amyloid involvement by other explorations is planned for this patient. Conclusion: Cardiac amyloidosis remains a rare disease. Keywords: Cardiac Amyloidosis, Syncope, SND Background g Chronic venous insufficiency (CVI) of the lower limb has great effect on quality of life. Untreated CVI results in variety of complications, the major ones being persistent pain and progressive skin changes ranging from small eritema to extensive areas of severe skin pigmentation that may ultimately lead to ulceration. The progressive unpleasant symptom itchiness that interfere daily activities is the prime cause for seeking treatment. Endovenous laser therapy (EVLT) is a less invasive procedure which is widely used for the treatment of CVI with excellent results. Conclusion In rural area with limited diagnostic testing must identifying more from clinical and physical significance. Cardiologist should be aware of cardiac amyloidosis to allow early treatment that can change the prognosis of this disease Keywords: Cardiac Amyloidosis, Syncope, SND Keywords: Cardiac Amyloidosis, Syncope, SND Keywords: Cardiac Amyloidosis, Syncope, SND Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Lower Limb Skin Pigmentation as Sequelae of Untreated Chronic Venous Insufficiency C. Adriana1, N. Anggriyani1 Faculty of Medicine, Diponegoro University, Semarang, Indonesia1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Case illustration A 48-year old woman presented with progressive skin pigmentation and itchy feeling on her right lower limb since 5 years ago. She has been plagued with fatigue leg, cramp, pain and burning sensation for 1 year. These symptoms worsen with prolonged sitting at work as administrative officer for 8 hours per day and obesity. She also had hypertension and dyslipidemia. Physical examination revealed inflammation skin discoloration appear as brown bruises on right tibial region. Lower limb arteries pulsation were equal and normal. Duplex ultrasound revealed severe CVI of right limbs without deep vein thrombosis. After performed EVLT, the patient symptom and skin discoloration improved and able to function at high capacity both at work and with her leisure activities. The extent complication of CVI can be reduced with non-surgical minimally invasive strategy. Conclusion Skin hyperpigmentation as a sequelae of underlying CVI required for treatment. EVLT has provided alte for CVI treatment, effective in reducing the associated symptoms and improved quality of life. for CVI treatment, effective in reducing the associated symptoms and improved quality of life. Keywords: chronic venous insufficiency, skin discoloration, skin hyperpigmentation, endovenous laser ablation for CVI treatment, effective in reducing the associated symptoms and improved quality of life. Keywords: chronic venous insufficiency, skin discoloration, skin hyperpigmentation, endovenous laser ablation Keywords: chronic venous insufficiency, skin discoloration, skin hyperpigmentation, ds: chronic venous insufficiency, skin discoloration, skin hyperpigmentation, endovenous laser ablation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Congenital Heart Disease (CHD) is the main cause of death in infants with 6.4 incidence per 1.000 birth. Complete Atrioventricular Septal Defect (CAVSD) which corelate much with Down Syndrome (DS) was 4-5.3 case per 10.000 birth. Here, we report a CAVSD patient with DS morphology. Case Illustration A 2-year-old girl was admitted to hospital for dyspnea, fever, and cough with history of tuberculosis one year ago and routine control to Public Health Center for malnutrition. She was born premature (32-33 weeks) due to inadequate antenatal care with breastfeeding difficulty after. DS morphology was found. She was severely wasted, underweight, and stunted (5kg, 76cm) with BP 63/41mmHg, HR 140, RR 40, SaO2 60-70%, T 38oC. Cyanosis were triggered by crying. We discovered 3/6 systolic ejection murmur at left sternal border and bilateral rhonchi along with increased CRP, leukocytosis, and hypothyroid. Perihilar and paracardial infiltrates were found. Transthoracic Echocardiography showed VSD inlet, ASD primum, and common AV valve. Antibiotics and conservative treatment were given besides Dobutamine and Norepinephrine for shock sepsis. After pneumonia resolved, chromosome test and cardiac surgery were made and investigate later in other paediatric or cardiology center. M. H. Trisnawan1, A. Alamsyaputra1, I. Uddin2, M.F. Ahnaf2 y p g y g Department Cardiology and Vascular Medicine, Dr. Kariadi General Hospital, Semarang, Indonesia2 Background: Anomalous Aortic Origin of Coronary Artery (AAOCA) are rare occurrence with incidence during routine coronary angiography approximately 0.6-1.3%. AAOCA presenting with STEMI are a rare clinical presentation and the management of an anomalous infarct-related coronary artery may be technically challenging. Case illustration: 60 years old man came to ED with chief complaint severe chest pain accompanied with cold sweating, nausea, and limp while he was cycling. Blood pressure was 121/73 mmHg, HR 64 bpm, RR 21 tpm and there were no presentation of heart failure nor cardiogenic shock. The ECG showed ST elevation in II, III, aVF, and ST depression in lead I and aVL. Laboratory results showed HSTroponin I >40000.0 ng/L. Patient was diagnosed by Acute STEMI inferior 5 hours onset Killip I. Primary PCI was performed via right femoral artery, we had difficulties to access the LCA. Surprisingly, there was an anomaly in the coronary arteries and the patient just told the operator at the table that he had previous single ostium coronary artery anomaly from the CCTA and elective coronary angiography result at the past. So we decided to access the RCA with JR 3.5/6F by aortography technique. The LCA have acute take-off angle, arising from ostial RCA and fortunately have retro-aortic course. Subtotal occlusion thrombus type as the culprit lesion at proximal RCA and we implanted 1 stent DES Firebird II 4.0 x 18 mm at 16 atm. Final result was TIMI flow 3 with residual stenosis 0% without other complications during the implantation. Conclusion: Cuspogram or aortography technique at the cusps of a target ostium can useful in emergency setting where short time has significant outcome where rapid identification of the culprit’s vessel is crucial to achieve successful primary PCI p y Keywords: aaoca, acute stemi, primary pci Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Complete Atrioventricular Septal Defect in Patient with Down Syndrome Morphology: Case Report I.B.U. Kramasanjaya1 RSUD Balaraja1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Complete Atrioventricular Septal Defect in Patient with Down Syndrome Morphology: Case Report I.B.U. Kramasanjaya1 RSUD Balaraja1 Complete Atrioventricular Septal Defect in Patient with Down Syndrome Morphology: Case Report I.B.U. Kramasanjaya1 RSUD Balaraja1 Conclusion Conclusion CAVSD is a rare CHD and usually symptomatic. Early diagnosis and prompt treatment prevent further fatal prognosis. Keywords: Complete Atrioventricular Septal Defect, Down Syndrome CAVSD is a rare CHD and usually symptomatic. Early diagnosis and prompt treatment prevent further fatal prognosis. K d C l A i i l S l D f D S d Keywords: Complete Atrioventricular Septal Defect, Down Syndrome Echocardiography Echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Pulmonary Embolism as an Insidious Manifestation in Post Tuberculosis Obstructive Disease with Pulmonary Hypertension : An Experience from East Nusa Tenggara, Indonesia Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Ventricular Septal Rupture after Extensive Anterior STEMI without Cardiac Surgical Intervention Facility: A Case Report M.R. Ramadhan1, D. Jovindho1 RS Bhayangkara Jambi1 Background: Ventricular Septal Rupture (VSR) is a rare but lethal complication of acute myocardial infraction.1 The VSR incidence was approximately 2% with significant mortality and morbidity.1-2 The management of VSR is stabilize the hemodynamic problems with reducing afterload to increase effective LV stroke volume by reducing left-to-right shunting.3-4 Case Illustration: A 52-years-old woman with complaint of shortness of breath since a day before admission. A history of epigastric pain radiating to the back accompanied by cold sweating felt two weeks ago. The patient had a history of uncontrolled hypertension. Cardiac auscultation revealed a systolic murmur on intercostal space 3 left-parasternal and rhonchi in medio-basal bilateral of the lung area. ECG showed ST-Elevation at the anterior extensive lead. From Echocardiography examination was found a rupture in intraventricular septal near the apex area with left-to-right shunt (Figure 1). From chest X-Ray was found cardiomegaly with increase of bronchovesicular pattern of lung. The patient was diagnosed with Late Onset Anterior-Extensive ST-Elevation Myocardial Infraction (STEMI) accompanied with Acute Lung Oedema complicated by Ventricular Septal Rupture. The patient got conservative therapy during treatment and close monitoring in ICU. One month post- treatment, the patient exhibited a relatively stable hemodynamic with light activity limitation. Discussion: The management of VSR patient with lack of hospital facility is a challenging case and conservative therapy become an option for focusing of maintaining the stability of hemodynamic. This aims to minimize hemodynamics thereby reducing the work of the heart until the rupture of the intraventricular are fixed. Conclusion: Due to the high risk of death, every clinician should be aware with potentially fatal complications. Discussion: The management of VSR patient with lack of hospital facility is a challenging case and conservative therapy become an option for focusing of maintaining the stability of hemodynamic. This aims to minimize hemodynamics thereby reducing the work of the heart until the rupture of the intraventricular are fixed. Conclusion: Due to the high risk of death, every clinician should be aware with potentially fatal complications. The keys to achieve optimal results are early diagnosis and prompt treatment. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Pulmonary Embolism as an Insidious Manifestation in Post Tuberculosis Obstructive Disease wit Pulmonary Hypertension : An Experience from East Nusa Tenggara, Indonesia M. Tombeng1, K. A. Nugraha1 Samuel J. Moeda Navy Hospital1 Background: Pulmonary embolism (PE) is an acute disease with high mortality, which is prone to be underdiagnosed, especially if it occurs concomitantly with pulmonary disease, such as post tuberculosis obstructive disease (PTOD). PE as a complication of pulmonary TB is rarely reported, but recently, there has been growing evidence supporting this association. Case Illustration: A 44-year-old man was admitted in our hospital because of worsening shortness of breath and chest discomfort. He had a history of completed and well responded to antitubercular therapy seven years ago. At admission, physical examination revealed normal blood pressure, tachycardia, decreased oxygen saturation 86% at room air, distended jugular vein, and accentuated pulmonary component of S2. No sign of DVT was found. ECG showed McGinn-White sign as S wave in lead I, Q wave, and inverted T wave in lead III. Chest X-ray revealed consolidation associated with collapse on bilateral upper lobe and hampton hump on left lung. Transthoracic echocardiography revealed good LV function, RV dysfunction, mild tricuspid regurgitation with high probability of PH without thrombus visualized. D-dimer level was elevated 0.9 mg/L, meanwhile other laboratory parameters were normal. CTPA revealed hazy appearance suspected as thrombus in left pulmonary artery and atelectasis with cavity lesion in bilateral upper lung that indicates POTD. The patient was given anticoagulant therapy with fondaparinux overlapping with optimal dose of warfarin. After five days of follow up, he showed significant improvement, his vital signs were normal, and could be discharged. g p g g Conclusion: This case report highlights the association between pulmonary embolism and post tuberculosis obstructive disease. This can lead to serious complication, which is potentially preventable by prompt diagnosis and treatment. Keywords: pulmonary embolism, post tuberculosis obstructive disease, pulmonary hypertension Fig 1. CTPA Axial and Coronal View Show LPA (red arrow) Thrombus Fig 1. CTPA Axial and Coronal View Show LPA (red arrow) Thrombus Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Ventricular Septal Rupture after Extensive Anterior STEMI without Cardiac Surgical Intervention Facility: A Case Report Background Ventricular tachycardia is defined as type of tachyarrhytmia with more than 3 consecutive beats as premature ventricular complex (PVC), rate more than 100 beat per minute, arised from ventricles, unrelated from atrial and atrioventricular nodal conduction. This arrhythmia can be caused by electrolyte imbalance such as hyperkalemia. CKD patients who have decreased renal function is susceptible to this potentially fatal condition. SUSTAINED MONOMORPHIC VENTRICULAR TACHYCARDIA (SMVT) CAUSED BY HYPERKALEMIA IN CHRONIC KIDNEY DISEASE (CKD) PATIENT : A CASE REPORT A. Siswanto1 RSD Balung Jember1 Case Illustration A-45-year-old woman was admitted in the ER because of severe dyspnea. There’s fluid build up in her stomach and all extremities. She had been diagnosed with cervical cancer stage III since 3 months ago. On physical examination, her skin looked pale and there’s coarse crackles in both lung fields. Ascites and edema in all extremities were also found. The ECG showed wide monomorphic QRS complexes, with no p wave thus confirmed Sustained Monomorphic Ventricular Tachycardia (SMVT). Then, the patient was given synchronized cardioversion twice with each dose 100 J and 125 J. The rhythm was converted to sinus rhythm and the symptoms was improved. Chest X-Ray showed cardiomegaly and congestive pulmonum. The laboratory studies showed high serum creatinin level 15.53 mg/dL and severe hyperkalemia 6.71 mmol/L. Then patient was given calcium gluconas and insulin intravenously to decrease serum potassium. If hyperkalemia persisted, the patient was scheduled for emergent dialysis. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964 doi: 10.30701/ijc.1537 SUSTAINED MONOMORPHIC VENTRICULAR TACHYCARDIA (SMVT) CAUSED BY HYPERKALEMIA IN CHRONIC KIDNEY DISEASE (CKD) PATIENT : A CASE REPORT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 SUSTAINED MONOMORPHIC VENTRICULAR TACHYCARDIA (SMVT) CAUSED BY HYPERKALEMIA IN CHRONIC KIDNEY DISEASE (CKD) PATIENT : A CASE REPORT Indonesian Journal of Cardiology y y g p Conclusion: Due to the high risk of death, every clinician should be aware with potentially fatal complications. The keys to achieve optimal results are early diagnosis and prompt treatment. eyword: Ventricular Septal Rupture, Myocardial Infraction, Conservative Therapy. The echocardiography showed rupture of the apex of septal area with left-to-right shunt flow The echocardiography showed rupture of the apex of septal area with left-to-right shunt flow p Case illustration A 64-year-old man complained of chest pain radiating to the jaw and frequently recurred over the past year. He had a history of CABG surgery seven years ago. On physical examination, all vital signs were normal. A 12-lead ECG examination showed no typical ischemic features. Chest X-ray showed cardiomegaly, and laboratory results showed critical e-GFR values. The patient was referred to the internal medicine department. The internist diagnosed him with end-stage renal disease (ESRD) and advised hemodialysis (HD). Angiography was then performed and showed total occlusion of mid-LAD, diffuse stenosis of LCX, long diffuse stenosis of RCA, total occlusion of osteal LAD graft, and total occlusion of osteal LCX graft, long diffuse stenosis of RCA graft. He was diagnosed with multiple coronary stenoses and grafted with ESRD. The patient was scheduled to undergo PCI. The operator chose to stent the SVG of the RCA using a drug-eluting stent. Then, he was prescribed multiple anti-platelets, nitrates, statins, and b-blockers. Percutaneous Coronary Intervention of Saphenous Vein Graft in Patients with End Stage Renal Disease - What to Consider? A. Nurjanah1, W. Putradana1, D. Herdianto2 Abdoel Wahab Sjahranie General Hospital, Samarinda, Indonesia1 A. Nurjanah1, W. Putradana1, D. Herdianto2 Abdoel Wahab Sjahranie General Hospital, Samarinda, Indonesia1 Department of Cardiology and Vascular Medicine, Abdoel Wahab Sjahranie General Hospital, Samarinda, Indonesia2 Conclusion One of complications of chronic kidney disease is high level of serum potassium. This electrolyte imbalance may pose clinical fatal consequences such as Ventricular Tachycardia. Therefore, individual screening in CKD patients should be routinely carried out especially for serum electrolytes. Besides that, all CKD patients must receive dialysis treatment right on time to prevent hyperkalemia or other complications. Keywords: sustained monomorphic ventricular tachycardia, hyperkalemia, chronic kidney disease Figure 1. ECG from the patient showed prolonged and uniform QRS complexes, absent of p wave indicating Monomorphic VT Figure 1. ECG from the patient showed prolonged and uniform QRS complexes, absent of p wave indicating Monomorphic VT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Percutaneous Coronary Intervention of Saphenous Vein Graft in Patients with End Stage Renal Disease - What to Consider? Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Percutaneous Coronary Intervention of Saphenous Vein Graft in Patients with End Stage Renal Disease - What to Consider? Background CABG surgery is considered the ideal treatment for multiple coronary stenoses. However, complications such as graft failure or re-stenosis often occur within days to years after surgery. Treatment for patients who develop complications with other comorbidities is quite limited. Thus, caution is needed in determining the therapy. Case illustration p Conclusion The failure rate of SVG within a decade is quite high. In favourable conditions, revascularization may be possible. PCI is generally preferred, as repeat bypass is considered unlikely due to the high risk of perioperative mortality. PCI using DES is thought to have a better prognosis. In cases of chronic total occlusion in the elderly, especially with low e-GFR values, treatment aims to reduce symptoms and improve quality of life. Keywords: Saphenous vein graft, Vein graft stenosis, Percutaneous coronary intervention in end-stage Renal Disease, Stenosis vein graft stenting. eywords: Saphenous vein graft, Vein graft stenosis, Percutaneous coronary intervention in end-stage Ren isease, Stenosis vein graft stenting. Figure 1. A Diffuse stenosis in RCA graft. B. Total occlusion in mid LAD and diffuse stenosis in LCX. Figure 1. A Diffuse stenosis in RCA graft. B. Total occlusion in mid LAD and diffuse stenosis in LCX. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Stroke During Primary Percutaneous Coronary Intervention Stroke During Primary Percutaneous Coronary Intervention G. Soemara1, C. Kurniawan1, Subiyanto1, D.U. Djafar1, J.A. Pangemanan1, G. Tangkudung2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R.D. Kandou General Hospital, Manado, Indonesia1 Department of Neurology, Faculty of Medicine, Sam Ratulangi University, Prof. Dr. R.D. Kandou General Hospital, Manado, Indonesia2 Background: Stroke is a rare but dangerous complication of Percutaneous Coronary Intervention (PCI). Potential risk factors for periprocedural stroke have been identified, namely advanced age, hypertension, diabetes mellitus, previous stroke history, congestive heart failure, use of an intra-aortic balloon pump, emergency catheterization, renal failure, and presence of coronary artery thrombus. Hereby, we present a case report on stroke during PCI procedure in Manado. Case Illustration: A 72-year-old male patient visited our emergency department with chief complaint of chest pain for 6 hours. Chest pain was like being crushed by a heavy object radiating to the back and accompanied by cold sweat. He had a history of hypertension for 10 years and did not routinely consume his hypertension drug. The ECG showed ST elevation in leads II, III, aVF, v7, v8, v9 and reciprocal in leads I and aVL. The patient was planned for primary PCI. At the time of procedure, he experienced an acute ischemic stroke with speech defect and right extremity hemiparesis. He was consulted to neurologist, Digital Subtraction Angiography (DSA) and intraarterial thrombolysis was performed with alteplase. After DSA and thrombolysis, the procedure was continued, a stent was placed on the right coronary artery then the procedure was completed with the patient's hemodynamic stability. After ninth day of hospitalization, patient was discharge with stabile condition and vital sign, also without any neurological disorders. Conclusion: Stroke is rare but can dangerously happened during PCI procedure. Maneuvers that have been shown to prevent periprocedural stroke are minimal. Some maneuvers like gentle removal of the wire from the catheter, washing the catheter after removing the wire, being cautious in using contrast injection and avoiding air bubbles can be performed to minimalization periprocedural stroke. p p p Keywords: Stroke, Primary Percutaneous Coronary Intervention, Acute Coronary Syndrome Stroke during PPCI Stroke during PPCI Stroke during PPCI I Ind pISS Role of External Counterpulsation in Wellens Syndrome F.B. Jaya1, E. Mulia2, E. Stroke During Primary Percutaneous Coronary Intervention Sari1 Sari Maranatha Sehat Clinic, Bandar Lampung, Indonesia1 Sari Maranatha Sehat Clinic, Ahmad Yani Hospital2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Post-Thrombotic Syndrome: Long Term Sequelae of Deep Vein Thrombosis Complicating by Right Heart Failure M. Reeiner1, B.A.P.D. Sutanegara2, A.P. Suwirya2 1Faculty of Medicine, Udayana University, Denpasar, Indonesia; 2Vascular Division, Department of Cardiology and Vascular Medicine, Prof. Ngoerah Hospital, Bali, Indonesia M. Reeiner1, B.A.P.D. Sutanegara2, A.P. Suwirya2 of Medicine, Udayana University, Denpasar, Indonesia; 2Vascular Division, Department of Cardiology and Vascular Medicine, Prof. Ngoerah Hospital, Bali, Indonesia M. Reeiner1, B.A.P.D. Sutanegara2, A.P. Suwirya2 1Faculty of Medicine, Udayana University, Denpasar, Indonesia; 2Vascular Division, Department of Cardiology and Vascular Medicine, Prof. Ngoerah Hospital, Bali, Indonesia Role of External Counterpulsation in Wellens Syndrome y y Keywords: External counterpulsation, Wellens syndrome, coronary system Electrocardiogram (ECG) showed inverted T waves which suited wellens syndrome Electrocardiogram (ECG) showed inverted T waves which suited wellens syndrome Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Post-Thrombotic Syndrome: Long Term Sequelae of Deep Vein Thrombosis Complicating by Right Heart Failure Background g Post-thrombotic syndrome (PTS) refers to the chronic manifestations of chronic venous insufficiency (CVI) following episodes of deep vein thrombosis (DVT), being an important and frequent long-term side effect of proximal DVT within two years in 20-50% of patients. Case Illustration Role of External Counterpulsation in Wellens Syndrome F.B. Jaya1, E. Mulia2, E. Sari1 Sari Maranatha Sehat Clinic, Bandar Lampung, Indonesia1 Sari Maranatha Sehat Clinic, Ahmad Yani Hospital2 F.B. Jaya1, E. Mulia2, E. Sari1 Sari Maranatha Sehat Clinic, Bandar Lampung, Indonesia1 Sari Maranatha Sehat Clinic, Ahmad Yani Hospital2 Background: Wellens syndrome describes an abnormal typical electrocardiogram (ECG) pattern, deeply inverted T waves in leads V2 and V3, that are secondary to proximal left anterior descending artery (LAD) stenosis that is more than 50%. It usually comes with a history of chest pain, normal or slightly elevated cardiac enzymes, isoelectric or minimally elevated ST segment, preservation of precordial R-wave progression, and no precordial Q waves. Enhanced external counterpulsation (EECP) is a non-invasive treatment for angina pectoris that is refractory to pharmacotherapy and revascularization. The popular concept is that EECP may promote collateral development and improve myocardial perfusion, which may give additional benefit on top of conventional treatment. Case illustration: A 47-year-old female patient came with typical chest pain and shortness of breath during activity since 1 month ago and worsened in the last 3 days. She had a history of hypertension and familial history of coronary artery disease. Physical examination showed normal blood pressure (126/72 mmHg) and normal heart rate (69 x/m). Electrocardiogram (ECG) showed inverted T waves which suited wellens syndrome. She was given clopidogrel 75 mg once daily, diltiazem HCl 100 mg once daily, rosuvastatin 20 mg once daily, ranolazine 375 mg twice daily. Echocardiogram showed preserved left ventricular ejection fraction with sign of left anterior descending wall motion abnormality. After EECP therapy sessions in combination with medical drug therapy, she was clinically improved. Her functional class and quality of life were improved. She could easily return to her daily work in the middle of the EECP therapy sessions. Abnormality in the ECG was also altered to sinus rhytm. The Seattle Angina Questionnaire scores was improved from 36 to 62. Conclusion: In this case, EECP has shown promising treatment for Wellens syndrome which gives some beneficial effects on coronary system. Indonesian Journal of Cardiology Complex Congenital Heart Defect Case of Infant with Interrupted Aortic Arch Type B and Double Outlet Right Ventricle p Case Illustration A 49-year-old male patient with a history of recurrent DVT on right lower extrimity, right heart failure (RHF) and coronary artery disease post stenting with normal ejection fraction, complained pain, heavy sensation, persistent swollen and hyperpigmentation of both legs with multiple ulcers since a year before. Duplex ultrasound shows compression ultrasound negative on the right and left lower extremity vein, positive augmentation on the squeeze- release test on the right and left popliteal vein, positive reflux with squeeze test right popliteal vein 1.7 s, right and left femoral vein 1.0 s and 0.9 s, no thrombus was seen in the right and left popliteal vein, left common femoral vein. In DVT, the inflammatory response and incomplete thrombus acts as a functional obstruction, causes reflux. Pulmonary hypertension and right heart failure are often overlooked, these consequences add to the patient's burden and give the effect of systemic venous hypertension which will lead to backward failure. Villalta score can help to diagnose PTS, while DUS can be an additional examination to diagnose chronic vein insufficiency as a main pathophysiology of PTS. RHF and CVI can be an additive factor in development of PTS, since RHF worsen the systemic venous hypertension. Lifestyle modification and elastic compression stocking are the mainstay therapy. While endovascular and surgical correction are still being considered by the patient. Conclusion mainstay therapy. While endovascular and surgical correction are still being considered by the patient. Conclusion Long-term sustained increase in venous pressure caused by deep vein obstruction and exacerbated by peripheral venous hypertension due to right heart failure results in the development of severe PTS. Keywords: post thrombotic syndrome, deep vein thrombosis, right heart failure Lower extrimities bilateral post thrombotic syndrome Long-term sustained increase in venous pressure caused by deep vein obstruction and exacerbated by peripheral venous hypertension due to right heart failure results in the development of severe PTS. Keywords: post thrombotic syndrome, deep vein thrombosis, right heart failure Lower extrimities bilateral post thrombotic syndrome Lower extrimities bilateral post thrombotic syndrome Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Complex Congenital Heart Defect Case of Infant with Interrupted Aortic Arch Type B and Double Outlet Right Ventricle Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 62-year-old woman presented with sudden loss of consciousness for 2 hours. Two days ago, she was taken to a “mantri” (orderly) for abdominal distension and chest tightness, then furosemide (1x20mg), metformin (2x500mg), glimepiride (2x2mg), ISDN (2x5mg), digoxin (2x0,25mg), and aspirin (1x100mg) were given. After taking these drugs, the patient complained of cold sweat. Upon examination, she was stuporous (GCS 1-1-3) and bradycardic 53x/min. Other vital signs were within normal limits. She was also severely hypoglycemic (20g/dl). She became fully awake after 3 flacons of D40% were administered. An AP chest x-ray showed cardiomegaly with suspicion of early lung edema. The ECG revealed sinus rhythm, with occasional PVCs, inverted T waves in V1-V2, and down-sloping ST depression as typical illustrations of digoxin effect namely "Salvador Dali sagging" in V5-V6. In the inpatient room, the patient complained of shortness of breath and malaise, but the blood sugar was normal (131g/dl). After 5 hours of treatment, the patient's condition suddenly deteriorated rapidly. The blood pressure became 60/palpation, with bradycardia 48x/min. Unfortunately, no repeated ECG was done. The patient's condition continued to worsen and she eventually died 30 minutes later after attempted resuscitation.Cardiac arrhythmias account for most deaths in digitalis intoxication cases and they can occur even when patients are asymptomatic. Suspicion for digoxin intoxication arises from the history of digoxin consumption twice daily as an initial dose without monitoring, which is inappropriate. However, its diagnosis couldn’t be established considering no serum examination was done. ase of Unexpected Death with Incidental Finding of Digoxin Effects on the ECG: who is the culprit? S.D. Rasti1, A.A.R. Sugiarto2, A.P.A. Nuryandi3 RS Tk. II Dr. Soepraoen, Malang, Jawa Timur1 RS Mawaddah Medika, Kab. Mojokerto, Jawa Timur2 RSUD Bhakti Dharma Husada, Surabaya, Jawa Timur3 Background g Although its current use in clinical practice is declining, digoxin is still known for its beneficial effects on the management of heart failure and atrial fibrillation cases. Digoxin has a narrow therapeutic window, hence requiring carefulness in its prescription. Complex Congenital Heart Defect Case of Infant with Interrupted Aortic Arch Type B and Double Outlet Right Ventricle M. Reeiner1, E. Gunawijaya2, V.K. Yantie2, 1Faculty of Medicine, Udayana University, Denpasar, Indonesia; 2Sanglah General Hospital, Denpasar, Indonesia Background: An interrupted aortic arch is defined as complete discontinuity of the aortic lumen between segments of the aortic arch, a rare and fatal anomaly, accounting for about 1% of congenital cardiovascular defects, these congenital abnormalities can coexist with other congenital heart defects and entails a very poor prognosis without surgical treatment. Case Illustration: A 11-month female with cyanotic congenital heart disease was referred to our center due to due to perioral and distal cyanosis when crying. Upon examination we found Interrupted aortic arch type B (Varians) with double outlet right ventricle doubly committed, dilated pulmonary artery, large patent ductus arteriosus, small atrial septal defect from echocardiography. On cardiac catheterization, the result was consistent with echocardiography result, with additional information of bicuspid pulmonary artery and severe pulmonary arterial hypertension. A biventricular approach will be carried out with 2 stages of the procedure. The first stage is repair of the interrupted aortic arch (end to side anastomosis + A. pulmonary autograft patch with a reverse flap of left subclavian artery or polytetrafluoroethylene graft), patent ductus arteriosus ligation, pulmonary artery banding, and will be followed by repeated cardiac catheterization examinations to assess pulmonary artery resistance. The second stage is pulmonary artery debanding and ventricular septal defect closure using an intraventricular tunneling patch or perforated patch. Conclusion: Type B IAA accompanied by several other congenital abnormalities is a rare case thus comprehensive management and involvement of cardiac surgery team is needed to determine the best treatment plan for the patient, in this case a biventricular approach with 2 stages of surgery will be carried out. Keywords: congenital heart defect, interrupted aortic arch, double outlet right ventricle, cyanotic heart disease ds: congenital heart defect, interrupted aortic arch, double outlet right ventricle, cyanotic heart disease Lower extrimities bilateral post thrombotic syndrome Lower extrimities bilateral post thrombotic syndrome Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Co c us o Physicians should be thoughtful of the subtle manifestations after digitalis administration attributable to its narrow therapeutic window. Keywords: digoxin effect, digitalis intoxication, unexpected death Physicians should be thoughtful of the subtle manifestations after digitalis administration attributable to its narrow therapeutic window. p Keywords: digoxin effect, digitalis intoxication, unexpected death p Keywords: digoxin effect, digitalis intoxication, unexpected death y g g p Figure 1. The ECG performed in the emergency room; the green arrow shows “Salvador Dali sagging”, a mark of digital effects. Figure 1. The ECG performed in the emergency room; the green arrow shows “Salvador Dali sagging”, a mark of digital effects. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: AORPA, TOF-PA, PA-VSD, MAPCAS ANOMALOUS ORIGIN OF THE RIGHT PULMONARY ARTERY FROM ASCENDING AORTA: A RARE CONGENITAL HEART DISEASE CASE F. Yuliani1, Kino1, D. Hariyanto1 RSUP Dr M DJAMIL PADANG1 Background: Anomalous origin of the right pulmonary artery from ascending aorta (AORPA) is a very rare congenital cardiac malformation that happened 0.1% of all congenital heart disease. Only 5% of this case reported in adult. The most common condition that related to AORPA is patent ductus arteriosus, Tetralogy of Fallot (TOF), atrial septal defect, ventricular septal defect (VSD), and coarctatio aorta. Case Illustration: a 11 years old girl, was referred to our hospital due to exertional dyspnoea worsened with time and mildly improve by resting in the last 2 months before admission. She also had cyanosis in extremities. Her body weight was 29 kg. The patient already known heart disease since young but never controlled to cardiologist. Physical examination revealed moderate condition. Heart rate was regular, and normal blood pressure and 80% oxygen saturation. Chest heart sound was pansystolic murmur grade 3/6 at the apex. Laboratory test concluded polycythemia with Hb 22.1 gr/dl and haematocrit 67%. Her electrocardiography showed sinus rhythm with right ventricular enlargement and chest radiograph showed cardiomegaly with boot shaped appearance. Echocardiography conclusion was Tetralogy of Fallot-Pulmonary Atresia (TOF-PA), Mayor Aortopulmonary Collateral Arteries (MAPCAS), good LV and RV function, and left arch. Initially patient was diagnosed with TOF-PA. After Aortography it was found that there were PA-VSD, AORPA, and arborisation left pulmonary from MAPCAS. Conclusion: Due to high mortality case, it is important to early diagnose patient with AORPA condition in order to treat patient as soon as possible with surgical correction and for better prognosis in the future. A high of clinical suspicion is required especially unexplained heart failure with TOF PA echocardiography as seen in this case Keywords: AORPA, TOF-PA, PA-VSD, MAPCAS Keywords: AORPA, TOF-PA, PA-VSD, MAPCAS Heart Catheterization of AORPA Heart Catheterization of AORPA Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 A Case Report: ST-Segment Elevation ECG on Hypertension and Diabetes Mellitus Patient with Chest Pain mimicking STEMI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Case Report: ST-Segment Elevation ECG on Hypertension and Diabetes Mellitus Patient with Chest Pain mimicking STEMI P.N. Permatasari1, I. Handirosiyanto2 General Practitioner, Baptis Batu Hospital, Malang, Indonesia1 Cardiologist, Baptis Batu Hospital, Malang, Indonesia2 P.N. Permatasari1, I. Handirosiyanto2 P.N. Permatasari1, I. Handirosiyanto2 General Practitioner, Baptis Batu Hospital, Malang, Indonesia1 Cardiologist, Baptis Batu Hospital, Malang, Indonesia2 Background: Electrocardiogram is crucial for diagnosing STEMI because “time is muscle”, the longer the infarct time, the greater the ischemia and necrosis myocardium. Early and correct diagnoses are needed to make better decisions, regarding pharmacological or mechanical revascularization. However, recording and interpretation of 12-lead ECG in patients remain challenging. ST-segment elevation also could cause by many non-cardiac conditions that mimic STEMI. Case Illustration: A 65-year-old Man was transferred to the Cardiology department because of chest pain within one week and ST-Elevation ECG. He had controlled DM and HT. There was no symptom of typical chest pain, DOE, or congestion. BP: 150/90 mmHg, HR: 56 times/minute. In general examination no rales, no abnormality of heart sound, or signs of congestion. The first record showed ST-Elevation in Lead II, III, aVR, aVL, and aVF. Cardiologist re-recorded a 12-lead ECG of the patient. The patient took off all metallic belonging including the metal belt. The ECG showed no ST-Elevation on all leads. Turn out that in the first record, the patient just took off his coin and car key from his pocket. He still used a metal belt that made ECG show ST-Elevation in Lead II, III, aVR, aVL, and aVF. Conclusion: Recording and interpretation of 12-lead ECG are very crucial. Every paramedic and doctor should be aware that there was no metal in the patient such as a belt, key, coin, or jewel. Metal material made an electrical disturbance to the recording ECG making it mimics STEMI. g g Keywords: Electrocardiogram, Mimicking STEMI, ST-Segment Elevation Figure 1: The First 12-Lead ECG Figure 1: The First 12-Lead ECG Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology ACUTE LIMB ISCHEMIC AS COMPLICATION OF FEMORALIS PUNCTURE : A CASE F.Q. Decroli1, W. Erlanda2, H. Kam3 Department of Emergency Medicine, M Djamil Hospital Padang1 Division of Cardiology Faculty of Medicine, Andalas University / M. Djamil Hospital Padang2 Division of Surgery Faculty of Medicine, Andalas University / M. Djamil Hospital Padang3 Background: Acute limb ischemia is a sudden decrease in limb blood perfusion with an onset of less than 14 days, commonly occurs in lower extremities. Iatrogenic complications related to cardiac catheterization is one of the potential embolic causes which are related to a sudden decrease in arterial perfusion in the limb. Local thrombosis of the femoral artery or lower extremity vessel related to the access site is unusual, reported to occur in less than 1%. In this case report we present who developed ALI due to prior femoralis puncture that had successful thrombectomy treatment. Case Illustration: 60 years old female patient brought to us with continuous pain on the right foot for three days and worsened in 24 hours prior to admission. Patient underwent Percutaneous Transluminal Coronary Angioplasty (PTCA) 4 days before the admission due to CAD2VD. No prior history of intermittent claudicaton before the procedure, and claudication worsened after the procedure. The right foot felt numb and weak. The fingers were pale and cold as well. The right foot had no pulsated from popliteal to dorsalis pedis artery, weak brachial pulse and undetected pulse oximetry result. Doppler Sonography revealed that there was no flow in the right common femoral artery. On the first day of hospitalization, the patient was scheduled for emergency thrombectomy at M Djamil Hospital Padang. Follow-up on the seventh-day post-surgery, the patient had no complaints of pain and pale in the right foot and the popliteal, tibialis, and dorsalis pedis arteries were palpable. Conclusion: Catheter-artery mismatch, the presence of peripheral vascular disease, advance age, cardiomyopathy and the presence of hypercoagulable states are risk factors of local thrombosis of the femoral artery. In the absence of predisposing factors, vessel dissection or spasm may also contribute. Early thrombectomy shows excellent prognosis in this case. p g Keywords: acute limb ischemia, femoralis puncture, thrombectomy Case Illustration A 70-year-old male patient was referred to the emergency room from a peripheral hospital for recurrent ventricular fibrillation. He has a history of gastritis and hypertension. Initially, he came with a seizure after vomiting and heartburn. He then experienced sudden chest pain and the first cardiac arrest where VF was found on the ECG. Advanced cardiac support was immediately started. During 4 days of treatment, VF episodes occurred daily and 22 defibrillation shocks were administered in total. Historically, some drugs such as furosemide, fondaparinux, corrective KCL drip, lidocaine, dobutamine, and norepinephrine pump were given. After referral, dobutamine and norepinephrine were immediately tapered off, while lidocaine was maintained. The patient's condition improved and the VF did not recur. His echocardiogram revealed decreased LV systolic function (EF 31%) and eccentric LVH with grade II diastolic dysfunction. He was assessed with recurrent cardiac arrest due to coronary artery disease with old myocardial infarct, ischemic cardiomyopathy, and hypertensive heart disease. A week later, he was well recovered and scheduled for ICD insertion and PCI at the central referral hospital. Malignant ventricular arrhythmias may be observed in dobutamine use at any dose. Patients with underlying arrhythmia, heart failure, or ischemic heart disease are at greater risk of proarrhythmia. The suspected involvement of inotropic in the development of recurrent VF in this patient is supported by clinical improvement after its tapering. Background Background Inotropic and vasopressor agents are becoming cornerstones of therapy for the treatment of important cardiovascular syndromes. These agents increase myocardial oxygen consumption and can exert proarrhythmic effects. A 70-years-old male who survived 22 times of defibrillation shocks after inotropes-induced recurrent ventricular fibrillation S.D. Rasti1, A.P.A. Nuryandi2, A.A.R. Sugiarto3, F. Hartono4 RS Tk. II Dr. Soepraoen, Malang, Jawa Timur1 RSUD Bhakti Dharma Husada, Surabaya, Jawa Timur2 RS Mawaddah Medika, Kab. Mojokerto, Jawa Timur3 Department of Cardiology and Vascular Medicine, RSPAL Dr. Ramelan, Surabay S.D. Rasti1, A.P.A. Nuryandi2, A.A.R. Sugiarto3, F. Hartono4 RS Tk. II Dr. Soepraoen, Malang, Jawa Timur1 RSUD Bhakti Dharma Husada, Surabaya, Jawa Timur2 RS Mawaddah Medika, Kab. Mojokerto, Jawa Timur3 t of Cardiology and Vascular Medicine, RSPAL Dr. Ramelan, Surabaya, Jawa Timur4 S.D. Rasti1, A.P.A. Nuryandi2, A.A.R. Sugiarto3, F. Hartono4 RS Tk. II Dr. Soepraoen, Malang, Jawa Timur1 RSUD Bhakti Dharma Husada, Surabaya, Jawa Timur2 RS Mawaddah Medika, Kab. Mojokerto, Jawa Timur3 Department of Cardiology and Vascular Medicine, RSPAL Dr. Ramelan, Surabaya, Jawa Timur4 Aneurysmatic Patient Presenting with ST-Elevation Myocardial Infarction: Role of Multimodality Imaging in Emergency Setting Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A 70-years-old male who survived 22 times of defibrillation shocks after inotropes-induced recurrent ventricular fibrillation S.D. Rasti1, A.P.A. Nuryandi2, A.A.R. Sugiarto3, F. Hartono4 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology A 70-years-old male who survived 22 times of defibrillation shocks after inotropes-induced recurrent ventricular fibrillation Aneurysmatic Patient Presenting with ST-Elevation Myocardial Infarction: Role of Multimodality Imaging in Emergency Setting Aneurysmatic Patient Presenting with ST-Elevation Myocardial Infarction: Role of Multimodalit Imaging in Emergency Setting Y.A. Fikri1, E.P.B. Mulia2, F.W. Nugroho2 g General Practitioner, Dr. Soetrasno Regional General Hospital Rembang1 Department of Cardiology and Vascular Medicine, Dr. Soetrasno Regional General Hospital Rem Background: Aortic dissection with concurrent ST-elevation myocardial infarction (STEMI) is rarely reported. As the proportion of myocardial infarction is higher in the emergency setting compared to aortic dissection, the diagnosis of aortic dissection may be overlooked, and it can be potentially fatal. By using bedside available information, detailed history taking and multimodality imaging in the emergency setting, it is possible to avoid a mistaken diagnosis. Here we present a case of aortic aneurysm presenting with anterior STEMI. Case Illustration: A 79-year-old woman was admitted to our emergency department with decreased consciousness. Shortly before the patient went unconscious, she had a short episode of dyspnea. Her ECG showed marked ST elevation in the anterior leads (Figure 1A). However, her chest radiograph revealed mediastinal widening and a prominent aortic knob (Figure 1B). Due to suspicion of aortic dissection from the chest radiograph and loss of consciousness, which may be a sign of malperfusion syndrome of aortic dissection, bedside handheld echocardiography was then performed. It revealed hypokinesis of anterior and anteroseptal walls, pericardial effusion, and dilated aortic root to ascending aorta with severe aortic regurgitation. The presence intimal flap can not be clearly excluded. Based on her imaging and clinical findings, aortic dissection was suspected and thrombolysis was postponed. The patient proceeded to undergo triple-rule-out computed tomography, from which the finding of ascending aortic aneurysm was noted, along with multiple stenosis of LAD (moderate-to-severe) and LCx (moderate), and there was no presence of false lumen. Conclusion: Acute aortic dissection should be considered as a differential diagnosis in patients presenting with symptoms suggesting acute coronary syndrome. A suspected case of acute aortic dissection should necessitate further imaging studies. Therefore, multimodality imaging plays a vital role in the emergency setting, as it may avoid fatal consequences of misdiagnosis and mistreatment. q g Keywords: aortic aneurysm, aortic dissection, myocardial infarction, chest pain, imaging, emergency A) ECG showed marked ST elevation in the anterior leads. B) Chest radiograph revealed mediastinal widening and a prominent aortic knob. A) ECG showed marked ST elevation in the anterior leads. B) Chest radiograph revealed mediastinal widening and a prominent aortic knob. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Electrolyte imbalances, ischemia, catecholamines, and inotropes may worsen arrhythmias and increase ventricular susceptibility to sustained arrhythmias. The likelihood of both successful resuscitation and survival of a cardiac arrest patient is inversely proportional to the total number of defibrillation shocks required for cardioversion. Therefore, be mindful of handling it. Keywords: ventricular fibrillation, inotropes, arrhythmias, recurrent VF, defibrillation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. The ECG 3 days upon referral; after dobutamine and norepinephrine were tapered off, bigeminy and occasional PVC were still found Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. The ECG 3 days upon referral; after dobutamine and norepinephrine were tapered off, bigeminy and occasional PVC were still found g p CASE ILLUSTRATION A 56-year-old woman presented with shortness of breath and leg swelling, as well as high fever and no other symptoms. Her vital signs showed a high heart rate and blood pressure, and her physical examination revealed lung rales, a systolic murmur, and edema in her legs. Chest X-ray revealed an enlarged heart. Echocardiography showed vegetation and mitral regurgitation (fig.1). Modified Duke criteria were used to diagnose vegetation and history of fever as 1 major and 1 minor criteria yield in diagnosing possible endocarditis. Blood cultures were performed with negative results. The previous use of antibiotics is thought to be the cause of the negative blood culture result. BLOOD CULTURE NEGATIVE IN NATIVE VALVE INFECTIVE ENDOCARDITIS N. Syauqi1 1Tarakan General Hospital, Jakarta, Indonesia Corresponding author email: dr.nafisadnan@gmail.com ABSTRACT BACKGROUND Infective endocarditis is a serious cardiac condition characterized by inflammation of the heart's endothelium, with a mortality rate of up to 30%. Diagnosis relies on symptoms, blood cultures, and echocardiography. However, diagnosing the disease becomes more complex when blood cultures yield negative results, which occurs in about 20% of cases, making prompt diagnosis and treatment difficult. CONCLUSION Diagnosing infective endocarditis (IE) is difficult due to patients lack of distinctive symptoms, despite the high mortality rate associated with IE. The results of supporting examinations and the establishment of diagnosis based on clinical criteria, supporting tests, and microbiological examinations remain crucial in diagnosing IE. Negative blood culture results still challenge establishing a diagnosis and initiating therapy. The presence of structural abnormalities and complications of heart failure are the main considerations for performing valve replacement. Keywords: invective endocarditis, valvular disease HYPERKALEMIA IN CHRONIC HEART FAILURE IS INVISIBLE, INSIDIOUS, AND DEADLY: A CASE REPORT A.I. Pramesti1, Z. Ramadhanty1 Faculty of Medicine, Sebelas Maret University1 Background:Hyperkalemia is an increased level of serum potassium >5,5 mmol/L that shows nonspecific signs such as bowel and muscle discomfort, paralysis, and arrhythmias that lead to cardiac arrest. ECG examination shows specific abnormalities and helps with the diagnosis. We report a case of a chronic heart failure patient with severe hyperkalemia who showed no specific signs, symptoms, and ECG abnormalities. Case Illustration:A 71-year-old woman with a history of chronic heart failure came to the polyclinic for monthly control, with the results of blood tests was severe hyperkalemia with a K+ value of 8.09mmol/L, increased ureum 126 mg/dl and creatinine 2 .87 mg/dL. The patient comes asymptomatic. Patient's heart rate was 63 bpm regular, blood pressure was 108/56mmHg, respiratory rate was 16breaths/min, and oxygen saturation was 98%. Physical examination of the heart revealed heart murmurs in the pansystolic mitral and tricuspid areas with grade 2/6, low pitch, without radiating. Other physical examination found no abnormalities. Electrocardiogram showed physiological left axis deviation, complete left bundle branch block, 2:1 advanced AV block degree 2, left ventricular hypertrophy, anterior T wave inversion, and poor r wave progression, and did not showed hyperkalemia. Echocardiography results showed significant improvement in left ventricular systolic function compared to the result three months earlier. Patients routinely take aspirin, simvastatin, amlodipine, bisoprolol, candesartan, spironolactone, thyrozol, and furosemide. The patient denied any history of kidney disease or diabetes mellitus. Severe hyperkalemia was successfully treated after potassium correction. Conclusion:Patients with heart failure with comorbidities in the form of kidney disease and receiving drugs with the effect of reducing excretion of potassium may cause hyperkalemia which does not show signs, symptoms, or typical ECG features. Assessment of risk factors, understanding the mechanism of the drugs, and periodic examination of serum electrolytes can prevent hidden hyperkalemia thereby reducing mortality and morbidity. erkalemia, Chronic Heart Failure, Acute Kidney Injury Figure 1. Patient’s Electrocardiogram Keywords: Hyperkalemia, Chronic Heart Failure, Acute Kidney Injury Figure 1. Patient’s Electrocardiogram Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Keywords: invective endocarditis, valvular disease Keywords: invective endocarditis, valvular disease Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Rare Case of Torsade de Pointes Following Dental Procedure: Idiosyncratic or Common Adverse Reaction? A Rare Case of Torsade de Pointes Following Dental Procedure: Idiosyncratic or Common Adverse Reaction? A Rare Case of Torsade de Pointes Following Dental Procedure: Idiosyncratic or Common Adverse Reaction? C.J. Purba1, S.D.T. Sitanggang2, A.E. Prasetia1 Putri Hijau Hospital, Medan1 RSUD Pakuhaji, Tangerang2 Background : Ventricular arrhythmia is one of most lethal complication following a heart attack. However, not all ventricular arrhythmia solely caused by primary cardiac problems yet it can also be precipitated by extrinsic factors. One of the most common extrinsic factors causing ventricular arrhythmia is drug-induced ventricular arrhythmia. In this report, we aim to present a rare case of polymorphic ventricular tachycardia, particularly Torsade de Pointes (TdP), that was induced during dental procedure after local anesthesia drug injection. Case Illustration : A 49-year-old male with pulp necrosis came into dental polyclinic to undergo tooth extraction. He had no previous medical history. Before the procedure, local anesthesia was performed using lidocaine- epinephrine solution (lidocaine HCl 40 mg, epinephrine 0.025 mg). The patient was frightened and anxious, thus the procedure took longer than expected. Second lidocaine-epinephrine injection of the same dose was administered after the patient started to feel pain. Several minutes after the second dose, he started to experience palpitations, tremor, and diaphoresis. The patient was then admitted to the emergency room and electrocardiography (ECG) examination was performed. ECG showed TdP waveform as depicted in Figure 1. Hemodynamic was stable and bedside monitor was placed. After <1 minute, his heart rate spontaneously recovered to a normal sinus rhythm. He was admitted to the intensive care unit and no recurrence of TdP was observed. Lab results were unremarkable. The patient was discharged after 3 days of hospitalization. Conclusion : This case demonstrates that a healthy patient without any heart disease may experience a brief episode of Torsade De Pointes even after receiving a clinical dose of epinephrine-lidocaine local injection. Catecholamines surge was suggested as the underlying mechanism. Therefore, patients who received epinephrine- containing anesthesia need to be monitored for any possible major cardiovascular side effects. eywords: Torsade de Pointes, catecholamine, arrhythmia, dental procedure, adverse reaction Documented TdP and patient’s baseline ECG, taken almost simultaneously. Keywords: Torsade de Pointes, catecholamine, arrhythmia, dental procedure, adverse reaction Documented TdP and patient’s baseline ECG, taken almost simultaneously. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Facing Up Complex Coronary Bifurcation Lesion with Left Main Involvement: The Upfront 2 Stents Method Subiyanto1, C. Kurniawan1, G. Soemara1, J.A. Pangemanan1, A.L. Panda1 Department of Cardiology and Vascular Medicine Sam Ratulangi University Prof. Dr. R.D. Kandou General Hospital Manado, Indonesia1 Subiyanto1, C. Kurniawan1, G. Soemara1, J.A. Pangemanan1, A.L. Panda1 Department of Cardiology and Vascular Medicine Sam Ratulangi University Prof. Dr. R.D. Kandou General Hospital Manado, Indonesia1 Background : Bifurcation lesion accounts for 15-20% of all percutaneous coronary intervention (PCI) leads to more complex procedures especially involving left main branch. Approaching this complex lesion, provisional or planned two stents are the choices. PCI procedure on bifurcation lesion with LM involvement by upfront two stents method, T- stenting will be shared through this case report. Case Illustration: A 57 years old male admitted into Emergency Department due to a typical angina chest pain during treadmill and his resting ECG 12-lead showed sinus rhythm with slight elevation at lead AVR and V1, ST depression at lead I, II, AVL, AVF, V4-6 as well as LVH with strain without any changes after serial ECG. Patient has been previously diagnosed of chronic coronary syndrome with recent echocardiography showed normal ejection fraction at 65%, global normokinetic. Cardiac markers of troponin T in normal range and not rising as well as the creatinine kinase level. During the procedure, coronary angiography revealed left main bifurcation lesion Medina 1-1-1 with significant lesion in proximal LAD (90%) and osteal LCx (80%). As the side branch (LCx) diameter is > 2.5mm, lesion length > 10mm, and bifurcation lesion angle > 70, It fulfilled the requirement of complex bifurcation lesion, which demands two-stents strategy. Our operator was, then, chose a T-stenting method by first preparing the lesion by dilating the main vessels, then deploying side branch stent followed by main vessel (LM-LAD) stent; lastly, kissing balloon inflation in the bifurcation site and Proximal Optimalization Technique of main vessel afterward. The procedure resulted in TIMI flow 3 without complications. C l i Th h d i b i d i l bif i l i T i h i i h Conclusion: The two-stents method is best suited in complex bifurcation lesion. T-stenting technique is the primary option when the angle of the bifurcation is close to 900. rds: Bifurcation Lesion, Intervention Cardiology, PCI, T-stenting, Two-stents method Figure 1. Coronary angiography results pre- and post stenting Keywords: Bifurcation Lesion, Intervention Cardiology, PCI, T-stenting, Two-stents method Figure 1. Facing Up Complex Coronary Bifurcation Lesion with Left Main Involvement: The Upfront 2 Stents Method Coronary angiography results pre- and post stenting Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Non-ST-segment Elevation Acute Coronary Syndrome Presenting with Acute Pulmonary Oedema and Gastro-Intestinal Bleeding: Thrombosis vs Bleeding Risk Management Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Non-ST-segment Elevation Acute Coronary Syndrome Presenting with Acute Pulmonary Oedema and Gastro-Intestinal Bleeding: Thrombosis vs Bleeding Risk Management Non-ST-segment Elevation Acute Coronary Syndrome Presenting with Acute Pulmonary Oedema and Gastro-Intestinal Bleeding: Thrombosis vs Bleeding Risk Management H.T. Abdurrahman1, Haikal2 Cardiology Resident, Faculty of Medicine, Airlangga University1 sultant, Department of Cardiology and Vascular Medicine, Gatot Soebroto Army Central Hospital2 H.T. Abdurrahman1, Haikal2 Cardiology Resident, Faculty of Medicine, Airlangga University1 Cardiology Consultant, Department of Cardiology and Vascular Medicine, Gatot Soebro Hospital2 H.T. Abdurrahman1, Haikal2 Cardiology Resident, Faculty of Medicine, Airlangga University1 Cardiology Consultant, Department of Cardiology and Vascular Medicine, Gatot Soebroto Army Central Hospital2 Background: Non-ST-segment Elevation Acute Coronary Syndrome (NSTE-ACS) presenting with acute pulmonary oedema has a high risk of mortality and morbidity and should be treated with an intervention strategy. However, some of these patients may develop gastro-intestinal bleeding (GIB) before an intervention strategy is performed. The risk of bleeding was higher in this case. Case illustration: A 52-year-old man came to the ED of Gatot Soebroto Army Central Hospital after complaining of severe shortness of breath. Several hours earlier, the patient had complained of epigastric pain accompanied by diaphoresis. His BP was 192/118 mmHg, oxygen saturation was 68%, and rales in half of his both lungs on physical examination. The ECG showed T inverted in lead II, III, and aVF. Laboratory findings showed an increase in Troponin I level. The patient was diagnosed with NSTEMI very high risk, acute pulmonary oedema, and hypertensive emergency. He was then intubated after initial treatment of acute pulmonary oedema failed to relieve his condition. DAPT was given using an NGT, and an intervention strategy was planned. However, NGT showed GIB, and the intervention strategy was postponed. He was managed by a multidisciplinary team with a gastroenterohepatologist. Conclusion: NSTE-ACS with acute pulmonary oedema carries a high risk of mortality, and GIB makes the condition more complex. The risk of thrombosis and bleeding should be managed by a multidisciplinary team. Even though this condition should be treated with an intervention strategy, the timing of the intervention itself should be managed to the patient’s condition. Keywords: NSTE-ACS, acute pulmonary oedema, GIB Indonesian J Cardiol Indonesian J Cardiol 2 pISSN: 2830-3105 / eI doi: 10.30701/ Sleep-Bruxism: Is it a New Sign of Hypertension and Other Cardiovascular Disease? S. Chandra1, A.A. Lukito1, R.A.P. Fernandes1, H. Hutabarat1, I. Sabrina1 Siloam Hospital Lippo Village1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Sleep-Bruxism: Is it a New Sign of Hypertension and Other Cardiovascular Disease? S. Chandra1, A.A. Lukito1, R.A.P. Fernandes1, H. Hutabarat1, I. Sabrina1 Siloam Hospital Lippo Village1 Background Sleep disordered breathing is a common and serious health problem. Accordingly to epidemiological data, it may affect about 20% of the adult population. The majorities are not aware of the disease. Bruxism is associated with increased masticatory muscle activity during sleep, which may be phased or tonic. It is estimated that the incidence of bruxism in the adult population is 13%. Sleep bruxism is associated with blood pressure fluctuations during sleep. Arousals and body movement often occur with sleep bruxism and can impact the magnitude of this blood pressure surge. g p g Case Illustration A 57-year-old woman presented with unstable blood pressure, with a previous medical history of hypertension for 2 years ago, and prediabetic state. On physical examination, blood pressure 130/77 mmHg, pulse 70 bpm regular, BMI scale was obese. Before diagnosed hypertension, patient did not snore when sleep, but had grinding her teeth when sleep since a long time ago. Treadmill Stress Test revealed negative ischemic response, hypertension at peak exercise around 220/62 mmHg. Therefore suggestion for this case was losing weight (exercise and intermittent fasting), and blood pressure medication. g ( g) p Conclusions Sleep bruxism is associated with external factors (such as stress, anxiety) and OSA. Most sleep bruxism episodes are under the transient influence of cardiac sympathetic activity (as a promoter of arousal), as shown in a rapid rise in heart rate at the onset of rhythmic masticatory muscle activity (time domain estimate of heart rate acceleration (tachycardia) or deceleration (bradycardia) during recurrent sleep arousal. In this case, this patient had a sleep bruxism since a long time ago but did not aware. Even though the patient tells did not has sleep apnea but her BMI was obese prone to OSA. Next examination for definitive diagnostic was polysomnography for OSA and sleep bruxism. rtension, Sleep-bruxism, Cardiovascular-disease Pathophysiologic Links Between Sleep-bruxism and Hypertension Keywords: Hypertension, Sleep-bruxism, Cardiovascular-disease Keywords: Hypertension, Sleep-bruxism, Cardiovascular-disease Pathophysiologic Links Between Sleep-bruxism and Hypertension THROMBOEMBOLECTOMY ON A RARE CASE OF ACUTE UPPER LIMB ISCHEMIC RUTHERFORD IIA WITH ATRIAL FIBRILATION D.B. Lululangi1, H.A.K. Cantika1, M.B.S. Ilham2, R.P. Wicaksono3, R. Putra4 General Practitioner, KRMT Wongsonegoro State Hospital, Semarang, Indonesia1 Internship Doctor, Bhayangkara Hospital, Denpasar, Indonesia2 Department of Cardiothoracic Surgery, KRMT Wongsonegoro State Hospital, Semarang, Indonesia3 Department of Cardiology, KRMT Wongsonegoro State Hospital, Semarang, Indonesia4 D.B. Lululangi1, H.A.K. Cantika1, M.B.S. Ilham2, R.P. Wicaksono3, R. Putra4 General Practitioner, KRMT Wongsonegoro State Hospital, Semarang, Indonesia1 Internship Doctor, Bhayangkara Hospital, Denpasar, Indonesia2 Department of Cardiothoracic Surgery, KRMT Wongsonegoro State Hospital, Semarang, Indonesia3 Department of Cardiology, KRMT Wongsonegoro State Hospital, Semarang, Indonesia4 Background: Acute Upper Limb Ischemia (AULI) is a rare condition compared to lower limb ischemia, accounting for less than 5% of all cases and predominantly associated with atrial fibrillation. Despite the management of AULI can be conservative, however, revascularization procedure remains the treatment of choice. Case Illustration: A 77-year-old female was referred to our emergency unit with the sudden, 8-hour onset of severe pain in the left arm accompanied by paresthesia and paralysis. The patient had a history of hypertension, underlying cardiac condition, and absence of prior traumatic incidents. Physical examination presented grade II hypertension with an irregular, weakened left radial pulse of 40 bpm, compared to a right radial pulse of 80 bpm. Neurovascular assessment of the left arm revealed poikilothermic and pallor extremity with finger oxygen saturation of 57%, 76%, -, -, and 83%, respectively. There were sensory deficits at the C4-C5 dermatome level with a subsequent decline of motoric arm strength at elbow level to fingertips. Cardiomegaly and atrial fibrillation were then found during further exams. From the doppler ultrasound, there was a thrombus in the left brachial artery hence patient confirmed the diagnosis of AULI grade IIA according to the Rutherford classification. Furthermore, the patient received anticoagulants, antiarrhythmic, and antihypertensive. Thromboembolectomy was then performed using a Fogarty catheter and a thrombus of approximately 5 ccs was obtained. The patient showed clinical improvement in the left arm, including optimal pulse oximetry level, adequate arterial pulse, and relief of symptoms. Postoperative transthoracic echocardiography reported no thrombi were recorded. The patient was discharged after being stabilized in ICU for further outpatient treatment. with a subsequent decline of motoric arm strength at elbow level to fingertips. Cardiomegaly and atrial fibrillation were then found during further exams. From the doppler ultrasound, there was a thrombus in the left brachial artery hence patient confirmed the diagnosis of AULI grade IIA according to the Rutherford classification. BACKGROUND Atrial septal defect and Ventricular septal defect are the most common type of congenital heart disease. There is an uncommon type of those communication anomaly, a left ventricle to right atrium connection, which is called the Gerbode defect. In this case we present incidental finding of VSD with Gerbode physiology. CASE ILLUSTRATION A term and fit 2-day-old neonate were referred from pediatrician for congenital heart disease screening. She came to ER with melena 1 day before admission. There is no cyanosis and dyspnea. On physical examination, vital sign is within normal limit, a holosystolic grade 3/6 murmur was found on the lower left sternal border. Trans-thoracic echocardiography showed a secundum ASD 2,1mm with left to right shunt and VSD 3,2mm with left to right shunt to right atrium and right ventricle. This condition matches the gerbode defect which caused by the connection between the left ventricle and the right atrium, resulting in high flow from the left ventricle into the right heart. A high doppler gradient is one of the hallmarks of the Gerbode (1) Without early intervention, VSD Gerbode and ASD may causing early-onset Eisemenger syndrome. y g y g y After stabilization, patient was referred to the tertiary hospital to undergo further assessment. 1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Diponegoro University- Dr. Kariadi Central General Hospital, Semarang, Indonesia D.J. Pesireron, D. Nugraha, P. Ardhianto, A.Y.A.B. Muchtar, S. Sungkar1, THROMBOEMBOLECTOMY ON A RARE CASE OF ACUTE UPPER LIMB ISCHEMIC RUTHERFORD IIA WITH ATRIAL FIBRILATION Furthermore, the patient received anticoagulants, antiarrhythmic, and antihypertensive. Thromboembolectomy was then performed using a Fogarty catheter and a thrombus of approximately 5 ccs was obtained. The patient showed clinical improvement in the left arm, including optimal pulse oximetry level, adequate arterial pulse, and relief of symptoms. Postoperative transthoracic echocardiography reported no thrombi were recorded. The patient was discharged after being stabilized in ICU for further outpatient treatment. g g p Conclusion: AULI represents a vascular emergency, which requires timely recognition and treatment. Revascularization without delay should be considered once there are signs of severe, progressive ischemia for enhancing long-term patient outcomes. eywords: acute upper limb ischemia, thromboembolectomy Comparison of finger oxygen saturation before thrombolembolectomy (left) and after (right) showed improvement. Keywords: acute upper limb ischemia, thromboembolectomy Keywords: acute upper limb ischemia, thromboembolectomy Comparison of finger oxygen saturation before thrombolembolectomy (left) and after (right) showed improvement. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Early Diagnosis of Rare Type Ventricular Septal Defect: A Gerbode Defect Case Report T.L.J. Putri1, M.R.M.P. Sinurat1 RSUD Cileungsi1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background: g Post Cardiac Injury Syndrome (PCIS) is an inflammatory syndrome secondary to cardiac injury that develop after percutaneous cardiac intervention such as catheter ablation. Acute post-ablation pericardial effusion as a manifestation of PCIS is a common complications of atrial fibrillation ablation but its occurrence following an uneventful PVC catheter ablation is rare. Case Illustration: 47-year-old women with complaints of frequent palpitations. Holter monitoring showed frequent premature ventricular contraction (PVC) with episodes of non-sustained ventricular tachycardia. Patient was referred for an electrophysiological study and catheter ablation. During the procedure ventricular tachicardia with right ventricular outflow tract (RVOT) origin was recorded. A total of four radiofrequency (RF) burns were applied to the anterior and posterior-mid septal region of RVOT. In the following day, the patient complained of severe chest discomfort with a slight dyspneu, relieved when she’s leaning forward. 12 lead ECG examinations showed ST elevation in almost all lead (lead I, II, II, avF, avL, V4-V6), with PR depression. Laborartory findings showed elevated CRP and Troponin, Transthoracic echocardiography revealed circumferential pericardial effusion. The patient was diagnosed with PCIS and initiated on NSAID, steroid and low-dose colchicine. At a 5-day follow- up, the patient was free of symptoms and the pericardial effusion was diminished. Conclusion: Pericarditis as a manifestation of Post Cardiac Injury Syndrome (PCIS) although rarely occurred following a PVC catheter ablation, can still happen. Early identification and prompt treatment is vital to mitigate the complication. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keyword: Post cardiac injury syndrome, inflammation, ablation, pericardial effusion CONCLUSION Trans-thoracic echocardiography can be used to detect CHD. CHD screening should be encouraged in any certain condition because some CHD might be asymptomatic. By doing early screening, we can lower the mortality and morbidity of CHD in the future. y y Keywords: Gerbode defect, Ventricular Septal Defect, Atrial Septal Defect, Congenital heart disease Echocardiography showed a left ventricle and right atrium connection which is calle a Gerbode defect Echocardiography showed a left ventricle and right atrium connection which is calle a Gerbode defect When the Cure Causes the Pain: Post-Cardiac Injury Syndrome Following Ventricular Tachycardia Ablation D.J. Pesireron, D. Nugraha, P. Ardhianto, A.Y.A.B. Muchtar, S. Sungkar1, J. Pesireron, D. Nugraha, P. Ardhianto, A.Y.A.B. Muchtar, S. Sungkar1, Background g ARNI (angiotensin receptor-neprilysin inhibition) is a new therapy for the treatment of heart failure with low EF. ARNI can cause ventricular remodeling within 12 months by increasing mean LVEF and clinically decreasing the LVEDV and LVESV indices. Case Illustration A 64-year-old female patient came to the emergency room of the Islamic Hospital Ibnu Sina Padang with complaints of shortness of breath since 4 months ago, which had increased since 2 days before hospitalization. Shortness of breath appears when doing moderate activity. The patient also complained of swelling in both legs and claimed to have lost 3–4 kg in one month. The patient had a smoking habit and a history of diseases such as hypertension, diabetes, and dyslipidemia. On Day 2 of hospitalization, therapy with ARB (angiotensin receptor blocker) was replaced with ARNI 2x50mg. The results of echocardiography on January 9, 2021, showed that the global LV systolic function decreased with an EF of 28%. Furthermore, on August 30, 2021, echocardiography showed an increase in EF to 41%. On February 18, 2022, echocardiography showed an increase in EF to 60%. The patient had a history of PCI 1 stent in mid LAD complete revascularization on April 1, 2022. REVERSE CARDIAC REMODELLING IN ADVANCED HEART FAILURE PATIENT R. Sebastian1, M. Putra2 1Departement of emergency, Ibnu Sina Padang Hostpital, West Sumatra, Indonesia. 2Departement of Cardiology, Ibnu Sina Padang Hostpital, West Sumatra, Indonesia. Keywords: Post cardiac injury syndrome, inflammation, ablation, pericardial effusion Keywords: Post cardiac injury syndrome, inflammation, ablation, pericardial effusion Keywords: Post cardiac injury syndrome, inflammation, ablation, pericardial effusion Conclusion Conclusion According to the trial of PARADIGM-HF and the related prospective observational study PROVE-HF, ARNI was presented. ARNIs play a role in remodeling of the ventricles by increasing levels of natriuretic peptides and blocking the effects of angiotensin II. According to the trial of PARADIGM-HF and the related prospective observational study PROVE-HF, ARNI was presented. ARNIs play a role in remodeling of the ventricles by increasing levels of natriuretic peptides and blocking the effects of angiotensin II. g g Keywords: Heart Failure, ARNI, Ejection Fraction, Ventricular Remodeling Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Acute Dyspnea in Young Obese Women with Pulmonary Hypertension Sign. What is Your Immediate Diagnosis? Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology ACUTE PULMONARY EMBOLISM AND BIVENTRICULAR THROMBUS IN PERIPARTUM CARDIOMYOPATHY M.R. Giovanno1, I.R. Yose2, M. Yanni3, M. Syafri4, M. Syukri5 Cardiology Resident of Cardiology and Vascular Medicine, Andalas University/ DR. M. Djamil General Hospital, Padang, West Sumatera, Indonesia. Department of Cardiology and Vascular Medicine, Andalas University/ DR. M. Djamil General Hospital, Padang, West Sumatera, Indonesia. M.R. Giovanno1, I.R. Yose2, M. Yanni3, M. Syafri4, M. Syukri5 Cardiology Resident of Cardiology and Vascular Medicine, Andalas University/ DR. M. Djamil General Hospital, Padang, West Sumatera, Indonesia. Department of Cardiology and Vascular Medicine, Andalas University/ DR. M. Djamil General Hospital, Padang, West Sumatera, Indonesia. Background: Venous thromboembolism (VTE) is a common cardiovascular syndrome that presents as deep vein thrombosis (DVT) or pulmonary embolism (PE). Peripartum cardiomyopathy (PPCM) is a rare cardiac disorder that leads to heart failure in the last month of pregnancy or up to five months postpartum. PPCM patients are at an increased risk of thromboembolic complications due to a hypercoagulable state. Case Illustration: we present a case of a 40-year-old female who presented with acute onset dyspnea and chest pain six weeks postpartum. Echocardiography revealed biventricular thrombi and an acute PE. The patient was started on heparin and thrombolytic and subsequently transitioned to warfarin. She was also started on heart failure therapy with angiotensin-converting enzyme inhibitors, beta-blockers, and diuretics. Follow-up echocardiography showed complete resolution of the thrombi and normalization of left ventricular function. The patient was discharged with recommendations for long-term anticoagulation and close follow-up. PPCM is a rare but serious condition that requires a high level of suspicion for timely diagnosis and management. VTE is a common complication of PPCM that can lead to significant morbidity and mortality. Anticoagulation therapy with heparin followed by oral anticoagulant is the standard of care. The use of thrombolytic therapy should be reserved for patients with massive or sub-massive PE and careful consideration of the potential risks and benefits is required. In addition to anticoagulation, heart failure therapy is also essential. Conclusion: This case highlights the importance of considering acute PE in the differential diagnosis of dyspnea and chest pain in PPCM patients. A multidisciplinary approach involving cardiology, obstetrics, and hematology is required for the optimal management of these patients. Anticoagulation therapy with heparin followed by oral anticoagulant and heart failure therapy is essential. Follow-up echocardiography is necessary to assess the resolution of thrombi and recovery of left ventricular function. Acute Dyspnea in Young Obese Women with Pulmonary Hypertension Sign. What is Your Immediate Diagnosis? Acute Dyspnea in Young Obese Women with Pulmonary Hypertension Sign. What is Your Immedi Diagnosis? S.J. Sitorus1, M.R. Amadis1 Department of Cardiology and Vascular Medicine, University Airlangga Dr Sutomo Hospital, Surabaya, Indonesia1 S.J. Sitorus1, M.R. Amadis1 Department of Cardiology and Vascular Medicine, University Airlangga Dr Sutomo Hospital, Surabaya, Indonesia1 S.J. Sitorus1, M.R. Amadis1 Department of Cardiology and Vascular Medicine, University Airlangga Dr Sutomo Hospital, Surabaya, Indonesia1 Background Acute pulmonary embolism is a prevalent and potentially life-threatening cardiovascular condition. It may not be easy to immediately diagnose for non-specific clinical presentations that mimic many other common diagnoses. This is a paradox with the availability of effective treatment, which should be started as soon as possible. Case Illustration A twenty-nine-year-old woman with acute dyspnea was consulted for cardiomegaly finding in a chest x-ray from the pulmonology department with suspected pneumonia in the emergency department. She denies chest pain, immobility, leg swelling, and any medical problem before. Her blood pressure was 102/69 mmHg, heart rate was 98 b.p.m, respiratory rate 30 b.p.m, with oxygen saturation was 78% on ambient air. Her weight was 90kg with a height of 152cm. From physical examinations, we found seems symmetrical leg size. An electrocardiogram (ECG) showed sinus rhythm 93x/' with a sign of right ventricular hypertrophy. Echocardiography revealed pulmonary hypertension signs with right ventricular dysfunction (TAPSE 1,2 cm). We suspicious the possibility of acute pulmonary embolism and relook at the ECG that found S1Q3T3. We take a photo of both legs to evaluate more objectively and found a slightly bigger size on the right side. We perform duplex ultrasonography and found a thrombus of the right femoral vein. We asked for CT-pulmonary angiography confirming a left pulmonary artery thrombus. The patient was taken over to the cardiology department, and anticoagulation was started immediately. Conclusion We report a case of an unprovoked acute pulmonary embolism in a young obese woman. This patient's absence of classical risk factors and masked led edema make the diagnosis challenging. Bedside echocardiography is valuable in the evaluation of pulmonary hypertension sign. However, a high level of suspicion is critical and clinically relevant so that cases should not be missed. Keywords: Pulmonary Embolism, Obesity, Woman, Pulmonary Hypertension ACUTE PULMONARY EMBOLISM AND BIVENTRICULAR THROMBUS IN PERIPARTUM CARDIOMYOPATHY Keywords: PPCM, Biventricular Thrombus, Pulmonary Embolism Left figure showing multipe left ventricle thrombus with size 1 cm x 1,2 cm, right figure showing multiple right ventricle thrombus. Left figure showing multipe left ventricle thrombus with size 1 cm x 1,2 cm, right figure showing multiple right ventricle thrombus. Background: g Ischaemic stroke is the most common cerebrovascular disease (CVD) which causes a high mortality and morbidity and leads to a disease burden globally. Nevertheless, its incidence in young age is scarce and remarkable. Atherothrombotic diseases are the most common cause of ischaemic stroke, and uncommonly by hypercoagulation disorder. Moreover, atrial fibrillation (AF) as the distinct underlying cause should be also considered. Case Illustration: A healthy sportaholic 43 years-old Caucasian man without any comorbidity presented to our emergency department with sudden onset of dysarthria and talked incoherently which lasted for a few seconds. Physical examinations revealed BP 104/78 mmHg; HR 71 bpm without any abnormalities. ECG presented was SR with LAE. Laboratory results were in normal limits. Echocardiography showed slight dilated LA, with normal LVEF. MRI/MRA brain and carotid ultrasonography were in normal limits. Diagnostic of TIA and suspicion of AF was confirmed. Further laboratory results showed a protein C and protein S deficiency (36.5 and 57.4), and also an AF on 7 days holter ECG. Oral anticoagulant (OAC) was initiated, and a routine follow-up as well as electrophysiology study with ablation was recommended. Natural Anticoagulants Deficiency Concomitant Silent Atrial Fibrillation in a Transient Ischemic Attack (TIA) Healthy Caucasian Sportaholic Male: A Case Report A.A. Lukito1, H. Hutabarat1, S. Chandra1, R.A.P. Fernandes1 Siloam Hospital Lippo Village1 Natural Anticoagulants Deficiency Concomitant Silent Atrial Fibrillation in a Transient Ischemic Attack (TIA) Healthy Caucasian Sportaholic Male: A Case Report A.A. Lukito1, H. Hutabarat1, S. Chandra1, R.A.P. Fernandes1 Siloam Hospital Lippo Village1 A SURVIVING CASE OF RECURRENT LETHAL ARRYTHMIA IN ADOLESCENT FULMINANT MYOCARDITIS WITH SUSPECTING GNAPS D.B. Lululangi1, H.A.K. Cantika1, L. Dewiyanti2, P.R. Dewi3, I.M. Handajaningrum4 General Practitioner, KRMT Wongsonegoro State Hospital, Semarang, Indonesia1 Department of Pediatric, KRMT Wongsonegoro State Hospital, Semarang, Indonesia2 Departement of Cardiology, KRMT Wongsonegoro State Hospital, Semarang, Indonesia3 Departement of Internal Medicine, Nefrology-Hipertension Subdivision, KRMT Wongsonegoro State Hospital, Semarang, Indonesia4 Background: Fulminant myocarditis (FM) is a rare condition of rapidly progressive cardiac inflammation with high mortality profile. Although considered uncommon, arrhythmias may present in FM, leading to sudden cardiac death. Case Illustration: A 14-year-old female was referred to our emergency department with shortness of breath, cough, and chest pain. The patient also had fever and sore throat five days prior. Physical examination revealed signs of heart failure (HF). Initial blood test showed mild anemia, leukocytosis, electrolyte imbalance, increased levels of serum urea, creatinine, and troponin. Urinalysis findings were in favor of nephritic involvement. Left ventricular dilatation with 30% ejection fraction and severe ventricular wall thickening were confirmed through echocardiography examination, directed to a suspicion of FM. The patient was admitted to the pediatric intensive care unit for further monitoring and received several medications to support cardiac function, treat infection and prevent end-organ damage. Unfortunately, for the next three days the patient experienced several episodes of cardiac arrest and lethal ventricular arrhythmia. Immediate administration of mechanical ventilators, high doses of steroids, continuous antiarrhythmic agents, and inotropics were given during resuscitation effort, followed by return of spontaneous circulation. The patient's clinical condition was dramatically improved, thus allowing the detachment of ventilation support. Our followed-up examination in pediatric ward resulted in resolved HF signs and symptoms with normal resting echocardiography. The patient was discharged on the eleventh day of hospitalization. Outpatient care is undergone for evaluation and assessing the etiology of the disease. Conclusion: The course of FM is heterogeneous, varying from partial or full clinical recovery in a few days to advanced HF with lethal arrythmia requiring mechanical circulatory support. Prompt clinical diagnosis and optimal treatment implementation from multidisciplinary approach will be beneficial to overt multiorgan system failure, moreover, increase patient’s prognosis. A SURVIVING CASE OF RECURRENT LETHAL ARRYTHMIA IN ADOLESCENT FULMINANT MYOCARDITIS WITH SUSPECTING GNAPS Keywords: fulminant myocarditis, lethal arrythmia, adolescent The patient's echocardiography shows left ventricular dilatation with 30% ejection fraction and severe ventricular wall thickening Keywords: fulminant myocarditis, lethal arrythmia, adolescent Keywords: fulminant myocarditis, lethal arrythmia, adolescent The patient's echocardiography shows left ventricular dilatation with 30% ejection fraction and severe ventricular wall thickening Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Natural Anticoagulants Deficiency Concomitant Silent Atrial Fibrillation in a Transient Ischemic Attack (TIA) Healthy Caucasian Sportaholic Male: A Case Report A.A. Lukito1, H. Hutabarat1, S. Chandra1, R.A.P. Fernandes1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case illustration: We present two cases of patients who were initially diagnosed with MINOCA but later found to have TTS. Our first case is a classic example of Takotsubo syndrome, based on systolic apical ballooning on transthoracic echocardiography (TTE). Within two days, she made a full recovery. In the second case, TTE revealed an atypical TTS characterized by hypokinesis of the basal segment and reserve of the mid-ventricular and apical segments. He suffered from cardiogenic shock and gradually recovered within 30 days. The international takotsubo (InterTAK) diagnostic score and speckle tracking TTE are two modalities that can be performed rapidly to diagnose TTS. InterTAK score is a scoring model that can differentiate TTS from ACS using all easily obtainable emergency department parameters. Speckle tracing echocardiography has acquired a fundamental role in the evaluation of TTS due to its numerous advantages over the Doppler method, such as early detection of heart muscle damage, angle independence, semi-automatic quantitative analysis, rapid image acquisition, and strain determination without temporal integration, which makes it less dependent on the operator. While higher initial levels of cardiac troponin-I and NT-proBNP are predictorss for worse clinical course and slower left ventricle recovery. MINOCA Presenting with STEMI: Two Distinct Types and Courses of Takotsubo Syndrom M.Y. Alsagaff1, T.T. E. Lusida1, R.I. Gunadi1 Faculty of Medicine, Universitas Airlangga – Dr. Soetomo General Hospital1 Background: Myocardial infarction with non-obstructive coronary arteries (MINOCA) was responsible for some patients presenting with ST-segment elevation myocardial infarction (STEMI). However, many people are unaware that Takotsubo syndrome (TTS) is no longer classified as MINOCA as of the 4th universal definition of myocardial infarction. Furthermore, recognition of atypical TTS (the basal, midventricular, and focal wall motion patterns) has increased in recent years. Conclusions: Due to the low incidence of CVD at young age, therefore a careful and thorough examinations are needed. Suspicion toward silent AF and trombophilic disorders such as protein C, protein S, AT III deficiency, factor V Leiden mutation, antiphospholipid antibodies syndrome, lupus anticoagulant and anticardiolipin should be considered. 7-days holter ECG can be used as a modality in detecting silent AF. OAC should be initiated if a positive result is found. Keywords: Natural Anticoagulants Deficiency, Atrial Fibrillation, TIA, Young Age Keywords: Natural Anticoagulants Deficiency, Atrial Fibrillation, TIA, Young Age Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 MINOCA Presenting with STEMI: Two Distinct Types and Courses of Takotsubo Syndrome M.Y. Alsagaff1, T.T. E. Lusida1, R.I. Gunadi1 Faculty of Medicine, Universitas Airlangga – Dr. Soetomo General Hospital1 Highlighting The Importance of Assessing Peripheral Stigmata in Early Diagnosis of Infective Endocarditis: A Case Report Highlighting The Importance of Assessing Peripheral Stigmata in Early Diagnosis of Infective Endocarditis: A Case Report T.S. Tartila1, I.F.A. Putra1, A.F. Prianda2, R. Istisakinah2 General Practitioner, Hasna Medika Kuningan Heart Hospital, West Java, Indonesia1 Cardiologist, Hasna Medika Kuningan Heart Hospital, West Java, Indonesia2 General Practitioner, Hasna Medika Kuningan Heart Hospital, West Java, Indonesia1 Cardiologist, Hasna Medika Kuningan Heart Hospital, West Java, Indonesia2 Background: The diverse spectrum of infective endocarditis (IE) often presents diagnostic challenge that can delay initiation of therapy. A thorough physical examination to assess cutaneous manifestations as classical signs of IE can help in early diagnosis especially at center without advance diagnostic modalities. We report a case of IE with cutaneous stigmata of Janeway lesions. Case Illustration: A 28-year-old previously healthy man was referred to our hospital with prolonged fever for 2 weeks, chest pain, cough, and myalgia. The patient had already been hospitalized at previous hospital with typhoid fever but showed no improvement. The patient later developed dyspnea and then suspected with acute rheumatic fever. The vital signs were in normal range, but on physical examination we discovered holosystolic murmur near the apex and the presence of irregular, flat, erythematous, and painless macules on the soles consistent with Janeway lesions that led us to the suspicion of IE. The laboratory results showed leukocytosis, renal insufficiency, and elevated titer of ASTO (anti streptolysin O). From chest x-ray we found cardiomegaly and reverse comma sign. The echocardiographic evaluation revealed the presence of vegetation on posterior mitral leaflet and severe mitral regurgitation. The patient met 1 major and 4 minor criteria of Duke Criteria and was classified as definite IE. We diagnosed the patient with IE and concurrent reactivation of rheumatic fever. The patient was treated with intravenous ceftriaxone and gentamycin and recovered well. Conclusion: Early recognition of peripheral stigmata is important to make early diagnosis of IE. We highlight that dermatologic examination should always be part of physical examination in evaluating the possibility of IE, especially in low-resource settings where diagnosis relies mainly on clinical findings. Keywords: infective endocarditis, peripheral stigmata, cutaneous manifestations, Janeway lesions, early diagnosis Conclusion: Early recognition of peripheral stigmata is important to make early diagnosis of IE. We highlight that dermatologic examination should always be part of physical examination in evaluating the possibility of IE, especially in low-resource settings where diagnosis relies mainly on clinical findings. y Conclusion: TTS is different from MINOCA. The InterTAK score and speckle TTE are efficient and rapid bedside diagnostic tool for distinguishing TTS from ACS. Higher troponin-I and NT-proBNP levels may be associated with a worse clinical course and slower recovery from LV dysfunction Keywords: Takotsubo syndrome, MINOCA, Echocardiography TTS is different from MINOCA. The InterTAK score and speckle TTE are efficient and rapid bedside diagnostic tool for distinguishing TTS from ACS. Higher troponin-I and NT-proBNP levels may be associated with a worse clinical course and slower recovery from LV dysfunction Keywords: Takotsubo syndrome, MINOCA, Echocardiography Indonesian Journal of Cardiology Highlighting The Importance of Assessing Peripheral Stigmata in Early Diagnosis of Infective Endocarditis: A Case Report K d i f i d di i i h l i if i J l i l ords: infective endocarditis, peripheral stigmata, cutaneous manifestations, Janeway lesions, early sis Keywords: infective endocarditis, peripheral stigmata, cutaneous manifestations, Janeway lesions, early diagnosis The appearance of Janeway lesions The appearance of Janeway lesions Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Lewis Lead: Revelation of the P Wave A. Laurentius1, K. Kohar1, E. Ferdinand1, S.N. Siagian2 Faculty of Medicine, Universitas Indonesia1 Division of Pediatric Cardiology and Congenital Heart Disease, Department of Cardiology and Vascular Medicine, National Cardiovascular Centre Harapan Kita, Universitas Indonesia2 A. Laurentius1, K. Kohar1, E. Ferdinand1, S.N. Siagian2 Faculty of Medicine, Universitas Indonesia1 Division of Pediatric Cardiology and Congenital Heart Disease, Department of Cardiology and Vascular Medicine, National Cardiovascular Centre Harapan Kita, Universitas Indonesia2 Background: Lewis lead is a method used in electrocardiography in order to magnify the recorded atrial activity in relation to that of ventricles. It values better visualization of P wave in differentiating some well-known arrhythmia, such as atrial fibrillation, atrial flutter, and multifocal atrial rhythm. Correct diagnosis of types of arrhythmia could render optimal treatment. y p Case Illustration:We present a case of a 67-year-old Eastern Belitung female who experienced weakness in all extremities while having breakfast two hours before coming to the ER. Her family admits that she was neither aware nor responsive to any call during the event, yet she did not experience syncope. She had uncontrolled chronic hypertension. Her family denied having history of diabetes mellitus, autoimmune disease, and cardiovascular or neurological events, as well as similar past symptoms. During physical examination, her airway was patent, respiratory rate was 19 breaths per minute with oxygen saturation of 96% on air. Heart rate was 59 beats per minute with blood pressure of 130/90 mmHg. Clinically, the patient appeared responsive to pain stimuli but neither speech nor sound with Glasgow coma scale of 13. Her axillary temperature was 37 degrees Celsius. Pulmonary assessment was normal. Cardiac auscultation disclosed slow heart sound with no murmur. Asymmetrical neurological deficit was found in her right arm and leg, as well as slight drooping of right oral commissure with intact forehead strength. Babinski reflex was positive on her right leg. The remainder of physical examination was normal with warm extremities. The electrocardiogram (ECG) showed narrow QRS complexes and irregular R-R interval with no observed P wave. However, slight ECG modification using Lewis lead exhibited coarse fibrillatory P wave, which emphasized diagnosis of atrial fibrillation. Conclusion: Lewis lead configuration is helpful to prevent misdiagnosis of types of arrhythmia with poor P waves visualization. Conclusion: Lewis lead configuration is helpful to prevent misdiagnosis of types of arrhythmia with poor P waves visualization. Keywords: Lewis lead, P waves, Electrocardiography Figure 1. SUCCESSFUL RESOLUTION OF TOTAL ATRIOVENTRICULAR BLOCK (TAVB) IN INFERIOR ST-ELEVATION NON REVASCULARIZATION WITH DIABETIC KETOACIDOSIS (DKA) : A CASE REPORT M. Marpaung1, D. Krisnawati2 General Practitioner Demang Sepulau Raya Regional Hospital, Lampung, Indonesia1 Cardiologist Abdul Moeloek General Hospital, Lampung Indonesia2 M. Marpaung1, D. Krisnawati2 Background: Total Atrioventricular Block (TAVB) is a common complication of inferior myocardial infarction (MI). An occlusion in the Right Coronary Artery (RCA) can also cause impedance to the cardiac conduction system, such as TAVB. Diabetic Ketoacidosis (DKA) may be the precipitating event for the occurrence of acute MI, serious arrhythmia, and pulmonary edema, while MI is also a well-known precipitant factor for DKA. Case Illustrations: A-48 years old-woman has typical chest pain accompanied by diaphoresis, syncope, dyspnoea, hypotension, and bradycardia starting 24 hours before admission. She had risk factors for hypertension and Diabetes Mellitus (DM). Electrocardiography (ECG) showed inferior ST-elevation and TAVB. The laboratory found CKMB 69 U/L; hyperglycemic (RBS 512 g/dl), HbA1C 14,8, ketonuria; BGA results of compensated metabolic acidosis; potassium 2.8 mmol/L; creatinine 2,8 mg/dL. Chest X-Ray: cardiomegaly and bronchopneumonia. A temporary pacemaker (TPM) and Percutaneous Coronary Interventions (PCI) cannot be done due to cost constraints. According to unstable hemodynamics, atropine sulfate 1 mg has been given. As an alternative for TAVB, we use dopamine 3-10mcg/kg bb/minute. Initial treatment for MI: oxygenation, DAPT, statin, and UFH 700IU-1000IU/hour. DKA has been treated with saline and insulin resuscitation, metabolic acidosis correction, and antibiotic for infections. After 10 days of treatment, we found ECG evolved from TAVB to 1st-degree AV block and after 14 days of treatment, ECG showed sinus rhythm without TAVB, metabolic acidosis clinically improved, hypokalemia has corrected (potassium: 4.7 mmol/L ), and RBS decreased to 141 g/dl. Conclusion: Hyperglycemia, acidosis, and electrolyte imbalance can directly affect the heart by inducing toxicity, impairing myocardial blood flow, autonomic dysfunction, and altering activation and conduction of electrical impulses throughout the heart, increasing the risk of arrhythmias and ischemia. When DKA is well treated, the arrhythmia in the patient is also resolved. Keywords: TAVB, inferior ST-elevation, percutaneous coronary interventions, diabetic ketoacidosis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Lewis Lead: Revelation of the P Wave Electrocardiography findings with classic 12-lead configuration (left) and Lewis lead configuration (right). Keywords: Lewis lead, P waves, Electrocardiography Figure 1. Electrocardiography findings with classic 12-lead configuration (left) and Lewis lead configuration (right). Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 62 years old male presented with palpitations and shortness of breath in emergency room (ER). Patient had history of congestive heart failure from 3 months before with poor compliance to take medicine. His blood pressure was 140/90 mmHg and heart rate was 130-140x/min. Pansistolic murmur grade 3/6 at apex radiating to axila was found in patient. ECG showed AF with rapid ventricular response. Transthoracal echocardiography showed bileaflet myxomatous mitral valve degeneration barlowish type with severe MR while in AF, LA size was 77mm, with EF 56% and good right ventricular function. Amiodarone intravenous was administered leading to control rhythm but AF still persistant with controlled in rate. The diuretics, anticoagulant, and optimal heart failure medicine was administered in high care unit. The Giant LA indicated the chronic process from MR severe in patient with poor compliance, that make AF became persistant as complicating burden of MR. After multidisciplinary evaluation, he was offered mitral valve repair, however he elected for medical management only. Background g Atrial fibrillation (AF) is the most common arrhythmia and associated with an adverse prognosis. Mitral regurgitation (MR) is of particular interest regarding the development of AF because MR is common particularly in elderly and tends to produce left atrial (LA) enlargement, high risk precursor of AF. We report a rare case barlowish type mitral regurgitation who had presented with AF. Atrial fibrillation in barlowish mitral regurgitation: a burden that never end A. Firmansyah1, C.K. Krevani2 A. Firmansyah1, C.K. Krevani2 y Cardiology and Vascular Medicine Residency, Andalas University, Padang, West Sumatera.1 Cardiology and Vascular Medicine Department, M. Djamil Hospital, Padang, West Sumatera.2 y Cardiology and Vascular Medicine Residency, Andalas University, Padang, West Sumatera.1 Cardiology and Vascular Medicine Department, M. Djamil Hospital, Padang, West Sumatera.2 The Challenge and Modification Towards Cardiovascular Rehabilitation Program on Post PCI Patient with Permanent Pacemaker: A Case Report D.Pasahari1, D. Sarvasti2, F.A. Wardani1, M.E. Saputra1, A.D. Lamara3 Airlangga University1 Widya Mandala Catholic University2 University of Airlangga3 Case illustration A-67-Years-Old-Women previously diagnosed with non-ST-segment elevation myocardial infarction (NSTEMI) with bradycardia, underwent Percutaneous-Coronary-Intervention, and PPM implantation was referred to our facility. Single-chamber PPM positioned at right ventricle, with 60-pace per minute. The patient had hypertension, diabetes, and dyslipidemia. At the first visit, the patient’s blood pressure was measured 110/70mmHg. Electrocardiogram recorded pacemaker rhythm 75-bpm, left-axis-deviation, and left-bundle- branch-block. Chest X-ray suggests cardiomegaly, and echocardiography shows left-ventricle-diastolic- dysfunction-grade-III. The 6-minute walk test (6-MWT) presented Metabolic Equivalents (METs) of 3.30, resembling the patient's exercise capacity. In every session, the patient undergoes moderate-intensity exercise, consisting of flat-track walking and a static bike. The difficulty increases with increasing progression. Training modifications for patients with PPM have specific instructions, regulating target heart rate and aerobic activities within limitation, having a preferred BORG Scale of 11-13. g At the 12th appointment, a stress test using a modified Bruce protocol is usually performed. Due to patient complaints of knee pain, and leg fatigue, the stress test is replaced with 6-MWT, showing the increment in patient’s METs to 3.87, with no chest pain and shortness of breath complaints during training. Based on the psychological questionnaire, the patient feels more confident and happier after the program. Conclusion Co c us o Cardiovascular rehabilitation programs, especially physical training, differ slightly for patients with PPM. However, the program could still improve patient quality of life physically and psychologically. Background g The cardiovascular rehabilitation program is eligible for patients who have undergone cardiovascular intervention to improve cardiovascular function and quality of life. This program is also beneficial for patients with a permanent pacemaker (PPM), which have slight modifications for the physical training. Conclusion The case highlights an important mechanism of MR which can be noted in AF patient. Poor compliance make MR and AF became a vicious cycle that increased morbidity to patient. The case highlights an important mechanism of MR which can be noted in AF patient. Poor compliance make MR and AF became a vicious cycle that increased morbidity to patient. Keywords: Atrial fibrillation, mitral regurgitation, barlowish disease eywords: Atrial fibrillation, mitral regurgitation, barlowish disease Keywords: Atrial fibrillation, mitral regurgitation, barlowish disease Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 The Challenge and Modification Towards Cardiovascular Rehabilitation Program on Post PCI Patient with Permanent Pacemaker: A Case Report Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 The Challenge and Modification Towards Cardiovascular Rehabilitation Program on Post PCI Patient with Permanent Pacemaker: A Case Report Case Illustration: A 56-year-old Asian female with a history of PPM (VVIR mode) implantation due to a high degree AV block was transferred to our hospital with a diagnosis of acute decompensated heart failure (ADHF). On arrival, she looked restless and had difficulty breathing, with a respiratory rate of 25 tpm and a body temperature of 39 degrees Celcius. Physical examination showed bilateral leg edema, and rhonchi were heard bilaterally. Laboratory tests revealed a white blood cell count of 33.760 /mm3 with 84% neutrophils, and NT pro BNP level was 9.233 pg/L. An electrocardiogram (ECG) showed atrial flutter with ventricular pacing rhythm 60 bpm RVOT origin. Chest x- ray revealed pulmonary edema, infiltrates, and cardiomegaly with a single lead PPM seen at RVOT. Evaluation of her PPM showed good condition with 99.5% RV pacing. Her echocardiogram showed all chamber dilatation, global hypokinetic, and reduced ejection fraction (EF) from 62% to 26% in less than two years. Coronary angiography was performed, which showed non-significant coronary artery stenosis with 20% stenosis at distal LAD. After three days of antibiotics and decongestive treatment, her complaints of fever, bilateral leg edema, and shortness of breath resolved. His-Bundle pacing (HBP) was scheduled to reduced LV dyssynchrony and improve LV function. Background: g Permanent right ventricular pacing (RVP) is a standard treatment for high-grade atrioventricular (AV) block. Still, it may cause abnormal ventricular activation, which results in an inefficient myocardial contraction pattern due to ventricular dyssynchrony and may lead to left ventricular (LV) dilatation, systolic dysfunction, and heart failure (HF). Chronic and high-burden RVP can cause pacing-induced cardiomyopathy (PICM) that may occur several months or years after permanent pacemaker (PPM) implantation. We describe a case of a PICM with a significant decrease in LV systolic function in less than two years after PPM implantation. Coronary angiography showed widely patent vessels and subsequently, His-Bundle pacing (HBP) was scheduled on the patient. Keywords y Cardiovascular rehabilitation, metabolic equivalent, percutaneous coronary intervention, permanent pacemaker, 6-minute walk test. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Rapid Degradation of Left Ventricular Function after Permanent Right Ventricular Pacing in Patients with High-Grade Atrioventricular Block D. Setiawan1,2, A. Rizal1,2, I. Prasetya1,2 1Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya, 2Dr. Saiful Anwar General Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rapid Degradation of Left Ventricular Function after Permanent Right Ventricular Pacing in Patients with High-Grade Atrioventricular Block D Setiawan1,2 A Rizal1,2 I Prasetya1,2 pid Degradation of Left Ventricular Function after Permanent Right Ventricular Pacing in Patients with High-Grade Atrioventricular Block D. Setiawan1,2, A. Rizal1,2, I. Prasetya1,2 1Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya, 2Dr. Saiful Anwar General Hospital Keywords: STEMI, Fibrinolysis, Cardiac Arrest Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Successful Fibrinolysis on a Cardiac Arrest Survivor Following Ventricular Tachycardia due to STEMI in Toraja Utara Conclusion: Single-chambered pacemakers create ventricular dyssynchrony, which can cause structural and molecular changes leading to cardiomyopathy. With early intervention, these changes can be reversible. Therefore, timely check-ups and awareness are necessary for patients on PPM. Keywords: Atrioventricular Block, Right Ventricular Pacing, Pacing-induced Cardiomyopathy Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Successful Fibrinolysis on a Cardiac Arrest Survivor Following Ventricular Tachycardia due to STEMI in Toraja Utara A 42-Year Old Male Patient with Wellen's Syndrome: A Rare Case Report Background: A characteristic electrocardiogram (ECG) pattern of T-waves in the precordial leads that were associated with a critical stenosis (>90%) of the proximal left anterior descending (LAD) coronary artery was described in 1982 by Wellens. Wellen Syndrome (WS) criteria include T-wave changes in precordial leads, plus a history of anginal chest pain without marked serum marker abnormalities and Q-waves. Background: A characteristic electrocardiogram (ECG) pattern of T-waves in the precordial leads that were associated with a critical stenosis (>90%) of the proximal left anterior descending (LAD) coronary artery was described in 1982 by Wellens. Wellen Syndrome (WS) criteria include T-wave changes in precordial leads, plus a history of anginal chest pain without marked serum marker abnormalities and Q-waves. Case Illustration: A 42-year-old male patient came to the emergency ward with intermittent moderate chest pain and shortness of breath over a few days. The patient has a history of smoker and uncontrolled hypertension. A history of the same symptoms was found, and misdiagnosed several times. The initial vital sign depicted 160/90 blood pressure and ECG displayed slight ST depression on V1-V5 lead. T-troponin was normal on initial finding. He was hospitalized and conducted ECG serial. At the pain-free, ECG displayed biphasic t wave on leads V2-V5, specific for Wellen’s Sign. He referred and an angiography study showed mid stenosis 90% on the left anterior descendent artery with the consecutively reduced coronary flow. The patient is treated by drug-eluting stent implantation and angioplasty. Case Illustration: A 42-year-old male patient came to the emergency ward with intermittent moderate chest pain and shortness of breath over a few days. The patient has a history of smoker and uncontrolled hypertension. A history of the same symptoms was found, and misdiagnosed several times. The initial vital sign depicted 160/90 blood pressure and ECG displayed slight ST depression on V1-V5 lead. T-troponin was normal on initial finding. He was hospitalized and conducted ECG serial. At the pain-free, ECG displayed biphasic t wave on leads V2-V5, specific for Wellen’s Sign. He referred and an angiography study showed mid stenosis 90% on the left anterior descendent artery with the consecutively reduced coronary flow. The patient is treated by drug-eluting stent implantation and angioplasty. Conclusion: Wellen's syndrome is a life-threatening case characterized by abnormal T-waves found on ECG. The ECG changes during pain relief. Successful Fibrinolysis on a Cardiac Arrest Survivor Following Ventricular Tachycardia due to STEMI in Toraja Utara F.J. Tandirerung1, Y. Iskandar1 RS Elim Rantepao Hospital1 F.J. Tandirerung1, Y. Iskandar1 RS Elim Rantepao Hospital1 Background. Cardiac arrest (CA) following ST-elevation myocardial infarction (STEMI) is associated with a higher mortality rate. Percutaneous Coronary Intervention (PCI) is crucial in lowering the mortality rate in such cases. However, PCI might not be accessible for hospitals in peripheral regions, leaving fibrinolysis as the only feasible option. Furthermore, prolonged/traumatic resuscitation is a relative contraindication for fibrinolysis, complicating the fibrinolysis decision for the post-resuscitation CA survivor. Here we present a case of successful fibrinolysis in a CA survivor secondary to STEMI in Toraja Utara. Case Illustration. A 66-year-old patient was admitted to the emergency room (ER) due to typical chest pain 30 minutes prior to hospital admission. The pain was described as heavy, radiating to the left shoulder, with diaphoresis and shortness of breath. The patient was a smoker with a history of hypertension. At the ER, the patient had a seizure, which was followed by CA. Cardiopulmonary resuscitation (CPR) was performed, followed by defibrillation due to ventricular tachycardia. Following a 5-minute CPR with defibrillation, the patient returned to spontaneous circulation, fully conscious with stable hemodynamics. A 12-lead electrocardiogram (ECG) assessment was carried out that showed ST-elevation on leads V1-V3, therefore assessed and treated as STEMI. However, the nearest PCI center is approximately 400 kilometers (8 hours) away in Makassar. Hence, fibrinolysis with streptokinase was selected for initial reperfusion and the patient was discharged after six days of hospitalization with no neurological or cardiopulmonary complications. The patient refused PCI referral during the hospitalization period and was only referred months later with favorable clinical outcomes. Conclusion. Despite reperfusion through PCI remains the stronger survival predictor in STEMI with CA, successful reperfusion with fibrinolysis is attainable with timely intervention following CPR. This is clinically substantial, especially in peripheral areas with difficult access to PCI. Keywords: STEMI, Fibrinolysis, Cardiac Arrest Indonesia Card Indonesian J Card pISSN: 2830-3105 doi: 10.30 A 42-Year Old Male Patient with Wellen's Syndrome: A Rare Case Report M.F.A. Akbar1, Y.B. Saragih1 Aceh Tamiang General Hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A 42-Year Old Male Patient with Wellen's Syndrome: A Rare Case Report In our case, the patient presents with biphasic T-wave on anterior leads after being pain-free, on this point, it is crucial to conduct an ECG serial. Therefore, the physician should be aware of recognizing ECG features to help identify high-risk patients who require immediate invasive strategy. An invasive strategy is a cornerstone of the therapy to make a good prognosis. Keywords: Keywords: Myocardial infarction, T-wave abnormality ST-segment elevation with biphasic T wave in lead V2-V5 Keywords: Keywords: Myocardial infarction, T-wave abnormality ST-segment elevation with biphasic T wave in lead V2-V5 Indonesian Jo Cardiolo Indonesian J Cardiol 20 pISSN: 2830-3105 / eIS doi: 10.30701/i Facing Junctional Bradycardia with Cardiogenic Shock Case in ER : A Case Report P. Yeantesa1, N.A. Byant1, S.D. Utami1 Awal Bros Panam Hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Facing Junctional Bradycardia with Cardiogenic Shock Case in ER : A Case Report P. Yeantesa1, N.A. Byant1, S.D. Utami1 Awal Bros Panam Hospital1 Background: Sinoatrial node is a default pacemaker in human’s heart, they trigger electrical activity, so the heart can beating. In some cases, someone’s heart using atrioventricular node to make rhythm, one of their rhythm is junctional bradycardia, this rhythm has rate below 60 bpm. This rhythm can decrease heart pumping and blood supply to the heart cells, so somebody can fall into cardiogenic shock. Case illustration: 54-Year-Old patient came into ER with feeling weakness and have no energy since 3 days before admission. Ten days before admission, she started felt nausea, then vomited until she didn’t know how many times it happened. Before that, she went to the clinic and checked peripheral blood, the result is she had anemia and trombositosis. She had diabetic mellitus type 2, hypertension heart disease, aorta regurgitation, atrial fibrilation, congestive heart failure. On physical examination, we obtained BP 88/55 mmHg, HR 44 bpm and pulse was so weak, RR 20 x/min, afebrile and SpO2 93% room air, conjunctival pallor, crackles(+), other examination were normal. Twelve leads ECG showed junctional bradycardia (44 bpm), echocardiography showed EF 66%, Global N, LVH+, MR mild, AR mild. She also had anemia, renal insufficiency, hyperkalemia and hyponatremia. Keywords: Junctional Bradycardia, Hyperkalemia, Cardiogenic Shock A 42-Year Old Male Patient with Wellen's Syndrome: A Rare Case Report In ER, twelve ampoules of atropine sulfate is just administered, but heart rate still between 40-50 bpm, so that, dopamine is administered, the normal rate and blood pressure is just achieved and then she transferred to HCU. Conclusion: Electrolyte imbalance such as hyperkalemia make biphasic effect for cardiac: action potential being short, the conduction getting faster, but in the end, this conduction being slowly and induce junctional bradycardia.As bradycardia running, our heart doesn’t ability to pump enough blood to the whole body and person would fall into condition like cardiogenic shock. Keywords: Junctional Bradycardia, Hyperkalemia, Cardiogenic Shock Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: STEMI, Ventricular Tachicardia, Malignant Arrhytmia Apical Hypertrophic Cardiomyopathy Mimicking Acute Coronary Syndrome Presentation: What is the Important Clue? M. Reeiner1, L.O.S. Suastika2, N.M.A.W. Sari2, I.B.R. Wibhuti2 1Faculty of Medicine, Udayana University, Denpasar, Indonesia; 2Sanglah General Hospital, Denpasar, Indonesia Background: Apical hypertrophic cardiomyopathy (ApHCM) is a rare variant of hypertrophic cardiomyopathy, characterized by a spade-like left ventricular cavity, and more commonly seen in the Asian population. the electrocardiographic changes and symptoms associated with apical hypertrophic cardiomyopathy often mimic acute coronary syndromes. Distinguishing between these conditions was important to determine early and definitive treatment. Case Illustration: A 56-year-old man came to our emergency room with severe chest pain three hours after the onset of symptoms. His chest pain was induced by physical activity but persisted at rest. Similar symptoms had occurred prior to the current event, he had done coronary angiography several years ago and the result was normal. His medical history was hyperlipidemia and family history of hypertension. His cardiac enzymes were not elevated. The electrocardiogram showed giant negative T wave inversion in the lateral precordial leads (V2–V6, I, and aVL). As a result of the typical symptoms of unstable angina pectoris, electrocardiographic repolarization disturbances indicative of acute myocardial ischemia, and his cardiovascular risk profile, the decision was made to carry out cardiac catheterization. Coronary angiography excluded significant coronary artery disease. However, echocardiography demonstrated a typical spade-like configuration of the left ventricular (LV) cavity. The wall thickness of the apical septal regions and the apical posterior region was 16 mm and 18 mm, respectively, and systolic LV function was normal. Apical myectomy was deferred in this patient and beta blocker was our first choice of treatment. Conclusion: ApHCM can mimic the symptoms and electrocardiographic changes of acute coronary syndromes and should be considered as a differential diagnosis, especially in patients with giant T wave negativity, normal cardiac marker and spade-like configuration of the LV cavity. Echocardiography of the apical segments, and CMR is helpful in establishing the diagnosis of ApHCM. Keywords: Apical hypertrophic cardiomyopathy, acute coronary syndrome, giant T wave inversion, echocardiography Keywords: Apical hypertrophic cardiomyopathy, acute coronary syndrome, giant T wave inversion, echocardiography Lower extrimities bilateral post thrombotic syndrome Lower extrimities bilateral post thrombotic syndrome Indonesian Cardio Indonesian J Cardiol pISSN: 2830-3105 / doi: 10.3070 Challenging STEMI with Ventricular Tachicardia Case in ER : A Case Report R.P. Putra1, S.D. Utami1, N.A. Ventricular Tachycardia During Balloon Atrial Septostomy in a Neonate with TGA: A Rare but Life- Threatening Complication A.Yudha1, A. Priyatno2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia1 Department of Pediatric, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia2 A.Yudha1, A. Priyatno2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia1 Department of Pediatric, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia2 A.Yudha1, A. Priyatno2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Central General Hospital, Semarang, Indonesia1 Department of Pediatric, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General H Semarang, Indonesia2 A.Yudha1, A. Priyatno2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia1 Department of Pediatric, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia2 Case Illustration : A 4-day-old male neonate who was diagnosed with complete TGA shortly after birth. Balloon atrial septostomy was planned as a palliative procedure to improve the patient's oxygenation. During the procedure, the patient developed ventricular tachycardia, which was initially managed with anti-arrhythmic medications. However, the patient remained hemodynamically unstable, and electrical cardioversion was required to restore normal sinus rhythm. Following the cardioversion, the patient's rhythm stabilized, the septostomy was successfully completed, and there was an improvement in oxygen saturation from 50% to 80%. The patient ultimately made a full recovery and was discharged from the hospital with stable cardiac function.The occurrence of ventricular tachycardia during balloon atrial septostomy is a rare but serious complication that requires prompt recognition and management. In our case, the prompt recognition of the arrhythmia and the.immediate administration of anti-arrhythmic medications and electrical cardioversion were crucial in stabilizing the patient's condition. With timely and appropriate intervention, such as anti-arrhythmic medications and electrical cardioversion, the patient's condition can be stabilized and the procedure can be successful. Background : Ventricular tachycardia is a rare but potentially life-threatening complication that can occur during balloon atrial septostomy procedures in neonates. In this case report, we describe the management of a term neonate with complete transposition of the great artery (TGA) who developed ventricular tachycardia during a balloon atrial septostomy procedure. Apical Hypertrophic Cardiomyopathy Mimicking Acute Coronary Syndrome Presentation: What is the Important Clue? Byant1 Awal Bros Panam Hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Challenging STEMI with Ventricular Tachicardia Case in ER : A Case Report R.P. Putra1, S.D. Utami1, N.A. Byant1 Awal Bros Panam Hospital1 R.P. Putra1, S.D. Utami1, N.A. Byant1 Awal Bros Panam Hospital1 Background: of the ACS cases ended in Cardiac Arrest. The presence of myocardial ischemia due to reduced coronary blood flow will cause the myocardium to become necrotic (myocardial infarction). In addition to myocardial infarction, ischemia can also cause myocardial contractility disorders. The occurrence of myocardial infarction will cause disturbances in the conductivity of the heart's electricity which often triggers the occurrence of malignant arrhythmias. However, not all types of ACS can trigger malignant arrhythmias, depending on the location of the infarction. Case Illustrations: 53-Year-Old patient presented into ER with left chest pain since 7 hours before admission. 30 minutes before admission, he complained of palpitations. He had uncontrolled hyperthension and a smoker. On physical examination, we obtained BP 80/60 mmHg, HR 242 bpm, RR 20 x/min, SpO2 97% room air with normal in other general physical examination. 12-lead ECG showed inferior STEMI with Ventricular Tachicardi, echocardiography showed EF 53% concentric LVH, Coronary Arterial Disease anterior, good valve. He also had renal insufficiency and hyperglikemia in type 2 DM is newly reocgnized. In ER, he got heart loading, dobutamine drip and also amiodarone drip until he was transferred to the ICU. Conclusion: Myocardial Infraction can cause serious complications, one of whic is malignant arrhytm even lead to cardiac arrest. Keywords: STEMI, Ventricular Tachicardia, Malignant Arrhytmia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Ventricular Tachycardia During Balloon Atrial Septostomy in a Neonate with TGA: A Rare but Life- Threatening Complication Arrythmias in Leptospirosis, too fast or too slow; a case series T. Pramitasari, S. Fatimah, A.E. Satuti1 RSUD Kota Yogyakarta1 Background g Leptospirosis is a zoonotic disease with significant morbidity and mortality. Although infrequent, leptospirosis can affect the heart. Some cardiac manifestations are arrythmias to myocarditis, pericarditis, endocarditis and cardiogenic shock. Electrocardiographic changes are not specific and may resolve after clinical recovery. Case illustration Case-1: A 70 years-old male came to ER complaining fever, nausea and vomiting frequently for 3 days. He had low blood pressure, oliguria, conjunctival suffusion and gastrocnemius pain on examination. The laboratory test showed leukocytosis, thrombocytopenia, increased level of urea and creatinine. The electrocardiogram was atrial fibrillation normal ventricular response. Hours later, he suffered from general seizure and serial electrocardiogram showed atrial fibrillation slow ventricular response with sinus pause. He was treated accordingly with temporary pacemaker. Case-2: A 52 years-old male came to ER complaining difficulty of breathing and diarrhea. He had history of fever for 4 days. He had low blood pressure, tachycardia (the heart rate was 148 bpm) and gastrocnemius pain. The laboratory findings were thrombocytopenia, elevated urea and creatinine level. The electrocardiogram showed atrial flutter with variable conduction. Amiodarone as antiarrhythmic drug was given to control the sepsis-related tachycardia. These two patients had evidence of acute kidney injury and reactive antibody of Leptospira, were treated by antibiotics, serial hemodialysis and vasopressor for maintaining the blood pressure. As the improving leukocyte count, urea and creatinine blood test on day 7, the heart rate was getting better. Temporary pacemaker for case-1 was removed on day 9 and amiodarone for case-2 was no longer used on day 8. The patients were significantly improved and discharged by day 12 and 11. Conclusion : In conclusion, ventricular tachycardia during balloon atrial septostomy in neonates is a rare but potentially life- threatening complication that requires immediate recognition and management. This case report emphasizes the potential for ventricular tachycardia during balloon atrial septostomy in neonates with transposition of the great arteries and emphasizes the importance of prompt recognition and management of this rare but potentially life- threatening complication. With timely and appropriate management, the outcome for neonates with this complication can be favorable. Keywords: balloon atrial septostomy, transposition of the great artery, ventricular tachycardia, neonates Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Ventricular Tachycardia occur during BAS procedure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Ventricular Tachycardia occur during BAS procedure Indonesian Jo Cardiolo Indonesian J Cardiol 20 pISSN: 2830-3105 / eIS doi: 10.30701/ Arrythmias in Leptospirosis, too fast or too slow; a case series T. Pramitasari, S. Fatimah, A.E. Satuti1 RSUD Kota Yogyakarta1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion A case of an 18-year-old male, diagnosed as Infective Endocarditis with echocardiography showed vegetation on the heart valve. The patient has been given antibiotic specifically sensitive to the blood culture. Surgery is the further option of therapy, so the patient was referred to another facility. Background g Infective Endocarditis is an infection of the endocardial lining of the heart that includes heart valves, mural endocardium, and endocardial covering of the implanted material.1 Based on the modified Duke criteria, a definitive infective endocarditis requires two major criteria, or at least one major plus three minor criteria.2 Positive blood culture results and endocardial damage detected by echocardiography provide the strongest evidence for the diagnosis of infective endocarditis and are considered the major criteria. Up to 90% of patients present with fever, often associated with systemic symptoms of chills, poor appetite and weight loss. Heart murmurs are found in up to 85% of patients.3 Infective endocarditis has a mortality rate of 20-25% at six months, even when treated appropriately. Infective Endocarditis in 18-year-old Male Adolescents P.G. Fernandez1, L. Tiluata1 Prof Dr. W. Z Johannes Hospital, Kupang1 Case Illustration An 18-year-old male admitted to the emergency room at Prof. Dr. W. Z. Johannes Hospital with chief complaint of fever for more than a month, accompanied by shortness of breath. Accordingly, He was firstly treated by a pulmonologist with suspicion of pneumonia. But, heart murmurs was also found, He was than referred to cardiologist with suspicion of rheumatic heart disease. Blood culture showed the growth of Staphylococcus aureus bacteria. Echocardiography showed, moderate mitral regurgitation, tricuspid regurgitation, left ventricular dilatation and vegetation on the valve. He than got antibiotic therapy : Gentamicin, Levofloxacin, and Erythromycin, and symptomatic therapy. p Conclusion Leptospirosis can affect the heart manifesting various range of arrythmia. Physician should be aware of cardiac involvement because the symptoms are not specific due to co-existence with multi-organ dysfunction. Keywords Arrythmia, Leptospirosis Keywords: Arrythmia, Leptospirosis Ind Indones pISSN: 2 d Infective Endocarditis in 18-year-old Male Adolescents P.G. Fernandez1, L. Tiluata1 Prof Dr. W. Z Johannes Hospital, Kupang1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Infective endocarditis, vegetation, fever Figure 1. Echocardiography showed vegetation on the heart valve. Figure 1. Echocardiography showed vegetation on the heart valve. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Troubleshooting A Permanent Pacemaker Lead Dislodgement in Patients with Persistent Left Superior Vena Cava Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Troubleshooting A Permanent Pacemaker Lead Dislodgement in Patients with Persistent Left Superior Vena Cava M. Reeiner1, I M.P.S. Antara2 Faculty of Medicine Udayana University1, lectrophysiology Division of Department of Cardiology and Vascular Medicine Prof. I.G.N.G. Ngoerah Hospital Bali2 M. Reeiner1, I M.P.S. Antara2 Faculty of Medicine Udayana University1, Electrophysiology Division of Department of Cardiology and Vascular Medicine Prof. I.G.N.G. Ngoerah Hospital Bali2 M. Reeiner1, I M.P.S. Antara2 Faculty of Medicine Udayana University1, Electrophysiology Division of Department of Cardiology and Vascular Medicine Prof. I.G.N.G. Ngoerah Hospital Bali2 Background: Persistent left superior vena cava (PLSVC) is often discovered incidentally during cardiac imaging or at the time of implantation of a cardiac implantable electronic device. It can be challenging because of the sharp angle between the subclavian vein and the PLSVC and, extreme angle that exists between the CS and the tricuspid valve. This anatomy not only makes right ventricular (RV) lead implantation difficult but also predisposes to lead dislodgement. Case Illustration: A 40-years-old man came for routine control and permanent pacemaker reprogramming. The patient's initial complaint was that he almost fainted few times in the last six months. This situation was felt when the patient was on the road trip, felt like dark and almost fainted lasted for a few seconds. Complaints of chest pain, tightness, palpitations, nausea, and vomiting were denied by the patient. A permanent single chamber VVIR pacemaker was installed a year ago. Placement of the ventricular leads was difficult because PLSVC was found on venographic examination, but finally it could be done through PLSVC. The result of last reprogram was output 4.5 mA with pulse with 0.76 ms, and impedance 522 ohm with intermittent pacing rhythm. The battery voltage was 0.76 V with battery life estimation 3 years. Permanent pacemaker repositioning was carried out by trying to reposition the leads using a stylet wire, but failed, so it was decided to extract the lead and install a permanent pacemaker through the right SVC with access to the right subclavian vein. Keywords: Infective endocarditis, vegetation, fever Conclusion: Technical problems are often encountered using standard technique in patient with PLSVC, therefore alternative approaches have been developed. For this reason, a thorough evaluation of the venous system is required in PLSVC patients prior to re-intervention of the pacemaker system to reduce the risk of further complications. p Keywords: Persistent left superior vena cava, permanent pacemaker, lead dislodgement p p p g Lower extrimities bilateral post thrombotic syndrome Lower extrimities bilateral post thrombotic syndrome Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Not too old to be Closed: Transcatheter ASD Closure in Elderly Using Zero Fluoroscopy Technique, First Experience in North Sulawesi C. Kurniawan1, G. Soemara1, Subiyanto1, G.P. Lumentut1, J.A. Pangemanan1, A.L. Panda1, G.E.H. Reppi1, M.P.F. Rotty1, R. Prakoso2, R. Ariani2 RSUP Prof RD Kandou Manado1 National Cardiovascular Center Harapan Kita, Department of Cardiology and Vascular Medicine, University of Indonesia2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Not too old to be Closed: Transcatheter ASD Closure in Elderly Using Zero Fluoroscopy Technique, First Experience in North Sulawesi Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Not too old to be Closed: Transcatheter ASD Closure in Elderly Using Zero Fluoroscopy Technique, First Experience in North Sulawesi Not too old to be Closed: Transcatheter ASD Closure in Elderly Using Zero Fluoroscopy Technique, First Experience in North Sulawesi C. Kurniawan1, G. Soemara1, Subiyanto1, G.P. Lumentut1, J.A. Pangemanan1, A.L. Panda1, G.E.H. Reppi1, M.P.F. Rotty1, R. Prakoso2, R. Ariani2 RSUP Prof RD Kandou Manado1 National Cardiovascular Center Harapan Kita, Department of Cardiology and Vascular Medicine, University of Indonesia2 Background: Transcatheter closure of secundum type atrial septal defect (ASD) has emerged to be a preference compare to conventional surgical methods, given its non-inferiority. Echocardiography guided Transcatheter ASD closure without fluoroscopy has become more popular worldwide. Hereby, we present a case report on the first successful ASD closure with zero fluoroscopy technique in Manado. Case Illustration: 69 years old female complains dyspnea worsening for the last 3 months. TTE (transthoracic echocardiography) showed 20 mm ASD with L-R shunt, enlarged right heart chamber with good RV function, high probability of pulmonary hypertension with normal LV function. The patient was treated for her PH (Pulmonary Hypertension) and sent to the Cath Lab for evaluation. Background Stanford Type A aortic dissection is a rapidly progressing disease process, often fatal without emergent surgical repair. A small proportion of Type A dissections go undiagnosed in the acute phase and found delayed presentation or incidentally. An increasing number of acute phase survivors and outlive initial hospitalization for many years. Late complications, driven predominantly by chronic false lumen degeneration and aneurysm formation, often require surgical, endovascular, or hybrid interventions to treat or prevent aortic rupture. Case Illustration A 54-year-old woman complained sharp chest pain penetrated her back 3 hours before admission. Physical examination showed different right and left pressure 95/56 mmHg right and 147/63mmHg left, pulse 120x/minute, SpO2 93%, and fine crackles in both lung fields. Her past medical history was long standing aortic dissection and severe aortic insufficiency since June 2018. Echocardiography showing flap dissection of the ascending aorta with moderate to severe aortic regurgitation, LVEF 64%. The patient was diagnosed with Stanford A aortic dissection from cardiac MSCT. She refused for surgical procedure and continued medical therapy. MSCT evaluation in July 2022 showed Stanford A Debackey I aortic dissection without contrast extravasation, dissection of the right common carotid artery and left subclavian artery, thin thrombus in the false lumen of the ascending aorta. The patient was given intravenous diuretics to reduce congestion, and optimize medical therapy due to refusal of surgical therapy. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 I.P. Farissa1, A.Z. Abidin1, N. Anggriyani1, M.A. Sobirin1 logy and Vascular Medicine, Faculty of Medicine Diponegoro University, Semarang1 I.P. Farissa1, A.Z. Abidin1, N. Anggriyani1, M.A. Sobirin1 I.P. Farissa1, A.Z. Abidin1, N. Anggriyani1, M.A. Sobirin1 tment of Cadiology and Vascular Medicine, Faculty of Medicine Diponegoro University, Semarang1 I.P. Farissa , A.Z. Abidin , N. Anggriyani , M.A. Sobirin Department of Cadiology and Vascular Medicine, Faculty of Medicine Diponegoro University, Semarang1 gg y Department of Cadiology and Vascular Medicine, Faculty of Medicine Diponegoro University, S Keywords: Infective endocarditis, vegetation, fever A diagnostic right heart catheterization revealed pulmonary and systemic blood flow ratio (Qp:Qs) was 1.66 and PVR 3.9 woods unit, thus ASD closure is safe to be performed. Pre-procedural TEE (Trans Esophageal Echocardiography) revealed secundum 14-16 mm secundum ASD with L-R shunt, adequate rims, and mild tricuspid regurgitation. Transcatheter ASD closure with zero fluoroscopy technique was planned. The procedure started with insertion of 5F MP catheter through 5F sheath in the right femoral vein, directed into LUPV (left upper pulmonary vein) under TEE guidance. Cera ASD occluder was engaged to 10 F delivery sheath to be delivered into LA (left atrium) after crossing the RA (Right atrium). Then, LA disc was deployed first followed by RA disc later. Wiggle test was done to confirm the device rest in stable position. Post-procedural TEE documented the device was successfully placed without any residual shunts or complications. Evaluation within 12 weeks reveal successful closure of interatrial communication, device in situ, preserved LV function, and improvement of patient’s symptoms. onclusion: Transcatheter closure of secundum ASD with zero fluoroscopy in elderly is feasible, safe, and mproving symptoms. Conclusion: Transcatheter closure of secundum ASD with zero fluoroscopy in elderly is feasible, safe, and improving symptoms. Keywords: transcatheter ASD closure, zero fluoroscopy p g y p Keywords: transcatheter ASD closure, zero fluoroscopy Keywords: transcatheter ASD closure, zero fluoroscopy y y Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Refusing Surgery for Chronic Type A Aortic Dissection: A Bold Choice or a Gamble with Life? g Conclusion Type A aortic dissection patients who are undiagnosed in the acute phase may become Chronic Type A Aortic Dissections (CTAD), that prone to additional dissection, enlargement, aneurysmal dilatation, rupture, and death. In patients who refuse surgery or non-surgical interventions, it is a medical challenge to prevent re- hospitalization, improve quality of life and reduce risk of death. Keywords: Aortic Dissection Heart Failure Aortic Regurgitation ts who refuse surgery or non-surgical interventions, it is a medical challenge to prevent re- zation, improve quality of life and reduce risk of death. ds: Aortic Dissection, Heart Failure, Aortic Regurgitation MSCT MSCT MSCT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Reverse Spider View for hidden occlusion in ostial Left Anterior Descending Artery : Case Series with a comprehensive review Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Report : Alchoholic Cardiomyopathy with LV Thrombus in Young Adult Patient F. Ladediska1, V. Rosady1, M. Syafri2 1Cardiology Resident, 2Cardiologist dr.M Djamil Hospital F. Ladediska1, V. Rosady1, M. Syafri2 1Cardiology Resident, 2Cardiologist dr.M Djamil Hospital Reverse Spider View for hidden occlusion in ostial Left Anterior Descending Artery : Case Series with a comprehensive review Syafri2 1Cardiology Resident, 2Cardiologist dr.M Djamil Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Reverse Spider View for hidden occlusion in ostial Left Anterior Descending Artery : Case Series with a comprehensive review D.A.K. Purba1, A. Sitepu2 Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumat Indonesia2 D.A.K. Purba1, A. Sitepu2 Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia2 Background: Bifurcation of the Left Main Coronary Artery (LMCA) is a complex area where occlusion in the vessels may not be visible on a single projection, including Spider View, due to the foreshortening effect. The Reverse-Spider view, oriented with Right Anterior Oblique (RAO) 90o- 120o and Hyper Cranial Angulation (CR) 30o- 40o, is a rarely used angiographic angle that has the potential to unravel occlusions. Case Illustration: We present five cases diagnosed as Anterior ST Elevation Myocardial Infarction (STEMI). Routine ECG and laboratory examinations, including cardiac enzymes, were carried out with CK-MB level 104±77,002 U/l, and the medial Troponin-T level was 1280 (40 - >2000) ng/L. All patients planned for Percutaneous Coronary Intervention (PCI). In angiography, we compare Reverse Spider View to Spider View and other routine projections to evaluate LMCA and its bifurcation. The reverse-spider view could diagnose sub- occlusive lesions and total occlusion in ostial Left Anterior Descendent (LAD) that were not previously seen or obscured in Spider View. After PCI, an echocardiographic examination was performed with an average Ejection Fraction of 46,60± 10,52%. All patients were discharged from the hospital in good condition. Conclusion: Reverse Spider View is good at detecting a hidden occlusion in the bifurcation area of LMCA, providing a clearer view of the ostial and proximal parts of the Left Anterior Descendent (LAD), Left Circumflex (LCx), and Intermediate Branch. The reverse-spider view is also recommended when the Spider view or other projection results seem dubious to diagnose an occlusion. Keywords: Reverse Spider View, Ostial Left Anterior Descendent, Coronary Artery Disease, Percutaneous Coronary Intervention, Angiography Figure 1. Comparison of Left Stem Coronary Angiography Evaluation in 5 patients; LM = Left Main Artery; LAD= Left Anterior Descending Artery; LCx= Left Circumflex Artery; IB= Intermediate Branch Figure 1. Comparison of Left Stem Coronary Angiography Evaluation in 5 patients; LM = Left Main Artery; LAD= Left Anterior Descending Artery; LCx= Left Circumflex Artery; IB= Intermediate Branch Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Case Report : Alchoholic Cardiomyopathy with LV Thrombus in Young Adult Patient F. Ladediska1, V. Rosady1, M. g Case Illustration 45 years old male, suffered from heavy like sensation chest pain and syncope. He had risk factor of hypertension and was an active smoker. 10 months ago, he has sluggish accompanied with weakness of the left limb but improved by itself. From physical examination the BP 160/92mmHg with HR 90bpm and ictus cordis was displaced without any murmur or gallop. ECG examination showed ST elevation at V1-V4, biphasic T in leads V2-V5, Inverted T at I, AVL, and QS pattern at III, AVF, V1-V4. The level of cardiac troponin rose from 0.6 to 2.2. With a crusade score of 40, he received loading DAPT and LMWH injections, underwent PPCI with result Acute Total Occlusion at mid LAD then implanted 1 DES Cre8 3.0x16mm. The patient experienced a general motor onset tonic-clonic seizure on the second day of treatment. A CT scan revealed a subacute CVA with hemorrhagic transformation and an NIHSS score of 5. The patient's DAPT and anticoagulant were stopped. For patients who underwent PCI, some studies advise restarting DAPT within one month of the ICH to reduce the risk of stent thrombosis. Background g In patients with STEMI who underwent percutaneous coronary intervention, DAPT and an anticoagulant have been recommended. However, antithrombotic therapy was also associated with an increased risk of hemorrhagic stroke or intracranial hemorrhage. Although it only occurs in 0.3% of patients receiving DAPT, intracranial hemorrhage has serious consequences. A RARE AND CHALLENGING CASE TO CONTROL: HEMORRHAGIC TRANSFORMATION STROKE DURING THE ACUTE PHASE OF MYOCARDIAL INFARCTION: A Case Report Y. Afifah1, A.Y. Suprapta1, A.F. Rahimah1, C.T. Tjahjono1 Rumah Sakit Saiful Anwar, Malang1 Keywords: Acute myocardial infarction, CVA hemorrhage, Anticoagulant, Antiplatelet, DAPT Background g Alcoholic cardiomyopathy (ACM) is a cardiac disease caused by chronic alcohol consumption. The major risk factor for developing ACM is chronic alcohol use. ACM is characterized by increased left ventricular mass, dilatation of the left ventricle, and heart failure (both systolic and diastolic). Left ventricular thrombus (LVT) is a serious complication of acute myocardial infarction (MI) and also of non-ischemic cardiomyopathies. Case illustration A 22 year old patient came to the Emergency Departement of dr. M Djamil hospital with worsening shortness of breath since 1 day ago. The patient smokes and drinks alcohol since 10 years ago. Physical examination found JVP: 5+2cmH2O, cor: cardiomegaly was found on palpation, pansystolic murmur grade 3/6 at RIC III LLSB, extremity warm and no oedema. Laboratory found normal liver function, moderate hypokalemia (2.9), hyperbilirubinemia. CXR showed cardiomegaly. The ECG showed sinus rhythm with LVH. Echocardiography revealed LV EF 27%, eccentric LVH, hypertrophi with restrictive disorders LV diastolic dysfunction, mild MR, moderate TR, intermediate probability PH, sec(+) in LV, multiple thrombus (+) apical. Patient was diagnosed with ADHF w/w on CHF ec susp alcoholic cardiomyopathy, Multiple LV thrombus, TR moderate high probe PH, Mild hypokalemia, Susp Community Acquired Pneumonia. Patients were given furosemide 40 mg iv followed by drip furosemide starting at 3 mg/hour, correction of KCL 35 meq and KSR 3x600 mg, Uperio 2x50 mg, spironolactone 1x25 mg, warfarin 1x4mg, Lansoprazole 1x30mg IV, laxantia 1x10mg and consulted to the lung department. The patient was discharged after being hospitalized for 6 days. Conclusion Anamnesis, physical and supporting examinations, especially TTE, are needed to establish the diagnosis of ACM. LVT is one of the serious complications of ACM to be aware of. Keyword y Alcoholic cardiomyopathy, thrombus, young adult Alcoholic cardiomyopathy, thrombus, young adult Keywords: Alchoholic cardiomyopathy, Thrombus, young adult Keywords: Alchoholic cardiomyopathy, Thrombus, young adult Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 P.N. Permatasari1, I. Handirosiyanto2 y General Practitioner, Baptis Batu Hospital, Malang, Indonesia1 Cardiologist, Baptis Batu Hospital, Malang, Indonesia;Karsa Husada Hospital, Malang, Indonesia2 Background: STEMI is life-threatening. The longer ischemic time increases morbidity and mortality in patients. It’s time-critical to recognize STEMI and decide to manage the patient with pharmacological or mechanical reperfusion. From the onset of ECG patients with ischaemic symptoms interpreted as STEMI, PCI must be done within 120 minutes, if longer, immediate fibrinolysis and transfer to PCI Centre if indicated. Background: STEMI is life-threatening. The longer ischemic time increases morbidity and mortality in patients. It’s time-critical to recognize STEMI and decide to manage the patient with pharmacological or mechanical reperfusion. From the onset of ECG patients with ischaemic symptoms interpreted as STEMI, PCI must be done within 120 minutes, if longer, immediate fibrinolysis and transfer to PCI Centre if indicated. Case Illustration: We describe 2 cases of typical chest patients with ST-segment elevation on ECG. Case 1: A 44-year-old man was referred from Primary Healthcare Centre with typical chest pain, nausea, and vomitus within 1 hour, ECG-12 leads showed STMI Inferior and got DAPT before being referred to ER. He had uncontrolled Hypertension. In ER, BP: 159/96 mmHg, HR: 83 times/minute without congestion or pulmonary edema. ECG showed no elevation ST-Segment. Thrombocyte aggregation was autolyzed by DAPT. Case 2: Women 53 years old came to ER with typical chest pain within 1 hour She had controlled HTN BP: Case Illustration: We describe 2 cases of typical chest patients with ST-segment elevation on ECG. Case 1: A 44-year-old man was referred from Primary Healthcare Centre with typical chest pain, nausea, and vomitus within 1 hour, ECG-12 leads showed STMI Inferior and got DAPT before being referred to ER. He had uncontrolled Hypertension. In ER, BP: 159/96 mmHg, HR: 83 times/minute without congestion or pulmonary edema. ECG showed no elevation ST-Segment. Thrombocyte aggregation was autolyzed by DAPT. Case 2: Women, 53 years old, came to ER with typical chest pain within 1 hour. She had controlled HTN. BP: 110/80 mmHg, HR: 60 times/minute. ECG showed STEMI inferior. The patient got DAPT and a fibrinolytic agent, then transferred to ICU. After 2 hours, ECG showed successful fibrinolytic. Conclusion: Time is muscle. Early reperfusion by pharmacological therapy is needed if access to PCI Centre is longer than 120 minutes from the first diagnosis of STEMI. Keywords: autolysis, DAPT, reperfusion, STEMI, fibrinolytic Important Roles of Early Loading DAPT and Fibrinolytic on STEMI Patients in Non-Capable PCI Hospital: Case Series Important Roles of Early Loading DAPT and Fibrinolytic on STEMI Patients in Non-Capable PC Hospital: Case Series P.N. Permatasari1, I. Handirosiyanto2 Conclusion One of the uncommon side effects for patients who received DAPT is hemorrhagic stroke. For some high-risk patient subsets, such as those who have had a previous stroke or TIA, the risk of ICH associated with DAPT must be carefully considered. Physicians may use risk prediction scores to outline this trade-off, which can improve the process of making decisions together with patients who are at high risk for bleeding. Keywords: Acute myocardial infarction, CVA hemorrhage, Anticoagulant, Antiplatelet, DAPT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Important Roles of Early Loading DAPT and Fibrinolytic on STEMI Patients in Non-Capable PCI Hospital: Case Series Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Unusual Case of Critical Limb Ischemia as a Complication of Blood Culture Negative Infective Endocarditis in Children: A Rare but Serious Condition A. Alamsyaputra1, S.Y. Pradana1, I.P. Farissa1, A. Priyatno2 1Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia 2Department of Pediatric, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital Semarang, Indonesia Case illustration: Case illustration: A 10 years old boy admitted to hospital with exertional dyspnea and orthopnea. He was indolent, low- grade fever, distended abdomen, and swollen legs. Pansystolic murmur was heard at the apex and Chest X-Ray showed left atrial and left ventricle cardiomegaly. Echocardiography revealed severe mitral insufficiency with vegetation at the posterior mitral leaflet. Blood culture was shown negative, but based on Clinical Criteria on Modified Duke Criteria he graded as Definite IE and managed with empirical antibiotics for a month. Half year after antibiotics, he developed right leg claudication, reddish, and swelling. Doppler Ultrasonography showed no flow from popliteal to tibialis anterior and posterior artery, no thrombus was seen. Arteriography was performed and shown total occlusion at proximal tibialis artery with collateral vessel filled the distal part unto pedis. Embolectomy was performed and revealed occlusive, crushed artery at the distal femoral arteries. Repair the damaged arteries was done afterward. Background: Blood Culture Negative Infective Endocarditis (BCN-IE) is a type of Infective Endocarditis (IE) characterized by lack of growth of bacteria or other microorganisms in blood cultures. One of IE complication are Embolic Events (EE) which caused by migrating cardiac vegetations. The incidence of EE ranged from 20-50%, and 6% happen in peripheral artery. P.N. Permatasari1, I. Handirosiyanto2 Loading DAPT and DAPT with Fibrinolytic made an important role in each case. Conclusion: Time is muscle. Early reperfusion by pharmacological therapy is needed if access to PCI Centre is longer than 120 minutes from the first diagnosis of STEMI. Loading DAPT and DAPT with Fibrinolytic made an important role in each case. Keywords: autolysis, DAPT, reperfusion, STEMI, fibrinolytic Keywords: autolysis, DAPT, reperfusion, STEMI, fibrinolytic Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology A Simplified Technique Ductal Stenting in A Complex Duct-Dependent Pulmonary Circulation Infant: The Transfemoral Venous Shortcut A. Alamsyaputra1, I.P. Farissa1, M. Helthayana1, A.Yudha1, A. Priyatno2 1Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital, Semarang, Indonesia 2Department of Pediatric, Faculty of Medicine, Diponegoro University – Dr. Kariadi Central General Hospital Semarang, Indonesia Background: Transcatheter Patent Ductus Arteriosus (PDA) stenting currently an acceptable palliative treatment in newborns suffering from duct-dependent pulmonary circulation requiring neonatal repair or palliation with a secure source of pulmonary blood flow until more definitive surgery can be performed. This procedure remains technically a challenge in complex ductal morphology, which may eventually lead to detrimental outcomes with a failure rate of 16-20%. g Conclusion: Multiple clinical series have shown that the risk of EE decreases generally to 10-15%, within 1 week after initiation of appropriate antibiotic therapy. In BCN-IE no pathogen was isolated in blood cultures or in other specimens (embolism, valve tissue), so choosing treatment regimen can be difficult. Empiric antimicrobial therapy can be started and epidemiologic features of each case should be evaluated to assist broader coverage in defining treatment regimen. We also have to distinguish BCN-IE with Non-Bacterial Thrombotic Endocarditis caused by systemic lupus erythematosus, antiphospolipid syndrome or malignancy so we can treat the underlying cause. Keywords: Infective Endocarditis, Peripheral Arterial Disease, Embolic Event Keywords: Infective Endocarditis, Peripheral Arterial Disease, Embolic Event Keywords: Infective Endocarditis, Peripheral Arterial Disease, Embolic Event Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 A Simplified Technique Ductal Stenting in A Complex Duct-Dependent Pulmonary Circulation Infant: The Transfemoral Venous Shortcut Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case illustration: A 3 months old girl admitted to hospital with cyanosis and shortness of breath. She appeared to be under weight, rapid breathing and bluish with the saturation is 64% room air. Continuous machinery murmur was heard on the left infraclavicular area. Transthoracal Echocardiography showed PA-VSD (Fallot Type), PFO and a vertical duct PDA under the aortic arch. We performed catheterization procedure through femoral vein, MPA2 catheter successfully passed through from IVC, RA, RV to the distal of aortic arch via Ventricular Septal Defect. Aortography was performed and revealed a long tortuous vertical duct PDA Ø 11 mm under the aortic arch with stenosis at the proximal part of RPA and LPA. There was no MPA. Wire crossed through the PDA to distal of RPA using JR3.5/4F catheter and a 4.0 x 19 mm DES was successfully deployed. The peripheral saturation increased to 88%. Later Unfractionated Heparin was given for two consecutive days and switch to acetylsalicylic acid the day after. There were no complications afterward. Conclusion: Ductus arteriosus morphology and location influences technique and determines complexity, safety, duration, and final outcome of ductus arteriosus stenting.2 In PDA with very proximal origin, a stable position of the Judkin’s right catheter tip in the PDA can be accomplished by the antegrade transvenous route, across the VSD and into the ascending aorta, however, negotiating multiple angles and curves in the PDA may still be cumbersome and arduous procedure. Keywords: PDA, PDA stenting, Pulmonary Atresia, Duct-Dependent Pulmonary Circulation Keywords: PDA, PDA stenting, Pulmonary Atresia, Duct-Dependent Pulmonary Circulation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Multiple Culprit Lesion in A Patient with ST-Segment Elevation Myocardial Infarction: A Double Trouble Rare Case Report S. Andrian1, W. Erlanda1, M. Prasetya1, C.K. Krevani1, M. Syukri1, S. Phalguna1 Universitas Andalas, Padang/RSUP Dr. M. Djamil Padang1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Brugada syndrome (BrS) is a rare autosomal-dominant inherited arrhythmic disorder characterized by coved- type ST-segment elevation with negative T wave in the right precordial leads ECG pattern without structural heart disease. Patients with Brugada syndrome are at risk for sudden cardiac death (SCD) due to ventricular arrhythmia with predominantly occur in men (5.5-fold risk) compared with women. The mean age of patients with ventricular arrhythmia episodes is 26-56 years old. Arrhythmic events observed at rest or while asleep with most frequently occur from 12 am to 6 am, and the least frequent occur during the daytime. It can be asymptomatic for life or present with syncope (range from 17 to 42%) in some patients. Case Illustration We present a 65 years-old woman with typical chest pain at rest in early morning (03.00 am) with ST-segment elevation in the right precordial leads (V1-V3) Brugada pattern. Patient with risk factor of uncontrolled diabetes mellitus type II, from laboratory examination we found a hyperglycemic state with FBG 227, RBG 302, and HbA1C 7,7. History of syncope or cardiac disease was denied. Serial ECG and cardiac enzyme examination was no resolution in ST-segment and no alternation in cardiac enzyme. From DCA examination was found a stenosis 20-30% at proximal RCA. Calculation on Shanghai Score System was 3.5 score, with conclusion possible Brugada Syndrome. Because there was no clinical criteria in this patient, we conclude the patient with asymptomatic Brugada pattern type 1. Patient was being closely monitoring and advised to do EP study to stratify the possibility of ventricular arrhythmia events in the future. Brugada in Elderly, What Should We Do? Brugada in Elderly, What Should We Do? I.V. Nugroho1, A.F. Rahimah2 Fakultas Kedokteran Universitas Airlangga1 RS Saiful Anwar2 Indonesian Journal of Cardiology S. Andrian1, W. Erlanda1, M. Prasetya1, C.K. Krevani1, M. Syukri1, S. Phalguna1 Universitas Andalas, Padang/RSUP Dr. M. Djamil Padang1 Background: The reported incidence of multiple thrombotic coronary occlusions reached 50% in an autopsy series of patients who died from sudden cardiac death and occur in about 4,8 % of the cases, suggesting that the incidence is low in clinical practice because most of the patients die before medical contact. Although for the most part, this occlusion involves a single coronary artery, simultaneous thrombotic occlusions in multiple coronary arteries in the setting of STEMI are rare, and poorly understood. g p y Case Illustration: A 57 years old man came to the emergency room of Dr. M Djamil Hospital with typical chest pain 11 hours before admission. The pain scale was 6/10. The patient also complained of fainting for less than 1 minute while in chest pain, then soon recover. The patient was compos mentis with BP 87/60 mmHg, HR 36 x/min regularly, SO2 98 % on room air. The ECG revealed TAVB with ST elevation at lead II, III, aVF, V1-V4, V3R-V4R, and V7-V9. Troponin-I was increased (14.499 ng/mL). The patient performed TPM and continued to primary PCI. The angiography result was CAD 2VD with total occlusion at proximal LAD and proximal RCA and normal LM, LCX. The patient continued with PCI then stented DES 3,0x46 mm at osteal-mid RCA, POBA at osteal-distal LAD with residual stenosis at proximal-distal LAD, TIMI flow 3 MBG 3. After being hospitalized for 6 days, the patient was discharged in stable condition. y p g Conclusion: Simultaneous thrombosis of the coronary artery in the course of AMI is an uncommon entity. A step- by-step approach in decision making about device utilization and pharmacotherapy, in adherence with the most recent and widely accepted guidelines, alongside a flexible interventional strategy, is crucial in effectively managing such complex and unusual cases. Keywords: Multiple thrombotic coronary occlusion, PCI Coronay Angiography Coronay Angiography Coronay Angiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Cor Triatriatum Dextra (CTD) is a rare congenital cardiac anomaly which can be found in adults and should be considered as a differential diagnosis when patients present with atrial tachycardia and heart failure. Although it is rare, there has been an increase in diagnosis due to developments in diagnostic imaging techniques. Echocardiography has an important place in the diagnosis of congenital pathologies in this rare group. Case Illustration A 25-year-old female patient was admitted to our hospital with the complaint shortness of breath was accompanied by palpitation. On physical examination, There were rales in the lung basals by auscultation. There was an apical 2/6 systolic murmur on cardiac auscultation. Blood pressure was 128/76 mmHg, respiratory rate was 32/ min and pulse was 180/ min. 12-lead ECG revealed atrial tachycardia with intermitten WPW. Ejection fraction was normal in transthoracic echocardiography. Left atrium and right atrium were large. Transthoracic echocardiography revealed moderate mitral regurgitation, mild tricuspid regurgitation. In addition, a fibromuscular membrane dividing the right atrium in two, compatible with the CTD was observed in the right atrium. Cardiac magnetic resonance imaging and multiclice computed tomography were recommended to determine additional congenital anomalies and to determine the need for surgery. Surgical treatment was recommended for the treatment of CTD because of symptomatic valvular diseases and the need for additional cardiac surgery. Cor triatriatum dextra: A rare disease with a common presentation T.Y. Nugraha1, A. Rizal1, C.T. Tjahjono1 saiful anwar hospital1 T.Y. Nugraha1, A. Rizal1, C.T. Tjahjono1 saiful anwar hospital1 Conclusion Patient with asymptomatic Brugada pattern type 1 could be advised to do close follow up and do EP study to stratify the possibility of ventricular arrhythmia events in the future without any medication therapy. Keywords: Keywords: Indonesian Jo Cardiol Indonesian J Cardiol 2 pISSN: 2830-3105 / eI doi: 10.30701/ Cor triatriatum dextra: A rare disease with a common presentation T.Y. Nugraha1, A. Rizal1, C.T. Tjahjono1 saiful anwar hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusions: RHD and severe MR patient who refuse to do surgery need a strategy for optimal medical therapy. Keywords: severe MR, RHD, optimal medical therapy Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Although CTD is a very rare congenital anomaly in the society, the development of diagnostic methods has increased the diagnosis of this anomaly. Symptoms and signs of right heart failure and supraventricular arrhythmias are common in this anomaly. The present case demonstrates the use of different modalities to evaluate CTD; each modality has its own limitation. In most cases, more than one diagnostic imaging modality is necessary to come to a definitive conclusion. Keywords: Keyword : Cor Triatriatum Dextra, Atrial Tachycardia Figure A. ECG at dr. Saiful Anwar Hospital showed atrial tachycardia with intermitten WPW, Figure B and C TTE Showed a fibromuscular membrane dividing the right atrium Keywords: Keyword : Cor Triatriatum Dextra, Atrial Tachycardia Keyword : Cor Triatriatum Dextra, Atrial Tachycardia Figure A. ECG at dr. Saiful Anwar Hospital showed atrial tachycardia with intermitten WPW, Figure B and C TTE Showed a fibromuscular membrane dividing the right atrium Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:su pISSN: 2830-3105 / eISSN: 2964 doi: 10.30701/ijc.1537 Severe Mitral Regurgitation (MR) due to Chronic Rheumatic Heart Disease (RHD) with Increase in Pulmonary Arterial Pressure : How to treat conservatively? J.E. Yulianto1, A.K. Syammarhan1 1 Severe Mitral Regurgitation (MR) due to Chronic Rheumatic Heart Disease (RHD) with Increase in Pulmonary Arterial Pressure : How to treat conservatively? J.E. Yulianto1, A.K. Syammarhan1 RSUD Inche Abdoel Moeis Samarinda1 Severe Mitral Regurgitation (MR) due to Chronic Rheumatic Heart Disease (RHD) with Increase in Pulmonary Arterial Pressure : How to treat conservatively? J E Yulianto1, A.K. Syammarhan1 J.E. Yulianto1, A.K. Syammarhan1 RSUD Inche Abdoel Moeis Samarinda1 Background : RHD is a sequel of ARF resulting from an abnormal immune response to GAS infection that triggered valvular damaged. MR is the most common valvular abnormality at the early RHD stages, associated with ongoing inflammatory rheumatic activity and may resolve with effective treatment of the acute carditis and continued prophylactic therapy. In the late time course, MR is often associated with stenosis owing to intrinsic valvular lesions like fibrosis with retracted leaflets, restricted mobility and commissural fusion. According to ESC guideline, valve surgery is indicated in patient with persisting severe MR and symptomps. But the most problem in Indonesia, especially in Samarinda, patients often refused to do surgery, so the Cardiologist should do improvisation for an optimal medical therapy without surgery. p p py g y Case Illustration: Mrs.E, 34th years old,come to the ER with her chief complain is shortness of breath, PND, Orthopneu and swelling in abdomen. She was diagnosed with VHD during her first pregnancy in 12 years ago. Since childhood, she often experienced fever, sorethroat, with joint swelling. Physical examination show normotensive, takipneu, irregular pulse, raised JVP, rheels in bilateral lungs, holosystolic murmur in apex, and ascites. Laboratory findings normal limit, except increase of ASTO titer (800 IU/mL). ECG shows AF RVR and RVH. Chest X-ray shows RAH, RVH, with pulmonary oedema. Echo results EF 49% with all chambers dilatation, severe MR (MR ERO 1,3 cm2) with SEC in LA, and severe TR. MV characteristic fit to definite RHD: MR seen in all view,jet length≥2 cm, velocity 4,20 m/s, chordal thickening and restrictive leaflet motion. She refused to do surgery, so we treat conservatively with triple diuretic, ACEi, BB, Anticoagulant, and GAS Eradication with erytromicin. Conclusions: RHD and severe MR patient who refuse to do surgery need a strategy for optimal medical therapy. Keywords: severe MR, RHD, optimal medical therapy Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background: ST Elevation Myocardial Infarction still become an emergency condition and need perform primary PCI in PCI Centre. However, we also see patients who present with significant ST-elevations on the initial electrocardiogram but show complete spontaneous resolution of ST-elevations and clinical symptoms. This clinical condition is referred to as ‘transient STEMI’ and is reported to occur in 6% of all patients presenting with acute coronary syndrome. Early invasive strategy was a procedure that perform for this case. We describe a case of a Transient STEMI which perform early invasive strategy. STEMI WITH COMPLETE SPONTANEOUS RESOLUTION OF ST-ELEVATIONS AND CLINICAL SYMPTOMS: FRIEND OR FOE V. Mayangsari1, N. Kurnianingsih1, C. Kahadi1 Brawijaya University - Saiful Anwar General Hospital1 Case Illustration: A 48-year-old Asian male come to ER dr. Saiful Anwar General Hospital with typical chest pain, with VAS 9/10, and 6 hour onset. He was an active smoker more than 20 years old. He in hemodynamic without support, with BP 100/60, HR 71bpm, RR 18tpm, SpO2 97%. At First Medical Contact (FMC) onset 1,5 hours, we found ST Elevation in Lead II (3mm), III (2.5mm), aVF (4mm); ST depression in lead V1-V4, aVR, I, aVL (reciprocal). He got Loading ASA 320mg from FMC, then at 3 hours onset his chest pain was release with VAS 2-3/10 then perform ECG again, that ST Elevation got resolution without ECG evolution. From laboratory, Troponin I level 1,9 (onset 6h), 4,6 (onset 10h) then 1,6mcg/L (onset 16h). He performs an early invasive strategy and did not any significant lesion with TIMI Flow 3 in all Coronary Artery, unlike other cases with significance lesion. He got heparinization, then after four days of care his chest pain was relieved and discharged in good condition. Conclusion: BRASH SYNDROME IN SECUNDUM ATRIAL SEPTAL DEFECT: LOST IN PLAIN SIGHT S. Manurung1, A.R. Pratama1 Sultan Suriansyah General Hospital1 Case Illustration : A 48-year-old female with chronic kidney disease stage 5, essential hypertension, chronic heart failure, and receiving bisoprolol presented to the Emergency Department with lethargy and weakness that started 3 days ago, with rapid deterioration into shock with bradycardia. She denied any family history of congenital heart disease. Laboratory findingshyperkalemia (6,33 mmol/L), and acute kidney injury (urea 86.8 mg/dL, creatinine 7.19 mg/dL, CrCl 8) was presented. The initial electrocardiogram was remarkable for junctional rhythm with heart rate of 39 bpm. Chest x-ray showed cardiac enlargement with features of pulmonary edema. The echocardiogram revealed large sized secundum Atrial Septal Defect (ASD) with a left to right (L to R) shunt and intermediate probability pulmonary hypertension. The right atrium, right ventricle were dilated suggestive of a significant L to R shunt. Her biventricular contractility was noted to be normal with ejection fraction 77%. Bisoprolol was immediately held. Potassium-lowering agents and diuretic were begun, as were dopamine for vasopressor support. sildenafil was given to treat pulmonary hypertension. Hyperkalemia can be overcome. Renal function improved and heart rate stabilized at 80 bpm. The patient was discharged and advised to avoid atrioventricular-blocking agents, with Cardiology follow-up. p Conclusion: ST-elevations with complete spontaneous resolution of ST-elevations and clinical symptoms is still be happen in patient with acute coronary syndrome. Early invasive strategy should be performed, although its significance or not significance lesion in coronary artery for definite treatment. Keywords: Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 BRASH SYNDROME IN SECUNDUM ATRIAL SEPTAL DEFECT: LOST IN PLAIN SIGHT Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background : Bradycardia, Renal failure, AV-Nodal blockers, Shock, Hyperkalemia Syndrome (BRASH syndrome) is fundamentally a synergistic process created by a combination of hyperkalemia and renal failure synergizes with AV-nodal blockers, consequently between the clinical condition of patients with the degree of hyperkalemia and bradycardia are inappropriate. Therefore, rapid diagnosis and management of this entity are crucial to reduce mortality. We report a case and the management of BRASH syndrome in the Emergency Department. Case Illustration : Case Illustration A 67 years old male presented with chest pain since 9 hours in emergency room (ER). Chest pain accompanied by diaphoresis, duration more than 30 minutes, not improved by rest. There was no shortness of breath and palpitation. Patient was an active smoker as a risk factor of coronary artery disease (CAD). ECG showed sinus rhythm with rate 94 beat per minute, Q pathologic at V1-V4 lead, ST elevation 1-4 mm at lead I, aVL, V1-V6, ST depression at lead II, III, AVF. Chest x-ray showed no pathologic finding. Blood laboratory showed troponin I 13.310 ng/L. Patient underwent primary percutaneous coronary intervention (PPCI) got implanted 1 stent at proximal left anterior descendant (LAD) on CAD 1 vessel disease (VD) TIMI flow 2 MBG 2. The patient got therapies ASA 1x100 mg, ticagrelor 2x90 mg, enoxaparin 1x0,4 cc SC for 3 days, atorvastatin 1x40 mg, and ramipril 1x2,5 mg. The patient had progressive massive pericardial effusion. The patient observed for 14 days and not underwent pericardiosentesis because not showing tamponade sign. The patient discharged with stable condition with therapies ASA 3x800 mg for one month tapering off, clopidogrel 1x75 mg, colchicine 1x0,5 mg, atorvastatin 1x40 mg, ramipril 1x2,5 mg, and lansoprazole 1x30 mg. Background g Acute pericarditis post myocardial infarction (MI) is one of post cardiac-injury syndrome (PCIS) type. Pericarditis post myocardial infarction divided into 2 : early onset and late onset. MI didn’t get successful reperfusion increased risk of post MI complication. Acute pericarditis post MI is not dangerous, however can be life threatening if accompanied with massive pericardial effusion that can develop into cardiac tamponade. Post Myocardial Infarction Acute Pericarditis I. Yaqzhan1, M. Syafri1 Universitas Andalas1 g Conclusion : BRASH syndrome guarantees increased awareness among clinicians because early recognition of this syndrome can initiate the proper management and recovery of the patient. BRASH syndrome guarantees increased awareness among clinicians because early recognition of this syndrome can initiate the proper management and recovery of the patient. Keywords: BRASH, ASD, hyperkalemia, bradycardia Keywords: BRASH, ASD, hyperkalemia, bradycardia Keywords: BRASH, ASD, hyperkalemia, bradycardia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Most of the symptoms related to acute heart failure (HF) are caused by congestion, and loop diuretics represent the mainstay of its treatment. Furosemide as a potent loop diuretic is one of the main therapies given to heart failure patients with congestion. The diuretic effect of furosemide can cause fluid and electrolyte depletion in the body, influenced by the dose and preparation given. Furosemide has been rarely reported as the cause of thrombocytopenia. Furosemide-Induced Thrombocytopenia in Heart Failure: A Case Report S.E. Laelaem1, I. Haryana2, S. Tiro3 Puskesmas Masohi1 Universitas Pattimura2 Siloam Hospitals Ambon3 Conclusion This case highlights complication that can happen in STEMI patient, moreover in failed revascularization. Acute pericarditis post MI can be life-threatening condition especially if accompany with massive pericardial effusion. Watchful monitoring is essential in these patients. Keywords: Myocardial infarction, acute pericarditis, pericardial effusion Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration In this report, we present a case of a 55-year-old female with congestive heart failure, who had been treated with Furosemide which was identified as the probable cause of her drug-induced thrombocytopenia. She had been receiving oral furosemide for her heart failure. She was brought to the Emergency Department (ED) caused of shortness of breath. In ED she received intravenous (IV) furosemide 20mg/day. The patient's labs revealed for leukocytosis, thrombocytopenia, high serum creatinine, hyponatremia and hypochlorhydria. At first, her platelet counts were 51x103/mm3, when the dose of IV furosemide increased to 120mg/day her platelet counts dropped to 33x103/mm3. After a dose reduction back to 60mg/day the patient's platelet count 38x103/mm. A peripheral blood smear found platelet count below the lower limit of normal and morphology was normal so its analyze as suspect drug-induced thrombocytopenia. Background : It is recognized that acute myocardial infarction (MI) can occur not attributable to atherothrombosis. The fourth universal definition of MI defined type 2 MI as an ischaemic MI in the context of mismatch oxygen supply and demand.1 Case illustration : A 59-year old female came to the emergency room with the aggravating dyspnea since 4 days, fever and phlegm cough before admission, no chest discomfort felt. The respiratory rate was 27 times per minute with 94% room air saturation. Rhonchi were heard but no others significant findings. Electrocardiogram (ECG) showed no remarkable results. The patient was diagnosed with pneumonia, chronic obstruction pulmonary disease (COPD) and chronic cor pulmonale. The saturation dropped below 80% on day 3, the dyspnea have worsened, also chest discomfort felt. Marked ST segment depression in lead V3-V6 and II, III, aVF have seen on repeated ECG with elevation of Troponin T (140 ng/L). We concluded the patient had type 2 MI as it met the criteria of cardiac biomarkers elevation and evidence of myocardial ischaemia2, also the patient not presumably having a history coronary artery disease (CAD) and there was evidence of oxygen supply deficit. It was also in coherent with the patient’s sex, old-age and COPD comorbid.2,3 No specific therapy was given, the treatments were for the underlying diagnosis resulting in type 2 MI. The key points are pretest probability of type 1 MI and risks of giving or witholding the type 1 MI treatments.4 On the day 4, the complaints were subsided and the oxygen saturation went back to 92-93% after antibiotics therapy. We repeated the ECG and it showed normalization. M. Septian1, T. Nandhika2, A.F. Prianda1, A.R. Ruhukail1 RS Hasna Medika Kuningan1 RSUD 45 Kuningan2 M. Septian1, T. Nandhika2, A.F. Prianda1, A.R. Ruhukail1 RS Hasna Medika Kuningan1 RSUD 45 Kuningan2 Conclusion Based on this case report, clinicians should consider furosemide as one of the drugs potentially causing thrombocytopenia. y p Keywords: Furosemide, heart failure, drug-induced, thrombocytopenia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Challenging Management of Sinus Valsava Rupture and Aortic Regurgitation as a Complication of Doubly Committed Subaortic Ventricular Septal Defect : A Case Report. W. Mubarok1, M.H. Trisnawan1, S. Sungkar2 g 1 Faculty of Medicine, Diponegoro University, Semarang, Indonesia 2 Departement Cardiology and Vascular Medicine, Dr. Kariadi General Hospital Semarang, Indonesia ackground Doubly committed subarterial ventricular septal defects account for 10% of ventricular septal defects. The management of aortic regurgitation and rupture of sinus valsava in the setting of ventricular septal defect has always been challenging. Case Illustration A 22 years old man was referred to our hospital because of shortness of breath, palpitation and deterioration in exercise capacity. The complains worsen in last 6 month. Cardiac ecamination found a thrill and loud continous murmur along the left sternal border. The ECG represent sinus rythm 89 bpm with multifocal PVC. Distended jugular venous pressure, ascites and edema at lower extremity were present. Chest x-ray showed pancardiomegaly and the echocardiography were found a doubly committed subaortic ventricular septal defect with the severe aortic regurgitation and rupture of sinus valsava. The management of this patient including diuretic, ACE-i, digoxin and drainage of ascites. For further management, the patient underwent surgical correction with ventricular septal defect closure and aortic valve replacement. Conclusion : Conclusion : We provide the case of MI that result from non-atherothrombotic cause, thorough investigations and therapeutics approaches are needed for better outcome. We provide the case of MI that result from non-atherothrombotic cause, thorough investigations and therapeutics approaches are needed for better outcome. Keywords: myocardial infarction, oxygen supply and demand mismatch Fig. (A) Electrocardiogram (ECG) of first patient came to the ER. (B) Repeated electrocardiogram (ECG) with depression of ST segment in lead V3-V6 and II, III, aVF Keywords: myocardial infarction, oxygen supply and demand mismatch Fig. (A) Electrocardiogram (ECG) of first patient came to the ER. (B) Repeated electrocardiogram (ECG) with depression of ST segment in lead V3-V6 and II, III, aVF Background g The culprit lesion is used to designate the coronary stenosis thought to be responsible for the acute coronary syndrome. Case Illustration A 54-year-old male with typical chest pain during badminton and called for ambulance. The patient had previous history of PCI 5 years ago and had permanent afib. There is no significant finding in physical examination and hemodynamic. Patients routinely take rivaroxaban 15mg once daily, aspirin 80mg once daily. The loading dose of aspirin 160mg, clopidogrel 300mg was given by the ambulance physician and ticagrelol 90 mg at the emergency room. ECG showed atrial fibrillation normoventricular response, rate 60 bpm,LAD,borderline ST elevation II, III and avf. Coronary angiogram showed right dominant, proximal classification in RCA, ectasis from proximal to distal, 80% lesion in middle RCA, multiple mild lesions in distal+middle RPL and proximal RPDA. Normal left main coronary. 20% lesion in proximal LCX, 80% lesion in distal LCX (small calibre), 30% lesion in OM2, 80% lesion in ostial LPL, TIMI 3 distal flow and in LAD: proximal calcification, 35% mild lesion intra old stent in proximal mid LAD, 50% calcified lesion in mid distal LAD (from old stent), TIMI 3 distal flow. All coronaries flow were normal TIMI 3. In this case, IVUS is not possible because it is under maintenance. Judging from the typical chest pain & history of previous heart attack and atrial fibrillation, it was decided to stent the RCA that looked as a stable lesion that create confusion which is the culprit lesion. Conclusion Ventricular septal defects can lead to aortic regurgitation and ruptur of sinus valsava because of ventury effect. Echocardiography provided a complete evaluation of such cases and prohibited missed diagnosis of other coexistent congenital heart defects. Management of such condition include therapy of heart failure and surgical correction. Keywords: aortic regurgitation, ventricular septal defect, rupture of sinus valsava Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 SUCCESSFUL PERCUTANEOUS CLOSURE OF ATRIAL SEPTAL DEFECT USING ZERO FLUOOROSCOPY TECHNIQUE IN PREGNANT WOMAN I. Kamelia1, H. Martini2, V. Yogibuana2, A.F. Rahimah2, A. Wicaksono1, E. Fathoni1 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia Conclusion In case with clinical and typical investigations of STEMI, normally it will be easy to find the culprit lesion. This case did not find a typical culprit lesion, IVUS can play a role in providing information of the stability of suspect lesion based on borderline ST changes of the ECG Keywords: STEMI, Culprit, AFib Angiography RCA Angiography RCA BACKGROUND Atrial septal defects (ASDs) are one of the most common acyanotic congenital heart lesions with the initial clinical presentation and diagnosis or getting worsening clinical symptoms during pregnancy. If indicated, percutaneous closure can be performed safely. We report successful percutaneous closure by zero fluoroscopy technique in pregnant patient with ASD-PH. p g p CASE ILLUSTRATION A 38-year-old woman with diagnosis ASD-PH. TEE was demonstrated a large ASD measuring 28 - 32 mm with adequate rim suitable for closure by device. Result from RHC was ASD secundum with pulmonary hypertension high flow high resistance (mPAP 58mmHg and PARi 2.4 wu.m2 post oxygen test). She scheduled to performed ASD closure by device. But before closure had been done, she had got pregnant. Doctor in charge planned to perform closure as soon and safe as possible in second trimester of pregnancy. The procedure then performed under general anesthesia, using TEE guidance. Femoral venous access was achieved with a 14 F sheath inserted into the right femoral vein. The defect was crossed with a guide wire using TEE guidance. Considering the defect size and not available balloon assist device, operator decided to use modified-catheter assisted technique. The defect was then closed percutaneously using a 40 mm ADO device with no residual shunting and sufficient rim attachment on evaluation by TOE. After procedure done, there is no complication and patient came home safely. Clozapine-induced Cardiomyopathy: A Reversible Outcome With Lethal Effects Y. Newary. D.L. Munawar1 Binawaluya Hospital1 p CONCLUSION Pulmonary hypertension who may develop in patient with congenital heart diseases is one of contraindication of pregnancy. In our case, in spite of unexpected pregnant condition in patient with ASD-PH, the team in charge can give prompt treatment by perform percutaneous closure using zero fluoroscopy technique at second trimester. The timing of ASD closure is important to minimize the risk of radiation exposure to the developing fetus. Keywords: Atrial septal defect (ASD), percutaneous closure, Pregnancy, Zero fluoroscopy technique. Transthoracal Echocardiography showed Atrial septal defect 31mm Transthoracal Echocardiography showed Atrial septal defect 31mm Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Clozapine-induced Cardiomyopathy: A Reversible Outcome With Lethal Effects Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background Background Acute pulmonary embolism (PE) and acute coronary syndrome (ACS) are considered to be potentially life hreatening. Both have signs and symptomps that are common to either presentation such as dyspneu, chest discomfort and hemodinamic instability. Background g Drug-induced cardiomyopathy is a potentially reversible form of acquired CM and a common consequence of exposure to numerous medically prescribed drugs. It is particularly a common serious adverse side effect of anticancer, antiretroviral and antipsychotic therapies. Rarely, clozapine, an antipsychotic drug used to treat schizophrenia, has been reported to cause cardiomyopathy, as we report in this case. Case Illustration A 17-year-old man who was known to have schizophrenia and was on clozapine therapy, presented in our emergency department with clinical heart failure worsening 6 hours before admission. On physical examination, he was tachycardia, tachypneu and . ECG demonstrated left ventricular hypertrophy and chest x-ray showed cardiomegaly with pulmonary congestive. Echocardiography showed a reduced left ventricular ejection with dilated all cardiac chamber. CT coronary showed no abnormality. Other laboratory results high NTproBNp with negative respiratory viral panel including COVID-19 PCR, negative blood cultures and BTA. Then the patient was performed CT thorax with result thrombus in left pulmonary artery and the branch of segmented left pulmonal. The patient received management of heart failure with reduced ejection fraction. He improved clinically and was continued on anticoagulant therapy for embolism pulmonary. Conclusion Several researches have discussed possible mechanisms to explain clozapine-related cardiac side-effects such as cardiomyopathy. Given the potential risk to patients, it is obviously important to make the diagnosis as early as possible. Careful monitoring of cardiac function in patients started on clozapine would therefore have to be considered an important part. p p Keywords: cardiomyopathy, clozapine, heart failure Keywords: cardiomyopathy, clozapine, heart failure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 P. Maulina1, M. Yanni2 Faculty of Medicine, Andalas University, Padang, Indonesia1 Department of Cardiology and Vascular Medicine, DR M Djamil Hospital, Padang, Indonesia2 P. Maulina1, M. Yanni2 Faculty of Medicine, Andalas University, Padang, Indonesia1 Pulmonary Embolism Mimicking NSTE-ACS P. Maulina1, M. Yanni2 Faculty of Medicine, Andalas University, Padang, Indonesia1 Department of Cardiology and Vascular Medicine, DR M Djamil Hospital, Padang, Indonesia2 Case Illustration A 56-year-old male admitted to emergency room with chest pain and dyspneu since 4 hours before admission. On physical examination, blood pressure was 123/66 mmHg, tachycardia 135 bpm, tachypneu 33x/minute, and oxygen saturation 92 % with NRM 15 L/minute. Coarse rales was heard in both of lungs. ECG showed Q pathologies and inversion of T wave in lead III and aVF. Troponin I level was remarkably high. Due to hemodynamic instability with signs of acute heart failure, patient was sent to cathlab for immediate invasive strategy as very high-risk NSTE-ACS. However, coronary angiogram demonstrated normal coronary arteries. Bedside echocardiography showed McConnell’s sign as a sign of pulmonary embolism. Patient was treated with systemic thrombolytic therapy using Streptokinase, and continued with subcutaneous Enoxaparin. Computed tomography pulmonary angiography (CTPA) demonstrated thrombus in both of pulmonary arteries (Figure 1), and Doppler ultrasound showed deep vein thrombosis in both of popliteal vein. After 10 days of Enoxaparin, an evaluation of CTPA showed decreased in the size of thrombus. He was discharged with Vitamin K antagonist therapy and CTPA evaluation after 3 months showed no residual thrombus in pulmonary arteries. Conclusion A Case of Autoimmune-induced Acute Limb Ischemia in Patient with Systemic Sclerosis Preceded by Secondary Raynaud's Phenomenon Autoimmune-induced Acute Limb Ischemia in Patient with Systemic Sclerosis Preceded by Secondary Raynaud's Phenomenon R. Istisakinah1, T.S. Tartila2, I.F.A. Putra2 General Practitioner, Hasna Medika Kuningan Heart Hospital, West Java, Indonesia2 Background: Secondary Raynaud's phenomenon (RP) is common first manifestation of connective tissue disease and presented in nearly 95% patients with systemic sclerosis (SSc). Secondary RP can indicate severe underlying vasculopathy with greater risk of tissue ischemia and ulceration. We present a case of autoimmune-induced acute limb ischemia in patient with SSc preceded by secondary RP as heralding manifestation. Case Illustration: A 44-year-old female came to our outpatient clinic with years history of episodic painless discoloration of fingers and toes on exposure to low temperature. The patient also developed skin dyspigmentation and stiffness in the last 1 year. General examination revealed skin hyperpigmentation and thickening, with the appearance of salt-and-pepper pigmentation around forehead, nose bridge, and upper chest. There was no ulceration found from initial encounter. Demographic and clinical features suggested secondary RP caused by SSc, further confirmed by positive antinuclear antibody. We gave oral nifedipine and nitrate for routine treatment. Three months later, the patient came to ER with a week history of digital ulcers accompanied by darkening of the skin and pain. Physical examination discovered multiple bilateral ulceration with gangrene on her toes, with absence of peripheral pulsation of both right and left dorsalis pedis arteries. Doppler ultrasound showed no flow with no thrombus in bilateral tibialis anterior and dorsalis pedis arteries. We concluded that ischemic injury was induced by autoimmune-mediated vasculopathy. The patient was treated with oral nifedipine, nitrate, cilostazol, and enoxaparin injection. There was only slight improvement, so we referred the patient to vascular surgery. Conclusion: We emphasize the need for early recognition of secondary RP and its underlying connective tissue disease as the condition can lead to severe ischemic injury. Once established, comprehensive management should be started early to prevent permanent tissue damage and disability Conclusion: We emphasize the need for early recognition of secondary RP and its underlying connective tissue disease as the condition can lead to severe ischemic injury. Once established, comprehensive management should be started early to prevent permanent tissue damage and disability. py Conclusion Physician in emergency room must be aware that pulmonary embolism can present as chest pain and missdiagnosed as ACS. After excluded ACS with coronary angiogram, in hemodynamic instability patient, bedside transthoracal echocardiography can be done to see the right ventricle dysfunction. And when CTPA was not available, we can treat the patient as high risk PE, and systemic thrombolytic was recommended (Class I B) , as demonstrated in this case. Keywords: Pulmonary embolism, NSTE-ACS Keywords: Pulmonary embolism, NSTE-ACS Figure 1. CTPA demonstrated thrombus in both of pulmonary arteries Figure 1. CTPA demonstrated thrombus in both of pulmonary arteries Myocardial Infarction With Nonobstructive Coronary Arteries (MINOCA) : a case series S. Fatimah, T. Pramitasari, A.E. Satuti1 RSUD Kota Yogyakarta1 S. Fatimah, T. Pramitasari, A.E. Satuti1 RSUD Kota Yogyakarta1 Background g Myocardial infarction with non obstructive coronary arteries (MINOCA) is a syndrome characterized by clinical evidence of MI and angiographically normal or almost normal coronary. Its albeit rare with prevalence only 5 to 6% of all MIs but the reported prevalence keeps rising due to better recognition of this syndrome. Case illustration Case 1 : A 65 years old female came to ER complaining chest pain, described as heavy pressure on her left chest radiating to the back accompanied by diaphoretic and dyspnea. Electrocardiogram showed ST segment elevation with frequent VES and elevated troponin I suggest for high lateral STEMI. She was treated with dual antiplatelet, heparin, diuretic, beta blocker, ARB, and statin. Slow flow in LAD coronary artery without any occlusion was found during coronary arteriography. In the fifth day of treatment patient was discharged without any complain. Case 2 : A 55 years old female came to ER complaining shortness of breath. Symptoms began 6 hours before and progressively worsened. She had high blood pressure, tachypnea and low oxygen saturation. There were rales and wheezing. Electrocardiogram showed some pathological q wave and laboratory test showed elevated troponin I. Chest radiograph showed mild pulmonary oedema. She was treated with dual antiplatelet, heparin, vasodilator, diuretic, statin and ARB. During two days of hospitalization patient continuous experiencing symptom and suddenly had a very low BP suggest for high risk NSTEMI. She was taken for coronary angiography which reported no signs of obstructive CAD. In the fifth day of treatment patient was discharged without any complain. Conclusion The causes of MINOCA are several and the optimal management depends mainly on it. Careful consideration and identification of the underlying cause is important to make a better prognosis and to improve the quality of life of the patient. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Case of Autoimmune-induced Acute Limb Ischemia in Patient with Systemic Sclerosis Preceded by Secondary Raynaud's Phenomenon K d t i l i t li b i h i t i (A) Salt-and-pepper appearance over forehead, nose bridge, and upper chest, (B) Dorsal and (C) Plantar view of ulceration and gangrene of the toes and-pepper appearance over forehead, nose bridge, and upper chest, (B) Dorsal and (C) Plantar view of ulceration and gangrene of the toes (A) Salt-and-pepper appearance over forehead, nose bridge, and upper chest, (B) Dorsal and (C) Plantar view of ulceration and gangrene of the toes Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Myocardial Infarction With Nonobstructive Coronary Arteries (MINOCA) : a case series S. Fatimah, T. Pramitasari, A.E. Satuti1 RSUD Kota Yogyakarta1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Risk factors for cardiovascular disease in patients with non ST segment elevation myocardial infarction : A case report Keywords: Risk factors for cardiovascular disease, Acute coronary syndrome. Keywords Myocardial infarction, MINOCA Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Risk factors for cardiovascular disease in patients with non ST segment elevation myocardial infarction : A case report Background Acute heart failure (AHF) commonly is an impending complication to acute myocardial infarction (AMI). AMI is statistically uncommon in the young adults. Heart failure following AMI is the major driver of late morbidity, mortality and healthcare cost. Acute Heart Failure Following Acute Myocardial Infarction in a 25 years-old Male: A Reality M.N. Hutapea1, N.P. Pranidya1, A.A. Hakim2, N.R. Utantyo2, A. Parlautan2, P. Wulandari2, F. Ahmad2, A. Nandiwardhana2 General Practitioner, Badan Pengusahaan Batam Hospital, Riau Island, Indonesia1 Cardiologist, Badan Pengusahaan Batam Hospital, Riau Island, Indonesia2 General Practitioner, Badan Pengusahaan Batam Hospital, Riau Island, Indonesia Cardiologist, Badan Pengusahaan Batam Hospital, Riau Island, Indonesia2 y Case Illustration A 25-year-old man was referred to our emergency room (ER) due to typical chest pain. He was a heavy smoker without any medical history before. Physical examinations were found normal during the initial assessment at the ER. ECG showed an ST elevation in anterior leads and elevated serum troponin I was reported. The chest x-ray and other laboratories were normal. A coronary angiogram (CAG) was carried out and revealed single vessel disease with total occlusion in the osteal-proximal part of left anterior descending (LAD) coronary artery. PCI was successfully done with one stent implanted at osteal-proximal of LAD without any complication. However during the monitoring in CVCU, the patient’s condition showed symptoms and signs of Heart Failure (HF). Echocardiography showed eccentric LVH and hypokinetic in anterior wall with EF 48%. IVC diameter were found to be dilated. We perform decongestion strategy using intravenous loop diuretic therapy. We iniated guideline‐directed HF therapy to achieve euvolemic state and good perfusion. Monitoring HF treatment includes careful measurement of fluid intake and output, vital signs, and clinical signs and symptoms of congestion and hypoperfusion is an essential part. Risk factors for cardiovascular disease in patients with non ST segment elevation myocardial infarction : A case report H. Junianto1, P. Jeremiah1 RS BK Woodward Palu1 Background : Non ST segment elevation acute coronary syndrome is a clinical syndrome consisting of unstable angina pectoris and non ST segment elevation acute myocardial infarction. Characteristic include chest pain typical of infarction accompanied by elevated cardiac enzymes ( Troponin ). Case illustration : A 63 year old man came with complain of left chest pain that had appeared since one day ago. The chest pain was felt to radiate to the shoulder, and felt like being hit by a heavy load. Tightness is also complained of when experiencing chest pain. Chest pain is felt to arise and disappear, appearing during light activities and at rest. Duration of chest pain is more than 20 minutes, nausea is present, previous history of hypertension and smoking, history of taking amlodipine 5 mg daily but not regularly. Physical examination, blood pressure 190/90, pulse 88, respiration 25, and SpO2 98%. Laboratory examination, total cholesterol 223, HDL 40, trigliserida 236, troponin 1,0. EKG examination showed NSTEMI V4-V5, T inversion and LVH. The patient was recommended for hospitalization and observation for complaint of chest pain. Drug therapy aspilet 80 mg, clopidogrel 75 mg, isdn 3 x 5 mg, atorvastatin 20 mg, amlodipine 5 mg, ramipril 5 mg, diviti 2.5 mg sc for 3 days. The patient experienced improvement and was educated to stop smoking, reduce high fat foods, take medicine regularly and healthy lifestyle. After 3 days of hospitalization, the patient was allowed to be outpatient and routine control at the outpatient clinic. p Conclusion : In this case, cardiovascular disease risk factors play an important role in patient suffering from acute coronary syndrome disease. Appropriate and routine treatment should be carried out, and healthy lifestyle can be implemented to prevent the risk of more severe complication. Keywords: Risk factors for cardiovascular disease, Acute coronary syndrome. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Breaking the Silence: An Insightful Case Series on Hypertensive Emergency in Acute Aortic Syndrome Patients Breaking the Silence: An Insightful Case Series on Hypertensive Emergency in Acute Aortic Syndrome Patients A. Alamsyaputra1, I.P. Farissa1, M.F. Ahnaf1, L.D. Pradipta1 RSUP Dr. Kariadi Semarang / Universitas Diponegoro1 Background g Hypertensive emergency is an abrupt and significant rise in arterial blood pressure in individuals with established hypertension or spontaneously, causing immediate end-organ damage and requiring fast-acting medications. Acute aortic syndromes are symptomatic thoracic and abdominal aorta disease that require immediate evaluation and treatment. Acute aortic dissection, intramural hematoma, and penetrating atherosclerotic ulcer are examples. Case illustration First case was 47 years old man with 5 years uncontrolled hypertension history, presented to emergency room complaining about high intensity and sharp abdominal pain 5 days prior to admission, continuous, and referred to the back. His blood pressure was 201/112 mmHg with symmetrical peripheral pulses. Electrocardiography and troponin were negative. Abdominal computed tomography angiography (CTA) showed abdominal aorta intramural hematoma. Second case was 52 years old man with 3 years uncontrolled hypertension history, presented to emergency room with a chief concern of 2 days continuous and tearing abdominal pain. His blood pressure was 182/99 mmHg, ECG and troponin were unremarkable. Abdominal CTA revealed dissection of descending thoracic aorta and abdominal aorta. We managed the patient with surgery preparation, intravenous nicardipine and morphine sulphate, oral beta blocker, targeting systolic blood pressure <120 mmHg and heart rate <60 bpm before surgery. Conclusion AMI in young patients remains a challenge in the emergency department. Knowledge of the mechanisms responsible for ventricular dysfunction after MI is major importance. Better identification of patients at risk of HF development after MI is needed because timely initiation of guideline‐directed HF therapy can reduce the risk of further LV remodelling, morbidity, and mortality. g y y Keywords: Acute Heart Failure, Acute Myocardial Infarction, Young patients Figure 1. Coronary angiogram showed total occlusion at osteal-proximal part of LAD Figure 1. Coronary angiogram showed total occlusion at osteal-proximal part of LAD Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Breaking the Silence: An Insightful Case Series on Hypertensive Emergency in Acute Aortic Syndrome Patients Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion The primary management in acute aortic syndrome is to normalize blood pressure and to reduce the force of left ventricular ejection (dP/dt), which are the main determinants of dissection extension and rupture, by using beta blocker, opiates for pain control, and non-nitrate vasodilator. The importance of timely diagnosis and intervention, and offers practical strategies for managing this complex and potentially life-threatening condition. Diagnosis and treatment of hypertensive emergency in acute aortic syndrome patients serve as valuable resource for clinicians seeking to improve their understanding and management of this critical condition. g p g g Keywords: hypertensive emergency, acute aortic syndrome, left ventricular ejection force Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Increasing Succesful Rate of Percutaneus Transluminal Angioplasty in Immadiate Diagnosis and Management Central Venous Stenosis in Hemodialysis Patient: Faster Better A.R. Akbar1,3, N. Kurnianingsih1,3, A. Gunawan2,3 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; 2Department of Internal Medicine, Faculty of Medicine Universitas Brawijaya; 3dr. Saiful Anwar General Hospital Indonesian Journal of Cardiology Increasing Succesful Rate of Percutaneus Transluminal Angioplasty in Immadiate Diagnosis and Management Central Venous Stenosis in Hemodialysis Patient: Faster Better A.R. Akbar1,3, N. Kurnianingsih1,3, A. Gunawan2,3 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; 2Department of Internal Medicine, Faculty of Medicine Universitas Brawijaya; 3dr. Saiful Anwar General Hospital Increasing Succesful Rate of Percutaneus Transluminal Angioplasty in Immadiate Diagnosis and Management Central Venous Stenosis in Hemodialysis Patient: Faster Better A.R. Akbar1,3, N. Kurnianingsih1,3, A. Gunawan2,3 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; 2Department of Internal Medicine, Faculty of Medicine Universitas Brawijaya; 3dr. Saiful Anwar General Hospital 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; 2Department of Internal Medicine, Faculty of Medicine Universitas Brawijaya; 3dr. Saiful Anwar General Hospital Background: g Stasis, thrombosis, and impared arteriovenous (AV) fistula are possible problems to manifest Central venous stenosis (CVS). Almost 25% dysfunctional fistulas at patient with dialysis are known to have these problems. Proper and immediate identification of CVS is critical to improve the succesful rate of percutaneus transluminal angioplasty (PTA) in hemodialysis patient. We present the immediate desicion of CVS in hemodialysis patients. Discussion: CVS is generally associated with long term AV fistula. The Endothelial injury from physical stimulation of the vein wall preceded the development of CVS and also developed smooth muscle proliferation and microthrombic. PTA as an immadiate procedure for CVS has high rate success although patentability is low in long-term condition. This patient is still needed to Follow-up for determining long-term patency. Case illustration: A 53 year old woman complaining of left arm swelling since 2 weeks. These Complaints are not combined by pain, fever, or accidental injuries at the arm. She routinely undergoes hemodialysis twice a week with AV fistula access in the left arm for 12 years. Hemodialysis can still run smoothly even with swelling in her left arm. PTA was performed without waiting hemodialysis problem as an immadiate management of CVS and resulting without any complications. Conclusion: Percutaneous transluminal angioplasty is one of immadiate managements for better result of CVS in hemodialysis patient Key words: Central venous stenosis, Percutaneous transluminal angioplasty, Hemodialysis patient Keywords: Percutaneus Transluminal Angioplasty, Central venous stenosis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background Background In recent decades, the incidence of Acute myocardial infarction (AMI) is increasing in younger women in the context of increasing metabolic syndrome, diabetes mellitus, and non-traditional risk factors. Compared with men, women have greater in-hospital, early and late mortality, as a result of baseline comorbidities. Case illustration A 29-year-old woman referred from regional hospital ICU to dr. Kariadi Hospital with chief complaints of typical chest pain for 44 hours prior to dr. Kariadi hospital admission. Patient was fully alert with blood pressure of 99/72, and heart rate was 108 bpm. ECG showed a Q wave in lead II III AVF, ST segment elevation in II III AVF, V5- V6, V7-V9, V4R-V6R, with ST Depression in I AVL; V1-V3 and T inverted in V7-V9, V4R-V6R. Chest X-Ray showed pulmonary oedema with suspected cardiomegaly. Blood panels showed Dyslipidemia, Diabetes, elevated Lactate and Transaminase. Echocardiography finding was LV dilatation with eccentric type LVH, positive RWMA in inferoseptal, inferior, inferolateral and apicoinferior segment. LVEF was 40%(B) with LV DD grade II. Percutaneous coronary intervention was performed and showed 80% stenosis in Mid RCA, 70% in LAD, and 70% LcX. Revascularization was successfully performed with 2 DES in RCA. 29 Years Old Woman with Triple Vessel Disease: A Case Report 29 Years Old Woman with Triple Vessel Disease: A Case Report W.N. Yuandika1, S. Sungkar1 1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Diponegoro University – Dr. Kariadi General Hospital, Semarang Keywords: Myocardial Infarction, Angiography, Triple Vessel Disease, Young Adults, Woman Conclusion Although rare, AMI in very young woman usually happen because of poorly controlled risk factors, as in this case a history of Gestational Diabetes, Dyslipidemia and Obesity. AMI has historically been regarded as a man’s disease, and for many years, women have been underdiagnosed and undertreated. This case reminds us how secondary prevention acts as a corner stone in managing coronary artery disease. Keywords: Myocardial Infarction, Angiography, Triple Vessel Disease, Young Adults, Woman eywords: Myocardial Infarction, Angiography, Triple Vessel Disease, Young Adults, Woman Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Echocardiography as A Tool in Differentiating Left Atrial Myxoma and Other Intracardiac Masses W. Valerian, M. Yanni, M. Syafri1 Universitas Andalas1 NEW ONSET ATRIAL FIBRILATION IN STEMI PATIENT: CASE REPORT Herlina, D.J. Tito1 Puri Husada Hospital Tembilahan1 Background: Atrial fibrillation is one of the abnormalities in the heart rhythm that causes irregular or arrhythmia as a result of abnormal impulses on heart. Atrial Fibrillation (AF) is a common arrhythmia in the setting of acute coronary syndrome and acute ST-elevation myocardial infarction (STEMI). A population-based study demonstrated that the incidence of AF in the setting of Acute Myocardial Infarction (AMI) tended to increase up to 13.3% during last decade. AF in the setting of AMI has a worse impact on the clinical course and prognosis of the disease. AF should be diagnosed, treated and convert to sinus rhythm as soon as possible, since its duration correlates with mortality. y Case illustration: A 55-year-old male patient presenting an acute STEMI as initial presentation with AF as complication. The electrocardiogram (ECG) showed AF with a rapid ventricular response with ST segment elevation in extensive anterior on the second day of hospitalization. Chest X-ray found a cardiomegaly and laboratory result found increased high sensitive cardiac troponin I (hs-cTnI) level on admission, 7032.4 ng/L. During hospitalization, patient was treated with dual antiplatelet therapy (DAPT), anticoagulant, vasodilator, beta blocker, and antiarrhythmic medication. Oral amiodarone 200 mg as antiarrhythmic agent, is effective in the conversion of AF to sinus rhythm in this patient. Patient showed an improvement of his clinical condition and discharged on the fourth day of hospitalization with controlled heart rhythm. g y p y Conclusion: An arrhythmia complication in STEMI patient was detected with ECG on second day hospitalization. In addition to acute coronary syndrome therapy, the patient was also given antiarrhythmic drugs and showed good results. Keywords: atrial fibrillation, acute myocardial infarction Figure 1. The electrocardiogram (ECG) showed AF with a rapid ventricular response with ST segment elevation in extensive anterior on the second day of hospitalization. Keywords: atrial fibrillation, acute myocardial infarction Keywords: atrial fibrillation, acute myocardial infarction Figure 1. The electrocardiogram (ECG) showed AF with a rapid ventricular response with ST segment elevation in extensive anterior on the second day of hospitalization. Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964- doi: 10.30701/ijc.1537 Echocardiography as A Tool in Differentiating Left Atrial Myxoma and Other Intracardiac Masses W. Valerian, M. Yanni, M. Syafri1 Universitas Andalas1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Echocardiography as A Tool in Differentiating Left Atrial Myxoma and Other Intracardiac Masses W. Valerian, M. Yanni, M. Syafri1 Universitas Andalas1 W. Valerian, M. Yanni, M. Syafri1 Universitas Andalas1 Background: Myxoma of the left atrium is the most common benign tumor that can be found, accountable for almost 70% of primary cardiac tumors. Myxoma is often described as a mobile, lobular mass located in the left atrium, particularly at the fossa ovalis, and rarely found in the right atrium or ventricle. The ability to differentiate tumors and other intracardiac masses is what makes echocardiography needed. Case Illustration: A 58-year-old female came to the emergency room with shortness of breath. The symptoms persisted for more than 1 year, which worsened with activity. Other complaints were denied, but the patient admitted to having uncontrolled diabetes for more than 10 years. From the physical examination, we found mid- diastolic murmur at the apex. ECG showed sinus rhythm with P wave terminal force at V1-V2 and low voltage with right ventricular hypertrophy. Echocardiography findings showed a mobile, regular mass, 29 mm x 45 mm in size, attached to the intra-atrial septum. The mass prolapsed into the left ventricle covering mitral inflow in the diastolic phase resulting in relative mitral stenosis with a transmitral gradient of 15 mmHg. Conclusion: Echocardiography has an important role in diagnosing myxoma and evaluating tumor sizes and locations, as it can be similar to thrombus, vegetations, papillary fibroelastosis, lipomas, sarcomas, and other intracardiac masses. It also has a role in evaluating the patient’s hemodynamic state, determining the location of the incision, and predicting the outcome of the surgery. g g y KEYWORD: myxoma, echocardiography, intracardiac masses Keywords: myxoma, echocardiography, intracardiac masses Keywords: myxoma, echocardiography, intracardiac masses Keywords: myxoma, echocardiography, intracardiac masses Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 6 year old male admitted to hospital for dyspnea exacerbating in the last 3 days and accompanied by fever and cough. He was easily tired and bluish even with light activities, since 2 years ago, severely wasted and stunted (14 kg, 105 cm), lethargic, BP 100/59, HR 145, RR 38, SpO2 75-78%. Central cyanosis, distended jugular vein, 4/6 pansystolic ejection murmur, palpable thrill on the right side of the chest, and clubbing fingers. Chest x-ray showed dextrocardia with increased pulmonary vascularity. Echocardiography exhibited large ASD, common AV Valve, single ventricle and hypoplastic pulmonary arteries. Thoracal CT scan showed dextrocardia with cardiomegaly, the left lung consists three lobes meanwhile the right lung consists two lobes, and spleen was not well visualized. His consciousness worsened, GCS E2M4V1, in day 6 hospitalization. Facial tic and jerking extremities appeared frequently. Brain CT scan showed multiple hypodense lesions with hyperdense areas at right basal ganglia and left parietal lobe were found. The patient was treated with broad spectrum antibiotics, diuretics, anticonvulsant, and other supportive therapies. He was prepared to craniotomy with abscess excision and aspiration but unfortunately he suffered cardiac arrest the next day. Background Background Heterotaxy Syndrome is an abnormality where the internal thoraco-abdominal organs demonstrate abnormal arrangement across the left-right axis of the body. Heterotaxy Syndrome divided into right isomerism and left isomerism. It occurs in 1:5.000-7.000 live birth with CHD. Cardiac Manifestation in Heterotaxy Syndrome: Case Report I.B.U. Kramasanjaya1, Y. Ardelia1, R. Aviana1, K. Rizky2 1) General Practitioner, Balaraja General Hospital 2)Cardiologist, Balaraja General Hospital S.G. Maharani1, F.I. Shahab1 Intensive Coronary and Cerebrovascular Care Unit, Sentra Medika Cibinong Hospital, Bogor, Indonesia1 S.G. Maharani1, F.I. Shahab1 Intensive Coronary and Cerebrovascular Care Unit, Sentra Medika Cibinong Hospital, Bogor, Indonesia1 S.G. Maharani1, F.I. Shahab1 ntensive Coronary and Cerebrovascular Care Unit, Sentra Medika Cibinong Hospital, Bogor, Indonesia1 Background: Catheter-induced coronary artery dissection (CICAD) is a rare but severe complication of coronary angiography (CAG), with the incidence <0.1%. In a retrospective 10-year cohort study of 56,968 patients undergoing CAG, catheter dissection was more common in the Right Coronary Artery (RCA) and mostly due to guiding catheter. Dissection without complications of acute ischemia will heal on its own. Whereas in a spiral dissection, a stent is needed to prevent the expansion of dissection. Case Illustration: A 40-year-old male patient came to the cardiac clinic with typical chest pain of angina. The patient had a history of smoking and dyslipidemia. The results of Coronary Multislice Computerized Tomography (MSCT) examination were found in Left Anterior Descending (LAD) 70% proximal stenosis, 50% mid-distal stenosis. On the Left Circumflex (LCX) 50% distal stenosis. ECG result was sinus rhythm. Echocardiogram showed EF 66% with global normokinetics. CAG was performed via radial access. There was aortic elongation which makes cannulation to the osteal coronary quite difficult. In LAD there is calcification with 70-80% stenosis in the osteal-mid. Normal LCX. When cannulated into the RCA, spiral dissection occurred in proximal-distal. The patient had progressive chest pain. Access was immediately transferred to the femoral. Two stents were implanted in the RCA to close the dissection with the results of Thrombolysis in Myocardial Infarction (TIMI) 3 flow. One day after PCI the patient's hemodynamics were good and sent home. For the management of LAD lesion with calcification that would involve the left main artery, we planned elective PCI with Intravascular ultrasound (IVUS) guided. g Conclusion: A 40-year-old male patient, underwent coronary artery spiral dissection due to a catheter during angiography. 2 stents were implanted in the dissection area with good results. Keywords: Spiral Coronary Artery Dissection onclusion: A 40-year-old male patient, underwent coronary artery spiral dissection due to a catheter durin ngiography. 2 stents were implanted in the dissection area with good results. y p y y Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Conclusion HS is a rare congenital disorder with high mortality rate in early life. Early diagnosis and prompt treatment in cardiology or paediatric center may give better outcome for HS. Keywords: Heterotaxy Syndrome Echocardiography HS is a rare congenital disorder with high mortality rate in early life. Early diagnosis and prompt treatment in cardiology or paediatric center may give better outcome for HS. Keywords: Heterotaxy Syndrome HS is a rare congenital disorder with high mortality rate in early life. Early diagnosis and prompt treatment in cardiology or paediatric center may give better outcome for HS. Keywords: Heterotaxy Syndrome Echocardiography Echocardiography Echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 PERIMYOCARDITIS, MIMICKING ACUTE CORONARY SYNDROME, ASSOCIATED WITH COVID-19 P. Habibie1, S. Aghnia1, C. Amalia1 Emergency Hospital of COVID-19 Wisma Atlet Kemayoran1 Background: Perimyocarditis is an acute inflammation involving both of pericardium and myocardium which is commonly caused by viral infection. Several investigatioons found that COVID-19 is much related to the perimyocarditis. Damage to the pericardium/myocardium in COVID-19 patients increases mortality and morbidity. The immediate awareness of this condition provides better treatment and prognosis. Case Illustration: A 46-year-old male inpatient room after five days treatment as COVID-19 confirmed patient presented with angina type chest pain lasted more than 20 minutes since about 2 hours ago. It was described as squeezing and tightness in substernal area, radiating to the left arm, accompanies by cold sweats and was not improving with rest. The patient did not complain of nausea and vomiting, dizziness nor shortness of breath. Previously, chest pain had never been felt. There was no prior medical history of hypertension, diabetes mellitus, heart disease in the patient. The patient was an active smoker. Physical examination showed stable hemodynamic condition. Laboratory examination showed leucocytosis. ECG showed ST elevation in II, III, aVF, V2-V6. The patient was diagnosed with inferolateral STEMI and immediately referred to ER. The patient was treated with nitrates, antiplatelet, anticoagulant, ACE inhibitor, beta blocker, and statins. p ov g w t est. e pat e t d d ot co p a o ausea a d vo t g, d ess o s o t ess o b eat . Previously, chest pain had never been felt. There was no prior medical history of hypertension, diabetes mellitus, heart disease in the patient. The patient was an active smoker. Physical examination showed stable hemodynamic condition. Laboratory examination showed leucocytosis. ECG showed ST elevation in II, III, aVF, V2-V6. The patient was diagnosed with inferolateral STEMI and immediately referred to ER. The patient was treated with nitrates, antiplatelet, anticoagulant, ACE inhibitor, beta blocker, and statins. A few hours later, chest pain was not improving. The serial ECGs had been performed with no ECG evolution of STEMI was found. PR depression was also shown in the ECG. Furthermore, the patient was diagnosed with probable perimyocarditis associated to COVID-19. The patient was referred to Harapan Kita Hospital for cardiac marker, echocardiography, and coronary angiography. Conclusion: Pericarditis as well as myocarditis can show ST segment elevation in ECG, mimicking acute A few hours later, chest pain was not improving. Case Series of Stent Thrombosis : Nightmare of The Interventional Cardiology in The Modern Era F.H. Alkaf1, S. Sungkar2, I. Uddin2, Y. Herry2, S. Rifqi2 l f di i i i i S d Department Cardiology and Vascular Medicine, Kariadi Hospital, Semarang, Indonesia2 Background: Coronary stents have marked an era in interventional cardiology and have significantly decreased the rates of stent thrombsis. But stent thrombosis is still a major problem of interventional procedures. Stent thrombosis after percutaneous coronary intervention (PCI) is a rare and usually poor prognostic event that might result in myocardial infarction and sudden death Case Illustration: A 74-year-old woman presented to the emergency department (ED) with typical chest pain. The first ECG showed Non-ST Elevation, with PCI procedure was performed approximately 2 weeks before admission with Everolimus-Eluting Stent in LAD, but didn’t consume DAPT (aspirin and clopidogrel) for 1 week before admission to the hospital. She developed multiple episodes of ventricular fibrillation and received defibrillator shocks. After ROSC, The second ECG showed ST-Elevation in the anterior lead. She underwent successful PCI with Plain Old Balloon Angioplasty (POBA). A 56-year-old woman presented to the ED with chest pain onset 1 hour, with the first and second ECG (H0/H1) showing Non-ST Elevation. The echocardiography examination was performed to rule out aortic dissection. The third ECG (H4) showed ST-Elevation in the anterior lead. She was performed with PCI with Everolimus-Eluting Stent in LAD 10 days before admission and didn’t consume ticagrelor for 1 day before admission. She underwent successful PCI with stent-in-stent. A 67-year-old man came to the ED with typical chest pain. He had a history of PPCI in bifurcation of RCA-PDA after Inferior STEMI 7 days ago. He had good compliance with DAPT (aspirin and clopidogrel). He underwent successful PCI with POBA. Conclusion: Stent thrombosis remains a significant complication in interventional cardiology, even in the modern era. In the present era, complications associated with stent thrombosis are rare but fatal if not managed appropriately. There are many factors related to stent thrombosis such as procedure, patient and lesion, antiplatelet, and thrombogenicity. Keywords: Stent thrombosis, PCI, ticagrelor, intervention mbosis, PCI, ticagrelor, intervention Before and After PPCI in Stent Thrombsis Event Before and After PPCI in Stent Thrombsis Event PERIMYOCARDITIS, MIMICKING ACUTE CORONARY SYNDROME, ASSOCIATED WITH COVID-19 The serial ECGs had been performed with no ECG evolution of STEMI was found. PR depression was also shown in the ECG. Furthermore, the patient was diagnosed with probable perimyocarditis associated to COVID-19. The patient was referred to Harapan Kita Hospital for cardiac marker, echocardiography, and coronary angiography. Conclusion: Pericarditis as well as myocarditis can show ST segment elevation in ECG, mimicking acute myocardial infarct that can cause inappropriate treatment. Therefore, it requires proper early diagnosis to the patient. p Keywords: Perimyocarditis, myocardial infarct, COVID-19 Keywords: Perimyocarditis, myocardial infarct, COVID 19 The serial ECGs Suspicion of Contrast-Induced Neurotoxicity following Coronary CT Angiography: A Case Report R.Ayuningtyas1, A. Novitasari1, S. Inayasari1, W. Pamungkas1, A. Muthmaina1, L. Pribadi1, M.G. Suwandi1, A. Ciptasari1 RSPAU dr. S. Hardjolukito1 The serial ECGs The serial ECGs The serial ECGs Suspicion of Contrast-Induced Neurotoxicity following Coronary CT Angiography: A Case Report R.Ayuningtyas1, A. Novitasari1, S. Inayasari1, W. Pamungkas1, A. Muthmaina1, L. Pribadi1, M.G. Suwandi1, A 1 Suspicion of Contrast-Induced Neurotoxicity following Coronary CT Angiography: A Case Report Suspicion of Contrast-Induced Neurotoxicity following Coronary CT Angiography: A Case Report R.Ayuningtyas1, A. Novitasari1, S. Inayasari1, W. Pamungkas1, A. Muthmaina1, L. Pribadi1, M.G. Suwandi1, A. Ciptasari1 RSPAU dr. S. Hardjolukito1 R.Ayuningtyas1, A. Novitasari1, S. Inayasari1, W. Pamungkas1, A. Muthmaina1, L. Pribadi1, M.G. Suwandi1, A. Ciptasari1 RSPAU dr. S. Hardjolukito1 Keywords: Unprovoke, Acute Coronary syndrome, acute pulmonary embolisme, shortness of breath. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Diagnosis, risk stratification, and treatment in unprovoked acute pulmonary embolism: a case report H. Martini1, N. Kurnianingsih1, L.H. Zunardi1 Dr.Saiful Anwar General Hospital/ Faculty of Medicine Brawijaya University1 Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Diagnosis, risk stratification, and treatment in unprovoked acute pulmonary embolism: a case report H. Martini1, N. Kurnianingsih1, L.H. Zunardi1 Dr.Saiful Anwar General Hospital/ Faculty of Medicine Brawijaya University1 Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Diagnosis, risk stratification, and treatment in unprovoked acute pulmonary embolism: a case report H. Martini1, N. Kurnianingsih1, L.H. Zunardi1 Dr.Saiful Anwar General Hospital/ Faculty of Medicine Brawijaya University1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background: Coronary CT Angiography (CCTA) has emerged as an essential diagnostic modality for the detection and assessment of coronary artery disease (CAD). Contrast-induced headache is an important clinical entity that must be considered during and after angiography. Background: Coronary CT Angiography (CCTA) has emerged as an essential diagnostic modality for the detection and assessment of coronary artery disease (CAD). Contrast-induced headache is an important clinical entity that must be considered during and after angiography. Case illustration: A 52-year-old male admitted to the ER with severe headache, nausea, and later vomitus. Patients was gotten CCTA with contrast and metoprolol 9 hours ago. Patient’s history were recurrent headache and ACS. Patients presented with bradycardia heart rate 43. Head MSCT result was normal. Thus, patient was diagnosed with contrast-induced headache, hypoglycemia ec adrenal insufficiency, bradycardia symptomatic, CAD1VD ec APS CCS II. Patient admitted in regular ward with intensive monitoring. Patient was having history of primary headache, thus contrast-induced headache can occur but relieved with symptomatic treatment. Conclusion: The occurrence of contrast-induced headache is common among patients with a history of primary headache. Therefore, this adverse effect was considered mild and treatable. , CAD, CCTA, Coronary CT Angiography AD, CCTA, Coronary CT Angiography Figure 1. Patient's Head MSCT showing normal result KEYWORD: Headache, CAD, CCTA, Coronary CT Angiography Keywords: Headache, CAD, CCTA, Coronary CT Angiography KEYWORD: Headache, CAD, CCTA, Coronary CT Angiography Keywords: Headache, CAD, CCTA, Coronary CT Angiography Figure 1. Patient's Head MSCT showing normal result Figure 1. Patient's Head MSCT showing normal result Background: g Dilated cardiomyopathy has been associated with left ventricular (LV) thrombosis, caused by myocardial systolic dysfunction which leads to substantial morbidity and mortality as a site for systemic emboli. We report a case of a stroke and acute limb ischemia secondary to a massive LV thrombus in a young female 20-year-old patient with dilated cardiomyopathy. Case Illustration: A 20-year-old female was referred to our hospital due to right lower extremities pain and right limb weakness. She was diagnosed with peripartum cardiomyopathy 5 years ago. A general physical examination found cold and pulseless in right lower limbs. From neurologic examination was found hemiparesis on the right side. Chest radiography showed cardiomegaly. Electrocardiography showed normal sinus rhythm with LVH. Transthoracic echocardiograms (TTE) revealed severe global LV systolic dysfunction with LVEF of 23%, LV dilatation with massive thrombus at LV. Duplex ultrasound showed a thrombus at the right dorsal pedis artery. A head CT scan revealed an acute infarct at capsula interna et externa sinistra and corona radiata sinistra. The patient was started on heparin and bridged with warfarin 5mg orally daily. The patient was uneventfully discharged one week after admission. Indonesian Journal of Cardiology Acute Stroke and Limb Ischemia Secondary to Massive Intracardiac Thrombus in a Young Female 20- Year-Old Patient with Dilated Cardiomyopathy D. Lestari1, C. T. Tjahjono2, N. Kurnianingsih2, W. Karolina, A. F. Rahimah ,2 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia Acute Stroke and Limb Ischemia Secondary to Massive Intracardiac Thrombus in a Young Female 20- Year-Old Patient with Dilated Cardiomyopathy D. Lestari1, C. T. Tjahjono2, N. Kurnianingsih2, W. Karolina, A. F. Rahimah ,2 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia ute Stroke and Limb Ischemia Secondary to Massive Intracardiac Thrombus in a Young Female 20- Year-Old Patient with Dilated Cardiomyopathy Diagnosis, risk stratification, and treatment in unprovoked acute pulmonary embolism: a case report H. Martini1, N. Kurnianingsih1, L.H. Zunardi1 Dr.Saiful Anwar General Hospital/ Faculty of Medicine Brawijaya University1 Background: Pulmonary embolisme (PE) are serious conditions that can be life threatening and have high rates of morbidity and mortality. Despite the high prevalence of PE, its diagnosis is still challenging, mainly due to the unpredictability of symptoms and physical signs and unexplained cause (Unprovoked PE). Risk Stratification of acute pulmonary embolism is important because it determines the right steps in decision-making. Case Illustration: A 45-year-old man, He presented to the ER private hospital with complaints of sudden shortness of breath after taking a bath. His family brought him to the private hospital 30 minutes after its onset. At Private hospital, He came with desaturation and shock condition. He was assessed as having an acute coronary syndrome (ACS). He got loaded with dual antiplatelets and was referred to our hospital. Because the patient's complaint is not angina but sudden shortness of breath accompanied by desaturation, we did not conclude that the patient was headed for ACS. we considered the possibility of a pulmonary embolism, so we performed bedside TTE in the ER and found RV dysfunction and the McConnell's sign leading to acute PE. We continue with the CT examination and found thrombus in the pulmonary artery. we assessed patients with high risk pulmonary embolism and performed thrombolysis with rTPA. The patient's complaints gradually improved. Conclusion: Every case of dyspnea that shows up at an emergency room should have acute PE taken into consideration in the differential diagnosis. In patients with suspected PE without obvious risk factors, we can use CT for the triple rule out (ACS, Aortic dissection and pulmonary embolism). Patients treated with thrombolytic therapy show rapid improvement which may lead to a lower rate of mortality and morbidity. Keywords: Unprovoke, Acute Coronary syndrome, acute pulmonary embolisme, shortness of breath. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Stroke and Limb Ischemia Secondary to Massive Intracardiac Thrombus in a Young Female 20- Year-Old Patient with Dilated Cardiomyopathy D. Lestari1, C. T. Tjahjono2, N. Kurnianingsih2, W. Karolina, A. F. Rahimah ,2 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia Background: Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: The common causes of thrombus formation in the left ventricle remain ischemic cardiomyopathy, LV aneurysm, and dilated cardiomyopathy, although, it can be associated with cardiac impairment, stroke, and thromboembolic risk. The benefits of anticoagulants in patients with dilated cardiomyopathy have been reported as a reduction of thromboembolic events. Keywords: Acute Stroke and Limb Ischemia, Massive Intracardiac Thrombus, Young Female, Dilated Cardiomyopathy Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964- doi: 10.30701/ijc.1537 Successful Arrhythmia Control in Electrical Storm with Acute Coronary Syndrome and High Probability Long QT Syndrome S. Phalguna1, S. Andrian1, W. Erlanda1, H.E. Rasyid1 RSUP dr. M Djamil Padang1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Patent Ductus Arteriosus (PDA) is a congenital heart defect which is the ductus arteriosus failed to close spontaneously after birth. Incidence of this condition is about 6 per 10,000 in full-term infants and 1 per 3 in preterm infants. Untreated PDA leads to Eisenmenger syndrome (ES). ES patients need special consideration especially for pregnancy because of complication and maternal mortality. Risk of foetal and neonatal are higher due to maternal cardiac output and hypoxaemia. Case Illustration A 19 years old female presented with shorthness of breath since 1 year before admission. Patient has already been diagnosed with PDA and pulmonary hypertension. Patient was planned to did right heart catheterization (RHC) procedure. On the second day of hospitalization, patient complained that shortness of breath was increased. From physical examination, the respiratory rate was 29 times/minute, saturation was 96% in the right upper extremity, 77% in the others with fines rales, increased second heart sound, pansystolic murmur 3/6 at left parasternalis border 3rd ICS, pretibial edema. On the 7th day, she didn’t feel shorthness of breath. RHC was done on that day. RHC result was PDA with eisenmengerization, low flow high resistance, not reactive oxygen test. On the next day, she went home with therapies slidenafil 3x20 mg, furosemide 2x20 mg, spironolactone 1x25 mg, ramipril 1x2,5 mg, bisoprolol 1x2,5 mg. We explained to her and family that PDA closure is a not recommended in patient with Eisenmenger syndrome and pregnancy should be avoided. Successful Arrhythmia Control in Electrical Storm with Acute Coronary Syndrome and High Probability Long QT Syndrome S. Phalguna1, S. Andrian1, W. Erlanda1, H.E. Rasyid1 RSUP dr. M Djamil Padang1 Background: Electrical storm (ES) defined clustering of three or more episodes of ventricular tachycardia(VT)/ventricular fibrillation(VF) within 24 hours. Patient with history of myocardial infarct and channelopathies will increase risk of ES as a life threatening condition. p g Case illustration: A 50-years-old male referred to emergency room with typical chest pain since 48 hours ago. Patient had seizure, unconscious, and got chest compression, patient return conscious before went to hospital. In referral hospital, patient returned seizure, electrocardiogram(ECG) showed VT unstable, patient got cardiopulmonary resuscitation(CPR), defibrillation, amiodaron bolus followed maintenance dose. In our emergency room, patient still chest pain, ECG revealed Q pathologic, ST elevation in V1-V4, T biphasic V1-V6, diagnosed with Recent ST Elevation Myocardial Infarct(STEMI) of Anterior wall, we decided primary percutaneous coronary intervention. Angiography revealed subtotal occlusion mid LAD, stenosis 70-80% mid RCA, we stenting proximal-mid LAD. Next day patient returned seizure, unconscious, monitor showed VT unstable. Patient underwent CPR and intubated caused of inadequate respiratory, we performed complete revascularization. After extube, patient felt anxiety, patient returned of seizure and was being ES. QT interval was prolong, schwartz score: 7, echocardiography revealed reduced ejection fraction. We gave lidocaine in acute phase of ES, but had no effect. We sedated, gave propranolol, patient still ES. We knockdown patient, down-titrated sedation, gave metoprolol, and optimal therapy for heart failure. Patient wasn’t had ES anymore and discharged. Conclusions: We report case 50 years old male with recent STEMI Anterior with complete revascularization and high probability LQTs. Administration of propranolol as in a recent study for ES is not always successful. In this case, the effect of metoprolol combined with sedation, anti anxiety, and optimal treatment of heart failure success for arrhythmia control. However, we should perform genetic test and cardiac MRI to find main problems of ES. Keywords: Electrical Strom, Recent STEMI Anterior, LQTs ECG patient with Ventricular Tachycardia ECG patient with Ventricular Tachycardia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Diagnosis and Treatment Patent Ductus Arteriosus with Eisenmenger Syndrome in Young Adult Female Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 P.M. Bahri1, C.K. Krevani2 Faculty of Medicine, Andalas University1 Andalas University2 Tackling Calcified Lesion with Orbital Coronary Atherectomy and IVUS: A Case Report D.N. Ghassani1, R.A. Fagi1, Y.H. Oktaviono1 Cardiology and Vascular Medicine Department, Medical Faculty of Airlangga University – Dr. Soetomo General Hospital, Surabaya, Indonesia1 D.N. Ghassani1, R.A. Fagi1, Y.H. Oktaviono1 Cardiology and Vascular Medicine Department, Medical Faculty of Airlangga University – Dr. Soetomo General Hospital, Surabaya, Indonesia1 Background: Percutaneous coronary intervention (PCI) is a common treatment for coronary artery disease (CAD), but it may not always be effective in cases with heavily calcified lesions. Orbital atherectomy is a novel technique that uses a diamond-coated burr to ablate calcified plaque within the vessel wall. Intravascular ultrasound (IVUS) is a high-resolution imaging modality that provides detailed information about coronary anatomy, including the location and extent of plaque build-up. The combination of orbital atherectomy and IVUS has shown promising results in the treatment of heavily calcified coronary lesions. p g y y Case illustration: Male, 56 years old, with type 2 diabetes, dyslipidemia, and chronic coronary syndrome CCS III, has a history of PCI at proximal right coronary artery (RCA) and distal left circumflex artery (LCx) in 2020. His coronary map showed diffuse calcified lesion up to 85% at left anterior descending artery (LAD). During follow-up, he was still symptomatic and he was referred to our hospital. His vitals were stable. The physical examination and ECG were unremarkable. TTE showed preserved ejection fraction (Biplane 50%) with hypokinetic segments at septal and inferior, and eccentric left ventricular hypertrophy. The patient underwent PCI at proximal-mid LAD with orbital atherectomy using Diamondback 360 Coronary OAS Micro Crown with the help of IVUS, resulting in complete revascularization. Conclusion: The combination of orbital atherectomy and IVUS in treating calcified coronary artery disease is promising to improve outcomes. Orbital atherectomy effectively modifies calcified plaque and prepares the vessel for stent placement. IVUS provides real-time information about vessel size, plaque burden, and stent deployment, which help optimize procedural outcomes. The combination of these two techniques can improve the success rate of stent placement and reduce the risk of complications. Further studies are needed to evaluate the long-term outcomes of this approach. Keywords: Orbital coronary atherectomy, IVUS, PCI, Chronic Coronary Syndrom Keywords: Orbital coronary atherectomy, IVUS, PCI, Chronic Coronary Syndrome Keywords: Orbital coronary atherectomy, IVUS, PCI, Chronic Coronary Syndrome Figure 1. Chest X-ray of the patient (A); Electrocardiogram of the patient (B); Pre-procedure (C); and Post- procedure (D) Figure 1. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion PDA closure is not recommended in patient with Eisenmenger syndrome and unreactive oxygen test. Pregnancy should be avoided because of maternal mortality rate. Keywords: Patent ductus arteriosus, Eisenmenger syndrome, pregnancy Tackling Calcified Lesion with Orbital Coronary Atherectomy and IVUS: A Case Report Chest X-ray of the patient (A); Electrocardiogram of the patient (B); Pre-procedure (C); and Post- procedure (D) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 An Unusual Case of Femoral Deep Vein Thrombosis with Bullae Formation R. Ayuningtyas1, A. Ciptasari1, A. Novitasari1, W. Pamungkas2, N.A. Novebri1, M. Mohamad1, A. Muthmaina1, L. Pribadi1, M.G. Suwandi1, S. Inayasari1 Rumah Sakit Pusat Angkatan Udara Suhardi Hardjolukito1 Adisutjipto Air Force Base2 Background: Blood clots may be formed in deep veins, most frequently the legs, and are medically referred to as deep vein thrombosis (DVT). One of the rarest complications of DVT is a blockage of large proximal vein, likely resulting in bullae formation and blistering, which can be confused with bullous skin disorder. Case illustration: A 63-year-old male patient in the ward complained about sudden hard swelling on his left leg that was accompanied by pain, discoloration, reduced range of movement, and multiple bullae. History of previous similar complaints, DVT, malignancy, and surgery were absent, either in the patient or his family. However, the patient has recurrent history of leg swelling after travelling long distances, and provides with grade I hypertension, obesity, and type II diabetes mellitus as risk factors. The Wells score on this patient was 5, and rising D-Dimer levels > 10.000, therefore digital ultrasound was performed with the result of total thrombus on the left femoral vein and patent flow of arteries and veins on both legs. The patient was treated with intravenous unfractionated heparin, antibiotic ointments, and saline compresses. The bullae were aspirated, and blister roof was maintained. After two weeks of treatment, swelling was reduced and no new bulla was formed. Warfarin therapy was continued on outpatient basis. Conclusion: Bulla formation as a result of DVT is a very rare complication that must be distinguished from skin disease based on clinical findings, physical examination, and imaging. It was challenging to determine the best treatment approach since there were no clear guidelines existed. However, the principle of treatment is to continue anticoagulant therapy while keeping the blisters' roofs intact to prevent secondary infection. g py p g p y KEYWORD: deep vein thrombosis, bullae formation, blisters Keywords: deep vein thrombosis, bullae formation, blisters KEYWORD: deep vein thrombosis, bullae formation, blisters Keywords: deep vein thrombosis, bullae formation, blisters KEYWORD: deep vein thrombosis, bullae formation, blisters Keywords: deep vein thrombosis, bullae formation, blisters , , Figure 1. Bulla formation due to deep vein thrombosis Figure 1. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Life-Threatening Effect of Doxorubicin: What We Should be Aware of? D. Lestari1, W. Karolina,2, A. F. Rahimah2 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia A Life-Threatening Effect of Doxorubicin: What We Should be Aware of? D. Lestari1, W. Karolina,2, A. F. Rahimah2 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia Background: Anthracyclines such as Doxorubicin is an antibiotic with a broad spectrum of activity in human neoplasms that have demonstrated an increased risk of HF and cardiomyopathy with significant morbidity and mortality. We present a case report of a patient on going chemotherapy with breast cancer treated with Doxorubicin who presented with symptoms of AHF. An Unusual Case of Femoral Deep Vein Thrombosis with Bullae Formation Bulla formation due to deep vein thrombosis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Life-Threatening Effect of Doxorubicin: What We Should be Aware of? D. Lestari1, W. Karolina,2, A. F. Rahimah2 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia Case Illustration: A 51-year-old man was admitted to ER with chest pain since 7 hours before admission, felt like squeezing, wasn’t accompanied by dyspnea or palpitation. It already felt since a day before admission, then getting worse in the next hours. Six months ago, he reported a similar condition and underwent TMT with normal result. He was smoker and had hypertension and dyslipidaemia. Physical finding was unremarkable. An ECG revealed sinus tachycardia, LAD without ST-T changes, Troponin I was 0.01 ng/ml. CXR showed found cardiomegaly. Patient was diagnosed as UAP. We administered DAPT, statin, beta-blocker, anticoagulant, nitrate and transfer him to HCU. ECG serial revealed no evolution. Echocardiography showed LVEF 65%, global normokinetic, LVH, mild AR. In the 2nd day, chest pain was slightly relieved but he complains neck stiffness with intermittent hypesthesia on left arm. In the 3rd day, we performed CCTA with result minimal plaque burden and stenosis at LAD & RCA. We consulted the patient to neurologist then was performed Cervical X Ray with result spondylosis cervicalis. We stop nitrate and patient was administrated with mecobalamine and eperisone. In the 4th day, the complaint was relieved and discharged from the hospital. Cervical Angina: Kindly Reminder that not All of Angina Come from the Heart I. Fauzie1, D. Arara1 Primaya Hospital Depok1 Background: g Cervical angina has been defined as chest pain that resembles true cardiac angina but originates from the disorders of the cervical spine. Cervical angina can be diagnosed according to negative cardiac workups, positive neurologic examination, and cervical radiographic findings. Cardiac imaging examination with coronary CTA should be the primary imaging test to define the possible cause of angina which it has high overall sensitivity about 95.2%. Case Illustration: A 37-year-old female patient with left-sided breast cancer on going chemotherapy presented with progressively worsening shortness of breath and worsened exercise tolerance for the past 1 month. On May 2022, her breast cancer was treated with chemotherapy including doxorubicin and cyclophosphamide. On physical examination was found bilateral crackles with jugular venous distention and lower extremity edema. Electrocardiography showed sinus tachycardia. Laboratory findings were elevated N-terminal pro-brain natriuretic peptide (NT- proBNP). Transthoracic echocardiograms (TTE) pre-chemotherapy showed normal LV systolic with LVEF 68% by biplane, global normokinetic. TTE after 4 months revealed diffuse left ventricular hypokinesis, left ventricular ejection fraction (LVEF) of 22%, and reduced right ventricular contractility. Given the clinical presentation, the patient was treated for acute heart failure and was admitted to the Cardiac Intensive Care Unit. The patient got ventilator support, loop diuretic and inotropes/vasopressors. Conclusion: We should be aware of the effect of doxorubicin that induces cardiomyopathy. It typically presents earlier. Prevention and management of doxorubicin-induced cardiotoxicity involve a multidisciplinary approach that entails careful assessment of cardiac function. Keywords: Doxorubicin, Cardiomyopathy, Breast Cancer Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Cervical Angina: Kindly Reminder that not All of Angina Come from the Heart Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Cervical Angina: Kindly Reminder that not All of Angina Come from the Hea Background : Atrial fibrillation and total atrioventricular block are two different type of arrhythmia. Although the two arrhythmias can co-exist. Atrial fibrillation (AF) is a most common type cardiac arrhythmia characterized by uncoordinated and dyssynchronous atrial contraction resulting in irregular and often rapid excitation. Total atrioventricular (AV) block is the complete dissociation between the atrium and the ventricle. There is limited cases had been reported of AF with total AV block, can make a misinterpreted. A correct recognition is essential due to the risk of sudden cardiac death. The Deadly Silent of Atrial Fibrillation with Total Atrioventricular Block : A Case Report. C.F. Montang; P. Jeremiah; Hassanuddin; V. Chairiadi*** Intern in Cardiology and Vascular Medicine Departement, Undata General Hospital, Palu Cardiologist in Cardiology and Vascular Medicine Departement, Undata General Hospital, Palu Cardiologist, Head of Cardiology and Vascular Medicine Departement, Undata General Hospital, Palu Intern in Cardiology and Vascular Medicine Departement, Undata General Hospital, Palu Cardiologist in Cardiology and Vascular Medicine Departement, Undata General Hospital, Palu Cardiologist, Head of Cardiology and Vascular Medicine Departement, Undata General Hospital, Palu Case Illustration : A 63-year-old man patient presented with dyspnea and fatigue. His symptom were consistent with a class 3 New York Health Association cardiac function. His blood pressure was 140/80, heart rate 33/beats, auscultation of the chest revealed murmur. ECG showed absences of P waves with a regular QRS complexes. Echocardiography showed MR severe, TR moderate intermediate probability of PH, EF 62%, TAPSE 1,94cm. LVH (+) consentric, diastolic dysfungtion (+). The patient was given a sulfas atropine 0,5mg injection, dopamine 5cc/hours up titration, furosemide 40mg 1x1, spironolactone 25mg 1x1, candesartan 4mg 1x1 and simarc 2mg 1x1. After administration of atropine sulfate and dopamine intravenous injection the heart rate increased to 62/beats. Conclusion : Conclusion: Coronary CTA is a modality of choice to define angina causes in low risk patient. Cervical angina is one of possible diagnosis after rule out the cardiovascular cause. Coronary CTA is a modality of choice to define angina causes in low risk patient. Cervical angina is one of possible diagnosis after rule out the cardiovascular cause. K d i l i i h di i i A is a modality of choice to define angina causes in low risk patient. Cervical angina is o nosis after rule out the cardiovascular cause. ervical angina, coronary ct angiography, cardiac imaging The coronary CTA showed minimal plaque burden and stenosis at LAD and RCA p g Keywords: cervical angina, coronary ct angiography, cardiac imaging The coronary CTA showed minimal plaque burden and stenosis at LAD and RCA p Conclusion : Electrocardiography is a diagnostic for co-existing AF with total AV block, a correct diagnosis will provide initiation of definitive management. Electrocardiography is a diagnostic for co-existing AF with total AV block, a correct diagnosis will initiation of definitive management. Keywords: Atrial fibrillation, total av block, bradycardia, sudden death Keywords: Atrial fibrillation, total av block, bradycardia, sudden death Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rapid Management of Unstable Bradycardia Reveals Inferoposterior STEMI and Hyperkalemia: A Case Report K A Fit 1 K A H t 1 P S B t l2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Rapid Management of Unstable Bradycardia Reveals Inferoposterior STEMI and Hyperkalemia: A Case Report Rapid Management of Unstable Bradycardia Reveals Inferoposterior STEMI and Hyperkalemia: A Case Report K.A. Fitra1, K.A. Hasyaputra1, P.S. Bustanul2 Departement of Cardiology and Vascular Medicine, Royal Prima Hospital, Jambi, Indonesia2 Background: Sinus bradycardia is a common arrhythmia in patients with inferior or posterior acute myocardial infarctions (AMIs). Sinus Bradycardia is unstable when is associated with hypotension or shock, slower heart rates, changeable morphology of the P wave. The incidence of bradyarrhythmias in patients with ACS is 0,3% to 18%. Case Illustration: A 49-year-old man complained of typical angina 1 hour before admission. Vital signs show hypotension (75/39 mmHg), severe bradycardia (27 beats/min) and RR : 40 breathes/min. An ECG revealed junctional bradycardia and prolonged QT interval . Junctional bradycardia can be caused by hyperkalemia, and according to laboratorium examination, hyperkalemia is caused by Acute Kidney Injury. Bradycardia improved after administration of dopamine and CA Gluconate, but the patient still complained of chest pain, so a repeat ECG examination was performed which showed the results Atrial Fibrillation Normal Ventricular Response with ST-Elevation in the inferoposterior and right leads (Figure 1). The patient was diagnosed with Inferoposterior STEMI with Right Ventricular infarction and AF NVR exacerbated by Cardiogenic Shock and Hyperkalemia ec Acute Kidney Injury. It can be stated that the greatest possibility of bradycardia is caused by acute myocardial infarction and electrolyte disturbances in the form of overlapping hyperkalemia, so PCI management is needed. The patient received DAPT and statins as management for STEMI before being referred to Primary PCI. Conclusion: Early identification of STEMI and potential complications, including bradycardia, is necessary for proper care of the chest pain patient. It is imperative that the EM physician continues to remain up to date with management of the unstable patient with STEMI and be prepared to intervene if complications arise. Keywords: Bradyarrhythmias, Myocardial Infaction, Hyperkalemia Figure 1. ECG shows ST-Elevation in inferoposterior and right leads after the bradycardia was resolved. Keywords: Bradyarrhythmias, Myocardial Infaction, Hyperkalemia igure 1. ECG shows ST-Elevation in inferoposterior and right leads after the bradycardia was resolved. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Management of Acute Coronary Syndrome in Limited Resource Setting: a Case Series V. Calosa1, M. Valeria2, N. Dewi3 General Practitioner, Bajawa Public Hospital, East Nusa Tenggara, Indonesia1 General Practitioner, Inerie Public Health Center, East Nusa Tenggara, Indonesia2 Department of Internal Medicine, Bajawa Public Hospital, East Nusa Tenggara, Indonesia3 V. Calosa1, M. Valeria2, N. Dewi3 Background: Acute coronary syndrome (ACS) is a spectrum of coronary heart disease (CHD), which includes ST-elevation myocardial infarction (STEMI), non-ST elevation myocardial infarction (NSTEMI), and unstable angina. Prevalence of CHD in Indonesia is 1.5% based on the 2018 Indonesia Baseline Health Research. It is estimated 12.9% of deaths in Indonesia is caused by CHD based on the Sample Registration System survey in 2014. Classic symptom of ACS, substernal pressure-like chest pain that radiates to the jaw and/or left arm, does not always present. Often, other symptoms such as difficulty breathing, light-headedness, isolated jaw or left arm pain, nausea, epigastric pain, diaphoresis, and weakness present instead of the classic symptoms. The main treatment is to achieve reperfusion, either by fibrinolytics or percutaneous coronary intervention (PCI). In rural area, treating patients with ACS in the unavailability of reperfusion therapy might be challenging. Hereby we present a case series of four patients with ACS treated in limited-resource settings. Case Illustration: A sixty-nine-year-old man (Patient A), a seventy-six-year-old woman (Patient B), a sixty-two- year-old woman (Patient C), and a forty-one-year-old man (Patient D) were admitted with complaints of typical chest pain. A history of hypertension or dyslipidemia were noted in all patients. Electrocardiogram (ECG) findings were elevated ST-segments (Patient A, C, D) and flattened/inverted T-waves (Patient B). Reperfusion therapy was unavailable, thus we administered a loading dose of aspirin and clopidogrel followed by a maintenance dose, heparin infusion of 10,000 units/day for three days, isosorbide dinitrate, bisoprolol, and atorvastatin. Improved conditions and ECG findings were achieved within three-to-four-day hospitalization. Patients were referred to have coronary angiography and further management. Conclusion: In limited-resource settings where reperfusion therapy is unavailable, heparin infusion of 10,000 units/day with a loading dose of aspirin and clopidogrel followed by maintenance therapy may improve the patient’s conditions and ECG findings. p g Keywords: acute coronary syndrome, limited resource, heparin infusion, reperfusion, rural area Figure 1. Clinical, electrocardiogram, and laboratory findings regarding four patients in the case series Figure 1. Clinical, electrocardiogram, and laboratory findings regarding four patients in the case series Figure 1. Clinical, electrocardiogram, and laboratory findings regarding four patients in the case series Background Increase of ageing population and emerging cardiovascular disease updates may impact on conditions that previously underdiagnosed are being increasingly identified. Cardiac amyloidosis is a rare, multiorgan disease characterized by deposition of insoluble amyloid fibrils throughout tissues and organs. This case of light chain (AL) amyloidosis presenting with highly clinical suspicion of amyloidosis, comprehensive evaluation on patient with high suspicion of infiltrative disease plays pivotal role for its diagnosis. Case Illustration A 57-years-old male admitted with shortness of breath without any chest pain. On initial physical examination, his blood pressure was 80/52 mmHg with heart rate of 84x/min. He presented with elevated jugular venous pressure, rales, gallops, cool extremities, and bilateral pitting edema. He also had several skin bruises around the trunk and extremities. ECG reveals sinus rhythm, poor R wave progression, and low voltage. Chest X-ray shows cardiomegaly, pulmonary edema with right pleural effusion. Lab findings shows high level of NT-pro BNP and creatinine. Echocardiography results was suggestive of cardiac amyloidosis with concentric LVH, reduced LVEF, LV diastolic dysfunction grade III, mild functional mitral regurgitation, mild pericardial effusion, typical LV granule myocardial sparkling pattern and apical sparing bull’s eye pattern (cherry on top) on GLS. We performed further workup cardiac MRI results consistent with cardiac amyloidosis, scintigraphy shows grade 3 cardiac update, and serum free light chain (sFLC) test was abnormal with IgA Lambda oligoclonal gammopathy on immunofixation electrophoresis. For final diagnosis, we perform endomyocardial biopsy the result was positive on congo red staining, confirming diagnosis of amyloid monoclonal immunoglobulin light chain cardiomyopathy (AL-CM). Two Phenotypes of Cardiac Amyloidosis Presenting in a Single Patient: A Case Report M.R.R. Natadikarta1, N.Y. Kusumawardhani1, H.S. Prameswari1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia1 Mitral Annulus Calcification: When It Came with Many Disturbances D. Azhari1, F. Yuliani2, M. Yanni3 Cardiology and Vascular Medicine Residency, Andalas University, Padang, West Sumatera1 Cardiology and Vascular Medicine Residency, Andalas University, Padang, West Sumatera.2 Cardiology and Vascular Medicine Department, M. Djamil Hospital, Padang, West Sumatera3 D. Azhari1, F. Yuliani2, M. Yanni3 Background: Mitral Annulus Calcification (MAC) is a mitral valve degenerative disorder. Prevalence of MAC is between 8% and 15%, but it significantly increases with age and in patients with multiple cardiovascular risk factors. MAC is associated with increased risk of atrial fibrillation, enlargement of left atrium, increased rate of cardiovascular disease, and mortality. Case illustration: A 71-years old male presented with shortness of breath increased 1 day before admission. The patient has a history of 2-day watery diarrhea, chough, and fever. Patient was an excessive smoker. Physical examination showed irregular pulse, increase of jugular venous pressure, opening snap and mid diastolic murmur on auscultation, and ankle oedema in both lower extremities. Electrocardiogram demonstrated atrial fibrillation with normal ventricular response. Laboratory results showed thrombocytopenia, prolonged INR, hypoalbuminemia, and electrolyte imbalance. Transthoracic echocardiography revealed decreased LV systolic function EF 39%, severe mitral stenosis, moderate mitral regurgitation ec MAC, left and right atrium dilation, and concentric left ventricular hypertrophy. The patient was assessed with community acquired pneumonia, gastro- enteritis acute, hematuria, and suspected cardiac cirrhosis. Patient was treated with optimalization of heart failure therapy, treatment of infection, vitamin K therapy, correction of hypoalbuminemia and electrolyte imbalance. After six days hospitalized, the patient was discharged with stable condition. Patient was planned to have mitral valve replacement but refused and choosed optimal medical therapy. Conclusion: MAC is often clinically silent but can be symptomatic depending on severity of the mitral valve disease. Infection can lead to acute heart failure presentation on this patient. Treatment of precipitant and comorbidities will reduce days of hospitalization. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 61 y.o man, who had CABG procedure due to CAD and left leg amputation due to irreversible ischemia, presented with resting pain and open wound on his right foot since 6 months ago. Duplex Ultrasound and CT Angiography showed diffuse significant stenosis and calcified appearance with WIfI stage II and GLASS stage II. Patient was performed PTA with DEB at femoropopliteal artery, then blood flow returned to normal and many collateral infrapopliteal arteries appeared. One day prior to treatment, open wound healed and resting pain disappeared. Second patient, A 78 y.o high-risk man was admitted to hospital with unhealing gangrenes at left foot since 2 months ago and resting pain in both legs dominant in the right. Doppler ultrasound examinations showed abnormal ABI and abnormal flow at left infrapopliteal artery. Patient who also had WIfI stage II and GLASS stage II was performed Angiography and PTA procedures at femoropopliteal and infrapopliteal arteries. Necrotic wound at left foot started healing and pulse started to be detectable. Both patients were given wound care management dan multidiscipline approach to control risk factors. Background CLTI is a highly morbid disease, causing significant mortality, limb loss, pain, and diminished quality of life among those afflicted. The CLTI outcomes depends on primary and secondary care. PTA is widely used to improve blood flow and quality of life in CLTI patients. A.I. Nurudinulloh1, A.J. Firdaus1, N. Kurnianingsih2 A.I. Nurudinulloh1, A.J. Firdaus1, N. Kurnianingsih2 Resident of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia1 Department of Cardiology and Vascular Medicine, Vascular Division, Saiful Anwar General Hospital, Malang, Indonesia2 A.I. Nurudinulloh1, A.J. Firdaus1, N. Kurnianingsih2 Resident of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia1 Department of Cardiology and Vascular Medicine, Vascular Division, Saiful Anwar General Hospital, Malang, Indonesia2 gy , y , j y , g, Indonesia1 Department of Cardiology and Vascular Medicine, Vascular Division, Saiful Anwar General Hospital, Malang, Indonesia2 Conclusion AL-CM with grade 3 scintigraphy was extremely rare. This case highlights the critical importance of ruling out AL amyloidosis in all patients with positive uptake on scintigraphy to ensure such patients are not assumed to have transthyretin (ATTR) cardiac amyloidosis. y y Keywords: cardiac amyloidosis, cardiomyopathy, endomyocardial biopsy, infiltrative disease Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Keywords: CLTI, wound healing, PTA Background: g A pseudoaneurysm (PSA) is a locally-contained hematoma outside an artery due to damage to the vessel wall which is also recognized as a false aneurysm. Over years, PSA incidence has improved in ranges from 0.2% to 8% since the catheterization procedures are commonly used by various specialties. A significant complication in the form of femoral artery pseudoaneurysm is undergone by patients with diagnostic or therapeutic catheterization. Ultrasound is the quickest and best modality for assessment. Ultrasound can also be used for therapeutic probe compression known as Ultrasound-Guided Compression Repair (UGCR). Case illustration: Three patients have complained of swollen and pain around groin area after catheter removal. They were stable angina pectoris patients undergoing diagnostic and/or therapeutic catheterization. UGCR was performed to all of them. The first UGCR was successfully performed to a 66-year-old woman. The second one performed to a 76- year-old woman was not successful but the PSA resolved on its own. The last UGCR was also unsuccessful to a 75-year-old man and the PSA did not resolve but it was eventually successful after being managed by surgery. Generally, factors require the technical success consisting of the ability of patients to tolerate prolonged compression, the skill of examiners to control the hand fatigue and the PSA itself. Case Series Report: Ultrasound-Guided Compression Repair (UGCR) as a Non-Invasive Treatment of Pseudoaneurysms Complication of Catheterization in Dr. Saiful Anwar General Hospital Malang Case Series Report: Ultrasound-Guided Compression Repair (UGCR) as a Non-Invasive Treatment of Pseudoaneurysms Complication of Catheterization in Dr. Saiful Anwar General Hospital Malang N. Kurnianingsih1, A. Christine1 RSUD Dr. Saiful Anwar Malang1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Revascularization is the appropriate decision for limb saving in CLTI patients to restore blood flow, reduce ischemic burden, and optimize wound healing, which ultimately improves quality of life. Either PTA femoropopliteal or infrapopliteal promises a similar wound-healing quality. Keywords: CLTI, wound healing, PTA Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Case Series Report: Ultrasound-Guided Compression Repair (UGCR) as a Non-Invasive Treatment of Pseudoaneurysms Complication of Catheterization in Dr. Saiful Anwar General Hospital Malang Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: UGCR is a treatment that highly identified as a valid alternative, safe and cost-effective for patients with catheter-related femoral pseudoaneurysms and a normal coagulation profile. It is non-invasive and easier to perform than percutaneous and surgical repair. The success rate reaches 75% to 98%. Follow-up duplex scans are usually performed three to seven days after successful treatment. Further treatment may be suggested if the pseudoaneurysm is not completely removed by UGCR treatment. Keywords: pseudoaneurysm, ultrasound-guided compression repair Myocardial Dysfunction in Ketoacidosis: Myocarditis or Secondary Dysfunction due to Severe Acidosis? M.M. Sari1, D. Arara1 RSU Bunda Margonda1 Myocardial Dysfunction in Ketoacidosis: Myocarditis or Secondary Dysfunction due to Severe Acidosis? M.M. Sari1, D. Arara1 RSU Bunda Margonda1 M.M. Sari1, D. Arara1 RSU Bunda Margonda1 Background: Acidosis is one of the major consequences of hemodynamic instability associated with multiple organ failure and death. Most studies on the hemodynamic consequences of acidosis have been experimental, and thus far from the most common clinical situations. Myocarditis, and later myocardial dysfunction, in acidosis patients were reported in previous publication on 1996 and 2009. Case Illustration: A 37-year-old man was admitted to Hospital with acute onset of chest pain accompanied by shortness of breath, palpitation, vomiting, sweating since 5 hours before admission. The patient had history of diabetes, dieting for two weeks and not taking his medicines regularly. From PE were found diaphoresis and tachycardia with BP 130/80, HR 150 bpm, RR 32x/m. His ECG in ER showed Sinus Tachycardia with HR about 150 bpm. The laboratory tests showed pH 6,91; pCO2 16.0 ;HCO3 3.2, with RBG 611 mg/dL and ketone urine 4+. Echocardiography showed EF was 41%, with global Hypokinetic, reduced RV contractility, and ERAP 15. The next lab exam showed increased CRP and ESD levels. Unfortunately myocardial biopsy couldn’t be performed in out hospital. We suggest the patient had a myocarditis dd/ global myocardial dysfunction due to severe acidosis. Patient’s consciousness was deteriorated then being intubated and admitted to ICU. Patient was administrated with ramipril, spironolactone, dapagliflozin for heart failure and then corrected the ketoacidosis condition with insulin and sodium bicarbonate. In 3 days, the echocardiography evaluation showed the increased of EF from 41% to 60%, TAPSE 1.2 to 1.7 with stable hemodynamic condition then he was extubated. Patient was discharged in the 7th day of hospitalization in stable condition. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Clinical Presentation of MINOCA in Female with Thyroid Cancer Post Thyroidectomy ; A Case Rep Hanifah1, A. Puryani1, I.H. Siahaan2 General Practitioner, Persahabatan Central General Hospital1 Cardiologist, Persahabatan Central General Hospital2 Background: Myocardial infarction with non-obstructive coronary arteries (MINOCA) happens in 5-10% of all myocardial infarction (MI), and one third of these patients have ST-segment elevation in ECG. Patients with MINOCA were found having cancer more frequent than MI and obstructive coronary artery disease (MI-CAD). In cancer patients, ST-segment elevation is more likely to be found in MINOCA rather than MI-CAD. Also, MINOCA predominantly happens in females and younger patients. In this report we present the distinct characteristic of MI in cancer patient. p Case illustration: A 53 year old woman with thyroid cancer post total thyroidectomy presented to emergency room due to chest pain 6 hours before admission. The patient had hypertension and no history of anti-cancer therapy. The physical examination showed high blood pressure, tachycardia, tachypnea, desaturation, and bibasilar crackels in lungs. The electrocardiograms showed ST elevations in leads V1-V6, I and aVL. Laboratory test showed elevation of troponin I Hs 1306,6, and CKMB 84,9.The echocardiography showed Regional Wall Motion Abnormality (RWMA) : hypokinetics in mid-basal anterior-anteroseptal-inferoseptal-inferior, mild aortic and moderate mitral regurgitation, and ejection fraction 42 %. The patient was initially diagnosed with extensive anterior STEMI Killip class 2 and got loading dose of dual antiplatelet, nitrat sublingual, statin, and heparinization. After underwent the primary PCI, no significant stenosis coronary lesion to be found. Patient was treated conservatively. Conclusion: MINOCA is frequently happened in cancer patients due to either the cancer-related processes or the side effect of therapies. In comparison with MI-CAD, MINOCA patients are more likely to be younger and female. Recognition of the MI characteristic in cancer patients is essential to decide the proper treatment and prevention. Keywords: MINOCA, Cancer, STEMI Figure 1. Coronary angiography showed normal coronary arteries Figure 1. Coronary angiography showed normal coronary arteries Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Myocardial Dysfunction in Ketoacidosis: Myocarditis or Secondary Dysfunction due to Severe Acidosis? M.M. Sari1, D. Arara1 RSU Bunda Margonda1 Indonesian Journal of Cardiology Acute Aortic Syndrome in Suspected Marfan Syndrome: A Neglected Case in Rural Area K. Luthfiyah1, M.R.M.P. Sinurat1 Cileungsi General Hospital1 Background. g Acute aortic syndromes are a life-threatening medical condition, including acute aortic dissection (AAD), intramural hematoma (IMH) and penetrating atherosclerotic aortic ulcer (PAU). Disruption of the media layer of the aorta is the common feature.1 Most patients in AAD are male, aged >70 years, presents with systemic hypertension, atherosclerosis and iatrogenic causes, whereas Marfan syndrome is seen predominantly in younger patients.2 In Marfan syndrome, the connective tissue disorder which caused by heterozygous mutations in FBN1, leads to progressive aortic dilatation, thus increasing the risk of acute aortic dissection, with the incidence ranges from 8.4% to 18%.3-5 It is important to recognize Marfan Syndrome early to provide appropriate treatment to prevent life-threatening complications. Case Illustration. A 17-year-old male presented with chest pain accompanied by shortness of breath since 3 days before admission. Physical examination showed increased arm-span to height ratio (>1.05) with arachnodactyly, positive wrist and thumb signs, pectus carinatum and ectopia lentis. The patient was afebrile, tachypneic and had wide pulse pressure. An early diastolic murmur high pitched grade 2/4 at upper right sternal border was found. Electrocardiography showed sinus tachycardia with lateral wall ischemia and Left Ventricular Hypertrophy. Chest x-ray showed cardiomegaly. Bedside echocardiography documented an intimal flap in the proximal ascending aorta and aortic root, suggestive Stanford Type A AAD. The patient was then referred to National Cardiovascular Center Harapan Kita Hospital to undergo surgical treatment. Conclusions The underlying pathology of Myocarditis and Myocardial dysfunction in acidosis patient remains uncleared. Multiple mechanism might play a role. Acidosis can cause cardiac contractile dysfunction and cause injury on the cellular level then lead to myocardial dysfunction. Keywords: Myocardial dysfunction, Myocarditis, Acidosis Keywords: Myocardial dysfunction, Myocarditis, Acidosis Picture 1. (a) Echocardiography showed EF was 41%, with global Hypokinetic. (b) Echocardiography showed TAPSE was 1.2. Picture 1. (a) Echocardiography showed EF was 41%, with global Hypokinetic. (b) Echocardiography showed TAPSE was 1.2. Indonesian Jour Cardiology Indonesian J Cardiol 2023 pISSN: 2830-3105 / eISSN doi: 10.30701/ijc.1 Acute Aortic Syndrome in Suspected Marfan Syndrome: A Neglected Case in Rural Area K. Luthfiyah1, M.R.M.P. Sinurat1 Cileungsi General Hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion. Diagnosing acute aortic syndrome in early-stage Marfan syndrome is quite difficult due to absence of typical features and lack of resources. This circumstances can lead to under-diagnosis which increase mortality rate. Surgical treatment is still the mainstay of management for Stanford Type A AAD and needed to reduce mortality rate. Keywords: Acute Aortic Syndrome, Aortic Dissection, Marfan Syndrome, Bentall Procedure Figure 1: Echocardiography documented an intimal flap in ascending aorta Figure 1: Echocardiography documented an intimal flap in ascending aorta Tetralogy of Fallot With Labiopalathoschiziz : Let’s Total Correction or Not? F.H. Alkaf1, A. Priyatno2 F.H. Alkaf1, A. Priyatno2 Faculty of Medicine, Diponegoro University, Semarang, Indonesia1 Department Cardiology and Vascular Medicine, Kariadi Hospital, Semarang, Indonesia2 , y Faculty of Medicine, Diponegoro University, Semarang, Indonesia1 Department Cardiology and Vascular Medicine, Kariadi Hospital, Semarang, Indonesia2 Case Illustration : An 8-year-old boy with Tetralogy of Fallot, polydactyly, history of 3-step surgery to the correction of labiopalatoschiziz. With chief complaints of cyanotic, the body weight is difficult to gain, get tired easily, have speech disorders, and tend to squat in daily life as well. Saturation oxygen was 66%/65%//71%/74%. S1 normal, S2 weakened, systolic ejection murmur grade III/6 with punctum maximum at the left parasternal line SIC II. With nutritional status 16.5 kg, 117 cm, WAZ: -3.54, HAZ: -2.06, BMI: -3.42. Impression: Poor nutrition, very less weight. Laboratory haemoglobin 21.8 g/dL and hematocrit 68.7%. Chest x-ray CTR 48%, booth shaped, narrowed retrosternal space, RV cardiomegaly. EKG Sinus rhythm, 81x/min, RAD, RVH. Echo results: overriding aorta <50%, VSD malalignment, RV hypertrophy, severe PS with PG 75 mmHg. He was a successful total correction procedure and the condition was improved. Conclusion : Background : g Tetralogy of Fallot is the most common cyanotic heart defect with an incidence of 5% to 10% of all CHD events. The four hallmark features of the malformation that consists of an interventricular communication, also known as a ventricular septal defect, obstruction of the right ventricular outflow tract, override of the ventricular septum by the aortic root, and right ventricular hypertrophy. Background: g Cardiac CT angiography (CTCA) examinations frequently include the calcium score, which is a highly effective predictor of coronary heart disease events. A small field of view that is focused on the heart is typically used for CTCA tests. Coronary artery calcification and aortic calcification share similar risk factors, including advanced age and hypertension, they both require screening. By acquiring a larger field of view, they can be obtained simultaneously using a CT Angiography and commonly diagnosed incidentally during imaging for other indications. Incidental Non-Cardiac Findings of a Cardiac CT angiography : Should We Only Concentrate on the Heart? Incidental Non-Cardiac Findings of a Cardiac CT angiography : Should We Only Concentrate on the Heart? A.P. Febrianto1, H. Arifianto1 Department Cardiology and Vascular Medicine, Sebelas Maret Hospital1 Case illustration: 74-year-old woman, with intermediate likelihood of significant coronary artery disease presented with atypical chest pain was referred for CTCA. The physical examination revealed a firm, pulsatile abdominal mass. She had documented history of hypertension, dyslipidemia and no known history of aortic aneurysm. To evaluate abnormality in abdominal aortic, we decided perform larger field of view, by using run-off computed tomography angiography (CTA) with region of interest (ROI) set of images over bifucartio of abdominal aorta. Then patient undergoing CTCA, the result was CACS 2559.4 dan AAS >10.000, with moderate stenosis at shaft LM and mid LAD due to calcified plaque. Incidentally, CTA abdominal aorta demonstrated a massive, tortuous and fusiform aneurysm of the abdominal aorta measuring up osteal aneurysm diameter of 1.77 cm, distal 2.15 cm, widest diameter 7.74 cm and 18.26 cm in the transverse diameter infrarenal arteries and extending to the aortic bifurcation with extensive calcification. total correction Conclusion : The management of patients with TOF has its own challenges both for diagnosis and management because not all areas have adequate human resources or equipment. It is now expected that the prognosis of TOF patients will improve substantially because of the advances in surgical and medical management that have occurred over the last few decades. The decision to proceed with total correction or not depends on various factors such as the severity of the condition, the age of the patient, the presence of other health issues, and the risks and benefits of the surgical procedure. Keywords: Tetralogy Of Fallot, Labiopalathoschiziz, Malnutrition, Total Correction Keywords: Tetralogy Of Fallot, Labiopalathoschiziz, Malnutrition, Total Correction Keywords: Tetralogy Of Fallot, Labiopalathoschiziz, Malnutrition, Total Correction ) Pulmonic Stenosis by Angiography, (B) Echocardiography with VSD malalignment, (C) Labiopalathoschiziz and Polydactyly, (D) Booth shaped by Thorax Xray ) Pulmonic Stenosis by Angiography, (B) Echocardiography with VSD malalignment, (C) Labiopalathoschiziz and Polydactyly, (D) Booth shaped by Thorax Xray Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Incidental Non-Cardiac Findings of a Cardiac CT angiography : Should We Only Concentrate on the Heart? Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background Left main coronary artery closure during DCA is a rare but catastrophic event with a poor prognosis. Emergency reperfusion of the LMCA with hemodynamic support should be the primary goal in this setting. We describe a patient with LMCA disease that developed acute chest pain and pulse VT and was treated with an emergency LM stenting. Left main coronary artery occlusion following diagnostic coronary angiography : A disaster in the cath lab A. Trissatharra1, Y.H. Oktaviono1 1.Department of Cardiology and Vascular Medicine, Faculty of Medicine, Airlangga University / Dr. Soetomo Hospital Surabaya A. Trissatharra1, Y.H. Oktaviono1 1.Department of Cardiology and Vascular Medicine, Faculty of Medicine, Airlangga University / Dr. Soetomo Hospital Surabaya A. Trissatharra1, Y.H. Oktaviono1 1.Department of Cardiology and Vascular Medicine, Faculty of Medicine, Airlangga University / Dr. Soetomo Hospital Surabaya Case Illustration A 58-year-old man was admitted for an elective DCA, having reported episodes of chest pain and dyspnea on light exertion within a few months prior. His past medical history included dyslipidemia, TIA, and smoking. From the ECG, an anteroseptal OMI was obsereved and echocardiography showed hypokinetic anterior segment. DCA was performed through access from femoral artery with 6F Judgkin Left 4.0 catheter to LMCA. During cineagiography acquisition, patient exclaimed of a sudden chest pain, which progressed into a decrease of consciousness, and hypotension. The monitor showed an ST-elevation of aVR, followed by an onset of VT with pulse. Cardiopulmonary resuscitation was performed and the angiography showed critical stenosis of LMCA. The LMCA ostium was engaged with 6F 3.5 guide catheter, followed by guidewire penetration into the lesion. Dilatation was performed with a 2.0 x 15 mm balloon followed by insertion of a drug eluting stent Amphilimus (3.00 x 20 mm) to the LMCA, the stent was successfully implanted. Due to high thrombus burden, the patient was also given Intravenous GPIIB/IIIA. Other than a short period of gross hematuria, the patient had quite an uneventful event and discharge 4 days later with 100 mg aspirin and 75 mg clopidogrel. Conclusion LMCA occlusion can be rapidly fatal, making a rapid assessment and action crucial in its management. Therefore, operator and personnel’s alertness in signs of acute LMCA occlusion is paramount during any coronary interventional settings. Conclusions: ons: report emphasizes the usefulness of CTCA with large field of view as a screening technique for g incidental non-cardiac abnormalities with the same risk factor. ds: Abdominal Aortic aneursym, Cardiac CT angiography Giant, Tortuous and fusiform aneurysm of the abdominal aorta This case report emphasizes the usefulness of CTCA with large field of view as a screening technique fo identifying incidental non-cardiac abnormalities with the same risk factor. Keywords: Abdominal Aortic aneursym, Cardiac CT angiography This case report emphasizes the usefulness of CTCA with large field of view as a screening technique for identifying incidental non-cardiac abnormalities with the same risk factor. Keywords: Abdominal Aortic aneursym, Cardiac CT angiography Giant, Tortuous and fusiform aneurysm of the abdominal aorta Giant, Tortuous and fusiform aneurysm of the abdominal aorta Giant, Tortuous and fusiform aneurysm of the abdominal aorta Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Keywords Keywords Left main coronary artery, coronary angiography, stents Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 The Deadly Peripartum Cardiomyopathy: A Case Report F. Rachmaningrum1, D.J. Sugeng1, G. Tryan1, R. Ekaputra1 1RSU Banyumanik 2, Semarang, Indonesia I p The Deadly Peripartum Cardiomyopathy: A Case Repor F. Rachmaningrum1, D.J. Sugeng1, G. Tryan1, R. Ekaputra1 1RSU Banyumanik 2, Semarang, Indonesia The Deadly Peripartum Cardiomyopathy: A Case Report F. Rachmaningrum1, D.J. Sugeng1, G. Tryan1, R. Ekaputra1 1RSU Banyumanik 2, Semarang, Indonesia The Deadly Peripartum Cardiomyopathy: A Case Report F. Rachmaningrum1, D.J. Sugeng1, G. Tryan1, R. Ekaputra1 1RSU Banyumanik 2, Semarang, Indonesia p y g g Case illustration : A 25-years-old primiparous woman came to the emergency room five months after giving birth, reporting dyspnea, palpitations, edematous extremities, nausea, and bloating that had lasted two months. During examination, the patient had a blood pressure of 130/82 mmHg, a pulse rate of 116 bpm, a respiratory rate of 28/minute, and oxygen saturation of 95% on room air. Auscultation of the lungs revealed rhonchi sound at the basal. Cardiovascular exam revealed regular heart sounds with murmur and JVD 5+4 H2O. Chest x-ray examination showed cardiomegaly with pulmonary congestion. A transthoracic echocardiogram showed an LVEF of 25%, global hypokinesis, dilated all heart chambers, and thrombus at LV apex. Patient was given diuretic followed by administering ARB, beta-blocker, MRA and anticoagulant. The next day, patient had a seizure, monitor ECG showed ventricular tachycardia. The fourth day of treatment, patient had cardiac arrest, cardiopulmonary resuscitation was performed, but the patient died. Background : g Peripartum Cardiomyopathy (PPCM) is the main cause of heart failure in pregnancy with high morbidity and mortality, marked by reduction of LVEF <45% with no other cause of heart failure. The clinical signs and symptoms are dyspnea, orthopnea, and paroxysmal nocturnal dyspnea. PPCM usually overlaps with physiological changes in pregnancy and makes it underdiagnosed. p Conclusion : Peripartum cardiomyopathy is a rare life-threatening cardiomyopathy. The prognosis for women with PPCM depends on improvement of left ventricular size and function within 6 months after delivery. Delayed recovery was associated with delayed diagnoses, higher NYHA functional class, LV thrombus, and coexisting medical illnesses. Mortality rate varies widely (7-50%). The common cause of death are progressive heart failure, arrythmia, or thromboembolism. Due to its morbidity and mortality, careful cardiac follow up is essential for pregnant women who develop symptomps. p g p y p p Keyword : Peripartum cardiomyopathy, heart failure, pregnancy, postpartum Keyword : Peripartum cardiomyopathy, heart failure, pregnancy, postpartum Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g May-Thurner syndrome, also known as iliac vein compression syndrome, is a rare medical condition in which the left iliac vein is compressed by the adjacent right iliac artery. This can lead to the development of deep vein thrombosis (DVT), usually in the leg. We report a case of how we encountered a suspected May-Thurner syndrome in a rural area. Case Illustration A 43 years old female, presented with acute unilateral left lower limb swelling 6 hours before admission. experienced erythema, pain, and tenderness alongside the limb, Homans sign (+). On physical examination, the vital sign is within normal limit. Peripheral pulse is difficult to assess due to swelling. The right and left ankle-brachial index (ABI) was 1.1 and 1.3, respectively. Glucose blood level was 289 mg/dL. ECG showed sinus rhythm. Doppler ultrasound showed high burden thrombus and no flow throughout the left lower limb vein system, suggestive compression of the left iliac vein. Patient was diagnosed with acute DVT and suspected May-Thurner Syndrome. After optimal anticoagulant therapy, she was referred to a tertiary hospital to undergo further assessment and mechanical thrombectomy/angioplasty. PATENT DUCTUS ARTERIOSUS SUCCESSFULLY CLOSED WITH INTRAORAL IBUPROFEN IN PRE-TERM NEONATE : A CASE REPORT FROM RURAL R.K. Ratri1, C. Saputra2, Sieeny3, J.A. Widodo3 General Practitioner of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara1 Cardiology and Vascular Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara2 Pediatrics and Perinatal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara3 Background: Patent ductus arteriosus (PDA) is a common cardiovascular condition in pre-term neonate, defined as the failure of ductus arteriosus closure within 48 hours after birth. Symptomatic PDA provides significant hemodynamic consequences which can lead to significant respiratory distress. We chose Ibuprofen as medical treatment of choice for PDA cases in preterm. Case Illustration: A ten days old boy was born via elective cesarean section at 35 weeks and 2 days of gestation, weight 2.250 grams, with an Apgar score of 6/9 body temperature was 36.9 ℃, heart rate was 148 beats/min and regular, respiratory rate was 67 times/min, and blood oxygen saturation level was 97%. Day ten after birth a continuous heart murmur at the left upper sternal border was noted. Initial echocardiography on admission demonstrated the presence of PDA. The treatment strategy was to administer a total of three doses of intraoral ibuprofen, which an initial dose of 10 mg/kg at 0 hour followed by 20 mg/kg at 24 and 48 hour then. 24 hours after the first ibuprofen administration, no continuous murmur was heard, heart rate from 160-180 beats/min to 128 beats/min, as well as in the respiratory rate from 64-68 breaths /min to 46 breaths/min, retractive breathing also showed a reduction, and without demonstrated side effects. The patient remains in good systemic condition and was discharged from the hospital on day 18th after birth. Conclusion: Pre-term neonate with symptomatic PDA has been successfully treated with intraoral ibuprofen. Ibuprofen administration might be a useful medical treatment option. The patient’s condition has been improved and PDA was spontaneously closed with medication. p y Keywords: Patent Ductus Arteriosus, Ibuprofen, Pre-term neonate Bedside Echocardiography Exam showed PDA LR shunt Bedside Echocardiography Exam showed PDA LR shunt Indonesian Jou Cardiolog Indonesian J Cardiol 2023 pISSN: 2830-3105 / eISSN doi: 10.30701/ijc. A Case Report: Acute Deep Vein Thrombosis in Suspected May-Thurner Syndrome M.R.M.P. Sinurat1, D.S. Kusumaningayu1 Cileungsi Hospital, Bogor, Indonesia1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology A Case Report: Acute Deep Vein Thrombosis in Suspected May-Thurner Syndrome M.R.M.P. Sinurat1, D.S. Kusumaningayu1 Cileungsi Hospital, Bogor, Indonesia1 eywords: Thyroid Heart Disease, Hyperthyroid, Atrial Fibrillation, Acute Decompensated Heart Failure Conclusion Unprovoked DVT can be triggered by May-Thurner syndrome. Early diagnosis and referral could lower long- term complications and mortality rates. Keywords: May-Thurner Syndrome, Deep Vein Thrombosis Keywords: May-Thurner Syndrome, Deep Vein Thrombosis Doppler ultrasound showed thrombus and positive compression ultrasound (CUS) Doppler ultrasound showed thrombus and positive compression ultrasound (CUS) Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 A 36 Years Old Female with Acute Decompensated Heart Failure (ADHF) and Atrial Fibrillation (AF) Associated with Hyperthyroidism : A Case Report A 36 Years Old Female with Acute Decompensated Heart Failure (ADHF) and Atrial Fibrillation (AF) Associated with Hyperthyroidism : A Case Report Tricuspid Valve Obstruction Caused by Giant Intracardiac Tumor in Children : A Time Bomb A.R. Pratama1 RS AR Bunda Lubuk Linggau1 Background: Intracardiac tumor in children are rare and commonly benign, rhabdomyomas and fibromas are the most common benign cardiac tumors in children, but tumor with originate from the cardiac valve usually is papillary fibroelastomas, although are benign but can compromise cardiac function because of their potential for pulmonary artery obstruction or pulmonary embolism. A 36 Years Old Female with Acute Decompensated Heart Failure (ADHF) and Atrial Fibrillation (AF) Associated with Hyperthyroidism : A Case Report K. Nisa1, A. Ferly2 Pupuk Kaltim Hospital, Bontang, East Borneo, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Indonesia ‐ Nationa Cardiovascular Center Harapan Kita, Jakarta, Indonesia2 K. Nisa1, A. Ferly2 Pupuk Kaltim Hospital, Bontang, East Borneo, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Indonesia ‐ National Cardiovascular Center Harapan Kita, Jakarta, Indonesia2 Background: Hyperthyroidism affects the cardiovascular system in the form of hemodynamic changes and direct effect on the myocardium, which can lead to heart failure (HF) and arrhythmias (mostly AF) if not treated properly. Case Illustration: A 36-year-old female came to the emergency room with shortness of breath accompanied by palpitation and tremors. The patient had a history of hyperthyroid disease since she was 26 years old that wasn’t treated routinely for the past 5 years. On physical examination, blood pressure was 153/118 mmHg, heart rate 140-150 bpm, respiratory rate 22-24 bpm, temperature 36.3oC. The cardiovascular examination showed the heart rate was 188 bpm with irregular rhythm. The EKG showed atrial fibrillation-rapid ventricular response (AF RVR). The result of thyroid function was hyperthyroid. Chest X-ray showed cardiomegaly, pleural effusion and pulmonary edema. Echocardiography showed dilatation of all four cardiac chambers, with EF:28% (global hypokinetic), TAPSE: 14 mm. The patient was diagnosed with ADHF and AF RVR ec Thyroid Heart Disease (THD) treated with Furosemide, Spironolactone, Ramipril, Extra Fargoxin, Simarc, Enoxrin, Tyrozol. The patient was monitored and showed improvement, and was discharged after the 4th day of treatment. Early and optimal control of thyroid levels is key in the management and prevention of THD. B-blocker group is the first choice for rate control in AF but in this case of ADHF with AF, digoxin is a better choice. Hyperthyroidism caused HF via two main pathways: (1) tachycardia-induced HF with LV dysfunction, (2) RV heart failure due to the RV volume overload. HF in hyperthyroidism has 3 phases: hyperkinetic, normokinetic, followed by hypokinetic. Conclusion: Hyperthyroidism that's not treated properly can cause HF and AF, which increases the risk of Conclusion: Hyperthyroidism that's not treated properly can cause HF and AF, which increases the risk of disability and death. It is important to raise patient’s awareness to take medications regularly and routinely check for hyperthyroid treatment and cardiac function. A 36 Years Old Female with Acute Decompensated Heart Failure (ADHF) and Atrial Fibrillation (AF) Associated with Hyperthyroidism : A Case Report Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Tricuspid Valve Obstruction Caused by Giant Intracardiac Tumor in Children : A Time Bomb A R P t 1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 DIFFERENT ETIOLOGIES EMBOLISM ON ACUTE LIMB ISCHEMIA PATIENTS A.P. Febrianto1, A. Yasa1, H. Sulastomo1 Department Cardiology and Vascular Medicine, Dr. Moewardi Hospital1 Background: g The etiology of acute limb ischemia are largely due to embolism and thrombosis. Most cases are cardiac embolism, among which atrial fibrillation accounts for the majority due to the formation of mural thrombi. Other causes include valvular diseases, thrombosis due to an occlusive atherosclerotic lesion, and paradoxical embolism. We report 2 cases of acute limb ischemia patient with different sources embolism. Case illustration: Case 1, a 41-year-old woman suffered from right lower limb pain since 6 hours. She had cold acral, low sensory, peripheral saturation right lower limb was zero, also no pulse femoral right artery. Waveform was dampened and monophasic with soft thrombus occlusion in the right femoral artery from Duplex Ultrasound . DSA was performed, total occlusion was revealed in the osteal of right common femoralis artery. Transthoracic echocardiogram revealed signs of pulmonary hypertension (PH), dilated right ventricle and atrium, also we found secundum Atrial septal secundum right-to-left shunt. We suspected source of thrombus was from paradoxical embolic event. Case 2, a 54-year-old woman suffered from left lower limb pain and his left foot purple-turned for 15 hours. No pulse at left poplitea artery and his peripheral saturation left lower limb was zero. ECG showed Atrial Fibrillation normoventricular response. No flow waveform in left poplitea artery was found during Duplex UltraSound. Total occlusion was revealed at the osteal of right common femoralis artery from DSA. Transthoracic echocardiogram showed Mitral regurgitation severe, dillated left cardiac chambers and spontaneous echo contrast. We revealed source of thrombus was from micro thromboembolic event. Conclusions: Case Illustration: A 19-months old boy had presented with fever and shortness of breath of four days duration, previously the patient often complained of dyspneu since the age of 6 months without signs of cyanosis. At admision, he had signs and symptoms febrile, irritable, tachypnea and tachycardia. His Chest X-Ray demonstrated cardiomegaly, from laboratory result showed thrombocytopenia (24.000/uL) and hypoalbuminemia, due to found cardiomegaly patient was consulted for Echocardiography. Transthoracic echocardiography showed RA dilatation, a large tumor attached tricuspid valve with largest measured 2.17 cm x 1.88 cm, the right ventricle was severely underloaded and had dyskinetic wall motion. LV function was normal with paradoxical movement seen. Pericardial effusion is seen arround the cardiac chambers. Due to the potential for life-threatening embolic events, the patient was referred to tertiary hospital for further examination and the possibility of surgical resection and analysis histopathology of tumor, but unfortunately two days before surgery, the patient passed away. Conclusion: Intracardiac tumors are rare and mostly benign in children, they can compromise cardiac function depending on their size and location. A clinician should be aware of symptoms of heart failure such as dyspnea and signs of murmurs because the presence of a tumor can increase the risk of a life-threatening embolic event. Keywords: Intracardiac, tumor, children, embolic, cardio-oncology Keywords: Intracardiac, tumor, children, embolic, cardio-oncology (A) Chest X ray showed cardiomegaly (B) Transthoracic echocardiography apical 4 chamber view (A) Chest X ray showed cardiomegaly (B) Transthoracic echocardiography apical 4 chamber view Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Hyperkalemia induced Pseudo-infarction pattern: A Case Report M.R.M.P Sinurat1, N.M. Asoehan1 Cileungsi hospital, Bogor, Indonesia1 Case Illustration A 64 years old female, experienced severe vomitus and shortness of breath since 1 day before admission. On physical examination, She was normotensive, slight bradycardia and tachypnea with severe desaturation (65% on room air). ECG showed left bundle branch pattern (Modified Sgarbossa = 0) with flattened P wave and peak and tall T in V1-3. Potassium, ureum and creatinine level 209 mg/dL and 7.3 mg/dl, respectively. Hyperkalemia may present as an ST segment elevation that lead to misinterpretation and delay definitive therapy. ECG changes in hyperkalemia include: peak of the T wave, PR interval prolongation, widening of the QRS complex, flattening of the P wave. Our patient refused to undergo urgent haemodialysis and unfortunately died due to malignant arrhythmia. Background Background Electrolyte imbalances can cause ECG (Electrocardiogram) abnormalities that are often ambiguous. We report a death case of acute untreated chronic kidney disease presenting with pseudo-infarction pattern. Conclusions: This serial case emphasized the necessity to consider possible causes in ALI-cases Keywords: Acute Limb ischemia, paradoxical embolism, Atrial Fibrillation Different etiologies embolism on acute limb ischemia patients Different etiologies embolism on acute limb ischemia patients Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Challenging Permanent Pacemaker Implantation in a Patient with Total AV Block and Rare Vascular Anomaly: A Case Illustration of an 81-Year-Old Woman with Left Persistent Superior Vena Cavae A. Kurniawan1, A. Rizal1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; dr. Saiful Anwar General Hospital1 A. Kurniawan1, A. Rizal1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; dr. Saif Anwar General Hospital1 Case illustration: An 81-year-old woman was admitted to the hospital due to a total atrioventricular block. The patient had previously been treated with temporary pacemaker placement, but it was decided to perform permanent pacemaker implantation due to the persistence of the AV-block. However, during the procedure, the lead encountered technical difficulties in reaching the right ventricle, resulting in continuous displacement toward the coronary sinus. After several maneuvers were attempted without success, a rare anatomical variant of left persistent superior vena cava (LPSCV) was suspected as the culprit. The LPSCV was confirmed by venography, and ultimately, alternative techniques were employed to successfully position the lead in the right ventricle. LPSCV is a rare anatomical variation that can pose significant challenges during permanent pacemaker implantation. In some cases, selective maneuvers may be required to position the lead in the right ventricle. Therefore, a thorough understanding of the specific handling and appropriate maneuvers is essential to overcome these challenges and ensure successful permanent pacemaker implantation in patients with LPSCV. Background: Permanent pacemaker implantation is a crucial procedure for patients with total AV block to avoid life-threatening complications. However, anatomical abnormalities like left persistent superior vena cavae (LPSCV) can pose a challenge during implantation. y Conclusion ST segment elevation is not always indicate a myocardial infarction. Other condition such as hyperkalemia could mimic the ECG changes, comprehensive evaluation is crucial in identifying and managing this similar but comprising an entirely distinct mechanistic cause. Keywords: Hyperkalemia, ST Elevation, ECG Keywords: Hyperkalemia, ST Elevation, ECG ECG showed left bundle branch pattern (Modified Sgarbossa = 0) with flattened P wave and peak and tall T in V1-3. ECG showed left bundle branch pattern (Modified Sgarbossa = 0) with flattened P wave and peak and tall T in V1-3. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Challenging Permanent Pacemaker Implantation in a Patient with Total AV Block and Rare Vascular Anomaly: A Case Illustration of an 81-Year-Old Woman with Left Persistent Superior Vena Cavae A. Kurniawan1, A. Rizal1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; dr. Saiful Background: g Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease characterized by fibrofatty infiltration and arrhythmias. The diagnosis of ARVC is based on a combination of clinical, electrocardiographic, imaging, and pathological criteria. The major criteria for the diagnosis of ARVC include regional wall motion abnormalities and/or RV dilatation or reduced ejection fraction, ventricular arrhythmias, and tissue characterization by cardiac MRI, which can reveal fibrofatty infiltration. Successful Catheter Ablation of Ventricular Tachycardia in a Patient with Arrhythmogenic Right Ventricular Cardiomyopathy: A Case Report Successful Catheter Ablation of Ventricular Tachycardia in a Patient with Arrhythmogenic Righ Ventricular Cardiomyopathy: A Case Report A. Kurniawan1, A. Rizal1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; dr. Saiful Anwar General Hospital1 A. Kurniawan1, A. Rizal1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya; dr. Saif Anwar General Hospital1 Case illustration: We report the case of a 39-year-old male with a history of recurrent ventricular tachycardia (VT) who had undergone multiple hospitalizations and cardioversion. A Holter monitor showed multifocal PVCs with a dominant RVOT anterior endocardial origin. The patient was suspected to have ARVC, and a cardiac MRI revealed late enhancement in the transmural basal anterior, basal anteroseptal, mid anterior, mid anteroseptal, and apical anterior regions. The patient had a hypokinetic left ventricle with an ejection fraction (EF) of 45.2% and a reduced RV EF of 31.5%. The EKG during VT was also consistent with one of the major criteria for ARVC. The patient underwent unsuccessful catheter ablation. During follow up patient still experienced palpitations, and a repeat ablation was performed using HD Mapping ablation and selective maneuvers were employed to successfully eliminate ventricular tachycardia. The HD Mapping ablation technique has been shown to improve the success rate of ablation procedures in patients with complex arrhythmias, including ARVC. Conclusion: Permanent pacemaker implantation in patients with LPSCV is challenging and requires specific handling and appropriate maneuvers. Selective maneuvers can be effective in overcoming technical difficulties during lead placement. Keywords: Permanent pacemaker, left persistent superior vena cavae, technical difficulties Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Successful Catheter Ablation of Ventricular Tachycardia in a Patient with Arrhythmogenic Right Ventricular Cardiomyopathy: A Case Report Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Stress myocardial computed tomography perfusion (CTP) is a novel examination that provides both anatomic and physiological assessment of myocardial ischemia. Stress myocardial CTP outperformed single-photon emission computerized tomography (SPECT) and echocardiography, and was comparable to magnetic resonance imaging (MRI) and positron emission tomography in terms of accuracy in measuring myocardial perfusion. computerized tomography (SPECT) and echocardiography, and was comparable to magnetic resonance imaging (MRI) and positron emission tomography in terms of accuracy in measuring myocardial perfusion. We present a smoker, 45 years old male, complained about typical chest pain, undergoing first stress CTP procedure in Indonesia. We present a smoker, 45 years old male, complained about typical chest pain, undergoing first stress CTP procedure in Indonesia. Myocardial CT Perfusion Stress Test: The First Experience in Indonesia S.D. Handari1,3, Y.W. Ashari2, A.F. Rahimah3, W. Karolina3, I. Kamelia2 1Cardiovascular Imaging Division, Premier Hospital, Surabaya, Indonesia; 2Cardiology Resident at Faculty of Medicine Universitas Brawijaya-Saiful Anwar General Hospital, Malang, Indonesia; 3Cardiovascular Imaging Division, Faculty of Medicine, Universitas Brawijaya-Saiful Anwar General Hospital, Malang, Indonesia; S.D. Handari1,3, Y.W. Ashari2, A.F. Rahimah3, W. Karolina3, I. Kamelia2 1Cardiovascular Imaging Division, Premier Hospital, Surabaya, Indonesia; 2Cardiology Resident at Faculty of Medicine Universitas Brawijaya-Saiful Anwar General Hospital, Malang, Indonesia; 3Cardiovascular Imaging Division, Faculty of Medicine, Universitas Brawijaya-Saiful Anwar General Hospital, Malang, Indonesia; S.D. Handari1,3, Y.W. Ashari2, A.F. Rahimah3, W. Karolina3, I. Kamelia2 1Cardiovascular Imaging Division, Premier Hospital, Surabaya, Indonesia; 2Cardiology Resident at Faculty of Medicine Universitas Brawijaya-Saiful Anwar General Hospital, Malang, Indonesia; 3Cardiovascular Imaging Division, Faculty of Medicine, Universitas Brawijaya-Saiful Anwar General Hospital, Malang, Indonesia; S.D. Handari1,3, Y.W. Ashari2, A.F. Rahimah3, W. Karolina3, I. Kamelia2 1Cardiovascular Imaging Division, Premier Hospital, Surabaya, Indonesia; Case Illustration A 45 years old male, with risk factor heavy smoker and premature coronary artery disease (CAD) in his family, came to the cardiac outpatient clinic with typical chest pain. There were no history of hypertension, diabetes mellitus, nor dyslipidaemia in this patient. Electrocardiography, laboratory findings, and chest radiograph showed normal result. Echocardiography evaluation and treadmill stress test were also normal. We assessed pre-test probability score in this patient, was 22%. Using GE Revolution Apex 512 slices, we performed a comprehensive cardiac computed tomographic protocol combining computed tomography angiography (CTA) and CTP to provide a simultaneous assessment of both coronary artery anatomy and ischemia. Stress-rest procedure using adenosine and iso-osmolar contrast media. Total calcium score was 22 with evidence of coronary soft plaque 20- 30% at LMS-osteal LAD (CAD-RADS 2/V) with no ischemia inducible. Total radiation during procedure was 14.96 mSv. Conclusion: In summary, the diagnosis of ARVC is based on a combination of clinical, genetic, and imaging criteria. Recurrent arrhythmias in ARVC patients pose a significant management challenge, but the use of HD Mapping ablation techniques may improve success rates. Keywords: Arrhythmogenic right ventricular cardiomyopathy, catheter ablation, HD mapping Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Stress myocardial CTP, which might serve as a "one-stop-shopping" tool for the diagnosis and overall care of CAD patients, is currently the only non-invasive modality that providing an integrated anatomical/functional analysis. It therefore helps reducing the possible over-indication to myocardial revascularization. Keywords: Computed tomography myocardial perfusion, Myocardial perfusion imaging, Stress perfusion, Non- invasive diagnostic, Coronary artery disease Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Pulmonary Atresia with Intact Ventricular Septum: A Nightmare in Neonatal Intensive Care Unit M.M. Sari1, D. Arara1 RSU Bunda Margonda1 Background g Infective endocarditis (IE) is a serious infection that affects the heart and can be life-threatening if not recognized and treated promptly. IE with multiple complications poses a high mortality rate. The management of IE involves a multidisciplinary approach. Treatment goals include eradication of the infectious agent, prevention of further complications, and a surgical approach. Pulmonary Atresia with Intact Ventricular Septum: A Nightmare in Neonatal Intensive Care Unit M.M. Sari1, D. Arara1 RSU Bunda Margonda1 (b) Echocardiography showed intact ventricular septum with Tricuspid Regurgitation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 "Rising to Challenge: A Case Report on Managing Infective Endocarditis with Multiple Complications and Re-expansion Syndrome" Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology sing to Challenge: A Case Report on Managing Infective Endocarditis with Multiple Complications and Re-expansion Syndrome" M.S. Goma1, S.Y. Pradana1, A.Z. Abidin1, D.Y. Kurniawan1, S. Sungkar1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Diponegoro University- Dr. Kariadi Central General Hospital, Semarang, Indonesia1 Case Illustration A 59-year-old woman presented with symptoms of shortness of breath, fatigue, and fever, and reported a history of chills and sweating at night. On examination, a cardiac murmur erb’s area was heard, and there was a janeway lesion on her palmaris, her electrocardiogram showed sinus rhythm with a normal axis. Further laboratory tests revealed, elevated inflammatory marker, and elevated creatinine levels. y A Chest X-ray showed left ventricular and left atrial enlargement. Echocardiography examination showed vegetation at the aortic valve, and perivalvular abscess. The patient was diagnosed with definite IE with severe aortic regurgitation.We gave her empiric antibiotics and heart failure medication. At 5 days hospitalisation the clinical condition worsened and hematopneumothorax was present, and patient proceeded to water-seal drainage (WSD) insertion. After 4 hours WSD insertion, the patient felt more takipneu with rales in both lungs indicating acute lung oedema, patient developed re-expansion syndrome. Due to progessive respiratory distress and hemodynamic instability, the patient was sent to the CVCU for further monitoring and management. Conclusion Pulmonary Atresia with Intact Ventricular Septum: A Nightmare in Neonatal Intensive Care Unit M.M. Sari1, D. Arara1 RSU Bunda Margonda1 M.M. Sari1, D. Arara1 RSU Bunda Margonda1 Background: Pulmonary atresia (PA) is a heart defect present at birth that's usually diagnosed soon after birth. The incidence of Pulmonary atresia was about 9.6% of all congenital heart defect. Pulmonary atresia (with Ventricular Septal Defects (VSD) or Intact Ventricular Septum (IVS)) is one of congenital heart disease that can be classified as a duct dependent pulmonary circulation. Babies with pulmonary atresia need immediate treatment which may include medications and one or more procedures or surgeries. Case Illustration: Newborn term-baby girl was consulted by Consultant Neonatal Intensive Care to Cardiologist due to desaturation since birth. The mother had a delivery by C-section due to high-risk pregnancy (multipara and the mother was 40 years old). From the clinical examination were found continuous murmur at LUSB with pansystolic murmur grade III/VI at LLSB. Other physical findings were unremarkable. From chest x ray was found a cardiomegaly. Echocardiography showed Pulmonary atresia with intact ventricular septum, 3 mm PDA and moderate tricuspid regurgitation with right ventricular hypertrophy. We administrated the patient with drip prostaglandin E1 to keep the ductus arteriosus still open. We also took off the oxygen supplementation and the baby saturation were stable between 75-85%. We also look for referral hospital with advance cardiac pediatric care to manage this patient. In the 4th days of hospitalization, the baby finally can be referred to other hospital and she was performed balloon pulmonary valvulotomy. Unfortunately, in a 7th day of hospitalization, the baby was passed away due to multi-organ failure. Conclusion: Recognizing the cardiac defects by physical examination, chest x ray and echocardiography are mandatory in baby with suspected congenital heart disease. Prostaglandin drip with procedures or surgery is a mandatory management in PA-IVS patient. eywords: Congenital Heart Disease, Pulmonary Atresia, Intact Ventricular Septum, Ductus Arteriosus, t Picture 1. (a) Echocardiography showed “no flow” from RV to PA with Ductus Arteriosus. (b) Echocardiography showed intact ventricular septum with Tricuspid Regurgitation Picture 1. (a) Echocardiography showed “no flow” from RV to PA with Ductus Arteriosus. Background ac g ou d Coronary Artery Disease (CAD) is still one of the cardiovascular diseases with high mortality rate, responsible for around 8.9 million deaths each year1. Acute Coronary Syndrome (ACS) requires immediate diagnosis within 10 minutes with an electrocardiography (ECG)2. Identification of ST-segment elevation is very important, including in aVR lead. However, sometimes the aVR lead is neglected lead to find ST-elevation in ACS cases3. Several studies shown that ST-elevation in the aVR lead can be associated with various conditions including Left Main disease with a high mortality rate.4,5 Case illustration A 72-year-old man presents to emergency room complaining severe typical chest pain and shortness of breath for approximately 2 days. Physical examination revealed rhonchi at the lung bases. ECG examination found ST- segment elevation in aVR lead and ST-segment depression in I, II, aVL, aVF, V5 and V6 leads. ST-elevation in aVR lead may associate with critical Left Main Coronary Artery (LMCA) occlusion. Its not always be total occlusion that leads to rapid cardiac arrest.6,7 Laboratory tests showed increased cardiac enzymes both CKMB and troponin I. Patients diagnosed with high risk NSTEMI and referred immediately to PCI on capable hospital. Prior to coronary angiography, an echocardiographic examination revealed decreased EF to 37.8% accompanied by global hypokinetic. Angiography revealed critical LMCA occlusion with some calcification and non-dominant Right Coronary Artery (RCA). Early PCI was performed by placing two-DES stent in the occluded blood vessel. After procedure, chest pain significantly relieved. Importance of aVR Lead in Patient with Acute Coronary Syndrome :A Case Report G.F. Ramadhan1, T. Rahadiyan2 RS DKT Dr Bratanata Jambi1 RS DKT Dr Bratanata Jambi dan RSUD Raden Mattaher Jambi2 y Conclusion Managing infective endocarditis with multiple complications is quite tough and requires a collaborative effort from multidisciplinary specialties.Re-expansion syndrome is a rare but potentially fatal iatrogenic complication of thoracocintesis or tube thoracostomy. Vigilant monitoring and early diagnosis are crucial to prevent adverse outcomes and improve patient outcomes. fective Endocarditis, Complications, Re expansion Syndrome There was a vegetation at the aortic valve and perivalvular aortic abscess Keywords: Infective Endocarditis, Complications, Re expansion Syndrome There was a vegetation at the aortic valve and perivalvular aortic abscess Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Importance of aVR Lead in Patient with Acute Coronary Syndrome :A Case Report G.F. Ramadhan1, T. Rahadiyan2 RS DKT Dr Bratanata Jambi1 RS DKT Dr Bratanata Jambi dan RSUD Raden Mattaher Jambi2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Marfan syndrome (MFS) is an autosomal dominant, age-related but highly penetrant condition with substantial intrafamilial and interfamilial variability. The most prominent manifestations of MFS are asymptomatic aortic root aneurysms and aortic dissections. Untreated aortic root aneurysms can progress to life-threatening acute aortic dissections. In type A dissection patients with or without Marfan syndrome, open surgery was the preferred treatment method Case Illustration A-31-year-old women admitted due to shortness of breath with chest discomfort. Physical examination revealed long arm span (1,078) compared to her height, high arched palate, Pulse pressure 109/53 mmHg, early diastolic murmur grade III/IV high pitched, loudest at Erbs area, pulsatile abdominal pulse periumbilical, with wrist sign and thumb sign suggesting Marfan syndrome with aortic insufficiencies (AI). Cardiomegaly, scoliosis vertebra thoracal with congestive pulmonum was noted by chest x-ray. Echocardiography revealed severe AI and moderate TR, dilatation in aorta 29 mm with intimal flap distal of sinotubular junction, decreased LV systolic function EF 26%. In MSCT Aorta revealed Intimal flap from ascending aorta and abdominal aorta at Th.12 vertebrae (± 3.23 cm long), suspected aortic dissection.We managed to optimalized medical therapy while preparing for surgical procedure. Conclusion From this case, it shows that the importance of holistic ECG interpretation including aVR lead in ACS cases. Electrocardiography not only utilized as diagnostic tool, but also as predictor for patient outcomes. So, as practitioners we can provide adequate therapy to avoid complications. Keywords: aVR lead, ST-elevation, electrocardiography, left main disease, acute coronary syndrome a: ECG found ST-elevation in aVR lead and ST-depression in multiple leads; b-e: Angiography revealed significant stenosis 90-95% LMCA, 70-80% LAD and 80-90% LCx, hence 2-DES Stenting performed a: ECG found ST-elevation in aVR lead and ST-depression in multiple leads; b-e: Angiography reveale significant stenosis 90-95% LMCA, 70-80% LAD and 80-90% LCx, hence 2-DES Stenting performed Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Medical Management in Standford A Aortic Dissection Complicating long Standing Severe Aortic Insufficiency in Marfan women : A Bridge too far I.P. Farissa1, F. Tandri1, Y. Herry1, R. Hidayatullah1, S. Sungkar1 RSUP dr Kariadi/Diponegoro University1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Medical Management in Standford A Aortic Dissection Complicating long Standing Severe Aortic Insufficiency in Marfan women : A Bridge too far I.P. Farissa1, F. Tandri1, Y. Herry1, R. Hidayatullah1, S. Sungkar1 RSUP dr Kariadi/Diponegoro University1 Keywords: myocarditis, heart failure, sglt2inhibitors Conclusion Aortic dissection and insufficiency are common complications of Marfan syndrome. Early diagnosis and prompt treatment are crucial in preventing life-threatening complications, including aortic rupture and death. Medical management of aortic dissection and insufficiency in Marfan syndrome requires a multidisciplinary approach. Although the management of aortic dissection and insufficiency in Marfan syndrome is complex, with appropriate medical care and support, patients can lead healthy and fulfilling lives. Keywords: aortic insufficiency, marfan syndrome, aortic dissection Keywords: aortic insufficiency, marfan syndrome, aortic dissection Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 SGLT2 Inhibitors as a Promising Treatment Option for Myocarditis-Related Heart Failure F.Q. Decroli1, M. Yanni2 RSUP M DJAMIL1 Division of Cardiology and Vascular Medicine , M. Djamil Hospital Padang2 SGLT2 Inhibitors as a Promising Treatment Option for Myocarditis-Related Heart Failure Background : Myocarditis-related heart failure is a condition that can cause significant morbidity and mortality. Although traditional treatments for heart failure are useful in many instances, a significant number of patients continue to experience symptoms and have unfavorable results. SGLT2 inhibitors, may have added advantages for individuals with myocarditis-induced heart failure accompanied by a reduced ejection fraction (HFrEF). This case study demonstrates a myocarditis patient who received supplementary SGLT2 inhibitor therapy, resulting in improved outcomes. Case Illustration : a 44-year-old female who exhibited symptoms of chest pain, fatigue, and shortness of breath. Physical examination was within normal limit, ECG Showed LAD with LVH, CXR showed CTR 63% and NT- Pro BNP had a level of 4001 pg/ml. Echocardiography revealed that the patient had a reduced ejection fraction of 32% and with global hypokinesia and dilation of LV, suggested as Myocarditis with reduced EF. Coronary angiography was performed, which yielded no blockages, leading to the conclusion that coronary occlusion has been ruled out. The patient was initially treated with standard heart failure therapy, However, due to the poor response to initial therapy, an SGLT2 inhibitor was added to the treatment regimen. The patient showed significant improvement in ejection fraction after the addition of the SGLT2 inhibitor. After 10 months of treatment, the patient's symptoms improved significantly, and her ejection fraction increased to 55%. In addition, NT-proBNP levels improved from 4001 pg/mL to 107 pg/mL within 10 months of therapy. The patient's condition also progressed positively, moving from NYHA Functional Class III to NYHA Functional Class I. Conclusion : This case highlights the potential benefits of SGLT2 inhibitors in the management of myocarditis- related HFrEF in a single patient. Further studies are needed to confirm these findings, but our experience suggests that SGLT2 inhibitors may represent a valuable addition to standard heart failure therapy. Keywords: myocarditis, heart failure, sglt2inhibitors Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 BACKGROUND Cardiomyopathy in young people especially those associated with HIV infection has been reduced since the era of Highly Active Antiretroviral Therapy (HAART). In the era of post-HAART, manifestations of HIV-associated cardiomyopathy with impaired LV systolic function approximately about 1-3% in HIV-infected people. In this case we presenting how to diagnose and proper management in such of patient. CASE ILLUSTRATION A 27-year-old male patient who works as a health worker, came to the emergency room with complaints of shortness of breath with light activity in the last 2 weeks accompanied by orthopnea and leg edema. Other risk factors besides smoking such as family history, hypertension, diabetes mellitus, and dyslipidemia was denied. He was diagnosed with HIV on ART since 3 years ago. From echocardiography showed all chamber dilatation, global hypokinetic and significant decrease of LV systolic function (LVEF 16%). The patient was then treated by optimizing HF therapy in collaboration with internal medicine to continue HIV therapy in the patient. A YOUNG MALE PATIENT WITH CARDIOMYOPATHY ASSOCIATED WITH HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION IN THE ERA OF HIGHLY ACTIVE ANTIRETROVIRAL THERAPY A. Wicaksono1, H. Martini2, A.F. Rahimah2, A. Kurniawan1 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia Background Atrial septal defects (ASDs) are the most prevalent congenital heart defects in adults. Transcatheter device closure has become the treatment of choice for secundum ASDs with suitable anatomy. Left ventricular disease in elderly may increase the risk of pulmonary oedema following ASD closure. Fluid challenge test can be used as an invasive hemodynamic test to unmask heart failure with preserved ejection fraction (HFpEF) and to assess the risk of acute LV failure post-ASD closure in elderly patients. PCWP > 15 mmHg confirms the presence of HFpEF; increased PCWP to a value of > 20 mmHg or > 10 mmHg from the baseline after fluid challenge test is expected as high risk of acute pulmonary edema and ASD closure should be abandoned. The Compulsory of Fluid Challenge Test in Elderly Patients Prior to Transcatheter Closure of Atrial Septal Defects The Compulsory of Fluid Challenge Test in Elderly Patients Prior to Transcatheter Closure of Atr Septal Defects C.P. Iswandi 1, C.J. Cool 1 , H.S. Prameswari 1 1Department of Cardiology and Vascular Medicine, University of Padjadjaran, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia C.P. Iswandi 1, C.J. Cool 1 , H.S. Prameswari 1 1Department of Cardiology and Vascular Medicine, University of Padjadjaran, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia C.P. Iswandi 1, C.J. Cool 1 , H.S. Prameswari 1 1Department of Cardiology and Vascular Medicine, University of Padjadjaran, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia Case illustration A 77-year-old man with a long-standing uncorrected secundum ASD had chief complaints of dyspnea on effort and leg edema for two years. This patient underwent echocardiography showing secundum ASD of 18 mm with favorable anatomy for percutaneous closure and likely HFpEF (Intermediate HFA-PEFF Score). Because of patient’s worsening of symptoms and ASD being hemodynamically significant, transcatheter ASD closure was considered to improve symptoms. Prior to ASD closure, fluid challenge test using normal saline 0,9% 5mL/kg BW showed non-significant increased PCWP (12 -> 14 mmHg), supporting the low risk of acute LV failure post ASD closure and no diastolic dysfunction. The results emphasized that patient’s symptoms were solely due to ASD without HFpEF. We decided to proceed for the ASD closure using an ASD Occluder device no.21 and the patient was discharged the next day without signs of pulmonary edema. CONCLUSION In HIV patients who have fallen into heart failure, a proper diagnosed using relevant tools could be a reference for clinician to make a right decision. Prompt treatment combination of optimal HF therapy and HIV therapy are becoming the keys to the treatment. Keywords: Cardiomyopathy, Heart Failure, HIV, Antiretroviral Echocardiographic picture of the patient with all chamber dilatation Keywords: Cardiomyopathy, Heart Failure, HIV, Antiretroviral Echocardiographic picture of the patient with all chamber dilatation Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 The Compulsory of Fluid Challenge Test in Elderly Patients Prior to Transcatheter Closure of Atrial Septal Defects Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Improvement Left VeNtricular Ejection Fraction in Cardiomyopathy: Is It CRT or PCI? Y. Newary. D.L. Munawar1 Binawaluya Hospital1 Case Illustration A 65-year-old man presented with chronic heart failure with NYHA class III/IV and comorbid type 2 DM and chronic kidney disease with electrocardiography showed second degree AV block and left bundle branch block. We performed echocardiography and the left ventricular ejection fraction (LVEF) was 18% at that time. Our patient was implanted with a cardiac resynchronization device with pacemaker. Right ventricular and left ventricular leads were also implanted. We also performed percutaneous intervention with implanted 3 stents at RCA which was total occluded. The patient did not complaint any symptomps after procedure and the outcome was good. After 4 moths of CRT with PCI optimization, our patient's clinical status had significantly improved with LVEF was 37% and NYHA class II of heart failure. Conclusion This clinical case illustrates the value of fluid challenge test before percutaneous closure of ASD in elderly patients to confirm HFpEF and assess the risk of acute LV failure post-ASD closure. Keywords: atrial septal defect, fluid challenge test, HFpEF, device closure This clinical case illustrates the value of fluid challenge test before percutaneous closure of ASD in elderly patients to confirm HFpEF and assess the risk of acute LV failure post-ASD closure. Keywords: atrial septal defect, fluid challenge test, HFpEF, device closure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Improvement Left VeNtricular Ejection Fraction in Cardiomyopathy: Is It CRT or PCI? Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Ischaemic Cardiomyopathy (ICM) represents an important cardiovascular condition associated with substantially increased morbidity and mortality. it is well recognized that postinfarction patients with heart failure face a greater mprtality risk than those with a nonischaemic pathogenesis of heart failure whereas heart failure itself is a high risk factor for sudden death. therefore accodring to ESC 2021 Heart Failure patients with these indications are recommended for CRT therapy to reduce the risk of sudden death. We do know performing CRT on patient with heart failure as a primary prevention for sudden cardiac death and the research improving cardiac function still lack of. In this case report, we will present the outcome of cardiomyopathy patient undergone PCI and CRT. Case Illustration Keywords: ischaemic cardiomyopathy, PCI, CRT Case illustration A 76-year-old woman presented to ER with history of typical chest pain and fever for two days. Physical examination showed BP 89/61 mmHg, HR 71 bpm, without any abnormalities. Initial ECG revealed ST-elevation in all leads except aVR (Figure 1). Laboratory tests were significant for CK (1690 U/L), CK-MB (109 U/L), TnT- hs (2579 pg/mL), NT-proBNP (24807 ng/L) and elevated CRP (156 mg/L). Echocardiogram showed RWMA, grade-1 diastolic dysfunction, mild mitral, pulmonary, and tricuspid insufficiency with reduced LVEF of 39%. DAPT and statin, and other symptomatically drugs were given. Catheterization was then performed and revealed non-obstructed coronary arteries. Due to these result, suspicion to myopericarditis was considered and colchicine was given additionally. Following 6-days follow-up revealed a complete resolution of the signs, symptoms in conjunction with her ECG results. Background g ECG is a simple and useful tools which help clinician in detecting heart diseases. Myopericarditis is one of the heart disease that could give a variety clinical presentation and represent numerous ECG results which could mimicking other diseases without certain specific pathognomonic. Therefore, clinical diagnosis is often challenging and could lead to a misdiagnosis. Here we present a case with ST-segment elevation with absence of coronary artery obstruction or coronary spasm mimicking ACS in myopericarditis. The Dramatic ECG Evolution in Suspected MI Type 2: A Case Report I. Sabrina1, A.A. Lukito1 Siloam Hospital Lippo Village1 Conclusion Diagnosing myopericarditis is quite challenging, as in this case, it was mimicking ACS with ST-elevation MI type 2. Several differential diagnosis must be considered in the case like these to prevent a misdiagnosis. Therefore, taking into account its variety signs and symptoms, thorough history taking, physical examination, and appropriate investigations such as careful analysis of ECG results are crucial to work-up the diagnosis and management of myopericarditis. Keywords: ECG evolution, MI type 2, Myopericarditis Initial ECG revealed ST-segment elevation in all leads except lead aVR (top). After 6 days follow-up, ECG revealed complete resolution into normal (bottom) Conclusion Patients with dilated cardiomyopathy who undergone PCI after CRT implantation could have significant improvement in cardiac function with improvement LVEF. Changes in LVEF were inversely associated with the risk of mortality. The extent of LVEF improvement aster revascularization might be a potential factor which defines clinical outcomes. Keywords: ischaemic cardiomyopathy, PCI, CRT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 63-year-old man was admitted to Emergency Room (ER) with main complaint of shortness of breath. This conditions got worsen in two days especially when doing daily activities. He was diagnosed with Congestive Heart Failure since some years ago. On physical examination, his heart beats was pounding irregularly and there was murmur heard at mitral valve projections. From the ECG, there was atrial fibrillation with a wide QRS which looked like a PVC, that appeared after long and short RR interval. This was known as Ashman phenomenon. Chest X-Ray’s patient showed cardiomegaly with congestive pulmonum and there was also protruding left atrial appendage. This was supposedly caused by long standing atrial fibrillation. Then, the patient was treated with digoxin to control the heart rate. Background g Ashman phenomenon is defined as the appearance of abberant conduction, shown in ECG as wide QRS complex after long RR interval and a short RR interval, respectively. It is commonly found during atrial fibrillation. It was first found in 1947 by Dr. Gouaux and Dr. Ashman. Although this is not clinically important because there is no influence in mortality or morbidity patients, Ashman phenomenon frequently is confused with PVC or rarely ventricular tachycardia as this condition has different prognosis and treatment. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 ASHMAN PHENOMENON IN ATRIAL FIBRILLATION, IS IT USEFUL IN CLINICAL PRACTICE? A CASE REPORT A. Siswanto1 RSD Balung Jember1 Keywords: ECG evolution, MI type 2, Myopericarditis Keywords: ECG evolution, MI type 2, Myopericarditis Initial ECG revealed ST-segment elevation in all leads except lead aVR (top). After 6 days follow-up, ECG revealed complete resolution into normal (bottom) Background g Right ventricular thrombus are a rare phenomenon, encountered almost exclusively in patients with suspected or proven pulmonary embolism and diagnosed by transthoracic echocardiography. Currently the optimal management of right ventricular (RV) thrombus not well established. We present a case of RV thrombus in complicated by pulmonary embolism and Hepatocellular Carcinoma. Case Illustration A-58-year-old man admitted to Emergency Department because shortness of breath when doing strenuous activity since 1 months and getting worse in last 1 week. Patients with history of hepatocellular carcinoma (HCC) and had done Transarterial chemoembolization (TACE). From physical examination, there was a mild leg edema and normal heart sounds, without murmur and gallop. The ECG revealed Sinus Rhythm, Right Axis Deviation and Right Ventricullar hypertrophy. Cardiomegaly was noted by chest x-ray. Echocardiography revealed normal Left Ventricle function with RV Thrombus, Dilated RA , RV, Smallish LV which lead to severe tricuspid regurgitation. He was diagnosed as suspect pulmonary embolism and RV Thrombus. MSCT Angiography revealed hypodense lesion in the left pulmonary artery (length ± 4.08 cm, thickness ± 0.47 cm) suspected thrombus. The patient was commenced on antithrombotic with enoxaparin injection, aspilet and clopidogrel. There was improvement and then patient discharge with rivaroxaban as antithrombotic. p g Three months later patient checks routinely into the outpatient department with relieved shortness of breath. Echocardiography was performed then revealed improving Left Ventricle function but RV Thrombus was bigger. Conclusion Conclusion As atrial fibrillation prevalence is high, there may be Ashman phenomenon appears often in daily clinical practice. Even though it doesn’t show any important message, but clinicians understanding to this phenomenon may help them to distinguish it from premature ventricular complexes. This is mainly because PVC or rarely ventricular tachycardia has more serious complications and needs to be treated agressively. Keywords: atrial fibrillation, ashman phenomenon, aberrant conduction Figure 1. ECG showed long interval RR (L) and short interval RR (S) followed by abberant QRS complex (A) As atrial fibrillation prevalence is high, there may be Ashman phenomenon appears often in daily clinical practice. Even though it doesn’t show any important message, but clinicians understanding to this phenomenon may help them to distinguish it from premature ventricular complexes. This is mainly because PVC or rarely ventricular tachycardia has more serious complications and needs to be treated agressively. Keywords: atrial fibrillation, ashman phenomenon, aberrant conduction Figure 1. ECG showed long interval RR (L) and short interval RR (S) followed by abberant QRS complex (A) re 1. ECG showed long interval RR (L) and short interval RR (S) followed by abberant QRS complex (A) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Right ventricular thrombus constitutes a rare yet potentially fatal situation, whose optimal management remains controversial. The described case illustrates the crucial role of transthoracic echocardiography as the election diagnostic tool in this setting, as it allows for the thrombus detection and characterization, with the inherent therapeutic strategy implications. Keywords: Right ventricular thrombus, pulmonary embolism Right ventricular thrombus Case Illustration : 59 years old female came to Emergency Room with chief complaint typical chest pain that occurred 12 hours before admission. She was once undergoing PCI through right radial artery 2 months before. She had hypertension and type 2 diabetes mellitus. Patient’s heart rate was 75 bpm, blood pressure 130/70 mmHg. There was pulsatile, palpable mass with thrill at the post site of catheterization with bruit.The electrocardiography result was old pathologic q wave at lead III and aVF. Transthoracic echocardiography showed EF 46%, TAPSE 1.9 cm, concentric LVH, and regional wall motion abnormalities. Vascular doppler ultrasound showed a radial-cephalic arteriovenous fistula on the right superior limb. Invasive strategy was performed through the right transradial access with conventional technique combined with balloon-assisted tracking (BAT) technique to navigate through AV fistula. WAYFINDING OF AN IATROGENIC AV FISTULA FOR PCI IN A PATIENT WITH ACUTE CORONARY SYNDROME O. Sofyan1, A. Hafid1 RSUP dr Wahidin Sudirohusodo1 Background: The current international Coronary Angiography guideline reccomendates transradial access (TRA) for diagnostic and intervention percutaneous coronary because of the decreasing major side effect compared to transfemoral access (TFA). However, it needs to aware that coronary angiography has occurring complications, including Arteriovenous Fistula (AV fistula). Radial Iatrogenic AV Fistula have been reported varies from 0% to 2% and a big study reported that the incidence was 0,04%. Coronary Angiogprahy and percutaneous coronary intervention (PCI) is increasingly performed in patients with Acute Coronary Syndrome and AV fistula, including end-stage renal disease (ESRD), however studies on vascular access are limited. Both iatrogenic and hemodialysis AV fistulae usually prevents ipsilateral transradial access (TRA) for coronary angiography. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Right ventricular thrombus, pulmonary embolism Keywords: Right ventricular thrombus, pulmonary embolism Keywords: Right ventricular thrombus, pulmonary embolism Right ventricular thrombus Right ventricular thrombus Conclusions : In conclusion, we described patient with a case of coronary angiography and percutaneous coronary intervention via left TRA with navigation through an iatrogenic AV fistula. This case report emphasize the technique that could be used to navigate invasive strategy through AV fistula, although radial access for coronary angiography may be limited in such patient. Keywords: Iatrogenic Arteriovenous Fistula, Transradial Access, Percutaneous Coronary Intervention, Acute Coronary Syndrome Coronary Syndrome Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: STEMI, diabetes mellitus, rural area, heart failure ST-Segment Depression Morphology Differences: Pearl and Pitfall for Untrained Eye N. Sukmadi1, M.G. Suwandi2, C. Saputra3, W. Wiriadisurya4 FKIK Atma Jaya / RSK Marianum Halilulik1 RSPAU dr. S. Hardjolukito2 RSUD Mgr. Gabriel Manek3 RSK Marianum / RSUD Mgr. Gabriel Manek4 N. Sukmadi1, M.G. Suwandi2, C. Saputra3, W. Wiriadisurya4 FKIK Atma Jaya / RSK Marianum Halilulik1 RSPAU dr. S. Hardjolukito2 RSUD Mgr. Gabriel Manek3 RSK Marianum / RSUD Mgr. Gabriel Manek4 Background: Electrocardiogram (ECG) is a powerful clinical tool to choose a proper treatment. ST-Segment depression is one of common feature of an ECG. Patient with ST-Segment depression has broad clinical spectrum, from ischemia and/or infarction to LVH, LBBB, or even electrolyte disturbance. In this report, we present a case series about 2 patients with different disease but with similar ECG features for untrained eye. Case illustration: Our first case, 61 years old male came to emergency room (ER) with sudden typical chest pain, and shortness of breath. Patient heart rate was 126 bpm, blood pressure 192/100 mmHg, with respiratory rate of 40/minute, and oxygen saturation of 75%. The ECG results showed an oblique ST elevation in aVR with global horizontal and down-slopping ST depression (I, II, III, aVF, V2-V6). NSTE-ACS very high risk was diagnosed. Our second case, 31 years old women came to ER with general weakness, epigastric pain, and vomiting. Patient heart rate was 84 bpm, blood pressure was 150/102 mmHg, with respiratory rate of 20/minute, and oxygen saturation of 99%. The ECG results showed a QT prolongation with plateau ST elevation in aVR with global horizontal and up slopping ST depression (I, II, aVF, V2-V6). Severe hypokalemia was suspected, and confirmed with potassium concentration of 1.3 mmol/L. Conclusions: ST-Segment depression could be tricky for untrained eye. Clinicians should know to assess the morphology of ST-Segment depression, which could have deleterious effect when wrong interpreted. Down slopping and horizontal ST-Segment depression suggestive an ischemia event, but up slopping suggest a non- ischemia event. Keywords: ST-Segment Depression, Morphology Keywords: ST-Segment Depression, Morphology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Challenges in Managing ST-Elevation Myocardial Infarction (STEMI) Anteroseptal with Diabetes Mellitus in a Rural Area Hospital: A Case Report Background: Periprocedural myocardial infarction (MI) is frequent complication of percutaneous coronary intervention (PCI) associated with an increased rate of cardiovascular events. However, the detection of periprocedural MI in calcified coronary lesion is still not clear. HOW TO IDENTIFY PERIPROCEDURAL MYOCARDIAL INFARCTION IN CALCIFIED CORONARY LESION: A CASE REPORT T. Aryanugraha1, M.S. Rohman1 1Departement of Cardiology and Vascular Medicine, Faculty of Medicine, Brawijaya University ‐ dr.Saiful Anwar General Hospital, Malang East Java, Indonesia T. Aryanugraha1, M.S. Rohman1 1Departement of Cardiology and Vascular Medicine, Faculty of Medicine, Brawijaya University ‐ dr.Saif Anwar General Hospital, Malang East Java, Indonesia Case Illustration: A 73 year-old female came to our hospital because of stable chest pain while doing moderate activity. Four month prior to admission, she had diagnostic coronary angiography procedure with calcified long stenosis 80-90% in proximal-mid left anterior descending (LAD) artery was reported. Clopidogrel and aspirin were routinely consumed as dual antiplatelet therapy. PCI procedure was conducted with predilatation Scorflex NC balloon 2.5 x 15 mm at proximal-distal LAD then implantation of 2 DES, Cre8 3.5 x 46 mm at osteal-distal LAD and Combo Plus 3.5 x 15 mm at distal LAD (overlapped). She felt chest pain after procedure that relieved by isosorbide dinitrat. Fifteen hours after the PCI procedure, the patient felt weakness, bradycardia with total atrioventricular block seen in the ECG monitor and increased of troponin I level (0.7 g/L  12.1 g/L) and the patient was diagnosed with myocardial infarction associated with PCI. We treated the patient with dual antiplatelet therapy and unfractionated heparin (UFH) bolus continued with continuous infusion until 2x24 hours. Coronary angiography evaluation showed stent under-expansion with septal branch occlusion because of shifting plaque. Temporary pace maker (TPM) was inserted for 7 days. Due to the patient still pacemaker dependent, permanent pace maker (PPM) was implanted. She also had complication of pneumonia during hospital care. No event of subsequent acute coronary syndrome was observed. Challenges in Managing ST-Elevation Myocardial Infarction (STEMI) Anteroseptal with Diabetes Mellitus in a Rural Area Hospital: A Case Report Challenges in Managing ST-Elevation Myocardial Infarction (STEMI) Anteroseptal with Diabetes Mellitus in a Rural Area Hospital: A Case Report C. Dharwindo1 Rumah Sakit Penyangga Perbatasan1 Background: ST-Elevation Myocardial Infarction (STEMI) is a medical emergency that requires prompt diagnosis and treatment to prevent significant morbidity and mortality. Diabetes Mellitus (DM) is a known risk factor for cardiovascular disease and increases the risk of developing a STEMI. In rural areas, the management of STEMI can be challenging due to limited resources, inadequate healthcare infrastructure, and delayed patient presentation. This case report describes the challenges encountered in the management of a patient with STEMI Anteroseptal and DM, who presented to a rural area hospital. Case Illustration: An 84-year-old male with a past medical history of diabetes mellitus presented to the emergency department with complaints of atypical chest pain that had been ongoing for 2 days. The patient reported that the pain was initially mild but had progressively worsened over the last few days. The patient denied any shortness of breath, dizziness, or syncope. Upon arrival, the patient's vital signs were stable, with a blood pressure of 125/85 mmHg, heart rate of 85 beats per minute, and oxygen saturation of 98% on room air. Physical examination revealed no significant findings, except for mild tenderness to palpation in the chest area. An electrocardiogram (ECG) was performed, which showed evidence of ST-segment elevation in the anterior and septal leads consistent with an acute ST-segment elevation myocardial infarction (STEMI). The patient was not a candidate for thrombolysis due to his advanced age and increased risk of bleeding. Therefore, he was managed conservatively because the family refuse transferred to a tertiary care center for further management. Conclusion: Diabetic patients with STEMI may not experience typical chest pain. Instead, they may present with no symptoms at all. This can make it more challenging for healthcare providers to identify and treat the condition. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: The periprocedural myocardial infarction in the calcified coronary lesion could be identified from the patient's clinical symptom, physical examination and further investigations such as electrocardiogram changes, increase in cardiac enzymes, echocardiography changes, and coronary angiography findings. This patient requires thorough observation and monitoring. Keywords: Periprocedural Myocardial Infarction, Percutaneous Coronary Intervention, Calcified Lesion, Complete Atrioventricular Block, Side Branch Occlusion Keywords: Periprocedural Myocardial Infarction, Percutaneous Coronary Intervention, Calcified Lesion, Complete Atrioventricular Block, Side Branch Occlusion Keywords: Periprocedural Myocardial Infarction, Percutaneous Coronary Intervention, Calcified Lesion Complete Atrioventricular Block, Side Branch Occlusion Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration Male, 47 years old, complained of back pain since 3 days ago and worsened 1 day ago. Back pain feels like being stabbed and slashed. He had a history of uncontrolled hypertension since 2 years ago and smoked 1 packed/day. Physical examination revealed irregular heart sound and without murmur. Transthoracic echocardiography revealed aortic root dilatation and seen intimal flap on the ascending aorta. CT-scan thoracoabdominal with contrast revealed fusiform aneurysm from ascending aorta until common iliac arteries accompanied by intraluminal thrombus and seen leakage contrast as high as thoracal 10, suspect intimal tear in distal descending thoracic aorta. Patient was treated in the cardiac intensive care unit and after heart team meeting, patient was planned for total aortic arch replacement with frozen elephant trunk (FET) procedure. G. S. Ambara1, B.A.P. Dwi Sutanegara1, A.P. Suwirya1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia G. S. Ambara1, B.A.P. Dwi Sutanegara1, A.P. Suwirya1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia Background Retrograde type A aortic dissection (RTAAD) is defined as a dissection with the primary entry tear occurs distal to the left subclavian artery (LSA) and the dissection propagates retrogradely into the ascending aorta. Discussion RTAAD can be spontaneous or iatrogenic, especially when the proximal aorta is already dilated over 40 mm. Management of spontaneous RTAAD is currently not standardized with reports of successful treatment with optimized medical therapy, open surgery and/or endovascular repair. Conservative medical therapy has been successful for cases with completely thrombosed false lumen in the ascending aorta, providing the aorta that it is not dilated beyond 55 mm. On the other hand, presentation with a patent false lumen in the ascending aorta, pericardial effusion, aortic regurgitation or malperfusion mandates expedited surgical treatment (i.e. total aortic arch replacement with FET procedure). RETROGRADE TYPE A AORTIC DISSECTION WITH AN ENTRY TEAR IN DISTAL DESCENDING THORACIC AORTA: A RARE CASE REPORT G. S. Ambara1, B.A.P. Dwi Sutanegara1, A.P. Suwirya1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia Transesophageal Echocardiography Evaluation of Patent Ductus Arteriosus Percutaneous Device Closure: a new window to the heart A.F. Ghaznawie1, A.T.S. Adam1, Y. Patimang1, A.A.U. Armyn1, M.I. Nurkholis1 Hasanuddin University1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Transesophageal Echocardiography Evaluation of Patent Ductus Arteriosus Percutaneous Device Closure: a new window to the heart Conclusion RTAAD has anatomical differences compared to antegrade TAAD that impact the management and prognosis. Management is yet not standardized. Aggressive treatment for RTAAD with the exclusion of the primary entry tear to prevent immediate-complications and mid-term complications. Keywords: RTAAD, optimized medical therapy, aortic arch replacement with FET procedure Keywords: RTAAD, optimized medical therapy, aortic arch replacement with FET procedure Keywords: RTAAD, optimized medical therapy, aortic arch replacement with FET procedure Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:su pISSN: 2830-3105 / eISSN: 2964 doi: 10.30701/ijc.1537 Transesophageal Echocardiography Evaluation of Patent Ductus Arteriosus Percutaneous Device Closure: a new window to the heart Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A.F. Ghaznawie1, A.T.S. Adam1, Y. Patimang1, A.A.U. Armyn1, M.I. Nurkholis1 Hasanuddin University1 Background: Transesophageal echocardiography (TEE) is a modality that can be used to monitor ductal flow before and after patent ductus arteriosus (PDA) closure, enabling a pediatric cardiologist to discover any residual shunts and providing ultimate confirmation of PDA closure. In this case report we present a 18-year-old girl with patent ductus arteriosus scheduled for device closure. Transesophageal echocardiography was performed to assess morphologic characterization of the ductus and surrounding structures, assess of the shunt magnitude, to estimate pulmonary artery pressures as well as to evaluate the device patency post-procedure. Case illustration: A 18-year-old girl (38 kg) diagnosed with PDA was scheduled for device closure. Pre- procedural transthoracic echocardiography (TTE) showed a PDA sized 8-9 mm with a left-to-right. The systolic pressure gradient was 87.96 mmHg and the diastolic gradient was 21.41 mmHg. Along with PDA, severe mitral regurgitation due to prolapse AML (Carpentier type II) and moderate pulmonary regurgitation was assessed. A single-plane TEE probe was inserted in the patient’s esophagus after tracheal intubation and general anesthesia. Pre-closure 2D TEE color doppler flow imaging showed a PDA sized 7.8 - 8 mm with a left-to-right shunt through the PDA (systolic gradient: 60.55 mmHg, diastolic gradient: 14.27 mmHg). A further evaluation using 4D TEE imaging, a dynamic change in the PDA throughout the cardiac cycle can be observed. Transcatheter closure was then performed and a PDA sized 7.78 mm was viewed. An occluder (size 14-16) was then inserted. Post-closure TEE monitoring revealed a well-seated PDA occluder with no residual shunt on color doppler imaging. The TEE probe was then withdrawn and no complications were encountered during the procedure. Conclusion: We reported a 18-year-old girl with PDA who underwent device closure procedure. Transesophageal echocardiography was performed to evaluate the shunt pre and post procedure. Studies report that TEE consistently outperformed TTE for PDA visualization, and when compared to the former, the latter revealed greater values for ductal dimensions. 2D and 4D TEE is a useful and efficient and precise tool for device size selection, having only small differences in measurements compared to catheterization. TEE also holds a great role in post-procedural evaluation of the device placed. Keywords: transesophageal echocardiography, patent ductus arteriosus, percutaneous device closure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Anomalous Coronary Artery From Opposite Sinus (ACAOS) LCx Originated from RCA with Coronary Artery Minor Disease. Does It Really Benign ? Indonesian Journal of Cardiology Sinus of Valsalva Aneurysm with Great Dilatation and Coarctation of Aorta in A Middle-Aged Adult Male : A Rare Case Report Anomalous Coronary Artery From Opposite Sinus (ACAOS) LCx Originated from RCA with Coronary Artery Minor Disease. Does It Really Benign ? Anomalous Coronary Artery From Opposite Sinus (ACAOS) LCx Originated from RCA with Coronary Artery Minor Disease. Does It Really Benign ? O.W. Firmansyah1, B.S. Pikir1 Universitas Airlangga1 Background: Anomalous coronary artery from opposite sinus (ACAOS) is the rare condition that can affect 1% population in general. Classification varies from benign to malign related to risk of sudden cardiac death (SCD) event. In the absence of the anatomical risk, Such cardiac event related to myocardial infarction can make difficult situation for interventionist to perform. Case Illustration: the 60 years old female come with chief complaint of chest pain that worsen at the moderate to high physical activity. CT cardiac was performed and revealed that the patient has Anomalous Coronary Artery Originated from Opposite Sinus (ACAOS), in which Left Circumflex Artery originated from Right Coronary Artery. The patient is also had Coronary Artery Disease (CAD) minor disease (CAD RADS-3). Although the anomalous condition is categorized as benign, The patient is still planned for secondary prevention such as lifestyle modification and adequate medication to stop the progression of CAD minor disease and improve the condition. Conclusion: ACAOS LCx originated from RCA is rare but benign condition with low risk of SCD. There is no need of intervention of this patient regarding on the anomaly, but the patient definitely need the intervention in secondary prevention to prevent further progression of CAD and avoid unwanted event in future. Keywords: Coronary Anomaly, CT Cardiac, CAD Minor Disease, Secondary prevention. ronary Anomaly, CT Cardiac, CAD Minor Disease, Secondary prevention. onary Anomaly, CT Cardiac, CAD Minor Disease, Secondary prevention. Keywords: Coronary Anomaly, CT Cardiac, CAD Minor Disease, Secondary prevention. Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Sinus of Valsalva Aneurysm with Great Dilatation and Coarctation of Aorta in A Middle-Aged Adult Male : A Rare Case Report Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Heparin Treatment in Patients Deep Vein Thrombosis with End-Stage Renal Disease: A Case Report A.A. Budiarto1, Ahadi2 Medical Intern, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.1 Cardiologist, Dr. R. Sosodoro Djatikoesoemo General Hospital, Bojonegoro, Indonesia.2 Background g Patient end-stage renal disease (ESRD) is associated with a 2.3-fold increased risk of deep vein thrombosis (DVT) compared with the general population. DVT is a potentially life-threatening condition that comes under the category of venous thromboembolism (VTE). ESRD with VTE was associated with increases in the risk of death, VTE recurrence, and major bleeding. This case describes and highlights the importance of the treatment of DVT in patients with ESRD. Case illustration A 50-year-old female patient, with a medical history of hypertension for the past 10 years without routine treatment and chronic kidney disease stage V for the past 7 months, was on maintenance hemodialysis two times a week. She had a problem with an arteriovenous shunt so the hemodialysis was performed via dialysis catheters in the femoral site. However, she had a complaint of swelling in her right leg after the removal of dialysis catheters. The swelling felt to getting more significant in a month and she was taken to our hospital. On physical examination show swollen tender right leg. The US investigation demonstrated non-compressible femoral veins. A diagnosis of acute deep vein thrombosis lower right extremities were made. Unfractionated heparin (UFH) is preferred in the clinical setting to LMWH or Fondaparinux and is given intravenously in the hospital with laboratory monitoring. UFH in a therapeutic dose was initiated with bolus 5,000 U followed by maintenance 24,000 U/24 hours. The right femoral vein thrombosis resolved after 2 weeks with twice dose adjustments. A haematological workup did not reveal the presence of any hypercoagulable disorder. During the administration of heparin therapy did not find any complications. Sinus of Valsalva Aneurysm with Great Dilatation and Coarctation of Aorta in A Middle-Aged Adult Male : A Rare Case Report R. Antonius1, J.K.P. Phie1, A.F. Ghaznawie1 Department of Cardiology Hasanuddin University1 Background : Sinus of Valsalva Aneurysm (SOVA) is an enlargement of the aortic root area between the aortic valve annulus and the sinotubular ridge. SOVA is a rare heart condition is well known for its potentially fatal complications when rupture occurred. The prevalence of congenital SOVA is estimated to be 0.09% of the general population. Case Illustration : We report a 47 years old male, admitted to the hospital with shortness of breath during exercise, at night and on lying down. History of hypertension for 10 years. Physical examination showed irregular pulse, distended jugular vein, pulsus Corrigan and diastolic murmur at upper right sternal border. Electrocardiogram presented an atrial fibrillation. Laboratory findings within normal limit. Screening for syphilis showed negative results. Echocardiography showed great dilatation of aortic annulus and aortic root mainly sinus of Valsalva RCC portion (7,1 cm), dilatation of brachiocephalic trunk and pulmonary arteries, suggestive coarctation of aorta, severe aortic regurgitation due to uncoaptation of cusps. The echocardiography findings was confirmed by Cardiac CT scan and Aortography. The patient was diagnosed with sinus of valsalva aneurysm, coarctation of aortae and aortopulmonary shunt, severe aortic regurgitation, congestive heart failure and atrial fibrillation. The patient was treated with diuretic, mineralocorticoid antagonist, vitamin-K antagonist, ACE- inhibitor, followed by beta-blocker. Bentall procedure surgery was further suggested for the defect structure correction. Conclusion : SOVA with intracardiac abnormalities is recommended in guidelines for further surgical intervention to repair the aortic root aneurysm. According to the 2010 American Guidelines on Thoracic Aortic Disease, surgical repair should be considered in those with aneurysms >5.5cm. Correcting the aortic valve and aortic root, alongside coarctation of aortae repair could be done with Bentall procedure by cardiothoracic surgeon. eywords: Sinus of Valsalva Aneurysm, Coarctation of Aorta, Severe Aortic Regurgitation Figure 1. TransThoracal Echocardiography and MSCT Cardiac Aortography Keywords: Sinus of Valsalva Aneurysm, Coarctation of Aorta, Severe Aortic Regurgitation Figure 1. TransThoracal Echocardiography and MSCT Cardiac Aortography Heparin Treatment in Patients Deep Vein Thrombosis with End-Stage Renal Disease: A Case Report Heparin Treatment in Patients Deep Vein Thrombosis with End-Stage Renal Disease: A Case Report Background g Atrial Fibrillation (AF) is defined as tachyarrhythmia originated from supraventricular, firing wildly electrical activation thus making atrium contracts inadequately. It has three distincts characteristics from Electrocardiogram (ECG), such as irregularly irregular R-R intervals, no p wave, and irregular atrial activations. It can be caused by endocrine disorders, one of them is Diabetes Mellitus (DM). Studies have shown that this life threatening arrhythmia may be related to hypoglycemia condition which can be induced by DM treatments. UNSTABLE ATRIAL FIBRILLATION WITH RAPID VENTRICULAR RESPONSE (RVR) IN HYPOGLYCEMIA PATIENT, A POSSIBLE AND RARE CAUSE : A CASE REPORT A. Siswanto1 RSD Balung Jember1 Case Illustration A 50-year-old woman was referred to our Emergency Room (ER) for feeling dyspneic and palpitations. Her family told that she had cardiac problem before, but did not know about her medications, including the possibility of having history of DM. Her vital signs showed tachycardia (197 beats/min) and oxygen saturation 88% despite using Non-Rebreather Mask 15 lpm. On physical examination, her jugular vein pressure was increased and her ictus cordis shifted laterally. This was confirmed by her Chest X-Ray that showed cardiomegaly. The cardiac monitor showed AFib then the patient was treated with synchronized cardioversion 125 J. The rhythm was converted to sinus rhythm with rate 159 beats/min. From the laboratory studies, serum glucose was 14mg/dL then d40% was given intravenously. After that, the monitor showed SVT with rate 300 beats/min so the patient was treated with synchronized cardioversion 50 J again. The rhythm was converted to sinus with PACs with rate 160 beats/min. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion The results of our case suggest that the treatment of DVT of the limbs with heparin in patients with ESRD is an effective and safe therapy. py Keywords: heparin, deep vein thrombosis, chronic kidney disease Keywords: heparin, deep vein thrombosis, chronic kidney disease Fig 1. The US investigation demonstrated non-compressible femoral veins Fig 1. The US investigation demonstrated non-compressible femoral veins BRASH Syndrome as an under-recognized life-threatening complication: A Case Report A.O. Aslani1, G. Octania2, A.U. Dewi1, R.H. Wibowo1, T.A. Sabrawi1 RSUD Kabupaten Sumedang1 Universitas Padjadjaran2 Background g Bradycardia, renal failure, atrioventricular (AV) node blockade, shock, and hyperkalemia (BRASH) syndrome occurs in patients consuming AV-blocking medication. Clinicians often missed diagnosing BRASH syndrome due to the under-recognition of this syndrome. Here we report a case of a patient with chronic heart failure (CHF) and atrial fibrillation (AF) who takes medications regularly that presented life-threatening conditions. Case Illustration A 72-year-old female patient presented with a chief complaint of gradually worsening dyspnea. The previous history of heart disease was confirmed. The patient was taking bisoprolol 2.5 mg once daily, sacubitril-valsartan (Uperio™) 100 mg twice daily, furosemide 40 mg PRN, spironolactone 50 mg once daily, and warfarin 3 mg every night. Physical examination revealed bradycardia, cold and cyanosis of the distal extremities with CRT (capillary refill time) longer than 2 seconds. Laboratory findings showed uremia and severe hyperkalemia. Findings from electrocardiogram (ECG) include junctional bradycardia with tall T wave. She was treated with lansoprazole, atropine sulfate, dopamine, and calcium gluconate infusion. All regular medications were stopped, except for warfarin, and the patient underwent emergency hemodialysis. After 6 days of hospitalization, the patient responded well and was discharged home. Conclusion Atrial Fibrillation is one of dangerous cardiac arrhythmia for it can form thrombus and causing stroke. Diabetic and hypoglycemia condition is now emerging as major risk factor for developing AFib. Therefore, patients with DM needs appropriate control of serum glucose and cardiac monitoring every once in a while. Keywords: atrial fibrillation, diabetes mellitus, hypoglycemia Figure 1. Monitor showed AF first (A), then SVT (B) after glucose administration intravenously Keywords: atrial fibrillation, diabetes mellitus, hypoglycemia Figure 1. Monitor showed AF first (A), then SVT (B) after glucose administration intravenously Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Infective endocarditis (IE) is an uncommon but potentially fatal infectious disease. One of the common complications of IE is the embolization of endocardial vegetations with subsequent intracerebral artery obstruction that causes an acute ischaemic stroke. Here we present a case report of a patient presented with a neurological manifestation that turned out to be the complication of IE. p Conclusion BRASH syndrome should always be considered in patients presenting with shock accompanied by bradycardia, uremia, hyperkalemia, and routine consumption of AV-nodal-blocking medications. We highlight the importance of recognizing BRASH syndrome, especially in emergency settings, to be able to administer rapid and appropriate management to the patients. Therefore, we can improve the patients’ prognosis. g p , p p p Keywords: BRASH, bradycardia, renal failure, hyperkalemia, shock Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Ischaemic Stroke and Subarachnoid Haemorrhage as the Complication of Infective Endocarditis in Young Male: A Case Report Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Ischaemic Stroke and Subarachnoid Haemorrhage as the Complication of Infective Endocarditis in Young Male: A Case Report A.O. Aslani1, Z. Haryawan2, A.U. Dewi1, R.H. Wibowo1, T.A. Sabrawi1 RSUD Kabupaten Sumedang1 RSUD Ciamis2 Case Illustration We present a patient with a chief complaint of left hemiplegia. Bloodwork showed signs of infection. Head CT scan showed extensive infarction on the right lobe and subarachnoid hemorrhage. Echocardiography showed vegetation on the aortic valve suggesting the diagnosis of IE as the source of the embolization. The patient was then treated with supportive stroke therapy, high-dose ampicillin and gentamicin, and physical rehabilitation. Conclusion IE can be considered as one of the causes of acute ischaemic or haemorrhagic stroke. Ruling out other common causes of stroke and noticing signs of infection and vascular phenomenon should help defining the diagnosis. Echocardiography could help identifying valvular vegetation. Treatment consists of high-dose penicillins and supportive therapy for stroke. Keywords: Infective endocarditis, stroke, young age Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Ventricular Septal Defect Complicated with Acute Pulmonary Embolism: How it can be Happened? Andrianto1, T. Santoso1, E. Rosmillah1, R.A. Nugraha1, B.P. Dharma1 General Hospital dr. Soetomo1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Sudden Total Atrioventricular Block, Is Pacemaker The Only Treatment? : A Case Report P.E.D. Putri1, A.P. Suwirya2 Faculty of Medicine, Universitas Hang Tuah, East Java, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Udayana, Prof. I.G.N.G. Ngoerah General Hospital, Bali, Indonesia2 P.E.D. Putri1, A.P. Suwirya2 Faculty of Medicine, Universitas Hang Tuah, East Java, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Uday Ngoerah General Hospital, Bali, Indonesia2 Ventricular Septal Defect Complicated with Acute Pulmonary Embolism: How it can be Happened? Andrianto1, T. Santoso1, E. Rosmillah1, R.A. Nugraha1, B.P. Dharma1 General Hospital dr. Soetomo1 Ventricular Septal Defect Complicated with Acute Pulmonary Embolism: How it can be Happened? Andrianto1, T. Santoso1, E. Rosmillah1, R.A. Nugraha1, B.P. Dharma1 General Hospital dr. Soetomo1 Background: Acute pulmonary embolism is one of cardiovascular emergency that occurs due to pulmonary artery occlusion. However, pulmonary embolism in this case is caused by right ventricle (RV) failure with congenital heart disease, which is VSD. The RV failure results in blood stagnation because of RV wall contraction impairment that accordance with Virchow’s triad as venous stasis, then induce thrombus formation. This case is rare because only 4% of all pulmonary embolism accompanied by RV thrombus, in addition it is also accompanied by VSD. This case begins with VSD and become RV failure. The stagnation of blood, accompanied by RV failure promotes RV thrombus. this thrombus can break off and result in an acute pulmonary embolism. Virchow's triad in which one of the predisposing factors is static flow. Case Illustration: A 79-year-old woman came to ED of main referral hospital presence worsening dyspnea. The physical examination showed hemodynamically unstable increasing JVP, continuous murmur in intercostal 2 right parasternal. The ECG displayed T inversion and Q wave in lead III, S in lead I. The CXR displayed dilated central PA. TTE displayed dilatated RA and RV, thrombus (1,4 x2,2 cm) in apex RV, Mc Conell sign, and decreasing RV systolic function, and IVS defect (0.9 cm diameter), and mPAP 47,5 mmHg. The patient was diagnosed obstructive shock e.c. acute pulmonary embolism (WELLS score 6 (moderate risk) RIETE score 2 (moderate), PESI 229 (very high risk)) with RV failure and VSD perimembran. the patient was sent to intensive care unit, follow by anticoagulation and thrombolityc. during the stabilization, the patient was gone. Conclusion: The case about VSD manifest pulmonary embolism through RV failure is rare. The mechanism is VSD leads to RV failure, which occures blood stagnation, then promotes RV thrombus formation, and manifest acute pulmonary embolism. eywords: Congenital Heart diseases; Pulmonary Embolism; Ventricular Septal Defect; Acute Cardiac Car Pathofisiology Pathofisiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background Total Atrioventricular Block (TAVB) is a rare disorder of the cardiac conduction system. Some electrolyte imbalances are thought to affect the heart conduction system, but TAVB is one of the uncommon presentations. This presentation aims to brief the management of conduction abnormalities caused by electrolyte imbalance. Case illustration A previously healthy 28-year-old male complained of epigastrial pain, a burning sensation, nausea, and vomiting one day before admission. The patient was alert with blood pressure (BP) of 120/70mmHg; heart rate (HR) of 75 bpm; respiratory rate (RR) of 22 x/min; and normal oxygen saturation. Laboratory studies revealed elevated White Blood Cells 19.33 x 10 3/ uL with predominantly neutrophil 84,5 %. Four hours after admission, his complaints of palpitation, nausea, and vomiting worsened. He had vomited more than ten times. His BP was 120/70 mmHg and his electrocardiography showed a Total Atrioventricular Block with a ventricular rate of 28 bpm. He received sulfa-atropin 0,5mg and dopamine 325 mcg drip. Further laboratory studies revealed a high C-Reactive protein (CRP) of 116.30 mg/dl, Erytrosite sedimentation rate (ESR) of 60 mm/hours, and electrolyte imbalance, with potassium serum 5.18 mmol/L and calcium serum 8.1 mg/dL. His troponin I and CKMB was normal. Echocardiography demonstrated a Left Ventricle Concentric remodeling without other anatomical and functional abnormalities. Based on those data, the patient was diagnosed with TAVB caused by electrolyte imbalance, then received calcium gluconate 1 gram/10mL three times a day and potassium polystyrene sulfonate 5 grams three times a day. After a week of hospitalization, he got fully recovered. Conclusion The treatment of emergency TAVB consists of medication and pacemaker implantation. Finding the etiology of TAVB is mandatory, which can be seen from the patient's clinical condition, laboratory findings, and comorbidities. Keywords: hyperkalemia, Hypocalcemia, High degree atrioventricular block, Electrolyte Imbalance Figure 1. ECG shows Total Atrioventricular Block Figure 1. ECG shows Total Atrioventricular Block Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 CASE SERIES OF SURVEILLANCE AFTER FONTAN PROCEDURE: THE ROLE OF MRI IN EVALUATING SINGLE VENTRICLE PHYSIOLOGY Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 CASE SERIES OF SURVEILLANCE AFTER FONTAN PROCEDURE: THE ROLE OF MRI IN EVALUATING SINGLE VENTRICLE PHYSIOLOGY Constrictive Pericarditis: A Forgotten Cause of Right Heart Failure P.D.P. Lestari1, M.A.W. Sari1, M.W. Asih2 1Department of Cardiology and Vascular Medicine, Prof. dr. I.G.N.G. Ngoerah Hospital Bali, Faculty of Medicine Udayana University1 P.D.P. Lestari1, M.A.W. Sari1, M.W. Asih2 1Department of Cardiology and Vascular Medicine, Prof. dr. I.G.N.G. Ngoerah Hospital Bali, Faculty of Medicine Udayana University1 2 artment of Radiology, Prof. dr. I.G.N.G. Ngoerah Hospital Bali, Faculty of Medicine Udayana University Background: Constrictive pericarditis is a well-known but rare and commonly forgotten cause of right heart failure. Early diagnosis of constrictive pericarditis is difficult due to absence of typical cardiopulmonary signs and multiple vague symptoms and its insidious course. Case illustration: A 34-year-old man presented with shortness of breath since 4 days ago accompanied with ascites and pitting edema. He had a history of heart failure due to coronary artery disease, but already done coronary angiography with no significant stenosis. Physical examination found signs of congestion with predominantly ascites and edema on both legs with dominant on the right leg. Echocardiography found biatrial enlargement and right ventricle dilatation, decreased right and left systolic function with uncoordinated septal wall motion, decreased left ventricle diastolic function grade II, mild tricuspid regurgitation with high probability of PH, and echo dense structure compress base of left ventricle, half of left atrium suspect extracardiac mass. Doppler ultrasound was consistent with deep vein thrombosis right lower extremity. CT Scan Thorax with contrast found heterogeneous density suspect benign pericardium mass dd/ pericardium thickening due to chronic inflammation, constrictive pericarditis. During hospitalization, there was decreasing renal function, so we planned hemodialysis. We planned to do an excision biopsy, but the patient refused. Constrictive pericarditis may have sign of systemic congestion like right heart failure. The right heart failure caused by several etiology with different pathophysiology that effect the management and prognosis, but constrictive pericarditis is a rare condition that may cause right heart failure. C l i W d ib d i i h i i i di i kid i j DVT Ri h h f il Conclusion: We described a patient with constrictive pericarditis, acute kidney injury, DVT. Right heart failure may be the first clinical symptom in constrictive pericarditis. Thorough examination is needed to diagnose constrictive pericarditis. p Keywords: Ascites, echocardiography, constrictive pericarditis, right heart failure. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 CASE SERIES OF SURVEILLANCE AFTER FONTAN PROCEDURE: THE ROLE OF MRI IN EVALUATING SINGLE VENTRICLE PHYSIOLOGY C. Abdillah1, O. Lilyasari1 Faculty of Medicine Universitas Indonesia1 Background: Fontan circulation is one of the most common procedure to treat complex congenital heart disease. Echocardiography had limitation due to poor acoustic window in older patients. Cardiac Magnetic Resonance Imaging (MRI) have been validated as an important modality to evaluate major complication of the Fontan circulation.The objective was to present serial cases of patient with history of Fontan and explain the role of MRI as an advanced imaging technique to evaluate long-term complications. Case Illustration: Case 1 illustrate a 15 years old female with history of Fontan fenestrated procedure came to ER with dyspnea on effort. Echocardiographic examination showed suspicion of intraconduit thrombus formation, which was confirmed with MRI examination. The patient underwent heparinization in intermediate ward and was given oral thrombolytic therapy. Case 2 illustrate a 13 years old female came to outpatient clinic with history of swelling in face and legs. She had already treated with Fontan fenestration procedure in June 2011. The echocardiographic study revealed the anastomoses was unclear with suspicion of Fontan conduit obstruction. MRI revealed stenosis in Fontan conduit at the level of diaphragm. She therefore may continue her previous medication while she was scheduled for cardiac catheterization in order to stent the stenotic area. Case 3 illustrate a 22 years old female with history of Fontan procedure in 2006. She didn’t have any major complain. The MRI examination was done as surveillance strategy to evaluate Fontan pathways. Stenosis at pulmonary artery bifurcation and origin of pulmonary artery branches was noted, and she may continue her medication with plans for reexamination in the following year. Conclusion: Advanced imaging modalities, particularly cardiac MRI, are of importance in recognition of failing hemodynamic of Fontan which is essential for long-term survival. Appropriate imaging protocol and surveillance strategy are needed to assess the anatomy, prognosis and complication in univentricular heart. Keywords: Fontan procedure, MRI, Single Ventricle, Surveillance Figure 1. Thrombus detected in MRI evaluation Figure 1. Thrombus detected in MRI evaluation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background Multiple valve heart disease (VHD) is most often acquired. In the Euro Heart Survey, rheumatic fever was the predominant pathogenesis (51%). Mitral valve disease and joint pains are commonly caused by rheumatic heart disease (RHD). Other causes of mitral valve disease and polyarthritis should be considered in the differential diagnoses. We report a rare case multiple and mixed valvular heart disease in a young woman with joint pains. Case Illustration A 22 year old lady presented with polyarthritis and morning stiffness involving bilateral knees, ankles, elbows, wrists, metacarpophayngeal and proximal interphalyngeal joints for one month. Patient had history of polyarthritis from 9 years before with inappropriate treatment because she did not go to a health care facility. Rheumatoid factor has raised. She came to us for evaluation of a mid-diastolic murmur heard loudest over the apex and a soft diastolic murmur heard loudest at 3rd – 4th intercostal space at the left sternal edge. Echocardiogram showed mitral stenosis (MVA 1.6 cm2), mitral regurgitation, tricuspid stenosis, tricuspid regurgitation and mild aortic stenosis, with EF 66% and good right ventricular function. After multidisciplinary evaluation, she has gotten mitral valve replacement, aortic valve replacement and tricuspid valve repair. Multiple and mixed valvular heart disease in a young woman with joint pains F. Resi1, C.K. Krevani2 1. Cardiology and Vascular Medicine Residency, Andalas University, Padang, West Sumatera. 2. Cardiology and Vascular Medicine Department, M. Djamil Hospital, Padang, West Sumatera. A Spontaneous Resolution of Acute Inferior MI Concomitant Complete AV Block in Rural Primary Health Care : A Case Report M. Aulia1, A.I. Pratiwi1, S.H. Aulia2, I.M. Sufiyah3 Public Health Center of Patani1 Student of Medical Faculty of Airlangga University, Surabaya - Indonesia2 General hospital Dr. Soetomo, Department of Cardiology and Vascular Medicine, Medical Faculty of Airlangga, Surabaya3 M. Aulia1, A.I. Pratiwi1, S.H. Aulia2, I.M. Sufiyah3 Public Health Center of Patani1 Student of Medical Faculty of Airlangga University, Surabaya - Indonesia2 General hospital Dr. Soetomo, Department of Cardiology and Vascular Medicine, Medical Facult Airlangga, Surabaya3 M. Aulia1, A.I. Pratiwi1, S.H. Aulia2, I.M. Sufiyah3 Public Health Center of Patani1 Student of Medical Faculty of Airlangga University, Surabaya - Indonesia2 General hospital Dr. Soetomo, Department of Cardiology and Vascular Medicine, Medical Faculty of Airlangga, Surabaya3 Background: In acute inferior MI, where the RCA is often the culprit, high-grade AV block has been described in up to 17% of cases. Complete AV block is known to be reversible in some cases of acute inferior MI. Spontaneous reperfusion in the setting of ST-elevation ACS is reported in up to 30% of patients. Case Illustration: A 56-year-old man was admitted with left chest pain 1 hour before he was taken to the ER of PHC. Chest pain felt tight and heavy, radiating to the left arm, accompanied by nausea and weakness. Thirty minutes earlier, he had fainted during the activity. He had uncontrolled hypertension and 30-year smoking history. He was fully conscious and revealed cold, clammy and pale skin. His BP 80/palpation mmHg, HR 32bpm, RR 18bpm and SaO2 97%. The ECG showed ST elevation in leads II, III, aVF and complete AV block. He was diagnosed with acute inferior MI, complete AV block, and cardiogenic shock. He received fluid loading RL 1000 cc within 30 minutes, maintenance fluid therapy 1500cc/24 hours, and Aspirin loading 320mg. The final BP 110/70mmHg and HR 68bpm. He was referred to the nearest hospital (a 5-hour drive) for further examination and treatment. Blood tests and CXR showed normal findings. The ECG showed that the STEMI and TAVB spontaneously resolved. p y Conclusion: This case highlights the importance of early prehospital care in the rural PHC to treat life- threatening conditions and improve outcomes in acute inferior MI and concomitant complete AV block. Prompt initial treatment is crucial to prevent from failing in hemodynamic instability and reduce mortality. A Spontaneous Resolution of Acute Inferior MI Concomitant Complete AV Block in Rural Primary Health Care : A Case Report Extreme geographic, distance and socio-economy were challenging for the doctors to be more concerned about the patient's clinical condition and vital signs while referring the patient to the hospital. Keywords: Acute Inferior MI, Complete AV Block, Spontaneous Resolution SEVERE RHEUMATIC MITRAL REGURGITATION WITH SIGNIFICANT PERICARDIAL EFFUSION IN A CHILD: A CHALLENGE IN RURAL AREA Y. Natanael1, A. Almira2, Hendrianus3 Ruteng Public Hospital1 Brawijaya Women and Children Hospital Antasari2 Conclusion Multiple and mixed VHD are highly prevalent conditions. Mixed mitral VHD in young women mainly results from rheumatic processes. RHD is the most common cause of mitral valve disease and joint pains in developing world but other rare causes including rheumatoid arthritis should be considered in differential diagnosis. This case highlights the importance of characteristics of rheumatic and non-rheumatic origin of valvular heart disease. Keywords: Multiple and mixed valvular heart disease, rheumatic heart disease, mitral valve disease, joint pains Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology A Spontaneous Resolution of Acute Inferior MI Concomitant Complete AV Block in Rural Primary Health Care : A Case Report Keywords: Acute Inferior MI, Complete AV Block, Spontaneous Resolution SEVERE RHEUMATIC MITRAL REGURGITATION WITH SIGNIFICANT PERICARDIAL EFFUSION IN A CHILD: A CHALLENGE IN RURAL AREA Y. Natanael1, A. Almira2, Hendrianus3 Ruteng Public Hospital1 Brawijaya Women and Children Hospital Antasari2 Keywords: Acute Inferior MI, Complete AV Block, Spontaneous Resolution SEVERE RHEUMATIC MITRAL REGURGITATION WITH SIGNIFICANT PERICARDIAL EFFUSION IN A CHILD: A CHALLENGE IN RURAL AREA Y. Natanael1, A. Almira2, Hendrianus3 Ruteng Public Hospital1 Brawijaya Women and Children Hospital Antasari2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Cardiology and Vascular Medicine Resident, Faculty of Medicine Universitas Indonesia, National Cardiovascular Center Harapan Kita3 Cardiology and Vascular Medicine Resident, Faculty of Medicine Universitas Indonesia, National Cardiovascular Center Harapan Kita3 Background: Mitral valve regurgitation (MR) is one of the most common valvular abnormality at the early rheumatic heart disease (RHD) stages, whereas pericardial effusion can be present although rarely large enough to present with tamponade physiology. These conditions may result in early heart failure manifestations at young age, which causes disability and premature death in many countries. Case Illustration: An 11 years old girl was referred from a rural public hospital with worsening dyspnea since the last one month. She commented on one month history of palpitation before admission and migrating arthralgia since 2018. Upon arrival, she was fully alert with the following vital sign: blood pressure 85/55 mmHg, radial pulse of 116 bpm, respiratory rate of 24 breaths/min, body temperature of 36.7oC and oxygen saturation of 99% on room air. Her height and weight were 137 cm and 28 kg respectively. Physical examination revealed a fast regular rhythm with III/VI pansystolic murmur at mitral area. A 12-lead electrocardiogram evaluation showed a sinus rhythm with ST segment depression at V5-V6 and the chest X-ray demonstrated cardiomegaly with a third mogul sign. Transthoracic echocardiography revealed a severe MR with moderate pericardial effusion, mild aortic regurgitation, left ventricle dilatation with ejection fraction of 63% and left atrial dilatation. The patient was hospitalized for 13 days with intravenous furosemide, bisoprolol, ramipril, spironolactone, oral methylprednisolone, and secondary rheumatic prophylaxis with erythromycin. Her condition gradually improved and the pericardial effusion significantly subsided. She then was prepared for referral to a tertiary cardiac center for surgical management. g g Conclusion: This case highlights the challenge of RHD with pericardial effusion management and the urgency to improve awareness of RHD in a limited access to pediatric cardiac services, thus reducing the complication and improving the patient’s quality of life. HYPERTENSION URGENCY IN SEVERE ABDOMINAL PAIN ET CAUSA HEPATIC HYDATID CYST: THE ETIOLOGY HAS BEEN REVEALED R.K. Ratri1, C. Saputra2, Y.S. Sugi3, R.S. Sihombing3, W. Wiriadisurya3, J.R.L. Sulle4 eral Practitioner of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara1 gy and Vascular Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara2 R.K. Ratri1, C. Saputra2, Y.S. Sugi3, R.S. Sihombing3, W. Wiriadisurya3, J.R.L. Sulle4 General Practitioner of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara1 Cardiology and Vascular Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara2 I l M di i D f M G b i l M k SVD P bli H i l f A b E N T 3 gg nternal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tengg Radiology Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara gg Internal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara3 Radiology Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara4 Background: Hepatic hydatid cyst is a cystic disease of the liver caused by the larva stage parasitic tapeworm of Echinococcus granulosus and Echinococcus multilocularis. The most common mode of transmission to humans is by accidental food that has been contaminated. Hydatic cyst can also cause hypertension due to external compression of renovascular. For the pharmacological treatment, Albendazole is a chosen drug for Hydatid cysts. Case Illustration: A 54 years old female, was referred to the Emergency Department of the Public hospital of Atambua with experienced abdominal pain in the epigastric region. The patient also complained of pain all over the body, headache, and fever. The patient had a history of hypertension. In the physical examination, blood pressure 189/110 mmHg, axillary temperature was 37.9 Celsius degree, there is epigastric tenderness. Laboratory examination showed results of a normal neutrophil segment and an increased eosinophil segment, in the abdomen sonography found a Rim Calcification 3x2,5 cm in the right upper lobes hepar (hydatid cyst) (Figure). The patient was treated with low doses anti-hypertension and albendazole 800 mg twice a day the duration of treatment is 28 days for 3 cycles, with a break of 14 days without taking medication between cycles. The patient has shown immediate improvement. The patient was discharged after 3 days of hospitalization, hemodynamically stable, and with no complaints of abdominal pain. Keywords: Acute Inferior MI, Complete AV Block, Spontaneous Resolution SEVERE RHEUMATIC MITRAL REGURGITATION WITH SIGNIFICANT PERICARDIAL EFFUSION IN A CHILD: A CHALLENGE IN RURAL AREA Y. Natanael1, A. Almira2, Hendrianus3 Ruteng Public Hospital1 Brawijaya Women and Children Hospital Antasari2 p g p q y Keywords: rheumatic heart disease, mitral regurgitation, pericardial effusion, heart failure p g p q y Keywords: rheumatic heart disease, mitral regurgitation, pericardial effusion, heart failure ACUTE RHEUMATIC FEVER INDUCED THYROID STORM IN PATIENT WITH RHEUMATIC HEART DISEASE: A RARE CASE FROM RI-RDTL CROSSBORDER R.K. Ratri1, C. Saputra2, Y.S. Sugi3, R.S. Sihombing3, W. Wiriadisurya3, J.R.L. Sulle4 General Practitioner of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara1 Cardiology and Vascular Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara2 Internal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara3 Radiology Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara4 R.K. Ratri1, C. Saputra2, Y.S. Sugi3, R.S. Sihombing3, W. Wiriadisurya3, J.R.L. Sulle4 General Practitioner of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara1 Cardiology and Vascular Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara2 Internal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara3 Radiology Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara4 R.K. Ratri1, C. Saputra2, Y.S. Sugi3, R.S. Sihombing3, W. Wiriadisurya3, J.R.L. Sulle4 neral Practitioner of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara1 gy and Vascular Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara2 gg nternal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tengg Radiology Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara gg Internal Medicine Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara3 Radiology Department of Mgr. Gabriel Manek, SVD Public Hospital of Atambua-East Nusa Tenggara4 Background: Thyroid storm is a life-threatening medical condition. This condition occurs in thyrotoxic patients and is manifested by the decompensation of multiple organs such as congestive heart failure and respiratory failure. In this case, thyroid storm induced by a recent streptococcal infection (acute rheumatic fever). Case Illustration: A 27 years old female, was referred to the Emergency Department of the Public hospital of Atambua with experienced fever, palpitation, and multiple joint pains. In the physical examination heart rate 128 times/min, respiratory rate 32 times/min, axillary temperature was 38 Celsius degree increased of JVP (5+4 mmHg), a heart murmur at the apex cordis, rales at the base of both lungs, and bilateral lower extremity edema. 12 lead ECG showed irregular narrow complex QRS indicating AF RVR and chest x-ray showed cardiomegaly with early sign of pulmonary edema. Patient was treated with optimal HF medication. For further diagnosis, on the second-day echocardiography result found mitral and aortic regurgitation with normal LV and RV systolic function. HYPERTENSION URGENCY IN SEVERE ABDOMINAL PAIN ET CAUSA HEPATIC HYDATID CYST: THE ETIOLOGY HAS BEEN REVEALED Conclusion: In hypertension patients, we must search for etiology that may reverse progression of disease. In this patient, there is external compression of renovascular because of hepatic hydatid cyst. Keywords: Hydatid cyst, Albendazole, Hypertension Rim Calcification in hepar (hydatid cyst) Rim Calcification in hepar (hydatid cyst) ACUTE RHEUMATIC FEVER INDUCED THYROID STORM IN PATIENT WITH RHEUMATIC HEART DISEASE: A RARE CASE FROM RI-RDTL CROSSBORDER Thyroid ultrasonography study showed grave’s disease appearance, laboratory examination ASTO titer 550 IU/ml, and Hyperthyroid condition with Burch-Wartofsky point scale of 70. From this working diagnosed become acute rheumatic fever induced thyroid storm. The patient has been treated with Thyroid storm protocol along with acute rheumatic fever therapy. The patient has shown improvement, hemodynamically stable, convert into NSR, relieved symptom and discharged after 8 days of hospitalization. C l i E l i i f h id d h i f i h li i l di i i i Conclusion: Early recognition of thyroid storm and acute rheumatic fever with clinical diagnosis criteria may lead into proper diagnosis and management. p p g g Keywords: Acute Rheumatic Fever, Thyroid Storm Clinical Diagnosis and Sonography Exam Clinical Diagnosis and Sonography Exam Indonesian Journa Cardiology Indonesian J Cardiol 2023:44:s pISSN: 2830-3105 / eISSN: 296 doi: 10.30701/ijc.1537 A Case Report of Premature Ventricular Complex in Long-Standing Hypertension Patient At Limited Primary Health Care : High or Low Level of Risk Feature ? S.D.T. Sitanggang1, C.J. Purba2 RSUD Pakuhaji, Tangerang1 Puri Hijau Hospital Medan2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Case Report of Premature Ventricular Complex in Long-Standing Hypertension Patient At Limited Primary Health Care : High or Low Level of Risk Feature ? A Case Report of Premature Ventricular Complex in Long-Standing Hypertension Patient At Limited Primary Health Care : High or Low Level of Risk Feature ? S.D.T. Sitanggang1, C.J. Purba2 RSUD Pakuhaji, Tangerang1 Puri Hijau Hospital, Medan2 Background : Premature ventricular complex (PVC) is a premature beat that occur spontaneously arising from the ventricle region. PVCs are common in general population with the prevalence between 1 – 4 % and are precipitated by many factors, including hypertension. PVCs could induce weakening of heart muscle if it is oftenly occur and untreated. This case report aims to present deeper insight about risk feature of PVC in patient with long- standing hypertension at limited primary health care. g yp p y Case Illustration : A 52 years old female came to primary health care (PHC) with a chief complain of palpitation especially at rest accompanied by shortness of breath (SOB) and headache since 2 days ago. 11 months earlier, she had came to PHC with similar symptoms and was suspected with hypertensive heart disease. Amlodipine was prescribed but she didn’t take it routinely. No chest pain or syncope were reported. She had no family history of sudden cardiac death or other heart diseases. Blood pressure was 149/73 mmHg with 110 bpm heart rate. No gallop (S3), murmur, peripheral edema or rales were found. Electrocardiography (ECG) shows normo-axis sinus rhythm with multiple PVCs as shown in figure 1. She was considered to have high risk feature of PVCs that may potentially cause cardiomyopathy then she was given captopril, bisoprolol and referred to cardiologist. she had came to PHC with similar symptoms and was suspected with hypertensive heart disease. Amlodipine was prescribed but she didn’t take it routinely. No chest pain or syncope were reported. She had no family history of sudden cardiac death or other heart diseases. Blood pressure was 149/73 mmHg with 110 bpm heart rate. No gallop (S3), murmur, peripheral edema or rales were found. Electrocardiography (ECG) shows normo-axis sinus rhythm with multiple PVCs as shown in figure 1. She was considered to have high risk feature of PVCs that may potentially cause cardiomyopathy then she was given captopril, bisoprolol and referred to cardiologist. Conclusion : Chronic hypertension in our patient may causes structural heart disease, particularly left ventricular hyperthrophy, and induces PVCs. A Case Report of Premature Ventricular Complex in Long-Standing Hypertension Patient At Limited Primary Health Care : High or Low Level of Risk Feature ? PVCs that arise due to structural heart disease have high risk feature/malignant if following features occurs in ECG such as polymorphic form or multifocal origin, consecutive run/salvoes, short coupling interval (< 300 ms), >5 PVCs/bpm and most importantly, symptomatic to the patient. While PVCs are common, it is vital to recognize its high-risk features and should warrant further cardiac evaluation if encountered. Keywords: premature ventricular complex, hypertension, arrhythmia, risk level, electrocardiogram Conclusion : Chronic hypertension in our patient may causes structural heart disease, particularly left ventricular hyperthrophy, and induces PVCs. PVCs that arise due to structural heart disease have high risk feature/malignant if following features occurs in ECG such as polymorphic form or multifocal origin, consecutive run/salvoes, short coupling interval (< 300 ms), >5 PVCs/bpm and most importantly, symptomatic to the patient. While PVCs are common, it is vital to recognize its high-risk features and should warrant further cardiac evaluation if encountered. Keywords: premature ventricular complex, hypertension, arrhythmia, risk level, electrocardiogram premature ventricular complex, hypertension, arrhythmia, risk level, electrocardiogram premature ventricular complex, hypertension, arrhythmia, risk level, electrocardiogram Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 A Case Report Post Peripartum Cardiomyopathy: Interrelation Marriage, Pivotal Factor for PPCM A.M.A. Kastiran1 RS wahidin sudirohusodo1 Keywords: Dyspneu, Delivery, Interrelation Marriage, Peripartum cardiomyopathy Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Case Report Post Peripartum Cardiomyopathy: Interrelation Marriage, Pivotal Factor for PPCM A.M.A. Kastiran1 RS wahidin sudirohusodo1 A.M.A. Kastiran1 RS wahidin sudirohusodo1 Background: Peripartum cardiomyopathy (PPCM) is a dilated cardiomyopathy defined as systolic cardiac heart failure in the last month of pregnancy or within five months of delivery. PPCM, which affects thousands of women each year in the US, was first described in the 1800s, but its etiology is still unclear. PPCM has many risk factors. One of them happened in this case, interrelation Marriage. Its diagnosis is often delayed because its symptoms closely resemble those within the normal spectrum of pregnancy and the postpartum period. When PPCM is misdiagnosed or its diagnosis is delayed, the consequences for patients are deadly: The disorder carries a high mortality rate Case Illustration: A 23 - year old woman came to the emergency unit with dyspneu. There is a history of giving birth about 3 months ago. Physical examination shows a regular heart rhythm accompanied by rhonchi sounds in the basal aspect of the lung. The M - mode analysis of ECG showed left ventricular systolic ejection function is decreased ( 26 % ) while the right ventricle is normal. In the hospital, the patient got treated with diuretics, aldosterone antagonists, angiotensin II receptor blockers, and fluid management. The patient gave a good response to the therapy regimen. Conclusion: The early clinical manifestations of PPCM are difficult to recognize. In the majority of patients, 78% of symptoms are found at 4 months after delivery, and only 9% of patients show symptoms in the last month of pregnancy. Clinical symptoms include signs of pulmonary edema, orthopnea, atypical chest pain, abdominal discomfort, and paroxysmal nocturnal dyspnea. In general, PPCM-related mortality ranges from less than 2% to 50%. This case teaches us about the unfavourable effects of Interrelation Marriage as a risk factor for PPCM. Keywords: Dyspneu, Delivery, Interrelation Marriage, Peripartum cardiomyopathy Keywords: Dyspneu, Delivery, Interrelation Marriage, Peripartum cardiomyopathy Indonesian Journa Cardiology Indonesian J Cardiol 2023:44:s pISSN: 2830-3105 / eISSN: 296 doi: 10.30701/ijc.1537 A Heart of Stone: Extensive Myocardial Calcification in End-Stage Renal Disease (ESRD) I. Pratiwi1, R. Ariani1 University of Indonesia1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Heart of Stone: Extensive Myocardial Calcification in End-Stage Renal Disease (ESRD) Case Illustration: A male, 66 years went to NCCHK with chest discomfort, burning sensation, fatigue, and dyspnea on exertion. The patient has a slight limitation of physical activity, and these symptoms were amplified on moderate physical activity. In past medical history, he experienced anterior STEMI on May 2022 and was diagnosed with CAD ISR after CAG from Pasar Rebo general hospital. However, because the ISR and the first stent wasn’t available and too small, therefore he was referred to NCCHK. He underwent PCI twice on 2017. Due to history of ISR LAD (60-70% at mid-proximal LAD) and multiple stents, therefore the patient underwent POBA with DEB-OCT. Background: Percutaneous coronary intervention (PCI) with drug-eluting stents (DES) is a treatment for patients with poor prognosis CAD because of the extensive myocardium. Bare-metal stent (BMS) and drug eluting stents (DES) are the two types of widely used stents for PCI. DES demonstrated superiority to BMS, it has a lower rate (4-8%) of restenosis than BMS implantation (20-30%). However, the prevalence of in-stent restenosis (ISR) remains relatively unchanged, encompassing about 10% of percutaneous coronary interventions despite of its evolution and iteration. In contrast to BMS, DES have a lower risk of early restenosis, but require a prolonged dual antiplatelet therapy, potentially leading to thrombus formation in case of early discontinuation. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 M.R. Priatama1, D. Firman2 M.R. Priatama1, D. Firman2 Universitas Indonesia, National Cardiovascular Center Harapan Kita1 Division of Invasive Diagnostic and Interventional Cardiology, National Cardiovascular Center Harapa A Heart of Stone: Extensive Myocardial Calcification in End-Stage Renal Disease (ESRD) I. Pratiwi1, R. Ariani1 University of Indonesia1 Background: Dysregulation of mineral metabolism with a high calcium load resulting poor calcium phosphorus balance in ESRD. Majority, cardiac conduction block presented as complication in ESRD Case Illustration: A 52-year-old woman came to emergency department with worsening fatigue and frequent syncope episode since last two weeks. She had end-stage renal disease (ESRD) and undergone routine hemodialysis for more than 4 years. On physical examination, the patient looked moderately ill with hypertension (194/70 mmHg), bradycardia (50 bpm). Rales were heard in lower third of pulmonary bases, Ascites with liver enlargement 2 cm below arcus costa. Electrocardiography showed total atrioventricular (AV) block. Chest X-ray showed cardiac enlargement, prominent aortic segment, normal pulmonary segment, flatten cardiac waist, downward apex, and pulmonary infiltrates in both lungs. Anemia and impaired renal function were detected through blood test. Echocardiography on parasternal long axis (PLAX) showed patchy calcification at septal right ventricle (RV) and left atrium (LA) wall (Fig. 1A). Parasternal short axis (PSAX) view shown heavily calcified of aortic cusps from basal annulus, moderate mitral stenosis (MS), moderate aortic stenosis (AS) with good left ventricle (LV) systolic function with left ventricle ejection fraction (LVEF) of 59% (Fig. 1B). Four chambers (Fig. 1C), three chambers (Fig. 1D), and two chambers (Fig. 1E) views had not detected other cardiac structural anomalies. Right ventricle (RV) contractility was good (TAPSE 17 mm). Apical parasternal short axis (PSAX) view showed diffuse mitral annular calcification (Fig. 1F). Echocardiography results were strongly suggestive of metastatic myocardial calcification. Temporary pacemaker (TPM) implantation for total AV block was successful then given angiotensin receptor blocker (ARB), and diuretics. Patient was successfully discharged after clinical improvement. Conclusion: Echocardiography is the first-line method that permit rapid and non-invasive detection of unrecognized or even unsuspected rare case of extensive myocardial calcification in ESRD. Keywords: End-Stage Renal Disease, Myocardial Calcification Keywords: End-Stage Renal Disease, Myocardial Calcification Keywords: End-Stage Renal Disease, Myocardial Calcification Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 How Do We Overcome ISR: Role of Intravascular Imaging M.R. Priatama1, D. Firman2 Universitas Indonesia, National Cardiovascular Center Harapan Kita1 Division of Invasive Diagnostic and Interventional Cardiology, National Cardiovascular Center Harapan Kita2 Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 How Do We Overcome ISR: Role of Intravascular Imaging Indonesian Journal of Cardiology A Case Report of Leukostasis Presenting as ST-Elevation Myocardial Infarct: Proceed Reperfusion or Not? Conclusion : In-stent restenosis can be characterized by a significant reduction in the luminal diameter in the stent segment after successful PCI. The incidence of ISR has been reduced with the newer DES technology compared to the era of BMS and only 5-10% PCI procedures performed in clinical practice. The use of intravascular imaging can guide the management of ISR through several stages. It can help optimize the stent placement procedure by predicting and avoiding ISR. Keywords: PCI, DES, OCT, ISR, DCB Figure 1. Pre Intervention OCT (Left Side) and Post POBA Intervention OCT (Right Side) Figure 1. Pre Intervention OCT (Left Side) and Post POBA Intervention OCT (Right Side) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 A Case Report of Leukostasis Presenting as ST-Elevation Myocardial Infarct: Proceed Reperfusion or Not? K. Saleh1, W.E. Putri1, A.I. Taim1 Cardiology and Vascular Medicine, FK UNHAS1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Case Report of Leukostasis Presenting as ST-Elevation Myocardial Infarct: Proceed Reperfusion or Not? K. Saleh1, W.E. Putri1, A.I. Taim1 Cardiology and Vascular Medicine, FK UNHAS1 Background: Leukostasis is a medical emergency caused by compromise of tissue perfusion secondary to hyperleukocytosis in acute myeloid leukemia. Typically it affects lungs and brain, with cardiac involvement being exceedingly rare. Acute myocardial infarction and acute myeloid leukemia are rarely reported as concomitant conditions. The management of ST-elevation myocardial infarction in patients who have AML is challenging due to the increased risk of bleeding. Case illustration: A 66-year-old man with suspected acute myeloid leukemia presented with typical anginal chest pain. ECG revealed ST elevation in lead V2-V6. Troponin I peaked at 3548.4 ng/L. Echocardiogram showed mildly abnormal LV systolic function with ejection fraction of 45% (TEICH) due to segmental akinesia and hypokinesia. Blood work was notable for white blood cell count of 242.700 103/μL and Hemoglobin of 8.7 gr/dl. Peripheral blood analysis result was suspected acute myeloid leukemia. Conclusion: The management of ST-elevation myocardial infarction in patients who have acute myeloid leukemia is challenging: the leukemia-related thrombocytopenia, platelet dysfunction, and systemic coagulopathy increase the risk of bleeding, and the administration of thrombolytic agents can be fatal. It is important to note that prognosis for patients with concomitant conditions of acute myeloid leukemia and acute myocardial infarction is worse than that of either condition. There is no management guideline regarding these conditions and invasive reperfusion strategy could be done when there is no absolute contraindication. Keywords: leukostasis, hyperleukocytosis, acute myeloid leukemia, ST-elevation myocardial infarct Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Ablation of Idiopathic Trigeminy Premature Ventricle Complex using Zero–Fluoroscopy Approach: A Case Report Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Ablation of Idiopathic Trigeminy Premature Ventricle Complex using Zero–Fluoroscopy Approach: A Case Report Ablation of Idiopathic Trigeminy Premature Ventricle Complex using Zero–Fluoroscopy Approach: A Case Report A.F. Chandranegara1, M.R. P. Adri1 General Regional Hospital of Pasar Rebo1 A.F. Chandranegara1, M.R. P. Adri1 General Regional Hospital of Pasar Rebo1 Background: In the last few decades, awareness of the risks associated with radiation to patients and medical staff has increased significantly. It is also a topic of discusiion in the cardiology world because fluoroscopy, which also used radiation, is widely used to orient the position of catheters in the heart during Radiofrequency Catheter Ablation. The existence of a 3-dimensional navigation system make the management of arrhythmias less complicated. 3D Electroanatomical Mapping (EAM) can minimize (near-zero fluoroscopy) or eliminate radiation dose (zero-fluoroscopy). Here we present our first zero-fluoroscopy case in a breast tumor patient with trigeminy PVC. Case Illustration: 52 years old woman was consulted by the surgical department for a planned operation for a breast tumor but found trigeminy PVC on electrocardiogram (ECG). In recent years, patients often complain of palpitations. There are no remarkable results in physical examination, laboratory investigation, and echocardiography. The patient was planned for ablation using 3D EAM. A breast tumor suspected of cancer provoked the electrophysiologist to adopt a zero-fluoroscopy approach. The 3D EAM system will create geometric contours from the IVC until Right Ventricle Outflow Tract (RVOT). Surface ECG showed bigeminy PVC with positive morphology in I and aVL, positive in II, III, aVF, and LBBB pattern QRS notch in V3-V4. The earliest activation was found in the posteroseptal region with -117ms LAT and -37ms EA. Multiple RFA for 60-120 seconds was done at that location. After 30 minutes of observation, no PVCs were found. Conclusion: Three-Dimensional PVC ablation with zero-fluoroscopy was successfully performed. RFCA with zero-fluoroscopy approach is becoming the treatment of choice in patients with malignancy. Keywords: Ablation, Premature Ventricular Complex, Zero-Fluoroscopy, 3D-Electroanatomical Mapping 3D EAM showed the earliest activation in posteroseptal with LAT -117 ms and EA -37ms 3D EAM showed the earliest activation in posteroseptal with LAT -117 ms and EA -37ms Bradycardia-Induced VT in Degenerative Sinus Node dysfunction: High Risk Feature of Life Threatening VT in Bradycardia R. Hendiperdana1, R.Y.I. Putri1, S. Laksono2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita1 Department of Cardiology and Vascular Medicine, Diagram Heart Hospital2 R. Hendiperdana1, R.Y.I. Putri1, S. Laksono2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita1 Department of Cardiology and Vascular Medicine, Diagram Heart Hospital2 R. Hendiperdana1, R.Y.I. Putri1, S. Laksono2 of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita1 p , , Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita1 p Department of Cardiology and Vascular Medicine, Diagram Heart Hospital2 p ( Conclusion Severe bradycardia can lead to QT prolongation and subsequent life threatening polymorphic VT. Several clinical predictors such as older age and female have higher risk of TdP occurrence. Profile of QT interval and T wave morphology like QT interval, QTc interval, Tpeak-Tend duration, and complex T wave morphology could predict high occurrence of polymorphic VT as seen in our patient’s ECG presentation. Pacing at higher rate in emergency setting could become a lifesaving since it can stabilize the repolarization from perturbation. Keywords: bradycardia, Ventricular tachycardia, Long-QT Background g Bradycardia has been linked with inverse relationship with QT duration and repolarization time, since it can induce polymorphic VT in severe bradyarrhythmia. The potentially life threatening arrhythmia explained by the possibility of the rhythm to degenerate into ventricular fibrillation. C Ill t ti A 76-year-old woman was admitted with recurrent fainting and post cardiac arrest resuscitation. She was somnolence with bradycardia (40 bpm) and recurrent non-sustained VT on electrocardiograph (ECG) monitor. Her blood pressure was 140/90 mmHg with 99 % peripheral oxygen saturation. Her initial ECG record showed sinus bradycardia with intermittent junctional rhythm with a rate of 48 bpm, deep T wave inversion at precordial lead with prolonged QT interval (QT 720 ms, QTc 670 ms, Tpeak-Tend (Tpe) 200 ms, Tpe/QT ratio 0.27. Her blood chemistry was normal. Bedside echocardiogram showed no structural heart disease. While being monitored, she developed recurrent unstable sustained polymorphic VT episode which required DC cardioversion for termination. Dopamine infusion starting on 5 micron/kg/min was given and initiation of transcutaneous pacing (TCP) for stabilization. The patient was very dependent to TCP, when the paddle was removed, the bradycardia ensue and VT was inevitable induced. Temporary transvenous cardiac pacemaker (TPM) was finally implanted in this patient for stabilization. Conclusion CARDIOGENIC SHOCK AS A RESULT OF RARE ANTIPSYCHOTICS-INDUCED DILATED CARDIOMYOPATHY IN YOUNG PATIENT S. Nurfitri1, H. Isnanijah1, T. Rahayu1 S. Nurfitri1, H. Isnanijah1, T. Rahayu1 Pasar Rebo Regional General Hospital, Jakarta, Indonesia1 Background: Schizophrenia patients often take antipsychotics for considerably long periods to prevent psychotic episodes and maintain stabilized condition. Albeit drug-induced cardiomyopathy is a rare side effect of antipsychotics, particularly Clozapine and Quetiapine, it is a life-threatening event that is usually overlooked and underestimated. Given its insidious nature, patients tend to come in a peril state with obscure presentation, making it harder to draw correlation between cardiac episode and antipsychotic consumption, hence correct diagnosis and treatment will be challenging. Case Illustration: A 17-year-old male presented with dyspnea and cough since 4 days before admission. He had been taking Clozapine, Risperidone, Quetiapine, THP, and Lithium Carbonate for a year as schizophrenia treatment. No history of diabetes, lung, and cardiac disease is known. Physical examination revealed hypotension, RR = 28x/min, SpO2 = 99%, rhonchi on auscultation, cold extremities, and CRT >2 seconds. ECG showed sinus tachycardia without signs of new ischemic changes or pericardial pathologies. USCOM examination revealed that CI (1.8 l/min/m2) and SVI (15 ml/m2) are low, while SVRI (3419 d·s·cm−5/m2) is high. CXR showed marked cardiomegaly. Echocardiogram revealed dilatation of all heart chambers, reduced EF (17%), mild tricuspid, and moderate mitral regurgitations. Patient underwent fluid resuscitation. He was treated with dobutamine then transferred to PICU where he received HF medications consisting of furosemide, spironolactone, captopril, and digoxin. After consultation with a psychiatrist, the antipsychotics were stopped and the patient achieved hemodynamic stability with better clinical outcome. Given the absence of other clear etiologies, antipsychotic consumption is the leading cause of cardiomyopathy which progressed into cardiogenic shock. Conclusion: Drug-induced cardiomyopathy is a rare yet dangerous side effect of antipsychotics. Early recognition should be considered in patients who take antipsychotics to prevent deterioration and provide correct treatment. Keywords: cardiogenic shock, antipsychotics-induced cardiomyopathy, schizophrenia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Bradycardia-Induced VT in Degenerative Sinus Node dysfunction: High Risk Feature of Life Threatening VT in Bradycardia Kounis Syndrome Secondary To Drug-Induced Hypersensitivity: Report Of A Devastating Case From Referral Hospital In Central Borneo A R Sudirman1 Y Galenta1 Kounis Syndrome Secondary To Drug-Induced Hypersensitivity: Report Of A Devastating Case From Referral Hospital In Central Borneo A.R. Sudirman1, Y. Galenta1 RSUD dr. Doris Sylvanus Provinsi Kalimantan Tengah1 BACKGROUND The concurrence of ACS and hypersensitivity reactions is defined as Kounis syndrome. It is often misdiagnosed and lead to inappropriate treatment with high subsequent mortality rate. The mechanism behind is related with excessive catecholamine production alongside with inflammatory-cytokine-released triggered by allergic reaction. Hereby we present the case of drug-hypersensitivity induced Kounis syndrome in our hospital. CASE ILLUSTRATION A 65-year-old woman presented with shortness-of-breath and chest tightness. Her past medical history was remarkable for asthma with multiple readmission and drugs hypersensitivity. There is no cardiac-related history. Upon arrival, her saturation-92%, respiratory rate-35x/min and unable to speak. She was in respiratory distress with expiratory-wheezing on both lungs. Laboratory studies was unremarkable and ECG showing OMI. The patient was suggestive for acute severe asthma exacerbation. Methylprednisolone and ventolin nebulizer were administered. Suddenly, her respiratory distress worsen, with loss of consciousness, RR-50x/min, saturation-60%- on-NRM, and BP-70/40. She was developed into anaphylactic shock. IM-epinephrine was administered. Her consciousness improved with saturation-98% and RR-30x/min. Unfortunately, she was developed into ventricular tachycardia and her BP-exceeds-220/110. This condition returned spontaneously without any drug administration. Later on, she developed typical chest pain similar with myocardial ischemia symptom. Her ECG evaluation showing ST elevation in anterior segment with RBBB. Her troponin level slightly increased from <0.01 to 0.6. The patient was suggestive for ACS in the settings of mast-cell and platelet activation from anaphylactic reaction. Dual-antiplatelet and anticoagulant were administered. We cannot perform primary-PCI because refusal of any further medical-intervention. Her condition worsen following days. Her echocardiography-evaluation demonstrated global hypokinetic with-EF-40%. On the-seventh-day, she was passed away due to heart failure and cardiac arrest. Keywords: bradycardia, Ventricular tachycardia, Long-QT Keywords: bradycardia, Ventricular tachycardia, Long-QT Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Kounis Syndrome Secondary To Drug-Induced Hypersensitivity: Report Of A Devastating Case From Referral Hospital In Central Borneo 1 1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Coronary Syndrome In Severe Aortic Stenosis and Aortic Regurgitation Patient: A Case Report G.F. Mantong1, Y. Iskandar1 Elim Rantepao Hospital, North Toraja1 G.F. Mantong1, Y. Iskandar1 Elim Rantepao Hospital, North Toraja1 Background: Angina pectoris is one of common symptoms in patient with aortic stenosis (AS) and aortic regurgitation (AR). To diagnose acute coronary syndrome (ACS) in patient with severe AS and AR is challenging especially in peripheral hospital. In this case will discuss ACS in patient with severe AS and AR, focus on diagnosis. Case illustration: A 69 y.o women came with shortness of breath felt since 4 hours ago when she rested. She also felt chest pain that more severe and intens than before, radiated to neck and accompanied with diaphoresis. Patienthad history of severe degenerative AS and AR, sometimes she felt chest pain and shortness of breath during activity but relieve with rest. We found to-and-fro murmur and was best heard at right upper sternal border. Electrocardiography showed left bundle brunch block (LBBB) patern. Based on brief examination in emergency room, patient was diagnosed as ACS with troponin level was known markedly elevated later on. Vital signs were unstable during the first day but later controlled during the rest six days of hospitalization. CONCLUSION Kounis syndrome should be suspected whenever patient presents with features of ACS and allergic symptoms simultaneously. The challenges lies in the fact that each treatment of associated entities may worsen the other injury. Keywords: Kounis sydnrome, Acute coronary syndrome, Drug Hypersensitivity Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 ECG Changes after anaphylactic reaction and ventricular tachycardia. The changes demonstrated ST elevation in anterior segment with RBBB. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 ECG Changes after anaphylactic reaction and ventricular tachycardia. The changes demonstrated ST elevation in anterior segment with RBBB. Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Acute Coronary Syndrome In Severe Aortic Stenosis and Aortic Regurgitation Patient: A Case Report G.F. Mantong1, Y. Iskandar1 Elim Rantepao Hospital, North Toraja1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Coronary Syndrome In Severe Aortic Stenosis and Aortic Regurgitation Patient: A Case Report G.F. Mantong1, Y. Iskandar1 Elim Rantepao Hospital, North Toraja1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Keywords: Balloon Transcatheter Pulmonary Valvotomy, Valvular Pulmonal Stenosis, Patent Foramen Ovale p Conclusion: ACS diagnosis in severe AS and AR group is challenging because it is often presenting with similar symptom likes angina pectoris. Proper history taking and physical examination is the main point for the diagnosis of ACS especially in peripheral hospital. Keywords: Acute Coronary Syndrome, Angina Pectoris, Aortic Stenosis, Aortic Regurgutation Figure 1. ECG Keywords: Acute Coronary Syndrome, Angina Pectoris, Aortic Stenosis, Aortic Regurgutation Figure 1. ECG Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Successful Balloon Transcatheter Pulmonary Valvotomy in Children with Severe Valvular Pulmonary Stenosis : A Case Series cessful Balloon Transcatheter Pulmonary Valvotomy in Children with Severe Valvular Pulmonary Stenosis : A Case Series D.G. Nugroho1, W. Mubarok1, A. Priyatno2 Cardiovascular Department, Faculty of Medicine Diponegoro University, Dr Kariadi Hospital, Semarang1 Pediatric Department, Faculty of Medicine Diponegoro University, Dr Kariadi Hospital, Semarang2 Background: Pulmonary stenosis (PS) is typically benign yet progressive in pediatric patients. Severe PS may result in outflow obstruction that progresses over a period of years despite body growth. An excellent prognosis can be achieved by appropriate intervention. Balloon Transcatheter Pulmonary Valvotomy (BTPV) has replaced surgery and become preferred mode of treatment. Case Illustration: Patients of age 2, 5, and 11 years old showed aggressive progression of PS with variety of symptoms with similar malnourished state. The auscultation of 4/6 ejection systolic murmur, best heard in second and third left intercostal space were found in all three patients. The electrocardiography showed right axis deviation and right ventricular hyperthrophy. They had dome-shaped valves morphology with pressure gradient (PG) between 78-136 mmHg by echocardiography. Patent foramen ovale were found during catheterization. BTPVs were performed using THYSAK mini and Nucleus with size of 1,4 times larger than their pulmonary annulus diameter. The insertion of balloon were quite challenging. It had to be precisely placed in order to fully dilate the stenotic valve. The 50% PG reduction of RV-PA were found in all cases with periprocedural transient bradycardia. Seven days evaluation showed improved symptoms, better food intake with PG as low as 30 mmHg. Conclusion : Our successful cases suggest that BTPV is a promising and safe intervention for children with severe valvular PS. Reversal shunt of interatrial defect can be prevented by reducing the right chamber pressures. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Does His Bundle Pacing Have a Good Outcome? A Case of 66 Years Old Man, Presenting With Recurrent Syncope D. Surya1, F. Yuliani1, H.E. Rasyid1, C.K. Krevani1 Dr. M. Djamil General Hospital1 A RARE CASE OF PAINLESS TYPE A AORTIC DISSECTION PRESENTING AS ACUTE ISCHEMIC STROKE M.I. Muzakky1, S.G.V. Hayon1, M.B.S. Ilham2, A.A. Kesumaningputri3, D. Hudiana4 General Practitioner, Dr. Sitanala Central Hospital, Tangerang, Indonesia1 Internship Doctor, Bhayangkara Hospital, Denpasar, Indonesia2 Department of Cardiology, Dr. Sitanala Central Hospital, Tangerang, Indonesia3 Department of Neurology, Dr. Sitanala Central Hospital, Tangerang, Indonesia4 M.I. Muzakky1, S.G.V. Hayon1, M.B.S. Ilham2, A.A. Kesumaningputri3, D. Hudiana4 General Practitioner, Dr. Sitanala Central Hospital, Tangerang, Indonesia1 Internship Doctor, Bhayangkara Hospital, Denpasar, Indonesia2 Department of Cardiology, Dr. Sitanala Central Hospital, Tangerang, Indonesia3 Department of Neurology, Dr. Sitanala Central Hospital, Tangerang, Indonesia4 Background: Stanford type A aortic dissection (AD) with ischemic stroke is a rare condition, accounting for approximately 1.7% of all acute stroke cases. Painless AD is typically identified through incidental findings on a chest x-ray or the presence of related symptoms and complications. Diagnosing painless AD in patients presenting as stroke can be challenging and potentially misdiagnosed. Case Illustration: A 68-year-old woman presented with a sudden onset of left-sided weakness without chest or back pain. Her past medical history revealed hypertension for five years with no history of acute stroke. On arrival, her blood pressure was 101/65 mmHg, heart rate was 55 beats per minute, respiration rate was 26 times per minute, and oxygen saturation was 90%. The patient was admitted to the hospital 2 hours after onset and was immediately triaged to the code stroke response protocol. Neurological examination disclosed left hemiparesis (left motor strength 1/5) with a positive Babinski reflex. The National Institutes of Health Stroke Scale (NIHSS) score was 14. A diastolic murmur was heard on the third intercostal space along the left sternal border. A non-contrast head computed tomography (CT) showed no evidence of hemorrhagic stroke, while the chest X-ray indicated mediastinal enlargement and cardiomegaly. Further evaluation by thoracic and abdominal CT angiograms showed a tear in the ascending part of the aorta. The patient was diagnosed with stroke infarction, type A aortic dissection, and severe aortic regurgitation. However, the patient refused to undergo a surgical procedure and was admitted to the intensive care unit for conservative management integrated with neurology, which included regulation of blood pressure and heart rate. Conclusion: AD with atypical symptoms requires timely recognition and immediate surgical repair. However, conservative management is associated with a high incidence of early mortality. Keywords: type A aortic dissection, stroke infarction Conclusion: AD with atypical symptoms requires timely recognition and immediate surgical repair. Does His Bundle Pacing Have a Good Outcome? A Case of 66 Years Old Man, Presenting With Recurrent Syncope D. Surya1, F. Yuliani1, H.E. Rasyid1, C.K. Krevani1 Dr. M. Djamil General Hospital1 Background: Syncope is a state of losing consciousness because of cerebral hypoperfusion that usually happens in rapid onset, short duration, and is followed by spontaneous recovery. Approximately 3-5% of emergency cases were presented as syncope. One of the causes was sinus node dysfunction (SND) which mostly needs to be treated with permanent pacing. His bundle pacing (HBP) starts to be widely used as a modality pacing in patients with SND because it was expected to have fewer clinical complications. We present a case of a 66-year-old man with sinus node dysfunction who was successfully treated with permanent HBP. y y p Case Illustration: A 66-year-old male presented with dizziness to the emergency room. The patient had a blackout and collapsed for 3 seconds but was still aware of the surrounding. The patient also reported having blackouts, dizziness, and syncope about 10 times in the last 2 years. The patient was admitted to the hospital 1 month ago because of having a syncope for 2 seconds. Electrocardiography showed a period of sinus paused and we noticed sinus node dysfunction (SND) with atrial flutter. The patient was put on Holter monitor for a couple of days and we used HBP as the permanent pacemaker for the patient. Conclusion: Sinus node dysfunction (SND) with bradyarrhythmia was one of the indications for placing a pacemaker in this patient. We chose HBP as it was suggested to be able to prevent cardiomyopathy and reduced the length of stay in the hospital if compared to other pacing. g y p p p g Keywords: his bundle pacing (HBP), recurrent syncope, sinus node dysfunction (SND), atrial flutter ECG from the first day of admission (above) and ECG after His Bundle Pacing placement (below) ECG from the first day of admission (above) and ECG after His Bundle Pacing placement (below) ECG from the first day of admission (above) and ECG after His Bundle Pacing placement (below) Case Illustration Case Illustration A 44-year-old male patient, without any history of cardiovascular disease, was referred to our hospital due to anterior wall STEMI Killip IV – cardiogenic shock 28 hours of onset after failed fibrinolytic. He remained chest pain on admission and was hemodynamically unstable. Rescue PCI was performed, on angiography we found acute total occlusion in mid LAD after D1 with high thrombus burden, LCX was not visualized on LCA angiography. RCA was dominant without stenosis or thrombus, LCX was seen originating from common ostia with RCA in RSV (type II), retro-aortic in course, traversing between the aortic root and left atrium, to its distribution territory. Based on ECG and clinical appearance, LAD had a culprit lesion which we decided to stent. Angiography evaluation revealed TIMI 2-3 flow with residual thrombus. During the operation, patient had an episode of pulseless VT/VF but ROSC achieved after CPR. He was intubated and mechanically ventilated. Optimal medical therapy including heparinization continued. On the next day the patient had worsening of symptoms and was diagnosed to have uncompensated respiratory alkalosis. We optimized critical care management and his hemodynamic status gradually improved on the 5rd day of hospitalization. He was discharged home with stable condition after 12 days hospitalization and came to routine check-up without significant symptoms. Background g Anomalous left circumflex coronary artery (ALCx), as part of coronary artery anomalies (CAA), is a rare phenomenon. We present an unusual case of an anomalous aortic origin of LCx in a middle-aged patient who presented with acute coronary syndrome (ACS). Rare Incidental Finding of Anomalous Left Circumflex Artery Arising from Right Sinus Valsalva in Acute Coronary Syndrome Patient F.A. Nurullah1, N.R. Utantyo1 Embung Fatimah Regional Public Hospital1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A RARE CASE OF PAINLESS TYPE A AORTIC DISSECTION PRESENTING AS ACUTE ISCHEMIC STROKE However, conservative management is associated with a high incidence of early mortality. Keywords: type A aortic dissection, stroke infarction Computed tomography angio of coronal and axial view-showing dissection in ascending aorta. Computed tomography angio of coronal and axial view-showing dissection in ascending aorta. Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964- doi: 10.30701/ijc.1537 Rare Incidental Finding of Anomalous Left Circumflex Artery Arising from Right Sinus Valsalva in Acute Coronary Syndrome Patient Case Illustration A 29 years-old G4P3 female at approximately 12 weeks gestational age presented after feeling palpitations and shortness of breath, several hours before admitted to emergency department. She denied chest pain and syncope. She denied any past medical history and taking any medications. Initial heart rate was 198bpm and blood pressure was 80/50 mmHg. Chest X ray showed normal cardiac size. ECG showed SVT with short RP (>90ms). Laboratory examination showed mild hypokalemia (3.03mEq/L) and mild hypocalcemia (1.05mEq/L). Electrical cardioversion was done from 50 joule to 200 joule but there was no effect. In our hospital, adenosine, intravenous beta blocker, intravenous verapamil were unavailable, also inability to do cardiac ablation. So the only choice was intravenous digoxin 0,5mg. After 2 dose digoxin given, heart rate started to slowing down (110bpm) and converted to sinus rhythm with delta wave (WPW pattern). After 2 days observation at ICU, heart rate was stable at 70-90bpm. This patient was discharged because of financial problem and planned for echocardiography examination at outward clinic. Background Background Compared to the non-pregnant population, cardiac arrhythmias are rare in pregnancy, with an incidence of about 1.2 per 1000. Supraventricular Tachycardia (SVT) is the most common form of arrhythmia during pregnancy, although often benign in nature, can be concerning. The management of SVT in pregnancy, varies slightly based on the trimester of pregnancy. Conclusion Lack of choice (availability and safety) of anti arrhythmia drug and failure of electrical cardioversion was quite challenging for this case. All antiarrhythmic drugs cross the placenta, but when necessary, medical treatment should be used. Electrical cardioversion is safe during pregnancy, and catheter ablation can be performed in selected patients, preferably with zero-fluoroscopy technique. A multidisciplinary approach is crucial to balance maternal cardiac, obstetric and fetal considerations. Keywords: SVT, Pregnancy, WPW, Arrhythmia al Cardioversion to Convert Hemodynamic Unstable Supraventricular Tachycardia in 1st Trimester Pregnancy, What Should We Do? : a Case Report A. Leisyah1, B. Setiawan1 RSUD Sayang Kabupaten Cianjur1 Failure of Electrical Cardioversion to Convert Hemodynamic Unstable Supraventricular Tachycardia in 1st Trimester Pregnancy, What Should We Do? : a Case Report A L i h1 B S ti 1 Failure of Electrical Cardioversion to Convert Hemodynamic Unstable Supraventricular Tachycardia in 1st Trimester Pregnancy, What Should We Do? : a Case Report Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 y p Conclusion Although type II ALCx was reported as benign variant, under several circumstances it posed to malignant risks. In the setting of ACS patient, recognizing this anomaly is crucial since failure to angiographically describe CAA can cause inappropriate clinical decisions and complications during invasive procedure. Keywords: Anomalous left circumflex coronary artery (ALCx), Percutaneous Coronary Intervention, ST Keywords: Anomalous left circumflex coronary artery (ALCx), Percutaneous Coronary Intervention, S segment elevation myocardial infarction (STEMI), Coronary artery anomalies (CAA), Congenital Heart Figure 1: RCA angiography showed LCX was seen originating from common ostia with RCA in RSV (type II) re 1: RCA angiography showed LCX was seen originating from common ostia with RCA in RSV (type II Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Failure of Electrical Cardioversion to Convert Hemodynamic Unstable Supraventricular Tachycardia in 1st Trimester Pregnancy, What Should We Do? : a Case Report Case Illustration A 44-year-old woman came to the Emergency Department (ED) complaining of palpitation. She denied any shortness of breath, cough, or fever on the day of admission and had no history of cardiovascular disease. The patient’s condition is compos mentis, with a pulse rate of 144 bpm. The physical examination revealed an irregular heart rhythm without a murmur. An electrocardiogram (ECG) examination in the ED showed atrial fibrillation. The patient’s thorax X-ray showed bronchitis. COVID-19 rapid antigen test showed a positive result, so the patient was planned for COVID-19 PCR swab in the isolation room. Three days later, the PCR swab showed a positive result. Laboratory examination showed a total leukocyte count of 7030/µL, neutrophil-lymphocyte ratio (NLR) of 0.73, and elevated D-dimer of 2200 ng/ml. The patient was treated with ceftriaxone, bisoprolol, azithromycin, n-acetylcysteine, favipiravir, and fondaparinux. ECG examination was done every two days. The patient underwent a PCR swab evaluation on the seventh day with negative results. Thorax X-ray evaluation showed no bronchitis compared to the previous. ECG showed a sinus rhythm with a frequency of 66 bpm. Other workups were not carried out due to limited facilities. The patient had no complaints and was discharged from the hospital without any complications. Background g Coronavirus Disease 2019 (COVID-19) has been infecting people around the world caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). COVID-19 generally presents with symptoms of fever, cough, and shortness of breath. Cardiovascular problems have been reported in some COVID-19 patients, including arrhythmia. Atrial fibrillation is one type of it. Paroxysmal Atrial Fibrillation in a COVID-19 Patient : A Case Report from Rural Area R. Ainunnahqi1, A.N. Famila2 1General Practitioner at Islamic Hospital Purwokerto, Indonesia 2Department of Cardiology and Vascular Medicine, Banyumas Regional Hospital, Indonesia R. Ainunnahqi1, A.N. Famila2 1General Practitioner at Islamic Hospital Purwokerto, Indonesia 2Department of Cardiology and Vascular Medicine, Banyumas Regional Hospital, Indonesia BACKGROUND Delayed presentation or latecomer of ST-elevation myocardial infarction (STEMI) and complicated by cardiogenic shock (CS-STEMI) is a challenging scenario that commonly encountered in developing countries which one in three patients does not survive. We present a successful comprehensive management in latecomer CS-STEMI patient in the setting of remote area. CASE ILLUSTRATION A 42 year-old woman with a typical chest pain since 4 days prior admission was referred to our hospital after being hospitalized in two hospitals. From first hospital, she was discharged with undiagnosed ACS. In second hospitalization, she was treated as ACS patient by a non-cardiologist then was referred to our hospital. At our ED, she was diagnosed as late onset extensive anterior STEMI KILLIP IV-cardiogenic shock, type-2 diabetes mellitus with severe uncompensated metabolic acidosis, chronic kidney disease and secondary hepatic injury. Echocardiography showed reduced LV contractility (LVEF of 37%) and hypokinetic anterior wall. Her condition quickly deteriorated as she had episodes of pulseless VT/VF terminated by successful CPR and DC shock. She was intubated, mechanically ventilated and transferred to other PCI-capable hospital due to administration issue. Although as a late onset STEMI with unstable hemodynamic status, high risk primary PCI was performed. Coronary angiography showed acute total occlusion of proximal LAD. Successful PPCI to LAD was performed with TIMI 3 flow result, hemodynamic status improved. She also underwent haemodialysis as her kidney function worsened due to hypoperfusion state and contrast induced nephropathy. Patient was discharged in stable condition after 22 days hospitalization. High Risk Percutaneous Coronary Intervention and Comprehensive Management in Life-Threatening Latecomer ST-Elevation Myocardial Infarction Complicated by Cardiogenic Shock in Remote Area High Risk Percutaneous Coronary Intervention and Comprehensive Management in Life-Threatening Latecomer ST-Elevation Myocardial Infarction Complicated by Cardiogenic Shock in Remote Area F.A. Nurullah1, N.R. Utantyo1 Embung Fatimah Regional Public Hospital1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion Atrial fibrillation can occur in COVID-19 patients. Several factors, including tissue injury, hypoxia, age, and systemic infection, probably contribute. However, clear pathomechanism of this condition needs further exploration. p Keywords: Atrial Fibrillation, COVID-19, Rural Area Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 High Risk Percutaneous Coronary Intervention and Comprehensive Management in Life-Threatening Latecomer ST-Elevation Myocardial Infarction Complicated by Cardiogenic Shock in Remote Area CONCLUSION “Time is muscle”, chances of recovery decrease with every passing minute, but timely reperfusion is challenging in remote area due to resource constraints, delayed patient presentation and system delays which associated with more complicated case, high-cost management and poorer outcome. Major integrated improvement is crucial to reduce mortality and morbidity in STEMI patients. Keywords: Acute coronary Syndrome, ST-Elevation Myocardial Infarction, Cardiogenic Shock, Percutaneous Coronary Intervention, Hemodialysis Figure 1: ECG showed ST elevation in lead II, III, aVF, V2-V6 and coronary angiography showed acute total occlusion of proximal left coronary artery (LAD) Figure 1: ECG showed ST elevation in lead II, III, aVF, V2-V6 and coronary angiography showed acute total occlusion of proximal left coronary artery (LAD) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Total Atrioventricular Block in Postpartum Total Atrioventricular Block in Postpartum A.S. Maulana1, A. Candra2, D. Ariyanti1, K.W. Putri3 RSD Dr Soebandi Jember1 Faculty of Medicine Universitas Jember2 Faculty of Medicine Airlangga University3 Background: Total atrioventricular block (TAVB) in postpartum is a rare case. Cases are few and management is not yet standardized. The aim of this study was to investigate the risk factors for TAVB in postpartum. Case illustration: A 23-year-old P2A1 of 37 weeks gestation presented with frequent fainting since 5 months ago. She had never any medical examination before. At presentation, her heart rate was 44 x/minute, and her blood pressure was 147/76 mmHg. A 12-lead electrocardiogram (ECG) revealed a total AV block. The serum potassium level was 2.6 mmol/L, and the chloride level was 113 mmol/L. Chest X-ray showed cardiomegaly. A temporary pacemaker (TPM) was implanted. A transthoracic echocardiogram (TTE) was performed, which showed a suspicion atrial septal defect (ASD). After 5 days of TPM, her intrinsic heart rate was still below 50 bpm and was not returned to a normal sinus rhythm. A reversible cause, such as hypokalemia, has been resolved. Transesophageal Echocardiography (TEE) is performed to confirm the size and location of the ASD. However, TEE revealed interatrial septum was intact. A permanent implant pacemaker (PPM) was recommended. Conclusion Background: Total atrioventricular block (TAVB) in postpartum is a rare case. Cases are few and management is not yet standardized. The aim of this study was to investigate the risk factors for TAVB in postpartum. Indonesian Journal of Cardiology Indonesian Journal of Cardiology When Learning ECG Turns Into A Nightmare: A Rare Case Report Study of PVCs Conclusion Early detection of risk factors and treatment of reversible causes of total AV block in postpartum women are needed to treat total AV block successfully Early detection of risk factors and treatment of reversible causes of total AV block in postpartum women are needed to treat total AV block successfully. y Keywords: Total atrioventricular block, pacemaker, postpartum Keywords: Total atrioventricular block, pacemaker, postpartum Figure 1. ECG showed a total atrioventricular block Figure 1. ECG showed a total atrioventricular block Background : Infective endocarditis (IE) is associated with a high risk of mortality and morbidity.1,2 Surgery has been the treatment of choice for IE because of severe complications. The optimal timing of the surgery has been unclear.3,4 In particular conditions early surgery brings certain benefits, so to avoid further deterioration yet performing surgery during the active phase of IE carries significant risk.4 Identification, risk stratification and early consultation with cardiac surgeon is capital to determine the best therapeutic approach.3 Case Illustration: A 20-years-old female was admitted to emergency room with complaints of breathlessness, tachypnea, palpitation, heaviness in chest at night, and dyspnoea on exertion, had undergone an appendectomy in other hospital with one month history of prolonged fever. Physical examination revealed a regular heart rate with systolic murmur sound heard on the apex. An electrocardiography (ECG) was sinus rhythm, heart rate 115x/minutes, bigeminy premature ventricular contraction (PVC). A blood examination revealed anaemia, leucocytosis, thrombocytosis, electrolyte imbalance. A transthoracic echocardiography (TTE) dilatation of left atrial and ventricle, an eccentric hypertrophic left ventricle, severe mitral regurgitation with possible vegetation on anterior and posterior mitral leaflet. Patient was diagnosed with Infective Endocarditis (IE), severe Mitral Regurgitation accompanied with acute heart failure, treated to IE protocol for 2 weeks with good response. The patient was decided to get early surgery to mitral valve repair. The result was remarkable, with subsided symptoms of heart failure and mild residual MR. Conclusion: Infective Endocarditis along with complications need apropriate treatment plan, The decision to perform early surgery to this patient resulting favorable end, opening possibility to do it in regards of capitalizing improvement of patient’s condition, shortening length of stay and cutting costs over scalable risk that present in this patient. Keywords: INFECTIVE ENDOCARDITIS, EARLY SURGERY Early Surgery In Complicated Infective Endocarditis: To Accelerate Is To Be Appropriate A.M. Pardede1, T.M. Haykal1 RS Jantung Jakarta1 A.M. Pardede1, T.M. Haykal1 RS Jantung Jakarta1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 When Learning ECG Turns Into A Nightmare: A Rare Case Report Study of PVCs J. Budiono1, P.P.D. Yamin2, Haikal2, V. Pratama2, M.I.A. Nugraha3 Faculty of Medicine and Health Science, Atma Jaya Catholic University, Jakarta, Indonesia.1 Department of Cardiology, Gatot Soebroto Central Army Hospital, Jakarta, Indonesia.2 Faculty of Medicine, Universitas Pembangunan Nasional Veteran Jakarta, Indonesia.3 Background: Premature ventricular contractions (PVCs) are triggered from ventricular myocardium in variety of circumstances and have been identified in broad-spectrum populations in which the vast majority of these patients may experience no symptoms. Frequent PVCs are associated with reversible cardiomyopathy and sudden cardiac death. However, research regarding post covid-19 infection and PVCs as a complication is still limited. Background: Premature ventricular contractions (PVCs) are triggered from ventricular myocardium in variety of circumstances and have been identified in broad-spectrum populations in which the vast majority of these patients may experience no symptoms. Frequent PVCs are associated with reversible cardiomyopathy and sudden cardiac death. However, research regarding post covid-19 infection and PVCs as a complication is still limited. Case illustration: A 22-year-old man, a medical student, accidentally found PVCs during his ECG course in cardiology clinical rotation. He was asymptomatic, had no personal history of syncope, did not take any medication, and had no family history of sudden cardiac death. Results of his physical examination were normal, and this was the first time he underwent ECG in which PVCs have been recognized (figure 1). History of covid- 19 infection with mild symptoms, 3 months before, was considered may be related to this condition. The patient was suggested to undergo some laboratory tests, transthoracic echocardiogram, 24-h ECG Holter, and cardiac MRI. All laboratory tests were within normal limits, but his echocardiogram and MRI showed reduced left ventricle contractility function. His Holter revealed many couplets, triplets PVCs, and some run ventricular tachycardias. Initial empirically guided medication with beta-blocker and other foundational therapy for LV dysfunction have been given. Radiofrequency ablation of the PVCs could be an excellent therapeutic option if there is no improvement in the patient’s follow-up ECG with medication only. Conclusion: This unique case report should prompt the performance of multicenter studies exploring the prevalence of PVCs in subjects with apparently asymptomatic and normal hearts, especially those with history of covid-19 infection. Keywords: Premature Ventricular Contractions, Post covid-19 infection, Electrocardiogram Figure 1. Patient’s ECG shows couplets PVCs and trigeminy PVCs Figure 1. Patient’s ECG shows couplets PVCs and trigeminy PVCs Keywords: INFECTIVE ENDOCARDITIS, EARLY SURGERY Pict 1. Echocardigraphy After Surgery Pict 1. Echocardigraphy After Surgery Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Recurrent Purulent Pericarditis with Impending Cardiac Tamponade during Pandemic Situation. A role of Intrapericardial Fibrinolysis Therapy: A Case Report g p Keywords: Purulent pericarditis, intrapericardial fibrinolysis, cardiac tamponade Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Dilated Cardiomyopathy (DCM), Cerebrovascular Accident (CVA), Heart Failure, decrease cerebral infarction Acute Recurrent Purulent Pericarditis with Impending Cardiac Tamponade during Pandemic Situation. A role of Intrapericardial Fibrinolysis Therapy: A Case Report B.B. Dharmadjati1, M.D. Pratama1, M.R. Amadis1, A.F.D. Raharja1, M. Ramadhan1, D.R. Firmanda1, M.S. Tiyantara1 Department of Cardiology and Vascular Medicine, Airlangga University, Surabaya, Indonesia.1 Tiyantara1 Tiyantara Department of Cardiology and Vascular Medicine, Airlangga University, Surabaya, Indonesia.1 Background: Purulent pericarditis(PP) is a localized pericardial space infection characterized by gross pus in the pericardium or microscopic purulence. PP is relatively uncommon but a potentially life-threatening disease. Fibrin formation in the first week is the key to the progression of PP to constrictive pericarditis and also cardiac tamponade with increased mortality and morbidity. Intrapericardial fibrinolysis is a promising therapy that can reduce the mortality risk and complications of PP. Case Illustration: A 36-year-old man was referred for the second time to our emergency department. The patient complained of dyspnea and chest palpitations for a week which had worsened in the last two days. Physical examination reveals hypotension, tachycardia, pulsus paradoxus, and elevated jugular venous pressure (JVP) as signs of cardiac tamponade. The electrocardiogram shows low voltage in the limb leads and alternating electricity. Chest X-ray shows Erlenmeyer's sign. Echocardiography showed massive pericardial effusion on the right and left laterals, apex, and base with right atrial collapse. Covid-19 rapid antigen swab was positive, but with negative PCR swab. Urgent pericardiocentesis was performed. Staphylococcus aureus was found as the causative etiology. NSAID, colchicine, and intravenous antibiotic was given, and we administered 500.000 units of Streptokinase, diluted in 20-50 mL normal saline through the catheter into the pericardial cavity via pigtail. This regimen was performed 3 times at 8 hours intervals with good response and improved clinical status, the catheter was removed when when the drain was <25 to 50 ml/24 hours, and echocardiography showed minimal or no effusion. The patient was discharged after 14 days. g p diluted in 20-50 mL normal saline through the catheter into the pericardial cavity via pigtail. This regimen was performed 3 times at 8 hours intervals with good response and improved clinical status, the catheter was removed when when the drain was <25 to 50 ml/24 hours, and echocardiography showed minimal or no effusion. The patient was discharged after 14 days. Conclusion: PP is a potentially life-threatening disease with high mortality and may cause serious complications. In this case, we performed comprehensive management of PP with the addition intrapericardial fibrinolysis that leads to good outcomes for the patient. Background: g Ventricular Septal Defect (VSD) is the most common of congenital heart malformation, accounting for 20-30% of all congenital heart defect. This non cyanotic defect is marked by a ventricular shunt and affects blood flow to the lung. Almost all types of VSD can be corrected. Surgery was once the first choice for VSD repair, but nowadays, catheterization closure is preferred because it is less invasive. In recent years, transcatheter device closure for ventricular VSD guided by transesophageal echocardiography (TEE) and fluoroscopy without contrast has been used in our center. These report aims to describe the effectiveness of zero contrast transcatheter VSD closure using double disk device in network hospital. Case Illustration: There were 7 cases of perimembranous VSD performed zero contrast transcatheter closure using double disk device in Dr Saiful Anwar General Hospital Malang during October 2022 till March 2023. Zero contrast transcatheter procedure was performed with Lifetech Konar-MF Occluder using double disk device with retrograde transarterial approach under TEE and fluoroscopy guided. The patient’s mean age was 19.7±9.7 years old (3-34 years old), and mean body weight was 49.5±18.4 kg (13-70kg). The median VSD size from TEE was 6.1 mm (4-9 mm), and the mean size of the occlusion device was 8.1±2.3 mm (5-12 mm). The mean fluoroscopy times was 28.8±18.4 minutes (2-53 minutes). Using double disk device in variant size range 7/5 in 1 patient, 9/7 in 2 patients, 10/8 in 2 patients, 12/10 in 1 patient and 14/12 in 1 patient. Device displacement, pericardial effusions, arrhytmia, residual shunt or new valve regurgitation were not observed during and after procedure. The patients showed significant clinical improvement after the procedure. Zero Contrast Transcatheter VSD Closure using Double Disk Device: A Case Series from East Java Network Hospital Zero Contrast Transcatheter VSD Closure using Double Disk Device: A Case Series from East Jav Network Hospital I.M. Suprayoga1, H. Martini1, V.P. Yogibuana1 I.M. Suprayoga1, H. Martini1, V.P. Yogibuana1 Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya1 I.M. Suprayoga1, H. Martini1, V.P. Yogibuana1 Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya1 Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya1 Dilated Cardiomyopathy with decreased eGFR as cause of cerebrovascular accident M.R.A. Putra1, D.N. Oetomo2, M. Maulydia3 Faculty of Medicine, Airlangga University, Surabaya, East Java, Indonesia 1 Dr. Soetomo General Academic Hospital, Surabaya, East Java, Indonesia 2, 3 Background : Dilated Cardiomyopathy (DCM) is characterized with left ventricle (LV) enlarged and poorly contractile with ischemic cardiomyopathy being the most common type. Usually it will lead to a heart failure or abnormal heart ryhtm, but sometimes it could lead to cerebrovascular accident (CVA) such as cerebral infarction and cerebral haemorhage. Decreased eGFR in patient with DCM, increase the risk of CVA to happen. g p , pp Case Illustration : A 40 years old woman was referred to our emergency room with sudden loss of conciusness after 2 weeks of hospitalization from another hospital. The patient presented with foot and hand edema, shortness of breath, desaturation, and fatigue, with history of type II diabetes and controlled hypertension. ECG shows no sign of LVH but found anterolateral ischemia (Inverted T in V1-V4), and CXR shows sign of cardiomegaly with CTR around 60% with pneumonia aspiration. POCUS echo were done to assess the heart. The result showed declined of LV sistolic function with ejection fraction 36%, LV remodelling (LVDMi 79.7, RWT 0.500), and minimum mitral regurgitation. CT scan showed cerebral infarction and minimum haemorrhage. Laboratory findings showed elevated serum creatinine and elevated ureum with eGFR 9ml/min/1.73m2. We also found elevated infection marker, hypoalbuminemia and hyperglicemia. Patient with DCM were susceptible to thrombosis due to blood clots (caused by systolic dysfunction and low eGFR) was associated with intracranial arterial stenosis that lead to CVA. Management and treatment of DCM are in concordance with the guideline of heart failure with treating any identifiable and reversible underlying causes. ACE-I and diuretic were already given to the patient, but due to worsening desaturation, the patient went into the intensive care unit. After couple days of treatment, the patient passed away because septic shock. Conclusion : DCM could lead to CVA besides heart failure or abnormal heart ryhtm, especially patient with decreased eGFR. Dilated Cardiomyopathy with decreased eGFR as cause of cerebrovascular accident Keywords: Dilated Cardiomyopathy (DCM), Cerebrovascular Accident (CVA), Heart Failure, decreased eGFR, cerebral infarction Keywords: Dilated Cardiomyopathy (DCM), Cerebrovascular Accident (CVA), Heart Failure, decreased eGFR, cerebral infarction Keywords: Dilated Cardiomyopathy (DCM), Cerebrovascular Accident (CVA), Heart Failure, decreased eGFR, cerebral infarction Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Zero Contrast Transcatheter VSD Closure using Double Disk Device: A Case Series from East Java Network Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: This serial cases has shown that zero contrast transcatheter VSD closure using double disk device is feasible, safe and effective to be done in network hospital with good result. Keywords: VSD, transcatheter closure, zero contrast, outcome Keywords: VSD, transcatheter closure, zero contrast, outcome Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background : Arrhythmogenic cardiomyopathy (ACM) affects the right ventricle (RV), the left ventricle (LV), or both, whose hallmark pathologic feature is the fibrofatty myocardial replacement that underlies the impairment of systolic ventricular function and predisposes to potentially lethal ventricular arrhythmias, regardless of the severity of pump failure. Recently, the interpretation of structural, functional, and tissue characterization by contrast- enhanced cardiac Magnetic Resonance Imaging (MRI) has become a crucial imaging tool for the diagnosis of ACM as a result of technological advancements and improved experience in this field. The objective of this case study is to demonstrate the role of cardiac MRI in the diagnosis phenotype of ACM C ill t ti ABSTRACT ABSTRACT A Case of Arrhythmogenic cardiomyopathy (ACM): the role of Cardiac MRI A.B. Maharjito Cardiology resident of The Department of Cardiology And Vascular Medicine Faculty of Medicine Universitas Indonesia Case illustration : A 66 year-old-woman experienced dyspnea on effort for 3 years. Despite normal coroangiography, the patient has a reduced left ventricular ejection fraction and multivocal PVC with non-sustained VT from holter examination. The patient underwent cardiac MRI that depict a reduced both RV and LV systolic function with dyskinesia in apical RV and hypokinesia basal-apicoseptal LV, no myocardial edema, microinﬂammation and fat inﬁltration at apex LV and RV. Padua criteria are criteria for diagnosis of the entire spectrum of phenotypic variants of ACM, which not only included RV morpho-functional abnormality but LV as well. Our present case here met the criteria. Our case also showed that there was a fat infiltration at apex RV and LV, although this finding was not the part of criteria for ACM. Co c us o Cardiac MRI is a crucial imaging tool for the diagnosis of ACM, especially in terms of ACM phenotype. Conclusion Cardiac MRI is a crucial imaging tool for the diagnosis of ACM, especially in terms of ACM phenotype. Keywords: arrhythmogenic cardiomyopathy Padua criteria, cardiac MRI Keywords: arrhythmogenic cardiomyopathy Padua criteria, cardiac MRI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 A Case report of ST Elevation Myocardial Infarction (STEMI) in Young Woman: Shifting the Paradigm A. Agustyana1, B.I. Maula2, Z. Muin2, M. Furqon1 Abdul Wahab Sjahranie General Hospital1 Abdul Rivai General Hospital2 BACKGROUND Total atrioventricular block (TAVB) is frequently become the conductive disturbance complication of acute reinfarction. Inferior MI has low long-term mortality and greater reversibility than anterior MI which has higher in-hospital and long-term mortality. Transient Symptomatic Total Atrioventricular Block Complicating Acute Anterior Reinfarction R. Hendiperdana1, I. Firdaus1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita1 CASE ILLUSTRATION 44 old years man underwent PPCI stenting at proximal LAD of his acute anterior MI 2 days before presentation at the previous hospital. Patient had acute stent thrombosis then underwent urgent PCI and developed TAVB with idioventricular escape. In emergency department Cardiovascular Centre Harapan Kita, patient develop loss of consciousness with blood pressure 57/30 mmHg, heart rate 20 -30 bpm and TAVB rhythm. Laboratory showed increased serum lactate level 5.2. Patient was diagnosed with Total AV block caused by anterior MI. Patient was planned for emergency temporary pacemaker (TPM) implantation. After 24 hours close monitoring, the patient intrinsic rhythm resolved with spontaneous recovery. Patient was hemodynamically stable until discharge. Stent thrombosis of proximal stent of LAD will cause TAVB because of the source of the distal portion of the AV node is originating from septal branch of LAD. Cardiogenic shock could be a manifestation of TAVB with idioventricular escape. It is caused by extensive necrosis with higher in-hospital and long-term mortality, often culminated in permanent pacemaker. However, spontaneous recovery of TAVB into sinus rhythm take place. This could be caused by transient reversible ischemia of infra nodal region of AV node which supplied by septal perforator branch in anterior infarction. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Case report of ST Elevation Myocardial Infarction (STEMI) in Young Woman: Shifting the Paradigm A. Agustyana1, B.I. Maula2, Z. Muin2, M. Furqon1 Abdul Wahab Sjahranie General Hospital1 Abdul Rivai General Hospital2 Background: Although STEMI is more commonly associated with older adults and men, it can occur in younger women as well. Young patients presenting with STEMI are an important and challenging subset, especially in women. Cardiovascular disease in women tend to be missed diagnosed and untreated, causing greater mortality in addition to more comorbidities. Case Illustration: A 29-years old woman with obesity (body mass index 48.4) presented in the emergency room with recurrent isolated epigastric pain for the past 3 days accompanied with dyspnea and atypical chest pain. No prior history of any life-threatening event such as cardiovascular event or syncope. An Electrocardiogram (ECG) showed an extensive anterior STEMI (elevation in V1-6, I and aVL leads). Laboratory examination showed an increase in troponins T, glucose level and LDL cholesterols in blood. There was a family history of type 2 diabetes mellitus and hypercholesterolemia. Echocardiogram demonstrated anteroseptal and anterolateral walls hypokinetic with decreased ejection fractions. After given initial treatment, patient was transferred to PCI-capable hospital to performed cardiac catheterization and total occlusion of proximal LAD was revealed. It was successfully treated with a balloon stent. y Conclusion: The suspicion for myocardial infarct (MI) in younger age should be increased especially for women with risk factors and careful examination should be performed to prevent misdiagnosis. Diabetes mellitus and obesity are common risk factors in women with young age. The necessity to improve of focused management and primary prevention in younger patients are much needed, especially women, was important due to worse outcome. Keywords: STEMI, Young Age, Women, Total Occlussion ECG examination during chest pain demonstrating elevation in extensive anterior/anterolateral ECG examination during chest pain demonstrating elevation in extensive anterior/anterolateral CONCLUSION This case reporting a TAVB case during the course of acute anterior reinfarction and developed spontaneous resolution to sinus rhythm. Mechanisms of spontaneous resolution of TAVB in the setting of acute MI is associated transient ischemia after occlusion of proximal LAD. Keywords: total AV block, Bradycardia, Stent Thrombosis ECG Presentation in Emergency Ward National Cardiovascular Centre Harapan Kita. The ECG showed Total AV Block with Wide Complex Escape Rhythm Keywords: total AV block, Bradycardia, Stent Thrombosis ECG Presentation in Emergency Ward National Cardiovascular Centre Harapan Kita. The ECG showed Total AV Block with Wide Complex Escape Rhythm Indonesian Journal of Cardiology Indonesian Journal of Cardiology timal Timing of Renal Replacement Therapy for favorable Outcome in Patients with Acute Kidney Injury (AKI) Following a Coronary Artery Bypass (CABG) Surgery: The Sooner, The Better? B. Dewanggi1, N.R. Muttaqiin1, N.A. Tafriend2, D. Bima3 1General Practitioner of Intensive Care Unit, Gatot Subroto Presidential Hospital, Jakarta, Indonesia 2Cardiologist, Gatot Subroto Presidential Hospital, Jakarta, Indonesia 3Anesthesiologist-Consultant of Intensive Care Gatot Subroto Presidential Hospital Jakarta Indonesia g p Anesthesiologist-Consultant of Intensive Care, Gatot Subroto Presidential Hospital, Jakarta, Indonesia Background: Acute kidney injury (AKI), defined as a rapid deterioration of kidney function, is a major complication associated with cardiac surgery. Its incidence ranges from 8.9 to 42.5% and was related to poorer outcomes, ICU length of stay, and mortality, especially in severe AKI when renal replacement therapy was needed (RRT-AKI). Some scores such as Cleveland Score and AKICS score were developed to predict CSA- AKI (post-cardiac surgery AKI) and RRT-AKI so that early recognition and treatment can be done. Here we present a case of a post-CABG CSA-AKI patient which getting better after early initiation of continuous renal replacement therapy (CRRT). p py Case illustration: a 44-year-old man with Coronary Arterial Disease following three vessels (CAD 3 VD), a history of CHF, COPD, LVEF 29%, and Diabetes Mellitus, underwent CABG with a total CPB time of 2 hours and 50 minutes. According to the AKICS Score (score=14~21.8% risk of AKI) and Cleveland Score (score=6~7.8 to 9.5% risk of dialysis), he was at risk of post-cardiac surgery AKI and RRT. So that, when twelve hours later, the creatinine level was increased 1.7-fold from baseline and he was oliguria which is unresponsive to the furosemide infusion, we decide to perform CRRT. The patient’s condition improved and the diuresis was increased to 1.6 ml/kg/hour. On the 3rd-day post operations, he was stable enough to discharge from the intensive care unit. Conclusion: Using scores to predict the patients who are at risk of CSA-AKI and RRT-AKI may improve prognosis and also help clinicians to decide the appropriate timing for initiating therapy to achieve a favourable outcome. eywords: acute kidney injury, Coronary artery bypass graft, continues renal replacement therapy Keywords: acute kidney injury, Coronary artery bypass graft, continues renal replacement therapy Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A male, 56 yo came to Rumah Sakit Saiful Anwar Malang with stable angina pectoris (Class III symptoms with medical therapy) planned for elective cardiac catheterization with routine medical treatment. During PCI, he complain the chest pain and from cineangiography evaluation showed no flow at the diagonal branch because of the acute thrombosis. Then got thrombosuction and got white thrombus. After PCI procedure he got fibrinolytic with streptokinase 1.5 million unit for 60 minutes. He transfered to CVCU to observation and discharge after 5 days. Discussion Stent thrombosis based on ARC criteria include based on gradation (definite, probable, and possible stent thrombosis), based on onset (early, late, and very late). Anticoagulant therapy during PCI procedure has been focus of several clinical trial related to occur complication during or after PCI, for example stent thrombosis. Based on previous study showed a similar degree of evidence of thrombolytic treatment compared with percutaneous angioplasty in the management of stent thrombosis from symptom onset to reperfusion was less than 2 hours A MALE 56 YEAR OLD WITH ACUTE STENT THROMBOSIS WHEN PERCUTANEOUS CORONARY INTERVENTION, WHAT ARE THE PREDICTORS? M.S. Rohman1, H.I. Millisani1 RSUD dr. Saiful Anwar1 Background Stent thrombosis is the complete occlusion of a coronary arteries of previously implanted stent which unusual complication that occur in percutaneous coronary intervention (PCI), development of myocardial ischemia and poor prognosis for the patient. The etiology of acute stent thrombosis were multifactorials and early detection can reduce mortality rate. Pathophysiology of Mitral Annular Disjunction: A Case Report I.W.P. Wisuda1, N.M.A.W. Sari2 1Cardiology Specialist Study Program, Faculty of Medicine Udayana University/Prof. Ngoerah Hospital, Denpasar, Indonesia 2Department of Cardiology and Vascular Medicine, Prof. Ngoerah Hospital, Denpasar, Indonesia I.W.P. Wisuda1, N.M.A.W. Sari2 1Cardiology Specialist Study Program, Faculty of Medicine Udayana University/Prof. Ngoerah Hospital, Denpasar, Indonesia 2Department of Cardiology and Vascular Medicine, Prof. Ngoerah Hospital, Denpasar, Indonesia I.W.P. Wisuda1, N.M.A.W. Sari2 Background: Mitral Annular Disjunction (MAD) is a structural abnormality defined as the separation of the junction of the mitral valve annulus-wall of the left atrium with the basal aspect of the posterolateral left ventricle. The incidence of MAD is estimated to be 51% in patients with myxomatous mitral valve, 32.6% in the context of MVP, and 25.9% in severe mitral valve regurgitation. Case Illustration: A 61-year-old woman came to the cardiology clinic at Prof. Ngoerah Hospital with a referral from oncology clinic with a diagnosis of right and left breast cancer post right Modified Radical Mastectomy pro chemotherapy AC-T (Adriamycin Cyclophosphamide-Taxol). Physical examination, laboratory, and chest X-ray were within normal limits. Echocardiographic examination found dilated left atrium, LV eccentric hypertrophy, EF Biplane 63.4%, LV diastolic function intermediate, TAPSE 24 mm, global normokinetic, mild MR with mitral annular displacement in PLAX view, eRAP 8 mmHg. The patient was then diagnosed with mild MR with mitral annular displacement with a Cardiotoxicity Risk Score of 5 (high) with therapy ramipril 1x2.5 mg, carvedilol 2x3.125 mg and simvastatin 1x20 mg with echocardiographic evaluation in 3-6 months. Mitral annular contractility contributes significantly to mitral valve function. Shortening of the annular diameter during systole facilitates coaptation of the mitral valve leaflets. The presence of MAD impairs valve coaptation. MAD has implications for ventricular arrhythmias and sudden cardiac death. Mitral valve repair in severe mitral regurgitation through surgery in the form of sewing a ring to attach the leaflet to the ventricular myocardium. In this case, mild MR was obtained, so medical management was carried out. Conclusion: The presence of MAD impairs valve coaptation, resulting in MR. Severe MR should be corrected by surgery, however milder cases can be managed conservatively. Keywords: mitral annular disjunction, mitral valve prolapse, MAD management Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Percutaneus Transluminal Angioplasty Utilizing Carbon Dioxide Contrast in Chronic Limb Threatening Ischemia patient with Renal Failure S.M.A. Sabara1, S. Adiarto1 S.M.A. Sabara1, S. Adiarto1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Indonesia1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Percutaneus Transluminal Angioplasty Utilizing Carbon Dioxide Contrast in Chronic Limb Threatening Ischemia patient with Renal Failure Conclusion Stent thrombosis is a feared complication during and after primary PCI because it is associated with high mortality. The mechanisms by which ST arises are complex and multifactorial and must be early detection. Keywords: Acute stent thrombosis, Percutaneous coronary intervention Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Percutaneus Transluminal Angioplasty Utilizing Carbon Dioxide Contrast in Chronic Limb Threatening Ischemia patient with Renal Failure Case Illustration A 20-year-old-man complained of shortness of breath, abdominal distention, and decrease urine output for 5 days before admission. His previous medical history was severe mitral stenosis (MVA by planimetri 0.6cm, Wilkins score 9) with preserved LV ejection fraction (63%) with reduced RV function (TAPSE 11 mm). Right sided pleural effusion, pulmonary edema, and cardiomegaly were noted by chest x-ray. Abdominal ultrasound showed normal sized kidney, massive ascites, and hepatomegaly accompanied with distention of hepatic vein. Reduced kidney function was noted (Ur 96 Cr 1,59 UO: 600-700 cc/day). y y We managed him with optimal continuous infusion dose of furosemide and dobutamine in combination with spironolactone within first week yet failed to improve symptoms, congestion, and urine output (650-750 cc/day) significantly. Further, pleurocentesis was performed, resulting 600cc plural fluid drained. Nevertheless remained unchanged the symptoms. Eventually, we decided to perform abdominocentesis with result of 2500 cc of fluid drained which decreased abdominal circumference from 121 to 96 cm. Two days after procedure, there were improvement of symptoms, congestion, and renal function (Ur 32 Cr 0,9) accompanied with increase urine output (1500-1700cc/day). Background Background Renal dysfunction in heart failure has been linked to both reduced perfusion and increased congestion (central venous pressure). Renal tamponade hypothesis said that compression of renal structures caused by the limited space for expansion (increase interstitial space) contributes to worsening renal function in heart failure. C i Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Indonesia1 Herry1 Department of Cardiology and Vascular Medicine, Kariadi General Hospital, Semarang, Indonesia1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 mprovement of Renal Function after Abdominal Decompression in Heart Failure Caused by Mitral Stenosis : The Renal Tamponade Hypothesis D.Y. Kurniawan1, I.P. Farissa1, F. Tandri1, M.F. Ahnaf1, Y. Herry1 Department of Cardiology and Vascular Medicine, Kariadi General Hospital, Semarang, Indonesia1 D.Y. Kurniawan1, I.P. Farissa1, F. Tandri1, M.F. Ahnaf1, Y. Herry1 Department of Cardiology and Vascular Medicine, Kariadi General Hospital, Semarang, Indonesia1 D.Y. Kurniawan1, I.P. Farissa1, F. Tandri1, M.F. Ahnaf1, Y. Herry1 Department of Cardiology and Vascular Medicine, Kariadi General Hospital, Semarang, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Indonesia1 Background: At the present times, more than 202 million patients suffered from peripheral arterial disease (PAD) worldwide. Chronic limb-threatening ischemia (CLTI) often need lower extremity amputation, and the aftermath could be worse. For the patient with CLTI and chronic renal disease (CKD) who need endovascular therapy, iodinated contrast could enhance the risk of contrast-induced nephropathy (CIN). In high-risk patients who were allergic to iodinated contrast material and for those with renal insufficiency Hawkins in the 1970s pioneered the intra-arterial application of carbon dioxide (CO2) gas angiography to reduce the volume use of iodinated contrast. Objective: This report is aimed to emphasize the usage of CO2 contrast agent in PTA for the patients with renal dysfunction in order to prevent further loss of renal function Case Illustration: An 80-years old man referred to National Cardiovascular Center Harapan Kita with the chief complaint of independent rest pain and non healing wound in his left forefinger, in accordance with the criteria CLTI. The duplex ultrasound showed total occlusion at left anterior tibialis artery. The patient then undergone percutaneous transluminal angioplasty (PTA) procedure using Carbon Dioxide (CO2) contrast agent and using Plain Old Balloon Angioplasty (POBA) technique for revascularization. The flow to the distal of left anterior tibialis artery returned using only 30 ml of Iodinated contrast. The follow up of this patient showed no increase of serum creatinine level and eGFR Conclusion: Endovascular therapy in patient with CLTI with high risk of operation is feasible to be performed in patient with CKD utilizing the carbon dioxide contrast agent in order to minimize the usage of iodinated contrast avoiding further loss of renal function. In this case report, the procedure has performed successfully without increase in serum creatinine and decrease of GFR. Keywords: carbon dioxide contrast, limb ischemia, percutaneous transluminal angioplasty Keywords: carbon dioxide contrast, limb ischemia, percutaneous transluminal angioplasty Percutaneous Transluminal Angiography Procedures on this patient shows occlusion in anterior tibialis artery with Carbon Dioxide contrast (left) and Iodized contrast (right) Percutaneous Transluminal Angiography Procedures on this patient shows occlusion in anterior tibialis artery with Carbon Dioxide contrast (left) and Iodized contrast (right) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Improvement of Renal Function after Abdominal Decompression in Heart Failure Caused by Mitral Stenosis : The Renal Tamponade Hypothesis D.Y. Kurniawan1, I.P. Farissa1, F. Tandri1, M.F. Ahnaf1, Y. Conclusion In this case, the Renal Tamponade Hypothesis proposes that abdominal decompression can lead to improved renal function in patients with heart failure. This finding suggests that further research is warranted to investigate the potential of this approach as a novel therapy for managing renal dysfunction in heart failure patients. Keywords: Mitral Stenosis, Heart Failure, Ascites, Abdominocentesis, Renal Tamponade words: Mitral Stenosis, Heart Failure, Ascites, Abdominocentesis, Renal Tamponade BACKGROUND Diabetes increases the need for pacemaker and other complications including hyperkalemia. In severe conditions, hyperkalemia induced cardiac conduction disturbances and arrhythmias, but the clinical symptoms that appear do not only depend on blood potassium levels but also due to the use of drugs and co-morbidities. CASE ILLUSTRATION A 69-year-old woman came to our hospital with signs of acute heart failure, she has history of type II DM and hypertension, no history of previous heart disease. The patient regularly consumes bisoprolol and other drugs. At the presentation the patient's vital signs was relatively stable except for decreased pulse up to 30 beats per minute. On ECG examination we found atrial to ventricular conduction block and disappearance of the P wave. Due to limited facilities, initial management was carried out by giving atropine but administration of a maximum 3 mg showed a poor response with a progression to cardiogenic shock. We decide to use dopamine then. Severe hyperkalemia is treated with triple drug therapy; calcium gluconate together with a combination of 40% dextrose infusion and insulin Aspart, as well as salbutamol’s nebulization, all of which are carried out under the supervision of the emergency room. Failure of an adequate response and worsening of shock led to sodium bicarbonate infusion and initiation of norepinephrine. The patient's condition improved 12 hours later with the return of hemodynamic stability. The patient was then referred to a higher center. M.A. Wakasaputra1, K. Saleh1, A.F. Ghaznawie1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Hasanuddin1 M.A. Wakasaputra1, K. Saleh1, A.F. Ghaznawie1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Hasanuddin1 Partial Resolution of LA Thrombus in Mitral Stenosis after Optimal VKA Treatment Dr. M. Archie Wakasaputra, Dr. dr. Khalid Saleh, Sp.PD-KKV, dr. Aussie F. Ghaznawie, Sp.JP(K) Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Hasanuddin Abstrak Background : Left Atrial (LA) thrombus until now still remains as the main reason of morbidity in mitral stenosis due to rheumatic heart disease. This condition caused by slow and static blood flow in left atrial due to stenosis in mitral valve and corresponding arrhytmia like atrial fibrillation can aggravate static blood flow. Some research claims that prevalence of LA thrombus is 5 – 27% in atrial fibrillation patient due to mitral stenosis Case Illustration : We reported a 37 years old man with mitral stenosis, LA thrombus and corresponding atrial fibrillation. Transthoracal echocardiography showed 4,6 x 2,3 cm thrombus in left atrial. Thus, we preceded to treat with warfarin 2 mg/day. After 3 months of anticoagulation, we re-examine transthoracal echocardiography, and we found the size of preexisting thrombus decreased to 2,4 x 2,3 cm. p g Conclusion : LA thrombus is a long term complication in mitral stenosis plus atrial fibrillation, this situation is caused by Virchow’s triad : abnormal blood flow, abnormal vessel structure and abnormal blood constituents. To treat this condition, we need a medicine that can work as thrombolytic. In this report, we found that the use of anticoagulant like Vitamin K antagonist (warfarin), has thrombolytic properties in addition to its anticoagulant properties through thrombin-activated fibrinolysis inhibitor (TAFI) mechanism. In one study, warfarin has an additional nature as thrombolytic by reducing plasma TAFI which then cause thrombolytic action in preexisting thrombus. Keywords : Left Atrial Thrombus, Mitral Stenosis, Atrial Fibrillation, Vitamin K Antagonist, Virchow’s triad, Warfarin, Fibrinolysis, Thrombin-Activated Fibrinolysis Inhibitor Keywords: left atrial thrombus, mitral stenosis, atrial fibrilation, vitamin K antagonist, warfarin Keywords: left atrial thrombus, mitral stenosis, atrial fibrilation, vitamin K antagonist, warfarin Indonesian Journal of Cardiology EMERGENCY TREATMENT OF THE RARE HYPERKALEMIA INDUCED SYMPTOMATIC BRADICARDIA IN A DIABETIC TYPE II PATIENT, WHATS TO USE? S. Ashari1, P.S. Bustanul1 RSUD H. Abdul Manap1 Keywords: Bradycardia, Hyperkalemia, DM. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 CONCLUSION In our patient, administration of dopamine and triple therapy alone was unable to control hemodynamics, so sodium bicarbonate and norepinephrine infusion were needed to restore the patient's general condition until finally referred to a higher center. KEYWORD : Bradycardia, Hyperkalemia, DM. Keywords: Bradycardia, Hyperkalemia, DM. Keywords: Bradycardia, Hyperkalemia, DM. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Inferior STEMI Induces Cardiogenic Shock, Sinus Bradycarida with First Degree AV-Block in Type 2 Diabetes Patient: How We Optimize the Treatment in Life-Threatening and Poor Outcome Patients in Type C Rural Hospital AORTIC DISSECTION IN MARFAN SYNDROME: A DIAGNOSTIC APPROACH R.G. Adam1, A.H. Alkatiri1, A.F. Ghaznawie1 Hasanuddin University1 Background: The prevalence of Marfan syndrome(MFS)cases is estimated to be 1 per 5,000 population and 26% of patients have no family history of similar disease.Aortic dissection (AoD) is the main cause of death in MFS patients, with a mortality rate of approximately 1-3%.Medical treatment might not cure nor restore anatomical and physiological functions,however it could reduce the progression and severity of symptoms.This case report discusses MFS and AoD diagnosis and their treatment. Case illustration: A 33-years old man came with a main complaint of intermittent shortness of breath in the last month and worsens for the last week. There was hypertension history and cardiovascular disease history in the family, where the patient's uncle died at the age of 40-years old with a tall stature.The patient also had a history of visual acuity problem.Physical examination using the Ghent criteria revealed a score of 10, and investigations showed widening of the aortic root on echocardiography.A thoracal computed tomographycan revealed AoD,hence the patient was diagnosed with a working diagnosis of congestive heart failure New York Heart Association (NYHA)class III,MFS, and AoD Stanford A.The patient did not approve for surgical treatment, therefore conservative treatment was opted with ACE-inhibitor and beta-blocker drugs as well as education for physical activity restrictions. Conclusion: Young patients <40-years old with AoD should be suspected of MFS.Aortic root disease occurs in approximately 50-60% of adult Marfan syndrome patients and could easily be detected on echocardiography.Aortic root disease causes progressive aortic root aneurysms and is a major cause of morbidity and mortality in MFS.Diagnosis and treatment of MFS requires multidisciplinary management.A thorough physical examination,eye exam, genetic evaluation, radiography,and echocardiography are routinely recommended for clinical diagnosis.The Ghent criteria is useful for MFS diagnosis.Treatment must be tailored to clinical manifestations, because each patient has varying degrees of organ involvement. Keywords: Marfan syndrome, aortic dissection, echocardiography, aortic root A Diagnostic Challenge in Peripartum Cardiomyopathy with Chronic Kidney Disease: A Case Report B.B. Pratiwi1, A.A. Asmiragani2 General Hospital dr. Soediran Mangun Soemarso1 General Hospital Universitas Brawijaya Malang2 A Diagnostic Challenge in Peripartum Cardiomyopathy with Chronic Kidney Disease: A Case Report B.B. Pratiwi1, A.A. Asmiragani2 General Hospital dr. Soediran Mangun Soemarso1 General Hospital Universitas Brawijaya Malang2 Diagnostic Challenge in Peripartum Cardiomyopathy with Chronic Kidney Disease: A Case Report A Diagnostic Challenge in Peripartum Cardiomyopathy with Chronic Kidney Disease: A Case Report B.B. Pratiwi1, A.A. Inferior STEMI Induces Cardiogenic Shock, Sinus Bradycarida with First Degree AV-Block in Type 2 Diabetes Patient: How We Optimize the Treatment in Life-Threatening and Poor Outcome Patients in Type C Rural Hospital Sinus Bradycardia with First Degree AV-Block and ST-segment elevation in leads II, aVF Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Inferior STEMI Induces Cardiogenic Shock, Sinus Bradycarida with First Degree AV-Block in Type 2 Diabetes Patient: How We Optimize the Treatment in Life-Threatening and Poor Outcome Patients in Type C Rural Hospital Inferior STEMI Induces Cardiogenic Shock, Sinus Bradycarida with First Degree AV-Block in Type 2 Diabetes Patient: How We Optimize the Treatment in Life-Threatening and Poor Outcome Patients in Type C Rural Hospital W. Fathurrachman1, A.Sunata2 Sambas General Hospital1 Department of Internal Medicine of Sambas General Hospital2 Background: Inferior ST Segment Elevation Myocardial Infarction (STEMI) is a life-threatening condition. Almost 40% of the STEMI cases are inferior STEMI. Some complications that can occur because of inferior STEMI are supraventricular Arryhmia, conduction blocks, right ventricular infarction. This condition can impair blood to the left ventricle and make the left ventricular underfilled, thus leading to Cardiogenic Shock. g g Case illustration: A 69-year-old woman was admitted to the emergency department from other hospital with dull retreosternal chest pain for 25 hours. She was in acute distress secondary to the pain. She had a history of diabetes. This patient was diagnosed befored in other hospital with inferior STEMI, type 2 diabetes and congestive heart failure and was treated with fluids, diuretics, PPIs, rapid-acting insulin, antiplatelets, ARBs, beta blockers, nitrates, lactulose, and sucralfate. The next day, patient's blood pressure dropped to 60/palpation. She was then referred to our hospital. On arrival, the patient was in pain with a blood pressure 70/palpation, a pulse of 51 bpm, 24 breaths per minute, 100% oxygen saturation with 4 lpm oxygen therapy via a nasal cannula. General examination revealed cold extremities. The ECG showed sinus bradycardia with ST elevation in leads II, aVF, ST depression in leads I, aVL and First-Degree AV Block. The patient’s diagnosis was Sinus Bradycardia with Inferior STEMI Killip IV (Dry and Cold) with 25 hours onset, Cardiogenic Shock, First Degree AV-Block, and DM Type-2. In the ED, the patient received fluids, Oxygen, Dual antiplatelet therapy, rapid-acting insulin, unfractionated heparin, ionotropes. Even without revascularization, the patient was alive and the discharge after 6 days treated in the intensive care unit. Conclusion: Adequeate and apropriate management inferior STEMI may prevent life-threatening complications such as cardiogenic shock, conduction blocks especially in rural Hospital without availability of revascularization facility. Keywords: Inferior STEMI, Cardiogenic Shock, AV-Block, DM Type 2, Rural. Sinus Bradycardia with First Degree AV-Block and ST-segment elevation in leads II, aVF Keywords: Inferior STEMI, Cardiogenic Shock, AV-Block, DM Type 2, Rural. Conclusion The diagnosis of PPCM should be considered anytime a woman presents with even mild symptoms of heart failure in the last month of pregnancy or within the first 5 or 6 postpartum months. Early diagnosis is important and effective treatment is needed to decrease further complications such as chronic kidney disease. Case Illustration A 34-years old woman with a history of preeclampsia pregnancy a year ago presented in the emergency department with symptoms of orthopnea, paroxysmal nocturnal dyspnea, and leg swelling for two days before admission. In 2 months postpartum, she also had similar symptoms and was hospitalized in a private hospital. The cardiologist prescribed her Clonidin and Propanolol as anti-hipertensive medications but she only took them for two months. The physical examination showed blood pressure of 200/120 mmHg, tachycardia, no jugular venous distension, bibasilar crackles with decreased air entry at bases, pitting edema of legs, and SpO2 (oxygen saturation) of 89%. A chest X-ray study showed pulmonary oedema with bilateral pleural effusion and cardiomegaly. Electrocardiography showed sinus tachycardia and poor R-wave progression. An echocardiogram performed on hospital day revealed aleft ventricular systolic dysfunction (ejection fraction 43%) and mitral regurgitation. The initial laboratory findings showed the levels of urea 212 mg/dl; creatinine 18.5 mg/dl; and eGFR 2.0 mL/min/1.73m2. Cardiac marker couldn’t be done because of limited facilities. The patient was monitored in the intensive care unit (ICU). IV (intravenous) Furosemide and Nefrosteril were administered for the intial treatment. She required three hemodialysis sessions. The patient continued to improve clinically after therapy and still routinely undergoing routine hemodialysis two times one week. Background g Peripartum cardiomyopathy is a potentially life-threatening pregnancy-associated disease that develops in the last trimester of pregnancy to 5 months postpartum. It is characterized by left ventricular dysfunction and heart failure. Symptoms of heart failure resemble those of a healthy pregnancy, making it challenging to diagnose and often resulting in a delay in diagnosis. Here, we present an unsusual case of PPCM complicated by chronic kidney disease. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 AORTIC DISSECTION IN MARFAN SYNDROME: A DIAGNOSTIC APPROACH Asmiragani2 A Diagnostic Challenge in Peripartum Cardiomyopathy with Chronic Kidney Disease: A Case Report B.B. Pratiwi1, A.A. Asmiragani2 General Hospital dr. Soediran Mangun Soemarso1 General Hospital Universitas Brawijaya Malang2 General Hospital Universitas Brawijaya Malang2 Background Background Up to 10% of all DVTs are UEDVT. AV graft failure which manifests as AV graft thrombosis is a form of secondary UEDVT. It was considered as a major clinical event because it is strongly associated with increased mortality. Arteriovenous Graft Thrombosis: Upper Extremity DVT in AV Graft Failure Post Covid-19 at Prof Dr. IGNG Ngoerah Hospital I.W. Gunawan1, B.A.P. Sutanegara1, A.P. Suwirya1 Division of Vascular Division, Department of Cardiology and Vascular Medicine, Prof Dr. IGNG Ngoerah Hospital, Denpasar, Indonesia1 I.W. Gunawan1, B.A.P. Sutanegara1, A.P. Suwirya1 Division of Vascular Division, Department of Cardiology and Vascular Medicine, Prof Dr. IGNG Ngoerah Hospital, Denpasar, Indonesia1 g Conclusion Management of predisposing factors & risk of thrombosis, maintain adequate HD were crucial on the future to improve strategy for early detection of thrombotic recurrence and subsequent management AV graft thrombosis for optimal treatment outcomes. Keywords: Peripartum Cardiomyopathy; Chronic Kidney Disease; Hypertension Peripartum Cardiomyopathy; Chronic Kidney Disease; Hypertension Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Arteriovenous Graft Thrombosis: Upper Extremity DVT in AV Graft Failure Post Covid-19 at Prof Dr. IGNG Ngoerah Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 y Case Illustration A male, 52 years old, complained swelling of the right hand since two weeks. There was a history of it one year before, DM with insulin, hypertension, CKD on regular HD. Venography one year ago revealed of total occlusion of the right innominate vein, also AVF RC pseudoaneurysm by duplex US. Then performed ligation of right AV BC shunt and Pseudoaneurysm, installation of left AV BC shunt. Temporary venous tunnelling access also experiencing recurrent thrombosis. That also aggravated by the patient was confirmed Covid-19 before. Physical examination revealed right upper extremity was oedema. Increased D-dimer (2.56 Ug/mL), decreased LV systolic function (EF BP 43.4%), with RWMA. Right upper extremity Duplex US shows thrombus appears in the right brachial vein with diameter of 7.4 x 6.8 mm (short axis), 19.2 x 4.3 mm (long axis) (Figure 1. Despite Well's score support the diagnosis was established with UEDVT, patients are treated by anticoagulant warfarin at a dose according to INR, failure, CAD, antihypertensives therapy, insulin, HD regular 3 times a week with heparin. Conclusion Respiratory Failure and Cardiogenic Shock in Advanced Heart Failure Patient: A Case of Survivor without Mechanical Circulatory Support Device W. Tussakinah, C.I. Rahayu, B. Widyantoro EMC Sentul Hospital, Sentul, Bogor Respiratory Failure and Cardiogenic Shock in Advanced Heart Failure Patient: A Case of Survivor without Mechanical Circulatory Support Device W. Tussakinah, C.I. Rahayu, B. Widyantoro EMC Sentul Hospital, Sentul, Bogor Respiratory Failure and Cardiogenic Shock in Advanced Heart Failure Patient: A Case of Survivo without Mechanical Circulatory Support Device W. Tussakinah, C.I. Rahayu, B. Widyantoro EMC Sentul Hospital, Sentul, Bogor y CASE ILLUSTRATION A 62 – year –old male admitted to ED with excessive shortness of breath from 30 minutes before. He felt shortness of breath graduating for a week. They were chest discomfort, fever, and wet cough reported. The patient was a smoker for 3 packs/day and had been diagnosed with heart failure while hospitalized 5 months before. He took medicine not regularly; spironolactone 25mg OD and valsartan 80mg OD. He denied history of myocardial infarction, stroke, hypertension, dyslipidemia and diabetes. In ED, The patient fully alert and gasping, his blood pressure was 90/50, HR 130 bpm, T 37.4 °C, RR 35x/m, SpO2 76-82. Physical examination found JVP 5+3 cmH2O, rales 2/3rd up in bilateral lungs, cold extremities and clammy skin without edema. ECG showed sinus tachicardia with old LBBB. Laboratory revealed leukocytosis, increased NT-proBNP and respiratory acidocis. Echocardiography showed dilated heart with low LVEF 12-15% and TAPSE of 12 cm. The patient was diagnosed with cardiogenic shock following episode of acute pulmonary edema, triggered by pneumonia in advanced biventricular HF patient. The respiratory failure was treated with mechanical ventilation. The use of inotropes, vasopressor and ventilator setting were adjusted by close hemodynamic monitoring by cardiac intensivist with non-invasive echocardiography in ICCU. The patient was then treated with optimal medical therapy with antibiotics, lung protective ventilation strategies with low PEEP, restrictive fluid management, inotropes and vassopressor agents. Patient was successfully extubated in day-7, have a recovery phase with cardiac rehabilitation, and discharged with stable condition after 12-days hospitalization with optimal goal directed medical therapy for HF. BACKGROUND Acute pulmonary edema is a common case of acute respiratory distress in emergency department (ED). It has a high mortality rate up to 40%. Pulmonary edema can cause severe hypoxia and hypoxemia leading to respiratory failure and multi organ dysfunction. In pre-existing chronic heart failure patient, this condition further deteriorated myocardial function and triggered cardiogenic shock Keywords: AV Graft Thrombosis, UEDVT, AV Graft Failure, Post Covid-19. Keywords: AV Graft Thrombosis, UEDVT, AV Graft Failure, Post Covid-19. Right upper extremity Duplex US Right upper extremity Duplex US Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Respiratory Failure and Cardiogenic Shock in Advanced Heart Failure Patient: A Case of Survivor without Mechanical Circulatory Support Device W. Tussakinah, C.I. Rahayu, B. Widyantoro EMC Sentul Hospital, Sentul, Bogor Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Unexpected Cerebellar Infarction in A Patient with Tetralogy of Fallot: A Very Rare Complication O. Sofyan1, Y. Patimang1 RSUP dr Wahidin Sudirohusodo1 Unexpected Cerebellar Infarction in A Patient with Tetralogy of Fallot: A Very Rare Complication O. Sofyan1, Y. Patimang1 RSUP dr Wahidin Sudirohusodo1 O. Sofyan1, Y. Patimang1 RSUP dr Wahidin Sudirohusodo1 O. Sofyan1, Y. Patimang1 RSUP dr Wahidin Sudirohusodo1 Background: Along with the increasing number of survival rate of Tetralogy of Fallot patients, the higher the incidence of complications of the disease. In TOF patients who do not undergo correction, the degree of RVOT stenosis is considered a factor that contributes to the risk of complications, one of which is hyperviscosity syndrome. incidence of cerebellar infarction in patients with TOF has not been previously reported. Case illustration: 21 years old male came to Emergency Room with chief complaint headache, frequent vomiting and lightheadedness. When the patient was 2 y.o, patient started to felt shortness of breath during activities, squatting while dyspnea and looks cyanotic. Parents then brought the patient to Cardiology Pediatric and explained the patient had been diagnosed with ToF in whe he was 15 y.o, then lost to follow up. Physical examination showed underweight and oxygen saturation of 86% with room air at average extremities. the cardiac auscultation presence of right ventricular heaves, systolic murmur at ULSB with Grade III/VI. On the extremities showed cyanosis, clubbing fingers and syndactyly in left hand. The laboratory examination showed secondary policytemia, coagulation abnormality, and elevated Fe serum. The electrocardiography showed extreme right axis deviation, RAE, RVH, abrupt R wave. The chest x-ray showed suggestive TOF. The echocardiography showed Tetralogy of Fallot and EF 40.1%. Brain MSCT scan and MRI showed cerebellum infarction. During the hospitalization, we treated the dehydration as the patient’s hyperviscosity trigger with adequate rehydration and beta blocker as conservative treatment. From neurology department, the patient got symptomatic therapy supportive treatment with neuroprotector agent. py CONCLUSION Treatment of respiratory failure and cardiogenic shock in advanced biventricular HF patient is challenging. Rapid assessment and cardiac intensive care management guided by non-invasive hemodynamic monitoring is needed to stabilize the patient. Keywords: Respiratory Failure, Cardiogenic Shock, Heart Failure Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964- doi: 10.30701/ijc.1537 Unexpected Cerebellar Infarction in A Patient with Tetralogy of Fallot: A Very Rare Complication O. Sofyan1, Y. Patimang1 RSUP dr Wahidin Sudirohusodo1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Unexpected Cerebellar Infarction in A Patient with Tetralogy of Fallot: A Very Rare Complication O. Sofyan1, Y. Patimang1 RSUP dr Wahidin Sudirohusodo1 Conclusion Cardiac rehabilitation can be a choice to improve functional capacity, quality-of-life as well as blood pressure, also increase prognostic in Patient with CAD and No Revascularization. Cardiac rehabilitation can be a choice to improve functional capacity, quality-of-life as well as blood pressure, also increase prognostic in Patient with CAD and No Revascularization. ( ) Case Illustration A 60-year-old Asian male was diagnosed as CAD 3VD with CTO at mid LAD and proximal RCA. He was suggest to CABG procedure, but because personal and family preference, he refuse taking that procedure. Sometimes he got chest discomfort when moderate to heavy activities, but it got better. He was in stable hemodynamic after discharge with BP 130/80, HR 70bpm, RR 20tpm, SPO2 99% Room Air. He got 6-minutes walking test and got 595m distance, 4,9 Mets and VO2 Max 17ml/Kg/min. Then he prescribed Phase II Rehabilitation with initial 3,6km/h and target 5,9km/h. After 4 weeks, patient taken Treadmill Evaluation with Bruce Protocol and got total 10.23min exercise time, max HR 96%, max BP 210/100, 13,5Mets, Functional Capacity I, DTS Score -12 (Risk Category High, 5 Years Survival 72%). After that he continue rehabilitation until target 5,9km/h. Treadmill evaluation in 8 weeks, he got increase his functional capacity and prognostic with total 11.42min exercise time, max HR 107%, max BP 170/90, 13,5Mets, Functional Capacity I, DTS Score -6 (Risk Category Moderate, 5 Years Survival 91%). C l i CARDIAC REHABILITATION IN NO REVASCULATION PATIENT WITH CORONARY ARTERIAL DISEASES AND CABG SUGESSTION C.T. Tjahjono1, V. Mayangsari1, C. Kahadi1, A.Y. Suprapta1 Brawijaya University - Saiful Anwar General Hospital1 Background ac g ou d Coronary heart disease (CHD) are still the leading cause of death throughout the world. An evidence-based medical studies have shown that cardiac rehabilitation can significantly reduce the days and cost of hospitalization, mortality, and cardiovascular events in patients with CHD. Studies showed that outpatient cardiac rehabilitation programmers could provide some benefits for patients, such as convenience, fitness, improved functional capacity, quality-of-life, as well as blood pressure. In this paper, we describe a case of patient with Coronary Arterial Disease (CAD) who refuse to Coronary Artery Bypass graft (CABG) and got Cardiac Rehabilitation. Case Illustration pp Conclusions : We described a patient with a diagnosis of Tetralogy of Fallot and discuss the main outcome, diagnostic, and management aspects of the complication of this disease, which is cerebellar infarction. Symptomatic supportive treatment was effective in improving patient’s complaint and motor condition. p g p p Keywords: Cerebellar Infarction, Hyperviscosity Syndrome, Tetralogy of Fallot Keywords: Cerebellar Infarction, Hyperviscosity Syndrome, Tetralogy of Fallot Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Dengue fever, Supraventicular tachycardia Keywords: Keywords: No Revascularization; CABG; Cardiac Rehabilitation Indonesian J Cardiol Indonesian J Cardiol 2 pISSN: 2830-3105 / eI doi: 10.30701 Supraventricular Tachycardi In Children With Dengue Fever Y.D. Amalia1, H.M. Rini1, D. Eunike1, A.J.C. Kamalo1, M. Mahbubi1, I. Fahmi1 RSUD RAA Soewondo Pati1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Supraventricular Tachycardi In Children With Dengue Fever Y.D. Amalia1, H.M. Rini1, D. Eunike1, A.J.C. Kamalo1, M. Mahbubi1, I. Fahmi1 RSUD RAA Soewondo Pati1 Supraventricular Tachycardi In Children With Dengue Fever Y.D. Amalia1, H.M. Rini1, D. Eunike1, A.J.C. Kamalo1, M. Mahbubi1, I. Fahmi1 RSUD RAA Soewondo Pati1 Background: Dengue fever (DF) is known to involve the cardiac. Cardiac complications in DF patients include atrioventricular conduction disorders, arrhythmias, myocardial disorders, and myocarditis. In this report, we present a case of supraventricular tachycardia (SVT) as the initial presentation of an children DF. present a case of supraventricular tachycardia (SVT) as the initial presentation of an children DF. Case illustration: A 16-year-old girl presented with sudden onset of palpitation and breathlessness. She had fever for 3 days. Her blood pressure was 86/58 mmHg, pulse rate was 194 beats/minute, regular rhythm. Capillary refilling time was less than two seconds. The laboratory result showed leukopenia (2.7x103/ul), trombositopenia (71x103/ul), dengue IgG was positive. Electrocardiogram (ECG) showed SVT with the rate of 187 beats/minute. The child was diagnosed with DF complicated by SVT. Because the patient's condition was unstable. A 50 Joule (1J/kg) synchronous cardioversion was performed, followed by 2nd 100 joule (2J/kg) cardioversion because there was no response. The ECG remains unchanged after the cardioversion, therefore pharmacological therapy is needed. Injection of Amiodarone 150 mg IV was given because Adenosine was not available. Second injection of Amiodarone was also given because there was still no response after the 1st dose. The cardiac rhythm reverted to sinus rhythm. SVT complication is less frequent in DF. Membrane potential changes as a result of inflammatory processes and cytokine storms affecting myocytes and the interstitium can lead Arrhythmias in DF. The treatment strategy of SVT depends on the hemodynamic condition. Cardioversion is needed to terminate SVT in children with unstable hemodynamic. In our case, SVT was no response to cardioversion, therefore pharmacological therapy was chosen. Adenosine is considered the drug of choice, if it’s not available, Amiodarone IV may be a therapeutic option. Conclusions: DF can lead to arrhythmias in some cases. Monitoring and managing cardiac arrhythmias are required in DF. g y p p ons: DF can lead to arrhythmias in some cases. Monitoring and managing cardiac arrhythmias are n DF. Indonesian Journal of Cardiology q Keywords: Dengue fever, Supraventicular tachycardia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Modified pharmacological cardioversion in atrial fibrillation patients without significant structural heart disease: Case series from remote area Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Modified pharmacological cardioversion in atrial fibrillation patients without significant structural heart disease: Case series from remote area Modified pharmacological cardioversion in atrial fibrillation patients without significant structural heart disease: Case series from remote area E. Zuhri1 Teluk Bintuni Hospital, Papua Barat, Indonesia1 Background. Based on the currently available evidence, the indication for rhythm control is to reduce atrial fibrillation-related symptoms and improve quality of life. In case of uncertainty, an attempt to restore sinus rhythm in order to evaluate the response to therapy may be a rational first step. Pharmacological cardioversion to sinus rhythm is a procedure indicated in haemodynamically stable patients. The choice of a specific drug is based on the type and severity of associated heart disease. But, in remote area, sometimes the only drug available is amiodarone. Amiodarone is given by giving a bolus of 5-7 mg/kg over 1-2 hour followed by an infusion of 50 mg/hour through a large vein. However, prolonged infusion of amiodarone can cause some serious side effects. Case illustration. We made modified pharmacological cardioversion by giving 150 mg of amiodarone intravenously over 30 minutes in combination with 2.5 mg of bisoprolol orally in four patients with atrial fibrillation without significant structural heart diseases. If within one hour the rhythm had not been converted to sinus rhythm, another 150 mg of amiodarone was given intravenously over 30 minutes. The rate of conversion to sinus rhythm was achieved in less than 3 hours after the first administration of intravenous amiodarone and bisoprolol orally. There was no recurrence within 6 months of follow-up. Background. Based on the currently available evidence, the indication for rhythm control is to reduce atrial fibrillation-related symptoms and improve quality of life. In case of uncertainty, an attempt to restore sinus rhythm in order to evaluate the response to therapy may be a rational first step. Pharmacological cardioversion to sinus rhythm is a procedure indicated in haemodynamically stable patients. The choice of a specific drug is based on the type and severity of associated heart disease. But, in remote area, sometimes the only drug available is amiodarone. Amiodarone is given by giving a bolus of 5-7 mg/kg over 1-2 hour followed by an infusion of 50 mg/hour through a large vein. However, prolonged infusion of amiodarone can cause some serious side effects. Case illustration. We made modified pharmacological cardioversion by giving 150 mg of amiodarone Case illustration. We made modified pharmacological cardioversion by giving 150 mg of amiodarone intravenously over 30 minutes in combination with 2.5 mg of bisoprolol orally in four patients with atrial fibrillation without significant structural heart diseases. Keywords: amiodarone, atrial fibrillation, bisoprolol Case Illustration A 13 years old boy presented with shortness of breath, peripheral edema and tiredness. He had no medical history of disease. His blood pressure was 145/90 mmHg, regular heart rate 124 bpm, high respiration rate, inadequate to oxygen mask therapy therefore intubation is necessary. Physical examination got rhonchi bilateral on his lung and peripheral edema. ECG was sinus tachycardia. Transthoracic echocardiograph found LVEF 23%, global hypokinesis, normal chamber dimensions, and no valve dysfunction. Chest x-ray were batwing appearance and bilateral pleura effusion.Laboratory found leukocytosis, increase creatinine levels, high ASTO and CRP levels, and in urinalysis got gross hematuria also proteinuria. Treatment that has been given was sacubitril-valsartan and bisoprolol per oral, injection of furosemide, ceftriaxone, omeprazole, and dexamethasone, and nebulizer with fluticasone, salbutamol. Treatment that has been given was sacubitril-valsartan and bisoprolol per oral, injection of furosemide, ceftriaxone, omeprazole, and dexamethasone, and nebulizer with fluticasone, salbutamol. In this case, significant improvement within 3 days after an adequate treatment and extubation within 4 days. In day 10 of treatment patient got resolution of symptoms and echocardiograph shows normal cardiac function with In this case, significant improvement within 3 days after an adequate treatment and extubation within 4 days. In day 10 of treatment, patient got resolution of symptoms and echocardiograph shows normal cardiac function with LVEF 68%. Background g Cardiorenal syndrome (CRS) is a clinical condition that impacts both the heart and the kidneys, dysfunction of one organ leads to acute or chronic dysfunction of the other and received very little research attention in children. This clinical case report is to highlight the unusual event of CRS in young patient with symptoms of acute heart failure and glomerulonephritis had a good outcome result. Cardiorenal Syndrome: A Case Report of Acute Decompensated Heart Failure and Glomerulonephritis in a Young Patient Cardiorenal Syndrome: A Case Report of Acute Decompensated Heart Failure and Glomerulonephritis in a Young Patient Suhardi1, A.E. Putri1, J.A. Samjaya1 RSUD Taman Husada Bontang1 Modified pharmacological cardioversion in atrial fibrillation patients without significant structural heart disease: Case series from remote area If within one hour the rhythm had not been converted to sinus rhythm, another 150 mg of amiodarone was given intravenously over 30 minutes. The rate of conversion to sinus rhythm was achieved in less than 3 hours after the first administration of intravenous amiodarone and bisoprolol orally. There was no recurrence within 6 months of follow-up. Conclusion. Combination intravenous amiodarone and bisoprolol showed its effectiveness in converting and maintaining sinus rhythm. However, a bigger sample of randomized control trial needs to be done to show the effectiveness combination intravenous amiodarone and bisoprolol orally. Keywords: amiodarone, atrial fibrillation, bisoprolol Keywords: amiodarone, atrial fibrillation, bisoprolol Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Cardiorenal Syndrome: A Case Report of Acute Decompensated Heart Failure and Glomerulonephritis in a Young Patient Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion CRS have a higher risk of death and morbidity than those with each disease entity alone. The heart and kidneys interact bidirectionally and interdependently through several mechanisms. However, in pediatric patient even the evidence rarely to be found, this case illustrates the prompt and adequate treatment leading to optimal patient outcome, restore ventricular function and can be used in similar cases in the future. K d C di l d A t H t F il Gl l h iti Keywords: Cardiorenal syndrome, Acute Heart Failure, Glomerulonephritis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 LARGE PERICARDIAL EFFUSION WITH TUBERCULOUS PERICARDITIS: A CASE REPORT A.I.R. Paputungan1, A.H. Nashar1, J. Kusumanegara2, I. Djaharuddin3 1Department of Cardiology and Vascular Disease, Medical Faculty, Hasanuddin University, Makassar 2Department of Thoracic Surgery, Medical Faculty, Hasanuddin University, Makassar 3Department of Pulmonology and Respiratory Medicine, Medical Faculty, Hasanuddin University, Makassar Background: Tuberculous (TB) pericarditis has an incidence of ≤ 4% of all pericardial disease in developing countries. Fifty to seventy percent of pericardial effusion cases are caused by TB in endemic countries. Assessment of TB infection possibility in pericarditis patients with epicardial effusion is important for cardiologists to provide adequate treatment. This case report discusses a case of large pericardial effusion with TB pericarditis. p Case illustration: A 19-years old female was referred to our hospital with a chief complaint of shortness of breath accompanied by intermittent fever and cough. Physical examination results revealed tachycardia and decreased breath sounds in right hemithorax. Echocardiographic examination revealed a large pericardial effusion. The patient underwent a pericardial window procedure then examined for pericardial fluid analysis and cytology. Chronic granulomatous inflammation was found with a specific process suggestive of TB pericarditis. The patient's Tygerberg score was 6, hence TB Pericarditis diagnosis could be established. The patient was treated for 18 days at Wahidin Sudirohusodo Hospital and was then given category 1 anti-tuberculosis drug treatment for 8 weeks and was sent home in stable condition. Conclusion: Pericarditis is the main cause of pericardial effusion. Infection is often the main cause, one of which is TB infection. Diagnosing TB pericarditis could be challenging. Pericardial window is a procedure that could be both diagnostic and therapeutic in pericardial effusion. The Tygerberg score could also help its diagnosis. A total score ≥6 could establish TB pericarditis diagnosis with sensitivity = 86% and specificity = 85%. Background g Prolonged QT interval is a condition where the heart takes longer than normal to recharge between beats, which can lead to increased risk of cardiac arrhythmia such as ventricular tachycardia and torsades de pointes. Meanwhile, atrial fibrillation is a type of arrhythmia that occurs when the electrical impulses that regulate the heartbeat become rapid and disorganized in the upper chambers of the heart. The combination of these two conditions might increase the risk of ventricular tachycardia, but whether it increase the risk of myocardial infarction is yet to be understood. Case Illustration A 73-year-old patient presented to the ED with general weakness, blurred vision, and epigastric pain, had seizure at home with duration of 5-10 minutes, with brief loss of consciousness. The patient had no neurological deficit, no headache, no palpitation. On presentation, patient had blood pressure of 140/90 mmHg, heart rate of 56 beats per minute. The patient has history of prolonged QT interval and atrial fibrillation. ECG revealed monomorphic ventricular tachycardia. The patient received synchronized cardioversion and lidocaine infusion. Later on, we found that the patient had elevated troponin-I levels, diagnosed with NSTEMI. Ventricular Tachycardia and NSTEMI in a Patient with Prolonged QT Interval and Atrial Fibrillation: A Case Report Ventricular Tachycardia and NSTEMI in a Patient with Prolonged QT Interval and Atrial Fibrillation: A Case Report R.R. Eri1, F. Setiyarizki1, F.S.A. Pradana1 RSUD Bagas Waras Klaten1 Conclusion Keywords: TB pericarditis, pericardial effusion, pericardial window, Tygerberg score Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Ventricular Tachycardia and NSTEMI in a Patient with Prolonged QT Interval and Atrial Fibrillation: A Case Report Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion This case highlights the potential increased risk of ventricular tachycardia and NSTEMI in patient with prolonged QT interval and atrial fibrillation. Further studies are needed to better understand the underlying mechanisms and potential increased risk of myocardial infarction in these patients. This case report adds to the limited literature on the potential risks associated with the combination of prolonged QT interval and atrial fibrillation and emphasizes the importance of careful monitoring and treatment in these patients. Keywords: Prolonged QT interval, Atrial fibrillation, Ventricular tachycardia, NSTEMI Atrial Septal Defect (ASD) with Eisenmenger Sydrome Manifested with Clinical Sign of Left Heart Failure: a Case Report Atrial Septal Defect (ASD) with Eisenmenger Sydrome Manifested with Clinical Sign of Left Hear Failure: a Case Report S. Azzahra1, M.J. Al-Farabi2, M.Q. Ayun2, T.R. Mahmudah2, E. Hindariati2 Faculty of Medicine, Universitas Airlangga - Dr. Soetomo General Academic Hospital, Surabaya, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga - Dr. Soetomo General Academic Hospital, Surabaya, Indonesia2 Indonesian Journal of Cardiology Indonesian Journal of Cardiology A Reversible Type 1 Brugada-Like ECG Pattern In 67 Years Old Woman : A Syndrome or A Phenocopy? A Reversible Type 1 Brugada-Like ECG Pattern In 67 Years Old Woman : A Syndrome or A Phenocopy? A.R. Ananda1, M. Amir2, A.F. Muzakkir2 Wahidin Sudirohusodo Hospital, Makassar1 Department of Cardiology Hasanuddin University2 A.R. Ananda1, M. Amir2, A.F. Muzakkir2 Wahidin Sudirohusodo Hospital, Makassar1 Department of Cardiology Hasanuddin University2 Background : Brugada Syndrome (BrS) is an autosomal-dominant inherited disorder characterized with a typical ST Elevation followed with negative T wave in the right precordial leads without any structural cardiac malformations. Brugada Phenocopy is a clinical condition characterized by an identical ECG pattern to congenital Brugada Syndrome in general triggered by various factors. It is important to differentiate both conditions, here we reported a case of a 67 years old woman with a typical Brugada-Like ECG Pattern. Case Illustration : A 67 years old woman admitted to the emergency unit due to her ongoing atypical chest pain with onset 5 hours accompanied with fever and cough 2 days prior her admission; her vitals showed elevated temperature 37.7 C; her ECG showed a typical coved typed ST segment elevation in V2 and inverted T wave in lead V1-V3 in accordance with type 1 Brugada’s ECG pattern. Laboratory test demonstrated normal level of high sensitive troponin I (10.9 ng/l) with slightly elevated WBC (11.400/uL). Echocardiography was normal with LVEF 67%. However her Chest CT showed infiltrates in her right lung confirming Pneumonia. The patient was treated with prompt antipyretic, antibiotics, and mucolytic. After 3 days of treatment her ECG converted to normal without any documented episode of life-threatening arryhtmias, she was then discharged with stable condition and hemodynamic. Conclusion : It is important to distinguish the diagnosis of Brugada Phenocopy to true Brugada Syndrome in order to achieve the right treatment. Pneumonia can be a factor in occurrence of Brugada Phenocopy. At this point, the right treatment for Brugada Phenocopy is to ameliorate the underlying conditions. Keywords: Brugada Pattern, Arrythmia, Phenocopy, Pneumonia, ECG Figure 1. TransThoracal Echocardiography and MSCT Cardiac Aortography Figure 1. TransThoracal Echocardiography and MSCT Cardiac Aortography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology BACKGROUND Total atrioventricular block (TAVB) is associated with poor clinical outcomes in ST-elevation myocardial infarction (STEMI). We compared two patients with chest pain and hemodynamic instability with different time delay to reperfusion. Case Series of Total Atrioventricular Block (TAVB) Complicating Acute ST-Elevation Myocardial Infarction (STEMI): Culprit Vessel-Only or Complete Revascularization Strategy? ase Series of Total Atrioventricular Block (TAVB) Complicating Acute ST-Elevation Myocardial Infarction (STEMI): Culprit Vessel-Only or Complete Revascularization Strategy? F.A. Nurullah1, N.R. Utantyo1 Embung Fatimah Regional Public Hospital1 CASE ILLUSTRATION Patient A, 61 year old male with history of uncontrolled hypertension, was referred to our hospital with inferior STEMI 28 hours after onset and TAVB. Patient B, 66 year old male, with unknown medical history, came to our hospital complaining 2 hours onset of typical chest pain and shortness of breath. Both patients were hemodynamically unstable and both laboratory results showed elevated troponin I, additionally patient B has elevated blood glucose, liver enzyme and creatinine level. Both were diagnosed as acute inferior STEMI, TAVB and cardiogenic shock, patient B also has diabetes, secondary hepatic and kidney injury. Coronary angiography showed acute total occlusion in proximal right coronary artery (RCA) and significantly stenosed left anterior descending (LAD). PPCI was performed to the RCA, as culprit vessel. Evaluation showed TIMI flow III on both patients. On the next day, patient A developed acute pulmonary oedema and significant pleural effusion, he was intubated, mechanically ventilated and ultrasound-guided thoracentesis was done. Both patients’ condition gradually improved, patient A was successfully being weaned from ventilator after 7 days, ECG returned to sinus rhythm even when temporary pacemaker was off on 8th day. Both patients were successfully discharged home in stable condition after 15 and 10 days of hospitalization. ABSTRACT Background : Atrial septal defect (ASD) is usually manifested with pulmonary hypertension and right heart failure. Interestingly, we found an Acuted decompensated heart failure (ADHF) situation which may related with the presence of long-term ASD and right heart failure which led into left heart failure.. Case illustration : Woman, 50 years old, complained shortness of breath during heavy activity, worsening over the last five days, orthopnea and paroxysmal noctural dyspneu. Patient found out having septal defect, often seek for treatment but do not routinely take medication. ECG showed atrial fibrillation with rapid ventricular response with normal frontal dan horizontal axis. From chest x-ray, found cardiomegaly with prominent conus pulmonum and inverted coma sign, lungs got parahillar hazziness. Echocardiography showed ASD (3.4 cm) with bidirectional flow dominant R to L with high probability of pulmonary hypertension (TRVMax 4.5m/s) and RV failure (TAPSE 1.4 cm) with B lines suggesting congestion due to LV failure. Patient then treated with diuretics, inotropes (milrinone), and prostacycline analog (iloprost) and relieved her symptoms. p p y g p y p Conclusion : ADHF can also occur in longstanding ASD which has been manifested with rapid atrial fibrilation, pulmonary hypertension crisis and RV failure which then followed by LV failure. Diuretics combined with pulmonary hypertension drug remained effective treatment for this case. Keywords: ADHF, ASD, Atrial Fibrilation, Eisenmenger Syndrome Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:su pISSN: 2830-3105 / eISSN: 2964 doi: 10.30701/ijc.1537 Case Series of Total Atrioventricular Block (TAVB) Complicating Acute ST-Elevation Myocardial Infarction (STEMI): Culprit Vessel-Only or Complete Revascularization Strategy? F.A. Nurullah1, N.R. Utantyo1 Embung Fatimah Regional Public Hospital1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 CONCLUSION In our case, PPCI was done to the culprit lesion in the acute emergency setting. Some literatures demonstrated there’s no significant benefit for STEMI-CS patient treated with complete revascularization compared with culprit vessel-only intervention as patients are at increased risk of developing acute renal failure after multi-vessel intervention. Time from symptom onset to reperfusion is an important predictor of patient outcome. Keywords: Acute coronary Syndrome, ST-Elevation Myocardial Infarction, Cardiogenic Shock, Total Atrioventricular Block, Culprit Vessel-Only Revascularization, Complete revascularization Indonesian Journ Cardiology Indonesian J Cardiol 2023:44 pISSN: 2830-3105 / eISSN: 29 doi: 10.30701/ijc.153 A Rare Case Report of ST Segment Elevation in The Presence of Sepsis Complicated by Acute Respiratory Failure : Myocardial Infarction or Not ? S D T Si 1 C J P b 2 N G Li 3 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Rare Case Report of ST Segment Elevation in The Presence of Sepsis Complicated by Acute Respiratory Failure : Myocardial Infarction or Not ? S.D.T. Sitanggang1, C. J. Purba2, N.G. Liman3 RSUD Pakuhaji, Tangerang1 Putri Hijau Hospital, Medan2 RSUD Pakuhaji,Tangerang3 Background : ST elevation (STE) is widely regarded as the sign of myocardial infarction (MI) along with typical symptoms of ischemia in many clinical settings. Furthermore, this sign was oftenly thought as acute coronary occlusion requiring urgent reperfusion. However, not all ST segment elevation is attributed to acute coronary occlusion, there are various etiologies that may cause STE. Sepsis is known to cause QRS changes and conduction block yet STE is rarely reported. This case report aims to present a rare case of STE in patient with sepsis complicated by acute respiratory failure. Case Illustration : A 67 years old female was admited to our emergency unit (EU) with loss of consciousness. Before unconscious, she complained worsening shortness of breath. Around a week ago, she had been hospitalized in our hospital due to pneumonia. No chest pain was reported. No history of cardiac disease. Vital sign on admission was BP 124/82, RR 22x/min, SpO2 80 % RA, HR 120x/bpm, coma state. Ronchi was found in physical examination. Laboratory findings showed leucocytosis 24.000/ul, increased ureum and creatinine, 77 mg/dL and 2,9 mg/dL, respectively. Arterial blood gas showed metabolic acidosis with partial compensation. Chest X-ray showed pneumonia. ECG showed inferior STE with ST depression in antero-lateral lead. She was diagnosed with impaired consciousness, sepsis, acute respiratory failure and pneumonia. Around 6 hours after admission, she had cardiac arrest then cardiopulmonary resucitation was initiated along with intubation. The patient passed away after 20 minutes of resucitation. Conclusion : STE in sepsis can be a misleading condition. STE in sepsis is suggested due to oxygen supply/demand imbalance to myocardium without any occluded artery. These mechanism may cause myocardial injury known as type II myocardial infarction. Therefore, ST elevation during sepsis could rarely occur yet it should not urge physician to immediate reperfusion therapy. Keywords: sepsis, myocardial infarction, respiratory failure, ST segment elevation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Double culprit ST elevation : Optimal guidance for revascularization completion Sahniriansa, A.A. Alkatiri, S.S. A Rare Case Report of ST Segment Elevation in The Presence of Sepsis Complicated by Acute Respiratory Failure : Myocardial Infarction or Not ? Danny Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Indonesia, Indonesian Cardiovascular Research Center, National Cardiovascular Center Harapan Kita, Jakarta, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Double culprit ST elevation : Optimal guidance for revascularization completion Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology “Heart in DRESS”, Heart Failure as Long-Term Effect of Drug Reaction Related Eosinophilia Systemic Syndrome (DRESS Syndrome) Related to Cefadroxil Use: Rare Sequelae of Rare Entity A.B. Aryaputra1, H.P. Bagaswoto2, P. Mawardi3 RSU Islam Klaten1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Public Health, and Nursing Universitas Gadjah Mada – Dr. Sardjito Hospital, Yogyakarta, Indonesia2 Department of Dermatology Venereology , Faculty of Medicine Universitas Sebelas Maret- Dr Moewardi Hospital, Surakarta, Indonesia3 A.B. Aryaputra1, H.P. Bagaswoto2, P. Mawardi3 RSU Islam Klaten1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Public Health, and Nursing Universitas Gadjah Mada – Dr. Sardjito Hospital, Yogyakarta, Indonesia2 Department of Dermatology Venereology , Faculty of Medicine Universitas Sebelas Maret- Dr Moewardi Hospital, Surakarta, Indonesia3 Case Illustration A forty-years-old male came to emergency department (ER) with fullbody rash and worsening itch after taking Cefadroxil 2 weeks before. We discovered wide, erythematous, maculopapular rash with areas of scaling on entire body. Both vital sign and chest x-ray were normal. Laboratory investigation showed leucocyte level of 41.600/πl with 24.7% of eosinophiles (normal 1-4%). Patient was diagnosed with probable DRESS Syndrome (RegiSCAR: 5) dd Steven-Jhonson Syndrome. He received with intravenous methylprednisolone and antihistamine as treatment. Patient was discharged with oral steroid provided. Two months later, patient had exertional dyspnea, fatigue, diaphoresis, and pleuritic chest pain for two weeks. He also felt palpitation 2 days before finally readmitted to ER. He had neither history of hypertension nor heart disease. All physical examination were normal, except for bibasilar crackles and heart rate of 200 bpm. The EKG showed SVT with aberrancy, accompanied by cardiomegaly and pulmonary edema on the chest x-ray. Laboratory test revealed elevated liver enzymes and normal troponin I level. The echocardiography revealed global hypokinetic with EF of 28% and TAPSE 9 mm. Although diltiazem successfully suppressed the heart rate, the dyspnea persisted, therefore furosemide was given a few more days, along with other drugs on heart failure regiment. Patient was discharged after seven days with subsided symptoms. Eosinophilia-related cardiac toxicity was suggested for the mechanism of the acute heart failure. Further investigation using CMR should be done. Background Background g Drug reaction related Eosinophilia Systemic Syndrome (DRESS Syndrome) presents as severe skin eruption, eosinophilia, fever, and internal organ damage. Cardiac involvement in DRESS Syndrome is quite rare. Case Illustration Conclusion onclusion ardiac complication of DRESS Syndrome are felt to be rare, but could also under-recognized and life- Cardiac complication of DRESS Syndrome are felt to be rare, but could also under-recognized and life- threatening Cardiac complication of DRESS Syndrome are felt to be rare, but could also under-recognized and life- threatening. Keywords DRESS Syndrome, Heart Failure, Arrhythmia Keywords: DRESS Syndrome, Heart Failure, Arrhythmia Cardiac complication of DRESS Syndrome are felt to be rare, but could also under-recognized and life- threatening. Keywords Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Double culprit ST elevation : Optimal guidance for revascularization completion Sahniriansa, A.A. Alkatiri, S.S. Danny Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Indonesia, Indonesian Cardiovascular Research Center, National Cardiovascular Center Harapan Kita, Jakarta, Indonesia Background: In ST-elevation acute myocardial infarction, only one epicardial artery commonly contains an occlusive thrombus, widely referred to as the culprit artery. However, it is rare for patients to present with more than one acutely thrombotic coronary artery finding or double culprit. y y y g p Case Illustration: A 54-year-old man 30 minute onset of STEMI anteroinferior- RV infarct. He underwent Primary Percutaneus Coronary Angiography (PPCI), angiography showed total occlusion in proximal Left Anterior Descending (LAD) and proximal Right Coronary Artery (RCA), then the implantation of 1 Drugs Eluting Stents (DES) in LAD was carried out. During observation at cardiovascular care unit (CVCU), he had an episode of chest pain accompanied by on ECG inferior ST elevation. Repeat angiography was perform, thrombosucction and another DES PCI was implanted in proximal RCA. Post PCI, the patient's physical condition improved, there were no complaints of chest pain. The patient was given optimal therapy and discharged on day 5 of treatment with well condition. Conclusion: The possibility of double culprit's STEMI should be considered during primary PCI, especially in unstable patients. Rapid restoration of coronary blood flow will save the myocardium and decrease the risk of death. There are currently no guidelines for the optimal management strategy in patients with acute multi-vessel or double culprit. The choice of therapy is based on the patient's clinical and TIMI flow at the time of coronary intervention PCI. Keywords: Acute ST Segment Elevation Myocardial Infarction, primary coronary intervention, double culprit, multi-vessel coronary thrombus. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 g Objective Objective To review and discuss the challenging treatment strategies in two patients with STEMI and concomitant bleeding event. p Discussion Coronary intervention and dual anti-platelet medication for high risk acute coronary syndrome may increase the risk of bleeding despite the condition's life-threatening nature. In order to prevent Gastrointestinal bleeding, SAPT is advised in these circumstances, along with extra proton pump inhibitor therapy, therefore it was administered in both cases. C l i Case Illustration First patient was 67-year-old female with inferior STEMI (14-h of onset) with concurrent of active GI bleeding, right ventricular infarction and TAVB (Total AV Block). He received inotropes, blood transfusions and PPIs to manage GI bleeding. PPCI was then carried out after stabilization of the patient. The Second patient was 64-year- old female with Antero-posterolateral STEMI with 4 hours onset, the patient underwent fibrinolytic successful therapy, the second day of treatment at CVCU the patient experienced GI bleeding, early routine PCI was delayed, treatment for GI bleeding was carried out, after the bleeding was resolved, the patient then underwent PCI with 2 DES implantation in LAD. Background In STEMI patients, bleeding considerably lengthens the stay in the intensive care unit and raises mortality. Morbidity and mortality in STEMI patients have been decreased by primary percutaneous coronary intervention (pPCI) and contemporary antithrombotic therapy. Nonetheless, bleeding issues may be linked to efficient treatment plans. Little literature exists on the risk of performing coronary intervention (PCI) on patients who have had recent gastrointestinal bleeding (GIB), although bleeding after PCI has been identified as a risk factor for long-term mortality. Obj ti Keywords DRESS Syndrome, Heart Failure, Arrhythmia DRESS Syndrome, Heart Failure, Arrhythmia Keywords: DRESS Syndrome, Heart Failure, Arrhythmia Keywords: DRESS Syndrome, Heart Failure, Arrhythmia Indonesian Journ Cardiology Indonesian J Cardiol 2023:4 pISSN: 2830-3105 / eISSN: doi: 10.30701/ijc.15 High Bleeding Risk in Acute Coronary Syndrome: Stratification and Management Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration A 57 year old female patient was admitted to the ER with loss of consciousness. Found during physical exam, blood pressure of 60/40, heart rate of 132 bpm, 38.5 temperature, rhonchi in both lungs, diastolic murmur nd edema in all limbs. The patient had a history of congestive heart failure, atrial fibrillation, type 2 diabetes, chronic kidney disease, and gangrene, with no familial history of heart disease. Electrocardiography showed AF with rapid ventricular response, and blood work result, anaemia of 6.1mg/dl. On norepinephrine, consciousness and vital signs showed improvement with 120/70 BP. Hours after improved condition, the patient experienced altered mental status and drop in BP. Medicines used to combat the condition were norepinephrine, dobutamin, digoxin, furosemide, warfarin, spironolactone and bisoprolol. Echocardiography taken during previous hospitalization showed myxoma in the left atrium, size round 5x5cm located near the mitral valve with LA enlargement sized 5.82cm, and EF of 63.32%. The patient had previously been referred to a tertiary institution for surgery but the patient refused. Background Myxomas, a kind of cardiac tumor, may exhibit obstructive symptoms, yet many patients are asymptomatic at the time of diagnosis as the tumor may be identified incidentally on imaging studies. Myxomas could cause complications, such as potentially life-threatening thrombus, even cardiogenic shock when the tumor causes blockage. R.F.F. Rukman1, H.W. Parlindungan2 Pematang Siantar Army Hospital1 Department of Cardiology and Vascular Medicine, Pematang Siantar Army Hospital2 R.F.F. Rukman1, H.W. Parlindungan2 Pematang Siantar Army Hospital1 Department of Cardiology and Vascular Medicine, Pematang Siantar Army Hospital2 Conclusion Both patients delayed PCI and were treated for GI bleeding and hemodynamic stabilization, the first patient had a malignant arrhythmia as a complication of ACS, while the second patient had revascularization successfully Keywords : Acute Coronary Syndrome, Gastrointestinal Bleeding, STEMI Both patients delayed PCI and were treated for GI bleeding and hemodynamic stabilization, the first patient had a malignant arrhythmia as a complication of ACS, while the second patient had revascularization successfully g y p p y ords : Acute Coronary Syndrome, Gastrointestinal Bleeding, STEMI Keywords : Acute Coronary Syndrome, Gastrointestinal Bleeding, STEMI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Myxoma and Its Complications: A Case Report Conclusion We planned supervised progressive exercise therapy and comprehensive cardiac rehabilitation, and after several visits, we could see dramatic improvement in 6MWT, SPPB, and quality of life. Case Illustration: Mr. RE, 59 yo, a former taekwondo athlete were consulted for cardiac rehabilitation therapy from outpatient clinic. He was diagnosed with CHF Fc II-III ec DCM (EF 12%, TAPSE 16), s/p ICD 2017, mod- severe MR ec functional, severe TR with high probability of PH, mild AR, T2DM, and renal insufficiency. He felt activity intolerance and fatigue, recently hospitalized for acute decompensation of heart failure. Initial 6MWT was 270 m (low) and we did the frailty assessments using FRAIL scale, SPPB, 5-m gait speed test, showing frailty in this patient. We planned supervised progressive exercise therapy and comprehensive cardiac rehabilitation, and after several visits, we could see dramatic improvement in 6MWT, SPPB, and quality of life. Conclusion: Frailty is common in heart failure population. Heart failure and frailty are bidirectionally inter— related. Multiple assessment tools are available to assess frailty, FRAIL scale can be used to screen frailty and SPPB is one of practical assessment tools may be used to assess frailty. Exercise rehabilitation therapy is the cornerstone therapy of frailty, improving the functional capacity and quality of life of the patients. Keyword: Heart failure; Frailty; Functional class; Cardiac rehabilitation; Quality of Life Conclusion Myxomas most often originate from the left atrium and can obstruct the flow of blood in the heart depending on the location and the size of the tumor. Obstruction can cause low output symptoms and also lead to complications such as thromboembolism. Determining the correct diagnosis and earlier disease management ensure prevention for further complications, with echocardiography as a significant tool in the process. K d i l fib ill i h k h b b li Figure 1. Echocardiography shows myxoma in the left atrium Figure 1. Echocardiography shows myxoma in the left atrium Recognizing and Managing Frailty in Patients with Advanced Heart Failure K.Triangto1, B. Dwiputra2, D.R. Desandri2, A.M. Ambari2, B. Radi2, K.M.H.J. Tandayu1 National Cardiovascular Center Harapan Kita, Jakarta, Indonesia1 National Cardiovascular Center Harapan Kita2 Background: By the fact of the aging population in developing and developed countries, the prevalence of cardiovascular disease especially heart failure has dramatically increased causing morbidity, hospitalization and mortality in older people worldwide. Frailty, a biological syndrome that reflects a state of decreased physiological reserve and vulnerability to stressors, has become a high-priority theme in cardiovascular medicine due to aging and increasingly complex nature of cardiovascular patients. Up to 44.5% of HF patients were considered frail. Unfortunately, frailty is often unrecognized and many physicians are unaware of this condition cardiovascular disease especially heart failure has dramatically increased causing morbidity, hospitalization and mortality in older people worldwide. Frailty, a biological syndrome that reflects a state of decreased physiological reserve and vulnerability to stressors, has become a high-priority theme in cardiovascular medicine due to aging and increasingly complex nature of cardiovascular patients. Up to 44.5% of HF patients were considered frail. Unfortunately, frailty is often unrecognized and many physicians are unaware of this condition Case Illustration: Mr. RE, 59 yo, a former taekwondo athlete were consulted for cardiac rehabilitation therapy from outpatient clinic. He was diagnosed with CHF Fc II-III ec DCM (EF 12%, TAPSE 16), s/p ICD 2017, mod- severe MR ec functional, severe TR with high probability of PH, mild AR, T2DM, and renal insufficiency. He felt activity intolerance and fatigue, recently hospitalized for acute decompensation of heart failure. Initial 6MWT was 270 m (low) and we did the frailty assessments using FRAIL scale, SPPB, 5-m gait speed test, showing frailty in this patient. Extremely Elevation of Troponin-I (cTnI) Values in Acute Myocardial Infarction Patients with Chronic Renal Failure Y.W. Parhusip1, M. Riski2 1. General Practioner, Dr. Esnawan Antariksa Indonesian Air Force Hospital; Jakarta; Indonesia. 2. Department of Cardiology and Vascular Medicine, Dr. Esnawan Antariksa Indonesian Air Force Hospital; Jakarta; Indonesia. Y.W. Parhusip1, M. Riski2 1. General Practioner, Dr. Esnawan Antariksa Indonesian Air Force Hospital; Jakarta; Indonesia. 2. Department of Cardiology and Vascular Medicine, Dr. Esnawan Antariksa Indonesian Air Force Hospital; Jakarta; Indonesia. Background: Coronary Heart Disease (CAD) is a multifactorial disease caused by atheromatous plaque deposition and progressive narrowing of the arteries, with the typical symptom of chest pain. The most frequently used diagnostic support for CAD besides electrocardiography (ECG) is Troponin I enzyme examination. In chronic kidney disease (CKD) case, the severity and prevalence of CAD increases 20 times compared to the normal population, due to the involvement of traditional risk factors (hypertension, smoking, diabetes mellitus, dyslipidemia, left ventricular hypertrophy, male sex), and uremia-related risk factors, which are often characterized by high Troponin I values. y g p Case Illustration: A 72 year old man came to the emergency department with complaints of left chest pain since the previous 4 days. Complaints of left chest pain that feels like a cut and gets worse with activity, usually get better with isosorbite. Since the last 10 years, the patient has a history of type II diabetes mellitus and impaired kidney function. Conscious awareness, blood pressure 90/61 mmHg, pulse 107 x/min, respiratory rate, 26 x/min, temperature 36 ̊C, oxygen saturation 99% and no abnormality was found on physical examination. Normal EKG examination, sinus rhythm, Troponin I > 15.5 ng/dl, urea 86 mg/dl and creatinine 1.8 mg/dl. Patient was diagnosed with NSTEMI with grade III CKD comorbidities. During treatment at the hospital, the patient received anti- platelet aggregation, anti-platelet and anticoagulant therapy. The results of serial ECG examinations on the 2nd and 3rd day of treatment showed anterior ischemia (inverted T) which returned to normal on the 5th day of treatment. This case report shows that not only due to infarction, but also exposure to high creatinine urea in the blood, causes increased damage to myocyte cells of the heart muscle in patients with CAD accompanied by CKD. Because the condition of uremia causes the Troponin I released gradually from myocyte cells. It is hoped that doctors will be more aware of the morbidity and mortality rates of patients who have cardiovascular disease accompanied by CKD. Keywords: Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Recurrent Ventricular Tachycardia from Myocardial Infarction Non-obstructive Coronary Artery : an unusual event Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Recurrent Ventricular Tachycardia from Myocardial Infarction Non-obstructive Coronary Artery : an unusual event O. Sofyan1, M. Amir1, I. Mappangara1 RSUP dr Wahidin Sudirohusodo1 Background: Myocardial Infarction Non-obstructive Coronary Artery (MINOCA) is a new entity which is currently trend at interventional cardiology. Whereas cases of MINOCA may not need intervention treatment because of the absence of coronary artery occlusion. However, patient with MINOCA may have a worse prognosis if the condition is not appropriately managed, including recurrent ventricular tachycardia. Case illustration: We reported a 55 year old male patient came to Emergency Room with typical severe precordial chest pain (VAS 8/10) accompanied with nausea within 2 hours and followed with palpitation. Upon admission, the HR was 200 bpm with wide QRS complex on monitor, initial defibrillation were prepared but then spontaneously convert to sinus rhythm. Initial blood sample shown increased hs-Troponin I. Surface ECG shown monomorphic ventricular tachycardia. Echocardiography findings showed mildly reduced LV & RV systolic function with EF 47.7%, and TAPSE 1.6 cm. Coronary angiography showed non-significant stenosis. On following days the patient was clinically deteriorating with recurrent ventricular tachycardia. Cardiac MRI showed positive late gadolinium enhancement at septal wall and confirmed microvascular obstruction. The antiarrythmic agent was admitted, then electrophysiology study and ablation were performed with successful result. Conclusion: MINOCA presenting as ventricular scars is sometimes fatal. Dramatic complication such as recurrent ventricular tachycardia leads this case to be more challenging. With guidance of cardiac MRI and ablation are important things to do with intensive optimal management strategy. Keywords: Keywords : MINOCA, recurrent ventricular tachycardia. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Extremely Elevation of Troponin-I (cTnI) Values in Acute Myocardial Infarction Patients with Chronic Renal Failure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 PCI with DES and DCB for Treatment of Mixed In-Stent Restenosis and De Novo Stenosis: Case Report h DES and DCB for Treatment of Mixed In-Stent Restenosis and De Novo Stenosis: Case Report D.A.K. Purba1, A. Sitepu2 Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera U Indonesia2 D.A.K. Purba1, A. Sitepu2 Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia2 Background: In-Stent Restenosis (ISR) is the re-narrowing condition of stented artery segment in around 20% of Percutaneous Coronary Intervention (PCI) patients. ISR significantly increases the risk of death, poorer outcomes, and Major Adverse Cardiovascular Events (MACE), which is challenging in daily practice. j g g y p Case Illustration: A 76-year-old man came with a clinical manifestation of Unstable Angina Pectoris. He had a history of hypertension and diabetes mellitus and had one stent placed in Left Anterior Descending Artery(LAD) 18 years ago. Through coronary angiography examination, there appears to be a Mixed ISR and De Novo Stenosis in LAD. Then an Intravascular Ultrasound (IVUS) examination was carried out with the results of fibrous plaque, stent under-expansion, and neo-atherosclerosis. Aggressive pre-dilation with Raiden non-compliant (NC) balloon 3,0x15mm-22 atm, cutting balloon Wolverine 3,5x25mm-14 atm and Ikazuchi 3,0x10mm-12 atm were performed before DCB Sequent Please 3,5x25mm-10 atm installed at the under-expansion and neo-atherosclerosis area. For de novo stenosis, DES Xience Xpedition 3,5x33mm-14 atm was utilized. The final results showed that LAD developed entirely with Minimal Stent Areas (MSA) 11,83 mm2 in the ostial LAD and 7,52 mm2 in the neo- atherosclerotic area. on: DEB and DES are comparable and provide excellent clinical outcomes for ISR. Conclusion: DEB and DES are comparable and provide excellent clinical outcomes for ISR. Keywords: In Stent Restenosis Percutaneous Coronary Intervention Drug eluting Stent Drug Conclusion: DEB and DES are comparable and provide excellent clinical outcomes for ISR. Keywords: In-Stent Restenosis, Percutaneous Coronary Intervention, Drug-eluting Stent, Drug-coated b C A R l i i Conclusion: DEB and DES are comparable and provide excellent clinical outcomes for ISR. Keywords: In-Stent Restenosis, Percutaneous Coronary Intervention, Drug-eluting Stent, Drug-coated balloon, Coronary Artery Revascularization Conclusion: DEB and DES are comparable and provide excellent clinical outcomes for ISR. Keywords: In-Stent Restenosis, Percutaneous Coronary Intervention, Drug-eluting Stent, Drug-coated balloon, Coronary Artery Revascularization y y Figure 1. Extremely Elevation of Troponin-I (cTnI) Values in Acute Myocardial Infarction Patients with Chronic Renal Failure Conclusion: The causes of damage to heart muscle myocyte cells can be caused by many factors. However, CAD patients accompanied by CKD, myocyte damage to heart muscle cells is also caused by exposure to high uremia conditions in the blood. Careful analysis of clinical data and laboratory findings can help to correctly diagnose and treat patients appropriately. Keywords: Keywords: coronary artery disease, Troponin I (cTnI), chronic kidney disease (CKD Keywords: Keywords: coronary artery disease, Troponin I (cTnI), chronic kidney disease (CKD). Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 PCI with DES and DCB for Treatment of Mixed In-Stent Restenosis and De Novo Stenosis: Case Report PCI with DES and DCB for Treatment of Mixed In-Stent Restenosis and De Novo Stenosis: Case Report (A) IVUS Assessment of ISR; (B) PTCA Balloon Catheter; (C) DES & DCB Implantation; (D) IVUS Assessment Post PCI & DCB Figure 1. (A) IVUS Assessment of ISR; (B) PTCA Balloon Catheter; (C) DES & DCB Implantation; (D) IVUS Assessment Post PCI & DCB Brugada Syndrome Induced By Fever In Pericarditis Tuberculosis Patient: A Case Report D. Amelia1, T. Erlanggawati2, W.N. Hamdani3, E. Zuhri4 Kanujoso Djatiwibowo General Hospital, East Kalimantan, Indonesia1 Panglima Sebaya General Hospital, East Kalimantan, Indonesia2 Abdul Wahab Sjahranie, East Kalimantan, Indonesia3 Teluk Bintuni General Hospital, West Papua, Indonesia4 D. Amelia1, T. Erlanggawati2, W.N. Hamdani3, E. Zuhri4 Kanujoso Djatiwibowo General Hospital, East Kalimantan, Indonesia1 Panglima Sebaya General Hospital, East Kalimantan, Indonesia2 Abdul Wahab Sjahranie, East Kalimantan, Indonesia3 Teluk Bintuni General Hospital, West Papua, Indonesia4 Background: Brugada syndrome (BrS) is defined as rare autosomal-dominant inherited disorder that might lead to sudden cardiac death. One of the triggers for this syndrome is fever. Case illustration: A 25-year-old man was admitted to the emergency department with complaints of chest pain and fever, with history of frequent syncope. There was a family history of sudden death on young age. During physical examination, patient had tachycardia and fever, and friction rub and decreased breath sound on left lung on auscultation. ECG showed coved-type ST-segment elevation with negative T wave in V1-V2 lead. Chest x- ray demonstrated minimal left pleural effusion, which proven to be granulomatous tuberculosis. Echocardiography revealed mild pericardial effusion. Patient was given daily maintenance IV fluid, anti pyretic, colchicine, antibiotic, and tuberculosis drug regimen. Fever is one of the triggers for Brugada Syndrome, but the pathophysiology is still not clear. There are some theories saying fever caused prematurely shorter period of action potential, accelerated inactivation of INa channels, decreased level of sodium channels expression, and the loss of sodium channel function. Conclusion: Patient fulfilled the diagnosis criteria for Brugada Syndrome which is induced by fever caused by tuberculous pericarditis. As a clinician, we should be more aware about the possibility of this syndrome in patients with history of fever and syncope. Keywords: Brugada Syndrome, Fever, Sudden Death Keywords: Brugada Syndrome, Fever, Sudden Death Figure 1. Patient’s electrocardiogram at the time of admission in emergency department (Body’s temperature 39,9 Celsius) Figure 1. Patient’s electrocardiogram at the time of admission in emergency department (Body’s temperature 39,9 Celsius) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Successful Percutaneous Balloon Mitral Valvuloplasty in a Case of Natural History of Rheumatic Mitral Valvular Stenosis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Successful Percutaneous Balloon Mitral Valvuloplasty in a Case of Natural History of Rheumatic Mitral Valvular Stenosis Successful Percutaneous Balloon Mitral Valvuloplasty in a Case of Natural History of Rheumatic Mitral Valvular Stenosis R.A. Rizki1, C.K. Krevani2, W. Valerian1, S.I. Herman1 Andalas University1 RSUP dr. M. Djamil Padang2 Background: Rheumatic Heart Disease (RHD) is a damage of the heart valve due to Acute Rheumatic Fever (ARF) which results from the body’s autoimmune response to Streptococcal infection. ARF is one of the most important causes of Mitral Stenosis (MS) in developing countries. Case illustration: A 41 years old woman came to the outpatient clinic with shortness of breath, fatigue, and ankle swollen. Symptoms had been felt since 3 days ago and worsened. The patient had a medical history of ARF at the age of childhood and was treated with antibiotic for 5 years, which developed into RHD with MS at the age of adult and routinely given medication of diuretics, beta blocker, and digitalis by cardiologist. The haemodynamic was stable. Chest examination found opening snap and mid-diastolic murmur at apex grade 2/4 did not spread to axilla and no gallop. Her extremities were warm with ankle swollen. An electrocardiogram showed no specific present. The results of blood laboratory have no specific present. Chest X-Ray showed cardiomegaly. Echocardiographic before admission found MS severe with Mitral Valve Area (MVA) 0.9 cm2 ec RHD with Wilkins Score 8, and no thrombus found. Due to MS NYHA fc III ec RHD with Wilkins Score 8, we decided to do Percutaneous Balloon Mitral Valvuloplasty (BMV). Post BMV evaluation, the echocardiography showed an enlargement of MVA from 0.9 cm2 to 1.2 cm2, which was a success procedure. The next day after the procedure, the symptoms were reduced and planned to discharge with medication. Conclusion: Rheumatic MS is a disease that progressive, usually with a latent phase of 20-40 years from the initial ARF to the development of symptoms. Symptoms gradually increase in severity over several years. Percutaneous or surgical intervention is the definitive treatment for relief MS. g Keywords: Mitral Stenosis, Rheumatoid Heart Disease, Percutaneous Balloon Mitral Valvuloplasty Fig. 1 Echocardiographic Findings Fig. 1 Echocardiographic Findings Fig. 1 Echocardiographic Findings Complete Heart Block in Multigravida Inpartu Patient : One of the pacing tools options for intraoperative sectio caesarian setting A.F. Abadi1, M. Amir2, A.I. Successful Percutaneous Balloon Mitral Valvuloplasty in a Case of Natural History of Rheumatic Mitral Valvular Stenosis Taim1 Faculty of Medicine, Hasanuddin University, Makassar, Indonesia1 Cardiac Center Wahidin Sudirohusodo, Makassar, Indonesia2 Complete Heart Block in Multigravida Inpartu Patient : One of the pacing tools options for intraoperative sectio caesarian setting A.F. Abadi1, M. Amir2, A.I. Taim1 Faculty of Medicine, Hasanuddin University, Makassar, Indonesia1 Cardiac Center Wahidin Sudirohusodo, Makassar, Indonesia2 Complete Heart Block in Multigravida Inpartu Patient : One of the pacing tools options for intraoperative sectio caesarian setting Case Illustration: A 55-year-old male presented with fever and sharp chest pain. Physical examination revealed high BP, tachycardia and desaturation. ECG showed sinus tachycardia. CXR showed pneumonia with pleural effusion. He was treated as pneumonia and antibiotics was administered but did not show significant improvement. Echocardiography evaluation showed dilated MPA with hazy appearance suspected as thrombus. clinical DVT was not found. Laboratory evaluation revealed normal troponin dan increased D-dimer level. CT Pulmonary Angioram (CTPA) then was planned but hemodynamic was unstable. He was diagnosed with acute PE. Optimal dose of heparin was given, unfortunately the patient did not survive. Second case, a 33-year-old obese male with medical history of Covid-19 and arthroscopy complained of dyspnea and pleuritic chest pain. Physical examination revealed high BP, tachycardia and desaturation. CXR showed bilateral pleural effusion and pneumonia. He was treated for pneumonia and an antibiotic was given but was not improved clinically. Initial echocardiography revealed normal findings. During treatment, he was clinically deteriorating. Laboratory evaluation revealed normal troponin dan increased D-dimer level. ECG evaluation showed S1Q3T3 pattern. Repeated echocardiography evaluation showed RA thrombus, dilatation of RV, MPA, LPA, RPA, with positive Mc-Connel Sign. He has absence of clinical DVT. CTPA confirmed thrombus PA. The patient then was treated as an acute PE with a fondaparinux but did not survive. Background: g Pulmonary Embolism (PE) is often hard to distinguish because it shares similar clinical features with pneumonia. This case series aims to show the importance of comprehensive evaluation for PE mimicking pneumonia which is frequently overlooked. Pulmonary Embolism Masquerading as Pneumonia: A Case Series Hapsari1, E.A.D Batubara1, L. Widianto1, D.R. Fauzan1, K.A. Nugraha2, R.U. Setiani2, Haryadi2, Yulianto I.A. Siregar2 G.P. Hapsari1, E.A.D Batubara1, L. Widianto1, D.R. Fauzan1, K.A. Nugraha2, R.U. Setiani2, Haryadi2, Yulianto2, I.A. Siregar2 Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine University of Indonesia / National Cardiovascular Center Harapan Kita1 p Cardiologist, Department of Cardiology and Vascular Medicine, Navy Hospital Mintohardjo, Jakarta, Indonesia2 Cardiologist, Department of Cardiology and Vascular Medicine, Navy Hospital Mintohardjo, Jakarta, In Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background: The prevalence of bradyarrhythmia in women of reproductive age is 1/20,000 and a significant majority are diagnosed prior to pregnancy. Initial diagnosis in pregnancy requires specific approach. Management of a pregnant woman with complete heart block presenting with parturient and without pacing is challenging. Transcutaneous cardiac pacing offers several advantages when compared to the placement of a transvenous cardiac pacemaker in the emergency setting. Case illustration: We report a case of successful management of a 23-year-old, pregnant woman with complete heart block who had an emergency caesarean delivery under spinal anesthesia. She was an unbooked patient detected with complete heart block during active stage of labour. An emergency transcutaneous cardiac pacing was used during surgical procedure under the supervision of cardiologist, anesthesiologist, and obstetrician. Emergency caesarean procedure was uneventful with uncompromised hemodynamic status. The transcutaneous cardiac pacing was removed shortly after surgery and the patient was scheduled for permanent pacemaker implantation. p Conclusion: Medical emergency has its own challenge for the management principles and implementation especially in pregnant woman. Known complete atrioventricular block patients with pregnancy should always be counselled and worked up for permanent pacemakers. However, temporary pacemaker is needed in emergency settings. Transcutaneous cardiac pacing is an ideal early and temporary intervention for patients requiring stabilizing cardiac pacing support until more invasive procedures can be arranged in the proper clinical setting. Keywords: Transcutaneous cardiac pacing, complete heart block, pregnancy, emergency caesarean sect Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: PE should be considered as the differential diagnosis in patients who are deteriorating even with optimal pneumonia treatment and no risk factors of PE. As these cases have a lethal condition, a comprehensive evaluation is necessary to prompt diagnosis of PE mimicking pneumonia to reduce mortality by appropriate treatment. CTPA are essential diagnostic tools to confirm the diagnosis. Keywords: Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Percutaneous Transluminal Angioplasty for Total Occlusion of Subclavian Artery with Clinically Manifested Subclavian Steal Syndrome H.O. Hermawan1, P.D.K. Devi1, P.M.H. Putri1, A. Halim1, R.M. Budiarto1 Universitas Airlangga / RSUD Dr. Soetomo1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Percutaneous Transluminal Angioplasty for Total Occlusion of Subclavian Artery with Clinically Manifested Subclavian Steal Syndrome H.O. Hermawan1, P.D.K. Devi1, P.M.H. Putri1, A. Halim1, R.M. Budiarto1 Universitas Airlangga / RSUD Dr. Soetomo1 H.O. Hermawan1, P.D.K. Devi1, P.M.H. Putri1, A. Halim1, R.M. Budiarto1 Universitas Airlangga / RSUD Dr. Soetomo1 Background: Subclavian steal syndrome (SSS) refers to a phenomenon of flow reversal from the vertebral artery to the subclavian artery due to hemodynamically significant lesion of the proximal subclavian artery. SSS can manifest in some patients related to circulation insufficiency to the central nervous system and/or upper extremities. The presence SSS is associated with increased total mortality and cardiovascular disease mortality. Percutaneous transluminal angioplasty is a minimally invasive strategy with a high success rate in managing SSS. Case Illustration: A 72 years old female comes with left arm claudication, aggravated with moderate activities. Physical examination showed a discrepancy of 51 mmHg in blood pressure taken in both upper extremities. Duplex ultrasonography revealed a caudalad/retrograde flow at the left vertebral artery and monophasic morphology at the left axillary, brachialis, ulnar, and radial arteries without significant plaque/thrombus along arteries. Catheter-based angiography confirmed total occlusion at the left proximal subclavian artery (Figure 1). The patient underwent percutaneous transluminal angioplasty with marked flow improvement at the left subclavian, vertebral, and internal mammary arteries. Re-evaluation of duplex ultrasonography showed cephalad/antegrade flow at the left vertebral artery. Background: Subclavian steal syndrome (SSS) refers to a phenomenon of flow reversal from the vertebral artery to the subclavian artery due to hemodynamically significant lesion of the proximal subclavian artery. SSS can manifest in some patients related to circulation insufficiency to the central nervous system and/or upper extremities. Conclusion: The presence SSS is associated with increased total mortality and cardiovascular disease mortality. Percutaneous transluminal angioplasty is a minimally invasive strategy with a high success rate in managing SSS. Case Illustration: A 72 years old female comes with left arm claudication, aggravated with moderate activities. Physical examination showed a discrepancy of 51 mmHg in blood pressure taken in both upper extremities. Duplex ultrasonography revealed a caudalad/retrograde flow at the left vertebral artery and monophasic Conclusion: Subclavian steal syndrome is related to arterial insufficiency in a branch of the subclavian artery due to stenosis at the proximal subclavian artery. Subclavian stenosis should be considered in any patient with arm claudication with a discrepancy of > 15 mmHg in blood pressure taken in both upper extremities, as well as judicious use of duplex ultrasonography and angiography can determine the need for invasive strategy. Percutaneous transluminal angioplasty has a high success rate with marked clinical improvement. Keywords: Subclavian steal syndrome, subclavian stenosis, percutaneous transluminal angioplasty Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 DIAGNOSTIC APPROACH BY SPECKLE TRACKING ECHOCARDIOGRAPHY IN CARDIAC AMYLOIDOSIS : A CASE REPORT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: This case underlines the importance of non-invasive diagnostic techniques such as STE for Cardiac Amyloidosis’s early diagnosis, especially when CMR cannot be performed. Keywords: Cardiac Amyloidosis, Speckle Tracking, Echocardiography Case illustration : A 42 year-old woman came to the hospital with chief complaints of substernal chest pain radiating to the back accompanied by cold sweat. Patient also experienced shortness of breath that has worsened since a day ago. ECG showed sinus rhythm with HR 100 bpm, normal heart axis (-10 degrees), poor R wave but no ST segment or T wave changes. Laboratory findings showed no increase in Hs Trop I and HbA1C was 10.8%. Chest x-ray displayed cardiomegaly. Coronary angiography did not show any significant stenosis. Cardiac Magnetic Resonance Imaging (CMR) was performed and EF was 33%. T1 mapping with a value of 1413 ± 338, R2 = 0.996, showing a positive Late Gadolinium Enhancement (LGE) indicating the presence of fibrosis or infarction of the myocardium, with overall conclusions of early cardiomyopathy, left ventricle enlargement, and abnormal LV systolic function. DIABETIC CARDIOMYOPATHY IN 42 YEAR-OLD WOMAN MIMICKING ACUTE CORONARY SYNDROME A. Surya1, A.F. Muzakkir1 Department of Cardiology and Vascular Medicine Faculty of Medicine, University of Hasanuddin Makassar.1 Background : g Diabetic cardiomyopathy is defined as changes in myocardial structure and function in diabetic patients, in the absence of other cardiovascular risks such as ischemic hypertension, or significant valvular disease. Case illustration : DIAGNOSTIC APPROACH BY SPECKLE TRACKING ECHOCARDIOGRAPHY IN CARDIAC AMYLOIDOSIS : A CASE REPORT W.E. Putri1, A.F. Ghaznawie1, A.F. Muzakkir1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Hasanuddin University/ Dr. Wahidin Sudirohusodo Hospital, Makassar, Indonesia1 Background: Cardiac amyloidosis (CA) is a form of infiltrative cardiomyopathy due to deposition of amyloid fibrils in the myocardium. Amyloid fibril infiltration impairs myocardial function, often with a normal appearing Left Ventricle ejection fraction (LVEF). Two-dimensional speckle-tracking (STE) imaging is used to measure myocardial deformation and is proven to be useful in the case of cardiac amyloidosis. Case Illustration: We report the case of a 55-years-old female referred to our Hospital with progressive shortness of breath and lower extremity oedema. The electrocardiogram showed: sinus rhythm of 85 bpm, LVH with low-voltage criteria. Chest radiography proved lung edema and cardiomegaly. Laboratory analysis showed mild hyponatremia (130). Echocardiography showed: increased left ventricular (LV) and right ventricular (RV) wall thickening, biatrial enlargement with increased atrial septal thickness, grade II diastolic dysfunction with elevated LV filling pressures, severely reduced mitral annular tissue doppler velocities, pericardial effusion, diffuse valve thickening and a preserved ejection fraction with low stroke volume index. Based on her basic echocardiographic, cardiac amyloidosis was strongly suspected. LV longitudinal strain obtained by STE, showed a reduced global longitudinal strain with apical sparing. A Cardiac MRI was planned but due to the patient’s intolerance of lying for long durations, the examination was not completed. GLS is a unitless measure of longitudinal deformation with more negative values denoting greater deformation. When it is abnormal, it reflects contractile dysfunction. Abnormal GLS in the mid and basal walls of the LV with normal values in the apex, when plotted on a bull’s-eye map, known as the apical sparing pattern (or "cherry on top" pattern), is 93% sensitive and 82% specific in identifying patients with CA. Conclusion: This case underlines the importance of non-invasive diagnostic techniques such as STE for Cardiac Amyloidosis’s early diagnosis, especially when CMR cannot be performed. Keywords: Cardiac Amyloidosis, Speckle Tracking, Echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rare Case Ruptured Sinus Valsava in Adolescence with Doubly Committed Subarterial Ventricular Septal Defect and Infective Endocarditis I. Setiadi1, I. Sakidjan1 1Department of Cardiology and Vascular Medicine, Universitas Indonesia/National Heart Center Harapan Kita, Jakarta, Indonesia Background: g Ruptured Sinus Valsava is a rare disorder, with incidence ranging from 0.1 to 3.5% of all congenital heart diseases, occurring between the ages of 20-40 years. Ruptured sinus valsava can be congenital or acquired as a result of a ventricular septal defect, endocarditis, or chest trauma. The occurrence of VSD and concurrent endocarditis infection can hasten sinus valsava rupture. Conclusion: There have been reported case of 15-year-olds with ruptured sinus valsava, in VSD SADC, and Infective Endoarditis. Both, can result in a faster occurrence of complications, such as ruptured of the valsava sinus. Monitoring and early management of patients with VSD SADC, and aortic regurgitation should be more strictly enforced in order to prevent severe complications or increase morbidity and mortality in patients. Case Illustration: 15-year-old girl present with fever for one month prior to admission to the hospital, and are known to have congenital heart disease since the age of nine, with no prior symptoms. From the physical examination revealed signs of heart failure symptoms such as increased JVP, rales in bilateral pulmonary basal, and to-and-fro murmur on the left sternal border. On the ECG and x-ray of the thorax revealed left ventricular hypertrophy. Echocardiography reveals ruptured of sinus valsava, VSD SADC, aortic regurgitation and vegetation. A typical infectious endocarditis bacteria, streptococcus mitis / streptococcus oralis, was discovered in the blood culture examination. The patient was then given antibiotics based on the IE bacteria, as well as surgery to repair the valsava sinus rupture, close the VSD, repair the aortic valve, and evacuate the vegetation. y Conclusions : Diabetic cardiomyopathy is a complication occur in patients with a history of diabetes mellitus. Diabetes mellitus mainly cause hyperglycemia, systemic insulin resistance, and impaired insulin metabolic signaling in the cardiac that induce dysfunction of ventricular contractility and finally result in heart failure or cardiomyopathy. The clinical symptoms of this patient resembling acute coronary syndrome, but from the examinations no significant signs of acute coronary syndrome were found. CMR was the choice of examination despite the limited use in daily practice. Keywords: Diabetic cardiomyopathy, acute coronary syndrome, diabetic mellitus Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Rare Case Ruptured Sinus Valsava in Adolescence with Doubly Committed Subarterial Ventricular Septal Defect and Infective Endocarditis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Ruptured sinus valsava, VSD SADC, Infective Endocarditis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Role Of Echocardiography In The Diagnosis Of Cardiac Amyloidosis I. Setiadi1, E. Rudiktyo1 1 Department of Cardiology and Vascular Medicine, Universitas Indonesia/National Heart Center Harapan Kita, Jakarta, Indonesia Background: g Amyloidosis is a rare group of protein-misfolding illnesses that usually affect the heart due to the deposition of amyloid fibrils. Cardiac Amyloidosis (CA) has a low survival rate, limited access to diagnostic modalities (endomyocardial biopsy, CMR, or Bone scintigraphy), and is often underdiagnosed. Echocardiography plays a major role in the non-invasive evaluation of CA. Boldrini et all, introduced a diagnostic algorithm using an echo parameter called increased wall thickness (IWT Score) which has high specificity in diagnosing CA Case Illustration: Case Illustration: First patient was 54-year-old male presented to the clinic due to shortness of breath, during activity and ascites. Initial clinical workup revealed ascites, elevated JVP, low voltage ECG tracing, and highly elevated NT Pro BNP. Echocardiography revealed right and left ventricular hypertrophy, sparkling granulation, average GLS -4,7%, ejection fraction strain rate of 8.3, and apical sparring pattern, with an IWT score of 10, which has a specificity of 98%. Second patient was 54-year-old male presented to the outpatient clinic due to a chief complaint of shortness of breath, during activity and bilateral peripheral edema. Initial clinical workup revealed bilateral peripheral edema, elevated JVP, low voltage ECG tracing, and highly elevated NT Pro BNP. Echocardiography revealed right and left ventricular hypertrophy, average GLS -6,1% with ejection fraction strain rate of 3.4 and apical sparring pattern, with an IWT score of 10, which has a specificity of 98%. CMR imaging revealed global transmural late gadolinium enhancement suggestive ATTR Cardiac Amyloidosis. Conclusion: The use of echocardiography and IWT diagnostic scoring can show high specificity in diagnosing CA, avoid unnecessary tests, and limit the time to diagnose CA Keywords: Echocardiography, Diagnosis, Cardiac Amyloidosis, IWT Score Keywords: Echocardiography, Diagnosis, Cardiac Amyloidosis, IWT Score Background g Peripartum cardiomyopathy (PPCM) is a cardiomyopathy related to pregnancy. Patient with previously diagnosed PPCM has a risk of relapse during subsequent pregnancy (SSP), especially those with persistent LV dysfunction. There is limited data regarding SSP. Thus the aim of this study is to describe the pattern of presentation, course of disease and outcome of SSP. Case illustration A prospective study of fourteen cases of SSP in PPCM patients was conducted at Hasan Sadikin General Hospital Bandung from January 2015 to January 2023. Age distribution, symptoms, risk factors, medical management and pregnancy outcome were documented. Echocardiography was obtained before, at onset of SSP, during SSP, and after delivery. In this study 3/14 (21%) classified as NYHA II. Pre-eclampsia occurs in 1/14 (7%), and 1/14 (7%) had co-existing hypertension. Five unplanned pregnancies (35%) occurred in women with persistent LV dysfunction. One of the 5 patients experienced worsening of the symptoms, dyspnea and peripheral edema, and also decreased LVEF >5%. Nine well planned pregnancies (65%) had improved LV function (LVEF >50%). Out of the 9 patients, none experienced worsening of the symptoms, and 1 patient experienced reduce LVEF >5%. All patients gave birth to live children, two of them were born prematurely and one was intrauterine growth restriction (IUGR). Ten out of fourteen patients were followed up for a period of one year out of which 30% (3/10) patients have not fully recovered. There was no maternal mortality and rehospitalisation during follow up. Subvalvular Aortic Stenosis Caused By Structural Abnormality Subaortic Membrane A.I.S Putra1, N.M.A.W Sari2, L.O.S. Suastika2, I.B.R. Wibhuti2 1Residence at Faculty of Medicine Udayana University/Prof. dr Ngoerah Hospital, Denpasar, 2Faculty of Medicine Udayana University/Prof. dr Ngoerah Hospital, Denpasar, Indonesia, Echocardiography Division A.I.S Putra1, N.M.A.W Sari2, L.O.S. Suastika2, I.B.R. Wibhuti2 1Residence at Faculty of Medicine Udayana University/Prof. dr Ngoerah Hospital, Denpasar, 2Faculty of Medicine Udayana University/Prof. dr Ngoerah Hospital, Denpasar, Indonesia, Echocardiography Division Background : Diagnosis and management of aortic valve abnormalities are still challenging in the field of cardiology, especially in some cases such as congenital aortic abnormalities. Subaortic membrane causing subvalvular stenosis and LVOT obstruction may impair person’s quality of life. This case report fully discusses the challenging diagnosis to appropriate treatment of this population. g g g pp p p p Case Illustration : A 30-year-old female referred to central referral hospital for TEE procedure for definite diagnosis. The patient was consulted to cardiology for cardiac clearance for thyroid open biopsy procedure. Patient with unremarkable physical examination except diastolic murmur at left sternal border was noted. TTE confirmed the moderate aortic regurgitation with suspected bicuspid valve. Due to unclear structural and aortic mechanism from TTE, patient then conducted TEE. Specific findings of the aortic valve showed 3 cusps, short LCC structure causing moderate AR with an eccentric jet. A protruding structure seen at the LVOT (subaortic membrane) causing LVOT obstruction with moderate aortic stenosis (AV Vmax 3.44 m/s, AV meanPG 22.8 mmHg, AV maxPG 44.7 mmHg). Thus, the patient was diagnosed with rare subvalvular aortic stenosis caused by structural abnormality subaortic membrane. According to the guideline, specific treatment does not required for this patient, unless the severity of the abnormal valve increases upon observation. Resection of the sub-aortic obstruction, concomitant repair of the aortic valve, can ideally delay or prevent surgical aortic valve replacement. Conclusion: This case demonstrated the challenging diagnosis of patient with rare structural abnormality of the aorta. Patient with subvalvular aortic stenosis caused by subaortic membrane. TTE showed good result for early diagnosis, however TEE shown to be effective in diagnosing such patient. Treatment of choice was based on clinical presentation, abnormal valvular severity and team discussion would be useful for the patient Keywords: Subvalvular Aortic Stenosis, Subaortic Membrane, Transesophageal Echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Subsequent Pregnancy in Previously Diagnosed Peripartum Cardiomyopathy: A Case-Series P. Arianeu1, T.I. Dewi2, M. Hasan2, H.S. Subvalvular Aortic Stenosis Caused By Structural Abnormality Subaortic Membrane Prameswari2 General Practitioner, Hasan Sadikin General Hospital, Bandung, Indonesia1 Cardiologist, Hasan Sadikin General Hospital, Bandung, Indonesia2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Subsequent Pregnancy in Previously Diagnosed Peripartum Cardiomyopathy: A Case-Series Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A ISSN 2830 3105 / ISSN 2964 7304 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 P. Arianeu1, T.I. Dewi2, M. Hasan2, H.S. Prameswari2 General Practitioner, Hasan Sadikin General Hospital, Bandung, Indonesia1 Cardiologist, Hasan Sadikin General Hospital, Bandung, Indonesia2 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Regular Exercise Impact for Myocardial Infarction: For A Better Future S. Chandra1, A.A. Lukito1, R.A.P. Fernandes1, H. Hutabarat1, I. Sabrina1 Siloam Hospital Lippo Village1 Regular Exercise Impact for Myocardial Infarction: For A Better Future S. Chandra1, A.A. Lukito1, R.A.P. Fernandes1, H. Hutabarat1, I. Sabrina1 Siloam Hospital Lippo Village1 Background: Regular exercise training found to be associated with improvement of functional capacity and mortality reduction in post-MI. Exercise training beneficial effects in the process of cardiopulmonary rehabilitation and LV remodelling in the LV dysfunction patients after MI, and the greatest effects were achieved when exercise began at the post MI acute phase. Some studies showed improvement in functional capacity and even reduction in LV volumes in post MI patients. Case Illustration: A 47-year-old men presented with chest pain and shortness of breath. Patient had regularly exercise "pencak silat" 1 hour daily. On ECG examination there was ST elevation at anterior lead. Coronary angiography shown right dominant, two vessels disease with culprit lesion 100% at proximal LAD with thrombus burden, decided to stenting and thrombectomy proximal-mid LAD with good results. Echocardiography result was reduced LVEF 30%, RWMA (+). Laboratory result shown NT-proBNP 5811 ng/mL. After several days observation, the patient was discharged, and the last result of NT-proBNP was 655 ng/mL. After several days at home, patient exercise aerobic 30-45 minutes daily. After approximately 2 months since stenting, echocardiography was done, LVEF 50% with improving wall motion. NT-proBNP level was 243 ng/mL. Last week, echocardiography shown improving LVEF 63%, with slight abnormal wall motion, no scar was detected. NT-proBNP level was 105 ng/mL. Conclusion: Physical activity can play crucial role in reducing mortality of post-MI patients. To achieve maximal anti remodeling benefits clinically stable patients after uncomplicated MI should begin aerobis exercise training earlier after hospital discharge (form one week) and should continue training for up to 6 months. Type of physical activity is low-to moderate intensity with 150 minutes/week. In conclusion, this patient had recovery post MI by regularly exercise aerobics, taking medicine and had positive benefits from exercise. Keywords: Myocardial infarction, Exercise, Cardiac rehabilitation Effects of Exercise Training for Myocardial Infarction Keywords: Myocardial infarction, Exercise, Cardiac rehabilitation Effects of Exercise Training for Myocardial Infarction Conclusion The impact of SSP on cardiac function vary one to another. Low LVEF prior to SSP can lead to deterioration of symptoms and LVEF during pregnancy. Counseling should be given prior to SSP and close monitoring throughout the pregnancy is a necessity Keywords: Subsequent pregnancy, Peripartum Cardiomyopathy, Heart failure Indonesi Car Indonesian J Car pISSN: 2830-310 doi: 10.3 Regular Exercise Impact for Myocardial Infarction: For A Better Future S. Chandra1, A.A. Lukito1, R.A.P. Fernandes1, H. Hutabarat1, I. Sabrina1 Siloam Hospital Lippo Village1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background: g Organ injury is commonly present in patients with heart failure due to organ hypoperfusion. Ischemic gastritis is a rare disease with poor prognosis in patients with hemodynamic disruption. Low output state and repeated episodes of hypotension, can lead to organ perfusion impairment, one of them occurs in the gaster which is rarely reported. We present a case report of ischemic gastritis in a patient with heart failure reduced ejection fraction (HFrEF). Case illustration: A 56-year-old-male came to the ER with major complaint of nausea, vomiting after every time he eats, and epigastric pain since 3 days ago with a history of chronic epigastric pain for the last 4 years. The pain was sharp and non-radiating. No chest pain, palpitations, nor dyspnea. He has history of CAD with 2 stents at LAD, CHF, and AF NVR. Blood pressure was 87/49 mmHg and heart rate was 60 bpm irregular. ECG showed AF NVR, LBBB, and prolonged QTc (605 ms). Physical examination revealed soft, non-distended abdomen with epigastric pain. Laboratory studies showed Hemoglobin 15.8, Creatinine 3.08, NT-pro-BNP 2678, and Kalium 2.6. Left ventricular ejection fraction was 24%. He underwent gastroscopy and had several episodes of hypotension during the procedure that required 1000ml total of fluid resuscitation. Gastroscopy showed mucosal break at the distal esophagus, pale inflamed gastric mucosa with multiple erosions at the corpus and antrum. Biopsy was concluded as chronic active gastritis. He was given aggressive PPI therapy and gastric mucosa protector. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Uncommon Case of Ischemic Gastritis in Heart Failure Reduced Ejection Fraction Uncommon Case of Ischemic Gastritis in Heart Failure Reduced Ejection Fraction Background ardiorenal syndrome (CRS) is a dysfunction of heart and kidney that occur simultaneously and overlap, dev to a vicious cycle in the form of a decrease in interrelated and lethal function in both organs. Discussion CRS-type 5’s risk factors include sepsis, vasculitis and systemic conditions (SLE, amyloidosis and obesity) lead to cardiac and renal dysfunction. CRS type-5 mechanism include systemic inflammation, increased central venous pressure (CVP), activation of the renin-angiotensin-aldosterone system (RAAS), sympathetic neurohormonal system (SNS), oxidative stress, dysfunction vascular endothelium lead to cardiac and renal fibrosis resulting cardiac and renal insufficiency. Case Illustration A 20-year-old female, complained shortness of breath since 3 days and getting worse since 1 day ago. She also complained of swelling in both legs since 1 month. The patient has a history of SLE since 1.5 years. Physical examination: BP 90/60 mmHg, pulse 100 x/min regular, JVP neck PR+5 cmH2O, rhonchi +++/-++, all four extremities cold, edema -+/-+. EKG: sinus rhythm, HR 99x/min, LAD axis, P wave normal, PR interval 160 ms, QRS complex <0.12 sec, R/S ratio in V1 <1, RV5+SV1 >35 mm and T-inversion non- specific at II, III, aVF, with an effect of SR, 99 bpm, LVH. Laboratory examination: HGB 8.35, BUN 34.3, SC 1.34, eGFR 55.88, Procalcitonin 58.96, pH 6.94, pCO2 77.4, HCO3- 16.40. UL results: leukocytes 2+, nitrites (-), ketones (-), protein (2+), sedimentary leukocytes 48, bacteria ++. The patient was diagnosed with ADHF Profile C ec SLE related cardiomyopathy, cardiogenic shock, EF 40%, ACKD ec pre-renal on CKD suspect lupus nephritis, cardiorenal suspect type-5(CRS type-5)?, and severe metabolic acidosis(6.94). The patient was admitted to the ICCU. CARDIORENAL SYNDROME (CRS) TYPE-5 IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE): A RARE CASE REPORT G.S. Ambara1, K.S.S Darma2 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia2 G.S. Ambara1, K.S.S Darma2 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia2 G.S. Ambara1, K.S.S Darma2 1Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. dr. I. G. N. G. Ngoerah, Denpasar, Indonesia2 Conclusion : Patients with HFrEF, repeated episodes of hypotension, history of CAD and AF could experience ischemic gastritis due to chronic gastric hypoperfusion. Early diagnosis with gastroscopy and aggressive therapy is required to increase survival by preventing the development of hematemesis or gaster necrosis. Heart failure treatment also plays an important role in improving patient’s outcome. Keywords: Heart failure reduced ejection fraction, ischemia gastritis, hypoperfusion words: Heart failure reduced ejection fraction, ischemia gastritis, hypoperfusion Keywords: Heart failure reduced ejection fraction, ischemia gastritis, hypoperfusion Gastroscopy showed mucosal break at the distal esophagus (A), pale inflamed gastric mucosa with multiple erosions at the corpus (B, C) and antrum (D). stroscopy showed mucosal break at the distal esophagus (A), pale inflamed gastric mucosa with multiple erosions at the corpus (B, C) and antrum (D). Case Illustration A 62-year-old man presented to emergency department with chest discomfort and palpitation. He had a history of CHF CF II ec IHD/HHD presenting with inferior posterior lateral STEMI Killip II onset 3 hours seven days ago. He was referred to a bigger hospital to undergo primary coronary intervention. His coroangiography showed 80% occlusion in mid LAD, hence 1 DES was implanted in LAD. During his hospitalization, his ECG was back to baseline. He was discharged after sevendays, six hours before his second attack. g y Six hours after discharge, he came back to our ER with the same presentation. He was compos mentis with irregular heart rate of 178 bpm, tachypnea with oxygen saturation 98% using NRM 10 lpm. His blood pressure was normal. His ECG showed lateral STEMI with atrial fibrillation with rapid ventricular response. The patient was assessed with Reinfarction of lateral STEMI Killip II onset 1-2 hours, AFRVR, CHF CF II (Low EF) ec IHD/HHD. He was then referred again for further investigations. At the referal hospital, he was given Metoprolol for rate control. His pulse slowed down and during so, his ST segment came back to baseline. Background g ST elevation in an ECG is most often attributable to an episode of STEMI. Nevertheless, we should consider other possibilities which can evoke this ECG finding. Not Every ST Elevation is a STEMI: Tachycardia induced ST Elevation in a Post-STEMI Patient R. Ayuningtyas1, A. Ciptasari1, S. Inayasari1, A. Novitasari1, N.A. Novembri1, W. Pamungkas2, M.G. Suw L. Pribadi1, A. Muthmaina1 Rumah Sakit Pusat Angkatan Udara Suhardi Hardjolukito, Bantul, Indonesia1 Adisutjipto Air Force Base, Bantul, Indonesia2 R. Ayuningtyas1, A. Ciptasari1, S. Inayasari1, A. Novitasari1, N.A. Novembri1, W. Pamungkas2, M.G. Suwandi1, L. Pribadi1, A. Muthmaina1 Rumah Sakit Pusat Angkatan Udara Suhardi Hardjolukito, Bantul, Indonesia1 Adisutjipto Air Force Base, Bantul, Indonesia2 R. Ayuningtyas1, A. Ciptasari1, S. Inayasari1, A. Novitasari1, N.A. Novembri1, W. Pamungkas2, M.G. Suwandi1, L. Pribadi1, A. Muthmaina1 Rumah Sakit Pusat Angkatan Udara Suhardi Hardjolukito, Bantul, Indonesia1 Adisutjipto Air Force Base, Bantul, Indonesia2 Conclusion CRS type-5 is a complex condition that includes broad systemic disorders which there is progressive concomitant and overlapping cardiac and renal dysfunction that can result from systemic conditions such as SLE. Keywords: CRS, SLE, heart dysfunction, kidney dysfunction Indonesian Journal of Cardiology Conclusion When faced with ECG findings of ST elevation and AFRVR, moreover there's a history of CAD1VD not long before, the first diagnosis comes to mind must be a reinfarction. But before hastily make the decision, we should eliminate another differential diagnosis that can mimick those of STEMI reinfarction. The process leading to the diagnosis of STEMI relies on integration of multiple clinical, laboratory and ECG findings, assessment prompted by single cues must be avoided. Keywords: Atrial fibrillation, STEMI, Electrocardiogram Background g HIV infection has role in triggering and accelerates atherosclerosis. Atherosclerosis associated with HIV infection etiological factors and clinical features is different from atherosclerosis in non-HIV. Discussion Risk factors for MI in HIV patients include age, male sex, smoking, hypertension, co-infection and also ART especially protease inhibitor agents (PIs). Endothelial dysfunction secondary to the use of ART occurs by decreased of nitric oxide (NO) production, increased reactive oxygen species (ROS), impaired cholesterol, and accelerated foam cell formation. HIV infection increase the risk of CVD through the role of viremia, viral translocation, other co-infection which will cause hypercoagulation, endothelial dysfunction and myocardial fibrosis. ST-ELEVATION MYOCARDIAL INFARCTION (STEMI) INFEROPOSTERIOR + RIGHT VENTRICLE INFARCT IN A PATIENT WITH HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION: A CASE REPORT G.S. Ambara1, H. Wirawan1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Udayana University, RSUP Prof. d G. N. G. Ngoerah, Denpasar, Indonesia1 Case Illustration A 41-year-old man, complained of left chest pain since 5 hours ago. The patient has a history of HIV and taking ART. Patient still smoking 2 packs/day. Blood pressure was 145/93 mmHg, normal S1S2, regular, no murmurs. On the serial ECG showed sinus rhythm, HR 56 x/minute, normal axis, normal P wave, normal PR interval (160 ms), complex QRS <0.12 sec, R/S ratio in V1 < 1, RV5+SV1 <35 mm and ST elevation in leads II, III, aVF, V4R- V6R, V7-V9 back to the isoelectric line, with STEMI Inferoposterior impression + RV Infarct (susp autolysis?). Troponin I 342.8 and CKMB 132.2. The patient was diagnosed with STEMI Inferoposterior + RV Infarct Killip I (onset 5h, TIMI 1/14, EF BP 57%), Stage I hypertension, Dyslipidemia, Hypertriglyceridemia and stage I HIV infection. The patient undergoes conservative treatment and ARV FDC(TDF/3TC/EFV). Conclusion HIV infection creates a pro-inflammatory environment that stimulates the progression of atherosclerosis, which correlates with increased risk MI. However, despite control of viremia with the use of ART, continued low levels of HIV replication resulting in ongoing inflammatory processes and immune activation facilitate the progression of CVD. Keywords: Myocardial Infaction, HIV infection, inflammatory processes, immune activation Keywords: Myocardial Infaction, HIV infection, inflammatory processes, immune activation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. ECG in ER (STEMI Inferoposterior + RV Infarct) Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. ECG in ER (STEMI Inferoposterior + RV Infarct) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Surviving VSR: When to Operate and What Vascular Complications to Expect? M.J. Al-Farabi1, R.R. Juwita1, R. Bagus1, Y.H. Oktaviono1, Andrianto1, M.Y. Alsagaff1, Y.E. Sembiring2, O. Revianto2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Airlangga University, Surabaya, Indonesia1 Department of Thorax Surgery, Faculty of Medicine, Airlangga University, Surabaya, Indonesia2 Department of Thorax Surgery, Faculty of Medicine, Airlangga University, Surabaya, Indonesia2 Background: Ventricular septal rupture (VSR) is one of the most lethal mechanical complications following acute myocardial infarction (AMI) with 94% mortality in one month without surgery. Since it is a rare case, only a few consensuses are available to guide us in managing VSR, determining the surgical timing, and understanding potential complications. Interestingly, in June 2022 we have 6 cases of VSR admitted to Soetomo General Academic Hospital. Herein we describe insightful lessons from these case series. Case Illustration: 6 cases of VSR were admitted to our hospital in June 2022. All cases are late-presentation of non-reperfused Anterior STEMI coming from a rural hospital. All cases developed recurrent congestion and circulatory failure. 4 cases used IABP (Intra-Aortic Baloon Pump) to prevent cardiogenic shock and further development of VSR. 4 cases underwent surgery with a 75% survival rate. Two cases failed to undergo surgery due to irreversible shock despite multiple hemodynamic support including IABP, another case was due to no insurance. VSR case who was operated on the 21st day had the best clinical outcome and was discharged immediately 4 days after surgery with NYHA Class II, however, we need to deal with sepsis since the patient had a prolonged stay. Another case who was operated at the 14th day was having progressive residual VSR (0.6-1.2 cm) and develops circulatory failure which ended up in progressive kidney and liver damage. However, the patient survived and was discharged 14 days after surgery with NYHA Class IV. Cases that were operated on the 7th day have conflicting outcomes, the first case was successfully corrected with NYHA Class II outcome but with Acute Limb Ischemia in both of her legs, thus they were amputated. The second case failed to be corrected since no scar was available thus VSR hole cannot be stitched and the patient died immediately after. Other interesting findings are all survivors developed vascular complications. 3 cases developed blue toe syndrome and one case develop Acute Limb Ischemia. Conclusion: Ventricular septal rupture (VSR) timing of surgery may determine the success rate, survival, type of complications, and NYHA class outcome. Delaying surgery until the 21st day may provide the best outcome and survival yet it will be challenging for the cardiologist due to the risk of sepsis and recurrent congestion. Earlier surgery may be challenging for thoracic surgeons since the risk of stitching failure and residual VSR is high. We also need to be aware of vascular complications when managing VSR which may be caused by macro or microemboli which may be triggered by IABP, interseptal patch, or blood turbulence. Keywords: STEMI, Surgical Timing, VSR, Vascular Complication Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Total AV Block and Sinus Node Dysfunction in Acute Myocardial Infarction: To Pace or Not to Pace? A.C. Zulfiqar, S.B. Case Illustration: Raharjo Departement of Cardiology And Vascular Medicine, Faculty of Medicine Universitas Indonesia Management of Clinically Suspected Rheumatic Heart Disease in Rural Area V. Calosa1, M. Valeria2, N. Dewi3 General Practitioner, Bajawa Public Hospital, East Nusa Tenggara, Indonesia1 General Practitioner, Inerie Public Health Center, East Nusa Tenggara, Indonesia2 Department of Internal Medicine, Bajawa Public Hospital, East Nusa Tenggara, Indonesia3 V. Calosa1, M. Valeria2, N. Dewi3 General Practitioner, Bajawa Public Hospital, East Nusa Tenggara, Indonesia1 General Practitioner, Inerie Public Health Center, East Nusa Tenggara, Indonesia2 Department of Internal Medicine, Bajawa Public Hospital, East Nusa Tenggara, Indonesia3 Background: Rheumatic heart disease (RHD) is a sequela of acute rheumatic fever (ARF). Although rarely found nowadays, RHD persists among low to middle and income countries (LMIC), including Indonesia. Approximately 82% of deaths occurred in RHD-endemic locations. Adequate treatment when Streptococcal infection or ARF occurred may prevent RHD. It is often not achieved due to low socioeconomic status and limited access to health care. Most patients seek medical treatment once complications occur, therefore RHD has to be recognized earlier. Diagnosis requires echocardiogram and treatment requires surgical intervention, which are often unfeasible in rural area. We present two cases regarding management of clinically suspected RHD in rural area. Case Illustration: A thirty-five-year-old (‘Patient A’) and a twenty-nine-year-old woman (‘Patient B’) had complaints of dyspnea, orthopnea, fatigue, palpitation, cough, and epigastric pain. ‘Patient A’ had history of heart failure (HF) for 11 years, while ‘Patient B’ had history of dyspnea since first trimester of pregnancy. Both had frequent sore-throat in childhood. Physical examinations revealed tachycardia, tachypnea, diastolic murmur at cardiac apex, bibasilar crackles, and leg edema. The predominant cause of mitral stenosis (MS) is rheumatic fever and two-thirds of patients with rheumatic MS are female. Both patients were female and had diastolic murmur at cardiac apex which suggested MS. Clinically-suspected RHD was diagnosed based on history of frequent sore-throat in childhood, LMIC, RHD- endemic area, MS, HF, and AF. We treated the HF and AF. We administered aspirin for the MS and AF instead of vitamin-K-antagonist anticoagulant due to its unavailability. Patients had improved conditions, therefore were discharged and referred to cardiologist for further assessment and management. Conclusion: It is possible to diagnose clinically-suspected RHD, particularly in LMIC and RHD-endemic area with evident past medical history and clinical findings. Treatments are aimed to overcome RHD complications. Eventually, referral to cardiologist is mandatory. rds: rheumatic heart disease, mitral stenosis, acute heart failure, atrial fibrillation, rural area Figure 1. AV Block and Sinus Node Dysfunction in Acute Myocardial Infarction: To Pace or Not to Pace? q j Departement of Cardiology And Vascular Medicine, Faculty of Medicine Universitas Indonesia Background : Conduction disorder are frequent in cases of myocardial infarction. It represents 2-5% of the population with myocardial infarction and are three times more frequent in inferior myocardial infarction than in other region of infarction. Current guidelines recommend permanent pacemakers only for “persistent” infra-nodal or symptomatic second- or third-degree AV block or sinus node dysfunction, however determining persistent conduction disorder after MI are frequently challenging. ctive : To present a case of cardiac conduction disturbance in SA and AV node following inferior myocardi ction and its reversibility during hospitalization. Case Illustration : A 62 years old male came to the emergency room with typical ischemia chest pain since 1 days prior to index hospitalization and evidence of inferior ST-segment elevation. Reperfusion was done percutaneously with 1 drug eluting stent at the right coronary artery. During observation he developed complete AV block on the second day without evidence of re-infarction followed by Sinus node dysfunction on the eighth day of hospitalization. The conduction disorder showed improvement after the tenth days of observation, thus cardiac pacemaker plan was deferred. The patient was discharged from hospital with good haemodyamic status, and with good improvement of the ECG rhythm. g p y Conclusion : STEMI especially inferior MI is frequently associated with conduction disorders due to its region of vascularization. Most of the conduction disorders in myocardial infarction are reversible. Careful observation and analysis of the 12-lead ECG in patient with conduction disorders will lead to better patient survival. Keywords: Acute myocardial infarction TAVB SND pacemaker Management of Clinically Suspected Rheumatic Heart Disease in Rural Area Clinical findings, electrocardiogram, and chest x-ray of Patient A and Patient B Keywords: rheumatic heart disease, mitral stenosis, acute heart failure, atrial fibrillation, rural area Figure 1. Clinical findings, electrocardiogram, and chest x-ray of Patient A and Patient B Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Deep Dive Into Aminophylline as Precipitating Factor of Atrial Fibrillation : A Case Report N.A. Novebri1, A. Ciptasari1, R. Ayuningtyas1, S. Inayasari1, S. Novitasari1, M. Mohamad2, L. Pribadi1, A. Muthmaina1, M.G. Suwandi1 RSPAU HARDJOLUKITO1 PUSKESMAS BANGUNTAPAN 22 N.A. Novebri1, A. Ciptasari1, R. Ayuningtyas1, S. Inayasari1, S. Novitasari1, M. Mohamad2, L. Pribadi1, Muthmaina1, M.G. Suwandi1 RSPAU HARDJOLUKITO1 PUSKESMAS BANGUNTAPAN 22 To justify aVR, a single lead ST-elevation with high-priority reperfusion E.R. Utami1, I. Pratanu1, M. Budiarto1, Y.H. Oktaviono1 1Medical Faculty Airlangga University, Soetomo General Hospital, Surabaya, Indonesia N.A. Novebri1, A. Ciptasari1, R. Ayuningtyas1, S. Inayasari1, S. Novitasari1, M. Mohamad2, L. Pribadi1, A. Muthmaina1, M.G. Suwandi1 RSPAU HARDJOLUKITO1 PUSKESMAS BANGUNTAPAN 22 Background: Atrial fibrillation (AF) can occur from many causes. One of which less familiar to general practitioners are pulmonary drugs can also induce atrial fibrillation, including Aminophylline. Case Illustration: a 57-year-old woman came to the emergency department with chief complaints of a palpitation since one day before admission, she also complaint of short of breath, and severe sweating. No sign of acute decompensated heart failure (ADHF), including rales and pitting oedema. She had medical history of diabetes type II, hypertension, acute bronchitis, CHF NYHA III and AF. the patient was taking routine medicines novorapid 8 units, candesartan 16 mg, aminophylline 200 mg, amlodipine 10 mg. her temperature was normal, blood pressure was 159/91 and her pulse rate was 203 bpm with Irregular heartbeat. The initial ecg showed a rapid atrial fibrillation response. Upon laboratory examination, there was slightly increase in leukocytosis, blood sugar, and markers of kidney function. The patient was diagnosed with CHF fc II et causa HHD dd IHD, AFRVR, Hypertension, Diabetes type II, and Renal Insufficiency. Initially, the patient received a slow bolus injection of 500 mcg digoxin and was admitted to the ICU for further treatment. Discussion: Aminophylline is nonselective adenosine receptor antagonist. It has been demonstrated that aminophylline reduces the effective and functional refractory period of human and canine myocardium. This electrophysiological effect will dispose to a disparity in the recovery of atrial excitability that may initiate multiple reentrant circuits leading to AF. In this case, there is no sign of ADHF, the patient had history of aminophylline treatment. This condition could be the reason of atrial fibrillation in this patient. Conclusion: in this patient could be induced by many factors. Based on the risk and historical treatment, the used of aminophylline could be the reason of AF in this patient. Keywords: Atrial Fibrillation, Aminophylline, Diagnose, Electrocardiogram Figure 1. The initial ECG at Emergency Department with occurrence of AF RVR right after the patient came. Keywords: Atrial Fibrillation, Aminophylline, Diagnose, Electrocardiogram ure 1. The initial ECG at Emergency Department with occurrence of AF RVR right after the patient came. Figure 1. The initial ECG at Emergency Department with occurrence of AF RVR right after the patient came. Background g The 12-lead electrocardiogram (ECG) is a crucial tool in the diagnosis and risk stratification of acute coronary syndrome (ACS). Unlike other 11 leads, lead aVR has been long overlooked until recent years. However, recent investigations have shown that an analysis of ST-segment shift in lead aVR provides useful information on the coronary angiographic anatomy and risk stratification in ACS. Case illustration A 42 years old male patient with unremarkable medical history presented to the emergency room with typical chest pain one hour before initial assessment. His only risk factor is history of heavy smoker for 30 years. Initial electrocardiogram (ECG) showed ST elevation in aVR accompanied by diffuse ST depression. Sequentially, ECG evaluation showed QS pattern in inferior lead. Echocardiography evaluation displayed regional wall motion abnormality in inferior and posterior basal region. Intriguingly, cardiac marker result showed normal troponin I level (0.1 ng/mL). Persistent chest pain occurred despite optimal medical therapy. Urgent coronary angiography than was performed resulting in significant stenosis in distal left main, total occlusion in obtuse marginal (OM) 1, chronic total occlusion in left anterior descending and diffuse disease in right coronary artery. A drug eluting stent then landed in OM 1. Clinical evaluation indicated a substantial decline in chest pain visual analogue score from 7 to 3. Keywords: ST elevation, aVR, reperfusion. Conclusion Conclusion Cardiologists should pay more attention to the tracing of lead aVR when interpreting the 12-lead ECG in clinical practice. Keywords man’s Phenomenon – An Unusual Electrocardiogram Pattern in Atrial Fibrillation: A Case Report E.A.D Batubara1, G.P. Hapsari1, L.Widianto1, D.R. Fauzan1, R.U. Setiani2, Haryadi2, Yulianto2, I.A. Siregar2 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine University of Indonesia / National Cardiovascular Center Harapan Kita / University of Indonesia Academic Hospital, Jakarta, Indonesia; 2Cardiologist, Department of Cardiology and Vascular Medicine, Navy Hospital Mintohardjo, Jakarta, Indonesia Background: g The Ashman phenomenon is a rare electrocardiogram(ECG) finding. It is easily misinterpreted as a premature ventricular contraction (PVC) or ventricular tachycardia. The following presented case is a fascinating specimen of the ECG findings of a symptomatic Ashman phenomenon. Therefore, it is engaging because of the rarity of the case and also brings proper attention to the study of rhythm disorder. Case Illustration: A 53-year-old male complained of chest discomfort 4 hours prior, palpitation, and nausea. He had a history of hypertension, coronary artery disease, and atrial fibrillation. Coronary angiography two years ago revealed non- significant coronary stenosis with echocardiography concluded hypertrophic cardiomyopathy with normal LV systolic function (Ejection fraction 73%). Clinical examination showed an irregular heartbeat with grade I hypertension. ECG showed atrial fibrillation with aberrant conduction (the Ashman phenomenon), which has an RBBB pattern and occurred when a short R-R interval followed a long R-R interval. Laboratory examination and troponin were all within normal limits. The patient was treated with ramipril, atorvastatin, simarc and amiodarone but later was discontinued following a slow heart rate. Differentiating the Ashman phenomenon vs PVC could be challenging, especially if the heart rate is fast. Fisch criteria may help to discern between typical Ashman phenomenon and PVC, yet some references said that electrophysiological data might not confirm this criterion. Symptoms of the Ashman phenomenon are related to the underlying cardiac condition. No treatment is needed explicitly for this condition. Therapy mandates controlling heart rate or rhythm in atrial fibrillation. p Keywords Keywords Keywords: ST elevation, aVR, reperfusion. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Ashman’s Phenomenon – An Unusual Electrocardiogram Pattern in Atrial Fibrillation: A Case Report E.A.D Batubara1, G.P. Hapsari1, L.Widianto1, D.R. Fauzan1, R.U. Setiani2, Haryadi2, Yulianto2, I.A. Siregar2 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine University of Indonesia / National Cardiovascular Center Harapan Kita / University of Indonesia Academic Hospital, Jakarta, Indonesia; 2Cardiologist, Department of Cardiology and Vascular Medicine, Navy Hospital Mintohardjo, Jakarta, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Background g Amiodarone is reported to cause a wide continuum of serious lethal side effects. These include Amiodarone- induced pulmonary toxicity (AIPT), serious bleeding when combined with warfarin, and prolonged QT Interval which can manifest into Torsade de Pointes. Case Illustration We describe a 57-year-old patient who developed severe Anterior STEMI which manifested with recurrent ventricular tachycardia. On admission, the patient developed recurrent VT (Ventricular tachycardia) 3 times, and Atrial Fibrillation, hence Amiodarone was given repeatedly until reached 8.2 grams, and warfarin was given to prevent stroke. The patient underwent Percutaneous Coronary Intervention due to hemodynamic deterioration. We found total occlusion on its Left Main Coronary Artery and we stented its Left Main Coronary Artery to mid-LAD. Two days after PCI and 7th days after amiodarone first injection, the patient developed recurrent Torsade de Pointes with prolonged QT Interval (QTc 521), massive epistaxis, worsening bilateral infiltrates, and nodular opacities on chest imaging. We suspected the patient had developed multiple manifestations of amiodarone toxicity thus we stopped amiodarone infusion. We have excluded possible other causes of these manifestations since the patient has normal electrolyte levels, no sign of congestion on echocardiography, and negative bacterial culture. We successfully reversed Torsade de Pointes through cardioversion, prevent its recurrence using Lidocaine, stopped massive epistaxis using bellocq tampon, reversed INR level with Vitamin K, and reversed interstitial lung damage using the steroid for 1 month. 2 months after discharge, the sign of amiodarone toxicity has been reversed proven with normal QTc Interval and no sign of bilateral infiltrates on chest imaging. Successful Reversal of Triple Manifestation of Amiodarone Acute Toxicity in Patient with Total Occlusion of LMCA Complicated with Recurrent Ventricular Tachycardia. M.J. Al-Farabi1, Y. Azmi1, A. Kamardikan1, R. Mahardika1, M.Y. Alsagaff1, A. Bachtiar2, Y.H. Oktaviono1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Airlangga University, Surabaya, Indonesia1 Department of Pulmonology, Faculty of Medicine, Airlangga University, Surabaya, Indonesia2 Keywords: Amiodarone Toxicity, Epistaxis, STEMI, Torsade de Pointes Conclusion: In principle, the Ashman phenomenon is related to its underlying cardiac condition. However, it is necessary to describe and discuss the interpretation of this rare electrocardiographic finding because it should be differentiated from wide-complex ventricular arrhythmia. Keywords: The Ashman phenomenon, Atrial Fibrillation, Electrocardiogram Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Conclusion To our knowledge, this is the first case of triple Amiodarone toxicity manifestation on LM Occlusion. This case also gives insight into the early identification and aggressive reversal of amiodarone toxicity, which has proven to improve clinical outcomes. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Atrioventricular Node Ablation with Permanent His Bundle Pacing in Patient in Concomitant Atrial Fibrillation and Hypertrophic Obstructive Cardiomyopathy 1 1 2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Atrioventricular Node Ablation with Permanent His Bundle Pacing in Patient in Concomitant Atrial Fibrillation and Hypertrophic Obstructive Cardiomyopathy Node Ablation with Permanent His Bundle Pacing in Patient in Concomitant Atrial Fibrillation and Hypertrophic Obstructive Cardiomyopathy Rufiat1, Muzakkir1,2 1Faculty of Medicine, Hasanuddin University, Makassar, Indonesia; 2Cardiac Center Wahidin Sudirohusodo, Makassar, Indonesia Background: g Atrioventricular node ablation combined with His bundle pacing (HBP) are feasible, safe, and effective in patients with refractory atrial fibrillation. Among hypertrophic cardiomyopathy with left ventricular outflow tract obstruction patients, atrial fibrillation is a common sustained arrhythmia, primarily caused by left atrial dilatation and remodelling. Case Illustration : We report a case of successful atrioventricular node ablation combined with His bundle pacing in a 35-year-old male with slow atrial fibrillation and hypertrophic obstructive cardiomyopathy with left ventricular outflow tract obstruction. Echocardiographic examination showed maximum left ventricle outflow tract gradient of 64.61 mmHg, severe mitral valve regurgitation due to prolapse anterior mitral leaflet, normal systolic left ventricular function with ejection fraction of 68% and normal systolic right ventricular function prior to procedure. Maximum left ventricle outflow tract gradient was significantly reduced to 36.5 mmHg after procedure. Conclusion : Conclusion : His-Purkinje conduction system pacing combined with atrioventricular node ablation system pacing combined with atrioventricular node ablation (AVNA) is a feasible and safe with a high success rate in persistent atrial fibrillation patients with heart failure and ICD indication, such as hypertrophic obstructive cardiomyopathy with left ventricular outflow tract obstruction. AVNA with HBP in this condition is associated to improve symptoms and quality of life through decrease in left ventricle outflow tract obstruction. Keywords: atrioventricular node ablation, permanent his bundle pacing, atrial fibrillation, hypertrophic obstructive cardiomyopathy Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 A Rare Case of Total Atrioventricular Block in Children: what we can do in remote district hospitals? Eliza1, K.D. Krisnayanti1, G.A.R. Pertiwi1 Waikabubak Regional District Hospital, West Sumba, East Nusa Tenggara, Indonesia1 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration: A 62-year-old man presented to emergency department with chest discomfort and cold sweats. He was compos mentis with irregular heart rate of 109 bpm, tachypnea with oxygen saturation 98% using NK 3 lpm. His blood pressure was normal. He had a history of heart failure with low EF and also stroke, not routinely controlled or monitored. His ECG showed lateral STEMI with new onset RBBB. The patient was assessed with Lateral STEMI Killip II onset 3 hours, AFRVR, CHF cf II (Low EF) ec IHD/HHD, PETA, renal insufficiency. Due to worsening chest pain and relative contraindicative for fibrinolysis, he was then referred for primary coronary intervention. His coroangiography at the referal hospital showed 80% occlusion in mid LAD, hence 1 DES was implanted. Background: ac g ou d: ST-segment elevations in two or more contiguous leads or either new left bundle branch block (LBBB) or right bundle branch block (RBBB) on ECG in a patient with acute onset chest pain are diagnostic criteria for acute myocardial infarction and generally condone coronary angiography and cardiac catheterization. A Rare Case of Total Atrioventricular Block in Children: what we can do in remote district hospitals? Eliza1, K.D. Krisnayanti1, G.A.R. Pertiwi1 Waikabubak Regional District Hospital, West Sumba, East Nusa Tenggara, Indonesia1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Newly documented right bundle branch block in presence of lateral STEMI: A predictor of poor prognosis? Newly documented right bundle branch block in presence of lateral STEMI: A predictor of poor prognosis? R. Ayuningtyas1, N.A. Novembri1, A. Ciptasari1, S. Inayasari1, A. Novitasari1, W. Pamungkas2, M.G. Suwandi L. Pribadi1, A. Muthmaina1 Rumah Sakit Pusat Angkatan Udara Suhardi Hardjolukito, Bantul, Indonesia1 Adisutjipto Air Force Base, Bantul, Indonesia2 s1, N.A. Novembri1, A. Ciptasari1, S. Inayasari1, A. Novitasari1, W. Pamungkas2, M L. Pribadi1, A. Muthmaina1 Rumah Sakit Pusat Angkatan Udara Suhardi Hardjolukito, Bantul, Indonesia1 Adisutjipto Air Force Base, Bantul, Indonesia2 A Rare Case of Total Atrioventricular Block in Children: what we can do in remote district hospitals? Eliza1, K.D. Krisnayanti1, G.A.R. Pertiwi1 Waikabubak Regional District Hospital, West Sumba, East Nusa Tenggara, Indonesia1 A Rare Case of Total Atrioventricular Block in Children: what we can do in remote district hospitals? Eliza1, K.D. Krisnayanti1, G.A.R. Pertiwi1 Waikabubak Regional District Hospital, West Sumba, East Nusa Tenggara, Indonesia1 Background: Myocarditis complicated total atrioventricular block (TAVB) is rare in children. Of the varied presentations of myocarditis, total atrioventricular block (TAVB) was thought to be an isolated feature, with rapid and full recovery if it was diagnosed early and treated with emergency pacemaker implantation. Case Illustration: A 16-year-old male came to the emergency department with recurrent syncope of 10-minute episodes at home. One week ago, he developed a fever, epigastric pain and repeated vomiting. Physical examination were unremarkable except heart rate (HR) of 24 bpm. An electrocardiogram (ECG) showed a total AV Block with ventricular escape rhythm (atrial rate 100 bpm, ventricular rate 27 bpm). The chest x-ray and echocardiogram findings were normal. The LED level was increased (30 mm/h). Acute myocarditis with alternative of electrolyte imbalance were considered as underlying causes. Despite of maximal atropine sulfate doses, followed by a dopamine infusion, the ventricular rate relatively unchanged. So we start epinephrine infusion, combined with salbutamol, and ibuprofen as anti-inflammation. The HR was improved after third day with evolution to 2nd degree AV Block type I and the patient was safely discharged at the 8th day. A TAVB is ideally treated with a temporary pacemaker. However, as it was not available in our area, treatment was limited to the oral and IV medications described above. The fever and increased LED pointed to myocarditis as the underlying cause. The vomiting indicated the possibility of an electrolyte imbalance and this could not be definitely ruled out as we did not have the modality to check for electrolyte levels. y y y Conclusion: Myocarditis should be considered as a transient TAVB in children. In remote area, the core management include hemodynamic support and adequate anti-inflammation. Keywords: TAVB in childhood, symptomatic bradycardia, syncope, myocarditis acute Total AV Block Echocardiogram Keywords: TAVB in childhood, symptomatic bradycardia, syncope, myocarditis acute Total AV Block Echocardiogram Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Newly documented right bundle branch block in presence of lateral STEMI: A predictor of poor prognosis? Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Conclusion: New-onset RBBB in a patient with typical STEMI and ischemic symptoms should raise suspicion of critical LAD coronary occlusion. It is increasingly being recognized as one of ECG patterns associated with worse outcome. It is important to minimize the delay in performing reperfusion therapy as they can improve outcome and survival. Keywords: STEMI, RBBB, Acute Coronary Syndrome New-onset RBBB in a patient with typical STEMI and ischemic symptoms should raise suspicion of critical LAD coronary occlusion. It is increasingly being recognized as one of ECG patterns associated with worse outcome. It is important to minimize the delay in performing reperfusion therapy as they can improve outcome and survival. Keywords: STEMI, RBBB, Acute Coronary Syndrome Clinical Outcome of Post Right Ventricular Outflow Tract Stenting in the Severe Cyanosis Tetralogy of Fallot Case With Down Syndrome I.S. Pradisa1, M. Pramudyo1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Padjadjaran, Bandung, Indonesia1 Clinical Outcome of Post Right Ventricular Outflow Tract Stenting in the Severe Cyanosis Tetralogy of Fallot Case With Down Syndrome I S Pradisa1 M Pramudyo1 Clinical Outcome of Post Right Ventricular Outflow Tract Stenting in the Severe Cyanosis Tetralogy of Fallot Case With Down Syndrome I.S. Pradisa1, M. Pramudyo1 Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Padjadjaran, Bandung, Indonesia1 Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Abstract Background. Surgical repair of Tetralogy of Fallot (TOF) in early infancy has become advances. A less invasive strategy such as Right Ventricular Outflow Tract (RVOT) stenting has many potential benefits in term of stabilizering the clinical condition. However in a critically condition with severe cyanosis, surgical repair is expected to carry a high morbidity and mortality risk. ToF represents 75% in cyanotic congenital heart disease and infrequent association with Down Syndrome (DS). A good corrective surgery of TOF with DS patients can prolong their life. Thus, in this case report, we would like to propose clinical outcome of post RVOT stenting in the TOF patient with DS. Abstract Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Management of Tuberculous Pericarditis with Cardiac Tamponade in Remote Area: A case report E. Zuhri1, W.S. Yahya1 Teluk Bintuni Hospital, Papua Barat, Indonesia1 Abstract Abstract Background. Surgical repair of Tetralogy of Fallot (TOF) in early infancy has become advances. A less invasive strategy such as Right Ventricular Outflow Tract (RVOT) stenting has many potential benefits in term of stabilizering the clinical condition. However in a critically condition with severe cyanosis, surgical repair is expected to carry a high morbidity and mortality risk. ToF represents 75% in cyanotic congenital heart disease and infrequent association with Down Syndrome (DS). A good corrective surgery of TOF with DS patients can prolong their life. Thus, in this case report, we would like to propose clinical outcome of post RVOT stenting in the TOF patient with DS. Case Illustration. A 6-month-old girl presented to Hasan Sadikin Hospital due to worsening shortness of breath and progressively cyanosis 30 minutes prior to arrival. Her weighed 5.5kg and length 48cm. She was saturating 65% and had severe cyanosis. The child was intubated on ventilator. On physical examination, she has short stature, flattened facies with depressed nasal bridge, almond-shaped eyes, all the symptoms refers to DS. Echocardiography was performed and showed TOF with subinfundibular stenosis. She was brought to the catheterization laboratory for RVOT stenting. In the middle of procedure, she has severe bradycardia and cardiac arrest, fortunately she was Return of Spontaneous Circulation (ROSC) without any complication and show clinical improvement without cyanosis.. Conclusion. TOF with DS has high risk of severe cyanosis and can be increase morbidity and mortality risk in surgical repair. RVOT stenting is relatively safe for TOF case with DS without any complication. Keywords. tetralogy of fallot, right ventricular outflow tract, down syndrome, clinical outcome Keywords: tetralogy of fallot, right ventricular outflow tract, down syndrome, clinical outcome Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:supp pISSN: 2830-3105 / eISSN: 2964-7 doi: 10.30701/ijc.1537 Management of Tuberculous Pericarditis with Cardiac Tamponade in Remote Area: A case report E. Zuhri1, W.S. Yahya1 Teluk Bintuni Hospital, Papua Barat, Indonesia1 a case of 20 years old man with acute pericarditis A. Mahadewa1 Departemen Kardiologi FK Unhas1 BACKGROUND Acute pericarditis is a syndrome of inflammation of the pericardium with or without pericardial effusion. The etiology of acute pericarditis consists of both infectious and non-infectious consequences. Infection can be caused by viruses, tuberculosis, and pyogenic bacteria. While non-infectious can come from post myocardial infarction, uremia, malignancy, radiation-induced, connective tissue disease, and drugs. The characteristics of pericarditis consist of 3 stages, namely 1) local vasodilatation through fluid transudation into the pericardial cavity; 2) increased vascular permeability causing protein leakage into the pericardial cavity; 3) leukocyte exudation, starting from neutophiles then followed by mononuclear cells. The most common symptoms are chest pain and fever. Chest pain may be severe and is usually localized to the retrosternal and left precordial areas. Dyspnea also usually occurs but is not affected by activity and may result from the patient's fear of taking a breath due to pleuritic pain. The clinical diagnosis can be established by 2 out of 4 criteria, namely typical sharp chest pain, pericardial friction rub, diffuse ST segment elevation on EKG examination, and pericardial effusion Management of Tuberculous Pericarditis with Cardiac Tamponade in Remote Area: A case report E. Zuhri1, W.S. Yahya1 Teluk Bintuni Hospital, Papua Barat, Indonesia1 Background. Tuberculosis accounts for up to 4% of acute pericarditis and 7% of cardiac tamponade cases. Cardiac tamponade is a life-threatening condition. Emergent pericardiocentesis should be performed. But, sometimes in remote area, the standard equipments to perform pericardiocentesis are not available. q p p p Case Illustration. A 66-year-old man presented to the emergency room with dyspnea and hypotension. Chest X- rays showed global cardiomegaly with a rounded heart shape. The echocardiogram showed a large pericardial effusion with sign of cardiac tamponade. Because of limited equipment, an emergent pericardiocentesis was performed using double lumen central venous catheter 7F through apical approach. There is no complication during and post procedure. The pericardial fluid was seroxantochrome and the nested real time-polymerase chain reaction (PCR) of pericardial fluid examination showed tuberculosis infection. Cytology revealed no malignant cell. The serology for HIV was negative. Anti-tuberculosis regimen in combination with corticosteroid and colchicine was then started. The patient’s condition improved and was discharged on the tenth day. After 6 months of therapy, no signs of constrictive pericarditis was found. Conclusion. Key management of tuberculous pericarditis is the administration of anti-tuberculosis regimen and anti-inflammatory drugs to prevent further complications such as constrictive pericarditis. If tuberculous pericarditis is accompanied by cardiac tamponade, emergent pericardiocentesis should be performed even with limited equipment. Keywords: Cardiac tamponade, pericarditis, pericardiocentesis, tuberculosis Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 CASE ILLUSTRATION Acute pericarditis is a form of pericardial inflamatory disease with idiopathic or viral as a common cause. We reported a case of 20 years old man with acute pericarditis of unknown cause. The patient has appearrance of clinical and typical electrocardiography finding for acute pericarditis. Inhospital management using ibuprofen and colchicine with improvement therapeutic result. Pasien had discharge 5 days later and constantly receiving colchicine to avoid further complication CONCLUSION y CONCLUSION It has been reported a male patient aged 20 years with complaints of painacute chest, pleuritic and aggravated by lying down and relieved bysitting position. Preceded by an episode of fever 5 days earlier which resolved withitself. Electrocardiographic examination showed ST segment elevatio diffuse and diffusesegment depression. Laboratory tests showleukocytosis, increased ESR and increased cardiac enzymes. Patient later diagnosed as acute pericarditis. Keywords: Keywords: Indonesian Jour Cardiology Indonesian J Cardiol 2023:4 pISSN: 2830-3105 / eISSN: doi: 10.30701/ijc.15 Catheter-directed Thrombolysis in Acute Lower Limb Ischemia Rutherford II B P.D.K. Devi1, L.H. Andira1, J.N.E. Putranto1 Faculty of Medicine Universitas Airlangga / RSUD Dr. Soetomo1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Background: Catheter-directed thrombolysis (CDT) is the treatment of choice for patients with relatively mild acute limb ischemia (ALI) (Rutherford categories I and II A) with no contraindications to thrombolytic therapy. However, patients with severe acute limb ischemia (Rutherford category IIb) also need emergent revascularization. CDT should be considered, nonetheless, if the relative risks compared with the primary operation are favorable. Case Illustration: A 66 years old female comes with progressive pain on the right lower extremity, concomitant with pulselessness, pallor, and mild muscle weakness. Physical examination showed pulseless on the right distal femoral, popliteal, tibialis anterior, tibialis posterior, and dorsalis pedis arteries. Duplex ultrasonography showed irregularity of arterial wall along right leg arteries, partially color-coded at mid superficial femoral artery (SFA), no color-coded at distal SFA, popliteal artery, anterior tibialis artery, proximal tibialis artery, and distal peroneal artery. Angiography revealed total occlusion at distal SFA, then the patient underwent percutaneous catheter- directed intra-arterial thrombolysis. Re-angiography evaluation showed flow resolution at distal SFA, consistent with clinical improvement. Conclusion: Thrombolysis is the treatment of choice for ALI patients with categories I and II A, whereas patients with progressive ischemia or category II B are indicative of surgical revascularization. However, some studies show Rutherford II B ischemia can be safely treated with endovascular intervention, which may be an attractive option for patients at high risk for surgery due to prohibitive medical risk or anatomic factors. eywords: Acute limb ischemia, Rutherford, catheter-directed thrombolysis, endovascular intervention Keywords: Acute limb ischemia, Rutherford, catheter-directed thrombolysis, endovascular intervention Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 SATISFYING OUTCOME IN PATIENT WITH LAUBRY-PEZZI SYNDROME WITH INFECTIVE ENDOCARDITIS FOLLOWING CARDIAC SURGERY: LESSON LEARNED IN CASE OF 22 MALE YEARS OLD I. Defriany1, Y. Patimang2, A.A.U. Armin2, M.N. Mallapasi3, J. Kusumanegara3 Cardiovascular Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia1 Pediatric Division, Cardiovascular Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia2 Cardiovascular and Thoracic Surgery Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia3 I. Defriany1, Y. Patimang2, A.A.U. Armin2, M.N. Mallapasi3, J. Kusumanegara3 Cardiovascular Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia1 Pediatric Division, Cardiovascular Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia2 Cardiovascular and Thoracic Surgery Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia3 I. Defriany1, Y. Patimang2, A.A.U. Armin2, M.N. Mallapasi3, J. Kusumanegara3 Cardiovascular Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia1 Pediatric Division, Cardiovascular Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia2 Cardiovascular and Thoracic Surgery Department, Faculty of Medicine, Hasanuddin University-Hasanuddin University Hospital, Makassar, Indonesia3 Background: Laubry-Pezzi syndrome combines a ventricular septal defect (VSD) and aortic regurgitation (AR) due to coronary cuspis prolapse. Inadequate correction can exacerbate existing aortic regurgitation, even triggering complications of infective endocarditis. gg g p Case illustration: A 22-year-old man came to the emergency room with complaints of fever three weeks ago, which did not improve with antipyretics. This complaint has been accompanied by coughing and shortness of breath during activities since 1-3 months ago. There was no history of sore throat, visits to the dentist, or history of blues as a baby before. The hemodynamic state was relatively stable. However, a body temperature of 38.20C, a harsh holosystolic murmur of degree III/VI at the lower left sternal border, a grade III/IV diastolic murmur at (Intercostal Space) and apex, and pathognomonic signs of aortic regurgitation was obtained. This clinical condition is supported by cardiomegaly with an R-L shunt on chest X-ray examination, leukocytosis, and neutrophilia. Echocardiographic examination showed a subaortic VSD, severe AR, vegetation on the aortic valve that exceeded 10 mm, left ventricular dilatation, and mild pericardial effusion but preserved systolic function in both ventricles. The diagnosis of Laubry-Pezzi Syndrome with infective endocarditis was made in this patient and followed by VSD Closure, Aortic Valve Replacement (AVR), and vegetation evacuation. The patient's condition gradually improved during treatment, and he was subsequently discharged in a stable hemodynamic state. In this case, a subaortic VSD is suspected, which can cause a venturi effect and lead to AR. This venture effect may also precipitate infective endocarditis in these patients. Hypertrophic Cardiomyopathy: a Case Report R.S. Ayuningtyas1, R. Wahyuni, Michael1, E. Mariska1, N. Primaditta1, N. Marliyanti1, M. Jufianti1, S. Hidayatullah1, P.P. Dewi2 1General Practitioner, International Central Army Hospital Gatot Soebroto, Jakarta, Indonesia; 2Cardiologist, International Central Army Hospital Gatot Soebroto, Jakarta, Indonesia Background: Hypertrophic cardiomyopathy (HCM) is a common inheritable cardiac disease defined by presence of increased left ventricular (LV) wall thickness >15mm in adults, often accompanied by left ventricular outflow tract obstruction (LVOTO), and malignant arrhythmia leading to sudden cardiac death (SCD). Annual incidence of SCD in adult patients with HCM is approximately 1-2%. p pp y Case Illustration: A 68-year-old female was referred to a cardiologist with complaints of shortness of breath with marked limitation of physical activity and occasional palpitation. Her blood pressure was 127/87 mmHg, pulse rate 67 bpm regular, respiratory rate 20 times/minute and temperature 36,6 ºC. Mid-systolic heart murmur at 6th left intercostal space was also present. ECG findings were not specific with sinus rhythm, normal axis, no pathological Q wave nor ST-T changes. CT coronary angiogram showed non-significant 10% stenosis in LAD, Calcium score was 111. Her NT-proBNP level was 1528 pg/mL. p pg Transthoracic echocardiogram (TTE) showed concentric left ventricular hypertrophy (LVH) with interventricular septal thickness in diastole (IVSD) of 25mm, systolic anterior motion (SAM) with moderate regurgitation of mitral valve, and Max LVOT gradient of 87 mmHg. Patient was treated with Bisoprolol 2,5mg SID, Furosemide 20mg SID, Spironolactone 25mg SID, Candesartan 8mg SID, Acetylsalicylic Acid 80mg SID and Atorvastatin 20mg SID. Patient’s shortness of breath was alleviated after given therapy, but she remained limited to minimal physical activity.Presence of LVOT gradient >=50mmHg and refractory symptoms despite medication indicates consideration for septal reduction therapy (SRT). Patient was offered alcohol septal ablation (ASA), but patient declined interventive therapy for now. 24-hour holter monitoring is scheduled to find presence of arrhythmia to determine 5-year risk of SCD and indication for prophylactic ICD. y y Conclusions: HCM is closely related to sudden cardiac death, therefore risk stratification is important to determine patient’s plan of therapy. Keywords: hypertrophic cardiomyopathy, left ventricular outflow tract obstruction, HCM, LVOTO rophic cardiomyopathy, left ventricular outflow tract obstruction, HCM, LV Echocardiogram showing Max LVOT Pressure Gradient of 87 mmHg Echocardiogram showing Max LVOT Pressure Gradient of 87 mmHg Hybrid Endovascular Procedure in Chronic Aortic Dissection: First Time Experience outside of Central Indonesia Endovascular Procedure in Chronic Aortic Dissection: First Time Experience outside of Central Indonesia A.M. Tahir1, I. Mappangara1, A.H. Nashar1, A.A.U. Armyn1, J. Kusumanegara2, A. Adil3 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, University of Hasanuddin 2Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Faculty of Medicine, University of Hasanuddin 3Division of Cardiovascular Anesthesia, Department of Anesthesiology and Intensive Care, Faculty of Medicine, University of Hasanuddin Background: Aortic arch repair and great vessel are complex procedures and carries a high risk of complications as well as mortality. Type A Aortic dissection is a life- threatening condition that occurs in 60% of cases of aortic dissection. There is no general consensus in classifying ‘fitness for for open arch surgery’, TEVAR or hybrid procedures, depends on the surgeon’s individual experience only. Hybrid endovascular procedures combines traditional open surgery with stent placement (Thoracic Endovascular Aneurysm Repair/TEVAR). p g y p y p Case Illustration: We reported a 58-year-old male patient presented with progressive shortness of breath due to a large aneurysm of distal aortic arch and descending aorta (max diameter 80 mm). The patient diagnosed with Chronic Stanford A debakey type I Aortic Dissection underwent total arch debranching and concomitant on-pump surgery. Operations were performed under deep hypothermic circulatory arrest (DHCA) and selective antegrade cerebral perfusion (SACP). During antegrade cerebral and lower body (distal) perfusion, the ascending aorta was resected and an ‘elephant trunk procedure’ with a 22-mm prosthetic graft (Gelweave™). The aortic valve was competent. Hybrid endovascular procedures (TEVAR) was performed during the same operation as the de- branching procedure. TEVAR of the transverse arch (after artery innominate) using one stent graft 28(24) x 150 mm (Seal®). TEVAR coverage extended aortic descendens, was successfully performed retrogradely via the femoral artery under fluoroscopy with good perfusion below followed by completion angiography. Discussion: The optimal treatment of Chronic Stanford A debakey type I Aortic Dissection remains unclear. Strategies to aortic arch treatment is the hybrid aortic repair, which combine open surgical (total arch debranching) and endovascular techniques simultaneously in a single single-stage procedure in high-risk patients. Hybrid and total endovascular approaches show promising results but are associated with the risk of complication in early postoperative period and the risk of multiple in the late postoperative period, therefore careful attention about this fact must be required during follow up. Keywords: Chronic Stanford A debakey type I Aortic Dissection, Hybrid Endovascular Procedures, Total Arch Debranching, Thoracic Endovascular Aneurysm Repair. SATISFYING OUTCOME IN PATIENT WITH LAUBRY-PEZZI SYNDROME WITH INFECTIVE ENDOCARDITIS FOLLOWING CARDIAC SURGERY: LESSON LEARNED IN CASE OF 22 MALE YEARS OLD Based on the size of the vegetation and severe AR on echocardiographic examination, vegetation evacuation is indicated with recommendation class IIa. Conclusion: Surgical correction with VSD closure, AVR, and vegetation evacuation can provide good outcomes in patients with Laubry-Pezzi Syndrome with infective endocarditis. Keywords: Aortic Regurgitation Infective Endocaritis Laubry Pezzi Syndrome Surgery Ventricular Septal words: Aortic Regurgitation, Infective Endocaritis, Laubry-Pezzi Syndrome, Surgery, Ventricular Septal ct Keywords: Aortic Regurgitation, Infective Endocaritis, Laubry-Pezzi Syndrome, Surgery, Ventricular Septal Defect Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-73 doi: 10.30701/ijc.1537 Hybrid Endovascular Procedure in Chronic Aortic Dissection: First Time Experience outside of Central Indonesia Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Acute Coronary Syndrome, STEMI inferior, RV Infarction Hybrid Endovascular Procedure in Chronic Aortic Dissection: First Time Experience outside of Central Indonesia Conclusion: Open arch surgery continues to be the current “gold standard” for treatment of aortic pathology, and currently other lesser invasive procedures such as hybrid endovascular procedures (including debranching techniques) was performed. Target treatment strategies for Aortic Dissection are treat the rupture, reverse malperfussion and prevent acute aneurysm formation. The First Hybrid Endovascular Procedure was successfully performed on a single-stage procedure in our centre. Keywords: Chronic Stanford A debakey type I Aortic Dissection, Hybrid Endovascular Procedures, Total Arch Debranching, Thoracic Endovascular Aneurysm Repair. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 3D RECONSTRUCTION Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 3D RECONSTRUCTION 3D RECONSTRUCTION A Rare Case: RV Infarction due STEMI Inferior R. Carolin1, M.S. Queen1, A.S. Shahab1, A.A. Indah2, S. Firdausia1, A.T. Faizal3 p logist, Department of Cardiology and Vascular Medicine, Raden Said Sukanto Hospital, Jakarta3 Cardiologist, Department of Cardiology and Vascular Medicine, Raden Said Sukanto Hospital, Jakarta3 Background:STEMI is an indicator of total coronary artery occlusion. Inferior STEMI is one of the most common types of ACS with 40% of the total incidence. This situation requires myocardial reperfusion as soon as possible. This case report describes inferior STEMI with a wide variety of clinical manifestations before and after treatment. p y Case Illustration:A 52th y.o. man presented with typical chestpain, diaphoresis, vomit since 3hours before hospitalization. During triage, the ECG finding was ST elevation II, III, aVF, 3rd degree AV Block. General examination revealed GCS E3M5V3, BP 60/40mmHg, HR 35x/m, CK-MB 26U/L, and diagnosed by inferior STEMI, cardiogenic shock, and treated with SA 1mg, Dopamin 5mcg, Norepinephrine 12mcg, Clopidogrel 300mg, ASA 160mg, Atorvastatin 40mg/24h, and plan transferred to have PCI. ECG during thrombolytics showed ST elevation II, III, aVF, atrial fibrillation, 1st degree AV Block, pathological Q waves II,III,aVF. After thrombolytic patient transferred for PCI. After PCI ECG showed 1st degree AV Block, pathological Q wave III,aVF, inverted T waves II,III,aVF. Inferior STEMI is diagnosed if there is >1-2mm of ST elevation in two contiguous leads (II, III, aVF) with ischemic chest pain. In this case, the ECG shows ST elevation in leads II,III,aVF, with ST elevation in lead III higher than lead II. Right ventricular infarction is observed in patients with inferior wall myocardial infarction. Assessment of right precordial lead is particularly helpful for diagnosis of RV infarction. ST elevation >1.00mm in lead V4R and V6R is highly suggestive of proximal RCA occlusion and RV infarction. Conclusion: In the case of RV infarction, right-sided ECG and posterior ECG needs to be done in order to confirm the diagnosis. For the treatment, PCI should be done in under 12 hours after the onset in order to maximize the reperfusion of heart and to increase the life expectancy. Keywords: Acute Coronary Syndrome, STEMI inferior, RV Infarction Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology The Essential of Serial Electrocardiography in Patient with Chest Pain: A Case Report R. Rasaki1, M. Fitra1, T. Heriansyah1, M. Muqsith1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia. Background: Electrocardiography (ECG) is the established diagnostic tool in the assessment of patients with typical chest pain. The ECG characteristic in the setting of NSTE-ACS includes ST-segment depression, transient ST-segment elevation, T-wave changes, or normal ECG. Guidelines recommend serial ECG evaluation in the routine management of NSTE-ACS. In clinical practice, serial ECG may also identify dynamic T-wave changes. Case Illustration: We reported a 44-year-old man presented to the emergency department with a chief complaint of chest pain 4 hours before admission. His cardiovascular risk factor was smoking. Physical examination was unremarkable. Initial ECG showed normal sinus rhythm without ST-T abnormalities and initial laboratory findings showed normal Troponin-T level. We managed him with double anti-platelet, anticoagulant, statin, and isosorbide dinitrate. After 3 hours of observation, we performed serial ECG showing a normal sinus rhythm with inverted T- waves in leads V2-V6. Then we performed a serial Troponin T testing showed a significant increase in Troponin-T level. We planned to perform an early invasive strategy for the management of high-risk NSTEMI. Several hours later, ECG showed deeper inverted T-waves in leads V2-V6 plus inverted T-waves in leads I and aVL. Then, we performed coronary angiography revealing critical stenosis in proximal LAD, stenosis of 90% in distal LCx, and stenosis of 60% in PDA. It was managed by percutaneous coronary intervention using DES implantation in proximal LAD. Conclusion: Electrocardiography is a feasible and useful tool to evaluate patients with chest pain. It provides an important tool for the diagnosis and risk stratification of patients with acute coronary syndrome. Furthermore, serial ECG and troponin-T testing are essential to determine the best management for patients with NSTE-ACS. Keywords: Chest Pain, Serial Electrocardiography, Troponin-T Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. Serial electrocardiography tracing (A). Sinus rhythm with a heart rate of 97 beats per minute and incomplete right bundle branch block without ST-T abnormality, (B). Keywords: Pulmonary Venous Aneurysm CT-Scan Right Superior Pulmonary Venous Aneurism: The Role of Computed Tomography to Reveal It – A Rare Case A. Achmad1, H.A.P. Lubis2, S. Tarigan3, I.A. Pratiwi4, A.C. Lubis5, C.A. Andra6, K Indonesian Journal of Cardiology After 3 hours of observation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. Serial electrocardiography tracing (A). Sinus rhythm with a heart rate of 97 beats per minute and incomplete right bundle branch block without ST-T abnormality (B) After 3 hours of observation Figure 1. Serial electrocardiography tracing (A). Sinus rhythm with a heart rate of 97 beats per minute and incomplete right bundle branch block without ST-T abnormality, (B). After 3 hours of observation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Right Superior Pulmonary Venous Aneurism: The Role of Computed Tomography to Reveal It – A Rare Case Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A. Achmad1, H.A.P. Lubis2, S. Tarigan3, I.A. Pratiwi4, A.C. Lubis5, C.A. Andra6, K.K. Ilyas7 A. Achmad1, H.A.P. Lubis2, S. Tarigan3, I.A. Pratiwi4, A.C. Lubis5, C.A. Andra6, K.K. Ilyas7 1,3Cardiology Resident at Cardiac Center H. Adam Malik Hospital, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2,4,5,6,7 Cardiologist at Cardiac Center H. Adam Malik Hospital, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia. Background Pulmonary venous aneurism (PVA) is a rare vascular anomaly characterized by local aneurysmal dilatation of the pulmonary vein. Contrast CT Scan can help diagnose of a rare case venous anomaly such as PVA without having to performed invasive diagnostic test. PVA may present as a pulmonary nodule or mediastinal mass, and often deviced from incidental findings. The etiology of PVA can be an acquired or a congenital origin. Patients often asymptomatic, while some patients could have hemoptysis, dyspnea, chest tightness or cerebral thromboembolism. The cause of PVA may be associated with mitral regurgitation and increase left atrial pressure. Proper diagnosis is crucial to avoid unnecessary testing or surgical procedures. Case illustration: A 62-years-old woman with atypical chest pain with history of hypertension. The physical examination was unremarkable. ECG showed sinus rhythm. Chest X-Ray showed a solid lesion on the right lower region of the lung, mild cardiomegaly and aortic dilatation. A transthorachic echocardiography showed preserved LV systolic function, TR mild, normal RV function and no sign of pulmonary hypertension. Contrast chest CT scan revealed right superior pulmonary venous aneurysm with ostium diameter 15.3mm and aneurism diameter 49.6 x 41.3 mm. Aneurysm defined as a 50% increase from normal size of PVA. We found large PVA with more than 50% enlargement from contrast CT. Conclusion: Pulmonary venous aneurism (PVA) is a rare vascular anomaly characterized by local aneurysmal dilatation of the pulmonary vein. CT Scan can help diagnose PVA without having to performed invasive diagnostic test. Our case report showed PVA with no sign of mitral regurgitation and we suggest surgery procedure if patient develop worsening of clinical symptoms. Keywords: Pulmonary Venous Aneurysm CT-Scan Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Intra-aortic Balloon Pump For Haemodynamic Stabilization in Acute Myocardial Infarction Complicated by Cardiogenic Shock after Primary Percutaneous Coronary Intervention : A Case Report L.H. Hutapea1, K.K. Ilyas2, A. Sitepu3, F. Habib4, C.A. Andra5 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia;2,3,4,5Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Intra-aortic Balloon Pump For Haemodynamic Stabilization in Acute Myocardial Infarction Complicated by Cardiogenic Shock after Primary Percutaneous Coronary Intervention : A Case Report L.H. Hutapea1, K.K. Ilyas2, A. Sitepu3, F. Habib4, C.A. Andra5 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia;2,3,4,5Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia Intra-aortic Balloon Pump For Haemodynamic Stabilization in Acute Myocardial Infarction Complicated by Cardiogenic Shock after Primary Percutaneous Coronary Intervention : A Case Report Background: Mortality rate of over 40% occurred in patients of Acute Myocardial Infarction complicated by Cardiogenic Shock (AMICS). Nonetheless, many patients with AMICS have potentially significant amounts of stunned myocardium that could recover within days post- revascularization. The role of mechanical support with intra-aortic balloon pump (IABP) after percutaneous coronary intervention (PCI), is still questionable but sometimes can be considered as a bridge therapy to maintain hemodynamic stability and support myocardium recovery. Case Illustration: A 40 – year – old male presented with ventricular tachycardia (VT) and cardiogenic shock in recent anteroextensive MI and complained of chest pain, palpitations, and diaphoresis three days prior. He was a smoker, with neither any other modifiable risk factor nor significant family history with cardiovascular disease. ECG findings were ST segment elevations with pathological Q wave in lead V1-V6, I, and aVL followed by sustained VT. Echocardiogram revealed left ventricular dysfunction with 38% ejection fraction. In less than 24 hours, we conduct angiography and found total occlusion in Left Anterior Descending (LAD) artery which were then stented in proximal – mid LAD. After revascularization, the patient remained hemodynamically unstable and had recurrent VT, which became more frequent when we titrate inotrope and vasopressor. Keywords : IABP, AMICS, Ventricular Arrhythmia, Heart Failure Indonesian Journal of Cardiology We decided to perform IABP placement, then the hemodynamic status gradually improved and VT incidence was markedly decreased. The patient was weaned from IABP after 5 days of care and hemodynamic improvement. Patient then discharged with HF therapy, DAPT, and antiarrhythmic medicine. Conclusion: Studies showed no benefit of IABP in clinical outcome and mortality, albeit some benefit in hemodynamic stability in AMICS patients. In this case, IABP improved patient’s hemodynamic status after PCI. Failure of various studies to establish the role of device support in AMICS management may have resulted from their more relevant use in other cause and heterogenity of CS classification criteria. Keywords: Chronic Total Occlusion, Percutaneous Coronary Intervention Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:su pISSN: 2830-3105 / eISSN: 296 doi: 10.30701/ijc.1537 PCI to CTO lesion in Intractable Heart Failure Patient, Is There any Benefit? F.A. Kusyanto1, H. Munirwan 2, T. Heriansyah2 1,2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala Banda Aceh Indonesia Background Ventricular septal rupture (VSR) is a rare yet serious complication of acute myocardial infarction and leading to ventricular remodeling and subsequent heart failure. Mechanical unloading plays an important role during critical period especially as bridge to definitive treatment in ventricular septal rupture. Pharmacotherapy is another essential modality to prevent deterioration and halting cardiac remodeling. Combining mechanical support and medical agents may help ensure satisfying outcomes. Case Illustration A 52 years old male admitted with the chief complaint shortness of breath. 7 days before admission to the hospital, the patient complained of chest pain. 3 days before admission, the patient was taken to the local hospital because of shortness of breath and got hospitalized for 1 day and then referred to Adam Malik hospital for further treatment. Patient diagnosed with recent Anterolateral MI. Echocardiography showed IVS rupture at the apical septum with tunneling morphology 19 mm length, left to right shunt with a diameter of 11-14 mm. Coronary angiography showed total occlusion after Diagonal 1, and 20-30% stenosis at distal OM3. LV graphy showed contrast filling of the LV and RV through the VSR with diameter 6-7 mm. Patient recieve therapy ACE-i, MRA, diuretic, DAPT, and high intensity statin. Low Dose Inotropic were also given to reduce shunting. An Intra-aortic balloon pump (IABP) was inserted for reduce afterload and augmentation of cardiac output on day 13th. PCI was performed on day 17th and percutaneous closure was done on day 21st after MI onset. The patient was discharged 3 days after percutaneous closure in good condition. "Combination of Mechanical Unloading and Pharmacotherapy for Management of Post Infarction Ventricular Septal Rupture" : A Case Report M. Ferhat1, A.C. Lubis2, K.K. Ilyas3, A.N. Nasution4, T.W. Ardini5, C.A. Andra6 1Cardiology Resident at Cardiac Center H. Adam Malik Hospital, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2 3 4 5 6C di l i t t C di C t H Ad M lik H it l F lt f M di i U i it 1Cardiology Resident at Cardiac Center H. Adam Malik Hospital, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2,3,4,5,6Cardiologist at Cardiac Center H. Adam Malik Hospital, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2,3,4,5,6Cardiologist at Cardiac Center H. Adam Malik Hospital, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia Indonesian Journal of Cardiology PCI to CTO lesion in Intractable Heart Failure Patient, Is There any Benefit? F.A. Kusyanto1, H. Munirwan 2, T. Heriansyah2 PCI to CTO lesion in Intractable Heart Failure Patient, Is There any Benefit? F.A. Kusyanto1, H. Munirwan 2, T. Heriansyah2 1,2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia. Background: A chronic total occlusion (CTO) percutaneous coronary intervention (PCI) is one of the most challenging procedures in interventional cardiology right now. Various angiographic scoring systems are being developed to assess the difficulty of guidewire crossing. The J-CTO score shows how challenging the CTO crossing is. Case Illustration: We present a case of a 53-year-old male who was submitted to the ER with typical chest pain and difficulty breathing. The patient had a history of prior PCI with a patent stent in the LM and LCx. Echocardiography and cardiac SPECT imaging showed RCA still viable. Based on those findings, we decided to perform PCI. Throughout the PCA procedure JCTO was 3, from PCI procedure we had difficulties to penetrate the lesion. In the end, using backup balloons, the lesion was successfully penetrated and a drug-eluting stent was placed in the RCA. A few months after the pci the patient's condition is getting better. A thorough history check, appropriate imaging techniques, and choosing the right equipment and approach of PCI are vital for the success rate of CTO PCI. Conclusion: In patients with chronic total occlusion, percutaneous coronary intervention is still a possible option, despite the difficulties. Preparation and the use of angiographic scoring systems, such as the J-CTO score, to estimate the probability of success and the type of approach is essential in the planning of CTO PCI. CTO PCI can reduce the incidence of cardiac death compared to optimal medical therapy in patients with CTO. The yield of complete revascularization for mortality is similar to that of CABG but the rate of short-term attacks is less with PCI. It was further explained that PCI in reducing morbidity and mortality is better than optimal medical administration Keywords: Chronic Total Occlusion, Percutaneous Coronary Intervention Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Conclusion Combining mechanical unloading and aggressive pharmacotherapy can help halting cardiac remodelling and a viable option to achieve hemodynamic stabilization allowing a delayed closure of VSR in MI patients and contributing to improved survival. Keyword : Ventricular septal rupture, Myocardial infarction, Intra-aortic balloon pump, Unloading d : Ventricular septal rupture, Myocardial infarction, Intra-aortic balloon pump, Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1 : Echocardiography showed IVS rupture at the apical septum with tunnel form 19 mm length, with a diameter of 11-14 mm. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1 : Echocardiography showed IVS rupture at the apical septum with tunnel form 19 mm length, with a diameter of 11-14 mm. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Considerations in Management of Atrial Septal Defect with Pulmonary Hypertension on A Young Woman: A Case Report of Treat and Repair F. Napitupulu1, A.N. Nasution2, T.W. Ardini2, C.A. Andra2 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 2Cardiologist, Cardiac Center H. Adam Malik Hospital, Medan, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Considerations in Management of Atrial Septal Defect with Pulmonary Hypertension on A Young Woman: A Case Report of Treat and Repair F. Napitupulu1, A.N. Nasution2, T.W. Ardini2, C.A. Andra2 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 2Cardiologist, Cardiac Center H. Adam Malik Hospital, Medan, Indonesia Considerations in Management of Atrial Septal Defect with Pulmonary Hypertension on A Young Woman: A Case Report of Treat and Repair F. Napitupulu1, A.N. Nasution2, T.W. Ardini2, C.A. Andra2 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 2Cardiologist, Cardiac Center H. Adam Malik Hospital, Medan, Indonesia Background: Atrial septal defect (ASD) is an adult congenital heart disease that is often found in Sumatera Utara and can occur at every social level. Closure of an ASD that caused functional limitations cannot be done immediately. There is consideration such as the pulmonary arterial hypertension (PAH) which has developed. The ‘treat and repair’ is the method used to treat ASD with PAH and a pulmonary vascular resistance (PVR) > 5WU. In this case, we present a young woman with PVR > 5WU, how we treat her. Case Illustration: A 27 years old woman was referred to H. Adam Malik Hospital with a history of fatigue, shortness of breath during light activity, and hoarse voice. She was diagnosed by ASD with severe PH based on transthoracic echocardiography (TTE) and right heart catheterization (RHC). Transesophageal echocardiography (TEE) showed secundum ASD 24-32mm, left to right shunt, rims were suitable for transcatheter device closure. Patient took PH therapy sildenafil citrate 3x20mg for two months. Functional class became better. Keywords : Pre-excitation Syndrome, Wolff-Parkinson-White Syndrome, Ablation. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 "Management Strategy of Paroxysmal Atrial Fibrillation in Pre-excitation Syndrome : A Life-threatening Arrhythmia Case." Indonesian Journal of Cardiology Percutaneous transcatheter closure by Amplatzer Septal Occluder (ASO) guiding TEE with zero fluoroscopy was done. Sildenafil therapy was continued. There was a period of two months without sildenafil due to unavailability. On follow-up, 8 months post closure, World Health Organization Functional Class (WHO-FC) was improved from 2 to 1, voice was soft. TTE showed no residual leakage, no more right atrium (RA) and right ventricle (RV) dilation. RHC showed reduction of mean Pulmonary Arterial Pressure (mPAP): 61 to 24 mmHg, Pulmonary Vascular Resistance (PVR): 6,6 to 4,57 WU, Flow Ratio (FR): 4 to 1,2. Conclusion: Clinical improvement, high flow to the lungs, and young age women can be considerations not to delay defect closure in ASD with PAH. Longterm follow up is still needed. Keywords: ASD, PAH, Treat, Repair Keywords: ASD, PAH, Treat, Repair Keywords: ASD, PAH, Treat, Repair Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 M.R. Asepty1, A.C. Lubis2, C.A. Andra3 M.R. Asepty1, A.C. Lubis2, C.A. Andra3 1 Cardiology Resident at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2 Cardiac Electrophysiologist at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 3 Cardiologist at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia Background : Atrial fibrillation becomes a potentially lethal arrhythmia when occured in pre- excitation syndrome, thus requiring further management such as ablation procedure. Rapid ventricular activation from the accessory pathway can lead to ventricular arrythmia and making atrial fibrillation extremely life-threatening. Case Illustration : A Male, 54 years old patient was referred to H. Adam Malik Cardiac Center with history of palpitations and weakness which experienced intermittently since 3 years ago. The ECG examination at the previous hospital shown irregular wide QRS complex tachycardia. The 24 Hours Holter ECG Monitoring at the previous hospital found episodes of narrow-complex Atrial Fibrillation. While at H. Adam Malik Cardiac Center, the ECG shown sinus rhythm with delta wave morphology . EASY-WPW algorithm was used to predict the location of the accessory pathway, which suggestive of an accessory pathway in the posterolateral mitral annulus. The patient was immediately scheduled for electrophysiology study and ablation procedure, then fusion waves morphology was found which suggestive of an accessory pathway that located in the posterolateral mitral annulus, and then the ablation procedure was done in that location. After the ablation procedure, the ECG shown sinus rhythm with no pre-excitation morphology was found. Conclusion : Appropriate diagnosis, approach, and management strategy of patients with atrial fibrillation with pre-excitation is vital in preventing patients from falling into life-threatening condition. ywords : Pre-excitation Syndrome, Wolff-Parkinson-White Syndrome, Ablation. Keywords : Pre-excitation Syndrome, Wolff-Parkinson-White Syndrome, Ablation. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 BACKGROUND Total occlusion of infarct-related coronary artery is an uncommon finding for Non ST-Elevation myocardial infarction (NSTEMI) during routine angiography, and usually found in STEMI cases. NSTEMI patients with Total occlusion of the artery (TOA) prevalence was 29% and usually characterized by higher cardiovascular risks, such as diabetes, active smoker, and hypertension. NSTEMI patient with TOA is associated with greater extent of necrosis and worsen prognosis, and thus, must be treated with emergent reperfusion therapy. Early reopening of a completely occluded coronary artery reduces myocardial damage, prevents heart failure, and improves clinical outcomes. CONCLUSION We reported a case of NSTEMI patient with TOA. NSTEMI patients should undergo strict evaluation for signs indicating possible TOA and then performed reperfusion therapy as needed. CASE ILLUSTRATION We present a patient admitted to the hospital with symptoms of chest pain since 3 days ago. He had previous history of uncontrolled hypertension and smoking. His ECG showed ST‐segment depression in lead II, III, AVF and V4‐V6. Chest X-Ray revealed Cardiomegaly, and high troponin level. Echocardiography revealed akinesia inferoseptum segment, hypokinesia other segment and decreased left ventricular systolic function. Then, he underwent trans-radial approach coronary angiography and the result was total occlusion of the right coronary artery (RCA). Stents were implanted to the lesion. No complication after the procedure and he was discharged several days later. The patient then planned to continue therapy at the outpatient clinic. NON ST-ELEVATION MYOCARDIAL INFARCTION WITH TOTAL OCCLUSION OF THE CORONARY ARTERY : CASE REPORT D. Khairunnisah , C.A. Andra , A.N. Nasution , H. Hasan 1 Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia; 2,3,4 Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia KEYWORD NSTEMI, Coronary Angiography, Total occlusion of the coronary artery. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 KEYWORD NSTEMI, Coronary Angiography, Total occlusion of the coronary artery. NSTEMI, Coronary Angiography, Total occlusion of the coronary artery. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. Coronary Angiography of the patient showed Total Occlusion of the Right Coronary Artery. Figure 1. Coronary Angiography of the patient showed Total Occlusion of the Right Coronary Artery. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Background Acute limb Ischemia (ALI) was characterized by sudden decrease limb perfusion, which threatens limb viability and had symptoms for less than 14 days. The incidence of ALI is estimated to be 15 cases per 100,000 population per year, and constitutes 10% -16% of all vascular cases. In the case of ALI, the decrease in tissue perfusion occurs suddenly and results in a rapid and threatening ischemia process and there is not enough time for the formation of new blood vessels as collateral to compensate for the perfusion decrease. Therefore rapid detection and proper handling are very influential on patient prognosis. Case Illustration A 65-year-old female patient came with pain on her left foot for 1 week during activity and worsen within 2 days where pain also occurs at rest. Pallor and poikilothermia of the left foot was also found. Patient has controlled type 2 diabetes with insulin treatment, a controlled hypertension, CAD post PCI and paroxysmal atrial fibrillation. Left foot saturation was 86% until 90%. Her ECG showed sinus rhythm and echocardiography showed good LV systolic function with 60% EF. doppler ultrasound showed monophasic waves from common femoral artery until dorsalis pedis artery, thrombus and plaque at SFA, arterial flow was not found in popliteal, posterior tibial artery and dorsalis pedis artery. She was diagnosed with Grade I ALI on left leg. Arteriography showed occlusion in DFA and SFA sinistra. Percutaneous Intra-arterial Thrombolytic (PIAT) was done with streptokinase 100.000 IU for 4 hours via femoral catheter continued with 5000IU/hour for 24 hours was given. Two days later, a follow-up arteriography showed no thrombus at SFA and with good flow in SFA. Acute limb ischemia – Effectivity of Percutaneous Intra-arterial Thrombolysis as a First Choice: A Case Report M.H.M. Putra , T.B. Haykal , Y.F. Siregar , A.C. Lubis , C.A. Andra , K.K. Ilyas 1 Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 2Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia Conclusion PIAT is the appropriate treatment in grade I and IIA ALI with no contraindications for thrombolytic and there is sufficient time for reperfusion to preserve tissue viability. Streptokinase was one of the drugs of choices for PIAT. Within 3 days of treatment, the patient showed a good response to PIAT. Keywords: acute limb ischemia, percutaneous intra-arterial thrombolysis, streptokinase Figure 1 Arteriography, at admission (Left); post PIAT (Right) Indonesian Journal of Cardiology Figure 1 Arteriography, at admission (Left); post PIAT (Right) Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Unraveling the Cause of Angina in Patient with Rare Malignant Right Anomalous Coronary Artery From the Opposite Sinus with Stenotic Lesion Y. Adani1, P.P.R. Gharini1, N. Taufiq1 Department of Cardiology and Vascular Medicine, Dr Sardjito Hospital, Yogyakarta/Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Unraveling the Cause of Angina in Patient with Rare Malignant Right Anomalous Coronary Artery From the Opposite Sinus with Stenotic Lesion Y. Adani1, P.P.R. Gharini1, N. Taufiq1 Department of Cardiology and Vascular Medicine, Dr Sardjito Hospital, Yogyakarta/Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia Background: g Anomaly of coronary artery from the opposite sinus (ACAOS) is a rare congenital disease (0.2-1.2%) with right coronary artery arise from left coronary sinus (R-ACAOS) is the most common form. Approximately 20% has interarterial course (malignant) and may experience life- threatening manifestations. The mainstay of therapy according to the guidelines is surgical revascularization. Meanwhile, stenotic lesion in the coronaries made this case complicated to acknowledge the source of angina. From a pathophysiological point of view, percutaneous coronary intervention may be able not only to remove the lateral compression of the interarterial segment, but also to treat stenosis. Case Illustration: A 62 year-old female came with the main complaint of recurrent chest pain with comorbidities diabetes, dyslipidemia, and menopause. Vital signs and physical examinations within normal. Both electrocardiography and echocardiography examination showed no signs of ischemia and ventricular systolic function was good. However, stress test showed a positive ischemia response. A coronary computerized tomography angiography (CTA) was carried out with the results of interarterial R-ACAOS and severe stenosis in all three coronary vessels. Coroangiography results were accordance with the CTA report. Integrated modality using fractional flow reserve (FFR)-dobutamine, intravascular ultrasound (IVUS)-dobutamine, or ischemia non-invasive imaging are the tools to determine hemodynamic relevance of an apparent malignant ACAOS. Unfortunately, those modalities were not performed due to unavailability. Based on guideline in patients with significant hemodynamic changes related to interarterial R- ACAOS, who have significant stenosis, the preferred option is coronary artery bypass surgery. Otherwise, stent implantation might become the treatment of choice. Conclusion: Our report aims to seek the best management strategy of malignant R-ACAOS with stenotic lesions. Strong evidence of ischemia must be present before opting for surgery. A simultaneous approach of using dobutamine stress and FFR/IVUS might help in the decision- making in patients with coronary anomalies. Keywords: R-ACAOS, interarterial course, PCI, CABG, FFR-dobutamine, IVUS-dobutamine Keywords: R-ACAOS, interarterial course, PCI, CABG, FFR-dobutamine, IVUS-dobutamine Discussion: AIVR is believed to be automatic and usually readily overdriven by faster supraventricular pacemakers. Because ventricular and sinus rate are often similar, isorhythmic dissociation is a common finding. AIVR is mostly seen in reperfusion phase after ACS. It’s also seen in patients with other conditions, such as myocardial disease, hypertensive, rheumatic, and congenital heart disease. Meanwhile, preexcitation may mimic idioventricular rhythms. There was one case report about a patient with WPW in which electrophysiology study showed idioventricular rhythm. The opposite can also occur when presence of accessory pathway simulates ventricular complex. Electrophysiology study is needed to confirm the diagnosis. Background: Accelerated idioventricular rhythm (AIVR) is a slow ventricular rhythm usually seen in reperfusion phase after myocardial infarction (MI). Presence of AIVR without MI is rarely discussed. Intermittent AIVR in MI with Non-Obstructive Coronary Arteries: What Is the Mechanism? S. Haidar1, E. Maharani2, F. Hidayati2 1Resident of Department of Cardiology and Vascular Medicine, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University, Yogyakarta, Indonesia 2Staff of Department of Cardiology and Vascular Medicine, Faculty of Medicine, Gadjah Mada University Mechanism? S. Haidar1, E. Maharani2, F. Hidayati2 1Resident of Department of Cardiology and Vascular Medicine, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University, Yogyakarta, Indonesia 2Staff of Department of Cardiology and Vascular Medicine, Faculty of Medicine, Gadjah Mada University Case illustration: A 53-year-old patient complained of chest pain and was frequently diagnosed with non-ST elevation MI in district hospital. Echocardiography showed inferoseptal hypokinesia, but coro- angiography only showed slow flow in RCA which conclude diagnosis of MI with non-obstructive coronary arteries. Patient was referred to our hospital for further investigation. Since ECG showed slurred upstroke of QRS complex resembling delta wave, ambulatory ECG was done. Interestingly the result showed complete right bundle branch block and intermittent AIVR with rate 80 bpm. The patient was asymptomatic during AIVR. Cardiac magnetic resonance imaging (CMR) showed subendocardial fibrosis in basal-apicoinferior segment, which still doesn’t explain the patient’s symptom. However, this incidental finding begs further questions, does intermittent AIVR correlate with CMR finding or is there a concealed accessory pathway that allows AV re-entry? We proceed by planning electrophysiology study to determine the presence of accessory pathway and to test inducibility of sustained arrhythmias. Keywords: Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Intermittent AIVR in MI with Non-Obstructive Coronary Arteries: What Is t Conclusion: There are other aetiologies of AIVR besides MI. Further tests are needed to confirm diagnosis and appropriate management. Keyword: AIVR, preexcitation syndrome Keyword: AIVR, preexcitation syndrome Background: g Ventricular septal defect is the most common congenital cardiac defect accounting for more than 20% of all CHDs.1 Perimembranous ventricular septal defects (PMVSDs) in infants and young children are the most common cause of a hemodynamically significant ventricular septal defect (VSD).2 Percutaneous closure of VSDs is performed under general anesthesia and with fluoroscopic and transesophageal echocardiographic guidance is now a choice.3 Complete atrioventricular block (cAVB) has been deemed a rare complication after transcatheter closure for ventricular septal defect (VSD). However, this serious event appears to be underrecognized and is worth being investigated further. Conclusion: Atrioventricular block could be a risk while performing percutaneous VSD closure. Conduction system injury from mechanical trauma/compression by the delivery system or device causing acute intraprocedural atrioventricular block has a reasonable probability of early resolution. Immediate and long-term follow-up is essential in patients who have had these devices implanted. Case Illustration: A 19 years old male, presented with incidental finding of perimembranous VSD during an army selection. He was 170 cm tall and weighed 60 kg. Physical examination was suggestive of VSD. Baseline 12-lead electrocardiogram (EKG) showed normal sinus rhythm with no conduction delays. Transthoracic 2D echocardiography showed a 6-7 mm perimembranous VSD with normal pulmonary arterial diameter and all of the cardiac chambers. All pulmonary veins were draining into left atrium. Transcatheter device closure of VSD was carried out under general anesthesia with KONAR-MF VSD Occluder No 10-8 mm (Lifetech). During the procedure, a complete atrioventricular block was evident both on monitoring screen and echocardiography screen. It followed by chest discomfort, and resolved shortly after 1 mg of atropine administration, with stable haemodynamics. The procedure was resumed, and the device was placed with no evidence of residual VSD from the transesophageal echocardiography, nor recurrent atrioventricular block after the procedure. Transient AV Block during VSD Device Closure Procedure: to Stop or Not to Stop? P. Indrihapsari1,2, V. Yogibuana1,2 P. Indrihapsari1,2, V. Yogibuana1,2 1Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya, 2Dr. Saiful Anwar General Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal Cardiology Indonesian J Cardiol 2023:44:su pISSN: 2830-3105 / eISSN: 2964 doi: 10.30701/ijc.1537 Transient AV Block during VSD Device Closure Procedure: to Stop or Not to Stop? P. Indrihapsari1,2, V. Yogibuana1,2 1Department of Cardiology and Vascular Medicine Faculty of Medicine Universitas Brawijaya, 2Dr. Saiful Anwar General Hospital Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Transient AV Block during VSD Device Closure Procedure: to Stop or Not to Stop? P. Indrihapsari1,2, V. Yogibuana1,2 Keywords: y Ventricular septal defect, Atrioventricular block, percutaneous device closure Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Transcatheter Closure of Very Tortuous Patent Ductus Arteriosus Using Snaring Technique and Balloon Sizing: A Case Report R. Rasaki1, R. Prakoso2, T. Heriansyah1, M. Muqsith1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia. 2Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita, Jakarta, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Transcatheter Closure of Very Tortuous Patent Ductus Arteriosus Using Snaring Technique and Balloon Sizing: A Case Report R. Rasaki1, R. Prakoso2, T. Heriansyah1, M. Muqsith1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia. 2Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita, Jakarta, Indonesia Background: Patent ductus arteriosus (PDA) is one of the most common congenital heart diseases in premature infants. PDA causes volume overload in the left heart and may lead to congestive heart failure, atrial arrhythmia, or pulmonary arterial hypertension. Recently, transcatheter device closure of PDA is an established and widely accepted treatment modality in adolescents, children, and older infants. Case Illustration: We reported a 17-year-old boy was referred to the National Cardiovascular Center Harapan Kita hospital from South Sumatra with a chief complaint of dyspnea on exertion in the past 2 years. He had a history of recurrent respiratory tract infections when he was a baby. He was born spontaneously, premature at 32 weeks of gestation, with a birth weight of 2300 grams. On physical examination, we found a normal first heart sound and a normal split of the second heart sound with a continuous murmur in the upper left sternal border radiating to the left infraclavicular. The transthoracic echocardiography showed a PDA with the left to right shunt. We diagnose the patient with patent ductus arteriosus according to clinical findings, and transthoracic echocardiography and planned to perform transcatheter PDA closure. In the cath lab, transesophageal echocardiography evaluation showed the PDA morphology was very tortuous. So, we decided to perform transcatheter PDA closure using snaring technique and balloon sizing guided by transesophageal echocardiography minimal fluoroscopy technique. The procedure was performed successfully without major complications. Keywords: Patent Ductus Arteriosus, Very Tortuous, Transcatheter Closure Keywords: Atrial Septal Stenting, Idiopathic Pulmonary Arterial Hypertension Keywords: Conclusion: Transcatheter device closure of a very tortuous patent ductus arteriosus is feasible and safe with minimal fluoroscopy time. Careful patient selection and good imaging quality are essential for procedural success and avoiding complications. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 TRANSCATHETER ATRIAL SEPTAL STENTING IN IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION: A CASE REPORT T.F. Hadi1, R. Prakoso2, T. Heriansyah1, M. Muqsith1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Zainoel Abidin General Hospital, Banda Aceh, Indonesia 2 Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Indonesia, National Cardiovascular Center Harapan Kita, Jakarta, Indonesia Background : The treatment of severe idiopathic pulmonary arterial hypertension (IPAH) remains challenging, despite improved medical options to address this life threating condition. Atrial septostomy is used as an additional therapeutic strategy for IPAH-associated right heart failure. Case Illustration : A 36-year-old female was referred to National Cardiovascular Center Harapan Kita hospital with a chief complaint dyspnea on exertion, swelling in abdomen and lower extremity in the past 10 years. She has diagnosed with idiopathic pulmonary arterial hypertension since 8 years ago. She was planned to undergo transcatheter closure of PDA guided by TEE and fluoroscopy technique. However, before the procedure, TEE evaluation was carried out, obtaining RA-RV dilation, LV smallish, D-shape (+), good RV contractility, reduce LV contractility, intact IAS, IAS bulging to the left, TR mild (+), PR (-), LVOT VTI 9 cm. We performed atrial septal stenting with omnilink elite 10 mm x 29 mm x 135 cm. We performed TEE evaluation after procedure. It revealed stent in stowed position, flow (+), LVOT VTI 12 cm. Post-procedure, descending aorta pressure was 93/53 mmHg and peripheral saturation was 92%. The fluoroscopy time in this procedure was just approximately 11 minutes without major complications. The patient was discharged after 24 hours and the patient did not show any symptoms at the time of subsequent follow-up. Conclusion : Transcatheter atrial septal stenting in idiopathic pulmonary arterial hypertension is feasible and safe. It reduced symptoms and increasing quality of life. Indonesian Journal o Cardiology Indonesian J Cardiol 2023:44:sup pISSN: 2830-3105 / eISSN: 2964- doi: 10.30701/ijc.1537 Figure 1. Omnilink elite 10 mm x 29 mm x 135 cm in stowed position Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. Omnilink elite 10 mm x 29 mm x 135 cm in stowed position Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Electrophysiology Study and Ablation in Atypical Atrioventricular Nodal Reentrant Tachycardia (Slow-Slow) Keywords: Atypical AVNRT, Electrophysiology Study, Ablation Electrophysiology Study and Ablation in Atypical Atrioventricular Nodal Reentrant Tachycardia (Slow-Slow) N.G. Wara1, P.N.P. Limen1, B.M. Setiadi1 tment of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Manado Background: Atrioventricular nodal reentrant tachycardia (AVNRT) was a subtype of supraventricular tachycardia (SVT). AVNRT was further divided into typical and atypical AVNRT. Atypical AVNRT occured in 6-7% of all AVNRT cases, and Slow-Slow Pathway was rarer of the two subtypes. Case illustration: A male patient, 60 years old, came to the Emergency Department with chief complaint of episodic palpitation since 1 month before admission. 2 weeks prior, patient was admitted to Prof RD Kandou General Hospital with the same complaint, and diagnosed with AV Nodal Re-entry Tachycardia (AVNRT). Physical examination was found to be unremarkable. ECG show paroxysmal supraventricular tachycardia rhythm (150 beats per minute), no P-wave visible, with normal axis. Laboratory examination was unremarkable aside from hyperuricemia (uric acid 8.5 mg/dL). Chest X-ray was within normal limits. Echocardiography result show mild regurgitation in mitral and pulmonic valve, other findings were within normal limits. Based on results from history-taking, physical examination, and diagnostic studies during current and previous admission, patient was diagnosed with AVNRT, controlled hypertension, hyperuricemia, and dyspepsia syndrome. Patient was given treatment of candesartan 8 mg, lansoprazole 30 mg/12 hours, allopurinol 100 mg, and atorvastatin 20 mg. Patient underwent Electrophysiology Study with result showing atypical AVNRT with slow-slow pathway, and had succesful ablation of slow pathway afterward. After the procedure, patient was observed in the ICCU for the first 24 hours, then transferred to the common ward, and got discharged when condition confirmed to be stable. Conclusion: Slow-slow AVNRT occurs because patient had an additional slow pathway contributing to re-entry phenomenon. Electrophysiology study and radiofrequency ablation show good outcome in this case. Keywords: Atypical AVNRT, Electrophysiology Study, Ablation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1 ECG during AVNRT episode Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1 ECG during AVNRT episode Keywords: Atrial Fibrillation, High Bleeding Risk, Anticoagulation, Thrombocytopenia, Mean Platelet Volume. ABSTRACT Background: Thrombocytopenia is estimated to account for 6-24% of patients with AF. Previous studies have reported that similar rates of stroke and death were observed in thrombocytopenic and normocytopenic patients. The mean platelet volume (MPV) also serves as an independent predictor of stroke and left atrial thrombus in patients with AF. Of note, several cases of NOAC-induced thrombocytopenia have been reported in various literature. Case Illustration: A 78-year-old male with difficulty of breathing in the last ten days ago. Patient also reported feel easily tired in the last few years. Patients look pale, he had black-colored stool on his defecation last week. Upon examination revealed conjunctiva anemic and rales in basal lung. An electrocardiogram showed atrial fibrillation with a rapid ventricular response of 149 bpm. Blood work at first admission revealed anemia (10.3 gr/dL), thrombocytopenia (65x103/mm3), high MPV (13.2 fL) and unexplained high INR (2.49). Chest X-ray showed cardiomegaly and transthoracic echocardiogram (TTE) revealed all chamber dilatation with left and right ventricular systolic dysfunction. Patient had moderate mitral, tricuspid, and aortic regurgitation. The patient was treated with fresh frozen plasma on hospital day 1st to correct his high INR. Patient was started with 10mg of Rivaoxaban with intensive monitoring of serial platelets and bleeding diathesis. Serial platelets showed no improvement, liver function test elevated (SGOT/SGPT 201/185 U/L) and bruises appeared in IV-line catheter installation area. Rivaroxaban was switched to Clopidogrel on hospital day 2nd due to liver problems and for stroke prevention. Conclusion: The need for anticoagulation in patients with thrombocytopenia is an intricate problem. Decision to administer or withhold anticoagulation depends on the assessed risks of thrombosis and bleeding. Several principles should be considered when deciding to start anticoagulation: 1) a low platelet count does not protect from thrombosis; and 2) in general, thrombotic complications are more dangerous than bleeding complications. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. Patient’s Trend of Thrombocytes, Mean Platelet Volume (MPV) and INR. Indonesian Journal of Cardiology Indonesian Journal of Cardiology gure 1. Patient’s Trend of Thrombocytes, Mean Platelet Volume (MPV) and INR. WELLENS’ SYNDROME : PCI IS THE TREATMENT GOAL AND SHOULDN’T BE DELAYED Taufiqurrahman1, M. Fitra1, T. Herianyah1, M. Muqsith1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia Taufiqurrahman1, M. Fitra1, T. Herianyah1, M. Muqsith1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia Background : Wellens’ syndrome is a clinical manifestation of coronary artery disease developed as NSTE-ACS. It describes a pattern of electrocardiographic changes, particularly deeply inverted or biphasic T waves in leads V2-V3, that is highly specific for critical proximal stenosis of the left anterior descending coronary artery. It is important to recognize this ECG patterns because patients with wellens’ syndrome are at high risk for impending large anterior wall acute myocardial infarction. Case Illustration : A 57 years old male presented to ER with chief complaint of chest pain since 2 weeks prior admission. Chest pain was felt like a heavy sensation and radiated to the back and accompanied with cold sweat. Chest pain was felt again around 5-6 hours before admission to ER but patient was pain-free when he presented to our hospital. He was a known case of uncontrolled hypertension. On presentation, the vital signs were measured with BP 164/79 mmHg, HR 85 beats/minute and RR 20 breathes/minute. Physical examination was normal. ECG showed deep T wave inversion in V1-V6. Troponin T did not elevated. Echocardiography showed normal ventricles function (LVEF 71%, TAPSE 2.5 cm). Patient was diagnosed with Wellen Syndrome type B and treated as UAP and then he was sent to ICVCU for intensive monitoring. The next day patient was sent to cathlab to perfomed corangiography and revealed a subtotal stenosis of proximal LAD and installed 1 stent DES of LAD. The next day patient discharged with good condition. Conclusion : Early recognizing from electrocardiography pattern of wellens’ syndrome is pivotal due to it can predict critical stenosis in proximal LAD. Wellens’ syndrome is very possibly to developed into STEMI, therefore early detection and prompt management is the key to prevent complication and mortality. Corangiography with PCI had to be immediately performed for further management. Keyword : Wellens’ syndrome, Proximal LAD, PCI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. Case Illustration An 8-year-old boy with Tetralogy of Fallot, polydactyly, history of 3-step surgery to the correction of labiopalatoschiziz. With chief complaints of cyanotic, the body weight is difficult to gain, get tired easily, have speech disorders, and tend to squat in daily life as well. Saturation oxygen was 66%/65%//71%/74%. S1 normal, S2 weakened, systolic ejection murmur grade III/6 with punctum maximum at the left parasternal line SIC II. With nutritional status 16.5 kg, 117 cm, WAZ: -3.54, HAZ: -2.06, BMI: -3.42. Impression: Poor nutrition, very less weight. Laboratory haemoglobin 21.8 g/dL and hematocrit 68.7%. Chest x-ray CTR 48%, booth shaped, narrowed retrosternal space, RV cardiomegaly. EKG Sinus rhythm, 81x/min, RAD, RVH. Echo results: overriding aorta <50%, VSD malalignment, RV hypertrophy, severe PS with PG 75 mmHg. He was a successful total correction procedure and the condition was improved. Background g Tetralogy of Fallot is the most common cyanotic heart defect with an incidence of 5% to 10% of all CHD events. The four hallmark features of the malformation that consists of an interventricular communication, also known as a ventricular septal defect, obstruction of the right ventricular outflow tract, override of the ventricular septum by the aortic root, and right ventricular hypertrophy. ords : Tetralogy Of Fallot, Labiopalathoschiziz, Malnutrition, Total Correction WELLENS’ SYNDROME : PCI IS THE TREATMENT GOAL AND SHOULDN’T BE DELAYED ECG showed LAD with deep T wave inversion in V1-V6 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. e 1. ECG showed LAD with deep T wave inversion in V1-V6 ECG showed LAD with deep T wave inversion in V1-V6 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Tetralogy of Fallot With Labiopalathoschiziz : Let’s Total Correction or Not? F.H. Alkaf1, A. Priyatno2 1Faculty of Medicine, Diponegoro University, Semarang, Indonesia 2Pediatric Department, Faculty of Medicine Diponegoro University, Dr Kariadi Hospital, Semarang Tetralogy of Fallot With Labiopalathoschiziz : Let’s Total Correction or Not? F.H. Alkaf1, A. Priyatno2 1Faculty of Medicine, Diponegoro University, Semarang, Indonesia 2Pediatric Department, Faculty of Medicine Diponegoro University, Dr Kariadi Hospital, Semarang Conclusion The management of patients with TOF has its own challenges both for diagnosis and management because not all areas have adequate human resources or equipment. It is now expected that the prognosis of TOF patients will improve substantially because of the advances in surgical and medical management that have occurred over the last few decades. As for all patients with congenital heart defects, the management of patients with tetralogy of Fallot does not end with complete improvement. Follow-up to the cardiologist needs to be carried out for further evaluation. Keywords : Tetralogy Of Fallot, Labiopalathoschiziz, Malnutrition, Total Correction Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Coronary Artery Ectasia Presenting with STEMI Inferior, How About Invasive And Non- Invasive Therapy; A Case Report Jefri.D.S 1, Haris M 2,M.Muqsith2,M.Ichsan2 1Faculty of Medicine, Syiah Kuala University/ Zainoel Abidin Hospital, Banda Aceh, Indonesia; 2Faculty of Medicine, Syiah Kuala University/ Zainoel Abidin Hospital, Banda Aceh, Indonesia; Background: Clinical manifestations of coronary Artery Ectasia(CAE) are heterogeneous, and often it is occasionally recognized during coronary angiography or computed tomography (CT). However, CAE may become clinically overt through different possible scenarios, including ACS, effort angina, and exercise-induced ischemia. The optimal management of patients with CAE is largely unknown. Case Illustration: a 59-year-old man was admitted to emergency department with complaint of chest pain since 3 Hours before admission. Patient has also history of Hypertension and Heavy Smoker. With blood pressure was 220/120 mmHg, pulse 42 beats per minute and respiratory rate was 22 times per minute. Electrocardiography (ECG) showed: Sinus rhythm, QRS rate 46 bpm ECG showed ST Elevation in lead II, III, and AVF (Figure 1). During percutaneous coronary intervention with result CAD 1 VD, total distal occlusion with thrombus (Grade V), impression of an aneurysm with a diameter of 8-10 mm in RCA(Figure 2,a), POBA has been done at RCA(Figure 2,b), good results with suggest double antiplatelet for 12 months, drip eptifibatide 12.0 ml/hours. Discussion: Percutaneous treatment of CAE is a valuable option in patients with suitable anatomical and clinical features. However, PCI of ectasia lesions presents several challenges, starting from lack of specific indications, in patients even though a stent is not installed, the PCI results are still good and there are no complications after the patient is discharged. Conclusion KEYWORD: Ectasia, STEMI, PCI KEYWORD: Ectasia, STEMI, PCI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1.a. from PAC Total distal occlusion with thrombus (Grade V), impression of an aneurysm with a diameter of 8-10 mm, 1.b.POBA has been done at RCA, good results Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1.a. from PAC Total distal occlusion with thrombus (Grade V), impression of an aneurysm with a diameter of 8-10 mm, 1.b.POBA has been done at RCA, good results 1.b.POBA has been done at RCA, good results A Case Of Fascicular Ventricular Tachycardia Previously Thought As Abberant SVT R. Faradilla1, M. Muqsith2, T. Heriansyah3 1 Cardiology Residen, Medical Faculty, Universitas Syiah Kuala 2,3Department of Cardiology and Vascular Medicine, Medical Faculty, Universitas Syiah Kuala A Case Of Fascicular Ventricular Tachycardia Previously Thought As Abberant SVT R. Faradilla1, M. Muqsith2, T. Heriansyah3 1 Cardiology Residen, Medical Faculty, Universitas Syiah Kuala 2,3Department of Cardiology and Vascular Medicine, Medical Faculty, Universitas Syiah Kuala Keywords: Fascicular Ventricle Tachycardia, aberrancy SVT, wide tachycardia, cardioversion ABSTRACT Background: The majority of ventricular tachycardia (VT) episodes that are seen in emergency rooms are ischemic in nature. Fascicular VT is a rare kind of VT. Its distinctive electrocardiographic (ECG) pattern of wide-complex tachycardia (WCT) with a right bundle branch block (RBBB) and left axis deviation is caused by a re-entry circuit mechanism in the Purkinje fibers of this benign VT. Case illustration: A 63-year-old female presented to emergency room (ER) for evaluation of palpitations, pleuritic chest pain and tightness. Physical examination were severe tachycardia. The remaining physical examination was unremarkable. Electrocardiographic (ECG) showed regular monomorphic ventricular tachycardia at 210 bpm with a wide QRS, complete right bundle branch block pattern and total posterior fascicular block. The patient was assessed as aberrant supraventricular tachycardia (SVT) and received a biphasic external electrical shock of 50 Joule, which was unsuccessful. However, later it was reassessed as fascicular VT and given 150 Joule shock, which reverted to sinus rhythm with multifocal premature ventricular contraction (PVC). Patient was also treated with verapamil and referred for catheter ablation. Discussion: Fascicular ventricular tachycardia may occur in structurally abnormal heart and it could be life threatening. Fascicular ventricle tachycardia is frequently misdiagnosed as aberrant supraventricular tachycardia. Fascicular ventricle tachycardia is characterized by: atypical V1 morphology,QRS width ≤140 ms, R/S Ratio in V6 ≤ 1, and positive aVR. Verapamil has become a cornerstone of treatment. Verapamil works by activating the slow inward calcium channel. Conclusion: Prompt recognition of this arrhythmia is necessary, 2 lead ECG shows a right bundle branch block picture with left axis deviation, a diagnosis of fascicular tachycardia should be considered. Despite, this type of VT is typically sensitive to verapamil, when symptoms are severe or when medical treatment is inadequate or poorly tolerated, catheter ablation is a recommended treatment option. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. (Top) ECGoindicatingotachycardiaoasoevidencedoby a rate of 210 bpm with a wide QRS. Figure 2. (Buttom) ECG afterosynchronized defibrillator 150 J, showingosinusorhythm, and complete RBBB, multifocal PVC Figure 1. (Top) ECGoindicatingotachycardiaoasoevidencedoby a rate of 210 bpm with a wide QRS. Figure 2. (Buttom) ECG afterosynchronized defibrillator 150 J, showingosinusorhythm, and complete RBBB, multifocal PVC Figure 1. (Top) ECGoindicatingotachycardiaoasoevidencedoby a rate of 210 bpm with a wide QRS. Figure 2. ABSTRACT (Buttom) ECG afterosynchronized defibrillator 150 J, showingosinusorhythm, and complete RBBB, multifocal PVC Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Routine Interventional Closure of Patent Ductus Arteriosus after PH Treatment: A Case Report M.A. Amanta1, A.N. Nasution2, C.A. Andra2 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Routine Interventional Closure of Patent Ductus Arteriosus after PH Treatment: A Case Report M.A. Amanta1, A.N. Nasution2, C.A. Andra2 Residen of Cardiovascular, Universitas Brawijaya, Saiful Anwar Hospital, Malang Residen of Cardiovascular, Universitas Brawijaya, Saiful Anwar Hospital, Malan ** Cardiologist at Cardiovascular Departement of Saiful Anwar Hospital, Malang Background Cardiac myxoma (CM) is the most prevalence of adult benign primary cardiac tumor that derived from mesenchymal cell precursor which are most commonly found in the left atrium (LA) attached by a stalk to the fossa ovalis. Clinical manifestations of CMs may vary from asymptomatic cases to unexpected sudden death. Intracardiac flow obstruction may occur and potentially life-threatening. The diagnosis of a CM is mostly done with echocardiography. On echocardiogram, a myxoma presents as a echogenic polypoid or papillary mobile mass within the atrial cavity remaining attached to the interatrial septum through a stalk. Their prognosis is excellent when treated with prompt surgical resection. Case Ilustration We reported three cases of LA myxoma. One case of fatal obstructive LA myxoma in female 39 years old who dead before the surgery, one case of successful surgery an obstructive LA myxoma in woman 52 years old, and one non obstructive LA myxoma that concomitant with coronary artery disease in male 77 years old. Routine Interventional Closure of Patent Ductus Arteriosus after PH Treatment: A Case Report M.A. Amanta1, A.N. Nasution2, C.A. Andra2 1 Cardiology Resident at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2 Cardiologist at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 1 Cardiology Resident at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2 Cardiologist at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia Background: Patients with congenital heart disease (CHD) now live well into adulthood because of advances in intervention techniques, improvements in medical management, and the development of novel therapeutic agents. Patent ductus arteriosus (PDA) occurs in 5% to 10% of all CHDs, excluding premature infants. PDA is an uncommon anomaly in adult patients, as it is usually identified in childhood. Patients can now receive therapies via nonsurgical and transcatheter techniques. In patients presenting with severe Pulmonal Hypertension (PH), transcatheter PDA closure combining with PH treatment will improve outcome of the procedure. There are few studies relating surgical and nonsurgical treatment to long-term prognosis in adults with patent ductus arteriosus. Case Illustration : A Male, 30 years old, patient was referred to H. Adam Malik Cardiac Center with chief complain was previous history of shortness of breath. From clinical finding, ECG, chest x-ray, echocardiography, and cardiac catheterization, he was diagnosed PDA with severe PH. He underwent PH treatment sildenafil citrate for 1 year more. After 1 year, he was planned to undergo cardiac catheterization, and showed changes such as; FR: 3,65 � 4,71 ; PAR: 7.88 � 6,76 ; PARI: 5.47 � 5. The estimation size of duct is 10.3 in isthmus diameter and 44.5 mm in ampulla. The procedure was done with amplatzer ductal occluder (ADO) size 20-22mm and placed successfully. A decrease in mPAP was found, 73�56 mmHg. Sildenafil therapy is continued. After ADO, the patient felt more better than before. Conclusion: In general, for the older adult with a PDA, transcatheter closure is the preferred approach due to the calcification that is routinely seen in the older adult aorta. Thus, any approach to close an adult PDA in the catheterization laboratory is likely to be of lower risk and safer. Keywords: PDA, ADO, PH. Keywords: PDA, ADO, PH. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Background Left Main Coronary Artery Spasm (LMCAS) is defined as a sudden vasoconstriction of Left Main coronary artery causing vessel occlusion or near occlusion. LMCAS commonly manifests as chest pain and transient ST elevation by coronary vasospasm, and generally follows a benign clinical course. Rarely, syncope induced by ventricular arrhythmia associated with transient myocardial ischemia can be developed by LMCAS. If the cause of syncope is not correctly diagnosed, patients are at risk of recurrent syncope. In particular, syncope without chest pain is an uncommon manifestation of coronary vasospasm. Thus, as in this case, it can be missed as the cause of syncope. Case Illustration A 49-year-old female patient with no significant cardiac history presented to the emergency department with history of syncope and palpitation. Initial Electrocardiogram (ECG) was within normal limit and stress tests are negative for inducible ischemia. Two dimensional (2D) echocardiogram showed no regional wall motion abnormalities with good biventricular function. There was no mitral or tricuspid regurgitation. During coronary-angiograhy showed a ventricular arrhythmia in ECG monitor. Conventional contrast coronary angiogram showed ostio-proximal LMCA 60 to 70% stenosis, with proximal left anterior descending coronary artery (LAD) 50% stenosis, and right coronary artery as normal. Percutaneous coronary intervention (PCI) was planned with intravascular ultrasound (IVUS) guidance. LMCA was normal in different views. Normalization of LMCAS suggests that there was completely only spasm in LMCA. On 24-hour Holter monitoring showed normal limit and there was no syncope and palpitation. Arrhytmia was not found, with minimal and maximal heart rate was 53 bpm and 69 bpm, respectively. Left Main Coronary Artery Spasm (LMCAS): Not Always Manifest as Chest Pain R. Abimanyu1, F. Habib2, C.A. Andra2 Left Main Coronary Artery Spasm (LMCAS): Not Always Manifest as Chest Pain R. Abimanyu1, F. Habib2, C.A. Andra2 Left Main Coronary Artery Spasm (LMCAS): Not Always Manifest as Ches R. Abimanyu1, F. Habib2, C.A. Andra2 1Cardiology Resident at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2Cardiologist at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 1Cardiology Resident at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia 2Cardiologist at H. Adam Malik Cardiac Center, Faculty of Medicine Universitas Sumatera Utara, Medan, Indonesia Conclusion This case demonstrates that syncope and ventricular arrhythmia could be related to LMCAS. We missed the diagnosis of LMCAS during the first admission because the patient did not complain of chest pain and we did not identify abnormal ST-segment elevation. Thus, syncopal patient of LMCAS without typical chest pain may be misdiagnosed as in this case. Therefore, in the event of an unexplained syncope, it is important to interpret Holter monitoring carefully and to consider coronary spasm provocation test for the diagnosis of LMCAS, if clinically indicated. Conclusion Only timely diagnosis and prompt surgery can reduce the morbidity and mortality of cardiac myxoma patients. Only timely diagnosis and prompt surgery can reduce the morbidity and mortality of cardiac myxoma patients. Keywords : Myxoma, Cardiac tumor, Obstruction, Echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Keywords LMCAS, Syncope, Ventricular Arrhythmia, Holter monitoring. Indonesian Journal of Cardiology Percutaneous Coronary Internvention of Infarct Related Artery vs Non Infarct Related Artery in Patient with Cardiogenic Shock Keywords : STEMI, Cardiogenic Shock, Multi vessel disease, PCI, Non Infarct related artery Keywords Keywords LMCAS, Syncope, Ventricular Arrhythmia, Holter monitoring. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indones Ca Indonesian J C pISSN: 2830-3 doi: 10 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Percutaneous Coronary Internvention of Infarct Related Artery vs Non Infarct Related Artery in Patient with Cardiogenic Shock A.S. Pratama1, N.Y. Kusumawardhani 2 A.S. Pratama1, N.Y. Kusumawardhani 2 1Faculty of Medicine, Padjadjaran University, Bandung, Indonesia 2Departement cardiology and Vascular Medicine, Hasan Sadikin Hospital, Bandung, Indonesia Background : Multivessel disease (MVD) was observed 40-65% of patients with STEMI. Non- infarct related artery stenosis (non-IRA) in patient with MVD was associated with increased mortality and MACE. Complete revascularization in STEMI with cardiogenic shock patients was recommended by guidelines but the supporting evidence has been scarce and it was only based on expert consensus considerations. Case Illustration : 63 years old male was admitted due angina. Onset 8 hours prior to admission. Patient appeared to be restless, hypotension with poor hypoperfusion on admission. ECG showed extensive anterior STEMI, CRBBB. Patient was diagnosed with extensive anterior STEMI with cardiogenic shock. After stabilization with inotropes, patient underwent cardiac catheterization. Angiongraphic findings showed acute total occlusion at LAD with severe calcification and significant lesion on ostial – distal LCx and ostial – distal RCA. PCI was performed in the proximal LAD as infarct related artery. On the next day, patient experienced another episode of angina. ECG monitor showed total AV block rhythm and patient was hemodynamically unstable. Another PCI procedure was performed to RCA. However, patient experienced cardiac arrest intra procedure, CPR was performed but to no avail. Conclusion : Reperfusion of culprit is main treatment in the STEMI patients, although multivessel PCI in stable STEMI patients can be considered in non-complex lesions. Culprit only approach should be performed in hemodynamic unstable patients such as cardiogenic shock. Complete revascularization still have a significant benefit in terms of mortality but the existence of MVD itself has a poorer outcome regardless appropriate approach to the patient. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rupture Sinus of Valsalva Aneurysm Associated With a Ventricular Septal Defect: The Importance of Intraoperative Transesophageal Echocardiography D.P. Sejati1, D.W. Anggraheni1, Noormanto2 1Department of Cardiology and Vascular Medicine, Dr Sardjito Hospital, Yogyakarta/Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia 2Department of Pediatric Medicine, Dr Sardjito Hospital, Yogyakarta/Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rupture Sinus of Valsalva Aneurysm Associated With a Ventricular Septal Defect: The Importance of Intraoperative Transesophageal Echocardiography D.P. Sejati1, D.W. Percutaneous Coronary Internvention of Infarct Related Artery vs Non Infarct Related Artery in Patient with Cardiogenic Shock Anggraheni1, Noormanto2 1Department of Cardiology and Vascular Medicine, Dr Sardjito Hospital, Yogyakarta/Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia 2Department of Pediatric Medicine, Dr Sardjito Hospital, Yogyakarta/Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia Background: Sinus of Valsava Aneurysm (SOVA) is a rare heart defect and is an acquired heart defect and/or a congenital heart defect. This case has a prevalence of 0.09% in the general population and only about 1-3.5% of congenital heart defects. The rupture sinus of valsava aneurysm (RSOVA) occurs most often towards RV(60%), then to RA(29%), LA(6%), LV(4%), and Pericardium(1%). Complications from a rupture that occurs suddenly can occur hemodynamic disorders that can lead to acute heart failure. The diagnosis of RSOVA can be established by multimodality imaging. Case Illustration: A 16-years-old male patient with an unconfirmed report of VSD since infancy, came with his parents to the outpatient clinic RSUP Sardjito, with the chief complaint of shortness of breath during sports activities and at rest. TTE showed RSOVA of Right Coronary Cuspis (RCC) into the right ventricle (RV) with a classic sign of Windsock Appearance, further confirmed by TEE. We further decide to proceed to for repair of the RSOVA. Unexpectedly, the preprocedural TEE revealed a Left to Right high PMO VSD that coexisted with the its RSOVA. Therefore, the surgical team proceeded to VSD closure along with the repair of RSOVA and it went successfully. During follow-up, the condition was improved with no complications and no residual defects was observed on TTE. Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 The prevalence of a coexisting VSD and SOVA is about 30-60%, in this case, the patient was late to come to our hospital and was diagnosed with RSOVA and High PMO VSD, and then we successfully repaired both lesion, with a good outcome. Performing TEE with and without sedation sometimes gives different flexibility in obtaining views, especially when certain maneuver is needed. Carefully caution is needed in looking at the results of multimodality imaging, especially if there were discrepancies in results to make a diagnosis of RSOVA with coexisting lesions. Discussion and Conclusion: Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 The prevalence of a coexisting VSD and SOVA is about 30-60%, in this case, the patient was late to come to our hospital and was diagnosed with RSOVA and High PMO VSD, and then we successfully repaired both lesion, with a good outcome. Performing TEE with and without sedation sometimes gives different flexibility in obtaining views, especially when certain maneuver is needed. Carefully caution is needed in looking at the results of multimodality imaging, especially if there were discrepancies in results to make a diagnosis of RSOVA with coexisting lesions. Keywords: y rupture sinus of valsava aneurysm, ventricular septal defect, intraoperative transesophageal echocardiography Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Patient with Patent Ductus Arteriosus and Infective Endocarditis Complicated with Worsening Left Ventricular Dysfunction Following Surgical Intervention A.N. Nurman1, C.J. Cool1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Padjadjaran, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Patient with Patent Ductus Arteriosus and Infective Endocarditis Complicated with Worsening Left Ventricular Dysfunction Following Surgical Intervention A.N. Nurman1, C.J. Cool1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Padjadjaran, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia Background: Patent ductus arteriosus (PDA) is a congenital heart disease that can cause left ventricular (LV) volume overload. PDA closure has been reported to cause deterioration of LV function. Case Illustration: A 23-year-old female was admitted with recurrent fever three months prior to arrival. She had history of PDA but never had routine follow-ups. She had cardiomegaly with continuous murmur grade III/VI at the 2nd left intercostal space. Her electrocardiography (ECG) showed sinus tachycardia and left ventricular hypertrophy. Transthoracic echocardiography (TTE) showed PDA (7-8 mm) continuous flow, left ventricular ejection fraction (LVEF) 46% with mild global hypokinetic, and multiple vegetations at main pulmonary artery. Blood culture showed positive for Staphylococcus hominis, Streptococcus anginosum, and Staphylococcus epidermidis. After two weeks of antibiotics administration, blood culture remained positive despite reduced vegetations size. Vegetation evacuation and PDA ligation were performed successfully. However, TTE evaluation showed worsening systolic function (LVEF 39%) without vegetation and residual PDA shunt. She received heart failure medications and was discharged in stable condition. Ten months thereafter, TTE evaluation showed improved systolic function (LVEF 44%). The deterioration of LV function can be caused by sudden reduction of LV volume after PDA closure which reduces muscle fibers stretch and decreases LVEF. Another explanation is increased afterload because LV ejects blood only to high-resistance systemic circulation after PDA closure. Several parameters, such as large PDA size, reduced preoperative LVEF, low fractional shortening, and high left ventricular end-diastolic diameter, have been shown to predict LV dysfunction after PDA closure. After closure, LV reverse remodelling can occur, resulting in improved LV function. Conclusion: PDA closure can cause deterioration of LV function. Keywords: Infective Endocarditis, Patent Ductus Arteriosus, Left Ventricular Dysfunction Background g Both acute ischemic stroke (AIS) and acute myocardial infarction (AMI) are life-threatening medical diseases with a short therapeutic window and a poor prognosis if not treated quickly. Management of both AIS and AMI at the same time is challenging. A delayed intervention of one infarcted zone for the other could result in irreversible morbidity, disability, and even death. Case Illustration A 48 years old woman came to ER with a chief complaint of chest pain, difficulty speaking, and had weakness in the right limb that she never felt before 27 hours before being admitted. The ECG was sinus tachycardia with RBBB-type ST elevation in the anterior lead. Her blood lab results showed leukocytosis and increased troponin levels. The chest x-ray showed cardiomegaly. The head CT-Scan revealed extensive infarction with vasogenic edema in the left temporal-parietal- occipital lobe. She was assessed as evolved STEMI anterolateral and non-hemorrhagic stroke. The patient was admitted to ICCU and got dual antiplatelet (DAPT) therapy, heparin as an anticoagulant, and antibiotics. After 5 days of treatment in the ICU, chest pain was reduced, weakness of the right limb improved and she was able to speak with limited vocabulary. Keywords : Stroke, Myocardial Infarction, Cardio-Cerebral Infarction Keywords: There are several parameters that can help to predict LV dysfunction. The LV dysfunction is mostly reversible and can take months to fully recover. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Appropriate Management of Simultaneous Acute Cardio-Cerebral Infarction: pp p g Are We There Yet? T.B. Prakoso1, S. Sungkar2, M.F. Ahnaf2, Retnaningsih3 1Faculty of Medicine, Diponegoro University, Semarang, Indonesia 2Department of Cardiology and Vascular Medicine, Kariadi Hospital, Semarang, Indonesia 3Department of Neurology, Kariadi Hospital, Semarang, Indonesia A Rare Entity Of Right-Sided Infective Endocarditis as a result of Uncorrected Ventricular Septal Defect: A Case Report A Rare Entity Of Right-Sided Infective Endocarditis as a result of Uncorrected Ventricular Septal Defect: A Case Report I. Munawarah1, T. Heriansyah1, Novita1 1) Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Zainoel Abidin General Hospital, Banda Aceh, Indonesia I. Munawarah1, T. Heriansyah1, Novita1 1) Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Zainoel Abidin General Hospital, Banda Aceh, Indonesia Conclusion Currently, there are no clinical trials or consensus guidelines for the management of CCI. It is necessary to identify the role of antiplatelet and thrombolytic drugs and a combination of percutaneous coronary and intravascular treatments of the brain. With current limited knowledge, the management approach needs to be individualized. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 A Rare Entity Of Right-Sided Infective Endocarditis as a result of Uncorrected Ventricular Septal Defect: A Case Report I. Munawarah1, T. Heriansyah1, Novita1 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology words: right-sided infective endocarditis, uncorrected ventricle septal defect, i ABSTRACT Background: Right-sided infected endocarditis (IE) has a lower prevalence and lower fatality rates as compared to left-sided IE. Infections of implantable cardiac devices, central venous catheters, and misuse of intravenous substances are commonly associated to right-sided IE. Congenital heart disease (CHD) are very less common to result in right- sided IE, among those, ventricular septal defects (VSD) has the highest percentage of up to two percent. p Case illustration: A 26-years-old male was presented to the emergency department with malaise, fever, fatigue and palpitation. The patient was diagnosed with CHD since a young age, but had refused surgical correction. Physical examination showed normal vital signs, abnormal auscultation: loud pansystolic murmur was heard at the lower left sternal border, systolic ejection murmur was heard in the upper left sternal border, and rales at the base of both lungs. Transthoracic echocardiography showed perimembranous VSD with left-to-right shunt and 24×10 mm vegetation on the septal leaflet of the tricuspid valve with mild tricuspid regurgitation. Heart failure treatment was given to the patient, IE was treated with ampicillin sulbactam 1 g t.i.d and gentamycin 80 mg b.i.d despite the blood culture results showed no bacteria were found. Uncorrected VSD is a predisposing factors of IE. In addition to the left-to-right shunt VSD contributes to the increased blood flow through pulmonary artery, promoting pathologic pulmonary vascular shear stress and circumferential stretch. This high flow causes endothelial disruption in the turbulence loci and triggers the vegetation formation during bacteraemia. The endothelial disruption facilitates fibrin deposition and aggregation, subsequently forming the vegetation. Conclusion: Despite the patient’s blood culture showed negative result, patient was still diagnosed with IE based on Modified Duke’s Criteria. Common pathology of the righ- sided IE, which proposes pathogen into the body, does not apply to this patient Task Keywords: right-sided infective endocarditis, uncorrected ventricle septal defect, vegetation Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. VSD Perimembranosus with flow L to R Shunt (left) and vegetation in septal leaflet of tricuspid leaflet (right). Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. VSD Perimembranosus with flow L to R Shunt (left) and vegetation in septal leaflet of tricuspid leaflet (right). PALPITATION IN YOUNG AND HEALTHY FEMALE : A CASE SERIES OF WOLFF- PARKINSON-WHITE SYNDROME A. Izzuddin 1, T. Heriansyah 2, M. Muqsith 2 Resident, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh. ardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala A. Izzuddin 1, T. Heriansyah 2, M. Muqsith 2 1) Cardiology Resident, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh. 2) Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala ABSTRACT Figure 1. VSD Perimembranosus with flow L to R Shunt (left) and vegetation in septal leaflet of tricuspid leaflet (right) Figure 1. VSD Perimembranosus with flow L to R Shunt (left) and vegetation in septal leaflet of tricuspid leaflet (right). Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 ABSTRACT Background: Wolff-Parkinson-White (WPW) syndrome refers to the presence of an overt (manifest) Accessory Pathways (AP), thus resulting in the so-called pre-excitation, in combination with usually recurrent tachyarrhythmias. y y y Case Illustration: Case 1: A 27-year-old female referred to ER with a chief complaint of shortness of breath that occurs since 2 hours. She had also experienced pre-syncopal event that occurred earlier that day. Symptoms included intermittent dizziness, lightheadedness, weakness, and palpitations of a one-hour duration. Currently, she is pregnant with a gestational age of 8 months. Her ECG showed Delta wave at lead V1-V6, III, and aVF. One day after admission, she had an episode of tachycardia. Patient then treated with vagal manouver, but the rhythm was not converted. We continued with IV Digoxin 0.5 mg, and the rhythm was converted to sinus completely. Case 2: A 31-year-old female referred to ER with a chief complaint of palpitation that occurs since 12 hours ago. Symptoms included intermittent dizziness, lightheadedness, and weakness. Her previously ECG showed a narrow complex tachycardia with retrograde P waves in II, III, aVF, and in V4 to V6. Patient treated with IV diltiazem, and after the rhythm converted, her ECG showed Delta wave at V2-V6, II, aVF. Conclusion: The hemodynamic and electrophysiological changes facilitate the development of arrhythmias. Early diagnosis and adequate multidisciplinary management are key factors to avoid complications. y p Keywords: Palpitation, Wolff-Parkinson-White Syndrome, Tachycardia, Accessories Pathway Keywords: Palpitation, Wolff-Parkinson-White Syndrome, Tachycardia, Accessories Pathway Keywords: Palpitation, Wolff-Parkinson-White Syndrome, Tachycardia, Accessories Pathway Indonesian Journal of Cardiology Indonesian Journal of Cardiology Cardiolog Indonesian J Cardiol 2023 pISSN: 2830-3105 / eISSN doi: 10.30701/ijc. Figure A. Case 1; Left-Sided AP in Type A WPW Syndrome, B. Case 2; Right-Sided AP in Type B WPW Syndrome Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure A. Case 1; Left-Sided AP in Type A WPW Syndrome, B. Case 2; Right-Sided AP in Type B WPW Syndrome Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Keywords: Buddy wire technique, PCI, Complex lesion. ST ELEVATION MYOCARDIAL INFARCTION INFERIOR AFTER PCI WITH SMALL VESSEL AT RIGHT CORONARY ARTERY, WHAT SHOULD WE DO? E. Mizwar1, A.M.Z Ismail1, A. Purnawarman1, T. Heriansyah1 1) Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Zainoel Abidin General Hospital, Banda Aceh, Indonesia Background: Cardiovascular diseases (CVDs) represents 31% of all global deaths. Acute Coronary Syndrome (ACS) constitute approximately 7 million deaths per year. Approximately 30% of ACS caused by ST Elevation Miocard Infarction (STEMI). STEMI after Percutaneous Coronary Intervention (PCI) is completely rare and not yet reported. Case Illustration: A 54-year-old woman was admitted to the hospital for elective PCI because of a history of recurrent chest pain since the previous 3 months. She had not previously undergone PCI because the onset was more than 12 hours and she was stable, so the patient was planned to undergo PCI in the next 3 months. The patient was admitted with a diagnosis of stable angina pectoris ec. Coronary Artery disease, acute on Chronic Kidney disease and hypertension. Lab results showed improvement in kidney function with serum creatinine 2.2 mg/dl. After corangiography, the results were CAD 2 VD with 70-80% stenosis in the proximal mid on the left anterior descendent (LAD) and small vessels with 80% stenosis on the right coronary artery (RCA). PCI 3 DES was performed in LAD. 6 days after PCI, she complained of severe chest pain. ECG showed ST elevation in II, III, AVF and reciprocal ST depression in I-AVL. We decided to perform fibrinolytic procedure using 1.5 million IU of streptokinase with close hemodynamic monitoring. After that, complaints of chest pain decreased and ST elevation decreased. she was discharged after hemodinamically stable and no chest pain. Conclusion: STEMI management after PCI with small vessels has not been widely reported, fibrinolytics can be considered for daily practice. Keywords: STEMI, small vessel, PCI, fibrinolitik Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. ECG at admission (above), ECG post PCI showing STEMI Inferior (Middle), and ECG 1 hour after fibrinolytic (below). Figure 1. ECG at admission (above), ECG post PCI showing STEMI Inferior (Middle), and ECG 1 hour after fibrinolytic (below). Figure 1. ECG at admission (above), ECG post PCI showing STEMI Inferior (Middle), and ECG 1 hour after fibrinolytic (below). PERCUTANEOUS CORONARY INTERVENTION MANAGEMENT WITH THE BUDDY WIRE TECHNIQUE, A SOLUTION TO SEVERAL CHALLENGING CIRCUMSTANCES: A CASE REPORT R. Novia1, H. Munirwan2 1)Faculty of Medicine, Universitas Syiah Kuala 2) Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Zainoel Abidin General Hospital, Banda Aceh, Indonesia Background: Percutaneous coronary intervention (PCI) as an invasive strategy has increased in use and reduced the mortality rate to ischemic heart disease from 17.2%(1995) to 6.3%(2015). Not all cases can be easily carried out PCI; occasionally, the operator will face problems during complex lesions. Case Illustration: A 60-year-old man was brought to the ED with a chief complaint of chest pain three days earlier. ECG showed complete RBBB, QS patterns in II, III, and AVF. Increased cardiac enzymes and decreased renal function in the laboratory. Echocardiography showed decreased LV function. An early invasive strategy performed, puncture through the dextra radial artery. Coronary angiography results; CTO of proximal RCA and distal LCx, 80-90% diffuse stenosis of proximal- distal LAD with severe calcification. PCI is conducted on the LAD, as it provides flow to the distal RCA. Balloon support was needed to allow the wire to penetrate the lesion, and coronary dilatation with a 2.5/12mm NC balloon to a pressure of 20 atm repeatedly. However, the stent could not pass through the lesion to the distal LAD. Stenting was performed first at the proximal LAD using a DES 2. 75/29mm was inflated to a pressure of 16 ATM. Finally, a buddy wire technique was performed to stent the distal LAD, using a DES 2.5/16mm inflated to a pressure of 18 ATM. With results, TIMI 3 flow and stable hemodynamics. The patient was discharged in good condition with improved LV function and increased renal function. The method using a second 0.014-inch coronary guide wire can assist in delivering balloons and stents through calcified or tortuous vessels. It delivered through radial access is also safe, switching access to femoral is unnecessary. Conclusion: The buddy wire technique is one of the simple solutions in assisting stent deployment in challenging situations, therefore improving PCI success. Keywords: Buddy wire technique, PCI, Complex lesion. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1 a. Diffuse stenosis of the LAD with severe calcification, b. stenting of the proximal LAD is carried out, c. Use of Buddy wire technique for mid-distal LAD stenting, d. TIMI 3 Flow final result. Cardiac Magnetic Resonance to diagnose Coronary Microvascular dysfunction with chronic myocarditis g g y y chronic myocarditis N. Widasari1,2, T. Heriansyah1, M. Muqsith1, Elen2 1Department of Cardiology and Vascular Medicine Universitas Syiah Kuala, Banda Aceh, Indonesia, 2Department of Cardiology and Vascular Medicine Universitas Indonesia/National Cardiovascular Centre Harapan Kita, Jakarta, Indonesia chronic myocarditis N. Widasari1,2, T. Heriansyah1, M. Muqsith1, Elen2 1Department of Cardiology and Vascular Medicine Universitas Syiah Kuala, Banda Aceh, Indonesia, 2Department of Cardiology and Vascular Medicine Universitas Indonesia/National Cardiovascular Centre Harapan Kita, Jakarta, Indonesia Case Ilustration A 72 years-old woman has been presented with frequent exertional chest pain since the last 2 months. Patient was breast cancer survivor with previous mastectomy and chemotherapy procedure 10 years ago. She also had history of hypertension. Coronary angiography revealed moderate stenosis on mid LAD, normal on other coronary arteries. CMR examination found hypokinetic at apico-anterior, basal apico-septal and basal apico inferior segments. Comparing rest and stress CMR sequences, there is a diffuse hypoperfusion at basal-apical LV on stress sequences. Intramyocardial LGE were found at basal-apico anterior, basal-mid septal, basal-mid inferior and basal inferolateral. Coronary microvascular dysfunction is defined as the clinical syndrome of angina with electrocardiographic ischemic changes in the absence of obstructive CAD. Microvascular dysfunction (MVD) are divided into four clinical setting, MVD without obstructive CAD and myocardial diseases, MVD in myocardial disease, MVD in obstructive CAD, and iatrogenic. The etiology of myocardial inflammation include viral infection, post mRNA COVID-19 vaccination, and anthracycline used as chemotherapy regiment. Anthracycline-induced cardiotoxicity and subsequent heart failure is very common in breast cancer patients. Control of traditional CV risk factors is the main therapeutic objective to prevent progression of microvascular disease and to reduce the frequency and intensity of ischemic symptoms. Introduction Microvascular dysfunction is responsible for chest pain in various clinical settting. Over 50% of symptomatic patients without flow-limiting coronary lesions have structural or functional abnormalities of the coronary microcirculation leading to impaired vasodilatation, which contributes to myocardial ischemia. In recent years, cardiovascular magnetic resonance have demonstrated to be emerging modalities for microcirculation assessment. PERCUTANEOUS CORONARY INTERVENTION MANAGEMENT WITH THE BUDDY WIRE TECHNIQUE, A SOLUTION TO SEVERAL CHALLENGING CIRCUMSTANCES: A CASE REPORT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1 a. Diffuse stenosis of the LAD with severe calcification, b. stenting of the proximal LAD is carried out, c. Use of Buddy wire technique for mid-distal LAD stenting, d. TIMI 3 Flow final result. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Case Illustration Women, 56 years old, came with complaints of shortness of breath that felt even during light activity and at rest. The patient admits that he often wakes up at night due to shortness of breath and has to use 2-3 pillows to sleep. DOE (+), OP (+), PND (+). Complaints are also accompanied by swollen feet. On physical examination, an increase in jugular venous pressure is found as high as R+3 cmH2O. On physical examination of the heart, there was irregular heart sound 1 and 2, accompanied by a murmur at the lower left sternal border which was difficult to assess, with a maximum punctum at LPAS. ECG results found atrial fibrillation rhythm with QRS rate 133x/i irregular, left anterior deviation, P wave difficult to assess, QRS duration 80 ms, Q path gel in III, avF, V1-V4, ST-T changes: T-inversion in I, avL. Impression: AF RVR + LAD. On the chest X- ray, a CTR of 76% was found, the Aortic segment was normal, Echocardiography results found: Atrial situs solitus, AV discordance, VA discordance, TR severe (PG 80 mmHg) and LA-RV dilatation. Patients were given treatment in the form of complete rest, IVFD NaCl 0.9% 10 tpm, inj furosemide 40 mg/8 hours, Spironolactone 1x25 mg, Digoxin 1x0.25 mg, Warfarin (2-3-2 mg), KSR 1x600 mg. Acute Heart Failure Caused by TR Severe and AF RVR with Congenitally Corrected Transposition of Great Arteries – A Case Report Heart Failure Caused by TR Severe and AF RVR with Congenitally Corrected Transposition of Great Arteries – A Case Report Acute Heart Failure Caused by TR Severe and AF RVR with Congenitally Corrected Transposition of Great Arteries – A Case Report Y.I. Taufik1 , R.M. Tamba2 , K.K. Ilyas3 , A.N. Nasution4 , C.A. Andra 1,2Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 3,4,5Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 1,2Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia 3,4,5Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia Background Acute heart failure refers to rapid onset or worsening of symptoms and/or signs of heart failure, requiring urgent evaluation and treatment. Heart failure results from disturbances in the structure and/or function of the heart resulting in increased intracardiac pressure and/or inadequate cardiac output. Disorders of the valves, pericardium, endocardium and disturbances of heart rhythm and conduction can also contribute to causing heart failure. Congenitally Corrected Transposition of Great Artery or CCTGA both ventricles (pumping chambers) of the heart are reversed. Fortunately, the arteries are reversed too, so the heart actually "corrects" the abnormal development, thus the name "congenitally corrected transposition of the great arteries." However, CCTGA is a complex malformation in which the heart is far from being normal. Conclusion CMR has shown promising results to distinguish epicardial versus microvascular impairment compared to invasive FFR assessment. Identification of MVD is important to manage medical strategies and improve prognosis. Keywords: Microvascular dysfunction, Myocarditis, CMR Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Acute Heart Failure Caused by TR Severe and AF RVR with Congenitally Corrected Transposition of Great Arteries – A Case Report Y I T fik1 R M T b 2 K K Il 3 A N N ti 4 C A A d 5 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology BACKGROUND Permanent Pacemaker implantation is a lifesaving procedure usually used in conditions of life- threatening bradycardia. Permanent Pacemaker (PPM) was associated with more favorable outcome in patients with cardiac conduction issues. However, the procedure of PPM implantation still resulted in operative associated complication, from superficial hematoma, until pneumothorax and lead dislodgement, several of these complications might resulted in mortality Associated with PPM implantation. In this paper we presented a case in-hospital monitoring for the patient underwent complication after PPM implantation. Post Procedural Complication after PPM Implantation : What should we do Post Procedural Complication after PPM Implantation : What should we do. S.S. Baskoro1, T. Aryanugraha1, M.A. Rosyidi1, M. Herbani1 , I. Kamelia1, S. Anjarwani2, A. Rizal2, A. Kurniawan1, F. Taufiq1 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia F. Taufiq 1Cardiology Resident, Faculty of Medicine Universitas Brawijaya- Saiful Anwar Hospital, Malang, Indonesia; 2Departement of Cardiovascular Medicine, Faculty of Medicine Universitas Brawijaya-Saiful Anwar Hospital, Malang, Indonesia CASE ILLUSTRATION A 62-year-old woman with chief complaint of syncope. Patient was having seizure multiple times on observation. From ECG was found with Total Atrioventricular Block (TAVB) with junctional escape rhythm and heart rate (HR) of 30-40 beats per minute (bpm). Due to unavailability of temporary or percutaneous pacemaker in the receiving hospital, epinephrine was used to increase the heart rate. After received in our hospital, patient was implanted with intravenous Temporary Pacemaker and previously administered epinephrine was stopped. Patient was in stable condition with BP 131/75 mmHg, HR 70x/m regular, and had insertion temporary pacemaker (TPM) at 70-5-3 configuration. Subsequent Work up ruled out electrolyte abnormalities (Hypo-hyperkalemia), and there was no regional wall motion abnormality from echocardiography and also no risk factor of coronary artery disease (CAD). Due to lack of apparent reversible causes, medical team decided on PPM implantation. The procedure was done without complication. However, at recovery room, she complained of typical chest pain, shortness of breath accompanied with hypotension (77/54 mmHg with HR 60 bpm appropriate RV pacing rhythm). Her acral was cold and wet. Echocardiography evaluation showed appropriate lead placement with minimal pericardial effusion and no sign of pneumothorax. There was no sign of tamponade. Sublingual Nitrate and Fluid resuscitation was given to the patient. The symptom was reduced, and her blood pressure slowly recovered. She was observed in the ward and discharged 3 days after. Conclusion Woman, 56 years old, diagnosed with Acute Heart Failure caused by severe TR and AF RVR with congenitally corrected transposition of great arteries based on history, physical examination and additional examinations. The patient was given therapy according to clinical manifestation and allowed to go home after 7 days of treatment. Keywords: Acute heart failure, Atrial fibrilation, CCTGA Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 CONCLUSION PPM implantation Is one of the core treatments for symptomatic bradycardia. Although its generally a safe procedure, complication regarding PPM Procedure needed to be anticipated in order to be treated accordingly. A close monitoring with trained personnel on standby is crucial to ensure patients well being after PPM implantation. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 KEYWORDS Total Atrioventricular Block (TAVB), Permanent pacemaker, post procedural monitoring Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 KEYWORDS ORDS rioventricular Block (TAVB), Permanent pacemaker, post procedural monitoring Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Flaring Osteal to Bail Out Proximal RCA Dissection during CTO PCI: A Case Report R.S. Bakry1, H. Munirwan1, T. Heriansyah1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia Indonesian Journal of Cardiology Flaring Osteal to Bail Out Proximal RCA Dissection during CTO PCI: A Case Report R.S. Bakry1, H. Munirwan1, T. Heriansyah1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia Background: The separation between two layers of coronary artery is recognized as coronary dissection. Deep engagement during catheter manipulation can induce an iatrogenic coronary dissection. Although the reported incidence was quietly few, 0.02-0.15%, it can be life threatening and need prompt surgery. Dissection in ostium RCA is more frequent than LCA with percentage 85% of all. Case Illustration: We reported a 57 years old age male with CAD 3VD post PCI 1 DES in LAD. Patient came for undergoing elective PCI. He has suffered chest pain during mild activities for last 6 months. His cardiovascular risk factor were hypertension and smoking. Physical examination revealed no abnormalities. ECG showed sinus rythm with rate 66 bpm, normoaxis, qs pattern in lead III aVF, and poor r wave progression in V1-4. We performed CTO PCI through femoral access. Angiography showed patent stent in LAD, diffuse stenosis 90% in osteal to distal LCx, and CTO with calcification in proximal RCA. Operator decided to perform CTO PCI in Proximal RCA using AL1 guide-catheter. During procedure, the dissection was occurred in proximal RCA. CONCLUSION We performed flaring the ostium using noncompliant balloon, protruding into the aorta flare the aortic portion of the stent using the stent balloon. Then, the angiography showed that RCA dissection was improved. The results of PCI were CAD 3VD + Stent patent in LAD and PCI 2DES in RCA. Conclusion: Flaring stent in the ostium of RCA by inflating balloon proximal to dissected segment was necessary for tack down the dissection flap resulting in flow improvement. It can be a solution for bailing out under the circumstances. Keyword: Flaring Osteal, CTO PCI, Coronary Dissection Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_ pISSN: 2830-3105 / eISSN: 2964-730 doi: 10.30701/ijc.1537 Figure 1. CTO in Proximal RCA with JCTO score 3, (A). Dissection In Proximal RCA, (B). Stenting in osteal-proximal RCA, (C). TIMI 3 Flow after flaring balloon in osteal RCA, (D). Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Figure 1. CTO in Proximal RCA with JCTO score 3, (A). Dissection In Proximal RCA, (B). Stenting in osteal-proximal RCA, (C). TIMI 3 Flow after flaring balloon in osteal RCA, (D). Transcatheter Post Infarct VSR Closure Using ASD Device : An Alternative Option in Scarcity y A.M.A.A. Lubis1, A.C. Lubis2 , A.N. Nasution3 , C.A. Andra4, T.W. Ardini5, K.K. Ilyas 6 1Cardiology Resident, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia; 2,3,4,5,6Cardiologist, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia Background: Transmural MI is a predisposition for ventricular septal rupture which occurs more frequently after STEMI than NSTEMI. Without surgical repair or percutaneous closure, the mortality rate can reach 94%. Although surgical repair has been the gold standard to correct the structural anomaly, percutaneous closure may represent a valuable therapeutic alternative in selected patient, with the purpose of immediate shunt reduction to prevent further hemodynamic deterioration. Case Illustration: A 60 – year – old woman with post infarction ventricular septal rupture and recent anterolateral STEMI was referred from Lhokseumawe with dyspnea as chief complaint which had been experienced for seventeen days before admission. At first, she refused a referral and eventually came with complicating pneumonia. She was admitted to the ICCU without hemodynamic instability and treated with afterload reduction strategy by using vasodilator. The CAG revealed a total occlusion in LAD after D2 and 70 – 80% stenosis in LCX after OM1. The left ventriculography showed VSR (Ø8 mm), L-R shunt. The TTE revealed a ventricular septal rupture Ø 6 x 12 mm. On the 21st day of the PI – VSR, she underwent a TEE and fluoroscopy guided transcatheter closure using the ASO Occlutech device No. 18 mm. The TEE showed that the device was well positioned with minimal central leakage. Shortly after the closure, the cardiac performance improved, proven by an increase of LVOT VTI from 6.3 cm to 13 cm. The patient then moved to the ward two days after the procedure. During the follow up, the patient unfortunately died of Pseudomonas aeruginosa infection four days after the closure despite a successful procedure and hemodynamic improvement. Conclusion: It is not yet clear which device is the best option for the treatment of a VSR due to anatomical difference. The P.I. Muscular VSD Occluder was specifically designed for postinfarction VSD closure. However, when it is not available, other device can become an alternative. Keyword : PI VSR, Transcatheter Closure Indonesian Journal of Cardiology Keywords: CHD, bifurcation lesion, PCI Introduction Chest pain is a common complaint in the emergency department that prompts accurate diagnosis and treatment. Aortic dissection (AD) and non-ST segment elevation acute coronary syndrome (ACS) are two of the most life-threatening diseases encountered in the emergency department that presented with chest pain. The failure in differentiating the two might resulted in improper and potentially harmful treatment. Here we presented a case of patient with acute chest pain diagnosed with ACS that turned out to be a large aortic root aneurysm with impending rupture. Rupture of Large Aortic Root Aneurysm Presenting as Acute Coronary Syndrome: The Importance of Triple Rule Out Computed Tomography F.A. Sihotang1, V. Mayangsari2, S. Anjarwani2, N. Kurnianingsih2 1Resident of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya, Malang, Indonesia 2Department of Cardiology and Vascular Medicine, Saiful Anwar General Hospital, Malang, Indonesia nt of Cardiology and Vascular Medicine, Saiful Anwar General Hospital, Malang, Dual Stent Strategy in Management of Bifurcation Lesion Kurnianingsih2 1Resident of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Brawijaya, Malang, Indonesia Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rupture of Large Aortic Root Aneurysm Presenting as Acute Coronary Syndrome: The Importance of Triple Rule Out Computed Tomography Dual Stent Strategy in Management of Bifurcation Lesion Dual Stent Strategy in Management of Bifurcation Lesion C. Kurniawan1, H.W. Rawung1, S.H. Rampengan1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Manado C. Kurniawan1, H.W. Rawung1, S.H. Rampengan1 1Department of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Manado C. Kurniawan1, H.W. Rawung1, S.H. Rampengan1 tment of Cardiology and Vascular Medicine, Faculty of Medicine, Sam Ratulangi University, Manado Background: Complex coronary lesions are frequently found and present with higher complication rates. Complex lesion include bifurcation lesion, calcified lesion, chronic total occlusion, left main coronary artery lesion, and ostial lesion. There are many different ways in managing a bifurcation lesion, and a standard guidance is not yet found. Case illustration: A male patient, 49 years old, was admitted with chief complaint of episodic chest pain with typical angina quality. Patient had a history of dyslipidemia, hypertension, and was a heavy smoker. Echocardiography 1 week prior show good left ventricular function. Treadmill test show positive ischemic response with high risk features. Laboratory examination show slightly increased LDL (102 mg/dL) and no remarkable increase in cardiac enzyme. ECG show T inversion in I and aVL. Chest X-ray was within normal limits. Based on history taking, physical examination, and diagnostic studies, patient was diagnosed with stable angina pectoris Canadian Cardiovascular Society (CCS) classification II-III, dyslipidemia, and hypertension. During the fourth day of admission, patient undergo coronary angiography revealing distal discrete stenosis 60% in Left Main (LM) with bifurcation lesion (medina 1-1-1), diffuse stenosis Left Anterior Descending (LAD) 90% osteal-proximal, diffuse stenosis Left Circumflex (LCx) 70-80% osteal-proximal, and normal RCA. Because of stenosis in LAD and LCx >= 50% with diameter >= 2.75 mm, dual stent strategy was chosen for this patient, in which stenting were done in main and side branches simultaneously. Patient had 2 stent implanted in LM-LAD and LM-LCx. Angiographic evaluation reveal TIMI Flow 3, no residual thrombus. Conclusion: Bifurcation lesion is a complex lesion with high complication during PCI procedure. Dual stent strategy is shown to be a safe technique in this case report, with acceptable long-term prognosis. Keywords: CHD, bifurcation lesion, PCI Keywords: CHD, bifurcation lesion, PCI Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Rupture of Large Aortic Root Aneurysm Presenting as Acute Coronary Syndrome: The Importance of Triple Rule Out Computed Tomography F.A. Sihotang1, V. Mayangsari2, S. Anjarwani2, N. Indonesian Journal of Cardiology Indonesian Journal of Cardiology ventricular ejection fraction. A CT-angiography of the aorta was suggestive of impending rupture of aneurysm. Patient was treated conservatively and was planned for referral to higher centre for surgical management of the aortic root aneurysm. Unfortunately, on the fifth day of care, an acute type A aortic dissection that extent superiorly to the carotid and inferiorly to the iliac occurred. Patient went into hypovolemic shock, oliguria and later acute lung oedema of cardiac type occurred. Breathing was managed with non-invasive ventilation and when the respiratory failure became severe and patient was planned for invasive ventilation, he went into cardiac arrest with unsuccessful resuscitation. Discussion In the setting of very high risk NSTEACS, the need of immediate revascularization is of outmost importance. However, this case demonstrated that aortic aneurysm can be falsely diagnosed as ACS; with the two are very different in the terms of treatment approach. Triple rule out computed tomography (TRO-CT) that noninvasively evaluates the coronary circulation, pulmonary arteries and thoracic aorta within a single scan might be of value in this scenario. Case Illustrations A 45-year old man with risk factors of active smoker and history of alcohol consumption presented to ER with acute de novo chest pain that occurred while the patient was resting accompanied with palpitation. At the ER, patient was tachycardic with electrocardiogram demonstrated a narrow complex tachycardia of 185 bpm suggestive of atrioventricular re-entry tachycardia. Patient was given electrical cardioversion and returned to sinus rhythm with ST elevation at aVR and ST depression at I, II, aVF, V4-V6 suggestive of very high risk NSTEACS and WPW pattern suggestive of right anteroseptal accessory pathway. An invasive strategy was performed that surprisingly demonstrated a normal coronary angiography. Echocardiography showed an aortic root aneurysm with moderate to severe aortic regurgitation without intimal flap and reduced left Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 ventricular ejection fraction. A CT-angiography of the aorta was suggestive of impending rupture of aneurysm. Patient was treated conservatively and was planned for referral to higher centre for surgical management of the aortic root aneurysm. Unfortunately, on the fifth day of care, an acute type A aortic dissection that extent superiorly to the carotid and inferiorly to the iliac occurred. Patient went into hypovolemic shock, oliguria and later acute lung oedema of cardiac type occurred. Breathing was managed with non-invasive ventilation and when the respiratory failure became severe and patient was planned for invasive ventilation, he went into cardiac arrest with unsuccessful resuscitation. Summary Triple rule out computed tomography (TRO-CT) should be considered in cases where the diagnosis of acute coronary syndrome and aortic dissection cannot be differentiated from clinical presentation and standard diagnostic tests. Keywords: acute coronary syndrome, aortic dissection, triple rule out CT Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Keyword: Adult Patent Ductus Arteriosus, Eisenmenger Syndrome, Pulmonary Hypertension, Transthoracic Echocardiography. Echocardiography Found Pulmonary Hypertension, Eisenmenger Syndrome in Adult Woman with Diminished Signs of Patent Ductus Arteriosus: A Case Report R. Hilman1, Novita1, T. Heriansyah1, M. Muqsith1 1 Department of Cardiology and Vascular Medicine, Medical Faculty, Syiah Kuala University Background: Patent Ductus Arteriosus is common congenital heart disease, commonly recognized and treated in childhood. However, 2% CHD patients did survive to adulthood undiagnosed. Prolonged patency of ductus arteriosus will cause pulmonary artery remodelling lead to pulmonary arterial hypertension and further into Eisenmenger Syndrome. In Eisenmenger Syndrome murmur, disappeared causing difficulty in diagnosis. Echocardiography can assess pulmonal hypertension severity, and PDA flow, since PDA closure might beneficial in pulmonary arterial hypertension setting, while not in Eisenmenger Syndrome. Case Illustration: A 29 years old women came to outpatient clinic with complain shortness of breath since 10 years ago and gradually worsen, and patient sometimes became cyanotic during exertion. Patient catch a cold often since her childhood and her symptoms got worsen during cold. The patient was the 7th alive child in her family, her mother experienced natural abortion 3 times. She was born during her mother 7th months of pregnancy. Physical examination found clubbing finger and no murmur was heard. ECG found incomplete RBBB with right axis deviation. Chest X-ray found increased vascularization in the central and widened intercostal space. Echocardiographic assessment found PDA bidirectional shunt, dominant right to left shunt with normal LV systolic and diastolic function, normal RV function and normal cardiac dimension. Pulmonary pressure was calculated by estimating systolic and diastolic pressure gradient with CW doppler and substracting this value from systemic systolic and diastolic pressure, Pulmonary pressure was found 117/65 mmHg which suggest severe pulmonary hypertension. Qp:Qs was 0.49 which represent Eisenmenger Syndrome. Conclusion: The presence of PDA was very obscured in this patient, because of the Eisenmenger syndrome, murmur was no longer heard. Without TTE, we could only notice the presence of cyanotic heart disease with pulmonary hypertension. Transthoracic echocardiography is reliable in finding congenital heart defect and its possibility for correction. Keyword: Adult Patent Ductus Arteriosus, Eisenmenger Syndrome, Pulmonary Hypertension, Transthoracic Echocardiography. Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian Journal of Cardiology Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Indonesian J Cardiol 2023:44:suppl_A pISSN: 2830-3105 / eISSN: 2964-7304 doi: 10.30701/ijc.1537 Undetected Large Patent Ductus Arteriosus from Transthoracic Echo I.G.B.B Wijaksana1, L.O.S Suastika2, N.L.M.A.W Sari2 I. B.R. Wibhuti2, M.S.Y. Dewangga2 E. Gunawijaya2, N. P.V.K Yantie2 Faculty of Medicine, Udayana University, Bali, Indonesia Prof. dr. Echocardiography Found Pulmonary Hypertension, Eisenmenger Syndrome in Adult Woman with Diminished Signs of Patent Ductus Arteriosus: A Case Report I.G.N.G Ngoerah, Bali, Indonesia General Hospital Background: Patent ductus arteriosus (PDA) presents as a persistent communication between the proximal descending aorta and the roof of the main pulmonary artery. Late presenting large PDA is a rare finding and could be undetected from initial transthoracic echocardiography (TTE). Case Illustration: We present a 32 y.o women with dyspnea on effort and orthopnea increasing in the past year. She had history of heart disease since childhood but did not seek medical attention. She had physical examination significant for differential cyanosis. ECG showed SR, RAE, RVH and T Inversion inferior and anterior leads. Chest X-ray showed cardiomegaly and signs of pulmonary conus dilatation. TTE showed RV dilatation, Mild MR, Mild TR with high probability of PH, Global hypokinetic with decreased LV and RV systolic function (EF BP 36.8% TAPSE 16 mm) and small muscular VSD with bidirectional shunt. Our working diagnosis was RHF caused by PH caused by suspected PDA, but no PDA was visible from initial TTE. Our next diagnostic approach was heart catheterization due to unavailability of CT scan at the time and to evaluate hemodynamic and feasibility of defect closure. Aortography revealed a large type A1 PDA with isthmus of 17 mm. Hemodynamic studies showed high pulmonary vascular resistance (PARI 24 WU.m2) which improved to 9 WU.m2 after acute vasoreactivity testing. PDA closure using ASO no 24 was successfully performed. Difficulty in PDA visualization from echocardiography could be explained by high PA pressure and resistance, poor echo view, or anatomic variation. In cases of conflicting clinical and TTE findings, heart catheterization (aortography) can be considered. Conclusion: TTE evaluation of PDA in late stages of presentation could be difficult. Differential cyanosis and unexplained signs of PH could be used as a clinical clue. Heart catheterization can be considered in cases with conflicting clinical and TTE findings Keywords: Patent Ductus Arteriosus, Differential Cyanosis, Transthoracic echo Keywords: Patent Ductus Arteriosus, Differential Cyanosis, Transthoracic echo Initial TTE showing no PDA from PSAX view meanwhile heart catheterization at LAO 90°, Cranial 0° showing large PDA Initial TTE showing no PDA from PSAX view meanwhile heart catheterization at LAO 90°, Cranial 0° showing large PDA
https://openalex.org/W2794806522	https://europepmc.org/articles/pmc5974482?pdf=render	English	null	Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection	Journal of virology	2,018	cc-by	17,173	VIRUS-CELL INTERACTIONS Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection Naﬁseh Chalabi Hagkarim,a Ellis L. Ryan,a* Philip J. Byrd,a Robert Hollingworth,a Neil J. Shimwell,a Angelo Agathanggelou,a Manon Vavasseur,a Viktoria Kolbe,b Thomas Speiseder,b Thomas Dobner,b Grant S. Stewart,a Roger J. Granda Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection Naﬁseh Chalabi Hagkarim,a Ellis L. Ryan,a* Philip J. Byrd,a Robert Hollingworth,a Neil J. Shimwell,a Angelo Agathanggelou,a Manon Vavasseur,a Viktoria Kolbe,b Thomas Speiseder,b Thomas Dobner,b Grant S. Stewart,a Roger J. Granda This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Grant S. Stewart, g.s.stewart@bham.ac.uk, or Roger J. Grand, r.j.a.grand@bham.ac.uk. * Present address: Ellis L. Ryan, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, United Kingdom. N.C.H. and E.L.R. made equal contributions to this work. jvi.asm.org 1 Received 23 November 2017 Accepted 9 March 2018 Received 23 November 2017 Accepted 9 March 2018 Received 23 November 2017 Accepted 9 March 2018 Accepted manuscript posted online 28 March 2018 Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection Naﬁseh Chalabi Hagkarim,a Ellis L. Ryan,a* Philip J. Byrd,a Robert Hollingworth,a Neil J. Shimwell,a Angelo Agathanggelou,a Manon Vavasseur,a Viktoria Kolbe,b Thomas Speiseder,b Thomas Dobner,b Grant S. Stewart,a Roger J. Granda aInstitute of Cancer and Genomic Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham, United Kingdom bHeinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany ABSTRACT Infection by most DNA viruses activates a cellular DNA damage re- sponse (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by target- ing a number of proteins for rapid proteasome-mediated degradation. We have now identiﬁed a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus sero- type 5 and adenovirus serotype 12. In both cases, degradation requires the ac- tion of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be sero- type speciﬁc, as the protein remains relatively stable following infection with ad- enovirus serotypes 4, 7, 9, and 11. We have gone on to conﬁrm that Tab182 is an integral component of the CNOT complex, which has transcriptional regula- tory, deadenylation, and E3 ligase activities. The levels of at least 2 other mem- bers of the complex (CNOT3 and CNOT7) are also reduced during adenovirus in- fection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early re- gion 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previ- ously unidentiﬁed major target for adenoviruses during infection. Received 23 November 2017 Accepted 9 March 2018 Accepted manuscript posted online 28 March 2018 Citation Chalabi Hagkarim N, Ryan EL, Byrd PJ, Hollingworth R, Shimwell NJ, Agathanggelou A, Vavasseur M, Kolbe V, Speiseder T, Dobner T, Stewart GS, Grand RJ. 2018. Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1, following adenovirus infection. J Virol 92:e02034-17. https://doi.org/10.1128/JVI.02034-17. Editor Lawrence Banks, International Centre for Genetic Engineering and Biotechnology Copyright © 2018 Chalabi Hagkarim et al. Accepted manuscript posted online 28 March 2018 There are in excess of 70 human adenovirus types, subdivided into 7 species designated groups A to G; the most commonly studied are the group C adenovirus serotypes 2 and 5 (Ad2 and Ad5) and the group A oncogenic Ad12. Adenoviruses have a linear double-stranded DNA ge- nome that is approximately 35 kbp in length. The ﬁrst gene to be expressed following infection is adenovirus early region 1A (AdE1A), which is present in two major forms, a long form and a short form, translated from 13S and 12S mRNAs, respectively. AdE1A induces the progression of the host cell into a “pseudo-S phase” through interactions with a number of cellular proteins, such as the retinoblastoma (Rb) family, CBP/p300, and components of the cellular transcriptional machinery (2–4). It is considered that this provides an environment conducive to viral replication. Adenovirus E1A is the major adenovirus oncogene and has long been known to transform cells in culture in combina- tion with a cooperating oncogene, such as mutant Ras or adenovirus E1B (3, 5). A d b Shortly after initial infection, the host cell initiates a DNA damage response (DDR), seen as the phosphorylation of a number of well-characterized ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) substrates (6–8). It is presumed that this may be due to the recognition of the viral genome as broken cellular DNA or perhaps due to stress caused by infection itself. The virus, in turn, is able to inhibit the DDR, primarily by the degradation and/or mislocalization of its key components (7–12). The cellular DDR comprises a series of pathways that have evolved to deal with different forms of DNA damage, such as double-strand breaks (DSBs), single-strand breaks (SSBs), and the formation of bulky adducts and base mismatches (13–15). The response to DSBs is based largely on the activities of three kinases, ATM, ATR, and DNA-dependent protein kinase (DNA-PK). DSBs can be detected by both the MRN complex (comprising MRE11, Rad50, and NBS1) and the Ku70/80 heterodimer, which can lead to repair by homologous recombination (HR) and nonhomologous end joining (NHEJ), respectively. The recognition of DSBs by the MRN complex is followed by the recruitment of ATM, which is activated by acetylation by Tip60, while the binding of Ku70/80 results in the autophosphorylation of DNA-PK that is required for NHEJ (16–18). Accepted manuscript posted online 28 March 2018 Citation Chalabi Hagkarim N, Ryan EL, Byrd PJ, Hollingworth R, Shimwell NJ, Agathanggelou A, Vavasseur M, Kolbe V, Speiseder T, Dobner T, Stewart GS, Grand RJ. 2018. Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1, following adenovirus infection. J Virol 92:e02034-17. https://doi.org/10.1128/JVI.02034-17. IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have conﬁrmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT pro- teins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during in- fection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the ﬁrst time that the CNOT complex has been identiﬁed as an adenoviral target. Editor Lawrence Banks, International Centre for Genetic Engineering and Biotechnology Copyright © 2018 Chalabi Hagkarim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Grant S. Stewart, g.s.stewart@bham.ac.uk, or Roger J. Grand, r.j.a.grand@bham.ac.uk. * Present address: Ellis L. Ryan, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, United Kingdom. * Present address: Ellis L. Ryan, Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, United Kingdom. N.C.H. and E.L.R. made equal contributions to this work. N.C.H. and E.L.R. made equal contributions to this work. YWORDS adenovirus E1B55K, Tab182, TNKS1BP1, CNOT complex, CNOT1, KEYWORDS adenovirus E1B55K, Tab182, TNKS1BP1, CNOT complex, CNOT1, AdE1B55K, adenoviruses KEYWORDS adenovirus E1B55K, Tab182, TNKS1BP1, CNOT complex, CNOT1, AdE1B55K, adenoviruses Journal of Virology June 2018 Volume 92 Issue 12 e02034-17 jvi.asm.org Chalabi Hagkarim et al. Journal of Virology A denoviruses, together with the papillomaviruses and polyomaviruses, are mem- bers of the small DNA tumor virus family (1). Accepted manuscript posted online 28 March 2018 Histone H2AX and multiple downstream targets are phosphorylated by ATM, which has the effect of recruiting a large number of components to the lesion to initiate repair as well as to cause cell cycle arrest so that damaged DNA is not replicated (13–17). ATR is activated in response to single-stranded DNA (ssDNA), which can arise as a result of DSB repair and stalled replication forks. Regions of ssDNA are coated with replication protein A (RPA), which in turn recruits ATR and the ATR-interacting protein (ATRIP). Further complexes, comprising Rad9-Rad1 and Hus1 (9-1-1) and Rad17- replication factor C2 (RFC2) clamp loader, together with TOPBP1 are recruited to ssDNA, RPA, and ATR, leading to cell cycle arrest and repair (17–19). It was originally shown that when cells were infected with an Ad5 mutant lacking the E4 region, viral genomes were joined end to end to form concatemers that could not be packaged into viral capsids (20). It was later demonstrated that during infection with a wild-type (wt) virus, cellular E3 ligases are hijacked by the virus and used to degrade key cellular DDR proteins; for example, p53 is degraded by both Ad5 and Ad12 and requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins (21–23). In the case of Ad5, the viral proteins recruit an E3 ligase, comprising elongins B and C, Rbx, and cullin 5 (Cul5), which ubiquitylates p53, and this is then degraded by the proteasome (9). Similarly, Ad12 also facilitates the degradation of p53 but through a cullin 2-based E3 ligase (12). Other DDR proteins degraded during Ad5 and Ad12 infection include MRE11, DNA ligase IV, and Bloom syndrome RecQ-like helicase (BLM) (10, 11, 24). In addition to DDR components, a number of other unrelated proteins are also degraded during Ad5 infection. These substrates include DAXX, integrin 3, and TIF1 (25–27). During infection, adenoviruses also cause the translocation of proteins associated with the DDR. For example, ATR, ATRIP, Rad17, 53BP1, BRCA1, TOPBPI, RPA, and hnRNPUL-1 have all been observed at sites of viral replication in the nucleus, known as viral replication centers (VRCs) (6, 8, jvi.asm.org 2 June 2018 Volume 92 Issue 12 e02034-17 Adenoviruses Target Tab182 for Degradation Journal of Virology 28). In addition, it is notable that certain DDR proteins, such as p53 and MRE11, are translocated to aggresomes, where they may be degraded (29–31). Accepted manuscript posted online 28 March 2018 Tab182 (also known as tankyrase 1 binding protein 1 [TNKS1BP1]) was previously shown to be an ATM and/or ATR substrate that is highly phosphorylated following exposure to ionizing radiation (IR) and to bind to tankyrase 1 (32, 33). It appears to be required for efﬁcient DSB repair and facilitates the poly(ADP-ribose) polymerase 1 (PARP1)-dependent autophosphorylation of DNA-PK, although its precise role is not clear at present (34, 35; our unpublished data). In addition, Tab182 has a role in the regulation of the actin cytoskeleton (36). Tab182 was previously suggested to be a component of the mammalian CNOT complex, although its role in this context is unknown. The CNOT complex is a multiprotein complex that is highly conserved in eukaryotes (37–39). In humans, the CNOT complex is composed of the components CNOT1 to CNOT11 (CNOT9 and CNOT11 have the alternative nomenclature RQCD and C2orf29, respectively) (40, 41). In yeast, with which most studies of CCR4-NOT have been performed, there are 9 core subunits, Cer4, Caf1, Caf40, Caf130, and NOT1 to NOT5, although no Tab182 ortholog has been identiﬁed (38, 42, 43). The human CNOT complex consists of a stable inner complex (CNOT1, CNOT2, CNOT3, CNOT9, and CNOT10), with CNOT6, its homolog CNOT6L, CNOT7, and CNOT8 being less strongly associated. CNOT4 seems to be weakly associated, whereas Tab182 and C2orf29 (CNOT11) are more strongly bound (40, 44, 45). Many different enzymatic activities have been attributed to the CCR4-NOT complex in yeast and CNOT in mammals. It is considered to be a major deadenylase that is responsible, with Pan2-Pan3, for the shortening of the poly(A) tails of cytoplasmic RNAs (38, 46, 47). The components CNOT7 and CNOT8, together with CNOT6 and CNOT6L, are deadenylase subunits. Further components of the complex have E3 ligase, translational repression, RNA export, and nuclear surveillance activities (38, 48–50). CNOT4 is the E3 ubiquitin ligase but seems to interact only weakly with the remainder of the complex (40). CNOT1 forms a scaffold on which the CNOT and deadenylase modules are formed (41, 51, 52). The central region of CNOT1 interacts with the deadenylase subunits, with CNOT7 forming a bridge between CNOT1 and CNOT6L (39). The C-terminal region of CNOT1 binds to the remainder of the NOT module, which comprises CNOT2 and CNOT3. A number of studies have implicated the CCR4-NOT complex in the DDR in yeast. Accepted manuscript posted online 28 March 2018 In the majority of those studies, sensitivity assays were performed by using yeast strains that had mutations of various CCR4-NOT components. For example, the loss of CCR4 and Caf1 renders the yeast sensitive to IR, hydroxyurea (HU), and camptothecin, an inhibitor of DNA topoisomerase I (53–55). Similarly, NOT1 to NOT5 mutant yeast strains have been shown to be sensitive to HU (53). These data suggest that the CCR4-NOT complex is involved in the response to a number of forms of DNA damage and replication stress, although the mechanisms involved remain unclear. Here we demonstrate that Tab182 is degraded during Ad5 and Ad12 infection in an E1B55K- and E4orf6-dependent manner. We have conﬁrmed that Tab182 is a compo- nent of the CNOT complex and that the levels of at least two other components of the complex are similarly reduced during adenovirus infection. Signiﬁcantly, the depletion of Tab182 or the disruption of the CNOT complex enhances the expression of adeno- virus E1A at the transcriptional level early in infection. RESULTS Tab182 is degraded during adenovirus infection. It was previously suggested that Tab182 may have a role in the DDR based on the observation that the protein has multiple potential ATM/ATR phosphorylation sites (SQ/TQ) and is phosphorylated following exposure to IR (33) as well as its recently proposed role in DSB repair (34, 35). In a screen to detect novel DDR components targeted by adenoviruses, the effect of viral infection on Tab182 was examined. It can be seen that during both Ad5 and Ad12 infection of HeLa cells, Tab182 levels decline rapidly after 24 h (Fig. 1A and B). It is particularly notable that the levels of Tab182 increase in the initial stages of infection by both serotypes (Fig. 1; see also succeeding ﬁgures). This appears to be a cell cycle jvi.asm.org 3 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. FIG 1 Degradation of Tab182 following infection with adenovirus serotype 5 or adenovirus serotype 12 is dependent on the adenovirus E1B55K protein. (A and B) HeLa cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. (C and D) HeLa cells were also infected with the adenovirus serotype 5 E1B55K-negative virus Ad5dl1520 (C) and the adenovirus serotype 12 E1B55K-negative virus Ad12dl620 (D) at 10 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 1 Degradation of Tab182 following infection with adenovirus serotype 5 or adenovirus serotype 12 is dependent on the adenovirus E1B55K protein. (A and B) HeLa cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. (C and D) HeLa cells were also infected with the adenovirus serotype 5 E1B55K-negative virus Ad5dl1520 (C) and the adenovirus serotype 12 E1B55K-negative virus Ad12dl620 (D) at 10 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. effect, since in nocodazole “shake-off” experiments, the Tab182 expression level is highest during S phase and mitosis and is reduced in the G1 phase of the cell cycle (data not shown). Reverse transcriptase PCR (RT-PCR) analysis demonstrated that increased protein expression coincides with increased Tab182 mRNA levels (data not shown). June 2018 Volume 92 Issue 12 e02034-17 RESULTS Tab182 degradation requires the E1B55K and E4orf6 viral proteins. Multiple previous studies have demonstrated the roles of adenovirus E1B55K (AdE1B55K) and AdE4orf6 in targeting cellular proteins for degradation (6–12). To determine whether these components are involved in the observed reduction in the level of Tab182, infection with a panel of mutant viruses was carried out. Infection with the Ad5 (Ad5dl1520) and Ad12 (Ad12dl620) EIB55K-negative viruses had no effect on the level of Tab182 (Fig. 1C and D), indicating a requirement for the larger AdE1B protein for degradation. Following infection with various Ad5E4-negative viruses, there was no reduction in the Tab182 level when the E4orf6 protein was not expressed, as in H5pm4154 and H5pm4155 (Fig. 2A and B). Viruses that fail to express other E4 proteins degrade Tab182 in a manner comparable to that of the wild type (Fig. 2). Thus, H5in351 (E4orf1 negative [E4orf1]), H5in352 (E4orf2), H5pm4166 (E4orf4), and H5pm4150 (E4orf3) are all able to cause the rapid degradation of Tab182 (Fig. 2). The H5dl356 virus, which is E4orf7 negative, appears to express E4orf6 at much lower levels than expected, which probably explains why the levels of Tab182 and MRE11 are reduced only very margin- jvi.asm.org 4 June 2018 Volume 92 Issue 12 e02034-17 Adenoviruses Target Tab182 for Degradation Journal of Virology FIG 2 Degradation of Tab182 following infection with adenovirus serotype 5 is dependent on the adenovirus E4orf6 protein. HeLa cells were infected with the Ad5 E4 mutants H5in351 (E4orf1) (A), H5pm4154 (E4orf6) (A), H5pm4155 (E4orf3 E4orf6) (B), H5pm4166 (E4orf4) (B), H5dl356 (E4orf6 E4orf7) (C), H5in352 (E4orf2) (C), and H5pm4150 (E4orf3) (D) at 10 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 2 Degradation of Tab182 following infection with adenovirus serotype 5 is dependent on the adenovirus E4orf6 protein. HeLa cells were infected with the Ad5 E4 mutants H5in351 (E4orf1) (A), H5pm4154 (E4orf6) (A), H5pm4155 (E4orf3 E4orf6) (B), H5pm4166 (E4orf4) (B), H5dl356 (E4orf6 E4orf7) (C), H5in352 (E4orf2) (C), and H5pm4150 (E4orf3) (D) at 10 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. ally (Fig. 2C). RESULTS HeLa cells were infected with Ad5 or Ad12 at 5 PFU/cell. Cells were harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cellular RNA was extracted from Ad5 (A)- and Ad12 (B)-infected cells, and ﬁrst-strand cDNA synthesis was carried out. RT-PCRs were performed by using Tab182-speciﬁc primers and real-time PowerUp SYBR green master mix. To determine the relative Tab182 gene expression level, calculated Tab182 CT values were normalized to CT values of GAPDH ampliﬁed from the same sample [ΔCT CT (Tab182) CT (GAPDH)], and the 2ΔΔCT method was used to calculate relative expression levels. Each experiment was performed in triplicate. Western blots of Ad5- and Ad12-infected HeLa cells were performed to conﬁrm Tab182 degradation (data not shown). FIG 3 Tab182 gene expression is enhanced in adenovirus-infected cells. HeLa cells were infected with Ad5 or Ad12 at 5 PFU/cell. Cells were harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cellular RNA was extracted from Ad5 (A)- and Ad12 (B)-infected cells, and ﬁrst-strand cDNA synthesis was carried out. RT-PCRs were performed by using Tab182-speciﬁc primers and real-time PowerUp SYBR green master mix. To determine the relative Tab182 gene expression level, calculated Tab182 CT values were normalized to CT values of GAPDH ampliﬁed from the same sample [ΔCT CT (Tab182) CT (GAPDH)], and the 2ΔΔCT method was used to calculate relative expression levels. Each experiment was performed in triplicate. Western blots of Ad5- and Ad12-infected HeLa cells were performed to conﬁrm Tab182 degradation (data not shown). to Ad5 and Ad12, infection of HeLa cells with Ad9 and Ad11 had no effect on Tab182 expression except at very late times, when host cell shutoff could be a contributory factor (Fig. 5B and C). Following Ad4 and Ad7 infection, there is a reduction in Tab182 levels at later times, and this is more pronounced than the effects seen with Ad9 and Ad11 but much less marked than the degradation after Ad5 and Ad12 infection (Fig. 5B and C). The effects of the viruses on Tab182 levels closely mirror those on MRE11 and, in the case of Ad4, on p53 (Fig. 5). RESULTS H5pm4155, which is E4orf3 and E4orf6 negative, expresses somewhat reduced levels of E1B55K compared to those of the other viruses shown here (Fig. 2C). The reasons for this are not apparent. Overall, we conclude that the degradation of Tab182 requires, in Ad5 at least, E1B55K and E4orf6. Signiﬁcantly, in all Western blots shown in Fig. 1 and 2 (see also Fig. 5A), the degradation of Tab182 occurs somewhat later than the degradation of MRE11 but at times similar to those for p53 degradation (data not shown). In addition, to conﬁrm that the reduction in Tab182 levels is not due to a reduction in mRNA levels, RT-PCR was carried out on Ad5- and Ad12-infected cells (Fig. 3). This clearly shows that Tab182 mRNA levels are equivalent to, or higher than, those in uninfected cells up to about 72 h postinfection, in contrast to the sharp reduction in Tab182 protein levels after 24 h (compare Fig. 1 and 3). We conclude that the loss of the Tab182 protein is due to active protein degradation and not host cell shutoff, which may occur after 72 h (Fig. 3A and B). To demonstrate that the E1B55K and E4orf6 proteins are solely responsible for the degradation of Tab182, plasmids encoding the two Ad5 and Ad12 proteins were transfected into HeLa cells. Cells were harvested after 48 h, and lysates were subjected to Western blotting for Tab182, MRE11, and the viral proteins (Fig. 4). E4orf6 proteins were tagged with hemagglutinin (HA) and detected with an anti-HA antibody. It can be seen that Tab182 and MRE11 were degraded in the presence of both the Ad5 and Ad12 E1B55K and E4orf6 proteins. These data conﬁrm that similar viral proteins are required for both Ad5- and Ad12-mediated degradation of Tab182. When the viral proteins were expressed singly, there was little reduction in Tab182 or MRE11 levels, conﬁrming that both E1B55K and E4orf6 are required for degradation (Fig. 4). Degradation of Tab182 is limited to certain virus serotypes. To determine how widespread the degradation of Tab182 is among other adenovirus serotypes, the levels of Tab182 were monitored by Western blotting following infection of HeLa cells with Ad4 (group E), Ad7 (group B1), Ad9 (group D), and Ad11 (group B2) (Fig. 5). In contrast jvi.asm.org 5 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. FIG 3 Tab182 gene expression is enhanced in adenovirus-infected cells. RESULTS It is now well established that NEDDylation is required for the activation of the cullin components of the E3 ligases during adenovirus infection (9). In the presence of the inhibitor, the degradation of Tab182 was appre- ciably reduced, as was that of p53, although it is interesting to note that the stabili- zation of MRE11 was appreciably less than was that of p53; this apparent difference may be due to the upregulation of p53 expression due to AdE1A (Fig. 7A and B). The active NEDDylated component of cullin 2 can be seen as a slower-migrating protein in the Western blots shown in Fig. 7A and B. This is markedly reduced in the MLN4924- treated samples. We conclude that active (NEDDylated) cullins are required for Tab182 degradation during adenovirus infection. by the proteasome, cells were treated with bortezomib, a well-characterized protea- some inhibitor, or dimethyl sulfoxide (DMSO) (as a negative control) and harvested after 48 h. In the presence of bortezomib, the degradation of Tab182 and MRE11 following viral infection was reduced but not completely inhibited; in the absence of the proteasome inhibitor (DMSO), the proteins were degraded in the presence of the viruses (Fig. 6). In a second experiment, it was shown that the inhibition of modiﬁcation with NEDD8 (NEDDylation) (with MLN4924) also results in the stabilization of Tab182 following Ad5 and Ad12 infection. It is now well established that NEDDylation is required for the activation of the cullin components of the E3 ligases during adenovirus infection (9). In the presence of the inhibitor, the degradation of Tab182 was appre- ciably reduced, as was that of p53, although it is interesting to note that the stabili- zation of MRE11 was appreciably less than was that of p53; this apparent difference may be due to the upregulation of p53 expression due to AdE1A (Fig. 7A and B). The active NEDDylated component of cullin 2 can be seen as a slower-migrating protein in the Western blots shown in Fig. 7A and B. This is markedly reduced in the MLN4924- treated samples. We conclude that active (NEDDylated) cullins are required for Tab182 degradation during adenovirus infection. Different adenovirus serotypes do not all make use of the same cullin components to degrade cellular proteins. Previously, it was shown that protein degradation follow- ing Ad5 infection utilizes a cullin 5-based E3 ligase, whereas Ad12 hijacks a cullin 2-based E3 ligase (9, 12). RESULTS (Ad7, Ad9, and Ad11 all markedly induce the expression of p53, as was reported previously [28 and 56].) We previously reported that Ad4 facilitates the rapid degradation of various DDR proteins (28) although perhaps to a lesser extent than Ad5 and Ad12. However, it appears to have only a relatively slight effect on Tab182 (Fig. 5B). While there is a limited reduction in protein levels, it seems likely that the group B1, B2, D, and E viruses do not cause a signiﬁcant degradation of Tab182. Degradation of Tab182 requires the proteasome and E3 ligases. A number of approaches were adopted to investigate the mechanism by which target proteins are degraded during Ad5 and Ad12 infection. Initially, to conﬁrm that Tab182 is degraded jvi.asm.org 6 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Adenoviruses Target Tab182 for Degradation FIG 4 Degradation of Tab182 during adenovirus serotype 5 and 12 infection is dependent on the adenovirus E1B55K and E4orf6 proteins. Two micrograms of plasmid DNA, as shown, was transfected into HeLa cells, and 48 h later, cells were harvested and subjected to SDS-PAGE and Western blotting using the indicated antibodies. The Ad5E4orf6 and Ad12E4orf6 proteins were detected with an antibody that recognized the HA tag. GAPDH is included as a loading control. FIG 4 Degradation of Tab182 during adenovirus serotype 5 and 12 infection is dependent on the adenovirus E1B55K and E4orf6 proteins. Two micrograms of plasmid DNA, as shown, was transfected into HeLa cells, and 48 h later, cells were harvested and subjected to SDS-PAGE and Western blotting using the indicated antibodies. The Ad5E4orf6 and Ad12E4orf6 proteins were detected with an antibody that recognized the HA tag. GAPDH is included as a loading control. by the proteasome, cells were treated with bortezomib, a well-characterized protea- some inhibitor, or dimethyl sulfoxide (DMSO) (as a negative control) and harvested after 48 h. In the presence of bortezomib, the degradation of Tab182 and MRE11 following viral infection was reduced but not completely inhibited; in the absence of the proteasome inhibitor (DMSO), the proteins were degraded in the presence of the viruses (Fig. 6). In a second experiment, it was shown that the inhibition of modiﬁcation with NEDD8 (NEDDylation) (with MLN4924) also results in the stabilization of Tab182 following Ad5 and Ad12 infection. RESULTS To examine whether this difference extends to the degrada- tion of Tab182, H1299 cells in which Cul2 or Cul5 expression had been ablated were infected with Ad5 and Ad12, and levels of Tab182 were monitored (Fig. 7C to E). In the Cul2-negative cells, Tab182 is more stable following Ad12 infection than in the control cell line, indicating that Cul2 is required for the degradation of Tab182 by this serotype (Fig. 7C and D). In contrast, in the Cul5-negative cells, Tab182 levels are comparable to those in cells following Ad12 infection, indicating that this cullin component is dis- pensable for Tab182 degradation (Fig. 7C and E). However, more subtle differences were observed in the degradation of Tab182 after Ad5 infection in Cul2-negative and Cul5-negative cells (Fig. 7D and E). During Ad5 infection, the loss of either cullin may result in some stabilization of Tab182 compared to control H1299 cells but does not clearly abrogate its degradation (Fig. 7C to E). As expected, MRE11 is stabilized in Cul5-negative cells but not in Cul2-negative cells after Ad5 infection. This suggests the possible involvement of Cul2, and/or perhaps an unidentiﬁed cullin, in Ad5-mediated Tab182 degradation. Further work will be required to determine if other proteins beside cullins 2 and 5 are involved in protein degradation by Ad5. jvi.asm.org 7 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. FIG 5 Tab182 levels following infection by group B, D, and E adenoviruses. HeLa cells were infected with Ad5 (group C) and Ad12 (group A) (A), Ad4 (group E) and Ad9 (group D) (B), and Ad11 (group B2) and Ad7 (group B1) (C) at 5 PFU/cell. Cells were harvested at 8, 24, 48, 72, 96, and 120 h postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using antibodies against Tab182, MRE11, p53, and -actin. Hexon expression was conﬁrmed, as a marker of viral infection, by Ponceau S staining of Western blots for total protein. FIG 5 Tab182 levels following infection by group B, D, and E adenoviruses. HeLa cells were infected with Ad5 (group C) and Ad12 (group A) (A), Ad4 (group E) and Ad9 (group D) (B), and Ad11 (group B2) and Ad7 (group B1) (C) at 5 PFU/cell. Cells were harvested at 8, 24, 48, 72, 96, and 120 h postinfection. June 2018 Volume 92 Issue 12 e02034-17 RESULTS Cells were treated with 0.5 M bortezomib or the DMSO control and harvested after 48 h. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 6 Downregulation of Tab182 protein levels during Ad5 and Ad12 infection can be rescued by the proteasomal inhibitor bortezomib. HeLa cells were infected with Ad5 or Ad12 at 5 PFU/cell. Cells were treated with 0.5 M bortezomib or the DMSO control and harvested after 48 h. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. were extracted prior to antibody staining; again, no colocalization of GFP-Tab182 with VRCs was observed (Fig. 8B). Tab182 associates with AdE1B55K proteins. As the adenovirus-mediated degra- dation of Tab182 is AdE1B55K dependent, we investigated whether the two proteins FIG 7 Degradation of Tab182 during Ad5 and Ad12 infection is dependent on cul Cells were treated with the Nedd8 inhibitor MLN4924 (4 M) 1 h before infecti various time points (0, 8, 24, 48, 72, and 96 h) postinfection. (A and B) Cell lysates w antibodies. (C to E) H1299 cells (C) or H1299 cells with an ablation of Cul2 (D) or Cu at 0, 8, 24, 48, 72, and 96 h postinfection. Cell lysates were subjected to SDS-PA FIG 7 Degradation of Tab182 during Ad5 and Ad12 infection is dependent on cullin function. HeLa cells were infected with Ad5 and Ad12 at 5 PFU/cell. Cells were treated with the Nedd8 inhibitor MLN4924 (4 M) 1 h before infection and retreated immediately postinfection. Cells were harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. (A and B) Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. (C to E) H1299 cells (C) or H1299 cells with an ablation of Cul2 (D) or Cul5 (E) expression were infected with either Ad5 or Ad12 and harvested at 0, 8, 24, 48, 72, and 96 h postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting with the antibodies shown. FIG 7 Degradation of Tab182 during Ad5 and Ad12 infection is dependent on cullin function. HeLa cells were infected with Ad5 and Ad12 at 5 PFU/cell. Cells were treated with the Nedd8 inhibitor MLN4924 (4 M) 1 h before infection and retreated immediately postinfection. Cells were harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. RESULTS Cell lysates were subjected to SDS-PAGE and Western blotting using antibodies against Tab182, MRE11, p53, and -actin. Hexon expression was conﬁrmed, as a marker of viral infection, by Ponceau S staining of Western blots for total protein. FIG 5 Tab182 levels following infection by group B, D, and E adenoviruses. HeLa cells were infected with Ad5 (group C) and Ad12 (group A) (A), Ad4 (group E) and Ad9 (group D) (B), and Ad11 (group B2) and Ad7 (group B1) (C) at 5 PFU/cell. Cells were harvested at 8, 24, 48, 72, 96, and 120 h postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using antibodies against Tab182, MRE11, p53, and -actin. Hexon expression was conﬁrmed, as a marker of viral infection, by Ponceau S staining of Western blots for total protein. Tab182 does not localize to viral replication centers. It was previously shown that a number of DDR proteins localize to the sites of adenovirus replication in the nucleus, known as VRCs (6, 8). To examine if this applies to Tab182, HeLa cells were transfected with green ﬂuorescent protein-tagged Tab182 (GFP-Tab182) and left for 24 h. The cells were then seeded onto glass coverslips and infected with Ad5 or Ad12. After a further 24 h, cells were ﬁxed and stained with antibodies that recognize VRCs (Fig. 8). In the case of Ad5, VRCs were visualized by using an antibody against the viral DNA binding protein (DBP), while RPA32 was used as a surrogate marker for Ad12 VRCs. No speciﬁc recruitment of Tab182 to viral replication centers was observed following infection with either adenovirus serotype (Fig. 8A). As the expression level of GFP- Tab182 was higher than that of the wt protein, in a further experiment, soluble proteins jvi.asm.org 8 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Adenoviruses Target Tab182 for Degradation FIG 6 Downregulation of Tab182 protein levels during Ad5 and Ad12 infection can be rescued by the proteasomal inhibitor bortezomib. HeLa cells were infected with Ad5 or Ad12 at 5 PFU/cell. Cells were treated with 0.5 M bortezomib or the DMSO control and harvested after 48 h. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 6 Downregulation of Tab182 protein levels during Ad5 and Ad12 infection can be rescued by the proteasomal inhibitor bortezomib. HeLa cells were infected with Ad5 or Ad12 at 5 PFU/cell. June 2018 Volume 92 Issue 12 e02034-17 RESULTS (A and B) Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. (C to E) H1299 cells (C) or H1299 cells with an ablation of Cul2 (D) or Cul5 (E) expression were infected with either Ad5 or Ad12 and harvested at 0, 8, 24, 48, 72, and 96 h postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting with the antibodies shown. June 2018 Volume 92 Issue 12 e02034-17 jvi.asm.org 9 Journal of Virology Chalabi Hagkarim et al. FIG 8 Tab182 does not localize to viral replication centers during adenovirus infection. GFP-Tab182 was transfected into HeLa cells, and 24 h later, cells were infected with Ad5 or Ad12. (A) Thirty hours later, cells were ﬁxed, extracted, and probed with the appropriate antibodies. (B) Thirty hours after infection, cells were preextracted as described in Materials and Methods before ﬁxing and then staining with antibodies. In both panels A and B, Ad5-infected cells were probed with DBP antibody, while Ad12-infected cells were probed with RPA32 antibody. Nuclear DNA is stained with DAPI. FIG 8 Tab182 does not localize to viral replication centers during adenovirus infection. GFP-Tab182 was transfected into HeLa cells, and 24 h later, cells were infected with Ad5 or Ad12. (A) Thirty hours later, cells were ﬁxed, extracted, and probed with the appropriate antibodies. (B) Thirty hours after infection, cells were preextracted as described in Materials and Methods before ﬁxing and then staining with antibodies. In both panels A and B, Ad5-infected cells were probed with DBP antibody, while Ad12-infected cells were probed with RPA32 antibody. Nuclear DNA is stained with DAPI. FIG 8 Tab182 does not localize to viral replication centers during adenovirus infection. GFP-Tab182 was transfected into HeLa cells, and 24 h later, cells were infected with Ad5 or Ad12. (A) Thirty hours later, cells were ﬁxed, extracted, and probed with the appropriate antibodies. (B) Thirty hours after infection, cells were preextracted as described in Materials and Methods before ﬁxing and then staining with antibodies. In both panels A and B, Ad5-infected cells were probed with DBP antibody, while Ad12-infected cells were probed with RPA32 antibody. Nuclear DNA is stained with DAPI. were associated, as is the case, for example, with Ad5E1B55K and p53 (57). To examine this possibility, glutathione S-transferase (GST) pulldown assays were initially carried out with puriﬁed GST-Tab182 (C-terminal region) and whole-cell lysates from E1B55K- expressing Ad12E1HER2 and Ad5E1HEK293 cells. RESULTS In both cases, the E1B55K protein was identiﬁed as a binding partner (Fig. 9A and B). As well as GST, GST-PRMT1 was included as an irrelevant (negative) control, as it has a molecular weight comparable to that of the Tab182 polypeptide. No binding of GST or GST-PRMT1 to E1B55K proteins was seen. In further experiments, using the same cell lines, E1B55K proteins were coimmunopre- cipitated by using an antibody against Tab182 (Fig. 9C and D). No coimmunoprecipi- tation was seen by using an irrelevant antibody against collagen IV. In a further experiment, Ad5E1HEK293 cells and Ad12E1HER2 cells were transfected with a con- struct encoding GFP-Tab182. The lysates were immunoprecipitated with antibodies against AdE1B55K proteins, and coprecipitated Tab182 was detected by Western jvi.asm.org 10 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Adenoviruses Target Tab182 for Degradation FIG 9 Adenovirus early region E1B55K interacts with Tab182 in vitro and in vivo. (A and B) Ad12E1HER2 (A) and Ad5E1HEK293 (B) cell lysates containing 500 g total protein were incubated with 5 g either GST-Tab182 or GST-PRMT1 or with GST alone. Protein complexes were captured by glutathione-agarose beads and subjected to SDS-PAGE and Western blotting (WB) with the antibodies indicated. (C and D) Ad5E1HEK293 (C) and Ad12E1HER2 (D) cell lysates (500 g total protein) were incubated with antibodies against Tab182 and collagen IV together with IgG (nonspeciﬁc binding controls). Immunocomplexes were isolated by using protein G-agarose beads and subsequently resolved by SDS-PAGE and Western blotting using antibodies against Ad5E1B55K/Ad12E1B55K proteins. IP, immunoprecipitation. (E) GFP-Tab182 was transfected into Ad5E1HEK293 and Ad12E1HER2 cell lines, which were harvested after 48 h. Cell lysates (500 g total protein) were incubated with Ad5E1B55K and Ad12E1B55K antibodies together with IgG. Western blotting was performed with an antibody against Tab182. (F) HeLa cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 g total protein) were immunoprecipitated with an antibody against Tab182 or rabbit IgG. Western blotting was performed with an antibody against HA. (G) Overexposed version of a portion of the Western blot shown in panel F. (H) Ad5E1HEK293 cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 g total protein) were immunoprecipitated with an antibody against HA or mouse IgG. Western blotting was performed with an antibody against p53. RESULTS (J and K) Ad5E1HEK293 (J) and Ad12E1HER2 (K) cell lysates (500 g total protein) were incubated with antibodies against CNOT1 and collagen IV together with IgG. Western blotting was performed with antibodies against Ad5E1B55K and Ad12E1B55K proteins. In all cases, the whole-cell lysates contained 15 g of protein. Although only limited areas of the Western blots are shown, no additional bands were seen in the original autoradiographs. FIG 9 Adenovirus early region E1B55K interacts with Tab182 in vitro and in vivo. (A and B) Ad12E1HER2 (A) and Ad5E1HEK293 (B) cell lysates containing 500 g total protein were incubated with 5 g either GST-Tab182 or GST-PRMT1 or with GST alone. Protein complexes were captured by glutathione-agarose beads and subjected to SDS-PAGE and Western blotting (WB) with the antibodies indicated. (C and D) Ad5E1HEK293 (C) and Ad12E1HER2 (D) cell lysates (500 g total protein) were incubated with antibodies against Tab182 and collagen IV together with IgG (nonspeciﬁc binding controls). Immunocomplexes were isolated by using protein G-agarose beads and subsequently resolved by SDS-PAGE and Western blotting using antibodies against Ad5E1B55K/Ad12E1B55K proteins. IP, immunoprecipitation. (E) GFP-Tab182 was transfected into Ad5E1HEK293 and Ad12E1HER2 cell lines, which were harvested after 48 h. Cell lysates (500 g total protein) were incubated with Ad5E1B55K and Ad12E1B55K antibodies together with IgG. Western blotting was performed with an antibody against Tab182. (F) HeLa cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 g total protein) were immunoprecipitated with an antibody against Tab182 or rabbit IgG. Western blotting was performed with an antibody against HA. (G) Overexposed version of a portion of the Western blot shown in panel F. (H) Ad5E1HEK293 cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 g total protein) were immunoprecipitated with an antibody against HA or mouse IgG. Western blotting was performed with an antibody against p53. (I) HEK293FT cell lysates (500 g protein) were incubated with antibodies against Tab182, collagen IV, or the IgG control. Western blotting was performed with an antibody against SV40T antigen. (J and K) Ad5E1HEK293 (J) and Ad12E1HER2 (K) cell lysates (500 g total protein) were incubated with antibodies against CNOT1 and collagen IV together with IgG. Western blotting was performed with antibodies against Ad5E1B55K and Ad12E1B55K proteins. In all cases, the whole-cell lysates contained 15 g of protein. June 2018 Volume 92 Issue 12 e02034-17 RESULTS (I) HEK293FT cell lysates (500 g protein) were incubated with antibodies against Tab182, collagen IV, or the IgG control. Western blotting was performed with an antibody against SV40T antigen. (J and K) Ad5E1HEK293 (J) and Ad12E1HER2 (K) cell lysates (500 g total protein) were incubated with antibodies against CNOT1 and collagen IV together with IgG. Western blotting was performed with antibodies against Ad5E1B55K and Ad12E1B55K proteins. In all cases, the whole-cell lysates contained 15 g of protein. Although only limited areas of the Western blots are shown, no additional bands were seen in the original autoradiographs. FIG 9 Adenovirus early region E1B55K interacts with Tab182 in vitro and in vivo. (A and B) Ad12E1HER2 (A) and Ad5E1HEK293 (B) cell lysates containing 500 g total protein were incubated with 5 g either GST-Tab182 or GST-PRMT1 or with GST alone. Protein complexes were captured by glutathione-agarose beads and subjected to SDS-PAGE and Western blotting (WB) with the antibodies indicated. (C and D) Ad5E1HEK293 (C) and Ad12E1HER2 (D) cell lysates (500 g total protein) were incubated with antibodies against Tab182 and collagen IV together with IgG (nonspeciﬁc binding controls). Immunocomplexes were isolated by using protein G-agarose beads and subsequently resolved by SDS-PAGE and Western blotting using antibodies against Ad5E1B55K/Ad12E1B55K proteins. IP, immunoprecipitation. (E) GFP-Tab182 was transfected into Ad5E1HEK293 and Ad12E1HER2 cell lines, which were harvested after 48 h. Cell lysates (500 g total protein) were incubated with Ad5E1B55K and Ad12E1B55K antibodies together with IgG. Western blotting was performed with an antibody against Tab182. (F) HeLa cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 g total protein) were immunoprecipitated with an antibody against Tab182 or rabbit IgG. Western blotting was performed with an antibody against HA. (G) Overexposed version of a portion of the Western blot shown in panel F. (H) Ad5E1HEK293 cells were transfected with pcDNA3 or pcDNA3 constructs expressing HA-tagged Ad9E1B55K or Ad16E1B55K. After 48 h, lysates (500 g total protein) were immunoprecipitated with an antibody against HA or mouse IgG. Western blotting was performed with an antibody against p53. (I) HEK293FT cell lysates (500 g protein) were incubated with antibodies against Tab182, collagen IV, or the IgG control. Western blotting was performed with an antibody against SV40T antigen. RESULTS Although only limited areas of the Western blots are shown, no additional bands were seen in the original autoradiographs. blotting (Fig. 9E). Both the slightly higher-molecular-weight GFP-Tab182 and endoge- nous Tab182 were seen in some lanes. These results strongly suggest that in both Ad5 and Ad12, the viral E1B55K proteins interact directly with Tab182. Although the degradation of Tab182 occurs to only a very limited extent during infection with adenoviruses other than Ad5 and Ad12 (Fig. 5), we considered the possibility that the E1B55K proteins from these other species may also associate with Tab182. Therefore, HeLa cells were transfected with constructs encoding HA-tagged Ad9E1B55K (group D) or HA-tagged Ad16E1B55K (group B1). After 48 h, Tab182 was immunoprecipitated, and the associated E1B55K protein was detected with an anti- jvi.asm.org 11 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. FIG 9 (Continued) FIG 9 (Continued) FIG 9 (Continued) body against HA (Fig. 9F). It can be seen that while the Ad9 protein bound strongly, the Ad16 equivalent could be seen only on overexposed Western blots, indicating a very weak association (Fig. 9G). Similar results were obtained when the constructs were transfected into Ad5E1HEK293 cells (data not shown). To check whether this differen- tiation extends to other adenovirus targets, the interaction with p53 was examined. After the transfection of both constructs into Ad5E1HEK293 cells, HA-tagged E1B55K proteins were immunoprecipitated, and bound p53 was detected by Western blotting (Fig. 9H). In contrast to Tab182, both the Ad9E1B55K and Ad16E1B55K proteins strongly interacted with p53. body against HA (Fig. 9F). It can be seen that while the Ad9 protein bound strongly, the Ad16 equivalent could be seen only on overexposed Western blots, indicating a very weak association (Fig. 9G). Similar results were obtained when the constructs were transfected into Ad5E1HEK293 cells (data not shown). To check whether this differen- tiation extends to other adenovirus targets, the interaction with p53 was examined. After the transfection of both constructs into Ad5E1HEK293 cells, HA-tagged E1B55K proteins were immunoprecipitated, and bound p53 was detected by Western blotting (Fig. 9H). In contrast to Tab182, both the Ad9E1B55K and Ad16E1B55K proteins strongly interacted with p53. It is now well established that the small DNA tumor viruses have many cellular targets in common, such as pRb, p53, and CBP/p300 (58, 59). RESULTS Proteins that were seen in both Tab182 and control IgG immunoprecipitations are not listed in Table 1. The proteins listed are the only ones that were consistently observed in ﬁve Tab182 immunoprecipitation experiments but not in controls. In a ﬁnal set of coimmunoprecipitations, we investigated whether AdE1B55K pro- teins were associated with other CNOT components. Using adenovirus E1-expressing cells, CNOT1 was immunoprecipitated, and associated E1B55K proteins were detected by Western blotting (Fig. 9J and K). It is possible that these results show a direct interaction of the viral proteins with CNOT1 but could also indicate an interaction with other, as-yet-unidentiﬁed, components of the intact CNOT complex or even Tab182. Adenovirus infection leads to reductions in the levels of other CNOT proteins. In light of the coimmunoprecipitation experiments shown in Table 1 and Fig. 9, we examined the levels of other CNOT proteins during adenovirus infection. Following infection of HeLa cells with either Ad5 or Ad12, levels of CNOT1, CNOT3, CNOT4, and CNOT7 were monitored by Western blotting (Fig. 10). In contrast to Tab182, the levels of CNOT1 and CNOT4 remained stable throughout the time course of infection (Fig. 10A and B). However, the levels of both CNOT3 and CNOT7 were markedly reduced after infection with both serotypes. In the case of Ad5, levels of CNOT3 declined prior to the observed decrease in the CNOT7 levels, whereas for Ad12, CNOT7 levels declined prior to the decline in CNOT3 levels (Fig. 10A and B). Further work will be required to determine whether these proteins are degraded in the same fashion as Tab182 and whether levels of other CNOT proteins are reduced during adenovirus infection, but these data suggest that the complex may be a major target for certain adenoviruses. Depletion of Tab182 and CNOT1 favors adenovirus infection. To determine what advantage adenoviruses might derive from the degradation of Tab182 and other CNOT complex proteins, a time course of infection was monitored in HeLa cells treated with Tab182 small interfering RNA (siRNA). In addition, the effect of the depletion of CNOT1 was also examined. CNOT1 forms a scaffold on which other members of the complex associate (41). We therefore reasoned that its depletion would cause a maximal disruption of CNOT complex activity. Cells were infected with Ad5 and Ad12 48 h after the addition of control, Tab182, or CNOT1 siRNAs. It can be seen from Fig. RESULTS It has already been reported that the E6 protein from human papillomavirus (HPV) genus beta species 2 (HPV17a and HPV38) associates with the CNOT complex (60). To examine if Tab182 interacts with proteins from other small DNA tumor viruses, a coimmunoprecipitation experiment was carried out with a Tab182 antibody by using HEK293FT cells, which express the simian virus 40 T antigen (SV40T). When Tab182 was immunoprecipitated, an appreciable amount of SV40T was associated with it (Fig. 9I). Tab182 is a component of the CNOT complex. It was previously noted that Tab182 can be coimmunoprecipitated with the CNOT complex from mammalian cells (40). To conﬁrm this association, Tab182 was immunoprecipitated by using a rabbit jvi.asm.org 12 June 2018 Volume 92 Issue 12 e02034-17 Adenoviruses Target Tab182 for Degradation Journal of Virology TABLE 1 Proteins identiﬁed by mass spectrometric analysis after coimmunoprecipitation with Tab182 antibodya Protein No. of peptides % coverage Mascot score Tab182 68 49.4 3,491 CNOT1 38 17.4 1,472 CNOT3 7 10.2 237 CNOT7 6 28.8 211 CNOT2 5 12.2 245 CNOT6L 1 1.8 21 CNOT10 1 1.3 29 C2orf29 (CNOT11) 1 2.4 56 RCD1 (CNOT9) 6 20.4 224 PRMT3 9 18.5 377 FHL2 12 52.3 470 aHeLa cells were immunoprecipitated with a rabbit antibody raised against the C-terminal fragment of Tab182 and analyzed as described in Materials and Methods. These data are representative of results from ﬁve independent experiments. TABLE 1 Proteins identiﬁed by mass spectrometric analysis after coimmunoprecipitation with Tab182 antibodya aHeLa cells were immunoprecipitated with a rabbit antibody raised against the C-terminal fragment of Tab182 and analyzed as described in Materials and Methods. These data are representative of results from ﬁve independent experiments. aHeLa cells were immunoprecipitated with a rabbit antibody raised against the C-terminal fragment of Tab182 and analyzed as described in Materials and Methods. These data are representative of results from ﬁve independent experiments. antibody raised against the C-terminal fragment, and the total immunoprecipitate was analyzed by mass spectrometry. Results from a representative coimmunoprecipitation experiment are presented in Table 1. In all cases, most components of the CNOT complex were detected, although there were limited variations from one experiment to the next. Speciﬁcally, CNOT4 was never detected in any Tab182 coimmunoprecipitate, and CNOT7 and CNOT8 were occasionally not identiﬁed. Signiﬁcantly, neither Tab182 nor CNOT proteins were detected in any of the control immunoprecipitates carried out with rabbit IgG (data not shown). June 2018 Volume 92 Issue 12 e02034-17 RESULTS 11A and B that in the absence of Tab182, expression levels of the E1A viral proteins were elevated to a limited extent compared to the controls during the time course of jvi.asm.org 13 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. FIG 10 Adenovirus serotypes 5 and 12 degrade components of the CNOT complex. HeLa cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. Cells were harvested at 0, 8, 24, 48, 72, and 96 h postinfection and subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 10 Adenovirus serotypes 5 and 12 degrade components of the CNOT complex. HeLa cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. Cells were harvested at 0, 8, 24, 48, 72, and 96 h postinfection and subjected to SDS-PAGE and Western blotting using the indicated antibodies. infection. Similar results were obtained with the E1B55K-negative viruses Ad5dl1520 and Ad12dl620 in that AdE1A proteins were expressed at higher levels in the absence of Tab182 (data not shown). In the samples treated with control siRNA, there was a reduction in the level of Tab182 as degradation proceeded. The expression levels of other viral proteins varied marginally between Tab182-depleted and control cells. However, in a further set of experiments, it was shown that when CNOT1 was depleted before infection with Ad5, there were notable increases in E1A and E1B55K expression levels compared to the controls (Fig. 11A). In Ad12-infected cells, there was an even more marked increase in the expression levels of the E1A and E1B55K proteins compared to those in control siRNA-treated cells (Fig. 11B). From the Western blots, it is clear that Tab182 depletion has its most marked effect 24 h after Ad12 infection. However, the depletion of CNOT1 causes a severalfold increase in the Ad12E1A expression level at 24 h, but notably, the level of protein stays consistently high up to 96 h. The effects on Ad5E1A were less pronounced, although, again, the loss of Tab182 had the greatest effect at 24 h postinfection, whereas the depletion of CNOT1 facili- tated AdE1A expression up to 96 h. It has long been known that adenovirus infection promotes cell cycle progression from G1 into a pseudo-S phase, accompanied by the enhanced expression of cyclin E (reviewed in reference 61, for example). June 2018 Volume 92 Issue 12 e02034-17 RESULTS In addition, it was also reported that AdE1A promotes the expression of the tyrosine phosphatase CDC25A, which is required for the G1-to-S-phase transition (62). In an attempt to examine whether the depletion of Tab182 and CNOT1 affects the ability of adenoviruses to initiate cell cycle progression, the expression of CDC25A was initially examined. It is notable that in Tab182- and CNOT1-depleted cells, there is only a very limited induction of CDC25A after infection, whereas this is appreciable in control infected cells (Fig. 11A and B). After 24 h in all cases, expression returns to a low level comparable to that in uninfected cells. We suggest that the low-level expression of CDC25A is required by the virus for the progression of infected cells into pseudo-S phase, but after that, to stop further progression, CDC25A may be detrimental to viral replication. It is possible that the reduction in the levels of CNOT components decreases CDC25A levels, retaining the cells in a cell cycle phase more conducive to viral early protein expression and viral replication. jvi.asm.org 14 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Adenoviruses Target Tab182 for Degradation FIG 11 AdE1A protein expression is enhanced in adenovirus-infected, Tab182- or CNOT1-depleted cells. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs. Forty-eight hours later, control, Tab182, and CNOT1 siRNA-treated cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 11 AdE1A protein expression is enhanced in adenovirus-infected, Tab182- or CNOT1-depleted cells. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs. Forty-eight hours later, control, Tab182, and CNOT1 siRNA-treated cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 11 AdE1A protein expression is enhanced in adenovirus-infected, Tab182- or CNOT1-depleted cells. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs. Forty-eight hours later, control, Tab182, and CNOT1 siRNA-treated cells were infected with adenovirus serotype 5 (A) or serotype 12 (B) at 5 PFU/cell. RESULTS Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 12 Expression of cyclin E and is enhanced in Tab182- and CNOT1-depleted cells. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs. Forty-eight hours later, control, Tab182, and CNOT1 siRNA-treated cells were mock infected (A) or infected with adenovirus serotype 12 (B) at 5 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. cycle, into a phase more favorable for adenovirus early protein expression, as suggested above. Ad5 infection of HeLa cells treated with the same siRNAs had little additional effect on cyclin E expression (data not shown). Depletion of Tab182 and CNOT1 enhances AdE1A mRNA expression. To deter- mine whether the depletion of Tab182 or CNOT1 affects AdE1A expression at the transcriptional level, cells depleted of either Tab182 or CNOT1 were infected with either Ad5 or Ad12 before the isolation of total RNA after 24 h. RT-PCR was performed following reverse transcription of total RNA to amplify Ad5 and Ad12 13S E1As using primers across the unique CR3 region of each protein; threshold cycle (CT) values were calculated, with normalization to the value for glyceraldehyde-3-phosphate dehydro- genase (GAPDH). The depletion of CNOT1 or Tab182 in Ad5- or Ad12-infected cells was veriﬁed by Western blotting (data not shown). The relative expression level of 13S E1A in infected cells with CNOT1 or Tab182 depleted was compared with that in mock- transfected, infected cells. It can be seen from the data presented in Fig. 13 that the depletion of Tab182 resulted in increases in both Ad5 and Ad12 13S E1A mRNA levels compared to controls. The depletion of CNOT1 had a more marked effect, consistent with the Western blots shown in Fig. 11. Depletion of Tab182 and CNOT1 favors the production of viral DNA during infection. To examine whether the advantage gained in the expression of early proteins in Tab182- and CNOT1-depleted cells extends to the production of viral genomes, HeLa cells were treated with appropriate siRNAs and infected with Ad5 and Ad12 48 h later. After a further 24 h, cells were harvested, and the DNA was isolated. RESULTS Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. Tab182 depletion favors progression into S phase after adenovirus infection. In view of the CDC25A Western blot data shown in Fig. 11, the effects of the depletion of Tab182 or CNOT1 on cyclin E expression were also examined. HeLa cells were again treated with control, Tab182, and CNOT1 siRNAs; mock infected or infected with Ad12; and then harvested at various times up to 96 h. In mock-infected cells treated with control siRNA, cyclin E is expressed at a constant low level, but in those cells treated with Tab182 and particularly CNOT1 siRNAs, there is an appreciable elevation in the cyclin E expression level (Fig. 12A). When a similar set of cells was infected with Ad12, elevated cyclin E levels were also observed (Fig. 12B). Thus, in control infected cells, there was a limited increase in the cyclin E expression level, but in the absence of Tab182 or CNOT1, the expression level of cyclin E was elevated to a much greater extent (Fig. 12B). It seems likely, therefore, that the effects seen in virally infected cells are attributable primarily to CNOT1 and Tab182 depletion rather than the virus itself. The advantage gained by the virus, facilitating E1A expression, could be due to the fact that the siRNA-treated cells had generally progressed slightly further through the cell jvi.asm.org 15 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. FIG 12 Expression of cyclin E and is enhanced in Tab182- and CNOT1-depleted cells. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs. Forty-eight hours later, control, Tab182, and CNOT1 siRNA-treated cells were mock infected (A) or infected with adenovirus serotype 12 (B) at 5 PFU/cell. Cells were then harvested at various time points (0, 8, 24, 48, 72, and 96 h) postinfection. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. FIG 12 Expression of cyclin E and is enhanced in Tab182- and CNOT1-depleted cells. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs. Forty-eight hours later, control, Tab182, and CNOT1 siRNA-treated cells were mock infected (A) or infected with adenovirus serotype 12 (B) at 5 PFU/cell. RESULTS The concentration of adenovirus DNA was measured by quantitative PCR, as outlined in Materials and Methods, using primers equivalent to hexon and GAPDH as a control. More viral DNA can be seen in the Tab182-depleted cells than in the control cells after both Ad5 and Ad12 infection (Fig. 14); similarly, there is an even greater increase after CNOT1 depletion, consistent with increased AdE1A expression. Interestingly, the effects June 2018 Volume 92 Issue 12 e02034-17 jvi.asm.org 16 Adenoviruses Target Tab182 for Degradation Journal of Virology FIG 13 The relative expression level of Ad13S E1A mRNA is increased in infected cells in the absence of CNOT1 or Tab182. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs, and 48 h later, they were infected with Ad5 (A) or Ad12 (B) at 5 PFU/cell. Cellular RNA was extracted from infected cells, and ﬁrst-strand cDNA synthesis was carried out. RT-PCRs were performed by using Ad13SE1A CR3 region-speciﬁc primers and real-time PowerUp SYBR green master mix. To check E1A relative gene expression levels, calculated E1A CT values were normalized to CT values of GAPDH ampliﬁed from the same sample [ΔCT CT (E1A) CT (GAPDH)], and the 2ΔΔCTmethod was used to calculate relative gene expression levels. Data are the means of results of 3 repeats. Statistical signiﬁcance was determined by using Student’s t test, and P values of less than 0.05 () or 0.01 () were considered signiﬁcant. Error bars represent standard errors of the means. FIG 13 The relative expression level of Ad13S E1A mRNA is increased in infected cells in the absence of CNOT1 or Tab182. HeLa cells were transfected with control, Tab182, or CNOT1 siRNAs, and 48 h later, they were infected with Ad5 (A) or Ad12 (B) at 5 PFU/cell. Cellular RNA was extracted from infected cells, and ﬁrst-strand cDNA synthesis was carried out. RT-PCRs were performed by using Ad13SE1A CR3 region-speciﬁc primers and real-time PowerUp SYBR green master mix. To check E1A relative gene expression levels, calculated E1A CT values were normalized to CT values of GAPDH ampliﬁed from the same sample [ΔCT CT (E1A) CT (GAPDH)], and the 2ΔΔCTmethod was used to calculate relative gene expression levels. Data are the means of results of 3 repeats. Statistical signiﬁcance was determined by using Student’s t test, and P values of less than 0.05 () or 0.01 (*) were considered signiﬁcant. RESULTS Error bars represent standard errors of the means. of the depletion of CNOT1 and Tab182 were very similar during Ad5 infection (Fig. 14A), whereas CNOT1 depletion had an appreciably greater effect than that of Tab182 depletion in Ad12-infected cells (Fig. 14B). DISCUSSION It is now well established that adenovirus infection triggers a cellular DDR (22). This is counteracted, in Ad5 and Ad12 at least, by the degradation of multiple cellular FIG 14 Viral DNA synthesis is increased in Tab182- and CNOT1-depleted cells after adenovirus infection. HeLa cells were treated with control, Tab182, and CNOT1 siRNAs for 48 h and then infected with Ad5 (A) or Ad12 (B) at 5 PFU/cell. After 24 h, cells were harvested, and the total DNA was isolated. Quantitative PCR was performed to determine the relative concentration of viral DNA. Hexon CT values were normalized to CT values for GAPDH DNA ampliﬁed from the same sample. Data are the means of results from 3 repeats. Statistical signiﬁcance was determined by using Student’s t test, and P values of less than 0.05 () or 0.01 (*) were considered signiﬁcant. Error bars represent standard errors of the means. FIG 14 Viral DNA synthesis is increased in Tab182- and CNOT1-depleted cells after adenovirus infection. HeLa cells were treated with control, Tab182, and CNOT1 siRNAs for 48 h and then infected with Ad5 (A) or Ad12 (B) at 5 PFU/cell. After 24 h, cells were harvested, and the total DNA was isolated. Quantitative PCR was performed to determine the relative concentration of viral DNA. Hexon CT values were normalized to CT values for GAPDH DNA ampliﬁed from the same sample. Data are the means of results from 3 repeats. Statistical signiﬁcance was determined by using Student’s t test, and P values of less than 0.05 () or 0.01 (**) were considered signiﬁcant. Error bars represent standard errors of the means. June 2018 Volume 92 Issue 12 e02034-17 jvi.asm.org 17 jvi.asm.org 17 Chalabi Hagkarim et al. Journal of Virology proteins. Initially, it was noted that p53 was a target for proteasome-mediated degra- dation during adenovirus infection (63, 64). This has been followed by demonstrations that other DDR proteins, such as MRE11, BLM, and DNA ligase IV, are targeted to the proteasome through the actions of the viral E1B55K and E4orf6 proteins (10, 11, 24). While this is the case for the group A and group C viruses, it certainly does not apply universally to all adenovirus serotypes (28, 56). DISCUSSION In particular, it has been shown that group B (for example, Ad7, Ad11, and Ad16) and group D (for example, Ad9) viruses target a much more limited set of DDR proteins, possibly not extending beyond DNA ligase IV. Furthermore, it seems that the E1B55K/E4orf6 complex is not always required, as the degradation of TOPBP1 requires only Ad12E4orf6, whereas DAXX degradation utilizes only Ad5E1B55K (12, 27). In a screen looking for additional DNA damage response proteins that might be targeted for adenovirus-mediated degradation, we identiﬁed Tab182 and, subse- quently, other members of the CNOT complex, CNOT7 and CNOT3, as probable targets. Tab182 was originally shown to interact with tankyrase 1 (32) and to be highly phosphorylated by ATM and/or ATR after DNA damage by IR (33). More recently, evidence has been presented to show that Tab182 plays a role in DSB repair and promotes the association of PARP1 with the DNA-PK catalytic subunit (34, 35). There had been suggestions that Tab182 was a peripheral component of the CNOT complex in mammals (40), and it was identiﬁed in various complexes in large-scale protein interactome screens (see, for example, references 65–67). We have now con- ﬁrmed that Tab182 is an integral component of the CNOT complex. The depletion of the protein increases the sensitivity of cells to damage induced by ionizing radiation, UV radiation, and HU and impairs the cell’s ability to form DNA repair foci following DNA replication stress (34, 35; our unpublished data). Here it has been shown that Tab182 is degraded during Ad5 and Ad12 infection (Fig. 1). In both cases, this requires the AdE1B55K and AdE4orf6 proteins but is independent of AdE4orf3, which has been shown to be required for the degradation of other cellular proteins (25) (Fig. 1 and 2). The degradation of Tab182 is inhibited by bortezomib, a proteasome inhibitor, and MLN4924, which inhibits cullin NEDDylation, preventing its activation (Fig. 6 and 7). As is the case for p53 degradation, Ad12 hijacks a cullin 2-based E3 ligase (Fig. 7), although it appears that the ablation of either Cul2 or Cul5 expression has a similar effect on Tab182 degradation during Ad5 infection (Fig. 7) in that the loss of either cullin causes partial protein stabilization. Clariﬁcation of this observation requires further investigation. June 2018 Volume 92 Issue 12 e02034-17 DISCUSSION To see if a similar effect occurred with other members of the CNOT complex, CNOT1, which is considered to be a scaffold protein required for the integrity of the complex, was depleted. During Ad5 infection, the E1A expression level was notably increased when CNOT1 was depleted, while in the case of Ad12, there was a greatly enhanced expression of E1A and a marked increase in the E1B55K protein level, following CNOT1 knockdown, compared to controls (Fig. 11). The increased effect of CNOT1 protein depletion on Ad12 compared to that on Ad5 appears to be consistent with Ad12’s somewhat enhanced ability to degrade Tab182. The difference in the expression of the E1A protein is due to an increase in the AdE1A mRNA level, as shown by RT-PCR (Fig. 13). Whether this effect is directly attributable to a reduction in the deadenylase activity of the CNOT complex will have to await further investigation. Interestingly, it was recently shown that the Ad5 E1B55K/ E4orf6 complex enhances E1A activity by stabilizing the protein, leading to increased levels, and by increasing the activation of E2F by E1A (68). It is possible that the effect of the same adenovirus complex on the CNOT complex, as demonstrated here, could contribute to the increased AdE1A level observed. In a further study, it was shown that the concentration of viral DNA is increased in Tab182- and CNOT1-depleted cells 24 h after both Ad5 and Ad12 infection (Fig. 14). More marked effects were seen with CNOT1 depletion than with Tab182 depletion, consistent with the observed increase in AdE1A expression (Fig. 11); however, the reduction in the Tab182 level had less of an effect on relative hexon DNA concentrations after Ad12 infection than after Ad5 infection; the reasons for this are not clear at present. The relationship between adenoviruses and the CNOT complex is not clear-cut, for while the virus is able to cause the degradation of various components, this occurs later than any initially enhanced increase in AdE1A expression seen after the depletion of CNOT proteins described here. It is notable that there is a sustained increase in AdE1A expression up to 96 h in the absence of CNOT1 (Fig. 11). However, increases in viral DNA concentrations were observed after the depletion of CNOT1 and Tab182, suggesting that the inactivation of the complex will facilitate viral replication to a limited extent. DISCUSSION To conﬁrm the results of the mutant virus infections, that Tab182 is targeted through AdE1B55K, coimmunoprecipitation assays were carried out, and it was found that Tab182 and both the Ad5 and Ad12 proteins could be immunoprecipitated together. Furthermore, both the Ad5E1B55K and Ad12E1B55K proteins bound to the GST-Tab182 C-terminal region, indicating a direct interaction (Fig. 9). Interestingly, Tab182 binds strongly to Ad9E1B55K but not Ad16E1B55K, although it does not appear to be degraded by either group B1 (Ad7 and Ad16) or group D (Ad9) viruses (Fig. 5 and 9). E1B55K proteins from both Ad9 and Ad16 interact with p53, as might be expected since it is transcriptionally inactive after Ad9 and Ad7 infection, even though it is present at a high level (28). These observations suggest that the interaction of E1B55K with Tab182 may be determined by factors other than a requirement for protein degradation. A more widespread examination of the interaction of Tab182 with E1B55K proteins from a number of adenoviruses may elucidate this point. Tab182 also associ- ates with the SV40T antigen in coimmunoprecipitation experiments, suggesting that the protein could be a target for the family of small DNA tumor viruses (Fig. 9I). Signiﬁcantly, previous studies have shown that the CNOT complex associates with HPV17a and HPV38 E6 proteins, although the consequences of this for the virus were not examined at the time (60). To see how extensive the relationship between adenoviruses and the CNOT com- plex was, the fate of other components of the complex following adenovirus infection was studied (Fig. 10). Although only a limited number of CNOT proteins were examined, jvi.asm.org 18 June 2018 Volume 92 Issue 12 e02034-17 Adenoviruses Target Tab182 for Degradation Journal of Virology it was seen that the levels of CNOT3 and CNOT7 were reduced during Ad5 and Ad12 infection, whereas the levels of CNOT4 and CNOT1 remained stable. Although a number of activities have been attributed to the CNOT complex, such as deadenylase, transcriptional regulation, and ubiquitin E3 ligase activity (37–39, 46–48), it is not clear what contribution Tab182 makes. To attempt to understand why adenovirus might target Tab182 (and other CNOT proteins), adenovirus infections were compared in control and siRNA knockdown cells. It was seen that the expression of E1A was enhanced, to a limited extent, in Tab182-depleted cells, although little or no difference was seen in the expression of late proteins (Fig. 11). June 2018 Volume 92 Issue 12 e02034-17 DISCUSSION It is also possible that the aim of the virus, in degrading and presumably inactivating the CNOT complex, is not necessarily just to facilitate AdE1A expression but to fulﬁll some other, as-yet-unidentiﬁed, role, perhaps linked to an effect on the DDR. It should be borne in mind, when considering the effects of CNOT1 depletion, that adenoviruses do not actually cause its degradation, and while its loss will probably indicate the effect of the inactivation of the CNOT complex, it does not necessarily coincide with what happens in vivo. It is also possible that other CNOT proteins could be targets for adenovirus-mediated degradation early in infection coincident with AdE1A expression, although we have no evidence of this. Signiﬁcantly, the degradation of Tab182 and CNOT7 occurs later in viral infection than is the case for MRE11 and BLM and is more similar to that seen for p53. The loss of components of the CNOT complex, for example, Tab182, appears to facilitate the progression of cells into late G1/early S phase, as evidenced by the enhanced expression of cyclin E and the transitorily enhanced expression of CDC25A (Fig. 11 and 12). This may provide an environment more conducive to the expression of early viral proteins, particularly E1A. For reasons that are not evident at present, the effect seems to be more marked with Ad12 than with Ad5. With relevance to the effects on cyclin E expression, it is worth noting that CNOT1 depletion has a more marked effect than does Tab182 depletion, suggesting that its loss enhances cell cycle pro- jvi.asm.org 19 June 2018 Volume 92 Issue 12 e02034-17 Chalabi Hagkarim et al. Journal of Virology gression to a greater extent. However, it is possible that the loss of other CNOT proteins could have a comparable effect. In summary, Ad5 and, in particular, Ad12 have been shown to target Tab182 and other CNOT proteins for proteasome-mediated degradation during viral infection. The loss of Tab182 and CNOT1 favors the enhanced expression of AdE1A and AdE1B55K In summary, Ad5 and, in particular, Ad12 have been shown to target Tab182 and other CNOT proteins for proteasome-mediated degradation during viral infection. The loss of Tab182 and CNOT1 favors the enhanced expression of AdE1A and AdE1B55K proteins in the early stages of infection. loss of Tab182 and CNOT1 favors the enhanced expression of AdE1A and AdE1B55K proteins in the early stages of infection. MATERIALS AND METHODS Cell lines, viruses, and plasmids. HeLa (obtained from ATCC), HEK293FT (Invitrogen), Ad5E1HEK293 (a generous gift from Frank Graham), and Ad12E1HER2 (69) cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 8% fetal calf serum (FCS). H1299-based cell lines in which Cul2 or Cul5 expression had been ablated were generous gifts from Paola Blanchette and Phil Branton. The cells were grown in DMEM supplemented with 8% FCS and 1 g/ml puromycin (Cul2) or 8% FCS, 1 g/ml puromycin, and 100 g/ml hygromycin (Cul5). Ad4, Ad5, Ad7, Ad9, Ad11, and Ad12 were obtained from the ATCC or were a generous gift from Jo Mymryk. The following Ad5 mutants were used: Ad5dl1520 (Ad5E1B55K) (70), H5in351 (E4orf1), H5pm4154 (E4orf6), H5pm4155 (E4orf3 E4orf6), H5pm4166 (E4orf4), H5dl356 (E4orf7), and H5in352 (E4orf2) (23, 26, 71–73). In addition, an Ad12E1B55K-negative mutant (Ad12dl620) was used (74). HeLa cells were generally infected at a multiplicity of infection of 5 PFU/cell. Ad5E1B55K and Ad12E1B55K DNAs were cloned into pcDNA3, and Ad5 and Ad12 E4orf6-HA tag DNAs were also cloned into pcDNA3 as previously described (75). NEDDylation was inhibited by the addition of MLN4924 to the cell culture medium at a concentration of 4 M, and proteasomal activity was inhibited with bortezomib (0.5 M). siRNA treatment to deplete Tab182 and CNOT proteins and protein transfections. HeLa cells were plated at a density of 4 105 cells per 6-cm dish. After 24 h, they were transfected with control or ON-TARGETplus Smart pool siRNAs (0.2 nmol/dish) (GE Dharmacon) directed against Tab182 or CNOT1 proteins by using Oligofectamine (Invitrogen) according to the manufacturer’s protocol. After 24 h, cells were split 1:3, and after a further 24 h, they were infected with virus. For protein transfections, cells were grown to 70% conﬂuence and then incubated with DNA constructs (2 g/6-cm dish or 5 g/10-cm dish) that had been previously mixed for 20 min with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco) according to the manufacturer’s protocol. After 24 h, cells were incubated with fresh medium and harvested 24 h later. Cloning of Tab182. Total cellular RNA was isolated from a lymphoblastoid cell line from a normal individual (cell line provided by the Coriell Institute for Medical Research) by using the Qiagen RNeasy minikit and was reverse transcribed into cDNA by using oligo(dT) primer d(T)23VN and the ProtoScript II ﬁrst-strand cDNA synthesis kit (New England BioLabs). June 2018 Volume 92 Issue 12 e02034-17 MATERIALS AND METHODS PCR was used to amplify the complete Tab182 cDNA sequence by using the forward primer 5=-GAGCGGGTCGACGATGAAAGTGTCTACTCTCAGG-3= (For 1) and the reverse primer 5=-CGTGATGTCGACTCAGACCTTCTTCTTCTTCAGTTT-3= (Rev13). Both primers contain the recognition sequence for the restriction enzyme SalI (underlined). The forward primer contains the translation initiation codon for Tab182 (italics), and the reverse primer contains the translation termination codon for Tab182 (italics) (strand antiparallel to the sense strand). The Tab182 cDNA sequence was ampliﬁed by using Q5 high-ﬁdelity DNA polymerase (New England BioLabs). An initial denaturation step at 98°C for 30 s was followed by 30 cycles of 98°C for 5 s, 62°C for 15 s, and 72°C for 4 min. A ﬁnal extension step for 5 min at 72°C followed the 30 cycles. The PCR products were analyzed by gel electrophoresis, and a product of the correct size (5,190 bp) was identiﬁed. The products were digested with SalI-HF, and the excised Tab182 band was puriﬁed by gel electrophoresis. Tab182 was cloned into the pEGFP-C3 plasmid. Sequence determination was performed by using an Applied Biosystems 3500 XL genetic analyzer. Sequences were analyzed online by using BLAST at the National Center for Biotechnology Information (NCBI) website. Sequences were all of the wild type. Codon 322 can encode threonine (ACT) or serine (AGT), and the ratio is approximately equal in the general population. The sequences isolated from the individual used to make the cDNA for this cloning exercise were all found to encode serine at amino acid 322. Isolation of RNA and cDNA synthesis. Cellular RNA was extracted by using the SV total RNA isolation system (Promega) according to the manufacturer’s protocol. To remove any DNA contamina- tion, RNA was treated with DNase I (Promega). RNA quantity and quality were evaluated by optical density measurements (ratios of optical densities at 260/280 nm) and by agarose gel electrophoresis. First-strand cDNA synthesis was performed by using SuperScript II reverse transcriptase (RT) (Invitrogen) and random primers according to the manufacturer’s instructions. Isolation of genomic DNA. Cellular DNA was extracted by using the QIAamp DNA minikit (Qiagen) according to the manufacturer’s protocol. In order to remove any protein or RNA contamination, 15 l proteinase K (10 mg/ml) (Sigma-Aldrich) and 4 l RNase A (20 mg/ml) (Invitrogen) were added to each sample. DNA quantity and quality were evaluated by optical density measurements (ratios of optical densities at 260/280 nm) and by agarose gel electrophoresis. Primer design and RT-PCR. MATERIALS AND METHODS performed for 10 min at 95°C for the precycling step to denature the cDNA and to activate Dual-Lock Taq DNA polymerase, which was then followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 1 min. To conﬁrm the expected ampliﬁcations, 2% agarose gel electrophoresis with ethidium bromide staining was performed. Viral AdE1A or hexon and host cell Tab182 and CNOT1 CT values were normalized to CT values of GAPDH ampliﬁed from the same sample [for example, ΔCT CT (Tab182) CT (GAPDH)], and the 2ΔΔCT method was used to calculate the relative expression level. Each experiment was performed in triplicate. Western blotting and antibodies. Cells were harvested after washing with ice-cold phosphate- buffered saline (PBS) and solubilized in a solution containing 8 M urea, 50 mM Tris HCl (pH 7.4), and 0.15 M -mercaptoethanol. Proteins were fractionated on polyacrylamide gels in the presence of 0.1 M Tris, 0.1 M bicine, and 0.1% SDS. For Western blotting, proteins were electrophoretically transferred to nitrocellulose membranes before incubation with antibodies overnight at 4°C. Antibodies used in the study were antibodies to Tab182 (an antibody raised in rabbits against GST-Tab182 [C-terminal frag- ment]), MRE11, CNOT3, CNOT4, CNOT7, (all from GeneTex), CNOT1 (Proteintech), cullin 2, cyclin E1, RPA32 (Abcam), p53 (raised in rabbits), cullin 5, GAPDH, collagen IV, SV40T (Santa Cruz Biotechnology), and -actin (Sigma-Aldrich). Rabbit antibodies against Ad5 hexon and Ad12 ﬁber proteins were gifts from Vivien Mautner and Paul Freimuth, respectively. A mouse monoclonal antibody against the Ad5 DBP was a gift from Pieter van der Vliet. Antibodies against Ad5E1A (M73), Ad12E1A (5DO2), Ad12E1B55K (XPH9), Ad5E1B55K (2A6), p53 (DO1), and HA (12CA5) were puriﬁed from monoclonal supernatants. GST pulldown assays and coimmunoprecipitation. The C-terminal fragment of Tab182 (amino acids 824 to 867 and 1221 to 1729) was expressed in Escherichia coli as a GST fusion protein, as described previously (49). For GST pulldown and coimmunoprecipitation assays, cells were harvested in ice-cold PBS and lysed in a solution containing 0.4 M NaCl, 40 mM Tris HCl (pH 7.4), 5 mM EDTA, and 1% NP-40. Insoluble protein was removed by centrifugation (45,000-rpm 30 min at 4°C). Lysates were incubated overnight with either the GST fusion protein or the appropriate antibody. Protein complexes were retrieved on glutathione-agarose beads or protein G-agarose beads as appropriate. MATERIALS AND METHODS Cellular RNA or DNA was extracted as described above. The sequences of the primers used for RT-PCR are shown in Table 2. The speciﬁcity of the primers was checked with NCBI Primer-BLAST. Primer design and RT-PCR. Cellular RNA or DNA was extracted as described above. The sequences of the primers used for RT-PCR are shown in Table 2. The speciﬁcity of the primers was checked with NCBI Primer-BLAST. RT-PCRs were performed with the Mx3005P system (Stratagene), using real-time PowerUp SYBR green master mix (Applied Biosystems). Quantitative RT-PCR was carried out with a ﬁnal volume of 20 l containing 2 g or 10 ng of cDNA or DNA, respectively, 5 pmol of the forward primer, 5 pmol of the reverse primer, and 10 l of PowerUp SYBR green master mix. The thermocycling program was jvi.asm.org 20 Adenoviruses Target Tab182 for Degradation Journal of Virology TABLE 2 Primers used in this study Gene Sequence (5=¡3=) Length (bp) Start position Stop position PCR product size (no. of bases) E1A-Ad5 (CR3) Forward, TAGATTATGTGGAGCACCCCG 21 990 1010 110 Reverse, GCCACAGGTCCTCATATAGCAA 22 1099 1078 E1A-Ad12 (CR3) Forward, AGTCCTGTGAGCACCACCG 19 980 1053 74 Reverse, GTAGGCTCGCAGATAGCACA 20 998 1034 Tab182 Forward, CTGCTCTGAGGGACTCCTTG 20 2310 2329 158 Reverse, CTGGGTCTCCTCTAGGGCTT 20 2448 2467 GAPDH (RNA) Forward, GAGTCAACGGATTTGGTCGT 20 53 72 183 Reverse, ACAAGCTTCCCGTTCTCAG 19 218 236 GAPDH (DNA) Forward, CGGCTACTAGCGGTTTTACG 20 6534369 6534388 188 Reverse, AGAAGATGCGGCTGACTGT 20 6534538 6534557 Hexon (Ad5) Forward, GCCACGGTGGGGTTTCTAAACTT 23 18862 18882 127 Reverse, GCCCCAGTGGTCTTACATGCACATC 25 18967 18989 Hexon (Ad12) Forward, GCCACGGTGGGGTTTCTAAACTT 23 17764 17784 127 Reverse, GCCCCAGTGGTCTTACATGCACATC 25 17869 17891 TABLE 2 Primers used in this study performed for 10 min at 95°C for the precycling step to denature the cDNA and to activate Dual-Lock Taq DNA polymerase, which was then followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 1 min. To conﬁrm the expected ampliﬁcations, 2% agarose gel electrophoresis with ethidium bromide staining was performed. Viral AdE1A or hexon and host cell Tab182 and CNOT1 CT values were normalized to CT values of GAPDH ampliﬁed from the same sample [for example, ΔCT CT (Tab182) CT (GAPDH)], and the 2ΔΔCT method was used to calculate the relative expression level. Each experiment was performed in triplicate. June 2018 Volume 92 Issue 12 e02034-17 REFERENCES Carson CT, Orazio NI, Lee DV, Suh J, Bekker-Jensen S, Araujo FD, Lak- dawala SS, Lilley CE, Bartek J, Lukas J, Weitzman MD. 2009. Mislocaliza- tion of the MRN complex prevents ATR signaling during adenovirus infection. EMBO J 28:652–662. https://doi.org/10.1038/emboj.2009.15. 24. Orazio NI, Naeger CM, Karlseder J, Weitzman MD. 2011. The adeno- virus E1b55K/E4orf6 complex induces degradation of the Bloom helicase during infection. J Virol 85:1887–1892. https://doi.org/10 .1128/JVI.02134-10. 25. 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We thank The Medical Research Council (MRC), through the University of Birming- ham, and Cancer Research UK (CRUK) for funding. We also thank Katie Grand for charitable donations. MATERIALS AND METHODS After washing with lysis buffer, bound proteins were released with either 25 mM glutathione (pH 8.2) (GST fusion proteins) or SDS sample buffer (immunoprecipitated samples) and fractionated by SDS-PAGE prior to Western blotting. Mass spectrometry. Proteins were immunoprecipitated as described above, except that the antibody-antigen complexes were released with a solution containing 8 M urea and 50 mM NH4HCO3 for 30 min at ambient temperature. Proteins were reduced in a solution containing 50 mM dithiothreitol (DTT) and 50 mM NH4HCO3 at 56°C for 30 min and then carboxymethylated in 100 mM iodoacetamide at ambient temperature in the dark for 30 min. Proteins were retrieved by using Amicon centrifugal ﬁlters (30K cutoff), which were washed four times with 50 mM NH4HCO3. The ﬁlters with the bound immu- noprecipitated proteins were incubated overnight at 37°C with trypsin (1 g) in 50 mM NH4HCO3. Tryptic peptides were retrieved by centrifugation, dried, and analyzed by using a Bruker amaZon ion trap mass spectrometer. Peptides were identiﬁed by using the ProteinScape central bioinformatics platform (Bruker). Immunoﬂuorescence microscopy. HeLa cells were grown on glass coverslips. After 24 h, cells were infected or mock infected with Ad5 or Ad12 (5 PFU/cell) for 30 h. Cells were ﬁxed in 3.6% paraformal- dehyde in PBS for 10 min and permeabilized in 0.5% Triton X-100 in PBS for 5 min. Fixed cells were stained with primary antibodies for 1 h, washed three times in PBS, and also stained with secondary antibodies for 1 h. DNA was stained with 4=,6-diamidino-2-phenylindole (DAPI). When preextraction was jvi.asm.org 21 June 2018 Volume 92 Issue 12 e02034-17 Journal of Virology Chalabi Hagkarim et al. used, cells were treated with preextraction buffer [10 mM piperazine-N,N=-bis(2-ethanesulfonic acid) (PIPES), 20 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 0.5% Triton X-100] for 7 min on ice before ﬁxing with 3.6% paraformaldehyde and antibody staining as described above. Fluorescence images were taken by using a Nikon E600 Eclipse 333 microscope equipped with a 60 oil lens, and images were acquired and analyzed by using Volocity software 334 v4.1 (Improvision). used, cells were treated with preextraction buffer [10 mM piperazine-N,N=-bis(2-ethanesulfonic acid) (PIPES), 20 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 0.5% Triton X-100] for 7 min on ice before ﬁxing with 3.6% paraformaldehyde and antibody staining as described above. REFERENCES Proc Natl Acad Sci U S A 107:12251–12256. https://doi.org/ 10.1073/pnas.0914605107. 29. 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https://openalex.org/W3211393156	https://orbi.uliege.be/bitstream/2268/295743/1/FINAL_s12934-022-01762-z.pdf	English	null	Xylanase Production By Thermobacillus Xylanilyticus is Impaired By Population Diversification But Can Be Mitigated Based On the Management of Cheating Behavior	Research Square (Research Square)	2,021	cc-by	10,752	Xylanase production by Thermobacillus xylanilyticus is impaired by population diversification but can be mitigated based on the management of cheating behavior Romain Bouchat1,2 , Frédéric Vélard3, Sandra Audonnet4, Damien Rioult5, Frank Delvigne2, Caroline Rémond1 and Harivony Rakotoarivonina1* Bouchat et al. Microbial Cell Factories (2022) 21:39 https://doi.org/10.1186/s12934-022-01762-z Bouchat et al. Microbial Cell Factories (2022) 21:39 https://doi.org/10.1186/s12934-022-01762-z Microbial Cell Factories Microbial Cell Factories Abstract Background: The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemi- cellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. Results: Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopula- tion exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. Conclusions: Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation condi- tions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium. *Correspondence: harivony.rakotoarivonina@univ-reims.fr 1 INRAE, FARE, UMR A 614, Chaire AFERE, Université de Reims Champagne Ardenne, 51097 Reims, France Full list of author information is available at the end of the article Backgroundh conditions e.g., for pulp and paper processing at high T° and high pH). The use of lignocellulosic biomass has been identified as a promising approach in the biorefinery field for the pro- duction of bioderived products like energy, fuels, chemi- cals and materials to substitute the fossil carbon sources causing global warming [1–3]. In this valorization, hemi- celluloses offer new possibilities of industrial applica- tions [4]. These ones are heteropolysaccharides and the most abundant are xylans formed by linear chains of d-xylopyranose linked by β-(1-4) glycosidic bonds [5, 6]. Some substitutes (depending on the plant origin) such as l-arabinofuranose, d-glucuronic and 4-O-methyl-d-glu- curonic acid and acetyl groups can be linked to the main chains by various bonds. In graminaceaous plant cell walls, the l-arabinofuranose residues can be esterified by phenolic compounds such as ferulic and p-coumaric acids which can interfere with the hydrolysis process but also present interesting applications [5–7]. Among the different processes that could be used for lignocellulose depolymerization, the use of enzymes from different sources is a very interesting alternative already reviewed [8–11]. In the context of this work, the production of hemicelluloses by the bacterium Thermobacillus xylani- lyticus will be considered. However, taking the microbial population out of its natural context for the industrial production of enzymes is still a challenge. Indeed, enzyme production is a highly regulated process, strongly dependent on the extracellu- lar conditions. Additionally, it is known that the release of these enzymes in the extracellular environment can lead to cheating behavior [19]. Cheaters are individuals among the population that benefit from the public goods released by the cooperators (i.e., in T. xylanilyticus case, microbial cells that are investing in enzyme production for the release of assimilable carbon sources), but that do not share the cost associated with the release of these public goods [20]. This is exactly what has been observed in this study, upon serial cultivations of T. xylanilyticus on xylan. Typically, these cell-to-cell heterogeneities are due to epigenetic mechanisms leading to fluctuations at the level of the biochemical reactions of the cells, and modulating gene expression [21, 22]. In some case, these population heterogeneities are reinforced by fitness advantage (i.e., in this study, benefiting from the release of assimilable sugars without investing in enzyme production) and can cause a decrease of global productivity due to the pres- ence of some specific non-producer (or low-producer) subpopulations [22, 23]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 17 Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Keywords: Hemicellulases, Xylanases, Thermobacillus xylanilyticus, Successive cultivation, Population diversification, Cell sorting, Substrate switch Successive cultivations of T. xylanilyticus on xylan leads to the progressive decline in xylanase activity Successive cultivations of T. xylanilyticus on xylan leads to the progressive decline in xylanase activity p g y y In order to characterize the behavior of T. xylanilyticus in presence of xylan and the resulting xylanase production, the growth rate (h−1) and its enzymatic activity (IU/mg) were measured during 107.7 generations. For this pur- pose, successive cultivation in sealed bottles was consid- ered even if the strain is aerobe. Indeed, in sealed bottles, carbon dioxide is added in the headspaces of the bottles. The carbon dioxide and the bicarbonate present in the medium are in equilibrium and create a buffer system with a pH at 7.75 necessary for the growth of the strain as described by Touzel [13]. It is with these conditions that production heterogeneity was observed. These con- ditions were then necessary to identify the factors influ- encing the enzymatic production of the strain along the generations. Backgroundh Microbial Cell Factories mg i.e., exhibiting a significant reduction of more than 90% by comparison with the first cultivation (p < 0.01).h bacteria. For this latter, whereas some cheater control strategies have been suggested [27, 28], no real imple- mentation of control/mitigation strategies have been considered so far. In this work, a simple and efficient control strategy will be implemented based on alterna- tive cultivation conditions on glucose and xylan. To date, this work is the first to study the subpopulations from an isogenic population of a hemicellulolytic bacterium and their impacts on the extracellular xylanase/enzymatic production. It is also the first work to implement a con- trol of the diversification process by switching between carbon sources during serial cultivations for the produc- tion of xylanase by a thermophilic bacterium. i The evolution of the tx-xyn11 gene expression, encod- ing the main xylanase GH11 secreted by T. xylanilyticus was also assessed along generations during cultivations on xylan and on glucose as a control. At the beginning of the cultivations, and as expected, the expression level of tx-xyn11 gene was very low in presence of glucose. On xylan, tx- xyn11 expression level was almost 30-fold higher than on glucose (Additional file 1: Fig. S1), this observation being in accordance with the induction level previously recorded for this strain [15]. Upon successive cultivations on xylan, a progressive decrease of tx-xyn11 expression over the generations was observed (Fig. 1B). This decrease accounted for 36.49% of the maximum level after 23.5 generations by compari- son with the tx-xyn11 expression level measured at the beginning of the cultivation tests which was significant (p < 0.01). After this first decrease, the expression level of tx-xyn11 remained close to 60% of the initial level for 108 generations. Interestingly, the evolution of the expres- sion level followed the same trend as the one recorded for the enzymatic activity during successive cultivations on xylan (Fig. 1A). Gene expression can be thus consid- ered as the main cause for the decrease observed for the xylanase activity. Considering the fact that the xylanase is an extracellular enzyme and that some cells can adopt a cheating strategy for avoiding the cost related to the syn- thesis of this molecule [29], single-cell experiments were then considered for determining the possible impact of cell-to-cell variability on xylanase production. Backgroundh y This bacterium is indeed a very promising natural producer of enzymes for the deconstruction of xylans [12]. This bacterium was originally isolated from a farm soil located under a manure heap in northern France) is aerobic, gram-positive, thermophilic and hemicellulo- lytic [13]. The hemicellulolytic enzymatic arsenal of this bacterium is composed by several enzymes including characterized two xylanases (GH10 and GH11 families, EC.3.2.1.8), one arabinofuranosidase (EC.3.2.1.55) and one feruloyl esterase (EC 3.1.1.73) [14–16]. This bacte- rium is able to mobilize a complex enzymatic arsenal according to the lignocellulosic biomass composition used as carbon source for promoting microbial growth [17]. The main hemicellulolytic activity of the strain is the GH11 xylanase secreted in the extracellular medium, Tx-xyn11 but it also produces debranching and exoen- zymes (such as esterase, xylosidase, and arabinosidase) activities mainly detected in the intracellular compart- ment [12, 17]. One important feature of the enzymes produced by T. xylanilyticus is that they are thermostable and active over a wide range of pH values i.e., from 5 to 8.5 as pointed out by previous studies involving enzymes obtained from cultivation on wheat bran and wheat straw [15, 16, 18] allowing their use under extreme reaction For addressing such population heterogeneities, high throughput single-cell analyses can be provided by flow cytometry (FC). In this work, two basic FC signals will be mainly used i.e., the forward and side scatter signals (FSC and SSC), accounting for cell size and cell internal struc- ture [24]. This approach can be complemented based on the utilization of metabolic sensors such as Redox Sen- sorGreen (RSG) that allows to discriminate subpopula- tions in function of their metabolic activities [25].hi The production of specific hemicellulose enzymatic cocktails is a key for improving the profitability of the lig- nocelluloses biorefineries by using all parts of the ligno- cellulose [3, 26]. Thermobacillus xylanilyticus represents an interesting source of thermostable and robust hemi- cellulolytic enzymes. A bottleneck for the utilization of this strain is the low enzyme production and the decrease of secreted xylanase activity with thime and over suc- cessive cultivations. Accordingly, the main goals of this study are then (1) to study and understand the dynamics of enzyme production by population of T. xylanilyticus; (2) to attempt improving the enzyme productions by the Page 3 of 17 Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Flow cytometry (FC) reveals two subpopulations exhibiting different light scattering properties The initial growth rate on xylan for the first cultiva- tion was 0.72 ± 0.12 h−1 (generation 0, Additional file 2: Table S1), and this value was considered as the reference (100%, generation 0, Fig. 1). We observed that the growth increased over the generations, this increase being more important and significant (p < 0.01) during the first gener- ations (increase of 31.56% after 23.5 generations). A stabi- lization was then noticed around a value of 0.95 h−1 until the end of the successive cultivation experiment. During this stabilization, no significant difference was observed. The xylanase activity, the main enzyme secreted by T. xylanilyticus, was also followed. At the beginning of the cultivations, xylanase activity was 141.36 ± 7.76 IU/ mg of proteins. The xylanase activities revealed a dras- tic decrease over generations, a significant decrease of almost 88.86% during the first 23.5 generations (p < 0.01) followed by a stabilization around a value of 28.10 ± 10.32 IU/mg (Additional file 2: Table S1). At the end of the suc- cessive cultivations (after more than 100 generations), the xylanase activity value represented only 18.51 ± 4.54 IU/ FC analyses were then performed for determining the possible occurrence of different subpopulations of T. xylanilyticus during the successive cultivation experi- ments. Results highlighted the presence of two sub- populations in T. xylanilyticus cultivations after 5 h of cultivations (Fig. 2A–C). Indeed, the segregation between the two subpopulations can be observed based on the forward scatter (FSC) signal which is proportional to the cell size. A FSC-A threshold value of 30,000 (Arbi- trary Unit) can discriminate two subpopulations. The subpopulation with values below 30,000 in FSC-A has been called low subpopulation (L) and the subpopulation with values above 30,000 in FSC-A has been called high subpopulation (H).h This clear segregation based on the FSC-A threshold allowed tracking the two subpopulations over the differ- ent generations (Fig. 3). At the beginning of the cultiva- tions (generation 0), the two subpopulations were present at a similar level (47.09 ± 11.84% and 52.91 ± 11.84% for Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 4 of 17 Fig. 1 Evolutions of growth rate and xylanase production characteristics of Thermobacillus xylanilyticus over generations. Flow cytometry (FC) reveals two subpopulations exhibiting different light scattering properties Evolution of relative growth rate (blue circles) and relative xylanase activity (green square) over generations normalized with the values at generation 0 (A); relative expression level of tx-xyn11 gene over generations (B). Absolute values of growth rate and enzymatic activity can be found in Additional file 2: Table S1. The level of tx-xyn11 gene expression was normalized with the 16 S rRNA gene expression before comparison along the generations during successive cultivations with xylan. The first cultivation (generation 0) was then defined as the 100% expression level and as reference sample. The expression levels over the generations were expressed as the fold increase of tx-xyn11 mRNA level over the generation 0 Fig. 1 Evolutions of growth rate and xylanase production characteristics of Thermobacillus xylanilyticus over generations. Evolution of relative growth rate (blue circles) and relative xylanase activity (green square) over generations normalized with the values at generation 0 (A); relative expression level of tx-xyn11 gene over generations (B). Absolute values of growth rate and enzymatic activity can be found in Additional file 2: Table S1. The level of tx-xyn11 gene expression was normalized with the 16 S rRNA gene expression before comparison along the generations during successive cultivations with xylan. The first cultivation (generation 0) was then defined as the 100% expression level and as reference sampl The expression levels over the generations were expressed as the fold increase of tx-xyn11 mRNA level over the generation 0 that it did not differ by their respective metabolic activity and membrane permeability. the low and the high subpopulation respectively). The evolution of the ratio between the subpopulations over the different generations was marked by an increase of the low subpopulation. This increase was more important during the 23.5 first generations, where the percentage rose significantly from 47.08 to 73.87% (p < 0.01) and then stabilized (with no more significant change) to 83.77% after 108 generations.hf Complementary FC analyses were performed on dif- ferent substrates (i.e., xylan, glucose and wheat bran) in order to better characterize the occurrence of the two subpopulations (Fig. 4). Wheat bran was selected as an interesting carbon source, considering its high xylan content [17]. Flow cytometry (FC) reveals two subpopulations exhibiting different light scattering properties At the beginning of the culture and during the exponential phase (4 h of cultivations) whatever the substrate used, the low subpopulation was dominant and accounted for 72.1, 87.3 and 84.9% of the total number The two subpopulations did not exhibit any differences upon staining respectively with RedoxSensor green and Propidium Iodide (FL1-A and FL3-A values), indicating Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 5 of 17 Fig. 2 Subpopulations of Thermobacillus xylanilyticus analyzed by flow cytometry during successive cultivations on xylan. The subpopulations were resolved based on the FSC-A values. The segregation between the two subpopulations was done by considering a gating value of 30,000 in FSC-A (orange line). The figures represent the SSC-A values (on the left), the FL1-A values (on the middle) and the FL3-A values (on the right) in function of the FSC-A values. Generations 0 (A); 23.5 (B) and 107.7 (C) respectively Fig. 2 Subpopulations of Thermobacillus xylanilyticus analyzed by flow cytometry during successive cultivations on xylan. The subpopulations were resolved based on the FSC-A values. The segregation between the two subpopulations was done by considering a gating value of 30,000 in FSC-A (orange line). The figures represent the SSC-A values (on the left), the FL1-A values (on the middle) and the FL3-A values (on the right) in function of the FSC-A values. Generations 0 (A); 23.5 (B) and 107.7 (C) respectively of cells on xylan, glucose and wheat bran respectively. At the end of exponential phase on glucose and xylan, an increase of the high subpopulation was observed (Fig. 4A, B). For wheat bran cultivations, the low subpopulation remained dominant during all the experiment. It is also on wheat bran that less sporulation was always observed. Then, the balance between the two subpopulations seems to be easier to achieve when rich carbon source (i.e., glu- cose) is considered instead of more difficult to assimi- late ones (i.e., xylan and wheat bran). Taken altogether, these data point out a possible occurrence of sporulation. This hypothesis is reinforced by the fact that cells trig- gering the formation of endospore tend to modify their light scattering properties as detected based on flow Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 6 of 17 Fig. 3 Evolution of cell percentage and xylanase activity for each subpopulation upon successive xylan cultivations. Cell sorting reveals that the high‑FSC subpopulations contains cells at the onset of sporulation In order to determine the impact of this diversification process on the production of extracellular xylanase by T. xylanilyticus, the low and the high subpopulations were sorted and subcultivated. The cell sorting was done at generation 0, after 5 h of cultivation during the exponen- tial growth phase (Fig. 6A) when the two subpopulations were present similarly at approximately the same ratio. In order to get more insights about the possible physio- logical differences between the low and the high subpop- ulations, cells collected from samples acquired during successive cultivations on xylan were sorted based on their FSC properties, and morphological analyses were performed with Scanning Electron Microscopy (SEM). More specifically, the generations for which a strong modification of the enzyme activity was recorded were analyzed i.e., the 20th generation (G20) and the 50th gen- eration (G50). The SEM pictures revealed that two types of morphologies were present at G0 after 5 h of cultiva- tion (Fig. 5A) i.e., either very long (or dividing) cells and sporulating cells. After cell sorting of the two subpopula- tions at G0, the high subpopulation contained cells with well-formed sporangia in central position (Fig. 5B). Some sporulating cells were also observed in low subpopulation but at an earlier sporulation stage (Fig. 5C). From G20 to G50, a predominance of the low subpopulation was observed by flow cytometry, most of the cells being in the vegetative state (at the same cultivation time) up to G50 (Fig. 5D). The cell sorting at G20 and G50 revealed the predominance of the low subpopulation with vegetative cells (Fig. 5E).hi The growth rate and the xylanase activity for each sub- population were assessed in the presence of xylan. A common feature observed for the two subpopulations was the lag phase, which was very long, probably due to the very low inoculum used after cell sorting. How- ever, the lag phase was longer for the low subpopulation (Fig. 6B). The growth began after 5 h for the high sub- population and after 10 h of incubation for the low sub- population. However, the duration of exponential growth phase was similar for the two subpopulations. (3, 5 and 4 h for the low and high subpopulation respectively) with growth rates of 0.55 ± 0.01 h−1 and 0.82 ± 0.02 h−1 respectively. Flow cytometry (FC) reveals two subpopulations exhibiting different light scattering properties The orange and blue bars represent the means of cells belonging to the low and high subpopulation respectively. The standard deviations are represented by white lines. The evolution of the enzymatic activity expressed in IU/mg of proteins (green lines) is also presented Fig. 3 Evolution of cell percentage and xylanase activity for each subpopulation upon successive xylan cultivations. The orange and blue bars represent the means of cells belonging to the low and high subpopulation respectively. The standard deviations are represented by white lines. The evolution of the enzymatic activity expressed in IU/mg of proteins (green lines) is also presented subpopulation with cells exhibiting advanced sporulation stage and the low subpopulation containing vegetative cells.i cytometry analyses. Accordingly, microscopy analyses will be considered in the next section. Cell sorting reveals that the high‑FSC subpopulations contains cells at the onset of sporulation About the xylanase activity, an important difference of xylanase activity was noticed at the beginning and at the onset of the exponential phase. Statistical analy- ses did not allow to conclude to a significant difference (p = 0.127) but the means xylanase activity produc- tion was 3.5-fold higher for the high subpopulation by comparison with the production level of the low sub- population at the beginning of the exponential phase (~ 0.52 ± 0.00 IU/mL vs. ~ 0.15 ± 0.21 IU/mL for the These observations confirmed the morphological and physiological diversification of the subpopulations upon successive cultivations and the progressive decrease of the high subpopulation. It also correlated the high Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 7 of 17 nd low subpopulations respectively) (Fig. 6C). At d of the exponential phase, a difference between o subpopulations was also observed as the xyla- 1.89-fold higher than the one observed for the low sub- population. It was not a significant difference, but it was more important than at the onset of sporulation Physiological changes of T. xylanilyticus detected by flow cytometry on different substrates. The substrates considered are either xylan (A), e (B) or wheat bran (C) Fig 4 Physiological changes of T xylanilyticus detected by flow cytometry on different substrates The substrates considered are either xylan (A) Fig. 4 Physiological changes of T. xylanilyticus detected by flow cytometry on different substrates. The substrates considered are either xylan (A), glucose (B) or wheat bran (C) 1.89-fold higher than the one observed for the low sub- population. It was not a significant difference, but it was more important than at the onset of sporulation (p = 0.0793). A sharp increase of xylanase production high and low subpopulations respectively) (Fig. 6C). At the end of the exponential phase, a difference between the two subpopulations was also observed as the xyla- nase activity produced by the high subpopulation was Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 8 of 17 Fig. 5 Morphological changes along the generations. Cells were observed by SEM at magnification 1500-fold (1), 5000-fold (2) and 15,000-fold (3) Generation 0 not sorted (A), generation 0 sorted to obtain high subpopulation (B), generation 0 sorted to obtain low subpopulation (C), G50 not sorted (D) and G50 after cell sorting (E) Fig. 5 Morphological changes along the generations. Cell sorting reveals that the high‑FSC subpopulations contains cells at the onset of sporulation Cells were observed by SEM at magnification 1500-fold (1), 5000-fold (2) and 15,000-fold (3). Generation 0 not sorted (A), generation 0 sorted to obtain high subpopulation (B), generation 0 sorted to obtain low subpopulation (C), G50 not sorted (D) and G50 after cell sorting (E) Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 9 of 17 6 Characterization of the two subpopulations of T. xylanilyticus. Growth and flow cytometry profiles of generation 0 cultivations before cell ing (A). The red boxes on the FSC-SSC dotplot represent the gates used for discriminating the two subpopulations; growth profiles of the subpopulations (B); xylanase activities for the two subpopulations at different growth phases (C). The blue lines and bars represent the high population, and the orange ones represent the low subpopulation Fi 6 Ch t i ti f th t b l ti f T l il ti G th d fl t t fil f ti 0 lti ti b f ll Fig. 6 Characterization of the two subpopulations of T. xylanilyticus. Growth and flow cytometry profiles of generation 0 cultivations before cell sorting (A). The red boxes on the FSC-SSC dotplot represent the gates used for discriminating the two subpopulations; growth profiles of the two subpopulations (B); xylanase activities for the two subpopulations at different growth phases (C). The blue lines and bars represent the high subpopulation, and the orange ones represent the low subpopulation Fig. 6 Characterization of the two subpopulations of T. xylanilyticus. Growth and flow cytometry profiles of generation 0 cultivations before cell sorting (A). The red boxes on the FSC-SSC dotplot represent the gates used for discriminating the two subpopulations; growth profiles of the two subpopulations (B); xylanase activities for the two subpopulations at different growth phases (C). The blue lines and bars represent the high subpopulation, and the orange ones represent the low subpopulation Page 10 of 17 Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories was observed between the end of exponential phase and stationary phase (1.33 ± 0.5 IU/mL to 3.89 ± 0.14 IU/mL and 2.52 ± 0.06 IU/mL to 4.43 ± 0.61 IU/mL for the low and high subpopulations respectively) with the two sub- populations exhibiting similar level of xylanase concen- tration after this phase. The cell sorting confirmed then the presence of different subpopulations related to xyla- nase production. Cell sorting reveals that the high‑FSC subpopulations contains cells at the onset of sporulation The difference is more noticeable with the required time for the production and not the produc- tion level itself. Indeed, both populations exposed xyla- nase production, but it required more time for the low population which needs to adapt its metabolism for pro- duction when cultivated alone. after 42.7 generations (Fig. 7A). In contrast to the experi- ments carried out on xylan, no significant loss of activity was observed when xylan was replaced by glucose. The level of xylanase activity was relatively high for all the generations when glucose was used as the main carbon source and subpopulations were switched to xylan at the end of each cultivation cycle. Globally the production of xylanase by T. xylanilyticus over successive cultivations was stabilized when glucose was used as the main carbon source (data from Additional file 3: Table S2, by compari- son with data from Additional file 2: Table S1). i The flow cytometry analyses performed during the suc- cessive cultivations on glucose pointed out the presence of the two previously observed subpopulations (low and high) such as for the successive cultivations on xylan. Fig- ure 7B showed that, at the beginning of the experiment, the low subpopulation represented 73.36 ± 7.13% of the total population. Over successive cultivations on glu- cose, the percentage of cells into the high subpopulation significantly increased up to 64.51% (p < 0.05) after 42.7 generations where the maximal level of xylanase activity was obtained. On the opposite, successive cultivations on xylan led to a drastic reduction of the high subpopulation. Improving the xylanase production based on a cheater management strategy As shown in the previous section, the presence of a sub- population exhibiting reduced extracellular enzyme pro- duction during successive cultivations led to a global decrease of xylanase production by T. xylanilyticus. This low, non-producing, subpopulation was considered as a cluster of cheater cells. In order to validate this hypoth- esis, and as an attempt to control the occurrence of cheater cells, successive cultivations on glucose instead of xylan were considered. Indeed, glucose being a more eas- ily usable carbon source, T. xylanilyticus does not require xylanase for its growth with this carbon source. The strat- egy was then to prevent the selection of one subpopula- tion along generations by providing an important pool of usable carbon source before switching to more complex substrates requiring enzymes to produce a usable form of carbon source. For this, T. xylanilyticus was cultivated during the successive cultivations experiment on glu- cose and carbon source shifts from glucose to xylan were done to evaluate the impact on xylanase production. The occurrence of the two subpopulations was done on flow cytometry analyses and xylanase production was also assessed (Additional file 3: Table S2 and Fig. 7).h Discussion Successive cultivation tests pointed out a population degeneration effect at the level of extracellular xylanase activity by T. xylanilyticus. This decrease was linked with a significant drop at the level of the transcriptional activity linked to this enzyme and was characterized for the first time for this bacterium. This kind of produc- tion decrease has already been observed for many other microorganisms (bacteria and fungi) of industrial interest [30]. The main question is to know whether this decrease can be attributed to a global reduction of enzyme pro- duction by all the cells within the population or due to a subpopulation of cells. Single-cell analyses revealed that two subpopulations were generated during the successive cultivations on xylan i.e., a subpopulation exhibiting a low FSC signal and the other exhibiting a high FSC signal. Such modulation of the FSC signal suggests a significant alteration of the cellular morphology [31]. According to microscopy observations, the high subpopulation com- prised cells in a premature stage of sporulation (onset to sporulation being triggered during the exponential phase). Upon cell sorting and subcultivation, the high subpopulation exhibited a shorter lag phase and an ear- lier xylanase production, suggesting that these sporu- lating cells are still able to produce the target enzyme. These differences at the level of the enzyme production capability suggest that a cheating mechanism can be at the origin of the population degeneration effect observed during successive cultivation. The cheater phenotype is i The growth rate on glucose at the beginning of the cul- tivations (generation 0) was 0.87 ± 0.05 h−1 and did not change significantly over the generations considering the standard deviations (Fig. 7A). At the beginning of the cultivation, the measured xylanase activity represented 217.07 ± 84.20 IU/mg of proteins (values closed to the ones obtained at genera- tion 0 on xylan successive cultivations, 141.36 ± 7.77 IU/ mg of proteins, considering the standard deviations). The xylanase enzymatic activity after the carbon source switch (from glucose to xylan) over the different genera- tions pointed out a stabilization of the production and the enzymatic levels quantified were comprised between 217.07 ± 84.20 and 288.55 ± 10.70 IU/mg between gen- erations 0 to 79.9 with a peak at 346.99 ± 47.07 IU/mg Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 11 of 17 Fig. Discussion xylanilyticus diversification at the population (A) and single-cell level (B) ig. 8 Model for T. xylanilyticus diversification at the population (A) and single-cell level (B) production from T. xylanilyticus along generations on xylan (decrease of 88.86% after 23.5 generations); (2) the presence of two different populations for this bacterium; (3) the difference between the two populations (the low population which is not sporulating but less producing and the high population which is sporulating but more producing) and the impact of balancing the two popu- lation on the xylanase production (selection of the low population along generations with xylanase production decrease); (4) the impact of substrate switching (from glucose to xylan) on populations stability according to xylanase activity (increase of 1.54-fold) and stability of this production with no more decrease of xylanase pro- duction along generations. All these elements open up new perspectives for the effective control of population diversification, notably by using advanced single-cell technologies. be easily detected based on single-cell technologies and could be used as a proxy for detecting population degen- eration. Some studies have already proven the presence of sporulating cells or morphologically different cells directly impacting the productivity of the process such as Clostridia [34, 35]. This work is the first to study bacte- rial subpopulations from an isogenic global population and highlights their impacts on an hemicellulase pro- duction. Some studies already exist for the application of flow cytometry for the identification and cell sorting (by FACS) of fungal cells (Aspergillus niger and Trichoderma reesei) to detect cellulase activity and select improved strains for cellulase production but not on hemicellu- lase production. Moreover, this study provides a strat- egy based on a cheater management which is a first for improving xylan hydrolysis of lignocellulosic biomasses. Unlike fungal studies, it is not based on mutagenesis, screening and cell sorting. This study is the first to date to highlight phenotypic diversification process as being responsible for the pro- gressive decrease in xylanase production by a thermo- philic hemicellulolytic bacterium. Also, based on the characterization of the diversification process, a mitiga- tion strategy has been derived in order to force the pop- ulation for producing the target enzyme. This strategy, relying on the alternance between glucose and xylan, was used effectively to mitigate the diversification of the pop- ulation based on the FSC signal, leading to an improved xylanase production during successive cultivation. Discussion 7 Thermobacillus xylanilyticus subpopulations evolution during glucose successive cultivations with xylanase activity being recorded after xylan switching. Relative evolution of the growth rate (blue circles) and enzymatic activity after substrate switch (green squares) over generation (A); evolution of cell subpopulations in percentage over generations after flow cytometry analyses (B). The xylanase activity was measured after a switch of carbon source from glucose to xylan at the same growth stage (green lines in A and B) Fig. 7 Thermobacillus xylanilyticus subpopulations evolution during glucose successive cultivations with xylanase activity being recorded after xylan switching. Relative evolution of the growth rate (blue circles) and enzymatic activity after substrate switch (green squares) over generation (A); evolution of cell subpopulations in percentage over generations after flow cytometry analyses (B). The xylanase activity was measured after a switch of carbon source from glucose to xylan at the same growth stage (green lines in A and B) phase. In this case, the premature sporulation can be due to the metabolic burden carried by the non-cheating cells. At the single-cell level, the picture is more complex (Fig. 8B). First, if there is a need for a better characteri- zation of the population dynamics, the transition rates between the main phenotypes (either productive or non- productive cells) and morphotypes (either sporulating or non-sporulating cells) need to be quantified. Addition- ally, a series of unknown remains i.e., some transitions that have not been considered in the initial view of the process (delineated by red arrows on Fig. 8B). However, a common observation is that the “sporulating” state can prone to appear when microbial population relies on an extracellular enzymatic production to degrade complex substrates into an usable [32, 33]. In T. xylanilyticus case, since xylanase is excreted to the extracellular medium, some cells can avoid the metabolic burden associated with the production of this enzyme but still take benefit from the monomers released by the active members of the population [19]. According to these observations, a preliminary model can be advanced (Fig. 8). At the population level (Fig. 8A), diversification and split into two subpopulations with dis- tinct FSC properties are observed during the exponential Bouchat et al. Microbial Cell Factories (2022) 21:39 Page 12 of 17 Bouchat et al. Microbial Cell Factories B) Fig. 8 Model for T. xylanilyticus diversification at the population (A) and single-cell level (B Fig. 8 Model for T. Discussion This strategy has been notably applied in adaptive evolution studies by switching between cellobiose and glucose in order to obtain Thermobifida fusca strain exhibit- ing higher cellulase production [39]. In T. xylanilyticus case, phenotypic diversification is more likely to occur, and such strategy could be implemented into a more Advanced single-cell technologies could be imple- mented in order to refine the resolution about microbial population dynamics. Among them, single-cell cultiva- tion microfluidics could be used for tracking cells and determining more precisely the transition rate between the different morphotypes [36]. Another alternative would be to use automated flow cytometry and feedback control. Such technologies have already been used for characterizing population dynamics of Gram-negative bacteria based on an approach called segregostat [37, 38]. Conclusionsh This study pointed out the importance of populations diversification processes during long-time cultiva- tion experiments. Four main observations can be high- lighted: (1) the strain degeneration at the level of xylanase Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories Page 13 of 17 Fig. 9 Schematic representation of the successive cultivations experiment of Thermobacillus xylanilyticus Fig. 9 previous one. The successive cultivation was performed every 5 h (Fig. 9). Each 4 cultivations (G20 or generation 23.5, G50 or generation 50.8, G80 or generation 80.3 and G100 or generation 107.7 for xylan), a following of the growth was done by measuring the OD600 nm. The growth rate was calculated during the exponential phase with the formula: µmax = ln (N2/N1)/t2 − t1 with N representing the bacterial population (here the optical density measured) and t the cultivation time. The generation numbers (n) were directly calculated with the formula: n = T/g where g represents the generation time (g = ln2/µmax) and T the total cultivation time. Successive cultivations experiment was performed in triplicate for 17 serial transfers (corre- sponding to more than 100 generations). sophisticated control procedure relying on automated single-cell measurement in order to prevent the appear- ance of cheaters during successive or long-term continu- ous cultivations. Strains and mediah Thermobacillus xylanilyticus XE9/11/91 isolated from a farm soil under a manure heap in northern France, already characterized by different studies and conserved in glycerol at − 80 °C was used in this work. The bacte- rium was cultivated on basal medium composed by three different solutions, a macro-mineral solution, a vitamin solution, and a metallic trace solution complemented with NH4Cl (1 g/L), yeast extract (2 g/L), NaHCO3 and supplemented with 10% CO2 as previously described by [13]. Cultivation volumes are 10 mL or 50 mL of media in sealed contents (100 mL or 500 mL bottles). Various carbon sources were used: xylan from beechwood 5 g/L (Roth), glucose 5 g/L (Sigma Aldrich) or destarched wheat bran 10 g/L (ARD Pomacle-Bazancourt, France). For the followed generations, population analyses were also done by cytometry, xylanase activity production and the xylanase (tx-xyn11) gene expression measured. Fig- ure 9 summarizes the main experiments performed dur- ing the successive cultivations. Successive cultivations of Thermobacillus xylanilyticush To evaluate the xylanase activity secreted by T. xylani- lyticus, for each followed generation, new 6 h (to reach the early stationary phase) cultivations on xylan in 10 mL were prepared. At the end of the cultivations, sam- ples were centrifuged at 3354×g for 10 min (Sorvall ST 8R centrifuge, Thermo Fisher Scientific) at 4 °C and the supernatant was recovered. The xylanase activity was determined in triplicate by using the reducing end sugars y y The strain was regenerated from glycerol stock and an overnight non sporulated preculture (OD600 nm between 1.5 and 2) was prepared on glucose basal medium at 50 °C and 150 RPM in glucose basal medium with Multi- tron shakers (Infors). The xylan media (in 50 mL volume) were inoculated with the preculture to reach a start opti- cal density of 0.1 for the cultivations start. The cultiva- tion was done at 50 °C, 150 RPM during 5 h in xylan basal medium. After 5 h, a new cultivation was started from the Page 14 of 17 Page 14 of 17 Bouchat et al. Microbial Cell Factories (2022) 21:39 Bouchat et al. Microbial Cell Factories (2022) 21:39 measurement according to the procedure described by [40]. measurement according to the procedure described by [40]. Quantitative PCR was realized with a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems™) to deter- mine the tx-xyn11 gene expression. Specific primers (for- ward primer: GACGGCACGCAGACGTTCCA, reverse primer: GCCTTCGGTTGCGAGCACCT) previously described [42] was used and yielded a specific 162 bp long product. Before utilization, the specificity of the primers was tested and confirmed. Briefly, 0.1 mL of the supernatants (diluted or not) are incubated in 0.9 mL beechwood xylan (Roth) at 0.5% w/v homogeneously suspended in 50 mM sodium acetate buffer (pH of 5.8) at 60 °C for 10 min. The reducing sugars were measured by following the absorbance at 420 nm on a Specord 200 Plus uv/vis spectrophotometer (Analytik Jena) and by comparing the values with a standard curve done with varying concentrations of xylose. The activity was expressed in IU/mL. One international unit (IU) of enzyme activity was defined as the quantity of enzyme (xylanase) required to liberate one µmol of equivalent xylose per minute at 60 °C. Measurements of the tx‑xyn11 gene expressionh The cultivation samples for gene expression (5 mL) were centrifuged at 3354×g for 10 min (Sorvall ST 8R centri- fuge, Thermo Fisher Scientific) at 4 °C. Cell pellets were kept in 1.5 mL of RNAlater at − 80 °C before the analyses. The cells pellets were used for RNA extraction with RNe- asy Mini Kit (Qiagen) by following the supplier recom- mendations. The obtained RNA solutions were treated with RNase-Free DNase Set (Qiagen) to prevent the pres- ence of DNA in the samples. Absence of DNA was veri- fied by polymerase-chain reaction with DreamTaq™ Hot Start Green PCR Master Mix (Thermo Fisher Scientific) by using specific primers for tx-xyn11 gene, followed by electrophoresis migrations on RNase-free TBE buffer 1× agarose gel 1% (Mupid® One Electrophoresis System, Eurogentec). The RNA concentrations were determined using a Qubit™ Fluorometer and Qubit® RNA Assay Kit (Invitrogen). First strand cDNAs were synthesized by using Super- Script® IV First-Strand cDNA Synthesis Reaction kit (Thermo Fisher Scientific) with 500 ng of total RNA in presence of random hexamer primers (50 µM), DNTP mix (0.5 mM of each), 1× SuperScript IV buffer, DTT (100 mM), RNAseOUT™ Recombinant RNase Inhibi- tor (2 U/µL) and 800 U (or 40 U/µL) of Superscript IV reverse transcriptase in a final volume of 20 µL. The syn- thesis was done at 23 °C for 10 min followed by a step at 52.5 °C for 10 min. An inactivation was done at 80 °C for 10 min. The generated cDNAs were kept at − 80 °C until utilization. Successive cultivations of Thermobacillus xylanilyticush i The amplifications were performed in 15 µL final vol- ume containing 7.5 µL of Absolute Blue qPCR SYBR green low ROX mix (Thermo Fisher Scientific), 1.4 µL of primers mix (280 nM), 1.1 µL of DNase/RNase-free water and 5 µL template cDNA (10-fold diluted). µ p The PCR program was the following: an initial dena- turation of 95 °C for 15 s followed by 40 cycles of 95 °C for 10 s, 60 °C for 45 s with a single fluorescence meas- urement before an elongation step of 72 °C for 30 s. The specificity of the PCR products was confirmed by melt- ing curve analysis (after a step at 95 °C for 15 s, the melt- ing curve analysis was performed between 60 and 95 °C with a heating rate of 0.1 °C/s). Different non-template controls and positive controls (with genomic DNA of T. xylanilyticus) were also included to confirm the specific- ity of the reactions. The tx-xyn11 transcripts were nor- malized by using the expression of T. xylanilyticus 16 S rRNA gene amplified by using the specific primers (for- ward primer: CGCGAGCGACGCAATCCCA, reverse primer: CGGTTACCCCACCGGCTTCG). For the rela- tive expression of tx-xyn11 gene, calculation was done using the 2−∆∆Ct method [43]. For the quantification of tx-xyn11 transcripts over generations, the expressions of tx-xyn11 at the beginning of the successive cultivations (generation 0) were used as reference samples. In order to compare, the expression of tx-xyn11 gene between glucose and xylan successive cultivations, the reference sample was defined as the first cultivation on glucose. The results were expressed as the fold increase of mRNA level over the reference samples. Quantitative PCR was realized in triplicates for each sample of successive cultivations. The IU values were normalized with the quantity (in mg) of total protein in the supernatants. The concentra- tions of total protein in the samples were determined by the Bradford procedure [41] with a commercial reagent 5× (Serva) as recommended by the supplier. After nor- malization, the activity was expressed in IU by milligrams of total proteins (IU/mg). Statistical testshf The different results of growth, xylanase activities, cell percentage and tx-xyn11 gene expression were com- pared for the different generations by statistical analysis with Student’s test from MATLAB statistics and machine learning toolbox. Differences were considered significant for p ≤ 0.05. The same statistical analyses were performed to compare the two subpopulations after cell sorting. Scanning electron microscopy analyses During the followed successive cultivations, samples were taken before and after the cell sorting for analy- ses. Before the cell sorting, drops of 20 µL were dried on glass coverslip. After the cell sorting, 10 mL of different sorted populations (at 500 cells by µL corresponding to 5 × 106) were concentrated by centrifugation at 16,000×g for 40 min. The cell pellets were solubilized in 500 µL and 22.5 µL drops were dried on glass slide (corresponding to 225,000 cells). Samples were also analyzed after staining with Bac- Light™ RedoxSensor™ Green (RSG, 1 µM) or propidium iodide (PI, 10 mg/L) (Invitrogen) to detect the metabolic activity and the membrane permeability respectively [37, 44]. After 488 nm laser excitation, green fluorescence were collected at 525 nm ± 30 nm for RSG (FL1-A) and the red fluorescence signal at 670LP (FL3-A) for PI. l Cells were also analyzed at different growth phases (exponential phase, stationary phase and sporulation) on different carbon sources (glucose, xylan and destarched wheat bran) with the same cytometric parameters. After drying, glass slides were washed 2-times in 1× PBS, then fixed in 2.5% (W/V in PBS) glutaraldehyde (Sigma-Aldrich) at room temperature for 1 h. After 2 distilled water washing, cells were dehydrated at room temperature in graded ethanol solutions (50, 70, 90, and twice with absolute ethanol) for 10 min and in a solution of ethanol (100%)/hexamethyldisilazane (V/V) for 5 min. Glass slides were finally desiccated with one final drop of HMDS. After air-drying at room temperature, samples were sputtered with a thin gold–palladium film using a JEOL ion sputter JFC 1100 instrument. Samples were then observed using a Schottky Field Emission Scanning Electron Microscope (JEOL JSM-7900 F). Successive cultivation on glucose l l Successive cultivations were also performed by using glucose as carbon source during 80 generations. For the generations 23.9, 42.7, 62.9 and 79.9 (due to a different growth rate than on xylan), a switch of the carbon source between glucose and xylan was performed and the same analyses (flow cytometry and xylanase activity) as those for xylan successive cultures were done. 5 × 106 events were collected for each population and the rate of sorting was around 2500 events/s with a 70 μm nozzle. Each cell sorting was performed in tripli- cate. The events were collected in 10 mL of PBS 1×. To check for a correct cell sorting, an analysis of each popu- lation collected was done with BD Accuri™ C6 plus flow cytometer. Each population solution was then centri- fuged at 12,108×g (Sorvall ST 8R centrifuge, Thermo Sci- entific) for 30 min at room temperature. The cell pellet conserved in 100 µL of 1× PBS was solubilized with 1 mL of xylan basal medium. All the cell solution was used to inoculate a new cultivation on xylan and characterize the separated populations. Population analyses by flow cytometryf Population analyses at the different generations described above (for the successive cultivations) were done by flow cytometry with a BD Accuri™ C6 (BD Biosciences) using the MOBICYTE core facilities (University of Reims Champagne-Ardenne). The analytical parameters were flow rate of 35 µL/minute, core size of 16 μm and the threshold were down to 30,000 in FSC-H. 20 000 cells were collected for each sample.f Samples taken at different generations (5 mL) were filtered with 5 μm cellulose syringe filters and diluted Bouchat et al. Microbial Cell Factories (2022) 21:39 Page 15 of 17 ST 8R centrifuge, Thermo Scientific). The supernatants were used for the measurements of xylanase activity and protein contents according to the procedures described above. with PBS 1× to reach maximum 2500 detected events/s (and events/µL) to prevent doublet reading. Samples were analyzed first unstained, and populations were detected by the forward and scatter signals (respectively FSC-A and SSC-A) to determine cell percentage of each detected population with a gating at 30 000 in FSC-A. The BD CSampler Software was used to acquire and treat the cytometric data. Cultivations of the sorted populations Population cultivations were performed as described in the previous parts for 19 h. The growth was followed with regular measurement of the OD600 nm. At the different growth phases determined (lag, beginning and end of the exponential phase and stationary phase), samples of 5 mL were taken and centrifuged at 3354×g for 10 min (Sorvall Cell sortingh The cell sorting was realized from T. xylanilyticus culti- vated on xylan basal medium at the generation 0 but also along generations (G20 and G50). The cell sorting was performed with a BD FACSAria™ II Cell Sorter coupled with the BD Accuri™ C6 Plus flow cytometer from the URCACyt technical platform facilities. In order to standardize the signals of the BD Accuri™ C6 Plus flow cytometer and BD FACSAria™ II cell sorter, an analysis of 2.5 μm microbeads (BD Biosciences) was done with both systems. FC: Flow cytometry; FSC: Forward scatter; GH: Glycoside hydrolase; HMDS: Hexamethyldisilazane; IU: International unit; OD: Optical density; PBS: Phos- phate buffer solution; PCR: Polymerase chain reaction; PI: Prodipidum iodide; Abbreviations FC: Flow cytometry; FSC: Forward scatter; GH: Glycoside hydrolase; HMDS: Hexamethyldisilazane; IU: International unit; OD: Optical density; PBS: Phos- phate buffer solution; PCR: Polymerase chain reaction; PI: Prodipidum iodide; Page 16 of 17 Page 16 of 17 Bouchat et al. Microbial Cell Factories (20 (2022) 21:39 RSG: RedoxSensor Green; SEM: Scanning electron microscopy; SSC: Side scatter. 3. 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J Biol Chem. 2011;286:39958–66. 40. Kidby DK, Davidson DJ. Ferricyanide estimation of sugars in the nanomole range. Anal Biochem. 1973;55:321–5. 40. Kidby DK, Davidson DJ. Ferricyanide estimation of sugars in the nanomole range. Anal Biochem. 1973;55:321–5. 41. Marion M, Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. 41. Marion M, Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. g 42. Rakotoarivonina H, Hermant B, Aubry N, Rabenoelina F, Baillieul F, Rémond C. Dynamic study of how the bacterial breakdown of plant cell walls allows the reconstitution of efficient hemicellulasic cocktails. Bioresour Technol. 2014;170:331–41. https://doi.org/10.1016/j.biortech. 2014.07.097. g 42. Rakotoarivonina H, Hermant B, Aubry N, Rabenoelina F, Baillieul F, Rémond C. Dynamic study of how the bacterial breakdown of plant cell walls allows the reconstitution of efficient hemicellulasic cocktails. Bioresour Technol. 2014;170:331–41. https://doi.org/10.1016/j.biortech. 2014.07.097. 43. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods. 2001;25:402–8. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W4299318067	https://zenodo.org/records/1461804/files/Zydorowicz_Hum_16_S.pdf	Polish	null	Ikonologiczne inspiracje w badaniu kultury współczesnej	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	5,995	Humaniora. Czasopismo Internetowe Nr 4 (16)/2016, ss. 25–37 Humaniora. Czasopismo Internetowe Nr 4 (16)/2016, ss. 25–37 jacek zydorowicz Uniwersytet im. Adama Mickiewicza w Poznaniu Instytut Kulturoznawstwa e-mail: jacek.zydorowicz@amu.edu.pl 2 Tak czy owak efekty pracy naukowej coraz częściej lądują dziś w sieci niż w drukarniach. Może się jednak okazać, że jedynie do pewnego momentu medium to będzie tolerować klasyczne e-booki przyjazne dla domowych drukarek. Internet przynosi nowe wyzwania i konieczność adaptacji do obowiązującej netykiety, logiki i obrazowej estetyki medium. W perspektywie tychże wyzwań znajdą się zatem rozmaite problemy związane z owym dopasowaniem. Nie są to jednak bariery, a właśnie wyzwania. Warto bowiem zauważyć, że przechodzenie z klasycznej, Ikonologiczne inspiracje w badaniu kultury współczesnej Abstract. There are many perspectives, turns, tendencies and paradigms in the history of cultural studies. Notwithstanding the considerable advance in civilization, there are also such viewpoints which draw on the iconological traditions worked out in earlier centuries. The contemporary reflection on visual culture makes references to such figures as Erwin Panofsky or Aby Warburg. The proclamation of the so called iconic turn in the humanities by W. J. T. Mitchell provoked many novel debates focused on the domination of images and various aspects of visual communication. It is worth stressing that apart from meta-dis courses, there started to emerge methodologies which made it possible to apply those theories in the research practice. This resulted in the emergence of such subdisciplines as visual sociology and visual anthropology and gave an opportunity the transdisciplinary testing of a new repertoire of research tactics based on images (photo/video essays, vid eo/photographic documentation, photoblog analyses, ethnography/sociology of the media etc.). It must be also noticed that the enthusiasts of those methods have a considerable awareness of the existence of many traps and limitations and the fact that solving one problem often leads to dozens of other problems. Keywords: visual studies, iconology, iconography, visual sociology, visual anthropology, imagology, iconic turn R ozmaite (tak współczesne, jak i historyczne) formy praktyk kulturowych – czy się to akademikom podoba, czy nie – są mocno zakorzenione w wizualności. Ktokolwiek jednak spróbuje zbliżyć się do zdefiniowania zjawiska obrazowości – utrzymuje William John Thomas Mitchell – nieuchronnie odkryje dwa fakty: po R – utrzymuje William John Thomas Mitchell – nieuchronnie odkryje dwa fakty: p 26 j a c e k z y d o r o w i c z pierwsze, zobaczy niezwykłą różnorodność przedmiotów określanych mianem obrazów (przedstawienia wizualne, pomniki, złudzenia optyczne, mapy, schematy, halucynacje, wspomnienia, myśli, spektakle, projekcje), po drugie, zrozumie, że nazywanie tych wszystkich zjawisk obrazami nie musi oznaczać, że cokolwiek je łączy1. Nie sposób jednocześnie zadowolić się konstatacją o niemożności spójnego uchwycenia tych zjawisk. Stawką jest bowiem istotna refleksja nad ko munikowaniem i symbolizowaniem. Stąd pośród rozmaitych dyscyplin (i działań interdyscyplinarnych) na polu badań kultury da się zauważyć pewne ożywienie. Z zainteresowaniem wzajemnym krzyżują się tu perspektywy antropologów, kulturoznawców, medioznawców, socjologów, psychologów, kognitywistów czy historyków sztuki. Analizy zjawisk kulturowych przeprowadzane tu są de facto za pomocą gotowych narzędzi, jakimi wyżej wymienieni dysponują. 1 W. J. T. Mitchell, Iconology, Image, Text, Ideology, University of Chicago Press, Chicago 1986, s. 9. Ikonologiczne inspiracje w badaniu kultury współczesnej Jednakże – jak to w przypadku badań interdyscyplinarnych bywa – wcale nie musi chodzić o wypracowanie jakiegoś konsensusu. Ujmując rzecz w nieco foucaultowskim duchu, tu i ówdzie pojawiają się również spory o reżimy produkcji wiedzy/władzy, dominację, kontrolę i legitymizację. Kultura obrazu z definicji doskonale wpisuje się w ten panopticon relacji władza – wiedza. Jej możliwości manipulowania sferą światopoglądową są dość powszechnie znane. Rzecz w tym, aby podchodzić do badań, mając te uwarunkowania na względzie. Należy tu nadmienić, że wprawdzie studia wizualne (visual studies) niespecjalnie roszczą sobie pretensje do bycia kolejną „twardą” dyscypliną badawczą, lecz z pewnością nie cechuje ich metodo logiczny nihilizm. Niniejszy szkic ma być właśnie próbą „spojrzenia z ukosa”: jak stosunkowo młoda dziedzina wiedzy radzi sobie z wyzwaniami współczesności, dynamicznie dopasowując tradycyjne i utrwalone teorie ikonologiczne do nowych metod i podejść nierzadko eksperymentujących. Obraz nie od dziś mediuje między różnymi wymiarami rzeczywistości, jednak sfera informacyjna w dzisiejszych mediach elektronicznych jest wyraźnie przezeń zdominowana. Nawet audycje radiowe w wydaniu internetowym coraz bardziej przypominają telewizję. Media się hybrydyzują i niespecjalnie należy boleć nad jakąś utratą integralności ich dotychczasowych tożsamości. Nie chodzi przy tej okazji o wzniecanie na nowo dysputy nad dylematem, czy słowo i obraz są dziś równymi partnerami w produkcji wiedzy o świecie. Stare chińskie przysłowie mówi: „jeden obraz wart tysiąca słów” – czy jesteśmy w stanie zaryzykować „lustrzaną” tezę, że oto jedno zdanie może być warte tysiąca obrazów?2 Złośliwi Ikonologiczne inspiracje w badaniu kultury współczesnej 27 powiedzą, że komunikacyjne operowanie na obrazkach warte było uwagi w cza sach, gdy większość stanowili analfabeci – by przywołać choćby biblię pauperum. Tymczasem tworzenie i interpretowanie wizerunków to pełnoprawny aspekt sfery komunikacyjnej, i jako taki od zarania dziejów domagał się stosownych kodyfikacji. Oprócz przeglądu rozmaitych historycznych i współczesnych podejść do ba dania obrazów warto jednak postawić pytanie natury stricte pragmatycznej: czy i jak metody badań wizualnych mogą przydać się w warsztacie badacza kultury współczesnej? Na pierwszą część pytania odpowiadam twierdząco, na drugą – postaram się w największym skrócie odpowiedzieć w tym tekście, przywołując wybrane strategie i taktyki badawcze. powiedzą, że komunikacyjne operowanie na obrazkach warte było uwagi w cza sach, gdy większość stanowili analfabeci – by przywołać choćby biblię pauperum. Tymczasem tworzenie i interpretowanie wizerunków to pełnoprawny aspekt sfery komunikacyjnej, i jako taki od zarania dziejów domagał się stosownych kodyfikacji. Ikonologiczne inspiracje w badaniu kultury współczesnej Oprócz przeglądu rozmaitych historycznych i współczesnych podejść do ba dania obrazów warto jednak postawić pytanie natury stricte pragmatycznej: czy i jak metody badań wizualnych mogą przydać się w warsztacie badacza kultury współczesnej? Na pierwszą część pytania odpowiadam twierdząco, na drugą – postaram się w największym skrócie odpowiedzieć w tym tekście, przywołując wybrane strategie i taktyki badawcze. linearnej struktury narracyjnej na hipertekst nie będzie najtrudniejsze. Jest to zwyczajnie dosko nalsza wersja tradycyjnych gloss i przypisów, co więcej, pewne nawyki internauci zdążyli już z powodzeniem nabyć. Co natomiast wydarzyć się może w warstwie wizualnej? Czy dyskurs naukowy skieruje się w stronę komiksu jak w przypadku rozprawy doktorskiej Nicka Sousanisa? (w 2015 r. na Columbia University przedstawił on dysertację zatytułowaną Unflattening; praca dotyczyła właśnie związków słowa i obrazu w literaturze, przy tym jej nowatorstwo polegało na ujęcie całości w formę komiksu, a ściślej powieści graficznej – graphic novel). Tradycyjnie usposobieni badacze nie powinni jednak wpadać w panikę – nie chodzi o defensywę języka werbalnego, lecz o jego stosowne wsparcie ze strony obrazów. Kierunkiem pożądanym byłoby zatem z jednej strony „myślenie obrazami” już na etapie badań, z drugiej uatrakcyjnienie (i swo ista „esencjalizacja”) sposobów prezentacji ich wyników. 3 Pełna wersja tytułu sugeruje to bowiem dość jednoznacznie: Ikonologia albo Opis Wize runków uniwersalnych, wydobytych z zabytków starożytności i z innych miejsc (...) Dzieło tyleż przydatne, co i niezbędne Poetom, Malarzom i Rzeźbiarzom przy przedstawianiu cnót, przywar, afektów i namiętności ludzkich. 1. Prekursorzy ikonologii Od czasów prehistorycznych malowideł naskalnych obrazowe komunikaty czło wieka stanowiły istotny element jego ekspresji. Tak przedstawienia obrazowe, jak i pismo piktograficzne tworzyły komunikaty do interpretowania. W XV wieku odkryto traktat greckiego filozofa Horapollona (V/VI wieku n.e.) pt. Hierogly phica, w którym zawarte zostały rozmaite spekulacje m.in. wokół egipskich hieroglifów oraz innych symboli i alegorii (np. ze starożytnej Grecji). Na gruncie średniowiecznego chrześcijaństwa nie brakowało również rozmaitych ksiąg obja śniających atrybuty świętych na obrazach czy też bestiariuszy. W 1593 r. wydana została Ikonologia Cersarego Ripy (Iconologia overo Descrittione dell’Imagini universali). Dla ścisłości – w tejże rozprawie nie chodziło o ikonologię w znaczeniu nadanym później przez Erwina Panofsky’ego, gdyż była ona raczej rozbudowanym katalogiem alegorii, swoistym wizualnym słownikiem czy wręcz poradnikiem dla artystów3. Pierwsze wydanie miało charakter wyłącznie tekstowy, ale wydana wkrótce druga edycja zawierała już mnogość drzeworytów. linearnej struktury narracyjnej na hipertekst nie będzie najtrudniejsze. Jest to zwyczajnie dosko nalsza wersja tradycyjnych gloss i przypisów, co więcej, pewne nawyki internauci zdążyli już z powodzeniem nabyć. Co natomiast wydarzyć się może w warstwie wizualnej? Czy dyskurs naukowy skieruje się w stronę komiksu jak w przypadku rozprawy doktorskiej Nicka Sousanisa? (w 2015 r. na Columbia University przedstawił on dysertację zatytułowaną Unflattening; praca dotyczyła właśnie związków słowa i obrazu w literaturze, przy tym jej nowatorstwo polegało na ujęcie całości w formę komiksu, a ściślej powieści graficznej – graphic novel). Tradycyjnie usposobieni badacze nie powinni jednak wpadać w panikę – nie chodzi o defensywę języka werbalnego, lecz o jego stosowne wsparcie ze strony obrazów. Kierunkiem pożądanym byłoby zatem z jednej strony „myślenie obrazami” już na etapie badań, z drugiej uatrakcyjnienie (i swo ista „esencjalizacja”) sposobów prezentacji ich wyników. j j ) p p j y 3 Pełna wersja tytułu sugeruje to bowiem dość jednoznacznie: Ikonologia albo Opis Wize runków uniwersalnych, wydobytych z zabytków starożytności i z innych miejsc (...) Dzieło tyleż przydatne, co i niezbędne Poetom, Malarzom i Rzeźbiarzom przy przedstawianiu cnót, przywar, afektów i namiętności ludzkich. 28 j a c e k z y d o r o w i c z Można tu zaryzykować tezę, że historia komunikacji zatoczyła swoiste koło – od obrazu poprzez abstrakcyjne idee i pojęcia, aż do ponownej potrzeby obrazowego zilustrowania tychże pojęć. Przytoczone dotąd dzieła miały w swych ambicjach raczej charakter ikonograficzny, stanowiły bowiem kompendia wiedzy koniecz nej do dekodowania obrazowego uniwersum. 4 A. Zeidler-Janiszewska, O tzw. zwrocie ikonicznym we współczesnej humanistyce. Kilka uwag wstępnych, „Dyskurs” 4, s. 151, http://www.asp.wroc.pl/dyskurs/Dyskurs4/AnnaZeidler Janiszewska.pdf [20.12.2016]. 1. Prekursorzy ikonologii Poważniejszy krok od ikonografii ku ikonologii poczynił dopiero niemiecki historyk sztuki i teoretyk kultury Aby Warburg (1866-1929), a kroki dalsze były już dziełem jego ucznia Erwina Pano fsky’ego (1882-1968). W książce Studies in Iconology (1939) wyłożył on swą metodę trójstopniowej analizy dzieła sztuki. W tymże ujęciu etap pierwszy miał stanowić opis preinkonograficzny, a zatem dostrzeżenie form tworzących rozma ite konfiguracje obiektów. Upraszczając nieco: chodziło o wstępne (bazujące na zwykłym, życiowym doświadczeniu) rozpoznanie, jakie obiekty i sytuacje widać na obrazie. W stadium drugim miała następować analiza ikonograficzna, czyli przypisanie owym obiektom i sytuacjom konwencjonalnych znaczeń w oparciu o zidentyfikowanie ich np. jako personifikacji czy alegorii. Ostatni etap, czyli analiza ikonologiczna, wymagał już czegoś, co można by ująć jako erudycja – tak wizualna, jak i pojęciowa. Panofsky określał ją mianem „intuicji syntetyzującej”, rozumianej jako „obycie z głównymi tendencjami ludzkiego ducha”. W tej fazie bowiem najsilniejszy akcent położono na kulturowy kontekst, czyli podłoże dzieła (filozoficzne, religijne, historyczne, regionalne, narodowe etc.). 2. Zwrot ikoniczny w humanistyce Tak się składa, że historię refleksji nad kulturą skłonni jesteśmy ujmować w kate gorii rozmaitych „zwrotów”. Jak pisze Anna Zeidler-Janiszewska, zwroty te [...] rekonfigurują pola badawcze, implikują nieoczekiwane zerwania i nowe sojusze, skłaniają do reinterpretacji tradycji myślowych. Dzieje się tak przynajmniej od czasu zwrotu lingwistycznego. Dwa ostatnie zwroty, krytyczne wobec dominacji perspektywy lingwistycznej w różnych dziedzinach badań nad kulturą (czy to w wersji struktural no-semiotycznej, czy dekonstrukcyjnej), odwołują się zarazem do diagnoz kultury współczesnej, która z jednej strony eksponuje rysy performatywne (stąd zwrot performa tywny), z drugiej natomiast – od dawna, tj. od czasów wynalezienia fotografii i technik masowej reprodukcji – nasyca się coraz bardziej różnego pochodzenia obrazami (stąd zwrot wizualny czy ikoniczny)4. Od zdiagnozowania symptomów takiego zwrotu do względnego utrwalenia niezależnej dyscypliny badawczej potrzeba jednak czasu. W marcu 1992 r. w cza Ikonologiczne inspiracje w badaniu kultury współczesnej 29 sopiśmie „ArtForum” pojawił się artykuł pt. The Pictorial Turn5. Jego autor, W.J.T. Mitchell, postanowił w znaczącym stopniu zakwestionować dominację strategii i metod badawczych ukształtowanych wokół zwrotu lingwistycznego. Ostrze jego krytyki wymierzone było przeciwko „ikonoklastycznemu” uprzywilejowa niu tekstów względem obrazów. Ponowne odkrycie obrazu miało służyć m.in. przemyśleniu na nowo jego statusu oraz poszukiwaniu nowych metod analizy i krytyki w obszarze wizualności. Tych ostatnich upatrywał właśnie w obszarze przepracowywanej przez siebie tradycji ikonologicznej. W późniejszej książce Picture Theory6 koncentrował się właśnie na mechanizmach wytwarzania znaczeń przez obrazy, a także ich działaniu i cyrkulacji w kulturowych obiegach. W do tychczasowych logocentrycznych teoriach obrazy stanowiły co najwyżej ilustrację dla wyrażanej tekstem myśli. Tymczasem sięgając do greckiej etymologii, teoria znaczy tyle, co patrzeć, a idea – widzieć. Mitchell poszukiwał więc sposobów na zwiększenie autonomii tego, co wizualne w zakresie nowych „gramatyk” dla filozoficznej refleksji. Tak pojęty obraz stałby się na nowo istotnym mediatorem pomiędzy podmiotem a światem. Dyskusja wokół studiów wizualnych i zwrotu ikonicznego wzbudzała spore zainteresowanie, jednak wbrew pozorom nie była pozbawiona punktów zapalnych. Czasopismo „October” poświęciło jeden z numerów właśnie tej problematyce, gdzie polemiczny głos zabrała Rosalind Krauss7. Nawiązując m.in. do Rolanda Barthes’a, stanęła ona w obronie intelektualnej spuścizny po zwrocie lingwistycz nym. W duchu krytycznym odniosła się natomiast do powszechnej nadprodukcji obrazów przez media masowe, które zabijają w odbiorcy zmysł zdystansowanego krytycznego osądu zjawisk. Krauss przy okazji dotknęła również innego czułego punktu – kwestii humanistyki zaangażowanej. 5 W. J. T. Mitchell, The Pictorial Turn, „ArtForum”, March 1992, s. 89 nn. 6 W. J. T. Mitchell, Picture Theory. Essays on Verbal and Visual Representation, University of Chicago Press, Chicago 1994. 7 R. Krauss, Welcome to the Cultural Revolution, „October” vol. 77, Summer 1996, ss. 83-96. 8 Por. F. Jameson, Postmodernizm, czyli logika kulturowa późnego kapitalizmu, Wyd. Uni wersytetu Jagiellońskiego, Kraków 2011. 5 W. J. T. Mitchell, The Pictorial Turn, „ArtForum”, March 1992, s. 89 nn. 2. Zwrot ikoniczny w humanistyce Parafrazując nieco Fredrica Jameso na, zasugerowała, że visual studies wcale nie muszą być tak radykalne i rewolucyjne (jak same chciałyby o sobie myśleć), gdyż w pewien sposób ideologicznie repli kują konsumpcyjną logikę kapitalizmu8. Niejako w kontrapunkcie do tej krytyki można jednak powiedzieć, że ten sam pakiet zastrzeżeń dałoby się sformułować np. względem artystów pop-artu, a jednak sztuka ta potrafiła skutecznie się obro nić, wytwarzając niezbędną warstwę postawy ironicznej. Jednocześnie wiemy o powszechnym przekonaniu, że artystom w diagnozowaniu świata wolno więcej niż badaczom: sztuka może sobie być zaangażowana, krytyczna, afirmatywna bądź ironiczna. 30 j a c e k z y d o r o w i c z Czy zatem faktycznie stoimy przed zagrożeniem, że oto ideologów zastąpią dziś sprytni (i równie żądni władzy) imagolodzy? Wszak od zawsze obrazowy język sztuki utrwalał lub wzmacniał jakiś określony dla danej kultury porządek symboliczny, dokonując tym samym waloryzacji panujących światopoglądów. Analizując te imagologiczne zagadnienia, Grzegorz Dziamski odsyła nas najpierw do diagnoz Milana Kundery zawartych w Nieśmiertelności, a dalej do Marka C. T l i E S i Ci i ili ć i l ii j k Czy zatem faktycznie stoimy przed zagrożeniem, że oto ideologów zastąpią dziś sprytni (i równie żądni władzy) imagolodzy? Wszak od zawsze obrazowy język sztuki utrwalał lub wzmacniał jakiś określony dla danej kultury porządek symboliczny, dokonując tym samym waloryzacji panujących światopoglądów. Analizując te imagologiczne zagadnienia, Grzegorz Dziamski odsyła nas najpierw do diagnoz Milana Kundery zawartych w Nieśmiertelności, a dalej do Marka C. Taylora i Esy Saarinena. Ci ostatni postanowili stworzyć zarys imagologii jako filozofii mediów, która nie tylko diagnozowałaby świat, ale również aktywnie i krytycznie uczestniczyła w jego przekształcaniu. Taylor powiada: Taylora i Esy Saarinena. Ci ostatni postanowili stworzyć zarys imagologii jako filozofii mediów, która nie tylko diagnozowałaby świat, ale również aktywnie i krytycznie uczestniczyła w jego przekształcaniu. Taylor powiada: [...] dostrzegamy radykalną zmianę i odnajdujemy w niej bogate możliwości [...]. Dlatego nasze zadanie jest podwójne: uświadamiać i przełamywać opory tych, którzy nie są świadomi zmiany lub jej się przeciwstawiają, a jednocześnie rozwijać krytycz ną świadomość i wzmacniać twórczy sprzeciw tych, którzy są całkowicie zanurzeni w nowoczesnym świecie9. [...] dostrzegamy radykalną zmianę i odnajdujemy w niej bogate możliwości [...]. Dlatego nasze zadanie jest podwójne: uświadamiać i przełamywać opory tych, którzy nie są świadomi zmiany lub jej się przeciwstawiają, a jednocześnie rozwijać krytycz ną świadomość i wzmacniać twórczy sprzeciw tych, którzy są całkowicie zanurzeni w nowoczesnym świecie9. 9 M. C. Taylor, E. Saarinen, Imagologies. Media Philosophy, Routledge, London 1994, cyt. za: G. Dziamski, Lata dziewięćdziesiąte, Wyd. Galerii Miejskiej Arsenał, Poznań 2000, ss. 183-184. 10 M. Bal, Wizualny esencjalizm i przedmiot kultury wizualnej, „Artium Quaestiones” XVII, Poznań 2006, s. 295. 11 Ibidem, s. 318. 15 Por. m.in.: M. Bogunia-Borowska, P. Sztompka (red.), Fotospołeczeństwo. Antologia tekstów z socjologii wizualnej, Znak, Kraków 2012; P. Sztompka, Socjologia wizualna jako metoda badawcza, Wyd. Naukowe PWN, Warszawa 2005; M. Krajewski, R. Drozdowski, Za fotografię! W stronę radykalnego programu socjologii wizualnej, Wyd. Bęc Zmiana, Warszawa 2010; J. Kaczmarek (red.), Do zobaczenia. Socjologia wizualna w praktyce badawczej, Wyd. Naukowe UAM, Poznań 2008; J. Kaczmarek (red.), Kadrowanie rzeczywistości. Szkice z socjo logii wizualnej, Wyd. Naukowe UAM, Poznań 2004. 2. Zwrot ikoniczny w humanistyce W tym ujęciu imagolog byłby figurą podwójną: badaczem obrazów i osobą wypowiadającą się za ich pomocą. Tak zaprojektowana filozofia mediów miałaby być z jednej strony krytyką myślenia „uwięzionego w abstrakcyjnych schematach”, z drugiej – dostosowaniem krytycznego dyskursu do komunikacyjnych praktyk nowych mediów. Tym samym filozof musi włączyć się w proces produkcji ob razowej i symbolicznej, wejść w krwiobieg kultury, zanurzając się w strumienie mediów masowych i logikę rynku. Oczywiście nie należy się w tym zatracić na tyle, by po drodze utożsamić kulturę wizualną (jako przedmiotu badań) ze studiami wizualnymi. Jak powiada Mieke Bal: „Nie można wydobyć się samemu z bagna, ciągnąc za własne włosy”10. W jej ujęciu pojęcie kultury jest zmienne, zróżnico wane, uwikłane w praktyki władzy i oporu, zaś termin „wizualny” siłą rzeczy musi być nieczysty, dyskursywny, synestetyczny. Zatem by zrozumieć „kulturę”, należy wyjść od „zrozumienia dyskursu, w którym używane jest to słowo lub jego dery waty, synonimy lub wyrazy pokrewne. W tej perspektywie izolowanie wizualności według przedmiotów, które są wizualne, samo uczestniczy w strategii dominacji”11. i Do problematyki zwrotu ikonicznego nawiązuje również niemiecki filozof i historyk sztuki Gottfried Boehm. Wychodząc z tradycji hermeneutycznej, zapro ponował on teoretyczne zaplecze dla Bildwissenschaften (nauk o obrazie) – choć sam preferował określenie „krytyka obrazu”. W tym kontekście interesują go za równo obrazy, jak i kulturowe konteksty patrzenia i przedstawiania. Zadaje sobie Ikonologiczne inspiracje w badaniu kultury współczesnej 31 zatem kluczowe pytanie o to, jak obrazy wytwarzają odmienny od językowego, czyli wizualny (ikoniczny) sens: Różnica ikoniczna, jako figura retoryczna, podejmuje próbę uwypuklenia tego, co iko niczne w logosie lub jako logos. [...] Każdy artefakt ikoniczny organizuje się w formie wizualnej, poznawczej, jak również deiktycznej, a to znaczy nie-językowej różnicy12. Takie ujęcie wymyka się zatem ujmowaniu obrazu jako „znaku ikonicznego” i w rezultacie kwestionuje roszczenia strategii semiotycznych względem obrazu. Szukając dalszych odpowiedzi, Boehm nie ogranicza się do jakiejś jednolicie „sfo kusowanej” teorii obrazu, lecz zaprasza do dyskusji architektów, muzykologów, literaturoznawców etc. To transdyscyplinarne podejście badacz zapowiada już na wstępie artykułu Powrót obrazów: „Kto pyta o obraz, pyta o obrazy, o nieprzej rzaną liczbę mnogą”13. 16 Por. w języku polskim m.in.: M. Banks, Materiały wizualne w badaniach jakościowych, Wyd. Naukowe PWN, Warszawa 2009; K. Olechnicki, Antropologia obrazu. Fotografia jako metoda, przedmiot i medium nauk społecznych, Oficyna Naukowa, Warszawa 200; K. Olechnicki, Obrazy w działaniu. Studia z socjologii i antropologii obrazu, Wyd. Uniwersytetu im. Mikołaja Kopernika, Toruń 2003. 14 Zob. numer czasopisma „Artium Quaestiones” XVII z 2006 r., gdzie oprócz dokonanych przez Mariusza Brylla przekładów klasyków studiów wizualnych (M. J. T Mitchel, Mieke Bal, Nicolas Mirzoef) polski czytelnik miał okazję zapoznać się ze środowiskową dyskusją. Osob nych aplikacji metodologicznych dokonywali również historycy. Por. P. Witek, Metodologiczne problemy historii wizualnej, „Res Historica” 37/2014. 13 Ibidem, s. 276. 2 G. Boehm, O obrazach i widzeniu. Antologia tekstów, Universitas, Kraków 2014, s. 308. 3 Ibidem, s. 276. 12 G. Boehm, O obrazach i widzeniu. Antologia tekstów, Universitas, Kraków 2014, s. 308. 13 Ibidem, s. 276. 14 Zob. numer czasopisma „Artium Quaestiones” XVII z 2006 r., gdzie oprócz dokonanych przez Mariusza Brylla przekładów klasyków studiów wizualnych (M. J. T Mitchel, Mieke Bal, Nicolas Mirzoef) polski czytelnik miał okazję zapoznać się ze środowiskową dyskusją. Osob nych aplikacji metodologicznych dokonywali również historycy. Por. P. Witek, Metodologiczne problemy historii wizualnej, „Res Historica” 37/2014. 15 Por. m.in.: M. Bogunia-Borowska, P. Sztompka (red.), Fotospołeczeństwo. Antologia tekstów z socjologii wizualnej, Znak, Kraków 2012; P. Sztompka, Socjologia wizualna jako metoda badawcza, Wyd. Naukowe PWN, Warszawa 2005; M. Krajewski, R. Drozdowski, Za fotografię! W stronę radykalnego programu socjologii wizualnej, Wyd. Bęc Zmiana, Warszawa 2010; J. Kaczmarek (red.), Do zobaczenia. Socjologia wizualna w praktyce badawczej, Wyd. Naukowe UAM, Poznań 2008; J. Kaczmarek (red.), Kadrowanie rzeczywistości. Szkice z socjo logii wizualnej, Wyd. Naukowe UAM, Poznań 2004. 16 Por. w języku polskim m.in.: M. Banks, Materiały wizualne w badaniach jakościowych, Wyd. Naukowe PWN, Warszawa 2009; K. Olechnicki, Antropologia obrazu. Fotografia jako metoda, przedmiot i medium nauk społecznych, Oficyna Naukowa, Warszawa 200; K. Olechnicki, Obrazy w działaniu. Studia z socjologii i antropologii obrazu, Wyd. Uniwersytetu im. Mikołaja Kopernika, Toruń 2003. 17 G. Rose, Interpretacja materiałów wizualnych. Krytyczna metodologia badań nad wizual nością, Wyd Naukowe PWN, Warszawa 2010. Autorka prowadzi również bloga poświęconego studiom wizualnym – zob. https://visualmethodculture.wordpress.com/ [20.12.2016]. 18 Por. M. Banks, Materiały wizualne w badaniach jakościowych, Wyd. Naukowe PWN, Warszawa 2009; S. Pink, Doing Visual Ethnography. Images, Media and Representation in Research, Sage, London 2001. 17 G. Rose, Interpretacja materiałów wizualnych. Krytyczna metodologia badań nad wizual nością, Wyd Naukowe PWN, Warszawa 2010. Autorka prowadzi również bloga poświęconego studiom wizualnym – zob. https://visualmethodculture.wordpress.com/ [20.12.2016]. 18 Por. M. Banks, Materiały wizualne w badaniach jakościowych, Wyd. Naukowe PWN, Warszawa 2009; S. Pink, Doing Visual Ethnography. Images, Media and Representation in Research, Sage, London 2001. 20 L. Pauwels, Zwrot wizualny w badaniach i komunikacji wiedzy. Kluczowe problemy roz wijania kompetencji wizualnej w naukach społecznych, w: M. Frąckowiak, K. Olechnicki (red.), Badania wizualne w działaniu. Antologia tekstów, Wyd. Bęc Zmiana, Warszawa 2011, s. 21. 19 Każda z tych taktyk zasługiwałaby tu na osobne omówienie, lecz wiele z tych metod zebrał i przejrzyście scharakteryzował zespół pod kierunkiem Marka Krajewskiego w ramach projektu archiwumwizualne.pl. 3. Ku pragmatyce – aplikacje teorii obrazów w obszar praktyk badawczych Zarysowane powyżej wybrane aspekty zwrotu ikonicznego w sposób oczywi sty zainspirowały przedstawicieli rozmaitych dyscyplin usiłujących uchwycić fenomeny współczesności. Historykom sztuki pozwoliło to na rozprawienie się z ogłoszonym w międzyczasie zmierzchem dyscypliny14, badaczom społeczeństwa i człowieka na ukształtowanie/rewitalizację odpowiednio takich subdyscyplin, jak socjologia wizualna15 czy antropologia wizualna16. Ową fascynację poznawczymi aspektami obrazu zarówno humaniści, jak i badacze z obszaru nauk społecznych 32 j a c e k z y d o r o w i c z sprowadzili zatem do bardzo konkretnych problemów i metodologii. Niemniej ważny jest fakt, iż na tym gruncie wkrótce zaczęły pojawiać się publikacje o cha rakterze metodyczno-podręcznikowym. Autorką jednego z takich opracowań17 jest Gillian Rose – feministycznie zorientowana geografka i socjolożka. Wyraża ona przekonanie, że obrazy oddziałują na odbiorcę odmiennie niż teksty i pozwalają dotrzeć do innego rodzaju doświadczeń. W swoim kompendium drobiazgowo omawia, w jaki sposób materiały wizualne mogą być wsparciem dla procesu ba dawczego, zarówno jako przedmiot, jak i metoda (np. fotowywiady, dokumentacje fotograficzne i filmowe). Bazując właśnie na ikonologicznych kryteriach, autorka (proklamująca z ducha krytyczne podejście) opowiada się za solidną oceną przy datności i dopasowania tych metod. Chodzi więc o uwzględnienie kulturowych kontekstów, praktyk społecznych, określonych reżimów wytwarzania znaczeń przez obrazy (z uwzględnieniem stosunków władzy/wiedzy determinujących te sposoby widzenia). Uchwycenie tychże kontekstów ma ułatwiać swoista sieć utkana z takich elementów, jak tworzenie obrazów i ich odbiór, na co z kolei nakładają się trzy modalności: technologiczna (narzędziowa), kompozycyjna (struktury wizualne) oraz społeczna (konkretne praktyki i relacje). Rose projektując ów podręcznik, zadbała jednocześnie o wskazanie wielu pułapek: problemów z policzalnością badanych zjawisk i trafnością procedur (w badaniach ilościowych), jak również z interpretacją, reprezentatywnością i powtarzalnością (w aspektach jakościowych). Dlatego w celu uniknięcia tych błędów i zakłóceń sugeruje gruntowne analizy dyskursów (pojmowanych w foucaultowskim duchu). Zakorzeniona w ikonologii teoretyczna podbudowa, stworzona tak przez socjo logów, jak i antropologów wizualnych18, zaowocowała całym szeregiem taktycz nych rozwiązań służących zarówno pracy w terenie, jak i analizom koncepcyjnym. Warto dla porządku wymienić choć kilka: i Warto dla porządku wymienić choć kilka: i – dokumentacja fotograficzna,i – dziennik fotograficzny/dziennik wideo,i – etnografia/socjologia mediów,i – etnografia/socjologia wideo,i – film dokumentalny,i – film socjologiczny, – fotoesej/wideoesej, – fotospacer, – obserwacja fotograficzna, 18 Por. M. Banks, Materiały wizualne w badaniach jakościowych, Wyd. Naukowe PWN, Warszawa 2009; S. Pink, Doing Visual Ethnography. Images, Media and Representation in Research, Sage, London 2001. Ikonologiczne inspiracje w badaniu kultury współczesnej 33 – sztuka w badaniach wizualnych, – sztuka w badaniach wizualnych, – sztuka w badaniach wizualnych, – oglądanie kolekcji zdjęć informatorów/found footage,ii – wywiad z użyciem filmu/fotografii, – wywiad z użyciem zdjęć zrobionych przez informatorów, – zdjęcia osobiste, albumy, fotoblogi, – zdjęcia robione przez respondentów, – werbalne odpowiedzi na wizualne stymulacje19. Wizualność może być zatem wehikułem takich aspektów wiedzy naukowej, których ujęcie w słowa może sprawiać trudności. Belgijski badacz komunikacji wizualnej Luc Pauwels zauważa ponadto, iż te reprezentacje wizualne pozwalają m.in. na zredukowanie złożoności zjawisk społecznych poprzez uchwycenie ich w zwięzłą formę, zobrazowanie abstrakcyjnych pojęć na etapie wnioskowania, osadzanie danych w szerszym kontekście (np. organizacji przestrzennej), asy stowanie werbalnemu i liczbowemu przekazowi wiedzy20. W swoich tekstach i wystąpieniach drobiazgowo omawia arsenał stosowanych przez siebie narzędzi. Bazując na doświadczeniu własnym, swoich współpracowników oraz studentów, dostrzega jednak potrzebę udoskonaleń w wielu perspektywach, by wymienić choćby takie jak: – rozwijanie wrażliwości na wizualność i różne typy reprezentacji wizualnych, tak w obserwacji bezpośredniej rzeczywistości, jak i reprezentacji (fotografia, film czy malarstwo wymagają zniuansowanych perspektyw); – rozwijanie u badacza umiejętności niezbędnych do wytwarzania materiałów wizualnych jako istotnej części dyskursu naukowego; przemyślane i umiejętne stosowanie tychże nowych technologii – sama technologia nie rozwiąże proble mów badacza; – wykorzystanie mimetycznego i ekspresyjnego potencjału narzędzi i praktyk reprezentacji wizualnej – rzekome przeciwieństwo tych potencjałów Pauwels uważa za mylące. „Wierna” reprodukcja nie wyklucza autorskiej ekspresji badacza – obie zawierają potencjał poznawczy, obie mogą być pożyteczne w pracy badawczej, wystarczy świadomość wzajemnych różnic wyniesiona z historii myśli estetycznej; – praca nad doskonaleniem teorii i metodologii wizualnych – Pauwels jest przeciwny stanowiskom głoszącym, że obrazy tracą na sile przy próbach ich porządkowania; – edukowanie akademickich i nieakademickich odbiorców w zakresie kompe tencji do nowych kodów, uzusów, np. czytania/pisania fotoesejów; – edukowanie akademickich i nieakademickich odbiorców w zakresie kompe tencji do nowych kodów, uzusów, np. czytania/pisania fotoesejów; 34 j a c e k z y d o r o w i c z – krzewienie interdyscyplinarnej wymiany myśli oraz eklektyzmu metodolo giczno-teoretycznego (teoria i historia filmu, techniki edukacyjne, nauki o komuni kacji, sztuce, psychologia, filozofia nauki, antropologia, socjologia etc.) – oczywi ście ze świadomością wielu pułapek i aporii na skrzyżowaniach tych dyscyplin21. – krzewienie interdyscyplinarnej wymiany myśli oraz eklektyzmu metodolo giczno-teoretycznego (teoria i historia filmu, techniki edukacyjne, nauki o komuni kacji, sztuce, psychologia, filozofia nauki, antropologia, socjologia etc.) – oczywi ście ze świadomością wielu pułapek i aporii na skrzyżowaniach tych dyscyplin21. 22 Na gruncie socjologii wizualnej w znacznym stopniu dylemat ten rozwiązuje Krzysztof T. Konecki. Zob. K. T. Konecki, Wizualna teoria ugruntowana. Podstawowe zasady i procedury, „Przegląd Socjologii Jakościowej” t. VIII, 1/2012, ss. 12-45, tekst dostępny on-line: http://www. przegladsocjologiijakosciowej.org/volume18_pl.php [20.12.2016]. 21 Ibidem, ss. 23-36. 4. Studia wizualne: obawy, opór i refleksja (auto)krytyczna Oprócz wielu ożywczych pomysłów metodologicznych zwrot ikoniczny wkrótce przyniósł spodziewany opór oraz krytyczną autorefleksję. W dobie digitalnych obrazów narzędzia fotograficzne stały się łatwe, dostępne, przyjazne użytkowni kom. Jest to z jednej strony cywilizacyjny „bonus” i przywilej powszechnego zeń korzystania, z drugiej natomiast – przyczynek do nadprodukcji, inflacji i banali zacji obrazów, w efekcie wiodący do „rozwadniania” i trywializacji przedmiotu badań. Przy okazji łatwość i taniość rejestrowania zdjęć i filmów tworzy pokusę bezkrytycznego i beztroskiego ich stosowania w procesie badawczym. Trudno przy takich okazjach, aby owa łatwość gromadzenia danych nie przerodziła się w amatorszczyznę, a ta z kolei nie rzutowała na kształt i wyniki badań. Dla bardziej konserwatywnie usposobionych akademików tworzy to momentalnie asumpt do zarzutów o „fajniactwo” metodologiczne studiów wizualnych bądź nazywania ich chwytliwą i chwilową modą. Pewne wątpliwości mogą również wynikać z postmodernistycznych diagnoz o kryzysie reprezentacji. Wszak Jean Baudrillard niejednokrotnie zapewniał nas, że fotografie nie są żadnymi reprezentacjami, lecz fikcją i symulakrą. Skoro są źródłem tylu nadużyć, nadinterpretacji, manipulacji, to jak mogą służyć za wiarygodne, obiektywne i naukowo prawomocne narzędzie?22 Na marginesie pojawia się również pytanie o ukonstytuowanie się nowych form komunikowania wyników badań. Oprócz pradawnego dylematu przerostu formy nad treścią, należy zadać pytanie, czy badacze wizualni nie są zmuszeni do swoistego „wykradania języka” artystom czy twórcom filmów dokumentalnych? Rodzi to kolejne dylematy natury etycznej, a w konsekwencji legislacyjnej, jak chociażby problem ochrony wizerunku, własność intelektualna etc. Wiadomo doskonale, że w podobnych kwestiach prawo zawsze jest opóźnione, będąc kilka kroków za kulturą wizualną. Z kolei przy innych okazjach ekspansja obrazów beztrosko wkracza na tereny chronione, by przywołać przykład funkcji „street view” oferowanej przez mapy Google. Sfotografowanie kogoś „w złym miejscu i czasie” niejednokrotnie prowadziło do kłopotów, toteż zaprojektowano specjalny Ikonologiczne inspiracje w badaniu kultury współczesnej 35 algorytm wykrywający i rozmazujący numery rejestracyjne samochodów i twarze przechodniów23. Pośród zarzutów stawianych studiom wizualnym przewija się również rozle niwienie badacza w zakresie opisów, szczególnie w sytuacjach, kiedy wydaje mu się, że obraz mówi sam za siebie. Paradoksalnie po części może to być pokłosie myślenia samego proroka zwrotu ikonicznego, czyli M. J. T. Mitchella. Dość konsekwentnie stosuje on bowiem mocno animistyczną retorykę, polegającą na przypisywaniu obrazom cech ludzkich. Owo wyposażanie wizerunków w nie zależną podmiotowość może być tyleż efektowne poznawczo, co – na dłuższą metę – irytujące. Nie umniejsza to wszakże trafności wielu spostrzeżeń i metafor autora książki Czego chcą obrazy?24. 4. Studia wizualne: obawy, opór i refleksja (auto)krytyczna Tak antropomorfizowany obraz może być bowiem zarówno skutecznym mediatorem pomiędzy różnymi wymiarami wartości i rzeczywistości, jak i krytycznym obserwatorem współczesnych ikonoklazmów. Te ostatnie w sposób bardzo przekonujący Mitchell uchwycił w rozprawie Clo ning Terror25. Po ataku na World Trade Center z 11 września 2001 r. obrazy po raz kolejny zyskały quasi-biologiczny byt, medialnie „klonując się” na wielką skalę zarówno w wizerunkach upadających wież, jak i równie ikonoklastycznych odpowiedzi George’a W. Busha (np. obalenie pomnika Saddama Husseina). Tym sposobem za sprawą obrazów terroryzm XXI wieku z taktyki walki przeobraził się w stan kolektywnej świadomości. 23 W konsekwencji poniekąd „wylano dziecko z kąpielą”, jeśli bowiem zapragniemy, nie ruszając się z domu, obejrzeć np. republikańskie lub lojalistyczne murale w Belfaście, okaże się, że ich bohaterom również cyfrowo rozmazano twarze. 25 W. J. T. Mitchell, Cloning Terror: The War of Images, 9/11 to the Present, University of Chicago Press, Chicago 2011. 26 W. J. T. Mitchell, Czego chcą obrazy?, s. 80. 27 Ibidem, s. 168. 23 W konsekwencji poniekąd „wylano dziecko z kąpielą”, jeśli bowiem zapragniemy, nie ruszając się z domu, obejrzeć np. republikańskie lub lojalistyczne murale w Belfaście, okaże się, że ich bohaterom również cyfrowo rozmazano twarze. 24 W. J. T. Mitchell, Czego chcą obrazy?, Narodowe Centrum Kultury, Warszawa 2015. 25 W. J. T. Mitchell, Cloning Terror: The War of Images, 9/11 to the Present, University of Chicago Press, Chicago 2011. 26 W. J. T. Mitchell, Czego chcą obrazy?, s. 80. 27 Ibidem, s. 168. Podsumowanie – słowo wobec obrazu, obraz wobec słowa Zdarza się, że metodom wizualnym wytyka się erudycyjną ubogość i niesamo dzielność względem tekstu. Mitchell utrzymuje jednak, że [...] widzenie pełni tak samo ważną jak język rolę pośrednika w relacjach społecznych i nie daje się sprowadzić ani do języka, ani do znaku, ani do dyskursu. Obrazy chcą takich samych praw, jakie posiada język, a nie przekształcania w język26. Dla bardziej wyrazistego ujęcia problemu dalej dorzuca: Dla bardziej wyrazistego ujęcia problemu dalej dorzuca: [...] przedstawienie wizualne nie tyle jest twierdzeniem lub aktem mowy, ile mówcą zdolnym do nieskończonej liczby wypowiedzi. Obraz nie jest tekstem przeznaczonym do czytania, lecz żołądkiem brzuchomówcy, w który projektujemy własny głos27. 23 W konsekwencji poniekąd „wylano dziecko z kąpielą”, jeśli bowiem zapragniemy, nie ruszając się z domu, obejrzeć np. republikańskie lub lojalistyczne murale w Belfaście, okaże się, że ich bohaterom również cyfrowo rozmazano twarze. 23 W konsekwencji poniekąd „wylano dziecko z kąpielą”, jeśli bowiem zapragniemy, nie ruszając się z domu, obejrzeć np. republikańskie lub lojalistyczne murale w Belfaście, okaże się, że ich bohaterom również cyfrowo rozmazano twarze. y 24 W. J. T. Mitchell, Czego chcą obrazy?, Narodowe Centrum Kultury, Warszawa 2015. 25 W. J. T. Mitchell, Cloning Terror: The War of Images, 9/11 to the Present, University of hicago Press, Chicago 2011. 36 j a c e k z y d o r o w i c z Jednakże owa radykalna „osobność” obrazów i tekstów w znacznym stopniu wydaje się nieporozumieniem, w dodatku nieco sztucznie i tendencyjnie podtrzy mywanym przez obie strony sporu. Inauguracji i rozwojowi nowego przedsięwzię cia zwanego studiami wizualnymi skutecznie sekundowała bowiem ikonologia. Ta natomiast od swego zarania operowała właśnie w języku, w nim formułując zarówno tezy, jak i wnioski. W tym kontekście wartym poważnego zainteresowania jest projekt „kompromisowy”, zaproponowany przez Mariannę Michałowską28. Dlaczego zamiast mało konkluzywnych dyskusji o osobności ontologii obrazu i tekstu, nie mielibyśmy uznać ich hybrydycznej natury jako foto-tekstów? Właśnie na przykładzie punktów stycznych i przenikania fotografii i narracji uchwyciła ona istotność ich wzajemnych relacji oraz bardzo szeroki horyzont kulturowych kontekstów, np. konstruowanie tożsamości czy pamięci społecznej. Jednakże owa radykalna „osobność” obrazów i tekstów w znacznym stopniu wydaje się nieporozumieniem, w dodatku nieco sztucznie i tendencyjnie podtrzy mywanym przez obie strony sporu. Inauguracji i rozwojowi nowego przedsięwzię cia zwanego studiami wizualnymi skutecznie sekundowała bowiem ikonologia. Przywołany w niniejszym szkicu repertuar badawczych perspektyw studiów wizualnych z pewnością nie wyczerpuje tematu, jednak pozwala choć w czę ści zilustrować ich adaptacyjny potencjał. Podsumowanie – słowo wobec obrazu, obraz wobec słowa Co więcej, dynamika tych adaptacji pozwala nam zaobserwować, że w diagnozowaniu kultury rozwój digitalnych technik obrazowania może być równie pomocny jak dorobek dawnych tradycji ikonologicznych. Na równi pokrzepiający jest fakt, że współcześni badacze obra zów nie tkwią wyłącznie w akademicko-teoretycznym samozachwycie, lecz nie ustają w taktycznym rozpoznawaniu potencjalnych wdrożeń i zastosowań swych metod w praktyce. Co warto podkreślić – czynią to ze sporą świadomością istnie nia licznych pułapek i ograniczeń, gdzie przezwyciężenie jednych problemów po częstokroć generuje tuziny kolejnych. 28 M. Michałowska, Foto-teksty. Związki fotografii z narracją, Wyd. Naukowe UAM, Poznań 2012. Literatura Bal M., Wizualny esencjalizm i przedmiot kultury wizualnej, „Artium Quaestiones” XVII, Poznań 2006. Banks M., Materiały wizualne w badaniach jakościowych, PWN, Warszawa 2009. Boehm G., O obrazach i widzeniu. Antologia tekstów, Wyd. Universitas, Kraków 2014. Do zobaczenia. Socjologia wizualna w praktyce badawczej, red. J. Kaczmarek, Wydaw nictwo Naukowe UAM, Poznań 2008, Dziamski G., Lata dziewięćdziesiąte, Wyd. Galerii Miejskiej Arsenał, Poznań 2000, Bogunia-Borowska M., Sztompka P. (red.), Fotospołeczeństwo. Antologia tekstów z socjo logii wizualnej, Znak, Kraków 2012. Horapollo, Hieroglyphica (V/VI wiek n.e.), w przekładzie na język angielski: The Hierogly phics of Horapollo Nilous, tłum. Alexander Turner Cory [1840], http://www.sacred-texts. com/egy/hh/index.htm [20.12.2016]. Kaczmarek J. (red.), Kadrowanie rzeczywistości. Szkice z socjologii wizualnej, Wyd. Na ukowe UAM, Poznań 2004. Ikonologiczne inspiracje w badaniu kultury współczesnej 37 Konecki K. T., Wizualna teoria ugruntowana. Podstawowe zasady i procedury, „Przegląd Socjologii Jakościowej”, t. VIII, 1/2012, http://www.przegladsocjologiijakosciowej. org/volume18_pl.php [20.12.2016]. i g p p p Krajewski M., Drozdowski R., Za fotografię! W stronę radykalnego programu socjologii wizualnej, Wydawnictwo: Bęc Zmiana, Warszawa 2010. Krauss R., Welcome to the Cultural Revolution, „October” vol. 77, Summer 1996. Kundera Milan, Nieśmiertelność, PIW, Warszawa 2002.i Michałowska M., Foto-teksty. Związki fotografii z narracją, Wydawnictwo Naukowe UAM, Poznań 2012. Mitchell W. J. T., Cloning Terror: The War of Images, 9/11 to the Present, University of Chicago Press, 2011. Mitchell W. J. T., Czego chcą obrazy?, Narodowe Centrum Kultury, Warszawa 2015. Mitchell W. J. T., Iconology, Image, Text, Ideology, University of Chicago Press, Chicag 1986. Mitchell W. J. T., Picture Theory. Essays on Verbal and Visual Representation, Universit of Chicago Press, 1994. Mitchell W. J. T., The Pictorial Turn, ArtForum (March 1992).i Olechnicki K., Antropologia obrazu. Fotografia jako metoda, przedmiot i medium nauk społecznych. Oficyna Naukowa, Warszawa 2003. Olechnicki Krzysztof, Obrazy w działaniu. Studia z socjologii i antropologii obrazu. Wy dawnictwo Uniwersytetu im. Mikołaja Kopernika, Toruń 2003. Panofsky E., Studies in Iconology, Westview Press, Oxford 1972 (pierwsze wydanie: 1939 Pauwels L., Zwrot wizualny w badaniach i komunikacji wiedzy. Kluczowe problemy rozwi jania kompetencji wizualnej w naukach społecznych, w: M. Frąckowiak, K. Olechnicki (red.), Badania wizualne w działaniu. Antologia tekstów, Wyd. Bęc Zmiana, Warszawa 2011. Pink S., Doing Visual Ethnography. Images, Media and Representation in Research, Sage London 2001. Ripa C., Ikonologia, wyd. Universitas, Kraków 2013. Rose G., Interpretacja materiałów wizualnych. Krytyczna metodologia badań nad wizual nością, Wydawnictwo Naukowe PWN, Warszawa 2010. Rose G., Interpretacja materiałów wizualnych. Krytyczna meto nością, Wydawnictwo Naukowe PWN, Warszawa 2010. Literatura Sztompka P., Socjologia wizualna jako metoda badawcza, Wyd. Naukowe PWN, Warszawa 2005. Taylor M. C., Saarinen E., Imagologies. Media Philosophy, Routledge, London 1994. Warburg A., Atlas obrazów Mnemosyne, wyd. II; Narodowe Centrum Kultury, Warszawa Kraków 2016. Witek P., Metodologiczne problemy historii wizualnej, „Res Historica” 37/2014. Zeidler-Janiszewska A., O tzw. zwrocie ikonicznym we współczesnej humanistyce. Kilka uwag wstępnych, „Dyskurs” 4, http://www.asp.wroc.pl/dyskurs/Dyskurs4/AnnaZeidler Janiszewska.pdf [20.12.2016].
https://openalex.org/W2124535291	https://europepmc.org/articles/pmc3504924?pdf=render	English	null	Identifying stage-specific protein subnetworks for colorectal cancer	BMC proceedings	2,012	cc-by	9,454	Abstract Background: In recent years, many algorithms have been developed for network-based analysis of differential gene expression in complex diseases. These algorithms use protein-protein interaction (PPI) networks as an integrative framework and identify subnetworks that are coordinately dysregulated in the phenotype of interest. Motivation: While such dysregulated subnetworks have demonstrated significant improvement over individual gene markers for classifying phenotype, the current state-of-the-art in dysregulated subnetwork discovery is almost exclusively limited to binary phenotype classes. However, many clinical applications require identification of molecular markers for multiple classes. Approach: We consider the problem of discovering groups of genes whose expression signatures can discriminate multiple phenotype classes. We consider two alternate formulations of this problem (i) an all-vs-all approach that aims to discover subnetworks distinguishing all classes, (ii) a one-vs-all approach that aims to discover subnetworks distinguishing each class from the rest of the classes. For the one-vs-all formulation, we develop a set-cover based algorithm, which aims to identify groups of genes such that at least one gene in the group exhibits differential expression in the target class. Results: We test the proposed algorithms in the context of predicting stages of colorectal cancer. Our results show that the set-cover based algorithm identifying “stage-specific” subnetworks outperforms the all-vs-all approaches in classification. We also investigate the merits of utilizing PPI networks in the search for multiple markers, and show that, with correct parameter settings, network-guided search improves performance. Furthermore, we show that assessing statistical significance when selecting features greatly improves classification performance. © 2012 Erten et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PROCEEDINGS Open Access * Correspondence: sinan.erten@case.edu 1Department of Electrical Engineering & Computer Science, Case Western Reserve University, Cleveland, OH, USA Full list of author information is available at the end of the article Identifying stage-specific protein subnetworks for colorectal cancer Sinan Erten1, Salim A Chowdhury2, Xiaowei Guan3,4,5, Rod K Nibbe3, Jill S Barnholtz-Sloan3,5, Mark R Chance3,5,6, Mehmet Koyutürk1,3,5 From Great Lakes Bioinformatics Conference 2012 Ann Arbor, MI, USA. 15-17 May 2012 From Great Lakes Bioinformatics Conference 2012 Ann Arbor, MI, USA. 15-17 May 2012 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Open Access Introduction led to identification of biomarkers for a range of complex diseases, including Parkinson’s disease [1], neuroblastoma [2], lung cancer [3] and breast cancer [4]. Genome-wide monitoring of mRNA expression, moni- tored using DNA microarrays and more recently deep sequencing, has proved quite useful in understanding the mechanistic bases of complex human diseases. Systematic analysis of differential gene expression in dif- ferent phenotypic classes leads to identification of novel biomarkers, which serve as features for phenotype classi- fication, as well as targets for therapeutic intervention. In previous studies, differential analysis of gene expression Traditional analyses generally take a univariate approach to study gene expression and identify genes with signifi- cant individual differential expression in the phenotype of interest. However, such univariate approaches are often limited in explaining the underlying mechanisms of com- plex diseases, which arise from the interplay among multi- ple genetic and environmental factors. For example, genes that cooperate or complement each other in pathogenesis may not necessarily be differentially expressed individually, but exhibit coordinated dysregulation when considered together. Correspondence: sinan.erten@case.edu 1Department of Electrical Engineering & Computer Science, Case Western Reserve University, Cleveland, OH, USA Full list of author information is available at the end of the article Page 2 of 12 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 In order to address the shortcomings of the univariate approaches, Chuang et al. develop an algorithm that integrates gene expression data with protein-protein interaction (PPI) networks to identify reproducible breast cancer metastasis markers composed of multiple interacting proteins (“dysregulated subnetworks”) [5]. They show that these subnetwork markers better predict breast cancer metastasis as compared to individually dysregulated genes. Motivated by the demonstrated pro- mise of this approach, several other algorithms are developed for network-based analysis of differential gene expression. In particular, Chowdhury et al. develop a set-cover based heuristic for identification of genes that complement each other in discriminating phenotype and control samples [6]. Phuong et al. further improve on these algorithms by introducing a biclustering algorithm that also accounts for the noise in PPI networks by incorporating reliability scores for PPIs [7]. More recently, recognizing the shortcomings of greedy algo- rithms in identifying dysregulated subnetworks, Phuong et al. introduce a color-coding based randomized algo- rithm to identify subnetworks that are highly discrimi- native of phenotype and control [8]. Introduction These methods are also extended to the identification of subnetwork expression signatures that can shed light into the regula- tory logic of the relationship between the dysregulation of multiple genes and the disease phenoype. In particu- lar, Chowdury et al. identify subnetworks whose combi- natorial expression states are indicative of phenotype by using a branch-and-bound algorithm [9], Dutkowski et al. grow network-guided forests by training decision trees using interacting proteins [10]. in identifying subnetworks that serve as indicators for specific classes. The algorithmic challenges, on the other hand, include unproportionately distributed samples across multiple classes. For these reasons, novel algo- rithms are needed that are robust and can work with datasets that are composed of different number of classes and sample distributions. Contributions of this study In this article, we introduce novel algorithms for net- work-based analysis of differential gene expression on applications that involve multiple phenotype classes. As an important application, we focus particularly on identi- fying subnetworks that can discriminate different stages of human colorectal cancer (CRC) according to Dukes’ classification. We first propose two formulations to gen- eralize information-theoretic measures of subnetwork dysregulation to multiple phenotype classes. These for- mulations differ in terms of how the target subnetworks discriminate phenotype classes from each other; namely we establish information-theoretic criteria for all-vs-all and one-vs-all discriminative subnetworks. Then, we extend the set-cover based algorithm by Chowdury et al. , NETCOVER, to identify one-vs-all discriminative sub- networks [6]. We also introduce a framework for asses- sing the statistical significance of the sub-networks identified by the set-cover based algorithm. Using public CRC datasets composed of samples labeled with Dukes’ four stages, we investigate the performance of the result- ing algorithm, COBALT, in identifying subnetworks that are useful in predicting the stages of colon cancer sam- ples. In particular we perform systematic computational experiments to investigate the following: All of the existing dysregulated subnetwork discovery algorithms are designed and validated for binary pheno- type classes (e.g. cancerous vs. non-cancerous, metastatic vs. non-metastatic, drug responders vs. non-responders) and prove to be promising in terms of accurate classifica- tion of samples. However, many progressive diseases such as glioblastoma, breast cancer and colorectal cancer require identification of molecular markers for multiple classes (such as the four stages in colorectal cancer according to Dukes’ classification) for effective prognosis and treatment. This implies the necessity of a framework that can also work on datasets with more than two phe- notype classes for network-based discovery of disease markers. Although most of the existing algorithms can be applied to multiple phenotype classes in principle, no tool is readily available for this purpose. Furthermore, subnetwork discovery on multi-class datasets requires additional design choices and poses novel algorithmic challenges. These choices include designing criteria to evaluate the dysregulation of a subnetwork; i.e., are we interested in identifying subnetworks that can distinguish all classes from each other at once, or are we interested • We compare the performance of all-vs-all and one- vs-all subnetworks in predicting phenotype and show that one-vs-all discriminative subnetworks are generally more reliable as features for classification. Contributions of this study • We compare the performance of all-vs-all and one- vs-all subnetworks in predicting phenotype and show that one-vs-all discriminative subnetworks are generally more reliable as features for classification. • We investigate the effect of using the PPI network to confine the space for searching groups of genes that are coordinately dysregulated subnetworks. We show that, while expansion of the search space through consideration of indirect interactions improve the classification performance of identified subnetworks, this improvement saturates after a point, demonstrating that PPI networks indeed pro- vide a shortcut to the identification of dysregulated groups of genes. We also show that our efficient set cover based algorithm renders network-free search feasible. • We investigate the effect of using statistically sig- nificant subnetworks (as opposed to high-scoring subnetworks) as features for classification and show that assessment of statistical significance facilitates Page 3 of 12 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 identification of more useful subnetwork features for classification. considered a subnetwork of interest if for all proteins gi Î S , there is at least one other protein gj Î S such that gi and gj are connected through at most ℓhops in the PPI network. Here, ℓis a parameter that adjusts the trade-off between functional relevance and computa- tional efficiency; a larger ℓallows searching for function- ally less related proteins at the cost of increasing the search space. In the next section, we start our discussion by propos- ing two alternate information-theoretic formulations of sub-network dysregulation. We also introduce our set- cover based algorithm, COBALT, for the identification of one-vs-all discriminative subnetworks and propose meth- ods for assessing the statistical significance of the identi- fied subnetworks. Subsequently, in Results Section, we provide comprehensive experimental results on the clas- sification performance of the subnetworks discovered by COBALT in predicting the stage of CRC on two gene expression datasets obtained from the Gene Expression Omnibus. We conclude the paper in Conclusion Section. For a given subnetwork S ⊆V , Chuang et al. define the subnetwork activity of S as ES = gi∈SEi/√\|S\|, that is the aggregate expression profile of the genes in S [5]. Using subnetwork activity, they define an infor- mation-theoretic measure to quantify the dysregulation of a subnetwork. Contributions of this study This “additive” definition of dysregula- tion limits the framework to the identification of subnet- works with all genes in the subnetwork dysregulated in the same direction (i.e., all up- or down-regulated in the phenotype of interest), and alternate approaches that compute combinatorial expression signatures are shown to be more powerful [9,10]. However, this additive for- mulation serves as a useful starting point to generalize subnetwork dysregulation to phenotypes that involve multiple classes. For this reason, we focus on additive subnetwork activity in this paper. All-vs-all discriminative power of a subnetwork All-vs-all discriminative power of a subnetwork It is straightforward to generalize the information-theo- retic measure for the dysregulation of a subnetwork [5] to multiple phenotype classes. Namely, the mutual infor- mation between the subnetwork activity of S and the multi-class phenotype vector, i.e., Δ all-vs-all (S) = I (ES, C) = H(C) −H(C\|ES ), provides a measure of the the reduction in the uncertainty about C given ES. Here, H(X) = − x∈χp(x) log(p(x)) denotes the Shannon entropy of discrete random variable X that can take over values from the set X . In our case, the support set for the random variable C is T , whereas the support set for the random variable ES is obtained by appropri- ately quantizing the expression levels. It is straightforward to generalize the information-theo- retic measure for the dysregulation of a subnetwork [5] to multiple phenotype classes. Namely, the mutual infor- mation between the subnetwork activity of S and the multi-class phenotype vector, i.e., Δ all-vs-all (S) = I (ES, C) = H(C) −H(C\|ES ), provides a measure of the the reduction in the uncertainty about C given ES. Here, H(X) = − x∈χp(x) log(p(x)) denotes the Shannon Dysregulation of subnetworks For a given set V of genes and U of samples, let Ei Î R\|u\| represent the properly normalized gene expression vector for gene gi ∈V , where Ei(j) denotes the relative expression of gi in sample sj ∈U . Assume that we have a set T , composed of different classes for the phenotype of interest (such as the four stages in colorectal cancer according to Dukes classification) and the phenotype vector C annotates each sample with one of the labels in T , i.e., C(j) = t where t Î T .We also define the set of all samples for a specific phenotype class t as U(t) = {sj ∈U : C(j) = t}. Methods In this section, we start by introducing the mathematical background of the information-theoretic formulation of coordinate dysregulation for a set of genes. Subsequently, we propose two alternate approaches for generalizing this notion to multiple phenotype classes. We then introduce COBALT, our set-cover based algorithm that is specifically designed to identify stage-specific discriminative subnet- works. Finally, we introduce a framework for assessing the statistical significance of the identified subnetworks, and describe how these subnetworks can be utilized for classi- fication of samples. One-vs-all discriminative power of a subnetwork Here, we propose an alternate measure to quantify the power of a subnetwork in discriminating multiple phe- notype classes from each other. This measure targets discriminating a particular phenotype class from all other classes. Namely, we define class-specific phenotype vector C(t) for class t Î T as Let G(V, ε) denote a PPI network where the product of each gene gi Î V is represented by a node and each edge gigj represents an interaction between the products of gi and gj. Given a PPI network and a gene expression dataset over multiple phenotype classes, we are inter- ested in finding sets of genes that can together discrimi- nate the phenotype classes with their gene expression signatures. In order to establish the functional relevance of these gene sets and search for these sets more effi- ciently, we confine the search space to PPI subnetworks, that is groups of proteins that are functionally interre- lated through PPIs. Formally, a set S ⊆V of proteins is c(t) j = 1 0 if Cj = t otherwise. (1) (1) Then, the mutual information between subnetwork activity and the class-specific phenotype vector C(t), i.e., (t) one - vs - all (S) = I(ES, C(t)), provides a measure of the reduction in the uncertainty about class t given ES. This formulation offers a number of benefits as compared to Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Page 4 of 12 the all-vs-all formulation of discriminative power: (1) The one-vs-all formulation may lead to identification of more interpretable markers, since, for example, it can provide stage-specific molecular signatures for colorectal cancer. (2) The one-vs-all formulation can extract class- specific molecular signatures that may be missed by the all-vs-all formulation because they do not discriminate other classes well. (3) The phenotype random variable takes a smaller number of values, thus offering more statistical power for the same number of samples. The concepts of all-vs-all and one-vs-all disciminative power of subnetworks and the computation of mutual informa- tion using these different formulations are illustrated in Figure 1. Identifying one-vs-all discriminative subnetworks The problems of identifying subnetworks with maximum all - vs - all(S) or one - vs - all(S) are intractable [5]. How- ever, it is straightforward to generalize the greedy algo- rithm by Chuang et al. to solve both problems efficiently [5]. This greedy algorithm initializes a subnetwork with a single protein. One-vs-all discriminative power of a subnetwork BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Page 5 of 12 Page 5 of 12 While the explained greedy algorithm is quite effective in efficiently discovering high-scoring subnetworks, it has several drawbacks [6]. First, this algorithm is biased toward identifying subnetworks with very few proteins that exhibit high dysregulation individually. This is because the algorithm lacks global awareness, i.e., it will stop expanding the subnetwork when the best candidate protein to add to the subnetwork has only marginal individual contribution, but may actually contribute a greater deal when additonal proteins are added. Second, this approach requires computation of mutual informa- tion for each and every candidate protein in the neigh- bourhood to be added to the growing subnetwork, which may prove to be costly when the algorithm needs to be run multiple times to assess statistical significance of identified subnetworks. Motivated by these obser- vations, Chowdhury et al. develop a set-cover based algorithm, NETCOVER, which is more effective in dis- covering proteins that complement each other in discri- minating phenotype and control samples [6]. However, NETCOVER is designed for binary phenotype classes and it assumes that the samples are paired. Here, we argue that the algorithmic insights introduced by NET- COVER suit particularly well to the identification of one-vs-all discriminative subnetworks. Based on this observation, we develop COBALT, which generalizes NETCOVER to handle unpaired samples and multiple phenotype classes to identify one-vs-all discriminative subnetworks. ˆEi(j) = ⎧ ⎨ ⎩ +1 if Ei(j) > μi + α ∗σi −1 if Ei(j) < μi −α ∗σi 0 otherwise (2) (2) Here, μi and si respectively represent the mean and standard deviation of expression value of gi across all sam- ples, i.e., μi = Sj∈UEi(j)/\|U\| and σi = Sj∈U(Ei(j) −μi)2. We define a as a user-defined threshold parameter for a gene’s dysregulation in a sample to be considered signifi- cant. We say that a gene gi positively covers a sample sj if ˆEi(j) = +1, and negatively covers sj if ˆEi(j) = −1, Follow- ing this definition, for a given gene gi and phenotype class t Î T , we define the positive cover set P(t) i as the set of all samples with phenotype t that are positively covered by gi, i.e., P(t) i = sj ∈U : C(j) = t and ˆEi(j) = +1 . One-vs-all discriminative power of a subnetwork Similarly, the negative cover set N (t) i contains all samples in class t that are negatively covered by gene gi. We illustrate these concepts in Figure 2. It can be shown that the mutual information of C(t) and Êi is a monotonically non-decreas- ing function of the cardinalities of P(t) i and N (t) i [6], i.e., one can maximize I(Êi,C(t)) by maximizing \|\|P(t) i \| −\|N (t) i \|\|. Using the negative and positive cover sets for each gene-class pair, COBALT identifies one-vs-all discrimi- native subnetworks for each phenotype class by using a greedy heuristic that is shown to be effective for the set- cover problem [11]. Namely, for each gene gi, we first identify the target phenotype class for that gene as the phenotype class with largest percentage of samples posi- tively (or negatively) covered by gi. Subsequently, we grow a subnetwork by systematically adding a gene in the neighborhood based on the coverage on the rest of the uncovered samples for that class. Without loss of generality, the algorithm identifies a minimal positive covering subnetwork seeded at gene gi as follows: One-vs-all discriminative power of a subnetwork It then grows the subnetwork by adding the protein in the neighborhood of the subnetwork (i.e., reachable from the subnetwork with ℓhops) that improves the objective function (all - vs - all(S) or one - vs - all(S)) the most. The algorithm stops either when there is no more protein in the neighborhood to add, or the best improvement provided by a protein in the neighborhood is below a user-defined threshold. Figure 1 Illustration of the difference between all-vs-all and one-vs-all discriminative subnetworks. Illustration of the difference between all-vs-all and one-vs-all discriminative subnetworks. The aggregate expression profiles of five hypothetical subnetworks are shown. Red and green respectively represent positive and negative expression, with intensity representing magnitude. The subnetworks S1, S2, S3 and S4 are one-vs-all discriminative, respectively indicating classes (CRC stages) A, B, C and D, since the expression profile of each subnetwork in samples that belong to the respective class can discriminate these samples from other classes. On the other hand, S5 is an all-vs-all discriminative subnetwork, since it discriminates all classes from each other. Figure 1 Illustration of the difference between all-vs-all and one-vs-all discriminative subnetworks. Illustration of the difference between all-vs-all and one-vs-all discriminative subnetworks. The aggregate expression profiles of five hypothetical subnetworks are shown. Red and green respectively represent positive and negative expression, with intensity representing magnitude. The subnetworks S1, S2, S3 and S4 are one-vs-all discriminative, respectively indicating classes (CRC stages) A, B, C and D, since the expression profile of each subnetwork in samples that belong to the respective class can discriminate these samples from other classes. On the other hand, S5 is an all-vs-all discriminative s bnet ork since it discriminates all classes from each other Figure 1 Illustration of the difference between all-vs-all and one-vs-all discriminative subnetworks. Illustration of the difference between all-vs-all and one-vs-all discriminative subnetworks. The aggregate expression profiles of five hypothetical subnetworks are shown. Red and green respectively represent positive and negative expression, with intensity representing magnitude. The subnetworks S1, S2, S3 and S4 are one-vs-all discriminative, respectively indicating classes (CRC stages) A, B, C and D, since the expression profile of each subnetwork in samples that belong to the respective class can discriminate these samples from other classes. On the other hand, S5 is an all-vs-all discriminative subnetwork, since it discriminates all classes from each other. Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Erten et al. COBALT:Cover-based algorithm for identifying one-vs-all discriminative subnetworks Recall that a one-vs-all discriminative subnetwork is defined as one with differential subnetwork activity in a specific phenotype class, as compared to all other classes. Since subnetwork activity is defined regularly, the genes in such a subnetwork have to be either all up-regulated or all down-regulated in the phenotype class of interest. Motivated by this observation, COBALT aims to identify subnetworks such that for each sample that belongs to the phenotype class of interest, there exists at least one gene in the subnetwork that is up-regulated (or down- regulated) in that sample. Such subnetworks are said to “cover” the entire patient population that represents the phenotype class of interest. 1. Initialize subnetwork: Si ¬ {gi} 1. Initialize subnetwork: Si ¬ {gi} 2. Define the target phenotype class t for this subnet- work as the class that has the maximum fraction of samples positively covered by gi: t ¬ argmaxt′ Î T 2. Define the target phenotype class t for this subnet- work as the class that has the maximum fraction of samples positively covered by gi: t ¬ argmaxt′ Î T In order to identify the genes that are up-regulated or down-regulated in each sample, we use the expression of that gene in all samples as the background distribu- tion. Subsequently, we identify samples in which the genes’ expression deviates significantly from this back- ground distribution. Namely, for a gene gi, consider the distribution of the expression values Ei across all sam- ples. We compute a quantized expression value for gi in sample sj as follows: \|P(t′) i \|/\|U(t′) i \| 3. Initialize the set of uncovered samples for class t: M(t) ←U(t)\P(t) i 3. Initialize the set of uncovered samples for class t: M(t) ←U(t)\P(t) i i 4. Initialize the set of network neighbors: Q ←{gj ∈V : δ(gi, gj) ≤ℓ} 5. For all genes gj Î Q , compute (P(t) j )′ ←P(t) j ∩M(t)and (N (t) j )′ ←N (t) j ∩M(t) Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Page 6 of 12 6. Find all the genes (can be multiple) in Q with maximum \|(P(t))′ \| −\| (N (t))′\| and let gk be the 7. Expand the subnetwork with gk: Si ¬ Si ∪{gk} 8. Update the set of uncovered positive samples for ( ( ( Figure 2 Illustration of the set-cover based algorithm for the identification of one-vs-all discriminative subnetworks. Here, the coverage provided by a hypothetical three-gene subnetwork is shown. In the left panel, the distribution of the expression levels of each gene across all samples is shown. We first compute the mean (μi) and the standard deviation (si) of this distribution for each gene gi. Subsequently, we identify samples that are positively or negatively covered by each gene. A gene gi with expression greater than μi + a * si in a sample is said to positively cover that sample, while a gene gi with expression less than μi - a * si in a sample is said to negatively cover that sample (we set a = 2 in our experiments). 7. Expand the subnetwork with gk: Si ¬ Si ∪{gk} 8. Update the set of uncovered positive samples for class t: ℳ(t) ¬ ℳ(t) \ P k (t) 9. Update set of neighbouring genes: Q ¬ Q ∪{gj Î V : δ(gk, gj) ≤ℓ} \ {gk} 10. If Q = ∅or M(t) = ∅, return Si; otherwise, go to step (5). p g g 8. Update the set of uncovered positive samples for class t: ℳ(t) ¬ ℳ(t) \ P k (t) 7. Expand the subnetwork with gk: Si ¬ Si ∪{gk} 8. Update the set of uncovered positive samples for class t: ℳ(t) ¬ ℳ(t) \ P k (t) 9. Update set of neighbouring genes: Q ¬ Q ∪{gj Î V : δ(gk, gj) ≤ℓ} \ {gk} 10. If Q = ∅or M(t) = ∅, return Si; otherwise, go to step (5). 9. Update set of neighbouring genes: Q ¬ Q ∪{gj Î V : δ(gk, gj) ≤ℓ} \ {gk} 7. Expand the subnetwork with gk: Si ¬ Si ∪{gk} 1. Initialize subnetwork: Si ¬ {gi} The negative and positive cover sets for each gene and the subnetwork composed of g1, g2 and g3 are shown on the right. In this example, this subnetwork negatively covers (all samples in) Stage B. Figure 2 Illustration of the set-cover based algorithm for the identification of one-vs-all discriminative subnetworks. Here, the coverage provided by a hypothetical three-gene subnetwork is shown. In the left panel, the distribution of the expression levels of each gene across all samples is shown. We first compute the mean (μi) and the standard deviation (si) of this distribution for each gene gi. Subsequently, we identify samples that are positively or negatively covered by each gene. A gene gi with expression greater than μi + a * si in a sample is said to positively cover that sample, while a gene gi with expression less than μi - a * si in a sample is said to negatively cover that sample (we set a = 2 in our experiments). The negative and positive cover sets for each gene and the subnetwork composed of g1, g2 and g3 are shown on the right. In this example, this subnetwork negatively covers (all samples in) Stage B. 6. Find all the genes (can be multiple) in Q with maximum \|(P(t) j )′ \| −\| (N (t) j )′\| and let gk be the gene among these genes with minimum t′∈T \{t} P(t)′ k (i.e., gk has minimum positive back- ground coverage). Page 7 of 12 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 This algorithm is also used for identifying the minimal negative covering subnetwork seeded at gi by simply replacing Q with P above. profiles of these subnetworks. This application also pro- vides a useful means for assessing the biological rele- vance, reproducibility, and utility of the identified subnetworks. In order to use stage-specific subnetworks in colorectal cancer to predict the stage of a patient, following steps are performed: Assessing statistical significance of subnetworks We then choose the top k positive and negative covering subnetworks for each phenotype class, giving us a total of 2k\|T \| features to be used in classification. Here, k is a user defined parameter to set the number of stage-specific subnetworks that are used for each class. Note also that the cover provided by a subnetwork for a target phenotype class depends on the size of the sub- network (i.e., the number of proteins in the subnetwork). In other words, if we construct subnetworks at random, we would expect larger subnetworks to have a higher coverage. Furthermore, in our experiments, we also observe that larger subnetworks tend to have higher dis- criminative power (Δ ). Motivated by these insights, we assess the statistical significance of a subnetwork as a function of its size. For this purpose, we stratify the sub- networks that compose each background distribution according to subnetwork size and compute the p-value of a subnetwork S by comparing Δ(S) to the discriminative power of the background subnetworks that have similar size to the subnetwork of interest. More precisely, the p-value of S is defined as the fraction of subnetworks with discriminative power greater than that of S among all subnetworks in the background set with size equal to that of S. A minimal covering subnetwork S discovered by COBALT is considered to be statistically significant if its p-value is less than the significance threshold for both background populations. (b) The second approach assumes that assess- ment of statistical significance will facilitate selection of biologically more meaningful subnet- works, also providing more power in classifica- tion as compared to high-scoring subnetworks. For this purpose, using the two proposed statisti- cal significance tests discussed in the previous section, we identify subnetworks that are signfi- cant according to both statistical tests and use all of these significantly discriminative subnetworks as features for classification. 3. Once we obtain a final list of features either using (a) or (b) at step (2), we compute the aggre- gate expression profiles (ES ) for each of these selected subnetworks and use these to construct feature vectors for each sample (where each fea- ture represents the aggreate expression of one subnetwork). 4. Finally, we use these feature vectors to train and test classifiers for predicting the class of the phenotype of interest. Assessing statistical significance of subnetworks In order to asses the significance of the identified sub- networks, we perform two distinct significance tests. In order to asses the significance of the identified sub- networks, we perform two distinct significance tests. Each significance test is performed by generating an empirical background distribution that carefully accounts for multiple hypothesis testing. The first back- ground distribution is obtained by randomly permuting the class labels. The second background distribution, on the other hand, is obtained by permuting the gene expression profiles (the rows of the gene expression matrix, thereby randomly reestablishing the relationship between the expression profiles and the nodes in the PPI network ). After generating a large number of these randomized datasets, we use COBALT to identify class- specific subnetworks for the randomized datasets as well. We then use these subnetworks as the background distribution to test the statistical significance of the dis- criminative power of the stage-specific subnetworks identified on the actual dataset. This approach implicitly handles multiple hypothesis testing, since the back- ground distribution is constructed using the most discri- minative subnetworks that could be identified on each randomized dataset. 1. We first identify both the positive and negative covering subnetworks for each gene gi Î V . 1. We first identify both the positive and negative covering subnetworks for each gene gi Î V . 2. In order to investigate the effect of statistical sig- nificance on the classification utility of subnetworks, we use two alternate strategies to extract a list of features from the sets of covering subnetworks found in (1): (a) The first approach assumes that high-scoring subnetworks are more useful for classification, as compared to significantly discriminative subnet- works. For each phenotype class t Î T , we sort the subnetworks based on their all-vs-all (all−vs−all(S)) or one-vs-all (all−vs−all(S)) dis- criminative power. We then choose the top k positive and negative covering subnetworks for each phenotype class, giving us a total of 2k\|T \| features to be used in classification. Here, k is a user defined parameter to set the number of stage-specific subnetworks that are used for each class. (a) The first approach assumes that high-scoring subnetworks are more useful for classification, as compared to significantly discriminative subnet- works. For each phenotype class t Î T , we sort the subnetworks based on their all-vs-all (all−vs−all(S)) or one-vs-all (all−vs−all(S)) dis- criminative power. Experimental design Colorectal Cancer (CRC) is one of the most common causes of cancer related deaths in the western civiliza- tion [12]. Diagnosis of CRC is often difficult as the symptoms appear only at the advanced stages of the dis- ease. Moreover, early diagnosis is very critical as the survival rate changes dramatically with the stage of the cancer. In fact, 5 year survival rates when diagnosis is made at the localized stage (cancer is confined in the primary site) and after cancer has metastasized are around 90% and 12% respectively [13]. These observa- tions suggest that, for effective diagnosis, prognosis and treatment, accurate determination of disease stage is crucial. COBALT is fully implemented in Matlab. We use this implementation to perform the following classification experiments: • Prediction of disease stage in GSE14333. Subnet- works discovered using GSE14333 are used to pre- dict the stages of samples in the same dataset in a 10-fold cross validation setting. Samples in each phe- notype class are randomly separated into ten similar- sized groups. In each iteration, one of the groups in each class is chosen to be the test data and the rest of the data is used to train the classifier. • Prediction of disease stage in GSE14333. Subnet- works discovered using GSE14333 are used to pre- dict the stages of samples in the same dataset in a 10-fold cross validation setting. Samples in each phe- notype class are randomly separated into ten similar- sized groups. In each iteration, one of the groups in each class is chosen to be the test data and the rest of the data is used to train the classifier. • Prediction of disease stage in GSE5206. Subnet- works discovered using GSE14333 are used to pre- dict the stages of samples in GSE5206. In this cross- classification setting, the classifier is trained on GSE14333 and tested on the other dataset, GSE5206. There are different classification systems for the pro- gression of colorectal cancer. Dukes’ famous staging sys- tem classifies patients based on how far the cancer is spread [14]. TNM is another staging method providing a more comprehensive framework including information about the size and localization of the tumor, as well as the involvement of lymph nodes [15]. CRC Datasets we use in our experiments are classified by Dukes’ staging system. For both of these settings, we use a naive Bayesian clas- sifier provided by Matlab’s classify function. Experimental design Using other classifier options provided by Matlab’s classifier proce- dure only marginally effects the results (data not shown). Results and discussion One application of identifying subnetworks that can dis- criminate multiple phenotype classes is to predict the phenotype class of a test sample using the expression In this section, we first give brief information about the colorectal cancer in human (CRC) and introduce the two stage-specific CRC datasets and the PPI network we Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Page 8 of 12 Table 1 Number of samples labeled with each colorectal cancer stage based on Dukes’ 4-stage classification in the datasets used in our experiments. Table 1 Number of samples labeled with each colorectal cancer stage based on Dukes’ 4-stage classification in the datasets used in our experiments. stage A B C D total GSE14333 44 94 91 61 290 GSE5206 12 32 33 21 98 use in our experiments. Subsequently, we describe in detail the experimental framework used. After introdu- cing the performance evaluation metrics used, we pre- sent our experimental results comparing one-vs-all and all-vs-all discriminative subnetworks, as well as the addi- tive and set-cover based algorithms that are used to dis- cover these subnetworks. Next, we analyze the effect of the network distance parameter (ℓ) that adjusts the search space size when growing the subnetworks. Finally, we compare the performance of high-scoring and statistically significant subnetworks in predicting the stages of samples. stage A B C D total GSE14333 44 94 91 61 290 GSE5206 12 32 33 21 98 Bi-oGrid, and BIND. We remove the nodes with no interactions to obtain a final PPI network that contains 8959 proteins and 33,528 interactions among these proteins. Datasets We use two CRC microarray datasets obtained from the Gene Expression Omnibus [16] in our experiments. These datasets that contain labeled CRC samples with Dukes’ 4-stage classification are the following: In order to obtain a comprehensive picture of the perfor- mance of different approaches, we list the precision and recall of the classification experiments for each pheno- type class separately. Precision refers to the percentage of the correct predictions over all samples predicted belong- ing to the respective CRC stage, whereas recall refers to the percentage of the correctly predicted samples over all samples that are clinically diagnosed to belong the respective CRC stage. Please note that we set the network distance parameter ℓ= 3 in all experiments unless other- wise noted, since it provides the best performance as shown in the next section. When quantizing the expres- sion values of a gene over all samples using Equation 2, we set a = 2 as the threshold parameter for the gene’s dysregulation in a sample to be considered positively or negatively covering. • GSE14333 contains the expression profiles of 54675 genes in 290 samples. • GSE5206 contains the expression profiles of the same 54675 genes in 98 samples. The distribution of the samples in each dataset with respect to CRC stage is shown in Table 1. The human protein-protein interaction data used in our experiments is obtained from NCBI Entrez Gene Database [17]. This database integrates interaction data from several other databases available, such as HPRD, Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Page 9 of 12 We compare COBALT with our implementation of the two additive greedy approaches explained in detail in the Methods section, namely additiveova and additiveava. additiveova and additiveava refer respectively to the algo- rithms that aim to identify one-vs-all and all-vs-all sub- networks by greedily maximizing the discriminative power of the subnetworks (one−vs−all and all - vs - all). In the first set of experiments, we use the 10-fold cross validation framework for prediction of disease stage of samples in GSE14333, using the high-scoring subnet- works extracted as features from the same dataset, i.e., we use the top scoring positive and negative subnetworks for each stage in COBALT (setting k = 1), top 2 subnet- works for each stage in additiveova and top 8 subnet- works for additiveava as features (a total of 8 features used in each method). Datasets reproducibility and the utility of identified stage-specific subnetworks. For this purpose, we systematically evaluate the classification performance of the subnetworks for varying network distance parameter (ℓ) that adjusts the search space size when growing the subnetworks using COBALT. We also compare the subnetworks identified by the network-guided algorithm with groups of genes that are identified by using the same algorithm in a net- work-free fashion. The set-cover based algorithm imple- mented by COBALT is quite efficient, therefore a network-free search for stage-specific groups of proteins is feasible. In the PPI network free approach, the next protein to be added to the subnetwork does not need to be in a cer- tain proximity (i.e., ℓis effectively set to ∞) to the pro- teins already in the subnetwork. This increases the search space for the algorithm, thus making it infeasible for most of the state-of-the-art algorithms to perform some complex analyses such as statistical significance compu- tations. The effect of the parameter ℓon the classification performance for each CRC stage is shown in terms of precision and recall in Figures 4 (a) and 4(b) respectively. As seen in the figures, the classification performance (hence the reproducibility) of identified subnetworks improves as PPI network neighborhood is defined more flexibly. This is expected, since the PPI network is incom- plete, thus consideration of indirect interactions accounts for missing interactions to a certain extent. However, as the search diameter reaches 3, the classification perfor- mance saturates and adding more flexibility to the search does not improve performance any more. This observa- tion suggests that incorporation of PPI networks is useful for increased efficiency of the search, as well as identifica- tion of more reproducible subnetworks. Thus we set As shown in Figure 3, COBALT provides better perfor- mance compared to both of the additive approaches in terms of the precision in prediction for all CRC stages. It also provides better recall values for all CRC stages except for samples in Stage A. COBALT achieves 0.84 weighted average precision over all stages where as addi- tiveova and additiveava respectively achieve 0.71 and 0.62 precision. Similarly, COBALT outperforms others by achieving 0.84 weighted average recall over all stages where as additiveova and additiveava respectively provide 0.69 and 0.60 recall. Datasets The effect of the PPI network on classification performance In this section, we discuss the effect of the PPI network in the classification performance of subnetworks identi- fied by COBALT. Since the use of the PPI network con- fines the search space to functionally related groups of proteins, these experiments provide insights into whether these functional constraints also improve the biological Figure 3 Precision and recall values for each stage in prediction of stages of samples in GSE14333. Precision and recall values for each stage in prediction of stages of samples in GSE14333 in a 10-fold cross validation framework are shown in (a) and (b) respectively. COBALT outperforms the additive approaches in terms of precision for all CRC stages. It also provides better recall values for all CRC stages except for Stage A. Figure 3 Precision and recall values for each stage in prediction of stages of samples in GSE14333. Precision and recall values for each stage in prediction of stages of samples in GSE14333 in a 10-fold cross validation framework are shown in (a) and (b) respectively. COBALT outperforms the additive approaches in terms of precision for all CRC stages. It also provides better recall values for all CRC stages except for Stage A Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Page 10 of 12 Figure 4 Precision and recall plots of each stage with respect to different values of the network distance parameter ℓ. Precision and recall plots of each stage, in prediction of CRC stages of samples in GSE14333 in a 10-fold cross validation framework are shown in (a) and (b) respectively, with respect to different values of the network distance parameter ℓ, as well as the PPI-free approach. Setting ℓ= 3 provides the best performance for most of the cancer stages in terms of both precision and recall, also providing a smaller search space compared to ℓ= 4 and the PPI-free approach. Figure 4 Precision and recall plots of each stage with respect to different values of the network distance parameter ℓ. Precision and recall plots of each stage, in prediction of CRC stages of samples in GSE14333 in a 10-fold cross validation framework are shown in (a) and (b) respectively, with respect to different values of the network distance parameter ℓ, as well as the PPI-free approach. The effect of using statistically significant subnetworks on classification performance Contingency tables for prediction of CRC stages of samples in GSE14333 with COBALT using (a) 9 statistically significant features composed of 139 unique genes, (b) 9 features with top mutual information scores composed of 144 unique genes. The weighted average precision scores are 0.86 and 0.81 for (a) and (b) respectively. Similarly, weighted average recall values are 0.85 and 0.81 respectively for (a) and (b). Contingency tables for prediction of CRC stages of samples in GSE14333 with COBALT using (a) 9 statistically significant features composed of 139 unique genes, (b) 9 features with top mutual information scores composed of 144 unique genes. The weighted average precision scores are 0.86 and 0.81 for (a) and (b) respectively. Similarly, weighted average recall values are 0.85 and 0.81 respectively for (a) and (b). Datasets Setting ℓ= 3 provides the best performance for most of the cancer stages in terms of both precision and recall, also providing a smaller search space compared to ℓ= 4 and the PPI-free approach. ℓ= 3 as it is the most reasonable choice in terms of both the classification performance and the computational efficiency of the algorithms. working on multi-class phenotypes, namely, one-vs-all and all-vs-all. We then introduced our cover-based algo- rithm for network-guided disease marker discovery, for identifying subnetworks with one-vs-all discriminative power. Moreover, we have introduced a framework for assessing the statistical significance of the identified sub- networks. Systematic experiments on real multi-staged CRC datasets show that the proposed algorithm outper- forms the additive algorithms in terms of providing higher precision and recall in prediction of sample The effect of using statistically significant subnetworks on classification performance The effect of using statistically significant subnetworks on classification performance In this section, we compare the classification perfor- mance of high-scoring subnetworks to that of statisti- cally significant subnetworks. On the GSE14333 dataset, COBALT identifies 9 statistically significant subnetworks with 139 unique genes (please see Additional File 1 for the list of genes and the covered stage of colorectal can- cer for these 9 subnetworks). Using these subnetworks as features, we predict the stage of the samples in GSE14333 in a 10-fold cross validation framework as previously explained. We choose the same number of features with top (one - vs - all(S). score and compare the classification performance of both approaches. Stage-specific precision and recall values for both approaches are shown in Table 2. As seen in the table, utilizing statistical significance computations when choosing features improve performance for predicting the stages of patients in GSE14333, with less number of unique genes used. Table 2 Contingency tables for prediction of CRC stages of samples in GSE14333. Predicted Classes stage A B C D recall Actual Classes A 37 3 0 4 0.84 B 3 74 11 6 0.78 C 5 5 77 4 0.84 D 4 7 3 47 0.82 precision 0.75 0.83 0.84 0.77 Predicted Classes stage A B C D recall A 38 1 2 3 0.86 Actual Classes B 4 75 8 7 0.79 C 3 2 83 3 0.91 D 2 3 4 52 0.85 precision 0.80 0.92 0.85 0.80 Contingency tables for prediction of CRC stages of samples in GSE14333 with COBALT using (a) 9 statistically significant features composed of 139 unique genes, (b) 9 features with top mutual information scores composed of 144 unique genes. The weighted average precision scores are 0.86 and 0.81 for (a) and (b) respectively. Similarly, weighted average recall values are 0.85 and 0.81 respectively for (a) and (b). Table 2 Contingency tables for prediction of CRC stages of samples in GSE14333. In the cross-classification framework, we use the statisti- cally significant features identified in GSE14333 to pre- dict the classes of samples in the GSE5206 dataset, i.e., the classifier is trained using GSE14333 and tested on GSE5206. The stage-specific precision and recall values are shown in Table 3. The weighted average precision and recall values are 0.57 and 0.56 respectively. Conclusions In this article, we have proposed two alternate formula- tions of the discriminative power of subnetworks when Page 11 of 12 Page 11 of 12 Page 11 of 12 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 Table 3 Contingency table for prediction of CRC stages of samples in GSE5206 using the statistically significant features identified from GSE14333. Table 3 Contingency table for prediction of CRC stages of samples in GSE5206 using the statistically significant features identified from GSE14333. Predicted Classes stage A B C D recall A 9 2 1 0 0.75 Actual Classes B 3 18 8 3 0.56 C 5 3 20 5 0.60 D 4 3 6 8 0.38 precision 0.42 0.69 0.57 0.50 Western Reserve University, Cleveland, OH, USA. 6Department of Genetics, Case Western Reserve University, Cleveland, OH, USA. Table 3 Contingency table for prediction of CRC stages of samples in GSE5206 using the statistically significant features identified from GSE14333. Predicted Classes stage A B C D recall A 9 2 1 0 0.75 Actual Classes B 3 18 8 3 0.56 C 5 3 20 5 0.60 D 4 3 6 8 0.38 precision 0.42 0.69 0.57 0.50 References S h stages. The efficient implementation of the cover-based algorithm enabled us to show that using statistically sig- nificant subnetworks as features improves classification performance compared to using same number of high- scoring subnetworks (in terms of mutual information with respect to the phenotype vector). We have also shown that guiding the subnetwork discovery search with the PPI network identifies subnetworks that are more informative (in terms of classification power) than the net-works identified without the PPI network. We have also investigated the impact of different values of the network distance parameter, ℓ, and concluded that using ℓ= 3 is the most reasonable choice in terms of both classification performance and computational efficiency. 1. Scherzer CR, Eklund AC, Morse LJ, Liao Z, Locascio JJ, Fefer D, Schwarzschild MA, Schlossmacher MG, Hauser MA, Vance JM, Sudarsky LR, Standaert DG, Growdon JH, Jensen RV, Gullans SR: Molecular markers of early Parkinson’s disease based on gene expression in blood. Proceedings of the National Academy of Sciences 2007, 104(3):955-960 [http://www.pnas. org/content/104/3/955.abstract]. 1. 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Additional material 6. Chowdhury A Salim, Koyutürk M: Identification of coordinately dysregulated subnetworks in complex phenotypes. Pacific Symposium on Biocomputing (PSB’10) 2010, 133-144 [http://dx.doi.org/10.1142/ 9789814295291_0016]. Additional file 1: List of 9 statistically significant subnetworks identified for GSE14333 dataset. All the gene products in the 9 statistically significant subnetworks identified for GSE14333 dataset are listed, as well as the covered colorectal cancer stage and cover direction of the corresponding subnetworks. Please note that these gene products might not be direct neighbours in the PPI network, as we set the network distance parameter ℓ= 3 in the experiments. 7. Dao P, Colak R, Salari R, Moser F, Davicioni E, Schanhuth A, Ester M: Inferring cancer subnetwork markers using density-constrained biclustering. Bioinformatics 2010, 26(18):i625-i631 [http://bioinformatics. oxfordjournals.org/content/26/18/i625.abstract]. 8. 8. Dao P, Wang K, Collins C, Ester M, Lapuk A, Sahinalp SC: Optimally discriminative subnetwork markers predict response to chemotherapy. Bioinformatics 2011, 27(13):i205-i213 [http://bioinformatics.oxfordjournals. org/content/27/13/i205.abstract]. 9. Chowdhury SA, Nibbe RK, Chance MR, Koyutürk M: Subnetwork state functions define dysregulated subnetworks in cancer. Journal of Computational Biology 2011, 18(3):263-281 [http://dx.doi.org/10.1089/ cmb.2010.0269]. Acknowledgements We would like to note the contribution of anonymous reviewers whose queries and suggestions have helped improve this article significantly. MK and SE are supported in part by the National Science Foundation grant CCF- 0953195. MK and MRC are also supported in part by the National Institutes of Health grant R01-HL106798. SE is also supported in part by Choose Ohio First Scholarship. JSB and XG are supported by Case Comprehensive Cancer Center Core Grant (5P30 CA043703). 10. Dutkowski J, Ideker T: Protein networks as logic functions in development and cancer. PLoS Comput Biol 2011, 7(9):e1002180 [http://dx. doi.org/10.1371%2Fjournal.pcbi.1002180]. 11. Chvatal V: A greedy heuristic for the set-covering problem. Mathematics of Operations Research 1979, 4(3):233-235 [http://dx.doi.org/10.2307/ 3689577]. This article has been published as part of BMC Proceedings Volume 6 Supplement 7, 2012: Proceedings from the Great Lakes Bioinformatics Conference 2012. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcproc/supplements/6/S7. 12. Macdonald F, Ford C, Casson A: Molecular biology of cancer Advanced text, BIOS Scientific Publishers; 2004 [http://books.google.com/books? id=SClAlGpFR-IC]. Supplement 7, 2012: Proceedings from the Great Lakes Bioinformatics Conference 2012. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcproc/supplements/6/S7. 13. Howlader N, Noone A, Krapcho M, Neyman N, Aminou R, Waldron W, Altekruse S, Kosary C, Ruhl J, Tatalovich Z, Cho H, Mariotto A, Eisner M, Lewis D, Chen H, Feuer E, Cronin K, Edwards Be: SEER cancer statistics review, 1975-2008. 2010 [http://seer.cancer.gov/csr/1975_2008/]. Competing interests No competing interests exist. 15. Sobin LH, Gospodarowicz MK, Wittekind C: TNM - classification of malignant tumours 2010. Author details 1 1Department of Electrical Engineering & Computer Science, Case Western Reserve University, Cleveland, OH, USA. 2School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA. 3Case Center for Proteomics & Bioinformatics, Case Western Reserve University, Cleveland, OH, USA. 4Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH, USA. 5Case Comprehensive Cancer Center, Case review, 1975-2008. 2010 [http://seer.cancer.gov/csr/1975_2008/]. 14. Dukes CE: The classification of cancer of the rectum. The Journal of Pathology and Bacteriology 1932, 35(3):323-332 [http://dx.doi.org/10.1002/ path.1700350303]. Page 12 of 12 Page 12 of 12 Erten et al. BMC Proceedings 2012, 6(Suppl 7):S1 http://www.biomedcentral.com/1753-6561/6/S7/S1 15. Sobin LH, Gospodarowicz MK, Wittekind C: TNM - classification of malignant tumours 2010. 16. Barrett T, Troup DB, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Muertter RN, Holko M, Ayanbule O, Yefanov A, Soboleva A: NCBI GEO: archive for functional genomics data sets-10 years on. Nucleic Acids Research 2010, 39(Database):D1005-D1010. 17. Maglott D, Ostell J, Pruitt KD, Tatusova T: Entrez Gene: gene-centered information at NCBI. Nucl Acids Res 2007, 35(suppl-1):D26-31 [http://nar. oxfordjournals.org/cgi/content/abstract/35/suppl_1/D26]. doi:10.1186/1753-6561-6-S7-S1 Cite this article as: Erten et al.: Identifying stage-specific protein subnetworks for colorectal cancer. BMC Proceedings 2012 6(Suppl 7):S1. 15. Sobin LH, Gospodarowicz MK, Wittekind C: TNM - classification of malignant tumours 2010. 16. Barrett T, Troup DB, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Muertter RN, Holko M, Ayanbule O, Yefanov A, Soboleva A: NCBI GEO: archive for functional genomics data sets-10 years on. Nucleic Acids Research 2010, 39(Database):D1005-D1010. 17. Maglott D, Ostell J, Pruitt KD, Tatusova T: Entrez Gene: gene-centered information at NCBI. Nucl Acids Res 2007, 35(suppl-1):D26-31 [http://nar. oxfordjournals.org/cgi/content/abstract/35/suppl_1/D26]. doi:10.1186/1753-6561-6-S7-S1 Cite this article as: Erten et al.: Identifying stage-specific protein subnetworks for colorectal cancer. BMC Proceedings 2012 6(Suppl 7):S1. 15. Sobin LH, Gospodarowicz MK, Wittekind C: TNM - classification of malignant tumours 2010. 16. Barrett T, Troup DB, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Muertter RN, Holko M, Ayanbule O, Yefanov A, Soboleva A: NCBI GEO: archive for functional genomics data sets-10 years on. Nucleic Acids Research 2010, 39(Database):D1005-D1010. 17. Maglott D, Ostell J, Pruitt KD, Tatusova T: Entrez Gene: gene-centered information at NCBI. Nucl Acids Res 2007, 35(suppl-1):D26-31 [http://nar. oxfordjournals.org/cgi/content/abstract/35/suppl_1/D26]. doi:10.1186/1753-6561-6-S7-S1 Cite this article as: Erten et al.: Identifying stage-specific protein subnetworks for colorectal cancer. Author details 1 BMC Proceedings 2012 6(Suppl 7):S1. 17. Maglott D, Ostell J, Pruitt KD, Tatusova T: Entrez Gene: gene-centered information at NCBI. Nucl Acids Res 2007, 35(suppl-1):D26-31 [http://nar. oxfordjournals.org/cgi/content/abstract/35/suppl_1/D26]. doi:10.1186/1753-6561-6-S7-S1 Cite this article as: Erten et al.: Identifying stage-specific protein subnetworks for colorectal cancer. BMC Proceedings 2012 6(Suppl 7):S1. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2162880594	https://repository.ubn.ru.nl/bitstream/handle/2066/109787/1/109787.pdf	English	null	The Effect of Chronic Deafferentation on Mental Imagery: A Case Study	PloS one	2,012	cc-by	7,790	The effect of chronic deafferentation on mental imagery: a case study Horst, A.C. ter; Cole, J.; Lier, R.J. van; Steenbergen, B. 2012, Article / Letter to editor (PLoS One, 7, 8, (2012), article e42742) Doi link to publisher: https://doi.org/10.1371/journal.pone.0042742 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/109787 Download date: 2024-10-24 Download date: 2024-10-24 Abstract Visual- and motor imagery rely primarily on perceptual and motor processes, respectively. In healthy controls, the type of imagery used to solve a task depends on personal preference, task instruction, and task properties. But how does the chronic loss of proprioceptive and tactile sensory inputs from the body periphery influence mental imagery? In a unique case study, we investigated the imagery capabilities of the chronically deafferented patient IW when he was performing a mental rotation task. We found that IW’s motor imagery processes were impaired and that visual imagery processes were enhanced compared to controls. These results suggest that kinaesthetic afferent signals from the body periphery play a crucial role in enabling and maintaining central sensorimotor representations and hence the ability to incorporate kinaesthetic information into the imagery processes. Citation: ter Horst AC, Cole J, van Lier R, Steenbergen B (2012) The Effect of Chronic Deafferentation on Mental Imagery: A Case Study. PLoS ONE 7(8): e42742. doi:10.1371/journal.pone.0042742 Editor: Nicholas P. Holmes, University of Reading, United Kingdom Editor: Nicholas P. Holmes, University of Reading, United Kingdom Received December 28, 2011; Accepted July 11, 2012; Published August 7, 2012 Received December 28, 2011; Accepted July 11, 2012; Published August 7, 2012 Copyright: 2012 ter Horst et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 ter Horst et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 ter Horst et al. This is an open-access article distributed under the term unrestricted use, distribution, and reproduction in any medium, provided the original author a Funding: No current external funding sources for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: a.terhorst@donders.ru.nl Competing Interests: The authors have declared that no competing interests exist. * E-mail: a.terhorst@donders.ru.nl and less accurate overall, but the influence of biomechanical constraints remained. Hence, the loss of kinaesthetic afferents alone is not sufficient to alter the embodied properties of MI processes. Consequently, it is likely that the postural and biomechanical conflicts arise (at least partly) from central processes. Introduction The ability to imagine is regarded as an extraordinary human capacity. Humans are able to mentally manipulate internal representations. This imagery capability is understood as a reconstruction of actual perceptual experience from the past. Two types of imagery that have been studied extensively are visual imagery (VI) and motor imagery (MI). In VI, participants mentally perform visual transformations of an object or scene without a retinal projection of that image [1]. In contrast, MI represents a mental movement of one’s own body parts from a first person perspective. MI is thus defined as a dynamic state during which a participant mentally simulates a given action without overt movement [2]. MI, but not VI, has been shown to be subject to postural manipulations [3,4,5] and biomechanical constraints [6,7]. These effects are thought to result from a conflict between the imagined movement and the body’s current posture and movement abilities [3,8]. From studies on amputees it is known that the (partial) loss of the effectors and hence both afferent and efferent kinaesthetic sensations, results in a lack of bodily influences on the MI processes [9,10,11]. These studies looked at the necessity of a present effector for the interaction between body representations and imagery processes [10], and the influence of a missing effector on MI processes [11]. There is, however, ambiguity as to the role of the mere kinaesthetic afferent or efferent sensations in the generation of these bodily influences on the MI processes. In a study with a peripherally deafferented patient, Mercier et al. (2008) argued that this conflict mainly arises from online afferent feedback, influencing the MI processes [8]. However, in a recent study, Silva et al. (2011) showed that during transient deafferen- tation due to local anaesthesia of the arm, MI processes are slower In order to answer this question, we performed two experiments with an individual suffering from a rare case of selective peripheral deafferentation - a condition of selective and complete chronic loss of proprioceptive and tactile afferents due to a sensory neurono- pathy [for a more elaborate description of the condition see: 16,17]. From the literature it is known that the deafferented subjects IW and GL are able to explicitly construct motor representations as both are able to perform accurate movements, although likely with a more visual cognitive supervision than controls [18,19]. Consequently, and in contrast to Mercier et al. Arjan C. ter Horst1,2, Jonathan Cole3, Rob van Lier1, Bert Steenbergen2,1 1 Radboud University Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, The Netherlands, 2 Radboud University Nijmegen, Behavioural Science Institute, Nijmegen, The Netherlands, 3 University of Bournemouth, Poole Hospital, Clinical Neurophysiology, Poole, United Kingdom 1 Radboud University Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, The Netherlands, 2 Radboud Univ Institute, Nijmegen, The Netherlands, 3 University of Bournemouth, Poole Hospital, Clinical Neurophysiology, Poole, United Kingdo Note: To cite this publication please use the final published version (if applicable). Abstract We know from previous studies that MI is dependent on centrally constructed body representations [4,12], which represent the body’s current posture and action abilities [13,14,15]. Therefore, we examine in the present study how the long-term loss of kinaesthetic afferents influences central imagery processes and specifically, the role of these kinaesthetic afferents on the interaction between the imagined movements and the body’s current posture and biomechanical constraints. By doing so, we provide new insight in the selective role of afferent information on (mental) motor processes. Participants p The study was approved by the ethics committee of the Faculty of Behavioural Sciences from the Radboud University Nijmegen and all participants gave written informed consent prior to the experiment, in accordance with the Helsinki declaration. The tasks in both experiments were performed by the deafferented person IW (age 59 years, male, left-handed), fifteen left-handed controls (mean age 57.1 years, range 51–61 years), denoted as CL, and fifteen right-handed controls (mean age 56.3 years, range 51–65 years), denoted as CR. All controls were neurologically healthy and age and sex matched to IW (z-score IW vs. CL: 0.58 and IW vs. CR: 0.45). Hand preference was assessed according to the Edinburgh Handedness Inventory [27]. Hand preference was found in all participants (laterality quotient: IW, 2100; left- handed participants, 254625.7 mean 6 SD and right-handed participants, 90618.5 mean 6 SD). All participants had normal or corrected-to-normal vision. Introduction (2008), we used an implicit mental rotation task to study the influence of long-term deafferentation on the implicit use of internal motor representations. We used mental rotation tasks in which MI (Experiment 1 and 2) and VI (Experiment 1) are implicitly induced. The mental rotation task is a well defined task to study imagery [7,20]. During the task, participants are presented with rotated pictures of corporeal or non-corporeal August 2012 \| Volume 7 \| Issue 8 \| e42742 1 August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org Effect of Deafferentation on Mental Imagery Figure 1. Examples of used stimuli. Examples of stimuli as used in Experiment 1 (letters and hands) and Experiment 2 (hands). Degrees represent the in-plane rotational angle. doi:10.1371/journal.pone.0042742.g001 Effect of Deafferentation on Mental Imagery Figure 1. Examples of used stimuli. Examples of stimuli as used in Experiment 1 (letters and hands) and Experiment 2 (hands). Degrees represent the in-plane rotational angle. doi:10.1371/journal.pone.0042742.g001 objects (i.e., hands or letters, respectively). In order to solve mental rotation tasks, participants use visual and motor based strategies. Without explicit task instructions on how to solve the task, the use of non-corporeal objects without accompanying motor represen- tations results in the implicit use of VI [21]. The presentation of corporeal objects results in the implicit use of MI [22]. In order to establish whether MI or VI was used, we manipulated the participants’ posture during both experiments and measured the influence of biomechanical constraints on the imagery processes. The influence of biomechanical constraints during the mental rotation can be defined as the difference in performance between hand stimuli rotated toward and away from the body’s midsagital plane [6,7,23,24,25]. Furthermore, in order to be able to ascribe possible effects to the deafferentation and not to handedness, we included both left and right handed age and sex matched controls as IW is strongly left handed. If IW is able to construct a representation of his current body posture and his action abilities, we expected to find postural and biomechanical influences on MI. If, on the other hand, the long-term loss of afferent information prevents IW constructing a postural and biomechanical represen- tation as controls, we expected to find a lack of postural and biomechanical influence on MI. Furthermore, we expected IW to outperform controls on the VI tasks as IW is used to visualizing not only his own movements prior to execution and movement rehearsal [26] but also the movements of others for anticipation in daily life. Figure 1. Examples of used stimuli. Examples of stimuli as used in Experiment 1 (letters and hands) and Experiment 2 (hands). Degrees represent the in-plane rotational angle. doi:10.1371/journal.pone.0042742.g001 Figure 1. Examples of used stimuli. Examples of stimuli as used in Experiment 1 (letters and hands) and Experiment 2 (hands). Degrees represent the in-plane rotational angle. doi:10.1371/journal.pone.0042742.g001 Procedure E i Experiment 1. The participants were placed in front of a computer screen. Stimulus presentation was controlled using custom developed software in Presentation (Neurobehavioral systems, Albany, USA). Prior to the stimulus a fixation cross was presented at the centre of the screen for a random time between 800 ms and 1200 ms. After this, the stimulus was presented and visible until a response was given. A response consisted of the words ‘‘left’’ or ‘‘right’’ for hand stimuli and ‘‘normal’’ or ‘‘mirror’’ for letter stimuli. RTs were automatically recorded by use of a microphone detecting supra-threshold responses. Response accu- racy was manually recorded by an experimenter during the experiment. After the response, a black screen was displayed for 800 ms. Stimuli were presented one at a time. Participants were instructed to judge the laterality of the hand-stimuli or the mirrored or canonical presentation of the letter-stimuli as fast and as accurately as possible, without explicit instructions on how to solve the task. Participants were tested in one experimental session consisting of eight blocks. For each stimulus type (i.e. letter and hands) the participants positioned their hands on their lap underneath the table with the palms oriented downward in two blocks. In the other two blocks, participants positioned their hands behind their back with their fingers intertwined [4]. Consequently, the participants performed four blocks with hand-stimuli and four blocks with letter-stimuli. All stimuli were repeated 4 times, resulting in eight blocks of 48 stimuli. The experiment was preceded by a test of 24 stimuli to familiarize the participants with the task. The order of hand position and stimulus type was randomized and counter-balanced per block. Results For the correct responses, the overall RT of IW did not differ from the control groups, see Figure 2a. The mixed design ANOVA revealed a significant main effect of Angle (F(3,84) = 90.270; p,0.001; g2 = 0.763). Despite a significant interaction between Angle and Group (F(3,84) = 3.214; p,0.05; g2 = 0.103), both groups showed a significant simple effect of Angle (CL: F(3,42) = 64.451; p,0.001; g2 = 0.822 and CR: F(3, 24) = 30.050; p,0.001; g2 = 0.682), see Figure 3. Furthermore, we obtained a significant interaction of Type by Posture (F(1,14) = 6.212; p,0.05; g2 = 0.307). Further simple effect anal- yses revealed no significant postural influences for neither of the stimulus types (all p.0.07). Crucially, we obtained a significant three-way interaction of Type by Posture by Group (F(1,28) = 4.412; p,0.05; g2 = 0.136). Further analyses for the CL group revealed no significant effect of posture or interaction of Type by Posture (all p.0.75). For the CR group, however, we obtained a significant interaction of Type by Posture (F(1,14) = 6.212; p,0.05; g2 = 0.307), which resulted in a signif- icant simple effect of Posture (F(1,14) = 6.643; p,0.05; g2 = 0.322) only for the hand stimuli and not for the letter stimuli (p.0.55), see Figure 4a. For IW we only obtained a significant effect of Angle for both the Hand stimuli (x2(3) = 10.275, p,.02) and Letter stimuli (x2(3) = 26.625, p,.001), see Figure 3. The postural influence found for the hand stimuli in the CR group differed significantly from the postural influence obtained for IW, see Figure 4a. The mixed design ANOVA on the biomechanical constraints revealed a significant mean effect of the DOR (F(1,28) = 26.288; p,0.001; g2 = 0.484). This effect was not modulated by Group (p.0.95), see Figure 4b. For IW, the influence of the biomechanical constraints was not significant (p.0.54). The influence of the biomechanical constraints for IW differed significantly from that of both control groups, see Figure 4b. Individual results of IW were analyzed using separate non parametric Friedman’s tests for both types of stimuli (i.e. hands and letters) with Angle as factor with 4 levels (0u, 60u, 120u and 180u). Wilcoxon Signed Rank Tests were used as post-hoc tests. The Wilcoxon Signed Rank test was also used to test for postural influences for both hand and letter stimuli separately. Effect of Deafferentation on Mental Imagery Effect of Deafferentation on Mental Imagery this second experiment we were interested in the effects of the postural manipulations, biomechanical constraints and the mod- ulation of visual feedback in the performance for hand stimuli in the different groups. As in Experiment 1, we used different mixed design ANOVAs. For testing the postural influence and the effect of changing the visual feedback we used a mixed design ANOVA with the following design: 1 between subject variable Group, with two levels: Control Left (CL) and Control Right (CR); 3 within- subject factors (Feedback, Congruency and Angle); with 2 levels for Feedback (Seen, Unseen), 2 levels for Congruency (Congruent, Incongruent) and 4 levels for Angle (0u, 60u, 120u and 180u). To test the influence of biomechanical constraints we used an identical test as in Experiment 1. Individual results of IW were analyzed as in Experiment 1 for the factors Angle, Feedback, Congruency and biomechanical constraints. Identical tests as in Experiment 1 were used to compare the results of IW with those of the left- and right handed controls. Accuracy data were analyzed using the same statistical designs as for the RT data. Post hoc analyses were Bonferroni corrected and alpha-level was set at p = 0.05. positioned their hands on their laps with the palms oriented downward or upward [28]. Furthermore, visual feedback was altered during the experiments. During four of the eight blocks, the participants’ hands were covered by a black cloth in order to prevent them from seeing their own hands. During the blocks in which visual feedback of the hands was impossible, the hand position of the participant was changed passively by the experimenter. In the case of IW, he then had no knowledge of the position of his hands, something checked verbally. All other parameters were identical to those of Experiment 1. Data analysis i Experiment 1. Reaction times smaller than 400 ms and larger than 3500 ms were excluded from further analyses (total loss 3.7% of all trials) in correspondence with former studies [20,29]. Reaction time analyses were then performed on correct responses. Incorrect responses were a ‘left’ response for a ‘right’ hand or a ‘mirrored’ response for a ‘canonically’ oriented letter and vice versa. Analyses on accuracy data were performed on the percentage of correct responses. The effect of the different conditions in the control participants was assessed using separate mixed design analyses of variance (ANOVA) for testing the influence of postural changes and biomechanical constraints. The rationale for using different ANOVAs is that letter stimuli in general, and 0u and 180u rotated hand stimuli, cannot be denoted as being laterally or medially rotated. Furthermore, this method provides a single numerical measure for the influence of the biomechanical constraints for comparing the biomechanical influences between the control groups and IW. In order to test the postural influence on performance of the controls we used an with the following design: 1 between subject variable Group, with two levels: Control Left (CL) and Control Right (CR); 3 within-subject factors (Type, Posture and Angle), with 2 levels for Type (Letter, Hand), 2 levels for Posture (on lap, behind back) and 4 levels for Angle (0u, 60u, 120u and 180u). The values labelled 60u and 120u are the averaged RTs of 60u and 300u, and 120u and 240u rotated stimuli, respectively. To test for the influence of biomechanical constraints on the performance for hand stimuli we used an ANOVA with 1 between subject variable (Group) with two levels (CL, CR) and 1 within subject variable Direction Of Rotation (DOR) with two levels (Lateral rotations, Medial rotations). Stimuli As corporeal stimuli we used a custom made 3D hand model designed in a 3D image software package (Autodesk Maya 2009, USA). From this realistic model we constructed all corporeal stimuli that were used in Experiment 1 and 2. The hand-stimuli were shown from both a back- and palm view, see Figure 1. Additionally, as non-corporeal objects, we used typographical character stimuli for Experiment 1 in Times New Roman font, shown in a canonical or mirrored orientation, see Figure 1. The stimuli were displayed on a 19’’ LCD computer screen, at a distance of approximately 70 cm from the participants’ eyes, resulting in a vertical visual angle of approximately 6u. All stimuli were shown in six different angles of in-plane rotation (i.e. 0u, 60u, 120u, 180u, 240u and 300u), resulting in 24 unique hand stimuli and 24 unique letter stimuli. Experiment 2. In the second experiment, the participants were presented with hand-stimuli identical to those used in Experiment 1. In contrast to Experiment 1, the participants Experiment 2. In the second experiment, the participants were presented with hand-stimuli identical to those used in Experiment 1. In contrast to Experiment 1, the participants PLOS ONE \| www.plosone.org August 2012 \| Volume 7 \| Issue 8 \| e42742 August 2012 \| Volume 7 \| Issue 8 \| e42742 2 Discussion In Experiment 1, we studied the influence of deafferentation on imagery capabilities. We expected IW’s MI to be impaired and his VI to be enhanced compared to controls. We found that controls showed the typical RT and accuracy profiles for mental rotation tasks for the letter and hand stimuli [6,7,20,22]. Hence, we can conclude, that the controls did use a mental rotation strategy to solve the task. For IW, though we also found significant Angle effects for both stimulus types, we only found a gradual increase in RTs as a function of rotational angle for the hand stimuli. The RT profile for the letter stimuli showed nearly equal RTs from 0u to 120u, all differing significantly from 180u, see Figure 3. Therefore, IW seemingly does not use a mental rotation strategy. However, during analysis if IW’s introspection, he reported that he mentally ‘‘placed the letter on an imaginary disc in order to rotate it upward’’. This implies that IW did use a mental rotation strategy, albeit a modified one. the task can result in the construction of a representation of the current posture from available visual feedback, thereby interfering with the imagined movement. In Experiment 1, the visibility of the participants’ hands was confined with the postural manipulation. That is, during the placing of the hands in the correct position in the ‘‘hands on lap’’ and the ‘‘hands behind the back’’ conditions, the participants’ hands were visible and invisible, respectively. Therefore, in Experiment 2, the participants’ posture and visibility of the hands were manipulated separately during a mental rotation task of hands. If IW is able to construct a visual representation of his hands’ current position and incorporates this representation into his planned movement, we would expect an influence of the hand posture on the performance only when IW is able to observe his own hands. In contrast, when IW does not construct a visual representation of his hands’ position, IW would not show any postural influence, irrespective of the ability to see his own hands. The postural manipulations did not influence IW’s performance during the mental rotation of letters and hands. For the letter stimuli, the difference scores for the postural manipulations between IW and the controls did not differ, see Figure 4a. This finding is intuitive and agrees with the literature; letter stimuli are non-corporeal objects and hence do not implicitly induce egocentric processing [20]. Results The same non-parametric test was used to test for differences between lateral and medial rotations. To compare the results of IW with those of the CL and CR groups, we used 95% confidence intervals (CI). We calculated difference scores across trials of the postural effects (i.e. hands behind the back.hands on the lap) for both stimulus types for IW, CL and CR. Furthermore, we also calculated difference scores for the DOR-effect for hand stimuli only (i.e., lateral.medial). From these (difference) scores, we calculated the 95% CI based on the t- distribution for CL and CR and determined whether IW’s difference scores fell outside the confidence intervals [8]. For the accuracy data we obtained a significant interaction of Angle by Group (F(3,84) = 6.050; p,0.005; g2 = 0.178). Further analyses revealed a decrease in accuracy as function of the angular rotation for CL (F(3,42) = 13.791; p,0.001; g2 = 0.496) and CR (F(3,42) = 32.416; p,0.001; g2 = 0.698). No further effects were found significant. In the comparison of the accuracy between the control groups and IW we found that IW was significantly more accurate than both control groups, see Figure 3b. Accuracy data were analyzed using the same statistical designs as for the RT data. Post hoc analyses were Bonferroni corrected and the alpha-level was set at p = 0.05. Experiment 2. For experiment 2, the same exclusion criteria were used as for Experiment 1, resulting in 4.2% loss of trials. In August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org 3 Effect of Deafferentation on Mental Imagery Figure 2. Mean reaction times and accuracy in Experiment 1. Reaction time (A) and accuracy data (B) for IW, CL and CR for Experiment 1. Error bars represent the 95% CI. # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g002 Discussion In Experiment 1 we studied the influence of deafferentation on Figure 2. Mean reaction times and accuracy in Experiment 1. Reaction time (A) and accuracy data (B) for IW, CL and CR for Experiment 1. Error bars represent the 95% CI. # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g002 Figure 3. Reaction times as function of rotational angle in Experiment 1. Results Reaction time data from Experiment 1 as function of angle for hand stimuli (A) and letter stimuli (B). Error bars represent Figure 2. Mean reaction times and accuracy in Experiment 1. Reaction time (A) and accuracy data (B) for IW, CL and CR for Experiment 1. Error bars represent the 95% CI. # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g002 Figure 3. Reaction times as function of rotational angle in Experiment 1. Reaction time data from Experiment 1 as function of angle for hand stimuli (A) and letter stimuli (B). Error bars represent standard error of the mean. doi:10.1371/journal.pone.0042742.g003 Discussion For the hand stimuli, the lack of postural influence for IW differed significantly from the CR group, but not from the CL group, see Figure 4a. The lack of postural influence for IW is in correspondence with Mercier et al. (2008), who showed that kinaesthetic afferents are an important factor in the modulation of the imagery processes. However, Mercier et al. (2008) also showed that the ability to see one’s own hand during Results In line with Experiment 1, we found for the correct responses that the overall RT of IW did not differ from the control groups, see Figure 5a. The mixed design ANOVA revealed a significant main effect of Angle (F(3,84) = 74.131; p,0.001; g2 = 0.726) and Congruency (F(1,28) = 4.621; p,0.05; g2 = 0.142), see Figure 6. Despite a significant interaction of Congruency by Angle August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org August 2012 \| Volume 7 \| Issue 8 \| e42742 4 Effect of Deafferentation on Mental Imagery Figure 4. Difference scores for postural and biomechanical influences in Experiment 1. Differences in reaction times between the ‘hands behind the back’ and ‘hands on lap’ postural conditions (A) and between lateral and medial rotations for IW, CL and CR (B). Error bars represent 95% CI. denotes significance at the p,0.05 level, ** denotes significant at the p,0.01 level and # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g004 Figure 5. Mean reaction times and accuracy in Experiment 2. Reaction time (A) and accuracy data (B) for IW, CL and CR for Experiment 1. Error bars represent the 95% CI. # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10 1371/journal pone 0042742 g005 Figure 4. Difference scores for postural and biomechanical influences in Experiment 1. Differences in reaction times between the ‘hands behind the back’ and ‘hands on lap’ postural conditions (A) and between lateral and medial rotations for IW, CL and CR (B). Error bars represent 95% CI. * denotes significance at the p,0.05 level, ** denotes significant at the p,0.01 level and # denotes that the mean score of IW falls outside the 95% CI of the control group. doi 10 1371/jo rnal pone 0042742 g004 Figure 4. Difference scores for postural and biomechanical influences in Experiment 1. Differences in reaction times between the ‘hands behind the back’ and ‘hands on lap’ postural conditions (A) and between lateral and medial rotations for IW, CL and CR (B). Error bars represent 95% CI. * denotes significance at the p,0.05 level, ** denotes significant at the p,0.01 level and # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g004 Figure 5. Results The postural influence found for the CR group differed significantly from the postural influence obtained for IW, see Figure 7a. The mixed design ANOVA on the biomechanical constraints revealed a significant mean effect of the DOR (F(1,28) = 35.138; p,0.001; g2 = 0.557). This effect was not modulated by Group (p.0.95), see Figure 7b. For IW, the influence of the biomechanical constraints was not significant (p.0.63), see Figure 7b. As in Experiment 1, the influence of the biomechanical constraints for IW differed significantly from that of both control groups, see Figure 7b. control groups and IW we found that IW was significantly more accurate than both control groups, see Figure 5a. Results Mean reaction times and accuracy in Experiment 2. Reaction time (A) and accuracy data (B) for IW, CL and CR for Experiment 1. Error bars represent the 95% CI. # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g005 (F(3,84) = 6.695; p,0.002; g2 = 0.193) we obtained significant simple effects of Angle for the Congruent (F(3,87) = 84.478; p,0.001; g2 = 0.744) and Incongruent conditions (F(3,87) = 40.696; p,0.001; g2 = 0.584). Crucially, we obtained a significant two-way interaction of Congruency by Group (F(1,28) = 6.040; p,0.02; g2 = 0.177). Further simple effect anal- yses for the CL group revealed no significant effect of Congruency (p.0.82). For the CR group, however, we obtained a significant simple effect of Congruency (F(1,14) = 10.048; p,0.01; g2 = 0.418), see Figure 7a. For IW we only obtained a significant effect of Angle (x2(3) = 16.350, p,.001) and no influence of the postural manipulation irrespective of the feedback manipulation (all p,0.09), see figures 6 and 7a, respectively. The postural influence found for the CR group differed significantly from the postural influence obtained for IW, see Figure 7a. The mixed design ANOVA on the biomechanical constraints revealed a significant mean effect of the DOR (F(1,28) = 35.138; p,0.001; g2 = 0.557). This effect was not modulated by Group (p.0.95), see Figure 7b. For IW, the influence of the biomechanical constraints was not significant (p.0.63), see Figure 7b. As in Experiment 1, the influence of the biomechanical constraints for IW differed significantly from that of both control groups, see Figure 7b. (F(3,84) = 6.695; p,0.002; g2 = 0.193) we obtained significant simple effects of Angle for the Congruent (F(3,87) = 84.478; p,0.001; g2 = 0.744) and Incongruent conditions (F(3,87) = 40.696; p,0.001; g2 = 0.584). Crucially, we obtained a significant two-way interaction of Congruency by Group (F(1,28) = 6.040; p,0.02; g2 = 0.177). Further simple effect anal- yses for the CL group revealed no significant effect of Congruency (p.0.82). For the CR group, however, we obtained a significant simple effect of Congruency (F(1,14) = 10.048; p,0.01; g2 = 0.418), see Figure 7a. For IW we only obtained a significant effect of Angle (x2(3) = 16.350, p,.001) and no influence of the postural manipulation irrespective of the feedback manipulation (all p,0.09), see figures 6 and 7a, respectively. Effect of Deafferentation on Mental Imagery Additionally, it has also been shown that a lack of experience in the sensation of a certain movement results in an inability to imagine that movement [37]. The emergence of postural and biomechanical conflicts from centrally generated conflicts between body representations and imagined movements is in line with recent experimental results [38]. Furthermore, it is also in line with the emulation theory [39], which states that body representations (represented in the ‘emulator’) play a crucial role in MI processes and are constructed from former experience of afferent sensations [40]. Consequently, by providing experimental evidence, our results give further rise to the notion that MI is a centrally generated, offline process [41]. Figure 7. Difference scores for postural and biomechanical influences in Experiment 2. Differences in reaction times between the ‘Incongruent’ and ‘Congruent’ postural conditions (A) and between lateral and medial rotations for IW, CL and CR (B). Error bars represent 95% CI. * denotes significance at the p,0.05 level, ** denotes significant at the p,0.01 level and # denotes that the mean score of IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g007 correspondence with the findings of Experiment 1, we found that IW was significantly more accurate than the CR and CL group and that the significant DOR effects for both control groups differed significantly from IW, see Figure 7b. For the postural manipulations we found that IW’s lack of postural influence differed significantly from the significant posture effect for the CR group, irrespective of the visual feedback. Consequently, these findings confirm that kinaesthetic feedback plays an important role in the emergence of a postural conflict irrespective of the visual feedback on the effectors involved. Furthermore, the ability to visually observe one’s own hand apparently does not evoke the use of a visually constructed representation of the hand by IW in a mental rotation task of hands. This does not imply that IW is unable to construct central representations of the body’s current position. IW is able to perform accurate movements by visualizing his movement from a first person perspective prior to the overt movement [16,42]. His ability to construct a representation of the body with only visual information is in line with the multimodal nature of body representations [13,40]. Consequently, IW is able to construct a ‘sensorimotor’ representation. Effect of Deafferentation on Mental Imagery Figure 7. Difference scores for postural and biomechanic influences in Experiment 2. Differences in reaction times betwe the ‘Incongruent’ and ‘Congruent’ postural conditions (A) and betwe lateral and medial rotations for IW, CL and CR (B). Error bars represe 95% CI. * denotes significance at the p,0.05 level, ** deno significant at the p,0.01 level and # denotes that the mean score IW falls outside the 95% CI of the control group. doi:10.1371/journal.pone.0042742.g007 IW’s MI to be impaired and that his VI might be enhanced compared to controls. In both experiments, we found that IW’s performance was not influenced by postural manipulations, irrespective of the ability to see his own hands. For the controls, we found that the CR group was influenced by the postural manipulations but not the CL group. This latter was expected and is considered to be related to differing internal representations of the hands between left- and right handed people [30,31]. Because both the CL group and IW show a lack of postural influence, one might argue that this is attributable to handedness alone, because IW is left handed. However, the influence of biomechanical constraints (as reflected in the DOR-effect) differed significantly between IW and both control groups in both experiments, see Figure 4 and 7. Consequently, the lack of embodied influences for IW cannot be solely attributed to handedness alone and is therefore likely to result from the lack of kinaesthetic afferents for IW. Collectively, our results show that the long-term loss of kinaesthetic afferents results in an inability to implicitly incorpo- rate kinaesthetic information into one’s centrally generated body representations. Clearly, due to deafferentation there is no direct kinaesthetic feedback to incorporate into a body representation. Interestingly it is likely that in addition, the long-term loss of kinaesthetic afferents also results in an inability to recall these sensations from memory in order to incorporate them into the body representation. Memory has been shown to play a role during MI [32]. The role of memory in MI processes is also evident from the sustained influence of biomechanical constraints during transient anaesthesia of the arm in a MI task [33]. Furthermore, it has been shown that even during transient peripheral deafferentation acute plastic changes occur in the brain [34,35], also leading to alterations in the central representation of the body in the brain [36]. Effect of Deafferentation on Mental Imagery However, this representation is phenomenologically different from the motor representations of controls [18,19] and is likely to be primarily based on visual perception. As MI consists of a mental transformation of visual and kinaesthetic percept’s [20], it is important to show that the observed lack of postural and biomechanical influences for IW results from affected central motor processes and not an affected ability to create and transform mental images. The results of the letter task show that IW is perfectly able to perform mental transformations of mental images. This is evidenced by the similar performance in reaction times and enhanced accuracy compared to controls. Therefore, it is likely that the observed lack of postural Discussion In this experiment, we were interested in the influence of visual feedback on the effect of postural manipulations for IW. In Figure 6. Reaction times as function of rotational angle in Experiment 2. Reaction time data from Experiment 1 as function of angle for hand stimuli. Error bars represent standard error of the mean. doi:10.1371/journal.pone.0042742.g006 Figure 6. Reaction times as function of rotational angle in Experiment 2. Reaction time data from Experiment 1 as function of angle for hand stimuli. Error bars represent standard error of the mean. doi:10.1371/journal.pone.0042742.g006 For the accuracy data we obtained a significant effect of Angle (F(3,84) = 26.492; p,0.001; g2 = 0.486). No further effects were found significant. In the comparison of the accuracy between the August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org 5 Effect of Deafferentation on Mental Imagery Effect of Deafferentation on Mental Imagery and biomechanical influences for IW results from affected central motor processes due to the chronic deafferentation. movements beforehand and uses visual imagery frequently and widely to maintain performance. In conclusion, this study provides new insights in the debate on the influence of afferent information in MI processes. In contrast to former studies on the influence of (congenital) amputations on MI processes, we selectively looked at the influence of afferent information on these processes. We found that kinaesthetic afferents play an important role in the conflict between imagined movement and the body’s current posture. The body’s current posture and biomechanical constraints are likely to be incorpo- rated in a structural body description and processed centrally during imagery. The long term loss of kinaesthetic afferents results in the loss of central kinaesthetic representations and hence impaired motor imagery. In order to compensate for this deficit, IW uses a visual construct of his body, together with online visual supervision to plan and control imagined motor acts. This extraordinary faculty developed over years for planning and indeed predicting movement is likely to explain IW’s enhanced visual imagery capabilities [25]. In addition, as IW controls movement with mental attention and close visual supervision, it is also likely that he developed visual mental images of his own body as well. Consequently, his mental transformation skills of visual images is not likely to be limited to letters only. Indeed, we found that IW outperformed both control groups on the accuracy level. with remarkably high accuracy levels between 95% and 100% for all angles. Because IW is unable to mentally simulate the kinaesthetic consequences of a movement, it is likely that IW used VI to solve the mental rotation tasks, irrespective of the used types of stimuli. Our results show that these VI abilities of IW are enhanced with respect to controls. This high accuracy may be a result of two processes. First, he uses visual imagery of movement in everyday life and used it in his rehabilitation and secondly he seems to have a high level of focused attention. It has already been shown that participants with higher focused attention scores have an increased performance in mental rotation tasks than participants with a lower focused attention score [43]. In daily life, IW has to continuously update his visual percept of the world and translate that knowledge into a motor plan. References 1. 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Tsakiris M, Haggard P (2005) The rubber hand illusion revisited: visuotactile integration and self-attribution. J Exp Psychol Hum Percept Perform 31: 80–91. 15. de Vignemont F, Ehrsson HH, Haggard P (2005) Bodily illusions modulate tactile perception. Curr Biol 15: 1286–1290. 34. Gandevia SC, Phegan CM (1999) Perceptual distortions of the human body image produced by local anaesthesia, pain and cutaneous stimulation. J Physiol 514 (Pt 2): 609–616. 16. Cole C, Paillard J (1998) Living without Touch and Peripheral Information about Body Position and Movement: Studies with Deafferented Subjects. In: Bermu´dez JL, Marcel A, Eilan N, editors. The Body and the Self: MIT Press. ( ) 35. Ziemann U, Corwell B, Cohen LG (1998) Modulation of plasticity in human motor cortex after forearm ischemic nerve block. J Neurosci 18: 1115–1123. y 17. Cole JD, Sedgwick EM (1992) The perceptions of force and of movement in a man without large myelinated sensory afferents below the neck. J Physiol 449: 503–515. 36. Martucci KT, Coghill RC (2011) Regional anesthesia: functional implications beyond the anesthetized nerve. Anesthesiology 114: 21–23. 37. Olsson CJ, Nyberg L (2010) Motor imagery: if you can’t do it, you won’t think it. Effect of Deafferentation on Mental Imagery Diminished attention or errors in the mental transformations are likely to result in improper movements and hence the risk of falling or not being able to grasp an object, for example. He is quite clear that he mentally rehearses Author Contributions Conceived and designed the experiments: ACTH JC RVL BS. Performed the experiments: ACTH JC. Analyzed the data: ACTH. Contributed reagents/materials/analysis tools: ACTH. Wrote the paper: ACTH JC RVL BS. Conceived and designed the experiments: ACTH JC RVL BS. Performed the experiments: ACTH JC. Analyzed the data: ACTH. Contributed reagents/materials/analysis tools: ACTH. Wrote the paper: ACTH JC RVL BS. General Discussion In the current case study we investigated the influence of deafferentation on imagery capabilities. We specifically looked at the role of kinaesthetic afferents and how the imagery processes are affected due to long term loss of afferent input. We expected August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org August 2012 \| Volume 7 \| Issue 8 \| e42742 6 Effect of Deafferentation on Mental Imagery 1. Guillot A, Collet C (2010) The neurophysiological foundations of mental and motor imagery. Oxford: Oxford University Press. 2. Decety J (1996) The neurophysiological basis of motor imagery. Behav Brain Res 77: 45–52. 43. Karadi K, Kallai J, Kovacs B (2001) Cognitive subprocesses of mental rotation: why is a good rotator better than a poor one? Percept Mot Skills 93: 333–337. 41. Jeannerod M (2001) Neural simulation of action: a unifying mechanism for motor cognition. Neuroimage 14: S103–109. 42. Miall RC, Haggard PN, Cole JD (1995) Evidence of a limited visuo-motor memory used in programming wrist movements. Exp Brain Res 107: 267–280. 42. Miall RC, Haggard PN, Cole JD (1995) Evidence of a limited visuo-motor memory used in programming wrist movements. Exp Brain Res 107: 267–280. 41. Jeannerod M (2001) Neural simulation of action: a unifying mechanism for motor cognition. Neuroimage 14: S103–109. References Scand J Med Sci Sports 20: 711–725. 18. Bosbach S, Cole J, Prinz W, Knoblich G (2005) Inferring another’s expectation from action: the role of peripheral sensation. Nat Neurosci 8: 1295–1297. 38. Vargas CD, Olivier E, Craighero L, Fadiga L, Duhamel JR, et al. (2004) The influence of hand posture on corticospinal excitability during motor imagery: a transcranial magnetic stimulation study. Cereb Cortex 14: 1200–1206. 19. Bosbach S, Knoblich G, Reed CL, Cole J, Prinz W (2006) Body inversion effect without body sense: insights from deafferentation. Neuropsychologia 44: 2950– 2958. 39. Grush R (2004) The emulation theory of representation: motor control, imagery, and perception. Behav Brain Sci 27: 377–396; discussion 396–442. 20. de Lange FP, Hagoort P, Toni I (2005) Neural topography and content of movement representations. J Cogn Neurosci 17: 97–112. 40. Maravita A, Spence C, Driver J (2003) Multisensory integration and the body schema: close to hand and within reach. Curr Biol 13: R531–539. 21. de Lange FP, Roelofs K, Toni I (2008) Motor imagery: a window into the mechanisms and alterations of the motor system. Cortex 44: 494–506. 7 August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org August 2012 \| Volume 7 \| Issue 8 \| e42742 Effect of Deafferentation on Mental Imagery Effect of Deafferentation on Mental Imagery Effect of Deafferentation on Mental Imagery August 2012 \| Volume 7 \| Issue 8 \| e42742 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8
https://openalex.org/W4302529897	https://zenodo.org/records/495753/files/45.pdf	English	null	A SURVEY ON WAVELET TRANSFORM TECHNIQUES	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	2,007	Abstract: Computational tool for a variety of signal and image processing applications do exist and the wavelet transform has become a useful tool among them. Smaller files are important for storing images using less memory and for transmitting images faster and more reliably and hence the wavelet transform is useful for the compression of digital image files. Compressing digitally scanned fingerprint images is done by the FBI using wavelet transforms. For compressing images acquired by their 18 cameras, NASA‟s Mars Rovers used wavelet transforms. The special requirements of deep-space communication to meet the wavelet-based algorithm implemented in software onboard the Mars Rovers is designed. Based on wavelet transforms JPEG2K (the newer JPEG image file format) compression technique is designed. „Cleaning‟ signals and „Images‟ are also done using Wavelet transforms which are also useful for reducing unwanted noise and blurring. Based on wavelet and wavelet-like transforms, some algorithms for processing astronomical images, for example do exist. Key Words: Wavelet Transform Image Processing Image Compression & Pipelining The wavelet transform, and how it is effective for noise reduction is illustrated by this Quick Study and it briefly describes several improvements of the basic wavelet transform and basic noise reduction method used in the illustration. What the wavelet transforms is, and the algorithms for processing a signal after its wavelet transform has been computed are described. Wavelet transform consists of two basic types of transformations. Easily reversible (invertible) is the nature of the one type of wavelet transform designed. It is the transform in which the original signal can be easily recovered after it has been transformed. Image compression and cleaning (noise and blur reduction) put these kinds of transforms into utilization. Typically the wavelet representation is then modified appropriately after computation of the wavelet transform and then the wavelet transform is reversed (inverted) to obtain a new image. Signal analysis is the use of the second type of the wavelet transform. The transform is used to detect faults in machinery from sensor measurements, to study EEG or other biomedical signals and to determine how the frequency content of a signal evolves over time. Modified form of the original signal is not needed in these cases and the wavelet transform need not be inverted. The inversion of the wavelet transform is possible but it requires a lot of computation time in comparison with the first type of wavelet transform. Abstract: Those wavelet transforms that are easily invertible will be the focus of this short survey. International Journal of Advanced Trends in Engineering and Technology (IJATET) Impact Factor: 5.665, ISSN (Online): 2456 - 4664 (www.dvpublication.com) Volume 2, Issue 1, 2017 A SURVEY ON WAVELET TRANSFORM TECHNIQUES J. Vinoth Kumar* & Dr. C. Kumar Charlie Paul** * St. Peter‟s University, Chennai Tamilnadu ** S.S.M College of Engineering, Bhavani, Tamilnadu Cite This Article: J. Vinoth Kumar & Dr. C. Kumar Charlie Paul, “A Survey on Wavelet Transform Techniques”, International Journal of Advanced Trends in Engineering and Technology, Page Number 72-73, Volume 2, Issue 1, 2017. International Journal of Advanced Trends in Engineering and Technology (IJATET) Impact Factor: 5.665, ISSN (Online): 2456 - 4664 (www.dvpublication.com) Volume 2, Issue 1, 2017 A SURVEY ON WAVELET TRANSFORM TECHNIQUES J. Vinoth Kumar* & Dr. C. Kumar Charlie Paul** * St. Peter‟s University, Chennai Tamilnadu ** S.S.M College of Engineering, Bhavani, Tamilnadu Cite This Article: J. Vinoth Kumar & Dr. C. Kumar Charlie Paul, “A Survey on Wavelet Transform Techniques”, International Journal of Advanced Trends in Engineering and Technology, Page Number 72-73, Volume 2, Issue 1, 2017. International Journal of Advanced Trends in Engineering and Technology (IJATET) Impact Factor: 5.665, ISSN (Online): 2456 - 4664 (www.dvpublication.com) Volume 2, Issue 1, 2017 A SURVEY ON WAVELET TRANSFORM TECHNIQUES J. Vinoth Kumar* & Dr. C. Kumar Charlie Paul** * St. Peter‟s University, Chennai Tamilnadu ** S.S.M College of Engineering, Bhavani, Tamilnadu 4. References: 1. W. Sweldens, "The lifting scheme: A custom-design construction of biorthogonal wavelets," Appl. Comput. Harmon. Anal., Vol. 3, no. 2, pp. 186- 200, Apr. 1996. 2. Daubechies and W. Sweldens, "Factoring wavelet transforms into lifting steps," J. Fourier Anal. Appl. Vol. 4, no. 3, pp. 247-269, 1998. 3. B. K. Mohanty and P. K. Meher, "Memory-efficient modular VLSI architecture for high-throughput and low-latency implementation of multilevel lifting 2-D DWT," iEEE Trans. Signal Process., vol. 59,no. 5,pp. 2072-2084, May 2011. 3. B. K. Mohanty and P. K. Meher, "Memory-efficient modular VLSI architecture for high-throughput and low-latency implementation of multilevel lifting 2-D DWT," iEEE Trans. Signal Process., vol. 59,no. 5,pp. 2072-2084, May 2011. 4. B. K. Mohanty, A. Mahajan, and P. K. Meher, "Area and power efficient architecture for high- throughput implementation of lifting 2-D DWT,"JEEE Trans. Circuits Syst. ii, Exp. Briefs, vol. 59, no. 7, pp. 434--438, July 2012. 4. B. K. Mohanty, A. Mahajan, and P. K. Meher, "Area and power efficient architecture for high- throughput implementation of lifting 2-D DWT,"JEEE Trans. Circuits Syst. ii, Exp. Briefs, vol. 59, no. 7, pp. 434--438, July 2012. pp y 5. C. Cheng and K. K. Parhi,"High-speed VLSI implementation of 2- D discrete wavelet transform," iEEE Trans. Signal Process., vol. 56,no. I,pp. 393-403,2008. 5. C. Cheng and K. K. Parhi,"High-speed VLSI implementation of 2- D discrete wavelet transform," iEEE Trans. Signal Process., vol. 56,no. I,pp. 393-403,2008. 6. Chiu, K.-B. Lee, and c.-W. Jen, “Optimal data transfer and buffering schemes for JPEG2000 encoder," in Proc. Signal Process. Syst., pp. 177-182, 2003. 6. Chiu, K.-B. Lee, and c.-W. Jen, “Optimal data transfer and buffering schemes for JPEG2000 encoder," in Proc. Signal Process. Syst., pp. 177-182, 2003. 7. C.-T. Huang, P.-C.Tseng, and L.-G. Chen,"Flipping structure: An efficient VLSI architecture for lifting-based discrete wavelet transform," IEEE Trans. Signal Process., vol. 52, no. 4, pp. 1080-1089, Apr. 2004. 7. C.-T. Huang, P.-C.Tseng, and L.-G. Chen,"Flipping structure: An efficient VLSI architecture for lifting-based discrete wavelet transform," IEEE Trans. Signal Process., vol. 52, no. 4, pp. 1080-1089, Apr. 2004. p 8. N. U. Bhanu and A. Chilambuchelvan,"High-speed systolic VLST architecture for 2-D forward lifting- based DWT ", Arab J Sci Eng 39, pp. 6125-6135, June 2014. 9. Y. Hu and C. C. Jong, "A memory-efficient scalable architecture for lifting-based discrete wavelet transform," IEEE Trans. Circuits Syst. II, Exp. Briefs, vol. 60, no. 8,pp. 502-506, August 2013. 10. W. Zhang, Z. International Journal of Advanced Trends in Engineering and Technology (IJATET) Impact Factor: 5.665, ISSN (Online): 2456 - 4664 (www.dvpublication.com) Volume 2, Issue 1, 2017 strip based scanning method for the first time is proposed by Chiu et al. [6]. Huang et al. [7] was the first to propose the Flipping method. The architecture to map last component with first component of same systolic array for column processing in a symmetric and cyclic manner was modified by Bhanu et al. [8]. Several parallel units in both row processor and column processor, achieving path delay of Ta + Tm with smaller size of the temporal memory of 3N has been implemented by Hu et at. [9]. The pipelined architecture with flipping method resulting in path delay of Tm only with the smaller transposition buffer is proposed by Zhang et at. [10]. The path delay of Tm only and smallest temporal memory of 2N with pipelined architecture is achieved by Darji et al. [11]. strip based scanning method for the first time is proposed by Chiu et al. [6]. Huang et al. [7] was the first to propose the Flipping method. The architecture to map last component with first component of same systolic array for column processing in a symmetric and cyclic manner was modified by Bhanu et al. [8]. Several parallel units in both row processor and column processor, achieving path delay of Ta + Tm with smaller size of the temporal memory of 3N has been implemented by Hu et at. [9]. The pipelined architecture with flipping method resulting in path delay of Tm only with the smaller transposition buffer is proposed by Zhang et at. [10]. The path delay of Tm only and smallest temporal memory of 2N with pipelined architecture is achieved by Darji et al. [11]. 3. Future Enhancement and Conclusion: 2-D lifting-based DWT that are recently implemented with existing memory efficient architectures are studied compared and analyzed for arithmetic resources utilized, on chip memory consumed and computation time required for processing with different delays. Parallel scan, block-based scan and strip scan with parallel computation, flipping and pipelining techniques are some of the architectures that are presented. Study and implementation of multi-level decomposition of images for real time applications will be the purpose of the survey. 4. References: 2. Literature Review: The architecture to map last component with first component of same systolic array for column processing in a symmetric and cyclic manner was modified by Bhanu et al. [8]. Several parallel units in both row processor and column processor, achieving path delay of Ta + Tm with smaller size of the temporal memory of 3N has been implemented by Hu et at. [9]. The pipelined architecture with flipping method resulting in path delay of Tm only with the smaller transposition buffer is proposed by Zhang et at. [10]. The path delay of Tm only and smallest temporal memory of 2N with pipelined architecture is achieved by Darji et al. [11]. 3 C i International Journal of Advanced Trends in Engineering and Technology (IJATET) Impact Factor: 5.665, ISSN (Online): 2456 - 4664 (www.dvpublication.com) Volume 2, Issue 1, 2017 2. Literature Review: The most promising tool for various applications such as image compression, bioinformatics, pattern recognition and video processing and many more) is the discrete wavelet transforms (DWT). Better image quality, coding efficiency, noise immunity are the suitable features of DWT making it more efficient as compared to Discrete Cosine Transform. VLSI implementation of DWT is done using convolution-based scheme and lifting-based scheme. Simplified computation, parallel and fast implementation, in place computation, integer to integer mapping, low memory requirement [1], [2], are the powerful features of lifting- based scheme which has more advantages compared to convolution-based scheme. Longer and critical delays for direct mapped architecture are the disadvantage of the lifting based scheme. Additionally it also needs extra memory for rearranging interleaved intermediate outputs, hence taking a toll on its hardware efficiency. The key points determining the efficiency of the architecture are the memory requirement and critical path delay. Intermediate results for column processing as soon as possible, with a transposition buffer of size 2.5N are proposed by Mohanty et al [3]. The architecture with parallel processed systolic arrays to make the intermediate results available immediately. Memory is divided into two categories. Temporal memory is used to store partial results and transposition memory is used to store intermediate outputs. The outputs of the previous step are used as inputs for the current step except the first step in the lifting based scheme. Addition and multiplication operation are the constituent of each lifting step. Critical path delay is contributed by the adder delay Ta and multiplication delay Tm. Temporal memory of large size is required to reduce the path delay. Increased size of transposition memory is the result if pipelining is used to reduce path delay. Architectures have been developed to achieve balance between path delay and memory requirement in recent years. To eliminate data transposition step between row and column processing Mohanty et al. [4] proposed architecture resulting into the small size of transposition memory. The block based scanning method is introduced by Cheng and Parhi et al. [5]. The 72 International Journal of Advanced Trends in Engineering and Technology (IJATET) Impact Factor: 5.665, ISSN (Online): 2456 - 4664 (www.dvpublication.com) Volume 2, Issue 1, 2017 strip based scanning method for the first time is proposed by Chiu et al. [6]. Huang et al. [7] was the first to propose the Flipping method. 4. References: Jiang, Z. Gao, and Y. Liu, "An efficient VLST architecture for lifting-based discrete wavelet transform," IEEE Trans. Circuits Syst. II, vol. 59, pp. 158-162, March 2012. 1. D. Darji and A. Limaye, "Memory efficient VLSI architecture for lifting-based DWT", 57th IEE International Midwest Symposium on Circuits and Systems, pp. 189-192, 2014. 73
https://openalex.org/W4360612638	https://www.nature.com/articles/s41598-023-30714-9.pdf	Latin	null	ABO blood types and SARS-CoV-2 infection assessed using seroprevalence data in a large population-based sample: the SAPRIS-SERO multi-cohort study	Scientific reports	2,023	cc-by	6,269	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports ABO blood types and SARS‑CoV‑2 infection assessed using seroprevalence data in a large population‑based sample: the SAPRIS‑SERO multi‑cohort study OPEN Mélanie Deschasaux‑Tanguy 1, Fabien Szabo de Edelenyi 1, Nathalie Druesne‑Pecollo 1, Younes Esseddik 1, Julien Allègre 1, Bernard Srour 1, Pilar Galan 1, Serge Hercberg 1, Gianluca Severi 2,3, Marie Zins 4, Emmanuel Wiernik 4, SAPRIS-SERO study group , Xavier de Lamballerie 5, Fabrice Carrat 6,7 & Mathilde Touvier 1 ABO blood type has been reported as a potential factor influencing SARS-CoV-2 infection, but so far mostly in studies that involved small samples, selected population and/or used PCR test results. In contrast our study aimed to assess the association between ABO blood types and SARS-CoV-2 infection using seroprevalence data (independent of whether or not individuals had symptoms or sought for testing) in a large population-based sample. Our study included 67,340 French participants to the SAPRIS-SERO multi-cohort project. Anti-SARS-CoV-2 antibodies were detected using ELISA (targeting the proteins spike (S) and nucleocapsid (NP)) and seroneutralisation (SN) tests on dried blood spots collected in May–November 2020. Non-O individuals (and especially types A and AB) were more likely to bear anti SARS-CoV-2 antibodies (ELISA-S, 2964 positive cases: ORnon-Ovs.O = 1.09[1.01– 1.17], ORAvs.O = 1.08[1.00–1.17]; ELISA-S/ELISA-NP/SN, 678 triple positive cases: ORnon-Ovs.O = 1.19 [1.02–1.39], ORAvs.O = 1.19[1.01–1.41], ORABvs.O = 1.43[1.01–2.03]). Hence, our results provided additional insights into the dynamic of SARS-CoV-2 infection, highlighting a higher susceptibility of infection for individuals of blood types A and AB and a lesser risk for blood type O. Since 2020, the world has been struggling with the COVID-19 (Coronavirus Disease 2019) pandemic caused by the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). The research community has mobilized to identify potential risk factors associated with SARS-CoV-2 infection and COVID-19 severity, in an effort to better understand the dynamic of infection, pinpoint individuals at higher risk and help prevent the disease and related adverse outcomes. Early reports comparing the distribution of ABO blood types between individuals infected or not by the SARS-CoV-2, suggested that ABO blood types could be related to the risk of SARS-CoV-2 infection1–4, as previously observed for SARS-CoV5 but also for other infectious diseases6,7. These studies and others published since then8–11 suggested that individuals with blood type O had a lower risk of SARS-CoV-2 1Inserm U1153, Inrae U1125, Cnam, Nutritional Epidemiology Research Team (EREN), Epidemiology and Statistics Research Center – Université Paris Cité (CRESS), Sorbonne Paris Nord University, Bobigny, France. 2UVSQ, Inserm, Gustave Roussy, “Exposome and Heredity” team, CESP UMR1018, Paris-Saclay University, Villejuif, France. ABO blood types and SARS‑CoV‑2 infection assessed using seroprevalence data in a large population‑based sample: the SAPRIS‑SERO multi‑cohort study OPEN 3Department of Statistics, Computer Science and Applications “G. Parenti”, University of Florence, Florence, Italy. 4Population‑based Cohorts Unit, INSERM, UMS 011, Paris Saclay University, Université de Versailles Saint-Quentin-en-Yvelines, Université Paris Cité, Paris, France. 5Unité des Virus Emergents (UVE), IRD 190, INSERM 1207, Aix Marseille Univ, Marseille, France. 6Inserm, Institut Pierre‑Louis d’Epidémiologie et de Santé Publique, Sorbonne Université, Paris, France. 7Département de Santé Publique, APHP, Sorbonne Université, Paris, France. A list of authors and their affiliations appears at the end of the paper. email: m.deschasaux@ eren.smbh.univ-paris13.fr 1Inserm U1153, Inrae U1125, Cnam, Nutritional Epidemiology Research Team (EREN), Epidemiology and Statistics Research Center – Université Paris Cité (CRESS), Sorbonne Paris Nord University, Bobigny, France. 2UVSQ, Inserm, Gustave Roussy, “Exposome and Heredity” team, CESP UMR1018, Paris-Saclay University, Villejuif, France. 3Department of Statistics, Computer Science and Applications “G. Parenti”, University of Florence, Florence, Italy. 4Population‑based Cohorts Unit, INSERM, UMS 011, Paris Saclay University, Université de Versailles Saint-Quentin-en-Yvelines, Université Paris Cité, Paris, France. 5Unité des Virus Emergents (UVE), IRD 190, INSERM 1207, Aix Marseille Univ, Marseille, France. 6Inserm, Institut Pierre‑Louis d’Epidémiologie et de Santé Publique, Sorbonne Université, Paris, France. 7Département de Santé Publique, APHP, Sorbonne Université, Paris, France. A list of authors and their affiliations appears at the end of the paper. email: m.deschasaux@ eren.smbh.univ-paris13.fr \| https://doi.org/10.1038/s41598-023-30714-9 Methodsh The SAPRIS (“SAnté, Perception, pratiques, Relations et Inégalités Sociales en population générale pendant la crise COVID-19”) study was set up in April 2020 to investigate various aspects of the COVID-19 crisis (COVID-19 infection/diagnosis and experience of lockdown), based on a consortium of existing French pro- spective cohort studies. In May 2020, participants from the Constances, E3N-E4N and NutriNet-Santé cohort studies answering SAPRIS questionnaires were also invited to take part in the SAPRIS-SERO project which aimed to estimate the seroprevalence of antibodies against SARS-CoV-2 at the population level, as previously described12,13. Briefly, between May and November 2020 (during or at the end of the first wave, pre-vaccine period), participants received self-sampling kits by mail to collect dried blood spots from which were detected anti-SARS-CoV-2 antibodies (IgG) directed against the S1 domain of the spike protein (S) and the nucleocapsid protein (NP) using ELISA (Euroimmun®, Lübeck, Germany), as well as neutralizing anti-SARS-CoV-2 antibodies (SN) using an in-house micro-neutralization assay. ELISA-S and ELISA-NP tests were considered to be posi- tive with an optical density ratio ≥ 1.1, indeterminate between 0.8 and 1.1, and negative < 0.8. The SN test was considered to be positive with a titre ≥ 40. ELISA-NP and SN tests were performed whenever the optical density ratio for ELISA-S was ≥ 0.7. Positivity (vs. negativity) to ELISA-S test and positivity (vs. negativity) to all three ELISA-S, ELISA-NP and SN tests (associated to more symptomatic COVID-19 in a previous study in SAPRIS- SERO13) were considered as outcomes. Characteristics of the participants including ABO and RhD blood types were collected from questionnaires as part of each cohort follow-up or from the shared SAPRIS questionnaire. Associations between the seroprevalence of antibodies against SARS-CoV-2 and blood types were studied using multi-adjusted logistic regression models stratified by cohort and including the following covariates: sex (men/ women), age (≥ 18– < 40, 40–49, 50–59, 60–69, ≥ 70 years old), month of blood collection, residential area during the lockdown: city size (rural, < 20,000, 20–100,000, ≥ 100,000 inhabitants) and French administrative region. Additional covariates linked to the risk of SARS-CoV-2 infection were included in sensitivity analyses: weight status, smoking status, educational level, professional activity during lockdown, socio-professional category, number of individuals at home during lockdown and alcohol intake. All tests were two-sided and P < 0.05 was considered statistically significant. Analyses were carried out using SAS 9.4 (SAS Institute Inc., USA). Ethics approval and informed consent. www.nature.com/scientificreports/ infection and that type A individuals would be more at risk, with yet some remaining inconsistencies, especially for B or AB types for which results are less robust. However, these results were obtained in selected populations (e.g., patients, blood donors) of various sample size (mostly under 15,000) and mostly using PCR test results (e.g., from questionnaires or registries) hence reflecting a population that sought for testing, with the exception of a recent report using seroprevalence data in ≈32,600 blood donors11. In contrast, this short report provides insights into the associations between ABO blood types and SARS-CoV-2 infection assessed from seroprevalence data obtained between May and November 2020 through the systematic screening for anti-SARS-CoV-2 antibodies in a large population-based sample of French adults. Methodsh The SAPRIS-SERO study (registered #NCT04392388) was con- ducted in accordance with the relevant guidelines and regulations and was approved by the ethics committee CPP Sud-Méditerranée III on April 27th 2020 and by the CNIL #920,193. All participants provided written or electronic informed consent for participation in each cohort and a specific electronic informed consent for par- ticipation in the SAPRIS-SERO study. Results d Our study included 67,340 participants, among which 2964 with a positive seroprevalence of anti SARS-CoV-2 antibodies according to the ELISA-S test and 64,376 participants who tested negative (respectively 678 triple positive and 64,011 triple negative to ELISA-S, ELISA-NP and SN tests). Characteristics of participants are shown in Table 1. Higher odds of presenting anti SARS-CoV-2 antibodies (Table 2) were observed in participants belonging to non-O blood types (ELISA-S: OR non-O vs. O = 1.09 [1.01–1.17], ELISA-S/ELISA-NP/SN: OR non-O vs. O = 1.19 [1.02–1.39]) and especially to blood type A (ELISA-S: ORA vs. O = 1.08 [1.00–1.17], ELISA-S/ELISA-NP/SN: OR A vs. O = 1.19 [1.01–1.41]) and AB (ELISA-S/ELISA-NP/SN: OR AB vs. O = 1.43 [1.01–2.03]). In analyses combin- ing ABO and RhD blood types, higher odds were observed in participants with blood types A- (ELISA-S: OR A- vs. O+ = 1.16 [1.00–1.35]), A + (ELISA-S/ELISA-NP/SN: OR A+ vs. O+ = 1.24 [1.03–1.48]) and AB + (which rep- resented the majority of AB participants, ELISA-S: OR AB+ vs. O+ = 1.24 [1.02–1.51], ELISA-S/ELISA-NP/SN: OR AB+ vs. O+ = 1.60 [1.11–2.30]). No association was observed comparing RhD positive and negative blood types overall. Additional adjustments in sensitivity analyses did not change the results of increased odds of positive seroprevalence in non-O participants (ELISA-S: OR non-O vs. O = 1.09 [1.01–1.18], ELISA-S/ELISA-NP/SN: OR non-O vs. O = 1.19 [1.02–1.39]). \| https://doi.org/10.1038/s41598-023-30714-9 \| https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 www.nature.com/scientificreports/ Discussion In this study, we used seroprevalence data obtained from screening a large population-based sample from 3 French prospective cohorts to assess the associations between ABO blood types and the risk of SARS-CoV-2 infection. Our results showed a decreased seroprevalence of anti-SARS-CoV-2 antibodies in type O individuals compared to all others. Among non-O blood types, types A and AB were more likely positive for SARS-CoV-2 infection. No association was observed with type B or RhD types.hi yp yp These results confirmed prior observations obtained in other population settings, namely a decrease risk of infection for type O individuals and an increase risk for type A individuals, with some indication of increased https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 \| www.nature.com/scientificreports/ Table 1. Characteristics of the population, SAPRIS-SERO multi-cohort study, 2020. a Missing va (2.4%) for weight status, N = 2606 (3.9%) for smoking status, N = 5275 (7.8%) for educational lev (1.1%) for city size and N = 7 (0.01%) for French administrative region. Table 1. Characteristics of the population, SAPRIS-SERO multi-cohort study, 2020. a Missing values: N = 1602 (2.4%) for weight status, N = 2606 (3.9%) for smoking status, N = 5275 (7.8%) for educational level, N = 766 (1.1%) for city size and N = 7 (0.01%) for French administrative region. Table 1. Characteristics of the population, SAPRIS-SERO multi-cohort study, 2020. a Missing values: N = 16 (2.4%) for weight status, N = 2606 (3.9%) for smoking status, N = 5275 (7.8%) for educational level, N = 766 (1 1%) for city size and N=7 (0 01%) for French administrative region ) g , ( ) g , ( ) , 1.1%) for city size and N = 7 (0.01%) for French administrative region. Table 1. Characteristics of the population, SAPRIS-SERO multi-cohort study, 2020. a Missing values: N = 16 ( %) f h N ( %) f k N ( %) f d l l l N Discussion This allowed us to test for SARS-CoV-2 infection in a single general population, regard- less whether the participants sought testing or had symptoms, allowing detecting a large panel of infected cases. This differential susceptibility to SARS-CoV-2 infection according to blood types could be due to the incorporation of ABH antigens on the envelop of SARS-CoV-2 virions, leading to a likelihood of infection from one individual to another following ABO incompatibility9,10. As type O individuals carry both anti-A and anti-B antibodies, they would be less susceptible to primary infection by SARS-CoV-2 viruses enveloped with type A or B-like patterns such as those excreted by non-O individuals. In contrast, type A individuals, carrying anti-B antibodies, would be susceptible to infection by viruses excreted by type O and type A individuals (i.e., 42% and 44% of the French population respectively), while type AB individuals carrying neither anti-A or anti-B antibodies would be susceptible to infection by individuals of all group types, which is why type A or AB are particularly at risk for SARS-CoV-2 infection. Type B individuals carrying anti-A antibodies are susceptible to infection by viruses excreted by type O and type B individuals but type B is less represented in the French population (10%) which may explain why no significant association was observed for these individuals. Another hypothesis, yet less supported by available data relates to the entry of SARS-CoV-2 in host’s cells, that would be facilitated by antigen A9,14. Finally, non-O individuals are at higher risk of thromboembolism, a common complication of COVID-19, hence beyond infection, non-O individuals could also be at higher risk of severe cases of COVID-199,10. p p g g y p g g g p This differential susceptibility to SARS-CoV-2 infection according to blood types could be due to the incorporation of ABH antigens on the envelop of SARS-CoV-2 virions, leading to a likelihood of infection from one individual to another following ABO incompatibility9,10. As type O individuals carry both anti-A and anti-B antibodies, they would be less susceptible to primary infection by SARS-CoV-2 viruses enveloped with type A or B-like patterns such as those excreted by non-O individuals. Discussion ELISA-S negative (N = 64,376) ELISA-S positive (N = 2,964) P N % N % Gender < 0.001 Male 21,174 (32.9) 832 (28.1) Female 43,202 (67.1) 2132 (71.9) Age (years) < 0.001 ≥ 18– < 40 6983 (10.8) 652 (22.0) 40–49 10,340 (16.1) 988 (33.3) 50–59 11,222 (17.4) 505 (17.0) 60–69 13,224 (20.5) 334 (11.3) ≥ 70 22,607 (35.1) 485 (16.4) Weight statusa 0.006 Underweight 1992 (3.1) 110 (3.7) Normal weight 37,189 (57.8) 1749 (59.0) Overweight 17,763 (27.6) 745 (25.1) Obesity 5917 (9.2) 273 (9.2) Smoking statusa < 0.001 Non smoker 29,586 (46.0) 1605 (54.1) Smoker 5958 (9.3) 237 (8.0) Ex-smoker 26,351 (40.9) 997 (33.6) Educational levela < 0.001 < High school 7262 (11.3) 171 (5.8) High school—undergraduate 28,063 (43.6) 1188 (40.1) Graduate or doctorate 24,074 (37.4) 1307 (44.1) Month of blood collection < .0001 May 8004 (12.4) 663 (22.4) June 427 (0.7) 25 (0.7) July 38,143 (59.3) 1556 (52.5) August 13,120 (20.4) 540 (18.2) September 4607 (7.2) 178 (6.0) October 72 (0.1) 1 (0.0) November 3 (0.0) 1 (0.0) Residential area during the lockdown : city sizea < 0.001 Rural area 14,876 (23.1) 555 (18.7) < 20,000 inhabitants 10,923 (17.0) 441 (14.9) 20–100,000 inhabitants 10,600 (16.5) 508 (17.1) ≥ 100,000 inhabitants 27,230 (42.3) 1441 (48.6) Residential area during the lockdown : French administrative regiona < .0001 Haut de France 4116 (6.4) 179 (6.0) Auvergne- Rhône-Alpes 8316 (12.9) 351 (11.8) Occitanie 6979 (10.9) 188 (6.3) PACA 4306 (6.7) 133 (4.5) Corse 77 (0.1) 3 (0.1) Normandie 2463 (3.8) 87 (2.9) Ile-de-France 10,492 (16.3) 818 (27.6) Grand-Est 4679 (7.3) 389 (13.1) Bretagne 5066 (7.9) 143 (4.8) Pays de la Loire 3952 (6.1) 144 (4.9) Centre-Val-de-Loire 4224 (6.6) 174 (5.9) Bourgogne 2463 (3.8) 111 (3.7) Nouvelle Aquitaine 7228 (11.2) 243 (8.2) DOM-TOM 8 (0.0) 1 (0.0) https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 \| www.nature.com/scientificreports/ Table 2. Associations between ABO and RhD blood types and the seroprevalence of anti-SARS-CoV-2 antibodies, SAPRIS-SERO multi-cohort study, 2020. a OR and 95%CI obtained from a multi-adjusted logistic regression model stratified by cohort and including the following covariates: sex (men/women), age (< 40ans, 40–49, 50–59, 60–69, ≥ 70 years old), month of blood collection, residential area during the lockdown: city size (rural, < 20,000, 20–10,000, ≥ 100,000 inhabitants) and French administrative region. b A participant was considered positive (respectively negative) if positive (respectively negative) to all three ELISA-S, ELISA-NP, and SN tests. Discussion a OR and 95%CI obtained from a multi-adjusted logistic regression model stratified by cohort and including the following covariates: sex (men/women), age (< 40ans, 40–49, 50–59, 60–69, ≥ 70 years old), month of blood collection, residential area during the lockdown: city size (rural, < 20,000, 20–10,000, ≥ 100,000 inhabitants) and French administrative region. b A participant was considered positive (respectively negative) if positive (respectively negative) to all three ELISA-S, ELISA-NP, and SN tests. Among the N = 2964 individuals with a positive seroprevalence of anti SARS Cov-2 antibodies according to the ELISA-S test, N = 1185 were also positive according to the ELISA-NP test and 678 were positive according to all three ELISA-S, ELISA-NP, and SN tests. Table 2. Associations between ABO and RhD blood types and the seroprevalence of anti-SARS-CoV-2 antibodies, SAPRIS-SERO multi-cohort study, 2020. a OR and 95%CI obtained from a multi-adjusted logistic regression model stratified by cohort and including the following covariates: sex (men/women), age (< 40ans, 40–49, 50–59, 60–69, ≥ 70 years old), month of blood collection, residential area during the lockdown: city size (rural, < 20,000, 20–10,000, ≥ 100,000 inhabitants) and French administrative region. b A participant was considered positive (respectively negative) if positive (respectively negative) to all three ELISA-S, ELISA-NP, and SN tests. Among the N = 2964 individuals with a positive seroprevalence of anti SARS Cov-2 antibodies according to the ELISA-S test, N = 1185 were also positive according to the ELISA-NP test and 678 were positive according to all three ELISA-S, ELISA-NP, and SN tests. Table 2. Associations between ABO and RhD blood types and the seroprevalence of anti-SARS-CoV-2 antibodies, SAPRIS-SERO multi-cohort study, 2020. a OR and 95%CI obtained from a multi-adjusted logistic regression model stratified by cohort and including the following covariates: sex (men/women), age (< 40ans, 40–49, 50–59, 60–69, ≥ 70 years old), month of blood collection, residential area during the lockdown: city size (rural, < 20,000, 20–10,000, ≥ 100,000 inhabitants) and French administrative region. b A participant was considered positive (respectively negative) if positive (respectively negative) to all three ELISA-S, ELISA-NP, and SN tests. Among the N = 2964 individuals with a positive seroprevalence of anti SARS Cov-2 antibodies according to the ELISA-S test, N = 1185 were also positive according to the ELISA-NP test and 678 were positive according to all three ELISA-S, ELISA-NP, and SN tests. Discussion Among the N = 2964 individuals with a positive seroprevalence of anti SARS Cov-2 antibodies according to the ELISA-S test, N = 1185 were also positive according to the ELISA-NP test and 678 were positive according to all three ELISA-S, ELISA-NP, and SN tests. Discussion N (%) ELISA-S ELISA-S, ELISA-NP, SN N positive/ negative OR [95%CI]a P N positive/ negativeb OR [95%CI] P ABO 0.16 0.08 O 28,752 (42.7) 1216/27,536 Ref 27,648 (42.7) 260/27,388 Ref A 28,742 (44.4) 1342/28,574 1.08 [1.00–1.17] 28,742 (44.4) 323/28,419 1.19 [1.01–1.41] B 5,874 (8.7) 275/5599 1.10 [0.96–1.27] 5,613 (8.7) 58/5555 1.06 [0.80–1.42] AB 2,798 (4.2) 131/2667 1.14 [0.95–1.38] 2,686 (4.2) 37/2649 1.43 [1.01–2.03] Non-O 38,588 (57.3) 1748/36,840 1.09 [1.01–1.17] 0.03 37,041 (57.3) 418/36,623 1.19 [1.02–1.39] 0.03 ABO/RhD 0.15 0.05 O+ 23,703 (35.2) 995/22,708 Ref 22,804 (35.3) 217/22,587 Ref O− 5,049 (7.5) 221/4828 1.03 [0.89–1.20] 4,844 (7.5) 43/4801 0.96 [0.69–1.33] A+ 25,315 (37.6) 1120/24,195 1.07 [0.98–1.17] 24,344 (37.6) 286/24,058 1.24 [1.03–1.48] A− 4,601 (6.8) 222/4379 1.16 [1.00–1.35] 4,398 (6.8) 37/4361 0.91 [0.64–1.29] B+ 4,886 (7.3) 224/4662 1.09 [0.94–1.27] 4,675 (7.2) 48/4627 1.04 [0.76–1.43] B− 988 (1.5) 51/937 1.21 [0.90–1.63] 938 (1.5) 10/928 1.14 [0.60–2.17] AB+ 2,363 (3.5) 118/2245 1.24 [1.02–1.51] 2,266 (3.5) 35/2231 1.60 [1.11–2.30] AB− 435 (0.6) 13/422 0.70 [0.40–1.22] 420 (0.6) 2/418 0.48 [0.12–1.93] RhD 0.48 0.08 + 56,267 (83.6) 2457/53,810 Ref 54,089 (83.6) 586/53,503 Ref − 11,073 (16.4) 507/10,566 0.97 [0.87–1.07] 10,600 (16.4) 92/10,508 1.22 [0.98–1.52] N (%) ELISA-S ELISA-S, ELISA-NP, SN N positive/ negative OR [95%CI]a P N positive/ negativeb OR [95%CI] P ABO 0.16 0.08 O 28,752 (42.7) 1216/27,536 Ref 27,648 (42.7) 260/27,388 Ref A 28,742 (44.4) 1342/28,574 1.08 [1.00–1.17] 28,742 (44.4) 323/28,419 1.19 [1.01–1.41] B 5,874 (8.7) 275/5599 1.10 [0.96–1.27] 5,613 (8.7) 58/5555 1.06 [0.80–1.42] AB 2,798 (4.2) 131/2667 1.14 [0.95–1.38] 2,686 (4.2) 37/2649 1.43 [1.01–2.03] Non-O 38,588 (57.3) 1748/36,840 1.09 [1.01–1.17] 0.03 37,041 (57.3) 418/36,623 1.19 [1.02–1.39] 0.03 ABO/RhD 0.15 0.05 O+ 23,703 (35.2) 995/22,708 Ref 22,804 (35.3) 217/22,587 Ref O− 5,049 (7.5) 221/4828 1.03 [0.89–1.20] 4,844 (7.5) 43/4801 0.96 [0.69–1.33] A+ 25,315 (37.6) 1120/24,195 1.07 [0.98–1.17] 24,344 (37.6) 286/24,058 1.24 [1.03–1.48] A− 4,601 (6.8) 222/4379 1.16 [1.00–1.35] 4,398 (6.8) 37/4361 0.91 [0.64–1.29] B+ 4,886 (7.3) 224/4662 1.09 [0.94–1.27] 4,675 (7.2) 48/4627 1.04 [0.76–1.43] B− 988 (1.5) 51/937 1.21 [0.90–1.63] 938 (1.5) 10/928 1.14 [0.60–2.17] AB+ 2,363 (3.5) 118/2245 1.24 [1.02–1.51] 2,266 (3.5) 35/2231 1.60 [1.11–2.30] AB− 435 (0.6) 13/422 0.70 [0.40–1.22] 420 (0.6) 2/418 0.48 [0.12–1.93] RhD 0.48 0.08 + 56,267 (83.6) 2457/53,810 Ref 54,089 (83.6) 586/53,503 Ref − 11,073 (16.4) 507/10,566 0.97 [0.87–1.07] 10,600 (16.4) 92/10,508 1.22 [0.98–1.52] Table 2. Associations between ABO and RhD blood types and the seroprevalence of anti-SARS-CoV-2 antibodies, SAPRIS-SERO multi-cohort study, 2020. Discussion risk for type AB individuals as well, thus strengthening the evidence towards a differential susceptibility of SARS-CoV-2 infection according to ABO blood types. Yet, most studies so far involved registry or hospital data of SARS-CoV-2 positivity from PCR test results in patients or blood donors in comparison to the general population8–10 and fewer studies used seroprevalence data, mostly in small samples, with the exception of a recent report in 32,600 blood donors11. In turn, our study used seroprevalence data and a screening design whereby a large population-based sample of 67,340 participants involved in the SAPRIS-SERO project received a dried blood spot sampling kit. This allowed us to test for SARS-CoV-2 infection in a single general population, regard- less whether the participants sought testing or had symptoms, allowing detecting a large panel of infected cases.hf risk for type AB individuals as well, thus strengthening the evidence towards a differential susceptibility of SARS-CoV-2 infection according to ABO blood types. Yet, most studies so far involved registry or hospital data of SARS-CoV-2 positivity from PCR test results in patients or blood donors in comparison to the general population8–10 and fewer studies used seroprevalence data, mostly in small samples, with the exception of a recent report in 32,600 blood donors11. In turn, our study used seroprevalence data and a screening design whereby a large population-based sample of 67,340 participants involved in the SAPRIS-SERO project received a dried blood spot sampling kit. This allowed us to test for SARS-CoV-2 infection in a single general population, regard- less whether the participants sought testing or had symptoms, allowing detecting a large panel of infected cases.hf risk for type AB individuals as well, thus strengthening the evidence towards a differential susceptibility of SARS-CoV-2 infection according to ABO blood types. Yet, most studies so far involved registry or hospital data of SARS-CoV-2 positivity from PCR test results in patients or blood donors in comparison to the general population8–10 and fewer studies used seroprevalence data, mostly in small samples, with the exception of a recent report in 32,600 blood donors11. In turn, our study used seroprevalence data and a screening design whereby a large population-based sample of 67,340 participants involved in the SAPRIS-SERO project received a dried blood spot sampling kit. References Viruses 13, 160 (2021). 1 Gallian P et al SARS-CoV-2 IgG seroprevalence surveys in blood donors before the vaccination campaign France 2020–2021 (2022) 1. Gallian, P. et al. SARS-CoV-2 IgG seroprevalence surveys in blood donors before the vaccination campaign, France 2020–2021. (2022) https://doi.org/10.1101/2022.07.29.22278190.iti p g 12. Carrat, F. et al. Age, COVID-19-like symptoms and SARS-CoV-2 seropositivity profiles after the first wave of the pandemic in France. Infection 50, 257–262 (2022). 12. Carrat, F. et al. Age, COVID-19-like symptoms and SARS-CoV-2 seropositivity profiles after the first wave of the pandemic in France. Infection 50, 257–262 (2022). f 13. Carrat, F. et al. Antibody status and cumulative incidence of SARS-CoV-2 infection among adults in three regions of France fo lowing the first lockdown and associated risk factors: A multicohort study. Int. J. Epidemiol. 50, 1458–1472 (2021). f 13. Carrat, F. et al. Antibody status and cumulative incidence of SARS-CoV-2 infection among adults in three regions of Fran lowing the first lockdown and associated risk factors: A multicohort study. Int. J. Epidemiol. 50, 1458–1472 (2021). gi y p 14. Schetelig, J. et al. Blood group A epitopes do not facilitate entry of SARS-CoV-2. J. Intern. Med. 290, 223–226 (2021).i g g p p p y 15. Patel, E. U. et al. Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titers. J. Clin. Microbiol. 59, 02257–02320 (2021). g g p p p y 15. Patel, E. U. et al. Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titers. J. Clin. Microbiol. 59, 02257–02320 (2021). 6. Rikhtegaran Tehrani, Z. et al. Performance of nucleocapsid and spike-based SARS-CoV-2 serologic assays. PLoS ONE 15, e0237828 (2020). Data availability Data from the study are protected under the protection of health data regulation set by the French National Com- mission on Informatics and Liberty (Commission Nationale de l’Informatique et des Libertés, CNIL). The data can be made available upon reasonable request to the corresponding author (m.deschasaux@eren.smbh.univ- paris13.fr), after a consultation with the steering committee of the SAPRIS-SERO study. The French law forbids us to provide free access to SAPRIS-SERO data; access could however be given by the steering committee after legal verification of the use of the data. Please, feel free to come back to us should you have any additional question. Received: 18 October 2022; Accepted: 28 February 2023 Acknowledgementsh The authors warmly thank all the volunteers of the Constances, E3N-E4N and NutriNet-Santé cohorts for their continuous participation in the study and for participating in the SAPRIS-SERO project. We thank the CEPH- Biobank staff for their adaptability and the quality of their work. In the virology department, we thank Dr Nadège Brisbarre and the technical staff for impeccable management of samples and serological assays. continuous participation in the study and for participating in the SAPRIS-SERO project. We thank the CEPH- Biobank staff for their adaptability and the quality of their work. In the virology department, we thank Dr Nadège Brisbarre and the technical staff for impeccable management of samples and serological assays. www.nature.com/scientificreports/ sample collection was performed between May and November 2020, the first and second waves of the pandemic occurring in spring and fall 2020 in France, so that antibodies should still be detectable in most participants. Finally, the observational nature of the study makes it difficult to assess the extent to which participants have actually been exposed to the virus. Although lifestyle-related exposure to the virus is mostly unlikely to have been differential according to blood types, we adjusted our models on several covariates related to the spatial distribution of the epidemic since it could also relate to blood types, considering the distribution of ABO blood types in different regions or populations. However, we did not have access to racial and ethnic data as the collection of such data is generally not permitted in the framework of French cohort studies. Finally, our data only reflect infections occurring before November 2020, that is, before the emergence of variants but also before the large-scale vaccination campaigns. g p g To conclude, this study adds to the current body of evidence providing data on the association between ABO blood types and the risk of SARS-CoV-2 infection from screening a large sample from the general population using seroprevalence data. Our results confirmed a higher susceptibility of infection for individuals of blood types A and AB and a lesser risk for blood type O, thereby providing additional insights into the dynamic of SARS-CoV-2 infection. However, considering the magnitude of association and the current mechanistic hypotheses related to blood type incompatibility (hence a collective rather than individual type of protection), type O individuals are not without risk of infection and protective measures remain the best way of preventing SARS-CoV-2 transmission and infection. Discussion In contrast, type A individuals, carrying anti-B antibodies, would be susceptible to infection by viruses excreted by type O and type A individuals (i.e., 42% and 44% of the French population respectively), while type AB individuals carrying neither anti-A or anti-B antibodies would be susceptible to infection by individuals of all group types, which is why type A or AB are particularly at risk for SARS-CoV-2 infection. Type B individuals carrying anti-A antibodies are susceptible to infection by viruses excreted by type O and type B individuals but type B is less represented in the French population (10%) which may explain why no significant association was observed for these individuals. Another hypothesis, yet less supported by available data relates to the entry of SARS-CoV-2 in host’s cells, that would be facilitated by antigen A9,14. Finally, non-O individuals are at higher risk of thromboembolism, a common complication of COVID-19, hence beyond infection, non-O individuals could also be at higher risk of severe cases of COVID-199,10. Our study strengths pertained to the systematic screening for anti-SARS-CoV-2 antibodies targeting a large well-characterized sample, hence providing data from positive and negative cases within the same population, as well as the use of seroprevalence allowing the detection of cases that may or may not have symptoms or sought testing. Yet, some limitations should also be acknowledged. First, although large, our sample size may have still been limited to have sufficient power in analyses combining ABO and RhD blood types, especially for rarer blood types. Next, misclassification of individuals as ‘negative’ may have arisen from imperfect sensitivity of the ELISA-S test (85–90%, the specificity being > 95%)15,16 and from the decrease of anti-SARS-CoV-2 antibodies over time (with no detectable antibodies 4–5 months post-infection in half of participants in a recent report11). Yet, the https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 \| www.nature.com/scientificreports/ References 1. Zhao, J. et al. Relationship between the ABO blood group and the coronavirus disease 2019 (COVID-19) susceptibility. Clin. Infect Dis. 73, 328–331 (2021).hf 2. Göker, H. et al. The effects of blood group types on the risk of COVID-19 infection and its clinical outcome. Turk J. Med. Sci. 50 679–683 (2020).h 3. Abdollahi, A., Mahmoudi-Aliabadi, M., Mehrtash, V., Jafarzadeh, B. & Salehi, M. The novel coronavirus SARS-CoV-2 vulnerability association with ABO/Rh blood types. Iran J. Pathol. 15, 156–160 (2020). yp 4. Gallian, P. et al. Lower prevalence of antibodies neutralizing SARS-CoV-2 in group O French blood donors. Antivir. Res. 181 104880 (2020). ( ) 5. Cheng, Y. et al. ABO blood group and susceptibility to severe acute respiratory syndrome. JAMA 293, 1450–1451 (2005). L. et al. Human susceptibility and resistance to Norwalk virus infe ndesmith, L. et al. Human susceptibility and resistance to Norwalk p y 7. Batool, Z., Durrani, S. H. & Tariq, S. Association of ABO and Rh blood group types to hepatitis B, hepatitis C, HIV and Syphillis infection, a five year’experience in healthy blood donors in a tertiary care hospital. J Ayub. Med. Coll. Abbottabad 29, 90–92 (2017). 8 B h lli F l Th l f ABO bl d i i i h SARS C V 2 i f i A i i J Cli M d 11 3029 7. Batool, Z., Durrani, S. H. & Tariq, S. Association of ABO and Rh blood group types to hepatitis B, hepatitis C, HIV and Syphillis infection, a five year’experience in healthy blood donors in a tertiary care hospital. J Ayub. Med. Coll. Abbottabad 29, 90–92 (2017). 8. Banchelli, F. et al. The role of ABO blood type in patients with SARS-CoV-2 infection: A systematic review. J. Clin. Med. 11, 3029 (2022) i 8. Banchelli, F. et al. The role of ABO blood type in patients with SARS-CoV-2 infection: A systematic review. J. Clin. Med. 11, 3029 (2022). 9. Pereira, E. et al. ABO blood group and link to COVID-19: A comprehensive review of the reported associations and their possible underlying mechanisms. Microb. Pathog. 169, 105658 (2022). y g g 10. Pendu, J. L., Breiman, A., Rocher, J., Dion, M. & Ruvoën-Clouet, N. ABO blood types and COVID-19: Spurious, anecdotal, or truly important relationships? A reasoned review of available data. Viruses 13, 160 (2021). yp p truly important relationships? A reasoned review of available data. Additional information Correspondence and requests for materials should be addressed to M.D.-T. Correspondence and requests for materials should be addressed to M.D.-T. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 © The Author(s) 2023 Competing interests h p g The authors declare no competing interests. Fabrice Carrat6,7, Pierre‑Yves Ancel8, Nathalie Bajos10, Marie‑Aline Charles9, Gianluca Severi2,3, Mathilde Touvier1 & Marie Zins4 8Obstetrical, Perinatal and Pediatric Research Team (EPOPé), Centre for Research in Epidemiology and StatisticS (CRESS), Inserm, INRAE, Université de Paris, Paris, France. 9Centre for Research in Epidemiology and StatisticS (CRESS), Inserm, INRAE, Université de Paris, Paris, France. 10IRIS, UMR CNRS 8156, EHESS, Inserm U997, Aubervilliers, France. Author contributionsh The authors’ contributions were as follows – M.D.T., M.T. conceptualized the study and defined the analytical strategy; F.S.E.: performed statistical analyses; M.D.T. supervised statistical analyses and drafted the manuscript; M.T.: supervised statistical analyses and writing; F.S.E., N.D.P., Y.E., J.A., P.G., S.H., G.S., M.Z., E.W., X.dL, F.C., M.T., SAPRIS-SERO study group: played a key role in the acquisition of the data. All authors critically helped in https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 \| www.nature.com/scientificreports/ the interpretation of results, revised the manuscript and provided relevant intellectual input. They all read and approved the final manuscript. M.D.T. and M.T. had primary responsibility for the final content. the interpretation of results, revised the manuscript and provided relevant intellectual input. They all read and approved the final manuscript. M.D.T. and M.T. had primary responsibility for the final content. Constances cohort Sofiane Kab4, Adeline Renuy4, Stéphane Le Got4, Céline Ribet4, Mireille Pellicer4, Emmanuel Wiernik4, Marcel Goldberg4 & Marie Zins4 Fundingh g The SAPRIS/SAPRIS-SERO projects received funding from ANR (Agence Nationale de la Recherche, #ANR-20- COVI-000, #ANR-10-COHO-06), Fondation pour la Recherche Médicale (#20RR052-00), and Inserm (Institut National de la Santé et de la Recherche Médicale, #C20-26). The NutriNet-Santé cohort study was supported by the following public institutions: Ministère de la Santé, Santé Publique France, Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Conservatoire National des Arts et Métiers (CNAM) and Université Sorbonne Paris Nord. The CONSTANCES cohort benefits from grant ANR-11-INBS-0002 from the French National Research Agency. CONSTANCES is supported by the Caisse Nationale d’Assurance Maladie, the French Ministry of Health, the Ministry of Research, and the Institut National de la Santé et de la Recherche Médicale (INSERM). CONSTANCES is also partly funded by AstraZeneca, Lundbeck, L’Oréal, and Merck Sharp & Dohme Corp. The E3N-E4N cohort is supported by the following institutions: Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation, INSERM, University Paris-Saclay, Gustave Roussy, the MGEN, and the French League Against Cancer. Study investigators are independent from the funders. Funders had no role in the study design, the collection, analysis, and interpretation of data, the writing of the manuscript, or the decision to submit the article for publication. Epipage2 and Elfe child cohorts Marie‑Aline Charles9, Pierre‑Yves Ancel8, Valérie Benhammou11, Anass Ritmi11, Laetitia Marchand11, Cecile Zaros12, Elodie Lordmi12, Adriana Candea12, Sophie de Visme12, Thierry Simeon12, Xavier Thierry12, Bertrand Geay12, Marie‑Noelle Dufourg12 & Karen Milcent12 Marie‑Aline Charles9, Pierre‑Yves Ancel8, Valérie Benhammou11, Anass Ritmi11, Laetitia Marchand11, Cecile Zaros12, Elodie Lordmi12, Adriana Candea12, Sophie de Visme12, Thierry Simeon12, Xavier Thierry12, Bertrand Geay12, Marie‑Noelle Dufourg12 & Karen Milcent12 8Obstetrical, Perinatal and Pediatric Research Team (EPOPé), Centre for Research in Epidemiology and StatisticS (CRESS), Inserm, INRAE, Université de Paris, Paris, France. 9Centre for Research in Epidemiology and StatisticS (CRESS), Inserm, INRAE, Université de Paris, Paris, France. 11EPIPAGE-2 Joint Unit, Paris, France. 12ELFE Joint Unit, Paris, France. NutriNet-Santé cohort Fabien Szabo de Edelenyi1, Julien Allègre1, Nathalie Druesne‑Pecollo1, Younes Esseddik1, Serge Hercberg1 & Mathilde Touvier1 E3N-E4N cohort Fanny Artaud2, Pascale Gerbouin‑Rérolle2, Mélody Enguix2, Camille Laplanche2, Roselyn Gomes‑Rimav2, Lyan Hoang2, Emmanuelle Correia2, Alpha Amadou Barry2, Nadège Senina2 & Gianluca Severi2,3 https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 \| www.nature.com/scientificreports/ NutriNet-Santé cohort Fabien Szabo de Edelenyi1, Julien Allègre1, Nathalie Druesne‑Pecollo1, Younes Esseddik1, Serge Hercberg1 & Mathilde Touvier1 Epipage2 and Elfe child cohorts Marie‑Aline Charles9, Pierre‑Yves Ancel8, Valérie Benhammou11, Anass Ritmi11, Laetitia Marchand11, Cecile Zaros12, Elodie Lordmi12, Adriana Candea12, Sophie de Visme12, Thierry Simeon12, Xavier Thierry12, Bertrand Geay12, Marie‑Noelle Dufourg12 & Karen Milcent12 8Obstetrical, Perinatal and Pediatric Research Team (EPOPé), Centre for Research in Epidemiology and StatisticS (CRESS), Inserm, INRAE, Université de Paris, Paris, France. 9Centre for Research in Epidemiology and StatisticS (CRESS), Inserm, INRAE, Université de Paris, Paris, France. 11EPIPAGE-2 Joint Unit, Paris, France. 12ELFE Joint Unit, Paris, France. IPLESP – methodology and coordinating data center Clovis Lusivika‑Nzinga6, Gregory Pannetier6, Nathanael Lapidus6, Isabelle Goderel6, Céline Dorival6, Jérôme Nicol6, Olivier Robineau6 & Fabrice Carrat6,7 Inserm Cindy Lai13, Liza Belhadji13, Hélène Esperou13 & Sandrine Couffin‑Cadiergues13 13Inserm, Paris, France. Institut de Santé Publique Jean‑Marie Gagliolo14 14Aviesan, Inserm, Paris, France. CEPH-Biobank Hélène Blanché15, Jean‑Marc Sébaoun15, Jean‑Christophe Beaudoin15, Laetitia Gressin15, Valérie Morel15, Ouissam Ouili15 & Jean‑François Deleuze15 15Fondation Jean Dausset-CEPH (Centre d’Etude du Polymorphisme Humain), CEPH-Biobank, Paris, France. Unité des Virus Emergents Stéphane Priet5, Paola Mariela Saba Villarroel5, Toscane Fourié5, Souand Mohamed Ali5, Abdenour Amroun5, Morgan Seston5, Nazli Ayhan5, Boris Pastorino5 & Xavier de Lamballerie5 CEPH-Biobank Stéphane Priet5, Paola Mariela Saba Villarroel5, Toscane Fourié5, Souand Mohamed Ali5, Abdenour Amroun5, Morgan Seston5, Nazli Ayhan5, Boris Pastorino5 & Xavier de Lamballerie5 https://doi.org/10.1038/s41598-023-30714-9 Scientific Reports \| (2023) 13:4775 \|
https://openalex.org/W2946981645	https://www.researchsquare.com/article/rs-313/v1.pdf	English	null	Effect of acupuncture on insomnia in menopausal women: a study protocol for a randomized controlled trial	Trials	2,019	cc-by	6,378	Effect of acupuncture on insomnia in menopausal women: A Study protocol for a Randomized Controlled Trial women: A Study protocol for a Randomized Controlled Trial Shanshan Li Shanghai Municipal Hospital of Traditional Chinese Medicine Ping Yin Shanghai Municipal Hospital of Traditional Chinese Medicine Xuan Yin Shanghai Municipal Hospital of Traditional Chinese Medicine Anna Bogachko Shanghai Municipal Hospital of Traditional Chinese Medicine Tingting Liang Shanghai Municipal Hospital of Traditional Chinese Medicine Lixing Lao University of Hong Kong Shifen Xu (  xu_doctor@hotmail.com ) Shanghai Municipal Hospital of Traditional Chinese Medicine https://orcid.org/0000-0001-5595-3480 Method Article Keywords: Menopause; Insomnia; Acupuncture; Clinical trial Posted Date: February 8th, 2019 DOI: https://doi.org/10.21203/rs.2.313/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on May 30th, 2019. See the published version at https://doi.org/10.1186/s13063-019-3374-8. Shanshan Li Shanghai Municipal Hospital of Traditional Chinese Medicine Ping Yin Shanghai Municipal Hospital of Traditional Chinese Medicine Xuan Yin Shanghai Municipal Hospital of Traditional Chinese Medicine Anna Bogachko Shanghai Municipal Hospital of Traditional Chinese Medicine Tingting Liang Shanghai Municipal Hospital of Traditional Chinese Medicine Lixing Lao University of Hong Kong Shifen Xu (  xu_doctor@hotmail.com ) Shanghai Municipal Hospital of Traditional Chinese Medicine https://orcid.org/0000-0001-5595-3480 Shanshan Li Shanghai Municipal Hospital of Traditional Chinese Medicine Ping Yin Shanghai Municipal Hospital of Traditional Chinese Medicine Xuan Yin Shanghai Municipal Hospital of Traditional Chinese Medicine Anna Bogachko Shanghai Municipal Hospital of Traditional Chinese Medicine Tingting Liang Shanghai Municipal Hospital of Traditional Chinese Medicine Lixing Lao University of Hong Kong Shifen Xu (  xu_doctor@hotmail.com ) Shanghai Municipal Hospital of Traditional Chinese Medicine https://orcid.org/0000-0001-5595-3480 Method Article Version of Record: A version of this preprint was published on May 30th, 2019. See the published version at https://doi.org/10.1186/s13063-019-3374-8. Page 1/17 Abstract Background: China today has high occurrences of insomnia in perimenopausal women. Some studies have shown that acupuncture might be effective in treating primary insomnia and insomnia related to depression and stroke. Although there are some programs supporting insomnia during the menopausal transition, there are few randomized controlled trials to provide evidence regarding their effectiveness. We design a randomized controlled trial of suitable sample size to verify the effectiveness of acupuncture in patients with insomnia during the menopausal transition and to form an optimized acupuncture treatment protocol. Method: In this randomized, single-site, single-blind, placebo-controlled trial, 84 eligible patients will be recruited and randomly assigned to either the acupuncture group (n = 42) or the sham control group (n = 42) in a 1:1 ratio. Participants will receive a total of 18 sessions of treatment for eight consecutive weeks. Treatments will be given three times per week in the first four weeks, twice a week for the next two weeks and finally treatment once weekly for the final two weeks. Treatment will utilize eight main acupoints (GV20, GV24, GV29, RN6, RN4, SP6, HT7, EX-HN22), and extra two acupoints based on syndrome differentiation. The primary outcome will be assessed using the Pittsburgh Sleep Quality Index (PSQI). The secondary outcomes will be measured by sleep parameters recorded in the Actigraphy (SE, TST, SA), Insomnia Severity Index (ISI), Self-Rating Anxiety Scale (SAS), Self-Rating Depression Scale (SDS) and Menopause Quality of Life (Men-QOL). The primary outcomes will be assessed at baseline, week four, week eight, and first and the third month following the end of treatment. Discussion: If the results confirm that acupuncture is effective and safe for the treatment on insomnia in menopausal women, this positive outcome could provide evidence for clinical application. Trial Registration: Chinese Clinical Trial Registry, ChiCTR1800018645, Date: 2018-10-01. URL: http://www.chictr.org.cn/showproj.aspx?proj=31482. Trial Registration: Chinese Clinical Trial Registry, ChiCTR1800018645, Date: 2018-10-01. URL: http://www.chictr.org.cn/showproj.aspx?proj=31482. Keywords: Menopause; Insomnia; Acupuncture; Clinical trial. Methods Hypotheses: Acupuncture treatment helps menopausal transition insomnia patients improve their sleep quality and other related symptoms. Objectives: Objectives: 1. To compare the differences in improvement of insomnia, assessed by the Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), between the intervention group and control group. 1. To compare the differences in improvement of insomnia, assessed by the Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), between the intervention group and control group. 2. To compare the differences in improvement of mood, measured by the Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS), between the intervention group and control group. 1. To compare the differences in improvement of insomnia, assessed by the Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), between the intervention group and control group. 2. To compare the differences in improvement of mood, measured by the Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS), between the intervention group and control group. 3. To compare the differences in improvement of Menopause Quality of Life (MenQOL), between the intervention group and control group. 1. To compare the differences in improvement of insomnia, assessed by the Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), between the intervention group and control group. 2. To compare the differences in improvement of mood, measured by the Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS), between the intervention group and control group. 3. To compare the differences in improvement of Menopause Quality of Life (MenQOL), between the intervention group and control group. 3. To compare the differences in improvement of Menopause Quality of Life (MenQOL), between the intervention group and control group. Design: This is a single-site, single-blind, randomized, placebo-controlled clinical trial that will be carried out in the Shanghai Municipal Hospital of Traditional Chinese Medicine. Eligible patients will be randomly divided into the acupuncture group and the sham acupuncture group in a 1:1 allocation ratio. All participants will sign the informed consent before proceeding with the trial. The flow chart of the study process is as follows in Fig. 1. Timing of treatment assessments and data collection are as follows in Table 1. Recruitment: Participants of the study will be recruited through the outpatient clinic, hospital-based Wechat advertising, and posters in Shanghai Municipal Hospital of Traditional Chinese Medicine in Shanghai, China. Background The menopausal transition (MT) is often accompanied by a myriad of symptoms, the most common of which being insomnia[1-3]. Insomnia is characterized by having difficulty falling asleep, frequent nocturnal wakings with difficulty falling back asleep[4] and unexplained pain. A study conducted by the National Institutes of Health estimates the prevalence of insomnia in menopausal women at 40-50%[5-8]. Women who experience poor sleep quality during the MT are more likely to have critical problems[9] such as depression, anxiety, memory and cognitive deprivation, cardiovascular disease and reduced quality of life[3, 4]. Yet, there are few effective therapies available for treating sleep problems during the MT. Yoga, therapeutic massage and exercise may be helpful for sleep disorders, but they have not yet been proven as an effective independent treatment[10]. Hormone therapy (HT) is currently the most effective treatment, but is also associated with a number of risks[11, 12]. HT can reduce hot flashes at night, and Page 2/17 Page 2/17 some studies have reported that it can reduce sleep disorders[13]. However, increasing numbers of women are refusing HT due to the risks (myocardial infarction, stroke, venous thromboembolism and breast cancer) outlined by trials published in the Women’s Health Initiative[14]. Moreover, these trails reported that HT has a weaker effect on insomnia than previously thought[15] . some studies have reported that it can reduce sleep disorders[13]. However, increasing numbers of women are refusing HT due to the risks (myocardial infarction, stroke, venous thromboembolism and breast cancer) outlined by trials published in the Women’s Health Initiative[14]. Moreover, these trails reported that HT has a weaker effect on insomnia than previously thought[15] . Acupuncture, an important part of Traditional Chinese Medicine (TCM), has been used for thousands of years in China to treat various diseases including sleep disorders[5]. Many studies have confirmed the efficacy of acupuncture for primary insomnia and related depressive disorders[16-18]. Based on the results that the efficacy of acupuncture for depression-related insomnia [19] and insomnia following stroke [20], we believe that acupuncture might be an effective treatment for insomnia during the MT. However, effects of acupuncture on sleep disorders remain uncertain because of flawed research methodology, such as improper sample size, treatment times and follow-up problems[21]. The main objective of this study is to determine whether acupuncture is effective as compared to a sham acupuncture control on insomnia in menopausal women. Background The findings of this trial will provide useful information in forming an optimal acupuncture treatment protocol. Methods Those who are interested in participating in the study will be screened either via Page 3/17 telephone or on-site. They will receive eight-week treatment of acupuncture and a series of assessments on sleep disorders free of charge. During the study, participants will be required to wear a wrist Actigraphy to record their sleep quality. The participants who meet the inclusion criteria will be informed of the research details and asked to sign the informed consent before the treatment commence. telephone or on-site. They will receive eight-week treatment of acupuncture and a series of assessments on sleep disorders free of charge. During the study, participants will be required to wear a wrist Actigraphy to record their sleep quality. The participants who meet the inclusion criteria will be informed of the research details and asked to sign the informed consent before the treatment commence. nclusion Criteria: Participants with the following conditions will be included: Inclusion Criteria: Participants with the following conditions will be included: (1) Menopausal women aged 45~60; (2) Participants who meets the diagnostic criteria of traditional Chinese medicine in accordance with the above perimenopausal and insomnia[22]; (2) Participants who meets the diagnostic criteria of traditional Chinese m above perimenopausal and insomnia[22]; (3) Participants in line with the international classification of sleep disorders (ICSD-3) for the diagnosis of insomnia; (4) Participants whose PSQI score is higher than five; (4) Participants whose PSQI score is higher than five; (4) Participants whose PSQI score is higher than five; (5) Participants who voluntarily agree to join the study and to sign the written informed consent form for the RCT. Exclusion Criteria: Participants with the following conditions will be excluded: (1) Participants who have severe mental illness such as depression, anxiety, schizophrenia and so on; (2) Participants with serious heart, brain, kidney or liver disease; (3) Participants with secondary insomnia caused by systemic diseases such as pain, fever, cough, surgery and external environment disturbance; (4) Participants who have taken hormones for 6 or more months; (5) Participants who are pregnant or currently lactating. Intervention: (5) Participants who are pregnant or currently lactating. Participants in each group will receive 18 real or sham treatments within an eight-week period. In each group, they will receive either acupuncture or sham acupuncture treatment three times per week for the first four weeks, twice per week for the next two weeks and once per week for the last two weeks. Methods Each treatment will last for 30 minutes. The each patient will be placed in a separate quiet space and lying supine. The acupuncturists will be registered practitioners with more than three years of experience in acupuncture practice. In order to increase the patients’ compliance, they will accept the intervention and assessment by reservation, and they will receive some financial subsidies (200 RMB) after the follow-up assessments. Page 4/17 Page 4/17 In the treatment group, acupuncture needles will be standard stainless steel, sterile, and disposable (0.25 × 40 mm and 0.30×40mm in length; Jia Jian, China). The control group will use the Streitberger Placebo- needle at the same acupoints. The treatment methods of acupuncture and acupoints are shown in Table 2. In the treatment group, acupuncture needles will be standard stainless steel, sterile, and disposable (0.25 × 40 mm and 0.30×40mm in length; Jia Jian, China). The control group will use the Streitberger Placebo- needle at the same acupoints. The treatment methods of acupuncture and acupoints are shown in Table 2. The acupuncture group: Participants in this group will receive acupuncture treatment during the first eight weeks. According to our in depth research on insomnia and acupuncture experience, Baihui (GV20), Shenting (GV24), Yintang (GV29), Qihai (RN6), Guanyuan (RN4) and bilateral Anmian (EX-HN22), Sanyinjiao (SP6), Shenmen (HT7) will be used as main acupoints[17]. Acupuncturists will be permitted to add acupuncture points based on TCM syndrome differentiation [23]. According to our own clinical practice, potentially relevant extraneous points include Mingmen (GV4) and Shenshu (BL23) for kidney yang deficiency, and TaiXi (KI3) and Fuliu (KI7) for kidney yin deficiency. The needles will be inserted into the skin to the depth of 10–30 mm and manipulated manually (including lifting, thrusting, and rotating) until the patient reports needling sensations (Deqi sensation). The needles on GV20 and GV29 will be connected to a G6805-2 Multi- Purpose Health Device (Huayi company), using continuous wave type, frequency at 2.5HZ, and intensity of 45 mA. Needles will be retained for 30 minutes before removal. The control group: Participants in this group will receive sham acupuncture treatment. We will use a non-invasive placebo control, the Streitberger Placebo-needle. Acupoints are the same as with acupuncture group, without insertion. An electroacupuncture apparatus (G6805-2 Multi-Purpose Health Device) will be set beside the patients and connected to the GV20 and GV29, without electrical pulse. Outcome measures We will make assessments at baseline, week four, week eight, post-treatment, and with follow-ups at the first and third month after the end of treatment. Primary outcome Primary outcome Pittsburgh Sleep Quality Index (PSQI): Pittsburgh Sleep Quality Index (PSQI): Pittsburgh Sleep Quality Index (PSQI): The PSQI is a self-rated questionnaire used to measure general sleep quality. It comprises 19 self-rated items and five other-rated items[20]. It assesses sleep based on seven domains in the past month: sleep duration, sleep disorders, sleep-onset latency, daytime dysfunction, sleep efficiency, use of medications to sleep, and overall sleep quality[24]. Each domain is rated from 0 to 3, and the accumulated scores of the seven domains constitute the total score of the PSQI (0–21). A total score of more than 5 indicates poor quality of sleep[25] . Secondary outcomes: Page 5/17 Page 5/17 Insomnia Severity Index (ISI): ISI is designed to assess the nighttime and daytime symptoms of insomnia. It includes seven items rated on a 0- to 4-point scale, total score ranges from 0–28[26]. All self-report questionnaires are conducted in Chinese. A higher score indicates a more severe insomnia. ISI classification: clinical insomnia (score 0-7), mild insomnia (8-14), moderate insomnia (score 15-21), and severe insomnia (score 22-28). Menopause Quality of Life (MenQOL): MenQOL is a questionnaire used to assess the quality of life in menopause, including 29 items, which are divided into four domains, Vasomotor (Items 1 to 3), Psychosocial (Items 4 to 10), Physical (Items 11 to 26), and Sexual (Items 27 to 29). For analyses, we convert the item scores to a score ranging from 1 to 8 (NO, 0-6). Each domain mean ranges from 1 to 8. The overall questionnaire score is the mean of the domain means. A higher score indicates a worse quality of life [29]. Self-Rating Depression Scale (SDS): The SDS is a self-rated scale for evaluating the severity of depression [28]. The main statistical index consists of 20 questions. We first calculate the standard score by ranking the participant’s responses to 20 questions. From this figure the total score determined as is 1.25 times of the standard score, making use of the integral part [17]. The demarcation point of depression is 50 points, the higher the score, the more significant the depression tendency. Actigraphy assessments: The wActiSleep-BT actigraph (Actigraph LLC, Pensacola, USA), will be worn on the patients’ wrist, which can record the sleep quality by noting sleep awakenings (SA), total sleep time (TST), sleep onset, sleep latency as well as the efficiency of sleep. The analysis of sleep condition and sleep quality will be performed by the software ActiLife6 (Version 6.8.1, Actigraph LLC) [17, 20]. Self-Rating Anxiety Scale (SAS): We will use SAS to assess the degree of anxiety in patients. The questionnaire includes 20 items, as follows: 15 negative and 5 positive descriptions, the opposite of the score[27]. The total score of the 20 items more than 40 points, indicating the state of anxiety. A higher score indicates more severe anxiety. Self-Rating Depression Scale (SDS): Estazolam dose: Patients will be allowed to take Estazolam (0.5mg-2.0mg) when they have difficulty in falling asleep for more than three consecutive days during the trial. They will be asked to record the dose and the time of taking Estazolam on the case report form (CRF). Sample size: Page 6/17 The sample size calculation was based on the change of PSQI scores. Systematic review[21] shows that at least 2.70 point difference in PSQI scores between acupuncture and sham-acupuncture has clinical significance. We expected that acupuncture would out-perform sham-acupuncture by three points, therefore a sample size of 35 participants should be recruited in each group. Considering about a 20% dropout rate, each group will take 42 cases. So a total of 84 participants should be recruited for this RCT. N= ；() Randomization and Blinding: We will use the block randomization method. We plan to use SPSS version 23.0 software to generate a random number table. The participants who meet the criteria will be randomly assigned to acupuncture group or control group with 1:1 ratio. The random allocation sequence will be generated in a block. The block size will be randomized to six. Then the independent researcher will pre-pack envelopes with the corresponding number according to the randomization schedule. Each patient will receive an envelope labeled with their name containing their numbered group assignment. Patients will be asked to wear an eye-patch when they receive treatment. Only the acupuncturists will know the group assignments. The participants and other researchers (including the data analysts, outcome assessors, statistician will be blinded to group allocation. In order to ensure the successful implementation of the blinding method, all researchers will be trained before the trial begins. Safety and undesirable effects: Safety and undesirable effects: Patients will be advised in the event of any undesirable effect after the application of acupuncture that they discontinue acupuncture and contact their doctors, the doctor will diagnose and treat the adverse reactions. They will also be asked to contact the researcher who will have completed an undesirable effect form as well as the undesirable effect record log. Adverse events such as fainting, allergies and pain may occur in acupuncture clinical trials[30]. If the participant has the reaction of fainting, the needle will be immediately withdrawn. The patient will be returned to a supine position and given warm water or sugar water. The patient will be allowed to rest until they have made a full recovery. In the case of pain and allergies, the needle should be taken out immediately and treated according to the specific circumstances. Any adverse event (include any discomfort, symptoms, or diseases occurring during the trial) will be reported by the patients and doctors after acupuncture treatment. All details of adverse events will be reported in the Case Report Form. At the end of the study we will analyze the influence of all events. Data management: Statistical analysis: All data will be analyzed by Intention-to-Treat (ITT), including data from any participants who have dropped out of the RCT during the trial. All data will be entered twice by two different researchers to ensure the accuracy. The statistical analysis of data will be carried out by SPSS 23.0 software. All data will be analyzed by Intention-to-Treat (ITT), including data from any participants who have dropped out of the RCT during the trial. All data will be entered twice by two different researchers to ensure the accuracy. The statistical analysis of data will be carried out by SPSS 23.0 software. Descriptive characteristics of baseline statistics will be reported as the mean ± standard deviation or median. The two study groups will be compared using t test for continuous measures and chi-square tests for categorical measures. To compare changes in sleep quality between two groups as the primary analysis, a repeated measures ANOVA (General Linear Model) will be used. P-Values ≤0.05 will be considered statistically significant. Monitoring: To improve the quality of this RCT, the whole process will be conducted under the supervision of a qualified clinical trial expert and be carried out by Shanghai Municipal Hospital of Traditional Chinese Medicine. The Clinical Research Center of Drugs of the Shanghai University of Traditional Chinese Medicine will provide data monitoring with access to any interim results and will make the final decision to terminate the trial if necessary. Clinical trial registration: This RCT was registered in the Chinese Clinical Trial Registry (ChiCTR1800018645), Date: 2018-10-01. Clinical trial registration: This RCT was registered in the Chinese Clinical Trial Registry (ChiCTR1800018645), Date: 2018-10-01. Clinical trial registration: This RCT was registered in the Chinese Clinical Trial Registry (ChiCTR1800018645), Date: 2018-10-01. URL: http://www.chictr.org.cn/showproj.aspx?proj=31482 RL: http://www.chictr.org.cn/showproj.aspx?proj=31482 Data management: Patient characteristics will be recorded in CRFs, which will be stored in the researcher’s work office. Quality of CRF completion will be monitored by the specified researcher. All the original data of the RCT will be entered into the ResMan research Manager of the Clinical Trial Management Public Platform. The Page 7/17 relevant users will be trained and the system will be tested before it is officially launched to ensure that the system meets the trial requirements. Only relevant personnel will receive the account number and password once the system is officially launched. The clinical supervisor will monitor the work of the clinical trial center at least once a month. relevant users will be trained and the system will be tested before it is officially launched to ensure that the system meets the trial requirements. Only relevant personnel will receive the account number and password once the system is officially launched. The clinical supervisor will monitor the work of the clinical trial center at least once a month. Statistical analysis: Discussion Acupuncture treatment has been practiced in China for more than two millennia[31] and in recent years has been widely used in western countries [32]. Recent studies have found that acupuncture can be used to relieve vasomotor symptoms during the MT [23], but have not yet established the effects of acupuncture on insomnia during the MT. This is a valuable clinical RCT aimed at treating insomnia in menopausal women. We use acupuncture and sham acupuncture[33] to evaluate the efficacy of acupuncture on improving sleep quality. In addition, we use a strictly randomized controlled trial, utilizing the control group method to reduce test bias and ensure the reliability of the RCT. The improvement on previous trials offered in this RCT is the method of point selection. Individualized treatment upon syndrome differentiation is the basis of TCM theory[23], and the selection of points is a question for the world acupuncture researchers [34]. To give some evidence for the effects of treatment upon syndrome differentiation, we set eight points as main points, and add two acupoints based on Page 8/17 Page 8/17 individual differences in the syndromes. Another advantage is the use of Actigraphy. This wrist-worn sleep monitor can provide an objective assessment for the treatment effect [35]. This is very useful for us to compare and evaluate the change of patients’ sleep quality before and after treatment. individual differences in the syndromes. Another advantage is the use of Actigraphy. This wrist-worn sleep monitor can provide an objective assessment for the treatment effect [35]. This is very useful for us to compare and evaluate the change of patients’ sleep quality before and after treatment. Another innovation in this trial is the design of the treatment frequency. In the first month we will give full dose (three times per week) treatment. After the initial month we will decrease the frequency of the acupuncture treatment. This will be beneficial for us to observe the acupuncture therapeutic effect, and to achieve the purpose of maintaining the minimum therapeutic dose for the long-term. However, this RCT still faces some challenges. (1) The single-blinded method. For clinical trials of acupuncture, it is inevitable to know about the acupuncture treatment or sham acupuncture treatment for the acupuncturists. All the participants will be asked to wear the eye-patch and arranged in a separate quiet space during the treatment. (2) The application of the sham acupuncture method. Discussion The acupuncturists will receive several sessions of strict training before the study begins to ensure the proper sham acupuncture techniques. (3) The challenge of participant compliance. We will provide patient outreach over the phone to arrange the treatment time reasonably and to improve the attendance rate of the patients. The researchers will conduct follow-up interviews by phone to record patient outcomes. To achieve our clinical goals, we will strive to standardize every step of the study, including acupoints selection, acupuncture operation, needle apparatus, and therapists’ clinical experience. We expect that this trial will provide strong positive evidence for acupuncture treatment of sleep disorders in the perimenopausal period. Abbreviations MT: Menopausal Transition; HT: Hormone Therapy; TCM: Traditional Chinese Medicine; PSQI: Pittsburgh Sleep Quality Index; ISI: Insomnia Severity Index; TST: Total Sleep Time; SAS: Self-Rating Anxiety Scale; SDS: Self-Rating Depression Scale; MenQOL: Menopause Quality of Life; ITT: Intention-to-Treat ; GV: Governor vessel; RN: Ren meridians; SP: Spleen meridian of foot Taiyin; HT: Heart meridian of hand Shaoyin; EX-HN: Extra acupoints on head; KI: Kidney meridian of foot Shaoyin; CRF: Case report form. References 1. Prairie BA, Wisniewski SR, Luther J, Hess R, Thurston RC, Wisner KL, Bromberger JT: Symptoms of depressed mood, disturbed sleep, and sexual problems in midlife women: cross-sectional data from the Study of Women's Health Across the Nation. Journal of women's health 2015, 24(2):119-126. 1. Prairie BA, Wisniewski SR, Luther J, Hess R, Thurston RC, Wisner KL, Bromberger JT: Symptoms of depressed mood, disturbed sleep, and sexual problems in midlife women: cross-sectional data from the Study of Women's Health Across the Nation. Journal of women's health 2015, 24(2):119-126. 2. Chiu HY, Hsieh YJ, Tsai PS: Acupuncture to Reduce Sleep Disturbances in Perimenopausal and Postmenopausal Women: A Systematic Review and Meta-analysis. Obstetrics and gynecology 2016, 127(3):507-515. 3. Yazdi Z, Sadeghniiat-Haghighi K, Ziaee A, Elmizadeh K, Ziaeeha M: Influence of sleep disturbances on quality of life of Iranian menopausal women. Psychiatry journal 2013, 2013:907068. 3. Yazdi Z, Sadeghniiat-Haghighi K, Ziaee A, Elmizadeh K, Ziaeeha M: Influence of sleep disturbances on quality of life of Iranian menopausal women. Psychiatry journal 2013, 2013:907068. 4. Sun D, Shao H, Li C, Tao M: Sleep disturbance and correlates in menopausal women in Shanghai. Journal of psychosomatic research 2014, 76(3):237-241. 4. Sun D, Shao H, Li C, Tao M: Sleep disturbance and correlates in menopausal women in Shanghai. Journal of psychosomatic research 2014, 76(3):237-241. 5. Cong Fu NZ, Zhen Liu,Lu-hua Yuan,Chen Xie,Wen-jia Yang,et al.: Acupuncture Improves Peri- menopausal Insomnia: A Randomized Controlled Trial. SLEEP 2017, 40:1-8. 5. Cong Fu NZ, Zhen Liu,Lu-hua Yuan,Chen Xie,Wen-jia Yang,et al.: Acupuncture Improves Peri- menopausal Insomnia: A Randomized Controlled Trial. SLEEP 2017, 40:1-8. 6. Slopien R, Wichniak A, Pawlak M, Slopien A, Warenik-Szymankiewicz A, Sajdak S: Disturbances of sleep continuity in women during the menopausal transition. Psychiatria polska 2015, 49(3):615- 623. 7. Berecki-Gisolf J, Begum N, Dobson AJ: Symptoms reported by women in midlife: menopausal transition or aging? Menopause 2009, 16(5):1021-1029. 8. Jin F, Tao M, Teng Y, Shao H, Li C, Mills E: Knowledge and attitude towards menopause and hormone replacement therapy in Chinese women. Gynecologic and obstetric investigation 2015, 79(1):40-45. 9. Philip S.Eichling JS: Menopause Related Sleep Disorders. Lournal of Clinical Sleep Medicine 2005, 1:291-300. 10. Joshua z SA, Claire L, Sara Nowakowski: Treatment of Insomnia, Insomnia Symptoms, and Obstructive Sleep Apnea During and After Menopause:Therapeutic Approaches. HHS Public Access 2015, 1:63-83. 11. H.Hachul TKPG, A. L. Maciel, F. Yagibara, S.Tufik, L. 2School of Chinese Medicine, University of Hong Kong, Hong Kong, China. 2School of Chinese Medicine, University of Hong Kong, Hong Kong, China. 3University of Maryland School of Medicine, Baltimore, MD, USA 21201 Declarations Trial status: The first investigators’ meeting took place on 31 August 2018. The RCT is in preparation now and will launch in November 2018. Recruitment is expected to end in late 2020. Ethics approval and consent to participate: This RCT was approved by the Ethics Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine on September 18, 2018 (certificate number 2018SHL-KY-09). The purpose, procedures, and Page 9/17 Page 9/17 potential risks of the RCT will be explained clearly to the participants. All participants shall give their written informed consent before joining the RCT. potential risks of the RCT will be explained clearly to the participants. All participants shall give their written informed consent before joining the RCT. Consent for publication: Consent for publication: Not applicable. Availability of data and material: The trial data will be available from the corresponding author upon reasonable request. Competing interests: The authors declare that they have no competing interests. The authors declare that they have no competing interests. The study is partly supported by grants from Shanghai Committee of Science and Technology, China (No 18401905200). Shanghai Hospital Development Center, China (No SHDC12016124), Shanghai Municipal Commission of Health and Family Planning, China (No 201640026) and State Administration of Traditional Chinese Medicine, China (Grant No. JDZX2015024). Authors' contributions: The trial was designed and developed by SFX and LXL. The manuscript was drafted by SSL. The protocol was carefully revised, edited by SSL and PY. XY, AB, and TTL contributed the discussion. All authors read and approved the final manuscript. Acknowledgements: Shifen Xu provided general support as the head of the Acupuncture Department in Shanghai Municipal Hospital of Traditional Chinese Medicine, and was responsible for the design of this RCT and for communicating important protocol modifications to relevant parties. We would like to thank Dr. Philippa Hazlewood from the International Education College, Shanghai University of Traditional Chinese Medicine, for her editorial support. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors' information: 1Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China. Page 10/17 2School of Chinese Medicine, University of Hong Kong, Hong Kong, China. References bittencourt: Acupuncture improves sleep in postmenopause in a randomized, double-blind, placebo-controiied study. CLIMACTERIC 2013, 16:36- 40. 12. Avis NE, Coeytaux RR, Levine B, Isom S, Morgan T: Trajectories of response to acupuncture for menopausal vasomotor symptoms. Menopause 2017, 24(2):171-179. 12. Avis NE, Coeytaux RR, Levine B, Isom S, Morgan T: Trajectories of response to acupuncture for menopausal vasomotor symptoms. Menopause 2017, 24(2):171-179. 13. Huang MI, Nir Y, Chen B, Schnyer R, Manber R: A randomized controlled pilot study of acupuncture for postmenopausal hot flashes: effect on nocturnal hot flashes and sleep quality. Fertility and sterility 2006, 86(3):700-710. 13. Huang MI, Nir Y, Chen B, Schnyer R, Manber R: A randomized controlled pilot study of acupuncture for postmenopausal hot flashes: effect on nocturnal hot flashes and sleep quality. Fertility and sterility 2006, 86(3):700-710. Page 11/17 Page 11/17 14. Jennifer Hays JK, Ockene, Robert, Jane M, Kotchenm, et al.: Effects of estrogen plus progestin on health-related quality of life. The NEW ENGLAND JONAL of MEDICINE 2003, 348:1839-1845. 15. Fredi K, Adriane Fugh-Berman: Complementary and alternative medicine for menopausal symptoms- a review of randomized, controlled trials. ACADEMIA AND CLININ 2002:805-814. 15. Fredi K, Adriane Fugh-Berman: Complementary and alternative medicine for menopausal symptoms- a review of randomized, controlled trials. ACADEMIA AND CLININ 2002:805-814. 16. Yin X, Xu J, Dong B, Ma J, Chen Z, Yin P, Wu J, Zhu B, Cao Y, Zheng H et al: Efficacy and Safety of Electroacupuncture on Treating Depression Related Sleep Disorders: Study Protocol of a Randomized Controlled Trial. Evidence-based complementary and alternative medicine : eCAM 2016, 2016:1069597. 17. Yin X, Gou M, Xu J, Dong B, Yin P, Masquelin F, Wu J, Lao L, Xu S: Efficacy and safety of acupuncture treatment on primary insomnia: a randomized controlled trial. Sleep medicine 2017, 37:193-200. 17. Yin X, Gou M, Xu J, Dong B, Yin P, Masquelin F, Wu J, Lao L, Xu S: Efficacy and safety of acupuncture treatment on primary insomnia: a randomized controlled trial. Sleep medicine 2017, 37:193-200. 18. Wing-Fai Yeung K-FC, Kwok-Chu Tso,Shi-Ping,Zhang-jin Zhang,Lai-Ming Ho: Electroacupuncture for residual insomnia associated with major depressive disorder- a randomized controlled trial. SLEEP 2011, 34:807-815. 19. Dong B, Chen Z, Yin X, Li D, Ma J, Yin P, Cao Y, Lao L, Xu S: The Efficacy of Acupuncture for Treating Depression-Related Insomnia Compared with a Control Group: A Systematic Review and Meta- Analysis. BioMed research international 2017, 2017:9614810. 20. References Cao Y, Yin X, Soto-Aguilar F, Liu Y, Yin P, Wu J, Zhu B, Li W, Lao L, Xu S: Effect of acupuncture on insomnia following stroke: study protocol for a randomized controlled trial. Trials 2016, 17(1):546. 20. Cao Y, Yin X, Soto-Aguilar F, Liu Y, Yin P, Wu J, Zhu B, Li W, Lao L, Xu S: Effect of acupuncture on insomnia following stroke: study protocol for a randomized controlled trial. Trials 2016, 17(1):546. 21. Cheuk DK, Yeung WF, Chung KF, Wong V: Acupuncture for insomnia. The Cochrane database of systematic reviews 2012(9):CD005472. 22. Sateia MJ: International classification of sleep disorders-third edition: highlights and modifications. Chest 2014, 146(5):1387-1394. 23. Avis NE, Coeytaux RR, Isom S, Prevette K, Morgan T: Acupuncture in Menopause (AIM) study: a pragmatic, randomized controlled trial. Menopause 2016, 23(6):626-637. 24. Jutta Backhaus KJ, Andreas Brocks, Dieter Riemann,Fritz Hohhagen: Test-retest reliability and validity of the Pittsburgh Sleep Quality Index in primary insomnia. Journal of Psychosomatic Research 2002, 53:737-740. 25. Daniel J. Buysse CFRI, Timmothy H. Monk, Susan R. Berman, David J. Kupfer: The Pittsburgh Sleep Quality Index- a new instrument for psychiatric practice and research. Psychiatry Research 1988, 28:193-213. 26. Célyne H. Bastien AV, Charles M.Morin: alidation of the Insomnia Severity Index as an outcome measure for insomnia research. Sleep medicine 2001, 2:297-307. 26. Célyne H. Bastien AV, Charles M.Morin: alidation of the Insomnia Severity Index as an outcome measure for insomnia research. Sleep medicine 2001, 2:297-307. 27. Li RX, Ma M, Xiao XR, Xu Y, Chen XY, Li B: Perimenopausal syndrome and mood disorders in perimenopause: prevalence, severity, relationships, and risk factors. Medicine 2016, 95(32):e4466. 28. WILLIAM W. K. ZUNG D: A Self-Rating Depression Scale. ARCHIVES OF GENNERAL PSYCHIATRY 1965, 12:63-70. Page 12/17 Page 12/17 29. Julie Lemieux MD, Wendy R, Paul E., James N. Kathleen I, et al.: Quality of Life From Canadian Cancer Trials Group MA.17R: A Randomized Trial of Extending Adjuvant Letrozole to 10 years. JOURNAL OF CLINICAL ONCOLOGY 2018, 36:563-571. 30. Li YQ, Lu YY, Zhang J, Guo Y: Analysis on the situation of adverse reaction to acupuncture and acupuncture risk. Chinese Acupuncture & Moxibustion 2011, 31(8):764-768. 31. ADAM BURKE DM, CLAIRE DYE, LAURA CHYU: Acupuncture use in the United States- findings from the National Health Interview Survey. THE JOURNAL OF ALTERNATIVE AND COMPLEEMENTARY MEDICINE 2006, 12:639-648. 32. References Yeung WF, Chung KF, Yung KP, Ho FY, Ho LM, Yu YM, Kwok CW: The use of conventional and complementary therapies for insomnia among Hong Kong Chinese: a telephone survey. Complementary therapies in medicine 2014, 22(5):894-902. 33. Lao L, Bergman S, Hamilton GR, Langenberg P, Berman B: Evaluation of acupuncture for pain control after oral surgery: a placebo-controlled trial. Arch Otolaryngol Head Neck Surg 1999, 125(5):567-572. 33. Lao L, Bergman S, Hamilton GR, Langenberg P, Berman B: Evaluation of acupuncture for pain control after oral surgery: a placebo-controlled trial. Arch Otolaryngol Head Neck Surg 1999, 125(5):567-572. 34. Albert Yeung DM, Jonathan Alpert, Yantao Ma, Maurizio Fava: Acupuncture for Major Depressive Disorder: Has Its Efficacy Been Disproved? LETTERS TO THE EDITOR 2007:1617-1622. 34. Albert Yeung DM, Jonathan Alpert, Yantao Ma, Maurizio Fava: Acupuncture for Major Depressive Disorder: Has Its Efficacy Been Disproved? LETTERS TO THE EDITOR 2007:1617-1622. 35. Short MA, Gradisar M, Lack LC, Wright H, Carskadon MA: The discrepancy between actigraphic and sleep diary measures of sleep in adolescents. Sleep medicine 2012, 13(4):378-384. 35. Short MA, Gradisar M, Lack LC, Wright H, Carskadon MA: The discrepancy between actigraphic and sleep diary measures of sleep in adolescents. Sleep medicine 2012, 13(4):378-384. Figure 1 Flowchart of the trial. Tables Table1. Timing of treatment assessments and data collection Table1. Timing of treatment assessments and data collection Page 13/17 STUDY PERIOD Enrolment Baseline Treatment phase Follow-up phase TIMEPOINT** -1week 0week 4week 8week 4week 12week ENROLMENT: Eligibility screen X Informed consent X Medical history X Allocation X INTERVENTIONS: Acupuncture X X X Sham Acupuncture X X X ASSESSMENTS: Primary outcome PSQI X X X X X Secondary outcomes ISI Actigraphy SDS SAS Men-QoL X X X X X X X X X X X X X Others Estazolam dose Adverse events X X X X X X X X X X Patients's satisfaction Success of blinding X X Page 14/17 Table 2. Details of intervention Table 2. Details of intervention Intervention group Control group Main Acupoints GV20, GV24, GV29, RN6, RN4, SP6, HT7, EX-HN22 GV20, GV24, GV29, RN6, RN4, SP6, HT7, EX-HN22 Added Acupoints Kidney yang deficiency GV4, BL23 Kidney yang deficiency GV4, BL23 Kidney yin deficiency KI3, KI7 Kidney yin deficiency KI3, KI7 Depth of insertion GV20, GV24, GV29, RN6, RN4, EX-HN22 10mm No insertion HT7, SP6, GV4, BL23, KI7, KI3 15mm Needle type Steel needle (Wuxi Jiajian Medical Co. Ltd. Wuxi, China) Blunt-tip needle (Streitberger Placebo-needle) Needle sensation With de-qi sensation Without de-qi sensation Electric stimulation Needle on GV20, GV29 connected to G6805-2 Multi- Purpose Health Device (Shanghai Medical Instruments High-Techno, China). with electric pulse at a frequency of 2.5 Hz and an intensity of 45 mA Needle on GV20, GV29, connected to G6805-2 Multi-Purpose Health Device (Shanghai Medical Instruments High-Techno, China). without electric pulse Frequency and duration Three times per week for the first four weeks Three times per week for the first four weeks Twice per week for the next two weeks Twice per week for the next two weeks Once per week for the final two weeks Once per week for the final two weeks Intervention group Figures Page 15/17 Page 15/17 Figure 1 Flowchart of the trial. Supplementary Files supplement1.dotx Supplementary Files Page 16/17 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to d Page 16/17 This is a list of supplementary files associated with this preprint. Click to download. supplement1.dotx supplement1.dotx Page 17/17
https://openalex.org/W4320062947	https://sistemascmc.ifam.edu.br/educitec/index.php/educitec/article/download/2015/872	Portuguese	null	pedagogia de projetos e a formação humana integral	Educitec	2,022	cc-by	7,602	Resumo Este artigo traz à discussão as relações entre a Pedagogia de Projetos e a Formação Humana Integral, buscando, de forma sistemática e crítica, superar a visão de ensino-aprendizagem desvinculada de uma formação crítica, inventiva e comprometida com a transformação social. Para tanto, se propôs a analisar a utilização da Pedagogia de Projetos como metodologia de ensino visando uma formação humana integral dos discentes, percebendo-os como sujeitos ativos de seu processo de aprendizagem. Durante a pesquisa, são destacadas as principais características da Pedagogia de Projetos e suas contribuições para a formação Humana Integral. Para tanto, foi desenvolvido um estudo de caso de uma turma do curso Técnico em Administração na Forma Integrada do IFAM campus Coari, de modo a apontar as descobertas realizadas e os resultados alcançados após a reflexão e análise do processo. O levantamento de dados foi feito a partir da aplicação de entrevista semiestruturada e observação remota dos participantes. Os resultados tiveram seus dados tabulados e submetidos ao tratamento da metodologia de análise Textual Discursiva, os quais indicam que a pedagogia de projetos contribuiu de forma significativa para a formação e o desenvolvimento das dimensões cultural, social, cognitiva, física e emocional dos discentes. Palavras-chave: Pedagogia de Projetos; Formação humana integral. A pedagogia de projetos e a formação humana integral: estudo de caso no município de Coari/AM Revista do Programa de Pós-Graduação em Ensino Tecnológico, do Instituto Federal do Amazonas ISSN: 2446-774X A pedagogia de projetos e a formação humana integral: estudo de caso no município de Coari/AM Revista do Programa de Pós-Graduação em Ensino Tecnológico, do Instituto Federal do Amazonas ISSN: 2446-774X Revista do Programa de Pós-Graduação em Ensino Tecnológico, do Instituto Federal do Amazonas A pedagogia de projetos e a formação humana integral: estudo de caso no município de Coari/AM 1 Mestrando em Educação Profissional e Tecnológica em Rede Nacional – (ProfFEPT)), Instituto Federal de Educação Ciência e Tecnologia do Amazonas – (IFAM), Coari, Amazonas, Brasil, ORCID: https://orcid.org/0000- 0003-4533-0807. E-mail: moyses.hassan@ifam.edu.br Abstract Abstract This article brings to the discussion the relationship between Project Pedagogy and Integral Human Education, seeking, systematically and critically to overcome the teaching-learning vision detached from a critical, inventive formation committed to social transformation. Therefore, it was proposed to analyze the use of Project Pedagogy as a teaching methodology aiming at an integral human formation of the students, perceiving them as active subjects of their learning process. During the research, the main characteristics of Project Pedagogy and its contributions the Integral Human Education are highlighted. To this end, a case study of a class of the Technical Course in Administration in the Integrated Form of the IFAM campus Coari was developed, to point out the discoveries made and the results achieved after the reflection and analysis of the process. Data collection was carried out through the application of semi-structured interviews and remote observation of the participants. The results had their data tabulated and submitted to the treatment of the Discursive Textual Analysis methodology, which indicate that the pedagogy of projects contributed significantly to the formation and development of the cultural, social, cognitive, physical and emotional dimensions of the students. Keywords: Pedagogy of projects. Integral human formation. Keywords: Pedagogy of projects. Integral human formation. La pedagogía de proyectos y la formación humana integral: estudio de caso en la ciudad de Coari/AM y 2 Doutor em Ciência da Computação (UFMG); Instituto Federal de Ciência e Tecnologia do Amazonas – (IFAM), Manaus, Amazonas, Brasil, ORCID: https://orcid.org/0000-0003-1498-2829. E-mail: pinheiro@ifam.edu.br Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 Resumen 1 Mestrando em Educação Profissional e Tecnológica em Rede Nacional – (ProfFEPT)), Instituto Federal de Educação Ciência e Tecnologia do Amazonas – (IFAM), Coari, Amazonas, Brasil, ORCID: https://orcid.org/0000- 0003-4533-0807. E-mail: moyses.hassan@ifam.edu.br 1 1 1 Este artículo trae a discusión la relación entre la Pedagogía de Proyectos y la Educación Humana Integral, buscando, de forma sistemática y crítica, superar la visión de enseñanza-aprendizaje desconectada de una formación crítica, inventiva y comprometida con la transformación social. Por lo tanto, se propuso analizar el uso de la Pedagogía de Proyectos como metodología de enseñanza con el objetivo de una formación humana integral de los estudiantes, percibiéndolos como sujetos activos de su proceso de aprendizaje. Durante la investigación se destacan las principales características de la Pedagogía de Proyectos y sus aportes a la Educación Humana Integral. Para ello, se desarrolló un estudio de caso de una clase del Curso Técnico en Administración en la Forma Integrada del IFAM campus Coari, con el fin de señalar los descubrimientos realizados y los resultados alcanzados luego de la reflexión y análisis del proceso. La recolección de datos se realizó mediante la aplicación de entrevistas semiestructuradas y observación remota de los participantes. Los resultados tuvieron sus datos tabulados y sometidos al tratamiento de la metodología Análisis Discursivo Textual, los cuales indican que la pedagogía de proyectos contribuyó significativamente en la formación y en el desarrollo de las dimensiones culturales, sociales, cognitivas, físicas y emocionales de los estudiantes. Palabras clave: Pedagogía del Proyecto; Formación humana integral. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 2 Introdução No atual mundo globalizado os constantes avanços da tecnologia e as exigências do mercado de trabalho trazem a necessidade de indivíduos que possuam habilidades e competências para atuar no mundo do trabalho e na sociedade, para além dos conhecimentos específicos da profissão. As mudanças destes perfis, algumas apontadas no levantamento das principais habilidades necessárias para o mundo do trabalho como: o pensamento analítico, a criatividade e a flexibilidade estão entre as habilidades necessárias apontadas na pesquisa sobre o futuro dos empregos (FORUM, 2020). Essas mudanças exigem uma nova postura das instituições escolares, principalmente nas escolas de educação profissional, pois de acordo com o Art., 6º das Diretrizes Curriculares Nacionais para a Educação Profissional Técnica de Nível Médio, cabe à Educação Profissional a preparação de profissionais com formação integral, com domínio das técnicas para atuarem no novo mundo do trabalho que está em constante mudança (BRASIL, 2012). Num mundo afetado pela pandemia da COVID-19 em que o mercado de trabalho mudou mais rápido do que o esperado, projetando novos desafios para a sociedade e não se trata do que a educação pode fazer por ela, mas de buscar indícios de como essa sociedade está impactando no uso do conhecimento, no modo do ensinar e do aprender. Em geral, mudanças disruptivas afetam a sociedade nos seus paradigmas, quebram padrões e modificam ou fortalecem valores, inclusive sobre a educação e a sua importância. Segundo Antunes (2008, p. 9), tais mudanças alteram em profundidade a função tradicional da família e o seu papel complementar na Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 2 2 2 educação e, em certa medida, transferem para a escola responsabilidades e papéis que antes não se preparam para realizar. As metodologias tradicionais de ensino, construída com base no modo de produção taylorismo/fordismo com métodos tradicionais de ensino, que privilegiam uma formação fragmentada, já não são suficientes para sustentar todas as mudanças sociais, econômicas, políticas e tecnológicas da atual sociedade e do mundo do trabalho. É consenso o quanto as tecnologias, de maneira geral, têm dinamizado o mundo do trabalho, exigindo profissionais que se posicionem frente às situações diversas e com proatividade na busca pela resolução de problemas, para além das questões tecnicistas. A ação docente impactada por essas mudanças precisa apontar caminhos que direcionem e rompam com os métodos tradicionais de ensino. Introdução Diante disso, há um crescente empenho, também motivado pelas Tecnologias de Informação e Comunicação, para o desenvolvimento de novas metodologias que coloquem o aluno como agente ativo no processo educacional. De fato, como a figura central, o ator principal, do processo de ensino e aprendizagem. Nesse sentido, este trabalho objetiva trazer à discussão as contribuições da pedagogia de projetos como metodologia que possibilita uma formação humana integral, utilizada como ferramenta pedagógica na melhoria do processo de ensino e aprendizagem, onde o aluno seja agente ativo na construção do conhecimento e o professor um mediador das atividades desenvolvidas no cotidiano escolar. Esta pesquisa se assenta a partir do entendimento de Weffort et al. (2019) acerca do conceito de formação humana integral e das dimensões que as compõe. Para este fim, embasado nas ideias de Amaral (2000), Araújo e Frigotto (2015), Behrens (2014), Berbel (2011), Moreira (2002) e Santos (2006) aplicou-se a pedagogia de projetos a uma turma de professores e alunos do ensino técnico integrado ao médio, extraindo desta experiência a percepção de ambos, alunos e professores, acerca da pedagogia de projetos e o seu impacto na formação humana integral. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 3 O contexto da pedagogia de projetos e a formação humana integral na educação profissional A história da educação no Brasil pode ser compreendida a partir de seu contexto político, econômico, social, cultural e valorativo que a envolve em determinado momento, ou seja, em conformidade aos aspectos sociais e históricos 3 3 que emolduram o desenvolvimento do processo de formação educacional no país. Gonçalves (2005, p. 13), ao buscar as bases históricas e filosóficas que compuseram e compõe o campo educacional brasileiro, cita que “é necessário observar em que moldes a educação no Brasil foi e é constituída”. Segundo o autor, é uma construção da coletividade e suas transformações acontecem de forma lenta e gradual, principalmente no campo das ideias, nunca de forma neutra e nem apolítica, pois envolve interesses que ultrapassam o âmbito escolar. Para Silva (2017, p. 98), “[...] a educação não se desenvolve de uma maneira positiva se mantida sob a lógica economicista, porque é mais uma ideologia e reforça uma lógica dual de educação”. A dualidade da educação marca historicamente a educação no Brasil. O Decreto nº 7.566 de 23 de setembro de 1909, cria a rede federal de educação profissional composta por várias escolas congêneres de ofícios. Essas instituições visavam principalmente “[...] amparar os órfãos e os demais desvalidos da sorte” (MOURA, 2007, p. 6). Os objetivos se caracterizavam pela necessidade de intervir na situação social dos jovens, criando aquilo que era um dos primeiros deveres do Governo da República, a saber, formar cidadãos úteis à Nação, não só habilitando-os com o conhecimento técnico e intelectual, como também “fazê-los adquirir hábitos de trabalho profícuo, que os afastará da ociosidade ignorante, escola do vício e do crime” (BRASIL, 1909, p. 1). Outra importante ação foi a Reforma Capanema, em 1942, que organizou o “ensino industrial, reformou o ensino comercial, reformulou o ensino secundário e criou o Serviço Nacional de Aprendizagem Industrial – SENAI”. Nesse período a dualidade educacional assumida pelo ensino médio se apresenta nas “diversas modalidades e pela segmentação e diferenciação dos processos educativos que tratam as diferentes classes sociais de forma desigual” (SILVA, 2017, p. 28). Já nos anos de 1990, com a edição do Decreto n.º 2.208/1997, a dualidade educacional foi reforçada, pois determinava a separação do ensino técnico do médio. Só em 2004, o Decreto n.º 5.154 revoga o Decreto anterior e abre a possibilidade de integração do ensino médio com a educação profissional e mantêm as formas de ofertas subsequente e concomitante. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 O contexto da pedagogia de projetos e a formação humana integral na educação profissional O primeiro sinal de intenção em mudar esta dualidade veio com as Diretrizes Curriculares Nacionais para a Educação Profissional Técnica de Nível Médio, aprovada pela Resolução nº 6, de 20 de setembro de 2012, que estabeleceram no Art. 6 dos princípios da Educação Profissional Técnica de Nível Médio a relação e a Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 4 4 articulação entre a formação desenvolvida no Ensino Médio e a preparação para o exercício das profissões técnicas, visando à formação integral do estudante. Claro que ainda se tinha a formação humana integral como proposta, mas se configurava um avanço. De acordo com as diretrizes, a proposta de educação que promova a formação humana integral deve pautar-se por princípios norteadores que garantam o desenvolvimento de todas as dimensões do ser humano. Dessa forma, nos apropriamos das ideias de Weffort et al. (2019, p.17) que, ao apresentarem a Educação Integral enquanto concepção educacional destacam que ela se sustenta por quatro princípios básicos: “equidade, inclusão, contemporaneidade e sustentabilidade”. Esses princípios apresentam a educação integral como uma proposta atual, e “fundamental para a superação das desigualdades”. A autora ainda destaca que a formação humana: [...] é um processo integral. Acontece o tempo inteiro, ao longo de toda a vida e em todos os espaços. É também trajetória social e trilha individual, em que valores, linhas de pensamento e formas de organização social se fundem com as escolhas, preferências e habilidades de cada um. A defesa da Educação Integral pressupõe garantir o desenvolvimento humano em todas as suas dimensões: intelectual, física, afetiva, social e cultural (WEFFORT et al. 2019, p. 16). Dessa forma, se entende que formação integral significa completo, inteiro, total, pleno, portanto, se trata de uma educação que idealize o ser humano por inteiro em todas as suas dimensões e ao longo de sua vivência. A proposta de educação integral traz o ser humano para o centro do processo educativo e o reconhece como ser social construtor da sua história, competente e multidimensional (WEFFORT et al. 2019, p. 16). A compreensão de cada dimensão que compõe o ser humano é fundamental no processo educativo, pois é preciso ter claro quais dimensões podem ser desenvolvidas através das diversas metodologias. Weffort et al. (2019, p. [...] é um processo integral. Acontece o tempo inteiro, ao longo de toda a vida e em todos os espaços. É também trajetória social e trilha individual, em que valores, linhas de pensamento e formas de organização social se fundem com as escolhas, preferências e habilidades de cada um. A defesa da Educação Integral pressupõe garantir o desenvolvimento humano em todas as suas dimensões: intelectual, física, afetiva, social e cultural (WEFFORT et al. 2019, p. 16). Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 5 Dimensão física: relaciona-se à compreensão das questões do corpo, do autocuidado e da atenção à saúde, da potência e da prática física e motora. Dimensão emocional ou afetiva: refere-se às questões do autoconhecimento, da autoconfiança e capacidade de auto realização, da capacidade de interação na alteridade, das possibilidades de auto reinvenção e do sentimento de pertencimento. Dimensão social: refere-se à compreensão das questões sociais, à participação individual no coletivo, ao exercício da cidadania e vida política, O contexto da pedagogia de projetos e a formação humana integral na educação profissional 27) ao definir e conceituar as dimensões da formação humana integral apresentam: Dimensão física: relaciona-se à compreensão das questões do corpo, do autocuidado e da atenção à saúde, da potência e da prática física e motora. Dimensão emocional ou afetiva: refere-se às questões do autoconhecimento, da autoconfiança e capacidade de auto realização, da capacidade de interação na alteridade, das possibilidades de auto reinvenção e do sentimento de pertencimento. Dimensão social: refere-se à compreensão das questões sociais, à participação individual no coletivo, ao exercício da cidadania e vida política, Dimensão física: relaciona-se à compreensão das questões do corpo, do autocuidado e da atenção à saúde, da potência e da prática física e motora. p Dimensão social: refere-se à compreensão das questões sociais, à participação individual no coletivo, ao exercício da cidadania e vida política, quisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 5 Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 5 5 5 5 ao reconhecimento e exercício de direitos e deveres e responsabilidade para com o coletivo. Dimensão intelectual: refere-se à apropriação das linguagens, códigos e tecnologias, ao exercício da lógica e da análise crítica, à capacidade de acesso e produção de informação, à leitura crítica do mundo. Dimensão cultural: diz respeito à apreciação e fruição das diversas culturas, às questões identitárias, à produção cultural em suas diferentes linguagens, ao respeito das diferentes perspectivas, práticas e costumes sociais. Na perspectiva da formação humana integral, existe a necessidade de os docentes buscarem novos modelos de ensino e novas metodologias que foquem no protagonismo dos estudantes e favoreçam a motivação e a promoção da autonomia. De acordo com Berbel (2011, p 28), os professores precisam adotar novas estratégias em suas práticas pedagógicas, o que exige mudanças de atitudes e criem as “condições básicas para o surgimento do estilo motivacional que promova a autonomia”, e conduzam a formação de futuros profissionais nas mais diversas áreas, o que pode ser estimulada por meio dos métodos ativos de aprendizagem. Dentre os métodos ativos de aprendizagem, destacamos a pedagogia de projetos, por permitir a ruptura com o modelo fragmentado de conhecimento onde o aluno busca informações, lê, conversa, anota dados, calcula, elabora gráficos e, por fim, transforma tudo em ponto de partida para o exercício ou aplicação na vida. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 6 O contexto da pedagogia de projetos e a formação humana integral na educação profissional Dentre as contribuições aos discentes pela vivência do método de projetos, podemos destacar: proporcionar um currículo vivo, seguir objetivos organizados, possibilitar a aprendizagem real e significativa, que seja ativa, interessante e atrativa; que se concentre na aprendizagem do aluno; que desenvolva o pensamento e ponto de vista diferente e desperte o desejo pela conquista, iniciativa, investigação, criação e responsabilidade, levam os alunos a se inserirem conscientemente na vida social e/ou profissional (BERBEL, 2011, p. 32). O que este trabalho aponta é a perspectiva de contribuir formação humana integral por meio da pedagogia de projetos. A pedagogia de projetos surgiu no início do século com John Dewey e outros representantes da chamada Pedagogia Renovada Progressivista, em oposição aos princípios da escola tradicional. “Foi na América do Norte que Dewey e seu discípulo William Kilpatrick criaram a ideia de projetos como uma atitude didática”. Esse movimento que teve início na Europa chegou ao Brasil conhecido como Escola Nova e foi divulgado por Anísio Teixeira e Lourenço Filho (AMARAL, 2000, p. 37). A pedagogia de projetos como método ativo de aprendizagem surge num contexto em que a formação dos estudantes tinha como base o modelo fordista que preparava para o mercado de trabalho. Diferencia-se das demais pelo diálogo entre 6 6 6 teoria e prática e o compromisso que assume com a formação humana integral e a transformação social (BEHRENS, 2014, p. 98). A pedagogia de projetos, em certa medida, inverte a base lógica do currículo – os conhecimentos formais – de tal modo que os conteúdos passam a ser estudados de forma ativa, onde o aluno parte de questões norteadoras, ou problemas do seu dia a dia, para resolvê-las no processo de aprendizagem através de projetos, sendo o aluno o motor e a figura central desse processo. Como explica Carlos Otávio Moreira, em estudo sobre o pensamento de John Dewey (MOREIRA, 2002), “o fundamento desses percursos se encontra na visão pragmática de que o conhecimento é o produto da relação entre uma questão que surge e a atividade que a soluciona ou explica” (MOREIRA, 2002, p. 95). Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 7 O contexto da pedagogia de projetos e a formação humana integral na educação profissional Trabalhar com a pedagogia de projetos, embora exija várias habilidades e competências do professor, faz com que a continuidade da prática e a reflexão sobre a mesma viabilizem muitos benefícios a ele e aos alunos, por que cria condições para que o aluno apresente os saberes prévios que possui sobre o assunto investigado e adquira novos saberes ao longo do projeto. Na pedagogia de projetos, o aluno se torna o principal agente da aprendizagem, primando por uma aprendizagem por descoberta ativa, orientado pela ação mediadora do professor. Nesse processo, a força criativa se desenvolve, principalmente por meio de “estratégias de problematização da realidade e dos conteúdos escolares”, gerando a busca por estratégias, teorias e práticas que sejam capazes de dar apoio aos discentes na resolução de problemas cotidianos e históricos (ARAÚJO; FRIGOTTO, 2015, p. 74). O projeto também permite ao discente a oportunidade de se mobilizar na busca e na construção de conhecimentos novos, exercitando a desenvoltura, a sociabilidade, a criatividade dentre outras competências, utilizando o método científico, que permite a formação do espírito científico, desenvolve a autoestima e a capacidade de gerir e solucionar problemas (SANTOS ,2006, p. 60). O raciocínio supracitado se alinha ao que Araújo e Frigotto (2015, p. 73), que tecem considerações a respeito da existência de diferentes possibilidades de procedimentos de ensino que podem favorecer o projeto de ensino integrado quando são organizados para promover a autonomia, por meio da valorização da atividade e da problematização, e para cultivar o sentimento de solidariedade, mediante o trabalho coletivo e cooperativo. Os autores destacam que a “pedagogia deweyana, de cunho liberal, também se caracteriza como uma pedagogia ativa”. Ela surge - de 7 7 acordo com os autores - da renúncia dos educadores à pedagogia tradicional, que trabalhava a passividade dos alunos e, em certa medida, também dos docentes. Abordam que o que distingue essa pedagogia das demais é o compromisso com a transformação social, através da formação integral dos estudantes. Neste contexto da pedagogia de projetos, o aluno desenvolve não apenas os conteúdos previstos no currículo, mas todas as habilidades e competências necessárias à sua execução, entre estas trabalhar em equipe, senso crítico, liderança, empatia, organização, resiliência, visão de mundo, entre outras que contribuem para a sua formação humana integral. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 8 Metodologia O sentido proposto neste trabalho foi o de não ficar apenas na elaboração teórica, ainda que a teoria seja o prumo usado, mas analisar de modo aplicado à teoria em busca de compreensões acerca do campo prático, uma vez que ao aplicar-se a teoria à realidade é possível que novas perspectivas sejam elaboradas e novos conhecimentos possam ser colocados em prática, permitindo novas soluções aos problemas enfrentados. O campo de investigação dessa pesquisa foi o Instituto Federal de Educação, Ciência e Tecnologia do Amazonas – IFAM, Campus Coari, mais especificamente uma turma do curso de Administração. Coari é um município que fica localizado a 365Km de Manaus, não possuindo ligação via rodovia com a capital, e cujo meio de deslocamento principal é via fluvial, de 1 a 2 dias, ou aéreo, 1h 25m. Atualmente, o Campus conta com um quadro funcional com corpo docente composto por 43 professores (sendo 38 efetivos e 05 substitutos), 35 técnicos-administrativos e 643 educandos matriculados, distribuídos nos vários cursos técnicos. Os procedimentos metodológicos utilizados nesta pesquisa centram-se numa abordagem de natureza aplicada, qualitativa. Os instrumentos adotados para a coleta de dados foram os seguintes: questionários, entrevista e observação participante. Foram utilizados dois questionários para os alunos e um para os professores. Os questionários utilizados foram estruturados com perguntas fechadas e abertas possibilitando maior liberdade de respostas. Os questionários dos alunos e o questionário dos professores foram aplicados através da plataforma Google Forms. Os questionários ajudaram na compreensão do perfil dos participantes, em relação aos trabalhos em grupos, familiaridade com projetos interdisciplinares, na 8 identificação de suas experiências com métodos ativos de aprendizagem e conhecimento sobre a formação humana integral. A entrevista foi outro instrumento para a coleta de dados que nos permitiu, de acordo com Gil (2008, p. 110), uma “interação social” com o intuito de obter informações dos alunos em relação ao tema da pesquisa. Nossa entrevista se deu de forma diferenciada, pois devido a pandemia COVID-19 e o distanciamento social não foi possível realizar a entrevista aos moldes convencionais. Realizamos primeiramente uma reunião via Aplicativo Google Meet com os alunos e orientamos sobre os procedimentos de como seria a entrevista. Enviamos um roteiro com as perguntas via grupo da rede social WhatsApp e os alunos gravaram um vídeo com base nas perguntas e encaminharam no grupo. É descrita como um processo que se inicia com uma unitarização em que os textos são separados em unidades de significado. Estas unidades por si mesmas podem gerar outros conjuntos de unidades oriundas da interlocução empírica, da interlocução teórica e das interpretações feitas pelo pesquisador. Neste movimento de interpretação do significado atribuído pelo autor exercita-se a apropriação das palavras de outras vozes para compreender melhor o texto. Depois da realização desta unitarização, que precisa ser feita com intensidade e profundidade, passa-se a fazer a articulação de significados semelhantes em um processo denominado de categorização. Neste processo reúnem-se as unidades de significado semelhantes, podendo gerar vários níveis de categorias de análise. A análise textual discursiva tem no exercício da escrita seu fundamento enquanto ferramenta mediadora na produção de significados e por isso, em processos recursivos, a análise se desloca do empírico para a abstração teórica, que só pode ser alcançada se Metodologia Cada vídeo foi transcrito e depois tabulado para ser analisado utilizando a técnica Análise Textual Discursiva (MORAES; GALIAZZI, 2006). Os sujeitos da pesquisa foram os alunos do 2º ano do curso Técnico de Nível Médio em Administração na forma integrada, e foi desenvolvido com os seguintes participantes: 35 alunos e 5 professores, sendo 1 professor da base comum e 4 professores da base técnica. A opção pela turma e série se justificou pelo fato de os alunos do 2º ano já estarem adaptados à instituição e possuírem mais disciplinas profissionais. No contexto específico de análise de dados, foi utilizado como instrumento de mediação prática a Análise Textual Discursiva (ATD), pelo fato de ser uma metodologia de análise de textos e de discursos no âmbito da pesquisa qualitativa muito utilizada em pesquisas nas ciências humanas, permitiu a análise do material produzido no contexto das entrevistas. A Análise Textual Discursiva (ATD), segundo Moraes e Galiazzi (2006, p. 118): É descrita como um processo que se inicia com uma unitarização em que os textos são separados em unidades de significado. Estas unidades por si mesmas podem gerar outros conjuntos de unidades oriundas da interlocução empírica, da interlocução teórica e das interpretações feitas pelo pesquisador. Neste movimento de interpretação do significado atribuído pelo autor exercita-se a apropriação das palavras de outras vozes para compreender melhor o texto. Depois da realização desta unitarização, que precisa ser feita com intensidade e profundidade, passa-se a fazer a articulação de significados semelhantes em um processo denominado de categorização. Neste processo reúnem-se as unidades de significado semelhantes, podendo gerar vários níveis de categorias de análise. A análise textual discursiva tem no exercício da escrita seu fundamento enquanto ferramenta mediadora na produção de significados e por isso, em processos recursivos, a análise se desloca do empírico para a abstração teórica, que só pode ser alcançada se squisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 9 9 o pesquisador fizer um movimento intenso de interpretação e produção de argumentos. Este processo todo gera meta-textos analíticos que irão compor os textos interpretativos. o pesquisador fizer um movimento intenso de interpretação e produção de argumentos. Este processo todo gera meta-textos analíticos que irão compor os textos interpretativos. Metodologia Buscou-se, portanto, abordar a análise dos dados no sentido de produzir novas perspectivas relacionada ao fenômeno examinado. Foi realizada, num primeiro momento, a consideração analítica dos textos e, em seguida, a categorização das unidades de análise no sentido de se chegar aos metatextos, o que, “A partir disso, possibilita-se a formação de novas estruturas de compreensão dos fenômenos sob investigação, expressas então em forma de produções escritas” (MORAES, 2003, p 101-2). Em função de a pesquisa tratar com seres humanos, o projeto de pesquisa foi submetido ao Comitê de Ética em Pesquisa com Seres Humanos (CEPSH) do IFAM, por meio da Plataforma Brasil, N. 4.640.483 recebeu parecer favorável para sua realização. Os participantes foram informados sobre a adesão voluntária à pesquisa mediante leitura e assinatura de Termo de Consentimento Livre Esclarecido pelos maiores de 18 anos ou por seus responsáveis legais. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/educitec.v8.2015 10 ec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 DOI: https://doi.org/10.31417/ educitec.v8.2015 Resultados e Discussão A pesquisa se iniciou com uma formação dos docentes participantes acerca da pedagogia de projetos, com uma oficina proferida por professor do Mestrado nacional em Educação Profissional Tecnológica (PROFEPT) – Polo IFAM, especialista no tema, visando nivelar o conhecimento de todos e torná-los aptos a desenvolverem a pedagogia de projetos com os alunos. A seguir, os projetos foram desenvolvidos conforme a Figura 1: 10 10 Figura 1 – Etapas de Desenvolvimento do Projeto Fonte: Adaptado de Santos (2006) Figura 1 – Etapas de Desenvolvimento do Projeto Fonte: Adaptado de Santos (2006) A escolha do tema do projeto foi discutida com os professores e coube-lhes provocar nos alunos a motivação necessária e envolvimento em todas as fases do projeto. De acordo com Amaral (2000, p. 39), nem sempre é possível trabalhar um tema escolhido exclusivamente pelos alunos. O tema do projeto foi “O papel das mídias digitais para o desenvolvimento da economia em tempos de pandemia”, supervisionado pelo Departamento de Ensino e Coordenação do curso, foi desenvolvido entre os dias 05 de abril a 19 de junho de 2021. O projeto foi desenvolvido de acordo com as seguintes etapas assim descritas: apresentação e discussão do tema, formação de Grupos de Trabalhos – GT’s, pesquisa bibliografia e de campo, discussão em grupo de trabalho e escrita da síntese, elaboração do projeto de vídeo, elaboração da comunicação científica – resumo, edição do vídeo de apresentação e avaliação do projeto. Todas as etapas do projeto foram apresentadas e discutidas de forma virtual pelo aplicativo Google Meet. Esses encontros virtuais foram os momentos em que os alunos foram instigados a apresentar suas opiniões e sugestões sobre a temática. A problematização foi fundamental para motivar os alunos e envolvê-los na proposição de estratégias e ações para a resolução do problema. A problemática proposta no projeto foi o quadro crítico que inúmeros microempreendedores e pequenos empresários passaram a vivenciar diante do cenário pandêmico do Covid-19. DOI: https://doi.org/10.31417/ educitec.v8.2015 11 11 Devido às necessárias restrições impostas pela pandemia, as mídias sociais passaram a assumir papel predominante na manutenção e continuidade das atividades comerciais em modalidade remota. Dessa forma, buscou-se compreender de que forma as mídias digitais contribuíram para a manutenção e continuação das atividades econômicas no município, tendo como resultado de cada projeto uma proposta de uso destas mídias para mitigar o problema. isas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X ec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 Resultados e Discussão Durante a execução dos projetos, os alunos realizaram uma pesquisa de campo, onde escolheram uma empresa local e realizaram uma entrevista com o proprietário procurando identificar as principais estratégias adotadas para superação da crise econômica ocasionada pela pandemia, e como as mídias digitais impulsionaram as vendas num período em que as atividades não essenciais como o comércio não eram permitidas. Após a pesquisa de campo, os alunos se reuniram para discussão em grupo, e de forma coletiva, realizaram uma análise crítica e reflexiva, onde foram desafiados a exporem seus textos individuais com o objetivo de produzir conhecimento coletivamente (BEHRENS, 2015, p. 110). Como acompanhamento do projeto, foram utilizadas estratégias de reuniões virtuais, discussão com os docentes orientadores em redes sociais, e apresentações periódicos em formato de pitch (apresentação resumida de 3 minutos). O projeto foi finalizado com uma apresentação no formato de evento científico, e estruturado virtualmente, sendo utilizadas as plataformas virtuais adequadas e recomendadas pela instituição. O evento teve início com a apresentação dos resultados no formato audiovisual por parte dos educandos, com uma carga horária de 8 horas, seguida da avaliação pelos docentes. Para além das questões cognitivas necessárias ao desenvolvimento dos projetos, os alunos foram desafiados a desenvolverem outras habilidades, tais como atuar em times, administrar conflitos, expor ideias e discutir de maneira adequada o contrário, se inserir no contexto socioeconômico do município, trazendo para si uma educação significativa, exercer liderança, senso crítico, entres outras habilidades socioemocionais que contribuem para uma formação humana integral. Os resultados foram coletados por um questionário inicial, aplicado a 35 alunos e composto de onze questões objetivas, sendo seis referentes a informações básicas como curso e idade, e outras cinco a respeito de conceitos como contextualização e metodologia de projetos, etc. Contudo, a avaliação mais interessante foi a entrevista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 12 ao término dos projetos, cujos dados foram sistematizados a partir da Análise Textual Discursiva (ATD), descrita em Moraes e Galiazzi (2006). Com isto, as respostas foram categorizadas, seguindo a ATD, cujo resultado se apresenta no Quadro 1. Quadro 1 – Processo de categorização das unidades de significados CATEGORIAS INICIAIS CATEGORIAS INTERMEDIÁRIAS CATEGORIAS FINAIS Desenvolvimento de competências e habilidades para o desenvolvimento profissional. Formação de valores e atitudes. Dimensão emocional ou afetiva Percepção da liderança como uma habilidade relacionada a ações e atitudes. ec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 Resultados e Discussão Auto cuidado com a saúde mental. Habilidades socioemocionais necessárias para o mundo do trabalho na atualidade. Saúde do corpo necessária para o desenvolvimento intelectual. Compreensão das questões do cuidado com o corpo. Dimensão Física Capacidade de compreender a realidade social diante dos problemas ocasionados pela pandemia. Conhecimento acadêmico necessário para a formação profissional. Dimensão Intelectual Habilidade para realizar pesquisa e produzir conhecimento. Construção de conhecimentos interdisciplinares e significativos. Uso da metodologia da projetos para a resolução de problemas da vida real de forma cooperativa. Métodos ativos de aprendizagem. Dimensão social Criação de vínculos de amizade e de sentimento de união entre os participantes do projeto. Compreensão das questões sociais, e reconhecimento da responsabilidade para com o coletivo. Desenvolvimento dos sentimentos de empatia e tolerância para com o outro. Respeito a diversidade cultural. Dimensão Cultural Desenvolvimento da consciência crítica que implique mudanças de pensamento ou comportamento. Fonte: Elaborado pelos autores (2022) As categorias que emergiram das falas dos participantes foram analisadas em forma de metatextos e fundamentaram a compreensão acerca das contribuições da metodologia de projetos interdisciplinares na formação humana integral na Educação Profissional e Tecnológica. Em todas as dimensões, ficou caracterizada uma evolução dos alunos, em maior ou menor grau, relativo às habilidades socioemocionais. DOI: https://doi.org/10.31417/ educitec.v8.2015 13 Como exemplos, um aluno quando questionado acerca do desenvolvimento das atividades, verbalizou que há a percepção de que “as atividades trazem valores pra gente como, por exemplo: experiência, conhecimento e compreensão; atividades que requer esforços, mas que geram um conhecimento dinâmico!”. Outros participantes deram ênfase no trabalho em times, um deles textualmente coloca que “eu consegui trabalhar bem em um grupo, foi produtivo, interessante e motivador”. Outro aspecto da formação humana integral, a percepção e visão de mundo, se evidenciou em algumas falas, como na fala de um aluno, ao afirmar que “o projeto foi muito importante para a construção de conhecimentos, ele também enriqueceu a nossa visão, a visão dos alunos de mundo”. Um bom exemplo de que precisaram desenvolver a administração de conflitos e a resiliência, se percebe na seguinte colocação de um aluno: “houve alguns contratempos, mas nada que o grupo não conseguisse resolver, conseguimos encaixar cada ideia na nossa atividade, que com esforço e dedicação concluímos”. Resultados e Discussão Em relação ao desenvolvimento das dimensões cognitiva, social e cultural os resultados da pesquisa demostraram que os alunos foram capazes de analisar a realidade a partir de conhecimentos interdisciplinares significativos, realizaram pesquisa, analisaram as informações coletadas e de forma coletiva respeitando as diversas opiniões tomaram decisões e buscaram soluções para as atividades e problemas propostos no projeto. Os professores participantes do projeto também foram entrevistados, e com tratamento similar de coleta e análise de dados que, no entanto, foram utilizados para a elaboração de um produto educacional, ainda não publicado, que auxilia professores a utilizarem a pedagogia de projetos como prática no processo de ensino e aprendizagem, visando uma formação humana integral. Considerações finais A história da educação profissional no Brasil é fortemente marcada pela dualidade distinta de educação oferecida às camadas mais pobres e outra oferecida às classes mais ricas da sociedade. A educação brasileira na atualidade deve buscar “romper com a dicotomia entre educação básica e técnica, resgatando o princípio da formação humana em sua totalidade” (RAMOS, 2014, p. 24). Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi org/10 31417/ educitec v8 2015 dos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 14 14 Compreendemos que a pedagogia de projetos como método ativo de aprendizagem se contrapõe aos métodos tradicionais de ensino que privilegiam uma formação fragmentada, pois assume um compromisso com a formação completa do ser humano e com a transformação social. Com essa visão nos debruçamos para analisar as contribuições da pedagogia de projetos na formação humana integral na Educação Profissional e Tecnológica de Nível Médio, a partir do contexto do IFAM, Campus Coari. A ideia foi de contribuir para a prática docente e para o processo de ensino-aprendizagem. A pesquisa nos mostrou após a análise dos dados que o projeto contribuiu para a formação de valores e atitude, bem como na compreensão do autocuidado com a saúde mental necessária para o mundo do trabalho e para uma vivência saudável. Mostrou ainda que o uso da pedagogia de projetos auxilia para a resolução de problemas da vida real de forma cooperativa permitindo a criação de vínculos de amizade e de sentimento de união entre os participantes do projeto. A pesquisa alcançou seu objetivo de realizar uma análise acerca das contribuições da pedagogia de projetos como prática de ensino que conduz ou que, pelo menos, contribui, na formação humana integral, a partir do contexto do IFAM, Campus Coari. Todo o processo teve dificuldades inerentes ao contexto da pandemia COVID 19, ao convencimento de professores para atuarem num projeto para além de suas atribuições do dia a dia, e das próprias dificuldades de um Campus situado em uma pequena cidade no interior do Amazonas. DOI: https://doi.org/10.31417/ educitec.v8.2015 ec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X Referências AMARAL, A. L. Um olhar sobre os projetos de trabalho. In: Salto para o Futuro: um olhar sobre a escola. Brasília: MEC/SEED, 2000. p. 37- 44. AMARAL, A. L. Um olhar sobre os projetos de trabalho. In: Salto para o Futuro: um olhar sobre a escola. Brasília: MEC/SEED, 2000. p. 37- 44. ANPED. Nota de Repúdio às Novas Diretrizes Curriculares Nacionais para Educação Profissional e Tecnológica. 2021. Disponível em: https://www.anped. org.br/sites/default/file s/images/manifesto_ do_gt09_contra_dcnept_jan _2021_fev.2021.docx: Acesso em: 10 jul. 2021. Considerações finais Entretanto, os resultados obtidos demonstraram que a pedagogia de projetos tem um poder expressivo de motivar alunos e professores, pois eles se percebem responsáveis por sua própria formação, entendem que há um significado e um objetivo tangível naquilo que estão aprendendo, e esta motivação dos próprios alunos foi o maior responsável pelo sucesso da pesquisa. A experiência de atuar em modo remoto também permitiu compreender limites e possibilidades da pedagogia de projetos. De maneira geral, propor um processo de ensino e aprendizagem por meio de um projeto oportuniza aos educandos desenvolverem as dimensões da formação humana integral: intelectual, física, emocional, social e cultural, como ficou evidenciado neste estudo de caso. A intenção aqui não foi o de esgotar o tema da pedagogia de projetos como método de ensino e aprendizagem, o que propomos é contribuir com o debate e 15 apresentar os indícios de um caminho que direcione e motive novos pesquisadores a se aprofundarem sobre novas formas de ensinar e de aprender, especialmente sobre a pedagogia de projetos e suas contribuições para formar o ser humano por completo. apresentar os indícios de um caminho que direcione e motive novos pesquisadores a se aprofundarem sobre novas formas de ensinar e de aprender, especialmente sobre a pedagogia de projetos e suas contribuições para formar o ser humano por completo. ANTUNES, C. Novas maneiras de ensinar, novas formas de aprender. Porto Alegre: Artmed, 2008. ARAUJO, R. M. de L.; FRIGOTTO, G. Práticas pedagógicas e ensino integrado. Revista Educação em Questão, [S. l.], v. 52, n. 38, p. 61–80, 2015. Disponível em: https://periodicos.ufrn.br/educacaoemquestao/article/view/7956. Acesso em: 29 jun. 2022. BEHRENS, M. A. Metodologia de projetos: aprender e ensinar para a produção do conhecimento em uma visão complexa. In: TORRES, P. L. (org.) Complexidade: Redes e Conexões na Produção do Conhecimento. Curitiba, SENAR, 2015. BEHRENS, M. A. Metodologia de projetos: aprender e ensinar para a produção do conhecimento em uma visão complexa. In: TORRES, P. L. (org.) Complexidade: Redes e Conexões na Produção do Conhecimento. Curitiba, SENAR, 2015. BERBEL, N. A. As Metodologias Ativas e a Promoção da Autonomia de Estudantes. N.Semina: Ciências Sociais e Humanas, Londrina, v. 32, n. 1, p. 25-40, jan./jun. 2011. Disponível em: http://www.uel.br/revistas/uel/index.php/se minasoc/article/view/10326/10999. Acesso em: 19 ago. 2021. BRASIL. Decreto nº 7.566, de 23 de setembro de 1909. Crêa nas capitaes dos Estados da Republica Escolas de Aprendizes Artifices, para o ensino profissional primário e gratuito. Diário Oficial da União. Brasília, DF, 26 de setembro de 1909. BRASIL. Resolução CNE/CEB 6/2012. Diário Oficial República Federativa do Brasil, Poder Executivo, Brasília-DF, 21 set. 2012. FÓRUM ECONÔMICO MUNDIAL. Future of jobs survey. Disponível em: https://www.weforum.org/reports/the-future-of-jobs-report-2020. Acesso em: 29 jun. 2022. GIL, A. C. Métodos e técnicas de pesquisa social. 6. ed. São Paulo: Atlas, 2008. GIL, A. C. Métodos e técnicas de pesquisa social. 6. ed. São Paulo: Atlas, 2 GONÇALVES, N. G. F. Fundamentos Históricos e Filosóficos da Educação Brasileira. Curitiba: IBPEX, 2005. ec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 DOI: https://doi.org/10.31417/ educitec.v8.2015 16 MORAES, R.; GALIAZZI, M. C. Análise textual discursiva: processo reconstrutivo de múltiplas faces. Ciência educ., Bauru, v. 12, n. 01, p. 117-128, abr. 2006. Disponível em: http://educa.fcc.org.br/scielo.php?script=sci_arttext&pid=S1516- 73132006000100009&lng=pt&nrm=iso. Acesso em: 29 jun. 2022. MOREIRA, C. O. Entre o indivíduo e a sociedade: um estudo da filosofia da educação de John Dewey. Bragança Paulista: EDUSF, 2002. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi org/10 31417/ educitec v8 2015 Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 MOREIRA, C. O. Entre o indivíduo e a sociedade: um estudo da filosofia da educação de John Dewey. Bragança Paulista: EDUSF, 2002. RAMOS, M. Trabalho e Educação no Brasil: um estudo a partir da formação de trabalhadores técnicos da Saúde. Rio de Janeiro: Ed. UFRJ e EPSJV/Fiocruz, 2010. RAMOS, M. "Metodologias ativas": entre movimentos, possibilidades e propostas. In: SOUZA, R. M. P; COSTA, P. P. (Org.). Redescola e a nova formação em saúde pública. Rio de Janeiro: ENSP/REDESCOLA, 2017. RAMOS, M. "Metodologias ativas": entre movimentos, possibilidades e propostas. In: SOUZA, R. M. P; COSTA, P. P. (Org.). Redescola e a nova formação em saúde pública. Rio de Janeiro: ENSP/REDESCOLA, 2017. SANTOS, G. R. C. M. dos. A metodologia de ensino por projetos. Curitiba: Ed. Ibpex, 2006. SANTOS, G. R. C. M. dos. A metodologia de ensino por projetos. Curitiba: Ed. Ibpex, 2006. SILVA, G. A. da. A formação humana integral e o ensino médio (integrado) no contexto das reformas educacionais (2016-2017): uma parada do velho novo? 2017. 102 f. Dissertação (Mestrado em Sociologia) - Universidade Federal de Goiás, Goiânia, 2017. Disponível em: http://repositorio.bc.ufg.br/ tede/handle/tede/8055. Acesso em: 10 jun. 2022. SILVA, G. A. da. A formação humana integral e o ensino médio (integrado) no contexto das reformas educacionais (2016-2017): uma parada do velho novo? 2017. 102 f. Dissertação (Mestrado em Sociologia) - Universidade Federal de Goiás, Goiânia, 2017. Disponível em: http://repositorio.bc.ufg.br/ tede/handle/tede/8055. Acesso em: 10 jun. 2022. WEFFORT, H. F; ANDRADE, J. P; COSTA, N. G. Currículo e educação integral na prática: uma referência para estados e municípios. São Paulo: Associação Cidade Escola Aprendiz, 2019. Recebido: 17/05/2022 Aprovado: 27/09/2022 Publicado: 17/10/2022 Como citar (ABNT): SILVA SOBRINHO, M. H.; QUEIROZ NETO, J. P. A pedagogia de projetos e a formação humana integral: estudo de caso no município de Coari/AM. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, v. 8, e201522, 2022. Contribuição de autoria: Moyses Hassan da Silva Sobrinho: Conceituação, curadoria de dados, análise formal, investigação, metodologia, administração de projeto, supervisão, validação, escrita (rascunho original). José Pinheiro de Queiroz Neto: Conceituação, metodologia, supervisão, validação, escrita (revisão e edição). Editor responsável: Iandra Maria Weirich da Silva Coelho. Direito autoral: Este artigo está licenciado sob os termos da Licença Creative Commons-Atribuição 4.0 Internacional Este artigo está licenciado sob os termos da Licença Creative Commons-Atribuição 4.0 Internacional. Educitec - Revista de Estudos e Pesquisas sobre Ensino Tecnológico, Manaus (AM), v. 8, e201522, 2022. ISSN: 2446-774X DOI: https://doi.org/10.31417/ educitec.v8.2015 17 17 17 18
https://openalex.org/W4391042822	https://www.qeios.com/read/CEEZ2C/pdf	English	null	Review of: "How E-School Counseling Has Supported Learners Facing Pandemic Challenges: Results and Perspectives for Counseling Professionals"	null	2,024	cc-by	119	Qeios, CC-BY 4.0 · Review, January 20, 2024 Qeios ID: CEEZ2C · https://doi.org/10.32388/CEEZ2C Jan Hauke1 1 Adam Mickiewicz University of Poznan Potential competing interests: No potential competing interests to declare. Review of: "How E-School Counseling Has Supported Learners Facing Pandemic Challenges: Results and Perspectives for Counseling Professionals" Jan Hauke1 Potential competing interests: No potential competing interests to declare. The article is devoted to analyzing the effectiveness (according to the title) of e-school counseling in helping students facing difficulties during the pandemic. The analysis and results are based on extensive surveys and interviews in the Casablanca-Settat region (Morocco). In my opinion, the paper contains interesting, valuable results and comments presented in an appropriate way. Qeios ID: CEEZ2C · https://doi.org/10.32388/CEEZ2C 1/1
https://openalex.org/W2127967821	https://journals.sub.uni-hamburg.de/hup2/ijrvet/article/download/18/11	English	null	VET workers’ problem-solving skills in technology-rich environments: European approach	International journal for research in vocational education and training	2,014	cc-by-sa	8,100	International Journal for Research in Vocational Education and Training (IJRVET) Vol.1, No. 1: 57-80, DOI: 10.13152/IJRVET.1.1.4 International Journal for Research in Vocational Education and Training (IJRVET) Vol.1, No. 1: 57-80, DOI: 10.13152/IJRVET.1.1.4 VET workers’ problem-solving skills in technology-rich environments: European approach Raija Hämäläinen* University of Jyväskylä, Finnish Institute for Educational Research, P.O.Box 35, 40014 Jyväskylä, Finland E-mail: raija.h.hamalainen@jyu.fi Sebastiano Cincinnato Ghent University Department of Educational Studies Henri Dunantlaan 2, B-9000 Ghent, Belgium E-mail: Sebastiano.Cincinnato@UGent.be Antero Malin University of Jyväskylä, Finnish Institute for Educational Research, P.O.Box 35, 40014 Jyväskylä, Finland E-mail: antero.malin@jyu.fi Bram De Wever Ghent University Department of Educational Studies Henri Dunantlaan 2, B-9000 Ghent, Belgium E-mail: Bram.DeWever@UGent.be * Corresponding author Received: 11 June 2014; Accepted: 12 August 2014; Published online: 31 August 2014 Abstract: The European workplace is challenging VET adults’ problem-solving skills in technology-rich environments (TREs). So far, no international large-scale assessment data has been available for VET. The PIAAC data comprise the most comprehensive source of information on adults’ skills to date. The present study (N=50 369) focuses on gaining insight into the problem-solving skills in TREs of adults with a VET background. When examining the similarities and differences in VET adults’ problem-solving skills in TREs across 11 European countries, two main trends can be observed. First, our results show that only a minority of VET adults perform at a high level. Second, there seems to be substantial variation between countries with respect to the proportion of VET adults that can be identified as “at-risk” or “weak” performers. For the future, our findings indicate the variations that can be used as a starting point to identify beneficial VET approaches. IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 58 Keywords: Adults with vocational education and training, Large-scale assessment, PIAAC, Technology-rich environments, Problem-solving, Workplace skills. Keywords: Adults with vocational education and training, Large-scale assessment, PIAAC, Technology-rich environments, Problem-solving, Workplace skills. Bibliographical notes: Adjunct Professor (Ph.D) Raija Hämäläinen works at the Finnish Institute for Educational Research, University of Jyväskylä. She has designed long-term research project that focuses on investigating new learning spaces for the future vocational education. Her research interests focus on technology-enhanced learning (TEL) at workplace contexts. Sebastiano Cincinnato works as a researcher at the Department of Educational studies at Ghent University, Belgium. He was one of the researchers for the OECD Programme for the International Assessment of Adult Competencies (PIAAC) in Belgium. His research interests focus on the learning divide in adult education. Ph.D Antero Malin is the professor in the field of quantitative methodology in educational research and the head of the Quantitative Methods and Data Management Unit in the Finnish Institute for Educational Research, University of Jyväskylä. Recently, Malin has been the National Project Manager of the OECD Programme for the International Assessment of Adult Competencies (PIAAC). Bram De Wever (Ph.D) works at the Department of Educational Studies. He got a tenure track position on Technology Enhanced Learning and Instruction in 2010. His research interests are related to computer-supported collaborative learning in (higher) education. He is also teaching a course on adult education at Ghent University. Acknowledgments: This research was supported by the Academy of Finland (Project n:o 258659). 1 Introduction The European workplace is going through a radical process of structural change, from mass-production technology to more flexible production methods. For adults with vocational education and training (VET), this structural change often means changes in their job descriptions (as routine tasks are taken over by technologies, Goos, 2013). As a direct result, many of today’s workplaces require creative problem-solving instead of in-routine activities in a variety of technology-rich settings. Therefore, the ability to work and solve problems in technology-rich environments (TREs) plays an increasingly important role (Frey and Osborne, 2013; Goos, 2013). Related to this change, adults’ problem-solving skills (or the lack thereof) may have short-term influence on how well workers are able to manage their work tasks and long-term influence on European welfare and competitiveness. As a direct result of this advancement, problem-solving in Europe’s TRE is one of the most important skills for future working life (Goos, 2013). In line with these emerging requirements, it is generally agreed that there is a need to develop adults’ skills and abilities to better meet the changing needs of the European workplace in terms of problem-solving in TRE, for example through formal education (Billett, 2008; Teichler, 2007; Fuller and Unwin, 2004) and lifelong (Loveder, 2011) and workplace learning (Billett, 2008; Tynjälä, 2013). g ( ) p g ( y j ) Currently, there is an emerging need for a greater understanding of the relationship between VET adults’ problem-solving skills in a TRE and their educational needs in these same realms at the European level. According to Lawn (2013), at best, it may be possible to benefit from the knowledge gathered from large-scale assessments in providing indicators of the skills of the adult population. The aim is that, when adults’ skills are analysed together with educational systems, educational approaches are better able to meet emerging societal needs. Recently, results of the Programme for the International Assessment of Adult Competencies (PIAAC) – the most comprehensive study of adult skills ever undertaken, led by the Organisation for Economic Co-operation and Development (OECD) – showed that problem-solving skills in TREs vary among the European countries (OECD, 2013a). In general, Nordic countries, such as Finland, had overall high proficiency levels in problem solving in TREs. IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach 59 2 Aims Europe’s workplace calls for a new understanding of VET adults’ problem-solving skills in TREs. So far, no international large-scale assessment data has been available for VET (cf. Programme for International Student Assessment (PISA) for 15-year student assessment and Assessment of Higher Education Learning Outcomes (AHELO) for higher education student assessment). Therefore, PIAAC data enables us to get new insight into the VET. Namely, this study aims to investigate VET adults’ performance in problem-solving in TREs and its relation to educational approaches. Specifically, our aim was to identify: x What is the level and distribution of problem-solving skills in TREs for adults with VET in Europe? x How is the level and distribution of problem-solving skills in TREs for adults with VET related to adults with other educational backgrounds? x How is the level and distribution of problem-solving skills in TREs for adults with VET related to adults with other educational backgrounds? 3 Method In practice, PIAAC data and Eurostat classification of the VET approaches were used for this study. Firstly, we used the OECD PIAAC data to obtain knowledge on the VET adults’ performance on problem-solving in TREs. The objective of the PIAAC was to determine whether adults’ basic skills were at a level that made them ready to respond to the unpredictable needs of the future, which might involve rapid changes. Secondly, calculations of the European Centre for the Development of Vocational Training (Cedefop) based on Eurostat classification for upper secondary VET on work-based approaches (for detailed description see European Commission, 2013, p. 7) grounded our analysis. 1 Introduction However, a recent analysis of the PIAAC data (Hämäläinen, De Wever, Malin and Cincinnato, 2014) highlighted the critical issue that, despite Finns’ high proficiency in general for problem-solving in TREs, the majority of adults with VET have weak skills or lack skills needed to solve problems in TREs. As a direct result, a crucial question for VET development involves investigating if the same trend holds true in other parts of Europe. Thus, we need to investigate what the level and distribution of problem-solving skills in TREs for adults with VET is in Europe and how well adults’ vocational skills and professional expertise match the changing needs of European society. As a starting point for investigating VET adults’ skills and abilities, we have to consider that, in general, VET education approaches vary between European countries (for a detailed description, see Eurydice Highlights 2012/2013; OECD, 2009), and this may have an influence on adults’ problem-solving skills in TREs. Specifically, VET has traditionally been based on either school- or work-based approaches (European Commission, 2013). In work-based approaches (applied, following the Eurydice Highlights 2012/2013, for example, in Germany, Austria and Switzerland) learning is intertwined with authentic work contexts, and learners spend a minimal amount of their time in a school setting. Alternatively, in school- IJRVET 2014 IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 60 based approaches (such as in France and Scandinavian countries, including Norway and Finland), learning more often takes place in traditional school settings. However, despite the differences in the approach of where vocational learning takes place, both approaches face the same challenge of structural changes in Europe’s workplace. For these reasons, critical discussions are currently underway about whether the new needs of the structural change (e.g. skills and abilities of creative problem-solving in TREs) have reached our VET adults in a satisfactory way. Furthermore, it is important to find out how different European VET education approaches are associated with VET adults’ problem-solving skills in TREs. 3.1 Data and analysis The PIAAC survey was conducted by an international consortium consisting of several research institutions and led by the Educational Testing Service (ETS, USA). Under the guidance of the consortium, each participating country implemented the survey (for a detailed description, see OECD, 2013b, chapters 10–13). The PIAAC survey contained, firstly, the background questionnaire including questions about skills used, and secondly, computer-based test results that captured the level of adults’ problem-solving skills (OECD, 2013b). A total of 66,322 16- to 65 year olds were involved (average weighted response rate of 60%, sample size of 113,744) in eleven European countries; 50,369 adults (males: n=24,812, females: n=25,556) took part in the test of problem-solving skills in TREs. IJRVET 2014 IJRVET 2014 61 VET workers’ problem-solving skills in technology-rich environments: European approach In PIAAC, proficiency is regarded as a continuum of abilities involving the mastery of information-processing tasks of increasing complexity. The PIAAC test design was based on a variant of matrix sampling, which used different sets of items as well as multistage adaptive testing. In addition, it used a variety of assessment modes where each of the respondents completed a subset of items from the total item pool. Since calculating a one-point estimate of proficiency for each respondent might lead to severely biased estimates of population characteristics, PIAAC uses ten plausible values that are drawn from an a posteriori distribution. This is done by combining the item response theory (IRT) scaling of the cognitive items with a latent regression model using information from the background questionnaire. The PIAAC Technical Report (OECD, 2013b, chapter 17) provides further details of the scaling of cognitive data. The results of problem-solving are represented on a 500-point scale, which was divided into four different proficiency levels. These levels are based on the skills and knowledge required to accomplish the tasks within each level. Level 1 (“weak performers”) included scores from 241 to 290. Level 2 (“moderate performers”) included scores from 291 to 340 and Level 3 (“strong performers”) included scores equal to or higher than 341. Additionally, the participants who scored below Level 1 were defined as “at risk”. Moreover, there is a “not- classified” group included in the sample. The reasons for this group not being classified are rather diverse (i.e. failed the ICT core test, no computer experience or opted out of the computer-based assessment, or fell out due to literacy-related non- response). As such, it is difficult to make proper statements about this group. Given the fact that there is no information at all with respect to their problem-solving proficiency level present in the database, we opted not to include them in our analyses, except for our first analyses presenting a general overview. The statistical analyses were implemented by taking the survey design into account using weights. The mean of regressions over ten plausible values was calculated, using Jack-knife replication for variance estimation (for further details, see OECD, 2013b, chapters 14-15). 3.3 Investigating the differences in problem-solving skills between VET and other adults As the first phase of the investigation of problem-solving skills differences, VET and other adults were categorized based on their problem-solving scores. In the second phase, the similarities and differences with respect to the relation between participants’ scores of the problem-solving test and educational background were discovered. Finally, a variety of adults with different educational backgrounds were compared. In our analysis, we used multinomial logit regression to estimate the likelihood of attaining an at-risk or weak proficiency level compared to a moderate or strong proficiency level (which are combined to act as the reference category). Additionally, in comparing the educational categories, adults with vocational education serve as the reference category, in order to compare VET adults with graduates from (at most) lower secondary education and graduates from (at least) upper secondary education. 3.2 Detecting the level of the problem-solving skills in TREs The aim of evaluating problem-solving in TREs was to assess how adults use technology in order to carry out a variety of complicated problem-solving tasks. The assessment framework as well as the tasks and the background questionnaire were designed by the international expert groups (OECD, 2012). In the PIAAC study, problem-solving in TREs is described below: "Problem solving in technology-rich environments involves using digital technology, communication tools and networks to acquire and evaluate information, communicate with others and perform practical tasks. The first PIAAC problem solving survey will focus on the abilities to solve problems for personal, work and civic purposes by setting up appropriate goals and plans, accessing and making use of information through computers and computer networks". (OECD, 2012, p. 47) In practice, during the test each participant solved tasks using various sources of information on a laptop computer. The computer environment offered several tools participants could use, such as an Internet browser and web pages, a computer-based reservation system, e-mail and word processing. To summarise, the main goal of the assessment was to explain to which extent adults use technology to solve today’s problems both proficiently and efficiently (OECD, IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 62 2012). In this study, the first step of the analysis was to calculate the level and distribution of participants’ problem-solving skills. 2012). In this study, the first step of the analysis was to calculate the level and distribution of participants’ problem-solving skills. 3.4 Examining the differences between the European countries applying school- or work-based approaches in VET To examine the differences between European countries we grounded our analysis on the orientation that European countries have taken in VET. Thus, we based our analysis on the Cedefop calculations of Eurostat data (see Table 1). In practice, we used the share of work-based practise as a starting point for dividing European countries that apply school- or work-based approaches in their educational systems. In this phase, we included all the countries listed on the Cedefop calculation (9 countries with low and 8 countries with high share of work-based learning; additionally for the rest of the European countries, no reliable data were available; for detailed information, see European Commission 2013; Eurydice Highlights 2012/2013; OECD, 2009). Next, we investigated all the countries in the Cedefop calculations that participate in the PIAAC study (13 countries) and selected those countries that had data on problem-solving in TREs (Spain and France were left out, as they did not participate in the problem-solving test of the PIAAC). Additionally, as the problem-solving proficiency scores are not available for the participants in the “not-classified” group, they were not included in statistical analyses. In practice, we included 6 countries with a low and 5 countries with a high share of work-based learning on our analyses (Table 1). Finally, to gain deeper insight into the problem-solving skills in TREs of European adults with a VET background, similarities and differences in VET adults’ problem-solving skills across the European countries applying school- or work-based approaches in their education were investigated. Table 1: Share of work-based learning in 11 countries Share of work-based learning High Low Austria, Slovak Republic, Czech Republic, Denmark, Germany Belgium, Finland, Ireland, Netherland, Norway, Poland Table 1: Share of work-based learning in 11 countries IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach 63 4.1 The general overview of problem-solving skills in TREs in Europe In the following section we further shed light on the similarities and differences in VET adults’ problem-solving skills across the European countries applying school- or work-based approaches. Before that, we will firstly give an overview over VET adults’ problem-solving skills, including the not-classified group, to give a general picture of the share of this group. Figure 1 illustrates that among VET adults, there is a tendency to have a lack of or low problem-solving skills in all 11 countries. Specifically, only a small minority of VET adults score on the highest level (level 3) and 63% or more of the adults with VET are below the moderate performance level (level 2). Additionally, in all of the countries more than 11% of the VET adults are at-risk performers in problem-solving. On average, one third of adults with VET are at Level 1. The pattern of distribution of adults at Level 1 is largely similar and close to the school- and work-based average in Austria, Czech Republic, Denmark and Finland. Furthermore, a large share of VET adults belongs to the not-classified group, on average almost one third of VET adults both in the school- and work-based countries. The lowest share is in the Netherlands (8%). Denmark and Norway (16%) as well as Germany (21%) are clearly below average. An interesting similarity between school- or work-based approaches can be noted: in both approaches there is one country with a high share of the VET adults belonging to the not-classified group (Slovak Republic and Poland, see Figure 1). These two countries clearly differ from the other countries in terms of having a large not- classified group, and because of that, fewer VET adults score the two highest levels of problem-solving skills (Figure 1). IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 64 Note. Due to rounding, the percentages do not always add up to exactly 100 percent. Note. Due to rounding, the percentages do not always add up to exactly 100 percent. Note. Due to rounding, the percentages do not always add up to exactly 100 percent. Figure 1: Percentage of VET adults scoring at each level of problem-solving skills by country 4.2 The main differences between VET and other adults’ problem-solving skills in TREs In all 11 countries, adults with VET perform lower on average than adults with other educational backgrounds. The average difference between work-based and school-based VET is very small (only 4 points, see Figure A1 in appendix). The difference between VET and non-VET adults’ problem solving skills varies between countries, ranging from 8 points in Austria to 32 points in Belgium (see Figure A1 in appendix). Thus, there is no indication of an association between VET adults’ problem-solving skills in TREs and education approaches applied in Europe. Instead, there is great variation in VET adults’ average problem-solving skills in TREs across 11 European countries, irrespective of whether they are applying school- or work-based approaches. The lowest mean performance scores are found in Poland and Belgium (255 points) and the highest in Austria and the Netherlands (280 points). When comparing VET adults to adults with at least upper secondary education, the latter strongly outperform the vocationally trained adults in all 11 countries (Figure 2). On average, graduates from upper secondary or higher score 26 points higher than VET graduates in work-based countries and 28 point higher in school-based countries. As the figures indicate, the difference between work- based and school-based countries is very small — although statistically significant (p < 0.05). However, there is variation between countries for both approaches. The difference is greatest in Belgium (42 points) and smallest in Ireland (17 points). IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach 65 Both of these countries apply the school-based approach. In the countries applying the work-based approach, the difference is greatest in Denmark (31 points) and smallest in Austria (18 points). When comparing the VET group with adults with lower secondary education, adults with lower secondary education score higher in 7 out of 11 countries (Figure 2). In this instance, the difference between work- and school-based countries is more pronounced: while in work-based countries, adults with VET have a 4-point- lower average performance score compared to adults with lower secondary education (p < 0.001), in the school-based countries there is no (statistically significant) difference in this respect (p = 0.554). The within-country variation is larger in school-based countries. The largest difference in favor of adults with lower secondary education is in Poland (25 points) and that in favor of VET adults is in Ireland (18 points). 4.2 The main differences between VET and other adults’ problem-solving skills in TREs Among the work-based countries, the greatest difference in favor of adults with lower secondary education is in the Slovak Republic (17 points) and that in favor of VET adults is in Austria (12 points). Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A1). Figure 2: Mean problem-solving scores by educational background for all t i Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A1). Figure 2: Mean problem-solving scores by educational background for all countries Figure 2: Mean problem-solving scores by educational background for all countries In Figures 3 and 4, we present the proportion of adults scoring at different proficiency levels of problem-solving in work- and school-based countries, excluding the not-classified group. When comparing VET adults to graduates from higher secondary school or higher, we observe that there is a clear difference in proportion among those scoring on lower (i.e. “at risk”, and “weak”) and higher IJRVET 2014 IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 66 levels (i.e. “moderate”, and “strong”). In fact, in work-based countries, more than two thirds (68%) of the VET adults perform on the lower levels compared to 42 % of the graduates of higher secondary school. In the school-based countries, 71% of the VET adults perform on the lower levels compared to 43% of the graduates of higher secondary. However, the differences between countries are rather large, even when controlling for the VET-system approach. In work-based countries, we can observe that the proportion of low-performing VET adults varies between 60% (Austria) and 78% (the Slovak Republic), whereas the proportion of low- performing graduates from upper secondary varies between 36% (the Czech Republic) and 55% (the Slovak Republic). In school-based countries, Belgium (Flanders) has the highest proportion of VET-qualified low performers (82%) and the Netherlands the lowest (60%); the proportion of low-performing higher secondary graduates varies between 34% (the Netherlands) and 54% (Ireland). y g ( ) ( ) Compared to lower secondary graduates (or less), VET adults show a slight tendency to have more adults performing on the low levels than the former. In work-based countries, we can observe that 61% of the lower secondary graduates have low levels of problem-solving skills (compared to 68% of the VET adults), and in school-based countries, 68% of the lower secondary graduates perform at low proficiency levels (compared to 71% of VET adults). However, once again between-country differences are rather large, even when controlling for the VET system; in work-based countries the proportion of low achievers among lower secondary graduates varies between 52% (the Czech Republic) and 69% (Austria). VET workers’ problem-solving skills in technology-rich environments: European approach 67 Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A2). Figure 3: Percentage of adults at each level of problem-solving skills in work- based countries Note. Figure 2: Mean problem-solving scores by educational background for all countries Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A2). Figure 3: Percentage of adults at each level of problem-solving skills in work- based countries Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A2). Figure 3: Percentage of adults at each level of problem-solving skills in work- based countries Figure 3: Percentage of adults at each level of problem-solving skills in work- based countries IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 68 Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A3). Figure 4: Percentage of adults at each level of problem-solving skills in school- based countries Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A3). Note. Due to rounding, the percentages do not always add up to exactly 100 percent. An extended tabulation can be found in the appendix (Table A3). Figure 4: Percentage of adults at each level of problem-solving skills in school- based countries In order to examine the impact of educational background on problem-solving skills in work-based countries, in Table 2, we present the regression coefficients (and corresponding odds ratios) of the model estimating the likelihood of scoring at an “at risk” or “weak” problem-solving level compared to a “moderate and strong” proficiency level (i.e. the reference category). The figures clearly demonstrate that VET adults are more likely, compared to graduates from upper secondary school (or more), to be “at risk” or “weak” performers in problem-solving than “moderate or strong performers”, independently from the country of origin. More precisely, VET adults are 3.2 (Austria) to 7.2 (Denmark) more likely to be “at-risk” performers, and 2.1 (Austria) to 2.8 (Denmark) more likely to be “weak” performers. Interestingly, the between-country variation is larger when comparing the problem-solving skills of VET adults and graduates from lower secondary school (or less). In fact, with the exception of the Czech Republic, VET adults and lower secondary graduates are equally likely to be “weak” performers since the regression parameters are non-significant at a level of α = 0.05. In the Czech Republic, VET adults are 1.6 times more likely to be “weak” performers than IJRVET 2014 IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach 69 lower secondary graduates. When estimating the likelihood of being an “at-risk” performer, the variation between countries is even more pronounced. More in particular, in the Czech Republic and the Slovak Republic, VET adults are respectively 1.9 and 3.7 times more likely to be “at-risk” performers. In Austria, on the contrary, lower secondary graduates are 2.3 times more likely than VET adults to be “at-risk” performers. In school-based systems, overall, we observe the same patterns as in work-based systems (Table 3). First, VET adults are clearly more likely to be “at-risk” or “weak” performers than the graduates of upper secondary education. In particular, VET adults are 3.1 (Ireland) to 14.7 (Belgium) times more likely to be “at-risk” 1 Notice that the figures of Belgium (Flanders) are potentially overestimated. In fact, many observations in the Belgian data are classified as “not stated or inferred” on the vocational education and training variable. This is due to the fact certain ISCED- categories, and more precisely the ISCED 3 (without distinction A – B – C) and ISCED 4, could not be uniquely identified as being vocationally oriented or not. As a consequence, these have been coded as ”not stated or inferred”. IJRVET 2014 In Denmark and Germany, the likelihoods are equal. Table 2: Likelihood of problem-solving skills proficiency in TREs by educational background in work-based countries (parameter estimates with standard errors and odds ratios) Table 2: Likelihood of problem-solving skills proficiency in TREs by educational background in work-based countries (parameter estimates with standard errors and odds ratios) educational background in work-based countries (parameter estimates with standard errors and odds ratios) At risk performers Weak performers b Odds ratio Inverse odds ratio b Odds ratio Inverse odds ratio Austria Intercept -1.04 *** (.085) 0.15 * (.067) Low Sec vs. VET 0.81 * (.165) 2.26 0.44 0.20 (.149) 1.22 0.82 Up Sec vs. VET -1.16 * (.200) 0.31 3.19 -0.74 * (.115) 0.48 2.10 Czech Republic Intercept -0.45 * (.093) 0.20 * (.084) Low Sec vs. VET -0.64 ** (.226) 0.52 1.91 -0.48 * (.183) 0.62 1.61 Up Sec vs. VET -1.86 * (.219) 0.16 6.43 -0.94 * (.158) 0.39 2.57 Denmark Intercept -0.13 (.079) 0.42 * (.079) Low Sec vs. VET -0.21 (.128) 0.81 1.24 -0.19 (.117) 0.83 1.21 Up Sec vs. VET -1.97 * (.125) 0.14 7.17 -1.01 * (.087) 0.36 2.75 Germany Intercept -0.37 * (.085) 0.24 * (.067) Low Sec vs. VET -0.08 (.157) 0.92 1.09 -0.19 (.142) 0.83 1.21 Up Sec vs. VET -1.59 * (.134) 0.20 4.90 -0.93*** (.101) 0.39 2.54 Slovak Republic Intercept 0.16 (.150) 0.87 * (.332) Low Sec vs. VET -1.31 * (.239) 0.27 3.71 -0.81 (.441) 0.45 2.24 Up Sec vs. VET -1.62 * (.199) 0.20 5.03 -0.92 * (.368) 0.40 2.50 Note. * p < 0.05, p < 0.01, * p < 0.001; Standard errors are in parentheses. In school-based systems, overall, we observe the same patterns as in work-based systems (Table 3). First, VET adults are clearly more likely to be “at-risk” or “weak” performers than the graduates of upper secondary education. In particular, VET adults are 3.1 (Ireland) to 14.7 (Belgium) times more likely to be “at-risk” IJRVET 2014 IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 70 performers than upper secondary graduates1; and the former are 1.9 (Ireland) to 5.2 (Belgium) times more likely to be “weak” performers than the latter. We observe, once again, between-country variation when comparing the problem-solving skills of VET adults to the skills of graduates from lower secondary education (or less). IJRVET 2014 Except for Ireland, Norway and the Netherlands, VET adults have a higher likelihood of being “weak” performers – ranging 1.3 (Finland) to 2.0 (Belgium). In the Netherlands, on the contrary, lower secondary graduates are 1.5 times more likely than VET adults to be “weak” performers. In Ireland and Norway, the differences are not statistically significant (α = 0.05). Estimating the likelihood of being an “at-risk” performer reveals an interesting picture as well. In fact, in Finland and Norway, VET adults have the same likelihood as lower secondary graduates to be “at-risk” performers. In Belgium and Poland, VET adults are more likely to be “at-risk” performers, and finally, in Ireland and the Netherlands, VET adults are less likely to achieve an “at-risk” problem-solving level. IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach 71 Table 3: Likelihood of problem-solving skills proficiency in TREs by educational background in school-based countries (parameter estimates with standard errors and odds ratios) educational background in school-based countries (parameter estimates with standard errors and odds ratios) At risk performers Weak performers b Odds ratio Inverse Odds ratio b Odds ratio Inverse Odds ratio Belgium a Intercept 0.70 * (.169) 1.04 * (.153) Low Sec vs. VET -0.43 * (.209) 0.65 1.54 -0.68 (.221) 0.51 1.98 Up Sec vs. VET -2.69 * (.197) 0.07 14.67 -1.64 * (.167) 0.19 5.15 Finland Intercept -0.42 * (.095) 0.28 *** (.069) Low Sec vs. VET -0.27 (.153) 0.76 1.31 -0.29 * (.141) 0.75 1.34 Up Sec vs. VET -1.80 * (.129) 0.16 6.06 -1.06 * (.080) 0.35 2.90 Ireland Intercept -0.25 (.131) 0.62 * (.114) Low Sec vs. VET 1.04 * (.218) 2.82 0.35 0.06 (.204) 1.06 0.95 Up Sec vs. VET -1.13 * (.138) 0.32 3.09 -0.67 * (.125) 0.51 1.94 Netherlands Intercept -0.96 * (.109) 0.14 (.087) Low Sec vs. VET 1.02 * (.141) 2.78 0.36 0.42 * (.113) 1.52 0.66 Up Sec vs. VET -1.46 * (.152) 0.23 4.32 -1.01 * (.110) 0.36 2.74 Norway Intercept -0.58 * (.100) 0.28 * (.073) Low Sec vs. VET 0.26 (.155) 1.29 0.77 -0.06 (.126) 0.95 1.06 Up Sec vs. VET -1.63 * (.135) 0.20 5.11 -0.96 * (.092) 0.38 2.61 Poland Intercept 0.52 * (.121) 0.53 * (.111) Low Sec vs. VET -1.32 * (.197) 0.27 3.76 -0.62 (.177) 0.54 1.86 Up Sec vs. VET -1.60 * (.125) 0.20 4.93 -0.78 *** (.128) 0.46 2.18 Note. 5 Discussion and Conclusions The PIAAC data comprise the most comprehensive source of information on adults’ skills to date. Therefore, it is now possible to investigate adults’ problem- solving skills in TREs in a novel way. The present study focuses on gaining more in-depth insight into the problem-solving skills in TREs of European adults with a VET background. At a general level, when examining the similarities and differences in VET adults’ problem-solving skills in TREs across 11 European countries, two main trends can be observed. First, our results show that only a minority of VET adults perform at a high level. Across all European countries in our study, adults with VET perform lower on average than adults with other educational backgrounds. Second, although adults with VET achieve a lower skill level throughout all countries, there seems to be substantial variation between countries with respect to the proportion of VET adults that can be identified as “at- risk” or “weak” performers. As problem-solving skills in TREs are becoming increasingly important in work life (Goos, 2013), our findings are in line with the critical notion that there seems to be a gap between what is learned in VET and what is required at European workplaces (Baartman and de Bruijn, 2011). Currently, European countries are developing their VET systems to better meet the emerging needs of European workplaces, and different trends can be observed. Namely, in 6 (Ireland, Finland, Norway, Netherland, Austria and Germany) of 11 countries investigated in this study, the share of workplace learning is increasing, while in 5 countries (Belgium, Poland, Czech Republic, Slovak Republic and Denmark), this share is decreasing (cf. European Commission, 2013; Eurydice Highlights 2012/2013; OECD, 2009). A clear pattern with regard to the association between educational background and problem-solving skills, typical for the work- or school-based vocational education systems, does not emerge from our analysis. Instead, our findings indicate the variations that can be used as a starting point to identify beneficial VET approaches. Our results suggest that when comparing adults with upper secondary education or higher with adults with a VET background, the same trend can be observed in all countries: VET adults are more likely to be at-risk or weak performers. However, when comparing VET adults with adults with lower secondary education or lower, three clusters of countries can be identified. IJRVET 2014 * p < 0.05, p < 0.01, * p < 0.001; Standard errors are in parentheses; a The Belgian estimates might be biased (see footnote 1). R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 72 5 Discussion and Conclusions In the first cluster, VET adults are more likely to be at-risk or weak performers than adults with lower secondary education or lower. This first cluster includes Finland, Czech Republic, Belgium, Poland and Slovak Republic, although for the latter this was only found for the likelihood of being an at-risk performer and for the former only for the likelihood of being a weak performer. In the second cluster, the reverse is true, namely VET adults perform better. This second cluster includes the Netherlands, Austria and Ireland, although for the latter two this was only observed for the at-risk performers. In the third cluster, containing Denmark, Germany and Norway, there is no difference between the level of TRE problem-solving skills of adults with VET background and lower secondary education backgrounds. These European variations hint towards influential differences between the countries. Namely, as VET is mediated by structural elements, such as the resources, norms, curriculum guidelines and externally mandated policies, future investigations need to focus on the constituent components of successful VET approaches within and across these three clusters of countries. IJRVET 2014 IJRVET 2014 73 VET workers’ problem-solving skills in technology-rich environments: European approach Workplaces and vocational schools are the two main components of VET (Schaap, Baartman and de Bruijn, 2012). When considering the findings of this study, as a whole it is important to consider that grouping national VET systems based on the Cedefop calculations of Eurostat data may lead to over-simplification in understanding the problem-solving skills in TREs of European adults with a VET background (as there are large between and within country variations of VET). Therefore, to develop VET systems that can better meet the needs of future workplaces, additional studies are needed to more clearly understand the combination of school-based and workplace learning experiences (see also Gessler, 2009). Recently, Billett (2014) argued that the emerging challenges of VET research involve integrating learners’ experiences in practice settings (i.e. workplaces) into educational programs. In the future, in addition to large-scale evaluations of VET adults’ problem-solving skills in technology-rich environments, it will be necessary to gain additional knowledge on successful problem-solving processes in TRE, e.g. how learners are able to build meaningful and adaptive problem-solving skills in workplaces and vocational schools (Billett, 2014). Therefore, future research needs to focus on the in-depth analysis of problem- solving processes in TREs. 5 Discussion and Conclusions In this respect, it could be beneficial to select the adults with a VET background from high-performing countries (e.g. Austria and the Netherlands) and the low-performing countries (e.g. Poland and Belgium) to understand why and where variations as well as low levels of problem-solving skills (cf. not classified group) exist in VET adults. ( g p) European countries are all facing a significant challenge in identifying approaches to enhance problem-solving skills for adults with a VET background. In the future, new technologies may be one way to support these higher TRE problem-solving skills. Recent studies have indicated that technology plays an increasingly important role in enabling flexible and adaptive life-long learning approaches. In practice, the role of technology is often to allow new learning experiences, for example, to learn problem-solving through game-based applications (Hämäläinen and De Wever, 2013), or to empower VET adults’ professional development (Boldrini and Cattaneo, 2012; Motta, Boldrini and Cattaneo, 2013). Furthermore, studies have indicated that VET technologies have many benefits for the development of vocational specific skills (Cuendet and Dillenbourg, 2013; Minnaert, Boekaerts, De Brabander and Opdenakker, 2011). Finally, recent research findings support the potential of designing new technologies and applying existing technologies in workplace settings to enhance problem-solving in TREs (Tynjälä, Häkkinen and Hämäläinen 2014). IJRVET 2014 R. Hämäläinen, S. Cincinnato, A. Malin, & B. De Wever 74 References Baartman, L. K. J., & De Bruijn, E. (2011). Integrating knowledge, skills and attitudes: Conceptualizing learning processes towards vocational competence. 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IJRVET 2014 76 Appendix Figure A1: Mean problem-solving scores in VET and NON-VET groups by countries n problem-solving scores in VET and NON-VET groups by countries IJRVET 2014 Appendix 76 76 IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach Table A1: Mean problem-solving scores by educational background for all countries Lower secondary or less VET Higher secondary or more n Mean SE n Mean SE n Mean SE Austria 557 268.84 2.24 2,056 280.58 0.90 1,242 298.32 1.66 Czech Republic 728 285.33 3.12 2,505 273.52 1.44 1,442 303.20 2.01 Denmark 915 270.10 1.74 1,651 267.25 1.23 3,267 298.27 0.96 Germany 713 273.64 2.44 1,949 271.54 1.31 1,877 299.69 1.20 Slovak Republic 425 281.31 2.13 751 263.99 1.61 2,361 286.13 1.03 Work-based 3,338 275.85 1.06 8,912 271.38 0.59 10,189 297.12 0.64 Belgium 661 259.75 2.14 475 255.03 2.25 1,624 297.42 1.16 Finland 640 277.03 2.19 1,307 272.71 1.30 2,556 302.27 1.00 Ireland 551 251.09 2.63 762 269.20 1.71 2,779 285.74 1.11 Netherlands 1,242 264.01 1.08 1,275 279.58 1.51 2,030 303.90 1.07 Norway 585 269.96 2.01 1,365 274.08 1.15 2,391 299.07 0.76 Poland 722 279.67 2.94 1,955 255.02 2.16 3,314 286.57 1.58 School-based 4,401 266.92 0.91 7,139 267.60 0.71 14,694 295.83 0.47 R. Hämäläinen, S. Cincinnato, A. Malin, & B. IJRVET 2014 De Wever 78 Table A2: Absolute number and (weighted) percentages of adults at each level of problem-solving skills (not classified included) in work-based countries Not classified Below level 1 Level 1 Level 2 Level 3 Total n % n % n % n % n % n % Austria Lower secondary or less 376 45.30 117 13.58 249 24.11 175 15.59 16 1.42 933 100.00 VET 666 24.65 278 10.61 921 34.80 756 26.75 101 3.20 2,722 100.00 Higher secondary or more 128 10.68 81 5.97 415 29.70 614 43.95 132 9.70 1,370 100.00 Czech Republic Lower secondary or less 206 35.65 95 10.33 268 23.21 301 25.05 63 5.75 934 100.00 VET 1,045 28.27 528 16.04 1,070 30.56 777 21.22 130 3.92 3,550 100.00 Higher secondary or more 154 8.14 103 5.84 436 27.65 666 43.36 237 15.02 1,596 100.00 Denmark Lower secondary or less 441 27.64 246 17.29 374 30.62 267 22.19 28 2.26 1,356 100.00 VET 387 16.12 493 21.68 750 37.47 380 22.86 29 1.87 2,038 100.00 Higher secondary or more 299 6.46 321 6.84 1,215 30.80 1,414 44.30 317 11.59 3,566 100.00 Germany Lower secondary or less 199 26.92 147 17.31 277 28.63 247 22.89 42 4.25 912 100.00 VET 478 21.29 419 18.37 827 33.85 619 23.64 83 2.86 2,427 100.00 Higher secondary or more 159 8.69 149 7.87 563 27.95 867 41.52 298 13.98 2,036 100.00 Slovak Republic Lower secondary or less 561 57.97 58 5.61 201 18.78 155 16.38 12 1.27 986 100.00 VET 1,020 56.86 196 11.14 390 22.53 158 8.96 8 0.51 1,771 100.00 Higher secondary or more 584 18.90 261 8.69 1,058 35.32 919 32.43 123 4.66 2,945 100.00 Work-based Lower secondary or less 1,783 38.70 663 12.82 1,369 25.07 1,145 20.42 161 2.99 5,121 100.00 VET 3,596 29.44 1,913 15.57 3,957 31.84 2,691 20.69 352 2.47 12,508 100.00 Higher secondary or more 1,324 10.57 914 7.04 3,688 30.28 4,480 41.11 1,108 10.99 11,513 100.00 IJRVET 2014 VET workers’ problem-solving skills in technology-rich environments: European approach 79 Table A3: Absolute number and (weighted) percentages of adults at each level of problem-solving skills (not classified included) in school-based countries Not classified Below level 1 Level 1 Level 2 Level 3 Total n % n % n % n % n % n % Belgium Lower secondary or less 361 36.86 218 22.04 254 24.22 168 15.16 20 1.72 1,022 100.00 VET 158 26.09 162 25.46 233 35.82 77 12.06 4 0.57 633 100.00 Higher secondary or more 87 5.17 127 7.73 523 30.94 772 44.67 203 11.48 1,711 100.00 Finland Lower secondary or less 338 34.56 125 13.13 253 25.96 231 23.15 32 3.19 978 100.00 VET 443 25.50 291 16.40 581 33.08 387 22.22 48 2.80 1,750 100.00 Higher secondary or more 180 7.10 175 6.42 757 27.11 1,232 45.02 392 14.35 2,736 100.00 Ireland Lower secondary or less 898 58.98 258 17.50 206 15.59 81 7.24 6 0.69 1,449 100.00 VET 325 31.07 171 14.80 391 35.21 186 17.47 14 1.45 1,087 100.00 Higher secondary or more 650 18.81 332 9.32 1,194 35.15 1,079 31.71 174 5.01 3,429 100.00 Netherlands Lower secondary or less 339 23.34 334 21.42 576 35.21 304 18.31 28 1.73 1,581 100.00 VET 103 8.05 189 13.95 580 41.77 456 32.71 49 3.52 1,378 100.00 Higher secondary or more 95 5.01 110 5.61 582 26.45 1,054 48.79 284 14.14 2,125 100.00 Norway Lower secondary or less 187 27.84 127 17.62 247 30.30 194 22.32 17 1.92 7,72 100.00 VET 236 15.77 255 16.41 626 38.62 448 27.01 37 2.19 1,601 100.00 Higher secondary or more 187 7.59 152 6.28 748 29.00 1,215 46.55 277 10.58 2,578 100.00 Poland Lower secondary or less 654 58.47 155 7.85 301 16.11 226 14.38 41 3.20 1,376 100.00 VET 1,865 64.42 594 13.62 791 13.82 492 7.12 78 1.02 3,820 100.00 Higher secondary or more 853 26.72 415 11.74 1,204 27.00 1,308 26.69 387 7.86 4,167 100.00 School-based Lower secondary or less 2,777 40.01 1,217 16.59 1,838 24.56 1,203 16.76 144 2.08 7,178 100.00 VET 3,130 28.48 1,661 16.77 3,203 33.06 2,046 19.76 230 1.92 10,269 100.00 Higher secondary or more 2,052 11.73 1,311 7.85 5,007 29.28 6,661 40.57 1,716 10.57 16,746 100.00 IJRVET 2014 IJRVET 2014 80 80
https://openalex.org/W2979586623	https://europepmc.org/articles/pmc6893303?pdf=render	English	null	Correction: APR-246 reactivates mutant p53 by targeting cysteines 124 and 277	Cell death and disease	2,019	cc-by	1,139	C O R R E C T I O N O p e n A c c e s s Zhang et al. Cell Death and Disease (2019) 10:769 https://doi.org/10.1038/s41419-019-1997-z Zhang et al. Cell Death and Disease (2019) 10:769 https://doi.org/10.1038/s41419-019-1997-z Cell Death & Disease Cell Death & Disease Correction: APR-246 reactivates mutant p53 by targeting cysteines 124 and 277 Qiang Zhang1, Vladimir J. N. Bykov 1, Klas G. Wiman 1 and Joanna Zawacka-Pankau2 Qiang Zhang1, Vladimir J. N. Bykov 1, Klas G. Wiman 1 and Joanna Zawacka-Pankau2 Correction to: Cell Death and Disease https://doi.org/10.1038/s41419-018-0463-7, published online 18 April 2018 corrections do not affect the original conclusions of this paper. The authors would like to apologize for any inconvenience caused. Since publication of this article, the authors have noticed that there was an error in Fig. 1d, third panel from left, “R273H + 200 μM MQ-H” should be “R273H + 200 μM MQ”. A corrected version of Fig. 1 is included here. Our Correspondence: Klas G. Wiman (Klas.Wiman@ki.se) 1Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, SE-17176 Stockholm, Sweden 2Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, SE-17177 Stockholm, Sweden Correspondence: Klas G. Wiman (Klas.Wiman@ki.se) 1Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, SE-17176 Stockholm, Sweden 2Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, SE-17177 Stockholm, Sweden © The Author(s) 2 © The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association Zhang et al. Qiang Zhang1, Vladimir J. N. Bykov 1, Klas G. Wiman 1 and Joanna Zawacka-Pankau2 Cell Death and Disease (2019) 10:769 Page 2 of 2 wtp53 R175H R273H 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 wtp53 + 50µM MQ R175H + 10µM MQ R273H + 50µM MQ R273H + 50µM MQ-H 22619 22756 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 wtp53 + 100µM MQ R175H + 25µM MQ R273H + 100µM MQ R273H + 100µM MQ-H % relative abundance 22756 22619 22893 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22893 22757 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22737 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22737 22600 22874 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22874 22737 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 22737 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22737 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 m/z wtp53 + 200µM MQ R175H + 50µM MQ R273H + 200µM MQ R273H + 200µM MQ-H 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22619 unmodified 1MQ 2MQ 22737 22875 N O MQ MQ-H N O a b c d e Adducts: Fig. 1 Mass measurement of wild-type, R273H and R175H p53 core domains by LTQ-MS. a mass spectra of p53 core domains. b–d Reaction titration with MQ or MQ-H. p53 core domains were incubated with MQ at 50–200 µM (wt and R273H) or 10–50 µM (R175H) concentration ranges. One MQ adduct increased the molecular mass of p53 core domains by 137 Da. Ofﬁcial journal of the Cell Death Differentiation Association Qiang Zhang1, Vladimir J. N. Bykov 1, Klas G. Wiman 1 and Joanna Zawacka-Pankau2 e Structure of MQ and MQ-H b R175H + 10µM MQ 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22737 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 wtp53 + 50µM MQ 22619 22756 e abundance b R273H + 50µM MQ 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22737 c 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 wtp53 + 100µM MQ % relative 22756 22619 22893 c R175H + 25µM MQ 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 22737 R273H + 100µM MQ-H 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 R273H + 100µM MQ 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22737 22600 22874 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22893 22757 wtp53 + 200µM MQ d d 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22601 R273H + 200µM MQ-H 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22600 22737 R175H + 50µM MQ 22875 22500 22600 22700 22800 22900 23000 0 20 40 60 80 100 120 22874 22737 m/z R273H + 200µM MQ N O MQ e e MQ-H N O MQ MQ-H Fig. 1 Mass measurement of wild-type, R273H and R175H p53 core domains by LTQ-MS. a mass spectra of p53 core domains. b–d Reaction titration with MQ or MQ-H. p53 core domains were incubated with MQ at 50–200 µM (wt and R273H) or 10–50 µM (R175H) concentration ranges. One MQ adduct increased the molecular mass of p53 core domains by 137 Da. e Structure of MQ and MQ-H Fig. 1 Mass measurement of wild-type, R273H and R175H p53 core domains by LTQ-MS. a mass spectra of p53 core domains. b–d Reaction titration with MQ or MQ-H. p53 core domains were incubated with MQ at 50–200 µM (wt and R273H) or 10–50 µM (R175H) concentration ranges. One MQ adduct increased the molecular mass of p53 core domains by 137 Da. e Structure of MQ and MQ-H Ofﬁcial journal of the Cell Death Differentiation Association Ofﬁcial journal of the Cell Death Differentiation Association
https://openalex.org/W2280839574	https://europepmc.org/articles/pmc5178111?pdf=render	English	null	Associação entre variáveis clínicas relacionadas à asma em escolares nascidos com muito baixo peso com e sem displasia broncopulmonar	Revista Paulista de Pediatria	2,016	cc-by	8,237	Rev Paul Pediatr. 2016;34(3):271---280 REVISTA PAULISTA DE PEDIATRIA www.rpped.com.br ORIGINAL ARTICLE Association between clinical variables related to asthma in schoolchildren born with very low birth weight with and without bronchopulmonary dysplasia Emília da Silva Gonc¸alves ∗, Francisco Mezzacappa-Filho, Silvana Dalge Severino, Maria Ângela Gonc¸alves de Oliveira Ribeiro, Fernando Augusto de Lima Marson, Andre Moreno Morcilo, Adyléia Aparecida Dalbo Contrera Toro, José Dirceu Ribeiro Departamento de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil Rev Paul Pediatr. 2016;34(3):271---280 REVISTA PAULISTA DE PEDIATRIA www.rpped.com.br ORIGINAL ARTICLE Association between clinical variables related to asthma in schoolchildren born with very low birth weight with and without bronchopulmonary dysplasia Emília da Silva Gonc¸alves ∗, Francisco Mezzacappa-Filho, Silvana Dalge Severino, Maria Ângela Gonc¸alves de Oliveira Ribeiro, Fernando Augusto de Lima Marson, Andre Moreno Morcilo, Adyléia Aparecida Dalbo Contrera Toro, José Dirceu Ribeiro Departamento de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil Rev Paul Pediatr. 2016;34(3):271---280 http://dx.doi.org/10.1016/j.rppede.2016.03.005 2359-3482/© 2016 Sociedade de Pediatria de S˜ao Paulo. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). REVISTA PAULISTA DE PEDIATRIA ORIGINAL ARTICLE ∗Corresponding author. E-mail: emiliasgoncalves@yahoo.com.br (E.S. Gonc¸alves). Association between clinical variables related to asthma in schoolchildren born with very low birth weight with and without bronchopulmonary dysplasia Association between clinical variables related to asthma in schoolchildren born with very low birth weight with and without bronchopulmonary dysplasia Emília da Silva Gonc¸alves ∗, Francisco Mezzacappa-Filho, Silvana Dalge Severino, Maria Ângela Gonc¸alves de Oliveira Ribeiro, Fernando Augusto de Lima Marson, Andre Moreno Morcilo, Adyléia Aparecida Dalbo Contrera Toro, José Dirceu Ribeiro Departamento de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Camp Received 22 June 2015; accepted 1 December 2015 Available online 13 June 2016 KEYWORDS Asthma; Bronchopulmonary dysplasia; Preterm birth Abstract Objective: To assess the prevalence, spirometry ﬁndings and risk factors for asthma in schoolchildren who were very low birth weight infants with and without bronchopulmonary dysplasia. Methods: Observational and cross-sectional study. The parents and/or tutors answered the International Study of Asthma and Allergies in Childhood questionnaire. The schoolchildren were submitted to the skin prick test and spirometry assessment. Results: 54 schoolchildren who were very low birth weight infants were assessed and 43 met the criteria for spirometry. Age at the assessment (bronchopulmonary dysplasia=9.5±0.85; without bronchopulmonary dysplasia=10.1±0.86 years) and birth weight (bronchopulmonary dysplasia=916.7±251.2; without bronchopulmonary dysplasia=1171.3±190.5g) were lower in the group with bronchopulmonary dysplasia (p<0.05). The prevalence of asthma among very low birth weight infants was 17/54 (31.5%), being 6/18 (33.3%) in the group with bronchopul- monary dysplasia. There was an association between wool blanket use in the ﬁrst year of life (p=0.026) with the presence of asthma at school age. The skin prick test was positive in 13/17 (76.5%) and 23/37 (62.2%) of patients with and without asthma, respectively. The schoolchildren with asthma had lower z-score values of forced expiratory ﬂow between 25% and 75% of forced vital capacity (n=16; −1.04±1.19) when compared to the group of patients without asthma (n=27; −0.380.93) (p=0.049). There was no difference between the spirometry variables in the groups regarding the presence or absence of bronchopulmonary dysplasia. Conclusions: Very low birth weight infants with and without bronchopulmonary dysplasia showed a high prevalence of asthma (33.3% and 30.6%, respectively). Pulmonary ﬂow in the small airways was lower in children with asthma. © 2016 Sociedade de Pediatria de S˜ao Paulo. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ∗Corresponding author. E-mail: emiliasgoncalves@yahoo.com.br (E.S. Gonc¸alves). ∗Corresponding author. Association between clinical variables related to asthma in schoolchildren born with very low birth weight with and without bronchopulmonary dysplasia http://dx.doi.org/10.1016/j.rppede.2016.03.005 Received 22 June 2015; accepted 1 December 2015 Available online 13 June 2016 Received 22 June 2015; accepted 1 December 2015 Available online 13 June 2016 KEYWORDS Asthma; Bronchopulmonary dysplasia; Preterm birth ∗Corresponding author. E il ili l @ h KEYWORDS Asthma; Bronchopulmonary dysplasia; Preterm birth Resumo Objetivo: Avaliar prevalência, espirometria e fatores de risco para asma em escolares que foram recém-nascidos de muito baixo peso com e sem displasia broncopulmonar. Métodos: Estudo observacional e transversal. Aplicou-se aos pais e/ou responsáveis o ques- tionário International Study of Asthma and Allergies in Childhood. Foi feito teste cutâneo de hipersensibilidade imediata e espirometria nos escolares. Resultados: Avaliados 54 escolares que foram recém-nascidos de muito baixo peso e 43 preencheram critérios para espirometria. A idade na avaliac¸ão (displasia broncopulmonar= 9.5±0,85; sem displasia broncopulmonar=10,1±0,86 anos) e o peso de nascimento (displasia broncopulmonar=9167±2512; sem displasia broncopulmonar=1171,3±1905g) foram menores no grupo com displasia broncopulmonar (p<0,05). A prevalência de asma entre os recém-nascidos de muito baixo peso foi de 17/54 (31,5%); no grupo com displasia broncopulmonar, de 6/18 (33,3%). Houve associac¸ão entre uso de cobertor de lã no primeiro ano de vida (p=0,026) com presenc¸a de asma na idade escolar. O teste cutâneo de hipersensibilidade imediata foi positivo em 13/17 (76.5%) e 23/37 (62,2%) nos grupos com e sem asma, respectivamente. Os escolares com asma apresentaram menores valores em z-score do ﬂuxo expiratório forc¸ado entre 25% e 75% da capacidade vital forc¸ada (n=16;−1,04±1,19) comparados com os do grupo de pacientes sem asma (n=27;−0,38±0,93) (p=0,049). Não houve diferenc¸a entre as variáveis da espirometria no grupo com relac¸ão à presenc¸a ou não de displasia broncopulmonar. Conclusões: Os recém-nascidos de muito baixo peso, com e sem displasia broncopulmonar, apre- sentaram prevalência elevada de asma (33,3% e 30,6%, respectivamente). Os ﬂuxos pulmonares das pequenas vias aéreas foram menores nos escolares com asma. © 2016 Sociedade de Pediatria de S˜ao Paulo. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http://creativecommons.org/licenses/by/4.0/). Asthma is a disease of great importance in global public health.23 The prevalence ranges from 2.1% to 32.2% and from 4.1% to 32.1% in children and adolescents, respectively.23 However, studies have not been able to determine which is the main risk factor for childhood asthma in VLBW infants, whether prematurity itself or BPD. Introduction Since 1960, with the improvement of perinatal care, there has been a reduction in mortality of newborns (NB) with birth weight (BW) of less than 1500g, termed very low birth weight (VLBW) infants.1 In contrast, there has been an increase of complications of prematurity, the most serious of which is bronchopulmonary dysplasia (BPD), the earliest chronic obstructive pulmonary disease that affects humans.1 Considering that preterm infants have been increasingly presenting a favorable outcome and that there are few stud- ies in Brazil on asthma prevalence in preterm infants with and without BPD,7,22 this study aimed to determine the pres- ence of asthma and atopy in children born with VLBW at school age and to assess lung function by spirometry, accord- ing to the presence or absence of BPD. During this period the prevalence of BPD is high, and it increases in lower gestational ages and smaller infants.2 BPD affects 30% of VLBW infants <1000g, 52% of those weighing between 501 and 750g, and 7% of those weighing between 1251 and 500g.2,3 The manifestations of prematurity include immune imbalance, increased susceptibility to viral infections,4,5 and allergen absorption.6 Infants with BPD present chronic lung inﬂammation, which predisposes them to infections and hospitalizations. These risk factors may be associated with the prevalence of wheezing in infants2 and asthma in child- hood and adolescence.7---14 Abstract Abstract Objective: To assess the prevalence, spirometry ﬁndings and risk factors for asthma in schoolchildren who were very low birth weight infants with and without bronchopulmonary dysplasia. Methods: Observational and cross-sectional study. The parents and/or tutors answered the International Study of Asthma and Allergies in Childhood questionnaire. The schoolchildren were submitted to the skin prick test and spirometry assessment. Results: 54 schoolchildren who were very low birth weight infants were assessed and 43 met the criteria for spirometry. Age at the assessment (bronchopulmonary dysplasia=9.5±0.85; without bronchopulmonary dysplasia=10.1±0.86 years) and birth weight (bronchopulmonary dysplasia=916.7±251.2; without bronchopulmonary dysplasia=1171.3±190.5g) were lower in the group with bronchopulmonary dysplasia (p<0.05). The prevalence of asthma among very low birth weight infants was 17/54 (31.5%), being 6/18 (33.3%) in the group with bronchopul- monary dysplasia. There was an association between wool blanket use in the ﬁrst year of life (p=0.026) with the presence of asthma at school age. The skin prick test was positive in 13/17 (76.5%) and 23/37 (62.2%) of patients with and without asthma, respectively. The schoolchildren with asthma had lower z-score values of forced expiratory ﬂow between 25% and 75% of forced vital capacity (n=16; −1.04±1.19) when compared to the group of patients without asthma (n=27; −0.380.93) (p=0.049). There was no difference between the spirometry variables in the groups regarding the presence or absence of bronchopulmonary dysplasia. Conclusions: Very low birth weight infants with and without bronchopulmonary dysplasia showed a high prevalence of asthma (33.3% and 30.6%, respectively). Pulmonary ﬂow in the small airways was lower in children with asthma. © 2016 Sociedade de Pediatria de S˜ao Paulo. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 272 Gonc¸alves ES et al. PALAVRAS-CHAVE Asma; Displasia broncopulmonar; Nascimento prematuro Associac¸ão entre variáveis clínicas relacionadas à asma em escolares nascidos com muito baixo peso com e sem displasia broncopulmonar Method This was an observational and cross-sectional study approved by the Institutional Review Board of the Univer- sity Hospital under No. 569/2010 and No. 605/2010. All parents and/or guardians signed an informed consent. Ini- tially, all records of VLBW infants born between October 2000 and November 2004 at the university hospital were selected. Deaths and cases with chronic diseases, genetic diseases, vascular ring, diaphragmatic hernia, pulmonary sequestration, chest wall deformity, and ciliary dyskinesia were excluded. Children with BPD have a high prevalence of recurrent wheezing.6 In contrast, the effects of BPD in the onset of atopy, asthma, and/or recurrent wheezing at older ages is controversial.3,4,9,13,15,16 The effect of BPD in lung func- tion in the long term has been evaluated in numerous age groups, including newborns,17 infants,18 preschoolers,19 schoolchildren,7---11 adolescents,12---14 and adults.20---22 Studies have shown reduced lung volumes in different age groups, but with low association to clinical symptoms. This fact has not been explained in the literature. The diagnosis of BPD was established in infants who, after 28 days of life, had respiratory failure and depended on oxy- gen at over 21% concentration to maintain partial pressure 273 Clinical variables related to asthma in schoolchildren born with very low birth weight between 25% and 75% of FVC (FEF 25---75%). A CPFS/D model spirometer (Medical-Graphics, Saint Paul, Minnesota, United States) was used. Spirometry followed the recommendations of the European Respiratory Society (ERS) and the Ameri- can Thoracic Society (ATS).24 The child took the test while standing and used the nose clip with open maneuver. The child was requested to perform a vigorous and prolonged expiratory maneuver to achieve the reproducibility criteria of the spirometry test software: Breeze PF version 3.8 for Windows 95/98/NT. The evaluation of FVC, FEV1, FEV1/FVC, and FEF 25---75% was conducted according to the standard reference equations by Quanjer et al.24 based on age, sex, weight, and ethnicity. Airway obstruction was described as FEV1/FVC ratios below the lower limit of normal; for sever- ity, the FEV1% z-score was used in two categories: (i) mild obstruction: z-score≥−2 or 70% of the predicted value; (ii) moderate obstruction: −2.5≤z-score<−2.0 or 60---69% of the predicted value.24,25 of oxygen >50mmHg.1 Two groups were considered: BPD and no BPD. Telephone calls were made to invite schoolchildren aged between 7 and 12 years to participate in the study. The aforementioned exclusion criteria were again applied. Method Asthma was deﬁned as positive answers to question 2 of the IQ: ‘‘In the last 12 (twelve) months, has your child had wheezing?’’ This question has a sensitivity of 100%, speciﬁcity of 78%, positive predictive value of 73%, and neg- ative predictive value of 100% for the diagnosis of asthma.23 The prevalence of severe asthma was considered in those who answered ‘‘yes’’ to at least two of the following IQ questions:23 ‘‘in the last 12 (twelve) months, how many wheezing crises has your child had?’’; ‘‘In the last 12 (twelve) months, how often was your child’s sleep disturbed by wheezing?’’; ‘‘In the last twelve (12) months, was the hiss of your child so strong as to prevent him/her from say- ing more than two words between every breath?’’; or ‘‘In the last 12 (twelve) months, has your child had wheezing after exercise?’’ g Data were processed using SPSS version 16.0 (SPSS Inc., Chicago, United States) and Epi-Info 6.04b (CDC, United States). To compare the ratios, the 2 test was used or, when indicated, Fisher’s exact test for 2×2 tables or the Fisher---Freeman---Halton exact distribution test for larger tables. In the case of the Fisher---Freeman---Halton test, bilateral probability was estimated using the Monte Carlo method. To compare the means of two independent groups, Student’s t-test was used. Prevalences and their 95% conﬁ- dence intervals were calculated using the exact method (binomial distribution), using the Epitable routine of Epi- Info. The Odds Ratio (OR) and its 95% conﬁdence interval for asthma in relation to BPD were determined using the StatCalc routine of Epi-Info. The mean, standard deviation, minimum, and maximum values of the quantitative variables of patients regarding the diagnosis of asthma and BDP were determined. A signiﬁcance level of p<0.05 was adopted. The participants underwent clinical examination, skin prick test (SPT), spirometry, and measurement of bronchial hyperresponsiveness (BHR) through a concentration of methacholine that produced a 20% decrease in the forced expiratory volume in one second (FEV1) at spirometry (PC20 methacholine). All procedures were performed in lung phys- iology laboratory of the Pediatrics Research Center, School of Medical Sciences. The SPT was performed by puncture23 technique on the forearm with plastic lancets (Alergoprick-FlexorTM, Sertãoz- inho, SP, Brazil). A set of lancets was used for each child, one for each extract. The following puriﬁed glycerinated extracts at 50%, manufactured by IPI-ASACTM (São Paulo, SP, Brazil), were used: Dermatophagoides pteronyssinus, D. Method A personal interview was conducted with parents and/or guardians, who answered the written International Study of Asthma and Allergies in Childhood (ISAAC) Phase I (IQ) and complementary ISAAC phase II (CIQ) questionnaires.23 The risk factors assessed by CIQ23 regarding the ﬁrst year of life and the past year were: breastfeeding duration; older sib- lings (regardless of the presence of atopy and/or asthma); age at start of daycare or primary school attendance; pater- nal and maternal atopy; contact with animals inside and outside the home, current smoking at home; current, pre- natal, and postnatal maternal smoking; presence of mold on the walls; type of ﬂoor, pillow, and blanket; and resi- dence in rural or urban area. Questions about family history of atopy in siblings, parents, and grandparents; pneumonia; atopic dermatitis; allergic rhinitis; bronchiolitis; heart dis- ease; hospitalizations; drug allergies; and relief and routine asthma medication were also asked. The BHR measurement followed the recommendations of ERS/ATS.24 Nebulizations with acetyl-beta-methylcoline (methacoline-chloride; code A2251, Sigma) were made with the following dilutions (in mg/mL): 0.125; 0.25; 0.5; 1; 2; 4; 8; 16; 32. Spirometry was performed at the start of the exam and 1min after each nebulization with methacholine. The exam was interrupted when a ≥20% decrease from base- line FEV1 was reached. Then, each subject received inhaled salbutamol (four jets of 100mcg) and after 30min, the last FEV1 was recorded. The test was considered positive when a 20% decrease in FEV1 occurred with less than 4mg of metha- choline, and was graded as (i) mild, at concentrations of 1---4mg/mL methacholine; (ii) moderate to severe, at con- centrations <1mg/mL. Method farinae, Blomia tropicalis, Blatella germanica, fun- gus mix (Alternaria alternata, Cladosporodium herbarum, Aspergilus fumigatus and Penicilium), Canis familiaris, Felis domesticus. The following controls were used: positive (his- tamine) and negative (0.9% saline solution). The results were measured after 15min; a reaction was considered positive if wheal size ≥3mm, with non-reactive negative control and positive control with wheal size ≥3mm.23 The presence of a positive response in the SPT was considered as atopy. For the present study, the sample power was calculated using G*Power 3.1.9.2, using as a parameter the group of patients with asthma; at Fisher’s exact test, the sample power was >0.8003402, above the cutoff value 80%. The calculation of sample power was made after the study. Results Fig. 1 presents the inclusion ﬂow diagram. Of the 54 patients included, 33/54 (61.1%) were male, aged 9.9±0.92 years, and 21/54 (38.9%) female, aged 9.9±0.90 years (p=0.810). Male and female VLBW infants had similar BW: 1065.1±243.8g and 1120±243g, respectively (p=0.423). Spirometry assessed the forced vital capacity (FVC), FEV1, Tiffeneau index (FEV1/FVC), and mean expiratory ﬂow 274 Gonc¸alves ES et al. 274 Gonc¸alves ES et al. 366 VLBW infants born between 2000 and 2004 297 survived 256 possible to reach 41 impossible to reach 81 reached 175 not reached 2 deaths 69 deaths 54 included ISAAC and SPT 2 refusals 43 perfomed spirometry 1 had moderate obstruction –2.5 ≤ z-score ≤–2.0 5 had no obstruction 37 had mild obstruction z-score ≥–2.0 25 refused to participate 8 did not reach reproducibility criteria 1 asthma exacerbation Figure 1 Inclusion ﬂow diagram of very low birth weight (VLBW) infants. ISAAC, International Study of Asthma and Allergies in Childhood; SPT, skin prick test. 366 VLBW infants born between 2000 and 2004 41 impossible to reach 256 possible to reach 175 not reached 54 included ISAAC and SPT 2 refusals 43 perfomed spirometry Figure 1 Inclusion ﬂow diagram of very low birth weight (VLBW) infants. ISAAC, International Study of Asthma and Allergies in Childhood; SPT, skin prick test. There was a higher prevalence of whites (47/54---87%). Table 1 shows the characteristics of VLBW infants with BPD. The age at the time the study was lower in the BPD group (p=0.014). Table 2 shows the characteristics of VLBW infants with and without asthma. positive in 13/17 (76.5%) and 23/37 (62.2%), respectively (p=0.364; Table 2). In both cases, a positive association was observed. Among the variables analyzed for the ISAAC question- naire, an association was observed with the use of wool blanket in the ﬁrst year of life (p=0.026; [With asthma and wool blanket: 6/17 individuals with asthma] and [with- out asthma and with wool blanket: 25/37 subjects without asthma]; OR=0.269; 95%CI=0.08---0.088). The prevalence of asthma in VLBW infants was 17/54 (31.5%). The BPD and no BPD groups presented similar prevalence of asthma: 6/18 (33.3%) and 11/36 (30.6%), respectively (p=0.836; Tables 1 and 2). p y (p ) BW in the BPD group (916.7±251.2g) was lower than in the no BPD group (1171.3±190.5g; p<0.001). No association between BPD and asthma severity (p=0.620) and between BPD and the number of pneumonias (p=1.0; Table 1) were observed. Clinical variables related to asthma in schoolchildren born with very low birth weight Variablesa With asthma (n=17/54) Without asthma (n=37/54) p-value OR (95%CI) Age at the test (years)b 9.7±0.6 (8.5---0.7) 10±1 (7.9---11.5) 0.322 --- Birth weight (g)b 1075.4±267.5 (420---1455) 1091.5±234.1 (600---1455) 0.824 --- Malec 9 (52.9%) 24 (64.8%) 0.404 0.615 (0.16---2.32) Whited 14 (82.3%) 33 (89.1%) 0.809 0.572 (0.08---4.42) Presence of severe asthmad 9 (52.9%) Prior history of pneumoniae None 10 (58.8%) 19 (51.3%) 0.638 1.346 (0.42---4.51) 1---3 5 (29.4%) 15 (40.6%) 0.515 (0.06---5.40) 4 or more 2 (11.7%) 3 (8.1%) 1.499 (0.16---11.06) Positive prick testc 13 (76.5%) 23 (62.16%) 0.364 1.954 (0.473---9.884) g, grams; n, number of patients; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; data with numerical distribution are presented as mean±standard deviation; minimum−maximum. b Student’s t-test. c 2 test. d Fisher’s exact test. g, grams; n, number of patients; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; data with numerical distribution are presented as mean±standard deviation; minimum−maximum. b Student’s t-test. c 2 test. d Fisher’s exact test. e Fisher---Freeman---Halton exact test d Fisher’s exact test. e Fisher---Freeman---Halton exact test of BPD and associated risk factors. Studies that aim to analyze the inﬂuence of lung diseases in early life and their repercussions in adolescence and adulthood have increased in recent decades. Many of them try to answer the question of whether the BPD and/or preterm birth are risk factors for asthma.4,11,21,26 As found in the present study, other authors have observed a higher prevalence of asthma in VLBW infants when compared with the prevalence of asthma in the world population,7,11,19,22,27 with values for extremely premature infants ranging between 26%5,19,27 and 40%.22 p=0.049; Fig. 2). There was no difference between spirom- etry variables in the BPD and no BPD groups, as well as in the groups with or without asthma. The values of PC20 methacholine in VLBW group with asthma were lower than in the group without asthma (p=0.022). In contrast, the values of PC20 methacholine in the VLBW BPD and no BPD groups were similar (p=0.72). Clinical variables related to asthma in schoolchildren born with very low birth weight Clinical variables related to asthma in schoolchildren born with very low birth weight 275 Table 1 Characterization of the population with birth weight lower than 1500g considering the presence of bronchopulmonary dysplasia (BPD). dysplasia (BPD). Variablesa BPD (n=18/54) No BPD (n=36/54) p-value OR (95%CI) Age at the test (years)b 9.5±0.85 (7.9---10.7) 10.1±0.86 (8.2---11.5) 0.14 --- Birth weight (g)b 916.7±251.2 (420---1320) 1171.3±190.5 (600---1465) <0.1 --- Malec 13 (72.2%) 20 (55.6%) 0.236 2.052 (0.54---8.97) Whited 16 (88.9%) 31 (86.1%) 1.000 1.284 (0.18---14.91) Presence of asthmac 6 (33.3%) 11 (30.6%) 0.836 1.134 (0.27---4.39) Presence of severe asthmad 4 (22.2%) 5 (13.8%) 0.620 2.278 (0.21---35.67) Prior history of pneumoniae None 9 (50.0%) 20 (55.6%) 1.000 0.803 (0.25---2.56) 1---3 7 (38.9%) 13 (36.1%) 0.815 (0.07---11.95) 4 or more 2 (11.1%) 3 (8.33%) 1.367 (0.10---13.22) Positive prick testc 10 (55.6%) 26 (72.2%) 0.239 0.488 (0.15---1.64) BPD bronchopulmonary dysplasia; g grams; n number of patients; OR Odds Ratio; 95%CI 95% conﬁdence interval a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; the data with numerical distribution are presented as mean±standard deviation; minimum−maximum. b Table 2 Characterization of the population with birth weight lower than 1500g considering the presence of asthma. Table 2 Characterization of the population with birth weight lower than 1500g considering the presence of asthma. Variablesa With asthma (n=17/54) Without asthma (n=37/54) p-value OR (95%CI) Age at the test (years)b 9.7±0.6 (8.5---0.7) 10±1 (7.9---11.5) 0.322 --- Birth weight (g)b 1075.4±267.5 (420---1455) 1091.5±234.1 (600---1455) 0.824 --- Malec 9 (52.9%) 24 (64.8%) 0.404 0.615 (0.16---2.32) Whited 14 (82.3%) 33 (89.1%) 0.809 0.572 (0.08---4.42) Presence of severe asthmad 9 (52.9%) Prior history of pneumoniae None 10 (58.8%) 19 (51.3%) 0.638 1.346 (0.42---4.51) 1---3 5 (29.4%) 15 (40.6%) 0.515 (0.06---5.40) 4 or more 2 (11.7%) 3 (8.1%) 1.499 (0.16---11.06) Positive prick testc 13 (76.5%) 23 (62.16%) 0.364 1.954 (0.473---9.884) g grams; n number of patients; OR Odds Ratio; 95%CI 95% conﬁdence interval zation of the population with birth weight lower than 1500g considering the presence of asthma. Table 2 Characterization of the population with birth weight lower than 1500g considering the p Table 2 Characterization of the population with birth weight lower than 1500g considering the presence of asthma. Clinical variables related to asthma in schoolchildren born with very low birth weight Table 4 describes the data regarding the ISAAC variables and the presence of asthma, and Table 5 describes the associa- tion of asthma severity with BPD and BW. Infants who develop BPD have a high prevalence of inﬂammation and decreased lung ﬂow in childhood and adulthood.1,4,28 The initial inﬂammatory manifestations result from genetic susceptibility, endocrine disorders, infections, volutrauma, barotrauma, oxygen toxicity, and a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; the data with numerical distribution are presented as mean±standard deviation; minimum−maximum. b g, grams; n, number of patients; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. Results Among the 43 children and adolescents who under- went spirometry, ﬁve showed no obstruction, 37 presented mild obstruction, and one teenager presented moderate obstruction.24 Table 3 shows the z-score distribution of spiro- metric values in patients with and without BPD compared to those who did or did not develop asthma. The mean z- score of FEF 25---75% in patients with asthma (−1.04±1.19) was lower than in the group without asthma (−0.38±0.82; The SPT was positive in 10/18 (55.6%) and 26/36 (72.2%) in the BPD and no BPD groups, respectively (p=0.239; Table 1). In the groups with and without asthma, the SPT was g, g ; , p ; , ; , a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; data with numerical distribution are presented as mean±standard deviation; minimum−maximum. b Student’s t-test. c 2 test. d Fisher’s exact test. e Fisher---Freeman---Halton exact test onchopulmonary dysplasia; g, grams; n, number of patients; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. Discussion These authors found no difference among VLBW infants with and without BPD regarding asthma.21 patent ductus arteriosus.3 The complexity of the presence and interaction of these factors, which contribute to the development and severity of BPD, can affect growth and lung function to a greater or lesser extent.3,4 In contrast, the inﬂuence of prematurity, BDP, or the interaction of both in the prevalence of asthma remains controversial.11,21,26 While most studies show a positive association between BPD2,7---14,17,20,22,26 or prematurity28,29 as a risk factor for asthma, one study did not observe this association.21 The present study observed a high prevalence of asthma; how- ever, it was similar in VLBW infants who developed BPD and those who did not. Possibly, prematurity was a more important factor than BPD for the development of asthma in schoolchildren in the present sample, a fact conﬁrmed by Bronstron et al.9 In addition to the present study, only one study failed to observe an association between BPD and asthma. Narang et al. studied 110 patients at 21 years, 60 patent ductus arteriosus.3 The complexity of the presence and interaction of these factors, which contribute to the development and severity of BPD, can affect growth and lung function to a greater or lesser extent.3,4 In contrast, the inﬂuence of prematurity, BDP, or the interaction of both in the prevalence of asthma remains controversial.11,21,26 While most studies show a positive association between BPD2,7---14,17,20,22,26 or prematurity28,29 as a risk factor for asthma, one study did not observe this association.21 The present study observed a high prevalence of asthma; how- ever, it was similar in VLBW infants who developed BPD and those who did not. Possibly, prematurity was a more important factor than BPD for the development of asthma in schoolchildren in the present sample, a fact conﬁrmed by Bronstron et al.9 In addition to the present study, only one study failed to observe an association between BPD and asthma. Narang et al. studied 110 patients at 21 years, 60 Another interesting ﬁnding is the inﬂuence of sex in the prevalence of obstructive lung diseases in children and ado- lescents. Discussion This study showed a high prevalence of asthma in VLBW infants at school age, regardless of the presence or absence 276 Gonc¸alves ES et al. Table 3 Distribution of the z-score of spirometry variables (FVC, FEV1, FEV1/FVC, and FEF 25---75%) of children and adolescents who were very low birth weight (VLBW) infants with and without bronchopulmonary dysplasia (BPD) in relation to those who did or did not develop asthma. Table 3 Distribution of the z-score of spirometry variables (FVC, FEV1, FEV1/FVC, and FEF 25---75%) of children and adolescents who were very low birth weight (VLBW) infants with and without bronchopulmonary dysplasia (BPD) in relation to those who did or did not develop asthma Variablesa BPD (12/43) 28% No BPD (31/43) 72% p-valueb FVC Asthma −0.37±1.83 −0.26±0.89 0.899 No asthma +0.21±1.07 −0.21±0.71 0.247 FEV1 Asthma −0.86±2.05 −0.49±1.08 0.641 No asthma −0.10±1.08 −0.11±0.88 0.978 FEV1/FVC Asthma −0.92±0.90 −0.44±0.95 0.364 No asthma −0.57±0.76 0.17±0.91 0.067 FEF25---75% Asthma −1.38±1.42 −0.89±1.11 0.463 No asthma −0.71±0.80 −0.27±0.96 0.292 Variables With asthma (16/43) 37.2% Without asthma (27/43) 62.8% p-valueb FVC BPD −0.37±1.83 +0.21±1.07 0.230 No BPD −0.26±0.89 −0.21±0.71 0.776 FEV1 BPD −0.86±2.05 −0.10±1.08 0.412 No BPD −0.49±1.08 −0.11±0.88 1.000 FEV1/FVC BPD −0.92±0.90 −0.57±0.76 0.648 No BPD −0.44±0.95 0.17±0.91 0.066 FEF25---75% BPD −1.38±1.42 −0.71±0.80 0.527 No BPD −0.89±1.11 −0.27±0.96 0.244 FVC, forced vital capacity; BPD, bronchopulmonary dysplasia; FEF 25% -75%, mean expiratory ﬂow between 25% and 75% of FVC; FEV1, forced expiratory volume in one second; FEV1/FVC, ratio of FEV1 and FVC. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; the data with numerical distribution are presented as mean±standard deviation. b Two-tailed Student’s t-test. forced expiratory volume in one second; FEV1/FVC, ratio of FEV1 and FVC. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; the data with numerical distribution are presented as mean±standard deviation. b Two-tailed Student’s t-test. full-term infants and 50 VLBW infants, and seven had BPD. Discussion In the present study, the high prevalence of smoking in parents (25%), attendance at daycare in the ﬁrst year of life (50%), low prevalence of breastfeeding, and history of maternal/paternal asthma in 20% of schoolchil- dren were noteworthy. Although the prevalence of asthma was high, the only risk factor associated with the CIQ was the use of wool blankets in the ﬁrst year of life (p=0.026). This may reﬂect a higher exposure to allergens factors, which, together with inﬂammatory alterations caused by prematurity, could facilitate the onset of asthma due to early allergic sensitization.6 Unlike the present ﬁndings, Palta et al. observed a higher prevalence of asthma in VLBW infants with BPD when compared with those without BPD. In that study, the BPD group had a higher frequency of risk factors for asthma, which included repeated respiratory infections in the ﬁrst year of life, family history of asthma, smoking parents, and family income.7 Kwinta et al. assessed risk factors for asthma in VLBW infants with or without BPD compared to healthy term infants. Those authors found that VLBW infants had more recurrent wheezing (OR=5.38, 95%CI 2.14---13.8) and a higher prevalence of risk factors, such as hospitalization and wheezing below 24 months.11 Ri k f f h h diff i h i Numerous researchers have aimed to determine whether diseases in the neonatal period affect lung function in childhood, adolescence, and in later ages.2,4,5,7---15,17---22,27 While the relationship between BPD and asthma remains controversial,4,15 decreased lung function in VLBW infants throughout life has been documented.8---10,26,28 Some studies,8,19,20,26 including the present, showed lower lung volumes in the peripheral airways in children with asthma who were VLBW infants. The presence or absence of BPD did not increase the prevalence of asthma or airway obstruction in the present patients. Thus, prematurity may have a higher probability of association with asthma than BPD. p y Gough et al. evaluated adults aged 24---25 years who had been VLBW infants with and without BPD. Those with BPD had twice as many episodes of wheezing and used three times more asthma medications.22 A prospective study showed that, at age 11, VLBW infants with BPD had lower lung volumes in spirometry and greater response to bron- chodilators when compared with those who did not develop BPD.14 Doyle et al. Discussion While male infants have narrower airways and increased prevalence of wheezing,6 the differences in lung function decrease with age and reverse in adolescence.2 It is believed that this occurs due to hormonal changes in women, with an increased risk of asthma at this age group.2 For the VLBW infants in the present study, there was no differ- ence between sexes for both BPD and for asthma. This result was similar to that obtained by Fawke et al.,14 who studied respiratory symptoms and lung function in VLBW infants at 11 years and found no differences between the sexes. This explains the reduction of lung function differences between the sexes observed with age. Clinical variables related to asthma in schoolchildren born with very low birth weight 277 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 –0.5 –1.0 –1.5 –2.0 –2.5 –3.0 –3.5 –4.0 With asthma Spirometry variables (expressed as z-scores) Without asthma FEV1 FVC FEV1/FVC FEF25-75 p=0.529 p=0.161 p=0.058 p=0.049 FVC FEV1 FEV1/FVC FEF25-75 Figure 2 Distribution of z-scores in relation to asthma in children and adolescents who were very low birth weight (VLBW) newborns regarding spirometry variables. FVC, forced vital capacity; FEV1, forced expiratory volume in one second; FEV1/FVC, ratio between FEV1 and FVC; FEF 25---75%, mean expiratory ﬂow between 25% and 75% of FVC; p, p-value. Medians are present as horizontal lines that divide the rectangles, which contain 50% of values. The bars extend to the maximum and minimum values. Two-tailed Student’s t-test. With asthma With asthma Without asthma Figure 2 Distribution of z-scores in relation to asthma in children and adolescents who were very low birth weight (VLBW) newborns regarding spirometry variables. FVC, forced vital capacity; FEV1, forced expiratory volume in one second; FEV1/FVC, ratio between FEV1 and FVC; FEF 25---75%, mean expiratory ﬂow between 25% and 75% of FVC; p, p-value. Medians are present as horizontal lines that divide the rectangles, which contain 50% of values. The bars extend to the maximum and minimum values. Two-tailed Student’s t-test. Most authors who have studied the association between prematurity/BPD and future asthma5,7,8,11,14,15,18,19,27 included the ISAAC23 risk factors and the Asthma Predictive Index (API).6 Although the present study did not speciﬁcally evaluate the API, all variables contained in that index, except for the number of eosinophils in peripheral blood, were analyzed, and no differences between both groups were observed. Discussion followed-up a cohort of 147 VLBW infants with and without BPD, assessed at ages 2, 5, 8, 11, 14, and 18 years. These authors observed that lung volumes showed greater reductions between the age range of 8---18 years, more pronounced in the BPD group.8 Recently, von Hove et al. assessed the respiratory symptoms and lung function in adolescents who were VLBW infants, 28 with and 28 with- out BPD. They concluded that those with BPD were more likely to present abnormalities in lung function.12 The main cause of recurrent wheezing in children is atopic asthma. The present study failed to demonstrate an association of allergy (proven by clinical history and SPT) with BPD or asthma. Several studies, including the review by Perez and Navarro, evaluated the associations between preterm birth with and without BPD and future atopy/allergies, demonstrating that BPD has no longitudinal relationship with atopy or BHR.2,3,9,13,30 One study found an association between BPD and future atopy.16 Subsequently, Risk factors for asthma may have different weights in children and adolescents who were VLBW or term newborn. While these factors have been thoroughly studied6 in chil- dren healthy at birth, the number of studies on VLBW infants is lower. Thus, the assessment of large cohorts following- up VLBW infants with and without BPD may clarify whether there are differences between them regarding the exposure to risk factors and development of future chronic diseases. 278 Gonc¸alves ES et al. Table 4 ISAAC risk factors in newborns with very low weight with and without asthma. Discussion Variablesa With asthma (17/54) 31.5% Without asthma (37/54) 68.5% p-value OR (95%CI) Breastfeeding durationb >6 months 4 (36.4%) 7 (63.3%) 0.672 1.312 (0.30---5.37) <6 months 8 (26.7%) 22 (73.3%) 0.589 (0.14---2.51) Absent 5 (38.5%) 8 (61.5) 1.498 (0.38---5.65) Older siblingsb ≥4 siblings 3 (42.9%) 4 (57.1%) 0.591 1.748 (0.29---9.55) 3 siblings 1 (33.3%) 2 (66.6%) 1.18 (0.02---24.73) 2 siblings 3 (50%) 3 (50%) 2.73 (0.41---18.29) 1 sibling 2 (16.7%) 10 (83.3%) 0.46 (0.04---2.98) No sibling 8 (30.8%) 18 (69.2%) 0.939 (0.29---3.04) Daycare attendanceb <12 months 1 (20%) 4 (80%) 0.416 0.521 (0.01---5.86) ≥12 months 8 (44.4%) 10 (55.6%) 2.26 (0.56---9.32) No 8 (25.8%) 23 (74.2%) 0.547 (0.15---2.02) Father with asthmac Present 1 (25%) 3 (75%) 1.000 0.891 (0.02---12.41) Mother with asthmac Present 2 (28.6%) 5 (71.4%) 1.000 0.803 (0.07---5.66) Parents with asthmac Present 0 1 (100%) 1.000 - Number of people sharing the child’s roomc 3 or more 3 (42.9%) 4 (57.1%) 0.665 1.748 (0.23---11.88) Indoor petd Present 10 (33.3%) 20 (66.7%) 0.743 1.21 (0.33---4.64) Maternal smoking during pregnancyc Present 5 (38.5%) 8 (61.55%) 0.733 1.498 (0.32---6.55) Maternal smoking during the ﬁrst year of the child’s lifec Present 5 (35.7%) 9 (64.3%) 0.745 1.290 (0.28---5.47) Maternal smokingc Present 7 (50%) 7 (50%) 0.103 2.933 (0.692---12.76) Household member smoking Present 4 (25%) 12 (75%) 0.506 0.646 (0.13---2.73) Current presence of moldd Present 6 (28.6%) 15 (71.4%) 0.713 0.803 (0.20---3.02) Presence of mold during the ﬁrst year of the child’s lifed Present 5 (35.7%) 9 (64.3%) 0.745 1.29 (0.28---5.47) Current presence of ﬁtted carpetsc Present 17 (31.5%) 37 (68.5%) --- --- Presence of ﬁtted carpets during the ﬁrst year of the child’s lifed Present 2 (66.7%) 1 (33.3%) 0.230 4.644 (0.23---290.8) Current tiled ﬂoorc Present 16 (37.2%) 27 (62.8%) 0.143 5.777 (0.70---273) Presence of tiled ﬂoor during the ﬁrst year of the child’s lifec Present 14 (35%) 26 (65%) 0.507 1.951 (0.42---12.69) Current hardwood ﬂoorc Present 0 2 (100%) 1.000 --- Presence of bare ﬂoor in the child’s bedroom during the ﬁrst year of the child’s lifec Present 1 (12.5%) 7 (87.5%) 0.411 0.273 (0.01---2.44) Current bare ﬂoor in the child’s bedroomc Present 1 (11.1%) 8 (88.9%) 0.244 0.232 (0.01---2.00) Use of foam pillow during the ﬁrst year of the child’s lifec Present 14 (35%) 26 (65%) 0.299 2.908 (0.52---30.67) Current use of foam pillowc Present 12 (34.3%) 23 (65.75) 0.547 1.451 (0.37---6.42) Use of synthetic ﬁber pillow during the ﬁrst year of the child’s lifec Present 0 6 (100%) --- --- Current use of synthetic ﬁber pillowc Present 3 (25%) 9 (75%) 0.732 0.672 (0.10 --- 3.29) Use of feather pillow during the ﬁrst year of the child’s lifec Present 1 (33.3%) 2 (66.7%) 1.000 1.163 (0.02---14.55) Current use of feather pillowc Present 2 (40%) 3 (60%) 0.645 1.499 (0.11---14.55) Use of wool blanket during the ﬁrst year of the child’s lifed Present 6 (19.4%) 25 (80.6%) 0.026 0.269 (0.08---0.88) Current use of wool blanketd Present 3 (20%) 12 (80%) 0.338 0.453 (0.07---2.09) Use of cotton blanket during the ﬁrst year of the child’s lifed Present 4 (33.3%) 8 (66.7%) 1.000 1.113 (0.21---5.13) Current use of cotton blanketd Present 4 (28.6%) 10 (71.4%) 1.000 0.834 (0.16---3.64) Living in a rural neighborhood during the ﬁrst year of the child’s lifed Present 2 (22.2%) 7 (77.8%) 0.703 0.578 (0.05---3.56) Living in an urbanneighborhood during the ﬁrst year of the child’s life/currentlyd Present 15 (33.3%) 30 (66.7%) 0.703 1.733 (0.28---19.11) Positive prick testc 13 (76.5%) 23 (62.2%) 0.364 1.954 (0.473---9.884) ISAAC, International Study of Asthma and Allergies in Childhood; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. Student s t test. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups. b Fisher---Freeman---Halton exact test. c Fisher’s exact test. d 2 ISAAC, International Study of Asthma and Allergies in Childhood; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. Student’s t-test. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups. b Fisher---Freeman---Halton exact test. c Fisher’s exact test. d 2 test. c Fisher’s exact test. d 2 test. References 1. Carraro S, Filippone M, Da Dalt L, Ferraro V, Maretti M, Bressan S, et al. Bronchopulmonary dysplasia: the earliest and perhaps the longest lasting obstructive lung disease in humans. Early Hum Dev. 2013;89:S3---5. 2. Pérez PG, Navarro NM. Broncopulmonary dysplasia and pre- maturity. Short-and long-term respiratory changes. An Pediatr. 2010;72:79.e1---16. 3. Cunha GS, Mezzacappa Filho F, Ribeiro JD. Maternal and neonatal factors affecting the incidence of bronchopulmonary dysplasia in very low birth weight newborns. J Pediatr (Rio J). 2003;79:550---6. Considering the diagnosis of asthma according to the ISAAC questionnaire, this study showed that VLBW infants, especially those of lower weight, had a higher prevalence of asthma, with no difference regarding the presence of BPD. Additional research including evolutionary comparison between infants, preschoolers, and schoolchildren through lung function by spirometry, lung clearance index, and bronchoalveolar lavage may bring light to the differences between the presence of BHR and asthma after prematu- rity and BPD. As these tools are difﬁcult to use in clinical practice, multi-center epidemiological follow-up studies should be encouraged to allow for the establishment of sam- ples that are adequate for comparisons in univariate and multivariate analyses. 4. El Mazloum D, Moschino L, Bozzetto S, Baraldi E. Chronic lung disease of prematurity: long-term respiratory outcome. Neona- tology. 2014;105:352---6. 5. Halvorsen T, Skadberg BT, Eide GE, Roksund O, Aksnes L, Oymar K. Characteristics of asthma and airway hyper- responsiveness after premature birth. Pediatr Allergy Immunol. 2005;16:487---94. 6. Lopez N, de Barros-Mazon S, Vilela MM, Condino Neto A, Ribeiro JD. Are immunoglobulin E levels associated with early wheez- ing? A prospective study in Brazilian infants. Eur Respir J. 2002;20:640---5. 7. Palta M, Sadek-Badawi M, Sheehy M, Albanese A, Weinstein M, McGuinness G, et al. Respiratory symptoms at age 8 years in a cohort of very low birth weight children. Am J Epidemiol. 2001;154:521---9. 8. Doyle LW, Victorian Infant Collaborative Study Group. Respi- ratory function at age 8---9 years in extremely low birth weight/very preterm children born in Victoria in 1991---1992. Pediatr Pulmonol. 2006;41:570---6. The authors conclude that preterm infants with and with- out BPD have higher prevalence of asthma in childhood and adolescence when compared with the data reported in the literature for individuals in the same age group. The impact of BPD in the prevalence and severity of asthma remains controversial. 9. Broström EB, Thunqvist P, Adenfelt G, Borling E, Katz-Salamon M. Clinical variables related to asthma in schoolchildren born with very low birth weight inical variables related to asthma in schoolchildren born with very low birth weight 279 Table 5 Characterization of the population born weighing less than 1500g considering asthma severity. Variablesa Severe asthma Mild asthma p-value OR (95%CI) Birth weight (g)b 1076.44±341.98; 1150 (420---1455) 1074.25±172.83; 1063 (805---1295) 0.987 BPD (present)c 4 (66.7%) 2 (33.3%) 0.620 2.278 (0.21---35.67) g, grams; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; the data with numerical distribution are presented as mean±standard deviation; median (minimum−maximum). b Student’s t-test. c Fisher---Freeman---Halton exact test. Table 5 Characterization of the population born weighing less than 1500g considering asthma severity. Variablesa Severe asthma Mild asthma p-value OR (95%CI) Birth weight (g)b 1076.44±341.98; 1150 (420---1455) 1074.25±172.83; 1063 (805---1295) 0.987 BPD (present)c 4 (66.7%) 2 (33.3%) 0.620 2.278 (0.21---35.67) g, grams; OR, Odds Ratio; 95%CI, 95% conﬁdence interval. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups; the data with numerical distribution are presented as mean±standard deviation; median (minimum−maximum). b Student’s t-test c Fisher---Freeman---Halton exact test. Acknowledgements several authors have failed to observe this association in children who were VLBW infants.9,13,30 One of the limitations of the present study and of that by Narang et al. was the small number of individuals included, compared to the total number of those who were eligi- ble. Among the eligible subjects in the present study, 25 (30.86%) of those aged between 7 and 12 years refused to participate. In contrast, the present study assessed the risk factors for childhood asthma, which was not studied by Narang et al. Another aspect is that the present patients used a surfactant, while the subjects of Narang et al.21 did not. Moreover, it is worth mentioning as a limitation of the present study the impossibility of obtaining data on BPD severity, duration of mechanical ventilation, and other respiratory diagnoses in the neonatal period. A difﬁcult-to- assess aspect that may constitute sample bias is whether the individuals who decided to participate in the present study had greater morbidities, including asthma, consider- ing that VLBW infants with BPD had lower birth weight and were younger at time of examination. The authors would like to thank the Maternity Ward of the University Hospital and the Pulmonary Physiology Laboratory of the University. References Obstructive lung disease in children with mild to severe BPD. Respir Med. 2010;104:362---70. 10. Kaplan E, Bar-Yishay E, Prais D, Klinger G, Mei-Zahav M, Mus- safﬁ H, et al. Encouraging pulmonary outcome for surviving, neurologically intact, extremely premature infants in the post surfactant era. Chest. 2012;142:725---33. Discussion Student’s t-test. a Data for variables with categorical distribution are represented as number of patients with variable/total number of individuals included in the group and percentage related to the variable analyzed within the possible groups. Table 4 ISAAC risk factors in newborns with very low weight with and without asthma. Funding This study did not receive funding. 11. Kwinta P, Lis G, Klimek M, Grudzien A, Tomasik T, Poplawska K, et al. The prevalence and risk factors of allergic and respiratory symptoms in a regional cohort of extremely low birth weight children (<1000g). Ital J Pediatr. 2013;39:4. Conﬂicts of interest Conﬂicts of interest 12. Vom Hove M, Prenzel F, Uhlig HH, Robel-Tillig E. Pulmonary out- come in former preterm, very low birth weight children with The authors declare no conﬂicts of interest. 280 Gonc¸alves ES et al. bronchopulmonary dysplasia: a case---control follow-up at school age. J Pediatr. 2014;164:40---5. bronchopulmonary dysplasia: a case---control follow-up at school age. J Pediatr. 2014;164:40---5. 22. Gough A, Linden M, Spence D, Patterson CC, Halliday HL, Mc Garvey LP. Impaired lung function and health status in adult survivors of bronchopulmonary dysplasia. Eur Respir J. 2014;43:808---16. 13. Lum S, Kirkby J, Welsh L, Marlow N, Hennessy E, Stocks J. Nature and severity of lung function abnormalities in extremely pre-term children at 11 years of age. Eur Respir J. 2011;37:1199---207. 23. Solé D, Camelo-Nunes IC, Wandalsen GF, Mallozi MC. Asthma in children and adolescents in Brazil: contribution of the Interna- tional Study of Asthma and Allergies in Childhood (ISAAC). Rev Paul Pediatr. 2014;32:114---25. 14. Fawke J, Lum S, Kirkby J, Hennessy E, Marlow N, Rowell V, et al. Lung function and respiratory symptoms at 11 years in children born extremely preterm: the EPICure study. Am J Respir Crit Care Med. 2010;182:237---45. 24. Quanjer PH, Stanojevic S, Cole TJ, Baur X, Hall GL, Culver BH, et al. Multi-ethnic reference values for spirometry for the 3---95- yr age range: the global lung function 2012 equations. Eur Respir J. 2012;40:1324---43. 15. Chatkin MN, Menezes AM. The association between low birth- weight and asthma: a systematic literature review. Rev Panam Salud Publica. 2005;17:102---9. 25. Quanjer PH, Pretto JJ, Brazzale DJ, Boros PW. Grading the severity of airways obstruction: new wine in new bottles. Eur Respir J. 2014;43:505---12. 16. Siltanen M, Savilahti E, Pohjavuori M, Kajosaari M. Respiratory symptoms and lung function in relation to atopy in children born preterm. Pediatr Pulmonol. 2004;37:43---9. 26. Gough A, Spence D, Linden M, Halliday HL, McGarvey LP. General and respiratory health outcomes in adult survivors of bronchopulmonary dysplasia: a systematic review. Chest. 2012;141:1554---67. 17. May C, Kennedy C, Milner AD, Rafferty GF, Peacock JL, Greenough A. Lung function abnormalities in infants devel- oping bronchopulmonary dysplasia. Arch Dis Child. 2011;96: 14---9. 27. Hacking DF, Gibson AM, Robertson C, Doyle LW, Victorian Infant Collaborative Study Group (VICS). Respiratory function at age 8---9 after extremely low birth weight or preterm birth in Victoria in 1997. Pediatr Pulmonol. 2013;48:449---55. 18. Conﬂicts of interest Sanchez-Solis M, Garcia-Marcos L, Bosch-Gimenez V, Pérez- Fernandez V, Pastor-Vivero MD, Mondéjar-Lopez P. Lung function among infants born preterm, with or without bronchopulmonary dysplasia. Pediatr Pulmonol. 2012;47:674---81. 28. Kotecha SJ, Edwards MO, Watkins WJ, Henderson AJ, Paranjothy S, Dunstan FD, et al. Effect of preterm birth on later FEV1: a systematic review and meta-analysis. Thorax. 2013;68:760---6. 19. Vrijlandt EJ, Kerstjens JM, Duiverman EJ, Bos AF, Reijneveld SA. Moderately preterm children have more respiratory problems during their ﬁrst 5 years of life than children born full term. Am J Respir Crit Care Med. 2013;187:1234---40. 29. Been JV, Lugtenberg MJ, Smets E, van Schayck CP, Kramer BW, Mommers M, et al. Preterm birth and childhood wheezing disorders: a systematic review and meta-analysis. PLoS Med. 2014;11:e1001596. 20. Northway WH Jr, Moss RB, Carlisle KB, Parker BR, Popp RL, Pitlick PT, et al. Late pulmonary sequelae of bronchopulmonary dysplasia. N Engl J Med. 1990;323:1793---9. 30. Guimarães H, Rocha G, Pissarra S, Guedes MB, Nunes T, Vitor B. Respiratory outcomes and atopy in school-age children who were preterm at birth, with and without bronchopulmonary dys- plasia. Clinics. 2011;66:425---33. 21. Narang I, Rosenthal M, Cremonesini D, Silverman M, Bush A. Lon- gitudinal evaluation of airway function 21 years after preterm birth. Am J Respir Crit Care Med. 2008;178:74---80.
https://openalex.org/W2603613273	https://revije.ff.uni-lj.si/MuzikoloskiZbornik/article/download/5532/5273	Slovene	null	Izdelovalci glasbil na Slovenskem 1606–1918	Muzikološki zbornik/Muzikološki zbornik	2,003	cc-by-sa	14,250	Instrument Makers in Slovenia 1606- tov, orgle, na, pihala, Keywords: instrument making, organ piano, harmonium, strings, violin, w brass, percussion Ključne besede: izdelovanje inštrum klavikord, klavir, harmonij, godala, vio trobila, tolkala Imena izdelovalcev izvirni obliki, ponekod pa so doda priimkov in imen, ki jih najdemo v p Pri rojstnih podatkih so navedene le smrti ter natančni datumi, kadar so z rih, kjer čas rojstva ni ugotovljen, letnice delovanja posameznika. Pri (Zeichen), Martin (1855-1863). Pri po čih več navedb, npr. izdelovalec orgel vec z glasbili ... Kadar so posamez manjših krajih, je zaradi boljše preg njeno bližnje večje mesto. Pri pome strih sta strnjena vrednotenje njih pomen za slovenski oz. širši evropsk vitalnimi podatki so rojstni kraj, pom vanja, pridobivanje meščanskih in m vic (pri mojstrih tudi okoliščine, ka obrtnih pravic: npr. nakup, poroka nega mojstra). Sledijo vrste izdelkov lovali v posamezni delavnici (npr. harmoniji ,„), območje delovanja ( omenjeni kraji in cerkve, kjer so bil ljene, kam so jih prestavljali in kje danes), velikost delavnice, proizvo likost opusa pri orglarjih), število p vajencev ter njihova imena. Nato izboljšave pri inštrumentih, razstave grade ter preimenovanje delavnice Posebna rubrika je namenjena nave inštrumentov v javnih zbirkah, pri števane muzejske in zasebne zbirk tujem. Orglarska dediščind je pre območju delovanja posameznika. S lavnice in morebitne selitve. Na kon loš]j:em redu navedeni viri, in sicer li stojne publikacije, članki v periodi dela, razstavni katalogi, zborniki in gesla), revije in časopisi ter arhivski a metodolo- eški leksiko- o zapisana v še različice marnih virih. ice rojstva in ani. V prime- rature. In order to achieve greater cessibility as regards the history making and our knowledge of indi at as of utmost importance to sum up results an the form of headwords, to most vita! <lata, so that users wall h cess to: desired information. pomembne er: CAJHEN, licu je mogo- organist, trgo- ki delovali v ednosti ome- bnejših moj- ega dela ter prostor. Med čniška poto- ojstrskih pra- o je prišel do vdovo obrt- ki so jih izde- lavirji, orgle, i orglarjih so orgle postav- o ohranjene nja (npr. ve- močnikov in iznajdbe in riznanja, na- n nasledniki. bi ohranjenih emer so upo- doma in na tavljena pri di naslov de- u so v krono- ratura (samo- , diplomska ksikografska ri. The formation of headwords has tak the methodology that has asserted it and English lexicographic, literature of individual makers are registered nal form, though an some cases var mes and first names, found in prima also added. Proučevanje zgodovine izdelovanja inš posameznih izdelovalcev na Slovenske ge tradicije. Na ta del glasbene preteklos no in drugo javnost prvi opozoril Josip 60-letnici ljubljanske Glasbene matice je ob tej priložnosti v Narodnem dom organizirala veliko razstavo Razvoj glasb cih, kjer so bili z izdelki predstavljeni starejši in sodobni domači izdelovalci in Med najmanj razvitimi segmenti muziko venskem sta organologija glasbenih inš zgodovina izdelovanja glasbil. Večje st zornosti so bile deležne predvsem org Orglarstvo in posamezne mojstre so od tih let naprej proučevali Ladislav Šaban, in Edo Škulj, v zadnjem desetletju pa zanimanje še za izdelovalce drugih inšt so v preteklih stoletjih delovali na ozem Slovenije. Rezultati posameznih študij v različnih publikacijah, revijah, zbornik nih, nekaj podatkov o izdelovalcih pa n v tuji literaturi. Da bi bilo poznavanje zgo lovanja glasbil in posameznih mojstrov no in dostopnejše, je nujno, da objavlj strnemo v obliki gesel z vsemi najbolj v datki, s čimer bodo imeli uporabniki b do informacij. mentov in nima dol- e strokov- antuani ob a 1932, ki v Ljubljani pri Sloven- di nekateri rumentov. ije na Slo- mentov in kovne po- in orglarji. demdese- ilko Bizjak stopnjuje mentov, ki u današnje objavljeni in leksiko- demo tudi vine izde- j pregled- e dosežke alnimi po- ši dostop Historical studies of instrument makin dual makers do not have a long tradit nia. Attention to this segment of our was for the first tirne drawn by Josip M the occasion of the both anniversary o na Glasbena matica (i.e. Musical associ fessional musicians, connoisseurs lovers); in 1932, when, in the Nation Ljubljana, a big exhibition - The Dev Music with the Slovenes - was organiz ing also severa! products of some old contemporary narive instrument maker least developed segments of musicolo nia are organology of musical instrum story of instrument making. Neverthele degree of professional attention has be organs and organ builders. Ever since ties, Ladislav Šaban, Milko Bizjak, an have done research in organ building vidual organ makers, whereas during t de there has been growing interest in ment makers, active in the past centurie territory of today's Slovenia. The resu studies have been printed in various p magazines, collections of scholarly pap cyclopaedias, while some of the <lata r strument makers can be found also in MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA Pri oblikovanju gesel je bila upoštev gija, ki se je uveljavila v nemški in an grafski literaturi. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Uvod MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Uvod 1 Josip Mantuani, Katalog razstave: Razvoj glasbe pri Slovencih, rokopis razmnožila Glasbena matica v Lj 2 J. Mantuani, n. d„ Predgovor. 3 Ugotovitve avtorjev so strnjene v: L. Šaban, Orgulje slovenskih graditelja u Hrvatskoj, v: Muzikološki zborn gical annual XV (1979), str. 13-41; M. Bizjak/E. Škulj, Orgle na Slovenskem, Ljubljana 1985; E. Škulj Knjižica Cerkvenega glasbenika, zv. 10, Ljubljana 1992; isti, Orgle v Ljubljani, Celje 1994. Prim. Darja Koter, Glasbilarstvo na Slovenskem, Maribor 2001. 5 Prvič je bil zgodovinski razvoj glasbilarstva na Slovenskem objavljen v Muzički enciklopediji (Zagreb 1974 geslo: Gradenje umetničkih muzičkih instrumenata). Geslo (avtor ni naveden) prlnaša kratek pregled inštrumentov s tipkami, nekaj goslarjev ter po enega izdelovalca tamburic in harmonik. Druga sinteza je bi deset let kasneje v Leksikonujugoslavenske muzike(Zagreb 1984, zv. 1, str. 286, geslo: Gradenje umetničk instrumenata), kjer pa ni bistvenih dopolnil. R. Vannes, Dictionnaire Universal des Luthiers, Les amis de la Musique, Bruxelles 1979. V abecednem redu ·>Baide Ivan, Blasich Giovanni, Blasich Louis, Boštjan Rihar, Demšar Blaž, Dolenz Giovanni, Dolenz Gius Jože, Ivančič August, Kantušar Jože, Lukas Gabriel, Lukeschitz Mathias, Mouchitch Mihael, Sancin Ivan, S milien in Kosovel Eugen"; C. Woodcock, Dictionary oj contemporary Violin and Bowmakers, Brighton navaja naslednje goslarje: "Baide Ivan, Blejz Alejz, Boštjan Anton Rihar, Ivančič August, Kantušar Joze, Lu Lukesic Mathias, Marolti Franjo, Modic Ivan, Music Mihael in Stendler Anton". Nekatere omenja tudi Rok K lina, Ljubljana 1996, str. 74-81). R. Vannes, Dictionnaire Universal des Luthiers, Les amis de la Musique, Bruxelles 1979. V abecednem redu ·>Baide Ivan, Blasich Giovanni, Blasich Louis, Boštjan Rihar, Demšar Blaž, Dolenz Giovanni, Dolenz Gius Jože, Ivančič August, Kantušar Jože, Lukas Gabriel, Lukeschitz Mathias, Mouchitch Mihael, Sancin Ivan, S milien in Kosovel Eugen"; C. Woodcock, Dictionary oj contemporary Violin and Bowmakers, Brighton navaja naslednje goslarje: "Baide Ivan, Blejz Alejz, Boštjan Anton Rihar, Ivančič August, Kantušar Joze, L Lukesic Mathias, Marolti Franjo, Modic Ivan, Music Mihael in Stendler Anton". Nekatere omenja tudi Rok lina, Ljubljana 1996, str. 74-81). The years of birth and d and, if possible, the exact <lata as well the <late of birth cannot be establis years of each individual are of gre Example: CAJHEN', (Zeichen), Mart As regards their trade, there as a v sibilities, e.g. organ builder, organi dealer . „ In cases when individuals smaller places, the first bigger town n tioned for the sake of clarity. With men, a condensed evaluation as giv account their role an Slovenia or, an se, within the European framework importance are <lata concerning the years of trave! as a trainee, acquisiti master rights (as for masters, also t ces under which they acquited thei ce: e.g. through; payment, or by widow of a deceased master craftsm followed by the type of products m dual workshops (e.g. pianos, org ums „.), region of activity (with org places and churches were the orga also where they were transferred to, a remain preserved today), worksho assistants and apprentices, as well a Further, as follows: inventions and i exhibitions, acknowledgements, pr renaming of the workshop, successor A special rubric is intended for stati that are preserved in public collect ums, as well as in private assemblage abroad. The heritage of organ build ted in connection with the region of follows the address of the workshop changes of dwellings. At the end, so stated in chronological order: litera dent publications, articles in periodica or rather diploma works, exhibition thologies and lexicographic headwor and newspapers, as well as archival 3 Ugotovitve avtorjev so strnjene v: L. Šaban, Orgulje slovenskih graditelja u Hrvatskoj, v: Muzikološki zborn gical annual XV (1979), str. 13-41; M. Bizjak/E. Škulj, Orgle na Slovenskem, Ljubljana 1985; E. Škulj Knjižica Cerkvenega glasbenika, zv. 10, Ljubljana 1992; isti, Orgle v Ljubljani, Celje 1994. 5 Prvič je bil zgodovinski razvoj glasbilarstva na Slovenskem objavljen v Muzički enciklopediji (Zagreb 1974 geslo: Gradenje umetničkih muzičkih instrumenata). Geslo (avtor ni naveden) prlnaša kratek pregled inštrumentov s tipkami, nekaj goslarjev ter po enega izdelovalca tamburic in harmonik. Druga sinteza je b deset let kasneje v Leksikonujugoslavenske muzike(Zagreb 1984, zv. 1, str. 286, geslo: Gradenje umetnič instrumenata), kjer pa ni bistvenih dopolnil. 2 J. Mantuani, n. d 3 1 Josip Mantuani, Katalog razstave: Razvoj glasbe pri Slovencih, rokopis razmnožila Glasbena matica v L 2 J. Mantuani, n. d„ Predgovor. 3 U i j j Š b O lj l kih di lj H k j M ik l ški b Proučevanje zgodovine izdelovanja inštrumentov in posameznih izdelov Slovenskem nima dolge tradicije. Na ta del glasbene preteklosti je strokovn go javnost prvi opozoril]osip Mantuani ob 60-letnici ljubljanske Glasbene m je ob tej priložnosti organizirala „prvi slovenski glasbeni festival v Ljubljani" gramu slovesnih dogodkov je bila spomladi leta 1932 v Narodnem domu v tudi velika razstava z naslovom Razvoj glasbe pri Slovencih. 1 To je bila pr predstavitev slovenskih glasbenih dosežkov. Kot je zapisal Manuani, »ni bilo sti še nikdar združenih toliko dokazil naše glasbene kulture[ ... ], četudi je v skica in oblika skrajno skromna".2 Na razstavi so bili z izdelki predstavljeni tu teri starejši in sodobni domači izdelovalci inštrumentov. Mantuanijev posku zametek predstavitve domače glasbene stroke, ki pa v naslednjih letih v obliki ni doživel širše nadgradnje. Žal tudi sedemdeset let po tem dogodku še nimamo zaokrožene muzejske zbirke, ki bi jo lahko imenovali „slovenski muzej". Kljub temu ugotavljamo, da je bilo na določenih glasbenozgod področjih do danes veliko storjenega, nekatere dele glasbene preteklosti pa mo šele zadnji čas. Med najmanj razvitimi segmenti muzikologije na Sloven gotovo organologija glasbenih inštrumentov in zgodovina izdelovanja glasb strokovne pozornosti so bile deležne predvsem orgle in orglarji, zato ne pr da sta zgodovina orglarstva in organologija orgel pri nas med najbolj raz Orglarstvo in posamezne mojstre so od sedemdesetih let naprej proučevali Šaban, Milko Bizjak in Edo Škulj.3 V zadnjem desetletju pa se stopnjuje zani za izdelovalce drugih inštrumentov, ki so v preteklih stoletjih delovali na današnje Slovenije.4 Rezultati posameznih študij so objavljeni v različnih cijah, revijah, zbornikih in leksikonih,5 nekaj podatkov o izdelovalcih pa tudi v tuji literaturi.6 Da bi bilo poznavanje zgodovine izdelovanja glasbil meznih mojstrov bolj pregledno in dostopnejše, je nujno, da objavljene dosež mo v obliki gesel z vsemi najbolj vitalnimi podatki, s čimer bodo imeli up boljši dostop do informacij. Navodila za u Pri oblikovanju gesel sem upoštevala metodologijo, ki se je uveljavila angleški leksikografski literaturi. Imena izdelovalcev so zapisana v iz ponekod pa so dodane še različice priimkov in imen, ki jih najdemo v virih. Pri rojstnih podatkih so navedene letnice rojstva in smrti ter natan kadar so znani. V primerih, kjer čas rojstva ni ugotovljen, so pomembne l vanja posameznika. Primer: CAJHEN, (Zeichen), Martin (1855-1863). Pr mogočih več navedb, npr. izdelovalec orgel, organist, trgovec z glasbili posamezniki delovali v manjših krajih, je zaradi boljše preglednosti omen večje mesto. Pri pomembnejših mojstrih sta strnjena vrednotenje njihov pomen za slovenski oz. širši evropski prostor. Med vitalnimi podatki so pomočniška potovanja, pridobivanje meščanskih in mojstrskih pravic ( tudi okoliščine, kako je prišel do obrtnih pravic: npr. nakup, poroka z vd ga mojstra). Sledijo vrste izdelkov, ki so jih izdelovali v posamezni del klavirji, orgle, harmoniji ... ), območje delovanja (pri orglarjih so omen cerkve, kjer so bile orgle postavljene, kam so jih prestavljali in kje so oh nes), velikost delavnice, proizvodnja (npr. velikost opusa pri orglarjih), šte nikov in vajencev ter njihova imena. Nato so iznajdbe in izboljšave pri in razstave, priznanja, nagrade ter preimenovanje delavnice in nasledniki. P brika je namenjena navedbi ohranjenih inštrumentov v javnih zbirkah, p upoštevane muzejske in zasebne zbirke doma in na tujem. Orglarska d predstavljena pri območju delovanja posameznika. Sledi naslov delavni bitne selitve. Na koncu so v' kronološkem redu navedeni viri, in sicer lit mostojne publikacije, članki v periodiki, diplomska dela, razstavni katalo in leksikografska gesla), revije in časopisi ter arhivski viri. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL št. ur. ZAL ZAP zap. št. zv. številka urednik Zgodovinski arhiv Ljubljana Zgodovinski arhiv Ptuj zaporedna številka zvezek MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL št. ur. ZAL ZAP zap. št. zv. številka urednik Zgodovinski arhiv Ljubljana Zgodovinski arhiv Ptuj zaporedna številka zvezek MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL št. ur. ZAL ZAP zap. št. zv. številka urednik Zgodovinski arhiv Ljubljana Zgodovinski arhiv Ptuj zaporedna številka zvezek Lit BETZ 1992: BETZ, Mariane, Der Czakan und seine Musik, Schneider, Tutz BIZJAK/ŠKULJ 1985: BIZJAK, Milko - ŠKULJ, Edo, Orgle na Slovenskem, 1985. CARDANA 1973: CARDANA, Iris, L'arcidiocesi di Gorizia, L'arte degli or Friuli Venezia Giulia, 1973. EBERSTALLER 1955: EBERSTALLER, Oskar, Orgeln und Orgelbauer in O Graz - Koln 1955. JEDRLINČIČ/KOMEL 1990:JEDRLINČIČ, JEDRLINČIČ/KOMEL 1990:JEDRLINČIČ, Alda-KOMEL, Zdenka, Ivan Kaci 1853)- orglar in Paul]. Šifler (1911) - skladatelj in organist (diplom ga), Univerza v Ljubljani, Pedagoška fakulteta (1990). KOTER 2001: KOTER, Darja, Glasbilarstvo na Slovenskem, Ma LAVRIČ LAVRIČ, l l bl k k f LAVRIČ 1988: LAVRIČ, Ana, Vloga ljubljanske likovni umetnosti J-II, SAZU, Ljubljana 198 LAVRIČ 1988: LAVRIČ, Ana, Vloga ljubljanskega škofa Tomaža Hrena v likovni umetnosti J-II, SAZU, Ljubljana 1988. MEDER 1992: MEDER, Jagoda, Orgulje u Hrvatskoj, Zag MOČNIK MOČNIK, MOČNIK 1996: MOČNIK, Janez, Gregor Rihar - I, v: Naši zbori 48 0996 PUNGARTNIK 1990 PUNGARTNIK A d j Sl ki i d l l i d PUNGARTNIK 1990: PUNGARTNIK, Andreja, Slovenski izdelovalci goda letju (diplomska naloga), Univerza v Ljubljani, Pedagoška fakulteta (1 ik l b l j blj i d l STESKA 1929: STESKA, Viktor, Javna glasbena šola v Ljubljani od leta 1816 v: Cerkveni glasbenik LII (1929). Š Š ŠABAN 1979: ŠABAN, Ladislav, Orgulje Slovenskih graditelja u Hrvatskoj, v loški zbornik/Musicological annual XV, Ljubljana 1979. Š 1992 Š d O l K jiži C k l b ik ŠKULJ 1992: ŠKULJ, Edo, Orglarstvo, Knjižica Cerkvenega glasbenika, zv Ljubljana 1992. Š Š ŠKULJ 1994: ŠKULJ, Edo, Orgle v Ljubljani, C ŠKULJ 2001 ŠKULJ Ed H k k ji ŠKULJ 2001: ŠKULJ, Edo, Hrenove korne knjige, Ljublj Kratice C. cerkev CG Cerkveni glasben Cod. Codex f. folija fasc. fascikel inv. št. inventarna števil kat. katalog l. leto NAL Nadškofijski arhi NAZ Nadškofijski arhi ok. okrog op. opus reg. register sign. signatura st. stoletje šk. škatla Revije i Cerkveni glasbenik (1929, 1940, Cerkveni glasbenik (1929, 1940, Cerkveni glasbenik (1929, 1940, Cerkveni glasbenik (1929, 1940, 1988, 1990). Kronika slovenskih mest I, II (1935). Laibacher Zeitung (1848). Naši zbori 48 0996), št. 1-2. Novice kmetijskih, rokodelskih in narodskih reči (1845-69 Zgodnja Danica (1864). Kronika slovenskih mes Kronika slovenskih mes Laibacher Zei Naši zbori 48 09 Novice kmetijskih, rokodelskih in narodskih reč Novice kmetijskih, rokodelskih in narodskih re Zgodnja Da MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNU CLINKSCALE 1999: CLINKSCALE, Novak Martha, Makers ojthe Piano, V 1860, Oxford University Press 1999. DOGULIN 1994: DOGULIN, Adriano, II Civico Museo Teatrale ·Carla Triest.e, /kat./, Triest 1994. EDITION BIZJAK 1992: Ptujska zbirka VII, Edition B HOPFNER 1999: HOPFNER, Rudolf, Wiener Musikinstmmentenmacher: Adressenverzeichnis und Bibliographie, Kunsthistorisches Museum ing 1999. Adressenverzeichnis und Bibliographie, Kunsthistorisches Museum ing 1999. g JALOVEC 1968: JALOVEC, Vinko, Encyclopedia oj Violin - Makers, vol. 1968. MANTUANI 1932: MANTUANI, Josip, Razvoj glasbe pri Slovencih, /kat matica Ljubljana, Ljubljana 1932. Publikacije ZAL 1971, 1972, 1977 Publikacije Zgodovinskega arhiva Ljubljana, zvezek 1, 2, 3, 4, Lju 1972, 1977. STRADNER 1986: STRADNER, Gerhard, Musikinstmmente in Grazer Wien 1986. STRADNER 1986: STRADNER, Gerhard, Musikinstmmente in Grazer S Wien 1986. SUPPAN 1962 1966 SUPPAN W l S i i h M ikl ik G 19 SUPPAN 1962-1966: SUPPAN, Walter, Steierische Musiklexikon, Graz VANNES 1979: VANNES, Renne, Dictionnaire Universal des Luthiers, Le Musique, Bruxelles 1979. WATTERHOUSE 1993: WATTERHOUSE, William, The New Langwill Ind nary oj Musical Wind Instrument Makers and Inventors, London 19 WITT de 1912: WITT de, Paul, Welt Adressbuch der gesamten Musikins Industrie, Leipzig 1912. WITT de 1925/26: WITT de, Paul, Welt Adressbuch der Musik - Indus Leipzig 1925/26. WOODCOCK 1965: WOODCOCK, Cyril, Dictionary oj Contemporary Bowmakers, Brighton Sussex England 1965. WOODCOCK 1965: WOODCOCK, Cyril, Dictionary oj Contemporary Bowmakers, Brighton Sussex England 1965. Razstavni katalogi, zborniki in le BOTSTIBER 1911: BOTSTIBER, Hugo, Musikbuch aus Osterreich, Wien - 1911. CLINKSCALE 1993 CLINKSCALE N k M h M k j h Pi V l CLINKSCALE 1993: CLINKSCALE, Novak Martha, Makers oj the Piano, Vol. 1820, Oxford University Press 1993. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL NAL, Nadškofijski arhiv - KAL, ma - Gornji grad A- folija 79, Korespondenca škofa Tomaža Hrena 15 - Gornji grad A- folija 79, Korespondenca škofa Tomaža Hrena 1 ZAL, Zgodovinski arhiv - ZAL, Code - ZAL, Code - ZAL, Codex XIII/36 - ZAL, Codex XIII/36 - ZAL, Reg. I, mapa 19, fol - ZAL, Reg. I, mapa 19, fo - ZAL, Burgerverzeichnis der Domstadt Laibach 1786-1899, s - ZAL, Burgerverzeichnis der Landeshauptstadt Laibach 1786-1867, Laib sign. 787, - ZAL, \;onscriptions Aufnahms Bogen vom Jahre 1830, škatla 29 (1 - ZAL, Einnahmen- und Ausgabenbuch 1581-1731, Codex XIII/120 (170 - ZAL, Einnahmen- und Ausgabenbuch 1581-1731, Codex XIII/224 (1707) - ZAL, Einnahmen- und Ausgabenbuch 1581-1731, Codex XIII/224 (170 Arhivs Arhiv mestne cerkve sv. Ju - Mrliške knjige 1805-1838 Arhiv mestne cerkve sv. Jur - Mrliške knjige 1805-1838 Arhiv mestne cerkve sv. Ju - Mrliške knjige 1805-1838 - Mrliške knjige NAZ, Nadškofijski arhi NAZ, Nadškofijski arhi - Acta Capit. Ant„ mapa 101 ZAL, Zgodovinski arhiv BILLICH, Jernej ( , j ( ) Orglar v Slovenj Gradcu. Doma je bil iz Trnovelj pri Celju. Leta 184 orgle za cerkev v Preboldu. Literatura: BIZJAK/ŠKUL Literatura: BIZJAK/ŠKUL BILLICH, Karl (Carl), Orglar in mizar iz Slovenj Gradca. Deloval je predvsem v Avstriji (186 je sin orglarja Jerneje Billicha. Izdeloval je enomanualne (6--8 registrov dvomanualne orgle (do 12 registrov). Leta 1864 je uglaševal orgle na Pes četrtku, kjer se je podpisal kot »Orglar v Slovenj Gradcu" (·Orgelbauer in Naročila je izpolnjeval tudi za cerkve na avstrijskem Koroškem in (Lawamtind, 1866; Wiel, 1876; Gaisgorn, 1884; Hohenburg, 1885; Graz berg, 1886; Kumberg, 1887; Neumarkt, 1887; Assach, 1892). g g ) Literatura: EBERSTALLER 1955, 134, 158, 160, 164, 170, 174, 179, 18 215, 229, 237; BIZJAK/ŠKULJ 1985, 50. ZAP, Zgodovinski - ZAP, Fond Bezirkshauptmannschaft Pettau 17 - ZAP, Grundbuch R - 501, Hau MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA BAJDE, Ivan ( Izdelovalec violin, samouk. Delal je po Stradivarijevih modelih. Ro Ljubljani, kjer je tudi umrl. Skonstruiral je "claviolino", inštrument, ki zdru in harmonij. Literatura: WOODCOC Literatura: WOODCOC BILLICH, Jernej (1840-1860) Orglar v Slovenj Gradcu. Doma je bil iz Trnovelj pri Celju. Leta 184 orgle za cerkev v Preboldu. Lit t BIZJAK/ŠKULJ 1985 120 DERNIČ, Anton (18 Orglar iz Radovljice. Deloval je predvsem na Gorenjskem. Njegove najve s tremi manuali so v romarski cerkvi na Brezjah na Gorenjskem (1913). S podjetja Mauracher iz St. Florijana v Zgornji Avstriji je prestavil iz altane na g in jih povečal oziroma izdelal nove s 47 registri. Drugi pomembnejši inštru Ljubljana Šentvid (33 registrov), Blejski otok (1912, 20 registrov), Brdo pri Kropa pri Kapelici in Besnica. Na Hrvaškem je postavil dvoje orgel (Trviž, Vid na Krku, 1913), ene pa na avstrijskem Koroškem (Maria Gail). Vse D orgle so pnevmatične z romantično dispozicijo. Literatura: EBERSTALLER 1955, 192; ŠABAN 1979, 34, 36; BIZJAK/ŠK 172, 174; ŠKULJ 1992, 13. Literatura: EBERSTALLER 1955, 192; ŠABAN 1979, 34, 36; BIZJAK/Š 172, 174; ŠKULJ 1992, 13. BRANDL, Josip (JoseD, ( p Orglar, izdelovalec harmonijev in klavirjev v Mariboru. Bil je eden na izdelovalcev orgel z romantično dispozicijo in pnevmatsko trakturo ter izdelovalcev klavirjev oziroma pianinov na Slovenskem od konca 19. d stoletja. V Maribor je prišel iz Eisendorfa na Bavarskem. Delavnico, ki je km v podjetje, je v Mariboru ustanovil l. 1893. V Brandlovem podjetju (·:Jos Orgelbau Anstalt / Marburg a(n)D(rau}.) so izdelali ok. 150 orgel, večino ve na slovenskem Štajerskem. Največje orgle so v Brestanici (Rajhenburg) (1914, 40 registrov). Brandl je pri teh orglah čisto pnevmatiko že zamenj mi na stožce (»Kegellade") in vgradil "Meidingetjev ventilator", kar je omogo brez mehača. Med pomembnejšimi so orgle v mariborski frančiškanski c op. 70, 34 registrov) in v tamkajšnji stolnici (30 registrov). Zaradi zastarel oziroma pnevmatike je bila večina njegovih izdelkov v drugi polovici 20. na oziroma uničena. Brandl je bil poslovno zelo uspešen tudi na Hrvaš podjetje postavilo več kot 50 orgel (Varaždin - c. sv. Nikole, 1895, 20 reg Marije, 1933, 28 registrov; Dubrovnik- c. sv. Vlaha, 1937, op. 144, 30 reg tat, 1938, op. 149). Nekaj manjših orgel (do 23 registrov) so izdelali tudi z avstrijskem Štajerskem (St. Georgen im Lavanttal, 1906; StrafS, 1910; Leuts Gamlitz, 1913; Wundschuh, 1914). Pianine so v Brandlovem podjetju izde vsem v 20. in 30. letih 20. st„ nekaj inštrumentov je evidentiranih v zase proizvodnja in dediščina harmonijev pa še nista raziskani. Kot pomočn Jakob Prašnik, ki je občasno samostojno popravljal orgle različnih izdelo 1904 je Brandl za svoje orgle v Neaplju prejel priznanje »Grand Prix". CAJHEN (Zeichen), Martin (18 CAJHEN (Zeichen), Martin (18 Orglar iz Polzele. Deloval je v širši okolici Celja. Izhaja iz Trbovelj. Orgla izučil pri Aloisu H6rbigetju (1810t1872), odličnem tirolskem mojstru, ki je Celju, nato pa na Dunaju. Cajhnova vajeniška doba je trajala od ok. l. 1842 nato pa je bil prib. šest let pomočnik (1848-1854 ali 1855). H6rbiger ga je z kar pottjujejo njegova priporočilna pisma (datirana za čas med l. 1848 in 1 je Cajhen označen kot izvrsten orglar in mojstrov najboljši pomočnik, pose poudarjeno, da je sposoben najzahtevnejših naročil doma, pa tudi v drugih Znane so samo ene Cajhnove orgle (Šoštanj, 1863, 16 registrov), največkrat je kot popravljalec inštrumentov. Literatura: BIZJAK/ŠKULJ 1985, 158; KOTER 2001, 106; Novice kmetijsk delskih in narodskih reči, XVII (1859), št. 7, 50. Literatura: EBERSTALLER 1955, 171, 189, 209, 222, 237; ŠABAN 1979, 3 ŠKULJ/BIZJAK 1985, 178, 204; MEDER 1992, 84, 91, 218; KOTER 2001, 108 Literatura: EBERSTALLER 1955, 171, 189, 209, 222, 237; ŠABAN 1979, 3 ŠKULJ/BIZJAK 1985, 178, 204; MEDER 1992, 84, 91, 218; KOTER 2001, 108 Literatura: EBERSTALLER 1955, 171, 189, 209, 222, 237; ŠABAN 1979, 3 ŠKULJ/BIZJAK 1985, 178, 204; MEDER 1992, 84, 91, 218; KOTER 2001, 108 MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA ene Devove orgle (Gerovo, 1856, 12 registrov). V njegovi delavnici se je Vrabec (1827t1898), kasneje samostojni orglar v Mokr ( ) j j g Naslov: Sevnica ob Savi (ok. 1854), Ljubljana, Breg (1 Literatura: STESKA 1929; BIZJAK/ŠKULJ 1985, 130, 136, 214; 102-103. Časopis: Novice kmetijskih, rokodelskih in narodskih reči, št. 63 (1863 (1869), 226--227. Časopis: Novice kmetijskih, rokodelskih in narodskih reči, št. 63 (18 (1869), 226--227. DEV (Deu), Franc Ksaver (1 Orglar, izdelovalec glasbenih avtomatov in harmonijev iz Ljubljane. Ro kronogu na Dolenjskem. Preden je prišel v Ljubljano, je imel delavnico v S Savi. Sodi med pomembnejše slovenske orglarske mojstre inštrumentov s kla dispozicijo. V zgodnjih 50. letih 19. st. se je učil na Dunaju, najverjetneje v Carla Hessa (1808t1882). V Ljubljano je prišel ok. l. 1854, ko je popravlja tamkajšnji Javni glasbeni šoli. Dev se je vodstvu šole ponudil, da bi izdelal n za 100 goldinatjev in popravil šolski klavir. Naročilo je dobil in ga naslednje izpolnil. Njegova delavnica je izdelala več dobrih inštrumentov (Rogaška Slati večina je uničenih ali predelanih, do danes pa so se ohranile le dvojne orgl Rob na Dolenjskem, ok. 1872, signatura: „f. X. DEU / ORGLARSKI IZDELOV LUBLANI·<). Iz časopisnih poročil je znano, da je izdeloval tudi glasbene av valji na več melodij za rabo na cerkvenih korih v manjših cerkvah, 'kjer ni b skih inštrumentov. Leta 1863 je Dev tovrstno „lajno" izdelal za cerkev v Pod Vipavskem. Takšni avtomati so posnemali orgelski zvok, uporabljali pa so jih ljavo k petju. Devova delavnica je izdelovala še harmonije. Na Hrvaškem EBNER, Leonhard Orglar v Mariboru. Deloval je na slovenskem Štajerskem in na Hrv daljeval je šolo Aloisa Horbigerja (1810t1872). Ebnerja prištevajo med n nejše štajerske orglarje romantičnih orgel. Njegovi izdelki so sloveli po i naciji posameznih registrov, cenili pa so tudi estetiko orgelskih omar. Iz ske, tako kot Alois Horbiger, ki je ok. l. 1842 ustanovil delavnico v Celju. nekaj let pri Horbigerju pomočnik, l. 1855, ko se je mojster odselil na Du osamosvojil in odprl lastno obrt v Mariboru, kjer je deloval vsaj do l. izdelal orgle za cerkev sv. Križa nad Mariborom. Prve orgle, ki so ene izm ohranjenih do danes, je postavil na Ponikvi pri Celju (1857, 22 registro značilen register »Schlagwerk· oz. t. im. »turška muzika•, ki sta ga na vgrajevala še Horbiger in Johann Gottfried Kunat (pred 1790t po 184 pomembnejše orgle so bile v stolni cerkvi v Mariboru (1859, 25 registro pri Celju (1868) in Ormožu. V slednjih je zapis o izdelovalcu orgel in ohiš 1868 im Monat November wurde dieses Werk durch Leonhard Ebner verfertigert. Die aussere Ausstatung durch den hiesigen Mahler l. K. Lieb pa so pripisani še šaljivi verzi na račun organista („Spiele mich fein, Be recht, Damit es tone nicht schlecht, Und Harmonie wird sein; Jede falsch Quint Sei dir, Organist, Todsund!·). Ebner je postavil orgle še v Framu p Sv. Jakobu v Slovenskih goricah, na Gori Oljki pri Celju in v Veliki Nedel žu. Na Hrvaškem sta znana dva njegova inštrumenta (Krapinske Topl Marija na Muri, 1870). Literatura: ŠABAN 1979, 31, 38; BIZJAK/ŠKULJ 1985, 94, 132, 140, 144 1992, 13-14. ECHTER, Johann Qanez), (1820) Izdelovalec kitar, citer, dud in igrajočih ur v Ljubljani v prvi tr Literatura: Publikacije ZAL 0977), zv. 4, 67; KOTER 2001, 14 Izdelovalec kitar, citer, dud in igrajočih ur v Ljubljani v prvi tr Literatura: Publikacije ZAL 0977), zv. 4, 67; KOTER 2001, 14 EISL (Eisel, Eysl), Johann Georg Qohannes Georgius), (1708t18. 11. Izdelovalec orgel in klavikordov v Ljubljani. Rodil se je v Salzburgu okolici, kjer se je najverjetneje tudi izučil. Njegove orgle kažejo na avstrij šolo in nekatere italijanske vzore, pripisujejo pa mu tudi poteze salzburšk Johanna Christiana Egedacherja (1664t1747). Eisl velja za osrednjo os orglarji, ki so delovali v Ljubljani v drugi polovici 18. st., in je edini znani klavikordov na Slovenskem. V Ljubljano je prišel vsaj l. 1740, ko je bil kr MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL prvi otrok. Od l. EBNER, Leonhard 1743 je bil pomočnik ljubljanskega orglarja Marka Gob 1710tpo 1751), ki je bil poročen z njegovo sorodnico Marijo Terezijo Eisl. P smrti (med l. 1751 in 1753) je delavnico nasledil Eisl in postal zelo uspeš Meščanske pravice je dobil šele 21. 2. 1763. Orgle je postavljal v različnih Slovenskem in Hrvaškem (Marija Gorica, 1759). Evidentiranih je 15 orgel vov. Najstarejše z ohranjeno etiketo s podpisom (•Joannes Georgius Eisl / cher / 1753·) so v cerkvi pri Treh Farah pri Metliki. Med pomembnejšimi so v Ljubljani (c. sv. Jakoba, 1780), v stolnici v Krki na Koroškem (1779-80) mestu (1764). Večina ohranjenih je iz 60. let 18. st. V Eislovi delavnici so tudi klavikorde. Ohranjena sta dva inštrumenta (eden ima ohranjeno etike som delavnice in letnico 1757), ki imata značilnosti izdelkov avstrijsko- prostora prve polovice 18. st. Izhajata iz ljubljanskega samostana uršulink. Inštrumenti: Ljubljana, Narodni muzej Slovenije, klavikord, inv. št. 16093 Literatura: ŠABAN 1979, 18--19; ŠKUIJ 1994, 147; KOTER 2001, 115--11 Inštrumenti: Ljubljana, Narodni muzej Slovenije, klavikord, inv. št. 16093 Literatura: ŠABAN 1979, 18--19; ŠKUIJ 1994, 147; KOTER 2001, 115--1 GOBL, Marko (Marc, Maks), (pred 1710t po 1751) Orglar v Ljubljani. Kot orglar ni imel meščanskih pravic, služboval je v st vi v Ljubljani. Podpisoval se je kot ·Orgelmacher aus Laybach•. Leta 1733 se FABER, Paulus (16 Orglar iz Šoštanja pri Celju. V času med letoma 1616 in 1618 je v Ljubljan ljal orgle v stolnici in orgelski rog na ljubljanskem gradu. Podpisal se je celjske grofije (»Orgelmacher aus der Grafschaft cilli„). Literatura: NAL, KAL, fasc. 290/1; ZAL, Codex XII/36b, ŠKULJ 2001, FALLER (Failler, Fahler), Joannis Oohann), (pred 1650 FALLER (Failler, Fahler), Joannis Oohann), ( pred 1650tpo 1717) Orglar v Ljubljani. Meščanske pravice je dobil l. 1665 tako kot Mathias L času med letoma 1671-1702 je bil edini orglar v Ljubljani z meščanskimi (»Burger und Orgelmacher in Laybach·). Njegovi inštrumenti kažejo na tiro ter na prepletanje italijanskih in nemških vplivov. Deloval je na Slovenskem kem. Leta 1676 je izdelal orgelski rog v stolpu na ljubljanskem gradu, l. 16 (5 registrov), ki je danes v cerkvi sv. Primoža nad Kamnikom in je najstarejši orgelski inštrument na Slovenskem. Na Hrvaškem je l. 1688 postavil orgle strov) v cerkvi sv .. Katarine v Zagrebu, l. 1690 je popravljal orgle v zagrebšk li, letnico 1699 pa imajo orgle, ki so danes v cerkvi v Sodražici na Dolenjsk p j g Naslov: Ljubljana, Virantov trg (1678--1687), Florjanska ulica 29 (1687 Literatura: ŠABAN 1979, 14--16; BIZJAK/ŠKUL GOSTL, Andrej (Andro), (1805t1885) Orglar v Nemški Loki pri Črnomlju. Ustno izročilo pravi, da je bil slep. je izdelal nekaj manjših orgel v krajih na Dolenjskem (Stara cerkev, Strug Straža), ene pa so znane tudi na Hrvaškem (Žakanje, 1858). GABRIEL, Lukas (17 Izdelovalec violin. Rojen je bil v Idriji. Obrti se je najverjetneje izučil v It je po Amatijevih modelih. Leta 1932 je bila njegova violina z letnico 1777 ra na razstavi Razvoj glasbe pri Slovencih v ljubljanskem Narodnem domu, k pravil Josip Mantuani. Literatura: MANTUANI 1932; WOODCOCK 1965, 53; VANNES 1 Literatura: ŠABAN 1979, 33, 37; BIZJAK/ŠKULJ 1985, 130; ŠKU Literatura: BIZJAK/ŠKULJ 1985, 142, 150, 196, 200, 218; ŠKULJ 1994, 47 KOTER 2001, 103-104. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA z Marijo Terezijo Eisl. Bila je v sorodu z Johannom Georgom Eislom (1 tl 780), ki je bil najprej Gablov pomočnik, po njegovi smrti pa je nasle delavnico. Eislovo sodelovanje pottjuje podpis v ohranjenih procesijsk Zapricah pri Kamniku, kjer je na pokrovu sapnice zapis izdelovalca o Gabl / Orgelmacher aus Laybach / 1743"), na meh pa se je podpisal ,:Jo gius Eisl / 1743". Vplivi Gablove delavnice so vidni tudi na orglah, ki jih neje izdeloval kot samostojni orglar. Z Gablovim podpisom je ohranjen letnico 1751 (Vesela Gora pri Šentrupertu), pripisujejo pa mu tudi dva po mi registri (župnišče stolne cerkve v Ljubljani, 1741? in Mišji Dol na Dole Literatura: EDITION BIZJAK, Ljubljana 1992; ŠKULJ 1994, 148. Časopis: Novice kmetijskih, rokodelskih in narodskih reči, št. 22 (1864) nja Danica, št. 17 (1864), 151-152; Kronika slovenskih mest, l. II (1935), GORŠIČ, Franc (21. 10. 1836t2 GORŠIČ, Franc (21. 10. 1836t2 Orglar v Ljubljani, najpomembnejši orglar v drugi polovici 19. st. D Slovenskem, občasno pa tudi na avstrijskem Koroškem in Štajerskem. R Ljubljani v Krakovem št. 18. Oče Martin Goršič je bil organist in orglar inštrumente Franca Goršiča je značilna romantična dispozicija, pri zad (Mokronog, 1898, op. 66) pa je takrat že zastarelo mehansko trakturo pnevmatiko. Orglarstva se je izučil pri Petru Rumplu (1787t1861), ki je i niku uspešno orglarsko podjetje, od ok. leta 1857 do 1864 je bil pomoč najskem mojstru Carlu Hesseju (1808tl882). Leta 1863 je dobil naročilo novih orgel v Ljubljani (c. v Trnovem, op. 1). Goršičeve prve orgle, ki so b času najmodernejše v Ljubljani, je prišel z Dunaja l. 1864 preizkusit celo m se. Svojega nekdanjega pomočnika je izjemno pohvalil („Gospod Goršič glami, ki ste jih tu postavili, ste mene, svojega mojstra, prekosili!"). Mešča ce je dobil l. 1879 kot "posestnik in izdelovalec orgel«. Za njegove zgodne značilna še sapnica na poteg, nato je uporabljal sapnice na stožce (Bo Ljubljani, 1884). Izdeloval je predvsem eno in dvomanualne orgle, edine ne so v Ljubljani (Uršulinska cerkev, 1891). V Avstriji je postavil dvojne or cu (Frančiškanska c., 1887; c. Srca Jezusovega, 1887) in v manjših krajih am Pickelbach, 1877; St. Jakob im Oberrosentale, 1877, Kaflach, 1884). Z v graški reviji Kirchenschmuk takoj po posvetitvi poročali, da so edinstv („ein Unicum im Lande«). V Goršičevi delavnici sta se za pomočnika i Kriegl, kasneje samostojni orglar v Ljubljani, in Ivan Milavec (1874t1915), v Ljubljani ustanovil lastno delavnico orgel in harmonijev. Literatura: BIZJAK/ŠKULJ 1985, 142, 150, 196, 200, 218; ŠKULJ 1994, 47 KOTER 2001, 103-104. Časopis: Novice kmetijskih, rokodelskih in narodskih reči, št. 22 (1864) nja Danica, št. 17 (1864), 151-152; Kronika slovenskih mest, l. II (1935), GOSTL, Andrej (Andro), (1805t1885) Orglar v Nemški Loki pri Črnomlju. Ustno izročilo pravi, da je bil slep. je izdelal nekaj manjših orgel v krajih na Dolenjskem (Stara cerkev, Strug Straža), ene pa so znane tudi na Hrvaškem (Žakanje, 1858). Š Š Š HEICHELE, Johann (Giovanni), (17 HEICHELE, Johann (Giovanni), (17 HEICHELE, Johann (Giovanni), (17 Izdelovalec čembal in klavirjev v Ljubljani in Trstu (Italija). Meščanske dobil najprej v Ljubljani (8. 3. 1790) kot izdelovalec klavirjev (»Klaviermacher Heichele je najstarejši doslej znani izdelovalec klavirjev v Sloveniji. Po predv se je iz ekonomskih razlogov kmalu po l. 1800 preselil v Trst, kjer je l. 1 meščanske pravice kot čembalist ter izdelovalec čembal in klavirjev („cem sia fabricator di cembali e forte piano•.) pod imenom Giovanni Heichele. P vanje krstnega imena iz nemške v italijansko različico je bilo v tistem času V Trstu je l. 1802 odprl lastno delavnico. Tržaški arhivi ga nazadnje omenja ohranjena dediščina štirih klavirjev pa kaže, da je deloval vsaj do dvajsetih le in ga uvršča med izdelovalce dunajske šole. Naslov. Trst, Via Boschetto št. 1058 (18 Naslov. Trst, Via Boschetto št. 1058 (18 Literatura: ZAL, Imenik meščanov stolnega mesta Ljubljane od 1786 sign. 767; Imenik mestjanov deželnega glavnega mesta ljubljanskega 1786- bach 1867, sign. 787; DUGULIN 1994, 73; CLINKSCALE 1993, 141-142; KOT 125-127. Inštrumenti: Bristol, Mobbs Keyboard Collection, inv. št. G 10; Hambur lung Beurmann: Utrecht, University of Utrecht; Trst, Museo Civico Teatr Schmidl" Trieste. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL na Dunaju, njegove največje dvomanualne orgle s 43 registri pa so v dun Altlerchenfelderkirche (1860 ali 1861). Znanih je okrog 60 inštrumentov celjski delavnici sta bila poleg brata Bartolomea pomočnika še Leonh (1855-1884) in Martin Cajhen (1855-1863). Prvi je imel po H6rbigerjeve na Dunaj samostojno obrt v Mariboru, drugi pa na Polzeli v Savinjski doli meo Horbiger je z bratom odšel na Dunaj, nekaj manjših pozitivov pa je pod svojim imenom. p j Naslov. Celje, Strassenheg (1 j g Literatura: BOTSTIBER 1911, 169; EBERSTALLER 1955, 140--142, 16 ŠKULJ 1985, 120, 122, 132, 146, 212, 216; ŠKULJ 1994, 100; KOTER 2001, Literatura: BOTSTIBER 1911, 169; EBERSTALLER 1955, 140--142, 16 ŠKULJ 1985, 120, 122, 132, 146, 212, 216; ŠKULJ 1994, 100; KOTER 2001, JANECHECK Qaneček),Joannes Franciscus Qanez Frančišek), (ok. 170 JANECHECK Qaneček),Joannes Franciscus Qanez Frančišek), (ok. 170 Orglar v Celju. Je najpomembnejši mojster slovenskega baroka in utem celjskega kroga orglarjev, ki so delovali od začetka 18. do sredine 19. st. porekla in predstavnik češke šole. Njegovi inštrumenti so se odlikoval mehaniki in čistem zvoku. Predvidoma izhaja iz znane češke rodbine iz inštrumentov. V Celju je prvič omenjen l. 1721, ko se je poročil z Marijo hčerko. Tedaj je omenjen tudiJanečkov oče Martin kot pokojnik. Janez F bil kot cenjen meščan v času med letoma 1759 in 1761 celo mestni sodnik Umrl je v času med letoma 1777, ko so znane zadnje orgle (Sela pri Sisku in 1779. Tega leta je v zapisniku celjskega mestnega sveta omenjen kot p zakonu z Marijo sta imela tri otroke. Meščanske pravice je dobil l. 1722, k imeti vsaj 24 let, kar je takrat pomenilo polnoletnost. V sapnice svojih orge no lepil etiketo z napisom „Joannes Franciscus / Genechek, Burger / Macher / in Zilla. 17~·. Napako v priimku je popravil s črnilom in dop številki kot letnico izdelave orgel. Imel je veliko in uspešno delavnico, v k izdelanih ok. 150 orgel. Do danes je na Slovenskem in Hrvaškem ohranj 30 inštrumentov. Večje orgle so bile dvomanualne, veličinama pa enoman njimi je bilo veliko manjših pozitivov (4-6 registrov), od katerih je nekaj t. sijskih orgel za prenašanje (Pokrajinski muzej Ptuj, 1739). Janeček je dobiv tudi v pomembnejših cerkvah, kot sta ljubljanska stolnica sv. Nikolaja Cl so ohranjene le omare) in c. sv. Marka v Zagrebu (1740--41). HORBIGER, Alois (1 Orglar v Celju in na Dunaju (Avstrija). Kot odličen izdelovalec klasicisti z značilnostmi nemške in italijanske šole je pomembno vplival na kvalite orglarstva na Slovenskem. Z njegovim odhodom ok. l. 1855 se je zaključila celjskega kroga orglarjev, ki ga je v začetku 18. st. utemeljil Čeh Janez Janeček (Joannes FranciscusJanechek, 1700t1777). Horbiger je bil rojen ko sin v kraju Tiersee pri Kufsteinu na Tirolskem (Avstrija), umrl je v Vršcu (Srb za samouka. Iz Tirolske je odšel najprej v vzhodno Avstrijo (Ost Osterreich se je naselil v Celju, kjer je tega leta dobil meščanske pravice kot orglar. bratom Bartolomeom in pomočniki je izpolnjeval naročila na širšem območju Slovenije. V celjskem obdobju je izdeloval predvsem enomanualne orgle z registri v slogu italijanske šole, ki je imela velik vpliv na tirolske orglarje nemški vpliv je opazen v dispoziciji registrov. Edine dvomanualne orgle o nih na Slovenskem, stojijo v opatijski cerkvi v Celju (1842), kjer se je še pod orglar iz Tirolske („Orgelbauer aus Tyrol•.), kasneje se je deklariral za Celjan bauer aus cilli„). Večje orgle je postavil v pomembnejših cerkvah, kot sta cerkev v Gornjem Gradu pri Kamniku in župnijska cerkev v Postojni (1854 pa v Slovenj Gradcu, Tinjah na Pohorju, Senožečah, Tolminu in v Cerknem dni po prihodu v Celje (1843) je izdelal orgle za cerkev laške skupnosti (Welsche Kirche). Okrog l. 1855 se je preselil v Atzgersdorf pri Dunaju, kje obrt uspešno nadaljeval. Vzrok za odhod iz Celja je bil ekonomskega zn »kmečko okolje", kakor je sam zapisal v pismu svojemu pomočniku Martin ni bilo dovolj spodbudno za njegovo delo. Nato je deloval na avstrijskem Š in Koroškem. Leta 1853 je postavil dvomanualne orgle v cerkvi sv. Janeza N MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA Na Slov ohranjene ene dvomanualne orgle (Olimje pri Podčetrtku,1764), največ pa nualnih pozitivov (Opatijska kapela v Celju, 1730; Crngrob pri Škofji Loki gatec, 1743; Ruše, 1755; Sladka Gora, Šmarje priJelšah, 1755; Čermožiše p 1763). Na Hrvaškem se je prvič pojavil l. 1730, ko je za zagrebško kated manjši pozitiv, nato je l. 1740 dobil naročilo za izdelavo orgel z 22 regis Marka na kapitlju, ki so bile v tistem času največje na Hrvaškem. Naročila s predvsem od cerkva, ki so bila pod protektoratom zagrebške kapiteljske sak, 1767; Sela pri Sisku, 1777) ter iz okolice Zagreba (Laduč, 1733, Kup Brezovica, 1771), od katerih so ohranjene vse razen tistih v Sisku. Ob Janezu je po l. 1765 kot njegov sodelavec znan orglar Janez Janeček. Sorodstv pojasnjena, vendar je delavnica do l. 1783, ko sta bila oba že pokojna, de istim imenom. Nato je družinsko obrt nadaljeval Anton Scholz (1756tl80 najverjetneje pri Janečku pomočnik. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Literatura: SUPANN 1962-66, 260; ŠABAN 1979, 21-23, 37; BIZJAK/ŠKU 32, 40, 42, 48, 50, 54, 56, 60; KOTER 2001, 39. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Literatura: SUPANN 1962-66, 260; ŠABAN 1979, 21-23, 37; BIZJAK/ŠKU 32, 40, 42, 48, 50, 54, 56, 60; KOTER 2001, 39. KACIN, Ivan (26. 8. 18 KACIN, Ivan (26. 8. 18 Izdelovalec orgel, harmonijev in pianinov ter organist in zborovodja. Bil glasbenik in vsestranski izdelovalec inštrumentov. Rodil se je v Otaležu pri Po končani orglarski šoli v Ljubljani 0900) je v času med letoma 1901-191 val kot organist in zborovodja v različnih krajih na Slovenskem in v bližini lija). Ob tem se je doma v očetovi delavnici izučil za izdelovalca harmon Ivan Kacin (1850) je bil samouk, izdelovalec harmonijev in mizar. Sin se j tudi za orglarstvo, ki se ga je učil v različnih delavnicah v Ljubljani, Bene Gradcu. Leta 1916 je v Ljubljani odprl lastno delavnico harmonijev, tri let 0919) se je preselil v Gorico (Italija) in odprl tovarno za orgle, harmonije in Kacinova tovarna je zaposlovala do 40 delavcev, njen uspeh pa pripisujem nim obnovam cerkva, ki so bile porušene v prvi svetovni vojni. Ob koncu imel podružnico v Celju, nato se je skupaj z nekaterimi delavci in njihovim mi iz Gorice preselil v Domžale. V tamkajšnji delavnici, ki je delovala d druge svetovne· vojne, so izdelovali le harmonije in pianine. Uveljavil se Italiji (Piazzola sul Brenta pri Padovi, c. Santo Redentore v Benetkah, Sacile nah) in na Hrvaškem (Tar pri Poreču), harmonije in pianine pa je uspešno doma ter v številnih krajih nekdanje Jugoslavije. V njegovi orglarski delavn zaposleni mojstri iz različnih evropskih držav in Slovenci. Kolavdacijo orgel ljala Vinko Vodopivec in Lojze Bratuž. Na Slovenskem ohranjene Kacin najdemo predvsem v manjših krajih na Primorskem v okolici Gorice (Av Gabrje pri Vipavi, Kal nad Kanalom, Kanal ob Soči, Kromberk, Ločnik, Miren selo). Med najpomembnejšimi pomočniki so bili Giorgio Bencz (izdeloval kasneje je postal samostojni orglar), piščali sta intoniralaJosip Valiček in Fr (oba vajenca orglarja Ivana Milavca, kasneje samostojna orglarja), za me skrbel Nemec Zitzman (kasneje družabnik J. Valička), strokovnjak za pian ČehJešek, glavni mizar pa Albin Kucler iz Ljubljane (kasneje orglar v firmi » Ivan Kacin je sodeloval tudi s strokovnjaki iz Nemčije, Avstrije in celo iz Izdelovali so predvsem pnevmatske, po naročilu pa tudi mehanske orgle. svetovni vojni je Kacin delal le občasno. Svoje izdelke je redno predstavlja sejmih v Ljubljani in na Reki (Hrvaška). Naslov: Ljubljana, Novi Vodmat (1916-1919); Gorica, Via Tommas 0920-1932); Domžale, Ljubljanska c. št. 108 0932-1940). KRIEGL, Karol 0 Orglar v Ljubljani. Deloval je na širšem območju današnje Slovenije. delavnici Franca Goršiča (1836t1898), ki je bil najpomembnejši orglar drugi polovici 19. st. in eden največjih na tem območju. Po učni dobi je pri Goršiču pomočnik, nato je delal skupaj z orglarjem Ivanom Milavcem ( ob koncu stoletja pa je odprl samostojno obrt. Za čas med letoma 189 znanih več orgel (Ljubljana, c. sv. Roka; Črni vrh nad Idrijo; Radovica pri M pri Kranju). Leta 1911 je bil še med aktivnimi orglarji. Naslov. Ljubljana, Kreuzgasse 8 08 Naslov. Ljubljana, Kreuzgasse 8 0 Literatura: BOTSTIBER 1911, 311; ŠKUL KREUGH (Krek), Thomas (Tomaž), (1602 Orglar v Gradcu in Ljubljani. Po rodu je bil Tirolec. V Ljubljano je priš iz Gradca. Leta 1649 je izdelal orgle za cerkev v Crngrobu pri Škofji Loki bile l. 1743 odstranjene in zamenjane z orglami Janeza Frančiška Janeč FranciscusJanechek) iz Celja. Kreugh je l. 1650 popravljal orgle v ljublja Literatura: BIZJAK/ŠKULJ 1985, 40, 151. KRANJC, Michael KRANJC, Michael Orglar v Mariboru. Na širšem območju Maribora je deloval deset let, pa ga najdemo tudi na Hrvaškem. Predvidoma izhaja iz graške rodbine orgel. Izučil se je na Dunaju pri Carlu Hesseju (1808t1882). Zanj so znač mehansko trakturo in sapnicami na poteg, kar kaže na čas pred nastop tike. Iz mariborskega obdobja je znanih 7 orgelskih inštrumentov, in sic krajih na Štajerskem (Maribor, c. sv. Magdalene, 1874; Slivnica, Prihova Mariboru; Hajdina in Destrnik pri Ptuju). Za Hrvaško so podatki o treh v manjših krajih (Mala Subotica, ok. 1873, 12 registrov; Donji K.raljevac, strov; Kuzminec, 1877, 12 registrov). Literatura: ŠABAN 1979, 32, 38; BIZJAK/ŠKULJ 1985, 158; CG 83 099 KREUGH (Krek), Thomas (Tomaž), (1602t MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Literatura: SUPANN 1962-66, 260; ŠABAN 1979, 21-23, 37; BIZJAK/ŠKU 32, 40, 42, 48, 50, 54, 56, 60; KOTER 2001, 39. Š Literatura: CARDANA 1973,42, 44, 62, 64, 83, 87, 97; BIZJAK/ŠKULJ 1 JEDRLINIČ/KOMEL 1990, 2-29. Literatura: CARDANA 1973,42, 44, 62, 64, 83, 87, 97; BIZJAK/ŠKULJ 1 JEDRLINIČ/KOMEL 1990, 2-29. KAFKA, Adalbe Orglar v Mariboru. Deloval je na slovenskem Štajerskem in v Prekmurju setih letih 19. st. Sodi k t. im. mariborskemu krogu orglarjev, ki so delovali polovice 18. do konca 19. st., zanje pa je značilen vpliv graških mojstrov. Le popravljal majhen baročni pozitiv neznanega orglarja v Loretski kapeli v MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA Mariboru („um 18 Juni / 1864 / Kafka / Orgelbauer / in Marburg.) in izd orgel (Maribor, c. Device Marije Pomočnice, 9 registrov; sv. Andraž v S Hajdoše,11 registrov; Laško, 22 registrov; Veržej, 10 registrov). Literatura: BIZJAK/ŠKUIJ 1985, 158; CG 83 0990), št. 7-9, 73. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Nakicu izučil za orglarja. Kot duhovnik je služboval v Vipavi, kjer je imel tu nico. Na Slovenskem so znane 3 njegove orgle. Prve je l. 1761 izdelal za R Dolenjskem (l. 1775 so v požaru zgorele). Po priporočilu tamkajšnjega ž l. 1762 izdelal enomanualne orgle z 32 registri za ljubljansko stolnico sv. (l. 1781 so jih odstranili). Pri delu mu je pomagalo osem obrtnikov. Leta 1763 je enomanualne orgle v ljubljanski uršulinski cerkvi, ki so imele 24 registrov (u bile v času Napoleonovih vojn). Križman je kot odličen orglar zaslovel, zat naročilo iz Avstrije in se l. 1770 tudi odselil. Še istega leta je postavil orgle v Sv v Gornji Avstriji (1770), iz časa med letoma 1774-79 so znane orgle v Steyer opatijski cerkvi v Admontu (1782), v letih 1782-85 je delal na Dunaju (St. La Schotenfeld), zadnje Križmanove orgle pa so v Rottenmanu (1794-95). Literatura: BIZJAK/ŠKULJ 1985 150 170; ŠKULJ 1994 31 38 42 71 72 Literatura: BIZJAK/ŠKULJ 1985, 150, 170; ŠKULJ 1994, 31-38, 42, 71 KUČERA (Kutschera), Josip Alojz (Joseph Alois), (1755?t pr KUČERA (Kutschera), Josip Alojz (Joseph Alois), (1755?t pred 1826) Orglar v Ljubljani. Bil je prvi predstavnik orglarjev, delujočih na Sloven so baročnim orglam dodajali klasicistične elemente (ok. l. 1780). Poročil se vo ljubljanskega orglarja Johanna Georga Eisla (1708t1780) in prevzel njeg Meščanske pravice je dobil l. 1782. Prvo večje delo je opravil za ljubljansko za katero je l. 1781 izdelal nove orgle. Kučera je baročni dispoziciji dod značilnosti klasicizma. Deloval je v številnih slovenskih krajih in na Hrv Sloveniji se je ohranilo malo njegovih inštrumentov (Dobrava pri Dobrniču, D 1783; Velike Bloke, 1787; Šmihel pri Žužemberku, 1795). Na Hrvaškem, kj venski orglarji do druge polovice 19. st. izdelali največ orgel med vsem delujočimi mojstri, je Kučera postavil orgle v Lipniku („Nro. 10, zu Ehere Go Joseph Alois Kutschera Burgl. Orgel Macher in Laybach, den 14 May 1785· Literatura: ŠABAN 1979, 19; ŠKULJ 1994, 34-38, 152-153. KUNAT, Johann Gottfried (Janez Gotfrid), (pred 1790t UN , Jo a Gott ed (Ja e Got d), ( p ed 790t po 8 6) Orglar v Ljubljani. Najpomembnejši predstavnik slovenskega klasicizma orgle so slovele po odličnem in mogočnem zvoku. Kot prvi orglar na Slove opustil baročno kratko oktavo, ki je bila pri nekaterih mojstrih pri nas obi sredine 19. st. Po rodu je bil Nemec. Njegov mentor je bil Blaž Potočnik, duh skladatelj. KRIŽMAN, Frančišek Ksaver ( Orglar na Slovenskem in v Avstriji. Prva leta svojega delovanja je pos na območju današnje Slovenije, kamor je prinesel vplive beneškega or l. 1770 pa je odšel v Avstrijo, kjer je v Rottenmanu tudi umrl. Velja za imenitne orglarske šole beneškega mojstra hrvaškega rodu Petra Nakica ( utemeljitelja celega niza menzur in jezičnih piščali, s katerimi je obog zvok italijanske orglarske tradicije. Njegovi številni nasledniki so nadalje do konca 19. st. Križman se je rodil v Braniku (Rihenberk) na Goriškem. bil posvečen za duhovnika, v času med letoma 1754 in 1758 pa se je v B MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA Friderik (1822-1912). Pri Kunatu se je orglarske obrti priučil Peter Rojc (* iz Tabora na Gorenjskem, kasneje samostojni orglar, verjetno pa tudi Pet Literatura: BIZJAK/ŠKULJ 1985, 16, 94, 98, 100, 112; ŠKULJ 1994, 38 71-72, 86-87, 153. Friderik (1822-1912). Pri Kunatu se je orglarske obrti priučil Peter Rojc (* iz Tabora na Gorenjskem, kasneje samostojni orglar, verjetno pa tudi Pet MALAHOVSKI (Malahovsky), Andrej Ferdinand (* Orglar in mizar v Ljubljani. Po rodu je bil Čeh, po predvidevanjih se Carlu Hessu (1808t1882). V Ljubljano je prišel z dvajsetimi leti, njegov m Gregor Rihar (1796t1863), organist in Regens chori ljubljanske stolnice te cerkvene glasbe, ki se je ukvarjal tudi s popravili orgel in klavirjev. Rihar Malahovskega večkrat pripravil dispozicijo. Meščanske pravice je dobil Njegove orgle imajo pretežno klasicistično dispozicijo. Prvi inštrument j križevniško cerkev v Ljubljani (1840), njegova večja dela so v cerkvi sv. Jak in sv. Petra (1853) v Ljubljani, drugi inštrumenti imajo letnice 1855, 1858, Uspešno je deloval tudi na Hrvaškem, kjer je za cerkve v manjših kraj enomanualnih orgel (1859, 1860, 1864, 1865 in 1870). Delavnica je izde manualne (6-14 registrov) in dvomanualne orgle (do 24 registrov). Malah tudi dober mizar in inovator stroja za rezanje furnirja. Literatura: ZAL, Imenik meščanov stolnega mesta Ljubljana od leta sign. 767, zap. št. 398; ŠABAN 1979, 20, 38; BIZJAK/ŠKULJ 1985, 138; M 190; ŠKULJ 1994, 41, 59--00, 81-82, 115-116, 122, 129, 153-154; Publikacije zv. 4, 67. Časopis: Novice kmetijskih, rokodelskih in narodskih reči, št. 5 ( p p Literatura: MANTUANI 1932; WOODCOCK 1965, 53; VANNES MALAHOVSKI (Malahovsky), Andrej Ferdinand (* MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Kunat je imel v Ljubljani skromno delavnico, po starejših vzorih p največkrat izdeloval kar na kraju naročila (Ljubljana Trnovo, 1818; Cerkni Ljubljana Šentvid, 1839; Ljubljana -uršulinska cerkev, 1842). Ohranjeni so šti menti. V nekatere izdelke je vgradil »Schlagwerk« oz. »turško muziko". Ta r na Slovenskem značilen še za Aloisa Horbigerja (1810t1872) in Leonhard (1855-1868). Kunat je izdelal nekaj kvalitetnih inštrumentov, med katerimi dvomanualne orgle z 32 registri za ljubljansko stolnico (1830), ki so njego opus, hkrati pa tudi najpomembnejši dosežek orglarskega klasicizma na Slo Čez trideset let jih je za dva registra povečal ljubljanski orglar Andrej Ferdi lahovski (1813t1887). To so bile največje slovenske orgle v 19. st. Kunat popravljal orgle v cerkvi sv. Petra v Trstu, kjer je zastopal Petra Rumpla (17 zelo uspešnega orglarja iz Kamnika. V Trstu je kot orglar deloval Kunatov si LINDTNER, Mathias ( Orglar v Ljubljani. Meščanske pravice je tako kot Johann Faller dobil letu 1671 ga ni več med ljubljanskimi meščani. Literatura: ZAL, Cod. XIIl/36, f. 37); Publikacije ZAL (1972), zv. 3, 59, 2001, 97. Literatura: ZAL, Cod. XIIl/36, f. 37); Publikacije ZAL (1972), zv. 3, 59, 2001, 97. LUKEŽIČ, Matija ( LUKEŽIČ, Matija ( Goslar, izdelovalec violin, samouk. Deloval je v Idriji v času med letoma Leta 1932 je bila njegova violina z letnico 1809 razstavljena v ljubljanskem domu na razstavi Razvoj glasbe pri Slovencih, ki jo je v okviru Glasbene pravil Josip Mantuani. p p Literatura: MANTUANI 1932; WOODCOCK 1965, 53; VANNES MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL kjer je postavljal večinoma enomanualne orgle (Trška Gora, Ajdovec, Otočec žica na Dolenjskem), med večjimi kraji pa sta Vipava in Pivka. Dvomanual pozitivom na komi ograji so ohranjene v Gorici pri Dolenjskih Toplicah in Kočevju. Na Hrvaškem, kjer sta znana le dva njegova inštrumenta, so zani kraju Kunic Ribnički na Kolpi (1867). Vanje je vgradil mehanizem harmonij organist lahko uporabljal povsem samostojno, ločeno od orgelskega zvoka uporabo istega mehovja. Druga posebnost je konstrukcija za pogon meho omogočala njegovo lažje gibanje. V Mandlinovi delavnici se je izučil And (1808t1883), kasneje samostojni orglar. Literatura: ŠABAN 1979, 33-34, 38; BIZJAK/ŠKULJ 1 Literatura: ŠABAN 1979, 33-34, 38; BIZJAK/ŠKULJ 1 MAROUT, A. (?) Izdelovalec klavirjev iz Ljubljane. Deloval v 20. letih 19. st. Literatura: KOTER 2001, 129-130. Inštrumenti: v Sloveniji je v zasebni lasti ohranjen klavir z značko „A M IN LAlBACH" ( k 1825) MAROUT, A. (?) Izdelovalec klavirjev iz Ljubljane. Deloval v 20. letih 19. st. Literatura: KOTER 2001, 129-130. Inštrumenti: v Sloveniji je v zasebni lasti ohranjen klavir z značko „A IN LAlBACH" (ok. 1825). Inštrumenti: v Sloveniji je v zasebni lasti ohranjen klavir z značko „A M IN LAlBACH" (ok. 1825). MARTHAL, Wenzel (Venceslav), (pred 1780t Orglar v Celju. Nadaljeval je tradicijo t. im. celjskega kroga, ki ga je ute glar češkega porekla Janez Frančišek Janeček (ok. 1700t ok. 1777), nad Anton Scholz (1756t1802). Marthal se je po slogu iz baroka usmerjal h kl dispoziciji. Po rodu je bil Čeh, v Celje pa je prišel vsaj l. 1804, ko je dokonč orgle Antona Scholza za cerkev sv. Jurija v Mozirju. Leta 1805 se je poročil svojega predhodnika Elizabeto ter si tako pridobil obrtne pravice in delavn go in odlično tradicijo češke šole, ki je bila za mojstre, delujoče v Celju zn prihoda Tirolca Aloisa Horbigerja l. 1842. Marthal je med drugim poprav svojih predhodnikov Janečka in Scholza, kot samostojnega orglarja pa ga po petih inštrumentih (Vitanje, 1803-1807; Mozirje, 1804; Strahinj pri Nakl Sv. Marjeta v Planini, 1821 in Šentilj pod Turjakom, 1821). Ko je l. 1825 umrl za lozo, je vdova Elizabeta obrt nekaj let vodila sama, nakar jo je prodala orgla stinu Quisierju, ta pa se je kot celjski obrtnik odjavil l. 1844. Literatura: BIZJAK/ŠKULJ 1985, 86, 88, 90; ŠKULJ MAZI, Jakob (1 Organist in orglar samouk v Preserjah. Doma je bil iz Brezovice pri Lju l. 1850 je bil organist na Rakitni, nato se je preselil v Preserje in skupaj z Florjanom in Martinom izdelal nekaj orgel (Rakitna, Preserje, Žalostna Gor serjah, 1845). Bratje Mazi, ki so znani tudi kot popravljalci orgel, so bili sr edini orglarji na območju Notranjske. Literatura: BIZJAK/ŠKULJ 1985, MANDLIN, Janez (* Orglar v Trebnjem na Dolenjskem. Deloval je predvsem v osrednj občasno pa tudi na Hrvaškem. Njegove orgle so imele klasicistično, za t zastarelo dispozicijo. Doma je bil v Ljubljani. Izučil se je pri Petru Rumplu (* v Kamniku, ki je imel eno najuspešnejših orglarskih podjetij na Slovenskem njeval pa se je na Bavarskem. Prve samostojno izdelane orgle je postavil cerkvi v Trebnjem. Naročila je dobival predvsem v manjših krajih na D Izdelovalec godal v Ljubljani. Deloval je od l. 1762, po l. 1786 ni več om najstarejši po imenu znani goslar, ki je deloval na območju današnje Slove MELLING, Johann (Janez), (17 Izdelovalec godal v Ljubljani. Deloval je od l. 1762, po l. 1786 ni več om najstarejši po imenu znani goslar, ki je deloval na območju današnje Slove MILAVEC, Ivan (* Orglar in izdelovalec harmonijev v Ljubljani. Deloval je na območju o venije in na Hrvaškem. Sodi med najboljše mojstre pnevmatičnih orgel skem na začetku 20. st. in je eden redkih izdelovalcev harmonijev pri nas v Cerkovski vasi pri Logatcu. V času med letoma 1888-1892 se je izučil za Francu Goršiču (1836t1898), ki je imel v Ljubljani eno najuspešnejših Slovenskem, do leta 1895 je bil tam pomočnik. Nato je delal pri Andreju izdelovalcu harmonijev na Vrhniki. Preden je l. 1900 odprl samostojno ob ni, je nekaj časa sodeloval z orglatjem Karolom Krieglom. Prve samosto izdelal za Škocjan na Notranjskem (1904), ki so edine imele še mehansk vse ostale so pnevmatične. Njegovo največje in najpomembnejše delo so ne orgle v ljubljanski stolnici sv. Nikolaja 0911, 52 registrov), ohranjen po velikosti sledijo dvomanualne (Žiri, Kamnik, Bled, Ljubljana Vič, Šmartin Idrija), nekaj pa je tudi manjših orgel (Ljubljana Rožnik). Milavec je delo Hrvaškem (Košljun, 1908, op. 10; Punat na Krku, 1912, op. 19; Karlovac, fr cerkev, 1914, op. 36). V njegovi delavnici je bilo izdelanih 37 orgel. Pri M izučil Franc Jenko (1894t1969), kasneje zelo uspešen samostojni orglar, nica deluje še danes. Med pomočniki je bil Josip Valiček, ki je nato del Kacinu (1884t1953), orglatju, izdelovalcu harmonijev in pianinov. Naslov-. Ljubljana, Kirchengasse 19 (19 Naslov-. Ljubljana, Kirchengasse 19 (19 Naslov-. Ljubljana, Kirchengasse 19 (19 Naslov-. Ljubljana, Kirchengasse 19 (19 Literatura: BOTSTIBER 1911, 311; BIZJAK/ŠKULJ 1985, 118, 168, 170; Š 42-43, 90-91, 122, 124. Literatura: BOTSTIBER 1911, 311; BIZJAK/ŠKULJ 1985, 118, 168, 170; 42-43, 90-91, 122, 124. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Literatura: ZAL, Reg. I, fasc.19, fol. 512 sl; MANTUANI 1932; Publikacije zv. 4, 33; KOTER 2001, 142. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA Literatura: ZAL, Reg. I, fasc.19, fol. 512 sl; MANTUANI 1932; Publikacije zv. 4, 33; KOTER 2001, 142. Naslov-. Celje, Arja vas (1862-1 Literatura: BIZJAK/ŠKULJ 198 Literatura: BIZJAK/ŠKULJ 198 NARAKS, Franc ( Orglar v Arji vasi pri Celju. Deloval je predvsem na Štajerskem (od 186 in sodi med mojstre, ki so bili kljub novejšim trendom privrženi tradiciji klasicizma. Izhaja iz Galicije pri Celju, delavnico pa je imel v Atji vasi. Nek imenujejo samouka, drugi pa v njegovih izdelkih vidijo naslednika Tir Horbigerja (1810t1872), ki je imel v času med letoma 1842 do ok. 18 delavnico v Celju, nato pa je odšel na Dunaj. Naraksove orgle prepričl vpliv Horbigerja, kar ga umešča med redke naslednike t. i. celjskega krog ki ga je na začetku 18. stoletja utemeljil Janez Frančišek}aneček (ok.1700 V Naraksovi delavnici so izdelali številne orgle (6-14 registrov). Največje ski cerkvi v Krškem, druge so v manjših krajih na Štajerskem (Žalec, Sliv no ob Paki, Kunigunda na Pohotju, Loče, Pernice, Šentjur pri Celju, Dramlj Peč, Šentilj pri Velenju, Draga, Majšperk, Poljčane). Naslednik Franca Na njegov sin Ivan, ki se je najprej izučil pri očetu, se izpopolnjeval v Nemčiji očetovi smrti l. 1906 prevzel domačo obrt. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA NARAKS, Ivan (1 NARAKS, Ivan (1 Orglar v Arji vasi pri Celju. Deloval je v osrednji Sloveniji. Že v času svoj vanja je veljal za odličnega mojstra v mehaniki, njegove orgle so kvalitetne Izučil se jev domači delavnici, ki jo je ustanovil oče Franc. Pomočniška pot ga vodila v Nemčijo, kjer se je izpopolnjeval štiri leta (Paderborn in Berlin). je delal skupaj z očetom, po njegovi smrti l. 1906 pa je obrt uspešno nadalje prvo svetovno vojno je zaradi vsesplošne krize v glavnem le popravljal st po njej pa je najprej nadomeščal orgelske principale, ki so bili med vojno z in uporabljeni kot material za potrebe vojaštva. Izdeloval je tudi nove orgl je dvanajst inštrumentov (Šmihel pri Mozirju, Celje - c. sv. Cecilije, Leskovec Šmohor nad Laškim, Ljubno v Savinjski dolini, Preddvor, Sv. Duh pri Krškem ter Dornava pri Ptuju kot zadnje delo). Največje orgle z dvema manualoma trov) so v Teharjih pri Celju, ki so ohranjene v izvirni obliki. Naslov. Celje, Arja vas (1906- Naslov. Celje, Arja vas (190 Literatura: BIZJAK/ŠKULJ 1985 Literatura: BIZJAK/ŠKULJ 1985, Literatura: BIZJAK/ŠKULJ 1985, OMERZU, Anton (1895) Orglar iz Šentvida pri Planini. Leta 1895 je povečal orgle v domači cerkv lal nov inštrument za dve manjši cerkvi v okolici (sv. Ožbalt in Žusem). Literatura: BIZJAK/ŠKULJ 1985, 158. OMERZU, Anton (1895) Orglar iz Šentvida pri Planini. Leta 1895 je povečal orgle v domači cerkv lal nov inštrument za dve manjši cerkvi v okolici (sv. Ožbalt in Žusem). Literatura: BIZJAK/ŠKULJ 1985, 158. Literatura: ŠABAN 1979, 25-28, 39; BIZJAK/ŠKULJ 1985, 72; CG 83 (19 71-72. MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA ce je dobil ok. l. 1792. Prvič je omenjen kot popravljalec očetovih orgel v na Hrvaškem (»Joseph Odonitsch Orgel und Instrument Macher in Mah Repafirt„). Kot samostojni obrtnik je sprva nadaljeval tradicijo inštrumen delavnice z baročnimi značilnostmi t. im. graške oz. štajerske šole, v zad pa je uvedel nekaj klasicističnih elementov (8-čeveljski principal). V prvi vanja je bil zelo uspešen, saj je letno izdelal tudi do štiri inštrumente. Evid okrog 97 orgel. Zaradi vojnih razmer in velikega pomanjkanja vajencev dobrega materiala je kvaliteta delavnice z leti precej upadla. Otonič se je zadolževati, zato nekaterih naročil ni izpolnil. Njegovo najstarejše samo so orgle iz l. 1796 (Sv. Jurij ob Ščavnici), orgle v Podsredi iz l. 1822 pome na klasicistično dispozicijo, zadnje (op. 97) pa so nastale l. 1832 (Kape Radencih). Kljub obsežnemu opusu je znanih le okrog ducat orgel, med dvojne dvomanualne (Lovrenc na Pohorju, 1809; Ludberg, Hrvaška, 1810 kem je evidentiranih 7 orgel, 4 so ohranjene (Hrastovljan, 1796, op. 18; Gorn 1800, op. 27; Veliko Trojstvo, 1802, op. 32; Močila, ok. 1807). Š Š Literatura: ŠABAN 1979, 28-32, 38; BIZJAK/ŠKULJ 1985, 78, 94, 9 PLEŠKO, Janez Kr Si i i d l l Sitar in izdelovalec bobnov. Deloval je v Ljubljani ok. l. 1848. Tega bobne za takrat ustanovljeno narodno gardo. Lit t A h L ib h Z i 6 4 1848 P blik ij Z Sitar in izdelovalec bobnov. Deloval je v Ljubljani ok. l. 1848. Tega le bobne za takrat ustanovljeno narodno gardo. Lit t A h L ib h Z i 6 4 1848 P blik ij ZAL Literatura: Anhang zur Laibacher Zeitung, 6. 4. 1848: Publikacije ZAL 4, 67; KOTER 2001, 168. Jnštrumentarij: Graz, Steierisches Volkskundemuseum, klarinet; Ptuj, muzej Ptuj, flavta, inv. št. GL 76 S. PAUHAUSS, Jurij U O l Lj blj i Orglar v Ljubljani. Meščanske pravice je dobil l. 1711. Po predvidev poročil z vdovo ljubljanskega meščana in orglarja Andreasa Sebastiana Wa (pred 1680tl 709) in si tako pridobil obrtne pravice. Leta 1712 je sklenil p popravilo velikih in malih orgel ter orgelskega roga za cistercijanski samos njevici na Krki. Š Literatura: ŠKU Literatura: STRADNER 1986, 107-108; KOTER 200 J š ij G S i i h V lk k d OMERZU, Anto Š OTONI ČER ( Ottonischer, Odtonischer, Ottonitscher, Odonitzer), Simon ( OTONI ČER ( Ottonischer, Odtonischer, Ottonitscher, Odonitzer), Simon (1 Orglar v Mariboru. Znan je kot prvi stalno naseljeni orglar v Mariboru, nik baročne tradicije in utemeljitelj mariborske orglarske tradicije za čas polovice 18. do 30. let 20. st. Deloval je na Štajerskem in v severni Hrvaški. je v Gradcu (Avstrija) v znani delavnici Cyriacha Wernerja (tl 747), kjer je bil pomočnik. Tega leta se je poročil z Wernerjevo hčerko Terezo. Leta 1764 sta se preselila v Maribor, kjer je Simon odprl orglarsko delavnico. Meščanske p dobil v času med letoma 1767 in 1774. V zakonu je imel tri otroke, od kate Joseph prevzel očetovo obrt. V Sloveniji sta iz delavnice Simona Otoničera rana le dva pozitiva s 6 registri (Ritoznoj pri Slovenski Bistrici, 1766; Sv. M Vurberku). Ohranjen je le prvi od omenjenih. Otoničer je bil prvi na Slo delujoči orglar, ki je postavljal igralnik na bočno stran orgelske omare, k tipična značilnost graškega kroga orglarjev tistega časa. Na Hrvaškem je ev nih pet orgel (Zelendvor, ok. 1765, pozitiv, 6 registrov; Sopot, 1766, pozitiv, 6 Radovan, 1774, pozitiv, 8 registrov; Petrijanec, 1777, 8 registrov; Hraščina, 177 strov). V njegovi delavnici se je izučil sin Joseph, ki se je podpisoval kot Otto j g j p j p p Literatura: ŠABAN 1979, 25-28, 39; BIZJAK/ŠKULJ 1985, 72; CG 83 (1990 71-72. OTONIČ (Odonitsch, Ottonitsch), Joseph (Josip), (17 Orglar in izdelovalec inštrumentov v Mariboru. Kot orglar je deloval n skem Štajerskem in na širšem območju severne Hrvaške. Rojen je bil v (krščen 8. 8. 1767) kot sin orglarja Simona Otoničera (1730tl 784). Meščans Naslov-. Maribor, Graško predmestje š MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL POHM, Michael (1759t14. Izdelovalec lesenih pihal, strugar in godbenik na Ptuju. Ptujski godbeniki mente kupovali predvsem na Dunaju in v Gradcu, med domačimi mojstri j Michael Pohm. Njegovi ohranjeni inštrumenti potrjujejo, da je bil dober obrtn je bil na Ptuju in izhaja iz družine strugarjev. Oče Johann Pohm je dobil me pravice l. 1759, Michael je kot meščan in strugar (•Drexlermeister„) zapri cembra l. 1782, njegov sin Johann pa je postal strugarski mojster l. 1812, v znano, če je izdeloval tudi glasbila. Michael je bil v času med letoma 179 vojak ptujskega meščanskega lovskega korpusa (»Bilrgerliche Jager Corps„). lovalec lesenih pihal (dokazane so oboa in flavte) je bil verjetno član vojaš tega korpusa, ki je na prehodu iz 18. v 19. st. z vojaško parado in »turško večkrat izkazala čast avstrijskim cesarjem. V mrliški knjigi je med ptujskim edini vpisan kot meščan strugar in izdelovalec inštrumentov (»bilrgerliche meister und Instrumentenmacher„). Naslov. Ptuj, hišna št. 167 (danes Cankarjeva ulica), 17 Literatura: Arhiv cerkve sv.Jurija Ptuj, Mrliška knjiga 1805-1838; ZAP, Fond glavarstvo Ptuj (Bezirkshauptmannschaft Pettau) 1788-1870; ZAP, Zemljišk R-501, h. št. 167; STRADNER 1986, 33; KOTER 2001, 156-158. Š Literatura: Arhiv cerkve sv.Jurija Ptuj, Mrliška knjiga 1805-1838; ZAP, Fond glavarstvo Ptuj (Bezirkshauptmannschaft Pettau) 1788-1870; ZAP, Zemljišk R-501, h. št. 167; STRADNER 1986, 33; KOTER 2001, 156-158. Š Literatura: Arhiv cerkve sv.Jurija Ptuj, Mrliška knjiga 1805-1838; ZAP, Fond glavarstvo Ptuj (Bezirkshauptmannschaft Pettau) 1788-1870; ZAP, Zemljišk R-501, h. št. 167; STRADNER 1986, 33; KOTER 2001, 156-158. Š Inštrumentarij: Gradec, Štajerski deželni muzej Joanneum (Steierma LandesmuseumJoanneum), oboa (izgubljena?), inv. št. KGW 1398; Maribor, ski muzej Maribor, flavta, inv. št. N. 4065; Milano, Museo <legli Strumenti Milano (inv. št. ni znana). POJE, M I d l Izdelovalec lesenih pihal v Mariboru in Gradcu(?) sredi 19. st. Ohran inštrumenta (flavta z značko »Poje/Marburg„ in klarinet »Poje/Gratz•), ki k bil dober obrtnik, izdelovalec godbenih glasbil. Leta 1846 je bival v Mari verjetno delal za potrebe tamkajšnje vojaške godbe. Viri ga tega leta o lastnika hiše, nato se sled za njim izgubi. Najverjetneje se je zaradi ekonom gov preselil v sosednji Gradec, kjer je sredi stoletja deloval izdelovalec le z istim imenom. l Naslov-. Maribor, Graško predmestje št STRADNER 1986 107 108 Naslov-. Maribor, Graško predmestje š Jnštrumentarij: Graz, Steierisches Volkskundemuseum, klarinet; Ptuj, muzej Ptuj, flavta, inv. št. GL 76 S. PRASSKY, Johann (1787t p Orglar in izdelovalec inštrumentov iz Ljubljane. Meščanske pravice je do 1787 kot „orodjar in orgljar« („Instrumenten und Orgelbauer„). Po tedanji terminologiji je beseda „orodjar" pomenila izdelovalca „drugih" inštrumento vi izdelki niso znani. Literatura: ZAL, Imenik meščanov stolnega mesta Ljubljana od leta 1786 sign. 767, zap. št. 568; ZAL, Imenik mestjanov deželnega glavarnega mesta skega 1786-1867, Laibach 1867, sign. 767, zap. št. 519; KOTER 2001, 101. QUESIER, Augustin (183 QUESIER, Augustin (1830?-1844) Orglar v Celju. Orglarsko obrt je odkupil od Elizabete Marthal, vdove orglarja Venceslava Marthala (pred 1780t1825), l. 1844 je svojo obrt že odjavil izdelki niso znani. Lit t BIZJAK/ŠKULJ 1985 90 Orglar v Celju. Orglarsko obrt je odkupil od Elizabete Marthal, vdove orglarja Venceslava Marthala (pred 1780t1825), l. 1844 je svojo obrt že odjavil izdelki niso znani. Š Literatura: BIZJAK/ŠKULJ Literatura: BIZJAK/ŠKULJ RAČIČ, Andrej (1808t1883) Organist in orglar v Cerkljah na Dolenjskem. Deloval je v domačem okol se je v delavnici Janeza Mandlina (1818t1891) v Trebnjem na Dolenjskem. orgle za domačo cerkev ter na Studencu, v Zagradu in Vodenicah, ene pa tudi na Hrvaškem (Petrovina, 1858). Njegovi izdelki imajo značilnosti orgel zg klasicizma. RAČIČ, Andrej (1808t1883) Organist in orglar v Cerkljah na Dolenjskem. Deloval je v domačem oko se je v delavnici Janeza Mandlina (1818t1891) v Trebnjem na Dolenjskem. orgle za domačo cerkev ter na Studencu, v Zagradu in Vodenicah, ene pa tudi na Hrvaškem (Petrovina, 1858). Njegovi izdelki imajo značilnosti orgel z klasicizma. Literatura: BIZJAK/ŠKULJ 1 RIHAR, Anton, Boštjanček ( RIHAR, Anton, Boštjanček ( Izdelovalec violin in godbenih glasbil, samouk. Sin Boštjana Riharja, izdelovalca različnih inštrumentov iz Polhovega Gradca. Leta 1932 je b violina razstavljena v ljubljanskem Narodnem domu na razstavi Razvo Slovencih, ki jo je v okviru Glasbene matice pripravil Josip Mantuani. j j p Literatura: MANTUANI 1932; KOTER 2001 Inštrumenti: pripisujejo mu violino, ki jo hrani Narodni muzej v Lj št. 9038. RIHAR, Boštjan (1773) Izdelovalec violin, klavirjev in harmonijev, samouk iz Polhovega Grad na prehodu iz 18. v 19. st. Literatura: WOODCOCK 1965, 2l;JALOVEC 1968, 159; VANNES 1979, 3 1996, 11-14; KOTER 2001, 143-144. Literatura: WOODCOCK 1965, 2l;JALOVEC 1968, 159; VANNES 1979, 3 1996, 11-14; KOTER 2001, 143-144. RIHAR, Gregor ( , g ( ) Organist, cerkveni skladatelj in vodja stolničnega kora v Ljubljani (o mladosti je očetu Boštjanu Riharju pomagal v domači delavnici inštrumen izdelala nekaj godal, klavir in harmonij. Mladi Gregor se je kmalu navd uglaševanje in popravljanje klavirjev, kar je v nuji za preživetje opravljal skih hišah. Z očetom Boštjanom sta skupaj izdelala klavir, ki naj bi ga Gre ljal vse življenje. Literatura: KOTE Časopis: CG LXIII (1940), št. 9-10, 130. Naši zbori 48 0996), št. RAKTELJ, Dominik ( Orglar v Mariboru, znan tudi kot potujoči orglar. Uvrščen je med ma glarje ob koncu 19. st. V času med letoma 1870 in 1907 je večkrat omenjen ljalec starih orgel, kot izdelovalec novih inštrumetnov pa je znan po štir Štajerskem (Lembah, 14 registrov; Makole, 12 registrov; Korena pri Mar 12 registrov; Limbuš, 1886, 14 registrov). Slednje veljajo za njegovo najv imajo v primerjavi s sodobniki, ki so delovali na Štajerskem, nekaj po sicer igralnik pred orgelsko omaro na komi ograji, slepi pozitiv ter radia Š 4 6 g p g g j p p Literatura: BIZJAK/ŠKULJ 1985, 158; CG 83 (1990), št. 4--6, 41; št MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNU RAKTELJ, Dominik ( RAKTELJ, Dominik ( ROJC, Peter ( Mizar in orglar samouk iz Tabora na Gorenjskem. Deloval je predv renjskem. Bil je sin mizarja in priučeni orglar, ki se je obrti naučil kot mla pri orglarju Janezu Kunatu (pred 1790t po 1846), ko je ta postavljal org domači vasi. Rojc je postal samostojen orglar in je izdelal okrog 45 inšt velikosti od 6 do 20 registrov. Najpomembnejša kolavdatorja orgel na Sl tistem času Gregor Rihar in Blaž Potočnik sta Rojčevo delo označila ko izjemno čisto in svojevrstno intonacijo. Evidentiranih je manjše število o niki, 1844 in 1875; Idrija, c. sv. Antona, 1847; Zapoge pri Vodicah, 1851; ROPAS Izdelovalec klavirjev in trgovec v Celju v drugi polovici 19. st. Delavnic no je imel l. 1865 na Ljubljanski cesti 16. Tam je delal tudi Martin Ropas. imelo l. 1912 podružnico v Zagrebu (Hrvaška). Izdelovalec klavirjev in trgovec v Celju v drugi polovici 19. st. Delavnico no je imel l. 1865 na Ljubljanski cesti 16. Tam je delal tudi Martin Ropas. P imelo l. 1912 podružnico v Zagrebu (Hrvaška). Naslov. Celje, Ljubljanska cesta Naslov. Celje, Ljubljanska cesta Naslov. Celje, Ljubljanska cesta Literatura: WITT de 1912, 333, 403; KOTER Literatura: WITT de 1912, 333, 403; KOTER Literatura: WITT de 1912, 333, 403; KOTER ROPAS, Martin, Izdelovalec klavirjev in trgovec v Celju. Leta 1865 je delal v delavnici Izdelovalec klavirjev in trgovec v Celju. Leta 1865 je delal v delavnici Ropasa v Celju, l. 1912 pa je imel trgovino v Zagrebu (Hrvaška). Naslov. Celje, Ljubljanska cesta 16 (1865); Zagreb, Jurišičeva ulica 24 ( Inštrnmenti: v Sloveniji je v zasebni lasti ohranjen klavir. Literatura: WITT de 1912, 333, 403; KOTER 2001, 134. p j , p j g g ( ) Naslov. Celje, Ljubljanska cesta 16 (1865); Zagreb, Jurišičeva ulica 24 MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL Bohinjska Bela, Leskovica, Sorica, Podbrezje, Tabor, Lesce, Kranj, Dobrova ljani), z enim inštrumentom pa je zastopan tudi na avstrijskem Koroškem. Literatura: BIZJAK/ŠKULJ 1985, 118, 128, 130, 150, 168. Bohinjska Bela, Leskovica, Sorica, Podbrezje, Tabor, Lesce, Kranj, Dobrov ljani), z enim inštrumentom pa je zastopan tudi na avstrijskem Koroškem. Literatura: BIZJAK/ŠKULJ 1985, 118, 128, 130, 150, 168. Bohinjska Bela, Leskovica, Sorica, Podbrezje, Tabor, Lesce, Kranj, Dobrov ljani), z enim inštrumentom pa je zastopan tudi na avstrijskem Koroškem. Š RUMPEL (Rumpl), Peter (1 RUMPEL (Rumpl), Peter (1 Orglar in izdelovalec klavirjev v Kamniku. Kot orglar je deloval v osred niji, na Hrvaškem in v Avstriji. Sodi med najpomembnejše izdelovalce o polovice 19. st., ki so delovali na območju današnje Slovenije; Njegov opu rega je značilna klasicistična usmeritev z nekaterimi romantičnimi potezam več kot 100 orgel. Sodobniki so cenili predvsem solidno izdelavo in dobe njegovih inštrumentov. Nekateri starejši viri poročajo, da se je izučil pri lju mojstru Janezu Kunatu (pred 1790t po 1846), novejše raziskave pa kažejo da se je šolal ali vsaj izpopolnjeval v kateri od boljših dunajskih delavn možnosti v prid je tudi Rumplov edini znani klavir z značko: »Peter Rumpl Instrumentm. /in Wien" (ok. 1820). Kaže, da je Rumpelsvoja pomočniška le na Dunaju, kjer je izdelal tudi klavir, ki po kvaliteti in mehaniki presega p mojstre tistega časa. S tem inštrumentom se je zapisal med redke izdelova jev na Slovenskem. Rumpel se je rodil in umrl v Kamniku, kjer je tudi po štiridesetih letih delovanja njegove orglarske delavnice je slovel kot odli občasno pa je popravljal tudi klavirje. Znano je, da je delavnico zasnoval nekega večjega podjetja. Dobro organizirano delo je teklo izredno hitro omare in mehaniko so izdelovali po določenih šablonah, vendar na sol Uspehu Rumplove delavnice, ki je bila v svojem okolju skorajda brez ko pripisujejo tudi ugodno lego delavnice, ki je bila v bližini kvalitetnih gozdo tirane orgle, največ jih je v Ljubljani ter v dekanijah Kranj, Kamnik, Litija, M Ljubljana z okolico, potrjujejo nekdanji sloves delavnice (Cerklje na Go Spodnji in Zgornji Tuhinj, c. sv. Valentina na Limbarski gori, Motnik, Hom pri Kamniku, Hotič, Čadež ob Savi, Dole, Češnjica, Ihan, Izlake, Svibno, Gora, Šmartno pod Šmarno goro, Ježica, Preska, Prežganje, Dolsko, Nov Skaručna, Šinkov Turn, Škofja Loka, Šmarjeta, Zali Log, Dobrnič). Na Hrv MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA evidentirane 4 orgle (Zagreb, c. sv. Katarine, 1830, 12 registrov; Mošče 1847, 12 registrov; Grobnik, 1848, Trsat nad Reko, 1850- danes ne obsto Avstriji je l. 1858 izdelal orgle pri Sv. Margareti v okolici Sv. Paula v Lab (St. Paul im Lavantthal), kar mu je prineslo naročilo za tamkajšnjo samo kev (1859), ki je bilo njegovo zadnje in največje delo (25 registrov). V delavnici je delalo precej pomočnikov, ki so kasneje postali samostojni o njimi sta bila Janez Mandlin (1818t1891) in Franc Goršič (1836t1898) delavnici sta se izučila tudi dva Petrova sinova, August in Johann. SALB, Josef Qosip), (ok. SALB, Josef Qosip), (ok. SALB, Josef Qosip), (ok. Orglar v Mariboru. V 40. in 50. letih 19. st. je bil edini orglar v Ma nadaljeval tradicijo Josepha Otoniča (1767t1835), pri katerem se je najve izučil. Po dispozicijah je bil predstavnik klasicizma. V orglarski obrti se izkazal. Najbolj je poznan po popravilih in uglaševanju orgel po Štajerske lovalec novih inštrumentov je zabeležen le dvakrat (Selnica ob Dravi, 1 Ormožu, 1853). Po njegovi smrti je obrt dedoval sin Joseph (1828), ki pa stvom ni ukvarja in je l. 1859 obrt opustil. Literatura: BIZJAK/ŠKULJ 1985, 116; ŠKU RUPNIK, Mihael ( Orglar v Pristavi pri Celju. Deloval je v času med letoma 1869 in 1885 ne so 3 orgle (Sv. Filip pri Olimju, 8 registrov; Zibika pri Šentjurju pri C strov; Vuzenica, 13 registrov), l. 1879 pa je povečal orgle na Pesku pri Po Literatura: BIZJAK/ŠKULJ 1985, 50, 158. Orglar v Pristavi pri Celju. Deloval je v času med letoma 1869 in 188 ne so 3 orgle (Sv. Filip pri Olimju, 8 registrov; Zibika pri Šentjurju pri Orglar v Pristavi pri Celju. Deloval je v času med letoma 1869 in 1885 ne so 3 orgle (Sv. Filip pri Olimju, 8 registrov; Zibika pri Šentjurju pri C strov; Vuzenica, 13 registrov), l. 1879 pa je povečal orgle na Pesku pri Po Literatura: BIZJAK/ŠKULJ 1985 50 158 ne so 3 orgle (Sv. Filip pri Olimju, 8 registrov; Zibika pri Šentjurju pri C strov; Vuzenica, 13 registrov), l. 1879 pa je povečal orgle na Pesku pri Po Literatura: BIZJAK/ŠKULJ 1985, 50, 158. RUMPEL (Rumpl), Peter (1 Prvi j časa (1868) samostojno orglarsko obrt na Dunaju, Johann pa je bil najpre do ok. 1858 pomočnik pri uglednem dunajskem mojstru Carlu Hesseju (* kasneje je postal naslednik Carla Friedricha Buckowa (1801), po krajšem Madžarskem pa je imel dve leti na Dunaju samostojno obrt (1870--1872) Naslov. Kamnik, Šutna (1 Naslov. Kamnik, Šutna (1 Literatura: EBERSTALLER 1955, 134, 214; ŠABAN 1979, 33, 39; BIZJAK/Š 106, 110, 112, 130, 182; ŠKULJ 1994, 130, 132; HOPFNER 1999, 411; KO 108-109, 127-129. Literatura: EBERSTALLER 1955, 134, 214; ŠABAN 1979, 33, 39; BIZJAK/ 106, 110, 112, 130, 182; ŠKULJ 1994, 130, 132; HOPFNER 1999, 411; K 108-109 127-129 Literatura: EBERSTALLER 1955, 134, 214; ŠABAN 1979, 33, 39; BIZJAK/Š 106, 110, 112, 130, 182; ŠKULJ 1994, 130, 132; HOPFNER 1999, 411; KO 108-109, 127-129. Inštrumenti: Grad Tuštanj pri Moravčah, klavir zasebna last 108-109, 127-129. Inštrumenti: Grad Tuštanj pri Moravčah, klavir, z Inštrumenti: Grad Tuštanj pri Moravčah, klavir, z STRČ>LIN, Johann (16 Orglar in izdelovalec drugih inštrumentov v Ljubljani. Bil je komorni str ljanskega škofa Tomaža Hrena, ki naj bi mu služil v času med letoma 1606-11 jetneje se je učil pri orglarju Vincenzu Colonnu v Benetkah. Leta 1611 je orgelski rog na stolp na ljubljanski grad, za kar je prejel plačilo, nato je na pr škofa Hrena odšel iz Ljubljane v drugo službo. Literatura: NAL, Gornji Grad A - f 79, Korespondenca škofa Tomaž 1597-1629; LAVRIČ 1988, 95, 354 (op. 733); KOTER 2001, 96. UNGLERTH (Ungelerth, Ungeleerth, Ungelert), Simon (1778t14. 11. 18 Strugar in izdelovalec inštrumentov v Ljubljani. Meščanske pravice je do STEINHOFFER (Steinhofer), Johann Michael (17 Orglar v Ljubljani. Po rodu je bil iz Westfalije. Obrtni davek je plačeval v letoma 1723-1729. Pripisujejo mu orgle v cerkvi v Mekinjah pri Kamniku (17 1727 je popravljal orgle v ljubljanski stolnici. Orglar v Ljubljani. Po rodu je bil iz Westfalije. Obrtni davek je plačeval v č letoma 1723-1729. Pripisujejo mu orgle v cerkvi v Mekinjah pri Kamniku (17 1727 je popravljal orgle v ljubljanski stolnici. Literatura: Publikacije ZAL 0972), zv. 3,105; ŠKULJ Literatura: Publikacije ZAL 0972), zv. 3,105; ŠKULJ SCHWEIZER, Jos Mizar in izdelovalec klavirjev iz Ljubljane. Deloval je v prvi polovic Meščanske pravice je dobil 29. l. 1808 kot mizar. Iz časopisnih oglasov v znano, da je izdeloval tudi klavirje. Ljubljanski knjigarnar Janez Giontini, ukvarjal tudi s prodajo inštrumentov, je l. 1852 preko oglasa ponujal Schw "star•, dobro ohranjen klavir s šestimi oktavami, kakršni so bili značilni za dru letje 19. st. Literatura: ZAL, Imenik meščanov stolnega mesta Ljubljane od 1786 767; KOTER 2001, 124. Literatura: ZAL, Imenik meščanov stolnega mesta Ljubljane od 1786-18 767; KOTER 2001, 124. 767; KOTER 2001, 124. Časopis. Kmetske in rokodelske novice, 1852, št. 54, Oglasn Časopis. Kmetske in rokodelske novice, 1852, št. 54, Oglasn Časopis. Kmetske in rokodelske novice, 1852, št. 54, Oglasnik STEINHOFFER (Steinhofer), Johann Michael (172 STEINHOFFER (Steinhofer), Johann Michael (17 MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUAL strov) v Janečkovem slogu. Nekaj jih je ohranjenih v manjših krajih na H (Dol Pribički, 1782; Poljanica Bistranska, 1784; Gornja Bistra, grad, ok. 1784 1785; Gotalovec, 1787; Novaki, 1794; Nedjelja, 1799). Večje orgle s 14 reg frančiškanski cerkvi v Kostanjki (Hrvaška), 1797, na Slovenskem pa so zn Hrovači pri Ribnici na Dolenjskem (1782). Z njegovo smrtjo se je v krogu orglarjev končalo tudi baročno orglarstvo. g j g Literatura: ŠABAN 1979, 21, 25, 39; BIZJAK/ŠKULJ 1985, 76, 88; ŠKULJ SCHOLZ, Anton ( Orglar v Celju. Deloval je na Slovenskem in Hrvaškem. Sodi v t. im. orglarjev, ki ga je na začetku 18. st. utemeljil Čeh Janez FrančišekJaneček ok. 1777). Scholz je ok. l. 1782 prevzel Janečkovo delavnico in nadalje češkega sloga. Kot izdelovalec orgel (»Orgel Macher") se je pojavljal med in 1785, nato pa je dobil meščanske pravice kot orglar (»Burger und Orge Cilli•). V zakonu z Elizabeto je imel dva sinova, ki sta zgodaj umrla in j potomcev. Umrl je za tuberkulozo pri šestinštiridesetih letih. Delavnic Čeh Venceslav Marthal (pred 1780t po 1825), ki se je poročil z vdov Znanih je okrog 10 orgel Antona Scholza, med katerimi je večina pozitivo Literatura: ZAL, Knjiga prejemkov in izdatkov 1581-1731, Cod. XIII/ fol. 36, Cod. XIII/224 (1707), fol. 39; NAZ, Acta Capit. Ant., fasc. 101/6 blikacije ZAL (1972), zv. 2, 105; ŠABAN 1979, 16-18; KOTER 2001, 98-99 VRABEC, Janko ( Orglar iz Mokronoga na Dolenjskem. Deloval je v okolici domačega k imel delavnico. Izdeloval je le manjše orgle (Gabrijele, 1870, Dolenj Drušče), ki kažejo za tisti čas zastarelo klasicistično smer. Izučil se je Franca Ksaverja Deva (1834t1872) v Ljubljani, ki je bil v svojem času n uspešen orglar klasicistične smeri. Literatura: BIZJAK/ŠKULJ 1985, 52, 130; ŠKULJ 199 Literatura: BIZJAK/ŠKULJ 1985, 52, 130; ŠKULJ 199 VALENTINČIČ, Anton ( Davče Ž VALENTINČIČ, Anton ( Davče Ž Orglar iz Davče pri Železnikih. Deloval je v času med letoma 1840 in je predvsem popravljal. Znani so le štirje njegovi inštrumenti, in sicer v ok (Preddvor, Kalobje, Bukovščica, Lesce), ki so bili za tisti čas že zastarel baročnem slogu. Literatura: ŠKU MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA 1801 kot izdelovalec inštrumentov (»Instrumentenmacher"). Uvrščamo ga membnejše izdelovalce lesenih pihal na Slovenskem. Izdeloval je flavt čakane, basetne rogove in flažolete. Ohranjeni inštrumenti ga povezuje odličnih dunajskih mojstrov njegovega časa. Rojstni kraj ni znan, umrl Ljubljani. Leta 1803 se je poročil z Lucijo Schlechter, s katero je imel sina Johanna (23. 9. 1811), ki je zgodaj umrl. Simon Unglerth je bil uspešen potrjuje lastništvo več hiš v Ljubljani. Na obrtno-industrijski razstavi v l. 1844 prejel bronasto medaljo. Naslov-. Ljubljana, Kapucinsko predmestje št. 40 in 41 ( Naslov-. Ljubljana, Kapucinsko predmestje št. 40 in 41 ( Literatura: ZAL, Conscriptions Aufnams Bogen der Kapuziner Vorsta 1830, Bogen 17, šk. 34; Publikacije ZAL 0977), zv. 4, 78-82, 84; BET KOTER 2001, 131, 150-156. Jnštrumentarij: K0benhavn, Musikhistorisk Museum og Carl Claudius, št. MMCCS CI 427; New York, Metropolitan Museum, The Crosby Brown basetni rog, inv. št. NY MM, 89.4.2143; Ptuj, Pokrajinski muzej Ptuj, flavta, i S, klarinet, inv. št. GL 18 S, del flažoleta, inv. št: GL 64 S, del flažoleta, inv. ŠTRUKELJ, Grego Orglar in organist v Zagrebu (Hrvaška). Po rodu izvira iz slovenske Šta Zagrebu je omenjen l. 1621 kot popravljalec orgel, sedem let kasneje kot sto nist in orglar, l. 1647 pa je izdelal orgle s 13 registri za zagrebško katedralo l. 1690 je njegov inštrument povečal orglar iz Ljubljane Johann Faller (pred 1717). Literatura: ŠABAN 1979, 15 ; MEDER 1 Literatura: ŠABAN 1979, 15 ; MEDER 1 WALLENSTEINER, Andreas Sebastian (pred Orglar v Ljubljani. Meščanske pravice je dobil l. 1702. Leta 1703 in 170 ljal orgelski rog na ljubljanskem gradu. V knjigi izdatkov je podpisan ko orglar („Burger und Orgelmacher"). V Ljubljani je živel do l. 1708. Tega le sal pogodbo za izdelavo orgel z 12 registri za frančiškansko cerkev v Zag ni končal, saj je l. 1709 med delom umrl in je pokopan v Zagrebu. Ohran zicija za omenjene orgle kaže, da bi bil lahko učenec Johanna Fallerja ( po 1717), orglarja iz Ljubljane. Literatura: ZAL, Knjiga prejemkov in izdatkov 1581-1731, Cod. XIII fol. 36, Cod. XIII/224 (1707), fol. 39; NAZ, Acta Capit. Ant., fasc. 101/6 blikacije ZAL (1972), zv. 2, 105; ŠABAN 1979, 16-18; KOTER 2001, 98-9 MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA Lastnik tovarne in trgovine klavitjev C>·Klavier Fabrik und Handlung•) v Najprej je deloval v Trstu, kjer je l. 1894 skupaj z Giuseppejem Schandlom firmo „Germania· („Pinoforte Gesellschaft / Germania•), ki je bila registr imenom »]. Schandl & R. Warbinek•. Izdelovali so klavirje, trgovali pa s harmoniji („Klavier-Fabrik, Klavier u.(nd) Harm.(onium) Handlung•. Leta Rudolf Warbinek preselil v Ljubljano in odprl tovarno za izdelovanje klav lastnim imenom. P. de Witt je to delavnico označil kot »prvo tovarno kla Kranjskem• (»Erste Krainische Pianoforte - Fabrik·). Po drugi svetovni vo Warbinekova tovarna nacionalizirana, stroje in orodje pa so prepeljali v tov bil v Mengšu, ustanovljeno l. 1946. Naslov. Trst, Via Felice Venecian 13 (1894); Ljubljana, Hilschergasse 5 (1 Naslov. Trst, Via Felice Venecian 13 (1894); Ljubljana, Hilschergasse 5 (189 Literatura: WITT de 1912, 350; MANTUANI 1932; PUNGARTNIK 1990 TER 2001, 124, 133. Literatura: BIZJAK/ŠKULJ 1985, 154; CG 81 (1988 WITTENZ (Bitenc), Andreas (Andrej) (18 WITTENZ (Bitenc), Andreas (Andrej) (18 Izdelovalec, uglaševalec in trgovec s klavirji v Ljubljani. Za obrtne prav prosil l. 1835, vendar je občinski odbor, pristojen za dodeljevanje meščans nih pravic, presodil, da njegova uveljavitev ljubljanskemu meščanstvu ne korist. Izučil se je v neznani delavnici na Dunaju, njegovi izdelki pa kažejo dober mojster. Ohranjena sta dva klavitja. Prvega hrani Narodni muzej v (ok. 1835), drugi pa je del zbirke glasbil v Pokrajinskem muzeju Ptuj (1856 Leta 1841 je razstavljal na obrtno-industrijski razstavi v Gradcu, l. 1844 pa v kjer je za svoj izdelek prejel srebrno odličje. Naslov. Ljubljana, Mesto št. 10 (»Stadt, Haus Nummer 1 Literatura: ZAL, Conscriptions Aufnahms Bogen von N. 1-100, šk. 29 (1) cije ZAL (1977), zv. 3, 67; MANTUANI 1932; KOTER 2001, 130-132. Literatura: ZAL, Conscriptions Aufnahms Bogen von N. 1-100, šk. 29 (1) cije ZAL (1977), zv. 3, 67; MANTUANI 1932; KOTER 2001, 130-132. Literatura: ZAL, Conscriptions Aufnahms Bogen von N. 1-100, šk. 29 (1) cije ZAL (1977), zv. 3, 67; MANTUANI 1932; KOTER 2001, 130-132. Inštrumenti: Ljubljana, Narodni muzej Slovenije, Oddelek za zgodovin rabno umetnost, inv. št. 21619; Ptuj, Pokrajinski muzej Ptuj, inv. št. GL 105 Inštrumenti: Ljubljana, Narodni muzej Slovenije, Oddelek za zgodovin rabno umetnost, inv. št. 21619; Ptuj, Pokrajinski muzej Ptuj, inv. št. GL 105 Inštrumenti: Ljubljana, Narodni muzej Slovenije, Oddelek za zgodovin rabno umetnost, inv. št. 21619; Ptuj, Pokrajinski muzej Ptuj, inv. št. GL 105 g j ( ) Orglar v Kropi. Velja za samouka. Deloval je v osrednji Sloveniji. Rod Kropi, kjer je tudi umrl. Izdeloval je manjše orgle (do 10 registrov) s kla dispozicijo. Prva leta je deloval sam (Javorje, Kropa, Koroška Bela, Ljubljan Šentjošt nad Horjulom, Lipoglav), l. 1880 je s sinovoma Ignacijem ml. (1853 Ivanom (1857t1900) ustanovil družinsko podjetje „Ignacij Zupan in sinovi•, p smrti pa se je podjetje preimenovalo v podjetje „Bratov Zupan•. Sinova sta p začela izdelovati sodobnejše inštrumente z romantično dispozicijo. Pod ime pan· je bilo izdelanih 127 orgel, kar je bila do takrat največja proizvodnja območju nekdanje Slovenije. Literatura: BIZJAK/ŠKULJ 1985, 154; CG 81 (1988 Literatura: ŠABAN 1979, 34, 40; BIZJAK/ŠKULJ 1985, 154; CG 81 09 MUZIKOLOŠKI ZBORNIK • MUSICOLOGICAL ANNUA ZUPAN, Ignacij ml. (21. 7. 1853 ZUPAN, Ignacij ml. (21. 7. 1853t Orglar v Kropi in Kamni Gorici. Deloval je v Sloveniji in na Hrvaškem je kot prvi orglar na Slovenskem uvedel sapnice na stožce, romantično d pnevmatsko trakturo. Rodil se je v Kropi, umrl pa v Kamni Gorici. (1825t1888) je bil orglar samouk. V domači delavnici se je skupaj z bra (1857t1900) izučil za orglarja. V poznih 70. letih 19. st. se je izpopolnjev Bratov Rieger v Jaegrndorfu v Šleziji (danes Krnov, Češka). Leta 1880 je o ma ustanovil podjetje „Ignacij Zupan in sinovi". Tega leta so Zupani preš vanje takrat sodobnih romantičnih orgel po vzoru podjetja Rieger ter najpomembnejše orglarsko podjetje na Slovenskem. Po očetovi smrti sta nico preselila v Kamno Gorico in jo preimenovala v »Brata Zupan", ki s tudi po Ivanovi smrti l. 1900. Ivan Zupan (*9. 12. 1857 v Kropi t4. 7. 1 Gorici) je v podjetju vodil dokumentacijo, bil pa je tudi organist in sklad nih skladb. Pod imenom „zupan" je bilo skupaj izdelanih 127 orgel. M slovenskih cerkvah si sledijo po velikosti: Poljane nad Škofjo Loko, Ig Bovec, Kranj, Bohinjska Bistrica, Maribor, Naklo, Šentilj, Radmirje, Sora, ta Gora pri Litiji, Gorje, Štorje, Dravograd, Dob in Sostro pri Ljubljani, Lju Od celotnega opusa je evidentiranih ok. 65 del, med njimi jih je na širš Hrvaške 15 (Reka, Omišalj, Drniš, Opatija, Čepič, Lovran, Gračišče, Žm Bogoviči, Roč, Šibenik, Volosko, Kringa, Lindar). Podjetje »Brata Zupan" mi slovenskimi orglarji, ki so zastopani na Hrvaškem, izdelalo največje š Literatura: ŠABAN 1979 34 40; BIZJAK/ŠKULJ 1985 154; CG 81 09
https://openalex.org/W2801042274	https://europepmc.org/articles/pmc5960530?pdf=render	English	null	Assessment of the Isoniazid Preventive Therapy Uptake and Associated Characteristics: A Cross-Sectional Study	Tuberculosis research and treatment	2,018	cc-by	7,221	Hindawi Tuberculosis Research and Treatment Volume 2018, Article ID 8690714, 9 pages https://doi.org/10.1155/2018/8690714 Hindawi Tuberculosis Research and Treatment Volume 2018, Article ID 8690714, 9 pages https://doi.org/10.1155/2018/8690714 1Department of Epidemiology and Biostatistics, School of Public Health of the College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda 1Department of Epidemiology and Biostatistics, School of Public Health of the College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda 2Faculty of Community and Health Sciences, University of Western Cape, Cape Town, South Africa 3Centre for International Child Health, University of Melbourne Department of Paediatrics and Murdoch Children’s, Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia 4International Union against Tuberculosis and Disease, Paris, France Correspondence should be addressed to Francine Mwayuma Birungi; fbirungi@nursph.org Received 11 August 2017; Revised 2 February 2018; Accepted 25 March 2018; Published 6 May 2018 Academic Editor: Vincent Jarlier Copyright © 2018 Francine Mwayuma Birungi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. To assess the uptake of isoniazid preventive therapy (IPT) by eligible children in Kigali, Rwanda, and associated individual, households, and healthcare systems characteristics. Methods. A cross-sectional study was conducted among child contacts of index cases having sputum smear-positive pulmonary tuberculosis. Data were collected from 13 selected primary health centres. Descriptive statistics were used to generate frequency tables and figures. Logistic regression models were performed to determine characteristics associated with IPT uptake. Results. Of 270 children (under 15 years), who were household contacts of 136 index cases, 94 (35%) children were less than 5 years old and eligible for IPT; and 84 (89%, 95% CI 81–94) were initiated on IPT. The reasons for not initiating IPT in the remaining 10 children were parents/caregivers’ lack of information on the need for IPT, refusal to give IPT to their children, and poor quality services offered at health centres. Factors associated with no uptake of IPT included children older than 3 years, unfriendly healthcare providers, HIV infected index cases, and the index case not being the child’s parent. Conclusion. The National Tuberculosis Program’s policy on IPT delivery was effectively implemented. Future interventions should find strategies to manage factors associated with IPT uptake. Francine Mwayuma Birungi ,1,2 Stephen Graham,3,4 Jeannine Uwimana,1,2 and Brian van Wyk2 1Department of Epidemiology and Biostatistics, School of Public Health of the College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda 2Faculty of Community and Health Sciences, University of Western Cape, Cape Town, South Africa 3Centre for International Child Health, University of Melbourne Department of Paediatrics and Murdoch Children’s, Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia 4International Union against Tuberculosis and Disease, Paris, France 1Department of Epidemiology and Biostatistics, School of Public Health of the College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda 2Faculty of Community and Health Sciences, University of Western Cape, Cape Town, South Africa 2. Methodology 2.1. Site Selection. A cross-sectional study was conducted at 13 primary health centres (PHCs) providing TB diagnostic and treatment services in Kigali. Kigali, the capital city of Rwanda, reports the highest prevalence of TB in Rwanda and around 30% of Rwanda’s total pulmonary TB (PTB) cases [9]. Thus, Kigali was selected as case study site. There are 35 primary health centres (PHCs) in Kigali whence 23 PHCs provide TB diagnostic and treatment services and represent entry points for TB cases. The criterion used to select 13 PHCs from the 23 PHCs providing TB diagnostic and treatment services was a record of at least 10 sputum smear-positive PTB cases report- ed between January and June 2015. Among the 13 PHCs se- lected for this study, nine (69%) were public-funded and four were faith-based (public and private funded). Of the 13 PHCs, three had two staff members, and ten (77%) had one staff member, working in TB services. All the staff members were trained in TB management and provided counseling to parents/caregivers on IPT before their children started the regimen. In Rwanda, medication for TB is provided free of charge. Moreover, all TB index cases are offered the oppor- tunity to choose the nearest healthcare facility or community health worker they wish to receive TB treatment or IPT. The uptake of IPT is defined as the proportion of children eligible to receive IPT according to the WHO recommen- dation [12]. Screenings conducted during this study identified all children as eligible for IPT according to the WHO recom- mendation [12]. To assess the uptake of IPT, every par- ent/caregiver of an eligible child was asked whether the child was initiated on the IPT or not. The IPT register at PHCs assisted to verify the information given by parents/caregivers. The sociodemographic characteristics and medical history of index cases, such as a result of smear microscopy, HIV status, residential address, and telephone number, were recorded. The data collected were validated by the index cases, parents, or caregivers of selected children once they were identified to ensure their accuracy. All children underwent a history, phys- ical, and chest X-ray (CXR) examination. Anteroposterior and lateral CXR were also performed on all children; they were read by two independent experienced general prac- titioners, trained in interpreting CXR and blinded to the clinical details of participants; an experienced radiologist verified all CXR to rule out any discordance. 1. Introduction most widely recommended regimen is isoniazid preventive therapy (IPT) that is provided as a daily dose for at least 6 months. Notwithstanding the potential benefits of contact screening for active case detection and initiation of IPT, these activities are rarely implemented in TB endemic settings [5, 6]. Furthermore, even when IPT is offered to eligible children, its uptake is often poor [7, 8]. In 2015, the World Health Organisation (WHO) estimates showed that 10% of the 9 million tuberculosis (TB) incident cases occurred in children, which resulted in 210,000 TB- related deaths including 170,000 in HIV-negative children [1]. Children exposed to index cases with TB, particularly sputum smear-positive pulmonary TB (PTB), are at risk of infection and when infected, infants and young children (<5 years old) are at high risk of developing the disease [2, 3]. WHO recom- mends routine screening of child contacts in resource-limited settings through a symptom-based screening approach that can be implemented in the community and provision of pre- ventive therapy for at-risk contacts after excluding TB [4]. The t Rwanda is a TB endemic resource-limited country, which had an estimated 6.6 [95% IC: 5.6–7.6] thousand new TB cases in 2015 [1]. According to the TB surveillance system, 5,763 TB cases, including 68.1% of new confirmed bacteriological pul- monary TB cases and 85.3% of sputum smear-positive PTB cases, were reported in the period between 2015 and 2016. Children under 15 years old represented 5.3% of all TB cases Tuberculosis Research and Treatment 2 reported [9]. The active contact screening and IPT are recom- mended by the National TB Programme (NTP) in Rwanda but TB case detection strategy is limited to passive screening. Although guidelines for IPT have been in existence since 2005, their implementation has not been assessed. For a few years, particular attention has been given to TB in children by Rwanda’s NTP since TB treatment is recognized as an oppor- tunity that prevents and addresses an important cause of child mortality [10, 11]. The NTP strategy has promoted the uptake and adherence of IPT as one of the 30 performance indicators since 2009. This paper reports about the uptake of IPT by eligible children in Kigali, the capital city of Rwanda, and evaluates the associated individual, households, and health- care systems characteristics. by parents or primary caregivers. 1. Introduction Ineligible child contacts included those born after the index cases were diagnosed and initiated on TB treatment, child contacts on TB treatment, and those that were not living in the same household with the index cases before the diagnosis. Moreover, child contacts older than 5 years, including those infected with HIV, were also excluded in accordance with Rwanda’s NTP policy. 2.3. Data Collection and Management. Data were collected in twofold, from patients’ records at the PHC facilities and from parents/caregivers of eligible child contacts interviewed by trained enumerators using a structured questionnaire. The questionnaire was developed, pretested during a pilot study in two selected sites, and modified for later use in data collection. Twelve selected data enumerators were trained to conduct interviews with parents/caregivers of selected child contacts. Additionally, 20 CHWs were trained to identify and enumerate all eligible children in the households as well as explain the study to parents/caregivers and sensitize them to take child contacts to the nearest PHCs for clinical evaluation and data collection. Furthermore, by screening each child and interviewing each parent/caregiver, data enumerators ensured that the child contact was eligible or otherwise excluded even if he/she has been declared eligible by CHWs.hi 3. Results Figure 1 shows the flow of recruitment of child contacts with 136 (39%) of 346 sputum smear-positive PTB index cases diagnosed and treated at 13 PHCs in Kigali during the 7- month study period. The index cases had at least one child contact aged between 0 and 14 years. Of the 136 index cases, 72 (53%) had at least a child contact who met the inclusion criteria. Of 94 (35%) children from 72 index cases who were eligible, 84 (89%) had started IPT. 2.4. Data Analysis. The data collected were double-entered into a Microsoft Excel worksheet and exported to Stata Soft- ware for statistical analysis after checking their consistency. The data were analyzed using descriptive and multivariate statistics. Continuous variables were dichotomized using the median as the cut-off. Categorical variables were described using frequency tables and proportionate methods. Univari- ate and multivariate logistic regression was performed to determine characteristics associated with IPT uptake. Where appropriate, Chi-square test or Fisher’s exact test was per- formed to assess the association between two variables. Vari- ables with a 𝑝value < 0.2 in univariate analysis were included in the logistical regression model using backward stepwise method. The final model included those factors that retained statistical significance. The odd ratios (OR) and adjusted OR (aOR) along with its 95% confidence interval were calculated using Stata Software (version 13). The associations were declared significant if 𝑝value < 0.05. Tables 1, 2, and 3 show the characteristics of the eligible index cases, their households, and child contacts by IPT uptake, respectively. The results show no significant differ- ence between children who started IPT and those who did not with regard to the characteristics of index cases (Table 1) and healthcare facilities (that are unfriendly versus friendly and satisfaction level of parents/caregivers on the quality of services offered). There were significant differences between children who started IPT and those who did not with regard to the sex of the head of the household (13/62 versus 5/10, 𝑝= 0.049) (Table 2) and the age of the child contact (≤3 versus >3 years, 𝑝= 0.007) (Table 3). Children living in households headed by female were more likely not to be initiated on IPT than those living in households headed by a male. Also, children aged > 3 years old were more likely not to be initiated on IPT than those ≤3 years old. 2. Methodology The components of all reports were agreed on before a final diagnosis was determined. Symptomatic children are treated with antibi- otics for seven days as recommended by the current TB diag- nostic algorithm in the country. Child contacts with persis- tent TB-related symptoms or abnormal CXR were referred to the District Hospital for further tests including smear microscopy, Xpert MTB/RIF assay, and solid culture using sputum collected through gastric lavage following the stan- dard procedure [13] in order to exclude TB disease.h 2.2. Study Population. This study was conducted among household contacts of index cases with sputum smear-posi- tive PTB in 13 selected PHCs from 1 August 2015 to 29 Febru- ary 2016. The criteria for selecting an index case of any gender were as follows: having at least one child under the age of 5 years, not belonging to a household having a previously selected index case, and providing proof of living in Kigali during the period of study. Identified index cases were re- quested, through either phone conversations or trained com- munity health workers (CHWs), to bring their children to the nearest PHC on a specific day that coincided with data enu- merators’ visits to the PHC. In case the index cases were not parents/caregivers of the children needed at the PHCs, they were requested to inform those children’s parents/caregivers to do so. The interview with eligible children’s parents/caregivers identified the sociodemographic and economic character- istics of the index cases, their households, and knowledge on how to prevent TB in child contacts. A parent/caregiver was considered knowledgeable about, first, IPT prevention if he/she knew that administering INH for 6 months would Eligible child contacts were aged below 5 years old and shared the same household with a selected index case within 3 months prior to the diagnosis of the latter. 2. Methodology The children were enrolled following the signing of written informed consent Tuberculosis Research and Treatment 3 176/270 child contacts not eligible for IPT: 17 children were not living with index case within 3 months prior to the diagnosis; 37 under ﬁve years old child contacts born afer diagnosis of index cases; and 122 living with index case within 3 months prior to the diagnosis, but ≥5 years old Child contacts who started IPT = 84 (89%) Child contacts who did not Start IPT = 10 (11%) Index with child contact who met the inclusion criteria = 72/136 (53%) Number of child contacts who met the inclusion criteria (<5 years old) and screened for TB 94/270 (35%) (39%). 270 child (<15 years) contacts Index cases with child contacts = 136/346 Number of Pulmonary TB cases sputum positive = 346 Figure 1: Flow of recruitment of child TB contacts. 176/270 child contacts not eligible for IPT: 17 children were not living with index case within 3 months prior to the diagnosis; 37 under ﬁve years old child contacts born afer diagnosis of index cases; and 122 living with index case within 3 months prior to the diagnosis, but ≥5 years old (39%). 270 child (<15 years) contacts Index cases with child contacts = 136/346 Index with child contact who met the inclusion criteria = 72/136 (53%) Number of child contacts who met the inclusion criteria (<5 years old) and screened for TB 94/270 (35%) Figure 1: Flow of recruitment of child TB contacts. Review Board of the University of Rwanda, College of Medi- cine and Health Sciences, approved the study protocol. Per- mission was obtained from Rwanda NTP to collect data from the eligible PHCs. protect the child contacts against TB. Second, prevention of child contacts from contracting TB if he/she knew three of the following pieces of information: using a mask when breast- feeding, avoiding to kiss him/her, avoiding to sleep in the same room or bed with him/her, opening windows and doors for good ventilation, and using arm protection when cough- ing. The interview with eligible children’s parents/caregivers also determined characteristics of the healthcare facility such as quality of services provided and the attitude of health providers towards patients. Those characteristics were assessed by asking parents/caregivers the level of satisfaction with the quality of services offered at the PHCs and whether the healthcare service providers were friendly. 3. Results Tuberculosis-related symptoms such as coughing, fever, and weight loss were reported by 25% (23/94) of child contacts, and those cases reported responded to generic antibiotics recommended by the current TB diagnostic algorithm in the country. Neither 2.5. Ethical Approval. The Biomedical Research Ethics Com- mittee of the University of the Western Cape and the Ethics Tuberculosis Research and Treatment Tuberculosis Research and Treatment 4 Table 1: Characteristics of the index cases of the child contacts by uptake of IPT. Table 1: Characteristics of the index cases of the child contacts by uptake of IPT. Table 1: Characteristics of the index cases of the child contacts by uptake of IPT. Characteristics Index cases (𝑁= 72) (%) Index case whose children took IPT (𝑁= 62) (%) Index case whose children did not take IPT (𝑁= 10) (%) 𝑝value Age group 0.716 ≤35 years old 39 (54) 34 (55) 5 (50) >35 years old 33 (46) 28 (45) 5 (50) Sex, female 33 (46) 28 (45) 5 (50) 0.776 Residence of the index case 0.641 Nyarugenge 12 (17) 11 (18) 1 (10) Kicukiro 20 (28) 16 (26) 4 (40) Gasabo 40 (55) 35 (56) 5 (50) Type of health facility used by index cases 0.262 Public 51 (71) 42 (68) 9 (90) Faith-based 21 (29) 20 (32) 1 (10) Marital Status 0.243 Never married 11 (15.5) 8 (13) 3 (30) Married 50 (69) 45 (73) 5 (50) Separated 11 (15.5) 9 (14) 2 (20) Index case Head of household 29 (40) 25 (40) 4 (40) 0.985 Index case tested for HIV 61 (85) 52 (84) 9 (90) 0.617 Result of HIV test Positive 18/61 (30) 13/52 (25) 5/9 (56) 0.063 IPT = Isoniazid Preventive Therapy; HIV = human immunodeficiency virus. years), HIV status of child contacts (HIV-positive versus HIV-negative), relationship between child contacts and index cases (child versus others), HIV status of index case (HIV- positive versus HIV-negative), household income (income < 50.000 Rwandan Franc versus ≥50.000 Rwandan Franc), and relationship between healthcare providers and par- ents/caregivers (friendly versus unfriendly). After adjust- ing the variables, the age of child contacts and relation- ship between healthcare providers and parents/caregivers remained associated with the uptake of IPT. Child contacts older than 3 years were more likely not to be initiated on IPT than those less than 3 years old (aOR 29, 95% CI 2.17–400; 𝑝< 0.011). 3. Results Moreover, the child contacts whose parents/care- givers found healthcare providers unfriendly were also more likely not to be initiated on IPT than those whose parents/ caregivers found them friendly (aOR 19, 95% CI 2.51–140; 𝑝< 0.017). The HIV status of index cases and the relationship between child contacts and index cases were also associated with no uptake of IPT in multivariate analysis. Child contacts living with HIV-positive index cases were more likely not to be initiated on IPT than those living with HIV-negative ones (aOR 8.1, 95% CI 2.53–537; 𝑝< 0.038). Furthermore, the child contacts who were not children of index cases were more likely not to be initiated on IPT than those who were index cases’ children (aOR 59, 95% CI 2.74–127; 𝑝< 0.009). of the children screened presented with an abnormal CXR nor a diagnosed TB disease [12, 14]. The majority of par- ents/caregivers of child contacts (66/72, 92%) had knowledge of at least one method on how to prevent transmission of TB to children and 32% (23/72) had knowledge of IPT prevention (Table 2). All the 94 eligible child contacts were screened for TB by PHC providers whence 84 (89%, 95% CI 81–94) were initiated on IPT for 6 months as per the national and WHO guidelines [12, 14]. The reasons given by parents/caregivers for not initiating child contacts on IPT were lack of awareness of the need to do so (5/10 or 50%); failure to initiate IPT (4 or 40%); and poor healthcare service experienced at the PHC (10%). In univariate analysis, the age of child contacts, sex of the household head, and relationship between healthcare provid- ers and parents/caregivers were associated with the uptake of IPT (Table 4). Child contacts over 3 years old were more likely not to be initiated on IPT than those who were below 3 years old (OR 7, 95% CI 1.65–29; 𝑝< 0.008). Children living in households headed by a female were more likely not to be initiated on IPT than those living in households headed by a male (OR 4.6, 95% CI 1.18–17.9; 𝑝< 0.028). Child contact whose parents/caregivers did not find friendly healthcare providers at the PHCs were also more likely not to be initiated on IPT than those whose parents/caregivers did. (OR 10, 95% CI 1.26–83; 𝑝< 0.029). 4. Discussion In multivariate analysis, the sex of households’ head had no significance. The final explanatory variables of no uptake of IPT were age group of child contacts (≤3 years versus >3 The primary aim of this study was to assess the uptake of IPT by child contacts and associated factors in order to Tuberculosis Research and Treatment 5 Table 2: Characteristics of the household of child contacts by uptake of IPT. Characteristics Total (𝑁= 72) (%) Child contact started IPT (𝑁= 62) (%) Child contact did not start IPT (𝑁= 10) (%) 𝑝value Head of household Age group 0.501 ≤37 years old 38 (53) 34 (55) 4 (40) >37 years old 34 (47) 28 (45) 6 (60) Sex, female 18 (25) 13 (21) 5 (50) 0.049 Household monthly income 0.149 ≤50.000 Rwandan Franc 47 (65) 38 (61) 9 (90) >50.000 Rwandan Franc 25 (35) 24 (39) 1 (10) Marital Status 0.967 Never married 7 (10) 6 (10) 1 (10) Married 56 (78) 48 (77) 8 (80) Separated 9 (12) 8 (13) 1 (10) Highest education level completed 0.625 Never attended school 9 (12) 7 (11) 2 (20) Primary school 43 (60) 38 (61) 5 (50) Secondary school and plus 20 (28) 17 (28) 3 (30) Household Number of people living in the house at the time of the diagnosis of the index case One person 19 (26) 16 (26) 3 (30) 0.717 Two persons or more 53 (74) 46 (74) 7 (70) Parents/caregivers had knowledge of prevention of transmission of 𝑇𝐵𝑎 66 (92) 58 (94) 8 (80) 0.192 Parents/caregivers have knowledge on the role of 𝐼𝑃𝑇𝑏 23 (32) 22 (35) 1 (10) 0.153 IPT = isoniazid preventive therapy; TB = tuberculosis; aaware about using a mask when breastfeeding, avoiding sleeping in the same room or bed with child contacts, opening windows and doors for improved ventilation, practicing hygiene while coughing. bKnowledgeable about the administration of INH for 6 months to protect child contacts against TB. Table 2: Characteristics of the household of child contacts by uptake of IPT. Table 2: Characteristics of the household of child contacts by uptake of IPT. The WHO provided the first estimates of preventive therapy coverage for eligible children under the age of 5 years in 2016 [1]. The estimates showed that only 5.6% of an estimated 440,000 child contacts received preventive therapy in 2015 in the African region. 4. Discussion Characteristics Under five years old child contacts (𝑁= 94) (%) Child contacts who started IPT (𝑁= 84) (%) Child contacts who did not start IPT (𝑁= 10) (%) 𝑝value Age group ≤3 Years 66 (70) 63 (75) 3 (30) 0.007 >3 Years 28 (30) 21 (25) 7 (70) Sex child contact 0.504 Female 43 (46) 37 (44) 6 (60) BCG scar Yes 90 (96) 80 (95) 10 (100) 1.000 Children tested for HIV 47 (50) 39 (46) 8 (80) 0.091 HIV test Result Positive 2 (4) 2 (5) 0 (0.0) 0.051 Relationship to the Index case Child 70 (75) 65 (77) 5 (50) 0.060 Others 24 (25) 19 (23) 5 (50) Had TB-related symptom during the screeninga 23 (24) 19 (23) 4 (40)/ 0.252 Share the same bedroom with index cases 48 (51) 42 (50) 6 (49) 1.000 Time spend with index cases ≥8 hours 75 (80) 66 (79) 9 (90) 0.681 BCG = Bacilli Calmette-Guerin; IPT = isoniazid preventive therapy, TB = tuberculosis; HIV = human immunodeficiency virus; aTB-related symptom = to have one of those symptoms (a cough, fever, and weight loss). Table 4: Risk factors for nonuptake of IPT. Factors OR (95% CI) 𝑝value aOR (95% CI) 𝑝value Child contacts >3 Years 7.0 (1.65–29) 0.008 29 (2.17–400) 0.011 HIV positive 5.0 (1.0–25) 0.050 10 (0.61–174) 0.105 Not child of the index case 3.4 (0.89–13) 0.072 59 (2.74–127) 0.009 Index cases HIV-positive 4.0 (0.97–16.77) 0.054 8.1 (2.53–537) 0.038 Household factors Sex of the head of the household 4.6 (1.18–17.9) 0.028 - Income < 50.000 Rwandan Franc 0.1 (0.22–1.57) 0.123 0.1 (0.01–1.01) 0.050 Heath facility factors Provider not friendly 10 (1.26–83) 0.029 19 (2.514–140) 0.017 IPT = isoniazid preventive therapy; HIV = human immunodeficiency virus; CI = confidence interval; OR = odds ratio; aOR = adjusted OR. Our study showed that most parents/caregivers of child only 8% of parents with sputum smear-positive TB took their Tuberculosis Research and Treatment 6 Table 3: Characteristics of child contacts eligible for IPT by IPT uptake. Table 3: Characteristics of child contacts eligible for IPT by IPT uptake. Table 3: Characteristics of child contacts eligible for IPT by IPT uptake. 4. Discussion Characteristics Under five years old child contacts (𝑁= 94) (%) Child contacts who started IPT (𝑁= 84) (%) Child contacts who did not start IPT (𝑁= 10) (%) 𝑝value Age group ≤3 Years 66 (70) 63 (75) 3 (30) 0.007 >3 Years 28 (30) 21 (25) 7 (70) Sex child contact 0.504 Female 43 (46) 37 (44) 6 (60) BCG scar Yes 90 (96) 80 (95) 10 (100) 1.000 Children tested for HIV 47 (50) 39 (46) 8 (80) 0.091 HIV test Result Positive 2 (4) 2 (5) 0 (0.0) 0.051 Relationship to the Index case Child 70 (75) 65 (77) 5 (50) 0.060 Others 24 (25) 19 (23) 5 (50) Had TB-related symptom during the screeninga 23 (24) 19 (23) 4 (40)/ 0.252 Share the same bedroom with index cases 48 (51) 42 (50) 6 (49) 1.000 Time spend with index cases ≥8 hours 75 (80) 66 (79) 9 (90) 0.681 BCG = Bacilli Calmette-Guerin; IPT = isoniazid preventive therapy, TB = tuberculosis; HIV = human immunodeficiency virus; aTB-related symptom = to have one of those symptoms (a cough, fever, and weight loss). Under five years old child contacts (𝑁= 94) (%) Child contacts who started IPT (𝑁= 84) (%) Child contacts who did not start IPT (𝑁= 10) (%) 𝑝va Table 4: Risk factors for nonuptake of IPT. Table 4: Risk factors for nonuptake of IPT. Factors OR (95% CI) 𝑝value aOR (95% CI) 𝑝value Child contacts >3 Years 7.0 (1.65–29) 0.008 29 (2.17–400) 0.011 HIV positive 5.0 (1.0–25) 0.050 10 (0.61–174) 0.105 Not child of the index case 3.4 (0.89–13) 0.072 59 (2.74–127) 0.009 Index cases HIV-positive 4.0 (0.97–16.77) 0.054 8.1 (2.53–537) 0.038 Household factors Sex of the head of the household 4.6 (1.18–17.9) 0.028 - Income < 50.000 Rwandan Franc 0.1 (0.22–1.57) 0.123 0.1 (0.01–1.01) 0.050 Heath facility factors Provider not friendly 10 (1.26–83) 0.029 19 (2.514–140) 0.017 IPT = isoniazid preventive therapy; HIV = human immunodeficiency virus; CI = confidence interval; OR = odds ratio; aOR = adjusted OR. only 8% of parents with sputum smear-positive TB took their children to a medical clinic for screening despite having clear information on the need to do so [8].h Our study showed that most parents/caregivers of child contacts had some general knowledge on how to prevent TB in children. That level of knowledge was higher than that reported in studies conducted elsewhere [26, 27]. 4. Discussion However, Rwanda was among a few countries in Africa that provided data to the WHO from routine surveillance of preventive therapy for young child contacts [1]. Therefore, the high uptake in our study may reflect the particular attention being given by Rwanda NTP to TB in children in accordance with the Rwanda government’s priority intervention aimed at preventing and addressing the most important causes of child mortality [10, 11]. Addi- tionally, the goal of Rwanda NTP strategies is to strengthen more than 30 indicators outlined in the Performance-Based Financing (PBF) since 2009. The outlined indicators include “number of children eligible for IPT who received it” and “number of children aged below 5 years old who completed IPT.” The funding of healthcare services according to the PBF is based on the performance of medical facilities in enhancing the quantity and quality of preventive and curative treatment to the people [22]. Hence, the PBF has improved the quantity and quality of healthcare in many countries [23–25]. inform the NTP on its implementation. Despite the diversity methodology designs, the IPT uptake established in this study (89%) was found to be higher than 6% [15], 18.7% [16], 26.8% [17], 33% [18], and 64.3% [19] reported in Malawi, Timor- Leste, South Africa, South India, and Ethiopia, respectively. In contrast, recent studies conducted in the Gambia [20] and Benin [21] have reported an 89% and 99% of IPT uptake, which is similar to and higher than this study’s findings, respectively. The integration of IPT into the programmatic delivery of healthcare might explain the high uptake reported in this study as well as in study findings reported in Gambia and Benin. This is in contrast to earlier studies that were con- ducted in a healthcare facility environment [15, 16]. Rwanda’s NTP strategy adopts the households’ visit of index cases by healthcare providers at the beginning of sputum smear PTB treatment. These visits allow for screening child contacts and initiating them on IPT. Our high IPT uptake finding corroborates results published in the 2013-2014 and 2015-2016 annual reports of Rwanda’s NTP revealing an uptake of 85% and 78%, respectively. However, these reports do not provide information on the actual number of eligible contacts who had access to IPT in the community. 6 Tuberculosis Research and Treatment Table 3: Characteristics of child contacts eligible for IPT by IPT uptake. 4. Discussion Informa- tion, Education, and Communication sessions are organized twice a week at healthcare facilities by service providers, at the community level by the CHWs and at local politico- administrative authorities level as well as in media, to inform the population about TB. Other studies have shown that low- level knowledge on TB could negatively affect the health- seeking behaviour of the people [27, 28]. Contrasting findings have been reported in a study conducted in Malawi, where The geographical accessibility can also explain the high uptake of IPT in Rwanda. Across the country, there has been an improvement in the ease of access to healthcare centres. Countrywide, the average time to access the nearest health- care centre is less than 1 hour [29]. Transport cost was not a limiting factor mentioned by any parent/caregiver whose children were not initiated on IPT in our study as was the case in the study conducted in Malawi [30]. Furthermore, a study carried out in Bangkok, Thailand, by Tornee et al. [31] shows 7 Tuberculosis Research and Treatment that the short distance from home to the nearest medical clinic was associated with adherence of the households’ con- tact to screening.i cases were followed up in public PHCs and 56% of them whose children were not started on IPT were HIV infected. This suggests that negative experience in government health- care facilities and social stigma among TB-HIV coinfected persons have a negative impact on the uptake of IPT among child contacts. This finding is a cause of concern in Rwanda since the healthcare-seeking behaviour of HIV-positive par- ents/caregivers influence TB screening and management in children.h In the univariate analysis, the significance of the sex of household heads was lost when it was adjusted for other variables. This finding suggests that the sex of index cases was a confounder variable in this study. Nevertheless, additional studies are needed to investigate the role of gender in the decision to initiate IPT. In multivariate analysis, our study established that the child contacts who were not children of index cases were more likely not to be initiated on IPT than those who were their children. This finding corroborates a study conducted in Timor-Leste [16] as well as a qualitative study in Bangkok, Thailand [31]. Both studies reported lack of screening of child contacts who were not children of index cases. 4. Discussion The approach is slightly different in Rwanda, whereby household visits of index cases with sputum smear-positive PTB by healthcare providers helps in screening child contacts and initiating them on IPT. Thus, child contacts who are not children of the index cases have a high possibility of being screened even though the initiation of IPT among these children may be low. When an index case is not the biological parent of a child contact, the latter may choose to be visited by a healthcare provider in the absence of the former. Often, healthcare providers inform the index cases about the intended home visit so that children can be screened but not necessarily start on IPT. This is because index cases may not inform the parents or caregivers of the children the need for initiating their children on IPT. This could explain the 100% screening of eligible child contacts in this study whence only 89% were initiated on IPT. The parents/caregivers of 50% of child con- tacts who were not initiated on IPT reported their lack of in- formation about its usefulness. The study has some limitations. First, the research was conducted in Kigali; thus, the findings might not be general- ized to the whole country, especially remote rural areas where healthcare-seeking behaviour may be different. Second, the sample size was small to enable comparative analyses that may have limited our statistical detection of small differences in the IPT uptake. 5. Conclusion Findings from our study reveal that the NTP policy on the provision of IPT has been effectively implemented in Rwanda under the set programmatic conditions. Despite differences in methods of study, the percent IPT uptake established in this study is higher than that reported in Malawi, Timor- Leste, South Africa, South India, and Ethiopia and similar to and lower than in Gambia and Benin, respectively. Special attention should be given to child contacts more than 3 years old, child contacts who are not children of index cases, and child contacts who are children of HIV infected persons in order to identify the challenges experienced in initiating the IPT. Future interventions should find strategies to (1) fight against social stigma, especially in TB coinfected patients, and (2) eradicate the unfriendly attitude of healthcare providers towards all patients in general and TB coinfected patients in particular. Our study also established that child contacts older than 3 years were more likely not to be initiated on IPT than those aged below 3 years old. These findings can be explained by the fact that parents/caregivers protect their younger children from contracting TB more than their older children. Similar findings were reported in a qualitative study conducted in Bangkok, Thailand, by Tornee et al. [31], which showed that parents/caregivers worry that their younger children might get TB infection and take them to healthcare facilities for screening.h Conflicts of Interest The authors declare that there are no conflicts of interest re- garding the publication of this article. Authors’ Contributions The parents/caregivers who found unfriendly healthcare providers at the PHCs were also more likely not to initiate their children on IPT than those who found them friendly. Child contacts living with HIV-positive index cases were less likely to be initiated on IPT than those living with HIV- negative index cases. Those two factors may be correlated. A study demonstrated that interactions and negative experience of people seeking treatment in government healthcare facili- ties contribute to a reduction in subsequent medical visits or follow-ups [32], which is mostly observed among the HIV- positive population. Francine Mwayuma Birungi conceived the idea, designed the research, and was involved in the collection, analysis, and interpretation of the data. She drafted and critically revised the paper. Brian van Wyk, Jeannine Uwimana, and Stephen Graham advised on the protocol and drafted and critically reviewed the paper for important intellectual content. All authors reviewed and approved the final manuscript for pub- lication. All authors read and agreed on the final manuscript. References [18] H. R. Shivaramakrishna, A. Frederick, A. Shazia et al., “Isoni- azid preventive treatment in children in two districts of South India: does practice follow policy?” The International Journal of Tuberculosis and Lung Disease, vol. 18, no. 8, pp. 919–924, 2014. [1] World Health Organisation (WHO), “Global Tuberculosis Re- port 2016,” 2016,http://apps.who.int/iris/bitstream/10665/250441/ 1/9789241565394-eng.pdf.h [2] B. J. Marais, R. P. Gie, H. S. Schaaf et al., “The natural history of childhood intra-thoracic tuberculosis: A critical review of literature from the pre-chemotherapy era,” The International Journal of Tuberculosis and Lung Disease, vol. 8, no. 4, pp. 392– 402, 2004. [19] Y. Tadesse, N. Gebre, S. Daba et al., “Uptake of isoniazid preventive therapy among under-five children: TB contact investigation as an entry point,” PLoS ONE, vol. 11, no. 5, Article ID e0155525, 2016. [20] U. Egere, A. Sillah, T. Togun et al., “Isoniazid preventive treat- ment among child contacts of adults with smear-positive tuber- culosis in the Gambia,” International Journal of Tuberculosis and Lung Diseases, vol. 6, no. 4, pp. 226–231, 2016. [3] M. Singh, M. L. Mynak, L. Kumar, J. L. Mathew, and S. K. Jindal, “Prevalence and risk factors for transmission of infec- tion among children in household contact with adults having pulmonary tuberculosis,” Archives of Disease in Childhood, vol. 90, no. 6, pp. 624–628, 2005. [21] M. Adjobimey, E. Masserey, C. Adjonou et al., “Implementation of isoniazid preventive therapy in children aged under 5 years exposed to tuberculosis in Benin,” The International Journal of Tuberculosis and Lung Disease, vol. 20, no. 8, pp. 1055–1059, 2016. [4] World Health Organisation (WHO), Guidance for National Tuberculosis Programmes on the Management of Tuberculosis in Children, vol. 2nd, World Health Organisation, Geneva, Switzerland, 2014. [22] The AIDSTAR-Two Project, The PBF Handbook: Designing and Implementing Effective Performance-Based Financing Programs, vol. 1, Cambridge: Management Sciences for Health, 2011. [5] P. C. Hill, M. E. Rutherford, R. Audas, R. van Crevel, and S. M. Graham, “Closing the policy-practice gap in the management of child contacts of Tuberculosis cases in developing countries,” PLoS Medicine, vol. 8, no. 10, Article ID e1001105, 2011.i [23] L. Rusa, J. D. D. Ngirabega, W. Janssen, S. Van Bastelaere, D. Porignon, and W. Vandenbulcke, “Performance-based financ- ing for better quality of services in rwandan health centres: 3- year experience,” Tropical Medicine & International Health, vol. 14, no. 7, pp. 830–837, 2009. [6] M. E. Rutherford, P. C. Hill, R. Triasih, R. References Sinfield, R. van Crevel, and S. M. Graham, “Preventive therapy in children exposed to Mycobacterium tuberculosis: problems and solutions,” Tropical Medicine & International Health, vol. 17, no. 10, pp. 1264–1273, 2012. [24] Management Sciences for Health, “Performance-based financ- ing improves quantity and quality of health services in Haiti,” 2017, https://www.msh.org/sites/msh.org/files/haiti pbf techni- cal brief 01feb2017.pdf. [7] K. T. Garie, M. A. Yassin, and L. E. Cuevas, “Lack of adherence to isoniazid chemoprophylaxis in children in contact with adults with Tuberculosis in Southern Ethiopia,” PLoS ONE, vol. 6, no. 11, Article ID e26452, 2011. [25] Management Sciences for Health, “Performance-based financ- ing improves health facility performance and patient care in the Democratic Republic of the Congo,” 2017, https://www.msh .org/sites/msh.org/files/drc pbf technical brief 01feb2017.pdf. [8] M. Nyirenda, R. Sinfield, S. Haves, E. M. Molyneux, and S. M. Graham, “Poor attendance at a child TB contact clinic in Malawi,” The International Journal of Tuberculosis and Lung Dis- ease, vol. 10, no. 5, pp. 585–587, 2006. [26] K. E. Agho, J. Hall, and B. Ewald, “Determinants of the know- ledge of and attitude towards tuberculosis in Nigeria,” Journal of Health, Population and Nutrition, vol. 32, no. 3, pp. 520–538, 2014. [9] Rwanda Biomedical Centre, “Tuberculosis and Other Respira- tory Diseases. Annual Report July 2015-June 2016,” Ministry of Health, Rwanda, 2016. [10] Ministry of Health Rwanda, “Success Factors for Women’s and Children’s Health Rwanda,” 2014. [27] N. P. Hoa, A. E. K. Thorson, N. H. Long, and V. K. Diwan, “Knowledge of tuberculosis and associated health-seeking behaviour among rural Vietnamese adults with a cough for at least three weeks,” Scandinavian Journal of Public Health, vol. 31, no. 6, supplement, pp. 59–65, 2003. [11] Rwanda Biomedical Centre, “Tuberculosis National Strategic Plan July 2013- June 2018,” 2014. [12] World Health Organisation (WHO), “Guidance for national tuberculosis programmes on the management of tuberculosis in children,” World Health Organisation, vol. WHO/HTM/TB, 2006. [28] D. G. Storla, S. Yimer, and G. A. Bjune, “A systematic review of delay in the diagnosis and treatment of tuberculosis,” BMC Public Health, vol. 8, article 15, 2008. [13] Global Laboratory Initiative, Mycobacteriology Laboratory Manual Editor, 1st edition, 2014. [29] National Institute of Statistics of Rwanda, “Rwanda Integrated Household Living Conditions Survey 2013/2014 B,” 2015. [14] Ministry of Health Rwanda, Handbook of Tuberculosis and TB- HIV, 5th edition, 2009. [30] R. Zachariah, M.-P. Spielmann, A. D. Acknowledgments Besides the negative experience from unfriendly health- care providers, the HIV-positive people have to cope with social stigma [33, 34]. A study revealed that women, who often take children to healthcare facilities, experience stigma- related problems more than men [35]. In our study, 71% index The authors would like to express their sincere gratitude to study participants and their parents or caregivers, NTP staff, TB focal points, head of PHCs, community health workers, and data enumerators involved in this study. They thank also Tuberculosis Research and Treatment 8 Mr. G´ed´eon Bahemuka Jino and Dr. Mary Nelima Ondiaka for editing the manuscript. This study was funded by UR- Sweden collaboration programme. Mr. G´ed´eon Bahemuka Jino and Dr. Mary Nelima Ondiaka for editing the manuscript. This study was funded by UR- Sweden collaboration programme. exposed to tuberculosis in Timor-Leste,” Tropical Medicine & International Health, vol. 20, no. 6, pp. 730–736, 2015. [17] M. Osman, A. C. Hesseling, N. Beyers et al., “Routine program- matic delivery of isoniazid preventive therapy to children in Cape Town, South Africa,” Public Health Action, vol. 3, no. 3, pp. 199–203, 2013. 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Sukijthamapan, R. dos Santos et al., “Challenges to delivery of isoniazid preventive therapy in a cohort of children Tuberculosis Research and Treatment Tuberculosis Research and Treatment 9 [32] S. Churcher, “Stigma related to HIV and AIDS as a barrier to accessing health care in Thailand: a review of recent literature,” WHO South-East Asia Journal of Public Health, vol. 2, no. 1, pp. 12–22, 2013. [33] S. X. Jittimanee, S. Nateniyom, W. Kittikraisak et al., “Social stigma and knowledge of tuberculosis and HIV among patients with both diseases in Thailand,” PLoS ONE, vol. 4, no. 7, Article ID e6360, 2009. [34] C. Burapat, W. Kittikraisak, K. P. Cain et al., “Health-seeking behavior among HIV-infected patients treated for TB in Thai- land,” Southeast Asian Journal of Tropical Medicine and Public Health, vol. 40, no. 6, pp. 1335–1346, 2009. [35] M. 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https://openalex.org/W2467984491	https://europepmc.org/articles/pmc4941516?pdf=render	English	null	Competition for Cooperation: variability, benefits and heritability of relational wealth in hunter-gatherers	Scientific reports	2,016	cc-by	6,706	Competition for Cooperation: variability, benefits and heritability of relational wealth in hunter- gatherers 4 March 2016 10 June 2016 OPEN received: 24 March 2016 accepted: 10 June 2016 Published: 12 July 2016 Nikhil Chaudhary1, Gul Deniz Salali1, James Thompson1, Aude Rey1, Pascale Gerbault2, Edward Geoffrey Jedediah Stevenson1, Mark Dyble1, Abigail E. Page1, Daniel Smith1, Ruth Mace1, Lucio Vinicius1 & Andrea Bamberg Migliano1 Many defining human characteristics including theory of mind, culture and language relate to our sociality, and facilitate the formation and maintenance of cooperative relationships. Therefore, deciphering the context in which our sociality evolved is invaluable in understanding what makes us unique as a species. Much work has emphasised group-level competition, such as warfare, in moulding human cooperation and sociality. However, competition and cooperation also occur within groups; and inter-individual differences in sociality have reported fitness implications in numerous non-human taxa. Here we investigate whether differential access to cooperation (relational wealth) is likely to lead to variation in fitness at the individual level among BaYaka hunter-gatherers. Using economic gift games we find that relational wealth: a) displays individual-level variation; b) provides advantages in buffering food risk, and is positively associated with body mass index (BMI) and female fertility; c) is partially heritable. These results highlight that individual-level processes may have been fundamental in the extension of human cooperation beyond small units of related individuals, and in shaping our sociality. Additionally, the findings offer insight in to trends related to human sociality found from research in other fields such as psychology and epidemiology. Many unique aspects of human sociality such as language, theory of mind and cultural norms have been pro- posed to provide the framework for human cooperative behaviour1–4, which stands alone in its scale and ubiquity between unrelated individuals5. Cooperation has been fundamental to the demographic success of our species - resource exchange, collective action and specialisation have increased our efficiency at surmounting a vast array of environmental pressures6,7. Therefore deciphering the context in which human cooperation and sociality evolved is invaluable to understanding what makes us unique as a species.fi Inter-individual differences in sociality have been reported to have fitness implications in numerous taxa. For instance, in various non-human primates, greater social integration or social capital has been associated with increased longevity, offspring survival or mating access8–10. However, in humans, the link between individual differences in social integration and reproductive fitness has received little attention. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 Competition for Cooperation: variability, benefits and heritability of relational wealth in hunter- gatherers 4 March 2016 10 June 2016 OPEN Therefore, if certain individuals are better able to accrue cooperative social relationships with others (relational wealth), differences in fitness at the individual level may emerge within groups. We hypothesise that individual level ‘competition for cooperation’ may have been an important driving force in human evolution and fundamental in shaping our sociality. p g p g y We attempt to identify the role of inter-individual variation in relational wealth in the dynamics of within-group competition among BaYaka hunter-gatherers. The BaYaka are simple and mobile hunter-gatherers - they con- sume food they forage soon after acquisition and lack storage mechanisms, and are also politically egalitarian; such societies are the best extant approximation of the ecological conditions under which our species evolved. Therefore, although the BaYaka are an extant population of cognitively modern humans, their forager lifestyle offers a valuable opportunity for inference regarding human evolutionary history. f Whereas in other subsistence modes food storage is an option, simple hunter-gatherers rely profoundly on food sharing to mitigate risks associated with the unpredictability of their foraging niche14,15; thus cooperation is at the heart of these populations. Although these societies are usually egalitarian16, social interaction and exchange is still structured within these populations15,17, and certain individuals may be better able to accrue cooperative links with others by means not dependent on formal hierarchy. In such a context, where individuals are so reliant on cooperative relationships, those with more relational wealth are likely to have an evolutionary advantage. Women may benefit from more access to allocare and provisioning, in turn increasing the health and survival prospects of their offspring, and aiding in the trade-off between childcare and foraging effort18. Men with more cooperative partners, may profit from biased resource allocation towards their families, and also have increased access to mates19. Therefore, in these societies where material wealth is absent, it may be relational wealth that drives documented patterns of individual fitness variance20. Indeed some evolutionary anthropolo- gists have noted the likely relevance of the related concept of social capital to hunter-gatherers21. Social capital is traditionally used in economics and sociology, and the term has been used to describe social relationships and interactions with others that generate returns for the individual22,23. Kaplan et al. Competition for Cooperation: variability, benefits and heritability of relational wealth in hunter- gatherers 4 March 2016 10 June 2016 OPEN There is substantial evidence that humans have a series of psychological and physiological reinforcement mechanisms encouraging the forma- tion and maintenance of social relationships11. The existence of these proximate mechanisms encouraging social integration, implies social ties must also have some ultimate fitness enhancing function within our species. We pay particular attention to the cooperative function of human social relationships, and investigate the importance of inter-individual differences in relational wealth12 (access to cooperation from group members) within the group. g p Substantial research into the evolution of human sociality and cooperation has focused on group-level expla- nations. Such explanations emphasise the importance of cooperation between unrelated individuals in large scale warfare and resource competition between groups13. These theories assert that human sociality includes a suite of traits such as tendencies to form in-group biases and internalise cultural norms, which evolved to help individuals 1Department of Anthropology, University College London, London WC1H 0BW, United Kingdom. 2Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, United Kingdom. Correspondence and requests for materials should be addressed to N.C. (email: charan.chaudhary.11@ucl.ac.uk) Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 1 www.nature.com/scientificreports/ Figure 1. The distribution of relational wealth. Kernel-Density Distributions of the number of honey-stick nominations received per individual for men (blue) and women (red) in three Mbendjele camps. Camp names are indicated above each graph. Figure 1. The distribution of relational wealth. Kernel-Density Distributions of the number of honey-stick nominations received per individual for men (blue) and women (red) in three Mbendjele camps. Camp names are indicated above each graph. function in highly cooperative groups4. However, competition and cooperation can also occur at the individual level between members of the same group; and as alluded to above, in numerous non-human taxa differences in sociality within the group have important implications for fitness. Therefore, if certain individuals are better able to accrue cooperative social relationships with others (relational wealth), differences in fitness at the individual level may emerge within groups. We hypothesise that individual level ‘competition for cooperation’ may have been an important driving force in human evolution and fundamental in shaping our sociality. function in highly cooperative groups4. However, competition and cooperation can also occur at the individual level between members of the same group; and as alluded to above, in numerous non-human taxa differences in sociality within the group have important implications for fitness. Competition for Cooperation: variability, benefits and heritability of relational wealth in hunter- gatherers 4 March 2016 10 June 2016 OPEN hypothesise that since activities such as food sharing are often not uniform in hunter-gatherer groups, markets for cooperative partners emerge and social capital is likely to become relevant for consumption patterns and fitness21.t p y p pi Here we use economic gift games to construct and compare individual cooperative networks in three BaYaka camps. Our results demonstrate the presence of individual variation in relational wealth, which is particularly striking among men. We show that those with more relational wealth receive food transfers from a larger number of individuals than their peers, and this is reflected in their significantly higher BMI; women with more relational wealth also have significantly higher age-specific fertility. The data also suggest there is a heritable component to relational wealth, and that cooperative alliances may be transmitted inter-generationally. While cooperation may have been important for increasing group resilience in warfare and resource competition, our findings indicate that individual level competition for cooperation within the group may also have been fundamental in shaping human sociality. Results In fact, acquisition of social benefits has been postulated as the driving force behind male specialisation in foraging for unpredictable resources that are widely shared27. Additionally, a central aspect of Mbendjele life is the process of undergoing sex-specific initiation rites in order to gain membership to various religious cults, which increase bonding and solidarity amongst members; there is greater variation in membership to these religious cults amongst men. Nevertheless there is substantial variation in gifts received by both sexes, suggesting that if these relationships translate to benefits related to sur- vival or reproduction, both men and women with more relational wealth can gain advantage over fellow camp members. receive substantially more honey sticks than their peers. Levene’s tests highlight a significantly larger variance in male than female relational wealth in two of the three camps – Longa (p = 0.023; n = 47, 25 female) and Ibamba (p = 0.011; n = 30, 18 female) (see Supplementary Table 2). The lack of significance in camp Masia (p = 0.123; n = 20, 9 female) is likely a result of the small sample that is concomitant with the camp size.hl This trend may reflect the fact that usually male hunting production is both more variable and shared more widely than female gathered foods in hunter-gatherer socities25,26, thus there are likely to be larger differences between men in opportunities to form alliances via food sharing. In fact, acquisition of social benefits has been postulated as the driving force behind male specialisation in foraging for unpredictable resources that are widely shared27. Additionally, a central aspect of Mbendjele life is the process of undergoing sex-specific initiation rites in order to gain membership to various religious cults, which increase bonding and solidarity amongst members; there is greater variation in membership to these religious cults amongst men. Nevertheless there is substantial variation in gifts received by both sexes, suggesting that if these relationships translate to benefits related to sur- vival or reproduction, both men and women with more relational wealth can gain advantage over fellow camp members. Relational wealth variation results in individual differences in overcoming environmental risk, and is associated with higher female fertility. Results Using multiple regression we found a significant asso- ciation between our measure of relational wealth (HSGG nominations) and the number of camp members from whom an individual receives food in real world transfers (β = 0.24; p = 0.005; n = 53) (see Supplementary Table 3 for full results). We also find a significant relationship between BMI and relational wealth for both men (β = 0.53; p = 0.032; n = 39) and women (β = 0.90; p = 0.003; n = 34) (see Fig. 2; see Supplementary Table 4 for full results). Hunter-gatherer subsistence is highly unpredictable, thus food transfers between households are vital in buffering this high acquisition risk14,28. Although these societies are often characterised by norms promoting widespread sharing29,30, research shows that food transfers are biased by kinship ties, reciprocal relationships and foraging effort of others31–33. The findings here indicate that those individuals with more relational wealth are better able to secure a stable nutritional income, and tackle this fundamental adaptive problem. p p Maintaining a healthy body weight may also be particularly important for female fertility as it avoids sec- ondary amenorrhea34. Indeed we find relational wealth is a significant predictor of female age-specific fertility (β = 0.19; p = 0.010; n = 49) (see Supplementary Table 5). The result cannot be explained by reverse causality i.e. HSGG participants preferably distributing their honey sticks to women who have more offspring and thus may be in greater need: Female participants were at different stages of their reproductive career and offspring of older participants may have already reached adulthood, thus a participant’s total fertility does not necessarily match their current number of dependent (under 16) offspring. We find no significant correlation between a wom- an’s current number of dependent offspring living in the household and relational wealth (G = 0.14, p = 0.280, n = 51), suggesting in the initial association between relational wealth and age-specific fertility relational wealth is affecting fertility rather than vice-versa. Nevertheless the result must be treated as preliminary since our measure of fertility and relational wealth reflect different timescales; specifically relational wealth is a measure reflecting one point in time (the data collection period), whereas fertility reflects the length of ego’s reproductive career thus far. Relational wealth is inherited from fathers. Results Individual variation in relational wealth. We constructed an adult-to-adult gift network by playing a honey stick gift game (HSGG)24 with all adults in three BaYaka camps (n = 97, 52 female), where each participant must choose the distribution of three honey sticks amongst other members of his/her camp. Figure 1 shows the distributions of total number of honey sticks received by an individual in the HSGG, which is our measure of relational wealth. It is clear that there is individual-level variation in number of gifts received for both sexes. It is noteworthy that the effect of individual differences in genetic relatedness to members of one’s camp on number of gifts received does not reach significance (p = 0.067; R2 = 0.036) (see Supplementary Table 1). The extent of male variation is particularly striking producing multi-modal distributions in all three camps, where certain men Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 2 www.nature.com/scientificreports/ Figure 2. Relational wealth and body mass index (BMI). Relationship between relational wealth and BMI z-score (standardised by sex and age category–pre/post reproductive age for women and over/under 45 for men). Shaded bands indicate 95% confidence intervals. Blue line and shaded band represent males, red line and shaded band represent females. Figure 2. Relational wealth and body mass index (BMI). Relationship between relational wealth and BMI z-score (standardised by sex and age category–pre/post reproductive age for women and over/under 45 for men). Shaded bands indicate 95% confidence intervals. Blue line and shaded band represent males, red line and shaded band represent females. receive substantially more honey sticks than their peers. Levene’s tests highlight a significantly larger variance in male than female relational wealth in two of the three camps – Longa (p = 0.023; n = 47, 25 female) and Ibamba (p = 0.011; n = 30, 18 female) (see Supplementary Table 2). The lack of significance in camp Masia (p = 0.123; n = 20, 9 female) is likely a result of the small sample that is concomitant with the camp size. This trend may reflect the fact that usually male hunting production is both more variable and shared more widely than female gathered foods in hunter-gatherer socities25,26, thus there are likely to be larger differences between men in opportunities to form alliances via food sharing. Discussioni We find that relational wealth varies by individual, provides health and fertility benefits and is partially herita- ble. These results highlight that in the absence of material wealth accumulation and social hierarchy, relational wealth may be an important determinant of individual fitness among simple hunter-gatherers. Individuals vary widely in their access to cooperation from fellow camp members, and those with more relational wealth are better equipped to overcome the high risk that characterises the hunter-gatherer lifestyle, since they have a significantly larger pool of food donors to insure against nutritional shortfalls. In addition to augmenting survival and health outcomes, social ties appear to increase reproductive rates of the BaYaka. Women with more relational wealth have higher age-specific fertility, a relationship which may be mediated by BMI since low body-weight disrupts ovulatory processes34. Additionally, we previously demonstrated that men with very high relational wealth are more likely to achieve polygyny in this group, which increases their reproductive rate19. Studies of other foraging societies have also reported positive associations between male social status and fertility, by examining the effect of hunting ability on mating access and reproductive outcomes37,38. g y g p Egalitarian hunter-gatherers lack the heritable hierarchical positions which are found in agricultural and industrialised societies16, however, our results indicate partial heritability of relational wealth. We did not inves- tigate the mechanism for this heritability explicitly, but there are several possibilities. Genetic factors have been shown to influence social network positioning in human and non-human primates39,40. Additionally, the inher- itance may operate via the direct transmission of cooperative alliances from parents to offspring. This may explain the significant association with paternal but not maternal relational wealth - if relationships are transmitted inter-generationally, an individual’s relational wealth would be more closely associated with the parent whose sex has higher variability in number of social relationships. This inheritance of social ties would increase the evolutionary advantage of strengthening one’s social network since the associated benefits can accrue over mul- tiple generations. Therefore, in hunter-gatherer groups which are often egalitarian and do not accumulate mate- rial resources, relational wealth may drive documented patterns of inter-individual fitness variance and fertility inheritance20,41, and may be the resource that is transmitted inter-generationally.hifihi y g y These findings offer a significant contribution to our understanding of human social evolution. Results In order to test if relational wealth is heritable, we con- ducted gamma correlations between the number of honey stick nominations of parents and their adult offspring in the HSGG (see Fig. 2). Ego’s (male or female) relational wealth as an adult is positively correlated with ego’s father’s (G = 0.65, p = 0.002; n = 14), and ego’s mother’s (G = 0.17, p = 0.294; n = 26) relational wealth (see Fig. 3); but these results are only significant for the former. Although this hunter-gatherer society is egalitarian29,35,36–no Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 3 www.nature.com/scientificreports/ Figure 3. The inheritance of relational wealth. Scatter plots representing the relationship between ego’s relational wealth and ego’s father’s (blue) and ego’s mother’s (red) relational wealth. Shaded bands indicate 95% confidence intervals. Figure 3. The inheritance of relational wealth. Scatter plots representing the relationship between ego’s relational wealth and ego’s father’s (blue) and ego’s mother’s (red) relational wealth. Shaded bands indicate confidence intervals. Figure 3. The inheritance of relational wealth. Scatter plots representing the relationship between ego’s relational wealth and ego’s father’s (blue) and ego’s mother’s (red) relational wealth. Shaded bands indicate 95 confidence intervals. Figure 3. The inheritance of relational wealth. Scatter plots representing the relationship between ego’s relational wealth and ego’s father’s (blue) and ego’s mother’s (red) relational wealth. Shaded bands indicate 95% confidence intervals. individuals can exert any authority over others, and there are no hierarchical positions - the results here indicate there is a degree of heritability of relational wealth; we explore the potential mechanisms in the discussion. Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 www.nature.com/scientificreports/ www.nature.com/scientificreports/ which are better able to promote cooperation and group beneficial behaviours outcompete others4. Our results do not undermine the possibility of selection at the group level, but draw attention to the importance of the role of cooperation in competition within the group. We find substantial inter-individual variability in access to cooperation (Fig. 1), which largely cannot be explained by kinship networks (Supplementary Table 1), and has meaningful consequences for health and fertility outcomes (Fig. 2/Supplementary Table 5). Cooperation is an integral means by which hunter-gatherers deal with their unpredictable environment, and extends across many activities including childcare, foraging and food sharing17,45,46. In the same way that groups with a greater capa- bility to harness cooperation performed well in warfare and resource competition4, here we show that individuals within groups who harness more cooperation have increased resilience against the unpredictable foraging niche typifying hunter-gatherer subsistence.i y y g g Our findings suggest consideration of within-group competition is crucial to a complete understanding of the evolution of human sociality. These results indicate that over their evolutionary history some hunter-gatherer individuals may have outcompeted other members of their group by expanding their cooperative networks beyond the small close kin units ubiquitous in the animal kingdom. Therefore, although we may have psycholog- ical tendencies to form in-group biases and internalise cultural norms as a result of inter-group competition4,47, many of our derived social traits may also reflect within-group competition. Research from psychology and epi- demiology on modern populations demonstrate a number of findings consistent with our results such as - posi- tive associations between individual social integration and mental and physical health48; a psychological tendency for individuals to evaluate their social positioning relative to their peer group49; and neuroendocrine mechanisms encouraging the formation and maintenance of friendships11. Methodsh This study has full approval from the Ethics Committee of University College London, and the methods were carried out in accordance with the approved guidelines. Informed consent was obtained from all participants and research permission granted by the Republic of Congo’s Ministry Of Scientific Research. The fieldwork took place between March and July 2014. Study population. Our study uses data from the Mbendjele BaYaka, a subgroup of the BaYaka who speak Mbendjele language and whose residence spans across the forests of Congo and Central African Republic. BaYaka subsistence techniques include hunting, trapping, fishing, gathering and honey collecting; as well as some trade with neighbouring farmer groups. Food sharing is an integral component of BaYaka subsistence and culture. The BaYaka live in langos - multi-family camps constituted of a number of fumas (huts) in which nuclear fam- ilies reside; camp size tends to vary from 10–60 individuals, and genetic relatedness within camps is low36. The BaYaka are predominantly serially monogamous, with some incidence of polygyny19. We visited three camps in the Likoula and Sangha regions of Congo’s Ndoki Forest (see Supplementary Fig. 2 for map). HSGG. This game was played with all willing members of a camp and was completed as quickly as possible, usually within 2–3 days in each camp. All instructions were spoken in French by the researcher, and then imme- diately repeated in Mbendjele by the translator. The game was based on the procedure of Apicella et al. 24.The key features of our protocol for the game were: 1. Participants were asked to accompany the researcher and translator to a private area. 1. Participants were asked to accompany the researcher and translator to a private area. 3. Participants were told they must decide to whom they would like us (the researchers) to give the honey sticks. 4 P ti i t t ld th ld i f l i ll th ti k t i di id l ti k t th diff t 3. Participants were told they must decide to whom they would like us (the researchers) to give the honey sticks. 4. Participants were told they could give freely i.e. all three sticks to one individual or one stick to three different 3. Participants were told they must decide to whom they would like us (the researchers) to give the honey sticks. 4. Participants were told they could give freely i.e. Methodsh all three sticks to one individual or one stick to three different individuals etc. . Participants were told they must decide to whom they would like us (the researchers) to give the honey sticks . Participants were told they could give freely i.e. all three sticks to one individual or one stick to three differen individuals etc. P i i ld h ld i d l i h i h h h l 5. Participants were told they could nominate any adult in their camp other than themselves.t 5. Participants were told they could nominate any adult in their camp other than themselves.t 6. After the games had been completed with all adults in camp, the honey sticks were distributed according to the results. 6. After the games had been completed with all adults in camp, the honey sticks were distributed according to the results. Food Transfer Observations. Households were observed by JT over a series of two to four hour time blocks, with households observed for a total of 24 or 36 hours depending on the camp. Observations were evenly distributed between 6 am and 6 pm and spread over several days. During observation periods, a record was made of all food produced by a focal household. If division of resource packages occurred, all recipient households were identified. For all food cooked and consumed by the household, the type and amount of food were recorded and all those who ate the food were identified. Anthropometrics. We measured height and weight of all willing and non-pregnant adults in each camp in order to calculate BMI. Height was measured to the nearest mm using a Harpenden anthropometer, and weight using a Philipps mechanical scale. Analyses. In all analyses, relational wealth is calculated as the number of nominations received in the HSGG standardised by camp and sex. Discussioni The benefits of social bonds and importance of individual differences in social positioning have been identified for numerous taxa including non-human primates, feral horses and bottlenose dolphins10,42,43. Social ties have been associ- ated with a variety of benefits in different species including increased longevity, offspring survival and mating access, enhanced dominance rank and reduced harassment8,9,42,44. However, similar research investigating the importance of inter-individual differences in sociality among humans is scant. This study differs from those in non-human taxa in its specific focus on cooperative networks (rather than proximity networks for example), nevertheless we still demonstrate that individual variation in an aspect of human sociality (relational wealth) has an important impact on health and fertility in hunter-gatherers.i Many investigations in to the evolution of human cooperation and sociality, specifically its widespread nature and extension beyond kin ties, have emphasised the importance of inter-group competition. These explanations highlight that human sociality evolved to facilitate group wide cooperation, since groups with cultural norms Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ We use multiple regression to analyse the relationship between relational wealth and BMI for each sex. We control for whether ego is post-reproductive (females)/over 45(males) since there is a significant decline in BMI for these age-groups in our sample. We also control for camp membership (categorical). g g p p p p ( g ) We use multiple regression to analyse the relationship between relational wealth and female fertility. We use age and age2 as controls to account for the quadratic relationship between age and fertility. To check whether reverse causality may explain the significant association found, we conduct a gamma correlation between female relational wealth and the number of dependent offspring in their household. The gamma correlation is conducted using the rococo package in R; and is selected as it is appropriate for variables which contain many ties, such as number of dependent offspring. pf p g For correlation analyses of ego’s and ego’s parents’ relational wealth we also use gamma correlations because they are appropriate for small sample sizes and data with ties. 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Author Contributions A.B.M. idealized the project; N.C., G.D.S., J.T., A.B.M., L.V. and R.M. designed the study; N.C., G.D.S., J.T. and A.R. collected the data with the help of P.G., E.S., M.D., A.P. and D.S. N.C. wrote the manuscript with the help of all other authors. All authors give their final approval for this version to be published. Scientific Reports \| 6:29120 \| DOI: 10.1038/srep29120 References Science 348, 796–798 (2015). 36. Dyble, M. et al. Sex equality can explain the unique social structure of hunter-gatherer bands. Scien y q y p q g ( ) 37. Smith, E. A. Why do good hunters have higher reproductive success? Hum. Nat. 15, 343–364 (2004).i y do good hunters have higher reproductive success? Hum. Nat. 15 38. Gurven, M. & von Rueden, C. Hunting, social status and biological fitness. Soc. Biol. 53, 81–99 (2006). g gi 39. Brent, L. J. N. et al. Genetic origins of social networks in rhesus macaques. Sci. Rep. 3, 1042 (2013). 40. Fowler, J. H., Dawes, C. T. & Christakis, N. 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Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Chaudhary, N. et al. Competition for Cooperation: variability, benefits and heritability of relational wealth in hunter-gatherers. Sci. Rep. 6, 29120; doi: 10.1038/srep29120 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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https://openalex.org/W3145427343	https://bmcinfectdis.biomedcentral.com/track/pdf/10.1186/s12879-021-06021-x	English	null	HIV voluntary counseling and testing uptake and associated factors among Ethiopian youths: evidence from the 2016 EDHS using multilevel modeling	BMC infectious diseases	2,021	cc-by	9,786	Nigatu et al. BMC Infectious Diseases (2021) 21:334 https://doi.org/10.1186/s12879-021-06021-x Nigatu et al. BMC Infectious Diseases (2021) 21:334 https://doi.org/10.1186/s12879-021-06021-x Open Access HIV voluntary counseling and testing uptake and associated factors among Ethiopian youths: evidence from the 2016 EDHS using multilevel modeling Mamo Nigatu1* , Teshome Kabeta1, Abonesh Taye2 and Merga Belina3 Abstract Background: Existing evidence showed that Human Immunodeficiency Virus counselling and testing uptake among Ethiopian youths is low, and factors contributing to it are not well studied. Therefore, this study aims to assess the status of uptake and identify its determinants using the 2016 Ethiopia Demographic and Health Survey data. Method: Data of 10,903 Ethiopian youths were extracted from the 2016 Ethiopian Demographic and Health Survey. The association between the response variable and the predictors was modeled by multilevel binary logistic regression, whereas adjusted odds ratio and confidence intervals were used to measure associations and their statistical significance. The variation in the uptake of counselling and testing of HIV across regions of Ethiopia was quantified by intra-class correlation. Result: The current study revealed that, overall, 34.9% (95% CI: 33.5, 36.2%) Ethiopian youths were ever tested for human immunodeficiency virus. Results show that about 9% of the variation in the probability of being tested for the disease was due to the regional variations. Moreover, having moderate and comprehensive HIV knowledge, being rich, having risky sexual behaviour, having a better educational level, having professional work, being married, owning of mobile, and having access to media were positively associated with human immunodeficiency virus voluntary counselling and testing uptake. On the other hand, being male, following protestant religion, following Muslim religion, and following other religions than orthodox religion were negatively associated with the uptake of human immunodeficiency virus counselling and testing. Conclusion: Voluntary human immunodeficiency virus counselling and testing uptake among Ethiopian youths is very low and varies across the regions which might hamper the ambitious plan of Ethiopia to end the disease as a public health threat by 2030. Emphasis should be given to promoting the youths’ HIV-related knowledge through community-based education, encouraging and empowering the youths to participate in professional works by giving due focus to poor youths, and promoting mass media utilization to better achieve the plan. Keywords: EDHS, Multilevel, Youths, Voluntary HIV counselling, And testing p g @g ; g @j 1Faculty of Public Health, Department of Epidemiology, Jimma Unive Institute of Health, Jimma, Oromia, Ethiopia Full list of author information is available at the end of the article Background East and Southern Africa is the most affected African region where 20.6 million people had been living with HIV and 800,000 were newly infected in 2018 [9]. The number of people living with HIV in Ethiopia was decreased from the 2016 WHO estimate of 710,000 to 690,000 in 2018 [7, 10]. However, 23,000 people were newly infected at the end of 2018 leaving Ethiopia far off achieving the 2020 target [7]. y The risk of contracting HIV infection among youths is highly associated with age at sexual initiation. Hence, abstaining from sexual intercourse and delaying the age at sexual debut is among the efficient HIV prevention ef- forts for young people [1]. Studies have also witnessed that HIV-related age at sexual debut, stigma, and dis- crimination, place of residence, educational level, age, marital status, number of a life sex partner, exposure to mass media, Having Antenatal Care (ANC) follow up, having sexually-transmitted disease (STI) symptoms, wealth index and knowing about HIV were among other variables significantly associated with HIV Voluntary Counselling and Testing (VCT) uptake among youths [13–17]. Effectively addressing the root causes that put young people at risk of new HIV infections is an over- arching intervention to achieve the ambitious plan of ending AIDS by 2030 [11]. Young people HIV counsel- ing and testing create the gateway to care, treat, and support them [2, 18]. To affirm that young people exer- cise their right to know their HIV status and benefitted from antiretroviral treatment (ART), HIV testing and counseling must be radically scaled up [19]. Pieces of evidence from the 2016 Ethiopian Demo- graphic and Health Survey (EDHS) report showed that 9% of young women and 1% of young men age 15–24 had started sexual intercourse before their 15 years of age. However, only 27% of young women and 29% of young men who had sexual intercourse in the past 12 months before the commencement of the survey was tested for HIV and received the results of the test [20]. Despite this low HIV VCT uptake among Ethiopian youths, to the best knowledge of the authors, there is no study done to address potential factors associated with it using nationally representative data. Moreover, there was also a dearth of study that tried to consider regional variations about the uptaking of youths’ HIV VCT. Background the illnesses related to AIDS every 10 min [11]. In 2018, 21 % of the total 37,832 newly diagnosed HIV cases in the US were among the youth [12]. In 2017, 290,000 youths were newly diagnosed with HIV infections in Eastern and Southern Africa which were the highest of all the diseases’ incidences that occurred among youths all over the world in the same year [11]. Any person who is in the age group of 14 to 24 years, ac- cording to the WHO, is considered as a youth [1]. Ac- cording to the Ethiopian Federal HIV/AIDS Prevention and Control Office (HAPCO) and Federal Ministry of Health guideline, youths are among the top priority population segments for VCT which is given free of charge since they are vulnerable to the Human Immuno- deficiency Virus (HIV) because of the strong influence of peer pressure and the development of their sexual and social identities which often lead to experimentation [2]. HIV, unlike many other diseases, continued to be a major challenging public health problem to prevent and control. Starting from the first occurrence of the pan- demic, more than seventy-five million people have been infected by the disease, and it has claimed more than 32 million lives [3–5]. The global community is committed to an ambitious plan of bringing the acquired immuno- deficiency syndrome (AIDS) to an end by the year 2030 [6]. In 2014, the United Nations Program on Acquired Immunodeficiency syndromes (UNAIDS) being with other stakeholders launched the three 90s targets of diagnosing 90 % of all HIV-positive persons, providing antiretroviral therapy (ART) for 90 % of those diagnosed, and achieving viral suppression for 90 % of those treated by the year 2020 [4]. However, according to the reports from the 2019 UNAIDS and World Health Organization (WHO), globally, 37.9 million people were living with the disease at the end of 2018, whereas, 1.7 million people and 770,000 people were respectively newly in- fected and died from the disease-related causes [3, 5]. Even though the global annual number of new infections and death have declined, reaching the 2020 milestone with the current achievement is unthinkable [5, 7]. The disease disproportionally affected Sub-Saharan Africa where more than 70% of the disease’s global burden has occurred. Two-third of the estimated 6000 new infec- tions that occur globally each day occur in SSA [8]. Background Therefore, the current study is aimed to determine the status of HIV VCT uptake and to identify factors associ- ated with HIV voluntary counseling and testing among Ethiopian youths using 2016 nationally representative EDHS data. In 2017, globally, 590,000 youths were newly diag- nosed with HIV disease and 3.9 million youths were liv- ing with the disease [11]. According to the evidence from 2020 WHO estimates, globally, often people who were newly diagnosed with HIV infection, three persons were youths [1]. Approximately, worldwide, one thou- sand six hundred youth contract HIV infection every single day, and one young person loss his/her life due to © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Data analysis S 14 2 Stata 14.2 statistical software was used for extracting relevant variables, data processing, summarizing descrip- tive data and running inferential statistics. To assess re- gional variation of HIV-VCT uptake among the youths and to identify its associated factors, the multilevel logis- tic regression model was fitted. As the surveys are based on multistage stratified cluster sampling, the DHS sur- veys often follow a hierarchical data structure [20]. The degree of dependency in the clustered data plays a great role in the estimation of the parameters in the analysis of multilevel regression and is captured by intra-class correlation (ICC), which reflects the proportion of total variation in the response variable which is accounted for by the between-group variation [25]. The weights used in the analysis were obtained from DHS and were ad- justed as per the recommendation by Adam [26]. The goodness of fit was checked after weighting the dataset by both candidate weights. A model with the smallest AIC and BIC with its significant variables is considered, and the results are presented in Table 3. Sampling design for the EDHS Ethiopia is divided into 9 regions and two city adminis- trations. The hierarchy of Ethiopian government struc- ture has four layers namely Regions, Zones, Weredas, and Kebele in order of higher to lower level respectively. The sampling frame used in the 2016 EDHS was ob- tained from the Population and Housing Census (PHC) conducted in 2007 by CSA. A random and representa- tive sample was obtained by two-stage sampling. The de- tails of the sampling procedure are found in the 2016 EDHS report [21]. In the 2016 EDHS dataset, the occupational status of the respondents was presented in too much detail and the authors have reclassified it as agricultural workers, professional workers, trade or sales workers, elementary occupation, and other workers [24]. Data sources Central Statistical Agency (CSA) of Ethiopia and other stakeholders by technical assistance of ICF International collected the 2016 EDHS data; processed and organized Page 3 of 11 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Page 3 of 11 the six domains of HIV stigma and discrimination summed to 0, Moderate HIV stigma if the sum of the re- sponse to the six domains is between 1 and 3, and high HIV stigma if the sum of the response to the six do- mains of HIV stigma and discrimination scores is be- tween 4 and 6. On the other hand, the youths were considered as practicing risky sexual behavior if they responded either as having multiple sex partners, i.e. more than one sex partner, in a lifetime or if they had multiple sex partners in the last 12 months excluding the spouse. it into different datasets. The authors have accessed the dataset from a public domain MEASUREDHS website by permission. The authors didn’t involve in any part of the sampling design and data collection. The authors used this as secondary data. Ethical consideration The Federal Democratic Republic of Ethiopia Ministry of Science and Technology and the Institutional Review Board of ICF International reviewed and approved The EDHS 2016 survey protocol. Written consent was ob- tained from each participant. The investigators received permission from the public domain MEASUREDHS website and reanalyzed the data set on youth respon- dents whose ages range from 15 to 24. Measurements and operational definitions The outcome variable was a single direct question asking whether the respondent has ever been tested for HIV or not (the response was Yes/No). In the context of this study, HIV VCT means that the person has already got both counseling and testing service. A questionnaire was used to get participants response on the following set of variables; sex, age, religion, wealth status, marital status, occupational status, sex of household head, education level, place of residence and region, and other variables like coverage status of health insurance, frequency of reading newspaper or magazine in a month, frequency of listening to the radio in a month, frequency of watch- ing television, use of the internet, frequency of using the internet within a month, decisions on personal health care, relationship with a most recent sex partner, owning of a mobile telephone, discussion about family planning with a health worker, decisions on large household pur- chases and decisions on how to spend respondent’s earnings, and respondent’s involvement during check- ups for the most recent child. Stigma status was mea- sured by six domains of stigma [22]. Similarly, HIV knowledge was also measured by another six domains of its measurement [23]. Based on its six domains of mea- surements the knowledge of HIV is categorized as low, moderate, and comprehensive. If the scores of the six domains of HIV knowledge measurement are summed from 0 to 2 it is categorized as low level of HIV know- ledge. If the sum of the response to the six domains is between 4 and 5, it is categorized as a moderate level of HIV knowledge. If the sum of the response to the six do- mains of HIV knowledge scores six, it is categorized as comprehensive HIV knowledge. Likewise, stigma to- wards HIV patients was categorized based on six do- mains of measurement. The youths were categorized as having no stigma towards HIV patients if the scores of HIV VCT uptake The current study revealed that the overall representa- tion of the prevalence of VCT uptake among Ethiopian youths was estimated to be 34.9% and a 95% CI [33.5, Page 4 of 11 Page 4 of 11 Nigatu et al. BMC Infectious Diseases (2021) 21:334 36.0%]. Among the youths ever been tested for HIV, the youths living in Dire-Dawa special district were the lead- ing where 55.9% of them were tested followed by Gam- bella and Addis Ababa youths where 54.7 and 51.6% were respectively tested while the Somali region’s youths registered the lowest testing figure where only 13.2% were tested for the disease (Fig. 1). them had low HIV knowledge. The Majority, more than two-thirds, 69.6% of them did not discuss FP with healthcare workers in the last few months before the survey. Regarding the coverage of health insurance among Ethiopian youths included in the current survey, only 5.4% of youths were covered with health insurance. From all married-youths included in the survey, 66.6 and 67.9% of youths were from households where re- spondents and their wives or their partners jointly de- cide on respondents’ health care and large households’ purchase respectively. The majority, 88.8% of the youths replied that the most recent sex partner relationship they had was a spousal relationship (Table 2). Characteristics of participants Ten thousand one hundred thirteen (10,903) Ethiopian youths participated in the 2016 EDHS. The mean age of the study participants was 19.1 (+ 2.8) years. Regarding their religion, orthodox Christian followers were the dominant religious group (43.9%) followed by Muslims (30.4%). Concerning their economic and marital status, nearly half (49.0%) of the participants were rich, and more than two-thirds (69.7%) of them were never in marital union. More than three-fourth (76.5%) of the youths who participated in the study were from male- headed households. Agriculture is the leading type of oc- cupation constituting 33.7% of all types of work captured in the survey. The majority, more than three-fourth, (78.0%) of the youths included in the study were rural dwellers (Table 1). The HIV VCT uptake and concomitant factors Multilevel logistic regression was fitted to identify inde- pendent predictors of HIV VCT uptake among Ethiop- ian Youths. The intra-class coefficient was calculated to assess the regional variation of HIV VCT uptake among the youths. Accordingly; knowledge of HIV, wealth sta- tus, risky sexual behavior, religion, educational level, marital status, occupational status, owning a mobile phone, frequency of listening to the radio, and gender were the predictors of HIV VCT uptake among Ethiop- ian youths. Of all Ethiopian youths who participated in the study, 11.4% of them had risky sexual behavior, whereas, 63.2 and 30.5% of youths had moderate to a high level of stig- matized attitude towards PLWH. Regarding their know- ledge of HIV, less than one-third, 30.3% of them had comprehensive HIV knowledge, whereas, one-fifth of The odds of getting tested for HIV for Ethiopian youths who had moderate and comprehensive HIV knowledge were nearly 1.78 (AOR = 1.78, 95% CI: 1.45– 2.19) and 2.16 (AOR = 2.16, 95% CI: 1.73–2.72) times higher compared to their peers who had low HIV Fig. 1 HIV VCT uptake among the Ethiopian youths by regions in the country, 2016 EDHS Fig. 1 HIV VCT uptake among the Ethiopian youths by regions in the country, 2016 EDHS Page 5 of 11 Nigatu et al. The HIV VCT uptake and concomitant factors BMC Infectious Diseases (2021) 21:334 Table 1 Socio-demographic characteristics of Ethiopian youths, EDHS 2016 Variables Categories Total (%) Ever been tested for HIV Yes No N % N % Religion Orthodox 4647 (43.9) 1980 42.6 2667 57.4 Protestant 2500 (23.6) 810 32.4 1690 67.6 Muslim 3222 (30.4) 846 26.3 2376 73.8 Other 299 (2.2) 57 24.9 172 75.2 Wealth Status Poor 3450 (32.6) 835 24.2 2615 75.8 Middle 1960 (18.5) 598 30.5 1362 69.5 Rich 5187 (49.0) 2260 43.6 2928 56.4 Marital Status Never in union 7388 (69.7) 2040 27.6 5349 72.4 Married 2684 (25.3) 1363 50.8 1322 49.2 Other 525 (5.0) 291 55.3 235 44.7 Sex of household head Male 8104 (76.5) 2756 34.0 5348 66.0 Female 2494 (23.5) 937 37.6 1557 62.4 Occupational Status Not working 4262 (40.2) 1358 31.9 2905 68.2 Agricultural Workers 3572 (33.7) 988 27.7 2584 72.3 Professionals 253 (2.4) 199 78.8 54 21.2 Trade/Sales 1014 (9.6) 529 52.2 485 47.9 Elementary work 968 (9.1) 334 34.5 634 65.5 Others 528 (5.0) 285 54.0 243 46.1 Residence Urban 2335 (22.0) 1200 51.4 1134 48.6 Rural 8263 (78.0) 2493 30.2 5770 69.8 Educational level No education 1773 (16.7) 411 23.2 1362 76.8 Primary 6076 (57.3) 1783 29.3 4294 70.7 Secondary 2095 (19.8) 1046 49.9 1048 50.1 Higher 654 (6.2) 453 69.3 200 30.7 Sex of the respondent Female 6143 (58.0) 2315 37.7 3828 62.3 Male 4455 (42.0) 1378 30.9 3077 69.1 knowledge. On the other hand, Ethiopian youths’ wealth status was also significantly associated with their HIV test uptakes. The odds of getting HIV tests for Ethiopian youths with middle and rich wealth sta- tus were nearly 1.28 (AOR = 1.28, 95%CI: 1.07–1.53) and 1.72 (AOR = 1.72, 95%CI: 1.39–2.14) times higher compared to their poor peers. The odds of getting HIV tests for youths who had risky sexual behavior were about 2.39 (AOR = 2.39, 95%CI: 2.04–2.80) times higher compared to youths who had no risky sexual behavior. Ethiopian youths’ religions were also inde- pendently predicted their HIV test uptake; for Prot- estant, Muslim, and other religions follower youths, the odds of getting HIV test were lower by 23% (AOR = 0.77, 95%CI: 0.63–0.95), 22% (AOR = 0.78, 95%CI: 0.66–0.91) and 34% (AOR = 0.66, 95%CI: 0.53–0.82) compared to Orthodox Christians youths, respectively. The HIV VCT uptake and concomitant factors For Ethiopian youths who completed primary, second- ary and higher education the odds of getting HIV test was 1.46 (AOR = 1.46, AOR = 1.13–1.87), 2.47 (2.47, 95%CI: 1.85–3.29), and 3.49 (AOR = 3.49, 95%CI: 2.57– 4.73) times higher compared to their Ethiopian unedu- cated peers. The odds of getting HIV tests for married youths and youths with other marital status were nearly 5.37 (AOR = 5.37, 95%CI: 3.72–7.74) and 4.60 (AOR = 4.60, 95%CI: 3.41–6.21) times higher compared to their peer youths who are not in marital union. For profes- sional worker youths, trade/sales worker youths, and youths who were engaged in other occupations, the odds of getting HIV test were 1.87 (AOR = 1.87, 95%CI = 1.14–3.07), 1.45 (AOR = 1.45, 95%CI: 1.03–2.06) and 1.60 (AOR = 1.60, 95%CI: 2.20–2.13) times higher com- pared to their Ethiopian peer youths who were not en- gaged in any kind of occupation. Regarding mobile telephone ownership, the odds of getting an HIV test for Nigatu et al. BMC Infectious Diseases (2021) 21:334 Page 6 of 11 Table 2 Behavioral and Individual Characteristics of Ethiopian Youths, 2016 EDHS Variables Categories Total (%) Ever been tested for HIV Yes No N % N % Risky Sexual Behavior No 9389 (88.6) 2935 31.3 6454 68.7 Yes 1209 (11.4) 758 62.7 451 37.3 Stigma Status No 635 (6.3) 280 44.2 355 55.8 Moderate 6347 (63.2) 2479 39.1 3868 61.0 High 3060 (30.5) 934 30.5 2127 69.5 Knowledge of HIV Low 2086 (20.9) 482 23.1 1605 76.9 Moderate 4863 (48.8) 1866 38.4 2997 61.6 Comprehensive 3023 (30.3) 1341 44.3 1683 55.7 Discussion about FP with HWs in a month No 1434 (69.6) 744 51.9 690 48.2 Yes 628 (30. The HIV VCT uptake and concomitant factors 5) 417 66.4 211 33.6 Covered by health insurance No 10,028 (94.6) 3450 34.4 6578 65.6 Yes 569 (5.4) 243 42.6 327 57.4 A person who should have a greater say: respondent’s health care Respondent alone 308 (13.4) 186 60.2 123 39.8 Respondent and wife/partner 1531 (66.6) 816 53.3 715 46.7 Wife/partner alone 436 (19.0) 170 39.0 266 61.0 Someone else 16 (0.7) 6 37.3 10 62.7 Other 6 (0.3) 2 28.3 4 71.7 A person who usually decides large on household purchases Respondent alone 183 (8.0) 98 53.4 85 46.6 Respondent and wife/partner 1561 (67.9) 860 55.1 701 44.9 Wife/partner alone 523 (22.8) 212 40.5 311 59.5 Someone else 26 (1.13) 7 25.2 19 74.8 Other 5 (0.2) 4 75.3 1 24.7 Relationship with the most recent sex partner Spouse 2226 (88.8) 1156 51.9 1070 48.1 Girlfriend/fiancé 161 (6.4) 111 68.8 50 31.2 Casual acquaintance 11 (0. 5) 6 50.4 6 49.6 Commercial sex worker 1 (0.1) 1 100.0 0 0.0 Live-in partner 107 (4.3) 55 51.8 51 48.3 Other 1 (0.03) 0 10.9 1 89.1 Table 2 Behavioral and Individual Characteristics of Ethiopian Youths, 2016 EDHS Variables Categories Relationship with the most recent sex partner Ethiopian youths at a national level would have resulted in a biased estimate had we had not considered the re- gional variation. The median odds ratio (MOR) was esti- mated to be 1.94 (Table 3). mobile-owner Ethiopian youths was 1.88 (AOR = 1.88, 95%CI: 1.61–2.20) times higher compared to their Ethi- opian peers who did not own mobile. Concerning access to media, the odds of getting an HIV test for Ethiopian youths who listen to the radio at least once a week was 1.33 (AOR = 1.33, 95%CI: 1.12–1.57) times higher com- pared to their Ethiopian peers who did not listen to the radio. Likewise, the odds of getting HIV tests for male Ethiopian youths were lower by 30% (AOR = 0.70, 95%CI: 0.60–0.83) compared to their female counterparts. Ethiopian youths at a national level would have resulted in a biased estimate had we had not considered the re- gional variation. The median odds ratio (MOR) was esti- mated to be 1.94 (Table 3). Discussion Nearly one-third, 34.9% of the Ethiopian youths have ever got HIV voluntary counseling and testing. This finding is almost consistent with the finding from the study done in Sub-Saharan Africa using secondary data analysis of DHS where 36.5% of youths were ever tested for HIV [27]. However, the current finding is lower than the findings from the studies done in Uganda, South Af- rica, and Tanzania where 81.8, 52.2, and 40% of youths were ever tested for HIV [28–30] and is greater than the finding from Nigeria where less than one-fourth (23.7%) The result of ICC showed that 9.0% (±4.7%) of vari- ation in the likelihood of HIV testing uptake among the Ethiopian Youths was explained by the regional variation which was statistically significant (95% CI:3.1–23.4%). This indicates that estimating HIV testing uptake among Page 7 of 11 Nigatu et al. BMC Infectious Diseases (2021) 21:334 y Logistic Regression Using Data from Ethiopians Youths, 2016 EDHS Robust Std. Err. Z P > z AOR [95% Conf. Int.] 0.190 5.4 0.0000 1.78 [CI (1.45, 2.19)] 0.249 6.7 0.0000 2.16 [CI (1.73, 2.71)] 0.114 2.8 0.0050 1.28 [CI (1.07, 1.53)] 0.189 5 0.0000 1.72 [CI (1.39, 2.14)] 0.195 10.7 0.0000 2.39 [CI (2.04, 2.80)] 0.081 −2.5 0.0130 0.77 [CI (0.63, 0.95)] 0.061 −3.2 0.0010 0.78 [CI (0.66, 0.91)] 0.073 −3.8 0.0000 0.66 [CI (0.53, 0.82)] 0.186 3 0.0030 1.46 [CI (1.13, 1.87)] 0.360 6.2 0.0000 2.47 [CI (1.85, 3.29)] 0.542 8 0.0000 3.49 [CI (2.57, 4.73)] 1.004 9 0.0000 5.37 [CI (3.72, 7.74)] 0.704 10 0.0000 4.60 [CI (3.41, 6.21)] 0.092 −0.6 0.5640 0.95 [CI (0.78, 1.14)] 0.473 2.5 0.0130 1.87 [CI (1.14, 3.07)] 0.260 2.1 0.0360 1.45 [CI (1.03, 2.06)] 0.194 0.6 0.5640 1.11 [CI (0.78, 1.56)] 0.233 3.2 0.0010 1.60 [CI (1.20, 2.13)] 0.151 7.9 0.0000 1.88 [CI (1.61, 2.20)] 0.070 1.7 0.0860 1.11 [CI (0.98, 1.26)] 0.114 3.3 0.0010 1.33 [CI (1.12, 1.57)] 0.057 −4.3 0.0000 0.70 [CI (0.60, 0.83)] 0.017 −11.4 0.0000 0.07 [CI (0.05, 0.12)] 0.044 0.14 [CI (0.75, 0.26)] of youths were ever tested for HIV [31]. The differences could be attributed to differences in awareness and had low HIV knowledge. This finding is consistent with the results from the study done using data from four Table 3 Results from Multilevel Binary Logistic Regression Using Data from Ethiopians Youths, 2016 EDHS VCT uptake Robust Std. Err. Z P > z AOR [95% Conf. Discussion Int.] Knowledge of HIV (Ref.:Low) Moderate 0.190 5.4 0.0000 1.78 [CI (1.45, 2.19)] Comprehensive 0.249 6.7 0.0000 2.16 [CI (1.73, 2.71)] Wealth Status (Ref.: Poor) Middle 0.114 2.8 0.0050 1.28 [CI (1.07, 1.53)] Rich 0.189 5 0.0000 1.72 [CI (1.39, 2.14)] Risky Sexual Behavior (Ref.: No) Yes 0.195 10.7 0.0000 2.39 [CI (2.04, 2.80)] Religion (Ref.: Orthodox) Protestant 0.081 −2.5 0.0130 0.77 [CI (0.63, 0.95)] Muslim 0.061 −3.2 0.0010 0.78 [CI (0.66, 0.91)] Other 0.073 −3.8 0.0000 0.66 [CI (0.53, 0.82)] Educational level (Ref: No Education) Primary 0.186 3 0.0030 1.46 [CI (1.13, 1.87)] Secondary 0.360 6.2 0.0000 2.47 [CI (1.85, 3.29)] Higher 0.542 8 0.0000 3.49 [CI (2.57, 4.73)] Marital Status (Ref.: Never in a union) Married 1.004 9 0.0000 5.37 [CI (3.72, 7.74)] Other 0.704 10 0.0000 4.60 [CI (3.41, 6.21)] Occupational Status (Ref.: Not working) Agricultural Workers 0.092 −0.6 0.5640 0.95 [CI (0.78, 1.14)] Professional Workers 0.473 2.5 0.0130 1.87 [CI (1.14, 3.07)] Trade/Sales 0.260 2.1 0.0360 1.45 [CI (1.03, 2.06)] Elementary occupation 0.194 0.6 0.5640 1.11 [CI (0.78, 1.56)] Others 0.233 3.2 0.0010 1.60 [CI (1.20, 2.13)] Own a mobile telephone Yes 0.151 7.9 0.0000 1.88 [CI (1.61, 2.20)] Freq. of listening to a radio (Ref.: No) Less than once a week 0.070 1.7 0.0860 1.11 [CI (0.98, 1.26)] At least once a week 0.114 3.3 0.0010 1.33 [CI (1.12, 1.57)] Sex of the youths (Ref.: Female) Male 0.057 −4.3 0.0000 0.70 [CI (0.60, 0.83)] _cons 0.017 −11.4 0.0000 0.07 [CI (0.05, 0.12)] Region var.(_cons) 0.044 0.14 [CI (0.75, 0.26)] AOR Adjusted Odds Ratio Table 3 Results from Multilevel Binary Logistic VCT uptake Robust Knowledge of HIV (Ref.:Low) Moderate 0.190 Comprehensive 0.249 Wealth Status (Ref.: Poor) Middle 0.114 Rich 0.189 Risky Sexual Behavior (Ref.: No) Yes 0.195 Religion (Ref.: Orthodox) Protestant 0.081 Muslim 0.061 Other 0.073 Educational level (Ref: No Education) Primary 0.186 Secondary 0.360 Higher 0.542 Marital Status (Ref.: Never in a union) Married 1.004 Other 0.704 Occupational Status (Ref.: Not working) Agricultural Workers 0.092 Professional Workers 0.473 Trade/Sales 0.260 Elementary occupation 0.194 Others 0.233 Own a mobile telephone Yes 0.151 Freq. Discussion p The odds of accepting HIV test among youths who had risky sexual behavior was about 2.4 times more likely than the odds of accepting HIV test among youths who had no risky sexual behavior. This finding is in line with the studies done in southeast Asia, China, Burkina Faso, and Cambodia where youths with higher risk sex- ual behavior received HIV testing more likely than youths with low risky sexual behavior [32, 34, 41, 52]. This could be due to the perceived risk of being infected for people who have risky sexual behavior. An explora- tory qualitative study done in Kampala, Uganda showed a low perceived risk of HIV infection is associated with low HIV test uptake among youths [53]. Regarding the occupational status, professional worker youths, trade/sales worker youths, and youths who were engaged in other occupations were 1.87, 1.45, and 1.60 times more likely to accept the voluntary HIV testing and counseling compared to their Ethiopian peer youths who were not engaged in any kind of occupation. The study in rural Ethiopia also showed that people who are engaged in professional work were more likely to accept HIV testing compared to their peer farmers [57]. How- ever, the study done in Guizhou province, China showed that farmers were more likely to receive HIV voluntary counseling and testing services than other professionals [52]. The discrepancy could be due to differences in HIV-related awareness between farmers in developing and developed countries. p g y Ethiopian youths’ religions were also independently predicted their HIV test uptake; for Protestant, Muslim and other religion follower youths, the odds of getting HIV test were lower by 23.0, 22.0, and 34.0% compared to Orthodox Christian youths respectively. This finding supports the finding from the study done among repro- ductive age Ethiopian women where Christian and Muslim women were less likely to accept HIV testing as compared to Orthodox Christian women [54]. The study in Tanzania among Catholic, Lutheran, and Pentecostal churches parishioners also showed that shame-related HIV stigma is strongly associated with religious beliefs such as the belief that HIV is a punishment from God or that people living with HIV/AIDS (PLWHA) have not followed the Word of God [55]. This could be due to the differences in beliefs and dogmas across different religions. Discussion their Ethiopian uneducated peers. This finding supports the findings from secondary data analysis of DHS which were done in Cambodia, Burkina Faso, Malawi, Tanzania, and Senegal where academic advancement was highly associated with receiving HIV testing [30, 32, 33, 41, 56]. This could be due to the reason that edu- cated youths have better HIV knowledge and have less stigmatized attitudes towards PLWH. The current study has shown that youths who have deep insight into HIV better accept HIV testing. p p g On the other hand, Ethiopian youths’ wealth status was also significantly associated with their HIV test up- takes. The odds of getting HIV tests for Ethiopian youths with middle and rich wealth status were nearly 1.28 and 1.72 times higher compared to their poor peers. This finding is similar to the results of many other stud- ies done in Southeast Asia and Africa where youths from households in the highest wealth quintile were more likely to opt-in for HIV testing as compared to youths from households in the poor wealth quintile [31, 33, 41, 42]. This can be explained by differences in access to media and access to health facilities for rich people than poor people. On the other hand, many studies have wit- nessed that exposure to mass media promotes HIV- related knowledge and better creates awareness on HIV [43–51]. The current study and many other aforemen- tioned studies witnessed that HIV-related knowledge boosts HIV test uptake. For married youths and youths with another form of marital status, the odds of getting HIV tests were nearly 5.37 and 4.60 times higher compared to their peer youths who are not in marital union. Studies from Cambodia, Malawi, Tanzania, and Senegal using second- ary DHS data analyses also showed that ever-married women more likely accept HIV testing as compared to never-married women which are consistent with the findings from the current study [30, 33, 41, 56]. There could be different possible reasons for this. Married youths seek health services, especially services related to pregnancy, and maybe counseled to receive HIV testing by their health care providers. On the other hand, mar- ried youth may also seek HIV counseling and testing services if they doubt their partner’s HIV serostatus. Married youths may also make joint-decisions to seek HIV counseling and testing to safeguard their marriage and the health of their current or future child/children. Discussion of listening to a radio (Ref.: No) Less than once a week 0.070 At least once a week 0.114 Sex of the youths (Ref.: Female) Male 0.057 _cons 0.017 Region var.(_cons) 0.044 AOR Adjusted Odds Ratio Primary 0.186 3 0.0030 1.46 [CI (1.13, 1.87)] Secondary 0.360 6.2 0.0000 2.47 [CI (1.85, 3.29)] Higher 0.542 8 0.0000 3.49 [CI (2.57, 4.73)] Marital Status (Ref.: Never in a union) Married 1.004 9 0.0000 5.37 [CI (3.72, 7.74)] Other 0.704 10 0.0000 4.60 [CI (3.41, 6.21)] Occupational Status (Ref.: Not working) Agricultural Workers 0.092 −0.6 0.5640 0.95 [CI (0.78, 1.14)] Professional Workers 0.473 2.5 0.0130 1.87 [CI (1.14, 3.07)] Trade/Sales 0.260 2.1 0.0360 1.45 [CI (1.03, 2.06)] Elementary occupation 0.194 0.6 0.5640 1.11 [CI (0.78, 1.56)] Others 0.233 3.2 0.0010 1.60 [CI (1.20, 2.13)] Own a mobile telephone Yes 0.151 7.9 0.0000 1.88 [CI (1.61, 2.20)] Freq. of listening to a radio (Ref.: No) Less than once a week 0.070 1.7 0.0860 1.11 [CI (0.98, 1.26)] At least once a week 0.114 3.3 0.0010 1.33 [CI (1.12, 1.57)] Sex of the youths (Ref.: Female) Male 0.057 −4.3 0.0000 0.70 [CI (0.60, 0.83)] _cons 0.017 −11.4 0.0000 0.07 [CI (0.05, 0.12)] Region var.(_cons) 0.044 0.14 [CI (0.75, 0.26)] AOR Adjusted Odds Ratio had low HIV knowledge. This finding is consistent with the results from the study done using data from four Sub Saharan countries: Burkina Faso, Senegal, South Af- rica, Southeast Asia, and Kenya, where youths who had higher knowledge of HIV opted in HIV test uptake [27, 29, 32–36]. This might be due to the reason that people with better HIV knowledge have less misconception on the disease and less stigmatized attitude towards PLWH and have higher odds of being tested for HIV. Studies of youths were ever tested for HIV [31]. The differences could be attributed to differences in awareness and knowledge of HIV between youths residing in different countries. In the current study, Youths’ HIV knowledge was in- dependently associated with their HIV VCT uptake. The odds of getting HIV tests for Ethiopian youths who had moderate and comprehensive HIV knowledge were nearly 1.78 times higher compared to their peers who Nigatu et al. BMC Infectious Diseases (2021) 21:334 Page 8 of 11 Page 8 of 11 have witnessed that people with comprehensive know- ledge of HIV have a less stigmatized attitude towards people living with HIV [37–40]. Conclusions Regardless of all efforts put in a place to prevent HIV/ AIDS in Ethiopia since the beginning of the disease in the country, still only about one-third (34.9%) of Ethiop- ian Youths have ever tested for HIV and know their HIV serostatus. The study also revealed that there are signifi- cant disparities in HIV voluntary counseling and testing uptake among Ethiopian youths living in different re- gions of the country. Youths who have less HIV know- ledge, who are from poor households, who do not have risky sexual behavior, who are protestant Christian and Muslims, who are male, who are less educated, who are not in marital union, who are not professional workers, who do not listen to the radio and who do not have a mobile phone are less likely to accept HIV counseling and testing as compared to their counterpart peers. One of the strategies launched by UNAIDS and other part- ners to control HIV was, diagnosing 90% of people living with HIV by 2020. The Democratic Republic of Ethiopia has also committed to ending AIDS as a public health threat by 2030. But with the current unacceptable lower magnitude of HIV counseling and testing uptake among Ethiopian youths, achieving this target is unthinkable. Therefore, any stakeholder working on HIV prevention and control in the country should give due emphases to promoting youths’ academic advancement by integrating and streamlining HIV-related education in academic curricula, expanding mobile and other media coverages, promoting HIV-related awareness creation through community-based educations and religious organiza- tions, and encouraging and strengthening youths to par- ticipate in professional works to boost the current unacceptably low level of VCT among them to meet the ambitious plan of ending HIV as a public health threat by 2030. The intra-class correlation was used to capture the magni- tude of variability concerning the prevalence of VCT uptake across the regions in Ethiopia. The result of ICC showed that 9.0% (±4.7%) of variation in the likelihood of HIV testing up- take among the Ethiopian Youths was explained by the re- gional variation which was statistically significant. This indicates that estimating HIV testing uptake among Ethiop- ian youths at a country level would have resulted in a biased estimate had we had not considered the regional variation. AIDS: Acquired immune deficiency syndrome; AOR: Adjusted Odds Ration; ART: Anti-Retroviral Therapy; CI: Confidence Interval; EA: Enumeration Area; EDHS: Ethiopian Demographic and Health Survey; FP: Family Planning; HIV: Human Immunodeficiency Virus; HW: Health Workers; ICC: Intra class correlation; MOR: Median Odds Ratio; OR: Odds Ratio; PLWHIV: People living with HIV; PPS: Probability Proportionate to Size; PPS: Probability Discussion The gender dynamics and perceptions of HIV test- ing study done in Lesotho showed that males usually say HIV testing is for females [58]. On the other hand, fe- males do have a high probability of getting HIV counsel- ing and testing during the antennal follow-up during pregnancy and during postnatal care after child delivery. Discussion This finding is consistent with the finding from the study done in Sub Saharan Africa using sec- ondary data analysis of DHS where the odds of getting tested for HIV among male youths was 68% less likely than the odds of getting tested among their female peers [27] and the study in South Africa where female Youths more likely accepted HIV testing than their male peers [29]. This could be due to the less risk perception and the reluctance of males to seek HIV counseling and test- ing. The gender dynamics and perceptions of HIV test- ing study done in Lesotho showed that males usually say HIV testing is for females [58]. On the other hand, fe- males do have a high probability of getting HIV counsel- ing and testing during the antennal follow-up during pregnancy and during postnatal care after child delivery. The intra-class correlation was used to capture the magni- tude of variability concerning the prevalence of VCT uptake across the regions in Ethiopia. The result of ICC showed that 9.0% (±4.7%) of variation in the likelihood of HIV testing up- take among the Ethiopian Youths was explained by the re- gional variation which was statistically significant. This indicates that estimating HIV testing uptake among Ethiop- ian youths at a country level would have resulted in a biased estimate had we had not considered the regional variation. From the multilevel model it was estimated that if a youth moved to another region with a higher probability of HIV testing uptake, the median increase in their odds of HIV test- ing uptake would be 1.94-fold (MOR = 1.94). For male Ethiopian youths, the odds of getting tested for HIV was lower by 30% compared to their female counterparts. This finding is consistent with the finding from the study done in Sub Saharan Africa using sec- ondary data analysis of DHS where the odds of getting tested for HIV among male youths was 68% less likely than the odds of getting tested among their female peers [27] and the study in South Africa where female Youths more likely accepted HIV testing than their male peers [29]. This could be due to the less risk perception and the reluctance of males to seek HIV counseling and test- ing. Conclusions From the multilevel model it was estimated that if a youth moved to another region with a higher probability of HIV testing uptake, the median increase in their odds of HIV test- ing uptake would be 1.94-fold (MOR = 1.94). The current study has its strengths and limitations. The EDHS data is the most reliable data as the survey method was methodologicaly rigorious. A large number of Ethiopian youths were proportionally selected from every region of the country to ensure national represen- tation of the youths who were included in the study. To minimize bias that could have been introduced as a re- sult of clustering effects, the authors weighted the DHS data before conducting further analyses. To account for the variation of levels of HIV counseling and testing across the regions in the country, moreover, multilevel logistic regression modeling was done. Discussion Regarding mobile telephone ownership, the odds of getting an HIV test for mobile owner Ethiopian youths was 1.88 times higher compared to their Ethiopian peers who did not own mobile. The finding is in line with the finding of the study done in Senegal using the secondary data analyses of DHS data where women and men mo- bile owners were respectively 40 and 90% more likely to accept HIV testing services as compared to non-mobile owners [33]. Ethiopian youths who listen to the radio at least once a week were 1.33 times more likely to accept HIV voluntary counseling and testing compared to their The odds of getting HIV tests for Ethiopian youths who completed primary, secondary, and higher educa- tion were 1.45, 2.47, and 3.49 times higher compared to Page 9 of 11 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Ethiopian peers who did not listen to the radio. The findings from secondary data analysis of DHSs data from 27 SSA countries and DHS data from India showed that people who frequently watch television and frequently listen to the radio had more comprehensive HIV know- ledge than people who do not [45, 47]. Many other stud- ies [29, 44, 46, 48, 49, 51] had also witnessed that exposure to mass media has an independent impact on acquiring comprehensive HIV knowledge. The current study and other aforementioned studies have witnessed that better knowledge of HIV was associated with better HIV counseling and testing uptake. On the other hand, the current study does not assure the temporal relationship between HIV test uptake among Ethiopian youths and its predictors as data re- lated to both the outcome and predictor variables were simultaneously collected, and the yielded evidence should be utilized cautiously. Furthermore, due to the absence of qualitative data from the 2016 EDHS, the in- vestigators failed to investigate the link between socio- cultural factors and HIV test uptake among the youths. Lastly, the only variable we have at the community level was ‘region’ for which intraclass correlation was reported. that better knowledge of HIV was associated with better HIV counseling and testing uptake. For male Ethiopian youths, the odds of getting tested for HIV was lower by 30% compared to their female counterparts. Consent for publication Not applicable. Not applicable. Availability of data and materials The Stata format datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The Stata format datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 18. WHO. HIV testing and counseling; consolidated ARV guidelines, June 2013, Geneva; 2013. 18. WHO. HIV testing and counseling; consolidated ARV guidelines, June 2013, Geneva; 2013. Abbreviations Page 10 of 11 Page 10 of 11 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Nigatu et al. BMC Infectious Diseases (2021) 21:334 Proportionate to the Size; SD: Standard Deviation; TV: Television; UNAIDS: United Nations Program on HIV/AIDS; VCT: Voluntary Counseling and Testing; WHO: World Health Organization 10. WHO. Ethiopia HIV Country Profile: 2016. Geneva; 2017. 10. WHO. 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Factors associated with knowledge and awareness of HIV/aids among married women in Bangladesh: evidence from a nationally representative survey. Sahara J. 2018;15(1):121–7. https://doi.org/10.1080/17290376.2018.1523022. 2018;15(1):121–7. https://doi.org/10.1080/17290376.2018.1523022. 52. Ma W, Detels R, Feng Y, Wu Z, Shen L, Yu L, et al. Acceptance of and barriers to voluntary HIV counselling and testing among adults in Guizhou province, China. AIDS. 2010;21(8):1–10. 53. Aluzimbi G, Lubwama G, Muyonga M, Hladik W. HIV testing and risk perceptions : a qualitative analysis of secondary school students in Kampala, Uganda. J Public Health Africa. 2017;8(577):1–6. 53. Aluzimbi G, Lubwama G, Muyonga M, Hladik W. HIV testing and risk perceptions : a qualitative analysis of secondary school students in Kampala, Uganda. J Public Health Africa. 2017;8(577):1–6. 54. Erena AN, Shen G, Lei P. Factors affecting HIV counselling and testing among Ethiopian women aged 15–49. 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https://openalex.org/W4283451637	https://zenodo.org/records/6717730/files/Abdullajonova%20Oyxon%20Sherzodbek%20qizi.pdf	Tagalog	null	CHINGIZ AYTMATOV VA LUQMON BO'RIXON IJODIDA MUSHTARAK G'OYALAR TALQINI ("BIRINCHI MUALLIM" VA "SIRLI MUALLIM" QISSALARI TAHLILI MISOLIDA)	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	991	Talqin va tadqiqotlar respublika ilmiy-uslubiy jurnali №5 Talqin va tadqiqotlar respublika ilmiy-uslubiy jurnali №5 Talqin va tadqiqotlar respublika ilmiy-uslubiy jurnali №5 Abdullajonova Oyxon Sherzodbek qizi Qo’qon davlat pedagogika instituti,talaba Annotatsiya: Ushbu maqolada jahon adabiyotining yetuk namoyandasi Chingiz Aytmatovning “Birinchi muallim” va XX asr o’zbek adabiyotining ist’edodli vakili Luqmon Bo’rixonnning “Sirli muallim” qissalaridagi mushtarak g’oyalar tahlilga tortilgan. Kalit so’zlar : mavzu,g’oya, janr, obraz, uslub, qiyosiy tahlil. Tabiat va jamiyatda asosan takrorlanuvchi, bir-biriga o’xshash yoki o’z tabiati, tuzilishi, mohiyati bilan bir-biriga zid bo’lgan narsa-hodisalar qiyoslash obyekti bo’lib xizmat qiladi. Bu hol adabiyotshunoslik fani, xususan qiyosiy adabiyotshunoslik uchun ham badiiy-estetik metod vazifasini bajaradi. Adabi- yotshunoslikda ma’lum bir yozuvchi yoki shoir ijodini milliy va xalqaro adabiy an’analar bilan qiyoslab o’rganish muhim ilmiy- nazariy ahamiyatga ega. Chunki, shunday qiyoslashlar orqali yozuvchining ijodiy mustaqilligi va uning ham milliy, ham jahon adabiyotidagi o’rni belgilanadi. Yozuvchi ijodining o’ziga xos qirralarini o’rganishda va anglab yetishda uni boshqa bir ijodkorning o’xshash asarlari bilan qiyoslab tahlil qilish muhimdir. Qaysi mashhur asarni olmaylik, u avvalo, ma’lum bir millat vakilining ijodiy merosi, u yoki bu xalqning ma’naviy boyligi sanaladi. Masalan, Chingiz Aytmatov asarlari qirg’iz xalqining ma’naviy boyligi bo’lishi bilan birga, butun jahon xalqlarining adabiy merosi sanaladi. Chunki, adib asarlarida ifodalangan insonparvarlik, mardlik, do’stlik, sadoqat kabi umumbashariy g’oyalar barcha xalqlar uchun birdek qadrli va ahamiyatlidir. Biz quyida Chingiz Aytmatovning “Birinchi muallim” va o’zbek adabiyotida o’z o’rniga ega Luqmon Bo’rixonning “Sirli muallim” qissalaridagi mushtarak g’oyalar va o’ziga xosliklar haqida so’z yuritishni maqsad qildik. Bir qarashda turli ijtimoiy-tarixiy davrda yashagan va turli milliy ma’daniy qatlamga mansub ikki qahramonning mushtarak jihatlari mavjudligi kishini hayratga soladi. “Birinchi muallim” va “Sirli muallim” qissalariga diqqat qaratadigan bo’lsak, ularda turli davr voqealari badiiy ifoda topgan bo’lsada, syujetining shakllantirilishida mushtaraklik bor. Ikkala asar ham realistik uslubda yozilganligi sababli voqealar sabab-oqibat zanjirida rivojlanib boradi. Har ikki asarning bosh qahramoni o’qituvchi. O’qituvchi bo’lganda ham millatning kelajagi bo’lgan, yoshlarning ziyoli bo’lishini chin dildan istagan va o’z imkoniyati darajasida kurashgan fidoyi o’qituvchi. “Birinchi muallim” 25 Talqin va tadqiqotlar respublika ilmiy-uslubiy jurnali №5 asarida Duyshen- kichkina qirg’iz ovulida katta voqealarga sabab bo’lgan. Egniga qora movutdan tikilgan shinel kiyib yuradigan baland bo’yli, burgut qovoq, salobatli bu yosh yigit: chala savod qirg’iz ovulini savodli qilishda, muallimlikdek ulug’ kasbni zimmasiga olgan. Allaqaysi boydan qolgan otxonani “maktab” dek ulug’ dargohga aylantirgan jasoratli inson. “Sirli muallim” asari qahramoni Najot G’aybulla- baland bo’yli, kelishgan, yuzlaridan allanechuk nur yog’ilib turadigan, qiroat bilan salomlashadigan, hamisha yangi va orasta kiyimlar kiyadigan, yurish- turishi viqorli bir yigit.Bu ikki muallimning tashqi ko’rinishida va ust boshida yer bilan osmoncha farq ko’rsakda, lekin ruhiy olami birdek. Duyshen va Najot G’aybullaning bir-biriga o’xshash sifatlari anchagina. Abdullajonova Oyxon Sherzodbek qizi Qo’qon davlat pedagogika instituti,talaba Muallimlikning eng oliy cho’qqisida turuvchi bu ikki inson bolalarni va o’z kasblarini chin yurakdan sevishi bilan atrofdagilardan ajralib turishadi. Ulardagi jasorat tirishqoqlik, maqsadlariga erishish yo’lida har qanday to’siqqa bosiqlik ila bas kelishlari, ulardagi mushtarak sifatlarning yana bir isbotidir. Duyshenning: “O’zim nimani bilsam, sizlarga ham shuni o’rgataman…” kabi so’zlaridan, yetti-pushti savodsiz o’tgan bu bolalarni, o’zi hijjalab o’qisada o’qish yozishga o’rgatishi qahramonlikdir. Najot muallim ham darsliklarsiz va sharoiti og’ir chekka qishloq bolalarini, darsliklar bilan ta’minlashi, ularga o’zlari o’rganayotgan fanni anglab yetishlari uchun turli imkoniyatlar yaratishi chinakam jasoratdir. Eng asosiysi ikkala o’qituvchi ham o’z kasbining “ustasi”, beg’ubor bolalarning hayollarini jonlantira olishadi. “Birinchi muallim” asarida Oltinoy tilidan aytilgan:”Duyshen, otasi temir yo’lda ishlab yurgan kezlari, Mixail Semyonovich degan bir keksa muallimning qo’lida ikki qish o’qigani, keyin armiyaga borib, oqlar bilan urishganlarini hikoya qilganda, biz ham u bilan yonma-yon turib jang qilganday bo’lardik”. Ushbu parcha fikrimiz isbotidir. “Sirli muallim” asarida esa Norxolning tilidan:”Najot G’aybullaning sokin, sohir ovozi sinfxona bo’ylab sirli sirli kezinadi. Xammayoq tinch ummoniga g’arq bo’lganday jim-jit…O’quvchilar allaqachon sinfxonadan “uchib” ketishgan. Ular ustozlariga qo’shilib olis-olis orollarda, suv dunyosining g’aroyibotlari ichida sayr etib yurishibdi…” kabi jumlalari ham bunga isbotdir. Muallimlarning nafaqat o’z shogirdlarining,balki, atrofdagi insonlarning ham o’zgarishlariga sabab bo’la olganliklari ularning o’xshash jihatlaridan biridir. Biroq, ikki muallimimizning farqli jihatlari ham mavjud. Ikki insoning yashash sharoitida judda katta farq sezish mumkin. Va albatta ikkisining muhiti ham farqli. Najot G’aybullaning qo’li barcha narsaga yetsa, Duyshen bundan farqli o’ta nochor deyishimiz mumkin. Najot G’aybullaning imkoniyatlarga boyligi va Duyshenning aqalli, yaxshiroq kiyim kiyishga imkoni yetmasligi buning isbotidir. Ikki asarda ham muallimlarning o’z oldilariga qo’ygan maqsadlari mavjud. “Birinchi muallim” asarida Duyshenning maqsadi bolalarning savodini chiqarish ularni ziyoli insonlarga aylantirish edi. Bolalarda o’zi ko’ra olgan iste’dod va 26 Talqin va tadqiqotlar respublika ilmiy-uslubiy jurnali №5 istiqboldan kelib chiqqan holda o’quvchilarni “katta odam” bo’lishiga ishongandi. Uning bu kabi maqsadini ro’yobga chiqarishda ko’plab to’siqlar bo’ldi, “ tushunchasiz insonlar” farzandining kemoliga ishonch bildirmadilar. Lekin muallimninh Oltinoy Sulaymonovaning “akademik” bo’lib yetishishida uning shunday o’quvchilar tarbiyalaganini va o’zining oliy maqsadiga qisman erishganini ko’ramiz. “Sirli muallim” asarida ham Najot G’aybullaning, o’qituvchilikdek ulug’ kasbni etagidan tutib, kasbiga jon- jahdi bilan yondashib, o’zidan ajoyib izdoshlar qoldirishni maqsad qilganini ko’ramiz. Va albatta, ularning eng oliy maqsadi vatanining, yurtining koriga yarovchi, ziyoli insonlarning sonini orttirishni, maqsad qilishlaridir.Muallimlar arofida yig’ilgan ba’zi insonlarni “gumrohlar” deb atashimiz mumkin. “Birinchi muallim” asarida Oltinoyning yangasi, Satimqul tajang va boshqalarning Duyshenga ters muomalalarini ko’rishimiz mumkin. Abdullajonova Oyxon Sherzodbek qizi Qo’qon davlat pedagogika instituti,talaba Ocharchilikda yashayotgan va ilmning asil mohiyatini anglolmagan bunday insonlar uchun tabiiy hol albatda.Muallimning bolalarni o’qitishi uchun ko’mak berish o’rniga, uni kamsitishdi.Albatta bunday “insonlar” makon va zamon tanlamaydilar. Najot G’aybullaning atrofidagilar esa o’zlari sezmagan xolda unga havas yoki xasad qilishdi. Uning salohiyatiga shubxa bilan qarashdi. X.Xurramovich, Ulash “agranom- shoir” kabi insonlar Najot muallimga doim shubha bilan qarashib, uning faoliyatini to’xtatishga urinishdi. Ularga bepisandlik bilan munosabatda bo’lishdi. Lekin, Duyshenga yordam qo’lini cho’zgan: Sayqal buvi va Qartangboy otani asl inson sifatida ko’rsata olamiz. Qiyoslangan mazkur ikki asar arxitektonasida farq bo’lsada, yozuvchilarning bosh qahramon tanlash tamoyili, ifodalagan mavzu va g’oya nuqtai nazaridan mushtaraklikni ko’rish mumkin. Turli tarixiy-ijtimoiy sharoit, turli mentalitet, turlicha hayot tarziga ega bo’lgan qahramonlarimizni yaqinlashtirgan narsa, bu ularning ruhiy olami, orzu- intilishlarining mushtarakligidir. Ya’ni ilm-ma’rifat orqali dunyoni jaholat va halokatdan asrab qolish. Foydalanilgan adabiyotlar: Foydalanilgan adabiyotlar: 1.Aytmatov Ch. Birinchi muallim.-Toshkent:” O’qituvchi”,1986. 1.Aytmatov Ch. Birinchi muallim.-Toshkent:” O’qituvchi”,1986 2. Luqmon Bo’rixon. Xizir ko’rgan yigit”.-Toshkent: “Sharq”, 2007. 3.Xalliyeva G. Qiyosiy adabiyotshunoslik.-Toshkent:”Akademnashr”,2020 Karimov B. Adabiyotshunoslik metodologiyasi.Toshkent: “Muharrir”2011 27 27 27
https://openalex.org/W2904714032	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0209315&type=printable	English	null	Stability of end-of-life preferences in relation to health status and life-events: A cohort study with a 6-year follow-up among holders of an advance directive	PloS one	2,018	cc-by	7,760	RESEARCH ARTICLE Background Stating preferences about care beforehand using advance care planning and advance directives has become increasingly common in current medicine. There is still lack of clarity what happens over the course of time in relation to these preferences. We wanted to deter- mine whether the preferences about end-of-life care of a person owning an advance direc- tive stay stable after the experience of a life-event; how often advance directives are altered and discussed with family members and physicians over time. Editor: Kevan Hartshorn, Boston University School of Medicine, UNITED STATES Editor: Kevan Hartshorn, Boston University School of Medicine, UNITED STATES Received: November 6, 2017 Accepted: December 4, 2018 Published: December 18, 2018 Matthijs P. S. van WijmenID1, H. Roeline W. Pasman1, Jos W. R. Twisk2, Guy A. M. Widdershoven3, Bregje D. Onwuteaka-Philipsen1 1 Department of Public and Occupational Health, Amsterdam Public Health research institute, VU University Medical Center, Amsterdam, The Netherlands, 2 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands, 3 Department of Medical Humanities, Amsterdam Public Health research institute, VU University Medical Center, Amsterdam, The Netherlands a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 m.vanwijmen@vumc.nl * m.vanwijmen@vumc.nl Design A longitudinal cohort study with a population consisting of people owning the most common advance directives in the Netherlands, with a follow-up of 6-years from 2005 until 2011. Respondents were recruited using two associations that provided the advance directives, Right to Die-NL (n = 4463) and the Dutch Patient Organisation (n = 1263). Each 1.5 year a questionnaire was sent. We analyzed the relationship between variables using generalized estimated equations. Copyright: © 2018 van Wijmen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. OPEN ACCESS Citation: van Wijmen MPS, Pasman HRW, Twisk JWR, Widdershoven GAM, Onwuteaka-Philipsen BD (2018) Stability of end-of-life preferences in relation to health status and life-events: A cohort study with a 6-year follow-up among holders of an advance directive. PLoS ONE 13(12): e0209315. https://doi.org/10.1371/journal.pone.0209315 * m.vanwijmen@vumc.nl Stability of end-of-life preferences in relation to health status and life-events: A cohort study with a 6-year follow-up among holders of an advance directive Matthijs P. S. van WijmenID1*, H. Roeline W. Pasman1, Jos W. R. Twisk2, Guy A. M. Widdershoven3, Bregje D. Onwuteaka-Philipsen1 Introduction Stating preferences about care beforehand using advance care planning and advance directives has become increasingly common in current medicine. Advance directives (ADs) are instru- ments aimed at ensuring medical care at the end of life according to someone’s wishes in case of incompetence. Yet, it is unclear what happens over the course of time in relation to these documents. Are the preferences they are supposed to represent stable, also after changes in health status of their owners or the experience of other life-events? Do their owners make alter- ations in their ADs? How often are these documents discussed, and with whom? Auriemma et al. did a systemic review on stability of preferences of mainly patients and older adults [1]. They found that preferences overall remain stable, with higher stability among patients suffering from a serious illness [1,2] and people who had completed an advance direc- tive [3–7]. However, they also stress the need for further research on this subject, especially on the relation between stability of preferences and a change in health status, because findings until now on this subject are inconclusive. Besides a change in a person’s health status, other type of life-events could also be relevant when it comes to stability of preferences. An indica- tion for this could be our finding in a previous study that the experience of someone suffering a serious illness or a death in a person’s close surroundings was linked to the motivation to draw up an AD [8]. While there is data on stability of preferences, it is not known whether owners of ADs make alterations in the actual documents over the course of time. Next to stability of preferences, communication about ADs is a relevant issue. In recent years it has become increasingly clear that an AD in itself is not enough, and that it is crucial to talk about preferences. This resulted in a focus on advance care planning (ACP), a process in which a person’s preferences are discussed with a caregiver and recorded. Research showed that ACP can improve care at the end of life [9]. Because communication seems to be pivotal for the suc- cess of ADs, communication surrounding ADs over time is an important issue for research. Stability of end-of-life preferences Conclusion The NVVE and NPV helped with the collection of the data by providing the addresses of the respondents and preparing the sending of the questionnaires. The other funders had no role in the data collection. None of the funders had a role in study design, data analysis, decision to publish, or preparation of the manuscript. Our results largely dispute criticism concerning usability of advance directives due to lack of stability of preferences. Whereas a change in health status and the experience of other life- events were not related to instability in preferences, they did increase the odds of communi- cation about advance directives. Because our results show that the possession of an advance directive does not neces- sarily result in frequent discussions between patients and caregivers, a more structured approach like advance care planning might be a solution. Competing interests: The authors have declared that no competing interests exist. Introduction With the study presented in this paper we aimed to fill in some of the blanks on the subjects of stability of preferences and communication by following a cohort of people who possess the most common ADs in the Netherlands for six years. We identified people who experienced a change of health, but also other life-events, and observed if this affected their preferences about the end of life. Besides stability of preferences, we also investigated whether they actually made alterations in their ADs. Finally we looked how often ADs were discussed, with whom and what were factors associated with communication. Results 96.9–98.1% of the respondents who had experienced a life-event had stable preferences. 89.9–93.7% of Right-to-Die-NL-members who had experienced a life-event didn’t make any alterations in their advance directives. During the 6-year course of our study, a minority of both groups didn’t discuss their advance directive with anyone (8.7–16.4%), while a majority didn’t discuss it with physicians (ranging 58.1–95.1%). Factors related to health, such as deterioration in experienced health, increased the odds to discuss advance directives. Funding: This study was funded by the Innovational Research Incentives Scheme VICI 2008 from the Netherlands Organisation for Scientific Research (NWO) (grant number: NWO VICI 916.96.628), Pieter van Foreest Stichting, Right to Die-NL (NVVE) and the Nederlandse Patie¨nten Vereniging (NPV) (all received by BO). PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 1 / 14 Stability of end-of-life preferences by any policy. Dutch physicians are obligated by law to adhere to the wishes put down in an AD, but at the same time they can determine if a treatment at the end of life is medically futile. In practice most decisions about continuing or forgoing care at the end of life are taken with physicians, patients and their family involved [13]. In order to include people with an AD in the cohort, we used two associations who publish the most common standard formats for ADs in the Netherlands. Right to Die-NL (Neder- landse Vereniging voor een Vrijwillig Levenseinde (NVVE) in Dutch), an association that makes an effort for patient autonomy and a self-chosen end of life, provides the most common types of ADs in the Netherlands. Among them a refusal of treatment document (ROTD), a do not resuscitate order (DNR), the appointment of a healthcare proxy and an advance euthanasia directive (AED). The second association, the Dutch Patient Association (translated from Nederlandse Patie¨nten Vereniging (NPV) in Dutch), is Christian orientated and provides one type of AD: the ‘wish-to-live statement’. The content of this AD states that its owner wants to receive proper care, meaning no excessive, medical futile treatments at the end of life, but also no actions with the purpose to actively end life. The membership files of these two associations were used to recruit a random sample of respondents for the cohort. They were given extensive information about the study before they agreed to participate. The return of a completed questionnaire was taken as consent to partici- pate. Individual participants were followed over time by repeatedly sending them question- naires and identifying them with the use of pseudonymous identification numbers. The researchers only received anonymized data, leaving names and addresses under sole control of the associations. The Medical Ethics Review Committee of the VU University Medical Center approved this study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Methods This paper draws on a cohort study on people owning an AD, which took place in the Nether- lands [10]. Only 7% of the Dutch population owns an AD [11,12]. Their use is not promoted 2 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Stability of end-of-life preferences The question about stability of preferences we used for this paper was only answered in connection to the questions about the experience of life-events. We asked if the respondents experienced a life-event in the 1,5 years between this questionnaire and the previous one. The question went as follows: ‘Could you indicate if you experienced one of the following events the past one and a half year?’ Followed by captions like ‘Health (both mental or physical, for instance the diagnosis, treatment or recovery of a disease)’ and then options like ‘A change in my own health’. Respondents could choose from the following options: a change in their own health or the health of a loved-one, the death of a loved-one, the birth of (grand)child or having a new partner. The last two were combined in the experience of a positive life-event for the analyses. Respondents also had the opportunity to describe a life-event that was not amongst the given options. If respondents indicated they had experienced a life-event the pas 1,5 years, the follow-up question was if this had led to changes in their preferences. To measure experienced quality of life we used the Eq5d, a set of questions mainly focused on health status, used to calculate a utility score. [15,16] The subject of adjustments in ADs or the completion of new ADs during the course of the study could only be investigated with NVVE-members, while the fixed nature of the AD of the NPV did not allow alterations. A translated version of the questionnaire used is added as a supplementary file. Statistical analyses We performed descriptive statistics for the main outcome variables used for this paper, which were stability of preferences about the end of life, alterations made in ADs and how often respondents discussed their AD. We then analyzed which factors were statistically associated with making alterations in ADs and communication about them. We looked at background characteristics (gender, age, marital status, the presence of offspring, place of residence, educa- tion and religion) several variables representing (a change in) health status, and the experience of life-events. To analyze above mentioned relationships, we used generalized estimated equations (GEE), which is suited for longitudinal analyses, because it takes into account the fact that multiple observations of one subject are used over time and corrects for within-subject correlation. To analyze the relationship between adjustments in ADs as dependent variable and background characteristics, (a change in) health status and the experience of life-events as independent var- iables, first univariable analyses were performed. We then put all independent variables that were significantly associated in a multivariable model. We removed variables with the highest P-values until only variables with a P-value below 0.05 remained in the model. We did the same for communication about ADs as dependent variable. Regarding instability of prefer- ences we only performed descriptive analysis, because the amount of subjects with instable preferences was too small to perform these analyses well. Population and questionnaires The respondents of the cohort answered questionnaires in Dutch every 1,5 year, the first sent in 2005. These questionnaires contained questions about background characteristics as well as about more specific topics surrounding end of life care and ADs. For this paper respondents were included who indicated they possessed a completed AD at the start of the cohort in 2005 (4463 NVVE-members and 1263 NPV-members). During the progress of the study respon- dents dropped out of the cohort, because they deceased, moved, ended their membership with the association, or choose no longer to participate. The response rates per wave of question- naires ranged from 82–87% for NVVE-members and 84–89% for NPV-members. Due to financial en practical reasons it was not possible for us to extend the cohort after 2011. For previous studies we compared the members of the cohort with the Dutch general public [10,14]. We found that NVVE-respondents were more often single, higher educated and non- religious, while amongst NPV-respondents there were a lot more Protestant-Christians com- pared to the Dutch public. When it came to their views on the end of life, NVVE-members were similar to the general public, but more outspoken. NPV-members differed from the gen- eral public because they more often preferred to continue medical treatment at the end of life. Because of the differences between the two groups in terms of religiosity and views on the end of life, we choose to perform analyses (and present the results) for both groups separately or correct for membership of association (NVVE or NPV) when performing analyses on the pop- ulation as a whole. The questions we focused on for this paper, about preferences, the experience of life-events, adjustments in ADs and communication, were included four times in the questionnaires from the second questionnaire in 2007 till the last in 2011. 3 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 https://doi.org/10.1371/journal.pone.0209315.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Respondent characteristics Table 1 shows the characteristics of the respondents of the cohort. In 2007, 63% of the NVVE- members experienced a life-event, of whom 43% experienced a change in own health, 34% a change in the health of a loved-one and 27% the death of a loved-one. Of the 66% of NPV- members who experienced a life-event, 41% experienced a change in own health, 42% a change in health of a loved-one and 41% a death of a loved-one. The answers to the questionnaires from other years showed similar results. 4 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Stability of end-of-life preferences Table 1. Respondent characteristics. Table 1. Respondent characteristics. Characteristic (measured in 2005, unless reported differently) NVVE-members (n = 4463) NPV-members (n = 1263) Age, mean (SD), in years 68.3 (12.4) 59.5 (17.7) Sex, No. (rounded %) - Male 1577 (36%) 493 (40%) - Female 2832 (64%) 745 (60%) Marital status - Married 2171 (50%) 844 (68%) - Living together 247 (6%) 8 (1%) - Partner otherwise 195 (4%) 18 (2%) - Divorced 341 (8%) 35 (3%) - Widowed 1057 (24%) 159 (13%) - Single otherwise 377 (9%) 173 (14%) Children - Children, good relation 2940 (67%) 838 (68%) - Children, bad relation with some or all 393 (9%) 102 (8%) - No children 993 (23%) 269 (22%) - Otherwise 54 (1%) 20 (2%) Residing in (2007) - Own home 3494 (96%) 1038 (96%) - Otherwise (e.g. Nursing-home, sheltered living) 158 (4%) 45 (4%) Education - Elementary or basic vocational 712 (16%) 436 (35%) - Secondary 1373 (31%) 422 (34%) - Higher 2290 (52%) 375 (30%) Belief and its importance in someone’s life - Important belief 861 (20%) 1212 (97%) - Not important belief or no belief 3464 (80%) 32 (3%) Suffering from a disease - No 1473 (34%) 567 (46%) - Yes 2916 (66%) 659 (54%) Change in experienced health(2007) - No Change 2245 (61%) 722 (66%) - Better 186 (5%) 68 (6%) - Much better 154 (4%) 37 (3%) - Worse 952 (26%) 232 (21%) - Much worse 151 (4%) 33 (3%) Life-events (2007) Any life-event 2221 (63%) 725 (66%) Of which: - Change in own health 945 (43%) 294 (41%) - Change in health of a loved-one 753 (34%) 304 (42%) - Death of a loved-one 596 (27%) 295 (41%) - Postive life-event: birth of a (grand)child or a new partner 441 (20%) 303 (42%) Respondents were asked how their health status was as compared to 1,5 years before. Percentages presented here represent the proportion of specific life events from the total of life events 5 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 201 Stability of end-of-life preferences The experience of life-events, end-of-life preferences and adjustments in ADs After experiencing a life-event, the preferences about the end of life of a vast majority of respondents remained unchanged or became stronger in both groups, ranging from 96.8% to 97.5% for the NVVE and from 97.4 to 98.1% for the NPV for the different intervals between questionnaires (Table 2). Over the four consecutive waves of questionnaires constantly small percentages indicated they were in doubt (ranging 1.4–1.9% for the NVVE and 1.1–1.6% for the NPV) and an even smaller percentages reported that their preferences had changed (rang- ing 1.2–1.6% for the NVVE and 0.4–1.1% for the NPV). Using GEE we tested if there were sig- nificant differences between the different waves when it came to instability of preferences, but there weren’t any (p = 0.514 for the NVVE, p = 0.932 for the NPV and p = 0.608 for both groups together). Because of the small percentages of instable preferences, it was not possible to analyze in what way different factors were associated with instability of preferences. When asked if they made adjustments to their existing ADs or if they drafted a new AD during the one and a half year previous to the questionnaire, a part of NVVE-members answered they did, ranging 6.3–10.1% for the four waves of questionnaires. These differences in percentages of respondents who made adjustments that were found between the four waves of questionnaires were statistically significant when analyzed with GEE (p<0.001). We found that only the experience of the death of a loved one remained significant in a multivariable model (OR = 1.3; 95%-CI 1.1–1.6), when we analyzed whether the experience of a life-event was statistically significantly associated with making adjustments in an AD or a new AD over time. Other variables that remained significant were higher age, having a bad relationship with some or all of your children, a higher education and an experienced health that was less than good (Table 3). Stability of end-of-life preferences Table 2. Changes in preferences and ADs of people who experienced a life-event in the previous 1,5 year. Spring 2007 Autumn 2008 Spring 2010 Autumn 2011 NVVE n = 2221 n = 2063 n = 1885 n = 1645 Did your views or preferences about the end of life change? No change or they became stronger 1815 (97.2%) 1711 (96.8%) 1797 (97.5%) 1561 (96.9%) I’m in doubt 30 (1.6%) 34 (1.9%) 25 (1.4%) 25 (1.6%) They changed 22 (1.2%) 23 (1.3%) 22 (1.2%) 25 (1.6%) Adjustments in existing AD or new AD? 198 (9.2%) 124 (6.3%) 138 (7.5%) 156 (10.1%) NPV n = 725 n = 597 n = 554 n = 482 Did your views or preferences about the end of life change? No change or they became stronger 550 (97.7%) 495 (97.4%) 527 (97.8%) 462 (98.1%) I’m in doubt 9 (1.6%) 8 (1.6%) 6 (1.1%) 7 (1.5%) They changed 4 (0.7%) 5 (1.0%) 6 (1.1%) 2 (0.4%) The subject of adjustments in ADs or the completion of new ADs during the course of the study could only be investigated with NVVE-members, while the fixed nature of the AD of the NPV didn’t allow alterations in it or the addition of new ADs besides it. Table 2. Changes in preferences and ADs of people who experienced a life-event in the previous 1,5 year. The subject of adjustments in ADs or the completion of new ADs during the course of the study could only be investigated with NVVE-members, while the fixed nature of the AD of the NPV didn’t allow alterations in it or the addition of new ADs besides it. The subject of adjustments in ADs or the completion of new ADs during the course of the study could only be investigated nature of the AD of the NPV didn’t allow alterations in it or the addition of new ADs besides it. Having a partner, children, being female and having a belief that is of importance in one’s life increased the odds to talk with family. Higher age, having a higher education and a lower experienced quality of life (measured using the Eq5D), all increased the odds to discuss ADs with a physician. As was also seen in the descriptive figures, membership of the NPV makes it less likely to talk about ADs, with family as well as physicians. Communication about ADs When asked with whom they had spoken about their AD the previous one and a half year, NVVE-members most often indicated their partner (ranging from 49.8% to 58.0% for the four intervals between the questionnaires), followed by their children (ranging from 33.3% to 42.4%), and general practitioner (GP, ranging from 13.7% to 22.2%; Table 4). The answers from the NPV- members showed similar results, with lower percentages: talking with their partner ranged from 30.9% to 44.8%; with their children from 17.2% to 27.3%; with their GP from 4.4% to 15.5%. A substantial part of the respondents did not speak with anyone about their AD in the pre- ceding one and a half year period (percentages ranging 28.7–35.9% for the NVVE and 40.6– 57.5% for the NPV). When analyzing the period as a whole, from 2005 till 2011, a majority talked to somebody about their AD as least once (91.3% of the NVVE-members and 83.6% of the NPV-members). For the whole period, the results show the same order as for the four separate one-and-a-half year periods when it comes to with whom respondents talked most frequently: 81.6% of NVVE-members and 71.9% of NPV-members talked at least once with their partner during the 6 year period, 67.5% (NVVE) and 44.4% (NPV) with their children, 41.9% (NVVE) and 27.9% (NPV) with their GP (Table 4). Almost all factors investigated were significantly associated with communication about ADs univariably, but a smaller number remained so when put in a multivariable model (Table 5). Suffering from a disease as well as a deterioration in experienced health both increased the odds of talking about ADs with family or a physician. The same was found for residing in a care-facility and experiencing the life-events of a change in own health and change in health or death of a loved-one. 6 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Discussion A vast majority (ranging 96.9–98.1%) of people with an AD who experienced a life-event indi- cated their preferences remained unchanged or became stronger. This corresponds with ear- lier findings in research about this subject, showing that stability of preferences is higher among the owners of ADs [3,4,6,7,17]. Nonetheless this is a noteworthy result because we know that part of the experienced life-events, concerning serious health issues of a loved one, are important for the decision to draw up an AD [8]. Also changes in a person’s own health do not seem to influence the stability of preferences. Many previous studies found no association between a change in health and instability of preferences [7,18–24]. From those that did find an association, some found that a change in health alters preferences towards wanting more treatment[3,6], while others found the oppo- site [2,25]. From this Auriemma et al. concluded in their review that the data on this subject was inconclusive [1]. Compared with the studies cited by Auriemma, ours had a longer follow- up, a bigger sample and the population consisted solely of people actually owning an AD. Our findings suggest no association between instability of preferences and the experience of life- events, including a change in health. Of the NVVE-members who had experienced a life-event, the proportion making alter- ations in their AD was larger than the proportion with instable preferences. However, these percentages still represent small minorities (ranging 6.3–10.1%). Combined with our finding that preferences overall remained stable or became stronger, the alterations made in ADs could well be a sharpening of previous statements. Another explanation for the difference between the number of instable preferences and alterations in ADs could be the fact that the NVVE published new versions of ADs during the 7 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 https://doi.org/10.1371/journal.pone.0209315.t003 The EQ5D is a validated set of questions on five domains (mobility, self-care, usual activities, pain and discomfort, anxiety and depression), which can be used to calculate a single index value representing someone’s perceived quality of life. A score of 100% represents full health and a score of 0 represents death. https://doi.org/10.1371/journal.pone.0209315.t003 course of our study. This may have been an incentive for respondents to draft a new one and is unrelated to instability of preferences. It could also explain the statistically significant differ- ences between the 4 waves of questionnaires when it came to the percentages of respondents that made alterations in ADs (ranging 6.3–10.1%). A third explanation of the difference between stability of preferences and alterations in ADs may be found in the way stability of preferences was assessed for this study. Respondents were asked if their views or preferences had changed in general, without specifying. We know from a study by Evans et al. that specific treatment preferences from elderly people do not always concur with their general end-of-life goals [26]. The majority of studies reviewed by Auriemma et al. assessed the stability of preferences by repeatedly asking about specific types of medical care over time [1]. This seems to be a more objective method as compared to the method used for this study. This has to be taken into account when interpreting the results of this study: the stability of preferences could be some- what overestimated. However, in the context of ACP and palliative care in practice the self- perceived preference stability is highly relevant. From the life-events only the experience of the death of a loved one remained significantly associated with alterations in ADs, next to a (subjectively reported) bad health and higher age among others. g People with an AD tend to talk about these documents, as shown by the fact that only small minorities indicated they did not talk at all about this subject during the six-year course of our People with an AD tend to talk about these documents, as shown by the fact that only small minorities indicated they did not talk at all about this subject during the six-year course of our Table 4. Talked about AD? Respondents who reported to have a partner or children at the time they answered the questionnaire that year. p y q y age of the data, we only could include the figures of the respondents who had answered this question in all 4 waves of the stud a partner or children at the time they answered the questionnaire that year. In order to prevent a distorted image of the data, we only could include the figures of the respondents who had answered Stability of end-of-life preferences Table 3. Factors associated with adjustments in ADs or a new AD (only NVVE). (n = 3980, OR’s, 95%-CI’s). Variable univariable multivariable Gender - Male 1.0 - Female 1.0 (0.9–1.2) Age (per year) 1.02 (1.01–1.02) 1.01 (1.01–1.02) Marital status - Married 1.0 - Living together 1.1 (0.8–1.6) - Partner otherwise 1.0 (0.7–1.5) - Divorced 1.3 (1.0–1.7) - Widowed 1.3 (1.1–1.6) - Single otherwise 1.1 (0.8–1.6) Children - Children, good relation 1.0 1.0 - Children, bad relation with some or all 1.5 (1.2–1.8) 1.4 (1.1–1.7) - No children 1.1 (0.9–1.3) 1.2 (1.0–1.4) - Otherwise 1.2 (0.7–2.0) 1.0 (0.6–1.9) Residing in - Own home 1.0 - Otherwise (e.g. Nursing-home, sheltered living) 1.4 (1.0–1.8) Education - Higher 1.0 1.0 - Secondary 0.9 (0.8–1.1) 0.8 (0.7–1.0) - Elementary or basic vocational 0.8 (0.6–1.0) 0.7 (0.5–0.9) Belief and its importance in someone’s life - Important belief 1.0 - Not important belief or no belief 0.9 (0.7–1.1) Experienced health - Very good 1.0 1.0 - Good 1.1 (0.9–1.3) 1.0 (0.8–1.3) - Less than good 1.8 (1.4–2.2) 1.6 (1.3–2.1) Suffering from a disease - No 1.0 - Yes 1.4 (1.2–1.6) Experienced quality of life (EQ5D tariff) Per 10% decrease in score 1.10 (1.06–1.13) Change in experienced health - No Change 1.0 - Better 0.9 (0.6–1.2) - Much better 1.3 (0.9–1.9) - Worse 1.5 (1.2–1.7) - Much worse 2.1 (1.6–2.8) Life-events Did not experience life-event 1.0 Change in own health 1.3 (1.1–1.5) Change in health of a loved-one 1.2 (1.1–1.5) Death of a loved-one 1.4 (1.1–1.5) 1.3 (1.1–1.6) Positive life-event 0.8 (0.7–1.1) (Continued) 8 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Stability of end-of-life preferences Table 3. (Continued) Variable univariable multivariable Any life-event 1.4 (1.2–1.6) The EQ5D is a validated set of questions on five domains (mobility, self-care, usual activities, pain and discomfort, anxiety and depression), which can be used to calculate a single index value representing someone’s perceived quality of life. A score of 100% represents full health and a score of 0 represents death. https://doi.org/10.1371/journal.pone.0209315.t003 Table 3. (Continued) Variable univariable multivariable Any life-event 1.4 (1.2–1.6) The EQ5D is a validated set of questions on five domains (mobility, self-care, usual activities, pain and discomfort, anxiety and depression), which can be used to calculate a single index value representing someone’s perceived quality of life. A score of 100% represents full health and a score of 0 represents death. Respondents who reported to have a partner or children at the time they answered the questionnaire that year. In order to prevent a distorted image of the data, we only could include the figures of the respondents who had answered this question in all 4 waves of the study. https://doi.org/10.1371/journal.pone.0209315.t004 https://doi.org/10.1371/journal.pone.0209315.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 https://doi.org/10.1371/journal.pone.0209315.t004 1/journal.pone.0209315.t004 Autumn 2005- spring 2007 Spring 2007- autumn 2008 Autumn 2008- spring 2010 Spring 2010- autumn 2011 Autumn 2005- spring 2011 NVVE (n = 3713) NPV (n = 1105) NVVE (n = 3510) NPV (n = 1027) NVVE (n = 3016) NPV (n = 915) NVVE (n = 2843) NPV (n = 825) NVVE (n = 2363) NPV (n = 748) With partner 1150 (53.6%) 326 (42%) 987 (49.8%) 324 (44.8%) 973 (58.0%) 265 (40.8% 885 (56.1%) 179 (30.9%) 1155 (81.6%) 389 (71.9%) With Children 1043 (37.4%) 220 (25.9%) 876 (33.3%) 217 (27.3%) 966 (42.4%) 166 (23.3%) 862 (40.6%) 113 (17.2%) 1196 (67.5%) 259 (44.4%) With other family members 262 (7.1%) 73 (6.6%) 226 (6.4%) 68 (6.6%) 244 (8.1%) 50 (5.5%) 203 (7.1%) 34 (4.1%) 429 (18.2%) 116 (15.5%) With GP 609 (16.4%) 107 (9.7%) 480 (13.7%) 159 (15.5%) 619 (20.5%) 101 (11.1%) 632 (22.2%) 36 (4.4%) 991 (41.9%) 209 (27.9%) With medical specialist 92 (2.5%) 18 (1.6%) 85 (2.4%) 17 (1.7%) 101 (3.3%) 17 (1.9%) 95 (3.3) 13 (1.6%) 160 (6.8%) 37 (4.9%) With Others 601 (16.2%) 91 (8.2%) 547 (15.6%) 102 (9.9%) 609 (20.2%) 97 (10.6%) 544 (19.1) 74 (9.0%) 917 (38.8%) 170 (22.7%) With no one 1334 (35.9%) 504 (45.6%) 1191 (33.9%) 417 (40.6%) 972 (32.2%) 413 (45.1%) 817 (28.7%) 474 (57.5%) 205 (8.7%) 123 (16.4%) Table 4. Talked about AD? PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 9 / 14 Stability of end-of-life preferences Table 5. (Continued) Table 5. (Continued) Talked with family Talked with physician Change in own health 1.3 (1.2–1.4) 1.2 (1.1–1.3) 1.8 (1.7–2.0) 1.4 (1.3–1.5) Change in health of a loved-one 1.3 (1.2–1.4) 1.2 (1.1–1.3) 1.2 (1.1–1.3) 1.1 (1.0–1.3) Death of a loved-one 1.3 (1.2–1.4) 1.4 (1.2–1.5) 1.2 (1.1–1.4) 1.2 (1.1–1.3) Positive life-event 1.1 (1.0–1.3) 0.8 (0.7–0.9) Any life-event 1.3 (1.3–1.4) 1.5 (1.4–1.7) For the analyses on communication using GEE, variables that were presented separately for the descriptive analyses in Table 2 were merged. ‘Talked with family’ consists of talking ‘With partner’, ‘With Children’ and ‘With other family members’. ‘Talked with physician’ consists of talked ‘With GP’ and ‘With medical specialist’. The EQ5D is a validated set of questions on five domains (mobility, self-care, usual activities, pain and discomfort, anxiety and depression), which can be used to calculate a single index value representing someone’s perceived quality of life. A score of 100% represents full health and a score of 0 represents death. https://doi.org/10.1371/journal.pone.0209315.t005 For the analyses on communication using GEE, variables that were presented separately for the descriptive analyses in Table 2 were merged. ‘Talked with family’ consists of talking ‘With partner’, ‘With Children’ and ‘With other family members’. ‘Talked with physician’ consists of talked ‘With GP’ and ‘With medical specialist’. The EQ5D is a validated set of questions on five domains (mobility, self-care, usual activities, pain and discomfort, anxiety and depression), which can be used to calculate a single index value representing someone’s perceived quality of life. A score of 100% represents full health and a score of 0 represents death. https://doi.org/10.1371/journal.pone.0209315.t005 study (8.7% for NVVE-members and 16.4% for NPV-members). However, taken into account that communication is essential to the success of ADs [9], there is still progress to be made, especially when it comes to the communication with physicians, which stays behind as com- pared to communication with loved ones. From qualitative studies we know that patients as well as caregivers experience barriers to discuss end-of-life preferences [8,27,28]. Talking about preferences may especially be relevant when there is a change in someone’s health. Our findings do show a change in health status is an incentive, because multiple variables linked to (a change in) health increased the odds to talk about ADs. Strengths of our study are the long study period and the large number of cohort members. Our study also had some limitations. Stability of end-of-life preferences Table 5. Factors associated with communicating about ADs. (n = 5111, OR’s, 95%-CI’s). Talked with family Talked with physician Variable univariable multivariable univariable multivariable Gender - Male 1.0 1.0 1.0 - Female 1.0 (0.9–1.1) 1.2 (1.1–1.3) 1.1 (1.0–1.2) Age (per year) 1.00 (1.00–1.01) 1.03 (1.03–1.04) 1.02 (1.02–1.03) Marital status - Married 1.0 1.0 1.0 - Living together 1.4 (1.2–1.8) 1.5 (1.2–1.9) 1.0 (0.8–1.3) - Partner otherwise 1.1 (0.9–1.4) 1.2 (0.9–1.4) 1.0 (0.7–1.3) - Divorced 0.6 (0.5–0.7) 0.5 (0.4–0.6) 1.5 (1.2–1.8) - Widowed 0.8 (0.7–0.8) 0.6 (0.5–0.6) 1.7 (1.5–2.0) - Single otherwise 0.3 (0.3–0.3) 0.4 (0.3–0.5) 1.2 (1.0–1.5) Children - Children, good relation 1.0 1.0 1.0 - Children, bad relation with some or all 0.9 (0.8–1.1) 0.9 (0.8–1.0) 1.4 (1.2–1.6) - No children 0.5 (0.4–0.5) 0.5 (0.5–0.6) 0.9 (0.8–1.0) - Otherwise 0.7 (0.5–1.0) 0.7 (0.5–0.9) 1.0 (0.7–1.5) Residing in - Own home 1.0 1.0 1.0 1.0 - Otherwise (e.g. Nursing-home, sheltered living) 1.2 (1.0–1.4) 1.3 (1.1–1.6) 2.0 (1.7–2.4) 1.3 (1.0–1.5) Education - Higher 1.0 1.0 1.0 - Secondary 1.0 (0.9–1.1) 0.8 (0.7–1.0) 0.8 (0.7–0.9) - Elementary or basic vocational 0.9 (0.8–1.0) 1.0 (0.8–1.1) 0.9 (0.8–1.1) Belief and its importance in someone’s life - Important belief 1.0 1.0 1.0 - Not important belief or no belief 1.4 (1.3–1.5) 0.9 (0.8–1.0) 1.3 (1.2–1.5) Memberschip of association - Member of NVVE 1.0 1.0 1.0 1.0 - Member of NPV 0.5 (0.5–0.6) 0.4 (0.4–0.5) 0.5 (0.4–0.5) 0.6 (0.5–0.6) Experienced health - Very good 1.0 1.0 - Good 1.1 (1.0–1.2) 1.5 (1.4–1.8) - Less than good 1.1 (1.0–1.3) 2.5 (2.1–2.9) Suffering from a disease - No 1.0 1.0 1.0 1.0 - Yes 1.2 (1.1–1.3) 1.1 (1.0–1.2) 2.0 (1.8–2.2) 1.3 (1.2–1.5) Experienced quality of life (EQ5D tariff) Per 10% decrease in score 1.04 (1.02–1.06) 1.19 (1.17–1.22) 1.08 (1.05–1.12) Change in experienced health - No Change 1.0 1.0 1.0 1.0 - Better 1.1(1.0–1.2) 1.2 (1.0–1.3) 1.2 (1.0–1.4) 1.1 (0.9–1.4) - Much better 0.8 (0.7–1.0) 0.8 (0.6–0.9) 1.3 (1.0–1.6) 1.2 (0.9–1.6) - Worse 1.2 (1.1–1.3) 1.1 (1.0–1.2) 1.9 (1.7–2.1) 1.2 (1.1–1.4) - Much worse 1.4 (1.2–1.7) 1.3 (1.1–1.6) 3.5 (2.9–4.3) 1.7 (1.4–2.2) Life-events Did not experience life-event 1.0 1.0 1.0 1.0 (Continued) Table 5. Factors associated with communicating about ADs. (n = 5111, OR’s, 95%-CI’s). PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 10 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 First, there may have been a recall bias when it comes to communication about ADs and alterations made in ADs. Second, our study in itself, by send- ing the questionnaires every one-and-a-half year, may have been an incentive for the respon- dents to think, talk about or make alterations in their ADs. Third, although we were able to follow our respondents over time in order to assess stability of preferences and the experiences of life-events, this study doesn’t provide data about the end of life of the participants. We know from a study by Bischoff et al. that 37% of patients change their code status near the end of life [29]. On the other hand Bolt et al. found that a change or deterioration in health near the end of life generally did not seem to influence preferences con- cerning euthanasia of people with an advance euthanasia directive [30]. Finally, one can question to which extend the results of our study can be generalized to the general population, due to the fact that ADs in the Netherlands are not promoted by any policy and therefore are not widespread (only 7% of the Dutch population owns an AD)[11,12]. Results of previous studies we performed, where we compared the members of the cohort with the Dutch general public, showed both groups, NVVE- and NPV-members, can be placed at opposite end of the spectrum when it comes to views on then end of life in the Netherlands [10,14]. While not representative for the general public both groups at opposite ends of the spectrum show similar results. Author Contributions Conceptualization: H. Roeline W. Pasman, Guy A. M. Widdershoven, Bregje D. Onwuteaka- Philipsen. Data curation: Matthijs P. S. van Wijmen. Formal analysis: Matthijs P. S. van Wijmen, Jos W. R. Twisk. Funding acquisition: Bregje D. Onwuteaka-Philipsen. Investigation: Matthijs P. S. van Wijmen, H. Roeline W. Pasman, Bregje D. Onwuteaka- Philipsen. Methodology: Matthijs P. S. van Wijmen, H. Roeline W. Pasman, Jos W. R. Twisk, Bregje D. Onwuteaka-Philipsen. Supervision: Bregje D. Onwuteaka-Philipsen. Writing – original draft: Matthijs P. S. van Wijmen. Writing – review & editing: H. Roeline W. Pasman, Jos W. R. Twisk, Guy A. M. Widdersho- ven, Bregje D. Onwuteaka-Philipsen. Supporting information Supporting information S1 File. Dataset used for the paper. SPSS-file, the data is structured long. (SAV) S2 File. Questionnaire used to collect the data for the paper, original version in Dutch. (DOC) S3 File. Questionnaire used to collect the data for the paper, translated version in English. (DOC) S1 File. Dataset used for the paper. SPSS-file, the data is structured long. (SAV) Conclusions The results of our study make an important contribution to disputing the criticism concerning the usability of ADs due to a lack of stability of preferences of their owners. Whereas a change in health status and the experience of other life-events were not related to instability of general self-reported preferences about the end of life, they did increase the odds of communication about ADs. Yet, communication remains an issue of concern. In order to translate general preferences about end of life put down in ADs to specific decisions in practice, communication 11 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209315 December 18, 2018 Stability of end-of-life preferences is vital. Our results show that possession of an AD does not necessarily lead to frequent discus- sions between patients and caregivers. A more structured approach, like ACP, might be useful to foster communication and improve the relevance of ADs in practice. is vital. Our results show that possession of an AD does not necessarily lead to frequent discus- sions between patients and caregivers. A more structured approach, like ACP, might be useful to foster communication and improve the relevance of ADs in practice. References 1. Auriemma CL, Nguyen CA, Bronheim R, et al. Stability of end-of-life preferences: A systematic review of the evidence. JAMA Intern Med. 2014; 174: 1085–1092. https://doi.org/10.1001/jamainternmed. 2014.1183 PMID: 24861560 2. Carmel S, Mutran EJ. Stability of elderly persons’ expressed preferences regarding the use of life-sus- taining treatments. Soc Sci Med. 1999; 49: 303–311. PMID: 10414816 3. Danis M, Garrett J, Harris R, Patrick DL. Stability of Choices about Life-Sustaining Treatments. 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https://openalex.org/W4281385127	https://journal.kdpu.edu.ua/ped/article/download/5446/4981	Ukrainian	null	CURRENT STATE OF THE PROBLEM OF FORMATION CULTURES OF COMMUNICATION OF THE FUTURE TEACHERS BY DIALOGICAL MEANS LEARNING SITUATIONS	Osvìtnìj vimìr	2,022	cc-by	1,608	выступает одной из важных характеристик управления механизмами ускорения познавательной деятельности. «Человек действует либо на основе потребностей своего организма, либо на основе интересов и целей своего общества, класса или социальных групп, если эти цели и интересы стали активной частью его собственных убеждений, либо его вынуждают действовать требования общества, государства и коллектива» [1,91]. Стремление студентов к постоянному приобретению новых знаний и умений, помогающих успешно решать познавательные задачи и приобретать практический опыт, является важным условием качественной педагогической подготовки и решающим фактором их профессионального роста. выступает одной из важных характеристик управления механизмами ускорения познавательной деятельности. «Человек действует либо на основе потребностей своего организма, либо на основе интересов и целей своего общества, класса или социальных групп, если эти цели и интересы стали активной частью его собственных убеждений, либо его вынуждают действовать требования общества, государства и коллектива» [1,91]. Стремление студентов к постоянному приобретению новых знаний и умений, помогающих успешно решать познавательные задачи и приобретать практический опыт, является важным условием качественной педагогической подготовки и решающим фактором их профессионального роста. выступает одной из важных характеристик управления механизмами ускорения познавательной деятельности. «Человек действует либо на основе потребностей своего организма, либо на основе интересов и целей своего общества, класса или социальных групп, если эти цели и интересы стали активной частью его собственных убеждений, либо его вынуждают действовать требования общества, государства и коллектива» [1,91]. Стремление студентов к постоянному приобретению новых знаний и умений, помогающих успешно решать познавательные задачи и приобретать практический опыт, является важным условием качественной педагогической подготовки и решающим фактором их профессионального роста. р ф Литература 1. Акофф Р.,Эмери Ф. О целеустремленных системах. - М., 1974. 1. Акофф Р.,Эмери Ф. О целеустремленных системах. - М., 1974. 2. Афанасьев В.В., Пидкасистый П.И. Управленческая проблема как объект педагогического исследования. - Педагогика. - №5. 2. Афанасьев В.В., Пидкасистый П.И. Управленческая проблема как объект педагогического исследования. - Педагогика. - №5. 3. Афанасьев В.Г. Научное управление обществом: опыт системного исследования. - М., 1973. 3. Афанасьев В.Г. Научное управление обществом: опыт системного исследования. - М., 1973. 4. Рубинштейн С.Л. Основы общей психологии. - М.: Учпедгиз, 1946. 4. Рубинштейн С.Л. Основы общей психологии. - М.: Учпедгиз, 1946. 5. Жариков Е.С. О действиях составляющих постановку научной проблемы. - Философские науки. - №1. 5. Жариков Е.С. О действиях составляющих постановку научной проблемы. - Философские науки. - №1. О.ВЛисевич СУЧАСНИЙ СТАН ПРОБЛЕМИ ФОРМУВАННЯ КУЛЬТУРИ СПІЛКУВАННЯ МАЙБУТНІХ ПЕДАГОГІВ ЗАСОБАМИ ДІАЛОГІЧНИХ НАВЧАЛЬНИХ СИТУАЦІЙ О.ВЛисевич СУЧАСНИЙ СТАН ПРОБЛЕМИ ФОРМУВАННЯ КУЛЬТУРИ СПІЛКУВАННЯ МАЙБУТНІХ ПЕДАГОГІВ ЗАСОБАМИ ДІАЛОГІЧНИХ НАВЧАЛЬНИХ СИТУАЦІЙ В статье раскрываются разнообразные проблемы и аспекты формирования культуры общения в диалогических учебных ситуациях. 155 The article deals with the various problems and aspects of communicational culture formation in dialogue training \| studying situation of professional orientation. y g fp f Наприкінці XX - початку XXI ст. у науці посилюється увага до методологічних, теоретичних, експериментальних проблем спілкування та ролі діалогу у процесах спілкування та пізнання. Увага до цих проблем спричинена зміною позицій у пізнанні світу не з традиційних монологічних позицій, а діалогічно; з появою нових технологій, які створюють нове середовище спілкування (комп’ютерні комунікації) і відповідно нові можливості розвитку навчання у діалозі. Спілкування, культура спілкування, глибоко досліджується у всьому світі, але до цього часу немає однозначного тлумачення поняття “спілкування”, його форм та механізмів. У зарубіжній літературі культура педагогічного спілкування розуміється як педагогічна технологія. Наприкінці 30-х років XX ст. розробляється проблема типології стилю поведінки вчителів. Психологи Левин та Андерсон стверджують, що від педагогічного стилю вчителя залежить поведінка учня. Якщо вчитель підтримує інтереси учнів, надає їм допомогу у вирішенні проблем, то вони стають більш активними, самостійними, а такий стиль називається інтегративним. У 70-х роках психологи звернули увагу на те, що лише 2% вчителів поводять себе інтегративно, тоді як більшість вибирає авторитарний стиль поведінки. За твердженням Леонтьева О.О., на Заході у 70-і роки писалося сотні робіт з педагогічного спілкування, в яких були опубліковані дослідження про суб’єктивність спілкування вчителя [8,14,15]. Вітчизняні вчені вивчали психологію спілкування (Страхов, Коломийський, Леонтьев, Гусєв); спілкування як наука та мистецтво (Власов, Ільїна; фундаментальні праці Леонтьева, Бодальова, Кан-Каліка, Лутоникіна, Рахматуліной, Курбанової). р ф Литература 156 Але треба відзначити, що проблема культури педагогічного спілкування розглядалась у зарубіжній та вітчизняній літературі у контексті спілкування вчителя школи та учнів. Проблема спілкування у вузівській практиці розроблена менше [2,35-38]. р р [ ] Комунікативну культуру майбутнього вчителя досліджували Архангельський, Кан-Калік В.А., Сластьонін В.О., Підласий І.П., Кондрашова Л.В., Грехньов B.C., Мандрикін Т.С. та інші. р Аналіз наукової літератури свідчить про те, що ефективність формування культури спілкування залежить не стільки від застосування нових засобів чи винайдення інших підходів до цієї проблеми, скільки від перебудови існуючих. Так, ми пропонуємо формувати культуру спілкування майбутніх педагогів-філологів засобами діалогічних навчальних ситуацій професійної спрямованості. Моделювання комунікативних ситуацій на заняттях містить у собі значні дидактичні можливості для підвищення професійного інтересу студентів, розвитку їх педагогічних та комунікативних здібностей і є засобом розвитку особистості студента [12,70]. Спробуємо розглянути педагогічну ситуацію в контексті навчального процесу - заняття. Вона обов’язково повинна містити мету, завдання, етапи і зміст, характер взаємодії викладача та студента. Залежно від етапу заняття педагогічна ситуація може бути ситуацією ознайомлення з навчальним матеріалом (орієнтація у ньому, осмислення), тренування (опрацювання загальних способів дії), контролю та оцінки способів дії, контролю і оцінки засвоєного знання способів дії. За динамікою співробітництва можуть бути визначені ситуації початку роботи, завершення. За характером навчальної взаємодії можугь бути ситуації співпраці, змагання, конфлікту, протистояння. За характером вирішення навчальних задань ситуація розглядається як нейтральна або проблемна [7,336-337]. 157 У спілкуванні виділяють структурні та функціональні одиниці. Структурна одиниця спілкування розглядається різними авторами по-різному. Вважають, що структурною одиницею діяльності спілкування є дія побудови висловлювання. Можна розглядати спілкування як форму взаємодії, але практично всі схеми мають вигляд - “той, хто говорить - той, хто слухає”. та Вперше про діалог, як умову спілкування, почав говорити М.М.Бахтін, але історія людства знає діалог з найдавніших часів (філософія та освіта Сходу, твори Платона, Сократа, розмови Конфуція з учнями). Сучасні науковці у своїх роботах по-різному розуміють діалог. Г.О.Ковальов розглядає діалог як рівність позицій учасників спілкування, які світ цінностей іншого здатні зрозуміти і прийняти як свої. Для О.Крутія та С.Ю.Курганова діалог є засобом інтелектуалізації старшокласників, Т.О.Флоренська розглядає діалогічний метод, який побудовано на безкорисливому ставленні до іншої людини. С.Ю.Степанов та І.І.Семенов формують поняття діалогу як особистісно-рефлексивне утворення, що вимагає спеціально створених суперечностей. В .В. Андрієвська і Г.О.Балл аналізують діалог як стратегію педагогічної діяльності. Оскільки діалогічність повинна стати умовою нашого життя, засобом розуміння сучасного світу, то формування культури спілкування у майбутніх педагогів- філологів засобами діалогічних навчальних ситуацій є необхідною умовою становлення професійного мислення. р ф Литература Конструювання діалогу в навчальній ситуації передбачає такі структурні компоненти: • діалог у розвитку суб’єктної взаємодії; • зіткнення протилежних позицій; • відкритий фінал, який вимагає самостійного пошуку відповідей. За ЛВ.Кондрашовою, діалог базується на принципах: • вибір найбільш цікавої та корисної ситуації для учасників; 158 • зв’язок ситуації з відомим матеріалом; • зв’язок ситуації з відомим матеріалом; • наявність сприятливого емоційного клімату; • колективно-консультативна роль викладача; • розподіл ролей з урахуванням інтересів та можливостей студентів. • розподіл ролей з урахуванням інтересів та можливостей студентів. у Використання діалогічних навчальних ситуацій, на нашу думку, сприятиме формуванню культури спілкування майбутніх педагогів. Література р ур 1. Баженова Л. Професійне самовдосконалення педагога // Психолог.- 2002.- №16.- С. 13-15. 2. Батищев Г.С. За воспитание, но другодоминантное // Вестник высшей школы.- 1989.- №11.- С. 35-38. 3. Болюбаш Я. Державна програма “Вчитель” - поступ назустріч учителю: Освіта XXI ст // Рідна школа. - 2002.- №12.- С.3-6. 4. Бурман Л. Формування діалогічних умінь студентів через залучення їх до активного навчального діалогу // Педагогіка вищої та середньої школи: 36. наук, праць.- Вип.4.- Кривий Ріг, 2002.- С. 103-110. 4. Бурман Л. Формування діалогічних умінь студентів через залучення їх до активного навчального діалогу // Педагогіка вищої та середньої школи: 36. наук, праць.- Вип.4.- Кривий Ріг, 2002.- С. 103-110. 5. Гаврилюк 3. Гуманістичні засади процесу формування комунікативної культури майбутніх вчителів // Педагогіка вищої та середньої школи- Вип.З.- Кривий Ріг, 2001.- С. 30-36. 5. Гаврилюк 3. Гуманістичні засади процесу формування комунікативної культури майбутніх вчителів // Педагогіка вищої та середньої школи- Вип.З.- Кривий Ріг, 2001.- С. 30-36. 6. Глузман А. Да зравствует университет: Особенности современного высшего образования за рубежом // Гуманітарні науки.- 2003.- №12.- С. 54-60. у р у 7. Державна програма “Вчитель” // Освіта України.- 2002. - 2 квітня. - С. 1-8. 8. Леонтьев О.О. Педагогическое общение.- М.: Знание, 1979. 9. Щербак О. Педагог професійної школи: пошук сучасних підходів // Освіта.- 2003,- 24-31 грудня.- С.З. 10. Корсак К., Поживілова О. Оцінювання якості роботи вищої школи: світової тенденції і національні традиції // Науковий світ.- 2002.- №12. - С. 8-9. 159 11. Кузь В.Г. Учитель, школа - пріоритети XXI ст.// Педагогіка і психологія.- 2002.** №1-2.- С. 11-19. 12. Процес обучения в высшей школе: Учебное пособие / Л.В. Кондрашова.- Кривой Рог: ИВИ, 2000. Г, ОЛнтрощеико ПІДГОТОВКА МАЙБУТНЬОГО ВЧИТЕЛЯ ДО ФОРМУВАННЯ В УЧНІВ ПОЧАТКОВИХ КЛАСІВ КУЛЬТУРИ МІЖНАЦІОНАЛЬНОГО СПІЛКУВАННЯ В статье рассматривается проблема подготовки будущего учителя к формированию в учеников начальных классов культуры межнационального общения. Указаны формы и методы, которые обеспечивают эффективное решение данного вопроса и сопутствуют активизации научно-воспитательного процесса в начальной школе. The preparation of the future teacher for the forming of the culture of the international communication in the pupils of the elementary school. This article examines the problem of the preparation of the future teacher for the forming of the culture of the international communication in the pupils of the elementary school. This article denotes the forms and methods which provide the effective decision of this problem and accompanies of the scientific-teacher process in the elementary school. f p y Державною програмою “Вчитель” [1] передбачені теоретико-практичні проблеми підготовки майбутніх вчителів до здійснення виховного процесу на всіх ступенях діяльності загальноосвітньої школи. Зокрема, наголошується на необхідності розвитку цілісного педагогічного процесу, культури взаємовідносин між педагогами та вихованцями. Програма є одним із складників втілення в життя Національної доктрини розвитку освіти України в XXI столітті [2]. Програма охоплює широкий комплекс завдань, вирішення яких сприятиме утвердженню нового, престижного суспільного статусу вчителя, інтенсифікації його ролі в навчально-виховному процесі, формуванню нової 160
https://openalex.org/W4245882340	https://oceanrep.geomar.de/id/eprint/49745/1/s12864-020-6735-5.pdf	English	null	Genomic variation among closely related Vibrio alginolyticus strains is located on mobile genetic elements	Research Square (Research Square)	2,020	cc-by	11,602	Chibani et al. BMC Genomics (2020) 21:354 https://doi.org/10.1186/s12864-020-6735-5 Chibani et al. BMC Genomics (2020) 21:354 https://doi.org/10.1186/s12864-020-6735-5 Open Access Genomic variation among closely related Vibrio alginolyticus strains is located on mobile genetic elements Cynthia Maria Chibani1,2, Olivia Roth3, Heiko Liesegang1 and Carolin Charlotte Wendling3,4* Abstract Keywords: Pangenome, Mega-plasmids, Mobile genetic elements, Horizontal gene transfer, Genomic islands, Niche- adaptation * Correspondence: carolin.wendling@env.ethz.ch 3GEOMAR, Helmholtz Centre for Ocean Research, Marine Evolutionary Ecology, Duesternbrooker Weg 20, 24105 Kiel, Germany 4Department of Environmental Systems Science, ETH Zürich, Universitätsstraße 16, 8092 Zürich, Switzerland Full list of author information is available at the end of the article * Correspondence: carolin.wendling@env.ethz.ch 3GEOMAR, Helmholtz Centre for Ocean Research, Marine Evolutionary Ecology, Duesternbrooker Weg 20, 24105 Kiel, Germany 4Department of Environmental Systems Science, ETH Zürich, Universitätsstraße 16, 8092 Zürich, Switzerland Full list of author information is available at the end of the article Abstract Background: Species of the genus Vibrio, one of the most diverse bacteria genera, have undergone niche adaptation followed by clonal expansion. Niche adaptation and ultimately the formation of ecotypes and speciation in this genus has been suggested to be mainly driven by horizontal gene transfer (HGT) through mobile genetic elements (MGEs). Our knowledge about the diversity and distribution of Vibrio MGEs is heavily biased towards human pathogens and our understanding of the distribution of core genomic signatures and accessory genes encoded on MGEs within specific Vibrio clades is still incomplete. We used nine different strains of the marine bacterium Vibrio alginolyticus isolated from pipefish in the Kiel-Fjord to perform a multiscale-comparative genomic approach that allowed us to investigate [1] those genomic signatures that characterize a habitat-specific ecotype and [2] the source of genomic variation within this ecotype. Results: We found that the nine isolates from the Kiel-Fjord have a closed-pangenome and did not differ based on core-genomic signatures. Unique genomic regions and a unique repertoire of MGEs within the Kiel-Fjord isolates suggest that the acquisition of gene-blocks by HGT played an important role in the evolution of this ecotype. Additionally, we found that ~ 90% of the genomic variation among the nine isolates is encoded on MGEs, which supports ongoing theory that accessory genes are predominately located on MGEs and shared by HGT. Lastly, we could show that these nine isolates share a unique virulence and resistance profile which clearly separates them from all other investigated V. alginolyticus strains and suggests that these are habitat-specific genes, required for a successful colonization of the pipefish, the niche of this ecotype. Conclusion: We conclude that all nine V. alginolyticus strains from the Kiel-Fjord belong to a unique ecotype, which we named the Kiel-alginolyticus ecotype. The low sequence variation of the core-genome in combination with the presence of MGE encoded relevant traits, as well as the presence of a suitable niche (here the pipefish), suggest, that this ecotype might have evolved from a clonal expansion following HGT driven niche-adaptation. * Correspondence: carolin.wendling@env.ethz.ch 3GEOMAR, Helmholtz Centre for Ocean Research, Marine Evolutionary Ecology, Duesternbrooker Weg 20, 24105 Kiel, Germany 4Department of Environmental Systems Science, ETH Zürich, Universitätsstraße 16, 8092 Zürich, Switzerland Full list of author information is available at the end of the article Background Especially multiple prophage arrays cannot be resolved using draft genome assemblies from short- read sequencing techniques. Due to the incomplete in- formation concerning the genome organization stored in draft genomes, it is not possible to study genome dy- namics of mobile genetic elements especially of pro- phages without long-read sequencing data [20]. Not only the size of pangenomes differs among species but also the relationship between the proportion of core-genes relative to the pangenome [7]. A large pan- genome with a proportionally small core-genome and with high rates of HGT is considered open, whereas a small pangenome with a high proportion of core-genes and low HGT rates is considered to be closed [8]. For a wide range of bacteria, the pangenome size is positively correlated with the effective population size (Ne) [7]. Species with a high Ne are likely to occupy different en- vironmental niches where they experience a larger diver- sity of selection pressures, which will drive selection for increased genome diversity and ultimately lead to larger pangenomes [8]. However, niche adaptation in species with high Ne can also select for distinct ecotypes (i.e. ecologically and evolutionary distinct subpopulations) and ultimately initiate a speciation process with a niche- specific pangenome [8]. It remains debated when eco- types are sufficiently diverged to be considered different species [9]. p g g q g The present study aimed to investigate how genome organization, as well as core-genomic signatures and accessory genes, differ within a group of closely related environmental Vibrio isolates. To do so, we performed a multi-scale comparative genomics approach using Vibrio alginolyticus as a model organism. V. alginolyticus, a ubi- quitous marine opportunistic pathogen can cause mass mortalities in shellfish, shrimp, and fish, resulting in se- vere economic losses worldwide [3, 21, 22]. Vibrio pathogenicity is a complex interaction of abiotic and bi- otic factors, including high temperatures [23, 24] low sa- linities [25], host and bacterial genotypes [26, 27]. To date only six closed alginolyticus genomes are available, while 28 remain fragmented with 2 to 75 contigs (last accessed: December 2019). A detailed comparative gen- omic characterization of the alginolyticus species, includ- ing plasmid content, distribution of MGEs and virulence factors of this species is as yet missing. Here we analysed An excellent model organism to study pan-core gen- omic signatures and niche adaptation resulting from HGT is the genus Vibrio. Background decreases considerably. For instance, within the cholerae clade, the pangenome is only 4.5 times larger than the core-genome [11]. The relatively small cholerae-pangen- ome (6923 genes) compared to the entire Vibrio-pangen- ome, might reflect a specific set of genes, required to occupy the cholera-specific niche. The genomic variation within a bacterial species is reflected by accessory genes. Together with strain- specific singletons (genes found in only one strain) and the core-genes that are shared by all strains of a species (such as housekeeping genes and genes required for es- sential cellular functions) the accessory elements form the pangenome [1]. Accessory genes are usually only found in a subset of genomes of a species and are attrib- uted to habitat-specific functions, such as strain-specific virulence factors [2], antibiotic resistance [3], or niche- adaptation [4]. As such, accessory genes are often lo- cated on mobile genetic elements (MGEs), including plasmids, bacteriophages, transposons, integrative or conjugative elements (ICEs) and genomic islands (GIs) and are shared between species via horizontal gene transfer (HGT) [5]. Even though HGT between strains of the same species is more frequent, some MGEs have a broad host range enabling them to move genes across species boundaries thereby blurring the definition of a species [6]. py p Niche adaptation and ultimately speciation in this genus has been suggested to be mainly driven by HGT [13]. Furthermore, MGEs, in particular filamentous phages, contribute significantly to the emergence of pathogenic vibrios from environmental populations [6] and the prophage repertoire of a given Vibrio species can account for a large fraction of the variation among strains [14, 15]. Our knowledge about the diversity and distribution of Vibrio MGEs is heavily biased towards human pathogens, e.g. V. cholerae and V. parahaemoly- ticus. While recent studies started to expand our know- ledge on Vibrio-specific MGEs to other clades, such as V. anguillarum [16, 17], V. fischerii [18], or V. alginolyti- cus [19], our understanding about the distribution of core-genomic signatures and accessory genes encoded on MGEs within specific Vibrio clades is still incomplete. This might be because most of the available genomes are draft genomes comprising multiple contigs, whereas only a few complete genomes of environmental vibrios are available to date [19]. It is challenging to generate high-quality genomes of vibrios due to repetitive gen- omic regions such as rRNA operons present in the two chromosomes [8–12] and arrays of multiple integrated prophages. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chibani et al. BMC Genomics (2020) 21:354 Chibani et al. BMC Genomics (2020) 21:354 Page 2 of 14 Page 2 of 14 Genome features of Vibrio alginolyticus isolated from the Kiel-Fjord We sequenced the genomes of nine Vibrio alginolyticus strains, previously isolated from the gut or gills of six different pipefish caught in the Kiel-Fjord (Germany, 54°75′57″N; 9°87′66″E) in May 2010 [28], using a com- bination of PacBio long- and Illumina short-read tech- nology. The assembly resulted in eight closed genome sequences and one draft genome (strain K09K1), where both chromosomes have been assembled into a single contig due to multiple copies of an integrated filament- ous phage. The replicon boundaries could not be re- solved experimentally based on PCR thus strain K09K1 has been assigned a “permanent draft” status. All V. algi- nolyticus genomes contain a ~ 3.47 Mbp chromosome 1 and a ~ 1.88 Mbp chromosome 2, with a %GC content of around 44% (Table 1), which is typical for the genus Vibrio as well as for the species V. alginolyticus [29, 32]. We found extra-chromosomal replicons including plas- mids and filamentous phages in seven isolates (Table 1). g y We extrapolated both, the within-Kiel and the global V. alginolyticus pangenome by fitting a least-squares curve based on Heaps’ Law [1] and found that the within-Kiel pangenome is closed (α = 1.12), whereas the global V. alginolyticus pangenome is open (α = 0.58). This means, that within the global V. alginolyticus pan- genome each newly sequenced V. alginolyticus isolate will reveal a unique set of genes, irrespective of the num- ber of strains included in the present analysis. This open V. alginolyticus pangenome reflects the diversity in habi- tats in which this species exists. These distinct environ- ments probably require different adaptations and/ or promote high levels of HGT. In contrast, a closed pan- genome, as has been detected for the within-Kiel V. algi- nolyticus pangenome, suggests that the number of genes that will be obtained from any newly sequenced isolate will converge to zero. Here, we assume, that at least the sequenced pipefish associated V. alginolyticus strains within the Kiel-Fjord contain the major part of the gene equipment that is requested to adapt to their habitat. Thus the genomes can be expected to be highly similar, potentially resulting from niche adaptation and strong selection thereof. Strong positive selection of such adap- tive genes might have led to a clonal expansion of the Kiel-alginolyticus ecotype. Indeed, we found no sequence divergence based on core-genomic signature among all nine isolates from Kiel (Fig. Genome features of Vibrio alginolyticus isolated from the Kiel-Fjord 2c), indicating a clonal ex- pansion of this ecotype. It remains to be investigated, whether free-living V. alginolyticus strains or isolates from other eukaryotic hosts from the Kiel-Fjord share the same gene-pool or are more divergent from the pipefish-associated strains. Species definition and phylogenetic relationship of the Kiel-Fjord isolates All nine strains isolated from the Kiel-Fjord share more than 98% average nucleotide identity (ANI) with other alginolyticus strains, suggesting that these strains belong, as previously suggested based on a multi-locus- sequencing-approach (MLSA) [23], to the species V. alginolyticus. All alginolyticus strains share ~ 92% ANI with two other closely related Vibrio species, i.e. V. dia- bolicus and V. antiquarius (Fig. 1), which is below the species threshold of ANI = 95% [33, 34], indicating that V. alginolyticus is a distinct species. Pangenome To compare the gene repertoire of the nine V. alginolyti- cus strains from the Kiel-Fjord with the global V. algino- lyticus gene repertoire, we calculated their pangenome including the amount of core-and accessory genes as well as strain-specific singletons, i.e. genes unique to sin- gle strains [35]. The analysis included in total 73,277 protein-sequences encoded on chromosomes and MGEs, including plasmids and extra-chromosomal phage repli- cons. Overall, we found that the size of the pangenome of V. alginolyticus increased stronger (from 4997 to 8843 Background These bacteria constitute one of the most diverse bacterial genera with a large pangen- ome comprising more than 100 species from different clades, that occupy different ecological niches [10, 11], where they can exist as pathogenic, opportunistic, mu- tualistic or free-living forms [12]. The considerable large pangenome (> 26,500 genes) of the genus Vibrio, which is more than 50 times larger than the Vibrio specific core-genome [11], indicates a large reservoir of genomic diversity for this genus. In contrast, within a specific Vibrio clade, the pangenome to core-genome ratio Chibani et al. BMC Genomics (2020) 21:354 Page 3 of 14 Page 3 of 14 gene clusters) with the sequential addition of each new genome when we included all 15 strains from diverse habitats (Fig. 2a). In contrast, we only observed a slight increase in the pangenome (from 5044 to 5679 gene clusters) when we only included the nine V. alginolyticus isolates from the Kiel-Fjord (Fig. 2b). An increase in the pangenome results from new strain-specific genes, found in each newly analyzed isolate. The much larger increase in the pangenome across all available V. alginolyticus isolates relative to the increase in the pangenome of Kiel-Fjord isolates suggests that the genomic diversity of V. alginolyticus within the Kiel-Fjord is very limited. However, the species V. alginolyticus per se has a vast reservoir of genomic diversity. core-and accessory-genomic signatures of V. alginolyti- cus by comparing nine closely related V. alginolyticus ge- nomes, isolated from the pipefish Syngnathus typhle in the Kiel-Fjord [28], (later named Kiel-alginolyticus eco- type) with less closely related V. alginolyticus isolates (Table 1). Results and discussion Genome features of Vibrio alginolyticus isolated from the Kiel-Fjord Core-genome and singletons g g We observed a stronger decrease in the core-genome when we included all V. alginolyticus isolates as opposed to when we performed the analysis only within the Kiel- alginolyticus ecotype. Comparative analyses between all 15 V. alginolyticus strains and the Kiel-alginolyticus eco- type only, revealed that the core genome (4708 gene Chibani et al. BMC Genomics (2020) 21:354 Page 4 of 14 Table 1 All V. alginolyticus strains used in the present study (* circular phage replicon) strain GC% Replicon Size [bp] CDS Ref Genbank Kiel-Fjord Vibrio alginolyticus genomes K01M1 44.6 Chromosome 1 3,468,303 3206 This study CP017889.1 Chromosome 2 1,883,748 1668 This study CP017890.1 pL9064 9064 8 This study CP028135.1 K04M1 44.31 chromosome 1 3,473,127 3213 This study CP017891.1 chromosome 2 1,870,775 1660 This study CP017892.1 pL19 19,690 28 This study CP017893.1 pL280 280,614 305 This study CP017894.1 vK04M1* 7079 11 This study CP017895.1 K04M3 44.31 chromosome 1 3,476,174 3219 This study CP017896.1 chromosome 2 1,903,830 1708 This study CP017897.1 pL294 294,086 325 This study CP017898.1 K04M5 44.31 chromosome 1 3,470,916 3211 This study CP017899.1 chromosome 2 1,900,618 1688 This study CP017900.1 pL294 294,721 320 This study CP017901.1 K05K4 44.34 chromosome 1 3,473,579 3218 This study CP017902.1 chromosome 2 1,875,554 1670 This study CP017903.1 pL289 289,065 315 This study CP017904.1 vK05K4_1* 21,012 34 This study CP017905.1 vK05K4_2* 13,327 23 This study CP017906.1 K06K5 44.31 Chromosome 1 3,471,297 3213 This study CP017907.1 Chromosome 2 1,879,729 1662 This study CP017908.1 pL29 29,688 20 This study CP017909.1 pL291 291,285 322 This study CP017910.1 K08M3 44.32 Chromosome 1 3,468,139 3214 This study CP017913.1 Chromosome 2 1,886,577 1675 This study CP017914.1 pL300 300,425 331 This study CP017915.1 K09K1 44.61 Chromosome 1 1,897,210 3209 This study CP017918.1 Chromosome 2 3,465,619 1704 This study CP017919.1 K10K4 44.6 Chromosome 1 3,494,647 3231 This study CP017911.1 Chromosome 2 1,894,531 1682 This study CP017912.1 all other closed V. alginolyticus genomes ATCC 33787 44.48 Chromosome 1 3,362,673 3190 Wang et al. 2016 [29] CP013484.1 Chromosome 2 1,851,538 1674 Wang et al. 2016 [29] CP013485.1 pMBL128 128,112 144 Wang et al. 2016 [29] CP013486.1 pMBL287 286,750 301 Wang et al. 2016 [29] CP013487.1 pMBL96 95,866 109 Wang et al. 2016 [29] CP013488.1 ZJ-T 44.67 Chromosome 1 3,535,128 3301 Deng et al. 2016 [30] CP016224.1 Chromosome 2 1,870,966 1657 Deng et al. 2016 [30] CP016225.1 ATCC 17749 44.7 Chromosome 1 3,334,467 3128 Liu et al. Core-genome and singletons 2015 [31] CP006718.1 Chromosome 2 1,812,170 1640 Liu et al. 2015 [31] CP006719.1 FDAARGOS_108 44.15 Chromosome 1 3,418,310 3047 Hoffmann, M (unpublished) CP014053 Chromosome 2 1,810,594 1587 Hoffmann, M (unpublished) CP014054.1 unnamed plasmid 217,123 231 Hoffmann, M (unpublished) CP014052 FDAARGOS_110 44.57 Chromosome 1 3,434,696 3156 Hoffmann, M (unpublished) CP014040.1 Chromosome 2 1,833,981 1622 Hoffmann, M (unpublished) CP014041.1 FDAARGOS_114 44.68 Chromosome 1 3,372,141 3054 Hoffmann, M (unpublished) CP014045.1 Chromosome 2 1,806,127 1593 Hoffmann, M (unpublished) CP014044.1 Chibani et al. BMC Genomics (2020) 21:354 Page 5 of 14 Chibani et al. BMC Genomics Fig. 1 Average nucleotide identity (ANI) percentage analysis of closed Vibrio genomes. ANI analysis based on MUMmer alignment of the genome sequences was performed and visualized using PYANI. ANI-values ranging from 0 to 70% no hits, 70–95% ANI colored in blue and higher than 95% ANI in red) All true V. alginolyticus (including the nine strains from the present study) can be separated from V. diabolicus and V. antiquaries (small red square, bottom left) Fig. 1 Average nucleotide identity (ANI) percentage analysis of closed Vibrio genomes. ANI analysis based on MUMmer alignment of the genome sequences was performed and visualized using PYANI. ANI-values ranging from 0 to 70% no hits, 70–95% ANI colored in blue and higher than 95% ANI in red) All true V. alginolyticus (including the nine strains from the present study) can be separated from V. diabolicus and V. antiquaries (small red square, bottom left) Fig. 1 Average nucleotide identity (ANI) percentage analysis of closed Vibrio genomes. ANI analysis based on MUMmer alignment of the genome sequences was performed and visualized using PYANI. ANI-values ranging from 0 to 70% no hits, 70–95% ANI colored in blue and higher than 95% ANI in red) All true V. alginolyticus (including the nine strains from the present study) can be separated from V. diabolicus and V. antiquaries (small red square, bottom left) Fig. 1 Average nucleotide identity (ANI) percentage analysis of closed Vibrio genomes. ANI analysis based on MUMmer alignment of the genome sequences was performed and visualized using PYANI. ANI-values ranging from 0 to 70% no hits, 70–95% ANI colored in blue and higher than 95% ANI in red) All true V. alginolyticus (including the nine strains from the present study) can be separated from V. diabolicus and V. Core-genome and singletons antiquaries (small red square, bottom left) In contrast, despite all being members of the same spe- cies, the global accessory gene-pool is highly variable (56% of the pangenome) and the V. alginolyticus core- genome constitutes only 44% of the pangenome. clusters) is four times larger than the accessory genome (971 gene clusters) when we only included the nine Kiel strains. However, when we extended the analysis and in- cluded all 15 V. alginolyticus, we found that the core genome (3876 gene clusters) becomes smaller than the accessory gene pool (4967 gene clusters). In other words, within the Kiel-Fjord, different V. alginolyticus isolates have a large core-genome (83% of the pangenome) with a limited accessory gene pool (17% of the pangenome). Habitat specific chromosomal regions The curve shows mean values of multiple iterations (> 100) where genomes were randomly added and the pan- and core-genome was calculated successively for all 15 steps (left: All 15 V. alginolyticus strains) and nine steps (right: Kiel-Fjord V. alginolyticus only). b Gene presence (dark blue) -absence (light blue) matrix showing the distribution of core and accessory genes for each of the 15 V. alginolyticus genomes. Each row corresponds to one strain, each column represents an orthologous gene family. Numbers on the right (#C) correspond to number of gene clusters per genome. The red square indicates the core-region specific for the Kiel isolates, the six mega-plasmids are indicated by white pL letters. c Bayesian phylogenetic tree of all 15 isolates based on the core-genome alignment K05K4, but present in some non-Kiel strains, such as ATCC17749 and FDAARGOS_114, suggesting that GR 5 is not unique for the Kiel isolates. Similarly, GR 15, which corresponds to a multiphage-cassette consisting of multiple repeats of a combination of the two filament- ous phages Vibrio phage VALGΦ6 and Vibrio phage VALGΦ8 [19], is absent in most Kiel strains, except K04M3 and K04M5 but in parts present in ATCC17749 and thus not unique for the Kiel system. Of the remaining 14 GRs, which do not correspond to inte- grated phages, we could identify four genomic islands: GR 2, GR 6, GR 7 and GR 8 (> 10 kb, presence of inte- grase/ transposase, different GC content). According to functional COG categories, these islands contain a var- iety of proteins, most of them involved in replication, re- combination, and repair [L] and transcription [K] (see Table S3 for functional annotation of all GRs). The high prevalence of those maintenance genes is however not surprising and might result from an identification bias, as they are usually better annotated for MGEs than accessory genes, which would provide a selective advan- tage to the host. As such, many proteins were classified as phage integrases, transposases or other proteins, K05K4, but present in some non-Kiel strains, such as ATCC17749 and FDAARGOS_114, suggesting that GR 5 is not unique for the Kiel isolates. Habitat specific chromosomal regions Similarly, GR 15, which corresponds to a multiphage-cassette consisting of multiple repeats of a combination of the two filament- ous phages Vibrio phage VALGΦ6 and Vibrio phage VALGΦ8 [19], is absent in most Kiel strains, except K04M3 and K04M5 but in parts present in ATCC17749 and thus not unique for the Kiel system. Of the remaining 14 GRs, which do not correspond to inte- grated phages, we could identify four genomic islands: GR 2, GR 6, GR 7 and GR 8 (> 10 kb, presence of inte- grase/ transposase, different GC content). According to functional COG categories, these islands contain a var- iety of proteins, most of them involved in replication, re- combination, and repair [L] and transcription [K] (see Table S3 for functional annotation of all GRs). The high prevalence of those maintenance genes is however not surprising and might result from an identification bias, as they are usually better annotated for MGEs than accessory genes, which would provide a selective advan- tage to the host. As such, many proteins were classified as phage integrases, transposases or other proteins, mechanism seems to be particularly important for bac- terial adaptation to new ecological niches but also for how bacteria diverge from each other, forming ecotypes and ultimately new species [37]. Genomic islands, which encode specific functions allowing for niche adaptation and maybe even speciation events are common within the genus Vibrio [38]. For the Kiel-alginolyticus ecotype, we could identify 19 chromosomal genomic regions (GRs) of which most are unique to the strains isolated from the Kiel-Fjord (Fig. 3, Table S2). Overall, these 19 GRs encode a total of 487 genes out of which 305 have only been found within the Kiel-alginolyticus ecotype. Out of these 19 GRs five could be assigned to integrated prophages. GR 13 and GR 14 correspond to Vibrio phage VALGΦ2/2b and Vibrio phage VALGΦ6, which are unique for the isolates from the Kiel-Fjord and have not been found elsewhere [19]. GR 3 corresponds to Vibrio phage VALGΦ1, which has so far only been de- tected in V. alginolyticus ATCC33787, with a relatively low query cover of 57%, thus making it a unique region for Kiel. Habitat specific chromosomal regions The acquisition of entire gene-blocks (genomic islands, plasmids, prophages) by HGT can rapidly alter the life- style of a bacterium in quantum leaps [36]. This Chibani et al. BMC Genomics (2020) 21:354 Page 6 of 14 Fig. 2 Pan-core-genome analysis of 15 closed Vibrio alginolyticus strains. a Number of gene clusters for pan (dashed line) and core genome (solid line) resulting from the sequential addition of genomes. The curve shows mean values of multiple iterations (> 100) where genomes were randomly added and the pan- and core-genome was calculated successively for all 15 steps (left: All 15 V. alginolyticus strains) and nine steps (right: Kiel-Fjord V. alginolyticus only). b Gene presence (dark blue) -absence (light blue) matrix showing the distribution of core and accessory genes for each of the 15 V. alginolyticus genomes. Each row corresponds to one strain, each column represents an orthologous gene family. Numbers on the right (#C) correspond to number of gene clusters per genome. The red square indicates the core-region specific for the Kiel isolates, the six mega-plasmids are indicated by white pL letters. c Bayesian phylogenetic tree of all 15 isolates based on the core-genome alignment Fig. 2 Pan-core-genome analysis of 15 closed Vibrio alginolyticus strains. a Number of gene clusters for pan (dashed line) and core genome (solid line) resulting from the sequential addition of genomes. The curve shows mean values of multiple iterations (> 100) where genomes were randomly added and the pan- and core-genome was calculated successively for all 15 steps (left: All 15 V. alginolyticus strains) and nine steps (right: Kiel-Fjord V. alginolyticus only). b Gene presence (dark blue) -absence (light blue) matrix showing the distribution of core and accessory genes for each of the 15 V. alginolyticus genomes. Each row corresponds to one strain, each column represents an orthologous gene family. Numbers on the right (#C) correspond to number of gene clusters per genome. The red square indicates the core-region specific for the Kiel isolates, the six mega-plasmids are indicated by white pL letters. c Bayesian phylogenetic tree of all 15 isolates based on the core-genome alignment Fig. 2 Pan-core-genome analysis of 15 closed Vibrio alginolyticus strains. a Number of gene clusters for pan (dashed line) and core genome (solid line) resulting from the sequential addition of genomes. Habitat specific chromosomal regions Genomic Regions (GRs) unique for Kiel isolates, which have additionally been predicted using IslandViewer are annotated on the outside involved with viral integration into host DNA or DNA repair, suggesting that HGT might have played an im- portant role during the acquisition of these GIs. GR 6 encodes the multi-drug transporter subunit MdtN and two other loci which were assigned to COG category [V] and predicted to be involved in defense mechanisms. This suggests that GR 6 might play a role in resistance. None of the other genomic islands could be identified as pathogenicity−/ metabolic- or resistance island. All other GRs were either smaller than 10 kb or did not contain integrases or transposases and are thus referred to as genomic regions instead of genomic island. Out of these GRs, GR 16 encoded three proteins associated with the type VI secretion system and GR 18 encoded a beta- lactamase protein suggesting an adaptive role in viru- lence and resistance, respectively. The acquisition of these unique GRs might have allowed the Kiel-alginolyti- cus ecotype to invade a new niche, which was then followed by clonal expansion of this ecotype. Clonal ex- pansion has been observed for several pathogens, in par- ticular within the genus Vibrio. The best characterized example of clonal expansion upon acquisition of viru- lence genes is V. cholerae. But also other Vibrio patho- gens, for instance V. parahaemolyticus have been shown to experience similar evolutionary dynamics (for a re- view see [39]). Genomic differences within Kiel-alginolyticus ecotype To investigate genomic differences between the nine strains from the Kiel-Fjord, we focused on all those gene-clusters from the Roary analysis, which could only be found within the Kiel-alginolyticus ecotype but were absent in all non-Kiel isolates. We found that all Kiel- specific core-genes (n = 412) were located exclusively on one of the two chromosomes (Fig. 4). In contrast, the majority of the Kiel-specific accessory gene clusters (89%) were encoded on mobile genetic elements, in par- ticular plasmids. These results support ongoing theory that accessory genes are predominately located on MGEs and shared by horizontal gene transfer (HGT) [5]. We found 490 gene clusters with no orthologous in any other Kiel strain, i.e. singletons out of which 40% were located on MGEs, in particular, extrachromosomal repli- cating elements (170 on plasmids and 27 on extrachro- mosomal phages) and 60% (n = 293) were chromosomal (Fig. 4). Habitat specific chromosomal regions In contrast GR 5, which corresponds to the fila- mentous phage Vibrio phage VALGΦ8 on chromosome 1, is absent in most Kiel strains, except K10K4 and mechanism seems to be particularly important for bac- terial adaptation to new ecological niches but also for how bacteria diverge from each other, forming ecotypes and ultimately new species [37]. Genomic islands, which encode specific functions allowing for niche adaptation and maybe even speciation events are common within the genus Vibrio [38]. For the Kiel-alginolyticus ecotype, we could identify 19 chromosomal genomic regions (GRs) of which most are unique to the strains isolated from the Kiel-Fjord (Fig. 3, Table S2). Overall, these 19 GRs encode a total of 487 genes out of which 305 have only been found within the Kiel-alginolyticus ecotype. Out of these 19 GRs five could be assigned to integrated prophages. GR 13 and GR 14 correspond to Vibrio phage VALGΦ2/2b and Vibrio phage VALGΦ6, which are unique for the isolates from the Kiel-Fjord and have not been found elsewhere [19]. GR 3 corresponds to Vibrio phage VALGΦ1, which has so far only been de- tected in V. alginolyticus ATCC33787, with a relatively low query cover of 57%, thus making it a unique region for Kiel. In contrast GR 5, which corresponds to the fila- mentous phage Vibrio phage VALGΦ8 on chromosome 1, is absent in most Kiel strains, except K10K4 and Page 7 of 14 Chibani et al. BMC Genomics (2020) 21:354 Fig. 3 Comparison of all nine Kiel isolates and six other fully closed V. alginolyticus isolates. Blast matches between all strains are shown as concentric colored rings on a sliding scale according to percentage identity (100 and 70%). For chromosome 1 (left) we used K10K4 as a reference (inner black circle), as this strain has more integrated prophages than the other Kiel isolates. The second ring (yellow) corresponds to strain K05K4 which has the same integrated prophages as K10K4, the third ring (orange) corresponds to all other strains from Kiel, which miss GR 5, i.e. Vibrio phage VALGΦ8. For the same reasons, we used strain K04M3 as a reference for chromosome 2. Here, the second ring (yellow) corresponds to strain K04M5, which has a similar prophage composition than K04M3 while the third ring (orange) corresponds to all other Kiel strains, which miss the multiphage-cassette consisting of repeats of both filamentous Vibrio phages VALGΦ6 and Vibrio phage VALGΦ8. Habitat specific chromosomal regions GC content is shown in black. Genomic Regions (GRs) unique for Kiel isolates, which have additionally been predicted using IslandViewer are annotated on the outside Fig. 3 Comparison of all nine Kiel isolates and six other fully closed V. alginolyticus isolates. Blast matches between all strains are shown as concentric colored rings on a sliding scale according to percentage identity (100 and 70%). For chromosome 1 (left) we used K10K4 as a reference (inner black circle), as this strain has more integrated prophages than the other Kiel isolates. The second ring (yellow) corresponds to strain K05K4 which has the same integrated prophages as K10K4, the third ring (orange) corresponds to all other strains from Kiel, which miss GR 5, i.e. Vibrio phage VALGΦ8. For the same reasons, we used strain K04M3 as a reference for chromosome 2. Here, the second ring (yellow) corresponds to strain K04M5, which has a similar prophage composition than K04M3 while the third ring (orange) corresponds to all other Kiel strains, which miss the multiphage-cassette consisting of repeats of both filamentous Vibrio phages VALGΦ6 and Vibrio phage VALGΦ8. GC content is shown in black. Genomic Regions (GRs) unique for Kiel isolates, which have additionally been predicted using IslandViewer are annotated on the outside Fig. 3 Comparison of all nine Kiel isolates and six other fully closed V. alginolyticus isolates. Blast matches between all strains are shown as concentric colored rings on a sliding scale according to percentage identity (100 and 70%). For chromosome 1 (left) we used K10K4 as a reference (inner black circle), as this strain has more integrated prophages than the other Kiel isolates. The second ring (yellow) corresponds to strain K05K4 which has the same integrated prophages as K10K4, the third ring (orange) corresponds to all other strains from Kiel, which miss GR 5, i.e. Vibrio phage VALGΦ8. For the same reasons, we used strain K04M3 as a reference for chromosome 2. Here, the second ring (yellow) corresponds to strain K04M5, which has a similar prophage composition than K04M3 while the third ring (orange) corresponds to all other Kiel strains, which miss the multiphage-cassette consisting of repeats of both filamentous Vibrio phages VALGΦ6 and Vibrio phage VALGΦ8. GC content is shown in black. Habitat specific chromosomal regions Left: Kiel-specific core genes (shared by all nine isolates), middle: Kiel-specific accessory genes (shared by two or more isolates, but not by all), and right: Kiel-specific singletons (unique for one isolate) distributed across replicons (CR1: chromosome 1, CR2: chromosome 2, Phages: extrachromosomal phages, Plasmids) and assigned to Clusters of Orthologous Groups of Proteins (COGs): black: information storage and processing; orange: cellular processes and signaling; blue: metabolism; green: poorly characterized; white: not assignable strains with a size of 0.9 to 2.9 kbp (Table 1). The fourth plasmid type was characterized as a mega-plasmid (Fig. 5a), which ranged between 280 and 300 kbp in size, and was present in six out of the nine strains (Fig. 5b). The six mega-plasmids share 296 core-genes, encode 129 accessory genes and between 5 and 26 singletons per plasmid (Fig. 5b). Together with the three small plasmids, plasmid-encoded singletons make up 34.5% of all 486 Kiel-specific singletons. The majority of the plasmid-specific singletons comprise hypothetical pro- teins. The remaining singletons include AAA proteins (K04M1_pL280), PFAM phosphoadenosine phosphosul- fate reductase and ATPases (K04M5_pL294), spore ger- mination and type IV secretion system (K5K4_pL289), endonuclease and site-specific methyl-transferase, poten- tially forming a restriction-modification system (K06K5_ pL291), and DNA polymerase (K08M3_pL300). V. algi- nolyticus ATCC 33787 contains as well three plasmids including plasmid pMBL287, which is similar in size as the Kiel mega plasmids [29]. However, a comparison of ATCC 33787 plasmids revealed no sequence similarity to any of the plasmids from the Kiel strains. majority of the singletons (37%) was predicted to be ded- icated to cellular processes/ signaling, while relatively small proportions of gene clusters (10 and 16%) belong to information storage/ processing and metabolism. In contrast, the largest proportion of gene-clusters encoded on core-genes was predicted to belong to information storage/ processing (24%), and only 16 and 13% of gene- clusters encoded on core genes belong to cellular pro- cesses/ signaling and metabolism. Among the gene- clusters encoded on the accessory genome 22% could be assigned to information storage/ processing as well as to cellular processes/ signaling and only 6% to the metabol- ism (Fig. 4). Within the accessory genome most of the genes are involved in replication, recombination, and re- pair (COG [L], Table S4). Habitat specific chromosomal regions All Kiel-specific alginolyticus gene clusters were further assigned to putative functional categories using the Clusters of Orthologous Groups of Proteins (COG) database [40] (Table S4). Even though a large fraction of the gene clusters could either not be assigned to a COG or was poorly characterized, we found differences in the relative distribution of super-functional COGs between core- and accessory genomes and singletons: The involved with viral integration into host DNA or DNA repair, suggesting that HGT might have played an im- portant role during the acquisition of these GIs. GR 6 encodes the multi-drug transporter subunit MdtN and two other loci which were assigned to COG category [V] and predicted to be involved in defense mechanisms. This suggests that GR 6 might play a role in resistance. None of the other genomic islands could be identified as pathogenicity−/ metabolic- or resistance island. All other GRs were either smaller than 10 kb or did not contain integrases or transposases and are thus referred to as genomic regions instead of genomic island. Out of these GRs, GR 16 encoded three proteins associated with the type VI secretion system and GR 18 encoded a beta- lactamase protein suggesting an adaptive role in viru- lence and resistance, respectively. The acquisition of these unique GRs might have allowed the Kiel-alginolyti- cus ecotype to invade a new niche, which was then followed by clonal expansion of this ecotype. Clonal ex- pansion has been observed for several pathogens, in par- ticular within the genus Vibrio. The best characterized example of clonal expansion upon acquisition of viru- lence genes is V. cholerae. But also other Vibrio patho- gens, for instance V. parahaemolyticus have been shown to experience similar evolutionary dynamics (for a re- view see [39]). Chibani et al. BMC Genomics (2020) 21:354 Page 8 of 14 Fig. 4 Genomic variation among all nine Kiel V. alginolyticus strains. Left: Kiel-specific core genes (shared by all nine isolates), middle: Kiel-specific accessory genes (shared by two or more isolates, but not by all), and right: Kiel-specific singletons (unique for one isolate) distributed across replicons (CR1: chromosome 1, CR2: chromosome 2, Phages: extrachromosomal phages, Plasmids) and assigned to Clusters of Orthologous Groups of Proteins (COGs): black: information storage and processing; orange: cellular processes and signaling; blue: metabolism; green: poorly characterized; white: not assignable Fig. 4 Genomic variation among all nine Kiel V. alginolyticus strains. Habitat specific chromosomal regions These include mainly proteins involved in HGT, such as transposases, integrases, trans- ferases, recombinases as well as proteins involved in im- munity, such as CRISPR associated helicase Cas3 and restriction modification methylases. This extensive rep- resentation of proteins involved in HGT on the accessory genome suggests that HGT was potentially one of the driving evolutionary mechanisms underlying the diversification of the nine V. alginolyticus strains from the Kiel-Fjord. Only 6.5% of the 870 Kiel-specific accessory genes and singletons are located on extrachromosomal phages. The within-Kiel variation caused by these phages can be ex- plained by the absence of extrachromosomal replicons of Vibrio phage VALGΦ8 in all but two strains (K04M1 and K05K4). However, four strains (K04M3, K04M5, K05K4, and K10K4) had an intra-chromosomal version The majority of the accessory gene-pool within the Kiel-alginolyticus ecotype is located on plasmids (Fig. 4). We could identify four different plasmids from all nine Kiel V. alginolyticus isolates. Three plasmid types iso- lated from the Kiel strains were unique for specific Chibani et al. BMC Genomics (2020) 21:354 Page 9 of 14 Fig. 5 Comparative analysis of the Kiel alginolyticus mega plasmid. a Plasmid map. ORFs are color-coded according to COG functional categories: Purple: S, yellow: D, pink: EH, pink: K, blue: L, black: NA, magenta: NU, red: O, light-blue: S, green: T, orange: U, sky-blue: V, grey: hypothetical proteins. b Gene presence (dark blue) -absence (light blue) matrix showing the distribution of core and accessory genes for each of the six mega- plasmids. Each row corresponds to one strain, each column represents an orthologous gene family. The red square represents the core genome of the mega-plasmids (296 genes) as opposed to the number of accessory genes of the mega-plasmids (129 genes). Numbers on the right correspond to (#S) number of singletons per plasmid and (#C) number of gene clusters per plasmid Fig. 5 Comparative analysis of the Kiel alginolyticus mega plasmid. a Plasmid map. ORFs are color-coded according to COG functional categories: Purple: S, yellow: D, pink: EH, pink: K, blue: L, black: NA, magenta: NU, red: O, light-blue: S, green: T, orange: U, sky-blue: V, grey: hypothetical proteins. b Gene presence (dark blue) -absence (light blue) matrix showing the distribution of core and accessory genes for each of the six mega- plasmids. Each row corresponds to one strain, each column represents an orthologous gene family. Habitat specific chromosomal regions The red square represents the core genome of the mega-plasmids (296 genes) as opposed to the number of accessory genes of the mega-plasmids (129 genes). Numbers on the right correspond to (#S) number of singletons per plasmid and (#C) number of gene clusters per plasmid (~ 165 virulence genes) and V. parahaemolyticus (162 virulence genes), but similar to what has been found in other V. alginolyticus strains, except (strain FDAARGOS_ 114: 165 virulence genes). Unique for the Kiel-Fjord iso- lates is the absence of genes involved in Vibriobactin bio- synthesis, which are present in almost all other V. alginolyticus strains. Similarly, In contrast to other V. algi- nolyticus isolates, the strains from the Kiel-Fjord miss the gene flaC, which is involved in the regulation of stringent- response-induced toxin production [41] and vscP, a gene involved in the type III secretion system. In contrast to other V. alginolyticus strains, the Kiel-alginolyticus eco- type encodes the type IV secretion system effectors, the phytotoxin coronatine and the thermolabile hemolysin (tlh), both toxins, which could not be found in any other V. alginolyticus strain. This unique virulence profile of the Kiel-Fjord isolates separates them from other Vibrio spe- cies including other V. alginolyticus isolates abut also an- other strain from the Kiel-Fjord: V. typhli, K08M4 (Chibani C, Hertel R, Goehlich H, Mertens V, Bunk B, Overmann J, et al. Complete Genome Sequence of Vibrio typhli K08M4, a fish pathogen isolated from the Kiel Fjord. in preparation) (Fig. 6). This clear separation was further supported by a hierarchical cluster analysis (Fig. 6), which indicates, that only based on the presence/absence of virulence genes the Kiel-alginolyticus ecotype can be distinguished from all other V. alginolyticus strains, se- quenced to date. The unique resistance and virulence pro- file within the Kiel-Fjord V. alginolyticus isolates might be a further indication for niche-adaptation followed by clonal expansion of this ecotype. of this phage, while strains K01M1, K06K5 and K08M3 did neither contain an intra- nor an extrachromosomal version of Vibrio phage VALGΦ8 (for a detailed analysis of Kiel vibriophages see [19]). Parts of Vibrio phage VALGΦ8 could be identified on both chromosomes of ATCC 17749T, and on chromosome 1 of FDAARGOS_ 114 but not on any other non-Kiel Vibrio. Genome ana- lyses of Vibrio phage VALGΦ8 revealed no significant virulence-associated genes nor any other genes that could be associated with a habitat-specific adaptation [19]. Virulence and resistance of the Kiel-Fjord V. alginolyticus isolates We found an identical virulence and resistance profile among the nine V. alginolyticus isolates from the Kiel- Fjord. The Kiel-Fjord isolates encode a total number of 17 homologues resistance genes out of which the majority (n = 13) is located on chromosome 1 and four resistance genes are encoded on chromosome 2 (Fig. 6, Table S5). In comparison with other V. alginolyticus strains (~ 25–130 resistance genes on Chromosome 1 and 11–12 resistance genes on Chromosome 2), isolates from the Kiel-Fjord have significantly less resistance genes (t-test: t8.85 = 3.51, p = 0.007; Fig. 6) and are missing in particular genes conferring resistance to tetracycline, aminoglycosides, and quinolones. In contrast to non-pathogenic strains, such as V. fischerii, which is a mutualist containing 112 virulence genes, the Kiel-Fjord isolates encode more virulence genes (150 virulence genes, exception strain K05K4, 149 viru- lence genes). This number is lower than what has been found for human pathogenic vibrios, such as V. cholerae We found an identical virulence and resistance profile among the nine V. alginolyticus isolates from the Kiel- Fjord. The Kiel-Fjord isolates encode a total number of 17 homologues resistance genes out of which the majority (n = 13) is located on chromosome 1 and four resistance genes are encoded on chromosome 2 (Fig. 6, Table S5). In comparison with other V. alginolyticus strains (~ 25–130 resistance genes on Chromosome 1 and 11–12 resistance genes on Chromosome 2), isolates from the Kiel-Fjord have significantly less resistance genes (t-test: t8.85 = 3.51, p = 0.007; Fig. 6) and are missing in particular genes conferring resistance to tetracycline, aminoglycosides, and quinolones. In contrast to non-pathogenic strains, such as V. fischerii, which is a mutualist containing 112 virulence genes, the Kiel-Fjord isolates encode more virulence genes (150 virulence genes, exception strain K05K4, 149 viru- lence genes). This number is lower than what has been found for human pathogenic vibrios, such as V. cholerae Page 10 of 14 Chibani et al. BMC Genomics (2020) 21:354 Fig. 6 Resistance and virulence genes. Resistance genes are shown for (a) chromosome 1 and (b) chromosome 2. (c) Virulence genes, presented as a presence (black) / absence (grey) matrix, are grouped into 10 different virulence classes (left). Those virulence genes, which contribute to the unique profile of the Kiel-alginolyticus ecotype are labeled on the right and if present, marked in turquoise. Virulence and resistance of the Kiel-Fjord V. alginolyticus isolates Hierarchical cluster analysis of the 22 strains (bottom), which were incorporated into this analysis shows that the Kiel-alginolyticus ecotype can be separated from all other V. alginolyticus strains solely based on presence/ absence of specific virulence genes n for (a) chromosome 1 and (b) chromosome 2. (c) Virulence genes, presented different virulence classes (left). Those virulence genes, which contribute to the right and if present, marked in turquoise. Hierarchical cluster analysis of the 22 that the Kiel-alginolyticus ecotype can be separated from all other V. virulence genes Fig. 6 Resistance and virulence genes. Resistance genes are shown for (a) chromosome 1 and (b) chromosome 2. (c) Virulence genes, presented as a presence (black) / absence (grey) matrix, are grouped into 10 different virulence classes (left). Those virulence genes, which contribute to the unique profile of the Kiel-alginolyticus ecotype are labeled on the right and if present, marked in turquoise. Hierarchical cluster analysis of the 22 strains (bottom), which were incorporated into this analysis shows that the Kiel-alginolyticus ecotype can be separated from all other V. alginolyticus strains solely based on presence/ absence of specific virulence genes Conclusions Third, niche adaptation and ultimately speciation in the genus Vibrio have been suggested to be mainly driven by HGT [13]. Likewise, unique GRs and a unique repertoire of MGEs within the Kiel-Fjord isolates (including unique plasmids and prophages) support ongoing theory that the acquisition of gene-blocks by HGT, one of the most im- portant mechanisms driving the evolution of niche adap- tation and clonal expansion also played an important role in the evolution of the Kiel-alginolyticus ecotype. Lastly, the unique virulence and resistance profile which clearly separates the Kiel-Fjord isolates from all other investigated V. alginolyticus strains, suggests, that these are essential By using a multi-scale comparative genomic approach, we conclude that the nine V. alginolyticus strains isolated from pipefish in the Kiel-Fjord belong to a unique ecotype, which evolved from a clonal expansion following niche adaptation. We came to this conclusion based on the fol- lowing observations. First, the nine strains from the Kiel- Fjord have a closed-pangenome reflecting low genomic di- versity within its niche. Second, even though these strains have been isolated from different eukaryotic host species, they did not differ based on core-genomic signatures which has been previously suggested based on MLSA [23]. Chibani et al. BMC Genomics (2020) 21:354 Page 11 of 14 Chibani et al. BMC Genomics (2020) 21:354 Chromosome 2) using the ANIm mode from the PyANI python module [42] based on MUMmer (https://github. com/widdowquinn/pyani). Briefly, nucleotide sequences were extracted from each GenBank file using Biopython (https://biopython.org/) and subsequently used as input for PyANI for genome sequence alignment. Chromosome 2) using the ANIm mode from the PyANI python module [42] based on MUMmer (https://github. com/widdowquinn/pyani). Briefly, nucleotide sequences were extracted from each GenBank file using Biopython (https://biopython.org/) and subsequently used as input for PyANI for genome sequence alignment. genes, required for a successful colonization of the pipe- fish, the niche of the Kiel-alginolyticus ecotype. To ultim- ately verify this theory, colonization experiments with knock-out mutants lacking the Kiel-specific virulence genes, or alternatively non virulent Vibrio strains equipped with the Kiel-specific virulence genes, would be required. We are aware that our conclusion is limited by the low number of non-Kiel V. alginolyticus strains available included in the comparative genomic analyses. We could have included draft genomes, to increase the number of sequences available for comparison. Phylogenetic analysis To further infer the phylogenetic relationship among all V. alginolyticus isolates we generated a phylogenetic tree based on the core-genome alignment for all 15 strains obtained from the Roary analysis (length for all strains ~ 3.84 Mbp, which corresponds to approximately 72% of the average size of the input genomes). To do so, we used the Bayesian Markov chain Monte Carlo (MCMC) method as implemented in MrBayes version 3.2.5 [47, 48]. The TN93 [49] model plus invariant sites (TN93 + I), as suggested by the Akaike information criterion (AIC) given by jModelTest [50], was used as a statistical model for nucleotide substitution. The MCMC process was repeated for 106 generations and sampled every 5000 generations. The first 2000 trees were deleted as Pan-Core genome prediction Pan Core genome prediction To determine the total gene-pool of V. alginolyticus within the Kiel-Fjord as well as the global gene reper- toire of V. alginolyticus, we performed two pan-core genome analyses. In the first analysis, we only included the nine strains from the Kiel-Fjord. In the second ana- lysis, we included all fully closed V. alginolyticus ge- nomes available from NCBI (accession date 10.10. 2019) which were isolated from various regions (Table 1), in addition to the nine Kiel strains. Genbank files of all closed V. alginolyticus isolates were converted to GFF3 files and a nucleotide alignment was generated using Roary 3.8.0 [43] with standard settings (minimum BLASTP identity of 90% with paralogue splitting). We used concatenated files for each isolate including both chromosomes and if existing, mobile genetic elements (plasmids and episomal phages). To estimate whether we have an open or a closed pangenome we used Heaps’ Law: η = κ ∗N−α [1], implemented in the R package ‘micropan’ [44], where η is the expected number of genes for a given number of Genomes (N), and κ and α are constants to fit the specific curve. The exponent α is an indicator whether the pangenome is open (α < 1) or closed (α > 1). Lastly, a functional characterization of all protein-coding sequences was performed using the eggnog-mapper v2 database v5.0 [45, 46]. We subse- quently determined the COG category (Cluster of Orthologous Groups of Proteins [40]) for Kiel-specific core- and accessory genes as well as singletons identified by the pan-core analysis based on the Roary clustering input table. The present study extends our current knowledge of pan-and core-genomic signatures in the genus Vibrio which is currently heavily biased towards human patho- gens, such as V. cholerae and V. parahaemolyticus. Based on the present results we suggest that also non- human pathogens might experience similar evolutionary dynamics, once relevant pathogenic traits (in the present case: Vibrio phage VALGΦ6 [19], plus three unique virulence genes (Fig. 6)) have been acquired. The avail- ability of a suitable niche, in the present case, presum- ably the pipefish, might have led to niche adaptation and clonal expansion of the Kiel-alginolyticus ecotype, clearly separating it from other V. alginolyticus species. Bacterial genome data Using a combination of PacBio and Illumina sequencing, we generated eight closed genomes and one permanent draft genome of nine Vibrio strains isolated from differ- ent pipefish in the Kiel-Fjord [28] and previously charac- terized as V. alginolyticus based on multi-locus-sequence typing [23]. For sequencing and annotation information please see [23]. We additionally sequenced prophages in- duced with mitomycin C from each strain, for details see [19]. We compared all replicons from the nine V. algino- lyticus strains to 10 closed Vibrio genome sequences downloaded from the NCBI nucleotide database; date of accession 1.12.2019 (Table S1). Conclusions However, due to the incomplete information especially for MGEs stored in draft genomes, the number of core-genomes drops con- siderably by approximately 50% compared to when only complete genomes are included in the analysis [20]. In favor of a more accurate analysis, we thus decided to only include complete genomes. Comparative genome analysis Average nucleotide identity The average similarity between the nine V. alginolyticus genomes from the present study, six other complete V. alginolyticus genomes as well as four other closely related vibrios (i.e. three V. diabolicus and one V. antiquarius) was measured as the average nucleotide identity (ANI) of all 19 Vibrio replicons (concatenated Chromosome 1 and Page 12 of 14 Chibani et al. BMC Genomics (2020) 21:354 Chibani et al. BMC Genomics (2020) 21:354 Chibani et al. BMC Genomics (2020) 21:354 burn-in processes and the consensus tree was con- structed from the remaining trees. Convergence was as- sured via the standard deviation of split frequencies (< 0.01) and the potential scale reduction factor (PSRF~ 1). The resulting phylogenetic tree and associated posterior probabilities were illustrated using FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-020-6735-5. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-020-6735-5. Additional file 1: Table S1. Vibrio genomes accessed from NCBI 3.12.2019 used for PyANI analysis. Additional file 1: Table S1. Vibrio genomes accessed from NCBI 3.12.2019 used for PyANI analysis. Additional file 2: Table S2. Genomic regions (GRs) unique for the nine V. alginolyticus strains from the Kiel-Fjord. For chromosome 1 (left) we used K10K4 as a reference, as this strain has more integrated prophages than the other Kiel isolates. For the same reason, we used strain K04M3 as a reference for chromosome 2. For a detailed comparison of GRs among all nine strains, see Fig. 3 main manuscript. Funding This project was funded by three consecutive DFG grants [WE 5822/ 1–1, WE5822/1–2, and RO RO462/4–2] within the priority programme SPP1819 given to given to CCW and OR, a grant from the Cluster of Excellence “The Future Ocean”, given to CCW, and a KAAD stipend given to CCM. The funding agency played no part in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors’ contributions CC d O d CCW, HL and OR designed the study. CMC and CCW analyzed the data. CCW wrote the manuscript. All authors commented on the manuscript and approved the final version. Consent for publication Not applicable. Not applicable. Not applicable. Genomic Islands We used IslandViewer [51] to predict putative genomic islands of all nine isolates from the Kiel-Fjord. The selection criterion for genomic islands was based on the presence of mobile-related genes (integrases or transpo- sases), a size > 8 kb, and a different GC content com- pared to the rest of the genome. To visualize the genomic islands, we used BLAST Ring Image Generator [52, 53] and created a circular map per chromosome of all fully closed V. alginolyticus genomes. Functional characterization of all protein-coding sequences found on the putative genomic islands was performed as de- scribed above using the eggNOG-mapper v2 database v5.0 [45, 46] where the resulting COG assignments were used for further analysis. Additional file 3: Table S3. Functional characterization of all protein- coding sequences found on the Genomic Regions (using the eggNOG- mapper v2 software) with the resulting COG assignments. Similar to Table S2 we used strain K10K4 as reference for chromosome 1 and strain K04M3 as reference for chromosome 2. The column Kiel Specific indicates whether the GR is unique for the Kiel alginolyticus ecotype. Additional file 4: Table S4. Functional annotation of Kiel specific gene clusters (core genome [C], accessory genes [A], singletons [S], i.e. gene cluster which was only found within the Kiel alginolyticus ecotype with the resulting COG assignments. Additional file 5: Table S5. Resistance genes found using IslandViewer for all nine V. alginolyticus strains from the Kiel-Fjord. Even though this table shows the locus tags for strain K04M3 only, all other strains have been found to contain the exact same resistance genes. Abbreviations GIs: Genomic islands; GRs: Genomic regions; ANI: Average nucleotide identity; MLSA: Multi-locus-sequencing-approach; COG: Clusters of Orthologous Groups of Proteins; MGEs: Mobile genetic elements; HGT: Horizontal gene transfer GIs: Genomic islands; GRs: Genomic regions; ANI: Average nucleotide identity; MLSA: Multi-locus-sequencing-approach; COG: Clusters of Orthologous Groups of Proteins; MGEs: Mobile genetic elements; HGT: Horizontal gene transfer Competing interests p g he authors declare that they have no competing interests. Acknowledgements f l We are grateful to Anja Poehlein for her support in sequencing the isolates. Plasmids We used Easyfig [54] for pairwise plasmid sequence comparisons and synteny comparisons with an E-value cut-off of 1e−10. Plasmid maps were generated using SnapGene Viewer (version 4.3.10) with annotated gen- bank files as input files. The output resulting from Roary was used to determine orthologous genes, while the out- put of eggNOG-mapper [45, 46] was used to assign a functional characterization to the coding protein se- quences of the six mega-plasmids detected in the Kiel- specific V. alginolyticus isolates. Availability of data and materials h f h The virulence of the nine Kiel-isolates was determined using controlled infection experiments in a previous study [19], and could in part be explained by the pres- ence and the coverage of a virulence-gene carrying fila- mentous phage Vibrio phage VALGΦ6. To determine other potential chromosomal encoded virulence factors, we scanned all 15 V. alginolyticus strains and seven other strains from different Vibrio clades using the Viru- lence Factor Database (https://www.mgc.ac.cn/VFs/ main.htm) [55]. We used IslandViewer [51] to identify homologues of resistance genes in all 15 V. alginolyticus strains. The genomic sequences of the nine Kiel V. alginolyticus strains analyzed during the current study are available at GenBank (CP017889-CP017919). The genomic sequences of the nine Kiel V. alginolyticus strains analyzed during the current study are available at GenBank (CP017889-CP017919). Ethics approval and consent to participate Ethics approval and consent to participate References 1. Tettelin H, Riley D, Cattuto C, Medini D. Comparative genomics: the bacterial pan-genome. Curr Opin Microbiol. 2008;11(5):472–7. 1. Tettelin H, Riley D, Cattuto C, Medini D. Comparative genomics: the bacterial pan-genome. Curr Opin Microbiol. 2008;11(5):472–7. 1. Tettelin H, Riley D, Cattuto C, Medini D. Comparative genomics: the bacterial pan-genome. Curr Opin Microbiol. 2008;11(5):472–7. 25. Poirier M, Listmann L, Roth O. Selection by higher-order effects of salinity and bacteria on early life-stages of Western Baltic spring-spawning herring. Evol Appl. 2017;10(6):603–15. 2. 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https://openalex.org/W3040491196	https://repository.ubn.ru.nl//bitstream/handle/2066/232886/232886.pdf	English	null	Consensus statements on PSMA PET/CT response assessment criteria in prostate cancer	European journal of nuclear medicine and molecular imaging	2,020	cc-by	7,312	Consensus statements on PSMA PET/CT response assessment criteria in prostate cancer Fanti, S.; Goffin, K.; Hadaschik, B.A.; Herrmann, K.; Maurer, T.; MacLennan, S.; Oprea-Lager, D.E.; Oyen, W.J.G.; Rouviere, O.; Mottet, N.; Bjartell, A. 2021, Article / Letter to editor (European Journal of Nuclear Medicine and Molecular Imaging, 48, 2, (2021), pp. 469-476) Doi link to publisher: https://doi.org/10.1007/s00259-020-04934-4 Doi link to publisher: https://doi.org/10.1007/s00259-020-04934-4 Doi link to publisher: https://doi.org/10.1007/s00259-020-04934-4 Consensus statements on PSMA PET/CT response assessment criteria in prostate cancer Fanti, S.; Goffin, K.; Hadaschik, B.A.; Herrmann, K.; Maurer, T.; MacLennan, S.; Oprea-Lager, D.E.; Oyen, W.J.G.; Rouviere, O.; Mottet, N.; Bjartell, A. 2021, Article / Letter to editor (European Journal of Nuclear Medicine and Molecular Imaging, 48, 2, (2021), pp. 469-476) Doi link to publisher: https://doi.org/10.1007/s00259-020-04934-4 Consensus statements on PSMA PET/CT response assessment criteria in prostate cancer Fanti, S.; Goffin, K.; Hadaschik, B.A.; Herrmann, K.; Maurer, T.; MacLennan, S.; Oprea-Lager, D.E.; Oyen, W.J.G.; Rouviere, O.; Mottet, N.; Bjartell, A. 2021, Article / Letter to editor (European Journal of Nuclear Medicine and Molecular Imaging, 48, 2, (2021), pp. 469-476) Doi link to publisher: https://doi org/10 1007/s00259-020-04934-4 Consensus statements on PSMA PET/CT response assessment criteria in prostate cancer Stefano Fanti1 & Karolien Goffin2 & Boris A Hadaschik3 & Ken Herrmann4 & Tobias Maurer5 & Steven MacLennan6 & Daniela E. Oprea-Lager7 & Wim JG Oyen8,9,10 & Olivier Rouvière11,12 & Nicolas Mottet13 & Anders Bjartell14 Stefano Fanti1 & Karolien Goffin2 & Boris A Hadaschik3 & Ken Herrmann4 & Tobias Maurer5 & Steven MacLennan6 & Daniela E. Oprea-Lager7 & Wim JG Oyen8,9,10 & Olivier Rouvière11,12 & Nicolas Mottet13 & Anders Bjartell14 Received: 27 May 2020 /Accepted: 17 June 2020 # The Author(s) 2020 / Published online: 2 July 2020 Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). https://doi.org/10.1007/s00259-020-04934-4 European Journal of Nuclear Medicine and Molecular Imaging (2021) 48:469–476 https://doi.org/10.1007/s00259-020-04934-4 European Journal of Nuclear Medicine and Molecular Imaging (2021) 48:469–476 ORIGINAL ARTICLE Abstract Purpose Prostate-specific membrane antigen (PSMA) positron emission tomography (PET)/computed tomography (CT) is used for (re)staging prostate cancer (PCa) and as a biomarker for evaluating response to therapy, but lacks established response criteria. A panel of PCa experts in nuclear medicine, radiology, and/or urology met on February 21, 2020, in Amsterdam, The Netherlands, to formulate criteria for PSMA PET/CT-based response in patients treated for metastatic PCa and optimal timing to use it. Methods Panelists received thematic topics and relevant literature prior to the meeting. Statements on how to interpret response and progression on therapy in PCa with PSMA PET/CT and when to use it were developed. Panelists voted anonymously on a nine-point scale, ranging from strongly disagree (1) to strongly agree (9). Median scores described agreement and consensus. Results PSMA PET/CT consensus statements concerned utility, best timing for performing, criteria for evaluation of response, patients who could benefit, and handling of radiolabeled PSMA PET tracers. Consensus was reached on all statements. PSMA PET/CT can be used before and after any local and systemic treatment in patients with metastatic disease to evaluate response to treatment. Ideally, PSMA PET/CT imaging criteria should categorize patients as responders, patients with stable disease, partial response, and complete response, or as non-responders. Specific clinical scenarios such as oligometastatic or polymetastatic disease deserve special consideration. Purpose Prostate-specific membrane antigen (PSMA) positron emission tomography (PET)/computed tomography (CT) is used for (re)staging prostate cancer (PCa) and as a biomarker for evaluating response to therapy, but lacks established response criteria. A panel of PCa experts in nuclear medicine, radiology, and/or urology met on February 21, 2020, in Amsterdam, The Netherlands, to formulate criteria for PSMA PET/CT-based response in patients treated for metastatic PCa and optimal timing to use it. p Conclusions Adoption of PSMA PET/CT should be supported by indication for appropriate use and precise criteria for inter- pretation. PSMA PET/CT criteria should categorize patients as responders or non-responders. Specific clinical scenarios deserve special consideration. Keywords Monitoring .Oligometastaticprostatecancer . PERCIST .PET/CT . Polymetastaticprostatecancer . Prostatecancer . Prostate-specific membrane antigen (PSMA) . PSMA ligand . RECIST Keywords Monitoring .Oligometastaticprostatecancer . PERCIST .PET/CT . Polymetastaticprostatecancer . Prostatecancer . Prostate-specific membrane antigen (PSMA) . PSMA ligand . * Anders Bjartell anders.bjartell@med.lu.se Introduction experts in the aforementioned specialties, to separately discuss these thematic topics in greater detail and develop statements related to the use of PSMA PET/CT. The breakout groups then reconvened, discussed all statements written by each subgroup, and created a combined list of statements organized under thematic topics. Prostate-specific membrane antigen (PSMA) positron emis- sion tomography (PET)/computed tomography (CT) is being used more frequently for prostate cancer (PCa) staging and for localization of recurrent disease [1]. For the scope of the present project, PSMA PET/CT refers to PET/CT imaging performed with PSMA-targeting radioligands (e.g., [68Ga]Ga-PSMA-11, [18F]F-PSMA-1007, [18F]DCFPyL). International guidelines, including the guideline of the European Association for Urology (EAU) [2], have incorpo- rated PSMA PET/CT and provide recommendation on its ap- propriate use. It is the consensus imaging modality in patients with rising PSA after radical treatment to confirm a diagnosis of oligorecurrent (metachronous) oligometastatic PCa [3]. In other clinical situations, such as PSA persistence after radical treatment, PSMA PET/CT is also recommended [2], while for initial staging of high-risk patients, the ultimate role of PSMA PET/CT is still debated. The most recent results of prospective trials are very promising [4, 5]. Each statement was singular and phrased so that panel members could agree or disagree with it. The resulting state- ments were uploaded to an online voting tool, and the panel- ists voted anonymously online [8]. Panelists voted on a nine- point scale, ranging from strongly disagree (1) to strongly agree (9) (i.e., 1–3 disagree, 4–6 uncertain, 7–9 agree). The results of the vote on each statement were conveyed to partic- ipants immediately after voting. There are procedure guidelines for performing PSMA PET/CT endorsed by European Association of Nuclear Medicine (EANM) [1]. PSMA PET/CT is considered a poten- tially useful tool for evaluating responses to therapy, but there is lack of data regarding criteria to be used for evaluating PSMA PET/CT findings in relation to therapy response as- sessment [2, 4]. In recognition of the growing importance of PSMA radiopharmaceuticals in the diagnosis and treatment of patients with PCa, the EAU in collaboration with EANM re- cruited a panel of international experts in PCa within the fields of nuclear medicine, radiology, and urology, who met on February 21, 2020, in Amsterdam, The Netherlands, for an European consensus panel to formulate criteria for PSMA PET/CT-based response and progression in patients treated for metastatic PCa and when to use it. Abstract RECIST This article is part of the Topical Collection on Oncology - Genitourinary * Anders Bjartell anders.bjartell@med.lu.se 1 Nuclear Medicine Division, Policlinico S Orsola, University of Bologna, Bologna, Italy 2 Department of Nuclear Medicine and Molecular Imaging, University Hospital Leuven and KU Leuven, Leuven, Belgium 3 Department of Urology, University of Duisburg-Essen and German Cancer Consortium (DKTK)-University Hospital Essen, Essen, Germany 4 Department of Nuclear Medicine, University of Duisburg-Essen and German Cancer Consortium (DKTK)-University Hospital Essen, Essen, Germany 5 Department of Urology and Martini-Klinik Prostate Cancer Center, Universitätsklinikum, Hamburg-Eppendorf, Hamburg, Germany 6 Academic Urology Unit, University of Aberdeen, Foresterhill, Aberdeen, UK 7 Department of Radiology and Nuclear Medicine, Amsterdam University Medical Centers, VU University, Amsterdam, The Netherlands 8 Humanitas University and Humanitas Clinical and Research Center, Milan, Italy 9 Department of Radiology and Nuclear Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands 10 Department of Radiology and Nuclear Medicine, Rijnstate Hospital Arnhem, Arnhem, The Netherlands 11 Department of Imaging, Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France 12 Faculté de Médecine Lyon Est, Université de Lyon, Université Lyon 1, 69003 Lyon, France 13 University Hôpital Nord, Saint Etienne, France 14 Department of Urology, Skåne University Hospital, Malmö, Sweden 7 Department of Radiology and Nuclear Medicine, Amsterdam University Medical Centers, VU University, Amsterdam, The Netherlands * Anders Bjartell anders.bjartell@med.lu.se 11 Department of Imaging, Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France 4 Department of Nuclear Medicine, University of Duisburg-Essen an German Cancer Consortium (DKTK)-University Hospital Essen, Essen, Germany 12 Faculté de Médecine Lyon Est, Université de Lyon, Université Lyon 1, 69003 Lyon, France 5 Department of Urology and Martini-Klinik Prostate Cancer Center, Universitätsklinikum, Hamburg-Eppendorf, Hamburg, Germany 13 University Hôpital Nord, Saint Etienne, France 14 Department of Urology, Skåne University Hospital, Malmö, Sweden 14 Department of Urology, Skåne University Hospital, Malmö, Sweden 470 Eur J Nucl Med Mol Imaging (2021) 48:469–476 Introduction The main output of this meeting was the development of sets of consensus statements concerning PSMA PET/CT criteria for response assessment in various clinical scenarios for patients with metastatic PCa, and these are presented here. The statistical analysis of the voting focused on the level of agreement (median score) with each statement and whether there was consensus (dispersion of scores around the median). This followed the methods proposed by the research and de- velopment project (RAND)/University of California, Los Angeles (UCLA) Appropriateness Method which has been shown to be robust for smaller panels and can be used in panels of any size [9]. For each statement, first the median score was calculated, then the 30th and 70th percentiles were calculated, which constitute the interpercentile range (IPR). The IPR was used to calculate the interpercentile range adjust- ed for symmetry (IPRAS), which is calculated using the for- mula: IPRAS = 2.35 + (asymmetry index [AI] * 1.5), where the AI is defined as the absolute difference between the central point of the IPR and 5 (i.e., the central point on the 1–9 scale). If the IPR < IPRAS, then this indicates there is no extreme dispersion of scores (i.e., there is “consensus”). The median agreement score (MAS) was used to determine the level of agreement: a MAS in the 1–3 range indicated the panel disagreed with the statement, a MAS in the 4–6 range indicated the panel were uncertain, and a MAS in the 7–9 range indicated the panel agreed with the statement. Then, the IPRAS was used to assess consensus. For a statement to be considered to have agreement and consensus, two condi- tions had to be met: a median score in the 7–9 category and an IPRAS < IPR. Materials and methods A modified nominal group technique was used to reach con- sensus statements on PSMA PET/CT response assessment criteria [6, 7]. Ten weeks prior to meeting, each panelist re- ceived thematic topics concerning PSMA PET/CT response assessment along with relevant literature suggested by the panelists. The panelists (Stefano Fanti, Karolien Goffin, Boris Hadaschik, Ken Herrmann, Tobias Maurer, Daniela Oprea-Lager, Wim JG Oyen, Olivier Rouvière, and Anders Bjartell), all authors of this paper, are PCa experts in nuclear medicine (five panelists), radiology (one panelist), or urology (three panelists). A one-day meeting was held, where the the- matic topics were again presented. The panelists were subse- quently divided into two groups, each containing a balance of Results Within the scope of each thematic topic, statements were de- veloped by the panelists during the one-day meeting concerning how to interpret PSMA PET/CT to define re- sponse and progression in patients treated for metastatic PCa and when to use it. These thematic topics and statements along Eur J Nucl Med Mol Imaging (2021) 48:469–476 471 are, for example, concerns regarding how to interpret uptake of tracer especially in hormone-naive patients starting andro- gen deprivation therapy (ADT) [11]. Consensus was reached on the statement that in principle, PSMA PET/CT can be used before and after any local and systemic treatment in patients with metastatic disease (including N1 disease). PSMA PET/ CT is actually the most sensitive technique to detect metasta- ses, and thus, it is likely that the prognosis of M0 and M+ patients based on PSMA PET/CT will be changed, as com- pared to the prognosis of M0 and M+ patients based on stan- dard work up (CT and bone scan). Nonetheless, in the absence of clinical trials assessing the prognosis of patients with me- tastases detected only by PSMA, it remains unclear whether PSMA PET/CT should be widely used in high-risk patients. with the corresponding MAS, IPR, and IPRAS from the vot- ing are reported in Table 1. Briefly, the statements covered the best timing for performing PSMA PET/CT, the criteria that should be used to evaluate the PSMA PET/CT response, the patients who could benefit from such evaluation from a clin- ical perspective, and how the various PSMA-targeting PET tracers should be handled. with the corresponding MAS, IPR, and IPRAS from the vot- ing are reported in Table 1. Briefly, the statements covered the best timing for performing PSMA PET/CT, the criteria that should be used to evaluate the PSMA PET/CT response, the patients who could benefit from such evaluation from a clin- ical perspective, and how the various PSMA-targeting PET tracers should be handled. Other relevant thematic topics were also addressed with statements: Available real-world data and/or trial data should be analyzed for response assessment with PSMA PET/CT before designing future prospective trials; Criteria for treat- ment assessment should be evaluated in test-retest trials to assess normal variability in the measurement. Results In every in- stance, the MAS fell in the 7–9 category and IPR as lower than IPRAS, so there was “agreement” and “consensus.” The panel is well aware that the proposed criteria are based on practical experience and authors’ opinions; indeed, there is very little data to ultimately support some of the statements that are made. Nonetheless, it is clear that some baseline criteria are proposed to be used for future studies and applied for clinical scenarios that are already happening. With expect- ed publication of more data from prospective studies, it will hopefully be possible to refine the statements proposed here, possibly with future further consensus events to be held for review of datasets with outcome measures. Once the criteria have been evaluated in clinical trials, they could surely be improved. As an antecedent to the discussion of the thematic topics, the panelists considered the utility and impact of PSMA PET/ CT in PCa and developed four statements and reached con- sensus on all four. • PSMA PET/CT is currently the most sensitive imaging staging technique for depicting metastases at any stage of the disease. MAS = 8 (IPR = 1; IPRAS = 7.6). • Use of PSMA PET/CT will cause stage migration, which is a problem for clinical decision-making. MAS = 8 (IPR = 0; IPRAS = 6.85). • PSMA PET/CT should be used as imaging tool at bio- chemical recurrence. MAS = 9 (IPR = 0; IPRAS = 8.35). Discussion All consensus statements were developed following the European Society for Radiotherapy and Oncology (ESRO) and European Organisation for Research and Treatment of Cancer (EORTC) consensus recommendations for classifica- tion of oligometastatic disease [10]. The panelists agreed that potential flare observed in a PSMA scan following initiation of ADT is an early event, and misinterpretation can be avoided by performing the scan not earlier than 3 months after the start of ADT (see also statement 2.3). Data concerning the flare phenomenon ob- served with ADT and second-generation anti-androgen thera- py are limited and not unequivocal [12–15]. The correlation between androgen signaling and PSMA expression seems to be quite complex and requires more study before practice- defining conclusions can be drawn. Nonetheless, the panelists, based on their practical experience, suggested 3 months as the minimal timing for performing PSMA PET/CT after initiation of hormonal intervention. A prospective study has shown that androgen deprivation therapy could impact the timing for PSMA PET/CT [16]. The study included 15 men, eight pa- tients with metastatic hormone sensitive PCa treated with lu- teinizing hormone-releasing hormone and bicalutamide and Timing for PSMA PET/CT • PSMA PET/CT should not be used in low-risk disease. MAS = 9 (IPR = 0; IPRAS = 8.35). With respect to the best timing for performing PSMA PET/ CT, some evident prerequisites were confirmed, such as that in case of primary PCa negative on PSMA PET/CT, further imaging with PSMA PET/CT is generally not adequate (state- ment 2.1), and if used for response assessment, baseline PSMA PET/CT should be performed before the start of treat- ment (statement 2.2). General statements Regarding the use of PSMA PET/CT for assessing response to therapy, it was confirmed that such an approach should be used only if a change of clinical management is expected from the results (statements 1.2 and 2.4) as already emphasized in EAU Guidelines [2]. Thus, in case of good PSA response after salvage treatment, there is no indication for performing PSMA PET/CT (statement 1.4); also, in case of late-stage disease where no further therapies can be initiated or hormone sensi- tive PCa with favorable PSA response under systemic therapy, there is no role for PSMA PET/CT (statements 1.5 and 1.6). Asking whether PSMA PET/CT can be used before, after, and during treatment is an important question, because there 472 Eur J Nucl Med Mol Imaging (2021) 48:469–476 Table 1 Consensus statements on PSMA PET/CT response assessment criteria along with their respective median agreement scores (MAS), interpercentile ranges (IPR), and interpercentile ranges adjusted for symmetry (IPRAS) Table 1 Consensus statements on PSMA PET/CT response assessment criteria along with their respective median agreement scores (MAS), interpercentile ranges (IPR), and interpercentile ranges adjusted for symmetry (IPRAS) Table 1 Consensus statements on PSMA PET/CT response assessment criteria along with their respective median agreement scores (MAS), interpercentile ranges (IPR), and interpercentile ranges adjusted for symmetry (IPRAS) Thematic topic and corresponding statement Statement MAS IPR IPRAS 1. Can PSMA PET/CT be used before and after any treatment? 1.1 In principal, PSMA PET/CT can be used before and after any local and systemic treatment in patients at risk of having metastatic disease (including N1 disease). 8 1 7.6 1.2 PSMA PET/CT should not be used if no change of clinical management is expected. 9 0 8.35 1.3 PSMA PET/CT should not be used if local disease within the prostate is expected. 8 1 7.6 1.4 After salvage treatment of oligometastatic PCa with curative intent, if PSA indicates complete response, PSMA PET/CT is to be avoided as an assessment tool. 9 0.6 7.9 1.5 Routine use of PSMA PET/CT should not be used in patients with hormone sensitive PCa and PSA response under systemic therapy. 8 0.6 7.3 1.6 Routine use of PSMA PET/CT should not be used in patients with late-stage disease where no further therapies can be initiated. 7 0 5.35 2. What is the best timing for performing PSMA PET/CT? General statements 2.1 In cases where the primary PCa is negative on PSMA PET/CT, further staging with PSMA PET/CT is generally not adequate, and other imaging should be performed. 9 0 8.35 2.2 If used for response assessment, then a PSMA PET/CT should be performed before the start of treatment (i.e., baseline PSMA PET/CT). 9 0 8.35 2.3 PSMA PET/CT should not be performed within three months after initiation of systemic therapy in hormone sensitive PCa. 9 1 7.6 2.4 PSMA PET/CT should not be performed for routine follow-up if management of the patient is unlikely to change. 9 0.6 7.9 3. Which PET criteria should be used to evaluate the PSMA response? 3.1 For clinicians, PSMA PET/CT should always be accompanied by evaluation of clinical and laboratory data. 9 0 8.35 3.2 For imaging specialists, only treatment response criteria for PSMA PET/CT are needed (not taking other parameters into account (e.g. PSA, etc.). 9 1 7.6 3.3 Ideally, PSMA PET/CT criteria should categorize patients as responders or non-responders. 9 0 8.35 3.4 Categories of responders should include patients with stable disease and partial and complete response on PSMA PET/CT imaging; non-responders should include patients with progressive disease on PSMA PET/CT. 8 1.2 6.85 3.5 In early recurrent PCa, appearance of any new lesion with high suspicion should be regarded as progressive disease. 8 1 7.6 3.6 In polymetastatic PCa, increase of uptake or tumor volume > 30% is defined as progressive disease. 8 1 7.6 3.7 In polymetastatic PCa, appearance of two or more new lesions should not be regarded as progressive disease, if total tumor volume or uptake does not increase > 30%. 9 1 7.6 3.8 PCWG3-criteria for evaluation of bone scintigraphy should not necessarily be used for PSMA PET/CT evaluation. 9 0.6 7.9 3.9 Complete response: complete disappearance of any lesion with tracer uptake; partial response: reduction of uptake and tumor volume by > 30%; SD: change of uptake and tumor volume ± ≤30% and no new lesions; progressive disease: appearance of two or more new lesions and/or increase of uptake or tumor PET volume > 30%. 9 0.6 7.3 4. Which patients could benefit from PSMA response assessment from a clinical perspective (other than those for whom a treatment change will have a significant impact on outcome)? 4.1 PSMA PET/CT response assessment should be evaluated in the context of clinical trials. General statements 9 0 8.35 4.2 In clinical practice, PSMA PET/CT response assessment may be performed in patients with inconsistent laboratory findings and/or clinical course of the disease if change of management is considered. 9 0 8.35 5. How should the various PET tracers be handled? 5.1 Different 68Ga and 18F radiolabeled PSMA tracers (e.g., [68Ga]Ga-PSMA-11, [18F]PSMA-1007, and [18F]DCFPyL) show similar performance even if there is lack of comparative data. 8 1 7.6 5.2 For response assessment the same PSMA PET tracers should be used. 9 0 8.35 5.3 Quality assurance is mandatory either for radiotracer production and image acquisition and should include EARL-harmonized protocols (scanner, reconstruction algorithms) regarding dosage, time of acquisition and quantification should be done using validated software. 9 0 8.35 6. Other relevant statements 6.1 Available real-world data and/or trial data should be analyzed for response assessment with PSMA PET/CT before designing future prospective trials. 9 0 8.35 6.2 Criteria for treatment assessment should be evaluated in test-retest trials to assess normal variability in the measurement. 9 1 7.6 EARL, European Association of Nuclear Medicine Research Ltd.; IPR, interpercentile range; IPRAS, interpercentile range adjusted for symmetry; MAS, Thematic topic and corresponding statement Thematic topic and corresponding statement Statement 1. Can PSMA PET/CT be used before and after any treatment? 1.3 PSMA PET/CT should not be used if local disease w 2. What is the best timing for performing PSMA PET/CT? 2.3 PSMA PET/CT should not be performed within three months after initiation of systemic therapy in hormone sensitive PCa. 2.4 PSMA PET/CT should not be performed for routine follow-up if management of the patient is unlikely to change. 3.1 For clinicians, PSMA PET/CT should always be accompanied by evaluation of clinical and lab 3.2 For imaging specialists, only treatment response criteria for PSMA PET/CT are needed (not taking other parameters into account (e.g. PSA, etc.). 3 Ideally, PSMA PET/CT criteria should categorize patients 3.4 Categories of responders should include patients with stable disease and partial and complete response on PSMA PET/CT imaging; non-responders should include patients with progressive disease on PSMA PET/CT. 8 1.2 3.5 In early recurrent PCa, appearance of any new lesion with high suspi ymetastatic PCa, increase of uptake or tumor volume > 30% is de 3.7 In polymetastatic PCa, appearance of two or more new lesions should not be r disease, if total tumor volume or uptake does not increase > 30%. PCWG3-criteria for evaluation of bone scintigraphy should no 3.9 Complete response: complete disappearance of any lesion with tracer uptake; partial response: reduction of uptake and tumor volume by > 30%; SD: change of uptake and tumor volume ± ≤30% and no new lesions; progressive disease: appearance of two or more new lesions and/or increase of uptake or tumor PET volume > 30%. 9 0.6 7.3 4.1 PSMA PET/CT response assessment should be evaluated in the context of clinical trials. 4.2 In clinical practice, PSMA PET/CT response assessment may be performed in patients with inconsistent laboratory findings and/or clinical course of the disease if change of management is considered. 9 0 8.35 r response assessment the same PSMA PET tracers should be us 6.2 Criteria for treatment assessment should be evaluated in test-retest trials to assess normal variability in the measurement. 9 1 7.6 Eur J Nucl Med Mol Imaging (2021) 48:469–476 473 seven patients with castration resistant PCa treated with enzalutamide or abiraterone, and showed that at 9 days post treatment, [68Ga]Ga-PSMA-11 PET/CT SUVmax decreased in the hormone-sensitive patients (median 30% (interquartile range 5–61) but increased in the castration-resistant PCa (me- dian 45% (interquartile range 12.7–66)). Criteria for interpretation of PSMA PET/CT Criteria to be used to evaluate PSMA response were thorough- ly discussed and addressed. Indeed, molecular response criteria have been proposed as better than morphological criteria in patients with metastatic castration resistant PCa [17], but the precise criteria to be used have not been identi- fied. There are no clear definitions for oligometastatic and polymetastatic disease, so, in the context of this report, pro- gression was defined in order to start creating guidance on how to use PSMA PET/CT. In general, there is confidence in drawing conclusions in cases with complete response as well as for detecting clear progression of disease and scoring of new lesions [18–20], but uncertainty remains for everything in between. Semi-quantitative evaluation of radiolabeled PSMA PET/ CT scans could be very useful when assessing response to treatment. Standardized uptake value (SUV) is the most com- monly used semi-quantitative parameter, providing a measure of radiotracer uptake. The main recommendation for using SUV parameters, (SUVmax, SUVmean, or SUVpeak) is to optimize reproducibility by limiting all variation factors (qual- ity of injection, uptake time, type of scanner, reconstruction algorithm, and others), namely to rigorously harmonize pro- cedures and parameters in all scans. Volumetric PET measure- ments (so called tumor burden values) based on available software packages can partly overcome such problems [24, 25], since they account for less variations across patients and are better reproducible. However, these parameters are still not widely used in clinical practice because they are technically demanding. Tools for quantitative estimation of the total tu- mor burden are under development, and these could reduce current problems with interobserver variability and low repro- ducibility with manual interpretation of images. The panelists agreed that PSMA PET/CT criteria should categorize patients as responders or non-responders (statement 3.3) for both, clinical use and trial incorporation. Given the nature of PCa, it was agreed that categories of responders should include patients with stable disease, partial response, and complete response on PSMA PET/CT imaging; non- responders should include patients with progressive disease on PSMA PET/CT (statement 3.3). In this light, the consensus statements were written with progression defined as a 30% increase of tumor burden, and this is in line with other studies and modified PET response criteria in solid tumors (PERCIST) [21–23]. Criteria for interpretation of PSMA PET/CT In general, the panel agreed on a definition of PSMA response as Complete in case of disappearance of any lesion with tracer uptake; Partial as reduction of uptake and tumor PET volume by > 30%; Stable as change of uptake and tumor PET volume ± ≤30% without evidence of new lesions; Progression as appearance of > 2 new lesions or increase of uptake or tumor PET volume >= 30% (statement 3.9). The use of uptake thresholds based on PERCIST could not be supported by the literature, because these have only been validated for FDG PET. Nonetheless, the panelists agreed that a threshold should be arbitrarily chosen as baseline for future studies; furthermore, it should be at least wider than repeat- ability coefficients (published only for the PSMA-targeted agent DCFPyL) and could be modified in the future to hope- fully arrive at the appropriate cut-offs for tumor response [25]. 1. Can PSMA PET/CT be used before and after any treatment? appearance of two or more new lesions should not be neces- sarily regarded as progressive disease, if total tumor PET vol- ume or uptake does not increase > 30% (statements 3.6 and 3.7). For the purpose of this project, the definition of a new lesion is based on the appearance of a new focal area of PSMA uptake at PET, with or without CT change. Disease progres- sion should not be based solely on PET/CT. The rationale here was to avoid misinterpretation of progressive disease in case of an overall good response of known lesions to systemic therapy with only appearance of a few minor lesions. Indeed, it would be a mistake to switch an active treatment too early in polymetastatic situations. Limit of the methodology Study concept and design: All authors Acquisition of data: Stefano Fanti, Karolien Goffin, Boris Hadaschik, Ken Herrmann, Tobias Maurer, Steven MacLennan, Daniela Oprea- Lager, Wim JG Oyen, Olivier Rouvière, and Anders Bjartel Analysis and interpretation of data: All authors Drafting of the manuscript: All authors Critical revision of the manuscript for important intellectual content: All authors Critical revision of the manuscript for important intellectual content: All authors Statistical analysis: Steven MacLennan Obtaining funding: Anders Bjartell Supervision: Anders Bjartell Statistical analysis: Steven MacLennan Obtaining funding: Anders Bjartell Supervision: Anders Bjartell Funding information Open Access funding provided by Lund University. The one-day EAU/EANM PSMA PET/CT Response Assessment Criteria meeting received an unrestricted grant from Janssen, and no influences on the content of the meeting or on the publication. Perspective Available real-world data and/or trial data should be analyzed for response assessment with PSMA PET/CT before design- ing future prospective trials. Furthermore, criteria for treat- ment assessment should be evaluated in test-retest trials to assess normal variability in the measurement. Compliance with ethical standards Conflict of interest Stefano Fanti reports personal fees from ANMI, Astellas, Bayer, BlueEarth Diagnostics, GE Healthcare, Janssen, Novartis, and Sofie Biosciences, and non-financial support from AAA, Bayer, GE Healthcare, Curium, Tema Sinergie, Sanofi, Telix, outside the submitted work; Karolien Goffin reports personal fees from Bayer, Blue Earth Diagnostics, GE Healthcare, and Lightpoint Medical, outside the submitted work; Boris Hadaschik reports personal fees from ABX, Bayer, Lightpoint Medical, Inc., Janssen R&D, Bristol-Myers-Squibb, and Astellas; research funding from Profound Medical, German Cancer Aid, German Research Foundation, Janssen R&D, Bristol-Myers- Squibb, MSD, Pfizer, and Astellas; and travel from AstraZeneca, Janssen R&D, and Astellas; Ken Herrmann reports personal fees from Bayer, other from Sofie Biosciences, personal fees from SIRTEX, non- financial support from ABX, personal fees from Adacap, personal fees from Curium, personal fees from Endocyte, grants and personal fees from BTG, personal fees from IPSEN, personal fees from Siemens Healthineers, personal fees from GE Healthcare, personal fees from Amgen, personal fees from Novartis, and personal fees from Y-mAbs, Need for standard interpretation of scans While these criteria are ready to be used in most situations, and should be evaluated in future studies, some specific clin- ical scenarios deserve special consideration. The definition of progression indicated by the panel is largely in line with the proposal of PSMA PET progression [18], largely based on the appearance of ≥2 new lesions, which is adequate in many cases. Nonetheless, the panelists agreed that in early recurrent PCa, appearance of any new lesion with high suspicion should be regarded as progressive disease (statement 3.5). The ratio- nale was to maximize the sensitivity of the approach in such settings, where additional local therapies may still be pro- posed. Conversely, in polymetastatic PCa, increase of uptake or tumor PET volume > 30% was regarded as progression; Quality standards for interpretation of PSMA PET/CT scans are needed. Visual, qualitative assessment of the attenuated and non-attenuated PET images is the basis of any PET study interpretation. The major concern regarding this assessment is the significant inter- and intra-observer variability of PET im- age interpretation. Therefore, quality standards for interpreta- tion of PSMA PET/CT scans are needed. Yet, the report from the nuclear medicine physician needs to adhere to quality standards, as recently proposed in the E-PSMA: the EANM standardized reporting guidelines for PSMA PET/CT. The quality of the procedure itself is standardized using existing Eur J Nucl Med Mol Imaging (2021) 48:469–476 474 procedures guidelines to obtain high-quality imaging [1], and the same applies to quantification software. performed within 3 months after initiation of systemic therapy in hormone sensitive PCa. Different PSMA tracers, including [68Ga]Ga-PSMA-11 and [18F]F-labeled PSMA-1007 and [18F]DCFPyL tracers, are supposed to perform similarly, even if there is lack of comparative data [26]. For response assessment, it is evident that the same PSMA PET tracers should be used for baseline and subsequent imaging. Quality assurance is mandatory for both radiotracer production and image acquisition. Ideally, PSMA PET/CT criteria should categorize patients as responders or non-responders: categories of responders in- clude patients with stable disease, partial response, and com- plete response on PSMA PET/CT imaging. Specific clinical scenarios such as oligometastatic or polymetastatic disease deserve special consideration. The use of PSMA PET/CT re- sponse assessment should be implemented and evaluated in the context of clinical trials. Limit of the methodology EAU and EANM endorse and promote high-quality stan- dards in performing and reporting PSMA PET/CT scans. There was consensus for every statement, and this is undoubt- edly related to the fact that the panel mostly created the state- ments on the meeting day and discussed them in depth before voting. In-group bias is therefore a potential limitation, but the panel were sampled from the two most directly relevant pro- fessional societies (EANM and EAU) and these individuals represent expertise on this topic. Furthermore, voting was anonymous, so had any participant disagreed with the state- ments, there was a channel to express disagreement. Acknowledgments The authors acknowledge John William Bean PhD (Bean Medical Writing, Halle, Belgium, and funded by EAU Research Foundation) for providing medical writing services and Wim Witjes MD PhD (Scientific and Clinical Research Director EAU Research Foundation) and Emily Spieker (Management Assistant, European Association of Urology) for project management. Author contributions Stefano Fanti and Anders Bjartell had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Author contributions Stefano Fanti and Anders Bjartell had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The panelists refrained from evaluating response to some particular therapies due to lack of data. As previously men- tioned, the proposed criteria were mainly based on practical experience. Lutetium PSMA, due to its theranostic nature, is the only treatment where PSMA PET/CT has been used more frequently to evaluate response to therapy [27, 28]. For this indication, PSMA PET/CT is mandatory for establishing the suitability of treatment, and prognostic information can be derived from baseline PSMA PET/CT [29]. It is also possible, that PSMA PET/CT could be used to describe duration of response and decide the frequency of treatment. Conclusions PSMA PET/CT is currently the most rapidly growing imaging technique in PCa, but its adoption should be adequately sup- ported by indication for appropriate use and precise criteria for interpretation. PSMA PET/CT holds great potential in several clinical situations. Use of PSMA PET/CT should not be con- sidered, if no change of clinical management is expected. For evaluation of response to treatment, PSMA PET/CT can be used before and after any local and systemic treatment in patients with metastatic disease. However, it should not be 475 Eur J Nucl Med Mol Imaging (2021) 48:469–476 8. Live interactive audience participation. Poll Everywhere n.d. https://www.polleverywhere.com (accessed Mar 5, 2020). outside the submitted work; Tobias Maurer reports personal fees from Astellas, Bayer, BlueEarth Diagnostics, Janssen Cilag ROTOP, Phillips, and grants from Acelity, outside the submitted work; Steven MacLennan reports no conflicts of interest; Daniela Oprea-Lager has no personal fees to report, except for the unrestricted grant received from Janssen for the one-day EAU/EANM PSMA PET/CT Response Assessment Criteria meeting; Wim JG Oyen has no personal fees to report, except for the unrestricted grant received from Janssen for the one-day EAU/EANM PSMA PET/CT Response Assessment Criteria meeting; OR reports per- sonal fees from Takeda and an unrestricted grant from Janssen for the one-day EAU/EANM SMA PET/CT Response Assessment Criteria meeting; NM reports personal fees as advisor with Astellas, Jansen, BMS, Bayer, IPSEN, Ferring, Sanofi, and Steba; Anders Bjartell reports personal fees from Astellas, Bayer, Ferring, IPSEN, and Janssen, and institutional research grants from Astellas and Ferring, outside the sub- mitted work. 9. 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https://openalex.org/W4387147774	https://journals.asmarya.edu.ly/jau/index.php/jau/article/download/1192/786	Chinese	null	من معالم المنهج في تفسير "أحكام القرآن" للقاضي أبي بكر محمد بن العربي (ت543 هـ)	Mağallaẗ al-ğāmiʿaẗ al-Asmariyyaẗ	2,020	cc-by	19,235	ﺎﻣﻌﺔ اﻷﺳﻤﺮﯾﺔ: اﻟﻌﻠﻮم اﻟﺸﺮﻋﯿﺔ واﻹﻧﺴﺎﻧﯿﺔ 33 ( اﻟﻌدد اﻷول ) ﯾوﻧﯾو2020 ( ﻣ ﻣﻌﺎﻟ اﻟﻬﺞ ﻓﻲ ﺗﻔ "أﺣﺎم ا"ﻟﻘ آن ﻟﻠﻘﺎﺿﻲ أﺑﻲ ! ﷴ ﺑ اﻟﻌ ﻲ )ت543 (ﻫـ ﺣﺎزم ز. -ﺎ ﻣ,ﻲ اﻟ+ﯾ ﻗ ﻋﻠم اﻟﻘآن واﻟﯾ، ﻠﺔ اﻟﻌﺔ، ﺟﺎﻣﻌﺔ دﻣ.، دﻣ، ﺳرﺎ Email: hazem9k@yahoo.com ﺎﻣﻌﺔ اﻷﺳﻤﺮﯾﺔ: اﻟﻌﻠﻮم اﻟﺸﺮﻋﯿﺔ واﻹﻧﺴﺎﻧﯿﺔ 33 ( اﻟﻌدد اﻷول ) ﯾوﻧﯾو2020 ( ﻣ ﻣﻌﺎﻟ اﻟﻬﺞ ﻓﻲ ﺗﻔ "أﺣﺎم ا"ﻟﻘ آن ﻟﻠﻘﺎﺿﻲ أﺑﻲ ! ﷴ ﺑ اﻟﻌ ﻲ )ت543 (ﻫـ ﺣﺎزم ز. -ﺎ ﻣ,ﻲ اﻟ+ﯾ ﻗ ﻋﻠم اﻟﻘآن واﻟﯾ، ﻠﺔ اﻟﻌﺔ، ﺟﺎﻣﻌﺔ دﻣ.، دﻣ، ﺳرﺎ Email: hazem9k@yahoo.com ﻣﺠﻠﺔ اﻟﺠﺎﻣﻌﺔ اﻷﺳﻤﺮﯾﺔ: اﻟﻌﻠﻮم اﻟﺸﺮﻋﯿﺔ واﻹﻧﺴﺎﻧﯿﺔ اﻟﻣﺟﻠد ) 33 ( اﻟﻌدد اﻷول ) ﯾوﻧﯾو2020 ( ﻣ ﻣﻌﺎﻟ اﻟﻬﺞ ﻓﻲ ﺗﻔ "أﺣﺎم ا"ﻟﻘ آن ﻟﻠﻘﺎﺿﻲ أﺑﻲ ! ﷴ ﺑ اﻟﻌ ﻲ )ت543 (ﻫـ ﺣﺎزم ز. -ﺎ ﻣ,ﻲ اﻟ+ﯾ ﻗ ﻋﻠم اﻟﻘآن واﻟﯾ، ﻠﺔ اﻟﻌﺔ، ﺟﺎﻣﻌﺔ دﻣ.، دﻣ، ﺳرﺎ Email: hazem9k@yahoo.com ﻗ ﻋﻠم اﻟﻘآن واﻟﯾ، ﻠﺔ اﻟﻌﺔ، ﺟﺎﻣﻌﺔ دﻣ.، دﻣ، ﺳرﺎ 23 ISSN: 2706-9524 (Print) ISSN: 2706-9532 (Online) اﻟ01ﻠ ﺗ0ﺎول اﻟ7 3ﺎﻟﻌض واﻟ5ﻠﻞ 3ﻌـ2 ﻣﻌـﺎﻟ اﻟ1ـ0ﻬﺞ ﻓـﻲ ﺗﻔـ "أﺣ'ـﺎم اﻟﻘـآن" ﻟﻠﻘﺎﺿـﻲ اﺑـــ: اﻟﻌ9ـــﻲ اﻟ1ـــﺎﻟ@ﻲ اﻷﻧﻟـــﻲ )ت543ــــﻫ ،(ـ و َﻋـــ ض ﺣﺎ ﺗـــﻪ وﺗ@0ـــﻪ اﻟﻌﻠ1ـــﻲ وأﺣـــاث ﻋIـــﻩ اﻟ5ﺎرQـﺔ اﻟ5ـﻲ ﺗﻔﺎﻋـﻞ ﻣﻌﻬـﺎ 3Pﻘـﺔ ﻓـOّة ﻣـ: ﺧـﻼل ﺗﻔـﻩ ، 1ـﺎ ﺗ0ـﺎول اﻟ7ـ اﻟ1ﻌـﺎﻟ اﻟ10ﻬRــﺔ اﻟ5ﺎﻟﺔ: اﺳ5ﻔﺎدﺗﻪ اﻟ0ﻘWﺔ ﻣ: اﻟ5اث اﻟ5ﻔV ﻗUﻠﻪ، وﻣ0ﻬRـﻪ ﻓـﻲ اﻟ5ﻌﺎﻣـﻞ ﻣـﻊ اﻟﻠﻐـﺔ، و ﻣ0ﻬRـﻪ ﻓـﻲ اﻟ5ﻌﺎﻣﻞ ﻣـﻊ ﻣIـ ﺎدر اﻟ5ﻔـ* 3ﺎﻟ1ـﺄﺛر، واﻷﺣﺎدﯾـ اﻟ0Uـﺔ، واﻹﺳـاﺋﻠﺎت، وﻣـZ اﻋ51ـﺎدﻩ ﻋﻠـﻰ :اﻟ5ﻔــ 3ــﺎﻟأV، وﻗــﺎﻧن اﻟ5ــﺟﺢ ﻓــﻲ اﻟ5ﻔــ، وﻣ0ﻬRــﻪ ﻓــﻲ اﻟ5ﻌﺎﻣــﻞ ﻣــﻊ ﻣQﺎﻟ_ــﻪ، وﻣﻗﻔــﻪ ﻣــ أﺣـاث ﻋIــﻩ ّ. وﻗـ ﺗــ .ﺗfـe اﻟ1ــ0ﻬﺞ اﻟﺻـﻔﻲ ﻟﻠcــﺎم 3ـﺈﺟاءات اﻟ7ــ وﺗﻘـ أﻫاﻓــﻪ وﻗــ وﺻﻞ اﻟ7 إﻟﻰ ﻧ5ﺎﺋﺞ :أﺑزﻫﺎ15اﻋ ﺎ ،د اﺑ: اﻟﻌ9ﻲ ﻋﻠﻰ اﻟ10ﻬﺞ اﻟ5ﻠﻠﻲ ﻓﻲ ﺗﻔـﻩ واﺳـ5ﻌﺎ3ﻪ ﺗاث اﻟ1ﻔ: ﻣ: ﻗUﻠﻪ، وﺗﻌﺎﻣﻠ ﻪ ﻣﻊ ﻫOا اﻟ5اث 3Pﻘﺔ ﺗ0ﱡ ﻋـ: اﺣ5ـام ﻋ1ـ ﻟـﻪ ﻣـﻊ اﺣ5ﻔﺎfـﻪ ِّﻟ0ﻔــﻪ ﻓــﻲ اﻟﻗــl ذاﺗــﻪ 3ــ اﻟ10ﺎﻗــﺔ واﻟ0ﻘــ واﻟــد ، 1ــﺎ اﻋ51ــ اﺑــ: اﻟﻌ9ــﻲ ﻋﻠــﻰ ﻣ0ﻬRــﻲ اﻷﺛــ واﻟأV ﻓﻲ ﺗﻔ ﻩ، ﻣﻊ ﻣﻼﺣpﺔ أﻧﻪ ﺎن أﻛn ﺗﺳﻌﺎً ودﻗﺔً وﻧﻘاً ﻓﻲ ﻣان اﻷﺛ واﻷﺧ7ـﺎر، ﻫـOا 3ﺎﻹﺿﺎﻓﺔ إﻟﻰ أﻧﻪ ﻗ اﺗQO ﻣﻗﻔﺎً ﻧﻘWﺎً إزاء اﻷﺣﺎدﯾ اﻟrqﻔﺔ واﻟ1ﺿﻋﺔ واﻹﺳـاﺋﻠﺎت :؛ وﻣـ ﻧ5ـﺎﺋﺞ اﻟ7ـ أWrـﺎً أن اﺑــ: اﻟﻌ9ـﻲ ـﺎن ﯾ05Iــ ﻟ1Ot7ـﻪ ﻋﻠـﻰ اﻟ1ــ5Z اﻟﻔﻘﻬـﻲ واﻟﻌﻘـV، وــﺎن ّ5W 23ﻌ اﻟﻲء ﻓﻲ اﻟدّ ﻋﻠﻰ ﻣQﺎﻟ_ـﻪ ، 1ـﺎ ،اﻣ5ـﺎز اﺑـ: اﻟﻌ9ـﻲ ﺑـ9u ﺗﻔـﻩ ﻟﻠﻘـآن اﻟ@ـ 23 ISSN: 2706-9532 (Online) ISSN: 2706-9524 (Print) اﻗـــﻊ ﻣـــﻠ1ﻲ ﻋIـــﻩ وﻣـــﺎﻛﻠﻬ، وﻫـــOﻩ اﻟ1ـــvة زادت ﻣـــ: ﻗـــرة ﻫـــOا اﻟ5ﻔـــ* ﻋﻠـــﻰ اﻟ5ﻔﺎﻋـــﻞ ﻣـــﻊ Iﻩ واﻟ5ﺄﺛ* ﻓﻲ أﺣاﺛﻪ. اﻟ ﻘ+ﻣﺔ اﻟ1 x وﺳﻼم ﻋﻠﻰ w7ﺎدﻩ اﻟOﯾ: اﺻPﻔﻰ ... ﯾﺟــ ﻓــﻲ ﺗــﺎرﺦ ــﻞِّ ﻋﻠــ اﺗRﺎﻫــﺎتٌ وﻣــارسُ أﺳﺎﺳــﺔ W1nِّﻠُﻬــﺎ أﻋــﻼم 3ــﺎرزون Wﻘﻣـــــن 3ﺎﺳ5rـــــﺎر ﺗـــــﺎرﺦ ﻫـــــOا اﻟﻌﻠـــــ واﺳـــــ5Qﻼص ﻋIـــــﺎرة ﺧUﺗـــــﻪ اﻟ10ﻬRـــــﺔ واﻟ1ﻌﺔ وﻓ رؤﺔ ﻣﺿwﺔ ﺗ5 3ﺎﻟ1اﺟﻌﺔ واﻟ0ﻘـ واﻹﺿـﺎﻓﺔ؛ وﻓـﻲ اﻟﻌـﺎدة ﻓـﺈن ﻫﻻء اﻷﻋﻼم ﯾدﻋن ﻧ5ﺎﺋﺞ ﻋ1ﻠﻬ ﻫOا ﻓﻲ 5ﺎبٍ ﻟﻬ .W1nِّﻞ ذروة ﻧ5ﺎﺟﻬ اﻟﻌﻠ1ﻲ ﻫOا اﻷﻣ ﯾ0PUـ 3ـ'ﻞ Uـ ﻋﻠـﻰ اﻟﻘﺎﺿـﻲ اﺑـ: اﻟﻌ9ـﻲ اﻟ1ـﺎﻟ@ﻲ )ت543 ه ،( وﺗﻔـــﻩ اﻟﻔـــOّ )أﺣ'ـــﺎم اﻟﻘـــآن(، اﻟـــOV ﺗـــﻌﻰ ﻫـــOا اﻟراﺳـــﺔ إﻟـــﻰ ﺑـــﺎن وﺗﻠـــﻞ أﻫـــ ﺧIﺎﺋIـــﻪ وﻣﻌﺎﻟ1ـــﻪ اﻟ10ﻬRـــﺔ اﻟ5ـــﻲ أﻛـــU5ﻪ اﻟ1'ﺎﻧـــﺔ اﻟﻌﻠ1ـــﺔ اﻟ1ﻣﻗـــﺔ ﻓـــﻲ ﺗـــﺎرﺦ اﻟ5ﻔ 3ﻌﺎﻣﺔ، وﺗﺎر .ﺦ اﻟ5ﻔ* اﻟﻔﻘﻬﻲ 3Qﺎﺻﺔ ﺗ 50ــﺎول ﻫــOﻩ اﻟراﺳــﺔ 3ــﻲء ﻣــ: اﻟ5ﻔIــﻞ واﻟ5ﻠــﻞ واﻟ5ﻘــ 3ﻌــ2 ﻣﻌــﺎﻟ اﻟ1ــ0ﻬﺞ "اﻟ5ﻔV اﻟ5ﻲ اﻟ5ـvم ﺑﻬـﺎ اﻟﻘﺎﺿـﻲ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﺗﻔـﻩ "أﺣ'ـﺎم اﻟﻘـآن ، وﻣ5ﺎ3ﻌـﺔ أﺛﻫﺎ اﻟ5PUﻘﻲ ﻪ. أﻫ+اف اﻟ+راﺳﺔ ﺗـــﻌﻰ ﻫـــOﻩ اﻟراﺳـــﺔ إﻟـــﻰ ﺗﯾـــ أﻫـــ اﻟﻌ0ﺎﺻـــ اﻟ10ﻬRـــﺔ ﻟ1ﻔِّ ـــ Uـــ ﻣـــ: أﺋ1ـــﺔ اﻟ5ﻔـــــ* اﻟﻔﻘﻬـــــﻲ، وزـــــﺎدة ﺣrـــــرﻫﺎ ﻓـــــﻲ وﻋـــــﻲ اﻟارﺳـــــ: ﻟﻌﻠـــــ اﻟ5ﻔـــــ ﻋ1ﻣـــــﺎ :وﻟﻠ15ﻐﻠ :nواﻟ7ﺎﺣ .ًﻓﻲ اﻟ5ﻔ* اﻟﻔﻘﻬﻲ ﺧIﺻﺎ ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن أﻫ3ﺔ اﻟ+راﺳﺔ ِّ'ﺗ ًﻞ ﻣﻌﺎﻟ اﻟ10ﻬﺞ اﻟ1روﺳﺔ ﻫ0ﺎ ﻧ1ذﺟﺎ W1': أن WﺄﺧO 3ﻪ اﻟارﺳن واﻟ1ﻔون اﻟ1ﻌﺎﺻــون اﻟ1ﻬ51ــن 3ﺎﻟ5ﻔــ* اﻟﻔﻘﻬــﻲ، و ــﺎﻫ ﻓــﻲ :اﻟــِّ ﻣــ 23ﻌــ اﻟﻘIــر اﻟﻌﻠ1ـــﻲ واﻟ10ﻬRـــﻲ اﻟـــOV ﻧـــﻬﻩ اﻟـــم ﻓـــﻲ اﻟﻌ1ـــﻞ اﻟ5ﻔـــV اﻟ1ﻌﺎﺻـــ ﻓـــﻲ ﻣRـــﺎل .اﻟ5ﻔ* اﻟﻔﻘﻬﻲ ﻣ:اﻟ+راﺳﺎت اﻟﺎ!ﻘﺔ ذات اﻟ;ﻠﺔ ﻣ:اﻟ+راﺳﺎت اﻟﺎ!ﻘﺔ ذات اﻟ;ﻠﺔ 2ﺗﺟـــ 3ﻌـــ اﻟ@5ـــ اﻷﻛﺎدW1ـــﺔ lاﻟ5ـــﻲ ﺗ0ﺎوﻟـــ اﺑـــ: اﻟﻌ9ـــﻲ و ﺗﻔـــﻩO، أﻛ5ﻔـــﻲ ﺑـــ اﻟ@5ﺎﺑ: اﻟ5ﺎﻟ*: ﻣ0ﻬﺎ: 2ﺗﺟـــ 3ﻌـــ اﻟ@5ـــ اﻷﻛﺎدW1ـــﺔ lاﻟ5ـــﻲ ﺗ0ﺎوﻟـــ اﺑـــ: اﻟﻌ9ـــﻲ و ﺗﻔـــﻩO، أﻛ5ﻔـــﻲ ﺑـــ اﻟ@5ﺎﺑ: اﻟ5ﺎﻟ: ﻣ0ﻬﺎ:  اﺑــ اﻟﻌ ــﻲ اﻟــﺎﻟ5ﻲ اﻹﺷــ? ﻠﻲ وﺗﻔــ ﻩ أﺣــﺎم اﻟﻘــ آن. tﻣIــPﻔﻰ إﺑــا اﻟ10ﻲ، 1، دار اﻟRﻞ، دار ﻋ1ﺎر، ﺑوت، ،ﻋ1ﺎن1991 .م  ﻣ;ـــﺎدر اﻟ4ﻔـــ اﻟﻔﻘﻬـــﻲ ﻋـــ+ اﺑـــ اﻟﻌ ـــﻲ اﻟـــﺎﻟ5ﻲ ﻓـــﻲ .4ﺎ!ـــﻪ "أﺣـــﺎم "اﻟﻘــــ آن. اﻟ9ــــﻊ ﷴ ﻣ0Iــــﻒ اﻟﻘ1ــــﺎﻲ، 1 ،، دار اﻟﻔﻔــــﺎء، ــــاﺑﻠ ،ﻟUـﺎ2009 .م وﻫـOا اﻟ@5ــﺎب ﻓـﻲ اﻷﺻــﻞ رﺳـﺎﻟﺔ ﻣﺎﺟـ5، ﻗُــ ّ ﻣl ﻓـﻲ ﻗــ اﻟراﺳﺎت اﻹﺳﻼﻣﺔ ﻓﻲ ،ﻠﺔ اﻵداب، ﺟﺎﻣﻌﺔ اﻟﻔﺎﺗﺢ2008 / 2009م. :وﻗ اﺳ5ﻔت ﻣ:ﻫOﯾ: اﻟ@5ﺎﺑ .7ﻓﻲ ﻫOا اﻟ ﺗــّ اﺳــ5Qام اﻟ1ــ0ﻬﺞ اﻟﺻــﻔﻲ اﻟــOV Wﻌ50ــﻲ 3ــﺎﻟ557ﻊ اﻟ1ــﻲ ﻟﻠ1ــﺎدة اﻟﻌﻠ1ــﺔ ﻟ5ﻔــ ."أﺣ'ﺎم اﻟﻘآن" 3Pﻘﺔ ﻋﻠ1ﺔ ﺗﻘم ﻋﻠﻰ اﻟ5I0e واﻟ5ﻠﻞ واﻻﺳ505ﺎج واﻟ5ﻘ    اﻟ 4ﻬ+ أﻧـــvل ﷲ :اﻟﻘـــآن اﻟ@ـــ ﻟ5ﻘـــ ﻣﻘﺎﺻـــ nـــة، W1'ـــ: ﺟ1ﻌﻬـــﺎ ﻓـــﻲ ﻣﻘIـــﯾ :ﻋp1: ﻫ1ﺎ ﺗــّ اﺳــ5Qام اﻟ1ــ0ﻬﺞ اﻟﺻــﻔﻲ اﻟــOV Wﻌ50ــﻲ 3ــﺎﻟ557ﻊ اﻟ1ــﻲ ﻟﻠ1ــﺎدة اﻟﻌﻠ1ــﺔ ﻟ5ﻔــ ."أﺣ'ﺎم اﻟﻘآن" 3Pﻘﺔ ﻋﻠ1ﺔ ﺗﻘم ﻋﻠﻰ اﻟ5I0e واﻟ5ﻠﻞ واﻻﺳ505ﺎج واﻟ5ﻘ    اﻟ 4ﻬ+ أﻧـــvل ﷲ :اﻟﻘـــآن اﻟ@ـــ ﻟ5ﻘـــ ﻣﻘﺎﺻـــ nـــة، W1'ـــ: ﺟ1ﻌﻬـــﺎ ﻓـــﻲ ﻣﻘIـــﯾ :ﻋp1: ﻫ1ﺎ ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ :أوﻟﻬــﺎ ﺗIــﺢ اﻟ5Iــر اﻟ7ــV ﺣــل ﻗrــﺔ وﺟــد ﷲ ،، وﻗrــﺎWﺎ اﻹﻧــﺎن ﻣ: ﺣ* اﻟ1Uأ واﻟ1I، وﺻﺎﻏﺔ رؤﺔ ﺟﯾة ﻟﻠ@ـن واﻟﻌـﺎﻟ، و9ـﺎن اﻟ1ـدات واﻟﻌ0ﺎﺻـــ اﻟ10ﻬRـــﺔ اﻟ@UـــZ ﻟﻠ5ﻌﺎﻣـــﻞ ﻣﻌﻬ1ــــﺎ وﺗـــQﻫ1ﺎ ﻓـــﻲ إـــﺎر ﻋUدWــــﺔ ﷲ -ﺳـــ7ﺎﻧﻪ- وﻣ0ﻬRـــﻪ، وﻣ0ﻔﻌـــﺔ ﺧﻠﻘـــﻪ أﺟ1ﻌـــ:. ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 6 أﻫ3ﺔ اﻟ+راﺳﺔ وﻗـــ ﺗ@ﻔﱠﻠـــl آWـــﺎت اﻟﻌﻘـــة واﻟ@ـــن واﻟ5ــﺎرﺦ واﻟﻘIــ اﻟ1ــOرة ﻓــﻲ اﻟﻘــآن اﻟ@ــ ﺑUــﺎن ــﻞ ﻣــﺎ ﯾ5ﻌﻠــ ﺑﻬــOا اﻟ1ﻘIــ .اﻷول :ﺛﺎﻧ ﻬــﺎ ،ًﺗﻘــ وﺻــﻼح ﺳــﻠك اﻹﻧــﺎن ﻓــﻲ ﻫــOﻩ اﻷرض ﻓــداً ــﺎن أم ﺟ1ﺎﻋــﺔ وﻣR51ﻌـﺎً ـﺎن أم أﻣـﺔً، وﻫاﯾ5ـﻪ إﻟـﻰ ﻣــﺎ Wـﺎﻋﻩ ﻋﻠـﻰ ﺗﻐ*ـ ﺣﺎﺗـﻪ وﺗﺟﻬﻬـﺎ ﻧــ .ُاﻷﻓrﻞ ﻓﻲ ﻞ اﻷﻣر، وﻋﻠﻰ ﺎﻓﺔ اﻟIُﻌ وﻗـــ ﺗ@ﻔﱠﻠـــl ﺑﻬـــOا اﻟ1ﻘIـــ آWـــﺎت اﻟ5ـــﻊ واﻷﺧـــﻼق واﻟ59ـــﺔ واﻟـــﻠك اﻟ5ـــﻲ ﻛﺎﻧـــl ﻣﺿـــﻊ اﻫ51ـــﺎم اﻟ1ﻔـــ: ﻣ0ـــO ﻧـــﺄة ﻋﻠـــ اﻟ5ﻔـــ* ﻧpـــاً ﻟﺎﺟـــﺔ اﻟ1R51ـــﻊ اﻹﺳــﻼﻣﻲ اﻟ1ﻠــﺔ ﻣ0ــO ﻧــﺄﺗﻪ ﻟ1ﻌﻓــﺔ ﺷــع ﷲ واﻻﻟ5ــvام 3ــ 5ﻪ ﻋﻠــﻰ ﻣــ Z اﻟq7ــﺎدات .واﻟﻌﻘد واﻟ1ﻌﺎﻣﻼت، وﻞ وﺟﻩ اﻟﻔﻌﻞ اﻹﻧﺎﻧﻲ ﻓﻲ ﻞ ﻣ0ﺎﺣﻲ اﻟﺎة وﻓــﻲ اﻟcﻘــﺔ ﻓﺈﻧــﻪ 3ــU f0ــﺔ دﻻﻟــﺔ nــ ﻣــ: آWــﺎت اﻷﺣ'ــﺎم ﻋﻠــﻰ أﺣ'ﺎﻣﻬــﺎ ﻓﻘــ lاﺧ5ﻠﻔl اﺳ507ﺎﺎت اﻟﻌﻠ1ﺎء ﻣ0ﻬﺎ، اﺑ5اءً ﻣ: ﻋIـ اﻟIـﺎ3ﺔ واﻟ5ـﺎ3ﻌ:، واﺧ5ﻠﻔـ 3PUﻌــــﺔ اﻟــــﺎل اﻻﺟ5ﻬــــﺎدات اﻟﻔ ﻘﻬــــﺔ اﻟ1ــــ50ة إﻟــــﻰ ﻫــــOﻩ اﻵWــــﺎت ﺣــــ ﻣ0ــــﺎﻫﺞ . ِّاﻻﺟ5ﻬﺎد وأﺻﻟﻪ اﻟ5ﻲ Wﻌ51ﻫﺎ و* ﻋﻠﻬﺎ ﻞ ﻓcﻪ وﻣﻔ Vوﻗ ﺳﺎر اﻷﻣ ﻋﻠﻰ ﻫOا اﻟ10ـال ﻓـﻲ ﻋIـ اﻻﺟ5ﻬـﺎد اﻟﻔﻘﻬـﻲ وﺗـو: اﻟﻌﻠـم، أ ﻓﻲ اﻟﻘون اﻟﻬRﺔ اﻟnﻼﺛﺔ اﻷوﻟﻰ، أﻣﺎ 3ﻌ ذﻟ، أV ﻓﻲ ﻋI اﺳـ5ﻘار اﻟ1ـOاﻫ اﻟﻔﻘﻬــﺔ، واﻛ51ـــﺎ ل أﺻــﻟﻬﺎ اﻻﺟ5ﻬﺎدWـــﺔ، و9ﻌــ fﻬـــر اﻟ1ﻟﻔــﺎت اﻟﻔﻘﻬـــﺔ واﻟ5ﻔـــﺔ ،اﻟ@UــــZ ﻓﻘــــ fﻬــــت ﻣR1ﻋــــﺔ ﻣــــ: اﻟ5ﻔﺎﺳــــ* اﺧ5Iــــl ﺑ5ﻔــــ* آWــــﺎت اﻟ5ــــﻊ واﺳــ507ﺎ اﻷﺣ'ــﺎم ﻣ0ﻬــﺎ، وﻓــ أﺻــل وﻣ0pــر اﻟ1ــOﻫ اﻟﻔﻘﻬــﻲ اﻟــOV ﯾ51ــOﻫ 3ــﻪ اﻟ1ﻔِّ ــ اﻟﻔcــﻪ ﻣــﻊ اﻧﻔ5ــﺎح وﺣــار وﻧﻘــ ﻟ7cــﺔ اﻟ1ــOاﻫ ﺑِ0َ ــ ﻣQ5ﻠﻔــﺔ ﺣ ــ ــﻞ .ﻣﻔِّ ، واﺳ51 ﻫOا اﻷﻣ ﺣ5ﻰ وﺻﻠ0ﺎ إﻟﻰ اﻟﻌI اﻟﯾ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 6 26 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن وﻫ'ــOا ﻓﺈﻧ0ــﺎ ﻧRــ ﺑــ: أﯾــﯾ0ﺎ اﻟــم ﻋــداً ﻣــ: 5ــ اﻟ5ﻔــ* اﻟﻔﻘﻬــﻲ1 ، اﻟ5ــﻲ ﺗــﻬ 31ــZ ﻋ0ﺎWــﺔ اﻷﻣــﺔ 3ــﺎﻟﻘآن اﻟ@ــ، وﻣــZ ﻋ0ﺎﯾ5ﻬــﺎ وﺣﺻــﻬﺎ ﻋﻠــﻰ ﺿــ7u وﺗ0pــ ﻛــﻞ ﺗﻔﺎﺻــﻞ ﺣــﺎة أﻓادﻫــﺎ وﻣR51ﻌﺎﺗﻬــﺎ وﻓــ ﻣ0pﻣــﺔ اﻷﺣ'ــﺎم وا ﻟcــ اﻟﻘآﻧــﺔ اﻟ5ــﻲ أﻧvﻟﻬﺎ ﷲ ﺗﻌﺎﻟﻰ ﻟ5@ن رﺣ1ﺔ وﺷﻔﺎء ﻟﻠ1ﻣ0: وﻟ5Qج اﻟ0ـﺎس ًﺟ1ﻌـﺎ ﻣـ: اﻟpﻠ1ـﺎت 0إﻟﻰ اﻟ:ر، Wﻘل ﷲ ﺗﻌﺎﻟﻰ  ِاﻟَﺮ ﻛِﺘَﺎبٌ أَﻧﺰَﻟْﻨَﺎهُ إِﻟَﻴْﻚَ ﻟِﺘُ%ْ ِجَ اﻟﻨ#ـﺎسَ ﻣِـﻦَ اﻟﻈﻠُﻤَـﺎتِ إَِـ اﻟﻨـﻮرِ ﺑِـِذْن ِرَﺑ43ِﻢْ إَِ 2ِ َاطِ اﻟْﻌَﺰِﻳﺰ ِاﻟْﺤَﻤِﻴﺪ 2. وﻗ ﺷﻬت ﺣﺔ اﻟ5ﻔ* اﻟﻔﻘﻬﻲ اﻟPﻠﺔ ﻣﻘﺎر9ﺎت ﻣ0ﻬRﺔ ﻣ50ﻋﺔ وﻣ5ﻌدة ﻣــ: ﺣــ اﻟ1ــZ واﻟ1Iــﺎدر وﻘــﺔ اﻟﻌــض واﻟ1ــ0ﻬﺞ 3ــU ﻋاﻣــﻞ ﻋﯾــة ﻣ0ﻬــﺎ ،اﻟQﻠ_ﺔ اﻟ1OﻫUﺔ اﻟ@ﻼﻣﺔ واﻟﻔﻘﻬﺔ ﻟﻠ1ﻔِّ ، واﺧـ5ﻼف ﻗIـ ـﻞ ﻣﻔِّ ـ ﻣـ: ﻋ1ﻠـﻪ 9ﻓrﻼً ﻋ: اﺧ5ﻼف ﻋIر اﻟ1ﻔ: و ،ﺎﺗﻬ اﻟﻌﻠ1ﺔ، وﺗ@0ﻬ اﻟﻌﻠ1ﻲ اﻟQﺎص واﻟﻌﻠـــ اﻟـــOV ﻏﻠـــ ﻋﻠـــﻰ ـــﻞ واﺣـــ ﻣـــ0ﻬ، وﻣـــZ ﺗـــﺄﺛﻩ واﻫ51ﺎﻣـــﻪ 3ﻌIـــﻩ وأﺣاﻟـــﻪ اﻟ5ﺎرQﺔ3. أﻫ3ﺔ اﻟ+راﺳﺔ .اﻟ1ﻌﺎﻓV، ﯾ05ﻬﻲ ﻧ7ﻪ إﻟﻰ ﻗPﺎن ) وُﻟـ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﻋـﺎم468 ـﻫــ ،( ﻓـﻲ ﻣﯾ0ـﺔ إﺷـUﻠﺔ، ﻓـﻲ 3ـﻼد اﻷﻧــﻟ ﻓﻲ ﺑl ﻋﻠٍ وﺳﺎﺳﺔ، ﻓاﻟﻩ أﺑ ﷴ ﻋU ﷲ ﺑ: اﻟﻌ9ﻲ، ﺎن ﻣ: وﺟﻬـﺎء وأwـﺎن وﻋﻠ1ــﺎء إﺷــUﻠﺔ اﻟ7ــﺎرز:، ﺣــ ــﺎن وزــاً ﻟﻠ1ﻌ51ــ ﺑــ: w7ﱠــﺎد آﺧِــ ﻣﻠــك إﺷــUﻠﺔ ﻗUـﻞ ﺳـﻘﻬﺎ ﻋﻠـﻰ ﯾـ اﻟ1ـا3P:، وـﺎن ﻣـ: أﻫـﻞ اﻵداب اﻟاﺳـﻌﺔ واﻟ5ﻔـ0: واﻟUاﻋـﺔ ًZﻓـﻲ ﻋﻠــم ﺷـ5ﻰ، وــﺎن ﻣــ: 7ـﺎر أﺻــﺎب اﺑـ: ﺣــvم اﻟpــﺎﻫV، وـﺎن ﺑ5ــﻪ ﻣ05ــ ﻟﻠﻌﻠ1ﺎء واﻷد3ﺎء، وﻟﻪ ﯾﺟﻊ اﻟﻔrﻞ 1ﺎ ﺣIﱠﻠﻪ اﺑ0ﻪ أﺑ ،3' ﻣ: ﻋﻠم وﻣﻌﺎرف ) وﻣﺎ ﺗﻬﺄ ﻟﻪ ﻣ: ﻟﻘﺎء أﺋ1ﺔ اﻟﻌﻠ واﻟﯾ: ﻓﻲ ﻋIﻩ، ﺗﻓﻲ ﻓﻲ ﻋﺎم493 ﻫــ.( ،ﺑــأ اﺑــ: اﻟﻌ9ــﻲ ﺗIــﻠﻪ اﻟﻌﻠ1ــﻲ ﻋﻠــﻰ ﯾــ واﻟــﻩ، ﻓﻌ0ــﻪ أﺧــO ﺗﻌﻠ1ــﻪ اﻷوﱠﻟــﻲ وﻟ@ــ: ﻟ@nــة أﺷــﻐﺎل واﻟــﻩ وارﺗ7ﺎــﻪ 31ﻬــﺎم اﻟوﻟــﺔ 3ﺎﻋ57ــﺎرﻩ وزــاً ﻷﻣــ إﺷــUﻠﺔ ﻓــﻲ lذﻟــ اﻟﻗــ- اﻟ1ﻌ51ــ ﺑــ: w7ــﺎد- u7ﻓﻘــ ﻋﻬــ 3ــﻪ إﻟــﻰ ﺛﻼﺛــﺔ ﻣﻌﻠ1ــ:، أﺣــﻫ ﻟrــ ،اﻟﻘــآن اﻟ@ــ 3ﺄﺣﻓــﻪ اﻟــ7ﻌﺔ، واﻟnــﺎﻧﻲ ﻟ5ﻌﻠ1ــﻪ اﻟﻌ9ــﺔ، واﻟnﺎﻟــ ﻟ17ــﺎد¤ اﻟــﺎب :ﻓOق اﻟﻘآن، وﻫ اﺑ: ﺗﻊ ﺳ0:، وﻟ ﯾUﻠـﻎ اﻟﺎدﺳـﺔ ﻋـة ﻣـ: ﻋ1ـﻩ ﺣ5ـﻰ أﺗﻘـ اﻟﻘاءات اﻟﻌ، وﺟ1ﻊ ﻓ0ﻧﺎً ﻣ: اﻟﻌ9ﺔ، وﻣَﻬَ ﻓﻲ اﻟﻠﻐﺔ واﻷدب واﻟﻌ1. :اﻟWﻠV اﻟZﺎﻧﻲ رﺣﻠ4ﻪ اﻟﻌﻠ3ﺔ إﻟﻰ اﻟ9 ق ﻣﺎ إن ﺑﻠﻎ اﺑ: اﻟﻌ9ﻲ اﻟﺎ3ﻌﺔ ﻋة ﻣ: ﻋ1ﻩ، ﺣ5ﻰ ﺷع 3Iـ7ﺔ واﻟـﻩ ﻓـﻲ رﺣﻠــﺔ ﻋﻠ1ــﺔ إﻟــﻰ ﺣاﺿــ اﻟﻐــب اﻹﺳــﻼﻣﻲ، وﻣاﻛــv اﻟﻌﻠــ ﻓــﻲ اﻟــق اﻹﺳــﻼﻣﻲ ) ﻟ1ـــة زادت ﻋﻠـــﻰ ﻋـــ ﺳـــ0ات485 - 495 Vه(. أﻫ3ﺔ اﻟ+راﺳﺔ  1 ( ﻧـــO ﻣـــ: ﺑـــ: أﺑـــز ﻫـــOﻩ اﻟ5ﻔﺎﺳـــ ﻋ0ـــ اﻟﻌﻠ1ـــﺎء اﻟ15ﻘـــﻣ:: "أﺣ'ـــﺎم اﻟﻘـــآن" ﻹﺳـــ1ﺎﻋﻞ ﺑـــ: إﺳـــ اﻟRﻬrـــ1ﻲ اﻟ1ــﺎﻟ@ﻲ )ت282 ،(ه "أﺣ'ــﺎم اﻟﻘــآن" ﻟﻠRIــﺎص اﻟ0ﻔــﻲ )ت371 ه(، "أﺣ'ــﺎم اﻟﻘــآن" ﻟﻠ@ــﺎ اﻟﻬاﺳــﻲ اﻟــﺎﻓﻌﻲ )ت405 ه(، "أﺣ'ــــﺎم اﻟﻘــــآن" ﻻﺑــــ: اﻟﻌ9ــــﻲ اﻟ1ــــﺎﻟ@ﻲ )ت543 ه(، "اﻟRــــﺎﻣﻊ ﻷﺣ'ــــﺎم اﻟﻘــــآن" ﻟﻠﻘUــــﻲ اﻟ1ــــﺎﻟ@ﻲ )ت671 ه(، "ﻛ0ـــv اﻟﻌﻓـــﺎن ﻓـــﻲ ﻓﻘـــﻪ اﻟﻘـــآن" ﻟ1ﻘـــاد اﻟـــرV اﻹﻣـــﺎﻣﻲ )ت826 ه(، "اﻟn1ـــات اﻟﺎﻧ ﻌـــﺔ واﻷﺣ'ـــﺎم اﻟاﺿﺔ اﻟﻘﺎﻌﺔ" ﻟﺳﻒ اﻟnﻼﺛﻲ اﻟvـV )ت832 :ه(. وﻧـO ﻣـ: ﺑـ: أﺑـز ﻫـOﻩ اﻟ5ﻔﺎﺳـ ﻓـﻲ اﻟﻌIـ اﻟـﯾ ،:"ﻧــﻞ اﻟ1ــام ﻓــﻲ ﺗﻔــ آWــﺎت اﻷﺣ'ــﺎم" ﻟ11ــ ﺻــﯾ ﺧــﺎن، "ﺗﻔــ* آWــﺎت اﻷﺣ'ــﺎم" ﻟ11ــ ﻋﻠــﻲ اﻟــﺎW وآﺧــ "" ،"ﺗﻔ* آWﺎت اﻷﺣ'ﺎم" ﻟ10ﺎع اﻟﻘPﺎن +آWـــﺎت اﻷﺣ'ـــﺎم،: ﺗﻔـــ* واﺳـــ507ﺎ" ﻟ0ـــر اﻟـــﯾ: ﻋ5ـــ. اﻧpـــ، ﷴ ﺣـــ: اﻟـــOﻫUﻲ ،اﻟ4ﻔـــ واﻟﻔـــ ون 6 ، ،)ﺑوت، ﻣ'57ﺔ وt7ﺔ1995 ،(م2 / 467 - 473 ؛+اﻟ4ﻔ واﻟﻔ ون ﻓﻲ ﺛJﻪ اﻟI+ﯾ ،، ﻋUـ اﻟﻐﻔـر ﺟﻌﻔـ 1 ،)اﻟﻘﺎﻫة، دار اﻟﻼم2007 ،(م525 - 532 ،؛ ﻋU رب اﻟ0Uﻲ ﻋﺎﻟ اﻟ4ﻔـ اﻟﻔﻘﻬـﻲ اﻟ,ـ+ﯾO: ﻗMـﺎLﺎ أﺻـJﻟ3ﺔ وﻓﻘﻬ3ﺔ ،، )ﻣ0رات ﺟﺎﻣﻌﺔ ﻋU اﻟ1ﺎﻟ اﻟﻌV، ﺗPان2005 ،( 1 / 12 - 37 . 2 ( ،tﺳرة إﺑا :اﻵWﺔ 1 . 3 ( ،ﻗــﺎرن ﺑــــــ: ﻓﻬــ اﻟوﻣــﻲ أﺻــJل اﻟ4ﻔــ وﻣﺎﻫIــﻪ ، 1 ،، ) اﻟــﺎض: ﻣ'57ــﺔ اﻟ59ــﺔ 1413 ( 91 - 95 ؛ ﻧﺎﺋﻠــﺔ ،Vاﻟـــــﻠ0ﻲ اﻟاﺿــــــ ﻗMـــــﺎLﺎ اﻷﺳــــــ ة واﺧــــــ4ﻼف اﻟ4ﻔﺎﺳــــــ ، 1) اﻟـــــار اﻟUrــــــﺎ ،ء ﺑـــــوت: اﻟ1ــــــv اﻟnﻘــــــﺎﻓﻲ ،اﻟﻌ9ﻲ2002 ،( 24 - 27 . 3 ( ،ﻗــﺎرن ﺑــــــ: ﻓﻬــ اﻟوﻣــﻲ أﺻــJل اﻟ4ﻔــ وﻣﺎﻫIــﻪ ، 1 ،، ) اﻟــﺎض: ﻣ'57ــﺔ اﻟ59ــﺔ 1413 ( 91 - 95 ؛ ﻧﺎﺋﻠــﺔ ،Vاﻟـــــﻠ0ﻲ اﻟاﺿــــــ ﻗMـــــﺎLﺎ اﻷﺳــــــ ة واﺧــــــ4ﻼف اﻟ4ﻔﺎﺳــــــ ، 1) اﻟـــــار اﻟUrــــــﺎ ،ء ﺑـــــوت: اﻟ1ــــــv اﻟnﻘــــــﺎﻓﻲ ،اﻟﻌ9ﻲ2002 ،( 24 - 27 . أﻫ3ﺔ اﻟ+راﺳﺔ ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ :اﻟU,O اﻷول اﻟﻘﺎﺿﻲ اﺑ اﻟﻌ ﻲ: ﺣ3ﺎﺗﻪ وﺗ5J-ﻪ اﻟﻌﻠﻲ وﻋ; ﻩ :اﻟWﻠV اﻷول وﻻدﺗﻪ وﻧ9ﺄﺗﻪ ﻫـــ أﺑـــ 3'ـــ ﷴ ﺑـــ: ﻋUـــ ﷲ ﺑـــ: ﷴ ﺑـــ: ﻋUـــ ﷲ ﺑـــ: أﺣ1ـــ ﺑـــ: اﻟﻌ9ـــﻲ .اﻟ1ﻌﺎﻓV، ﯾ05ﻬﻲ ﻧ7ﻪ إﻟﻰ ﻗPﺎن ) وُﻟـ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﻋـﺎم468 ـﻫــ ،( ﻓـﻲ ﻣﯾ0ـﺔ إﺷـUﻠﺔ، ﻓـﻲ 3ـﻼد اﻷﻧــﻟ ﻓﻲ ﺑl ﻋﻠٍ وﺳﺎﺳﺔ، ﻓاﻟﻩ أﺑ ﷴ ﻋU ﷲ ﺑ: اﻟﻌ9ﻲ، ﺎن ﻣ: وﺟﻬـﺎء وأwـﺎن وﻋﻠ1ــﺎء إﺷــUﻠﺔ اﻟ7ــﺎرز:، ﺣــ ــﺎن وزــاً ﻟﻠ1ﻌ51ــ ﺑــ: w7ﱠــﺎد آﺧِــ ﻣﻠــك إﺷــUﻠﺔ ﻗUـﻞ ﺳـﻘﻬﺎ ﻋﻠـﻰ ﯾـ اﻟ1ـا3P:، وـﺎن ﻣـ: أﻫـﻞ اﻵداب اﻟاﺳـﻌﺔ واﻟ5ﻔـ0: واﻟUاﻋـﺔ ًZﻓـﻲ ﻋﻠــم ﺷـ5ﻰ، وــﺎن ﻣــ: 7ـﺎر أﺻــﺎب اﺑـ: ﺣــvم اﻟpــﺎﻫV، وـﺎن ﺑ5ــﻪ ﻣ05ــ ﻟﻠﻌﻠ1ﺎء واﻷد3ﺎء، وﻟﻪ ﯾﺟﻊ اﻟﻔrﻞ 1ﺎ ﺣIﱠﻠﻪ اﺑ0ﻪ أﺑ ،3' ﻣ: ﻋﻠم وﻣﻌﺎرف ) وﻣﺎ ﺗﻬﺄ ﻟﻪ ﻣ: ﻟﻘﺎء أﺋ1ﺔ اﻟﻌﻠ واﻟﯾ: ﻓﻲ ﻋIﻩ، ﺗﻓﻲ ﻓﻲ ﻋﺎم493 ﻫــ.( ،ﺑــأ اﺑــ: اﻟﻌ9ــﻲ ﺗIــﻠﻪ اﻟﻌﻠ1ــﻲ ﻋﻠــﻰ ﯾــ واﻟــﻩ، ﻓﻌ0ــﻪ أﺧــO ﺗﻌﻠ1ــﻪ اﻷوﱠﻟــﻲ وﻟ@ــ: ﻟ@nــة أﺷــﻐﺎل واﻟــﻩ وارﺗ7ﺎــﻪ 31ﻬــﺎم اﻟوﻟــﺔ 3ﺎﻋ57ــﺎرﻩ وزــاً ﻷﻣــ إﺷــUﻠﺔ ﻓــﻲ lذﻟــ اﻟﻗــ- اﻟ1ﻌ51ــ ﺑــ: w7ــﺎد- u7ﻓﻘــ ﻋﻬــ 3ــﻪ إﻟــﻰ ﺛﻼﺛــﺔ ﻣﻌﻠ1ــ:، أﺣــﻫ ﻟrــ ،اﻟﻘــآن اﻟ@ــ 3ﺄﺣﻓــﻪ اﻟــ7ﻌﺔ، واﻟnــﺎﻧﻲ ﻟ5ﻌﻠ1ــﻪ اﻟﻌ9ــﺔ، واﻟnﺎﻟــ ﻟ17ــﺎد¤ اﻟــﺎب :ﻓOق اﻟﻘآن، وﻫ اﺑ: ﺗﻊ ﺳ0:، وﻟ ﯾUﻠـﻎ اﻟﺎدﺳـﺔ ﻋـة ﻣـ: ﻋ1ـﻩ ﺣ5ـﻰ أﺗﻘـ اﻟﻘاءات اﻟﻌ، وﺟ1ﻊ ﻓ0ﻧﺎً ﻣ: اﻟﻌ9ﺔ، وﻣَﻬَ ﻓﻲ اﻟﻠﻐﺔ واﻷدب واﻟﻌ1. :اﻟWﻠV اﻟZﺎﻧﻲ رﺣﻠ4ﻪ اﻟﻌﻠ3ﺔ إﻟﻰ اﻟ9 ق ﻣﺎ إن ﺑﻠﻎ اﺑ: اﻟﻌ9ﻲ اﻟﺎ3ﻌﺔ ﻋة ﻣ: ﻋ1ﻩ، ﺣ5ﻰ ﺷع 3Iـ7ﺔ واﻟـﻩ ﻓـﻲ رﺣﻠــﺔ ﻋﻠ1ــﺔ إﻟــﻰ ﺣاﺿــ اﻟﻐــب اﻹﺳــﻼﻣﻲ، وﻣاﻛــv اﻟﻌﻠــ ﻓــﻲ اﻟــق اﻹﺳــﻼﻣﻲ ) ﻟ1ـــة زادت ﻋﻠـــﻰ ﻋـــ ﺳـــ0ات485 - 495 Vه(. ﻓـــvار وأﺧـــO اﻟﻌﻠـــ ﻋﻠـــﻰ أﯾـــ 1ﻋﻠ ،ﺎء: ﻣﺎﻟﻘﺔ، ﻏﻧﺎﺔ ، اﻟ1ـﺔ، 3RﺎWـﺔ، ﻋ0ﺎ3ـﺔ، ﺳﺳـﺔ، اﻟﻘـوان، ﺑﻗـﺔ، اﻟﻘـﺎﻫة 1 ( ،اﻧp، أﺣ1 ﺑ: ﷴ ﺑ: ﺧﻠ@ﺎن وa3ﺎت اﻷ`3ﺎن وأﻧUﺎء أﺑﺎء اﻟ^ﻣﺎن ،، ﺗﻘ إﺣﺎن w7ﺎس 1، ﺑوت، ،دار ﺻﺎدر1971م ، 4 / 296 - 297 ،. ﺷ1 اﻟﯾ: اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، ﺗﻘ: ﺷﻌ* اﻷرﻧ ﺎؤو ¦ﷴ ،ﻧq اﻟﻌﻗﺳﻲ 1 ،، ﺑوت، ﻣﺳﺔ اﻟﺳﺎﻟﺔ1985م ، 20 / 198 . أﻫ3ﺔ اﻟ+راﺳﺔ ﻓـــvار وأﺧـــO اﻟﻌﻠـــ ﻋﻠـــﻰ أﯾـــ 1ﻋﻠ ،ﺎء: ﻣﺎﻟﻘﺔ، ﻏﻧﺎﺔ ، اﻟ1ـﺔ، 3RﺎWـﺔ، ﻋ0ﺎ3ـﺔ، ﺳﺳـﺔ، اﻟﻘـوان، ﺑﻗـﺔ، اﻟﻘـﺎﻫة ) وُﻟـ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﻋـﺎم468 ـﻫــ ،( ﻓـﻲ ﻣﯾ0ـﺔ إﺷـUﻠﺔ، ﻓـﻲ 3ـﻼد اﻷﻧــﻟ ﻓﻲ ﺑl ﻋﻠٍ وﺳﺎﺳﺔ، ﻓاﻟﻩ أﺑ ﷴ ﻋU ﷲ ﺑ: اﻟﻌ9ﻲ، ﺎن ﻣ: وﺟﻬـﺎء وأwـﺎن وﻋﻠ1ــﺎء إﺷــUﻠﺔ اﻟ7ــﺎرز:، ﺣــ ــﺎن وزــاً ﻟﻠ1ﻌ51ــ ﺑــ: w7ﱠــﺎد آﺧِــ ﻣﻠــك إﺷــUﻠﺔ ﻗUـﻞ ﺳـﻘﻬﺎ ﻋﻠـﻰ ﯾـ اﻟ1ـا3P:، وـﺎن ﻣـ: أﻫـﻞ اﻵداب اﻟاﺳـﻌﺔ واﻟ5ﻔـ0: واﻟUاﻋـﺔ ًZﻓـﻲ ﻋﻠــم ﺷـ5ﻰ، وــﺎن ﻣــ: 7ـﺎر أﺻــﺎب اﺑـ: ﺣــvم اﻟpــﺎﻫV، وـﺎن ﺑ5ــﻪ ﻣ05ــ ﻟﻠﻌﻠ1ﺎء واﻷد3ﺎء، وﻟﻪ ﯾﺟﻊ اﻟﻔrﻞ 1ﺎ ﺣIﱠﻠﻪ اﺑ0ﻪ أﺑ ،3' ﻣ: ﻋﻠم وﻣﻌﺎرف ) وﻣﺎ ﺗﻬﺄ ﻟﻪ ﻣ: ﻟﻘﺎء أﺋ1ﺔ اﻟﻌﻠ واﻟﯾ: ﻓﻲ ﻋIﻩ، ﺗﻓﻲ ﻓﻲ ﻋﺎم493 ﻫــ.( ،ﺑــأ اﺑــ: اﻟﻌ9ــﻲ ﺗIــﻠﻪ اﻟﻌﻠ1ــﻲ ﻋﻠــﻰ ﯾــ واﻟــﻩ، ﻓﻌ0ــﻪ أﺧــO ﺗﻌﻠ1ــﻪ اﻷوﱠﻟــﻲ وﻟ@ــ: ﻟ@nــة أﺷــﻐﺎل واﻟــﻩ وارﺗ7ﺎــﻪ 31ﻬــﺎم اﻟوﻟــﺔ 3ﺎﻋ57ــﺎرﻩ وزــاً ﻷﻣــ إﺷــUﻠﺔ ﻓــﻲ lذﻟــ اﻟﻗــ- اﻟ1ﻌ51ــ ﺑــ: w7ــﺎد- u7ﻓﻘــ ﻋﻬــ 3ــﻪ إﻟــﻰ ﺛﻼﺛــﺔ ﻣﻌﻠ1ــ:، أﺣــﻫ ﻟrــ ،اﻟﻘــآن اﻟ@ــ 3ﺄﺣﻓــﻪ اﻟــ7ﻌﺔ، واﻟnــﺎﻧﻲ ﻟ5ﻌﻠ1ــﻪ اﻟﻌ9ــﺔ، واﻟnﺎﻟــ ﻟ17ــﺎد¤ اﻟــﺎب :ﻓOق اﻟﻘآن، وﻫ اﺑ: ﺗﻊ ﺳ0:، وﻟ ﯾUﻠـﻎ اﻟﺎدﺳـﺔ ﻋـة ﻣـ: ﻋ1ـﻩ ﺣ5ـﻰ أﺗﻘـ اﻟﻘاءات اﻟﻌ، وﺟ1ﻊ ﻓ0ﻧﺎً ﻣ: اﻟﻌ9ﺔ، وﻣَﻬَ ﻓﻲ اﻟﻠﻐﺔ واﻷدب واﻟﻌ1. :اﻟWﻠV اﻟZﺎﻧﻲ رﺣﻠ4ﻪ اﻟﻌﻠ3ﺔ إﻟﻰ اﻟ9 ق ﻣﺎ إن ﺑﻠﻎ اﺑ: اﻟﻌ9ﻲ اﻟﺎ3ﻌﺔ ﻋة ﻣ: ﻋ1ﻩ، ﺣ5ﻰ ﺷع 3Iـ7ﺔ واﻟـﻩ ﻓـﻲ رﺣﻠــﺔ ﻋﻠ1ــﺔ إﻟــﻰ ﺣاﺿــ اﻟﻐــب اﻹﺳــﻼﻣﻲ، وﻣاﻛــv اﻟﻌﻠــ ﻓــﻲ اﻟــق اﻹﺳــﻼﻣﻲ ) ﻟ1ـــة زادت ﻋﻠـــﻰ ﻋـــ ﺳـــ0ات485 - 495 Vه(. ﻓـــvار وأﺧـــO اﻟﻌﻠـــ ﻋﻠـــﻰ أﯾـــ 1ﻋﻠ ،ﺎء: ﻣﺎﻟﻘﺔ، ﻏﻧﺎﺔ ، اﻟ1ـﺔ، 3RﺎWـﺔ، ﻋ0ﺎ3ـﺔ، ﺳﺳـﺔ، اﻟﻘـوان، ﺑﻗـﺔ، اﻟﻘـﺎﻫة 1 ( ،اﻧp، أﺣ1 ﺑ: ﷴ ﺑ: ﺧﻠ@ﺎن وa3ﺎت اﻷ`3ﺎن وأﻧUﺎء أﺑﺎء اﻟ^ﻣﺎن ،، ﺗﻘ إﺣﺎن w7ﺎس 1، ﺑوت، ،دار ﺻﺎدر1971م ، 4 / 296 - 297 ،. ﺷ1 اﻟﯾ: اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، ﺗﻘ: ﺷﻌ* اﻷرﻧ ﺎؤو ¦ﷴ ،ﻧq اﻟﻌﻗﺳﻲ 1 ،، ﺑوت، ﻣﺳﺔ اﻟﺳﺎﻟﺔ1985م ، 20 / 198 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 28 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن اﻟﻘـــس، دﻣـــ، 3ﻐـــاد، ﻣ'ـــﺔ اﻟ1'ﻣـــﺔ، اﻟ1ﯾ0ـــﺔ اﻟ10ـــرة، اﻹﺳـــ'0رﺔ. أﻫ3ﺔ اﻟ+راﺳﺔ وﻓـــﻲ ﻫـــOﻩ اﻟ1اﺋ: اﻟﻌﻠ1ﺔ ﺣIﱠﻞَ اﺑ: اﻟﻌ9ﻲ ﻗﺎﻋﺗﻪ اﻟﻌﻠ1ﺔ اﻟاﺳQﺔ واﻟﺎﻣﻠﺔ ﻓﻲ ﻞ اﻟﻌﻠم .اﻹﺳﻼﻣﺔ واﻟﻠﻐـﺔ اﻟﻌ9ـﺔ وآداﺑﻬـﺎ ،ﻓﻘـ ﺗ7ﱠـ ﻓـﻲ ﻋﻠـم اﻟﻘـآن واﻟ5ﻔـ واﻟﻘـاءات وﻋﻠـــم اﻟـــﯾ دراWـــﺔً ورواWـــﺔً، وﻋﻠـــ اﻟ@ـــﻼم واﻟﻌﻘﺎﺋـــ واﻟﻔِـــَق، واﻟ5Iـــف، وﻋﻠـــم اﻟ0ـــ واﻟIـــف واﻟ7ﻼﻏـــﺔ، واﻟـــﻌ واﻷدب واﻟ5ـــﺎرﺦ. وﻋـــﺎد ﻣـــ: رﺣﻠ5ـــﻪ إﻟـــﻰ 3ـــﻼد اﻷﻧﻟ ﺑvادٍ ﻋﻠ1ﻲ ﻟ WU أن ﻋـﺎد 3ـﻪ أV ﻋـﺎﻟ أﻧﻟـﻲ آﺧـ زار وﻠـ اﻟﻌﻠـ ﻓﻲ 3ﻼد اﻟق اﻹﺳﻼﻣﻲ1. :ﻗـﺎل ﻋ0ـﻪ اﻟـﻲ ﻓـﻲ 5ﺎ3ـﻪ 7ﻘـﺎت اﻟ1ﻔـ : ".. وﻋـﺎد]اﺑـ: اﻟﻌ9ـﻲ[ إﻟـﻰ ﺑﻠـﻩ 3ﻌﻠ n ﻟ ﯾُﺧﻠﻪ أﺣٌ ﻣ: ﻗUﻠﻪ ﻣ1: ﺎﻧl ﻟﻪ رﺣﻠﺔ إﻟﻰ اﻟ1ق، وﺎن ﻣ: أﻫﻞ اﻟ5ﻔ0: ﻓﻲ اﻟﻌﻠم واﻻﺳ57ﺎر ﻓﻬـﺎ واﻟR1ـﻊ ﻟﻬـ.ﺎ ﻣﻘـﱠﻣﺎً ﻓـﻲ اﻟ1ﻌـﺎرف ﻠﻬـﺎ أﺣـ ْ:ﻣـ ﺑﻠــﻎ رﺗ7ــﺔ اﻻﺟ5ﻬــﺎد، وأﺣــ ًﻣــ: اﻧﻔــد 3ﺎﻷﻧــﻟ 3ﻌﻠــ اﻹﺳــ0ﺎد، ﺛﺎﻗــ اﻟــOﻫ: ﻣﻼزﻣــﺎ "ﻟ0 اﻟﻌﻠ، ﺻﺎرﻣﺎً ﻓﻲ أﺣ'ﺎﻣﻪ ﻫ9ﺎ ﻋﻠﻰ اﻟpﻠ1ﺔ2 . ﻲ ﻹ ﻲ 3 :ﻗـﺎل ﻋ0ـﻪ اﻟـﻲ ﻓـﻲ 5ﺎ3ـﻪ 7ﻘـﺎت اﻟ1ﻔـ : ".. وﻋـﺎد]اﺑـ: اﻟﻌ9ـﻲ[ إﻟـﻰ ﺑﻠـﻩ 3ﻌﻠ n ﻟ ﯾُﺧﻠﻪ أﺣٌ ﻣ: ﻗUﻠﻪ ﻣ1: ﺎﻧl ﻟﻪ رﺣﻠﺔ إﻟﻰ اﻟ1ق، وﺎن ﻣ: أﻫﻞ اﻟ5ﻔ0: ﻓﻲ اﻟﻌﻠم واﻻﺳ57ﺎر ﻓﻬـﺎ واﻟR1ـﻊ ﻟﻬـ.ﺎ ﻣﻘـﱠﻣﺎً ﻓـﻲ اﻟ1ﻌـﺎرف ﻠﻬـﺎ أﺣـ ْ:ﻣـ ﺑﻠــﻎ رﺗ7ــﺔ اﻻﺟ5ﻬــﺎد، وأﺣــ ًﻣــ: اﻧﻔــد 3ﺎﻷﻧــﻟ 3ﻌﻠــ اﻹﺳــ0ﺎد، ﺛﺎﻗــ اﻟــOﻫ: ﻣﻼزﻣــﺎ "ﻟ0 اﻟﻌﻠ، ﺻﺎرﻣﺎً ﻓﻲ أﺣ'ﺎﻣﻪ ﻫ9ﺎ ﻋﻠﻰ اﻟpﻠ1ﺔ2 . :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ ذﻧﺎ ﻓﻲ اﻟﻔﻘة اﻟﺎ3ﻘﺔ أن اﺑ: اﻟﻌ9ﻲ ﺗﻠﻘﻰ اﻟﻌﻠ ﻋﻠﻰ أﻛﺎﺑ ﻋﻠ1ﺎء ﻋIـﻩ اﻟـــOﯾ: اﻟ5ﻘـــﻰ ﺑﻬـــ ﻓـــﻲ 3ـــﻼد اﻟـــق اﻹﺳـــﻼ ﻣﻲ، وﻧـــ: ﻫ0ـــﺎ ﻧـــO ﻣـــ: ﺑـــ: ﻫـــﻻء اﻟﻌﻠ1ــﺎء اﻷﺳــ1ﺎء اﻟ5ﺎﻟــﺔ ﻓﻘــu: اﻟﻔcــﻪ أﺑــ 3'ــ اﻟPﺷــﻲ اﻟ1ــﺎﻟ@ﻲ، واﻟﻘﺎﺿــﻲ أﺑــ اﻟـــ: ﻣ'ـــم ﺑـــ: ﻣـــزوق، وأﺑـــ اﻟﻔـــ5ﺢ اﻟ1ﻘﺳـــﻲ اﻟـــOV أﺧـــO ﻋ0ـــﻪ 5ـــ اﻟIـــﺎح وﻏﻫﺎ، وأﺑ اﻟﻔrﻞ اﻷﺑردV اﻷدﯾ اﻟ1ﻬر، وأﺑ ﷴ اﻟ: اﻟPUـV، وأﺑـ :3'ــــ ﷴ ﺑــــ أﺣ1ــــ اﻟﺎﺷــــﻲ رﺋــــ اﻟــــﺎﻓqﺔ ﻓــــﻲ 3ﻐــــاد وﻣــــ: ﻋﻠ1ــــﺎء اﻟ1رﺳــــﺔ ،اﻟ0pﺎﻣــﺔ، وﺣRــﺔ اﻹﺳــﻼم أﺑــ ﺣﺎﻣــ اﻟﻐvاﻟــﻲ ﺣــ ﻗــأ ﻋﻠــﻪ ﻣR1ﻋــﺔً ﻣــ: 57ــﻪ :وﻫ اﻟOV أَدﺧﻞَ إﻟﻰ اﻷﻧﻟ 5ﺎ3ـﻪ إﺣـﺎء ﻋﻠـم اﻟـﯾ:؛ وأﺑـ اﻟﻘﺎﺳـ إﺳـ1ﺎﻋﻞ ﺑـ 1 ( ،اﻧp، ﺷ1 اﻟﯾ: اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 197 - 199 . ،Vأﺣ1 ﺑ: ﷴ اﻷدﻧﻪ و bUﻘﺎت - اﻟﻔ ،VvQ، ﺗﻘ* ﺳﻠ1ﺎن ﺑ: ﺻﺎﻟﺢ اﻟ 1 ، اﻟ1ﯾ0ﺔ اﻟ10رة: ﻣ ،''57ﺔ اﻟﻌﻠم واﻟ1417 /ه1997م ، 180 - 181 . 2 ( ،ﺟﻼل اﻟﯾ: اﻟﻲ- bUﻘﺎت اﻟﻔ ،1، ﺗﻘ ﻋﻠﻲ ﷴ ﻋ 1 ، ،اﻟﻘﺎﻫة: ﻣ'57ﺔ وt7ﺔ1396 / 1976 ، 105 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 29 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ VvﻋU اﻟ1ﻠ اﻟﺎﻛ1ﻲ اﻟPﺳﻲ، وأﺑ زﺎ Wﻰ ﺑ: ﻋﻠﻲ اﻟ5Uـ ﺷـﺦ 3ﻐـاد ﻓـﻲ ،Vاﻟﻠﻐﺔ و اﻷدب، واﻟﺎﻓª أﺑـ ﻋـﺎﻣ ﷴ ﺑـ: ﺳـﻌون اﻟ1ـرﻗﻲ اﻟ7ﻐـادV اﻟpـﺎﻫ :وأﺑــ اﻟــ: ﷴ ﺑــ: Wﻌﻠــﻰ اﻟﻔــاء اﻟ0Uﻠــﻲ، واﻟــﺎﻓª أﺑــ ﷴ ﺟﻌﻔــ ﺑــ: أﺣ1ــ ﺑــ اﻟـاج اﻟ0Uﻠـﻲ ﻣﻘـ¤ 3ﻐــاد1 . 1 ( ،pاﻧ ،اﺑ: ﺧﻠ@ﺎن وa3ﺎت اﻷ`3ﺎن، 4 / 296 ،؛ اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 198 - 199 . 2 ( ،اﻧp، اﺑ: ﺧﻠ@ﺎن وa3ﺎت اﻷ`3ﺎن ، 4 / 297 ،؛ اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 200 - 201 . :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ وﻫ'ـOا ﻓﺈﻧ0ـﺎ ﻧــرك ﻣـ: ﺗ0ـّع ﺷــخ اﺑـ: اﻟﻌ9ـﻲ، ﻣــﺎ ﺑ: ﺣ0ﻔﻲ وﺷﺎﻓﻌﻲ وﺣ0Uﻠ ،ٍﻲ وﻣﺎﻟ@ﻲ وfﺎﻫV، وﻣ: ﺗ0ّع ﻣﺎ اﻣ5ﺎزوا 3ـﻪ ﻣـ: ﻗـاءة َوﺗﻔـــ، وﺗـــﯾ، وﻓﻘـــﻪ، وأﺻـــل، وﻟﻐـــﺔٍ، وأدب، وﺗIـــفٍ ، وﻋﻠـــ ـــﻼم، ﺣRـــ .وﺗ0ّعَ وﺷ1ل اﻟﻌﻠ اﻟOV أﺣﺎ 3ﻪ أﺛ0ﺎء رﺣﻠ5ﻪ اﻟﻌﻠ1ﺔ اﻟ1Oرة :اﻟWﻠV اﻟ ا!ﻊ 3ﻋﻠﻪ ﻓﻲ اﻟﻘMﺎء واﻟ4ﻌﻠ ﻋﺎد اﺑ: اﻟﻌ9ﻲ إﻟﻰ ﺑﻠـﻩ إﺷـUﻠﺔ 3ﻌـ أن ﺗـvوﱠد ﻣـ: ﻣاﻛـv اﻟﻌﻠـ ﻓـﻲ اﻟﻌـﺎﻟ اﻹﺳــﻼﻣﻲ ﻓــﻲ ﻋIــﻩ، وأﺧــO ﻧIــ7ﻪ ﻣــ: ﻣﻌــﺎرف زﻣ0ــﻪ، وﻓــﻲ اﻟcﻘــﺔ ﻓﻘــ ﺳــ7ﻘ5ﻪ ﺳ1ﻌ5ﻪ اﻟﻌﻠ1ﺔ اﻟ@Uة إﻟـﻰ و0ـﻪ، ﺣـ ﺗـﺎﺑ اﻟ0ـﺎس إﻟـﻰ اﻟ5ﺣـ 3ـﻪ واﻻﻟ5ﻘـﺎء .3ﻪ، وﺷﱠ ﻼب اﻟﻌﻠ ﻣ: ﻞ أﻧﺎء اﻷﻧﻟ اﻟﺣﺎل إﻟـﻪ ﻟﺄﺧـOوا اﻟﻌﻠـ ﺑـ: ﯾWـﻪ وﻗ ﻋف ﻗرﻩ اﻟﻌﻠ1ﻲ – ًأWrﺎ- ﺣ'ﺎم وأﻣاء و0ﻪ، ووﻻﱠ :ﻩ أﻣ* إﺷUﻠﺔ "ﺳ ﺑـ أ ﺑـــﻲ 3'ـــ اﻟﻠ15ـــﻧﻲ" ﻣ0Iـــ اﻟـــرZ ﻟـــﻪ ، وﻫـــ ﻣ0Iـــ رـــﻊ ﻻ WIـــﻞ إﻟـــﻪ إﻻ ،اﻟIـﻔة ﻣــ: اﻟﻌﻠ1ـﺎء واﻟﻔﻘﻬــﺎء، وRﻌﻠﻬــ ﻓـﻲ ﻣIــﺎفِّ اﻟــزراء وUـاء رﺟــﺎل اﻟوﻟــﺔ وﻣــ: ﻫ0ــﺎ ﻓﻘــ وﺟــﻧﺎ 3ﻌــ2 ﻣﻌﺎﺻــﻪ WPﻠــ ﻋﻠــﻪ ﻟﻘــ اﻟــز. ﺛــ وﻟــﻲﱠ اﻟﻘrــﺎء ،:ﻋﻠــــﻰ إﺷــــUﻠﺔ، ﻓ@ﺎﻧــــl ﻟــــﻪ ﺳــــرةٌ ﻣﻫ9ــــﺔ ﻓــــﻲ اﻟpــــﺎﻟ1:، ﻣــــﻊ اﻟﻓــــ 3ﺎﻟ1ــــﺎﻛ وﺻـــاﻣﺔٌ 3ـــﺎﻷﻣ 3ـــﺎﻟ1ﻌوف واﻟ0ﻬـــﻲ ﻋـــ: اﻟ10@ـــ2 ،وﻟ@ـــ: و9ـــU ﻫـــOﻩ اﻷﺳـــ7ﺎب ﻣR51ﻌــﺔ ﻟــ ﯾ5ــ ﻟــﻪ اﻟ7ﻘــﺎء ﻣــة ــﺔ ﻓــﻲ ﻫــOا اﻟ10Iــ ﻟ@nــة اﻟ15rــر: ﻣ0ــﻪ اﻟOﯾ: ﻟ ﯾﺗﺎﺣا ﻟﻠة اﻟﻌﺎدﻟ ﺔ اﻟ5ﻲ ﺳﻠ@ﻬﺎ ﻫOا اﻟﻘﺎﺿﻲ اﻟ0vﻪ اﻟOV ﻻ WQﻰ ﻓـﻲ اﻟــ ﻟﻣــﺔ ﻻﺋــ، ﻟــOﻟ ﻋ1ــﻞ ﻫــﻻء اﻟ15rــرون ﻋﻠــﻰ ﺗ05ــﻪ، وﻫــOا ﻟﻸﺳــﻒ ﻣــﺎ ﺗﻘــ ﻟﻬــ 3ﻌــ ﺛ1ﺎﻧــﺔ أﺷــﻬ ﻣــ: ﺗﻟــﻪ ﻫــOا اﻟ10Iــ. وﻟ @ــ: إذا اﺳــ5Pﺎع ﻫــﻻء اﻟ0ــﺎس ﻋvﻟــﻪ ﻋــ : ﻣ0Iــ اﻟﻘrــﺎء، ﻓــﺈﻧﻬ ﻟــ Wــ5Pﻌا إ3ﻌــﺎدﻩ ﻋــ: ﺣﻠﻘــﺎت اﻟﻌﻠــ 1 ( ،pاﻧ ،اﺑ: ﺧﻠ@ﺎن وa3ﺎت اﻷ`3ﺎن، 4 / 296 ،؛ اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 198 - 199 . 2 ( ،اﻧp، اﺑ: ﺧﻠ@ﺎن وa3ﺎت اﻷ`3ﺎن ، 4 / 297 ،؛ اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 200 - 201 . :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 30 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن واﻟ5ﻌﻠــ: وﻧــ اﻟ1ﻌــﺎرف ﺑــ: أﺑ0ــﺎء اﻷﻧــﻟ، ﺑــﻞ ﻋﻠــﻰ اﻟﻌ'ــ ﻣــ ذﻟــ ﻓﻘــ ﺗﻔــﱠغ 3ﻌــ ﻋvﻟــﻪ، أﻛnــ ﻓــﺄﻛn، ﻟﻬــOﻩ اﻟ1ﻬ1ــﺔ اﻟﻌﻠ1ــﺔ، وﺧﻠّــﻒ ﻟ0ــﺎ ﺟــﻼً ﻣــ: اﻟﻌﻠ1ــﺎء ﺣ1ــﻞ ًﻟاء اﻟﻌﻠ 3ﻌﻩ ﻓﻲ اﻷﻧﻟ، 1ﺎ أﻧﻪ ﺗك ﻟﻠrﺎرة اﻹﺳﻼﻣﺔ ﻣR1ﻋﺔ :ﻧ_ﺔ ﻣ اﻟ1ﻟﻔﺎت ﻓﻲ ﻣQ5ﻠﻒ ﻓوع اﻟﻌﻠم اﻹﺳﻼﻣﺔ، ﺗ ِّـﻬ ﻋﻠـﻰ ﻋﻠـ q7ـﻪ ورـﻊ ﻣﻘﺎﻣـﻪ ورﺳــخ ﻓﻬ1ــﻪ، ودﻗــﺔ ﺗcﻘــﻪ ﻓــﻲ ــﻞ اﻟﻘrــﺎWﺎ واﻟ1ــﺎﺋﻞ اﻟ5ــﻲ ﺗﻌــﱠض ﻟﻬــﺎ 3ﺎﻟ7ــ .eواﻟ5ﺄﻟ :dاﻟWﻠV اﻟ1ﺎﻣ ﻣeﻟﻔﺎﺗﻪ اﻟﻌﻠ3ﺔ :ﻗﻠ0ﺎ إنّ اﺑ اﻟﻌ9ﻲ ﻗ ﺗك ﻟﻠ1ﻠ1: ﻣ: 3ﻌﻩ ﺛـوة ﻋﻠ1ـﺔ ﺿـQ1ﺔ، ﺳـﺎﻋﻩ ﻋﻠـــﻰ إﻧRﺎزﻫـــﺎ اﻻﺑ5ﻌـــﺎد ﻋـــ: اﻟ10ﺎﺻـــ 3ﻌـــ ﻋvﻟـــﻪ ﻋـــ: اﻟﻘrـــﺎء، وﺗﻔّﻏـــﻪ ﻟﻠ5ﻌﻠـــ ًواﻻﺷــ5ﻐﺎل 3ﺎﻟ5ــﺄﻟe اﻟﻌﻠ1ــﻲ. وﻓــﻲ اﻟcﻘــﺔ ﻓــﺈن ﻣﻟﻔﺎﺗــﻪ ﺗvــ ﻋﻠــﻰ اﻟ5ــﻌ: ﻣﻟﻔــﺎ ،ﻓﻲ اﻟ5ﻔ* واﻟﯾ واﻟﻌﻘـة واﻟﻔﻘـﻪ واﻷﺻـل واﻟ5ـﺎرﺦ واﻟ0ـ واﻷدب واﻟﺎﺳـﺔ وﻟ@: ﻟﻸﺳﻒ اﻟ،ﯾ، ﻓﻘ ﻓﻘﻧﺎ ﻗ1ﺎً ﻻ 3ﺄس 3ﻪ ﻣـ: ﻫـOﻩ اﻟ1ﻟﻔـﺎت ٌوﻣـﺎ زال ﻗـ ﻛU ﻣ0ﻬﺎ ﺣUـ اﻟ1QPـﺎت وﺧـvاﺋ: اﻟ@5ـ ﺣ5ـﻰ اﻵن، وﻟـ Wﻌـف ﻘـﻪ إﻟـﻰ اﻟ0ـــ ﻣ0ﻬـــﺎ إﻻ 3rـــﻌﺔ 5ـــ1 :. ﻏـــ أن ﻫـــOﻩ اﻟ@5ـــ اﻟ10ـــرة، وﻋﻠـــﻰ اﻟـــﻏ ﻣـــ ﻗﻠّ5ﻬــﺎ، ﺎــﺔٌ ﻟﻠﻻﻟــﺔ ﻋﻠــﻰ اﻟ1ــﺎﻫ1ﺔ اﻟﻌﻠ1ــﺔ اﻟ@Uــة واﻷﺻــﻠﺔ :اﻟ5ــﻲ ﻗــﺎم ﺑﻬــﺎ اﺑــ اﻟﻌ9ـــﻲ ﻓـــﻲ ﻣـــان اﻟﻌﻠـــم اﻹﺳـــﻼﻣﺔ ﻓـــﻲ 3ـــﻼد اﻷﻧـــﻟ 3ـــ'ﻞ ﺧـــﺎص، واﻟﻌـــﺎﻟ اﻹﺳﻼﻣﻲ 3'ﻞ ﻋﺎم ﻓﻲ ﻋIﻩ، واﻟ5ﻲ ﻣﺎزاﻟl ﺣﺎﺿة 3c15ﻬﺎ وﺛﻘﻠﻬﺎ اﻟﻌﻠ1ـﻲ ﻓـﻲ .ﺣﺎﺗ0ﺎ اﻟﻌﻠ1ﺔ اﻟ1ﻌﺎﺻة ﺳﺄﻛ5ﻔﻲ ﻫ0ﺎ 3ﺎﻹﺷﺎرة إﻟﻰ ﻣﻟﻔﺎﺗﻪ ﻓﻲ ﻣان ﺗﻔ اﻟﻘآن وﻋﻠﻣﻪ2: 1 ( ،اﻧp، اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 199 ، 201 ،؛ ﺳﻌ* أﻋـاب ﻣـﻊ اﻟﻘﺎﺿـﻲ أﺑـﻲ !ـ ﺑـ اﻟﻌ ـﻲ ، 1 ، ،ﺑــــوت، دار اﻟﻐــــب اﻹﺳــــﻼﻣﻲ1407 / 1987 :، ص121 - 173 ) . :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ ﺣــــ أﺣIــــﻰ اﻟ1ﻟــــﻒ92 :( 5ﺎ3ــــﺎً ﻻﺑــــ .اﻟﻌ9ﻲ 2 ( ﻣــﻊ ﺿــورة اﻹﺷــﺎرة إﻟــﻰ أن اﺑــ: اﻟﻌ9ــﻲ ــﺎن، ﻓrــﻼً ﻋــ: ﺟﻬــدﻩ ﻓــﻲ اﻟ5ﻔــ* وﻋﻠــم اﻟﻘــآن، ﻣــ: أﺑــز ﻋﻠ1ــﺎء اﻟﯾ اﻟ0UV وﺷاﺣﻪ ﻓﻲ اﻷﻧﻟ ﻓﻲ ﻋIﻩ، ﻣ: أﺑز ﻣﻟﻔﺎﺗﻪ ﻓﻲ ﻫـOا اﻟ1Rـﺎل: ﻋﺎرﺿـﺔ اﻷﺣـذV 3ـح ﺟـﺎﻣﻊ اﻟ5ﻣـــOV، اﻟcــــ7 ﻓــــﻲ ﺷــــح ﻣــــﺄ ﻣﺎﻟــــ ﺑــــ: أﻧــــ، ﺗﺗــــ اﻟ1ــــﺎﻟ ﻓــــﻲ ﺷــــح ﻣــــﺄ ﻣﺎﻟــــ، اﻟ0ــــ: ﻓــــﻲ ﺷــــح .:Iاﻟ ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 31 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ أﺣﺎم اﻟﻘ آن: وﻫ ﺗﻔﻩ اﻟOV ﻧQIﻪ ﻫ0ﺎ ﺑﻬOﻩ اﻟراﺳﺔ؛ اﻷﺣ'ﺎم اﻟIﻐZ: وﻫ ﻣQ5Iــ ﺗﻔــﻩ" أﺣ'ــﺎم اﻟﻘــآن"؛ اﻟ0ﺎﺳــﺦ واﻟ10ــخ ﻓــﻲ اﻟﻘــآن؛ ﻗــﺎﻧن اﻟ5ﺄوــﻞ ﻓــﻲ ﺗﻔ* اﻟﻘآن؛ اﻟ1ﻘ57 ﻣ: اﻟﻘاءات؛ أﻧار اﻟﻔR ﻓﻲ ﻣRﺎﻟ اﻟـO، وﻫـ ﺗﻔـ :ﻛﺎﻣﻞ ﻟﻠﻘآن اﻟ@، ﻗﺎم اﺑ: اﻟﻌ9ﻲ ﺑ5ﺄﻟﻔﻪ ﻋـ ـ إﻟﻘﺎﺋـﻪ دروﺳـﺎ ﻓـﻲ ﻣRـﺎﻟ ﻋﺎﻣﺔ ﻓﻲ اﻟ1ﺎﺟ ﺧـﻼل ﻋـ: ﺳـ0ﺔ، وﻫـOا اﻟ5ﻔـ* Wﻘـﻊ ﻓـﻲ ﺛ1ـﺎﻧ: ﻣRﻠـا، ﻓـﻲ .ﻛﻞ ﻣRﻠ أﻟﻒ ورﻗﺔ. )ﻣﻔﻘد(؛ اﻟ1ُ'ﻠ:. أV ﻣُ'ﻞ اﻟﻘآن اﻟ@ واﻟ0ﺔ اﻟﻔﺔ ()ﻣﻔﻘـــد(؛ ﺷـــح ﺣـــﯾ "أﻧـــvل اﻟﻘـــآن ﻋﻠـــﻰ ﺳـــ7ﻌﺔ أﺣـــف" )ﻣﻔﻘـــد1 V؛ "ﺗﺗـــ آ اﻟﻘآن" ذﻩ ﻓﻲ 5ﺎ :3ﻪ "اﻟ0ﺎﺳﺦ واﻟ10خ" أﺛ0ﺎء ﺗIـﯾﻩ ﻟـرة اﻷﻧﻌـﺎم، ﺣـ ﻗـﺎل "واﻷﺣ'ـــﺎم ﻓﻬـــﺎ ﻗﻠـــﻞ، ﻟﻌـــﺎرض ﺑ0ـــﺎ وﺟﻬـــﻪ ﻓـــﻲ "ﺗﺗـــ آV اﻟﻘـــآن"، وﻫـــ 5ـــﺎب أﺧﻔ0ﺎﻩ 3ﻌ أن ﺟ1ﻌ0ﺎﻩ، ﻟ1ﺎ رأﯾ0ﺎ ﻪ ﻣ: ﻋﻠﻩ ﻋﻠـﻰ أﻗـار أﻫـﻞ اﻟvﻣـﺎن، وأﻧـﻪ ﻟـ "ﻟﻪ ﻓﻲ ﻫOﻩ اﻷﻗPﺎر ﺣﻔﻲّ ﻓﺿﻌ0ﺎﻩ ﻓﻲ ﺳَ َب ﺧﻔﻲ2 . VاﻟWﻠ :اﻟﺎدس ﻣg+َiﻫV اﺑ اﻟﻌ ﻲ اﻟﻔﻘﻬﻲ واﻟﻌَﻘ ﻋــﺎش اﺑــ: اﻟﻌ9ــﻲ ﻓــﻲ اﻟــور اﻟQــﺎﻣ ﻣــ: أدوار اﻟﻔﻘــﻪ اﻹﺳــﻼﻣﻲ اﻟــOV ﯾUــأ ﻣــ: ﻣ05Iــﻒ اﻟ1ﺎﺋــﺔ اﻟا3ﻌــﺔ إﻟــﻰ ﺣــود أواﺳــu اﻟﻘــن اﻟــﺎ3ﻊ اﻟﻬRــV، وﻫــ 3ــ'ﻞ ﻋﺎم ﻋI اﻟد واﻟ5ﻘﻠ اﻟﻔﻘﻬﻲ، ﻓﻠـ Wـﺄت 3ﻌـ أﺑـﻲ ﺟﻌﻔـ اﻟPUـV )ت310 (ه ٌإﻣـــﺎم ﻣـــ: أﺋ 1ـــﺔ اﻻﺟ5ﻬـــﺎد اﻟ1Pﻠـــ، ﻟ@ـــ: fﻬـــ رﺟـــﺎل ﻟـــ Wﻘﻔـــا ﻋ0ـــ ﺣـــود اﻟ5ﻘﻠـــ اﻟ1ــ2، ﺑــﻞ ﺳــﺎروا ﻋﻠــﻰ ﺑِّ0ــﺔ ﻣــ: اﻷﻣــ، ﻓR1ﻌــا اﻵ ،ﺛــﺎر اﻟ0Uــﺔ وأﻗــال اﻟــﻠﻒ و ﻣّIا اﻟواWﺎت، واﺳ507Pا اﻷﺣ'ﺎم ﻣ: ﯾ0ﺎﺑﻌﻬـﺎ، وﺣـﺎوﻟا أن ﯾـدوا اﻟﻔـوع إﻟـﻰ أﺻﻟﻬﺎ، وﺎﻧl ﻟﻬ ﻣاﻗﻒ ﻣﻬرة ﻓﻲ ﺑـﺎن اﻟـ واﻟ ﻻﻟـﺔ ﻋﻠـﻪ. :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ و1'ـ: ﻟ0ـﺎ أن ﻧﻌــﱠ ﻣــ: ﻫــOﻩ اﻟP7ﻘــﺔ ﻣــ: اﻟﻌﻠ1ــﺎء، اﻟﻘﺎﺿــﻲ أﺑــ 3'ــ ﺑــ: اﻟﻌ9ــﻲ اﻟــOV ﺗﻠﻘﱠــﻰ اﻟﻌﻠــ - ﻋﻠﻰ اﻟﻏ ﻣـ: أﻧـﻪ ﻣـﺎﻟ@ﻲ اﻟ1ـOﻫ- ﻋﻠـﻰ أﯾـV ﻋﻠ1ـﺎء ﻣﻌpـ اﻟ1ـارس اﻟﻔﻘﻬـﺔ 1 ( ،اﻧp، ﺑﻫﺎن اﻟﯾ: إﺑاt ﺑ: ﻓﺣنVاﻟ+ﯾUﺎج اﻟiﻫV ﻓـﻲ ﻣﻌ ﻓـﺔ أ`3ـﺎن اﻟـiﻫ،.، د، ﺑـوت: دار اﻟ@5ـ ،اﻟﻌﻠ1ﺔ) (د.ت، 281 - 284 ،؛ ﻣIPﻔﻰ إﺑاt اﻟ10ﻲ اﺑـ اﻟﻌ ـﻲ اﻟـﺎﻟ5ﻲ اﻹﺷـ? ﻠﻲ وﺗﻔـ ﻩ أﺣـﺎم اﻟﻘـ آن ، 1، ﺑوت- ﻋ1ﺎن، دار اﻟRﻞ- ،دار ﻋ1ﺎر1411 / 1991 ، 27 - 34 . 2 ( ،ﺟﻼل اﻟﯾ: اﻟﻲ اﻹﺗﻘﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن ،، ﺗﻘ* ﻣv اﻟراﺳﺎت اﻟﻘآﻧﺔ 1 ، ﻣR1ﻊ اﻟ1ﻠ ﻓﻬ ،ﻟP7ﺎﻋﺔ اﻟ1Iﻒ اﻟe، اﻟ1ﯾ0ﺔ اﻟ10رة1426 ه ، 5 / 1837 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 32 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن ﻓﻲ ﻋIﻩ ﻣ: ﺷﺎﻓqﺔٍ وأﺣ0ﺎفٍ وﻣﺎﻟ@ـﺔٍ وﺣ0ﺎﺑﻠـﺔٍ وfﺎﻫـﺔٍ، 1ـﺎ أﻧـﻪ اﺣـ5ﻔª ﻟ0ﻔـﻪ ﻓﻲ ﻗrﺎWﺎ nـ ة 3ـ اﻻﺧـ5ﻼف ﻋـ: إﻣـﺎم ﻣOt7ـﻪ، 3ﺎﻹﺿـﺎﻓﺔ إﻟـﻰ ﻣ0ﺎﻗـﺎﺗﻪ اﻟـة .Zواﻟ0ﻘWﺔ ﻟ@ﻞ اﻟ1Oاﻫ اﻷﺧ وأﻣﺎ ﻣ: ﺣـ ﻣOt7ـﻪ اﻟﻌَﻘَـV، ﻓﻘـ ـﺎن اﺑـ: اﻟﻌ9ـﻲ أﺷـﻌV اﻟ1ـOﻫ، ﯾـOﻫ إﻟـﻰ ﻣﺎ ذﻫ إﻟﻪ أﻫـﻞ اﻟـ0ﺔ واﻟR1ﺎﻋـﺔ، وـد ﻋﻠـﻰ أﺻـﺎب اﻟﻔـق اﻷﺧـZ، وﻬـﺎﺟ اﻟﻔﻼﺳــــﻔﺔ وﻏــــﻼة اﻟ15Iـــــﻓﺔ، 1ــــﺎ :Wpﻬــــ ذﻟـــــ ﺟﻠــــﺎ ﻓــــﻲ 5ﺎ3ـــــﻪ "اﻟﻌاﺻــــ ﻣـــــ اﻟﻘاﺻـــ"، وpﻬـــ 3Iـــرة أو 3ـــﺄﺧZ ﻓـــﻲ ﺗﻔـــﻩ أﺣ'ـــﺎم اﻟﻘـــآن 1ـــﺎ ﺳـــ0Z 3ﻌـــ .ﻗﻠﻞ1 :اﻟWﻠV اﻟﺎ!ﻊ ا ﻷﺣ+اث ا ﻟ4ﺎر-13ﺔ اﻟ4ﻲ ﻋﺎﺻ ﻫﺎ اﺑ اﻟﻌ ﻲ ،وُﻟــ اﺑــ: اﻟﻌ9ــﻲ ﻓــﻲ اﻟﻗــl اﻟــOV ﺎﻧــl ــﻪ 3ــﻼد اﻷﻧــﻟ ﻣ1vﱠﻗــﺔ ﺿــqﻔﺔ ﯾ0ﻔــد ــﻞﱡ أﻣــ ﻓﻬــﺎ 3ﺈﻣــ ﺎرة ﺧﺎﺻــﺔ 3ــﻪ، وqــ° ﻓــﻲ ﺣﺎﻟــﺔ ﻧــvاعٍ وﺷِ ــﻘﺎق ﻣــﻊ 3cــﺔ ،اﻹﻣـــﺎرات، وـــﺧﻞ أﺣﺎﻧـــﺎً ﻓـــﻲ ﻋﻘـــد ﺗـــﺎﻟﻒ وﻧIـــة ﻣـــﻊ اﻹﻓـــﻧﺞ ﻟـــ5ﻔª 31ُﻠ@ـــﻪ 3ﻐــــ2 اﻟ0pــــ ﻋــــ: اﻟﻌاﻗــــ اﻟﺧ1ــــﺔ اﻟ5ــــﻲ W1'ــــ: أن ﺗــــﻞﱠ 3ــــﻪ و9ﺈﻣﺎرﺗــــﻪ و9'ــــﻞ اﻟ1ﻠ1: ﻓﻲ اﻷﻧﻟ ﻣ: ﺟﱠاء ﻫOا اﻟ5ﺎﻟﻒ واﻟ0Iة، وﻗ أﻠ اﻟ1رﺧـن ﻋﻠـﻰ ﻫOﻩ اﻟc7ﺔ ﻣ: ﺗـﺎرﺦ اﻟ1ـﻠ1: ﻓـﻲ اﻷﻧـﻟ، اﺳـ ﻋﻬـ ﻣﻠـك اﻟPاﺋـﻒ. 1 ( ،اﻧpـ، ﻣﻘﻣـﺔ ﺳــﻌ* أﻋـاب ﻟ@5ـﺎب اﺑــ: اﻟﻌ9ـﻲ l اﻷﺣــﺎم اﻟ;ـﻐ ، 1 0، ﻣ ـرات اﻟ10p1ــﺔ اﻹﺳـﻼﻣﺔ ﻟﻠ59ــﺔ ،واﻟﻌﻠم واﻟnﻘﺎﻓﺔ1409 / 1989 . :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ وﻣـﺎ إن ،ﺑﻠﻎ اﺑ: اﻟﻌ9ﻲ اﻟﺎ3ﻌﺔ ﻋة ﻣ: ﻋ1ﻩ ﺣ5ﻰ ﺷﻬ 3ﺄم ﻋ0ﻪ ﺳﻘ ﻫOﻩ اﻟ11ﺎﻟـ اﻟاﺣة إﺛ اﻷﺧZ ﻋﻠـﻰ ﯾـ اﻟ1ـا3P: 3cـﺎدة زwـ1ﻬ ﯾﺳـﻒ ﺑـ: ﺗﺎﺷـﻔ:، ووﻻدة دوﻟـــﺔ اﻟ1ـــا3P: ﻓـــﻲ اﻟ1ﻐـــب واﻷﻧـــﻟ، وﻗـــ اﺳـــ 5ﻌﺎد اﻟ1ـــﻠ1ن ﻋـــ: ـــ ﻫـــOﻩ :اﻟوﻟﺔ ﺷ* ﺎً ﻏ* ﻗﻠﻞ ﻣ: وﺣﺗﻬ وﻗﺗﻬ وﻫUـ5ﻬ اﻟﺎﺳـﺔ، ﻟـOﻟ ﻓﻘـ ﺳـﺎﻫ اﺑـ اﻟﻌ9ﻲ وواﻟﻩ رﺣ1ﻬ1ﺎ ﷲ 0ﻓﻲ ﺗ* أرﺎن ﺣ' ﻫOﻩ اﻟوﻟﺔ، ﻋ ﻣﺎ ﻗﺎﻣﺎ أﺛ0ـﺎء :وﺟدﻫ1ـــﺎ ﻓـــﻲ 3ﻐـــاد ﺑ5@ﻠـــe ﻣـــ: اﻷﻣـــ اﺑـــ: ﺗﺎﺷـــﻔ 3ﺈfﻬـــﺎر وﺗﻘـــW وﻻء :اﺑـــ :ﺗﺎﺷـﻔ وﺎﻋ 5ـﻪ ﻟQﻠﻔـﺔ اﻟ1ـﻠ1: آﻧــOاك" اﻟ1ـ5pﻬ 3ـﺎ±"، وﺣIـﻟﻬ1ﺎ ﻣ0ـﻪ ﻋﻠــﻰ ّﺧPـــﺎب رﺳــــ1ﻲ Wُﻘـــ ُاﻟQﻠﻔــــﺔ 31ﺟ7ـــﻪ 3ــــwﺔ إﻣـــﺎرة اﺑــــ: ﺗﺎﺷـــﻔ: ﻋﻠــــﻰ اﻟ1ﻐــــب 1 ( ،اﻧpـ، ﻣﻘﻣـﺔ ﺳــﻌ أﻋـاب ﻟ@5ـﺎب اﺑــ: اﻟﻌ9ـﻲ l اﻷﺣــﺎم اﻟ;ـﻐ ، 1 0، ﻣ ـرات اﻟ10p1ــﺔ اﻹﺳـﻼﻣﺔ ﻟﻠ59ــﺔ ،واﻟﻌﻠم واﻟnﻘﺎﻓﺔ1409 / 1989 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 33 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ واﻷﻧـﻟ، وﻓــﻲ ﻫــOا اﻟQPــﺎب ﻣـﺎ ــﻪ ﻣــ: ﺗnUــl ﺣ'ـ دوﻟــﺔ اﻟ1ــا3P:، وﻋPــﺎﺋﻬ اﻟwﺔ اﻟ@ﺎﺔ ﻟﻼﺳ51ار ﻪ، ﻣ: ﺟﻬـﺔٍ، وﺗﺛـ ﻋُـZ وَﺣَـة اﻟ1ـﻠ1: ﻓـﻲ ذﻟـ :اﻟﻌIــ، ﻣــ: ﺟﻬــﺔ أﺧــZ. وﻫ0ــﺎ WRــ أن ﻧــ* إﻟــﻰ أن ﺣﺎﻟــﺔ اﺳــ5داد ﺷــﻲءٍ ﻣــ اﻟﻘة واﻟﺣة اﻹﺳﻼﻣﺔ اﻟ5ـﻲ ﻋﻓ5ﻬـﺎ 3ـﻼد اﻟ1ﻐـب واﻷﻧـﻟ ﻓـﻲ ذﻟـ اﻟﻗـl، ﻓـﻲ اﻟ9ﻊ اﻷﺧ* ﻣ: اﻟﻘن اﻟQﺎﻣ اﻟﻬRV، ﺎﻧl ﺗﻘﺎﺑﻠﻬـﺎ ﺣﺎﻟـﺔ ﻣـ: اﻟ5ﻔـﱡق واﻟ5ـذم واﻟrـﻌﻒ ﻓــﻲ 3ـﻼد اﻟــق اﻹﺳـ ﻼﻣﻲ ﺗــl ﺣ'ـ اﻟــﻠRﻗ*:، اﻟـOﯾ: ــﺎن أﻣــاؤﻫ Qﻓﻲ ﺣب داﺋ1ﺔ ﻟﻠPة ﻋﻠﻰ دﻓﺔ اﻟ'ـ، وـﺎن ﻋﻠـﻰ اﻟ ﻠﻔـﺔ اﻟ1ﻐﻠـب ﻋﻠـﻰ أﻣـﻩ ﻓــﻲ 3ﻐــاد أنْ ﯾﻗِّــﻊ ﻟ@ــﻞ ﻏﺎﻟــ وﻣ5ﻐﻠِّــ ﻋﻠــﻰ اﻟ'ــ ﻣﺳــﻣﺎً Wﻘrــﻲ 3ﺎﻟQP7ــﺔ ﻟــﻪ 3Rﺎﻧــ اﺳــ1ﻪ ﻣــ: ﻓــق ﻣ0ــﺎﺑ 3ﻐــاد. وــﺎن ﻣــ: أﺳــأ آﺛــﺎر ﻫــOا اﻟ5ﻐﺎﻟــ واﻟ50ــﺎزع ﻟــ ا ﻠRﻗﻲ ﻋﻠــﻰ اﻟــﻠPﺔ وﺿــﻌﺎف اﻟQﻼﻓــﺔ ،أنْ اﺳــ5ﻟﻰ اﻟIــﻠUن ﻋﻠــﻰ اﻟﻘــس ) وﺣﱠﻟﻫــﺎ إﻟــﻰ إﻣــﺎرةٍ ﺻــﻠUﺔٍ ﻋــﺎم492 /ه1098م ( ﻓــﻲ أWــﺎم اﻟQﻠﻔــﺔ اﻟ1ــ5pﻬ ْ3ـﺎ±، اﻟــOV ﻟــ Wــ5Pﻊ آﻧـOاك، 3ــ ﻣــﺎ آﻟــl إﻟـﻪ أوﺿــﺎﻋﻪ وأوﺿــﺎع اﻟQﻼﻓــﺔ، أن Wــِّك ﺳــﺎﻛ0ﺎً ﻓــﻲ ﻣاﺟﻬــﺔ اﻟ1Rــﺎزر اﻟﺣــﺔ اﻟ5ــﻲ اﻗ .5ﻓﻬــﺎ اﻟIــﻠUن ﻓــﻲ اﻟﻘــس U وأﻣﺎ آﺧ ﺣََث ﺳﺎﺳﻲ ﺷﻬﻩ اﺑ: اﻟﻌ9ﻲ ﻓﻲ أواﺧ ﺣﺎﺗﻪ ﻓﻬـ ﺳـﻘ دوﻟـﺔ اﻟ1ــا3P: أWrــﺎً ﻋﻠـــﻰ ﯾــ أﻣــ اﻟ1ﺣِـــﯾ: ﻋUــ اﻟ1ـــﻣ: ﺑــ: ﻋﻠــﻲ. 1 ( ،اﻧpــVﻣﻘ+ﻣــﺔ ﻣ,ــV اﻟــ+ﯾ اﻟ1W ــ" ، اﻟ5ــﻲ 5Uﻬــﺎ ﻟ5cﻘــﻪ ﻟ@5ــﺎب اﺑــ: اﻟﻌ9ــﻲاﻟﻌJاﺻــ ﻣــ اﻟﻘJاﺻــ W"، ﺗﻘــ ،ﺧﺎﻟــ اﻟﻌـــ، دﻣـــ، دار اﻟ7ـــﺎﺋ)د.ت. ،( 31 - 54 ،. ﺳـــﻌ أﻋـــاب ﻣـــﻊ اﻟﻘﺎﺿـــﻲ اﺑـــ اﻟﻌ ـــﻲ:، ص56 - 57 . ،وﻣIPﻔﻰ اﻟ10ﻲ اﺑ اﻟﻌ ﻲ وﺗﻔ ﻩ ، 39 - 40 . 2 ( ،اﻧp، اﻟOﻫUﻲ ﺳ أﻋﻼم اﻟUﻼء ، 20 / 203 . 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، ﺗﻘ* ﻋﻠﻲ ﷴ اﻟ7RﺎوV، ﺑوت، دار ،اﻟ1ﻌﻓﺔ1987م، 1 / 8 ، 2 / 893 ، 4 / 1971 . 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 8 ، 266 . :OاﻟWﻠV اﻟZﺎﻟ ﺷ Jﺧﻪ وﻗـــ ــﺎن ﻫـــ ﺷQIــﺎً ﻋﻠــﻰ رأس وﻓــ إﺷــUﻠﺔ اﻟــOV ﺳــﺎﻓ إﻟــﻰ ﻣــاﻛ° ﻋﺎﺻــ1ﺔ اﻟ1ﺣــﯾ: ﻋــﺎم ) 542 ـﻫ ( ﻟ5ﻘW اﻟﻻء.واﻟPﺎﻋﺔ واﻟUﻌﺔ ﻷﻣ* اﻟوﻟﺔ اﻟRﯾة1 أﺳــﻠََ اﺑــ: اﻟﻌ9ــﻲ روﺣــﻪ اﻟPــﺎﻫة إﻟــﻰ 3ﺎرﺋﻬــﺎ أﺛ0ــﺎء وﺟــدﻩ ﻓــﻲ اﻟ1ﻐــب ﻟ5ﺄدWﺔ اﻟUﻌﺔ ﻷﻣ* اﻟ1ﺣﯾ:، ﻓﻲ ﻣ0Pﻘﺔ ﻗ7ﺔ ﻣ: ﻓﺎس ﺗُﻋﻰ "ﻣﻐﻠـﺔ"، ﻓـﻲ ﺳـ0ﺔ ) 543 ه(، وﺣُ1ﻞ إﻟﻰ ﻓﺎس ﺣ دُﻓـ: ﻫ0ـﺎك، رﺣ1ـﻪ ﷲ :، وﺟـvاﻩ ﻋـ اﻹﺳـﻼم واﻟﻌﻠ ﺧ* اﻟRvاء2. ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 34 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 8 ، 266 . 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، ﺗﻘ* ﻋﻠﻲ ﷴ اﻟ7RﺎوV، ﺑوت، دار ،اﻟ1ﻌﻓﺔ1987م، 1 / 8 ، 2 / 893 ، 4 / 1971 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ ﻗUﻞ اﻟQـض ﻓـﻲ 3ﻌـ2 ﻗrـﺎWﺎ اﻟ1ـ0ﻬﺞ ﻋ0ـ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﺗﻔـﻩ" أﺣ'ـﺎم اﻟﻘآن"، ﻧZ ﻣ: اﻟrورV اﻹﺷﺎرة اﻟﻌﺎﻣﺔ إ .ﻟﻰ أﺳﻠ9ﻪ وﻘ5ﻪ ﻓﻲ ﺗﻔﻩ 1 ( ﺗ0ﺎول ﻓﻲ ﺗﻔﻩ ﺎﻣﻞ ﺳر اﻟﻘآن اﻟ@ وﻓـ ﺗﺗـ اﻟ1Iـﻒ اﻟﻌn1ـﺎﻧﻲ ﻣـﺎ ،ﻋـــا 3rـــﻊ ﺳـــر ﻓﻘـــu )اﻟﻘ1ـــ، اﻟﺎﻗـــﺔ، اﻟ0ﺎزﻋـــﺎت، اﻟ5@ـــ، اﻻﻧﻔPـــﺎر، اﻟﻘﺎرﻋـــﺔ Zاﻟﻬ1vة، اﻟ@ﺎﻓون(، وﻟ@0ﻪ ﺎن Wﻘ5I ﻓﻲ ﺗﻔ ﻞ ﺳرة ﻋﻠﻰ اﻵWﺎت اﻟ5ـﻲ ﯾـ .أﻧﻬﺎ ﺗ0PV ﻋﻠﻰ أﺣ'ﺎم ﺷwﺔ 2 ( ﺎن، ﻓﻲ ﺣﺎﻻت :ﻗﻠﻠﺔ، ﻗUﻞ أن Wع ﺑ5ﻔ ﺳرة ﻣﺎ، ﯾO ﻣﻘﻣﺔ ﻋﺎﻣﺔ ﻋ اﻟـــرة ﯾ50ـــﺎول ﻓﻬـــﺎ ﻋﻠـــﻰ ﺳـــUﻞ اﻟ1nـــﺎل: ﻣ'ﺎﻧ5ﻬـــﺎ وﻓrـــﻠﻬﺎ، واﺳـــ1ﻬﺎ أو أﺳـــ1ﺎﺋﻬﺎ وﻣﻌﺎﻧﻲ ﻫOﻩ اﻷﺳ1ﺎء، وأﺳ7ﺎب اﻟ51ﺔ ﺑﻬﺎ، وﺗﺎرﺦ ﻧvول اﻟرة وﻣ'ﺎﻧ ﻬﺎ1. 3 ( ﯾUأ ﺑ5ﯾ ﻋد آWﺎت اﻷﺣ'ـﺎم اﻟ5ـﻲ ﯾ0ـV أن WﻘIـ ﺗﻔـﻩ ﻋﻠ ﻬـ ﺎ ﻓﻘـu. ﻓﻘـ ذ ﻋﻠﻰ ﺳUﻞ اﻟ1nﺎل أنﱠ ﺳرة اﻟ7ﻘة ﺗr ﺗﻌ: آWﺔ ﻣ: آWﺎت اﻷﺣ'ـﺎم، وﺳـرة uاﻟ59ﺔ ﺗr ﺳ5ﺎً وﻋ: آWﺔ ﻓﻘ2. و90ﺎء ﻋﻠﻰ ﻫOا اﻟ5ﯾـ ﻓـﺈنّ ﻣR1ـع ﻣـﺎ ﻓﱠ ـ ﻩ ﻣ: اﻟﻘآن اﻟ@ ﻟ ﯾ5Rﺎوز .uأر9ﻌﺎً وﺛﻼﺛ: وﺳ51ﺎﺋﺔ آWﺔ ﻓﻘ 4 ( ،3ﻌــ أن Wــد ﻋــد اﻵWــﺎت اﻟ5ــﻲ Wﻘــِّر ﺗﻔــﻫﺎ، ﯾUــأ، وﻓــ اﻟ1ــ0ﻬﺞ اﻟ5ﻠﻠــﻲ :3ﺎﻵWﺎت واﺣةً إﺛ أﺧZ، ﺣ ﯾUأ 3ﻌض اﻟ1ﺎﺋﻞ اﻟ5ﻲ W1'ـ: اﺳـ507ﺎﻬﺎ ﻣـ :ﻛﻞ آWﺔ، ﻣﻘِّ 1ﺎً ﻞ واﺣة ﻣ0ﻬﺎ إﻟﻰ ﻣﺎﺋﻞ، Wﻌﺎﻟﺞ ﻓﻲ ﻞ ﻣﺄﻟﺔٍ ﻣ0ﻬـﺎ ﻗrـﺔ ﻣـ ،اﻟﻘrﺎWﺎ اﻟ5ﻲ ﺗ50ﺎوﻟﻬﺎ اﻵWـﺔ اﻟ1روﺳـﺔ ﻣnـﻞ : ،ﺳـU ﻧvوﻟﻬـﺎ، وأوﺟـﻪ اﻟﻘـاءات ﻓﻬـﺎ وﻣﻌﺎﻧﻬـــﺎ اﻟﻠﻐـــﺔ واﻟ7ﻼ²ـــﺔ، واﻵﺛـــﺎر اﻟـــاردة ﻓﻬـــﺎ، واﻟ1ـــﺎﺋﻞ اﻟﻔﻘﻬـــﺔ واﻷﺻـــﻟﺔ ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 35 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ اﻟ15ﻔﺎدة ﻣ0ﻬﺎ، وﻗ Wﻌـِّج إﻟـﻰ ﻣـﺎﺋﻞ ﺗ5ﻌﻠـ 3ﺎﻟﻌﻘـة واﻟ5Iـف، واﻟ5ـﺎرﺦ، وواﻗـﻊ ﻋIﻩ ﻋ0 وﺟد أدﻧﻰ إﺷﺎرة إﻟﻬﺎ ﻓﻲ ﺳﺎق اﻵWﺔ اﻟ5ﻲ Wﻔِّ ﻫﺎ1 . 5 ( ،ﻛــﺎن Wُ@nــ ﻣــ: اﻹﺷــﺎرة إﻟــﻰ 57ــﻪ اﻟــﺎ3ﻘﺔ، ﻣnــﻞ: ﺗﻔــﻩ اﻟ@Uــ أﻧــار اﻟﻔRــ ،واﻟ0ﺎﺳﺦ واﻟ10خ، واﻟ1Iل ﻓﻲ أﺻل اﻟﻔﻘﻪ، واﻹﻧIﺎف ﻓـﻲ ﻣـﺎﺋﻞ اﻟQـﻼف ¤واﻟ1ـــ'ﻠ:، وﻣﻠR ـــﺔ اﻟ15ﻔﻘﻬـــ: إﻟـــﻰ ﻣﻌﻓـــﺔ ﻏـــاﻣ2 اﻟ0ـــ:؛ وﺣﺎﻟـــﺔ اﻟﻘـــﺎر ﻟﻠ5ﺳﻊ واﻻﺳ5vادة ﻣ0ﻬﺎ ﻓـﻲ nـ ﻣـ: اﻟ1ـﺎﺋﻞ اﻟ5ـﻲ ﺗﻌـﱠض ﻟﻬـﺎ ﻓـﻲ ﺗﻔـﻩ 3ـ'ﻞ vﻣﺟ2. 2 ( ،اﻧp ﻋﻠﻰ ﺳUﻞ اﻟ1nﺎل، اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 8 ، 32،35 ، 36 ، 37 ، 77 ، 79 - 80 ؛2 / 533 ، 561 ، 899 . 1 ( اﻧp ﻋﻠﻰ ﺳUﻞ،اﻟ1nﺎل، اﺑ: اﻟﻌ9ﻲ ،nاﻟ;+ر اﻟﺎﺑ 2 / 776 - 781 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ ون ﺎن ﻟﻬOﻩ اﻹﺷﺎرة واﻹﺣﺎﻟﺔ ﻣ: دﻻﻟﺔ، ﻓﺈﻧﻬ ﺎ ﺗل ﻋﻠﻰ أن اﺑـ: اﻟﻌ9ـﻲ ﻗـــ ﺻـــ0ﱠﻒ ﺗﻔـــﻩ ﻫـــOا ﻓـــﻲ ﻓ5ـــة اﺳـــ5@1ﺎﻟﻪ ﻷدواﺗـــﻪ اﻟﻌﻠ1ـــﺔ، واﻛ51ـــﺎل ﺧUﺗـــﻪ ﻓـــﻲ ﻣــان اﻟ5ــﺄﻟe، وﻫـــ اﻷﻣــ اﻟـــOV ﻧﻠ1ــﻪ 3'ــﻞ وﺿـــح وﺟــﻼء ﻓـــﻲ ــﻞ ﺻـــﻔﺎت .ﺗﻔﻩ و Rــر ﺑ0ــﺎ ﻗ Uــﻞ ﺗ0ــﺎول 3ﻌــ2 ﻣﻌــﺎﻟ اﻟ1ــ0ﻬﺞ ﻋ0ــ اﺑــ: اﻟﻌ9ــﻲ أن ﻧــ إﻟــﻰ أﻧﻪ ﻗ ﻧّ 3'ﻞ ﻋﺎم ﻓﻲ ﻣﻘﻣﺔ ﺗﻔﻩ ﻋﻠـﻰ اﻟﻌ0ﺎﺻـ اﻟ10ﻬRـﺔ اﻷﺳﺎﺳـﺔ اﻟ5ـﻲ اﻋ51ــﻫﺎ ﻓــﻲ ﻋ1ﻠــﻪ، واﻟ5ــﻲ ﺗ51nــﻞ ﺑــ ـ: ﺗﻠــﻞ اﻟ@ﻠ1ــﺎت اﻟ1ﻔــدة وﺗﻘــ ﻣﻌﺎﻧﻬــﺎ، ﺛــ ﺗﻠﻞ اﻟ5اﻛ وﺗﯾ ﻣﻌﺎﻧﻬﺎ، واﻟﻌ0ﺎWﺔ ﺑﺟﻩ اﻟ7ﻼﻏﺔ ﻟﻠ0p اﻟﻘآﻧﻲ، ﺛ اﺳ507ﺎ اﻷﺣ'ـــﺎم اﻟـــwﺔ ﻣـــﻊ اﻟﻔـــﺎ³ ﻋﻠـــﻰ اﻻﻧـــRﺎم واﻻﺗـــﺎق ﺑـــ: اﻟ1ﻌـــﺎﻧﻲ واﻷﺣ'ـــﺎم ،اﻟ1507Pﺔ، ﺛ ذ ﻣﺎ ﺟﺎء ﻓﻲ اﻟﻘآن اﻟ@ واﻟ0ﺔ اﻟIﺔ 1ﺎ ﯾ5ﻌﻠ 3ﺎﻵWﺔ ﺛـــ إﯾـــاد 3ﻌـــ2 اﻟﻘrـــﺎWﺎ واﻟ1ـــﺎﺋﻞ اﻟ5ـــﻲ ﺗnﻫـــﺎ اﻵWـــﺔ ﻣـــﻊ ﻋ0ﺎWـــﺔ ﺧﺎﺻـــﺔ 3ﺎﻟ5ـــﺎرﺦ وﻣﺎﻫاﺗﻪ وأﺣـ اث ﻋIـﻩ. Wﻘـل اﺑـ: اﻟﻌ9ـﻲ: ".. ﻓ0ـO اﻵWـﺔ، ﺛـ ﻧﻌPـﻒ ﻋﻠـﻰ ،ﻛﻠ1ﺎﺗﻬـــﺎ ﺑـــﻞ ﺣوﻓﻬـــﺎ، ﻓ0ﺄﺧـــO 31ﻌﻓ5ﻬـــﺎ ﻣﻔـــدة، ﺛـــ ﻧUﻬـــﺎ ﻋﻠـــﻰ أﺧاﺗﻬـــﺎ ﻣrـــﺎﻓﺔ ،وﻧﻔــª ﻓــﻲ ذﻟــ ﻗــ اﻟ7ﻼﻏــﺔ، وﻧ5ــﱠز ﻋــ: اﻟ10ﺎﻗrــﺔ ﻓــﻲ اﻷﺣ'ــﺎم واﻟ1ﻌﺎرﺿــﺔ 3 وﻧ5ــﺎ ﻋﻠــﻰ ﺟﺎﻧــ اﻟﻠﻐــﺔ، وﻧﻘﺎﺑﻠﻬــﺎ ]أV اﻵWــﺔ اﻟ@1ــﺔ[ ﻓــﻲ اﻟﻘــآن 1ــﺎ ﺟــﺎء ﻓــﻲ اﻟــ0ﺔ اﻟIــﺔ، وﻧ5ــZ وﺟــﻪ اﻟR1ــﻊ، إذ اﻟ@ــﻞ ﻣــ: ﻋ0ــ ﷲ، وﻧ1ــﺎ 3ُﻌــ ﷴ ـ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 36 ﺻﻠﻰ ﷲ ﻋﻠﻪ وﺳﻠ ـ ّﻟUـ: ﻟﻠ0ـﺎس ﻣـﺎ ﻧـvِّل إﻟـﻬ، وﻧﻌﻘِّـ ﻋﻠـﻰ ذﻟـ ﺑ5ا3ـﻊ ﻻ ﺑـ "ﻣ: ﺗIﻞ اﻟﻌﻠ ﺑﻬﺎ ﻣ0ﻬﺎ1. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 1 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ ﻧﻼﺣـــª ﻣـــ: ﻫـــOا اﻟـــ0ِّ أن اﺑـــ:ﱠ اﻟﻌ9ـــﻲ ﯾـــﻟﻲ اﻟﻠﻐـــﺔ ﻣﻔـــداتٍ وﺗاﻛـــَ ووﺟ ًﻫــــﺎ 3ﻼ²ـــﺔ ﻣ'ﺎﻧـــﺔ اﻟIـــارة ﻓـــﻲ ﻣ0ﻬRـــﻪ، ﺛـــ ﯾ0Pﻠـــ 3ﻌـــ ذﻟـــ إﻟـــﻰ اﺳـــ507ﺎ اﻷﺣ'ـــﺎم اﻟـــwﺔ ﻣﻌ51ـــاً 3ـــ'ﻞ رﺋـــ ﻋﻠـــﻰ اﻟﻘـــآن اﻟ@ـــ واﻟـــ0ﺔ اﻟـــﻔﺔ ﻣـــﻊ اﻻﻫ51ـــﺎم 23ﻌIــــﻩ وﻣـــــﺎﻛﻠﻪ.وﻓﻲ 3n0ـــــﺎ ﻫــــOا ﺳ0ﻘ5Iـــــ ﻓﻘـــــu ﻋﻠــــﻰ دراﺳـــــﺔ 3ﻌـــــ اﻟ1ﻌـــــﺎﻟ اﻟ10ﻬRﺔ اﻟ1Oرة أﻋﻼﻩ، وﻓ اﻟ1Pﺎﻟ :اﻟ5ﺎﻟﺔ Wاﻟ :ﻠV اﻷول اﻻﺳ4ﻔﺎدة ا ﻟﻘ+Lﺔ ﻣ ﺗ اث اﻟﻔ - ﻣ ﻗ?ﻠﻪ Wاﻟ :ﻠV اﻷول اﻻﺳ4ﻔﺎدة ا ﻟﻘ+Lﺔ ﻣ ﺗ اث اﻟﻔ - ﻣ ﻗ?ﻠﻪ اﻋ51ـ اﺑــ: اﻟﻌ9ــﻲ ﻓــﻲ ﺗﻔــﻩ ﻋﻠــﻰ ﺟﻬــد 3ﻌــ2 اﻟ1ﻔــ: ﻣــ: ﻗUﻠــﻪ، ﺷــﺄﻧﻪ ،ﻓﻲ ذﻟ ﺷﺄن ﻞ ﻣﻔِّ ﻟﻠﻘآن اﻟ@، وذﻟ أﻣ ﺿـورV ﻣـ: اﻟ0ﺎﺣـﺔ اﻟ10ﻬRـﺔ ﻓﻼ W1': أن ﯾUأ أVﱡ ﻣﻔِّ ﻣ: ﻧﻘPﺔ اﻟIﻔ، ﻓ@ﻞ 5ﺎب ﻓﻲ اﻟ5ﻔ ﻫ 3Iـرة :أو 3ﺄﺧZ ﺣIﻠﺔ ﻣﺎ ﺳ7ﻘﻪ ﻣ: ﺟﻬـد ﻓـﻲ ﺣﻘـﻞ اﻟ5ﻔـ، 3ﺎﻹﺿـﺎﻓﺔ إﻟـﻰ ﻣـﺎ W1'ـ أن Wُrﻔﻪ ﺻﺎﺣ ﻫOا اﻟ5ﻔـ* إﻟـﻰ ﻫـOﻩ اﻟRﻬـد، وﻫـOا أﻣـ Wﻌـد إﻟـﻰ ﻣـZ ﻋﻠـ وﺛﻘﺎﻓــﺔ ﻫــOا اﻟ1ﻔِّ ــ وﻣــZ ﺗ1ﱡــv واﺳــ5ﻘﻼل ﺷQIــ5ﻪ اﻟﻌﻠ1ــﺔ. وﻫــOا ﻣــﺎ ﺣــ ث ﻣــﻊ اﺑ: اﻟﻌ9ﻲ، ﻓﻘ ﻧﻘﻞ وﺳRﱠﻞ 3ﺄﻣﺎﻧﺔ ﻋﻠ1ﺔ آراء 23ﻌ ،ﻋﻠ1ﺎء اﻟ5ﻔ* اﻟOﯾ: ﺳ7ﻘﻩ وﺗﻌﺎﻣــﻞ ﻣﻌﻬــﺎ ﻓــﻲ اﻟﻗــl ﻧﻔــﻪ ﺗﻌــﺎﻣﻼً ﻧﻘــWﺎً ﻋ0ــ اﻟﺎﺟــﺔ. أﺷــﺎر اﺑــ: اﻟﻌ9ــﻲ إﻟــﻰ ﻋ1ﻠــﻪ ﻫــOا ﻓــﻲ ﻣﻘﻣــﺔ ﺗﻔــﻩ أﺣ'ــﺎم اﻟﻘــآن ﻋ0ــﻣﺎ ﻗــﺎل: ".. وﻟ1ّــﺎ ﻣــ:ﱠ ﷲ ﺳــ7ﺎﻧﻪ 3ﺎﻻﺳ57Iـﺎر ﻓــﻲ اﺳــ5nﺎرة اﻟﻌﻠــ م ﻣــ: اﻟ@5ــﺎب اﻟﻌvـv ﺣــ ﻣــﺎ ﻣﻬﱠﺗــﻪ ﻟ0ــﺎ اﻟ1ــQﺔ ،اﻟـــOﯾ: ﻟﻘ0ـــﺎ، ﻧpﻧﺎﻫـــﺎ ﻣـــ: ذﻟـــ اﻟ1Pـــح، ﺛـــ ﻋﺿـــ0ﺎﻫﺎ ﻋﻠـــﻰ ﻣـــﺎ ﺟﻠ7ـــﻪ اﻟﻌﻠ1ـــﺎء وﺳUﻧﺎﻫﺎ 3qﺎر اﻷﺷﺎخ، ﻓ1ﺎ اﺗﻔـ ﻋﻠـﻪ اﻟ0pـ أﺛU50ـﺎﻩ، وﻣـﺎ ﺗﻌـﺎرض ـﻪ ﺷـRﻧﺎﻩ "]أ3ﻌﻧﺎﻩ[، وﺷOﻧﺎﻩ ﺣ5ﻰ ﺧﻠ ﻧُrﺎرﻩ، وورق ﻋارﻩ2 . وﻓ ﻫOﻩ اﻻﺳ5ﻔﺎدة اﻟ0ﻘWـﺔ رأﯾ0ﺎﻩ ﯾ5ﻌﺎﻣﻞ ﻣﻊ آراء ﺷﺦ اﻟ1ﻔ: اﻟPUV )ت310 ه(، واﻟﻘﺎﺿﻲ أﺑـﻲ إﺳـ إﺳــــــ1ﺎﻋﻞ اﻟRﻬrــــــ1ﻲ اﻟ1ــــــﺎﻟ@ﻲ )ت282 ه(، وــــــOﻟ اﻷﻣــــــ ﻣــــــﻊ اﻟRIــــــﺎص 2ا ا،ﺑ: اﻟﻌ9ﻲ أﺣﻜﺎم اﻟﻘﺮآن،1/1 ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 37 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ اﻟ0ﻔــــﻲ)ت370 ه(، واﻟ@ــــﺎ اﻟﻬاﺳــــﻲ اﻟــــﺎﻓﻌﻲ )ت504 ه( اﻟﻠــــOﯾ: ﺳــــ7ﻘﺎﻩ ﻓــــﻲ اﻟ5ــﺄﻟe ﻓــﻲ ﻣــان أﺣ'ــﺎم اﻟﻘــآن وﻓــ أﺻــل ﻣــOﻫUﻬ1ﺎ1. 1 ( ،اﻧp ﻋﻠﻰ ﺳUﻞ اﻟ1nﺎل اﻟ;+ر،nاﻟﺎﺑ 1 / 394 ، 397 - 398 ، 469 ؛3 / 1466 . 1 ( ،اﻧp ﻋﻠﻰ ﺳUﻞ اﻟ1nﺎل اﻟ;+ر،nاﻟﺎﺑ 1 / 394 ، 397 - 398 ، 469 ؛3 / 1466 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 1 / 1 . ( ،اﻧp ﻋﻠﻰ ﺳUﻞ اﻟ1nﺎل اﻟ;+ر،nاﻟﺎﺑ 1 / 394 ، 397 - 398 ، 469 ؛3 / 1466 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 1 / 1 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ وﻟ0rــب ﻣnــ ﺎﻻً ﻋﻠــﻰ ذﻟ، ﻧO ﻣﻗﻔﻪ ﻣ: اﻟPUV اﻟـOV ـﺎن W5ﻣـﻪ، وـﻗﻩ ﺗـﻗاً ﻋﻠ1ـﺎً ﻗﺎﺋ1ـﺎً ﻋﻠـﻰ إﻗارﻩ ﻟﻪ 3ﺎﻹﻣﺎﻣﺔ، ورﺳخ اﻟﻘم ﻓﻲ ﻣان اﻟ5ﻔ، ﻓﻬـﺎ ﻫـ Wﻘـل ﻋ0ـﻪ ﻓـﻲ ﻣﻘﻣـﺔ nﻛ5ﺎ3ﻪ: "اﻟPUV ﺷﺦ اﻟﯾ:، ﻓRﺎء ﻪ ]WﻘI ﻓﻲ اﻟ5ﻔ[ 3ﺎﻟﻌR اﻟﻌُRﺎب، وﻧ 5ــﻪ أﻟ7ــﺎب اﻷﻟ7ــﺎب، وﻓــ ﺢ ــﻪ ﻟ@ــﻞِّ ﻣــ:ْ ﺟــﺎء 3ﻌــﻩ إﻟــﻰ ﻣﻌﺎرﻓــﻪ اﻟ7ــﺎبَ ، ﻓ@ــﻞﱡ أﺣــ "ﻏـف ﻣ0ـﻪ ﻋﻠـﻰ ﻗـْر إﻧﺎﺋـﻪ، وﻣـﺎ ﻧﻘIــl ﻗPـةٌ ﻣـ: ﻣﺎﺋـﻪ2 :. و9ﺎﻟﻔﻌـﻞ ﻓﻘـ أﻛnـَ اﺑــ :اﻟﻌ9ــﻲ ﻣــ: اﻟ0ﻘــﻞ واﻻﻏ5ــاف ﻣــ: 3ــ اﻟPUــV ﻓــﻲ ﻣQ5ﻠــﻒ ﻣRــﺎﻻت اﻟ5ﻔــ* ﻣــ أﺳـــ7ﺎب ﻧـــvولٍ ، وﻗـــاءاتٍ ، وأﺧ7ـــﺎرٍ وآﺛـــﺎرٍ ، إﻟـــﻰ ﺗcﻘـــﺎت ﻟﻐـــﺔ وﻧـــﺔ ٍ، إﻟـــﻰ آراء وأﺣ'ــﺎمٍ ﻓﻘﻬــﺔ..، وﻟ@0ــﻪ ﻟــ W'ــ: W'5ﻔــﻲ 3ﺎﻟ0ﻘــﻞ اﻟــﻗ* واﻷﻣــ: ﻋ0ــﻪ، وﻣاﻓﻘ5ــﻪ ﻓــﻲ ﻣﻌpــ اﻷﺣــﺎن ؛ ًﺑــﻞ ــﺎن ﻓــﻲ 3ﻌــ2 اﻷﺣــﺎن ﯾ0ﺎﻗــﻪ، و05ﻘــﻩ ﻧﻘــاً ﻋﻠ1ــﺎً ﺻــﺎرﻣﺎ .ﻛﺎن WIﻞ 3ﻪ أﺣﺎﻧﺎ إﻟﻰ درﺟﺔ ﻏ ﻣﻘUﻟﺔ ﻣـــ: اﻷﻣnﻠـــﺔ ﻋﻠـــﻰ ﻫـــOا اﻟ0ﻘـــﺎش اﻟﻌﻠ1ـــﻲ اﻟ11ـــvوج 3ﺎﻟـــة واﻟ5ﻘـــﻊ، ردﱡ اﺑـــ: اﻟﻌ9ـــﻲ :ﻟ5ﻔــ* اﻟPUــV ﻟﻬRــ اﻟ0ــﺎء ﻓــﻲ ﻗﻟــﻪ ﺗﻌــﺎﻟﻰ ِوَا<ْﺠُــﺮُو<ُﻦ# ; ِــ: اﻟْﻤَﻀَ ــﺎﺟِﻊ ]اﻟ0ــﺎء، 34 [، ﻋ0ﻣﺎ اﺧ5ﺎر أن Wﻔﻩ 3ﺄن "ﯾُ9P: 3ﺎﻟﻬِRَﺎر، وﻫ اﻟَUْـﻞ ﻓـﻲ اﻟUـت، وﻫـﻲ اﻟ1ــاد 3ﺎﻟ1rــﺎﺟﻊ، إذ ﻟــ ﻟ@ﻠ1ــﺔ "اﻫRــوﻫ:" إﻻ أﺣــ ﺛﻼﺛــﺔ ﻣﻌــﺎن. ﻓــﻼ WIــﺢ أن W'ــن ﻣــ: اﻟﻬَRْــ اﻟــOV ﻫــ اﻟﻬــOWﺎن، ﻓــﺈن اﻟ1ــأة ﻻ ﺗُــاوZ ﺑــOﻟ، وﻻ ﻣــ: اﻟﻬُRْــ اﻟـــــOV ﻫـــــ ﻣـــــ5ﻔ° ﻣـــــ: اﻟﻘـــــل، ﻷن ﷲ ﻻ Wـــــﺄﻣ 3ـــــﻪ، ﻓﻠـــــ ﻟـــــﻪ وﺟـــــﻪ إﻻ أن ﺗ9Pﻫ: 3ﺎﻟﻬِRَﺎر." وﻫ0ﺎ ردﱠ ﻋﻠﻪ اﺑ: اﻟﻌ9ﻲ 3ﻘﻟﻪ: "Wﺎ ﻟﻬﺎ ﻫﻔة ﻣ: ﻋﺎﻟٍ 3ﺎﻟﻘآن واﻟــ0ﺔ، وﻧــﻲ ﻷَﻋRــ7' ﻣــ: ذﻟــ، إن اﻟــOV أﺟــأﻩ ﻋﻠــﻰ ﻫــOا اﻟ5ﺄوــﻞ، وﻟــ ﯾُــد أن lWIِّح 3ﺄﻧﻪ أﺧOﻩ ﻣ0ﻪ، ﻫ ﺣﯾٌ ﻏ رواﻩ اﺑ: وﻫ ﻋـ: ﻣﺎﻟـ أن أﺳـ1ﺎء ﺑ0ـ :أﺑﻲ 3' اﻟIﯾ اﻣأة اﻟv9 ﺑ: اﻟﻌام، ﺎﻧl ﺗQج ﺣ5ﻰ ﻋُﺗ ﻓـﻲ ذﻟـ. ﻗـﺎل ،وﻋ5 ﻋﻠﻬﺎ وﻋﻠـﻰ ﺿَ ـّﺗﻬﺎ ،ًﻓﻌﻘـ ﺷـﻌ واﺣـة 3ـﺎﻷﺧZ، وﺿـ9ﻬ1ﺎ ﺿـ9ﺎ ﺷـﯾا ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 38 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن ،وﺎﻧl اﻟrة أﺣ: اﺗﻘﺎء، وﺎﻧl أﺳ1ﺎء ﻻ ﺗ5ﻘﻲ، ﻓ@ﺎن اﻟrب ﺑﻬـﺎ أﻛnـ وآﺛـ ،ﻓ'5ﻪ إﻟﻰ أﺑﻬـﺎ أﺑـﻲ 3'ـ، ﻓﻘـﺎل ﻟﻬـﺎ: أV ﺑُ0ﱠـﺔ اﺻـUV، ﻓـﺈن اﻟv9ـ رﺟـﻞ ﺻـﺎﻟﺢ 1وﻟﻌﻠﻪ أن W'ن زوﺟَـ ﻓـﻲ اﻟR0ـﺔ. وﻟﻘـ ﺑﻠﻐ0ـﻲ أن اﻟﺟـﻞ إذا اﺑ5@ـ 3ـﺎﻟ أة ﺗvوﱠﺟﻬـﺎ ،ﻓــﻲ اﻟR0ــﺔ. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن، 1 / 418 - 419 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ ﻓــأZ ]أV اﻟPUــV[ اﻟــ9u واﻟﻌﻘــ ﻣــﻊ اﺣ51ــﺎل اﻟﻠﻔــª ﻣــﻊ ﻓِﻌْــﻞ اﻟv9ــ ،ﻓﺄﻗم ﻋﻠﻰ ﻫـOا اﻟ5ﻔـ* ﻟـOﻟ. وﻋR7ـﺎً ﻟـﻪ ﻣـﻊ ﺗ7ﱡـﻩ ﻓـﻲ اﻟﻌﻠـم، وﻓـﻲ ﻟﻐـﺔ اﻟﻌـب ﻛe 3ﻌُـ ﻋﻠـﻪ ﺻـاب اﻟﻘـل، وﺣـﺎد ﻋـ: ﺳـاد اﻟ0pـ، ﻓﻠـ W'ـ: ﺑـﱞ، واﻟﺎﻟـﺔ ﻫـOﻩ ِrﻣ: اﻻﺟ5ﻬﺎد اﻟ1ُﻔ َﺔ 3ﺎﻟ@ﻬﺎ إﻟﻰ اﻟاد، ﻓ0pﻧـﺎ ﻓـﻲ ﻣـارد )ه ﺟـ( ﻓـﻲ :ﻟــﺎن اﻟﻌــب ﻋﻠــﻰ ﻫــOا اﻟ0pــﺎم، ﻓﺟــﻧﺎﻫﺎ ﺳــ7ﻌﺔ: ﺿــ اﻟﺻــﻞ، ﻣــﺎ ﻻ ﯾ07ﻐــﻲ ﻣــ اﻟﻘــل، ﻣRﺎﻧ7ــﺔ اﻟــﻲء، وﻣ0ــﻪ اﻟﻬRــة، ﻫــOWﺎن اﻟ1ــ2، اﻧ5Iــﺎف اﻟ0ﻬــﺎر، اﻟــﺎب pاﻟ:، اﻟUْﻞ اﻟOV Wُﱡ ﻓﻲ ﺣﻘ اﻟ7ﻌ* ﺛ Wُﱡ ﻓﻲ أﺣ رُﺳْ µﻪ. وﻧ ﻧﺎ ﻓﻲ ﻫOﻩ اﻟ1ارد، ﻓﺄﻟﻔ0ﺎﻫﺎ ﺗور ﻋﻠﻰ ﺣف واﺣـ، وﻫـ اﻟ7ُﻌـ ﻋـ: اﻟـﻲء، ﻓـﺎﻟﻬR ﻗـ 3َﻌُـ ﻋ: اﻟﺻﻞ اﻟOV ﯾ07ﻐﻲ ﻣـ: اﻷُﻟﻔـﺔ وﺟ1ـﻞ اﻟIـ7ﺔ، وﻣـﺎ ﻻ ﯾ07ﻐـﻲ ﻣـ: اﻟﻘـل ﻗـ 3ﻌُـ ﻋــ: اﻟIـاب، وﻣRﺎﻧ7ــﺔ اﻟـﻲء 3ُﻌْــٌ ﻣ0ـﻪ وأﺧــO ﻓـﻲ ﺟﺎﻧــ آﺧـ ﻋ0ــﻪ، وﻫــOWﺎن ،اﻟ12 ﻗ 3ﻌُ ﻋ: ﻧpﺎم اﻟ@ﻼم :واﻧ5Iﺎف اﻟ0ﻬﺎر ﻗ 3ﻌُ ﻋ: ﻪ اﻟ11دﯾ ﻓـﻲ اﻋ5ـال اﻟﻬـاء وﻣ'ـﺎن اﻟ5Iـف، واﻟــﺎب اﻟـ: ﻗـ 3ﻌُـ ﻋـ: اﻟﻌـﺎب، واﻟUــﻞ :اﻟــOV Wُــﱡ 3ــﻪ اﻟ7ﻌــ ﻗــ أ3ﻌــﻩ ﻋــ: اﺳ5ﺳــﺎﻟﻪ ﻓــﻲ ﺗIــﻓﻪ واﺳ5ﺳــﺎل ﻣــﺎ رُ9ــu ﻋــ :ﺗﻘﻠﻘﻠـــﻪ وﺗــــﻪ. وذا ﺛUـــl ﻫــــOا وـــﺎن ﻣﺟــــﻊ اﻟR1ـــﻊ إﻟــــﻰ اﻟ7ُﻌـــ، ﻓ1ﻌ0ــــﻰ اﻵWــــﺔ 3أ ﻌــوﻫ: ﻓــﻲ اﻟ1rــﺎﺟﻊ. وﻻ Wُ5ــﺎج إﻟــﻰ ﻫــOا اﻟ5@ﻠﱡــﻒ اﻟــOV ذــﻩ اﻟﻌــﺎﻟِ، وﻫــ ﻻ "!Vﯾ07ﻐــﻲ ﻟ1nــﻞ اﻟــV واﻟ@ﻠUــﻲ ﻓ@ــe أن WQ5ــﺎرﻩ اﻟPUــ1 0ﻧﻼﺣــª ﻣــ: ﻫــOا اﻟــ اﻟPﻞ e أن ﻫ7ﺔ اﻟPUـV وﻣ'ﺎﻧ5ـﻪ اﻟﻌﻠ1ـﺔ اﻟﻌـﺔ اﻟ5ـﻲ Wﻘـﱡ ﺑﻬـﺎ اﺑـ: اﻟﻌ9ـﻲ ﻟــــ ﺗ10ﻌــــﻪ ﻣــــ: ﻧﻘــــﻩ واﻟــــد ﻋﻠــــﻪ ﻣــــ5Qﻣﺎً ﻣــــ0ﻬ ﺞ اﻟ5ﻠــــﻞ اﻟﻠﻐــــV اﻟــــﻗ ﻟ1ــــﺎدة )ﻫR(، ﻟ@: ﻣﻊ ﺟ0ﺣﻪ ﻓﻲ ردﻩ ﻋﻠﻪ إﻟﻰ أﺳﻠب اﻟ5ﻘﻊ ﻏ* اﻟﻼﺋ. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن، 1 / 418 - 419 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 39 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ :اﻟWﻠV اﻟZﺎﻧﻲ ﻣﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻟﻠﻐﺔ ﻻﺣp0ﺎ 1ﺎ ﻗﺎﻟﻪ اﺑ: اﻟﻌ9ﻲ ﻓﻲ ﻣﻘﻣﺔ ﺗﻔﻩ أنﱠ ﻋ0ﺎﯾ5ﻪ 3ﺎﻟﻠﻐﺔ ﺗ'ِّﻞ ﺣR اﻟvاوﺔ ﻓﻲ ﻋ1ﻠﻪ اﻟ5ﻔV. وﻗ ﺎن ﺗﻌﺎﻣﻠﻪ ﻣﻊ ﻟﻐﺔ 5اﻟﻘآن ﺗﻌﺎﻣﻼ ﯾ 3ﺎﻟ1ل ﻣﻊ إWRﺎز واﺧ5Iﺎر ﻓﻲ أﻏﻠ اﻟﺎﻻت. 2 ( ،اﺑ: اﻟﻌ9ﻲ اnﻟ;+ر اﻟﺎﺑ ، 1 / 10 . 1 ( ،ﺳرة اﻹﺳاء100 . 1 ( ،ﺳرة اﻹﺳاء100 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ ﻗـــﺎل اﺑـــ: اﻟﻌ9ـــﻲ ﻓـــﻲ ﺗﻔﻩ ﻟﻘﻟﻪ :ﺗﻌﺎﻟﻰ  َوَاﺗ#ﺒَﻌُـﻮاْ ﻣَـﺎ ﺗَﺘْﻠُـﻮاْ اﻟﺸ# ـﻴَﺎﻃِZYُ ﻋَWَـ ﻣُﻠْـﻚِ ﺳُـﻠَVَْن: "اﺧ5ﻠـﻒ اﻟ0ـﺎس ﻓـﻲ .ﺣف )ﻣﺎ(: ﻓ10ﻬ ﻣ: ﻗﺎل: إﻧﻪ ﻧﻔﻲٌ، وﻣ0ﻬ ﻣ: ﻗﺎل: إﻧﻪ ﻣﻔﻌل، وﻫ اﻟIـﺢ وﻻ و ﺟﻪ ﻟﻘل ﻣـ: Wﻘـل: إﻧـﻪ ﻧﻔـﻲٌ، ﻻ ﻓـﻲ ﻧpـﺎم اﻟ@ـﻼم وﻻ ﻓـﻲ ﺻـﺔ اﻟ1ﻌ0ـﻰ، وﻻ ﯾ5ﻌﻠ ﻣ: ﻧﻪ ﻣﻔﻌﻻً ﺳﺎقُ اﻟ@ﻼم 31ُﺎل ﻋﻘﻼً، وﻻ W150ﻊ ﺷﻋﺎً، وﺗﻘـ ﯾﻩ : واﺗ7ـﻊ اﻟﻬد ﻣﺎ ﺗﻠ5ﻪ اﻟﺎ:ُ ﻣ: اﻟ ﻋﻠﻰ ﻣُﻠ ﺳﻠ1ﺎن أV ﻧـU5ﻪ إﻟـﻪ وأﺧUـت 3ـﻪ :ﻋ0ــﻪ، ﻘﻟــﻪ ﺗﻌــﺎﻟﻰو َْﻣَــﺎ أَرْﺳَــﻠ :ﻨَﺎ ﻣِــﻦ ﻗَﺒْﻠِــﻚَ ﻣِــﻦ ر#ﺳُــﻮلٍ وَﻻَ ﻧَdِــ`ٍّ إِﻻ# إِذَا ﺗَﻤَa#ــ` أَﻟْ_َــ اﻟﺸ# ــﻴْﻄَﺎنُ ; ِــ ِUأُﻣْﻨِf#ﺘِــ ِّ7، أV إذا ﺗــﻼ أﻟﻘــﻰ اﻟــPﺎن ﻓــﻲ ﺗﻼوﺗــﻪ ﻣــﺎ ﻟــ ﯾُﻠﻘــﻪ اﻟ0Uــﻲﱞ، Wﺎﻛــﻪ وﻠــ "ﻋﻠﻰ اﻟﺎﻣﻌ: 3ﻪ ﺣ71ﺎ ﺑّ0ﺎﻩ2. 3 ( :ﻣ ﺣ O اﻟUﻼﻏﺔ ﻻ ﯾﻗﻰ اﻫ51ﺎم اﺑ: اﻟﻌ9ﻲ ﺑ5ﻠﻞ وﺟﻩ 3ﻼﻏﺔ اﻟﻘـآن إﻟـﻰ اﻫ51ﺎﻣﻪ ﺑ5ﻠﻞ ﻣﻔدات اﻟﻘآن وﺗاﻛ7ـﻪ، وﻟ@00ـﺎ ﻋﻠـﻰ اﻟـﻏ ﻣـ: ذﻟـ ﻧRـ أﻧـﻪ ﻓـﻲ 3ﻌــ2 اﻟــﺎﻻت Wــ5Qم اﻟ5ﻠــﻞ اﻟ7ﻼﻏــﻲ ﻟ5ﻘــ ﻣﻌ0ــﻰ ﻣــﺎ. ﻓﻬــﺎ ﻫــ Wــ إﻟــﻰ ﻓIﺎﺣﺔ 3ﻌـ2 اﻟ5اﻛـ واﻵWـﺎت اﻟﻘآﻧـﺔ 3ـ'ﻞ ﺳـﻊ وﺧـﺎﻒ ﻟIـﻞ إ ﻟـﻰ ﺗﻘـ اﻟ1ﻌ0ــﻰ اﻟ1ــاد، وــ5ﻌ: ﻋﻠــﻰ ذﻟــ–أWrــﺎ- 3ﺎﻟ0pــﺎﺋ اﻟﻘآﻧــﺔ. ﻣــ: اﻷﻣnﻠــﺔ ﻋﻠــﻰ ذﻟ ﻗﻟﻪ ﻓﻲ ﺗﻔ* ﻗﻟﻪ ﺗﻌﺎﻟﻰ:  .. َ..ﻓَﻌِﱠةٌ ﻣِّ:ْ أَWﱠـﺎمٍ أُﺧَـ ]اﻟ7ﻘـة، 184 [ : "ﻗـﺎل Oﻋﻠ1ﺎؤﻧﺎ: ﻫ ا اﻟﻘل ﻣ: ﻟPe اﻟﻔIﺎﺣﺔ، ﻷن ﺗﻘـ ،ﯾﻩ: ﻓـﺄﻓP ﻓﻌـة ﻣـ: أWـﺎم أﺧـ ﻛ1ﺎ ﻗﺎل ﺗﻌﺎﻟﻰ :  ٌﻓَﻤَﻦ iَﺎنَ ﻣِﻨﻜُﻢ ﻣ#ﺮِhﻀﺎً أَوْ ﺑِUِ أَذًى ﻣ4ﻦ ر#أْﺳِUِ ﻓَﻔِﺪْﻳَﺔ "، ﺗﻘﯾﻩ ﻓَﻠَ ﻓﻔWﺔ3 اﻟ1ﻘــــس، وﻫــــ اﻷﻗــــ ZZ، ﻟﻘﻟــــﻪ ﺗﻌــــﺎﻟﻰ ﻓــــﻲ اﻵWــــﺔ اﻷﺧــــ  َﻓَﻘَﻌُــــﻮاْ ﻟَــــUُ ﺳَــــﺎﺟِﺪِﻳﻦ ،R]اﻟ29 [ ،p، وﻟ W': ﻋﻠﻰ ﻣﻌ0ﻰ اﻟ5ﻌ وﻧ1ﺎ ﺻر ﻋﻠﻰ و ﺟﻪ اﻹﻟـvام ﻟﻠq7ـﺎدة واﺗQﺎذﻩ ﻗUﻠﺔ، "وﻗ ﻧﺦ ﷲ ﺟ1ﻊ ذﻟ ﻓﻲ ﻫOﻩ اﻟ1ﻠﺔ1 . وnاً ﻣﺎ ـﺎن Wﻌ51ـ ﻋﻠـﻰ اﻹﻋاب واﻟﺎق وﻧp اﻟ@ﻼم واﻟ0pﺎﺋ اﻟﻘآﻧﺔ ﻟ دﻻﻟﺔ ﻣﻌ0ﻰ ﺗ ﻣﺎ 3ﻌـ ﻣ0ﺎﻗﺔ ﻟﻼﺣ51ﺎﻻت اﻷﺧZ، ﺛـ Wﻘـم ﺑ5ﻘـ ﻣﻌ0ـﻰ اﻟR1ﻠـﺔ اﻟﻘآﻧـﺔ اﻟ5ـﻲ و رد ﻓﻬـﺎ ﻫـــOا اﻟ5ـــ ﻓـــﻲ ﺿـــء اﻟ1ﻌ0ـــﻰ اﻟـــاﺟﺢ اﻟـــOV وﺻـــﻞ إﻟـــﻪ. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 16 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 28 . 3 ( ،اﺑ: اﻟﻌ9ﻲ أ ﺣﺎم اﻟﻘ آن ، 1 / 78 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ وﻧﻘI 3ﺎﻟ1ل ﺗ0ﺎوﻟَﻪ ﻟﻠﻐﺔ 0اﻟﻘآن اﻟ@ 3'ﻞ ﻣ5ﺎﺗﻬﺎ اﻟ1ﻔدات واﻟ5اﻛ* واﻟ0p واﻟ7ﻼﻏﺔ، وﻟ@: ﺑ .ﻣ5ﻔﺎوﺗﺔ 1 ( :ﻣ ﺣ O اﻟﻔ دات ﻛـﺎن Wﻘـم ﻓـﻲ اﻷﻏﻠـ ﺑ5ﻠـﻞ اﻟ@ﻠ1ـﺎت اﻟ1ﻔـدة وﺗﻘـ ًﻣﻌﺎﻧﻬﺎ وﺿﻌﺎ واﺳ5ﻌ1ﺎﻻً ﻋ0 اﻟﻌب، وﻗ Wﻌِّج ﻋﻠﻰ اﺳ5ﻌ1ﺎل اﻟﻘآن ﻟﻬﺎ، وﻘـم ﻓــﻲ أﺛ0ــﺎء ذﻟــ ﺑ5ﯾــ اﻟ1ﻌ0ــﻰ اﻟ1ــاد ﻣ0ﻬــﺎ ﻓــﻲ اﻵWــﺔ اﻟ5ــﻲ وردت ﻓﻬــﺎ، ــﻞ ذﻟــ 3ﺈWRــﺎز وﺗ@nــe ﯾــﻋ إﻟــﻰ اﻹﻋRــﺎب 31ﻘــرة اﺑــ: اﻟﻌ9ــﻲ ﻋﻠــﻰ اﻹﺣﺎــﺔ 31ﻌ0ــﻰ اﻟ1ﻔدة 3'ﻠ1ﺎت ﻗﻠﻠﺔ ﺎﺔ وواﺔ. ﻣ: اﻷﻣnﻠﺔ ﻋﻠﻰ ذﻟـ ﺗﯾـُﻩ ﻣﻌ0ـﻰ اﻟ0ﻔﻘـﺔ ﻓـﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ:  ِوَﻣِﻤ#ﺎ رَAَﻗْﻨَﺎ<ُﻢْ ﻳُﻨﻔ َﻘُﻮن ]اﻟ7ﻘة، 3[ : "اﻟ1ﺄﻟﺔ اﻷو ﻟـﻰ ،ﻓـﻲ اﺷـ5ﻘﺎق اﻟ0ﻔﻘـﺔ وﻫــــﻲ w7ــــﺎرة ﻋــــ: اﻹﺗــــﻼف وﻟ5ــــﺄﻟe )ﻧَﻔَــــ( ﻓــــﻲ ﻟــــﺎن اﻟﻌــــب ﻣﻌــــﺎنٍ ، أﺻــــﻬﺎ :اﻹﺗــﻼف، وﻫــ اﻟ1ــاد ﻫــﺎ ﻫ0ــﺎ، Wُﻘــﺎل: ﻧَﻔِــ اﻟــvادُ ﯾ0ﻔــ إذا ﻓ0ِــﻲ، وأﻧﻔﻘــﻪ ﺻــﺎﺣ7ﻪ أﻓ0ﺎﻩ، وأﻧﻔ اﻟﻘم: ﻓ0ﻲ زادﻫ، وﻣ0ﻪ ﻗﻟﻪ ﺗﻌﺎﻟﻰ ِإِذاً ﻷ# َﻣْﺴَﻜْﺘُﻢْ ﺧَﺸْﻴَﺔَ اﻹِDﻔَﺎق 1" 2. 2 ( ﻣ ﺣ O اﻟ4 اﻛ V اﻟﻠﻐJ-ﺔ وﻧr اﻟ5ـﻼم: ،ﻷن اﻟ@ﻠ1ـﺔ اﻟ1ﻔـدة ﻻ ﺗُﻘIـ ﻟـOاﺗﻬﺎ وﻻ ﺗلّ ﻋﻠﻰ اﻟ1ﻌ0ﻰ ﺑOاﺗﻬﺎ، ﺑﻞ ﻣ: ﺧﻼل إﺿـﺎﻓ5ﻬﺎ ﻟﻐﻫـﺎ وﺗUﻬـﺎ وﻧp1ﻬـﺎ ﻣﻌـﻪ ،3Pﻘﺔ ﺗﺎﻣﺔ ﻣﻔة ﻟ1ﻌ0ﻰ ﻣد، ﻟOﻟ اﻋ50ﻰ اﺑ: اﻟﻌ9ﻲ ﺑUـﺎن وﺗﻠـﻞ اﻟ5اﻛـ وﻗـــ ﺗ5ﻌـــد اﻟ1ﻌـــﺎﻧﻲ ﻘـــم ﺑـــOﻫﺎ ﺛـــ ﯾﻠRـــﺄ إﻟـــﻰ اﻟ0pـــﺎﺋ اﻟﻘآﻧـــﺔ ﻟ5ﯾـــ اﻟ1ﻌ0ـــﻰ :اﻟاﺟﺢ ﺑ0ﻬﺎ. ﻣ: ذﻟ ﺗﯾﻩ ﻟ1ﻌ0ﻰ ﺗ )اﺳRوا ﻵدم( ﻓـﻲ ﻗﻟـﻪ ﺗﻌـﺎﻟﻰ ْوَإِذ َِﻜَـــﺔِ اﺳْـــﺠُﺪُواْ ﻵدَمَ ﻓَﺴَ ـــﺠَﺪُواْ إِﻻ# إِﺑْﻠِـــﻴﺲÒَﻗُﻠْﻨَـــﺎ ﻟِﻠْﻤَﻼ ] ،اﻟ7ﻘـــة 34 [: "اﺗﻔﻘـــl اﻷﻣـــﺔ ﻋﻠـــﻰ أن اﻟـــRد ﻵدم ﻟـــ W'ـــ: ﺳـــRد w7ـــﺎدة، وﻧ1ـــﺎ ـــﺎن ﻋﻠـــﻰ أﺣـــ وﺟﻬـــ:: إﻣـــﺎ ﺳـــﻼم lاﻷﻋـــﺎﺟ 3ـــﺎﻟ5@ﻔِّﻲ واﻻﻧ0ـــﺎء واﻟ5ﻌpـــ، وﻣـــﺎ وَﺿْ ـــﻌﻪ ﻗِUْﻠـــﺔ ﺎﻟـــRد ﻟﻠ@q7ـــﺔ و9ـــ 2 ( ،اﺑ: اﻟﻌ9ﻲ اnﻟ;+ر اﻟﺎﺑ ، 1 / 10 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 40 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن اﻟ1ﻘــــس، وﻫــــ اﻷﻗــــ ZZ، ﻟﻘﻟــــﻪ ﺗﻌــــﺎﻟﻰ ﻓــــﻲ اﻵWــــﺔ اﻷﺧــــ  َﻓَﻘَﻌُــــﻮاْ ﻟَــــUُ ﺳَــــﺎﺟِﺪِﻳﻦ ،R]اﻟ29 [ ،p، وﻟ W': ﻋﻠﻰ ﻣﻌ0ﻰ اﻟ5ﻌ وﻧ1ﺎ ﺻر ﻋﻠﻰ و ﺟﻪ اﻹﻟـvام ﻟﻠq7ـﺎدة واﺗQﺎذﻩ ﻗUﻠﺔ، "وﻗ ﻧﺦ ﷲ ﺟ1ﻊ ذﻟ ﻓﻲ ﻫOﻩ اﻟ1ﻠﺔ1 . وnاً ﻣﺎ ـﺎن Wﻌ51ـ ﻋﻠـﻰ اﻹﻋاب واﻟﺎق وﻧp اﻟ@ﻼم واﻟ0pﺎﺋ اﻟﻘآﻧﺔ ﻟ دﻻﻟﺔ ﻣﻌ0ﻰ ﺗ ﻣﺎ 3ﻌـ ﻣ0ﺎﻗﺔ ﻟﻼﺣ51ﺎﻻت اﻷﺧZ، ﺛـ Wﻘـم ﺑ5ﻘـ ﻣﻌ0ـﻰ اﻟR1ﻠـﺔ اﻟﻘآﻧـﺔ اﻟ5ـﻲ و رد ﻓﻬـﺎ ﻫـــOا اﻟ5ـــ ﻓـــﻲ ﺿـــء اﻟ1ﻌ0ـــﻰ اﻟـــاﺟﺢ اﻟـــOV وﺻـــﻞ إﻟـــﻪ. :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ ﻗـــﺎل اﺑـــ: اﻟﻌ9ـــﻲ ﻓـــﻲ ﺗﻔﻩ ﻟﻘﻟﻪ :ﺗﻌﺎﻟﻰ  َوَاﺗ#ﺒَﻌُـﻮاْ ﻣَـﺎ ﺗَﺘْﻠُـﻮاْ اﻟﺸ# ـﻴَﺎﻃِZYُ ﻋَWَـ ﻣُﻠْـﻚِ ﺳُـﻠَVَْن: "اﺧ5ﻠـﻒ اﻟ0ـﺎس ﻓـﻲ .ﺣف )ﻣﺎ(: ﻓ10ﻬ ﻣ: ﻗﺎل: إﻧﻪ ﻧﻔﻲٌ، وﻣ0ﻬ ﻣ: ﻗﺎل: إﻧﻪ ﻣﻔﻌل، وﻫ اﻟIـﺢ وﻻ و ﺟﻪ ﻟﻘل ﻣـ: Wﻘـل: إﻧـﻪ ﻧﻔـﻲٌ، ﻻ ﻓـﻲ ﻧpـﺎم اﻟ@ـﻼم وﻻ ﻓـﻲ ﺻـﺔ اﻟ1ﻌ0ـﻰ، وﻻ ﯾ5ﻌﻠ ﻣ: ﻧﻪ ﻣﻔﻌﻻً ﺳﺎقُ اﻟ@ﻼم 31ُﺎل ﻋﻘﻼً، وﻻ W150ﻊ ﺷﻋﺎً، وﺗﻘـ ﯾﻩ : واﺗ7ـﻊ اﻟﻬد ﻣﺎ ﺗﻠ5ﻪ اﻟﺎ:ُ ﻣ: اﻟ ﻋﻠﻰ ﻣُﻠ ﺳﻠ1ﺎن أV ﻧـU5ﻪ إﻟـﻪ وأﺧUـت 3ـﻪ :ﻋ0ــﻪ، ﻘﻟــﻪ ﺗﻌــﺎﻟﻰو َْﻣَــﺎ أَرْﺳَــﻠ :ﻨَﺎ ﻣِــﻦ ﻗَﺒْﻠِــﻚَ ﻣِــﻦ ر#ﺳُــﻮلٍ وَﻻَ ﻧَdِــ`ٍّ إِﻻ# إِذَا ﺗَﻤَa#ــ` أَﻟْ_َــ اﻟﺸ# ــﻴْﻄَﺎنُ ; ِــ ِUأُﻣْﻨِf#ﺘِــ ِّ7، أV إذا ﺗــﻼ أﻟﻘــﻰ اﻟــPﺎن ﻓــﻲ ﺗﻼوﺗــﻪ ﻣــﺎ ﻟــ ﯾُﻠﻘــﻪ اﻟ0Uــﻲﱞ، Wﺎﻛــﻪ وﻠــ "ﻋﻠﻰ اﻟﺎﻣﻌ: 3ﻪ ﺣ71ﺎ ﺑّ0ﺎﻩ2. 3 ( :ﻣ ﺣ O اﻟUﻼﻏﺔ ﻻ ﯾﻗﻰ اﻫ51ﺎم اﺑ: اﻟﻌ9ﻲ ﺑ5ﻠﻞ وﺟﻩ 3ﻼﻏﺔ اﻟﻘـآن إﻟـﻰ اﻫ51ﺎﻣﻪ ﺑ5ﻠﻞ ﻣﻔدات اﻟﻘآن وﺗاﻛ7ـﻪ، وﻟ@00ـﺎ ﻋﻠـﻰ اﻟـﻏ ﻣـ: ذﻟـ ﻧRـ أﻧـﻪ ﻓـﻲ 3ﻌــ2 اﻟــﺎﻻت Wــ5Qم اﻟ5ﻠــﻞ اﻟ7ﻼﻏــﻲ ﻟ5ﻘــ ﻣﻌ0ــﻰ ﻣــﺎ. ﻓﻬــﺎ ﻫــ Wــ إﻟــﻰ ﻓIﺎﺣﺔ 3ﻌـ2 اﻟ5اﻛـ واﻵWـﺎت اﻟﻘآﻧـﺔ 3ـ'ﻞ ﺳـﻊ وﺧـﺎﻒ ﻟIـﻞ إ ﻟـﻰ ﺗﻘـ اﻟ1ﻌ0ــﻰ اﻟ1ــاد، وــ5ﻌ: ﻋﻠــﻰ ذﻟــ–أWrــﺎ- 3ﺎﻟ0pــﺎﺋ اﻟﻘآﻧــﺔ. ﻣــ: اﻷﻣnﻠــﺔ ﻋﻠــﻰ ذﻟ ﻗﻟﻪ ﻓﻲ ﺗﻔ* ﻗﻟﻪ ﺗﻌﺎﻟﻰ:  .. َ..ﻓَﻌِﱠةٌ ﻣِّ:ْ أَWﱠـﺎمٍ أُﺧَـ ]اﻟ7ﻘـة، 184 [ : "ﻗـﺎل Oﻋﻠ1ﺎؤﻧﺎ: ﻫ ا اﻟﻘل ﻣ: ﻟPe اﻟﻔIﺎﺣﺔ، ﻷن ﺗﻘـ ،ﯾﻩ: ﻓـﺄﻓP ﻓﻌـة ﻣـ: أWـﺎم أﺧـ ﻛ1ﺎ ﻗﺎل ﺗﻌﺎﻟﻰ :  ٌﻓَﻤَﻦ iَﺎنَ ﻣِﻨﻜُﻢ ﻣ#ﺮِhﻀﺎً أَوْ ﺑِUِ أَذًى ﻣ4ﻦ ر#أْﺳِUِ ﻓَﻔِﺪْﻳَﺔ "، ﺗﻘﯾﻩ ﻓَﻠَ ﻓﻔWﺔ3 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 28 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 41 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ وــﺎن Wــ5ﻌ: 3ﺎﻟ7ﻼﻏــﺔ ﻓــﻲ 3ﻌــ2 اﻟــﺎﻻت ﻟ5ــﺟﺢ ﻣﻌ0ــﻰ ﻣــ: ﺑــ: ﻋــة ﻣﻌــﺎن ﻣ51ﻠــﺔ ﻟ@ﻠ1ــﺔ أو ﺗــ ﻗآﻧــﻲ وذﻟــ 3ـــﺎﻟ0p إﻟــﻰ أن اﻟﻘــآن اﻟ@ــ ﻧــvل ﺑﻠـــﺎن ﻋ9ﻲ ﻣU: وأﻧﻪ ﺟﺎر ﻋﻠﻰ أﺳﺎﻟ* اﻟﻌب ﻓﻲ اﻟﻔIﺎﺣﺔ واﻟUﺎن1 . ﻣ: اﻷﻣnﻠﺔ ﻋﻠﻰ ذﻟ ﺗﻔﻘُﻪ ﺑ: ﻣﻌ0ﻰ اﻟ7ﺎﻏﻲ واﻟﻌـﺎدV، وﺗﯾـ دﻻﻟـﺔ ـﻞ ﻣ0ﻬ1ـﺎ ﺑﻗﺔ ﻓﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ :  ِإِﻧ#ﻤَﺎ nَ #مَ ﻋَﻠَﻴْﻜُﻢُ اﻟْﻤَfْﺘَـﺔَ وَاﻟـﺪ#مَ وَﻟَﺤْـﻢَ اﻟْﺨِﻨﺰِﻳـﺮِ وَﻣَـﺎ أُ<ِـﻞ# ﺑِـUِ ﻟِﻐَZْـjِ اﻟﻠّـUِ ﻓَﻤَـﻦ َاﺿْ ــpُ# ﻏ ٍZْــjَ ﺑَــﺎغٍ وَﻻَ ﻋَــﺎد ]اﻟ7ﻘــة، 173 [ ، eﺧــﺔَ اﻟﻗــع ﻓــﻲ اﻟ5@ــار اﻟــOV ﻻ Wrــ @ﻣﻌ0ﻰ، وﻫOا أﻣ ﻻ ﯾﻠ 3ﻔIﺎﺣﺔ اﻟﻘآن اﻟ . 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 4 / 1935 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 57 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ أَوْ ﺻَ ِWﻘ ]اﻟ0ر، 61 [ : nﻣَ:ْ ﻟﻲ 31: ﯾ ــــــــــ اﻟﻔادُ ﺑُدِّﻩ وذا ﺗﺣﱠ ـــــــــــ ﻞ ﻟ ﯾvغْ ﻋ: ﻋﻬﻩ Wﺎ ﺑس ﻧﻔﻲ ﻣِ:ْ أخٍ ﻟﻲ 3ﺎذل ﺣُْ :َ اﻟﻓ ـ ـــــــــــــ ﺎء 3ِﻘُْ9ﻪ ﻻ 3ُﻌِْﻩ ﯾُﻟﻲ اﻟIﻔﺎء ﺑ0ُPْﻘِﻪ ﻻ ﺧُﻠ ــــــ ﻘـــــ ﻪ وُسﱡ ﺻ ــــــ ﺎ3ﺎً ﻓﻲ ﺣﻼوة ﺷﻬْﻩ ﻓَﻠﺎﻧﻪ ﯾُUV ﺟ ــــــــــــــــــ ْاﻫ ﻋِﻘ ــ ﻩ وﺟَ0َﺎﻧ ـــــــــــــــــ ﻪ ﺗﻐﻠﻲ ﻣاﺟِﻞُ ﺣِﻘْﻩ ُﻻ ﻫُﱠ إﻧﻲ ﻻ أ ـــــــــــــــ ً ﻓاﺳﺔ 3 أﺳ5ﻌO ﻣ: اﻟد وَِْﻩ1 :OاﻟWﻠV اﻟZﺎﻟ ﻣﻬIﻪ ﻓﻲ اﻟ 4ﻌﺎﻣﻞ ﻣﻊ ﻣ;ﺎدر اﻟ4ﻔ !ﺎﻟﺄﺛJر ،ﻻ ﺷﱠ أن اﻟ5ﻔـ* 3ﺎﻟ1ـﺄﺛر ﻫـ أﻓrـﻞ وأﻗـم ﻣ0ـﺎﻫﺞ اﻟ5ﻔـ ﻷﻧـﻪ إﻣـﺎ أن W'ن ﺗﻔاً ﻟﻠﻘـآن اﻟ@ـ 3'ـﻼم ﷲ–ﺗﻌـﺎﻟﻰ- ﻧﻔـﻪ، ¦ﷲ–ﺗﻌـﺎﻟﻰ- .أﻋﻠـ 31ـادﻩ وﻣــﺎ أن W'ــن ﺗﻔــاً ﻟــﻪ 3'ــﻼم رﺳــل ﷲ- ﺻــﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ- :ِّوﻫــ اﻟ1Uــ ،اﻟــﺎرح ﻟ@ــﻼم ﷲ ﺗﻌــﺎﻟﻰ. وﻣــﺎ أن W'ــن 3ــﺄﻗال اﻟIــﺎ3ﺔ اﻟــOﯾ: ﺷــﺎﻫوا اﻟ50vــﻞ وﻫ أﻫﻞ اﻟﻠﺎن ا :ﻟOV ﻧـvل 3ـﻪ اﻟﻘـآن اﻟ@ـ، وﺗ1ـvوا ﻋـ: ﻏـﻫ 31ـﺎ ﺷـﺎﻫوﻩ ﻣـ اﻟﻘاﺋ: واﻷﺣال ﺣ: ﻧـvول اﻟـﺣﻲ. وﻣـﺎ 3ـﺄﻗال اﻟ5ـﺎ3ﻌ: ﺗﻼﻣـOة اﻟIـﺎ3ﺔ، وﻧَﻘَﻠـﺔ .ﻋﻠﻣﻬ2 وﻓﻲ اﻟcﻘﺔ ﻓﺈنّ اﻟ7ﺎﺣ ﻓﻲ ﺗﻔ اﺑ: اﻟﻌ9ﻲ WR اﻟ5vاﻣﺎً واﺿﺎً ﻣ0ﻪ 3ﺎﻻﻋ51ﺎد ﻋﻠـــﻰ ﻣIـــﺎدر اﻟ5ﻔـــ* 3ﺎﻟ1ـــﺄﺛر: اﻟﻘـــآن اﻟ@ـــ، واﻟـــ0ﺔ اﻟ0Uـــﺔ اﻟـــﻔﺔ، وأﻗـــال .:اﻟIﺎ3ﺔ واﻟ5ﺎ3ﻌ :OاﻟWﻠV اﻟZﺎﻟ ﻣﻬIﻪ ﻓﻲ اﻟ 4ﻌﺎﻣﻞ ﻣﻊ ﻣ;ﺎدر اﻟ4ﻔ !ﺎﻟﺄﺛJر :OاﻟWﻠV اﻟZﺎﻟ ﻣﻬIﻪ ﻓﻲ اﻟ 4ﻌﺎﻣﻞ ﻣﻊ ﻣ;ﺎدر اﻟ4ﻔ !ﺎﻟﺄﺛJر ،ﻻ ﺷﱠ أن اﻟ5ﻔـ* 3ﺎﻟ1ـﺄﺛر ﻫـ أﻓrـﻞ وأﻗـم ﻣ0ـﺎﻫﺞ اﻟ5ﻔـ ﻷﻧـﻪ إﻣـﺎ أن W'ن ﺗﻔاً ﻟﻠﻘـآن اﻟ@ـ 3'ـﻼم ﷲ–ﺗﻌـﺎﻟﻰ- ﻧﻔـﻪ، ¦ﷲ–ﺗﻌـﺎﻟﻰ- .أﻋﻠـ 31ـادﻩ وﻣــﺎ أن W'ــن ﺗﻔــاً ﻟــﻪ 3'ــﻼم رﺳــل ﷲ- ﺻــﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ- :ِّوﻫــ اﻟ1Uــ ،اﻟــﺎرح ﻟ@ــﻼم ﷲ ﺗﻌــﺎﻟﻰ. وﻣــﺎ أن W'ــن 3ــﺄﻗال اﻟIــﺎ3ﺔ اﻟــOﯾ: ﺷــﺎﻫوا اﻟ50vــﻞ وﻫ أﻫﻞ اﻟﻠﺎن ا :ﻟOV ﻧـvل 3ـﻪ اﻟﻘـآن اﻟ@ـ، وﺗ1ـvوا ﻋـ: ﻏـﻫ 31ـﺎ ﺷـﺎﻫوﻩ ﻣـ اﻟﻘاﺋ: واﻷﺣال ﺣ: ﻧـvول اﻟـﺣﻲ. وﻣـﺎ 3ـﺄﻗال اﻟ5ـﺎ3ﻌ: ﺗﻼﻣـOة اﻟIـﺎ3ﺔ، وﻧَﻘَﻠـﺔ ﻋﻠﻣﻬ2 وﻓﻲ اﻟcﻘﺔ ﻓﺈنّ اﻟ7ﺎﺣ ﻓﻲ ﺗﻔ اﺑ: اﻟﻌ9ﻲ WR اﻟ5vاﻣﺎً واﺿﺎً ﻣ0ﻪ 3ﺎﻻﻋ51ﺎد ﻋﻠـــﻰ ﻣIـــﺎدر اﻟ5ﻔـــ* 3ﺎﻟ1ـــﺄﺛر: اﻟﻘـــآن اﻟ@ـــ، واﻟـــ0ﺔ اﻟ0Uـــﺔ اﻟـــﻔﺔ، وأﻗـــال .:اﻟIﺎ3ﺔ واﻟ5ﺎ3ﻌ Zإنّ ﻣUــأ ﺗﻔــ اﻟﻘــآن 3ــﺎﻟﻘآن واﻟــ0ﺔ ﻫــ ﻣــ: اﻟﻌ0ﺎﺻــ اﻟ10ﻬRــﺔ اﻟ@Uــ اﻟ5ﻲ ﻻ ﯾ0ﻔﱡ أﺣـﻫﺎ ﻋـ: اﻵﺧـ ﻓـﻲ ﻋ1ﻠـﺔ ﺗﻔـ* اﺑـ: اﻟﻌ9ـﻲ ﻟﻠﻘـآن اﻟ@ـ، وﻗـ أﺷﺎر إﻟﻰ ﻫـOا اﻟ1Uـأ اﻟ5ـﺄوﻠﻲ اﻟ1ـvV وأﻓIـﺢ ﻋ0ـﻪ ﻓـﻲ ﻣﻘﻣـﺔ ﺗﻔـﻩ 1ـﺎ أﺷـﻧﺎ إﻟﻰ ذﻟ ﻣ: ﻗUﻞُ. :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ Wﻘــل اﺑــ: اﻟﻌ9ــﻲ : "ﻗﻟــﻪ ﺗﻌــﺎﻟﻰ:  ٍﻏَZْــjَ ﺑَــﺎغٍ وَﻻَ ﻋَــﺎد ــﻪ :أﻗــال nــة ﻧQU5ﻬــﺎ اﺛ0ــﺎن اﻷول : أن اﻟ7ــﺎﻏﻲ ﻓــﻲ اﻟﻠﻐــﺔ ﻫــ اﻟPﺎﻟــ ﻟQــ ــﺎن أو ﻟــ، إﻻ أﻧــﻪ ﺧُــّ ﻫ0ــﺎ 3Pﺎﻟ اﻟ، وﻣ: ﺎﻟ اﻟ اﻟQﺎرجُ ﻋﻠﻰ اﻹﻣﺎم اﻟ1ﻔﺎرقُ ﻟﻠR1ﺎﻋـﺔ، وﻫـ اﻟ1ـاد 3ﻘﻟﻪ ﺗﻌﺎﻟﻰ : ﻓَِن َﻐَﺖْ إِﺣْﺪَا<ُﻤَﺎ ﻋَWَ اﻷْ ُsْ َى واﻟﻌﺎدV، وﻫ اﻟ1Rﺎوز ﻣﺎ WRز إﻟـﻰ ﻣـﺎ ﻻ WRـــز، وﺧُـــّ ﻫ0ـــﺎ 3ﻘـــﺎﻊ اﻟـــUﻞ، وﻗـــ ﻗﺎﻟـــﻪ ﻣRﺎﻫـــ واﺑـــ: ﺟُUـــ. اﻟnـــﺎﻧﻲ: أن اﻟ7ــﺎﻏﻲ آﻛــﻞ اﻟ15ــﺔ ﻓــق اﻟﺎﺟــﺔ، واﻟﻌــﺎدV آﻛﻠﻬــﺎ ﻣــﻊ وﺟــد ﻏﻫــ ﺎ ، ﻗﺎﻟــﻪ ﺟ1ﺎﻋــﺔ ﻣ0ﻬ ﻗ5ﺎدة واﻟ: وﻋ'ﻣﺔ. وﺗﻘ اﻟﻘل ﻓﻲ ذﻟ أنﱠ اﻟﻌﺎدV 3ﺎغٍ، ﻓﻠ1ﺎ أﻓد ﷲ ًﺗﻌﺎﻟﻰ ﻞ واﺣ ﻣ0ﻬ1ﺎ 3ﺎﻟO ﺗﻌّ: ﻟﻪ ﻣﻌ0ﻰً ﻏ ﻣﻌ0ﻰ اﻵﺧـ، ﻟـ ﻼ W'ـن ﺗ@ـارا WQــج ﻋــ: اﻟﻔIــﺎﺣﺔ اﻟاﺟ7ــﺔ ﻟﻠﻘــآن. واﻷﺻــﺢ واﻟﺎﻟــﺔ ﻫــOﻩ أن ﻣ ٍﻌ0ــﺎﻩ ﻏــ ﺎﻟــ ﺷـاً وﻻ ﻣ5Rـﺎوز ّﺣـ اً، ﻓﺄﻣـﺎ ﻗﻟـﻪ : )ﻏـ ﺎﻟـ ﺷـاً( ﻓـﺧﻞ ﺗ5ـﻪ ـﻞ ﺧـﺎرج ﻋﻠـﻰ اﻹﻣـــﺎم وﻗـــﺎﻊ ﻟﻠPـــ وﻣـــﺎ ﻓـــﻲ ﻣﻌ0ـــﺎﻩ. وأ ﻣـــﺎ )ﻏـــ ﻣ5Rـــﺎوز ﺣـــاً( ﻓ1ﻌ0ـــﺎﻩ ﻏـــ "ﻣ5Rﺎوز ﺣﱠ اﻟrورة إﻟﻰ ﺣ اﻻﺧ5ﺎر2 . 4 ( : اﻟ9ــﻌ وﻓــﻲ ﻫــOا اﻟــﺎق ﻻ ﺑــّ أن ﻧــ إﻟــﻰ أن اﺑــ: اﻟﻌ9ــﻲ ــﺎن W5ــﻬ 3ﺎﻟﻌ وOﻩ ﻠ1ﺎ ﺳ0l ﻟﻪ ﻓﺻﺔ إﻟﻰ ذﻟ، إﻣﺎ ﻣ: أﺟﻞ ﺗﺿـﺢ ﻣﻌ0ـﻰ ﻠ1ـﺔ ﻣ: اﻟ0ﺎﺣـﺔ اﻟﻠﻐـﺔ، وﻣـﺎ ﻣـ: أﺟـﻞ أن ﯾvـ ﻣﻌ0ـﻰً أﺷـﺎرت إﻟـﻪ اﻵWـﺔ اﻟ5ـﻲ Wﻔِّ ـﻫﺎ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 2 42 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن وﺿـﺣﺎً وﻗـةً. ﻣـ: ﻣnـﻞ ﻣــﺎ ذـﻩ ﻣـ: أﺑـﺎت ﺷـﻌﺔ ﻧp1ﻬــﺎ ﺑ 0ﻔـﻪ Wـ' ﻓﻬـﺎ ﻧــرة :اﻟIﯾ اﻟcﻘﻲ، ﻋ0 ﺗﻔﻩ ﻟﻘﻟﻪ ﺗﻌﺎﻟﻰ .. ُْ@ِ.. أَوْ ﺻَ ِWﻘ ]اﻟ0ر، 61 [ : nﻣَ:ْ ﻟﻲ 31: ﯾ ــــــــــ اﻟﻔادُ ﺑُدِّﻩ وذا ﺗﺣﱠ ـــــــــــ ﻞ ﻟ ﯾvغْ ﻋ: ﻋﻬﻩ Wﺎ ﺑس ﻧﻔﻲ ﻣِ:ْ أخٍ ﻟﻲ 3ﺎذل ﺣُْ :َ اﻟﻓ ـ ـــــــــــــ ﺎء 3ِﻘُْ9ﻪ ﻻ 3ُﻌِْﻩ ﯾُﻟﻲ اﻟIﻔﺎء ﺑ0ُPْﻘِﻪ ﻻ ﺧُﻠ ــــــ ﻘـــــ ﻪ وُسﱡ ﺻ ــــــ ﺎ3ﺎً ﻓﻲ ﺣﻼوة ﺷﻬْﻩ ﻓَﻠﺎﻧﻪ ﯾُUV ﺟ ــــــــــــــــــ ْاﻫ ﻋِﻘ ــ ﻩ وﺟَ0َﺎﻧ ـــــــــــــــــ ﻪ ﺗﻐﻠﻲ ﻣاﺟِﻞُ ﺣِﻘْﻩ ُﻻ ﻫُﱠ إﻧﻲ ﻻ أ ـــــــــــــــ ً ﻓاﺳﺔ 3 أﺳ5ﻌO ﻣ: اﻟد وَِْﻩ1 وﺿـﺣﺎً وﻗـةً. ﻣـ: ﻣnـﻞ ﻣــﺎ ذـﻩ ﻣـ: أﺑـﺎت ﺷـﻌﺔ ﻧp1ﻬــﺎ ﺑ 0ﻔـﻪ Wـ' ﻓﻬـﺎ ﻧــرة :اﻟIﯾ اﻟcﻘﻲ، ﻋ0 ﺗﻔﻩ ﻟﻘﻟﻪ ﺗﻌﺎﻟﻰ .. ُْ@ِ.. :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ واﻷدﻟﺔ ﻋﻠﻰ اﻟ5vاﻣﻪ ﺑﻬOا اﻟ1Uأ أﻛn ﻣ: أن ﺗُIﻰ ﻓﻲ ﺗﻔﻩ .uإذ ﻫ W'ِّﻞ ﻗام ﻣ0ﻬRﻪ اﻟ5ﻔV، ﻟ@00ﺎ ﻫ0ﺎ ﺳ0@5ﻔﻲ ﺑO أﻣnﻠﺔ ﻗﻠﻠﺔ ﻋﻠﻪ ﻓﻘ 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ، 3 / 1406 ،ً. واﻧp أWrﺎ2 / 777 ، 818،910 - 912 ؛3 / 1549 ؛4 / 1742 . 2 ( ،اﻧp، ﻓﻬ اﻟوﻣﻲ أﺻJل اﻟ4ﻔ و ﻣﺎﻫIﻪ ، 71 - 72 ،؛ ﷴ ﻟPﻔﻲ اﻟI7ﺎغ !,Jث ﻓﻲ أﺻـJل اﻟ4ﻔـ ، 1 ، ﺑوت، اﻟ1'5 اﻹ ،ﺳﻼﻣﻲ1988م ، 67 - 76 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 43 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ :ﻣ: اﻷﻣnﻠﺔ ﻋﻠﻰ ﺗﻔﻩ اﻟﻘآن 3ﺎﻟﻘآن ﺗﻔُﻩ "اﻻﺳ5n0ﺎء" ﻓﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ  إﻻ ﻣﺎ 5ذﱠ ]اﻟ1ﺎﺋة، 3[ 3ﻘﻟﻪ: "ﻪ ﺛﻼﺛﺔ أﻗال: اﻷول: أﻧﻪ اﺳ5n0ﺎء ﻣﻘPـع ﻋ1ـﺎ ﻗUﻠـﻪ ﻏ* ﻋﺎﺋ إﻟﻰ ﺷﻲء ﻣـ: اﻟ1ـOرات، و ذﻟـ ﻣـﻬر ﻓـﻲ ﻟـﺎن اﻟﻌـب، وRﻌﻠـن :إﻻ 31ﻌ0ﻰ ﻟ@:، ﻣ: ذﻟ ﻗﻟﻪ  ًوَﻣَﺎ iَﺎنَ ﻟِﻤُﺆْﻣِﻦٍ أَن hَﻘْﺘُﻞَ ﻣُﺆْﻣِﻨﺎً إِﻻ# ﺧَﻄَﺌﺎ ْﻣﻌ0ﺎﻩ: ﻟ@: إن ﻗ5ﻠـــ"...ﻪ ﺧPـــﺄ، وﻗـــ ﺗﻘـــﱠم ﻼﻣ0ـــﺎ ﻋﻠـــﻪ1. وﻗـــ ﻓﱠ ـــ ﻠ1ـــﺔ "اﻟIـــﺎدﻗ:" ﻓـــﻲ ﻗﻟـــﻪ :ﺗﻌﺎﻟﻰ َUّﻳَﺎ أَﻳ3َﺎ اﻟ#ﺬِﻳﻦَ آﻣَﻨُﻮاْ اw#ﻘُﻮاْ اﻟﻠ َYZِوَﻛُﻮﻧُـﻮاْ ﻣَـﻊَ اﻟﺼ# ـﺎدِﻗ ]اﻟ59ـﺔ ، 119 [ 3ـﺎﻟﻘآن ﻋ0ـﻣﺎ :ﻗــﺎل :"اﻟIــﺎدﻗن ﻫــ اﻟ1ﻓــن 31ــﺎ ﻋﺎﻫــوا، وذﻟــ 3ﻘﻟــﻪ ﺗﻌــﺎﻟﻰ ٌرِﺟَــﺎل ﺻَ ــﺪَﻗُﻮا ﻣَــﺎ ِUْﻋَﺎ<َﺪُوا اﷲَ ﻋَﻠَﻴ ." 2 وﻣــ: أﻣnﻠــﺔ ﺗﻔــ اﺑــ: اﻟﻌ9ــﻲ اﻟﻘــآن اﻟ@ــ 3ــﺎﻟﻘآن وe9ﺎﻟــﯾ اﻟــ lﻓــﻲ وﻗــ واﺣ ﻓﻲ ﻣﻌp اﻟﺎﻻت ُ، ﺗﻔ:ﻩ "ﺳUﻞ ﷲ" ﻓﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ  ِUوَﻗَﺎﺗِﻠُﻮاْ ; ِ: ﺳَـ\|ِﻴﻞِ اﻟﻠّـ ]اﻟ7ﻘــة، 244 [ 3ﻘﻟــﻪ: "ﻣــﺎ ﻣــ: ﺳــUﻞ ﻣــ: ﺳُ ــUُﻞ ،ﷲ ﺗﻌــﺎﻟﻰ إﻻ Wُﻘﺎﺗــﻞ ﻋﻠﻬــﺎ وﻓﻬــﺎ :وأوﻟﻬــﺎ وأﻋp1ﻬــﺎ دﯾــ: اﻹﺳــﻼم، ﻗــﺎل ﷲ ﺳــ7ﺎﻧﻪﻗُــﻞْ <َـــﺬِهِ ﺳَــ\|ِﻴWِ: أَدْﻋُــ ﻮ إَِــ اﻟﻠّــUِ ﻋَWَــ ٍَﺼِــZjَة، وزاد ﺻــﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ ﺗ1ﺎﻣــﺎً ﻓﻘــﺎل: )ﻣَــ:ْ ﻗﺎﺗــﻞ ﻟ5@ــن ﻠ1ــﺔ ﷲ اﻟﻌﻠــﺎ ﻓﻬ ﻓﻲ ﺳUﻞ ﷲ(" 3 P. وOﻟ ﺗﻔﻩ ﻣﻌ0ﻰ اﻟ :ﺎﻋﺔ ﻓﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ َ:Wَﺎ أَﯾﱡﻬَﺎ اﻟﱠOِﯾ ُْ@0آﻣَ0ُــــاْ أَِ ﻌُــــاْ ·َّ وَأَِ ﻌُــــاْ اﻟﱠﺳُ ــــلَ وَأُوْﻟِــــﻲ اﻷَﻣْــــِ ﻣِــــ ]اﻟ0ــــﺎء، 59 [ :، 3ﻘﻟــــﻪ "ﺣcﻘـــﺔ اﻟPﺎﻋـــﺔ، وﻫـــﻲ اﻣ5nـــﺎل اﻷﻣـــ" ، 1ـــﺎ أن اﻟ1ﻌIـــﺔ ﺿـــﻫﺎ، وﻫـــﻲ ﻣQﺎﻟﻔـــﺔ .. اﻷﻣــ. واﻟPﺎﻋـــﺔ ﻣــﺄﺧذة ﻣـــ: ــﺎع إذا اﻧﻘـــﺎد، واﻟ1ﻌIــﺔ ﻣـــﺄﺧذة ﻣــ: ﻋIـــﻰ ﻓ1ﻌ0ــﻰ ذﻟــ اﻣ5nﻠــا أﻣــ ﷲ ﺗﻌــﺎﻟﻰ وأﻣــ رﺳــﻟﻪ ﺻــﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 2 / 539 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ، 2 / 1027 . 3 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 229 . 4 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 451 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ وﻗــ ﻗــﺎل اﻟ0Uــﻲ- ﺻــﻠﻰ ﷲ ﻋﻠــﻪ و ﺳــﻠ-: )ﻣَــ ْ: أــﺎع أﻣــV ﻓﻘــ أــﺎﻋ0ﻲ، وﻣَــ ْ: أــﺎﻋ0ﻲ ﻓﻘ أﺎع ﷲ ْ:ﺗﻌﺎﻟﻰ، وﻣ ﻋIﻰ أﻣV ﻓﻘـ ﻋIـﺎﻧﻲ، وﻣَـ ْ: ﻋIـﺎﻧﻲ ﻓﻘـ ﻋIـﻰ (ﷲ ﺗﻌــــﺎﻟﻰ4 :. وﻗــــ ﻓّ ــــ اﺑــــ: اﻟﻌ9ــــﻲ "اﻹﺣــــﺎن" ﻓــــﻲ ﻗﻟــــﻪ ﺗﻌــــﺎﻟﻰ  ﻫَــــﻞْ ﺟَــــvَاء ُاﻹِْﺣَْ ــﺎنِ إِﻻﱠ اﻹِْﺣَْ ــﺎن ] :1اﻟــﺣ، 60 [ 3ﺎﻻﻋ51ــﺎد ﻋﻠــﻰ اﻟــﯾ اﻟــe، ﺣــ 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 2 / 539 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ، 2 / 1027 . 3 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 229 . 4 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 451 . 4 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 451 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 4 44 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن ﻗـــﺎل: "وﻗـــ ﺛUـــl ﻓـــﻲ اﻟـــﯾ اﻟIـــﺢ أن ﺟUـــﻞ ﺳـــﺄل اﻟ0Uـــﻲ- ﺻـــﻠﻰ ﷲ ﻋﻠـــﻪ وﺳﻠ- ﻋ: اﻹﺣﺎن ﻓﻘﺎل: أن ﺗﻌU ﷲ ﺄﻧ ﺗاﻩ ﻓﺈن ﻟ ﺗ@: ﺗاﻩ ﻓﺈﻧﻪ ﯾاك. ﻓﻬOا إﺣـــﺎن اﻟﻌUـــ. وأﻣـــﺎ إﺣـــﺎن ﷲ ﻓﻬـــ دﺧـــل اﻟـــ0ﻰ و ﻫـــﻲ اﻟR0ـــﺔ ، وﻟﻠـــ0ﻰ ... درﺟﺎت :وﻫOا ﻣ: أﺟﻠﻬﺎ ﻗراً و أﻛﻣﻬﺎ أﻣاً، وأﺣ0ﻬﺎ ﺛا3ﺎً، ﻓﻘ ﻗﺎل ﷲ ﺗﻌﺎﻟﻰ  َﻟ4ﻠ#ﺬِﻳﻦَ أ ٌﺣْﺴَﻨُﻮاْ اﻟْﺤُﺴْ aَ` وَزِﻳَـﺎدَة "ﻓﻬـOا ﺗﻔـﻩ1 . وﻫ'ـOا ﻓﺈﻧ0ـﺎ ﻧﻼﺣـª ﻣـ: ﺧـﻼل اﻷﻣnﻠـﺔ اﻟـــﺎ3ﻘﺔ أنّ اﺑـــ: اﻟﻌ9ـــﻲ WR1ـــﻊ ﻓـــﻲ ﺗﻔـــﻩ ﻟﻶWـــﺎت اﻟﻘآﻧـــﺔ ﺑـــ: اﻟﻘـــآن واﻟـــﯾ اﻟIــﺢ ﻓـــﻲ آنٍ واﺣـــ، وﺟ1ﻌــﻪ ﻫـــOا WﻌP0ـــﺎ دﻻﻟـــﺔ واﺿــﺔ ﻋﻠـــﻰ اﻟ5vاﻣـــﻪ اﻟـــﯾ .31Uأ ﺗﻔ* اﻟﻘآن 3ﺎﻟﻘآن واﻟ0ﺔ أﻣــــﺎ اﻷﻣnﻠــــﺔ اﻟ5ــــﻲ ﺗــــ* إﻟــــﻰ اﻋ51ــــﺎد اﺑــــ: اﻟﻌ9ــــﻲ اﻟ@Uــــ ﻋﻠــــﻰ أﻗــــال اﻟIــــﺎ3ﺔ واﻟ5ﺎ3ﻌ: ﻓﻬﻲ ﺗ@ﺎد ﺗ@ـن ﻣﺟـدة ﻓـﻲ ـﻞ ﺻـﻔﺎت ﺗﻔـﻩ ، ﻟـOﻟ ﻓـﺈﻧﻲ ﺳـﺄﻛ5ﻔﻲ .uﺑO ﻣnﺎل واﺣ ﻣ0ﻬﺎ ﻓﻘ ﻗـــﺎل: "وﻗـــ ﺛUـــl ﻓـــﻲ اﻟـــﯾ اﻟIـــﺢ أن ﺟUـــﻞ ﺳـــﺄل اﻟ0Uـــﻲ- ﺻـــﻠﻰ ﷲ ﻋﻠـــﻪ وﺳﻠ- ﻋ: اﻹﺣﺎن ﻓﻘﺎل: أن ﺗﻌU ﷲ ﺄﻧ ﺗاﻩ ﻓﺈن ﻟ ﺗ@: ﺗاﻩ ﻓﺈﻧﻪ ﯾاك. ﻓﻬOا إﺣـــﺎن اﻟﻌUـــ. وأﻣـــﺎ إﺣـــﺎن ﷲ ﻓﻬـــ دﺧـــل اﻟـــ0ﻰ و ﻫـــﻲ اﻟR0ـــﺔ ، وﻟﻠـــ0ﻰ ... درﺟﺎت :وﻫOا ﻣ: أﺟﻠﻬﺎ ﻗراً و أﻛﻣﻬﺎ أﻣاً، وأﺣ0ﻬﺎ ﺛا3ﺎً، ﻓﻘ ﻗﺎل ﷲ ﺗﻌﺎﻟﻰ  َﻟ4ﻠ#ﺬِﻳﻦَ أ ٌﺣْﺴَﻨُﻮاْ اﻟْﺤُﺴْ aَ` وَزِﻳَـﺎدَة "ﻓﻬـOا ﺗﻔـﻩ1 . 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ، 4 / 1736 . 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ، 4 / 1736 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 226 - 227 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ و1'ــ :ﺗﻠQ ﻋ0ﺎﺻ ﻫOا اﻟ10ﻬﺞ ﻣ: ﺧﻼل اﻟ0ﻘﺎ اﻟ5ﺎﻟﺔ اﻟWﻠV اﻟ ا!ﻊ: ﻣﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻷﺣﺎدﯾO اﻟ?J-ﺔ ﻣــﱠ ﻣﻌ0ــﺎ ﻓــﻲ ﻓﻘــة ﺳــﺎ3ﻘﺔ أن اﺑــ: اﻟﻌ9ــﻲ ــﺎن ﻣــ: اﻟﻔّــﺎ³، و7ــﺎر ﻋﻠ1ــﺎء وﺷــﱠاح اﻟــﯾ اﻟ0UــV، وﻣــ: اﻟ1ﻠ5ــvﻣ: 31ــ0ﻬﺞ ﻣرﺳــﺔ اﻷﺛــ ﻓــﻲ اﻟ5ﻔــ، ﻟــOﻟ ﻛـــﺎن ﻣـــ: اﻟPUﻌـــﻲ أن ﻧRـــ ﻟـــﻪ ﻋ0ﺎWـــﺔ ﺧﺎﺻـــﺔ 3ﺎﻻﺳ5ـــﻬﺎد واﻻﺳـــ5ﻻل 3ﺎﻟـــﯾ اﻟ0UــV ﻣــ: أوﺛــ ﻣIــﺎدر ﻩ اﻟ1ﻌ51ــة ﻓــﻲ اﻟ1ــﺎﺋﻞ اﻟﻔﻘﻬــﺔ اﻟ5ــﻲ ﺗﻌــّض ﻟﻬــﺎ، وﻧRــ ﻋ0ــﻩ :ﻣ0ﻬRــﺎً ﻣ51ّــvاً ﻓــﻲ اﻟ5ﻌﺎﻣــﻞ ﻣــﻊ اﻷﺣﺎدﯾــ اﻟ5ــﻲ ﯾردﻫــﺎ ﻓــﻲ ﺗﻔــﻩ. و1'ــ :ﺗﻠQ ﻋ0ﺎﺻ ﻫOا اﻟ10ﻬﺞ ﻣ: ﺧﻼل اﻟ0ﻘﺎ اﻟ5ﺎﻟﺔ 1 ( Wــﻌﻰ ﺟﻬــﻩ أن ﻻ ﯾــO ﻣــ: اﻷﺣﺎدﯾــ إﻻ اﻟIــﺔ اﻟnﺎﺑ5ــﺔ ﻣ0ﻬــﺎ، اﻟ1ــOرة Vﻓـــﻲ 5ـــ اﻟـــ0ّﺔ اﻟIـــﺔ اﻟ15ﻠﻘـــﺎة 3ـــﺎﻟﻘUل ﻋ0ـــ اﻷﻣـــﺔ، وﻫـــﻲ ﺻـــﺎ اﻟ7Qـــﺎر وﻣـﻠ واﻟ1ـﺄ و5ــ اﻟـ0: اﻷر9ﻌـﺔ1، ﻓﻬــﺎ ﻫـ ﻓـﻲ ﺗﻌﻠﻘــﻪ ﻋﻠـﻰ اﻟواWـﺎت اﻟــاردة ﻓـــﻲ اﻟIــــﻠات اﻹﺑاt1ــــﺔ ﯾــــﻋ اﻟ1ــــﻠ1: إﻟـــﻰ اﻟ5nUــــ l واﻟ51ــــv ﺑــــ: اﻟIــــﺢ واﻟﻔﺎﺳ أﺛ0ﺎء ﺗﻌﺎﻣﻠﻬ ﻣﻊ اﻟواWﺎت 3'ﻞ ﻋﺎم: "ﻣ: ﻫـOﻩ اﻟواWـﺎت ﺻـﺢ، وﻣ0ﻬـﺎ ﺳـــc... وﻧ1ـــﺎ ﻋﻠـــﻰ اﻟ0ـــﺎس أن ﯾ0pـــوا ﻓـــﻲ أدWـــﺎﻧﻬ ﻧَpَـــﻫ ﻓـــﻲ أﻣـــاﻟﻬ، وﻫـــ ﻻ WﺄﺧOون ﻓﻲ اﻟUـﻊ دﯾ0ـﺎراً ﻣﻌ7ـﺎً، وﻧ1ـﺎ WQ5ـﺎرون اﻟـﺎﻟ اﻟPـ، ـOﻟ ﻓـﻲ اﻟـّﯾ: ﻻ ﯾﺧــO ﻣــ: اﻟ :واWــﺎت ﻋــ- اﻟ0Uــﻲ ﺻــﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ- إﻻ ﻣــﺎ ﺻــﺢﱠ ﺳــ0ﻩ ﻟـــ ﻼ ﯾﺧﻞ ﻓﻲ ﺧU اﻟ@Oب ﻋﻠﻰ رﺳل ﷲ- ﺻﻠﻰ ﷲ ﻋﻠﻪ وﺳﻠ- ، ﻓU01ـﺎ ﻫـ WPﻠـ ".:U1اﻟﻔrﻞ إذا 3ﻪ ﻗ أﺻﺎب اﻟ0ﻘ، ﺑﻞ ر91ﺎ أﺻﺎب اﻟQان اﻟ2 ﺞ 1 ( Wــﻌﻰ ﺟﻬــﻩ أن ﻻ ﯾــO ﻣــ: اﻷﺣﺎدﯾــ إﻻ اﻟIــﺔ اﻟnﺎﺑ5ــﺔ ﻣ0ﻬــﺎ، اﻟ1ــOرة Vﻓـــﻲ 5ـــ اﻟـــ0ّﺔ اﻟIـــﺔ اﻟ15ﻠﻘـــﺎة 3ـــﺎﻟﻘUل ﻋ0ـــ اﻷﻣـــﺔ، وﻫـــﻲ ﺻـــﺎ اﻟ7Qـــﺎر وﻣـﻠ واﻟ1ـﺄ و5ــ اﻟـ0: اﻷر9ﻌـﺔ1، ﻓﻬــﺎ ﻫـ ﻓـﻲ ﺗﻌﻠﻘــﻪ ﻋﻠـﻰ اﻟواWـﺎت اﻟــاردة ﻓـــﻲ اﻟIــــﻠات اﻹﺑاt1ــــﺔ ﯾــــﻋ اﻟ1ــــﻠ1: إﻟـــﻰ اﻟ5nUــــ l واﻟ51ــــv ﺑــــ: اﻟIــــﺢ واﻟﻔﺎﺳ أﺛ0ﺎء ﺗﻌﺎﻣﻠﻬ ﻣﻊ اﻟواWﺎت 3'ﻞ ﻋﺎم: "ﻣ: ﻫـOﻩ اﻟواWـﺎت ﺻـﺢ، وﻣ0ﻬـﺎ ﺳـــc... :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ وﻫ'ـOا ﻓﺈﻧ0ـﺎ ﻧﻼﺣـª ﻣـ: ﺧـﻼل اﻷﻣnﻠـﺔ اﻟـــﺎ3ﻘﺔ أنّ اﺑـــ: اﻟﻌ9ـــﻲ WR1ـــﻊ ﻓـــﻲ ﺗﻔـــﻩ ﻟﻶWـــﺎت اﻟﻘآﻧـــﺔ ﺑـــ: اﻟﻘـــآن واﻟـــﯾ اﻟIــﺢ ﻓـــﻲ آنٍ واﺣـــ، وﺟ1ﻌــﻪ ﻫـــOا WﻌP0ـــﺎ دﻻﻟـــﺔ واﺿــﺔ ﻋﻠـــﻰ اﻟ5vاﻣـــﻪ اﻟـــﯾ .31Uأ ﺗﻔ اﻟﻘآن 3ﺎﻟﻘآن واﻟ0ﺔ :ﻓّ اﺑ: اﻟﻌ9ﻲ اﻟﻘ0ت ﻓﻲ ﻗﻟﻪ ﺗﻌـﺎﻟﻰ  َYZِوَﻗُﻮﻣُﻮاْ ﻟِﻠّUِ ﻗَﺎﻧِﺘ ]اﻟ7ﻘـة، 238 [ :3ﻘﻟـﻪ "اﻋﻠ1ا وﻓﻘ@ ﷲ ﺗﻌﺎﻟﻰ أن اﻟﻘ0ت ﯾد ﻋﻠﻰ ﻣﻌﺎ ،ن، أﻣﻬﺎﺗﻬﺎ أر9ﻊ: اﻷول: اﻟPﺎﻋﺔ ﻗﺎﻟﻪ اﺑ: w7ﺎس. اﻟnﺎﻧﻲ: اﻟcﺎم، ﻗﺎﻟﻪ اﺑ: ﻋ1، وﻗـأ:  ًأَﻣ#ﻦْ <ُﻮَ ﻗَﺎﻧِﺖٌ آﻧَﺎء اﻟﻠ#ﻴْﻞِ ﺳَﺎﺟِﺪا ًِﻤﺎÒوَﻗَﺎ، وﻗﺎل اﻟ0Uﻲ ﺻﻠﻰ ﷲ ﻋﻠﻪ وﺳﻠ: )أﻓrﻞ اﻟIﻼة ل اﻟﻘ0ت( :. اﻟnﺎﻟ إﻧــﻪ اﻟــ'ت، ﻗﺎﻟــﻪ ﻣRﺎﻫــ. وﻓــﻲ اﻟIــﺢ ﻗــﺎل زــ: 0ــﺎ ﻧــ5@ﻠ ﻓــﻲ اﻟIــﻼة ﺣ5ــﻰ :lﻧvﻟــ َYZِوَﻗُﻮﻣُــﻮاْ ﻟِﻠّــUِ ﻗَــﺎﻧِﺘ ، ﻓﺄُﻣﻧــﺎ 3ﺎﻟــ'ت. اﻟا3ــﻊ: أن اﻟﻘ0ــت اﻟQــع. وﻫــOﻩ اﻟ1ﻌﺎﻧﻲ ﻠﻬﺎ WIﺢ أن W'ن ﺟ1ﻌﻬﺎ ﻣاداً، ﻷﻧﻪ ﻻ ﺗ0ﺎﻓ ﻪ إﻻ ا ﻟcﺎم ﻓﺈﻧـﻪ ﯾ7ﻌـ أن W'ن ﻣﻌ0ـﻰ اﻵWـﺔ: وﻗﻣـا x ﻗـﺎﺋ1:، إﻻ ﻋﻠـﻰ ﺗ@ﻠّـﻒ. وﻗـ ﺻـﻠﻰ اﺑـ: w7ـﺎس اﻟIــ7ﺢ وﻗ0ــl ﻓﻬــﺎ، ﻓﻠ1ــﺎ ﻓــغ ﻣ0ﻬــﺎ ﻗــﺎل: ﻫــOﻩ ﻫــﻲ اﻟIــﻼة اﻟﺳــPﻰ، وﻗــأ اﻵWــﺔ إﻟــﻰ ﻗﻟــﻪ ﺗﻌــﺎﻟﻰ YZﻗــﺎﻧﺘ :. واﻟIــﺢُ رواWــﺔ زــ ﺑــ: أرﻗــ ﻷﻧﻬــﺎ ﻧــﱞ ﺛﺎﺑــl ﻋــ اﻟ0Uﻲ ﺻﻠﻰ ﷲ ﻋﻠﻪ وﺳﻠ، ﻓﻼ ﯾُﻠ5ﻔl إﻟﻰ ﻣ51َﻞ ﺳاﻫﺎ" 2 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 226 - 227 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 45 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ ﻣــــ: ﻫــــOا اﻟ1nــــﺎل ﻧﻼﺣــــª 3Rــــﻼء ــــe Wــــ5ﻌ: اﺑــــ: اﻟﻌ9ــــﻲ 3ﺎﻟ1Iــــﺎدر اﻷر9ﻌــــﺔ ﻟﻠ5ﻔـ 3ﺎﻟ1ـﺄﺛر ﻟﻔِّ ـ ﻟ0ـﺎ ﻣﻌ0ـﻰ ﻠ1ـﺔ ﻗآﻧـﺔ واﺣـة. و ﻣـ: ﻫ0ـﺎ W1'00ـﺎ أن ﻧــ 3ــ اﻧ5ــﺎب اﺑــ: اﻟﻌ9ــﻲ إﻟــﻰ ﻣرﺳــﺔ اﻟ5ﻔــ* 3ﺎﻟ1ــﺄﺛر، ون ﻟــ W'5ــﻒ ﺑﻬــﺎ، 1ــﺎ ﺳ0ﻼﺣª ذﻟ.1ﺎ 3ﻌ اﻟWﻠV اﻟ ا!ﻊ: ﻣﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻷﺣﺎدﯾO اﻟ?J-ﺔ ﻣــﱠ ﻣﻌ0ــﺎ ﻓــﻲ ﻓﻘــة ﺳــﺎ3ﻘﺔ أن اﺑــ: اﻟﻌ9ــﻲ ــﺎن ﻣــ: اﻟﻔّــﺎ³، و7ــﺎر ﻋﻠ1ــﺎء وﺷــﱠاح اﻟــﯾ اﻟ0UــV، وﻣــ: اﻟ1ﻠ5ــvﻣ: 31ــ0ﻬﺞ ﻣرﺳــﺔ اﻷﺛــ ﻓــﻲ اﻟ5ﻔــ، ﻟــOﻟ ﻛـــﺎن ﻣـــ: اﻟPUﻌـــﻲ أن ﻧRـــ ﻟـــﻪ ﻋ0ﺎWـــﺔ ﺧﺎﺻـــﺔ 3ﺎﻻﺳ5ـــﻬﺎد واﻻﺳـــ5ﻻل 3ﺎﻟـــﯾ اﻟ0UــV ﻣــ: أوﺛــ ﻣIــﺎدر ﻩ اﻟ1ﻌ51ــة ﻓــﻲ اﻟ1ــﺎﺋﻞ اﻟﻔﻘﻬــﺔ اﻟ5ــﻲ ﺗﻌــّض ﻟﻬــﺎ، وﻧRــ ﻋ0ــﻩ :ﻣ0ﻬRــﺎً ﻣ51ّــvاً ﻓــﻲ اﻟ5ﻌﺎﻣــﻞ ﻣــﻊ اﻷﺣﺎدﯾــ اﻟ5ــﻲ ﯾردﻫــﺎ ﻓــﻲ ﺗﻔــﻩ. :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ وﻧ1ـــﺎ ﻋﻠـــﻰ اﻟ0ـــﺎس أن ﯾ0pـــوا ﻓـــﻲ أدWـــﺎﻧﻬ ﻧَpَـــﻫ ﻓـــﻲ أﻣـــاﻟﻬ، وﻫـــ ﻻ WﺄﺧOون ﻓﻲ اﻟUـﻊ دﯾ0ـﺎراً ﻣﻌ7ـﺎً، وﻧ1ـﺎ WQ5ـﺎرون اﻟـﺎﻟ اﻟPـ، ـOﻟ ﻓـﻲ اﻟـّﯾ: ﻻ ﯾﺧــO ﻣــ: اﻟ :واWــﺎت ﻋــ- اﻟ0Uــﻲ ﺻــﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ- إﻻ ﻣــﺎ ﺻــﺢﱠ ﺳــ0ﻩ ﻟـــ ﻼ ﯾﺧﻞ ﻓﻲ ﺧU اﻟ@Oب ﻋﻠﻰ رﺳل ﷲ- ﺻﻠﻰ ﷲ ﻋﻠﻪ وﺳﻠ- ، ﻓU01ـﺎ ﻫـ WPﻠـ ".:U1اﻟﻔrﻞ إذا 3ﻪ ﻗ أﺻﺎب اﻟ0ﻘ، ﺑﻞ ر91ﺎ أﺻﺎب اﻟQان اﻟ2 1 ( ،أﺷــﺎر اﻷﺳــ5ﺎذ اﻟ9ــﻊ اﻟﻘ1ــﺎﻲ إﻟــﻰ أن اﻟ1Iــﺎدر اﻟﯾnــﺔ اﻷﺳﺎﺳــﺔ اﻟ5ــﻲ اﻋ51ــ ﻋﻠﻬــﺎ اﺑــ: اﻟﻌ9ــﻲ ﻓــﻲ ﺗﻔــﻩ .ﻫﻲ اﻟ1Iﺎدر اﻟ5ﺎﻟﺔ: اﻟ1ﺄ ﻟﻺﻣﺎم ﻣﺎﻟ، وﺻﺎ اﻹﻣﺎﻣ: اﻟ7QﺎرV وﻣـﻠ، وﺳـ0: أﺑـﻲ داود وﻣـﻠ واﻟ0ـﺎﺋﻲ ،اﻧpــ،ﻣ;ــﺎدر اﻟ4ﻔــ اﻟﻔﻘﻬــﻲ ﻋــ+ اﺑــ اﻟﻌ ــﻲ ﻓــﻲ .4ﺎ!ــﻪ أﺣــﺎم اﻟﻘــ آن ،اﻟ9ــﻊ ﷴ ﻣ0Iــﻒ اﻟﻘ1ــﺎﻲ 1 ، اﺑﻠ، دار اﻟﻔﻔﺎء2009 ، 143 . 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 3 / 1584 . 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 3 / 1584 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 46 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن 2 (ﻛــﺎن ﺷــﯾ اﻟ0ُﻔــة ﻣــ: اﻷﺣﺎدﯾــ اﻟrــqﻔ ﺔ واﻟ1ﺿــﻋﺔ، وــﺎن Wﻘــم 3ﺎﻧ5ﻘﺎدﻫــ ﺎ ، وﺗOﯾ اﻟ0ﺎس ﻣ0ﻬ ﺎ 2. ﻓﻬﺎ ﻫ Wﻘل ﻷﺻﺎ3ﻪ وﻼ3ﻪ 3ﻌـ أن ﺗﻌـّض ﻟـO 3ﻌـ اﻷﺣﺎدﯾـ اﻟrـqﻔﺔ اﻟـاردة ﻓــﻲ وﺿـء اﻟ0Uـﻲ ،: "ﻗﻠ0ـﺎ: ﻫــOﻩ اﻷﺣﺎدﯾـ ﻟـ ﺗIــﺢ وﻗ أﻟﻘـlُ إﻟـ' وﺻـ5ﻲ ﻓـﻲ ـﻞ وﻗـl وﻣRﻠـ أﻻﱠ ﺗـ5ﻐﻠا ﻣـ: اﻷﺣﺎدﯾـ 31ـﺎ ﻻ "WIﺢ ﺳ0ﻩ1. وﻘل ﻓﻲ ﻣ'ﺎن آﺧ :ﻓﻲ ﺳـﺎق ﺗﻔـﻩ ﻟﻘـل ﷲ ﻋـv وﺟـﻞﱠ ِUوَﻟِﻠّـ ِْUِ ﺳَـﻴُﺠÒاﻷَﺳْﻤَﺎء اﻟْﺤُﺴْ aَ` ﻓَﺎدْﻋُﻮهُ ﺑِ3َﺎ وَذَرُواْ اﻟ#ﺬِﻳﻦَ ﻳُﻠْﺤِﺪُونَ ; ِ: أَﺳْـﻤَﺂ َﺰَوْنَ ﻣَـﺎ iَـﺎﻧُﻮاْ hَﻌْﻤَﻠُـﻮن ]اﻷﻋـاف ، 180 [ ﻣﺻﺎً أﺻﺎ3ﻪ 3ﺎﻻﻗ5Iﺎر ﻋﻠﻰ 5 اﻟ0ﺔ اﻟIﺔ ﻓﻘـu ﻓـﻲ دﻋـﺎء ﷲ ﺳـــ7ﺎﻧﻪ: "Wﻘـــﺎل: أﻟـــ ََـــ وﻟ : إذا ﻣـــﺎل، ،واﻹﻟـــﺎد W'ـــن ﺑـــﺟﻬ:: 3ﺎﻟvـــﺎدة ﻓﻬـــﺎ ًواﻟ0ﻘIــﺎن ﻣ0ﻬـــﺎ، 1ــﺎ Wﻔﻌﻠـــﻪ اﻟRﻬﱠــﺎل اﻟـــOﯾ: WQ5ﻋــن أدwـــﺔ VWــ1ﱡن ﻓﻬـــﺎ اﻟ7ـــﺎر ،3ﻐــ أﺳــ1ﺎﺋﻪ، وOوﻧــﻪ 31ــﺎ ﻟــ ﯾــOﻩ ﻣــ: أﻓﻌﺎﻟــﻪ إﻟــﻰ ﻏــ ذﻟــ ﻣ1ــﺎ ﻻ ﯾﻠــ 3ــﻪ َOﻓـــ ِار ْﻣ0ﻬـــﺎ، وﻻ ﯾـــ ُﻋ َنﱠ أﺣـــٌ ﻣـــ0@ إﻻ 31ـــﺎ ﻓـــﻲ اﻟ@5ـــ اﻟQ1ـــﺔ، و ﻫـــﻲ 5ـــﺎب ،اﻟ7QﺎرV وﻣﻠ و اﻟ5ﻣOV و أﺑﻲ داود و اﻟ0ﺎﺋﻲ، ﻓﻬOﻩ اﻟ@5 ﻫـﻲ ﺑـء اﻹﺳـﻼم ّ:وﻗ دﺧﻞ ﻓﻬﺎ ﻣﺎ ﻓﻲ اﻟ1ـﺄ اﻟـOV ﻫـ أﺻـﻞ اﻟ5Iـﺎﻧe، وذروا ﺳـاﻫﺎ، وﻻ Wﻘـﻟ أﺣٌ: أﺧ5ﺎر دﻋﺎء Oا، ﻓﺈن ﷲ ﻗ اﺧ5ﺎر ﻟﻪ، وأرﺳﻞ ﺑOﻟ إﻟﻰ ا "ﻟQﻠ رﺳﻟﻪ2. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن، 2 / 583 . 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن، 2 / 583 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 2 / 816 . 3 ( ،اﺑ: اﻟﻌ9ﻲ اﻟ;+ر nاﻟﺎﺑ ، 2 / 872 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 2 / 816 . 3 ( ،اﺑ: اﻟﻌ9ﻲ اﻟ;+ر nاﻟﺎﺑ ، 2 / 872 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ 3 ( إذا ذــ ﺣــﯾnﺎً ﻓﺈﻧــﻪ nــاً ﻣــﺎ ﯾــO ﻣQِّﺟــﻪ وراوــﻪ ﻣﻌــﺎً، ﻗــﺎل اﺑــ: اﻟﻌ9ــﻲ ﻓــﻲ :ﺗﻔﻩ ﻟﻘﻟﻪ ﺗﻌﺎﻟﻰ ِوَأَﻋِﱡواْ ﻟَﻬُ ﻣﱠﺎ اﺳْ 5َPَﻌْ5ُ ﻣِّ: ﻗُﱠةٍ وَﻣِ: رِّ9َﺎِ اﻟْQَْﻞ ]اﻷﻧﻔـﺎل ، 60 [ : ".. روZ اﻟ7QـــﺎرV ﻋـــ: ﺳـــﻠ1ﺔ ﺑـــ: اﻷﻛـــع ، ﻗـــﺎل: ﻣـــﱠ ﻋﻠـــﻰ َﻧﻔـــ ﻣـــ: أﺳْ ـــﻠ ﯾ05rــﻠن 3ﺎﻟــﻬﺎم، ﻓﻘــﺎل اﻟ0Uــﻲ ﺻــﻠﻰ ﷲ ْﻋﻠــﻪ وﺳــﻠ: )ار ﻣُــ َا ﺑ 0ِــ َﻲ إﺳــ1ﺎﻋﻞ ، ﻓــﺈنﱠ ُأ3ﺎﻛ َ ﺎن ِراﻣ .(ﺎً، وأﻧﺎ ﻣﻊ ﺑ0ﻲ ﻓﻼن ْﻗﺎل: ﻓﺄﻣ َ َ ُأﺣ اﻟﻔﻘ: 3ﺄﯾﯾﻬ ، ﻓﻘﺎل )ﻣـﺎ َﻟ ُ@ ْ ُﻻ ﺗﻣ َن ؟( ﻗﺎﻟا َe: و َﻧ ْ َlﻣﻲ وأﻧ َﻣﻌ ُﻬ ْ ْ! ﻓﻘﺎل رﺳل ﷲ: )ار ُﻣ َا وأﻧ 'ﺎ ﻣﻌ ُﻛ ِّﻠ ُ@ ( " 3 :. و'5ﻔﻲ أﺣﺎﻧﺎً ﺑO درﺟﺔ اﻟﯾ، ﻓﻌ0ﻣﺎ ﻓّ ﻗﻟﻪ ﺗﻌﺎﻟﻰ وَاﻋْﻠَﻤُﻮاْ أَﻧ#ﻤَﺎ 3 ( ،اﺑ: اﻟﻌ9ﻲ اﻟ;+ر nاﻟﺎﺑ ، 2 / 872 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ ِﻏَﻨِﻤْﺘُﻢ ﻣ4ﻦ َ`ْءٍ ﻓََن# ﻟِﻠّUِ ﺧُﻤُﺴَ Uُ وَﻟِ#ﺳُـﻮل ]اﻷﻧﻔـﺎل، 41 [ ) :ﻗـﺎل: "وﻓـﻲ اﻟIـﺢ إﻧ1ـﺎ أﻧـﺎ ِﻗﺎﺳ، 3ُﻌnl أن أﻗ َ ِﺑ0@، ﻓﺎ± ﺣﺎﻛ ٌ ، واﻟ0Uﻲﱡ ٌﻗﺎﺳ ، واﻟﱡ ﻟﻠQﻠ" ( 1. 4 ( ﻟ W': اﺑ: اﻟﻌ9ﻲ ﻣRد ﻧﺎﻗﻞ ﻟﻸﺣﺎدﯾـ، ﺑـﻞ ـﺎن ـ ًأWrـﺎ ـ 0ﻧﺎﻗـاً ﺧUـاً ﻟﻠـ :واﻟ15: إذا اﻗ5rﻰ اﻷﻣ ذﻟـ اﻟ0ﻘـ. ﻓﻌﻠـﻰ ﺳـUﻞ اﻟ1nـﺎل ﻋ0ـ ﺗﻔـﻩ ﻟﻘﻟـﻪ ﺗﻌـﺎﻟﻰ  ﻳَﻮْمَ ﻳُﺤْﻤَﻰ ﻋَﻠَﻴْ3َﺎ ; ِ: ﻧَﺎرِ ﺟَ3َﻨ#ﻢَ ﻓَﺘُﻜْﻮَى ﺑِ3َﺎ ﺟِﺒَﺎ<ُ3ُﻢْ وَﺟُﻨﻮﺑُ3ُﻢْ وَﻇُ3ُﻮرُ<ُﻢْ <َـﺬَ ا ﻣَـﺎ ﻛَﻨَـﺰْﺗُﻢْ ﻷَDﻔُﺴِـﻜُﻢْ ﻓَـﺬُوﻗُﻮاْ ﻣَـﺎ ُﻛُﻨــﺘُﻢْ ﺗَﻜْﻨِــﺰ َون ]اﻟ59ــﺔ ، 35 [ ، ﻗــﺎل رﺣ1ــﻪ ﷲ:" رُوV ﻋــ: أﺑــﻲ ﻫــة ﻗــﺎل: ﻣــ: ﺗــك .ﻋــة آﻻف درﻫــ ﺟﻌﻠــl ﺻــﻔﺎﺋﺢ WُﻌــOﱠب ﺑﻬــﺎ ﺻــﺎﺣUﻬﺎ ﯾــم اﻟcﺎﻣــﺔ ﻗUــﻞ اﻟﻘrــﺎء وروV ﻋ: اﺑ: ﻣﻌد ،ًأﻧﻪ ﻗﺎل: و ﷲ ﻻ WﻌOب ﷲ رﺟﻼً 3'0v 1 درﻫ درﻫ1ﺎ .وﻻ دﯾ0ــﺎر دﯾ0ــﺎراً، و ﻟ@ــ: ﯾﺳــﻊ ﺟﻠــﻩ ﺣ5ــﻰ ﯾﺿــﻊ ــﻞ دﯾ0ــﺎر ودرﻫــ ﻋﻠــﻰ ﺣﺗــﻪ وﻋ: ﺛ9ﺎن ﻗـﺎل: ﻗـﺎل رﺳـل ﷲ 2: )ﻣـﺎ ﻣـ: رﺟـﻞ W1ـت وﻋ0ـﻩ أﺣ1ـ أو أﺑـ ُ إﻻ ﺟُﻌﻞ ﻟﻪ 3'ﻞ ﻗا ﺻ_ﺔ ﻣ: ﻧﺎر @Z ﺑﻬﺎ ﻣ: ﻓَْﻗِﻪ إﻟﻰ ٌﻗﻣـﻪ، ﻣﻐﻔـر ﻟـﻪ 3ﻌــ ذﻟــ أو ﻣﻌــOﱠ(ب ُ0. ﻗــﺎل اﻟﻘﺎﺿــﻲ: ﻫــOﻩ اﻷﺣﺎدﯾــ ﻟــ WIــﺢ ﺳــ ﻫﺎ ُ، وﻫــﻲ 3ﻌــ ٌﻣ1ﻟﺔ ﻋﻠﻰ ﻣﺎ ﻟ ّﺗُد ُزﺎﺗ"ﻪ2 . 1 ( ،اﺑ: اﻟﻌ9ﻲ ، أﺣﺎم اﻟﻘ آن2 / 858 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 2 / 935 . 3 ( ، اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 3 / 1114 . 4 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ، 1 / 7 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ وﻗ ﺿﻌﱠﻒ اﺑ: اﻟﻌ9ـﻲ ﺣـﯾ: )أﻧـﺎ ﻣﯾ0ـﺔ اﻟﻌﻠـ وﻋﻠــﻲ 3ﺎﺑﻬــﺎ( ﻣــ: ﺣــ ﻣ50ُــﻪ، ﻋ0ــﻣﺎ ﻗــﺎل: "وﻫــ ﺣــﯾ 3ﺎــﻞ، اﻟ0Uــﻲ ﺻــﻠﻰ ﷲ ﻋﻠـﻪ وﺳــﻠ ﻣﯾ0ــﺔُ ﻋﻠـٍ وأﺑ uاﺑﻬــﺎ أﺻــﺎ3ﻪ، وﻣــ0ﻬ اﻟ7ـﺎب اﻟ10ﻔــﺢ، وﻣــ0ﻬ اﻟ15ﺳــ "ﻋﻠﻰ ﻗْر ﻣ0ﺎزﻟﻬ ﻣ: اﻟﻌﻠم3 . 5 ( ًأﻣﺎ 1ﺎ ﯾ5ﻌﻠ 3ﺄﺣﺎدﯾ ﻓrﺎﺋﻞ اﻟر ﻓﻘ رﻓ2 اﺑـ: اﻟﻌ9ـﻲ ﻣﻌp1ﻬـﺎ ﻣﻌ5Uـا إWﺎﻩ ﻏ* ﺻﺢ، ﺣـ Wﻘـل: "وﻟـ ﻓـﻲ اﻟﻘـآن ﺣـﯾ ﺻـﺢ ﻓـﻲ ﻓrـﻞ ﺳـرة إﻻ ﻗﻠـــﻞ ﺳ0ـــ* إﻟـــﻪ، و9ﺎﻗﻬـــﺎ ﻻ ﯾ07ﻐـــﻲ ﻷﺣـــ" ﻣـــ0@ أن ﯾﻠ5ﻔـــl إﻟﻬـــﺎ4 . و9ﺎﻟﻔﻌـــﻞ ﻓﻌ0ﻣﺎ ﺷع ﻓـﻲ ﺗﻔـ* ﺳـرة اﻟ7ﻘـة، أﺷـﺎر 3'ـﻞ وﺿـح إﻟـﻰ أﻧـﻪ "ﻟـ ﻓـﻲ ﻓrـﻠﻬﺎ ﺣـﯾ ﺻــﺢ إﻻ ﻣــ: ــ أﺑـﻲ ﻫــة ﻋــ: اﻟ0Uــﻲ- ﺻـﻠﻰ ﷲ ﻋﻠــﻪ وﺳــﻠ- أﻧــﻪ 1 ( ،اﺑ: اﻟﻌ9ﻲ ، أﺣﺎم اﻟﻘ آن2 / 858 . 2 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 2 / 935 . 3 ( ، اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 3 / 1114 . 4 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ، 1 / 7 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 48 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن ﻗـــﺎل: )ﻻ ﺗRﻌﻠـــا ﺑـــﺗ@ ﻣﻘـــﺎﺑ، ون اﻟUـــl اﻟـــOV ﺗُﻘـــأ ـــﻪ ﺳـــرة اﻟ7ﻘـــة ﻻ ﯾﺧﻠـــﻪ ﺷPﺎن(. ﺧﱠ"VOﺟﻪ اﻟ5ﻣ1. VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت رﻓــ2 اﺑــ: اﻟﻌ9ــﻲ اﻻﺳ5ﺳــﺎل ﻓــﻲ رواWــﺔ اﻹﺳــاﺋﻠﺎت 3ــ'ﻞ ﻋــﺎم، و9ــ'ﻞ ﺧــﺎص اﻟواWــﺎت اﻟ5ــﻲ ﺗQــﺎﻟﻒ fــﺎﻫ اﻟﻘــآن واﻟــ0ﺔ اﻟIــﺔ اﻟnﺎﺑ5ــﺔ، أو اﻟ5ــﻲ ﻻ ،Wﻬان ﻟﻬﺎ 3ﺎﻟIﺔ، واﻋ5Uﻫـﺎ رواWـﺎت ﺳـﺎﻗPﺔ ﻻ أﺻـﻞ ﻟﻬـﺎ وﻻ ﺗIـﺢ ،أﺳـﺎﻧﻫﺎ ﻓﻼ WIﺢ اﻻﺳ5ﻻل وﺗﻔ* اﻟﻘآن ﺑﻬﺎ. Wﻘل اﺑ: اﻟﻌ9ـﻲ ﻓـﻲ ﺳـﺎق ﺗﻔـﻩ ﻟﻘﻟـﻪ ﺗﻌــﺎﻟﻰ:  ْوَدَاوُودَ وَﺳُــﻠَVَْنَ إِذْ ﻳَﺤْﻜُﻤَــﺎنِ ; ِــ: اﻟْــَ ْثِ إِذْ Dَﻔَﺸَ ــﺖْ ﻓِﻴــUِ ﻏَــﻨَﻢُ اﻟْﻘَــﻮْمِ وَﻛُﻨ#ــﺎ ﻟِﺤُﻜ َﻤِ3ِــﻢْ ﺷَــﺎ<ِﺪِﻳﻦ ]اﻷﻧUــﺎء، 78 [: "أن ﷲ ذــ ﻟﺳــﻟﻪ ﻣــﺎ ﺟــZ ﻣــ: اﻷﻣــ وﻋﻠﻬــﺎ، وأﻗــال اﻷﻧUــﺎء وأﻓﻌﺎﻟﻬـﺎ ﻓﺄﺣـ:َ اﻟﻘIــ، ّوﻫـ أﺻــﻗﻪ، ﻓـﺈن اﻹﺳـاﺋﻠﺎت ذوﻫــﺎ ﻣUﱠﻟـﺔ و9vــﺎدة °3ﺎﻠــﺔ ﻣﺻــﻟﺔ، أو ﺑ0ﻘIــﺎن ﻣــِّف ﻟﻠ1ﻘIــ ﻣ0ﻘﻟــﺔ، وﻣــﺎ ﻧُﻘــﻞ ﻣــ: ﺣــﯾ ﻧﻔْــ اﻟﻐــ0، وﻗrــﺎء داود وﺳــﻠ1ﺎن ﻓﻬــﺎ، اﻧpــوا ــﻪ ﻓ1ــﺎ واﻓــ ﻣ0ــﻪ fــﺎﻫ اﻟﻘــآن ﻓﻬــ ﺻﺢ، وﻣﺎ ﺧﺎﻟﻔﻪ ﻓﻬ 3ﺎﻞ، وﻣﺎ ﻟ ﯾد ﻪ ذ ﻓﻬ ﻣ51ﻞ ، ر9ـ أﻋﻠـ 3ـﻪ" 2 . uوﻫ ﻻ WﻘUـﻞ ﻣـ: اﻹﺳـاﺋﻠﺎت إﻻ اﻟ5ـﻲ ﺗُQUِـ ﻋـ: ﺑ0ـﻲ إﺳـاﺋﻞ أﻧﻔـﻬ ﻓﻘـ، ﻻو ﺗ ّQﺎﻟﻒ fﺎﻫ اﻟﻘآن، ﻷﻧﻬﺎ ﻓﻲ ﻫـOﻩ اﻟﺎﻟـﺔ ﺗُﻌـ ،ﻣـ: 3ـﺎب إﻗـار اﻟ1ـء ﻋﻠـﻰ ﻧﻔـﻪ وﻫOا ﻣﻘUل. 2 ( ،اﺑ: اﻟﻌ9ﻲ ،أﺣﺎم اﻟﻘ آن3 / 1266 . 1 ( ،اﺑ: اﻟﻌ9ﻲ ،أﺣﺎم اﻟﻘ آن 1 / 8 . :اﻟU,O اﻟZﺎﻧﻲ ﻣ ﻗMﺎLﺎ اﻟﻬﺞ ﻋ+ اﺑ اﻟﻌ ﻲ أﻣ ِUQُﺎ 1ﺎ ﺗ َ 3ﻪ ﻋ: ﻏﻫ ﻓﻬـﻲ ﻣـدودة، ﻷن إﺧ7ـﺎر ﻫ ﻋـ: ﻏـﻫ ٌﻣُﻔ5ﻘـ إﻟــﻰ ر0ــﻲ اﻟQUــ اﻟIـﺢ، وﻫ1ــﺎ اﻟﻌاﻟــﺔ واﻟnUــت إﻟـﻰ أﺻــﻞ اﻟQUــ. Wﻘــل :اﺑ: اﻟﻌ9ﻲ ﻓﻲ ﺳﺎق ﺗﻔﻩ ﻟﻘﻟﻪ ﺗﻌﺎﻟﻰ ْوَإِذْ ﻗَـﺎلَ ﻣُﻮَـ` ﻟِﻘَﻮْﻣِـUِ إِن# اﻟﻠّـUَ ﻳَـْﻣُﺮُﻛُﻢْ أَنْ ﺗَـﺬْﺑَﺤُﻮا ًََ َة َYZِﻗَﺎﻟُﻮاْ أَﺗَﺘ#ﺨِﺬُﻧَﺎ <ُﺰُواً ﻗَﺎلَ أَﻋُﻮذُ ﺑِﺎﻟﻠّـUِ أَنْ أَﻛُـﻮنَ ﻣِـﻦَ اﻟْﺠَـﺎ<ِﻠ ]اﻟ7ﻘـة، 67 [: "ﻛnُـ اﺳ5ﺳـﺎل اﻟﻌﻠ1ــﺎء ﻓــﻲ اﻟــﯾ ﻋــ0ﻬ ﻓــﻲ ــﻞ ــ، وﻗــ ﺛUــl ﻋــ: اﻟ0Uــﻲ ﺻــﻠﻰ ﷲ ﻋﻠــﻪ ُوﺳــﻠ أﻧــﻪ ﻗــﺎل: )ﺣــِّﺛا ﻋــ: ﺑ0ــﻲ إﺳــاﺋﻞ وﻻ ﺣــج(. وﻣﻌ0ــﻰ ﻫــOا اﻟQUــ اﻟــﯾ 0ﻋ ﻬ 31ﺎ WQUون 3ﻪ ﻋ: أﻧﻔﻬ وﻗIIـﻬ ﻻ 31ـﺎ WQUـون 3ـﻪ ﻋـ: ﻏـﻫ، ﻷن أﺧ7ﺎرﻫ ﻋ: ﻏﻫ ﻣﻔ5ﻘة إﻟﻰ اﻟﻌاﻟﺔ واﻟnUت إﻟﻰ ﻣ05ﻬﻰ اﻟQU، وﻣﺎ WQUون 3ﻪ 1 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 1 / 23 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 1 / 30 . 3 ( ،اﻧp، ﷴ ﻋU اﻟﻌp اﻟvرﻗﺎﻧﻲ ﻣﺎﻫﻞ اﻟﻌ ﻓﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن ، 3 ،، ﺑوت، ار إﺣﺎء اﻟ5اث اﻟﻌ9ﻲ )د.ت. ،( 1 / 501 - 529 ،؛ ﻓrﻞ ﺣ: w7ﺎس إﺗﻘﺎن اﻟ? ﻫﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن ، 2 ،، اﻷردن، دار اﻟ0ﻔﺎﺋ 2010 ،م2 / 198 - 201 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 1 / 30 . 1 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 1 / 23 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 49 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ ﻋــ: أﻧﻔــﻬ 'ــن ﻣــ: 3ــﺎب إﻗــار اﻟ1ــ ء "ﻋﻠــﻰ ﻧﻔــﻪ أو ﻗﻣــﻪ، ﻓﻬــ أﻋﻠــ ﺑــOﻟ1 . وﻗ وﺟﻧﺎﻩ ﻓﻲ 3ﻌ2 اﻷﺣﺎن ﯾـO ﺷـ ﺎً ﻣـ: اﻹﺳـ اﺋﻠﺎت ﺑﻬـف اﻟ5ـOﯾ ﻣ0ﻬـﺎ و9ـــﺎن ﻓـــﺎدﻫﺎ، 1ـــﺎ ﻓﻌـــﻞ ﻋ0ـــﻣﺎ ذـــ اﻟواWـــﺔ اﻹﺳـــاﺋﻠﺔ ﻋـــ: اﻟ1َﻠَ@ـــ: ﻫـــﺎروت وﻣﺎروت، 7ﻌـ أن ذﻫـﺎ 3'ﺎﻣـﻞ ﻧIِّ ـ:ﻬﺎ اﻟPـﻞ، ﻗـﺎل "... وﻧ1ـﺎ ﺳـﻘ0ﺎ ﻫـOا اﻟQUـ ﻷن اﻟﻌﻠ1ﺎء روَوْﻩ ودوﱠﻧﻩ ﻓQ0ﺎ أن Wﻘﻊ ﻟِ1َ:ْ Wrﻞﱡ 3ﻪ. وﺗﻘـ اﻟﻘـل ـﻪ أﻧـﻪ ﻟـ IW "ﺢ ﺳ0ﻩ2 . :اﻟWﻠV اﻟﺎدس gﻣ+l اﻋ4ﺎدﻩ ﻋﻠﻰ اﻟ4ﻔ !ﺎﻟ أ ﻣـــ: اﻟ1ﻌﻠـــم أن اﻟ5ﻔـــ 3ـــﺎﻟأV أV 3ﺎﻟﻌﻘـــﻞ واﻻﺟ5ﻬـــﺎد ﻣﻘUـــل ﻋ0ـــ ﻋﻠ1ـــﺎء اﻟﻘآن اﻟ@ واﻟ5ﻔ* ﺎﻟ1ﺎ ﺎن ﻫOا اﻟ5ﻔ* ﯾﻠ5vم 3و اﻟـأV اﻟ11ـد، وﻫـﻲ إﻋPـــﺎء اﻷوﻟـــﺔ ﻓـــﻲ اﻟ5ﻔـــ* ﻟﻠ@5ـــﺎب واﻟـــ0ﺔ، وﻋـــم ﻣQﺎﻟﻔ5ﻬ1ـــﺎ :3ـــﺄV ﺷـــ'ﻞ ﻣـــ اﻷﺷ'ﺎل، واﻷﺧO 31ﻘ5rﺎت اﻟﻠﻐﺔ اﻟﻌ9ﺔ ودﻻﻻﺗﻬـﺎ، وأﺳـﺎﻟ* اﻟﻌـب، وﻣﻌﻬـدﻫ ﻓـﻲ اﻟQPـﺎب، واﻟﻌﻠـ 3ﺄﺻـل اﻟﻔﻘـﻪ، واﻟ7ﻌـ ﻋـ: اﻟR0ـح ﻟﻠﻬـZ، واﻟ1ـﻞ ﻣﻌـﻪ ﺣــ ﻣﺎل3 . وﻧ: إذا دﻗﻘ0ﺎ اﻟ0p ﻓﻲ ﺗﻔ* اﺑـ: اﻟﻌ9ـﻲ وﺟـﻧﺎﻩ 3ﺎﻹﺿـﺎﻓﺔ إﻟـﻰ اﻟ5vاﻣـﻪ 31ـــ0ﻬﺞ اﻟ5ﻔـــ* 3ﺎﻟ1ـــﺄﺛر، Wـــ 5ﻌ: 3ـــ'ﻞ واﺿـــﺢ 3ﺎﻟ5ﻔـــ 3ـــﺎﻟأV اﻟ11ـــد ﻓـــﻲ اﺳـــ507ﺎﺎﺗﻪ اﻟﻔﻘﻬـــﺔ ﻣـــ: اﻵWـــﺎت اﻟﻘآﻧـــﺔ اﻟ5ـــﻲ ﻓّ ـــﻫﺎ، VوـــOِّر ﻣـــ: اﻷﺧـــO 3ـــﺎﻟأ :اﻟ1ـOﻣم. ﻓﻘـ ﻗــﺎل ﻋ0ـ ﺗﻔــﻩ ﻟﻘﻟـﻪ ﺗﻌــﺎﻟﻰ ْوَﻟَﻘَـﺪْ ﺟَــﺎءتْ رُﺳُــﻠُﻨَﺎ إِﺑْــﺮَا<ِﻴﻢَ ﺑِﺎﻟْﺒُـــْ َى ﻗَــﺎﻟُﻮا َﺳَــﻼَﻣﺎً ﻗَــﺎلَ ﺳَــﻼَمٌ ﻓَﻤَــﺎ ﻟَﺒِــﺚ ٍأَن ﺟَــﺎء ِﻌِﺠْــﻞٍ ﺣَﻨِﻴــﺬ ]ﻫــد، 69 [ l:"ﻗــﺎل 3ﻌــ2 ﻋﻠ1ﺎﺋ0ــﺎ: ﺎﻧــ ،ﺿﺎﻓﺔ ﻗﻠﻠﺔ ﻓ'ﻫﺎ اﻟU* ﻣ: اﻟU، وﻫOا ﺗ'ﱡـ 3ـﺎﻟp:، ﻓـﻲ ﻣﺿـﻊ اﻟﻘPـﻊ و9ﺎﻟcــﺎس ﻓــﻲ ﻣﺿــﻊ اﻟ0ﻘــﻞ، ﻣـــ: أﯾــ: ﻋَﻠِــ أﻧــﻪ ﻗﻠـــﻞ؟ ﺑــﻞ ﻗــ ﻧﻘــﻞ اﻟ1ﻔـــون أن 'اﻟ1ﻼﺋ@ــﺔ ــﺎﻧا ﺛﻼﺛــﺔ: ﺟUــﻞ وﻣ ﺎﺋــﻞ وﺳــاﻓﻞ ِ، وﻋ Rْــ ٌﻞ ﻟnﻼﺛــﺔ ﻋpــ، ﻓ1ــﺎ ﻫــOا ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 50 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن اﻟ5ﻔــ* ﻓــﻲ 5ــﺎب ﷲ 3ــﺎﻟأV؟ ﻫــOا 3ﺄﻣﺎﻧــﺔ ﷲ ﻫــ اﻟ5ﻔــ* اﻟ1ــOﻣم، ﻓــﺎﺟ50Uﻩ ﻓﻘــ "ﻋﻠ151ﻩ1. اﻟ5ﻔــ* ﻓــﻲ 5ــﺎب ﷲ 3ــﺎﻟأV؟ ﻫــOا 3ﺄﻣﺎﻧــﺔ ﷲ ﻫــ اﻟ5ﻔــ* اﻟ1ــOﻣم، ﻓــﺎﺟ50Uﻩ ﻓﻘــ "ﻋﻠ151ﻩ1. :وﻣ: أﻣnﻠﺔ اﺳ5Qام اﺑ: اﻟﻌ9ﻲ اﻟأV ﻓﻲ ﺗﻔﻩ، ﻣﺎ ﻗﺎﻟﻪ ﻓﻲ ﺗﻔـﻩ ﻟﻘﻟـﻪ ﺗﻌـﺎﻟﻰ  ْوَاﺳْﺘَﺸْ ــ3ِﺪُواْ ﺷَــ3ِﻴﺪَﻳْﻦِ ﻣــﻦ ر4ﺟَــﺎِﻜُﻢ ]اﻟ7ﻘــة، 282 [ .: "ﻗــﺎل ﻣRﺎﻫــ: أراد ﻣــ: اﻷﺣــار :واﺧ5ﺎرﻩ أﺑ إﺳ وأ0ـ ـﻪ. 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 3 / 1063 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﻗـﻞ: اﻟ1ـاد 3ـﻪ ﻣـ: اﻟ1ـﻠ1:، ﻷن ﻗﻟـﻪ ﺗﻌـﺎﻟﻰ ﻣــﻦ اﻟﺮﺟــﺎل ــﺎن Wﻐ0ــﻲ ﻋ0ــﻪ، ﻓــﻼ ﺑــّ ﻟﻬــOﻩ اﻹﺿــﺎﻓﺔ ﻣــ: ﺧIIــﺔ، وﻫــﻲ إﻣــﺎ أﺣار وﻣﺎ ﻣﻣ0، واﻟ1ﻣ0ن 3ﻪ أﺧـ ﻣـ: اﻷﺣـار، ﻷن ﻫـOﻩ اﻹﺿـﺎﻓﺔ ﻫـﻲ إﺿـــﺎﻓﺔ اﻟR1ﺎﻋـــﺔ، و ﻻ ﻓَ1ـــ:ْ ﻫـــ اﻟـــOV WR1ـــﻊ اﻟـــ5ﺎت وُـــ ِّp0 ُ َاﻟـــ1ﻞ َp0اﻟـــ VOاﻟـــ WIـــــﺢﱡ ِﻣ0ـــــﻪ اﻹﺿـــــﺎﻓﺔ. واﻟIـــــﺢ ﻋ0ـــــV أن اﻟ1ـــــاد 3ـــــﻪ اﻟ7ـــــﺎﻟﻐن ﻣـــــ: ذـــــر 1اﻟ1ﻠ نO، ﻷن اﻟPﻔﻞ ﻻ Wُﻘـﺎل ﻟـﻪ رﺟـﻞ، وـ ًا اﻟ1ـأة ﻻ Wُﻘـﺎل ﻟﻬـﺎ رﺟـﻞ أWrـﺎ . وﻗـ ﺑـــﱠ: ﷲ ﺗﻌـــﺎﻟﻰ 3ﻌـــ ذﻟـــ ﺷـــﻬﺎدة اﻟ1ـــأة، وﻋـــﱠ : 3ﺎﻹﺿـــﺎﻓﺔ ﻓـــ ﻲ ﻗﻟـــﻪ ﺗﻌـــﺎﻟﻰ:  ﻣـــﻦ رﺟــﺎﻜﻢ اﻟ1ــﻠَ، وﻷن اﻟ@ــﺎﻓ ﻻ ﻗــلَ ﻟــﻪ، وﻋَ0َــﻰ اﻟ@Uــَ أWrــﺎً، ﻷن اﻟIــﻐ ﻻ َVِّﻣIل ﻟﻪ. وﻧ1ﺎ أﻣ ﷲ ﺗﻌﺎﻟﻰ 3ﺈﺷـﻬﺎد اﻟ7ـﺎﻟﻎ، ﻷﻧـﻪ اﻟـOV WIـﺢ أن ﯾُـد اﻵن اﻟــﻬﺎدة، ﻓﺄﻣــﺎ اﻟIــﻐ* ــﻔª اﻟــﻬﺎدة، ﻓــﺈذا أدﱠاﻫــﺎ وﻫــ رﺟــﻞ ﺟــﺎزت، وﻻ ﺧــﻼف "ﻪ2 ﻣ: ﻫOا اﻟ1nﺎل ﻧﻼﺣª e اﺳ5ﻌض اﺑ: اﻟﻌ9ﻲ اﻷﻗال اﻟاردة ﻓﻲ ﺗﻔ ﻛﻠ1ـــﺔ اﻟﺟـــﺎل ﻓـــﻲ اﻵWـــﺔ اﻟ1ـــOرة ً، ﺛـــ ـــe ﻧﺎﻗـــﻬﺎ ﻣ0ﺎﻗـــﺔ د»ﻘـــﺔ ﻣﻌ51ـــا ﻋﻠـــﻰ اﻟ1ﻌ0ــﻰ اﻟﻠﻐــV ﻟ@ﻠ1ــﺔ اﻟﺟــﺎل ، وﻘــﺔ إﯾادﻫــﺎ )أﺳــﻠب اﻹﺿــﺎﻓﺔ( وــOﻟ ﺳــﺎق ( اﻵWﺔ )ذُِْ ﺷﻬﺎدة اﻟ1ـأة 1ـﺎ 3ﻌـ، ﻟIـﻞ ﻣـ: ﺧـ ﻼل ذﻟـ ﻠـﻪ إﻟـﻰ رأWـﻪ اﻟﻌﻠ1ـﻲ ،اﻟ15ﻘﻞ و.ًاﻟ1ﻘUل ﻟﻐﺔً وﺷﻋﺎ :اﻟWﻠV اﻟﺎ!ﻊ ﻗﺎﻧJن اﻟ4 ﺟ3ﺢ ﻓﻲ ﺗﻔ ﻩ ،إنﱠ ﻗة ﺷQIﺔ اﺑ: اﻟﻌ9ﻲ اﻟﻌﻠ1ﺔ واﺳ5ﻘﻼﻟﻬﺎ ﻣﺄﻟﺔ ﺷﯾة اﻟﺿـح ﻓـﻲ ﺗﻔـﻩ Z5وﻻ ﻏا3ـــﺔ ﻓـــﻲ ذﻟـــ، ﻓﻬـــ ﻋـــﺎﻟٌ راﺳـــﺦٌ ﻓـــﻲ ـــﻞ اﻟﻌﻠـــم اﻟ5ـــﻲ ﺗﻫﻠـــﻪ ﻟﻬـــOا اﻟ1ـــ :اﻟﻌﻠ1ﻲ، ﻣ 5ﺎبٍ وﺳ0ﺔٍ و ﻟﻐـﺔٍ وﻓﻘـﻪٍ وأﺻـل دﯾـ: وأﺻـل ﻓﻘـﻪ وﺗـﺎرﺦ وﺧUـة ﻓـﻲ 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 3 / 1063 . ﻲ 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 251 - 252 . ﻲ 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 251 - 252 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ اﻟﻘrـــﺎء ..، ﻓﻬــــ ﺣــــ: Wﻌــــض اﻟواWــــﺎت واﻷﺧ7ــــﺎر Wﻘــــم ﺑ5cﻘﻬــــﺎ وﺗ1Iــــﻬﺎ ، وﻋ0ﻣﺎ ﯾO اﻵراء واﻷﻗال اﻟﻔﻘﻬﺔ ﯾ0ﺎﻗﻬﺎ ود ﻋﻠﻬﺎ ﻋ0ـ اﻟﺎﺟـﺔ، و05ﻬـﻲ ﻓـﻲ 1ﺧﺎﺗ1ﺔ اﻟ1Pﺎف إﻟﻰ ﺗﻘ وﺗﺟﺢ ﻣﺎ Wﻌ5ﻘ أﻧـﻪ ﺻـﺢ. و 'ـ: اﻟﻘـل ﻋﻠـﻰ وﺟـﻪ .اﻹﺟ1ﺎل إﻧﻪ Wﻘم 3ﺎﻟ5ﺟﺢ ﺑ0ﺎءً ﻋﻠﻰ اﻟﻘآن واﻟ0ﺔ واﻟﻠﻐﺔ وﻋ0ﻣﺎ ﯾO اﻵراء واﻷﻗال اﻟﻔﻘﻬﺔ ﯾ0ﺎﻗﻬﺎ ود ﻋﻠﻬﺎ ﻋ0ـ اﻟﺎﺟـﺔ، و05ﻬـﻲ ﻓـﻲ 1ﺧﺎﺗ1ﺔ اﻟ1Pﺎف إﻟﻰ ﺗﻘ وﺗﺟﺢ ﻣﺎ Wﻌ5ﻘ أﻧـﻪ ﺻـﺢ. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 451ـ452 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 2 VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت و 'ـ: اﻟﻘـل ﻋﻠـﻰ وﺟـﻪ .اﻹﺟ1ﺎل إﻧﻪ Wﻘم 3ﺎﻟ5ﺟﺢ ﺑ0ﺎءً ﻋﻠﻰ اﻟﻘآن واﻟ0ﺔ واﻟﻠﻐﺔ وﻣــ: أﻣnﻠـــﺔ ﺗﺟـــﻪ 3ــﺎﻟﻘآن اﻟ@ـــ دون أن Wﻐﻔـــﻞ دور اﻟــﯾ و اﻟـــأV واﻻﻟ5ﻔـــﺎت :ﻟﻠاﻗﻊ ، ﺗﻔُﻩ "أوﻟـﻲ اﻷﻣـ" ﻓـﻲ ﻗﻟـﻪ ﺗﻌـﺎﻟﻰ ْﻳَـﺎ أَﻳ3َـﺎ اﻟ#ـﺬِﻳﻦَ آﻣَﻨُـﻮاْ أَﻃِﻴﻌُـﻮاْ اﻟﻠّـUَ وَأَﻃِﻴﻌُـﻮا ْاﻟﺮ#ﺳُﻮلَ وَأُوِْ: اﻷَﻣْﺮِ ﻣِـﻨﻜُﻢ ]اﻟ0ـﺎء، 59 [: :"ﻓﻬـﺎ ﻗـﻻن: اﻷول: ﻗـﺎل ﻣ1ـن ﺑـ: ﻣﻬـان :ﻫ أﺻﺎب اﻟاWﺎ، وروZ ﻓﻲ ذﻟ ﺣﯾnﺎً، وﻫـ اﺧ5ـﺎر اﻟ7QـﺎرV. وروZ ﻋـ: اﺑـ w7ﺎس أﻧﻬﺎ ﻧvﻟl ﻓﻲ ﻋU ﷲ ﺑ: ﺣُOاﻓـﺔ، إذ 3ﻌnـﻪ اﻟ0Uـﻲ ﺻـﻠﻰ ﷲ ﻋﻠـﻪ وﺳـﻠ ﻓـﻲ ﺳَ ــِﱠﺔ. اﻟnــﺎﻧﻲ: ﻗــ :ﺎل ﺟــﺎﺑ: ﻫــ اﻟﻌﻠ1ــﺎء، واﺧ5ــﺎرﻩ ﻣﺎﻟــ، ﻗــﺎل ﻣPــّف واﺑــ: ﻣــﻠ1ﺔ ﺳ1ﻌ0ﺎ ﻣﺎﻟ@ﺎً Wﻘـل: ﻫـ اﻟﻌﻠ1ـﺎء. وﻗـﺎل ﺧﺎﻟـ ﺑـ: ﻧـvار: وﻗﻔـlُ ﻋﻠـﻰ ﻣﺎﻟـ ﻓﻘﻠـlُ : Wـﺎ :أ3ﺎ ﻋU ﷲ، ﻣﺎ ﺗZ ﻓـﻲ ﻗﻟـﻪ ﺗﻌـﺎﻟﻰ  ْوَأُوِْـ: اﻷَﻣْـﺮِ ﻣِـﻨﻜُﻢ ؟ ﻗـﺎل: وـﺎن ﻣُ5Uـﺎً ﻓـﻞﱠ ﺣUْﺗــﻪ، وــﺎن ﻋ0ــﻩ أﺻــﺎب اﻟــﯾ ﻓﻔــ ،5ﺢ ﻋ0ــﻪ ﻓــﻲ وﺟﻬــﻲ، وﻋﻠ1ــlُ ﻣــﺎ أراد ْ:وﻧ1ﺎ ﻋَ0ﻰ أﻫﻞ اﻟﻌﻠ. واﺧ5ﺎرﻩ اﻟPUV، واﺣ5ﺞ ﻟﻪ 3ﻘﻟﻪ ﺻﻠﻰ ﷲ ﻋﻠﻪ وﺳﻠ: )ﻣ أــﺎع أﻣــV ﻓﻘــ أــﺎﻋ0ﻲ ...( اﻟــﯾ. واﻟIــﺢ ﻋ0ــV أﻧﻬــ اﻷﻣــاء واﻟﻌﻠ1ــﺎء ﺟ1ﻌــﺎً، أﻣـــﺎ اﻷﻣـــاء ﻓــﻸن أﺻـــﻞ اﻷﻣـــ ﻣــ0ﻬ، واﻟ'ـــ إﻟـــﻬ. وأﻣــﺎ اﻟﻌﻠ1ـــﺎء ﻓـــﻸن ﺳــ اﻟﻬ واﺟــ ﻣ5ﻌَــ ِّ* : ﻋﻠــﻰ اﻟQﻠــ، وﺟــاﺑﻬ ﻻزم، واﻣ5nــﺎل ﻓ5ــاﻫ واﺟــ، وــﺧﻞ ﻪ اﻟvوج ﻟﻠvوﺟﺔ، ﻻﺳ1ﺎ وﻗ ﻗـﻣ0ﺎ أن ـﻞ ﻫـﻻء ﺣـﺎﻛ ، و ﻗـ ﺳـ1ﺎﻫ ﷲ ﺗﻌـﺎﻟﻰ ﺑOﻟ:، ﻓﻘﺎل  َﻳَﺤْﻜُﻢُ ﺑِ3َﺎ اﻟﻨ#\|ِﻴﻮنَ اﻟ#ﺬِﻳﻦَ أَﺳْﻠَﻤُﻮاْ ﻟِﻠ#ﺬِﻳﻦَ <َﺎدُواْ وَاﻟﺮ#ﺑ#ﺎﻧِﻴﻮن ُوَاﻷَﺣْﺒَﺎر . ﻓﺄﺧU ﺗﻌـﺎﻟﻰ أن اﻟ0Uﻲ ﺣﺎﻛ، واﻟ9ﺎﻧﻲ ﺣﺎﻛ، واﻟَUْ ﺣﺎﻛ. واﻷﻣ ﻠﻪ ﯾﺟﻊ إﻟﻰ اﻟﻌﻠ1ﺎء، ﻷن اﻷﻣ ﻗ أﻓrﻰ إﻟﻰ اﻟRﻬﺎل، و ﺗﻌّ: ﻋﻠﻬ ﺳـال اﻟﻌﻠ1ـﺎء، ﻟـOﻟ ﻧpـ ﻣﺎﻟـ إﻟـﻰ ﺧﺎﻟـ ﺑـ: ﻧــvار ﻧpـةً ﻣ0@ــة، ﺄﻧـﻪ Wـ* ﺑﻬــﺎ إﻟـﻰ أن اﻷﻣــ ﻗـ وﻗـﻒ ﻓــﻲ ذﻟـ ﻋﻠــﻰ اﻟﻌﻠ1ـــﺎء ٌ، وزال ﻋـــ: اﻷﻣـــاء ﻟRﻬﻠﻬـــ واﻋ5ـــاﺋﻬ، واﻟﻌـــﺎدل ﻣـــ0ﻬ ﻣﻔ5ﻘـــ إﻟـــﻰ اﻟﻌـــﺎﻟ "ﻛﺎﻓ5ﻘﺎر اﻟRﺎﻫﻞ1. 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 451ـ452 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 52 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن :وأﻣﺎ ﺗﺟﻪ 3ﺎﻟﯾ اﻟe، ﻓ1nﺎﻟﻪ ﻓﻲ ﺗﻔﻩ ﻟﻘﻟﻪ ﺗﻌـﺎﻟﻰ ِhَﺴْ ـَﻟُﻮﻧَﻚَ ﻋَـﻦِ اﻟْﺨَﻤْـﺮ وَاﻟْﻤَﻴِْ ِ ﻗُﻞْ ﻓِﻴ3ِﻤَﺎ إِﺛْﻢٌ ﻛَﺒِZjٌ وَﻣَﻨَﺎﻓِﻊُ ﻟِﻠﻨ#ﺎسِ وَإِﺛْﻤُ3ُﻤَﺎ ُjأَﻛَْـ ﻣِـﻦ D#ﻔْﻌِ3ِﻤَـﺎ.. 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 149 / 150 . 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 1 / 149 / 150 . 2 ( ،اﺑ: اﻟﻌ9ﻲ أ ﺣﺎم اﻟﻘ آن ، 1 / 448 . ،أ ﺣﺎم اﻟﻘ آن ، 1 / 448 . 2 ( ،اﺑ: اﻟﻌ9ﻲ أ ﺣﺎم اﻟﻘ آن ، 1 / 448 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت  ]اﻟ7ﻘـة ، 219 [ ، "اﻟ1ـﺄﻟﺔ ::اﻟnﺎﻧﺔ: ﻓﻲ ﺗﻘ* اﺳ اﻟQ1ـ وﻣﻌ0ـﺎﻩ: وﻗـ اﺧ5ﻠـﻒ اﻟﻌﻠ1ـﺎء ﻓـﻲ ذﻟـ ﻋﻠـﻰ ﻗـﻟ أﺣﻫ1ﺎ: أن اﻟQ1ـ ﺷـاب Wُﻌ5Iـ ﻣـ: اﻟﻌ0ـ ﺧﺎﺻـﺔ، وأﻣـﺎ ﻣـﺎ اﻋُ5Iـ ﻣـ: ﻏـ اﻟﻌ0ـــ ﺎﻟv9ـــO واﻟ51ـــ وﻏﻫ1ـــﺎ Wﻘـــﺎل ﻟﻬ1ـــﺎ ﻧUـــ . ﻗﺎﻟـــﻪ أﺑـــ ﺣ0ﻔـــﺔ و أﻫـــﻞ .اﻟ@ﻓـــﺔ اﻟnﺎﻧﻲ ِ: أن اﻟQ1 ﻞﱡ ﺷاب ﻣُﻠ ِPُOّ ﻣ ب. ﻗﺎﻟﻪ أﻫﻞ اﻟ1ﯾ0ـﺔ وأﻫـﻞ ﻣ'ـﺔ، وﺗﻌﻠﱠـ أﺑـ ﺣ0ﻔـــﺔ 3ﺄﺣﺎدﯾـــ ﻟـــ ﻟﻬـــﺎ ﺧُPُـــ، وﻻ أزﻣﱠـــﺔ ذﻧﺎﻫـــﺎ ﻓـــﻲ ﺷـــح اﻟـــﯾ وﻣـــﺎﺋﻞ اﻟQﻼف ﻓﻼ ﯾُﻠ5ﻔl إﻟﻬﺎ. واﻟIـﺢ ﻣـﺎ روZ اﻷﺋ1ـﺔ أنﱠ :أﻧـﺎً ﻗـﺎل) ُِّﺣ ﻣـl اﻟQ1ـ ُِّﯾم ﺣlﻣ، وﻣـ ﺎ 3ﺎﻟ1ﯾ0ـﺔ ﺧ1ـ اﻷﻋ0ـﺎب إﻻ ﻗﻠـﻞ، وﻋﺎﻣـﺔ ﺧ1ﻫـﺎ اﻟ7ُْ ـ واﻟ51ـ( . Vﺧّﺟــﻪ اﻟ7Qــﺎر ُِّ. واﺗﻔــ اﻷﺋ1ــﺔ ﻋﻠــﻰ رواWــﺔ أن اﻟIــﺎ3ﺔ إذ ﺣ ﻣــl اﻟQ1ــ ﻟــ :W'ــ 0ﻋ0ﻫ ﯾﻣ O ﺧ1 ﻋ ، وﻧ1ﺎ ﺎﻧا W9ن ﺧ1 اﻟ0UO، ﻓ@وا دِﻧﺎﻧﻬ، و9ﺎدروا اﻻﻣ5nﺎل ﻻﻋ5ﻘﺎدﻫ أن ذﻟ ﻠﻪ ﺧ1. وﺻﺢّ ﻋـ: ﻋ1ـ أﻧـﻪ ﻗـﺎل ﻋﻠـﻰ اﻟ10Uـ: إن ﺗـــــ اﻟQ1ـــــ ﻧـــــvل، وﻫـــــﻲ ﻣـــــ: ﺧ1ـــــﺔ: اﻟﻌ0ـــــ و اﻟ51ـــــ واﻟﻌـــــﻞ واﻟ0Pـــــﺔ و "اﻟﻌ1. :وأﻣـــﺎ ﻣnـــﺎل ﺗﺟـــﻪ 3ﺎﻟﻠﻐـــﺔ pﻬـــ ﻓـــﻲ ﺗcﻘـــﻪ ﻣﻌ0ـــﻰ اﻟIـــﻌ* ﻓـــﻲ ﻗﻟـــﻪ ﺗﻌـــﺎﻟﻰ  َﻓ ً5ََ1ﱠ1ُــاْ ﺻَ ــﻌِاً َِّ7ــﺎ ]اﻟ0ــﺎء، 43 [ : "ﻗﻟــﻪ ﺗﻌــﺎﻟﻰ ًﺻــﻌﻴﺪا :ــﻪ أر9ﻌــﺔ أﻗــال اﻷول :وﺟﻪ اﻷرض :، ﻗﺎﻟﻪ ﻣﺎﻟ. اﻟnﺎﻧﻲ: اﻷرض اﻟ15ﺔ، ﻗﺎﻟﻪ اﺑ: زـ. اﻟnﺎﻟـ اﻷرض اﻟ1ﻠﺎء. اﻟا3ﻊ: اﻟ5اب، ﻗﺎﻟﻪ اﺑ: w7ـﺎس واﺧ5ـﺎرﻩ اﻟـﺎﻓﻌﻲ. واﻟـOV Wﻌrـﻩ اﻻﺷــ5ﻘﺎق، وﻫــ ﺻــﺢ اﻟﻠﻐــﺔ، أﻧــﻪ وﺟــﻪ اﻷرض ﻋﻠــﻰ أV وﺟــﻪ ــﺎن ﻣــ: رﻣــﻞ أو "ﺣR أو ﺗاب2. ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 53 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ :اﻟWﻠV اﻟZﺎﻣ ﻣﻬﺞ اﺑ اﻟﻌ ﻲ ﻓـ1ﻲ ﺗﻌﺎﻣﻠـﻪ ﻣـﻊ ﻣ ﺎﻟy3ـﻪ ﻓـﻲ ﻣixUـﻪ اﻟﻔﻘﻬـﻲ g+َواﻟﻌَﻘ ﻓﻲ اﻟcﻘﺔ إن اﺑ: اﻟﻌ9ﻲ ﻗ وﻗﻊ ﻓﻲ n* ﻣ: اﻟﺎﻻت ﺗl ﺗﺄﺛ* اﻟ0pة ،اﻟ1OﻫUـﺔ اﻟrــﻘﺔ، ﺑــﻞ ﻗــ دﻓﻌ5ـﻪ ﻫــOﻩ اﻟ0pــة ﻓــﻲ 3ﻌـ2 اﻷﺣــﺎن إﻟــﻰ 3ــu ﻟــﺎﻧﻪ ورﻣﻲ ﻣQﺎﻟ_ﻪ 3ﺄﻟﻔﺎ³ٍ ﻻ ﯾﻠ* أن ﺗIر ﻣ: ﻋﺎﻟٍ Uـ 3ـ ﻋﻠ1ـﺎء آﺧـ: ﺣ5ـﻰ وﻟ اﺧ5ﻠﻔا ﻣﻌﻪ ﻓﻲ اﻟ1Oﻫ واﻟأV، ﻓﻠ@ﻞٍ ﻣ0ﻬ اﺟ ª5ﻬﺎدُﻩ وأدﻟ5ُﻪ. VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﻟ@00ﺎ ﻧﻼﺣ ﻓ ﻲ ﻣﻘﺎﺑﻞ ذﻟ أن اﺑ: اﻟﻌ9ﻲ ﺎن ﻓـﻲ 3ﻌـ2 اﻷﺣـﺎن Wﻘـم 3ﺈﻧIـﺎف ﻣQﺎﻟ_ـﻪ ﻋ0ـﻣﺎ ًﯾ5ﻘــ ﻣــ: ﻗــة دﻟــﻠﻬ، وﻫــOا ﻣ0ــvعٌ ﺣــ: 0ــﺎ ﻧ510ــﻰ أن ﻧــاﻩ 3ــ'ﻞ أﻛnــ وﺿــﺣﺎ :ﻓــﻲ ﺗﻔــﻩ، وRــ أن ﻧــ ﻫ0ــﺎ أWrــﺎً إﻟــﻰ أن اﺑــ: اﻟﻌ9ــﻲ ــﺎن ﻋﻠــﻰ اﻟــﻏ ﻣــ ﺗﻌIّ ــ ،7ﻪ اﻟاﺿــﺢ وﻧIــﺗﻪ اﻟــﯾة ﻟ1Ot7ــﻪ اﻟ1ــﺎﻟ@ﻲ، ــﺎن Wﻘــم ﻓــﻲ أﺣــﺎن ﻗﻠﻠــﺔ 310ﺎﻗــﺔ ﻣOt7ــﻪ، وﺗــﺟﺢ ﻏــﻩ ﻋﻠــﻪ إذا ﺗUــ: ﻟــﻪ ﺿــﻌﻒ دﻟــﻞ ﻣOt7ــﻪ، وﻗــة دﻟــﻞ اﻟ1ــOﻫ اﻵﺧــ، وﻫــOا ﻟﻌ1ــ اﻟــ ﻣﻗــﻒ Wُ1َــ ﻟــﻪ، وُﻘــﱠ ر ﻟــﻪ ﻓــﻲ ﻣــvان ﺣــ0ﺎﺗﻪ .اﻟﻌﻠ1ﺔ واﻟ10ﻬRﺔ 1ﻣـــ: اﻷﻣnﻠـــﺔ ﻋﻠـــﻰ ﺗﻌIِّ ـــ7ﻪ ﻹﻣﺎﻣـــﻪ، ورﻓﻌـــﻪ ﻟ :ﻘﺎﻣـــﻪ ﻋﻠـــﻰ ﺣـــﺎب ﻣﻘـــﺎم ﻏـــﻩ ﻣـــ :اﻷﺋ1ﺔ، ﻗﻟﻪ ﻋ: ﺗﻔ اﻹﻣﺎم اﻟﺎﻓﻌﻲ ﻟﻠﻌل ﻓﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ  ْذَﻟِﻚَ أَدْ َ أَﻻ# wَﻌُﻮﻟُﻮا ]اﻟ0ﺎء، 3[ 3'nة اﻟqﺎل: "ﻗﻠ0ﺎ أُﻋR أﺻﺎب اﻟﺎﻓﻌﻲ 3'ﻼﻣـﻪ ﻫـOا، وﻗـﺎﻟا : ﻫـ ﺣُRــﺔٌ ﻟ10vﻟــﺔ اﻟــﺎﻓﻌﻲ ﻓــﻲ اﻟﻠﻐــﺔ، وﺷُ ــﻬﺗﻪ ﻓــﻲ اﻟﻌ9ــﺔ، واﻻﻋ5ــاف ﻟــﻪ 3ﺎﻟﻔIــﺎﺣﺔ ،ﺣ5ﻰ ﻟﻘ ﻗﺎل اﻟR0ﻲ: ﻫ أﻓIـﺢ ﻣَـ:ْ ﻧPـ 3ﺎﻟrـﺎد، ﻣـﻊ ﻏﺻـﻪ ﻋﻠـﻰ اﻟ1ﻌـﺎﻧﻲ وﻣﻌﻓ5ـــﻪ 3ﺎﻷﺻـــل ... ﻗـــﺎل اﺑـــ: اﻟﻌ9ـــﻲ: ـــﻞ ﻣـــﺎ ﻗﺎﻟـــﻪ اﻟـــﺎﻓﻌﻲ أو ﻗـــﻞ ﻋ0ـــﻪ أو ،ًوُﺻﻒ 3ﻪ ﻓﻬ ﻠﻪ ﺟvء ﻣـ: ﻣﺎﻟـ وﻧَµْ7ـﺔ ]ﺟﻋـﺔ[ ﻣـ: 3ـﻩ، وﻣﺎﻟـ أوﻋـﻰ ﺳـ1ﻌﺎ وأﺛﻘـ ﻓﻬ1ــﺎً، و أﻓIـﺢ ﻟــﺎﻧﺎً، وأﺑـع ﺑﺎﻧــ ﺎً، وأﺑــع وﺻـﻔﺎً، وــﻟﱡ ﻋﻠـﻰ ذﻟــ ﻣﻘﺎﺑﻠــﺔ "ﻗــلٍ 3ﻘــلٍ ﻓــﻲ ــﻞ ﻣــﺄﻟﺔ و ﻓَIْ ــﻞ1 . وﻣــ: اﻷﻣnﻠــﺔ أWrــﺎً، ردﱡﻩ ﻋﻠــﻰ اﻹﻣــﺎم أﺑــﻲ :ﺣ0ﻔﺔ ﻋ0ﻣﺎ ﻓﱠ )اﻷﺷّ( ﻓﻲ ﻗﻟﻪ ﺗﻌﺎﻟﻰ و َْﻻَ wَْ َﺑُﻮاْ ﻣَﺎلَ اﻟْfَِﻴﻢِ إِﻻ# ﺑِﺎﻟ#ِ` ِ:َ أَﺣ `ﺴَﻦُ ﺣَ#ـ ُﻳَﺒْﻠُـﻎَ أَﺷُـﺪ#ه ]اﻷﻧ ﻌـﺎم، 152 [ :3Q1ـﺔ وﻋــ: ﻋﺎﻣـﺎً، ﺣـ ﻗـﺎل: "ﻗـﺎل أﺑـ ﺣ0ﻔــﺔ 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 314 . 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 314 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 54 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن اﻷﺷــّ ﺧ1ــﺔ وﻋــون ﻋﺎﻣــﺎً، وﻋR7ــﺎً ﻣــ: أﺑــﻲ ﺣ0ﻔــﺔ ﻓﺈﻧــﻪ ﯾــZ أن اﻟ1ﻘــﱠ رات ﻻ ﺗnUl ﻧpاً وﻻ »ﺎﺳﺎً، وﻧ1ﺎ ﺗnUl ﻧﻘﻼً ﻋﻠـﻰ ﻣـﺎ ﺑﱠ 0ـﺎﻩ ﻓـﻲ أﺻـل اﻟﻔﻘـﻪ، وﻫـ ﯾnU5ﻬـﺎ I3ﺎﻷﺣﺎدﯾ اﻟrqﻔﺔ، وﻟ@0ﻪ ﺳ': دار اﻟrب ]Wﻘ ،3ﻐاد[ ﻓ@n ﻋ0ـﻩ اﻟ1ـﻟ وﻟــ ﺳــ': دار اﻟ1ﻌــن ]WﻘIــ اﻟ1ﯾ0ــﺔ اﻟ10ــرة[ 1ــﺎ »ــﱠ2 ﷲ ﻟ1ﺎﻟــ، ﻟَ1َــﺎ ﺻــر "ﻋ0ﻪ إﻻ إﺑv اﻟﯾ:، وﻛ اﻟ1ﻠﺔ 1ﺎ ﺻر ﻋ: ﻣﺎﻟ1. VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﻗــ ﺧــَ ا ﺑــ:ُ اﻟﻌ9ــﻲ أ3ــﺎ 3'ــ اﻟRIــﺎصَ اﻟ0ﻔــﻲ ﺑــدود ﻋﯾــة اﻣ5ــvج ﻓﻬــﺎ اﻟــد اﻟﻌﻠ1ﻲ 3ﺎﻷﻟﻔﺎ³ اﻟﻘﺎ ،ﺳﺔ اﻟﻼذﻋـﺔ ﻋﻠـﻰ ﺳـUﻞ اﻟ1nـﺎل، ﻧﻘـﻞ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﺗﻔـﻩ ﻟﻘﻟﻪ ﺗﻌﺎﻟﻰ:  وَﻣَﻦ ﻟ#ﻢْ hَﺴْﺘَﻄِﻊْ ﻣِﻨﻜُﻢْ ﻃَﻮْﻻً أَن ﻳَﻨﻜِﺢَ اﻟْﻤُﺤْﺼَﻨَﺎتِ اﻟْﻤُﺆْﻣِﻨَﺎتِ ﻓَﻤِﻦ ﻣ4ﺎ ﻣَﻠَﻜَـﺖْ أَﻳْﻤَـﺎﻧُﻜُﻢ ﻣ4ـﻦ ﻓََﻴَﺎﺗِﻜُﻢُ اﻟْﻤُﺆْﻣِﻨَـﺎتِ وَاﻟﻠّـUُ أَﻋْﻠَـﻢُ ﺑِِﻳﻤَـﺎﻧِﻜُﻢْ َﻌْﻀُ ـﻜُﻢ ﻣ4ـﻦ َﻌْـ ٍﺾ ]اﻟ0ـﺎء، 25 [ ، ﻗـلَ اﻟRIـﺎص 3ﺄﻧﻪ "ﻟ ﻧ@ﺎح اﻷﻣَـﺔ ﺿـورة، ﻷن اﻟrـورة ﻣـﺎ WُQـﺎف ﻣ0ـﻪ ﺗﻠـﻒ اﻟـ0ﻔ أو ﺗﻠـﻒ ﻋr، وﻟ ﻓﻲ ﻣﺄﻟ50ﺎ ﺷﻲء ﻣ: ذﻟ. ﻗﻠ0ﺎ: ﻫOا ﻼم ﺟﺎﻫﻞ 310ﻬـﺎج اﻟـع أو ﻣ5ﻬ'ِّ ﻻ ﯾ7ﺎﻟﻲ 31ﺎ ﯾَد اﻟﻘل. ﻧ: ﻟ ﻧﻘﻞ إﻧﻪ ﺣ'ٌ ﻧu 3ﺎﻟrورة، إﻧ1ﺎ ﻗﻠ0ﺎ: إﻧـﻪ ُﺣ ْ@ ٌ ِّﻋﻠ 3ﺎﻟﺧIﺔ اﻟ1ﻘوﻧﺔ 3ﺎﻟﺎﺟﺔ، وﻟ@ﻞِّ واﺣ ﻣ0ﻬ1ﺎ ﺣ' WQـ5ﱡ ٌ3ـﻪ، وﺣﺎﻟـﺔ ِّWُﻌ5Uــ ﻓﻬــﺎ، وﻣــ: ﻟــ Wﻔــ ق ﺑــ: اﻟrــورة واﻟﺎﺟــﺔ اﻟ5ــﻲ ﺗ@ــن ﻣﻌﻬــﺎ اﻟﺧIــﺔ ﻓــﻼ Wُﻌ0ــﻰ 3ــﺎﻟ@ﻼم ﻣﻌــﻪ، ﻓﺈﻧــﻪ ﻣﻌﺎﻧــٌ أو ﺟﺎﻫــﻞ، وﺗﻘــﯾ ذﻟــ إﺗﻌــﺎب ﻟﻠــ0ﻔ ﻋ0ــ ﻣــ: ﻻ "ﯾ05ﻔﻊ 3ﻪ2 . اﻷﺷــّ ﺧ1ــﺔ وﻋــون ﻋﺎﻣــﺎً، وﻋR7ــﺎً ﻣــ: أﺑــﻲ ﺣ0ﻔــﺔ ﻓﺈﻧــﻪ ﯾــZ أن اﻟ1ﻘــﱠ رات ﻻ ﺗnUl ﻧpاً وﻻ »ﺎﺳﺎً، وﻧ1ﺎ ﺗnUl ﻧﻘﻼً ﻋﻠـﻰ ﻣـﺎ ﺑﱠ 0ـﺎﻩ ﻓـﻲ أﺻـل اﻟﻔﻘـﻪ، وﻫـ ﯾnU5ﻬـﺎ I3ﺎﻷﺣﺎدﯾ اﻟrqﻔﺔ، وﻟ@0ﻪ ﺳ': دار اﻟrب ]Wﻘ،3ﻐاد[ ﻓ@n ﻋ0ـﻩ اﻟ1ـﻟ وﻟــ ﺳــ': دار اﻟ1ﻌــن ]WﻘIــ اﻟ1ﯾ0ــﺔ اﻟ10ــرة[ 1ــﺎ »ــﱠ2 ﷲ ﻟ1ﺎﻟــ، ﻟَ1َــﺎ ﺻــر "ﻋ0ﻪ إﻻ إﺑv اﻟﯾ:، وﻛ* اﻟ1ﻠﺔ 1ﺎ ﺻر ﻋ: ﻣﺎﻟ1. : ر * ﯾ: و إﺑv إ وﻗــ ﺧــَ ا ﺑــ:ُ اﻟﻌ9ــﻲ أ3ــﺎ 3'ــ اﻟRIــﺎصَ اﻟ0ﻔــﻲ ﺑــدود ﻋﯾــة اﻣ5ــvج ﻓﻬــﺎ اﻟــد اﻟﻌﻠ1ﻲ 3ﺎﻷﻟﻔﺎ³ اﻟﻘﺎ ،ﺳﺔ اﻟﻼذﻋـﺔ ﻋﻠـﻰ ﺳـUﻞ اﻟ1nـﺎل، ﻧﻘـﻞ اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﺗﻔـﻩ ﻟﻘﻟﻪ ﺗﻌﺎﻟﻰ:  وَﻣَﻦ ﻟ#ﻢْ hَﺴْﺘَﻄِﻊْ ﻣِﻨﻜُﻢْ ﻃَﻮْﻻً أَن ﻳَﻨﻜِﺢَ اﻟْﻤُﺤْﺼَﻨَﺎتِ اﻟْﻤُﺆْﻣِﻨَﺎتِ ﻓَﻤِﻦ ﻣ4ﺎ ﻣَﻠَﻜَـﺖْ أَﻳْﻤَـﺎﻧُﻜُﻢ ﻣ4ـﻦ ﻓََﻴَﺎﺗِﻜُﻢُ اﻟْﻤُﺆْﻣِﻨَـﺎتِ وَاﻟﻠّـUُ أَﻋْﻠَـﻢُ ﺑِِﻳﻤَـﺎﻧِﻜُﻢْ َﻌْﻀُ ـﻜُﻢ ﻣ4ـﻦ َﻌْـ ٍﺾ ]اﻟ0ـﺎء، 25 [ ، ﻗـلَ اﻟRIـﺎص 3ﺄﻧﻪ "ﻟ ﻧ@ﺎح اﻷﻣَـﺔ ﺿـورة، ﻷن اﻟrـورة ﻣـﺎ WُQـﺎف ﻣ0ـﻪ ﺗﻠـﻒ اﻟـ0ﻔ أو ﺗﻠـﻒ ﻋr، وﻟ ﻓﻲ ﻣﺄﻟ50ﺎ ﺷﻲء ﻣ: ذﻟ. ﻗﻠ0ﺎ: ﻫOا ﻼم ﺟﺎﻫﻞ 310ﻬـﺎج اﻟـع أو ﻣ5ﻬ'ِّ ﻻ ﯾ7ﺎﻟﻲ 31ﺎ ﯾَد اﻟﻘل. 1 ( ،اﺑ: اﻟﻌ9ﻲ ،nاﻟ;+ر اﻟﺎﺑ 2 / 771 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت ﻧ: ﻟ ﻧﻘﻞ إﻧﻪ ﺣ'ٌ ﻧu 3ﺎﻟrورة، إﻧ1ﺎ ﻗﻠ0ﺎ: إﻧـﻪ ُﺣ ْ@ ٌ ِّﻋﻠ 3ﺎﻟﺧIﺔ اﻟ1ﻘوﻧﺔ 3ﺎﻟﺎﺟﺔ، وﻟ@ﻞِّ واﺣ ﻣ0ﻬ1ﺎ ﺣ' WQـ5ﱡ ٌ3ـﻪ، وﺣﺎﻟـﺔ ِّWُﻌ5Uــ ﻓﻬــﺎ، وﻣــ: ﻟــ Wﻔــ ق ﺑــ: اﻟrــورة واﻟﺎﺟــﺔ اﻟ5ــﻲ ﺗ@ــن ﻣﻌﻬــﺎ اﻟﺧIــﺔ ﻓــﻼ Wُﻌ0ــﻰ 3ــﺎﻟ@ﻼم ﻣﻌــﻪ، ﻓﺈﻧــﻪ ﻣﻌﺎﻧــٌ أو ﺟﺎﻫــﻞ، وﺗﻘــﯾ ذﻟــ إﺗﻌــﺎب ﻟﻠــ0ﻔ ﻋ0ــ ﻣــ: ﻻ "ﯾ05ﻔﻊ 3ﻪ2 . وﻣــ: أﻣnﻠــﺔ إﻧIــﺎف اﺑــ: اﻟ ﻌ9ــﻲ ﻟ1Qﺎﻟ_ــﻪ ﻗﻟــﻪ ﻓــﻲ أﺑــﻲ ﺣ0ﻔــﺔ ﻋ0ــﻣﺎ ﻓﱠ ــ :ﻗﻟﻪ ﺗﻌﺎﻟﻰ ِiُﻠُﻮاْ ﻣِﻦ ﺛَﻤَﺮِهِ إِذَا أَﺛْﻤَﺮَ وَآﺗُﻮاْ ﺣَﻘ#Uُ ﻳَﻮْمَ ﺣَﺼَﺎدِه ]اﻷﻧﻌﺎم، 141 [ 3ﻘﻟـﻪ: "وأﻣـﺎ أﺑـ ﺣ0ﻔــﺔ ﻓRﻌــﻞ اﻵWــﺔ ﻣآﺗــﻪ ﻓﺄ3Iــ اﻟــ، وﻗــﺎل: إن ﷲ أوﺟــ اﻟvــﺎة ﻓــﻲ اﻟ1ــﺄﻛل ﻗُﺗـــﺎً ـــﺎن أو ﻏـــﻩ، و9ـــﱠ: اﻟ0Uـــﻲ ذﻟـــ ﻓـــﻲ ﻋ1ـــم) :ﻗﻟـــﻪ 1ـــﺎ ﺳـــﻘl اﻟـــ1ﺎء ُْاﻟﻌ( 3 . :وﻣ: أﻣnﻠﺔ ﻣQﺎﻟﻔ5ﻪ ﻹﻣﺎﻣﻪ، ﻗﻟﻪ ﻓﻲ ﺗﻔ ﻗﻟﻪ ﺗﻌﺎﻟﻰ #وَﻟَﻘَﺪْ آﺗَfْﻨَﺎ دَاوُودَ ﻣِﻨ ﺎ َﻓَﻀْﻼً ﻳَﺎ ﺟِﺒَﺎلُ أَو4ِ: ﻣَﻌَUُ وَاﻟﻄ#Zْjَ وَأَﻟَﻨ#ﺎ ﻟَUُ اﻟْﺤَﺪِﻳﺪ ]ﺳ7ﺄ، 10 [: "ﻗﻟﻪ ًﻓﻀﻼ ... ﻪ دﻟﻞ ﻋﻠﻰ 1 ( ،اﺑ: اﻟﻌ9ﻲ ،nاﻟ;+ر اﻟﺎﺑ 2 / 771 . 2 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 394 . 3 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 2 / 759 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ اﻹﻋRﺎب 3: اﻟIت... واﺳ5: n ﻣ: ﻓﻘﻬﺎء اﻷﻣIﺎر اﻟﻘـاءة 3ﺎﻷﻟـﺎن واﻟ5ﺟﻊ، وﻫﻪ ﻣﺎﻟ. وﻫ ﺟﺎﺋv ﻟﻘل أﺑﻲ ﻣﺳﻰ ﻟﻠ0Uﻲ ﻋﻠﻪ اﻟﻼم: ﻟ ُl1ﻋﻠ ِأﻧ ﺗ1ﻊ ﻟَUّﺗﻪ ﻟ ﺗUاً، ﯾـ ﻟَRﻌﻠ5ـﻪ ﻟـ أﻧاﻋـﺎً ﺣ ـﺎﻧﺎ، وﻫـ اﻟ5ﻠـ: ﻣـﺄﺧذ ﻣـــ: اﻟnـــب اﻟ1Uﱠـــ وﻫـــ اﻟ1QPـــu 3ـــﺎﻷﻟان.. واﻷﺻـــات اﻟـــ0ﺔ ﻧﻌ1ـــﺔ ﻣـــ: ﷲ ﺗﻌـــﺎﻟﻰ، وزـــﺎدة ﻓـــﻲ اﻟQﻠـــ وﻣ0ّـــﺔ، وأﺣـــ ﻣـــﺎ ﻟ7ـــl ﻫـــOﻩ اﻟﻠـــﺔ اﻟ0_ـــﺔ واﻟ1t7ـــﺔ َاﻟ@1ﺔ 5ﺎب ﷲ، ﻓَ0ِﻌ ﷲ ُإذا ﺻُ ﻓl ﻓﻲ اﻟPﺎﻋﺔ ﻓﻘ ﻗ"rﻲ ﺑﻬﺎ ﺣﱡ اﻟ0ﻌ1ﺔ1 . وأﻣﺎ أﺳﻠ9ﻪ ﻣ: ﺣ* اﻟ5ﻌﺎﻣﻞ ﻣﻊ ﻣQﺎﻟ_ﻪ ﻓﻲ ﻣOt7ﻪ اﻟﻌﻘـV، ﻓﻘـ أﺷـﻧﺎ ﻓـﻲ ﻓﻘـة ،(ﺳـــﺎ3ﻘﺔ إﻟـــﻰ أنّ اﺑـــ: اﻟﻌ9ـــﻲ ـــﺎن ﻋﻠـــﻰ ﻋﻘـــة أﻫـــﻞ اﻟـــ0ﺔ واﻟR1ﺎﻋـــﺔ )اﻷﺷـــﺎﻋة واﻋ5ﻘﺎدﻩ ﻫOا واﺿﺢٌ ـﻞ اﻟﺿـح ﻓـﻲ ﺗﻔـﻩ، ﻓﻬـ WR5ﻬـ ﻓـﻲ ﺗﻔـ* اﻵWـﺎت وﻓـ ﻣﻘ5rــﺎت وأﺻــل اﻷ ،ًﺷــﺎﻋة ﻠ1ــﺎ وﺟــ إﻟــﻰ ذﻟــ ﺳــUﻼ وﻘــم ﻓــﻲ اﻟﻗــl ﻧﻔــﻪ 31ــﻼت ردٍ ﻗــﺔ ﻋﻠــﻰ ﻣQﺎﻟ_ــﻪ ﻣــ: أﻫــﻞ اﻟ1ــOاﻫ اﻷﺧــZ، و9ــ'ﻞ ﺧــﺎص ﻋﻠــﻰ اﻟ1ﻌ5vﻟــﺔ. 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 4 / 1596 - 1597 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 2 / - 997 . 1 ( ،اﺑ: اﻟﻌ9ﻲ nاﻟ;+ر اﻟﺎﺑ ، 4 / 1596 - 1597 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﻣــ: اﻷﻣnﻠــﺔ ﻋﻠــﻰ ذﻟــ ﺗﻔــﻩ ﻟؤــﺔ ﷲ :ﻓــﻲ ﺗﻔــﻩ ﻟﻘﻟــﻪ ﺗﻌــﺎﻟﻰ وﺳـــZjى اﷲ ﻋﻤﻠﻜـــﻢ ]اﻟ59ـــﺔ، 94 [: "اﻟ7ـــﺎرV راءٍ ﻣﺋـــﻲ، ﯾـــZ اﻟQﻠـــ ووﻧـــﻪ ، ﻓﺄﻣـــﺎ رؤ5ﻬ ﻟﻪ ﻓﻔﻲ ﻣﻞ ﻣQIص]WﻘI ﻓﻲ اﻟR0ﺔ[، :وﻣ: ﻗمٍ ﻣQIﺻ] IWﻘ :0اﻟ1ــﻣ[ ، وأﻣــﺎ رؤ5ــﻪ ﻟﻠQﻠــ ﻓاﺋ1ــﺔٌ، ﻓﻬــ ﺗﻌــﺎﻟﻰ Wﻌﻠــ وــ :Z. وﻗــﺎل ﺟ1ﺎﻋــﺔ ﻣــ اﻟ1U5ﻋﺔ: إﻧﻪW ٌﻌﻠ وﻻ ﯾُZ، وﻣ5ﻰ أﺧU ﻋ0ﻪ 3ﺎﻟؤﺔ ﻓﺈﻧﻬـﺎ راﺟﻌـﺔ إﻟـﻰ اﻟﻌﻠـ، وﻗـ دﻟﻠ0ـﺎ ﻓـﻲ 5ـ اﻷﺻــل ﻋﻠـﻰ أﻧـﻪ راءٍ ﺑؤــﺔ، 1ـﺎ أﻧـﻪ ﻋـﺎ :ﻟ 3ﻌﻠــ، ﻷﻧـﻪ أﺧUـ ﻋــ ﻧﻔﻪ ﺑOﻟ ُU، وﺧﻩ ﺻﺎدق، وﻟ ﻟ :'W Vراﺋﺎً ﻟَ@ﺎن ﻣوﻓﺎً ]أﺻﺎﺣ ﻋﺎﻫﺔ[، ﻷن اﻟــﻲ إذا ﻟــ W'ــ: ﻣُــْرِﺎً ــﺎن ﻣوﻓــﺎً، وﻫــ اﻟ15ﻘــِّس ﻋــ: اﻵﻓــﺎت واﻟ0ﻘــﺎﺋ، وﻫــOﻩ اﻟﻌُ1ة اﻟﻌﻘﻠﺔ ﻟﻌﻠ1ﺎﺋ0ﺎ، ﻓﻘ أﺧU ﺳ7ﺎﻧﻪ ﻋ: ﻧﻔﻪ 31ﺎ WR ﻟﻪ ﻣـ: ﺻـﻔ5ﻪ، وﻗـﺎم "اﻟﻟﻞ ﻋﻠﻪ ﻣ: ﻧﻌ5ﻪ، ﻓﻠvﻣ0ﺎ اﻋ5ﻘﺎدﻩ واﻹﺧ7ﺎر 3ﻪ2. ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 56 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن :اﻟWﻠV اﻟ4ﺎﺳﻊ ﻣJ{z اﺑ اﻟﻌ ﻲ ﻣ أﺣ+اث ﻋ; ﻩ أﻧــvل ﷲ 5ﺎ3ــﻪ اﻟ@ــ ﻟُِQــج اﻟ0ــﺎس ﻣــ: اﻟpﻠ1ــﺎت، وﻟﻬــﯾﻬ إﻟــﻰ أﻗــم اﻟُ ــUُﻞ اﻟ5ــﻲ ﺗﺄﺧـــO 3ﺄﯾــﯾﻬ إﻟـــﻰ ﺗﻘــ ﻣIــﺎﻟﻬ، ودﻓـــﻊ اﻟ1ﻔﺎﺳــ ﻋـــ0ﻬ ﻓــﻲ ﻫـــOﻩ ،اﻟـــﻧﺎ واﻟـــار اﻵﺧـــة ﻋﻠـــﻰ اﻟـــاء. وآWـــﺎت اﻟﻘـــآن اﻟ5ـــﻲ ﺗـــ إﻟـــﻰ ﻫـــOﻩ اﻟcﻘـــﺔ واﺿﺔٌ ﻓﻲ دﻻﻻﺗﻬﺎ، وﻻ ﺗ51ﻞ أVﱠ ﺗﺄوﻞ. ﻣnﻞ ﻗﻟﻪ ﺗﻌﺎﻟ :ﻰ إِن# <َــﺬَ ا اﻟْـُ ْآنَ ﻳِ3ْـﺪِي ًﻟِﻠ#ِ` ِ:َ أَﻗْﻮَمُ وَﻳُﺒَ4 ُ اﻟْﻤُﺆْﻣِﻨِZYَ اﻟ#ﺬِﻳﻦَ hَﻌْﻤَﻠُـﻮنَ اﻟﺼ# ـﺎﻟِﺤَﺎتِ أَن# ﻟَ3ُـﻢْ أَﺟْـﺮاً ﻛَﺒِZـjا ]اﻹﺳـاء، 9[ . ﻟـOﻟ أدرك أﻫﻞ اﻟ ﻣ: ﻋﻠ1ﺎء اﻹﺳﻼم أنّ ﻋﻠﻬ واﺟ7ﺎً Uاً، ﯾ51nﻞ ﻓﻲ أدﻧﻰ درﺟﺎﺗﻪ ﻓﻲ ﺑﺎن أﺣ'ﺎم اﻹﺳﻼم ﻓﻲ اﻟﻘrـﺎWﺎ اﻟ@UـZ اﻟ5ـﻲ ﯾ5ﻌـّض ﻟﻬـﺎ اﻟ1ـﻠ1ن، و9ـ'ﻞ ﺧﺎص ﻓﻲ أوﻗﺎت اﻷزﻣﺎت اﻟ5ﻲ ﺗﻌIﻒ 3ـﺎدﺗﻬ ﻋﻠـﻰ أراﺿـﻬ، وﺗﻬـد وﺟـدﻫ V3'ﻞ ﻋﺎم، وﻓﻲ دﻋﺗﻬ إﻟﻰ ﻧُIة وﻣازرة 3ﻌrﻬ 3ﻌrﺎً إذا أﺣق اﻟQP 3ﺄ فٍ ﻣ0ﻬ. VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﻫOا 3ﺎﻟﻔﻌﻞ ﻣـﺎ W1'ـ: أن ﻧﻠ1ـﻪ 3Rـﻼء ﻋ0ـ اﺑـ: ا ﻟﻌ9ـﻲ، وﻗـ ﻋﻓ0ـﺎ أﻧﻪ ﻋﺎﺻ أﺣاﺛﺎً ﺗﺎرQﺔً وﺳﺎﺳـﺔً داﻣـﺔً، ﻓﻘـ أدﱠZ أﻣﺎﻧـﺔ اﻟUـﺎن واﻟ0Iـﺔ اﻟ5ـﻲ أﻧﺎﻬــــﺎ ﷲ :3ﺎﻟﻌﻠ1ــــﺎء، ﻋ0ــــﻣﺎ وﺟــــﻧﺎﻩ ﻓــــﻲ أﻛnــــ ﻣــــ: ﻣ'ــــﺎن ﻓــــﻲ ﺗﻔــــﻩ ﯾُUــــ ﻟﻠ1ﻠ1: أﺣ'ـﺎم ﷲ و9Pﻬـﺎ 3ـﺄﺣاﻟﻬ، وـﻋﻫ ﻟﻠﻗـف ﯾـاً واﺣـةً ﻓـﻲ وﺟـﻪ vاﻟﻔﻧRـــﺔ اﻟـــOﯾ: ﻏـــ وا 3ﻼدﻫـــ، وﻓ5@ـــا 3ـــﺄرواﺣﻬ، ﻓﻬـــﺎ ﻫـــ Wﻘـــل ﻓـــﻲ ﺗﻔـــﻩ ﻟﻘﻟـــﻪ :ﺗﻌــﺎﻟﻰ  ُاDْــِ ُواْ ﺧِﻔَﺎﻓــﺎً وَِﻘَــﺎﻻً وَﺟَﺎ<ِــﺪُواْ ﺑِــَﻣْﻮَاِﻜُﻢْ وَأَDﻔُﺴِــﻜُﻢْ ; ِــ: ﺳَــ\|ِﻴﻞِ اﻟﻠّــUِ ذَِﻜُــﻢْ ﺧَZْــjٌ #ﻜُــﻢْ إِن ﻛُﻨــﺘ ْﻢ َwَﻌْﻠَﻤُــﻮن ]اﻟ59ــﺔ، 41 [: ...:"اﺧْ5ُﻠِــﻒ ﻓــﻲ إﺣ'ــﺎم ﻫــOﻩ اﻵWــﺔ أو ﻧــQﻬﺎ ﻋﻠــﻰ ﻗــﻟ :واﻟIـــﺢ أﻧﻬـــﺎ ﻏـــ ﻣ0ـــﺧﺔ، وﻗـــ ﺗ@ـــن ﺣﺎﻟـــﺔٌ WRـــ ﻓﻬـــﺎ ﻧﻔـــ اﻟ@ـــﻞ إذا ﺗﻌـــﱠ ،ْاﻟRﻬـــﺎد ﻋﻠـــﻰ اﻷwـــﺎن 3ﻐﻠ7ـــﺔ اﻟﻌـــو ﻋﻠـــﻰ ﻗPـــٍ ﻣـــ: اﻷﻗPـــﺎر، أو 3ﻠﻟـــﻪ 3ـــﺎﻟﻌُﻘ R ﻋﻠﻰ ﺎﻓﺔ اﻟQﻠ اﻟRﻬﺎد واﻟQـوج إﻟـﻪ؛ ﻓـﺈن ﻗIﱠ ـوا ﻋIـا. وﻟﻘـ ﻧـvل ﺑ0ـﺎ ،اﻟﻌو-ﻗI1ﻪ ﷲ- ،ﺳ0ﺔ ﺳ7ﻊ وﻋ: وﺧ11ﺎﺋﺔ، ﻓRـﺎس دWﺎرﻧـﺎ، وأﺳـ ﺟﺗ0ـﺎ َوﺗﺳﱠ ــu 3ﻼدﻧــﺎ ﻓــﻲ ﻋــدٍ ﻫــﺎل ،اﻟ0ــﺎس ﻋــدُﻩ، وــﺎن nــاً، ون ﻟــ ﯾUﻠــﻎ ﻣــﺎ ﺣــﱠدوﻩ :@5ﻓﻘﻠl ﻟﻠاﻟﻲ واﻟ1ﻟﻰ ﻋﻠﻪ: ﻫـOا ﻋـو ﷲ، وﻗـ ﺣIـﻞ ﻓـﻲ اﻟﱠ ـَك واﻟﱠ ـ7'ﺔ، ﻓﻠـ 0ﻋ0 ﺑﺔ، وﻟ5pﻬ ﻣ @ إﻟﻰ ﻧIة دﯾ: ﷲ اﻟ15ﻌِّ0ﺔ ﻋﻠ' ﺣﺔ، ﻓﻠQج إﻟـﻪ ﺟ1ــﻊ اﻟ0ــﺎس ﺣ5ــﻰ ﻻ ﯾ7ﻘــﻰ ﻣــ0@ أﺣــﱞ ﻓــﻲ ﺟ1ــﻊ ﻫــOﻩ اﻷﻗPــﺎر، ُــﺎ 3ــﻪ، ﻓﺈﻧــﻪ ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 57 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ ،ﻫﺎﻟـــٌ ﻻ ﻣﺎﻟـــﺔ إن Wﱠ ـــ ﷲ ﻟـــﻪ؛ ﻓﻐﻠUـــl اﻟـــOﻧب، ورﺟﻔـــl اﻟﻘﻠـــب 3ﺎﻟ1ﻌﺎﺻـــﻲ 1وﺻﺎر ﻞ أﺣ ﻣ: اﻟ0ﺎس ﺛﻌﻠ7ﺎً WﺄوV إﻟﻰ وِﺟَـﺎرﻩ، ون رأZ اﻟ x 'ـوﻩ 3Rـﺎرﻩ؛ ﻓﺈﻧـﺎ وﻧــﺎ إﻟــﻪ راﺟﻌــن، وﺣــU0ﺎ ﷲ وﻧﻌــ اﻟــﻞ... إذا ــﺎن اﻟ0ﻔــ ﻋﺎﻣــﺎً ﻟﻐﻠ7ــﺔ اﻟﻌــو ًﻋﻠــﻰ اﻟــزة أو اﺳــ5ﻼﺋﻪ ﻋﻠــﻰ اﻷﺳــﺎرZ، ــﺎن اﻟ0ﻔــ ﻋﺎﻣــﺎ ووﺟــ اﻟQــوج ﺧِﻔﺎﻓــﺎ وﺛِﻘﺎﻻً، ورُﻛ7ﺎﻧﺎً ورﺟﺎﻻً، ﻋUاً وأﺣاراً، ﻣ:ْ ﺎن ﻟﻪ أب ﻣ: ﻏ إذﻧـﻪ، وﻣـ: ﻻ أب ﻟﻪ O، ﺣ5ﻰ Wpﻬ دﯾ: ﷲ، وﺗُ1ﻰ اﻟUrﺔ، وﺗُﻔª اﻟزة، وQvZ اﻟﻌو، وـ50ﻘ اﻷﺳZ، وﻻ ﺧﻼف ﻓـﻲ ﻫـOا. وﻟﻘـ رُوV أن 3ﻌـ2 اﻷﻣـاء ﻋﺎﻫـ ﻔـﺎرا أﻻﱠ W7ـا أﺳــاً، ﻓــﺧﻞ رﺟــﻞ ﻣــ: ﺟﻬ5ــﻪ 3ﻼدﻫــ، ﻓ1ــﱠ ﻋﻠــﻰ ﺑــl ﻣﻐْﻠــ، ﻓ0ﺎدﺗــﻪ اﻣــأة: إﻧــﻲ أﺳـــة، ﻓـــﺄﺑﻠِﻎْ ﺻـــﺎﺣ7 ﺧUـــV. VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت ﻓﻠ1ـــﺎ اﺟ51ـــﻊ 3ـــﻪ، اﺳـــ5Pﻌ1ﻪ ﻋ0ـــﻩ، وﺗRﺎذ3ـــﺎ ذﯾـــﻞ اﻟﯾ، ﺣ5ﻰ اﻧ5ﻬﻰ اﻟQUـُ إﻟـﻰ ﻫـOﻩ اﻟ1ﻌOﱠ3ـﺔ، ﻓﺄﻟﻘـﺎﻩ إﻟـﻪ، ﻓ1ـﺎ أﻛ1ـﻞ ﺣﯾnـﻪ ﺣ5ـﻰ ﻗـــﺎم اﻷﻣـــ ﻋﻠـــﻰ ﻗﻣـــﻪ، وﺧـــج ﻏﺎزـــﺎً ﻣـــ: ﻓـــرﻩ، وﻣـــﻰ إﻟـــﻰ اﻟUﻠـــ ﺣ5ـــﻰ أﺧـــج اﻷﺳة، واﺳ5ﻟﻰ ﻋﻠﻰ اﻟ1ﺿﻊ. ﻓ@ـe ﺑ0ـﺎ وﻋ0ـﻧﺎ ﻋﻬـُ ﷲ أﻻﱠ ﻧـﻠِّ إﺧاﻧ0ـﺎ إﻟـﻰ اﻷﻋ اء، وﻧَ0ْﻌ وﻫ ﻓﻲ اﻟﻘﺎء، أو ﻧ1ﻠ اﻟﺔ وﻫ أرﻗﱠـﺎء. Wـﺎ x، وﻟﻬـOا اﻟQPـ ّ01اﻟR! ﻧﺄل ﷲ اﻟ5ﻓ ﻟﻠR1ﻬر، واﻟ"ﺔ 3Iﻼح اﻵﻣ واﻟ1ﺄﻣر1 . وﺎن Wﻐ50 أWﺔ ﻣ0ﺎﺳ7ﺔ ﺗﻠح ﻟﻪ، ﻟ* ﻣ: ﺧﻼﻟﻬﺎ إﻟﻰ ﻣﻗﻊٍ ﺟﻐاﻓﻲ رآﻩ، أو واﻗﻊ اﺟ51ﺎﻋﻲ ﺷﺎﻫﻩ، أو ﺣارٍ أو ﺣﺎدﺛﺔٍ ﻋ ًﻠ1ﺔ أو ﺳﺎﺳﺔ أو ﺗﺎرQﺔ، ﺎن ﻫ ﻓﺎ ﻓﻬـــﺎ. وﻧpـــاً ﻟ@nـــة إﺷـــﺎراﺗﻪ ﻫـــOﻩ، W1'ـــ: ﻷV ﻣـــرخٍ إذا ﻗـــﺎم 3ﺎﺳ5ﻘIـــﺎﺋﻬﺎ وﺗﻠﻠﻬـــﺎ ،ور9Pﻬﺎ 3ﺎﻗﻬﺎ اﻟﻌﻠ1ﻲ واﻟ5ـﺎرQﻲ اﻟﻌـﺎم، أن Wﻘـِّم ﻟ0ـﺎ ﺻـرةً، وﻟـ ﺎﻧـl ﻣـودة :ﻋـــ: اﻟU* ـــﺔ اﻟﻌﻠ1ـــﺔ واﻟ5ﺎرQـــﺔ واﻟRﻐاـــﺔ اﻟ5ـــﻲ ﻋـــﺎش وﺷـــﺎرك ﻓﻬـــﺎ اﺑـــ اﻟﻌ9ـــﻲ 3ﻔﻌﺎﻟـــﺔٍ، ﺳـــاء ﻓـــﻲ 3ـــﻼد اﻟـــق اﻹﺳـــﻼﻣﻲ اﻟ5ـــﻲ ﻋـــﺎش ﻓ5ـــةً ﻓـــﻲ ﻣـــﻧﻬﺎ وﻣاﻛvﻫـــﺎ اﻟﻌﻠ1ﺔ، أم ﻓﻲ ﺣاﺿ اﻷﻧﻟ ﻓﻲ ﻧﻬﺎWـﺔ اﻟﻘـن اﻟQـﺎﻣ اﻟﻬRـV، واﻟnﻠـ اﻷول :ﻣ: اﻟﻘن اﻟﺎدس اﻟﻬRV. وﻣ: اﻷﻣnﻠﺔ ﻋﻠﻰ ذﻟـ، ﻗﻟـﻪ ﻓـﻲ ﺗﻔـ ﻗﻟـﻪ ﺗﻌـﺎﻟﻰ  ْﻳَﺎ أَﻳ3َﺎ اﻟ#ﺬِﻳﻦَ آﻣَﻨُﻮا ًإِﻧ#ﻤَﺎ اﻟْﻤُْ ِﻛُﻮنَ ﻧَﺠَﺲٌ ﻓَﻼَ hَْ َﺑُـﻮاْ اﻟْﻤَﺴْـﺠِﺪَ اﻟْـَ َامَ َﻌْـﺪَ ﻋَـﺎﻣِ3ِﻢْ <َــﺬَ ا وَإِنْ ﺧِﻔْـﺘُﻢْ ﻋَﻴْﻠَـﺔ ٌﻓَﺴَ ــﻮْفَ hُﻐْﻨِــﻴﻜُﻢُ اﻟﻠّــUُ ﻣِــﻦ ﻓَﻀْ ــﻠِUِ إِن ﺷَــﺎء إِن# اﻟﻠّــUَ ﻋَﻠِــﻴﻢٌ ﺣَﻜِــﻴﻢ ]اﻟ59ــﺔ، 28 [: Z"وﻟﻘــ 0ــlُ أر ،ﻫﺎﻟـــٌ ﻻ ﻣﺎﻟـــﺔ إن Wﱠ ـــ ﷲ ﻟـــﻪ؛ ﻓﻐﻠUـــl اﻟـــOﻧب، ورﺟﻔـــl اﻟﻘﻠـــب 3ﺎﻟ1ﻌﺎﺻـــﻲ 1وﺻﺎر ﻞ أﺣ ﻣ: اﻟ0ﺎس ﺛﻌﻠ7ﺎً WﺄوV إﻟﻰ وِﺟَـﺎرﻩ، ون رأZ اﻟ x 'ـوﻩ 3Rـﺎرﻩ؛ ﻓﺈﻧـﺎ وﻧــﺎ إﻟــﻪ راﺟﻌــن، وﺣــU0ﺎ ﷲ وﻧﻌــ اﻟــﻞ... إذا ــﺎن اﻟ0ﻔــ ﻋﺎﻣــﺎً ﻟﻐﻠ7ــﺔ اﻟﻌــو ًﻋﻠــﻰ اﻟــزة أو اﺳــ5ﻼﺋﻪ ﻋﻠــﻰ اﻷﺳــﺎرZ، ــﺎن اﻟ0ﻔــ ﻋﺎﻣــﺎ ووﺟــ اﻟQــوج ﺧِﻔﺎﻓــﺎ وﺛِﻘﺎﻻً، ورُﻛ7ﺎﻧﺎً ورﺟﺎﻻً، ﻋUاً وأﺣاراً، ﻣ:ْ ﺎن ﻟﻪ أب ﻣ: ﻏ* إذﻧـﻪ، وﻣـ: ﻻ أب ﻟﻪ O، ﺣ5ﻰ Wpﻬ دﯾ: ﷲ، وﺗُ1ﻰ اﻟUrﺔ، وﺗُﻔª اﻟزة، وQvZ اﻟﻌو، وـ50ﻘ اﻷﺳZ، وﻻ ﺧﻼف ﻓـﻲ ﻫـOا. وﻟﻘـ رُوV أن 3ﻌـ2 اﻷﻣـاء ﻋﺎﻫـ ﻔـﺎرا أﻻﱠ W7ـا أﺳــاً، ﻓــﺧﻞ رﺟــﻞ ﻣــ: ﺟﻬ5ــﻪ 3ﻼدﻫــ، ﻓ1ــﱠ ﻋﻠــﻰ ﺑــl ﻣﻐْﻠــ، ﻓ0ﺎدﺗــﻪ اﻣــأة: إﻧــﻲ أﺳـــة، ﻓـــﺄﺑﻠِﻎْ ﺻـــﺎﺣ7 ﺧUـــV. ﻓﻠ1ـــﺎ اﺟ51ـــﻊ 3ـــﻪ، اﺳـــ5Pﻌ1ﻪ ﻋ0ـــﻩ، وﺗRﺎذ3ـــﺎ ذﯾـــﻞ اﻟﯾ، ﺣ5ﻰ اﻧ5ﻬﻰ اﻟQUـُ إﻟـﻰ ﻫـOﻩ اﻟ1ﻌOﱠ3ـﺔ، ﻓﺄﻟﻘـﺎﻩ إﻟـﻪ، ﻓ1ـﺎ أﻛ1ـﻞ ﺣﯾnـﻪ ﺣ5ـﻰ ﻗـــﺎم اﻷﻣـــ ﻋﻠـــﻰ ﻗﻣـــﻪ، وﺧـــج ﻏﺎزـــﺎً ﻣـــ: ﻓـــرﻩ، وﻣـــﻰ إﻟـــﻰ اﻟUﻠـــ ﺣ5ـــﻰ أﺧـــج اﻷﺳة، واﺳ5ﻟﻰ ﻋﻠﻰ اﻟ1ﺿﻊ. VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت ﻓ@ـe ﺑ0ـﺎ وﻋ0ـﻧﺎ ﻋﻬـُ ﷲ أﻻﱠ ﻧـﻠِّ إﺧاﻧ0ـﺎ إﻟـﻰ اﻷﻋ اء، وﻧَ0ْﻌ وﻫ ﻓﻲ اﻟﻘﺎء، أو ﻧ1ﻠ اﻟﺔ وﻫ أرﻗﱠـﺎء. Wـﺎ x، وﻟﻬـOا اﻟQPـ ّ01اﻟR! ﻧﺄل ﷲ اﻟ5ﻓ ﻟﻠR1ﻬر، واﻟ"ﺔ 3Iﻼح اﻵﻣ واﻟ1ﺄﻣر1 . وﺎن Wﻐ50 أWﺔ ﻣ0ﺎﺳ7ﺔ ﺗﻠح ﻟﻪ، ﻟ* ﻣ: ﺧﻼﻟﻬﺎ إﻟﻰ ﻣﻗﻊٍ ﺟﻐاﻓﻲ رآﻩ، أو واﻗﻊ اﺟ51ﺎﻋﻲ ﺷﺎﻫﻩ، أو ﺣارٍ أو ﺣﺎدﺛﺔٍ ﻋ ًﻠ1ﺔ أو ﺳﺎﺳﺔ أو ﺗﺎرQﺔ، ﺎن ﻫ ﻓﺎ ﻓﻬـــﺎ. وﻧpـــاً ﻟ@nـــة إﺷـــﺎراﺗﻪ ﻫـــOﻩ، W1'ـــ: ﻷV ﻣـــرخٍ إذا ﻗـــﺎم 3ﺎﺳ5ﻘIـــﺎﺋﻬﺎ وﺗﻠﻠﻬـــﺎ ،ور9Pﻬﺎ 3ﺎﻗﻬﺎ اﻟﻌﻠ1ﻲ واﻟ5ـﺎرQﻲ اﻟﻌـﺎم، أن Wﻘـِّم ﻟ0ـﺎ ﺻـرةً، وﻟـ ﺎﻧـl ﻣـودة :ﻋـــ: اﻟU* ـــﺔ اﻟﻌﻠ1ـــﺔ واﻟ5ﺎرQـــﺔ واﻟRﻐاـــﺔ اﻟ5ـــﻲ ﻋـــﺎش وﺷـــﺎرك ﻓﻬـــﺎ اﺑـــ اﻟﻌ9ـــﻲ 3ﻔﻌﺎﻟـــﺔٍ، ﺳـــاء ﻓـــﻲ 3ـــﻼد اﻟـــق اﻹﺳـــﻼﻣﻲ اﻟ5ـــﻲ ﻋـــﺎش ﻓ5ـــةً ﻓـــﻲ ﻣـــﻧﻬﺎ وﻣاﻛvﻫـــﺎ اﻟﻌﻠ1ﺔ، أم ﻓﻲ ﺣاﺿ اﻷﻧﻟ ﻓﻲ ﻧﻬﺎWـﺔ اﻟﻘـن اﻟQـﺎﻣ اﻟﻬRـV، واﻟnﻠـ اﻷول :ﻣ: اﻟﻘن اﻟﺎدس اﻟﻬRV. وﻣ: اﻷﻣnﻠﺔ ﻋﻠﻰ ذﻟـ، ﻗﻟـﻪ ﻓـﻲ ﺗﻔـ ﻗﻟـﻪ ﺗﻌـﺎﻟﻰ  ْﻳَﺎ أَﻳ3َﺎ اﻟ#ﺬِﻳﻦَ آﻣَﻨُﻮا ًإِﻧ#ﻤَﺎ اﻟْﻤُْ ِﻛُﻮنَ ﻧَﺠَﺲٌ ﻓَﻼَ hَْ َﺑُـﻮاْ اﻟْﻤَﺴْـﺠِﺪَ اﻟْـَ َامَ َﻌْـﺪَ ﻋَـﺎﻣِ3ِﻢْ <َــﺬَ ا وَإِنْ ﺧِﻔْـﺘُﻢْ ﻋَﻴْﻠَـﺔ ٌﻓَﺴَ ــﻮْفَ hُﻐْﻨِــﻴﻜُﻢُ اﻟﻠّــUُ ﻣِــﻦ ﻓَﻀْ ــﻠِUِ إِن ﺷَــﺎء إِن# اﻟﻠّــUَ ﻋَﻠِــﻴﻢٌ ﺣَﻜِــﻴﻢ ]اﻟ59ــﺔ، 28 [: Z"وﻟﻘــ 0ــlُ أر 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 2 / 954 - 955 . ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 8 58 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن 3 ﺑﻣ ﻋR7ﺎً، ﺎن ﻟRﺎﻣﻌﻬﺎ ،ﺎ3ﺎن: 3ﺎب ﺷﻗﻲ، وﻫـ 3ـﺎب ﺟَْـون، و9ـﺎب ﻏ9ـﻲ وﺎن اﻟ0ﺎس WRﻌﻠﻧﻪ ﻘﺎً W1ن ﻋﻠﻬﺎ ﻧﻬﺎرﻫ ﻠّـﻪ ﻓـﻲ ﺣـاﺋRﻬ، وـﺎن اﻟـOﻣﻲﱡ :ّإذا أراد اﻟ1ور، وﻗﻒ ﻋﻠـﻰ اﻟ7ـﺎب ﺣ5ـﻰ W1ـّ 3ـﻪ ﻣـﻠ، ﻣR5ـﺎز، ﻘـل ﻟـﻪ اﻟـOﻣﻲ Wﺎ ﻣﻠ أﺗﺄذن ﻟﻲ أن أﻣـّ ﻣﻌـ؟ ﻘـل: ﻧﻌـ، ﻓـﺧﻞ ﻣﻌـﻪ، وﻋﻠـﻪ اﻟµَِـ ﺎر ﻋﻼﻣـﺔ ،أﻫﻞ اﻟOﻣﺔ، ﻓﺈذا رآﻩ اﻟcِّ، ﺻﺎح 3ﻪ: ارﺟﻊ، ارﺟﻊ، ﻘل ﻟﻪ اﻟ1ﻠ: أﻧـﺎ أذِﻧـlُ ﻟـﻪ "ّcﻓ5ﻪ اﻟ1 :. وﻣ: اﻷﻣnﻠﺔ ﻋﻠﻰ ذﻟ أWrﺎً، ﻗﻟﻪ ﻓﻲ ﺗﻔ* ﻗﻟﻪ ﺗﻌـﺎﻟﻰ وَﻣِﻨْ3ُﻢ َﻣ#ﻦْ ﻋَﺎ<َﺪَ اﻟﻠّUَ ﻟَِﻦْ آﺗَﺎﻧَﺎ ﻣِﻦ ﻓَﻀْﻠِUِ ﻟَﻨَﺼ#ﺪ#ﻗَﻦ# وَﻟَﻨَﻜُﻮﻧَﻦ# ﻣِﻦ َYZِاﻟﺼ#ﺎﻟِﺤ ِUﻓَﻠَﻤ#ﺎ آﺗَـﺎ<ُﻢ ﻣ4ـﻦ ﻓَﻀْ ـﻠِUِ ﺑَﺨِﻠُـﻮاْ ﺑِـ َوَﺗَﻮَﻟ#ﻮاْ و#<ُـﻢ ﻣﻌْﺮِﺿُـﻮن ْﻓَـَﻋْﻘَﺒَ3ُﻢْ Dِﻔَﺎﻗـﺎً ; ِـ: ﻗُﻠُـﻮﺑِ3ِﻢْ إَِـ ﻳَـﻮْمِ ﻳَﻠْﻘَﻮْﻧَـUُ ﺑِﻤَـﺎ أَﺧْﻠَﻔُـﻮاْ اﻟﻠّـUَ ﻣَـﺎ وَﻋَـﺪُوهُ وَﺑِﻤَـﺎ iَـﺎﻧُﻮا َﻳَﻜْــﺬِﺑُﻮن ]اﻟ59ــﺔ75 - 77 [: "... 1 ( ،اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 2 / 914 . 2 ( ،اﺑ: اﻟﻌ9ﻲnاﻟ;+ر اﻟﺎﺑ ، 2 / 987 - 988 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﻋﻠــﻰ ذــ ﻫــOﻩ اﻵWــﺔ أﻧUــ ُ@ أﻧــﻲ 0ــlُ 31Rﻠــ اﻟز اﻟﻌـﺎدل أﺑـﻲ ﻣ0Iـر ﺑـ: ﺣ1ـ ﻋﻠـﻰ رُﺗ7ـﺔ ﺑﱠ0ﺎﻫـﺎ ﻓـﻲ 5ـﺎب ) ﺗﺗـ اﻟﺣﻠـﺔ ﻟﻠ5ﻏ 3ﺎﻟ1ﻠﺔ:¤(، ﻓﻘأ اﻟﻘﺎر  ٌﺗَﺤِf#ﺘُ3ُﻢْ ﻳَﻮْمَ ﻳَﻠْﻘَﻮْﻧَUُ ﺳَـﻼَ م ]اﻷﺣـvاب، 44 [ ، و0ـlُ ﻓـﻲ اﻟIــﻒ اﻟnــﺎﻧﻲ ﻣــ: اﻟﻠَﻘــﺔ، ﻓpﻬــ أﺑــ اﻟﻓــﺎء ﻋﻠــﻲ ﺑــ: ﻋ ﻘــﻞ إﻣــﺎم اﻟ0Uﻠــﺔ ﺑﻬـــﺎ ]3ﻐاد[، وﺎن ﻣﻌ5vﻟﻲ اﻷﺻل، ﻓﻠ1ﺎ ﺳ1ﻌlُ اﻵWﺔ ﻗﻠlُ ﻟIﺎﺣٍ ﻟـﻲ WRﻠـ ﻋﻠـﻰ ُlWــﺎرV: ﻫــOﻩ اﻵWــﺔ دﻟــﻞٌ ﻋﻠــﻰ رؤــﺔ ﷲ ﻓــﻲ اﻵﺧــة، ﻓــﺈنﱠ اﻟﻌــب ﻻ ﺗﻘــل: ﻟﻘــ ﻓﻼﻧﺎً، إﻻ إذا رأﺗﻪ، ﻓIف وﺟﻬﻪ أﺑ اﻟﻓﺎء اﻟ1ـOر إﻟ0ـﺎ ﻣـﻋﺎً، وﻗـﺎل: ﺗ05Iـ v5ﻟ1Oﻫ اﻻﻋ :ال ﻓﻲ أنﱠ ﷲ ﻻ ﯾُZ ﻓﻲ اﻵﺧة، ﻓﻘ ﻗـﺎل ﻓََﻋْﻘَﺒَ3ُﻢْ Dِﻔَﺎﻗـﺎً ; ِـ: ﻗُﻠُـﻮﺑِ3ِﻢْ إَِـ ُUَﻳَﻮْمِ ﻳَﻠْﻘَﻮْﻧ :، وﻋ0ك أن اﻟ10ﺎﻓﻘ: ﻻ ﯾون ﷲ ﻓـﻲ اﻵﺧـة. وﻗـ ﺷـﺣ0ﺎ وﺟـﻪ اﻵﯾ5ـ ".(ﻓﻲ اﻟ1ُ'ِﻠ: )WﻘI ﻓﻲ 5ﺎ3ﻪ اﻟ1'ﻠ:: أV ﻣُ'ِﻞ اﻟ@5ﺎب واﻟ0ﺔ2    ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ OاﻟU,O اﻟZﺎﻟ : أﺛ اﻟ4ﻔ و{34ﻪ :اﻟWﻠV اﻷول ﻣ+l ﺗﺄﺛ اﺑ اﻟﻌ ﻲ ﻋﻠﻰ اﻟﻔِّ - ﻣ !ﻌ+ﻩ ﻣـــ: اﻟ1ﻌـــﺎﯾ* اﻟ15ﻔـــ ﻋﻠﻬـــﺎ ﻟ1ﻌﻓـــﺔ »1ـــﺔ أV ﻣﻔِّ ـــ، وﻣ'ﺎﻧ5ـــﻪ ﻓـــﻲ ﺗـــﺎرﺦ :اﻟ5ﻔ، ﻫ ﻣZ 3ﻘﺎء وﺣrر ﻣﺎ أﻧRvﻩ ﻓـﻲ ﺗﻔـﻩ، ﻓـﻲ إﻧRـﺎزات اﻟ1ﻔِّ ـ :ﻣـ 3ﻌــﻩ ًﻗــW1ﺎً وﺣــﯾnﺎ ، وﻧwــﺔ ﻫــOا اﻟrــر. وpﻬــ ذﻟــ ﻣــ: ﺧــﻼل اﺗQــﺎذﻫ إWــﺎﻩ ﻣﺟﻌــﺎً ﻻ ﻏ0ــﻰ ﻋ0ــﻪ ﻓــﻲ ﺗﻔﺎﺳــﻫ، WﺄﺧــOون ﻣ0ــﻪ، و0ﻘﻠــن ﻋ0ــﻪ، وRﺎدﻟﻧــﻪ، ﺑــﻞ ،و0ﻘوﻧﻪ. ﻞ ﻫOﻩ اﻟPاﺋ ﻣ: اﻟ5ﻔﺎﻋﻞ ﺗُﻔIﺢ ﻋ: ﻗة ﻫOا اﻟ5ﻔ* وﻗة ﺻﺎﺣ7ﻪ و»15ﻬ1ﺎ اﻟﻌﻠ1ﺔ. وﻧ: إذا أردﻧﺎ أن ﻧPU ﻫOا اﻟ1qﺎر ﻋﻠﻰ ﺗﻔـ ،اﺑـ: اﻟﻌ9ـﻲ ﻓﺈﻧ0ﺎ ﺳ0R أن ﻣﻌp اﻟ1ﻔ: اﻟOﯾ: أﺗا ﻣ: 3ﻌﻩ اﺳ5ﻔﺎدوا ﻣ0ـﻪ ﺑﺟـﻪ ﻣـ: اﻟﺟـﻩ اﻟــﺎ3ﻘﺔ. وﻟ@ــ: ﻟﻠــ ﻧﻘــل: إن أﻛnــ ﻣَــ:ْ اﺳــ5ﻔﺎد ﻣ0ــﻪ، وﺗــﺄﺛ 3ــﻪ، و910ﻬRــﻪ ﻫــ اﻹﻣــﺎم اﻟﻘUــﻲ )ت671 ه( ﺻــﺎﺣ ﺗﻔــ "اﻟRــﺎﻣﻊ ﻷﺣ'ــﺎم اﻟﻘــآن"، وﻫــOا أﻣــ ﻣ5ﻗﻊ، ﻓﺎﻟﺟﻼن أﻧﻟﺎن، وﻣﺎﻟ @ﺎن، وﻓق ﻫOا وذاك ﺻ7ﱠﺎ اﻫ51ﺎﻣﻬ1ﺎ ﻓـﻲ ﻣـان اﻟ5ﻔـــ ﻋﻠـــﻰ ﻣـــﺎ W1'ـــ: اﺳـــ507ﺎﻪ ﻣـــ: أﺣ'ـــﺎم اﻟﻘـــآن اﻟ@ـــ. وﻣـــ: ﻫ0ـــﺎ ﻓﺈﻧـــﺎ ﻟـــ ًﻧــ5ﻐب أﺑــاً ﻋ0ــﻣﺎ ﻻﺣp0ــﺎ اﻫ51ــﺎم واﺣ5ﻔــﺎء اﻟﻘUــﻲ ﺑ5ﻔــ* اﺑــ: اﻟﻌ9ــﻲ اﺣ5ﻔــﺎء Wpﻬــــ ﻟﻠــــارس ﻓــــﻲ ــــﻞ آWــــﺔ ﻓﱠ ــــﻫﺎ اﺑــــ: اﻟﻌ9ــــﻲ، وﺗ0ﺎوﻟﻬــــﺎ 3ﺎﻟ5ﻔــــ* ﻣــــ: 3ﻌــــﻩ اﻟﻘUﻲ، ﺳاء ﺻـﱠح ﻓـﻲ ذﻟـ اﻟﻘUـﻲ أم ﻟـ WIـِّح. 1 ( ،اﻟﻘUﻲ اﻟIﺎﻣﻊ ﻷﺣﺎم اﻟﻘ آن ،ﺑوت، دا ر إﺣﺎء اﻟ5اث اﻟﻌ9ﻲ ، 2 / 249 ،. واﻧp اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 62ـ63 . VاﻟWﻠdاﻟ1ﺎﻣ: ﻣ ﻬIﻪ ﻓﻲ اﻟ4ﻌﺎﻣﻞ ﻣﻊ اﻹﺳ اﺋ ﻠ3ﺎت وﺗ@ـﺎد ﺗ@ـن وﺟـﻩ اﺳـ5ﻔﺎدة اﻟﻘUﻲ ﻣ: اﺑ: اﻟﻌ9ﻲ ﺗ1ﻞ ﻞ ﻣﺎدﯾ: اﻟ5ﻔ، ﻓﻬ ﻗ اﺳ5ﻔﺎد ﻣ0ﻪ ﻓـﻲ اﻟ1ﻘـﺎم اﻷول ﻓﻲ ﻣRﺎل اﻷﺣ'ﺎم اﻟﻔﻘﻬﺔ، وﻣ: ﺛَﱠ ﻓﻲ اﻟ1Rﺎﻻت اﻷﺧZ ﻣ: أﺳ7ﺎب ﻧـvول وﻗـــ اءات وﻟﻐـــﺔ وﻧـــ وأﺧ7ـــﺎر وأﺻـــل ﻓﻘـــﻪ . وﺗRـــ اﻹﺷـــﺎرة ﻫ0ـــﺎ إﻟـــﻰ أن اﺳـــ5ﻔﺎدة اﻟﻘUﻲ ﻣ: اﺑ: اﻟﻌ9ﻲ، ون ﺎﻧl ﻓﻲ ﻣﻌp1ﻬـﺎ اﺳـ5ﻔﺎدة ﻧﻘـﻞ وﻣاﻓﻘـﺔ، وﻟ@0ﻬـﺎ ﻟـ :ﺗﻘ5Iـــ ﻋﻠـــﻰ ذﻟـــ ﺑـــﻞ ـــﺎن اﻟﻘUـــﻲ، وﻫـــ اﻹﻣـــﺎم اﻟ1ﻔِّ ـــ اﻟ@Uـــ، ﯾ5ﻌﻘﱠـــ اﺑـــ . I5اﻟﻌ9ﻲ ﻓﻲ 3ﻌ2 اﻷﺣﺎن 3ﺎﻟ0ﻘ واﻟ وﻣ: اﻷﻣnﻠﺔ ﻋﻠـﻰ ﻫـOا اﻟ5ﻌﻘﱡـ، ﻗـل اﻟﻘUـ :ﻲ ﻓـﻲ ﺗﻔـﻩ ﻟﻘﻟـﻪ ﺗﻌـﺎﻟﻰ ﻳَـﺎ أَﻳ3َﺎ اﻟ#ﺬِﻳﻦَ آﻣَﻨُﻮاْ ﻛُﺘِﺐَ ﻋَﻠَﻴْﻜُﻢُ اﻟْﻘِﺼَﺎصُ ; ِ: اﻟْﻘَﺘْWَـ اﻟْـُ ﺑِـﺎ ِﻟُْ 4 وَاﻟْﻌَﺒْـﺪُ ﺑِﺎﻟْﻌَﺒْـﺪ ]اﻟ7ﻘـة، 78 [ : "ﻗـﺎل اﺑ: اﻟﻌ9ﻲ : ﻟﻘـ ﺑﻠﻐـl اﻟRﻬﺎﻟـﺔ 3ـﺄﻗام إﻟـﻰ أن ﻗـﺎﻟا Wُﻘ5ـﻞ اﻟـ 3ﻌUـ ﻧﻔِ ـ ﻪ، ورووا ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 60 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن ﻓﻲ ذﻟ ﺣﯾnﺎً ﻋ: اﻟـ: ﺑـ: ﺳـ1ة أن رﺳـل ﷲﻗـﺎل: ْ:)ﻣـ َﻗ5ـﻞ ﻋUـ (ﻩ ﻗ5ﻠ0ـﺎﻩ وﻫــOا ﺣــﯾ ﺿــqe. ﻗﻠــlُ : ﻫــOا اﻟــﯾ اﻟــOV ﺿــﻌﱠﻔﻪ اﺑــ: اﻟﻌ9ــﻲ، ﻫــ ﺻــﺢ ْ:أﺧﺟــﻪ اﻟ0ــﺎﺋﻲ وأﺑــ داود، وﺗ51ــ ﻣ50ــﻪ: )وﻣــ ْ:ﺟــع أﻧــﻒ ﻋUــﻩ ﺟــﻋ0ﺎﻩ، وﻣــ :أﺧIﺎﻩ أﺧI0ﺎﻩ(، وﻗﺎل اﻟ7QﺎرV ﻋ: ﻋﻠـﻲ ﺑـ: اﻟ1ـﯾ0ﻲ ﺳـ1ﺎع اﻟـ: ﺑـ: ﺳـ1ة ﺻــﺢ، وأﺧــO ﺑﻬــOا اﻟــﯾ، وﻗــﺎل اﻟ7QــﺎرV: وأﻧــﺎ أذﻫــ إﻟــﻪ. ﻓﻠــ ﻟــ WIــﺢ ﻟ1ــﺎ "ذﻫ إﻟﻪ ﻫOان اﻹﻣﺎﻣﺎن، وﺣ7ُ ﺑﻬ1ﺎ1 . VاﻟWﻠ اﻟZﺎﻧﻲ : ﻣ,ﺎوﻟﺔ ﻓﻲ اﻟ3 4ﻘ 3ﻌ ﻫOﻩ اﻟRﻟﺔ اﻟ1ﺟvة ﻣﻊ اﻟﻘﺎﺿﻲ اﺑ: اﻟﻌ9ﻲ و9ﻌ2 ﻣﻌﺎﻟ اﻟ1ـ0ﻬﺞ ﻓـﻲ :'1W ،"ﺗﻔﻩ "أﺣ'ﺎم اﻟﻘآن:ﻟ0ﺎ، ﻋﻠﻰ ﺳUﻞ اﻟ5ﻘ، اﻹدﻻء 3ﺎﻟ1ﻼﺣpﺎت اﻟ5ﺎﻟﺔ 1 ( اﺳ5Pﺎع اﺑ: اﻟﻌ9ﻲ أن ﯾﻠ5vم 31ﺎ أﺧOﻩ ﻋﻠﻰ ﻧﻔﻪ ﻣ: ﻋ0ﺎﺻ ﻣ0ﻬRﺔ أﻓIﺢ ﻋ0ﻬــﺎ ﻓــﻲ ﻣﻘﻣــﺔ 5ﺎ3ــﻪ 1ــﺎ ذﻧــﺎ ﻣــ: ﻗUــﻞُ. و9ﺎﻟﻔﻌــﻞ، و1ــﺎ ﺗُnUــl ذﻟــ اﻟﻔﻘــات اﻟﺎ3ﻘﺔ، ﻓﻘ أﻋPﻰ اﺑ: اﻟﻌ9ﻲ ﻞﱠ ﻋ0ﺎﺻ ﻣ0ﻬRﻪ ﺣﻘﻬﺎ ﻣ: اﻹﻋ1ﺎل وا.UP5ﻟ 2 ( ﻋﻠﻰ اﻟﻏ ﻣ: أن اﻋ50ﺎء اﺑـ: اﻟﻌ9ـﻲ ﻓـﻲ ﺗﻔـﻩ اﻧIـﱠ ﻋﻠـﻰ ﻧﺎﺣـﺔ اﻷﺣ'ـﺎم ،اﻟwﺔ، إﻻ أﻧﻪ ﻟـ Wـﺄلُ ﺟُﻬـاً ﻓـﻲ ﺗ0ـﺎول اﻵWـﺎت اﻟ5ـﻲ ﻓﱠ ـﻫﺎ ﻣـ: ﺟ1ـﻊ اﻟ0ـاﺣﻲ واﻟﻗــف ﻋﻠــﻰ ﺟ1ــﻊ دﻻﻻﺗﻬــﺎ وWﺎءاﺗﻬــﺎ، 3ﺎﻹﺿــﺎﻓﺔ إﻟــﻰ ذــ ــﻞ ﻣــﺎ ﯾ5ﻌﻠــ ﺑﻬــﺎ ﻣ: أﺣﺎدﯾ وأﺧ7ﺎر وأﻗال ﺳﻠﻒ ،. و9ﻬOا ﻧ5Pﻊ أن ﻧﻘل: إن ﺗﻔ* اﺑ: اﻟﻌ9ﻲ 0وﻋﻠـــﻰ اﻟـــﻏ ﻣـــ: fـــﺎﻫ ﻋ اﻧـــﻪ اﻟـــOV ﯾـــﺣﻲ 3ﺎﻟPـــﺎ3ﻊ اﻟﻔﻘﻬـــﻲ ، :وﻋﻠـــﻰ اﻟـــﻏ ﻣـــ ،ًاﻗ5Iﺎرﻩ ﻋﻠـﻰ اﻵWـﺎت اﻟ5ـﻲ ﻗـّر أﻧﻬـﺎ ﺗ5rـ1: أﺣ'ﺎﻣـﺎ W1'00ـﺎ أن ﻧُرﺟـﻪ ﻓـﻲ 5ـ اﻟ5ﻔــ* اﻟ5ﻠﻠــﻲ اﻟﻌــﺎم، ﻣــﻊ ﻟﻔــl اﻷﻧpــﺎر إﻟــﻰ اﻫ51ﺎﻣــﻪ وﻋ0ﺎﯾ5ــﻪ اﻟ@Uــة 3ﺎﻷﺣ 'ــﺎم .اﻟﻔﻘﻬﺔ 3 ( ﺗUـــأ اﻟﻘﺎﺿـــﻲ اﺑـــ: اﻟﻌ9ـــﻲ ﻣ'ﺎﻧـــﺔً ﻣﻣﻗـــﺔً ﻓـــﻲ ﺗـــﺎرﺦ اﻟ5ﻔـــ، واﺳـــ5Pﺎع أن Wﻔض ﺗﻔﻩ 1ﺟﻊ ﺿورV ﻟ@ﻞ ﻣ: ﺗIـﱠZ ﻟ5ﻔـ* اﻟﻘـآن اﻟ@ـ 3ﻌـﻩ وﺣ5ـﻰ أWﺎﻣ0ــﺎ اﻟاﻫ0ـــﺔ، ﻋﻠـــﻰ اﻷﻗـــﻞ ﻣـــ: ﺣـــ اﻟراﺳـــﺔ اﻟﻔﻘﻬـــﺔ ﻵWـــﺎت اﻟﻘـــآن اﻟ@ـــ اﻟ5ـــﻲ 1 ( ،اﻟﻘUﻲ اﻟIﺎﻣﻊ ﻷﺣﺎم اﻟﻘ آن ،ﺑوت، دا ر إﺣﺎء اﻟ5اث اﻟﻌ9ﻲ ، 2 / 249 ،. واﻧp اﺑ: اﻟﻌ9ﻲ أﺣﺎم اﻟﻘ آن ، 1 / 62ـ63 . ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 61 ﺗ0ﺎوﻟ :'1ﻬﺎ ﻓﻲ ﺗﻔﻩ. و اﻋ57ﺎر ﺗﻔﻩ ﻋﻼﻣﺔ 3ـﺎرزة ﻣـ: ﻋﻼﻣـﺎت ﺗPـر ﻣرﺳـﺔ "اﻟ5ﻔــ* اﻟﻔﻘﻬــﻲ، وﺧPــة رﺋــﺔ ﻣﻬﱠــت اﻟPــ ﻟpﻬــر اﻹﻣــﺎم اﻟﻘUــﻲ وﺗﻔــﻩ اﻟRﺎﻣﻊ ﻷﺣ'ـﺎم اﻟﻘـآن" اﻟـOV Wُﻌـ ﻋﻼﻣـﺔ ﻧrـﺞ ﻣرﺳـﺔ اﻟ5ﻔـ اﻟﻔﻘﻬـﻲ ﻓـﻲ ﺗـﺎرﺦ .اﻟ5ﻔ* 3'ﻞ ﻋﺎم    اﻟ4ﺎﺋﺞ اﻟ4ﺎﺋﺞ ﻣـــ: ﺧـــﻼل رﺣﻠ50ـــﺎ اﻟ15اﺿـــﻌﺔ ﻣـــﻊ اﻹﻣـــﺎم اﺑـــ: اﻟﻌ9ـــﻲ رﺣ1ـــﻪ ﷲ وﺗﻔـــﻩ :"أﺣ'ﺎم اﻟﻘآن"، W1': ﻟ0ﺎ أن ﻧRﻞ اﻟ05ﺎﺋﺞ اﻟ5ﺎﻟﺔ 1 ( VOاﻋ51ـــ اﺑـــ: اﻟﻌ9ـــﻲ ﻋﻠـــﻰ اﻟ1ـــ0ﻬﺞ اﻟ5ﻠﻠـــﻲ ﻓـــﻲ ﺗﻔـــﻩ، وﻫـــ اﻟ1ـــ0ﻬﺞ اﻟـــ ،ﻣــﺎزال Wpــﻰ 3ﺎﻟrــر اﻟﻘــV ﺣ5ــﻰ اﻵن ﻓــﻲ ﻣــان ﺗﻔــ* اﻟﻘــآن اﻟ@ــ ﻓﻘـ اﻗ5Iــ اﺑــ: اﻟﻌ9ــﻲ ﻓــﻲ 5ﺎ3ــﻪ ﻋﻠـﻰ ﺗﻔــ* آWــﺎت اﻷﺣ'ــﺎم اﻟاﺣــة 3ﻌــ اﻷﺧــZ ﺣــ ﺗﺗUﻬــﺎ ﻓــﻲ ﺳــرﻫﺎ، ﻣِّــvاً ﺟﻬــﻩ ﻋﻠــﻰ ﺗﻔــ 3ُﻌْــﻫﺎ ا ﻟﻔﻘﻬــﻲ 53'ﻞ ﻋﺎم، ﻣﻊ ﻣﻼﺣpﺔ أﻧﻪ ﺎن ﯾﻬ ـ ًأWrﺎ ـ :3ﺎﻷ3ﻌﺎد اﻷﺧZ ﻟﻶWﺎت ﻣ ﺣـــــ ﺗﻔـــــﻫﺎ اﻟﻌـــــﺎم، وﻣـــــﺎ ﻓﻬـــــﺎ ﻣـــــ: دﻻﻻت ﻋﻘWـــــﺔ و ﺷـــــﺎرات ٍﺗﺎرQـــــﺔ .ٍوﺳﺎﺳﺔ ﻣـــ: ﺧـــﻼل رﺣﻠ50ـــﺎ اﻟ15اﺿـــﻌﺔ ﻣـــﻊ اﻹﻣـــﺎم اﺑـــ: اﻟﻌ9ـــﻲ رﺣ1ـــﻪ ﷲ وﺗﻔـــﻩ :"أﺣ'ﺎم اﻟﻘآن"، W1': ﻟ0ﺎ أن ﻧRﻞ اﻟ05ﺎﺋﺞ اﻟ5ﺎﻟﺔ 2 ( اﺳ5ﻋ اﺑ: اﻟﻌ9ﻲ ﺗاث اﻟ1ﻔ: ﻣ: ﻗUﻠﻪ واﻧ5p ﻓﻲ داﺧﻠﻪ، وﺗﻌﺎﻣﻞ ﻣﻊ ﻫOا اﻟ5اث 3Pﻘﺔ ﺗ0ﱡ ﻋ: اﺣ5ـام ﻋ1ـ ﻟـﻪ ﻣـlﻊ اﺣ5ﻔﺎfـﻪ ﻟ0ﻔـﻪ ﻓـﻲ اﻟﻗـ .ذاﺗﻪ 3 اﻟ10ﺎﻗﺔ واﻟ0ﻘ واﻟدِّ ﻋ0ﻣﺎ ﯾZ ﻣﺟ7ﺎً ﻟOﻟ 2 ( اﺳ5ﻋ اﺑ: اﻟﻌ9ﻲ ﺗاث اﻟ1ﻔ: ﻣ: ﻗUﻠﻪ واﻧ5p ﻓﻲ داﺧﻠﻪ، وﺗﻌﺎﻣﻞ ﻣﻊ ﻫOا اﻟ5اث 3Pﻘﺔ ﺗ0ﱡ ﻋ: اﺣ5ـام ﻋ1ـ ﻟـﻪ ﻣـ lﻊ اﺣ5ﻔﺎfـﻪ ﻟ0ﻔـﻪ ﻓـﻲ اﻟﻗـ .ذاﺗﻪ 3 اﻟ10ﺎﻗﺔ واﻟ0ﻘ واﻟدِّ ﻋ0ﻣﺎ ﯾZ ﻣﺟ7ﺎً ﻟOﻟ 3 ( اﻋ51 اﺑـ: اﻟﻌ9ـﻲ ﻋﻠـﻰ ﻣ0ﻬRـﻲ اﻷﺛـ واﻟـأV ﻓـﻲ ﺗﻔـﻩ، ﻣـﻊ ﻣﻼﺣpـﺔ أﻧـﻪ ﻛﺎن أﻛn ﺗﺳﻌﺎً ودﻗﺔً وﻧﻘـاً ﻓـﻲ ﻣـان اﻷﺛـ واﻷﺧ7ـﺎر، ﻫـOا 3ﺎﻹﺿـﺎﻓﺔ إﻟـﻰ أﻧﻪ ﻗ اﺗQO ﻣﻗﻔﺎً ﻧﻘWﺎً إزاء اﻷﺣﺎد.ﯾ اﻟrqﻔﺔ واﻟ1ﺿﻋﺔ واﻹﺳاﺋﻠﺎت 4 ( ﻛــﺎن ﯾ05Iــ ﻟ1Ot7ــﻪ وﻣﺎﻣـــﻪ ﻋﻠــﻰ اﻟ1ــ5Z اﻟﻔﻘﻬـــﻲ واﻟﻌﻘــV، وــﺎنªWﻐﻠـــ 3ﻌ2 اﻟﻲء ﻓﻲ اﻟدّ ﻋﻠـﻰ ﻣQﺎﻟ_ـﻪ ﻓـﻲ 3ﻌـ2 اﻟـﺎﻻت ، ﻣـﻊ fﻬـر روح 2اﻟ5ﻘﯾ واﻹﻧIﺎف ﻋ0ﻩ ﻟﻠﻌﻠ1ﺎء اﻵﺧ: ﻓﻲ 3ﻌ اﻷﺣﺎن. ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 2 62 ﺣﺎزم زﻛرﯾﺎ ﻣﺣﻲ اﻟدﯾن 5 ( fﻬـــت ﺷQIـــﺔ اﺑـــ: اﻟﻌ9ـــﻲ اﻟﻌﻠ1ـــﺔ اﻟ5ﻔـــﺔ 3ـــ'ﻞ واﺿـــﺢ ﻣـــ: ﺧـــﻼل ﺟ1ﻌــــﻪ ﻟﻸﺧ7ــــﺎر واﻵﺛــــﺎر، واﻟ5ﻌﺎﻣــــﻞ ﻣﻌﻬــــﺎ 3Pﻘــــﺔ ﻧﻘWــــﺔ، وﻋﺿــــﻪ ﻷﻗــــال اﻟﻔﻘﻬﺎء وﻣ0ﺎﻗ5ﻬﺎ وﺗﺟﺢ اﻷﻗZ دﻟﻼً ﻓﻬﺎ، ﺣ5ﻰ وﻟـ ـﺎن ﻣQﺎﻟﻔـﺎً ﻟ1Ot7ـﻪ ﻓﻲ 3ﻌ2 اﻟﺎﻻت، وﻟRﺋﻪ، ﻓﻲ 3ﻌ2 اﻷﺣﺎن، إﻟﻰ اﺳ507ﺎ ﺣ'ـٍ ﺧـﺎص V3ـــﻪ ﻓـــﻲ ﻋـــد ﻣـــ: اﻟ1ـــﺎﺋﻞ واﻟﻘrـــﺎWﺎ. اﻟ4ﺎﺋﺞ وأﻛUـــ اﻟpـــ: ﻋ0ـــ أن أﺳـــﺎس ﻗـــة ًاﻟQIــﺔ اﻟﻌﻠ1ــﺔ ﻋ0ــﻩ، Wﻌــد إﻟــﻰ ﺗ1'ﱡ0ــﻪ ﻣــ: ﻋﻠــم اﻟــﯾ وﻧﻘــﻩ رواWــﺔ ودراWــﺔً، 3ﺎﻹﺿــﺎﻓﺔ إﻟــﻰ رﺳــخ ﻗﻣــﻪ ﻓــﻲ ﻋﻠــم اﻟﻠﻐــﺔ واﻷﺻــل، وﻫــOﻩ ﻠﻬــﺎ أدوات ﻻ ﻏ0ـــﻰ ﻋ0ﻬـــﺎ ﻟ@ـــﻞ ﻣـــ: ﯾ5IـــﱠZ ﻟﻼﺟ5ﻬـــﺎد أو ﻋﻠـــﻰ اﻷﻗـــﻞ ﻟﻠ5ـــﺟﺢ اﻟﻌﻠ1ﻲ. اﻟﻌﻠ1ﻲ. 6 ( اﻣ5ــﺎز اﺑــ: اﻟ ﻌ9ــﻲ ﺑــ9u ﺗﻔــﻩ ﻟﻠﻘــآناﻟ@ــ ًﻠ1ــﺎ وﺟــ إﻟــﻰ ذﻟــ ﺳـــUﻼ ﺑاﻗﻊ ﻣﻠ1ﻲ ﻋIﻩ وﻣﺎﻛﻠﻬ، وﻫOﻩ اﻟ1vة اﻹﺻﻼﺣﺔ زادت ﻣ: ﻗرة ﻫـOا اﻟ5ﻔ ﻋﻠﻰ اﻟ5ﻔﺎﻋـﻞ ﻣـﻊ ﻋIـﻩ واﻟ5ـﺄﺛ* ﻓـﻲ أﺣاﺛـﻪ، وﺟﻌﻠـl ﻣ0ـﻪ 3ﺎﻟ0ـ7ﺔ ﻟ0ـــــﺎ ﺻــــــرةً ﺻـــــﺎدﻗﺔ ﻋــــــ: 3ﻌـــــ2 أﺣــــــال ﻋIـــــﻩ ﻣــــــ: اﻟ0ﺎﺣـــــﺔ اﻟﺎﺳــــــﺔ wواﻻﺟ51ﺎ ــﺔ، ﻓrــﻼ ◌ًــ ـ 3PUﻌــﺔ اﻟــﺎل ـ:ﻋــ .اﻟ0ﺎﺣــﺔ اﻟﻌﻠ1ــﺔ واﻟﻔ@ــﺔ وﻗــﱠﻣl ﻟ0ــﺎ ـ ًأWrــﺎ ـ ﻧ1ذﺟــﺎً Wُﻘ5ــZ 3ــﻪ ﻟِ1ــﺎ WRــ أن Wﻘــم 3ــﻪ أVﱡ ﻣﻔِّ ــ .ﯾ51ﻞ ﻣوﻟ5ﻪ اﻟﻌﻠ1ﺔ واﻟﯾ0ﺔ إزاء اﻟﻌI اﻟOV Wq° ﻪ وﻓﻲ ﻧﻬﺎWﺔ ﻫOا اﻟ7 ﻧﻋ :nاﻟ7ﺎﺣ إﻟﻰ ﺑOل اﻟ1v ﻣـ: اﻟ7ـ واﻻﺟ5ﻬـﺎد ﻓــــﻲ:اﺳــــ5Qاج ﻣــــﺎ W1'ــــ: اﺳــــ5Qاﺟﻪ ﻣــــ اﻟﻌ0ﺎﺻــــ اﻟ10ﻬRــــﺔ و اﻟﻘاﻋــــ واﻷﺻــــل اﻟ5ﻔﺔ اﻟ5ﻲﻗﺎم ﻋﻠﻰ ﻫV ووﺣﻲ ﻣ0ﻬﺎ ﺗاﺛ0ﺎ اﻟ5ﻔV اﻟﻌـ، اﻟﻘـW واﻟـﯾ ،ﻋﻠـﻰ اﻟــاءوذﻟــ ِّ'ﻧpـاً ﻷن ﻫــOا اﻻﺳــ5Qاج Wــ ﻞ ﺧPـة ﺿــورﺔ ﻋﻠــﻰ ــ ا ٍ*ﺳ5@1ﺎل ﺗ ﻋﻠ1ﻲ ُﻣ َ' ﻟU0ﺎء ﻋﻠ أﺻل اﻟ5ﻔ اﻟOV ﻣـﺎ زال ﯾ05pـ اﻟ@nـ cUP5ﻣـــ: اﻟRﻬـــد اﻟ0pـــﺔ واﻟ ﺔ ـــ ﻹﻧrـــﺎﺟﻪ وﺗﺳـــQﻪ، وﻋPﺎﺋـــﻪ اﻟ1'ﺎﻧـــﺔ اﻟﻌﻠ1ـــﺔ اﻟ1رﺔ اﻟ@Uة اﻟ5ﻲ W5ﻘﻬﺎ. اﻟ4ﺎﺋﺞ ا ﻟ;ﺎدر واﻟ اﺟﻊ اﺑ: ﺧﻠ@ﺎن ، ) أﺣ1 ﺑ: ﷴ1971 .(وa3ـﺎت اﻷ`3ـﺎن وأﻧUـﺎء أﺑـﺎء اﻟ^ﻣـﺎن ،، ﺗﻘـ: إﺣـﺎن w7ـﺎس 1، دار ﺻﺎدر، ﺑوت ، ﻟU0ﺎن. اﺑــ: اﻟﻌ9ــﻲ، أﺑــ 3'ــ ﷴ ﺑــ: ﻋUــ ﷲ ) 1409 /ه1989 .( l اﻷﺣــﺎم اﻟ;ــﻐ ،، ﺗﻘــ: ﺳــﻌ أﻋــاب 1 .، ﻣ0رات اﻟ10p1ﺔ اﻹﺳﻼﻣﺔ ﻟﻠ59ﺔ واﻟﻌﻠم واﻟnﻘﺎﻓﺔ .ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ) 1407 /ه1987(. أﺣـــﺎم اﻟﻘـــ آن:، ﺗﻘـــ ،Vﻋﻠـــﻲ ﷴ اﻟ7Rـــﺎو 3 ، دار اﻟ1ﻌﻓﺔ، ﺑوت ، ﻟU0ﺎن. .(ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ. )د.تاﻟﻌJاﺻ ﻣ اﻟﻘJاﺻ :W، ﺗﻘـ وﻣﻘﻣـﺔ: ﻣـ اﻟـﯾ: اﻟQPـ، ﺗﻘـ ﺧﺎﻟ اﻟﻌ، دار اﻟ7ﺎﺋ، دﻣ ، ﺳرﺎ. .(اﺑــ: ﻓﺣــن، ﺑﻫــﺎن اﻟــﯾ: إﺑــاt )د.تVاﻟــ+ﯾUﺎج اﻟــiﻫV ﻓــﻲ ﻣﻌ ﻓــﺔ أ`3ــﺎن اﻟــiﻫ.، د. ، دار اﻟ@5 اﻟﻌﻠ1ﺔ، ﺑوت ، ﻟU0ﺎن. ) اﻷدﻧﻪ وV، أﺣ1ـ ﺑـ: ﷴ1417 /ه1997 .( - bUﻘـﺎت اﻟﻔـ ،Vv، ﺗﻘـ: ﺳـﻠ1ﺎن ﺑـ: ﺻـﺎﻟﺢ اﻟQـ 1، ﻣ'57ﺔ اﻟﻌﻠم واﻟ'، اﻟ1ﯾ0ﺔ اﻟ10رة ، اﻟﻌدWﺔ . أﻋــا) ب، ﺳــﻌ1407 /ه1987 .(ﻣــﻊ اﻟﻘﺎﺿــﻲ أﺑــﻲ !ــ ﺑــ اﻟﻌ ــﻲ، 1 ،، دار اﻟﻐــب اﻹﺳــﻼﻣﻲ ﺑوت ، ﻟU0ﺎن . ) '3 اﻟﻲ، ﺟﻼل اﻟﯾ: ﻋU اﻟﺣ1: ﺑ: أﺑﻲ1426 .(ه اﻹﺗﻘﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن v، ﺗﻘ: ﻣـ .اﻟراﺳــــﺎت اﻟﻘآﻧــــﺔ، د. ، ﻣR1ــــﻊ اﻟ1ﻠــــ ﻓﻬــــ ﻟP7ﺎﻋــــﺔ اﻟ1Iــــﻒ اﻟــــe، اﻟ1ﯾ0ــــﺔ اﻟ10ــــر ة ، اﻟﻌدWﺔ. اﻟ4ﺎﺋﺞ 6 ( اﻣ5ــﺎز اﺑــ: اﻟ ﻌ9ــﻲ ﺑــ9u ﺗﻔــﻩ ﻟﻠﻘــآناﻟ@ــ ًﻠ1ــﺎ وﺟــ إﻟــﻰ ذﻟــ ﺳـــUﻼ ﺑاﻗﻊ ﻣﻠ1ﻲ ﻋIﻩ وﻣﺎﻛﻠﻬ، وﻫOﻩ اﻟ1vة اﻹﺻﻼﺣﺔ زادت ﻣ: ﻗرة ﻫـOا اﻟ5ﻔ* ﻋﻠﻰ اﻟ5ﻔﺎﻋـﻞ ﻣـﻊ ﻋIـﻩ واﻟ5ـﺄﺛ* ﻓـﻲ أﺣاﺛـﻪ، وﺟﻌﻠـl ﻣ0ـﻪ 3ﺎﻟ0ـ7ﺔ ﻟ0ـــــﺎ ﺻــــــرةً ﺻـــــﺎدﻗﺔ ﻋــــــ: 3ﻌـــــ2 أﺣــــــال ﻋIـــــﻩ ﻣــــــ: اﻟ0ﺎﺣـــــﺔ اﻟﺎﺳــــــﺔ wواﻻﺟ51ﺎ ــﺔ، ﻓrــﻼ ◌ًــ ـ 3PUﻌــﺔ اﻟــﺎل ـ:ﻋــ .اﻟ0ﺎﺣــﺔ اﻟﻌﻠ1ــﺔ واﻟﻔ@ــﺔ وﻗــﱠﻣl ﻟ0ــﺎ ـ ًأWrــﺎ ـ ﻧ1ذﺟــﺎً Wُﻘ5ــZ 3ــﻪ ﻟِ1ــﺎ WRــ أن Wﻘــم 3ــﻪ أVﱡ ﻣﻔِّ ــ .ﯾ51ﻞ ﻣوﻟ5ﻪ اﻟﻌﻠ1ﺔ واﻟﯾ0ﺔ إزاء اﻟﻌI اﻟOV Wq° ﻪ وﻓﻲ ﻧﻬﺎWﺔ ﻫOا اﻟ7 ﻧﻋ :nاﻟ7ﺎﺣ إﻟﻰ ﺑOل اﻟ1v ﻣـ: اﻟ7ـ واﻻﺟ5ﻬـﺎد ﻓــــﻲ:اﺳــــ5Qاج ﻣــــﺎ W1'ــــ: اﺳــــ5Qاﺟﻪ ﻣــــ اﻟﻌ0ﺎﺻــــ اﻟ10ﻬRــــﺔ و اﻟﻘاﻋــــ واﻷﺻــــل اﻟ5ﻔﺔ اﻟ5ﻲﻗﺎم ﻋﻠﻰ ﻫV ووﺣﻲ ﻣ0ﻬﺎ ﺗاﺛ0ﺎ اﻟ5ﻔV اﻟﻌـ، اﻟﻘـW واﻟـﯾ ،ﻋﻠـﻰ اﻟــاءوذﻟــ ِّ'ﻧpـاً ﻷن ﻫــOا اﻻﺳــ5Qاج Wــ ﻞ ﺧPـة ﺿــورﺔ ﻋﻠــﻰ ــ ا ٍ*ﺳ5@1ﺎل ﺗ ﻋﻠ1ﻲ ُﻣ َ' ﻟU0ﺎء ﻋﻠ أﺻل اﻟ5ﻔ اﻟOV ﻣـﺎ زال ﯾ05pـ اﻟ@nـ cUP5ﻣـــ: اﻟRﻬـــد اﻟ0pـــﺔ واﻟ ﺔ ـــ ﻹﻧrـــﺎﺟﻪ وﺗﺳـــQﻪ، وﻋPﺎﺋـــﻪ اﻟ1'ﺎﻧـــﺔ اﻟﻌﻠ1ـــﺔ اﻟ1رﺔ اﻟ@Uة اﻟ5ﻲ W5ﻘﻬﺎ. ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 63 ا ﻟ;ﺎدر واﻟ اﺟﻊ اﺑ: ﺧﻠ@ﺎن ، ) أﺣ1 ﺑ: ﷴ1971 .(وa3ـﺎت اﻷ`3ـﺎن وأﻧUـﺎء أﺑـﺎء اﻟ^ﻣـﺎن ،، ﺗﻘـ: إﺣـﺎن w7ـﺎس 1، دار ﺻﺎدر، ﺑوت ، ﻟU0ﺎن. اﺑــ: اﻟﻌ9ــﻲ، أﺑــ 3'ــ ﷴ ﺑــ: ﻋUــ ﷲ ) 1409 /ه1989 .( l اﻷﺣــﺎم اﻟ;ــﻐ ،، ﺗﻘــ: ﺳــﻌ أﻋــاب 1 .، ﻣ0رات اﻟ10p1ﺔ اﻹﺳﻼﻣﺔ ﻟﻠ59ﺔ واﻟﻌﻠم واﻟnﻘﺎﻓﺔ .ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ) 1407 /ه1987(. أﺣـــﺎم اﻟﻘـــ آن:، ﺗﻘـــ ،Vﻋﻠـــﻲ ﷴ اﻟ7Rـــﺎو 3 ، دار اﻟ1ﻌﻓﺔ، ﺑوت ، ﻟU0ﺎن. .(ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ. )د.تاﻟﻌJاﺻ ﻣ اﻟﻘJاﺻ :W، ﺗﻘـ وﻣﻘﻣـﺔ: ﻣـ اﻟـﯾ: اﻟQPـ، ﺗﻘـ ﺧﺎﻟ اﻟﻌ، دار اﻟ7ﺎﺋ، دﻣ ، ﺳرﺎ. .(اﺑــ: ﻓﺣــن، ﺑﻫــﺎن اﻟــﯾ: إﺑــاt )د.تVاﻟــ+ﯾUﺎج اﻟــiﻫV ﻓــﻲ ﻣﻌ ﻓــﺔ أ`3ــﺎن اﻟــiﻫ.، د. ، دار اﻟ@5 اﻟﻌﻠ1ﺔ، ﺑوت ، ﻟU0ﺎن. ) اﻷدﻧﻪ وV، أﺣ1ـ ﺑـ: ﷴ1417 /ه1997 .( - bUﻘـﺎت اﻟﻔـ ،Vv، ﺗﻘـ: ﺳـﻠ1ﺎن ﺑـ: ﺻـﺎﻟﺢ اﻟQـ 1، ﻣ'57ﺔ اﻟﻌﻠم واﻟ'، اﻟ1ﯾ0ﺔ اﻟ10رة ، اﻟﻌدWﺔ . أﻋــا) ب، ﺳــﻌ1407 /ه1987 .(ﻣــﻊ اﻟﻘﺎﺿــﻲ أﺑــﻲ !ــ ﺑــ اﻟﻌ ــﻲ، 1 ،، دار اﻟﻐــب اﻹﺳــﻼﻣﻲ ﺑوت ، ﻟU0ﺎن . ) '3 اﻟﻲ، ﺟﻼل اﻟﯾ: ﻋU اﻟﺣ1: ﺑ: أﺑﻲ1426 .(ه اﻹﺗﻘﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن v، ﺗﻘ: ﻣـ .اﻟراﺳــــﺎت اﻟﻘآﻧــــﺔ، د. ، ﻣR1ــــﻊ اﻟ1ﻠــــ ﻓﻬــــ ﻟP7ﺎﻋــــﺔ اﻟ1Iــــﻒ اﻟــــe، اﻟ1ﯾ0ــــﺔ اﻟ10ــــر ة ، اﻟﻌدWﺔ. ا ﻟ;ﺎدر واﻟ اﺟﻊ اﺑ: ﺧﻠ@ﺎن ، ) أﺣ1 ﺑ: ﷴ1971 .(وa3ـﺎت اﻷ`3ـﺎن وأﻧUـﺎء أﺑـﺎء اﻟ^ﻣـﺎن ،، ﺗﻘـ: إﺣـﺎن w7ـﺎس 1، دار ﺻﺎدر، ﺑوت ، ﻟU0ﺎن. اﺑــ: اﻟﻌ9ــﻲ، أﺑــ 3'ــ ﷴ ﺑــ: ﻋUــ ﷲ ) 1409 /ه1989 .( l اﻷﺣــﺎم اﻟ;ــﻐ،، ﺗﻘــ: ﺳــﻌ أﻋــاب 1 .، ﻣ0رات اﻟ10p1ﺔ اﻹﺳﻼﻣﺔ ﻟﻠ59ﺔ واﻟﻌﻠم واﻟnﻘﺎﻓﺔ .ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ) 1407 /ه1987(. أﺣـــﺎم اﻟﻘـــ آن:، ﺗﻘـــ ،Vﻋﻠـــﻲ ﷴ اﻟ7Rـــﺎو 3، دار اﻟ1ﻌﻓﺔ، ﺑوت ، ﻟU0ﺎن. .(ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ. )د.تاﻟﻌJاﺻ ﻣ اﻟﻘJاﺻ:W، ﺗﻘـ وﻣﻘﻣـﺔ: ﻣـ اﻟـﯾ: اﻟQPـ، ﺗﻘـ ﺧﺎﻟ اﻟﻌ، دار اﻟ7ﺎﺋ، دﻣ ، ﺳرﺎ. ) اﻷدﻧﻪ وV، أﺣ1ـ ﺑـ: ﷴ1417 /ه1997 .( - bUﻘـﺎت اﻟﻔـ ،Vv، ﺗﻘـ: ﺳـﻠ1ﺎن ﺑـ: ﺻـﺎﻟﺢ اﻟQـ 1، ﻣ'57ﺔ اﻟﻌﻠم واﻟ'، اﻟ1ﯾ0ﺔ اﻟ10رة ، اﻟﻌدWﺔ . ) '3 اﻟﻲ، ﺟﻼل اﻟﯾ: ﻋU اﻟﺣ1: ﺑ: أﺑﻲ1426 .(ه اﻹﺗﻘﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن v، ﺗﻘ: ﻣـ .اﻟراﺳــــﺎت اﻟﻘآﻧــــﺔ، د. ، ﻣR1ــــﻊ اﻟ1ﻠــــ ﻓﻬــــ ﻟP7ﺎﻋــــﺔ اﻟ1Iــــﻒ اﻟــــe، اﻟ1ﯾ0ــــﺔ اﻟ10ــــر ة ، اﻟﻌدWﺔ. ) .ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ1396 ه/ 1976 .(م - bUﻘــﺎت اﻟﻔــ ،، ﺗﻘــ: ﻋﻠــﻲ ﷴ ﻋ1ــ 1 ، ﻣ'57ﺔ وt7ﺔ، اﻟﻘﺎﻫةI، ﻣ. ﻣ'57ﺔ وt7ﺔ، اﻟﻘﺎﻫةI، ﻣ. 64 اﻟvرﻗﺎﻧﻲ، ﷴ ﻋU اﻟﻌpـ )د.ت. .(ﻣﺎﻫﻞ اﻟﻌ ﻓﺎن ﻓﻲ ﻋﻠJم اﻟﻘـ آن ، 3، دار إﺣـﺎء اﻟ5ـاث اﻟﻌ9ـﻲ ، ﺑوت، ﻟU0ﺎن. ،اﻟﻘUﻲ أﺑ ﻋU ﷲ ﷴ ﺑ: أﺣ1 )د.ت. .( اﻟIﺎﻣﻊ ﻷﺣﺎم اﻟﻘ آن ، دار إﺣﺎء اﻟ5اث اﻟﻌ9ﻲ، ﺑـوت ، ﻟU0ﺎن . ،اﻟﻘ1ــﺎﻲ ) اﻟ9ــﻊ ﷴ ﻣ0Iــﻒ2009 .( ﻣ;ــﺎدر اﻟ4ﻔــ اﻟﻔﻘﻬــﻲ ﻋــ+ اﺑــ اﻟﻌ ــﻲ ﻓــﻲ .4ﺎ!ــﻪ أﺣــﺎم ،اﻟﻘ آن 1، دار اﻟﻔﻔﺎء، اﺑﻠ ، ﻟU0ﺎن. اﻟ10ﻲ، ) tﻣIPﻔﻰ إﺑا1411 /ه1991 ،(. اﺑ اﻟﻌ ﻲ اﻟـﺎﻟ5ﻲ اﻹﺷـ? ﻠﻲ وﺗﻔـ ﻩ أﺣـﺎم اﻟﻘـ آن 1، دار اﻟRﻞ- دار ﻋ1ﺎر، ﺑوت- .ﻋ1ﺎن ) ﺟﻌﻔــ، ﻋUــ اﻟﻐﻔــر1428 /ه2007 .( +اﻟ4ﻔــ واﻟﻔــ ون ﻓــﻲ ﺛJــﻪ اﻟI+ﯾــ، 1 ،، دار اﻟــﻼم اﻟﻘﺎﻫةI، ﻣ. ) ﻋﺎﻟ، ﻋUـ رب اﻟ0Uـﻲ2005 .(اﻟ4ﻔـ اﻟﻔﻘﻬـﻲ اﻟ,ـ+ﯾO: ﻗMـﺎLﺎ أﺻـJﻟ3ﺔ وﻓﻘﻬ3ـﺔ ، 1 ، ﻣ0ـرات ﺟﺎﻣﻌﺔ ﻋU اﻟ1ﺎﻟ اﻟﻌV، ﺗPان ، اﻟ1ﻐب. ) :w7ﺎس، ﻓrﻞ ﺣ2010 .(إﺗﻘﺎن اﻟ? ﻫﺎن ﻓﻲ ﻋﻠJم اﻟﻘ آن ، 2 .، دار اﻟ0ﻔﺎﺋ، اﻷردن ) اﻟﻣﺟﻠد33 ( اﻟﻌدد اﻷول )ﯾوﻧﯾو2020 ( 65 ﻣن ﻣﻌﺎﻟم اﻟﻣﻧﮭﺞ ﻓﻲ ﺗﻔﺳﯾر "أﺣﻛﺎم اﻟﻘرآن" ﻟﻠﻘﺎﺿﻲ اﺑن اﻟﻌرﺑﻲ Some Methodical Aspects of Tafsīr "Ahkam Al Quran" by Judge Abu Bakr Muhammad bin Al-Arabi (D.543 H.) Hazem Zakaria Mohieddin Keywords: Ahkam Al Quran, Ibn Al Arabi, Tafsir, methodology. Abstract This study presents and analyses some methodical aspects of Tafsīr "Ahkam Al Quran by judge Ibn Al Arabi Maliki al-Andalusi (543 H), and his scientific formation as well as the historical events of his era that he interacted with in a brilliant way through his interpretation. In addition, this research studies some methodological aspects as follows: enhancing his knowledge of criticism by studying preceding Interpretive heritage, how he deals with language, how he deals with the sources of interpretation based on maxims, Prophetic Hadiths, and Jewish narrations, how much he depends on rational exegesis, and the rules of weighting in interpretation, his way of treating his opponents, and his attitude towards historical events. Descriptive method was utilized to achieve the goals of this study. The findings of the study reveals that Ibn Al Arabi relies on the analytical method in his exegesis, assimilating respectably and critically the heritage of the previous interpreters, as well as Ibn Al Arabi relies on methods of tradition and opinion interpretations. Also, he depends on the methodologies of tradition and opinion in his interpretation, and he emphasizes on textual criticism especially when dealing with weak, forged, and Jewish narrations. Moreover, he has a tendency to his doctrine on levels doctrine and jurisprudence. Finally, his interpretation of Quran is connected to Muslims issues in his era. Thus this feature augmented the status of this exegesis and increased its impact on its historical context Keywords: Ahkam Al Quran, Ibn Al Arabi, Tafsir, methodology. ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ ﻣﺟﻠﺔ اﻟﺟﺎﻣﻌﺔ اﻷﺳﻣرﯾﺔ : اﻟﻌﻠوم اﻟﺷرﻋﯾﺔ واﻹﻧﺳﺎﻧﯾﺔ 66
https://openalex.org/W2162294485	https://jbiomedsci.biomedcentral.com/counter/pdf/10.1186/s12929-014-0096-9	English	null	Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells	Journal of biomedical science	2,014	cc-by	5,299	Abstract Background: Hyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status. We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL). Results: The growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent. Conclusion: Higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions. Keywords: Glucose, Helicobacter pylori, CagA Keywords: Glucose, Helicobacter pylori, CagA Keywords: Glucose, Helicobacter pylori, CagA may be an important cofactor to influence H. pylori mediated gastric carcinogenesis [8]. * Correspondence: sheubs@mail.ncku.edu.tw 1Institute of Basic Medical Sciences, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 2Department of Medicine, College of Medicine, National Cheng-Kung University, Tainan, Taiwan Full list of author information is available at the end of the article © 2014 Sheu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells Shew-Meei Sheu1,2, Hsin Cheng2, Cheng-Ye Kao1,4, Yao-Jong Yang3, Jiunn-Jong Wu1,4 and Bor-Shyang Sheu1,2,5* Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Open Access Background Helicobacter pylori infection in the human stomach causes chronic inflammation, leading to peptic ulcers and gastric malignancy [1-3]. Other cofactors may be involved in the H. pylori related gastric carcinogenesis, because not all H. pylori-infected patients develop gastric cancer. Some reports have shown that there is a significantly higher prevalence of H. pylori infection in patients with type 2 diabetes mellitus (DM), who also have a lower eradication rate [4-6]. In particular, a higher fasting plasma glucose level in H. pylori-seropositive patients may correlate with an increased risk of gastric cancer up to a nearly 3.5-4.2 fold increase [7]. These data suggest that hyperglycemia To establish H. pylori colonization and chronic inflam- mation at the gastric epithelium, adherence can serve as the first step of infection and then deliver effectors to induce inflammation [9-11]. The major adhesion, blood group antigen binding adhesin (BabA), binds to Lewis b (Leb) and related ABO antigens to initiate bacterial adhesion and stimulate IL-8 secretion [12-14]. More- over, cytotoxin-associated gene A antigen (CagA), an onco-protein, is translocated into gastric epithelial cell through type IV secretion system of H. pylori [15-18]. The tyrosine in the EPIYA motif of CagA C-terminal could be phosphorylated [19] and thus can change the cell morphology, increase cell motility, and promote cell proliferation [20-23]. * Correspondence: sheubs@mail.ncku.edu.tw 1Institute of Basic Medical Sciences, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 2Department of Medicine, College of Medicine, National Cheng-Kung University, Tainan, Taiwan Full list of author information is available at the end of the article Hyperglycemia leads into an increased risk of gastric cancer in the H. pylori-infected patients [7]. Moreover, high glucose can enhance cancer-associated Wnt/β- Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Page 2 of 6 catenin signaling [24]. These results suggest a link between hyperglycemia, H. pylori and gastric cancer. High glucose could also increase endothelial permeability and altered basement membrane composition and structure [25,26], which may make H. pylori infection occur in a high glucose condition, such as the patients with hyperglycemia. There- fore, it is worth investigating whether or not glucose can influence the expressions of H. pylori virulence to promote carcinogenesis. This study is original to illustrate that higher glucose concentrations can promote bacterial growth of H. pylori isolates, facilitate bacterial adhesion to gastric epithelial cells, and up-regulate the expression of CagA protein to facilitate more virulence related to type IV secretion system. Western blotting for detection H. pylori BabA, CagA and CagA phosphorylation The bacterial suspension adding to detect adhesion ability under different glucose concentration was also collected to determine protein concentration. An equal amount of bacterial protein (1.5-2 μg) was used to detect the expres- sion of BabA and CagA by performing western blotting. Moreover, the well of co-cultured H. pylori and AGS cells incubated for 4 h was washed 3 times to collect cell lysates for detection of CagA and CagA phosphor- ylation. Antibodies (Ab) used in western blotting included the anti-BabA as applied in our previous report [27], anti-phospho-tyrosine (PY99) (Santa Cruz Biotechnology), anti-CagA (Santa Cruz Biotechnology) and anti-beta Actin (Minipore) Abs. Assessment to the H. pylori growth under different glucose levels Strain J99 and ATCC 43504 were purchased from ATCC and stored at –70°C in BHI with 30% glycerol until testing was conducted. H. pylori strains were cultured on CDC anaerobic blood agar (BBL, Microbiology Systems, Cock- eysville, MD, USA). When determining growth curve, H. pylori strains were cultured in Brucella broth containing 10% horse serum and different concentration of glucose (100, 150, 200 mg/dL) at 37°C for 3 days in a microaero- philic conditions, with shaking at 50-52 rpm. The optical density (OD) at 600 nm of bacterial growth was evaluated at 20, 48, and 72 h. Bacterial viability was detected at 48 and 72 h of growth curve and analyzed by serious dilution to count colony formation unit (CFU) per ml. H. pylori adhesion assay to AGS cells under different glucose levels In order to calculate the percentage of bacterial adhesion, lysate of H. pylori adhering to AGS cells and the original suspension of H. pylori adding to adhesion assays were seriously diluted respectively and grown on CDC plates to count bacterial CFU. The adhesion CFU divided by the original CFU is the adhesion percentage of H. pylori. Co-culture of H. pylori & AGS cells under different glucose levels The human gastric adenocarcinoma cell line, AGS, was purchased from Food Industry Research and Development Institute in Taiwan and was grown in the cell medium, Ham’s F-12 medium (GIBCO BRL, Grand Island, NY) containing 10% FCS. The cells were sub-cultured every second to third day. To conduct the H. pylori adhesion assays in AGS cells, the AGS cells (5.5 × 105/well) were seeded to one well of 6-well plates for 22 h and then replaced the cell medium to cell medium supplemented with different concentrations (100, 150, 200 mg/dL) of glucose for 3.5-4 h. After washing with the mixture of F-12 medium and 1× PBS (1:1) to the AGS cells cultivated in medium with specific concentrations of glucose, the serum-free F-12 medium (SFM) with the same glucose concentration were added again. As well, the both H. pylori strain J99 and 43504 were cultured for 20 hours in Brucella broth including 10% horse serum under three glucose concentrations, including 100, 150, 200 mg/dL, respectively. The bacteria shall be washed twice with the SFM containing the same glucose contraction before Background adding to cells. After washing, these bacterial suspensions in the SFM will be then applying to the well that con- tained AGS cells with the same concentration of glucose treatment at Multiplicity of infection (MOI) as 30-40. In each glucose level, there should be co-cultured with H. pylori and AGS cells for two wells, including the first one for adhesion assay, the second one for the detection of CagA phosphorylation. The first well was washed three times with the SFM 30 min later to detect bacterial adhesion and the second well was incubated for 4 h to detect CagA phosphorylation. Statistics The statistical analysis was performed by using a paired t test. The differences were considered to be significant at p < 0.05. Effect of glucose level on H. pylori growth curve and viability Strain J99 and 43504, grown in broth containing three concentrations of glucose (100, 150, 200 mg/dL), were applied to detect the growth curve during 72 h (Figure 1A and B). Optical density of strain J99 at 200 mg/dL were kept on increasing at 72 h, however, OD value was decreased in 100 and 150 mg/dL of glucose conditions at the same time (Figure 1A). The significant difference between 3 growth curves of strain J99 was observed at 48 h (J99-100 vs. J99-150, p < 0.05; J99-100 vs. J99-200, Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Page 3 of 6 Figure 1 H. pylori growth and viability was increased under higher glucose level after 48 hr. (A, B) Strain J99 and 43504 grew in Brucella broth containing 10% horse serum and different level of glucose (100, 150, 200 mg/dL). The optical density (OD) at 600 nm of bacterial growth was evaluated during 72 h. (C, D) Colony formation unit (CFU) of strain J99 and 43504 was determined at growth curve of 48 and 72 h. * indicated p < 0.05 (paired t test). Figure 1 H. pylori growth and viability was increased under higher glucose level after 48 hr. (A, B) Strain J99 and 43504 grew in Brucella broth containing 10% horse serum and different level of glucose (100, 150, 200 mg/dL). The optical density (OD) at 600 nm of bacterial growth was evaluated during 72 h. (C, D) Colony formation unit (CFU) of strain J99 and 43504 was determined at growth curve of 48 and 72 h. * indicated p < 0.05 (paired t test). bacteria (strain J99 on Figure 2A and C; strain 43504 on Figure 2B and D). p < 0.05; J99-150 vs. J99-200, p > 0.05) and 72 h (J99-100 vs. J99-150, p = 0.05; J99-100 vs. J99-200, p < 0.05; J99-150 vs. J99-200, p < 0.05). Growth of strain 43504 in higher glucose concentrations (150 and 200 mg/dL) had the trend of higher optimal density, as compared to low glucose (100 mg/dL), except 48 h. Besides strain 43504 at 48 h, H. pylori viability was increased by the increasing glucose concentration (Figure 1C and D). Especially at 72 h, the viability of two strains cultured in 150 and 200 mg/dL were consistently increased as compared to that in 100 mg/dL (p < 0.05). Effect of glucose level on H. pylori growth curve and viability Viability showed 3-4 fold increases in 200 mg/dL as compared to that in 150 mg/dL (p < 0.05). To check that the effect of glucose on increasing bac- terial growth and adhesion not due to change osmolality, we used L-glucose without biological activity to assess the same experiment. Our data confirmed L-glucose did not increase OD value at 72 h. Additionally, the adhesion ability of both strains in either 150 or 200 mg/dL of glu- cose were similar to that in 100 mg/dL (data not shown). Effect of glucose level on H. pylori type IV secretion system-related virulence BabA expression of H. pylori, in either strain J99 or 43504, was similar among three glucose concentrations including 100, 150, and 200 mg/dL. However, for either one isolates, the expression of CagA can be increased as the bacteria exposed to glucose concentration which was raised from 100 to 200 mg/dL (Figure 3A). When H. pylori infected to AGS cells, the cell-associated CagA of strain J99 pre- sented with a significant stepwise increase by the glucose treatment in level up to 150 and 200 mg/dL (p < 0.05), as compared to the glucose treatment as 100 mg/dL (Figure 3B). Also in Figure 3B, the cell-associated CagA of strain 43504 had the similar scenario to that of strain J99. Strain 43504 had a significantly increased trend of phosphorylated CagA levels ranking in order parallel to the elevation of glucose levels from 100, 150, and up to 200 mg/dL (p < 0.05, Figure 3B). Higher glucose level treated to H. pylori isolates enhance the bacterial adhesion Higher glucose level treated to H. pylori isolates enhance the bacterial adhesion With the treatment of 200 mg/dL of glucose to H. pylori and AGS cells, the adhesion ability of strain J99 and 43504 was significantly enhanced as compared with that of 150 and 100 mg/dL of glucose treatment (Figure 2A and B). In order to test whether cellular factors play some role in adhesion enhancement, strain J99 and 43504 growth in 100, 150, and 200 mg/dL of glucose adhered to AGS cells with pretreatment of with contrary con- centration (200, 150, 100 mg/dL), respectively (shown in Figure 2C and D). Nevertheless, the adhesion abilities within each strain were similarly disclosed with an in- creasing trend based on the glucose level treated to the Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Page 4 of 6 Figure 2 Adhesion ability of H. pylori increased under the treatment of higher glucose concentration. Strain J99 and 43504 growth in different level of glucose (100, 150, 200 mg/dL) was added to AGS cells with the pretreatment of the same glucose concentration (A, B) or the pretreatment of contrary concentration (200, 150, 100 mg/dL) (C, D). Relative adhesion ratio indicates that the adhesion of H. pylori in 100 mg/dL of glucose serve as the reference, and the bacterial adhesion value in 150 and 200 mg/dL of glucose was divided by the value of reference. indicated a significant difference between the treatment of different glucose concentrations (paired t test, p < 0.05). Figure 2 Adhesion ability of H. pylori increased under the treatment of higher glucose concentration. Strain J99 and 43504 growth in different level of glucose (100, 150, 200 mg/dL) was added to AGS cells with the pretreatment of the same glucose concentration (A, B) or the pretreatment of contrary concentration (200, 150, 100 mg/dL) (C, D). Relative adhesion ratio indicates that the adhesion of H. pylori in 100 mg/dL of glucose serve as the reference, and the bacterial adhesion value in 150 and 200 mg/dL of glucose was divided by the value of reference. indicated a significant difference between the treatment of different glucose concentrations (paired t test, p < 0.05). Discussion (B) The level of cell-associated CagA and phosphorylated CagA after H. pylori infecting AGS cells for 4 h. Thee glucose concentrations were 100, 150 and 200 mg/dL, respectively. indicated a significant difference between the treatment of different glucose concentrations (paired t test, p < 0.05). higher glucose concentrations (Figure 2A and B). We pro- vide data supporting the possibility that glucose may influ- ence H. pylori density colonizing on the gastric epithelium. H. pylori colonization could further interact with gastric epithelium to induce gastric inflammation [32,33]. In dia- betic patients, H. pylori infection was significantly associated with chronic gastritis, but not in dyspeptic patients [34]. This study should be considered as being original to illustrate that bacterial adhesion to gastric epithelial cells can be enhanced by a higher glucose condition. H. pylori growth in 100, 150 and 200 mg/dL of glucose is shown to have an increasing trend after being added to AGS cells pretreated with contrary concentrations of glucose (Figure 2C and D). It indicates that bacterial factors may play a more important role in increasing adhesion. When we analyzed BabA expression of H. pylori growth in three different glucose conditions, it did not show significant differences (Figure 3A). However, CagA expres- sion was elevated as H. pylori growth in the increasing concentration of glucose (Figure 3A). Cell-associated CagA of strain J99 and phosphorylated CagA of strain 43504 were significantly increased in higher glucose conditions of infection (150 and 200 mg/dL) (Figure 3B). Therefore, the enhancement of cell-associated or phos- phorylated CagA could be due to the elevated adhesion ability and CagA expression of H. pylori in higher glucose conditions (Figures 2A, B and 3A). Figure 3 Figure 3 H. pylori CagA and phosphorylated CagA increased in higher glucose concentration. (A) BabA and CagA expression of H. pylori grown in three glucose concentrations. (B) The level of cell-associated CagA and phosphorylated CagA after H. pylori infecting AGS cells for 4 h. Thee glucose concentrations were 100, 150 and 200 mg/dL, respectively. indicated a significant difference between the treatment of different glucose concentrations (paired t test, p < 0.05). Ishijima et al. [33] demonstrated that BabA-mediated adherence increases the effectiveness of H. pylori type IV secretion activity, implying the promoting role of H. pylori adhesion in type IV secretion activity. Moreover, phosphor- ylation of CagA is essential for induction of gastrointestinal neoplasm in transgenic mice [35]. Discussion conditions. These results support the second possible mechanism. The identification of cofactors involved in H. pylori- related gastric carcinogenesis is important due to the fact that not all H. pylori-infected patients developing gastric cancer. Higher fasting plasma glucose levels in H. pylori-seropositive patients significantly increased the risk of gastric cancer, suggesting that hyperglycemia may contribute to H. pylori-related gastric carcinogenesis [7,8]. The possible mechanisms by which hyperglycemia affects H. pylori-related gastric carcinogenetic process include: (1) glucose itself may activate Wnt/β-catenin pathway or increase reactive oxygen species to induce DNA damage, which contribute to H. pylori pathogenic effect; (2) hyper- glycemia influences H. pylori and H. pylori-related infec- tious status or stimulates its carcinogenic effect. However, at present, the detailed mechanism is remains unclear. In the present study, we demonstrate that higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA expression was further increased to facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose Reynolds and Pann demonstrated that glucose enhances H. pylori growth in a defined medium [28]. Albertson et al. [29] found that the presence of glucose did not show growth enhancement on H. pylori. This discrepancy may be due to strain variations and detection methods utilized. In the study of Albertson et al, growth and yield of H. pylori (numbers of CFU per milliliter) was shown to have no difference by adding 0.2 or 1.0% glucose (200 or 1000 mg/dL) to the defined medium supplemented with 0.5% BSA. This could be due to the high bacterial density (107 CFU/ml) at the beginning of inoculation. Glucose was rapidly consumed and could not provide for further growth because of growth and yield only increased before 20 h. In our study, we found that higher glucose concentrations (200 mg/dL) could keep up the growth curve detected by OD at 600 nm after 48 h of inoculation (Figure 1A and B). Moreover, the viability of H. pylori at the same concentration of glucose was obviously higher than that of 100 or 150 mg/dL at 72 h Page 5 of 6 Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Page 5 of 6 Figure 3 Figure 3 H. pylori CagA and phosphorylated CagA increased in higher glucose concentration. (A) BabA and CagA expression of H. pylori grown in three glucose concentrations. Conclusions In summary, we provide in vitro evidence that H. pylori growth, viability, CagA and phosphorelated CagA after infecting gastric epithelial cells could be enhanced in higher glucose condition, supporting that hyperglycemia could be a cofactor to increase a risk of H. pylori related gastric carcinogenesis. The evidence would indicate that it is important to conduct large-scale H. pylori screening and eradication to control the increased risk of gastric cancer for DM patients, especially in high H. pylori-infected or gastric cancer prevalent countries. (Figure 1C and D). This data was supported by Albertson et al. [29]. They found that viability of H. pylori in the pres- ence of glucose obviously retains after inoculation of 2 days, as compared to non-glucose addition. We further provide the result that glucose can enhance H. pylori viability with a dose-dependent effect at 72 h (Figure 1C and D). Whether or not an infection rate of H. pylori is higher in DM patients remains controversial. Some studies report that the prevalence of H. pylori in DM patients shows no significant difference as compared to controls [30,31]. In these articles, only the H. pylori infection rate was studied, but no more detailed analysis was conducted to trace the H. pylori density-related histology or virulence severity. Our study revealed that H. pylori adhesion was enhanced in (Figure 1C and D). This data was supported by Albertson et al. [29]. They found that viability of H. pylori in the pres- ence of glucose obviously retains after inoculation of 2 days, as compared to non-glucose addition. We further provide the result that glucose can enhance H. pylori viability with a dose-dependent effect at 72 h (Figure 1C and D). Whether or not an infection rate of H. pylori is higher in DM patients remains controversial. Some studies report that the prevalence of H. pylori in DM patients shows no significant difference as compared to controls [30,31]. In these articles, only the H. pylori infection rate was studied, but no more detailed analysis was conducted to trace the H. pylori density-related histology or virulence severity. Our study revealed that H. pylori adhesion was enhanced in (Figure 1C and D). This data was supported by Albertson et al. [29]. They found that viability of H. pylori in the pres- ence of glucose obviously retains after inoculation of 2 days, as compared to non-glucose addition. Discussion Due to the elevated phosphorylated CagA in higher glucose conditions, it may further contribute to facilitate a more evident gastric carcinogenesis in the H. pylori-infected DM patients. Figure 3 Figure 3 H. pylori CagA and phosphorylated CagA increased in higher glucose concentration. (A) BabA and CagA expression of H. pylori grown in three glucose concentrations. (B) The level of cell-associated CagA and phosphorylated CagA after H. pylori infecting AGS cells for 4 h. Thee glucose concentrations were 100, 150 and 200 mg/dL, respectively. indicated a significant difference between the treatment of different glucose concentrations (paired t test, p < 0.05). Figure 3 Figure 3 H. pylori CagA and phosphorylated CagA expression of H. pylori grown in three glucose concentrations. (B) The level of cell-associated CagA and phosphorylated CagA after H. pylori infecting AGS cells for 4 h. Thee glucose concentrations were 100, 150 and 200 mg/dL, respectively. indicated a significant difference between the treatment of different glucose concentrations (paired t test, p < 0.05). Conclusions We further provide the result that glucose can enhance H. pylori viability with a dose-dependent effect at 72 h (Figure 1C and D). Whether or not an infection rate of H. pylori is higher in DM patients remains controversial. Some studies report that the prevalence of H. pylori in DM patients shows no significant difference as compared to controls [30,31]. In these articles, only the H. pylori infection rate was studied, but no more detailed analysis was conducted to trace the H. pylori density-related histology or virulence severity. Our study revealed that H. pylori adhesion was enhanced in Competing interests The authors declare that they have no competing interests. References 1. 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J Biol Chem 2004, 279:17205–17216. Authors’ contributions d d h SMS conducted the experiment and prepared the draft. SBS developed the study concept and refined the draft. CH, KCY, YYJ and WJJ assisted with the gathering of data. All authors have read and approved the final manuscript. Page 6 of 6 Sheu et al. Journal of Biomedical Science 2014, 21:96 http://www.jbiomedsci.com/content/21/1/96 Acknowledgement h d protein in gastric epithelial cells by a type IV secretion apparatus. Cell Microbiol 2000, 2:155–164. g This study was supported by grants 101-2314-B- 006-016-MY3 from the National Scientific Council, and DOH102-TD-C-111-003 from Department of Health, Taiwan. 17. Odenbreit S, Püls J, Sedlmaier B, Gerland E, Fischer W, Haas R: Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 2000, 287:1497–1500. Author details 1 18. 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Rad R, Gerhard M, Lang R, Schöniger M, Rösch T, Schepp W, Becker I, Wagner H, Prinz C: The Helicobacter pylori blood group antigen-binding adhesin facilitates bacterial colonization and augments a nonspecific immune response. J Immunol 2002, 168:3033–3041. 15. Asahi M, Azuma T, Ito S, Ito Y, Suto H, Nagai Y, Tsubokawa M, Tohyama Y, Maeda S, Omata M, Suzuki T, Sasakawa C: Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med 2000, 191:593–602. doi:10.1186/s12929-014-0096-9 Cite this article as: Sheu et al.: Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells. Journal of Biomedical Science 2014 21:96. 16. Backert S, Ziska E, Brinkmann V, Zimny-Arndt U, Fauconnier A, Jungblut PR, Naumann M, Meyer TF: Translocation of the Helicobacter pylori CagA
https://openalex.org/W2061914542	https://hts.org.za/index.php/hts/article/download/2530/4343	Afrikaans	null	Oor die metodiek van ’n holistiese geskiedenis van Oud-Israel	HTS teologiese studies	1,993	cc-by	11,284	Oor die metodiek van n holistiese geskiedenis van Oud-Israel E H SchefQpr Universiteit van Suid-Afrika Abstract On the methodology of a holistic history of ancient Israel In this article certain issues relating to the concept ‘holistic historiography’ are clarified. Thereafter follows a reflection on methodology that would be appropriate for holistic historiography, involving the critical use of biblical and extra-biblical sources, Syro-Palestinian archaeology, historical geography, sociology, psychology and secondary literature. Abstract On the methodology of a holistic history of ancient Israel In this article certain issues relating to the concept ‘holistic historiography’ are clarified. Thereafter follows a reflection on methodology that would be appropriate for holistic historiography, involving the critical use of biblical and extra-biblical sources, Syro-Palestinian archaeology, historical geography, sociology, psychology and secondary literature. 1. INLEIDING In ’n vorige bydrae (kyk Scheffler 1988a:665-683) het ek betoog dat indien historio- grafie reg wil laat geskied aan die voile spektrum van menslike lewe, dit komprehen- sief of omvattend moet wees. Dit geld ook veral wat die historiografie van Israel in die Ou-Testamentiese tydvak betref, aangesien laasgenoemde in die verlede hoof- saaklik op politieke aspekte gekonsentreer het. Hierdie aspekte het hoofsaaklik betrekking op gebeure aan die koninklike hof, die lewens van die konings of hoog- geplaastes en die berig van militêre veldslae.i Kulturele en godsdienshistoriese stu dies is wel apart onderneem, maar ’n komprehensiewe geskiedskrywing beoog egter om gesamentlik en geintegreerd ’n geskiedenis aan te bied waarin politieke, sosio- kulturele, godsdienstig-ideologiese en literêre (kommunikatiewe) dimensies van die werklikheid oor alle vlakke van die samelewing heen in hulle onderlinge samehang ondersoek word. Alvorens ek nou (soos voorheen voorsien, vgl Scheffler 1988a:682) ’n gepaste metodiek vir so ’n geskiedskrywing gaan ondersoek (waarin die gebruik 908 HTS 49/4 (1993) E S c h ^ van primêre en sekondêre literatuur, argeologie, geografie, sosiologie en psigologie aan die bod sal kom) verdien verskeie sake rakende die begrip ‘holistiese historio- grafie’ eers ons aandag van primêre en sekondêre literatuur, argeologie, geografie, sosiologie en psigologie aan die bod sal kom) verdien verskeie sake rakende die begrip ‘holistiese historio- grafie’ eers ons aandag 2. CX)R DIE BEGRIP‘HOUSnESE HISTORIOGRAFIE’ Omdat geskiedenis egter per slot van sake die totale werklikheid raak, ook as daar gefokus word op ’n voorafgedefinieerde deel(tjie) van die geskiedenis van die totale werklikheid (soos bv die geskiedenis van Israel), het die holistiese perspektief juis implikasies vir geskiedskrywing. Dit sou byvoorbeeld beteken dat verskeie dimensies van Israel se geskiedenis nie slegs in isolasie ondersoek word nie, maar ook op mekaar betrek sal word. Dit sou verder beteken dat die ondersoek na die politieke dimensie juis nie volledig sou wees indien dit nie bevra word in terme van die sosio-kulturele, godsdienstig-ideologiese en literêre (kommunikatiewe) dimen sies nie. Daar moet toegegee word dat die ‘betrekking’ van die dimensies op mekaar ’n interpretatiewe stap deur die historikus behels. Nogtans is dit nood- saaklik omdat belangrike insigte in die geskiedenis as geheel (wat juis ter wille van kenbaarheid in verskeie dimensies onderverdeel is) andersins verlore sou gaan. ) g Daar word toegegee dat die vier dimensies wat vir Oud-Israel se geskiedenis onderskei is, nie absoluut is nie. Bloot die term ‘samelewingsgeskiedenis’ (vgl Thompson 1970:423-430; Deist 1987:20-31; Heyns 1986) sou ook ’n holistiese bena- dering kon impliseer. Aan die ander kant is die term ‘samelewingsgeskiedenis’ ook vir misverstand vatbaar aangesien dit enersyds ’n benadering kan impliseer waarin politieke aangeleenthede steeds die fokus het, of andersyds juis met die oog op gewone mense wat dan die samelewing sou uitmaak, onderbeklemtoon word. Laas- genoemde gevaar dreig vir my juis wanneer (in reaksie teen eensydige politieke ge skiedenis) die stelling gemaak word dat geskiedenis nie politieke geskiedenis behels nie maar geskiedenis Van mense’ is.^ ’n Holistiese benadering tot die geskiedenis sou egter sodanige swaai van die pendulum wou ondervang. Deurdat geskiedenis nou baie breër gesien word, impliseer glad nie dat die politieke dimensie onderspeel moet word nie - inteendeel, die ingrypende en in baie opsigte selfs bepalende in- vloed van politieke aangeleenthede opsigself, én op die lewe van die alledaagse mens word steeds erken. Daarom sou dit sin maak om enige aanbieding van ’n holistiese geskiedenis steeds met die politieke dimensie (as die breedste moontlike konteks) te laat begin. Dit is ’n vraag oor hoe die interaksie tussen geheel en dele in ’n holistiese geskiedskrywing hanteer moet word. Moet politiek, kultuur, ideologie en literatuur alles tegelyk aangebied word of eers afsonderlik? 2. CX)R DIE BEGRIP‘HOUSnESE HISTORIOGRAFIE’ Met ‘komprehensiewe’ en ‘holistiese’ geskiedskrywing word dieselfde saak bedoel. Ek reken egter dat dit tans meer van pas sou wees om te praat van holistiese geskiedskrywing en wel om verskeie redes. Eerstens word die term ‘holisties’ meer en meer in die geesteswetenskappe gebruik wanneer daar uitdrukking gegee wil word aan interdissiplinêre samewerldng (vgl Van Aarde 1988:49-64). Holistiese geskiedskrywing maak vandag toenemend van ander dissiplines (as sogenaamde hulpwetenskappe) in die volvoering van sy taak gebruik. Die besef van die sosiologiese, antropwlogiese en psigologiese dimen- sies van geskiedenis beinvloed geskiedskrywing so wesenlik dat die kunsmatige grense tussen die vakwetenskappe eintlik vervaag. Tweedens is die term ook van pas as daar gepoog word om ’n sintetiese perspek- tief op ’n bepaalde vakgebied te verkry. Die behoefte aan sodanige perspektief ont- wikkel juis wanweë toenemende spesialisasie in ’n vakgebied wat veroorsaak dat ’n geheelbeeld op die gebied (of selfs spesifieke onderwerp) verlore gaan. Die skrywe van die geskiedenis van Israel vanuit ’n uitsluitlik politieke perspektief hou dan ook in ’n bepaalde sin ’n eensydige beeld van Israel as geheel voor. Derdens is daar aspekte van die filosofie van holisme soos oorspronklik deur Smuts ([1926] 1987) ontwikkel, wat myns insiens uiters relevant is vir die skryf van holistiese geskiedenis. Holisme behels naamlik dat die geheel van die dele afhank- lik is en dat die dele opsigself ook eers hulle sin vind in die geheel. Dit is die ‘philo sophical view that nature combines units into organic wholes and that these units, although each functioning by its own laws, are functionally subordinate to the whole’ (Deist 1990:116). Of soos Smuts dit self saamvat ([1926] 1987:317): Holism has been presented...as the ultimate synthetic, ordering, orga nising, regulative activity in the universe which accounts for all the structural groupings and syntheses in it, from the atom and the phy sico-chemical structures, through the cell and organisms, through Mind in animals, to Personality in man. The ail-pervading and ever- increasing character of synthetic unity or wholeness in these structures leads to the concept of Holism as the fundamental activity underlying and co-ordinating all others, and to the view of the universe as a Holistic Universe. ISSN 0259 9422 = 909 HoKtiicsc gcsticdcms Smuts het sy besinning uiteraard nie binne die konteks van geskiedsfilosofie gedoen nie. 2. CX)R DIE BEGRIP‘HOUSnESE HISTORIOGRAFIE’ ’n Betoog waarin die vier dimen sies van die begin af geintegreerd aangebied word, sou sekerlik die ideaal wees en teoreties ook sekerlik die beste reg laat geskied aan wat met die term holisties ver- staan word." Vanweë die beperktheid van die mens se rede en die behoefte aan analise, is sodanige aanbieding egter nie altyd prakties, en dus wenslik nie, veral as die geskiedenis kompleks raak vanwee onder andere die spekulatiewe aard daarvan 910 HTS 49/4 (1993) E Scheffler E Scheffler as gevolg van die feit dat daar met die verre verlede gehandel word. Die aangewese weg om te volg sou dus wees om die dimensies eers afsonderlik te behandel, waama aan die einde van die afsonderlike behandelings en ook almal saam aan die einde, die dimensies op mekaar betrek sal word ten einde ’n insig te verkry in hulle onder- linge wisselwerking. In die aanbieding van die afsonderlike dimensies sal die histo- rikus egter juis nie sy oe sluit vir die ander dimensies nie en, soos wat sy aanbieding dit vir hom toelaat, die wedersydse invloed van die dimensies op mekaar vermeld. Slegs op hierdie wyse kan hy reg laat geskied aan die diversiteit binne die geskie- denis sowel as die uiters komplekse aard daarvan. As voorbeeld van die wisselwerking tussen verskeie faktore wat deur holistiese geskiedskrywing onderskei word, kan ons verwys na Salomo se koningskap^. Dat hy as koning (politieke dimensie) verantwoordelik was vir groot bouwerke (kulturele dimensie) is ’n bekende gegewe (vgl o a Miller 1986:204-213). Dat hy daarvoor uitermate hoe belastings gehef het en van dwangarbeid gebruik moes maak, impli- seer ’n bepaalde wisselwerking tussen die politieke dimensie (gebruik van mag) en sosio-kulturele dimensie (ekonomiese en fisiese uitbuiting van die gewone burger ter wille van kulturele ontwikkeling). Die saak word nog meer insiggewend indien die godsdienstig-ideologiese dimensie ook hierby betrek word. Wat sou Salomo se ideologiese beweegredes vir sy optrede gewees het? Ons weet dat die tempel in Jerusalem (‘vir die Naam van die Here’ - vgl 1 Kon 5:5) een van sy belangrikste bouwerke was. Sou Salomo gereken het dat die opbou van die land deur groot bou werke ook tot eer van God gedien het, omdat Israel immers God se land was? Was hierdie godsdienstig-ideologiese beskouing van Salomo so sterk dat hy sy optrede teenoor Jan Alleman in die lig hiervan kon regverdig? 2. CX)R DIE BEGRIP‘HOUSnESE HISTORIOGRAFIE’ Of wou Salomo ten diepste - soos dit maar so dikwels die geval is met heersers van destyds sowel as vandag - homself maar net verewig deur bouwerke, en het hy die tempelgodsdiens gebruik om dit te regverdig? Het hy dus die godsdiens voor sy eie karretjie ingespan soos dit gewoonlik voor oorloë gebeur? Die wisselwerking van die godsdienstig-ideologiese dimensie met die politieke en sosiale kan egter ook van onder gedoen word. Hoe het Jan Alleman Salomo se optrede beleef en religieus verwerk? Is God nie beskou as aan die kant van die arme, eenvoudige en geringe nie? Het die koningsvyandige tradisie in Israel (wat sou beweer dat God alleen Israel behoort te regeer) nie juis ontwikkel as gevolg van Salomo se optrede nie? Dit is goed moontlik dat die godsdienstige perspektief van die gewone onderdaan (as kritiek op die koningskap van Salomo) juis beslag gevind het in Deuteronomium 17:14-20 (vgl met 1 Kon 3-11) waarin oor die beperkings wat ten opsigte van koningskap behoort te geld, gehandel word. . ISSN 0259 9422 = HTS 49/4 (1993) 911 Hoiisticsc geskicdcnis Vierdens behels holistiese geskiedenis dat by die bestudering van ’n sekere periode in die geskiedenis van ’n bepaalde land of volk die totale (strukturele geskiedenis of geskiedenis van ‘langer duur’) geskiedenis van daardie land of volk juis insigte kan verskaf wat, indien dit in ag geneem sou word, dit nie alleen lig op ’n bepaalde periode kan werp nie maar juis kan help om teoretiese oplossings aan te bied vir die moeilikste historiese probleme van ’n bepaalde periode. So byvoor- beeld benader Coote & Whitelam (1987), op voetspoor van Braudel (1972) se beginsel van la longue durée, die moeilike probleem van Israel se ontstaan: The emergence of Israel, datable to about 1250-920 BCE, is, to use concepts popularized by Braudel, a surface event understandable only in terms of the wider, slower movement of much longer duration, what Braudel styles la longue durée. Human beings in history are con strained by climate, topography, vegetation, animal population, agri cultural potentialities, and the like. Surface events, primarily those events which are most often the focal point of traditional political his tories, are played out around more permanent elements such as urban sites, trade routes, harbours, and climate. 2. CX)R DIE BEGRIP‘HOUSnESE HISTORIOGRAFIE’ The realizations of the various potentialities of Palestine have been governed throughout its agrarian history by the presence (or more rarely absence) of outside powers and the complex interaction of external world events. (Coote & Whitelam 1987:23) (Coote & Whitelam 1987:23) Die land Palestina het met ander woorde ’n bepaalde ritme (vgl Alt [1944] 1969:1- 19; Weippert 1988:XV) wat oor ’n baie lang tydperk onderskei kan word. Voordat hulle enigsins aandag aan Israel se ontstaan self gee, ondersoek Coote & Whitelam (1987:27-116) eers die vestigingspatrone, streekshandel, geografie en sosiale ver- houdings in Palestina vanaf die Vroeë Bronstydperk (3150 v C) tot vandag. Die ver- onderstelling is dat insig in hierdie ‘long duration’ aan die historikus die raamwerk verskaf om te kan onderskei wat in ’n bepaalde periode moontlik was (vgl ook Coote 1990). Die hierbo genoemde voorbeelde van Salomo se koningskap, sowel as die histo riese probleem van Israel se ontstaan in die lig van die totale geskiedenis van Pales tina, toon myns insiens duidelik aan dat holistiese geskiedenis - as gevolg van die vrae wat dit aan die geskiedenis vra, nooit as afgehandel beskou kan word nie. Dui- sende permutasies van moontlike vrae deur duisende moontlike historic! met uit- eenlopende moontlikhede van antwoorde op die gestelde vrae, is moontlik. In elk geval is die ‘afgehandeldheid’ van sogenaamde ‘feite’ of “barre gegewens’, in die lig van die voortgang en sukses van argeologiese opgrawings in Palestina (vgl Thomas 1%7; Dever 1990; Kenyon 1988; Mazar 1990; Weippert 1988; Ben-Tor 1992) ook ’n 912 HTS 49/4 (1993) EScheffler mite. Soos wat ons hieronder sal betoog, werp die gebruik van ander sosiale weten- skappe ook aansienlike lig op die historiografie as ’n interpretatiewe wetenskap. mite. Soos wat ons hieronder sal betoog, werp die gebruik van ander sosiale weten- skappe ook aansienlike lig op die historiografie as ’n interpretatiewe wetenskap. In wat volg word daar besin oor hoe daar te werk gegaan kan word in die holis- tiese bestudering van Israel se geskiedenis. Aangesien die historikus nie as tabula rasa sy taak aanpak nie en alreeds ’n bepaalde voorkennis het ten opsigte van die meeste sake wat hieronder aangeroer word, behels wat volg, nie ’n uiteensetting van stappe wat die navorser chronologies móét volg nie. Juis omdat inligting uit verskil- lende ‘bronne’ onderling op mekaar betrek word, is dit nodig dat ter eniger tyd aan enige bron of sosiaal-wetenskaplike insig aandag gegee kan word. Die volgorde van aanbieding verraai egter wel ’n relatiewe prioriteit van die hulpmiddele wat tot die historikus se beskikking is, soos wat dit by elke onderafdeling ook sal blyk. 3.1 Die Bybelse geskrifte Die Bybel (die Protestantse en Joodse historikus moet die apokriewe boeke hierby insluit) bly myns insiens steeds die belangrikste bron van inligting vir die geskied- skrywing van Oud-Israel, aangesien dit self van die oudste geskiedskrywing van Israel bevat (Noth 1960:42-44; Herrmann 1980:54-59; Donner 1984:22-27; Miller & Hayes 1986:58-79; Deist 1987:5-17). Uiteraard kom hierdie uitgangspunt vandag toenemend onder die loep, veral in die lig van die moontlikheid wat al hoe meer geopper word om ’n geskiedenis van Israel te skiyf sonder gebruikmaking van die Bybel (vgl Miller 1991:93-102). Dat die Bybel geen geskiedenisboek is nie, is ’n stel- ling wat holruggery is - veral as in terme van politieke geskiedenis geredeneer word. Holistiese geskiedskrywing bring egter juis meer dimensies in spel: die feit dat die Bybel juis literatuur is wat godsdienstig-ideologiese opvattings weerspieël, is vandag nie meer ’n struikelblok vir geskiedskrywing nie, maar juis ’n uitdrukking daarvan. Ten opsigte van die politieke en sosio-kulturele dimensies geld die beperktheid van en ’n kritiese benadering jeens die Bybel egter steeds. Die Deuteronomistiese geskiedswerk dek die geskiedenis vanaf die intog tot en met die Babiloniese Ballingskap* terwyl die Kronis in sy eerste nege hoofstukke (1 Kron 1-9) deur middel van geslagsregisters ’n voëlvlugoorsig oor die geskiedenis vanaf die patriarge tot ná die Ballingskap gee, waama hy dan vollediger berig oor die Verenigde Koninkryk, die Noordryk en die Suidryk tot teen die einde van die Ballingskap (1 Kron 1 0 - 2 Kron 36). Esra-Nehemia berig dim uitvoerig oor sekere sake in die Persiese tydvak (vir ongeveer 200 jaar het ons egter geen bron nie) ter wyl die Makkabeerboeke weer berig oor die Hellenistiese tydvak. Die ander Bybel- BSN (E » 9«22 - HT5 «>/¥ (1993) 913 Hofisticflc se boeke (Pentateug, profete, poëtiese literatuur, wysheidsboeke) behoort opsigself nie tot die genre van geskiedskrywing nie. As produkte van die geskiedenis kom hulle egter nie slegs tereg by die literêre aspek van die geskiedenis nie, maar hulle bevat ook belangrike historiese inligting wat op ’n kritiese wyse deur die historikus verdiskonteer kan word. By die gebruik van die Bybel vir ’n hedendaagse holistiese konstruksie van die geskiedenis van Israel moet ’n paar prominente sake egter in gedagte gehou word. Eerstens bevat die Bybel die oudste berigte oor Oud-Israel. Dit tel in die Bybel- se geskiedskrywers se guns dat hulle hulle in ’n situasie bevind het wat chronologies baie nader was aan die gebeure waaroor hulle berig. 3.1 Die Bybelse geskrifte Hierdie diskrepansies vereis uit die aard van die saak ’n siftende, kritiese benadering by die geskiedskrywer wat nie die oneerlike weg van blote harmoniëring van die gegewens wil volg nie. Vierdem besit die uiteenlopende geskrifte wat in die Bybel voorkom, ook ver- skillende grade van betroubaarheid. Die datering van Bybelse materiaal is geen seker saak nie en veral wat die pre-monargale periode (waaroor ons feitlik geen lite- ratuur het nie en ouer tradisies moet probeer onderskei wat moontlik in latere teks- te opgeneem is) betref, beweeg ons op losse grond. Daarbenewens is materiaal wat wel onderskei kan word, ook histories onbetroubaarder as materiaal wat dateer uit ’n tyd toe geskiedskrywing reeds minstens ’n aanvang geneem het. Die Pentateug se berig oor die eksodus kan nie summier sonder meer as histories betroubaar aanvaar word, bloot omdat daaroor berig is nie. Die berig stam naamlik uit ’n latere tydvak en verskaf in velerlei opsigte meer inligting oor die tyd wanneer berig word ( m a w die tyd toe die verhaal ontstaan het) as oor die tyd waaroor berig word. Die Bybelse materiaal weerspieël dus minstens die periode waarin dit ontstaan het (of redaksio- neel verwerk is) en is daarom vir ons ’n spieël op die geskiedenis van Oud-Israel. Ten einde die Bybel as bron vir geskiedskrywing te kan gebruik, moet die teks dus krities ondersoek word. Die historikus is in die eerste plek eksegeet en sal alle inligting wat eksegete in kommentare geboekstaaf het, krities nagaan en met die cog op ’n ruimer insig in die geskiedenis verdiskonteer. Die histories-kritiese metode is hier van groot hulp. Deur middel van literêre kritiek word die bronne wat aan ’n teks ten grondslag lê, onderskei en gesitueer binne die tydvak waaruit dit kom. Indien twee verskillende tekste oor dieselfde gebeure berig, moet hulle krities met mekaar vergelyk word ten einde so na as moontlik aan die gebeure self te kom (vgl Jos 1-12 en Rig 1 - kyk De Vaux 1978:593-596). Deur middel van oorlewerings- kritiek word selfs die voorfases van die tekstuele neerslag ondersoek en tradisies wat sodanig onderskei is, sal dus moontlik inligting bevat oor die vroegste fases van die geskiedenis en as sodanig vorm hulle self deel van daardie geskiedenis (vgl Barth & Steck 1980:47). Redaksionele toevoegings tot bestaande tekste verskaf eweneens inligting van die tyd toe die toevoegings of veranderings aan die teks gemaak is. 3.1 Die Bybelse geskrifte Maar dit is juis hulle antieke gesitueerdheid wat verhoed dat hulle noodwendig meer betroubare beriggewers as modeme geskiedskrywers kan wees. Bybelse geskiedskrywers het hulle geskiedskry wing nie krities beoefen nie, maar wel met duidelike subjektiewe vooronderstellings voor oë. ’n Tweede belangrike saak hang hiermee saam. Hierbo is reeds vermeld dat dikwels gesê word (met die oog op mitologiese en ‘onhistoriese’ inhoude in die Bybel wat die ‘gesag’ en ‘waarheid’ van die ‘Woord van God’ sou aantas) dat die Bybel nie ’n ‘geskiedenishandboek’ is nie. Weinig word egter gereflekteer oor wat ’n geskiedenishandboek sou wees en oor wat die aard van die geskiedenisboeke sou wees wat ons wel in die Bybel aantref. Die Deuteronomistiese en Kronistiese geskiedswerke en die Makkabeerboeke bevat naamlik pre-kritiese en (veral wat die Koningsboeke betref) annalistiese geskiedskrywing'^. Die verhale sentreer nie slegs rondom die persoon en dade van die konings nie, maar word doelbewus gerig deur ’n ideologiese of godsdienstige doelwit. So word byvoorbeeld algemeen aanvaar dat die Deuteronomis die geskie denis verhaal om aan te dui dat beide Israel en Juda se ballingskappe plaasgevind het as gevolg van die verkeerde godsdienstige wee wat die Israeliete bewandel het (vgl 2 Kon 17:7-23). As gevolg van sodanige godsdienstig-ideologiese oogmerke kan belangrike historiese inligting eenvoudig weggelaat word (die Deuteronomis berig bv slegs in agt versies oor die belangrike regeringstyd van Omri* - vgl 1 Kon 16:15- 28), terwyl sekere inligting weer oorbeklemtoon kan word omdat dit inpas by die skrywer se religieuse perspektief (bv die uitvoerige berigte oor die optrede van Elia en Elisa in 1 Kon 1 7 - 2 Kon 9). Hiermee word geensins beweer dat die religieuse intensie wat die skrywer aan sy verhaal gee, onbelangrik is nie. Holistiese geskied skrywing bestudeer juis ook die godsdiens en ideologie van Oud-Israel. Laasge- noemde moet daarom krities ontsluit word wanneer daar uit godsdienstige perspek tief oor politieke aangeleenthede berig word. 914 NTS 49/4 (1993) E Sduffler E Sduffler Derdens bevat die Bybelse verhale - ook in die sogenaamde geskiedswerke - verskeie historiese teenstrydighede (vgl bv Jos 8:3 teenoor Jos 8:12; Jos 15:63, Rig 1:21, 2 Sam 5 teenoor Rig 1:8; 1 Sam 16:14-23 teenoor 1 Sam 17:55-58; 1 Sam 17:1, 49 teenoor 2 Sam 21:18-19 teenoor 1 Kron 20:5; 2 Kon 21:16-18 teenoor 2 Kron 33: 12-20; vgl verder Deist 1979). 7)2 Buite-Bybelse geskrifie Naas die Ou Testament bestaan daar ’n magdom literatuur uit die ou Nabye Ooste wat met die gegewens in die Ou Testament vergelyk kan word. Hierdie literatuur moet net so krities as die tekste van die Ou Testament self benader word. Dit is belangrik, veral omdat binne die Ou-Testamentiese kritiese wetenskap die neiging begin posvat het cm die Ou-Testamentiese tekste uiters krities (tereg) te evalueer maar dan ’n haas fundamentalistiese werkswyse te volg indien ons met buite Bybelse tekste te doen het. Die buite-Bybelse literatuur handel ook meestal oor die hooggeplaastes in die samelewing (soos bv die Assiriese koninklike annale - vgl Pritchard 1%9:274-301) en bevat dus slegs karige en indirek afleibare gegewens oor die gewone burgers van die land. Hierdie literatuur moet dus op meriete en van geval tot geval beoordeel word terwyl veral die genre telkens in aanmerking geneem word. So byvoorbeeld is daar heelwat af te lei oor die sosiale toestande in Palest tydens die laat-Bronstydperk (1550-1200) uit die ongeveer 400 Amarna-tablette (slegs ’n seleksie in Pritchard 1969:483-490) wat bestaan uit amptelike korrespon- densie tussen die Egiptiese owerhede en die vasalvorste van die Kanaanitiese stad- state. Hierdie literatuur het, uit ’n historiese hoek gesien, die voordeel dat hulle oor praktiese sake uit die alledaagse lewe handel en ook kontemporêr stam uit die tyd waaroor hulle berig. Heel anders is die geval van die sogenaamde Merneptah-stele (ongeveer 1230 v C, of Israel-stele - Pritchard 1969:376-378), ’n oorwinningsgedig van die Egiptiese koning, Merneptah, waarin die oorwinnings van die koning oor ander volkere in Klein-Asie besing word. Hierdie gedig bevat in velerlei opsigte oordrywings en onakkuraathede, omdat ons uit ander broime weet dat Merneptah uit ’n militaristiese oogpunt ’n ‘swak’ farao was. Die Memeptah-stele het egter nie daarom geen historiese waarde nie. In die stele kom die naam Israel (as bevolkings- groep) vir die eerste keer in buite-Bybelse literatuur voor. Uit die oogpunt van holistiese historiografie is dit belangrik dat baie buite- Bybelse literatuur godsdienstig (bv die Babiloniese skeppingsepos, Gilgamesh-epos, die wysheid van Amen-em-opet en Agikar, die Babiloniese teodisee - vgl veral Beyerlin 1975) en sosiaal (bv die kode van Hammurabi, die wette van Eshnunna, Lipit-Ishtar en Ur-Nammu) relevant is. 3.1 Die Bybelse geskrifte Die historikus is veral gemteresseerd in die historiese omstandighede wat hierdie veranderings aan die teks gefasiliteer het (bv die Priesterlike redaksie van die Pentateug tydens die Babiloniese Ballingskap). ISSN 0259 9422 = HTS 49/4 (1993) 915 HoiisticK gcskiedciiis 7)2 Buite-Bybelse geskrifie Hierdie literatuur verskaf nie slegs ’n goeie vergelykingsbasis vir die Israelitiese literatuur deurdat die historikus daardeur ’n breëe kompetensie opbou van die godsdienstige en sosiale toestande in die ou Nabye Ooste nie, maar verteenwoordig in baie gevalle die oorspronklike voedbo- dem van die Israelitiese literatuur. ’n Toenemende keimis van hierdie literatuur lei onteenseglik tot die historiese insig dat Oud-Israel nie net geografies nie, maar ook 916 HTS 49/4 (1993) E Sduffler E Sduffler op sosio-kulturele vlak integraal ingebed was in die leefwêreld van die ou Nabye Ooste. Die omvangrykheid van die beskilcbare literatuur (veral wanneer mens vanuit ’n holistiese perspektief alles as relevant beskou) veroorsaak egter vir die historikus ’n probleem. Daar bestaan byvoorbeeld ongeveer 20 000 spykerskriftablette uit die Mesopotamiese stad Mari (18e eeu v C) waaronder 5 000 briewe waarvan slegs vier in Pritchard (1969:482-483) opgeneem is. Die historikus staan dus onvennydelik voor die situasie dat hy beperkte toegang tot hierdie tekste het (waarvan baie nog nie ontsyfer is nie) en dat hy moet selekteer. Seleksies wat reeds gedoen is en in vertaalde vorm gepubliseer is, verskaf dus ’n goeie vertrekpunt. Pritchard (1969) het sekerlik die omvangrykste seleksie gedoen van die vemaamste tekste uit die ou Nabye Ooste wat op die Ou Testament betrekking het. Dit is van kort inleidende notas voorsien wat ’n wegwyser tot die totale korpus bied. Korter seleksies (met iet- wat meer uitvoerige inleidings as Pritchard) is gedoen deur Thomas (1961) en Beyerlin (1975 - laasgenoemde hoofsaaklik religieuse tekste). Gottwald (1985:52-55; vgl ook Scheffler 1988b:67) is die oningev^ryde tot verdere hulp met ’n geseleketeerde lys van geskrifte wat volgens tema, literêre genre of historiese verband gekorreleer is met die Bybelse literatuur. By die naam van elke geskrif word die oorspronklike taal aangedui sowel as die bladsynommers soos dit in Pritchard (1969) of die Engelse vertaling van Beyerlin (1975 = Near Eastern religious texts relating to the Old Testament) voorkom. Nie alle buite-Bybelse geskrifte werp noodwendige enige of tot dieselfde mate lig op die geskiedenis van Israel nie. In sommige gevalle kan daar inderdaad literê- re afhanklikheid tussen Ou-Testamentiese geskrifte en sommige van hierdie geskrif te bestaan. Literêre afhanklikheid is egter nie ’n voorwaarde vir historiese belang nie. 7)2 Buite-Bybelse geskrifie Dit is dus doenlik om weer eens te beklemtoon dat indien die historikus van hierdie bronne gebruik maak om die geskiedenis van Israel te bestudeer, hy elke geskrif ad hoc, op meriete en krities moet beoordeel ten einde vas te stel of dit sy spesifieke doel dien al dan nie. 4. DIE GEBRUIK VAN ARGEOLOGIESE GEGEWENS 4. DIE GEBRUIK VAN ARGEOLOGIESE GEGEWENS Die geskiedenis van die besondere verband wat daar bestaan tussen argeologie en die Bybelwetenskappe is ’n veelbewoë een. Aan die een kant is die buitengewone belangstelling wat daar bestaan in die argeologiese tydvakke van die laat-Bronstyd- perk tot die Romeinse tyd, te danke aan diegene wat vanuit Bybels-teologiese oor- wegings ’n aktiewe belangstelling in argeologie toon. Aan die ander kant is dit juis ’n oorgretige, romantiese belangstelling wat poog om die Bybelse gegewens met die ISSN 0259 «H22 •= HTS 49/4 (1993) 917 Hofiiticie gpikiedcBÍi hulp van argeologie te bewys wat tot onwetenskaplike spekulasies aanleiding gegee het (vgl bv die boek van Keller 1955). In die gesldedenis van opgrawings in Palestina was daar dus gewoonlik drie fak- tore ter sprake, te wete die Bybel, die geskiedenis en argeologie. Indien die Bybel as vertrekpunt geneem is, is daar dan gepoog om vanuit die argeologie aanknopings- punte te soek wat, indien dit gevind sou word, die historiese waarde van die Bybel sou onderstreep. In hierdie verband kan veral die werk van die Albright-skool genoem word (vgl Albright 1949; 1957; Wright 1960; 1962; Unger 1954).9 Indien die geskiedenis as vertrekpunt geneem word, word daar eerlik gepoog om die geskiede nis te konstrueer waarvoor die Bybel sowel as die argeologie as bronne kan dien. Indien Bybelse (en buite-Bybelse) gegewens en argeologiese data gekorreleer word, hang die wetenskaplikheid daarvan af of dit krities gedoen word en nie miskien sir- kelredenasies ten prooi val nie (vgl Miller 1976:46; Smith 1970:33). ’n Gangbare konklusie op grond van argeologiese gegewens sou slegs gesteun kon word indien daar genoegsame argeologiese data beskikbaar is en wanneer altematiewe interpre- tasies verreken is (vgl Nel 1987:23). Indien so ’n interpretasie op eie meriete gedoen kan word, sonder inagneming van ander getuienis (soos bv die Bybel), des te beter (vgl Barr 1977:138). 5. DIEGEBRUIKVANGEOGRAFIESEGEGEWENS 5. DIEGEBRUIKVANGEOGRAFIESEGEGEWENS Naas geskrifte soos die Bybel, ander Oud-Oosterse literatuur en argeologiese arte- fakte, is die land Palestina by uitnemendheid dit wat ’n skakel verskaf tussen die hede en die verlede. Vertroudheid met die geografie van Palestina is dus vir die navorser van Oud-Israel se geskiedenis onontbeerlik, nie slegs omdat mens met geo- grafiese kennis ’n beter beeld kan vorm van die gebeure wat hulle in Palestina afge- speel het nie, maar juis ook omdat sodanige kennis dikwels bydra tot die vorm van historiese oordele (vgl Rainey 1982; Olivier 1991; Burger 1992). So kan iemand wat geen geografiese kennis het nie, die gegewe dat Moses op die berg Nebo gestaan en Palestina aanskou het (Dt 34:1-4), goedskiks aanvaar sender cm te besef dat die menslike oog nie van die berg Nebo tot in Palestina kan sien nie. ’n Verdere voor- beeld is die situasie rondom die vestiging van die Israeliete in die heuwellande. Hulle moes die Kanaaniete in die vlaktes vermy, maar ook op sodanige plekke hulle vestig dat hulle minstens ’n bestaan kon maak. ’n Kennis van die Palestynse topo- grafie is dus noodsaaklik. Historiese geografie behels ’n tweerigtingverkeer tussen geografie en geskie denis. Aan die een kant stel die land die moontlikhede en beperkinge daar waar- binne die geskiedenis kan plaasvind. Aan die ander kant verander menslike optrede ook aan die land se voorkoms (veral wanneer stede ter sprake is). Die bou van ter- rasse vir landboudoeleindes in die berge, die dreinering van die Hulehmeer, sowel as die moderne boerdery in die Jordaanvallei tussen die See van Galilea en die Dooie See (wat tot gevolg het dat die Jordaanrivier eintlik nie meer daar vloei nie), is voorbeelde van laasgenoemde. Daar bestaan verskeie standaardwerke wat die historikus help in sy bestudering van die geografie van Palestina en wat terselfdertyd poog om die band te lê met die geskiedenis self. So verskaf Baly (1974; vgl ook Noth 1962:1-95; Eybers 1988) ’n ‘suiwere’ geografie (met egter ook verwysings na die Bybel), terwyl die Macmillan Bible Atlas van Aharoni en Avi-Yonah (1977; vgl ook Pritchard 1989) histories-geo- grafies te werk gaan deurdat dit die geskiedenis van Israel oor die verskillende periodes heen deur middel van landkaarte illustreer. In sy The Land of the Bible: A historical geography (1979) behandel Aharoni in twee dele die land self en ‘Palestine during the ages’. ISSN 0259 9422 = HTS 49/4 (1993) 7)2 Buite-Bybelse geskrifie Oor die regte of verkeerde gebruik van argeologie in sy relasie tot die Bybel is al so ruim besin dat hier nie verder daarop ingegaan word nie.i® Vanuit ’n holistie- se perspektief op die geskiedenis van Israel is argeologie egter nie slegs van belang in so verre vondse met die Bybeltekste gekorreleer kan word of illustratiewe waarde ten opsigte van laasgenoemde het nie. Dit is veral belangrik omdat dit inligting ver- skaf oor sake wat nie in die tekste aangeroer word nie, maar wat as sosio-kulturele dimensie ’n integrale deel van die geskiedenis uitmaak. Argeologiese vondste is dus op sigself belangrik vir wat uit die aard van die vondste self (wat baie meer met die alledaagse leefwyse te doen het) afgelei kan word. Dit lê byvoorbeeld die tipe huise bloot waarin die Israeliete in ’n bepaalde tydvak in hulle geskiedenis gebly het (vgl Callaway 1983; Stager 1985), hoe hulle hulleself van water voorsien het (vgl Schee- pers 1984), en gee insig in hulle landboumetodes (De Geus 1975; Edelstein & Gib son 1982). Insig in die ontwikkeling van Ou-Israelitiese kuns word ook dikwels ver- kry van erdeware wat tekeninge bevat en beeldjies en ander artefakte wat opgegra- we word. Holistiese historiografie baat dus grootliks by die insigte van die soge- naamde ‘New Archaeology’ (vgl Dever 1990; Coggins 1990:54-55) wat geinteres- seerd is in die sosio-kulturele geskiedenis van Palestina en nie in die eerste plek daarop uit is om die historiese korrektheid van sekere belangrike politieke gebeur- tenisse te bewys nie. 918 HTS 49/4 (1993) E Sduffler 6. DIEGEBRUIKVANSOSIOIjOGIESEINSIGTE Aangesien die modeme historikus veral belangstel in die geskiedenis van Oud-Israel as samelewingsgeskiedenis, verskaf sosiologiese of sosiaal-wetenskaplike modelle wat uitgewerk is om samelewings te ontleed, gewoonlik belangrike hulp in die inter- pretasie van gegewens. Hierdie interpretasie van historiese gegewens kan dan weer tot verdere historiese insigte lei (vgl Herion 1986; Braudel 1968:64-82; 1980). Alhoewel holistiese geskiedskiywing meer is as bloot samelewingsgeskiedenis, is dit veral vanweë die sosio-kulturele dimensie van die geskiedenis dat die gebruik van sosiologiese insigte en modelle minstens net so relevant is.H Dit is belangrik om in gedagte te hou dat daar nie net een sosiologiese benade- ring bestaan nie. Dit sou ook verkieslik wees dat die historikus hom nie aan een model uitverkoop nie, soos wat dit van Gottwald in sy magistrale werk The tribes of Yahweh (1979) ten opsigte van ’n Marxistiese model waar blyk te wees nie. Afge- sien van die Marxistiese, is die volgende drie myns insiens die bruikbaarste in ge skiedskiywing (vgl Scroggs 1980; Wilson 1984): • Rolanalise. Hiervolgens word persone, partye, instansies, organisasies en bewe- gings in die samelewing ondeed aan die hand van die ml wat hulle in die same- lewing speel. Die onderlinge verhouding tussen persone en instansies is derhalwe van groot belang. Wat was byvoorbeeld in die vroeë monargale tyd die rolle wat Saul, Samuel en Dawid gespeel het? Wat sou die rol van Josua gewees het gedurende die intog en wat sou die rol van die Kanaaniete en Fihstyne wees in die klaarblyklike feit dat die intog nie glad verloop het nie? Die vraag kan ook gevra word of en in welke mate daar in die samelewing ’n ommekeer van rolle kan plaasvind. * Konflikmodel. Aan die hand van insigte van sosioloë soos Weber, Mannheim en Troeltsch (laasgenoemde eintlik ’n teoloog) word daar uitgegaan van die veron- derstelling dat ’n diep gesetelde konflik inherent deel van elke samelewing is. Elke gemeenskap het maghebbers en magteloses wat met mekaar in konflik is. Die maghebbers wil gewoonlik die status quo bewaar terwyl die magteloses dit wil omverwerp. Hanson (1979) het byvoorbeeld hierdie insigte met vrug toege- pas op die post-eksiliese samelewing in Juda. Volgens Hanson het die konflik tussen die sogenaamde hiërokrate (geassosieer met Esegiël en die terugge- keerde ballinge) en die visionêres (geassosieer met die arm deel van die bevol- king wat in Palestina agtergebly het) neerslag gennd in Jesaja 56-66 (Trito-Jesa- ja). 5. DIEGEBRUIKVANGEOGRAFIESEGEGEWENS Gesien vanuit die perspektief van holistiese historiografie, spreek dit vanself dat die geografie belangrik is. Dit gee insig in die verhoog enersyds waarop ingrypende politieke gebeurtenisse plaasgevind het en andersyds waarop die gewone mense van die samelewing hulle alledaagse bestaan gevoer het. ISSN 0259 9422 = HTS 49/4 (1993) 919 HolMticaic gegkiedcans 6. DIEGEBRUIKVANSOSIOIjOGIESEINSIGTE ISSN 02S9 9C2 - HTS 49/4 (1993) 6. DIEGEBRUIKVANSOSIOIjOGIESEINSIGTE Die hiërokrate was diegene wat met die goedkeuring van Kores uit balling- skap teruggekeer het om die tempel te herbou. Die armes in die land se toe- komsverwagtinge is dus genegeer en hulle het ’n gevoel van vervreemding be- leef - vandaar die gemeenskapskonflik. 920 HTS 49/4 (1993) EScheíPer EScheíPer * Funksionalisme. Volgens die funksionalisme (geassosieer met die naam van Talcott Parsons) hoef ’n samelewing nie tot so ’n mate op konflik gebaseer te wees dat dit slegs in terme van konflik beskryf kan word nie. Volgens hierdie model is daar egter in elke samelewing bepaalde funksies of instansies wat elk sy eie deel bydra tot die globale funksioneríng van die samelewing. Parsons (vgl De Klerk & Schnell 1987:144) onderskei vicr sodanige fanksies - kultuw, sosia- le stelsel, gedragsorganisme en indiwiduele persoonlikheid - wat elk weer uit substelsels bestaan en so ’n bydrae tot die geheel lewer. Die onderskeie funk sies staan in wisselwerking met mekaar. Die samelewing is soos ’n lewende organisme en die verandering aan een aspek beinvloed die geheel (vir verdere subfunksies, vgl Parsons & Platt 1973:236). Parsons se model is myns insiens suksesvol deur Heyns toegepas op Israel van die agste eeu v C (vgl Heyns 1986). In die lig van die beskikbaarheid van verskillende modelle ontstaan die vraag na watter model die historikus moet kies en op welke gronde. Alhoewel ’n model wat ’n geskiedskrywer kies, sekerlik blyke sal gee van sy eie vooronderstellings, is ’n aan- gewese kriterium om van daardie model se insigte gebruik te maak wat die beste ’n gegewe tydvak of bepaalde historiese situasie verklaar. Die geskiedenis (of tekste) self moet dus as’t ware bepaal watter model gekies word. So sou die funksibnalisme miskien die aangewese model wees om die interne Israelitiese samelewing ten tye van die Rigters en Dawid te beskryf, terwyl ’n konflikmodel weer die aangewese sou wees vir die tyd van Amos (konflik tussen arm en ryk) of die tyd ná ballingskap (stryd tussen hiërokrate en visioenêres). 7. DIE GEBRUIK VAN PSIGOLOGIESE INSIGTE Indien die sosiologie die historikus van hulp kan wees, so ook die psigologie. Waar sosiologie te make het met mense in huUe samelewingr/eThand (die samelewing is dus die uitgangspunt), bestudeer sielkunde die motivering van individuele menslike gedrag asook die interaksies tussen mense. Daar sou byvoorbeeld gevra kan word watter psigologiese dinamiek daar in die verhouding tussen Saul en Samuel aanwesig was. Dit blyk uit die teks van 1 Samuel dat Samuel nie juis ten gunste van die koningsk^ was nie en nog minder was hy Saul goedgesind. Telkens sê Samuel dan ook vir Saul dat die Here van hom gewyk het. E>ie vraag wat gevra kan word, is of Saul se selfmoord nie in ’n groot mate deur Samuel se houding veroorsaak is nie. Saul intemaliseer later die boodskap wat die ‘profeet’ tot hom rig en begin in homself twyfel. So kom hy dan tot die gevolgtrek- king dat die Here inderdaad van hom gewyk het en hy pleeg s e lf m o o r d . ^2 ISSN 02S9 9C2 - HTS 49/4 (1993) 921 921 Holistieie gBskicdcilit Bestaande konstruksies kan goedskiks as die korrekte aanvaar word 922 HTS 49/4 (1993) ESchiffler ESchiffler (vgl bv die aanvanklike grootskaalse aanvaarding van Noth se amfiktíonieteoríe), met die gevolg dat die historikus ’n blindevlek kan ontwikkel wat nuwe perspektíe- we op die geskiedenis belemmer. Die bestudering van die geskiedenis mag dus nooit uit slegs die lees van sekondêre literatuur bestaan nie. Aan die ander kant is die sekondêre literatuur ’n goeie toets vir nuwe hipoteses wat die navorser op grond van sy primêre bronne maak. Nuwe teorieë moet teen bestaandes opgeweeg word, want dikwels sal dit blyk dat nuwe hipoteses nie opweeg in die lig van bestaande kennis (wat weer verdere navorsing stimuleer) nie. Hierdie artikel kan nouliks aan die magdom van sekondêre literatuur reg laat geskied. Daar word derhalwe met enkele kursoriese opmerkings volstaan. Naas Alt (1969) se opstelle in sy Kleine Schriften, en die belangrike versamel- bundel van Hayes & Miller (1977), is die tien belangrikste ‘geskiedenisse’ van Israel myns insiens (chronologies weergegee, d w s nie in die volgorde van belangrikheid nie) dié van Noth (1958); Bright ([1959] 1980); Gunneweg (1972); De Vaux (1978); Jagersma (1979; 1985); Herrmann (1980); Fohrer (1982); Donner (1984; 1986); Mil ler & Hayes (1986) en Lemche (1988). Daar moet egter gewys word op die feit dat hierdie ‘geskiedenisse’ (alhoewel die beste tans beskikbaar) steeds ‘politieke’ geskie denisse is en nie reg laat geskied aan die holistiese perspektief nie. Laasgenoemde kom meer tot sy reg in die huidige tendens onder historid om op ’n spesifieke perio- de te konsentreer (vgl bv Coote & Whitelam 1987; Coote 1990; Gottwald 1979; vgl verder die literatuur vermeld by Hayes & Miller 1977). Vir die sosio-kulturele dimensie, is naas die boek van De Vaux (1%1), wat ongelukkig nie altyd tussen die verskillende periodes onderskei nie, die literatuur wat afdeling 4 hierbo vermeld is, ter sake en vir die godsdienstig-ideologiese dimensie dié vermeld in afdeling 8. Wat die literêre (kommunikatiewe) dimensie betref, is die holistiese geskiedskrywer aan- gewys op die standaard ‘Inleidings’ waarvan die stof telkens binne die geskiedenis gesitueer moet word. Gottwald se Hebrew Bible: A socio-literary introduction (1985) verdien hier spesiale vermelding aangesien die Bybelse literatuur juis binne hul sosiaal-historiese konteks (d w s ook chronologies) behandel word. Holistieie gBskicdcilit Holistieie gBskicdcilit Uit ’n holistiese perspektief is hierdie gebeure wat tussen Saul en Samuel afge- speel het, self deel van die geskiedenis en dit kan goedskiks deur middel van siel- kundige insigte (’n groot aantal modelle bestaan ook hier: bv, klassieke psigo-ana- lise, Jungiaanse analitiese sielkunde, behaviourisme, leerteorie en kognitiewe siel- kunde - vgl Scheffler 1988c:355-364) ontleed word^^. As verdere voorbeelde sou gevra kon word wat die psigologiese motivering en effek van offers in Oud-Israel was of wat die sielkunde te sê het vir Prediker se sinloosheidsgedagtes, of vir die lyding wat ons in die boek Job aantref (vgl Jung 1%5; 1972; Scheffler 1991; 1993). Alhoewel die psigologie nog nie op ’n grootskaalse wyse ten behoewe van die historiografie ingespan is nie (vgl egter Spiegel 1972; Drewermann 1984; 1985; Fromm 1966; Freud [1913] 1980; [1939] 1975; 1967), kan die plek daarvan nie betwis word nie. In Parsons se vierfunksieparadigma waarna hierbo verwys is (vgl afd 6), kan gesien word dat die ‘indiwiduele persoonlikheid’ (sy persoonlike beweeg- redes, drange en gedrag dus ingesluit) ’n belangrike rol in die samelewing en dus ook in die geskiedenis speel. 8. DIE GEBRUnC VAN GODSDDENS-WETENSKAPUKE INSIGTE Indien die godsdienstig-ideologiese dimensie van die geskiedenis bestudeer word, is dit raadsaam vir die historikus om fenomenologies te werk te gaan en so objektief moontlik die instellings en idees te beskrywe soos wat hy dit waameem. ’n Fenome- nologiese vertrekpunt beteken nie hier absolute objektiwiteit nie maar veral die strewe daarna. Die historikus sal hom/haar dus vergewis van uitgangspunte en metodes wat deur godsdienswetenskaplikes (vgl bv Kruger 1982) gebruik word. Vanuit die perspektief van holistiese historiografie eindig die ondememing egter nie by die beskiywing van die godsiens nie (vgl die bruikbare werke van Ringgren 1963; Schmidt 1982; Fohrer 1969; Garbinni 1987 en selfs Von Rad 1960; 1968), maar word daar gepoog om die godsdienstige of ideologiese opvattings op die politieke en sosio-kulturele en kommunikatiewe (literêre) dimensies van die geskiedenis (binne ’n bepaalde periode) te betrek. 9. DIE GEBRUIK VAN SEKONDêREUTERATUUR Die navorsing van Jsrael se geskiedenis vind nie in isblasie plaas nie, maar juis in gesprek met medenavorsers. Tientalle geskiedenisse van Israel het reeds die lig gesien. Ingewikkelde hipoteses en konstruksies is reeds gemaak en het baie by gedra tot die historikus se begrip van die geskiedenis. Dit hou ’n voordeel in, maar ook ’n gevaar. 10. DIE INTEGRERING VAN GEGEWENS Die vraag is hierbo (afd 2) reeds aangesny oor hoe die historikus (nadat uit alle bronne geput is) die gegewens (wat binne die hohstiese perspektief op die verskil lende dimensies betrekking het) gaan integreer. Daar is geen vasgestelde reel nie en die historikus sal hom deur sy materiaal self (en wat hy deur die aanbieding daar- van wil bereik) moet laat lei. Hoe hy dan op die ou end sy verhaal gaan uiteensit, hang met ander woorde af van wat sy eie siening is van die geskiedenis wat hy besig ISSN 0 2 » 9422 - NTS 49/4 (1993) 923 HnBrtirw gratinilnMi is om te skryf. Die historikus gee ook nie meganies een-vir-een aan bogenoemde bronne of sosiaal-wetenskaplike insigte aandag nie, maar bou vir hom/haarself met- tertyd ’n bevoegdheid of kompetensie op wat sy beeld van die geskiedenis bepaal. Sy verhaal sal uiteraard met daardie beeld korreleer. Die beoefening van historio- grafie geskied inderdaad nie volgens ’n kitsresep nie. Dit is egter noodsaaklik dat die geskiedskrywer al bogenoemde aspekte en vergaarde gegewens uit bronne voortdurend in gedagte sal hou en sover moontlik sal verdiskonteer in sy aanbie- ding. 1. Dat antieke geskiedskrywing meestal gekonsentreer het op gebeure wat aan ’n koninklike hof afgespeel het, is deels te verstane aangesien dit gewoonlik aan 11. SLOT Sonder om weer op die sin en doel van Oud-Israelitiese historiografie in te gaan (sien Scheffler 1988a:666-669), kan dit nie anders nie as om hier te beklemtoon dat die ontmoeting met die geskiedenis ’n kommunikasie met die verlede is wat ten diepste ook ’n ontmoeting met die lewe self is. Hierdie ontmoeting laat die histori kus nie onaangeraak nie. Hiermee word nie betoog dat die historikus doelbewus sy persoonlike vooronderstellings oor die lewe in al sy fasette in die beoefening van sy vak moet indra en uit die geskiedenis moet probeer aflees nie. Die afwysing van ’n positiwistiese benadering maak nie die strewe na objektiwiteit as ’n belangrike waar- de in die beoefening van historiografie ongedaan nie. Aan die ander kant, as gevolg van die groot interpretatiewe inset wat die historikus as kennende subjek lewer, kan so ’n ‘indra’ eintlik nie vermy word nie. Anders gestel: juis omdat die historikus interpreteer en subjektief betrokke is, moet hy/sy poog om onnodige uitings van sy/ haar subjektiwiteit tee te werk. Nooit moet en kan hy/sy hom/haar probeer voor- hou as so ’n suiwer objektiewe ondersoeker wat net sowel ’n robot kon gewees het nie. ’n Dialektiese balans is hier van pas: As subjektiewe mens beoefen die histori kus ’n (‘objektiewe’) wetenskap. Die beste teenvoeter vir onnodige subjektiwiteit wat steeds die historikus se menswees respekteer, is die onderlinge bespreking van hipo- teses en resultate met vakgenote en belangstellendes. Teologies uitgedruk: Oud- Israelitiese historiografie is op sy beste ’n ekumeniese wetenskap. Daarom hou die feit dat die geskiedenis van Oud-Israel nie bestaan, kan bestaan, sal bestaan en hoef te bestaan nie, geen bedreiging in nie. Asimptoties word die waarheid egter wel benader (vgl Van.Wyk Louw se gedig Groot Ode in Tristia 1962:125). Eindnotas 1. Dat antieke geskiedskrywing meestal gekonsentreer het op gebeure wat aan ’n koninklike hof afgespeel het, is deels te verstane aangesien dit gewoonlik aan 924 HTS 49/4 (1993) ESchegier ESchegier koninklíke howe en tempels (die fokuspunte van handel en besigheid) was waar antieke geskiedskrywers ’n heenkome kon vind. Fensham (1982:10-11) wys in die lig hiervan daarop dat die patriargale verhale (wat familiegeskiedenisse is) dus nouliks deur kontemporêre antieke geskiedskrywers opgeteken kon gewees het. koninklíke howe en tempels (die fokuspunte van handel en besigheid) was waar antieke geskiedskrywers ’n heenkome kon vind. Fensham (1982:10-11) wys in die lig hiervan daarop dat die patriargale verhale (wat familiegeskiedenisse is) dus nouliks deur kontemporêre antieke geskiedskrywers opgeteken kon gewees het. 2. Die onderhawige besinning (afd 2) oor die begrip holistiese historiogrc^ is meer as ’n blote samevatting van wat voorheen (kyk Scheffler 1988a) gesê is. Laasge- noemde word hier veronderstel en verdere refleksies sedert genoemde artikel word hier verdiskonteer. Aangesien die fokus in hierdie artikel primêr gerig is op die metodologie van geskiedskrywing, word die meriete van ’n holistiese benadering nie hier binne die konteks van algemene geskiedskrywing of ge- skiedsfilosofie beredeneer nie (vgl bv Braudel 1968; 1980; Carr 1961; Dray 1966; Dreyer 1974; Stem 1970; Van Jaarsveld 1980). 3. Dieselfde gevaar dreig myns insiens by die benadering van sominige voorstan- ders van die sogenaamde ‘demokratiese geskiedenis’ of ‘peoples histor/. Soos gewoonlik die geval, hang die geldigheid van die term ook hier af van die begrip waaraan dit probeer uitdrukking gee. Die term ‘demokratiese geskiedenis’ hoef dus nie noodwendige ongeldig te wees nie, mits dit steeds alle invloede op die geskiedenis erken en nie in die eerste plek daarop gemik is om geskiedskrywing vir ’n bepaalde eksklusiwistiese ideologic te probeer kaap nie (vgl in hierdie verband Van Aarde [1988] se kritiek op Mosala [1986]). Vanuit ’n holistiese perspektief (waarin gepoog word om vanuit ’n eensydige politieke ‘establish- ment’-geskiedenis weg te beweeg), sou die term ‘demokratisering’ van die geskiedenis nie net verkieslik nie, maar selfs in ’n groot mate nuttig wees. 4. In sy boek Die noodlottige band: Kerk en stoat in Oud-Israel (1975) slaag Deist (as gevolg van die tema van die boek) myns insiens geredelik daarin om ’n stuk gekiedenis te skryf waarin verskeie aspekte geïntegreerd weergegee word. Eindnotas Daar word hier uitgegaan van die periodisering van die geskiedenis soos voor- gestaan in my vroeëre artikel (Scheffler 1988a:681) met enkele aanpassings: Aartsvadertyd (1800-1300), Eksodus (1300-1250), vestiging in Palestina (1250- 1150), Rigtertyd (1200-1020), Verenigde Koninkryk (1020-922), Noordryk (922- 721), Suidryk (721-587), Babiloniese Ballingskap (587-538), Persiese tyd (538- 322), Hellenistiese tyd (322-63), Romeinse tyd (63v C-135n C). Hiermee is niks gesê oor die historisiteit van die vroeëre tydperke (bv Aartsvaders en Eksodus) nie. 6. Daar word hier uitgegaan van die periodisering van die geskiedenis soos voor- gestaan in my vroeëre artikel (Scheffler 1988a:681) met enkele aanpassings: Aartsvadertyd (1800-1300), Eksodus (1300-1250), vestiging in Palestina (1250- 1150), Rigtertyd (1200-1020), Verenigde Koninkryk (1020-922), Noordryk (922- 721), Suidryk (721-587), Babiloniese Ballingskap (587-538), Persiese tyd (538- 322), Hellenistiese tyd (322-63), Romeinse tyd (63v C-135n C). Hiermee is niks gesê oor die historisiteit van die vroeëre tydperke (bv Aartsvaders en Eksodus) nie. 7. Volgens Deist (1987) word die geskiedenis van geskiedskrywing gewoonlik onderverdeel in twee hooffases, te wete die pre-historiese (mites, sages, tradi- sies) en historiese fase. Die historiese fase word onderverdeel in ’n annalistiese (‘koningsrekords’: bv lyste, briewe, berigte van militêre veldslae, bouprojekte) en ’n beskrywende periode. Laasgenoemde word onderverdeel in ’n pre-kritiese (meer klem op gebeure en persone naas die koning) en ’n kritiese fase wat op sy beurt weer in inpre- enpost-Verligting fase verdeel word. Laasgenoemde (in die tradisie van Von Ranke se “wie es eigentlich gewesen ist’) vermy alle romantise- ring in geskiedskrywing en is baie meer krities ten opsigte van sogenaamde feite. Vergelyk ook Hayes (1977:1-69). 8. Vgl in die lig hiervan Miller & Hayes (1986:250-287) se poging om uit ’n histo riese hoek meer reg te laat geskied aan die Omri-dinastie. 8. Vgl in die lig hiervan Miller & Hayes (1986:250-287) se poging om uit ’n histo riese hoek meer reg te laat geskied aan die Omri-dinastie. 8. Vgl in die lig hiervan Miller & Hayes (1986:250-287) se poging om uit ’n histo riese hoek meer reg te laat geskied aan die Omri-dinastie. 9. 5. Wat hier volg, geld alleen as voorbeeld om die moontlike integrering van die verskillende dimensies van die geskiedenis te illustreer en is op sigself geen uit- spraak oor die historisiteit van die Salomoniese verhale of Israel se sogenaamde ‘goue eeu’ nie (vgl bv Millard 1991a; 1991b; Miller 1991). Eindnotas Die skryf van die geskiedenis aan die hand van ’n bepaalde tema kan egter ook beperkend inwerk (as gevolg van die selektiewe werkwyse) indien mens aan die ideaal van holistiese geskiedskrywing (wat per definisie die verskeie dimensies móét omvat) reg wil laat geskied. Dit impliseer dat holisitiese geskiedskrywing in die streng sin van die woord (waarin bewustelik uitgegaan word van die verskillende dimensies) nie voorgehou kan word as die enigste manier waarop historiografie bedryf kan word nie. Tematiese geskiedenis bly steeds geldig en selfs nodig (ook vir holistiese geskiedskrywing). Tematiese geskiedskrywing sou egter ook in die uitvoering van sy taak by holistiese geskiedskrywing baat deur in die ondersoek na ’n bepaalde saak aan die mees moontlike lewensdimensies (soos wat die saak vereis en toelaat) aandag te gee. 4. In sy boek Die noodlottige band: Kerk en stoat in Oud-Israel (1975) slaag Deist (as gevolg van die tema van die boek) myns insiens geredelik daarin om ’n stuk gekiedenis te skryf waarin verskeie aspekte geïntegreerd weergegee word. Die skryf van die geskiedenis aan die hand van ’n bepaalde tema kan egter ook beperkend inwerk (as gevolg van die selektiewe werkwyse) indien mens aan die ideaal van holistiese geskiedskrywing (wat per definisie die verskeie dimensies móét omvat) reg wil laat geskied. Dit impliseer dat holisitiese geskiedskrywing in die streng sin van die woord (waarin bewustelik uitgegaan word van die verskillende dimensies) nie voorgehou kan word as die enigste manier waarop historiografie bedryf kan word nie. Tematiese geskiedenis bly steeds geldig en selfs nodig (ook vir holistiese geskiedskrywing). Tematiese geskiedskrywing sou egter ook in die uitvoering van sy taak by holistiese geskiedskrywing baat deur in die ondersoek na ’n bepaalde saak aan die mees moontlike lewensdimensies (soos wat die saak vereis en toelaat) aandag te gee. ISSN 0259 9422 = HTS 49/4 (1993) 925 HoBtíieic geskicdcoie 5. Wat hier volg, geld alleen as voorbeeld om die moontlike integrering van die verskillende dimensies van die geskiedenis te illustreer en is op sigself geen uit- spraak oor die historisiteit van die Salomoniese verhale of Israel se sogenaamde ‘goue eeu’ nie (vgl bv Millard 1991a; 1991b; Miller 1991). 6. Eindnotas Ben-Tor (1992:8) merk myns insiens tereg op dat Albright self nie binne die kategorie val van diegene wat die Bybel ten alle koste reg wou bewys nie (’n indruk wat dikwels geskep word - vgl Scheepers 1992:124), maar dat hy eerder in reaksie was teen ‘the German school of biblical criticism, which at that time cast grave doubts on the antiquity of many biblical texts relating to the antiquity of the Israelites’. Die waarheid van hierdie stelling kan getoets word aan Albright se (kritiese) siening oor die bekende historiese probleem rondom die verwoesting van Jerigo en Ai (vgl Albright 1957:276-280). 10. Vgl Pritchard (.1958); Noth (1959:49-53; 1962:%-167); Wright (1962:17-28); De Vaux (1970); Harrison (1970:13-18); Wright (1971); Fensham & Oberholzer (1978:311-313); Herrmann (1980:42-49); Eybers (1980); Moorey (1981:110- 125); Fensham & Pienaar (1982:14-15); Miller (1982; 1987); Sauer (1982); Deist & Le Roux (1987:41-43); Dever (1982; 1990:1-36); Dever (1991:103-115); Donner (1984:27-29); Gottwald (1985:49-63); Nel (1987); Weippert (1988:1-68); 926 HTS 49/4 (1993) E S c h ^ E S c h ^ Lemche (1988:71-73); Coggins (1990:42-55); Ahlstrom (1991:116-142); Schee- pers (1990; 1992); Burger (1992); Ben-Tor (1992:1-9). 11. ’n Samelewingsgeskiedenis kan wel die politieke en godsdienstige dimensies veronderstel, maar ’n holistiese perspektief wil juis verhoed dat laasgenoemde dimensies onderspeel word. Aangesien ’n holistiese benadering (waarin die sosio-kulturele dimensie prominent figureer) die noodsaak van ’n sosiologiese invalshoek duidelik laat blyk en onderstreep, word daar nie hier in debat getree met diegene wat die gebruik van sosiologiese modelle bevraagteken nie. 11. ’n Samelewingsgeskiedenis kan wel die politieke en godsdienstige dimensies veronderstel, maar ’n holistiese perspektief wil juis verhoed dat laasgenoemde dimensies onderspeel word. Aangesien ’n holistiese benadering (waarin die sosio-kulturele dimensie prominent figureer) die noodsaak van ’n sosiologiese invalshoek duidelik laat blyk en onderstreep, word daar nie hier in debat getree met diegene wat die gebruik van sosiologiese modelle bevraagteken nie. 12. Vergelyk ook Scheffler (1990:263-273) vir ’n vollediger ontleding van die verhouding tussen Saul en Samuel met gebruikmaking van transaksionele analise (Beme 1967). Vir ’n psigo-analitiese analise van nog vyf ander Bybelse leiersfigure (naas Samuel en Saul), vergelyk Zeligs (1974). 12. Vergelyk ook Scheffler (1990:263-273) vir ’n vollediger ontleding van die verhouding tussen Saul en Samuel met gebruikmaking van transaksionele analise (Beme 1967). Vir ’n psigo-analitiese analise van nog vyf ander Bybelse leiersfigure (naas Samuel en Saul), vergelyk Zeligs (1974). 13. Eindnotas Soos in die voorbeeld van Salomo se koningskap (hierbo vermeld) word die ver houding tussen Samuel en Saul hier as illustrasie genoem en word geen klakke- lose aanvaarding van die historisiteit van die tekste veronderstel waama verwys word nie. UteratuuTverwysings Aharoni, Y 1979. The land of the Bible: A historical geography. Philadelphia: West minster. Aharoni, Y & Avi-Yonah, M 1977. The Macmillan Bible atlas. New York: Macmil lan. Ahlstrom, G W 1991. The role of archaeological and literary remains in reconstruc ting Israel’s history, in Edelman 1991:116-142. 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https://openalex.org/W4386543846	https://www.qeios.com/read/E8NCXI/pdf	English	null	Review of: "International Clinicians and Academics Supporting the Refugee Journey- personal narratives from the field."	null	2,023	cc-by	105	Qeios, CC-BY 4.0 · Review, September 8, 2023 Qeios ID: E8NCXI · https://doi.org/10.32388/E8NCXI Review of: "International Clinicians and Academics Supporting the Refugee Journey- personal narratives from the field." Shaharior Rahman Razu1 Shaharior Rahman Razu1 1 Khulna University 1 Khulna University Potential competing interests: No potential competing interests to declare. Hi Dr Clive, happy to review the new version of submission. Just wondering if the title has been changed. I am a bit unsure about it. Could you please confirm? Also, please may I request you to highlight what changes were made in the new version as well? Thank you. Qeios ID: E8NCXI · https://doi.org/10.32388/E8NCXI 1/1
https://openalex.org/W3042256888	https://europepmc.org/articles/pmc7357834?pdf=render	English	null	Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model	Acta Cirúrgica Brasileira	2,020	cc-by	5,034	Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model1 başiI , Hasan Ali KirazII , Emin Tunç DemirIII , Yasemen AdaliIV , Sait ElmasV Hasan Oğuz KapicibaşiI , Hasan Ali KirazII , Emin Tunç DemirIII , Yasemen AdaliIV I Assistant Professor, Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Thoracic Surgery, Çanakkale, Turkey. Design of the study, technical procedures, manuscript writing, critical revision, final approval. i II Assistant Professor, Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Anesthesiology and Reanimation, Çanakkale, Turkey. Design of the study, technical procedures, manuscript writing, critical revision, final approval.i i II Assistant Professor, Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Anesthesiology and Reanimation, Çanakkale, Turkey. Design of the study, technical procedures, manuscript writing, critical revision, final approval.i ,i pp III MD, Trakya University, Faculty of Medicine, Department of Anesthesiology and Reanimation, Division of Intensive Care, Edirne, Turkey. Technical procedures, animal care, critical revision, final approval. i pp III MD, Trakya University, Faculty of Medicine, Department of Anesthesiology and Reanimation, Division of Intensive Care, Edirne, Turkey. Technical procedures, animal care, critical revision, final approval. ii IV Associate Professor, Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Medical Pathology, Çanakkale, Turkey. Analysis and interpretation of data, histopathological examinations, critical revision, final approval. i IV Associate Professor, Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Medical Pathology, Çanakkale, Turkey. Analysis and interpretation of data, histopathological examinations, critical revision, final approval. i ,i pp V Veterinarian Doctor, Çanakkale Onsekiz Mart University, Experimental Research Application and Research Center, Çanakkale, Turkey. Technical procedures, animal care, critical revision, final approval. i pp V Veterinarian Doctor, Çanakkale Onsekiz Mart University, Experimental Research Application and Research Center, Çanakkale, Turkey. Technical procedures, animal care, critical revision, final approval. ACTA CIRÚRGICA BRASILEIRA ORIGINAL ARTICLE Experimental Surgery ■Introduction Sepsis is defined as life-threatening organ dysfunction caused by an unregulated host response to infection1. The pathology of sepsis involves complex interactions between host organs and invading pathogens. Ultimately tissue injury and organ failure are due to negative effects of systemic activation of host immunity2,3. Sepsis forms a large problem in intensive care and is the main cause of death occurring there4,5. According to a report published by the Global Burden of Disease, each year nearly 10 million people die from infections and this is much greater than the number of people who die from cancer annually6. Lungs are the first organs affected by sepsis and sepsis causes severe injury to lung tissue7. In spite of advanced antibiotherapy, supportive treatments and all technological opportunities, sepsis continues to be a situation progressing with morbidity and mortality8. The earliest target-directed basic treatment principles for sepsis comprise determining high-risk patients, ensuring appropriate cultures and source control, and beginning appropriate antibiotherapy without delay9.i Implementation and surgical procedure Twenty-four hours after Escherichia coli administration, rats in the 1st, 2nd and 3rd groups were arranged in groups following skin cleaning for asepsis and the following treatments began. Care was taken to administer treatments at the same time every day. Additionally, an ozone generator (device name and model: Turkozone Blue S, device serial no: BG-19144427046) was used to produce the ozone/ oxygen mixture for ozone therapy. 1st group: This group was the antibiotic group, so after Escherichia coli was administered by the intraperitoneal route with the aim of inducing sepsis, cefepime 50 mg/kg dose was administered subcutaneously at the same time for 7 days. 2nd group: Rats in this group had single dose of 0.6 mg/kg ozone therapy administered for 1 min intraperitoneal simultaneous to the 50 mg/kg subcutaneous cefepime for 7 days. 3rd group: Rats in this group had single dose of 1.1 mg/kg ozone therapy, different to the 2nd group, administered for 1 min intraperitoneal simultaneous to the 50 mg/kg subcutaneous cefepime for 7 days. Abstract Purpose: This experimental sepsis model created with Escherichia coli aimed to investigate the histopathological effects of two different doses of ozone combined with antibiotherapy on lung tissue. Methods: Rats were divided into 5 groups. Then sepsis was induced intraperitoneally in the first 4 groups. The 1st group was treated with cefepime, the 2nd and 3rd groups were treated with cefepime combined with ozone at a dose of 0.6 mg/kg and 1.1 mg/kg. Lung tissue sections were stained with hematoxylin-eosin and assessed under light microscope and scored between 0-4 in terms of histopathological findings. Results: In the comparisons between Group 1 and Group 4 in terms of cellular damage (p=0.030), inflammation (p=0.000) and overall score (p=0.007), statistically significant positive effects were observed in favor of Group 1. In the comparisons of Groups 2 and 3 with Group 4, only positive effects were observed in terms of inflammation (p=0.020, p=0.012, respectively). Conclusion: Although negative histopathological effects of ozone on tissue injury were detected, it was noteworthy that the increase in the ozone dose reduced the number of damaged parameters. Key words: Lung. Ozone. Sepsis. Rats. Key words: Lung. Ozone. Sepsis. Rats. DOI: http://dx.doi.org/10.1590/s0102-865020200060000004 Acta Cir Bras. 2020;35(6):e202000604 Acta Cir Bras. 2020;35(6):e202000604 Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. (File Registration Number: 2018/1800080971 & Decision number: 2018/06-06). Our study included 40 male Sprague-Dawley rats with mean weight 350-400 g in appropriate condition. During the study, rats were kept in ÇOMÜ Experimental Research Center in wire cages with 12-hour night-12-hour day circadian rhythm, environmental temperature 24-26°C and humidity 50-60%. Rats were fed with standard commercial feed and municipal drinking water. Rat feed was stopped 12 hours before the study; however, water was given freely during this time. All rat care was performed in accordance with the “Regulation on the Welfare and Protection of Animals Used for Experimental and Other Scientific Purposes” (13.12.2011-28141) prepared by the Ministry of Food, Agriculture and Livestock. Experimental groups Experimental animals were randomly divided into 5 groups with equal numbers (n=8). Sepsis was induced in rats in the first 4 of these groups with 2.1x109 CFU/mL Escherichia coli ATCC 25922 administered in 1 mL saline via the intraperitoneal route in 32 rats. Rats in the 5th group only had 1 mL intraperitoneal saline administered. i Medical ozone therapy is an alternative treatment model linked to the administration of ozone and gas mixture with oxygen to body fluids or cavities. To date, clinical and experimental studies have shown that ozone therapy is beneficial for inflammation-mediated diseases like infected wounds, chronic skin ulcers, burns and advanced ischemic diseases10. Ozone/ oxygen mixtures are reported to display a variety of effects on the immune system like phagocytic activity modulation11. Additionally, ozone therapy is proposed to cause increased antioxidant enzyme expression12. Additionally, it is known that ozone is also an immune system modulator. Though antiseptic properties of ozone are well known, it may harm not just the pathogen but also the patient linked to dose and exposure duration13. Ozone is one of the strongest oxidants and may show unwanted effects through a variety of mechanisms like free radical formation, lipid peroxidation, enzymatic activity loss, changes in membrane permeability, direct organ inflammation and injury14. Based on the known positive and negative effects of ozone, in our study we aimed to assess the effect of two different doses of ozone therapy added to antibiotic treatment in an experimental sepsis model induced with Escherichia coli on the histopathologic findings observed in the inflammatory process in the lungs. Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. ■Methods Ethical approval was obtained from Çanakkale Onsekiz Mart University Ethical Board of Animal Studies 2 Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. Acta Cir Bras. 2020;35(6):e202000604 Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. 4th group: Rats in this group were not given any treatment after administration of Escherichia coli by the intraperitoneal route with the aim of inducing sepsis. accessing the thorax cavity with median sternotomy and rats were sacrificed. In the blood samples, the diagnosis of sepsis was confirmed by working with the rats Escherichia coli protein (E.coli P) enzyme-linked immunosorbent assays (ELISA) kits (Shangai Coon Koon Biotech Co, China). In view of the data on the researched serums, the very low level of E.coli P in the rats of the control group was construed and reported as the absence of sepsis. The statistically significant differences concerning the presence of E.coli P in the first four groups in comparison with the control groups signify that sepsis was successfully induced in these groups (Fig. 1). 5th group: Rats in this group did not have a sepsis model induced, but were administered 1 mL saline intraperitoneal at the start and were only administered subcutaneous saline at equivalent times to the treatments in the other groups. Twenty-four hours after the final treatment in our study, rats were administered high-dose anesthetics (xylazine - 10 mg/kg and ketamine - 80 mg/kg) and lung tissue samples and blood samples were taken by Randomized Male Sprague Dawley Rats n = 40 n = 8 n = 8 n = 8 n = 8 n = 8 n = 0 n = 0 n = 1 n = 0 n = 0 n = 8 n = 8 n = 7 n = 8 Analyzed Analyzed Analyzed Analyzed Analyzed Died Died Died Died (Day 6) Died Group II Group III Group IV Group V Figure 1 - Study flow chart. ■Methods Hemorrhage, cellular injury, alveoloseptal thickening and inflammation were scored from 0-4 and a general injury score (total score) was obtained by summing all scores per case. ■Methods Randomized Male Sprague Dawley Rats n = 40 n = 8 n = 8 n = 8 n = 8 n = 8 n = 0 n = 0 n = 1 n = 0 n = 0 n = 8 n = 8 n = 7 n = 8 Analyzed Analyzed Analyzed Analyzed Analyzed Died Died Died Died (Day 6) Died Group II Group III Group IV Group V Figure 1 - Study flow chart. Lung tissue samples were fixated for 24 hours in 10% buffered formalin solution. Later, tissue monitoring procedures were performed appropriate to groups and tissues were submerged in paraffin. Sections with 4-micron thickness were taken from the prepared paraffin blocks and stained with hematoxylin & eosin (H&E) before being investigated with a light microscope (Olympus BX46, Japan) at magnification above 10 fields15. The tissues were assessed by the parameters used by Yamanel et al.16 with some modification and their scoring system was used. Hemorrhage, cellular injury, alveoloseptal thickening and inflammation were scored from 0-4 and a general injury score (total score) was obtained by summing all scores per case. Statistical analysis Maximum care was taken to use the lowest number of animals necessary to gain scientific quality from the methods applied in the study and the obtained results. Analysis of data used the Statistical Package for the Social Sciences for Windows Evaluation v. 15.0 (SPSS, Chicago, IL, USA) program. Results are given as median and minimum-maximum values. Comparisons of groups used the Mann-Whitney U test. Tests were completed in the 95% confidence interval and values with p lower than 0.05 were accepted as statistically significant. Randomized Male Sprague Dawley Rats Group III Group II n = 0 Died Died (Day 6) Died Died n = 0 Analyzed Analyzed Figure 1 - Study flow chart. Lung tissue samples were fixated for 24 hours in 10% buffered formalin solution. Later, tissue monitoring procedures were performed appropriate to groups and tissues were submerged in paraffin. Sections with 4-micron thickness were taken from the prepared paraffin blocks and stained with hematoxylin & eosin (H&E) before being investigated with a light microscope (Olympus BX46, Japan) at magnification above 10 fields15. The tissues were assessed by the parameters used by Yamanel et al.16 with some modification and their scoring system was used. Statistical analysis Lung tissue samples were fixated for 24 hours in 10% buffered formalin solution. Later, tissue monitoring procedures were performed appropriate to groups and tissues were submerged in paraffin. Sections with 4-micron thickness were taken from the prepared paraffin blocks and stained with hematoxylin & eosin (H&E) before being investigated with a light microscope (Olympus BX46, Japan) at magnification above 10 fields15.i Maximum care was taken to use the lowest number of animals necessary to gain scientific quality from the methods applied in the study and the obtained results. Analysis of data used the Statistical Package for the Social Sciences for Windows Evaluation v. 15.0 (SPSS, Chicago, IL, USA) program. Results are given as median and minimum-maximum values. Comparisons of groups used the Mann-Whitney U test. Tests were completed in the 95% confidence interval and values with p lower than 0.05 were accepted as statistically significant. The tissues were assessed by the parameters used by Yamanel et al.16 with some modification and their scoring system was used. Hemorrhage, cellular injury, alveoloseptal thickening and inflammation were scored from 0-4 and a general injury score (total score) was obtained by summing all scores per case. 3 Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. Acta Cir Bras. 2020;35(6):e202000604 Histopathological findings When Group 4 is compared with Group 2 and Group 3, positive effects were only observed in Groups 2 and 3 for inflammation (p=0.020, p=0.012, respectively), with no statistical significance for the other parameters (p>0.05).iii As the histopathologic findings observed in the study are categoric data, the median and interval values are presented in Table 1. When Group 4 is compared with the control group of Group 5, Group 4 was identified to have more statistically significant injury in terms of cellular damage (p=0.030), alveoloseptal thickening (p=0.037), inflammation (p=0.001) and total score (p=0.009), with no significant difference between the groups in terms of hemorrhage (p=0.663). While there was no statistical significance for hemorrhage between Group 1 and Group 2 (p=0.655), there were clear positive effects in Group 1 for cellular damage (p=0.002), alveoloseptal thickening (p=0.044), inflammation (p=0.002) and total score (p=0.017). When Group 1 is compared with Group 3, there was no statistical significance for bleeding (p=0.901), alveoloseptal thickening (p=0.081) and inflammation (p=0.054), but positive effects were pronounced in Group 1 for cellular injury (p=0.002) and total score (p=0.035). Histopathologic findings of the groups are demonstrated in Figure 2. Comparison of Groups 2 and 3 did not have any statistical significance (p>0.05). When Group 4 is compared with Group 1, there was no statistical significance for bleeding (p=0.428) and alveoloseptal thickening (p=0.085), while there were clear positive effects in Group 1 for cellular injury (p=0.030), inflammation (p=0.000) and total score (p=0.007). Table 1 - Statistical significance with value intervals and median values for histopathological findings. Group 1 Median (Min-Max) Group 2 Median (Min-Max) Group 3* Median (Min-Max) Group 4 Median (Min-Max) Group 5 Median (Min-Max) Bleeding 2/2 (1-3) 1/1 (1-4) 2 (1-3) 2/2 (1-4) 2/2 (1-4) Cellular Damage 0/0 (0-1)b 1/1 (1-3)a,c 2 (1-3)a,c 1/1 (0-3)a,c 0/0 (0-1)b Alveoloseptal Thickening 0/0 (0-1) 1/2 (0-3)a,c 1 (0-3)a 1/1 (0-3)a 0/0 (0-1)b Inflammation 0/0 (0-1)b 1/1 (1-3)a,b,c 1 (0-3)b 2/3 (2-4)a,c 0/0 (0-1)b Total Score 2/2 (1-5)b 5/8 (3-11)a,c 6 (2-11)c 7/8 (3-12)a,c 3/3 (1-7)b a: p<0.05, when compared with Group 5, b: p<0.05, when compared with Group 4, c: p<0.05, when compared with Group 1. : As Group 3 only included 7 rats there is one median value, the other groups comprised 8 rats and had 2 median values. nificance with value intervals and median values for histopathological findings. ■Discussion Sepsis-linked acute lung injury is an important cause of morbidity and mortality in adults and children and significantly contributes to intensive care costs17-20. Many studies have created pioneering ideas for the use of ozone as a strong antimicrobial agent21-24. Ozone is a strong oxidizing agent and important disinfectant. According to literature data, exposure of bacteria, spores and viruses to ozone for only a few minutes causes inactivation25-26. Studies by Ricevudi et al.27 proposed oxygen-ozone treatment as a new immunotherapeutic treatment method for the viral agent COVID-19 causing this pandemic and that patients with COVID-19 using this in combination with other treatment methods would benefit from the assisting and synergic effects of ozone therapy. Again, as stated by Ricevudi et al.27 the need for more studies is clear. The bactericidal effect of ozone is linked to attacking the biological material in microorganisms through the oxidation pathway. In fact, the antibacterial effect of ozone is said to be more effective than iodine and chlorine23,28. Previous experimental studies have shown that ozone therapy has experimental benefits on pathologic processes16,23,29. In many organs like pancreas, peritoneum, liver, mesenteric lymph nodes and cecum, ozone therapy has proven reducing effects on bacterial translocation16. In an experimental necrotizing pancreatitis model, ozone therapy was seen to be more effective to reduce oxidative stress levels, tissue injury and bacterial translocation rates compared to hyperbaric oxygen treatment30. Schulz et al.31 observed a decrease in polymicrobial peritonitis after they administered intraperitoneal ozone. Çakır et al.32 reported the systemic inflammatory response markers of TNF-alpha and IL-1B levels reduced after ozone treatment. An experimental sepsis study by Yamanel et al.16 compared hyperbaric oxygen treatment with ozone therapy and identified that both methods lowered oxidative stress indices, myeloperoxidase activity and serum proinflammatory cytokine levels. As a result, histopathologic injury reduced in the lung tissue of septic rats and ozone therapy showed to provide more benefits according to histopathologic injury scores and IL-1B levels. As a result of the experimental study, they proposed that ozone therapy may be a complementary medicine treatment method that could be applied together with antibiotherapy for sepsis. Additionally, studies have shown that ozone causes ii In our study, positive effects were observed in terms of inflammation in Group 2 and Group 3 with added ozone therapy (p=0.020, p=0.012, respectively). However, it is considered that these effects are due more to antibiotic effects rather than to ozone therapy. Histopathological findings ble 1 - Statistical significance with value intervals and median values for histopathological findings. a: p<0.05, when compared with Group 5, b: p<0.05, when compared with Group 4, c: p<0.05, when compared with Group 1. : As Group 3 only included 7 rats there is one median value, the other groups comprised 8 rats and had 2 median values. a: p<0.05, when compared with Group 5, b: p<0.05, when compared with Group 4, c: p<0.05, when compared w 3 only included 7 rats there is one median value, the other groups comprised 8 rats and had 2 median values. 1 2 3 4 5 6 Figure 2 - Some histopathologic section samples from the groups. 1. Group 5: Lung tissue at morphological limits (H&E, x100); 2. Group 4: Vascular congestion, severe intraalveolar hemorrhage and parenchymal inflammation (H&E, x100); 3. Group 4: Peribronchial inflammation (H&E, x200); 4. Group 1: Vascular congestion, moderate intraalveolar hemorrhage and parenchymal inflammation (H&E, x100); 5. Group 2: Vascular congestion and moderate parenchymal inflammation (H&E, x100); 6. Group 3: Vascular congestion and moderate parenchymal inflammation (H&E, x100). 1 2 3 4 5 6 Figure 2 - Some histopathologic section samples from the groups. 1. Group 5: Lung tissue at morphological limits (H&E, x100); 2. Group 4: Vascular congestion, severe intraalveolar hemorrhage and parenchymal inflammation (H&E, x100); 3. Group 4: Peribronchial inflammation (H&E, x200); 4. Group 1: Vascular congestion, moderate intraalveolar hemorrhage and parenchymal inflammation (H&E, x100); 5. Group 2: Vascular congestion and moderate parenchymal inflammation (H&E, x100); 6. Group 3: Vascular congestion and moderate parenchymal inflammation (H&E, x100). 4 Acta Cir Bras. 2020;35(6):e202000604 Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. Pulmonary effects of ozone therapy at different doses combined with antibioticotherapy in experimental sepsis model Kapicibaşi HO et al. desquamation and alveolar septal injury results in emphysema and airway hyperresponsiveness33,34. Ozone-linked injury and inflammation is linked to the dose and frequency of ozone exposure35. if ■Discussion The source point for this thought is that the cellular injury, inflammation, alveoloseptal thickening and general histopathologic scores in Group 1, only administered antibiotics, were at statistically significantly low levels compared to Group 2 (p=0.002, p=0.002, p=0.044, p=0.0017, respectively). Similarly, the cellular injury and general histopathologic score were observed at statistically significantly low levels in Group 1, administered only with antibiotic, compared to Group 3, administered with high-dose ozone (p=0.002, p=0.035, respectively).i When the results of our study are investigated, it was identified that ozone increased the negative histopathologic effects induced in the lung by inflammation. However, as the dose of ozone increased, the fall in the number of parameters with statistical significance confirms that positive effects may occur if the dose of ozone used was increased. Additionally, a limitation of our study is that there was no group administered with ozone therapy alone after sepsis, so the histopathologic effects of ozone on lung tissue in sepsis could not be assessed. ■Conclusion Data obtained as a result of our study lead to the consideration that ozone therapy administered in addition to antibiotherapy may cause negative effects on lung tissue damaged due to sepsis. ■References Does leflunomide attenuate the sepsis-induced acute lung injury? Pediatr Surg Int. 2008;24(8):899–905. doi: 10.1007/s00383-008-2184-y.i 22. Steinhart H, Schulz S, Mutters R. Evaluation of ozonated oxygen in an experimental animal model of osteomyelitis as a further treatment option for skull-base osteomyelitis. Eur Arch Otorhinolaryngol. 1999;256(3):153–7. doi: 10.1007/s004050050130.ti 8. Rooney Z, Nadel S. Optimizing intensive care management in paediatric sepsis. Curr Opin Infect Dis. 2009;22(3):264– 71. doi: 10.1097/QCO.0b013e328329c493. 9. 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PLoS One. 2015;10(7):e0131236. doi: 10.1371/journal.pone.0131236. inflammation in mice. PLoS One. 2015;10(7):e0131236. doi: 10.1371/journal.pone.0131236. 35. Zhang M, Fei X, Zhang GQ, Zhang PY, Li F, Bao WP, Zhang YY, Zhou X. Role of neutralizing anti-murine interleukin- 17A monoclonal antibody on chronic ozone-induced airway inflammation in mice. Biomed Pharmacother. 2016;83:247–56. doi: 10.1016/j.biopha.2016.06.041. 35. Zhang M, Fei X, Zhang GQ, Zhang PY, Li F, Bao WP, Zhang YY, Zhou X. Role of neutralizing anti-murine interleukin- 17A monoclonal antibody on chronic ozone-induced airway inflammation in mice. Biomed Pharmacother. 2016;83:247–56. doi: 10.1016/j.biopha.2016.06.041. 34. Mathews JA, Kasahara DI, Ribeiro L, Wurmbrand AP, Ninin FMC, Shore SA. γδ T cells are required for M2 macrophage polarization and resolution of ozone-induced pulmonary 34. ■References Mathews JA, Kasahara DI, Ribeiro L, Wurmbrand AP, Ninin FMC, Shore SA. γδ T cells are required for M2 macrophage polarization and resolution of ozone-induced pulmonary Correspondence: Hasan Oğuz Kapicibaşi Çanakkale Onsekiz Mart University Faculty of Medicine, Department of Thoracic Surgery 17100, Kepez, Çanakkale Turkey Phone: +90 532 377 99 07 hokan@comu.edu.tr Received: Feb 15, 2020 Reviewed: Apr 13, 2020 Accepted: May 11, 2020 Conflict of interest: none Financial source: none 1Research performed at Canakkale Onsekiz Mart University, Faculty of Medicine, Experimental Research Application and Research Center, Canakkale, Turkey. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conflict of interest: none Financial source: none p 34. Mathews JA, Kasahara DI, Ribeiro L, Wurmbrand AP, Ninin FMC, Shore SA. γδ T cells are required for M2 macrophage polarization and resolution of ozone-induced pulmonary 33. Mathews JA, Krishnamoorthy N, Kasahara DI, Cho Y, Wurmbrand AP, Ribeiro L, Smith D, Umetsu D, Levy BD, Shore SA. IL-33 drives augmented responses to ozone in obese mice. Environ Health Perspect. 2017;125(2):246–53. doi: 10.1289/EHP272. Conflict of interest: none Financial source: none Correspondence: Hasan Oğuz Kapicibaşi Çanakkale Onsekiz Mart University Faculty of Medicine, Department of Thoracic Surgery 17100, Kepez, Çanakkale Turkey Phone: +90 532 377 99 07 hokan@comu.edu.tr 1Research performed at Canakkale Onsekiz Mart University, Faculty of Medicine, Experimental Research Application and Research Center, Canakkale, Turkey. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 7 7
https://openalex.org/W3141258368	https://www.rau2.ufscar.br/index.php/rau/article/download/351/307	Portuguese	null	Fazendo corpos e parentes	R@u	2,020	cc-by-sa	12,923	1 Agradecemos ao apoio financeiro propiciado pela Fundação de Apoio à Pesquisa do Distrito Federal, que possibilitou a apresentação oral deste trabalho no GT 22 da XII Reunião de Antropologia do Mercosul. R@U, 12 (2), jul./dez. 2020: 131-155. R@U, 12 (2), jul./dez. 2020: 131-155. Carolina Perini de Almeida Doutoranda em Antropologia Social pela Universidade de Brasília carol_perini@yahoo.com.br Carolina Perini de Almeida Doutoranda em Antropologia Social pela Universidade de Brasília carol_perini@yahoo.com.br Cíntia Engel Doutora em Antropologia Social pela Universidade de Brasília1 cintiaengel@gmail.com Cíntia Engel Doutora em Antropologia Social pela Universidade de Brasília1 cintiaengel@gmail.com Fazendo corpos e parentes: como grafar cotidianos? Carolina Perini de Almeida Doutoranda em Antropologia Social pela Universidade de Brasília carol_perini@yahoo.com.br Abstract We both decided to go back to our ethnographic fields – kinship in Terena villages in the Pantanal of Mato Grosso do Sul (Brazil) and family reorganizations for caring of dementia in the urban Federal District of Brazil. Not to go further, but to try again. We realized that we needed to write about the various times and cadences of daily life, from idleness to that which sounds invisible. We thus seek to bring effects of the sensible and to produce effects by writing, trying to consider that “language is never the simple attire of a thought that knows itself with all clarity” (Merleau-Ponty 2012: 12). This has multiple and unforeseen implications, the main one being the construction of unfinished texts, permeable to the extent that they do not contain all the possibilities of questions and answers and require of themselves to be rewritten, and therefore, to be transformed into something else to remain engaged with their effects. Keywords: everyday life; body; kinship; care; ethnography. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Resumo Ambas decidimos voltar aos nossos campos etnográficos – a feitura de parentesco em aldeias Terena no pantanal sul-mato-grossense e as reorganizações familiares para o cuidado da demência em Brasília – não para ir além, mas para tentar de novo, experimentar e escrever sobre algo que facilmente escapa: os diversos tempos e cadências do cotidiano, do ócio àquilo que soa invisível. Buscamos, assim, trazer efeitos do sensível e produzir efeitos pela grafia, tentando levar em conta que “a linguagem jamais é a simples vestimenta de um pensamento que conhece a si mesmo com toda a clareza” (Merleau-Ponty 2012: 12). Isso possui implicações múltiplas e imprevistas, sendo a principal delas a construção de textos-tentativas, permeáveis na medida em que não contenham todas as possibilidades de perguntas e respostas e exijam de si serem reescritos, e, portanto, transformarem-se em outra coisa, para continuarem engajados com seus efeitos. Palavras-chave: cotidiano; corpo; parentesco; cuidado; etnografia. 1 Agradecemos ao apoio financeiro propiciado pela Fundação de Apoio à Pesquisa do Distrito Federal, que possibilitou a apresentação oral deste trabalho no GT 22 da XII Reunião de Antropologia do Mercosul. Fazendo corpos e parentes: como grafar cotidianos? 132 Carolina Perini de Almeida e Cíntia Engel múltiplas e imprevistas, sendo a principal delas a construção de textos-tentativas, permeáveis na medida em que não contenham todas as possibilidades de perguntas e respostas e exijam de si serem reescritos, e, portanto, transformarem-se em outra coisa, para continuarem engajados com os seus efeitos. Por ser um diálogo, algumas vezes escrevemos juntas, ao mesmo tempo, e, assim, usamos a primeira pessoa do plural. Evocamos juntas. Mas, às vezes, falamos na primeira pessoa do singular, aquela que orientou nossas escritas em momentos divorciados. Trazemos uma para a outra as nossas tentativas de texto e as colocamos em diálogo para evocar junto. Não queremos comparar campos e, assim, produzir qualquer tipo de análise comparativa ou transcultural; queremos, sim, lidar com um desafio que nos afeta ambas e nos situa em determinados debates em torno dos projetos de conhecimento e criação com a etnografia. Eu, Carolina, busco pensar sobre alguns aspectos do cotidiano de meus interlocutores Terena – povo indígena da família linguística Aruak, habitante do pantanal sul-mato-grossense – nas aldeias Argola (Akulea) e Cachoeirinha (Mbokoti), da Terra Indígena Cachoeirinha, em Miranda-MS (e também nas cidades sul-mato-grossenses para as quais se deslocam com frequência). Para tanto, tomo como ponto de partida a noção que me foi apresentada e com a qual venho trabalhando até o momento: xuve ko’ovokuti. Como tentarei apontar, o termo xuve pode se referir tanto à posição social alcançada a partir do investimento pessoal e do reconhecimento público, quanto ao coletivo de pessoas e a lugares. Na leitura indígena, tal como a planta, o xuve tem raízes e sementes, nasce, cresce e morre. O que me fez insistir no palpite de que poderia se tratar de uma metáfora vivida, que diz sobre crescimento por multiplicação, proliferação e, sobretudo, relações. Em certos pontos, minhas tentativas de escrita sobre os troncos têm passado a ressoar a partir do diálogo com Cíntia, especialmente quando ela escreve buscando encontrar espaço para a conformação de redes de solidariedade e para modulações da escuta. Desse diálogo, ao invés de construir uma ponte entre realidades tão distintas, tenho procurado compreender nossas grafias como o próprio rio sobre o qual a ponte passa. Como propõe Coelho de Souza (2017:111), a capilaridade do rio e de suas margens abertas na terra, sem fronteiras ou limites, seriam imagens mais adequadas para a evocação da vida como simbiose, co-existência e co-constituição. Introdução A tentativa e modo de ser deste artigo é um diálogo entre perspectivas e campos etnográficos distintos. Em alguma medida, eles causam em nós reflexões que se articulam. Talvez pelo modo de ser das relações, pela oportunidade de ver relações criando e fazendo mundo, e pelos desafios que diferentes mundos enfrentam em fazer-viver junto, em articulação e interdependência com pessoas, terra, substância, medicamentos, cuidados. Ambas decidimos voltar aos nossos campos etnográficos – a feitura de parentesco em aldeias Terena no pantanal sul-mato-grossense e as reorganizações familiares para o cuidado da demência em Brasília – não para ir além, mas para tentar de novo, experimentar. Existem questões que apreendemos em campo e que foram (e, em alguma medida, ainda são) de difícil articulação pela escrita. Em campo, partilhamos certos gestos, olhares, jeitos de fazer, ser, conversar; além de paisagens e espaços feitos disso tudo. Por nossos sentidos entramos em contato com delicadezas de convivência, com demandas e possibilidades de mundo. Articular tais processos por meio da escrita é um desafio. Ainda nos parece fundamental nos engajarmos com os efeitos dos nossos textos e experiências anteriores e continuar tentando expressar. Esse texto é uma experiência-desafio. Vamos tomar a liberdade de tentar, encarando de forma aberta que “a linguagem jamais é a simples vestimenta de um pensamento que conhece a si mesmo com toda a clareza” (Merleau-Ponty 2012: 12). Isso possui implicações Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 133 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 2 Seguindo a proposta do texto, consideramos importante destacar que ele foi escrito, debatido e refeito entre o final de 2017 e início de 2019 e que, de lá para cá, foram alguns os efeitos e engajamentos gerados. Em parte, este diálogo inspirou uma discussão sobre ética, cuidado e escrita sistematizada no interlúdio da tese da Cíntia (Engel 2020:228). De outro lado, esta experiência tem mobilizado a busca de sentido para minha escrita de tese durante uma pandemia com bebê em casa e sem rede de apoio. As questões: por quê e para quê escrever persistem e podemos dizer que o artigo em si foi e continua sendo reescrito e transformado em outras coisas – segue, pois, como um rascunho de futuros rascunhos e tentativas etnográficas. Fazendo corpos e parentes: como grafar cotidianos? Fazendo corpos e parentes: como grafar cotidianos? 134 de cuidado e manejo de substâncias. Convivi com técnicas, exercícios cotidianos de cooperação e lidas com efeitos de medicamentos, interações entre eles, entre médicos, entre sintomas e, em conjunto, com relações de divisão, partilha, cooperação e conflito entre lógicas, pessoas e mundos. Com esse campo, tenho tido alguns desafios, especialmente para articular em escrita diversas relações complexas que compõem corpos, demências, pessoas e redes sociotécnicas – deixando que o cotidiano e suas cadências ordinárias tenham protagonismo. Gostaria de narrar sem estabilizar o que vem a ser demência, ou cuidado, ou uso de medicamentos, ou mesmo corpos, direitos, interações e partilhas. Aqui, nesse texto, quero aprender com Carolina a pensar sobre os modos de narrar experiências etnográficas de convivência. Nós iremos, a partir de agora, (re)escrever sobre esses dois campos para, em seguida, tentar articular juntas as questões que nos motivaram a refletir sobre a grafia, em suas potencialidades e desafios. A partir da tentativa de escrita desse texto, voltamos a colocar em jogo os efeitos com os quais queremos continuar engajadas em nossos campos, com os desafios narrativos de fazer isso e com o que pensamos ser importante nesse exercício por ora: por quê, e para quê, escrever2. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Já eu, Cíntia, tenho pesquisado a vida com demências no Distrito Federal junto de um centro multidisciplinar de geriatria e de mundos que se articulam com ele ou em algum tipo de relação com ele. Parte do meu campo envolve as lógicas de cuidado e manejo de substâncias por esse centro, outra parte me relaciona a algumas famílias e seus cotidianos Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 135 Da primeira vez que encontrei com Seu Calixto – xuve morador da aldeia urbana Marçal de Souza, meu primeiro amigo e interlocutor Terena (que, infelizmente, nos deixou em 2017, com 87 anos) – para apresentar e discutir com ele o tema da minha pesquisa de mestrado, ele argumentou como era importante haver pessoas que estudassem e tentassem entender o que os índios realmente precisam. Disse-me que, se quisesse saber sobre os parentes dele, teria que ir para a aldeia, pois sua família “levava” para lá. E, por fim, acrescentou que os purutuye3 precisavam ir viver um pouco com os índios para que pudessem “sentir” a realidade indígena. Sua última colocação chamou de súbito minha atenção por tocar exatamente no que acredito estar em jogo no engajamento antropológico. Em campo sentimos com o nosso corpo tudo o que queremos mais tarde transformar em texto. É o nosso corpo engajado que permite o saber antropológico, e isso vai além da simples emoção e empatia, ainda que não as exclua. De sorte que, motivada por ele, parti para a aldeia Argola para viver com seus parentes e “sentir” um pouco daquela realidade. Sem saber direito explicar o que é que queria estudar daquela vez, nessas primeiras viagens me limitava a dizer que queria entender a “família Terena”4. Pensando e perguntando sobre as ressonâncias da noção de “família”, cheguei à noção de xuve. Pude compreendê-la na medida em que me vi envolvida em diversas situações cotidianas das quais participei e nas quais aprendi não apenas sobre os troncos, mas sobre outros tipos especiais de laços que as pessoas estabelecem umas com as outras. Inspirada por Carsten (2000), penso que, conquanto tais laços, formas de relatedness, possam às vezes ser descritos em termos genealógicos, também podem ser apreendidos de outras maneiras. De modo que, com o dia a dia vivido na aldeia, desvelaram-se “novos significados e novas experiências de estar relacionado” (Carsten 2000: 34, tradução minha). Tentarei resumir minha compreensão da questão até aqui. Tendo em vista que “O ideal Terena é, pois, viver entre parentes” (Pereira 2009: 79), as ideias em torno de o que é, quem é e como deve se portar um parente são centrais para se entender qualquer processo social relacionado aos Terena. 4 É preciso dizer que no idioma Terena não existe palavra que equivalha literalmente à “família”, sendo a melhor aproximação a noção de iyenoxapa, traduzida para mim como “meu grupo”, “meu pessoal” e “meus parentes”. A ideia de “casa” (ovokuti) também é fortemente associada à de “família”; mais do que isso, a associação entre coletivo de parentes e lugares revela o quanto as dinâmicas de parentesco são espacializadas nesse contexto, por isso considero a possibilidade de se pensar esses modos de se relacionar expressos pela metáfora do tronco como também constituidores de “paisagens” Os troncos como metáforas vividas: crescer, conviver e multiplicar DESPALAVRA Hoje eu atingi o reino das imagens, o reino da despalavra. Daqui vem que todas as coisas podem ter qualidades humanas. Daqui vem que todas as coisas podem ter qualidades de pássaros. Daqui vem que todas as pedras podem ter qualidades de sapo. Daqui vem que todos os poetas podem ter qualidades de árvores. Daqui vem que os poetas podem arborizar os pássaros. Daqui vem que todos os poetas podem humanizar as águas. Daqui vem que os poetas devem aumentar o mundo com as suas metáforas. Que os poetas podem ser pré-coisas, pré-vermes, podem ser pré-musgos. Daqui vem que os poetas podem compreender o mundo sem conceitos. Que os poetas podem refazer o mundo por imagens, por eflúvios, por afeto. (Manoel de Barros 2001) 2 Seguindo a proposta do texto, consideramos importante destacar que ele foi escrito, debatido e refeito entre o final de 2017 e início de 2019 e que, de lá para cá, foram alguns os efeitos e engajamentos gerados. Em parte, este diálogo inspirou uma discussão sobre ética, cuidado e escrita sistematizada no interlúdio da tese da Cíntia (Engel 2020:228). De outro lado, esta experiência tem mobilizado a busca de sentido para minha escrita de tese durante uma pandemia com bebê em casa e sem rede de apoio. As questões: por quê e para quê escrever persistem e podemos dizer que o artigo em si foi e continua sendo reescrito e transformado em outras coisas – segue, pois, como um rascunho de futuros rascunhos e tentativas etnográficas. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 3 Termo da língua Terena derivado de “português” para se referir primeiramente aos eur posteriormente, aos não indígenas brasileiros. Fazendo corpos e parentes: como grafar cotidianos? 136 esferas da vida desse povo. A partir de suas formulações, pude orientar a interpretação do meu material etnográfico e chegar aos xuve ko’ovokuti. Ou, na realidade, partir deles, como me instaram sempre meus instrutores Terena, aliás. esferas da vida desse povo. A partir de suas formulações, pude orientar a interpretação do meu material etnográfico e chegar aos xuve ko’ovokuti. Ou, na realidade, partir deles, como me instaram sempre meus instrutores Terena, aliás. Xuve é o termo empregado para tronco de árvore. Ovokuti, da raiz ovoku (casa/ home, que Seu Aristides traduziu como “casa dele”), acrescido da partícula ti seria “casa nossa” (ovoku-ti), “nossa casa”, “nosso lar”. O sentido corrente do termo, como o traduziram para mim, seria o de “casa da família”, e é essa extensão de tradução que tenho utilizado. No cotidiano da aldeia, o termo xuve pode se referir ao “tronco da casa”, “tronco que chama os filhos” ou ao “chefe de casa/família”, designando tanto uma posição social dependente do investimento pessoal e do reconhecimento público, quanto coletivos de pessoas e lugares. Ademais, como dito acima, esta noção faz render a imagem vegetal a que o termo remete. Inicialmente, havia tomado como “confusão” o fato de que não conseguia encontrar na bibliografia sobre os Terena, e muito menos nas conversas com as pessoas em campo, um consenso sobre se a noção dos troncos fazia referência a coletivos de pessoas, a lugares ou a sujeitos em uma determinada posição. Formulei, então, a questão que hoje percebo que de tão ruim era boa por exigir a reformulação de seus próprios termos: é o xuve pessoa ou coletivo? Autor que mais detidamente refletiu sobre o tema, Pereira (2009) definiu os troncos, de modo geral, como um grupo de pessoas unidas por relações de parentesco e amizade sob a liderança de um indivíduo ou casal de idosos. Ainda que a fala transcrita por ele se reporte ao tronco como sujeito (“O tronco tem que saber conversar, saber fazer a convivência boa, fazer agrado, aí o nome dele vai correndo longe” PEREIRA, 2009: 57) e que tenha esclarecido que os anciãos são também identificados como tronco, analiticamente, o antropólogo optou pela expressão de “líder do tronco” ao fazer menção ao sujeito individual. Não obstante, o que antes tomei como “certa imprecisão na bibliografia” (Perini de Almeida, 2013) era, na verdade, reflexo do fato de que nosso vocabulário não acompanha o vernáculo. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Por isso considero que as elaborações de Pereira sobre os troncos, intrinsecamente relacionados a um ethos e à concepções muito particulares de civilidade e etiqueta – já que engendram “estilos de vida” –, foram fundamentais para minha percepção dessa dinâmica articulada a outras Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? Carolina Perini de Almeida e Cíntia Engel 137 formam esses coletivos. Nesse sentido, as ponderações de Pereira sobre a relação dos troncos com “estilos de vida” e padrões comportamentais são bastante sugestivas, abrindo questões para além da obsessão sociologista com os grupos e seus governantes, com as unidades sociais e as suas chefias. Na mesma direção, atentando para o caráter dinâmico – vivo – do xuve, e para as implicações da metáfora vegetal por meio da qual seu significado se desdobra, é que quero buscar uma maior compreensão das noções e das vidas dos meus interlocutores. Parafraseando Seu Aristides – senhor de cerca de 70 anos, um de meus anfitriões na aldeia e quem me concedeu uma das explicações mais sistemáticas sobre os troncos até aqui – o xuve é “orientador”, é o tronco que orienta os filhos e tudo vem da orientação dele, inclusive o processo de construção de mais famílias e formação de novos xuve. É dele que partem os rumos (os ramos) para a família. Ele também deve prezar pela manutenção da ordem e da harmonia; deve ser sábio e experiente para falar bem e dar bons conselhos; deve, com suas atitudes, dar o “exemplo” de boa conduta; deve ter bons modos e evitar o conflito aberto; deve saber se adaptar e tirar o melhor proveito das mais diversas e adversas situações; além de organizar e liderar a vida econômica da família, especialmente na lida com a terra. Com esses atributos pode se considerar o responsável por todos que convivem em sua área. O tronco que gera sementes também é raiz, encarna a história e partilha memória. Assim, o tronco bem-sucedido torna-se um colecionador de boas relações e coloca-se em constante movimento, seu nome “corre longe”. Da forma pela qual venho expondo a noção de xuve até o presente o/a leitor/a poderá ter a impressão de que essa figura é essencialmente masculina. Porém, tal impressão não corresponde inteiramente ao que observei na aldeia. A importância do casamento e da condição de homem casado para o xuve, além de evidenciar o aspecto processual da formação do tronco, revela a importância da mulher nesse universo. O tronco, como processo iniciado com o casamento, tem como lócus de sua força o casal, por isso a êno ko’ovokuti (êno-ko-ovokuti, “mãe”; 3º pessoa singular; casa; “mãe da família/nossa casa”) é crucial ao xuve. Fazendo corpos e parentes: como grafar cotidianos? Talvez por esse motivo, não ter conseguido decidir entre pessoa e coletivo foi sentido por mim, a princípio, como “inconsistência”. Estamos, todavia, diante de um modo de vida – pensamento – que não se pauta pelo regime do um ou muitos como em outras partes da América indígena (cf. Stolze Lima 1996; Sztutman 2002). O xuve não apenas pode ser tanto um e muitos, como também pode ser dois – um par! Assim, minha percepção foi a de que meus interlocutores, ao falarem em troncos, além de se referirem aos agrupamentos domésticos familiares ou locais, remetiam principalmente ao próprio indivíduo ou mesmo casal de idosos em torno de quem se Carolina Perini de Almeida e Cíntia Engel Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Como disse Seu Aristides, “já sai da família do xuve para fazer outra família, constituir mais família para ser xuve”. O xuve é, por conseguinte, construído pelos dois: “os dois já formam um xuve, os dois são”. De modo que a mulher também é “tronco”, é “pé”, ainda que haja, de fato, ênfase diferencial no polo masculino. Talvez resida aí a centralidade do casamento e da noção de orientação. Explicou Seu Aristides: “o casamento tem que ser preparado antes de casar, para formar outra família. O processo é assim. Tudo vem da orientação do xuve, que é o pai. Porque não pode morar assim, casar sem responsabilidade. Aí não dá, não convive. (...) Tem que preparar.”. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? 138 Pegando emprestado aqui as analogias nativas, uma das principais responsabilidades dos troncos originários, a fim de se tornarem raízes importantes, é preparar o terreno para suas sementes. Ou seja, é preparar o casamento para que, na boa convivência, dele forme-se outro tronco. De tal maneira que o “processo” de proliferação dos troncos, como ressaltou Seu Aristides, é marcado pela preocupação com o bem conviver e com a estabilidade do casal: por meio de um investimento cotidiano. Nessa direção, o final de uma conversa com Seu Aristides, na qual ele responde a uma provocação boba da minha parte, pode ser bastante instrutivo. C.: Será que eu vou ser Xuve um dia? Eu já casei, Seu Aristides. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 139 Aqui, sobretudo no que tange a ideia de orientação, já podemos vislumbrar, portanto, uma imagem diferente daquela da planta cujo crescimento é orientado pelo sol. Para me valer de um vocabulário botânico, diria que se trata antes de um geotropismo positivo (a penetração da raiz solo abaixo) e de uma espécie de tigmotropismo (como o do enrolamento de gavinhas de maracujá, chuchu e videira em torno de esteios de madeira, por exemplo), do que de um fototropismo positivo (crescimento das plantas em direção a luz do sol). Assim, um de meus objetivos seria, justamente, refletir sobre esse aspecto da metáfora e seus efeitos possíveis, como indicarei mais adiante5. Sendo mais explícita, portanto, creio que a metáfora arbórea vivida e evocada pelos Terena com quem venho trabalhando diz respeito a um modelo vegetal mais rizomático (Deleuze & Guattari 1995)6. O Xuve não é uma coisa ou outra: se é o tronco e se vive em troncos. Talvez seja um coletivo investido de qualidades pessoais concretizadas na figura do (in)divíduo (o casal) tronco. Talvez seja o chefe investido de qualidades coletivas concretizadas no seu ovokuti. Também pode ser toda a história e a memória que encarna, todas as relações que o compõem. Partindo, pois, do pressuposto de que a linguagem e suas metáforas são vividas, gostaria de pensar, a partir de agora, sobre algumas questões que surgiram ao se levar a sério as implicações dessa imagem vegetal (os troncos) como modo de vida constituidor de mundo. Quero, portanto, me deter a partir de agora em duas questões que me parecem fundamentais: i) quais os efeitos criativos da linguagem? e ii) quais as possíveis implicações político-ontológicas de se viver em troncos, de se recusar a alternância do um e muitos? Estou, por fim, querendo pensar aqui no que nos incita Manoel de Barros com o poema que escolhi como epígrafe desta parte: como aumentar o mundo com nossas metáforas? Como compreender o mundo sem conceitos (ou com outro conceito de conceito)? Como, enfim, refazer o mundo por imagens, por eflúvios, por afeto? Comecemos, em ordem, pela primeira questão, que, em certo sentido, relaciona- se com a própria escolha em dizer de outra maneira o que já havia dito na dissertação de mestrado para que o trabalho pudesse se transformar em “outra coisa”. C.: Será que eu vou ser Xuve um dia? Eu já casei, Seu Aristides. A.: Pensa. Você também Carol você já está no processo de ser xuve, porque você já casou. Mas o seu trabalho não é questão de, assim, de mandar numa família. Assim, como nós aqui, a gente está aqui. Eu não saio e fico direto por aqui. Você não! Você é uma pessoa volante, que anda, procurando dialogar com alguém. Ou você é capaz de orientar, você já aprendeu conviver com o seu companheiro, que é esposo, marido. (...) Então, como no caso aqui sou xuve e você é orientador e está escrevendo. Não é que você não sabe que é, assim, responsável da família. Você já sabe. Você já casou. Você é uma responsável do trabalho seu para criar bem- estar daqui para frente. C.: Hum... Tem que parar quieta, né? A.: É. C.: Hum... Tem que parar quieta, né? Ao invés de simplesmente responder “porque você não é Terena!”, Seu Aristides se esforçou para pensar como minha situação pessoal se encaixaria, ou não, nesse contexto. E de sua reflexão ficam evidentes características indispensáveis para poder ser xuve, para além do status de casado. Com muito cuidado e elegância, ele procurou me explicar como meu estilo de vida “volante” de estudante de antropologia – diferente do dele, que não fica direto “por aí” – não me permitem aglutinar pessoas perto de mim e criar raízes. Por outro lado, ressaltou alguns outros atributos que reúno que possibilitariam um caminho futuro como xuve (“para criar bem-estar daqui para frente”), como a responsabilidade, a capacidade de orientar e de saber conviver. Tornar-se tronco é, pois, dependente de um investimento pessoal, que ultrapassa desdobramentos automáticos do casamento e procriação. Casar-se e ter boas raízes não fazem sozinhos um xuve. O tronco aglutina e orienta, para isso é preciso “estar aqui” – “como nós aqui, a gente está aqui”. Ou seja, o tronco não apenas cresce e prolifera, mas também atrai. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Estou, assim, 5 Vale dizer que representar e experimentar relações (relatedness) por meio de metáforas arbóreas é algo bastante arraigado na cultura ocidental e, como argumenta Mary Bouquet (1996), embasa o tom moral e a influência visual do diagrama genealógico utilizado pelos antropólogos. 6 É importante notar que há, com efeito, uma discussão a ser amadurecida sobre a correlação entre a produção do parentesco e o crescimento vegetal na etnologia a partir de contextos etnográficos em que essa espécie de “linguagem vegetal” é recorrente na conceituação de pessoas e coletivos (Guerreiro Junior 2011, 2012a, 2012b; Costa 2007; Veiga 2000; Rival 1993). Desse modo, com vistas a situar meu material etnográfico em relação a questões mais gerais da etnologia indígena, será objeto de reflexão futura afinar o diálogo com essa bibliografia, levando em conta, especialmente, a possibilidade de se pensar no tronco como “pessoa-relação”, tal como sugere Guerreiro Junior (2011) quando discute a relação entre tronco, corpo e parentesco entre os Kalapalo. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? 140 de princípio, partilhando do entendimento daquilo que Merleau-Ponty expressou na citação que reproduzimos de início (2012:12). Nessa leitura, a linguagem atua como um devir, uma antecipação, uma transgressão, uma operação violenta. Isso porque ela tem o poder de superar a ela própria em mais do que se diz termo a termo “(...) quer se trate de lançar o outro em direção ao que sei e que ele ainda não compreendeu ou de orientar-me em direção ao que vou compreender” (Merleau-Ponty 2012: 216-217). É, portanto, por meio da linguagem que os sujeitos se tornam o que são e as coisas transformam-se nelas mesmas. Nesse sentido, entendo que a linguagem também faz mundo. Daí a importância vital da escolha das metáforas e suas operações. Em alguma medida, estou inspirando-me na proposta de Roy Wagner (2010) de levar às últimas consequências o poder inovador das metáforas (tanto coletivas, expressões socializantes, quanto individuativas, expressões que conferem poder). Desse modo, quando tento pensar junto com meus interlocutores Terena na metáfora dos troncos (xuve), e nos problemas dela decorrentes, tento fazer o exercício de entender que não há um estado anterior aos termos e contextos. Ou seja, não haveria um estágio pré-referencial antes das relações. São as relações, em suas estéticas, que compõem referentes e seus termos. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 141 antes ocupado pelo casal. Ela passou a ocupar o quarto de uma das suas filhas mais novas, que havia se mudado para a casa do namorado/marido assim que S. Calixto faleceu. Dona Hilda disse que a filha iria se casar, já tendo até juntado o dinheiro para a festa, porém, com a morte do pai, ela desistiu. “Quem vai entrar comigo na igreja?”. A filha havia começado a namorar um vizinho enquanto S. Calixto estava doente. D. Hilda, percebendo o movimento, conversou com ela e disse que aquilo “não estava certo”. O pretendente deveria ir conversar com o pai da moça, pedi-la em casamento. O pai – xuve junto à esposa – tinha que autorizar a relação. No dia seguinte a essa conversa, contou que o moço apareceu na porta de sua casa, sozinho e “com a roupa do serviço ainda”, perguntando se ela queria falar com ele. Aquilo a enfureceu. “Onde já se viu?! Não é assim que se faz as coisas!”. Disse a ele: “não sou eu que quero falar com você, é você que tem que querer falar comigo e com o Calixto!”. Por sorte, S. Calixto nesse dia não estava se sentindo bem e havia ido se deitar, não tendo ficado sabendo de nada. D. Hilda falou novamente com a filha, que ficou muito envergonhada pelo comportamento do companheiro: “não acredito que ele fez isso e sem eu estar junto... Vou falar com ele de novo!”. No outro dia, aí sim, o companheiro apareceu junto com a filha, de banho tomado e camisa, e pediu para falar com S. Calixto. Aí deu tudo certo e o Calixto ficou feliz. “Tem um jeito certo de fazer as coisas!”, ela me explicou. Há um jeito certo de fazer as coisas e, somente assim, criam-se os efeitos (e mundos) desejados. Voltamos, pois, a nossa questão sobre os efeitos criativos da linguagem. Se nossas metáforas fazem mundos, que mundos possíveis são esses? Queria, instigada pela “boa comvivência” que sempre defendem os meus amigos Terena, poder pensar nas metáforas dos troncos como constitutiva de mundos de confluências e coexistências. O estímulo agora vem da análise resolutiva elaborada por Antônio Bispo dos Santos (2015: 89)7. Conforme o autor, confluência (e convivência) é estar junto, mas não misturado. 7 Agradeço ao amigo e colega de doutorado Lucas Coelho Pereira por me chamar atenção para essa possibilidade de diálogo com o pensamento de Bispo neste ponto. Carolina Perini de Almeida e Cíntia Engel Voltando aos xuve, o tronco – seja planta, seja gente, seja lugar – é assim constituído por serem estabelecidas com ele relações do tipo específico (a forma, o “como se”) que se tem com troncos (lugares/parentes/plantas). É tronco/xuve aquilo que tem afeto de tronco. Este é o momento em que quero passar à segunda questão, usando como gancho algo da reflexão de Strathern (2014: 358-360) sobre a forma quando descreve os processos de reificação (objetificação e personificação) entre os Hagen. Nessa ocasião, a autora argumenta que a demasiada atenção conferida aos efeitos estéticos das entidades vem do que lhe parece ser um contraste útil entre os pressupostos euro-americanos sobre a naturalidade das coisas e a maneira como os melanésios se concebem como tendo que trabalhar arduamente para fazer com que as coisas apareçam em seus aspectos apropriados. “O escrutínio da forma ressaltava que, ipso facto, uma forma só pode aparecer com suas propriedades apropriadas – ou então ela não aparece” (Strathern 2014: 360). Em consequência, seria a estética o que tornaria as relações visíveis. Nesse sentido, estou entendendo aqui que as metáforas, sendo estéticas, são as formas pelas quais as relações aparecem, ao mesmo tempo em que criam com elas mundos possíveis. Ou seja, viver de determinada forma tem implicações e efeitos político-ontológicos. Em minha última visita a Dona Hilda Francelino, agora viúva de Seu Calixto, ela me contava sobre seu luto, que a obrigava a mudar de quarto, deixando vazio o cômodo Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 um é sempre muitos. Para Deleuze e Guattari (1995), o rizoma é o oposto de uma estrutura composta por pontos e posições. O rizoma é feito somente de linhas. Algo como uma terceira margem do rio: “A árvore impõe o verbo ‘ser’, mas o rizoma tem como tecido a conjunção ‘e...e...e...” (DELEUZE & GUATTARI, 1995:36). Pensando com esses autores, se o tronco tem uma orientação de crescimento rizomática, portanto com ênfase na multiplicidade, temos que entender, que ao se afirmar como um e muitos ele, na realidade, nega a ambos. Igualmente, como um modo de falar sobre relações, xuve também é um modo de viver com, logo de criar. E o que se cria com os troncos passa por como se cria com os troncos. Poucos dias antes de ir embora da aldeia, em março de 2017, perguntei a uma amiga como eu poderia xingar alguém no idioma Terena. Ela disse prontamente: “mokere!”, traduzindo-me o termo como “bobo”. Mas logo em seguida acrescentou: “ou surdo”. Foi aí que notei de que maneira a falta de audição pode ser uma das piores condições a se apresentar para o convívio Terena. Ironicamente, mokere (em seu duplo sentido) é exatamente como sempre me sinto lá, tendo que pedir diversas vezes para as pessoas repetirem o que me dizem, mesmo em português. O contrário raramente ocorre. Geralmente sou compreendida de primeira, mesmo quando julgo estar falando rápido demais ou sussurrando algo que deveria ter sido ouvido apenas por alguém próximo, mas que acaba sendo escutado por todos ao redor. Assim, lembro-me agora de Bernardo, o personagem principal de Manoel de Barros, e do seu silêncio como modo de comunicação, um modo próprio de fazer um mundo seu. Acredito que dialogue com isso, em algum nível, a insistente denúncia de Davi Kopenawa (2015: 226) contra a surdez dos brancos: “Nossos maiores amavam suas próprias palavras. (...) Suas mentes não estavam fixadas em outro lugar. (...) Hoje, todas essas falas a respeito dos brancos atrapalham nossos pensamentos. A floresta perdeu seu silêncio. Palavras demais vêm das cidades.”. Para ele, em alguma instância, esse conflito entre mundos, o dos Yanomami e o dos brancos, seria uma patologia da comunicação. Bernardo que era homem, era árvore e era pássaro, que era um, era muitos e não era ambos, produziu escuta com seu silêncio. Carolina Perini de Almeida e Cíntia Engel Ao contrapor saberes orgânicos a um sintético, Bispo está igualmente enfatizando a diferença entre o conhecimento de elaboração e estruturação vertical versus conhecimentos de orientação circular. Justamente, o que os saberes biointerativos reivindicariam para si é o “e... e... e...”, a “confluência de todas as experiências” (a diversidade); ao passo em que o saber sintético/desenvolvimentista operaria por meio do “ou”, ou seja, da negação do outro para que possa se estabelecer. E aqui me volto ao ideal do bom conviver Terena ao qual dedica-se o tronco, pensando que pode ser uma espécie de confluência, de se estar junto sem se misturar – um viver com. Lanço, mais uma vez, a hipótese de que podemos estar diante de um fazer mundo que nega o “ou” e afirma que o 7 Agradeço ao amigo e colega de doutorado Lucas Coelho Pereira por me chamar atenção para essa possibilidade de diálogo com o pensamento de Bispo neste ponto. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? 142 um é sempre muitos. Grafar cotidianos em meio a relações de cuidado e convivência com demências Em 2012, passei a conviver com pessoas diagnosticadas com diferentes tipos de demência, com uma terapêutica gestada por um centro de medicina coordenado por geriatras e formado por uma equipe multidisciplinar e com cuidadoras, especialmente aquelas que eram categorizadas e se autodenominavam como cuidadoras familiares e principais. Nessa aproximação, circulei com mais intensidade em grupos de acolhimento multidisciplinar e psicológico para pessoas recentemente diagnosticadas com demência e grupos para cuidadoras de pessoas diagnosticadas (Engel 2013). Entre 2017 e 2018 voltei ao centro, acompanhei consultas, reuniões interdisciplinares, um coral, fiz algumas entrevistas e li prontuários, foi uma nova imersão a partir de outros circuitos desse mesmo lugar. Desde que comecei a me envolver com redes da demência no Distrito Federal queria poder saber mais dos dias, das horas dos dias, das coisas por fazer, dos modos de conviver e se relacionar em casa, da busca e manejo diário das substâncias receitadas. Por isso, nesse novo período de campo, passei também a frequentar semanalmente a casa de três famílias e acompanhar as coisas que eram feitas, a dinâmica das relações e do uso de substâncias, especialmente os medicamentos – parte central dos dias. Abandonei as entrevistas, os espaços de narrativa da condição e de feitura do que é Alzheimer e como cuidar dele e de outras demências e passei a dar carona para ir até a Farmácia de Alto Custo8, fazer caminhadas, mexer nas coisas pela casa, lavar a louça, apreender os ritmos da casa e me engajar para estabelecer comunicações significativas com as pessoas que conheci. – parte central dos dias. Abandonei as entrevistas, os espaços de narrativa da condição e de feitura do que é Alzheimer e como cuidar dele e de outras demências e passei a dar carona para ir até a Farmácia de Alto Custo8, fazer caminhadas, mexer nas coisas pela casa, lavar a louça, apreender os ritmos da casa e me engajar para estabelecer comunicações significativas com as pessoas que conheci. Em relação à primeira imersão de campo e tentativa de elaborar sobre o que via – a dissertação – a categoria cuidado ou, mais especificamente, care (Tronto 1993; Lagarde 2003; Molinier 2004; Hirata & Guimarães 2012) me orientou. 8 Por meio desse campo, conheci algo sobre como o Sistema Único de Saúde organiza institucionalmente o cuidado de pessoas com demência, desde o atendimento em um centro especializado até o Programa de Saúde da Família e a lista de remédios que podem ser acessados ou em Farmácias do Estado/Distrito, Farmácias Populares e Farmácias de Alto Custo. Carolina Perini de Almeida e Cíntia Engel 143 um é sempre muitos. Inspirada por ele, quero com essa pesquisa pensar os troncos – que são pessoas, coletivos, lugares, corpos, relações – como uma vida possível, um mundo que se cria com metáforas, eflúvios e afetos. Quero, junto com os Terena, pensar que “viver como” não é “virar um”, é antes de tudo um comviver. E, assim, quem sabe, ao escrever sobre os possíveis efeitos de se viver em troncos e se formar terra possa ajudar a criar mais escuta do que palavras. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? Fazendo corpos e parentes: como grafar cotidianos? 144 Leibing 2006). Preocupei-me com isso também, mas, apesar de diversas tentativas, não consegui me engajar com tal trabalho. O que acabou tomando o texto foram as tentativas e dilemas em se estabelecer relações significativas por meio de trocas e relações envolvendo diversas dinâmicas de cuidado e convivência. Leibing 2006). Preocupei-me com isso também, mas, apesar de diversas tentativas, não consegui me engajar com tal trabalho. O que acabou tomando o texto foram as tentativas e dilemas em se estabelecer relações significativas por meio de trocas e relações envolvendo diversas dinâmicas de cuidado e convivência. Na época da dissertação, conceituei essa habilidade de se relacionar de forma significativa como “alteridade perceptiva” – um processo de aquisição de talentos, a partir de relações atentas, que buscam comunicações mesmo quando não partilham de experiências e conexões perceptivas com o mundo. Entendi que era pelo reconhecimento de diferenças no modo de se relacionar com o espaço, corpo e ambiente, que pessoas envolvidas no cuidado e convivência com as demências passam a desenvolver suas habilidades técnicas e afetivas que promoviam potenciais relações significativas, apesar das diferenças. Estava muito tomada pelas discussões sobre composições do corpo e pelo modo contínuo do corpo se fazer a partir de atos perceptivos relacionados aos mundos, ambientes, objetos e seres (Merleau-Ponty 1945). Não sei mais se a ideia de alteridade perceptiva é uma boa conceituação, ou se aproxima do que eu gostaria de narrar. De toda forma, o texto teve certos efeitos a partir do modo como foi construído. Quando apresentava algum dos meus textos com trechos sobre essas tentativas de estabelecimento de relações em congressos, eventos ou em diálogos esparsos entre acadêmicos, alguém me parava em um canto depois, ou mesmo intervinha para dialogar com a minha fala dizendo algo como: “eu lembrei muito da minha vó”, “da minha mãe”, “do meu tio”, “a gente passou por isso”. Por mais que eu tivesse vontade de dialogar ali sobre o que vinha refletindo a partir do campo e com os conceitos que tinha intentado, nos congressos e nos encontros acadêmicos meu texto interpelava algo sobre outro lugar – das lembranças de casa – e era sobre a casa que meus interlocutores queriam conversar e ser ouvidos. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Grafar cotidianos em meio a relações de cuidado e convivência com demências Naquele momento, mesmo com possíveis limitações do termo em alguns contextos – como a ideia de que cuidado é prestado de uma pessoa para outra de certa forma e de modo passivo a partir de uma relação de dependência (conceito já questionado em Winance 2010; Taylor 2017; Fonseca & Fietz 2018) – era o único termo que me levava ao que parecia fundamental aos meus interlocutores: os modos de estabelecer relações significativas. O campo da demência é, com razão, ocupado com determinados conceitos e modos de reformulá-los para reconhecer a manifestação da pessoa em sujeitos com demência, iniciativa que ficou conhecida como personhood movement (Kitwood 1997; Kontos 2005; 8 Por meio desse campo, conheci algo sobre como o Sistema Único de Saúde organiza institucionalmente o cuidado de pessoas com demência, desde o atendimento em um centro especializado até o Programa de Saúde da Família e a lista de remédios que podem ser acessados ou em Farmácias do Estado/Distrito, Farmácias Populares e Farmácias de Alto Custo. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 145 falta, como transformá-los, também perdi um pouco do que levava as pessoas para esse outro lugar com o texto. Ao insistir em como melhorar minha possibilidade de falar sobre as demências e as relações que as envolvem a partir de uma escolha qualificada de certos autores e palavras, o mais ordinário e cotidiano perdeu espaço. Recentemente li algo que se comunicou com tal experiência a partir da produção de pequenas histórias em um site8: Pols et al. (2018) sugerem que um rumo complementar aos trabalhos sobre demência seja pensar junto com as pessoas que conhecemos em campo e menos sobre elas e sobre o que viria a ser a demência, ou as demências. As autoras recuperam o termo cuidado para essa empreitada. Mol, Moser e Pols (2010) sugerem que o cuidado deve ser seguido em suas muitas feituras, incluindo toda a articulação necessária para que ele se realize e investigando quais são as eficácias que surgem dessas articulações. Não faria sentido, nesse modo de compor as coisas, separar o cuidado do uso de medicamentos, acoplamentos com tecnologias, organização dos espaços, feitura de alimentos e por aí vai. Ademais, não se pretende que o cuidado seja compreendido como algo feito de uma parte para outra, mas sim como uma relação de cooperação complexa e de elementos interconectados em certas relações, que podem ser atravessados por conflitos e faltas de cooperação, especialmente porque existe um povoamento enorme de elementos actantes sendo articulados. Sendo o cuidado essencialmente composto de relações e cooperações, abordá-lo não seria simplesmente adotar o ponto de vista daquelas que são chamadas de cuidadoras e os seus relatos, mas acompanhar as feituras de relações e interações diversas que forjam vida, morte e a lida com adoecimentos. Essa é uma reflexão bastante conhecida dos estudos de deficiência (Winance 2010; Fonseca & Fietz 2018), e que, contemporaneamente, tem ganhado mais espaço dentro do universo da demência9, retomando algumas de suas discussões clássicas: como o debate sobre personhood (Jenkins 2014; Halewood 2016). Passo, então, a dar espaço para um excerto dos meus dias de campo, isso para tentar me comunicar e retomar a preocupação com a convivência e com a criação de relações significativas a partir de muitos cuidados. Quando cheguei pela primeira vez na casa de D. 9 Para acessar a iniciativa, consulte: http://somatosphere.net/thinking-with-dementia Fazendo corpos e parentes: como grafar cotidianos? “Forcei a mão” em algumas oportunidades, tentando melhorar conceitualmente o que vinha tentando dizer: aproximei-me de estudos de ciência e tecnologia (Latour 2005; Mol 2002; Ingold 2010; Gane & Haraway 2010), de sua relação com mais recentes estudos de care (Mol, Moser e Pols 2010) e da demência (Lock 2013; Leibing 2018; Feriani 2017; Vianna 2013). O que, nos circuitos que frequentei no doutorado, parecia me aproximar mais de alguns critérios de qualidade da reflexão conceitual. Sentia que precisava melhorar meu arcabouço e palavras para falar do que me interpelava em campo. Foi um bom aprendizado, importante, mas ao orientar meu esforço para destrinchar e focar no que os conceitos ou as chaves analíticas podem fazer, o que lhes Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? 146 relações intensas com ele, mas é parte do Estado do Goiás. No telefone, a filha me disse que a D. Terezinha10, quando recebe visitas, ou conhece pessoas estranhas e novas para sua convivência, tem algo que elas chamam de ‘surto de sanidade’: ela lembra das coisas, conversa sem esquecer ou mostrar os esquecimentos; o que normalmente leva as pessoas a pensarem que as duas são mentirosas ou exageradas dizendo que a mãe tem Alzheimer. Por esse motivo, achou melhor me deixar previamente avisada. De fato, D. Terezinha me contou muitas histórias sobre as filhas, a casa no interior do Maranhão, dos bichos de lá e da doença dela: “eu tenho Alzheimer, é uma coisa que tá dentro de mim (fechou a mão e passou-a na frente do rosto, da garganta e do peito), é ruim, minha filha, mas eu quero muito viver, então é desse jeito que vai ser. Com os poder de Jesus!”. A filha mais velha, Marineide, contou que, no começo, foi difícil aceitar que a mãe tinha essa doença. D. Terezinha era a ‘costela da casa’, contaram, era ela quem agregava todo mundo, cuidava, dava conselhos e mantinha os laços familiares bem cuidados. Agora, já depois de dez anos convivendo com os vai e vens dos dias com a mãe, aprenderam a lidar melhor com tudo, recompor as relações. Continua difícil, disseram, mas no começo foi pior. Contou ainda que, se eu ficasse indo assim toda semana, logo a D. Terezinha ia se acostumar comigo, se soltar mais, falar dos menininhos que ela via pela casa e mexer em tudo, mudar de lugar, fazer as arrumações dela na minha frente. Todos entenderam de pronto minha pesquisa, achei que ia ser mais difícil explicar e conseguir autorização para ficar indo para dentro de casa. Disse que eu queria ver como era no dia a dia, porque vi os médicos passando remédios e vi os grupos de cuidadoras, mas não sabia bem como “as coisas são feitas todo dia, né?” – completou o neto, André. Era isso! As duas filhas com quem convivi mais – Marineide e Marilene – passaram a me incluir no que elas acreditavam que eu precisava saber e também nas coisas em que eu poderia ajudar (ou, ao menos, não atrapalhar): como dando caronas, fazendo companhia para a D. 10 Utilizo nomes fictícios, conforme combinado com meus interlocutores. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Terezinha9 – uma senhora maranhense de 80 anos, que veio para a Cidade Ocidental/GO porque quatro de suas cinco filhas mulheres estavam morando ali e acharam melhor trazê- la, junto do marido, para o cuidado ser mais dividido –, estavam ela, duas filhas, um neto, o marido e os dois cachorros preparados para me receber. Eu liguei antes, me expliquei um pouco e disse que gostaria de encontrá-los pessoalmente. A Cidade Ocidental faz parte de uma região conhecida como “Entorno”, isso porque ela circunda o Distrito Federal, tem Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 147 Também passei muitos dias em casa, na rotina dos dias sem eventos (e nos dias com eventos, como os desmaios). Nesse passar dos dias, aprendi o que elas faziam para dar os remédios, testar se eram bons, compreender as vontades de D. Terezinha. D. Terezinha me ensinou também o que a incomodava nessas trocas, o que achava de ir aos médicos, quem eram os meninos que andavam pela casa, em qual casa ela morava e o medo que tinha de passar mal, morrer e deixar seus filhos pequenos todos por criar. Errei muito, fiz muitas bobagens na convivência com elas e com D. Terezinha, e foi a partir desses erros, das conversas com as filhas e a cuidadora da casa, que fui me aproximando mais de adquirir um tipo (ainda que, no meu caso, muito precário) de experiência. Uma experiência para conviver, conviver de modo mais leve e atento com um mundo cheio de altos e baixos, troca de remédios, seu manejo e testagem, brigas por uma divisão maior das tarefas, lutos e lidas afetivas. Um desses erros foi crucial para minha convivência com D. Terezinha melhorar e para que eu conseguisse ouvir de forma mais atenta algumas dicas sobre a melhor convivência que as filhas e a cuidadora estavam tentando me fazer entender. Quando precisávamos ir até a consulta com o geriatra, era uma maratona! Isso porque tanto D. Terezinha como o seu marido consultavam no mesmo dia. Era preciso acordar de madrugada, tomar café, banho, vestir, lidar com o nervoso de ir ao médico, chegar cedo, pegar fila, esperar, lembrar de muitas coisas que aconteceram nos meses que antecederam as consultas, preparar os papéis e lidar com o lugar. Por isso as duas irmãs precisavam ir juntas, e, se tivesse uma terceira pessoa, era ainda melhor. Eu ia e ficava com D. Terezinha enquanto a consulta do seu marido acontecia, para que ambas as filhas pudessem participar da consulta. No primeiro dia em que fiquei sozinha com ela, foi complicado. Marineide me perguntou: “você dá conta?” Disse que sim, confiante! Nessa época ela já me conhecia, sabia meu nome eventualmente, mas sempre sabia que eu era próxima da casa e parecia gostar de mim. Mas ao ficar pela primeira vez sozinha comigo, estranhou: perguntava o tempo todo pelas meninas, me dizia que queria ir embora porque precisava arrumar o almoço delas e do marido. Fazendo corpos e parentes: como grafar cotidianos? Terezinha, indo até os médicos e farmácias, ajudando a lembrar de todas as receitas, pesquisando na internet datas de provas de concurso público para conseguir um emprego fixo, coisas assim. Elas me levaram no médico geriatra de Brasília, no médico do posto de saúde para trocar as receitas de uma Secretaria de Saúde para outra e assim conseguir os medicamentos pela Farmácia da prefeitura; foram comigo na cidade vizinha para pagar as contas – que ali o banco tinha ido embora por conta de muitos assaltos; na emergência por causa de picos de pressão e depois por desmaios constantes. Não só me mostraram o que precisava ser feito, mas onde ir, os circuitos e articulações de lugares, além de porque e o que tinha relação fundamental com os ajustes dos dias e humores. Carolina Perini de Almeida e Cíntia Engel Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? Fazendo corpos e parentes: como grafar cotidianos? 148 uso do espaço que era um hospital e com o uso que ela queria fazer dele era um problema meu (e dos outros), não dela. Deixei ela andar, andei com ela conversando solto. O clima foi melhorando entre a gente, prestei atenção nos detalhes da parede, no labirinto que era para achar qualquer saída, no jeito que as outras pessoas olhavam para a D. Terezinha. Ela me levou a ficar atenta à uma série de detalhes e à minha inexperiência. Minha agonia não passou completamente nesse dia e me senti bastante frustrada com ele por semanas. Mas no próximo dia em que passamos por uma experiência parecida, foi bem melhor, tínhamos acertado alguns ponteiros e confianças mútuas. uso do espaço que era um hospital e com o uso que ela queria fazer dele era um problema meu (e dos outros), não dela. Deixei ela andar, andei com ela conversando solto. O clima foi melhorando entre a gente, prestei atenção nos detalhes da parede, no labirinto que era para achar qualquer saída, no jeito que as outras pessoas olhavam para a D. Terezinha. Ela me levou a ficar atenta à uma série de detalhes e à minha inexperiência. Minha agonia não passou completamente nesse dia e me senti bastante frustrada com ele por semanas. Mas no próximo dia em que passamos por uma experiência parecida, foi bem melhor, tínhamos acertado alguns ponteiros e confianças mútuas. Foi aí que entendi uma dica que a Marilene tinha me dado sobre conviver e cuidar: “ela só faz o que ela quer, não adianta mandar. E ela malina! Tem que ficar de olho para ela não arrancar a torneira da pia, tirar os espelhos das tomadas elétricas, mas tem que deixar ela à vontade”. Esse equilíbrio entre tomar cuidado para nada machucar D. Terezinha e, eventualmente, causar prejuízos para a casa em época de desemprego e, ainda, deixar ela à vontade, era um aprendizado fino, que as filhas foram desenvolvendo com o tempo, na interação com D. Terezinha, a partir do que a deixava mais satisfeita com a convivência e com o que a deixava mais incomodada. Nem sempre essa técnica dava certo e precisava constantemente de ajustes – processo comum às lógicas de cuidado e chamado por Mol, Moser e Pols (2010: 14) de tinkering. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Passou a abrir as portas do hospital, algumas dessas levavam a salas de consulta. Procurava pela cozinha, pela saída, ou por outro lugar. Eu, meio sem saber o que fazer e assustada com uma possível retaliação dos médicos ou funcionários, falei para a D. Terezinha que estávamos em um hospital, que a gente não poderia abrir as portas, porque os médicos iriam se incomodar. Segui um pouco a cartilha dos grupos de cuidado: “olha no olho, explica, se não der certo, engana, vai distraindo”. Não deu certo. Ela, irritada, me disse: “pois se eles brigarem comigo, eu brigo com eles, por que não posso abrir uma porta?” Entendi ali que a minha agonia com o Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel alguma demência, aparecem aos olhos sem que sejam lidas e entendidas como culpa da demência ou da tarefa de cuidado, simplesmente. Cuidado, inclusive, parece ser parte de um modo de conviver amplo e cotidiano, e não um conjunto de atividades, ou mesmo uma perspectiva unívoca. Nesse sentido, parece-me relevante olhar para esses acoplamentos e relações necessárias às cooperações de cuidado, junto com suas condições de possibilidade e suas conexões parciais. Donna Haraway (1991a), na tentativa de pensar biopolítica, saúde e estabelecimento de fronteiras entre saúde e doença, afirma que corpos são produzidos em meio a determinados contextos, ou melhor, co-textos, multifacetados, mas hierarquicamente formulados como regimes de verdade e feitura de padrões. Pensar em compósitos, que por vezes Haraway (1991b) chama de cyborgues, traz a atenção para as conexões estabelecidas e o nascimento de entidades actantes. A singularidade dos compósitos está nas articulações parciais estabelecidas dentro de historicidades localizadas. Relações interafetadas por práticas cooperativas de cuidado fazem gerar corpos, mundos e pessoas. As estratégias e experiências de partilha de certos processos de sofrimento, adoecimento, cuidado e feitura de vida nos levam a acompanhar as coisas sendo feitas (Ingold 2011). Minha aposta é constituir uma estratégia ao individuar que não seja adotar o ponto de vista de cuidadoras, ou de pessoas com demência, ou de profissionais de saúde, colocando em foco, no lugar disso, na experiência e narrativa das práticas e relações que formam acoplamentos, atentando, assim, para as condições de fazimento deles, parcialidades envolvidas e limitações das cooperações – várias delas, historicamente e localmente constituídas. Mais para o meio do percurso da minha convivência na casa de D. Terezinha, estávamos tomando o lanche da tarde, ela, com muita dificuldade de engolir a comida – cuscuz com leite – começou a tirar pedaços da boca e brincar com eles no prato. Marilene, tentando que a mãe parasse com aquilo, disse: “Ô, mãe, olha a Cíntia aqui, vai ficar fazendo essas sujeiras na frente da visita?”; “Visita? Nah, essa aí vive aqui, tá aqui já tem é tempo”. Rimos todas, Marilene comentou: “é, não dá mais pra usar essa”. Lembrei daquele primeiro dia e do ‘surto de sanidade’ com pessoas estranhas. Fazendo corpos e parentes: como grafar cotidianos? Isso poderia se dar por conta de razões das mais variadas: desde um remédio novo que bagunçou as dinâmicas, até um lugar estranho que exigia de todo mundo posturas não conhecidas; ou mesmo por razão nenhuma, ou que não se sabia. Ou até por falta de paciência e imposições diversas de vontades conflitantes. Isso tudo exigia versatilidade de habilidades e de conhecimentos que iam sendo agregados e desafiados constantemente. Também não era possível simplesmente falar como uma nova pessoa envolvida com o cuidado deveria fazer, elas orientavam, mas a afinação vinha com a convivência. No fim, me pareceu que o equilíbrio vem a ser mais uma tentativa constante de compreensão mútua e ajustes de vontades do que um modo específico de agir. Ou, como Moser (2010) observa em seu campo sobre o cuidado da demência em uma instituição da Holanda: trata-se de um conjunto de técnicas para desenvolver algo que foi chamado, naquele contexto, de co-presença. E, nesse sentido, me convenci de novo que, nesses circuitos da demência, a partir de uma série de engajamentos práticos, são as relações e seus manejos que ganham centralidade no cotidiano. Manejo, inclusive, das interações entre diversas substâncias, médicos, serviços de saúde e espaços da cidade. A partir delas e das dificuldades e não cooperações explícitas ou implícitas que dilemas e silenciamentos das pessoas engajadas em circuitos da demência, sejam cuidadoras ou pessoas com Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 149 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Fazendo corpos e parentes: como grafar cotidianos? Fazendo corpos e parentes: como grafar cotidianos? 150 o que é manejar os dias, os erros, os aprendizados ao longo do tempo. Em uma bonita autoetnografia sobre o cuidado de sua mãe que tem Alzheimer e mora nos EUA, Taylor (2010) reflete sobre a insistência da pergunta que as pessoas sempre lhe fazem: “ela reconhece você?” Para a autora, essa pergunta se articula com uma série de discursos e noções sobre o Alzheimer que circulam e informam como os esquecimentos vão afetar a capacidade da pessoa em reconhecer ou não pessoas próximas, especialmente da família, a partir da evocação de seus nomes e de lembranças específicas do passado. Tal foco no que a demência faz com a pessoa e no que é ou deixa de ser o Alzheimer, incomoda Taylor, e ela nos diz que gostaria de falar sobre a vida com demência e os desafios de mantê-la a partir de outras questões, gostaria que lhe perguntassem algo relacionado ao cuidado, que lhe perguntassem como ela faz para manejar tudo e que também tentassem se comunicar com sua mãe por quem ela é, não pelo luto sobre o que ela deixou de nominar ou lembrar. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Naquele dia me senti alegre por poder participar da brincadeira com o cuscuz: ela não precisava fingir que se importava com certas coisas e, assim, eu aprendi bem mais sobre as coisas com as quais eu devia me importar menos para conviver melhor. Para falar desse processo de aprendizagem para conviver melhor, acho que é importante fazer aparecer na narrativa o ócio, os tempos, os desafios em prática, Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel 151 Não é recriando coisas extraordinárias que vamos nos engajar com a experiência sensível que partilhamos. Como fazer? Talvez o como fazer esteja muito conectado com outra questão: para que fazer? Para quê insistir na densidade, repetição e dimensão das relações? Responderíamos agora que é para deixar elas transcenderem de nós e para, assim, fazer uma boa etnografia, grafar cotidianos e, quiçá, ajudar na abrangência das possibilidades de ser deles. Talvez um caminho importante seja fazer o texto conviver, cuidar, abrir espaço. Apostando que forma é conteúdo e que, se estamos falando de relações, convivência e partilha, um jeito possível de fazer é construir um texto que cuida das relações. Que não se fecha, que não disputa um espaço, um campo intelectual, que discorda de si mesmo, que levanta questões sem destruir possibilidades contrárias de levantá-las. Que tenta não se isolar, que tenta estabelecer relações de entendimento e solidariedade com mundos, pessoas, corpos diversos. Erra e traz o erro para pensar. Um constante rascunho, quem sabe? Se precisamos abrir nossos corpos para partilhar de relações sensíveis que envolvem conviver bem e compreender para além do que tomamos como normal, ou igual, talvez um dos caminhos seja abrir nossos textos e modos de argumentar para delicadezas e convivências. Quem sabe localizamos em nossos diálogos a potência de criar e partilhar sensibilidades como um ponto de partida, no sentido de aprimorar nossa capacidade mesmo de escuta, nosso silêncio, nosso engajamento de relação. Um texto-povoamento! Podemos pensar a etnografia como extensão de mundos, modos de existência e pensamentos. Para isso, entendemos que talvez seja preciso se desafiar a fazer com a linguagem uma descrição que não feche, uma grafia não resolutiva. Ou seja: produzir uma descrição em profundidade que não encerre o assunto e proponha um caminho melhor, pelo contrário, que abra espaços de contínuo refazimento. Ao pensar nas modalidades de participação com nossos campos e constituição da escrita, podemos voltar a falar sobre plantas, e dessa vez gostaríamos de emprestar algo da reflexão botânica-filosófica de Emanuele Coccia, especialmente quando o autor toma como princípio que toda forma de vida pressupõe a existência de vida no mundo para construir uma filosofia feita pelas plantas (uma metafisica da mistura) – “Viver é essencialmente viver da vida de outrem: viver na e através da vida que outros souberam construir ou inventar” (2018: 14). Afinal, como recriar com a linguagem – o que criar junto com ela? Ambas convivemos com certos fazimentos de coisas muito básicas na relação com as pessoas, lugares e substâncias e nos pareceu que esses fazimentos, por eles mesmos, eram centrais para os nossos interlocutores e para o que seria importante escrever com eles e para as pessoas interessadas neles. Mas continuamos em dúvida se o que havíamos escrito conseguia produzir e se engajava de forma justa com esses efeitos. Não é que discordamos do que fizemos antes, a partir de limitações que julgamos, agora, superar; mas é porque a escrita parece demandar uma continuidade. Tentamos, até aqui, fazer dialogar, afinar e adequar nosso referencial para reescrever, escrever de novo, ou até repensar a escrita. Toda vez que tentamos descobrimos novas coisas, elementos que nos fazem desconfiar das palavras com as quais escolhemos dialogar. Talvez porque esse momento de parada da reflexão, de afinamento do que foi dito, de adequação final ao que faz sentido dizer a partir dos critérios atuais de qualidade e conceituação teórica nos nossos circuitos de produção não seja central para o que nos engaja com nossos campos. Eles nos dizem mais de um ajuste contínuo, sem parada, sem receita final e com certos engajamentos para com as relações e convivência. Nos parece que é difícil criar o que estamos querendo com a linguagem porque precisamos remontar o cotidiano. Dar o tom do tempo, do espaço, dos modos de fazer, das delicadezas mínimas deles, do jeito de montar possibilidades de convivência. E isso é difícil porque um texto é quase como um episódio, um evento, um caso, um encontro. Mas a gente precisa falar da vida diária, das horas correndo, do fluxo intenso de articulações. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Carolina Perini de Almeida e Cíntia Engel Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 11 Agradecemos a professora Iara Maria de Almeida Souza pela sugestão de diálogo com Stengers e a ecologia das práticas. Fazendo corpos e parentes: como grafar cotidianos? Fazendo corpos e parentes: como grafar cotidianos? 152 partilhar sensibilidades como modos de aprimorar nossa capacidade de engajamento de relação, leva a nos perguntarmos sobre os supostos dilemas trazidos pelos conceitos de estranhamento e alteridade. A oposição entre nós e eles pode não apenas nos levar a folclorizar e romantizar um eu ou um outro, mas também nos conduzir a apresentar propostas, respostas prontas e fechamentos. Não se trata, assim entendemos, de sistematizar conclusões, mas de se engajar em possibilidades não resolvidas, partilhar com nossos interlocutores de seus problemas, para os quais nem sempre há uma “solução”. partilhar sensibilidades como modos de aprimorar nossa capacidade de engajamento de relação, leva a nos perguntarmos sobre os supostos dilemas trazidos pelos conceitos de estranhamento e alteridade. A oposição entre nós e eles pode não apenas nos levar a folclorizar e romantizar um eu ou um outro, mas também nos conduzir a apresentar propostas, respostas prontas e fechamentos. Não se trata, assim entendemos, de sistematizar conclusões, mas de se engajar em possibilidades não resolvidas, partilhar com nossos interlocutores de seus problemas, para os quais nem sempre há uma “solução”. Por isso também resolvemos não estabelecer uma comparação entre um modo de constituir convivência e outro, mas sim iluminar tecnologias de convivência para trocarmos reflexões acerca da escrita. Seguindo Stengers (2005)11, queremos olhar para as práticas no sentido de animar cada vez mais os mundos constituídos com elas, fortalecendo elementos, actantes, seres e vidas se fazendo, não as atomizar, explicá-las ou usar delas por meio de uma comparação injusta para assegurarmo-nos de um ponto. Em conjunto, julgamos importante (ou fomos interpeladas a) falar sobre os modos pelos quais relações significativas das mais complexas se estabelecem, ou não, a partir de tentativas; dando espaço para o que, em nossos campos, nos aproxima dessas convivências e dilemas ao estabelecê-las. Para tanto, escolhemos a forma de escrita que nos pareceu dialogar com esse processo: textos-tentativas ou, simplesmente, rascunhos. Referências BARROS, Manoel de. 2001. Ensaios Fotográficos. Rio de Janeiro: Editora Record. BISPO DOS SANTOS, Antônio. 2015. Colonização, Quilombos: modos e significados. Brasília: INCTI/UnB/INCT/CNPq/MCTI. BOUQUET, Mary. 1996. “Family Trees and Their Affinities: The Visual Imperative of the Genealogical Diagram”. The Journal of the Royal Anthropological Institute, 2(1):43-66. CARSTEN, Janet. 2000. “Introduction: cultures of relatedness”. In: ______ (org.), Cultures of Relatedness: new approaches to the study of kinship. 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https://openalex.org/W3048177760	https://dro.deakin.edu.au/articles/journal_contribution/Editorial_Placebo_and_Nocebo_Effects_in_Psychiatry_and_Beyond/20689705/1/files/36904141.pdf	English	null	Editorial: Placebo and Nocebo Effects in Psychiatry and Beyond	Frontiers in psychiatry	2,020	cc-by	4,992	Edited and reviewed by: Stefan Borgwardt, University of Basel, Switzerland Edited and reviewed by: Stefan Borgwardt, University of Basel, Switzerland Correspondence: Katja Weimer katja.weimer@uni-ulm.de Editorial on the Research Topic Placebo and Nocebo Effects in Psychiatry and Beyond Editorial: Placebo and Nocebo Effects in Psychiatry and Beyond Katja Weimer 1, Paul Enck 2, Seetal Dodd 3,4,5,6 and Luana Colloca 7,8,9 1 Department of Psychosomatic Medicine and Psychotherapy, Ulm University Medical Center, Ulm, Germany, 2 Department of Psychosomatic Medicine and Psychotherapy, University Hospital Tübingen, Tübingen, Germany, 3 The Institute for Mental and Physical Health and Clinical Translation, Deakin University, Geelong, VIC, Australia, 4 Centre for Youth Mental Health, University of Melbourne, Parkville, VIC, Australia, 5 Department of Psychiatry, University of Melbourne, Parkville, VIC, Australia, 6 University Hospital Geelong, Barwon Health, Geelong, VIC, Australia, 7 Department of Pain and Translational Symptom Science, School of Nursing, University of Maryland, Baltimore, MD, United States, 8 Departments of Anesthesiology and Psychiatry, School of Medicine, University of Maryland, Baltimore, MD, United States, 9 Center to Advance Chronic Pain Research, University of Maryland, Baltimore, MD, United States Keywords: placebo effect, nocebo effect, learning, expectancy, conditioning, psychotherapy, psychiatr Keywords: placebo effect, nocebo effect, learning, expectancy, conditioning, psychotherapy, psychiatry INTRODUCTION The placebo effect is part of every medical intervention and plays a crucial role in randomized placebo-controlled trials (RCTs). It is beneﬁcial to maximize the placebo effect when treating patients, but it should be minimized in RCTs to estimate the true drug effect (1). Studies have shown that the placebo effect is formed by learning mechanisms (2), and an expert consensus has suggested that the beneﬁcial effects of placebo can be harnessed for clinical use to improve patient outcomes (3). In contrast to the placebo effect, adverse events can occur and symptoms can get worse through a negative placebo effect, the so-called nocebo effect (4). Yet, to exploit placebo mechanisms in clinical practice a lot of questions remain unanswered. For this Research Topic Issue, we called for the latest research articles in the ﬁeld of placebo and nocebo research. The issue comprises 38 articles from “Hypothesis and Theory” to “Reviews” and to “Original Research” articles. Correspondence: Katja Weimer katja.weimer@uni-ulm.de Correspondence: Katja Weimer Specialty section: This article was submitted to Psychosomatic Medicine, a section of the journal Frontiers in Psychiatry Received: 18 July 2020 Accepted: 24 July 2020 Published: 07 August 2020 Citation: Weimer K, Enck P, Dodd S and Colloca L (2020) Editorial: Placebo and Nocebo Effects in Psychiatry and Beyond. Front. Psychiatry 11:801. doi: 10.3389/fpsyt.2020.00801 Specialty section: This article was submitted to Psychosomatic Medicine, a section of the journal Frontiers in Psychiatry Received: 18 July 2020 Accepted: 24 July 2020 Published: 07 August 2020 After giving an overview about the underlying mechanisms of the placebo effect, such as conditioning, expectations and inﬂuencing factors, Friesen summarizes ethical views regarding the use of the placebo effect. Until recently, it has been assumed that placebos take only effect when patients are deceived, but she encourages considering placebos as a “source of agency”, without deception and in agreement with patients’ autonomy. Babel complements the current view about classical conditioning in the placebo effect. In fact, many studies use a combination of classical conditioning and verbal suggestions to induce placebo and nocebo effects. Due to recent studies using hidden and subliminal conditioning procedures, Babel argues that classical conditioning is a EDITORIAL published: 07 August 2020 doi: 10.3389/fpsyt.2020.00801 EDITORIAL published: 07 August 2020 doi: 10.3389/fpsyt.2020.00801 EDITORIAL published: 07 August 2020 doi: 10.3389/fpsyt.2020.00801 THE PLACEBO EFFECT IN PSYCHOTHERAPY Particularly in psychiatry, patients are not only treated with pharmacotherapy but often with different forms of psychotherapy. The role and mechanisms of the placebo effect in psychotherapy has been repeatedly discussed, and Enck and Zipfel point to the challenges of disentangling speciﬁc effects of the different psychotherapeutic approaches including unspeciﬁc and the placebo effect. This is even more challenging when considering that many psychotherapeutic approaches are equally effective and there is still a debate within psychotherapy research about the speciﬁc, common and unspeciﬁc factors (also known as the “Dodo bird verdict”). Enck and Zipfel encourage psychotherapy researchers as well as therapists to understand that the placebo effect exists and provide a framework that acknowledges context, common, and speciﬁc factors for further research. With her Mini Review, Blease attempts to provide greater clarity in the deﬁnition of the placebo effect in psychotherapy and gives insights into controversial views such as “psychotherapy is a placebo”. She argues that the problem could be solved when placebos and the placebo effect are clearly deﬁned the same way as they are deﬁned in clinical trials: as control interventions and the effect they induce. In the ﬁrst instance, it seems to be contradictory that Blease recommends using a clear deﬁnition of the placebo effect, whereas Jonas states that “the placebo response is a myth” and does not exist. According to his arguments it is contradictory that an inert treatment will produce a response and votes for a broader understanding of this response that should be called “meaning response” or “healing response”. However, these two views are compatible and in line with the deﬁnitions of “placebo effect” as the effect elicited by placebo mechanisms, and “placebo response” as all health changes after administration of an inert treatment, as stated by expert consensus of placebo researchers published in 2018 (3). To evaluate how and how often oncologists make use of empathy expressions by practitioners, van Vliet et al. assessed video-taped consultations between oncologists and patients with advanced breast cancer in an observational study. Overall, oncologists often provided information about expectancy and used several empathic behaviors such as understanding, respecting, supporting and exploring, whereas a lack of empathy was less often observed. Further studies should evaluate effects of empathic expressions on treatment outcomes and (nocebo) side effects. THE ROLE OF CONTEXT FACTORS IN PLACEBO AND NOCEBO EFFECTS In psychotherapy research, context factors such as the patient- provider interaction are considered a common factor, albeit they are considered to be part of the placebo response in other treatments. In their systematic review, Daniali and Flaten found that aspects of a positive patient-provider interaction such as higher conﬁdence in the provider, perceived higher competence and professionalism, and positive nonverbal behaviors were associated with lower pain reports and higher placebo effects in patients and participants. In contrast, negative nonverbal behaviors led to higher pain reports and nocebo THE PLACEBO EFFECT IN PSYCHOTHERAPY Not only physicians are aware of the effect of unspeciﬁc factors on treatments, patients are aware of them, too: In their large online survey among Italian patients with musculoskeletal pain, Rossettini et al. found that patients believe that contextual factors such as an empathetic alliance, and verbal and non-verbal communication are effective and work through mind-body connections. Furthermore, they have positive attitudes towards their use in clinical practice if they are not used in a deceptive way. One of the challenges in placebo research is to disentangle the placebo effect from other effects through elaborate study designs. To differentiate the placebo effect from the psychosocial context, Gruszka et al. as well as Curkovic et al. recommend outsourcing some parts of the psychosocial context via smartphone applications. Such an app could be used for standardized recruitment, randomization and the provision of treatment information to induce positive expectations. Furthermore, it could be used to assess expectations, symptom severity, or physiological data via smartphone sensors (e.g., heart rate) without personal interaction and in daily life. Additionally, Curkovic et al. suggest that studies should rigorously investigate and report aspects of research plans to the better investigate which aspects of an intervention at which dose is relieving symptoms, and this could also be achieved through an app. Citation: Weimer K, Enck P, Dodd S and Colloca L (2020) Editorial: Placebo and Nocebo Effects in Psychiatry and Beyond. Front. Psychiatry 11:801. doi: 10.3389/fpsyt.2020.00801 August 2020 \| Volume 11 \| Article 801 Frontiers in Psychiatry \| www.frontiersin.org Editorial: Placebo and Nocebo Effects in Psychiatry Weimer et al. effects. Howe et al. delve deeper in speciﬁc aspects of the patient-provider-interaction and differentiate between competence and warmth. They provide a framework for researchers and practitioners about how patients perceive competence and develop the feeling that the physician “gets it”, and how they perceive warmth when the physician “gets them”. However, non-speciﬁc effects of treatments comprise many aspects, and Gerger et al. translated and validated the ﬁrst German version of the Healing Encounters and Attitudes Lists (HEAL-D) and its short form (HEAL-D-SF). This set of questionnaires assesses patients’ views on the patient-provider interaction, the healthcare environment, treatment expectations, positive outlook, spirituality, and attitudes towards complementary and alternative medicine. It may help to turn non-speciﬁc into speciﬁc effects, and therefore may be usable for research purposes and clinical practice. distinct mechanism that works without conscious expectations. However, there are only a few studies limited to the area of pain and further studies are needed. THE PLACEBO EFFECT ON DEPRESSION, ANXIETY, PAIN, AND OTHER SYMPTOMS Irving Kirsch published several studies and meta-analyses about the placebo response and placebo effect in treatments with August 2020 \| Volume 11 \| Article 801 Frontiers in Psychiatry \| www.frontiersin.org 2 Editorial: Placebo and Nocebo Effects in Psychiatry Weimer et al. antidepressants and questioned whether the placebo response and the drug effect in RCTs are additive (5, 6). In his recent article, Kirsch summarizes the results of these and other meta- analyses clearly demonstrating that “most (if not all) of the beneﬁts of antidepressants in the treatment of depression and anxiety are due to the placebo response”. However, RCTs cannot answer the question how patients’ symptoms evolve without any treatment or how they should be treated instead. Kirsch reports several alternative treatments such as psychotherapy, physical exercise, omega-3 supplements, and yoga that has been shown to be as effective as antidepressants but with less side effects, and in some cases with better long-term effects than antidepressants. To further evaluate how expectancy could inﬂuence outcomes in antidepressant trials, Laferton et al. performed a re-analysis of a double-blind RCT in major depression comparing escitalopram, S-adenosyl-L-methionine (SAMe) and placebo. Results show that the patients’ perceived treatment assignment during the trial changed, was predicted by symptom improvements, and contributed more to treatment outcomes than actual treatment. Finally, there was no difference between groups. Furthermore, expectations of pain decrease were a mediator for placebo analgesia, but higher age and higher warmth-detection thresholds were associated with lesser placebo analgesia. g A large proportion of our knowledge about the placebo effect and its underlying mechanisms stems from experimental studies with pain, but there is little knowledge whether the same mechanisms apply to other symptoms. To elucidate this question, Wolters et al. reviewed the literature about placebo and nocebo effects in dyspnea, fatigue, nausea, and itch. They can conﬁrm that in general the same mechanisms as in pain are at work in these symptoms, such as the combination of verbal suggestions and conditioning, and that subjective symptoms are more prone to elicit a placebo effect than are physiological measures. However, there are also some differences as the inﬂuence of individual characteristics varies between symptoms. Evidence can be added by an experimental study by Meeuwis et al. who investigated placebo and nocebo effects through verbal suggestions on itch. THE PLACEBO EFFECT ON DEPRESSION, ANXIETY, PAIN, AND OTHER SYMPTOMS Participants received the respective information either in an open-label condition knowing that the applied tonic was a placebo (a pink-colored skin disinfectant), or in a closed-label condition in which they were deceptively told that the tonic was effective. Whereas suggestions did not affect itch reports during histamine iontophoresis, participants in both positive suggestion groups reported lower itch and lower skin temperature increase after the iontophoresis compared to the negative suggestion groups. Interestingly, their open-label suggestion was as effective as the deceptive information about the effectiveness of the placebo, and they found a symptom speciﬁc physiological reaction to itch. But patients do not only “feel better” through the placebo effect, several neuroimaging studies could demonstrate neurophysiological changes in the brain. Brown and Pecina underline these results and provide an overview of neuroimaging studies of the antidepressant placebo effect. They show that this effect is comparable to the placebo effect on pain. This ﬁnding implies common underlying mechanisms involving brain areas associated with cognitive control, the representation of expectations, and reward and emotional processes. Still, pain is the best investigated symptom in placebo research. Complementary to neuroimaging studies, Reicherts et al. present an electroencephalography (EEG) study combining the motivational priming hypothesis and the conditioning of placebo and nocebo effects. Participants who were told that unpleasant pictures decrease pain, indeed reported less pain, and consequently, somatosensory evoked potentials were decreased when they watched unpleasant pictures compared to neutral pictures. They conclude that the well-known modulation of pain by emotions is inﬂuenced by expectations. Another underreported areas are placebo and nocebo effects on cardiac symptoms and physiology. In an experimental study with patients with Takotsubo cardiomyopathy—a rare, reversible form of cardiomyopathy after stressful psychosocial life events— and heart-healthy controls, all participants received a saline infusion three times together with the information that it has no effect, a positive (placebo) or negative (nocebo) effect on cardiac functions, respectively. Olliges et al. report that before and during the nocebo condition subjective stress rating, heart rate, and systolic blood pressure increased, whereas the latter also increased after placebo information. However, there were no differences between patients and controls. The experimental pain study by Zhou et al. found interactional effects of different expectations, sex of participants and personal characteristics such as dispositional optimism and state anxiety on pain reports in a complex manner. THE PLACEBO EFFECT ON DEPRESSION, ANXIETY, PAIN, AND OTHER SYMPTOMS After a conditioning procedure with electrical pain, women in the low expectancy group reported decreased pain compared to the No or High expectancy groups, whereas men reported decreased pain in the High expectancy group in the test session. Whether optimism or state anxiety predicted placebo effects was dependent on the expectancy level, but independent of sex. To explore other predictors of placebo analgesia, Wang et al. used latent class analyses (LCA) to identify learning patterns during a conditioning procedure in an experimental pain study. LCA revealed that greater or increased differences between high and low pain ratings in combination with red and green light signaling stimuli during conditioning were associated with greater placebo analgesia in the subsequent testing phase. Frontiers in Psychiatry \| www.frontiersin.org AREAS RELATED TO MENTAL DISORDERS The placebo effect could not only be helpful to directly decrease symptoms of a disorder, but also when it is used to inﬂuence functions related to mental disorders such as cognitive functioning or appetite regulation. Participants in the study of Fuhr and Werle were randomized to listen to a mental training or philosophy lecture both audio-taped for 20 min, and half of the participants of each group were told that they listen to an effective or control tape. All participants improved their cognitive performance as measured with a d2-test, but those August 2020 \| Volume 11 \| Article 801 Frontiers in Psychiatry \| www.frontiersin.org 3 Editorial: Placebo and Nocebo Effects in Psychiatry Weimer et al. participants who experienced a greater improvement rated the received treatment as effective irrespective of group assignment. This, at least, shows that healthy persons can rate their cognitive performance without being inﬂuenced by (bogus) verbal suggestions, and thus, could be indicative of a healthy function. Winkler and Hermann chose a different study design: two groups received a nasal spray along with the suggestion of a cognitive improvement (placebo) or impairment (nocebo) effect, and one group served as a control (without nasal spray or suggestions). Similar to the study by Fuhr and Werle, verbal suggestions did not affect actual cognitive performance. However, participants in the placebo group rated their cognitive improvement better and felt less tired compared to the nocebo group. The authors conclude that these subjective effects may explain why so-called neuroenhancers are still popular among college students. For their study about placebo and nocebo effects of a sham transcranial magnetic stimulation (sTMS), Höﬂer et al. employed women who turned out to be placebo or nocebo responders, respectively, in previous studies. According to their responsiveness they received the information that the sTMS will increase (placebo) or decrease (nocebo) their left-sided visual attention in an eye-tracking experiment. As in the above-mentioned studies, the placebo instruction did not affect actual visual attention, but subjectively improved attention. In contrast, nocebo responders showed the opposite to the expected reaction. the “bad brother” of the placebo effect have shown that known placebo mechanisms such as conditioning, expectations, and social learning can also have negative outcomes. Faasse et al. deﬁne “nocebo effects as unpleasant or adverse outcomes triggered by the treatment context”. AREAS RELATED TO MENTAL DISORDERS The authors differentiate between primary nocebo effects and nocebo side effects, and the misattribution of regular symptoms to an (inert) treatment. Furthermore, they describe how experimental studies should be designed to investigate the nocebo effect appropriately. While Faasse et al. focus on studies with treatments involving drugs or medical devices, Locher et al. emphasize that the nocebo effect could also occur in psychotherapy. They provide two examples where a nocebo or nocebo-related effect could evolve: In patients with chronic primary pain or other symptoms without a clear physiological etiology, and in relation to trauma debrieﬁng to prevent post- traumatic stress disorders (PTSD). To prevent nocebo (side) effects it would be helpful if nocebo responders could be detected in advance. In a re-analysis of experimental endotoxemia studies, Benson and Elsenbruch investigated predictors of the nocebo effect. Nocebo responders, deﬁned as participants in the placebo arms of RCTs who believed they were allocated to the verum arm, reported signiﬁcantly more physical symptoms but did not differ from non-responders in psychological or physical parameters. Within nocebo responders, physical symptoms correlated with greater state anxiety, negative mood, catastrophizing and neuroticism. Their study demonstrates that it is difﬁcult to predict who will be a nocebo responder, but that perceiving nocebo side effects could affect perceived treatment allocation—another reason why nocebo side effects should be reduced. Webster and Rubin provide a systematic review of RCTs investigating brief psychological interventions to reduce or avoid nocebo side effects in medical treatments. In the 27 studies found, omitting side effect information was most successful to reduce nocebo side effects, whereas other communication strategies such as priming, distraction, and altering the branding of drugs showed mixed effects. De-emphasizing of side effects was not effective. Finally, they discuss that it could be challenging to balance the reduction of nocebo side effects with informed consent. Pan et al. investigated another strategy to reduce nocebo side effects in an experimental study: Participants with weekly headaches received a placebo pill and were randomized to read a bogus medication leaﬂet only or to read additionally an explanation about the nocebo effect. Two minutes after pill intake, the group that had received the explanation about nocebo reported less nocebo symptoms than the other group. This effect was moderated by baseline symptoms, perceived sensitivity to medicine, and expectations. Furthermore, most participants evaluated the nocebo information as helpful. AREAS RELATED TO MENTAL DISORDERS In another eye-tracking study from the same work group, Potthoff et al. did not directly target visual attention, but a placebo pill that claimed to reduce appetite was given to healthy, mostly normal-weight women, and their reactivity to food cues was registered. Participants reported decreased appetite which was related to decreased visual attention for food, e.g., ﬁxation and dwell time on high and low-caloric food images compared to non-food pictures. The experimental study by Hoffmann et al. conﬁrms these results: healthy normal-weight participants reported decreased appetite after ingesting a placebo pill that should increase satiety compared to a control group. They additionally assessed an objective marker of hunger and found that the opposite information—that a placebo pill claimed to enhance appetite - increased plasma ghrelin levels but did not affect appetite itself. In a third study of placebo effects on food consumption, Panayotov showed that the information about a calorie-reduced diet decreased body mass, body mass index (BMI), and fat tissue in overweight and obese participants of a weight loss program. Although participants did not strictly adhere to their diet programs and the sample size was small, this preliminary study shows that weight regulation could be directly addressed through manipulating expectations of patients. NOCEBO EFFECTS Most of the articles in this Research Topic deal with the placebo effect and response after typical applications of treatments such In conjunction with studies about the placebo effect, the nocebo effect has already been mentioned above. Previous studies about August 2020 \| Volume 11 \| Article 801 Frontiers in Psychiatry \| www.frontiersin.org Editorial: Placebo and Nocebo Effects in Psychiatry Weimer et al. and systolic blood pressure, and decreased pain perceptions in relaxed participants in a pain paradigm with rectal distensions. Such experimental studies show promising ways to harness the placebo effect for patients’ treatments in ethical and legal ways. Beneﬁts for patients are clear as they experience symptom as well as side effect reductions, but the placebo effect is rarely used systematically. Showing that harnessing the placebo effect is not only effective but also cost-efﬁcient could improve its visibility and acceptability. A systematic review by Hamberger et al. investigated if placebo interventions are also cost-efﬁcient but showed that there is a lack of health economic evaluations and encourage placebo researchers to report costs of placebo interventions. as pills or ointments. However, disentangling the true treatment effect from the placebo response and placebo effect is also challenging in other forms of treatments, e.g., psychotherapy (see above). Chae et al. discuss in particular two aspects that could lead to a high placebo response in acupuncture: the fact that even sham acupuncture may elicit physiological responses, and the difﬁculty of effective blinding of provider and patient. They suggest more appropriate alternative control strategies in acupuncture treatment. There is less research about the placebo effect in children (7) and this Research Topic comprises only two further articles about it: one involved an experimental design with healthy children, and one discusses the inﬂuence of the so-called placebo-by-proxy effect. The placebo-by-proxy effect was introduced by Grelotti and Kaptchuk (8) in 2011 and describes the effect where people in the social environment of a patient (parents, siblings, relatives, peers) feel better when the patient receives an effective treatment. Czerniak et al. complement this concept with the corresponding “nocebo- by-proxy” effect and discusses the impact of these two concepts particularly on children’s symptoms and treatments. Their review of the available literature opens an important research ﬁeld. The inﬂuence of parents or other proxies on placebo and nocebo responses has rarely been studied. The experimental study by Watolla et al. CONCLUSION: MORE QUESTIONS THAN ANSWERS? In summary, the multifaceted articles in this Research Topic issue show that placebo and nocebo effects are complex phenomena. There is still a debate about the role of placebo and nocebo effects in psychotherapy research and their relation to common and context factors. In contrast, context factors such as the patient-provider interaction have already been acknowledged as part of the placebo effect in other treatments. Research about the placebo effect on depression, anxiety, and pain reveals a high placebo effect showing symptom improvement and neurophysiological changes in the brain. However, there is less research about other symptoms such as itch or heart-related diseases, among others. Recent studies aim to harness the placebo effect to improve functions that are related to mental disorders, such as cognitive functioning or appetite regulation, and may be an interesting research area for further studies. There are several other underreported research ﬁelds such as: appropriate control conditions for treatments other than pills, placebo and nocebo effects in children, and the role of genetics and heritability. An increasing amount of articles investigate the nocebo effect and nocebo related adverse effects, their mechanisms and strategies to avoid or reduce them. Finally, all research aims to improve treatments of patients and recent studies show promising results by employing techniques that enhance the placebo effect or reduce the nocebo effect. However, more research is needed to transfer knowledge about placebo and nocebo effects into clinical practice to beneﬁt patients in an ethical and broadly accepted manner (10). NOCEBO EFFECTS investigated the effect of a suggested ginkgo patch on cognitive performance in children and one parent. While they found only a poor overall placebo effect, neither the cognitive performance nor the expectations of children and their parents were interrelated. This may imply that shared information and heritability have a low impact on the placebo effect. Although it should be taken into account that the participants were all healthy and without need for cognitive improvement. This ﬁnding is supported by the ﬁrst study involving a classical twin design: Weimer et al. employed healthy mono- and dizygotic twin pairs in an experimental study with a heat pain paradigm. After conditioning the effectiveness of an ointment, twins reported a signiﬁcant placebo analgesic effect in the test condition. This effect was mainly related to the personal learning experience during the conditioning procedure, but not to the effect of their co-twin, suggesting that heritability and shared environment play a minor role. In contrast, ﬁrst studies show a genetic component in the placebo effect, but these results are still inconclusive (9) and twin studies should be combined with genetic analyses to further elucidate this area. AUTHOR CONTRIBUTIONS Elsenbruchetal. tie in with ﬁrst evidence that psychophysiological responses, such as an increase of parasympathetic activation, to placebo interventions could play a role in the establishment of a placebo effect. In their study, a brief progressive muscle relaxation exercise but not a control task reduced heart rate KW wrote the ﬁrst draft of the manuscript. PE, SD, and LC provided critical revision of the manuscript and important intellectual contributions. All authors contributed to the article and approved the submitted version. August 2020 \| Volume 11 \| Article 801 Frontiers in Psychiatry \| www.frontiersin.org Editorial: Placebo and Nocebo Effects in Psychiatry Weimer et al. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 4. Dodd S, Walker AJ, Brnabic AJM, Hong N, Burns A, Berk M. Incidence and characteristics of the nocebo response from meta-analyses of the placebo arms of clinical trials of olanzapine for bipolar disorder. Bipolar Disord (2019) 21:142–50. doi: 10.1111/bdi.12662 Copyright © 2020 Weimer, Enck, Dodd and Colloca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Weimer, Enck, Dodd and Colloca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Weimer, Enck, Dodd and Colloca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 5. Kirsch I. Are drug and placebo effects in depression additive? Biol Psychiatry (2000) 47:733–5. doi: 10.1016/s0006-3223(00)00832-5 5. Kirsch I. Are drug and placebo effects in depression additive? Biol Psychiatry (2000) 47:733–5. doi: 10.1016/s0006-3223(00)00832-5 6. Kirsch I, Sapirstein G. Listening to Prozac but hearing placebo: a meta- analysis of antidepressant medication. Prev Treat (1998 1(2):Article 2a. doi: 10.1037/1522-3736.1.1.12a 6. Kirsch I, Sapirstein G. Listening to Prozac but hearing placebo: a meta- analysis of antidepressant medication. Frontiers in Psychiatry \| www.frontiersin.org REFERENCES 7. Weimer K, Gulewitsch MD, Schlarb AA, Schwille-Kiuntke J, Klosterhalfen S, Enck P. Placebo effects in children: a review. Pediatr Res (2013) 74:96–102. doi: 10.1038/pr.2013.66 1. Enck P, Bingel U, Schedlowski M, Rief W. The placebo response in medicine: minimize, maximize or personalize? Nat Rev Drug Discovery (2013) 12:191– 204. doi: 10.1038/nrd3923 8. Grelotti DJ, Kaptchuk TJ. Placebo by proxy. BMJ (2011) 343:d4345. doi: 10.1136/bmj.d4345 9. Colloca L, Wang Y, Martinez PE, Chang YP, Ryan KA, Hodgkinson C, et al. OPRM1 rs1799971, COMT rs4680, and FAAH rs324420 genes interact with placebo procedures to induce hypoalgesia. Pain (2019) 160:1824–34. doi: 10.1097/j.pain.0000000000001578 2. Colloca L, Miller FG. How placebo responses are formed: a learning perspective. Philos Trans R Soc Lond B Biol Sci (2011) 366:1859–69. doi: 10.1098/rstb.2010.0398 3. Evers AWM, Colloca L, Blease C, Annoni M, Atlas LY, Benedetti F, et al. Implications of Placebo and Nocebo Effects for Clinical Practice: Expert Consensus. Psychother Psychosom (2018) 87:204–10. doi: 10.1159/ 000490354 10. Colloca L, Barsky AJ. Placebo and Nocebo Effects. N Engl J Med (2020) 382:554–61. doi: 10.1056/NEJMra1907805 August 2020 \| Volume 11 \| Article 801 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Prev Treat (1998 1(2):Article 2a. doi: 10.1037/1522-3736.1.1.12a August 2020 \| Volume 11 \| Article 801 Frontiers in Psychiatry \| www.frontiersin.org
https://openalex.org/W4313437646	https://japer.in/storage/files/article/92521c61-180f-4ae3-86c2-c195b3f4fe8c-jgdDjv3JyxX8rLC5/japer-vol-13-iss-1-62-65-8078.pdf	English	null	Innovative and motivational SDT-based approach to promote Iranian women&#39;s physical activity	Journal of advanced pharmacy education and research	2,023	cc-by	3,230	Introduction The new perspective on health does not define health only as the absence of illness or disability but also defines health as complete physical, mental, and social well-being [1]. Therefore, the effect of numerous factors such as background, lifestyle, social, economic conditions, and the like, which are referred to as "social determinants of health (SDH)", are determined on the health status of individuals. SDHs include all the conditions in which a person is born, lives, works, and strives, and these conditions affect his / her opportunity to enjoy a healthy and productive life [2]. Recent models of health promotion have chosen SDH as a key component of the economic, political, and social environment that is effective on lifestyle and behavior as ABSTRACT Despite proving the undeniable role and importance of physical activity in maximizing physical, mental, and social health and social health from childhood to old age, different societies are still suffering from inactivity, globesity, and subsequent health problems and despite the importance of women's health and maternal roles in achieving the family and community health, women are generally more sedentary and less activate than men, so that 45.3% of Iranian men and 61.9% of Iranian women do not have adequate physical activity. This is even though routine-based interventions in some countries about increasing physical activity in line with implementing the recommendations of the World Health Organization by 2030 are not effective. This mini-review intends to introduce barriers and drivers in addressing women's physical activity, suggest motivational and new methods and explain the psychological facilitators of behavior and pay attention to the importance of supportive environments and the SDT conceptual framework in improving self- regulation and promoting physical activity. Keywords: Women's health, Physical activity, Self determination theory, Lifestyle well as life quality [3]. Findings from a study on women's lifestyle and quality of life indicate that sedentary women experience higher levels of stress than active women and experience higher rates of depression and suicidal ideation. Active women also have a higher quality of life [4]. Any bodily movement produced by the skeletal muscles that require more energy than the resting state of the body is called physical activity (PA) [5]. PA has also been mentioned as the basis of energy balance and weight control [6]. Innovative and motivational SDT-based approach to promote Iranian women's physical activity Mohamad Ezati Asar1, Elahe Saleh1, Mohammadreza Ghaneapur1* 1 Department of Health Education & Health Promotion, School of Public Health, Semnan University of Medical Sciences, Semnan, Iran. Correspondence: Mohammadreza Ghaneapur, Department of Health Education & Health Promotion, School of Public Health, Semnan University of Medical Sciences, Semnan, Iran. mrghaneapur@semums.ac.ir A novel approaches According to some studies, 62% of participants cited "laziness" as the most important obstacle to participation in physical activity Iranian adults mentioned meeting new people (74%), happiness (71.8%), and meeting friends (67.73%) as the most common reasons for participating in physical activity and referred to social support from family and friends to be effective in physical activity [15]. In studies conducted for this purpose, "pleasure and interest" [16, 17], "fitness" [16], and "pleasure and happiness" [18] have been mentioned as important drivers of women's desire for physical activity. It is important to note that despite the large number of qualitative studies that have examined and explained the facilitators and barriers to physical activity among adults, limited quantitative studies have been conducted in this area [19]. Despite the high prevalence of sedentary lifestyle among women, in many cases, women have acknowledged and believed in the health benefits of increased physical activity, and research has shown that people with higher knowledge have a greater perception of PA benefits and self-efficacy and are more likely to engage in physical activity [15]. However, the fundamental question is why interventions to improve physical activity do not lead to continued behavior in the long run, and individuals are transferred to the position before the intervention or even lower than the previous position [21, 22]. Although more than four decades have passed since the design of motivational approaches and SDT theories which are based on internal and external motivation and autonomous behavior and individual competence in the psychological and clinical fields, they are not properly included in health promotion interventions. SDT categorizes a person's motivation levels as 1. Motivation 2. Internal regulation and 3. External regulation. Intrinsic motivation is when a person performs a behavior due to intrinsic pleasure and external motivation occurs when a person performs a behavior due to the consequent results [24]. SDT, as an Organismic Theory, assumes that individuals are inherently prone to growth and mental integration and as a result learning, mastery, and communication with others. However, these tendencies do not manifest themselves automatically and supportive conditions are needed to strengthen them. SDT specifically states that for healthy growth, we need support for basic psychological needs, including autonomy, competence, and relatedness [25]. Autonomy involves a sense of initiative and ownership in one's actions which are supported by interesting and valuable experiences. Autonomy is undermined by controlled external experiences, whether reward or punishment. Relationship between diseases and physical activity In an age when overweight, the global obesity epidemic (Globesity), and its subsequent health problems are increasing in the developing world [7], PA has promised to achieve a balanced body mass index (BMI) [8]. As a result, it can significantly reduce non-communicable diseases (NCDs) such as cardiovascular diseases, metabolic diseases, diabetes and cancer and so on which are caused by a sedentary lifestyle [9]. PA can also play a significant role in reducing 63% of morbidity and mortality from NCDs, which are referred to as avoidable deaths [10]. This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non Commercial- ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non Commercial- ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. 62 Ezati Asar et al.: Innovative and motivational SDT-based approach to promote Iranian women's physical activity Ezati Asar et al.: Innovative and motivational SDT-based approach to promote Iranian women's physical activity class and education, revealing the characteristics through which the fundamental distinction between cultures is made possible [21]. The results of studies carried out have focused on some cultural factors like cultural identity, cultural perspectives, gender norms associated with physical activity (PA), cultural perspectives on health associated with PA, family cultural expectations, lack of sports facilities appropriate to culture as cultural factors related to PA [22]. Hence, besides other elements, PA level can drastically be affected by the cultural values prevailing the society, so that the girls, women, the elderly, the disadvantaged groups, and those with disabilities and chronic diseases still have fewer opportunities for safe, cost- effective, and proper places to engage in PA in many countries [11]. Thus, removing conservative socio-cultural barriers before considering PA as a norm for women has to be on the agenda [23]. Prevalence of physical activity Despite the numerous evidence and benefits of performing PA, only one in four adults and three in four adolescents follow the WHO global recommendations for increasing physical activity. With the economic growth of societies, the trend of sedentary lifestyle is increasing [11] and based on evidence sedentary lifestyles in 50 years and older women are significantly more than men [12]. The findings of the study showed a high prevalence of sedentary lifestyle in the Iranian population (54.7%) so 45.3% of Iranian men and 61.9% of Iranian women do not have adequate physical activity [13]. The findings of some studies in Iran indicate that the physical activity of women aged 30-45 years is 214± 4524 steps per day [14], which is less than half of the WHO recommended values for physical activity [11]. A novel approaches Competence is about the feeling of dominance, the feeling that one can succeed and grow. The need for competence is best met in well-structured environments that provide optimal challenges, growth opportunities, and positive feedback. Relatedness means the sense of belonging and connection. This is facilitated by the transfer of respect and care. Disabling any of these three basic References 1. Charter O. Ottawa Charter for health promotion. First international conference on health promotion; 1986. pp.17-21. 1. Charter O. Ottawa Charter for health promotion. First international conference on health promotion; 1986. pp.17-21. Given the undeniable role of a sedentary lifestyle in the occurrence and exacerbation of many non-communicable diseases, the methods and strategies used have not yet achieved significant success. Although educating people has been considered as the key approach to increasing physical activity over long years given the role of "self-awareness" in people's health [23] and reasons like lack of awareness and limited investment in this area have been emphasized despite the global progress in increasing PA, this can not be ignored that the increase in PA among the women, on the one hand, needs a system-based approach and there is no single political solution for all countries [11], and training-based interventions in the development of sports behavior and continuous physical activity have not been very successful on the other hand [25]. Hence, given the many components that affect PA, using interventions associated with PA based on SDT can facilitate and cultivate the basic psychological needs (Autonomy, Competence, and Relatedness), not only in enhancing and promoting PA but also in creating preventive behavior and maintaining healthy behaviors, combating emerging diseases such as Covid-19 [30] and even in teaching and learning environments can be helpful and used [31]. The implementation guidelines of SDT-based interventions in changing the behaviors resulting in health and behavioral regulation like the maintenance and continuation of PA in various countries like Iran have been reviewed and localized and their effects have been significantly proven in recent years [14, 16, 32, 33]. 2. Labonté R, Schrecker T. Globalization and social determinants of health: the role of the global marketplace (part 2 of 3). Glob Health. 2007;3(6):1-17. 3. Krumeich A, Meershoek A. Health in global context; beyond the social determinants of health? Glob Health Action. 2014;7(1):23506. 4. Lim HS, Yoo JJ. Association between health indicators and health-related quality of life according to physical activity of older women. Healthcare; 2020: Multidisciplinary Digital Publishing Institute. 5. Gagne M. Autonomy support and need satisfaction in the motivation and well-being of gymnasts. J Appl Sport Psychol. 2003;15(4):372-90. 6. Organization WH. Global strategy on diet, physical activity, and health. 2004. 7. Cecchini M, Sassi F, Lauer JA, Lee YY, Guajardo-Barron V, Chisholm D. components Despite the importance of women's health and maternal roles in achieving family and community health, for reasons such as factors related to a physical condition (gender, age, and health status of women), psychosocial factors (social determinants of health, including interpersonal factors, financial income, employment, level of education, family and social support) and factors related to the environment of sports and physical activity (security, environmental characteristics at home, work and regular access to sports facilities), women compared to men have lower physical activity [19]. Although research has identified knowledge, perceived benefits, and self-efficacy as predictors of PA [15]. Regarding this, the findings of another study show that the perception of healthy behaviors in the girls' lives is drastically affected by the determinants of personal and environmental components, which need to be considered in creating interventions in doing so [20]. Among the environmental components, one can state the cultural determinants involving elements like ethnicity, race, country of origin, language, non- verbal communication, acculturation, gender, age, sexual orientation, values, behavioral norms, laws, etiquette, social grouping and relations, religious and spiritual beliefs, economic 63 ournal of Advanced Pharmacy Education & Research \| Jan-Mar 2023 \| Vol 13 \| Issue 1 Journal of Advanced Pharmacy Education & Research \| Jan-Mar 2023 \| Vol 13 \| Issue 1 Ezati Asar et al.: Innovative and motivational SDT-based approach to promote Iranian women's physical activity ds is detrimental to motivation and health [26]. Providing ditions to satisfy these basic needs facilitates the individual's ivation and independent health, and the frustration of these ds leads to illness and is accompanied by low-quality ivation and in many cases with a highly controlled motivation . sociocultural context and based on SDT. Thus, STD-based interventions focusing on increasing PA steadily and as a service package with high quality and “cost-effective” will be able to be integrated with the program to combat non-communicable diseases lie non-communicable disease control plan and in limited resources conditions can bring about quality care as health care system of the Islamic Republic of Iran [34]. sociocultural context and based on SDT. Thus, STD-based interventions focusing on increasing PA steadily and as a service package with high quality and “cost-effective” will be able to be integrated with the program to combat non-communicable diseases lie non-communicable disease control plan and in limited resources conditions can bring about quality care as health care system of the Islamic Republic of Iran [34]. components needs is detrimental to motivation and health [26]. Providing conditions to satisfy these basic needs facilitates the individual's motivation and independent health, and the frustration of these needs leads to illness and is accompanied by low-quality motivation and in many cases with a highly controlled motivation [25]. Results and Discussion Acknowledgments: Thanks and appreciation to Dr. Ruhollah Rostami, Vice Chancellor for Research, Damghan School of Health, Semnan University of Medical Sciences, IRI. As a country in epidemiological transition, Iran has a great share of disability-adjusted life years (DALYs) to non-communicable diseases. About 78% of deaths are because of non-communicable diseases so about 312,000 deaths occur as a result of non- communicable diseases out of 380,000 annual deaths, and the death rate from non-communicable diseases is predicted to reach 650,000 deaths by 2040. Accordingly, it will lead to about $ 47 billion annual direct and indirect costs to implement non- communicable disease care programs that confirm the opinion of the World Health Organization that “One of the main reasons for the depletion of the health system's resources is a non- communicable disease” [27-29]. Conflict of interest: None Financial support: None Financial support: None Financial support: None Ethics statement: None Ethics statement: None References Tackling of unhealthy diets, physical inactivity, and obesity: health effects and cost-effectiveness. Lancet. 2010;376(9754):1775-84. 8. Fararouei M, Brown I, Toori MA, Haghighi RE, Jafari J. Happiness and health behaviour in Iranian adolescent girls. J Adolesc. 2013;36(6):1187-92. 9. Burnham J. Exercise is medicine: health benefits of regular physical activity. J La State Med Soc. 1998;150(7):319-23. 10. Bloom DE, Cafiero E, Jané-Llopis E, Abrahams-Gessel S, Bloom LR, Fathima S, et al. The global economic burden of noncommunicable diseases. Program on the Global Demography of Aging; 2012. Journal of Advanced Pharmacy Education & Research \| Jan-Mar 2023 \| Vol 13 \| Issue 1 Conclusion Nonetheless, the general acceptance of some health education systems is still based on traditional and knowledge-based approaches that call for reflection, solutions, removal of cultural barriers, and designing PA interventions in line with the 11. Organization WH. Global action plan on physical activity 2018-2030: more active people for a healthier world: World Health Organization; 2019. 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College Students’motivation For Physical Activity: University of Louisiana; 2006. 14. Ghaneapur M, Eftekhar H, Montazeri A, Garmaroudi G, Yaseri M, Saleh E, et al. Changes in physical activity and behavioral regulation of women receiving a sdt-based motivational physical activity intervention. J Organ Behav Res. 2019;4(s2):1-13. 27. Asar ME, Haghi M. Obesity in Iran: Challenges Arising and Policy Options. Iran J Public Health. 2019;48(11):2112. 28. Aminorroaya A, Fattahi N, Azadnajafabad S, Mohammadi E, Jamshidi K, Khalilabad MR, et al. Burden of non- communicable diseases in Iran: past, present, and future. J Diabetes Metab Disord. 2020:1-7. 15. Salehi L, Shojaeizadeh D, Eftekhar H, Mohammad K, Taghdisi MH. Physical activity among a sample of Iranians aged over 60 years: an application of the transtheoretical model. Arch Iran Med. 2010;13(6):528-36. 29. Emamgholipour S, Sari AA, Pakdaman M, Geravandi S. Economic Burden of Cardiovascular Disease in the Southwest of Iran. Int Cardiovasc Res J. 2018;12(1):6-12. 16. Ghaneapur M, Eftekhar H, Montazeri A, Saleh E, Ezati Asar M. A Randomized Clinical Trial of a SelfDetermination Theory-Based Intervention on Physical Activity of Women Aged 30-45 Years. Arch Pharma Pract. 2020;11(S1):182-9. 30. Neufeld A, Malin G. Journal of Advanced Pharmacy Education & Research \| Jan-Mar 2023 \| Vol 13 \| Issue 1 Conclusion Twelve tips to combat ill-being during the COVID-19 pandemic: A guide for health professionals & educators. MedEdPublish. 2020;9. 31. Ryan RM, Deci EL. Intrinsic and extrinsic motivation from a self-determination theory perspective: Definitions, theory, practices, and future directions. Contemp Educ Psychol. 2020;61:101860. 17. Luong A, Hall A, McMillian D. Common Experiences and Beliefs among Highly Active Individuals. 2018. 18. do Nascimento Júnior JRA, Granja CTL, da Silva DB, de Oliveira DV, Gonçalves MP, Carvalho FO. Is motivation to physical activity practice associated with the lifestyle of individuals who undergo weight training in the context of higher education? Acta Sci Health Sci. 2018;40:e39109. 32. Ghaneapur M, Eftekhar H, Montazeri A, Garmarudi G, Yaseri M, Ahvanoei AR. Effectiveness of a Self- Determination Theory (SDT) Based Intervention on Physical Activity, Quality of Life, and Happiness: A Protocol for a Randomized Clinical Trial. Biochem Technol Soc. 2019(2):108-17. 19. Alyafei A, Albaker W. Barriers and facilitators for regular physical exercise among adult females narrative review 2020. J Community Med Public Health. 2020;4(2). 20. Boyington JE, Carter-Edwards L, Piehl M, Hutson J, Langdon D, McManus S. Cultural attitudes toward weight, diet, and physical activity among overweight African American girls. Prev Chronic Dis. 2008;5(2):A36. 33. Mahmoodabad SSM, Tonekaboni NR, Farmanbar R, Fallahzadeh H, Kamalikhah T. The effect of motivational interviewing-based intervention using self-determination theory on promotion of physical activity among women in reproductive age: a randomized clinical trial. Electron Physician. 2017;9(5):4461. 21. Cultural Determinants CdIKW, editor. Encyclopedia of Public Health. Dordrecht: Springer Netherlands; 2008. p. 185. 34. Damari B, Heidari A. Implementation of integrated management of non-communicable disease prevention and control in Iran: A proposal. Payesh (Health Monitor). 2020;19(1):7-17. 22. Mathew Joseph N, Ramaswamy P, Wang J. Cultural factors associated with physical activity among U.S. adults: An integrative review. Appl Nurs Res. 2018;42:98-110. 23. Friedman HS, Kern ML. Personality, well-being, and health. Annu Rev Psychol. 2014;65:719-42. 65 Journal of Advanced Pharmacy Education & Research \| Jan-Mar 2023 \| Vol 13 \| Issue 1
W4251709441.txt	https://www.qeios.com/read/9ZUJ31/pdf	null		Hypoattenuation	Definitions	2,020	cc-by	55	Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Hypoattenuation National Cancer Institute Source National Cancer Institute. Hypoattenuation. NCI T hesaurus. Code C124041. Describes areas on an x-ray or CT scan that show up as whiter or brighter than normal. Qeios ID: 9ZUJ31 · https://doi.org/10.32388/9ZUJ31 1/1
https://openalex.org/W3209809826	https://europepmc.org/articles/pmc8556977?pdf=render	English	null	Identifying competencies for integrated knowledge translation: a Delphi study	BMC health services research	2,021	cc-by	12,956	Abstract Background: Considerable progress has been made to advance the field of knowledge translation to address the knowledge-to-action gap in health care; however, there remains a growing concern that misalignments persist between research being conducted and the issues faced by knowledge users, such as clinicians and health policy makers, who make decisions in the health care context. Integrated knowledge translation (IKT) is a collaborative research model that has shown promise in addressing these concerns. It takes advantage of the unique and shared competencies amongst researchers and knowledge users to ensure relevance of the research process and its outcomes. To date, core competencies have already been identified to facilitate training in knowledge translation more generally but they have yet to be prioritized for IKT more specifically. The primary aim of this study was to recruit a group of researchers and knowledge users to identify and prioritize core competencies for researchers and knowledge users to engage with IKT. Methods: We recruited health care knowledge users (KUs) and researchers with experience and knowledge of IKT for a quantitative, cross-sectional study. We employed a modified Delphi approach consisting of three e-survey rounds to establish consensus on competencies important to IKT for KUs and researchers based on mean rating of importance and agreement between participants. Results: Nineteen (73%) of the initial 26 participants were researchers (response rate = 41% in the first round; retention in subsequent rounds > 80%). Participants identified a total of 46 competencies important for IKT (18 competencies for KUs, 28 competencies for researchers) under 3 broad domains. Technical research skills were deemed extremely important for researchers, while both groups require teamwork and knowledge translation skills. Conclusions: This study provides important insight into distinct and overlapping IKT competencies for KUs and researchers. Future work could focus on how these can be further negotiated and contextualized for a wide range of IKT contexts, projects and teams. Greater attention could also be paid to establishing competencies of the entire team to support the research co-production process. Keywords: Integrated knowledge translation, Knowledge user, Researcher, Competence, Delphi challenges with the uptake of evidence into health care policy and practice are not only attributed to underutilization of research evidence, but may also stem from failures in the knowledge production process. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Scholars have voiced a growing concern that misalign- ments may exist between the research conducted and the issues faced by knowledge users, such as clinicians and policy makers, who make decisions in the health care context [1–4]. These incongruences have led to Identifying competencies for integrated knowledge translation: a Delphi study Euson Yeung1, Stephanie Scodras1, Nancy M. Salbach1, Anita Kothari2 and Ian D. Graham3,4 Euson Yeung1, Stephanie Scodras1, Nancy M. Salbach1, Anita Kothari2 and Ian D. Graham3, Background Over the last few decades, considerable progress has been made to advance the field of knowledge translation to address the knowledge-to-action gap in health care. Among these advances is the increasing recognition that * Correspondence: Euson.yeung@utoronto.ca * Correspondence: Euson.yeung@utoronto.ca 1University of Toronto, 160-500 University Avenue, Toronto, Ontario M5G 1V7, Canada Full list of author information is available at the end of the article 1V7, Canada Full list of author information is available at the end of the article * Correspondence: Euson.yeung@utoronto.ca 1University of Toronto, 160-500 University Avenue, Toronto, Ontario M5G 1V7, Canada Full list of author information is available at the end of the article Yeung et al. BMC Health Services Research (2021) 21:1181 https://doi.org/10.1186/s12913-021-07107-7 Yeung et al. BMC Health Services Research (2021) 21:1181 https://doi.org/10.1186/s12913-021-07107-7 (2021) 21:1181 Yeung et al. BMC Health Services Research https://doi.org/10.1186/s12913-021-07107-7 Open Access Participants and recruitment p We considered two stakeholder groups to be relevant in this study: knowledge users (KUs) and researchers who are familiar and experienced with partnered research using an IKT approach. We define a KU as “an individ- ual who is likely to be able to use the knowledge gener- ated through research to make informed decisions about health policies, programs and/or practices” [22]. Know- ledge users in the health care system, such as organizational or system-level decision-makers in health care settings (clinical managers, health care managers, policy-makers), and point of care health care providers, were eligible to participate. Individuals who were a KU or researcher and who had worked in the health sector for at least 2 years were considered eligible. Potential study participants were identified through the Integrated Knowledge Translation Research Network (IKTRN), and through the research team’s professional network. The IKTRN is a network of “knowledge users and re- searchers committed to studying, teaching and prac- ticing integrated knowledge translation” in Canada [23] or are known to the research team to have experience in the field. Given the uniqueness of the patient perspec- tives with different health conditions and healthcare set- tings, and the growing focus specifically on patient engagement in health research, we opted to focus this study exclusively on clinician and health system know- ledge users and therefore did not include patients as study participants. Since sample sizes for Delphi panels remain variable [19, 24], we prioritized the need for par- ticipants to reflect the full range of KU and researcher stakeholders to optimize the credibility and acceptability Although the ideals of research co-production are not new, conceptualization of the researcher and knowledge user roles within the research process requires greater attention, especially with respect to how capacity can be built to improve and sustain the practice of IKT. For ex- ample, since ongoing collaborative relationships between researcher and knowledge user is a key feature of the IKT process [16], it follows that both groups may need to develop competence in forming, contributing, and sustaining collaborative working relationships for the purpose of co-producing research. A range of enabling conditions have been identified to support organizations to succeed in IKT. Categorized as organizational, professional and individual-level condi- tions, these constitute a framework by which organiza- tions can develop and monitor their capacity for IKT [17]. Yeung et al. BMC Health Services Research (2021) 21:1181 Page 2 of 18 Page 2 of 18 views that research results are inaccessible and have lim- ited applicability to everyday clinical practice. the competencies identified by Mallidou et al. [18] per- tain to either or both researchers and knowledge users. The primary aim of this study was to recruit a group of researchers and knowledge users to identify and prioritize core competencies for researchers and know- ledge users to engage with IKT. Integrated knowledge translation (IKT) is a collabora- tive research model that has shown promise in address- ing the aforementioned views [3, 4]. This approach involves researchers and knowledge users (clinicians, managers, policy makers, etc.) “engaging in a mutually beneficial research project or program of research” wherein knowledge users identify a problem and have the authority to implement the research recommenda- tions [5, 6]. Thus, the IKT approach considers re- searchers and knowledge users as co-producers of knowledge [7–11]. Methods We used a modified Delphi approach [19, 20] to identify and prioritize a set of IKT competencies for the two key groups of stakeholders involved in the research process: knowledge users (KU) and researchers. The Delphi tech- nique is an established and widely used methodology in healthcare research that enables consensus building among individuals with pertinent experience and poten- tially diverse views [21]. Results from Delphi studies are considered data generated at a single point in time for a particular group of individuals, and are therefore intended for further testing and verification to ultimately inform theory and practice [19]. Research co-production has been described as “a col- laborative endeavour of academic and non-academic ac- tors” [12], characterized by “a space where science intersects with non-science” [13]. Academic and non- academic participants in the research endeavour interact throughout the research process to co-produce know- ledge rather than only interacting during dissemination of the research results. The nature of such collaborations is sustained for a period of time, and roles and responsi- bilities between researcher and knowledge user are at times necessarily indistinct. As such, IKT approaches take advantage of the unique as well as shared compe- tencies amongst researchers and knowledge users [14, 15]. Consequently, the knowledge that is co-produced through these collaborative efforts is more likely to be contextually-relevant and therefore applied in health care practice and policy. Round 2 y g p The initial list of competencies for KUs and researchers was developed based on the ‘Knowledge Translation Pathways (KTP)’ tool that arose from a scoping review of competencies for knowledge translation [18]. This is in keeping with other Delphi studies that utilized pre- existing information for the initial Delphi round [29]. Two members of the research team (EY, SS) reviewed and identified competencies that were most relevant to IKT. EY is an Integrated Knowledge Translation Expert member of the IKTRN and SS is a healthcare profes- sional and research trainee with graduate level education in knowledge translation. They synthesized the list into 29 competencies for KUs and 30 competencies for The aims of Round 2 were to (1) provide feedback (me- dian rating and percent of participants who rated the competency as either ‘Very Important’ or ‘Extremely Im- portant’) on the competencies rated in Round 1, (2) re- rate the importance of all competencies from the initial list to assess for consistency between rounds, (3) rate the importance of newly added competencies, and (4) gener- ate new competencies based on participants’ comments if they arise. Ethical considerations Participants demonstrated implied consent by respond- ing to the online questionnaire and provided their email address if they agreed to be contacted in subsequent rounds. The University of Toronto Research Ethics Board approved the study protocol. Defining consensus for core competencies Our a priori definition of a highly rated competency consisted of an overall median rating of ‘Very Important’ or ‘Extremely Important’ in addition to at least 70% of respondents agreeing with either of those ratings within a round. In combination, median score and percent agreement is the most commonly used criteria for con- sensus based on a systematic review of health care Del- phi studies [24]. We considered there to be consensus for a core competency if that competency was highly rated in two rounds. Competencies that did not meet the highly rated criteria for two rounds were excluded from the final list of core competencies. Competencies that were rated twice and received inconsistent ratings between rounds (e.g., highly rated in Round 2 and not highly rated in Round 3) were resolved through group discussion by members of the research team (EY, SS and NMS). Modified Delphi methodology Our modified Delphi methodology was established a priori and consisted of three rounds, which is considered to be the optimal number of rounds [21]. We asked par- ticipants to rate the importance of competencies needed by KUs and researchers who are engaging in IKT on a five-point ordinal scale (Not Important, Slightly Import- ant, Important, Very Important, Extremely Important) through an online questionnaire developed using Sur- veyMonkey™. In addition, participants were asked to identify new competencies not already included in the questionnaire. Participant feedback was provided be- tween rounds as this is an important aspect of develop- ing group consensus [27, 28]. In rounds two and three, we provided participant feedback in the form of the overall median rating and the percent of the participants who rated the competency as either ‘Very Important’ or ‘Extremely Important’ from the previous round for each competency being rated. Data were collected over a course of 2 months (April – June, 2019), and participants had 2 weeks to complete each round with 1 week separ- ating the rounds. Round 1 The aims of Round 1 were to (1) characterize the partic- ipants in terms of stakeholder group (KU or researcher) as well as work role and setting, (2) rate the importance of all initially identified competencies for KUs and re- searchers to engage in IKT, and (3) generate new com- petencies based on participants’ comments. The Round 1 questionnaire was reviewed by a member of the re- search team (NMS), and minor adjustments were made to improve content and clarity prior to administering the survey. Participants and recruitment Of the individual-level conditions identified, acqui- sition of adequate IKT-specific knowledge and skills, or competence, were deemed as essential to the IKT process. To date, core competencies have already been identified to facilitate training in knowledge translation more generally [18]; however competencies have yet to be prioritized for research co-production more specific- ally. What remains to be clarified is the extent to which Yeung et al. BMC Health Services Research (2021) 21:1181 Page 3 of 18 of the Delphi results [24], with an ultimate pragmatic target sample size of 20–25 panelists. researchers through discussion. In keeping with the ori- ginal ‘KTP’ tool, the competencies were divided into do- mains of ‘Evidence’, ‘Teamwork’, and ‘Knowledge Translation (KT) Activities’ rather than discrete know- ledge, skills and attitudes. We believe this categorization accounts for the integrated nature of competencies, and allows us to better visualize the competencies relevant to the key processes critical to IKT. Following a modified Dillman approach for recruit- ment [25], we sent a pre-notice email prior to the first invitation to participate in round one of the survey, followed by two thank you/reminder emails and a final reminder email for each survey round. Sending multiple email reminders is an appropriate method of increasing response rates in Delphi studies [26]. Data analysis f h After each round, we calculated the overall median rat- ing and the percent of the participants who rated each competency as ‘Very Important’ or ‘Extremely Import- ant’ (percent agreement). We also conducted a post-hoc analysis of the median rating and percent agreement for the individual KU and researcher groups since there was an unplanned difference between the number of KUs and researchers in the sample. To determine whether one group influenced the overall ratings of competen- cies, we followed the steps outlined by Elwyn et al. [30] to calculate the equimedian for competencies where there was discordance between the median rating for the KU and researcher groups (i.e., one group median was “Very Important” or above, and the other group median was below “Very Important”). The equimedian is the median of a cumulative distribution function that gives equal weight to the two groups’ responses. We used Microsoft Excel 2016 to perform the data analysis. Round 3 The aims of Round 3 were to (1) provide feedback (me- dian rating and percent of participants who rated the Page 4 of 18 Yeung et al. BMC Health Services Research (2021) 21:1181 settings (n = 4, 57%), and researchers primarily worked in academic institutions (n = 11, 58%) or hospital (n = 5, 26%) settings. competency as either ‘Very Important’ or ‘Extremely Im- portant’) on the competencies rated in Round 2 that were being rated again, (2) re-rate the importance of ini- tial competencies that received inconsistent ratings be- tween Round 1 and Round 2, and (3) re-rate the importance of competencies that were added after Round 1 based on participants’ comments. Round 1 Of the 59 initial competencies (KU competencies =29, researcher competencies = 30), 43 (72.9%) met the highly rated criteria (KU competencies = 16, researcher compe- tencies = 27). The majority of the KU competencies that did not meet the highly rated criteria (9/16, 56.3%) were from the ‘Evidence’ domain. Based on the participants’ comments, we identified seven new competencies (KU competencies = 5, researcher competencies = 2) to in- clude in subsequent rounds (Table 2). Round 2 Of the 43 competencies that met the highly rated criteria from Round 1, nine (20.9%) (KU competencies = 2, re- searcher competencies = 7) fell below the threshold after Round 2 and were subsequently eliminated. All 16 (100%) competencies that did not meet the highly rated criteria in Round 1 also did not meet these criteria in Round 2, demonstrating consistency between rounds. One (14.3%) of the seven new competencies that were added after Round 1 was rated highly. Participants did not identify new competencies after Round 2. Competencies eliminated during the Delphi process A total of 20 competencies from the initial list were eliminated (Table 4). Sixteen of these were KU compe- tencies (Evidence = 11, Teamwork = 3, Knowledge Translation Activities = 2) and 4 of these were researcher competencies (Evidence = 1, Teamwork = 1, Knowledge Translation Activities = 2). Table 1 Characteristics of participants (Rounds 1, 2, and 3) competencies that received discordant ratings between KU and researcher groups by calculating the equime- dian, or weighted median, for each competency rating [30]. Equimedian values are presented in Supplemental Table 1 (core competencies) and Supplemental Table 2 (eliminated competencies). Based on this analysis, we concluded that use of the equimedian rather than the overall median as an a priori criterion for a high rating, would have resulted in very few (3%) of the competen- cies receiving a higher median rating. Therefore, the group influence on the overall ratings of the competen- cies was very small. Additionally, because a high rating was also dependent on the percent agreement with ‘Very Important’ or ‘Extremely Important’, using the equime- dian would not have changed the outcomes of this study. competencies that received discordant ratings between KU and researcher groups by calculating the equime- dian, or weighted median, for each competency rating [30]. Equimedian values are presented in Supplemental Table 1 (core competencies) and Supplemental Table 2 (eliminated competencies). Based on this analysis, we concluded that use of the equimedian rather than the overall median as an a priori criterion for a high rating, would have resulted in very few (3%) of the competen- cies receiving a higher median rating. Therefore, the group influence on the overall ratings of the competen- cies was very small. Additionally, because a high rating was also dependent on the percent agreement with ‘Very Important’ or ‘Extremely Important’, using the equime- dian would not have changed the outcomes of this study. Round 3 Sixty-three potential participants were invited to partici- pate in this study, 26 of whom responded to the first round of the questionnaire, representing a 41.3% re- sponse rate (Table 1). One participant did not provide an email address and therefore could not be contacted in subsequent rounds. Twenty-two out of the 26 partici- pants from round 1 completed the questionnaire for the second round (84.6% retention rate) and 18 of 22 partic- ipants responded to the third round of the survey, repre- senting an 81.8% retention rate. The nine (100%) competencies that showed inconsist- ency between Round 1 and Round 2 (i.e., met the highly rated criteria in Round 1 but not in Round 2), were rated highly in Round 3, and were therefore retained in the final list of core competencies (Table 3). Of the 7 new competencies that were added in Round 2, one (14.3%) remained consistently highly rated, four (57.1%) remained consistently below the highly rated threshold, and two (28.6%) met the highly rated criteria in Round 3 despite missing the threshold in Round 2. Members of the research team discussed the two competencies that demonstrated inconsistency between Rounds 2 and 3 and resolved to include them in the final list of priori- tized competencies for IKT. Participants were asked to identify the predominant role that they fill in the IKT process, either KU or re- searcher. Researchers represented approximately 68 to 73% of the participants in each round (Table 1). In an open-ended comment section in Round 1, two partici- pants reported that they consider themselves to fill the role of KU and researcher, although both participants se- lected researcher as their predominant role. Core competencies for IKT A total of 46 competencies were identified as highly im- portant for IKT in our study (Table 3). Eighteen compe- tencies were deemed core competencies for KUs (Evidence = 3, Teamwork = 6, Knowledge Translation Activities = 9), and there were 28 core competencies for researchers (Evidence = 8, Teamwork = 8, Knowledge Translation Activities = 12). In round 1, 7 KUs and 19 researchers responded. The most common type of work was program development/ evaluation (n = 2, 29%) and research support (n = 2, 29%) for KUs, with research (n = 16, 84%) being the most common type of work for researchers. KUs worked primarily in government (n = 2, 29%) and ‘Other’ Yeung et al. Discordance between KU and researcher groups To determine if the unequal number of KUs and re- searchers had an impact on the outcome of the Delphi study, we conducted a post-hoc analysis for Table 2 New competencies identified by participants in Round 1 1 Knowledge users 1. Advocate for inclusion of appropriate knowledge users in the IKT process (evidence domain) 2. Understand the resource implications (e.g. funding, time) of the IKT process (evidence domain) 3. Implement actionable strategies to ensure all team members remain accountable for their expected contributions throughout the process (evidence domain) 4. Differentiate between evaluation, research, and quality improvement (evidence domain) 5. Utilize various resources (e.g., resource librarians) for evidence- gathering activities (evidence domain) 6. Clarify the roles of individual team members (teamwork domain) Researcher 7. Understand how intellectual property considerations may impact the dissemination process Knowledge users 1. Advocate for inclusion of appropriate knowledge users in the IKT process (evidence domain) 2. Understand the resource implications (e.g. funding, time) of the IKT process (evidence domain) 3. Implement actionable strategies to ensure all team members remain accountable for their expected contributions throughout the process (evidence domain) 4. Differentiate between evaluation, research, and quality improvement (evidence domain) 5. Utilize various resources (e.g., resource librarians) for evidence- gathering activities (evidence domain) 6. Clarify the roles of individual team members (teamwork domain) Researcher 7. Understand how intellectual property considerations may impact the dissemination process Discussion This study provides important insights regarding KU and researcher competencies that are perceived by study participants as important to engaging in IKT. Both KU and researcher participants agreed on a total of 46 com- petencies, with 18 competencies deemed important for KUs in the domains of Evidence (n = 3); Teamwork (n = 6), and Knowledge Translation Activities (n = 9); and 28 competencies rated as important for researchers under the same domains (Evidence (n = 8), Teamwork (n = 8), and Knowledge Translation Activities (n = 12)). Round 3 BMC Health Services Research (2021) 21:1181 Page 5 of 18 Table 1 Characteristics of participants (Rounds 1, 2, and 3) Round 1 (n = 26) Round 2 (n = 22) Round 3 (n = 18) No. of respondents (%) IKT stakeholder Group Knowledge user 7 (26.9) 7 (31.8) 5 (27.8) Researcher 19 (73.1) 15 (68.2) 13 (72.2) Predominant type of work Research 16 (61.5) 14 (63.6) 13 (72.2) Clinical work (patient care) 2 (7.7) 1 (4.6) 1 (5.6) Program evaluation 1 (3.9) 1 (4.6) 0 (0) Education and training 1 (3.9) 0 (0) 0 (0) Other (e.g., program development, research support, policy analysis) 6 (23.1) 6 (27.3) 4 (22.2) Predominant work setting Research – academic institution 7 (26.9) 6 (27.3) 5 (27.8) Research – hospital 5 (19.2) 5 (22.7) 5 (27.8) Education – academic institution 5 (19.2) 3 (13.6) 3 (16.7) Administration – government 2 (7.7) 2 (9.1) 1 (5.6) Clinical – hospital 1 (3.9) 0 (0) 0 (0) Administration – hospital 1 (3.9) 1 (4.5) 0 (0) Other (e.g., community organizations, research foundations, professional associations) 5 (19.2) 5 (22.7) 4 (22.2) Table 1 Characteristics of participants (Rounds 1, 2, and 3) Researcher 7. Understand how intellectual property considerations may impact the dissemination process Yeung et al. BMC Health Services Research (2021) 21:1181 Page 6 of 18 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) Knowledge User Evidence Domain 1 Apply different types of knowledge to inform decision- making Extremely Important (71.4) Very Important (84.2) Very Important (80.8) Very Important (100) Very Important (80.0) Very Important (86.4) 2 Identify decision- makers’ information needs and priorities Very Important (85.7) Very Important (84.2) Very Important (84.6) Very Important (100) Very Important (93.3) Very Important (95.5) 3 Understand how local healthcare system factors (e.g. Very Important (100.0) Researcher health services, health literacy) impact decision making processes Extremely Important (71.4) Very Important/ Extremely Important (94.4) n = 18 Extremely Important (88.0) n = 25 Very Important (57.1) Very Important (73.3) Very Important (68.2) Very Important (100) Very Important (100) Very Important (100.0) Teamwork Domain 1 Build healthy working relationships with other team members Extremely Important (100) Extremely Important (94.7) Extremely Important (96.2) Very Important (85.7) Extremely Important (86.7) Extremely Important (86.4) 2 Foster productive networks of researchers and decision makers Important (28.6) Extremely Important (94.7) Very Important (76.9) Very Important (85.7) Very Important (73.3) Very Important (77.3) 3 Create opportunities to learn and share knowledge through informal and formal means Extremely Important (71.4) Extremely Important (94.7) Extremely Important (88.5) Very Important (85.7) Very Important (66.7) Very Important (72.7) 4 Demonstrate and promote appropriate attitudes and behaviours when working with marginalized or vulnerable populations Extremely Important (85.7) Extremely Important (78.9) Extremely Important (80.8) Extremely Important (83.3) n = 6 Extremely Important (80.0) Extremely Important (81.0) n = 21 5 Value and contribute to knowledge sharing activities Extremely Important (85.7) Extremely Important (89.5) Extremely Important (88.5) Very Important (85.7) Very Important (80.0) Very Important (81.8) 6 Advocate for inclusion of appropriate knowledge users in the IKT process Very Important (71.4) Very Important (73.3) Very Important (72.7) Very Important (100) Very Important (100) Very Important (100.0) KT Activities Domain 1 Address barriers and facilitators to applying knowledge Very Important (71.4) Very Important (89.5) Very Important (84.6) Very Important (85.7) Very Important (80.0) Very Important (81.8) es for knowledge users and researchers to engage in IKT after 3 survey rounds KT Activities Domain Yeung et al. BMC Health Services Research (2021) 21:1181 Page 7 of 18 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) to policy/decision- making 2 Interact with knowledge brokers (an intermediary who links knowledge sources, and knowledge itself to organizations in its network) to assist with developing and/or finding and implementing evidence Very Important (71.4) Very Important (78.9) Very Important (76.9) Very Important (100) Very Important (66.7) Very Important (77.3) 3 Identify practice gaps and opportunities to use relevant evidence to improve practice Very Important (100) Extremely Important (89.5) Very Important/ Extremely Important (92.3) Very Important (85.7) Very Important (80.0) Very Important (81.8) 4 Identify and address inconsistencies between research findings and expertise or patient preferences Extremely Important (85.7) Very Important (84.2) Very Important (84.6) Very Important (71.4) Very Important (60.0) Very Important (63.6) Very Important (100) Very Important (92.3) Very Important (94.4) 5 Describe how the patient’s values affect the balance between potential advantages and disadvantages of available healthcare/ policy options Very Important (85.7) Very Important (84.2) Very Important (84.6) Very Important (85.7) Very Important (73.3) Very Important (77.3) 6 Appropriately involve the patient in decision-making Extremely Important (71.4) Extremely Important (84.2) Extremely Important (80.8) Extremely Important (71.4) Extremely Important (80.0) Extremely Important (77.3) 7 Promote the use of research and outcome data to formulate, evaluate and/or revise policy and practices to improve care Extremely Important (100) Extremely Important (89.5) Extremely Important (92.3) Extremely Important (100) Very Important (100) Very Important (100.0) 8 Adapt and apply the evidence for the local practice context/ environment Extremely Important (85.7) Extremely Important (100) Extremely Important (96.2) Extremely Important (85.7) Very Important (86.7) Very Important/ Extremely Important (86.4) 9 Understand the resource implications (e.g. KT Activities Domain funding, time) of the IKT process Very Important (57.1) Very Important (53.3) Very Important (54.5) Very Important (80.0) Very Important (69.2) Very Important (72.2) ROUND 1 ROUND 2 ROUND 3 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) to policy/decision- making 2 Interact with knowledge brokers (an intermediary who links k l d Very Important (71.4) Very Important (78.9) Very Important (76.9) Very Important (100) Very Important (66.7) Very Important (77.3) evidence 3 Identify practice gaps and opportunities to use relevant evidence to improve practice Very Important (100) Extremely Important (89.5) Very Important/ Extremely Important (92.3) Very Important (85.7) Very Important (80.0) Very Important (81.8) 4 Identify and address inconsistencies between research findings and expertise or patient preferences Extremely Important (85.7) Very Important (84.2) Very Important (84.6) Very Important (71.4) Very Important (60.0) Very Important (63.6) Very Important (100) Very Important (92.3) Very Important (94.4) 5 Describe how the patient’s values affect the balance between potential advantages and disadvantages of available healthcare/ policy options Very Important (85.7) Very Important (84.2) Very Important (84.6) Very Important (85.7) Very Important (73.3) Very Important (77.3) 6 Appropriately involve the patient in decision-making Extremely Important (71.4) Extremely Important (84.2) Extremely Important (80.8) Extremely Important (71.4) Extremely Important (80.0) Extremely Important (77.3) 7 Promote the use of research and outcome data to formulate, evaluate and/or revise policy and practices to improve care Extremely Important (100) Extremely Important (89.5) Extremely Important (92.3) Extremely Important (100) Very Important (100) Very Important (100.0) 8 Adapt and apply the evidence for the local practice context/ environment Extremely Important (85.7) Extremely Important (100) Extremely Important (96.2) Extremely Important (85.7) Very Important (86.7) Very Important/ Extremely Important (86.4) 9 Understand the resource implications (e.g. KT Activities Domain funding, time) of the IKT process Very Important (57.1) Very Important (53.3) Very Important (54.5) Very Important (80.0) Very Important (69.2) Very Important (72.2) ROUND 1 ROUND 2 ROUND 3 Yeung et al. BMC Health Services Research (2021) 21:1181 Page 8 of 18 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) Median (% agree) Median (% agree) Median (% Agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) Researcher Evidence Domain 1 Understand how different types of knowledge (e.g. KT Activities Domain research, practice, theory) are generated and used in KT Extremely Important (100) Extremely Important (94.7) Extremely Important (96.2) Extremely Important (71.4) Extremely Important (86.7) Extremely Important (81.8) 2 Apply appropriate research methodologies to examine the determinants of knowledge use across different settings and stakeholder groups Extremely Important (100) Extremely Important (94.7) Extremely Important (96.2) Extremely Important (100) Extremely Important (86.7) Extremely Important (90.9) 3 Design and evaluate the impact, effectiveness and sustainability of KT strategies in different settings Very Important (85.7) Extremely Important (94.7) Extremely Important (92.3) Extremely Important (100) Extremely Important (73.3) Extremely Important (81.8) 4 Respond to questions by stakeholders regarding the evidence generated to inform decision- making Extremely Important (71.4) Extremely Important (100) Extremely Important (92.3) Extremely Important (100) Very Important (80.0) Very Important (86.4) 5 Apply the most appropriate dissemination tool for communicating with different audiences/ stakeholders Very Important (57.1) Very Important (84.2) Very Important (76.9) Very Important (85.7) Very Important (73.3) Very Important (77.3) 6 Incorporate the most relevant stakeholder perspectives into the research process and implementation cycle Very Important (100) Extremely Important (100) Extremely Important (100.0) Extremely Important (100) Extremely Important (86.7) Extremely Important (90.9) 7 Select appropriate KT models or Very Important Extremely Important Extremely Important Very Important Extremely Important Very Important Evidence Domain Yeung et al. BMC Health Services Research (2021) 21:1181 Page 9 of 18 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) frameworks of knowledge dissemination and implementation for the context being considered (85.7) (83.3) n = 18 (84.0) n = 25 (85.7) (73.3) (77.3) 8 Help transform clinical, management or policy questions into research questions Very Important (85.7) Extremely Important (94.7) Very Important/ Extremely Important (92.3) Very Important (85.7) Very Important (80.0) Very Important (81.8) Teamwork Domain 1 Use effective communication strategies within the context being considered Extremely Important (100) Extremely Important (100) Extremely Important (100.0) Very Important (85.7) Very Important (73.3) Very Important (77.3) 2 Use effective strategies to set priorities and manage/resolve conflict between stakeholders with differing interests Important (42.9) Very Important (84.2) Very Important (73.1) Very Important (71.4) Very Important (60.0) Very Important (63.6) Very Important (80.0) Very Important (69.2) Very Important (72.2) 3 Evaluate the impact of knowledge brokering to connect evidence to practice/policy Very Important (85.7) Very Important (68.4) Very Important (73.1) Very Important (71.4) Very Important (60.0) Very Important (63.6) Very Important (100) Very Important (61.5) Very Important (72.2) 4 Demonstrate and promote appropriate attitudes and behaviours when working with marginalized or vulnerable populations Extremely Important (66.7) n = 6 Extremely Important (89.5) Extremely Important (84.0) n = 25 Extremely Important (85.7) Extremely Important (73.3) Extremely Important (77.3) 5 Form sustainable working relationships with relevant partners (e.g. Evidence Domain government, industry, academia, funders etc) Extremely Important (85.7) Extremely Important (94.7) Extremely Important (92.3) Extremely Important (100) Very Important (80.0) Very Important/ Extremely Important (86.4) 6 Advocate for appropriate change or action(s) Very Important (57.1) Extremely Important (78.9) Very Important (73.1) Very Important (57.1) Very Important (60.0) Very Important (59.1) Very Important (80.0) Very Important (76.9) Very Important (77.8) 7 Form collaborative networks of relevant stakeholders to effectively generate, disseminate, and collate knowledge Very Important (100) Extremely Important (89.5) Very Important/ Extremely Important (92.3) Very Important (85.7) Very Important (66.7) Very Important (72.7) Teamwork Domain Page 10 of 18 Yeung et al. Teamwork Domain BMC Health Services Research (2021) 21:1181 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) throughout the KT process 8 Implement actionable strategies to ensure all team members remain accountable for their expected contributions throughout the process Very Important (71.4) Very Important (53.3) Very Important (59.1) Very Important (60.0) Very Important (76.9) Very Important (72.2) KT Activities Domain 1 Identify the most appropriate approach (es) to closing the knowledge-to-action gaps in the context being considered Very Important (57.1) Extremely Important (84.2) Extremely Important (76.9) Very Important (71.4) Extremely Important (73.3) Extremely Important (72.7) 2 Develop and prioritize the steps in a dissemination plan within the research design Very Important (71.4) Very Important (89.5) Very Important (84.6) Very Important (71.4) Very Important (66.7) Very Important (68.2) Very Important (100) Very Important (100) Very Important (100.0) 3 Consider the individual, organizational and system-level barriers and facilitators to knowledge uptake in planning KT activities Very Important (71.4) Extremely Important (94.7) Extremely Important (88.5) Very Important (71.4) Extremely Important (86.7) Extremely Important (81.8) 4 Create KT plans that are closely linked to the goals of the research project Very Important (57.1) Very Important/ Extremely Important (94.4) n = 18 Very Important (84.0) n = 25 Very Important (100) Extremely Important (80.0) Very Important/ Extremely Important (86.4) 5 Incorporate patient’s values into KT plan by balancing potential advantages and disadvantages of available options Very Important (85.7) Extremely Important (89.5) Very Important (88.5) Very Important (85.7) Extremely Important (86.7) Very Important (86.4) 6 Conduct stakeholder analyses to understand the target audiences, interest in and capacity to engage with the evidence Very Important (71.4) Extremely Important (84.2) Very Important/ Extremely Important (80.8) Extremely Important (71.4) Very Important (60.0) Very Important (63.6) Very Important (100) Very Important (92.3) Very Important (94.4) 7 Work collaboratively Very Extremely Extremely Very Very Very KT Activities Domain 1 Identify the most appropriate approach (es) to closing the knowledge-to-action gaps in the context being considered Very Important (57.1) Extremely Important (84.2) Extremely Important (76.9) Very Important (71.4) Extremely Important (73.3) Extremely Important (72.7) 2 Develop and prioritize the steps in a dissemination plan within the research design Very Important (71.4) Very Important (89.5) Very Important (84.6) Very Important (71.4) Very Important (66.7) Very Important (68.2) Very Important (100) Very Important (100) Very Important (100.0) 3 Consider the individual, organizational and system-level barriers and facilitators to knowledge uptake in planning KT activities Very Important (71.4) Extremely Important (94.7) Extremely Important (88.5) Very Important (71.4) Extremely Important (86.7) Extremely Important (81.8) 4 Create KT plans that are closely linked to the goals of the research project Very Important (57.1) Very Important/ Extremely Important (94.4) n = 18 Very Important (84.0) n = 25 Very Important (100) Extremely Important (80.0) Very Important/ Extremely Important (86.4) 5 Incorporate patient’s values into KT plan by balancing potential advantages and disadvantages of available options Very Important (85.7) Extremely Important (89.5) Very Important (88.5) Very Important (85.7) Extremely Important (86.7) Very Important (86.4) 6 Conduct stakeholder analyses to understand the target audiences, interest in and capacity to engage with the evidence Very Important (71.4) Extremely Important (84.2) Very Important/ Extremely Important (80.8) Extremely Important (71.4) Very Important (60.0) Very Important (63.6) Very Important (100) Very Important (92.3) Very Important (94.4) 7 Work collaboratively with decision/policy makers to Very Important (85.7) Extremely Important (100) Extremely Important (96.2) Very Important (85.7) Very Important (80.0) Very Important (81.8) Very Important (100.0) KT Activities Domain Page 11 of 18 Yeung et al. BMC Health Services Research (2021) 21:1181 Table 3 List of core competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) Median (% agree) Median (% agree) Median (% Agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) synthesize and develop tailored messages for the target audience 8 Create strategies to collect, collate and package evidence in an accessible and relevant manner for policy and practice Very Important (71.4) Extremely Important (94.7) Very Important (88.5) Very Important (71.4) Very Important (73.3) Very Important (72.7) 9 Develop a systematic and inclusive KT plan that addresses the critical aspects of project implementation and management Very Important (57.1) Extremely Important (89.5) Very Important (80.8) Important (42.9) Extremely Important (73.3) Very Important (63.6) Very Important (100) Very Important (100) Very Important (100.0) 10 Identify various roles of KT partners and practitioners in enhancing user engagement Important (28.6) Very Important (89.5) Very Important (73.1) Very Important (57.1) Very Important (66.7) Very Important (63.6) Very Important (100) Very Important (84.6) Very Important (88.9) 11 Design KT strategies that include program-level and organizational-level KT Very Important (57.1) Extremely Important (89.5) Very Important (80.8) Very Important (71.4) Very Important (73.3) Very Important (72.7) 12 Use tools to support knowledge production processes such as: ethics approval, collaboration agreements, and shared decision- making structures Very Important (71.4) Very Important (84.2) Very Important (80.8) Very Important (85.7) Very Important/ Extremely Important (71.4) n = 14 Very Important (76.2) n = 21 “% Agree” refers to the percentage of participants who agreed with either “Very Important” or “Extremely Important” ‘Evidence’ domain in decision-making. This is consistent with assertions that a deep understanding of the context for KT ac- tivities and an appreciation for a range of evidence resources are critical for successful knowledge transla- tion [18]. This is also in keeping with the conceptualization of evidence-based practice in which the application of different types of knowledge, in- cluding tacit knowledge, has been previously recog- nized as central to decision-making within a range of health care contexts [31, 33–35]. For IKT in particu- lar, KUs are particularly well-positioned to bring unique context-related knowledge and perspectives to The competencies prioritized for KUs and researchers in the ‘Evidence’ domain were complementary and support the sentiment that the partnerships formed between KUs and researchers are greater than the sum of its parts in the IKT context [31, 32]. Knowledge user competencies The competencies from the ‘Evidence’ domain that were rated as highly valuable skills for KUs pertain to having a working familiarity with the local health care system and the use of different forms of knowledge Page 12 of 18 Yeung et al. BMC Health Services Research (2021) 21:1181 Table 4 List of eliminated competencies for knowledge users and researchers to engage in IKT after 3 survey rounds Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) Knowledge User Evidence Domain 1 Critically appraise research findings Very Important (71.4) Important (31.6) Important (42.3) Very Important (57.1) Important (6.7) Important (22.7) 2 Identify and select the most appropriate evidence for the context being considered Very Important (57.1) Very Important (68.4) Very Important (65.4) Very Important (85.7) Important/ Very Important (50.0) n = 14 Very Important (61.9) n = 21 3 Develop research questions for literature searches Very Important (71.4) Important (31.6) Important (42.3) Important (0.0) Important (7.1) n = 14 Important (4.8) n = 21 4 Create and execute an efficient search strategy within relevant electronic databases Slightly Important (42.9) Slightly Important (26.3) Slightly Important (30.8) Slightly Important (0.0) Slightly Important (0.0) n = 14 Slightly Important (0.0) n = 21 5 Keep up to date on relevant literature for the context being considered Important (42.9) Important (47.4) Important (46.2) Important (28.6) Important (28.6) n = 14 Important (28.6) n = 21 6 Explain the different types of knowledge (e.g. research, practice, theory) that contribute to decision-making Important (42.9) Important/ Very Important (50) n = 18 Important (48) n = 25 Important (0.0) Important (14.3) n = 14 Important (9.5) n = 21 7 Examine and interpret selected evidence from a literature search Important (42.9) Important (47.4) Important (46.2) Important (0.0) Important (7.1) n = 14 Important (4.8) n = 21 8 Use implementation resources (e.g. Knowledge user competencies knowledge translation tools) to apply evidence for multiple audiences Very Important (85.7) Very Important (57.9) Very Important (65.4) Very Important (71.4) Very Important (64.3) n = 14 Very Important (66.7) n = 21 9 Describe the research process (e.g. research question, research ethics, different methodologies, data collection and analyses) Important (14.3) Important (31.6) Important (26.9) Important (14.3) Slightly Important (0.0) n = 13 Slightly Important (5.0) n = 20 10 Differentiate between Very Important Slightly Important/ Important (42.9) Very Important Important (23.1) Important (33.3) Yeung et al. Knowledge user competencies BMC Health Services Research (2021) 21:1181 Table 4 List of eliminated competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) Teamwork Domain 1 Understand how intellectual property considerations may impact the dissemination process Very Important (71.4) Important (46.7) Very Important (54.5) Very Important (80.0) Very Important (61.5) Very Important (66.7) KT Activities Domain 1 Articulate the difference between research and evaluation for a particular context Very Important (71.4) Very Important (63.2) Very Important (65.4) Very Important (71.4) Important/ Very Important (50.0) n = 14 Very Important (57.1) n = 21 2 Understand the role and practices of social media in the KT process Important (42.9) Very Important (63.2) Very Important (57.7) Very Important (85.7) Important (42.9) n = 14 Very Important (57.1) n = 21 “% Agree” refers to the percentage of participants who agreed with either “Very Important” or “Extremely Important” Teamwork Domain “% Agree” refers to the percentage of participants who agreed with either “Very Important” or “Extremely Important” “% Agree” refers to the percentage of participants who agreed with either “Very Important” or “Extremely Important” one of knowledge co-production involving KUs and re- searchers [38, 39]. Greater attention will need to be paid to interventions and curricula that facilitate attitudinal shifts toward beliefs and values that support the entire IKT process rather than simply focusing on knowledge transfer between distinct groups of researchers and knowledge users [18, 37]. the development of research priorities, and the co- production and application of research evidence [31, 36]. Competencies in the ‘Evidence’ domain that were not prioritized for KUs included the ability to critically ap- praise research findings, create and execute an efficient search strategy within relevant electronic databases, examine and interpret selected evidence from a litera- ture search, and keep up to date on relevant literature. Knowledge user competencies This was a notable yet surprising finding given the im- portance attributed to developing research knowledge and skills for knowledge users such as health care pro- fessionals in the context of evidence-based practice [37]. Due to the low number of KUs in this study and their diverse roles, some participants may not have perceived these competencies as important for their day-to-day work, and competency requirements may vary across contexts. For example, while KUs serving in clinical roles may be expected to learn continuously about new clin- ical approaches, the same expectations may not be ap- plied to KUs working in managerial roles with respect to the explicit use of research evidence. Issues of feasibility may also prevent all stakeholders from engaging with the entire IKT process in every circumstance [18]. More- over, the competencies not prioritized for KUs may have been perceived as more relevant to the researcher role; suggesting that the knowledge-to-action gap may still be perceived as a knowledge transfer issue [36] rather than Knowledge user competencies BMC Health Services Research (2021) 21:1181 Page 13 of 18 Table 4 List of eliminated competencies for knowledge users and researchers to engage in IKT after 3 survey rounds (Continued) Competency ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) evaluation, research, and quality improvement (85.7) Important (21.4) n = 14 n = 21 (60.0) 11 Utilize various resources (e.g., resource librarians) for evidence- gathering activities Important (42.9) Slightly Important (0.0) n = 14 Important (14.3) n = 21 Important (40.0) Slightly Important (7.7) Slightly Important (16.7) Teamwork Domain 1 Help others access and appraise evidence Important (28.6) Important (42.1) Important (38.5) Important (14.3) Slightly Important/ Important (0.0) n = 14 Important (4.8) n = 21 2 Actively seek opportunities to engage in the research process Important (42.9) Important (47.4) Important (46.2) Important (14.3) Slightly Important/ Important (0.0) n = 14 Important (4.8) n = 21 3 Clarify the roles of individual team members Very Important (57.1) Important (26.7) Important (36.4) Very Important (60.0) Important (33.3) n = 12 Important (41.2) n = 17 KT Activities Domain 1 Utilize appropriate KT framework(s) for the context being considered Very Important (71.4) Important (47.4) Very Important (53.8) Very Important (71.4) Important (30.8) n = 13 Important (45.0) n = 20 2 Lead the team in synthesizing research evidence to improve healthcare services Important (28.6) Important (36.8) Important (34.6) Important (42.9) Slightly Important (0.0) n = 14 Slightly Important (14.3) n = 21 ROUND 1 ROUND 2 ROUND 3 Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) Median (% agree) KU Researcher Pooled KU Researcher Pooled KU Researcher Pooled n = 7 (unless otherwise indicated) n = 19 (unless otherwise indicated) n = 26 (unless otherwise indicated) n = 7 (unless otherwise indicated) n = 15 (unless otherwise indicated) n = 22 (unless otherwise indicated) n = 5 (unless otherwise indicated) n = 13 (unless otherwise indicated) n = 18 (unless otherwise indicated) Researcher Evidence Domain 1 Identify and set priorities regarding policy needs and research options Important (28.6) Very Important (73.7) Very Important (61.5) Very Important (71.4) Important/ Very Important (50.0) n = 14 Very Important (57.1) n = 21 Page 14 of 18 Yeung et al. Researcher competencies The competencies in the ‘Evidence’ domain that were prioritized for researchers relate directly to the research process. This is not surprising since researchers are gen- erally academic researchers who are directly involved in and skilled at carrying out the research process [32, 39]. Notably, “Identify and set priorities regarding policy needs and research options” was not a highly rated com- petency for researchers. In keeping with IKT theory and practice [17, 32], this may be because this competency was considered by study participants to be a role of the KUs who may be better suited to set priorities given their deep understanding of the context. ‘Teamwork’ domain Our study findings elaborate on the competencies sug- gested by Mallidou et al. [18] who described collabora- tive skills in managing and leading teams as central to knowledge translation. Competencies within the Page 15 of 18 Page 15 of 18 Page 15 of 18 Yeung et al. BMC Health Services Research (2021) 21:1181 Yeung et al. BMC Health Services Research (2021) 21:1181 ‘teamwork’ domain in our study were not only highly rated in all 3 Delphi rounds in this study, but were con- sidered important for both KUs and researchers. They pertain to navigating the ‘boundary organizations’ occur- ring at the intersection between science and practice [13] and promoting ongoing partnerships between KUs and researchers. Consistent with other participatory knowledge co-production approaches, many of the highly rated ‘Teamwork’ competencies support KU and researchers’ roles in knowledge sharing and in forming and maintaining healthy relationships between KUs and researchers [14, 15, 40–42]. The knowledge co- production process is particularly vulnerable to the im- pact of power relationships amongst researchers and KUs, especially for issues of sustainability, and the inte- gration of diverse perspectives and ideas [12]. Data from the present study support the development of specific ‘teamwork’ competencies to address these shortcomings and to minimize these common vulnerabilities, particu- larly when marginalized populations are involved. Among these are competencies related to the formation and maintenance of healthy relationships and networks throughout the research cycle to optimize team func- tioning; these include recognizing and valuing the ex- pertise of all team members, and for KUs to advocate for their involvement in the IKT process. These competen- cies are well-aligned with community-based participa- tory approaches in which efforts aimed at fostering the team’s capacity for shared ownership of research results afforded opportunities for all team members to engage in meaningful dialogue [43]. However, the full potential of ‘teamwork’ competencies may only be optimally real- ized if the underlying values and commitment of team members are sufficiently oriented toward supporting IKT [18]. themselves as agents of change in a formal manner and may not recognize the implicit ways in which they are already applying KT frameworks in policy and practice. However, there is growing evidence to suggest that KUs can be effective leaders and change agents for imple- menting innovations, particularly in contexts where KUs are highly committed and proactively forming working relationships with community partners [45]. ‘Teamwork’ domain Thus, while team leadership in synthesizing research evidence was not prioritized as a KU competency in this study, it may still be worthwhile to examine the need to build capacity among individual KUs as well as organizations, noting specific conditions under which KUs may best serve as change agents and leaders over knowledge translation activities [17]. Researcher competencies There is emerging evidence to suggest that there are po- tential benefits to blurring the lines between research, quality improvement (QI) and evaluation which has im- portant implications for partnership formation between academic and health organizations [36]. Thus, it is sur- prising that recognizing the difference between research and evaluation in a particular context was not prioritized as a researcher competency. This is perhaps due to a lack of understanding of the potential opportunities afforded by overlapping research with QI and evaluation efforts. When research, QI and evaluation are conceptu- alized as distinct activities within an organization, it can inadvertently lead to an artificial separation of roles and responsibilities to the detriment of the IKT process. Thus, future work is warranted to explore how trad- itional conceptions of research could be reimagined [36] and the competencies required of KUs and researchers for this reconceptualization. Limitations We acknowledge that of the use of previously identified general KT competencies may have inadvertently de- emphasized or excluded competencies relevant to IKT, including those that underpin social processes important to IKT. Hence, future work could explore specific com- petencies, such as negotiation skills, that are critical to managing the evolving roles of KU and researcher throughout the research cycle. We also recognize that while some competencies did not meet the criteria in this study, they may still hold relevance for researchers and knowledge users in some contexts, and hence should not be ignored altogether. While we sought ap- propriate representation from researchers and know- ledge user groups, participants were recruited from a single international IKT network that is dominated by Canadian knowledge users who might share a particular worldview about IKT. We also acknowledge the smaller number of KU participants in this study and the absence of data regarding gender identities. Future studies could examine the external validity of the competencies priori- tized in this study for a wide range of research contexts, research partnerships and research co-production team roles assumed by KUs. This should include establishing competencies to support the formation of patient part- ners for projects that involve patient oriented research. p p Since a key ingredient for successful IKT is establish- ing a shared understanding of language, roles and re- sponsibilities, and ultimately how decisions are made, it stands to reason that KUs and researchers should also be equipped to negotiate the IKT process. While negoti- ation was not an explicit competency in this study, we believe it is implicit in the KU competencies prioritized in this study, such as to ‘address inconsistencies between research findings and expert or patient preferences’, and ‘advocate for inclusion of appropriate KUs in the IKT process’. Similarly, strong negotiation skills are implicit in researcher competencies including setting research priorities and identifying the roles of partners in enhan- cing user engagement. Moreover, negotiation of roles and responsibilities to enable successful teamwork is im- portant across diverse organizational and non- organizational contexts, as well as teams that represent multiple stakeholders. In a study of how nurse practi- tioners were integrated into primary health care teams, role negotiation and clarification was a critical determin- ant in the performance of health care teams [48]. ‘Knowledge translation activities’ domain Knowledge user competencies Our study highlights the criticality of teamwork compe- tencies to enable KUs and researchers from both sides of the ‘boundary organizations’ to interact effectively and efficiently to co-produce knowledge [12]. In the face of long-standing hierarchical traditions and power differen- tials within the research context, one possible way for- ward is to build team members’ capacity to create and/ or contribute to research environments in which team- work competencies and practices are promoted, normal- ized and made explicit. Creating a safe space for exchange of dialogue by setting ground rules, ensuring participant interactivity, and encouraging shared owner- ship of study results are examples of strategies to assist in managing power relations [43]. Central to these strat- egies is KUs and researchers’ capacity to be reflexive about one’s own values and assumptions, as well as the ability to communicate clearly and effectively [43, 46]. The KU competencies prioritized under the ‘KT Activ- ities’ domain support activities typically executed to- wards the end of the research process that promote the application of research evidence to the local context. Notably, the two KU competencies that were eliminated from the ‘KT Activities’ domain were 1) utilizing appro- priate KT framework(s) for the context being considered and 2) leading the team in synthesizing research evi- dence to improve healthcare services. Until more re- cently, both of these competencies have been traditionally associated with the researcher role [3]. Owing to their familiarity with stakeholder needs and the local environment in which knowledge is to be ap- plied, some have argued for KUs, and specifically man- agers, to be at the helm for planning and executing KT interventions [38, 44]. Indeed, KUs may not view Page 16 of 18 Page 16 of 18 Yeung et al. BMC Health Services Research (2021) 21:1181 Yeung et al. BMC Health Services Research (2021) 21:1181 reflective of persistent traditional paradigms of research in which leadership over KT activities is held by re- searchers. While the rewards of more collaborative models of leadership have been purported in the health care practice context [46, 47], wider adoption of similar models in IKT must be considered. As we strive to understand ways to improve implementation processes, a way forward may be to explore the extent to which do- mains of the Implementation Leadership Scale (pro- active, knowledgeable, supportive and perseverant leadership) may be relevant to and fostered amongst KUs [49, 50]. ‘Knowledge translation activities’ domain Knowledge user competencies Pursuing this line of research can poten- tially yield opportunities to invest in alternative means for KUs to contribute meaningfully to the IKT process. Notably, competency in reflexivity was not included in the initial Delphi survey, nor did study participants rec- ommend this as a new competency to include in subse- quent rounds. Thus, a potential focus of competency development to support IKT could be to build reflexive skills among KUs and researchers. Evidence is also accumulating from studies of health care teams to suggest that training efforts should add- itionally attend to the collective competence of the en- tire team or of the organization [18, 46, 47]. Building individual competence may be necessary but insufficient to address the multitude of contextual factors facing each unique IKT scenario [46, 47]. Similarly, teams working in the IKT context must simultaneously address the expertise relevant to the task at hand as well as the processes unique to that context that allow for optimal collective functioning. Thus, further research is war- ranted to determine if and to what extent the notion of collective competence plays a role in the IKT context, and how team-based learning approaches may benefit the development of expertise in IKT. Limitations In this case, role clarification was most effective when individ- uals mobilized their negotiation skills within the con- straints of their respective organizational processes. Therefore, it may be prudent to draw on organizational development and transdisciplinary literatures, and health care team practices to identify ideal approaches to build capacity among KUs and researchers to negotiate effectively. Acknowledgements We would like to thank Chris McCutcheon for his insights and assistance during development of the manuscript. 7. Elliot H, Popay J. How are policy makers using evidence? Models of research utilization and local NHS policy making. J Epidemiol Community Health. 2000;54(6):461–8. https://doi.org/10.1136/jech.54.6.461. Funding h d 10. Salbach NM, Guilcher SJT, Jaglal SB, Davis DA. Determinants of research use in clinical decision making among physical therapists providing services post-stroke: a cross-sectional study. Implement Sci. 2010;5(1):77. https://doi. org/10.1186/1748-5908-5-77. 10. Salbach NM, Guilcher SJT, Jaglal SB, Davis DA. Determinants of research use in clinical decision making among physical therapists providing services post-stroke: a cross-sectional study. Implement Sci. 2010;5(1):77. https://doi. org/10.1186/1748-5908-5-77. This study was supported by a subgrant from a Canadian Institutes of Health Research (CIHR) Foundation Grant to IDG (FDN# 143237). The CIHR had no role in the project. 11. Walter I, Davies H, Nutley S. Increasing research impact through partnerships: evidence from outside health care. J Health Serv Res Policy. 2003;8(S2):58–60. https://doi.org/10.1258/135581903322405180. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 12. Pohl C, Rist S, Zimmerman A, Fry P, Gurung GS, Schneider F, et al. 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Boundary organizations in environmental policy and science: an introduction. Sci Technol Hum Values. 2001;26(4):399–408. https://doi.org/1 0.1177/016224390102600401. Ethics approval and consent to participate Abbreviations IKT I d k IKT: Integrated knowledge translation; IKTRN: Integrated Knowledge Translation Research Network; KTP: Knowledge Translation Pathway; KT: Knowledge translation; KU: Knowledge user 4. Kothari A, McCutcheon C, Graham ID. Exploring the frontiers of research co- production: the Integrated Translation Research Network concept papers. Collection published in Research Involvement and Engagement, Health Resarch Policy and Systems, Implementation Science, Implementation Science Communications, BMC Medical Research Methodlogy, BMC Health Services Research, and Systematic Reviews. https://www.biomedcentral.com/ collections/IKT. Accessed 19 July 2021. Conclusion Development of highly skilled KUs and researchers is critical if IKT teams are to effectively and efficiently co- produce research to address gaps in health care policy and practice. While core competencies have previously been identified to facilitate training in knowledge trans- lation more generally [15], this study begins to clarify the competencies that are most relevant to KUs and re- searchers to support successful IKT. The results from this Delphi study specify competencies in three domains (‘evidence’, ‘knowledge ‘teamwork’ and ‘knowledge trans- lation activities’) that are of particular importance for This study led to the exclusion of ‘leadership’ over KT activities as a KU competency in despite of literature pointing to the important role of KUs, such as managers, in implementation leadership [44, 45]. This could be Page 17 of 18 Yeung et al. BMC Health Services Research (2021) 21:1181 Yeung et al. BMC Health Services Research (2021) 21:1181 Yeung et al. BMC Health Services Research (2021) 21:1181 KUs and researchers to attend to when engaging in IKT. The competencies identified reflect the dynamic and complex nature of IKT wherein both KUs and re- searchers must be skilled in managing team dynamics and functions throughout all phases of the research process. Although competencies have been specified sep- arately for KU and researchers in this study, future work could focus on how these can be further negotiated and contextualized due to the wide variations in IKT context, project and team Received: 6 April 2021 Accepted: 27 September 2021 Received: 6 April 2021 Accepted: 27 September 2021 Competing interests 16. Kothari A, McCutcheon C, Graham ID. Defining integrated knowledge translation and moving forward: a response to recent commentaries. Int J Health Policy Manag. 2017;6(5):2–1. https://doi.org/10.15171/ijhpm.2017.15. The authors declare that they have no competing interests” in this section. Supplementary Information y The online version contains supplementary material available at https://doi. org/10.1186/s12913-021-07107-7. 5. Kothari A, McCutcheon C, Graham ID. Defining integrated knowledge translation and moving forward: a response to recent commentaries. Int J Health Policy Manag. 2017;6(5):299–300. https://doi.org/10.15171/ijhpm.201 7.15. Ethics approval and consent to participate The University of Toronto Research Ethics Board approved the study protocol. Protocol ID: 37448. All methods were performed in accordance with the guidelines and regulations of this research ethics board. Informed consent was obtained from all participants in this research study. 14. Jull J, Giles A, Graham ID. Community-based participatory research and integrated knowledge translation: advancing the co-creating of knowledge. Implement Sci. 2017;12(1):150. https://doi.org/10.1186/s13012-017-0696-3. p p with the guidelines and regulations of this research ethics board. 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https://openalex.org/W2780713752	https://researchonline.lshtm.ac.uk/id/eprint/4646018/1/Illness%20recognition%20and%20care-seeking%20for%20maternal%20and%20newborn%20complications%20in%20rural%20eastern%20Uganda_Gold%20VoR.pdf	English	null	Illness recognition and care-seeking for maternal and newborn complications in rural eastern Uganda	Journal of health, population and nutrition	2,017	cc-by	7,992	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: To enhance understanding of the roles of community-based initiatives in poor rural societies, we describe and explore illness recognition, decision-making, and appropriate care-seeking for mothers and newborn illnesses in two districts in eastern Uganda where in one implementation district, a facility and community quality improvement approach was implemented. Methods: This was a cross-sectional study using qualitative methods. We conducted 48 event narratives: eight maternal and newborn deaths and 16 maternal and newborn illnesses. Additionally, we conducted six FGDs with women’s saving groups and community leaders. Qualitative data were analyzed thematically using Atlas.ti software. Results: Women and caretakers reported that community initiatives including the presence of community health workers and women’s saving groups helped in enhancing illness recognition, decision-making, and care-seeking for maternal and newborn complications. Newborn illness seemed to be less well understood, and formal care was often delayed. Care-seeking was complicated by accessing several stations from primary to secondary care, and often, the hospital was reached too late. Conclusions: Our qualitative study suggests that community approaches may play a role in illness recognition, decision-making, and care-seeking for maternal and newborn illness. The role of primary facilities in providing care for maternal and newborn emergencies might need to be reviewed. is available and provided. However, as described by Thaddeus and Maine [3] for maternal deaths and Waiswa et al. [4] for newborn deaths, care-seeking delays are common and result in severe morbidity and mortality. Understanding how illness recognition, timely decision-making, and appropriate care-seeking can be achieved effectively and sustainably, and at scale is important for building efficacious national programs. Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 DOI 10.1186/s41043-017-0125-x Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 DOI 10.1186/s41043-017-0125-x Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 DOI 10.1186/s41043-017-0125-x * Correspondence: mokuga@musph.ac.ug 1Department of Health Policy, Planning and Management, Makerere University School of Public Health, Kampala, Uganda 2Center of Excellence for Maternal, Newborn and Child Health, Makerere University, Kampala, Uganda Full list of author information is available at the end of the article Illness recognition and care-seeking for maternal and newborn complications in rural eastern Uganda Monica Okuga1,2, Peter Waiswa1,2,3, Rogers Mandu1,2, Juddy Wachira4, Claudia Hanson3,5 and Fatuma Manzi6 Background With the adoption of the Sustainable Development Goals, new and ambitious targets have been set for reducing ma- ternal and neonatal deaths globally: by 2030, the global maternal mortality rate (MMR) should not be higher than 70 per 100,000 live births and neonatal mortality rate (NMR) not more than 12 per 1000 live births [1]. In Uganda, the MMR is 368 deaths per 100,000 live births, while the NMR is 27 per 1000 live births [2]. Achieving these global targets will require that maternal and new- born illness or complication is recognized early, a decision to seek care is appropriately made followed by timely care-seeking, and when facilities are reached, quality care The primary delivery of health care and evidence- based interventions for maternal and newborn health is mandated in Uganda to administrative districts as per decentralization policy [5]. Particular implementation focus is on rural districts, as they often have the highest mortality rates [6]. However, there is limited evidence in the literature on how districts best operationalize access to care including illness recognition, timely decision- making and appropriate care-seeking. Correspondence: mokuga@musph.ac.ug 1Department of Health Policy, Planning and Management, Makerere University School of Public Health, Kampala, Uganda 2Center of Excellence for Maternal, Newborn and Child Health, Makerere University, Kampala, Uganda Full list of author information is available at the end of the article * Correspondence: mokuga@musph.ac.ug 1Department of Health Policy, Planning and Management, Makerere University School of Public Health, Kampala, Uganda 2Center of Excellence for Maternal, Newborn and Child Health, Makerere University, Kampala, Uganda Full list of author information is available at the end of the article In Mayuge district of eastern Uganda, a district-wide maternal and newborn intervention study, the Expanded Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Page 76 of 96 Quality Management Using Information Power (EQUIP) project [7] implemented a community quality improve- ment approach based on national community health worker (CHW) strategy. EQUIP aimed to improve ma- ternal and newborn care. The neighboring Namaingo district served as a comparison district for this plausibility trial which is explained elsewhere in detail [7]. In short, the EQUIP quality improvement approach (2010– 2014) was based on the collaborative model of im- provement [8] and linked communities, health facil- ities, and district health management teams. Background At the community level, quality improvement teams (QIT) were formed by CHW at the village level to work on local problems which hamper the implementation of WHO essential interventions [9]. Teams were encour- aged to analyze locally identified problems––including delayed care-seeking for maternal and newborn care––and to generate possible solutions. The solu- tions, called “change ideas” were then tested within the community, and their effects were assessed using locally generated data on maternal and newborn indi- cators. Examples of change ideas included visiting of pregnant women to counsel women, identify maternal and newborn danger signs and refer if necessary, using an approach and materials developed as part of the Uganda Newborn Study (UNEST) and the estab- lishment of community savings funds (women’s sav- ings groups) [7, 10]. Table 1 Sample size of each category of respondents in each district Case type Mayuge (intervention) Namaingo (control) Total cases/ interviews Maternal Women who perceived to have had excessive bleeding after birth and survived (maternal illness) 8 8 16 Women who died within 42 days of birth regardless of cause (maternal death) 4 4 8 Newborn Women who perceived their newborn became ill before 28 days of life and survived (newborn illness) 8 8 16 Women whose newborn became ill and died within 28 days of life (newborn death) 4 4 8 FGDs with community leaders 2 2 4 FGDs with women’s groups 2 2 Total number of interviews 28 26 54 Table 1 Sample size of each category of respondents in each Table 1 Sample size of each category of respondents in each district caretakers of women and babies (spouses, neighbors, friends, siblings) who fell ill or died from four purposefully selected parishes (two rural and two peri-urban). In each selected parish, with the help of the CHWS, we identified one maternal death, two cases of excessive bleeding, two newborn illnesses, and one newborn death. The selection criteria for the cases were that they must have been home when the illness was recognized. Cases arising while in hospital were excluded. Case definitions are further de- scribed in the protocol paper in this supplement [13]. Six focus group discussions (FGDs) were conducted in both the intervention and comparison districts (Table 1) to gain perspectives on illness recognition and care-seeking with community leaders and women’s savings groups. Background Women’s savings groups are groups of 50–70 women within the community who make a monthly contribution of $0.30 that can be borrowed to facilitate health care-seeking. These savings groups were part of the change ideas initi- ated and used in the Mayuge district only. Potential cases for event narratives were identified with the help of CHWs in the selected parishes. In Mayuge district, the CHWs keep updated registers where they record all preg- nant women, maternal deaths, newborn deaths, deliver- ies, and women who have had maternal complications such as post-partum hemorrhage, newborn illnesses, and prematures within their parishes. However, in Namaingo, even though the CHW structure is in place, it is not fully operational. Further screening of identified cases was conducted using a checklist to determine true cases. A screening checklist was used to screen cases and determine eligibility. In view that EQUIP provided a novel approach of col- laborative quality improvement using CHWs to improve illness recognition and care-seeking, we aimed to under- stand how maternal and newborn illness recognition, timely decision-making, and appropriate care-seeking were enhanced. We did not set out to explore perceived quality of maternal and newborn care, although this in- advertently emerged during the interviews. Study design, area, and intervention This was a qualitative cross-sectional study conducted in the EQUIP intervention (Mayuge) and comparison (Namaingo) districts in eastern Uganda. Both are typical rural districts. Mayuge has a population of 460,000 while Namaingo has approximately 233,000 people [11]. Both districts lie on the Northern shores of Lake Victoria. This region has a fertility rate of 6.8, greater than the na- tional average of 5.9 [12]. Namaingo has 22 health cen- ters and no hospital, and Mayuge district has 40 health centers and one private-not-for-profit hospital that charge fees for services. Study population and sampling We selected 48 mothers; 24 each from Mayuge and Namaingo districts (Table 1) aged 18–49 years, and Page 77 of 96 Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Page 77 of 96 Ethics approval and consent to participate pp p p This research was approved by the Makerere University School of Public Health Higher Degrees Research and Ethics Committee and Uganda National Council of Sci- ence and Technology. Permission was also obtained from the Mayuge and Namaingo District Health Offices. Written informed consent was obtained from all partici- pants using a consent form translated to Lusoga for clear understanding. All data was treated with confidentiality and presented only in aggregate form or anonymized. Confidentiality was ensured throughout the study. Audio recordings of the illness narratives and focus group dis- cussions were kept in a locked room and will be destroyed after 5 years in accordance with the IRB rules. For the FGDs, eight community leaders/members of the women’s groups were identified and selected as respon- dents per FGD with the help of the CHWs. Informed written consent was obtained from each respondent. Quality assurance was maintained through a number of steps. Prior to the study, the RAs underwent a 4-day train- ing on study objectives, methods, and ethical consider- ations. Qualitative data collection tools were translated into Lusoga and back translated to English by different people to ensure their accuracy. All discussion guides for narratives and FGDs were pretested in one of the parishes not included in the sampling frame. To ensure quality, audio recordings were checked against the transcripts. All translations were double checked. Daily field notes were also used for quality checks. Intermittently, the study manager supervised data collection in the field. Results We conducted 48 event narratives (8 maternal deaths, 16 maternal illnesses, 8 newborn deaths, and 16 newborn ill- nesses) and 6 FGDs. There were two refusals (cases related to one newborn death and one maternal death) for the event narratives, but these were replaced. Focal persons (key caretakers) in the maternal illness and death cases were mostly the husbands and mothers-in-law. In some cases, sisters and friends were involved. All the respon- dents for the newborn event narratives were female, and the majority were the biological mothers. Demographic characteristics of the focal person are presented in Table 2. Data analysis Interviews were transcribed verbatim. The qualitative data analysts read through all transcripts and field notes from group interviews and FGDs multiple times to familiarize themselves with the data. Complete event narratives were written for each index case using data from the interviews. Content analysis methodology was used to code the transcripts using Atlas.ti software [14]. The responses were coded by case type. Any code with less than 100% inter-coder agreement was re-evaluated and dropped if there was no agreement. This analysis included coding within cases and comparing across cases and between intervention and comparison areas, identi- fying crisis recognition categories and broad themes. Key We grouped findings in three domains: (1) illness recog- nition and care-seeking for maternal and newborn cases, (2) role of community initiatives in illness recognition and care-seeking, and (3) patient experiences with care. Data collection narrative domains and sub-domains include perceived signs and symptoms (what and by whom), care-giving at home (what, by whom, when in relation to signs, and symptoms), care-seeking decision-making (whether it occurred or not, involving whom, what were the consid- erations), care-seeking (from whom, in what sequence, barriers encountered), perceived cause of the illness, and contextual factors that may have influenced recognition of and response to the illness. To provide depth to and illustrate the model, we also coded narrative quality, noting the presence, content, and resolution of contra- dictory statements among informants in the group. The analysis model was in the form of a table with four do- mains that contained key themes and sub-themes which were explained chronologically by multiple factors to authenticate the phenomena. The interviews and FGDs were conducted from April to July 2015 by four trained Research Assistants (RAs): two data collectors and two note takers, chosen on the basis of prior experience with qualitative interviews. All the qualitative interviews were conducted in the local language, Lusoga. The research team screened the previously identified households to locate cases of maternal excessive bleed- ing after delivery, maternal deaths, neonatal illness, and neonatal deaths using the inclusion criteria as described above in the study population. Willing participants were then asked to identify 2–3 individuals who were present and provided support during the maternal/newborn ill- ness or death. In some cases, where respondents were available, the group interview was conducted immedi- ately, while in other cases, an appointment had to be made. In most of the cases, the narratives were done in a single interview with all the care givers in the family. However, in some cases, for example, where the mother seemed uncomfortable speaking in the presence of other relatives, an additional separate interview was conducted with her. Maternal and newborn illness recognition and care- seeking We noted differences in terms of illness recognition, decision-making, and care-seeking between mothers and newborns (Table 3). Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Page 78 of 96 Table 2 Demographics of maternal respondents Variable Category Maternal death (number/%age) N = 8 Maternal illness (number/percentage) N = 16 Respondents for newborn illnesses (number/percent) N = 16 Respondents for newborn death (number/%) N = 8 Age, 0–18 years 0 (0) 2 (12.5) 0 (0) 1 (12.5) 19–24 years 2 (25) 5 (31.25) 10 (62.5) 5 (62.5) > 25 years 6 (75) 9 (56.25) 6 (37.5) 2 (25) Parity 1 3 (37.5) 1 (6.25) 3 (18.75) 2 (25) 2 1 (12.5) 6 (37.5) 4 (25) 4 (50) 3 and above 5 (62.5) 9 (56.25) 9 (56.25) 2 (25) Marital status Married 8 (100) 14(87.5) 13 (81.25) Single/widowed/separated 0 (0) 2 (12.5) 3 (18.75) Table 2 Demographics of maternal respondents Other maternal illness symptoms recognized included loss of consciousness, headaches, swelling of the body during pregnancy, dizziness, being bed ridden, and in- ability to do household chores. All the maternal illness cases in the intervention area were quickly recognized as opposed to four of the eight cases in the comparison area. Unlike post-partum bleeding, newborn illnesses which are a much broader category of conditions were not easily nor quickly recognized. The newborn illness signs that were commonly recognized included baby cry- ing excessively, fever, failure to breast feed, difficulty in breathing, and diarrhea. Yellowing of the skin was one of the signs less easily recognized. The respondents who Care sought from multiple points up to 4 steps Most cases sought care from government health facilities and private clinics Care sought from 1 to 2 points Care sought mainly from government facilities and informal providers Maternal and newborn illness recognition In both the intervention and comparison areas, recogni- tion of maternal complications and danger signs was common. Maternal illness symptoms of excessive bleed- ing were generally quickly recognized as heavy bleeding with clots, bright red blood gushing out, having clothes and mattresses drenched with blood and frequently changing sanitary pads. “…..I was bleeding so much that whenever i put a pad, it would immediately get soaked with blood and I would have to change. I then knew that there was a problem” (maternal illness, 21-year-old mother, intervention). Table 3 Differences between maternal and newborn cases Domain Maternal cases Newborn cases Intervention area Comparison area Illness recognition Illnesses fairly quickly recognized Delay in recognition of illness––symptoms Illness seems to be more quickly recognized Interviews reveal better knowledge of danger signs Knowledge attained from the VHTs and Health workers Minimal influence of culture Larger delay in Illness recognition Less knowledge of danger signs Culture plays big role in delay in recognition Cause of illness and death mainly reported as biological Cause of illness and death unknown and mainly reported as God’s will. Other causes reported are witchcraft Decision makers The focal woman, husband, and mother in law are key decision makers The mother and mother in law are key decision makers Husband only contacted when a referral is required or the illness is complicated Care-seeking Care sought mainly from formal health care providers Although informal in a few cases First care-seeking point is home. Home remedies tried first before others Care sought from m points up to 4 step Most cases sought government health and private clinics Care-seeking relatively fast Delay in care-seeking Community initiatives influence Women report abo helpfulness of VSLA groups to facilitate Care sought from multiple points up to 4 steps Most cases sought care from government health facilities and private clinics Care-seeking relatively fast Delay in care-seeking Women report about the helpfulness of VSLAs/women’s groups to facilitate care-seeking Page 79 of 96 Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 illness. The role of the husbands was to provide finances for care-seeking. have babies with jaundice had initially thought the ba- bies were well until they worsened. In both intervention and comparison areas, respon- dents were able to mention the biomedical causes of maternal illnesses and death such as excessive bleeding after delivery, high blood pressure in pregnancy, and anemia. Maternal and newborn illness recognition For example, excessive bleeding was stated as the cause of a mother being anemic. “Most times our husbands left responsibilities to us as mothers, there are few men who care that the baby is sick and they have to be taken to the health center but most times it is us the mothers who have to look for a way you get your family health care” [FGD, women’s group, intervention] “They told me the bleeding was too much and the patient was bleeding non-stop so to me I feel the problem was this excessive bleeding, because they tried to give her the injection which stops bleeding but the bleeding continued so that was caused the woman to become anemic” [Maternal death, 31-year-old husband, intervention] Decision makers The key decision makers for maternal cases were the mothers-in-law, the focal woman herself, and her hus- band. In most cases, mothers-in-law were present at the time of the illness and influenced the decision-making process. Husbands were usually consulted even though they were sometimes not present in the home. In cases where the husband was away from home, the use of mo- bile phones was seen as critical in enhancing communi- cation between husbands and the caretakers; however, this caused delays in making decisions. The first point of care was at home for four and at a health center for three of the eight newborn deaths (Fig. 2). Remedies used at home included tak- ing baths, tepid sponging, and use of herbs among other home remedies. Four of the eight cases visited a health facility as the second step of care-seeking. Only one baby was brought to a hospital. Two ba- bies died before reaching the hospital, while four ba- bies died at the health center, one at home and one at an informal care provider’s (herbalists) practice. Care-seeking involved in one case, only one step; in four cases, two steps; in two cases, three steps; and in one case, four steps. “Based on the phone calls I (the husband) was getting from the caretaker I knew the condition was not good at all and at every point of progression they were calling me but her condition was instead worsening. I asked them to proceed to the health facility while I also tried to find my way there to meet with them” [Maternal death, 40-year-old husband, intervention] Care-seeking for maternal and newborn illnesses and deaths Generally, care-seeking patterns were similar for mater- nal illnesses and deaths and likewise for newborn ill- nesses and deaths studied. Families reported that care was sought from several providers before death (Fig. 1). For example, mother 1 first tried to manage the compli- cations at home and she died on the way to a health center. Mother 2 went first to seek care from an infor- mal provider, then to a health center, and died then fi- nally while seeking care in a hospital. The first point of care-seeking was the informal sector (1 of 8), the health center (1 of 8), and the private clinics (2 of 8) in the intervention district. In the comparison district, one mother sought care first at home, one in the informal sector, and one at the health center, and one died on the way to a health center. None of the mothers, either in the intervention or the comparison area, went straight to the hospital to seek care. The care-seeking steps in- volved for two mothers’ one step, four mothers’ two steps, and two mothers’ three steps. Four of the eight mothers died on the way to the health facility to seek care either during the first, second, or third steps. Two of the mothers died in a health facility, one in a hospital and one in a private clinic. On the contrary, the perceived causes of newborn ill- nesses and death were reported as unknown or associ- ated with non-medical influences including “God’s will” and witchcraft. In very few cases, medical causes, for ex- ample prematurity, were cited. “I think it was traditional because there were some women who came to see me after I had delivered and I think they are the ones who caused the death of my baby as one is a well-known witch” [Newborn death, 20-year-old mother, comparison] Patient experiences with care Regarding newborn illnesses, the key decision makers were the mothers of the sick newborns and the mothers- in-law (husband’s mother). They determined where the baby would be taken for care and at what point of the Quality of care was defined by the respondents often in terms of availability of drugs, presence of a health worker at all times, waiting time, health worker attitudes, user fees, and interpersonal relations. Provider-patient relationship Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Page 80 of 96 Fig. 1 Care-seeking patterns for maternal deaths “I prefer going to the clinic because they always have drugs. At the government health facility, they always ask us to buy drugs from the pharmacy in town” [Maternal illness, 22-year-old mother, intervention] was mentioned as one of the factors affecting utilization of services. Some respondents mentioned that the reason why they do not seek care from certain facilities is because the health workers are rude and unwelcoming. Notably, private health facilities were mentioned by nine of the respondents to have more client-focused care and friendly health workers as compared to public health facilities. Respon- dents mentioned that at the private facilities, they were likely to find health workers who were kind to them and gave them the necessary drugs required as opposed to public facilities. “At the government facility, the nurses are always very rude and abuse you for no reason” [Maternal illness, 24-year-old mother, comparison] The waiting time was reported by most respondents as a determinant of which facility to go to. In some facilities, Fig. 2 Care-seeking patterns for newborn illnesses Fig. 2 Care-seeking patterns for newborn illnesses Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Page 81 of 96 mothers had to queue for many hours before receiving services, especially in the public health facilities. This re- sulted in care-seeking from other places such as drug shops, private facilities, and in some cases, home remedies or no care sought at all. CHWs were also mentioned as key influencers of care- seeking especially in the intervention area. Patient experiences with care All respondents in the intervention area and half of the respondents in the comparison area stated that usually when they experienced an illness, they would first consult with the CHW who would either refer them or give them the necessary medi- cation. Consultation was either by phone or in person. “You can line in a queue from morning to 1:00 pm, and that is when the health workers go for lunch, you sit and wait for them to come back at 3:00 pm and they write for you that there are no drugs, so most times I do not go there, the only time that I go there is when am pregnant.” [Newborn illness, 28-year-old mother, intervention] “The VHTs/CHWs are very helpful. We always ask them when we have problems and they tell us what to do…either to go buy certain medicines or go to the health facility.” [FGD, community, intervention] In both the intervention and comparison areas, gener- ally, community members like neighbors/friends also fa- cilitated care-seeking by being caregivers, looking after the home or children when the sick mother sought care, or contributing food and money for care-seeking as well as for institutional delivery. Role of community initiatives in illness recognition and care-seeking for maternal and newborn illnesses The role of the community initiatives in maternal and newborn illness recognition and care-seeking was more apparent in the intervention than the comparison area (Table 3). These community initiatives included the women’s saving groups initiated in the intervention area as a quality improvement change idea to help women access funds for health care. “The people in the village helped and gave me money for the transport I used to go to the health facility to deliver and to come back home. When I came back people brought for me sugar and soap.” [Maternal illness, 38-year-old mother, comparison] The respondents from the 11 of the 12 event narra- tives (interviews) in the intervention area reported that they obtained knowledge about maternal and newborn danger signs mainly from formal health workers at the health facility, the CHW, or the Village Health Team (VHT) through village sensitization meetings and home visits. They reported further that CHWs used a visual aid to convey information on the danger signs, which was not the case in the comparison district. Discussion “In our women savings groups, when a woman reaches a time to deliver or has any sickness or her child is sick, she goes and she is given money and after she refunds it and it is given to another.” [FGD, women’s group, intervention] Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Page 82 of 96 Page 82 of 96 Page 82 of 96 advised and even referred patients to the formal facilities for care. Such community initiatives have been successfully implemented elsewhere for neonatal illnesses leading to an increase in care-seeking behaviors and improved newborn survival [16, 19]. symptoms prompting women and families to rather go immediately to a hospital. This could prevent delays. The review of referral and referral transport system was also mentioned as a key priority in the recently published Lancet Maternal Health series [27]. In order for a mother to seek care, decision-making was influenced by both the male partner or by older women such as a mother-in-law. However, men had lim- ited influence in care-seeking for newborn illness. This being a patriarchal society, men still dominate economic power and related decision-making [20]. Therefore, for most cases, the spouse/husband had to be contacted even when he was away from home. The mobile phones played a key role in communication with husbands who were away from home especially with regard to maternal illnesses. On the contrary, interviews for neonatal ill- nesses found that the mother of the sick newborn and her mother-in-law were the chief decision makers. This may have been due to the fact that informal care sought for the newborns has a less bearing on economic resources. In this setting, perhaps based on presumed experience, the mothers-in-law or older women seem to wield influence in terms of illness recognition and decision-making on whether to seek care or not and from where to seek care. This finding is similar in other patriarchal settings [21]. Despite some differences in car-seeking patterns for mothers and newborns, we found that the factors influ- encing care-seeking were similar for both maternal and newborn and also for the intervention and comparison areas. These ranged from availability of finances for health care, availability of transport, distance to facility, quality of care provided, attitudes of the health workers, and other community support systems. These are similar to findings elsewhere [22, 24, 28]. Discussion However, interviews in the intervention area reported that the financial bar- riers were mitigated through the formation of Women’s savings groups from which funds to seek care could be borrowed. These saving groups were formed as a result of community quality improvement change ideas in the intervention district. In these groups, monthly contribu- tions of 1000 Uganda shillings (about $0.3) are made, and members needing financial support can borrow and pay back later in installments. Members of these groups were therefore reported to borrow money to facilitate care in the event of illness or at the time of delivery, hence a big boost for care-seeking. Our findings support the value of community programs which are gaining popularity for improving care-seeking for maternal and newborn health, especially in LMICs [29–34]. Our quali- tative findings also support that CHWs can link the community and formal health facilities by referring patients to facilities for adequate treatment. Care-seeking for maternal and newborn illnesses was influenced by differences in illness recognition and per- ceived cause of the illness. Home-based care, including home remedies such as baths, herbal tonics, and prayers, were more often described as the first step of care for newborns. This is a similar finding to other studies done in India, Nepal, Bangladesh, and Ethiopia where home- based care is frequently tried first for newborn illnesses [22–24]. For maternal cases, care-seeking was described as more proactive after illness recognition probably as the recognition of severity was easier [15, 16, 20]. One strength of our study is that it provides insight to perceptions and experiences around illness recognition and care-seeking for maternal and newborn illnesses and deaths. This could inform health delivery improvement within the country. Our main limitation is that while our qualitative interviews covered an intervention and comparison area where a community approach was im- plemented, our study design prevents us from making comparative conclusions. Our sample was small. An- other limitation is the possibility of recall bias of the events that could have occurred. We mitigated this by conducting group interviews with the people who were available when the event occurred. Finally, our study setting was rural with few peri-urban communities, and hence, it may not be possible to generalize these findings to purely urban locations. Sick mothers and newborns are usually referred from one level to another following Uganda’s pyramidal health care system. Discussion In this study, we observed that illnesses such as post- partum hemorrhage which typically presents with clearly noticeable signs and symptoms were well recognized. In contrast, respondents reported greater difficulties in interpreting the more unspecific signs and symptoms of newborn illnesses. Care-seeking for both maternal and newborn death involved several steps and referrals. Often, a pattern from care-seeking at one or more facil- ities belonging to the primary level was described. In no case was the first point of care-seeking a hospital. “I learned about danger signs in mothers and babies through the VHT who visited my home while I was pregnant. So when my baby started breathing badly, I knew I had to go to a health facility” [Newborn illness, intervention] Illness recognition is a critical step to making a deci- sion to seek care. From our study findings, newborn illness was not well understood and formal care often delayed. The challenge in recognition of newborn ill- nesses may have been due to lack of specific symptoms or even masking of symptoms. This finding was similar in other settings in Uganda [4, 15, 16] and in Tanzania [17]. It may also be that the symptoms are actually seen, but severity is not recognized. Mothers and families seemed to be able to recognize bleeding after birth (post-partum hemorrhage), lack of blood (anemia), fever (which they called malaria), and hypertension in pregnancy (which they identified with swelling of the feet and the face). Respon- dents from the intervention districts explained that CHWs facilitated recognition of illnesses through sensitization of communities on maternal and newborn danger signs and hence reducing delay one [15, 18]. These CHWs often Respondents from the case interviews and FGDs in the intervention area mentioned factors such as presence of vil- lage saving groups/women’s groups, CHW, and community support systems as influencers of care-seeking. The pres- ence of village saving groups, especially the women’s saving groups, was reported as a chief facilitator of care-seeking in the intervention district. Saving groups were not present in the comparison district as this was one of the change ideas initiated by the EQUIP project at community level. Discussion However, in 50% maternal death cases and five of the eight cases of newborns deaths, the caretakers were advised to seek care at a higher level while referral with an ambulance to the hospital could not be organized. This finding is similar to other low- and middle-income countries (LMICs) such as Ethiopia where health centers were found to be physical barriers to access [25, 26]. Indeed, in many cases, mothers died on the way during this “empty referral.” These findings call for the need to revisit the role of primary facilities for emergency care. Ideally, primary facility should provide emergency care and prompt referral if needed. However, our interviews in- dicate that the primary facilities were not able to do so. While upgrading of facilities to provide better emergency care and organize referral is paramount, care-seeking advice during counseling might also talk about signs and Conclusions Our qualitative findings propose that CHWs and community-based interventions may play a role in early illness recognition and care-seeking for sick mothers and babies. Interviews suggested that recognition of newborn Page 83 of 96 Page 83 of 96 Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Competing interests The authors declare that they have no competing interests. illness was difficult, and care-seeking often limited or delayed. Maternal danger signs were more consistently reported and recognized. The role of primary facilities in providing care for maternal and newborn emergencies needs to be reviewed to prevent delays by following the pyramidal health system. Competing interests Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abbreviation 1Department of Health Policy, Planning and Management, Makerere University School of Public Health, Kampala, Uganda. 2Center of Excellence for Maternal, Newborn and Child Health, Makerere University, Kampala, Uganda. 3Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden. 4School of Medicine/AMPATH, Moi University, Nairobi, Kenya. 5London School of Hygiene and Tropical Medicine, London, UK. 6Ifakara Health Institute, Dar es Salaam, Tanzania. CHW: Community health worker; EQUIP: Expanded Quality Management Using Information Power; FGDs: Focus group discussions; LMICs: Low- and middle-income countries; QI: Quality improvement; QIT: Quality improvement team; UNEST: Uganda Newborn Study Ethics approval and consent to participate This research was approved by the Makerere University School of Public Health Higher Degrees Research and Ethics Committee (125th meeting, protocol no. 116) and Uganda National Council of Science and Technology (HS1085). Permission was also obtained from the Mayuge and Namaingo District Health Offices. Written informed consent was obtained from all participants using a consent form translated to the local language-Lusoga for clear understanding. 17. Hanson C, et al. Health system support for childbirth care in Southern Tanzania: results from a health facility census. BMC research notes. 2013;6(1):435. 17. Hanson C, et al. Health system support for childbirth care in Southern Tanzania: results from a health facility census. BMC research notes. 2013;6(1):435. 18. Waiswa P, et al. Effect of the Uganda Newborn Study on care-seeking and care practices: a cluster-randomised controlled trial. Glob Health Action. 2015;8 18. Waiswa P, et al. Effect of the Uganda Newborn Study on care-seeking and care practices: a cluster-randomised controlled trial. Glob Health Action. 2015;8 19. Roy SS, et al. Improved neonatal survival after participatory learning and action with women's groups: a prospective study in rural eastern India. Bull World Health Organ. 2013;91(6):426–433B. 19. Roy SS, et al. Improved neonatal survival after participatory learning and action with women's groups: a prospective study in rural eastern India. Bull World Health Organ. 2013;91(6):426–433B. Funding Th d This study and publication were funded by the US Agency for International Development through the Translating Research into Action (TRAction) project under cooperative agreement no. GHS-A-00-09-00015-00. 8. Kilo CM. A framework for collaborative improvement: lessons from the Institute for Healthcare Improvement’s Breakthrough Series. Qual Manag Healthcare. 1998;6(4):1–14. 8. Kilo CM. A framework for collaborative improvement: lessons from the Institute for Healthcare Improvement’s Breakthrough Series. Qual Manag Healthcare. 1998;6(4):1–14. Availability of data and materials 9. World Health Organisation, The Aga Khan University, and The Partnership for Maternal Newborn & Child Health, Essential Interventions, commodities and guidelines. A global review of key interventions related to reproductive, maternal, newborn and child health (RMNCH). Geneva: PMNCH; 2011. 9. World Health Organisation, The Aga Khan University, and The Partnership for Maternal Newborn & Child Health, Essential Interventions, commodities and guidelines. A global review of key interventions related to reproductive, maternal, newborn and child health (RMNCH). Geneva: PMNCH; 2011. The datasets used in this study are not yet publicly available due to institutional restrictions on data use during the first 3 years after finalization of the study. However, data and materials may be made available upon reasonable request from the corresponding author. 10. Waiswa P, et al. Poor newborn care practices-a population based survey in eastern Uganda. BMC pregnancy and childbirth. 2010;10(1):1. 10. Waiswa P, et al. Poor newborn care practices-a population based survey in eastern Uganda. BMC pregnancy and childbirth. 2010;10(1):1. g g y 11. UBOS. National population and housing census 2014. Uganda: Uganda Bureau of Statistics; 2014. 11. UBOS. National population and housing census 2014. Uganda: Uganda Bureau of Statistics; 2014. About this supplement This article has been published as part of Journal of Health, Population and Nutrition Volume 36 Supplement 1, 2017: How Households Identify and Respond to Illness: A Systematic Qualitative Study of Recognition and Care-Seeking in Seven Countries. The full contents of the supplement are available online at https://jhpn.biomedcentral.com/articles/supplements/volume-36-supplement-1. 12. Uganda Bureau Of Statistics, Uganda Demographic and Health Survey 2011, Uganda Bureau Of Statistics and ICF International Inc, Editor. 2012: Kampala, Uganda and Calverton, Maryland. Uganda; 2012. 12. Uganda Bureau Of Statistics, Uganda Demographic and Health Survey 2011, Uganda Bureau Of Statistics and ICF International Inc, Editor. 2012: Kampala, Uganda and Calverton, Maryland. Uganda; 2012. 13. Moran AC, Charlet D, Madhavan S, Aruldas K, Donaldson M, Manzi F, et al. Methodology for a mixed methods multi-country study to assess recognition of and response to maternal and newborn illness. Journal of Health, Population and Nutrition. (41043–36-S1-S1 in this issue). Published: 21 December 2017 Published: 21 December 2017 Published: 21 December 2017 References 1 WHO H 1. WHO. Health in 2015: from MDGs, millenium development goals to SDGs, Sustainable Development Goals. France: WHO; 2015. 1. WHO. Health in 2015: from MDGs, millenium development goals to SDGs, Sustainable Development Goals. France: WHO; 2015. 1. WHO. Health in 2015: from MDGs, millenium development goals to SDGs, Sustainable Development Goals. France: WHO; 2015. 2. UBOS, Uganda Demographic and Health Survey 2016, UBOS and ICF Inc, Editor. 2016: Kampala, Uganda. 2. UBOS, Uganda Demographic and Health Survey 2016, UBOS and ICF Inc, Editor. 2016: Kampala, Uganda. 3. Thaddeus S, Maine D. Too far to walk: maternal mortality in context. Soc Sci Med. 1994;38(8):1091–110. 4. Waiswa P, et al. 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The findings of this study are the sole responsibility of the authors and do not necessarily reflect the views of USAID or the US government. 5. Villadsen, S. and Lubanga, F., Democratic decentralisation in Uganda: a new approach to local governance. 1996: Fountain Publishers. 6. Kananura RM, et al. The neonatal mortality and its determinants in rural communities of eastern Uganda. Reprod Health. 2016;13(1):1. 7. Hanson C, et al. Expanded Quality Management Using Information Power (EQUIP): protocol for a quasi-experimental study to improve maternal and newborn health in Tanzania and Uganda. Implement Sci. 2014;9(1):41. Authors’ contributions MO, PW, CL, and FM designed the study. MO, PW, RM, and JW adapted the tools and collected the data. MO, RM, and JW analyzed the data with support from PW, CL, and FM. MO, JW, and PW wrote the first draft of the manuscript. All authors contributed to the subsequent revisions and approved the final manuscript. 14. Muhr T. 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This work is part of a multi-country research conducted under the Translating Research into Action (TRAction) project funded by the US Agency for International Development (USAID). The countries involved include Uganda, Tanzania, Ethiopia, Nigeria, Indonesia, India, and Nepal. Special thanks go to TRAction for the opportunity and funding to conduct this work. We also wish to extend our gratitude to TRAction and AMPATH (Kenya) for the technical support provided. Consent for publication 20. Timsˇa L, et al. Strategies for helping families prepare for birth: experiences from eastern central Uganda. Glob Health Action. 2015;21(31):37. 20. Timsˇa L, et al. Strategies for helping families prepare for birth: experiences from eastern central Uganda. Glob Health Action. 2015;21(31):37. All the study participants were informed about the aim of the research project and gave their consent to participate and publish the findings. Page 84 of 96 Okuga et al. 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Recognizing childhood illnesses and their traditional explanations: exploring options for care-seeking interventions in the context of the IMCI strategy in rural Ghana. Tropical Med Int Health. 2003;8(7):668–76. 29. Baqui AH, et al. Community-based validation of assessment of newborn illnesses by trained community health workers in Sylhet district of Bangladesh. Okuga et al. Journal of Health, Population and Nutrition 2017, 36(Suppl 1):47 Consent for publication Tropical Med Int Health. 2009;14(12):1448–56. 30. Bhutta ZA, et al. Community-based interventions for improving perinatal and neonatal health outcomes in developing countries: a review of the evidence. Pediatrics. 2005;115(2 Suppl):519–617. 31. Bhutta ZA, et al. Improvement of perinatal and newborn care in rural Pakistan through community-based strategies: a cluster-randomised effectiveness trial. Lancet. 2011;377(9763):403–12. 32. Kumar V, et al. Community-driven impact of a newborn-focused behavioral intervention on maternal health in Shivgarh, India. Int J Gynaecol Obstet. 2012;117(1):48–55. 33. Lassi ZS, Haider BA, Bhutta ZA. Community-based intervention packages for reducing maternal and neonatal morbidity and mortality and improving neonatal outcomes. Cochrane Database Syst Rev. 2010;11:CD007754. 34. Dynes M, et al. Home-based life saving skills in Matlab, Bangladesh: a process evaluation of a community-based maternal child health programme. Midwifery. 2011;27(1):15–22. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W4212795930	https://durham-repository.worktribe.com/file/1207950/1/Published%20Journal%20Article	English	null	Fast reconstruction of centre of mass and foot kinematics during a single-legged horizontal jump: A point-cloud processing approach	Journal of biomechanics	2,022	cc-by	5,199	Fast reconstruction of centre of mass and foot kinematics during a single-legged horizontal jump: A point-cloud processing approach Fast reconstruction of centre of mass and foot kinematics during a single-legged horizontal jump: A point-cloud processing approach S.J. McLaren a,b, W. Evans c, B. Galna d,e, M.D. Portas g,h, M. Weston f, I.R. Spears d,* S.J. McLaren a Newcastle Falcons Rugby Club, Newcastle upon Tyne, UK b Department of Sport and Exercise Sciences, Durham University, Durham, UK c Faculty of Health Sciences and Wellbeing, Exercise, Sport and Rehabilitative Therapies, University of Sunderland, Sunderland, UK d School of Biomedical, Nutritional, and Sport Sciences, Newcastle University, Newcastle, UK e College of Science, Health, Engineering and Education, Murdoch University, Western Australia, Australia f Institute for Sport, Physical Education & Health Sciences, University of Edinburgh, UK g Technical Directorate, The Football Association, St. George’s Park, Burton upon Trent, UK h Center for Rehabilitation, School of Health and Life Sciences, Teesside University, Middlesbrough, Teesside, UK A R T I C L E I N F O Keywords: Movement screening Gait analysis Centre of mass Markerless Horizontal jumps are discrete, fast, over-ground movements requiring coordination of the centre of mass (CoM) and base of support and are routinely assessed in sports settings. There is currently no biomechanics-based system to aid in their quick and objective large-scale assessment. We describe a practical system combining a single low-cost depth-sensing camera and point-cloud processing (PCP) to capture whole-body CoM and foot kinematics. Fourteen participants performed 10 single-leg horizontal jumps for distance. Foot displacement, CoM displacement, CoM peak velocity and CoM peak acceleration in the anterior-posterior direction of movement were compared with a reference 15-segment criterion model, captured concurrently using a nine-camera motion capture system (Vicon Motion Systems, UK). Between-system Pearson’s correlations were very-large to near- perfect (n = 140; foot displacement = 0.99, CoM displacement = 0.98, CoM peak velocity = 0.97, CoM peak acceleration = 0.79), with mean biases being trivial–small (-0.07 cm [0.12%], 3.8 cm [3.5%], 0.03 m⋅s−1 [1.6%], 0.42 m⋅s−2 [7%], respectively) and typical errors being trivial–small for displacement (foot: 0.92 cm [0.8%]; CoM: 3.8 cm [3.4%]) and CoM peak velocity (0.07 m⋅s−1 [4.3%]), and large for CoM peak acceleration (0.72 m⋅s−2 [15%]). Limits of agreement were −1.9 to 2.0 cm for foot displacement, −11.3 to 3.6 cm for CoM displacement, −0.17 to 0.12 m⋅s−1 for CoM peak velocity and −2.28 to 1.43 m⋅s−2 for CoM peak acceleration. The practical system captured CoM and foot kinematics during horizontal jumps with acceptable precision. Further work to improve estimates of CoM accelerations and different populations are warranted. * Corresponding author at: School of Biomedical, Nutritional and Sport Sciences, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK. E-mail address: Iain.Spears@Newcastle.ac.uk (I.R. Spears). Journal of Biomechanics 135 (2022) 111015 Journal of Biomechanics 135 (2022) 111015 a Newcastle Falcons Rugby Club, Newcastle upon Tyne, UK b Department of Sport and Exercise Sciences, Durham University, Durham, UK c Faculty of Health Sciences and Wellbeing, Exercise, Sport and Rehabilitative Therapies, University of Sunderland, Sunderland, UK d School of Biomedical, Nutritional, and Sport Sciences, Newcastle University, Newcastle, UK e College of Science, Health, Engineering and Education, Murdoch University, Western Australia, Australia f Institute for Sport, Physical Education & Health Sciences, University of Edinburgh, UK g Technical Directorate, The Football Association, St. George’s Park, Burton upon Trent, UK h Center for Rehabilitation, School of Health and Life Sciences, Teesside University, Middlesbrough, Teesside, UK Available online 22 February 2022 0021-9290/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.jbiomech.2022.111015 Accepted 17 February 2022 Available online 22 February 2022 0021-9290/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author at: School of Biomedical, Nutritional and Sport Sciences, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK. E-mail address: Iain.Spears@Newcastle.ac.uk (I.R. Spears). https://doi.org/10.1016/j.jbiomech.2022.111015 Accepted 17 February 2022 1. Introduction Skeletal tracking, in which arti ficial intelligence (AI) is used on images to infer on whole-body joint positions (Colyer et al., 2018), provides accurate estimates of kinematic parameters in some poses (Galna et al., 2014; Eltoukhy et al., 2017). The errors for foot position, however, can be quite high (>10 cm (Xu and McGorry, 2015)). In contrast, point cloud processing (PCP), in which raw depth data is converted directly into 3D landmark coordinates, has been shown to achieve greater levels of accuracy. Notably, studies using PCP have consistently reported errors of <1 cm for the foot (Paolini et al., 2014), ankle (Geerse et al., 2019), pelvis (MacPherson et al., 2016) and knee (Timmi et al., 2018). In addition, PCP has also been applied (albeit using multiple cameras) to measure CoM kinematics with similar levels of accuracy (Kaichi et al., 2019). To date, PCP has so far been restricted to the analysis of cyclical, slow and relatively stationary activities. Whether this technology can track simultaneously the kinematics of the foot and CoM during discrete, fast over-ground movements involved in the horizontal jump remains to be Fig. 1. a) Schematic representation of the capture setup (i) used by the two systems to capture concurrently the movements of athlete during a single (right to left). Also shown is the global origin (0,0,0) of the criterion system and the rigid calibration frame (P1, P2, P3 and P4). Jumping was per positive y-direction (anterior) towards the low-cost camera. In the sagittal view (lower image), the ‘clean’ point cloud along with the trajectories of th CoM (long-dashed line) and the left foot marker (short-dashed line) are shown. Note the two localised minima of the y-position of the whole-body CoM t to anchor the data from the two systems (20% and 55% of the jump cycle). Also shown are the ‘clean’ point clouds in frontal view (z-x plane) in c infrared (iii). The calculation of whole-body CoM uses all these points, whereas calculation of foot marker position uses only the point at the virtual midp the 2 strips of reflective (highlighted with a cross on the infrared image (iii). b) Time-normalised kinematics from Vicon (blue) and PCP (yellow) (mean CoM in the y-direction (n = 1200) are shown. Overlapping regions of the standard deviations are shown in green. Note that all y-axes are scaled to s between maximal and minimal data points on the time-series. 1. Introduction plates or marker-based motion capture (Colyer et al., 2018). In field settings, jump performance is assessed using a tape measure (McCubbine et al., 2018) and technique assessed visually (Padua et al., 2015). Such methods are time-consuming and often with low inter-rater reliability (Lindblom et al., 2021). There are other commercial systems used in the field based on planar switches (e.g., Optojump (Microgate, Italy)) but these are can only track the feet, potentially missing important features of jump performance. Movement screening forms a regular component of athlete moni toring, providing important information on general movement skills and physical performance potentials (Read et al., 2017). Horizontal jumps are common to many screening batteries as a proxy measure of explosive ability (e.g., Strokosch et al., 2018). These tests involve a coordinated pattern of countermovement, body rotation and arm swing to generate maximal anterior-posterior displacement, velocity, and acceleration of the centre of mass (CoM) on take-off and then control CoM above the new landed position of the feet (Wakai and Linthorne, 2005). In research settings, these kinematic outcomes are quantified directly using force There are several emerging technologies for the simultaneous mea surement of foot and whole-body CoM kinematics which have potential for monitoring jump performance. Studies using multi-segment inertial measurement units have reported errors for feet and CoM positions of Journal of Biomechanics 135 (2022) 111015 S.J. McLaren et al. <1 cm and <2.57 cm, respectively (Fasel et al., 2017). While likely to be acceptable for the present purposes, the costs and ease-of-use for large- scale screening programmes are prohibitive. A potential alternative is computer vision (Colyer et al., 2018). Skeletal tracking, in which arti ficial intelligence (AI) is used on images to infer on whole-body joint positions (Colyer et al., 2018), provides accurate estimates of kinematic parameters in some poses (Galna et al., 2014; Eltoukhy et al., 2017). The errors for foot position, however, can be quite high (>10 cm (Xu and McGorry, 2015)). In contrast, point cloud processing (PCP), in which raw depth data is converted directly into 3D landmark coordinates, has <1 cm and <2.57 cm, respectively (Fasel et al., 2017). While likely to be acceptable for the present purposes, the costs and ease-of-use for large- scale screening programmes are prohibitive. A potential alternative is computer vision (Colyer et al., 2018). 1. Introduction c) Limits of agreements (Bland and Altman, 1986) for the two systems (±1.96SD) for foot (i), CoM displacement (ii), CoM peak velocity (iii) and CoM peak acceleration (iv). Fig. 1. a) Schematic representation of the capture setup (i) used by the two systems to capture concurrently the movements of athlete during a single-legged jump (right to left). Also shown is the global origin (0,0,0) of the criterion system and the rigid calibration frame (P1, P2, P3 and P4). Jumping was performed in the positive y-direction (anterior) towards the low-cost camera. In the sagittal view (lower image), the ‘clean’ point cloud along with the trajectories of the whole-body CoM (long-dashed line) and the left foot marker (short-dashed line) are shown. Note the two localised minima of the y-position of the whole-body CoM that were used to anchor the data from the two systems (20% and 55% of the jump cycle). Also shown are the ‘clean’ point clouds in frontal view (z-x plane) in colour (ii) and infrared (iii). The calculation of whole-body CoM uses all these points, whereas calculation of foot marker position uses only the point at the virtual midpoint between the 2 strips of reflective (highlighted with a cross on the infrared image (iii). b) Time-normalised kinematics from Vicon (blue) and PCP (yellow) (mean ± SD) for the CoM in the y-direction (n = 1200) are shown. Overlapping regions of the standard deviations are shown in green. Note that all y-axes are scaled to span the range between maximal and minimal data points on the time-series. c) Limits of agreements (Bland and Altman, 1986) for the two systems (±1.96SD) for foot displacement (i), CoM displacement (ii), CoM peak velocity (iii) and CoM peak acceleration (iv). 2 S.J. McLaren et al. Journal of Biomechanics 135 (2022) 111015 representation of the anterior surface of the athlete (Fig. 1a). The posi tion of the CoM (i.e., the point about which the summed moment of all points in the cloud was zero) was then calculated in the x-, y- and z- directions (i.e., xCoM, yCoM, zCoM) using the following: determined. This study will describe the development and examine the criterion validity of PCP for the quantification of single-leg horizontal jump performance (Fig. 1ai) in terms of displacement, velocity and ac celeration outcomes. 2. Methods The camera was tilted by −30◦ about the x-axis. This configuration was considered optimal in terms of maximising the capture volume, as determined by trial-and-error. Before the tests, a rigid calibration frame (600 × 2000 mm) positioned 740 mm anterior to the global origin was used to create a transformation matrix. Specifically, four strips (5 × 5 mm) of retroreflective tape were glued to the apices of the frame (Fig. 1ai, Superior View) giving four coplanar points (P1, P2, P3 and P4) in both systems. The calibration frame was then removed from the testing area, and subsequent point cloud data were transformed from the camera to the criterion global system. y Statistical Analysis: Since our aims are to assess the agreement be tween two measurement systems, rather than to examine any biological outcomes, data from all participants (n = 14) and their trials (n = 10 pp) were treated as independent measures (i.e., n = 140 datapoints per outcome measure). We used separate linear regressions (SPSS Version 24, IBM Corp., Armonk, NY, USA) to examine the criterion-related validity of the foot displacement and whole-body COM kinematics. Criterion-derived values of the outcome measures were entered as separate dependent variables and the corresponding PCP-derived values were entered as independent variables. Relationship strength was quantified with Pearson’s product moment correlation coefficient (r), with the associated R2 value (coefficient of determination) used to ex press the proportion of explained variance. Additionally, the intraclass correlation coefficient was calculated using a two-way mixed effects model (ICC3,1), but these are not reported given that the values were all within ± 0.0002 of the Pearson’s r for displacement and velocity and ±0.0274 for accelerations. Typical errors ([TE], or standard errors of the estimate) were used to represent unexplained (random) bias. The mean difference between PCP and the criterion was used to represent sys tematic (mean) bias. Finally, Bland & Altman’s 95% limits of agreement were calculated by adding and subtracting 1.96 times the standard de viation of the difference (PCP–criterion) in paired measurements (Bland and Altman, 1986). i Following Paolini et al. (2014), coloured markers were attached to the feet of each participant (Fig. 1aii), enabling to reconstruct the foot position from the point cloud data. These foot markers included two retroreflective strips spaced 70 mm apart, causing two regions of localised overexposure of the infrared image (Fig. 1aiii). Following MacPherson et al. 2. Methods where MTotal is the mass of the participant, n is the number of points in the ‘clean’ point cloud, mk is the mass of each point averaged across the surface (i.e., mass of the participant / number of points) and xk, yk and zk are global (i.e., transformed) coordinates of individual points (Fig. 1aii). The study received ethical approval from The University of Sunder land’s Ethics committee. Fourteen physically active males (age: 28 ± 10 years, stature: 181 ± 9 cm, body mass: 82 ± 10 kg, BMI: 24.9 ± 2.7 kg⋅m−2) volunteered and provided written informed consent. All par ticipants were free from injury and, after a warm-up, performed single- legged horizontal jumps at one-minute intervals within the capture volume of the PCP and laboratory systems (Fig. 1ai). Data processing: The displacement data from both systems were differentiated to yield velocities and accelerations. All data were then time-normalised to a percentage of the jump cycle (Fig. 1b), using the first (20% of jump cycle) and second trough (55% of jump cycle) of the zCoM time-series data as anchor points (shown as dashed line in Fig. 1ai, Sagittal View). The normalised data in the y-direction (anterior-poste rior) were processed to yield outcome measures of jump performance, which were: displacement of the feet (cm) defined as the distance be tween the right and left foot markers at 20% and 55%, respectively; displacement of the CoM (cm) defined as the distance between 20% and 55%; peak velocity and acceleration defined as the highest positive ve locity (m⋅s−1) and acceleration (m⋅s−2) in the y-direction throughout the cycle. Criterion three-dimensional system: The criterion method of quanti fying foot and CoM kinematics was a nine-camera optoelectronic system (Bonita B10, Vicon motion systems, Oxford, UK) at 100 Hz. Using a 19- segment plug-in gait model, markers were placed bilaterally on anatomical landmarks (Vicon motion systems, Oxford, UK). Trajectory data were low-pass filtered using a fourth-order Butterworth filter with cut-off frequency of 6 Hz. Depth sensor system: The PCP-based system created is based on custom-written algorithms developed by Pro-Football Support Ltd using C# script in the Unity3D gaming engine. A low-cost depth sensing camera (Kinect™ V2, Microsoft, USA) was positioned at 0 mm, 1850 mm, and 3740 mm in the medial–lateral (x-axis), superior-inferior (z- axis) and anterior-posterior (y-axis) directions relative to the global origin of the criterion system (Fig. 1ai). 1. Introduction This single-legged jump is a more challenging version of the standing long jump, requiring the athlete to jump as far as possible horizontally from one foot to the other - requiring them to control their CoM in relation to a small base of support on landing. The specific aim of our study is to quantify the criterion validity of the displacement, velocity and acceleration outcomes based on PCP against those from a laboratory-grade system for the single-legged jump. xCoM = ∑n k=0 mk.xk MTotal yCoM = ∑n k=0 mk.yk MTotal zCoM = ∑n k=0 mk.zk MTotal (1 yCoM = ∑n k=0 mk.yk MTotal zCoM = ∑n k=0 mk.zk MTotal (1) 3. Results Between 0 and 20% of the jump cycle, the CoM moves laterally to above the position of the standing foot and then in a shallow counter movement (i.e., trunk, hip, knee and ankle flexion) the z-position of the CoM falls (Fig. 1ai). At the same time, the athlete begins to shift the CoM anteriorly relative to the base of support, thus creating anterior misalignment between the COM and base of support. The athlete is then able to move CoM horizontally (Fig. 1bi) during the push-off (20–30%) by extending the joints. The peak velocity of the CoM in the y-direction occurs between 30 and 40% of the cycle (Fig. 1bii) and the CoM is decelerated abruptly thereafter (Fig. 1biii). The athlete attempts to control the CoM above the base of support provided by the landed foot and hold this position until the end of the trial. There was general agreement between the systems for all three kinematic variables in the y- direction, although the PCP tended to overestimate positive accelera tions during take-off and underestimate the negative accelerations during landing (Fig. 1biii). There are important limitations to this study. First, the current single camera was only able to capture at 30 Hz, a possible reason for the only large typical error for peak accelerations (~15%). Further improve ments such as higher sampling or multiple cameras may be required to accurately quantify acceleration-based CoM variables. Second, camera position and orientation relative to the movements were optimised to capture volume, but not accuracy. Further work to optimise position and orientation (e.g., Yeung et al., 2021) may assist in reducing the errors present. Third, our sample was quite homogeneous in terms of sex and training status. Further work using female and/or highly trained (elite) athletes may produce different results, possibly being susceptible to differences in body compositions and/or kinematics. Fourth, we have not modelled all possible performance outcomes related to the foot and CoM relationship: it is not known how these errors propagate when other measures, such as dynamic balance (Hrysomallis, 2011), are calculated. The results of the validity analysis are shown in Table 1 and Fig. 1c. The association (r) between the systems for outcome measures were near perfect for foot displacement, CoM displacement and peak velocity, and very large for peak acceleration. Table 1 Table 1 Validity analysis between point-cloud processing (PCP) and criterion-derived estimates of jump performance during the single-leg jump. Outcome Measure Performance* (mean ± SD) r (±90% CL)* R2 Mean bias (±90% CL) Typical Error (£/÷90% CL) Raw Units Standardized (d)a Raw Units Standardized (d)b Total Foot Displacement (cm) 140.5 ± 27.2 0.999; ±0.0002 0.999 −0.07 (0.15) 0.00 (0.01) 0.92 (1.12) 0.03 (1.17) Total CoM Displacement (cm) 126.5 ± 21.2 0.983; ±0.005 0.967 3.84 (0.6) 0.18 (0.03) 3.83 (1.12) 0.18 (1.17) CoM Peak Velocity (m⋅s−1) 1.84 ± 0.30 0.973; ±0.009 0.946 0.03 (0.01) 0.09 (0.04) 0.07 (1.12) 0.24 (1.18) CoM Peak Acceleration (m⋅s−2) 5.49 ± 1.46 0.792; ±0.059 0.627 0.42 (0.15) 0.38 (0.13) 0.72 (1.12) 0.86 (1.23) CL, confidence limits. * from the PCP. a < 0.2 = trivial, 0.2–0.6 = small, 0.6–1.2 = moderate, >1.2 = large. b < 0.1 = trivial, 0.1–0.3 = small, 0.3–0.6 = moderate, >0.6 = large. Table 1 Validity analysis between point-cloud processing (PCP) and criterion-derived estimates of jump performance during the single-leg jump. Outcome Measure Performance* (mean ± SD) r (±90% CL)* R2 Mean bias (±90% CL) Typical Error (£/÷90% CL) Raw Units Standardized (d)a Raw Units Standardized (d)b Total Foot Displacement (cm) 140.5 ± 27.2 0.999; ±0.0002 0.999 −0.07 (0.15) 0.00 (0.01) 0.92 (1.12) 0.03 (1.17) Total CoM Displacement (cm) 126.5 ± 21.2 0.983; ±0.005 0.967 3.84 (0.6) 0.18 (0.03) 3.83 (1.12) 0.18 (1.17) CoM Peak Velocity (m⋅s−1) 1.84 ± 0.30 0.973; ±0.009 0.946 0.03 (0.01) 0.09 (0.04) 0.07 (1.12) 0.24 (1.18) CoM Peak Acceleration (m⋅s−2) 5.49 ± 1.46 0.792; ±0.059 0.627 0.42 (0.15) 0.38 (0.13) 0.72 (1.12) 0.86 (1.23) CL confidence limits ng (PCP) and criterion-derived estimates of jump performance during the single-leg jump ween point-cloud processing (PCP) and criterion-derived estimates of jump performance during the single-leg jump. communicate effect magnitude. depend largely on how accurate the system needs to be. Accordingly, we provided a more standardized interpretation of our findings for this task and found trivial mean biases for all outcome measures, with typical errors being trivial for displacement, small for velocity and large for accelerations. Furthermore, the 95% limits of agreement of foot displacement (−1.9 to 2.0 cm), for example, are small compared the variation of performance between young active adults (group standard deviations, male: 19.3; female: 12.8 cm) (Meylan et al., 2009). 3. Results Mean biases were trivial for total foot displacement (<0.2%) and CoM peak velocity (~1.5%), CoM total displacement (3.5%), and small for CoM peak acceleration (~7%). The typical errors were trivial for total foot displacement (~1%), small for total CoM displacement (~3.5%) and CoM peak velocity (~4%), and large for CoM peak acceleration (~15%). The limits of agreement (Fig. 1c) for foot displacement (−1.9 cm to 2.0 cm), CoM displacement (−11.3 cm to 3.6 cm), CoM peak velocity (−0.17 to 0.12 m⋅s−1) and CoM peak acceleration (−2.28 to 1.43 m⋅s−2). Table 1 Our data therefore suggest that, although not perfect, both foot and CoM displacement may be quantified with acceptable precision to detect small but worthwhile changes. However, velocities and accelerations may need further work, and this may entail different camera orienta tions, higher resolution, multiple cameras and/or higher sampling frequency. CRediT authorship contribution statement S.J. McLaren: Conceptualization, Resources, Validation, Writing – original draft, Writing – review & editing, Methodology, Data curation. W. Evans: Investigation, Validation, Data curation. B. Galna: Concep tualization, Validation, Writing – review & editing, Methodology. M.D. Portas: Conceptualization, Funding acquisition, Project administration, Resources, Writing – review & editing. M. Weston: Validation, Writing – review & editing, Methodology. I.R. Spears: Conceptualization, Fund ing acquisition, Investigation, Project administration, Software, Vali dation, Visualization, Writing – review & editing. 4. Discussion The project received government funding from a Knowledge Transfer Partnership (Innovate UK) to Pro Sport Support Ltd and Teesside Uni versity (KTP 009965). Biomechanical analysis of movement screening tests could play an important role in both athletic and clinical settings. In these areas, ex pediency and validity are highly valued. Despite its simplicity, the PCP- system showed excellent criterion validity with a 3D motion analysis system in tracking whole-body CoM and feet markers simultaneously during a single-legged horizontal jump. Typical errors between the systems in foot displacements were 0.94 cm (<1%) which are consid ered acceptable in field-testing (McCubbine et al., 2018). The errors in CoM displacement were 3.8 cm, being similar to other practical mea sures used in gait research (3 cm, Yang and Pai, 2014; 4 cm Huntley et al., 2017), but slightly larger than those from inertial suits (2.6 cm, Fasel et al., 2017). A key advantage of the PCP over other technologies is the simplicity of data collection. Following a ten-minute setup, the system was able run continuously to capture and display outcome measures within 300 ms of task completion. 2. Methods (2016), a virtual midpoint between these regions was created (Fig. 1aiii, inset), enabling to identify a pixel at the centre of the foot marker. These pixel coordinates (2D) were then fed into the 3D point cloud data to acquire the relevant 3D position of the foot marker. The whole-body CoM reconstruction used markerless PCP on the entire point cloud (Fig. 1aii and iii). The processing involved 5 stages con ducted on a frame-by-frame basis. First, points visible in the current frame and before the tests were identified and removed. Second, points with less than 5 neighbouring points within a radius of 5 cm were identified and removed. Third, a mean 3D centroid position of all remaining points was calculated and used to position a cylindrical vol ume around the athlete (shown as a blue circle and rectangle in Superior and Sagittal view, respectively [Fig. 1ai]). The dimensions of the cyl inder (height = 2 m, diameter 1.2 m) were determined prior to testing and considered to be optimal in terms of maximising the number of points used, whilst minimising the risk of random clusters (due to camera artefacts, reflection etc) in the calculation of CoM. Points outside of this volume were removed, thus leaving a ‘clean’ point cloud Uncertainty in all estimates were expressed using 90% confidence limits (CL), calculated from the t-distribution for mean differences, the z- distribution for (transformed) correlation coefficients and the chi- squared distribution for SEE. We declared the magnitude of correla tion coefficients as small moderate, large, very large and near perfect based on standardized anchors of 0.1, 0.3, 0.5, 0.7 and 0.9, respectively (Hopkins et al., 2009). To provide a standardized interpretation of mean bias (i.e., d), we used 0.2, 0.6 and 1.2 of the pooled between-participant standard deviation for each outcome measure (Table 1) to represent small, moderate and large differences (Hopkins et al., 2009). These thresholds were then halved to declare practical magnitudes of SEEs (Smith & Hopkins, 2011). 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https://openalex.org/W2596478656	https://europepmc.org/articles/pmc5355995?pdf=render	English	null	Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis	Scientific reports	2,017	cc-by	8,437	Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis Xingchao Liu, Yaqing Cheng, Mengya Lyu, Ying Wen, Yuan Song, Zhi Chen & Jilun L received: 22 November 2016 accepted: 09 February 2017 Published: 17 March 2017 gchao Liu, Yaqing Cheng, Mengya Lyu, Ying Wen, Yuan Song, Zhi Chen & Jilun Li The regulatory role of redox-sensing regulator Rex was investigated in Streptomyces avermitilis. Eleven genes/operons were demonstrated to be directly regulated by Rex; these genes/operons are involved in aerobic metabolism, morphological differentiation, and secondary metabolism. Rex represses transcription of target genes/operons by binding to Rex operator (ROP) sequences in the promoter regions. NADH reduces DNA-binding activity of Rex to target promoters, while NAD+ competitively binds to Rex and modulates its DNA-binding activity. Rex plays an essential regulatory role in aerobic metabolism by controlling expression of the respiratory genes atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, rex-hemAC1DB, hppA, and ndh2. Rex also regulates morphological differentiation by repressing expression of wblE, which encodes a putative WhiB-family transcriptional regulator. A rex-deletion mutant (Drex) showed higher avermectin production than the wild-type strain ATCC31267, and was more tolerant of oxygen limitation conditions in regard to avermectin production. Streptomyces, a genus of filamentous Gram-positive soil-dwelling bacteria, are obligate aerobes1,2. During growth, they encounter a variety of environmental stresses resulting from the complex nature of soil. Growth in wet soil is particularly challenging because little or no oxygen is present. Streptomyces strains are widely used in industrial production of various antibiotics, and oxygen supply is a key parameter determining product yield during the antibiotic fermentation process3. It is important to understand how Streptomyces strains sense and respond to oxygen limitation. yg Rex is a redox-sensing regulator widely distributed in Gram-positive bacteria4,5. NAD (H) plays a central role in redox metabolism. NAD+ is reduced to NADH by accepting electrons during substrate oxidation, and NADH is then reoxidized by the electron transport chain. Changes of oxygen status are reflected by the intracel- lular ratio of NADH to NAD+. When NADH/NAD+ ratio is low, Rex binds to target genes and represses tran- scription of genes involved in NAD+ regeneration. In contrast, high NADH/NAD+ ratio inhibits DNA-binding activity of Rex and derepresses transcription of its target genes4,6–8. Rex was first identified in Streptomyces coelicolor and shown to regulate expression of cytochrome bd terminal oxidase (cydABCD operon) and heme biosynthesis (rex-hemACD operon)4. In Bacillus subtilis, a facultative aerobe, Rex represses expression of cytochrome bd oxidase (cydABCD), NADH dehydrogenase (yjlC-ndh), NADH-linked fermentative lactate dehydrogenase (lctP-ldh), and a formate-nitrate transporter (ywcJ)6,9,10. In Staphylococcus aureus, Rex directly regulates at least 19 genes. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis Xingchao Liu, Yaqing Cheng, Mengya Lyu, Ying Wen, Yuan Song, Zhi Chen & Jilun L It acts as a central regulator of anaerobic metabolism leading to anaerobic NAD+ regeneration, which includes lactate, formate, and ethanol fermentation (adh1, adhE, lctP, ldh1, pflBA) and nitrate respiration (narG, nirC, nirR)7. The binding sequence of Rex (Rex operator; ROP) is highly conserved in Gram-positive bacteria. Reported consensus sequence in S. coelicolor (5′-TGTGAACNNNTTCACA-3′)4, in B. subtilis (5′-WWTGTGAANTNNTNNNCAAW-3′; W represents either A or T)10, and in S. aureus (5′-TTGTGAAWWWWTTCACAA-3′)7 are very similar to the palindromic sequence in S. coelicolor. State Key Laboratory of Agro-biotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China. Correspondence and requests for materials should be addressed to Z.C. (email: chenzhi@cau.edu.cn) Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 www.nature.com/scientificreports/ Figure 1. Construction and growth curve of rex-deletion mutant. (A) Method (schematic) used for rex deletion. Long arrows: genes and their directions. Short black arrows: positions of primers used for cloning of exchange regions and confirmation of gene deletion (see M&M). Rectangles: exchange regions used for rex deletion. (B,C) Growth (B) and avermectin production (C) of rex-related mutant strains in liquid fermentation medium II. Values shown are mean ± SD from three replicate flasks. Figure 1. Construction and growth curve of rex-deletion mutant. (A) Method (schematic) used for rex deletion. Long arrows: genes and their directions. Short black arrows: positions of primers used for cloning of exchange regions and confirmation of gene deletion (see M&M). Rectangles: exchange regions used for rex deletion. (B,C) Growth (B) and avermectin production (C) of rex-related mutant strains in liquid fermentation medium II. Values shown are mean ± SD from three replicate flasks. Even though Rex was first characterized in S. coelicolor and its regulatory mechanism has been extensively studied, few target operons/genes of Rex in Streptomyces have been confirmed4, and the overall regulatory func- tion of Rex in this genus remains to be elucidated. S. avermitilis is an important species used for industrial produc- tion of avermectins, a group of anthelmintic antibiotics widely used in the medical, veterinary, and agricultural fields11. We investigated the regulatory role of Rex in the expression of operons/genes involved in aerobic metab- olism, morphology, and secondary metabolism of S. avermitilis. Our findings have potential application to novel genetic engineering strategies for high antibiotic-producing strains and hypoxia-tolerating strains of this genus. Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 Results E i Expression of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB is negatively regu- lated by Rex. The rex gene is conserved within the genus Streptomyces and is cotranscribed with the heme synthesis genes hemACD4. To evaluate the regulatory role of Rex in S. avermitilis, we constructed a rex-deletion mutant (termed Drex) by homologous recombination in wild-type strain ATCC31267. rex deletion had no effect on growth in liquid fermentation medium (Fig. 1).h The promoter regions of the operons atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB in S. avermitilis all contain a putative Rex-binding motif, 5′-TTGTGAANNNNTTCACAA-3′ (Table 1). nuoA1-N1 (SAV4837-4850) encodes putative NADH dehydrogenase I (complex I), cydA1B1CD (SAV4260-4258) encodes putative cytochrome bd-I oxidase (cytochrome bd complex), atpIBEFHAGDC (SAV2888-2880) encodes putative F-type proton-transporting ATPase, and hemAC1DB (SAV4739-4742) is cotranscribed with rex and encodes heme synthesis enzymes (Fig. S1). NADH dehydrogenase I, cytochrome bd-I oxidase, and F-type proton-transporting ATPase are essential components of the respiratory chain. Heme is most abundant in cytochromes, which are electron transfer proteins involved in the final reduction of oxygen during aerobic respi- ration. We performed qRT-PCR to determine whether expression of these genes involved in aerobic respiration is regulated by Rex.f Expression of these genes differed greatly when cells were static-cultured following 3 days’ culture on a rotary shaker (250 rpm). Transcription level of cydA1 under oxygen limitation condition in ATCC31267 increased steadily during 60 min, whereas the level in Drex increased to a maximal value during the first 30 min, then gradually declined during the subsequent 30 min (Fig. 2). Results E i Transcription levels of cydA1, nuoA1, hemA, and atpI were consistently higher for Drex than for ATCC31267 under equivalent treatments, confirming that these genes are negatively regulated by Rex.i oxygen limitation condition is mediated by Rex. Expression of nuoA1 and hemA under oxygen limitation increased slightly in the first 10 min, then declined during the subsequent 50 min, in both ATCC31267 and Drex. In contrast, expression of atpI under oxygen limitation declined steadily during 60 min in both ATCC31267 and Drex (Fig. 2). Transcription levels of cydA1, nuoA1, hemA, and atpI were consistently higher for Drex than for ATCC31267 under equivalent treatments, confirming that these genes are negatively regulated by Rex.i i C-terminal His6-tagged Rex fusion protein was overexpressed in E. coli and purified for DNA binding analy- sis. EMSAs were performed to evaluate interactions between Rex and the promoters in vitro. Rex-His6 bound to the promoter regions of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB operons (Fig. 3A). ChIP assays were performed to assess interactions in vivo. ATCC31267 and Drex cells were treated with formaldehyde at days 2 and 6 to cross-link Rex to its DNA targets. Cross-linked DNA was extracted, fragmented by sonication, and immunoprecipitated by anti-Rex antibodies for screening of Rex-bound DNA fragments. In comparison to control hrdB promoter, PCR products of rex, cydA1, atpI, and nuoA1 promoter regions were selectively enriched from immunoprecipitated DNA of ATCC31267, whereas no such PCR bands were amplified from immunopre- cipitated DNA of Drex (Fig. 3B). Results of EMSAs and ChIP assays revealed that Rex binds specifically to the promoter regions of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB operons. Determination of Rex operator (ROP) sequences on promoter regions of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB. Rex binding sequences in 5′-end fluorescein-labeled pro- moter regions of the above operons were determined by DNase I footprinting analysis. One protected region was detected in the rex promoter region in the presence of 1.2 or 2.4 μΜ Rex-His6. The region extends for 23 nucleotides from positions −39 to −17 relative to the transcriptional start site (TSS) of rex. A consecutive ROP site (5′-TTGTGCACGCGTTCACAA-3′) was found in the protected region; the site is located between −35 region and −10 region and encompasses −35 region (Fig. S2A). A 28-nt protected region (positions −3 to + 25 relative to TSS) was detected in the cydA1 promoter region. One ROP site (5′-ATGTGAACGCGTTCACAA-3′) was found in the protected region downstream from TSS. Results E i These findings suggest that induction of cydA1 under Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 2 www.nature.com/scientificreports/ Gene Function Nucleotide positiona PMW Scoreb Sequence ATG Distancec Start End cydA1B1CD (SAV4260-4258) putative cytochrome bd-I oxidase (cytochrome bd complex) 5226395 5226412 1.00 ATGTGAACGCGTTCACAA 81 rex-hemAC1DB (SAV4738-4742) redox-sensing transcriptional repressor; heme biosynthetic enzymes 5776959 5776976 1.00 TTGTGCACGCGTTCACAA 77 atpIBEFHAGDC (SAV2888-2880) putative F-type proton-transporting ATPase 3533332 3533349 2.00 TTGTGATACGGTTCACGA 137 wblE (SAV3016) putative WhiB-family transcriptional regulator 3771378 3771395 2.00 ATGTGAACGCTTTCACGA 43 hppA (SAV4616) putative inorganic H+ pyrophosphatase 5632612 5632629 2.00 TCGTGAATCAATTCACGA 195 nuoA1-N1 (SAV4837–4850) putative NADH dehydrogenase I (complex I) 5880239 5880256 2.00 ATGTGAAGCAGGTCACAA 147 pbp3-4 (SAV3603-SAV3604) putative penicillin-binding protein 4460250 4460267 2.00 TTCTGAACGTGTTCAGAA 37 SAV828 putative rhamnosidase 982811 982828 3.00 CTGTGAATCGATTCACCT 137 echA7 (SAV2316) putative enoyl-CoA hydratase 2820643 2820660 3.00 TCGTGACGACAGTCACAA 66 SAV2652 putative regulatory protein 3252402 3252419 3.00 TTGTGCACCGCTTCACCC 288 ndh2 (SAV3529) putative NADH dehydrogenase (complex I) 4369478 4369495 3.00 TTGTGAAGGGGCGCACGA 119 ectABCD (SAV6398-6395) putative L-2,4-diaminobutyrate acetyltransferase, ectoine biosynthesis 7673586 7673603 3.00 TTGTGATCGACTCCACAT 155 SAV6368 putative multiple sugar ABC transporter permease protein 7638483 7638500 3.00 TGGTGAAGCGCTTCGCGT 81 avaB- avaL2 (SAV2267-2268) putative gamma-butyrolactone- dependent transcriptional regulator 2766273 2766290 3.00 TCGTGAACGAATTCTAAT 29 SAV3213 putative nitroreductase family protein, NADH dehydrogenase/NAD(P)H nitroreductase 4004553 4004570 3.00 TGGTGATCGGCTTCACAG 96 SAV1351 putative fatty acid-CoA racemase 2820643 2820660 3.00 TCGTGACGACAGTCACAA 66 tmk (SAV4622) putative thymidylate kinase 5643744 5643761 3.00 GTGTGGAGGCGTCCACAA 73 folD2 (SAV543) putative methylenetetrahydrofolate 688370 688387 3.00 TTGTGTGTGAGTTCAGAA 231 parA (SAV6508) putative partitioning or sporulation protein 7797460 7797477 3.00 ATGTCGACTCATTCACAA 114 SAV7415-7416 putative sugar isomerase putative simple sugar ABC transporter 8846427 8846444 3.00 TCGTGAAAGGTTTCAACT 203 SAV897 putative alpha-amylase inhibitor 1075479 1075496 3.00 TTGCGAAAGTTGTCGCAA 73 Table 1. Putative Rex target genes. aGenomic position. bNumber of mismatches with respect to the consensus. PWM, positive weight matrix. cValues are distances (in nucleotides) to the predicted start codon of the downstream gene. Table 1. Putative Rex target genes. aGenomic position. bNumber of mismatches with respect to the consensus. PWM, positive weight matrix. cValues are distances (in nucleotides) to the predicted start codon of the downstream gene. oxygen limitation condition is mediated by Rex. Expression of nuoA1 and hemA under oxygen limitation increased slightly in the first 10 min, then declined during the subsequent 50 min, in both ATCC31267 and Drex. In contrast, expression of atpI under oxygen limitation declined steadily during 60 min in both ATCC31267 and Drex (Fig. 2). Results E i A half-site ROP (5′-TTGTGAA-3′) was also found in the protected region; it is located upstream from the ROP site and encompasses TSS (Fig. S2B). EMSA revealed two retarded bands between Rex and the cydA1 promoter region (Fig. 3A), suggesting that Rex can interact with the half-site ROP. A 29-nt protected region (positions −50 to −22 relative to TSS) containing a ROP site (5′-TTGTGATACGGTTCACGA-3′) was detected in the atpI promoter region (Fig. S2C). Rex-His6 pro- tected a 27-nt region extending from positions −42 to −16 relative to TSS of nuoA1, which contains a ROP site Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 3 www.nature.com/scientificreports/ Figure 2. RT-qPCR analysis of transcription levels of cydA1, nuoA1, hemA, atpI, and wblE in ATCC31267 and Drex. RNA was prepared from cells grown in fermentation medium for 3 days on a rotary shaker (250 rpm) and then static-cultured for the indicated time. Quantitative data were normalized to hrdB (SAV2444) expression value. Values shown are mean ± SD from three replicates. Figure 2. RT-qPCR analysis of transcription levels of cydA1, nuoA1, hemA, atpI, and wblE in ATCC31267 and Drex. RNA was prepared from cells grown in fermentation medium for 3 days on a rotary shaker (250 rpm) and then static-cultured for the indicated time. Quantitative data were normalized to hrdB (SAV2444) expression value. Values shown are mean ± SD from three replicates. Figure 3. Binding of Rex-His6 to promoter regions of rex, cydA1, atpI, nuoA1, and wblE. (A) EMSAs using Rex-His6 protein at the indicated concentrations, and the probes indicated below the panels. Arrow: free probe. (B) ChIP assay analysis of Rex binding to promoter regions of rex, cydA1, atpI, nuoA1, and wblE in vivo. Rex- DNA complexes were immunoprecipitated by anti-Rex antibodies from formaldehyde-treated ATCC31267 and Drex cells. DNAs used for PCR were: total DNA prior to immunoprecipitation (positive control: lanes “+”), immunoprecipitated DNA (experimental sample: lanes “S”), and DNA without antibody (negative control: lanes “−”). hrdB promoter region was used as control. Figure 3. Binding of Rex-His6 to promoter regions of rex, cydA1, atpI, nuoA1, and wblE. (A) EMSAs using Rex-His6 protein at the indicated concentrations, and the probes indicated below the panels. Arrow: free probe. (B) ChIP assay analysis of Rex binding to promoter regions of rex, cydA1, atpI, nuoA1, and wblE in vivo. Rex- DNA complexes were immunoprecipitated by anti-Rex antibodies from formaldehyde-treated ATCC31267 and Drex cells. Results E i In comparison to ATCC31267, Drex showed delayed morphogenesis on SFM agar at day 2, when aerial mycelium was initiated. Spore formation at day 6 did not dif- fer notably between the two strains. Morphogenesis of the Drex complementation strain was similar to that of ATCC31267 (Fig. 5), indicating that the delayed morphogenesis was due solely to rex deletion.h The promoter region of wblE in S. avermitilis contains a putative Rex-binding motif (Table 1). wblE encodes a putative WhiB-family transcriptional regulator, which may be involved in morphological differentiation12,13. qRT-PCR analysis revealed notable increases of wblE transcription level in Drex. Levels under oxygen limitation condition declined gradually during 60 min for both ATCC31267 and Drex, and were consistently higher for Drex than for ATCC31267 (Fig. 2). EMSAs showed that Rex-His6 bound to the wblE promoter region in vitro (Fig. 3A). In in vivo ChIP assays, PCR product of the wblE promoter region was selectively enriched from immu- noprecipitated DNA of ATCC31267, whereas no such PCR band was amplified from immunoprecipitated DNA of Drex (Fig. 3B). These findings indicate that wblE is negatively regulated by Rex. Rex binding sequence in the wblE promoter region was determined by DNase I footprinting analysis. A 28-nt region protected by Rex-His6 was detected, extending from positions + 108 to + 135 relative to TSS of wblE (Fig. S4). The protected region contains a consecutive ROP site (5′-TCGTGAAAGCGTTCACAT-3′) and a half-site ROP (5′-TTCACAA-3′) located downstream of TSS. Rex may inhibit the progress of RNA polymerase by binding to ROP downstream of the wblE promoter, and thereby repress transcription. To test the possibility that overexpression of wblE in Drex results in delayed morphogenesis, we attempted to delete wblE in S. avermitilis. However, this attempt was unsuccessful. wblE is evidently an essential gene in Streptomyces; an attempt to delete it in S. coelicolor was also unsuccessful13. When wblE was overexpressed in ATCC31267, the resulting strain had a phenotype similar to that of Drex (Fig. 5), suggesting that Rex regulates morphological differentiation through its effect on wblE expression. Rex negatively regulates avermectin production. The overexpression of rex caused a decrease in aver- mectin production to 33% of ATCC31267 level. Drex had avermectin production ~3-fold higher than that of ATCC31267. The mycelial dry weight of Drex was similar to that of ATCC31267, indicating that the improved avermectin yield was not achieved by improved growth. Results E i In the Drex complementation strain, avermectin pro- duction was similar to that of ATCC31267 (Fig. 1; Fig. 6A). These findings indicate that rex negatively regulates avermectin production in S. avermitilis. We also measured avermectin production under oxygen limitation con- dition. In ATCC31267, lower agitation speed (230 rpm; control speed was 250 rpm) resulted in a 20% reduction of avermectin production, and static culture for 2 h on day 5 resulted in a 35% reduction. In Drex, avermectin production was reduced by 3.8% and 25%, respectively, under the above two conditions (Fig. 6B). Thus, rex deletion resulted in increased tolerance of S. avermitilis to oxygen limitation in regard to avermectin production. yg g p qRT-PCR analysis was performed to determine whether Rex regulates avermectin production at the transcrip- tional level. Drex showed significantly increased transcription levels of pathway-specific regulatory gene aveR and biosynthetic genes aveA1 and aveD, relative to ATCC31267. Oxygen limitation for 60 min reduced expression of these genes in ATCC31267; however, Drex showed lower fold repression, and a slight induction of aveA1 and aveD (Fig. 6C). EMSAs revealed that Rex-His6 did not bind to the aveR promoter region or the aveD-A1 inter- genic region (Fig. S5). Although no retarded band was observed when aveR promoter region was probed with Rex-His6 protein, DNase I footprinting analysis showed one protected region extending for 15 nucleotides on the aveR coding strand in the presence of 10 or 15 μΜ Rex-His6 (Fig. 7). No consecutive ROP site was observed in the protected region; however, two adjacent half-site ROP (5′-TCGTGAA-3′ and 5′- TTGTGGA-3′) were found in the protected region and downstream region. Rex can evidently interact with the half-site ROP; however, because the interaction is weak and easily dissociated in vitro, EMSA did not reveal a clear shifted band. Confirmation of putative Rex target genes. A genome-wide search of consensus motif 5′-TTGTGAANNNNTTCACAA-3′ using the genome sequence of ATCC31267 revealed the presence of 36 motifs up to 350 bp upstream of predicted genes: 2 motifs with one mismatch, 10 motifs with two mismatches, and 24 motifs with three mismatches. Our previous experiments showed that wblE, cydA1B1CD, rex-hemAC1DB, atpIBEFHAGDC, and nuoA1-N1 are directly controlled by Rex. To investigate whether Rex binds to promoter regions of other putative target genes, we selected 16 genes with predicted gene function for EMSAs (Table 1). Results E i DNAs used for PCR were: total DNA prior to immunoprecipitation (positive control: lanes “+”), immunoprecipitated DNA (experimental sample: lanes “S”), and DNA without antibody (negative control: lanes “−”). hrdB promoter region was used as control. (5′-TTGTGACCTGCTTCACAT-3′) (Fig. S2D). ROP in the nuoA1 and atpI promoter regions is located between −35 region and −10 region, and encompasses −35 region. These findings suggest that Rex blocks attachment of RNA polymerase to the promoters or inhibits the progress of RNA polymerase by binding to ROP in or (5′-TTGTGACCTGCTTCACAT-3′) (Fig. S2D). ROP in the nuoA1 and atpI promoter regions is located between −35 region and −10 region, and encompasses −35 region. These findings suggest that Rex blocks attachment of RNA polymerase to the promoters or inhibits the progress of RNA polymerase by binding to ROP in or Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 4 www.nature.com/scientificreports/ downstream of the promoters of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB, and blocks transcription of these operons. downstream of the promoters of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB, and blocks transcription of these operons. DNA-binding activity of Rex is modulated by NADH/NAD+ ratio. In S. coelicolor, NADH at con- centrations <5 μM inhibits DNA-binding activity of Rex, whereas 1 mM NAD+ has no inhibitory effect. NAD+ competes with NADH for Rex binding4. In B. subtilis and S. aureus, NAD+ enhances binding of Rex to putative Rex-binding sites, while NADH competes with NAD+ for Rex binding and reduces Rex activity6,7. We examined the effects of NAD+ and NADH on DNA-binding activity of Rex to upstream regions of cydA1 in S. avermit- ilis. DNA-binding activity of Rex was reduced by addition of NADH, but not by NAD+ concentrations up to 1 mM (Fig. 4A,B; Fig. S3). NADH and NAD+ were added to EMSA binding buffer to assess the effect of NAD+/ NADH ratio on DNA-binding activity of Rex in vitro. At NAD+ concentration 0.2 mM, 5 μM NADH was suffi- cient to dissociate the Rex-DNA complex (Fig. 4C). At NAD+ concentration 1 mM, dissociation of DNA-Rex complex required 25 μM NADH, suggesting that Rex-binding activity was recovered by addition of increasing amounts of NAD+ (Fig. 4D). These findings indicate that NAD+ and NADH bind competitively to Rex and mod- ulate its DNA-binding activity. These findings also imply that Rex exploits the similar regulatory mechanism in Streptomyces. Rex regulates morphological differentiation. Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 Results E i Of these, Rex bound to the probes of hppA (encodes an inorganic H+ pyrophosphatase), ndh2 (encodes a NADH Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 5 www.nature.com/scientificreports/ Figure 4. DNA-binding activity of Rex is modulated by NADH/NAD+ ratio. (A) EMSAs of cydA1 region using Rex-His6 and 0.1 or 1 mM pyridine nucleotides. (B) EMSAs of cydA1 promoter region u His6 with various NADH concentrations. (C,D) Assay mixtures contained NADH at indicated concen and 0.2 mM (C) or 1 mM (D) NAD+. Arrow: free probe. Figure 4. DNA-binding activity of Rex is modulated by NADH/NAD+ ratio. (A) EMSAs of cydA1 promoter region using Rex-His6 and 0.1 or 1 mM pyridine nucleotides. (B) EMSAs of cydA1 promoter region using Rex- His6 with various NADH concentrations. (C,D) Assay mixtures contained NADH at indicated concentration and 0.2 mM (C) or 1 mM (D) NAD+. Arrow: free probe. 6 Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Phenotypes of rex- and wblE-related mutant strains. Growth of indicated strains on SFM agar for 2 and 6 days. WT, wild-type ATCC31267; Drex, rex-deletion mutant; Drex-C, complementation strain of Drex; Drex-pSET152 and 31267-pKC1139, empty plasmid-containing controls; OwblE, wblE overexpressing strain. Figure 5. Phenotypes of rex- and wblE-related mutant strains. Growth of indicated strains on SFM agar for 2 and 6 days. WT, wild-type ATCC31267; Drex, rex-deletion mutant; Drex-C, complementation strain of Drex; Drex-pSET152 and 31267-pKC1139, empty plasmid-containing controls; OwblE, wblE overexpressing strain. dehydrogenase [complex I]), echA7 (encodes an enoyl-CoA hydratase), ectABC (encodes ectoine biosynthesis enzymes), SAV828 (encodes a rhamnosidase), and SAV2652 (encodes a regulatory protein). Probes whose bind- ing motif had one or two mismatches showed higher affinity than probes whose binding motif had three mis- matches (Fig. 3A, Fig. 8). These findings demonstrated that 5′-TTGTGAANNNNTTCACAA-3′ is the consensus motif of Rex in S. avermitilis. Discussion l f h Agitation speed 230 rpm (control, 250 rpm); Static culture for 2 h at day 5 during fermentation (250 rpm). (C) RT-qPCR analysis of aveR, aveA1, and aveD transcription levels in ATCC31267 and Drex. RNA samples were the same ones used for experiments shown in Fig. 1. Quantitative data were normalized to hrdB expression value. Values shown are mean ± SD from three replicates. Statistical significance of differences was determined using Student’s t-test. *P < 0.001; NS, not significant. Figure 6. Effect of rex deletion on avermectin production in S. avermitilis. (A) Avermectin production in rex-related mutant strains. ATCC31267, wild-type; Drex, rex-deletion mutant; 31267 (pSET152) and 31267 (pKC1139), empty plasmid-containing controls; Drex-C, rex-deletion complementation strain 31267 (pSET- rex); Orex, rex overexpressing strain 31267 (pKC-rex). (B) Avermectin production in ATCC31267 and Drex with various oxygen limitation conditions. Agitation speed 230 rpm (control, 250 rpm); Static culture for 2 h at day 5 during fermentation (250 rpm). (C) RT-qPCR analysis of aveR, aveA1, and aveD transcription levels in ATCC31267 and Drex. RNA samples were the same ones used for experiments shown in Fig. 1. Quantitative data were normalized to hrdB expression value. Values shown are mean ± SD from three replicates. Statistical significance of differences was determined using Student’s t-test. *P < 0.001; NS, not significant. Figure 6. Effect of rex deletion on avermectin production in S. avermitilis. (A) Avermectin production in l d ld d l ( ) d Fowler-Goldsworthy et al.13 reported that wblE could not be deleted in various strains of S. coelicolor, and we made a similar observation in S. avermitilis. Thus, wblE appears to be essential in this genus. The homolog of wblE in Mycobacterium tuberculosis is whiB1, which encodes an essential transcription factor in response to nitric oxide exposure28. We demonstrated that wblE is directly negatively regulated by Rex, and that wblE overexpres- sion results in delayed morphogenesis similar to that of Drex. Expression of wblE, like that of atpIBEFHAGDC, is downregulated by oxygen limitation in both ATCC31267 and Drex, suggesting that (i) wblE is jointly regulated by Rex and some other regulator, or (ii) wblE itself responds to low oxygen concentration via its own redox-sensitive [4Fe-4S] cluster. Under oxygen limitation condition, wblE expression in Streptomyces is downregulated, with consequent stimulation of sporulation and production of a large number of spores to maintain viability under conditions of little or no oxygen. Discussion l f h Results of this study show that Rex in S. avermitilis acts as a repressor of aerobic metabolism, morphological differentiation, and secondary metabolism (summarized schematically in Fig. 9). Results of EMSAs demon- strated that at least 11 genes/operons are directly regulated by Rex. Among these, atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB operons encode key components of the electron transfer chain and play crucial roles in aerobic metabolism14–17. hppA encodes a putative pyrophosphate-energized proton pump that converts energy from pyrophosphate hydrolysis into active H+ transport across the plasma membrane18. ndh2 encodes a NADH dehydrogenase involved in NAD+ regeneration19,20. echA7 encodes an enoyl-CoA hydratase that catalyzes the second step of the β-oxidation pathway of fatty acid metabolism21. SAV828 encodes a rhamnosidase that hydrolyzes L-rhamnose from L-rhamnoside22. Under oxygen limitation condition, the increase of intracellular NADH/NAD+ ratio in S. avermitilis dissociates binding of Rex from its target binding sites and derepresses its target genes/operons, and upregulation of cydA1B1CD, nuoA1-N1, rex-hemAC1DB, ndh2, and hppA increases oxygen utilization, NAD+ regeneration, and ATP synthesis (Fig. 9). On the other hand, expression of atpIBE- FHAGDC in ATCC31267 and Drex is downregulated by oxygen limitation, suggesting that this operon is also directly controlled by regulators other than Rex. The F0F1-ATPase operon in Corynebacterium glutamicum is reg- ulated by ECF σH 23. A sigH homolog is present in Streptomyces; whether it regulates atpIBEFHAGDC expression remains to be tested. WhiB-like family transcription factors are widely present in actinomycetes, but not found in other bacte- rial orders. WhiB was first identified as a small transcription factor-like protein essential for sporulation in S. coelicolor24. Genome sequencing revealed that Streptomyces species have multiple whiB-like genes (designated “wbl”). Eleven wbl genes (including whiB and whiD) have been identified in S. coelicolor13. Among these, wblA, whiB, and whiD are essential for sporulation, and WblA also negatively regulates antibiotic biosynthesis in Streptomyces13,25–27. Other wbl genes are not involved in morphological development, with the exception of wblE. Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 7 www.nature.com/scientificreports/ p / Figure 6. Effect of rex deletion on avermectin production in S. avermitilis. (A) Avermectin production in rex-related mutant strains. ATCC31267, wild-type; Drex, rex-deletion mutant; 31267 (pSET152) and 31267 (pKC1139), empty plasmid-containing controls; Drex-C, rex-deletion complementation strain 31267 (pSET- rex); Orex, rex overexpressing strain 31267 (pKC-rex). (B) Avermectin production in ATCC31267 and Drex with various oxygen limitation conditions. Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 Materials and Methods Bacterial strains and growth conditions. The S. avermitilis strains used were ATCC31267 (wild-type), Drex (rex-deletion strain), Drex-C (rex-deletion complementary strain harboring plasmid pSET-rex), and Orex (ATCC31267 harboring rex overexpressing plasmid pKC-rex). E. coli strains JM109 and BL21 (DE3) were used for routine cloning and protein expression, respectively. YMS medium and SFM medium were used for sporula- tion and phenotype studies30,31. Culture conditions for mycelial growth, protoplast preparation, and regeneration of S. avermitilis were as described previously30. Seed medium and fermentation medium FM-I were used for aver- mectin production and for RNA isolation, and soluble fermentation medium FM-II was used for ChIP analysis32. Gene deletion, complementation, and overexpression. A rex (SAV4738) gene deletion mutant was generated through targeted gene deletion mediated by homologous recombination. A 566-bp fragment upstream of rex (position −460 to + 87 from start codon) was amplified by primers rex-up-Fw and rex-up-Rev, and a 579-bp fragment downstream of rex (position +539 to +1098) was amplified by primers rex-dw-Fw and rex-dw-Rev, using ATCC31267 genomic DNA as template (Table S1; Fig. 1). The two fragments, after recovery, were digested respectively by BamHI/HindIII and BamHI/EcoRI, and ligated together into EcoRI/HindIII-digested pKC113933 to produce rex-deletion vector pKCD-rex. pKCD-rex was introduced into ATCC31267 protoplasts. Double-crossover recombinant strains were selected as described previously34,35. The rex-deletion mutant (termed Drex) was confirmed by PCR using one pair of external primers (rex-V-Fw/rex-V-Rev) and one pair of internal primers (rex-V2-Fw/rex-V2-Rev) (Table S1; Fig. 1). Use of the external primers yielded a 1.3-kb band from Drex and a 1.8-kb band from ATCC31267. Use of the internal primers yielded a 225-bp band from ATCC31267 and no band from Drex (data not shown).i A 1038-bp DNA fragment carrying the rex ORF and its putative promoter was amplified by PCR using prim- ers rex-E-Fw and rex-E-Rev (Suppl. Table 1), and then ligated into EcoRI/XbaI-digested pSET152 or pKC1139 to produce vector pSET-rex or pKC-rex. For complementation analysis of Drex, pSET-rex was transformed into Drex protoplasts. For overexpression of Rex, pKC-rex was introduced into ATCC31267 protoplasts. RNA extraction and qRT-PCR analysis. RNA was isolated using Trizol reagent (Tiangen; China) from S. avermitilis mycelia grown in FM-I as described previously32. Transcription levels of various genes were deter- mined by qRT-PCR using the primer pairs listed in Table S1. An RNA sample without prior reverse transcription was used as negative control to rule out chromosomal DNA contamination. hrdB gene (SAV2444) was used as internal control. Figure 7. Determination of Rex binding site on aveR promoter region by DNase I footprinting assay Figure 7. Determination of Rex binding site on aveR promoter region by DNase I footprinting assay. (A) Fluorograms correspond to control DNA fragment and to protected reactions (with 10 and 15 µM Rex-His6). (B) Nucleotide sequences of aveR promoter region. Shaded boxes: sequences protected from DNase I digestion. Dotted boxes: ROP. Dotted line: half-site ROP. Black bent arrows with boldface letters: TSSs. Boxes: presumed −35 and −10 elements of promoters. Gray bent arrows with boldface letters: translational start codons. Discussion l f h Another Rex target, ectABC, encodes enzymes for biosynthesis of ectoine (a compatible solute) that serves as an osmolyte and promotes survival under osmotic or temperature stress29. By regulating ectABC transcription, Rex facilitates ectoine biosynthesis to enhance viability under these types of stress. In Drex, expression of regulatory gene aveR and biosynthetic genes, and avermectin production, were notably increased. Although EMSA showed no clearly retarded band between aveR promoter region probe and Rex-His6, DNase I footprinting analysis revealed one 15-nt protected region consisting of two adjacent half-site ROP on the coding strand of aveR by Rex-His6. Thus, Rex may directly regulate aveR expression by interacting with the half-site ROP in the aveR promoter region. Expression of electron transfer chain components was enhanced in Drex, thus promoting aerobic respiration rate, ATP production, and secondary metabolism. The notable increase of atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, and rex-hemAC1DB expression in Drex enhanced the tolerance of cells to oxygen limitation. The findings described here provide a basis for construction of new Streptomyces strains with high antibiotic production and hypoxia tolerance. 8 Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 www.nature.com/scientificreports/ ure.com/scientificreports/ Figure 7. Determination of Rex binding site on aveR promoter region by DNase I footprinting assay. Figure 7. Determination of Rex binding site on aveR promoter region by DNase I footprinting assay. (A) Fluorograms correspond to control DNA fragment and to protected reactions (with 10 and 15 µM Rex-His6). (B) Nucleotide sequences of aveR promoter region. Shaded boxes: sequences protected from DNase I digestion. Dotted boxes: ROP. Dotted line: half-site ROP. Black bent arrows with boldface letters: TSSs. Boxes: presumed −35 and −10 elements of promoters. Gray bent arrows with boldface letters: translational start codons. igure 7. Determination of Rex binding site on aveR promoter region by DNase I footprinting assay. Materials and Methods Chromatin Immunoprecipitation (ChIP) assay. ChIP assay was performed as described previously36. In brief, S. avermitilis cultures grown in FM-II for 2 or 6 days were fixed in cross-linking buffer (0.4 M sucrose, 10 mM Tris·Cl [pH 8.0], 1 mM EDTA) containing 1% formaldehyde for 20 min at 28 °C. ChIP was performed using anti-Rex antibody. After DNA extraction, pellets were washed with 70% ethanol and resuspended in 50 μl Tris·EDTA buffer. 1 μl DNA solution was subjected to PCR using the primer sets listed in Table S1. Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 9 www.nature.com/scientificreports/ ture.com/scientificreports/ Figure 8. Binding of Rex-His6 to promoter regions of putative Rex targets. Rex (μM): Rex-His6 concentrations. Arrow: free probe. Figure 8. Binding of Rex-His6 to promoter regions of putative Rex targets. Rex (μM): Rex-His6 concentrations. Arrow: free probe. Figure 8. Binding of Rex-His6 to promoter regions of putative Rex targets. Rex (μM): Rex-His6 concentrations. Arrow: free probe. Overexpression and purification of Rex-His6. The rex coding region was amplified by PCR using primers His-rex-Fw and His-rex-Rev. The purified fragment was cut with NcoI/HindIII and cloned into NcoI/HindIII-digested pET28a (+) to generate expression plasmid pET-rex. pET-rex was introduced into E. coli BL21 (DE3) for overexpression of C-terminal His6-tagged Rex. Rex-His6 was induced by 0.2 mM IPTG at 37 °C and purified from whole-cell lysate by Ni-NTA agarose chromatography (Bio-works; Sweden) according to the manufacturer’s instructions. Electrophoretic mobility gel shift assays (EMSAs). EMSAs were performed according to the manu- facturer’s instructions (DIG Gel Shift Kit, 2nd Generation, Roche) as described previously35. DNA probes were obtained by PCR using the primers listed in Table S1, and labeled with Digoxigenin-11-ddUTP at the 3′ end using recombinant terminal transferase. DIG-labeled DNA probe was incubated with various quantities of Rex-His6 for 30 min at 25 °C in a total volume of 20 μl containing 1 μg poly[d(I-C)]. Electrophoresis (5.0% native polyacryla- mide gel; 0.5 × TBE as running buffer) was performed to separate protein-bound probes from free probes. DNA was electroblotted onto a positively charged nylon membrane, and retarded and unbound bands were detected by chemiluminescence and recorded on X-ray film. DNase I footprinting assays. A fluorescent labeling procedure was used for DNase I footprinting assays37. DNA fragments were obtained by PCR using FAM-labeled primers (Table S1), and purified from agarose gel. Prediction of Rex putative targets. To search for putative Rex target genes, Rex consensus motif 5′-TTGTGAANNNNTTCACAA-3′ was used to scan the intergenic regions of the S. avermitilis genome using Virtual Footprint software38. Prediction of Rex putative targets. To search for putative Rex target genes, Rex consensus motif 5′-TTGTGAANNNNTTCACAA-3′ was used to scan the intergenic regions of the S. avermitilis genome using Virtual Footprint software38. References Journal of Bacteriology 194, 1145–1157, doi: 10.1128/JB.06412-11 (2012). J f gy , , J ( ) 6. Gyan, S., Shiohira, Y., Sato, I., Takeuchi, M. & Sato, T. Regulatory loop between redox sensing of the NADH/NAD(+) ratio by Rex (YdiH) and oxidation of NADH by NADH dehydrogenase Ndh in Bacillus subtilis. Journal of Bacteriology 188, 7062–7071, doi: 10.1128/JB.00601-06 (2006). 6. Gyan, S., Shiohira, Y., Sato, I., Takeuchi, M. & Sato, T. Regulatory loop between redox sensing of the NADH/NAD(+) ratio by Rex (YdiH) and oxidation of NADH by NADH dehydrogenase Ndh in Bacillus subtilis. Journal of Bacteriology 188, 7062–7071, doi: 10.1128/JB.00601-06 (2006). 7. Pagels, M. et al. Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus. Molecular Microbiology 76, 1142–1161, doi: 10.1111/j.1365-2958.2010.07105.x (2010).h 7. Pagels, M. et al. Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus. Molecular Microbiology 76, 1142–1161, doi: 10.1111/j.1365-2958.2010.07105.x (2010).h gy j 8. Bitoun, J. P., Liao, S., Yao, X., Xie, G. G. & Wen, Z. T. The redox-sensing regulator Rex modulates central carbon metabolism, stres l d bi fil f i b St t t Pl S O 7 44766 d i 10 1371/j l 0044766 (2012) gy j 8. Bitoun, J. P., Liao, S., Yao, X., Xie, G. G. & Wen, Z. T. The redox-sensing regulator Rex modulates central carbon metabolism, stress tolerance response and biofilm formation by Streptococcus mutans. PloS One 7, e44766, doi: 10.1371/journal.pone.0044766 (2012). 9 L J T R A & W h f ld C C di d f h bd d l d h d i i 8. Bitoun, J. P., Liao, S., Yao, X., Xie, G. G. & Wen, Z. T. The redox-sensing regulator Rex modulates central carbon metabolism, stress tolerance response and biofilm formation by Streptococcus mutans. PloS One 7, e44766, doi: 10.1371/journal.pone.0044766 (2012). 9. Larsson, J. T., Rogstam, A. & von Wachenfeldt, C. Coordinated patterns of cytochrome bd and lactate dehydrogenase expression in tolerance response and biofilm formation by Streptococcus mutans. PloS One 7, e44766, doi: 10.1371/journal.pone.0044766 (2012) 9. Larsson, J. T., Rogstam, A. & von Wachenfeldt, C. Coordinated patterns of cytochrome bd and lactate dehydrogenase expression in Bacillus subtilis. Microbiology 151, 3323–3335, doi: 10.1099/mic.0.28124-0 (2005). gy 10. Wang, E. et al. Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex. ermentation and HPLC analysis of avermectin production. Fermentation of S. avermitilis strains nd estimation of avermectins yields by HPLC analysis were performed as described previously32. Fermentation and HPLC analysis of avermectin production. Fermentation of S. avermitilis strains and estimation of avermectins yields by HPLC analysis were performed as described previously32. Determination of transcriptional start sites. Transcriptional start sites (TSS) of rex and wblE were mapped by 5′-RACE using a 5′/3′ RACE Kit (2nd Generation, Roche). Total RNA was extracted from ATCC31267 grown in FM-I for 2 days. A gene-specific primer (sp1) was used to synthesize cDNA, and template RNA was degraded with RNase H. A homopolymeric A-tail was purified and added to the 3′-end of cDNA using terminal transferase. Tailed cDNA was PCR amplified through 35 cycles with a specific nested primer (sp2) and an oligo (dT)-anchor primer (Table S1). PCR products were electrophoresed, purified using a DNA agarose gel recovery kit (BioTek; China), and sequenced. Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 Materials and Methods Labeled DNA fragments (400 ng) and various quantities of Rex-His6 were incubated in a 25-μl volume for 30 min at 25 °C. DNase I digestion was performed for 40 sec at 37 °C, and terminated by addition of 10 μl 0.2 M EDTA Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 10 www.nature.com/scientificreports/ Figure 9. Regulatory network (schematic) whereby Rex controls major genes involved in aerobic metabolism, morphological differentiation, and secondary metabolism. Solid arrows: direct induction. Solid lines with blunt end: repression. Dashed arrows: indirect induction. Figure 9. Regulatory network (schematic) whereby Rex controls major genes involved in aerobic metabolism, morphological differentiation, and secondary metabolism. Solid arrows: direct induction. Solid lines with blunt end: repression. Dashed arrows: indirect induction. (pH 8.0). Samples were subjected to phenol/chloroform extraction, ethanol precipitation, and capillary electro- phoresis. Electrophoregrams were analyzed using GeneMarker software v2.2.0. References 1. van Keulen, G., Alderson, J., White, J. & Sawers, R. G. The obligate aerobic actinomycete Streptomyces coelicolor A3(2) survives extended periods of anaerobic stress. Environmental Microbiology 9, 3143–3149, doi: 10.1111/j.1462-2920.2007.01433.x (2007). 2. Fischer, M., Falke, D. & Sawers, R. G. A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe 1. van Keulen, G., Alderson, J., White, J. & Sawers, R. G. The obligate aerobic actinomycete Streptomyces coelicolor A3(2) survive extended periods of anaerobic stress. Environmental Microbiology 9, 3143–3149, doi: 10.1111/j.1462-2920.2007.01433.x (2007). 2. Fischer, M., Falke, D. & Sawers, R. G. A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe l l ( ) l l b l d ( ) 1. van Keulen, G., Alderson, J., White, J. & Sawers, R. G. The obligate aerobic actinomycete Streptomyces coelicolor A3(2) survives extended periods of anaerobic stress. Environmental Microbiology 9, 3143–3149, doi: 10.1111/j.1462-2920.2007.01433.x (2007). F h M F lk D & S R G A d l l f h bl b gy j 2. Fischer, M., Falke, D. & Sawers, R. G. A respiratory nitrate reductase active exclusively in resting Streptomyces coelicolor A3(2). 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Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 43–49, doi: Doi 10.1016/0378-1119(92)90627-2 (1992). 4. Zhang, X. et al. Deletion analysis of oligomycin PKS genes (olmA) in Streptomyces avermitilis. Chinese Science Bulletin 49, 350, doi 10.1360/03wc0469 (2004). 35. Liu, Y. et al. Characterization of SAV7471, a TetR-family transcriptional regulator involved in the regulation of coenzym metabolism in Streptomyces avermitilis. Journal of Bacteriology 195, 4365–4372, doi: 10.1128/JB.00716-13 (2013).hi 36. Guo, J. et al. Acknowledgementsh g This study was supported by a grant (No. 31470190) from the National Natural Science Foundation of China. The authors are grateful to Dr. S. Anderson for English editing of the manuscript. g This study was supported by a grant (No. 31470190) from the National Natural Science Foundation of China. The authors are grateful to Dr. S. Anderson for English editing of the manuscript. 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Scientific Reports \| 7:44567 \| DOI: 10.1038/srep44567 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. How to cite this article: Liu, X. et al. Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis. Sci. Rep. 7, 44567; doi: 10.1038/ srep44567 (2017). How to cite this article: Liu, X. et al. Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis. Sci. Rep. 7, 44567; doi: 10.1038/ srep44567 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W2605874837	https://figshare.com/articles/journal_contribution/A_Transcriptomic_Comparison_of_Two_Bambara_Groundnut_Landraces_under_Dehydration_Stress/4884575/1/files/8197979.pdf	English	null	A Transcriptomic Comparison of Two Bambara Groundnut Landraces under Dehydration Stress	Genes	2,017	cc-by	14,409	1 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK; stxfk5@nottingham ac uk 1 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK; stxfk5@nottingham.ac.uk g 2 Crops for the Future, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia; huihui.chai@nottingham.edu.my 3 Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK; bhzia@exmail.nottingham.ac.uk (I.A.); charlie.hodgman@nottingham.ac.uk (C.H.) g g 4 School of Animal Rural and Environmental Sciences, Nottingham Trent University, Clifton Campus, Nottingham NG11 8NS, UK; chungui.lu@ntu.ac.uk * Correspondence: sbzsm4@exmail.nottingham.ac.uk; Tel.: +44-0115-951-8062 Academic Editor: Qingyi Yu Academic Editor: Qingyi Yu Received: 20 February 2017; Accepted: 10 April 2017; Published: 18 April 2017 Received: 20 February 2017; Accepted: 10 April 2017; Published: 18 April 2017 Abstract: The ability to grow crops under low-water conditions is a signiﬁcant advantage in relation to global food security. Bambara groundnut is an underutilised crop grown by subsistence farmers in Africa and is known to survive in regions of water deﬁcit. This study focuses on the analysis of the transcriptomic changes in two bambara groundnut landraces in response to dehydration stress. A cross-species hybridisation approach based on the Soybean Affymetrix GeneChip array has been employed. The differential gene expression analysis of a water-limited treatment, however, showed that the two landraces responded with almost completely different sets of genes. Hence, both landraces with very similar genotypes (as assessed by the hybridisation of genomic DNA onto the Soybean Affymetrix GeneChip) showed contrasting transcriptional behaviour in response to dehydration stress. In addition, both genotypes showed a high expression of dehydration-associated genes, even under water-sufﬁcient conditions. Several gene regulators were identiﬁed as potentially important. Some are already known, such as WRKY40, but others may also be considered, namely PRR7, ATAUX2-11, CONSTANS-like 1, MYB60, AGL-83, and a Zinc-ﬁnger protein. These data provide a basis for drought trait research in the bambara groundnut, which will facilitate functional genomics studies. An analysis of this dataset has identiﬁed that both genotypes appear to be in a dehydration-ready state, even in the absence of dehydration stress, and may have adapted in different ways to achieve drought resistance. This will help in understanding the mechanisms underlying the ability of crops to produce viable yields under drought conditions. In addition, cross-species hybridisation to the soybean microarray has been shown to be informative for investigating the bambara groundnut transcriptome. Keywords: Bambara groundnut; landraces; dehydration stress; cross-species microarray analysi genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes Faraz Khan 1, Hui Hui Chai 2, Ishan Ajmera 3, Charlie Hodgman 3, Sean Mayes 1,2,* and Chungui Lu 4 Faraz Khan 1, Hui Hui Chai 2, Ishan Ajmera 3, Charlie Hodgman 3, Sean Mayes 1,2,* and Chungui Lu 4 Faraz Khan 1, Hui Hui Chai 2, Ishan Ajmera 3, Charlie Hodgman 3, Sean Mayes 1,2,* and Chungui Lu 4 1 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK; stxfk5@nottingham.ac.uk 1. Introduction Dehydration is one of the major stresses that inhibits plant growth and can reduce crop productivity. Hence, drought resistance is a key target in helping to ensure global food supply. Plants respond to dehydration stress in three broad approaches: (1) Dehydration escape; (2) Dehydration avoidance; and (3) Dehydration tolerance. Such mechanisms are seen in a range of leguminous Genes 2017, 8, 121; doi:10.3390/genes8040121 www.mdpi.com/journal/genes 2 of 19 Genes 2017, 8, 121 species, including the mung bean [1] and pigeon pea [2]. Dehydration escape is the ability of plants to complete their growth cycle and reach maturity with successful reproduction before the shortage of water reaches damaging levels [3]. Mechanisms of avoidance include improved root traits for a greater extraction of soil moisture, stomatal closure, a decreased radiation absorption through leaf rolling, a decreased leaf area for reduced water loss, and the accumulation of osmoprotectants such as proline, trehalose, and dehydrins [4]. Dehydration tolerance allows plants to survive through improved water-use efﬁciency, i.e., performing all of the biological, molecular, and cellular functions with minimal water. Numerous studies on the effects of dehydration stress on staple crops have been reported [1,2,4–10]. p Reduced water availability causes the production of abscisic acid (ABA), the phyto-hormone which initiates stomatal closure and inﬂuences other aspects of plant growth and physiology. It is responsible for regulating a broad range of genes during the dehydration response. The SNF1-related protein kinase, AREB (ABA-responsive element)/ABF are the key regulators of ABA signalling [11]. Improving the dehydration tolerance has also been linked to a reduction in shoot growth, while root growth is maintained, leading to an altered partition between the root and shoot. This process is achieved by cell-wall synthesis and remodelling. The formation of reactive oxygen species (ROS) and lignin peroxidases are the key steps involved in cell wall thickening. Stomatal closure limits the CO2 uptake by leaves, which leads to a reduction in photosynthesis as the leaf’s internal CO2 is depleted. Severe dehydration stress also limits photosynthesis by down-regulating the expression of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), fructose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase (PPDK), and NADP-malic enzyme (NADP-ME) [12]. Plant responses to dehydration affect vegetative growth by reducing the leaf-area expansion and total dry matter, which in turn decreases light interception [13]. Under dehydration stress, wheat (Triticum dicoccoides) shows a reduction in the number of grains, grain yield, shoot dry weight, and harvest index [8]. 1. Introduction In soybean specimens (Glycine max), the loss of seed yield was reported to be greatest when dehydration appeared during anthesis and the early reproductive stages [6–9]. A range of dehydration stress-related genes have been identiﬁed in Arabidopsis thaliana, rice (Oryza sativa), and other model plants [14]. These can be classiﬁed into two main groups: (i) Effector proteins, whose role is to alleviate the effect of the stress (such as water channel proteins, detoxiﬁcation enzymes, LEA proteins, chaperones, and osmoprotectants); and (ii) Regulatory proteins, which alter the expression or activity of effector genes and modify plant growth, such as the transcription factors DREB2 and AREB, and also protein kinases and phosphatases [15]. In recent years, plant breeders have turned to landraces (i.e., locally adapted genetically mixed populations) for trait improvement in various crops, including barley [16], sorghum [17], sesame [18], and soybean [19]. An early attempt to investigate the use of landraces in addressing the problem of dehydration tolerance has been carried out in wheat [20], although this did not delve into the speciﬁc genetics conferring the desirable traits. An alternative approach to identifying the genes conferring dehydration avoidance and tolerance is to study species that are already resilient under arid conditions. In this regard, bambara groundnut (Vigna subterranea (L) Verdc.) is a potential candidate. It is an underutilised, drought-resistant African legume, which is mainly grown in sub-Saharan Africa [5–21] and is sometimes used as an intercrop with major cereals, such as maize, because of its nitrogen ﬁxing potential [22]. Bambara groundnut is considered as a drought resistant crop with a reasonable protein content (18% to 22%), a high carbohydrate content (65%), and some level of lipids (6.5%) [23], with a similar overall composition to chickpea. A number of bambara groundnut landraces have well-developed tap roots which grow up to a height of 30–35 cm [24]. From the results of Mabhaudhi et al. [25], bambara groundnut has been shown to adopt dehydration-escape mechanisms, including a shortened vegetative growth period, early ﬂowering, a reduced duration of the reproductive stage, and early maturity under dehydration stress. Such responses are likely to be employed where the initial plant growth is based on stored soil water, 3 of 19 Genes 2017, 8, 121 but further rain is unlikely. 1. Introduction It has been reported that bambara groundnut responds to dehydration stress by partitioning more assimilate into the root, relative to the shoots, so that a greater soil volume can be exploited [26,27]. Nyamudeza [27] also observed that bambara groundnut allocated a greater fraction of its total dry weight to the roots than the groundnut, irrespective of the available soil moisture. This would suggest that bambara groundnut commits a greater supply of assimilates to root growth, irrespective of the soil moisture status. This strategy may have clear advantages when water subsequently becomes limited, but there could be a trade-off with the yield under benign environments. A greater root dry-weight was also reported when the bambara landrace, Burkina, was subjected to dehydration stress [28]. Dehydration-avoidance traits have also been observed, especially the accumulation of proline [21] and a reduced leaf area [29]. This study aims to investigate the effects of dehydration on gene expression in this reportedly drought-resistant species. The transcriptomes of two genotypes (DipC and Tiga Nicuru (TN)) were sampled, to identify what is common and how they differ in their response to a prolonged, but slowly intensifying, dehydration treatment. The climatic conditions in their native regions (Botswana and Mali, respectively) suggest that they are likely to have evolved in regions which would select for drought resistance, while potentially exhibiting some variation in the mechanisms employed to deal with dehydration, as they are morphologically and phenologically distinct [30]. Chai et al. [30,31] reported that transgressive segregation was observed in the segregating F5 population derived from the TNxDipC cross. The contrast between the two parental lines for a number of traits such as the days-to-maturity, stomatal conductance, 100-seed weight, leaf area, internode length, peduncle length, pod number per plant, and leaf carbon (delta C13) isotope analysis, suggest that some of these mechanisms for adaptation to dehydration could be non-identical in the two genotypes. For example, delta C13 was associated with a higher yield as observed in DipC, compared to TN [30]. In addition, the results showed that there were lines in the segregating population that performed better in terms of the ability to produce higher yields under drought conditions than the parental genotypes. Hence, evaluating the transcriptome of the two parental lines under dehydration stress could be a good indicator to investigate the molecular mechanism occurring in the two genotypes and its relationship to phenology and phenotype. 1. Introduction As a complete genome sequence is not available and microarray tools are still to be developed in this species, cross-species hybridisation with the Affymetrix Glycine-max microarray was investigated to test if this approach is acceptable for bambara groundnut transcriptomics, as it has been successful for other species [32–34]. 2.1. Plant Materials In this study, the experiment was conducted in the FutureCrop controlled tropical glasshouses at the School of Biosciences, Sutton Bonington Campus, University of Nottingham, UK. Two genotypes of bambara groundnut, DipC and TN, were planted in both ‘Water-limited’ and ‘Water-sufﬁcient’ control plots. 2.4. cRNA and Genomic DNA Affymetrix Labelling and Hybridisation The above RNA extracts were reverse transcribed to synthesize double stranded complementary DNA (cDNA). After puriﬁcation of the double-stranded cDNA products, the sample was transcribed in vitro to generate Biotinylated complementary RNAs (cRNAs), followed by puriﬁcation and fragmentation. The puriﬁed and fragmented cRNAs were then hybridised to the Affymetrix Soybean Gene Chip array (ThermoFisher Scientiﬁc, Lutterworth, UK). The scanned arrays produced CEL raw data ﬁles that were loaded onto Genespring GX version 13.1 (Agilent Genomics, Santa Clara, CA, USA) for further analysis. The extraction of genomic DNA (gDNA) from the two genotypes was performed using the DNA extraction Qiagen kit according to manufacturer’s instructions. Extracted DNA was labelled and hybridised to the Affymetrix Soybean TEST3 array and resulted in the generation of gDNA cell-intensity ﬁles (CEL ﬁles), after scanning. To identify probe pairs that efﬁciently hybridise to the gDNA, a series of user deﬁned threshold values were evaluated for the signal intensity. The perfect match (PM) probes were selected for interpreting the GeneChip arrays challenged with RNA from the species of interest [35]. 2.2. Site Descriptions and Experimental Design Plants were grown over a period of ﬁve months. A 12-hour photoperiod was created using an automated blackout system (Cambridge Glasshouses, Newport, UK), with day and night temperatures set at 28 ◦C and 23 ◦C respectively. Trickle tape irrigation with PVC micro-porous tubing was placed beside each plant row. The plants were irrigated at 06:00 h and 18:00 h for 20 min, with a measured flow rate of 1 L/h per tube, and each tube was 5 m in length. Two independent soil pits (5 m × 5 m × 1 m) containing sandy loam soil were used in the glasshouses. These were isolated from the surrounding soil by a Butyl liner and concrete pit structure with gravel drainage for separate water-limited and water-sufﬁcient plots. The PR2 water proﬁle probe (Delta-T devices, Cambridge, UK) was used to 4 of 19 Genes 2017, 8, 121 measure the soil moisture content. A randomised block design (RBD) with three blocks for each soil pit was implemented for this experiment. Three replicate plants for the water-sufﬁcient plot (continuously irrigated) and four replicates for the water-limited treatment plot were used. Three seeds were sown per replicate at a depth of 3–4 cm with a spacing of 25 cm × 25 cm between each ﬁnal plant position and multiple plants were later thinned to one plant per replicate at 20 days after sowing (DAS). Figure S1 shows the treatment regime. The irrigation system for the water limited treatment plot was turned off at 50 DAS and resumed at 92 DAS for plant recovery (in total, six weeks of treatment after 100% ﬂowering). Normal irrigation continued for the water-sufﬁcient plot throughout. The water-limited treatment was continued until an average of a 50% reduction in stomatal conductance was observed. Leaves from water-sufﬁcient and water-limited plants were collected at 92 DAS before recommencing irrigation, while those from ‘recovered’ plants were collected at 107 DAS after watering was resumed at 92 DAS. Labelled aluminium foil was used to wrap the harvested leaves, which was then transferred into liquid nitrogen for long term storage. All samples were stored in a −80 ◦C freezer before RNA extraction. DNA extraction from the two parental genotypes was completed using the DNA extraction Qiagen kit handbook. 2.3. RNA Extraction RNA was extracted using the RNeasy Qiagen kit (Qiagen, Manchester, UK), according to the manufacturer’s instructions. DNA was eliminated using DNase. A total of 80 µL of DNase I incubation mix, containing 10 µL DNase I stock solution and 70 µL buffer RDD, was added and incubated at room temperature for 15 min. Nanodrop readings and gel electrophoresis were performed to check the quality and quantity of RNA, as RNA samples required 100 ng/µL for 10 µL for microarray analysis. To make sure that the samples were free from active RNAse, 0.63 µL of 40 U/µL RNasin (Promega, Southampton, UK) was added for every 25 µL of the RNA sample. All samples were tested on an Nanodrop and Agilent bioanalyser for integrity (looking at the quality (ratio of 2.0) and integrity (a ratio of 2 for 28S/18S) for respective quantitation) before preparation for the microarray. .4. cRNA and Genomic DNA Affymetrix Labelling and Hybridisation 2.7. Expression Validation of Differentially Expressed Genes Using Real-Time qPCR Four genes which were potential candidate dehydration-associated genes (based on their functional annotations) with a differential expression level of >2-fold change and FDR corrected p-value ≤0.05 from the differential expression analysis, were chosen for quantitative PCR (qPCR) validation. The actin-11 from the available bambara groundnut transcriptome sequence was used as a housekeeping gene. The actin-11 gene is known to be one of the most stable reference genes for gene expression normalisation and has been used in soybean and rice specimens [41,42]. PCR forward and reverse primers were designed using Primer-BLAST [43] for the chosen genes. The primers were designed in three steps. Firstly, the target gene sequence to which the primers needed to be designed was downloaded from the soybean database. Secondly, the soybean-speciﬁc target gene sequence was blasted against a bambara groundnut transcriptome generated from RNA-sequencing data for a low-temperature stress experiment [44], by creating a BLAST database. Thirdly, the target gene sequence obtained from the bambara groundnut BLAST database was used to search through the BLAST database at NCBI to add weight to the selection of this sequence. Once the gene sequence was identiﬁed in the BLAST database, it was utilised to design primers with an appropriate primer size, GC content, and melting temperature (Tm) using Primer-BLAST. PCR was performed to check the quality of all the primers designed for the four dehydration-associated genes. PCR analysis was performed using the 7000 Sequence Detection System (Applied Biosystems, Cheshire, UK). The annealing temperature was set to 60 ◦C for the primer designed for the genes for PAL1 (Phenylalanine ammonia-lyase 1) and COMT (3-Caffeic acid o methyltransferase), and 58 ◦C for the Beta-fructofuranosidase and UBC-2 (ubiquitin conjugating enzyme-2) genes. The cycling parameters were set as: 95 ◦C for 10 min, 40 cycles of denaturing at 95 ◦C for 30 s, annealing at 60 ◦C/58 ◦C for 30 s, and extension at 72 ◦C for 30 s. First strand cDNA synthesis for all the RNA samples was carried out using a SuperScript III First-Strand Synthesis kit (ThermoFisher Scientiﬁc, Lutterworth, UK). The ﬁrst-strand cDNA was prepared for analysis by qPCR using PerfeCta SYBR Green SuperMix (Quantabio, Beverly, MA, USA) containing 2X reaction buffer (with optimized concentrations of MgCl2), dNTPs (dATP, dCTP, dGTP, dTTP), AccuStart Tag DNA Polymerase (Quantabio, Beverly, MA, USA) SYBR Green 1 dye, and stabilizers. 2.5. Probe Selection and Identiﬁcation of Differentially Expressed Genes The soybean array contained 37,500 probe sets, each containing 11 probe pairs per probe-set. For each genotype, custom CDF ﬁles were obtained, with reference to their gDNA hybridisation signal strength [36] for a subsequent estimation of the transcript levels. RNA CEL ﬁles were normalised in GeneSpring [37] using the Robust Multi-array Average. Differentially expressed genes (DEGs) were calculated using a t-test test (corrected by Benjamin Hochberg false discovery rate (FDR) multiple testing). Probe-sets with a FDR corrected p-value ≤0.05 and fold change of >2 were considered to be differentially expressed (either up or down regulated). Principal Component Analysis (PCA) was also 5 of 19 Genes 2017, 8, 121 carried out in GeneSpring and Bioconductor package “prcomp”. BINGO was used for discovering (from input gene lists) over-represented terms from the Gene Ontology [38]. 2.6. Construction of the Co-Expression Network Co-expression network analysis was carried out using the DeGNserver [39] and cytoscape 3.4 [40]. Separate networks were generated for each genotype. The input probe-sets were restricted to those that were differentially expressed between each pair of treatments (water-limited, water-sufﬁcient and recovery) and RMA (Robust Multi-Array Average)-normalised values were used across all samples. Links were assigned between pairs of nodes (i.e., probe-sets) when their Spearman’s Rank correlation was 0.9. The co-expression network was imported into cytoscape for visual representation and network analysis. For each genotype, another input ﬁle was made which, for each probe-set, deﬁned the parent (DipC or TN), the direction of differential expression caused by dehydration (up or down), and the role identiﬁed through homology in relation to drought resistance. This aided the interpretation of the combined network derived from both genotypes. 3.1. Probe Selection Based on gDNA to TN gDNA) Number of DEGs in DipC Number of DEGs in TN 20 61,072 61,072 670,388 670,388 6165 6165 60 60,877 60,895 479,538 482,352 6927 6814 100 59,782 59,835 302,834 304,708 7183 7159 150 56,266 56,511 190,570 193,522 7036 7159 200 51,071 51,319 129,806 132,521 6638 6731 300 37,813 38,000 66,907 68,106 5275 5345 500 17,469 18,176 23,464 24,693 2784 2911 600 12,258 12,930 15,701 16,650 2089 2170 700 8896 9566 11,193 12,061 1574 1673 800 6687 7208 8415 9070 1195 1291 900 5140 5657 6559 7140 958 1057 1000 4085 4482 5304 5733 802 877 gDNA: genomic DNA; DEG: differentially expressed gene; TN: Tiga Nicuru; Hyb: Hybridisation. Table 1. Retained probe-sets and probe-pairs at different threshold values. g p bl d b d b d ff h h ld l Table 1. Retained probe-sets and probe-pairs at different threshold values. Threshold Value Number of Probe Sets (Soybean Chip Hyb. to DipC gDNA) Number of Probe Sets (Soybean Chip Hyb. to TN gDNA) Number of Probe Pairs (Soybean Chip Hyb. to DipC gDNA) Number of Probe Pairs (Soybean Chip Hyb. to TN gDNA) Number of DEGs in DipC Number of DEGs in TN 20 61,072 61,072 670,388 670,388 6165 6165 60 60,877 60,895 479,538 482,352 6927 6814 100 59,782 59,835 302,834 304,708 7183 7159 150 56,266 56,511 190,570 193,522 7036 7159 200 51,071 51,319 129,806 132,521 6638 6731 300 37,813 38,000 66,907 68,106 5275 5345 500 17,469 18,176 23,464 24,693 2784 2911 600 12,258 12,930 15,701 16,650 2089 2170 700 8896 9566 11,193 12,061 1574 1673 800 6687 7208 8415 9070 1195 1291 900 5140 5657 6559 7140 958 1057 1000 4085 4482 5304 5733 802 877 gDNA: genomic DNA; DEG: differentially expressed gene; TN: Tiga Nicuru; Hyb: Hybridisation. Table 1. Retained probe-sets and probe-pairs at different threshold values. Threshold Value Number of Probe Sets (Soybean Chip Hyb. to DipC gDNA) Number of Probe Sets (Soybean Chip Hyb. to TN gDNA) Number of Probe Pairs (Soybean Chip Hyb. to DipC gDNA) Number of Probe Pairs (Soybean Chip Hyb. 2.7. Expression Validation of Differentially Expressed Genes Using Real-Time qPCR The synthesized cDNA was cleaned from the remaining RNA using the enzyme mix included in the kit (Escherichia coli RNase H). The qPCR components were prepared for 10 µL reactions and Melt-curve analysis was performed. The sample cycle threshold (Ct) was standardized for each template based on the actin-11 gene control amplicon behaviour. The 2−∆∆Ct method was used to analyse the relative changes in gene expression from the qRT-PCR experiment [45]. To validate whether the right PCR product was generated for the expression studies, the desired fragment of intact cDNA for all genes was sent for sequencing after the gel extraction using a QIAquick Gel Extraction Kit (Qiagen, Manchester, UK). 6 of 19 Genes 2017, 8, 121 3.1. Probe Selection Based on gDNA 3.1. Probe Selection Based on gDNA The genomic DNA of both genotypes was individually hybridised to the Affymetrix Soybean GeneChip array to study the global genome hybridisation for probe selection. The numbers of retained probe-pairs and probe-sets are shown in Table 1. With increasing threshold values, the number of probe-pairs retained in the probe mask ﬁle started decreasing rapidly (Figure 1), while the number of probe-sets (representing genes) decreased at a slower rate. This suggests that, even at higher gDNA hybridisation thresholds, at least some of the gene-designed oligonucleotides are cross-hybridising for many of the probe-sets and that the cross-species array approach is a reasonable approach for bambara groundnut transcriptomics. Genes 2017, 8, 121 6 of 19 higher gDNA hybridisation thresholds, at least some of the gene-designed oligonucleotides are cross- hybridising for many of the probe-sets and that the cross-species array approach is a reasonable h f b b d t t i t i Table 1. Retained probe-sets and probe-pairs at different threshold values. Threshold Value Number of Probe Sets (Soybean Chip Hyb. to DipC gDNA) Number of Probe Sets (Soybean Chip Hyb. to TN gDNA) Number of Probe Pairs (Soybean Chip Hyb. to DipC gDNA) Number of Probe Pairs (Soybean Chip Hyb. to TN gDNA) Number of DEGs in DipC Number of DEGs in TN 20 61,072 61,072 670,388 670,388 6165 6165 60 60,877 60,895 479,538 482,352 6927 6814 100 59,782 59,835 302,834 304,708 7183 7159 150 56,266 56,511 190,570 193,522 7036 7159 200 51,071 51,319 129,806 132,521 6638 6731 300 37,813 38,000 66,907 68,106 5275 5345 500 17,469 18,176 23,464 24,693 2784 2911 600 12,258 12,930 15,701 16,650 2089 2170 700 8896 9566 11,193 12,061 1574 1673 800 6687 7208 8415 9070 1195 1291 900 5140 5657 6559 7140 958 1057 1000 4085 4482 5304 5733 802 877 gDNA: genomic DNA; DEG: differentially expressed gene; TN: Tiga Nicuru; Hyb: Hybridisation. pp g p Table 1. Retained probe-sets and probe-pairs at different threshold values. Threshold Value Number of Probe Sets (Soybean Chip Hyb. to DipC gDNA) Number of Probe Sets (Soybean Chip Hyb. to TN gDNA) Number of Probe Pairs (Soybean Chip Hyb. to DipC gDNA) Number of Probe Pairs (Soybean Chip Hyb. 3.1. Probe Selection Based on gDNA to TN gDNA) Number of DEGs in DipC Number of DEGs in TN 20 61,072 61,072 670,388 670,388 6165 6165 60 60,877 60,895 479,538 482,352 6927 6814 100 59,782 59,835 302,834 304,708 7183 7159 150 56,266 56,511 190,570 193,522 7036 7159 200 51,071 51,319 129,806 132,521 6638 6731 300 37,813 38,000 66,907 68,106 5275 5345 500 17,469 18,176 23,464 24,693 2784 2911 600 12,258 12,930 15,701 16,650 2089 2170 700 8896 9566 11,193 12,061 1574 1673 800 6687 7208 8415 9070 1195 1291 900 5140 5657 6559 7140 958 1057 1000 4085 4482 5304 5733 802 877 gDNA: genomic DNA; DEG: differentially expressed gene; TN: Tiga Nicuru; Hyb: Hybridisation. Figure 1. Effect of intensity thresholds. Number of probe pairs (blue line) and probe sets (magenta line) retained for DipC (top) and Tiga Nicuru (TN) (bottom) respectively at different genomic DNA (gDNA) intensity thresholds. Figure 1. Effect of intensity thresholds. Number of probe pairs (blue line) and probe sets (magenta line) retained for DipC (top) and Tiga Nicuru (TN) (bottom) respectively at different genomic DNA (gDNA) intensity thresholds. Figure 1. Effect of intensity thresholds. Number of probe pairs (blue line) and probe sets (magenta line) retained for DipC (top) and Tiga Nicuru (TN) (bottom) respectively at different genomic DNA (gDNA) intensity thresholds. Figure 1. Effect of intensity thresholds. Number of probe pairs (blue line) and probe sets (magenta line) retained for DipC (top) and Tiga Nicuru (TN) (bottom) respectively at different genomic DNA (gDNA) intensity thresholds. The number of retained probe-sets and probe-pairs on the Soybean chip for both the DipC and TN gDNA hybridisations were determined, corresponding to each threshold value (Table 1). A custom CDF file with a threshold of 100 was chosen for differential expression analysis in both genotypes, as it allowed for a good sensitivity to detect the maximum number of differentially- The number of retained probe-sets and probe-pairs on the Soybean chip for both the DipC and TN gDNA hybridisations were determined, corresponding to each threshold value (Table 1). A custom CDF ﬁle with a threshold of 100 was chosen for differential expression analysis in both genotypes, as it 7 of 19 Genes 2017, 8, 121 allowed for a good sensitivity to detect the maximum number of differentially-expressed transcripts (Table 1). Furthermore, both genotypes were found to be highly similar in terms of the probe-sets detected at this threshold. 3.2. Principal Component Analysis The PCA plot (Figure 2) shows that, under water-sufﬁcient treatment, the two genotypes appear to have similar transcriptomes. The ﬁrst two Principal Components account for 25.45% and 17.11% of the variance, respectively, suggesting that it is due to a range of hybridisation/expression differences between the chips. Recovery after dehydration stress, however, caused the most variation and suggests that the recovery transcriptome does not return to the water-sufﬁcient state (control). The DipC water-limited treatment sample ‘D.DipC.Rep2’ could be a potential outlier and this needs to be borne in mind in further analysis. The 3D PCA plots of genotype-speciﬁc data showed a good separation of the three treatments (conditions) and better PCA scores (see Figure S2). Genes 2017, 8, 121 7 of 19 differences between the chips. Recovery after dehydration stress, however, caused the most variation and suggests that the recovery transcriptome does not return to the water-sufficient state (control). The DipC water-limited treatment sample ‘D.DipC.Rep2’ could be a potential outlier and this needs to be borne in mind in further analysis. The 3D PCA plots of genotype-specific data showed a good separation of the three treatments (conditions) and better PCA scores (see Figure S2). Figure 2. Principal component analysis (PCA) plot of the expression data from the microarrays. The principal components PC1 and PC2 values for each chip have been placed on a scatter plot. Each chip result is defined by a three-part character string consisting of the treatment, genotype, and replicate number. IR, D, and REC refer to water-sufficient, water-limited, and recovery treatment, respectively; the genotypes are DipC and TN; and Rep1–4 refers to the specific biological replicate. Note, the water- sufficient and recovery treatments have only three replicates, while dehydration has four. 3.3. Gene Expression Under Water-Sufficient Conditions Figure 2. Principal component analysis (PCA) plot of the expression data from the microarrays. The principal components PC1 and PC2 values for each chip have been placed on a scatter plot. Each chip result is deﬁned by a three-part character string consisting of the treatment, genotype, and replicate number. IR, D, and REC refer to water-sufﬁcient, water-limited, and recovery treatment, respectively; the genotypes are DipC and TN; and Rep1–4 refers to the speciﬁc biological replicate. Note, the water-sufﬁcient and recovery treatments have only three replicates, while dehydration has four. Figure 2. Principal component analysis (PCA) plot of the expression data from the microarrays. 3.1. Probe Selection Based on gDNA A total of 59,533 probe-sets were common to both genotypes at the threshold of 100, while 249 and 302 probe-sets were speciﬁc to DipC and TN, respectively. These results therefore suggest a high sequence similarity (>99%) at this level of sequence sampling. 3.3. Gene Expression Under Water-Sufﬁcient Conditions 3.3. Gene Expression Under Water-Sufﬁcient Conditions It is pertinent to consider the state of the genotype transcriptomes before any dehydration treatment has taken place. However, owing to the high background noise in microarray studies, it is unclear what intensity level deﬁnes a gene as being transcribed. Figure S3 shows that the ranked intensity values follow a roughly sigmoidal curve. The point of inﬂection (at which the declining gradient is at its shallowest) covers the top two-thirds of the probe-sets, and corresponds to an RMA value of 0.97. This may be a stringent cut-off, given that an RMA value of one corresponds to the average across all probe-sets on the array, but it ensures that there were few, if any, false positives. This left 39,855 probe-sets for DipC and 39,890 for TN. There are 26,496 probe-sets in common between the two genotypes, suggesting differences in the general transcriptional behaviour of the two genotypes. Each genotype had a little over 90 probe sets with functional annotations related to ABA signalling and dehydration responses (see Tables S1 and S2), of which 60 were common to both. These include homologues of much of the ABA synthesis and response network, the DREB1 transcription factor, Early-Response to Dehydration proteins 3, 4, 8, 14–16, and 18, four osmoprotectant genes, two dehydration-response genes inﬂuencing photosynthesis, and 21 other probe sets corresponding to dehydration-associated proteins of an unknown function (see Table S3). Table S4 lists the genes differentially expressed between the two water-sufﬁcient treated genotypes, but at this stage, nothing stands out as remarkable. 3.2. Principal Component Analysis The principal components PC1 and PC2 values for each chip have been placed on a scatter plot. Each chip result is defined by a three-part character string consisting of the treatment, genotype, and replicate number. IR, D, and REC refer to water-sufficient, water-limited, and recovery treatment, respectively; the genotypes are DipC and TN; and Rep1–4 refers to the specific biological replicate. Note, the water- sufficient and recovery treatments have only three replicates, while dehydration has four. Figure 2. Principal component analysis (PCA) plot of the expression data from the microarrays. The principal components PC1 and PC2 values for each chip have been placed on a scatter plot. Each chip result is deﬁned by a three-part character string consisting of the treatment, genotype, and replicate number. IR, D, and REC refer to water-sufﬁcient, water-limited, and recovery treatment, respectively; the genotypes are DipC and TN; and Rep1–4 refers to the speciﬁc biological replicate. Note, the water-sufﬁcient and recovery treatments have only three replicates, while dehydration has four. Genes 2017, 8, 121 8 of 19 3.4. Identiﬁcation of Differentially Expressed Genes Mostly, the upregulated genes under dehydration stress in DipC relate to the secondary metabolism of cell-wall components, while the TN genes include transcription-related factors, most notably a CONSTANS-like gene. Furthermore, GO term overrepresentation analysis for both DipC and TN showed an emphasis on various metabolic processes related to cellular amino acids and their derivatives, secondary metabolites and carbohydrates (Table S10). Hence, despite the genomic hybridisation mask demonstrating that the pure hybridisation was very similar between the two genotypes, there is a very different transcriptional response to dehydration stress by each genotype. Microarray data has a limited dynamic range, even when within species, so it is important to validate a small set of microarray observations. Hence, validation through qRT-PCR was performed. Table 3. Top upregulated genes in DipC and TN. Gene Name FDR Fold Change Gene Description References UP-Regulated Genes in DipC PAL1 (Phenylalanine ammonia-lyase 1) 0.018 3.901 Key enzyme involved in the biosynthesis of isoprenoid antioxidative and polyphenol compounds such as lignin and is involved in defense mechanism. [53] ATEP3/AtchitIV 0.001 3.845 Encodes an EP3 chitinase that is stimulated under abiotic stress. [54] TXR1(Thaxtomin A resistant 1)/ ATPAM16 6.87 × 10−5 3.718 TXR1 is a component of a dispensable transport mechanism. Involved in negative regulation of defense responses by reducing reactive oxygen species (ROS). [55] Acetyl-CoA C-acyltransferase, putative/ 3-ketoacyl-CoA thiolase 0.001 3.554 Functions in Jasmonic acid synthesis which plays a role in plant response to mechanical and abiotic stress. [56] UBC-2 (ubiquitin-conjugating enzyme 2) 0.004 3.407 Ubiquitination plays a part in increasing rate of the protein breakdown. Arabidopsis plants overexpressing UBC-2 were more tolerant to dehydration stress compared to the control plants. [57] Rho GDP dissociation inhibitor 2 0.001 3.348 Involves in the regulation of Rho protein and small GTPase mediated signal transduction. [58] Histidine amino acid transporter (LHT1) 0.001 3.256 Amino acid transmembrane transporter involved in apoplastic transport of amino acids in leaves. [59] COMT (3-Caffeic acid o methyltransferase) 0.006 3.234 Involved in lignin biosynthesis. High activation of lignifying enzymes was found in dehydration-stressed white clover (Trifolium repens L.), which lead to reduced forage growth. [60] Glycine decarboxylase complex H 0.005 3.113 Functions in photo respiratory carbon recovery. Carbon dioxide is found to be low in plants subjected to dehydration stress due to the closing of stomata in order to prevent water loss. 3.4. Identiﬁcation of Differentially Expressed Genes For DipC and TN, the numbers of genes differentially expressed as a result of the dehydration and recovery treatments, and detected by the cross-species microarray approach, are shown in Table 2, with the full lists of probe-sets and functional annotations presented in Tables S5–S8. The top upregulated and downregulated genes in DipC and TN are shown in Tables 3 and 4, respectively. The numbers for DipC were consistently higher than for TN, and the water-limited treatment caused more down- than upregulation, while recovery had the reverse effect. Table 2. Differentially expressed gene numbers. Water-Limited versus Water-Sufﬁcient Water-Limited versus Recovery Up-Regulated under Dehydration Down-Regulated under Recovery Down-Regulated under Dehydration Up-Regulated under Recovery Up-Regulated Down-Regulated DipC 80 68 109 94 340 146 Tiga Nicuru 28 22 53 42 294 97 Table 2. Differentially expressed gene numbers. Recovery led to many more differentially expressed genes (486 and 391) than dehydration stress (189 and 81). There were six possible system effects that can be gleaned from these data (Figure S4). The upregulated genes under the water-limited treatment that returned to a water-sufﬁcient state on recovery and the downregulated genes that returned to a normal expression at recovery are the strictly dehydration-responsive genes (~75% in both genotypes), while those that signiﬁcantly changed and did not return to the pretreatment levels (~25%) correspond to a dehydration-induced state change. The latter may be due to epigenetic effects, such as a change in the methylation state of gene-regulatory regions. The larger numbers of differentially expressed genes from water-limited conditions to recovery may be accounted for by aging and other highly variable factors (see Figure 2), such as the soil conditions in each pit. The fold changes of the upregulated genes under dehydration stress in both genotypes are relatively small (mostly < 4-fold). Furthermore, there were only nine differentially expressed genes which were common to both genotypes (see Table 5). The only common upregulated gene was beta-fructofuranosidase, which hydrolyses sucrose to provide more glucose, hence playing a potential role in osmoprotection and energy production. In contrast, half of the common downregulated genes Genes 2017, 8, 121 9 of 19 were related to transcription and also play roles in stomatal regulation. Excluding the potential outlier, ‘D.DipC.Rep2’ had little effect upon the common gene analysis (Table S9), so it has been included in subsequent analyses. 3.4. Identiﬁcation of Differentially Expressed Genes [61] Up-Regulated Genes in TN Clp amino terminal domain-containing protein, putative 0.035 3.778 Protein and ATP binding. CONSTANS-LIKE 1 0.025 3.294 Transcription factor regulating ﬂower development and response to light stimulus. [62] DRB3 (DSRNA-BINDING PROTEIN 3) 0.020 2.984 Assists in miRNA-targeted RNA degradation. [63] SIGE (SIGMA FACTOR E) 0.032 2.808 Responds to effects of abiotic stresses. Phosphorylation of major sigma factor SIG1 in Arabidopsis thaliana inhibits the transcription of the psaA gene, which encodes photosystem-I (PS-I). This disturbs photosynthetic activity. [64,65] Reticulon family protein 0.029 2.772 Playing a role in promoting membrane curvature Cytochrome c oxidase family protein 0.025 2.727 Essential for the assembly of functional cytochrome oxidase protein. DNA-binding S1FA family protein 0.049 2.717 Binds to the negative promoter element S1F. DNA photolyase 0.032 2.667 DNA repair enzyme. Zinc knuckle (CCHC-type) family protein 0.040 2.567 Zinc ion binding. Monosaccaride transporter 0.025 2.547 Plays a role in long-distance sugar partitioning or sub-cellular sugar distribution. Nodulin MtN3 family protein 0.025 2.376 Key role in the establishment of symbiosis. Serine acetyltransferase, N-terminal 0.040 2.302 Catalyzes the formation of a cysteine precursor. Table 3. Top upregulated genes in DipC and TN. Table 3. Top upregulated genes in DipC and TN. 10 of 19 Genes 2017, 8, 121 Table 4. Top downregulated genes in DipC and TN. Table 4. Top downregulated genes in DipC and TN. Gene name FDR Fold Change Gene Description References Down-Regulated Genes in DipC Dihydroxyacetone kinase 0.003 6.489 Glycerone kinase activity. Phosphoglucomutase, putative/glucose phosphomutase, putative 0.007 6.471 Involved in controlling photosynthetic carbon ﬂow and plays essential role starch synthesis. Down regulation of photosynthesis-related gene will lead to signiﬁcant reduction in plant growth. [66] Auxin-induced protein 22D AUXX-IAA 0.003 4.627 Involved in stress defense response. Many AUXX-IAA genes were found to be down-regulated in Sorghum bicolor under drought conditions. [67] CP12-1, putative 0.014 4.390 Involved in calvin cycle, therefore linked to photosynthesis. Most drastic down-regulated genes which were photosynthesis-related was observed in barley (Hordeum vulgare L.). [68] PHS2 (ALPHA-GLUCAN PHOSPHORYLASE 2). 0.014 4.375 Encodes a cytosolic alpha-glucan phosphorylase. APRR5 (PSEUDO-RESPONSE REGULATOR 5), Pseudo ARR-B family 0.001 4.145 Linked to cytokinin-mediated regulation. Thiamine biosynthesis family protein 0.002 4.132 Catalyses the activation of small proteins, such as ubiquitin or ubiquitin-like proteins. Zinc ﬁnger (C3HC4-type RING ﬁnger) 0.007 3.611 Mediate ubiquitin-conjugating enzyme (UBC-2) dependent ubiquitation. [69] WRKY40 0.033 3.104 Regulator of ABA signalling. 3.4. Identiﬁcation of Differentially Expressed Genes [69] WRKY40 0.033 3.104 Regulator of ABA signalling. It inhibits the expression of ABA-responsive genes ABF4, AB14, AB15, DREB1A, MYB2 and RAB18. [70] Down-Regulated Genes in TN AGL83 (AGAMOUS-LIKE 83) 0.025 4.374 DNA-binding transcription factor. CRR23 (chlororespiratory reduction 23) 0.025 3.625 A subunit of the chloroplast NAD(P)H dehydrogenase complex, involved in PS-I cyclic electron transport. Located on the thylakoid membrane. Mutant has impaired NAD(P)H dehydrogenase activity. Part of dehydration repressing photosynthesis. [71] MYB30 (MYB DOMAIN PROTEIN 30) 0.032 3.250 Acts as a positive regulator of hypersensitive cell death and salicylic acid synthesis. Involved in the regulation of abscisic acid (ABA) signalling. [72] Photosystem II family protein, putative 0.029 3.158 Linked to photosynthesis. Down-regulation of photosynthesis-related genes during dehydration stress was observed in maize (Zea mays), which in turn leads to signiﬁcant reduction in plant growth. [73] Phosphoesterase 0.047 3.136 Hydrolase activity, acting on ester bonds. Zing-ﬁnger (C3HC4-type) 0.045 2.947 Mediate ubiquitin-conjugating enzyme (UBC-2) dependent ubiquitation. [69] NHX2 (Sodium proton exchanger 2) 0.040 2.742 Involved in antiporter activity. Also involved in potassium ion homoeostasis and regulation of stomatal closure. Involved in the accumulation of K+ that drives the rapid stomatal opening. Down-regulation of genes related to stomatal regulation has been observed in soybean, which appears to be a part of dehydration response, leading to a reduction in the amount of stomata in leaves. [74] Inositol 1,3,4-trisphosphate 5/6-kinase 0.035 2.090 Part of IP3 signal transduction pathway. [75] Table 5. Overlapping up- and downregulated genes. Gene Name FDR Fold Change Gene Description References Up-Regulated Genes Beta-fructofuranosidase 8.90 × 10−4 3.193 Catalyses the hydrolysis of sucrose. A rise in monosaccharide content caused by the Beta-fructofuranosidase can compensate for the decline in photosynthetic carbon assimilation indicated by the decrease in net photosynthesis. [46,47] Down-Regulated Genes MEE59 (maternal effect embryo arrest 59) 8.94 × 10−4 8.580 Embryo development ending in seed dormancy. Calcineurin-like phosphoesterase family protein (CPPED1) 6.72 × 10−4 5.857 Plays inhibitory role in glucose uptake. Down-regulation of CPPED1 improves glucose metabolism. [48] Putative lysine-speciﬁc demethylase JMJD5 Jumonji/Zinc-ﬁnger-class domain containing protein 0.003 4.971 Plays role in a histone demethylation mechanism that is conserved from yeast to human. Down-regulation may lead to an increase in methylated histones and hence general down-regulation of transcription. [49] MYB-like transcription factor 0.024 4.103 Arabidopsis homolog is known to regulate stomatal opening, ﬂower development, and plays role in circadian rhythm. [50] Table 5. Overlapping up- and downregulated genes. 3.4. Identiﬁcation of Differentially Expressed Genes It inhibits the expression of ABA-responsive genes ABF4, AB14, AB15, DREB1A, MYB2 and RAB18. [70] Down-Regulated Genes in TN AGL83 (AGAMOUS-LIKE 83) 0.025 4.374 DNA-binding transcription factor. CRR23 (chlororespiratory reduction 23) 0.025 3.625 A subunit of the chloroplast NAD(P)H dehydrogenase complex, involved in PS-I cyclic electron transport. Located on the thylakoid membrane. Mutant has impaired NAD(P)H dehydrogenase activity. Part of dehydration repressing photosynthesis. [71] MYB30 (MYB DOMAIN PROTEIN 30) 0.032 3.250 Acts as a positive regulator of hypersensitive cell death and salicylic acid synthesis. Involved in the regulation of abscisic acid (ABA) signalling. [72] Photosystem II family protein, putative 0.029 3.158 Linked to photosynthesis. Down-regulation of photosynthesis-related genes during dehydration stress was observed in maize (Zea mays), which in turn leads to signiﬁcant reduction in plant growth. [73] Phosphoesterase 0.047 3.136 Hydrolase activity, acting on ester bonds. Zing-ﬁnger (C3HC4-type) 0.045 2.947 Mediate ubiquitin-conjugating enzyme (UBC-2) dependent ubiquitation. [69] NHX2 (Sodium proton exchanger 2) 0.040 2.742 Involved in antiporter activity. Also involved in potassium ion homoeostasis and regulation of stomatal closure. Involved in the accumulation of K+ that drives the rapid stomatal opening. Down-regulation of genes related to stomatal regulation has been observed in soybean, which appears to be a part of dehydration response, leading to a reduction in the amount of stomata in leaves. [74] Inositol 1,3,4-trisphosphate 5/6-kinase 0.035 2.090 Part of IP3 signal transduction pathway. [75] Table 4. Top downregulated genes in DipC and TN. Gene name FDR Fold Change Gene Description References Down-Regulated Genes in DipC Dihydroxyacetone kinase 0.003 6.489 Glycerone kinase activity. Phosphoglucomutase, putative/glucose phosphomutase, putative 0.007 6.471 Involved in controlling photosynthetic carbon ﬂow and plays essential role starch synthesis. Down regulation of photosynthesis-related gene will lead to signiﬁcant reduction in plant growth. [66] Auxin-induced protein 22D AUXX-IAA 0.003 4.627 Involved in stress defense response. Many AUXX-IAA genes were found to be down-regulated in Sorghum bicolor under drought conditions. [67] CP12-1, putative 0.014 4.390 Involved in calvin cycle, therefore linked to photosynthesis. Most drastic down-regulated genes which were photosynthesis-related was observed in barley (Hordeum vulgare L.). [68] PHS2 (ALPHA-GLUCAN PHOSPHORYLASE 2). 0.014 4.375 Encodes a cytosolic alpha-glucan phosphorylase. APRR5 (PSEUDO-RESPONSE REGULATOR 5), Pseudo ARR-B family 0.001 4.145 Linked to cytokinin-mediated regulation. Thiamine biosynthesis family protein 0.002 4.132 Catalyses the activation of small proteins, such as ubiquitin or ubiquitin-like proteins. Zinc ﬁnger (C3HC4-type RING ﬁnger) 0.007 3.611 Mediate ubiquitin-conjugating enzyme (UBC-2) dependent ubiquitation. 3.5. Conﬁrmation of Candidate Dehydration-Associated Genes by Real-Time qRT-PCR a small set of microarray observations. Hence, validation through qRT PCR was per 3 5 C fi ti f C did t D h d ti A i t d G b R l Ti RT PCR Four differentially expressed genes (PAL1, Beta-fructofuranosidase, COMT and UBC-2) were chosen for further analysis, as they showed high levels of expression under water-limited treatment [46–53,57–60] (Tables 4 and 5) and are dehydration-associated genes based on their functional annotations. Figure 3 shows the results of qPCR analysis. The transcript levels of Beta-fructofuranosidase, COMT, and UBC-2 conﬁrmed the expression trends seen in the microarray data. PAL1 showed the expected increase in DipC, and an increase in TN was observed, which was not observed in the microarray results. The reason for this is unclear. 3.5. Confirmation of Candidate Dehydration-Associated Genes by Real-Time qRT-PCR Four differentially expressed genes (PAL1, Beta-fructofuranosidase, COMT and UBC-2) were chosen for further analysis, as they showed high levels of expression under water-limited treatment [46–53,57–60] (Tables 3 and 4) and are dehydration-associated genes based on their functional annotations. Figure 3 shows the results of qPCR analysis. The transcript levels of Beta- fructofuranosidase, COMT, and UBC-2 confirmed the expression trends seen in the microarray data. PAL1 showed the expected increase in DipC, and an increase in TN was observed, which was not observed in the microarray results. The reason for this is unclear. Figure 3. Comparison of qPCR and microarray intensity values: Rows (A) and (B) respectively refer to results for DipC and TN. The left- and right-hand pairs of columns correspond to the qPCR and microarray values for DipC and TN, respectively. The gene under study is named at the top of each panel. In order, the investigated genes are Beta-fructofuranosidase, COMT, UBC-2, and PAL1. qPCR and Microarray values are shown as fold changes with respect to the water-sufficient treatment (Irrigated). Error bars denote the standard error. Single and double asterisks indicate that p-value is less than 0.05 and 0.01, respectively, which was assessed by the paired t-test between groups. Irrigated and Drought refer to water-sufficient and water-limited treatments, respectively. 3 6 T i ti F t A i t d ith D h d ti St Figure 3. Comparison of qPCR and microarray intensity values: Rows (A) and (B) respectively refer to results for DipC and TN. The left- and right-hand pairs of columns correspond to the qPCR and microarray values for DipC and TN, respectively. The gene under study is named at the top of each panel. In order, the investigated genes are Beta-fructofuranosidase, COMT, UBC-2, and PAL1. 3.4. Identiﬁcation of Differentially Expressed Genes Gene Name FDR Fold Change Gene Description References Up-Regulated Genes Beta-fructofuranosidase 8.90 × 10−4 3.193 Catalyses the hydrolysis of sucrose. A rise in monosaccharide content caused by the Beta-fructofuranosidase can compensate for the decline in photosynthetic carbon assimilation indicated by the decrease in net photosynthesis. [46,47] Down-Regulated Genes MEE59 (maternal effect embryo arrest 59) 8.94 × 10−4 8.580 Embryo development ending in seed dormancy. Calcineurin-like phosphoesterase family protein (CPPED1) 6.72 × 10−4 5.857 Plays inhibitory role in glucose uptake. Down-regulation of CPPED1 improves glucose metabolism. [48] Putative lysine-speciﬁc demethylase JMJD5 Jumonji/Zinc-ﬁnger-class domain containing protein 0.003 4.971 Plays role in a histone demethylation mechanism that is conserved from yeast to human. Down-regulation may lead to an increase in methylated histones and hence general down-regulation of transcription. [49] MYB-like transcription factor 0.024 4.103 Arabidopsis homolog is known to regulate stomatal opening, ﬂower development, and plays role in circadian rhythm. [50] 11 of 19 Genes 2017, 8, 121 11 of 19 Table 5. Cont. Table 5. Cont. Gene Name FDR Fold Change Gene Description References Down-Regulated Genes F-box family protein (FBL14) 0.001 3.744 Functions in signal transduction and regulation of cell cycle. BRH1 (BRASSINOSTEROID- RESPONSIVE RING-H2) 0.007 2.899 BRH1 is known to inﬂuence stomatal density. [51] Bundle-sheath defective protein 2 family/bsd2 family 0.003 2.441 Protein required for post-translational regulation of Rubisco large subunit (rbcL). [52] Mitochondrial substrate carrier family protein. 0.030 2.435 Involved in energy transfer. FDR: false discovery rate. Genes 2017, 8, 121 11 of 19 and TN showed an emphasis on various metabolic processes related to cellular amino acids and their derivatives, secondary metabolites and carbohydrates (Table S10). Hence, despite the genomic hybridisation mask demonstrating that the pure hybridisation was very similar between the two genotypes, there is a very different transcriptional response to dehydration stress by each genotype. Microarray data has a limited dynamic range, even when within species, so it is important to validate 3.6. Transcription Factors Associated with Dehydration Stress 3.6. Transcription Factors Associated with Dehydration Stress 3.6. Transcription Factors Associated with Dehydration Stress 3.6. Transcription Factors Associated with Dehydration Stress The DEGs genes from both genotypes identiﬁed various transcription-related factors (TFs). Common to both genotypes are the downregulation of BRH1, an MYB, MEE59, and JMJD5. The latter is a histone demethylase, so could suggest changes at the epigenetic level of gene expression. Its downregulation could result in the indirect repression of multiple genes. On top of the common genes, DipC shows the upregulation of two TFs (WRKY51 and a bHLH TF) and the downregulation of four others (ATAUX2-11, WRKY40, a C2H2 Zn-ﬁnger, and three probe-sets for GIGANTEA). TN, on the other hand, shows the upregulation of genes for CONSTANS1-like, S1FA DNA-binding, and a double-strand RNA-binding protein (which can aid microRNA-mediated RNA degradation). The downregulated TFs in TN are MYB60 and a second MEE59. Co-expression networks were individually built for DipC and TN (see Tables S11 and S12), and the dehydration-speciﬁc network of each were merged. This resulted in more TFs being included, which are features of recovery treatment. By looking at the number of links that each node has in the genotype-speciﬁc and merged networks, it is possible to rank the potential importance of the different TFs (see Table 6). The DipC TFs had a higher number of links than TN. In the case of DipC, WRKY40 stands out as being the TF with the most co-expressed genes, with ATAUX2-11, PRR7, and a Zinc-ﬁnger protein (GmaAffx.33796.3.S1_at) also looking relevant. For TN, however, the TFs are not ranked so highly, with CONSTANS-like 1 and MYB60 showing the greatest involvement. For this genotype, the differentially expressed TFs in common with DipC seem almost as important. Table 6. Vertex degrees of differentially expressed transcription factors. 3.6. Transcription Factors Associated with Dehydration Stress DipC TN Probe-Set Name V◦Whole V◦Drought Probe-Set Name V◦Whole V◦Drought Gma.16733.1.S1_at WRKY40 68 17 GmaAffx.45249.1.S1_at CONSTANS-like 1 16 3 Gma.6670.1.S1_at PRR7 49 7 GmaAffx.84566.2.S1_at MYB60 8 3 GmaAffx.33796.3.S1_at Zinc-ﬁnger like C2H2 45 7 GmaAffx.86517.1.S1_at AGL83 6 1 GmaAffx.92679.1.S1_s_at ATAUX2-11 41 9 Gma.1576.1.S1_at Zinc-ﬁnger C3HC4 5 1 GmaAffx.35309.1.S1_s_at GRF2 35 6 GmaAffx.65059.1.S1_at bHLH 32 7 GmaAffx.90399.1.S1_at C3HC4 Zinc-ﬁnger 31 9 Gma.15774.1.S1_at Zinc-ﬁnger C3HC4 26 3 GmaAffx.53180.1.S1_at PRR7 25 9 GmaAffx.80492.1.S1_at PRR5 9 2 GmaAffx.73009.2.S1_at WRKY51 7 5 Common TFs GmaAffx.60283.1.S1_at BRH1 42 6 GmaAffx.9286.1.S1_s_at MYB 27 4 Gma.17248.1.A1_at JMJD5 26 3 GmaAffx.10162.1.S1_at MEE59 13 3 V◦refers to the number of links of each transcription factor (TF) node, in either the whole genotype-speciﬁc network, or merged dehydration-speciﬁc network. Table 6. Vertex degrees of differentially expressed transcription factors. V◦refers to the number of links of each transcription factor (TF) node, in either the whole genotype-speciﬁc network, or merged dehydration-speciﬁc network. 3.5. Conﬁrmation of Candidate Dehydration-Associated Genes by Real-Time qRT-PCR a small set of microarray observations. Hence, validation through qRT PCR was per 3 5 C fi ti f C did t D h d ti A i t d G b R l Ti RT PCR qPCR and Microarray values are shown as fold changes with respect to the water-sufﬁcient treatment (Irrigated). Error bars denote the standard error. Single and double asterisks indicate that p-value is less than 0.05 and 0.01, respectively, which was assessed by the paired t-test between groups. Irrigated and Drought refer to water-sufﬁcient and water-limited treatments, respectively. Figure 3. Comparison of qPCR and microarray intensity values: Rows (A) and (B) respectively refer to results for DipC and TN. The left- and right-hand pairs of columns correspond to the qPCR and microarray values for DipC and TN, respectively. The gene under study is named at the top of each panel. In order, the investigated genes are Beta-fructofuranosidase, COMT, UBC-2, and PAL1. qPCR and Microarray values are shown as fold changes with respect to the water-sufficient treatment (Irrigated). Error bars denote the standard error. Single and double asterisks indicate that p-value is less than 0.05 and 0.01, respectively, which was assessed by the paired t-test between groups. Irrigated and Drought refer to water-sufficient and water-limited treatments, respectively. Figure 3. Comparison of qPCR and microarray intensity values: Rows (A) and (B) respectively refer to results for DipC and TN. The left- and right-hand pairs of columns correspond to the qPCR and microarray values for DipC and TN, respectively. The gene under study is named at the top of each panel. In order, the investigated genes are Beta-fructofuranosidase, COMT, UBC-2, and PAL1. qPCR and Microarray values are shown as fold changes with respect to the water-sufﬁcient treatment (Irrigated). Error bars denote the standard error. Single and double asterisks indicate that p-value is less than 0.05 and 0.01, respectively, which was assessed by the paired t-test between groups. Irrigated and Drought refer to water-sufﬁcient and water-limited treatments, respectively. 12 of 19 Genes 2017, 8, 121 12 of 19 4. Discussion Landraces are a potentially valuable resource for ﬁnding genes conferring useful agricultural and processing traits. Bambara groundnut is an underutilised African legume whose landraces are adapted, in many cases, to arid conditions. We have developed single genotypes derived from landraces for analysis. There have been several dehydration studies carried out on bambara groundnut, but the molecular mechanisms of how the crop responds and adapts to dehydration stress are still under investigation. This study has carried out transcriptomic comparisons in two genotypes of bambara groundnut, DipC and TN, in an attempt to identify potential genes conferring advantageous traits for crop growth and yields in marginal environments. Cross-species hybridisation to the soybean microarray has been shown to be informative for investigating the bambara groundnut transcriptome, as good gene (probe-set) retention was observed at high gDNA hybridisation thresholds. In support of the results, Bonthala et al. [44], reported a high 13 of 19 13 of 19 Genes 2017, 8, 121 correlation between cross-species microarray data and RNA-sequencing approaches for detecting differentially expressed genes under a cold temperature stress experiment in bambara groundnut. Probe-sets retained by the mask after genomic hybridisation are almost identical (>99%), suggesting that, at this level of resolution, the two genotypes are highly similar at the sequence level. Four known dehydration-associated genes, seen to be differentially expressed in these data, were subjected to qPCR, and supported the notion that the observed trends in the microarray data are valid. The 26,496 probe sets common between the two genotypes, under irrigated conditions, (with a RMA cut-off of 0.97), include some sixty dehydration- and ABA-related genes. The latter include genes for producing osmoprotectants. They might provide two components of the dehydration avoidance capability of these genotypes, by retaining normal cell functioning when water access becomes limited. Clearly, if the plant has already activated part of the dehydration response, it could have multiple effects. The presence of osmoprotectants might draw in even more water than otherwise might be the case, and there will be a greater proportion of biomass devoted to root growth, resulting in even deeper roots that are better able to survive dehydration later on. Bambara groundnut is known to allocate a greater fraction of its dry weight to the roots than to the shoots, irrespective of the soil moisture status [27]. 4. Discussion This strategy may have clear advantages when water subsequently becomes limited, suggesting an adaptation to harsh environments and a decision to prioritise survival. In addition, as bambara groundnut is grown in harsh environments and has not undergone intensive breeding for the yield and above ground biomass, this suggests that it still allocates more effort to developing root architecture to handle dehydration when it happens. Moreover, Nayamudeza [27] also stated that the fraction of total dry weight allocated to the roots in bambara groundnut is greater than that allocated to the groundnut. In addition, a relatively higher expression of dehydration-associated genes in both genotypes under water-sufﬁcient treatment including ABI1 (ABA Insensitive 1), ABF1 (ABRE binding factor 1), ERD4 (Early responsive to dehydration 4), and RD19 (Response to dehydration 19), compared to other species such as Soybean [76] (see Figure S5), suggest that bambara groundnut could at least be in a partially ready state for dehydration, even in the absence of dehydration stress. However, further research is needed to validate this hypothesis. Given that 59,782 and 59,835 probe-sets were used to evaluate the transcriptome changes after probe-masking in DipC and TN, respectively, there were only very small numbers of genes signiﬁcantly differentially expressed (189 in DipC and 81 in TN) under water-limited treatment. It could be speculated that the slow and progressive dehydration stress might not cause signiﬁcant shock to the plants. The upregulated genes in both genotypes were subdivided into ~75% dehydration responsive (with expression levels returning to normal after recovery) and ~25% dehydration perturbed (where the expression levels remained altered). In the case of downregulated genes, 80–85% of the expression levels returned to being comparable with the non-stressed state. The dehydration-perturbed expression levels might be caused by changes at the chromatin level, through DNA methylation or histone modiﬁcation, and it is therefore interesting to note that a protein-lysine demethylase is repressed by dehydration. The above observations show that the two genotypes appear to be very similar in terms of their genotype (validating the comparability of the transcriptome data compared using the microarray), while exhibiting differences in their general transcriptional behaviour in water-sufﬁcient conditions and in response to dehydration stress. However, when the sets of differentially expressed genes are compared, there is almost no overlap. 4. Discussion They have been coloured according to their activity in relation to the dehydration response: red (transcription), orange (cell wall), yellow (lignin synthesis), green (photosynthesis), blue (transporters), indigo (hormone signalling), pink (osmoprotection), black (oxidative stress), and grey (others). Node borders have been coloured red and blue to denote up- and downregulation under stress. Nodes have been arranged in seven horizontal bands with probe-sets in common in the middle flanked by TFs and hormone-signalling genes, other genes that play various roles in response to dehydration, and others. Nodes have been linked by the criteria of the co-expression analysis. Figure 4. Comparison of genotype co-expression networks. Cytoscape has been used to layout the merged dehydration-responsive network of co-expressed probe-sets. Node shapes are triangles, diamond circles, and squares, respectively, for the differential expression of the probe-sets of TN, DipC, both (i.e., common), and both but affecting stomata. They have been coloured according to their activity in relation to the dehydration response: red (transcription), orange (cell wall), yellow (lignin synthesis), green (photosynthesis), blue (transporters), indigo (hormone signalling), pink (osmoprotection), black (oxidative stress), and grey (others). Node borders have been coloured red and blue to denote up- and downregulation under stress. Nodes have been arranged in seven horizontal bands with probe-sets in common in the middle ﬂanked by TFs and hormone-signalling genes, other genes that play various roles in response to dehydration, and others. Nodes have been linked by the criteria of the co-expression analysis. Several transcription factors that seem likely to play a role in the bambara groundnut dehydration response and which are common to both genotypes are BRH1 and an MYB transcription factor, which are known to affect the stomata in Arabidopsis thaliana [50], and JMJD5. DipC shows a more significant response, with changes to WRKY40, and is of particular interest. It is a well-known member of plant dehydration-response networks [67] and is the most highly linked TF node in the co-expression networks. For DipC, the network also reveals the importance of PRR7, a core circadian clock component known to play a complex role in abiotic stresses [77]. It is somewhat surprising that TN does not show a >2-fold change in the expression of WRKY40, but it may have roles for CONSTANS-like 1 (another clock-related gene associated with flowering in rice that may be associated with abiotic stress in bambara groundnut [78]) and MYB60, which affect stomatal closure in A. 4. Discussion Out of 189 and 91 genes differentially expressed in DipC and TN, respectively, only nine were common between the two genotypes, suggesting that some of the mechanisms for adaption to dehydration are substantially different in the two genotypes. Of these, Beta-fructofuranosidase contributes to osmoprotection [46,47], an MYB gene is associated with the stomatal opening in Arabidopsis thaliana [50], BRH1 affects the stomatal density [51], and bsd2 affects photosynthesis in maize [52], while JMJD5 plays an epigenetic role [49], as mentioned above. Figure 4 illustrates how two genotypes with very similar genomes may have adapted to achieve dehydration 14 of 19 Genes 2017, 8, 121 response traits (transcriptional and hormone signalling to affect cell-wall modiﬁcation, lignin synthesis, photosynthesis, transporters, hormone signalling, osmoprotection, oxidative stress) through largely different sets of effector genes. Genes 2017, 8, 121 14 of 19 Figure 4. Comparison of genotype co-expression networks. Cytoscape has been used to layout the merged dehydration-responsive network of co-expressed probe-sets. Node shapes are triangles, diamond circles, and squares, respectively, for the differential expression of the probe-sets of TN, DipC, both (i.e., common), and both but affecting stomata. They have been coloured according to their activity in relation to the dehydration response: red (transcription), orange (cell wall), yellow (lignin synthesis), green (photosynthesis), blue (transporters), indigo (hormone signalling), pink (osmoprotection), black (oxidative stress), and grey (others). Node borders have been coloured red and blue to denote up- and downregulation under stress. Nodes have been arranged in seven horizontal bands with probe-sets in common in the middle flanked by TFs and hormone-signalling genes, other genes that play various roles in response to dehydration, and others. Nodes have been linked by the criteria of the co-expression analysis. Figure 4. Comparison of genotype co-expression networks. Cytoscape has been used to layout the merged dehydration-responsive network of co-expressed probe-sets. Node shapes are triangles, diamond circles, and squares, respectively, for the differential expression of the probe-sets of TN, DipC, both (i.e., common), and both but affecting stomata. They have been coloured according to their activity in relation to the dehydration response: red (transcription), orange (cell wall), yellow (lignin synthesis), green (photosynthesis), blue (transporters), indigo (hormone signalling), pink (osmoprotection), black (oxidative stress), and grey (others). Node borders have been coloured red and blue to denote up- and downregulation under stress. 4. Discussion Nodes have been arranged in seven horizontal bands with probe-sets in common in the middle ﬂanked by TFs and hormone-signalling genes, other genes that play various roles in response to dehydration, and others. Nodes have been linked by the criteria of the co-expression analysis. Figure 4. Comparison of genotype co-expression networks. Cytoscape has been used to layout the merged dehydration-responsive network of co-expressed probe-sets. Node shapes are triangles, diamond circles, and squares, respectively, for the differential expression of the probe-sets of TN, DipC, both (i.e., common), and both but affecting stomata. They have been coloured according to their activity in relation to the dehydration response: red (transcription), orange (cell wall), yellow (lignin synthesis), green (photosynthesis), blue (transporters), indigo (hormone signalling), pink (osmoprotection), black (oxidative stress), and grey (others). Node borders have been coloured red and blue to denote up- and downregulation under stress. Nodes have been arranged in seven horizontal bands with probe-sets in common in the middle flanked by TFs and hormone-signalling genes, other genes that play various roles in response to dehydration, and others. Nodes have been linked by the criteria of the co-expression analysis. Figure 4. Comparison of genotype co-expression networks. Cytoscape has been used to layout the merged dehydration-responsive network of co-expressed probe-sets. Node shapes are triangles, diamond circles, and squares, respectively, for the differential expression of the probe-sets of TN, DipC, both (i.e., common), and both but affecting stomata. They have been coloured according to their activity in relation to the dehydration response: red (transcription), orange (cell wall), yellow (lignin synthesis), green (photosynthesis), blue (transporters), indigo (hormone signalling), pink (osmoprotection), black (oxidative stress), and grey (others). Node borders have been coloured red and blue to denote up- and downregulation under stress. Nodes have been arranged in seven horizontal bands with probe-sets in common in the middle ﬂanked by TFs and hormone-signalling genes, other genes that play various roles in response to dehydration, and others. Nodes have been linked by the criteria of the co-expression analysis. Figure 4. Comparison of genotype co-expression networks. Cytoscape has been used to layout the merged dehydration-responsive network of co-expressed probe-sets. Node shapes are triangles, diamond circles, and squares, respectively, for the differential expression of the probe-sets of TN, DipC, both (i.e., common), and both but affecting stomata. 5. Conclusions Understanding the mechanisms underlying the ability of crops to produce viable yields under drought conditions is a priority for global food security. This study has examined the transcriptomic reponse to dehydration and recovery in two genotypes derived from landraces of bambara groundnut, in an attempt to investigate the molecular mechanisms occurring in the two landraces. In addition, this study also tested whether the cross-species hybridisation to the soybean microarray is suitable for investigating the bambara groundnut transcriptome. It was shown that many potential dehydration-responsive genes are expressed, even under water-sufﬁcient conditions, in both landraces, suggesting that bambara groundnut could at least be in a partially ready state for dehydration, even in the absence of dehydration stress. In terms of differential expression, there were only a very small number of genes differentially expressed under water-limited treatment in both landraces, suggesting that the slow and progressive dehydration stress might not cause a signiﬁcant shock to the plants. Although the transcription factors and dehydration-response genes were largely different between the two landraces, they may achieve the same effect in terms of survival under drought conditions. The DipC genotype displayed the differential expression of some well-known dehydration-associated transcriptions factors (especially WRKY40), while TN showed the differential expression of CONSTANS-LIKE 1 and MYB60. Cross-species hybridisation to the soybean microarray has been shown to be informative for investigating the bambara groundnut transcriptome, as good gene retention was observed at high gDNA hybridisation thresholds. Supplementary Materials: The following are available online at www.mdpi.com/2073-4425/8/4/121/s1. Figure S1: Treatment Regimes, Figure S2: Genotype speciﬁc 3D PCA scatter plots, Figure S3: Ranked mean intensities of water-sufﬁcient genotype samples, Figure S4: Categories of system response in gene expression, Figure S5: Comparison between bambara groundnut and soybean for expression levels for dehydration-associated genes under water-sufﬁcient conditions. Table S1: Dehydration-associated genes expressed under water-sufﬁcient condition in DipC, Table S2: Dehydration-associated genes expressed under water-sufﬁcient condition in TN, Table S3: Dehydration-associated genes expressed under water-sufﬁcient condition in both DipC and TN, Table S4: Comparison of water-sufﬁcient conditions transcriptomes of DipC and TN, Table S5: Upregulated DipC genes as a result of water-limited treatment (p-value <= 0.05, Abs. F.C > 2), Table S6: Downregulated DipC genes as a result of water-limited treatment (p-value <= 0.05, Abs. F.C > 2), Table S7: Upregulated TN genes as a result of water-limited treatment (p-value <= 0.05, Abs. 4. Discussion thaliana [79], and AGL-83, a MADS-Box protein with an uncertain role. Several transcription factors that seem likely to play a role in the bambara groundnut dehydration response and which are common to both genotypes are BRH1 and an MYB transcription factor, which are known to affect the stomata in Arabidopsis thaliana [50], and JMJD5. DipC shows a more signiﬁcant response, with changes to WRKY40, and is of particular interest. It is a well-known member of plant dehydration-response networks [67] and is the most highly linked TF node in the co-expression networks. For DipC, the network also reveals the importance of PRR7, a core circadian clock component known to play a complex role in abiotic stresses [77]. It is somewhat surprising that TN does not show a >2-fold change in the expression of WRKY40, but it may have roles for CONSTANS-like 1 (another clock-related gene associated with ﬂowering in rice that may be associated with abiotic stress in bambara groundnut [78]) and MYB60, which affect stomatal closure in A. thaliana [79], and AGL-83, a MADS-Box protein with an uncertain role. Genes 2017, 8, 121 15 of 19 15 of 19 5. Conclusions F.C > 2), Table S8: Downregulated TN genes as a result of water-limited treatment (p-value <= 0.05, Abs. F.C > 2), Table S9: List of differentially expressed genes (water-limited versus water-sufﬁcient) common to DipC and TN when sample D.DipC.rep2 is excluded from the analysis, Table S10: GO-term overrepresentation of all the gene-sets in Soybean GeneChip array to compare DipC, TN, and Soybean datasets, Table S11: DipC Co-expression network, Table S12: TN Co-expression network. Acknowledgments: Faraz Khan thank Kevin Ryan and Jiang Lu for their guidance and comments on the manuscript. Faraz Khan thank Crops for the Future (CFF) and the University of Nottingham for providing funding for the research. Faraz Khan thank the NASC arabidopsis centre for Microarray hybridisation and Presidor Kendabie for the help in the dehydration experiment. Charlie Hodgman thanks the support from the European Research Council Advanced Grant funding (FUTUREROOTS 294729). Author Contributions: F.K. analysed the microarray data, created custom CDF ﬁles, performed qPCR experiments, constructed and analysed the co-expression network, interpreted the results, and drafted the manuscript. C.H. created the merged dehydration-responsive network, co-analysed the co-expression network, interpreted the results, and critically reviewed the manuscript. H.H.C. performed the dehydration experiment and RNA extraction. S.M. and C.L. conceived the project and critically reviewed the manuscript. 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Tripathi, P.; Rabara, R.C.; Reese, R.N.; Miller, M.A.; Rohila, J.S.; Subramanian, S.; Shen, Q.J.; Morandi, D.; Bucking, H.; Shulaev, V.; et al. A toolbox of genes, proteins, metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes. BMC Genomics 2016, 17, 102. [CrossRef] [PubMed] 75. Xia, H.J.; Guang, Y. Inositol 1,4,5-trisphosphate 3-kinases: Functions and regulations. Cell Res 2005, 15, 83–91. [CrossRef] [PubMed] 76. Le, D.T.; Nishiyama, R.; Watanabe, Y.; Tanaka, M.; Seki, M.; Ham le, H.; Yamaguchi-Shinozaki, K.; Shinozaki, K.; Tran, L.S. Differential gene expression in soybean leaf tissues at late developmental stages under drought stress revealed by genome-wide transcriptome analysis. PLoS ONE 2012, 7, e49522. [CrossRef] [PubMed] Grundy, J.; Stoker, C.; Carré, I.A. Circadian regulation of abiotic stress tolerance in plants. Front. Plant Sci 2015, 6, 648. [CrossRef] [PubMed] 78. Liu, J.; Shen, J.; Xu, Y.; Li, X.; Xiao, J.; Xiong, L. References Ghd2, a constans-like gene, confers drought sensitivity through regulation of senescence in rice. J. Exp. Bot. 2016, 67, 5785–5798. [CrossRef] [PubMed] 79. Oh, J.E.; Kwon, Y.; Kim, J.H.; Noh, H.; Hong, S.-W.; Lee, H. A dual role for myb60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress. Plant Mol. Biol. 2011, 77, 91–103. [CrossRef] [PubMed] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3097634534	https://www.matec-conferences.org/articles/matecconf/pdf/2020/18/matecconf_matbud2020_01009.pdf	English	null	Effect of limestone powder on strength and permeability of cementitious mortars	MATEC web of conferences	2,020	cc-by	2,320	Effect of limestone powder on strength and permeability of cementitious mortars Wei-Ting Lin1,, An Cheng1, and Robert Černý2 1National Ilan University,,Department of Civil Engineering, Shennong Rd., I-Lan 260, Taiwan 2Czech Technical University in Prague, Faculty of Civil Engineering, Department of Materials Engineering and Chemistry, Thákurova 7, 166 29 Prague 6, Czech Republic Wei-Ting Lin1,, An Cheng1, and Robert Černý2 Abstract. This study investigated the mechanical properties and durability of mortar specimens containing various quantities of waste limestone powder (0%, 10%, 20%, and 30% of the weight of cement). The mechanical properties were evaluated in terms of flowability, compressive strength, and splitting tensile strength. Permeability was evaluated in terms of resistivity, absorption, and rapid chloride penetration (total charge- passed). Test results revealed that replacing 10% of the cement with limestone powder improves both mechanical performance and durability. However, reducing the cement content reduced the availability of hydration products to fill the pores in the microstructure. The hydration product of limestone powder was mainly calcium hydroxide, which undermines the development of strength in the paste. Nonetheless, our results indicate that waste limestone powder is suitable for the partial replacement of cement. 1 Introduction The processing of stone by cutting, carving, and grinding produces stone waste in the form of sludge with a composition similar to that of the original stone [1, 2]. This waste material has a potentially considerable economic value [3, 4]. The stone-processing industry in Taiwan is centred in the city of Hualien, where it generates an annual economic output of nearly 40 billion yuan. Despite this enormous output, the efficiency of resource utilisation is somewhat limited [5, 6]. It seems worth investigating whether stone sludge could serve as a raw material in cement products, lightweight building materials, and fibreboard. At present, most stone waste goes into the fabrication of permeable bricks and bituminous mixtures [11-13]. Note that the use of limestone powder in cementitious materials, such as fly ash and granulated blast furnace slag, is being phased out in Taiwan. New uses must be found for this valuable resource. In this study, we examined the feasibility of using waste limestone powder as a replacement for cement in concrete. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: wtlin@niu.edu.tw MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 https://doi.org/10.1051/matecconf/202032201009 * Corresponding author: wtlin@niu.edu.tw https://doi.org/10.1051/matecconf/202032201009 MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 2 Materials and test procedures The chemical composition of the waste limestone powder used in this study (An Jung Mineral Chemical Corporation) is summarised in Table 1. The white-grey limestone powder had a specific gravity of 2.70 and the main constituent was calcium oxide. Table 1. Chemical composition of waste limestone powder. Table 1. Chemical composition of waste limestone powder. Item Cinc./% Sigma/% RSD/% LLD/% Net area Backgr. Chi Ca 99.43 0.100 0.1 0.02 917,292 2,961 5.16 Mn 0.099 0.003 2.5 0.003 2,796 1,051 1.39 Fe 0.345 0.003 1.0 0.003 12,401 1,053 0.81 Sr 0.129 0.001 1.0 0.002 15,182 4,752 1.16 In accordance with ASTM C150 standards, the cement used in this study was type I Portland cement with a specific gravity of 3.15. The properties of the fine aggregate were specific gravity (2.65), water absorption (1.93%), and fineness modulus (2.25). The mix designs used for the mortar specimens are listed in Table 2. Limestone powder was used as a partial replacement for cement (0%, 10%, 20%, and 30%) with a water/binder ratio (W/B) of 0.40 and flowability controlled at 115%. Table 2. Mix designs of mortar specimens (kg/m3). Mix no. W/B Water Cement Limestone powders Fine aggregates OPC 0.40 210 525.0 0 1,684 LP10 210 472.5 52.5 1,684 LP20 210 420.0 105.0 1,684 LP30 210 367.5 157.5 1,684 Table 2. Mix designs of mortar specimens (kg/m3). OPC denotes ordinary Portland cement and LP refers to limestone with a number to indicate the percentage replacement. Table 3 lists test methods, dimensions of the test specimens, and testing standards. Table 3. Test parameters. Test methods Specimen dimensions (mm) Standard Testing period (days) Compressive strength test 50x50x50 ASTM C109 3, 7, 28, 56 Splitting tensile strength test φ100x200 ASTM C496 3, 7, 28, 56 Resistivity test φ100x200 ASTM WK37880 3, 7, 28, 56 Water absorption test φ100x200 ASTM C642 3, 7, 28, 56 Rapid chloride penetration test φ100x50 ASTM C1202 56 2 https://doi.org/10.1051/matecconf/202032201009 MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 3 Results and discussion The results of compressive strength for all mixes are shown in Fig. 1. The addition of limestone powder was shown to increase the strength of all samples, regardless of the curing duration. At 3 and 7 days, the LP10 specimens presented the highest strength (34.21 MPa) followed by LP20 (31.55 MPa). At 28 and 56 days, the compressive strength of the experiment specimens still exceeded that of the control specimens: LP10 (+51%) and LP20 (+42.62%). It seems that the inclusion of limestone powder in cement-based composites can increase compressive strength; however, the magnitude of the improvements drops at replacement values exceeding 10%. Fig. 1. Compressive strength development curves versus limestone content. The results of splitting tensile strength are illustrated in Fig. 2. At 3 days, the addition of limestone powder was shown to increase the splitting tensile strength, compared to OPC specimens. At 28 days, the splitting strength of the samples was as follows: OPC (4.30 MPa), LP10 (4.32 MPa), LP20 (4.21 MPa), and LP30 (4.12 MPa). At 56 days, the splitting strength of the LP specimens still exceeded that of the OPC specimen; however, the magnitude of the improvements dropped at replacement values exceeding 10%. The gradual decrease in the amount of cement may result in a lack of aluminate (such as C3A) and hydrated limestone powder [4]. Fig. 1. Compressive strength development curves versus limestone content. The results of splitting tensile strength are illustrated in Fig. 2. At 3 days, the addition of limestone powder was shown to increase the splitting tensile strength, compared to OPC specimens. At 28 days, the splitting strength of the samples was as follows: OPC (4.30 MPa), LP10 (4.32 MPa), LP20 (4.21 MPa), and LP30 (4.12 MPa). At 56 days, the splitting strength of the LP specimens still exceeded that of the OPC specimen; however, the magnitude of the improvements dropped at replacement values exceeding 10%. The gradual decrease in the amount of cement may result in a lack of aluminate (such as C3A) and hydrated limestone powder [4]. The results of splitting tensile strength are illustrated in Fig. 2. At 3 days, the addition of limestone powder was shown to increase the splitting tensile strength, compared to OPC specimens. At 28 days, the splitting strength of the samples was as follows: OPC (4.30 MPa), LP10 (4.32 MPa), LP20 (4.21 MPa), and LP30 (4.12 MPa). 3 Results and discussion At 56 days, the splitting strength of the LP specimens still exceeded that of the OPC specimen; however, the magnitude of the improvements dropped at replacement values exceeding 10%. The gradual decrease in the amount of cement may result in a lack of aluminate (such as C3A) and hydrated limestone powder [4]. 3 3 https://doi.org/10.1051/matecconf/202032201009 MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 Fig. 2. Splitting tensile strength development curves versus limestone content. Fig. 2. Splitting tensile strength development curves versus limestone content. The Wenner four-electrode resistance index is used to evaluate the quality of cement- based materials in terms of permeability. Essentially, this index measures the resistivity of specimens saturated with water as an indication of microstructure development, wherein higher resistivity values indicate a denser internal microstructure. The resistivity results are illustrated in Fig. 3. The resistivity of the LP specimens was significantly lower than that of the control specimens (i.e., OPC). Furthermore, the time- dependent increase in microstructure density was more pronounced in the OPC specimens, as indicated by a growing discrepancy in the resistivity values of specimens with or without limestone powder. At 56 days, the resistivity of the LP30 specimen was 43% below that of the OPC specimens. Clearly, the addition of limestone powder hindered the formation of the internal microstructure. This can presumably be attributed to reactions between the hydration products of limestone and cement resulting in greater porosity and a looser microstructure. Fig. 3. Resistivity development curves versus limestone content. Saturated water absorption tests are used as an index of permeability indicating the overall quality of cement-based materials, wherein water absorption is proportional to the number of pores. Materials with fewer pores possess a denser internal microstructure Fig. 3. Resistivity development curves versus limestone content. Saturated water absorption tests are used as an index of permeability indicating the overall quality of cement-based materials, wherein water absorption is proportional to the number of pores. Materials with fewer pores possess a denser internal microstructure Saturated water absorption tests are used as an index of permeability indicating the overall quality of cement-based materials, wherein water absorption is proportional to the number of pores. Materials with fewer pores possess a denser internal microstructure 4 4 MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 https://doi.org/10.1051/matecconf/202032201009 capable of resisting the penetration of harmful substances which might otherwise damage the material over time. 4 Conclusions Our conclusions are as follows: 1. The LP10 specimens presented the highest compressive strength and splitting tensile strength, closely followed by LP20. 1. The LP10 specimens presented the highest compressive strength and splitting tensile strength, closely followed by LP20. 2. The inclusion of limestone powder as a replacement for cement had a negative effect on permeability and durability in a dose-dependent manner. In rapid chloride penetration tests, the total charge passed through LP30 specimens was 2.47 times higher than that of LP10. These results are consistent with those pertaining to resistivity and absorption. 3. Replacing more than 10% of the cement with limestone powder was shown to hinder the formation of hydration products required to fill the capillary pores and gel pores, resulting in a microstructure of lower density. 3. Replacing more than 10% of the cement with limestone powder was shown to hinder the formation of hydration products required to fill the capillary pores and gel pores, resulting in a microstructure of lower density. 4. In terms of mechanical properties and permeability, it appears that up to 10% of the cement could be replaced with limestone powder without adverse effects. 4. In terms of mechanical properties and permeability, it appears that up to 10% of the cement could be replaced with limestone powder without adverse effects. Acknowledgements: This work has been financed by Polish National Agency for Academic Exchange under the International Academic Partnership Programme within the framework of the grant: E- mobility and sustainable materials and technologies EMMAT. This work also has been supported by the Ministry of Science and Technology (MOST-108-2221-E-197-006) in Taiwan and the Grant Agency of the Czech Technical University in Prague, Project No. SGS19/143/OHK1/3T/11. 3 Results and discussion The results of water absorption tests are illustrated in Fig. 4. At 3 and 7 days, LP samples absorbed less water than the control specimens did. This can be attributed to limestone powder reacting more quickly than the cement in the formation of hydration products to fill in pores. Over time, the benefits of the limestone powder in terms of reducing porosity diminished. In later stages, only the LP10 sample outperformed the OPC in terms of water absorption: 28 days (+3%) and 56 days (+2%). Clearly, adding more than 10% limestone powder would be of no benefit in reducing the porosity of composites. Fig. 4. Water absorption development curves versus limestone content. Fig. 4. Water absorption development curves versus limestone content. The rapid chloride penetration test (RCPT) measures the resistance of composite specimens to chloride ion penetration as an indicator of durability. Table 4 lists the total charge-passed (cumulative coulombs) and total current at 56 days. Under a test duration of 6 hours, the LP10 specimens presented the lowest total charge-passed (2,272 coulombs), followed by LP20 (5,216 coulombs), and LP30 (5,623 coulombs). It appears that the inclusion of more than 10% limestone powder increased the number of fine pores during hydration with a corresponding increase in chloride ion penetration. Table 4. RCPT test results. Mix no. Time (min) Measured current (mA) LP10 LP20 LP30 30-60-90 0 0 0 120-150-180 0 0 12 210-240-270 64 884 1,884 300-330-360 1,544 2,600 1,228 total charge-passed (cumulative coulombs) 2,272 5,216 5,623 Table 4. RCPT test results. 5 5 https://doi.org/10.1051/matecconf/202032201009 MATEC Web of Conferences 322, 01009 (2020) MATBUD’2020 References 1. R. Zulcão, J. L. Calmon, T. A. Rebello, D. R. Vieira, Constr. Build. Mater. 257, 119523 (2020) 1. R. Zulcão, J. L. Calmon, T. A. Rebello, D. R. Vieira, Constr. Build. Mater. 257, 119523 (2020) 2. W. A. Moura, J. P. Gonçalves, R. S. Leite, Sitientibus 26, 49 (2002) 2. W. A. Moura, J. P. Gonçalves, R. S. Leite, Sitientibus 26, 49 (2002) 3. M. C. Juenger, R. Snellings, S. A. Bernal, Cem. Concr. Res. 122, 257 (2019) 4. D. K. Panesar, R. Zhang, Constr. Build. Mater. 251, 118866 (2020) 5. L. D. Ellis, A.F. Badel, M. L. Chiang, R. J. Y. Park, Y. M. Chiang, Proc. Natl. Acad. Sci. 117, 12584 (2020) 6. Q. D. Nguyena, A. Castel, Cem. Concr. Res. 132, 106051 (2020) 7. R. D. Hooton, Cem. Concr. Res. 78, 165 (2015) 8. I. G. Richardson, Cem. Concr. Res. 38, 137 (2008) 9. R. S. Macedo, C. Ulsen, A. Mueller, Constr. Build. Mater. 229, 116875 (2019) 10. S. Palm, T. Proske, M. Rezvani, S. Hainer, C. Müller, C. A. Graubner, Constr. Build. Mater. 119, 308 (2016) 11. M. Ghrici, S. Kenai, M. Said-Mansour, Cement Concr. Compos. 29, 542 (2007) 12. Á. Fernández, J. G. Calvo, M. C. Alonso, Cement Concr. Compos. 89, 238 (2018 13. D. Vaičiukynienė, I. Pundienė, A. Kantautas, A. Augonis, E. Janavičius, V. Vaičiukynas, J. Alobeid, J. Clean. Prod. 244, 118493 (2020) 6 6
https://openalex.org/W3213041089	https://www.research.unipd.it/bitstream/11577/3405535/2/s41598-021-01349-5.pdf	English	null	Multidisciplinary investigation reveals the earliest textiles and cinnabar-coloured cloth in Iberian Peninsula	Scientific reports	2,021	cc-by	8,049	Multidisciplinary investigation reveals the earliest textiles and cinnabar‑coloured cloth in Iberian Peninsula N Margarita Gleba 1, M. Dolores Bretones‑García 2, Corrado Cimarelli 3, Juan Carlos Vera‑Rodríguez 4 & Rafael M. Martínez‑Sánchez 5 Textile production is among the most fundamental and more complex technologies in human prehistory, but is under-investigated due to the perishable nature of fibrous materials. Here we report a discovery of five textile fragments from a prehistoric (fourth-third millennium cal BC) burial deposit located in a small cave at Peñacalera in Sierra Morena hills, near Córdoba, Southern Spain. These textiles accompanied a set of human remains as grave goods, together with other organic elements such as fragments of wood and cork, and some pottery vessels. They were characterized and dated using digital microscopy, Scanning Electron Microscopy, Energy Dispersive Spectroscopy and Accelerator Mass Spectrometry. Two of the fragments described here are the oldest examples of loom-woven textiles in the Iberian Peninsula, dating from the second half of the fourth millennium cal BC. This correlates chronologically with the first appearance of loom weights in the archaeological record of this region. The more recently dated textile is the earliest preserved cloth intentionally coloured with cinnabar in the western Mediterranean. The Peñacalera finds are a key reference for understanding the development of textile technologies during the Neolithic and Copper Age in western Europe and beyond. Textiles have been an essential element of human material culture since at least the Neolithic period, but their poor preservation often hampers our understanding of the development of this fundamental technology. The discovery of uncharred textile or basketry remains, preserved by desiccation, is particularly rare in archaeological contexts of the Western Mediterranean, and finds dating to the Neolithic period and Copper Age are exceptional. In the Iberian Peninsula, these finds are usually concentrated in the south-eastern region, which currently has a sub-arid climate1. Among the best-known cases, are the outstanding Neolithic examples of basketwork made of esparto grass and wooden objects from the Los Murciélagos cave in Albuñol (Granada), dated to fifth mil- lennium cal BC2,3, and textiles from the site of Los Millares (Almeria) dated two millennia later4. Examples of cordage, esparto basketry and linen textiles are also known from various Bronze Age contexts of El Argar culture (c. 2200 cal BC and 1550 cal BC) in Almería, Murcia and Alicante4–6. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2021) 11:21918 Multidisciplinary investigation reveals the earliest textiles and cinnabar‑coloured cloth in Iberian Peninsula N Among the most unique prehistoric Iberian textile finds are the remains of two linen tunics found in the so-called Cueva Sagrada I (Lorca, Murcia), alongside other organic materials such as esparto and wood, and the remains of five individuals, dated c. 2300 cal BC7–9.i g pi Here, we present five textile fragments dated to the Late Neolithic and Copper Age (fourth-third millennium cal BC), found in a burial context inside a small cave in a rocky outcrop located in the Province of Córdoba, in the southwest of Spain. The cave was discovered at the end of 2014 by speleologists, who immediately recognised the significance of the find. In March 2016, an archaeological intervention was carried out (directed by M.D.B.G.), which included full planimetry of the cavity’s space and stratigraphic excavation.h p y y p g p The so-called Cueva de la Peña de La Calera (or Peñacalera Cave) is a small cavity formed by a fracture ori- ented W–E, located in one of the rocky outcrops of the so-called Cerro de La Calera, which is situated southwest 1Department of Cultural Heritage, Università Degli Studi di Padova, Piazza Capitaniato 7, 35139 Padova, Italy. 2Territorial Delegation of Cultural Heritage, C/Martínez‑Montañés 8, 23007 Jaén, Spain. 3Department of Earth and Natural Sciences, Ludwig-Maximilians-Universität München, Theresienstrasse 41, 80333 Munich, Germany. 4Department of History, Geography and Anthropology, Universidad de Huelva, Campus de El Carmen, Avda. de las Fuerzas Armadas, s/n, 21071 Huelva, Spain. 5Department of History, Universidad de Córdoba, P/Hospital Cardenal Salazar 3, 14071 Córdoba, Spain. email: margarita.gleba@unipd.it \| https://doi.org/10.1038/s41598-021-01349-5 Scientific Reports \| (2021) 11:21918 www.nature.com/scientificreports/ Figure 1. (A) Location of the Peñacalera site in the south of the Iberian Peninsula. (B) Guadalbarbo Creek Valley, with limestone outcrops behind, where the cavity is located, marked with a red arrow. (C) Image of the burial context of Peñacalera, sector 2, where Textile 1 was found. (D) Plan 2 of the burial context with detailed Plan 8; red numbers indicate relative depths, black numbers are inventory numbers of the finds; the black star in Plan 2 indicates the approximate location of Textile 5’s find spot; the black star in Plan 8 indicates the find spot of Textile 1. Maps generated by Inkscape (https://inkscape.org/). Figure 1. (A) Location of the Peñacalera site in the south of the Iberian Peninsula. (B) Guadalbarbo Creek Valley, with limestone outcrops behind, where the cavity is located, marked with a red arrow. Multidisciplinary investigation reveals the earliest textiles and cinnabar‑coloured cloth in Iberian Peninsula N (C) Image of the burial context of Peñacalera, sector 2, where Textile 1 was found. (D) Plan 2 of the burial context with detailed Plan 8; red numbers indicate relative depths, black numbers are inventory numbers of the finds; the black star in Plan 2 indicates the approximate location of Textile 5’s find spot; the black star in Plan 8 indicates the find spot of Textile 1. Maps generated by Inkscape (https://inkscape.org/). f the village of Obejo (Córdoba, Andalusia, Spain), next to the river bed of the Guadalbarbo stream, in the Guadalquivir River basin (38°6′19.24″N, 4°49′56.13″W) (Fig. 1A,B).h q g The archaeological remains were concentrated under the east wall, scattered amongst the stone blocks of different sizes that formed a kind of cairn up to 1 m thick (Fig. 1C). This deposit was to some extent altered by post-depositional processes, such as percolation and bioturbation, small carnivores having used the space as a den. Underneath and among the stone blocks, were the disarticulated bones of a minimum of five human individuals and various burial goods. The latter consisted of at least eight ceramic vessels, a bone awl, a lithic polisher, and various fragments of cork tree bark (Fig. 1D). Together with these materials, five textile fragments were recovered. All of the fragments were found in sector 2, in a very small space and at a short distance from each other. One of the fragments (PC16 S-2 215-I, hereafter Textile 1) was found during excavation and was associated with two complete ceramic vessels deposited together and directly related to highly fragmented human remains (Fig. 1D Plan 8). Fragment PC16 S-2 34-I (hereafter Textile 5) was recovered with a sample of vegetal turf material excavated together with a ceramic vessel (34) and subsequently identified as a textile (Fig. 1D Plan 2). The other three fragments (PC16 S-2 C-TEX-1A, PC16 S-2 C-TEX-1B, PC16 S-2 C-ESP-3, hereafter Textiles 2, 3 and 4), were recovered during manual sieving of the sediment, and were isolated once they had been identified as textiles. The spatial data collected during the excavation revealed that Textile 5 was recovered from a shallow depth, in the most superficial stone layer of the assemblage. Textile 1 was found in the same sector, but at a depth of approximately 0.64 m with respect to the level of Textile 5.hi pp y p The five textile fragments (Fig. Multidisciplinary investigation reveals the earliest textiles and cinnabar‑coloured cloth in Iberian Peninsula N 2) were preserved by desiccation in the stable and dry environment of the cave and are still organic, with much of their original texture, colour and elasticity intact. These are finds of great importance, since direct evidence of the prehistoric textile production in the region has, until now, not been attested before the third millennium cal BC. The finds were characterized using digital microscopy for structural https://doi.org/10.1038/s41598-021-01349-5 Scientific Reports \| (2021) 11:21918 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Textiles 1–5 recovered from Pañacalera at the same scale. For technical details of all fragments, see Table 1. Figure 2. Textiles 1–5 recovered from Pañacalera at the same scale. For technical details of all fragments, see Table 1. textile analysis, Scanning Electron Microscopy (SEM) for fibre determination, Energy Dispersive Spectroscopy (EDS) for the chemical characterization of the colourant. Four of the textiles were dated using Accelerator Mass Spectrometry (AMS) radiocarbon dating. The results of this multidisciplinary investigation are presented below. textile analysis, Scanning Electron Microscopy (SEM) for fibre determination, Energy Dispersive Spectroscopy (EDS) for the chemical characterization of the colourant. Four of the textiles were dated using Accelerator Mass Spectrometry (AMS) radiocarbon dating. The results of this multidisciplinary investigation are presented below. Results il Textile characterisation. All five textiles are woven in plain weave or tabby (Fig. 3). Plain weave is one of the earliest loom-woven structures as it is the simplest textile binding attainable with two thread systems on a loom, with passive warp and active weft threads alternating one over one in each direction. Textile 4 is a balanced plain weave, with approximately the same number of threads per unit of length in warp and weft. Textile 1 is a warp-faced plain weave, with twice as many warps as wefts per unit of length; the warp direction is indicated by the small area of preserved simple selvedge (see detail in Fig. 2(1)). In the selvedge, the weft turns every fourth thread, possibly indicating the use of two wefts during weaving. The remaining three textiles are likely warp-dominant or warp-faced as well, although they do not preserve any selvedges or borders for a definitive identification of warp and weft systems. it y Based on the thread counts and thread diameters (Fig. 3 and Table 1), the textiles can be divided into three groups, with two coarser (Textiles 1 and 4), two finer (Textiles 2 and 3) and one exceptionally fine fabric (Textile 5). With 43–45 warp threads and 21–22 weft threads per cm, the latter is the finest prehistoric fabric discovered to date in the Iberian Peninsula, as demonstrated by comparative structural data from textiles discovered at other Copper and Bronze Age sites (section in italics of Table 1).hit The five textiles are woven in spliced rather than draft-spun yarn (Fig. 4). Splicing is a technique used to convert plant fibre into yarn that, until recently, has been assumed to have been used exclusively in ancient Egypt and East Asia10,11. In contrast to draft spinning, during which the combed and prepared fibres are fixed on a distaff and are continuously drawn to receive a twist through the rotation of a spindle, in splicing, the ends of pre-formed fibre bundles stripped from plant stalks are joined, so that the ends of the fibres would overlap in bunches. Splicing has recently been identified in Neolithic, Bronze and Iron Age textiles in Italy, Greece, Spain Scientific Reports \| (2021) 11:21918 \| https://doi.org/10.1038/s41598-021-01349-5 www.nature.com/scientificreports/ Table 1. Results il Textile structural data summary for the five textiles from Peñacalera and (in italics) other Copper and Bonze Age Iberian textile finds (data for Lorca from8,9; for Los Millares, El Argar and El Oficio from4,5). Object Weave Warp count Weft count Warp twist Weft twist Warp diameter Weft diameter Pañacalera textile 1 (Inv. PC16 S-2 215-I) Warp-faced plain weave 15 7 Z2s Z2s 0.7–0.8 0.6–1 Pañacalera textile 2 (Inv.PC16 S-2 C-TEX-1A) Warp-dominant plain weave 18–20 12 Z2i Z2i 0.3–0.4 0.4–0.6 Pañacalera textile 3 (Inv. PC16 S-2 C-TEX-1B) Warp-faced plain weave 28–30 14–16 Z2i Z2i 0.2–0.3 0.2–0.4 Pañacalera textile 4 (Inv. PC16 S-2 C-ESP-3) Balanced plain weave 6 6 Z2s Z2s 0.8–0.9 0.9–1 Pañacalera textile 5 (Inv. PC16 S-2 34-I) Warp-faced plain weave 43–45 21–22 Z2i Z2i 0.1–0.3 0.1–0.3 Lorca tunic A Warp-dominant plain weave 12–18 9–15 Z2i-s Z2i-s 0.3–0.5 0.2–0.7 Lorca tunic B Weft-dominant plain weave 14–16 23–24 Z2i-s Z2i-s 0.3–0.5 0.2–0.4 Lorca textile frag. B Weft-dominant plain weave 21 28 Z2i-s Z2i-s 0.1–0.2 0.1–0.2 Lorca textile frag. C Warp-dominant plain weave 20 13 Z2i-s Z2i-s 0.4–0.7 0.4–0.7 Los Millares (various burials Plain weave 11–14 12–16 Z2i-s Z2i-s 0.2–0.3 0.2–0.3 El Argar (various burials) Plain weave 8–27 8–27 Z2i-s Z2i-s 0.2–0.9 0.2–0.9 El Oficio (various burials) Plain weave 8–19 8–19 Z2i-s Z2i-s 0.3–1 0.3–1 Table 1. Textile structural data summary for the five textiles from Peñacalera and (in italics) other Copper and Bonze Age Iberian textile finds (data for Lorca from8,9; for Los Millares, El Argar and El Oficio from4,5) and more widely across Europe and western Asia12. All of the prehistoric woven linen textiles found in Spain to date appear to be made using spliced yarn. In the two coarser fragments (Textiles 1 and 4), the single threads have a clear counterclockwise- or s-twist, while in the other three fragments no twist is discernible in the single threads, or only a very faint s-twist. This may be a chronological difference as observed in Egypt10, since Textiles 1 and 4 are the oldest (see below on Dating). Yarn for the earlier textiles was likely made using continuous splicing, while the latter appears to have end-to-end splices12, although the fragments are too small to identify the splicing technique definitively. Fibre characterisation. All five textiles are made of plant bast (stem) fibres, which are present in ultimates bundles (Fig. Results il 4 bottom right), and in Textile 4 also preserve remains of epidermal and parenchymal tissues. The fibres display nodes/dislocations and polygonal cross-sections characteristic of plant bast fibre (Supplementary Material Table 1 and Fig. 1). The cracks in the fibres of Textiles 3 and 5 are S or counterclockwise, possibly indicating s-microfibrillar orientation. Fibre diameter measurements range between 6.9 and 25.9 µm and mean diameters in the five fragments are between 13.1 and 19.7 µm. All the diagnostic features are consistent with flax (Linum sp.)13. Colourant characterisation. Elemental composition analysis of warp and weft samples from Textile 5, which has a distinct and homogeneously reddish pink colour unequivocally showed the spatial association of clear peaks of Hg (mercury) and S (sulphur), which are elements of the red mineral cinnabar (mercury sulphide, HgS). This association of elements is evident both on EDS maps of the whole sample (Fig. 5A–D), as well as single-point spectra executed locally on the portion of the samples where the mineralization was more consist- ent (see Supplementary Material Fig. 2). No traces of cinnabar were found on the other four textile samples (see Supplementary Material Fig. 3). Dating. Direct dating of four of the five textiles by radiocarbon-AMS resulted in them grouping into two distinct chronological ranges (Fig. 6; Supplementary Material Table 2). The earlier pertains to Textiles 1 and 4. Both dates (Beta-491868, 4620 ± 30 BP and Beta-561185, 4450 ± 30 BP) are circumscribed within the second half of the fourth millennium cal BC, which also fits with a well defined pottery typology of some of the grave goods found in the cave. The dates pertaining to the earliest period of cave use thus point to a mean chronology of calibrated dates (IntCal20 atmospheric curve) of c. 3400 cal BC. The dating of the finer Textiles 3 (Beta-498433, 3980 ± 33 BP) and 5 (Beta-586167, 3940 ± 30 BP), yielded an average of c. 2500–2300 cal BC, being almost a mil- lennium later than the other two dated textiles. Scientific Reports \| (2021) 11:21918 \| https://doi.org/10.1038/s41598-021-01349-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Micrographs of textile weaves. Scale bar in each panel is one millimeter. Figure 3. Micrographs of textile weaves. Scale bar in each panel is one millimeter. Figure 3. Micrographs of textile weaves. Scale bar in each panel is one millimeter. Discussion Th l fi d The textile finds from Peñacalera are important in terms of their raw materials, thread structure, production technique and decoration (use of colour), since they are the earliest examples known to date of loom-woven and cinnabar-coloured textiles from the Iberian Peninsula. Flax is believed to have been introduced to the Iberian Peninsula as part of the Neolithic package5,15. Before its introduction no similar bast fibres appear to have been used in the region. Until now, the earliest linen textiles known in Spain were found in the Copper Age contexts of southeastern Iberia, such as the site of Los Millares (3200–2300 BC) and other contemporary settlements4, the Cueva Sagrada I at Lorca8,9, as well as the various Bronze Age cemeteries of the Argaric Culture4,5. The textiles from Peñacalera are thus significantly older, pushing the date for the use of linen in textiles back in time and making Textile 1 the oldest loom-woven textile in Spain made of flax fibre. As such, it provides the direct evidence of flax use for linen textile production in Iberian Pen- insula during the Late Neolithic period and indicates that flax was introduced as a fibre crop by this point in time. li Like other analysed Copper and Early Bronze Age linen textiles found in Iberian Peninsula, the textiles of Peñacalera are woven with spliced yarn, which is in agreement with contemporaneous plant fibre textiles across Europe, Egypt and western Asia12. The use of splicing may also be indirectly indicated by the rarity or almost https://doi.org/10.1038/s41598-021-01349-5 Scientific Reports \| (2021) 11:21918 \| www.nature.com/scientificreports/ Figure 4. SEM micrographs of the threads showing the spliced nature; bottom right image shows a closeup of fibre bundles. igure 4. SEM micrographs of the threads showing the spliced nature; bottom right image shows a closeup of i bre bundles. complete absence of spindle whorls in the Neolithic and Copper Age period in southern Iberia16, although they could have been made in perishable materials and did not survive.h The regular plain weave structure of all textiles and the partly preserved selvedge of Textile 1 from Peñacalera indicate that they were woven on a loom, thus constituting the earliest direct evidence of this technology in the Iberian Peninsula. Until the discovery of these textiles, the earliest evidence for the use of this technology were the third-millennium cal BC tunics of Cueva Sagrada 1, which preserve a fringed finishing border, typical for loom-woven textiles9. Discussion Th l fi d In Central Europe, particularly in the Alpine lake dwellings in Switzerland, loom-woven linen textiles began to appear from the second quarter of the fourth millennium cal BC17,18, just a few centuries before the earliest Peñacalera textile.h The earliest archaeologically-attested looms in Iberia and more widely across Europe were of the vertical warp-weighted type, which utilizes loom weights to put tension on the warp threads19,20. Having frequently been made of fired clay, loom weights survive well in the archaeological record and serve as proxies for the use of the warp-weighted loom technology. The earliest evidence for the use of the warp-weighted loom comes from Central Europe and dates to the late sixth millennium BC21. In southern Iberia, loom weights appear by Scientific Reports \| (2021) 11:21918 \| https://doi.org/10.1038/s41598-021-01349-5 www.nature.com/scientificreports/ Figure 5. Elemental composition analysis of warp sample from Textile 5: (A) composite EDS elemental map; (B) EDS elemental map of Hg (mercury); (C) EDS elemental map of S (sulphur); (D) spectrum of the composite EDS map. Figure 5. Elemental composition analysis of warp sample from Textile 5: (A) composite EDS elemental map; (B) EDS elemental map of Hg (mercury); (C) EDS elemental map of S (sulphur); (D) spectrum of the compos EDS map. Figure 5. Elemental composition analysis of warp sample from Textile 5: (A) composite EDS elemental map (B) EDS elemental map of Hg (mercury); (C) EDS elemental map of S (sulphur); (D) spectrum of the compo EDS map. Figure 5. Elemental composition analysis of warp sample from Textile 5: (A) composite EDS elemental map; (B) EDS elemental map of Hg (mercury); (C) EDS elemental map of S (sulphur); (D) spectrum of the composite EDS map. Figure 6. Multi-plot with the AMS dates obtained on four of the textiles. On top, sum of probabilities. Calibrated from the IntCal20 curve14. For details, see Supplementary Material Table 2. Figure 6. Multi-plot with the AMS dates obtained on four of the textiles. On top, sum of probabilities. Calibrated from the IntCal20 curve14. For details, see Supplementary Material Table 2. the middle of the fourth millennium cal BC, shortly before the appearance of the so-called “Carinated Bowls Complex”, which marks the beginning of the Copper Age in the region22. In the second half of the fourth and during the third millennium cal BC, the loom weights are of crescent or rectangular plaque shape, with perfo- rations at the ends23. www.nature.com/scientificreports/ Red soils, resulting from the presence of powdered cinnabar, have been documented on the chamber floors of some Andalusian megalithic monuments, such as Alberite (Villamartín, Cádiz)40 and the menhir of Casas de Don Pedro (Belmez, Córdoba)41, both dated to the fifth millennium cal BC. From more recent times (first half of the fourth millennium cal BC), it is reported in the Velilla dolmen (Osorno, Palencia) and in the monuments of southern Portugal, such as Anta Grande do Zambujeiro (Alentejo)42.i g j j From the Copper Age onwards (3300–2200 cal BC), its use increased, as demonstrated by the finds at the Dolmen of Casas de Don Pedro in the Guadiato Valley (Córdoba)43–46. The cinnabar powder has been found on and under the skeletal remains of numerous funerary deposits, their number rising considerably during the third millennium cal BC. In the Montelirio passage tomb (Castilleja de Guzmán, Seville), dated c. 2800 cal BC, cinnabar is present on various artefacts, on human remains and the chamber stones, sharing space with exotic luxury materials such as ivory, gold, amber, rock crystal and ostrich eggs44. In that tomb, and specifically in its so-called Great Chamber, at least 20 individuals have been identified, mostly women, wearing hundreds of thou- sands of white discoidal beads, interpreted as embellishment of the funerary clothing or textiles45. In some cases, cinnabar powder was found covering beads or directly associated with the skeletal remains, a possible evidence of cinnabar-coloured textiles within the same assemblage. In other cases, direct association with human skeletal remains has been explained as body paint47. y More than a century ago, Louis Siret hypothesised the presence of textiles coloured with cinnabar in tomb 356 of the El Argar necropolis (2200–1700 cal BC) on the basis of the red bands observed on the skull48,49, and textile imprints in clay preserving cinnabar particles35. The recently discovered skeletal remains and grave goods documented in the Bell Beaker culture necropolis of Humanejos (Parla, Madrid), display direct impregnation with cinnabar. An interesting case is that of an elderly male (individual 1) from tomb 5, who displays three linear bands of cinnabar transferred directly onto the skull, indicating original presence of cinnabar-coloured textile bands or headwear decorated with linear motifs. Furthermore, alongside the face of this individual, a large cinnabar stain was identified, which has been interpreted as evidence of a bag or textile coloured with or containing cinnabar50. www.nature.com/scientificreports/ (Illora, Granada)25, Casa del Tabaco (El Carpio, Córdoba)26, while a later variant with horizontal perforations constitutes an important element of the material culture in many settlements of southwestern Iberia. They are abundant in Perdigões (Alentejo, Portugal)27, São Pedro (Redondo, Portugal)28, Castillejos de Montefrío (Gra- nada)29, and Polideportivo-La Alberquilla (Martos, Jaén)30 throughout the second half of the fourth millennium BC, and continue to be used in this region during the third millennium BC. The earliest loom weight evidence in southern Iberia is thus contemporaneous with the earliest Peñacalera textile. Cinnabar is a natural mineral of volcanic or hydrothermal origin composed of mercury and sulphur (HgS), with a characteristic scarlet-red colour. It has been mined in Europe, Asia and the Americas to be used as pigment for pottery decoration and body paint, in burials, metallurgy and for medicinal purposes. Bands of cinnabar are present on a Pre-Pottery Neolithic B (ninth-eighth millennium BC) child skull at Tel Abu Hureira in Syria31 and a woman’s skull at Çatalhöyük in Turkey, where cinnabar was used not only in burial contexts but also as pigment in wall paintings32. Almadén de la Plata (Ciudad Real) in central Spain is one of the largest natural sources of cinnabar in the world33, and analytical studies have demonstrated this site as one of the main sources of cinnabar present at other archaeological sites34. The exploitation and use of cinnabar as a pigment in Iberian prehistory is a well documented phenomenon. It has been mined with certainty since Classical antiquity, when this exploitation is referenced in written sources, until today35, but a recent study indicates that the use of the cinnabar deriving from the region during the Late Neolithic and Copper Age period in Portugal was sufficiently extensive to cause mild to severe mercury poisoning in the prehistoric population36.h y p g p p p The earliest evidence of human use of cinnabar in Iberia dates back to the Early Neolithic (5500–4800 cal BC), when its use as a colouring material to fill impressions and incisions on ceramic vessels and stone bracelets is documented at Cueva de Los Murciélagos (Zuheros, Córdoba)37. Cinnabar powder was found inside a Glycymeris shell container in Cova de l’Or (Beniarrés, Alicante), dated to the same period38, while a flint blade covered with cinnabar has been reported in the Casa Montero flint mine (Madrid), exploited between 5300–5100 cal BC39. Discussion Th l fi d They are ubiquitous in settlement sites, sometimes found in groups attesting their use in sets24. Slightly flattened crescents, with vertical perforations at the ends, have been found for example at La Loma the middle of the fourth millennium cal BC, shortly before the appearance of the so-called “Carinated Bowls Complex”, which marks the beginning of the Copper Age in the region22. In the second half of the fourth and during the third millennium cal BC, the loom weights are of crescent or rectangular plaque shape, with perfo- rations at the ends23. They are ubiquitous in settlement sites, sometimes found in groups attesting their use in sets24. Slightly flattened crescents, with vertical perforations at the ends, have been found for example at La Loma https://doi.org/10.1038/s41598-021-01349-5 Scientific Reports \| (2021) 11:21918 \| www.nature.com/scientificreports/ Methods Textile structural analysis. The structural analysis was carried out using autopic observation, portable Dino-Lite AM7115MZT digital microscope at different magnifications (× 20, × 50, × 230) and a binocular micro- scope Leica M80 with optical magnifications until × 60, with built-in-camera EC3 Hd, focus × 0.5, in the Labora- tory of Archaeometry A. Arribas-Palau, University of Granada, Spain. Textile analysis involved determination of structural parameters such as weave and thread count per cm, thread twist, diameter and angle, presence of edges and any other diagnostic features51. Due to the small size of Textile 5, warp thread count was calculated based on the count carried out in several areas 2.5 mm wide. Fibre identification and analysis. Fibre identification was carried out using Hitachi TM3000 TableTop Scanning Electron Microscope at the McDonald Institute for Archaeological Research, University of Cambridge, UK (Textiles 1–4) and Hitachi SU 5000 at the Ludwig Maximilian University of Munich, Germany (Textile 5). The samples were analysed to determine the morphological characteristics of the fibre and to acquire more detailed surface information for fibre species identification. The following instrumental settings were used: vari- able vacuum conditions, analytical condition mode at 15.00 kV accelerating voltage, compositional imaging and working distance of 5–10 mm. The samples were not coated. The observed features were compared with M.G.’s fibre reference collection. The diameter of fibres was measured using the SEM utility tool at × 400 magnification. Elemental composition analysis. All textiles and a sample of sediment from the cave were analysed using a Scanning Electron Microscope equipped with an Energy Dispersive Spectrometry analyser (SEM–EDS) at the Department of Earth and Natural Sciences at the Ludwig Maximilian University of Munich. The EDS sensor uses a Ultim®Max Silicon Drift Detector (SDD) produced by Oxford Instruments, which allows chemical and elemental point and map analysis of the sample. The analysis of the samples has been conducted in variable vacuum condition using an acceleration voltage of 15 kV and a current intensity of 0.14 mA. Both chemical EDS maps and single-point EDS spectra have been collected for the characterization of the samples. Radiocarbon dating. Four samples of fibres from Textiles 1, 3, 4 and 5 were radiocarbon dated by Beta Analytic Inc. in Miami, Florida, USA. Due to financial constrains and technical similarity with Textile 3, Textile 2 was not dated. Methods The amounts analysed were 2.5 mg for Textile 1, 0.39 for Textile 3, 2.3 for Textile 4 and, 1.1 for Textile 5. No coating products, preservatives or hardener of any type were used on the remains. All samples were simply pretreated with a cleaning solution of acid/ alkali/ acid before dating. The samples were at first gently crushed then dispersed in deionized water. They were then washed with hot HCl acid to eliminate carbonates, followed by an alkali wash (NaOH) to remove secondary organic acids. The alkali wash was followed by a final acid rinse to neutralize the solution before drying. Each chemical solution was neutralized before the application of the next. During these serial rinses, mechanical contaminants such as associated sediments, organic residues, fulvic acid and rootlets were eliminated. www.nature.com/scientificreports/ Despite the continuing discussion regarding the original use of cinnabar as either body paint, a ritual burial element or textile colourant, until now there has been no direct evidence of cinnabar use to colour textiles in prehistoric Iberia. The discovery of cinnabar on Textile 5 from Peñacalera cave provides the first and earliest unequivocal proof of the use of this substance as a textile colourant at least by the Copper Age in the Iberian Peninsula. The geological ambiance of the Peñacalera cave is sedimentary limestones of the Namurian (Carbon- iferous), a rock of marine origin with fossils of crinoids, with no possibility of the natural presence of cinnabar. Since the mineral is absent from the other textiles and on any other objects found in the cave, and the textile is completely impregnated with the mineral powder, it could not have been coloured by transfer; rather we sug- gest it was intentionally coloured with the mineral. Cinnabar is not a dye which chemically binds to fibre but a mineral pigment. The method of applying it to textiles remains unknown but clearly involves grinding it to a fine powder and its physical application to the textile surface either by rubbing or soaking it in a suspension.i i p p y pp y g g p Although small and fragmentary, the textile finds from Peñacalera add important new information to our understanding of the development of textile technologies in the Iberian Peninsula and Europe during the Late Neolithic period and Copper Age. By the second half of the fourth millennium cal BC, loom-woven textiles were being used by the south Iberian populations, as also indirectly confirmed by the loom weight evidence. The thread was produced using splicing technique, which was refined over time allowing to produce exceptionally fine threads and textiles by the middle of the third millennium cal BC. Also by this time, the use of cinnabar to colour textiles was well developed as demonstrated by the extremely homogeneous distribution of the mineral on Textile 5. Scientific Reports \| (2021) 11:21918 \| https://doi.org/10.1038/s41598-021-01349-5 www.nature.com/scientificreports/ References e e e ces 1. Rivas-Martínez, S. Memoria del mapa de Series de Vegetación de España (Publ. Ministerio Agricultura, 1987). 1. Rivas-Martínez, S. Memoria del mapa de Series de Vegetación de España (Publ. Ministerio Agricultura, 1987). lf d d l l d d d l d l l ( lb l d 2. Alfaro, C. 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Scientific Reports \| (2021) 11:21918 \| https://doi.org/10.1038/s41598-021-01349-5 www.nature.com/scientificreports/ Aproximación al fenómeno megalítico en el Alto Valle del Guadiato, in d Hi t i d A d l í I 133 146 (C j í d C l M di A bi d l J d A d l í & C j S de Historia de Andalucía, I, 133–146 (Consejería de Cultura y Medio Ambiente de la Junta de Andalucía & CajaSur, 1994) j y j 47. Díaz-Guardamino Uribe, M., Wheatley, D. W., Williams, E. F. & Garrido Cordero, J. A. Arquitectura, estratigrafia y depósitos del Tholos de Montelirio, in Montelirio. Un gran monumento megalítico de la Edad del Cobre (eds Fernández Flores, Á. et al.) 345–364 (Junta de Andalucía, 2016). 48. Siret, E. & Siret, L. Las Primeras Edades del Metal en la Península Ibérica (Dirección General de Cultura, Museo Arqueológico de Murcia, 1890). 49. LópezPadilla, J. A. et al. Ocre y cinabrio en el registro funerario de El Argar. Trab. Prehist. 69(2), 273–292 (2012). d l d l l f d ( l 50. Garrido Pena, R., Flores Fernández, R. & Herrero-Corral, A. M. Las sepulturas campaniformes de Humanejos (Parla, Ma (Comunidad de Madrid, 2019).hi 51. Emery, I. The Primary Structures of Fabrics. An Illustrated Classification. (Textile Museum, 1966). https://doi.org/10.1038/s41598-021-01349-5 Scientific Reports \| (2021) 11:21918 \| www.nature.com/scientificreports/ Acknowledgementsh g The site was discovered by speleologists A. Aljama and R. Bermúdez, who, together with M.J. Martínez-Fernández and G.M. Lara we thank for their cooperation throughout the fieldwork. Dating was carried out thanks to the Spanish Ministry of Science, Innovation and Universities-Universidad de Granada, Juan de la Cierva-Incor- poración Agreement IJCI-2016-27812 (2016, by R.M.M.S.), and the funding provided in the framework of the project “Archaeobiology of the Neolithic of the Southern Iberian Peninsula” (NeArqBioSI) A-HUM-460-UGR18 funded by Consejería de Economía, Conocimiento, Empresas y Universidad, FEDER Programme—Junta de Andalucía-Universidad de Granada, Call 2019. M.G.’s work was supported by the European Research Council (Grant Agreement FP/2007-2013-312603). Competing interests h The authors declare no competing interests. Author contributions M.G. designed the study, coordinated sample analysis, performed structural and fibre analysis and wrote the initial report, which formed the core of the manuscript. M.D.B.G. and J.C.V.R. directed the fieldwork, excavating this site and the burial context. R.M.M.S. identified and separated the remains, coordinated radiocarbon dating and contributed to the manuscript about Iberian contexts, parallels and chronology. C.C. performed elemental composition analysis of cinnabar and contributed to the manuscript. M.G. and R.M.M.S. wrote the main manu- script text. All authors discussed the results and reviewed the manuscript. Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-01349-5. Correspondence and requests for materials should be addressed to M.G. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-01349-5 Scientific Reports \| (2021) 11:21918 \|
https://openalex.org/W3212500202	https://jurnal.umk.ac.id/index.php/simet/article/download/4886/2867	Indonesian	null	APLIKASI FORECASTING KEHADIRAN SISWA DI SMP 2 JEKULO MENGGUNAKAN METODE REGRESI LINEAR	Simetris: Jurnal Teknik Mesin, Elektro dan Ilmu Komputer/Simetris	2,021	cc-by	4,895	Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 ABSTRAK Kehadiran siswa memegang peranan penting dalam kegiatan belajar mengajar. Keterlibatan siswa dalam kegiatan belajar mengajar secara langsung akan menimbulkan interaksi belajar mengajar berjalan dengan baik, memudahkan terjadinya transfer ilmu pengetahuan secara langsung dari guru ke siswa. Siswa yang hadir memungkinkan untuk lebih aktif dalam interaksi belajar mengajar, berbeda dengan siswa yang tidak hadir, hal ini akan berimplikasi pada prestasi belajar siswa. Kehadiran siswa juga berpengaruh pada kelancaran siswa dalam melanjutkan ke jenjang kelas selanjutnya, karena untuk bisa naik kelas maka siswa harus memenuhi prosentase kehadiran yang ditetapkan oleh sekolah termasuk oleh SMP 2 Jekulo. SMP 2 Jekulo menetapkan siswa dapat naik kelas jika memenuhi kehadiran 85%. Dimana siswa tidak dapat naik kelas jika terdapat Alpha (tidak hadir tanpa keterangan) lebih dari 14. Ketidakhadiran siswa terdiri dari tiga kategori yaitu sakit, izin dan alpha. Tujuan penelitian adalah untuk membuat aplikasi forecasting yang dapat digunakan untuk memprediksi ketidakhadiran siswa yaitu dengan kategori sakit, izin dan alpha. Metode analisis prediksi ketidakhadiran siswa menggunakan metode analisis regresi linear sederhana. Tahapan penelitian yakni pengumpulan data, analisis kebutuhan sistem, perancangan sistem dan implementasi. Aplikasi forecasting kehadiran siswa ini berbasis web, dibuat dengan menggunakan bahasa pemrograman PHP. Hasil penelitian adalah dibangun aplikasi yang dapat digunakan untuk memprediksi keketidakhadiran siswa pada bulan tertentu menggunakan data pada periode sebelumnya. Kata kunci: forecasting, web, analisis regresi linear sederhana, kehadiran siswa APLIKASI FORECASTING KEHADIRAN SISWA DI SMP 2 JEKULO MENGGUNAKAN METODE REGRESI LINEAR Peni Retnowati Fakultas Teknik, Program Studi Teknik Informatika Universitas Muria Kudus Email: 201751218@std.umk.ac.id Tutik Khotimah Fakultas Teknik, Program Studi Teknik Informatika Universitas Muria Kudus Email: tutik.khotimah@umk.ac.id Kata kunci: forecasting, web, analisis regresi linear sederhana, kehadiran siswa 1. PENDAHULUAN Belajar adalah suatu aktivitas atau suatu proses untuk memperoleh pengetahuan, meningkatkan keterampilan, memperbaiki perilaku, sikap dan mengkokohkan kepribadian. Berkaitan dengan hal tersebut, kegiatan belajar siswa tidak akan berjalan dengan baik, apabila siswa tidak meluangkan dan membagi waktunya untuk belajar dengan sebaik-baiknya. Melihat hal ini, pemanfaatan waktu yang baik oleh siswa untuk belajar akan menimbulkan kesadaran terhadap pentingnya keterlibatan siswa secara aktif hadir dalam kegiatan belajar mengajar sehingga memungkinkan terjadinya interaksi belajar mengajar dengan baik [1]. Kondisi di lapangan guru atau sekolah dihadapkan pada permasalahan bahwa siswa seringkali dengan mudah meninggalkan kegiatan belajar mengajar dengan alasan izin atau tanpa izin. Hal yang sama terjadi di SMP 2 Jekulo, dimana masih banyak siswa – siswa di SMP 2 Jekulo yang tidak masuk sekolah tanpa keterangan ketidakhadiran secara jelas. Siswa yang sering tidak mengikuti kegiatan pembelajaran akan berdampak buruk tertinggal pelajaran dengan siswa yang lain. Dan jika jumlah ketidakhadiran melewati batas yang ditetapkan maka sebagai konsekuensinya menjadi salah satu pertimbangan untuk siswa yang bersangkutan tidak naik kelas. Mengingat pentingya kehadiran siswa dalam kegiatan belajar mengajar, maka sekolah perlu mengadakan langkah antisipatif agar ketidakhadiran siswa dapat diminimalisir. Langkah antisipatif meminimalisir ketidakhadiran siswa akan lebih akurat jika sekolah mempunyai dasar pertimbangan dengan melakukan forecasting atau peramalan ketidak hadiran siswa dalam waktu tertentu, dengan kategori ketidak hadiran yaitu sakit, izin dan alpha. Ada banyak metode yang terdapat dalam forecasting, salah satunya metode regresi linear sederhana. forecasting dan perhitungan metode regresi linear telah diterapkan dalam penelitian diberbagai bidang. Metode Regresi Linear sangat membantu dalam perhitungan peramalan pembukaan kelas, dimana metode ini sangat mudah dan sederhana dalam mengimplementasikan kedalam sistem [2]. Metode regresi linear juga diterapkan dalam memprediksi jumlah penjualan Batik Lasem pada KUB Sarwo Endah [3]. Penggunaan metode regresi linear selanjutnya digunakan untuk memprediksi anggaran penjualan [4], hasil penelitian menyatakan anggaran penjualan yang disusun PD. Idaman melalui peramalan penjualan secara kualitatif dalam upaya meningkatkan penjualan perusahaan berjalan cukup baik, Hasil analisis efektivitas menemukan bahwa target penjualan yang disusun dalam anggaran penjualan PD. Idaman selama tahun 2012-2016 cukup efektif karena selisih antara target dan hasil penjualan sudah cukup optimal. Metode Linier Regresi juga digunakan untuk memprediksi mahasiswa registrasi secara tepat [5] serta diterapkan pada prediksi penjualan properti berdasarkan data penjualan properti pada PT XYZ [6]. Penelitian tersebut memiliki persamaan dengan penulis, yaitu mengenai konsep dan metode prediksi yaitu sama-sama melakukan peramalan atau forecasting dengan menggunakan metode regresi linear. 1. PENDAHULUAN Sejalan dengan penelitian-penelitian sebelumnya tersebut, penulis tertarik untuk membangun aplikasi yang dapat memudahkan dalam prediksi ketidakhadiran siswa dengan kategori sakit, izin dan alpha. Aplikasi dibangun menggunakan pemrograman PHP. ABSTRACT The presence of students plays an important role in teaching and learning activities. The involvement of students in teaching and learning activities directly will lead to interaction of teaching and learning going well, facilitating the transfer of knowledge directly from teacher to student. Students who attend make it possible to be more active in teaching and learning interactions, in contrast to students who are absent, this will have implications for student achievement. The presence of students also affects the smoothness of students in continuing to the next level of class, because to be able to move up the class, students must meet the percentage of attendance set by the school, including by SMP 2 Jekulo. Junior High School 2 Jekulo determined that students could move up if they meet 85% attendance. Where students cannot graduate if there is an Alpha (absent without information) more than 14. Student absence consists of three categories, namely illness, permission and alpha. The purpose of this study is to create a forecasting application that can be used to predict student absence from the sick, permission and alpha categories. The prediction analysis method for student absence uses the simple linear regression analysis method. The stages of research are data collection, system requirements analysis, system design and implementation. This student attendance forecasting application is web based, created using the PHP programming language. The results of the study are built applications that can be used to predict the absence of students in a particular month using data in the previous period. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Keywords: forecasting, web, simple linear regression, student attendance 2.1 Tinjauan Pustaka a. Kehadiran Siswa dalam Proses Belajar Mengajar a. Kehadiran Siswa dalam Proses Belajar Mengajar Pengertian kehadiran di sekolah bukan hanya berarti peserta didik secara fisik ada di sekolah, melainkan ialah keterlibatan siswa dalam kegiatan-kegiatan sekolah, seperti di sebutkan dalam “dictionary of education”, good carter: “attendance at school not merely being bodily presence but incluiding actual participation in the work and activities of the school” [7]. Dari definisi di atas, dapat disimpulkan bahwa kehadiran peserta didik ialah keikutsertaan peserta didik secara fisik dan mental, serta keterlibatan mereka dalam kegiatan-kegiatan sekolah. Sedangkan ketidakhadiran peserta didik bisa di kata, tidak terlibatnya peserta didik dalam kegiatan sekolah. Pada umumnya ketidakhadiran siswa dapat dibagi kedalam tiga bagian: Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 a) Alpa, yaitu ketidakhadiran tanpa keterangan yang jelas, dengan alasan yang tidak bisa dipertanggungjawabkan; a) Alpa, yaitu ketidakhadiran tanpa keterangan yang jelas, dengan alasan yang tidak bisa dipertanggungjawabkan; p gg gj b) Izin, ketidakhadiran dengan keterangan dan alasan tertentu yang bisa dipertanggungjawabkan, biasanya disertai surat pemberitahuan dari orang tua; b) Izin, ketidakhadiran dengan keterangan dan alasan tertentu yang bisa dipertanggungjawabkan, biasanya disertai surat pemberitahuan dari orang tua; c) Sakit, ketidakhadiran dengan alasan gangguan kesehatan, biasanya disertai surat pemberitahuan dari orang tua atau surat keterangan sakit dari dokter. c) Sakit, ketidakhadiran dengan alasan gangguan kesehatan, biasanya disertai surat pemberitahuan dari orang tua atau surat keterangan sakit dari dokter. b. Forecasting Forecasting merupakan teknik untuk menduga, memperkirakan suatu kondisi di masa yang akan datang berdasarkan pada kondisi masa lalu dan sekarang, sehingga dapat dengan tepat memperkirakan suatu tindakan atau keputusan. Fungsi dari forecasting ini dapat dikatakan sebagai tindakan dasar pada saat seseorang melakukan tahap perencanaan, misalnya anggaran, jumlah penjualan, perencanaan produksi dan persediaan, sumber daya misalnya jumlah tenaga kerja, serta perencaan kebutuhan bahan baku. Dua hal pokok yang harus diperhatikan dalam proses forecasting atau peramalan yang akurat dan bermanfaat adalah pengumpulan data yang relevan berupa informasi yang dapat menghasilkan peramalan yang akurat dan pemilihan teknik peramalan yang tepat yang akan memanfaatkan informasi data yang diperoleh semaksimal mungkin [8]. Dengan melakukan peramalan, para perencana dan pengambil keputusan akan dapat mempertimbangkan alternatif-alternatif strategi yang lebih luas daripada tanpa peramalan. Dengan demikian berbagai rencana strategi dan aksi dapat dikembangkan untuk menghadapi berbagai kemungkinan yang bisa terjadi di masa mendatang. Terdapat tiga jenis metode peralaman, yaitu metode time series, metode regresi, dan metode kualitatif : Terdapat tiga jenis metode peralaman, yaitu metode time series, metode regresi, dan metode kualitatif : a) Metode Time Series merupakan salah satu teknik statistik yang menggunakan data permintaan historis untuk memprediksi masa depan permintaan. b) Metode Regresi, yaitu sebuah metode statistik juga yang melakukan peramalan dengan menggunakan pengembangan hubungan matematis antara variable yaitu variable dependen (Y) dengan variable Independen (X). Dalam metode regresi suatu model perlu dispesifikasikan sebelum dilakukan pengumpulan data. c) Metode Kualitatif, merupakan sebuah metode yang digunakan untuk memutuskan hal-hal yang berhubungan dengan proses perencanan yang panjang dengan menggunakan penilaian manajemen, keahlian, dan pendapat sehingga dapat dihasilkan suatu perkiraan. c) Metode Kualitatif, merupakan sebuah metode yang digunakan untuk memutuskan hal-hal yang berhubungan dengan proses perencanan yang panjang dengan menggunakan penilaian manajemen, keahlian, dan pendapat sehingga dapat dihasilkan suatu perkiraan. c. Metode Regresi Linear Metode Analisa Regresi Linear Sederhana adalah Metode Statistik yang berfungsi untuk menguji sejauh mana hubungan sebab akibat antara Variabel Faktor Penyebab (X) terhadap Variabel Akibatnya. Faktor Penyebab pada umumnya dilambangkan dengan X atau disebut juga dengan Predictor sedangkan Variabel Akibat dilambangkan dengan Y atau disebut juga dengan Response. Regresi Linear Sederhana atau sering disingkat dengan SLR (Simple Linear Regression) juga merupakan salah satu Metode Statistik yang dipergunakan dalam melakukan peramalan ataupun prediksi . Secara matematis model analisis regresi linier sederhana dapat digambarkan sebagai berikut:      = = = = = − − = n i n i i i n i n i n i i i i i n X X n X Y X Y 1 1 2 2 1 1 1 1 ) ( ) )( (       = = = = = − − = n i n i i i n i n i n i i i i i n X X n X Y X Y 1 1 2 2 1 1 1 1 ) ( ) )( (  (1) (1) n 𝜷𝟎=𝜸̅ − 𝜷𝟏 ×̅ 𝜸 = 𝜷𝟎+ 𝜷𝟏× (2) (3) (3) 𝜸 = 𝜷𝟎+ 𝜷𝟏× Keterangan: Keterangan: Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Y = variabel terpengaruh Y = variabel terpengaruh 𝛽0= konstanta 𝛽1 = gradien garis X = variable pemberi pengaruh Secara praktis analisis regresi linier sederhana memiliki kegunaan sebagai berikut: p g g g a) Model regresi sederhana dapat digunakan untuk forecasting atau memprediksi nilai Y. Namun sebelum melakukan forecasting, terlebih dahulu harus dibuat model atau persamaan regresi linier. Ketika model yang fit sudah terbentuk maka model tersebut memiliki kemampuan untuk memprediksi nilai Y berdasarkan variabel Y yang diketahui. a) Model regresi sederhana dapat digunakan untuk forecasting atau memprediksi nilai Y. Namun sebelum melakukan forecasting, terlebih dahulu harus dibuat model atau persamaan regresi linier. Ketika model yang fit sudah terbentuk maka model tersebut memiliki kemampuan untuk memprediksi nilai Y berdasarkan variabel Y yang diketahui. b) Mengukur pengaruh variabel X terhadap variabel Y. Hubungan antara variabel dalam analisis regresi bersifat kausalitas atau sebab akibat. 2.2 Kerangka Pemikiran Kerangka pemikiran penelitian dapat dilihat pada gambar 1. Kerangka pemikiran penelitian dapat dilihat pada gambar 1. Kerangka pemikiran penelitian dapat dilihat pada gambar 1. Kerangka pemikiran penelitian dapat dilihat pada gambar 1. Gambar 1. Kerangka Pemikiran 3 M t d P liti apat dilihat pada gambar 1. Gambar 1. Kerangka Pemikiran Gambar 1. Kerangka Pemikiran 3. HASIL DAN PEMBAHASAN 3.1. Analisis a. Entity Relationship Diagram Pada perancangan sistem yang dibuat agar dapat menjadi sistem database yang rapi dan terstruktur, maka membutuhkan Entity Relationship Diagram (ERD), yaitu tools yang digunakan untuk melakukan pemodelan data secara abstrak dengan tujuan untuk mendeskripsikan atau menggambarkan struktur dari data yang digunakan [9]. Entity Relationship Diagram (ERD) juga model untuk menyusun database agar dapat menggambarkan data yang mempunyai relasi dengan database yang akan dirancang seperti yang ditunjukkan pada Gambar 2 Gambar 2. Entity Relation Diagram Gambar 2. Entity Relation Diagram 2.3 Metode Penelitian Penelitian ini adalah penelitian rekayasa perangkat lunak (research and development). Metode yang digunakan adalah Software Development Life Cycle (SDLC). Tahapan penelitian sebagai berikut: a. Pengumpulan Data Pada tahap ini dilakukan pengumpulan data berupa rekapitulasi absensi siswa SMP 2 Jekulo pada semester 1 Tahun Pelajaran 2019 / 2020 dengan kategori ketidak hadiran yaitu sakit, izin dann alpha. b. Analisa Setelah dilakukan pengumpulan data, tahap berikutnya adalah melakukan analisa kebutuhan sistem. c. Perancangan Pada tahap ini dilakukan perancangan sistem dengan menggunakan Entity Relationship Diagram, Data Flow Diagram dan flowchart. Selain itu juga dilakukan perancangan antarmuka (input/output). Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 d. Implementasi Tahap implementasi adalah tahap pengkodean terhadap rancangan-rancangan yang telah dirancang dan didefinisikan sehingga akan membentuk sistem yang diharapkan. Pengkodean sistem dilakukan dengan menggunakan bahasa pemprograman PHP. Pada sistem ini, MySQL digunakan sebagai basis data. Rancangan basis data juga diimplementasikan menjadi sebuah basis data yang dapat digunakan untuk menyimpan data sesuai kebutuhan. Aplikasi dibangun dengan software XAMPP versi 5.6.33, PHP 4.9.4, Google Chrome 72.0 dan Notepad ++. Pada tahap implementasi, dilakukan penyusunan View dan Logic sesuai perancangan yang telah dibuat. d. Implementasi Tahap implementasi adalah tahap pengkodean terhadap rancangan-rancangan yang telah dirancang dan didefinisikan sehingga akan membentuk sistem yang diharapkan. Pengkodean sistem dilakukan dengan menggunakan bahasa pemprograman PHP. Pada sistem ini, MySQL digunakan sebagai basis data. Rancangan basis data juga diimplementasikan menjadi sebuah basis data yang dapat digunakan untuk menyimpan data sesuai kebutuhan. Aplikasi dibangun dengan software XAMPP versi 5.6.33, PHP 4.9.4, Google Chrome 72.0 dan Notepad ++. Pada tahap implementasi, dilakukan penyusunan View dan Logic sesuai perancangan yang telah dibuat. b. Pembuatan Tabel Data Pada tahap ini, dibuat tabel data yang nantinya akan dimasukkan ke dalam basis data. Adapun tabel data tersebut antara lain sebagai berikut: 2. Tabel Kelas Berisi identitas kelas yang akan dilakukan prediksi kehadiran, ditunjukkan pada Tabel 4. Tabel 4. Tabel Kelas No Field Tipe Data Panjang 1 Id Kelas Int 2 2 Kelas Varchar 10 Tabel 4. Tabel Kelas 3. Tabel Admin Berisi identitas pengguna (admin) yang dapat mengakses aplikasi. Username dan Password diperlukan untuk masuk ke dalam sistem. Ditunjukan pada tabel 5 Tabel 5. Tabel Admin No Field Tipe Data Panjang 1 User Nama Varchar 25 2 Password Varchar 25 Tabel 5. Tabel Admin Tabel 5. Tabel Admin 1. Tabel Kehadiran Terdiri dari berbagai macam data yang ditulis untuk membuat data forecasting. Mulai dari data kelas, bulan, semester, tahun, keterangan (S/I/A) dan jumlah dari (S/I/A), ditunjukkan pada Tabel 3. Tabel 3. Tabel Kehadiran Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 No Field Tipe Data Panjang 1 No Int 4 2 Id_ Kelas Int 2 3 Bulan Int 2 4 Semester Varchar 6 5 Tahun Int 4 6 Keterangan Varchar 1 7 Jumlah Int 3 2. Tabel Kelas Berisi identitas kelas yang akan dilakukan prediksi kehadiran, ditunjukkan pada Tabel 4. 2. Tabel Kelas Berisi identitas kelas yang akan dilakukan prediksi kehadiran, ditunjukkan pada Tabel 4. c. Data Flow Diagram Penggunaan Aplikasi Forecasting Kehadiran Siswa ini dimulai dengan Admin melakukan login pada aplikasi yaitu dengan memasukan User Name dan Password, jika data yang dimasukan benar maka akan berhasil masuk pada halaman utama, jika User Name dan Password yang dimasukan tidak sesuai maka akan muncul peringatan dari sistem. Proses Login ditunjukkan pada Gambar 6. Flowchart Login. Gambar 5. DFD Level 2 Gambar 5. DFD Level 2 c. Data Flow Diagram Setelah relasi dari basis data (ERD) dan tabel data telah dibuat, selanjutnya adalah membuat Data flow diagram (DFD) . Data Flow Diagram adalah representasi grafik yang menggambarkan aliran informasi dan transformasi yang diaplikasikan sebagai data yang mengalir dari masukan dan keluaran [10]. Dengan demikian Data flow diagram (DFD) untuk menggambarkan proses alur aplikasi yang dipadukan dengan data yang telah ada sehingga dapat berinteraksi antara data dengan alur programnya. Dalam DFD Level 0, menjelaskan alur Admin akan melakukan proses login pada aplikasi, jika proses login berhasil maka sistem akan memberikan konfirmasi login, dan Admin akan diarahkan masuk pada halaman utama. Pada proses ini maka Admin akan melakukan kegiatan seperti memasukan Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 data-data kehadiran (Absensi), mencari data absensi dan melakukan proses prediksi (forecasting). Dan sistem akan menampilkan input data-data serta forecasting yang dilakukan oleh Admin. DFD Level 0 ditunjukkan pada Gambar 3 Gambar 3. DFD Level 0 Gambar 3. DFD Level 0 Dalam DFD Level 1, dijelaskan admin melakukan proses login dengan memasukan Username dan Password, jika data admin yang dimasukan bisa diterima sistem, maka admin akan diarahkan pada halaman utama yang berisi menu Input Data, Tampil Data dan Prediksi Data. Admin bisa melakukan input data absensi, pencarian data absensi dan pencarian data forecasting, jika proses berhasil maka data absensi terbaharui, tampil data absensi dan tampil hasil forecasting. DFD Level 1 ditunjukan pada Gambar 4. Gambar 4. DFD Level 1 Dalam DFD level 2, dijelaskan lebih detail alur proses admin melakukan input data kelas berdasarkan data kelas yang akan menampilkan informasi kelas dan input data absensi berdasarkan data absensi menampilkan informasi tentang kehadiran. DFD Level 2 ditunjukan pada Gambar 5. Gambar 4. DFD Level 1 Gambar 4. DFD Level 1 Dalam DFD level 2, dijelaskan lebih detail alur proses admin melakukan input data kelas berdasarkan data kelas yang akan menampilkan informasi kelas dan input data absensi berdasarkan data absensi menampilkan informasi tentang kehadiran. DFD Level 2 ditunjukan pada Gambar 5. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Gambar 5. DFD Level 2 d. Flowchart Sistem Setelah Entity Relationship Diagram dan Data Flow Diagram beserta rancangan antar muka dibuat, selanjutnya adalah membuat flowchart untuk mengetahui proses alur dari sistem aplikasi yang dipadukan dengan data yang telah ada sehingga dapat berinteraksi antara data dengan alur programnya. d. Flowchart Sistem Setelah Entity Relationship Diagram dan Data Flow Diagram beserta rancangan antar muka dibuat, selanjutnya adalah membuat flowchart untuk mengetahui proses alur dari sistem aplikasi yang dipadukan dengan data yang telah ada sehingga dapat berinteraksi antara data dengan alur programnya. Penggunaan Aplikasi Forecasting Kehadiran Siswa ini dimulai dengan Admin melakukan login pada aplikasi yaitu dengan memasukan User Name dan Password, jika data yang dimasukan benar maka akan berhasil masuk pada halaman utama, jika User Name dan Password yang dimasukan tidak sesuai maka akan muncul peringatan dari sistem. Proses Login ditunjukkan pada Gambar 6. Flowchart Login. Gambar 6. Flowchart Login Input Data menunjukkan proses data-data yang harus dimasukankan oleh admin. Data-data yang harus dimasukkan antara lain: Data Kelas, Bulan, Semester, Tahun, Keterangan (Sakit, Izin, Alpha) dan jumlah ketidak hadiran. Jika proses memasukkan data berhasil maka akan disimpan dalam database. Proses Input Data ditunjukkan pada Gambar 7. Flowchart Input Data. Gambar 6. Flowchart Login Gambar 6. Flowchart Login Gambar 6. Flowchart Login Input Data menunjukkan proses data-data yang harus dimasukankan oleh admin. Data-data yang harus dimasukkan antara lain: Data Kelas, Bulan, Semester, Tahun, Keterangan (Sakit, Izin, Alpha) dan jumlah ketidak hadiran. Jika proses memasukkan data berhasil maka akan disimpan dalam database. Proses Input Data ditunjukkan pada Gambar 7. Flowchart Input Data. Input Data menunjukkan proses data-data yang harus dimasukankan oleh admin. Data-data yang harus dimasukkan antara lain: Data Kelas, Bulan, Semester, Tahun, Keterangan (Sakit, Izin, Alpha) dan jumlah ketidak hadiran. Jika proses memasukkan data berhasil maka akan disimpan dalam database. Proses Input Data ditunjukkan pada Gambar 7. Flowchart Input Data. Input Data menunjukkan proses data-data yang harus dimasukankan oleh admin. Data-data yang harus dimasukkan antara lain: Data Kelas, Bulan, Semester, Tahun, Keterangan (Sakit, Izin, Alpha) dan jumlah ketidak hadiran. Jika proses memasukkan data berhasil maka akan disimpan dalam database. Proses Input Data ditunjukkan pada Gambar 7. Flowchart Input Data. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Gambar 7. Flowchart Input Data Gambar 7. Flowchart Input Data Pada Tampil Data akan menampilkan hasil perhitungan (forecasting) berdasarkan data-data yang sudah dimasukan pada Input Data.Ditunjukan pada Gambar 8. Flowchart Tampil Data. Pada Tampil Data akan menampilkan hasil perhitungan (forecasting) berdasarkan data-data yang sudah dimasukan pada Input Data.Ditunjukan pada Gambar 8. Flowchart Tampil Data. Gambar 8. d. Flowchart Sistem Flowchart Tampil Data Prediksi Data menampilkan alur proses prediksi dimana proses dimulai dengan memilih bula dan kriteria kehadiran (S/I/A) yang akan diprediksi, jika proses berhasil maka ssstem akan menampilka hasil prediksi Ditunjukkan pada Gambar 9 Flowchart Prediksi Data Gambar 8. Flowchart Tampil Data Prediksi Data menampilkan alur proses prediksi dimana proses dimulai dengan memilih bulan dan kriteria kehadiran (S/I/A) yang akan diprediksi, jika proses berhasil maka ssstem akan menampilkan hasil prediksi. Ditunjukkan pada Gambar 9. Flowchart Prediksi Data. Prediksi Data menampilkan alur proses prediksi dimana proses dimulai dengan memilih bulan dan kriteria kehadiran (S/I/A) yang akan diprediksi, jika proses berhasil maka ssstem akan menampilkan hasil prediksi. Ditunjukkan pada Gambar 9. Flowchart Prediksi Data. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Gambar 9. Flowchart Prediksi Data Gambar 9. Flowchart Prediksi Data 3.2. Implementasi Implementasi adalah tahap penerapan sekaligus pengujian aplikasi yang telah dibangun, untuk melangkah ke implementasi yang harus didukung dengan tools dan fasilitas yang tepat. Berikut merupakan tampilan antar muka aplikasi forecasting kehadiran siswa di SMP 2 Jekulo.. Implementasi adalah tahap penerapan sekaligus pengujian aplikasi yang telah dibangun, untuk melangkah ke implementasi yang harus didukung dengan tools dan fasilitas yang tepat. Berikut merupakan tampilan antar muka aplikasi forecasting kehadiran siswa di SMP 2 Jekulo.. a. Halaman Login i dib Sistem dibangun hanya mempunyai fitur login untuk admin. Admin bertugas sebagai pemegang penuh dan bertanggung jawab terhadap data yang ada didalam sistem. Login pada level ini dapat melakukan pengelolaan data, menambah, mengedit, dan menghapus data ketidak hadiran siswa dengan kategori sakit, izin dan alpha Admin adalah guru Bimbingan Konselingr. Halaman login merupakan halaman yang digunakan untuk admin login ke sistem. Jika username dan password di ketik dengan benar maka admin akan masuk ke dalam sistem. Halaman Login ditunjukkan pada Gambar 10 . Gambar 10. Halaman Login b. Halaman Utama Gambar 10. Halaman Login Gambar 10. Halaman Login b. Halaman Utama b. Halaman Utama Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Setelah mengisi username dan password agar bisa login, admin akan diarahkan pada halaman utama, terdapat menu pilihan yang bisa diakses yaitu : input data, tampil data, prediksi data dan logout. Input Data akan mengarahkan pada jenis data yang akan dimasukkan. Tampil Data akan menampilkan hasil masukan data, sedang Prediksi Data akan memproses dan menampilkan hasil prediksi. Halaman utama ditunjukkan pada gambar 11. Gambar 11. Halaman Utama c. Halaman Input Data Pada halaman input data, admin akan diarahkan untuk mengisi form berupa data-data yang diperlukan untuk melakukan perhitungan prediksi kehadiran. Adapaun data yang diisi antara lain : kelas, bulan, semester, tahun, keterangan (sakit, izin, alpha) dan jumlah. Data-data yang dimasukan adalah dari data kehadiran siswa yang dapat ditambah, diubah, maupun dihapus oleh admin. Halaman input data ditunjukkan pada gambar 12. Gambar 12. Halaman Input Data d. Halaman Tampil Data Setelah mengisi lengkap data yang diperlukan pada halaman input data, maka admin dapat menampilkan data yang dimasukkan pada halaman tampil data. Pada halaman Tampil Data, admin juga dapat melakukan perubahan pada data yang sudah dimasukan dengan melakukan aksi hapus data. Halaman tampil data ditunjukkan pada gambar 13. Gambar 12. Halaman Input Data Gambar 12. Halaman Input Data e. Halaman Prediksi Data dan Hasil Prediksi Data e. Halaman Prediksi Data dan Hasil Prediksi Data Untuk bisa melakukan prediksi pada halaman prediksi data, maka admin harus memasukkan bulan dan keterangan seperti sakit, izin atau alpha yang akan diprediksi. Halaman prediksi data ditunjukkan pada gambar 14 Gambar 14. Halaman Prediksi Data d. Halaman Tampil Data p Setelah mengisi lengkap data yang diperlukan pada halaman input data, maka admin dapat menampilkan data yang dimasukkan pada halaman tampil data. Pada halaman Tampil Data, admin juga dapat melakukan perubahan pada data yang sudah dimasukan dengan melakukan aksi hapus data. Halaman tampil data ditunjukkan pada gambar 13. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Gambar 13. Halaman Tampil Data Gambar 13. Halaman Tampil Data Gambar 14. Halaman Prediksi Data Dan pada halaman hasil prediksi data menampilkan hasil prediksi kehadiran siswa berdasarkan data yang dimasukan saat akan melakukan prediksi yaitu bulan dan keterangan berupa sakit, izin atau alpha. Halaman hasil prediksi data ditunjukkan pada gambar 15 Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 f. Pengujian Pengujian yang dilakukan pada Aplikasi Forecasting Kehadiran Siswa di SMP 2 Jekulo ini adalah pengujian BlackBox. Pengujian dilakukan untuk mengetahui apakah rumus yang digunakan dalam aplikasi sudah sesuai atau belum. Daftar pengujian dapat dilihat pada Gambar 16 adalah perhitungan forecasting menggunakan Microsoft Excel dan Gambar 17 perhitungan menggunakan aplikasi, bertujuan memprediksi jumlah siswa sakit pada bulan selanjutnya sebagai berikut: Gambar 16. Perhitungan Prediksi Siswa Sakit (Microsoft Excel) Gambar 16. Perhitungan Prediksi Siswa Sakit (Microsoft Excel) Gambar 17. Perhitungan Prediksi Siswa Sakit (Aplikasi) Gambar 17. Perhitungan Prediksi Siswa Sakit (Aplikasi) Dari Daftar pengujian pada Gambar 16 yaitu perhitungan forecasting menggunakan Microsoft Excel dan Gambar 17 perhitungan menggunakan aplikasi, dengan tujuan memprediksi jumlah siswa sakit pada bulan selanjutnya diperoleh hasil perhitungan yang sama. j y p p g y g Berikutnya pengujian prediksi jumlah siswa izin pada satu bulan berikutnya dapat dilihat pada Gambar 18 yaitu perhitungan forecasting menggunakan Microsoft Excel dan Gambar 19 perhitungan menggunakan aplikasi, dan diperoleh hasil perhitungan yang sama. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Gambar 18. Perhitungan Prediksi Siswa Izin (Microsoft Excel) Gambar 18. Perhitungan Prediksi Siswa Izin (Microsoft Excel) Gambar 18. Perhitungan Prediksi Siswa Izin (Microsoft Excel) Gambar 18. Perhitungan Prediksi Siswa Izin (Microsoft Excel) Gambar 19. Perhitungan Prediksi Siswa Izin (Aplikasi) Gambar 19. Perhitungan Prediksi Siswa Izin (Aplikasi) Gambar 19. Perhitungan Prediksi Siswa Izin (Aplikasi) Demikian pula pada pengujian prediksi jumlah siswa alpha pada satu bulan berikutnya dapat dilihat pada Gambar 20 yaitu perhitungan forecasting menggunakan Microsoft Excel dan Gambar 21 perhitungan menggunakan aplikasi, dan diperoleh hasil perhitungan yang sama pula. Demikian pula pada pengujian prediksi jumlah siswa alpha pada satu bulan berikutnya dapat dilihat pada Gambar 20 yaitu perhitungan forecasting menggunakan Microsoft Excel dan Gambar 21 perhitungan menggunakan aplikasi, dan diperoleh hasil perhitungan yang sama pula. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 Gambar 20. Perhitungan Prediksi Siswa Alpha (Microsoft Excel) Gambar 21. Perhitungan Prediksi Siswa Alpha (Aplikasi) Gambar 20. Perhitungan Prediksi Siswa Alpha (Microsoft Excel) Gambar 20. Perhitungan Prediksi Siswa Alpha (Microsoft Excel) Gambar 21. Perhitungan Prediksi Siswa Alpha (Aplikasi) Gambar 21. Perhitungan Prediksi Siswa Alpha (Aplikasi) Gambar 21. Perhitungan Prediksi Siswa Alpha (Aplikasi) 4. KESIMPULAN Kesimpulan yang dapat ditarik berdasarkan hasil dan pembahasan diatas tentang aplikasi forecasting kehadiran siswa di SMP 2 Jekulo adalah sebagai berikut: a. Telah dihasilkan Aplikasi Forecasting Kehadiran Siswa berbasis teknologi informasi (web) yang dapat digunakan untuk melakukan prediksi kehadiran siswa berdasarkan data pada periode sebelumnya selama waktu tertentu. a. Telah dihasilkan Aplikasi Forecasting Kehadiran Siswa berbasis teknologi informasi (web) yang dapat digunakan untuk melakukan prediksi kehadiran siswa berdasarkan data pada periode sebelumnya selama waktu tertentu. b. Aplikasi Forecasting Kehadiran Siswa berbasis teknologi informasi (web) memberikan manfaat yaitu prediksi ketidakhadiran siswa berdasarkan keterangan sakit (S), izin(I) dan alpha (A) dapat dilakukan dengan cepat dan mudah sehingga Guru BK mampu merancang langkah-langkah secara efektif dalam meningkatkan kedisiplinan siswa Dalam pembuatan aplikasi forecasting kehadiran siswa di SMP 2 Jekulo masih ada banyak hal yang dapat dikembangkan, seperti: Dalam pembuatan aplikasi forecasting kehadiran siswa di SMP 2 Jekulo masih ada banyak hal yang dapat dikembangkan, seperti: a. Fitur dan tampilan yang tersedia masih sangat terbatas dan sederhana harapan kedepannya fitur dan tampilan bisa diperbaharui dengan desain tampilan yang lebih menarik dan fitur yang lebih lengkap dan memadai. Jurnal SIMETRIS, Vol 11 No 2 November 2020 P-ISSN: 2252-4983, E-ISSN: 2549-3108 b. Aplikasi masih terbatas pada rekap data hadir dan tidak hadir dengan keterangan sakit, izin dan alpha, harapannya aplikasi forecasting kehadiran siswa bisa terintegrasi dengan sistem absensi (presensi) disekolah DAFTAR PUSTAKA [1] Suryono dan Hariyanto, (2014). Belajar dan Pengajaran: Teori dan Konsep Dasar. Bandung: PT Remaja Rosdakarya. Sukamto, & Shalahuddin. (2013). Analisa dan Desain Sistem Informasi. Yogyakarta: Andi Offset. (1) gy [2] Zain, Mirrah, Ni Ketut Dewi Ari Jayanti, Yohanes Priyo Atmojo. 2013. “Implementasi Forecasting Pada Perancangan Sistem Pembukaan Kelas di STIKOM Bali dengan Menggunakan Metode Regresi Linear”, Eksplorasi Informatika. Vol. 3. No. 1, pp 17-28. (2) [3] Khotimah, Tutik, Ratih Nindyasari. 2017. “Forecasting Dengan Metode Regresi Linier Pada Sistem Penunjang Keputusan Untuk Memprediksi Jumlah Penjualan Batik (Studi Kasus Kub Sarwo Endah Batik Tulis Lasem)”, Jurnal Mantik Penusa, Vol 1 No 1, pp 72-75 (3) [4] Iik, Ade .2017. “Analisis Penerapan Forecasting Dalam Penentuan Anggaran Penjualan Dan Implikasinya Terhadap Peningkatan Penjualan Perusahaan”. Jurnal Ekonologi, Vol 4. No 1. (4) [5] Amiruddin, Rezqiwati Ishak, 2018. “Prediksi Jumlah Mahasiswa Registrasi Per Semester Menggunakan Linier Regresi Pada Universitas Ichsan Gorontalo “. ILKOM Jurnal Ilmiah. Vol 10. No 2, pp 136-143. (5) [6] Ayuni, Ghebyla Najlai, Devi Fitriana. 2019 “Penerapan Metode Regresi Linear Untuk Prediksi Penjualan Properti pada PT XYZ”. Jurnal Telematika, Vol.14. No. 2, pp 79-85. (6) Imron, Ali Tahun: 1994. Manajemen Peserta Didik Jilid 1. Malang: Proyek OPF IKIP Malang {7 [8] Makridakis. 1999. Metode dan aplikasi peramalan. Edisi 2. Jakarta: Binarupa Aksara. (8) [9] Mulyani, Dr. Sri. (2016). Metode Analisis dan Perancangan. Sistem. Bandung: Abdi SisteMatika. [9] [10] Sukamto, & Shalahuddin. (2013). Analisa dan Desain Sistem Informasi. Yogyakarta: Andi Offset.
https://openalex.org/W2150868856	https://europepmc.org/articles/pmc3127786?pdf=render	English	null	Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane	Lipids in health and disease	2,011	cc-by	12,172	© 2011 Corsetto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane * Correspondence: angelamaria.rizzo@unimi.it Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Italy Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane Paola A Corsetto, Gigliola Montorfano, Stefania Zava, Ilaria E Jovenitti, Andrea Cremona, Bruno Berra and Angela M Rizzo* Abstract Background: PUFAs are important molecules for membrane order and function; they can modify inflammation- inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells. Methods: Two lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane. Results: We have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR. N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa. Conclusions: Our results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Antibodies The mouse monoclonal anti-Bcl2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the C20 goat polyclonal anti-procaspase-8 p18 antibody were used to study the n-3 PUFA induction of the apoptosis process. The 1005 rabbit polyclonal anti-EGFR antibody and the 11C2 mouse monoclonal anti-pEGFR antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to investigate the alterations of EGFR receptors after treat- ment with PUFAs. The monoclonal anti-actin (AC-40) antibody (Sigma-Aldrich, USA) was used to normalize gel loading. Nevertheless the mechanism by which n-3 PUFAs inhibit the growth of breast cancer cells is not well understood, but it has been suggested that these fatty acids might change the fluidity and structure of the cell membrane. In fact, changes in the structural characteris- tics of the plasma membrane in mammalian cells can modify the activity of proteins that function as ion chan- nels, transporters, receptors, signal transducers or enzymes [21-25]. Bound primary antibodies were visualized by second- ary horseradish peroxidase (HRP)-linked antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and immunoreactivity was assessed by chemilumines- cence (ECL, Amersham). In this study, we have investigated the impact of EPA, DHA and AA on breast cancer cell growth, on cell sig- nalling in apoptosis and on epidermal growth factor receptor (EGFR) activity. We hypothesize that the alteration of cellular cycle, of gene expression and the induction of apoptosis determined from n-3 PUFAs are also a consequence of membrane architecture modifica- tions. For these reasons we have analyzed PUFA incor- poration in breast cancer membrane and their PL- specific enrichment. PUFAs EPA (cis-5,8,11,14,17-eicosapentaenoic acid sodium salt), DHA (cis-4,7,10,13,16,19-docosahexaenoic acid sodium salt) and AA (arachidonic acid sodium salt) were pur- chased from Sigma-Aldrich, USA. The PUFAs were dis- solved in ethanol and stored at -80°C under nitrogen gas. Cell viability assay Th b f i The numbers of viable cells exposed to fatty acids were evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) colorimetric assay [26]. Initially, cells were seeded and cultured in 96-well plates for 48 h to allow adhesion to the plate and to reach 50- 60% confluence. After this period, the culture medium was changed to the experimental medium supplemented with EPA or DHA or AA then cultured for 72 h. We stu- died the effects of different concentrations of PUFAs (50-300 μM). The final concentration of ethanol (<1%) in the culture medium had no antiproliferative effect on any cell line tested; therefore, 10 μl of MTT stock solution (5 mg/ml in PBS, pH 7.5) was added to each well and incubated for 4 h as a control. Then 100 μl of solubilizing solution (10% SDS in 0.01M HCl) was added and incu- bated overnight. Plates were read at 540 nm in a plate reader. All reagents were purchased from Sigma-Aldrich, Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 2 of 16 process estradiol via cytoplasmic estrogen receptors. The MDA-MB-231 cells over-express EGFR. cells [10,11]. Similarly, in vitro treatment with EPA is reported to arrest the growth of K-562 human leukemic and many other cancer cells accompanied by down- regulation of cyclin expression in some instances [12-14]. These cell lines were maintained in DMEM (Gibco- BRL, Life Tecnologies Italia srl, Italy) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. In addition, recent studies of human breast cancer have shown that n-3 PUFAs up-regulate syndecan 1 (SDC-1), which has been shown to play a role in cell adhesion [15,16], inhibit matrix metalloproteinases [17] and decrease invasion of tumour cells. SDC-1 induces apoptosis in myeloma cells and some studies suggest a similar property in breast cancer cells [18,19]. The tran- scriptional pathway for the n-3 PUFA regulation of SDC-1 expression involves the nuclear hormone recep- tor peroxisome proliferator-activated receptor gamma (PPARg) [20]. Moreover n-3 PUFAs down-regulate the expression of HER2/neu, a well characterized oncogene that plays a key role in aetiology, progression and che- mosensitivity of various types of human cancer in which this oncogene is over-expressed. HER2/neu encodes transmembrane tyrosine kinase orphan receptor p185Her2/neu, which regulates biological functions includ- ing cellular proliferation, differentiation, motility and apoptosis [21]. Medium for treatments (MFT) was DMEM supple- mented with 10% FBS. Cells were grown at 37°C in a 5% CO2 atmosphere with 98% relative humidity. Background actions of n-3 PUFAs. The most prominent mechanism for the chemopreventive action of n-3 PUFAs is their suppressive effect on the production of arachidonic acid (AA)-derived prostanoids, particularly prostaglandin E2 (PGE2), which has been implicated in the immune response to inflammation, cell proliferation, differentia- tion, apoptosis, angiogenesis and metastasis [6]. Breast cancer is the most common cancer among women worldwide, with an estimated 1.4 million new breast can- cer cases only in 2008. Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and subsequent progression of breast can- cer [1-3]. The role that long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play in the aetiology of cancer has been highlighted by animal experiments and in vitro studies [4,5]. A number of mechanisms have been proposed for the anticancer The n-3 PUFAs might alter the growth of tumour cells by influencing cell replication, by interfering with compo- nents of the cell cycle or by increasing cell death either by way of necrosis or apoptosis [7,8]. For example, these fatty acids are involved in regulating the tumour p53 proapop- totic signal and superoxide dismutase (SOD) levels, telo- mere shorting and tumour angiogenesis [9]. In vitro treatment with DHA arrested cell-cycle progression in human-derived breast cancer and malignant melanoma * Correspondence: angelamaria.rizzo@unimi.it Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Italy Cell treatment Cell culture experiments were done with the MDA-MB- 231 and MCF-7 cell lines to determine the concentra- tions of EPA (230 μM), DHA (200 μM), required to inhibit growth by 20-30%, and AA (200 μM). Cells were seeded at 1.5 × 104 cells/cm2 for MDA-MB-231 and at 3 × 104 cells/cm2 for MCF-7 in 18 ml of medium con- taining 10% FBS and allowed to adhere for 48 h, then the medium was replaced with 18 ml of fresh medium (DMEM, 10% FBS) containing the experimental fatty acids and incubated for 72 h without changing the med- ium. Experiments included untreated cells that were not exposed to any exogenous fatty acids but to an equal content of ethanol during the incubation to serve as controls. After 72 h, cells were harvested using trypsin- EDTA and centrifuged at 900 rpm for 10 min. The supernatant was removed and the pellets were subjected to lipid analysis. After elution, the eluate was split with one part going to the detector and nine parts to a Gilson fraction col- lector model 201 to collect the different PL classes for GC analysis. Total fatty acids and PL fatty acids were determined as methylesters by gas chromatography (GC). The methyl esters were obtained by reaction with 3.33% (w/v) sodium methoxide in methanol and injected into an Agilent Technologies (6850 series II) gas chromatograph equipped with a flame ionization detector (FID) and a capillary column (AT Silar) (length 30 m, film thickness 0.25 μm). The carrier gas was helium, the injector tem- perature was 250°C, the detector temperature was 275°C, the oven temperature was set at 50°C for 20 min and then increased to 200°C at 10°C min-1 for 20 min. Cell lines and culture conditions Human breast cancer cell lines MDA-MB-231 (ER-nega- tive) and MCF-7 (ER-positive) were kindly provided by Dr P. Degan from the IST (Italian National Cancer Research Institute, Genoa Italy, Laboratory of Molecular Mutagenesis and DNA Repair). Both cell lines are derived from human mammary adenocarcinoma; the MCF7 line retains several characteristics of differen- tiated mammary epithelium, including the ability to Page 3 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 USA. Data points represent the mean of eight wells and the results are expressed as relative growth rate (RGR) in comparison to controls that were exposed to a concen- tration of ethanol equal to that in the samples exposed to fatty acids. USA. Data points represent the mean of eight wells and the results are expressed as relative growth rate (RGR) in comparison to controls that were exposed to a concen- tration of ethanol equal to that in the samples exposed to fatty acids. time was 40 min/sample; 23 min analysis, 12 min to restore initial conditions and 5 min for re-equilibration. Eluent A was chloroform/methanol/water (80:19.5:0.5, by vol.) and eluent B was chloroform/methanol/water (60:34:6, by vol.) and the flow rate of the eluent was 1.0 ml/min. An evaporative light-scattering detector (ELSD) was used to detect and quantify the separated PL species. Western blot analysis for Bcl2, caspase-8, EGFR and pEGFR For western blot analysis, cells were harvested by scrap- ing in phosphate-buffered saline containing 0.4 mM Na3VO4. Cells were centrifuged and then suspended in 1.4 ml of lysis buffer (1% Triton X-100, 10 mM Tris buf- fer, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 75 mU/ml aproti- nin), kept on ice for 20 min and then disrupted by 10 strokes in a tight-fitting Dounce homogenizer. The cell lysate was centrifuged (5 min at 1300 g) and the supernatant was transferred to an eppendorf tube. Total protein was determined by the Lowry assay [27]. Control and treated cell lysates (100 μg protein/lane) were separated by SDS-PAGE (10% polyacrylamide gel), transferred to a polyvinylidene difluoride (PVDF) mem- brane and analysed by western blot with anti-Bcl2 (1:100), anti-caspase-8 (1:500) and b-actin (1:1800) anti- bodies. The PVDF membrane was blocked for 1 h in blocking buffer 5% (w/v) dried non-fat milk in Tris-buf- fered saline (T-TBS: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween®20) followed by incubation with an appropriate primary antibody in blocking buffer at room temperature for 2 h. The blots were washed with T-TBS and then incubated with the proper second- ary antibody in blocking buffer at room temperature for 1 h. The protein bands were visualized using ECL wes- tern blot detection reagents (PerkinElmer, USA). DHA and EPA induce apoptosis in breast cancer cells DHA and EPA induce apoptosis in breast cancer cells In order to delineate the possible mechanism(s) by which EPA and DHA induce apoptosis we examined the cytoplasmic levels of the Bcl2 protein. Figure 2A and 2B indicate that there was a slight reduction of Bcl2 level in MCF7 cells after treatment with 200 μM DHA, whereas treatment with 230 μM EPA determined the loss of sig- nal; the expression of Bcl2 is also decreased when MDA-MB-231 cells are treated with 230 μM EPA and the protein is not detectable after incubation with 200 μM DHA. The expression of procaspase-8 was determined by western blot analysis. In Figure 3A and 3B it is possible to observe a reduction of the proform of caspase-8 for both cell lines treated with EPA and DHA; the reduc- tion was statistically significant after DHA treatment in both cell lines, and also after EPA treatment of MCF7 cells. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 blots were probed under the same conditions with primary antibody b-actin diluted 1:1800 to confirm equal protein loading. blots were probed under the same conditions with primary antibody b-actin diluted 1:1800 to confirm equal protein loading. 0 50 100 150 50 100 150 200 220 240 260 280 300 EPA (M) RGR (%) MDA MCF7 ** * 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA (M) RGR (%) * ** 0 50 100 150 50 100 150 200 250 300 AA (M) RGR (%) ** A B C Figure 1 Effects of PUFA on viability of breast cancer cells. The effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). * p < 0.05; p < 0.01 compared to control cells. 0 50 100 150 50 100 150 200 220 240 260 280 300 EPA (M) RGR (%) MDA MCF7 * A The relative intensities of band signals were deter- mined by digital scanning densitometry and b-Actin was used to normalize the results to protein content. Statistical analysis The data are presented as mean ± SD. Student’s unpaired t-test was used for comparisons between trea- ted and control cells and the level of statistical signifi- cance was set at P < 0.05 and P < 0.01. 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA (M) RGR (%) * ** ** B B Lipid composition analysis Cell lipids were extracted with three different chloro- form/methanol mixtures (1:1, 1:2 and 2:1, v/v) and parti- tioned with a theoretical upper phase (chloroform/ methanol/water, 47:48:1, by vol.) and then with water. The organic phase was dried and then suspended in chloroform/methanol (2:1, v/v) for the analysis of total and PL fatty acids. For the analysis of EGFR and p-EGFR, cells treated or not with DHA and EPA were cultured in MFTs supple- mented with 10 nM EGF (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C for 15 min of stimulation. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed as described above. Equal amounts of protein (100 μg/lane) from each treatment were sepa- rated by SDS-PAGE (10% polyacrylamide gel) and trans- ferred onto a PVDF membrane then blocked in blocking buffer at room temperature for 1 h. Primary antibodies to EGFR and p-EGFR were diluted 1:200 in blocking buffer at room temperature for 2 h and then with an appropriate secondary antibody at room temperature for 1 h. Parallel Purification of single PL moieties was achieved with an HPLC-ELSD system (Jasco, Japan) equipped with one pump, a SCL-10 Advp, a degasser module and a Rheo- dyne manual injector with 20 μl sample loop and a col- umn (length 250 mm, I.D 4.6 mm and film thickness 5 μm) packed with silica normal-phase LiChrospher Si 60 (LiChroCART 250-4, Merck, Darmstadt, Germany). The chromatographic separation was achieved with a linear binary gradient of 0% B to 100% B in 14 min and then 100% B for 9 min. The total chromatographic run Page 4 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 The effects of treatment with PUFAs on breast cancer cell growth * p < 0.05; p < 0.01 compared to control cells. From these experiments, we extrapolated the dose to be used in successive experiments to assess n-3 PUFA incorporation into cell membrane PLs: 230 μM for EPA, 200 μM for DHA, which correspond to 70~80% viability for both cell lines, and 200 μM for AA. The effects of treatment with PUFAs on breast cancer cell growth To evaluate the effects of PUFAs on breast cancer pro- liferation, cells were incubated for 3 days in medium supplemented with n-3 and n-6 PUFAs (EPA, DHA and AA). 0 50 100 150 50 100 150 200 250 300 AA (M) RGR (%) ** C C The effect on cell viability of MDA-MB-231 and MCF7 cells was assessed and quantified by the MTT assay. As shown in Figure 1, cells were treated with var- ious concentrations of n-3 and n-6 PUFAs in the range 50-300 μM. DHA and EPA induce a dose-dependent reduction of cell viability at concentrations > 200 μM (Figure 1A and 1B). Figure 1 Effects of PUFA on viability of breast cancer cells. The Figure 1 Effects of PUFA on viability of breast cancer cells. The effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). * p < 0.05; p < 0.01 compared to control cells. In contrast, AA (Figure 1C), the major n-6 PUFA, had no significant effect on MCF7 cell viability. The MCF7 cell line was more resistant than the MDA-MB-231 cell line to all treatments with PUFAs. effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). EPA and DHA alter the EGFR and pEGFR levels in MDA- MB-231 cells p < 0.01 compared to control cells; n = 3 Bcl2 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT A MCF7 Bcl2 ȕ-actin CTR EPA CTR DHA CTR EPA CTR DHA Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT ProCASPASE 8 A MCF7 -actin CTR EPA CTR DHA B MDA-MB-231 CTR EPA CTR DHA Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT B MDA-MB-231 -actin ProCASPASE 8 Figure 3 Effects of n 3 PUFAs on caspase 8 expression The CTR EPA CTR DHA B MDA MB 231 -actin ProCASPASE 8 Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT B MDA-MB-231 CTR EPA CTR DHA Bcl2 ȕ-actin Figure 2 Effects of n-3 PUFA on Bcl2 expression. Both cell lines B MDA-MB-231 CTR EPA CTR DHA Bcl2 ȕ-actin B Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment with DHA (200 μM) and EPA (230 μM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. B: MDA-MB-231 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. Semi-quantitative analysis performed by plate scanning. b-actin was used to normalize results of protein content. p < 0.01 compared to control cells; n = 3 Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment with DHA (200 μM) and EPA (230 μM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. B: MDA-MB-231 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. Semi-quantitative analysis performed by plate scanning. b-actin was used to normalize results of protein content. p < 0.01 compared to control cells; n = 3 Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase 8 after treatment Figure 2 Effects of n-3 PUFA on Bcl2 expression. EPA and DHA alter the EGFR and pEGFR levels in MDA- MB-231 cells Both cell lines were treated with DHA (200 μM) or EPA (230 μM) for 72 h. Control and treated cell lysates are separated on 10% SDS-PAGE and transferred to PVDF membrane. The expression of the anti-apoptotic protein Bcl2 was assessed by western blot and semi-quantitative analysis performed by plate scanning. b actin was used to normalize results of protein content. A: MCF7 cells, B MDA-MB-231 cells. of EGF. EPA did not modify EGFR expression in breast cancer cells; while EGF stimulation significantly increase EGFR phosphorylation to about 140%; co-treatment with EPA/EGF significantly inhibit EGFR activation down to about 40% compared to control non stimulated cells (Figure 4A). As shown in Figure 4B, DHA significantly reduces the EGFR level (70%) compared to control cells and com- pletely inhibit EGFR activation in cells treated with DHA or DHA/EGF. mitogenic signals to downstream target signalling cas- cades that involve cell survival and proliferation, such as phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-acti- vated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) [29]. In human breast cancer cell lines, such as MDA-MB- 231, the EGFR level is elevated compared with that in other breast cancer cells, such as MCF7 [30]; for this reason, we have studied the effects of DHA and EPA on EGFR activity mainly in MDA-MB-231 cells. EPA and DHA alter the EGFR and pEGFR levels in MDA- MB-231 cells Furthermore, apoptosis involves the activation of pro- caspase-8 (55 kDa) by its cleavage to caspase-8 (18 kDa); this smaller protein together with caspase-3 med- iates the rapid dismantling of cellular organelles and architecture [28]. EGFR is usually activated in response to extracellular ligands (EGF) by its phosphorylation; ligand binding leads to homo- or heterodimerization with another ligand-bound ErbB receptor, and transmits extracellular Page 5 of 16 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Bcl2 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT A MCF7 Bcl2 ȕ-actin B MDA-MB-231 CTR EPA CTR DHA Bcl2 ȕ-actin CTR EPA CTR DHA Figure 2 Effects of n-3 PUFA on Bcl2 expression. Both cell lines were treated with DHA (200 μM) or EPA (230 μM) for 72 h. Control and treated cell lysates are separated on 10% SDS-PAGE and transferred to PVDF membrane. The expression of the anti-apoptotic protein Bcl2 was assessed by western blot and semi-quantitative analysis performed by plate scanning. b actin was used to normalize results of protein content. A: MCF7 cells, B MDA-MB-231 cells. CTR EPA CTR DHA Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT CTR EPA CTR DHA Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT B MDA-MB-231 -actin ProCASPASE 8 ProCASPASE 8 A MCF7 -actin Figure 3 Effects of n-3 PUFAs on caspase-8 expression. The determination of the integrity of the procaspase-8 after treatment with DHA (200 μM) and EPA (230 μM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. B: MDA-MB-231 cells treated with DHA (200 μM) or EPA (230 μM) for 72 h. Semi-quantitative analysis performed by plate scanning. b-actin was used to normalize results of protein content. Total fatty acid profile after treatment with PUFA y p Treatment with AA, EPA or DHA alters the FA profile in MDA-MB-231 and MCF7 cells compared with con- trol cells (Table 1). Treatment of both cell lines with AA resulted in a sig- nificant increase of AA content in total cell lipids, from 14.40% to 46.85% in MDA-MB-231 and from 12.73% to Figure 4 reports the effects of EPA and DHA treat- ments on expression and activation of EGFR in presence Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 6 of 16 EGFR 0 50 100 CTR EPA EGF EGF/EPA EGFR 0 50 100 CTR DHA EGF EGF/DHA p-EGFR 0 50 100 150 CTR EPA EGF EGF/EPA * ** p-EGFR 0 50 100 150 CTR DHA EGF EGF/DHA * EGF CTR DHA DHA/EGF B pEGFR EGFR -actin CTR EGF EPA/EGF EPA pEGFR EGFR -actin A Figure 4 EGFR expression and EGF stimulation (p-EGFR) in MDA-MB-231 breast cancer cells after n-3 PUFAs treatment. A: MDA-MB-231 treated with 230 μM EPA for 72 h, lane 1 control, lane 2 230 μM EPA, lane 3 EGF, lane 4 EGF+EPA. B: MDA-MB-231 treated with 200 μM DHA for 72 h, lane 1 control, lane 2 200 μM DHA, lane 3 EGF, lane 4 EGF+DHA. The relative intensities of band signals, reported in graphics, are determined by digital scanning densitometry. b-actin was used to normalize results of protein content. * p < 0.05; ** p < 0.01 compared to control cells; n = 3. A B p-EGFR 0 50 100 150 CTR DHA EGF EGF/DHA * p-EGFR 0 50 100 150 CTR EPA EGF EGF/EPA * ** Figure 4 EGFR expression and EGF stimulation (p-EGFR) in MDA-MB-231 breast cancer cells after n-3 PUFAs treatment. A: MDA-MB-231 treated with 230 μM EPA for 72 h, lane 1 control, lane 2 230 μM EPA, lane 3 EGF, lane 4 EGF+EPA. B: MDA-MB-231 treated with 200 μM DHA for 72 h, lane 1 control, lane 2 200 μM DHA, lane 3 EGF, lane 4 EGF+DHA. The relative intensities of band signals, reported in graphics, are determined by digital scanning densitometry. b-actin was used to normalize results of protein content. * p < 0.05; p < 0.01 compared to control cells; n = 3. Total fatty acid profile after treatment with PUFA Table 1 Total fatty acid composition of PUFA-treated breast cancer cells MDA-MB-231 MCF-7 CTR AA EPA DHA CTR AA EPA DHA C:16:0 13.91 ± 2.19 13.48 ± 2.91 6.57 ± 0.91 8.66 ± 1.25 16.03 ± 4.06 15.52 ± 1.56 14.7 ± 1.76 11.14 ± 3.59 C 16:1 1.61 ± 0.85 1.19 ± 0.87 0.87 ± 0.58** 0.77 ± 0.42* 8.25 ± 3.27 2.97 ± 0.86 3.95 ± 0.54 2.94 ± 1.53** C 18:0 17.57 ± 1.97 10.46 ± 2.05* 5.15 ± 1.03 6.39 ± 1.30 14.83 ± 1.58 15.73 ± 1.76 10.41 ± 2.49 9.46 ± 0.89 C 18:1 26.14 ± 4.74 15.91 ± 4.21* 8.15 ± 0.95 9.65 ± 1.29 30.14 ± 2.91 12.12 ± 1.19 12.21 ± 1.94 9.77 ± 1.12 C 18:2 6.05 ± 2.38 3.07 ± 0.65 2.38 ± 0.17 2.46 ± 0.32 4.23 ± 0.73 2.48 ± 1.27 2.26 ± 0.42 1.66 ± 0.21 C 18:3 1.54 ± 4.10 0.68 ± 0.37 0.22 ± 0.16 0.32 ± 0.10 1.32 ± 0.87 0.96 ± 0.53* 0.59 ± 0.36* 0.63 ± 0.61* C 20:3 1.64 ± 0.21 1.60 ± 0.10 0.72 ± 0.37** 1.18 ± 0.70* 2.41 ± 0.67 1.28 ± 0.11 0.67 ± 0.09 0.78 ± 0.16** C 20:4 (AA) 14.40 ± 2.92 46.85 ± 10.48* 2.56 ± 0.87 3.91 ± 0.50 12.73 ± 2.90 44.26 ± 3.80 3.13 ± 0.57 3.20 ± 0.55 C 20:5 (EPA) 2.42 ± 0.70 0.70 ± 0.23 38.75 ± 3.52 1.98 ± 0.43 3.22 ± 0.98 0.74 ± 0.75 34.16 ± 3.89 4.90 ± 0.51 C 22:5 (DPA) 7.04 ± 1.10 3.61 ± 0.44 33.30 ± 1.83 2.94 ± 0.62 1.10 ± 0.71 1.29 ± 0.45 15.80 ± 3.32 0.96 ± 0.14 C 22:6 (DHA) 7.68 ± 1.55 2.45 ± 0.60 1.29 ± 0.35 61.76 ± 3.93 5.73 ± 2.36 2.65 ± 0.68 2.12 ± 0.31 54.55 ± 6.02 SFA 33.35 ± 5.50 23.94 ± 4.88* 11.76 ± 1.90 15.05 ± 2.52 30.86 ± 3.21 31.24 ± 2.89 25.11 ± 3.81 20.60 ± 4.18 MUFA 27.74 ± 4.69 17.10 ± 4.61* 9.01 ± 0.82 10.41 ± 1.06 38.39 ± 4.36 15.09 ± 1.62 16.16 ± 2.02 12.71 ± 2.27 n-3 PUFA 18.68 ± 3.93 7.44 ± 0.56 73.56 ± 3.16 67.00 ± 3.45 11.37 ± 3.71 5.63 ± 1.58 52.67 ± 6.24 61.05 ± 5.96 n-6 PUFA 22.09 ± 3.15 51.52 ± 9.81 5.67 ± 1.37 7.55 ± 0.70 19.38 ± 3.58 48.03 ± 4.00 6.06 ± 0.76 5.64 ± 0.59** Breast cancer cells were treated with solvent (ethanol) as control, AA (20:4, n-6), EPA (20:5, n-3), and DHA(22:6, n-3). Discussion After treatment with AA, the content of this n-6 PUFA was increased significantly in PE and PC in MDA-MB-231 (Figure 5, Table 2). The treatment induced a reduction of omega-3 PUFAs, particularly EPA, that was significantly decreased in all phospholi- pids but SM; while DHA content was decreased after AA treatment only in PE and PC, the other two omega- 3 fatty acids namely DPA and ALA (C18:3) were decreased in all phospholipids but SM. Breast cancer is the leading cause of the death among women in the world. The principal effective endocrine therapy for treatment on this type of cancer is anti- estrogens, but therapeutic choices are limited for estro- gen receptor (ER) negative tumor, which are more aggressive. Moreover the development of ER positive cancer cells that are resistant to chemotherapeutic agents is a major factor responsible to the successful treatment of breast cancer. This is a strong input to dis- cover new approaches in vitro. Incubation with EPA caused an increase of EPA con- tent in all PLs in MDA-MB-231 cells. In particular, the incorporation of EPA was different in relation to the PL moiety with highest levels of incorporation in PI and PC. There was a decrease of monounsaturated FA in PE and PC, and a significant increase of polyunsaturated FA in PI and PC. Furthermore, an increase of DPA con- tent was found in all PLs, especially PC. The content of AA was significantly decreased in all phospholipids but SM. Several epidemiologic and clinical studies have shown that n-3 PUFAs are able to provide beneficial effects in a wide variety of pathologies ranging from autoimmune and inflammatory diseases to neurological and psychia- tric disorders and, in particular, to several types of malignancy, including ovarian, pancreatic, prostate, renal, colorectal and breast cancer [31-33]. This study was prompted by the observation that MDA-MB-231 and MCF7 breast cancer cell lines showed a significant reduction in cell number following treat- ment with n-3 PUFAs. The same conclusion is not possi- ble for the AA incubation. We hypothesize that this reduction in cell number results from both proliferation reduction and induction of apoptosis. Apoptosis is a genetically controlled form of cell death that is conserved from worms to humans. Deregulation of apoptosis is a hallmark of all cancer cells and the agents that activate apoptosis in cancer cells could be considered as anti- cancer therapeutics [34]. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 7 of 16 Page 7 of 16 PC. The concentration of DHA was significantly decreased in PE and PI, whereas the AA content was significantly reduced in PE, and PI. 44.26% in MCF7. Furthermore, the data for MDA-MB- 231 show a significant decrease of EPA, docosapentae- noic acid (DPA) and DHA, whereas the data for MCF7 cells show a significant reduction of only EPA and DHA. The exposure of MCF7 cells to DHA determined a significant increase of DHA in all PLs, but not in SM, and of EPA content in PE, PI, and PC, whereas the con- tent of AA was significantly reduced only in PE, PI and PS. When both cell lines were treated with EPA, the con- tent of this FA in total cell lipids was increased signifi- cantly and we observed a significant reduction of AA. Unexpectedly, we found an increase of DPA content, indicating that EPA is incorporated into cells and is further metabolized by elongation. Also in these cells a significant decrease of monounsa- turated fatty acids is always present when the cells are treated with n-6 and n-3 PUFAs; while saturated fatty acids are in most cases constant. The treatment with DHA determines a significant increase of DHA content in both cell lines and an increase of EPA content in MCF7, probably due to a retro conversion; a significant reduction of AA content was also measured. Moreover also 18:2, 18:3 (n-3) and 20:3 (n-6) are sig- nificantly decrease after PUFA treatment. As far as phospholipids content concerns, it is worth noting the significant decrease of SM content (from 11.32% to 9.02%, data not shown) in MCF7 after treat- ment with DHA even if, sphingomyelin is the phospholi- pid less influenced in its fatty acid composition by PUFA treatment. The other treatments did not modify the distribution of PL in both cell lines. Effects of treatment with PUFAs on PL composition in breast cancer cells Table 2 and 3 give the fatty acid composition of specific PLs in MDA-MB-231 and MCF7 cells treated with n-3 or n-6 PUFAs; to simplify the tables SD, are reported as plain numbers above the bold mean value. Total fatty acid profile after treatment with PUFA Fatty acid composition was analyzed and expressed as percentage of total fatty acids (mean ± SD). Asterisks indicate the significant differences between treated- and control cells (n=10, P < 0.05; P < 0.01). Table 1 Total fatty acid composition of PUFA-treated breast cancer cells MDA-MB-231 Discussion In some mammalian cells, apoptosis can be triggered by members of the Fas/TNF receptor family. When activated by receptor aggregation, After treatment of MDA-MB-231 cells with DHA, the content of this fatty acid was significantly increased in all cell membrane PLs, but not in SM. We measured a significant decrease of the content of EPA in PE, PS, PI and PC. The concentration of AA was significantly reduced in PE and PI and SM as the result of treatment with DHA. In MCF7 cells (Figure 6, Table 3), the treatment with AA induced a significant increase of this fatty acid in all PLs, except in SM; a significant reduction of EPA and of DHA in PE, PI, PS and PC was also measured. Discussion PE MDA 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * SM MDA 0 2 4 6 8 10 12 14 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * Figure 5 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 cells treated with LCPUFAs. MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Discussion After treatment with EPA, the EPA and DPA content was significantly increased, especially in PE, PI, PS and MDA MB 231 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/Omega-3 AA/EPA AA/DHA 3.65 6.14 7.85 24.71 18.90 53.34 35.08 18.26 1.95 8.96 3.93 0.90 1.01 1.06 3.16 2.80 4.03 3.42 2.01 0.30 2.99 0.79 0.66* 3.75* 3.09* 28.76* 16.93* 51.08* 43.39* 7.70* 5.69* 81.49* 13.38* 0.49 0.74 0.45 1.73 0.52 0.89 1.16 0.80 0.61 37.02 1.79 22.52* 18.86* 1.62* 26.09 14.23* 53.96 10.63* 43.34* 0.25* 0.36* 5.10 1.33 2.80 0.26 5.28 1.07 3.47 1.13 3.78 0.04 0.07 1.14 2.10* 2.26* 42.31* 26.31 10.57* 56.55 13.46* 47.24* 0.26* 6.41 0.25* 0.71 0.45 3.04 5.38 1.95 7.81 3.21 2.96 0.01 3.60 0.02 1.44 4.65 3.98 40.05 14.35 41.67 30.34 11.33 3.23 22.84 8.98 0.65 1.12 1.74 5.37 5.32 5.09 6.33 3.34 1.68 11.79 9.39 0.40* 2.16* 2.94 41.95 15.26 37.99 32.11 5.87* 5.51* 75.95* 10.23 0.04 0.15 0.36 3.09 2.39 2.53 2.70 0.40 0.69 15.180 1.75 14.30* 16.40* 1.32* 30.55* 15.93 48.87* 15.02* 33.85* 0.45* 0.84* 10.35 5.28 4.92 0.74 2.27 6.31 6.89 2.60 5.10 0.07 0.15 6.91 0.96* 2.55* 20.43* 42.96 13.89 39.71 16.48* 22.57* 0.81* 10.77* 0.53* 0.24 0.36 2.58 8.07 4.59 6.02 6.13 4.12 0.48 3.14 0.15 2.50 5.74 7.52 32.40 24.94 38.00 20.12 16.73 1.25 6.03 1.90 1.20 1.69 3.71 9.19 7.87 11.64 10.49 4.72 0.68 3.59 1.64 0.41* 1.82* 6.07 34.38 29.80 26.37* 17.81 8.56* 2.12* 47.07* 2.86 0.23 0.63 1.75 3.20 3.57 4.01 4.27 0.98 0.70 29.02 1.92 16.53* 23.17* 2.48* 27.92 19.97 48.44 10.06* 38.38* 0.29* 0.48* 2.18 0.34 7.45 0.59 13.86 4.06 13.24 4.27 12.21 0.14 0.27 0.83 1.23* 2.41* 30.84* 36.77 12.26* 45.48 10.63 34.49* 0.32* 9.65 0.33* 0.40 0.83 8.78 17.15 3.15 11.23 3.27 8.40 0.09 3.81 0.05 1.20 1.88 2.29 37.73 38.64 22.62 16.04 6.58 2.50 7.50 3.86 0.44 0.64 0.72 3.25 3.55 2.82 2.26 1.15 0.47 3.56 1.43 0.10* 1.45* 1.33* 38.50 24.87* 34.80* 31.29* 3.51* 8.89* 277.85* 18.21* 0.01 0.23 0.28 1.43 2.03 3.27 3.13 0.15 0.56 34.12 2.17 15.71* 14.26* 1.36 34.14 22.36* 40.96* 8.77* 32.17* 0.28* 0.24* 3.42 2.09 5.43 0.93 4.74 1.92 6.92 0.36 6.75 0.05 0.04 1.29 1.78* 1.80 23.90* 37.41 20.77* 39.77* 11.80* 27.96* 0.43* 4.12* 0.33* 0.45 0.66 4.15 4.53 1.62 3.56 0.42 3.75 0.08 0.54 0.09 ttp://www.lipidworld.com/content/10/1/73 p p 1.34 11.07 1.73 0.45 7.33 0.83 1.76* 30.33* 1.86 0.23 10.50 0.26 0.90 1.78* 3.47 0.45 0.83 2.00 0.79* 5.76* 0.70* 0.37 3.21 0.37 http://www.lipidworld.com/content/10/1/73 p MCF7 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/Omega-3 AA/EPA AA/DHA 5.28 0.72 5.71 29.77 27.73 42.50 30.09 12.41 2.47 4.75 4.58 0.69 0.56 1.37 1.86 3.83 3.88 2.66 1.96 0.37 0.50 1.04 0.70* 1.03 2.56* 41.68* 9.57* 48.75* 44.19* 4.57* 9.97* 76.64* 17.85* 0.47 0.39 0.81 1.44 1.57 2.19 1.94 0.86 1.80 30.68 5.10 33.91* 7.68* 1.07* 36.87* 10.77* 52.36* 9.40* 42.96* 0.23* 0.22* 5.42 4.88 2.26 0.50 1.58 4.00 5.13 1.76 6.77 0.08 0.06 0.45 9.45* 0.52 36.92* 34.65* 8.33* 57.02* 9.73* 47.30* 0.21* 0.71* 0.19* 2.19 0.33 7.43 4.38 2.66 6.35 1.95 6.27 0.05 0.13 0.08 2.22 1.64 8.99 42.79 20.65 36.56 23.02 13.54 1.80 8.04 1.84 1.65 1.00 3.07 5.35 7.91 7.50 4.66 3.77 0.47 4.32 1.20 0.96* 1.52 2.83* 43.44 7.83* 48.73* 43.12* 5.61* 8.34* 69.09* 23.36* 0.80 1.41 1.77 2.35 4.03 2.81 3.70 1.65 2.63 49.53 17.92 24.41* 8.60* 2.13* 41.84 8.51* 49.65* 14.07* 35.57* 0.40* 0.44* 4.76 4.29 1.69 2.18 1.73 2.04 2.96 1.61 3.59 0.08 0.10 3.02 7.71* 0.87* 26.82* 33.66* 14.77* 51.57* 15.26* 36.31* 0.43* 1.16* 0.31* 3.67 0.76 6.04 6.91 5.63 7.46 5.64 5.29 0.19 0.48 0.17 3.18 2.19 6.14 35.75 31.45 32.81 20.43 12.37 1.83 5.17 2.49 1.91 2.00 3.36 8.35 8.32 8.96 7.21 4.27 0.84 4.46 1.57 1.21* 1.69 3.18* 41.78* 14.89* 43.33* 36.80* 6.53* 6.49* 38.97* 12.22* 0.63 2.12 1.47 5.84 7.35 9.32 10.79 2.15 3.45 31.62 6.48 33.04* 8.11* 3.05 36.93 8.37* 54.70* 10.25* 44.45* 0.22* 0.22* 4.62 12.94 3.14 3.04 11.06 3.52 14.23 3.84 10.79 0.06 0.06 4.12 5.00 1.55 31.06* 34.56 15.35* 50.10* 11.82* 38.28* 0.32* 1.85* 0.22* 2.94 0.59 2.17 6.22 3.88 4.33 5.70 4.53 0.18 1.50 0.12 1.18 0.59 2.23 34.93 49.35 15.73 10.89 4.83 2.61 5.70 3.91 0.62 0.92 2.01 3.83 4.82 5.01 2.61 2.95 0.85 3.65 3.51 0.19* 0.29 1.21* 39.16* 21.65* 39.19* 37.11* 2.08* 18.12* 222.12* 30.38* 0.10 0.07 0.33 0.63 1.61 1.91 1.61 0.33 2.44 100.56 8.64 20.12* 1.75 1.07 41.95* 27.66* 35.69* 6.92* 23.48* 0.34* 0.21* 4.37 8.27 1.39 0.63 0.91 6.34 0.53 1.68 9.30 0.17 0.10 2.54 10.01* 0.39 21.34* 39.03* 18.62* 42.35* 9.93 32.42* 0.32* 0.51* 0.26* 1.36 0.20 5.63 4.11 5.19 6.44 1.47 6.01 0.07 0.06 0.08 1.60 4.45 6.27 1.01 4.12 7.52 1.50 6.56 3.41 0.48 5.17 1.33 0.59* 1.49* 0.74* 0.22 0.52 0.37 0.94 4.00 1.05* 0.41 2.60 0.47 http://www.lipidworld.com/content/10/1/73 p p Corsetto et al. Discussion Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 12 of 16 PE MDA 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * * PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * SM MDA 0 2 4 6 8 10 12 14 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * Figure 5 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 LCPUFAs. MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an H PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Discussion PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * Figure 6 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated w cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fat determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are exogenous fatty acids. * p < 0.01; n = 3. Discussion PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * SM MDA 0 2 4 6 8 10 12 14 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * Figure 5 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 cells treated with LCPUFAs. MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Discussion Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 13 of 16 PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * Figure 6 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated with LCPUFAs. MCF7 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 14 of 16 Fas and TNFR1 induce the activation of a set of cysteine proteases called caspases. Studies designed to elucidate the mechanism(s) by which Fas and TNFR1 stimulation lead to caspase activation are underway. In the case of Fas, receptor aggregation by the Fas ligand induces the formation of a death-inducing signalling complex (DISC) of proteins comprising Fas itself, the adaptor protein FADD and the inactive zymogen form of caspase-8. Shortly after formation of the DISC, procaspase-8 is cleaved and the active protease is released. Once acti- vated, caspase-8 is thought to activate other downstream caspases by proteolytic cleavage of their zymogen forms, thus amplifying the caspase signal [35]. Our results demonstrate the activation of caspase-8 in response to incubation with n-3 PUFAs by a reduction of the levels of its zymogen form in both cell lines. In many cells, over-expression of either Bcl2 or Bcl-xl inhibits apopto- sis, affecting the release of cyt-c and apoptosis-inducing factor (AIF) from the mitochondrial intramembrane space to the cytosol. Once released, AIF is translocated to the nucleus where it is capable of inducing nuclear chromatin condensation and large-scale DNA fragmenta- tion that mediates a caspase-independent mitochondrial apoptotic pathway [36]. Cyt-c, together with dATP, binds to apoptotic proteinase activating factor-1 (Apaf-1) and this complex promotes procaspase-9 autoactivation. The active forms of caspase-8 and caspase-9 might activate the downstream effectors caspase-3, -6 and -7, resulting in the cleavage of crucial cellular proteins and apoptosis. We have observed a significant difference in the amount of Bcl2 present in the DHA-treated MDA-MB-231 cells and EPA-treated MCF7 cells compared to the control group. The absence of Bcl2 when compared to the con- trol is suggestive that the cell might be more likely to proceed to apoptosis. The entire mechanism by which n-3 PUFAs exert their beneficial effects is not fully understood. We have hypothesized that the induction of apoptosis, the reduc- tion of cell proliferation and the inhibition of EGFR activity by these fatty acids might be the consequences of cell membrane alterations induced by FA. Our data indicate that EPA and DHA are incorporated in breast cancer membrane. In particular the EPA treatment determines an increase of EPA and DPA content, and a reduction of SFA, MUFA and n-6 PUFA concentration in both cell lines. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 This suggests an incorporation of EPA which is further metabolised. In fact, EPA is converted to 22:5 n-3 (DPA) by elongase (Elovl)-5 and then by Elovl-2 to 24:5, n-3. The next step requires desaturation of 24:5 by Δ6 desaturase to produce 24:6, n-3. This pro- duct is translocated from the endoplasmic reticulum to the peroxisome, where the b oxidation pathway involves acyl chain shortening of C2 to produce DHA [40]. y g p Also DHA incubation determines an increase of EPA, of DHA, and in general of the unsaturation degree in both cell lines. We have also observed that PUFAs are incorpo- rated into the breast cancer membrane with different spe- cificity for each PL moiety. The enrichment is significant, especially in PE, PI and PC. The transbilayer distribution of lipids across biological membranes is asymmetric. The choline-containing lipids PC and SM are enriched primar- ily on the external leaflet of the plasma membrane or the topologically equivalent luminal leaflet of internal organelles. In contrast, the amine-containing glyceropho- spholipids PE and PS are located preferentially on the cytoplasmic leaflet. Other minor PLs, such as phosphatidic acid (PA), PI and phosphatidylinositol-4,5-bisphosphate (PIP2) are also enriched on the cytofacial side of the mem- brane. Specific alterations of the molecular composition of the plasma membrane occur during apoptosis. Hence, cells undergoing apoptosis express signals, including lipids, proteins and modified sugar moieties that facilitate recog- nition and ingestion by macrophages. Loss of transmem- brane PL asymmetry, with consequent exposure of PS in the external monolayer, occurs in both normal and patho- logic conditions. PS externalization is induced early in the process of apoptosis. On the basis of our findings, the data suggest that the incorporation of n-3 PUFAs is mainly into cytofacial leaflet PLs, altering the membrane environ- ment to impact on the activation of cell signalling. More- over, a significant decrease of SM was evident in cells treated with DHA. EGFR is an interesting target for tumour therapy, because it is over-expressed in many human tumours such as lung and breast cancers [37]. MDA-MB-231 cells express high levels of EGFR and are a good model to study EGFR modulation by n-3 PUFAs. This receptor is a member of the ErbB receptor tyrosine kinase family, which consists of EGFR (or HER1 or ErbB1), HER2/ ErbB2, HER3/ErbB3 and HER4/ErbB. Discussion PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * Figure 6 Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated with LCPUFAs. MCF7 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p < 0.01; n = 3. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 References 1. Bartsch H, Nair J, Owen RW: Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20:2209-18. 1. Bartsch H, Nair J, Owen RW: Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20:2209-18. Moreover PUFA incorporation induces an alteration of SFA, MUFA and PUFA content in membrane phos- pholipids; these data suggest a metabolic rearrangement in cells in order to try to balance the ratio between satu- rated and unsaturated fatty acids. 2. Rose DP, Connolly JM: Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther 1999, 83:217-44. 3. Hardman WE: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132:3508S-12S. 4. Lee CY, Sit WH, Fan ST, Man K, Jor IW, Wong LL, Wan ML, Tan-Un KC, Wan JM: The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 2010, 36(4):991-8. 4. Lee CY, Sit WH, Fan ST, Man K, Jor IW, Wong LL, Wan ML, Tan-Un KC, Wan JM: The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 2010, 36(4):991-8. In addition membranes constitute a meeting point for lipids and proteins. Thousands of cellular proteins inter- act with membranes in different ways, for example inte- gral (transmembrane, as EGFR) proteins are embedded in the lipid bilayer and their activity is sensitive to changes in the lipid environment. Recently, multiple stu- dies demonstrated very rapid ERa actions at level of the plasma membrane [45]. O’Malley and collaborators have demonstrated that ERa on the membrane initially acti- vates cytoplasmic kinases, which in turn phosphorylate and activate coactivators proteins in the cytoplasm. These coactivators then travel to the nucleus and modu- late ERa-mediated transcriptional events [46]. 5. Habermann N, Schön A, Lund EK, Glei M: Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo. Apoptosis 2010, 15:621-630. 5. Habermann N, Schön A, Lund EK, Glei M: Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo. Apoptosis 2010, 15:621-630. 6. Larsson SC, Kumlin M, Ingelman-Sundberg M, Wolk A: Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Authors’ contributions PAC carried out cell treatments, MTT tests, WB assays and drafted the manuscript, GM performed lipid analysis, SZ was responsible for cell cultures, IEJ performed lipid analysis, AC performed lipid analysis, BB Coordinated the study, AMR conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. PAC carried out cell treatments, MTT tests, WB assays and drafted the manuscript, GM performed lipid analysis, SZ was responsible for cell cultures, IEJ performed lipid analysis, AC performed lipid analysis, BB Coordinated the study, AMR conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. All authors have read and approved the final manuscript. References Am J Clin Nutr 2004, 79:935-945. 7. Serini S, Piccioni E, Merendino N, Calviello G: Dietary polyunsaturated fatty acids as inducers of apoptosis: implications for cancer. Apoptosis 2009, 14(2):135-52. 8. Field CJ, Schley PD: Evidence for potential mechanisms for the effect of conjugated linoleic acid on tumor metabolism and immune function: lessons from n-3 fatty acids. Am J Clin Nutr 2004, 79:1190S-8S. 8. Field CJ, Schley PD: Evidence for potential mechanisms for the effect of conjugated linoleic acid on tumor metabolism and immune function: lessons from n-3 fatty acids. Am J Clin Nutr 2004, 79:1190S-8S. lessons from n 3 fatty acids. Am J Clin Nutr 2004, 79:1190S 8S. 9. Das UN: A radical approach to cancer. Med Sci Monit 2002, 8:RA79-92. K hh SK D PP S RH S SS Gh h SN Eff f y 9. Das UN: A radical approach to cancer. Med Sci Monit 2002, 8:RA79-92. 10. Kachhap SK, Dange PP, Santani RH, Sawant SS, Ghosh SN: Effect of n-3 fatty acid (docosahexaenoic acid) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem 2001, 16:257-63. 10. Kachhap SK, Dange PP, Santani RH, Sawant SS, Ghosh SN: Effect of n-3 fatty acid (docosahexaenoic acid) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem 2001, 16:257-63. Then n-3 PUFAs, modifying the unsaturated degree, the permeability, the flip-flop process and the fluidity of the plasma membrane, might alter the activity of these proteins. This hypothesis will be investigated in our laboratory. 11. Albino AP, Juan G, Traganos F, Reinhart L, Connolly J, Rose DP, Darzynkiewicz Z: Cell cycle arrest apoptosis of melanoma cells by docohexaenoic acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60:4139-45. 11. Albino AP, Juan G, Traganos F, Reinhart L, Connolly J, Rose DP, Darzynkiewicz Z: Cell cycle arrest apoptosis of melanoma cells by docohexaenoic acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60:4139-45. 12. Chiu LCM, Ooi VEC, Wan JMF: Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 2001, 19:845-9. 12. Chiu LCM, Ooi VEC, Wan JMF: Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 2001, 19:845-9. Acknowledgements This paper is dedicated to the memory of Prof. Bruno Berra, who dedicated his life to the biochemistry of lipids. This paper is dedicated to the memory of Prof. Bruno Berra, who dedicated his life to the biochemistry of lipids. Financial support to Dr Angela M. Rizzo came from VI PQ: Nutra Snack Project and from the Italian Space Agency (ASI). Abbreviations PL: (phospholipid); FA: (fatty acid); PUFA: (polyunsaturated fatty acid); MUFA: (monounsaturated fatty acid); SFA: (saturated fatty acid); DHA: (docosahexaenoic acid); EPA: (eicosapentaenoic acid); AA: (Arachidonic acid); PE: (phosphatidylethanolamine); PI: (phosphatidylinositol); PC: (phosphatidylcholine); PS: (phosphatidylserine); SM: (sphingomyelin). Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 6 April 2011 Accepted: 12 May 2011 Published: 12 May 2011 Received: 6 April 2011 Accepted: 12 May 2011 Published: 12 May 2011 Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Page 15 of 16 Page 15 of 16 described that are responsible for the ATP-dependent transport of lipids. The best characterized activity is flip- pase, which transports PS from the outer monolayer to the cytoplasmic surface of the plasma membrane and requires ATP and Mg2+ but its activity is inhibited by Ca2+. A second ATP-dependent activity, catalysed by flip- pases, transports lipids in the opposite direction. The third class of lipid transporter consists of the Ca2+-acti- vated scramblases that catalyse the PS externalization [41,42]. Growing evidence indicates that excessive con- centrations of FA affect cell functions by altering the activity of various ion transporters and channels, includ- ing Ca2+. Zhang et al. have found that PUFAs, but not monounsaturated or saturated FAs, cause [Ca2+]i mobili- zation in NT2 human tetracarcinoma cells by causing release of this proton from mitochondria [43]. Further- more, Djemli-Shipkolye et al. showed that FA modifica- tions in membranes could be correlated with the variations observed in the activity of ATPase, for instance of Mg-ATPase [44]. This effect could influence the flip- pase and scramblase activities, and thus the transbilayer lipid asymmetry. Corsetto et al. Lipids in Health and Disease 2011, 10:73 http://www.lipidworld.com/content/10/1/73 Ligand binding to EGFR induces its dimerization with another EGFR or with other members of the ErbB family, and activates tyrosine kinase residues on the intracellular domains of the protein through autophosphorylation. EGFR recruits downstream signalling proteins, triggering signal cas- cades along a number of pathways that eventually lead to cell growth, migration and apoptosis resistance [38,39]. We found that the phosphorylated EGFR levels are reduced after treatment with n-3 PUFAs (EPA or DHA) in MDA-MB-231 cells, whereas the EGFR level was decreased only after incubation with DHA. 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J Nutr 2011, 14(2):201-6. doi:10.1186/1476-511X-10-73 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: g 34. 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